JOURNAL OF FOOD, AGRICULTURE & ENVIRONMENT, Print ISSN:1459-0255 / Online ISSN:1459-0263

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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014
WFL Publisher
www.world-food.net

Vol. 12, No. 3&4
CONTENTS
July-October 2014
Chemical composition of pumpkin (Cucurbita maxima D.)

flesh flours used for food
Food & Health

Reduction of foodborne pathogens in parsley by an improved
formulation containing lime and oregano extracts
Jurgita Kulaitien, Elvyra Jarien, Honorata Danilenko, Judita

erniauskien, Agata Wawrzyniak, Jadwiga Hamulka and
Edita Jukneviien
61
Alany Celestino, Brianda Jaime, Ricardo Luvano, Luisa Sols, Santos

Garca and Norma Heredia
6
Training a sensory panel for describing texture in peach and

nectarines
Food security of Northwest China under current water
Loreto Contador, Paulina Shinya, Andrea Bunger, Carmen Senz and

Rodrigo Infante
65
resources and food consumption patterns

Jianping Li, Jing Chen and Zhouping Shangguan
12
Modified atmosphere storage of banana (Musa acuminata)

using diffusion channel and mathematical modelling of steadystate oxygen concentration within the package
Arulselvam Karthiayani, Nachimuthu Varadharaju and
Madasamy Siddharth
19
Anti-inflammatory and anti-cancer effects of -carotene,

extracted from Dunaliella bardawil by milking
24
Effects of some technological parameters on chemical and

sensory qualities and free fatty and amino acids of various
probiotic cultures in Beyaz cheese during ripening process
32
77
The in vitro antibiofilm activity of Rosmarinus officinalis L.

essential oil against multiple antibiotic resistant
Pseudomonas sp. and Staphylococcus sp.
Effect of storage time and temperature on the quality

characteristics of chicken eggs
Yeasmin Akter, Azhar Kasim, Hishamuddin Omar and Awis Qurni
Sazili
87
40
Improving the layout of ventilation ports in packaging for fresh

produce using computational fluid dynamics
Hiroaki Kitazawa and Naoko Hasegawa
Inhibitory effect of gamma radiation in degrading and

preventing fungal toxins
Ozgur Ceylan, Aysel Uur, Nurdan Sara, Filiz Ozcan and Tuba
Baygar
82
The effects of mint (Mentha spicata) essential oil fortified

edible films on the physical, chemical and microbiological
characteristics of lor cheese
Gkhan Kavas and Nazan Kavas
Muhammad T. Sultan, Masood S. Butt, Saeed Akhtar, Atif N. Ahmad,

Mubasher Rauf, Muhammad S. Saddique and Ambreen Naz
71
Amira Hassan Abdullah Al-Abdalall
Abeer M. Badr, Effat F. Shabana, Hoda H. Senousy and

Hend Y. Mohammad
Filiz Yanglar and Salih Ozdemir
Antioxidant and antimicrobial potential of dried cumin

(Cuminum cyminum L.), caraway (Carum carvi L.) and
turmeric powder (Curcuma longa L.)
46
Evaluation of the antioxidant activity of extracts from Psidium

guajava L. and Anacardium occidentale L. leaves obtained by
different extraction methods
Suzara R. C. Sena, Theresa R. F. Dantas and Camila G. Pereira
93
Validation of ELISA-based detection of L. monocytogenes and

E. coli O157:H7 in fresh cut vegetables
Peanut protein isolates improve the nutritional quality of

muffins that can be handy tool to cure protein energy
malnutrition in developing economies
Muhammad Sibt-e-Abbas, Masood Sadiq Butt, Muhammad Tauseef
Sultan, Atif Nisar Ahmad, Muhammad Abrar and Mir Muhammad
Nasir Qayyum
51
Assessment of microbiological quality of ready-to-eat foods in

Istanbul, Turkey
Vecdet z, Sukriye Karadayi, Hseyin akan, Beytullah Karadayi and
Filiz Ekim evik
56
Marina Cavaiuolo, Antonio Ferrante, Spiros Paramithiotis, Agni

Hadjilouka, Periklis Tzamalis and Eleftherios H. Drosinos
98
Composition and content of selected elements of Croatian

blackberry wines
Ivana Alpeza, Tatjana Varga and Veronika Kubanovi
100
Physicochemical properties of honey samples from Ondo state,

Nigeria, and their bioactivity against spoilage and pathogenic
organisms
Funmilola Oluyemi Omoya, Oluwatosin Ademola Ijabadeniyi and
Olayemi Bosede Ogonnoh
104
ii
Shigatoxin-producing Escherichia coli in raw cow milk from

small farm producers and phylogenetic subtype determination
Genotypic variability for nutrient, antioxidant, yield and yield

contributing traits in vegetable amaranth
Ivo Sirakov, Ralitsa Popova, Hristo Daskalov, Iskra Slavcheva, Eva

Gyurova and Boyko Mitov
108
Umakanta Sarker, Md. Tofazzal Islam, Md. Golam Rabbani and

Shinya Oba
168
Pre-processing glutinous rice effects on textural and

morphological changes
Respiratory activity of rice seeds stored for 10 years at
Viboon Pansa-ead, Kannika Huaisan, Supan Yangyeun and

Songchai Wiriyaumpaiwong
different temperatures
115
Agriculture
Effect of dietary crude palm oil on quality and oxidative stability

of chicken eggs
Assessment of performance ability of Cabernet Sauvignon,

Merlot and Syrah wine cultivars on Southeast region of Turkey
Dilek Deirmenci Karata and Hseyin Karata
122
Dry matter accumulation trend on corn (TWC 647) as affected

by plant density and planting pattern
Ali Reza Saberi and Siti Aishah Hassan
Fabola de Oliveira Krger, Dario Munt de Moraes, Daniel Fernandes

Franco, Caroline Jcome Costa, Chaiane Fernandes Vaz and
Paula Rodrigues Gayer Ribeiro
175
127
Yeasmin Akter, Azhar Kasim, Hishamuddin Omar and Awis Qurni

Sazili
179
Evaluation a polyvalent vaccine against abscess disease of

sheep from pathogenic bacteria isolated from Saudi Arabia
K. B. Alharbi
Performance evaluation of the Telagasari Irrigation Scheme

(TIS) of Karawang Regency, Indonesia
Productivity and gas exchange parameters of selected pasture

grasses under drought stress
Sangam Shrestha, Foyya Yusufu Aquino and Vishnu P. Pandey
Anna Koco and Mariola Staniak
Biodegradation of imidacloprid in an open compost pile
131
Effect of sulphur fertilization on fatty acid composition of faba

bean (Vicia faba L.), white lupin (Lupinus albus L.) and pea
(Pisum sativum L.) grains
Eugenio Cazzato, Vito Laudadio, Edmondo Ceci and
Vincenzo Tufarelli
136
Yield and quality of Cenchrus ciliaris (L.) affected by nitrogen

and phosphorus fertilization
Ihsan Abu-Alrub, Ahmed Aran, Omar Hamad and
Abdelaziz Awaga
139
Meo Maksim, Murrja Arif, Ndregjoni Agim and Cerpja Teuta
143
Improved water use efficiency in rice under limited water

environment through microbial inoculation
Numerical investigation into optimal agricultural water

management for typical soils using HYDRUS-1D model
Po Li, Feiqing Wu and Kefeng Zhang
155
Effects of allelopathic crop water extracts and their

combinations on weeds and yield of rainfed wheat
Shahbaz Hussain, Fayyaz-ul Hassan, Muhammad Rasheed, Safdar Ali
and Mukhtar Ahmed
161
198
Potential methane production from manure of cattle fed diet

supplemented with wet brewery grain
Larissa S. Mallmann, Simone Damasceno, Maximiliane A. Zambom,
Mnica S. S. M. Costa, Douglas G. B. Torres and
Jefferson L. G. Silva
203
Influence of the auxin-like activity of humic acid on bio and

microbiometric parameters of Pisum sativum L. by in vitro
cultures of pea plants
209
Biotransformation of 2-(4-methoxybenzyl)cyclopentanone by
Solanum aviculare and Rheum palmatum plant cells
Petr Soudek, Zdenk Wimmer and Tom Vank
Mohamad Husni Omar, Zulkarami Berahim, Norazrin Ariffin, Mohd

Razi Ismail, Halimi Mohd Saud, Nurul Amalina, S. H. Habib and
H. Kausar
149
187
eljko Herner, Renata Baok and Felicita Briki
Andrzej Gawlik, Danuta Kulpa, Dorota Gobiowska and

Romualda Bejger
Some alternatives of improvement the cow milk production

efficiency in Albania: Cash flow analysis
182
213
Impact of nitrogen fertilisation and irrigation on water utilization

efficiency, N accumulation, growth and yields of Zea mays L.
Waleed Hassan Abou El-Hassan, Emad Maher Hafez, Alaa A. Ahmed
Ghareib, Mohamed Ragab Freeg and Mahmoud Fathy Seleiman 217
Effect of water stress on yield components of sorghum

(Sorghum bicolor)
Cndido Ferreira de Oliveira Neto, Ricardo Shigueru Okumura, Ismael
de Jesus Matos Vigas, Herclito Eugnio Oliveira da Conceio, Lucila
Elizabeth Fragoso Monfort, Raimundo Thiago Lima da Silva, Jackeline
Arajo Mota Siqueira, Luma Castro de Souza, Roberto Cezar Lobo da
Costa and Daiane de Cinque Mariano
223
iii
Linseed response to treatment with swine wastewater as

biofertilizer
Jhonatas Antonelli, Cleber Antonio Lindino, Reginaldo Ferreira
Santos, Samuel Nelson Melegari de Souza, Willian Czar Nadaletti,
Paulo Cremonez, Eduardo Rossi and Flvio Gurgacz
229
Thermic sum and crop coefficient of canola (Brassica napus L.)

for the region of Tangar da Serra, Mato Grosso State, Brazil
Kssio De Marco, Rivanildo Dallacort, Adalberto Santi, Ricardo
Shigueru Okumura, Mirian Hiroko Inoue, Joo Danilo Barbieri,
Dejnia Vieira de Araujo, Roberto Antnio Savelli Martinez and Willian
Fenner
232
Soil health sustainability and organic farming: A review

Sudarsan Biswas, Md. Nasim Ali, Rupak Goswami and Somsubhra
Chakraborty
237
Ergonomic assessment of traditional and improved methods of

paddy threshing for drudgery reduction of hill region
Divya Singh and Deepa Vinay
244
Phenology and green leaf yield of coriander at different sowing

dates and harvesting times
Sagarika Guha, Amit Baran Sharangi and Sandip Debnath
251
Applications of xerophytophysiology in plant production:

Potato yield increase induced by drying the cut trace of seed
tuber blocks
Feifei Su, Hui-lian Xu, Fenglan Li, Feifei Qin, Yili Chen, Dianqiu L,
Linshuang Hu, Yong Li, Shaopeng Wang and Ying Shi
255
Hydric and physiological monitoring of soybean in oxisol and

oxisol incorporated with biodegradable waste residue
Alexandre C. Salvestro, Paulo Sergio Loureno de Freitas, Roberto
Rezende, Erci Marcos Del Quiqui, Cludia Regina Dias Arieira, Jos
Carlini Junior, Magnun Rodrigo Silva, Vinicius Hicaro Frederico
Abe and Matheus A. Mendes
265
Sowing dates and plant density of peanut cultivars in different

soil and climatic conditions of Mato Grosso state, Brazil
Joo Danilo Barbieri, Rivanildo Dallacort, Adalberto Santi, Kssio
De Marco, Alcir Jos Modolo, Santino Seabra Jnior, Ronicely
Pereira Rocha and Rafael Cesar Tieppo
269
Effect of composted sewage sludge on durum wheat:

Productivity, phenolic compounds, antioxidant activity, and
technological quality
Antonella Pasqualone, Laura Nunzia Delvecchio, Giovanni Lacolla,
Luciana Piarulli, Rosanna Simeone and Giovanna Cucci
276
Genetic diversity and presence of DREB gene in watermelon

cultivars and wild type of watermelon based on molecular
markers
Abdullah S. Alsohim and Mohamed I. Motawei
281
Impact of PAR interception at different time points on total

dry matter production in rice (Oryza sativa L.) crop
transplanted on different dates
Shrabani Basu, Srijani Maji, Swaraj Kumar Dutta , Sarika Jena,
Rajib Nath and Prodip Kumar Chakraborty
285
Growth and visual symptoms of nutrient deficiencies in young

Mentha piperita plants
Diocla Almeida Seabra Silva, Mrio Lopes da Silva Jnior, Ismael de
Jesus Matos Vigas, Allan Klynger da Silva Lobato, Vnia Silva de
Melo, Snia Maria Arajo Botelho, George Rodrigues da Silva, Joze
Melisa Nunes de Freitas, Cndido Ferreira de Oliveira Neto, Milton
292
Leite Alves da Cunha and Ana Regina da Rocha Araujo
Decrease in photosynthetic pigments promotes negative

consequences on carbon compounds in young Euterpe oleracea
plants submitted to progressive water deficiency
Emilly dos Santos Pereira, Allan Klynger da Silva Lobato, Odyone
Nascimento da Silva, Argemiro Pereira Martins Filho, Carla Leticia
Figueredo de Carvalho Souza, Tiago Rodrigues Ferreira, Gustavo
Antonio Ruffeil Alves, Elaine Maria Silva Guedes, Ismael de Jesus Matos
Vigas, Ricardo Shigueru Okumura, Augusto Jos Silva Pedroso, Roberto
Cezar Lobo da Costa and Benedito Gomes dos Santos Filho
297
Environment
Microbes and dietary values of some major fish sources
in Nigeria
Olajide Adedayo Ajayi, Emmanuel I. Adeyeye and Anthony I.
Okoh
303
Impact of micro credit and training on efficiency of smallscale entrepreneurs: Evidence from National Directorate
of Employment (NDE) loan/training programmes in
Nigeria
Olumide Oyewole Akinrinola, M. M. Fasoranti and Oluyede
Adeleke Aturamu
307
Modulation of micronutrients and antioxidants defenses in

Conocarpus lancifolius under abiotic stress
Amina Redha, Redha Al-Hasan and Mohammad Afzal
312
Heavy metal distribution in Fagonia indica and Cenchrus

ciliaris
native vegetation plant species
Adel M. Ghoneim, Soud M. Al-Zahrani, Salem E. El-Maghraby
and Abdullah S. Al-Farraj
320
Performance of lateral move type sprinkler irrigation

system
Jonathan Dieter, Silvio C. Sampaio, Gisele Vogel , Mrcio A. Vilas
Boas, Elisandro P. Frigo and Alvaro Mari Junior
325
Improving water quality in the Nile Delta irrigation

network by regulating reuse of agricultural drainage
water
Abd Elhamed Khater, Yoshinobu Kitamura, Katsuyuki Shimizu ,
Hiroaki Somura and Waleed H. Abou El Hassan
329
iv
Antioxidant potential and secondary metabolite content of

grape berries influenced by microclimate
Trophic State Index (TSI) applied in the assessment of anthropic
Mustafa Ozden
Adir Otto Schmidt, Slvio Cesar Sampaio, Ralpho Rinaldo dos Reis, Camila
Jussara Schmidt, Edison Barbosa da Cunha and Lisdefferson Hamann
Andrade
400
338
Tourists perception on local economy of Terengganu state

in Malaysia
Md. Anowar Hossain Bhuiyan, Chamhuri Siwar, Shaharuddin
Mohamad Ismail and Aini Aman
345
Identification of brownfields in China: Concept, procedure

and practice
Hongbin Xie, Mingshui Lin , Changchun Zhou, Yang Zhang and
Liangjin Zhou
349
Evaluation of the microbiological and physicochemical quality

of artesian well water used for irrigation in ArRiyadh
Sulaiman Ali Alharbi, M. E. Zayed, Arunachalam Chinnathambi, Naiyf
S. Alharbi and Milton Wainwright
355
X-ray diffraction (XRD) and x-ray fluorescence (XRF) analysis

of ancient bricks from Sungai Batu Temple (site SB1), Bujang
Valley, Kedah
impacts on the surface water of a watershed
Seasonal elemental variations of Fe, Mn, Cu and Zn and

conservational management of Rastrelliger kanagurta fish from
Karachi fish harbour, Pakistan
Quratulan Ahmed, Farzana Yousuf, Maliha Sarfraz, Nor Kartini Abu
Bakar, Mansour A. Balkhour and Muhammad Aqeel Ashraf
405
The use of urea molasses multinutrient block on pica symptom

of cattle
Haili Li, Keling Wang, Limin Lang, Yindi Xu, Qinxian Zhang, Wenhao
Zhu, Lixian Zhang, Yi You, Feng Xu and Wan Lu
415
Views and attitudes of mulberry cultivators according to the

Regulation (EC) No 1257/99: The case of Evros prefecture
S. Ch. Tsiantikoudis , A. Parissi , Z. Papadopoulou , A-M. Fidani-Mantzoula,
420
G. Kourtelis and Z. Andreopoulou
Zuliskandar Ramli, Nik Hassan Shuhami Nik Abdul Rahman, Abdul

Latif Samian, Muhammad Rizal Razman, Sharifah Zarina Syed Zakaria
and Hossein Sarhaddi Dadian
360
A proposal to standardize herbicide sorption coefficients in

Brazilian tropical soils compared to temperate soils
Estimating residents willing to pay using contingent valuation

for ecological restoration and recreational benefits of AL-Prespa
Kassio Ferreira Mendes, Marcelo Rodrigues Dos Reis, Ana Carolina

Ribeiro Dias, Jos Ari Formiga , Pedro Jacob Christoffoleti and Valdemar
Luiz Tornisielo
424
protected area in Albania

Dorina Grazhdani
365
Jatropha cake as a fertilizer for the growth of Blc. Amy Wakasugi

Yamanashi orchid
Roberto A. Ribeiro, Maria Flvia R. Starling and Luiza A. R.
Rossi-Barbosa
371
In vitro regeneration of Acacia crassicarpa A. Cunn Ex Benth

through organogenesis from juvenile sources
Griffin Akeng, Sures Kumar Muniandi and Nor Aini Ab Shukor
375
Assessing salt-affected degraded soils using remote sensing.

Case study: Al-Qassim region, Saudi Arabia
Abdulla S. Modaihsh, Abdelazeem Sh. Sallam, Adel M. Ghoneim and
Mohamed O. Mahjoub
383
Determination of pond water quality for aquaculture and

ecosystem management
Umme Shahina Khanom, Sabrina Sharmeen, Jannatul Ferdouse, Wahhida
Shumi, Arifin Abdu, Hazandy Abdul Hamid and Md. Aktar Hossain 389
The relocation of undisturbed soil in long-term experiment

impacts organo-mineral complex degree and combined humus of
black soil
Fengqin Chi, Enjun Kuang, Baoku Zhou, Jiiuming Zhang, Qingrui Su
and Shanshan Cai
434
Environmental guidelines of So Francisco Verdadeiro river

according to Brazilian standards
Kayla W. Garmus, Silvio C. Sampaio, Maria Hermnia F. Tavares, Adir
Otto Schmidt and Marcelo Remor
439
Isolation and identification of microorganisms from soil in a

young oil palm plantation
Nur Masirah Mohd Zain, Rosli B. Mohamad, Kamaruzaman Sijam and
Yahya Awang
443
Monitoring larval populations of Aedes aegypti in different

residential districts of Jeddah governorate, Saudi Arabia
Khalid M. Al-Ghamdi , Abbas M. Al-Azab, Hassan M. Khormi, Lalit
Kumar and Jazem A. Mahyoub
448
Impact of pre-sowing treatment on seed germination and seedlings

growth attributes of Calamus longisetus Griff. at nursery and
field conditions
M. Rafiqul Haider, Md. Sah Alam, Md. Aktar Hossain and
Nor Aini Ab. Shukor
395
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Meri-Rastilantie 3 B, FI-00980
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e-mail: info@world-food.net
Journal of Food, Agriculture & Environment Vol.12 (3&4): 6-11. 2014
www.world-food.net
Reduction of foodborne pathogens in parsley by an improved formulation containing

lime and oregano extracts
Alany Celestino, Brianda Jaime, Ricardo Luvano, Luisa Sols, Santos Garca and Norma Heredia *
Departamento de Microbiologa e Inmunologa, Facultad de Ciencias Biolgicas, Universidad Autnoma de Nuevo Len, Apdo.
Postal 124-F, San Nicols, Nuevo Len, Mxico 66451. *e-mail: norma@microbiosymas.com
Received 18 May 2014, accepted 24 September 2014.
Abstract
Parsley (Petroselinum crispum-Apiaceae) has been reported as a vehicle for Salmonella and E. coli O157:H7; and Shigella sonnei has been responsible
for shigellosis outbreaks. Plant-derived extracts have been proposed as alternatives to reduce food contamination without modifying food properties,
and several extracts have decreased pathogenic bacterial growth in vegetables. The objective of this study was to reduce E. coli O157:H7, Salmonella
and Shigella levels inoculated in parsley, after washing with an improved formulation of edible vegetable extracts. Extracts from five edible plants,
resuspended in ethanol, were tested for antimicrobial activity and the minimum bactericidal concentration (MBC) determined. Extract mixtures were
analysed for synergistic activity. The mixture exhibiting synergism was used to wash parsley samples previously inoculated with 105 bacterial cells/
g. Following 0, 1, 3, 5, and 7 d, viable bacterial counts were determined. Chlorine, Citrol, and ethanol were used as controls. Mexican lime and Mexican
oregano extracts (4.3 - 4.8 and 1.5 - 2.0 mg/ml MBC, respectively) were selected. Synergistic antimicrobial effects were observed under a 1.25:0.19
mg/ml mixture. The mixture exhibited a > 2 log reduction in the bacterial level in parsley on the first day. In this study, we followed a simple, low cost,
and laboursaving extraction system. The antimicrobial efficacy of the improved formulation was clearly demonstrated on parsley. Considering human
health and environmental hazards associated with chlorine use, the Mexican lime:oregano mixture provides a viable alternative to chlorine, and is
equally effective at significantly reducing bacterial pathogens associated with outbreaks stemming from leafy green vegetables.
Key words: Escherichia coli, Salmonella, Shigella, produce contamination, natural antimicrobials, parsley.
Introduction
Foodborne diseases (FD) caused by pathogenic microorganisms
remain a major cause of morbidity and mortality worldwide. Fresh
produce consumption has increased as a result of healthier
lifestyles. However, fresh produce is often consumed minimally
processed or raw, and raw foods are known vehicles for human
disease 1, 2. In 2008, WHO placed leafy greens at its highest priority
for fresh produce safety at a global level due to its potential to
cause widespread FD outbreaks 3. The most notably, Escherichia
coli O157:H7, Salmonella sp. and Shigella sp. have been
associated with FD following contaminated leafy green vegetable
consumption. Parsley (Petroselinum crispum-Apiaceae) has been
reported as a vehicle for Salmonella and E. coli O157:H7 4, 5; and
Shigella sonnei was responsible for eight shigellosis outbreaks
in Canada and the US 6.
A great variety of compounds and intervention measures have
been tested and used to reduce or eliminate foodborne pathogens
from produce. Chlorine is a common disinfectant used to
decontaminate fresh produce 7; however, a chlorine wash cannot
completely remove or inactivate microorganisms, and it can
generate chlorinated organic compounds, which result in safety
concerns for humans 8.
Synthetic chemical compounds have been applied by the food
industry to control microbial contamination, however, the new
trends of consumers include ingesting fewer synthetic food
additives, and consuming more natural or all-natural foods 9, 10.
The antimicrobial activities of many essential oils have been
reported, and studies have demonstrated these compounds exhibit
activity against foodborne pathogens 10. Yet, essential oils can be
6
costly to extract, many are not acceptable for industrial processing

and some alter the organoleptic properties of foods 3, 10. However,
plant-derived extracts have been proposed as alternatives to reduce
food contamination without modifying food properties 11, 12, and
several extracts have decreased pathogenic bacterial growth in
vegetables 3, 11, 13. In the present study, we examined the efficacy
of six edible plant extracts in reducing Salmonella, Shigella, and
E. coli O157:H7 in parsley, developed an improved formulation,
and compared the extract performance to chlorine and a commercial
citrus-base sanitizer.
Materials and Methods
Bacterial cultures: Escherichia coli O157:H7 ATCC 43890 GFP
(modified with green fluorescent protein gen as marker), E. coli
ATCC 43895, E. coli O157:H7 ATCC 43894, Shigella sonnei F2353
(modified with green fluorescent protein gen as marker), S. flexneri
ATCC12022, Salmonella Typhi ATCC19430, and S. Typhimurium
ATCC 14028 (serovars of Salmonella enterica subsp. enterica)
were used in this study. All strains were maintained at -80C in
Brain Heart Infusion (BHI) broth (Difco) containing 20% (v/v)
glycerol. Fresh cultures were prepared by inoculating an aliquot
of stored culture in fresh BHI broth, and subsequently incubated
at 37C for 24 h. An aliquot of this culture was plated onto BHI
agar, and incubated at 37C for 24 h. Colonies were suspended in
saline solution, and adjusted to a 1.5 x 105 CFU/ ml concentration.
Plant extracts: Five edible plant species (Table 1) were purchased
from retail markets in the metropolitan area of Monterrey, Nuevo
Table 1. Plants analysed, common names and parts used.

Scientific name
Citrus aurantifolia (Christm.) Swingle
Hibiscus sabdariffa L.
Lippia graveolens Kunth
Origanum majorana L.
Tamarindus indica L.
Common name
Mexican lime
Roselle
Oregano
Sweet marjoram
Tamarind
Part used
Peel
Flower
Leaves
Leaves
Peel / Pulp of fruit
Leon, Mexico. Plant material was surface washed with distilled

water and dried for 24 h at 50C. Twenty grams of each sample
were immersed in 100 ml of 96% ethanol, and ground using a mixer
to extract soluble plant material. Extracts were macerated at room
temperature for 48 h 14. Macerated samples were filtered through
Whatman #1 paper, and placed on glass plates to facilitate complete
ethanol evaporation (approximately 48 h at room temperature).
Dried extracts were resuspended in 10-15 ml of 96% ethanol,
centrifuged (Eppendorf Model 5414C) at 14,000 rpm for 15 min,
supernatants removed, and filter-sterilized using nitrocellulose
membranes (Millipore, 0.45-m pore size). Samples were collected
in sterile amber flasks, and maintained at 4C until experimental
procedures were performed (maximum 6 mos) 12. An aliquot of
each extract was used to determine dry weight.
Antimicrobial activity test: Preliminary screening for antimicrobial
activity was conducted using the agar diffusion well test 12. Briefly,
Petri dishes (150 mm) were filled with 25 ml Mueller-Hinton (MH)
agar (Difco), and surface inoculated with aliquots (100 l) of the
bacterial culture (1.5 105 CFU/ ml). Five holes (5 mm in diameter)
were subsequently made on the inoculated agar plate, and each
filled with 100 l of each extract. Ethanol (96%) was used as a
negative control. The plates were incubated at 37C for 48 h.
Inhibitory activity was visualised as the reduction or cessation of
bacterial growth in the area surrounding the holes, and quantified
by measuring the inhibition zone diameter.
Determination of minimum bactericidal concentration (MBC):
A range of extract concentrations (10 to 1 mg/ml) 13, 15 was added
to sterile 96-well polystyrene U-microtiter plates (BD Falcon) filled
with 150 l of 2X MH broth (Difco), then distilled sterile water was
added to reach a final volume of 300 l. Active cultures of each
pathogen (3 l, 1.5 105 CFU/ml) were used to inoculate each
plate, and subsequently incubated at 37C for 24 h. Following
incubation, the content of each well was plated on MH agar and
incubated at 37C for 48 h. The MBC was regarded as the lowest
extract concentration that prevented any visible bacterial colony
growth (total absence of colonies) on the MH agar plate after the
48 h incubation period.
Extract mixture analysis: The efficacy of the extract mixtures
was established by evaluating the natural antimicrobials
individually or in combination with other extracts. The extracts
exhibiting the highest antimicrobial activities were mixed, and the
effects against bacteria were determined by the checkerboard
method following Orhan et al. 16, with minor modifications. Sterile
96-well microtiter plates were filled with 50 l of 2X MH broth,
plus 50 l of an extract mixture (containing lower concentrations
than the MBC of each extract) (Table 2). Plates were inoculated,
incubated, and plated as described above. Mixtures and synergism
effects were evaluated by the fractional inhibitory concentration
index (FIC) 13. The FIC was defined as the sum of the MBC of each
Table 2. Extract combinations at different concentrations

determined by the checkerboard method following Orhan
et al. 16.
Mexican
lime extract
(mg/ml)
lower row
0.05
0
0.05
0.312
0.05
0.625
0.05
1.25
0.05
2.5
0.05
5
0.05
10
Oregano extract
(mg/ml) upper row
0.1
0.19
0.38
0
0
0
0.1
0.19
0.38
0.312 0.312 0.312
0.1
0.19
0.38
0.625 0.625 0.625
0.1
0.19
0.38
1.25
1.25
1.25
0.1
0.19
0.38
2.5
2.5
2.5
0.1
0.19
0.38
5
5
5
0.1
0.19
0.38
10
10
10
0.75
0
0.75
0.312
0.75
0.625
0.75
1.25
0.75
2.5
0.75
5
0.75
10
1.5
0
1.5
0.312
1.5
0.625
1.5
1.25
1.5
2.5
1.5
5
1.5
10
3
0
3
0.312
3
0.625
3
1.25
3
2.5
3
5
3
10
extract when applied in combination, divided by the MBC of each

extract applied individually 16. A synergistic effect was defined as
a FIC value < 0.5, an indifferent effect was defined as a FIC value
between 0.5 and 2, and an antagonistic effect was defined as a FIC
value 2 16.
Determination of leafy green decontamination effectiveness:
The method for assessing decontamination effectiveness
followed Lang and Harris 17, and Orue et al. 13, with minor
modifications.
Inoculum preparation: The assay was performed using E. coli
O157:H7 ATCC 43890, S. sonnei F2353, and S. Typhimurium ATCC
14028. A sterilised loopful of fresh cultures grown on BHI agar
was inoculated into tubes with 10 ml tryptic soy broth (TSB, Difco),
and incubated at 37C for 18 h. Cells were collected by
centrifugation (3000 rpm, 10 min at room temperature), and the
pellet resuspended in 1% peptone water. Similar volumes of each
strain were mixed into a cocktail, and the suspension (1.5 105
CFU/ml) was used as inoculum.
Parsley inoculation: Parsley was purchased from local markets,
and stored at 4C for a maximum of 2 d before inoculation. The
initial microbe content of the purchased samples was determined
by microbiological analyses (total viable counts and E. coli
O157:H7, Shigella sp., and Salmonella sp. presence) according
to the Bacteriological Analytical Manual 18. When these
pathogenic bacteria were detected in the samples, the vegetables
were discarded.
For the assays, 25 g of parsley samples were cleaned with gentle
washing using running tap water followed by gentle rinsing in
sterile distilled water, and dried for 2 h in a class II biosafety
cabinet (Labconco) at room temperature. Twenty-five aliquots of
10 l of the cocktail strains (adjusted al 1.5 x 105 CFU/ml) were
spotted over the previously disinfected parsley surface 17 in a
biosafety cabinet. The inoculated vegetables were subsequently
air-dried for 15 min at room temperature.
Determination of effectiveness of decontamination agents:
Inoculated parsley was submerged in 225 ml of a respective
1.25:0.19 mg/ml (final concentration) lemon peel and oregano for
20 min. Chlorine (200 ppm) and a grapefruit-based commercial

disinfectant (Citrol K Ultra, Corpo Citrik, S.A. de C.V. Mxico,
D.F.; [200 ppm dissolved in water]) were used in place of plant
extracts and as positive controls. Non-rinsed and water-rinsed
parsley were also used as controls. Parsley was transferred to
sterile bags and stored at 4C for 7 d.
Parsley samples were removed at 0, 1, 3, 5, and 7 d, and the
pathogenic bacteria presence was determined. A 5 g sample was
placed in 45 ml 0.1% peptone water and homogenised. Following
homogenisation, and decimal dilutions, the samples were streaked
in duplicate on XLD agar (for Salmonella, Difco), TSB
supplemented with 0.05 mM isopropyl--D-thiogalactopyranoside
(IPTG, for E. coli O157:H7 GFP) 19 and MacConkey agar (for S.
sonnei GFP). These plates were incubated at 37C for 24 - 48 h.
The TSB and MacConkey agar plates were examined under
ultraviolet light (356 nm, UV SL-58 Ultra-Violet Products), and
green fluorescent colonies were counted. For Salmonella, pink
colonies with or without black centers on XLD agar were
enumerated.
Statistical analysis: All experiments were performed twice, and at
least three replicates were conducted for all samples. SPSS 17.0
was used to compare and analyse efficacy among treatments (SPSS
Inc. Chicago, Illinois). Non parametrical test and Kruskal-Wallis
test were used. ANOVA and Tukey test (P 0.05) were used to
multiple comparison of means.
Results and Discussion
Plant extracts might serve as important alternatives to develop
antimicrobial formulations to control microorganisms, and as food
preservatives 20. Results of our preliminary antimicrobial activity
testing five extracts against growth of E. coli, Salmonella and

Shigella strains narrowed the panel to four active extracts: Mexican
lime, roselle, Mexican oregano, and thyme, which showed growth
inhibition zones for most bacteria ranging from 1.4 to 2.7 cm in
diameter (Table 3). Mexican lime and oregano activity was
congruent with Orue et al. 13; however, inhibition diameter was
smaller than observed in the present study. The resuspension
solvent (PBS) used in Orue et al. 13 might contribute to these
inconsistencies. Furthermore, differences in antimicrobial effects
of the same vegetable type may be attributed to a variety of factors,
including vegetable quality, growth stage at extraction, and field
growth (ecological) conditions 21, 22.
MBC analyses (Table 4) indicated that the most active ethanolic
extracts were Mexican lime (MBC of 4.3 to 4.8 mg/ml) and oregano
(MBC of 1.5 to 2.0 mg/ml). Orue et al. 13 reported MBCs from 5.3 to
8.1 mg/ml from lime extracts resuspended in PBS against
Salmonella, Shigella, and E. coli strains, and MBC of 5.2 to 6.4
mg/ml from oregano extracts against the same bacteria. MBC values
were lower from the extracts applied in the present study. The
solvents used to resuspend the extract (PBS or ethanol) can
increase the efficacy of plant extracts. For example, ethanol,
methanol, and acetone are superior to water in the extraction of
most plant bioactive compounds 23.
Different mixed concentrations of oregano and lime extracts
were tested based on the checkerboard method (Table 2), and the
respective 1.25:0.19 mg/ml lime:oregano mixture exhibited a FIC
synergistic effect. Subsequently, parsley artificially contaminated
with E. coli O157:H7 was washed with this mixture, Citrol, and
chlorine. At time 0 (immediately after washing), chlorine resulted
in a pathogen reduction of 2.4 log CFU/g, Citrol showed a decrease
of 1.9 log CFU/g, and 1.7 log CFU/g was observed with the
Table 3. Antimicrobial effects of plant extracts against Salmonella, E. coli, and Shigella bacteria determined
by the agar well diffusion method. All extracts were prepared using 96% ethanol.
Scientific name
Citrus aurantifolia
(Christm.) Swingle
Hibiscus
sabdariffa L.
Lippia graveolens
Kunth
Origanum
majorana L.
Tamarindus
indica L.
Fruit/
Vegetable
Part
used
Mexican
lime
Diameter of growth inhibition

(cm standard deviation)
S.
S.
E. coli
Typhi
Typhi
43895
murium
19430
14028
S.
flexneri
12022
S.
sonnei
F2353
GFP
1.90.0
2.21.0
2.40.7
1.60.3
1.70.1
2.00.7
1.60.4
2.40.2
1.50.1
1.70.1
2.70.2
2.10.5
NI
NI
NI
NI
NI
E. coli
43890
(GFP)
E. coli
43894
Peel
2.10.1
1.70.2
2.00.3
1.80.3
Roselle
Flower
1.70.2
1.40.2
1.40.2
Oregano
Leaves
2.60.1
2.50.2
Sweet
marjoram
Leaves
NI
NI
Tamarind
Peel
Pulp of
fruit
NI
NI
NI
NI
NI
NI
NI
1.70.4
1.40.2
1.50.4
1.60.2
1.50.1
2.30.4
2.10.8
* NI indicates no inhibition.
Table 4. Minimum bactericidal concentration (MBC) of the selected extracts against E. coli
O157:H7, Salmonella Typhimurium, and Shigella sonnei.
Plant
(common name)
Roselle
Mexican lime
Oregano
Tamarind (pulp)
E. coli
O157:H7
43890 (GFP)
>10
4.30.3*
2.00.1
>10
E. coli
O157:H7
43894
>10
4.70.3
2.00.1
>10
MBC (mg/ml)
S.
E. coli
Typhimurium
43895
47028
>10
>10
4.80.3
4.50.0
1.50.1
2.00.1
>10
>10
S.
Typhi
19430
>10
4.50.0
2.00.1
>10
S.
flexneri
12022
>10
4.50.0
1.50.1
>10
S. sonnei
F2353
(GFP)
>10
4.30.3
1.50.1
>10
* Standard deviation.
lime:oregano extract. The control, washing with water alone,

reduced the pathogen by 0.9 log CFU/g (Table 5). S. Typhimurium
was reduced 2.9 log CFU/g with chlorine, 2.7 log CFU/g using
Citrol, 2.4 log CFU/g with lime:oregano extract, and the water
control resulted in a reduction of 0.9 log CFU/g. Results for S.
sonnei showed chlorine decreased the pathogen by 1.9 log CFU/
g, Citrol by 1.8 log CFU/g, lime:oregano extract reduced the
pathogen by 2.2 log CFU/g, and the water control showed a 1.0
log CFU/g decrease. Significant differences (P 0.05) were
observed among all antimicrobial treatments and water controls
(washed and not-washed); however, significant differences were
not detected among antimicrobial treatments. The number of
microorganisms exhibited a decrease over the seven-day test
period, including the not-washed treatments; however, the
reduction in microorganisms was more notable in the chlorine,
Citrol, and lime:oregano extract treatments (Table 5).
The mesophilic microorganism number was reduced immediately
following all treatments, but increased during the study period.
At day five of the study, these species returned to log CFU levels
comparable to the water washed or not-washed samples (Table 6).
Orue et al. 13 reported that no significant difference in mesophilic
microorganism levels were observed at seven days among samples
washed with aqueous extracts of lime or oregano, and the control,
in accordance with our results.
The synergistic effect between both extracts found in this study
(1.25:0.19 mg/ml, lime:oregano mixture), allowed to reduce
significantly the amount of extracts for the washing solution with
similar efficacy. Previous studies used individual extracts dissolved
in PBS at levels > 5.0 mg/ml of each extract and no synergistic
effect was found 13.
In this study, the formulation was tested against
enterohemorrhagic and non-enterohemorrhagic strains of E. coli,
S. sonnei, S. flexneri and two serovars of Salmonella. The results
exhibited the efficacy of these compounds between different
strains/species.
Chlorine (200 ppm) reportedly removes approximately 1.5 to 2
log CFU/g of background or pathogenic microflora on lettuce,
cilantro, and parsley 8, 24. The chlorine efficacy in our study was
Table 6. Mesophilic organism enumeration on parsley following

different washing treatments.
Log CFU/g
Day
0
1
3
5
7
Not washed
H2O
Chlorine
Citrol
4.70.09*
5.70.18
5.70.11
6.10.16
6.10.16
4.20.25
4.50.06
5.10.16
6.00.05
6.10.16
3.20.16
3.90.67
5.20.24
6.00.05
6.40.09
3.80.14
4.40.31
5.40.09
6.20.31
6.00.0
Mexican
Lime-Oregano
mixture
4.20.16
4.40.09
4.70.04
5.00.00
6.20.16
slightly higher (reducing 2.9 log), and interestingly, the

lime:oregano extract mixture was comparable (P < 0.05) to chlorine
in reducing S. Typhimurium, E. coli O157:H7, and S. sonnei.
The antimicrobial activity of different Citrus species has been
documented. Lemon extract showed activity against Bacillus
licheniformis, Saccharomyces cerevisiae, and Pichia
subpelliculosa 10. Citrus sp. rind has antimicrobial activity against
E. coli and S. aureus growth 25. Studies have demonstrated lime
extract activity against several virulent microorganisms 13, 26.The
antimicrobial activity of Citrus sp. extracts has been established,
however, few applications with the exception of commercial oils
have been developed. Valtierra et al. 12 showed lime extracts alone,
or in a mixture with other edible fruits reduced viability of C. jejuni
and C. coli in vitro, and in chicken skin by more than 4 log CFU/
g. Orue et al. 13 also demonstrated that aqueous lime and oregano
extracts reduced Salmonella, Shigella, and E. coli O157:H7
viability by 2 log CFU/g from cilantro, parsley, and spinach.
Several plants that share similar odor and flavor attributes are
known as oregano. Origanum vulgare L. (Lamiaceae) and
Lippia graveolens Kunth (Verbenaceae) (synon. L. berlandieri
Schauer), are the most studied oregano that exhibit antimicrobial
activity. These plants are commonly used spices with medicinal
properties for the treatment of gastrointestinal and respiratory
illnesses 27 . Lippia graveolens exhibits antioxidant and
antimutagenic activities 28, and the essential oil is active against
Gram-positive, Gram-negative bacteria, and phytopathogenic
molds 27, 29, 30. Extracts of L. graveolens contains high amounts of
phenolics compounds, with flavonoids
as the major constituents. Thymol and
Table 5. Enteropathogenic bacteria enumeration in parsley immediately after washing
carvacrol, and the oil precursors, i.e., pwith different agents (Day 0) or washing and storage at 4C and counts at Days
cymene and -terpinene are considered
5
1, 3, 5, and 7. Inoculum 1.5 10 CFU/ml.
the antimicrobial compounds 30, 31. These
Mexican Lime-Oregano
Not
compounds affect the structure and
mixture
Bacteria
Day
H2O
Chlorine
Citrol
washed
functionality of the bacterial membrane,
(1.25:0.19 mg/ml)
provoking changes in intracellular pH, and
Log CFU/g
0
3.90.02* 2.70.06 1.00.0 1.20.14
1.50.02
alterations in membrane potential and ATP
1
3.70.07 2.40.09 1.00.0 1.50.41
1.40.17
synthesis 32, 33.
Salmonella
3
2.40.58 2.20.19 1.00.0
1.00.0
1.10.18
Typhimurium
Formulations containing grape seed
5
1.50.58 1.10.12 1.00.0
1.00.0
1.00.06
and olive extracts have been proposed
7
1.90.03 1.00.06 1.00.0
1.00.0
1.00.0
as alternatives to chlorine-based
0
3.50.07 2.60.01 1.10.16 1.60.22
1.80.09
1
3.50.13 2.50.02 1.00.0 1.50.19
1.60.27
solutions for washing fresh produce 13, 3436
E. coli O157:H7
3
3.30.23 2.50.04 1.00.0
1.00.0
1.00.02
.
Shigella sonnei
5
7
0
1
3
5
7
3.00.12
3.00.02
3.90.03
3.70.19
3.50.15
2.40.76
1.00.0
2.40.06
2.20.12
2.90.0
3.00.19
2.60.11
1.90.14
1.00.0
1.00.0
1.00.0
2.00.10
1.00.0
1.00.0
1.00.0
1.00.0
1.00.0
1.00.0
2.10.22
1.10.16
1.00.0
1.00.0
1.00.0
1.00.0
1.00.0
1.70.09
1.30.4
1.00.0
1.00.0
1.00.0
Conclusions
In this study, we followed a simple, low cost, and laboursaving
extraction system. The antimicrobial efficacy of the improved
formulation was clearly demonstrated on parsley. Considering
human health and environmental hazards associated with chlorine
use, the Mexican lime:oregano mixture provides a viable alternative
to chlorine, and is equally effective at significantly reducing
bacterial pathogens associated with outbreaks stemming from leafy
green vegetables.
Acknowledgements
This research was supported by the Consejo Nacional de Ciencia
y Tecnologa de Mxico (CONACYT) grant #105389.
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31
11
WFL Publisher
Helsinki, Finland
www.world-food.net
Food security of Northwest China under current water resources and food
consumption patterns
Jianping Li 1, Jing Chen 1 and Zhouping Shangguan 2*
1
School of Agriculture, Ningxia University, Yinchuan Ningxia, 750021, P. R. China. 2 State Key Laboratory of Soil Erosion and
Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation,Chinese Academy of Sciences, Yangling
Shaanxi, 712100, P. R. China. *e-mail: shangguan@ms.iswc.ac.cn
Received 22 June 2014, accepted 24 September 2014.
Abstract
This study investigated the effects of water resources and food consumption patterns on the food security of Northwest China. A regional water
requirement model (RWRM) and a food security model (FSM) were set up to evaluate the water shortage and food security of Northwest China,
respectively. The results showed that the water resource shortage of Northwest China is severe and thus the status of food security is unsafe without
food import; the urban food security of the region is better than the rural food security, and more water and energy are needed for the urban
population; and the water shortage of Northwest China has increased dramatically since 1983 and will continue to increase in the future, having
already reached 170 billion m3 in 2010 and will reach 400 billion m3 in 2050. Finally, some countermeasures that should be taken to safeguard the food
and water securities of Northwest China are as follows: control the population growth according to the local conditions and the population and
structure of the ethnic (minority) peoples; promote calorie-appropriate and energy-efficient diets instead of unhealthy diets; eliminate food wastes;
and develop water-saving agriculture and breed water-saving crop varieties. The models and proposed countermeasures are expected to provide
theoretical foundation and practical guidance for sustainable development and food security of Northwest China.
Key words: Food security, agricultural water resource, food consumption pattern, Northwest China, model.
Introduction
In addition to population growth, industrial development and
uncontrolled economic growth, water shortage is more and more
recognized as a major threat to food security due to its restriction
of agricultural production 1, 2. The changes in consumption
patterns, i.e., the increasing proportion of water-intensive food
(e.g. meat), may become the main cause of water shortage 3.
Currently, approximately one third of the worlds population lives
in countries suffering water shortage, including north China, west
Asia, and Libya and Saudi Arabia, who have used water for
irrigation that greatly exceeds their annual total water resources 4.
Many authors estimate that a large part of the worlds population
- up to two-thirds - will be affected by water shortage over the
next decades 5, 6.
In China, water resources uses can be divided into four forms:
agricultural water (62% of the total amount), industrial water (24%),
domestic water (12%), and eco-environmental water (2%) 7. Nearly
all agricultural water is freshwater 8 and a shortage of freshwater
exists all over the world 9. So water shortage fundamentally results
from insufficient freshwater for food production 3, 10. Generally
speaking, different amounts of water are required to produce
different foods. For example, about 1 - 3 m3 of water is required to
produce 1 kg of cereal, and about 13.5 m3 of water is required to
produce 1 kg of beef in California 11. Consequently, different food
consumption patterns require different amounts of water
resources. With economic development and improved living
standards, the proportion of water-intensive foods has been
growing in food consumption patterns, so that more water
resources are required to meet human food demands. For example,
12
a typical American diet which includes red meat requires twice as

much water as a vegetarian diet to provide the same nutritional
intake 12. Also, climate change can be a significant factor that
affects agriculture and food production, exerting either a positive
or a negative influence on food security. For instance, higher CO2
concentrations can have a positive effect on many crops by
enhancing their biomass accumulation and final yields. However,
extreme weather conditions due to climate change can have
negative effect on food security by blocking food distribution,
and causing food supplies to be unstable and stored foods to
decompose 13.
In arid regions, the difference between water resource supply
and water demand is increasingly becoming acute due to
increasing water requirements and unchanging or decreasing water
supply. China is a drought-prone country suffering severe water
shortage. Although the total water resources amount of China is
2.8 trillion m3, ranking sixth in the world, its per-capita water amount
is only 2300 m3, 1/4th of the average level in the world. However,
China is one of the 13 countries suffering water shortage 14. In
terms of their spatial distribution, the water resources of China
tend to decrease from its northeast coastal area to northwest inland
area. Meanwhile, the agricultural water use efficiency of China is
low and farmland soil salinization and environmental pollution are
severe, particularly in the rural area of China 15. With its population
growth and rapid industrialization, the industrial water and
domestic water needs of China are increasing and their combined
demand restricts the agricultural water use for food production.
In the meantime, the food consumption pattern of China has shifted
200'0" N
200'0" N
300'0" N
300'0" N
400'0" N
400'0" N
500'0" N
500'0" N
towards animal product-dominated patterns, particularly meatMaterials and Methods

dominated ones, which mean that the agricultural water needs of Profile of study area: With the longitude ranging within 7341'E China have further increased. The result will be that the water 11115'E and latitude from 3142'N - 4933'N, Northwest China has
supply for food production will be insufficient to meet all demands a total area of 3,045,600 km2, accounting for 30.8% of the total area
which may result in food insecurity. Relevant data collected in the of China. It covers five provinces and autonomous regions from
2010 China Agriculture Yearbook 16 showed that China uses 48% southeast to northwest: Shaanxi, Ningxia Hui Autonomous
of irrigation farmland to produce 75% of grain yield and more than Region, Gansu, Qinhai and Xinjiang Uygur Autonomous Region
90% of cotton and vegetables. Therefore, the agricultural water (Fig. 1). In late 2010, Northwest China had its peak population of
use of China is a key factor that affects its food security.
96.6 million, which accounted for 7.2% of the total population of
Northwest China is the region suffering the most severe water China and of which the minority population accounted for 19%.
shortage, where the water and agricultural land resources are The landscape of Northwest China includes plateaus, basins and
generally spatially unevenly distributed and the ecosystem is mountains. Characterized by low rainfall and high evaporation,
fragile. The data collected from the Main Data Communique of the Northwest China is an arid and semi-arid region. The annual mean
Sixth National Population Census 17 showed that Northwest China precipitation of Northwest China decreases from 400 mm in the
has a population growth rate of more than 10, higher than the east to 200 mm in the middle part to less than 50 mm in the
average national level of 5.7. Also, the food consumption pattern northwest. Northwest China has an annual total water resources
of Northwest China differs greatly from the other regions of China. (surface and aquifer water) amount of 230 billion m3, which
For instance, the animal products consumption of the former is accounts for 9% of that of China. Since a majority of agricultural
higher compared with that in other regions. Northwest China is a irrigation facilities are poorly developed and most of farming
district, where there are many minority ethnic peoples including practice is dry land farming, northwest China depends on
Uygur, Hui, Tibetan, Mongolian and so on. Since these ethnic precipitation for the majority of crop production.
peoples consume more fresh milk and meat-dominated foods, and
only use grains as dietary supplements, Northwest China needs Methods:
more agricultural water than other regions to produce foods in RWRM and its parameters: A RWRM was established, in terms of
order to maintain its food security for its diverse population. water amount necessary to produce a unit of product and per
Meanwhile, with the acceleration of implementing the Great capita food consumption, to calculate the amount of water resource
Western Development Strategy in China where Northwest China that was used in agriculture to produce food, and to calculate the
was zoned as one industrial development region, more and more total amount of water resource requirement using a proportional
freshwater will be used by industry, which will probably rate of agricultural water use. The model is expressed as follows:
n
additionally limit water resources used by agriculture. In a word,
1
people will need more and more water resources to safeguard WRWR = ( M iWi )( Pr + Pu )
i =1
their food security, but there are limited water resources, which
are insufficient to produce food for food security, in Northwest
where WRWR represents the regional water requirement under
China, as well as the limited water resources will
800'0" E
1000'0" E
1200'0" E
1400'0" E
be used by industry and other aspects instead of
agriculture. Therefore, the sharp conflict between
the water resources and food security are
becoming acute, and how to tackle these conflicts
N
is a highly important topic that attracts attention
from all the fields.
Focusing on the water resource and food
consumption patterns of Northwest China, the
objectives of this study were to establish a
regional water requirement model (RWRM) and a
food security model (FSM) to evaluate the status
of its water shortage and food security, respectively;
develop models for predicting its future water and
food security depending on the current status of
water and food security, which were calculated
by RWRM and FSM; and provide sustainable
development-oriented countermeasures to tackle
the conflicts between the water shortage and food
security of the region depending on the models
and analyses. The results will lay a theoretical
Provincial capital
foundation and provide practical guidance for the
Study area
sustainable development and food security of
Provincial boundary
Northwest China.
900'0" E
1000'0" E
1100'0" E
1200'0" E
Figure 1. Location of the study area.

13
the current food consumption pattern, 1/() is ratio of water

consumption for food production to the total water resources,
is the proportion of water supply to total water resource, is the
proportion of agricultural water to total water supply, is
agricultural water use efficiency, M i is annual per-capita
consumption of the i th food (kg), Wi is water amount necessary
for to produce the i th food (m3 kg-1), n is food number, Pr is rural
population, and Pu is urban population. The parameters of Wi are
listed in Table 1 and those of Mi in Table 2.
Wtotal is assumed to represent the total water resources . If WRWR >
Wtotal, then there will be insufficient water resources and insufficient
water supply to produce enough food for consumption under the
current food consumption patterns. Otherwise, food and water
security will be guaranteed. Different regions have different
over time; the average is about 83% from 1980 to 2010. The
average is within 80 - 86%, which comes from the China Statistics
Yearbook 13. The value of is from the published papers 18-20,
ranging from 0.4 to 0.6. Since rural and urban food consumption
patterns differed greatly, the rural and urban populations were
Table 1. Actual water consumptions and energy water
productivities for mostly consumed food items.
Food items
Cereals and roots
Rice
Wheat
Maize
Other
Potatoes and other starchy roots
Sugar and sweetener
Oil corps and vegetable oils
Soybeans and other oil crops
Vegetable oils
Vegetables and fruits
Vegetables
Fruits
Animal products
Beef
Pork
Poultry
Mutton and goat meat
Fish and sea food
Eggs
Milk
Alcoholic
-1
Ei (kcal kg )
1.31
0.98
0.84
1.24
0.23
1.02
3625
2633
2872
2709
699
3481
3.2
5.08
3314
8720
0.19
0.5
188
413
12.56
4.46
2.39
4.5
5
3.55
1
0.18
2021
3500
1708
2005
497
1455
670
490
FSM and its parameters: A FSM was established to convert food

intake into energy intake to estimate food security. It was defined
as:
n
E EI = M i Ei
-1
Wi (m kg )
separately introduced into the RWRM so that the estimation and

evaluation would not be biased.
Food consumption patterns greatly influence water security.
For example, beef production needs 13 times more water than
wheat (Triticum aestivum L.) production to produce same amount
of weight, and 17 times more water than wheat to supply same
amount of energy 21. The study divided food consumption patterns
into two types: rural and urban patterns, because rural people and
urban people have different purchasing powers, dietary habits,
and food consumption habits. Thus, the comparison errors
resulting from ignoring these differences were reduced. Table 2
presents the annual per capita food consumption in the past 30
years (1980 - 2010), which indicates that the per capita annual
consumption of all food items has increased, while the
consumption of cereals has decreased. It also indicates that the
per capita food consumption of the rural population was less than
that of the urban population, again except for cereal consumption.
i =1
where EEI is per capita energy intake per day (kcal), Mi is daily
per-capita consumption of the food (kg), Mi = Mi /365, and Ei is
the energy of the i th food (kcal kg-1), n is foods number (Table 1).
Energy intake, recommended as the main indicator for measuring
food security by FAO 24, consists of four requirements:
i. Basal Metabolic Rate (BMR) for adults: 1300 - 1700 kcal per
person per day.
ii. Allowance for light activities: 1720 - 1960 kcal per person per
day.
iii. Allowance for appropriate activities: 2000 - 2310 kcal per person
per day.
iv. Allowance for labours or activities above the average intensity
or surpassing appropriate activities: 2600 - 2950 kcal.
This study adopted the average-weighted caloric requirement
of 2300 kcal/person/day to measure food security, which was the
calorie number required for appropriate activities. If EEI>2300 kcal,
then the food security was adequate; and if 1700 kcal<EEI< 2300
kcal, the food security was low; and if EEI<BMR, the energy
supply was insufficient and people were in the state of
malnourished and starvation. However, with development of
economic and improvement of the peoples living standard, the
Sources: Actual water consumptions of cereals, soybean, vegetables and fruits from Liu
et al. 21, fish and seafood from Zimmer and Renault 22, other food items from Chapagain
et al. 23.
Note: Wi is water amount necessary for to produce the i th food (m3 kg-1), Ei is the energy
of the i th food (kcal kg-1).
Table 2. Rural and urban food consumption patterns of northwest China over time.
Food items
Cereals and roots
Vegetables
Vegetable oils
Pork
Beef & goat meat
Poultry
Eggs
Fish and sea food
Milk
Fruits
Alcoholic
1980
Urban Rural
130
257
60
40
5
2
15
8
2
0
2
1
4
1
2
0
3
0
18
3
0
2
1985
Urban Rural
135
257
74
47
6
4
17
11
2
2
3
1
7
2
7
1
5
2
25
5
5
4
1990
Urban Rural
131
262
89
68
6
5
18
11
3
2
3
1
7
2
8
2
9
2
34
6
5
6
1995
Urban Rural
97
259
116
85
7
6
17
11
2
3
4
2
10
3
9
3
14
2
40
14
6
7
2000
Urban Rural
82
248
115
86
8
8
17
12
3
3
5
1
11
2
10
0
17
4
46
16
5
2
2005
Urban Rural
76
214
123
95
9
5
23
21
4
5
10
3
8
3
7
1
19
4
52
14
6
9
2010
Urban Rural
70
195
125
99
10
5
28
25
5
5
15
4
8
2
10
1
16
8
51
17
12
10
Source: National Bureau of Statistics (1981-2011)

Notes: China Statistical Yearbooks generally tell consumption data by classifying food items into food groups. One example is the consumption of cereals and roots. So, the study divided the cereal and
roots into there parts: 65% of wheat, 35% of rice, and 10% of roots, according to the food consumption habits of northwest China.
14
per capita energy intake should increase as well as the measurement

of food security in underdeveloped countries and developing
countries.
Data sources: The data for this study, including water resources,
population and food consumption patterns, were from China
Statistics Yearbook 16 and China Agriculture Yearbook 25. The
parameters of the RWRM and FSM were extracted out of the
published papers and China Statistics Yearbook 16 and China
Agriculture Yearbook 25. The parameters of the predicted models
(shown in Table 3) come from the regression models, which were
developed by the data (from 1980 to 2010) that were calculated by
RWRM and FSM.
3500
Population (104)
6000
5250
Rural population
Per capita water resource
Urban population
4500
3750
3250
3000
2750
3000
2500
2250
1500
2250
1980
1985
1990
1995
Year
2000
2005
Per capita water resource (m3)
Analysis and Results

Current water resources and population: The annual total water
resources for Northwest China were almost stable around 230
billion m3 in the past 30 years, without much change from year to
year (Fig. 2). Because of the exploding population, the annual per
capita total water resource sharply decreased (Fig. 3) from 3400 m3
at the end of 1980 to 2300 m3 in 2010. In the meantime, the per
capita water supply also decreased dramatically from 1019 m3 in
1980 to only 699 m3 at the end of 2010, which was far below the
water shortage-warning line (1000 m3 per capita).With the increased
water pollution and reduced usable fresh water resource, fresh
2010
Total water requirement and total

water resources in northwest
China (cu.m billion )
Figure 2. Population and per capita water resource reserves

in the different years.
400
water resource for the people was significantly reduced 26.

Figure 3 shows that the urban population has been growing
linearly since 1980. Although it has a large base, the rural
population has increased slowly since 1980. Because the
urbanization and industrialization have caused many rural
residents to migrate to the urban areas, and the natural increase in
the rural population was offset by the amount of rural population
who migrated to urban areas, the rural population has remained at
approximately 53 million in the past 30 years. So, the urban
population growth can be viewed as the population growth of
Northwest China. Meanwhile, Northwest China will be a main
area that has a population growth faster than the other regions of
China, because the population growth rate is 14.05 (average
value in the past 30 years), compared with the national average of
5.7. Therefore, more population, especially the urban population
who consume more energy than rural residents, will threaten water
security and food security.
Status of water security: The regional water requirement is defined
as the amount of water resources required for the present food
consumption patterns and all other water use. The water
requirement of Northwest China has been larger than its supply
since 1982, and the gap between the former and the latter has
increased dramatically year by year (Fig. 2). In 1980, the total
water resources of 240 billion m3 were larger than the water
requirement of 225 billion m3. This indicated that there was
sufficient water for producing food and other uses and water
resources were not a key constraining factor in agriculture and
industry. However, the difference between the water requirement
and the water supply has increased sharply from 5 billion m3 in
1982 to 170 billion m3 in 2010. By 2010, more than 80% of the total
water resource was used for agriculture. Due to water shortage,
the agriculture production will be reduced and food supply will be
insufficient to meet food consumption, and food insecurity will
occur without the importation of food. In the meantime, how to
properly distribute the limited total water resource among
agriculture, industry, life and ecology will be a big problem, and
an irrational distribution will have a negative effect on the regional
economy and society.
Total water requirement

Total water resources
Status of food security: The average per capita energy intakes per
day were obtained by the FSM model. The rural per capita energy
320
intake per day was less than the urban one during the past 30
years (Fig. 4). After 1983, the urban per capita energy intake per
280
day was over 2300 kcal, indicating that the urban food security of
240
Northwest China was safeguarded. The urban per capita energy
intake per day reached a high record of 3100 kcal in 2000 and
1980 1985 1990 1995 2000 2005 2010
generally decreased from 2000 to 2010 but remained above 2700
Year
kcal, surpassing the food security threshold of 2300 kcal. This
Figure 3. Total water resources and total water requirement
decrease resulted from the changes in food consumption patterns,
of northwest China in the different years.
which was the consumption of less cereal and more
meat and vegetables. However, the rural food security
Table 3. Forecast models and water securities in the following 40 years.
was poor. The rural per capita energy intake per day
was 1400 kcal in 1980, which was lower than that
needed for the Basal Metabolic Rate for adults. The
rural population suffered malnutrition and starvation
in 1980s. The per capita energy intake per day has
generally increased from 1400 kcal in 1980 to 2290 kcal
Note: P is population, E is per capita energy intake per day (Kcal), W is amount of total water need for food, e is the base
in 1998 but has remained below the food security
360
of the natural logarithm (approximately 2.7183), x is year, and R2 is determination coefficient.
15
Per capita energy intake per day

(Kcal/person /day )
3000
2700
2400
2100
1800
Urban area
Rural area
Food security line
1500
1980
1985
1990
1995
Year
2000
2005
2010
Figure 4. Per capita energy intakes per day of northwest

China in the different years.
threshold; the per capita energy intake per day has generally
fluctuated around 2300 kcal after 1998, being 2400 kcal in 2000,
2250 kcal in 2005, and 2330 kcal in 2010. Thus, the food security
for the rural population was nearly average. Therefore, the food
security of Northwest China was safeguarded from 1998 to 2010,
but this sufficient food security may not last long because of the
population growth, water shortage, and living standard
improvement.
Food security and water security in the future: This study
developed the Population Forecast Model (P), the Energy Intake
per Capita per Day Model (E), and the Water Resource Requirement
Forecast model (W) depending on the populations, per capita
energy intakes per day, and total water resources and water
requirement from 1980 to 2010. W Wtotal, the differences between
the water requirements and the existing total water resources, was
used to evaluate the future water situation (Table 3). The forecast
models depended on the economies, population and food patterns
in the past 30 years to forecast the future.
Table 3 shows that the population of northwest China is
projected to grow exponentially from 110.5 million at the end of
2020 to 135.9 million in 2050, a 25.4 million increase, and more than
80% of the population will become urban residents in 2050.
Meanwhile, the per capita energy intake per day and the regional
water requirement will reach 3762 kcal and 625.5 m3 in 2050,
respectively. The water gap (W Wtotal) will increase sharply in the
next 40 years. The total water resources will meet only 50% of the
water requirement of 463.5 billion m3 in 2020 and the gap will reach
400.1 billion m3 in 2050. Consequentially, the population growth,
sharp water gap increase and energy intakes of northwest China
will threaten the food security because of the insufficient water
for producing food in the future.
Discussion
Water shortage and food security have become primary factors
that restrict the national economic progress of China. If water
shortage evolves into a crisis, the effects may be far more severe
than the crisis of oil shortage that we have experienced so far 27.
Water security is the basis for food security, which is the basis for
modern agriculture. Agriculture can save water resources to
safeguard food security by consuming less water to produce more
food 28, 29. At present, water resources for food security of China is
facing challenges resulting from the water shortage, due to
increasing industrial and urban water uses, lack of extensive water
resources management, and water loss and soil erosion.
Agricultural water use is crucial to China, because China has a big
16
population and suffers poverty. Two-thirds of the undernourished

population (not enough food to eat) of the world live in seven
countries (Bangladesh, China, the Democratic Republic of Congo,
Ethiopia, India, Indonesia and Pakistan) and over 40% of the
undernourished population live in China and India 30. Thus, water
for food production cannot be diverted for other uses, especially
in the rural area of Northwest China where much of the
undernourished population lives. The typical water distribution
pattern of China is that it is dry in its northwest and humid in its
southeast, and its water supply differs across regions and time.
Because the water supply of Northwest China represents a much
smaller portion of Chinas total water resource, it faces very serious
problems with agricultural water shortage. Since agriculture is
now the largest branch of water user, it is important to develop
modern water-saving agriculture to safeguard the food security,
water security, and ecology security of China. The measures for
this purpose have been recognized by the scientists and
governments 31, 32. In addition, most reserve cultivated-land
resources of China are located in Northwest; it is difficult to exploit
them because of water shortage. So, the option to safeguard food
security by expanding the amount of cultivated land will be difficult
to achieve.
Insufficient food resulting from water shortage in Northwest
China can be made up through trade 23 or food distribution. Our
study showed that in 2010, the total water requirement of Northwest
China was 390 billion m3, but the total water resources were only
230 billion m3. Also, the agricultural water requirement of 72.5
billion m3 is more than agricultural water supply of 42.5 billion m3,
so that obviously the region does not have enough water to
produce food under the current food consumption patterns.
Nevertheless, both the rural and urban food securities of northwest
China were good in 2010 mainly because reasonable food trade
and grain circulation offset its food shortage. Where trade is
possible at a reasonably low water cost, the crucial food security
issue is whether the monetary and non-monetary resources at the
disposal of the population are sufficient to allow everyone to get
access to adequate food supplies rather than not whether the
foods are sufficient, that is to say, food availability is not a key
factor affecting food security. An important corollary to this is
that regional self-sufficiency is neither necessary nor sufficient
to guarantee food security at the regional level. It is noted that
Hong Kong and Singapore are not self-sufficient (they dont have
agriculture) but their populations are food-secure, meanwhile,
India is self-sufficient but a large part of its population is not
food-secure 13. However, more and more people will suffer from
food insecurity, if there are insufficient food supplies for trading
or circulation. In other words, both food production and trading
are important for safeguarding food security.
The results of this study showed that the water requirement for
food production has almost doubled from 1980 to 2010 (Fig. 3),
largely due to an increase in animal products consumption in the
recent decades. The models indicate that the future total water
requirement for food production will be likely to increase in the
next four decades. Even under the low modernization scenario,
food consumption pattern shifts along with population growth
will probably cause the total water requirement to reach 400 billion
m3 per year in 2050, even taking into consideration relevant
technological advances. This will undoubtedly put enormous
pressure on limited water resources in Northwest China. The
RWRM model showed that there were five key factors affecting
the regional water requirement of Northwest China: population,
food consumption pattern, agricultural water, water supply
capacity, and crop water use efficiency. The following
countermeasures in terms of the five factors probably could
guarantee the future water security and food security of Northwest
China.
First, the population growth of Northwest China should be
regulated according to the local conditions. At present, the
population of northwest China is mainly rural with many ethnic
peoples living together, and thus the possibilities of the economic
security, societal security and cultural security should be
considered in a balanced way. Proper population countermeasures
should be taken and timely adjusted according to the population
and structure of the ethnic peoples to ensure common development
and prosperity of all the nationalities concerned. While the
population size will be controlled, the populations quality of life
should be improved.
Second, caloric-appropriate diets and energy-efficient food
consumption patterns instead of unhealthy diets should be
promoted, and food wastes should be prohibited. In general, food
consumption patterns are closely related to increasing affluence.
However, eating habits probably plays a role in affecting the food
preference of Chinas population. For example, the meat
consumption of China now exceeds what is recommended by China
Nutrition Society 33, and Northwest China has higher meat
consumption than other regions. In addition, the current diet shifts
of Northwest China may be detrimental to the populations health,
and could cause higher incidences of diet-related diseases 34.
Raising public consciousness and promoting the diet recommended
by the CNS may help mitigate the future water shortage of China.
Meanwhile, food losses of China resulting from seeding, feeding,
harvesting, food processing, storage, transportation, and cooking
are large, making up 30% of the total food production of the
country. Consequentially, to eliminate all such food losses by
educating the population in healthy diet, food producing and
nutrition are wise measures to save food and water resources.
Third, Northwest China should develop water-saving agriculture
to guarantee the food and water securities. It is necessary for
Northwest China to increase water use efficiency and then water
supply as the total water resources are limited in the arid area. The
key approaches for increasing the water supply of Northwest
China can be summarized as follows: constructing reservoirs and
implementing water diversion projects; reasonably exploiting and
scientifically managing urban groundwater resources under the
precondition of sustainable development economy and society;
and trying to recycle waste water. Water-saving agriculture should
be promoted to safeguard water security. Water-saving agriculture
as an integrated system should include four aspects: rational uses
of agricultural water resources, water-saving irrigation, agronomic
water-saving techniques, and agricultural management 24.
Governmental supports and encouragements will be necessary to
achieve this system.
Fourth, biotechnological as well as traditional breeding methods
should be adopted as the efficient method to develop water-saving
crops for the arid areas of northwest China to feed its growing
population. Water-saving crops developed by modern biotechnology
breeding cannot only improve water use efficiency but also can
increase food production. In other words, more foods will be
produced without additional or with lowered consumption of the

currently existing water resources. Water-saving crop varieties,
such as drought resistant wheat in Australia, drought tolerant
cotton (Gossypium hirsutum L.) in America and drought tolerant
fruit trees in Israeli 35, have good drought tolerance ability, stable
yields and good qualities.
Conclusions
In brief, improving water use efficiency through biological
approaches in combination with other water-saving methods and
projects and establishing sustainable agriculture with limited water
resources have become, and will continue to be, the challenges
for agricultural scientists in the future.
Acknowledgements
This study was funded by the Startup Project of Doctor Scientific
Research of Ningxia University (BQD2012008) and Key Project of
the Knowledge Innovation Program of the Chinese Academy of
Sciences (KZCX2-YW-JC408).
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18
18
WFL Publisher
Helsinki, Finland
www.world-food.net
Modified atmosphere storage of banana (Musa acuminata) using diffusion channel

and mathematical modelling of steady-state oxygen concentration within the package
Arulselvam Karthiayani 1*, Nachimuthu Varadharaju 2 and Madasamy Siddharth 3
1
Department of Food Engineering, College of Food and Dairy Technology, Chennai-52, India. 2 Department of Food and
Agricultural Process Engineering, College of Agricultural Engineering, Coimbatore-3, India. 3 College of Food and Dairy
Technology, Chennai-52, India. *e-mail: mankarthi@yahoo.com
Received 22 April 2014, accepted 4 September 2014.
Abstract
Banana (Musa acuminata), variety Poovan (Group AB) fruits of commercial maturity were stored inside the air-tight container under modified
atmosphere conditions using diffusion channels. The gaseous exchange was made through the glass channels called as diffusion channels of different
geometries viz., length 5, 10, 15 and 20 cm and inner diameter 3, 5 and 7 mm. The fruits were packed with rated quantities of moisture and ethylene
absorbents and the containers were stored at three different temperatures (RT-Room temperature, 24C and 14C). Gas samples were drawn from
the container and was analysed for oxygen concentration for every 24 h up to a period of 20, 30 and 40 days for RT, 24C and 14C storage,
respectively. Oxygen concentration was noted for the storage containers with different dimensions of diffusion channel and storage temperature. A
mathematical model was developed using non-linear multiple regression to fit the observed data. The developed model was found to be useful in
determining the length of the diffusion channel for the required steady-state oxygen concentration for banana. The shelf life of banana can be increased
to three to four folds by adopting diffusion channel technique compared to control fruits at all the respective storage temperatures.
Key words: Modified atmosphere storage, diffusion channel, banana, modelling, oxygen.
Introduction
Indias contribution to world production of banana is about
15.02% 9. The Indian banana growers are losing around 20 to 40%
of their production on account of existing handling practices 14.
The post harvest losses in India are much higher due to tropical
climate and also due to lack of technology for handling and
processing 18. Hence an attempt is made to enhance the shelf life
of banana using diffusion channels and to develop a mathematical
model for determining the steady-state oxygen concentration.
The principle behind modified atmosphere packaging is
reduction in respiration rate of fruits by modifying the gas
composition of the storage atmosphere and thus enhancing the
shelf life of the fruits 17. If the availability of oxygen is restricted,
the rate of respiration could be slowed down; thereby quality of
the commodities is preserved with enhancement of shelf life 11.
The appropriate atmosphere is made to evolve within the storage
chamber by the respiration of the produce and maintaining that
atmosphere by selective permeability of the gases through
membrane or by diffusion through channels 12, 19. Diffusion channel
is a hollow tube or channel fitted to an airtight storage chamber in
which produce is stored, and the other end of the tube is exposed
to the ambient air. The diffusion channel storage system is one of
the methods of modified atmosphere packaging in which the
storage chamber is impermeable to gas of concern and the exchange
of gas takes place only through the diffusion channels. The
presence of diffusion channel controls the flow of gases on either
direction (i.e., from package atmosphere to ambient atmosphere
and vice versa), thus maintaining the gas composition.
The gas composition surrounding the fruit material within the
package plays an important role in determining shelf life of fruits
under MA Storage. Low oxygen and elevated carbon dioxide levels

are known to influence flavour by reducing loss of acidity 7, starch
to sugar conversion, sugar inter conversion and biosynthesis of
flavour volatiles. However, levels of oxygen below 2% and carbon
dioxide above 15% have been found to result in irreversible effects
on the fruit quality 4. Elevated carbon dioxide atmosphere inhibited
the activity of ACC synthesis (key regulatory site for ethylene
synthesis) and retarded biosynthesis of carotenoids and
anthocyanins synthesized during fruit ripening 3. This also slowed
down the activity of cell wall degrading enzyme which influenced
softening of fruit tissue 13.
Experimental containers for storage study: The containers made
of Poly Ethylene Teraphthalate (PET) with 8.5 litres capacity and
transparent bodies were selected for the study. Three holes were
made in the lid. In the first hole, a silicon septum was fitted using
brass fittings to draw gas samples for gas analysis. The second
hole was used for flushing nitrogen gas into the storage chamber
and the third one was used to fix diffusion channel using rubber
cork. The diffusion channels of rigid type were made up of glass
with the different geometries viz., length 5, 10, 15 and 20 cm and
inner diameter 3, 5 and 7 mm (Table 1), were selected for the study.
The geometry of the diffusion channels was arbitrarily fixed based
on the information available in the literature cited 1, 20. All the
experimental containers were washed well with soap water and
rinsed with chlorinated water (500 ppm) and tested for air tightness
using soap solution to detect any leaks before starting the
experiment. An advantage of this design is the containers provide
19
quick and easy opening and closing procedures and good

visibility to assess the product during the storage period. The
schematic view of the experimental container is depicted in Fig. 1.
Table 1. Treatment details for banana and mango.
T1
Channel dimensions
L
d
5
3
T2
10
T3
15
T4
20
T5
T6
10
T7
15
T8
20
T9
T10
10
T11
15
T12
20
Treatment
till the required oxygen concentration of 5% was reached. The

containers were stored in clean and dry place in three different
temperatures viz., room temperature (27 - 33C, 50 - 70% relative
humidity-RH), 24C and 14C. The samples were kept in walk-in
cold room to store at 24C and 14C and RH 90 - 95%. Considering
the recommended cold storage temperature and evaporative cooler
temperature for banana, the storage temperatures of 14C and
24C were chosen, respectively.
Preliminary studies on MA storage of banana: Preliminary
experiments were done to ascertain the position of the diffusion
channel and best fruit to free volume ratio to be kept inside the
container. Three position viz., top (bottom of the channel touching
the top of the lid), middle (middle of the channel touching the top
of the lid) and bottom (top of the channel touching the top of the
lid) and five levels of fruit to free volume ratios viz., 1:2, 1:3, 1:4, 1:5
and 1:6 were investigated. The experimental procedure was
repeated. Visual as well as gas analysis was done at 24 h interval
to check the ripening of fruits and gas composition inside the
package, respectively.
L - length of diffusion channel (cm). d - inner diameter of diffusion channel (mm).
Determination of gas composition within the package and

modelling: Oxygen concentration within the container was found
out using MAP analyser (PBI Dansensor Model, Checkmate).
The needle of MAP analyser was inserted into gas sampling septum
of the storage chamber and the pump button was put on. The
vacuum pump inside the MAP analyser sucks about 5 ml of gas
from the storage container and oxygen concentration was
calculated by the sensor and the percentage was displayed in the
digital screen. The gas concentration was analysed for all the
containers with different treatments as described in Table 1.
Mathematical model for length of diffusion channel: Based on
the review of previous work done, a mathematical model was
developed and represented as follows:
rmsV f
v

yO container
KAc DO2 N 2 DO2 N 2 2
ln(
)
rmsV f
v

yO2 air
KAc DO2 N 2 DO2 N 2
(1)
Equation (1) can be further be simplified as:
Figure 1. Schematic view of diffusion channel storage.
Experimental procedure: Banana (Musa acuminata), variety

Poovan (Group AB) was selected for the study. The fresh banana
bunches with 90% maturity were procured from farm orchard,
Coimbatore, Tamilnadu, India. The bunches were carefully
dehanded and washed in tap water and were dipped in 500 ppm of
benomyl fungicide solution for 2 - 3 min 5 and then shade dried for
about 4 - 5 h to facilitate evaporation of surface moisture. This
could prevent the fruits from fungal infection during storage.
Calcium hydroxide, CO2 and moisture absorbent (@100 g/kg of
fruit) were kept at the bottom of the storage container. A perforated
plastic plate was placed above the absorbent in order to keep the
fruit samples for storage so that there was no direct contact
between the fruits and the absorbent. Known quantity of fruit
was kept over the perforated plastic tray along with the ethylene
absorbent (@20 g/kg of fruit) pouch. The containers were then
closed with the lids and nitrogen gas was flushed into the container
20
DO2 N 2
rm V a a y
1
ln( s f 2 1 O2 container )
a1
rmsV f a2 a1 yO2 air
where:
a1
a2
(2)
v
DO
N2
1
KAc DO2 N 2
where:
L - Length of the diffusion channel (cm)
DO2 - N2 - Diffusivity of O2 in N2 (cm2 h-1)
Ac - Cross sectional area of the diffusion channel (cm2)
yO2 - Oxygen molar fraction
k - Total mole of gas within the storage container (mole)
Vf - Free volume inside the storage container (cm3)
r - Respiration rate of the stored produce (mg O2 kg-1 h-1)

ms - Mass of the stored produce (kg)
v - Velocity gradient of O2 in N2 (cm s-1).
Equation (1) is used to find out the length of the diffusion
channel for given mass of sample and free volume inside the
storage chamber. The model parameters a1 and a2 were obtained
by fitting the experimental data by non-linear regression using
the software package Sigma-plot 8.0.
It is evident from the results that, the steady-state oxygen

concentration was directly proportional to diameter and inversely
to length of the channel. The results obtained are in accordance
with Stewart et al. 16 for the modified atmosphere packaging of
Dwarf Cavendish banana using diffusion channel. From Fig. 3, it
may also be observed that the steady-state oxygen concentration
was higher at lower storage temperature. This may be due to less
respiration of fruits at lower storage temperature.

Position of the channel and fruit to free volume ratio: Based on
the preliminary studies, it is found that the top position of the
diffusion channel and fruit to free volume ratio of 1:5 was the most
suitable to maintain the required oxygen concentration of 2 - 5% 5,
6, 10
. Hence, further experiments were conducted on top position
of channel and fruit to free volume ratio 1:5.
Values of constants fitted with the developed mathematical model:

The mathematical model was developed as described. The
parameters viz., a1 and a2 described in the model were determined
using the experimental data by non-linear regression using Sigmaplot 8.0 software and presented in Table 2.
Oxygen concentration in storage container: Oxygen concentration

attained by banana inside the storage chamber was recorded at one
day interval throughout the storage period at different temperatures
and presented in Fig. 2. The initial gas level was kept at 5% oxygen
for all the treatments. In all the cases, the oxygen level increased
initially and stabilised at that level for about three to five days and
the concentration subsequently decreased and attained a steady
state. Also, a sudden decrease in oxygen concentration was seen
during the later period of storage in most of the samples.
From the Fig. 2, it is observed that the attainment of steadystate oxygen concentration took about 5 - 10 days, 6 - 8 days
and 12 - 15 days for the fruit samples stored at RT, 24C and
14C, respectively.
Oxygen concentration (%)
Steady-state oxygen concentration attained by the fruits: The

steady-state oxygen concentration attained by the fruits is
presented in Fig. 3. At ambient storage temperature, the maximum
steady state oxygen concentration of 4.8 and minimum of 2.1%
was noted for T9 and T4, respectively. Also for all the storage
temperatures, the maximum and minimum values of oxygen
concentration were noted for T9 and T4, respectively. Kader 8 and
Kim et al. 2 observed that the optimum modified atmosphere
conditions for most of the tropical fruits were 3 to 8% oxygen
which indicated that all the samples were containing recommended
oxygen concentration.
Ambient
Am
24
24C
14
14C
Table 2. Values of constants a1 and a2 for banana at different

storage temperatures.
Variety
Poovan
d
(mm)
3
5
7
Ambient
a2
a1
0.4927 0.0222
0.3676 0.0165
0.4075 0.0183
Determination of best shelf life of banana: The samples stored

for determining the best shelf life and the data is represented in
Table 3. The oxygen concentration attained by the best treatments
ranged between 2.3 and 5.2% which is in conformation with the
results obtained by Isaak et al. 5, 6 and Kudachickar et al. 10 for the
storage of banana. It is inferred from the above results that the
shelf life of banana can be increased to three to four folds by
adopting diffusion channel technique compared to control fruits
at all the respective storage temperatures.
14C
Table 3. Best treatment for banana at different storage

temperatures.
Sample
Storage
temperature
Banana
Ambient
24qC
14qC
2
1
T1
T2
14qC
a1
a2
0.2208 0.0297
0.1788 0.0217
0.2012 0.0225
The convective flow of oxygen and nitrogen within the chamber

is taken care of the constant a1 which is described as the velocity
of oxygen flow per unit diffusivity coefficient of oxygen in nitrogen
(DO2-N2). The parameter a2 represented the reciprocal of the product
of total mole of gas within the storage container, cross sectional
area of the diffusion channel and the diffusivity coefficient of
oxygen in nitrogen (DO2-N2). Since DO2-N2 is assumed as constant,
the parameter a1 is directly proportional to the velocity of air flow
and a2 is inversely proportional to the cross-sectional area of the
diffusion channel. The length of the diffusion channel may be
determined by substituting the values of a1 and a2 in Equation (2).
24qC
a1
a2
0.1745 0.0128
0.1725 0.0126
0.1724 0.0125
T3
T4
T5
T6
T7
Treatment
T8
T9
T10
T11
T12
Fruits under diffusion

channel storage
Best
Shelf life
treatment
(days)
T 11
15
T4
27
T3
38
Control
(fruits stored in air)
Shelf life
(days)
4
7
11
Figure 3. Effect of different channel diameters, lengths and storage

temperatures on steady state oxygen concentration of banana.
21
00
88
1010
l = 5cm
cm
I-5
10
10
Storage period (days)
15
15
10
10
1515
l = 10cm
cm
I-10
l = 15cm
cm
I-15
Storage
period (days)
Storage
periods
(days)
55
20
20
l = 20cm
cm
I-20
2020
20
20
d = 7 mm
d = 7 mm
Storage periods (days)
55
d = 5 mm
d = 5 mm
d = 3 mm
d = 3 mm
10
10
1515
55
Ambient
00
22
44
66
88
00
0
0
00
1010
00
22
44
66
88
1010
00
22
44
66
88
1010
l I-5
= 5 cm
cm
55
55
15
15
l=
15 cm
I-15
cm
25
25
l =I-20
20 cm
cm
25
25
d = 7 mm
30
30
30
30
30
30
d = 5 mm
d = 7 mm
10
15
20
20
15
10
lI-10
= 10cm
cm
25
25
d = 3 mm
d = 3 mm
d = 5 mm
20
20
10
10
10
15
20
10
20
15
24C
q
8
10 10
0
0
00
0
0
10 10
lI-5
=5
cm
cm
5
5
55
55
10
10
10
10
10
10
14C
15
15
20
20
25
25
20
20
25
25
l I-10
= 10cm
cm
35
35
lI-20
= 20cm
cm
35
35
40
40
40
40
40
40
d = 7 mm
d = 7 mm
30
30
30
30
l I-15
= 15cm
cm
15
15

Storage
periods (days)
35
35
d = 3 mm
d = 3 mm
d = 5 mm
d = 5 mm
15
20
25
30
20
25
15
30
Storage
(days)
Storageperiod
periods
(days)
g 2: Oxygen concentration attained by Poovan at different channel lengths diameters and temperatures during storage (where d is
00
22
44
66
00
00
00
22
44
66
88
1010
00
22
44
66
88
Oxygen concentration (% )
1010
Figure 2. Oxygen concentration attained by Poovan at different channel lenghts,diameters and temperstures during storage.
O xyg en co n cen tratio n (% )
Oxygen concentration (% )


Oxygen concentration(%)


22
Conclusions
The smallest channel length of 5 cm attained the steady state
quickly compared to largest channel length of 20 cm at all the
storage temperatures. Also, the variation in attainment of steadystate period was more pronounced for smaller channel diameter of 3
mm compared to 5 and 7 mm. The maximum and minimum values of
steady-state oxygen concentration were noted for T9 and T4,
respectively, for all the storage temperatures which represented
that the steady-state oxygen concentration was directly
proportional to diameter and inversely to length of the channel.
The developed mathematical model will be a useful tool for
determining the length of the diffusion channel for said oxygen
concentration. Also the shelf life of banana can be increased to
three to four folds by adopting diffusion channel technique
compared to control fruits at all the respective storage
temperatures.
Highlights:
The time of attainment of steady-state oxygen concentration
within the package increased as the storage temperature
increased.
The attained of steady-state oxygen concentration was directly
proportional to the diameter and inversely proportional to the
length of the channel.
The developed mathematical model will be a useful tool for
determining the length of the diffusion channel for banana for
the required steady-state concentration.
Acknowledgements
The authors sincerely express their gratitude towards the funding
agency viz., University Grants Commission, New Delhi, India for
carrying out the research work.
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8
23
WFL Publisher
Helsinki, Finland
www.world-food.net
Anti-inflammatory and anti-cancer effects of -carotene, extracted from

Dunaliella bardawil by milking
Abeer M. Badr 1*, Effat F. Shabana 2, Hoda H. Senousy 2 and Hend Y. Mohammad
Zoology Department, 2 Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt.
e-mail: abeerbadr@gmail.com, dr-effatshabana@hotmail.com, hodasenousy1@hotmail.com, hendgooonet@hotmail.com
Received 10 July 2014, accepted 22 September 2014.
Abstract
Natural -carotene was continuously extracted from Dunaliella bardawil in a two phase system by milking with the biocompatible solvent dodecane
(20% v/v) at 20,000 lux and 170 rpm for 15 days. The b-carotene yield was 23.30 g/ml at the end of the experiment. High performance liquid
chromatography (HPLC) analysis showed that b-carotene is formed of the two isomers 9-cis and all trans in a ratio of 1.13. We aimed to assess the
protective and therapeutic effects of natural b-carotene at various doses (30, 140 and 350 g/kg) compared with synthetic b-carotene at a fixed dose
(350 g/kg) on lipopolysaccharide (LPS)-induced inflammatory cytokines in CD1 mice, and also to test the cytotoxicity of natural b-carotene on
breast cancer (MCF-7) and hepatoma (HepG2) human cell lines in vitro. In pre- and post-treatment of LPS injected mice, low dose (30 g/kg) of
natural b-carotene treatment caused a significant reduction in the levels of interleukin (IL)-1a, interferon (IFN)-g and tumor necrosis factor (TNF)-a
compared with LPS-treated control group. Administration of natural b-carotene at dose of 140 g/kg exhibited a significant decrease in the levels of
IL-1a and IFN-g as protective and IL-1a as therapeutic. In contrast, high dose (350 g/kg) of natural of b-carotene failed to exert anti-inflammatory
effect either in pre or post-treatment. Synthetic b-carotene pretreatment induced protective inhibition of IL-1a and IFN-g levels while post-treatment
had no influence. IC50 of natural b-carotene was 14.58 and 7.44 g/ml, while IC50 of doxorubicin was 7.51 and 2.67 g/ml for HepG2 and MCF-7 cell
lines, respectively. Hence, natural b-carotene is capable of enhancing anti-inflammatory activity in vivo and is a promising anti-cancer drug.
Key words: Dunaliella bardawil, -carotene, milking, lipopolysaccharides, anti-inflammation, pro-inflammatory cytokines, breast cancer MCF-7 cell
line, HepG2 cell line, anti-cancer, acute inflammation.
Introduction
Drug research in marine environment has shown that algae are an
important source of innovative biochemically active compounds.
Carotenoids biosynthesized by algae have commercial
applications in food science, pharmaceutical industry, cosmetics,
and nutritional health 1. -carotene, as an important carotenoid, is
metabolized in the mammalian cells and acts as a natural source of
vitamin A 2. It is considered as a powerful antioxidant and has
been suggested to inhibit the development of inflammationassociated diseases such as rheumatoid arthritis 3, inflammatory
bowel disease 4, and atherosclerosis 5.
Dunaliella bardawil, a marine unicellular halotolerant green
alga, showed an environmental adaptation through excess
production of -carotene and glycerol to maintain its osmotic
balance 6. Natural -carotene produced by D. bardawil is
composed of equimolar mixture of two stereoisomers, all-trans
and 9-cis -carotene 7. Milking, a kind of in-situ extraction process
in which the starting cell mass is reused for continuous production
to overcome the low productivity of algal cultures in producing
high-value compounds, i.e. the first step in the experiment did not
need to be repeated. Milking has been found successful in carotene production from D. salina 8-10. Production of carotenoids
from D. salina by milking is selective since mainly secondary
carotenoid was extracted when gentle mixing was used 10.
Production of -carotene under stress conditions was higher than
24
under normal conditions 11. A 9-cis stereoisomer in D.bardawil is

still highest among all other natural carotenoid sources 12. A 9-cis-carotene isomer has a valuable metabolic activity as it has a
better antioxidant property, a higher liposolubility than the alltrans isomer, and accumulates more effectively in animal tissue 13.
Early epidemiological evidence has suggested that intake of carotene may introduce protection against certain types of
cancers 14, 15. However, administration of synthetic all trans carotene implied no effects on cancer and cardiovascular
diseases 16 and sometimes affected negatively on smokers with
lung cancer 17 and atherosclerosis 18.
Inflammation is a complex process mediated by action of various
immune cells, such as natural killer cells, neutrophils, and
macrophages. It is characterized with overexpression of
inflammatory mediators (IL-1, IL-6, and TNF-) 19. Lipopolysaccharide (LPS), a bacterial endotoxin, presents in the outer
membrane of Gram-negative bacteria that induces toxic effects 20,
and able to promote inflammatory responses. LPS treatment is a
way to mimic inflammatory state 21.
The present study aimed to assess the efficacy of natural carotene extracted from D. bardawil by milking on beneficial
inhibition of pro-inflammatory cytokines during acute inflammation
of LPS-stimulated mice comparing to synthetic -carotene. We
also aimed at investigating the potential cytotoxicity of natural -
carotene on hepatoma (HepG2) and breast cancer (MCF-7) human

cell lines in a dose dependent manner in vitro.
Organism and growth conditions: D. bardawil was isolated from
the salt marshes near El-Bardawil Lake, Northern part of SinaiArish Governorate, Egypt. It was grown in batch cultures in MH
nutrient medium 22 with modification of NaCl concentration to 1.5
M (pH 7.5, 251C) and light intensity of 39 Em-2s-1 (2000 lux) by
white fluorescent lamps, the initial algal inoculum was adjusted to
be 5106 cells/ml. Three replicates were used for each treatment.
Extraction ability of different organic solvents to -carotene
and cell membrane integrity of Dunaliella: Some non-polar
organic solvents with log P octane (oct) more than 6 (dodecane, decane,
tetradecane, hexadecane, ethyl oleate) (Sigma-Aldrich) were used
in this experiment to choose the suitable biocompatible one 23.
Each treatment consisted of 80% aqueous phase and 20% organic
phase. D. cultures were incubated at 15,000 lux, 251C and mixing
rate 200 rpm. The extracellular -carotene in the organic phase
was measured after 24 h. Cells were harvested and the chlorophyll
a content, intracellular -carotene and cell count were measured.
The organic solvent phase was protected from light by covering
it with aluminum foil in all solvent experiments. The cell membrane
integrity was expressed as the absorbance of the Evans blue
extraction solution samples at 600 nm 24.
Milking of D. bardawil for continuous production and extraction
of -carotene in two-phase system: After preliminary experiments,
algal culture with initial cell count of 5106 cells/ml was
supplemented with 20% (v/v) dodecane and incubated at
20,000 lux (light intensity) and 170 rpm mixing rate. Every
three days, chlorophyll a content, -carotene (intracellular
& extracellular), and cell count were measured till the end of
the experiment (15 days) 8. Cells were counted by Sedqwick
Rafter cell after the addition of 1-2 drops of Lugols solution.
Chlorophyll a was determined as described previously 25, 26.
-carotene was quantified photometrically using as a molar
extinction coefficient 450 nm 134, 500 L mol-1 cm-1 27.
conditions for 7 days before the experiment. All experimental

procedures in this study were approved (CUFS/S-PHY/11/14) by
the Institutional Animal Care and Use Committee (Faculty of
Science, Cairo University, Egypt).
Induction of inflammation and experimental design: For
induction of inflammation, mice were injected intraperitoneally
(i.p.) with a single acute dose of 2.5 mg/kg body weight of LPS
(Escherichia coli 0.26.B6, Sigma-Aldrich Co, USA) 28, euthanized
after 6 h of LPS injection. The doses of -carotene were chosen
according to Murthy et al. 29. Natural and synthetic -carotenes
were dissolved in olive oil. Mice were divided into 15 groups (n=6
per each) in two sets for assessment of the protective (pretreatment) and therapeutic (post-treatment) effects of -carotene
as shown in Fig. 1. In protective set mice were orally administered
with olive oil once in a day for 15 days as vehicle control (Group
1) followed with LPS injection (Group 2). Mice were orally
administered with synthetic -carotene at only one dose of 350
g/kg (Group 3) and three doses of natural -carotene, 30 g/kg
(Group 4), 140 g/kg (Group 5), and 350 g/kg (Group 6), once in a
day for 15 days. Oral administration of synthetic -carotene (Group
7) and all doses of natural -carotene (Group 8, 9 and 10) once in
a day for 15 days, followed with LPS injection. In therapeutic set
mice were injected with LPS at once and immediately i.p. injected
with a single dose of olive oil as control LPS (Group 11). LPS
injection at once and immediately i.p. injected with a single dose
of synthetic -carotene (350 g/kg, Group 12) and three doses of
natural -carotene: 30 g/kg (Group 13), 140 g/kg (Group 14),
Detection of -carotene by high performance liquid

chromatography (HPLC) analysis: The -carotene extract
from D. bardawil was lyophilized and transferred to hexane
with the addition of water, dried completely under N2, and
re-dissolved in methylene chloride. Then, it was analysed
by HPLC (Agilent 1100, USA, with Thermo Electron
Corporation Hypersil Gold DC8 column, particle size 5 m,
150 mm 4.6 mm and flow rate of 1 ml/min). The HPLC
preparative time for pigment analysis did not exceed 15 min.
The synthetic -carotene (Sigma type I, Sigma-Aldrich,
USA) was used as standard and dissolved prior to
chromatography in methylene chloride 7.
Animals: Male CD1 mice weighing about 20-22 g were
obtained from the breeding animal centre of National
Research Centre (Giza, Egypt). Mice were kept at room
temperature (222C), 555% humidity and a 12 h dark/light
cycle with free access to regular laboratory chow and water
ad libitum. Mice were allowed to acclimate to these
Figure 1. Diagram of experimental design.

LPS injected mice (2.5 mg/kg) euthanized after six hours of injection. (A) Protective set: synthetic
(synth) at single dose (350 g/kg) and natural (nat) -carotene at various doses (30, 140, and 350 g/
kg) were administered for 15 days before LPS injection. (B) Therapeutic set: LPS injected mice were
treated with synthetic -carotene at single dose (350 g/kg) and natural -carotene at various doses
(30, 140, and 350 g/kg).
25
and 350 g/kg (Group 15). Mice were euthanized with isoflurane.
The blood was collected by cardiac puncture and centrifuged at
3500 rpm for 10 min. The collected sera were stored at - 20C for
further analysis.
Quantitative determination of cytokines: Levels of cytokines
(IL-1, IFN- and TNF-) were assessed by an indirect enzymelinked immunosorbent assay (ELISA) (eBioscience, GmbH, Vienna,
Austria). ELISA was performed following the instructions of the
ELISA test kits. The levels of cytokines were expressed as pg/ml.
In vitro cytotoxicity assay of -carotene extract: Cytotoxic
activity of natural extract of -carotene was determined by
sulphorhodamine-B assay (SRB) 30. HepG2 of hepatoma cancer
and MCF-7 of breast cancer human cell lines were maintained at
the National Cancer Institute (Cairo, Egypt). -carotene was
prepared in dimethyl sulphoxide (DEMSO) and tested as negative
control. The cells were seeded in 96-well microplates (5104-105
cells/well) for 24 h. The monolayer cells were incubated with serial
dilutions of -carotene (5, 12.5, 25 and 50 g/ml) for 72 h at 37C in
a humidified incubator with 5% CO2. Triplicate wells were prepared
for each concentration. After 72 h, the cells were fixed, washed
and stained with 0.4% SRB stain dissolved in 1% acetic acid.
Excessive dye was washed with 1% acetic acid, the plates were
air-dried, and protein-bound dye was solubilized with 10 mM Tris
base solution. Colour intensity of each well was measured at 564
nm with microplate reader. The anticancer drug doxorubicin was
used as a positive control. IC50 is the concentration necessary to
cause 50% cell inhibition (or 50% cytotoxicity).
Statistical analysis: The present data were analysed by one-way
analysis of variance (ANOVA) using SPSS statistical package
version 20 to test the effect of -carotene on the investigated
parameters. In addition, the comparison between various studied
groups was performed using post ANOVA Duncans test of

homogeneity. The data values were expressed as meanstandard
error of mean (SEM). Regression analysis and correlation
coefficient were used to fit the relationship between various
studied variables.
Results
Extraction ability of different organic solvents to -carotene
and cell membrane integrity of Dunaliella: Table 1 reveals that
decane has the highest extraction ability (extracellular -carotene)
among all solvents used (5.6 g/ml) followed by dodecane (3.3
g/ml) while tetradecane, hexadecane and ethyl oleate have less
and similar ability to extract -carotene from D. bardawil cells.
Measurement of growth parameters showed that most organic
solvents used caused a remarkable decrease in values of growth
parameters after 24 h of extraction at 200 rpm mixing rate when
compared to their initial values. The highest values of chlorophyll
a and cell count were recorded in cells treated with hexadecane
while the least values were recorded in cells treated with decane.
The highest intracellular and total -carotene (intracellular +
extracellular) were observed with dodecane. The cell membrane
integrity measurements (Table 1) showed that optical density (O.
D.) readings increased with increase in log Poct of the solvent
indicating increase in the cell membrane damage. The decane
recorded the least O. D. reading which increased with other
solvents; the highest one was for ethyl oleate. Different organic
solvents have a significant effect on growth, -carotene
production and extraction as well as cell membrane integrity.
Milking of D. bardawil for continuous production and extraction
of -carotene: In milking experiment, chlorophyll a and cell count
decreased drastically by increasing time of continuous extraction
(Table 2). In contrast, both of intracellular and extracellular carotene increased gradually by increasing the time of extraction,
Table 1. Extraction ability of different organic solvents to -carotene; membrane integrity of

Dunaliella bardawil incubated for 24 h at 15,000 lux and 20 % (v/v) solvent at mixing
rate 200 rpm.
Growth
parameters
Solvents
Decane
Dodecane
Tetradecane
Hexadecane
Ethyl oleate
log Poct
Chlorophyll a
(g/ml)
Cell count
(x106 cell /ml)
6.25
6.6
7.6
8.8
8.51
6.050.08a
7.030.01b
8.750.06c
9.310.08e
8.990.07d
2.220.04a
3.890.02b
4.140.03c
5.020.06e
4.900.04d
Intracellular
-carotene
(g/ml)
2.510.20a
5.610.11d
4.080.02b
4.910.05c
4.020.06b
Extracellular
-carotene
(g/ml)
5.600.15d
3.300.05c
1.390.02b
1.030.02a
1.330.04b
Cell membrane
integrity (O.D.)
0.190.01a
0.270.01b
0.280.02b
0.300.01b
0.860.01c
Initials: chlorophyll 9.99 g/ml, cell count 5.31106 cell/ml, -carotene 6.52 g/ml. Means marked with the same superscript letters are not-significant
(P>0.05). Data are average of three replicates; each value represents the mean SEM.
Table 2. Milking of Dunaliella bardawil for continuous production and extraction of carotene using dodecane (20% v/v) at 20,000 lux and 170 rpm.
Growth paramerters
Mixing
rates (rpm)
170
Time
(days)
0
3
6
9
12
15
Chlorophyll a
(g/ml)
Cell count
(x 106 cell /ml)
Intracellular
-carotene
(g/ml)
Extracellular
-carotene
(g/ml )
Total
-carotene
(g/ml)
9.940.03
7.19 0.04
3.580.06
2.010.03
1.910.01
1.280.04
5.840.06
3.110.01
2.340.05
0.790.07
0.620.02
0.190.05
6.400.08
8.820.05
9.610.03
10.350.03
11.290.01
12.340.03
___
6.190.11
8.100.07
9.070.05
9.650.09
10.960.04
6.40
15.01
17.71
19.42
20.94
23.30
Data are average of three replicates; each value represents the mean standard error of mean.
26
the highest value of total -carotene was obtained after 15 days

(23.30 g/kg). Since, chlorophyll a and cell count recorded minimum
values after 15 days and so continuous production and extraction
of -carotene in this experiment cannot be completed more than
15 days due to the negative effect on the growth of the cells.
Detection of -carotene by HPLC analysis: HPLC chromatogram
of the standard synthetic -carotene revealed one peak of all trans
-carotene at retention time 3.929 min (Fig. 2A). Chromatogram of
natural -carotene extracted from D. bardawil by dodecane
showed two peaks. These two peaks of stereoisomers 9-cis and
all trans -carotene appeared at retention times 3.480 and 3.882
min, respectively (Fig. 2B). The ratio of 9-cis to all trans -carotene
was 1.13.
mAU
A
2000
1500
1000
500
0
0
mAU
1000
4
6
Retention time (min)
min
800
600
400
200
0
0
min
Retention time (min)
Figure 2. High performance liquid chromatography (HPLC)

chromatograms of (A) synthetic -carotene as standard and (B) -carotene
extracted from D. bardawil. -carotene was detected at 450 nm
absorbance. Rt: retention time.
Protective effect of -carotene: Table 3 shows that either in

presence or absence of LPS, the various concentrations of natural
-carotene (30, 140 and 350 g/kg) have no significant effect on
the levels of IL-1 and TNF-, whereas the level of IFN- was
significantly affected. In absence of LPS, administration of
synthetic (350 g/kg) or natural -carotene at different doses
showed non-significant change on the levels of studied cytokines
compared to vehicle control. Pre-treatment with natural -carotene
in LPS injected groups exhibited a significant reduction in IL-1
and IFN- at both doses of 30 and 140 g/kg. Non-significant
difference was observed in Group 8 compared to LPS control for
all investigated cytokines. Synthetic -carotene administration
for 15 days in the presence of LPS (Group 7) caused a significant
reduction in the levels of IL-1 and IFN- compared to LPS control,
whereas it has no marked effect on the level of TNF-. All

concentrations of natural -carotene showed a positive correlation
with the levels of cytokines (Table 3).
Therapeutic effect of -carotene: One-way ANOVA analysis after
LPS injection showed that various concentrations of natural carotene (30, 140 and 350 g/kg) had no significant effect on the
levels of IL-1, IFN-, and TNF- (Table 4). Administration of low
dose (30 g/kg) of natural -carotene after LPS injection exhibited
a significant reduction in the levels of IL-1, IFN- and TNF-
compared to control LPS. Moreover, the levels of IL-1 revealed a
significant reduction in Group 14 compared to control LPS,
however, the levels of IFN- and TNF- were not affected. Posttreatment with synthetic or natural -carotene at high dose (350
g/kg) failed to induce significant reduction in the levels of IL-1,
IFN- and TNF- compared to control LPS. The levels of IL-1,
IFN- and TNF- were positively correlated with various
concentrations of natural -carotene and their correlation
coefficients were 0.92, 0.70 and 0.94, respectively.
The cytotoxicity of natural -carotene against human cancer
cell lines: Percentages of cell inhibition of HepG2 cell line were
positively correlated with various concentrations of -carotene (r
= +0.90) and doxorubicin (r = +0.76). The value of IC50 was 14.58
g/ml for natural -carotene in comparison with IC50 of doxorubisin
which was 7.51 g/ml (Fig. 3A). The various concentrations of
natural -carotene showed a positive correlation with percentage
of cell inhibition of MCF7 cells (Fig. 3B). The correlation coefficient
of natural -carotene (r = +0.85) was lower than doxorubicin (r =
+0.99). IC50 of natural -carotene mounted 7.44 g/ml while IC50 of
doxorubicin was 2.67 g/ml.
Discussion
A biphasic aqueous/organic two-phase system offers a solution
in which both production and extraction occurs simultaneously.
Two of the most important criteria for solvent selection are high
product recovery capacity and biocompatibility. Many previous
studies mentioned that log Poct is used to predict the activity
retention of biocatalyst in organic media. The retention of the
biocatalyst activity is high in a polar organic solvent having log
Poct > 4 31. It appeared that when log Poct increased, extraction
ability of solvent to -carotene decreased as the solubility of
solvent in water decreased. In the present study, decane has the
highest extraction ability among all solvents used as it recorded
highest extracellular -carotene value, but after more than 24 h
cultures turned white. So, dodecane was chosen as the most
suitable biocompatible solvent for extraction of -carotene from
D. bardawil due to its higher extraction ability (it was the second
after decane) and highest -carotene content (intracellular +
extracellular). Our results were in agreement with Leon et al. 23 as
they found that decane and dodecane were more compatible to D.
salina cells than tetradecane and hexadecane. Most milking
studies have used dodecane as a biocompatible solvent 8, 32. As in
the present study, there is no significant difference between
extraction ability of most of the different biocompatible solvents
used 33. Moreover, -carotene productivity per cell with dodecane
was highest and phase separation was rapid producing -carotene
with high purity.
The cell membrane seems to be the primary target of solvent
27
Table 3. The levels of IL-1, IFN- and TNF- (pg/ml) in serum of mice
orally administered an olive oil, synthetic -carotene (synth, 350
g/kg) and various concentrations of natural -carotene (nat, 30,
140, and 350 g/kg) in absence and presence of lipopolysaccharide
(LPS, 2.5 mg/kg).
action. The modification of the cell membrane

characteristics is the more relevant cellular adaptation
mechanism due to stress caused by organic solvent 34.
Solvent toxicity has been associated to the ability of
solvent to penetrate the cell membrane, altering its fluidity
and tampering with normal cellular functions 9. In this
Cytokines levels (pg/ml)
Group
work, decane and dodecane recorded the lowest optical
IL-1
IFN-
TNF-
olive oil
55.089.15a
28.068.28a
80.0022.06a
densities indicating lower cell membrane integrity and so
olive oil+LPS
156.3325.27d
132927.41c
606.16157.23c
lower cell membrane damage.
In absence of LPS
In a two-phase milking experiment, we noticed that the
a
a
a
synth 350
35.104.9
328.22
38.384.54
increase in continuous extraction time caused an increase
nat30
65.055.80a
14.562.57a
55.314.25a
in -carotene content whereas the total amount of nat140
59.513.76a
15.864.24a
86.338.39a
nat350
69.264.36a
56.0912.69a
88.5018.06a
carotene produced (-carotene content of the cells +
ANOVA
F2,15=0.244,P>0.05 F2,15 =9.01, P<0.05 F2,15=2.494, P>0.05
extracted part to the organic phase) is highest at the end
r
+0.80
+0.95
+0.58
of the experiment, which is in agreement with Hejazi et
In presence of LPS
al. 35, whereas higher production of -carotene is caused
synth350+LPS
76.938.53c
898.00144.78b
487.1677.69bc
abc
b
b
nat30+LPS
109.3530.25
615.16196.14
351.6617.41
by the continuous extraction, and the presence of the
nat140+LPS
118.3631.44bc
867.41247.98b
432.16100.93bc
bio-compatible solvent (dodecane) in the culture medium
nat350+LPS
281.809.62d
1297.3323.03c
660.66140.87c
may induce the -carotene production pathway. Mixing
ANOVA
F2,15=14.16, P>0.05 F2,15=6.62, P<0.05 F2,15=2.541, P>0.05
causes some shear stress to the cells and also can increase
r
+0.99
+0.99
+0.96
Data were represented as mean of six mice standard error of mean (SEM). In the columns: Means marked with the
contact of the cells with the organic phase. Our
same superscript letters are similar (insignificant difference, P>0.05).
continuous milking experiment here could not be run for
: the effect of various concentrations of natural -carotene on the levels of cytokines (pg/ml).
P>0.05: insignificant effect of various natural -carotene concentrations on the levels of cytokines (pg/ml).
more than 15 days due to decreased growth levels of
r: correlation coefficient between the concentrations of natural -carotene and the levels of cytokines (pg/ml).
cells (the decreased levels of chlorophyll a and cell count).
It has been found that the mixing of aqueous and organic
Table 4. The levels of IL-1, IFN- and TNF- (pg/ml) in serum of mice
phase may enhance extraction but can also enhance
administered with lipopolysaccharide (LPS, 2.5 mg/kg) followed
damage caused by organic solvent, so a compromise
by olive oil, synthetic -carotene (synth, 350 g/kg) and various
between toxicity and extraction ability has to be taken
concentrations of natural -carotene (nat, 30, 140, and 350 g/
into consideration when choosing the organic solvent 35.
kg) after 6 h of LPS injection.
It was obvious from the present data that extracted Cytokines (pg/ml)
Group
carotene
is composed of two isomers; these are 9-cis IL-1
IFN-
TNF-
carotene and all trans -carotene. The ratio of 9-cis to all
lps+olive oil
127.57.90b
1203.097.01b
959.1615.8c
trans -carotene is 1.13, which is in harmony with
LPS+synth350
120.619.70b
1038.4263.75b
520.0 96.5bc
LPS+nat30
68.49.20a
407.473.57a
321.2 42.8a
Kleinegris et al. 10, who reported that when high light
LPS+nat140
65.023.80a
1129.4193.26b
737.6 95.4bc
intensity was used to stress the cells, the ratio of 9-cis to
LPS+nat350
114.012.50b
1056.4229.11b
966.2 109.9c
ANOVA
F2,12=0.543, P>0.05 F2,12=3.464, P>0.05 F2,12=13.941, P>0.05 all-trans -carotene increased from 0.55 to 1.27. It was
observed that the increase in 9-cis to all-trans -carotene
r
+0.92
+0.70
+0.94
Data were represented as mean of six mice standard error of mean (SEM).
ratio was mainly due to accumulation of carotenoid
In the columns: Means marked with the same superscript letters are similar (insignificant difference, P > 0.05).
containing globules (over 50% 9-cis -carotene) and a
: the effect of various concentrations of natural -carotene on the levels of cytokines (pg/ml).
P > 0.05: insignificant effect of various natural -carotene concentrations on the levels of cytokines (pg/ml).
decrease in thylakoid-bound carotenoids (mainly all-trans
r: correlation coefficient between the concentrations of natural -carotene and the levels of cytokines (pg/ml).
-carotene) 36.
W
y = 5.2237ln(x) + 60.535, r= +0.76
y = 21.134ln(x) - 7.0242, r= +0.90
-carotene
Doxorubicin
12
22
32
42

52
100
90
80
70
60
50
40
30
20
10
0
W
100
90
80
70
60
50
40
30
20
10
0
-carotene
Doxorubucin
y = 32.752ln(x) - 32.246,
r= +0.99
y = 29.433ln(x) - 34.149, r= +0.85
12
22
32
42
52

Figure 3. Dose-response curve of natural -carotene and doxorubicin cytotoxicity against HepG2 and MCF-7 human
cell lines using SRB assay.
Percentages of cells inhibition of (A) HepG2 and (B) MCF-7 cell lines after 72 h of incubation with natural -carotene and doxorubicin at various
concentrations (5, 12.5, 25, 50 g/ml). Each value was a mean of three samples standard error of mean. r: correlation coefficient.
28
During inflammation, there is recruitment of various immune

cells to the injury or inflammatory site, the activated cells release
pro-inflammatory cytokines. Hosts defense immune system
functions a pivotal role in controlling the acute inflammatory
response. Overproduction of pro-inflammatory cytokines is
associated with pathogenesis of many diseases 37. Here, treatment
with natural -carotene was evaluated in order to sustain the
preventive efficacy on pro-inflammatory cytokine production in
CD1 mice. Natural -carotene at different doses was investigated
in parallel with synthetic -carotene at only one fixed dose of 350
g/kg.
In the current work, mice treated with either synthetic or natural
-carotene groups at various doses for a period of 15 days showed
no significant effect on the levels of IL-1, IFN- and TNF-
compared to vehicle group. Our findings were in agreement with
Novoselova et al. 28 who reported that antioxidant feeding showed
no considerable effect on cytokine milieu of healthy animals. Upon
induction of inflammation, we observed an inflammatory response
that resulted in enhancement of serum levels of pro-inflammatory
cytokines, whereas the levels of IL-1, IFN- and TNF- were
significantly elevated in LPS control compared to vehicle group.
Within aspects of protective assessment, we implied that serum
IL-1 and IFN- levels were significantly inhibited in natural carotene pretreated inflamed mice compared to LPS control at
doses of 30 and 140 g/kg. This tendency towards normalization
of serum cytokine levels has been previously reported 28. Moreover,
D. bardawil powder rich diet showed reduction in mice liver
inflammation by decreasing the expression of IL-1 18.
TNF- is a pleotropic cytokine implicated in immunomodulation 38. TNF- is a crucial inflammatory cytokine released
by monocytes and macrophages, involved in the pathogenesis of
many inflammatory disorders 39. Pre-treatment of inflamed mice
with natural -carotene caused a marked reduction in TNF- at
low dose of 30 g/kg. It is clear that low dose was accompanied
with beneficial suppression of this key inflammatory mediator,
however, the other doses failed. This result has been demonstrated
by Liu et al.40, whereas low doses of natural -carotene have been
suggested to induce protection on Ferrets lungs exposed to
cigarette smoke. Treatment with D. bardawil alga containing both
all-trans and 9-cis-carotene diet has potential ability to inhibit
atherosclerosis progression, particularly in high-fat diet regime 18.
Additionally, natural -carotene could reduce pro-inflammatory
cytokines in mononuclear cells of Alzheimers disease patients in
vitro 41.
In the present study, we found that high dose of natural carotene pretreatment failed to suppress the release of IL-1, IFN, and TNF-. In addition, levels of IL-1 and TNF- to some
extent showed elevation compared to LPS control. Thereby, high
dose of natural -carotene didnt provide a protective effect and
moreover could provoke inflammatory condition. These results
were confirmed in vitro, whereas the high dose of -carotene
exerted pro-inammatory effects by signicantly increasing the
secretion of TNF- from LPS-stimulated murine macrophage cells 42.
Also, it is suggested as a stimulator of an inflammatory process in
peripheral blood mononuclear cells from healthy donors 43.
In our investigation, when therapeutic efficacy of natural carotene identified in a dose dependent manner, we found that
low dose of natural -carotene post-treatment in inflamed groups,
exerted immunosuppressive effect on IL-1, IFN-, and TNF-
production compared to the control LPS. These findings were

supported by previous study in vivo, which found that elevated
serum levels of TNF- and IL-1 induced by LPS administration
were significantly suppressed by administration of natural carotene 44. Furthermore, dietary administration of rich 9-cis carotene employed inhibition of adipose tissue inflammation in
diabetic mice 45. Recently, the algal carotenoid extract of D. salina
demonstrated a potent suppression of IL-1, IL-6 and TNF-
production in initial inflammatory response of LPS-stimulated
macrophage cells; low doses showed higher effective inhibition
on IL-1 production as compared to all trans--carotene 19. Indeed,
post-treatment with either high dose of natural or synthetic carotene failed to promote significant inhibition of IL-1, INF-
and TNF-. These data explain that with the increase in the
concentration of natural -carotene, its ability to inhibit the release
of pro-inflammatory cytokines decreased.
It has been documented that carotenoids induced antiproliferative activity on cancer cells, introducing alterations in
pathway mechanisms, led to cancer cell death 46. D. bardawil 9cis--carotene is up to ten times more effective at cancer
prevention than all trans -carotene 47. Cytotoxicity of the natural
extract of -carotene was determined in comparison to the
anticancer drug doxorubicin in vitro. Natural -carotene showed
cytotoxicity against HepG2 and MCF-7 cells in a dose dependent
manner. Our results showed that natural -carotene exhibited
cytotoxicity to HepG2 and MCF-7 cell lines, whereas IC50 of carotene was nearly two- and threefolds of IC50 of doxorubicin,
respectively, that was a stronger cytotoxic drug. These findings
were supported by Fujii et al. 48 indicating to the inhibitory effect
of -carotene rich D. bardawil on mammary tumor progression
cell line. IC50 of D. salina algae extracted under stress was lower
than normal condition 11. Moreover, -carotene revealed inhibitory
effect on cell growth 49 inducing apoptosis 50 in MCF7 cells.
Conclusions
Our results suggest that low dose of natural -carotene extracted
from D. bardawil is capable of enhancing beneficial antiinflammatory activity by exerting immuno-suppressive effects on
proinflammatory cytokines in vivo. Hence, natural -carotene may
be a useful therapeutic tool for treatment of various inflammatory
diseases; providing protection against inflammation. Additionally,
this study demonstrates the cytotoxic effect of natural -carotene
on human cancer cell lines in vitro. Further studies will be important
to consider the possible use of -carotene to inhibit cancer cell
survival in vivo.
Acknowledgements
This work was supported by Faculty of Science, Cairo University,
Egypt.
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31
WFL Publisher
Helsinki, Finland
www.world-food.net
Effects of some technological parameters on chemical and sensory qualities and free
fatty and amino acids of various probiotic cultures in Beyaz cheese during
ripening process
Filiz Yanglar 1* and Salih Ozdemir 2
1
Department of Food, Engineering Faculty, Ardahan University, 75000, Ardahan, Turkey. 2 Department of Agricultural, Food
Engineering, Atatrk University, 25000, Erzurum, Turkey. *e-mail: f_yangilar@hotmail.com, filizyangilar@ardahan.edu.tr
Received 16 May 2014, accepted 2 October 2014.
Abstract
It was aimed in the present study to assess the effects of four probiotic strains on the chemical, physical and organoleptic properties of Beyaz cheese.
For all experimental samples taken into consideration in the study, total chemical parameters were found to increase during ripening (p<0.05) process
in the study. The FFAs concentration in control cheeses was significantly higher than that of made with probiotic dahi cheese samples (p<0.05). In
addition, probiotic B. bifidum BB-12 appeared to increase the production of FFAs (caproic, 1.67; caprilic, 0.16 and capric, 2.57%). Bifidum BB
12+L. acidophilus LA5 increased significantly the rate of nitrogen compounds with low molecular weight and individual free amino acids
(p<0.05). Length of ripening period contributed to a significant increase in the content of free amino acids. For the sensorial characteristics, cheese
sample E was found to the least preferred by the panellists.
Key words: Beyaz cheese, probiotics, free fatty acids, free amino acids.
Introduction
Today consumers are concerned in great majority with not only
food security and its nutritional values, but also its health
benefits 1, 2. Such kind of demands helped shape new concepts in
food industry, e.g. functional food, where probiotic ingredients
take important parts 3. Probiotics are defined in an overview to be
living non-pathogenic microorganisms used in food industry to
be dietary supplements due to their health benefits 4-9, such as
increasing the intake rate of low-fat dairy products and the
reduction of the incidence of cardiovascular diseases (CVD),
preclinical atherosclerosis, and cardiovascular risk factors in
middle-age and older age persons 3, 10-16. Although the number of
probiotic bacteria that provides health benefits has not been
firmly established, levels between 106 and 109 cfu/g have been
suggested 17, 18. Various parameters must be considered when
adding probiotic bacteria to foods: type of culture to use, addition
level required to obtain a physiological effect, survival during
process parameters, stability during storage, and effect on the
sensory properties 8, 19, 20.
Dairy products have been used as carrier foods for probiotic
bacteria, as many of them had already been optimized for survival
of lactic cultures 21, 22 , while new products including milk, yoghurt,
fermented milk, desserts, fruit juice and some cheese types have
also taken their places among probiotic products 23. Cheese owns
a deserved fame to be a good carrier for probiotic bacteria by
allowing them to survive throughout gastrointestinal tract 15, 24-32.
Beyaz cheese (Turkish acronym of white cheese) is a soft or semihard cheese type produced from sheep or cow milk or mixture of
them 26. Cheese may offer several advantages over fermented milk
products such as yoghurt by serving as a delivery system for
32
viable probiotic in gastrointestinal tract; tending to increase fat

content; and offering protection to probiotic bacteria during
storage and passage through the gastrointestinal tract. Cheese
can also exhibit larger buffering capacity than yoghurt 33. Various
chemical and biochemical reactions can be seen during the ripening
process of Beyaz cheese including glycolysis, lipolysis and
especially proteolysis, which is an important process and plays a
direct role in the development of cheese flavour and texture 34.
Being among the most widely used probiotic bacteria, lactobacilli
and bifidobacteria can contain proteolytic and peptidolytic enzyme
types and therefore affect proteolysis 35-40. Addition of lactic acid
bacteria to dairy products was stated to contribute to the
production of free fatty acids (FFAs) causing the lipolysis of milk
fat 41-43.
The present study was conducted to evaluate the implications
of different probiotics [B. bifidum BB-12 (B), B. bifidum BB-12+L.
acidophilus LA-5 (C), B. bifidum (D) and B. longum (E)] in addition
to commercial lactic culture [L. lactis and L. cremoris (A)] in the
chemical and physical composition and sensory performance; and
determine the fatty acid composition and related properties of
Beyaz cheese during storage at 4C.
Cultures: Research and Application Farm of Atatrk University
provided cow milk and Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. cremoris frozen to dry were obtained
from DSM Food Specialties Pty. Ltd. (Moorebank, NSW, Australia)
and used in the preparation of starter culture. Strains of various
probiotic bacteria were used as adjunct cultures, among which B.
bifidum BB-12 and B. bifidum BB-12+L. acidophilus LA-5 were

obtained from Peyma Hansen (Gayrettepe, Istanbul, Turkey), while
B. bifidum and B. longum were obtained from Christian Hansen
(Christian Hansen, Valinnhos, Brazil). The organisms were
activated using the method by Martensson et al. 44 and
experimental cheese material was produced in the pilot dairy plant
of the Agricultural Faculty of Atatrk University.
Cheese manufacture: In the study, probiotic Beyaz cheese
material manufactured by taking the rules of Demirci and imek 45
into consideration was divided into five experimental groups (one
control and four probiotic containing groups). Cheese
manufacturing process was initiated by taking 500 L raw milk and
adjusting its fat concentration to 3%. Milk sample was pasteurized
at 65C for 30 min, cooled to the incubation temperature of 35C
and divided into five equal parts (batch). A solution of 20 g/100 L
CaCl2 was added to each batch and a control batch was prepared
using 1 mL/100 mL commercial culture mix consisting of L. lactis
and L. cremoris and a four batch mix [B (Bifidobacterium bifidum
BB12), C (B. bifidum BB12+L. acidophilus LA5), D (B.bifidum)
and E (B. longum)] was produced using equal concentrations of
the probiotic and commercial mixes. Experiments on cheese
production were conducted at the beginning with 100 L of raw
milk, which was pasteurized at 65C for 30 min, cooled to 35C,
CaCl2 (20 g/100 L) and a commercial culture mix inoculum (1 mL/
100 mL;1% v/v) of cheese starter (Lactococcus lactis and
Lactococcus cremoris) was added. Probiotic strains were added
into samples B, C, D and E to inoculate at a level up to 107 cfu/mL.
Then 12 mL chymosin (Peyma Hansen, Turkey) dispersed in 100
mL water was added in each cheese vat in a sufficient rate to
coagulate them in 90 min. At the end of curdling process about 1
cm3 small curd blocks were left for storage and compressed, after
which formed cheese material was cut into 8 cm3 cubes salted in
pasteurized brine (12% w/v, NaCl) for 6 h. After brine-salting, cheese
samples in the form of blocks were taken to store at room
temperature for 12 h and then transferred to plastic bags containing
brine water by leaving ripening at 41C. Such manufacturing
process of cheese was triplicated and the samples were left to
ripen for 2 months by analysing at the 2nd, 15th, 30th and 60th days of
ripening.
Chemical and physical analysis: Moisture, fat, dry matter fat,
salt, dry matter salt (%), ash (%), protein (%), water-soluble protein
(WSN; %), ripening degree (%) and titratable acidity (SH) of
probiotic Beyaz cheeses were measured three times according to
Kurt et al. 46. The pH was measured by adding and mixing 20 mL of
distilled water in grated cheese (10 g) using a digital pH meter
(WTW 3401 47).
Nitrogen fractions: Probiotic Beyaz cheese fractions soluble in
12% trichloroacetic acid-soluble nitrogen (TCA-SN) were
determined according to Polychroniadou et al. 48 and the microKjeldahl method by IDF 49. In this process, homogenisation of
grated cheese samples (20 g) was performed by adding and
blending 40 mL of H2O in the samples an Ultraturrax blender (IKA,
Wilmington, NC) for 2 min after which the mixture (homogenate)
was stored at 40C for 1 h and centrifuged at 3000 g for 30 min at
4C. The fatty slick was removed and the supernatant was filtrated
through filter paper (Scleicher & Schuell 589/2). Twenty five mL
extract prepared for WSN was taken in an equal volume of 24%

(w/v) and TCA was added in the mixture to fragment nitrogenous
compounds. The mixture samples were left for incubation for 2 h
at ambient temperature. Precipitates were filtered through white
ribbon filter paper (Schleicher & Schuell, 589/2).
Analysis of free fatty acids (FFAs): Direct transesterification gas
chromatography was used to define FFA profile of milk and dahi
samples as in Akaln et al. 50. One mL of methanol : benzene (3:2
ratio) was added to an aliquot of 100 mL of milk or 100 mg of dahi
samples and freshly prepared 1 mL acetyl chloride : methanol
(5:1000) was also added in the mixture and the tubes were capped
tightly. The mixture tubes were taken in methanolysis process at
100C for 1 h, cooled to room temperature and 1 mL methylated
penta decanoic acid-hexane solution (500 mg dissolved in 1 mL of
hexane) and 1 mL water were added. After that, the tubes were
shaken and stored at 4C until the gas chromatography. GC-Agilent
6890 N (USA) equipment had a glass column of 60 m 0.25 mm ID
packed with 10% DC200 on chromosorb, and a flame ionisation
detector. Nitrogen was the carrier gas with the flow rate of 28 mL
min-1. The injector port temperature was fixed at 100C for 2 min by
reaching gradually up to 250C. Peak rates of each FFA were
identified according to the retention times of the reference
standards (Sigma Chemical Co., St. Louis, MO, USA).
Free amino acid (FAA) analysis: Free amino acids were extracted
from probiotic Beyaz cheese curd slurry samples conveniently to
the method in Standara et al. 51. Cheese slurry (10 g) was added in
90 mL trichloroacetic acid (TCA) by mixing to homogenize the
mixture. Fatty top layer of the mixture was removed and the
remaining sample was stored at 3C to separate cream from the
mixture at more advanced level and then the mixture was centrifuged
at 3C for 10 min at 8000 g to remove fully the cream remnant and
vacuum filtered (No. 1 filter paper, Whatman International Ltd,
Maidstone, UK). The isolated free amino acids were derivatized
according to protocol of EZ: FaastTM kit (Phenomenex, Torrance,
CA, USA) and subjected to gas chromatography (GC-Agilent 6890
N (USA) with a split injection port and flame ionization detector
(FID)). Amino acid samples were injected and separated on a
Zebron ZB-PAAC column (10 m 0.25 mm, Phenomenex) and
helium was carrier gas at 60 kPa and injection port and detector
were set to 250C and 320C, respectively, by increasing oven
temperature 35C in one minute from 110C to 320C and leaving
at 320C for one minute. Amino acid standards included in EZ:
FaastTM kits were used for the identification of amino acids in the
samples.
Sensorial analysis: Experimental samples stored at 41C for 60
days were evaluated by trained and experienced panellists
considering sensorial properties and the principles in Lyne 52.
Each panellist gave scores, ranging from 1 (poor) to 9 (excellent)
to the cheese samples taking five sensory properties into account
including colour, texture, taste and aroma, foreign flavour and
aroma, saltiness and general acceptability. Panellists were
permitted to have water and bread to screen the tastes of each
sample.
Statistical analysis: The randomized complete block design was
adopted for the experiments in the study. Data obtained was
33

Probiotic Beyaz cheese samples were produced from the skimmed
milk material containing dry matter, fat, protein, ash, % acidity and
pH in the rates of 11.680.45%; 3.210.38%; 3.310.17%;
0.590.20%; 8.880.71SH and 6.400.03, respectively. Mean yields
of cheese samples A, B, C, D and E were 15.94ab, 16.85b, 18.15b,
15.48a and 17.60 b, respectively. Cheese sample D yielded
significantly lower than B, C and E (p<0.05) possibly caused by
the different acidification processes employed in sample D. It was
stated by Kindstedt et al. 54 and Buriti et al. 55 that when the direct
milk acidification process is used in common cheese production,
higher yield rate, higher moisture content, as well as improved
durability can be obtained due to delayed acidification.
Physical and chemical composition: Table 1 gives the changes
observed in the physico-chemical parameters obtained from
probiotic Beyaz cheese samples A, B, C, D and E throughout
during 60-day storage period. Length of the ripening period was
found to have statistically significant effects on the chemical
composition of experimental samples. The highest rate of dry
matter (44.43%) was determined in control sample (Lactococcus
lactis+Lactococcus cremoris) even though the dry matter content
of all cheese samples increased during the ripening period. Such
a situation may be expressed by the positive correlation between
dry matter and salt 56, 57. Differences in the rates of dry matter
content between the samples were found to be statistically
significant (p<0.01) in the present study, while Atasever et al. 58,
Topcu and Saldaml 59 and ksz et al. 60 found mean dry matter
rates of 35.41 to 39.48%, 39.80 to 41.75% and 30 to 61%,
respectively, which are in a similar range with those found in the
present study. The highest fat (22.48%) and dry-matter fat
(52.57%) contents were in B. bifidum BB-12 sample.

In the present study, differences in fat rates between samples
were statistically significant (p<0.01) during ripening period in
which fat contents of all cheese samples increased. A general
decrease in the titratable acidity rates of all probiotic cheese
samples was observed throughout ripening period (Fig. 1). The
acidity rate of B. bifidum BB12+L. acidophilus LA-5 (19.12 SH)
sample was lower than those in others in the present study, which
may be related to the salt contents of C cheese sample. Higher salt
and moisture contents might have reduced the activities of lactic
acid bacteria found in cheese 61, 62. During the ripening period in
the present study, pH rates of the cheese samples showed a slight
increase and the highest increase in pH value was seen in C
probiotic cheese sample (6.44), which is convenient with literature,
e.g. Atasever et al. 58 reporting a mean pH rate ranging from 4.98
to 5.68. The protein content of cheese samples ranged from 13.44
to 16.14% resulting from the hydrolysis of proteins to water-soluble
nitrogenous compounds and to brine 62, 63. Mean protein content
of Beyaz cheese ranged from 12.78 to 17.27% by Hayalolu 64,
being in convenience with the present study.
25
Titratable acidity (SH)
transferred and evaluated in SPSS Statistical Software (version

15.0). Standard deviations of mean chemical and biochemical
values were also calculated and statistically significant differences
in mean values were compared using Duncans multiple range
tests. Each sample was subjected to triplicate analysis 53.
24
23
22
21
20
19
18
2
15
30
Ripening time (day)
60
Figure 1. Changes in titratable acidity (SH) of probiotic Beyaz

cheese samples.
Nitrogen fractions: Table 1 and Fig. 2 summarizes the results of

the assessment of proteolysis in the control and probiotic Beyaz
cheese samples through the determination of water-soluble
Table 1. Some chemical and physical properties of probiotic Beyaz cheese.

Beyaz
cheese
samples
A
Ripening
time
(days)
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
Dry
matter (%)
Protein
(%)
Fat (%)
Salt (%)
Ash (%)
Fat in dry
matter (%)
Salt in dry
matter (%)
pH
WSN (%)
40.600.18
41.330.13
42.450.11
44.430.08
40.460.15
41.900.18
42.060.44
43.600.71
37.490.62
38.950.31
40.760.58
41.920.65
39.850.69
41.590.78
42.200.86
43.230.12
39.950.93
39.990.24
41.590.50
42.910.41
13.750.14
14.070.12
14.500.05
15.200.16
13.440.11
13.530.50
13.720.44
15.010.29
14.030.42
14.150.51
15.030.85
15.040.29
16.140.58
15.530.42
15.620.26
15.660.32
15.440.71
15.320.25
15.660.43
15.900.20
19.370.30
20.560.28
21.230.43
22.180.52
20.260.37
21.310.65
22.110.91
22.480.76
18.410.85
19.450.19
20.200.16
20.240.34
18.500.29
20.060.27
19.870.61
20.630.74
18.600.36
18.540.61
19.070.25
19.470.48
4.370.42
5.660.38
6.070.39
6.420.34
4.150.47
4.330.58
5.500.54
6.270.32
4.300.46
4.580.82
5.340.51
5.750.46
4.560.53
5.460.36
6.170.62
6.340.48
5.450.39
5.830.53
6.480.42
7.050.57
4.890.24
6.110.42
6.690.50
6.770.38
4.700.63
4.840.37
5.910.28
6.790.45
4.830.47
5.080.32
5.520.35
6.260.29
5.100.37
6.020.26
6.660.63
6.840.56
5.980.61
6.120.23
6.820.74
7.510.32
47.710.52
49.740.70
49.990.59
49.920.41
50.080.35
50.860.26
52.570.47
51.550.39
49.110.64
49.940.43
49.550.34
48.280.56
46.410.39
48.070.45
47.090.17
47.720.23
46.550.47
46.350.39
45.860.70
45.380.55
10.760.50
13.680.71
14.310.32
14.450.68
10.260.41
10.340.60
13.070.77
14.370.35
11.480.39
11.770.23
13.090.18
13.710.46
11.440.70
13.200.52
16.630.38
14.660.67
13.630.49
14.580.20
15.580.38
16.440.41
5.320.69
5.460.76
5.870.39
6.080.43
5.030.46
5.130.37
5.630.62
5.740.25
6.060.28
6.260.42
6.440.31
6.230.49
5.360.16
5.430.32
5.660.15
5.720.43
5.580.64
5.830.39
5.950.26
6.140.57
1.920.14
2.030.20
2.660.18
3.120.37
2.840.26
2.450.52
2.820.47
2.750.24
2.930.02
2.990.40
2.970.18
3.260.20
2.150.11
2.190.37
2.180.45
2.210.38
2.110.31
2.240.60
2.230.12
2.650.29
Ripening
degree
(%)
13.970.09
14.440.24
18.380.52
20.560.13
21.170.17
18.100.35
20.550.42
18.360.51
20.860.78
21.150.05
19.800.11
21.400.85
13.350.41
14.090.03
13.960.64
14.100.07
13.700.32
14.610.03
14.250.99
16.650.83
** The values presented are the average of three recurrences. A(Control): Only lactic culture. B: B. bifidum BB-12+lactic culture. C: B. bifidum BB-12+L. acidophilus LA-5+lactic culture. D: B. bifidum+lactic culture.
E: B. longum+lactic culture.
34
Table 2. Free fatty acids (FFAs) composition of probiotic Beyaz

cheeses during storage.
2.5
TCA-SN (%)
Beyaz
Cheese
Samples
1.5
1
A
0.5
B
0
15
30
Ripening time (day)
60
Figure 2. Changes in trichloroacetic acid-soluble nitrogen (TCASN) of probiotic Beyaz cheese samples.
C
D
E
Chemical analysis of all types of cheese samples in the present

study showed that the addition of tested probiotic microorganisms
in Beyaz cheese has no adverse effect on cheese composition
and similar results were reported in previous studies, e.g. Gardiner
et al.29, Ong et al.66 and Kilic et al.26.
Free fatty acid (FFA) composition: Results of free fatty acid
analysis of probiotic Beyaz cheese during ripening period are
given in Table 2. The rate of caproic acid in the present study
ranged from 1.01 to 1.74% (Fig. 3), which is convenient with the
results found in Prandini et al. 67 where this rate was stated to be
1.14% in Alpine cheese samples. Total rate of FFA, capric acid
(1.822.85%), was higher in probiotic dahi in the present study
(Fig. 4). Corbo et al. 19 reported that the rate of capric acid in
Bifidobacterium sp. added Italian cheese was higher than that
found in control samples, which is in agreement with the findings
in the present study. It can be seen in literature that butyric acid is
produced largely through lipolytic activity of LAB 43, 68 and the
butyric acid content of food may contribute to medicinal properties
of the dahi product 43, 69.
Free amino acid (FAA) composition: Proteolysis in ripening
process can contribute to the formation of flavour thanks to
peptides and free amino acids. Presence of free amino acid in
Butiric
(C4)
0.560.63
0.530.32
0.530.56
0.520.35
0.580.50
0.520.42
0.540.28
0.500.45
0.550.29
0.520.64
Free fatty acids (%)

Caproic
Caprilic
(C6 )
(C8)
1.010.70 0.210.16
1.610.52 0.210.21
1.740.34 0.220.10
1.670.43 0.160.16
1.640.56 0.170.39
1.360.17 0.150.25
1.370.25 0.130.13
1.510.41 0.150.34
1.380.53 0.140.18
1.290.59 0.130.31
Capric
(C10)
2.290.32
2.850.24
2.510.32
2.570.22
2.290.30
1.920.24
1.820.43
2.200.29
2.100.50
1.970.21
Caproic acid (%)
** The values presented are the average of three recurrences.
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
60
Ripening time (day)
Figure 3. Changes in caproic acid of probiotic Beyaz cheese samples.

3
2.8
Capric acid (%)
nitrogen (WSN) and trichloroacetic acid-soluble nitrogen (TCASN) over 60-day ripening period at 4C. The ratio of WSN to total
nitrogen (TN) in all samples increased consistently. Cheese
produced from B. bifidum BB-12+L. acidophilus LA-5 (3.26%)
revealed the highest water-soluble protein rate. WSN rates
reported by Ong and Shah 65 in the samples of probiotic Cheddar
cheese are similar to those found in the present study.
The rate of TCA-SN also increased continuously and intensified
more until 60th day (Fig. 2). Ong and Shah 65 found that the levels
of SN-TCA in probiotic Cedar cheeses increased during ripening
period similarly with those in the present study. Reason for such
a situation may be the responsibility of the starter and probiotic
bacteria proteinases for the formation of TCA-SN. Cheese samples
produced from B. longum (2.27%) in general showed the highest
rate of TCA-SN. Such a condition may show that upon the
formation of soluble peptides by rennet and starter culture, the
peptidases and proteinases of probiotic adjuncts hydrolysed these
peptides and released more intermediate- and smaller-size peptides.
This situation was easily seen at the end of 12 weeks when the
primary proteolysis gave products as substrates for the
subsequent proteolysis by the probiotic organisms.
Ripening
time
(days)
2
60
2
60
2
60
2
60
2
60
2.6
2.4
2.2
2
1.8
1.6
1.4
60
Ripening time (day)
Figure 4. Changes in capric acid of probiotic Beyaz cheese samples.
probiotic Beyaz cheese can be attributed to the proteolytic activity

of the bacteria during ripening. Proline was found to increase
until the 30th day and then decreased until the 60th day while glycine,
serine and isoleucine were stable following the 60th day. The
production of free proline was determined to be associated with
flavour Swiss cheese and attributed to the metabolic activity or
the propionibacterium in some studies 70, 71. Such results are
convenient with our study. Free methionine increase in ripening
period may also increase the rate of sulphur volatile flavour
compounds. Free amino acids may exhibit various taste
characteristics depending on their side chains. When the effects
of the presence of probiotic bacteria (Table 3) on free amino acid
rate were taken into consideration, only the effects of lysine and
serine were found to be significant (p<0.05). It was found when
considered 5 sample types treated with 4 different probiotic bacteria
contents (Table 4) that free amino acid rate exhibited a higher
increase in 4 bifidobacteria added cheese curd slurries in 60-day
ripening period compared to B. bifidum BB12+L. acidophilus
LA5, B. bifidum and the B. longum, B. bifidum BB12 and control
sample. In Beyaz cheese curd slurries, proteolytic activity
35
Table 3. Effect of ripening time on free amino acid

contents in Beyaz cheese.
Free Amino
Acid a
Glycine
Alanine
Valine
Isoleucine
Methionine
Proline
Phenylalanine
Tyrosine
Tryptophan
Serine
Threonine
Cystine
Lysine
Histidine
Aspartic acid
Glutamic acid
Asparagine
Glutamine
2
216.9a
321.2a
125.6a
78.3a
115.6a
295.7a
139.5a
179.3a
357.6a
1424.2a
175.9
1582.7
479.6a
301.4a
92.5a
318.6 a
392.1a
1729.8a
probiotic cheese. The results of sensory analysis in the present

study showed that there might have been a common effect
between the test culture mix and commercial starter culture and
the combination of test probiotic culture and commercial starter
culture might be suggested to have positive effects on the sensory
characteristics of Beyaz cheese.
Ripening Time (days)

15
30
60
936.1b
1461.2b
1744.0c
2310.5b
3159.4c
3865.9c
b
c
12.260.7
17.100.2
19.213.6d
5114.0b
6635.8b
7064.0b
b
c
5291.7
8276.9
8954.5cd
291.6c
715.3b
827.9d
1578.2b
3891.4c
3759.4b
154.3a
1205.1b
951.6c
914.8b
1890.3c
2162.0c
17.100.2b 17.383.6b 18.940.9b
233.9
321.8
357.6
1374.0
959.2
925.4
5170.3b
7532.9c
5333.7b
1251.7b
2413.6c
3100.9c
1621.2b
2258.0c
2937.4c
b
d
6413.4
9475.3
9978.1b
4720.1b
5465.8b
5873.6d
14.396.1b 20.417.1b 22.645.2b
Conclusions
Objective of this study was to investigate chemical and
organoleptic characteristics of probiotic bacteria added Beyaz
cheese. In B. bifidum BB-12+L. acidophilus LA-5+lactic culture
cheese samples, increase in FFA was found to be higher than that
in probiotics added cheese samples at the end of ripening process.
It was thought by considering the increased content of some free
amino acids in cheese samples that when probiotic bacteria were
added in cheese samples, they might have accelerated the ripening
process. It may be concluded from the results of the study that
Beyaz cheese bears the ability of being one of the most suitable
tools, which can be used to vehicle for delivering the tested strains
of probiotic bacteria via human diet.
a: Values are given in nmoles g-1 cheese slurry and are means of triplicate analyses.
increased with the free amino acid content when bifidobacteria is

presented in the media. Bergamini et al. 2 stated that compared to
control samples larger rate of free amino acid in probiotic bacteria
added semi-hard cheese samples may be caused by higher
proteolytic activity of the samples. These findings are convenient
with the results of the present study.
Acknowledgements
This work was supported financially by the Atatrk University
Research fund (Project No: 2008/64).
Sensory analysis: Table 5 shows the mean scores given to the

sensory parameters for probiotic cheese samples during their
storage period. Panellists gave nine points to all probiotic cheese
groups and the results of sensory analysis indicated that C cheese
batch (B. bifidum BB-12 and L. acidophilus LA-5) received the
highest sensory scores. E samples were given the lowest score
for saltiness parameter (5.72), which might have been due to high
salt concentration of E probiotic cheese. Previous studies (e.g. 26,
33, 72, 73
) reported no negative effects on sensorial properties of
Table 4. Effect of probiotic bacteria treatment on free amino acid contents in
Beyaz cheese samples.
Free Amino
Acid a
Control
Glycine
Alanine
Valine
Isoleucine
Methionine
Proline
Phenylalanine
Tyrosine
Tryptophan
Serine
Threonine
Cystine
Lysine
Histidine
Aspartic acid
Glutamic acid
Asparagine
Glutamine
642.5
2041.0
7149.6
3962.8
4503.4
736.2
1257.0
272.8
959.0
10.143.6a
551.3
2261.9
3125.4a
1252.8
1412.8
4131.5
3511.9
9984.6
Probiotic bacteria treatment

B. bifidum B. bifidum BB12+
B. bifidum
BB12
L. acidophilus LA5
829.1
872.3
1213.5
1627.3
1468.2
2751.0
9359.4
9837.4
10.274.7
4065.7
4261.0
2236.8
4284.1
474.3
5085.4
776.3
752.8
916.3
1860.4
2026.9
2429.5
587.2
607.7
610.2
1492.9
1104.9
1004.7
13.758.2cd
14.861.0bc
13.838.3c
216.9
36.2
272.0
1723.2
1588.7
1492.5
4248.0c
4517.3b
4127.3bc
1142.5
1092.7
1850.4
1698.2
1427.3
1147.2
5254.6
6263.5
5913.1
3227.9
3717.9
4067.2
12.264.7
14.058.0
13.021.9
B. longum
503.9
1167.4
7835.1
3219.0
3606.4
791.2
1245.7
267.4
804.3
7340.6ab
39.4
1522.6
2819.2a
1293.1
1192.4
4629.5
2020.1
8752.0
a: Values are given in nmoles g-1 cheese slurry and are means of triplicate analyses.
36
Table 5. Results of sensory analysis of probiotic Beyaz cheese samples for 60 days.
Beyaz Cheese
Samples
A
Ripening
time (days)
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
Colour
Texture
8.030.28
7.620.45
7.310.37
7.190.63
7.000.74
7.660.32
7.330.21
7.000.61
6.330.47
6.330.35
7.000.29
6.660.32
7.330.19
7.000.31
6.660.03
7.000.15
8.000.56
7.660.63
7.330.37
8.000.26
7.550.21
7.440.14
7.330.08
7.440.32
7.190.18
7.550.32
7.490.63
7.550.07
7.440.49
7.440.24
7.580.12
7.600.57
7.440.84
7.520.29
7.380.33
7.270.69
6.460.28
6.450.12
6.660.55
6.530.19
Taste and
Aroma
7.600.41
7.440.70
7.300.76
7.440.12
7.210.18
7.600.06
7.660.24
7.440.35
7.410.91
7.350.27
7.520.14
7.710.65
7.380.16
7.490.49
7.330.07
6.800.12
6.940.18
6.910.36
6.770.25
6.580.08
Foreign taste
and odour
8.250.80
7.610.43
7.490.62
7.720.54
7.300.10
7.940.13
7.670.05
7.330.11
7.980.62
7.380.56
7.690.32
7.810.45
7.300.33
7.750.68
7.550.16
7.240.64
7.430.44
7.190.30
7.190.55
6.900.39
Saltiness
7.440.98
7.410.63
7.220.76
7.490.15
7.050.80
7.660.63
7.490.43
7.330.49
7.550.24
7.440.77
7.220.90
7.380.21
7.300.52
6.910.43
6.470.28
6.530.78
7.020.27
6.500.19
5.800.63
5.720.19
General
acceptability
7.600.65
7.470.37
7.360.43
7.220.52
7.270.30
7.550.28
7.660.55
7.550.44
7.360.33
7.550.20
7.410.58
7.490.42
7.300.13
7.440.35
7.210.18
7.240.62
6.000.93
6.760.58
6.000.39
6.230.37
** The values presented are the average of three recurrences.
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39
WFL Publisher
Helsinki, Finland
www.world-food.net
The effects of mint (Mentha spicata) essential oil fortified edible films on the physical,
chemical and microbiological characteristics of lor cheese
Gkhan Kavas 1* and Nazan Kavas
Department of Dairy Technology, Faculty of Agriculture, Ege University, 35100 zmir, Turkey.
Dairy Products Programme, Ege Vocational Training School, Ege University, 35100 zmir, Turkey.
*e-mail: gokhan.kavas@ege.edu.tr, nazan.kavas@ege.edu.tr
1
Abstract
Sorbitol (S) + whey protein isolate (WPI) and 4 different edible films containing mint (Mentha spicata) essential oil were prepared in 1% (v/v) (Me1),
2% (v/v) (Me2), 3% (v/v) (Me3) and 4% (v/v) (Me4) concentrations. These films were used for coating lor cheese (the traditional whey cheese of
Turkey) samples and stored at +4C for 15 days. Microbiological and physico-chemical analyses were carried out on the 1st, 7th, 10th and 15th days
of storage. Coating the lor cheese with edible film had a positive effect on the weight loss. The increase in the concentration of essential oil had a
significant effect on the fat content, titratable acidity, water vapour permeability, weight loss and antimicrobial activity. For the determination of
antimicrobial activity, the cheese samples were artificially contaminated with Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus
aureus, and yeast-mold enumeration was made immediately after the cheese production and accepted as the initial value. E. coli O157:H7 was the
most resistant microorganism to the essential oil, and the most sensitive ones were S. aureus, L. monocytogenes and yeasts and molds, respectively.
The general results showed that addition of mint essential oil to S+WPI based film in 2% (v/v), 3% (v/v) and 4% (v/v) concentrations had a positive
effect on the extension of the shelf life of lor cheese.
Key words: Lor cheese, edible film, essential oil, mint.
Introduction
Whey is revealed during cheese production and its composition
depends on the milk processed to cheese and the cheese
production method, and it is processed to cheese types such as
Mysost, Ricotta, Serac and Requezjao in the world and to lor
cheese in our country. Lor is a traditional cheese type. For the
production of lor cheese, whey is boiled for the precipitation of
serum proteins and separation from whey, kept for 24 hours, and
drained (for 1-4 days) with the help of fine-mesh cloth with weights
placed on. When the desired consistency was obtained, 2-3%
salt was added to the product and offered to consumption as
fresh 1. Despite the elimination of the natural microbial flora with
the heat treatment applied in production, contaminations create
risks after heat treatment and generally the shelf life of the cheese
is limited to 7 days. Microflora of the lor cheese changes even in
storage at low temperatures, microbial load increases in terms of
Enterobacter, yeasts, E. coli and Staphylococcus species. Also,
due to not using lactic acid bacteria in the production, Listeria
monocytogenes, Escherichia coli O157:H7 and Staphylococcus
aureus may grow in the product after contamination 2-4. In this
study, for the extension of the shelf life of lor cheese, which is a
subject that few studies were made on, lor cheese was coated
with Sorbitol (S) + (WPI) based film. WPI films are used in edible
films for their properties such as low oxygen permeability,
gelatinization, thermal stability, foaming and forming polymers
with carbohydrates with covalent binding, as well as their high
nutritional value and functional properties 5, 6. However, due to
their low moisture barrier properties related to their hydrophilic
groups, plasticizers (glycerol, propylene, glycol and sorbitol) are
40
added to WPI based films 7. In many studies, sorbitol is used as a

plasticizer in WPI based films due to its low moisture adsorption
and high-dissolving abilities 8-10. Many studies report that
antioxidant and anti-microbial active compounds may be added
to edible films and coatings. In this regard, many studies show
that essential oils are widely used to take pathogenic bacteria
growth and degradation in foods under control 11-15. In this study,
mint (Mentha spicata) essential oil with antimicrobial (especially
against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis,
Enterococcus faecium, Klebsiella pneumoniae, Escherichia
coli) 16, antifungal (especially against Pseudomonas solanacearum,
Aspergillus niger, Alternaria alternata and Fusarium
chlamydosporum) 17-19, antiviral (against influenza, herpes and
other viruses) 20, 21 properties are used. Menthol found in the
composition of mint essential oil is a potential agent for the
elimination of plasmid resistance of bacteria 22, 23, additionally, it has
an antiviral property 20, and menthone has an antifungal property 24,
25
. Furthermore, eugenol which is found in the oil composition of
some mint species and accepted in GRAS status has medium, and
carvone has high antimicrobial properties against pathogens 26, 27.
Hussain et al. 28 reported that the main components responsible
for the antimicrobial activity of mint essential oil were cis-carveol
and carvone.
In this study, edible films were produced by adding mint essential
oils to S+WPI based film in different concentrations. A portion of
lor cheese was coated with these films in different concentrations,
while a portion is left uncoated (control group). In all cheese
samples, E. coli O157:H7, L. monocytogenes, S. aureus and yeast-
mold enumerations were carried out. Lor cheese samples were

stored at +4C and their microbiological and physico-chemical
properties were examined on the 1st, 7th, 10th and 15th days of the
storage.
Whey protein isolate, sorbitol and essential oil: For the
preparation of the coating film, WPI was obtained from Bipro,
Davisco Foods International, Le Sueur, MN, USA. Sorbitol, which
was used as plasticizer, was obtained from Merck Company. Oil,
which was obtained from Mentha spicata species by using
Klavenger hydro distillation method contained carvone (40.5%),
1,8-cineole (23.3%), cis-carveol (19.1%), -caryophyllene (3.9%),
-pinene (2.8%), trans-dihydrocarvone (2.6%), carvyl acetate (2.1%),
trans-sabinen hydrate (1%), menthone (0.4%) and mint essential
oil, was obtained from a local business. The active substances of
the essential oils were determined with Shimadzu GC-9A Model,
Thermon 600 T (50 ml, 0.25 mm ID) column, flow ratio of 60:1,
temperature program 70-180C ,10-30 min, split ratio 60:1, injection
temperature 250C, 1 ml injection rate and with the nitrogen as
carrier gas GC-MS (gas chromatography-mass spectrometry).
Lor cheese: In this study, lor cheese produced from whey, which
was left over from a kashar cheese production by a private
company, was used.
Preparation of edible film solution: Edible films were prepared
according to Pintado et al. 29 and Mchugh and Krochta 30, with
some modifications. Accordingly, 5% w/v whey protein isolate
was prepared, and after the addition of 1.5 g/100 ml sorbitol
plasticizer to the homogeneous solution, a homogenization process
was carried out in a homogenizer. The mixture pH was adjusted
to 8 and kept in a water bath at 902C for 30 min in order to
improve the mechanical properties of the film solution. The
solution was cooled to room temperature and sorbitol-amended
PAS protein isolate film (WPI) was obtained. The cooled film was
filtered and divided into five equal parts; mint essential oil was
added to the first four parts in different concentrations (1% (v/v)
(Me1), 2% (v/v) (Me2), 3% (v/v) (Me3) and 4% (v/v) (Me4)), the
fifth part was artificially contaminated but not coated, and it
constituted the control sample (K). Mint essential oils were added
by using Tween 20 15 for the homogeneous distribution of oil in
the solution. Solution was re-homogenized (20,000 rpm/min) 10
and cooled to room temperature. Cheese samples coated with
these films were left to dry at room temperature for 24 hours. For
microbiological examination, 30 ml film solution was moved to
Petri dishes and evaporated at room temperature for 24 hours and
stored at 41C 31.
Preparation and storage of samples: E. coli O157:H7 (ATCC 25922),
L. monocytogenes (ATCC 19118) and S. aureus (ATCC 6538) strains
used for the artificial contamination of kashar cheese samples were
obtained from Hemakim. Yeast-mold enumeration was made
immediately after the cheese production and accepted as the initial
value. Inocula of 106 cfu/g (6 Log cfu/g) were used for the artificial
contamination. Lor cheese samples of 50 g were immersed in
inocula and kept at 4C for 15 min in a sterile cabinet for bacterial
adhesion and absorption of inocula. Artificially contaminated
cheese samples were packaged by immersing in mint essential oil
solutions at different concentrations. Following the immersion

process, the samples were left to dry in a cooled incubator set
to 10C (Nve; ES 500) for 4-5 hours. The prepared samples
were stored at +41C refrigerator conditions for 15 days and E.
coli O157:H7, L. monocytogenes and S. aureus counts were
calculated in terms of Log10 cfu/g on the 1st, 7th, 10th and 15th days
of the storage.
Physico- chemical analysis: The pH values were examined with a
SS-3 Zeromatic pH meter (Beckman Instruments Inc., California,
USA). Titratable acidity (SH) values were analysed according to
AOAC 32. Film thicknesses were measured with a micrometer at
0.001 precision (Digimatic Micrometer/Japan). Water vapour
permeability of films was determined using ASTM E96-80 (1983)
method gravimetrically at 25C.
Microbiological analysis: Enrichments of the bacterial strains
were carried out prior to artificial contamination of the cheese
samples. FDA method for E. coli O157:H7 enumeration 33 and ISO
method 34 for L. monocytogenes enumeration were implemented.
For the enumeration of E. coli O157:H7, Sorbitol MacConkey Agar
(CT-SMAC) containing Cefixime-Tellurite-Supplement (CT) was
used with incubation at 35-37C for 24-48 hours. For the
enumeration of L. monocytogenes, Palcam Listeria Selective Agar
(Base) (Merck 1.11755) was used and inoculated by spreading to
the surface at 37C for 48 hours. For the enumeration S. aureus,
initial enrichment was implemented with Brain Hearth Infusion
Broth (Oxoid CM225) under anaerobic conditions (37C/48 hours).
For the enumeration, 5% egg yolk tellurite emulsion (Oxoid SR 0054)
solution was added to Baird Parker Agar (Oxoid CM 0275
Basingstoke, U.K.) and incubated under aerobic conditions at 3537C for 24-48 hours 35. In yeast and mold enumeration, Yeast-extractglucose chloramphenicol agar (YGC) (Merck 1.16000) was used and
incubated at 25C for 3-5 days.
Statistical analysis: Five different cheese samples were examined
with 3 parallels and 2 repetitions. For this purpose, SPPS version 15
statistical analysis package software was used. Data significance
as a result of analysis of variance (ANOVA) was tested according
to the Duncan multiple comparison test at p < 0.001 level.
The pH and titratable acidity (SH) values of cheese samples,
which were coated with mint essential oil fortified S+WPI based
films and K sample, which was left uncoated, are given in Fig. 1,
fat (%) and weight loss values are given in Tables 1 and 2.
The pH values in all samples decreased during storage. The
greatest decrease in pH value was determined in K sample. With
the effect of increase in mint essential oil concentrations, decrease
in pH value was lower in Me3 on the 10th and 15th days and in Me4
on the 7th, 10th and 15th days of storage.
The relationship between film coating and pH values was
significant in terms of both storage and the samples (p<0.01). The
pH results were similar to those of titratable acidity, the highest
increase was in K sample during storage and the increases in Me3
and Me4 samples were close to each other (Fig. 1).
Fat values increased during storage depending on the
concentration of essential oils (Fig. 2A), and the relationship
between the two parameters was significant (p < 0.01). The effect
41
Fat (%)
Me1
Me2
Me3
6.562.46 bB 6.592.78c B 6.620.32 dB
6.613.99 bB 6.684.57 cB 6.753.78 dB
6.680.29 bB 6.780.24cB 6.922.54 dB
6.701.00 bB 7.000.08 cB 7.070.02 dB
Weight loss (%)
14.920.23B 13.450.18B 12.050.33B
11.90.32B 10.540.22B 7.680.24B
6.090.41B 5.420.15B 3.450.16B
Day/
sample
K
1
6.52.42 aA
7
6.523.97 aA
10
6.542.70 aA
15
6.553.50 aA
1
7
10
15
15.212.27A
12.453.90A
6.741.72A
Me4
6.6911.8 eB
6.9124.5 eB
7.1116.8 eB
7.2415.7 eB
Fat (%)
Table 1. The average weight loss (%), fat (%) and SH values and
standard deviations in mint essential oil film coated samples
and K sample (p < 0.01), (n = 3).
11.140.29B
7.040.12B
3.250.14B
Me1
Me2
Me3
Me4
Thickness
(mm)
0.179 0.013
0.180 0.010
0.181 0.012
0.182 0.014
Weight loss (%)
Sample
Water vapour permeability

(g mm m-2 h-1 kPa-1)
8.64 A
8.62 B
8.60 C
8.57 D
pH
Me1
Me2
Me2
Me3
Me3
10
Acidity (SH)
K
K
Me1
Me1
Me2
Me2
15
Me3
Me3
7
10
Storage time (days)
Me4
Me4
15
Figure 1. The average pH and SH values in mint essential

oil film coated samples and K sample.
of coating with sorbitol-amended film on fat values was significant

(p < 0.01). The high fat content of samples coated with mint essential
oils in different concentrations are associated with better
dissolution of fat in lipid fraction of the cheese due to its
hydrophobic properties 36 and good barrier properties of whey
based films against fat 37.
Weight losses in film coated cheese samples during storage
42
Me3
Me3
Me4
Me4
7
10
Storage time (days)
Me1
Me1
Me2
Me2
Me3
Me3
15
Me4
Me4
7
10
Storage time (days)
15
Me4
Storage time (days)

159
154
149
144
139
134
129
124
119
114
109
Me2
Me2
Figure 2. The average fat and weight loss values in mint

essential oil fortified film coated samples and K sample.
5.85
5.8
5.75
5.7
5.65
5.6
5.55
5.5
5.45
5.4
5.35
5.3
1
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Me1
Me1
A, B, C, D, E: The differences between the values in the same column are statistically
significant (p < 0.01).
KK
K
K
K
K
a, b, c, d, e: The differences between the values in the same column are statistically significant (p < 0.01).
A, B, C, D: The differences between the values in the same line are statistically significant (p < 0.01).
Table 2. The average film thickness and vapour

permeability values of films containing mint
essential oils in different concentrations
and standard deviations (n = 3).
7.3
7.2
7.1
7
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
6.1
were lower than K sample. The effect of film coating on weight

loss was significant (p < 0.01), and S+WPI based film is a good
water barrier. In all concentrations of essential oil and K sample,
the weight loss was highest on the 7th day of the storage. The
weight losses in K and Me1 were close to each other on the 7th
day (Fig. 2B) and the relationship between increase in the
concentration and the decrease in weight loss was significant
(p < 0.01). In many studies, film coating prevented the passage
of water vapour and thus decreased the economic losses 8, 38, also
using whey based films together with lipids is effective on
preventing the weight losses in products due to their high moisture
permeability 37. The results regarding S+WPI based film in this
study were consistent with those studies.
Film thickness values of mint essential oil fortified films in
different concentrations were 0.179 mm for Me1, 0.180 mm for
Me2, 0.181 mm for Me3 and 0.182 mm for Me4. The differences
between the film thicknesses were not significant (p > 0.001). The
lowest water permeability value was found in Me4, this highest
water permeability value was in Me1, the difference was significant
(p < 0.01). Film thicknesses increased as the essential oil
concentrations increased and vapour permeability and
correspondingly weight losses decreased.
Cheese samples were contaminated with pathogenic microorganisms at 106 (6 Log cfu/g) levels, yeast-mold counts were 6.21
log10 cfu/g. Antimicrobial effects increased related to the increase
in essential oil concentrations, and mint essential oil had
bacteriostatic and bactericidal effects on the chosen
microorganisms. The results obtained in our study showed
similarities with many studies 16-19. The relationship between the
increase in concentration and antimicrobial effect was significant
among the samples in terms of storage (p < 0.01). Moreover,
antimicrobial effect in all concentrations increased with the
duration of storage (Table 3).

This result was explained with the slow passage of antimicrobial
substances from film layer to food in edible film systems and the
longer lasting effect of high concentrations of antimicrobial
substance left in film and on food surface against microorganisms 39, 40. Additionally, it was associated with the increase
in hydrophobic properties of essential oil related to the decrease
in pH, thus the increase in antimicrobial activity due to higher
dissolution in lipid phase of cell membrane of food and the
bacteria 36.
Bacteriostatic effects were determined in all concentrations of
essential oils on microorganisms. Bactericidal effect was

determined in Me2 for S. aureus, Me3 for L. monocytogenes and
yeast-mold and Me4 for E. coli O157:H7. The highest
microorganism growth was determined in K sample, while the
bactericidal effect of mint essential oil was effective on all
microorganisms in Me4. Accordingly, the highest increases of the
average storage days in K sample were yeast-mold (7.55 Log10
cfu/g with 1.34 Log10 cfu/g increase), S. aureus (7.65 Log10 cfu/g
with 0.92 Log10 cfu/g increase), E. coli O157:H7 (7.71 Log10 with
0.81 Log10 cfu/g increase) and L. monocytogenes (7.56 Log10 cfu/
g with 0.78 Log10 cfu/g increase), respectively. The highest
antimicrobial activities of the average storage days
Table 3. The average microorganism counts of in mint essential oil fortified
in Me3 sample were S. aureus (0.83 Log10 cfu/g with
film coated samples and K sample (n = 3); (p < 0.01).
5.9 Log10 cfu/g decrease), yeast-mold (1.41 Log10 cfu/
g with 4.8 Log10 cfu/g decrease), L. monocytogenes
Mint essential oil concentration (%)
(2.25 Log10 cfu/g with 4.53 Log10 cfu/g decrease) and
K
Me1
Me2
Me3
Me4
Microorganisms Day
Microorganism counts (Log10 cfu/g)
E. coli O157:H7 (2.89 Log10 cfu/g with 4.01 Log10
1 7.120.11aA 7.090.05aB 6.140.25 aC 3.850.35 aD
cfu/g decrease), respectively. In general, E. coli
7 7.880.23bA 7.631.12bB 5.910.88 bC 3.210.36bD
O157:H7 was the most resistant microorganism to
E. coli O157:H7
cA
cB
cC
cD
7.672.47
5.680.54
2.520.12
10 7.910.09
all concentrations of essential oil, and the most
dA
dB
dC
dD
15 7.930.05
7.711.54
5.280.23
2.010.09
sensitive one was S. aureus. The bacteriostatic effect
1 7.242.36 aA 5.932.87 aB 5.870.99 aC 3.350.01 aD
of essential oil on E. coli O157:H7 was started to
7 7.421.12bA 5.241.47bB 3.920.14 bC
S. aureus
10 7.761.08 cA 5.482.10bB 3.020.03 cC
observe in Me2 and Me3 (in higher concentrations),
15 7.92.05 dA 3.811.15dB
however, no bactericidal effect was observed in these
1 7.090.24 aA 7.010.53 aB 5.860.33 aC 3.800.02 aD
concentrations. Bactericidal effect for E. coli
bA
bB
bC
bD
7 7.030.33
7.050.64
5.470.42
3.040.01
L. monocytogenes
O157:H7 was determined from the 1st day of the
cA
cB
cC
cD
10 7.530.09
7.230.64
4.210.09
2.170.89
dA
dB
dC
storage in Me4 concentration (Fig. 3A).
5.320.10
3.870.08
15 7.710.07
The bacteriostatic effect of essential oil on S.
1 6.881.13 aA 6.740.04 aB 3.541.28 aC 3.120.91 aD
7 7.580.95bA 6.81.49 bB 3.092.87bC 2.520.12bD
aureus
was observed in Me1 from the first days of
Yeast - Mold
10
15
7.760.33 cA 5.210.99 cB 2.851.54 cC

7.870.33dA 5.010.87dB 2.411.54 dC
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
E. colli O157:H7
A
Log10 (cfu/g)
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
7
10
Storage time (days)
15
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
Staphylococcus aureus
B
Log10 (cfu/g)
Log10 (cfu/g)
Log10 (cfu/g)
a, b, c, d: The differences between the values in the same column are statistically significant (p < 0.01).
A, B, C, D: The differences between the values in the same line are statistically significant (p < 0.01).
7
10
Storage time (days)
15
K
K
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
Me1
Me1
Listeria monocytogenes
10
77
10
Storage time (days)
D
Yeast - Mold
11
Me2
Me2
15
15
10
77
10
Storage time (days)
Me3
Me3
15
15
Me4
Me4
Figure 3. E. coli O157:H7 (A), S. aureus (B), L. monocytogenes (C) and yeast-mold (D) growth in samples coated with
mint essential oil added film and C sample.
43
the storage, related to the increase in concentration, and

bactericidal effect was acquired on the 15th day in Me2 and on
the 7th day in Me3 (Fig. 3B). The bacteriostatic effect of essential
oil on L. monocytogenes was observed on the 10th day in Me1 and
on the 7th day in Me2. The bacteriostatic effect was observed from
the 1st day in Me3 and the bactericidal effect was determined on
the 15th day. The bactericidal effect was observed from the 1st day
of the storage in Me4 (Fig. 3C). All concentrations of essential
oils showed a bacteriostatic effect on yeast-molds. The
bacteriostatic effect was observed on the 7th day in Me1 and on
the 1st day in Me2 and Me3 (in higher concentrations), the
bactericidal effect was determined on the 10th day in Me3 and on
the 1st day in Me4 (Fig. 3D).
The bacteriostatic effect of essential oil on microorganisms was
determined in Me2 and the bactericidal effect was determined in
Me4 from the 1st day of the storage. The results obtained in this
study for E. coli O157:H7 and S. aureus are consistent with those
of many other studies on antimicrobial activity of various essential
oils 6, 15, 41-43.
Conclusions
In our study, fortification of S+WPI with mint essential oil had a
significant effect on extension of the shelf life of lor cheese, and
antimicrobial effect was obtained especially in Me2 and Me3
concentrations. Bactericidal effect was observed on all
microorganisms in Me4 concentration. Additionally, the fat values
increased with the increase in essential oil concentrations and
S+WPI based film proved to be a good water, oxygen and fat
barrier. It was concluded that coating with film decreased weight
losses of lor cheese and created a positive effect in terms of quality.
Acknowledgements
The authors wish to thank O.Turer for expert assistance during
essential oil extraction and Boyacoglu Dairy Factory Co., Turkey,
for assistance during production of lor cheese.
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31
45
WFL Publisher
Helsinki, Finland
www.world-food.net
Improving the layout of ventilation ports in packaging for fresh produce using
computational fluid dynamics
Hiroaki Kitazawa * and Naoko Hasegawa
Food Engineering Division, National Food Research Institute, NARO, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
e-mail: ktz@affrc.go.jp, naohase@affrc.go.jp
Abstract
Packaging for fresh produce must sometimes include features that ensure uniform and rapid airflow to enable cooling and gas exchange. For this reason,
such packaging usually has ventilation ports. However, attempting to understand cooling and ventilation efficiency by actually manufacturing various
types of packaging in order to develop packaging with maximum ventilation efficiency would require substantial time and effort. Therefore, the size
and layout of the vent ports in the packaging of fresh produce is presumably not determined based on any scientific rationale. Computational fluid
dynamics (CFD) is a method to analyse the behaviour of fluids in space. By setting appropriate parameter values, no great difference was found
between the simulation value and the measured value for temperature change when air passes through the packaging. Consequently, in this study, to
optimise the layout of ventilation ports in packaging made of corrugated fibreboard for fresh produce, we used CFD simulation to propose an
improved vent layout for uniform internal ventilation of one-layer packaging for strawberries. Simulations were conducted in three steps: the first
step involved estimating airflow of packaging with various port layouts; the second step was proposing improved port layouts for the packaging; and
the last step was the estimation of the packagings ventilation ability based on improved port layouts. Our results suggested that, with the improved
port layout resulting from CFD simulation, it is possible to induce large airflow in the lower level of one-layer packaging for strawberries. However,
further study to investigate the shape and number of ventilation ports is needed to achieve uniform ventilation in the upper layer of packaging.
Therefore, in future studies, differences in the number and shape of ports that affect airflow in this space will be considered.
Key words: Computational fluid dynamics (CFD), fresh produce, packaging, ventilation port.
Introduction
Packaging for fruits and vegetables must sometimes include
features that ensure uniform and rapid airflow to enable cooling
and gas exchange 1. For this reason, such packaging usually has
ventilation ports. However, attempting to understand cooling and
ventilation efficiency by actually manufacturing various types of
packaging in order to develop packaging with maximum ventilation
efficiency would require substantial time and effort. Moreover, it
would be difficult to measure the flow of air within each part of a
closed and narrow space. Therefore, the size and layout of the
vent ports in the packaging for fruits or vegetables are presumably
not determined based on any rigorous or tested rationale.
Computational fluid dynamics (CFD) is a method used to analyse
the behaviour of fluids in space. CFD is employed for analysis in
a wide range of fields, including meteorology 2, automotive
engineering 3, and agriculture 4. Therefore, CFD simulation may
be a useful technique for improving ventilation efficiency in fruit
and vegetable packaging. In fact, CFD has been used in several
reports analysing changes in airflow and/or temperature in
packaging containers for certain fruits, such as apples 5, 6 and
strawberries 7-11.
In a previous study 12, the authors used CFD simulation to
study the effect of inlet velocity and port size on airflow in onelayer packaging for strawberries 13; this type of packaging had a
single port in the centre of the boxs long side, and this study
46
revealed factors hindering ventilation efficiency. Furthermore, by

setting appropriate parameter values, no great difference was found
between the simulation value and the measured value for
temperature change when air passed through the packaging 1.
In the present study, we used CFD simulation to obtain port
layouts that enables uniform airflow inside one-layer packaging
for strawberries.
Sequence of analysis: The analysis sequence for the present study
is shown in Fig. 1. The scope of this study was to use CFD analysis
to identify issues related to airflow inside packaging with the
normal port layout and, subsequently, to propose an improved
layout. Therefore, evaluation of compression stress and ventilation
efficiency of actual packaging was not performed in this study.
Simulation conditions for CFD: The model for this analysis is a
corrugated fibreboard box for one-layer packaging of strawberries;
the internal dimensions of this box are 210 290 45.25 mm3, as
shown in Fig. 2. In this type of packaging, two trays filled with
strawberries are placed inside the box, and the edges of these
trays touch the boxs inner walls; therefore, the plurality of the
interior space is divided horizontally. A three-dimensional shape
resembling such a space was created using a fluid analysis
In this equation, U is the inlet velocity [m s-1] and L is the

representative length [m]. From this equation, if the port diameter
is set to a typical length of 19 mm, Re for the inlet portion in this
analysis will be approximately 260. Consequently, this analysis
assumes a simple laminar flow state, and the numerical
computations were conducted accordingly. Normal calculation
was performed in the simulation analysis, and when the residual
numerical calculations for the inlet velocity became less than 10-3,
the numerical calculation was judged to have converged. A
personal computer (Vostro 260S, Dell Japan CPU: Intel CoreTM
i5-2400 3.10 GHz, RAM: 4.00 GB) was used for the calculation.
Figure 1. Simulation sequence in this study.
Estimation of ventilation ability:

Estimation with various port layouts (Simulation 1): The diameter
of the port was set to 19 mm (which is close to the limiting size),
regardless of whether the port was placed in the upper or lower
layer of the box. There were two ports made on each of the boxs
long sides; a pair of ports was the inlet, and the other was the
outlet. Furthermore, considering the various situations in which
packaging might actually be stored, the boxs centre of gravity
was taken to be the point of symmetry, and ports were then made
at distances of 0, 24, 48, 72, 96, and 120 mm from the centre of the
long side such that the ports were symmetrical with respect to
both the inlet and the outlet sides. Based on these different layouts,
the twenty patterns of port layout that were analysed in this study
are shown in Fig. 3.
Figure 2. Type of packaging for CFD analysis.
preprocessor (GAMBIT 2.4.6, ANSYS, Inc., PA, USA). The number

of meshes in each shape was approximately 130,000. The ratio of
the mesh to the volume of the internal space was equivalent to the
ratio in a previous study 12, and furthermore, it has also been
verified that this ratio matches the actual measurements 1. The
number of ports and their diameter and layout will be described
later. The airflow in the box when air was allowed to flow from one
side was reproduced using CFD analysis software (ANSYS Fluent
14.5, ANSYS Inc., PA, USA). In their CFD analysis of forced
aeration in apple packaging, Zou et al. 5 calculated the inlet velocity
to be 0.9 m s-1. In a previous study 12, the analysed inlet velocity
ranged between 0.3 and 1.5 m s-1. In the present study, to examine
the ventilation capability when the airflow was very small, the
inlet velocity was set to 0.2 m s-1. The physical properties of air
at 15C were used (viscosity = 1.7894 10-5 Pa s and density
= 1.225 kg m-3).
The Reynolds number Re for the flow field in the box is expressed
with the following equation, where Re 2000 is considered to be
turbulent:
Re = U L ( -1) -1
96 48 0 48 96
96 48 0 48 96
120 72 24 24 72 120
120 72 24 24 72 120
Distance from the centre of the
long side [mm]
long side [mm]
shows inlet ports on the front side.
shows outlet ports on the other side.
shows that the position of inlet ports overlaps with outlet ports.
Figure 3. Basic port layouts for CFD analysis.
This section seeks to determine the port layout in which the

airflow in the box becomes most uniform. First, the effect of
differences in port layout on the average velocity distribution in
lines 1-8 (shown in Fig. 4) was studied. Next, the average and
standard deviation in lines 1-5 in the upper layer and in lines 6-8 in
the lower layer of the box were calculated. Then, the port layout in
which the variation in the average velocity was small and the
average velocity was large in both the upper and the lower layers
was selected. In a previous study 12, researchers concluded that it
was possible to adjust the magnitude of the velocity to a certain
extent by changing the size of the ports. However, it was also
47
Estimation of ventilation ability with improved port layouts

(Simulation 3): Minimising port size is essential for maintaining
the strength of the box. In each of the port layouts discussed in
Simulation 1, the air entering the ports is divided in two directions
by the vertical tray placed inside the box. Consequently, to ensure
that the cross-sectional area of the ports located in both the upper
and lower portions were half the size of the port with the 19-mm
diameter, the ports were replaced with smaller ports with a 13.44mm diameter. These ports were then enlarged by factors of 1.05,
1.10, 1.20, 1.30, and 1.40 (which resulted in diameters of 14.10,
14.78, 16.12, 17.46, and 18.80 mm, respectively), and upon placing
the enlarged ports at the positions proposed in Simulation 2, the
airflow in the interior was simulated. The analyses relating to
differences in port diameter were conducted separately for the
lower and upper layers. The analyses of other conditions were
performed according to Simulation 1.
Figure 4. Position for calculating air velocity distribution.
suggested that controlling the airflow distribution by changing

either the size of the ports or the inlet velocity of air through the
ports was very difficult. For this reason, it was decided that, in the
port layout, smaller variation in the average flow velocity would
be prioritised over the maximum velocity, and this reasoning was
followed in subsequent analyses.
Proposing improved port layouts (Simulation 2): From the
results of the analysis in Simulation 1, several port layouts in
which airflow in the upper and lower layers was large and mostly
uniform were selected, and based on these layouts, the airflow in
the interior of the box was simulated. Using the results of
Simulation 1, ports were made in the upper and lower layers.
Therefore, the four ports were made on the inlet and outlet ends
of the long sides of the box. The port diameter and the analysis
method from Simulation 1 were also applied to this simulation.

Estimation with various port layouts (Simulation 1): The airflow
distributions in the interior space for several port layouts are
shown in Table 1. Whenever a port was located in the centre of
the long side of the box (Cases 1-5), the variation in the velocity
for both the lower and upper layers was large [hereafter, this
variation is referred to as the standard deviation (SD) value], and
in these port layouts, while some places exhibited strong airflow,
there would presumably be places where only limited airflow
occurred (Fig. 5). Based on Cases 6-10 (in which a port was
located 24 mm from the centre of the long side), despite the trend
toward a greater SD for the average airflow in the upper layer in
Case 8 and in the lower layer in Case 10, the SD in the average
velocity was smaller in both the lower and upper layers in other
cases. In Cases 11-14, in which a port was located 48 mm from the
centre of the long side, both the average velocity and its SD in the
lower layer appeared largely unaffected by the port layout.
However, a trend toward a larger average velocity was observed
in Case 13 in the upper layer, and a trend toward a small SD was
seen in Case 14. In Cases 15-17, in which a port is located 72 mm
from the centre of the long side, despite the large average velocity
in the upper layer, a tendency toward a large SD in velocity was
Table 1. Airflow distribution in packaging with various port layouts.

Analysis
pattern
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Case 11
Case 12
Case 13
Case 14
Case 15
Case 16
Case 17
Case 18
Case 19
Case 20
Line 1
0.020
0.015
0.011
0.009
0.006
0.008
0.003
0.004
0.005
0.006
0.008
0.006
0.007
0.007
0.011
0.009
0.010
0.015
0.010
0.039
Upper layer (local velocity [m/s])

Line 2 Line 3 Line 4 Line 5 Average
0.020
0.079
0.012
0.010
0.028
0.034
0.077
0.016
0.004
0.029
0.085
0.079
0.021
0.004
0.040
0.016
0.085
0.010
0.004
0.025
0.012
0.099
0.008
0.003
0.026
0.013
0.013
0.021
0.022
0.015
0.016
0.014
0.031
0.015
0.016
0.018
0.013
0.098
0.010
0.029
0.009
0.021
0.014
0.008
0.011
0.011
0.032
0.011
0.006
0.013
0.032
0.014
0.020
0.020
0.019
0.035
0.015
0.032
0.014
0.020
0.034
0.016
0.109
0.010
0.035
0.014
0.016
0.01
0.007
0.012
0.106
0.011
0.026
0.022
0.035
0.109
0.011
0.034
0.014
0.035
0.110
0.013
0.112
0.010
0.051
0.011
0.008
0.011
0.028
0.015
0.016
0.008
0.015
0.011
0.012
0.010
0.006
0.010
0.039
0.021
Z: Standard deviation of average value.
48
SDZ
0.026
0.026
0.035
0.030
0.037
0.005
0.009
0.035
0.005
0.010
0.008
0.011
0.038
0.004
0.036
0.038
0.049
0.007
0.003
0.015
Lower layer (local velocity [m/s])

Line 6 Line 7 Line 8 Average
SD
0.043
0.108
0.024
0.058
0.036
0.025
0.114
0.022
0.054
0.042
0.024
0.119
0.019
0.054
0.046
0.020
0.116
0.016
0.050
0.046
0.011
0.115
0.011
0.046
0.049
0.033
0.043
0.044
0.040
0.005
0.029
0.051
0.027
0.036
0.011
0.025
0.052
0.026
0.034
0.012
0.022
0.054
0.022
0.032
0.015
0.014
0.069
0.014
0.033
0.026
0.039
0.038
0.046
0.041
0.004
0.034
0.047
0.030
0.037
0.007
0.031
0.049
0.030
0.036
0.009
0.027
0.052
0.028
0.036
0.011
0.041
0.036
0.045
0.041
0.004
0.036
0.04
0.034
0.038
0.005
0.032
0.047
0.033
0.037
0.007
0.042
0.033
0.050
0.042
0.007
0.035
0.040
0.036
0.037
0.002
0.053
0.025
0.052
0.043
0.013
0.30
0.27
0.24
0.21
0.18
0.15
(Z = 34.6 mm)
96 48 0 48 96
120 72 24 24 72 120
long side [mm]
0.12
0.09
0.06
96 48 0 48 96
120 72 24 24 72 120
long side [mm]

0.03
Figure 6. Improved port layouts for CFD analysis.
0.00
Velocity [m/s]
(Z = 10.5 mm)
Figure 5. Simulated airflow in Case 3.

Arrows show the positions of inlet ports.
also observed. Furthermore, a similar trend in both the velocity

and its SD in the lower layer were observed in Cases 11-14. For
Cases 18-19, in which a port was located 96 mm from the centre of
the long side, both the average velocity in the upper layer and the
SD for the average velocity in the lower layer were observed to be
small; this trend was similar to the tendency observed in Cases
11-17. In Case 20, in which all ports were located 120 mm from the
centre of the long side, the observed trend was that the SD for the
average velocity was large in the upper layer.
In summary, these results reveal that, when a port is in a position
24-120 mm from the centre of the long side of the box and is
positioned such that the inlet port and the outlet port do not
overlap, the SD in the velocity was generally reduced (even though
the SD for the average velocity in the upper layer may be assumed
to be greater in some portions of the layout), and the velocity in
the interior space may be considered to approach uniformity.
Based on this fact, ten patterns of port layout (shown in Fig. 6)
were selected to simulate the effect of port layout on the velocity
distribution in the interior space in the next section.
Proposing improved port layouts (Simulation 2): Among Cases
21-30 that were analysed, in Cases 22, 25, 28, and 29, a tendency
toward greater average velocity in the upper layer was observed
(Table 2). However, the SD for the average velocity was also large,
and it was therefore expected that, in areas of large velocity, a bias
may occur in favour of the accompanying port layouts. The next
instances of large average velocity in the upper layer occurred in

Cases 26 and 27. The SD in the upper layer of Case 27 was the
smallest among the cases analysed in this section.
For the lower space, the largest average velocity and the smallest
SD occurred in Cases 27 and 29. Because the SD for the average
velocity in the upper layer was large in Case 29, Case 27 was
deemed to present the best port layout in this section. Therefore,
in the following section, ten patterns with changing port diameters
(shown in Fig. 7) were simulated based on the port layout of Case 27.
96 48 0 48 96
120 72 24 24 72 120
long side [mm]
96 48 0 48 96
120 72 24 24 72 120
long side [mm]

Figure 7. Various port diameters with improved port layouts

for airflow simulation.
Estimation of ventilation ability with improved port layouts

(Simulation 3): In Cases 31-35, in which the effect of differences
in the lower layers port diameter on average velocity in this layer
was analysed, average velocity increased with an increase in port
diameter. This finding is clear from comparing the 1.4-fold increase
in port diameter with the 1.05-fold increase, which results in a
1.78-fold increase in average velocity, as shown in Fig. 8 and
Table 3.
Table 2. Airflow distribution in packaging with improved port layouts.

Analysis
pattern
Case 21
Case 22
Case 23
Case 24
Case 25
Case 26
Case 27
Case 28
Case 29
Case 30
Line 1
0.003
0.004
0.004
0.005
0.006
0.004
0.006
0.010
0.024
0.026

Line 2 Line 3 Line 4 Line 5 Average
0.006
0.015
0.008
0.005
0.008
0.019
0.010
0.112
0.007
0.031
0.016
0.010
0.025
0.012
0.013
0.010
0.011
0.012
0.026
0.013
0.030
0.011
0.118
0.007
0.034
0.032
0.011
0.028
0.011
0.017
0.026
0.012
0.017
0.027
0.018
0.030
0.008
0.119
0.006
0.035
0.021
0.009
0.124
0.009
0.037
0.007
0.006
0.008
0.013
0.012
SDZ
0.004
0.041
0.007
0.007
0.043
0.011
0.008
0.043
0.044
0.008

Line 6 Line 7 Line 8 Average
SD
0.025
0.057
0.02
0.036
0.015
0.028
0.056
0.028
0.037
0.013
0.030
0.054
0.030
0.038
0.011
0.043
0.047
0.033
0.041
0.006
0.032
0.049
0.032
0.038
0.008
0.033
0.047
0.033
0.038
0.006
0.046
0.042
0.037
0.042
0.004
0.036
0.046
0.036
0.039
0.005
0.039
0.039
0.047
0.042
0.004
0.040
0.037
0.047
0.041
0.004
Z: refer to Table 1.
49
Table 3. Effect of differences in port diameter on the airflow distribution in packaging with improved port layouts.
Port diameter (mm)
Upper
Lower
layer
layer
13.44
14.10
13.44
14.78
13.44
16.12
13.44
17.46
13.44
18.80
14.10
13.44
14.78
13.44
16.12
13.44
17.46
13.44
18.80
13.44
Analysis
pattern
Case 31
Case 32
Case 33
Case 34
Case 35
Case 36
Case 37
Case 38
Case 39
Case 40
Z: refer to Table 1. y: not simulated.
Lower layer
Line 1
Line 2
Line 3
Line 4
Line 5
Average
SDZ
Line 6
Line 7
Line 8
Average
SD
0.032
0.035
0.041
0.048
0.053
0.012
0.012
0.012
0.012
0.012
0.019
0.020
0.022
0.025
0.028
0.027
0.027
0.027
0.025
0.024
0.019
0.020
0.022
0.024
0.026
0.009
0.010
0.012
0.013
0.015
0.052
0.057
0.067
0.079
0.091
-
0.048
0.052
0.062
0.073
0.084
-
0.041
0.045
0.054
0.064
0.075
-
0.047
0.051
0.061
0.072
0.083
-
0.005
0.005
0.005
0.006
0.007
-
0.007
0.008
0.009
0.010
0.011
layers) without limiting the strength of packaging, it will be

necessary to conduct strength tests on actual packaging.
0.30
0.27
0.24
0.21
0.18
0.15
0.12
0.09
0.06
0.03
0.00
Velocity [m/s]
Acknowledgements
The simulations using GAMBIT 2.4.6 and ANSYS Fluent 14.5
were performed with the assistance of the Agriculture, Forestry
and Fisheries Research Information Technology Center for
Agriculture, Forestry and Fisheries Research, MAFF, Japan.
References
Kitazawa, H. 2012. Optimisation of packaging for fresh produce by
applying computational fluid dynamics (CFD). Food Packag. 53(5):1923 (in Japanese).
2
Flaherty, J. E., Stock, D. and Lamb, B. 2007. Computational fluid
dynamic simulations of plume dispersion in urban Oklahoma City. J.
Appl. Meteor. Climatol. 46:2110-2126.
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Fu, L., Ishima, T., Long, W. Q. and Tian, J. P. 2009. Research on the
ignition-chamber GDI engine combustion system. J. Therm. Sci. Tech.
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4
Kacira, K., Short., T. H. and Stowell, R. R. 1998. A CFD evaluation of
naturally ventilated multi-span, sawtooth greenhouses. Trans. ASAE
41:833-836.
5
Zou, Q., Opara, L. U. and McKibbin, R. 2006. A CFD modeling system
for airflow and heat transfer in ventilated packaging for fresh foods: II.
Computational solution, software development, and model testing. J.
Food Eng. 77:1048-1058.
6
Opara, L. and Zou, Q. 2007. Sensitivity analysis of a CFD modeling
system for airflow and heat transfer of fresh food packaging: Inlet air
flow velocity and inside-package configurations. Int. J. Food Eng.
3(5):article No. 16.
7
Ferrua, M. J. and Singh, R. P. 2009a. Modeling the forced-air cooling
process of fresh strawberry packages. Part I: Numerical model. Int. J.
Refrig. 32:335-348.
8
Ferrua, M. J. and Singh, R. P. 2009b. Modeling the forced-air cooling
process of fresh strawberry packages. Part II: Experimental validation
of the flow model. Int. J. Refrig. 32:349-358.
9
Ferrua, M. J. and Singh, R. P. 2009c. Modeling the forced-air cooling
process of fresh strawberry packages. Part III: Experimental validation
of the energy model. Int. J. Refrig. 32:359-368.
10
Ferrua, M. J. and Singh, R. P. 2009d. Design guidelines for the forcedair cooling process of strawberries. Int. J. Refrig. 32:1932-1943.
11
Ferrua, M. J. and Singh, R. P. 2011. Improved airflow method and
packaging system for forced-air cooling of strawberries. Int. J. Refrig.
34:1162-1173.
12
Kitazawa, H., Funaki, T., Nakao, M., Ohshiro, Y., Hiruta, M. and
Ishikawa, Y. 2012. Air flow visualization for fresh produce packaging
by CFD analysis. Food Sci. Tech. Res. 18:525-534.
13
Kitazawa, H., Ishikawa, Y., Lu, F., Hu, Y., Nakamura, N. and Shiina, T.
2010. Alleviation of strawberry bruising due to vibration using 1-layer
packaging with cushioning. J. Packag. Sci. Tech. 19:33-42.
1
Y
X
(Z = 10.5 mm)
Figure 8. Effect of port diameter (d [mm]) on airflow in Cases 31-35.

Arrows show the direction of airflow.
In comparison with Case 1 (analysed in Simulation 1, in which

the average velocity in this space is greatest), when the port
diameter was increased 1.4 times, the average velocity was
predicted to increase 1.43 times. Furthermore, with the increase in
port size, there is, conversely, a tendency for the SD of the average
velocity in this space to decrease. In Cases 36-40, in which the
upper layer was analysed, although the average velocity increased
as the port diameter increased, the maximum increase was only 1.35
times. Therefore, compared with the lower layer, the increase in
average velocity in the upper layer was predicted to be small,
even when the port diameter increased. Furthermore, when
compared with Cases 1-20 in Simulation 1 (Table 1), the average
velocity in most cases was found to be less than the corresponding
values in that space. Furthermore, it was assumed that, in this
space, the SD for the average velocity would increase with
increasing port size. Consequently, in this space, either an increase
or uniformity in average velocity could not be expected to result
from increasing the port size.
Conclusions
According to the aforementioned simulation, it is possible to
create large uniform airflow in the lower layer of produce packaging
by increasing the port diameter in the port layout of Case 27,
which was proposed in Simulation 2. For the upper layer,
increasing the average velocity or eliminating the distribution may
not be possible with this port layout. In future studies, differences
in both the number and the shape of ports that affect the airflow in
this space will be considered. To examine how much the number
and size of ports can be increased (in both the upper and lower
50
WFL Publisher
Helsinki, Finland
www.world-food.net
Peanut protein isolates improve the nutritional quality of muffins that can be handy
tool to cure protein energy malnutrition in developing economies
Muhammad Sibt-e-Abbas 1*, Masood Sadiq Butt 1, Muhammad Tauseef Sultan 2, Atif Nisar Ahmad 2,
Muhammad Abrar 1 and Mir Muhammad Nasir Qayyum 3
1
2
National Institute of Food Science & Technology, University of Agriculture, Faisalabad 38000, Pakistan.
Bahauddin Zakariya University, Multan 60000, Pakistan. 3 Karakram University, Gilgit 15100, Pakistan.
*e-mail: abbas_fst14@yahoo.com
Abstract
The developing economies are facing the menace of malnutrition particularly due to inadequate intake of quality proteins. The people using wheat
and rice as staples need to increase the intake of quality proteins, e.g. nuts and animal proteins. In the present research, proteins extracted from
partially defatted peanut flour (DPF) of indigenous varieties, i.e. GOLDEN and BARI 2011, were supplemented with straight grade wheat flour in
various proportions. These flours were subjected to rheological characteristics studies. After rheology muffins were prepared using these flour
blends. Muffins were then tested for physical characteristics including colour, texture and volume. At the end, the sensory evaluation was performed
by trained panellists. Results regarding the rheological properties, i.e. farinograph and mixograph, revealed that peanut protein isolates positively
affected the rheology of dough. Best results for water absorption (%) were shown by T4. Similar results were noted for dough development time (min)
and dough stability time (min). The physical characteristics of muffins indicated an increase in quality and nutritional status by the addition of protein
isolates. T3 showed notable result for colour. Furthermore, sensory evaluation of muffins showed remarkable results on 15% supplementation of
wheat flour with peanut protein isolates. Conclusively, the protein isolates obtained from defatted peanut flour or meal left after oil extraction can be
effectively utilized for the supplementation of bakery products, i.e. muffins. As these bakery products are gaining much popularity in the developing
economies, hence these can play an imperative role to curtail the increasing risks of malnutrition.
Key words: Peanut, protein isolates, composite flour, muffins.
Introduction
Malnutrition is a major nutritional dilemma in the developing
countries. It persists due to insufficient intake of nutrients resulting
in adverse effects on body building and function. Malnourished
people either do not have enough calories in their diet or are
eating a diet that lacks protein, vitamins or trace minerals 1. Protein
malnutrition is one of such example that causes severe effects on
immune functions, growth and development of children, their
learning ability and work efficiency. Approximately 70% of the
worlds malnourished children live in Asia, resulting in the region
having the highest concentration of childhood protein energy
malnutrition 2, 3.
The role of proteins in human nutrition is substantial. According
to Modern Nutrition Recommendations, human beings should
rely mostly on vegetable and legume proteins to meet the protein
requirement in their diet. In addition to their nutritional value,
proteins provide great potential as functional food ingredients
enhancing the useful properties when incorporated into food
commodities. In order to utilize a byproduct as a protein source, it
should contain high protein content and protein value (quality)
based on well-balanced essential amino acids.
Peanuts (Arachis hypogaea) are among the most vital sources
of vegetable oil throughout the world. Peanuts also contain
appreciable quantity of valuable proteins. Peanut protein isolates
generally contain 47-55% high quality protein with high essential
amino acid content, which lends itself being used in many food
applications 4-6. These protein isolates can be utilized as functional
ingredients in various food products to improve the nutritional
and textural properties of the product 7.
The supplementation of various food products with peanut
protein isolates can play a vital role in the reduction of protein
deficiency 8. As the demand of bakery products is increasing at
the rate of 10.07% per annum so these are considered as excellent
vehicle for fortification, value addition and feeding at mass scale.
Among the bakery products, muffins (cupcake) prove to be an
excellent tool for the supplementation of peanut protein isolates
as their consumption in world is more than 46% of all other savoury
foods 9. The present research project explicated the role of peanut
protein isolates as a tool against protein deficiency and potential
source for supplementation of baked products.
Procurement of raw materials: Two varieties of peanut (GOLDEN
and BARI 2011) were procured from Barani Agriculture Research
Institute Chakwal. Chemicals and other consumables were
purchased from local market. The protein isolates were prepared
in the post graduate laboratories of National Institute of Food
Science & Technology, University of Agriculture, Faisalabad,
Pakistan.
51
Preparation of peanut protein isolates supplemented blends:

Peanut protein isolates of the two varieties (GOLDEN and BARI
2011) were supplemented with straight grade flour in various
proportions as given in Table 1.
Table 1. Composition of different composite flours.
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
Straight
grade flour (%)
100
95
90
85
80
95
90
85
80
Peanut protein
isolates (%)
(GOLDEN)
0
5
10
15
20
---------
Peanut protein
isolates (%)
(BARI 2011)
----------5
10
15
20
Sensory evaluation of protein enriched muffins: The muffins

were evaluated for taste, colour, flavour, texture, aroma, and overall
quality on a sensory evaluation Performa. All evaluations were
conducted at room temperature on the same day, in the National
Institute of Food Science and Technology (NIFSAT), University
of Agriculture, Faisalabad according to the procedure described
by Meilgaard et al. 15.
Statistical analysis: The data obtained for each parameter was
subjected to statistical analysis in order to determine the level of
significance as described by Steel et al. 16.
The present study was designed to explore the nutritional value
of peanut protein isolates with special reference to supplementation
in bakery products, i.e. muffins.
Rheological characteristics of composite flours: The physical

properties of flour supplemented with different levels of peanut
protein isolates such as water absorption, dough development
time, dough stability time, mixing tolerance index and softening of
dough was studied by Brabender Farinograph (Method No. 54-21)
and mixing time and peak height percentage was determined by
running the flour samples through Mixograph (Method No. 5440A) according to their respective methods as outlined in AACC 10.
Preparation of protein enriched muffins: Muffins were prepared
with supplemented blends and of control treatment as mentioned
in Table 1 by following the method described by Shearer and
Davies 11 with some modifications.
Physical analysis of protein enriched muffins: The colour of
muffins was determined with the help of hand held tristimulus
colormeter II (Mod, Neuhaus Neotec, Germany, Colormeter,
Colortest 11 serial no. 95808) as described by Baixauli et al. 12. The
textural study was conducted by using texture analyser (Model
TA-XT2, Stable Microsystems, Surrey, UK) with a 5 kg load cell
as described by Piga et al. 13. It gives the measurements of the
hardness (firmness) and resistance (fracturability) of the muffins
to bend or snap. Muffin volume was measured after baking by
rapeseed displacement method according to procedure as
described in AACC 10. The muffin was placed in the container
filled with rapeseeds and the volume of rapeseeds displaced by
the muffin was recorded according to Keskin et al. 14.
Rheological analysis: The rheological characteristics of straight

grade wheat flour containing different levels of peanut protein
isolates from two peanut varieties (GOLDEN and BARI 2011) were
studied for the parameters such as water absorption, dough
development time, dough stability time, mixing tolerance index
and softening of dough by using Brabender Farinograph. T1 to T8
treatments were prepared using different concentrations of peanut
protein isolates while T0 was control treatment with no addition of
protein isolates. First four treatments were formulated using protein
isolates from GOLDEN peanut variety (T1 5%, T2 10%, T3 15%,
T4 20%) while the other four treatments were prepared using
protein isolates from BARI 2011 peanut variety (T5 5%, T6 10%,
T7 15%, T8 20%).
The data representing the effects of various levels of peanut
protein isolates on the farinographic characteristics of the dough
are given in Table 2. The data regarding water absorption indicated
that the values are not significantly different from each other. It
was obvious from the results that water absorption was higher in
T8 (66.900.30) followed by T7 (65.400.30) and T4 (65.100.20), while
lowest water absorption was observed in T5 (62.100.30). The
results revealed that the water absorption increased with the
increasing levels of protein isolates. These results are in conformity
with the findings of Azizi et al. 17 and Ravi et al. 18. They observed
that the percent water absorption increases with the addition of
protein isolates.
Dough development time is defined as the time required for the
development of gluten. The present results for this trait indicated
a highly significant difference among the treatments. Mean values
for the dough development time of different treatments (Table 2)
Table 2. Farinographic characteristics of flour blends.

Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
Water
absorption (%)
62.760.15a
64.900.20a
63.400.20a
64.100.20a
65.100.20a
62.100.30a
62.300.20a
65.400.30a
66.900.30a
Dough development
time (min)
6.700.10a
5.400.10d
4.700.20e
5.300.20d
5.800.30c
6.500.20ab
5.600.25cd
6.800.30a
6.200.30b
Dough stability
time (min)
11.500.20d
14.500.20a
13.600.20b
12.200.20c
14.200.30a
11.900.20c
13.300.20b
11.300.20d
14.400.20a
Mixing tolerance
index (BU)
30.002.51e
35.002.00d
40.002.08c
50.002.52a
20.002.00f
33.002.00de
40.002.00c
35.002.00d
44.003.00b
Softening
of dough (BU)
47.002.51ef
63.002.52cd
71.002.52b
61.003.05d
48.003.00ef
75.002.00a
50.002.00e
66.003.00c
46.002.00f
* Means sharing the same letter in a column are not significantly different. T0 = 100% straight grade wheat flour (SGF). T1 = 95% SGF and 5% peanut protein
isolate (GOLDEN). T2 = 90% SGF and 10% peanut protein isolate (GOLDEN). T3 = 85% SGF and 15% peanut protein isolate (GOLDEN). T4 = 80% SGF
and 20% peanut protein isolate (GOLDEN). T5 = 95% SGF and 5% peanut protein isolate (BARI 2011). T6 = 90% SGF and 10% peanut protein isolate (BARI
2011). T7 = 85% SGF and 15% peanut protein isolate (BARI 2011). T8 = 80% SGF and 20% peanut protein isolate (BARI 2011).
52
indicated that the highest value (6.800.30 min) was found Table 4. Physical characteristics of muffins supplemented with peanut
in T7 followed by T0 (6.700.10) while it was lowest for T2
protein isolates.
(4.700.20 min). These results are in agreement with the
Firmness
Fracturability
Volume
Treatments Colour (CTn)
findings of Azizi and Rao 19, Sim et al. 20. The mean values
(g)
(mm)
(cm3)
T0
159.334.16cd
216.6728.86a
865.2731.24a
86.740.14a
showed that the highest value for dough stability time
T1
162.678.73bcd 116.6714.43de 769.2224.24b 87.610.33b
was observed in T1 (14.500.20 min) and the lowest value
162.674.93bcd 133.3314.43bcd 715.238.07c
84.580.10c
T2
was obtained in T7 (11.300.20 min). The above findings
a
cde
d
181.974.72
116.6714.43
675.3316.94
84.380.15c
T3
are in close agreement with the results of Ravi et al. 18 and
161.674.04bcd
83.33314.43e
589.5610.10e 83.760.10d
T4
Indrani and Rao 21.
159.6719.00d 116.6738.18cde
514.138.15f
83.140.09e
T5
d
bc
g
153.3311.54
141.6738.18
478.1315.13
81.170.13f
T
6
The data for the analysis of variance for mixing tolerance
abc
de
h
174.009.00
91.66728.87
429.418.65
80.140.07g
T7
index indicate a highly significant difference among
176.009.00ab
166.6728.87b
412.3713.36h 79.310.12h
T8
treatments. The mean values for mixing tolerance index * Means sharing the same letter in a column are not significantly different.
show the highest value for T3 (50.002.52 BU) followed
by T8 (44.003.00 BU) and T2 (40.002.00 BU) while it was lowest and fracturability. Hardness (firmness) was calculated in terms of
for T4 (20.002.00 BU). These results are in conformity with the maximum force (g) and fracturability was determined in terms of
values as observed by Ravi et al. 18 and Azizi and Rao 19. The distance (mm). Hardness can be defined as the peak force during
mean values for softening of dough showed a maximum value for the first compression cycle (first bite). It is the force required to
T5 (75.002.00 BU) and a minimum value for T8 (46.002.00 BU). attain a given deformation. The values for the hardness of muffins
The above mentioned results are in conformity with the findings ranged from 412.37 g to 865.27 g. The mean values (Table 4)
indicated maximum hardness value for T0 (865.2731.24 g) and
of Asghar et al. 22.
The results of mixographic studies are shown in Table 3. These minimum value for T8 (412.3713.36 g). The results are in agreement
results showed that maximum mixing time was observed for T3 with the observations of Azizi and Rao 19 and Ashwini et al. 25.
(3.000.20 min) while it was minimum for T5 (1.150.02 min). The Fracturability (also known as brittleness) is the force at first
present results are in harmony with the findings of Indrani and significant break in the curve. It is the force with which the material
Rao 21 and Asghar et al. 22. Mean values for peak height percentage or the product fractures. The mean values for the fracturability of
indicated that ranged between 31% and 50%. The mean values muffins are given in Table 4. It is obvious from the mean values
explicated maximum peak height percentage for T5 (50.003.00%) that maximum fracturability was observed in T1 (87.610.33 mm)
followed by T3 (43.006.55%) and T7 (41.500.20%) while the followed by T0 (86.740.14 mm) and T2 (84.580.10 mm) while
minimum value was observed for T6 (31.500.10%). These results minimum value for fracturability was observed in T8 (79.310.12
mm). These results are similar to the findings of Ashwini et al. 25.
are in concurrence with the findings of Indrani and Rao 21.
Table 3. Mixographic characteristics of flour blends.
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
Mixing time (min)

1.450.02f
2.150.20d
2.300.03c
3.000.20a
1.300.02g
1.150.02h
2.000.02e
2.450.02b
2.000.04e
Peak height (%)

41.500.10b
40.004.00bc
34.500.20cd
43.006.55b
33.002.00d
50.003.00a
31.500.10d
41.500.20b
41.500.10b
* Means sharing the same letter in a column are not significantly different.
Measurement of physical characteristics of muffins: Colour value

was determined by using colour meter II. It was first calibrated
with the standards having lower and upper limits (51-200),
respectively. The mean values for colour (Table 4) showed that
the highest value was observed in T3 (181.974.72 CTn) and the
lowest in T6 (153.3311.54 CTn). The above mentioned colour
values are in agreement with Azizi and Rao 19 and Abu-Ghoush
et al. 23. Volume of muffins is affected by various factors such as
quality of flour, type of ingredients and processing conditions.
The mean values for the volume of muffins given in Table 4
indicated that the maximum value for volume was observed in T0
(216.6728.86 cm3) followed by T8 (166.6728.87 cm3) and T6
(141.6738.18 cm3) while the minimum value was observed in T4
(83.33314.43 cm3). The current findings are in concord with Azizi
and Rao 19, Kaur et al. 24 and Ashwini et al. 25.
Texture of muffins was measured in terms of hardness (firmness)
Sensory evaluation of muffins: Sensory characteristics are much

significant towards the liking and disliking of product, i.e. muffins.
Muffins with light brown and creamy colour with soft texture are
usually preferred by the consumers. Sensory evaluation of muffins
was carried out by a trained panel for the attributes such as colour,
taste, flavour, tenderness, moistness and overall acceptability.
The results pertaining to all sensory parameters of muffins (Table 5)
are discussed in detail hereunder. The mean values indicated that
the highest score for colour (6.660.51) was assigned to the
treatments T3 and T7 while the lowest score (4.830.75) for this
attribute was given to T4. These findings revealed that the muffins
prepared by using 15% protein isolates from GOLDEN (T3) and
BARI 2011 (T7) showed highest results for the colour score 6.66.
The mean scores for flavour of muffins indicated that the muffins
prepared from T0 and T5 got the highest scores for flavour
(7.000.89) followed by T2 which obtained 6.660.51 score for
flavour. Muffins prepared from T4 were found to be disliked by
the judges regarding their flavour score, i.e. 5.330.51. The current
results are also similar to the findings of McGuire et al. 9 who
declared that addition of protein isolates in any product caused
changes in taste, flavour, texture and overall acceptability scores.
The mean scores assigned by the panellists to the texture of
muffins revealed that the control muffins (T0) got 6.830.75 score
for texture. The muffins prepared from T3 and T7 were considered
more acceptable by the panellists regarding the texture as 6.500.54
and 6.660.51, respectively. Mean scores for tenderness of muffins
indicated that the highest tenderness score (6.660.51) was
assigned to T3 and T7 followed by T1, T2 and T6 (6.330.51). The
53
Table 5. Sensory evaluation of muffins.

Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
Colour
6.500.54a
6.000.63a
6.500.54a
6.660.51a
4.830.75b
6.160.75a
6.160.40a
6.660.51a
5.000.63b
Flavour
7.000.89a
6.330.81a
6.660.51a
6.500.54a
5.330.51b
7.000.89a
6.330.81a
6.500.54a
5.500.54b
Texture
6.830.75ab
6.500.54ab
6.160.75bc
6.500.54ab
5.500.54cd
7.000.89a
6.500.54ab
6.660.51ab
5.000.89d
Tenderness
6.000.63ab
6.330.51a
6.330.51a
6.660.51a
5.330.51bc
6.160.75a
6.330.51a
6.660.51a
5.000.63c
Moistness
6.500.54a
5.660.51bc
6.160.40ab
6.500.54a
5.660.81bc
6.660.51a
5.500.54c
6.500.54a
5.330.51c
Shape
6.830.98ab
6.330.51ab
6.160.75b
7.160.75a
4.660.81c
6.500.83ab
6.160.75b
7.000.89ab
4.830.75c
Acceptability
5.161.16c
5.500.54bc
6.331.03ab
6.500.54a
5.000.89c
6.160.98ab
5.500.54bc
6.500.54a
5.000.63c
* Means sharing the same letter in a column are not significantly different.
results further indicated that T8 muffins got the lowest tenderness

score 5.000.63. The mean values for this attribute showed that
the highest value (6.660.51) was observed in T5 followed -by T0,
T3 and T7 having moistness score of 6.500.54. The lowest
moistness score (5.330.51) was recorded in T8. Mean values for
shape ranged from 4.830.75 (T8) to 7.160.75 (T3).
Muffins prepared from T4 were assigned the lowest score
4.660.81. Mean values indicated that the overall acceptability
scores of muffins showed a slight variation with the addition of
varying concentrations of peanut protein isolates. The highest
score (6.500.54) was assigned to T3 and T7. The judges slightly
disliked muffins prepared from T4 and T8 and assigned overall
acceptability score of 5.000.89 to both. The current results are in
agreement to the findings of Shearer and Davies 11 and Ramcharitar
et al. 26.
The current results regarding sensory evaluation of muffins
prepared using different concentrations of peanut protein isolates
from two peanut varieties (GOLDEN and BARI 2011) revealed
that the treatments T3 (15% protein isolates from GOLDEN peanut
variety) and T7 (15% protein isolates from BARI 2011 peanut
variety) showed the best results and were assigned maximum
scores by the panellists in terms of likeness.
Conclusions
The overall results that peanut is a vital source of protein and the
protein isolates exhibit remarkable rheological properties when
blended with straight grade wheat flour. In the limelight of these
properties, peanut protein isolates can be utilized for improving
the quality of muffins and value addition. The physical and
sensory attributes of muffins prepared using different levels of
protein isolates revealed that the treatment with 15%
supplementation showed better results. Thus, muffins enriched
with peanut protein isolates can be a handy tool to cope with
protein deficiency among the vulnerable groups.
Acknowledgements
The authors are thankful to National Institute of Food Science
and Technology, University of Agriculture, Faisalabad for
providing instrument facilities to carry out analyses. The authors
are also thankful to Dr. Muhammad Shahid (Assistant Professor)
for their valuable suggestions and inputs regarding the research.
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WFL Publisher
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www.world-food.net
Assessment of microbiological quality of ready-to-eat foods in Istanbul, Turkey

Vecdet z 1, Sukriye Karadayi 2*, Hseyin akan 1, Beytullah Karadayi 3 and Filiz Ekim evik
Department of Microbiology, Forensic Science Institute, Istanbul University, 34098, Istanbul, Turkey. Department of
Microbiology, Public Health of Agency, 34020, Zeytinburnu, Istanbul, Turkey. 3 Cerrahpasa Faculty of Medicine, Forensic
Medicine Department, Istanbul University, 34098, Istanbul, Turkey. *e-mail: skaradayi2000@yahoo.com
1
Abstract
The purpose of this study is to assess the microbiological quality of ready-to-eat (RTE) foods produced at catering companies in Istanbul, Turkey.
RTE food was collected from May 2009 to May 2010 in Istanbul and a total of 750 samples including 13 food types were examined for the presence
of coliform bacteria, E. coli, S. aureus, B. cereus, Salmonella spp. and L. monocytogenes. Comparison with the Microbiological Turkish Food Codex
(TFC) shows that 3.6% of 532 meats, 18.3% of 120 salads, and 50% of 12 pastries were at an unacceptable level of microbiological quality. However,
all pasta (32 samples), puddings (41 samples) and patties (14 samples) were at an acceptable level of microbiological quality according to the TFC.
In the samples that were examined coliform bacteria, E. coli, B. cereus and S. aureus, respectively, were isolated to 4.8%, 2.4%, 2.25% and 6.4%.
Salmonella spp. and L. monocytogenes were not detected. RTE food should be served to the consumer in a microbiologically safe form. The results
from this study can be used to assess of microbiological risks in food safety. The present study proposes that to minimize bacterial level in RTE foods
in Turkey regular microbiological quality control programs and good hygiene practices are necessary. The use of microbiologic results of ready-to-eat
foods in the Istanbul will provide an example for similar regions.
Key words: Ready-to-eat foods, foodborne pathogen, microbiological quality, food safety.
Introduction
RTE food production has recently increased due to the fast growth
in population density, which brings rapid, on the go consumption
of food in both in the world and Turkey, especially in Istanbul 1-3.
Catering companies producing RTE food offer cheap, economical
and easily accessed products. Therefore, many private or official
foundations have been using such a service at hospitals, nursing
homes, nursery schools and military posts. The inclination to buy
in services from catering companies brings a new changed lifestyle
and consequently increases the importance of microbiological
analysis in food safety 4. Therefore, assessment of the
microbiological quality of RTE food based on the initial bacteria
level becomes very important 5.
Foodborne diseases due to bacteria occur with the consumption
of food contaminated by pathogen microorganisms 6. At present,
the spectrum of foodborne diseases is increasing and these
diseases create a major health problem 7. Different foodborne
pathogens have been associated with outbreaks of foodborne
diseases 8. Many studies have shown that the most common
bacteria associated with RTE food are Salmonella spp., Listeria
monocytogenes, Campylobacter jejuni, Staphylococcus aureus,
Bacillus cereus and Clostridium perfringens 9, 10. Foodborne
diseases have been studied in many national and international
researches. A study which was done in Taiwan has shown that
the percentage of foodborne diseases, which occurred due to
bacterial pathogens between the years of 1986 and 1995 was 65% 11.
In France in the years of 1999 to 2000, 17,378 people were tested,
and foodborne disease was found in 1,267 people and the deaths
of ten people were determined 12. In Turkey, according to the
56
statistics of the Turkish Ministry of Health, 108,246 people were

hospitalized due to foodborne poisoning in 1993-2005 13.
This study aimed to analyse the bacteriological profile of RTE
food produced by various catering companies, in Istanbul, Turkey.
Furthermore, it was also examined whether catering companies
were compatible according to the Turkish Food Codex 14.
Collection of samples: RTE food samples were collected from
May 2009 to May 2010 in Istanbul, Turkey. All food sampled in
the studies was aseptically collected. A total of 750 samples were
transferred from 14 different catering companies to the National
Public Health Association using thermo boxes. Appropriate RTE
food samples were studied at the Istanbul University Forensic
Science Institute and Microbiology Laboratory. The samples
consisted of soup, rice, Turkish kebabs, vegetable meals, food
legumes, meatballs, chicken meals, meat dishes, pastries, puddings,
salads, patties, and pasta. Food samples encountered in the study
contained many various ingredients. For example, 120 soups were
composed of 23 different ingredients (yogurt, rice, spinach, potato,
tomato, and so on) (Table 2).
Sample processing and microbiological analysis: The samples
were examined in terms of the existence of microorganisms such
as coliform bacteria, Escherichia coli, Bacillus cereus,
Staphylococcus aureus, Salmonella spp. and Listeria
monocytogenes. Not all samples were tested for all parameters.
Selection of parameters depended on the type of food and criteria
of the TFC. For coliform bacteria, Escherichia coli,

Staphylococcus aureus and Bacillus cereus analysis, 10 g or 10
ml of each of the samples was weighed aseptically into sterile
stomacher bags and homogenized in a stomacher (AES) for about
two min. For Salmonella spp. and Listeria monocytogenes
analysis, 25 g or 25 ml of each of the samples was weighed
aseptically and the process mentioned above was applied. Bacteria
were studied as described in Table 1 15-20. In determining the
microorganisms, biochemical specifications of the bacteria and
identification test kits that were commercially available were used
(Table 1).
Results
In this study, specimens belonging to 14 firms producing RTE in
different regions of Istanbul were used. A total of 750 samples
including 13 food types (soup, rice, Turkish kebabs, vegetable
meals, chicken meals, ready-to-eat salads, and so on) and 218
different kinds of food were examined (Table 2). RTE food whose
sample numbers were highest were 120 soups and salads and 100
vegetable meals (Table 2). Four different micro-organisms were
isolated from the food sampled. These were coliform bacteria, E.
coli, S. aureus and B. cereus (Table 3). Salmonella spp. and L.
monocytogenes were not isolated from any of the samples
analysed.
In the study, coliform contamination was detected in about 4.8%
of samples (Table 3). When 750 RTE foods were researched in 13
food categories, E. coli was determined in 18 of them (2.4% of
samples) (Table 3). The microbiological level changed from 101 to
106 CFU/g (Table 4). In rice samples, the highest E. coli and coliform
bacteria levels were 105-106 CFU/g (Table 4). The pastries were in
the group whose Escherichia coli percentage was highest with
33.33% and that the Escherichia coli percentage of the salads
was 5% (Table 4). E. coli was not isolated in the samples of legumes,
meatballs, meat dishes, pies or pasta (Table 4).
In 718 RTE food samples, 46 (6.4% of samples) contained S.
aureus with levels ranging from 101 to 106 CFU/g (Tables 3 and 4).
The highest values were determined in pastry (33.3% of pastry
samples), chicken meals (6.3% of chicken samples), rice (5.8% of
rice samples) and meatballs (5.8% of meatball samples) (Table 4).
In this study in 13 (2.25% of samples) of the food samples analysed,
B. cereus was detected in 577 samples (Table 3). In the ten
categories studied, B. cereus was detected in all food groups
Table 2. Food types encountered in the study.

Food type
Soup
Rice
Turkish Kebab
Vegetable meals
Food legumes
Meatball
Chicken meals
Meat dishes
Pastry
Pudding
Salad
Patty
Pasta
Total
Number
120
70
23
100
20
52
48
98
12
41
120
14
32
750
Kind
23
8
10
23
4
2
27
32
5
10
34
7
12
218
Table 3. Number and percentage of food samples.

Microorganisms
Coliform bacteria
E. coli
S. aureus
B. cereus
Sample n
709
750
718
577
Positive n
34
18
46
13
Positive %
4.8
2.4
6.4
2.25
except for food legume, patty and pasta.

In this study, in the pastry samples, coliform bacteria ranged
from 102 to 104 CFU/g, E. coli from 101 to 103 CFU/g and S. aureus
from 102 to 105 CFU/g (Table 4). B. cereus was not detected in the
pastry samples. Also, soup and rice samples were the groups
which had the highest levels of B. cereus (104 to 105 CFU/g) (Table
4). Neither indicator nor pathogenic bacteria were detected in the
food legume samples.
In assessing the microbiological results of the RTE foods, TFC
microbiological quality criteria were used (Table 5). When RTE
food was assessed according to the TFC, these results were
obtained: 512 of 532 general meal samples (96.4% of samples), 98
of 120 salads (81.7% of samples), six of 12 pastries (50% of samples),
and all the 32 pasta samples, 41 puddings and 14 cakes were
acceptable in terms of microbiological criteria (Table 6).
Discussion
Recently, the RTE sector which is becoming an important industry
has also brought some problems along with it 1. Every year, the
consumption of RTE has increased and consequently, catering
company sectors have also increased in parallel. Therefore, an
error, which may occur at any step of this sector, can cause
Table 1. Microbiological techniques used for the testing of food samples.

Incubation
Media
Temp (C) Time (h)
Coliform
Violet Red Bile (VRBL) agar (Oxoid CM0968)
Pour plate
37
24
bacteria
purplish-red colonies (ISO 4832, 2006)
Tryptone Bile X-glucuronide (TBX) agar (Oxoid
E. coli
Pour plate
44
18-24
CM0945) blue colonies (ISO 16649-2, 2001)
Baird Parker agar (Oxoid CM0275) black or gray
S. aureus
Spread plate
37
24-48
colonies (ISO 6888-1, 1999)
Mannitol egg Yolk Polymixin (MYP) agar
B. cereus
Spread plate
30
18-24
(Merck 1.05267) pink colonies (ISO 7932, 2004)
Pre-enrichment
37
18
Buffered peptone water (Oxoid CM0509)
Enrichment
41.5
24
Rappaport Vassiliadis (HiMedia 1137)
Salmonella
Xylose Lysine Deoxycholate (XLD) agar (Merck
spp.
Spread plate
37
24
1.05287) black centered colonies
(ISO 6579, 2002)
Pre-enrichment
30
24
Half Fraser Broth
Listeria
Enrichment
37
48
Fraser Broth
monocyt.
Palcam agar (CM0877) Green-gray colonies with
Spread plate
37
24-48
black halo and core (ISO 11290-2, 1998)
Bacteria
Analysis
method
Confirmation technique
Brilliant green lactose bile broth (BGB) (Oxoid
CM0031)
Coagulation +, catalase +, gram+

Hemolysis+
Biochemical tests: triple sugar iron agar (HiMedia

M021), urea broth (Merck 1.08483) Final test;
VIDAS Identification system (Biomerieux)
API Listeria strip (Biomerieux)
57
Table 4. Microbiological analysis results of coliform, E. coli, S. aureus, B. cereus, Salmonella spp. and Listeria
monocytogenes.
Food type (detailed)
Soup (120)
Rice (70)
Turkish Kebab (23)
Veg. meal(100)
Food legume (20)
Meatball (52)
Chicken meal (48)
Meat dish (98)
Pastry (12)
Pudding (41)
Salad (120)
Patty (14)
Pasta (32)
Coliform
1(%0.8)
4(%5.7)
3(%13)
4(%4.2)
ND
ND
2(%4.2)
ND
4(%33.3)
NT
14(%11.7)
1(%7.1)
1(%3.13)
CFU/g
101-102
105-106
101-102
101-103
103-104
102-104
101-104
102-103
102-103
Salmo. spp.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
E. coli
1 (%0.8)
1 (%1.5)
1 (%4.4)
2 ( %2)
ND
ND
2 (%4.2)
ND
4 (%33.3)
1 (%2.4)
6 (%5)
ND
ND
CFU/g
101-102
105-106
101-102
101-103
103-104
101-103
101-103
101-104
L. monoc.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
S. aureus
2(%1.7)
4(% 5.8)
1(%4.4)
5(%5)
1(%5)
3(%5.8)
3(%6.3)
4(%4.1)
4(%33.3)
2(% 4.9)
17(%4.2)
ND
NT
CFU/g
102-103
101-106
101-103
102-104
102-103
102-104
101-103
102-103
102-105
102-103
102-105
B. cereus
1(%0.8)
1(%1.4)
3 (%13)
3 ( %3)
ND
1 (%1.9)
2(%4.2)
2 (%2)
NT
NT
NT
ND
ND
CFU/g
104-105
104-105
102-104
102-103
102-103
102-104
101-103
ND: Not detected. NT: Not tested.
Table 5. Microbiological quality of RTE (meal, salad, pasta and pudding) in Turkish Food Codex.
RTE meals (CFU/g)
Microbiological
Criterion
quality
E. coli
<101
S. aureus
103
B. cereus
Salmonella spp.
103
Not detected in
25 g-mL
RTE salads (CFU/g)

Microbiological
Criterion
quality
E. coli
101
S. aureus
103
Not detected in
Salmonella spp.
25 g-mL
Not detected in
Listeria monocyt.
25 g-mL
RTE pasta (CFU/g)

Microbiological
Criterion
quality
E. coli
<101
B. cereus
103
Not detected in
Salmonella spp.
25 g-mL
Table 6. Acceptable/unacceptable RTE foods according to

Turkish Food Codex.
Food type
Meal (n=532)
Salad (n=120)
Pasta (n=32)
Patty (n=14)
Pudding (n=41)
Pastry (n=12)
Total (n=750)
Acceptable
512
98
32
14
41
6
703
%
96.43
81.67
100
100
100
50
93.3
Unacceptable
19
22
0
0
0
6
47
%
3.6
18.33
0
0
0
50
6.7
foodborne disease and great economic loss, even death.

The most common known causes of foodborne diseases are
pathogenic bacteria. In this study, the aim was to analyse the
bacteriological profile of RTE food produced by various catering
companies in Istanbul, Turkey. Therefore, the potential risks, which
RTE products could cause for health care, were studied.
In Korea, coliforms were detected in approximately 50% of the
samples at levels up to 105 CFU/g 21. In our study, coliforms were
detected at levels up 106 CFU/g. The incidence of the fecal indicator
organism E. coli in salad vegetables was found to be 102 CFU/g
(3.7% of samples) in a study prepared in the UK 22. According to
the Public Health Laboratory Service (UK) criteria, the amount of
E. coli in RTE food was defined as <20 CFU/g (satisfactory), 20
<100 CFU/g (acceptable), 100 CFU/g (unsatisfactory) 8. In our
study, E. coli was detected from 10 CFU/g to 106 CFU/g in 18
(2.4%) of the 750 samples tested and was found as 10 CFU/g to
104 CFU/g (5% of samples) in salads. Microbial contamination of
RTE can occur due to the environment, from contact with
contaminated containers, equipments and utensils, hands,
aerosols or pets 23, 24. As a result, the detection of coliform bacteria
and E. coli shows the possibility of fecal contamination. RTE
foods, especially salads have been implicated in foodborne illness
outbreaks because these foods are often prepared by hand 25.
The decontamination activity of the washing system for bacteria
removal is mostly unknown in vegetables 26. We think that
58
RTE pudding (CFU/g)

Microbiological
Criterion
quality
Yeast and mold
103
S. aureus
103
Not detected in
Salmonella spp.
25 g-mL
Not detected in
Listeria monocyt.
25 g-mL
approximately one in five salads is unacceptable for consumption

due to the reasons mentioned above.
The presence of S. aureus in RTE food is an indication of poor
hygiene practices. S. aureus in RTE food is associated with crosscontamination occurring during processing and storage or through
the contamination of raw ingredients 27. In a study in Cyprus in
1991-2000, 1382 RTE samples were analysed and in 2% of them S.
aureus (> 104 CFU/g) was determined 28. In a study in Turkey,
among a total of 512 RTE samples, S. aureus was detected in eight
of 32 Russian salads (25% of samples) and in nine of 75 vegetable
salads (12% of samples) 29. In the present study, S. aureus was
detected in 6.4% of 718 RTE samples and 4.2% of 120 salads.
In a study of sauces in England, B. cereus and/or other
pathogenic Bacillus spp. were detected in 3.8% of 1208 samples
(from 104 CFU/g to 105 CFU/g) 22. In another study in Taiwan,
164 RTE samples were studied and in noodles (a traditional meal),
B. cereus was detected at a high percentage (66.7%) 11. In the
present study, the percentage of B. cereus (from 102 CFU/g to
104 CFU/g) was 13%, in traditional Turkish kebabs. These levels
are significant, potentially hazardous levels. As a result of all our
analysis, the level of B. cereus determined in RTE foods changes
from 101 CFU/g to 105 CFU/g. These results are similar to those
of Meldrum et al. 22 but are much lower in comparison with those
of Fang et al. 11. We consider that these differences possibly
resulted from discrepancies in meal preparation culture in different
geographical regions and hygienic conditions during the
production and storage of the food.
Salmonella spp. and Listeria monocytogenes are the most
important factors in foodborne disease and in our study these
pathogens were not detected in any RTE food. However, in some
examinations, Salmonella spp. wasnt detected in the studies done
on RTE in 2000 and 2004 in the UK 23, 30. The results of our studies
are a positive finding in terms of hygiene conditions.
When 750 RTE samples in total were assessed according to the
TFC, 6.7% of them were not acceptable (Table 6). In the pastry
products, the bacterial level was found to be higher when

compared with other RTE food. In this study, 50% of pastries,
18.33% of salads and 3.6% of the meat samples were detected as
unacceptable according to the TFC (Table 6). Despite poor
environmental hygiene, all RTE food samples except for pastry,
salad and meat were detected to be microbiologically safe food
(Table 6). Similar results were reported for soup, rice, general meat
and salad in Ankara 31.
Because the samples were determined to be unacceptable in
half of the pastries analysed, these foods are potentially a big risk
to public health if necessary measures arent taken. Pastries have
the property of increasing bacterial level with their water activity
and specific pH 5. Also, we think that the reason for this finding is
the fact that pastry is exposed to lower heat processes compared
with others as well as the many more steps during the preparation.
S. aureus was isolated from the pudding samples (Table 4).
However, these samples were assessed to be acceptable, because
the borderline limit in S. aureus for pudding samples was
considered to be 103 CFU/g as cited by the TFC (Table 5).
In Turkey, the number of studies about the microbiological level
of RTE foods is very limited. We encourage increasing the number
of studies about this subject and controls according to the TFC in
Turkey. We conclude that companies who provide such a service
should be careful with personnel training and should always be
up to date with all recent developments and innovations in the
field in order to provide the best practice. It is crucial that all
employees including administrators and the people who work in
the kitchen should receive theoretical and practical training.
According to the results of our studies, we think that if RTE food
meets the required criteria, the risk to public health posed by
microorganisms can be reduced.
Conclusions
We reached the conclusion that pastries, salads and chicken meals
served in Istanbul constitute a risk to public health. Crosscontamination and infected food handlers were reported generally
as important factors in the determination of these microorganisms.
RTE food should be served to consumer in a microbiologically
safe manner. The production of safe RTE food is the responsibility
of the producer. At the same time, maintenance of correct
transportation is important for the safety of RTE food as well. The
results from this study can be used to assess microbiological
risks in food safety. The present study proposes that to minimize
bacterial levels in RTE foods in Turkey regular microbiological
quality control programs and good hygiene practices are
necessary. The use of microbiologic results of ready-to-eat foods
in the Istanbul will provide an example for similar regions.
Acknowledgements
The authors wish to thank the Forensic Science Institute
Microbiological Laboratory for technical support and Public Health
Laboratory. This work was supported by the Scientific Research
Projects of the Istanbul University with project number 5601.
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60
WFL Publisher
Helsinki, Finland
www.world-food.net
Chemical composition of pumpkin (Cucurbita maxima D.) flesh flours used for food
Jurgita Kulaitien 1, Elvyra Jarien 1, Honorata Danilenko 1, Judita erniauskien 1, Agata Wawrzyniak 2,
Jadwiga Hamulka 2 and Edita Jukneviien 1*
1
Institute of Agriculture and Food Sciences, Agronomy Faculty, Aleksandras Stulginskis University, Studentu str. 11, LT 53361
Akademija, Kaunas distr, Lithuania. 2 Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences
(WULS-SGGW), 02-776 Warsaw, 159C Nowoursynowska str., Poland. e-mail: jukneviciene.edita@gmail.com,
judita.cerniauskiene@asu.lt, jurgita.kulaitiene@asu.lt, honorata.danilcenko@asu.lt, elvyra.jariene@asu.lt
Abstract
Pumpkin flours (Cucurbita maxima D.) are alternative horticultural products and functional properties of food components. The main aim of this
study was to investigate the quality parameters of the pumpkin (Cucurbita maxima D.) fruit flesh flours of different cultivars: Justynka F1,
Karowita and Amazonka. Standard methods were applied to determine dry matter, crude fibre, crude protein, crude fat, crude ash, the neutral
dietary fiber (NDF), modified acid-detergent fibre (MADF), water-soluble carbohydrates (WSC) and carotenoids (-carotene, lutein + zeaxanthin,
lycopene). The pumpkin fruit flours of Justynka F1 accumulated significantly highest content of dry matter, crude ash, crude fiber, water-soluble
carbohydrates (WSC). The highest amount of crude protein, crude fat and lutein + zeaxanthin were in the pumpkin fruit flours of Karowita. The
maximum of neutral detergent fiber (NDF) and modified acid-detergent fibre (MADF) was accumulated in Amazonka pumpkin flour, respectively,
21.37, 20.13% DM, so that the flour of this pumpkin variety is most suitable to enrich food with dietary fiber.
Key words: Pumpkin, flesh, flours, quality, nutritional value.
Introduction
Pumpkins can be processed into flour which has a longer shelflife. This flour can be used for its flavour, sweetness, deep yelloworange color and considerable amount of dietary fiber. It can be
also used to supplement cereal flours in bakery products, soups,
sauces, instant noodles and also as a natural coloring supplement
for food 28, 29.
Currently Lithuanian consumers also buy more vegetables that
are grown in small farms and have exclusive properties (organic
products), vegetables of exceptional quality. In this way changes
are inevitable in the cultivation of raw materials, their processing
and marketing. Lithuanian climate is suitable for growing pumpkins
as well. They grow well in the soil which is sheltered from the
winds, in sandy loam or in clay, warming soil.
Pumpkins produce high yields in comparison with other
vegetables and they are rated for the simple production
technology14. The breeders have already created shrubby type of
pumpkin plants. Cucurbita maxima is cultivated for flesh and
seeds for human nutrition, either for direct consumption or for
preparation of other foods such as syrups, jellies, jams, and purees.
This vegetable can be processed in different ways. It can be baked,
frozen, dried, crystallized, marinated or lyophilized 10. The fruits of
pumpkins have a lot of biologically active compounds : vitamin C,
vitamin E, minerals, pectins and carotenoids. The beneficial
influences of carotenoids on human health have proven by many
researchers. In the human body carotenoids keep same chemical
reactivity as in plants - catching free radicals and active atomic
oxygen 11. Carotenoids also potentially play an important role in
human health by acting as biological antioxidants, protecting cells
and tissues from the damaging effects of free radicals and singlet
oxygen. The protective role of xanthophyll pigments lutein and
zeaxanthin have been recently added to the list of potentially
beneficial nutrients for coronary heart diseases and stroke, cataract
and macular degeneration (AMD) 18. In China, Yugoslavia,
Argentina, India, Mexico, Brazil, and America pumpkins are utilized
in the pharmaceutical industry 30.
Pumpkins are good sources of proteins and fibre. Proteins are
irreplaceable, because other nutrients dont have nitrogen or amino
acids. Many investigations have been reported which concerning
the health benefits or the quantities of the fibre found in fruits and
vegetables 13, 25. Most of the research has concentrated on the
physiological properties of fibres and how they influence the
gastrointestinal tract. The fibre plays an important role in the
prevention and cure of diabetes, obesity, atherosclerosis, heart
diseases and colon cancer 7, 8. The structural polysaccharides are
the major part of plant cell walls. The types of plant material that
are included within the definitions of dietary fiber may be divided
into two forms based on their water solubility: insoluble dietary
fiber, which includes celluloses, some hemicelluloses and lignin
and soluble dietary fiber which includes -glucans, pectins, gums,
mucilages and some hemicelluloses 4.
Minerals play an important role for the human body. They affect
the utilization of dietary vitamins and are integral parts of bones,
teeth, soft tissues, muscles, blood and nerve cells. At least 22
mineral elements are required for the well-being of humans and
these can be supplied by a balanced diet 33.
Pumpkin flour is currently the main processed product of
61
pumpkin fruit, because it can be easily stored for long time and
conveniently used in manufacturing formulated foods. Adding
pumpkin flour in the processing of noodles, breads and cakes,
not only enhances the content of various nutrients, but also
improves the flavour of products 6.
The main aim of this study was to investigate the quality
parameters of the pumpkin (Cucurbita maxima D.) fruit flesh flours
of cultivars Justynka F1, Karowita and Amazonka.
Three pumpkin (Cucurbita maxima D.) cultivars Justynka F1,
Karowita and Amazonka were grown in the experimental field
of ecological farm (Lithuania, Kaunas distr.). The field was not
fertilized. Pumpkins were sown in the plastic cups in the end of
April, 20122013 (23 seeds were put into one hole of 24 cm
depth) and were considered in the glasshouse. Into the constant
growing place of the field shoots were planted on the middle of
May, 2012-2013. Plants were grown in four replications. Pumpkins
were harvested at the end of September.
Plant material and flour preparation: The pumpkin fruits were
washed, halved and the seeds were removed. The flesh and peel
were sliced, and dried at 60C in the thermostat (Termaks, Norway).
Dried slices of pumpkin were grinded using ultra centrifugal mill
(ZM 200, Retsch, Germany) to produce flours, which were kept
chilled in an air-tight container at 1218C temperature, until the
laboratory analysis. The samples were evaluated in triplicate for
each analysis.
quality and output of the recycled products. Depending on the

type and cultivar, the amount of the above mentioned substances
in pumpkin flesh can fluctuate from 4.15% to 23.1% 16, 27. The fruits
of great pumpkins accumulate higher amounts of dry matter
compared with the amount of the fruits of oil pumpkins. This is
due to the relatively high sugar content in the flesh of Cucurbita
maxima fruits 1, 26. Great pumpkins that are grown in Lithuania can
accumulate 7.4122.20% of dry matter. Plant fertilization with
complex and humus fertilizers increases dry matter content in the
fruits of pumpkins 17.
The amount of dry matter in different great pumpkin fruit
flesh flours ranged from 7.57 to 12.44% (Table 1). The significant
higher quantities of the above mentioned substances have been
found in Justynka F1 12.44%.
Protein is important for tissue repair and cell growth. They
provide the building block for just about every tissue in human
body (i.e. muscle, hair skin, blood, enzymes, etc.). They affect
transport through the cell membranes of various vitamins and
minerals. The content of crude protein in dry matter of tested
pumpkin fruit flesh flours was in the range from 8.35 to 11.33%
(Table 1). Amazonka flesh accumulated the lowest amounts of
crude protein (8.35%). This can be explained by the shortest
vegetation period of this cultivar. In the pumpkin Karowita flesh
flours had twice higher amount of crude protein (Table 1).
Quantities of minerals in pumpkins are influenced by numerous
complex factors including genotype, soil, environmental
conditions and nutrition interactions 32. It is very beneficial to
consume food with sufficient amounts of these substances. The
content of crude ash in the fruit flesh flours of tested pumpkins
was in the range from 6.61 to 8.89% (Table 1). The highest amount
of crude ash was accumulated in the flesh flours of Justynka.
Cucurbits are among the most important plant families supplying
with edible products and useful fibres 2. The fibre is mainly present
as cell-wall polysaccharides, which have cholesterol-lowering
properties. Antioxidative effects of these pumpkin components
have been also reported 24. On the average, most of the crude fibre
in dry matter was accumulated in Justynka F1 flesh flours (6.66%)
(Table 1). The fruit flesh of analysed cvs. accumulated similar
amounts of crude fibre.
Amount of crude fat in pumpkin fruit flesh flours ranged from
2.45 to 3.21%, between cultivars there were insignificant differences
(Table 1). Pumpkin flesh flours have very low amount of crude fat.
Foods produced from plants abounds with natural biologically
active compounds, such as polyphenols, vitamin C or b-carotene,
have antioxidant properties and are the great value to human
health 3. The most notable positive effect of processing on the
overall quality or health capacity of food is the increased
bioavailability of b-carotene resulting in an increased antioxidant
status. According to scientists, Pumpkin varieties with high lutein
content and low carotene content show a bright yellow color in
seeds 19.
Methods of sample preparation and chemical analyses: The

studies were carried out at the laboratories of Lithuanian Research
Centre of Agriculture and Forestry, Agriculture and Food Sciences
Institute of Agronomy Faculty of Aleksandras Stulginskis
University and Faculty of Human Nutrition and Consumer
Sciences, Warsaw University of Life Sciences (WULS-SGGW).
Dry matter (DM) content of the pumpkin flesh flours was
determined by drying samples to the constant weight at 105C 20.
Amounts of crude protein, crude fibre 22, crude ash and crude fat
were also determined 22.
The amount of water-soluble carbohydrate (WSC) was
determined using anthrone method 35. Samples were subjected to
the analyses of fiber components: neutral detergent fiber ((NDF)
cellulose, hemicellulose and lignin) and modified acid-detergent
fibre (MADF) using cell wall detergent fractionation method
according to Faithfull 5 and Van Soest et al. 34.
Carotenoids (-carotene, lutein + zeaxanthin, lycopene) content
were detected according to the methods Konings and Roomans 15
and Helsper et al. 12. Analyses were performed with Shimadzu
HPLC 10A system.
The experimental data was statistically processed by the
analysis of variance (ANOVA), software STATISTICA 7.0
(StatSoft, USA). Arithmetical means and standard
errors of means of the experimental data were Table 1. Dry matter, crude protein, ash, fibre and fat contents (mean s.d.) in the
great pumpkin flesh flours.
calculated. Tukey test (p<0.05) estimated statistical
reliability of mean differences.
Cultivars
Dry matter
Crude protein Crude ash Crude fibre
Crude fat
One of the most important chemical content quality
indicator is the amount of dry matter. It ensures the
62
'Justynka F1'
'Karowita'
'Amazonka'
%
12.441.89b*
8.351.33a
7.570.08a
% DM
9.911.1ab
11.331.33b
8.350.16a
% DM
8.890.62b
6.851.61a
6.610.02a
% DM
6.660.82a
6.090.71a
5.610.47a
% DM
2.450.40a
3.211.35a
2.901.19a
*Means in column with different letters are significantly different (p < 0.05) for the different cultivars.
Our results show that great pumpkin flesh flours are rich in
source of carotenoids, especially in lutein and zeaxanthin. Of
course there are other carotenoids that are good precursors of
vitamin A. It was established that the amount of lutein and
zeaxanthin was significantly different in all cvs. pumpkin fruit flours
(Table 2). According to our results significantly highest amount
of lutein and zeaxanthin was in pumpkin flours of Karowita 12.31
mg/100 g.
Murkovic et al. 23 reported that three species of pumpkin (C.
pepo, C. maxima and C. moschata) consisted of beta-carotene
(0.06-7.4 mg/100 g), alpha-carotene (0-7.5 mg/100 g) and lutein (017 mg/100 g).
Lycopene concentration is lower than that of other carotenoids.
The fruit flesh of cv. accumulated lycopene amount varied from
0.72 to 0.81 mg/100 g, and the significant highest was identified in
Justynka F1 flours (Table 2).
Table 2. Carotenoids content (mean s.d.) in great
pumpkin flesh (mg/100 g).
Cultivars
'Justynka F1'
'Karowita'
'Amazonka'
Lutein+zeaxanthin
7.960.07a*
12.310.03c
7.960.02b
Lycopene
0.810.01c
0.790.01b
0.720.02a
- carotene
2.420.02b
1.860.02a
2.440.02b
The content of WSC varied depending on cultivars. Pumpkin

flours displayed high content of total WSC (44.73% DM) in
Justynka F1and lowest content in Karowita (36.40% DM)
(Table 3).
Conclusions
The pumpkin fruit flours of Justynka F1 accumulated significantly
highest content of dry matter, crude ash, crude fiber, water-soluble
carbohydrates (WSC). The highest amount of crude protein,
crude fat and lutein+zeaxanthin was in fruit flours of Karowita.
The maximum neutral detergent fiber (NDF) and modified aciddetergent fibre (MADF) was accumulated in Amazonka pumpkin
flour, respectively, 21.37, 20.13% DM, so the flour of this pumpkin
variety could be most suitable to enrich manufactured food with
dietary fiber.
Acknowledgements
This publicatios is funded by European Social Fund and the
Budget of the Republic of Lithuania (project Eureka E! 6855
ECORAW Higher functionality food products from organic
vegetable raw materials).
*Means in column with different letters are significantly different (p < 0.05) for the different
cultivars.
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Cultivars
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'Karowita'
'Amazonka'
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MADF
WSC
amount, % DM
18.800.10a* 16.430.06a 44.730.12c
18.870.25a 18.860.28b 36.400.26a
21.370.47b 20.130.15c 39.830.06b
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33
WFL Publisher
Helsinki, Finland
www.world-food.net
Training a sensory panel for describing texture in peach and nectarines

Loreto Contador 1, Paulina Shinya 1, Andrea Bunger 2, Carmen Senz 1 and Rodrigo Infante 1*
1
Facultad de Ciencias Agronmicas, Universidad de Chile, Casilla 1004, Santiago, Chile. 2 Facultad de Ciencias Qumicas y
Farmacuticas, Universidad de Chile, Casilla 223, Santiago, Chile. *e-mail: rinfante@uchile.cl
Abstract
Analysing the texture of fresh fruit is a fundamental aim in the food industry because it is the main factor that affects consumer acceptance, and it must
therefore be measured objectively. However, as it is a sensory property, human beings should be involved in assessing it. To achieve this goal, panels
of trained judges describe and quantify certain previously defined textural attributes for a particular food. For fresh peaches and nectarines, there are
no established methodologies for training a specific panel to describe texture. Therefore, the aims of this investigation were (1) to select sensory
attributes, develop lexicon and intensity scales for training a sensory panel and (2) to describe peach and nectarine cultivars according to their textural
properties. An appropriate lexicon was generated using five descriptors: hardness, juiciness, melting, crispness and crunchiness. The panel
was able to describe twelve peach cultivars and segregate them according to their textural properties in three groups: melting flesh, non melting flesh,
and an intermediate group formed by MF and NMF genotypes.
Key words: Peach, nectarine, sensory properties, trained panels, texture attributes, lexicon, descriptive analysis, principal components analysis,
hierarchical clustering.
Introduction
The most important attributes that define the quality of a food are
appearance, flavour, aroma, texture and nutritional attributes.
However, from the consumers point of view, texture is the main
quality attribute that influences acceptance or rejection of food 1,
and for most fresh fruit, texture is more important than the aromatic
properties 2. Therefore, when the food satisfies consumers
psychological and physiological expectations, the perception of
texture is placed on a subconscious level, but if there is a defect,
it becomes the main cause for rejection 3.
There are two approaches to study texture of foods: rheological
properties and sensory analysis 1, 4. However, the so-called texture
testing instruments can detect and quantify only certain physical
parameters which must be interpreted in terms of sensory
perception 1. For any kind of food, descriptive profiling is an
essential tool that involves the evaluation of both qualitative and
quantitative sensory characteristics of a product by a panel 5. The
beginning of this process is product familiarization and
development of a lexicon that comprehensively and accurately
describes the product dimension 5. The lexicon of texture can be
so varied that there are studies examining the differences between
languages, such as between English and Finnish 6, English, French,
Japanese and Chinese 7, and a study by Hayakawa et al. 8 that
reports the complexity of Japanese and classifies 445 different
terms to describe components of texture. There are also studies
that go more deeply into the description of the specific language
for texture in red apple 9, tomato 10 and mango 11.
Publishing lexicons is beneficial because it promotes
standardization of sensory vocabulary across multiple panels,
companies and countries. Ideally, a published lexicon has the
complete list of products from which it was developed; all attribute
terms, definitions for every attribute and references for every
attribute 12.
For fresh foods such as fruits and vegetables, textural properties

such as firmness are widely used as indices of readiness to harvest
(maturity) to meet requirements for long-term handling, storage
and assuring acceptability of consumers 13. It should be considered
that fresh fruits are prone to biological heterogeneity, therefore
variations in the data may be due to assessor differences and/or
product heterogeneity 14, 15. For this reason, the analysis of the
fresh fruit texture is more complex than in other processed foods.
Moreover, according to Hampson et al. 16 who studied the apple
genotype differences from a sensory point of view, the
heterogeneity of the fruit may cause difficulty in differentiating
samples. Due to this fact inherent to fresh fruits, it is necessary to
find the most effective methods to homogenize the samples.
The sensory analysis of fresh peaches and nectarines has been
approached using the descriptors appearance, aroma, firmness
sweetness and acid taste 17-20. No researches have delved more
deeply, into the sensory analysis of the texture of these types of
fruits.
The aims of this study were (1) to select sensory attributes,
develop lexicon and intensity scales for training a sensory panel
and (2) to describe peach and nectarine cultivars according to
their textural properties.
Training of the panel for descriptive analysis (DA): The training
process was conducted in 16 sessions of approximately 1.5 hours
each. All of the sessions were conducted in a sensory evaluation
laboratory, equipped with individual booths and a conference
room, following the protocols based on ISO 8586-1 (1993) 21.
Recruitment and selection: Twenty-three people were recruited
for the following training process. The participant ages ranged
65
between 21 and 35 years. To ensure that all of the participants

were suitable for training, they had to have previously passed a
test of recognition of basic tastes and a test of visual ranking 21.
As the purpose of the training was to quantify textural variables,
it was necessary to determine the selected candidates ability to
correctly define the textural properties of certain foods used in
the reference scales. To focus the scales toward fresh fruits, the
anchors for texture evaluation compiled by Harker et al. 22 were
applied. The assessors had to test pairs of samples and describe
their texture. With this exercise, the skill to discriminate differences
and express textural descriptors with suitable language was
determined. All participants correctly determined the basic
characteristics that differentiated the two samples.
Qualitative stage: Open discussions took place regarding those
descriptors that best define the texture of peaches. Finally, each
participant was asked to write down the parameters that they
considered the most important. The most frequently mentioned
attributes were hardness (13 mentions), juiciness (13 mentions),
smoothness/melting (13 mentions) and crispness/crunchiness
(9 mentions). According to the description of the assessors
smoothness and melting were considered similar, as the sensation
of breakup of the fruit in the mouth without chewing the sample.
Other concepts named less frequently were turgidity,
consistency, fibrousness and compression. During the
discussion turgidity, consistency and compression were
considered less relevant and were attributed to the overall concept
of hardness. The fibrousness of the flesh was defined as an
attribute that is not always present in peach, and therefore, it was
considered a defect that does not define the texture of the flesh.
Quantitative stage: Participants were taught to quantify the 5
textural descriptors defined in the previous stage: hardness,
juiciness, melting, crunchiness and crispness, definitions
and the evaluation protocols are shown in Table 1. Fruit and
vegetable samples were obtained from a supermarket in Santiago,
under normal retail conditions. Sample size for sensory evaluation
was standardised in 2 cm thick slices. Samples were prepared
immediately before tasting.

For each textural descriptor, panellists, according to the ranking
method, scored each product from lowest (-) to highest (+)
intensity on the proper intensity scale 23. This task improves
assessors skill in recognising different intensities and therefore
in developing their ability in building a hierarchy according to a
given characteristic (Table 2).
For hardness, the scale used was proposed by Harker et al. 22.
For crunchiness and crispness, the standard scale for wet
foods created by Chauvin et al. 24 was applied with some
modifications. For the attributes juiciness and melting there is
not reference scales for fresh fruits sensory training, so new scales
are proposed in this work. The scales were constructed in open
discussions 25 and using the products for filling the ends of the
scale suggested by Harker et al. 22.
After finishing each session, an open debate took place where
each panellist could discuss their ranking compared with the panel
average. Scores on the 15-cm-scale were assigned for each product
(Table 1). Sample tasting was repeated as many times as necessary
until reaching consensus. The purpose of this phase was to reduce
the inherent individual differences between assessors 26.
Cultivar selection: Fruits were picked at a pre-climacteric but
physiologically mature stage from an experimental orchard located
near Santiago, Chile (334814.85S; 70406.54W). The cultivars
were the melting flesh (MF) nectarines Artic Snow, Andes Nec 1,
Nectaross, Venus and Mara Dolce; the melting flesh (MF) peach
September Sun; and the non-melting flesh (NMF) peaches Dr.
Davis, Andross, Hesse, Malherbe, Corona and 11A1.
Uniform size fruits were selected when the skin background colour
was green-yellow 17, 27-30.
Immediately after harvest, the fruits were transported to the lab
and kept in a ripening chamber at 20C and 80% RH for 4 days
until the flesh had reached an adequate firmness value for
consumption (approximately 10 20 N). Fruits of the NMF cultivars
were also kept in the same chamber even if a firmness value of 10
20 N was not reached, as NMF genotypes are characterised as
having a slow softening rate 31, 32.
Table 1. Definition, evaluation technique and references for fresh peach and nectarine textural descriptors.
Medium
reference
(medium
intensity = 7.5)
High
reference
(extreme
high= 15)
Ripe banana
Granny Smith
apple
Raw carrot
Ripe banana
Apricot
Raw carrot
Ripe banana
Radish
Green pepper
Ripe banana
Strawberry
Watermelon
Raw carrot
Ripe Nectarine
Canned sliced
mango
Low reference
Descriptor
Definition
Crispness
Unique, strong, clean and acute

sound produced in the first bite of
the food with incisors and open
lips.
Hardness
Force required for compress the

sample between the molars.
Crunchiness
Multiple and full sound perceived

as a series of events, evaluated
with molars and closed lips.
Juiciness
Amount of fluid released during

chewing.
Melting
Ease with which the flesh

disintegrates under a slight
pressure exerted between the
tongue and the palate.
66
Technique
Place the sample between the
incisors (front teeth) and penetrate
it. Evaluate the sound intensity
produced at the first bite.
molars and evaluate the force
necessary to compress the food
until the molars are joined.
molars and chew it three times, and
evaluate the intensity of the sound
produced.
Place the food between the molars,
chew it 3 times and evaluate the
amount of juice released.
Place the sample on the tongue and
press it against the palate. Evaluate
how the sample flows.
(absent/ extreme
low = 0)
Table 2. Products used for training each texture descriptor.

Descriptor
Crispness
Hardness
Crunchiness
Juiciness
Melting
ripe banana
ripe banana
ripe banana
ripe banana
raw carrot
red apple var. 'Royal Gala'

mushroom
red apple 'var. Royal Gala'
raw carrot
banana
Descriptor of lower intensity (-) to high (+)

pickle
radish
Peach
apricot
radish
green apple var. 'Granny Smith'
strawberry
grape
ripe nectarine (1-2kg-f)
canned mango
Sensory analysis: The fruit was assessed at consumption ripeness.

Evaluations were performed at normal light and temperature
conditions, in individual booths and following the standard
protocols 26. To reduce the enzymatic reactions caused by cutting
when the samples were prepared, each panellist was instructed to
slice the fruit by cutting with a sharp knife a piece of peach
lengthwise along the seam, extracting a quarter of the fruit. It was
specified that the sample was to be a bite of the equatorial zone of
the quarter. This procedure also homogenised the samples, as the
ripeness of the peaches is not homogenous within the same fruit 15.
Each fruit was provided on a white plate marked with a three-digit
code, with the same code in the score sheet. The attributes
evaluated were hardness, juiciness, melting, crunchiness
and crispness on an unstructured scale from 0 to 15. In all
sessions, assessors had the information of Table 1 available.
Statistical analysis: To analyse the discrimination capacity of
each panellist, the inversion number of the assessor was
calculated using the subtraction (in absolute value) between the
correct rank of the sample evaluated (according to its intensity)
and the rank given by the assessor, and thus, the inversion
number of each assessor was obtained (Table 3).
The inversion number of each assessor must be equal to or
less than n + 1 (n being the number of samples) to be considered
discriminating. This procedure was performed for all attributes.
Likewise, the analytical ability of the sensory panel as a single
entity had to be confirmed. The inversion number of the panel
as a whole was calculated by taking the total sum of the partial
values of each sample tasted by all of the assessors. Then, this
total sum was multiplied by the correct rank value of each sample
within the series. These results were added, and the total inversion
number of the panel was obtained 33. If the alternative hypothesis
is greater than null hypothesis, the panel was considered to
have significantly discriminated the difference in concentration
between the samples evaluated 21.
The panel result consisted on the average of eight trained
panellists whose scores were centred to take into account scale
effects. A principal components analysis (PCA) and hierarchical
clustering using average linkage with Euclidean distance method
were conducted to describe cultivars. The statistical program
InfoStat v. 2011 (InfoStat Group, Crdoba, Argentina) was used 34.
green pepper
red apple var. 'Royal Gala'
raw carrot
watermelon
raw carrot

Training the panel for DA: Once the participants had been
recruited and selected, the qualitative stage of this work consisted
of the creation, understanding and unification of the specific
sensory lexicon of texture in peaches. This phase represents an
important challenge in the study of the textural properties of foods
because of the large number of terms associated with the sensory
description of texture, which is also specific to each food and to
each country 35. If creating an adequate texture lexicon for a certain
food is a difficult objective, the task is made even more arduous
when the terms crispness and crunchiness are defined as
descriptors. In this study, both descriptors were associated with
the sound that occurs when biting and chewing a fruit. Although
a diligent definition was not required to create the lexicon, when
unifying the criteria during the discussion sessions, it was
necessary to spend some extra time for understanding and
differentiating both descriptors. The term crispness alone has
more than one meaning and is difficult to define with exactitude 36.
Both crispness and crunchiness are associated with the fracture
properties of the food 37, 38 as they occur in materials that are
essentially non-deformable and that, therefore, are broken with
relative ease 37. Most likely, to determine and quantify crispness,
the initial bite suffices, whereas crunchiness requires a
succession of fracture events that occur during chewing 37, 39. The
correct use of both terms is so important that some studies have
concentrated on analysing the use of these descriptors among
consumers in different Spanish-speaking countries 35, 40. As both
descriptors are relevant in the textural characterisation of fresh
fruit 22, strategies have been developed to correlate the sensory
perception of the sound with the wavelengths produced when
the food is penetrated by a probe 24, 41-45 and also relate to the
analysis of the force-deformation curve to the sensory values
recorded through a sensory panel 46.
One of the most frequently identified descriptors was melting,
which has also been identified as a main attribute in the texture of
mango flesh 25. There is a certain consensus in its definition that
has to do with the way in which the sample disintegrates in the
mouth, often without chewing 22.
The last descriptor defined was juiciness, for which there was
general agreement among the panellists that it is associated with
freshness 22, 47, 48. As a textural parameter, juiciness is related to
Table 3. Inversion number of each panellist and each sample, and L value of the complete panel for the attribute hardness.
ASSESSORS
order
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23
Ripe banana 1* 2 1 1 1 1 1 1 1
1
1
2
2
1
1
1
1
1
1
1
1
3
1
1
X 28
28
Mushroom
3 1 2 2 2 2 2 2 3
2
2
1
1
2
3
3
2
2
2
2
3
2
2
2
X 48
96
Peach
2 3 4 3 3 3 3 3 2
3
3
4
3
4
2
2
3
3
5
3
2
1
3
3
X 67 201
Apricot
4 4 3 4 5 5 5 5 4
5
4
3
4
3
4
5
4
4
3
5
4
5
5
4
X 97 388
Apple
5 5 5 5 4 4 4 4 5
4
5
5
5
5
5
4
5
5
4
4
5
4
4
5
X 105 525
Raw carrot
6 6 6 6 6 6 6 6 6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
X 138 828
Inversion
2 2 2 0 2 2 2 2 2
2
0
4
2
2
2
4
0
0
4
2
2
6
2
L 2066
number
Sample
67
PC 2 (17.1%)
5
Juiciness
the free water content 49 or moisture in the sample 1,
and it is often included in the sensory evaluation of
fruits, although the texture might not necessarily be
3
11A1
the main goal of the investigation 50-52.
Nectaross
Corona
Crispness
September Sun
The quantitative stage of the training process
Hardness Hesse
Andross
Andes
Nec
1
0
Crunchiness
began with the descriptor hardness and to obtain
Maria Dolce Malherbe
Melting
Dr Davis
Artic Snow
the association between the definition and the
Venus
physical sensation, the food scale was ordered as
-3
proposed by Szczesniak 3 and adapted to the
availability of local brands. Despite the ease with
-5
which the group correctly ordered the samples during
-5.0
-2.5
0
5.0
2.5
the discussion, this scale was not appropriate for the
PC 1 (76.8%)
aim of this research. This result is extremely important,
Figure 1. PCA biplot for the sensory attributes of texture of 12 varieties of peaches
as for the panels performance to be optimal in terms of
and nectarines.
discriminating ability, consistency, reproducibility and
precision, the products used on the scales during training must be
Table 4. Pearsons correlation coefficients among the sensory
similar to the food being studied 5. Therefore, based on panellist
attributes, based on the results of panel trained to
input and literature on fresh fruit, the scales used for the attributes
evaluate peaches and nectarines.
defined in this investigation were created.
Crispness
Hardness
Crunchiness Juiciness
For hardness, the L value with the L = 2066, number of
Hardness
0.88
products = 6 and judges = 23 was 10 (Table 3). Therefore, HA was
Crunchiness
0.91
0.97
accepted both for 5% and 1%. This means that the panel was
Juiciness
-0.27
-0.4
-0.42
being able to discriminate the intensities of hardness correctly.
Melting
-0.77
-0.91
-0.89
0.27
The scores of the inversion number of each panellist were less
than 7 (n + 1); therefore, all panellists determined the order correctly.
Hierarchical cluster analysis identified 3 groups with similar
This procedure was followed for the rest of the attributes, and for texture sensory characteristics (Fig. 2). The first group is composed
each, it was indicated that each panellist and the panel as a whole of the MF cultivars Artic Snow Nectaross, Andes Nec 1 and
ordered the given samples correctly at random. In total, the training September sun. It was observed that such cultivars are closer to
process was performed in 24 hours, divided into 16 sessions, and the descriptor melting (Fig. 1). A second group was formed by
a panel of trained judges was formed to evaluate texture in fresh the NMF cultivars Dr. Davis and Andross and the MF cultivars
peaches and nectarines. Generally, there is no literature that details Mara Dolce and Venus. Finally, the third group is composed
panel training methodology for DA or quantitative descriptive only by NMF cultivars Corona, 11A1, Hesse and Malherbe
analysis (QDA), which are intimately related 5. There are studies characterised by reaching higher scores for crispness, hardness
that detail training methodologies for mango 25, fibre 53 and bread 54. and crunchiness.
However, knowledge of the textural properties of foods is a goal of
Fruit quality is a complex trait and is not determined by any
primary importance for both the actors of the food industry as single attribute but rather by a combination of sensory
well as researchers in the area 13.
characteristics (e.g., appearance, texture, flavour and aroma),
nutritional value, and chemical, mechanical and functional
Sensory evaluation: PC1 and PC2 explained 93.9% of the total properties 2. This is how the sensory evaluation of fresh peaches
variation in the model with respect to sensory parameters (Fig. 1), and nectarines has become an important goal in research, using
indicating that cloud of data was effectively bi-dimensional.
trained or consumer panels 57. In general, the aim of sensory tests
Crispness, hardness and crunchiness were strongly has been to evaluate the effects of postharvest treatments 30, 58, to
correlated between them and with the PC1 (Table 4), whereas establish differences between states of maturity 20, 59 and to
melting was correlated negatively with PC1 and with hardness determine differences between cultivars 17, 19, 60-64 , with
(r = -0.91), crispness (r = -0.77) and crunchiness (r = -0.89)
Artic Snow
(Table 4).
Nectaross
These results are similar to those obtained by Valente et
Andes Nec 1
25
al. , and therefore, PC1 can be considered to be associated September Sun
with the viscoelastic (elastic and plastic) nature of peach
Dr Davis
flesh, whereas PC2 is associated with (the viscous nature)
Andross
juiciness. High degrees of correlations were also found Maria Dolce
Venus
between the attributes hardness, crispness and
Hesse
55
fracturability in apples and pears , between hardness
Malherbe
and fracturability when evaluating reference scales for
Corona
texture 56 and between firmness, crunchiness and melting
11A1
on mangos 25, which eliminates redundancy in the terms
0
1
2
3
4
used. Furthermore, the definition and evaluation methodology
Dissimilarity
for each of these descriptors are different, so the decision
Figure 2. Hierarchical clustering of peaches and nectarines according to their
was made to keep the three descriptors.
sensory texture characteristics perceived by the panel.
68
appearance, aroma, flavour, firmness or hardness,

sweetness, acidity and juiciness being the most frequently
used attributes. There are very few sensory investigations of
texture in peach. It has been approached from the point of view of
the physical behaviour of the fruit, as in the studies of apricots
conducted by Haciseferogullari et al. 65 and Missang et al. 66 or
through the characterisation of the changes in the components of
the cell wall during the softening process 27, 67, 68. Texture has also
been considered a unique attribute 17 , but given its complexity, it
must be disaggregated into its particular components.
Conclusions
An appropriate lexicon was generated for describing the texture
of peaches and nectarines, which considers five descriptors:
hardness, juiciness, melting, crispness and crunchiness.
The scales used for the training process for these attributes allowed
the panel to describe twelve peach and nectarine cultivars and
segregate them according to their textural properties in three
groups: MF and NMF genotypes and an intermediate group formed
with both genotypes.
From the biological standpoint, peach and nectarine flesh are
classified as MF and NMF; however, these categories are
insufficient for describing and analysing the growing supply of
new cultivars released annually to market that exhibit novel flesh
typologies. Due to this dynamism, sensory evaluation is a tool
that allows more efficient classification of the different types of
flesh, which is important, as measuring only the firmness of the
pulp appears to be insufficient.
Acknowledgements
This work was conducted with funding from CONICYT through
the scholarship Doctorado nacional 2010 and FONDECYT
Project 1130198.
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53
WFL Publisher
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Antioxidant and antimicrobial potential of dried cumin (Cuminum cyminum L.),

caraway (Carum carvi L.) and turmeric powder (Curcuma longa L.)
Muhammad T. Sultan 1*, Masood S. Butt 2, Saeed Akhtar 1, Atif N. Ahmad 3, Mubasher Rauf 3,
Muhammad S. Saddique 1 and Ambreen Naz 2
Department of Food Sciences, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan.
National Institute of Food Science and Technology, University of Agriculture, Faisalabad. 3 Faculty of Veterinary Sciences,
Bahauddin Zakariya University, Multan, Pakistan. *e-mail: tauseefsultan@bzu.edu.pk
1
Abstract
The process of oxidation is vital for energy metabolism but it is also coupled with the production of oxygen free radicals (OFRs). The excessive
production of OFRs results in oxidative stress and such conditions demand the supplementation of antioxidants. The bioactive components present
in common spices and condiments are of imperative significance as they scavenge OFRs along with acting as antimicrobial agents. The current study
aimed to explore the antimicrobial and antioxidant potential of dried cumin (Cuminum cyminum L.), caraway (Carum carvi L.) and turmeric
(Curcuma longa L.) powders. The results elaborated the importance of aforementioned spices as they all contain significant amounts of cruder
protein, crude fats, fiber and carbohydrates. Cumin seeds contain the highest amounts of fats, while appreciable amounts of carbohydrates were
observed in turmeric (43.87 1.41). Vitamin C was present in turmeric and caraway seeds. The results regarding minerals indicated that the cumin
contains appreciable quantities of calcium, magnesium, sodium and iron. The results regarding antioxidant potential indicated that the maximum total
polyphenol was present in caraway seeds (1016.72 63.68 mg GAE/100 g) that can also be correlated with higher DPPH and -carotene inhibition
activities (57.71 0.77 and 47.65 0.74%, respectively). The caraway seeds were more effective antimicrobial agent as compared to cumin and
turmeric. Overall, the results indicated the potential of dried condiments as natural antioxidants and antimicrobial agent.
Key words: Natural antioxidants, antimicrobial agents, spices, cumin, caraway, turmeric.
Introduction
The process of oxidation is essential for the vitality of humans to
generate sufficient amount of energy from important food
components like carbohydrates, proteins, and lipids. However,
such processes are also associated with the production of free
radicals 1. These highly reactive radicals, especially oxygen free
radicals (OFRs), can disturb the human metabolisms through
destruction of membranous structures. The process of oxidation
can also result in quality deterioration of different food products.
Although, humans inherited immune defense systems but
antioxidants are required to be supplemented through diet to
control the excessive amounts of OFRs. The natural sources of
antioxidants include fruits, vegetables, spices, and herbs of some
medicinal value 2. In the recent past, the old concepts of food
pyramids and dietary guidelines were modified in the light of
modern research that explored the significant association of diet
and health. The dietitians and nutritionists researched the role of
various plants and their metabolites to control the menace of
oxidative stress and allied complications. The trends also
witnessed the increased share of such health foods in global food
chains owing to their higher acceptability by the consumers 3.
Nutraceutical and functional foods are generic terms used to
categorize foods with certain health claims. Most of these foods
possess multiple health benefits, especially against lifestyle related
and degenerative disorders 4. Natural compounds found in fruits,
cereals, vegetables and spices hold antioxidant activity. The
bioactive antioxidative compounds present in them are usually
classified in different categories like carotenoids, tocopherols,

polyphenols, anthocyanins, alkaloids, and aged glycated peptides 5.
Cumin (Cuminum cyminum L.) belongs to the Apiaceae family
and is annual herb that is native to the Mediterranean region. It is
one of the most commonly used condiment to add flavor and
specific aroma to the foods. The cumin seed contains several
bioactive compounds belonging to the categories of polyphenols,
flavonoids, cumin aldehydes, and terpenoids 6. Some of these
bioactive components are antioxidants and might be useful in
health disorders pertaining to the OFRs. Cumin seeds are reported
to be effective against various ailments including diabetes mellitus
and cardiovascular disorders. Caraway (Carum carvi L.) belongs
to the Umbelliferae family and is biennial herb and used as
condiment to add flavor and specific aroma to the foods. The
caraway seeds are also helpful in mitigating the free radical
production and thus controlling some health disorders. Caraway
seeds contain several bioactive components that belong to the
category of terpenoids, limonene, thymol, glycosides, and
flavonoids. The safety and biochemical efficacy of caraway and
its essential oil has been already tested in various research
interventions 7. Turmeric (Curcuma longa L.), belonging to the
family of Zingiberaceae, is perennial herb usually called as golden
spice due to its specific color tone. Although it is native to Indian
continent, various countries are cultivating it for routine use in
cuisines. The turmeric contains more than 500 bioactive
components and most of them hold some medicinal value. Turmeric
71
is effective against various ailments including cancer insurgence,

cardiovascular disorders, diabetes mellitus, infectious, and
degenerative disorders 8, 9.
Globally, the aforementioned plants are explored in various
studies and several herbal supplements containing bioactive
components from these plants are available in the global food
chain 10. Although, fewer research studies are undertaken in
Pakistan, yet such sort of studies have certain limitations. The
present study aimed at exploring the antioxidant and antimicrobial
potential of sun-dried cumin, caraway, and turmeric. The results
of this intervention are providing supporting evidences to claim
these spices as health promoting entities. The limitation of the
present research included the utilization of unidentified strains of
microorganisms, thus later studies should be conducted to check
the influence of these nutritionally rich commodities on identified
microbial strains.
The present research intervention was completed in the
Department of Food Sciences and Department of Pathobiology,
Bahauddin Zakariya University, Multan. For the purpose, three
different spices (cumin, caraway and turmeric) were collected from
local market of Layyah, Punjab, Pakistan. The cleaning of the
samples was carried out through the operations like sieving and
washing. Later, the samples were ground into a fine powder for
analytical purpose.
Proximate analysis: The cumin, caraway, and turmeric were
examined for their nutritional components that include protein,
fats, ash, fiber, and moisture contents. Nitrogen free extract (NFE)
was calculated using standard equation mentioned in AACC 11
that indicates the presence of carbohydrates. The AACC Method
No. 44 - 01, Method 08 - 01, Method No: 46 - 13, Method 30 - 10,
and Method 32 - 10 were followed for the said purpose 11.
NFE % = 100 (Total ash % + crude protein % + crude fat % + crude fiber %)
The ash collected was further utilized for the analysis of macro
and micro minerals. Sodium, potassium, and calcium were
determined using flame photometry and rests of the minerals were
analyzed using atomic absorption spectrophotometry.
Determination of total sugars and vitamin C: Total sugar is the
sum of reducing and non-reducing sugars and was determined
using the volumetric method (LaneEynon method) mentioned in
AOAC (1990). Vitamin - C content was estimated using 2,6dichlorophenolindophenol dye using the procedures outlined in
the standard method 12.
Determination of antioxidant potential: The antioxidant rich
extracts of selected spices were prepared by slurring the samples
with aqueous ethanol for a period of one hour. The facilities of
National Institute of Food Science and Technology, University of
Agriculture, Faisalabad, were used in this context. Briefly, the
samples were slurred with aqueous ethanol using mechanical
shaker and centrifuged for 15 min at 7000 rpm at 0C. The
supernatant was further filtered with Whatman filter paper No. 1.
The solvent from the supernatant was separated at 50C in a rotary
vacuum evaporator (EYELA, N-N series, Japan). The extracts were
72
further analyzed for their antioxidant potency through different

parameters like total phenolic contents, antioxidant activity and
free radical scavenging activity (DPPH assay).
Determination of total phenolic content: Total phenolic contents
(TPC) were measured according to Mustafa et al. 13. Firstly, 5 ml of
DMSO dissolved 5 mg of dried extracts of spices. Then, 0.5 ml of
the resulting aliquot was added to 1 ml of 50% Folin-Ciocalteau
reagent and incubated for 3 min at room temperature (20 - 25C).
Next, 3 ml of 1% Na2CO3 was added to the mixture, thoroughly
vortex-mixed and incubated for further 30 min. Absorbance of the
mixture was read at 760 nm, using a spectrophotometer (Thermo
Scientific Genesys 20, USA). Results were expressed as mg of
gallic acid equivalents per 100 g of sample (mg GAE/g).
-carotene bleaching assay: The -carotene bleaching assay that

indicates the antioxidant activity based on coupled oxidation of
-carotene and linoleic acid following the procedures was outlined
by Taga et al. 14. For the purpose, 5.0 mg of -carotene was
dissolved in 50 ml of chloroform. Later, linoleic acid (40 mg) and
Tween 20 (400 mg) were added and solvent was removed using
nitrogen gas. The oxidative changes in -carotene emulsion were
measured using spectrophotometer (absorbance at 470 nm) at
specific time intervals. The degradation rate of the extracts was
calculated according to first order kinetics antioxidant activity
(AA) expressed as % inhibition relative to the control.
DPPH free radicals scavenging assay: The DPPH assay was
carried out using the method described by Prabhasankar et al. 15
with slight modification. Briefly, 0.10 mM of DPPH was dissolved
in 100 ml of 99.9% ethanol. Stock solutions of freeze-dried extracts
(0.5 g) were dissolved in 100 ml distilled water on the day before
analysis to allow the extract to be finely dissolved. Serial dilutions
with varying concentrations (50, 100, 500, 1000, 3000 and 5000
ppm) were made from the stock solution of each sample by
adjusting the volume up to 10 ml using volumetric flasks with
distilled water. A 2 ml DPPH solution was mixed with 2 ml of each
dilution in the test tubes and shaken well for at least 15 s. Finally,
2 ml of 99.9% ethanol was mixed with 2 ml of DPPH solution (used
as blank) and test tubes were kept in the dark for 1 hour. The
absorbance of blank, control and all the samples were taken at 517
nm. The scavenging effect (%) was calculated according to the
following equation:
% Inhibition DPPH = (AbsDPPH Abssample) x 100
AbsDPPH
AbsDPPH is the absorbance of the DPPH solution without extracts.

Abssample is the absorbance of sample solution.
Determination of antimicrobial potential of extracts:
Antimicrobial potential of medicinal plant extracts was evaluated
against the bacterial strains, i.e. E. coli, Salmonella,
Campylobacter, Listeria, coliforms, Clostridium and
Staphylococcus, and fungi like Candida. The microorganisms
were isolated and identified by the method described by Koneman
et al. 16. The results are not mentioned in the research project as
this work was carried out in collaboration with Dr. Atif Nisar
Ahmad. Later, the minimum inhibitory concentrations (MIC) of
medicinal plants against these bacterial genera were determined

and mentioned as under. Briefly, isolated strains were cultured on
their specific media, i.e. MacConkey agar (E. coli), SalmonellaShigella agar (Salmonella and Shigella spp.), blood agar
(Pseudomonas, Listeria and Streptococcus spp.), mannitol salt
agar (Staphylococcus spp.), modified charcoal cefeperazone
deoxycolate agar (Campylobacter spp.), different reinforced
Clostridium medium (Clostridium spp.), Sabouraud agar (Candida
spp.). The agar disc diffusion method was used to determine the
antimicrobial activity. Sterile discs (6 mm, Hi-media, India) were
loaded with 50 l of (30 mg/ml) cumin, turmeric and caraway powder
extract dissolved in 5% dimethyl sulfoxide (DMSO). Bacterial
suspensions were also diluted to match the 0.5 McFarland
standard scales (approximately 1.5 x 108 CFU/ml). Further, MllerHinton agar (MHA) was poured into Petri dishes to give a solid
plate and inoculated with 100 l of suspension containing 1.5 x 1
08 CFU/ml of bacteria. The plates were incubated at 37C for 24 to
36 h and inhibition zones diameter around each of the discs was
measured and recorded. Minimum inhibition concentrations of
the plant extracts was tested by the checkerboard assay method.
Statistical analysis: The data presented in the paper is the mean
and standard deviation and whole experiment was carried out in
triplicates. The dried spices were further compared through
analysis of variance technique to determine the level of
significance 29. The means were compared through Least
Significance Difference (LSD) test using Statistical Analysis
System (SAS Institute, Cary, NC) version 9.1 and Microsoft Excel
2007.
Results
The mean values regarding moisture contents indicated that
moisture contents varied from 7.91 0.15 to 11.80 0.57% (Table
1). The maximum moisture contents were recorded in turmeric,
while minimum was recorded in cumin seed (7.91 0.15%). The
protein contents in different plants varied significantly and
maximum protein contents of 21.01 1.12% were recorded in
caraway seeds followed by cumin seeds (19.29 1.04%), whilst
minimum protein contents of 7.54 0.05% were observed in
turmeric. Crude fat in cumin seeds (24.90 0.57%) was higher
than in caraway (15.69 0.28%) and turmeric (9.92 0.57%). Cumin
contains the highest amount of ash contents (7.16 0.39%) and
turmeric ranked at the bottom (5.67 0.21%). Caraway and turmeric
contain higher amount of fiber, i.e. 39.83 1.94 and 21.52 0.44%,
respectively. In comparison, nitrogen free extract (NFE) was higher
in turmeric (43.87 1.41%) followed by cumin (31.70 0.21%). The
mean values regarding sugars indicated the total sugars ranged
from 0.62 0.02% to 2.93 0.08% with the maximum in turmeric
and the minimum in caraway 0.62 0.02%. Vitamin C content was
recorded highest in turmeric (24.31 0.45 mg/100 g) followed by
caraway (19.33 0.21 mg/100 g), however, cumin was ranked at
the bottom regarding this trait (Table 1).
The mean values regarding calcium content (Table 2) indicated
that the values for the said trait ranged from 202.45 7.09 to
1004.38 27.58 mg/100 g. The maximum calcium and magnesium
contents were observed in cumin followed by caraway whereas
the minimum was found in turmeric. Overall, phosphorus content
ranged from 270.21 9.12 to 645.56 7.35 mg/100 g. The maximum
phosphorus content was observed in caraway (645.56 7.35 mg/
Table 1. Means for proximate composition of dried cumin,

turmeric and caraway powder.
Selected dried spices
Cumin
Turmeric
Caraway
Moisture (%)
7.91 0.15 11.80 0.57 10.34 0.05
Protein (%)
19.29 1.04 7.54 0.05 21.01 1.12
Fat (%)
24.90 0.57 9.92 0.57 15.69 0.28
Ash (%)
7.16 0.39 5.67 0.21
5.68 0.32
Crude fiber (%)
11.21 0.14 21.52 0.44 39.83 1.94
NFE (%)
31.70 0.21 43.87 1.41 8.07 0.42
Total sugars (%)
2.38 0.07 2.93 0.08
0.62 0.02
Vitamin C (mg/100 g) 8.33 0.04 24.31 0.45 19.33 0.21
Parameter
*NFE = Nitrogen free extract (It represents the carbohydrate fraction.)
100 g) followed by cumin (511.00 11.04 mg/100 g). Mean values

regarding potassium content explicated that it ranged from 1332.57
27.54 to 2574.28 55.10 mg/100 g. The maximum potassium
contents were observed in turmeric, whereas the minimum
potassium contents were recorded in caraway. The sodium
contents varied significantly and the highest sodium contents
were observed in cumin (172.93 4.09 mg/100 g). The turmeric
(34.50 1.51 mg/100 g) and caraway (17.48 0.82 mg/100 g) were
down in the ladder (Table 2).
Table 2. Means for macromineral profile (mg/100 g) of dried
cumin, turmeric and caraway powder.
Cumin
Turmeric
Caraway
Calcium (Ca)
1004.38 27.58 202.45 7.09
767.25 3.36
Magnesium (Mg) 406.15 16.45
201.49 6.75
235.16 12.26
Phosphorus (P)
511.00 11.04
270.21 9.12
645.56 7.35
Potassium (K)
1607.71 93.80 2574.28 55.10 1332.57 27.54
Sodium (Na)
172.93 4.09
34.50 1.51
17.48 0.82
Parameter
The results regarding microminerals indicated that spices contain

appreciable amounts but differed significantly from each other
(Table 3). The maximum iron contents were examined in cumin
(60.60 3.71 mg/100 g) and caraway was ranked in the last (15.62
0.50 mg/100 g). Zinc contents were ranged from 4.66 0.22 to
5.29 0.11 mg/100 g in different plants with the maximum in caraway
(5.29 0.11 mg/100 g) and the minimum in turmeric (4.66 0.22 mg/
100 g). Mean values regarding copper depicted that cumin seeds
contain the maximum copper contents (0.92 0.03 mg/100 g), while
the minimum contents were recorded in turmeric (0.66 0.04 mg/
100 g). The mean values regarding manganese varied from 1.30
0.06 to 8.02 0.35 mg/100 g. It is important to write for the readers
that all plants were devoid of fluorides.
Table 3. Means for micromineral profile (mg/100 g) of
dried cumin, turmeric and caraway powder.
Cumin
Turmeric
Caraway
Iron (Fe)
60.60 3.71 44.98 2.07 15.62 0.50
Zinc (Zn)
4.98 0.27 4.66 0.22
5.29 0.11
Copper (Cu)
0.92 0.03 0.66 0.04
0.85 0.04
Manganese (Mn) 3.16 0.11 8.02 0.35
1.30 0.06
Parameter
The statistical results regarding TPP indicated that the plants

differed significantly (Table 4). The means indicated that the TPP
contents varied from 329.26 6.41 to 1016.72 63.68 mg GAE/100
g. The maximum amount of TPP was observed in caraway while
least amounts were recorded in cumin. The DPPH inhibition as
perceived through the statistical analysis was significant and all
plants were effective in this respite, however, the percentage varied
73
Table 4. Means for antioxidant potential of dried cumin, turmeric

and caraway powder.
free extract (NFE) Moreover, vitamin C contents were recorded

higher in turmeric and caraway seeds. The results regarding
proximate components were quite conclusive that the spices hold
Parameter
important nutritional value but their consumption in slight amounts
Cumin
Turmeric
Caraway
TPP (mg GAE/100 g)
329.26 6.41 980.20 47.18 1016.72 63.68 results in their lower worth as energy source. The results regarding
DPPH (% inhibition)
39.01 2.11
47.26 0.32
57.71 0.77
proximate constituents and mineral contents are in agreement with
-carotene (% inhibition) 38.96 0.89
36.17 2.09
47.65 0.74
the scientific literature available. In this regard, Ghosh-Das and
TPP (total polyphenols), GAE: Gallic acid equivalent, DPPH Diphenyl picryl hydrazine
Savage 17 reported that turmeric contains abundance of medicinally
with each plant. The results regarding percent DPPH inhibition, active chemicals. The chemical constituents present in turmeric
the caraway exhibited maximum DPPH inhibition (57.71 0.77%) holds significant antioxidant capabilities. Curcumin as major
followed by turmeric (47.26 0.32%). However, least DPPH ingredient of turmeric reported to be effective antioxidant and
inhibition was recorded in cumin (39.01 2.11%). The percentage helps to fight cancer insurgence and inflammation 8.
Spices are the building blocks of flavor in foods and the presence
inhibition of free radicals through -carotene bleaching method
indicated that antioxidant activity of the plants varied. The means of polyphenols in them is positively correlated with their
regarding the said trait depicted that cumin and turmeric inhibited antioxidant potential that may include DPPH inhibition and lipid peroxidation non-significantly with mean inhibition of 38.96 carotene bleaching assay 18. Various scientists studied the
0.89% and 36.17 2.09%, respectively, whilst the caraway was nutritional profile and antioxidant potential of cumin and overall it
most effective in reducing the extent of lipid peroxidation, i.e. 47.65 can be observed that cumin contains bioactive components like
cymene, cuminlaldehyde, cuminal, -terpinene, -pinene, carveol,
0.74 %.
The results regarding antioxidant potential indicated that and myrtenal as major volatile components. Moreover, cumin
turmeric is most effective against all microorganisms tested in the contains 2.520.11% of essential oil that possesses DPPH
present study (Fig.1). The turmeric powder had MICs of 35.00 inhibition activities of 85.440.50% 2, 6. Hajlaoui et al. 19 also reported
1.77, 21.35 1.21, 41.18 1.01, 37.14 0.96, 32.05 1.18, 38.01 the antioxidant potential in various assay, i.e. -carotene bleaching
1.39 and 42.54 1.56 mm against E. coli, Salmonella, test and DPPH inhibition. Recently, Kedia et al. 2 reported strong
Campylobacter, Listeria, Clostridium, Staphylococcus, and antioxidant potential of cumin essential oil with mean IC50 value of
Candida, respectively. The caraway ranked second but cumin 0.092 l/ml. Lekshmi et al. 20 reported that the antioxidant capacity
seed powder showed minimum MICs for the tested of turmerin (a bioactive compound present in turmeric) is of
significant importance as it inhibits DPPH, superoxide, and ABTS
microorganisms.
radicals with mean IC50 values of 29, 48, and 83 g/ml, respectively.
Caraway seeds essential oil and its bioactive components are
Discussion
The bioactive compounds present in spices make them an ideal effective antioxidants and Kapoor et al. 21 showed their strong
candidate for the extraction of natural antioxidants and antioxidant activity that can be slightly comparable with BHA and
antimicrobial agents and thus can also be effective in lowering BHT. The same authors claimed antioxidant activity through
the risk of various maladies. Amongst different spices, cumin, various assay DPPH assay, -carotene bleaching assay, and total
caraway, and turmeric are important in Asian cuisines and are polyphenols. Samojlik et al. 7 reported that essential oil extracted
globally recognized as condiment and flavor enhancer or taste from caraway seeds inhibits lipid peroxidation and DPPH free
modifier 10. In the present research, sun-dried cumin, caraway, and radical with mean IC50 value of 2.5 l/ml. De Martino et al. 22 reported
turmeric powders were analyzed for their basic proximate and that essential oil of caraway seeds exhibit significant antioxidant
mineral composition, antioxidant potential, and antimicrobial activity owing to the presence of several bioactive compounds
perspectives. The results indicated that the cumin and caraway like anethole, carvacrol, and estragol. Curcumin
contain appreciable amounts of protein and fat. Fiber contents (diferuloylmethane), golden color pigment, is main constitute of
were higher in turmeric and caraway, while turmeric contains turmeric that holds antioxidant and antimicrobial potential 9.
Antimicrobial agents are important to preserve the food products
significant amounts of carbohydrates as indicated from nitrogen
from spoilage. Moreover, some of them are of
Cumin
Caraway
Turmeric
45
significant value for curing infectious disorders.
Kedia et al. 2 reported that cumin essential oil
40
can inhibit aflatoxin production @ 0.6 and 0.5 l/
35
ml, concentrations. Although, cumin seed
30
powder was least effective in inhibiting the growth
25
of microorganisms but several researchers reported
20
its effectiveness against Aspergilus flavus,
Aspergillus niger, Bacillus subtilis, Staphylococcus
15
epidermidis, Saccharomyces cerevisiae and
10
Candida albicans23. Like cumin, turmeric holds
5
several health benefits and its incorporation in
0
different products have been tested, e.g. bread 24
E. coli
Salmonella Campylobacter Listeria
Clostridium Staphylococcus Candida
to enhance the well being of the individuals.
Species
Turmeric and its active ingredients are effective
Figure 1. Minimum inhibitory concentration (mm) of selected spices against various
in inhibiting the growth of Aspergillus niger,
isolated mictobial strains.
Caraway
Caraway
Turmeric
Turmeric
Minimum inhibitory concentration (mm)
Cumin
Cumin
74
Fusarium moniliformes, S. aureus, Bacillus subtilis and

Fusarium oxysporium 25, 26. Caraway seeds are also effective in
reducing the growth of Staphylococcus aureus, Saccharomyces
cerevisiae and Aspergillus niger in different food products 27.
Previously, De Martino et al. 22 also reported that essential oil of
caraway seed is effective in inhibiting the growth of Bacillus
cereus, Pseudomonas aeruginosa, Escherichia coli and
Penicillium citrinum. Caraway seeds are effective in enhancing
the efficiency of drug to control pathogenic microorganisms 28.
In the nutshell, it can be claimed that spices and their bioactive
components are important in human nutrition as they act as
antioxidants and scavenge free radicals along with acting as
antimicrobial agents to inhibit the growth of pathogenic
microorganisms 4.
Conclusions
The spices evaluated in the present research showed significant
antioxidant activity and all of them could be further explored for
their potential application. Similar to antioxidant potential, the
antimicrobial activities of all spices were of significant importance
and should be tested at industrial level. In the nutshell, the
antioxidant and antimicrobial potential of the selected spices is
of commercial significance, however, safety assessment through
animal modelling is essential for warranting them commercial
antioxidant and antimicrobial agents.
Acknowledgements
Corresponding author is thankful to Directorate of Research,
Bahauddin Zakariya University, Multan, Pakistan, for providing
essential funds for running this project. Moreover, the authors
are thankful to the National Institute of Food Science and
Technology, University of Agriculture, Faisalabad, for providing
instrumental facilities to conduct the experiments regarding
antioxidant potential of selected spices.
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www.world-food.net
Inhibitory effect of gamma radiation in degrading and preventing fungal toxins

Amira Hassan Abdullah Al-Abdalall
Department of Biology, Faculty of Science, Dammam University El-Dammam, P. O. Box 838, Dammam 31113,
Kingdom of Saudi Arabia. e-mail: dr2000amira@hotmail.com
Abstract
The aim was to study the effect of ionizing radiation on prevention of fungal growth and degradation of toxins in food materials. Ffungal strains
(Aspergillus niger, A. alliaceus, A . melleus, A. flavus and Fusarium solani) used in this study were isolated from coffee beans. Fungi differed in their
response to different doses of gamma radiation on a decline in the ability to produce biological active materials. Treatment with 3 kGy stopped
production of these materials. F. solani did not lose completely the ability to produce these materials even when using a dose of 10 kGy. Boiling
culture filtrates led to the blocking of active biological materials, or did reduce the inhibitory of bacterial growth. In all the fungi tested in liquid media
and even in the control one, ochratoxin A did not appear, and when exposed to 10 kGy, the fungal cells died and were not able to grow and produce
mycotoxins. On the other hand, the ability to production of aflatoxin by F. solani was increased after treatment with 1 and 3 kGy and decreased
thereafter. Irradiation of culture filtrates with gamma rays may have significantly reduced the inhibitory activity of the growth of bacteria B. subtilis.
The increase of inhibition was affected positively with the dose, and the effect was different with different fungal strains.
Key words: Aspergillus, ochratoxin, aflatoxin, gamma radiation, B. subtilis.
Introduction
Some fungal species belonging to genera Penicillium and
Aspergillus are toxin-producing organisms as food contaminants.
They are leading to health problems for human and animals and
economic loss. Some species of genus Aspergillus are natural
contaminants to medicinal plants and the effect of gamma
radiation on fungal toxin production was studied 1 . Van Dyck et
al. 2 proved that presence of water accelerates destruction of
aflatoxin when gamma radiation is used. Radiation in the presence
of water produces free radicals, which may destroy AFB1 in the
furan ring recycling in products of low biological activities. The
decrease of AFB1 activity in aqueous solution was 34, 44, 47 and
100% after exposure to -radiation doses of 2.5, 5, 10 and 20 kGy,
respectively. Aziz and Abd El-Aal 3 found that -radiation at 10
kGy can stop toxication of coffee seeds and some foodstuffs with
aflatoxin. Gamma-radiation lowered contamination of maize seeds
97.6% at 2 kGy and to 94% at 5 kGy. AFB1 decreased by 68.9 %
using -dose of 2 kGy, 46% at 5 kGy, and at 10 kGy, there was a
complete inhibition of AFB1and AFB2. Aziz et al. 4 studied the
effect of -radiation and maize fats on aflatoxin production by
A.flavus in contaminated maize and found a reversible relationship
between growth of the fungus and amount of -rays. At 3 kGy,
complete inhibition of aflatoxin B1 was recorded, even after storage
of maize until 45 day. Aflatoxin B1 production was activated at 0.1
and 1.5 kGy radiation dosage.
The fungal flora in fruits was sensitive to -radiation, complete
sterilization was obtained at 5 kGy, and there was a reduction of
AFB1 toxins from 380-500 to 20 mg/kg at 20 kGy 5. Complete
degradation of aflatoxin B was found after exposure to 20 kGy radiation 6.
Refai el al. 7 could isolate fungal colonies of about 103-106 CFU/

g in summer from Egyptian pastrami, which is made of meat with
salt and a layer of pepper, garlic, fenugreek and cumin, while in
winter they could isolate 102-105 CFU/g. Then 2.8 - 47 mg/kg of
aflatoxins was recorded from meat, but using 5 kGy radiation led
to complete inhibition of toxin production.
Aziz and Mahrous 8 studied the effect of -rays on aflatoxin
production by A. flavus and the chemical composition of wheat,
common beans and soy beans. They recorded increase in protein
content and decrease in lipids and carbohydrates after infection
by A. flavus. On the other hand, using -radiation at 5 kGy led to
reduction in fungal toxin production.
Kim et al. 9 and Wi et al. 10 proved that exposure to ionic radiation produce free radicals within molecules leading to changes
in shape, anatomy, and biochemical reactions within the plants.
Changes also included cell metabolism as expansion of membranes
and disorders of photosynthesis and accumulation of phenolic
compounds. Aquino et al. 11 mentioned that aflatoxins B1 and B2
decreased to levels that cannot be detected after treatment at 10
kGy.
Seed contamination by spores and fungi from the storage
environment would affect the quality and nutrient contents of
seeds. So, irradiation proved to be effective in crop and seed
production 12.
Irradiation of biological material to keep it from contamination
is important in marketing. Irradiation at 40 kGy kept coffee seeds
sterile from contaminants 13. Also, Chelack et al. 14 and Sajilata
and Singhal 15 recommended irradiation of foodstuffs with the
ionizing radiations.
77
The aim was to study the effect of ionizing radiation on

prevention of fungal growth and degradation of toxins in food
materials.
Test pathogens: The microorganisms (Aspergillus niger, A.
alliaceus, A . melleus, A. flavus and Fusarium solani) used in this
study were isolated from coffee beans. The developing fungal
colonies were identified up to the species level by microscopic
examination according to the keys of several researchers 16-19.
Source of gamma irradiation: The source of irradiation used for
the tested fungal species was Cobalt- 60 gamma Atomic Energy
Research Institute of King Abdulaziz City for Science and
Technology (KACST).
Exposure of the spores of the tested fungal species to gamma
radiation and radiosensitivity test of fungi: Fungi growth from
pure 14 days old culture of each of the tested fungal species were
irradiated by gamma radiation at dose levels of 1, 3, 5, 7 and 10
kGy. Three replicates were used for each dose as well as for the
control (non-irradiated). One cm discs of fungal growth in
irradiated and non-irradiated spore suspension were plated on
Potato Dextrose agar medium in a Petri dish. The plates were
incubated at 27C for 14 days. One-cm discs of fungal species
were used to inoculate yeast extract-glucose medium, incubated
at 25C for 14 day, with 3 replicates, followed by filtration. The
filtrate was incubated in a water bath at 100C for 20 min followed
by centrifugation at 300 rpm for half an hour. The inhibitory effect
of the filtrate on B. subtilis growth was measured. Tubes were
incubated in a water bath at 100C for half an hour to degrade the
active biologically substances. B. subtilis was used for bioassays.
Mycotoxin analysis:
Apparatus: The High Performance Liquid Chromatography (HPLC)
system consisted of Waters Binary pump Model 1525, a Model
Waters 1500 Rheodyne manual injector, a Waters 2475 MultiWavelength Fluorescence Detector, and a data workstation with
software Breeze 2. A phenomenex C18 (250 x 4.6 mm i.d), 5 m from
Waters corporation (USA) for aflatoxins. A HyperClone 5 ODS
column (C18) 120 , DIM: 250 x 4.60 mm (Phenomenex).
Extraction of aflatoxins by VICAM 20 method: Twenty-five g
sample with 5 g sodium chloride was weighed and placed in a
blender jar. Then, 125 ml of methanol: water (70:30) was added,
covered and blended at high speed for 1 min. Cover was removed
from the jar and the extract poured into fluted filter paper. Filtrate
was collected in a clean vessel. Then 15 ml filtered extract was
poured into a clean vessel. Extract was diluted with 30 ml of purified
water and mixed well. Diluted extract was passed through the
glass microfiber filter into a glass syringe barrel using markings
on barrel to measure 4 ml.
Immunoaffinity chromatography: Fifteen ml of filtered diluted
extract (15 ml = 1 g sample equivalent) was passed completely
through AflaTest -P affinity column at a rate of about 1-2 drops/
s until the air comes through the column. Five ml of purified water
was passed through the column at a rate of about 2 drops/s.
Affinity column was eluted by passing 1.0 ml HPLC grade methanol
78
through the column at a rate of 1-2 drops/s and collecting all of

the sample eluate (1 ml) in a glass vial. Eluate was evaporated to
dryness under a stream of nitrogen and sample was determined
by HPLC.
Detection and determination of aflatoxins by HPLC:
Derivatization: The derivatives of samples and standard were
done as follow:100 l of trifluoracetic acid (TFA) was added to
samples and mixed well for 30 s and the mixture stand for 15 min.
900 l of water: acetonitrile (9:1 v/v) was added and mixed well by
vortex for 30 s and the mixture was used for HPLC analysis.
HPLC conditions: The mobile phase consists of acetonitile/water/
methanol (1:6:3). The separation was performed at ambient
temperature at a flow rate of 1.0 ml/min. The injection volume was
20 l for both standard solutions and sample extracts. The
fluorescence detector was operated at an excitation wavelength
of 365 nm and an emission wavelength of 450 nm. AFB 1
concentration in samples was determined from the standard curve,
using peak area for quantification.
Ochratoxin analysis:
HPLC equipment: The HPLC system consisted of Waters Binary
pump Model 1525, a Model Waters 1500 Rheodyne manual injector,
a Watres 2475 Multi-Wavelength Fluorescence Detector, and a
data workstation with software Breeze 2.
Chemicals and reagents: OTA standard, Chartist, microfiber filter
1.5 m, and filter papers were purchased from VICAM, Milford,
MA USA. Acetonitrile, glacial acetic acid HPLC grade were
obtained from BDH, England. Sodium chloride, sodium bicarbonate,
sodium hydrogen phosphate, potassium dihydrogen phosphate
and potassium chloride were purchased from (BDH, Merck
chemicals). Tween -20 was obtained from Sigma (St. Louis, MO,
USA).
HPLC condition: A Symmetry C18 (5 m particle size, 150 mm x 4.6
mm i.d.) from the Waters Corporation (USA) was used along with
a mobile phase of acetonitile/water/ acetic acid (55:43:2). The
separation was performed at ambient temperature at a flow rate of
1.0 ml/min. The injection volume was 50 l for both standard
solutions and sample extracts. The fluorescence detector was
operated at an excitation wavelength of 330 nm and an emission
wavelength of 470 nm. OTA concentrations in coffee extracts were
determined from the standard curve, using peak area for
quantitation.
Statistical analysis: The results obtained in this research were
analysed statistically using the 16th version of SPSS16 program
where transaction averages were compared at the abstract level
(0.05) using the least significant difference test (LSD) designed
by Norusis 21.
Results
Inhibitory effect of -radiations on fungal toxins: Table 1 gives
the effect of -irradiation on fungal toxin production. A. melleus,
A. flavus and A. niger lost ability to produce biologically active
toxins in variable degrees after exposure to 3 kGy, although mycelia
growth was realized. Using 1 kGy reduced toxin production of A.
melleus by 34.19%, A. flavus by 36.09%, and A.alliaceus by 3.69%.

F. solani did not lose its ability for toxin production even after
exposure to 10 kGy. However, rate of production decreased after
using 1, 3, 5, 7 and 10 kGy, reduction rates were 15.16, 87.31, 96.35
and 75.62%. Boiling of the filtrate after irradiation leads to complete
degradation of fungal toxins at 10 kGy. Aspergillus showed inability
to inhibit bacterial growth after boiling of its filtrate.
Quantitative and qualitative estimates of fungal toxins after
irradiation: Biological effect of fungal filtrate was proved by
B.subtilis growth inhibition. Effects of rays on these substances
are in Table 2. Fungal filtrate of A.niger proved to concentration
8.086 mg/l of G 1 and B1 aflatoxin. After exposure to 1 kGy, G1 toxin
disappeared, while B1 decreased to 1.279 mg/l. Exposure of
A.alliaceus to 1 kGy led to increase in toxin production, from 0.45
mg/l, in control experiment, to 0.616 mg/l. The same finding was
recorded to A. melleus, concentration of all toxins changed after
exposure from 1.255 to 1.275 mg/l. B1 toxin increased with
disappearance of G1.
No fungal filtrates showed ochratoxin production and exposure
to 10 kGy led to death of cells. Table 3 shows that F. solani
production of aflatoxin decreased after exposure to 1 kGy from
1.635 to 0.189 mg/l, but by exposure to 3 kGy toxin production
increased to 9.902 mg/l. Doses at 5, 7 and 10 kGy led to decrease in
toxin production to 0.868, 0.607 and 0.453 mg/l, respectively.
Effect of -rays on the production of biologically active
substances in fungal filtrate: Exposure to -rays affects the active
biological substances in fungal filtrate, which inhibits B.subtilis
growth. The inactivation was directly proportional with dose
increase, it also valued according to type of fungus. The rate of
inactivation reached 35.77% for A. niger, in comparison to control
experiment after exposure to 1 kGy. Inactivation reached 39.41,
45.66, 47.05 and 55.99% after exposure to 3, 5, 7, 10 kGy,
respectively. Exposure of A.alliaceus to 1, 3, 5, 7 and 10 kGy led to
inactivation percentages as 24.995, 34.69, 43.89, 49.75 and 52.70%,
respectively. This fungus was less affected than A.melleus in
Table 3. Effect of gamma rays on the production of aflatoxins

and ochratoxin A by Fusarium solani.
Dose level
(kGy)
Control
1
3
5
7
10
Average
L.S.D
B1
0.972
0.189
3.791
0.734
0.545
0.450
1.114
2.039
Aflatoxin conc. (g/ litre)

B2
G1
G2
0.203 0.460
ND
ND
ND
ND
ND
3.347 1.664
ND
0.084 0.050
ND
0.042 0.020
ND
ND
0.020
0.34 0.665 0.292
1
1.207 1.065
Total
1.635
0.189
8.802
0.868
0.607
0.453
2.092
1.542
Ochratoxin
ND
ND
ND
ND
ND
ND
-
decreasing inhibitory activity of its filtrate until exposure to 10

kGy, while the biological inhibitory substance in A.melleus filtrate
lost its biological inhibitory effect after exposure to 10 kGy followed
by A. alliaceus (1) which showed complete inhibition at 7 kGy,
while A. flavus (2) lost its inhibitory action after exposure to 10
kGy.
Discussion
Results proved the variable decrease in inhibitory action of fungi
after exposure to different doses of -rays. For A.melleus, A.flavus
and A.niger after exposure to 3 kGy stopped the inhibitory activity
and toxin production of these fungi although they can give
mycelia. Exposure to 1 kGy decreased the ability for inhibition of
bacterial growth. Similar finding was recorded by Afifi et al. 22 on
A. flavus, A. fumigatus, A. niger and P. expansum exposed to 0.5,
1, 1.5 and 2 kGy. The dose of 0.5 kGy stimulated toxin production
but 1 and 2 kGy decreased the aflatoxin formation. Although the
doses affected the fungi, no damage was recorded on apple fruits.
The ionizing effect of -rays, leaving free redicals with the
molecules, was explained by Schmidt-Heydt et al. 23 and Boonchoo
et al. 24. These radicals react and do changes in plant morphology,
anatomy and biochemical properties depending on the irradiation
dose. Changes may include in photosynthesis balance,
antioxidant system and accumulation of phenolic compounds.
For F. solani exposure to rays decreased its ability for producing
Table 1. Effect of gamma rays on the production of aflatoxins and ochratoxin A by tested fungi.
Tested
fungi
A. niger
A. alliaceus
A. flavus
A. melleus
F. solani
Average
L.S.D #
Control
41.86
40.37
38.67
33.88
29.61
36.82
16.45
kGy1
33.33
-20.03
41.86
+3.69
25.45
-34.19
21.66
36.09
25.12
-15.16
29.48
8.11
Before boiling
kGy3 kGy5
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
20.43 18.99
-31
-35.87
4.09
3.80
1
1
kGy7
0
-100
0
-100
0
-100
0
-100
10.08
-65.96
2.02
1
kGy10
0
-100
0
-100
0
-100
0
-100
7.22
-75.62
1.44
1
Control
29.39
-%
35.78
-%
5.995
-%
3.52
-%
7.92
-%
16.52
2.48
kGy1
22.63
-23
32.5
-9.17
4.33
-27.78
0
-100
7.91
-0.13
13.47
2.21
After boiling
kGy3
kGy5
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
5.37
4.52
-32.20 -42.93
1.07
0.90
1
1
kGy7
0
-100
0
-100
0
-100
0
-100
0
-100
-
kGy10
0
-100
0
-100
0
-100
0
-100
0
-100
-
Table 2. Quantitative and qualitative of aflatoxins and ochratoxin A in fungal filtrates before and after irradiation.
Tested
fungi
A. niger
A. alliaceus
A. flavus
A. melleus
Average
L.S.D #
B1
4.739
0.450
0.758
0.806
1.688
1.66
B2
ND
ND
0.094
0.140
0.059
1.67
Control
G1
G2
3.347
ND
ND
ND
0.251 0.126
0.209 0.100
0.952 0.057
1.19
1.71
Total
8.086
0.450
1.229
1.255
2.757
1.54
OTA
ND
ND
ND
ND
-
B1
1.279
0.616
0.569
1.161
0.906
4.95
B2
ND
ND
ND
0.109
0.027
1
1 kGy
G1
G2
ND
ND
ND
ND
ND 0.050
ND 0.005
0.014
1.13
Total
1.279
0.616
0.619
1.275
0.947
4.98
OTA
ND
ND
ND
ND
-
B1
ND
ND
ND
ND
-
B2
ND
ND
ND
ND
-
10 kGy
G1 G2
ND ND
ND ND
ND ND
ND ND
-
Total
ND
ND
ND
ND
-
OTA
ND
ND
ND
ND
-
79
Table 4. Effect of gamma rays on the active biological substance

in the filtrates of fungi.
Tested fungi
A. niger
A. alliaceus (1)
A. flavus (1)
A. alliaceus (2)
A. flavus (2)
A. melleus (1)
A. melleus (2)
Average
L.S.D #
Control
14.13
-%
49.01
-%
49.01
-%
19.29
-%
29.54
-%
34.90
-%
20.96
-%
34.83
7.51
Inhibition zone (cm2)

kGy1
kGy3
kGy5
26.42
24.92
22.35
-35.77 -39.41 -45.66
36.76
32.01
27.5
-24.995 -34.69 -43.89
27.99
23.04
19.78
-42.89 -52.99 -59.64
10.28
8.38
8.04
-46.71 -56.56 -58.32
11.67
3.98
3.69
-60.49 -86.53 -87.51
0
0
0
-100
-100
-100
12.36
4.65
3.98
-41.03 -77.82 -81.01
17.93
13.85
12.19
3.72
2.93
2.99
kGy7
21.78
-47.05
24.63
-49.75
16.91
-65.50
0
-100
0.695
-97.65
0
-100
3.41
-83.73
9.63
2.31
kGy10
18.10
-55.99
23.18
-52.70
8.81
-82.02
0
-100
0
-100
0
-100
3.35
-84.02
7.63
2.12
biologically active materials, but did not stop it completely even

after exposure to 10 kGy. After exposed the culture filtrate to the
-radiation at 7 and10 kGy. and then boiled it, the mycotoxins
were degradated. The same finding was recorded for filtrate of
Aspergillus.
Control filtrates, without exposure, showed high concentrations
of aflatoxins. These toxins degraded in various degrees after
exposure to 1 kGy. G 1 aflatoxin was highly affected by radiation,
but in case of A.alliaceus, the dosage of 1 kGy stimulated toxin
formation.
In all tested fungi, ochratoxin was not produced even in the
control experiment.
Exposure to 10 kGy led to death of fungal cells. Toxin production
of F.solani increased after exposure to irradiation although this
strain is known not to produce aflatoxin as mentioned by Mokobia
and Anomohanran 25, who could isolate F. kyushuence strain
capable to produce aflatoxin G1 and B1. They used
chromatographic and gene sequence techniques to prove the
sequence of aflatoxin encoded genes which appeared similar to
that of A. flavus. Comparing results of specific microarray covering
oligonucleotide sequencing, they realized similarity in sequence
of toxin nucleotides of A. flavus and F. kynshuense. They also
mentioned that Fusarium fungus had low expression of the toxin
trichothecene in the liquid media. High expression appeared in
solid media. It seems that, the media used is an important factor in
secondary metabolism.
This study proved that pasteurization of coffee seeds using
doses of -rays can sterilize these seeds from any fungal infection.
The recommended doses are 3, 5, 7 and 10 kGy. We could not
isolate any fungal growth even after one month storage period.
Results also showed that at 1 kGy, infection decreased between
Herrary, Habashy, Lokmaty and wild tested types. These findings
are in agreement with Nemtanu et al. 13, who could stop
contamination of green coffee seeds after exposure to 40 kGy
without any damage of seed constituents or its antioxidants. This
technology was applied on malt plant 14, 24, and on rice, food and
drugs 25.
Sterilization using -rays with doses 1-10 kGy is recommended
by FAO, IAEA and WHO 26, 27. Gunckel and Sparrow 28 cleared that
exposure to -rays may affect plant physiology, biochemistry and
morphology. Several reseachers 29-31 mentioned that exposure to
80
low doses of -rays might stimulate growth.

Conclusions
The fungi differed in their response to different doses of gamma
radiation, and a decline in the ability of fungi on the production of
mycotoxins. In all the fungi exposed to a dose of 10 kGy. the
fungal cells died and were not able to grow and produce
mycotoxins, with the exception of Fusarium solani with increased
toxin production. The increase of inhibition is affected positively
with the dose that was used, and in addition it is affected differently
with the different fungal strains used.
Recommendations
This study proved in habitation of fungal toxins after exposure to
-rays. We recommend using this type of irradiation to sterilize
our food, but after more detailed studies about the fate of
degraded.
Acknowledgements
The author deeply thanks for the expert technical assistance of
Atomic Energy Research Institute of King Abdulaziz City for
Science and Technology (KACST).
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81
WFL Publisher
Helsinki, Finland
www.world-food.net
The in vitro antibiofilm activity of Rosmarinus officinalis L. essential oil against

multiple antibiotic resistant Pseudomonas sp. and Staphylococcus sp.
Ozgur Ceylan 1*, Aysel Uur 2, Nurdan Sara 3, Filiz Ozcan 3 and Tuba Baygar 3
Apiculture Program, Ula Ali Kocman Vocational School, Mugla Stk Koman University, Ula, Mugla, Turkey.
Department of Basic Sciences, Section of Medical Microbiology, Faculty of Dentistry, Gazi University, Emek, Ankara, Turkey.
3
Department of Biology, Faculty of Arts and Sciences, Mugla Stk Koman University, Kotekli, Mugla, Turkey.
*e-mail: ozgceylan@hotmail.com
1
Abstract
Rosmarinus officinalis (rosemary) is widely used as a flavouring agent for food and well known medicinally for its chemical composition. The aim of
this study was to investigate the antibiofilm activity of the essential oil from Rosmarinus officinalis against biofilm formation of Staphylococcus
aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus cereus and Bacillus subtilis. Essential oil was
obtained from the aerial parts of the plant by using a Clevenger apparatus for 4 h. The antibacterial activity of the obtained essential oil from
Rosmarinus officinalis was determined using disc diffusion technique, minimum inhibitory concentrations (MICs) and minimum bacterial concentration
(MBC). Staphylococcus sp. and Pseudomonas sp. were evaluated as the resistant microorganisms in the antibacterial assays. The antibiofilm effect
of MBC, MIC, MIC/2, MIC/4, MIC/8 and MIC/16 concentrations of Rosmarinus officinalis essential oil was assessed by the microplate biofilm
assay. The essential oil exhibited significant antibacterial activity against all tested bacteria. In contrast to the antibacterial activity, MBC and
subinhibitory concentrations of essential oil showed limited antibiofilm activity. MBC concentrations of essential oil attenuated the biofilm
formation at 60.76% and 74.7% for Staphylococcus aureus MU 47 and Pseudomonas aeruginosa MU 187, respectively. Direct observation by
scanning electron microscope (SEM) analysis further revealed an exact reduction for the bacterial biofilm formation in response to the effective
concentration. This study has demonstrated that the Rosmarinus officinalis essential oil may be considered as a potent agent for the prevention of
biofilm-related applications that are increasingly problematic in the food processing environments and medical industries.
Key words: Rosmarinus officinalis, antimicrobial, antibiofilm, Staphylococcus, Pseudomonas.
Introduction
Rosmarinus officinalis L. (rosemary) is a spice and medicinal herb
widely used around the world 1. The fresh and dried leaves are
frequently used in traditional Mediterranean cuisine as an additive.
They have a bitter, astringent taste, which complements a wide
variety of foods. A tisane can also be made from them. They are
extensively used in cooking, and a distinct mustard smell gives
off while they are burned, therefore, they often are used to flavor
foods while barbecuing 2. As medicinal plant rosemary belongs to
the pool of herbs, which probably more than others, lies at the
boundary between myth, superstition and traditional popular
usages, but at the same time, its efficacy is largely acknowledged,
being, in fact, listed in the official pharmacopoeia of several
countries 3. Historically, rosemary has been used as a medicinal
agent to treat renal colic and dysmenorrhea. It has also been used
to relieve symptoms caused by respiratory disorders and to
stimulate the growth of hair. Extracts of rosemary are used in
aromatherapy to treat anxiety-related conditions and to increase
alertness 4.
The latest research related with rosemary essential oil has mainly
focused on its antibacterial 1, 2, 5-18, antioxidant 18-27, antifungal 28, 29,
anticancer 30, 31 and antibiofilm properties 12-14, 32.
82
In many ecosystems, bacteria are growing in surfaces as a layer

that is called biofilm 33. Bacterial biofilms are associated with a
large number of infections. Biofilms are ubiquitous, for example in
dental plaque, endocarditis, lung infections, and infections related
to the use of medical devices, such as catheters and stents. Many
persistent and chronic bacterial infections are now thought to be
linked to biofilm formation, over 60% of all bacterial infections
have been estimated to involve biofilms 34. Also, outside the
medical field, biofilms cause a host of problems such as surface
fouling and blocking of equipment 35. Biofilm-dwelling bacteria
are particularly resistant to antibiotics, making it hard to eradicate
biofilm-associated infections. Earlier investigations on plants and
their active constituents have almost exclusively focused on their
effects on planktonic bacteria with little emphasis on the more
antimicrobial resistant pathogens and difficult to control biofilm
forms 32.
There are a number of studies focusing on the biological
activities of R. officinalis essential oil in recent years, but to our
knowledge, fewer comparative studies on antibiofilm activity
including against human clinical isolates have been reported. In
these studies, there are no study that reveals the antibiofilm activity
of test bacteria used in our study. The present study reports the
antimicrobial and antibiofilm activity of R. officinalis essential oil
against multi-antibiotic resistant Staphylococcus spp. and
Pseudomonas spp. that cause clinical problems with high biofilm
formation.
Plant material: Leaves and flowers of R. officinalis were collected
from Mugla, Turkey, in May-July 2012 and a voucher specimen
has been deposited in the Herbarium of Mugla Sitki Kocman
Univesity. Samples were air-dried at room temperature for 2-4 days.
Extraction of essential oil: One hundred gram of dried plant was
submitted to hydro-distillation for 4 h using a Clevenger apparatus.
Oil was recovered directly, using a micro-pipette from above the
distillate without adding any solvent, and stored in dark vials at
4C.
Bacterial strains and culture conditions: The antimicrobial
activity of the essential oil was individually tested against a group
of bacteria including Bacillus subtilis ATCC 6633, Bacillus cereus
RSKK 863, Staphylococcus aureus ATCC 25923, Pseudomonas
aeruginosa ATCC 29212 and Pseudomonas aeruginosa ATCC
27853. Five clinically relevant microorganisms (Staphylococcus aureus
MU 38, MU 40, MU 46, MU 47 and Staphylococcus epidermidis
MU 30) and three marine microorganisms (Pseudomonas
aeruginosa MU 187, MU 189 and Pseudomonas fluorescens MU
180) provided from Mugla Sitki Kocman University Culture
Collection (MUKK) were also studied.
The above-mentioned bacteria were cultured in nutrient broth
(NB) (Difco, USA) at 370.1C for 24-48 h except from P. aeruginosa
and P. fluorescens strains which were incubated at 300.1C for
18-24 h. Inocula were prepared by adjusting the turbidity of the
medium to match the 0.5 McFarland Standard Dilutions. The strains
were maintained in their appropriate agar slants at 4C throughout
the study and used as stock cultures.
Disc diffusion assay: The antibacterial activity was based on the
disc diffusion method 36 using a bacterial cell suspension whose
concentration was equilibrated to the 0.5 McFarland standard
dilutions. Of each bacterial suspension 0.1 ml was spread on a
Mueller-Hinton agar plate. Sterile 6 mm paper discs (Schleicher
and Schuell) were impregnated with 10 l of essential oil. The
discs were allowed to dry and were then placed on the inoculated
agar. The plates were incubated at appropriate temperature and
time for the microorganisms, as mentioned above. At the end of
the incubation periods, diameters of no-growth zones around the
disks were measured to the nearest 0.1 mm using vernier calipers.
The experiments were performed in triplicate.
Determination of minimum inhibitory concentration (MIC) and
minimum bactericide concentration (MBC): The inhibitory and
bactericidal activities of the rosemary essential oils were
determined by the tube dilution method 37. The MIC was defined
as the lowest antibiotic concentration that yielded no visible
growth. Mueller-Hinton Broth was used as the test medium and
the density of bacteria was 5105 colony-forming units (CFU)/ml.
Cell suspensions (100 l) were inoculated into the wells of 96-well
microtitre plates (Nunc F96 MKroWell plates; NunclonTM ,
Denmark) in the presence of essential oil with different final

concentrations (0.312-80 l/ml). The essential oil was dissolved in
DMSO (Sigma, USA) and serially diluted 2-fold in MHB to give
final concentrations. Negative controls (bacteria+MHB), positive
controls (bacteria+MHB+essential oil), vehicle controls
(bacteria+MHB+DMSO) and media controls (MHB) were
included. The inoculated microplates were incubated at 37C for
Staphylococcus spp., Bacillus spp. and at 30C for Pseudomonas
spp., for 24 h. All experiments were performed in triplicate.
The MBC was obtained by subculturing 100 l of the culture
from each tube, in which the MIC assay showing no apparent
growth, onto substance-free Mueller-Hinton agar plates. The
plates were incubated at 37C or 30C for 24 h and the MBC was
defined as the least concentration that produced subcultures
growing maximum five colonies on each plate.
Effect of essential oil on bacterial biofilm formation: The effect
of subinhibitory concentration of R. officinalis essential oil on
biofilm-forming ability of bacteria was tested with a microplate
biofilm assay 38. Bacterial strains were inoculated in 2-5 ml of
trypticase soy broth (TSB) and growed up to stationary phase.
Cultures diluted to 1:100 in TSB, and 100 l of each dilution was
pipetted to four wells in a sterile flat bottom micotiterplate. After
incubation at 37C for 48 h, the wells were washed with distilled
water twice to remove the planktonic bacteria. The remaining
bacteria were subsequently stained with 125 l of 0.1% crystal
violet solution (Sigma Chemical Co.) at room temperature. Wells
were washed once again to remove the crystal violet solution that
is not specifically staining the adherent bacteria. The plates were
air-dried and 200 l of 95% ethanol and 33% glacial acetic acid
(Sigma Chemical Co.) were added to each Gram-negative and Grampositive bacteria wells, respectively. Biofilm stains solubilized at
room temperature. After shaking and pipetting of wells, 125 l of
the solution from each well was transferred to a sterile tube and
the volume was made up to 1 ml with distilled water. Finally, optical
density of each well was measured at 550 nm wavelength (Thermo
Scientific Multiskan FC, Vantaa, Finland). Negative controls
(cells+TSB), positive control (cells+TSB+essential oil), vehicle
control (cells+TSB+DMSO), and media controls (TSB) were
included. Positive controls for essential oil of 0.312-80 l/ml were
prepared via serial dilution techniques.
Each strain was tested for biofilm formation in duplicate and the
assay was repeated three times. Replicate absorbance readings
for each concentration were averaged and the average of the media
control was subtracted. This value was divided by the mean
absorbance of the (cell+TSB) and multiplied by 100.
Scanning electron microscopy (SEM): To observe the biofilm

formation, glass coverslips were prepared for SEM observation.
Coverslips at size of 3 mm x 3 mm were placed in Eppendorf tubes
containing 1.5 ml TSB supplemented with 1% glucose and sterilized.
Then 300 l bacterial suspension and 200 l essential oil were
added to reach the final concentration of 0.078-80 l/ml. Eppendorf
tubes were incubated at 37C for Bacillus and Staphylococcus
and at 30C for Pseudomonas, for 48 h. Prior to imaging, the
bacteria were fixed and dehydrated. Briefly, the coverslips were
83
gently rinsed twice with 0.01 M PBS and then initially fixed by
2.5% glutaraldehyde at 4C for 2 h. The surfaces were washed
twice with 0.01 M PBS for 1 h. The coverslips were post-fixed with
0.1% osmium tetroxide for 1 h. The bacteria were dehydrated by
replacing the buffer with increasing concentrations of ethanol
(30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min for each.
After critical point drying and coating by gold sputter, samples
were examined with a scanning electron microscope (JEOL JSM7600F; JEOL Ltd., Tokyo, Japan).
Statistical analysis: Differences between groups were statistically
analyzed using analysis of variance (ANOVA). All experiments
were performed in triplicate.
The antimicrobial activity of R. officinalis essential oil was
evaluated in vitro against 13 microorganisms which are known to
cause human diseases. The measured inhibition zones and MIC/
MBC results of the rosemary essential oil against the test bacteria
are given in Table 1. Based on their MIC and MBC values obtained
from antimicrobial tests and the percentage inhibition of biofilm
formation against the test bacteria are given in Table 2. The
antibiofilm activity of different concentrations of R. officinalis
essential oil for Gram-positive and Gram-negative test bacteria is
shown in Figs. 1 and 2, respectively.
The results indicated that the R. officinalis essential oil showed

anti-bacterial activity mainly against the Gram-positive bacteria
(S. aureus and S. epidermidis), similar to Jiang et al. 2, Jordan et al. 10,
Okoh et al. 11, Zaouali et al. 18. The highest antibiofilm activity for
the Gram-positive test bacteria was determined against S. aureus
MU 47 with 1.25 l/ml essential oil concentration (MBC) with the
inhibition rate by 60.76%. S. aureus MU 47 biofilm formation was
reduced to 48.14% by MIC as 0.625 l/ml. Due to the decrease in
the concentration of essential oil, the biofilm formation of S. aureus
MU 47 was inhibited by 33.45%, 14.11% and 8.07% in MIC/2, MIC/
4 and MIC/8, respectively. The MBC and MIC of S. aureus ATCC
25923 were 1.25 and 0.312 l/ml, respectively. R. officinalis essential
oil in MBC and MIC concentrations reduced the S. aureus ATCC
25923 biofilm formation to 58.02% and 27.6%. For S. aureus MU
38, MBC and MIC were 10 and 5 l/ml and biofilm formation was
reduced to 53.83% and 26.43% in these concentrations. Quave et
al. 12 reported the effect of R. officinalis extract on planktonic
growth, biofilm formation and adherence of methicillin-resistant S.
aureus. In this study, MIC50 value of 512 g/ml and IC50 value of 16
g/ml were reported for R. officinalis extract. Here we provide the
first report, to our knowledge, of the demonstration of the
antibiofilm activity of R. officinalis essential oil against S. aureus
and S. epidermidis.
Microorganism
B.s. ATCC 6633
B.c. RSKK 863
S.a. ATCC 25923
S e. MU 30
S.a. MU 38
S.a. MU 40
S.a. MU 46
S.a. MU 47
P.a. ATCC 27853
P.a. ATCC 29212
P.f. MU 180
P.a. MU 187
P.a. MU 189
Inhibition zone
15
16
14
12
17
13
12
12
11
10
MIC
5
5
0.312
0.625
5
2.5
0.312
0.625
20
1.25
1.25
20
1.25
% Inhibition
Table 1. Determination of MIC, MBC (l/ml) and disc

diffusion (mm) assay of R. officinalis essential oil.
MBC
10
10
1.25
1.25
10
5
0.625
1.25
80
5
2.5
80
2.5
l//ml))
Essential oil concentration (
Figure 1. The percent inhibition of R. officinalis essential oil for biofilm

formation in Gram positive bacteria.
B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P.

aeruginosa; P.f.: P. fluorescens; -: No inhibition.
Table 2. The effect of R. officinalis essential oil on tested bacteria

biofilm formation expressed as percentage inhibition.
MBC
B.s. ATCC 6633

B.c. RSKK 863
S.a. ATCC 25923
S.e. MU 30
S.a. MU 38
S.a. MU 40
S.a. MU 46
S.a. MU 47
P.a. ATCC 27853
P.a. ATCC 29212
P.f. MU 180
P.a. MU 187
P.a. MU 189
56
38.77
58.02
35.1
53.88
25.89
32.33
60.76
58.3
22.88
30.69
74.7
46.51
Essential oil concentration

MIC/2 MIC/4 MIC/8
Percentage (%) inhibition
51.3
37.09
19.3
6.1
18.52
27.6
21.29
17.76
16.61
5.2
26.43
3.14
27.22
6.81
48.14 33.45
14.11
8.07
21.83
7.73
6.16
18.55
1.04
39.49 19.91
14.38
5.39
35.57
3.22
MIC
MIC/16
-
B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P. aeruginosa; P.f.: P. fluorescens;
-: No inhibition.
84
% Inhibition
Microorganism
Essential oil concentration (

l//ml))
Figure 2. The percent inhibition of R. officinalis essential oil for biofilm

formation in Gram negative bacteria.
Essential oil of R. officinalis showed similar antimicrobial activity

against B. cereus RSKK 863 and B. subtilis ATCC 6633, but the
antibiofilm activity of essential oil was more effective against B.
subtilis ATCC 6633. Our results were similar to those previously
reported by Celiktas et al. 8, Jiang et al. 2, Okoh et al. 11, Zaouali et
al. 18 for the antimicrobial activity of the R. officinalis essential oil
against B. subtilis. A good to moderate antimicrobial activity of R.
officinalis essential oil against B. cereus has been reported by
Genena et al. 1 and Zaouali et al. 18.
The R. officinalis essential oil has also exhibited an
antibacterial effect against the Gram-negative bacteria (P.
aeruginosa and P. fluorescens). However, this effect was less
efficient than that presented against the Gram-positive bacteria,
since a higher MIC value was obtained with the Gram-negative
bacteria. In this study, the highest MBC and MIC were 80 and 20 l/
ml against P. aeruginosa ATCC 27853 and P. aeruginosa MU 187,
respectively. P. aeruginosa MU 187 biofilm formation has been
reduced to 74.71% in MBC and 39.49% in MIC. For the same
bacteria, the biofilm formation reduced by 19.91% in MIC/2, by
14.38% in MIC/4 and by 5.39% in MIC/8. MIC/16 concentrations
of the rosemary essential oil did not reduce the bacterial biofilm
formation of the tested bacteria. Biofilm formation of P. aeruginosa
ATCC 27853 was inhibited by 58.3% in MBC, by 21.83% in MIC
and by 7.73% in MIC/2. The results of the antimicrobial activity
on Gram-negative bacteria are similar to Celiktas et al. 8 and Jiang
et al. 2. In contrast to our results, Zaouali et al. 18 reported that
the essential oil of R. officinalis has no antimicrobial activity
against P. aeruginosa. The antimicrobial activity of R. officinalis
extracts against P. aeruginosa have also been reported by Genena
et al. 1 and Sandasi et al. 14. Sandasi et al.14 showed that R.
officinalis extract inhibited 56% of P. aeruginosa biofilm
formation. Contrary to this, they also reported that R. officinalis
extract was unable to inhibit the growth and development of a
pre-formed biofilm of P. aeruginosa.
To evaluate the relevance of biofilm formation assay, scanning
electron microscopy was employed. Direct observation by
scanning electron microscopy of S. aureus MU 47 showed that,
in the absence of rosemary essential oil (Fig. 3), bacterial cells
formed evident biofilms with matrix material. In the presence of
rosemary essential oil at concentrations of 1.25 l/ml (MBC)
bacterial cells grew as looser colonies, and the amount of biofilm
was reduced to 60.76% (Fig. 4).
Figure 4. Scanning electron micrographs showing reduction in S.aureus

MU 47 biofilm with 1.25 l/ml R.officinalis essential oil.
Conclusions
R. officinalis is a spice and medicinal herb widely used around the
world 1. It is widely found in the lands of Aegean and
Mediterranean regions of Turkey 8. In this study, the antimicrobial
and antibiofilm activities of the R. officinalis essential oil was
evaluated. The results obtained are confirmed by SEM observation.
According to the results of antimicrobial activity, the R. officinalis
essential oil is more active against Gram-positive than Gramnegative bacteria, as evidenced by the lower MIC values for the
former. Data showed that R. officinalis essential oil is not only
able to kill Staphylococcus spp. and Pseudomonas spp. cells
efficiently but also inhibits biofilm formation, but the results also
indicate that good antimicrobial activity against planktonic microorganisms does not imply good antibiofilm activity. However, the
results of this study show that rosemary essential oil is capable of
affecting S. aureus biofilm formation significantly. Here it is
suggested that R. officinalis essential oil may be considered as a
potential substance in the development of novel antimicrobial
and antibiofilm agents that could play a solution oriented role in
the field of food and pharmaceutical industries.
Acknowledgements
The authors would like to thank to Prof. Dr. Nazime Mercan Dogan
and Prof. Dr. Omur Baysal for their constructive comments and
suggestions.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Effect of storage time and temperature on the quality characteristics of chicken eggs
Yeasmin Akter 1, 2*, Azhar Kasim 1*, Hishamuddin Omar 3 and Awis Qurni Sazili
Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Department of Animal Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200,
Bangladesh. 3 Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
*e-mail: yesakter@yahoo.com, azharkasim@putra.upm.edu.my
1
Received 10 March 2014, accepted 30 August 2014.
Abstract
The experiment was conducted to evaluate the effect of storage time and temperature on external and internal qualities of eggs and lipid peroxidation
in the yolk during refrigeration and room storage. Total of eighty ISA Brown hens were used for this experiment. The diet used for laying hen was
prepared as iso-protein and iso-calorie. Eggs were collected daily and stored in refrigerator (4C) and room temperature (28-31C) for 7, 14, 21 and 28
days. At the designated day, the eggs were processed to evaluate their internal and external qualities. Both refrigeration and room storage increased egg
weight loss, percent yolk weight, yolk pH, albumen pH and lipid peroxidation and reduced Haugh units, percent albumen weight at 28 days of
storage. Yolk colour was unchanged during the whole storage period. However, eggs stored in refrigerator showed better quality up to 28 days and in
room temperature up to 14 days.
Key words: Chicken eggs, storage time, egg quality, lipid oxidation.
Introduction
Eggs are highly versatile and easily available foods for all
categories of people 1. Recently, people are very concerned about
the quality of eggs. So the scientists are giving more emphasis
to maintain the quality of eggs. The quality of eggs would be
deteriorated when the eggs are stored for long time that may be
unsuitable for human consumption. As a result, appropriate
technology for storage of eggs is essential to retain quality. The
main degradation factors for eggs are storage time 2, temperature,
humidity, air movement, and handling 3, 4. Interior quality
deterioration of eggs can be retarded significantly by maintaining
storage temperature, because quality deterioration occurs faster
at high temperatures than at refrigerated temperatures during
storage 5, 6.
The major difference between fresh and stored eggs is in
albumen pH and quality 7. During storage, the pH of the egg
albumen increases, which is related to the deterioration of albumen
quality or Haugh unit 3, 8, 9. The albumen of an AA egg is firm and
thick, the air cell is very small, and the albumen and yolk contain
no blood or meat spots. The increase of albumen pH mainly
depends on the buffering capacity of the albumen, not only this,
it also depends on the temperature, storage duration, gaseous
environment in the storage room and conductance of the
eggshell 7, 10-12. In fresh albumen, the buffering capacity is weakest
in pH 7.0 to 9.0 10. It is impossible to maintain albumen pH within
the range of 8.3 to 8.5 during prolonged storage of eggs without
modification of storage conditions. Another important change
that can be observed during storage is the flattening of the yolk
caused by the weakening of the vitelline membrane 13. After eggs
are laid, water moves from the albumen to the yolk due to
differences in osmotic pressure, and this may change the yolk
index and may cause the weakening of the vitelline membrane.
Fromm 14 reported that yolk with high water content showed high
yolk index, when the albumen pH was maintained at or below pH
8.0. So, the albumen pH is the most important factor that affects
the strength of the vitelline membrane and yolk index. Walsh et al. 7
concluded that one requirement for successful long-term storage
was the prevention of water loss from the egg. During storage,
water from egg is lost through evaporation, and the rate of
evaporation is influenced by the length of storage, temperature,
humidity and the surface and porosity of the shell 15. Lipid oxidation
is also an important deterioration that occurs during storage, and
it may produce toxic compounds. Oxidative products in eggs can
reduce its nutritive value 16. So, it is essential to prevent and
minimize the lipid oxidation in order to maintain egg quality and
fatty acid stability during storage,
Therefore, the present study has been undertaken to determine
the effects of storage time and temperature on some external
and internal characteristics for reducing the deterioration of egg
quality during refrigerated and room storage.
Experimental birds and diet: The experiment was conducted
at the poultry unit, Department of Animal Science, University
Putra Malaysia. A total of 80 ISA Brown layers, 44 weeks of age
were used for this study to evaluate egg quality characteristics.
The layer diets were isonitrogenous and isocaloric and formulated
according to NRC 17 recommendations for dietary need of laying
hens for various nutrients. Experimental diets were prepared weekly
to avoid oxidation.
Sampling and storing of eggs: Immediately after collection,
eggs were labeled according to date of production and hen
87
Evaluation of egg quality: Characteristics were evaluated in

individual eggs for internal and external quality traits. The external
characteristics of eggs were egg weight (g), egg length and width
(cm), specific gravity, shell weight (g) and shell thickness (mm),
whereas internal quality parameters include albumen weight (g),
albumen height (mm), yolk width (cm), yolk weight (g), and
Haugh unit. Other parameters measured included egg shape index,
albumen pH and yolk pH. Egg weight was measured by weighing
egg individually using sensitive balance. Egg length and width
(cm) and shell thickness were measured by using Digital Vernier
Calipers. Egg shape index was obtained as a ratio of the egg width
to the length. Egg shell thickness was determined by the mean
of three measurements taken from three different sides of the
shell. Specific gravity was determined through saline solutions
method of the same temperature, but with different densities.
The analysed egg is passing, one time each, from a pot to another,
being well drained from the previous solution; the pot in which
the egg rise to the surface shows the specific gravity, by its
solutions; density. Albumen height, yolk colour and Haugh unit
were measured automatically by egg multitester (Orka Food
Technology). The yolk was separated from the albumen and
weighed. Egg yolk width was measured by compass. Egg shells
from individual eggs were cleaned, dried at room temperature and
weight recorded 18. Shell percentage was calculated by dividing
shell weight with egg weight and multiplying by 100 19. Albumen
weight was calculated from the difference between egg weight
and weight of the yolk and shell. The pH of the albumen and yolk
were measured immediately using a pH meter.
Water loss measurements during storage: Eggs were weighed
at each sampling time. The same eggs were weighed over the 4
week study. Egg weight loss was calculated as follows:
thiobarbituric acid, and was further incubated at 100C for 30 min

to develop pink colour. Following incubation, the mixture was
cooled under tap water and centrifuged at 3000 g for 10 min.
Absorbance of supernatant was measured at 532 nm using a
spectrophotometer. The concentration of MDA in analysed
samples (mg/kg yolk) was calculated from a standard curve of 1, 1,
3,3 tetra-ethoxypropane. All measurements were conducted in
triplicate.
Statistical analysis: Analyses were conducted using the SAS
software program 21. Significant differences among the means
of the treatment groups were determined by Duncans multiplerange test. Variability in the data was expressed as standard error
(S.E.) and a probability level of p<0.05 was considered as
statistically significant.
Effect of storage time and temperature on external qualities
of chicken eggs: The effect of storage time and temperature on
egg weight loss is shown in Fig. 1. The percent egg weight loss
values showed significant (p<0.05) variations during storage.
Total egg weight loss increased when storage duration increased,
and the highest weight losses were recorded at 28 days of storage
in both temperatures. Weight loss occurs due to loss of solvents
from the egg content through the shell by evaporation. These
results are supported by Altan et al. 22, Fasenko et al. 23, Tilki and
Inal 24, Hassan et al. 25, Reijrink et al. 26, Gonzalez-Redondo 27
and Alsobayel and Albadry 28, who reported that with increase
the length of storage, egg weight losses increase.
Egg weight loss (%)
number and weighed by using sensitive balance. Fresh eggs were

analysed within 2 h of being laid. To study the effect of storage on
egg quality parameters, eggs were stored at 4C and room
temperature (28-31C) for 7, 14, 21 and 28 days. The stored
eggs were identified and analysed at each corresponding storage
time and temperature.
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
4C
28-31C
14
21
28
Storage time (days)
Total egg weight loss (g) = Initial weight (g) - final weight (g)
Weight loss percentage was calculated in relation to day 0 egg

weight (g):
Weight loss (%) =
Weight loss (g)

Initial weight
(g)
100
Thiobarbituric acid (TBA) determination: The TBA values were

determined for the malonaldehyde (MDA) formed in fresh eggs
and those that were stored at 4C and room temperature for
different time periods (7, 14, 21 and 28 days of storage). This
MDA value was measured according to the TBA method described
by Botsoglou et al. 20 with some modifications. Yolk samples were
homogenized by Polytron homogenizer in the presence of 8 ml 5%
aqueous trichloroacetic acid (TCA), and 5 ml of 0.8% butyrate
hydroxytoluene in hexane was immediately added and the mixture
centrifuged. The top layer was discarded and a 2.5 ml aliquot from
the bottom layer was mixed with 1.5 ml of 0.8% aqueous 288
Figure 1. Effect of storage time and

temperatures on egg weight loss (%).
Similarly egg weight loss depends on the temperatures. As a

result, eggs stored at 4C showed significantly (p<0.05) lower
weight loss than room temperature. This may be due to the less
loss of solvents (water and other gaseous products) from egg
contents than those in room temperature. These results are in
agreement with Shanawa 29, Samli et al. 4 and Hasan and Okur 30
who noticed a decrease in egg weight within 10 days of storage at
29C.
The effect of storage time and temperature on shape index of
egg is shown in Table 1. Shape index did not affect significantly
(P>0.05) by storage time and temperature. These results are also
supported by Woodard 31, Song et al. 32 and Tilki and Saatci 33,
who observed no effect of storage time and temperature on shape
index of eggs.
Similarly, no significant difference was found in the shell
thickness during storage of eggs at two different temperatures
Table 1. Effect of storage time and temperature on shape index, shell thickness and shell
weight of chicken eggs.
Shell thickness (mm)

Shell weight (%)
0
76.590.53
76.510.33
0.340.005
0.350.002
11.470.43
11.470.43
Storage time (days)

7
14
21
76.590.53 76.340.34 75.950.76
76.480.31 76.240.34 75.880.95
0.340.004 0.340.004 0.340.003
0.340.005 0.340.003 0.340.003
11.450.35 11.440.27 11.440.23
11.440.35 11.430.35 11.420.35
Specific gravity
(Table 1). These findings on shell thickness are also in agreement

with Dudusola 34 and alayan et al. 35, who did not find any
effect of storage time and temperature on shell thickness in
partridges and Japanese quail eggs.
Shell weight as percentage was not affected significantly
(P>0.05) by storage time and temperature (Table 1). These findings
are in line with Silversides and Scott 36, Tilki and Inal 24, Akyurek
and Okur 37 and alayan et al. 35, who reported no effect of
storage time on egg shell weight. In contrast, Samli et al. 4 noticed
significant (p<0.05) change in shell weight during storage at
different time and temperature.
On the other hand, specific gravity of eggs declined (p<0.05)
gradually with the advancing storage time as a result maximum
(p<0.05) decreases at 28 days of storage in both temperatures, but
the specific gravity of eggs kept at room temperature declined
more rapidly (p<0.05) than that of refrigerated eggs (Fig. 2) . This
might be due to the size of the air cell because with increase of
storage time and temperature the size of the air cell increased.
These results are in agreement with Akyurek and Okur 37, who
observed the size of the air cell exceeded 5 mm in 7 days at all
storage temperatures.
1.09
1.085
1.08
1.075
1.07
1.065
1.06
1.055
1.05
1.045
1.04
4C
28-31C
14
21
28
Storage time (days)
Figure 2. Effect of storage time and temperature

on specific gravity of chicken eggs.
Effect of storage time and temperature on internal qualities of

chicken eggs: The results of Haugh unit (HU) of eggs are
presented in Fig. 3. HU of fresh eggs declined significantly (P<0.05)
with the increasing storage time. Haugh unit reduction was
happened due to the decrease in thick albumen height, because
during storage, the ovomucin-lysozyme complex breaks down,
which helps to increase the pH of eggs. The results of this study
were supported by Morais et al. 38 who observed the reduction in
the Haugh unit of eggs at 21 days of storage. Similarly storage
temperatures can affect the HU of eggs. High storage
temperatures promote the breakdown of ovomucin-lysozyme
complex. As a result, HU of eggs stored at room temperature was
reduced significantly (P<0.05) compared to refrigeration. The results
are in agreement with Campos and Baio 39, Sauveur 40 and Samli
28
75.930.43
75.710.45
0.340.003
0.340.002
11.430.27
11.410.27
90
4C
80
Haugh unit (HU)
Shape index (%)
Temperature
(C)
4
28-31
4
28-31
4
28-31
28-31C
70
60
50
40
30
20
10
0
7
14
21
Storage time (days)
28

on Haugh unit (HU) of chicken eggs.
et al. 4, that storage time and temperature adversely affected HU

of eggs. Consequently, other researchers like Tona et al. 41 and
Akyurek and Okur 37 reported that the rate of water loss from egg
is influenced by the rate of evaporation from egg content.
Figure 4 also indicates that storage of eggs at different time
intervals showed a significantly (P<0.05) higher pH value than
fresh eggs, which may occur due to evaporation and exchange of
carbon dioxide from eggs; the equilibrium of the carbonatebicarbonate buffer system is thought to be shifted towards
production of CO2. Maximum increase occurred during the first 7
days of storage followed by progressively slower rate of increase
throughout the rest of the storage period. These findings are
supported by Scott and Silversides 18, Samli et al. 4 and Akyurel
and Okur 37. Along with storage time, the pH of egg albumen can
also be influenced by temperature. As a result the albumen pH
was significantly (P<0.05) higher at room temperature than in
refrigeration during the whole storage period, which may be due
to higher amount of evaporation from eggs. Increase in albumen
pH with storage was also reported by Moula et al. 42 and Silversides
and Budgell 43. In contrast, Walsh et al. 7 revealed that neither
temperature nor storage time influenced the pH of egg albumen.
Albumen pH
Parameter
9.4
9.2
9
8.8
8.6
8.4
8.2
8
7.8
7.6
7.4
4C
28-31C
14
21
28
Storage time (days)

temperature on albumen pH of chicken eggs.
89
Albumen weight (%)
64
4C
62
28-31C
60
58
56
54
52
14
21
28
Storage time (days)

on albumen weight (%) of chicken eggs.
The effect of storage time and temperature on the pH of egg

yolk is shown in Fig. 6. The results revealed that the pH of egg
yolk increased significantly (p<0.05) at 28 days of storage, the pH
of the yolk was significantly higher (p<0.05) in room temperatures
than in refrigeration. This may occur due to the loss of carbon
dioxide from egg by diffusion, which helps to increase the yolk
pH. The present results are in agreement with Samli et al. 4 and
Akyurel and Okur 37, that increases in yolk pH were significantly
affected by storage time. This increase in pH value was lower
than the value of Samli et al. 4 who found that yolk pH differed
from 5.75 to 6.08 during 10 d of storage at 29C. This may be due
to lower diffusion rate of carbon dioxide from the egg.
6.5
Yolk pH
Yolk weight (%)
28
27
26
4C
28-31C
25
24
23
14
21
28
Storage time (days)

on yolk weight (%) of chicken eggs.
and Barbosa et al. 47. On the other hand, when the storage
temperature was higher, the rate of increase in yolk weight was
significantly (p<0.05) higher than in refrigeration (4C). These
results are supported by Davis and Stephenson 48, Morais et al.38
and Leandro et al. 49, who reported that the most important factors
that affect egg quality during storage are temperature and relative
humidity.
From Fig. 8, it is noticed that the duration and temperatures of
storage significantly (P<0.05) increased the value of yolk width.
The increase in yolk width observed in this study could be due to
decrease of the strength of vitelline membrane. When eggs are
stored under room temperature for long periods, the strength of
vitelline membrane breaks and makes the yolk to spread into the
albumin. These results are in agreement with Kirunda and McKee 50,
who reported that vitelline membrane strength (VMS) decreases
during storage and makes the yolk more susceptible to breaking,
as a result, water slowly enters into the yolk from the albumen, so
this creates a mottled appearance in yolk, and the yolk becomes
flattened. The yolk width values were higher in room temperature,
because at a very high temperature, the amount of water migration
from the albumen to the yolk is high, which helped to increase
yolk width 51.
40.5
40
39.5
39
38.5
38
4C
37.5
37
28-31C
14
21
28
Storage time (days)
6.3

temperature on yolk width of chicken eggs.
6.2
4C
6.1
28-31C
6
0
14
21
28
Storage time (days)

temperature on yolk pH of chicken eggs.
Yolk weight and yolk weight as % of egg weight (Fig. 7) showed

significant (p<0.05) changes during storage, and increased linearly
with storage time, which might be due to the diffusion of water
from the albumen to the yolk. These results concur with Haugh 46
90
30
29
36.5
36
6.4
5.9
31
Yolk width (mm)
Effect of storage time and temperature on albumen weight (%)

of eggs is presented in Fig. 5. The albumin weight was significantly
(p<0.05) affected by storage time. As a result highest albumen
weight losses were recorded for 28 days of storage in both storage
temperatures. This loss of albumen weight happened due to loss
of solvents from albumen, which may decrease the weight of the
albumen in egg by increasing the weight of yolk. These results
are inconsistent with Siyar et al. 44 and Tabidi 45, who reported that
the loss in albumen weight is attributed to loss of humidity from
inside the egg due to evaporation. Consequently, albumen weight
loss was significantly (p<0.05) higher at room temperature than in
refrigeration at 28 days of storage. This may be due to the higher
amount of water loss from the albumen to the yolk. Similar results
were demonstrated by Tona et al. 41 and Akyurek and Okur 37,
that water loss from eggs may be influenced by storage time,
temperature, relative humidity and porosity of the shell.
In this study, the pigmentation (Fig. 9) was observed for both

temperatures at different storage periods, but no significant
(P>0.05) difference was for any period and temperature studied;
this proves that the temperature did not influence the yolk
pigmentation. This might be due to the antioxidant content of
layer ration, because layer diet was formulated by using crude
palm oil (CPO), which was rich in carotenoids and vitamin E, which
delayed or prevented the oxidation of carotenoid pigments
contained in feed and yolk 52; while in another study, it was
demonstrated that the addition of vitamin E (20 and 40 mg/kg) to
hen diets reduced yolk colour intensity, in comparison with the
Yolk colour score
5.4
4C
28-31C
5.35
5.3
5.25
5.2
5.15
7
21
14
Storage time (days)
28
Figure 9. Effect of storage time and temperature on

yolk colour score of chicken eggs.
control group 53. Spada et al. 54 found increased pigmentation of

the red in yolks after 28 days of storage at room temperature
(25C) and stabilizing on the 36th day. No recent data was found
regarding the relationship between yolk colour and storage
temperature or time.
Yolk lipid oxidation increased significantly (p<0.05) with
storage time and temperature (Fig. 10), because storage of egg
for longer periods at different temperatures may reduce the
antioxidant activity. This result agrees with Franchini et al. 55 and
Lakins et al. 56, that the TBARS values increased when the eggs
were stored for 30-90 days at 4C. On the contrary, the storage at
room temperature increased TBARS due to reduction in the
amount of vitamin E in stored eggs 55.
1.4
TBARS (mg/kg)
1.2
1
0.8
0.6
4C
0.4
28-31C
0.2
0
14
21
28
Storage time (days)

temperature on TBARS value (mg/kg of
yolk) of chicken eggs.
Conclusions
This study has confirmed that the quality characteristics of eggs
were not adversely affected when eggs were stored in refrigerators,
but room temperatures negatively affect some egg quality
characteristics by increasing weight loss, yolk weight, yolk pH,
albumen pH, yolk lipid oxidation and by reducing HU and albumen
weight during storage for different time intervals. It has been
concluded that egg should be kept in refrigerators up to 28 days
and at room temperature up to 14 days.
Acknowledgements
The authors would like to thank Universiti Putra Malaysia for
providing financial support for the publication of this manuscript.
The authors are also grateful to Organization for Women in
Science for the Developing World (OWSD) for providing the
scholarship for PhD student.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Evaluation of the antioxidant activity of extracts from Psidium guajava L. and

Anacardium occidentale L. leaves obtained by different extraction methods
Suzara R. C. Sena, Theresa R. F. Dantas and Camila G. Pereira *
Laboratory of Separation Process in Foods, Department of Chemical Engineering (Federal University of Rio Grande do NorteUFRN), CEP 59072-970 Natal, Brazil. *e-mail: camila@eq.ufrn.br
Received 20 February 2014, accepted 28 September 2014.
Abstract
The presence of bioactive compounds in foods presents the possibility to improve public health through the diet. However, the obtaining of active
compounds from food for posterior application depends on the processing and fractionating methodologies used. Several functional properties have
been attributed to compounds presented in different parts of the plant, including the leaves and fruit. Parallel, the discovery of new applications of
by-products improves regional economy and makes value on the regional products. The aim of this work was to evaluate the antioxidant activity of
extracts from guava (Psidium guajava L.) and cashew (Anacardium occidentale L.) leaves and quantify the total phenolic compound concentrations
of different extracts. Extracts were obtained by employing three different techniques: low-pressure solvent, Soxhlet, and ultrasound extraction
methods. The effect of extract concentration on the percentage of inhibition (antioxidant activity) was analysed. Higher total phenolic compound
concentrations were obtained through Soxhlet extraction for guava leaf extracts (44.02 mg GAE/g extract) and low pressure solvent for cashew leaf
extracts (29.21 mg GAE/g extract). The results indicated that all extracts had good antioxidant activity due to the presence of phenolic compounds and
other compounds present in the extracts, with greater than 70% inhibition for all guava leaf extracts at concentrations greater than 5 mg/ml. The
percentage inhibition was also dependent on extract concentration, which can be attributed to differing concentrations of phenolic compounds and the
possible presence of other compounds that enhance or decrease the antioxidant activity of the extracts.
Key words: Guava leaves, cashew leaves, phenolic compounds, antioxidant activity.
Introduction
Antioxidants are now being included in functional diets due to
their potential beneficial characteristics for human physiology.
The ingestion of food products high in antioxidants is important
not only to combat malnutrition, but also to protect against
degenerative diseases caused by free radical-mediated oxidative
reactions. It may be possible to improve the nutritional value of
traditional food products and protect against damage caused by
free radicals by manufacturing new food products with
supplemental antioxidant compounds.
Northeast Brazil is a region rich in natural products. Some natural
products are locally consumed. However, the use of them in large
scale, commercial applications, such as food, cosmetic or medicinal
industries, is uncommon. These products could be used to
formulate new commercial products. In fact, the use of food as a
source of bioactive compounds has increased in recent years.
The bioactivity of the foods arises due to several classes of
compounds, including essential oils, flavonoids, carotenoids and
alkaloids. These compounds could be used industrially in different
formulations, thereby creating a demand for regional natural
products. In addition, novel compounds may be discovered,
potentially resulting in new products. Hence, tropical food species
are important in Northeast Brazil not only for the high nutritional
value as a food source, but as a potential contributor to the local
economy.
Special attention has been paid to natural products with
antioxidant activity as antioxidants play a role in the maintenance
of healthy organisms. Studies have revealed that antioxidant
activity is mainly due to the presence of phenolic compounds 1-4.

Among regional species of great economic importance in
Northeast Brazil is Psidium guajava L. (Myrtaceae), commonly
known as guava. Guava trees are native to Mesoamerica and are
found in Southeast and Northeast Brazil. The fruit of guava
trees is tasty and highly nutritious, and has important activities
such as anti-inflammatory, hydrophilic and lipophilic antioxidant
activities 5, 6. In addition, the leaves of this species exhibit
intestinal anti-spasmodic, hypoglycaemic, hypotensive,
antimicrobial, anti-nociceptive, anti-diabetic and hepato-protective
effects 7-12.
Another economically important species in Northeast Brazil is
Anacardium occidentale L. (Anacardiaceae), commonly known
as cashew. This tree is originally from tropical America, and is
native to Northern and Northeast Brazil 13. This species produces
fruits (nuts) and pseudo-fruits that are widely consumed by local
populations. Although the principal use of this species is as a
food source, A. occidentale can also be used for other purposes.
Previous studies have found that the branches of A. occidentale
have antimicrobial activity against different microorganisms 14-16.
The cashew nut has demonstrated pharmacological effects, such
as antibacterial activity, mutagenicity, antioxidant potential, and
anti-mutagenic activity against hydrogen peroxide activities 17, 18.
In addition, extracts from the leaves of A. occidentale are also
effective for the treatment of leishmanial ulcers due to the presence
of flavonoids and tannins 19 and antibacterial action against some
selected microorganisms 20.
93
Although several investigations had been done with these

species in last decades, recent studies concerning on their
pharmacological effects and applicability studies continue being
made 21-27, indicating that several information still needs to be
revealed about these species.
In order to use these species in the industry, it is necessary to
evaluate what kind of process is more appropriate to obtain the
desired product. The recovery of active compounds in plant
extracts depends on the type of extraction and fractionation
methodologies used in the extraction process. Optimisation of
the extraction of specific compounds is essential for the
development of commercial uses for these species. In this scenario,
the objective of this study was to evaluate the concentrations of
phenolic compounds present in extracts from cashew (Anacardium
occidentale L.) and guava (Psidium guajava) leaves obtained
using different extraction techniques, and to determine the
antioxidant activity of these extracts.
Preparation and characterisation of the raw plant materials:
Cashew and guava leaves were collected in the Lagoa Nova region
in Natal, Brazil. The material was dried at ambient conditions (1720C) in the shade with air circulation for 25 days and subsequently
packed in plastic bags before storage in a refrigerator (Cnsul,
Modelo VU28A0, Santa Catarina, Brazil). The dried leaves were
ground using a knife mill (Tecnal, Model TE-631/2, Piracicaba,
Brazil). The processed material was classified according to size
using an agitator (Bertel, model NOVO 110/220, So Paulo, Brazil)
with standard Tyler series sieves.
Methodology of the analysis: Extracts obtained in previous studies
in the Laboratory of Separation Processes in Foods (LAPSEA/DEQ/
UFRN) were analysed to determine the concentration of phenolic
compounds and antioxidant activity 28, 29.
The extracts were obtained using the following methods: low
pressure solvent extraction (LPSE) at 60C, Soxhlet extraction at
78C, and ultrasound extraction at 40C. The temperatures were
chosen based on optimised conditions reported in previous
studies 28, 29. In all extraction processes, ethanol was used as the
solvent using a solid: solvent ratio of 1:10. Table 1 shows the
global yield of the extracts used in this study.
Table 1. Global yield of extracts from guava leaves
(P. guajava) and cashew leaves (A.
occidentale) obtained by different
extraction methods.
Extraction method
LPSE
Soxhlet
Ultrasound
Global yield (%)

Guava leaves 28 Cashew leaves 29
12
10.3
21
14.3
5.9
18.5
Quantification of total phenolic compounds: The total

concentration of phenolic compounds was determined using
the methodology described by Singleton and Rossi 30 modified
as described below. Approximately 1 ml of the extract and 1
ml of Folin-Ciocalteau reagent were mixed. After 5 min, 1 ml
of a saturated solution of Na2CO3 (approximately 35% in
methanol; PA ACS, Lot 0904809, Vetec) was added and the
94
total sample volume was made up to 25 ml with methanol. The

mixture was protected from light for 90 min before reading the
absorbance at 725 nm using a spectrophotometer (Varian 50, UVvis). A standard curve was generated using gallic acid standard
(Lot 0806387, Vetec) diluted in methanol at 20, 40, 60, 80, 100 and
120 mg/l with 1 ml of Folin-Ciocalteau reagent 31.
Measurement of antioxidant activity: Antioxidant activities of
the extracts were determined using the methodology of BrandWilliams et al. 32 as modified by Herrero et al. 33. In this method,
free radicals of DPPH (2,2-difenyl-1-picryl-hydrazil, Lot 07717TH,
Sigma-Aldrich) are neutralised with antioxidants in the extracts
resulting in a decrease in absorbance at 516 nm. To carry out the
reaction, a solution of 0.0214 mg/ml DPPH in methanol (Lot
0904809, Vetec) was prepared. Of the DPPH solution 3.9 ml was
mixed with 0.1 ml of the extract solutions at concentrations from
10 to 0.5 mg/ml. The total reaction volume was 4 ml. The reaction
was left to proceed for 4 h at ambient temperature (21C). The
absorbance was then determined at 516 nm using a
spectrophotometer. The antioxidant activities (AAs) were
calculated in terms of percentage of inhibition using Eq. 1:
AA (%) = 100 {[(Abssample Abscontrol) x 100] / AbsDPPH}
(1)
where Abssample is the absorbance of the sample, Abscontrol is the

absorbance of the control solution and AbsDPPH is the absorbance
of the DPPH.
Characterisation of the raw plant materials: The results of
characterisations of the raw plant materials are shown in Table 2.
After the initial preparation stage, the raw plant materials had
humidity, particle diameters (Dst), real and apparent densities,
granulometries and porosities suitable for use with extraction
processes to obtain bioactive compounds from natural products34.
The choice of the best technique should not only be based on the
global yield. An analysis of the chemical profile is also essential.
Total phenolic compounds: Quantification of the total phenolic
compounds was performed for each extract using a standard curve
from measurements of gallic acid standards (20-120 g/ml), and
expressed as mg of GAE (gallic acid equivalent) per g of extract.
The total phenolic compounds found in the guava and cashew
leaf extracts are shown in Table 3. Guava leaf extracts obtained by
Soxhlet extraction yielded higher amounts of total phenolic
compounds (44.02 mg GAE/g extract) than the other extraction
methods. For cashew leaves, the extracts obtained by LPSE yielded
higher concentrations of total phenolic compounds (29.21 mg
GAE/g extract).
Table 2. Characterisation of guava (P. guajava) and cashew (A.

occidentale) leaves.
Properties
Granulometry-in mesh (%)
Dst (mm)
Humidity (%)
Real density (g/cm)
Apparent density (g/cm)
Porosity (H)
Guava leaves
24 (52.0%), 28 (16.9%)
32 (4.1%), 48 (27.0%)
0.657
10.4 r 0.4
0.439 r 0.004
0.424 r 0.005
0.035
Cashew leaves
24 (70.4%), 28 (12.1%)
32 (2.8%), 48 (14.7%)
0.682
14 r 3
0.295 r 0.001
0.218 r 0.004
0.263
Extraction method
LPSE
Soxhlet
Ultrasound
Total phenolic compounds

(mg GAE/g extract)
Guava leaves Cashew leaves
12.72
29.21
44.02
25.36
11.74
23.87
100
90
80
Inhibition (%)
Table 3. Total phenolic compounds present in

extracts from guava (P. guajava)
and cashew (A. occidentale)
leaves obtained by different extraction
methods.
70
60
50
40
30
20
10
0
Antioxidant activity of the extracts: The antioxidant activity of

each guava and cashew leaf extract was determined using DPPH
radicals and is expressed in terms of inhibition percentage, which
indicates the antioxidant activity of the extract. Figs. 1 and 2 show
the percentage of inhibition as a function of the concentration of
the diluted extracts.
As shown in Fig. 1, extracts from guava leaves obtained by
Soxhlet extraction had more than 70% of inhibition at low extract
concentrations. The inhibitory effect increased slightly with
increasing concentration (73.6-79.0%). These results indicate that,
for these extracts, the AA is independent of the concentration of
bioactive compounds. In other words, the AA of the extract is
associated with the presence of an unidentified antioxidant
compound, in a manner independent of the concentration of the
antioxidant present in the extract. This is consistent with results
from a previous study 35. In contrast, extracts obtained by
ultrasound extraction had a low inhibition (31.5%) at a low extract
concentration but increased to 72.1% inhibition as the extract
concentration was increased from 0.5 to 5 mg/ml. In this case, the
inhibitory effect is dependent on the concentration of bioactive
compounds. These results are consistent with the total phenolic
compound contents shown in Table 3, as the extracts with higher
4
6
8
Concentration (mg/ml)
10
12
Figure 1. Inhibition (%) by guava leaves extracts obtained

by LPSE (z), Soxhlet (*) and ultrasound (S) extraction
methods.
100
90
80
Inhibition (%)
Previous studies have presented only qualitative data for

extraction methods studied here 28, 29. In these studies, TLC analysis
showed that the amount of flavonoids obtained by Soxhlet
extraction and LPSE were similar to that for other techniques
studied. Flavonoids are a sub-group of phenolic compounds.
Based on these results, it could be inferred that the amount of
phenolic compounds in the Soxhlet and LPSE extracts may also
be similar. However, the results of this study show that this is not
the case. In our study, the total phenolic content was determined
quantitatively so it was possible to determine that there are
significant differences (p<0.05) between extraction methods,
especially for the guava leaf extracts. This indicates that nonflavonoid phenolic compounds are also present in the extracts.
It is also important to notice that the global yield (Table 1) is
useful only as a first analysis to guide the choice of extraction
methods for further study. Although the global yield and amount
of total phenolic compounds were highest for the Soxhlet extracts
of guava leaves, the opposite was true for the cashew leaf extracts
where the highest total phenolic compound content was obtained
for the LPSE extract, which had the worst global yield. These
results reinforce the importance of evaluating more than the global
yield of an extraction method in order to avoid selecting an
extraction method that does not give a high yield of the desired
compounds.
70
60
50
40
30
20
10
0
4
6
8
10
12
Figure 2. Inhibition (%) by cashew leaves extracts obtained

by LPSE (z), Soxhlet (*) and ultrasound (S) extraction
methods.
total phenolic compound concentrations, such as Soxhlet extracts,

showed higher AA activity. In general, AA was observed for all
guava extracts, with observed inhibitions higher than 70% for all
extracts at concentrations greater than 5 mg/ml. It is important
also to note that LPSE extracts showed high AA (60.0-72.5%)
even though the LPSE extracts had low concentrations of total
phenolic compounds (12.72 mg GAE/g extract). Based on these
results, there must be non-phenolic compounds in the LPSE extract
with AA.
Figure 2 shows that the cashew leaf ultrasound extract had an
AA effect different from that observed for guava leaf ultrasound
extract. The percentage of inhibition increased (21.73 to 74.96%)
as the extract concentration increased from 0.5 to 5 mg/ml, reaching
a plateau above 5 mg/ml. The effect of concentration on the AA
was not evident for the Soxhlet extracts of cashew leaves. However,
the inhibition percentage was high (74.6%) for the lowest extract
concentration (0.5 mg/ml). Interestingly, for cashew leaf LPSE
extracts, high concentrations of the extract yielded lower inhibition
percentages than low concentrations. This may occur if other
compounds present in the extracts counteract the AA of the
bioactive compounds. For instance, the results showed that for
guava extracts obtained by Soxhlet extraction, the extract
concentration had little effect on AA, indicating the presence of a
bioactive compound for the AA independent of concentration.
The AA of the bioactive compound may increase with increasing
extract concentration, but other compounds may also be present
95
that react with the antioxidant compound, thereby decreasing the

observed AA. Another possibility is the presence of compounds
in the extracts that promote oxidation, thereby opposing the AA
of the extract.
Figure 2 also shows that all extracts exhibited AA over the range
of extract concentrations studied here. LPSE and Soxhlet extracts
had high AA at low concentrations (0.5-1.0 mg/ml), while
ultrasound extracts had high AA for concentrations higher than 5
mg/ml.
Conclusions
In our study, it was observed that when developing methods for
the extraction of specific compounds from natural products, the
global yield should only be used as an initial guide as high global
yields do not necessarily guarantee efficient extraction of specific
target compounds. Higher total phenolic compound concentrations
were observed in extracts obtained by Soxhlet extraction and LPSE
for guava and cashew leaves, respectively. Antioxidant activity
was observed for all extracts, with greater than 70% inhibition for
all guava leaf extracts at concentrations greater than 5 mg/ml. The
percentage inhibition was also dependent on extract concentration
for cashew leaf extracts. Variability in AA with extract concentration
can be attributed to differing concentrations of phenolic
compounds and the possible presence of other compounds that
enhance or decrease the AA of the extracts.
Acknowledgements
The authors are grateful to FAPERN (PPP003/2007) for financial
support.
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www.world-food.net
Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in

fresh cut vegetables
Marina Cavaiuolo 1, Antonio Ferrante 1*, Spiros Paramithiotis 2, Agni Hadjilouka 2, Periklis Tzamalis
and Eleftherios H. Drosinos 2
Dept. Agricultural and Environmental Sciences, Universit degli Studi di Milano, via Celoria 2, 20133 Milano, Italy.
Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University
of Athens, Iera Odos 75, GR-118 55 Athens, Greece.
e-mails: marina.cavaiuolo@unimi.it, antonio.ferrante@unimi.it, sdp@aua.gr, agni_xatz@aua.gr, pertzam@otenet.gr,ehd@aua.gr
1
Abstract
Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in
minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and
evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the
production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the
pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing
similar sensitivity, specificity, negative predictive values and negative likelihood ratios.
Key words: Leafy vegetables, melon, vegetables, ELISA, ISO.
Introduction
Ready to eat fresh-cut vegetables (RTEs) are convenient foods
that have increased the volume and value of commercialisation
among different European countries. Nevertheless, the economic
crisis in the recent years has slightly affected the fresh-cut fruits
and vegetables market. This trend can be explained considering
the higher quality of the products, which must be convenient and
safe 1. Most of the leafy vegetables used for the fresh-cut
preparation are grown in the soil and bacteria contamination can
easily occur. Moreover, their short growing cycles require an
higher supply of organic matter in order to keep the soil fertility.
The organic matter supply is usually performed by manure and as
such represents a possible source of contamination of human
pathogenic bacteria like Escherichia coli O157 L. and Listeria
monocytogenes L. 2.
Washing treatments are able to reduce the bacteria load and
allow to preserve the RTE products for long time 3. The official
standard procedures for the detection of bacterial pathogens
require 4-5 days. Unfortunately, the shelf life of leafy vegetable
products is limited to 5-7 days that include two days of processing
and five days of shelf life. Therefore, faster and reliable methods
are required to detect the presence of pathogens without losing
the period of commercialisation. In the framework of the EU project
QUAFETY - Comprehensive Approach to Enhance Quality and
Safety of Ready to Eat Fresh Products different detection methods
based on ELISA approach was developed and compared with the
standard official ISO procedures.
Samples: Rocket and mixed salads were provided by Agronomia
s.r.l., (San Paolo dArgon, BG, Italy) and EuroCatering S.A. (Greece),
98
while Piel De Sapo melons were provided by NoviFruits (Portugal).

Samples were collected from raw materials, processed materials
(immediately after packaging) and three days shelf-life materials.
Thirty-nine samples were analysed corresponding to 2 different
batches of melons (Portugal), 7 different batches of rocket (Italy
and Greece) and 4 different batches of mixed salad (Italy and
Greece).
ELISA-based detection of L. monocytogenes and E. coli
O157:H7: Five g of material was homogenised in 1PBS (1 mM
KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.2). Isolation of
bacteria, ELISA detection and data analysis were performed
according to the protocol described in Cavaiuolo et al. 4. For the
detection anti-E. coli O157 (ab20976), anti-L. monocytogenes (LZA2)
(ab11439) and Polyclonal Goat anti-Mouse IgG+IgM H&L (HRP)
antibody (ab47827) were purchased from Abcam (Cambridge, UK).
ISO procedures for L. monocytogenes and E. coli O157:H7
detection: Twenty-five g of each sample was used to detect the
presence of L. monocytogenes and E. coli O157:H7 according to
ISO 11290-1:1996/Amd 1:2004 and ISO 16654:2001 5, 6, respectively.
A sample was characterised as positive for the presence of L.
monocytogenes when the identity of typical colonies isolated from
ALOA agar was verified by series of biochemical tests (Gram stain,
catalase test, oxidase reaction, hemolysis, fermentation of
rhamnose, xylose, mannitol and methyl -D-mannopyranoside,
and a motility test). In any other case (i.e. identity not verified,
absence of typical colonies), the sample was characterised as
negative for the presence of L. monocytogenes. A sample was
characterised as positive for the presence of E. coli O157:H7 when
the identity of typical colonies was verified by successive

subculturing in BH agar and submittion to E. coli O157:H7 Latex
Test (Remel, Lenexa, USA). In any other case (i.e. identity not
verified, absence of typical colonies) the sample was characterised
as negative for the presence of E. coli O157:H7.
While all melon samples were negative for both bacteria (average
OD450nm 0.09, negative control (NC) OD450nm 0.06), in fresh cut
salads the ELISA method identified few suspected samples. Based
on the NC absorbance values (average OD450nm 0.101), on the
coefficient of variation (CV) among samples and assays and on
the OD450nm values of different concentrations of E. coli O157:H7
and L. monocytogenes reported in the work of Cavaiuolo et al. 4,
the thresholds were set for classifying the samples as sure
negative, suspect and sure positive. Samples with and OD450nm0.3
were considered sure negative and samples with OD450nm0.5 were
considered sure positive for E. coli O157:H7. Only three samples
were considered as suspect: Rocket raw (OD450nm 0.36), mix raw
(OD450nm 0.321) and mix three days-shelf (OD450nm 0.322). Samples
with and OD450nm0.5 were considered sure negative and samples
with OD450nm0.6 were considered sure positive for L. monocytogenes
(Table 1). Based on these thresholds, two suspected samples were
isolated: Mix salad raw (OD450nm 0.52) and mix three days shelf
(OD450nm of 0.566).
Table 1. Detection of E. coli O157:H7 and L. monocytogenes in
rocket and mix salad samples using the ELISA method.
E.coli 0157:H7
L. monocytogenes
OD450nm
OD450nm
Stdev
Stdev
Sample
mean
mean
Rocket raw
0.360 0.204 Suspect
0.449 0.181 Negative
Rocket processed 0.152
0.08 Negative 0.203 0.138 Negative
Rocket shelf-life
0.202 0.105 Negative 0.285 0.171 Negative
Mix raw
0.321
0.08 Suspect
0.519 0.148 Suspect
Mix processed
0.242
0.05 Negative 0.419 0.202 Negative
Mix shelf-life
0.322
0.06 Suspect
0.566 0.228 Suspect
Negative
0.101 0.017
0.095 0.030
Melon raw
Melon processed
Melon shelf-life
Negative
0.061 0.003
0.063 0.001
The use of classical microbiological approaches verified the

presence of E. coli O157:H7 in mixed salad raw and rocket three
days shelf-life and absence in the rest of the samples. Absence of
L. monocytogenes was verified in all samples.
If the samples classified as suspects are considered positives,
then a 22 contingency table summarising the results obtained
by the ELISA can be formed and the samples can be differentiated
into true positives, true negatives, false positives and false
negatives (Table 2). Furthermore, the performance indices of the
newly developed methods used to detect L. monocytogenes and
E. coli O157:H7 can be calculated (Table 3).
Table 3. Performance indices of the newly ELISA method

used to detect L. monocytogenes and E. coli
O157:H7.
Sensitivity
Specificity
Positive predictive value
Negative predictive value
Positive likelihood ratio
Negative likelihood ratio
L. monocytogenes
1
0.94
0.5
1
19.5
0
E. coli O157:H7
0.75
0.94
0.06
0.97
13.87
0.26
Conclusions
Results obtained exhibited the technical efficiency of the
developed methods. More accurately, all methods compared had
similar sensitivity, specificity, negative predictive values and
negative likelihood ratios. False positive results obtained by the
ELISA method resulted in the reduction of positive predictive
values. Regarding their efficacy and viability for routine analysis
it is mostly dependent upon available equipment and technical
expertise.
Acknowledgements
The research leading to these results has received funding from
the European Union Seventh Framework Programme (FP7/20072013) under grant agreement no. 289719 (Project QUAFETY). All
authors contributed equally to this publication.
References
Rico, D., Martn-Diana, A. B., Barat, J. M. and Barry-Ryan, C. 2007.
Extending and measuring the quality of fresh-cut fruit and vegetables:
A review. Trends Food Sci. Tech. 18(7):373-386.
2
Oliveira, M., Usall, J., Vinas, I., Anguera, M., Gatius, F. and Abadias,
M. 2010. Microbiological quality of fresh lettuce from organic and
conventional production. Food Microbiol. 27(5):679-684.
3
Goodburn, C. and Wallace, C. A. 2013. The microbiological efficacy of
decontamination methodologies for fresh produce. Food Control
32(2):418-427.
4
Cavaiuolo, M., Paramithiotis, S., Drosinos, E. H. and Ferrante, A. 2013.
Development and optimization of an ELISA based method to detect
Listeria monocytogenes and Escherichia coli O157 in fresh vegetables.
Anal. Methods 5(18):4622-4627.
5
International Organization for Standardization (ISO) 1996. Microbiology
of Food and Animal Feeding Stuff - Horizontal Method for the
Detection and Enumeration of Listeria monocytogenes. Part 1:
Detection Method. Geneva, Switzerland.
6
International Organization for Standardization (ISO) 2001. Microbiology
of Food and Animal Feeding Stuffs - Horizontal Method for the
Detection of Escherichia coli O157. Geneva, Switzerland.
1
Table 2. The 2 2 contingency table obtained from the

application of the newly ELISA method used to
detect L. monocytogenes and E. coli O157:H7.
L. monocytogenes
E. coli O157:H7
TP
2
3
TN
37
35
FP
2
2
FN
0
1
TP: True positive. TN: True negative. FP: False positive. FN: False negative.
99
WFL Publisher
Helsinki, Finland
www.world-food.net
Composition and content of selected elements of Croatian blackberry wines

Ivana Alpeza *, Tatjana Varga and Veronika Kubanovi
Croatian Centre for Agriculture, Food and Rural Affairs, Institute of Viticulture and Enology, Jandrieva 42, 10000 Zagreb,
Croatia. *e-mail: ivana.alpeza@hcphs.hr
Abstract
The blackberry wine is recognised as a natural source of many bioactive molecules and essential elements that play an important role in health
promotion and disease prevention. The wine is traditionally popular medicine for anemia and iron deficiency. The aim of this work was to evaluate
quality physical and chemical characteristics and concentration of macro elements Ca, K, Mg, Na, essential elements Cu, Fe, Mn, Zn, soil associated
elements Li, Rb, Sr and toxic elements Al, Co, Pb of selected 22 blackberry wines from different regions of Croatia. Basic quality characteristics of
all fruit wines were determined as follows: alcoholic strength (% vol), total sugars (g L-1), total extract (g L-1), ash (g L-1), pH, total acidity (g L-1, malic
acid) and volatile acidity (g L-1, acetic acid). The results indicate that blackberry wines represent high quality beverage. For determination of elements,
fast and precise method of inductively coupled plasma optical emission spectrometry (ICP-OES) was used. Following elements were detected in
investigated blackberry wines in different amounts: macro elements Ca, K, Mg, Na (mg L-1), essential elements Cu, Fe, Mn, Zn (mg L-1), soilassociated elements Li, Rb, Sr (g L-1) and toxic elements Al, Co and Pb (g L-1). It can be concluded that moderate consumption of blackberry wines
may contribute to daily dietary intake of essential elements and wines can be considered as health safe, because potentially toxic elements are kept
under allowable limits.
Key words: Blackberry, fruit wine, elements, ICP-OES.
Introduction
Blackberry (Rubus fruticosus L.) does not get enough credit when
it comes to its health benefits. Ancient civilisations prised
blackberries as a food and traditionally used them for medicinal
and health purposes as well. The ancient Greeks used blackberries
and their juice to treat gout and the Chinese used blackberries to
treat kidney and urinary problems 5. Blackberries were used in
Europe during the16th century as a medicinal plant to treat
infections of mouth and eyes 1. Blackberries are notable for their
health benefits based on high nutritional contents of dietary fiber,
vitamins, folic acid, the essential mineral manganese and other
bioactive compounds 2. Blackberry fruit contains high level of
anthocyanins and other phenolic compounds, mainly flavonols
and ellagitannins, which contribute to its high antioxidant potential
and other biological activities 3, 4. In sense of nutritional value,
daily consumption of blackberry wine in recommended quantities
(about 250 ml) can be a significant dietary source of essential
minerals and could improve health 5, 6. Influence of fermentation
process on aroma composition 7, phenolic, antioxidant capacity
and volatile compounds in blackberry wine 8 were studied. Not
only blackberries, but also their fruit wines rank highly for
antioxidant strength 9, particularly due to their high contents of
phenolic compounds, such as quercetin, gallic acid and
anthocyanins 10. Potent in vitro antioxidant and vasodilatory
effects of Croatian commercial blackberry wines that are roughly
comparable to those of red wines were confirmed 11. On the other
hand, several metals in wine and fruit wine, such as cadmium, lead
and arsenic, are toxic and harmful 26. There is not much available
data on mineral and heavy metal contents in blackberry wines.
Therefore, metal content in wine is regulated according to the
100
national legislation and the legislation of the European Union. As

the production of blackberry wine has been increasing in Croatia
for the last few years, the aim of this study was to determine the
content of macro elements Ca, K, Mg, Na, essential elements Cu,
Fe, Mn, Zn, soil associated elements Li, Rb, Sr and toxic elements
Al, Co, Pb in 22 blackberry fruit wines from different Continental
and Coastal sub-regions of the Republic of Croatia by ICP-OES
method.
Samples: In this study, 22 market ready blackberry wines from
Continental and Coastal region of Croatia were analysed. Ten
wines were in the category of fruit wine and twelve in the category
of dessert fruit wine 12. The vintage comprised the years 2011 and
2012.
Physical and chemical analysis: Alcoholic strength (% vol), total
sugars (g L-1), total extract (g L-1), ash (g L-1), pH, total acidity (g L-1
malic acid) and volatile acidity (g L-1 acetic acid) were determined
by methods described by International Organization of Vine and
Wine OIV 13.
Sample preparation for ICP-OES determination: Four mL HNO3
60 % (Ultra-pure, 101518, Merck, Darmstadt, Germany) was added
to 50 mL of fruit wine sample. This solution was evaporated in a
water bath at 90-95C to reduce the volume to approximately 30 mL,
in order to remove ethanol and therefore, minimise matrix
interferences during analysis and diminish plasma instability,
caused by introduction of organics into the plasma. The sample
residue was quantitatively transferred to volumetric flask and

volume was set to 50 mL with 2% nitric acid 14.
Blanks and standards: HNO3 ( 2%) solution was prepared from
60% HNO3 and ultra-pure water 18 M/cm (obtained from Easy
pure RF, Barnstead, Dubuque, IA, USA), which was used as blank.
Standard solutions containing 2% HNO3 were prepared with
appropriate dilutions to cover the concentration range of each
element in the wines. Standard solutions of Al (13-119), Co (1410), Cu (13-145), Fe (14-17), Mn (13-146), Mg (13-119), Pb (14-18),
Zn (14-47), Li (15-31), Sr (14-90), Rb (14-134), K (14-102), Ca (14-37)
and Na (14-44) were PerkinElmer (Shelton, USA).
ICP-OES method: The validation parameters of method used for
determination of 14 elements is fully described 15 and samples
were analysed using a direct calibration curve.
Instrumentation: Multi-element determinations were carried out
with Perkin-Elmer Optima 2000 DV instrument equipped with a
Meinhard spray chamber, nebulizer and peristaltic sample delivery
system. The instrument was controlled by the ICP WinLab 1.35
Perkin Elmer software. The operating conditions and the
wavelength used for the analysis of each element are given in
Table 1. The flow conditions for plasma gas, auxiliary gas and
nebulizer gas were 15.0, 0.2 and 0.8 L/min, respectively. Power was
set at 1300 W.
Table 1. Operating conditions and wavelengths used
for quantification of each element by ICP
OES method.
Element
Al
Ca
Co
Cu
Fe
K
Li
Mg
Mn
Na
Pb
Zn
Rb
Sr
Wave length (nm)

396.153
317.933
228.616
327.393
238.204
766.490
670.784
285.213
257.610
589.592
220.353
206.200
780.023
407.771
Plasma view
Axial
Radial
Axial
Axial
Axial
Radial
Axial
Radial
Axial
Radial
Axial
Axial
Axial
Axial

The quality physico-chemical characteristics of investigated
blackberry wines are presented in Table 2. The alcoholic strength
for all wines varied between 10.5 and 15.6% vol, which corresponds
to the range published for other Croatian blackberry wines 6, 11.
Values of alcoholic strength were in accordance with the Croatian

Regulation on fruit wine 12, where the fruit wine is defined as a
food product produced by fermentation of fruit juice or must with
a minimum of 1.2% by volume alcoholic strength and dessert fruit
wine is allowed to have addition of sugar or alcohol of fruit origin,
to extent the actual alcoholic strength from 13% vol to 22.0% by
volume for total alcoholic strength.
The content of total sugars in all wines varied between 19.1 and
146.2 g L-1 that was within range published for other Croatian
blackberry wines 16. The content of total extract for all wines ranged
from 44.3 to 174.9 g L-1 while the content of ash ranged from 1.6 to
4.7 g L-1. The pH values for all wines varied between 3.11 and 3.99.
The range of pH values published for the blackberry wines
produced in Croatia is 3.11-3.56 with an average of 3.33 5. The pH
values of Croatian blackberry wines obtained by fermentation
using commercial yeasts (Fermol Rouge and Fermol Mediterranee)
were 3.34 and 3.33 17, respectively, which are the same as published
values 16. The total acidity expressed as g L-1 of malic acid for all
wines varied between 8.0 and 16. Published data of Croatian
blackberry wines total acids 6 ranged from 6.0 to 16.2 g L-1 and
were within the range obtained in this study. The volatile acidity
expressed as g L-1 acetic acid for all wines varied between 0.3 and
1.5 g L-1. Croatian Regulation on fruit wines 12 has a requirement
that fruit wines can contain maximum of 1.5 g L-1 volatile acidity,
meaning that wines submitted to this study had about two times
lower volatile acidity.
Metals are unique nutrients because of their important role in
metabolism. They are essential part of many important enzymes
and they also play roles as catalysts and antioxidants. The majority
of literature data refers to K, Na, Ca and Mg as major elements of
wine and fruit wine. As expected, in this study K was the most
predominant element present in blackberry wines at an average of
1250 mg L-1 (Table 3).
Potassium is known to play an important role in the regulation
of osmotic pressure and neuromuscular, enzymatic, hormonal and
other metabolic activity 18. The concentration of K varied over the
range from 615 to 1760 mg L-1, which is rather wider than range 924
to 1507 mg L-1 published in the earlier study 6. All considered
samples contain a high amount of Ca and Mg, but little quantities
of Na. The concentration of Ca varied from 115 to 555 mg L-1 and
was higher than range from 86.4 to 457.1 mg L-1 obtained in the
earlier study 6. Magnesium is an important element, because it is
essential for many biosynthetic processes, as well as nucleic acid
and protein metabolism. Together with Ca, Mg is known to be
associated with the regulation of heart muscles 19. The
concentration of Mg in blackberry wines ranged from 77 to 238
mg L-1 that was much narrower than published 6 range from 183.2
to 381.2 mg L-1. Furthermore, detected Na concentrations varied
from 3.0 to 13.5 mg L-1. Those concentrations were much lower
Table 2. Quality physico-chemical characteristics of fruit wines (n=10), dessert fruit wines (n =12) and all wines (n=22).
Physico-chemical parameter
Alcoholic strength (% vol)
Total sugars (g L-1)
Total extract (g L-1)
Ash (g L-1)
pH value
Total acidity (g L-1 malic acid)
Volatile acidity (g L-1 acetic acid)
Fruit wine
MeanSD
Min
12.60.9
10.5
97.834.9
44.3
134.944.2
75.8
3.40.6
2.5
3.370.22
3.18
11.71.8
10.0
0.80.3
0.5
Max
13.9
146.2
174.9
4.7
3.62
13.6
1.4
Dessert fruit wine

MeanSD
Min
Max
14.10.9
13.0
15.6
78.941.8
19.1 140.3
112.437.2
44.3 175.0
3.00.6
1.6
4.6
3.420.11
3.11
3.99
11.31.1
8.0
16.0
0.80.2
0.3
1.5
All wines
MeanSD
Min
13.41.2
10.5
87.139.3
19.1
122.241.2
44.3
3.20.6
1.6
3.400.18
3.11
11.41.51
8.0
0.80.3
0.3
Max
15.6
146.2
174.9
4.7
3.99
16.0
1.5
101
Table 3. Concentrations of macro elements Ca, K, Mg and Na in blackberry fruit wines (n=10),
dessert fruit wines (n=12) and all wines (n=22).
Element
(mg L-1)
Ca
K
Mg
Na
Fruit wine
MeanSD
Min
13419
115
1333238
955
16424
122
9.82.3
6.1
Dessert fruit wine

MeanSD
Min
155129
51
1181269
615
14841
77
8.87.0
3.0
Max
171
1760
207
13.3
Max
555
1585
238
13.5
than range from 11.81 to 120.10 mg L-1 obtained for blackberry

wines originated from three different Croatian regions Slavonija,
Prigorje-Bilogora and Zagorje-Medimurje 6. Sodium is required in
human body for the regulation of osmotic pressure and the acidbase balance.
Moreover, as it can be seen in Table 4, detected concentrations
of essential elements Cu, Fe, Mn and Zn in blackberry wines were
0.051-3.83 mg L-1, 0.093-5.49 mg L-1, 0.472-11.3 mg L-1 and 0.3041.96 mg L-1, respectively.
Copper and iron are essential in blood formation and copper is
involved in normal carbohydrate and lipid metabolism. Copper
acts as a cofactor of enzymes involved in various metabolic
processes within living cells 20. Reported range 6 of copper 0.0580.767 mg L-1 was much narrower than results of this study, while
concentrations of iron ranged from 0.082 to 6.273 mg L-1 and Mn
from 1.47 to 11.53 mg L-1 and they were very close to the values
obtained in this study. Zinc is an important microelement, because
it acts as a cofactor of enzymes involved in metabolic reactions.
The published data 21 of Zn concentration that ranged from 0.557
to 3.569 mg L-1 were much higher in comparison to our results.
The concentration of three elements that are soil-associated
elements, Li, Rb and Sr in investigated blackberry wines were 140, 90-1470 and 165-1445 g L-1, respectively (Table 5).
Red wines from the Province of Chieti and the Province of
Teramo 22 had Rb concentrations of 1815.36 and 1599.7 g L-1 ,
respectively, that are much higher than those obtained for
blackberry wines. At the same time, concentration of Sr in red
All wines
MeanSD
Min
14695
115
1250261
615
15635
77
9.235.3
3.0
Max
555
1760
238
13.5
wines varied from 395.58 in the Province of Chieti to 458.36 g L-1 in

the Province of Teramo 22 and were close to the concentration
obtained for investigated blackberry wines (439 g L-1). The
concentration of Sr for red wines from two wine producing Spanish
regions (Ribera del Guardiana and Mntrida) 23 were 379 and
981 g L-1. The concentration of Sr of blackberry wines was closer
to that for red wines from region Ribera del Guardiana. The
concentrations of Li in red wines from two regions 23 were 48 and
69 g L-1, which is much higher than concentration of 21 g L-1 in
blackberry wines.
In general, the toxic effects of aluminium result from its
competition with other metal ions in enzymes and proteins. As the
aluminium ion substitutes the metal at its binding site, the function
of the protein is changed and the metabolism of the cell is altered,
consequently affecting the organism gravely 24. The concentration
of toxic elements Al, Co and Pb in the investigated blackberry
wines were 37-1110, 2-40 g L-1 (six samples had concentration
below the detection limit of the method) and 17-154 g L-1,
respectively (Table 6). The concentration of Al found in the
previous study 25 ranged from 6.01 to15.73 mg L-1 , much higher
than concentration obtained in this study. Concentration of Co
varied from 1.29-4.43 g L-1, which is far below concentration found
in this study and concentration of Pb varied from 13.56 to 52.81 g
L -1 , where maximum value was three times higher than
concentration obtained in this study. It should be noted that
concentration of Pb was below the internationally established
maximum allowed values.
Table 4. Concentrations of essential elements Cu, Fe, Mn and Zn in blackberry fruit wines (n=10), dessert
fruit wines (n=12) and all wines (n=22).
Element
(mg L-1)
Cu
Fe
Mn
Zn
Fruit wine
MeanSD
Min
0.6551.127
0.076
1.8391.412
0.731
5.7483.069
1.12
0.9440.425
0.304
Max
3.83
5.49
10.56
1.96
Dessert fruit wine

MeanSD
Min
0.2540.155
0.051
1.0580.628
0.093
4.3623.483
0.472
0.8310.375
0.360
Max
0.53
2.22
11.3
1.74
All wines
MeanSD
Min
0.4360.774
0.051
1.4131.104
0.093
4.9923.299
0.472
0.8820.393
0.304
Max
3.83
5.49
11.3
1.96
Table 5. Concentrations of Li, Rb and Sr in blackberry fruit wines (n=10), dessert fruit wines (n=12) and all
wines (n=22).
Element
(g L-1)
Li
Rb
Sr
Fruit wine
MeanSD
Min
2618
2
414173
90
385114
240
Max
40
716
576
Dessert fruit wine

MeanSD
Min
Max
1815
1
40
631415
160
1470
483345
165
1445
MeanSD
2116
532340
439266
All wines
Min
1
90
165
Max
40
1470
1445
Table 6. Concentrations of toxic elements Al, Co and Pb in blackberry fruit wines (n=10), dessert fruit wines
(n=12) and all wines (n=22).
Element
(g L-1)
Al
Co
Pb
102
MeanSD
290138
139
5020
Fruit wine
Min
136
4
30
Max
490
30
100
Dessert fruit wine

MeanSD
Min
297290
37
1411
2
6143
17
Max
1110
40
154
MeanSD
294229
1411
5634
All wines
Min
37
2
17
Max
1110
40
54
Conclusions
The selected quality physical and chemical characteristics indicate
that blackberry wines were in accordance with the Croatian fruit
wine legislation. Therefore, they represent high quality beverage.
The concentration of most of the investigated elements is different
among blackberry wines. Results of Cu, Fe, Mn and Zn, the most
important essential elements, lead to the conclusion that blackberry
wines can be considered as good source of these elements. The
concentrations of toxic elements, such as Co and Pb, did not
exceed the limits given by the Croatian regulations, which leads
to the conclusion that organic and mineral fertilizers, inorganic
pesticides and other means of blackberry growing and winemaking
practice are safe. All these parameters suggest that Croatian
blackberry wine is safer than the wines mentioned above. One
explanation might be the absence of environment pollutants, such
as heavy industry and automobiles exhaust gases around orchard
where blackberry was grown. It can be concluded that moderate
consumption of blackberry wines may contribute to daily dietary
intake of essential elements and blackberry wines can be
considered as health safe, because potentially toxic elements are
under allowable limits.
Acknowledgements
The authors would like to thank to the producers for the support
of this study.
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Gropper, S. S., Smith, J. L. and Groff, J. L. 2009. Advanced Nutrition
and Human Metabolism. 5th edn. Wadsworth, Cengage Learning,
Belmont, CA, pp. 443-450.
20
Arredondo, M. M. and Nez, T. 2005. Review: Iron and copper
metabolism. Mol. Aspects Med. 26:313-327.
21
Amidi Klari, D., Klari, I., Veli, D. and Vedrina Dragojevi, I. 2010.
Blackberry wine as a good source of essential mineral nutrients. In
ubari, D. (ed.). Book of Abstracts of the International Scientific and
Professional Conference 13th Ruika Days Today Science-Tomorrow
Industry. Faculty of Food Technology Editions, Osijek, pp. 45-45.
22
Cichelli, A. 2013. ICP-MS analysis for the characterization of the origins
of wines. Agro. Food Industries 24(1):30-34.
23
Garca-Rodrguez, G., Hernndez-Moreno, D., Soler, F. and PrezLpez, L. 2011. Characterization of Ribera del Guadiana and
Mntrida Spanish red wines by chemometric techniques based on
their mineral contents. J. Food Nutr. Res. 50(1):41-49.
24
Exley, C. and Birchall, D. 1992. The cellular toxicity of aluminium. J.
Theor. Biol. 159(1):83-98.
25
Amidi Klari, D., Klari, I., Veli, D. and Vedrina-Dragojevi, I. 2010.
Determination of trace elements content in blackberry wines by graphite
furnace atomic absorption spectrometry. In Zorc, B. (ed.). Knjiga
saetaka 4. hrvatski kongres farmacije s meunarodnim sudjelovanjem,
Zagreb. Hrvatsko farmaceutsko drutvo, 199 p.
26
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Meta-analysis of target hazard quotients reveals health risks. Chemistry
Central Journal 2:22.
11
103
WFL Publisher
Helsinki, Finland
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Physicochemical properties of honey samples from Ondo state, Nigeria, and their
bioactivity against spoilage and pathogenic organisms
Funmilola Oluyemi Omoya 1, Oluwatosin Ademola Ijabadeniyi
1
2, 3
* and Olayemi Bosede Ogonnoh
Department of Microbiology, Federal University of Technology, Akure, Nigeria. Department of Biotechnology and Food
Technology, Durban University of Technology, South Africa. *e-mail: tosynolu@yahoo.com
2, 3
Received 22 January 2014, accepted 6 September 2014.
Abstract
Honey can be defined as the natural sweet substance produced by honeybees (Apis mellifera) from the nectar of blossoms or from the secretion of
living parts of plant or plant sucking insects living on parts of plants. The medicinal property of honey has been an area of interest to researchers in
recent times. This study focused on assessing the physicochemical components of honey samples and their bioactivity on some food spoilage
organisms. One hundred samples of honey were collected from different locations in Ondo state, Nigeria. Their physicochemical components which
include conductivity, ash content, moisture content, pH, mineral contents and colour were determined. There was variation in the physicochemical
components of some of the honey samples with reference to international standards. The assessment of the honey samples as an antibacterial agent
revealed it inhibitory potency on both bacteria and fungi isolated from food sample. The inhibitory effect was compared with that of standard
antibiotic. The honey samples were seen to display a higher inhibitory effect on the tested organisms than the employed antibiotic.
Key words: Bioactivity, honey, physicochemical, food spoilage organisms, antibiotics.
Introduction
Honey is a sweet food made by bees using nectar from flowers 18.
The variety produced by honey bees (the genus Apis) is the most
commonly referred to and is the type of honey collected by
beekeepers and consumed by humans 12. Honey produced by
other bees and insects has distinctly different properties. Honey
bees form nectar into honey by a process of regurgitation, and
store it as a primary food source in wax honeycombs inside the
beehive. Beekeeping practices encourage overproduction of
honey so the excess can be taken from the colony. Honey gets its
sweetness from the monosaccharides fructose and glucose, and
has approximately the same relative sweetness as that of
granulated sugar 18. It has attractive chemical properties for baking,
and a distinctive flavor that makes some people to prefer it over
sugar and other sweeteners. Most microorganisms do not grow
in honey because of its low water activity of 0.6 13. However,
honey sometimes contains dormant endospores of the bacterium
Clostridium botulinum, which can be dangerous to infants, as
the endospores can transform into toxin-producing bacteria in
the infants immature intestinal tract, leading to illness and even
death 22.
Honey has a long history of human consumption, and is used
in various foods and beverages as a sweetener and flavoring 12. It
also has a role in religion and symbolism. Flavors of honey vary
based on the nectar source, and various types and grades of
honey are available. It is also used in various medicinal traditions
to treat ailments. The study of pollen and spores in raw honey can
help to determine floral sources of honey. Furthermore, because
bees carry an electrostatic charge, and can attract other particles,
the same technique of its pollen and spores study can be used in
area environmental studies of radioactive particles, dust or
particulate pollution 16. The term traditional medicine (indigenous
104
medicine or folk medicine) essentially represents a natural form of

health care which has been used through generations 1.
Traditional medicine practices existed in Africa and other cultures
for centuries since man came into being but until recently, has
been neglected or even outlawed in some cases due to undue
pressure from practitioners of modern medicinal practice and
unscientific background of its method of operation 8. The 21st
century is witnessing serious scientific effort to discover major
active ingredients in medicinal plants through research and
development. According to Osermene et al. 20, this could help
orthodox medicine to comprehensively address most disease
conditions plaguing mankind or it may be a response to the clarion
call by the World Health Organization 17 that developing countries
should endeavour to develop and utilize local medications that
are most appropriate to their local circumstances especially for
Primary Health Care (PHC) in order to reduce the cost associated
with incessant drug importation 17. Traditional medicine is the
sum of all knowledge and practical application, whether explicable
or not used in diagnosis, prevention and elimination of physical,
mental or social imbalances and relying exclusively on practice
and experience and the sociological environment 5, 23. Most of
these medicines produced traditionally entail herbs from different
plants, honey, Aloe vera, and from bitter tasted plants 15. This
present study was aimed at investigating the physicochemical
properties of honey samples in Ondo state, Nigeria and their
inhibitory potency on spoilage bacteria isolated from food samples.
Study area: The Ondo state of Nigeria is one of the states in
south west Nigeria (7100" N and 5 50"E). It covers 15,500 km2,
with an estimated population in the 2006 census of 3,440,000
inhabitants. The climate condition of this state is classified as

tropical, with a mean temperature ranging from 25C to 29C year
round. It has a minimum and maximum temperature between 21.6
and 33.8C respectively; and a mean relative humidity of 85%.
Sample collection: The honey samples were purchased from
different honey sellers from various locations in the Ondo state of
Nigeria namely; Akure, Owo, Akungba, Ile-oluji, Ondo town, IkareAkoko, Ilara, Igbara Oke, Itaogbolu and Iju. They were taken to
the laboratory and screened for their microbiological and
physicochemical properties. The local food samples namely rice,
beans moin-moin, pounded yam and amala were collected into
sterile Petri dishes from selected cafeteria in Akure and were taken
to the laboratory for microbiological examination.
Antibacterial activity of honey samples and antibiotic sensitivity

tests: An agar diffusion method was used to access the
antibacterial activity of the selected honeys against the 5 isolated
bacterial strains. A cell load of 107-108 cfu/ml in their log phase of
isolated bacteria were inoculated in Mueller- Hinton broth and
incubated for 10 h. Petri dishes containing 1ml of each bacterial
isolate in the broth and Mueller-Hinton agar were prepared. Wells
were bored with a sterile cork borer on the seeded agar plates. The
honey, water and antibiotics were aseptically pipetted into the
bored well and the plates were incubated uninverted at 37C for 24
h. The water and antibiotics served as negative and positive
controls, respectively. The sensitivity of the tested organisms
was indicated by zones around the wells and diameter was taken
as an index of degree of sensitivity.
Microbiological examination of honey and food samples: The

fresh honey samples were conveyed to microbiology research
laboratory of Federal University of Technology, Akure. The honey
samples were filtered separately with sterile Seitz filter and the
obtained filtrates were aseptically streaked on nutrient agar and
potato dextrose agar plates and incubated at 37C for 24 h and
272C for 72 h, respectively, to observe for bacterial and fungal
growth. After incubation, the honey plates where no growth were
observed were dispensed into sterile Pyrex sample bottles and
kept at room temperature prior to further analyses. One g of each
of the food samples was weighed and with the use of sterile spatula
homogenized inside 9 ml of sterile distilled water. This was serially
diluted and 0.1 ml was pour plated on nutrient agar and potato
dextrose agar plates and incubated at 37C for 24 h and 272C for
72 h, respectively, to observe for bacterial and fungal growth.

Bacteria and fungi isolated from the tested food samples included
Bacillus cereus, Bacillus subtilis, Escherichia coli, Aspergillus
fumigates, Salmonella typhi and Varicosporium elodea (Table 1),
most of which are pathogenic microorganisms. However, Bacillus
subtilis is also a spoilage bacterium. Varicosporium elodea is
spoilage fungus while Aspergillus fumigates, a fungus, has a
potential to produce mycotoxins.
Most foods contain viable bacteria and fungi, which could be
result of improper handling, exposure to dust, air, flies or result of
the food being under heated. These food spoilage organisms can
also be pathogenic to consumers. Endospores of Bacillus species
are more resistant to heat which could be the reason why this
species occurred most in the food samples. Physiologically, these
organisms could produce chemical changes in foods, such as
breaking down of proteins to polypeptides, amino acids, fats to
Physico-chemical analyses of honey samples: The pH of the glycerol and fatty acids, and hydrolysis of complex carbohydrates
samples was measured using a pH meter according to the method to simple ones. Unpleasant odours resulting from gases formation
described by Gonnet 9. The moisture content was obtained by may occur. Spoilt rice is by nature slimy and rotten. This nature
drying the granulated pulp in a hot air oven under vacuum at readily provides a veritable and suitable environment for the
105C until a constant weight was attained and the ash content growth of fungal isolate like Aspergillus fumigates. This was in
by incinerating dried samples in a muffle furnace at 550C for 6 h. agreement with the work of Leveen et al. 14. Vit et al.26 reported
They were then cooled in a desiccator and weighed immediately 4. that the pH and temperature of food substrate readily support the
The mineral contents were analyzed using the atomic absorption growth of mesophilic food spoilage organisms such as fungal isolates.
and spectrophometer method as described by Paulwels et al. 21.
The physicochemical properties of the different samples of
The minerals analyzed included calcium, potassium, sodium and honey obtained from different locations in Ondo, Nigeria, are
phosphorus. Vitamin C content was also analyzed. Electrical reported in Table 2. The color ranged from light amber to completely
conductivity was determined by measuring 20 g dry matter of dark. The pH values were in the range of 2.90 to 4.40. Although
honey in 100 ml of ultra pure water. This was mixed thoroughly to there was significant variation between honey obtained from
form a solution. The electrical conductivity cell was immersed at Itaogbolu and the remaining locations. The values were within
20C while reading was expressed in mhos. The colour of the the reference value and in agreement with White 11 who reported
honey samples was determined by using the P- fund scale (mm). that honey was characteristically quite acidic. The pH of honey is
Two ml of the honey samples was poured in a beaker, the instrument low enough to inhibit the growth of many species of bacteria but
was calibrated, dipped into the sample and the reading was taken. this acidity is neutralized in the body by buffering fluids. The
moisture content investigated varied from 13.28-17.98%. The
lowest was recorded in Igbara Oke sample while the highest was
from Ikare Akoko samples. This could
Table 1. Frequency of microorganisms isolated from selected food samples.
be as a result of the composition and
floral origin of the honey samples. The
Food samples
Bacillus
Bacillus Escherichia Aspergillus Salmonella
Varicosporium
low moisture content property of
cereus
subtilis
Coli
fumigatus
typhi
elodea
honey serves as a protection from
Beans
+
+
+
+
Yam
+
+
+
+
attack by microorganisms 25. When the
Moin- moin
+
+
+
+
+
moisture content is high, it is an
Amala
+
+
+
indication of adulteration. According
Rice
+
+
105
106
15.21.03
0.55.01
0.1801
0.28.02
0.11.02
0.13.02
0.05.01
0.60.0.2
0.10.0.1
0.70.0.1
0.30.0.1
Ash
(%)
38.50.02
3.60.0.1
21.00.0.2
4.00.0.1
17.60.0.1
19.20.0.1
45.00.0.2
12.50.0.1
51.50.0.2
23.00.0.2
Electrical
conductivity
(mhos)
1.50.01
2.00.01
1.60.02
2.90.02
2.60.01
2.10.01
1.00.03
1.40.02
1.80.01
1.90.03
Ca2+
0.68.01
0.19.01
1.32.02
0.70.02
1.60.01
0.20.01
1.36.02
0.65.01
1.11.03
0.50.01
K+
22.451
21.241
20.012
22.981
22.012
32.422
50.431
18.611
44.901
50.002
Mineral content(g/g)
20.28
9.68
20.30
20.28
17.69
40.22
42.515
75.26
30.91
30.31
Na+
NR
102
mm
NR
102
mm
NR
NR
NR
C
112
mm
122
mm
152
mm
B
132
mm
102
mm
162
mm
A
142
mm
132
mm
182
mm
21
mm
162
mm
NR
NR
NR
NR
Selected honey samples

D
E
F
G
122
132
122
112
mm
mm
mm
mm
132
122
142
102
mm
mm
mm
mm
152
202
182
152
mm
mm
mm
mm
21
NR
NR
NR
mm
102
152
102
102
mm
mm
mm
mm
NR
102
mm
NR
H
132
mm
122
mm
152
mm
NR
I
152
mm
162
mm
212
mm
102
mm
182
mm
NR
102
mm
NR
J
92
mm
112
mm
182
mm
NR
102
mm
NR
AM
102
mm
102
mm
112
mm
NR
182
mm
NR
CPX
132
mm
102
mm
172
mm
NR
112
mm
NR
51
mm
NR
202
mm
NR
71
mm
NR
NR
NR
GRI
NR
PEF
122
mm
122
mm
202
mm
35.121
32.341
30.323
31.331
32.422
32.422
33.453
30.234
35.542
34.231
32.422
Glucose
Key: NR= No zone of inhibition A=Akure, B= Owo, C= Akungba, D= Ile-oluji, E= Ondo town, F=Ikare- Akoko, G=Ilara, H=Igbara Oke, I=Itaogbolu, J=Iju, AM=Ampiclox, CPX=Ciprofloxacin, S=Streptomycin,
PEF=Pefloxacin, GRI= Griseofulvin.
Tested
organisms
Bacillus
Cereus
Bacillus
subtilis
Escherichia
Coli
Aspergillus
fumigatus
Salmonella
typhi
Varicosporium
elodea
Light
Amber
Light
Amber
Amber
Amber
Amber
Amber
Amber
Dark
Amber
Dark
Amber
Light
Amber
Color
Antibiotics
S
102
mm
122
mm
132
mm
1.40.02
4.30.01
3.40.01
1.80.02
2.35.03
1.90.02
3.40.01
1.25.01
3.75.01
2.00.02
Vitamin C
(mg/10ml)
Table 3. Antibacterial activity of honey samples and antibiotic sensitivity test on food spoilage organisms.
Reference values from Tchoumboue et al. 24, Vinda-Martos et al .2,Anon., 3; Crane 6,7.
3.12.20
Iju
13.28.03
3.89.11
14.14.02
13.61.01
4.00.21
2.90.07
17.98.01
3.21.12
Itaogbolu
16.00.02
16.75.01
16.21.01
3.27.05
4.40.01
Ile-oluji
Ondo
town
IkareAkoko
Ilara
Igbara
Oke
4.40.02
Akungba
17.00.01
13.40.02
4.35.15
3.28.02
Moisture
(%)
pH
Owo
Akure
Honey
samples
Table 2. Mean valuesSE of physicochemical properties of selected honey samples.
32.422
33.661
30.121
28.422
27.524
29.123
31.003
31.225
30.112
29.421
33.122
Fructose
Color=light to
amber
Vitamin C = ___
Na+ = 0.6- 40
P=2.0-60.0
K =1.0-47.5
Ca2+ =4.0-30.0
Electrical
conductivity= __
Ash=0.020-1.030
Moisture(%)=13.426.6
pH=3.4-6.1
Glucose=
22.0-40.7
Fructose= __
Reference values
to the international regulatory standard for honey 10, honey with

high water content aids fermentation or deterioration. Values for
the ash content ranged from 0.10% to 0.70%. This variability in
ash content could be explained by the floral source of the honey 26.
Obtained values are within the reference value. The mineral content
from Akungba Akoko and Ondo town turned out to be in
decreasing order of Na, P, Ca and K while in samples from the
remaining locations, the phosphorus content is highest followed
by sodium and the least mineral content is potassium. The presence
of these minerals makes it nutritionally suitable for both children
and elderly people. Variation in mineral content was recorded from
the reference value in some honey samples for instance the sodium
ion content samples from Akungba Akoko and Ile Oluji did not
agree with reference value. The electrical conductivity values
varied from 3.60 to 51.50 mhos. According to International Standard
of honey (2002), the electrical conductivity of honey is near zero
hence higher conductivity indicates adulteration of the honey sample.
The bioactivity of honey samples compared favourably with
the tested conventional antibiotics. The honey sample showed
more inhibitory potency against the tested bacteria than the fungal
isolates (Table 3). Generally honey sample from Itaogbolu
displayed high inhibitory potency against all the tested organisms
with the highest zone of inhibition recorded against Escherichia
coli (212 mm), this in no doubt could be because of the
physicochemical quality this sample possessed, although its
bioactivity against fungal isolates was low compared to the
conventional antibiotics. In all the honey samples tested none
was able to inhibit Varicosporium elodea. The antibiotic
griseofulvin, was able to inhibit Varicosporium elodea with 52
mm zone of inhibition while (71mm) was recorded for Aspergillus
fumigatus, however, honey samples from Itaogbolu showed
greater zone of inhibition than this antibiotic (10 2 mm). This
study revealed that the honey samples possess different
antimicrobial activities, which agreed with the study conducted
by Omoya and Akharaiyi 19 that honey in its saturated solution of
sugar will cause osmotic effect on bacteria.
Conclusions
The results confirm that the physicochemical parameters, such as
moisture content, pH level and sugar content of different honey
samples, influence their bioactivity against microorganisms. The
honey samples were able to establish variable inhibitory zones in
vitro. Favourably comparison was established between the honey
samples and the conventional antibiotics employed on the tested
spoilage organisms. Honey may therefore be a potential eco-friendly
intervention to control food spoilage and pathogenic microorganisms.
Acknowledgements
Acknowledgements to Omoya and Associates for funding of this
study. Acknowledgements also to Fred Akharaiyi of Microbiology
Department FUTA and Department of Biochemistry FUTA for their
technical assistance.
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3
107
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Shigatoxin-producing Escherichia coli in raw cow milk from small farm producers
and phylogenetic subtype determination
Ivo Sirakov, Ralitsa Popova, Hristo Daskalov *, Iskra Slavcheva, Eva Gyurova and Boyko Mitov
National Diagnostic and Research Veterinary Institute, Blvd. Pencho Slaveykov 15, 1606 Sofia, Bulgaria.
*e-mail: hdaskal@hotmail.com
Abstract
Cases of unauthorised direct sale of raw cow milk from small farms do exist in Bulgaria. In 2012, we tested 80 samples of raw cows milk from small
farms in south-western Bulgaria for the presence of shigatoxin-producing Escherichia coli strains. Twenty-three of these samples were taken from
cows with subclinical mastitis. The tests included isolation on selective solid medium, biochemical detection and conventional PCR for detection of
the shigatoxin genes (stx1, stx2), the intimin gene (eae) and the enterohemolysin gene (hlyA). The stx1 gene was detected in three Escherichia coli
isolates from three cows milk samples from animals with subclinical mastitis. An Escherichia coli strain with the intimin eae gene was isolated from
a sample of normal cows milk from a small farm. Two of the isolates with the stx1 gene were also found to harbour the enterohemolysin hlyA gene.
The presence of virulence factors in the four Escherichia coli isolates was additionally validated using Real-time PCR. The amplified virulence genes
were sequenced and a phylogenetic analysis based on amino acid sequences was performed. The cytotoxic effects of the E. coli isolates were studied
on Vero cells. The potential role of shigatoxin-producing E.coli strains for provoking food-borne illness is discussed.
Key words: Escherichia coli, raw cow milk, mastitis milk, PCR, virulence factors.
Introduction
Raw milk produced in very small farms which do not meet the EU
quality and safety requirements could be a risk factor for consumer
health. Raw cows milk from such farms is on the market in violation
of the regulations for the sale of raw milk. There is also a hazardous
practice of mixing milk from healthy animals and subclinical mastitis
milk. Escherichia coli is a microbial agent which is often found in
raw milk, especially in cases of poor hygiene during milking and
primary processing. E. coli causes mastitis or concomitant
infections in lactating cows. According to Schoonderwoer 1, E.
coli is naturally found in the gastrointestinal tract in animals, but
some strains, such as shiga-toxin-producing E. coli, are associated
with diarrhea in humans and animals. Other authors 2, 3 emphasise
that verotoxin-producing E. coli (VTEC) can cause serious illness
in humans, beginning with mild diarrhea and leading to hemorrhagic
colitis and hemolytic uremic syndrome. Mainil 4 point out that a
key factor for E.coli infections in humans are ruminants excreting
E.coli in their feces. VTEC may be transmitted through
consumption of undercooked meat, unpasteurised milk products,
vegetables and water contaminated with the feces of carrier animals.
Cases of person-to-person transmission are also known 3, 5, 6. Animals
intended for food are considered to be the main source of VTEC/
EPEC (enteropathogenic E.coli) strains. In humans, life-threatening
infections associated with consumption of milk and milk products
contaminated with VTEC/EPEC have been reported 7. The main
etiological agent in infections with enterohemorrhagic E.coli is
generally thought to be Escherichia coli O157:H7. It is still unclear,
which other factors in addition to verotoxin production are
involved in the transformation of shigatoxin-producing-E.coli
(VTEC) into enterohemorrhagic E. coli (EHEC). Each VTEC isolate
should be considered potential EHEC and the detection methods
108
should be targeted at detection of verotoxin rather than of the

serotype 8. Another key factor in the virulence of VTEC isolates is
enterohemolysin (Ehly), which is coded by the enterohemolysin
ehxA gene, also known as EHEC-HlyA. The relation of EHEC
hemolysin and verotoxin production in pathogenic E.coli
describes the possible role of EHEC hemolysin in bacterial
virulence 9. Subclinical mastitis caused by E. coli brings about
substantial economic losses in dairy cattle herds because of
reduced amount and quality of the produced milk, incurred
veterinary treatment costs and in some cases death of the diseased
animal 10. Milk can be contaminated with pathogenic microorganisms
through fecal contamination as well as from colonized teats or
from an infected udder in the case of clinical or subclinical mastitis
during the milking process from the environment or from
contaminated water 11. Polymerase chain reaction (PCR) is an
effective method for detection of extremely low quantities of
nucleic acids in various types of samples. That is why PCR is a
common technique used for detection of various food-borne
pathogens.
The aim of the present study was to analyse samples of raw
cows milk, including milk taken from cows with subclinical mastitis,
for the presence of E. coli isolates with specific virulence markers
(stx1, stx2 and eae) by means of PCR and to evaluate their possible
role in provoking food-borne illness in consumers.
A total of 80 raw cow milk samples were analysed. The milk was
collected from January to December 2012 from unauthorised
sources (small farms or individual sellers).
Mastitis assay: All milk samples were analysed according to an

official reference method, the microscopic method for counting
somatic cells described in the International Standard ISO 133661|IDF 148-1 12.
Isolation of Escherichia coli: To obtain Escherichia coli
isolates, 10 g of each sample were weighed and added to 90 ml
of buffered peptone water and the resulting suspensions were
incubated at 37C for 24 h. Then, the broth was used for plating
on selective nutrient media: Sorbitol McConkey Agar (SMAC,
Merck, Darmstadt, Germany), Tryptone Bile X-Glucuronide Agar
(TBX, Merck, Darmstadt, Germany) and McConkey Agar
(HiMedia, India). The plates were incubated for 18 h to 24 h at 37C.
Following cultivation, the selective media were observed for
typical growth. Escherichia coli isolates were simultaneously
analysed for the presence of the stx1, stx2 and eae genes.
Biochemical identification: For biochemical identification typical
colonies of Escherichia coli from the selective media were plated
on nutrient agar and further identified with Kovacs reagent
(Merck, Darmstadt, Germany) for the presence of indole and with
the oxidase test (Oxidase test, HiMedia, India). The isolates which
were positive for stx1 and eae genes by conventional and RT PCR
were also characterised based on 21 specific biochemical
reactions 11 MICRONAUT-E plates (Merlin, Germany).
Identification of serotype O: Whether the isolates identified as
E. coli belong to serotype O was determined with E. coli OK
Pool 1 (O26, O103, O111, O145 and O157) sera obtained from the
Staten Serum Institute (Copenhagen, Denmark) based on
agglutination reaction.
DNA extraction: A minimum of 10 colonies with typical E.coli
morphology were selected from the selective agar plates and were
re-plated on nutrient agar (HiMedia, India) for 18 h to 24 h at 37C.
For conventional PCR amplification of the stx1, stx2 and eae genes,
DNA extracted from the isolated colonies, was resuspended in
100 l of sterile distilled water and heated to 100C for 5 min. DNA
was extracted again from the three stx1 positive colonies and the
eae positive colony with InstaGene DNA Purifica Matrix (BioRad,
Hercules, CA, USA) to validate the positive amplification results
by means of real-time PCR.
Conventional PCR: DNA extracted form E. coli isolates was
amplified by conventional multiplex PCR with primers specific for
the stx1, stx2 and eae gene described in previous research 13.
The PCR reaction was performed with the HotStart-ITFideliTaq
USB Mastermix (Affymetrix, Santa Clara, CA, USA), according to
the manufacturers instructions. The final reaction mixture volume
was 25 l. The primers were added at a final concentration of 10
pmol/l each. All samples were compared with a positive control
strain C210-03 provided by WHO in Copenhagen, which harbours
the three genes. The samples were proved to be contamination
free with a negative control containing distilled water instead of
DNA.
The amplification of stx1, stx2 and eae genes was performed as
described by Paton and Paton 13 with some modifications for this
study. Amplification of the stx1, stx2 and eae genes was performed
in a Techne thermocycler as follows: 95C/5 min, 40 cycles of
95C/1 min, 62C/1.30 min, 72C/1.30 min and 72C/7 min. The
amplification for HlyA was according to Schmidt et al. 9. The
electrophoresis of the amplified products was run in 2% Agarose
(Genshun Biotech, Guangzhou, China), 1Tris-EDTA buffer,
pH 8.1, 120 V, 40 mA for 30 min. A 100 bp molecular weight
marker (Genshun Biotech, Guangzhou, China) was used. Ethidium
bromide 1 g/ml (Sigma-Aldrich, EU) was used for staining.
Real time PCR: The sequences of the primers and probes used in
the real-time PCR assay are according to Perelle et al. 14 and Nielsen
and Andersen 15.
A 5 nuclease PCR assay was used with probes labeled with
FAM as a reporter dye at the 5-end and TAMRA as a quencher
dye at the 3-end. The Maxima Probe/RoxqPCR Master Mix (2)
(Fermentas, Lithuania) was used, following the manufacturers
instructions. The reaction mixture was with a final volume of 20 l.
For amplification of the stx1 and the stx2 gene the following
reagents were used: 4 mM MgCl2, 1 M of each primer, 200 nM
probe and 2 l of template DNA, according to Perelle et al. 14. For
amplification of the eae gene, the final concentration of the probe
was 200 nM and that of each primer was 600 nM, according to
Nielsen and Andersen 15. The pUC19 plasmid (New England
BioLabs, MA, USA) was employed as an internal amplification
control with the primers and the probe described by Fricker et al. 16
and IAC concentration of about 100 copies per PCR reaction.
Amplification was performed in a Step One Plus apparatus
(Applied Biosystems, Foster City, CA, USA), according to Perelle
et al. 14 for the stx genes and Nielsen and Andersen 15 for the eae
gene.
Cell culture assay: Stocks of Vero cell line purchased from Robert
Koch Institute were used after trypsinisation and growth as a
monolayer at 37C in rotation. The procedure by Konowalchuk et
al. 17 was used with some modifications. MEM-Eagle in Earles
BSS (GIBCO) was used as a medium for Vero cells with addition of
0.2 M L-glutamine, 0.075% NaHCO3 for pH 7.4 and fetal calf serum,
10% for growth medium and 2% for supporting medium (GIBCO)
and Gentamycine 50 mg/mL. A quantity of 0.05 mL of bacterial
filtrate from a 24 h bacterial culture was added to the cells for toxin
activity assay. Cultures were incubated at 37C. Morphological
effects of the cultures were screened on 18, 24, 48, 72 and 96 h.
16S rRNA gene amplification, sequencing and data processing:
As additional identification we used 16S rRNA gene sequencing
as described before 18. Nucleotide sequence alignment was
performed with the Muscle algorithm 19. The sequencing data of
the 16S rRNA gene were analysed for close homology using the
Basic Local Alignment Search Tool (BLAST) available at the
National Center for Biotechnology Information (NCBI, Bethesda,
MD) (http // :www .nbi .nlm. nih. Gov /BLAST) 20, 21.
Only nonsynonymous substitutions could lead to changes in
the amino-acid structure of proteins. Because of that, for the
purpose of amino-acid analysis of the PCR products, we used the
nucleotide sequences with a length of 180 bp (between 746-925
bp determined by reference sequence AF461169.1) and 357 bp
(between 40-396 396 bp determined by reference sequence
AJ877229.1), respectively, as previously described 22, corresponding
to the stx1 and eae genes, the number for the eae gene nucleotide
sequence in GenBank being KC196849. The predicted amino-acid
109
sequences were obtained using the MEGA 5 software 23.

The protein sequencing data was compared with sequences
deposited in NCBI GenBank (Table 1). These sequences were
used to build amino acid phylogenetic trees.
To find the best model for the construction of phylogenetic
trees based on amino acid sequences, ProtTest 2 24 was used.
PhyML 3.0 software 25 by Phylemon 2 26 and 1000 bootstrap
replications were used in building the phylogenetic trees. The
graphical view of the phylogenetic trees was generated with
FigTree1.4.0 software (http://tree.bio.ed.ac.uk/software/figtree/).
The amino acid sequence of the eae gene region of isolate
number 7 has an ID: AGC29881.1 for access in NCBI. The amino
acid sequence of the stx1 gene was not sent to NCBI due to its
short length.
Results
Escherichia coli was isolated from 56 of the 80 analysed raw
cows milk samples, whereas typical growth was not observed
in 24 samples. An increased somatic cell count (500,000 SC/
cm3) was scored in 23 cows milk samples. These 23 samples were
also shown to contain E. coli. The appearance of the milk and the
results from the microscopic reference method confirmed that the
animals were suffering from subclinical inflammation of the
mammary gland.
The 56 isolates showed the indole-positive and oxidasenegative biochemical profile typical for Escherichia coli. Next,
the 56 Escherichia coli isolates were assayed by conventional
PCR for amplification of the stx1, the stx2 and the eae gene. None
of these three genes was detected in 52 of the isolates. A specific
band for the stx1 gene, however, was obtained for three Escherichia
coli isolates originating from three different cows milk samples
(lab numbers 6, 298-3 and 298-4). The eae gene was detected in an
Escherichia coli strain isolated from a sample of normal cows
milk (lab number 7) (Fig. 1). In two of the E. coli strains positive
for stx1, presence of the gene for enterohemolysin hlyA (lab
numbers 6, 298-3) was found. The positive results in the
conventional PCR assay were validated by real-time PCR. The
isolates of Escherichia coli that were positive for stx1 and eae
genes were further validated with the MICRONAUT-E plates
(Merlin).
In addition, the four Escherichia coli strains that were found to
be positive for virulence factor genes (stx1 or eae) were tested for
the presence of serogroups O111, O145, O103, O157 and O26 most
frequently related to human disease. The isolates were not found
to belong to any of these serotypes.
Some differences were observed in the MICRONAUT-E
biochemical profiles of the four Escherichia coli strains positive
for virulence factors (stx or eae). The eae-positive isolate was
esculin-positive and ornithin decarboxylase-negative. The three
stx1-positive strains (6, 298-3, 298-4) were esculin-negative and
Figure 1. Amplification of vtx1 (180 bp) and eae (384 bp) by

conventional PCR.
A: Lane 1: 100 bp DNA molecular weight marker. Lane 2: Negative control. Lane 3:
Sample 6. Lane 4: Sample 2398-3. Lane 5: Sample 298-4. Lane 6: Sample 7. Lane 7:
Control strain harbouring the stx1, the stx2 and the eae gene.
ornithin-decarboxylase-positive. The stx1-positive strain 298-4

was arginine-dehydrogenase-positive, whereas the other three
isolates (6. 298-3 and 7) were arginine-dehydrogenasenegative.
The first destructive effects of the bacterial filtrates tested on
Vero cells were obvious at different sampling times for the different
isolates. The first evident effect on the monolayer was for the
reference strain that was also included on 24 h. The effect of our
isolates on the monolayer was visible at 48 h after inoculation:
two of the strains positive for the stx 1 gene (6, 298-3) showed the
strongest effect and the third strain positive for stx1 (298-4) had a
slightly weaker effect on the Vero cells. The monolayer inoculated
with the strain positive for the eae gene (lab number 7) appeared
normal after 48 h and showed effect on the cells at 72 h (Fig. 2).
After sequencing of the PCR fragments for the 16S rRNA gene,
sequencing products with a length of 600 to 632 bp were obtained.
After alignment of the sequences, a product with a length of 590
bp was used for analysis. BLAST analysis gave an identity value
of 99% with E. coli. The sequences of the 16S rRNA gene were
deposited in GenBank with the following accession numbers:
KF429755, KF 429756, KF 429757 and KF 429758 for isolates 7, 6,
298-3 and 298-4, respectively. Our results from sequencing 16S
rRNA as well as the biochemical characterisation of the strains
confirmed that the isolates were E. coli.
The part of the eae gene which was examined covers codons
14-132. In comparison with the reference strain 210-03 for the
eae gene our isolate with number 7 showed amino acid differences
(in the part examined by us) in codons 20, 24, 25, 26, 36, 48, 56, 57,
58, 59, 61, 75, 76, 81, 83, 86, 105, 118, 119 and 126. The amino acid
Table 1. NCBI reference numbers for amino-acid sequences of E. coli stx1 and eae proteins included in this study.
NCBI protein ID
stx1 protein source
subtype
NCBI protein ID
stx1 protein source
subtype
NCBI protein ID
eae protein source
CAC85628.1
E.coli ovine
stx1 c
AFM85307.1
E.coli feces
stx1 a
CAI46996.1
human
CAC85553.1
E.coli human
stx1 c
AAO19475.1
E.coli bovine
stx1 d
ABF69117.1
no data
CAA85370.1
E.coli Australia
stx1 c
BAD08527.1
E.coli bovine
stx1 d
AAC32028.2
rabbit
ABE02587.1
E.coli Hungary
stx1 c
AAY43857.1
E.coli shellfish
stx1 d
AAB97764.1
bovine
AAM70037.1
E.coli human
stx1 a
AAY43852.1
E.coli shellfish
stx1 d
BAL47133.1
Hirundo rustica
AAA26538.1
S.dysenteriae
stx1 a
BAD08529.1
E.coli bovine
stx1 d
AAK48432.1
goat kids
AAA98347.1
S.dysenteriae
stx1 a
C210-03
BAL47187.1
human
Positive control strain C210-03 kindly provided by WHO in Copenhagen.
110
Figure 2. Destructive effect of strains 298-3, 298-4, 7 on the

monolayer at 24 h and monolayer control of Vero cells.
sequence in these codons for the reference strain was

LRCRQGHDSYNDLDNTAPFL and for our isolate was
FSAGNSQNAADNIGSVGSVV.
The part of the stx1 gene which was examined, covers codons
152-211 of SubunitA. Our data from a previous study showed that
in our isolates only the point mutation at position 791 is
nonsynonymous, where C is replaced with T. In this region, the
protein analysis revealed that our isolates differ from the reference
strain C210-03 at codon 167, where there was Y (tyrosine) in
contrast to H (histidine) in the reference strain.
The ProtTest 2.4 software, considering the length of the
analysed fragments, their number and existing amino-acids showed
that the most appropriate models for constructing amino acid
phylogenetic trees for the eae and stx1 genes were MtArt+F and
JTT, respectively.
The amino-acid based phylogenetic analysis differentiated three
groups based on the sequences predicted from the eae gene (Fig. 3)
and two groups based on the sequences predicted from the stx1
gene (Fig. 4).
Discussion
Verotoxigenic Escherichia coli can cause serious illness in
humans, such as hemorrhagic uremic syndrome and hemorrhagic
colitis. However, it is still not clear whether all VTEC variants are
equally pathogenic to humans, having in mind that the presence
of stx1 alone may not be sufficient for VTEC to cause illness in
humans. It has been considered that eae-negative VTEC are not a
serious threat to human health, unlike classical EHEC 27. The results
from our study showed four isolates with virulence factors: Three
stx1-positive strains and an eae-positive one. Both these genes
together, which would pose a higher potential risk to consumer
health, were not found in any of the isolates studied by us. Some
of the infectious potential of E.coli is due to its ability to survive
for a few hours in low acidic conditions (pH<3.0) and thus to
survive in the stomach and further pass in the intestine 28-30. It has
been determined that the high acid resistance of E.coli depends
on genes for decarboxylation of arginin AdiA 31. These data may
suggest that our strain 298-4 (arginine-dehydrogenasepositive)
could in certain conditions show higher pathogenic potential in
humans. During cheese ripening, the pH decreases and pathogens
Figure 3. Phylogenetic tree of the eae gene sequence from the milk
isolate (Protein ID in NCBI AGC29881.1) in comparison with Escherichia
coli reference strains.
The tree was calculated from 119 amino acid aligned positions in the final data set, using the
MUSCULE algorithm. One thousand bootstrap replications were used to build a
phylogenetic tree.
Figure 4. Phylogenetic tree of the vtx1 gene sequences from the milk
isolates in comparison with Escherichia coli reference strains and
Shigella dysenteriae.
The tree was calculated from 60 amino acid aligned positions in the final data set, using the
MUSCULE algorithm. One thousand bootstrap replications were used to build a
phylogenetic tree.
111
which can survive in low acid environment may become a real

threat when consumed by humans. For example, Muehlherr et
al.32 reported that 16% of the raw goats milk in Switzerland is stxpositive. The results of Orden et al. 33 show that stx1 is the
predominant stx genotype in healthy goats. Interestingly, in our
experiments with raw cows milk, three stx1-positive isolates were
obtained from milk with subclinical mastitis, whereas the eaepositive strain was isolated from a sample of raw cows milk that
met the somatic cell count standards. Since the stx1-positive
isolates were obtained from milk samples with subclinical mastitis,
it could be suggested that bacteria from the teats probably
contaminate the milk following colonisation of the milk duct or
from the infected mammary gland in cases of clinical or subclinical
mastitis. Another possible scenario is for contamination to happen
at some stage of the milking and milk processing technology 11, 34.
There is evidence that mastitis in cows can be caused by E.coli 35,
but there are few data about mastitis caused by E. coli O157 or
VTEC, although they could also contaminate the milk from the
udder of cows with mastitis 36. In dairy herds, fecal contamination
of the udder poses a risk for pathogen contamination of milk. That
is why, it is important to prevent fecal contamination of the udder
in order to reduce VTEC and other pathogens in raw milk. There
are data that other dairy domestic animals apart from cows, e.g.
goats and ewes, also excrete these pathogens with their feces,
which could also lead to milk contamination 37.
The Escherichia coli isolates obtained from raw cows milk in
our experiments are in agreement with the results of Hassan and
Elmalt 38, who isolated Escherichia coli from 38 out of 50 samples.
The authors carried out a PCR assay and identified stx2 in three of
the isolates, stx1 in one of the isolates and the intimin-coding eae
gene in none. Unlike their results, our experiments did not reveal
any isolates harbouring the stx2 gene, but showed one with the
eae gene. Pradel et al. 7 also observed a prevalence of stx1-positive
isolates in dairy products. Since most STEC can be found in the
intestines of ruminants, it is possible for fecal contamination to
happen during the milking process or as a result of crosscontamination from other animals in the farm, which could become
a means for VTEC transmission 39.
Some reports 7 indicate that shiga-toxin production may be
associated with production of hemolysin as a virulence factor,
which is confirmed by our results.
The eae gene, which codes for the outer membrane protein
intimin, was amplified in only one of our raw cows milk samples,
whereas the stx1 gene was identified in three of the mastitis milk
samples. The eae gene was detected in 14 Escherichia coli isolates
from raw yaks milk in India 40. Six of these 14 eae-positive isolates
were found to harbour only the eae virulence factor gene without
stx genes, identifying the strains as enteropathogenic Escherichia
coli (EPEC). In an earlier study, the same authors determined a
considerably lower EPEC counts in rectal swab samples collected
from animals 41.
The phylogenetic groups determined on the basis of aminoacid sequences predicted from the nucleotide sequences of the
stx1 and the eae gene differ from those obtained in our previous
study, where nucleotide phylogenetic analysis was carried out 22.
The results of the amino acid phylogenetic analysis differed from
those of the nucleotide phylogenetic analysis for the stx1, where
two main groups were formed as well as 2 and 3 subgroups. One
of the branches included a group of isolates with stx1a and one
112
group with stx1c. The other branch included three groups with
isolates possessing stx1d (our isolates were placed in a separate
group). This is due to the fact that 8 mutations were determined
(compared to the reference strain). The difference in the topology
of our isolate and the reference strain C210-03 regarding the eae
gene on the basis of the amino acid sequences was determined by
20 nonsynonymous mutations out of 47 mutations in the
nucleotide chain of the examined part. Moreover, a change in the
primary amino acid sequence is a prerequisite for a change in the
secondary and tertiary structure of the protein molecule (the toxin).
This, in turn, incurs a phenotypic change, which in our particular
case affects probably the toxicity of the isolates. Our suggestion
could be supported by the phylogenetic analysis of Scheutz et al. 42,
who demonstrated how specific sequences of stx2 variants could
affect the biological activity of the toxin. The authors determine
Shiga toxin 1 and their nomenclature as stx1a, stx1c and stx1d.
They establish higher percentage similarities between stx1a and
stx1c in contrast with stx1d. This could be confirmed with the
phylogenetic analysis which proved stx1d to be the most distant
and different outbranch of the tree, compared to other two variants.
The nonsynonymous substitutions are important for the structure
and function of the amino-acid chain, as they change the primary
structure of proteins. That is why we performed amino acid
phylogenetic analysis. The result corresponds to Scheutz et al. 42
data; however, there were some differences as compared with the
nucleotide analysis: particularly, two branches were formed with
no additional groups. Stx1a and stx1c were part of one of the
branches and stx1d formed a separate branch. These data show
that the nucleotides in the region of the stx gene could be
appropriate for subtyping of isolates after sequencing and
phylogenetic analysis, while amino acid analysis could clearly
differentiate only stx1d. According to these data, we can conclude
that our isolates belong to stx1d.
Conclusions
Our study showed occurrence of the stx1 gene in Escherichia
coli isolates in milk from cows with subclinical mastitis (3 out of
20 samples). The obtained results indicate that some Escherichia
coli may harbour the intimin-coding eae gene. The existing
unauthorised sales of raw cows milk (this is how the samples
analysed by us were collected) raise the question as to the safety
of the consumed milk and dairy products. Escherichia coli is a
well-known causative agent of mastitis in ruminants. Our study
showed that stx1-positive E. coli strains are also isolated and
could apparently be involved in mastitis development. The results
obtained by us also throw more light on the genetic diversity of
the four isolates and on their phenotypic changes, especially those
associated with the toxicity of the isolates.
The amino-acid phylogenetic analysis separated the studied
strains into two branches on the basis of nonsynonymous
substitution in codon 169 of stx1. This clearly shows that this
mutation is a key factor that determines the stx1d subtype and
differentiates this subtype from stx a and stx1c, but does not
differentiate the latter two subtypes from each other.
Acknowledgements
The authors thank EU RL VTEC, Rome, Italy and WHO in
Copenhagen for providing reference strain and competent support.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Pre-processing glutinous rice effects on textural and morphological changes

Viboon Pansa-ead 1, Kannika Huaisan 2, Supan Yangyeun 1 and Songchai Wiriyaumpaiwong 1*
Faculty of Engineering, Mahasarakham University, Khamrieng, Kantarawichai, Mahasarkham, 44150, Thailand.
Faculty of Agro Industrial Technology, Rajamangala University of Technology Isan, Kalasin Campus, Kalasin 46000, Thailand.
*e-mail: songchai.w@msu.ac.th
1
Abstract
Glutinous rice is frequently enjoyed at snack and meal time in Southeast Asia. Khaowong Kalasin is a cultivar of glutinous rice in Thailand, and its
name is a geographical indicator of Kalasin province in Northeast Thailand. Its quality can be described as outstandingly soft and sticky. This study
addresses the soaking and cooking methods that can be used for Khaowong Kalasin glutinous milled rice to achieve a uniform texture. Textural and
sensory evaluations were compared between Khaowong Kalasin and RD6 cultivars in a container stored overnight. More specifically, the effects of
drying and temperature conditions on rehydration, texture, and external visualization for instant Khaowong Kalasin rice were investigated. Soaking
times were tested from 30 to 90 min. Three cooking methods were used: firewood with bamboo basket and earthenware steamer, LPG with bamboo
basket, and aluminium pot steamer and a modern programmable rice cooker. Temperatures from 40 to 60C with hot air drying and freeze drying were
evaluated. To reduce variations of hardness and stickiness, glutinous milled rice was soaked for 60 min and cooked in a programmable rice cooker
instead of using two traditional methods: firewood with bamboo basket and earthenware and LPG with bamboo basket and aluminium pot.
Khaowong Kalasin cooked rice could be stored in a container for a longer period of time than the RD6 cultivar. A 9-point hedonic test showed
differences between both varieties in softness and colour, but adhesiveness, odour and overall acceptance were very similar. An evaluation of the
effects of drying and temperature conditions on the rehydration and texture of Khaowong Kalasin showed hardness of the rehydrated rice after hot
air drying at 40C was closer, and the stickiness higher, than the control (fresh cooked rice). Rehydrated rice after hot air drying between 50 and 60C
and freeze drying (-80C) was too soft. The instant rice after drying at 40C needed the longest rehydration time but had no significant change in
rehydration maximum weight gain. On external microscope visualization, testers specifically preferred hot air drying below 60C because the higher
temperature led to more fractures inside the kernel and they were hidden by the opaque surface after freeze drying.
Key words: Glutinous rice, soaking, drying, instant glutinous rice, rehydration, sensory evaluations.
Introduction
Glutinous rice (Oryza sativa var. glutinosa) is a cultivar widely
found in Southeast Asia. The Khaowong District of Kalasin
Province in Northeast Thailand is home to one of the most popular
glutinous rice cultivars called Khaowong Kalasin. It originally
derived from RD6, which is cultivated in many parts of the upper
and middle provinces of North-eastern Thailand 1. It has very low
amylose content resulting in a sticky and dense quality when
cooked 2. Glutinous rice after husking is generally opaque and
white. In traditional glutinous rice cooking, the rice is soaked for
approximately 1 hour in water and then steamed using a straining
cloth, bamboo basket and earthenware steamer. During steaming,
the opaque and white kernels gradually become transparent and
clear. In conventional steaming by firewood, the rice becomes
soft and fragrant but needs more time to cook, and it is more
difficult to control the cooking temperature. Thai people still use
traditional steaming techniques rather than modern techniques 3.
Recently, farmers, growing RD6 in Khaowong, has found that
after cooking their rice was softer and more fragrant than the
original cultivar. Even stored in a woven bamboo container for
several hours until it cooled down, its texture remained stable. To
date, no scientific studies have confirmed the better properties of
the Khaowong Kalasin cultivar.
In todays modern world, competitive social pressures cause

people in large cities to turn to meals that save time and can be
eaten quickly. Many of the most favourite meal types are the
ready to eat and instant meals, for instance, rice ready meals,
instant rice and instant hot beverages. These commercial ready to
eat meals require an efficient method of cooling quickly. Vacuum
cooling, air blast cooling and cold room cooling are examples of
quick cooling processes that cool immediately to reduce the weight
loss of cooked rice 4. Similarly, ready to eat glutinous rice loses
weight in the quick cooling processing and often it becomes harder
and less fragrant. However, the instant process probably improves
its texture.
Sung et al. 5 found that storing glutinous rice (often referred to
as waxy rice) over six months was more effective toward increasing
hardness than using gamma radiation. Cooked rice stored at 37C
was harder and less adhesive than cooked rice stored at 4C 6.
Recent research with quick cooking rice 7, cooked rice 8, 9 and
sticky rice 10 has involved soaking and processing using high
hydrostatic pressure (or HHP soaking). HHP soaking leads to
lower hardness and higher cohesiveness for all kinds of rice. The
texture after cooking Jasmine rice, at various pressures and
temperatures 11, revealed that temperature significantly affected
115
pore size and texture, whereas pressure had little or no effect. For
Thai glutinous rice starch, pressure and temperature on the
gelatinization rate followed the Arrhenius and Eyring equations 12.
The effects of spray drying 13 and microwave heating 14 on
properties of glutinous rice starch have been determined. Spray
dried glutinous rice starch was almost amorphous and formed a
hydrophilic matrix barrier while microwave heating had a high
effect on starch granules re-aggregation. In addition, changes of
head rice yield and textural and digestive properties of glutinous
rice during hot air fluidized-bed drying have been investigated 15,
16
. Higher temperature drying led to higher head rice yield, and
cooked glutinous rice had reduced hardness and higher stickiness
and more easily digested starch.
Cooking, drying and pre-treatment methods play important roles
in achieving the desired quality for preparing instant rice. It was
found that instant rice, after rehydration in a rice cooker, was
harder and less sticky than freshly cooked rice while rehydrated
rice that had passed a freezing pre-treatment was insignificantly
different 17. Several models were developed to predict drying and
rehydration of instant rice kinetics using combined microwave
and hot air drying 18. It was recommended that a combination of
300 W microwave power and 80C hot air temperature was optimal
in terms of drying time, rehydration time and colour. Moreover,
the V-type pattern amylase-lipid complexes were developed by
instant jasmine rice processing 19.
In this study, we focused on cooking methods and instant
processing of glutinous rice cv. Khaowong Kalasin to determine
optimum conditions for cooking, storage and drying preparation.
The cooked rice, i.e., moisture, texture (hardness and stickiness),
and sensory qualities were evaluated. In addition, texture and
visualization of rehydrated rice was assessed.
Premium grade glutinous milled rice cv. Khaowong Kalasin was
collected from Khaowong rice mill at Kalasin Province,
Northeastern Thailand. This rice variety has a special characteristic
and is registered as a Geographical Indication (GI) by the Thai
Department of Intellectual Property (DIP). Supreme RD6 glutinous
milled rice was purchased from a common market. Both varieties
of rice were to be stored for approximately six months.
Experiments:
Rice soaking and cooking: Firstly, the effects of soaking time
and cooking method on the textural properties of cooked glutinous
milled rice (Khaowong Kalasin) were studied. Milled rice (500 g)
was soaked in water at Thai ambient conditions (30C and 50-60%
RH) with soaking times of 30, 60 and 90 min. The three cooking
methods used were firewood earthenware steamer, LPG aluminium
pot steamer and programmable cooker. In the firewood
earthenware steamer and the LPG aluminium pot steamer, rice was
soaked in a straining cloth and then steamed above boiling water
in a bamboo basket until kernels appeared clear. In the
programmable cooker, rice was soaked and cooked with 1:1 rice
and water ratio. Cooked rice samples from all cooking methods
were mixed well and stored in woven bamboo containers until
they cooled down before texture measurement.
Storage tests in a woven bamboo container: Samples of 500 g
glutinous rice cultivars, Khaowong Kalasin and RD6, were soaked
116
in pure water at ambient temperature for about 60 min until

absorbed a constant amount of water. Then, they were cooked in
a programmable cooker (SHARP model KS-ZT18) with rice and
water 1:1 by volume. Khaowong Kalasin glutinous rice acquires
its reputation from storage in a bamboo container. Therefore, the
two varieties were compared after storage in woven bamboo
containers for times from 0 to 24 hours. After the grain cooled
down to room temperature, it was hypothesized that the Khaowong
Kalasin rice texture remained softer. The textural and sensory
properties of cooked glutinous rice were evaluated.
Drying processing: After cooking, the rice became agglomerated
by gelatinization. The rice was spread on a wire mesh tray in a
single layer to facilitate washing in water at room temperature for
about 30 s. After that, it was taken out and left until almost dry
prior to drying in a hot air oven (Memmert ULE500, Germany) at
40, 50 and 60C temperatures compared to -80C freeze drying
(HetoPowerDry PL6000, UK). The rice from the drying process
was instant rice with a final moisture content of 10-12% dry basis
for hot air drying and 2-3% dry basis after freeze drying. Instant
rice samples were kept in desiccators pending rehydration.
Rehydration: Instant glutinous rice was rehydrated in a
transparent glass beaker with rice: water 1:2 by weight in an 850
W microwave oven (SANYO model EM-S 2088W). Samples were
weighed every minute until the weight reached a maximum. Weight
gain on rehydration (WGR) was calculated using equation 1 as
modified allowing for use of a microwave oven. Textural properties
of the rehydrated rice were measured and also visually examined
by microscopy.
WGR = (Wt Wi) / Wi
(1)
where Wt and Wi are weight at any time (t) compared to initial

state (i).
Moisture content determination: Moisture content of cooked,
instant and rehydrated glutinous rice was measured. A 50 g sample
was placed on a disposable aluminium foil pan and was then placed
in a digital moisture analyser (A&D company, Limited, MX-50,
Japan). Triplicates were analysed.
Texture measurement: Hardness and stickiness of cooked and
rehydrated glutinous rice were tested using a texture analyser
(Stable Micro System, TA AT Plus, and UK). Ten rice kernels were
randomized and put on a base plate in 2 rows and pressed by a 30
mm diameter cylindrical probe. The compressive test deformed
rice to 90% at 0.5 mm/s and a post-test speed of 1 mm/s. Ten replicates
were measured for each sample. Hardness of glutinous rice was
recorded from the maximum compressive force of the first peak
and stickiness for the negative force when the probe pulled up.
Sensory evaluations: Five attributes assessed taste (softness),
smell (aroma), touch (adhesiveness) and sight (colour) and overall
acceptance. Softness, aroma, adhesiveness, colour and overall
acceptance were evaluated by a 9-point hedonic scale using 30
trainees as explained in Table 1. Each trainer tested both cultivars.
Fifteen grams warm rice samples stored in woven bamboo
containers were served to trainees who scored on each attribute.
Table 1. Terminology, definitions, and how to evaluate sensory level.

Attributes
Softness
Definition
Force required to press
the sample with molars
Adhesiveness
Smell of cooked rice

seeds
Stickiness
Colour
Overall acceptance
Clarity
-
Aroma
Assessment
Place the samples between
the molars and evaluated
force to bite
Sniffing samples
Moulding samples by hand
Visualizing inside a cup of
sample
-
External visualization: The sample was prepared by the paraffin

section method before external visualization. The external surfaces
and cross sectional area appearances of dried and rehydrated rice
samples, prepared by hot air or freeze drying, were examined
using stereomicroscopy (Meiji Techno EMT-1-P, USA) at 200X
magnification.
1
2
3
4
5
6
7
8
9
=
=
=
=
=
=
=
=
=
Scale
Dislike extremely
Dislike very much
Dislike moderately
Dislike slightly
Neither like nor dislike
Like slightly
Like moderately
Like very much
Like extremely
and stickiness. In order to cook uniformly, the programmable

cooker was used in the following sections. In addition, a 60 min
soaking time was used because it was close to the equilibrium
moisture (Fig. 1).
100
Statistical analysis: Variance analysis (ANOVA) data processed

the means and deviations. Duncans multiple range tests were
applied to determine significant differences between each
treatment 20.
Effects of soaking time and cooking method on textural
properties: Even though the tested rice was stale, cooked
Khaowong Kalasin rice had better softness than the other
varieties. Soaking and cooking were important factors which
affected the textural properties. To ensure cooking uniformity, the
variations of soaking time and cooking method were investigated
prior to drying and rehydration. Table 2 shows the hardness and
stickiness of cooked Khaowong Kalasin rice at various soaking
times and cooking methods. The results in all cooking methods
showed that the hardness and stickiness were reduced when the
soaking time increased. The longer soaking time seems to lead to
higher water uptake into the rice which, in turn, leads to the cooked
rice being softer and less sticky. However, replacement of steam
by water inside the kernel, by different steaming rates and cooking
times, may be the cause of a reduction of hardness and stickiness.
Visual observation, both of firewood and LPG combined with a
traditional cooking steamer, showed that these cooking methods
lead to non-uniform cooked rice which, in turn, leads to a wide
range of values of hardness and stickiness. In contrast, the rice
cooked by the programmable cooker was quite uniform in both
visual observation and had a narrow range of values of hardness
Table 2. Hardness and stickiness of steamed glutinous rice at
various soaking times and cooking methods.
Cooking methods
Charcoal earthenware
LPG earthenware
Programmable rice cooker
Soaking
times
(min)
30
60
90
30
60
90
30
60
90
Hardness
(kg force)
Stickiness
(kg force)
2.9 + 0.1a
2.4 + 0.1b
1.3 + 0.0c
5.4 + 0.7b
1.7 + 0.5c
6.7 + 0.6 a
3.7 + 0.1a
3.5 + 0.0b
3.4 + 0.0c
-0.20 + 0.20b
-0.40 + 0.04c
-0.02 + 0.01a
-0.30 + 0.2a
-0.40 + 0.4b
-0.50+ 0.3c
-0.04 + 0.02b
-0.40 + 0.01c
-0.03 + 0.01a
Means with the different letters in the same column are significantly difference (p0.05) by Duncans
multiple range tests.
Moisture content (%)
90
80
70
60
50
40
30
20
10
0
0
30
60
90
120
150
Soaking time (min)
180
210
240
Figure 1. Relationship between moisture content and soaking time.
Textural and sensory evaluations of two cultivars during storage

in a container: Storage was an important motive for achieving a
textural change of the cooked rice 6, 22. Cooked rice texture also
directly affected consumer sensory evaluation. In this section,
RD6 and Khaowong Kalasin glutinous rice effect textural and
sensory evaluations during storage in a woven bamboo container
as listed in Tables 3 and 4. Also shown in Table 3, the hardness of
cooked rice during storage at 0 to 24 h significantly changed at 6
h in RD6 and 12 h in Khaowong Kalasin cultivars. For 0-12 h
storage, the cooked rice was clear and warm, but the cooked rice
kernel left 24 h overnight was more opaque and cool. However,
the hardness of the Khaowong Kalasin cooked rice at 12 and 24 h
storage was significantly lower than the hardness of RD6. The
stickiness of cooked rice changed significantly at 6 h storage for
both cultivars. However, when comparing stickiness after 24 h
storage, the RD6 value was higher than that of Khaowong Kalasin.
These results indicate that the cooked glutinous rice of Khaowong
Kalasin had constant hardness until 12 h in a container - far longer
Table 3. Results of the period keeping in bamboo container shift
towards grain hardness and stickiness in both cultivars.
Storage
duration (h)
0
1
6
12
24
Hardness (kg force)

Khaowong
RD6
Kalasin
b
3.5+0.3
2.6+0.8 c
3.7+0.8 b
3.6 +0.7 c
3.5+0.3 b
3.5+0.4 c
3.7+0.2b
5.6+0.2 b
5.2 +0.3a
14.1 +0.5 a
Stickiness (kg force)

Khaowong
RD6
Kalasin
b
0.03+0.00
0.03+ 0.00 c
0.04+0.01 b 0.04 +0.01 c
0.03+0.00 b 0.04+0.01 c
0.04+0.00 b 0.06+0.00 b
0.20+ 0.01a 0.40+ 0.02 a
Means with the different letter in the same column are significantly difference (p0.05) by Duncans
multiple range tests.
117
Table 4. Sensory evaluation at different storage durations in a container of both varieties.

Time
(h)
0
1
6
12
24
Khaowong Kalasin
RD6
Colour
Softness
Aroma
Adhesiveness
7.81.3g
7.51.3fg
6.71.2ef
6.11.4cde
5.41.4bc
7.81.0e
7.11.0de
5.71.4c
4.51.6b
3.21.8a
7.01.5f
6.91.2f
6.31.4def
5.61.3ed
4.71.5ab
6.91.8cd
7.31.3cd
6.41.8bc
5.61.6b
4.31.8a
Overall
acceptance
7.61.4d
7.41.1d
6.31.5c
5.71.3bc
4.31.9a
Colour
Softness
Aroma
Adhesiveness
6.41.5de
6.31.5de
5.81.5bcd
5.11.5ab
4.41.6a
7.01.5de
6.41.9cd
5.51.7c
4.62.0b
3.21.8a
6.81.3ef
6.61.4ef
6.01.6cde
5.51.3bc
4.61.8a
7.41.6d
7.11.5cd
6.61.5bcd
5.71.8b
4.31.9a
Overall
acceptance
7.61.0d
7.21.0d
6.41.4c
5.41.5b
3.91.9a
Means with the different letter in the same column are significantly difference (p0.05) by Duncans multiple range tests.
KW = -0.62x + 8.56
R2 = 0.985
10
4
2
6
Storage duration (h)
12
KW = -1.18x + 9.2
R2 = 0.990
Softness
6
12
24
Aroma
Figure 4. Aroma of cooked RD6 and Khaowong Kalasin rice during

storage in bamboo rice containers.
KW = -0.69x + 8.17
R2 = 0.841
10
RD6 = -0.76x + 8.5

R2 = 0.921
KW
RD6
KW
RD6
8
6
4
2
0
6
12
24
Figure 5. Adhesiveness of cooked RD6 and Khaowong Kalasin rice

during storage in bamboo rice containers.
KW = -0.83x + 8.75
R2 = 0.949
KW
RD6
KW
RD6
RD6 = -0.92x + 8.86

R2 = 0.953
8
6
4
2
0
6
12
24
Figure 6. Overall acceptance for cooked RD6 and Khaowong Kalasin

rice during storage in bamboo rice containers.
KW
RD6
KW
RD6
RD6 = -0.66x + 7.6

R2 = 0.945
8
6
testing increased. It is likely that manually sensed adhesiveness

was based on bulk cooked rice which had a little deformation. On
the contrary, stickiness from the texture analyser was from the
compression test which was set at the 90% deformation of 10
kernels.
4
2
0
0
6
12
24
Figure 3. Softness of cooked RD6 and Khaowong Kalasin rice during

storage in bamboo rice containers.
118
24
Figure 2. Colour evaluation of cooked RD6 and Khaowong Kalasin

rice during storage in bamboo rice containers.
10
4
2
Overall acceptance
Colour
RD6
KW
RD6
KW
RD6
10
RD6 = -0.55x + 7.55

R2 = 0.957
KW = -0.59x + 7.87
R2 = 0.940
KW
RD6
KW
RD6 = -0.52x + 7.16

R2 = 0.945
10
Adhesiveness
than RD6. In other words, Khaowong Kalasin cooked rice lost

water at a slower rate than RD6. Hardness and stickiness, in later
periods of storage, increased because water was expelled from
retrograded amylose and amylopectin molecules, which acquired
a stronger structure due to retro gradation. The amylose structure
rearranged and formed hydrogen bonds during retro gradation
leading to the hardness and stickiness.
Table 4 shows the sensory evaluations for different storage
durations in containers of both varieties. To assess textural
properties, sensory evaluation was based on a 9-points hedonic
scale for five attributes - colour, odour, adhesiveness, softness
and overall acceptance. Figs. 2-6 show the scores for each
attribute for both varieties at various elapsed storage times in a
woven bamboo container.
All attributes of sensory evaluations in Figs. 2-6 declined with
storage duration for both varieties. Comparing both varieties, we
found that both colour and softness were different throughout
storage (Figs. 2 and 3) while the other attributes of aroma,
adhesiveness and overall acceptance differed insignificantly (Figs.
4-6). This indicated that human senses can differentiate the
softness and colour, but the overall acceptance for both varieties
appeared similar. The measured softness during storage correlated
with the hardness from the texture analyser. However, adhesiveness
decreased during storage whereas stickiness from compressed
Effects of drying temperature and method on instant rice

rehydration and textural properties: Instant glutinous rice was
made by using hot air drying at different temperatures and freeze
drying processes compared to cooked glutinous rice. Both drying
temperature and drying methods probably play an important role
on the final instant glutinous rice properties. Effects of drying
temperature are shown in Table 5. The moisture content of cooked

rice decreased from 121 down to 10.1-11.8% d.b. by hot air drying
and 2.7% d.b. by freeze drying. The drying time using hot air
decreased when the temperature increased. It was particularly
noticed that hot air drying at 50C can reduce drying time by half
compared to 40C. The higher temperature resulted in a higher
heat transfer rate into the rice kernel as well as a higher water
removal rate from the kernel.
All drying temperature and methods insignificantly affected
the maximum weight gain on rehydration (WGR) but had some
effect on the rehydration time. Rehydrated glutinous rice had a
maximum weight gain at 7 min for 40 and 50C hot air drying and
the maximum weight gain at 6 min for 60C hot air drying and -80C
freeze drying process. However, the final moisture content after
rehydration significantly increased with the drying temperature
and method. Drying at higher temperature made more rice kernels
crack leading to increased moisture content after rehydration.
Therefore, the cracking results were confirmed by external
visualization (Fig. 7 (a-c). Freeze-dried rice was opaque and had
loose structure (Fig. 7d) whereas the dried rice was clear with
dense structure and cracking (Fig. 7b). Therefore, the rehydrated
freeze-dried rice had a moisture content and WGR close to that by
60C hot air drying.
From the point of view of textural properties, the rehydrated
rice was considerably harder in hot air drying at 40C for 14 h.
Little cracking appeared due to the length of time for drying. Higher
cracking densities and lower hardness occurred with higher drying
temperature and shorter drying time. In addition, the hardness of
rehydrated rice from the freeze drying process was lower than
cooked rice but insignificantly different from hot air drying at 50
and 60C. For the freeze drying process, the loose structure was
caused by rapid water sublimation inside the rice kernel leading to
a lower hardness value. The stickiness after rehydration lay in the
range of 34 to 370 g force for all drying temperatures and methods.
Only 50C hot air-dried rice was similar in stickiness to freshly
cooked glutinous rice. The remarkable stickiness (370 g force)
was obtained from treatment of drying at 40C. This may be a
consequence of heat treatment over a long period of time, which
is an important key to attaining the remarkable hardness and
stickiness due to retrograde action.
External visualization: The external surface and cross sectional
area of dried and rehydrated Khaowong Kalasin rice in all treatments
were examined (Figs. 7 (a-d) and 8 (a-d). The hot air-dried rice was
glassy and clear whereas freeze-dried rice seemed white and
opaque. However, almost all external surfaces and cross sectional
200 m
200 m
(a) Cooked glutinous rice dried by hot air at 40C
200 m
200 m
(b) Cooked glutinous rice dried by hot air at 50C
200 m
200 m
(c) Cooked glutinous rice dried by hot air at 60C
200 m
200 m
(d) Cooked glutinous rice dried by freeze drying at -80C
Figure 7. Overall and cross-sectional views of dried kernels at

200X magnification by stereomicroscope.
Table 5. Weight gain after rehydration and textural properties of instant glutinous rice in
different drying temperatures and methods.
Glutinous rice
Khaowong Kalasin
Dried by HA at 40oC (14 h)
Rehydrated (7 min)
Rehydrated (7 min)
Rehydrated (6 min)
Dried by FD at -80oC
Rehydrated (6 min)
Cooked (control)
Moisture content
(% d.b.)
11.8 0.9e
134.4 1.2c
10.7 0.3e
141.7 0.8b
10.1 0.8e
149.3 1.4a
2.7 1.1f
150.8 0.8a
121.0 3.4d
Maximum WGR
(decimal)
NA
1.1 0.1ns
NA
1.2 0.2 ns
NA
1.2 0.4 ns
NA
1.1 0.3 ns
NA
Textural properties (kg force)

Hardness
Stickiness
NA
NA
2.2+0.7b
0.4+0.1a
NA
NA
0.4+0.2c
0.06+0.03bc
NA
NA
0.5+0.2c
0.08+0.02b
NA
NA
0.4+0.2c
0.05+0.01b
3.4+0.3a
0.03+0.00c
Note: HA denotes hot air drying and FD freeze drying process. Values are mean and standard deviation. Different superscripts (a, b, c, d, e and
f) in the same column imply that the values are significant different (p0.05) by Duncans multiple range tests.
119
200 m
Conclusions
Traditional glutinous rice (cv. Khaowong Kalasin) cooking
techniques resulted in a large variation of hardness and stickiness.
The variation of these textural properties could be reduced by
using a programmable rice cooker and soaking for at least 60 min.
If it is necessary to leave the cooked rice overnight in a woven
bamboo container, the textural properties of Khaowong Kalasin
and RD6 cultivars significantly change at 12 and 6 h, respectively.
A paired test between both cultivars based on a 9-point hedonic
test showed that softness and colour had distinct differences
whereas adhesiveness, odour and overall acceptance were not
significantly different. The cooked rice was processed by drying
to produce instant glutinous rice. The best conditions for
rehydration, using the microwave at the highest power (850 W)
and water and rice ratio of 2:1, was hot air drying at 40C. The
rehydrated rice had a low fracture density and hardness similar to
the control - fresh cooked rice. The rehydrated rice at other
conditions was too soft and less sticky. However, this best
condition had a longer rehydration time than the fastest conditions
of approximately 1 min.
200 m
(a) Rehydrated glutinous rice after dried at 40C
200 m
200 m
(b) Rehydrated glutinous rice after dried at 50C
Acknowledgements
We would like to thank the Thai Research Fund and Higher
Education Commission for financial support.
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14
121
WFL Publisher
Helsinki, Finland
www.world-food.net
Assessment of performance ability of Cabernet Sauvignon, Merlot and Syrah wine

cultivars on Southeast region of Turkey
Dilek Deirmenci Karata * and Hseyin Karata
Department of Horticulture, Faculty of Agriculture, Dicle University, 21210 Diyarbakr, Turkey.
*e-mail: degirmencidilek@yahoo.com
Abstract
In this study we evaluated the viticultural performance of Cabernet Sauvignon, Merlot and Syrah wine cultivars on dry-hot climate conditions in the
Southeast Region of Turkey. Vine yield components (bud survival (%), cluster number/vine (n), cluster number/shoot ratio (n), cluster weight (g),
yield/vine (kg) and berry weight (g), fruit composition as Brix (soluble solids %), and pH and titratable acidity (TA g/l) were measured during two
growing seasons between the years 2009 and 2010. The maximum mean bud survival rate (0.96%) was measured in the Cabernet Sauvignon grape
cultivar in 2009, and the minimum rate (0.77%) was measured in the Syrah grape cultivar in 2010. The highest yield in 2009 was found in the Cabernet
Sauvignon grape cultivars trained with Guyot system, with 7.63 kg. Mean Brix ranged between 24-27%, and mean pH between 3.64 and 3.86,
whereas TA was between 12.9 and 14.5 g/l. Based on the results, it can be concluded that Cabernet Sauvignon, Syrah, and Merlot are appropriate for
the Southeastern Anatolian conditions in Turkey.
Key words: Vitis vinifera, grapevine, yield components, fruit composition, training system.
Introduction
Grapevine (Vitis vinifera L.) is one of the oldest and most important
perennial crops in the world. Turkey is an important centre of
origin of the cultivated grapevine. The cultivation of grapes in
Anatolia began approximately 7000 - 8000 years ago 1, 16. Anatolia
has long been linked with the origin of viticulture and wine making,
especially in its eastern and southeastern regions, which are
commonly referred to as the epicenter of grapes 2. Canopy
management is widely accepted as an important tool for highquality wine production, however, little information is available
on its effectiveness under warm, dry climatic conditions 12. Jackson
and Lombard 10 argue that each cultivar and training system in a
region should be investigated to obtain the optimum yield that
will produce quality wine. Temperature is one of the primary
microclimatic factors in the driving rates of growth 3, 14, 17. Light
and temperature are the most important climatic factors for
inflorescence induction and differentiation. Environmental
regulations of fruitfulness are the most important climatic factors
for inflorescence induction and differentiation. High temperatures
have been found to promote fruitfulness in developing grapevine
buds 16. It is known that the terroir (the combination of soil, sub
soil, and climatic conditions) has a direct effect on the quantity
and quality of the grapes and on the wines produced 6, 9, 15.
In this study, we aimed to evaluate the viticultural performance
of Cabernet Sauvignon, Merlot and Syrah wine cultivars under
dry-hot climate conditions in the Southeastern Region of Turkey.
Vine yield components (bud survival, cluster number/vine, cluster
number/shoot ratio, cluster weight, yield/vine and berry weight),
and fruit composition (Brix, pH, titratable acidity) were measured
during two growing seasons between 2009 and 2010.
122

Experimental site: The experiment was conducted during 2009
and 2010 in Turkey (elevation 677 m, 3755.2' N- 40 13.8E). The
trial site was located within a commercial vineyard in the Province
of Diyarbakr. The study was carried out on wine grape cultivars
of Syrah, Merlot and Cabernet Sauvignon that were grafted on
110R rootstock. Bilateral cordon and Guyot canopy training
systems were characterized. The vines were spaced at intervals of
1.5 m, in north-south oriented rows with 2.0 m spacing.
The experiments were carried out on 25 vines for each grape
variety and studied in four replications. Trial data were taken from
the experimental area in 2009 and 2010. The soil in this region
shows clayey texture. In terms of soil properties, the soil consists
of 22% silt, 56.6% clay, 21.4% sand, 14% lime, and 1.8% organic
material. The vines were trained in two trunks and in bilateral
cordons at a height of 80 cm and winter-pruned to two-bud spurs,
leaving ~20 buds (excluding basal buds) per vine. Two trunks
were Guyot trained at a height of 80 cm and winter-pruned to 7-8
buds, leaving ~25 buds (excluding basal buds) per vine. The shoots
were loosely positioned between two pairs of foliage wires placed
at 40 cm and 50 cm in both systems.
Shoot thinning, hedging, and other canopy management
practices were applied. The vineyard was drip irrigated using
pressure-compensated emitters with a flow rate of 3 L/h, spaced
90 cm apart. Irrigation was applied, as needed, between the bud
break and bloom to avoid plant water stress. The water was
withheld through mid-July to achieve the control of shoot growth.
Irrigation was applied approximately once a week throughout
summer and less frequently during the cooler ripening period.
Nitrogen fertilizer (NH2NO3 x (NH2)2 CO) was applied annually at a
rate of ~15 kg N/ha by fertigation. All fertilizer applications and

pest and disease management practices were commercially applied
across the vineyard as uniformly as possible.
The growing season in the Province of Diyarbakr, Turkey, is
characterized by an absence of rainfall and dry-warm days. During
the 2009 and 2010 growing seasons, the phenological stages (bud
break, bloom, fruit set, veraison, and harvest) were recorded
(Table 1). Fig. 1 presents the long-term mean values of mean
temperature (C), mean temperature max. (C), and mean
temperature min. (C) at Diyarbakr conditions. Accordingly, longterm mean values of mean temperature max. (C) from the fruit set
period until the harvest period in June, July, August, and September
were 33.6 C, 37.2 C, 37.6 C, and 31.2 C, respectively.
Table 1. Phenological stages of 2009 and 2010 growing seasons
of the trial site.
Cultivar/Season
Cabernet Sauvignon
2009
2010
Syrah
2009
2010
Merlot
2009
2010
Budbreak
Bloom
Veraison
Harvest
10 April
08 April
26 May
29 May
01 July
03 July
08 August
07 August
30 March
03 April
20 May
22 May
28 June
05 June
01 August
06 August
02 April
05 April
20 May
23 May
02 July
04 July
05 August
07 August
50
40
Mean temperature
30
20
Mean temperature max.
10
0
-10
10
11
12
Mean temperature min.
-20
-30
Figure 1. Long-term average values of mean temperature (C), mean

temperature max. (C), mean temperature min (C) at Diyarbakr conditions.
Statistical analysis: Data were analyzed using IBM SPSS 20.0.

The analysis of variance was performed separately for each
variable, and the significance of each factor was evaluated. A
three-way (year x training system x cultivar) analysis of variance
(ANOVA - GLM Univariate procedure) was used to analyze the
effects of year, training system, and cultivar. When significant
effects (p<0.05) were detected, Duncans multiple comparison tests
were used to separate the means.
Determination of yield components: Yield components (bud
survival (%), cluster number/vine (n), cluster number/vine, cluster
number/shoot ratio (n), cluster weight (g), yield/vine (kg) and
berry weight (g) were measured for each year.
Bud survival (%): Data on bud survival were collected based on
per vine bud break each year, and the percentage of bud survival
was calculated by dividing the number of live nodes by the total
number of nodes and multiplied by 100.
Cluster number/shoot ratio (n): The cluster number/shoot ratio
was studied calculating the clusters on each shoot of each vine
on the experiment field.
Cluster weight (g): Cluster weight (g) was determined for each
vine, randomly selected and weighed by a balance.

Yield/vine (kg): The yield was found by measuring the product
of each vine on the experiment field.
Berry weight (g): A subsample of 100 berries was randomly
removed and weighed by a balance.
Determination of fruit composition: Samples were collected from
each grape cultivar for analyzing fruit compositions (Brix (%), pH,
and titratable acidity (TA) (g/l). Soluble solids (Brix%) were
measured using a refractometer. The pH was determined with a
temperature-compensated pH meter. TA was determined using 0.1
N NaOH. Ten ml of juice was mixed with 20 ml of distilled water,
and titrated to a pH 8.2. With this titration, the TA (g/l) was
calculated and reported as the amount of tartaric acid (g)/L juice.
Results
Yield components:
Bud survival (%):Survival of buds was high both in the training
systems and in all cultivars (Table 2). Bud survival decreased in
the Syrah cultivar in 2010. This result may be due to the fact that
the Syrah cultivar is susceptible to winter bud necrosis 5. The
maximum bud survival rate (0.98%) was measured in the Cabernet
Sauvignon grape cultivar on Cordon training system in 2009, and
the minimum rate (0.70%) was measured in the Syrah grape cultivar
on Guyot training type in 2010. Statistical difference was observed
among cultivars (Fcultivar 2.218, df 1, p>0.114). However, differences
were not significant among the training systems (Ftraining 7.523, df
1, p<0.007) and years (Fyear 12.876, df 1, p<0.000) (Table 2). The
statistical data revealed that the survival rate differed depending
on year and training system. The effects of other factors and their
interactions are not important with an exception of interaction
between training systems and years (Fyear x training 0.931, df 1, p>0.337)
(Table 2).
Cluster number/vine (n): While Merlot grape cultivar on Cordon
training system was found to show the highest production with
42.90 clusters per vine in 2009, the Cabernet Sauvignon grape
cultivar produced the highest number of clusters per vine in 2009,
with 51.60 on Guyot trellis type. From this comparison, it could be
said that the Cabernet Sauvignon grape cultivars gave the highest
values for both years. All of the factors cultivar (Fcultivar 34.792, df
1, p<0.000), training system (Ftraining4.928, df 1, p<0.029) and year
(Fyear121.595, df 1, p<0.000) and interactions were not statistically
significant (Table 2).
Cluster number/shoot ratio (n): In the cluster/shoot ratio, the
highest rate was obtained in the Syrah grape cultivar on Cordon
training system in 2010. The minimum value of cluster number/
shoot (1.94) was observed in Merlot grape cultivars on Cordon
trellis type in 2009. In Syrah grape cultivars, the cluster number/
shoot ratio was higher when it is on the Cordon training system.
No statistical significant differences were established in training
system (Ftraining 2.826, df 1, p<0.096) and year (Fyear48.356, df 1,
p<0.000); however, cultivar (Fcultivar 0.954, df 1, p>0.388) was found
effective on the number of clusters (Table 2). The interaction
between year and training system (Fyear x training0.357, df 1, p>0.551)
was significant (Table 2).
123
3.53 2.263
3.61 2.666
2.54 1.377
2.35 0.703
4.87 2.228
Means within columns followed by different letters differ significantly at p < 0.05 by Duncans new multible range test.
92.22 22.117
3.87 2.134
3.21 2.358
5.47 2.575 b
1.75 0.827 b
131.66 9.047
98.62 11.217
86.72 7.166
2.48 0.842 ab
2.22 0.544 ab
63.05 3.797
6.33 2.095 ab
3.42 1.177 ab
2.44 0.959 b
2.65 1.750 b
96.74 23.445
87.70 19.885
138.02 8.137 b
125.19 4.065 b
105.23 12.645 b
92.01 2.921 b
90.45 3.305 ab
82.99 8.162 ab
63.24 3.842 ab
62.86 3.948 ab
180.90 18.908 c
190.99 3.672 c
185.94 14.230
161.60 27.152 b
179.31 22.820 b
170.45 26.046
149.50 10.179 b
129.81 22.842 b
139.65 19.957
253.23 59.429 c 166.97 52.156

177.99 52.325 c 161.09 36.138
215.61 66.780
157.58 33.790
2.57 0.719
2.40 0.686
2.49 0.705
2.39 0.604 a
1.58 0.743 a
1.99 0.779
2.75 0.391 a
2.88 0.672 a
2.82 0.539
1.94 0.359 a
1.97 0.206 a
1.95 0.286
3.19 1.118 a
2.93 0.448 a
3.06 0.840
24.12 13.265
21.03 16.187
22.58 14.817
13.50 4.882 c
13.80 9.090 c
13.65 7.103..
15.20 4.517 b
12.40 2.459 b
13.80 3.820..
42.90 14.813 b
26.40 8.822 b
34.65 14.576 ..
0.94 0.110 a
0.85 0.232 a
0.90 0.183
0.86 0.111 a
0.92 0.161 a
0.89 0.137..
0.91 0.058 a
0.91 0.108 a
0.91 0.084 .
21.50 9.132 c
10.40 4.648 c
15.95 9.064..
0.91 0.116
0.84 0.188
0.87 0.160
0.85 0.114 a
0.70 0.204 a
0.77 0.178
Mean
2010
Syrah
Merlot
2009
2010
2009
Cultivars
Cabernet Sauvignon
Years
2009
2010
Bud survival (%)
Cordon
0.98 0.032 a
0.90 0.132 a
Guyot
0.93 0.078 a
0.70 0.270 a
Mean
0.96 0.064 ..
0.80 0.196..
Cluster numer/vine
Cordon
33.20 5.391 a
18.40 6.022 a
Guyot
51.60 8.996 a
11.60 3.627 a
Mean
42.40 11.883
15.00 5.965..
Cluster number/shoot ratio
Cordon
2.39 0.282 a
2.76 0.625 a
Guyot
2.46 0.208 a
2.57 0.497 a
Mean
2.42 0.244
2.67 0.559
Cluster weight (g)
Cordon
117.25 14.672 a 139.37 21.008 a
Guyot
148.15 16.903 a 140.33 32.410 a
Mean
132.70 22.101
139.85 26.587
Berry weight (100) (g)
91.20 4.365 a
92.29 3.421 a
Cordon
77.60 7.018 a
85.44 3.880 a
Guyot
Mean
84.41 9.000
88.87 5.003
Yield / vine (kg)
3.88 0.767 a
2.60 0 .921 a
Cordon
7.63 1.542 a
1.60 0.509 a
Guyot
Mean
5.75 2.256
2.10 0.888
Table 2. Main effect of training system on yield components of Cabernet Sauvignon, Merlot and Syrah grapevines in Diyarbakr, Turkey, 2009 to 2010.
124
Cluster weight (n): The highest mean cluster weight (253.23

g) was observed in 2010 in the Syrah grape cultivar by
Cordon training system. This is a highly optimal result for
the Syrah grape genotype in the climatic conditions in the
Southern Turkey. The lowest mean cluster weight (139.37 g)
was observed in the Cabernet Sauvignon grape cultivar in
2009. No statistical difference was established among the
varieties (Fcultivar 25.577, df 1, p<0.000) and the training
systems (Ftraining 6.615, df 1, p<0.012) effective on the cluster
weight; however, year was (Fyear 0.274, df 1, p>0.602)
effective on the cluster weight (Table 2).
Yield (kg/vine): The highest mean yield (5.75 kg) was
observed in the Cabernet Sauvignon grape cultivar in 2009.
However, the same cultivar produced mean yield of 2.10 kg
in 2010. The highest mean yield (kg /vine) in 2009 was found
in the Cabernet Sauvignon grape cultivar trained with Guyot
system, with 7.63 kg. In the same year, Cabernet Sauvignon
with Cordon training system produced only 3.88 kg. In 2009,
the highest yield (5.75 kg/vine) was revealed in the Cabernet
Sauvignon grape cultivar. However, according to 2010 data,
yield per grape vine obtained by the Cordon training
system was 2.60 kg and 1.60 kg by the Guyot training system.
The highest yield in the Merlot grape cultivars (6.33 kg)
was obtained in 2009 when it was trained with the Cordon
system. While mean yield was 4.87 kg in 2009, it was 2.35 kg
in 2010. The Cordon training system was applied in the
Syrah grape cultivars in 2010 and significant results were
obtained. In 2010, the Cordon training system produced
5.47 kg, but the Guyot training system yielded 3.42 kg of
production. This result can be explained by the fact that
the lowest buds failed to shoot when leaving many buds
with Guyot training system. Statistical differences were not
significant among cultivars (Fcultivar 4.001, df 1, p<0.021),
training systems (Ftraining 6.990, df 1, p<0.009) and years
(Fyea46.925, df 1, p<0.000). Also, the interactions among
varieties (p<0.000), training systems (p<0.000) and years
(p<0.000) were not statistically significant (p<0.000) (Table
2).
Berry weight (g): Berry weight was based on the weight of
100 berries. The Syrah cultivars produced the highest value
with 138.02 g in 2010 with the Cordon training system. The
weight of 100 berries in the Syrah cultivar was higher in
both years compared to other cultivars. The Syrah grape
variety has a genetic tendency to produce bigger berries
and bigger clusters compared to other cultivars. No statistical
difference was found among cultivars (Fcultivar 452.881, df 1,
p<0.000), training systems (Ftraining 64.776, df 1, p<0.000) and
years (Fyea 329.577, df 1, p<0.000). Only the year x training
system interaction (Table 3) (Fyearxtraining 0.001, df 1, p>0.980)
was found significant (p<0.000) (Table 2).
Fruit composition: Mean Brix (%), pH, and TA values for
grape cultivars in both years are presented in Table 3. Mean
Brix (%) ranged beetween 24.13% and 27.01%. No statistical
difference was established among the cultivars (Fcultivar
16.578, df 1, p<0.000) and years (Fyear 4.097, df 1, p<0.045).
However, the statistical difference between the training
Table 3. Main effect of training system on fruit composition of Cabernet Sauvignon, Merlot and Syrah grapevines in
Diyarbakr, Turkey, 2009 to 2010.
Years
Brix
Cordon
Guyot
Mean
pH
Cordon
Guyot
Mean
TA (g/l)
Cordon
Guyot
Mean
Cabernet Sauvignon
2009
2010
Merlot
Syrah
2009
2010
2009
2010
Mean
25.19 0.997 b
26.51 1.218 b
25.85 1.278
23.95 1.703 b
24.31 1.552 b
24.13 1.596
26.37 1.247 a
25.08 1.379 a
25.72 1.441
26.80 1.250 a
27.21 1.020 a
27.01 1.131
25.75 0.916 b
25.07 1.057 b
25.41 1.025
24.08 0.978 b
24.81 1.533 b
24.45 1.307
25.36 1.585
25.50 1.620
25.43 1.597
3.53 0.156 c
3.77 0.111 c
3.65 0.182
3.56 0.161 c
3.59 0.325 c
3.58 0. 250
3.97 0.169 a
3.73 0.087 a
3.85 0.182
3.85 0.306 a
3.86 0.137 a
3.85 0.232
3.71 0.451 b
3.57 0.131 b
3.64 0.331
3.82 0.096 b
3.90 0.115 b
3.86 0.112
3.74 0.290
3.74 0.206
3.74 0.250
13.57 0.501 a
13.02 0.622 a
13.29 0.618
14.59 0.956 a
14.47 1.191 a
14.53 1.0538
13.00 0.895 c
13.58 0.426 c
13.29 0.744
12.79 0.501 c
12.98 0.721 c
12.88 0.613
13.44 1.103 b
13.81 0.720 b
13.62 0.926
14.03 0.663 b
13.53 1.160 b
13.78 0.955
13.57 0.983
13.56 0.961
13.57 0.968
Means within columns followed by different letters differ significantly at p < 0.05 by Duncans new multible range test.
systems (Ftraining 0.383, df 1, p>0.537) was significant (Table 3). The

survival rates differed depending on the training systems. Year
and training interactions (Fyear x training 2.455, df 1, p>0.120) and
training and cultivar interactions (Ftraining x cultivar 2.628, df 1, p>0.077)
were both statistically significant (Table 3).
Mean pH ranged between 3.64 and 3.86. No statistical difference
was found among the cultivars (Fcultivar 12.075, df 1, p<0.000).
However, significant differences were observed between the
training systems (Ftraining 0.002, df: 1, p>0.963) and among the years
(Fyear 1.548, df 1, p>0.216). Year and training interactions (p>0.266)
were also significant, but the other interactions were not.
TA (titratable acidity) (g/l) values ranged between 12.88 and
14.53 g/l among cultivars. No statistical difference was found
among the cultivars (Fcultivar 10.681, df 1, p<0.000) and the years
(Fyear 4.694, df 1, p<0.032). However, statistical differences between
years and the training system interactions (Fyear x training 0.838, df 1,
p> 0.362) were significant (Table 3).
Discussion
In this study, we evaluated the viticultural performance of Cabernet
Sauvignon, Merlot and Syrah wine cultivars under dry-hot climate
conditions in the Southeastern Region of Turkey. Mean clusters
per vine ranged between 11.60 and 33.20 for Cabernet Sauvignon,
12.40 to 42.90 for Merlot and 10.40 to 21.50 in Syrah. From these
results, it is wise to claim that better yielding results were obtained
in the Syrah grape cultivars because they were trained by the
Cordon system. Mean yields (kg/vine) in all three cultivars were
examined. The yields of Cabernet Sauvignon (5.75 kg) and Merlot
(4.87 kg) were highest in 2009, whereas similar yields were obtained
in the Syrah cultivars in 2009 (2.54 kg) and 2010 (3.61 kg). The
training methods produced different yield capacities in the Syrah
grape cultivars in 2010. Bilateral Cordon training gave best yield
(5.47 kg) per vine in 2010. We found that the Cabernet Sauvignon
produced 5.75 kg/vine in 2009 and 2.10 kg/vine in 2010. Similar to
our study, Keller et al. 12 found that the crop level (kg/vine) changes
by years (3.54-9.81 kg) in the Cabernet Sauvignon grape cultivar
in Yakima Valley, Washington. Analyses showed that the lowest
cluster weight was observed in 2009 and 2010 as 132.70 g, 139.85
g in Cabernet Sauvignon grape cultivar and 139.65 g at Merlot
grape cultivar in 2009. The differences in cluster weights were
mainly due to the differences in berry weights.
Snhez and Dokoozlian 16 reported that the buds of Cabernet
Sauvignon increases with high-temperature climates. Similarly, we
also found higher values of cluster number/shoot ratio for Cabernet

Sauvignon. The vines had high numbers of clusters per shoot.
Climate was shown to exert an important influence over both the
timing of initiation and the extent of IP (inflorescence primordia)
differentiation, with IP development being far more advanced in a
hotter climate, compared with a cooler climate 7, 13, 21. In the same
way, in our study, the mean cluster number per vine was high
(42.40) in Cabernet Sauvignon and Merlot (34.65) in 2009. This
data is critical for improving the understanding of the
environmental effects on the formation of yield potential in
grapevines and how these may be modified by the temperature
increases related to climate change 21. Additionally, statistical
analyses showed that the cultivars were not effective on the pH
and TA (g/l) levels but the training systems were found effective
on the pH and TA (g/l) levels (Table 3). Brix (%), pH, and TA (g/l)
may show different values depending on environmental factors.
The warmth of the environment over the period of fruit set
could be related to the seasonal differences in the relative
proportions of seeded and seedless berries and live green ovaries
within the bunches in the Merlot grapes at New Zealand conditions
8
. In our study, we also observed low rates of shoot berry formation
in the Merlot grape variety, and we considered this as an effect of
high temperatures (38C) which caused live green ovary during
fruit set in June, 2009. Similar to our study, the differences in
vegetative growth, yield formation and fruit composition within
the cultivars were mostly due to the season (including weather
and soil moisture) rather than to yield 11-13, 19.
Conclusions
High temperatures have been found to promote fruitfulness in
developing grapevine buds. Cabernet Sauvignon grape cultivar
produced highest number of clusters for per vine on Guyot trellis
type with 51.60 in 2009 year. Our results for the Cabernet
Sauvignon, Syrah, and Merlot support the studies conducted in
some other countries where grape farming is rather common. It
can be concluded that Cabernet Sauvignon, Syrah, and Merlot
are appropriate for the Southeastern Anatolian conditions in
Turkey.
Acknowledgements
The authors would like to thank DUBAP (Dicle University Scientific
Research Project Coordination) for financial support of this work.
125
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15:3707-3714.
2
Aaolu, Y. S. and elik, H. 1985. Conservation of germplasm of Vitis
vinifera L. in Turkey. 4th International Symposium on Grapevine
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3
Alleweldt, G. and Hofcker, W. 1975. Einfluss von Umweltfaktoren auf
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Angelov, L. and Stalev, B. 2011. Study on the quality of wines produced
from Syrah and Tempranillo cultivars planted in two microregions
in Southern Bulgaria. Folia Hort. 23(1): 49-53.
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Collins, C. and Rawnsley, B. 2005. Factors influencing primary bud
necrosis (Pbn) in Australian vineyards. Acta Horticulturae (ISHS)
689:81-86.
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Crespy, A., 1992. Viticulture d aujourdhui. Lavoisier, France.
7
Dunn, G. M. and Martin, S. R. 2000. Do temperature conditions at
budburst affect flower number in Vitis vinifera L. var. Cabernet Sauvignon?
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8
Friend, A. P. and Trought, M. C. T. 2007. Delayed winter spur-pruning
in New Zealand can alter yield components of Merlot grapevines.
9
Galet, P., 1993. Prcis de viticulture. Dehan, Montpellier, France.
10
Jackson, D. I. and Lombard, P. B. 1993. Environmental and management
practices affecting grape composition and wine quality: A review.
American Journal of Enology and Viticulture 55:35-50.
11
Keller, M., Mills, L. J. and Harbertson, J. F. 2011. Rootstock effects on
deficit-irrigated winegrapes in a dry climate: Vigor, yield formation, and
fruit ripening. American Journal of Enology and Viticulture 63:29-39.
12
Keller, M., Mills, L. J., Wample, R. L. and Spayd, S. E. 2005. Cluster
thinning effects on three deficit-irrigated Vitis vinifera cultivars.
American Journal of Enology and Viticulture 56:91-103.
13
Keller, M., Tarara, J. M. and Mills, L. J. 2010. Warm spring temperatures
alter reproductive development in grapevines. Australian Journal of
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14
Lebon, E., Pellegrino, A., Louarn, G. and Lecoeur, J. 2006. Branch
development controls leaf area dynamics in grapevine (Vitis vinifera)
growing in drying soil. Annals of Botany 98:175-185.
15
Penkov, M. 2005. Choosing the most suitable lands for establishing
new vineyards and fruit orchards in Bulgaria. University Press, UASG,
Sofia.
16
Snchez, L. A. and Dokoozlian, N. K. 2005. Bud microclimate and
fruitfulness in Vitis vinifera L. American Journal of Enology and
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17
Seleznyova, A. N. and Greer, D. H. 2001. Effects of temperature and
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This, P., Lacombe, T. and Thomas, M. R. 2006. Historical origins and
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2004. Influence of training/trellis system and rootstock selection on
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L. cv. Chardonnay from hot and cool climates. Australian Journal of
Grape and Wine Research 14:46-53.
1
126
WFL Publisher
Helsinki, Finland
www.world-food.net
Dry matter accumulation trend on corn (TWC 647) as affected by plant density and
planting pattern
Ali Reza Saberi 1* and Siti Aishah Hassan
1
Agricultural & Natural Resources Research Center of Golestan, Gorgan, 4915677555, Iran. 2 Department of Crop Science,
University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. *e-mail: alireza_sa70@yahoo.com
Abstract
To examine the effect of plant density and sowing pattern on some characteristics of corn (hybrid T. W. C. 647), a field experiment was conducted
at Agricultural Research Station of Iranshahr. This experiment was laid out in a randomized complete block design arranged in a factorial with four
replications. This experiment had four levels of plant densities (D1 = 70,000, D2 = 80,000, D3 = 90,000 and D4 = 100,000) with three levels of
planting arrangements (p1 = single row, p2 = double row 15 cm space apart and p2 = double row 20 cm space apart). The results showed increasing
accumulation trend of leaf dry matter on different levels of planting arrangement till receiving 1560 degree day and fixation and after that decreasing.
With increasing density and using double row planting arrangement till before grain milking stage, the most leaf dry weight produced and after that
decreased. With increasing plant density amount of leaf dry matter will increase, as high plant density (100,000 plant ha-1) has the highest leaf dry
matter. The results showed till douching stage (1658 degree day) logarithmical increasing of assimilate accumulation reach to its highest amount and
after a time fixes and later that because of the leaves become old and transferring assimilates from source to sink, amount of leaf dry matter decrease.
Stem dry weight changes started from 607 growth degree day and continued till milking-doughty stage (1357 - 1560 GDD), and decreased later
because of assimilate repeated transferring during seed becoming old. Trend of changes in stem dry weight such as leaf is sigmoid. It means with
passing time stem dry weight have increased and decreased safter reaching to maximum weight. With plant entering to reproductive stage and after
getting 1123 GDD emergence of flowering started which ended to ear production. Study of ear dry matter accumulation trend at different levels of
planting arrangement showed there was significant difference between double row planting pattern and single row, but there is no significant difference
on two levels of planting arrangement. It means at minimum and medium plant density, especially on one double-row pattern, the plants can grow
better and produce a good ear. The changes on husk dry weight from 1123 GDD started and after getting 1357 GDD reached to its maximum very fast
and later there was gradually reduction at the growth season because of grain formation and filling. With increasing plant density amount of husk in
the unit area increased. Husk has chlorophyll and due to closing to grain plays effective role to filling, at single row planting pattern it is able to transfer
its assimilates to ear store sinks more than in other planting patterns. During growth season after pollination and grain formation grain dry weight
increased. Its trends at initial stage after getting 1123 - 1357 GDD is very fast and at the end because of losing assimilate and filling sinks and loading
becomes slow. The highest grain yield (1400 g m-2) was got from 90,000 plants h-1 density and double row with 15 cm space apart treatment at 1797
GDD.
Key words: Sowing density, planting arrangement, hybrid T. W. C. 647, grain yield.
Introduction
With the increase in world population, demand for food
consequently will grow. It is expected that human population will
increase to over 8 billion by the year 2020 and this will worsen the
current scenario of food security. Improved crop productivity over
the past 50 years has resulted in increasing world food supplies
up to 20% per person and reducing proportion of food-insecure
people living in developing countries from 57% to 27% of total
population 5. It is predicted that at least 10 million people will be
hungry and malnourished in the world by the end of this century 5.
Thus, to reduce the food insecurity, crop production will have to
be doubled, and produced in more environmentally sustainable
ways 2. This can be achieved by expanding the area of crop
production, increasing per hectare yield and improving crop
quality. Furthermore, during the second half of the past century,
rise in per hectare crop productivity was due to improved or high
yield potential 1.
The relationship between growth of corn under different
planting pattern and plant density is not well understood. Many

changes take place in plants to enable them to compete and
maintain photosynthetic activity. A consideration of the
adaptation mechanisms by which density affects photosynthesis
would aid the improvement of growth conditions and crop yield
and would provide useful tools for future genetic engineering.
Research in the late 1980s demonstrated that yields can be raised
two to three-fold by using available improved varieties and
appropriate agronomic techniques. However, these findings need
to be refined, improved and tested for local climatic, soil and crop
conditions 8.
These include in the aspects of to what extent of planting pattern
and plant density affect the yield and morpho-pysiological
parameters of corn. In addition, no comprehensive database is
available on corn under combination of pattern and density at
north of Iran. Thus, studies are still needed to improve
understanding of the effects of pattern and density for corn.
127
Hence, the present study was designed with the following

objectives:
1. To determine dry matter accumulation rate of corn at different
levels of plant density.
2. To study the effect of planting arrangement on dry matter
accumulation trend of corn.
3. To identify how interaction of planting pattern and plant
density affect dry matter accumulation trend of corn.

The results of comparing morphological parameters of corn at
four plant densities showed, that most of the corn characters
including leaf dry weight, stem dry weight, ear dry weight, husk
dry weight and grain dry weight were significantly different (P < 0.05)
between plant densities. Generally corn forage parameters increased
with increasing plant density but increasing of ear and grain is
limited and depended to planting arrangement could be increased.
The highest leaf dry weight, stem dry weight, ear dry weight and
grain dry weight were got from double row planting pattern (Fig. 1),
while the greatest husk dry weight was related to single row
planting pattern.
Growth indices and accumulation rate of dry matter were
analyzed based on GDD.
Accumulation rate of leaf dry matter: Fig. 2 shows increasing
accumulation trend of leaf dry matter on different levels of planting
arrangement till receiving 1560 degree day and fixation and after
that decreasing. With increasing density and using double row
planting arrangement till before grain milking stage, the most leaf
dry weight produced and after that decreased. At the end season
there was no significant difference between treatments.
Figure 3 shows with increasing plant density amount of leaf dry
matter increased, as high plant density (100,000 plant ha-1) has the
highest leaf dry matter. Till douching stage (1658 degree day)
logarithmical increasing of assimilate accumulation reached to its
128
Figure 1. General pictures from experiments, showing canopy

of double row planting pattern.
300
250
L - DM (g/m2)

A field experiment was conducted at Agricultural Research Station
of Iranshahr, Southern Iran. The experiment was laid out in a
randomized complete block design arranged in a factorial and
replicated four times. The experiment consisted of 12 treatments
outlined as follows: four levels of plant densities (D1 = 70,000, D2
= 80,000, D3 = 90,000 and D4 = 100,000) with three levels of planting
arrangements (p1 = single row, p2 = double row 15 cm space apart
and p2 = double row 20 cm space apart). The inter row spacing
was fixed at 75 cm while within row spacing was adjusted
according to plant densities and planting arrangement. Each
treatment combination was replicated in four blocks using a
randomized complete block design. Each plot comprised of six
raised beds of 6 m length and plants were harvested at the
physiological reach stage.
Sufficient number of plants was sown for each treatment to
facilitate destructive sampling for determining relative growth rates
at the various growth stages. The field was previously under
wheat which was harvested on 15 June. The land was plowed to a
depth of 20 - 25 cm followed by harrowing before planting. Data
were analyzed using the analysis of variance (ANOVA)
procedure 10 and means between the treatments were compared
using Duncan Multiple Range Test at P < 0.05.
200
A1
150
A2
100
A3
50
607
880 1123 1357 1560 1657 1797 1908

GDD
Figure 2. Effect of planting arrangement on leaf dry matter

accumulation trend of corn at different growth stages (average
of 2 years results).
highest amount and after a time fixes and later that because of the
leaves become old and transferring assimilates from source to
sink, amount of leaf dry matter decreased (Fig. 3).
Accumulation rate of stem dry matter: Stem dry weight changes
started from 607 growth degree day, continued till milking-doughty
stage (1357 - 1560 GDD), and later decreased because of assimilate
repeated transferring during seed becoming old (Fig. 4). Trend of
changes in stem dry weight such as leaf is sigmoid. It means with
passing time stem dry weight have increased and after reaching
to maximum weight, decreases.
350
L - DM (g/m2)
300
250
D1
200
D2
150
D3
100
D4
50
0
607
880 1123 1357 1560 1657 1797 1908
plant density amount of husk in the unit area increased. Husk has
chlorophyll and due to closing to grain plays effective role to
filling, at single row planting pattern is able to transfer its
assimilates to ear store sinks more than in other planting patterns
(Figs. 6 and 7).
200
H - DM (g/m2)
400
150
Figure 3. Effect of plant density on leaf dry matter accumulation

trend of corn at different growth stages (average of 2 years results).
800
A3
607
880 1123 1357 1560 1657 1797 1908

GDD
Figure 6. Effect of planting pattern on ear husk dry matter

700
600
500
D1
400
D2
160
300
D3
140
200
D4
120
100
0
607 880 1123 1357 1560 1657 1797 1908
GDD
H - DM (g/m2)
ST - DM (g/m2)
A2
50
GDD
100
Figure 4. Effect of plant density on stem dry matter accumulation

trend of corn at different growth stages (average of 2 years results).
Accumulation rate of ear dry matter: With entering plant to

reproductive stage and after getting 1123 GDD emergence of
flowering started which ended to ear production. Fig. 5 shows
bigger angle that means high speed of ear dry matter increasing,
but gradually with close to end season because of limited
assimilate and fill out sinks lead to decrease angle of curve. Study
of ear dry matter accumulation trend at different levels of planting
arrangement (Fig. 5) shows there is significant difference between
double row planting pattern and single row, but there is no
significant difference on two levels of planting arrangement. It
means at minimum and medium plant density especially on one
double - row pattern, the plants can grow better and produce a
good ear 7, 9, 11.
2000
1500
A1
1000
500
D2
60
D3
40
D4
0
607 880 1123 1357 1560 1657 1797 1908
GDD
Figure 7. Effect of plant density on ear husk dry matter

accumulation trend of corn at different growth stages (average of
2 years results).
Accumulation rate of grain dry matter: During growth season

after pollination and grain formation grain dry weight increased.
Its trend is very fast at initial stage after getting 1123 - 1357 GDD
and at the end because of losing assimilates and filling sinks and
loading become slow. The highest grain yield (1400 g m-2) got
from 90,000 plant h-1 density and double row with 15 cm space
apart treatment at 1797 GDD. Increasing the yield at high plant
density due to double row pattern is because of closing to square
planting arrangement. The yield at low plant density due to lacking
number of plants per surface and at high plant density because of
competition for absorption growth elements and interference of
male and female flowers become limited 3, 4, 6 (Fig. 8).
A2
1600
A3
1400
0
607
D1
80
20
880 1123 1357 1560 1657 1797 1908

GDD
Figure 5. Effect of planting pattern on ear dry matter

Accumulation rate of husk dry matter: The changes on husk dry

weight from 1123 GDD started and after getting 1357 GDD reached
to its maximum very fast and later gradually reducted at the growth
season because of grain formation and filling. With increasing
GR - DM (g/m2)
E - DM (g/m2)
A1
100
1200
1000
A1
800
A2
600
A3
400
200
0
607 880 1123 1357 1560 1657 1797 1908
GDD
Figure 8. Effect of planting pattern on grain dry matter

129
Conclusions
It might be concluded that by using double row planting pattern
the interplant competition could be decreased and higher yield
might be produced.
Acknowledgements
The authors would like to thank the Agricultural & Natural
Resources Research Center of Blochestan and the Seed & Plant
Improvement Institute of Iran for the financial support of this
research.
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A review of management strategies for salt-prone land and water
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Sri Lanka, IWMI Working Paper 125, 30 p.
9
Saberi, A. R., Mazaheri, D. and Heidari Sharif Abad, H. 2006. Effect of
density and planting pattern on yield and some agronomic
characteristics of maize (hybrid T. W. 647). Agricultural and Natural
Resources Science 1:67-76 (In Persian, Abstract in English).
10
SAS Institute 2004. SAS/STAT Users Guide. Release 9.0. 4th edn.
Statistical Analysis Institute, Inc., Cary, NC.
11
Sprague, G. D. and Dudley, J. W. (eds). 1988. Corn and Corn
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Agronomy, Inc., Madison, Wisconsin, USA, 986 p.
1
130
WFL Publisher
Helsinki, Finland
www.world-food.net
Productivity and gas exchange parameters of selected pasture grasses under

drought stress
Anna Koco and Mariola Staniak *
Department of Forage Crop Production, Institute of Soil Science and Plant Cultivation State Research Institute, Czartoryskich
Str. 8, 24-100 Puawy, Poland. e-mail: Mariola.Staniak@iung.pulawy.pl, Anna.Kocon@iung.pulawy.pl
Abstract
The objective of the study was to determine the effects of water stress on productivity, leaf greenness index and gas exchange parameters of selected
pasture grasses. A pot experiment was performed in a greenhouse of the Institute of Soil Science and Plant Cultivation, State Research Institute in
Puawy, at two soil moisture levels: 70% (optimum moisture content) and 40% (water stress) of field water capacity to study the responses of four
pasture grasses: Lolium multiflorum (Lam.), Festuca pratensis (L.), Festulolium braunii ((K. Richt.) A. Camus) and Dactylis glomerata (L.) to water
stress. The study showed that water stress caused a significant reduction of yield of all tested grasses. The greatest decrease in the yield of dry matter
was observed in L. multiflorum, while the lowest one was found in D. glomerata. Among the studied species of grasses, a higher resistance to stress
was recorded for the D. glomerata and F. braunii (drought susceptibility index (DSI) < 1.0). The water content in the soil modified the relative level
of chlorophyll in the leaves of tested grasses. Under the conditions of soil drought, greenness index in all species of grasses was higher on average by
19% compared to control objects. The lowest value of greenness index was found in the F. pratensis. Under water stress conditions, all studied species
were characterized by lower net photosynthesis (PN) and transpiration (E) rate as compared to control. The mean values of water-use efficiency
(WUE) were statistically higher under stress conditions in D. glomerata and F. pratensis than in L. multiflorum and F. braunii.
Key words: Grasses, drought stress, dry matter yield, net photosynthesis, transpiration rate, drought susceptibility index, leaf greenness index, wateruse efficiency.
Introduction
Crop performance and yield are the results of genotypic expression
as modulated by continuous interactions with the environment.
Water is one of most widely limiting environmental factors for
crop production on a global basis. Drought is strongly affecting
the growth processes and productivity of plants 1-4. The reaction
of forage grasses to drought is first of all strong inhibition of
growth and developmental perturbations, which leads to reduction
of yield. Grass species differ in tolerance for moisture conditions
of habitat and in adaptability to changeable conditions 5.
Chlorophyll is the most common photosynthetic pigment. It
can be also considered an indicator of the annual yield of grasses
and grass regrowth, since there exists a positive correlation
between its content in the leaf blades and dry matter yield 6-8.
Chlorophyll concentration is a reliable indicator of plant vigor
and resistance to environmental stressors. The chlorophyll
contents of leaves depend on species, variety, climatic and soil
conditions, nutrient availability and development stage of plant 9.
Plant growth and yield are the outcome of numerous processes,
photosynthesis being the key one. A rapid decrease in water supply
makes plants to close their stomata. This enables rapid reduction
of water losses during transpiration, but this process is
accompanied by increased diffusion resistance for CO2, leading
to photosynthesis inhibition. This allows the plant to survive
drought, but leads to yield decrease 4, 10, 11.
Droughts in Poland are hardly predictable. It is difficult to
forecast the term of their occurrence, duration, territorial range
and intensity 12. Therefore, searches for greater utilization of forage
plant tolerance to drought are desirable. In this study, an attempt

was made to verify the hypotheses: productivity and physiological
processes of grasses depend on soil moisture conditions. The
objective of the study was to determine the effects of water stress
on productivity, leaf greenness (SPAD) and gas exchange
parameters of selected pasture grasses.
A pot experiment was performed in a greenhouse of the Institute
of Soil Science and Plant Cultivation, State Research Institute in
Puawy (5125'N, 2158'E), at two soil moisture levels: 70%
(optimum moisture content) and 40% (water stress) of field water
capacity to study the responses of four pasture grasses: Lolium
multiflorum (Lam.) cultivar Gisel, Festuca pratensis (L.) cv.
Fantazja, Festulolium braunii (K. Richt.) A. Camus cv. Sulino
tetraploid hybrid between Lolium multiflorum and Festuca
pratensis and Dactylis glomerata (L.) cv. Amera to water stress.
Soil moisture content was differentiated 6 weeks after sowing. In
order to maintain the appropriate soil moisture, water losses were
made up on a daily basis, to achieve a specified weight of the pot
with soil. Mitscherlich pots were filled with 7 kg of mineral soil.
The available nutrient contents (mg/100 g) of the soil were P 32.0,
K 12.3 and Mg 5.1. Soil pHKCl was 6.2. The experiment was
performed in four replications. Two to three seeds were sown at
30 points of each pot. After outcrop poorly developed seedlings
were removed, leaving fifteen plants per pot. Nitrogen fertilization,
in the amount of 3.6 g per pot, was applied at three rates, before
131
Table 1. Dry matter yield of grasses species [g pot-1].

Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%
III
26.3 a
11.9 d
32.2 b
20.7 e
33.0 b
22.6 e
41.8 c
25.2 e
Total
yield
111.8 a
72.0 c
121.7 a
74.8 c
121.6 a
77.3 c
152.0 b
89.5 d
19.1 a
26.4 b
27.8 b
33.5 c
91.9 a
98.2 ab
99.5 b
120.7 c
33.4 a
20.1 b
126.8 a
78.4 b
0.422
0.41
0.404
0.399
0.388
0.377
0.366
0.355
0.344
0.33
0.322
DSI = [1 (Dn Kn-1)] [1 (Dx Kx-1)]-1
D. glomerata
YR = 1 (Yry Ypy-1)
where Yry is the reduced yield due to water deficiency, while Ypy is
the potential yield under optimal soil moisture.
The results presented in the paper are means of particular cuts.
They were analysed statically using STATISTICA 6.0 software.
The significance of differences was verified by the Tukey test at
a significance level p < 0.05
Results
Soil moisture is an important factor shaping the size of crop yield.
Under optimal moisture conditions, the highest yield was obtained
from L. multiflorum, while the lowest one from D. glomerata. F.
pratensis and F. braunii yielded at a similar level (Table 1). All the
compared species responded to lower soil moisture with a
significant yield decrease. The smallest decrease in the total yield
(35%) was observed at D. glomerata, while it was greatest at L.
multiflorum (41%). Hybrid F. braunii reacted to drought with a
lower decrease in the total yield than both parent species, L.
multiflorum and F. pratensis (Fig. 1). Considering individual cuts,
D. glomerata and L. multiflorum yielded best in the first cut,
F. braunii
F. pratensis
L. multiflorum
Grasses species
Figure 1. Yield Reduction (YR) of grasses species in condition of water

deficit in the soil.
while F. pratensis and F. braunii in the second. The yield of the

third cut was smallest in all studied species of grasses. The
calculation of drought susceptibility index (DSI) allowed for
ranking the tested grasses according to their susceptibility to
drought. The lowest sensitivity to water shortages in the soil was
recorded for D. glomerata, while L. multiflorum was least resistant
to stress conditions. F. braunii hybrid proved to be more drought
tolerant than the two parent species, which is proved by a less
than one value of the DSI index (Fig. 2).
The water content in soil was a factor modifying the chlorophyll
index in the leaves in all tested species of grasses (Table 2). Under
the conditions of soil drought, leaf greenness index (SPAD)
1.101
1.088
1.066
1.044
DSI
where Dn and Kn are the dry matter of the stressed and non-stressed
object, while Dx and Kx are the mean dry matter averaged over all
objects under stress and non-stress conditions, respectively. Thus,
a low DSI-value (< 1.0) is indicative for a relative tolerance, while
a high DSI-value (> 1.0) for its stress sensitivity.
Yield Reduction (YR) was calculated by abdzkis method 12
using the following equation:
132
Regrowth
Soil
moisture
I
II
70%
49.8 a* 35.6 a
40%
33.2 df 28.9 d
70%
40.1 b 48.0 b
40%
24.5 e 30.4 d
70%
42.5 b 46.1 b
40%
28.3 ef 27.3 d
70%
56.9 c
53.2 c
40%
34.9 f
30.9 d
Mean for species
32.3 a
41.5 a
32.3 b
39.2 b
35.4 b
36.7 b
45.9 c
42.1 c
Mean for soil moisture
47.35 a 45.8 a
30.22 b 29.4 b
* Values marked with the same letter did not differ statistically.
YR
sowing, after the first and second cuttings. Nitrogen was applied
in the form of NH4NO3 solution. Phosphorus, potassium and
magnesium were applied pre-sowing, in the forms of solutions:
KH2PO4, K2SO4 and MgSO4, respectively, at the following rates
[g pot-1]: P 1.0, K 1.5 and Mg 0.5.
Over the vegetation season, the leaf greenness index was
measured with an optical chlorophyll meter SPAD-502Plus (Konica
Minolta, Japan). This device measures the difference between
light absorption by leaf at wavelengths of 650 and 940 nm, and the
quotient of these values represents indexed leaf greenness, i.e.
indexed relative chlorophyll content. The obtained results are
shown in SPAD units (Soil and Plant Analysis Development) in
the range from 0 to 800. The measurements were taken for the first
time 8 weeks after sowing (2 weeks after water stress). Four
measurements were performed for each regrowth and readings
were taken at one-week intervals. The rate of photosynthesis and
transpiration was measured two times over the growing season
(first and third regrowth) with a LI6400XT portable gas analyser
(LI-COR Environmental, USA). The measurements were performed
at a constant concentration of CO2 390 ppm, photosyntetic active
radiation (PAR) of 1200 mol m-2 s-1 and temperature between 23
and 27C. The chlorophyll concentration and the rate of
photosynthesis and transpiration were measured on the youngest,
fully developed leaves selected randomly of each pot. The plants
were defoliated three times over the growing season.
Drought Susceptibility Index (DSI) was computed by Fisher
and Maurer 13 method using the following equation:
1.022
1.001
0.988
0.966
0.944
0.922
D. glomerata
F. braunii
F. pratensis
L. multiflorum
Grasses species
Figure 2. Drought susceptibility index (DSI) of grasses species.
Table 2. Leaf greenness index (SPAD) of grasses species.

Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%
Regrowth
Soil
moisture
I
II
III
70%
460 a* 574 ad 596 ac
40%
556 b 591 abd 598 ac
526 a
529 ab
70%
469 a
40%
622 b
638 bc
632 c
70%
452 a
503 a
515 b
40%
548 b
652 c
613 c
70%
415 a
515 a
508 b
40%
546 b
604 cd
626 c
Mean for species
583 a
597 a
508 a
545 ab
582 a
581 a
500 a
578 a
564 a
481 b
560 a
567 a
449 a
530 a
537 a
568 b
621 b
617 b
Table 3. Intensity of net photosynthesis (PN) of

grasses species [mol (CO2) m-2 s-1].
Mean
Species
543 a
582 ce
508 a
631 d
490 b
605 de
479 b
592 e
D. glomerata
F. pratensis
F. braunii
L. multiflorum
563 a
569 a
547 ab
536 b
D. glomerata
F. pratensis
F. braunii
L. multiflorum
505 a
602 b
70%
40%
Regrowth
Soil
moisture
I
III
70%
11.20 c* 7.40 e
40%
7.03 a
4.17 c
70%
13.80 e 10.27 g
40%
7.87 b
2.53 a
8.87 f
70%
12.53 d
40%
7.03 a
3.37 b
70%
14.08 f
9.37 f
40%
7.93 b
6.80 d
Mean for species
5.78 a
9.12 a
10.83 c
6.40 b
9.78 b
6.12 ab
11.00 d
8.08 c
12.90 b
8.46 b
7.47 a
4.89 a
Mean
9.33 c
5.57 a
12.03 e
5.23 a
10.70 d
5.17 a
11.73 e
7.40 b
7.45 a
8.63 c
7.93 b
9.57 d
10.95 b
5.84 a
increased on average by 19% compared to plants grown under

optimal moisture conditions. The smallest increase was recorded
for D. glomerata - an average of 7%, with significant differences
occurring only in the first cut. In other species, leaf greenness
index increased on average by 24%, and the individual cuts ranged
from 17 to 32%. Under conditions of optimum soil moisture, lower
chlorophyll index was observed in L. multiflorum and F. braunii,
and significantly higher in D. glomerata and F. pratensis. In water
deficiency conditions, F. pratensis showed the highest values of
leaf greenness factor. In this study, the lowest chlorophyll index
were recorded in the first regrowth. The calculated values of the
correlation coefficient and regression equations showed a
significant negative correlation between dry matter yield and leaf
greenness index (Fig. 3).
Measurements of photosynthetic activity revealed significant
differences between species of grasses. The mean PN was highest
in L. multiflorum and lowest in D. glomerata. This process was
more intensive in the leaves of all grasses in the first regrowth
than in the third one (Table 3). Water stress considerably reduced
F. braunii
40
30
20
DM yield (g pot-1)
DM yield (g pot-1)
y = 84.526 - 0.0936 x
R 2 = 0.59
50
y = 85.474 - 0.0914 x
R 2 = 0.52
50
40
30
20
10
10
400
450
500
550 600
SPAD
650
400
700
500
550
600
650
700
F. braunii
40
30
20
DM yield (g pot-1)
60
y = 105.464 - 0.1213 x
2
R = 0.66
50
450
SPAD
L. multiflorum
60
DM yield (g pot-1)
F.p pratensis
60
60
photosynthetic activity (on average by 46%). The strongest

response was recorded in F. pratensis, where PN decreased by
56%, as compared with the control treatments (70% FWC). The
decrease in photosynthetic rate, caused by water deficit in the
soil, was accompanied by reduced transpiration in the experimental
species, since both processes are interrelated, as confirmed by
high correlation coefficients. On average, drought stress reduced
E by 54%. Among the tested species, D. glomerata showed the
lowest E under moisture optimal and deficiency conditions (Table
4). The heaviest water losses were recorded in the first regrowth.
In all tested species, the values of water use efficiency were
significantly higher under stress conditions in the first regrowth
(ranged from 5.45 to 7.98) but in the third regrowth it was generally
lower in such conditions (Table 5). The mean values of WUE were
significantly higher under stress conditions in D. glomerata and
F. braunii.
y = 84.526 - 0.0936 x
R 2 = 0.59
50
40
30
20
10
10
400
450
500
550 600
SPAD
650
700
400
450
500
550 600
SPAD
650
700
Figure 3. Relationship between leaf greenness index (SPAD) and dry matter yield of grasses
species.
Discussion
The results showed that the grass grown
under water deficit conditions yielded on
average by about 38% lower compared to
those grown under optimal moisture
conditions. According to Olszewska 14, the dry
matter yield of L. perenne and D. glomerata
was on average by 35% lower compared to
control treatments. A significant decrease in
yield, caused by water deficit in forage
grasses was also reported 15, 16. A typical
response of plants to water stress is to reduce
the yield, because there are limitations to the
intensity of photosynthesis and plant growth
processes 10, 11. According to Starck 17 and
Rawson et al. 18, crop yield is also affected
by other factors, such as the efficiency of
transport and distribution of assimilates in
plants. Ardiani et al. 19 reported, that changes
in soil moisture during the season have a
greater impact on the productivity of forage
grasses than the photosynthetic efficiency
of individual species.
133
Table 4. Intensity of transpiration (E) of grasses

species [mmol (H2O) m-2 s-1].
Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%
Regrowth
Soil
moisture
I
III
70%
2.49 c* 1.77 bc
40%
0.88 a
1.12 a
2.36 d
70%
4.26 f
40%
1.23 b 1.57 abc
4.02 e
70%
3.15 d
40%
1.28 b
1.32 a
70%
3.63 e
2.03 cd
40%
1.41 b
1.85 bc
Mean for species
1.44 a
1.68 a
2.43 c
1.96 b
2.22 b
2.67 c
2.52 c
1.94 b
3.38 b
2.84 b
1.34 a
1.19 a
chlorophyll content in leaves of grasses in each regrowth. The

lowest leaf greenness values were recorded in the first regrowth,
confirming studies by Gbork 6 and Olszewska 14 of higher relative
chlorophyll concentrations in the second and third regrowth
compared to the first one. The level of chlorophyll in leaves is a
reliable indicator of plant life and their response to changing
habitat conditions 7. It can be assumed that larger leaf greenness
index in the leaves of crops grown in stressful conditions is the
result of defence against stress. Under the conditions of water
deficit, there is a decrease in the density of cells and tissues of the
leaf. Therefore, there is an increase in their concentrations of microand macromolecules, including chlorophyll 5.
Decrease in the photosynthesis and transpiration rate in grasses
and cereals resulting from water stress were also reported by other
authors 19, 24, 25. Olszewska 14 showed that a decrease in soil moisture
from 80% to 40% of FWC in L. perenne and D. glomerata reduced
the rate of photosynthesis by on average 42% and the rate of
transpiration by on average 66%. According to Jones et al. 10,
reduced transpiration rate of grasses exposed to moisture stress
is related to changes in leaf structure. Under water deficit
conditions leaves are shorter, their surface is smaller, tissue is
more compact, and stomata are shorter, but densely arranged.
Such leaf morphology limits transpiration, which makes plants
more resistant to drought.
Mean
2.13 d
1.00 a
3.00 e
1.40 bc
3.59 f
1.30 b
2.83 e
1.63 c
1.56 a
2.20 b
2.44 c
2.23 b
2.88 b
1.33 a
Table 5. Water use efficiency (WUE) of grasses

species [mol (CO2) mmol (H2O) m-2 s-1].
Regrowth
Soil
moisture
I
III
D. glomerata
70%
4.51 b* 4.21 d
40%
7.98 e 3.72 cd
4.36 d
F. pratensis
70%
3.80 a
40%
6.38 d
1.66 a
F. braunii
70%
3.99 a 2.20 ab
40%
5.45 c 2.76 bc
L. multiflorum
70%
3.87 a
4.63 d
40%
5.64 c 3.67 cd
Mean for species
3.96 b
D. glomerata
6.24 c
F. pratensis
5.09 b
3.01 a
4.72 a
2.48 a
F. braunii
L. multiflorum
4.76 a
4.15 b
70%
4.04 a
3.85 b
40%
6.36 b
2.95 a
Species
Mean
4.36 bc
5.85 d
4.08 b
4.02 b
3.10 a
4.11 b
4.25 bc
4.65 c
5.10 d
4.05 b
3.60 a
4.45 c
3.95 a
4.66 b
A quick and easy method to determine the content of the green

pigment in leaves is to use a chlorophyllometer. A close correlation
between the readings of a chlorophyllometer and relative
chlorophyll content calculated by the traditional laboratory method
was found. This is shown by Jordi et al. 20 (r = 0.974), Gregorczyk
and Raczyska 21 (r = from 0.947 to 0.973) and Kozowski et al. 7 (r
= 0.970). Soil water deficit was a factor which significantly affected
the level of chlorophyll in the leaves of forage grasses. All tested
species showed a higher chlorophyll index in conditions of water
deficit in the soil. Also, Olszewska 14 showed that under water
stress conditions, relative chlorophyll content in leaves of L.
perenne and D. glomerata was significantly higher compared to
control. At the same time L. perenne varieties were characterized
by higher leaf greenness index than varieties of D. glomerata.
The author demonstrated similar correlations in the case of F.
pratensis and P. pratense 22. A significant influence of weather
conditions on the increase of chlorophyll content in leaves was
also reported by Michaek and Sawicka 9, who showed that, in the
conditions of lower amount of rainfall and higher air temperatures
in July and August, the plants accumulate more of this pigment in
leaves. Goliski and Xi 23 showed significant differences of relative
134
Conclusions
Water stress caused a significant reduction of yield of D.
glomerata, F. pratensis, L. multiflorum and F. braunii. The
greatest decrease in the yield of dry matter was observed in
L. multiflorum, while the lowest one was found in D. glomerata.
Among the studied grass species, a higher tolerance to stress
was recorded for D. glomerata and F. braunii (DSI index < 1.0). F.
pratensis and L. multiflorum were more sensitive to drought index
(DSI) > 1.0.
The water content in the soil modified the relative level of
chlorophyll in the leaves of tested grasses. Under the conditions
of soil drought, leaf greenness index in all species of grasses was
higher compared to control object. The lowest values of
chlorophyll index were found in F. pratensis.
A decrease in moisture from 70% to 40% of field water capacity
reduced the activity of photosynthesis, on average 46% and the
rate of transpiration 54%. The mean values of water-use efficiency
(WUE) were statistically higher under stress conditions in D.
glomerata and F. braunii.
Acknowledgements
The studies have been supported by Ministry of Agriculture and
Rural Development of Poland within the multi-annual program of
Institute of Soil Science and Plant Cultivation, State Research
Institute, task 3.4. Analysis and evaluation possibilities of
shaping the quality of plant materials taking into account the
different directions of use and regional conditions and task 3.1
The support activities in the field of fertilizer in Poland.
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135
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Effect of sulphur fertilization on fatty acid composition of faba bean (Vicia faba L.),
white lupin (Lupinus albus L.) and pea (Pisum sativum L.) grains
Eugenio Cazzato 1*, Vito Laudadio 2, Edmondo Ceci 3 and Vincenzo Tufarelli
1
Department of Agro-Environmental and Territorial Sciences, University of Bari Aldo Moro, 70125 Bari, Italy. 2 Department of
DETO, University of Bari Aldo Moro, 70010 Valenzano, Italy. 3 Department of Veterinary Medicine, University of Bari Aldo
Moro, 70010 Valenzano, Italy. *e-mail: eugenio.cazzato@uniba.it
Abstract
Winter legume grains are suited as a significant source of vegetable protein for both human and livestock species, due to high protein content and the
high level of unsaturated fatty acids. This research reported the effect of S fertilization on the quality of three different pulses (faba bean, lupin and
pea) in terms of lipid content and fatty acid profile. For each species, randomized complete block design with three replicates was used, and three S
doses (0, 30 and 60 kg ha-1, respectively) were applied. The S fertilization was split in two times: 50% before sowing and 50% in the early of March
as K2SO4. Our findings indicated that the S fertilization in faba bean, lupin and pea grains led to a significant improvement of the fatty acid profile.
Furthermore, the S fertilization enhanced the legume grains oil composition through the increase of unsaturated fatty acids, and in particular the
remarkable decrease of the erucic acid in lupin grains.
Key words: Faba bean, lupin, pea, sulphur fertilization, fatty acids.
Introduction
Grain legumes are important crops in Mediterranean area and in
other countries of the world. Legume seeds exhibit high levels of
protein, essential amino acids, and important dietary minerals.
They are used in a popular breakfast food, and also used as a
vegetable green, fresh or canned. Also, they are important crops
for soil improvement and used as break crop in cereal rotation to
keep the soil fertile and productive 1, 2. Sulphur (S) is one of the
elements known to be essential for the legume-rhizobium system
with specific physiological and biochemical roles 3. The S demand
of legume crops is higher than that of cereal crops. Studies on
different legumes have shown that the concentration of the Scontaining amino acids was markedly declined with decreasing S
supply 4. Sulphur fertilization was also found to increase N
accumulation and yield of legumes on S-deficient soils. Among
the winter legume grains, the seed of lupin contains 9 - 14% oil,
whose composition includes in particular oleic acid, linoleic acid
and -linolenic acid 5. The increasing selection of varieties has
recently widened the possibility of increase the use of the legume
grains in human or livestock nutrition as a replacement for either
animal/vegetable proteins 6-8. However, to date studies on the
effects of S fertilization on grain legumes fatty acid contents are
limited. Therefore, the present field trial was conducted in Southern
Italy under Mediterranean conditions to evaluate the effect of S
fertilization on the fatty acid composition of faba bean, lupin and
pea grains.
A field trial was carried out in Southern Italy at Gaudiano di Lavello
Potenza (4106' N; 1551' E; 145 m above sea level) on a sandyclay soil, characterised as sub-alkaline, low in total N (0.77
136
Kjeldahl method) and high in available P (83 mg kg-1; Olsen

method), exchangeable K (482 mg kg-1; BaCl2; TEA method) and
low soluble-S as SO4-S (4.4 mg kg-1; KH2PO4 as extractant). The
experimental site was characterised by a summer-dry climate with
a total annual rainfall of 560 mm distributed from autumn to spring
and a mean temperature of 16C. During the experimental period
(November - June), the total rainfall was 402 mm and temperatures
did not show any significant variation from the average. Faba
bean (Vicia faba L. cv. Prothabat 69), low alkaloids variety of
lupin (Lupinus albus L. cv. Multitalia) and pea (Pisum sativum L.
cv. Spirale) were sown on November maintaining a row distance
of 20 cm for each species at seed rate of 300 kg ha-1 (faba bean and
lupin) and 270 kg ha-1 (pea), respectively. For each legume species,
a randomized complete block design with four replicates and plot
area of 15 m2 were utilized. Three S fertilization levels (0, 30 and 60
kg/ha) were applied. The S fertilization was split in two times: 50%
before sowing and 50% in the early of March as K2SO4. The K
contained in K2SO4 was compensated with KCl for the plots not
treated with S. Crops were grown under rainfed conditions and no
irrigation was used. At physiological maturity, plants were
harvested. These plant parts were oven-dried at about 70C for 48
h. The plant material was ground to pass through a 0.2 mm sieve.
Total lipids were extracted 9. In preparation for the analysis of FA
composition, samples of the different legume species (1 g each)
were freeze-dried and ground. Methyl heptadecanoate (No. 51633,
Fluka, USA) was dissolved into n-hexane (1 mg/ml) as an internal
standard. Methyl esters of the FA were prepared; samples (300
mg each) and 5 ml internal standard were incubated with methanolic
acetyl chloride in a total volume of 9 ml. After cooling to room
temperature, 7 ml of 7% (w/v) K2CO3 was added with mixing, and
the organic phase was collected after centrifuging at 1500 g for 2

min at 4C. Fatty acid methyl esters were fractionated over a CPSIL883 column (100 m 0.25 mm i.d., film thickness 0.20 m fused
silica; Varian, Palo Alto, CA, USA) in a Shimadzu (Model 2GC17A)
gas chromatograph with a Hewlett-Packard HP 6890 gas system
and using flame ionization detection. Helium was used as carrier
gas at a constant flow rate of 1.7 ml/min. The oven temperature
was programmed as follows: 175C, held for 4 min; 175 - 250C at
3C/min; and then maintained for 20 min. The injector port and
detector temperature were 250C. Samples (1 l) were injected
with an auto-sampler. Output signals were identified and quantified
from the retention times and peak areas of known calibration
standards. Composition was expressed as percentages of the total
FA.
Data was analysed by analysis of variance using (ANOVA)
CoStat version 1.03 Software (CoHort Software Inc., Monterey,
CA, USA). The statistical analysis was applied to data following
the one-way ANOVA design. Unless otherwise stated, significance
was declared at P < 0.05.
Results
The results of total lipids and FA analysis (% on total FA) of lupin,
faba bean and pea grains obtained under different S fertilization
are reported in Table 1. Our results indicated that the different S
fertilization rates did not influence the total lipid content of faba
bean (1.83%) and pea grains (1.09%); whereas, the different S
fertilization improved significantly the total lipid content in lupin
grain (from 8.13 to 8.75%; for S0 to S60, respectively). The results
of FA analysis (% on total FA) of lupin seeds obtained under
different S fertilization are presented in Table 1. Overall, total
monounsaturated FA (MUFA) decreased significantly from 67.3
to 65.5% (S0 and S30, respectively). In particular, among the
individual MUFA, a significant decrease was observed for erucic
acid, whereas palmitoleic, oleic and eicosenoic acids resulted
higher when S fertilization increased up to S30. Particularly, in our
cultivar of lupin the main MUFA was the cis9 n-9 oleic acid
representing about the 86% of total MUFA. Total polyunsaturated
FA (PUFA) content increased significantly from 21.9 to 23.1% (S0
and S30, respectively); the higher S application rate did not result
in further increase of PUFA level compared to the intermediate S
dose. The main PUFA was the linoleic acid that represented 93%
of total PUFA. In faba bean grains, the overall total saturated FA
(SFA) decreased significantly from 24.41 to 18.86% (S0 and S30,
respectively), whereas S60 application did not lead further variation
(18.47%) compared with S30 rate. In particular, among the
individual SFA, significant decrease was observed for myristic,
heptadecanoic, stearic and tetracosanoic acids when S
fertilization increased up to S30. No effects of S fertilization were
reported for total monounsaturated FA in faba bean grains that
in overall represented about 25% of total FA. Conversely, total
polyunsaturated FA (PUFA) content increased significantly from
51.35 to 56.12% (S0 and S30, respectively); the higher S application
rate did not result in a further increase of PUFA level compared
with the intermediate S dose. Particularly, in our cultivar of faba
bean seeds, the main PUFA was the n-6 linoleic acid representing
about the 90% of total PUFA. Data on FA content of pea grains
grown under different S fertilization levels are reported in Table 1. In
overall, the total saturated FA (SFA) resulted 16.33% and no
significant differences were reported among S fertilization levels
Table 1. Effect of sulphur fertilization on fatty acid (FA) in lupin,

faba bean and pea grains.
Fatty acids (% on total FA)
14:0 Myristic
16:0 Palmitic
16:1 n-7 Palmitoleic
18:0 Stearic
18:1 cis9 n-9 Oleic
18:2 n-6 Linoleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
20:1 n-11 Eicosenoic
22:1 n-9 Erucic
SFAa
MUFAa
PUFAa
Total lipids
12:0 Lauric
14:0 Myristic
15:0 Pentadecanoic
16:0 Palmitic
16:1 n-7 Palmitoleic
17:0 Heptadecanoic
18:0 Stearic
18:1 cis9 n-9 Oleic
18:2 n-6 Linoleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
20:1 n-11 Eicosenoic
20:4 n-6 Arachidonic
22:0 Docosanoic
24:0 Tetracosanoic
SFAa
MUFAa
PUFAa
Total lipids
16:0 Palmitic
18:0 Stearic
18:1 trans9 n-9 Elaidic
18:1 cis9 n-9 Oleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
SFAa
MUFAa
PUFAa
Total lipids
Treatment
S30
Lupin
0.19a 0.13b
6.59c 7.57a
0.33
0.37
3.98a 3.77ab
55.01b 58.04a
19.96b 21.89a
0.23a 0.14b
1.73
1.42
4.34b 4.85a
7.64a 2.22c
10.76b 11.47a
67.32a 65.48c
21.92c 23.05b
8.13b 8.72a
Faba bean
0.02
0.02
6.61a 0.50b
0.10
0.27
11.93b 13.26a
0.01
0.04
0.52a 0.19b
3.01
2.83
22.31c 23.24b
44.85c 49.82b
0.46a 0.11b
4.94
5.03
1.94
1.75
1.10
1.16
1.01a 0.80b
1.21
0.99
24.41a 18.86b
24.26 25.03
51.35b 56.12a
1.83
1.82
Pea
11.50a 11.38a
5.27
5.16
0.10b 0.26b
15.76b 17.20b
53.16a 51.90b
14.21a 14.10a
16.77 16.54
15.86c 17.46b
67.37a 66.00b
1.11
1.07
S0
S60
Mean SEM P-value
0.11c
7.30b
0.36
3.06b
58.46a
21.90a
0.12b
1.30
4.93a
2.46b
10.47c
66.21b
23.32a
8.75a
0.14
7.15
0.35
3.47
57.17
21.25
0.16
1.48
4.71
4.11
10.90
66.34
22.76
8.63
0.03
0.32
0.05
0.17
0.95
0.41
0.05
0.10
0.15
0.18
0.22
1.10
0.43
0.05
0.045
0.035
0.076
0.037
0.027
0.019
0.034
0.055
0.022
0.005
0.018
0.025
0.009
0.047
0.01
0.49b
0.26
13.30a
0.09
0.17b
2.80
23.87a
50.07a
0.01c
4.96
1.65
0.87
0.84b
0.60
18.47b
25.61
55.91a
1.84
0.02
2.53
0.21
12.83
0.05
0.29
2.88
23.14
48.25
0.19
4.98
1.78
1.04
0.88
0.93
20.58
24.97
54.46
1.83
0.01
0.11
0.05
0.21
0.02
0.04
0.09
0.38
0.45
0.04
0.12
0.08
0.05
0.13
0.15
0.28
0.37
0.49
0.09
0.255
0.014
0.101
0.042
0.095
0.052
0.069
0.045
0.014
0.011
0.052
0.069
0.051
0.039
0.058
0.021
0.117
0.012
0.115
10.25b
5.42
4.37a
22.76a
44.77b
12.43b
15.67
27.13a
57.20c
1.11
11.04
5.28
1.58
18.57
49.94
13.58
16.33
20.15
63.52
1.09
0.15
0.12
0.09
0.29
0.41
0.22
0.15
0.16
0.50
0.04
0.047
0.185
0.009
0.011
0.007
0.013
0.053
0.005
0.006
0.196
applied for both total and individual SFA. Significant effects of S

fertilization were reported for total MUFA in pea grains that in
overall represented about 20% of total FA, and in particular the
MUFA detected (elaidic and oleic acids) increased with the increase
of the S fertilization level. The total polyunsaturated FA (PUFA)
content decreased significantly from 67.37 to 57.20% (S0 and S60,
respectively). Particularly, in our cultivar of pea, the main PUFA
was the -linolenic acid representing about 50% of total PUFA.
Discussion
We could not locate any literature concerning the effects of S
fertilization on FA composition of faba bean, lupin and pea grains;
thus, this subject should be considered in new investigations. In
the present study, the experimental treatments affected FA profile
of the legume species investigated. Our findings are confirmed by
Abramovic and Abram 10 that the differences in the composition
of the FA in seed oil can be caused not only by the different
cultivars, but also by environmental conditions. Similarly, Zubr 11
137
concluded that the variations are due to the combined effects of

climatic and soil conditions on the crop, and the same study
revealed an effect of S fertilizer application on FA composition.
The S fertilization caused the highest contents of both palmitic
and oleic acids. The concentration of linolenic acid, which is the
important FA with respect to nutrition in humans and animals,
remained unaffected by the fertilizer treatments applied. This
indicates that oil of our faba bean variety had a good level of
unsaturation and a high linoleic content 12. In the present study,
the experimental treatments affected FA profile of lupin. We could
not locate any literature concerning the direct effect of S fertilization
on fatty acid composition of white lupin seeds. Our findings are
confirmed by Boschin et al. 5 who reported differences in the
composition of the FA in lupin oil content. In our study S
fertilization caused the highest contents of palmitic, oleic and
linoleic acids. Moreover, a significant reduction of erucic acid
content in lupin oil was found in fertilized crops. This work indicates
that a drawback of lupin is the presence of significant levels of
erucic acid 1, 2, 5. Using the same definition already applied to
rapeseed oil, only lupin oil from fertilized crops could be classified
as low-erucic acid oil (erucic acid 2%). However, this negative
feature appears to be marginal since lupin oil is not a commercial
product; on the other hand, the selection of erucic-free or lowerucic acid genotypes would be certainly desirable 5. The lipid
content of peas is low and it was in overall 1.1%; however, the low
crude oil content in pea may be of importance in the flavour of
peas 13. This general composition in pea coincides with the results
obtained by Coxon and Wright 14. Our findings in terms of fatty
acid composition of pea grains are in agreement with those reported
by Murcia and Rincn 15.
albus L. cv. Multitalia) as the main protein source for broilers: Influence
on growth performance, carcass traits and meat fatty acid composition.
J. Sci. Food Agric. 91:2081-2087.
7
Laudadio, V. and Tufarelli, V. 2012. Effect of treated field pea (Pisum
sativum L. cv Spirale) as substitute for soybean extracted meal in a
wheat middlings-based diet on egg production and quality of early
laying brown hens. Arch. Geflgelkd. 76:1-5.
8
Laudadio, V. and Tufarelli, V. 2010. Treated faba bean (Vicia faba var.
Minor) as substitute for soybean meal in diet of early phase laying
hens: Egg-laying performance and egg quality. Poult. Sci. 89:22992303.
9
Folch, J., Lees, M. and Sloane-Stanley, G. H. A. 1957. A simple methods
for the isolation and purification of total lipids from animal tissues. J.
Biol. Chem. 226:497-507.
10
Abramovic, H. and Abram, V. 2005. Physico-chemical properties,
composition and oxidative stability of Camelina sativa oil. Food
Technol. Biotech. 43:63-70.
11
Zubr, J. 2003. Dietary fatty acids and amino acids of Camelina sativa
seed. J. Food Quality 26:451-462.
12
Hossain, M. S. and Mortuza, M. G. 2006. Chemical composition of
Kalimatar, a locally grown strain of faba bean (Vicia faba L.). Pak.
J.Biol. Sci. 9:1817-1822.
13
Mccurdy, S. M., Drake, S. R., Swanson. B. G., Leung, H. K. and
Powers, J. R. 1983. Influence of cultivars, soak solution, blanch method
and brine composition on canned dry pea quality. J. Food Sci. 48:394399.
14
Coxon, D. T. and Wright, D. J. 1985. Analysis of pea lipid content by
gas chromatographic and microgravimetric methods. Genotype
variation in lipid content and fatty acid composition. J. Sci. Food
Agric. 36:847-856.
15
Murcia, M. A. and Rincn, F. 1991. Fatty acid composition of pea
(Pisum sativum L., var. Citrina) during seed growth. Grasas Aceites
42:444-449.
Conclusions
Under Mediterranean environment and sub-alkaline soil, the S
fertilization in winter legume grains led to an improvement of the
fatty acid profile. Furthermore, the S fertilization enhanced the
legume grains oil composition through the increase of unsaturated
fatty acids, and in particular the remarkable decrease of the erucic
acid in lupin grains.
Acknowledgements
The author would like to thank the laboratory technicians involved
in the chemical analysis.
References
Cazzato, E., Tufarelli, V., Ceci, E., Stellacci, A. M. and Laudadio, V.
2012. Quality, yield and nitrogen fixation of faba bean seeds as affected
by sulphur fertilization. Acta Agric. Scand. Sect. B-Soil Plant Sci.
62:732-738.
2
Cazzato, E., Laudadio, V., Stellacci, A. M., Ceci, E. and Tufarelli, V.
2012. Influence of sulphur application on protein quality, fatty acid
composition and nitrogen fixation of white lupin (Lupinus albus L.).
Eur. Food Res. Technol. 235:963-969.
3
Scherer, H. W. 2009. Sulfur in soils. J. Plant Nutr. Soil Sci. 172:326-335.
4
Gaylor, G. C. and Sykes, G. E. 1985. Effect of nutritional stress on the
storage proteins of soybeans. Plant Physiol. 78:582-585.
5
Boschin, G., DAgostina , A., Annicchiarico, P. and Arnoldi, A. 2007.
The fatty acid composition of the oil from Lupinus albus cv. Luxe as
affected by environmental and agricultural factors. Eur. Food Res.
Technol. 225:769-776.
6
Laudadio, V. and Tufarelli, V. 2011. Dehulledmicronised lupin (Lupinus
1
138
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Yield and quality of Cenchrus ciliaris (L.) affected by nitrogen and phosphorus
fertilization
Ihsan Abu-Alrub *, Ahmed Aran, Omar Hamad and Abdelaziz Awaga
Research and Development, Abu Dhabi Food Control Authority, P. O. Box 52150, Abu Dhabi, United Arab Emirates.
*e-mail: ihsan.joma@adfca.ae
Received 20 February 2014, accepted 20 April 2014.
Abstract
Forage and nitrogen content responses of common buffelgrass (Cenchrus ciliaris L.) to nitrogen (N) and phosphorus (P) fertilization were evaluated
for two successive years in the UAE. Treatments consisted of five rates of N (0, 250, 500, 750 and 1000 kg ha-1 yr-1) and four rates of P (0, 200, 400
and 600 kg ha-1 yr-1), applied via weekly fertigation. All N fertilizer treatments resulted in significant increase in forage dry yield, relative to control.
However, no yield increase was observed from N treatments greater than 250 kg N ha-1. There were neither significant yield responses to P
fertilization treatments over the two years of the study, nor significant N-P treatment interactions for yield. Forage N concentration progressively
increased with increasing levels of N fertilizer, reaching maximum levels under the 1000 kg ha-1 N treatment. A seasonal pattern of forage N
concentration occurred, being higher in winter and lower in summer.
Key words: Cenchrus, nitrogen, phosphorus, fertilizer rate, UAE, forage yield, N concentration.
Introduction
Cenchrus ciliaris (buffelgrass) is a perennial grass native to dry
areas of the African continent, West Asia, and India, and has
been widely introduced to arid and semi-arid regions worldwide 1.
Indigenous to the UAE, it occurs in areas having average annual
rainfall as low as 100 mm 2. However, natural species distribution
is in decline, largely due to selective overgrazing.
Currently, there is huge demand for forage production in the
UAE to support 3.7 million head of livestock, with inadequate
available forage supplies to maintain the livestock population 3. It
has been reported that Cenchrus ciliaris is a valued forage grass
for dry areas, due to its high biomass production and tolerance to
low moisture conditions 4, 5. Therefore, Cenchrus ciliaris is among
species having the greatest potential for forage production in
the UAE.
Soil of the UAE is predominately sandy textured, with very low
organic matter content, resulting in deficiencies in essential plant
nutrients. A number of studies have found forage yields to be
increased by fertilization on sandy lands 6. Nitrogen (N) fertilization
typically increases grass dry matter 7, 8, forage nitrogen
concentration 9, and seed yield 10. Generally, phosphorus (P)
fertilization alone does not sustainable increase forage yields 9, 11,
but combined application of P with N often does 12.
Nutritional quality of unfertilized buffelgrass in rangeland varies
seasonally 13. Improvements in forage quality with fertilization
have been shown in many studies 14-17. Significant increase in
forage N content, digestibility, and mean daily live mass gain per
sheep was found as nitrogen fertilization rates increased 18.
Fertilization has also been shown to increase soil water extraction
by forages, and improve water use efficiency 19, 20.
Limited data are available concerning production of irrigated
buffelgrass forage as affected by the fertilizer applications under
UAE conditions. The objective of this research was to evaluate
the influence of a range of N and P fertilization applications on

forage yield and quality.
Field plots were established 23 January 2011 in Al Ain, UAE (24
00'N, 5400' E). Climate is hot and arid, with daily high temperatures
ranging from 24 to 45C, and average annual rainfall 100 - 150
mm. Soil of the study area was classified as sandy. Nitrate,
available P, and extractable K were 164.2, 21.6 and 128 ppm,
respectively. Soil pH was 7.8, electrical conductivity of the
saturation extract (ECe) 10.2 dSm-1, organic matter 1.0%, and CEC
7.1 meq/100 g.
The experiment was laid out in a split plot arrangement within a
randomised complete block design with four replications. The
main plot was assigned to N treatments, with P treatments as
subplots. Individual plot size was 3 2.5 m, consisting of 4
rows, with distance between rows 75 cm. Seeding rate was 40 kg
ha-1 of Cenchrus ciliaris cv. Laredo.
N treatments were 0, 250, 500, 750 and 1000 kg N ha-1 yr-1 and P
treatments 0, 200, 400 and 600 kg P ha-1 yr-1. K fertilization was
constant for all plots at rate of 250 kg ha-1 yr-1. N was applied as
urea (N 46%), P as triple super phosphate (P2O5 46%), and K as
potassium sulphate (K2O 50 %). N and K fertilizers were applied
weekly via fertigation, and P was top dressed once before planting.
All plots were harvested 5 times during 2011, and 7 times during
2012, when the grass reached maturity. After cutting and weighing
the whole plot, 500 g green samples of grass from each plot were
dried at 85C for 24 h for dry matter determination. Prior to cutting,
the lengths of 10 randomly selected plants were measured from
the ground to the tip of the shoot. Forage samples were analysed
for N and P. Plant N concentration was determined by Kjeldahl
digestion and P concentration determined colorimetrically in a
139
Results
Forage yield: The effect of fertilizer treatments on forage yield is
presented in Table 1. For both years, all N treatments significantly
increased yields, relative to control. Maximum yields were achieved
with the 250 kg N ha-1 yr-1 treatment, with no further increase in
yield resulting from higher N rates. Yield of treatments N 250 kg
ha-1 yr-1 were 55 and 57% higher than control for the first and
second years, respectively. During the first year, N treatment of
1000 kg ha-1 yr-1 resulted in yield decrease, relative to 500 and 250
kg ha-1 yr-1; however, in second year there was no significant
difference among N application rates. Neither phosphorus
treatments, nor N P treatment interactions were significant in
either year for forage yield or plant height. Average forage yield
per harvest for 2011 was higher than for 2012 (data not shown), as
seasonal harvest progressed average yield per harvest declined.
The reduction in yield per harvest in 2012 for N treatment 0, 250,
500, 750 and 1000 kg ha-1 yr-1 was 22.4, 19.1, 15.4, 4.5 and 1.5,
respectively.
Table 1. Total dry matter yield and average
plant height as affected by N and P
treatment level.
Treatments
-1
Plant height
at harvest (cm)
2011
2012
Dry matter
yield (t ha-1)
2011 2012
64.2
92.4
92.5
96.2
88.7
56.9
87.1
89.7
89.9
88.6
16.3
36.5
34.7
32.3
30.3
17.7
41.3
41.1
43.2
41.8
84.4
87.4
86.9
88.5
80.614
83.747
81.905
83.675
28.5
30.1
31.1
30.6
35.6
38.9
37.8
36.0
3.5
ns
ns
3.3
ns
ns
2.4
ns
ns
3.4
ns
ns
N concentration (%)
0 N (kg/ha)
500 N (kg/ha)
750 N (kg/ha)
1000 N (kg/ha)
2
1.5
1
0.5
0
May
Jul. Aug. Oct. Dec. Feb. Apr. Jun.

Harvest date
2011
Jul. Aug. Oct. Nov.

2012
Error bars represent standard errors of the mean.
harvest dates. N concentration averaged 1.1, 1.5 and 1.6% at 0, 250

and 500 kg N ha-1 yr-1, respectively. In general, 1000 kg ha-1 yr-1 of N
produced significantly higher forage N concentration, relative to
other N treatments.
A seasonal pattern of forage N level occurred, with higher N
concentrations during the winter season. Summer depression in
N concentration was observed from May till August. With
increasing P treatment rates resulting in significantly increased
forage N contents. Application of 600 kg ha-1 yr-1 P producing the
highest forage N content in most harvests (Fig. 2).
1.54
a
b
1.52
1.5
1.48
1.46
1.44
1.42
1.4
0
-1
200
400
P fertilizer level (kg ha-1 yr-1)
600
Figure 2. Forage average N concentrations of buffelgrass at different P

application rates.
Bars with the different letters are significantly different based on LSD test (P < 0.05).
Forage P concentration: The largest significant differences in

forage P content were generally observed between control and
200 kg ha-1 yr-1 P application rate (Fig. 3). Application of 600 kg
ha-1 yr-1 P, relative to lower rates, resulted in significantly higher
forage P content on 5 harvest dates. N application rate had no
significant effect on forage P content. N-P treatment interaction
for forage P concentration occurred only at the June, July and
October harvest dates.
0 P (kg/ha)
200 P (kg/ha)
400 P (kg/ha)
600 P (kg/ha)
Plant height trends followed yield, with all N treatments

significantly increasing height, relative to control. Maximum height
was generally achieved at 250 kg N ha-1 yr-1, with little effect of
higher N rates.
During both years there were a highly significant linear and
quadratic relationship between forage yield and the amount of N
applied (2011, DM Yield = 17.31 + 0.61 (N) 0.0051 (N2), R2 = 0.68,
2012, DM Yield =18.94 + 0.75 (N) 0.0054 (N2), R2 = 0.76).
P concentration (%)
N (kg ha yr )
0
250
500
750
1000
P (kg ha -1 yr-1)
0
200
400
600
LSD (P<0.05)
Nitrogen
Phosphorus
Interaction
250 N (kg/ha)
2.5
Figure 1. Forage N concentration of buffelgrass at different N application

rates averaged across P application rates for the various harvest dates
(averaged across P application rates).
N concentration (%)
nitric acid system 21. Weeds were manually controlled.

An analysis of variance for balanced data with GLM model
(SAS, 1994) was used to determine differences between treatments.
Interaction that did not make a significant contribution to the
variance was omitted in subsequent analyses. Comparison among
treatment differences was determined by Least Significant
Difference (LSD) at P < 0.05. The regression procedure was used
to determine the linear and/or quadratic effects of N fertilizer.
Forage N concentration: Fig. 1 illustrates the influence of five

levels of N fertilizer on forage N concentration. Total N
concentration increased with increasing levels of N fertilizer at all
Figure 3. Forage P concentration of buffelgrass at different P application

rates averaged across N application rates for the various harvest
dates(averaged across N application rates).
140
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
May
Jul. Aug. Oct. Dec. Feb. Apr. Jun.

Harvest date
2011
Jul. Aug. Oct. Nov.

2012
Error bars represent standard errors of the mean.
Discussion
The response pattern of forage yield to N and P fertilization was
similar in both growing seasons. Though N fertilization increased
yield in both seasons, maximum yield occurred at 250 kg ha-1 yr-1,
with no further yield increase with higher N application rates,
suggesting that forage production had peaked. Quadratic forage
yield response to increasing N fertilization rate has been reported
in several studies 8, 22.
Nitrogen fertilization consistently had a marked influence on
the N concentration of forage in both years. Increased N
concentration in forage associated with increasing N fertilization
has been previously reported 8, 17. However, Rai 9 reported a 17.5%
increase in N concentration of buffelgrass with a single application
of 60 kg ha-1 year-1.
A summer depression in N concentration occurred is consistent
with findings of Johnson et al. 16, who reported a 15% depression
in forage N concentration in July, the hottest month of the
climatological area of the study. Though forage yields peaked in
summer in this study, forage quality declined. This trend may be
related to higher environmental temperature, which encourages
lignifications, rapid physiological development and metabolic
activity, resulting in decline in forage quality 23, 24.
The lack of response of yield to P fertilization was likely due to
the moderate level of P pre-existing in the soil, as indicated by soil
analysis. That N, and not P, was limiting to forage yield is consistent
with several previous studies 8, 25. Ocumpaugh et al. 22 reported
insignificant N-P interactions for forage yield over three years.
However, other research has shown that combined application of
N and P may significantly enhance forage yield 12. Though P
fertilization did not affect yield in this study, it did improve forage
quality by increasing N content. Thus, to maintain maximum yield
responses to N, as well as improved forage quality, P application
may be necessary in the long term under our conditions.
Our results suggest no effect of N application on forage P
content of buffelgrass. Conversely, Wiedenfeld et al. 8 reported
that P content of buffelgrass decreased significantly with
increasing N fertilization, but not observed for Pretoria 90. In
general, plant responsiveness to nutrients is related to genetic
potential for growth 26.
Conclusions
Nitrogen fertilisation had significant effects on the yield and
quality of buffelgrass grown under irrigated condition. The study
shows that buffelgrass can perform well at annual applications of
250 kg ha-1 N. Though P fertilization had no effect on yield, it did
improve forage quality by increasing forage N concentration.
Further studies, on the role of P as related to plant growth
specifically in the area where soil P levels are low, are needed
before concrete fertilizer recommendations can be made.
Acknowledgements
The authors would like to thank Abu Dhabi Food Control Authority
for funding and supporting this research. The authors are grateful
to Dr. Kenneth B. Marcum, United Arab Emirates University, for
reviewing and valuable suggestions.
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Kering, M. K., Guretzky, J. A., Funderburg, E. and Mosali, J. 2011.
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142
WFL Publisher
Helsinki, Finland
www.world-food.net
Some alternatives of improvement the cow milk production efficiency in Albania:

Cash flow analysis
Meo Maksim 1*, Murrja Arif 1, Ndregjoni Agim 2 and Cerpja Teuta
1
Economy and Agribusiness Faculty, Agricultural University of Tirana,Tirana, Albania. A. Moisiu University of Durres,
Durres, Albania. *e-mail: maksmec@yahoo.com
Abstract
Any major new capital investment, such as the purchase of land, machinery, buildings or animals, can have a large effect on cash flows, particulary
if additonal capital is borrowed to finance the purchase. Borrowed capital requires principal and interest payments. The questions to answer before
making the new investment is: Will the investment generate enough additional cash income to meet its additional cash requirements? In other words,
is the investment financially feasible, as opposed to economically profitable? Farms with high production efficiency are more successful due to three
factors. High levels of production result in: (1) low cost per unit of output, (2) with an increase in the effective size of the farm business, and (3) with
an increased effectiveness of labor and machinery. The high level of production provides a simple and effective method of increasing farm size. Farms
with high agricultural productivity and high levels of productivity per head resulting in a large volume of business compared with farms with the same
size but with lower production levels. This additional business volume is the result of working more effectively, without increasing the surface of the
land or the size of the activities. The main methods of raising the level of productivity of livestock production are: i) selection and improvement of
breed; ii) choice of a balanced food ration in relation to the level of production, iii) sheltering conditions and health care, iv) appropriate and timely
nutrition and; v) a good combination between use of pasture and concentrated food.
Key words: Cash flow, cow farms, milk quality, net farm income.
Introduction
The Republic of Albania is currently in the process of preparing
for the EU Instrument for Pre-Accession Assistance on Rural
Development (IPARD). The IPARD Programme should be based
on analyses of the current situation in the rural areas and on indepth analysis of the sectors concerned in the country.
The objective of this dairy sector analysis is to give a
quantitative and qualitative description of the Albanian dairy
sector, addressing both production and processing.
The methods to carry-out of the study had been desk studies,
meetings and collection of detailed information of 104 dairy farms
with cows. A questionnaire has been designed and data collected
during several focus group meetings.
The results of the analyses is prioritising key areas for potential
intervention to contribute upgrading to EU standards, strengthening
the overall competitiveness and performance of Albanian milk
sector as well as fostering the sustainable development of the
sector in the EU accession context.
The current structure of dairy sector of Albania is characterised
by large number of small milk producers. About 226 thousand
dairy cow farms with total 360 thousand cows and 82 thousand
farms with small ruminants (SR) with some 1.9 million milking sheep
and goats are producing in total about 1 million ton of milk.
About 86% of milk production is coming from cow milk. The
average number with 1.58 milking cows per farm or 23 milking
ewes or does per SR farm is very low in comparison with EU-27
average. The land privatization initiated in 1991 created a structure
of primary production that is characterized by extremely small plot
and herd sizes. Private farm business re-started in 1991 quasi
from zero.
Most milk producers are semi-subsistence households. Only

up to 46% of milk production is delivered to milk processors. The
rest is used for self consumption, direct sale to consumer or for
feeding of animals. The average size of farms in the country is
just 1.2 ha and the size of single plots about 0.3 ha. Typical farm
feature is a high level of land fragmentation.
About 3 thousand dairy cow farms have more than 5 cows per
farm and about 12 thousand SR farms have more than 50 sheep or
goats per farm. However, the structural change started during the
last years particularly in the cow milk production. Some farms
became more commercial oriented and specialised on milk
production with the modernisation and extension of the dairy
farm business. With the support of the IPARD programme those
farms can achieve further steps to come closer to EU standards.
In general milk production is labour intensive in comparison with
other sectors. Therefore the IPARD investment support should
give the dairy sector priority. The analysed investments on animal
production are in most cases based on scenarios which keep the
same quantity of animals and try to improve economic performance,
following also the IPARD approach, which is focused in improving
quality and performance of animal production, rather than aiming
at increasing the number of heads. However, one scenario is also
simulating the increase in the number of milking cows from 5 to 10,
using the natural growth of the herd, as IPARD resources cannot
be used to buy livestock.
Investment scenarios are inter-combined with gross margin
analysis in the context of dynamic analyses.
143
Cattle dairy farm, investing in improving quality of milk quality,

with constant quantity (The first scenario):
Key cost figures and basic assumptions: Assumptions and
parameters used for the simulations are listed in Table 1.
Table 1. Assumptions and parameters used for the
simulation of the first scenario.
Key cost figures and basic assumptions
Size
Yield
Total production
cost
5 milk cows
5,000 lit/head
591,250 ALL
4 calves per year
available for sale
305 days lactation
Land required for
Farm gate sales
Primary target
animal feed
prices
production
owned
Milk: 50 ALL per
Quality
lit.
Calf: 25,000 ALL
per head
Investment cost
Annual Percentage
Loan duration
Rate
10,000 Euro, of
1 scenario: 12 %
Scenarios ranging
which 50% is a grant
between 3 years
and 50% is obtained
(shortest) and
through a loan
5 years ( longest)
The investment is not focused on maximising quantity of milk,

so that it is assumed that the animals are properly fed, and
genetically good, but are not according the best performing ones.
At present, the average production per cow is about 2,500 lit,
but it may range up to 4,000. The assumptions of raising it to 5,000
litres/year is therefore already an ambitious target.
Table 2 shows the feed requirements for optimising milk
production. The yearly cash flow generated by the breeding
activity has been calculated as shown in Table 3.
Table 2. Feed requirements to produce 5,000 litres milk.
No
1
2
3
Item
Concentrate
Hay
Silage
Quantity/head (kg)
5
5
25
Table 3. Yearly cash flow budget for a milking cow farm (5 cows).
Item
I. Income
Milk
Calves sold
Total income
II. Expenses
Concentrate
Straw
Silage
Veterinary services
Electric power
Stable maintenance
Expendable equipment
Total expenses
Net farm Income
1 Euro = 135 ALL
Unit
Quantity
Price
Amount
ALL
Euro
litre
Head
25,000
4
40
25,000
1,000,000
100,000
1,100,000
7,407
741
8,148
kg
kg
kg
9,125
9,125
45,625
25
8
5
228,125
73,000
228,125
25,000
12,000
10,000
15,000
591,250
508,750
1,690
541
1,690
185
89
74
111
4,380
3,769
Table 4 shows the cash flow scenarios of the holding, including

the surplus or deficit after paying for the costs, loan and the
equivalent of two minimal wages.
The quality milk improvement is a long-lived capital investment,
144
Table 4. Cash flow analysis for a milking cow farm (size 5 cows).
Item
Cash inflow (in Euro)
Net farm Income (milk
& Calf)
Cash outflow (in Euro)
Two full time minimal
wages
Principal payments
Interest payments
Total outflow (in Euro)
Net cash flow (in Euro)
1 Euro = 135ALL
Year
3
4,848.8
4,848.8
4,848.8
4,848.8
4,848.8
3,352
3,352
3,352
3,352
3,352
1,667
600
5,619
-770.2
1,667
400
5,419
-570.2
1,666
200
5,218
-369.2
3,352
1,496.8
3,352
1,496.8
so it is important to look at the cash flow over a number of years,

rather than month by month for one year as done for the wholefarm cash flow budget.
To guarantee two full times minimal wages requires 3352 Euro
and the new loan requires a principal payment of 1667 Euro each
year of the three-year loan, plus interest on the unpaid balance.
This obligation generates a large cash outflow requirement during
the first three years, causing a negative net cash flow for these
years. Once the loan is paid off in the third year, there is a positive
net cash flow in the following years. This result is common when
a large part of purchase price is borrowed and the loan must pay
off in relatively short time.
This investment is obviously going to cause a cash flow problem
the first three years. Does this mean the investment is a bad one?
Not necessarily. Investment on improvement of milk quality should
last for more than the five years shown in the table and will continue
to generate a positive cash flow in later years. Over the total life of
the investment, there would be a positive net cash flow, perhaps
a substantial one. The problem is how to get by the first three
years.
1. At this point, the investment on improvement milk quality
should be incorporated into a cash flow budget for the entire
farm. This budget may show that other parts of farm business are
generating enough excess cash to meet the negative cash flow
that would result from investment on improvement milk quality.
2. If not, one possibility would be to negotiate with the lender
for a longer loan with smaller annual payments. This solution
would help reduce the cash flow problem but would extend
principal and interest payments over a longer period and increase
the total amount of interest paid.
3. Another alternative is that cash flow for the third first years
required to guarantee one full time minimal wages and not two
how is planned in initially scenarios.
Key findings and comments: This farm cannot afford such
investment, unless additional sources of income are available.
Otherwise, the farm size should be larger.
Cattle dairy farm, investing in increasing quantity of milk per
milking cow, with land owned by beneficiary (The second
scenario):
The main methods of raising the level of productivity of
livestock production are: i) selection and improvement of breed;
ii) choice of a balanced food ration in relation to the level of
Table 5. Assumptions and parameters used for the simulation

of the second scenario.
Size
Yield
5 milk cows
From 5,000 to 6,100

lt/head
4 calves per year
available for sale
305 days lactation
Farm gate sales prices
Land required for

animal feed
production
owned
Milk: 40 ALL per lit.

Calf: 25,000 ALL per
head
Annual Percentage
Rate
1 scenario: 12 %
Investment cost
10,000 Euro, of which
50% is a grant and
50% is obtained
through a loan
Total production
cost
682,500 ALL
Primary target
Quantity
Loan duration
Scenarios ranging
between 3 years
(shortest) and 5
years (longest)
production, iii) sheltering conditions and health care, iv)

appropriate and timely nutrition and; v) a good combination
between use of pasture and concentrated food.
In view of this objective, the applicant aims at increasing farm
mechanization. The applicant wants to buy a mini tractor and its
equipments and to improve the storage conditions of feed, building
of a new storage facility. With this number of animals, purchasing
an animal feed a mixer cannot be profitable.
production. The yearly cash flow generated by the breeding
activity has been calculated as shown in Table 7 below. Table 8
shows the cash flow scenarios of the holding, including the
surplus or deficit after paying for the costs, loan and the equivalent
of two minimal wages.

Net farm Income (milk &Calf)
Two full time minimal wages
Principal payments
Interest payments
1 Euro = 135 ALL
Item
Concentrate
Hay
Silage
Quantity/head (kg)
5
5
25
Total
2,555
1,825
9,125
I. Income
Milk
Calf
Total income
II. Expenses
Concentrate
Straw
Silage
Veterinary services
Electric power
Stable maintenance
Expendable equipment
Total expenses
Net farm Income
1 Euro=135 ALL
Unit
Quantity
Price
Amount
ALL
Euro
litre
Head
30,500
4
40
25,000
1,220,000
100,000
1,320,000
9,037
741
9,778
kg
kg
kg
12,775
9,125
45,625
25
8
5
319,375
73,000
228,125
25,000
12,000
10,000
15,000
682,500
637,500
2,366
541
1,690
185
89
74
111
5,056
4,722
Key findings and comments: The improvement quantity of milk

per cow is a long-lived capital investment, so it is important to
look at the cash flow over a number of years, rather than month by
month for one year as done for the whole-farm cash flow budget.
Year
3
5319.9
5319.9
5319.9
5319.9
5319.9
3352
1667
600
5619
-299.1
3352
1667
400
5419
-99.1
3352
1666
200
5218
101.9
3352
3352
3352
1967.9
3352
1967.9
Cattle dairy farm, investing in increasing quantity of milk per

milking cow with rented land (The third Scenario):
of the third scenario.
Size
5 milk cows
Land required for

animal feed production
Rented (3ha)
Table 7. Yearly cash flow budget for a milking cow farm (5 cows).
Item

and the new loan requires a principal payment 1667 Euro each
the first and second year, causing a negative net cash flow for
these years. Once the loan is paid off in the second year, there is
a positive net cash flow in the following years. This result is
common when a large part of purchase price is borrowed and the
loan must pay off in relatively short time.
The alternatives of solving the problem (negative cash flow for
the threeyear) for this scenario are the same as the first scenario,
but the possibility of solutions is easier.
Table 6. Feed requirements to produce 6,100 litres milk.

No
1
2
3
Item
Investment cost
50% is a grant and 50%
is obtained through a
loan
Yield
From 5,000 to 6,100
lt/head
4 calves per year
available for sale
305 days lactation
Total production cost

742,500 ALL
Milk: 40 ALL per

litre
head
Annual Percentage
Rate
1 scenario: 12%
Quantity
Primary target
Loan duration
Scenarios ranging
between 3 years
(shortest) and 10 years
(longest)
A farmer is keeping five milking cows, which produce 5000 litres

per head (305 days lactation). He wants to produce feed for cows
in the farm. No land is owned, but, he is leasing the land. The rent
that he is paying is 150 Euro/ha. To produce the necessary feeds
for cows, he is leasing three hectares of land. He wants to invest
in improvement of quantity of milk per milking cow from 5000 to
6100 litres per milking cow (20 litres/day per milking cow).
In view of this objective, he aims to increase the level of farm
mechanization. The farmer wants to buy a mini tractor and its
equipments and to improve the storage conditions of feed through
the building of a new store. Table 10 shows the feed requirements
for optimising milk production.
145
Table 10. Animal feed inputs.

No
1
2
3
Item
Concentrate
Hay
Silage
Quantity/head (kg)
5
5
25
Cattle dairy farm, investing to increase the number of milking

cows (The fourth scenario):
Total
2,555
1,825
9,125
Table 11 shows the yearly cash flow generated by the breeding

activity. Table 12 shows the cash flow scenarios of the holding,
including the surplus or deficit after paying for the costs, loan
and the equivalent of two minimal wages.
Table 11. Yearly cash flow budget for a milking cow farm (5
cows).
Item
I. Income
Milk
Calf
Total income
II. Expenses
Concentrate
Straw
Silage
Rent of land
Veterinary services
Electric power
Stable maintenance
Others
Total Expenses
Net farm income
1 Euro = 135 ALL
Unit
litre
Head
kg
kg
kg
ha
Quantity
Price
ALL
30,500
4
12,775
9,125
45,625
3
1,220,000
100,000
1,320,000
25
8
5
20,000
319,375
73,000
228,125
60,000
25,000
12,000
10,000
15,000
742,500
577,500
9,037
741
9,778
0
2,366
541
1,690
444
185
89
74
111
5,500
4,278
Investment cost
50% is a grant and 50%
is obtained through a loan
Item
Net farm Income (milk
&Calf)
Two full time minimal
wages
Principal payments
Interest payments
1 Euro = 135 ALL
Year
3
4878.7
4878.7
4878.7
4878.7
4878.7
3352
3352
3352
3352
3352
1667
600
5619
-740.3
1667
400
5419
-540.3
1666
200
5218
-339.3
3352
1526.7
3352
1526.7
Key findings and comments: The improvement quantity of milk

per cow is a long-lived capital investment, so it is important to
look at the cash flow over a number of years, rather than month by
month for one year as done for the whole-farm cash flow budget.
and the new loan requires a principal payment of 1667 Euro each
the first three years, causing a negative net cash flow for these
years. Once the loan is paid off in the third year, there is a positive
net cash flow in the following years. This result is common when
a large part of purchase price is borrowed and the loan must pay
off in relatively short time.
The alternatives of solving the problem (negative cash flow for
the threeyear) for this scenario are the same as the first scenario.
146
Size
Initial: 5 milk cows
Final: 10 milk cows
Land required for animal

feed production
Owned; sufficient for 10
cows
Amount
ALL
Euro
40
25,000

of the fourth scenario.
Yield
From 5,000 to 6,100
lt/head
305 days lactation
90% fertility
20% replacement of
animals
Total production cost

Initial: 682,500 ALL
Final:1,403,000 ALL
Milk: 40 ALL per

litre
head
Annual Percentage
Rate
2 scenarios: 10 and 15
%
Quantity
Primary target
Loan duration
Scenarios ranging
between 3 years
(shortest) and 10
years (longest)
The scenario foresees a gradual increase in the number of

milking cows, either keeping the heifers or using the sale revenues
of calves to buy more heifers.
production. In the first years, the expenses are higher than in
other scenarios, as young cows are eating, but not producing,
while revenues are lower, as part of the value of calves sales is
balanced by the investments in buying more cows. Table 15 shows
the evolution of parameters over the years. Based on the above,
the cash flow has been calculated, as shown in Table 16.
Table 14. Animal feed inputs.
No
1
2
3
Item
Concentrate
Hay
Silage
Quantity/head
(kg)
5
5
25
Total
2,555
1,825
9,125
Price/kg
ALL
25
8
5
In any scenario, the cash flow is not sufficient to cover the

costs of the loan and to pay for 2 persons minimum wages in the
first four years, when expenses for animal feed are high and output
is low. In particular, in the third year, when all the young cows are
eating, but not producing milk and the income form sale of calves
is lower, the cash flow is even not sufficient to repay the loan
instalment, apart from any wage.
However, the business is sustainable and profitable in the
medium term. The accrued cash flow becomes positive only from
the seventh year (i.e. the total income obtained from the beginning
of the investment exceeds total expenditures from the seventh
year on), as shown in Table 17.
The figure changes if a 15% annual percentage rate on the loan
is applied, but the conclusion is the same: the yearly cash flow
turns positive from the fifth year.
Table 18 shows the cash flow scenario of the holding, including
the surplus or deficit after paying for the costs, loan and the
equivalent of two minimal wages in the case of cattle dairy farm,
applying investment related to increased number of milking cows.
Table 15. Expected evolution of parameters used to calculate the yearly cash flow.
Item
I. Stock
Cows at the beginning of the year
Cows at the end of the year
Of which: Milking cows in full productivity
Cows not yet in full production
Cows not yet in production
Calves born
Calves retained for expansion/replacement
II. Outputs
Calves sold
Milk produced (litres)
III. Inputs
Concentrate (kg)
Straw (kg)
Silage (kg)
Veterinary services (cost ALL)
Electric power (cost ALL)
Stable maintenance
Others
Year 1
Year 2
Year 3
Year 4
Year 5
Year 6
Year 7-10
5
7
5
0
2
5
3
7
9
5
0
4
5
3
9
10
5
0
5
5
2
10
10
5
2
3
7
1
10
10
7
2
1
8
2
10
10
9
1
0
9
2
10
10
10
0
0
9
2
2
30,500
2
30,500
2
30,500
3
39,650
5
51,850
6
59,475
7
61,000
12,775
9,125
45,625
25,000
12,000
10,000
15,000
17,885
12,775
63,875
35,000
12,000
11,000
16,500
22,995
16,425
82,125
63,000
12,000
12,100
18,150
25,550
18,250
91,250
126,000
12,000
13,310
19,965
25,550
18,250
91,250
126,000
12,000
13,310
19,965
25,550
18,250
91,250
126,000
12,000
13,310
19,965
25,550
18,250
91,250
126,000
12,000
13,310
19,965
Table 16. Cash flow evolution.

Item
I. Incomes
Calves
Milk
Total Incomes
II. Expenses
Concentrate (ALL)
Straw (ALL)
Silage (ALL)
Veterinary services (cost ALL)
Electric power (cost ALL)
Stable maintenance
Others
Total Expenses
Gross cash flow
Equivalent in Euro
1 Euro = 135 ALL
Year 1
Year 2
Year 3
Year 4
Year 5
Year 6
Year 7-10
50,000
1,220,000
1,270,000
50,000
1,220,000
1,270,000
75,000
1,220,000
1,295,000
125,000
1,586,000
1,711,000
150,000
2,074,000
2,224,000
175,000
2,379,000
2,554,000
175,000
2,440,000
2,615,000
319,375
73,000
228,125
25,000
12,000
10,000
15,000
682,500
587,500
4,352
447,125
102,200
319,375
35,000
12,000
11,000
16,500
943,200
326,800
2,421
574,875
131,400
410,625
63,000
12,000
12,100
18,150
1,247,900
47,100
349
638,750
146,000
456,250
126,000
12,000
13,310
19,965
1,402,000
309,000
2,289
638,750
146,000
456,250
126,000
12,000
13,310
19,965
1,403,000
821,000
6,081
638,750
146,000
456,250
126,000
12,000
13,310
19,965
1,403,000
1,151,000
8,526
638,750
146,000
456,250
126,000
12,000
13,310
19,965
1,403,000
1,212,000
8,978
Table 17. Net yearly and accrued cash flow (three year loan repayment period,
10% interest).
Item
Gross cash flow
Loan repayment
2 minimum wages
Net cash flow
Year 1
4,352
1,936
3,200
- 784
Year 2
2,421
1,936
3,200
-2,715
Financial flow in Euro

Year 3 Year 4 Year 5
349
2,289
6,081
1,936
3,200
3,200
3,200
-4,787
-911
2,881
Year 6
8,526
Year 7-10
8,978
3,200
5,326
3,200
5,778
Table 18. Cash flow analysis for a milking cow farm (size growing from 5 to10 cows).
Duration of the loan in years
Gross cash flow in the period
10 % annual percent rate loan
Loan repayment
Accrued two people minimal wages
Net cash flow
15 % annual percent rate loan
Loan repayment
Accrued two people minimal wages
Net cash flow
3
7,121
4
9,410
5
15,492
6
24,018
7
32,996
8
41,973
9
50,951
10
59,929
5,808
9,600
-8,287
6,087
12,800
-9,477
6,374
16,000
-6,882
6,669
19,200
-1,852
6,972
22,400
3,623
7,284
25,600
9,090
7,602
28,800
14,549
7,929
32,000
20,000
6,240
6,679
9,600 12,800
-8,718 -10,069
7,137
16,000
-7,645
7,612
19,200
-2,794
8,105
22,400
2,491
8,614
25,600
7,760
9,139
28,800
13,012
9,680
32,000
18,249
147
Key findings and comments: The cash-flow in the first years of

business is negative, so that if the investment period is shorter
than 5 years, the business is not able to generate enough cash to
pay for two minimal wages and repay the loan. Two more years are
necessary to recover the unbalances of the previous years and to
obtain a profit exceeding the minimal cost/opportunity of labour.
The main constraint is linked to the initial investment: 10,000
euro are not sufficient to increase the size of the stable, buy the
necessary equipment and purchase new adult cows; as a result,
the number of cows must be gradually increased and in the first
years the income is lower, as production is lower.
For this kind of business it is better to invest more from the
beginning and buy adult cows, entering immediately in full
production. Yearly repayments will be higher, but so will be the
income from the first year. Costs will be not minimised, but profits
will be maximised.
However, in the long run, a family farm with two permanent
work force can manage 10 milking cows, getting a better profit
than with 5 milking cows.
References
Leoneti, L., Sena, S. and Meo, M. 2010: Cash flow scenarios of the
selected agriculture activities in the field of livestock, September 2010.
2
Schrder, E., Urui, M. and Meo, M. 2010. Capacity Building for
Implementing the Rural Development Strategy. Milk Sector Study.
3
An Assessment of the Competitiveness of the Dairy Food Chain in
Albania 2009. AgriPolicy Enlargement Network for Agripolicy
Analysis.
4
MAFCP - Agriculture and Food Sector Strategy 2007-2013.
1
148
WFL Publisher
Helsinki, Finland
www.world-food.net
Improved water use efficiency in rice under limited water environment through
microbial inoculation
Mohamad Husni Omar 1*, Zulkarami Berahim 1, Norazrin Ariffin 1, Mohd Razi Ismail 1, 2, Halimi Mohd Saud 1, 3,
Nurul Amalina 1, S. H. Habib 2 and H. Kausar 1
Laboratory of Food Crops, Institute of Tropical Agriculture, 2 Department of Crop Science, Faculty of Agriculture,
Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
*e-mail: mohdhusni@upm.edu.my
1
Abstract
An experiment was conducted to evaluate the potential of plant growth promoting rhizobacteria (PGPR) for enhancing yield and yield contributing
characters of MR219 rice variety in limited water environment. A total of 20 bacteria were isolated from different dry rhizosphere soil samples. Nine
isolates (A1, A4, A5, B4, C2, C3, D1, D2a and D3) were found to fix nitrogen by changing the colour of malate media into pale blue. Six isolates (A4,
B2b, B4, C2, D2a and D4) developed halo zones on phosphate growth medium showing their ability to solubilize phosphate on such media. The
significantly highest IAA was produced by the isolate D2a. Based on the results of in vitro screening test, two best performed isolates were further
evaluated on rice variety MR219 under drought condition. The results of the glass house study revealed that, both the selected isolates B4 and D2a
were effective in enhancing plant physiological and yield contributing characteristics over control treatment indicating their potential to be used as
PGPR in rice variety MR219 under drought condition.
Key words: Oryza sativa, PGPR, water stress, nitrogen fixation, phosphate solubilization, IAA.
Introduction
Rice (Oryza sativa L.) is one of the staple foods for more than half
of the worlds population. It accounts about 23% of the worlds
caloric intake. The demand for rice production is increasing with
the ever increasing world population 1, 2. About two-thirds of the
total rice production comes from irrigated paddy land 3. Therefore,
the present and future food security depends largely on irrigated
rice production system. Rice is a profligate water user and semi
aquatic in nature. Roughly it takes about 3,000 - 5,000 litres of
water to produce 1 kg of rice, which is 2 to 3 times more than to
produce 1 kg of any other cereal crops like wheat or maize 4. The
conventional system of rice cultivation is flooded condition, which
provides water and nutrient supply in anaerobic conditions and
uses large amounts of water. However, about half of the rice
growing area in the world does not have enough water to maintain
flooded conditions, and yield is therefore reduced, to some extent,
by drought. Drought at critical stage may result in considerable
yield reduction and crop failure 2. In rain fed ecosystems, drought
is a major limitation for rice production where timing and duration
of drought is related to phenological process and rice yields 5. A
challenge for sustainable rice cultivation is to decrease the usage
of water while increasing or maintaining the yield. However, rice
plants have relatively little adaptations to limited water and are
extremely sensitive to drought 6, 7. In near future, many countries
will face water problems for shortage, poor quality or flood that
will increase regional tension. By 2030, without more efficient
management of water resources, the present problem will hinder
food production in many countries 8.
Many breeding and genetic engineering strategies have been
proposed to develop drought resistant variety but the approaches
are slower than expectation due to genetic complexity to stress

responses. Therefore, different alternatives rather than irrigation
strategy should be explored to achieve the aim. Microorganisms
existing in plant rhizosphere offer new opportunity for agricultural
biotechnology 9. Plant growth promoting rhizobacteria (PGPR)
influence plant health and productivity by solubilization of mineral
nutrients, stimulation of root growth and suppression of root
diseases 10. Recently, few reports have been published on PGPR
as elicitors of tolerance to abiotic stresses such as drought, salt
and nutrient deficiency or excess 11-13.
Beneficial microbes or PGPR are free-living soil bacteria that
can either directly or indirectly facilitate or confer beneficial effects
to plants, such as increased plant growth and reduced
susceptibility to diseases caused by plant pathogenic fungi,
bacteria, viruses and nematodes. These PGPR include Azospirillum,
Pseudomonas, Bacillus, and Agrobacterium species. PGPR also
can protect plants from the deleterious effects of some
environmental stresses including heavy metals, flooding, salt, and
phytopathogens. Relatively, few mechanisms have been clearly
demonstrated to explain the increased resistance to environmental
stresses including water stress of plants treated with PGPR 14.
Previous research by using PGPR that confer resistance to water
stress in tomatoes and peppers showed that the PGPR can facilitate
plant growth in stress environment 15. Herman et al. 16 also reported
enhanced physiological responses of sweet pepper with
inoculation of PGPR and mycorrhiza. However, there are lack of
information in rice growth and production by using beneficial
PGPR under water stress environment. So, the aim of this study
was to evaluate the effectiveness of PGPR to improve water use
149
efficiency of rice under limited water environments.

Isolation and screening of bacterial strains: Plant growth
promoting rhizobacteria (PGPR) were isolated from dry rhizosphere
soil samples collected from Tanjung Karang paddy field, Selangor,
Malaysia. Soil samples consisted of five cores randomly taken
from five cm depth and pooled into clean plastic bags. Samples
were stored at 4C until use.
Ten grams of soil sample was added with 90 ml of sterile saline
water (0.85% NaCl) and agitated at 150 rpm for 30 min. Serial
dilutions from 10-2 to 10-7 were prepared by sequentially transferring
1 ml diluted sample into each test tube containing 9 ml of sterile
saline water. A 100 l of sample at selected dilutions was transferred
onto Yeast Extract Mannitol (YEM) agar plate [1.0 g yeast extract,
10.0 g mannitol, 0.5 g K2HPO4, 0.2 g MgSO4.7H2O, 0.1g NaCl, 20.0
g agar and 1.0 L distilled water; pH 6.5] and incubated at 28 - 30C
for 24 h. The samples were spread over media using a sterilized
bent glass rod. The plates were examined regularly. Bacterial single
colonies were prepared by touching and streaking on Yeast Extract
Mannitol (YEM) agar plates in a third streak pattern. Isolated
single colony was picked up and re-streaked onto fresh YEM agar
plates and incubated similarly. Pure cultures were kept as stock
at 4C until required for further studies.
In vitro screening for plant growth promoting activities: Isolated
bacterial strains were tested for plant growth promoting activities
such as nitrogen fixation, phosphate solubilization and indole-3acetic acid (IAA).
Determination of nitrogen-fixing ability: Nitrogen fixing ability
was tested using the media proposed by Jha et al. 17. All isolated
bacteria were cultured in N-free semisolid malate medium
containing per litre 5 g malic acid, 0.5 g K2HPO4, 0.2 g MgSO4.7H2O,
0.1 g NaCl, 0.02 g CaCl2.2H2O, 4.0 g KOH, 1.8 g agar with addition
of 2 ml micronutrient, 4 ml FeEDTA, 1 ml vitamin solution, pH 6.5].
The agar will turn into pale blue with growth of nitrogen-fixing
bacteria.
Determination of phosphate solubilization activity: Phosphate
(P) solubilization test was conducted using NBRIP media [10 g
glucose, 5 g Ca3(PO4)2, 5 g MgCl2.6H2O, 0.25 g MgSO4.7H2O, 0.2 g
KCl, 0.1 g (NH4)2SO4, 15 g agar and 1.0 L distilled water, pH 6.5]
containing tri-calcium phosphate. This medium was chosen as it
was reported to be more efficient in screening phosphate
solubilizing microorganisms than Pikovskaya medium 18. Bacterial
isolates were cultured in NB for two days and 10 l was spotted
on the surface of media plates by using a micro pipette. The
plates were incubated at 30C for one week. Halo zones around
the bacterial colonies indicate phosphate solubilization. The size
of the halo zones was determined by measuring the radius formed
around colonies.
Determination of indole-3-acetic acid (IAA): All bacteria isolates
were grown in Nutrient Broth (NB) on an incubator shaker (150
rpm) at room temperature (28 2C) for 24 h. After the 24 h
incubation, 1 ml of bacterial culture was inoculated into 100 ml of
sterile NB amended with 5 ml L-tryptophan solution and allowed
to grow for 48 h. Flasks containing un-inoculated media were
150
used as controls or blanks.

To determine the amount of IAA produced from the isolates,
1.5 ml of aliquot was sampled and centrifuged at 12,000 rpm for 5
min. One ml of the supernatant was added to 2 ml of Salkowski
reagent (ferric chloride and perchloric acid). After 25 min of
incubation the colour density of the mixtures was read using
UV-spectrophotometer (Model UV-3600, Shimadzu) at 530 nm
absorbance. The amount of IAA produced was determined using
the standard curve.
Plant material and experimental design: The experiment was
conducted at glass house, Universiti Putra Malaysia, from April
2011 to August 2011. Rice seeds of variety MR219 obtained from
GeneBank, MARDI Research Station, Pinang, Malaysia, were used
in this experiment. Seeds were surface sterilized, soaked in water
and Zappa Plus for 24 h, subsequently sown in silty clay soil for
germination.
The soil used in this study was collected from Tanjung Karang,
Selangor, Malaysia, belongs to Bernam soil series. The soil was
rich in P, K, Ca, Mg and Zn and with a pH of around 5.01 which is
suitable for rice cultivation. The soil was spread on the green
house floor, air dried, crushed and sieved through a 5.0 mm mesh
to remove gravels and large debris. A 2.5 kg of air-dried soil was
packed into plastic bags and sterilized by gamma ray (40 kGy).
Then the soil with plastic bag was placed in the plastic pot (25 cm
17 cm) and the soil was soaked with distilled water for 2 days. Each
pot was fertilized with 120-70-80 kg ha-1 N-P-K, and the sources
were urea, triple super phosphate, and muriate of potash,
respectively, in four instalments at 15, 35, 55 and 70 DAT. After 15
days of germination rice seedlings were transplanted into pots
according to the treatments (Table 1). After transplanting, plants
were maintained under well-watered condition until 15 days and
the drought treatments (watering interval) were started.
Table 1. Treatments used in this experiment.
Treatments
T1
T2
T3
T4
T5
T6
T7
Flooded condition
Watering at 5 days interval
Watering at 5 days interval + Isolate B4
Watering at 5 days interval + Isolate D2a
Watering at 10 days interval
Watering at 10 days interval + Isolate B4
Watering at 10 days interval + Isolate D2a
The experiment was laid out in a randomised complete block design

(RCBD) with three replications. Selected PGPR was grown in
YEMA medium on rotary shaker. At log phase of growth, bacterial
suspension was centrifuged at 10,000 rpm for 4 min and washed
three times in phosphate buffer. Bacterial concentration was
adjusted at 108 cfu ml-1 19, 20. Two ml of each bacterial isolate (B4
and D2a) was applied to respective plant at the transplanting time
and 10 days later.
Harvesting, data recorded of the plants and analysis: Data on
chlorophyll content and photosynthesis rate were also recorded
by using SPAD meter and Li-Cor LI-6400XT portable photosynthesis system, respectively. At the end of the experiment, plants
were harvested and rice yield components, such as total panicles,
length of panicles, total spikelets, and total filled spikelets, were
recorded.
Statistical analysis: The data was subjected to analysis of variance

(ANOVA) and tested for significance using least significant
difference (LSD) by PC-SAS software 9.0 at 5% probability.
Results
Isolation, screening and plant growth promoting (PGP) activities:
A total of 20 bacteria were isolated from different rhizosphere soil
samples. All isolates were analysed based on their cultural and
morphological characteristics, i.e., colony morphology, colour,
shape and growth patterns. All bacterial isolates exhibited different
efficiency to nitrogen fixation, phosphate solubilization and IAA
production when tested on N-free semisolid malate medium,
phosphate growth medium and NBRIP phosphate growth medium
with L-tryptophan (Table 2, Figs. 1-3).
Table 2. Ability of PGPR isolates of nitrogen

fixation, solubilization of phosphate, and
production of IAA on N-free semisolid
malate medium, phosphate growth
medium and NBRIP phosphate growth
medium with L-tryptophan.
Isolates
A1
A2(a)
A2(b)
A3
A4
A5
B1(a)
B1(b)
B2(a)
B2(b)
B3
B4
C1
C2
C3
D1
D2(a)
D2(b)
D3
D4
Plant growth promoting characteristic

Nitrogen
Phosphate
IAA
fixation
solubilization
production
++
+
+++
+
+
++
+
+
+++
++
++
+
+
+++
++
+
++
+++
++
++
+++
+
+++
+
+
+++
+
+++
+
+++
++
- = No production; + = weak producer; ++ = medium producer; and +++ = good

producer.
Nine isolates were found to turn N-free semisolid malate medium

into pale blue. Among them, four isolates (A5, B4, C2 and D3)
showed higher ability to fix nitrogen by changing the color of
malate media into pale blue. Three isolates (A1, A4 and C3) showed
intermediate potential whereas two isolates (D1 and D2a) showed
the lowest potential to fix nitrogen in in-vitro screening test (Table
2, Fig. 1).
Six isolates (A4, B2b, B4, C2, D2a and D4) developed halo zones
on phosphate growth medium showing their potency to solubilize
phosphate on such media. Among them, isolate D4 appeared as
best and produced the biggest halo zone on NBRIP growth
medium. Isolate B4 and C2 were categorized as intermediate while
B2b and D2a produced the smallest halo zone on NBRIP growth
Figure 2. Ability of PGPR isolates to solubilize phosphate on phosphate
growth medium.
medium (Table 2, Fig. 2).
Out of 20 bacterial isolates, 15 were found to
25
produce IAA. The significantly highest IAA
was produced by the isolate D2a (20.7 g/ml)
20
which was closely followed by the isolate A2a
(20.4 g/ml). The least amount of IAA was
produced by the isolate D2d, followed by B2a,
15
A1 and A3. On the other hand, four isolates
(A5, B2b, B3 and B4) did not produce IAA on
10
L-tryptophan added NBRIP phosphate growth
medium (Table 2, Fig. 3).
Different treatments exhibited significant
5
differences in chlorophyll content and
photosynthesis (Table 3). The results showed
0
that plants treated with T4 and T1, showed the
A 1 2(a) 2(b) A 3 A 4 A 5 B1(a) 1(b) B2(a) 2(b) B3 B4 C1 C2 C3 D 1 2(a) 2(b) D 3 D 4
B
A A
B
D D
significantly highest chlorophyll content and
Isolate
photosynthesis compared to other treatments.
In all plants, chlorophyll content ranged from
Figure 3. Ability of PGPR isolates to produce IAA on NBRIP phosphate growth medium
34 to 44. Treatments T1 and T4 showed the
with L-tryptophan.
IAA concentration (g/ml)
Figure 1. Ability of PGPR isolates to fix nitrogen on N-free semisolid

malate medium.
151
Table 3. Effect of different treatments on

chlorophyll content and
photosynthesis of rice.
Treatment
T1
T2
T3
T4
T5
T6
T7
Chlorophyll content
(Time, unit)
44 a
37 b
43 a
44 a
34 b
35 b
36 b
Photosynthesis
(Time, unit)
15 a
13 b
14 b
16 a
12 b
13 b
13 b
Values having the same letter(s) in a column do not differ significantly at the
5% level of probability.
significantly highest chlorophyll content followed by T3and T7.

On the other hand, the significantly lowest chlorophyll content
was recorded in plants treated with T4 which was closely followed
by T6.
Similar to chlorophyll content, the significantly highest
photosynthesis was recorded in plants treated with T4 followed
by T1. However, the least photosynthesis was recorded in plants
treated with T5 treatment.
Results show that panicle number, panicle length, spikelet
number and percentage of filled spikelets were higher in plants
treated with T4 (Table 4). The highest panicle number was recorded
in treatment T4, followed by T1, T3 and T6, respectively, whereas
it was lowest in treatments T5 and T7, respectively. The highest
panicle length was found in treatment T4, closely followed by T1,
T3 and T6, respectively, whereas it was least in treatment T5. The
significantly highest spikelets number was recorded in plants
receiving treatment T4, followed by T5, T6 and T7, respectively.
The significantly least spikelets number was found in plants
receiving treatment T1. The highest number of filled spikelets was
found in treatment T4 closely followed by T1 and T3, whereas it
was least in treatment T6.
Table 4. Effect of different treatments on growth and
yield contributing characters of rice.
Treatment
T1
T2
T3
T4
T5
T6
T7
Number of
panicles
16 a
15 ab
16 a
17 a
14 b
15 ab
14 b
Panicle
length
(cm)
26 a
25 a
26 a
27 a
24 a
25 a
26 a
Number of
spikelets
174 c
185 bc
196 bc
229 a
209 b
209 b
204 b
Filled
spikelets
(%)
70.3 a
60 .0 b
70.1 a
70.9 a
50.8 bc
50.0 c
52.8 c
Values having the same letter(s) in a column do not differ significantly at the 5% level
of probability.
Discussion
Plant growth-promoting microorganisms (PGPM) are known to
influence plant growth by various direct mechanisms through
production of plant hormones, improved mineralisation, increased
iron uptake and promoted plant growth and indirect mechanisms
through the production of siderophores, antibiosis, lysis of
pathogen cell walls and elicit induced systemic resistance (ISR) in
various crops including cereals 21.
The tropical soils of Malaysia harbour diverse groups of plant
growth promoting bacteria. In this study, out of 20 isolates, nine
were found to turn N-free semisolid malate medium into pale blue
152
indicating they have the ability to fix atmospheric nitrogen in

such medium. Large amounts of nitrogen derived from biological
fixation have been shown to be present in rice plants 22. They utilize
rhizosphere carbon compounds for growth and development and
subsequently fix nitrogen for the plant. Besides consequences of
nitrogen fixation, bacteria exhibit plant growth enhancement
activities such as production of phytohormones, antifungal or
antibacterial agents, siderophore and induction of systemic
acquired host resistance and increased availability of mineral
nutrients to plants 23. Azorhizobium caulinodans enters into the
root system of cereals by intercellular invasion between epidermal
cells and the xylem. The xylem colonization might provide a nonnodular niche for endosymbiotic nitrogen fixation in rice, wheat,
maize, sorghum and other non-legume crops 24. Vascular tissue is
an ideal niche for endophytic colonization as there is low partial
pressure of oxygen (pO2) and allocation of photosynthate.
Application of Rhizobium leguminosarum bv. trifolii has been
shown to successfully colonize rice roots and supplied 25 - 33%
of the recommended rate of N fertilizer 25.
In vitro screening results revealed that the production of IAA
varied with all the isolates tested. Isolate D2a produced the
significantly highest amount of IAA. IAA is one of the
phytohormones, considered as the most physiologically active
auxin in plants that influences root and shoot dry matter
partitioning, root and shoot elongation through cell wall
extension 26. These results were in agreement with Mirza et al. 27
who reported variations among different species and strains, as
well as the effects of culture conditions, growth stage and substrate
availability. The capability of PGPM in producing IAA appears to
be important mechanisms involved in promoting plant growth.
The application of Trichoderma spp., Enterobacter spp. and
Bacillus spp. as PGPM to enhance plant growth has been
reported to be related to the synthesis of plant growth hormones
like IAA 28. These findings are also supported with those by Zarea
et al. 29 who demonstrated that seeds inoculated with IAAproducing microorganisms significantly enhanced early seedling
establishment. IAA is an important phytohormone produced by
PGPM that increased shoot growth, root hair density and length 30,
enhanced rice seed germination and improved growth 31. Plant
water and nutrient uptake potential is closely related to root growth
such as root surface area especially under dry cultivation systems.
Mantellin and Touraine 32 and Mia et al. 33 who demonstrated that
phytohormone produced by microbes caused morphological and
physiological changes in root, and resulted in increased nutrients
and water uptake from the soil. The greater growth stimulation
and vigour of young seedlings with pre-inoculation would result in
better productivity and higher yields at maturity 34.
Application of phosphate-solubilizing microorganisms (PSM)
is essential for increasing P uptake for plant growth. In this study,
15 out of 20 bacterial isolates showed positive P-solubilization.
Among them D4 appeared as the most promising PSM on NBRIP
growth medium. Most Pseudomonas and Bacillus isolates are
involved in solubilization of inorganic phosphorus 35. However,
according to Eusuf Zai et al. 36, although P-solubilizing bacteria
outnumber P-solubilizing fungi in soil, fungi isolates generally
exhibit greater P-solubilizing ability than bacteria in both liquid
and solid media. Phosphorus (P) is an essential plant nutrient for
plant growth. However, only a small part of P is utilized by plants,
while the rest is converted into insoluble fixed forms 37. P deficiency
is usually the consequence of low intrinsic P fertility due to

weathering, in combination with intensive, and nutrient-extracting
agricultural practices. Moreover, phosphate diffusion to plant
roots may be too low to meet the requirements of the crop if soils
have low P solubility and a high P fixation capacity 38. Almost 75
to 90% of added P fertilizer in agricultural soils is precipitated by
iron, aluminium and calcium complexes present in soils 39.
Furthermore, phosphatic fertilizers are expensive, and excessive
use of rock phosphate (RP) is potentially and environmentally
undesirable. Among microbial populations in soils, phosphate
solubilizing bacteria (PSB) constitute solubilization potential of 1
to 50% 40. The mechanism involves solubilization of the phosphate
in the presence of organic acids released by microorganisms 41.
The most powerful P solubilizers were reported to be bacterial
strains from the genera Pseudomonas, Bacillus, Rhizobium and
Enterobacter along with Penicillium and Aspergillus fungi 42.
Rice plant inoculated with isolates B4 and D2a demonstrated
higher effects in plant growth promoting characteristics, such as
chlorophyll content and photosynthesis rate, resulting in yield
increments of rice variety MR219. In in-vitro screening test both
of the isolates were found to fix nitrogen, solubilize phosphate
and produce phytohormone IAA on N-free semisolid malate
medium, phosphate growth medium and NBRIP phosphate growth
medium with L-tryptophan. The results obtained in this study
confirmed that rice plants treated with either T4 or T3 accumulated
higher dry matter which contributed higher yields. These results
(obtained from treatment T4) were comparable with normal flooding
condition (treatment T1). However, an integration of several
microorganisms may provide a more significant reliable effect in
both protections against pathogens and also as plant growth
promoter 43. The use of organic amendments is also recommended
to support the growth of the inoculums. In the current study,
therefore, isolates B4 and D2a could be selected as microbial
consortium to promote plant growth and drought tolerance.
Conclusions
The results indicate that the plant growth promoting rhizobacteria
isolated from different rhizosphere soil samples were able to
enhance growth promoting, physiological and yield contributing
characteristics of rice variety MR219. Out of 20 different isolates,
two isolates, B4 and D2a, showed optimum plant growth
promoting activities on N-free semisolid malate medium, phosphate
growth medium and NBRIP phosphate growth medium with Ltryptophan. Glass house trial also showed that both of the selected
isolates enhanced plant growth, yield and yield contributing
characters in drought condition compared to control.
Acknowledgements
The authors would like to acknowledge the support of Universiti
Putra Malaysia (UPM) Research University Grant and the Ministry
of Education, Malaysia Long Term Research Grant Scheme (LRGS)
- Food Security-Enhances sustainable rice production for financial
support without which this research would have been impossible.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Numerical investigation into optimal agricultural water management for typical soils
using HYDRUS-1D model
Po Li, Feiqing Wu and Kefeng Zhang *
Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China. *e-mail: kfzhang@hotmail.com
Abstract
Quantitative research of water circulation and utilization in the soil-plant system is the basis for rational use of agricultural water. In this study, a
systematic investigation into the optimal irrigation rate and time by numerical simulations for various soils was carried out using HYDRUS-1D
model. Three typical and contrasting European soils (coarse, medium and fine) were used in the simulations. Surface runoff during irrigation was
simulated, and so was water content distribution in the soil profile immediately after irrigation as well as 24 hours after irrigation were simulated.
Results reveal that the coarse soil could subject to a great irrigation rate. Even for the irrigation rate of 25 mm/h no significant surface runoff occurred
after 5 h irrigation. However, for both the medium and fine soils, the maximum irrigation rates without significant runoff were greatly reduced. The
maximum rate was about 5.0 and 3.0 mm/h for the medium soil and fine soil, respectively. The soil wetting depth 24 h after the maximum irrigation
rate for 5 h reached to 60 cm in the coarse soil, compared with about 20 and 16 cm in the medium and fine soil, respectively. This indicates that
irrigation could wet the root zone of deep-rooted crops in the coarse soil, while it could only wet the root zone for shallow-rooted crops in the medium
and fine soils. Furthermore, it was simulated that the soil wetting depth 24 h after irrigation was considerably greater than that immediately after
irrigation, suggesting that consideration should be taken in estimating soil wetting depth caused by soil water re-distribution. The results from this
study are helpful for irrigation planning for crops grown in different soils with different rooting depths.
Key words: Optimal irrigation, HYDRUS-1D model, irrigation simulation, soil water content distribution, soil wetting depth.
Introduction
Nowadays, 40% of the worlds food is produced by irrigated
agriculture, which consumes 70% of fresh water worldwide 1.
However, 60% of irrigated water is wasted 2. With the increase of
global population, the area of irrigated land is expected to increase
by another 15% by 2050 2. The threat of overuse of water to the
environment becomes increasingly acute, and therefore urgent
actions are required to save use of water in agriculture. One cause
of overuse of agricultural water is due to the unwisely-designed
irrigation plans. Inappropriate irrigation rate and time could result
in waste of water via surface runoff and water loss via percolation
to the deep soil.
Optimising irrigation rate and time is critically important to save
water use in agriculture. However, it is a difficult task, partly due
to uncertainty in soil texture and the associated soil hydraulic
properties. As a result the irrigation practice is commonly nonscientific, leading to low water use efficiency. Recently, studies
have been reported on use of soil sensors to control irrigation 3-9.
Many of such irrigation systems used soil water sensors at a
depth of approximately 250 mm to indicate soil water status in the
root zone, while some devised multi-sensor systems and used
sensors in the subsoil as a detector to locate the wetting front in
the soil. Although the systems as such ensured the minimum soil
wetting depth, , they were unable to locate the final wetting depth
caused by soil water redistribution.
With the advances in plant and soil sciences and in computing
power, numerous numerical models have been put forward, such
as HYDRUS-1D 10 and HYDRUS-2D/3D 11 for studying soil water
movement and solute transport, and the agro-hydrological models

of SWAP-1D 12, FUSSIM2-2D 13, 14 and RSWMS-3D 15 for
investigating water and nutrient cycle in the soil-crop system.
The models of this kind have played an important role in precision
agriculture, and are now reliable enough to make reasonable
predictions, given the accurate inputs of the models 16.
HYDRUS models are widely known for the simplification of its
data preparation, and have been used for various purposes 17. Its
effectiveness and accuracy in predicting soil water movement
and solute transport have been well demonstrated in hundreds of
scientific papers 17. However, as far as we can see, there is no
systematic investigation into optimal irrigation rate and time for
various soils and crops with different rooting depths using
HYDRUS-1D. With the availability of typical soil hydraulic
properties based on a great number of soil samples across Europe
and because the majority of irrigated farmland is still using sprinkler
irrigation, it is highly the time to employ HYDRUS-1D model to
conduct a numerical investigation for the purpose of assisting
irrigation planning to save agricultural water use.
The principal purpose of this study was to systematically
simulate the soil water content distribution immediately after
irrigation and 24 h after irrigation for the typical European soils 18
based on HYDRUS1D model, to find out the maximum allowable
irrigation rate and time without significant surface runoff for
different soils and the associated wetting depth. The results could
be used as guidance for administrating irrigation under different
circumstances of the soil and rooting depth.
155

Governing flow equations: HYDRUS-1D model simulates
variably-saturated soil moisture migration using the Richards
equation which is solved numerically by the element-free Galerkin
method 19. The model has flexible boundary conditions (BC) like
atmospheric BC with surface layer and runoff for the upper BC,
and free drainage and deep drainage for the lower BC. Irrigation
volume can be treated as precipitation as the input of the model.
The soil is assumed to be as a homogeneous and isotropic
medium. One-dimensional equilibrium water movement in a partially
saturated porous medium is described by a modified form of the
Richards equation where the role of the air phase in the liquid
flow process and water flow due to thermal gradients are
neglected 10. The general form of the Richards equation with a
sink term is given as follows:
wT
wt
w
wh
K T cos D S w
wz
wz
(1)
where denotes the volumetric water content in soil (cm3 cm-3),

z is the vertical coordinate with its origin at the soil surface
(positive upward; cm), h is the soil water pressure head (cm), K()
is the soil hydraulic conductivity (cm d-1), S is a sink term (cm3
cm-3 d-1), and is the angle between the flow direction and the
vertical axis (i.e., = 0 for vertical flow, 90 for horizontal flow,
and 0 < a < 90 for inclined flow) 10.
In this study, the simulations were carried out for only a short
period of time for the purpose of determining soil water content in
the profile after irrigation, and therefore the root water uptake was
not taken into consideration. Also no horizontal flow was
considered, i.e = 0 and S = 0, leading to Eq. (1) to be simplified
as:
K()
h
+1
z
s- r
h<0
(3)
h0
K(h) = KsS el [1 - ( 1 - Sel/m )m]2
(4)
where
Se =
r
s r
Definition
Clay <18% and sand > 65%
18% < clay < 35%, 15% < sand or clay < 18% and
15% < sand < 65%
35% < clay < 60%
Medium
Fine
The simulated soil column is assumed to have a topsoil with a

depth of 30 cm (Fig. 1). In each column a higher percentage of
sand and a lower percentage of clay are contained in the subsoil
compared to the topsoil, and the adopted soil hydraulic property
values used in the simulations are given in Table 2 18. The upper
boundary condition was specified as the influx water flow, while
the lower boundary condition was set as free drainage.
z(cm)
Z(cm)
Soil surface
surface
Soil
Topsoil
0-30 cm
Subsoil
30-100 cm
Table 2. Mualem and van Genuchten hydraulic parameters for the

soils used in the simulations 18.
(cm3 cm-3)
(cm3 cm-3)
(cm-1)
n
(-)
Ks
(cm d-1)
l
(-)
0.403
0.439
0.520
0.025
0.010
0.010
0.0383
0.0314
0.0367
1.3774
1.1804
1.1012
60.0
12.1
24.8
1.2500
-2.3421
-1.9772
0.366
0.392
0.481
0.025
0.010
0.010
0.0430
0.0249
0.0198
1.5206
1.1689
1.0861
70.0
10.8
8.5
1.2500
-0.7437
-3.7124
Ts
Topsoil
Coarse
Medium
Fine
Subsoil
Coarse
Medium
Fine
m = 1 - 1/ n
(5)
Tr
The initial soil water content in the profile was assumed to be as

follows:
crit = 0.5 (FC + PWP)
in which r and s (cm3 cm-3) are the residual water content and
the saturated water content, , m and n are the shape parameters,
Ks is the saturated hydraulic conductivity, Se is the relative
saturation. The pore-connectivity parameter l in the hydraulic
conductivity function was estimated to be about 0.5 as an average
for many soils 21.
156
Name
Coarse
Figure 1. Simulated soil column.
(1 + h )m
Table 1. Texture classes used to classify the available

data 18.
(2)
Soil hydraulic functions: HYDRUS-1D implements the soil

hydraulic functions of van Genuchten 20 who used the statistical
pore-size distribution model of Mualem 21 to obtain a predictive
equation for the unsaturated hydraulic conductivity function in
terms of soil water retention parameters. The expressions are given
by van Genuchten 20 and Mualem 21:
(h) =
Model parameterization: In this study three different soils, which

are classified as coarse, medium and fine according to the soil
classification by Wsten et al. 18 were tested (Table 1).
(6)
where crit is the critical soil water content, a threshold for

irrigation, FC and PWP are the soil water content at field capacity
and at the permanent wilting point, respectively.
The assumption for irrigation (Eq. 6) was based on the
suggestion by Allen et al. 22. For most crops, when soil water
content in the root zone is below crit, root water uptake cannot
meet the demand of water for maximum growth of crops, and thus,
irrigation is required 22. The values of fc, pwp and crit for different
soils used in the simulations are shown in Table 3.
0.059
0.151
0.279
0.114
0.220
0.342
Subsoil
Coarse
Medium
Fine
Tpwp
Tcrit
0.110
0.274
0.407
0.037
0.150
0.298
0.074
0.212
0.353
The simulations started with soils subject to various irrigation

rate and time to observe soil water content distribution in the
profile immediately after irrigation, and then the changes in soil
water content in the profile 24 h after irrigation. For the coarse soil
with a big capacity ability of water infiltration, the irrigation rate
was set to be 17.5, 20 and 25 mm h-1, respectively. The irrigation
time ranged from 1 to 5 h. However, due to a smaller infiltration
capacity, the corresponding values of irrigation rate were 2.5, 5.0
and 7.5 mm h-1 for the medium soil and 2.0, 4.0 and 5.0 mm h-1 for
the fine soil, respectively. The irrigation time remained the same.
Also, the surface runoff was simultaneously simulated during
irrigation.
In light of the fact that the irrigation time is relatively short,
both crop transpiration and soil evaporation in the numerical
experiments were ignored.
Maximum irrigation rate and water runoff on the soil surface:
The surface runoff for various soils under different irrigation rate
and time was simulated (Fig. 2). It is evident that for the coarse
soil virtually no runoff was simulated, even for the situation where
the soil was irrigated with the rate of 25 mm h-1 for 5 h. This
suggests that the coarse soil has a great ability of drainage and a
small water holding capacity. Water can move in the soil easily
under gravity due to big pore sizes and the entire root zone for a
wide range of crop species could easily be wetted. However, for
both the medium soil and the fine soil, significant runoff was
simulated for the irrigation rate above 7.5 and 5 mm h-1. The surface
runoff accounted for about 32% of irrigated water for the medium
soil, while the corresponding figure was about 20% for the fine
soil. The maximum allowable irrigation rates without significant
runoff were 5.0 and 4.0 mm h-1 for the medium soil and fine soil,
respectively. It is probably worth pointing out that the margin in
the maximum allowable irrigation rate between the medium soil
and the fine soil was actually rather small. This might be attributed
to the fact the adopted saturated soil hydraulic conductivity was
smaller in the medium soil, compared to that in the coarse and fine
soils (Table 2). Furthermore, the simulations reveal that the amount
of surface runoff increased with the increase in the irrigation time
in an approximately linear manner.
(a)
(a)
10
5
0
22
11
20
33
Time(h)
Time (h)
2.5 mm/h
2.5mm/h
7.5 mm/h
7.5mm/h
15
44
5
5
5 mm/h
5.0mm/h
(b)(b)
10
5
0
11
22
33
Time (h)
Time(h)
44
55
20
2.0mm/h
2 mm/h
55.0mm/h
mm/h
15
4.0mm/h
4 mm/h
(c)
(c)
10
5
0
1
3
Time (h)
Figure 2. Comparison of soil surface runoff with different

irrigation rates during irrigation for coarse soil (a), medium soil
(b), and fine soil (c).
Water Content (cm3 cm-3)

0
0.1
0.2
0.3
0
0
(a)
mmhh-1-1
(a)10
10mm
-20
11hh
22hh
33hh
44hh
55hh
-60
-80
11hh
22hh
33hh
44hh
55hh
-60
-80
-3
(c)
mmhh-1-1
(c)20
20mm
-20
Water content (cm3 cm-3)

0.1
0.2
0.3
(d)25
25mm
(d)
mm hh-1-1
-20
-40
-60
(b)
mmhh-1-1
(b)15
15mm
-40
Water content (cm cm )

0
0.1
0.2
0.3
3
Water content (cm3 cm-3)

0.1
0.2
0.3
-20
-40
-80
Water content distribution in the soil profile: Figs. 3 to 5 illustrate

soil water content distribution in the profile 24 h after irrigation
under various irrigation rate and time for different soils.
For the coarse soil, the applied irrigation rate was 10, 15, 20, 25
mm h-1, and the irrigation time was from 1 h to 5 h. Generally, soil
water content distribution bears a very similar pattern (Fig. 3). In
a large part of the wetted soil near the surface, soil water content
was approximately identical. However, in a small region at the
bottom of the wetted soil, soil water content varied drastically.
20 mm/h
20mm/h
30 mm/h
30mm/h
Depth (cm)
0.169
0.289
0.404
Tfc
15 mm/h
15mm/h
25 mm/h
25mm/h
15
Depth (cm)
Tcrit
Runoff (mm)
Tpwp
Runoff (mm)
Tfc
Depth (cm)

Topsoil
Coarse
Medium
Fine
Depth (cm)
Table 3. Values of fc, pwp and crit for the soils used in the
simulations.
Runoff (mm)
20
11hh
22hh
33hh
44hh
55hh
-40
-60
-80
11h
h
22hh
33h
h
44h
h
55h
h
Figure 3. Comparison of water content in the soil profile

24 hours after irrigation with irrigation rates of 10 mm h-1
(a), 15 mm h-1 (b), 20 mm h-1 (c) and 25 mm h-1 (d) and
different irrigation time for coarse soil.
The reason for the entire wet zone with fairly constant soil
water content is due to the fact that the coarse soil has a great
capacity of drainage. Water penetration in the soil is mainly
induced by gravity and to a much lesser extent by the difference
in soil water potential.
157

00
0.25
0.3
0.35
20
-20
(a) 2.5 mm h-1
(a) 2.5mm h-1
Depth (cm)
-10
-10
11hh
22hh
33hh
44hh
55hh
40
-40
50
-50
-20
-20
Depth(cm)
Depth (cm)
(a)
(a)
00
10
-10
30
-30
20

0.2
0.25 0.3
0.35 0.4
0.4
Wetting depth (cm)
0.2
-30
-30
(b) 5.0 mm h-1
(b) 5.0mm h-1
11hh
22hh
33hh
44hh
55hh
-40
-40
-50
-50
00
0
-30
0
-40
-50
0
Wetting depth (cm)

0.3 0.35 0.4 0.45 0.5
(a) 2.0mm h-1
(a) 2.0 mm h
-1
11hh
22hh
33hh
44hh
55hh
-20
-20
-30
-30
-40
-40
-50
-50
(b) 4.0mm h-1
(b) 4.0 mm h
-1
11hh
22hh
33hh
44hh
55hh
3
Time (h)
15mm/h
mm/h
15
25mm/h
25
mm/h
20
(b)
(b)
15
10
2.5 mm/h
mm/h
2.5
5.0 mm/h
mm/h
5.0
5
0
3
Time (h)
(c)
(c)
15
10
5
0
2 mm/h
2.0
cm/h
4 mm/h
4.0
cm/h
3
Time (h)
Figure 6. Comparison of soil wetting depth 24 hours

after irrigation for coarse soil (a), medium soil (b) and
fine soil (c).
Figure 5. Comparison of water content in the soil profile 24

hours after irrigation with irrigation rates of 2.0 mm h-1 (a), and
4.0 mm h-1 (b) and different irrigation time for fine soil.
However, contrary to that in the coarse soil, soil water content

decreased with the increasing depth in the profile in a much more
gentle way in both the medium and fine soils (Figs 4 and 5). Soil
water content increased with the amount of the irrigated water at
a given depth. For example, when the medium soil was irrigated
with 5.0 mm h-1 for 1 h, the water content at the 10 cm depth was
about 0.2358 cm3 cm-3, while the corresponding value was about
0.3498 cm3 cm-3 for the soil irrigated with the same rate for 5 h.
The simulated results also revealed markedly differences in the
way the irrigated water was stored in the soil. If the coarse soil
was irrigated with a large quantity, a considerable amount of
irrigated water was stored in the subsoil (Fig. 3a-b). However, in
the medium and fine soils, the increase in irrigated water stored in
the soil was mainly implemented by an increase in soil water
content in the wetted area (Figs. 4b and 5b). Such a phenomenon
could be attributed to the greater drainage capacity and the smaller
water holding ability in the coarse soil as explained the above.
Wetting depth: Fig. 6 shows the variation of the soil wetting depth
24 h after irrigation with irrigation rate and time for all the simulated
soils.
Clearly there are some noticeable differences in the variations
amongst various soils. Under a small irrigation rate, the increase
in the wetting depth with the irrigation time for a given irrigation
rate in the coarse soil is somewhat similar with that of the medium
and fine soil. The wetting depth increase rate is much greater in a
shorter irrigation period (Figs. 3a, 4 and 5). For the coarse soil
under the irrigation rate of 10 mm h-1, the wetting depth was about
15 cm for the irrigation time of 1 h, and increased to only 36 cm for
the irrigation time of 5 h (Fig. 3a). The wetting depth under the
158
10mm/h
10
mm/h
mm/h
20
20mm/h
20
Wetting depth (cm)
0
-20
-10
-10
Depth
(cm)
Depth(cm)
Depth (cm)
0
-10
10
Figure 4. Comparison of water content in the soil profile 24

hours after irrigation with irrigation rates of 2.5 mm h-1 (a), and
5.0 cm h-1 (b) and different irrigation time for medium soil.
0.3 0.35 0.4 0.45 0.5
00
15
irrigation rate of 2.5 and 5.0 mm h-1 was about 8 cm and 10 cm for
the irrigation time of 1 h, and increased to 15 cm and 20 cm for the
5 h irrigation in the medium soil, respectively. Correspondingly the
figures were about 5 and 7 cm, and 12 and 16 cm for the irrigation
rate of 2.0 and 4.0 mm h-1 in the fine soil. However, under a greater
irrigation rate in the coarse soil, the increase in the wetting depth
with the irrigation time appears to be more linear, for example,
under the irrigation rate of 20 mm h-1, the wetting depth was about
19 cm for the irrigation time of 1 h, and increased to 56 cm for the
irrigation time of 5 h.
Movement of the wetting front: The locations of the wetting
front immediately after irrigation and 24 h after irrigation were
simulated, and some examples for different soils are shown in Fig. 7.
In all the cases, considerable expansion of the wetted area with
varying degrees was simulated due to soil water re-distribution.
The expansion depth depends on the soil type, irrigation rate and
time. It can be found that the increase in the expansion depth in
the coarse soil can be much greater than that in the other soils.
When the coarse soil was irrigated with the rate of 20 mm h-1 for 5
h, the soil wetting front could move 37 cm deeper 24 h after
irrigation, while for both the medium and fine soils under the
maximum irrigation rate, the wetting front could move downwards
about 10 and 9 cm only, respectively. There was no big difference
in terms of the wetting depth between the medium and fine soil.
Also, for a given soil and the irrigation rate, the movement of the
wetting front was positively related to irrigation time. For example,
the increase in the wetting depth in the coarse soil irrigated with
the rate of 20 mm h-1 for 1 h was about 19 cm, and the value increased
to 37 and 56 cm for the irrigation time of 3 and 5 h, respectively.

0 0.1 0.2 0.3 0.4 0.5
Depth (cm)
-20
-40
-20
-20
a
-60
-80
Depth (cm)

0.3
0.4
0.5
0.6
0
-10
-20
-30
-80
-80

0.1 0.2 0.3 0.4 0.5
-10
-10
h
time 1h
1h
--- irri.
irri.time
after irri.
24
24hh after
irri.
--- irri.
irri.time
time 5h
5h
h after
after irri.
24
24h
irri.
-80
-80
00

0.1 0.2 0.3 0.4 0.5
-10
-10
f
-20
-20
irri. time3h
3h
--- irri.time
24 hafter
after irri.
irri.
24h
-30
-30

0.3
0.4
0.5
0.6
00
-10
-10
to only 5.0 and 4.0 mm h-1, respectively. The wetting

depth 24 h after the maximum irrigation rate for 5 h,
i.e 25 mm irrigated water for the medium soil and 20
mm for the fine soil, was about 20 and 16 cm,
respectively, indicating that the irrigation could only
wet the entire root zone for the crops with shallow
and shallow-median roots.
-60
-60
--- irri.
irri.time
time 3h
3h
24
24hh after
irri.
after irri.
-20
--- irri.time
irri. time1h
1h
24 hafter
after irri.
irri.
24h
-40
-40
d
-20
-30
Depth (cm)
-40
-40
00
00

0 0.1 0.2 0.3 0.4 0.5
-20
-20
-60
-60
--- irri.
irri.time
time1h
1h
24
24h
irri.
h after
after irri.

0.1 0.2 0.3 0.4 0.5
0
-10
0
0

0
0.1 0.2 0.3 0.4 0.5
-20
-20
-30
-30
irri. time5h
5h
--- irri.time
24 hafter
after irri.
irri.
24h
-30
-30

0.6
0.3
0.4
0.5
0
-10
-10
i
time3h
3h
--- irri.
irri.time
after irri.
24
24hh after
irri.
-20
-20
-30
-30
time5h
5h
--- irri.
irri.time
24 h after
after irri.
24h
irri.
Figure 7. Water content in the profile immediately and 24 hours after irrigation for the
irrigation rate of 20 mm h-1 for coarse soil (a-c), 5.0 mm h-1 for medium soil (d-f) and
4.0 mm h-1 for fine soil (h-j).
Implications in irrigation planning: The principal tasks in

irrigation planning are to decide when to irrigate and how much
water is required to apply 23, i.e determination of irrigation
scheduling and the required amount. In commonly adopted
practices, when certain amount of readily available water (RAW)
in the root zone, dependent on the crop species and their growth
stages, is depleted, irrigation is triggered 22, 23. For most of crop
species irrigation is applied when a half of RAW is depleted 22.
The studies as such recommend the time of amount of irrigation,
but do not specify how the irrigation is administrated.
The results from this study could be useful in three aspects:
1) to decide the maximum irrigation rate without significant surface
water runoff to avoid water loss for a given soil; 2) to determine
the possibility of applying the recommended irrigation amount;
and 3) to estimate the wetting depth and thus to know the
possibility of wetting the entire root zone or potential water loss
to percolation. It is evident that the coarse soil could subject to a
much greater irrigation rate. The soil could basically infiltrate any
amount of required water without water loss to surface runoff,
and the irrigated water could penetrate to a great depth. Since for
a wide range of crop species the rooting depth is no more than 60
cm 21, the entire root zone could be wet after a single irrigation
event. However, for both the medium and fine soil, the maximum
irrigation rates without significant runoff were reduced drastically
Conclusions
The coarse soil could infiltrate a great amount of
water with the irrigation rate up to 25 mm h-1. The
maximum irrigation rate for the medium and fine soils,
however, is greatly reduced. The maximum irrigation
rate is 5.0 mm h-1 and the maximum irrigation amount
is about 25 mm in a single irrigation event for the
medium soil, while the 4.0 mm h-1 and 20 mm are for
the fine soil, respectively.
The maximum wetting depth 24 h after irrigation
could reach to 60 cm, enough to wet the entire root
zone for a wide range of crop species in the coarse
soil. On the other hand, the maximum wetting depth
could only reach to 20 and 16 cm for the medium and
fine soils, capable of wetting the entire root zone for
the crops with shallow and shallow-median roots
only.
The re-distribution of soil water content in the
profile after irrigation is important in predicting the
wetting depth and should therefore be taken into
consideration in devising irrigation planning.
Acknowledgements
The authors are grateful to the financial support
from the National Natural Science Foundation of
China (Grant No. 51379187) for carrying out the work.
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10
160
WFL Publisher
Helsinki, Finland
www.world-food.net
Effects of allelopathic crop water extracts and their combinations on weeds and
yield of rainfed wheat
Shahbaz Hussain 1, Fayyaz-ul Hassan 1, Muhammad Rasheed 1, Safdar Ali 2 and Mukhtar Ahmed 1*
1
Department of Agronomy, PMAS-Arid Agriculture University, Rawalpindi 46300, Pakistan. 2 Department of Soil Science and
Soil Water Conservation, PMAS-AAU, Rawalpindi 46300, Pakistan. *e-mail: ahmadmukhtar@uaar.edu.pk
Abstract
Efficacy of allelopathic crop water extracts to control weeds can be enhanced by integrating them with other phytotoxins. The effects of allelopathic
crop water extracts and their combinations along with lower rates of Atlantis 3.6 WG were studied to control weeds in rainfed wheat through field
experimentation during 2009-2010 and 2010-2011. Treatments included weedy check, hand weeding, Atlantis 3.6 WG @ 14.4 g a.i. ha-1, sorghum
water extract, sunflower water extract, sorghum + sunflower water extract and combinations of sorghum + sunflower water extracts with 25, 50 and
75% rates of Atlantis 3.6 WG arranged in randomized complete block design. Results revealed that combination of allelopathic extracts with lower
rates of herbicide decreased weed density, fresh and dry weights by 42-70%, 38-62% and 37-63%, respectively. Atlantis 3.6 WG @ 14.4 g a.i. ha-1
reduced these parameters by 59-66%, 52-53% and 52-56%. The maximum numbers of fertile tillers, number of grains spike-1 and grain yield were
recorded from hand weeding. Statistically, similar yields were obtained from allelopathic water extracts combined with 50-75% rates of herbicide and
full dose of herbicide. Relatively lower wheat grain yields were recorded during second year compared with first due to moisture stress. The maximum
net benefits were achieved from sorghum and sunflower water extracts mixed with 75% dose of Atlantis 3.6 WG followed by mixture of allelopathic
extracts with 50% herbicide and recommended rates of Atlantis 3.6 WG (14.4 g a.i. ha-1). Marginal analysis exhibited dominance of all treatments
except combination of extracts with lower doses of herbicides. The study concluded that sorghum and sunflower water extracts can be used in
combination with 50-75% rates of Atlantis 3.6 WG to control weeds economically in wheat crop under rainfed conditions.
Key words: Allelopathic, weeds, herbicide, wheat, marginal analysis.
Introduction
Wheat (Triticum aestivum L.) is an important cereal crop and staple
food of Pakistan. Crop is grown all over the country including
irrigated and rainfed areas. Share of rainfed wheat in total domestic
wheat production is about one third (1/3). Average yield of wheat
crop in Pakistan is 2714 kg /ha, which is very low as compared to
other advanced wheat producing countries of the world 1. There
are many factors contributing towards low yields in rainfed system
like, i.e. lack of moisture, availability of certified seed, sowing too
early or late, imbalanced nutrition, irrigation and plant protection,
etc. Weeds infestation is one of the major cause of low yields of
this crop. Weeds decrease the yield of wheat by 20-50% 9. Manual,
mechanical, cultural and chemical methods of weed control are
commonly being used by farmers. Chemical method has gained
popularity during past few years due to its effectiveness, quick
response and decreasing farm labour. Although herbicides provide
satisfactory weed control, yet their use poses environmental and
health hazards 25 along with affecting the quality of the produce 21.
It also causes herbicide resistance in weed species 22. There is
need to decrease herbicide usage and harness other means to
cope with the risks involved with herbicides. Allelopathy is a
natural and safe approach, which offers a great potential to manage
weeds 8. It deals with the direct influence of allelochemicals
produced by one living plant on the growth and development of
neighboring plant species 16. These chemicals are present in

conjugated form in almost all plants and in many tissues like roots,
stems, leaves, flowers, fruits and seeds 27. The discharge of
allelochemicals into environment occurs by dissolving toxins of
fresh and decomposed litter in water 19.
Sorghum herbage incorporation in soil and sorghum water
extract application can decrease fresh and dry weights of weeds
and increase yield of wheat upto 30% 4. Sorghum water extract
suppressed density and dry weight of weeds by 21-38% and 2648% in irrigated wheat 7. Sunflower extracts inhibited growth of
Chenopodium album L., Avena fatua L., Phalaris minor Retz.
and Coronopus didymus L. in wheat 20. Ashraf and Naeem 3 found
that the combination of sorghum and sunflower water extracts
suppressed density and fresh and dry weight of weeds more
strongly than their sole extracts. Awan et al. 5 reported that mixed
crop water extracts worked in concert with each other and decreased
weed biomass in a higher degree. Furthermore, allelochemicals
and allelopathic extracts can be used along with herbicides to get
the better weed control 12. Mushtaq et al. 18 and Jabran et al. 11
reported a reduction in herbicide rates by integrating them with
allelopathic crop water extracts under irrigated conditions.
Studies reported above and many others have been conducted
under irrigated conditions, where moisture availability at the time
161
of herbicide application was amply available. However, weed

management strategies are different in rainfed wheat production
system where availability constrained to wait for its ample presence
at the time of herbicide or combined application. Keeping in view
the moisture availability constraints under rainfed conditions,
present study aimed to document the sole and combined effects
of sorghum and sunflower water extracts with each other and with
low doses of herbicides to get effective weed control in wheat.
The study aims at the use of farm produced resources efficiently
and reduce the expenses on herbicides. The findings of the study
may help the farmers to use allelopathic crops to manage the
weeds and ultimately improve yield and environment.
Site description: The effects of sorghum and sunflower water
extracts alone and combined with lower rates of Atlantis 3.6 WG
on weeds in wheat were studied through field experiments
conducted at PMAS Arid Agriculture University, Research Farm,
Chakwal Road, from 2009 to 2011 on sandy loam soil having organic
matter 0.67% with soil pH 7.5. Available phosphorus and potassium
were 3.5 and 80 mg kg-1, respectively.
Preparation of crop water extracts: The herbage of sorghum
(Sorghum bicolor L. cv. Chakwal sorghum) and sunflower
(Helianthus annuus L. cv. Shamas) collected at their maturity were
chopped into small pieces. The chopped herbage of each crop
was soaked in tap water in a ratio of 1:10 (w/v) for 24 hours at room
temperature and filtered with the help of sieves. The filtrate was
boiled to reduce the volume up to 20 times. These concentrated
crop water extracts were used in respective treatments in the study.
The experiments were arranged in randomized complete block
design (RCBD) with four replications in plots of size 6 m 5 m on
a summer fallow field. Treatments in the experiments included
control (weedy check), hand weeding, Atlantis 3.6 WG @ 14.4 g
a.i. ha-1 spray at 40 days after sowing (DAS), sorghum water extract
@ 20 L ha-1 spray at 40 and 60 DAS, sunflower water extract @ 20 L
ha-1 spray at 40 and 60 DAS, sorghum + sunflower water extract @
10 L ha-1 each spray at 40 and 60 DAS, sorghum + sunflower water
extract @ 10 L ha-1 each + Atlantis 3.6 WG @ 3.6 g a.i. ha-1 spray at
40 DAS, sorghum + sunflower water extract @ 10 L ha-1 each +
Atlantis 3.6 WG @ 7.2 g a.i. ha-1 spray at 40 DAS and sorghum +
sunflower water extract @ 10 L ha-1 each + Atlantis 3.6 WG @ 10.8
g a.i. ha-1 spray at 40 DAS. Wheat (Triticum aestivum L. cv.
Chakwal 50) was sown on November 14, 2009 during first year and
on October 30, 2010 during the second year with rabi drill using
seed @ 100 kg ha-1 in 25 cm apart rows. Fertilizers were applied
uniformly in all treatments @ 75, 60, 60 NPK kg ha-1 at time of
sowing in the form of urea, DAP and SOP. Knapsack hand sprayer
fitted with flat fan nozzle was calibrated (375 L water ha-1) and
used for spraying.
Data on weed dynamics at 75 days after sowing (DAS), yield
and yield components of wheat were recorded at maturity.
Meteorological data were also obtained from the nearest station.
The data were subjected to analysis of variance and the means
were compared by the least significant difference test (LSD) at 5%
level of significance 17. An economic analysis was carried out on
the basis of variable costs and market prices of commodities and
marginal analysis were performed as suggested by Byerlee 6.
162
Results
Total weeds density (No. m-2) at 75 DAS: The weed flora of the
experimental site comprised of Convolvulus arvensis L. (field bind
weed), Fumaria indica L. (fumitory), Chenopodium album L.
(lambsquarters), Vicia sativa L. (common vetch) and Avena fatua
L. (wild oat). Field bind weed was the major one to infest the field.
All weed control treatments suppressed total weed density
recorded at 75 days after sowing (DAS). Relatively more weeds
per unit area were recorded during 2009-2010 compared with 20102011. The highest weed infestation was observed from weedy
check. Sorghum and sunflower water extracts depressed it by 2327% while their combination improved weeds inhibition.
Combination of allelopathic water extracts with lower rates of
Atlantis 3.6 WG (7.2 and 10.8 g a.i. ha-1) gave statistically similar
reduction of total weed density as was achieved by application of
Atlantis @ 14.4 g a.i. ha-1. However, the maximum control of weeds
was observed in hand weeding treatment, which decreased total
weed density from 78 to 84% during both years.
Total weed fresh weight (g m-2) at 75 DAS: Weed control
treatments reduced total weed fresh weight by 21-86% as compared
to control during both years at 75 DAS (Table 1). The maximum
reduction in total weed fresh weight was calculated from hand
weeding (83-86%) followed by allelopathic water extracts
combined with Atlantis 3.6 WG @ 10.8 g a.i. ha-1 (62%). Sorghum
and sunflower water extracts alone and their mutual combinations
reduced total weed fresh weight by 21-32% and were statistically
at par with each other. Full dose of Atlantis 3.6 WG (14.4 g a.i. ha-1)
reduced total weed fresh weight by 52%, which was statistically at
par with sorghum and sunflower water extracts combined with 5075% rate of Atlantis during both years. Results revealed that hand
weeding was the most effective method for reducing total weed
fresh weight, but it was a labour intensive and time consuming
approach.
Total weed dry weight (g m-2) at 75 DAS: All treatments reduced
total weed dry weight significantly as compared to control (T1) at
75 DAS (Table 1). Allelopathic extracts controlled total weed dry
weight by 21-32% at statistically equal level among each other.
Combination of extracts with lower rates of herbicide decreased
total weed dry weight by 37-63% as compared to control. The
data revealed that combinations of allelopathic extracts with half
to three fourth dose of herbicide were equally effective to reduce
total weed dry weight as compared with full dose of herbicides.
The maximum reduction of weed biomass was again recorded from
hand weeding (85-87%) during both years.
Number of fertile tillers m-2: Most of the treatments increased
the number of fertile tillers m-2 significantly compared with control.
It varied from 158 to 201 during both years (Tables 2 and 3). More
tillers were observed during 2010-2011 compared with 2009-2010.
The lowest numbers of tillers were recorded from control treatment
during both seasons. The maximum numbers of fertile tillers (187.1
and 201 tillers m-2) were obtained from hand weeding. Those were
statistically at par with T3, T8 and T9 during both years. Crop
water extracts produced statistically similar number of fertile tillers;
however, their combination with each other and with herbicides
improved it according to increasing concentrations of Atlantis.
Herbicide (Atlantis 3.6 WG) was applied at recommended dose,
Table 1. Effect of weed control treatments on weed dynamics in wheat at 75 DAS.

Treatments
T1 Control (weedy check)
T2 Hand weeding
T3 Atlantis 3.6 WG@ 14.4 g a.i. ha-1 spray at 40 DAS
T4 Conc. sorghum water extract @ 20 L ha-1 at 40 and 60 DAS
T5 Conc. sunflower water extract @ 20 L ha-1 at 40 and 60 DAS
T6 Conc. sorghum + sunflower water extract each @ 10 L ha-1 at
40 and 60 DAS
T7 Conc. sorghum + sunflower water extract each @ 10 L ha-1 +
Atlantis 3.6 WG @ 3.6 g a.i. ha-1 at 40 DAS
atlantis 3.6 WG @ 7.2 g a.i. ha-1 at 40 DAS
LSD at 5%
Total weeds density

(No. m-2)
2009
2010
54.5 a
41.75 a
8.5 e
9f
(84.40)
(78.44)
18.5 d
17 de
(66.06)
(59.28)
41.75 b
30.5 b
(23.39)
(26.95)
42 b
31 b
(22.94)
(25.75)
39.25 b
29.25 b
(27.98)
(29.94)
27 c
24 c
(50.46)
(42.51)
21.25 d
20 d
(61.01)
(52.10)
16.5 d
16.8 e
(69.72)
(59.88)
5.07
3.04
Total weeds fresh

weight (g m-2)
2009
2010
43.18 a
55.58 a
7.33 g
7.64 e
(83.03)
(86.26)
20.82 ef
26.29 cd
(51.79)
(52.71)
33.40 b
40.37 b
(22.66)
(27.37)
34.26 b
39.68 b
(20.65)
(28.62)
31.55 bc
37.72 b
(26.92)
(32.14)
26.84 cd
29.78 c
(37.85)
(46.41)
22.5 de
26.76 cd
(47.90)
(51.85)
16.46 f
21.82 d
(61.88)
(60.75)
5.43
6.01
Total weeds dry

weight (g m-2)
2009
2010
8.15 a
11.52 a
1.25 f
1.49 g
(84.70)
(87.07)
3.90 e
5.05 ef
(52.18)
(56.18)
6.21 b
8.32 b
(23.91)
(27.77)
6.47 b
8.32 b
(20.66)
(27.79)
5.76 bc
7.82 bc
(29.30)
(32.10)
5.11 cd
6.69 cd
(37.34)
(41.93)
4.52 de
5.74 de
(44.57)
(50.20)
3.62 e
4.31 f
(55.58)
(62.60)
0.96
1.14
Table 2. Effect of weed control treatments on yield and yield components of wheat during 2009-2010.
Treatments
T2 Hand weeding
40 and 60 DAS
LSD at 5%
36.13 d
38.63 a
38.35 a
36.70 cd
36.83 cd
1000 grain
weight (g)
32.90 b
33.40 ab
33.47 ab
32.97 ab
32.78 b
Grain yield
(Kg ha-1)
1751.1 e
2305.9 a
2201.8 ab
1945.6 cd
1874.1 de
172.4 bcd
36.92 cd
33.08 ab
2018.2 cd
178.1 abc
37.10 bc
33.28 ab
2094.1 bc
184.0 ab
37.28 bc
33.63 ab
2233.9 ab
186.1 a
37.88 ab
34.03 a
2353.2 a
12.97
0.94
1.07
182.40
No. of fertile
tillers m-2
158.3 e
187.1 a
181.8 ab
167.3 cde
163.3 de
Grains spike-1
Table 3. Effect of weed control treatments on yield and yield components of wheat during 2010-211.
Treatments
T2 Hand weeding
40 and 60 DAS
LSD at 5%
27.17 c
31.83 a
30.85 ab
29.27 abc
28.55 bc
1000 grain
weight (g)
32.50
33.50
33.20
32.70
32.70
Grain yield
(Kg ha-1)
1508.1 e
2087.7 a
1982.3 a
1690.2 cd
1635.5 de
178.5 bcd
29.35 abc
32.85
1759.5 cd
180.0 bcd
30.27 ab
32.78
1802.4 bc
193.8 abc
30.6 ab
33.00
1933.3 ab
198.4 ab
31.2 ab
33.25
2052.5 a
20.10
2.76
NS
162.22
No. of fertile
tillers m-2
171.4 d
200.8 a
195.7 ab
175.1 cd
173.4 d
Grains spike-1
163
its one half and three fourth rates mixed with allelopathic extracts
were equally effective in increasing number of fertile tillers of
wheat during both years.
Number of grains spike-1: Most of the treatments increased the
number of grains per spike significantly as compared to control. It
ranged from 27.17 to 38.63 (Tables 2 and 3) during the study.
Relatively, the lower number of grains was counted during the
second year. The maximum (31.83 to 38.63) number of grains spike-1
was recorded from hand weeding and it was at par with Atlantis @
14.4 g a.i. ha-1 and crop extracts mixed with Atlantis @ 10.8 g a.i.
ha-1 during both seasons. Although crop water extracts increased
the number of grains per spike, they were statistically similar with
control treatment. Control treatment produced the least number
of grains spike-1 during both seasons.
Thousand (1000) grains weight (g): Thousand grains weight
varied from 32.5 to 34.03 g (Tables 2 and 3) in all treatments during
both years. Sorghum and sunflower water extract along with
Atlantis @ 10.8 g a.i. ha-1 produced the heaviest grains (34.03 g)
during 2009-2010, which was at par with all other treatments. A
non significant effect of different weed control treatments was
found during the second year.
Grain yield (kg ha -1): All treatments except T5 produced
significantly higher grain yields as compared with control during
both years. It varied from 1508 to 2353 kg ha-1 (Tables 2 and 3) in
all weed control treatments. Sole water extracts of sorghum and
sunflower increased wheat grain yields (7-12%), but their mutual
combination produced 17% higher yields than T1. Statistically,
similar grain yields were recorded from T2, T3, T8 and T9 during
2009-2010 and 2010-2011. Herbicide application at full dose, hand

weeding, combination of sorghum and sunflower water extracts
with Atlantis @ 7.2 and 10.8 g a.i. ha-1 increased grain yields of
wheat and these were statistically at par with each other.
Economic and marginal analyses: All weed control treatments
increased net benefits of wheat crop over control during both
years (Tables 4 and 5). The highest variable cost was calculated
from T2 due to expensive manual labour. The maximum net benefits
were achieved from T9 (Rs. 60889 and 52721 ha-1 during the first
and second year, respectively) followed by T8 during both
seasons. These results demonstrated that combined application
of allelopathic extracts with 50 and 75% dose of herbicide gave
higher net benefits than its recommended rate.
Marginal analysis showed that all treatments except T7, T8 and
T9 were not profitable due to lower net benefits and higher variable
costs during both seasons (Tables 6 and 7). The higher marginal
rate of return (MRR) were calculated from T7 (532 and 442%)
followed by T8 (513 and 474%) and T9 (423%) during both years.
Discussion
Allelopathic aqueous extracts reduce weed dynamics by affecting
several physiological processes including inhibition of nutrient
uptake, mitotic inhibition, photosynthetic inhibition, effects on
membrane permeability and production of reactive oxygen species.
Atlantis 3.6 WG (mesosulfuron+iodosulfuron) is a sulfonyl urea
herbicide possessing amino lactate synthase (ALS) inhibition
mode of action. Sorghum water extracts suppressed total weed
density by 21-38% in irrigated wheat 7. Ashraf and Naeem 3 reported
stronger inhibitory effects of mixed allelopathic extracts of
sorghum and sunflower. Allelopathic extracts mixed with lower
Table 4. Economic analysis 2009-2010.

Treatments
Total grain yield
10 percent less
Adjusted yield
Gross income
Hand weeding
T1
1751.06
175.106
1575.95
47278.6
T2
2305.88
230.588
2075.29
62258.8
T3
2201.75
220.18
1981.58
59447.3
T4
1945.62
194.562
1751.06
52532.0
T5
1874.06
187.406
1686.65
50599.5
T6
2018.25
201.825
1816.43
54492.8
T7
2094.13
209.413
1884.72
56541.5
T8
2233.94
223.394
2010.55
60316.4
T9
2353.19
235.319
2117.87
63536.1
7500
Cost of herbicide
2462.5
615.62
1231.25
1846.88
Cost of water extracts
1100
900
1000
500
500
500
47278.6
7500
54758.8
50
300
2812.5
56634.8
100
600
1800
50732.0
100
600
1600
48999.5
100
600
1700
52792.8
50
300
1465.62
55075.9
50
300
2081.25
58235.1
50
300
2696.88
60839.3
Sprayer rent
Spray labour
Cost that vary
Net benefit
Remarks
Kg ha-1
Kg ha-1
To bring at farmer level
Rs.30 Kg-1
30 man day ha-1,
Rs.250 per man day
Rs. 985/5.76 g a.i.
Rs. 27.5 & 22.5 L-1
SWE & SunWE
Rs.50/spray
Rs.300/man/day/ha
Rupees ha-1
Rupees ha-1
Table 5. Economic analysis 2010-2011.

Treatments
Total grain yield
10 percent less
T1
1508.05
150.81
T2
2087.70
208.77
T3
1982.30
198.23
T4
1690.20
169.02
T5
1635.50
163.55
T6
1759.50
175.95
T7
1802.45
180.24
T8
1933.30
193.33
T9
2052.5
205.25
Remarks
Kg ha-1
Kg ha-1
Adjusted yield
1357.25
1878.93
1784.07
1521.18
1471.95
1583.55
1622.21
1739.97
1847.25
To bring at farmer level
Gross income
40717.4
56367.9
53522.1
45635.4
44158.5
47506.5
48666.2
52199.1
55417.5
Hand weeding
7500
Cost of herbicide
2462.5
615.62
1231.25
1846.88
Cost of water extracts
1100
900
1000
500
500
500
40717.4
7500
48867.9
50
300
2812.5
50709.6
100
600
1800
43835.4
100
600
1600
42558.5
100
600
1700
45806.5
50
300
1465.62
47200.5
50
300
2081.25
50117.8
50
300
2696.88
52720.6
Rs.30 Kg-1
30 man day ha-1,
Rs.250 per man day
Rs. 985/5.76 g a.i.
Rs. 27.5 & 22.5 L-1
SWE & SunWE
Rs.50/spray
Rs.300/man/day/ha
Rupees ha-1
Rupees ha-1
Sprayer rent
Spray labour
Cost that vary
Net benefit
164
Table 6. Marginal analysis of wheat crop 2009-2010.

Treatments
T1
T7
T5
T6
T4
T8
T9
T3
T2
Cost that vary

(Rs.)
0
1465.63
1600
1700
1800
2081.25
2696.88
2812.5
7500
Net benefit
(Rs.)
47278.62
55075.89
48999.50
52792.75
50732.00
58235.13
60839.26
56634.75
54758.76
Marginal cost
(Rs.)
Marginal net benefit

(Rs.)
1465.63
7797.26
615.62
615.63
3159.11
2604.13
Marginal rate of return

(%)
532.00
513.16
423.00
-
Table 7. Marginal analysis of wheat crop 2010-2011.

Treatments
T1
T7
T5
T6
T4
T8
T9
T3
T2
Cost that vary

(Rs.)
0
1465.63
1600
1700
1800
2081.25
2696.88
2812.5
7500
Net benefit
(Rs.)
40717.35
47200.53
42558.5
45806.5
43835.4
50117.85
52720.63
50709.6
48867.9
Marginal cost
(Rs.)
Marginal net benefit

(Rs.)
1465.63
6483.18
615.63
615.63
2917.33
2602.78
Marginal rate of return

(%)
442.35
473.88
422.79
-
T1 Control (weedy Check), T2 Hand Weeding, T3 Atlantis 3.6 WG @ 14.4 g a.i. /ha spray at 40 DAS, T4 Concentrated sorghum water extract @ 20 L/ha
spray at 40 and 60 DAS, T5 Concentrated sunflower water extract @ 20L/ha spray at 40 and 60 DAS, T6 Concentrated sorghum + sunflower water extract
at 10 L/ha each spray at 40 and 60 DAS, T7 Concentrated sorghum + sunflower water extract at 10 L/ha each + Atlantis 3.6 WG @ 3.6 g a.i. /ha spray at
40 DAS, T8 Concentrated sorghum + sunflower water extract at 10 L/ha each + Atlantis 3.6 WG @ 7.2 g a.i. /ha spray at 40 DAS, T9 Concentrated sorghum
+ sunflower water extract at 10 L/ha each + Atlantis 3.6 WG @ 10.8 g a.i. /ha spray at 40 DAS.
Marginal rate of return (MRR %): (Marginal net benefits/Marginal costs)*100.
rates of herbicides decreased weed density equivalent to full

herbicide dose 18. Razzaq et al. 24 showed a decline of weed density
by 96% with application of Atlantis @ 14.4 g a.i. ha-1 under irrigated
conditions. Present study showed reduction of total weed density
by individual and combined aqueous extracts of sorghum and
sunflower due to their phytotoxic effects under rainfed conditions.
Combined water extracts worked in concert with each other and
inhibited total weed density more than sole extracts. Allelopathic
extracts combined with lower rates of Atlantis 3.6 WG depressed
total weed density at par with full dose of Atlantis. Hence, our
results confirm the findings of above mentioned studies.
Total weed fresh weight in present study was affected by
different weed control treatments. Sorghum and sunflower water
extracts either applied alone or combined with each other or with
lower rates of herbicides reduced fresh weight of total weeds.
Allelochemicals and herbicide together reinforced their phytotoxic
effects. Allelopathic extracts mixed with lower dose of Atlantis 3.6
WG suppressed weed fresh weight at par with full dose of the
herbicide alone. Similar findings were reported by Jabran et al. 11,
who found that combination of sorghum and sunflower water
extract @ 15 L ha-1 with pendimethalin @ 0.6 kg a.i. ha-1 gave
similar control of weeds fresh weight as that of label dose. Awan
et al. 5 found that combined sorghum and sunflower extracts were
effective in decreasing fresh weight of weeds.
The study demonstrated inhibition of total weed dry weight by
different weed control treatments in varying degrees. Sole extracts
of sorghum, sunflower and their mutual combinations decreased
weed biomass, but the control of weed biomass was lower than
full dose of herbicide. Efficacy of extracts was improved by
combining them with lower rates of herbicides. The results are in
accordance with those of Jabran et al. 11, who reported similar
decrease of weed biomass with sorghum + sunflower water extracts
combined with low doses of herbicides and their recommended
rates. Furthermore, Awan et al. 5 found that combined sorghum

and sunflower extracts were effective in decreasing dry weight of
weeds. Our results confirmed findings of previous studies, which
expressed that Atlantis controlled weed dry weight at 75 DAS as
compared to weedy check 14. The findings suggested that herbicide
usage in wheat crop under rainfed conditions may be reduced by
combining its lower rates with sorghum and sunflower aqueous
extracts.
Increase of fertile tillers in weed control treatments of present
study may be ascribed to low weeds competition for resources as
compared with control. The findings are in agreement with those
of Rajpar et al. 23, who reported that hand weeding increased the
number of fertile tillers. Other studies supporting our results
concluded that herbicide application 26, one and two sprays of
sorghum water extract 15, sorghum and sunflower water extracts
alone and their combinations 10 and allelopathic extracts of
sorghum and sunflower combined with lower rates of herbicides
raised number of tillers in wheat 24. Higher number of fertile tillers
per unit area during 2010-2011 was recorded due to sufficient
moisture availability at germination, which helped better crop
establishment.
Weed control treatments increased the number of grains spike-1
due to the reduction of competition for resources like moisture,
light and nutrients. Sorgaab @ 12 L ha-1 mixed with lower rates of
herbicides 26 and combination of sorghum, sunflower and brassica
with bromoxynil+ MCPA 10 increased the number of grains spike-1
in wheat. Relatively lower numbers of grains spike-1 were observed
during the second year due to the moisture stress crop experienced
at 100 DAS.
Thousand grains weight was improved by various treatments
insignificantly. Awan et al. 5 reported the increase of 1000 grains
weight with herbicide application and hand weeding. Our results
are contrary to the findings of Mahmood et al. 15 and Iqbal et al. 10,
165
Grain yield (kg ha-1)
who found that the combination of sorghum and sunflower water

extracts with low doses of herbicides also enhanced 1000 grain
weight. These studies were conducted under irrigation while ours
in stress prone rainfed conditions.
Weed control treatments in the study enhanced wheat grain
yields. Allelopathic extracts mixed with Atlantis 3.6 WG @ 7.210.8 g a.i. ha-1, hand weeding and Atlantis 3.6 WG @ 14.4 g a.i.
ha-1 produced similar grain yields, but they were significantly
higher compared to weedy check. Similar findings were presented
by Ashraf and Akhlaq 2 who reported that grain yield increased
with herbicide application and hand weeding. Mixtures of sorghum
and sunflower water extracts 5 and combination of allelopathic
crops water extracts with low doses of herbicides 13 also increased
wheat grain yield. Comparatively higher grain yields during 20092010 may be attributed to higher number of grains spike-1. A negative
relationship between total weed biomass and wheat grain yields was
observed during both seasons (Figs. 1 and 2). Coefficients of
determination during 2009-2010 and 2010-2011 recorded were 0.83
and 0.92, respectively. These figures reflected the increase of grain
yields with various weed control treatments which depressed weed
biomass.
y = -94.42x + 2558
R2 = 0.835
Total weeds biomass (g m-2)
Figure 1. Relationship between weeds biomass (g m-2) and grain

yield (kg ha-1) during 2009-2010.
Grain yield (kg ha-1)
y = -66.53x + 2266
R2 = 0.924
Total weeds biomass (g m-2)
Figure 2. Relationship between weeds biomass (g m-2) and grain

yield (kg ha-1) during 2010-2011.
Mixtures of allelopathic water extracts with 50-75% dose of

Atlantis achieved higher net benefits. Although hand weeding
was an effective treatment in depressing weed biomass and
increased yield, it could not attain higher levels of net benefits
due to more variable cost. Similar findings were reported by Razzaq
et al. 24 who obtained higher benefits by combining allelopathic
extracts with reduced rates of herbicides under irrigated system.
Conclusions
Sorghum and sunflower crop water extracts decreased weed
biomass and improved crop yields. Their combined effects either
166
mutual or with lower rates of Atlantis 3.6 WG strongly inhibited

weeds and thereby increased wheat yields. Allelopathic extracts
mixed with 50-75% rates of Atlantis 3.6 WG produced similar or
even better results on weeds and crops and hence these treatments
can be used in rainfed wheat to control weeds economically and
increase yields.
Acknowledgements
The first author is highly grateful to Pakistan Agricultural Research
Council (PARC) for providing opportunity of PhD studies.
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167
WFL Publisher
Helsinki, Finland
www.world-food.net
Genotypic variability for nutrient, antioxidant, yield and yield contributing traits in
vegetable amaranth
Umakanta Sarker 1*, Md. Tofazzal Islam 2, Md. Golam Rabbani 3 and Shinya Oba 4
Department of Genetics and Plant Breeding, 2 Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural
University, Gazipur - 1706, Bangladesh. 3 Department of Horticulture, Bangladesh Agricultural University, Mymensingh, Bangladesh.
4
Laboratory of Field Science, Faculty of Applied Biological Science, Gifu University, Gifu, Japan.
.
*e-mail: umakanta_sarker@yahoo.com
1
Abstract
This study aimed to evaluate genotypic variability in 30 vegetable amaranth genotypes for nutrient composition, antioxidant content, and 12 yield
contributing traits. The trials were in the field of Bangabandhu Sheikh Mujibur Rahman Agricultural University in Bangladesh for consecutive two
years (2012 and 2013) implementing Randomized Complete Block Design (RCBD) with three replications. High mean value, high range of variability
and high genotypic variance were observed for all the traits except content of Ca, protein and betacarotenoid. Close differences between genotypic
and phenotypic variances and genotypic and phenotypic coefficient of variations were observed for all the traits. Considering all genetic parameters,
selection based on contents of potassium, manganese, and ascorbic acid, plant height, leaves/plant, diameter of stem base, fiber content, leaf area and
foliage yield/plot seemed to be effective for the improvement of vegetable amaranth. Foliage yield had significant positive correlation with plant
height, leaves per plant, diameter of stem base, fiber content and leaf area. Nutrient content and antioxidant traits exhibited interesting results, i.e., had
insignificant genotypic correlations with foliage yield and most of the studied traits indicating that selection with these traits might be possible
without compromising any yield loss. Therefore, concomitant selection for high nutrient, antioxidant and high foliage yield would be effective for
improvement of the vegetable amaranth. Based on mean, range, genetic parameters, correlation coefficient and path coefficient values, direct selection
through three traits, i.e., fiber content, leaf area and diameter of stem base would significantly improve the foliage yield of vegetable amaranth. On the
other hand, concomitant selection based on high nutrient and antioxidant content and high foliage yield would be effective selection method for
improvement of vegetable amaranth.
Key words: Antioxidant, nutrient, foliage yield, genotypic variance, heritability, genetic advance, genotypic and phenotypic coefficient of variation,
correlation, path coefficient.
Introduction
Amaranth was important food crop in the Aztec, Mayan, and Incan
civilizations. National Academy of Sciences of the U.S in 1975
elected amaranths as the worlds most promising crops with
promising economic value 1-3. The main vegetable type of
amaranth, Amaranthus tricolor L., seems to have originated in
South or Southeast Asia 4 and then spread through the tropics
and the temperate zone 5. Leafy vegetables are a valuable part of
the diet owing to their nutritive values which plays an important
role in the human diet 6, 7. Unlike other leafy vegetables, vegetable
amaranth is cultivated during hot summer months when no other
green vegetables are available in the market 8. Among 60 species,
vegetable amaranth (Amaranthus tricolor) is now very popular
as vegetable in many Asian and African countries. It contains
high amount of protein, dietary fiber, dietary minerals and
antioxidant compounds like ascorbic acid and betacarotenoid 9-13.
It has been rated equal or superior in taste to spinach and is
considerably higher in protein (14 - 30% on dry weight basis),
minerals (Fe, Mn and Zn) and antioxidants like betacarotenoids
(90 - 200 mg/kg) and ascorbic acid (about 28 mg/100 g) compared
to any other leafy vegetables 9, 14-21. Some metalloenzymes like
catalase (Fe) and superoxide dismutase (Mn and Zn) required Fe,
Mn and Zn minerals for their antioxidant activity. Antioxidant
neutralizes or removes free oxygen radicals in the body and helps
to protect many diseases including cancer, cardiovascular diseases,
168
neurodegenerative diseases and inflammation and prevent aging 22,

23
. It has high adaptability under varied soil and agro-climatic
conditions and great amount of genetic variability and phenotypic
plasticity 24, 25. Although vegetable amaranths contain precious
nutritional and antioxidant properties, very little attention has been
paid for genetic improvement of this underutilized crop plant 11, 26.
Large bodies of literature are available on genetic variability and
interrelationship studies among various traits such as growth,
nutrient contents and antioxidants in many other crops 27-29.
However, reports on vegetable amaranth are rare 30, 31. A plant
breeding program can be divided into three stages, viz. building
up a gene pool of variable germplasm, selection of individuals
from the gene pool and utilization of selected individuals to evolve
a superior variety 32. The available variability in a population can
be partitioned into heritable and non heritable parts with the aid
of genetic parameters such as genetic coefficient of variation,
heritability and genetic advance 33. Correlation coefficient helps
to identify the relative contribution of component characters
towards yield 34. The correlation between yield and a component
character may sometimes be misleading. Thus splitting of total
correlation into direct and indirect effects would provide a more
meaningful interpretation of such association. Path coefficient,
which is a standard partial regression coefficient, specifies the
cause and effect relationship and measures the relative importance
of each variable 35. Therefore, correlation in combination with path

coefficient analysis will be an important tool to find out the
association and quantify the direct and indirect influence of one
character upon another 36. Improvement of foliage yield of
vegetable amaranths with high antioxidant and nutrient content
through the knowledge of variability, association among various
antioxidants, nutrients and yield contributing traits along with
direct and indirect influence of these component traits on yield
has so far been lacking. Therefore, objectives of the present study
were to i) analyze variability in genetic parameters, association
among different antioxidants, nutrients and yield contributing
traits on yield of 30 promising genotypes of vegetable amaranth
available in Bangladesh; ii) determine contribution of the
component traits towards yield potential; and finally iii) find out
appropriate selection parameters for the improvement of vegetable
amaranth.
Experimental materials and cultural practices: Thirty germplasm
accessions of the vegetable amaranth (Amaranthus tricolor)
collected from different eco-geographical regions of Bangladesh
were grown in March 2012 and 2013, consecutively in a Randomized
Complete Block Design (RCBD) with three replications in the
experimental field of Bangabandhu Sheikh Mujibur Rahman
Agricultural University, Bangladesh. The experimental site was
located in the centre of the Madhupur Tract (AEZ-28) at about 2423
north latitude and 9008 east longitude having a mean elevation of
8.4 m above mean sea level. The plot size for each treatment was 4
m2 for foliage yield and 1 m2 for nutrient and antioxidant and yield
contributing traits. Spacing was maintained with row-to-row and
plant-to-plant distance 25 and 5 cm, respectively. The soil of the
experimental field was slightly acidic (pH 6.4), with about 0.87%
organic matter, 0.09% total N, and 0.13 cmol kg-1 K. The levels of P,
S and Zn were 14, 14 and 0.2 mg kg-1, respectively, and that of Ca
was 0.2 cmol kg-1. Day temperature during experimental period
ranges between 25 and 40C. Irrigation was provided at 5 - 7 days
interval. Weeding and hoeing was done at 7 days interval.
Adequate fertilizer and compost doses, appropriate cultural
practices were maintained in both years.
Data collection on plant traits: Data were collected at 30 days
after sowing the seeds for both the years. Ten randomly selected
plants were considered from each replication for measuring plant
height (cm), leaves/plant, leaf area (cm2), fiber (%) and diameter of
stem base (cm). Foliage yield was harvested on whole plot basis.
Above ground foliage part of the plants were cut and used for
three nutrient traits viz., K (g/100 g), Ca (g/100 g) and protein (mg/
100 g), and three antioxidant traits viz., betacarotenoid (mg/g),
ascorbic acid (mg/100 g) and Mn (mg/kg) estimation.
Extraction and estimation of antioxidant and nutrient traits:
Protein: Protein was estimated following the method of Lowry et
al. 37. Briefly, 500 mg fresh vegetable amaranth leaves were washed
and grinded in 1 ml of 20% trichloro acetic acid and placed over
night. Next day supernatant was discarded and the residue washed
thoroughly 2 3 times with distilled water. The chlorophyll was
removed from the residue by adding sufficient amount of 80%
acetone solution and centrifugation. After the removal of
chlorophyll, the sample was dried in vacuum to evaporate the
acetone. The pellet was digested with 1 ml of 0.5 N NaOH at 80C

for 10 min in water bath. Further, 4 ml of distilled water was added
and the sample was centrifuged at 7500 rpm. An aliquot of 0.5 ml
was taken and 5 ml B.C. reagent (The B.C. reagent was prepared
by adding 50 mg CuSO4.5H2O in 10 ml of 2% sodium tartrate and 1
ml of this solution was added to 50 ml of 2% sodium carbonate
prepared in 0.1 N NaOH) was added. After 10 min the colour was
developed by the addition of 0.5 ml 1 N Folin- Ciocalteus reagent
in the sample. The absorbance values were taken at wavelength
of 640 nm on spectrophotometer. The standard graph was plotted
against concentration of protein and absorbance values, using
bovine albumen serum protein of 0.2, 0.4, 0.6, 0.8 and 1 g/ml
concentrations. The amount of protein in the sample can be
calculated by comparing (interpolation) with the standard graph
and expressed as mg/100 mg of fresh sample weight taken initially.
Betacarotenoid: The estimation of betacarotenoid was done
according to Jensen 38. To carry out the extraction process, 500
mg of fresh leaf sample was grinded in 10 ml of 80% acetone and
centrifuged at 10,000 rpm for 3 4 min. The supernatant was taken
and volume was made up to 20 ml in a volumetric flask. The
absorbance values were taken at 510 and 480 nm. The betacarotenoid
was calculated by the following equation:
Amount of betacarotenoid = 7.6(Abs.at 480) - 1.49(Abs.at 510) Final
volume/(1000 fresh weight of leaf taken).
Fibre: Fibre content was estimated using the method proposed

by Watson 39. The 500 mg dried leaf sample was extracted by
boiling for 30 min in 50 ml of 5% H2SO4 and 75 ml of distilled water.
The sample was filtered through linen cloth after 1 h with the
addition of some cold distilled water and residue was washed
twice with distilled water. In the residue, 50 ml of 5% KOH was
added and volume was made up to the original volume. Further,
the solution was boiled for 30 min and allowed to stand for some
time after adding little cold distilled water and filtered through
linen cloth. The residue was again washed with hot distilled water
followed by a mixture of dilute HCl (HCl:H2O in ratio of 1:2) and 5
ml ethyl alcohol. The residue was finally dried in a crucible at 80 100C
and dried weight was measured and represented as percentage of
initial material taken.
Ascorbic acid: Ascorbic acid was analyzed by the method given
by Glick 40. To extract the sample, 5 g fresh leaves was grinded
with 5% H3PO3 10% acetic acid (5% meta phosphoric acid (H3PO3)
10% acetic acid was prepared by dissolving 50 g of H3PO3 in 800
ml of distilled water + 100 ml of glacial acetic acid and volume was
made up to 1 litre with distilled water) for 1 3 min. The amount of
extracting fluid was taken such that it should yield 1 10 g of
ascorbic acid/ml. In the solution, 1 2 drops of bromine was
added and stirred until the solution became yellow. The excess
bromine was decanted into bubbler and air was passed till bromine
colour disappeared. The bromine oxidized solution was placed in
2 matched tubes. In first tube 1 ml of 2,4-DNP thiourea reagent
(2,4-dinitrophenyl hydrazine-thiourea reagent was prepared by
dissolving 2 g 2,4-DNP in 100 ml of 9 N H2SO4. Four g thiourea was
added and dissolved in this solution. The filtered solution was
added and the tube was placed in water at 37C for 3 h. Five ml of
85% H2SO4 (100 ml distilled water + 900 ml conc. H2SO4; sp.gr. 1.84)
169
was added dropwise by the burette in the tube, placed in a beaker

of ice water. In second tube, 1 ml of 2,4-DNP thiourea reagent was
only added to prepare blank solution. After 30 min, the absorbance
reading of the sample was taken at the wavelength of 540 nm by
spectrophotometer. The blank solution was used for setting the
zero transmittance of the spectrophotometer. The standard solution
was prepared by dissolving 100 mg ascorbic acid of the highest
purity in 100 ml of 5% H3PO3 10% acetic acid. The solution was
oxidized with bromine water as above. Ten ml of this dehydroascorbic
acid was pipetted in 500 ml volumetric flask and the solution was
made up to 500 ml with the 85% H2SO4 solution. The solutions of
different dilution was prepared by pipetting 5, 10, 20, 30, 40, 50
and 60 ml of the above solution into 100 ml volumetric flasks and
volumes were made up to 100 ml of each by addition of 85%
H2SO4. Four ml solution of each flask was taken separately and
further the procedure was followed as discussed above for the
sample. The calibration curve was prepared by plotting
absorbance values against concentration of ascorbic acid (in g).
The amount of ascorbic acid (mg/100 g) was calculated as follows:
Ascorbic acid content (mg/100 g) = (g from curve)/1000 (ml of extract
taken)/4 100/(sample wt. in g)
For determination of mineral nutrients and antioxidant

composition, the leaves were first oven dried and then digested in
a 1:4 mixture of HClO3 and HNO3. Calcium and potassium were
determined by flame photometry and manganese using atomic
absorption spectrophotometer (Perkin Elmer 5100) 41, 42.
Statistical analysis: The raw data were compiled by taking the
means of all the plants taken for each treatment and replication for
different traits. The mean data of both years were averaged and
the average mean values were statistically and biometrically
analyzed. Analysis of variance was done according to Panse and
Sukhatme 43 for each character. Genotypic and p henotypic
variances, phenotypic (PCV) and genotypic coefficient of variation
(GCV), heritability in broad sense (h2b) and expected genetic
advance (GA %) were estimated according to Johnson et al. 44.
Correlation coefficient was analyzed following Hayes et al. 45.
Path coefficient analysis was calculated according to the formula
given by Dewey and Lu 36.
Genetic parameters: Mean values, range, genotypic variance (Vg)
and phenotypic variance (V p), genotypic and phenotypic

coefficient of variations (GCV and PCV), heritability in broad sense
(h2b), genetic advance (GA) and genetic advance in percent of
mean (GAPM) for 12 traits of 30 vegetable amaranth genotypes
including contents of nutrients and antioxidants, yield and yield
contributing characters are presented in Table 1. Variability plays
a vital role for the selection of superior genotypes in crop
improvement program. Pronounced variation in the breeding
materials is prerequisite for development of varieties for existing
demand. Selection of genotypes on the basis of their phenotypic
variation (mean and range) is impractical for a breeder. Actual
genetic variation may be masked by its environmental influence.
Therefore, partitioning the phenotypic variation into genotypic
and environmental effects is essential for selection of suitable
genotypes. The genotypic variance was highest for ascorbic acid
content (995.75) followed by Mn content (712.50) indicating greater
scope of selection (Table 1). K, Mn content, plant height, leaves/
plant, stem base diameter, fibre content, leaf area and foliage yield
exhibited desirable genotypic variances for greater scope of
selection based on these traits (Table 1). On the other hand, the
lowest genotypic variance was found for protein content (0.13)
followed by Ca content (0.16). The phenotypic variances for all
the traits were little bit higher but close to the genotypic variances
indicating preponderance of additive gene effects for these traits.
Genotypic coefficient of variation (GCV) considers the best relative
amount of genetic variation and it takes into account the mean
value as well as the unit of measurement. Genotypic coefficient of
variation values ranged from 9.87 to 52.01% (Table 1). The PCV
values showed similar trends as GCV values and ranged from
10.46 to 55.18%. The values of PCV were little bit higher but close
to the corresponding GCV values for all the traits (Table 1). The
small differences between PCV and GCV for all the traits indicated
that the variability was predominately due to genotypic differences,
i.e. little environmental influences. Similar results have also been
reported by Shukla et al. 11, Rastogi et al. 46, Revanappa et al. 47
and Bhargava et al. 48, 49. Fibre content had low coefficient of
variation, which validated the chances of getting substantial gains
under selection are possibly to be less for these traits. On the
other hand, high values of coefficient of variation for betacarotenoid,
foliage yield, leaves/plant, K, stem base diameter, plant height,
protein, leaf area, ascorbic acid content, Mn and Ca content
indicated great scope for improvement though these traits by
selection to improve the potentiality of foliage yield. In present
study, The mean and range for K, Mn, ascorbic acid content,
Table 1. Genetic parameters in 30 vegetable amaranth genotypes for nutrients, antioxidants, yield and its
contributing traits.
Character
Ca (g/100 g)
K (g/100 g)
Protein (mg/100g)
Mn (mg/kg)
Beta carotenoid (mg/g)
Ascorbic acid (mg/100g)
Plant height (cm)
Leaves/plant
Stem base diameter (cm)
Fibre (%)
Leaf area (cm2 )
Foliage yield/plot (kg)
Mean
1.70
3.98
1.25
112.25
0.85
115.001
21.77
9.75
6.41
8.17
26.12
4.57
Range
0.76-2.15
1.60-6.65
1.06-1.51
63.75-156.75
0.60-1.15
65.50-178.55
9.50-40.72
4.92-22.25
2.6-12.54
6.64-9.76
16.24-42.77
3.75-5.95
Vp
0.18
2.50
0.17
715.00
0.22
999.50
53.90
16.15
5.61
0.73
61.49
5.79
Vg
0.16
2.35
0.13
712.50
0.19
995.75
53.55
16.12
5.56
0.65
61.16
5.65
PCV
24.96
39.73
32.98
23.82
55.18
27.49
33.72
41.22
36.95
10.46
30.02
52.65
GCV
23.53
38.52
28.84
23.78
51.28
27.44
33.61
41.18
36.79
9.87
29.94
52.01
h2b (%)
88.89
94.00
76.47
99.65
86.36
99.62
99.35
99.81
99.11
89.04
99.46
97.58
GA (5%)
0.87
3.26
0.85
55.08
0.97
65.13
15.12
8.28
4.88
1.76
16.15
4.96
%GAPM
51.41
81.84
67.95
49.07
113.67
56.63
69.47
84.91
76.12
21.54
61.84
108.47
Vp = Phenotypic variance, Vg = Genotypic variance, PCV = Phenotypic coefficient of variation, GCV = Genotypic coefficient of variation, h2b = heritability in broad sense, GA = Genetic
advance, GAPM = Genetic advance in percent of mean, Ca = Calcium, K= potassium, Mn = manganese.
170
plant height, leaves/plant, stem base diameter, fibre content, leaf

area and foliage yield were observed pronounced indicating the
scope of selection of these traits. Shukla et al. 11 also observed
similar result for protein, ascorbic acid and fibre content, plant
height, stem diameter, leaf size and foliage yield.
The values of heritability estimates were high for all the traits
and ranged from 76.47% for protein content to 99.81% for leaves/
plant. Shukla et al. 11 and Revanappa et al. 47 also observed high
heritability values in Amaranthus. The high value of heritability
for all the traits suggests that all these traits are under genetic
control, i. e., less environmental influence. However, it will be
relevant to divulge here that the total genotypic variance is made
up of additive genetic variance and non-additive or non-fixable
variance. High heritability alone is not enough to make sufficient
improvement through selection generally in advance generations
unless accompanied by substantial amount of genetic advance 50.
The efficacy of heritability is increased with the estimation of
genetic advance, which indicates the degree of gain in a trait
obtained under a particular selection pressure. Thus, genetic
advance is yet another important selection parameter that aids
breeder in a selection programme 51. It has been emphasized that
without genetic advance, the heritability values would not be of
practical importance in selection based on phenotypic appearance.
So, the genetic advance should be considered along with
heritability in coherent selection breeding programmes 52. Bayer
and Becker 53 obtained comparatively higher phenotypic variance
values than genotypic variance for most of the traits in the crop
Vernonia galamensis, which was solely due to the involvement
of high error variance. So, they narrated that due to large difference
in the phenotypic variation between different traits, the GA was
not directly related to heritability. But, in our study, the magnitude
of genotypic variance; and phenotypic variances was quit closer
due to lesser role of environmental effect (Ve = Vp-Vg) therefore all
the traits were under the control of genotypic variance (additive +
non-additive). However, in general, it is considered that if a trait is
governed by non-additive gene action, it may give high heritability
but low genetic advance, whereas if the trait is governed by
additive gene action, heritability and genetic advance both would
be high. The traits, which had high heritability along with high
expected genetic advance, could be substantially considered for
making selections as these traits were mainly influenced by the
major effects of additive gene action. The expected genetic
advance as percent of mean varied from 21.54 for fibre content to
113.67% for betacarotenoid content. It was revealed that only
fibre content had the major role of non-additive gene action in the
transmission of this trait from parents to offspring. The highest
expected genetic advance was exhibited for betacarotenoid
(113.67) followed by foliage yield (108.47), leaves/plant (84.91), K
(81.84), stem base diameter (76.12), plant height (69.47), protein
(67.95), leaf area (61.84), ascorbic acid content (56.63), Ca content
(51.41) and Mn (49.07). All the traits except fibre content also
showed moderate to high coefficient of variation and high
heritability values, which indicated a major role of additive gene
action in the inheritance of these traits and their amenability for
improvement in the population for foliage yield and its component
traits. Earlier Shukla et al. 11 showed high values of heritability
and genetic advance for ascorbic acid, plant height, leaves/plant,
stem diameter, foliage yield and leaf size and in another
investigation Shukla et al. 25 also obtained high values of
heritability and genetic advance for foliage yield and leaves/plant.

It was concluded from the present investigation that considerable
amount of variability exists in the experimental material, in terms
of nutrient (K), antioxidants (Mn and ascorbic acid), yield as well
as its all contributing traits. The investigation revealed that
vegetable amaranth is rich in Ca, K, Mn, protein, betacarotenoid
and ascorbic acid, which could be the good and economical source
of nutrients in human diet especially for the vegetarian people in
developing countries. However, considering all genetic parameters,
selection could be made on the basis of contents of potassium,
manganese, and ascorbic acid, plant height, leaves/plant, diameter
of stem base, leaf area and foliage yield/plot seemed to be effective
for the improvement of vegetable amaranth.
Correlation studies: The phenotypic and genotypic correlations
among nutrients, antioxidants, yield and yield contributing traits
are presented in Table 2.
The genotypic correlation coefficients were little bit higher but
closer to the corresponding phenotypic values for all the traits
indicating additive type of gene action for these traits. The higher
magnitude of genotypic correlation than respective phenotypic
correlations between various characters in amaranth have also
been reported by Shukla et al. 31. Foliage yield had significant
positive correlation with plant height (0.442*), leaves per plant
(0.405*), diameter of stem base (0.494**), fiber content (0.518**)
and leaf area (0.522**) and considerable positive correlation with
manganese (0.317) and ascorbic acid (0.235). Shukla et al. 31
observed similar significant positive association in vegetable
amaranth for foliage yield with plant height, diameter of stem base
and fiber content. Leaf area had significant positive association
with plant height (0.440*) and leaves/plant (0.475**). This trait
observed considerable positive relationship with protein (0.325),
diameter of stem base (0.341) and fiber (0.312) content. Diameter
of stem base showed significant negative association with Ca (-0.530**)
and significant positive association with plant height (0.462*). Leaves/
plant showed significant negative association with Ca (-0.402*)
and significant positive association with plant height (0.550**).
Plant height had significant negative association with manganese
(-0.396*) and ascorbic acid (-0.397**) while this trait exhibited
significant positive association with betacarotenoid (0.395*).
Significant negative association was observed between protein
and Ca. Rest of the association among different nutrients,
antioxidants and yield and yield contributing traits were
insignificant. The genotypic correlation analysis presented in
Table 2 exhibited some interesting results of this study. Nutrient
traits (K and protein) had insignificant genotypic correlation
among foliage yield and most yield contributing traits indicated
that selection for high nutrient content might be possible without
compromising yield loss. Similarly, no significant association was
found between any antioxidant traits (Mn, betacarotenoid,
ascorbic acid) and all the traits except plant height indicated that
concomitant selection for high antioxidant and yield contributing
traits lead to develop high foliage yielding vegetable amaranth
varieties. On the other hand, all yield contributing traits had
significant positive correlation with foliage yield indicating
selection based on higher values for all yield contributing traits
lead to develop high foliage yielding verities also. Similar trend of
correlation has also been observed in A. tricolor by Shukla et al. 14. In
the present investigation, correlation study revealed that nutrient
171
-0.016
-0.015
0.008
0.007
0.009
0.008
0.162
0.160
-0.096
-0.095
0.007
0.006
-0.112
-0.110
0.151
0.150
-0.296
-0.295
-0.272
-0.270
0.071
0.070
0.325
0.322
0.157
0.155
0.150
0.150
0.374
0.372
0.440*
0.440*
0.475**
0.474**
0.341
0.340
0.312
0.310
Foliage
yield/plot
(kg)
-0.194
-0.192
0.232
0.230
0.087
0.085
0.317
0.315
0.172
0.170
0.235
0.236
0.442*
0.440*
0.405*
0.404*
0.494**
0.494**
0.518**
0.518**
0.522**
0.521**
Leaf area
(cm2)
Fibre
(%)
Stem base
diameter
(cm)
-0.530**
-0.528**
0.162
0.160
0.111
0.110
-0.190
-0.188
0.019
0.017
0.045
0.044
0.462*
0.460*
0.207
0.206
-0.132
-0.130
0.114
0.112
0.161
0.160
0.126
0.125
0.052
0.050
-0.322
-0.320
0.172
0.170
-0.271
-0.270
-0.396*
-0.395*
0.395*
0.393*
-0.397*
-0.395
-0.402*
-0.401*
0.309
0.308
0.080
0.078
-0.121
-0.120
0.312
0.312
-0.016
-0.014
0.550**
0.548**
Leaves/ plant
Plant height
(cm)
Ascorbic acid
(mg/100g)
Beta
carotenoid
(mg/g)
0.024
0.022
0.120
0.119
-0.215
-0.213
0.080
0.078
Mn
(mg
/kg)
0.177
0.175
-0.070
-0.069
-0.160
-0.158
Protein
(mg
/100g)
-0.454*
-0.452*
0.241
0.240
* Significant at 5% and ** significant at 1%
Leaf area (cm2)
Fibre (%)
Leaves/ plant
Plant height (cm)
Ascorbic acid (mg/100 g)
Mn (mg/kg)
Protein (mg/100g)
K (g/100 g)
Ca (g/100 g)
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
rg
rp
K
(g
/100g)
-0.015
-0.012
Character
Table 2. Genotypic and phenotypic correlation co-efficient (rg and rp) in 30 vegetable amaranth genotypes for nutrient, antioxidant, yield and its contributing traits.
172
and antioxidant traits had no linkage with foliage yield.

So, concomitant selection for high nutrient and,
antioxidant (viz., K, protein, manganese, betacarotenoid,
ascorbic acid) and high foliage yield would be effective to
develop high yielding vegetable amaranth with high
nutrient and antioxidant content. High linkage between
foliage yield and its contributing traits were found due to
highly significant correlation coefficient values. Selection
based on high yield contributing traits (viz., plant height,
leaves per plant, diameter of stem base, fiber content and
leaf area) foliage yield of vegetable amaranth would be
improved.
Path coefficient studies: Path coefficient analysis was
carried out using genotypic correlation coefficient among
twelve nutrients, antioxidants, yield and its contributing
traits to estimate the direct and indirect effect on foliage
yield (Table 3). The fiber content (0.616), leaf area (0.464),
diameter of stem base (0.420) and betacarotenoid (0.347)
had high positive direct effect on foliage yield. High
positive direct effect for fiber and carotenoid content,
moderate positive direct effect for plant height and
negative direct effect for leaf size in vegetable amaranth
has been reported 31.
The plant height (0.285), K (0.230), Mn (0.225) and
leaves/plant (0.200) exhibited considerable positive direct
effect on foliage yield. On the other hand, high negative
direct effect was observed in Ca content (-0.368) and
negligible positive direct effect was found in protein
content (0.010). Shukla et al. 31 also found similar results
for protein content in same crop. The ascorbic acid (0.172)
showed little positive direct effect on foliage yield. It was
interesting that path coefficient analysis results confirmed
the similarity of the correlation coefficient analysis results.
Calcium had high negative direct effect and insignificant
negative correlation. Potassium had considerable positive
direct effect and insignificant positive correlation. Protein
exhibited negligible positive direct effect and negligible
positive correlation. Direct selection based on these three
nutrient traits (Ca, K and protein) would not be effective
for the improvement of foliage yield of vegetable amaranth.
Concomitant selection based on high nutrient content
and high foliage yield would be effective for the
improvement of vegetable amaranth. Manganese showed
considerable positive direct effect with considerable
positive genotypic correlation, whereas betacarotenoid
exhibited high positive direct effect but its negative
indirect effect via plant height made negligible genotypic
correlation on foliage yield. Ascorbic acid had little
positive direct effect with negligible genotypic correlation
on foliage yield. Direct selection based on three antioxidant
traits (Mn, betacarotenoid and ascorbic acid) would not
be effective for improving foliage yield. Rather,
concomitant selection with high antioxidant and high
foliage yield would be effective selection method for
improvement of vegetable amaranth. Fiber content, leaf
area, diameter of stem base had high positive direct effect
along with highly significant positive genotypic
correlation with foliage yield. Shukla et al. 31 observed
Table 3. Partitioning of genotypic correlation into direct (bold phase) and indirect effect in 30 vegetable amaranth genotypes for
nutrient, antioxidant, yield and its contributing traits.
Character
Ca (g/100 g)
K (g/100 g)
Protein (mg/100g)
Mn (mg/kg)
Ascorbic acid (mg/100g)
Plant height (cm)
Leaves/plant
Fibre (%)
Leaf area (cm2)
Ca
-0.368
0.006
0.168
-0.066
-0.010
0.049
0.120
0.147
0.196
0.004
-0.100
-0.004
0.230
0.055
-0.017
0.029
0.027
0.041
0.071
-0.037
0.001
0.017
Protein
-0.004
0.002
0.010
-0.002
-0.002
0.002
-0.003
0.001
0.001
0.000
-0.121
Mn
Beta
carotenoid
Ascorbic
acid
Plant
height
0.040
-0.016
-0.037
0.225
0.018
0.028
-0.090
-0.028
-0.04
0.037
0.036
0.009
0.043
-0.075
0.028
0.347
0.019
0.138
0.109
0.006
-0.034
-0.051
-0.023
0.020
0.028
0.022
0.009
0.172
-0.068
-0.003
0.007
0.000
0.065
0.093
-0.051
0.079
0.114
-0.113
0.114
0.285
-0.157
-0.133
0.029
0.011
Leaves
/plant
Stem
base
diameter
-0.040
0.031
0.008
-0.013
0.031
-0.002
0.055
0.200
0.021
0.016
0.008
-0.011
0.003
0.002
-0.004
0.000
0.001
0.009
0.004
0.420
-0.006
0.001
Fibre
Leaf
area
-0.007
0.002
0.002
0.100
-0.061
0.001
-0.063
0.097
-0.184
0.616
0.192
0.125
-0.035
-0.152
-0.074
-0.071
-0.175
0.018
-0.035
-0.006
-0.145
0.464
Foliage
yield/
plot
(kg)
-0.190
0.236
0.089
0.315
0.178
0.234
0.442*
0.405*
0.494**
0.518**
0.522**
* significant at 5% and ** significant at 1%.
similar findings for plant height, fiber and betacarotenoid content

in vegetable amaranth. On the other hand, plant height and leaves/
plant contained considerable positive direct effect but their indirect
positive effects via Ca and betacarotenoid made the significant
genotypic correlation on foliage yield for both traits, respectively.
Direct selection on the basis of fiber content, leaf area and stem
base diameter would significantly improve the foliage yield of
vegetable amaranth. Selection based on plant height and leaves/
plant concomitantly required considering Ca and betacarotenoid
content of the genotypes.
Lot of variability in respect of nutrients, antioxidant contents
and yield contributing traits were observed among the germplasm
while analyzing genetic parameters, correlation and path
coefficient values and interpretation of these results. Breeder may
utilize the present findings for developing high yielding varieties
with high nutrient and antioxidant content in future. Further
investigation may be carried out to confirm the study in different
locations of Bangladesh for their stability analysis. Breeder can
improve the foliage yield without compromising high nutrient and
antioxidant content.
Conclusions
Considering all genetic parameters nutrient trait K, antioxidant
traits Mn and ascorbic acid five yield and its contributing traits
viz., plant height, leaves/plant, diameter of stem base, leaf area
and foliage yield would be selected for the improvement of 30
vegetable amaranth genotypes. However, correlation study
revealed that selection based on plant height, leaves/plant,
diameter of stem base, fiber and leaf area could lead to increase
the foliage yield of vegetable amaranth genotypes. Based on mean,
range, genetic parameters, correlation coefficient values and path
coefficient values direct selection through fiber content, leaf area
and diameter of stem base would significantly improve the foliage
yield of vegetable amaranth. Concomitant selection based on high
nutrient and antioxidant content and high foliage yield would be
effective for improvement of vegetable amaranth. Tremendous
variability was observed among different nutrients, antioxidants
and yield contributing traits in the studied germplasm by analyzing
and interpreting genetic parameters, correlation and path
coefficient results. Breeder can utilize this variability in future for
developing high yielding varieties with high nutrient and
antioxidant content. The investigation was carried out in single
location but in future investigation may be carried out in multi
location for confirmation of the present study to develop stable
vegetable amaranth varieties. Association of nutrient and

antioxidant and yield contributing traits revealed that breeder can
improve the foliage yield without compromising high nutrient and
antioxidant content.
Acknowledgements
The authors are thankful to National Science and Technology
(NST) authority of Ministry of Science and Technology,
Bangladesh. for providing partial financial support by special
allocation to carry out the present investigation.
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Fabola de Oliveira Krger 1, Dario Munt de Moraes 2, Daniel Fernandes Franco 3,
Caroline Jcome Costa 3, Chaiane Fernandes Vaz 4 and Paula Rodrigues Gayer Ribeiro
Biologist, PhD Student Postgraduate Plant Physiology, Federal University of Lavras - UFLA P. O. Box 3037, 37200-000
Lavras, MG, Brazil. 2 Agronomist, Professor Department of Botany, University Federal de Pelotas - UFPel, P. O. Box 354, 96010900 - Pelotas, RS, Brazil. 3 Agronomist, Research Embrapa Temperate, highway BR-392, Km 78, P. O. Box 403, 96010-971 Pelotas, RS, Brazil. 4 Biologist, Masters Graduate Program in Plant Physiology, Federal University of Lavras - UFLA, P. O. Box
3037, 37200-000 - Lavras, MG, Brazil. e-mail: fabiolaoliveirakruger@gmail.com, dariommoraes@gmail.com,
daniel.franco@embrapa.br, caroline.costa@embrapa.br, cha.fvaz@hotmail.com, paulinhagayer@hotmail.com
1
Abstract
The use of seeds of great physiological potential, which is influenced by several factors from field to storage, is necessary to achieve high yields.
Storage, when irregularly performed, may lead the seeds to deterioration, promoting changes on the cell membrane system and respiratory activity
that may cause problems to general activities of protein synthesis, thus, to seed germination and vigor. Our objective was to evaluate the physiological
potential and respiratory activity of rice seeds stored for 10 years at different temperatures. Rice seeds of the cultivars BRS Pelota, BRS Atalanta,
and BRS Firmeza were used, which were placed in impermeable packaging, and stored for 10 years at -15; 1; and 18C. The physiological potential
was evaluated by germination, seedling emergence under greenhouse conditions, germination rate, length and dry matter of aerial part and roots, and
respiratory activity. Rice seeds of BRS Pelota cultivar stored at -15C presented better physiological potential. For BRS Atalanta and BRS Firmeza
cultivars, the storage temperature of 1C maintained the seed physiological potential, confirming the results observed for respiratory activity. We
concluded that it is possible to maintain the physiological potential of BRS Pelota, BRS Atalanta, and BRS Firmeza rice seeds after 10 years of storage
at -15 and 1C. The evaluation of seed respiratory activity indicated that it presents relationship with vigor of seeds stored at different temperatures.
Key words: Oryza sativa L., viability, vigor, deterioration, physiological, germination.
Introduction
Rice is one of the most cultivated cereals in the world, comprising
an area of approximately 158 million hectares 1. Brazil, which is
considered the main rice producer in the Americas, falls among
the ten largest world producers 2. For the achievement of great
yields, the use of seeds of high potential is necessary, and storage
is considered one of the main stages for conservation of seed
physical, physiological, and sanitary quality 3.
The seed lot quality comprises several characteristics or
attributes that determine its value for sowing, so that an
inappropriate storage may result in serious seed damage.
In this sense, we may highlight the deterioration, an irreversible
process 4 which changes, when caused in the initial germination
stages such as water absorption and metabolism activation, may
modify the cell membrane system 5 and respiratory activity 6, 7; the
latter is one of the most frequent changes. Therefore, for longterm seed conservation, it is necessary to maintain its respiratory
activity at low levels via reducing the ambient temperature and
seed water content.
The objective of this study was to evaluate the physiological
quality and respiratory activity of rice seeds stored for 10 years at
different temperatures.

This study was conducted in the Official Laboratory of Seed
Analysis (LASO) belonging to Embrapa Temperate Climate
(Pelotas, Rio Grande do Sul State, Brazil) and located at Terras
Baixas Experimental Station (ETB), and in the Laboratory of Seed
Physiology belonging to the Department of Botany of the Federal
University of Pelotas (UFPel), campus in Capo do Leo, Rio
Grande do Sul State.
Seeds of three irrigated rice cultivars were used: BRS Pelota,
BRS Atalanta, and BRS Firmeza, which were collected from the
ETB seed production area during 2001/2002 harvest. After harvest,
seeds were dried in a forced air circulation heater up to 13% of its
water content. Seeds were then submitted to another drying stage
up to 9.9% of its water content for BRS Pelota and BRS Atalanta,
and 11.3% for BRS Firmeza. After drying, approximately 0.5 kg
seeds of each cultivar was placed in aluminium containers of 1.0
kg capacity. Each container was air-tight sealed and seeds were
stored for 10 years at different temperatures: -15; 1; and 18C.
After storage, seeds of the three cultivars were submitted to
the following evaluations:
Germination There were four replications of 100 seeds for
each treatment, which were sown in paper rolls moistened with
the distilled water equivalent of 2.5 times their dry weight, and
175
maintained in a germinator at 25C. Evaluations were performed

at 5 and 14 days after sowing (DAS) 8. Results were expressed in
percentage of normal seedlings.
Tetrazolium test To verify embryo viability, this test was
applied only for seeds that did not present germination. It was
performed with two subsamples of 50 seeds for each treatment,
which were preconditioned on germination paper, germitest type,
moistened, and maintained for 18 h at 25C. Seeds were then placed
in plastic cups (50 ml capacity) with 2,3,5-triphenyltetrazolium
chloride solution at 0.05% concentration, and maintained in a dark
chamber for 4 h at 25C. After this period, seeds were washed
under running water and individually evaluated from a longitudinal
cut at the embryonic axis 8.
Seedling emergence (E) There were four replications of 100
seeds for each treatment, which were sown in plastic trays of 40
cm width x 60 cm length, under greenhouse conditions, using, as
the substrate, soil from a rice cultivation area. Evaluations were
daily performed until number of emerged plants was constant.
The obtained results were expressed in percentage of emerged
seedlings at 21 days after sowing (DAS).
The germination rate (GR), verified during the seedling
emergence test, was calculated according to Maguire 9.
Length of seedling roots and aerial part determined during
the seedling emergence test, at 21 DAS 10. Means of length of
aerial part and roots were obtained via the sum of all subsample
measures divided by the number of measured seedlings. Results
were expressed in cm seedling-1.
Dry matter of seedling aerial part and roots obtained from
the seedling emergence test. Each sample was placed in paper
bags, and maintained in a forced air circulation heater at 70C until
constant weight. The dry matter of seedlings was determined using
a precision scale with accuracy of 0.001 g 10. Results were
expressed in g seedling-1.
Respiratory activity (RA) determined in the Pettenkofer
apparatus, using 100 g seeds of each rice cultivar and storage
temperature. Seeds were soaked in 80 ml distilled water for 60 min
to accelerate the respiratory process. After this period, seed
respiration was measured according to the methodology described
by Mendes et al. 11. Results of respiratory rate were expressed in
mg released CO2 mg seeds-1 h-1. Calculation of respiratory activity
was based on the following equation: ND22, where N is the
normality of the used acid (0.1 N HCl); D, the difference between
the volume of used HCl for titration of the blank reagent, and
volume of used HCl for sample titration; and 22, the CO2 normality.
The experimental design was entirely randomized, with four
replications. The obtained data were submitted to variance
analysis, and means were compared by the Tukey test at 5%
probability, using WinStat software, version 2.0 12. The linear
correlation was used to define the efficiency of a vigor test 13.
Seeds of BRS Pelota, BRS Atalanta, and BRS Firmeza cultivars
presented, respectively, 97, 96, and 96% of germination before
storage. After 10 years under this condition, we observed that the
germination percentage at different storage temperatures (Table 1)
was high (80%) at the lowest temperatures, and still within the
germination standards required for commercialization of irrigated
rice seeds 14. Seeds of BRS Pelota, BRS Atalanta, and BRS Firmeza
stored at -15C presented 91, 92, and 94% of germination,
176
Table 1. Germination, seedling emergence under greenhouse

conditions, germination rate (GR), and respiratory activity
(Respiration) of seeds of three irrigated rice cultivars
stored for 10 years at different temperatures.
Variables
Germination
(%)
Emergence
(%)
GR
Respiration
Cultivar
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
-15C
91 Aa
92 Aa
94 Aa
88 Aa
96 Aa
93 ABa
18.81 Ba
21.99 Aa
17.72 Ba
36.66 Bb
70.88 Ab
15.64 Cb
1C
87 Bb
93 Aa
96 Aa
89 Ba
96 Aa
93 ABa
17.88 Aa
19.26 Ab
17.85 Aa
65.63 Ba
116.35 Aa
35.93 Ca
18C
66 Ab
0 Bb
0 Bb
80 Ab
0 Bb
0 Bb
15.05 Ab
0 Bc
0 Bb
38.86 Ab
0 Bc
0 Bc
CV (%)
3.02
8.77
7.31
18.28
The same uppercase letters in the column and lowercase letters in the line do not differ from each other
by the Tukey test (p0.05).
respectively, while those stored at 1C had 87, 93, and 96% of

germination, respectively.
Although there was a reduction in the seed initial germination
of all cultivars at the different storage temperatures, the most
pronounced decrease was verified for those seeds stored at 18C.
In this case, seed germination of BRS Pelota decreased to 66%,
while seeds of BRS Atalanta and BRS Firmeza cultivars completely
lost their embryo viability (Table 1), what was confirmed by the
tetrazolium test (Fig. 1).
(a)
(b)
Figure 1. Tetrazolium test in seeds of the cultivars BRS Atalanta

(a) and BRS Firmeza (b) stored for 10 years at 18C.
Regarding seedling emergence, we verified that seeds of the

three cultivars stored at -15 and 1C presented the same
performance; however, those stored at 18C did not show the
same results (Table 1). Similarly, GR also demonstrated an inferior
performance for seedlings of all cultivars after storage at 18C.
These results corroborate the observed for germination, as seeds
of all cultivars stored at 18C presented a lower performance than
recommended; seeds of BRS Atalanta and BRS Firmeza cultivars
did not present germination and seedling emergence after storage
at 18C for 10 years (Table 1).
For all studied cultivars, the respiratory activity was higher
when seeds were stored at 1C in comparison with the other storage
temperatures (Table 1). Similarly, seeds of BRS Pelota, BRS
Atalanta, and BRS Firmeza presented a superior respiratory activity

when stored at 1C. Studies on seed lots of soybean, bean, maize 15,
and kidney bean 16 also detected greater respiratory activity from
lots of higher vigor.
It is known that the respiratory process is the first metabolic
activity promptly activated soon after seed imbibition 17. Therefore,
greater CO2 release characterizes the integrity of cell membranes,
including mitochondrial ones, and it is an indicator of higher
capacity for reorganization of seed cell systems, thus of greater
vigor.
The evaluation of length of aerial part from BRS Pelota and BRS
Atalanta seedlings, obtained during the emergence test, presented
the best performance after seed storage at -15 and 1C (Table 2).
However, seedlings of BRS Firmeza from seeds stored at -15C
had greater length of aerial part, achieving 24.82 cm, than those
obtained from seeds stored at 1C that reached 20.10 cm. Seeds of
BRS Pelota stored at 18C showed a different behaviour than
other materials regarding length of aerial part (LAP) and roots
(LR), with values of 18.87 and 5.11 cm, respectively; however,
seeds of BRS Atalanta and BRS Firmeza cultivars stored at 18C
did not have LAP and LR values, thus demonstrating sensibility
for separation into levels of lower vigor (Table 2).
The LR for BRS Atalanta and BRS Firmeza cultivars obtained
from seeds stored at -15C was 8.60 and 6.64 cm, respectively;
seeds stored at 1C had 6.18 and 8.10 cm, respectively (Table 2).
The dry matter of aerial part (DMAP) of BRS Atalanta and BRS
Firmeza from seeds stored at -15C was 357.0 and 264.7 mg,
respectively; for those stored at 1C, DMAP was 293.0 and 248.0
mg, respectively (Table 2).
The dry matter of roots (DMR) of BRS Atalanta and BRS Firmeza
was 34.75 and 72.25 mg, respectively, for seeds stored at -15C,
and 64.75 and 68.78 mg for those stored at 1C (Table 2).
The higher respiratory activity verified from seeds of BRS
Atalanta and BRS Firmeza stored at 1C did present a relationship
with the results found in the other tests for evaluation of seed
physical quality; the latter, along with high respiratory activity,
characterizes seeds of these cultivars as the most vigorous ones,
since greater CO2 release reflects more integrity of cell membranes,
including mitochondrial ones.
The use of linear correlation to define the efficiency of a vigor
test is described by Marcos Filho 13. The author emphasizes that,
among the criteria to assess the reliability of a certain test for seed
quality evaluation, the correlation of this test with field seedling
emergence is one of the most used. Such test is considered the
best indicator for inferences on seed lot vigor because, during its
execution, conditions that simulate those that seeds will be
subjected to in the field must be applied 18.
Responses of the respiratory activity test were correlated with
tests of germination, seedling emergence, germination rate, length
of aerial part and roots, and dry matter of aerial part. We observed
that these correlations were significantly high (1%), what revealed
that it is possible to estimate viability and vigor of rice seeds
stored for 10 years at different temperatures via the respiratory
activity test. The only test that it was not significant at 1% was
the dry matter of roots (Table 3).
We verified that there was significant correlation (r = -0.67)
Table 2. Length of aerial part (LAP) and roots (LR), and dry matter of
between
the tests of respiration and seedling emergence
aerial part (DMAP) and roots (DMR) of seedlings obtained
under
greenhouse
conditions (Table 3). This confirms the
from the emergence test for seeds of BRS Pelota, BRS
test
sensibility
to
detect differences in the physiological
Atalanta, and BRS Firmeza cultivars stored for 10 years at
potential
of
rice
seeds
stored at different temperatures for a
different temperatures.
long time.
Variables
LAP (cm)
LR (cm)
DMAP (mg)
DMR (mg)
Cultivar
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
BRS Pelota
BRS Atalanta
BRS Firmeza
-15 C
27.61 Aa
26.21 Aa
24.82 Aa
5.98 Ab
8.60 Aa
6.64 Aa
253.5 Ba
357.0 Aa
264.7 Ba
57.25 Aa
34.75 Ba
72.25 Aa
1 C
26.84 Aa
26.05 Aa
20.10 Bb
11.94 Aa
6.18 Ba
8.10 Ba
273.5 Aa
293.0 Aa
248.0 Aa
56.25 Aa
64.75 Aa
68.75 Aa
18 C
18.87Ab
0 Bb
0 Bc
5.11 Ab
0 Bb
0 Bb
127.75 Ab
0 Bb
0 Bb
69.50 Aa
0 Bb
0 Bb
CV (%)
Conclusions
It is possible to maintain the physiological quality of rice
seeds of BRS Pelota, BRS Atalanta, and BRS Firmeza cultivars,
submitted to drying and impermeable packaging, after 10
years of storage at -15 and 1C. The respiratory activity
presents a relationship with vigor in seeds stored at different
temperatures.
9.15
33.93
19.74
Acknowledgements
To CAPES for financial support and research scholarship.
39.45
The same uppercase letters in the column and lowercase letters in the line do not differ from each other by the
Tukey test (p0.05).
References
Table 3. Correlation coefficient among the analysed variables in the
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WFL Publisher
Helsinki, Finland
www.world-food.net
Effect of dietary crude palm oil on quality and oxidative stability of chicken eggs
YeasminAkter 1, 2*, Azhar Kasim 1*, Hishamuddin Omar 3 and Awis Qurni Sazili
Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Department of Animal Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200,
Bangladesh. 3 Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
*e-mail: yesakter@yahoo.com,azharkasim@putra.upm.edu.my
1
Abstract
The experiment was conducted to evaluate the effect of different levels of dietary crude palm oil (CPO) on quality characteristics and oxidative
stability of chicken eggs. A total of ninety six ISA Brown hens was assigned randomly into four dietary treatments containing 0 (control), 1.5, 3 and
5% CPO with four replications pre-treatment. Experimental diets were iso-nitrogenous and iso-calorie. Eggs were collected daily analysed for its
quality characteristics and lipid peroxidation. Yolk colour and oxidative stability were markedly improved (p<0.05) with corresponding increased
CPO in the diets. On the basis of this study it may be concluded that dietary CPO reduced lipid peroxidation in egg yolk without affecting egg quality
characteristics.
Key words: Chicken eggs, crude palm oil, egg quality, lipid oxidation.
Introduction
Oils and fats consist of fatty acids and glycerol, which have high
caloric value among the all nutrients 1. These are also important
sources of some essential fatty acids, fat-soluble vitamins,
decreases dustiness of diets, increases palatability of diets and
increases the efficiency of consumed energy. Moreover, it reduces
the rate of passage which may increase the retention time of feed
in the digestive tract, and increase the digestion and absorption
of feed nutrients 2. Generally commercial poultry producers use
fats and oils in poultry diets to supply higher amount of energy at
an economically justifiable cost. Many studies have demonstrated
that vegetable oils such as soybean, rapeseed, sunflower and
corn oils, can be successfully used in poultry feeding 1. These
oils are rich in linoleic acid which is an essential PUFA 3. It is well
known that oils containing PUFA are more prone to oxidation
than SFA which proceeds by a free radical chain mechanism 4.
Poultry diets with a higher concentration of vegetable oil should
be supplemented with antioxidants which can protect the poultry
and its products 5. Crude palm oil (CPO) is a potential source of
dietary energy and it may be used as a perfect substitute for other
vegetable oils. It will provide some additional benefits over other
vegetable oils when it will be incorporated in poultry ration
because it contains a vast amount of natural antioxidants such as
carotenoids, tocopherols, and tocotrienols. The combined effects
of high levels of carotenoids, tocopherols, and tocotrienols, and
a 50% saturation of fatty acids give palm oil higher oxidative
stability than other conventional vegetable oils 6. In this context,
the current experiment was arranged to investigate the effects of
dietary CPO on quality characteristics and lipid peroxidation of
chicken eggs.
Experimental birds and diets: The experiment was conducted at
the poultry unit, Department of Animal Science, University Putra
Malaysia. A total of ninety six ISA Brown layers, 44 weeks of age

were used for this study to evaluate quality characteristics of
eggs. The experimental birds were randomly distributed into four
dietary treatments with four replicates under conventional
conditions with access to feed and water ad libitum and dietary
treatments were control (diet without CPO) and diet with 1.5, 3
and 5% CPO. All diets were formulated according to NRC 7
guideline.
Evaluation of internal egg quality: Egg weight was measured
by weighing egg individually using sensitive balance. Egg length
and width (cm) and shell thickness were measured by using digital
Vernier calipers 8. Egg shape index was obtained as a ratio of the
egg width to the length. Characteristics evaluated in individual
eggs for internal quality traits. Yolk colour and Haugh unit were
measured by automatic egg multi tester (Orka Food Technology).
The yolk was separated from the albumen and weighed. Albumen
weight was calculated from the difference between egg weight
and weight of the yolk and shell.
Determination of lipid oxidation in yolks: The TBARS values
were determined for the malonaldehyde (MDA) formed in yolk.
The MDA value was measured by the TBARS method described
by Buege and Aust 9 with minor modifications. About 1 g of yolk
samples were homogenized by polytron homogenizer and mixed
with 5 ml of 0.375% thiobarbituric acid, 15% trichloroacetic acid
(TCA) and 0.25 N HCl stock solutions in a glass test tube. The
mixture was further incubated at 100C for 30 min to develop pink
colour. Following incubation, the mixture was cooled under running
tap water and centrifuged at 3000 g for 5 min. Absorbance of
supernatant was measured at 532 nm using a spectrophotometer.
The concentration of MDA in analysed samples (mg/kg of yolk)
was calculated from a standard curve of 1,1,3, 3- tetraethoxypropane.
179

Shape index, egg weight, egg shell weight as percentage and shell
thickness of laying hen did not show (P>0.05) any significant
differences among the all dietary treatment groups (Table 1). These
results were supported by the findings of Hosseini-Vashan and
Afzali 11 who did not notice any significant effect of dietary
inclusion of palm oils on shape index, shell thickness and egg
weight. Shakoor et al. 12 reported that dietary oils did not influence
egg quality and egg shell thickness. However, lower shell
percentage values were obtained in this study compared to 15%
reported by Yildirim 13 probably indicating less optimum calcium
metabolism for shell production in the birds used in the present
study.
Albumen weight as percentage of egg weight and Haugh Unit
were unaffected (P>0.05) by dietary CPO (Table 1). Rowghani et
al. 14 showed that the egg white weight was not modified by the
addition of different levels of oil supplementation to hens. Albumen
weight as well as albumen height and HU were not affected by
different levels of CPO because all diets had similar energy and
protein content according to the hens requirements. Therefore,
no effect of diet was expected in this aspect. Many scientists
reported that dietary fat sources had no significant effect on egg
quality parameters 15. Mazalli et al. 16 studied the effect of various
fats/oils on egg quality and did not notice any significant
differences for HU value of eggs.
There were no significant (P>0.05) effects of CPO fed diets on
yolk weight as percentage of egg weight among the treatment
groups (Table 1). These results are consistent with Shafey et al. 17
and Steinhilber 18 who revealed no effect of dietary fats on yolk
weight. Similarly, Hosseine -Vashan and Afzali 11 did not get any
change in the yolk weight percent with different levels of palm oil.
Some researchers reported that the rate of hepatic synthesis of
fats is sufficient to supply the amount of lipid needed to achieve
optimum performance, egg and yolk weight and exogenous fat
has no influence to meet these requirements 15, 17. Similarly feeding
of soybean oil and linseed oil in the ration did not affect the yolk
weight in laying hens 18. Yolk colour is an important trait of egg
that affects consumers choice 19. Carotenoids are used as
Dietary treatments
Control
1.5% CPO
3% CPO
Shape index
76.91 0.33 76.69 0.33 76.46 0.43
Egg weight (g)
59.45 0.40 59.74 0.73 60.39 0.75
11.51 0.85 11.47 0.43 10.76 0.58
Shell weight (%)
0.35 0.01
0.35 0.01 0.34 0.02
Shell thickness (mm)
63.04 0.80 62.27 0.59 63.71 0.89
Albumen weight (%)
5.89 0.02 5.91 0.03
Albumen Height (mm) 5.88 0.02
75.69 1.32 76.19 1.41 76.92 1.29
Haugh unit (H.U)
25.60 0.64 25.66 0.92 26.27 0.58
Yolk weight (%)
180
4
3
2
1
0
Control
1.5% CPO
3% CPO
Dietary treatments
5% CPO
Figure 1. Effect of dietary CPO on yolk colour score

of eggs.
a, b, c, d: Means with different letters each column differs significantly
P<0.05.
The CPO has much carotenoids increasing the yellow colour

of yolk. These findings were in agreement with Ng et al. 21 who
revealed that palm oil has much carotenoids and has positive
effect on yolk colour. The yolk colour is related to the
xanthophylls 22 content of feeds and CPO supplemented diet might
exhaust the soluble antioxidant pigments (carotene and
xanthophylls). According to Goh et al. 23, CPO contains about
90% carotenes and 10% xanthophylls. These carotenoids had
great contribution in yolk pigmentation. These results are in
agreement with Ng et al. 21 and Hosseini-Vashan and Afzali 11 who
reported that PO contains much carotenoids and increases the
yellow colour of egg yolk. The -carotene in control egg yolk was
lowest due to less proportion of carotenoids compare to CPO
supplemented groups and levels of increase were two times higher
in 3% and 2.5 times higher in 5% CPO supplemented group. These
results confirmed the findings of Hammershoj et al. 24 who reported
that after adding carrots in layer diets significantly increased yolk
colour compared to control. Kotrbacek 25 reported that Chlorella
supplemented layer diets increased the colour characteristics of
yolks.
Egg yolks from hens fed the control diet had higher (p<0.05)
TBARS values than CPO fed groups (Fig. 2). The higher TBARS
values in the yolks of control group might be due to higher amount
Table 1. Effects of different levels of CPO on egg quality characteristics of

chicken.
Parameters
5% CPO
76.42 0.67
59.31 0.49
10.79 0.40
0.34 0.02
63.54 0.43
5.90 0.03
75.48 1.48
25.98 0.49
1.6
TBARS (mg/kg) of yolk
Statistical analysis: Analyses were conducted using the SAS

software program 10. Significant differences among the means of
the treatment groups were determined by Duncans multiple-range
test. Variability in the data was expressed as standard error (S.E.)
and a probability level of p<0.05 was considered as statistically
significant.
pigmenting agents in poultry diets to get desirable yolk colours20.

Yolk colour was markedly improved (p<0.05) with corresponding
increased CPO in the diets (Fig. 1), as a result 5% CPO
supplemented group showed highest yolk colour (5.69) followed
by 3% (4.80), 1.5% (3.69) CPO and control (2.19), respectively.
Yolk colour score
All measurements were conducted in triplicate.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Control
1.5% CPO 3% CPO

Dietary treatment
5% CPO
Figure 2. Effect of dietary CPO on TBARS (mg/

kg) value of yolk of egg.
a, b, c: Means with different letters each column differs significantly
P<0.05.
of polyunsaturated fatty acids present in it.

Polyunsaturated fatty acids are more prone to oxidation than
saturated fatty acids 26. Cherian et al. 27 also reported high lipid
oxidation in eggs from hen fed diets containing conjugated linoleic
acid or fish oil, which contain polyunsaturated fatty acids. The
lower TBARS value in CPO supplemental groups resulted in an
increase in tocopherol, tocotrienol, and retinol contents in eggs 28.
However, lower TBARS values of egg yolk from CPO birds may
suggest an added protection of lipids provided by tocotrienols
and other antioxidants present in CPO. The antioxidant activity of
tocotrienol has been reported to be higher than that of tocopherol 29, 30. Lipid oxidation is a major cause of food
deterioration affecting colour, flavor, texture, and nutritional value
of poultry. Incorporation of more powerful and natural antioxidants
by dietary means may be more effective and practical in controlling
lipid-oxidation-related products and providing nutritious products
to health conscious consumers.
Conclusions
Like other vegetable oils, crude palm oil can be used effectively in
chicken diets. The results of this study confirmed that dietary
CPO significantly improved colour and oxidative stability of egg
yolk without affecting the quality characteristics of chicken eggs
which may help in providing a quality product that is acceptable
to consumers.
Acknowledgements
The authors would like to thank Universiti Putra Malaysia for
supporting this research.The authors are also grateful to
Organization for Women in Science for the Developing World
(OWSD) for providing the scholarship for Ph.D student.
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11
181
WFL Publisher
Helsinki, Finland
www.world-food.net
Evaluation a polyvalent vaccine against abscess disease of sheep from pathogenic

bacteria isolated from Saudi Arabia
K. B. Alharbi
Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, P. O. Box 6622
Buraidah 51452, Saudi Arabia. e-mail: kbhrby@qu.edu.sa, aboabdelelah17@hotmail.com
Abstract
A locally-prepared polyvalent vaccine (C. pseudotuberculosis and Staphylococcus aureus subsp. anaerobius) against abscess disease of sheep was
investigated. Twenty Najdi ewes were divided into 4 groups of 5 animals each. The animals were mixed with 5 rams infected with abscess disease as
a source of infection. The results show that 1 ml of the vaccine is protective for 6 months while higher doses were protective for 8 months. The results
showed also that the vaccine was safe and vaccination raised antibody titres against the disease as shown by the ELISA results.
Key words: Abscess disease, sheep, local vaccine.
Introduction
Abscess disease, commonly known as Morels disease and
caseous lymphadenitis (pseudotuberculosis) deserves interest
because of its contagious nature, worldwide distribution and lack
of effective control measures. It is primarily a disease of sheep
and goats, and once introduced into a flock, it is very difficult to
control because of its poor response to treatment, its ability to
persist in the environment and the limitations in detecting subclinically infected animals 9, 20.
Morels disease is caused by Staphyloccocus aureus subsp.
anaerobius, that exists as a single bacterial clone worldwide 2, 6.
Caseous lymphadentitis is characterized by formation of abscesses
in external and internal lymph nodes and is caused by
Corynebacterium pseudotuberculosis 1. Some sheep breeds were
found to be more susceptible to the disease than others 15.
The losses caused by CLA may become important when the
prevalence is high, particularly in countries with large numbers of
sheep such as Australia. The losses are caused by condemnation
and downgrading of carcasses and skin in abattoirs as well as
reduction in wool growth 11, 12, 20.
Previous studies 3, 11 have indicated spread of CLA mostly
occurred around the time of shearing. Several modes of
transmission of CLA during shearing have been suggested and
there are evidence incriminating the lungs in the transmission of
the disease 7. C. pseudotuberculosis was isolated from the tracheae
of sheep with lung abscesses, which indicated that the lung
transmits CLA through aerosol contamination of skin cuts of
uninfected sheep.
There is no available vaccine that is known to confer solid
immunity against abscess disease. Production of effective vaccines
targeting all known C. pseudotuberculosis antigens are advised 4.
In some countries where vaccines are available, vaccination failure
is blamed on improper implementation of CLA vaccination
program 13.
182
Eggleton et al. 5 carried out a field trial to evaluate a whole cell

vaccine for the prevention of caseous lymphadenitis (CLA) in
sheep and goats was performed in one goat herd and one sheep
flock over a period of three years. In goats, there was a
nonstatistically significant trend for fewer cases of CLA in the
vaccinated animals compared to the controls. In sheep, from six
months to 36 months postinitial vaccination, the proportion of
vaccinated sheep that developed CLA was significantly less (p <
0.05) than in the control sheep. The antibody titers to
Corynebacterium pseudotuberculosis as detected by
microagglutination assay were significantly different (p < 0.0001)
at all times except at the initial vaccination. Swellings occurred at
the vaccination site at an incidence level of 29.6% in goats and
34.1% in sheep. The vaccine appeared to be efficacious in reducing
the proportion of sheep that developed CLA when challenged
naturally in a field situation.
Abscess disease is endemic in Saudi Arabian sheep and goat
farms, and despite genuine efforts to control it by vaccination
and culling, its prevalence remained high. Sheep farmers complain
of poor response to available vaccines (killed formalized bacterins).
The disease is threatening the development of the sheep industry
in the Kingdom mainly because of the low sale value of infected
sheep in the market.
Experimental animals and protocol: Twenty healthy Najdi ewes
were ear tagged and divided into 4 four groups of 5 animals each.
Group ewes were numbered 81, 82, 83, 84, and 85. Group 2 ewes
were tagged with the numbers 86, 87, 88, 89 and 90. Group 3 ewes
were tagged 91, 92, 93, 94 and 95. Group 4 ewes were tagged 96, 97,
98, 99 and 100. The ewes were fed on berseem (alfalfa), a
concentrate diet made of barley and wheat bran and had free
access to drinking water.
A killed formalized vaccine containing 7 x109 bacteria per ml was

prepared from C. pseudotuberculosis and Staphylococcus aureus
subsp. anaerobius using standard methods. This was injected
into ewes of groups 1, 2 and 3 at the rate of 1, 2 and 3 ml, respectively.
Group 4 ewes were kept as unvaccinated controls. After one month,
a booster dose of the vaccine was given to groups 1, 2 and 3
ewes. Then all the animals were mixed with 5 infected rams that
acted as a natural source of infection.
All the ewes were observed for the development of abscess
disease after vaccination to record the effectiveness of
vaccination. The ewes were bled before and 2 weeks after
vaccination and after 2 weeks post booster injection. Three
samples of blood were collected. One sample was collected in
EDTA-tubes for hematological studies. Another sample was
collected in plain tubes and allowed to clot overnight to obtain
serum. Serum was used for the determination of serum
constituents and for immunological studies. A third sample was
collected in citrated blood to study lymphocyte activity.
The EDTA-blood was used for the determination of packed cell
volume (PCV) and haemoglobin concentration (Hb). The PCV was
determined by the micro-haematochrit centrifuge. The
concentration of haemoglobin was determined by an automated
auto-analyser.
The activity of the enzyme AST and the concentrations of
albumen, globulin and creatinine in serum were determined by
commercial kit sets using a spectrophotometer. The concentration
of globulin was determined by subtracting total protein from
albumen concentrations.
Microbiological methods: Pus samples were collected from
external lymph node abscesses in Najdi sheep. Pus samples were
also obtained from infected lymph nodes containing fluid pus
collected from the slaughterhouse. About 5-10 ml pus was
aspirated using sterile syringe and transported while cold to the
laboratory.
Each pus sample, collected in a syringe, was put into a sterile
plate, and a sterile cotton swab was used to mix the sample
thoroughly. Pus was then streaked onto blood agar plates.
Inoculated plates were incubated at 37C and checked daily for
up to 1 week. Bacterial colonies were identified on the basis of
colonial morphology according to Quinn et al. 18. Smears were
prepared from each colony type and stained with Grams method
for microscopic examination. Isolates suspected as Corynebacterium
pseudotuberculosis and S. aureus anaerobius were further
identified to prepare the vaccine. Coryne API strips were used
according to the manufacturer instructions concerning culture
preparation, inoculation, indicator application, and interpretation.
Each strip contained 20 tests + catalase which was applied in the
same strip using 3% H2O2. Additionally, different other indicators
were separately supplied from the same company.
Vaccine preparation: Single colonies of an identified
Corynebacterium pseudotuberculosis and S. aureus anaerobius
isolates were inoculated into 250 ml of brain heart infusion broth
with 1% Tween 80. The inoculated broth was incubated at 37C
with shaking for 18 hours followed by centrifugation for 15 min at
2000 xg at 4C. The supernatant was discarded and the bacterial
cell pellet was washed twice with acetone and twice with diethyl
ether and allowed to dry in air. The bacterial mass was weighed
and re-suspended in 1% formol saline in concentration of 20 mg

of cell pellet/1 ml formol saline. Tween 80 was added to the
suspension till the final concentration of 3%.
Enzyme linked immunosorbent assay (ELISA): ELISA was
standardized using antigen of local C. pseudotuberculosis and
Staphylococcus aureus subsp. anaerobius isolates to detect the
subclinical infection as well as to evaluate the vaccine potency.
Standardization was done as follows:
Antigen preparation: Test antigen was prepared from a PLDpositive C. pseudotuberculosis and Staphylococcus aureus
subsp. anaerobius isolates. The bacterial isolate was grown in
brain heart infusion broth at 37C for 48 hours. The bacterial cells
were spun down at 3000 rpm for 10 min. The bacterial pellet was
washed 3 times in phosphate buffered saline (PBS) and suspended
in 5 ml of PBS after the last wash. The bacterial suspension was
sonicated for many 30 s medium pulses.
Standardization of the antigen by the Checker-Board titration:
The whole bacterial sonicate was titrated in different dilutions
against different dilution of control positive serum from an animal
vaccinated with Glanvac vaccine. The antigen titration was
performed following a Checker-Board method as follows:
Antigen coating: Fifty l of carbonate-bicarbonate buffer (pH
9.6) was delivered into each well of a 96-well flat bottom
polystyrene plate. Fifty l of C. pseudotuberculosis and
Staphylococcus aureus subsp. anaerobius sonicate (diluted 1/4
in the same buffer) was delivered in each well of the first row (row
A). The antigen was serially 2-fold diluted using a 12 multichannel
micropipette from rows A to G (1/8 to 1/512). Row H was left without
antigen (serum control). The coated plate was left undisturbed for
overnight at 4C.
Blocking: The coated plate was washed 3 times in PBS containing
0.01% Tween 20 (PBS/T) after which 50 l of blocking buffer (PBS/
T with 1% bovine serum albumin) was added. The blocked plate
was left at room temperature for 15 min and washed 3 times in
PBS/T.
Serum dilution and distribution: Fifty l of PBS/T was delivered
into each well of the antigen-coated plate. Twenty five l of the
positive serum sample was delivered into each well of the column 1
(Wells A to H). The serum was serially diluted 3 fold from column 1
to column 11 (rows of column 12 were left as antigen control)
and the plate was incubated at room temperature for 30 min after
which it was washed 3 times with PBS/T.
Addition of conjugate and substrate: Fifty l of the antisheephorse radish peroxidase conjugate, diluted 1/4000 in PBS, was
delivered into all wells and the plate was kept at 37C for 30 min.
After 3 washings with PBS/T, 50 l of the chromogen-substrate
mixture was delivered into all wells and the plate was kept for 10
min at a dark place.
Reaction stoppage, reading and interpretation: After 10 min
incubation, the plate was taken out and 25 l of 1 M H2SO4 was
delivered into each well of the plate. The optical densities of the
183
reaction wells were read at a wavelength of 405 nm using the

ELISA plate reader.
The ELISA test proper: To test serum samples of vaccinated
animals as well as animals of flocks with history of abscesses,
ELISA was carried out using C. pseudotuberculosis and
Staphylococcus aureus subsp. anaerobius antigens at the proper
dilution as indicated by the results of the checker-board titration.
Each well of 50 ELISA plates were coated with 50 l of the antigen
at the proper dilution (in carbonate/bicarbonate buffer). The coated
plates were kept at 4C for overnight, washed 3 times with PBS/T,
dried and kept at 4C in tight plastic bags. When needed, an
ELISA plate was taken out of the refrigerator and 50 l of PBS/T
was distributed into each well. Twenty five l of each one of the
test serum samples, of different animals, was delivered into a
corresponding well of row A (A1-A12). The samples were 2-fold
serially diluted in the direction A-H and the plate was kept at room
temperature for 30 min. The test steps were completed following
the same steps of the antigen titration from blocking to reading
and interpretation. Negative and positive control samples were
tested in parallel with the test samples.
Results
Vaccination reaction: Following vaccination, ewes of groups 2
and 3 had pyrexia and were not very active. The enthusiasm to
take food was less marked compared to ewes of the group 1 and
the control group animals (group 4). The injection sites of all
vaccinated animals were examined 24 hours post vaccination. With
exception of a mild red swelling, no adverse reaction to tissues
was seen.
Protective effect of the vaccine: Table 1 shows the development
of abscess disease in the experimental ewes. Three ewes of the
control group developed abscesses on the head at the second, 5th
and 7th month of the experiment (Fig. 1). Two ewes from group 1
developed abscesses on the 7th and 10th months of the experiment.
Two ewes from groups 2 and 3 each developed abscesses at the 9th
and tenth week of the experiment. This shows that 1 ml of the
vaccine is protective for 6 months while higher doses were protective
for 8 months.
Table 1. Development of abscesses in the experimental
animals after vaccination with 9 109 bacteria,
29109 bacteria and 39109 bacteria.
Weeks after
vaccination
First month
Second month
Third month
Fourth month
Fifth month
Sixth month
Seventh month
Eighth month
Ninth month
Tenth month
G1 (n=5)
9 x109
bacteria
1
1
G2 (n=5)
2x 9 x109
bacteria
1
1
G3 (n=5)
3x 9 x109
bacteria
1
G4 (negative
control)
(n=5)
1
1
1
-
Table 2. Effect of vaccine dose on the blood picture before, 7 days

after vaccination and 7 days post booster with abscess
disease vaccine.
Weeks after
vaccination
Hb (g/dl)
Before vaccination
7 days after vaccination
7 days post booster
PCV (l/l)
Before vaccination
7 days post booster
G1 (n=5)
9x109
bacteria
G2 (n=5)
2x9x109
bacteria
G3 (n=5)
3x9x109
bacteria
G4
(negative
control)
(n=5)
8.61.2
9.81.2
10.01.6
9.81.2
9.41.6
10.21.2
8.61.6
10.01.6
9.41.4
8.81.4
10.41.2
9.21.4
0.330.02
0.330.02
0.300.06
0.340.02
0.320.04
0.320.04
0.320.06
0.340.02
0.310.02
0.0300.04
0.0290.02
0.0320.02
Table 3. Effect of vaccine dose on serum constituents before, 7

days after vaccination and 7 days post booster with
abscess disease vaccine.
Weeks after
vaccination
AST (IU)
Before vaccination
7days after vaccination
7 days post booster
Total protein (g/dl)
Before vaccination
7 days post booster
Creatinine (mol/l)
Before vaccination
7 days post booster
G1 (n=5)
9x109
bacteria
G2 (n=5)
2x9x109
bacteria
G3 (n=5)
3x9x109
bacteria
G4
(negative
control)
(n=5)
18.4 1.4
16.2 2.4
18.6 1.4
16.6 2.2
16.8 3.2
20.6 2.0
14.2 2.2
16.2 3.4
14.43.2
16.2 2.2
14.8 2.4
14.8 2.6
9.2 3.4
10.4 2.2
11.0 2.0
10.0 2.2
10.6 1.2
10.6 2.2
10.0 2.6
10.8 4.6
11.0 1.8
9.8 2.2
10.8 2.0
10.4 2.2
60.0 3.2
56.0 3.6
60.4 2.4
62.2 2.0
56.4 2.2
54.0 3.2
52.0 4.2
52.2 2.4
54.0 3.2
48.2 2.2
54.2 2.4
48.8 3.2
which indicated that the injected vaccine was safe and did not
affect blood, liver or kidney functions.
Blood and serum analysis results: Hematological and serum

constituents results showed no significant changes before and
after vaccination in all groups of ewes (Tables 2 and 3). The results
showed no variation from normal values in the vaccinated ewes,
184
Figure 1. Control sheep developing an abscess on the head after 2

months from the start of the experiment.
Vaccine preparation: Methods adopted in the study for vaccine

preparation from bacteria resulted in production of good harvest
of the bacterial pellet after washing with diethyl ether and dryness.
From one litre of culture, 1.8 g of dry pellet was obtained and
reconstituted in 1% formol saline to give a final concentration of
20 mg pellet/ml, i. e. the pellet was dissolved in 90 ml of formol
saline.
Table 4. Antibody titres in sheep serum 1/20 diluted

as detected by adapted ELISA.
Treatment groups
Post -Vaccination
After booster
Autogenous vaccine
0.73* 0.69 0.70
0.85 0.84 0.82
Control
0.36
0.28
0.3
*Value = OD value of vaccinated animal serum OD value of control well mean (no
serum). Values 0.4 is considered significant.
Antibody levels to CLA vaccines as detected by ELISA: As

shown in Table 4, vaccination with the autogenous vaccine
administration resulted in the highest antibody titres as compared
to control non-vaccinated animals. All vaccination trials resulted
in positive seroconversion. Administration showed improvement
in the antibody titre levels. Boostering resulted in little increase in
the antibody titre at two weeks post immunization as compared to
the titres two weeks after the first immunization.
Discussion
Vaccination against CLA in Saudi Arabia is not effective in
minimizing the incidence of abscesses in sheep flocks. Although
several bacterial organisms have been isolated from the lesions,
C. pseudotuberculosis and Staphylococcus aureus subsp.
anaerobius have been implicated as the major pathogens in this
condition 19.
Local strains of Corynebacterium pseudotuberculosis are
shown to confer better immunity when used to prepare vaccines
against abscess disease 8. Using a virulent United Kingdom
Corynebacterium pseudotuberculosis isolate, an ovine
experimental model of caseous lymphadenitis was developed, in
which the manifestation of disease was equivalent to the naturally
observed infection in this country. Subsequently, the capacity of
several experimental vaccines to protect against experimental
challenge was determined. Sheep were immunised with a
recombinant derivative of phospholipase D, deriving from the
virulent UK isolate, a formalin-killed bacterin of the same strain, or
a bacterin supplemented with recombinant phospholipase D.
Following homologous experimental challenge, the phospholipase
D and bacterin vaccines were observed to confer statistically
significant protection against infection, and appeared to restrict
dissemination of challenge bacteria beyond the inoculation site
in the majority of animals. More importantly, the combined vaccine
succeeded in providing absolute protection against infection,
whereby challenge bacteria were eradicated from all vaccinates.
In a study carried in Australia, it was reported that although
43% of producers used vaccination to control CLA in their sheep,
only about 12% had recommended CLA vaccination programs
that would decrease the prevalence of CLA in their sheep. The
finding that only 9% of producers were sending effectively
vaccinated sheep to abattoirs at the time of the survey was
disappointing. However, this could be expected to increase to
14% overall and nearly double from 15 to 27% in WA two to four
years after the study when ewes vaccinated according to
recommended CLA programs would be culled for age 10.
Enzyme linked immunosorbent assay (ELISA) tests have been
developed to study the humoral response to the immunodominant
PLD in experimentally infected sheep 14, 15, 17 . Antibodies develop
from the fifth day following challenge and reach a plateau
approximately three weeks post-challenge (pi) after which the titre
slowly decreases 14, 16.
In a similar study, lambs and kids were inoculated with a bacterin
containing C. pseudotuberculosis (1 mg whole cells and 50 g

muramyl dipeptide in 10% light mineral oil) twice I.M. in the thigh
1 month apart. Animals were then exposed to naturally infected
adults under field conditions. Serum antibody titers to C.
pseudotuberculosis were determined regularly in all vaccinated
animals, which rose sharply after vaccination and remained higher
in vaccinated animals after that.
Conclusions
We examined the efficacy of a polyvalent bacterin for the
prevention of naturally occurring abscesses in sheep. It induced
antibody responses and protected sheep in experimental for a
period of 6-8 months at doses providing 7910 and 14910 bacteria
per ml.
Acknowledgements
This research work was financed by the Deanship for Research,
Qassim University. The professional help of Professor Osama
Mahmoud is highly acknowledged. The help of Mr. Ahmed
Aldubeey, Mr. Abdullah Alhawas and Mr. Barood Khan in caring
for experimental animals is also acknowledged.
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12
186
WFL Publisher
Helsinki, Finland
www.world-food.net
Performance evaluation of the Telagasari Irrigation Scheme (TIS) of Karawang

Regency, Indonesia
Sangam Shrestha 1*, Foyya Yusufu Aquino 2 and Vishnu P. Pandey
1
Water Engineering and Management, School of Engineering and Technology, Asian Institute of Technology, P. O. Box 4 Klong
Luang, Pathum Thani 12120, Thailand. 2 Directorate of Irrigation Water Management, Directorate General of Agricultural
Infrastructure and Facility, Ministry of Agriculture, Republic of Indonesia. 3 International Research Centre for River Basin
Environment (ICRE), University of Yamanashi, 4-3-11Takeda, Kofu, Yamanashi 400-8510, Japan.
*e-mail: sangamshrestha@gmail.com, sangam@ait.ac.th
Abstract
This study evaluates the performance of five categories of irrigated areas of the Telagasari Irrigation Scheme (TIS) in Indonesia for the period 20072011 by using a set of twelve indicators representing irrigation performances in terms of service delivery, finance, and production efficiency. The
quality of irrigation at farm level was also evaluated using a set of structured questionnaires focusing on four aspects of service: equity, reliability,
flexibility, and adequacy. The results indicate that most areas in all five categories receive enough irrigation to meet crop water demand. Efficiency was
measured as the standardised gross value of production (SGVP) per unit of water consumed, and decreased from 2008-2011 as a result of reduced
world rice prices. The service quality of irrigation decreases downstream; the highest being in category area I and the lowest in V. Such evaluation helps
to determine the degree of influence of various factors on irrigation management, and supports the decision making process towards improved
irrigation performance.
Key words: Indonesia, irrigation management, performance evaluation, Telgasari Irrigation Scheme (TIS).
Introduction
Global water consumption has so far doubled every 20 years along
with population growth, urbanisation, and expanding economic
activities, and this increase has intensified pressure on water
resources. On a global scale, the irrigation sector is by far the
largest water user, which accounts for more than 70% of the total
water use and contributes to most of the worlds food production.
Water usage in Asia and the Pacific is primarily for agriculture, it
accounts for 79% of total withdrawals in 2002 compared to 13% for
industrial use and only 8% for domestic use 1. Investment in water
for agriculture has made a positive contribution to rural livelihoods,
food security, and poverty reduction 2. However, the majority of
irrigation systems are proven to be inefficient, resulting in
significant water wastage 3 through water logging and salinity. On
the other hand, with the fact of increasing fresh water scarcity,
many countries have inadequate water supplies to meet their
current urban, environmental, and agricultural needs. These needs
will continue to grow 4. The challenge therefore is to produce
enough food for two billion extra people over the next 50 years
while satisfying growing urban and environmental water
requirements 5. Some analysts have estimated that 60% of the
additional food required will come from irrigation 6. Raising food
production to support this larger global population requires an
improved and sustainable irrigation performance.
Optimising the use of irrigation water is vital in conserving land
and water resources as well as maximising yield with the water
available. Performance evaluation of irrigation systems is a major
component of proper management, which in turn forms the basis

for optimal use of land and water resources. It is also seen as the
information system, which enables the performance management
process to function effectively and efficiently 7. In todays market,
farmers should be able to optimise each part of their operation so
that production is maximised at minimal cost. Merely modernising
the irrigation system does not guarantee high performance. The
efficiency of an irrigation system depends as much, or more, on
the capability of the irrigator as on the quality of the system.
Irrigation systems require resources such as labour, water, energy,
and time. Hence, optimising an irrigation system by combining
the correct type, design, and operation of the system allows the
use of these resources to be minimised. From an operation and
maintenance viewpoint, irrigators need to know how much water
their systems are applying and how well they are performing.
Evaluating performance of irrigated systems is becoming
increasingly important as a basis of improvement in order to
achieve optimal productivity in the context of increased food
demand, open global markets, and competition for limited
freshwater resources 8, 9. In recent decades, many researchers have
developed and applied irrigation performance indicators and
benchmarking techniques to identify the best irrigation practices
and compare different and complex irrigation systems. Most of
the researches have assessed the performance of the irrigation
management process using financial and physical indicators 9, 10-18.
A few researchers have also conducted studies to evaluate irrigation
187
water management from the farmers perspective 19-22. Any

irrigation performance assessment should analyse the productive
and hydrological impacts of internal irrigation processes to assist
agents involved in crop production, water management, and water
policy to improve the performance of irrigated areas 23, 24. In order
to assess the performance of irrigation systems, a concept of
benchmarking performance in the irrigation and drainage sector
was introduced 20, 25. Benchmarking is defined as the identification
and application of organisation specific best practices for the
purpose of improving competitiveness, performance, and
efficiency.
Irrigation performance indicators range from water distribution
to agricultural, economic, social, and environmental aspects 12.
Selecting the type of performance measurement varies according
to the purpose of performance evaluation. Performance can be
assessed for a variety of purposes 9, 26, 27, such as improving system
operation, assessing progress against strategic goals as an integral
part of performance-oriented management, assessing the general
health of a system, assessing intervention impacts, diagnosing
constraints, gaining better understanding of determinants of
performance, and comparing the performance of a system with
others or with the same system over time. In this study, we
evaluated the performance of the Telagasari Irrigation Scheme
(TIS) at Karawang Regency in the Republic of Indonesia. The
purpose of evaluation was to compare the performance of five
different category areas of TIS with each other as well as with the
time period (2007-2011). We tried to use both quantitative (using
measurable indicators) and qualitative (using a structured
questionnaire) measures to evaluate performance of the irrigation
system in this study. Twelve indicators under three performance
categories (service delivery, financial, and production efficiency)
are compared for five categories of irrigation area in the TIS during
the period 2007-2011. Similarly, a structured questionnaire survey
is carried out with irrigation staff and farmers to assess the quality
of the irrigation service in terms of adequacy, reliability, equity,
and flexibility.
Study area:
Karawang Regency: Karawang Regency, located in the western
part of West Java Province, is about 52 km from Jakarta, the capital
city of Indonesia (Fig. 1). Geographically, it is located between
10702 to 10740 east longitude, and 556 to 643 south latitude.
Karawang Regency covers an area of approximately 1,700 km2,
which is 3.73% of the West Java Provinces area.
Topographically, the region lies in flat and low elevation land
with an altitude ranging from 0-1,279 m above mean sea level
(masl). The slope range is 0-40%, with flat slopes dominant in the
major part of Karawang. Generally, the soil type in Karawang
Regency is alluvial in the lowland area, and podsolik and latosol
in the highland area. The average annual temperature of this region
is about 27C.
Telagasari Irrigation Scheme (TIS): The Telagasari Irrigation
Scheme (TIS) covers an area of 410 km2 in Karawang Regency.
Irrigation water in the TIS is distributed from the Walahar Dam
and this receives water from the Jatiluhur Dam. There are three
main primary canals, which deliver water from the Walahar Dam:
(i) The Tarum Timur Canal, which is 67 km long and delivers water
to the Subang District and surrounding area, (ii) The Tarum Utara
Canal that delivers water to the Karawang District and surrounding
area through the Walahar Dam, and (iii) The Tarum Barat Canal,
which is 70 km long and delivers water to the Bekasi District, and
a drinking water supply company in Jakarta.
Irrigation activity in the primary and secondary canals in this
area is managed by Perum Jasa Tirta II, a state-owned enterprise,
Figure 1. Location of Karawang Regency in the Republic of Indonesia.

188
while the tertiary level is managed by the farmers. The total

command area of the TIS is approximately 84,700 ha, covering 15
sub-districts, with the field irrigated area of 41,000 ha. In order to
achieve reliability and adequacy in irrigation, the TIS is divided
into five categories based on the sequence of water distribution;
Category I receives water first and Category V receives last.
Category I has an irrigated area of about 5,200 ha. It is followed
by Category II with an area of 8,800 ha, Category III with an area
of 10,800 ha, Category IV with an area of 7,200 ha and Category V
with an area of 9,000 ha (Fig. 2).
Water distribution in the TIS is managed by the Walahar Dam
Authority. Water is distributed to several secondary canals and
then to tertiary canals. There are 42 secondary canals conveying
water to an area of 41,000 ha. For the irrigation area in Category I,
water is distributed through 7 secondary canals and 1 tertiary
canal, whereas for Category II through 14 secondary canals and 2
primary canals; for Category III through 16 secondary canals; for
Category IV through 12 secondary canals and 1 primary canal;
and for Category V through 17 secondary canals before the water
reaches the tertiary canal and field. The total canal length in the
TIS is approximately 215 km. Details of the irrigation network are
illustrated in Fig. 3.
There are 71 Water User Associations (WUAs) in the TIS, which
are spread in each secondary level network, with a total number of
approximately 8,607 persons. The working area of the WUAs is
only 11,230 ha. This condition shows that the requisite amount of
WUAs in this irrigation scheme is still far from ideal. There are
many secondary canal areas without WUAs being involved in

irrigation management. In such circumstances, irrigation is
managed by an Ulu-Ulu; a village representative chosen by the
head of the village 28.
Climate and crop: The local climate of the area is tropical and
strongly influenced by monsoons. The dry season starts from
April to September and the wet season is from October to March.
The analysis of 5 years (2007-2011) of meteorological data from
the Halim Perdana Kusumah station shows the average
temperature ranges from 22C to 34C. The minimum temperature
ranges from 22.2C (in 2011) to 25.1C (in 2010), whereas the
maximum temperature ranged from 34.5C (in 2007) to 29.3C (in
2008). The area receives an average annual rainfall of 2,300 mm
and tends to be erratic. During 2007-2011, the lowest average
annual rainfall was 1,300 mm (in 2011) and the highest 3,000 mm (in
2007). In terms of monthly rainfall, lowest at 0 mm occurred in
August 2011 and highest (1,081 mm) was in February 2007. The
Ciherang rice variety is the dominant crop grown (98% of total
area) in the TIS. The average rice yield is 6 tons/ha, and the
potential yield is 8.5 tons/ha. Rice planting periods for the different
categories of irrigation area for rainy and dry seasons are reported
in Table 1.
Methodology: For performance evaluation of the TIS, an indicatorbased approach was applied. Indicators for irrigation performance
range from water distribution to agricultural, economic, social, and
environmental aspects 12. To enable comparison
of the system with different infrastructures,
management types and environment, the
International Water Management Institute
(IWMI) has suggested the use of a minimum
set of comparative indicators, to give a broad
overview of the hydrological, agronomical,
financial, and environmental performance of
irrigation systems. The selection of a suitable
set of indicators varies depending on the
purpose of performance evaluation. After a
careful review of the indicators suggested in
certain literature 2, 9, 12, 26, 29, 30, twelve indicators,
under three types of performance (namely,
service delivery, financial, and production
efficiency) were selected for quantitative
evaluation of the TIS performance. We also
conducted a structured questionnaire survey
with irrigation staff and farmers to evaluate the
quality of irrigation service. The questionnaire
focused on evaluating performance from the
following four aspects: adequacy, reliability,
Figure 2. Category of irrigated area in the TIS based on water distribution. Category I
equity, and flexibility.
receives water first and Category V last.
Table 1. Characteristics of different irrigation area categories in the TIS.
Cat.
Command
area (ha)
I
II
III
IV
V
22,506
20,050
17,800
16,500
34,894
Irrigated
area
(ha)
5,200
8,800
10,800
7,200
9,000
Primary
canal
Secondary
canal
1
2
0
1
0
7
14
16
12
17
Canal
length
(km)
32.7
53.0
42.3
53.5
33.8
Canal
performance
(%)
70
73
70
69
67
Rice planting
Rainy season
Dry season
Early-Mid Oct.
Mid Oct.- Early Nov.
Early-Mid Nov.
Mid Nov. Early-Dec.
Early-Mid Dec.
Early-Mid April
Mid April-Early May
Early-Mid May
Mid May Early June
Early-Mid June
*Rice yield (t/ha)

Rainy
Dry
season season
7.0
6.9
7.2
6.9
6.8
6.7
6.7
6.6
6.6
6.6
*Average of 5 years (2007-2011). Cat. = Category.
189
SC. Talunpare
Primary canal: Tarum

Utara Barat (TUB)
Walahar DAM
Primary canal: Tarum

Utara Timur (TUT)
Legends:
Primary Canal (PC)
Secondary Canal (SC)
DAM
Branch/Division box
Category I
Category II
Category III
Category IV
Category V
Figure 3. Irrigation canal network of the Telagasari Irrigation Scheme (TIS) (not to scale).
Service delivery performance: This enables us to understand the

performance of an irrigation system in terms of water allocation in
the head system and water consumption by crops in the field. The
following five indicators were used to examine service delivery
performance in the TIS. Indicators of crop water demand were
measured as actual crop evapotranspiration (ETc) and calculated
by the water balance method using CROPWAT 8.0 of FAO 31. The
climatic data was taken from the weather station located within
Karawang Regency.
Irrigation water delivery per unit of irrigated area (IWDA; m3/
ha): The volume of water delivery per unit area for a given crop.
IWDA =
Total amount water delivery Q total

=
Irrigated area
A
(1)
Relative water supply (RWS): RWS provides information about

whether the total amount of water (rainfall and irrigation) delivered
to the crop during its growth cycle has been excessive, sufficient
or deficient 32. It also reveals how much water supply is adjusted
to meet the demand 9. It is expressed as a ratio of net water supply
to actual crop water demand (Eq. 2), where the net water supply
consists of irrigation water delivered (I) plus groundwater
abstraction (G) plus total effective rainfall (Pe), excluding any recirculating internal water within the system.
RWS =
Water supply
Actual crop evapotranspiration
(2)
RWS =1 reflects that a sufficient amount of water is diverted to

meet the crop water demand, whereas RWS <1 refers to insufficient
diversion, and > 1 to excessive water diversion beyond that
required. RWS >1 suggests inefficient water delivery to the field 22,
33
, which needs to be evaluated by an irrigation operator for the
190
next water delivery. RWS is useful because it serves as a basis for

the comparative study and analysis of irrigated areas located in
different regions with diverse characteristics.
Relative irrigation supply (RIS): RIS, similar to RWS, also
provides information about scarcity or excess water and how well
irrigation supply and demand are matched. The optimal value of
RIS is around one, meaning that water requirements not covered
by rainfall are met. A value over one suggests too much water is
being supplied, possibly causing waterlogging and a negative
impact on yields, and a value below one indicates water scarcity
for the crops. This indicator provides information concerning the
quality of the irrigation water delivered to the crop as it relates
water supply demand to net water requirements. RIS is calculated
as below (Eq. 3):
RIS =
Irrigation water supply

Crop water requirements
(3)
Relative rainfall supply (RRS): RRS relates the effective rainfall

to the total amount of water needed for production (Eq. 4). This
indicator, which only takes rainfall into account, determines the
extent to which crop water requirements have been met naturally.
When RRS is equal to RWS in the same period, it means that
rainfall is the only water supplied to the crop and irrigation does
not take place.
RRS =
Effective rainfall
Pe
=
Actual crop evapotranspiration ETc
(4)
Water delivery capacity (WDC): WDC is measured as the amount

of water delivery capacity at the head of the irrigation system and
expressed as Eq. 5. WDC is targeted as equal to one, meaning that
the canal capacity can deliver the required water during peak
irrigation demand. Values close to one indicate that there may be
difficulties in meeting short-term peak demand. Frequently,

additional capacity is designed (at additional cost) to allow for
more flexible water delivery, or to improve management:
WDC =
Capacity to deliver water at the system head

Qh
=
Peak consumptive demand
Qpeak
(5)
Financial performance: Three indicators were selected to

evaluate performance of the TIS in financial terms. They include:
(i) management, operation and maintenance costs per unit area
(US$/ha), (ii) staff per unit area (person/ha), and (iii) cost per person
(US$/person) employed in the irrigation system.
Management, operation, and maintenance cost per unit area
(MOMPA; US$/ha): The total cost of management, operation,
and maintenance (MOM) per unit area is calculated using the
budget allocation for management, operation, and maintenance
activities. Three institutions are engaged in irrigation operation in
the TIS: i) Perum Jasa Tirta II is a major operator, allocating the
budget for management, operation and maintenance; ii) Balai Besar
Wilayah Sungai Citarum (BBWSC) is responsible for managing
the river basin; and iii) The Ministry of Public Works allocates the
budget for maintenance only. The farmer does not have to be
involved in allocating the budget for operation and maintenance.
The government provides a subsidy for the operation and
management of an irrigation scheme through those three
institutions.
Area per staff (ha/person): This is used to estimate the ideal
number of irrigation agency workers engaged in the irrigation
system.
Cost per person employed in the irrigation system (CPP; US$/
person): This item includes the cost of all personnel employed by
the organisation including contractors and contract employees
engaged in the administration, management, and operation. The
total cost of personnel engaged in the irrigation system is
calculated using the salary of all staff involved in irrigation
activities at the TIS. The total number of staff involved in irrigation
management at the TIS comprises: one section head, three subsection head, nine administrative staff, four field supervisors, 27
field coordinators (of each field), and 105 irrigation staff/walkers.
Production efficiency: Productivity or production efficiency of
the TIS was assessed using the following four indicators: (i) output
per unit of service area; (ii) output per unit of irrigated area; (iii)
output per unit of irrigation supply; and (iv) output per unit of
water consumed. To enable comparison of system output from
various parts of the world and those growing different types of
crops, total annual output is expressed as the standardised gross
value of production (SGVP). SGVP makes it possible to compare
the performance of irrigation systems, no matter where they are or
what kind of crops are being grown. Output per unit of water used
to grow oranges in Mexico can be compared with that of apples
grown in Nepal. The SGVP captures both local preferences - for
example, specialised crops that may have a low international price,
but a high local value - and the value of non-traded crops. SGVP
is calculated as follows (Eq. 6):
Pi
SGVP = crops Ai * Yi *
Pworld
Pb
(6)
where, Ai is the area containing cropi, Yi is the yield of cropi, P is

the local price of cropi, Pb is the local price of the base crop (the
predominant locally grown, internationally traded crop) and Pworld
is the value of the base crop traded at world price.
SGVP per unit service area (SPSA; US$/ha): SGVP per unit
service area is used to calculate all the five category areas in the
TIS and compared for a five-year period. Higher output value per
unit of service area resulted from the high economic value crops
grown within the irrigation projects.
SGVP per unit irrigated area (SPIA; US$/ha): SGVP per unit
irrigated area is used to calculate all the five category areas in the
TIS and compared for a five-year period. Higher output per unit of
irrigated area resulted from the high economic value crops grown
within the irrigated area.
SGVP per unit irrigation supply (SPIS; US$/m3): SGVP per unit
of irrigation supply is used to calculate all the five category areas
in the TIS and compared for a five-year period. Higher output per
unit of irrigation supply resulted in the areas with low irrigation
requirements.
SGVP per unit water consumed (SPWC; US$/m3): SGVP per unit
of water consumed is used to calculate all the five category areas
in the TIS and compared for a five-year period. The water consumed
is defined as the actual evapotranspiration from irrigated crops.
The value of output per unit of water consumed is higher when
the cropping pattern and crop calendar are suited to the irrigation
schedule.
Quality of irrigation services: The quality of irrigation service in
the TIS was evaluated by interviewing irrigation agency staff and
farmers based on a structured questionnaire. Twenty-seven
irrigation agency staff responsible for water distribution activity
and sixteen Water User Association (WUA) representatives were
interviewed for this purpose. The questionnaire focused on the
following aspects:
Adequacy: Adequacy of irrigation services to meet crop water
requirements was assessed based on the percentage of areas
having a water deficit during periods of high water demand.
Reliability: Reliability of irrigation services was assessed based
on the following three indicators: reliability of the person in charge
of water distribution (20% weight), inundation risk (40% weight),
and canal condition (40% weight).
Equity: Quality of irrigation services in terms of equity was
assessed based on the following four indicators: organisational
rules (30% weight), physical constraints for uniform distribution
(30% weight), rotation of secondary canal for first irrigation (20%
weight), and rotation order after the first irrigation (20% weight).
Flexibility: Flexibility of the irrigation services was assessed based
on the degree of freedom the farmers have in controlling the
duration, flow rate, and frequency of the irrigation supply as
suggested in Malano and van-Hofwegen 34.
191

Service delivery performance:
Irrigation water delivery per unit irrigated area (IWDA; m3/ha):
The total annual volume of irrigation water delivery per unit area
(including delivery in both dry and wet planting seasons) varied
in each category from 12,000 to 18,000 m3/ha during 2007-2011
(Table 2). This delivery is quite high compared to ideal crop water
requirements for rice throughout its growing period, which is
estimated at 4,000 7,000 m3/ha by FAO 35. On average, Categories
I and II (i.e., those in the upstream) receive more irrigation water
than the other categories. This is attributed to the efficiency of
water distribution from upstream to the downstream area. It was
found that the canal condition in the upstream area was relatively
better than the downstream area during 2007-2009, which is also a
reason for better water distribution efficiency in the upstream.
During 2010 and 2011, an increase in irrigation water delivery is
observed in category areas in the downstream.
Relative water supply (RWS): RWS in the five category areas for
the period 2007-2011 was in the range of 0.94-1.79 in the dry season
and 0.77-1.48 in the wet season (Table 3). The five-year average
RWS suggests that Category I-III areas during the dry season
receive excess water, whereas Categories IV and V receive just
adequate water to meet crop water demand; however, during the
wet season, water supply (rain plus irrigation) seems adequate to
meet crop water demand in Categories I-IV and deficient in
Category V. Taking a closer look at RWS values in the wet season
for all 5 years (i.e., 2007-2011) reveals the values to be less than
one in most cases for Categories III-V. This indicates a case of
water scarcity or deficiency which could lead to a reduction in
crop yield. Irrigation management during the dry season in
Categories I-III was inefficient due to an excessive supply of water.
Relative irrigation supply (RIS): The 2007-2011 average RIS in
the five category areas varies from 0.84 to 1.14 in the dry season
(January-June) and from 0.76 to 0.86 in the wet season (JulyDecember) (Table 4). Due to high evaporation during the wet
season, the irrigation supply cannot meet crop water demand,
except for Category II in 2008, Categories II and III in 2009, and
Categories IV and V in 2010. For Categories I-III, irrigation supply
can meet crop water demand for the period 2007-2011. For
Categories IV and V, the RIS during dry seasons 2007-2010 is less
than one, which suggests irrigation water supply is inadequate to
meet the crop water demand.
Relative rainfall supply (RRS): The contribution of rainfall to
total water demand decreases during the dry season as evidenced
from the values lower than 1 RRS in all the categories during 20072011 (Table 5). For Categories IV and V, even RIS values for all the
years are lower than one (Table 4), which suggests an inadequate
irrigation water supply to meet crop water demand. The 2007-2011
average RRS values in the dry season for all five categories of
irrigation area are in the range of 0.44 to 0.66; whereas in the wet
season they vary from 0.83 to 1.04. From Table 5, it is evident that
there is a larger reduction in rainfall contribution to total water
demand during 2011 in all five categories of irrigated area.
Further analysis of RWS, RIS and RRS values in both dry and
wet seasons for all categories of irrigation area during 2007-2011
(Figs. 4 and 5) shows that dry season RRS values are lower than
192
one in all categories and for all years, which suggests a reduced
rainfall contribution to total water demand. A lower than one RIS
value in Categories IV and V in all years suggests that water
supplied through irrigation is not enough to meet the crop water
demand. On the other hand, values lower than one RIS in all
categories in all years during wet seasons reveal that the irrigation
water supply is not sufficient to meet the crop water demand.
However, it is supplied through rainfall in Categories III-V during
2007-2010 as the values of RRS are greater than one.
In addition, the evolution of wet and dry season RWS, RIS and
RRS values were also calculated for each category from 2007 to
2011. During the dry season, in Category I-III, irrigation water is
sufficient to meet the crop water demand; whereas it was
inadequate in Categories IV and V. During the wet season, in
Category III-V, rainfall is the only source of water supplied to meet
the crop water demand.
Water delivery capacity (WDC): Water delivery capacity indicates
if the system design is a constraint to meet the maximum irrigation
water requirement. A value greater than one indicates capacity is
not a constraint to meet irrigation water demands. Generally, water
in the conveyance system of the TIS can meet the irrigation water
demand even in the peak season. The maximum discharge capacity
that can be delivered from the canal intake is 38 m3/s, while peak
crop water demand in the five category areas during 2007-2011 is
in the range 17 24 m3/s. WDC values for all years from 2007-2011
are greater than one (Table 2), indicating adequate capacity of the
conveyance system intake to deliver water to each category area.
Financial performance: Financial performance was analysed in
order to understand the performance of management, operation,
and maintenance in the TIS in relation to budget and cost
allocation. In this assessment, the relevant budget and
management costs include: staff, office equipment, operation, and
maintenance but not the operation and maintenance cost of tertiary
canals and costs associated with farmers participation.
Management, operation, and maintenance (MOM) cost per unit
area (US$/ha): The MOM cost per unit area is calculated using
the budget allocation for those activities. As seen in Table 2, this
indicator varies over the years (i.e., 2007-2011). This is because
differences in canal conditions, currency fluctuations, and
management policy. The highest MOM budget per unit area (US$
7.69/ha) in the TIS was allocated in 2010 and the lowest (US$ 2.06/
ha) in 2008. The maintenance component contributes substantially
to the increase or decrease in the total MOM cost per unit area in
each year, while management and operation component cost is
relatively stable.
Area per staff (APS; ha/person): This indicator was analysed to
compare whether the number of staff to control the irrigation
system is appropriate for the best efficiency of the system. In the
TIS, one irrigation staff managed approximately 280 ha in 2007,
2010, and 2011 (Table 2). Due to staff retirement in 2008 and 2009,
the staff per unit area decreased to one person per 293 ha and one
person per 285 ha, respectively (Table 2). If compared to the
optimum staff number of one person per 333 ha to control an
irrigation system 36, the TIS may benefit from decreasing staff
numbers for optimum performance. The most appropriate number
193
Q
MCM
93.3
156.2
162.5
89.8
109.0
93.6
180.7
185.5
90.5
120.6
91.3
153.7
190.0
105.7
114.2
82.0
138.4
158.7
112.1
145.7
81.6
140.7
175.9
117.3
155.9
A
Ha
5200
8800
10800
7200
9000
5200
8800
10800
7200
9000
5200
8800
10800
7200
9000
5200
8800
10800
7200
9000
5200
8800
10800
7200
9000
132.7
132.7
132.7
132.7
132.7
263.8
263.8
263.8
263.8
263.8
247.5
247.5
247.5
247.5
247.5
231.4
231.4
231.4
231.4
231.4
301.3
301.3
301.3
301.3
301.3
P
mm
74.4
125.8
154.5
102.9
128.7
67.7
114.6
140.6
93.7
117.2
64.7
109.5
134.5
89.6
112.0
69.8
118.1
145.0
96.7
120.8
71.1
120.4
147.8
98.5
123.2
7.36
7.15
7.11
6.79
6.53
7.16
7.45
7.37
7.50
7.52
6.92
7.16
7.01
7.11
7.32
6.94
6.94
6.53
6.53
6.59
6.69
6.47
5.72
5.27
5.18
540.5
540.5
540.5
540.5
540.5
550.2
550.2
550.2
550.2
550.2
480.4
480.4
480.4
480.4
480.4
515.9
515.9
515.9
515.9
515.9
470.5
470.5
470.5
470.5
470.5
Input Parameters
CWD Yield LRP
MCM
t/ha
US$/t
551.7
551.7
551.7
551.7
551.7
520.6
520.6
520.6
520.6
520.6
589.4
589.4
589.4
589.4
589.4
700.2
700.2
700.2
700.2
700.2
335.9
335.9
335.9
335.9
335.9
WRP
US$/t
19.7
32.9
40.7
24.3
27.7
18.9
34.1
41.3
28.3
35.9
22.3
39.7
46.5
31.9
40.6
26.7
47.1
52.7
35.4
44.9
11.7
19.2
20.8
12.8
15.7
SGVP
MUS$
15.7
15.9
16.2
16.3
17.3
15.7
15.7
14.7
15.5
16.2
17.5
17.4
17.6
14.6
12.7
18.0
20.5
17.1
12.5
13.4
17.9
17.7
15.0
12.4
12.1
IWDA
*m3/ha
1.10
1.12
1.14
1.14
1.21
1.21
1.21
1.13
1.20
1.24
1.41
1.40
1.41
1.18
1.02
1.34
1.53
1.28
0.94
1.00
1.31
1.30
1.10
0.91
0.89
RWS
-
RRS
-
RIS
-
1.60
1.60
2.16
1.82
WDC
1.57
6.59
7.69
3.81
2.06
2.81
281
285
293
184
169
134
134
Performance indicators
MOMPA APS
CPP
US$/ha
ha/p US$/p
3.35
281
122
1469
1639
2287
1476
1272
1412
1701
2322
1714
1647
1665
1980
2615
1939
1862
3786
3735
3769
3384
3080
3639
3875
3827
3927
3989
4291
4511
4310
4444
4511
5147
5358
4882
4918
4990
2253
2179
1927
1775
1744
874
956
1169
775
720
1997
2352
2962
2146
2060
SPIA
US$/ha
SPSA
US$/ha
0.24
0.23
0.23
0.21
0.18
0.23
0.25
0.26
0.25
0.25
0.24
0.26
0.24
0.33
0.42
0.29
0.26
0.38
0.48
0.55
0.14
0.12
0.13
0.15
0.19
SPIS
US$/m3
0.26
0.26
0.26
0.24
0.22
0.28
0.30
0.29
0.30
0.31
0.34
0.36
0.35
0.36
0.36
0.38
0.40
0.36
0.37
0.37
0.16
0.16
0.14
0.13
0.13
SPWC
US$/m3
Relative irrigation supply (Dry season)

2007 2008 2009 2010 2011 Average
1.1
1.2
1.3
1
1.1
1.14
1.1
1.2
1.1
0.9
1.2
1.10
1.0
1.1
1.1
0.9
1.2
1.06
0.7
0.8
0.9
0.8
1.0
0.84
0.7
0.9
0.8
0.9
1.1
0.88
Relative irrigation supply (Wet season)
2007 2008 2009 2010 2011 Average
0.90 0.90 0.80 0.80 0.60
0.80
0.90 1.10 1.00 0.70 0.60
0.86
0.70 0.90 1.10 0.80 0.60
0.82
0.70 0.60 0.90 1.00 0.70
0.78
0.70 0.60 0.70 1.00 0.80
0.76
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
I
II
III
IV
V
Category
Relative water supply (January June, Dry season)

2007
2008
2009
2010
2011
Average
1.43
1.52
1.79
1.36
1.49
1.52
1.47
1.58
1.53
1.41
1.57
1.51
1.36
1.40
1.46
1.19
1.55
1.39
0.99
1.10
1.19
1.11
1.35
1.15
0.94
1.22
1.12
1.15
1.46
1.18
Relative water supply (July December, Wet season)
2007
2008
2009
2010
2011
Average
1.20
1.16
1.07
1.05
0.77
1.05
1.14
1.48
1.29
0.99
0.75
1.13
0.87
1.16
1.37
1.06
0.80
1.05
0.83
0.77
1.17
1.29
0.97
1.01
0.84
0.78
0.93
1.34
1.01
0.98
Category
Table 4. Relative irrigation supply (RIS) during 20072011 in the TIS.
Table 3. Relative water supply (RWS) during 2007-2011

in the TIS.
I
II
III
IV
V
I
II
III
IV
V
Category
Relative rainfall supply (Dry season)

2008 2009 2010 2011 Average
0.72 0.83 0.71 0.46
0.66
0.64 0.74 0.67 0.41
0.62
0.55 0.60 0.67 0.35
0.55
0.47 0.51 0.72 0.30
0.49
0.41 0.44 0.77 0.22
0.44
Relative rainfall supply (Wet season)
2007 2008 2009 2010 2011 Average
0.77 0.74 0.97 1.13 0.55
0.83
0.91 0.85 1.04 1.11 0.65
0.91
1.09 1.04 1.14 1.08 0.77
1.02
1.06 1.14 1.16 1.06 0.79
1.04
1.03 1.18 1.13 1.01 0.75
1.02
2007
0.58
0.62
0.55
0.43
0.36
Table 5. Relative rainfall supply (RRS) during 20072011 in the TIS.
Cat. is category; A is coverage/service area; ha is hectare; Q is total volume of irrigation water delivered; MCM is million-cubic-metres; P is total amount of rainfall; mm is millimetres; CWD is crop water demand (or actual evapotranspiration);Yield is rice
yield; t is ton; LRP is local rice price; WRP is world rice price (source: World Bank database); SGVP is standardised gross value of production; MUS$ is US Dollars in Million; xx/p is xx per person; full form of performance indicators are in Section-3; *IWDA
values are in 1000 m3/ha.
Cat.
area
Unit
2007
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
2008
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
2009
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
2010
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
2011
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
Refer to Table 4 for RIS values in dry and wet seasons
Table 2. Summary of input parameters and performance indicators.
Refer to Table 5 for RRS values in dry and wet seasons
RWS/RIS/RRS
RWS/RIS/RRS
RWS/RIS/RRS
RWS/RIS/RRS
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2011
2010
2009
2008
2007
II
II
II
II
II
III
Category
III
III
III
III
IV
IV
IV
IV
IV
a) Dry season (Jan - June)
RWS
RIS
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
RRS
2011
2010
2009
2008
2007
II
II
II
II
II
Category
III
III
III
III
III
IV
IV
IV
IV
IV
b) Wet season (July - Dec)
a) Dry season (January June) and b) Wet season (July-December).
Figure 4. Evolution of RWS, RIS and RRS in all categories (I-V) from 2007-2011.
RWS/RIS/RRS
RWS/RIS/RRS
RWS/RIS/RRS
RWS/RIS/RRS
RWS/RIS/RRS
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2008
2008
2008
2007
2008
Category V
2007
Category IV
2007
Category III
2007
Category II
2007
2008
Year
2009
2009
2009
2009
2009
2010
2010
2010
2010
2010
a) Dry season (Jan - June)

Category I
RWS
2011
2011
2011
2011
2011
RIS
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
-
2008
2008
2008
RRS
2007
2008
Category V
2007
Category IV
2007
Category III
2007
Category II
2007
2008
Year
2009
2009
2009
2009
2009
a) Dry season (January June) and b) Wet season (July-December).
2010
2010
2010
2010
2010
b) Wet season (July - Dec)

Category I
Figure 5. Evolution of RWS, RIS and RRS from 2007-2011 in each category.
RWS/RIS/RRS
194
2011
2011
2011
2011
2011
Cost per person employed in the irrigation system (CPP; US$/

person): The total cost of personnel engaged in irrigation
management is calculated using the salary of all the staff involved
in irrigation activity in the TIS. The cost per person is increasing
year by year; from US$ 163 in 2007 to US$ 184 in 2011 (Table 2).
The average incremental cost for this component is about 10%
per year.
Productive efficiency:
SGVP per unit service area (SPSA; US$/ha): The 2007-2011
average SGVP per unit of service area is approximately US$ 1,720/
ha and varies between US$ 720 to US$ 2,962/ha from 2007 to 2011
(Table 2). The highest output of US$ 2,962/ha was observed for
Category III in 2008, and during that time, the rice production was
140,600 tons and the world rice price reached US$ 700/ton. The
lowest output of US$ 720/ha was observed for Category V in
2007. These results show that the factors that contribute to the
output per unit of service area are paddy yield, command area,
local rice price, and world rice price.
SGVP per unit irrigated area (SPIA; US$/ha): The SGVP per
unit of irrigated area in the five category areas varied from US
$1,700/ha in 2007 to US$ 5,100/ha in 2008 (Table 2). The highest
output was from Category II in 2008 when paddy yield reached 7.6
tons/ha and the world rice price reached US$ 700/ton. The lowest
output was from Category V in 2007 when the paddy yield reached
5.2 tons/ha and the world rice price reached US$ 335/tons.
areas (Table 2). It tends to decrease from 2008-2011 in each

category area (Fig. 6) due to the influence of world rice prices and
fluctuation of crop water requirements during the period. The
highest output per unit of water consumed was in 2008 when the
world rice price reached US$ 700/tons and rice productivity was 7
tons/ha. World rice price fluctuation significantly influenced the
output per unit of water consumed, while the fluctuation in total
crop water demand was relatively stable. The lowest value was in
2007 due to a low rice yield of only 6 tons/ha and reduced world
rice price of US$ 335/tons. The results of the TIS are similar to the
findings of Sakthivadivel et al. 37, which, based on a study of 40
irrigation systems throughout the world, reported that the SGVP
per unit of irrigation supply ranged from US$ 0.20 to 0.60 in
irrigation schemes for orchard and industrial crops.
Quality of irrigation services: The TIS quality at service level
was assessed based on a structured questionnaire survey
focusing on the following four aspects of the service: equity,
reliability, adequacy, and flexibility. The results of the survey are
presented in Table 6 and discussed below.
Output per unit water consumed
(US$/m3)
of staff for an irrigation system, however, is expected to vary

depending upon location, environment, and other conditions.
0.5
0.45
SGVP per unit water consumed (SPWC; US$/m3): This indicator

value varies from US$ 0.13/m3 to US$ 0.40/m3 in the five category
2008
2009
2010
2011
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
SGVP per unit irrigation supply (SPIS; US$/m3): The highest

value in this indicator suggests that besides the yield and world
crop price, the volume of irrigation water delivered significantly
contributes to output. The results show that the indicator value
varies between the five category areas. The output ranges from
US$ 0.12/m3 (in Category II) to 0.19 (in Category V) in 2007, from
0.26 (in Category II) to 0.55 (in Category V) in 2008, from 0.24 (in
Categories I and III) to 0.42 in 2009 and from 0.23 to 0.26 in 2010
and 2011 (Table 2). The highest SGVP per unit of irrigation supply
was from Category V in 2008 (US$ 0.55/m3), where the irrigation
water delivery was lower than any other category. The lowest
output was from Category II in 2007 (US$ 0.12/m3).
2007
0.4
Cat. I
Cat. II
Cat. III
Cat. IV
Cat. V
Figure 6. SGVP per unit water consumed in TIS (Cat. = Category).
Equity: The quality of the TIS service from the perspective of

equity differed in the upstream and downstream irrigated
categories; with Category I having the highest score (0.67) and
Categories IV and V the lowest ones (0.50). In Category I, the
farmers obey the operation and maintenance rules and the
guidelines provided by irrigation authority, while this was less so
in Categories II-V. Farmers determine the planting time themselves
without taking into account suitable planting schedules
recommended by the local government and irrigation agency. In
Categories IV and V, 0-25% areas had difficulty in obtaining water.
Few fields in this category were located above the tertiary canal
and farmers had to use pumps to divert water from the canal to the
Table 6. Assessment of equity, reliability, flexibility, and adequacy of the TIS at service level.
Indicators for quality of services
1. Equity
1.1 Organisational rules
1.2 Physical constraints for uniform distribution
1.3 Rotation of secondary canals for first irrigation
1.4 Rotation order after first irrigation
2. Reliability
2.1 Reliability of the person in charge of irrigation distribution
2.2 Inundation risk
2.3 Canal state
3. Flexibility
3.1 Duration, flow rate, and frequency of irrigation
4. Adequacy
4.1 Water deficit in high water demand period
Weight
0.30
0.30
0.20
0.20
0.20
0.40
0.40
1.00
1.00
Cat. I
0.67
0.22
0.30
0.05
0.10
0.68
0.08
0.40
0.20
0.00
0.00
1.00
1.00
Cat. II
0.60
0.15
0.30
0.05
0.10
0.68
0.08
0.40
0.20
0.00
0.00
1.00
1.00
Cat. III
0.60
0.15
0.30
0.05
0.10
0.68
0.08
0.40
0.20
0.00
0.00
1.00
1.00
Cat. IV
0.50
0.15
0.20
0.05
0.10
0.58
0.08
0.30
0.20
0.00
0.00
0.75
0.75
Cat. V
0.50
0.15
0.20
0.05
0.10
0.38
0.08
0.20
0.10
0.00
0.00
0.75
0.75
Cat. = Category
195
field. Delay in water supply from upstream to downstream also

influenced water consumption in that area. For equitable
distribution of water between upstream and downstream farmers,
it is necessary to make and implement a penalty for those who do
not comply with the rules set by the government and irrigation
agency through stakeholder engagement.
Reliability: The reliability score ranged from 0.38-0.78. The highest
score was for Category I, and the lowest for V. The reliability of
the person in charge of irrigation distribution was very high in
each category area. There are inundation risks in some parts of
Categories IV and V where canal drainage conditions are poor.
Water disposal from upstream fields sometimes entered the
downstream fields even though it was still in the same category
area. Some parts of the area V located close to the coastal area
sometimes received sea water too. The average canal condition is
about 70% of normal (100%) in Category I-III, while in IV and V are
69% and 67%, respectively.
Flexibility: The freedom level for farmers in fixing the irrigation
water supply rate, frequency, and duration is determined by the
level of flexibility in an irrigation system. The flexibility score in
the five category areas of the TIS is 0 (i.e., no flexibility at all),
because water distribution is managed by the irrigation agency
(i.e., Perum Jasa Tirta II) based on the crop water demand estimated
by the agency. Therefore, farmers have no control over it.
Adequacy: Without considering the efficiency of water supply,
water distribution can satisfy all the users in general. Only a few
in Categories IV and V suffered a severe deficit. The adequacy
score in the five category areas ranged from 0.75 - 1.00. Categories
I-III had scores of 1.00, indicating no severe water deficit for any
plots in the category areas. In Categories IV and V, a few plots
suffered from severe water deficit due to a delay in water
distribution and the field location, which was difficult to reach by
the water conveyance system.
Conclusions
In this study, the performance of the five category areas of the
Telagasari Irrigation Scheme (TIS) for the period 2007-2011 was
evaluated quantitatively and qualitatively. Quantitative
assessment included the evaluation of twelve indicators
representing service delivery, financial performance, and
productive efficiency of the TIS; whereas qualitative assessment
included a structured questionnaire survey focusing on the equity,
reliability, adequacy, and flexibility aspects of the irrigation service.
A variation in performance was observed in different categories
over the period 2007-2011. The key conclusions drawn from the
performance assessment are as follows:
Service delivery performance indicators showed that upstream
areas (Categories I and II) are getting an excess of water over crop
water demand; whereas downstream (Categories III-V) water
delivery is just adequate to meet paddy water demand. The majority
of all five category areas, except some areas in Categories IV and
V, receive enough irrigation water to meet crop water demand.
Productive efficiency varied during 2007-2011 in all categories of
irrigated areas. The output, measured as SGVP, per unit of water
consumed decreased from 2008-2011 as a result of reduced world
rice price.
196
Although service delivery performance and quality of irrigation

services were relatively poor, the productive efficiency was higher
in Categories III-V.
Quality of irrigation service was high in Category I located
upstream and gradually decreasing in the downstream areas II,
III, IV and V.
By evaluating the irrigation performance indicators, it is possible
to determine the influence of various factors on irrigation
management and take subsequent measures to improve irrigation
performance.
Acknowledgements
The authors would like to extend sincere thanks to the Directorate
of Irrigation Water Management, Indonesia, for providing relevant
data for this study and also wish to thank the irrigation agency
staff and sixteen Water User Association (WUA) representatives
of the Telagasari Irrigation Scheme.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Biodegradation of imidacloprid in an open compost pile

eljko Herner 1*, Renata Baok
and Felicita Briki
Ministry of Agriculture, Ulica grada Vukovara 78, HR-10000 Zagreb, Croatia. 2 Faculty of Agriculture University of Zagreb,
Svetosimunska 25, HR-10000 Zagreb, Croatia. 3 Faculty of Chemical Engineering and Technology University of Zagreb,
Marulicev trg 19, HR-10000 Zagreb, Croatia. *e-mail: zeljko.herner@mps.hr
Abstract
The aim of the study was to determine in which way and at what speed a composting process affects the degradation of imidacloprid N-{1-[(6-chloro3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl} nitramide, one of the most effective and most widely used insecticide in the world, but also at the same
time one of the most accused insecticide for bee colony collapse disorder. The process of imidacloprid degradation through composting was monitored
at four experimental open piles, with an additional fifth pile composed of random green parts of plants and soil and used as a simulation of natural
and spontaneous degradation of organic matter in a field. Two of the compost piles were kept under aerobic conditions and forced aerated through
perforated pipes with a ventilator, while two were kept under anoxic conditions (the compost was wrapped in impermeable foil). Pseudomonas
aeruginosa FN culture was added in two composting piles, one under aerobic conditions and one under anoxic conditions. It has been established that
the half-life of imidacloprid occurred after 29 days in the pile inoculated with bacterial cells P. aeruginosa FN under anoxic condition, which is
considerably faster than in spontaneous degradation in the soil, which was simulated in the fifth pile (60 days). The physical and chemical properties
of compost piles did not significantly affect the dynamics of degradation rate, which was faster in the mesophilic and thermophilic phase and slower
in the maturation phase. The half-life of imidacloprid was shorter in piles with anoxic condition. The added culture, P. aeruginosa FN, has accelerated
the imidacloprid degradation rate in both piles with anoxic and aerobic conditions, faster than in the piles without the P. aeruginosa FN culture. That
is lesser influence than was expected, probably because of the abundance of microorganisms from animal manure, which created the competitive
relations with P. aeruginosa FN cultures.
Key words: Imidacloprid, composting process, dynamics of degradation, half-life time, aerobic, anoxic, Pseudomonas aeruginosa FN.
Introduction
Imidacloprid belongs to the group of pesticides called
neonicotinoids and it has been highly efficient in pests control in
agriculture. However, there are doubts about the negative effects
of imidacloprid and some other active ingredients belonging to
the group of neonicotinoids on pollinators 11. Many crops are
routinely treated with neonicotinoid insecticides as a seed
dressing or as a foliar treatment 12. Soil or seed applied plant
protection products (PPPs) aim at bringing the amount of active
substance involved to the only parts of the plant that have to be
protected 2. These compounds are systemic, migrating in the sap
to all parts of the plant and providing protection against insect
herbivores 18. Despite a reduced exposure of non-target organisms
by this way, an exposure of honey bees through residues in pollen
and/or nectar may not be excluded for substances that migrate
towards the upper plant parts 11. In modern cereal farming systems,
honey bees are readily exposed to pesticides because they rely
heavily on common blooming crops, like oilseed rape (Brassica
napus), maize (Zea mays) or sunflower (Helianthus annuus), which
are now routinely treated against insect pests 13. The most widely
used of these compounds is imidacloprid, which is routinely used
on most major crops including cereals, oilseed rape, corn, cotton,
sunflower and sugar beets. Potentially, bees could come in touch
with neonicotinoids through plant (nectar, pollen, guttation water,
propolis and plant surface such as leaves, petioles, axils, etc.) and
198
non-plant (droplets of spray and solid particles in air, water and/

or soil compartments) routes of exposure 11. Applying to the seeds,
imidacloprid protects the roots and seedling of field crops after
germination. The whole plant is protected during growth because,
as a systemic pesticide, imidacloprid moves into all parts of the
plant in various stages of growth. However, because of this
mobility, bees become exposed to imidacloprid through pollen
and nectar when they feed. Even at sub-lethal doses imidacloprid
may cause the inhibition of acetylcholine receptors in the bees,
and further have an impact on their orientation and colony collapse
disorder 17. Imidacloprid, which is used as a seed treatment in
maize and oilseeds as well as other field crop production, can
accumulate in soils with low humus content through the years 9.
In the flowering stage of subsequent crops grown in contaminated
soils, it can provide a serious threat to the bees and other beneficial
insects 4.
Composting of organic materials is the decomposition by which
microorganisms rapidly consumes organic compounds. Main
products of biological metabolism in presence of oxygen are CO2,
H2O, microbial biomass, heat and compost 10. The composting of
organic materials in agriculture is the topic of many studies, which
examine the influence of different materials 8 on composting
process and quality of compost 16. The subject of the study is the
influence of different materials 7 on composting process and
quality of compost 16. In a line with strong development of pesticide

application, these studies tend to explain pesticides degradation
mechanisms in soil 6 and the composting process. Studies have
shown that microorganisms can use pesticides and imidacloprid
as carbon source very successfully 6. Published studies on
Pseudomonas sp. 1G isolated from soil indicate that microorganisms have a significant impact on nitro group degradation
within neonicotinoids 14.
Composting agricultural crop residues that had been treated
with imidacloprid were subjected to ecological and cost-effective
way to reduce the accumulation of imidacloprid in the soil. At the
same time, by adding compost during fertilization of crops, one
increases microbial activity, which further contributes to
degradation of imidacloprid and other pesticides accumulated in
agricultural soils.
Raw material for composting: The research was carried out at
the municipality bio waste disposal ZG Holding, Zrinjevac Office,
PJ Markuevac. All fresh and dry components were taken from
biowaste disposal. The material for composting consisted of 1.53
m3 horse manure, 0.32 m3 vegetable waste, 0.84 m3 compacted wet
leaves, 4.8 m3 dry leaves and grass and 0.2 m3 soil. The total amount
of composite material was 7.69 m3. Raw materials for compost
piles were selected from the plant material and manure manner as
to ensure favourable C/N ratio (25-30:1) and acceptable humidity
compost material of 60% RH 5. This ratio is determined by the
calculator for measuring compost (Compost Mixture Calculation
Spreadsheets, Cornell Waste Management Institute). Laboratory
measurements determined a lower C/N ratio of 23.74:1 that is close
to the optimum C/N ratio (25:1) in the use of horse manure 1 .
Composting conditions: Composting was set up in five wooden
containers, 1m 0.96 m 1 m, four of them contained mixed raw
compost materials, and one contained randomly selected plant
material and soil (Fig. 1). Nutrient broth (5 L) was inoculated with
bacterial cells P. aeruginosa FN and the overnight culture was
grown on a rotary shaker at 37C and 160 rpm. Inoculum was
diluted 1:1 w/w in tap water and applied to the composting material,
for two composting piles. The first composting pile was aerated
without the addition of P. aeruginosa FN (AO). The second
composting pile was aerated with the addition of P. aeruginosa
AO
a)
APS
ANPS
d)
ANO
b)
BL
c)
e)
Figure 1. Design of composting piles: a) AO aerobic conditions

without bacterium, b) APS aerobic conditions with bacterium, c)
ANO anoxic conditions without bacterium, d) ANPS anoxic
conditions with bacterium, e) BL spontaneous degradation without
bacterium and forced aerating.
FN (APS). The third composting pile was kept in anoxic conditions

without bacterium (ANO) while in the fourth composting pile, P.
aeruginosa FN (ANPS) was added under anoxic condition. The
aeration was carried out by an air compressor (model Einhel BTAC 270/50, 1.8 kW, Landau/Isar, Germany) connected to PVC tubes
(6 cm and 3 m long), perforated in two diametrically opposite
lines, and placed at the bottom of the pile. Additionally, through
the tubes water was added to maintain optimal moisture 60% during
the composting process. Anoxic conditions were kept by means
of a waterproof and photo-impermeable foil into which compost
material was placed. The fifth compost pile (BL) was prepared for
the simulation of spontaneous degradation of imidacloprid on
agricultural land. It was composed of 0.3 m3 dry leaves and grass,
0.3 m3 compacted wet leaves, 0.1 m3 vegetable waste and 0.2 m3
soil with initial C/N ratio 1:32.7. Neither manure as a composting
process activator 3 nor bacterium were added in the fifth compost
pile. Formation of H2O is one of the products of composting
process. To collect drainage water under each compost pile glass
containers were placed to determine the loss of imidacloprid.
Dimensions of composting containers enabled the smooth running
of the thermophilic phase in composting process 5.
Imidacloprid: A plant protection product with 20% of active
substance imidacloprid, the Boxer 200 SL (ChromosAgro, Croatia)
was used. Four compost piles were prepared by dividing well
mixed composting material and spread in a thin layer on the plastic
sheet. The insecticide diluted in 250 ml of water was applied as 0.1
concentration. According to the manufacturers instructions, the
applied amount of 250 ml of diluted insecticides is enough to
cover 40 m2 of plant surface. The application of insecticide on the
composting material carried out with an applicator nozzle, which
allowed an even application. The treated material was several times
mixed, to obtain good distribution of imidacloprid in the compost.
Monitoring and sampling: Monitoring of the composting process
and sampling was planned to last 60 days. Temperature was
monitored daily (at 10, 60 and 80 cm depth from the top) using a
thermometer with probe (model TM 200, Kimo Instruments,
Montpon, France). Removable device, mode SPH, Agra, akovec,
Croatia, was used to measure pH and the presence of oxygen was
measured with oxygen meter model Greisinger GOX 100,
Regenstauf, Germany, respectively. During the sampling period,
laboratory tests were also carried out as a correction of the field
measured results. During the composting process, samples were
taken on a weekly basis. Samples (200 g, fresh weight) were taken
after mixing the composting materials. Standard probe for
pedological research of 1.20 m length with a transverse groove of
60 cm to collect samples was used. A composite sample was used
to determine the amount of imidacloprid, and the other one to
determine the changes of the pH and C/N ratio in composting
material. All collected samples were stored at -18C until laboratory
processing. All analyses were carried out for two replicate samples.
The analytical procedures: Imidacloprid standard (purity 99.4%)
molecular weight 255.7 g/ mol, supplied from the manufacturer
SAF in Germany, was used as an external standard. For extraction
process, a standard method 15 was used. Composting material (25
g) after drying at room temperature was ground in a mortar and
sieved through 200 m sieve. As extraction solvent, a mixture of
199
Statistical analysis: First-order kinetics analysis was used to

estimate the degradation rate of imidacloprid. The statistical
analysis of the results was performed using MicroMath Scientist
Model File (Scientist for Windows 3.0, MicroMath, Saint Louis,
USA).
The initial temperature in the compost material ranged between
37C (ANO) and 39C (ANPS), depending on the composting pile
(Fig. 2). Mesophilic phase (<45C) lasted for four days which was
followed by thermophilic phase. The highest temperature (62C)
in a thermophilic phase was recorded in the compost pile with
anoxic conditions (ANO) at 60 cm depth. Maximum oxygen
consumption was observed in the pile with anoxic condition and
P. aeruginosa FN (ANPS) after three days when the oxygen
content was only 3%. After the initial turbulent microbial activity
and peak thermophilic phase, the measured oxygen content ranged
between 15% (ANPS) and 20% (APS).
The initial C/N ratio in compost material was 23.74 (Fig. 3) and
fell on 19.81 (ANPS) where lowest was recorded 16.8 (APS),
respectively. Humidity of compost pile was kept by periodic
addition of water through perforated pipes for aerating or by
moistening.
The pH reaction in the starting material was 7.86 (Fig. 4). The
increase of pH was recorded after 1 week in all piles up to a
70
C/N ratio
32
30
28
26
24
22
20
18
16
% Oxygen
Temperature (C)
5
0
2
4
5
Time (weeks)
AO temperature
APS temperature
ANO temperature
ANPS temperature
BL temperature
AO oxygen
APS oxygen
ANO oxygen
ANPS oxygen
BL oxygen
Figure 2. Relation between changes of temperature and oxygen consumption in

composting piles during the monitoring period.
AO aerobic conditions without bacterium, APS aerobic conditions with bacterium, ANO anoxic
conditions without bacterium, ANPS anoxic conditions with bacterium, BL spontaneous
degradation without bacterium and forced aerating.
200
APS
ANO
ANPS
BL
APS
4
5
Time (weeks)
ANO
ANPS
BL
maximum of 9.1 (ANPS). Between the compost piles, there were

no significant differences in pH value. The initial pH value
increased in the beginning but thereafter slightly decreased
between 8 and 9.
The fifth pile (BL) of randomly selected plant material and soil
was also monitored. In this pile changes of temperature, pH and
C/N ratio were recorded, which indicated the activity of
microorganisms from soil and moist vegetables. The results
obtained show difference from those in composting piles. The
temperature increased only slightly from the start, and the pH
range of 8.2 was reached after 2 weeks. After 60 days C/N ratio
was 19.1. These results indicate that the process of degradation
of organic matter was also present in this pile, although
significantly slower than in the compost piles.
10
1
AO
10
Figure 4. Changes in pH reaction in composting piles during

eight weeks.
15
9.1
8.9
8.7
8.5
8.3
8.1
7.9
7.7
7.5
0
40
20
Figure 3. Changes of C/N ratio in the composting piles during

eight weeks.
20
30
AO
60
50
Time (weeks)
pH
acetonitrile (HPLC grade) and water in a ratio 80:20 v/v was used.
Extraction was performed on a shaker for two hours. After 10 min
when the precipitate had settled, the extract was separated by
decantation and filtered on a system for rapid filtration through
blue ribbon filter paper. The extraction was repeated once more.
The entire sample was filtered through the same system and the
residues were rinsed with a solvent. The combined filtrate was
evaporated for the most part on a rotary evaporator. Then the
sample was filtered and injected in a HPLC-MS/MS system. For
detection and quantification of imidacloprid in samples, HPLCMS/MS method was developed on Agilent 1200 HPLC, which
was connected to the mass spectrometer with triple quadrupole
Agilent 6410 (Agilent Technologies, Santa Clara, CA). All samples
were analysed in two replicates to ensure the accuracy and
repeatability.
Degradation rate of imidacloprid: The initial

concentration of imidacloprid (Fig. 5) in primary
composting material was 1.104 mg kg-1. In the aerated
compost pile (AO), at the end of eighth week records
showed 0.404 mg kg-1, in the aerated pile with the addition
of P. aeruginosa FN (APS) 0.390 mg kg-1, in the anoxic
pile 0.286 mg kg-1 and in the anoxic pile with the addition
P. aeruginosa FN it was 0.288 mg kg-1. In pile with random
stacks of various plants and soil, the final concentration
of imidacloprid was 0.695 mg kg-1, which was significantly
higher than in the other compost piles, shown in Table 1.
When comparing degradation rate within piles in aerobic
condition and in anoxic condition, it is obvious that added
bacterium slightly influenced on faster reduction of
imidacloprid as is shown in Table 1. The half-life of
imidacloprid in three compost piles was four weeks while
in the compost pile with aerobic conditions without
bacteria (AO) it was five weeks. In the pile with random
Concentration
(mg kg-1)
AO
10
20
30
40
50
Time (days)
Concentration
(mg kg-1)
z experiment data
60
70
0.5
20
40
Time (days)
z experiment data
20
40
Time (days)
1.5
z experiment data
1.5
ANPS
first-order kinetics
ANO
1.2
1
0.8
0.6
0.4
0.2
0
80
Concentration
(mg kg-1)
Concentration
(mg kg-1)
1.2
1
0.8
0.6
0.4
0.2
0
60
80
Concentration
(mg kg-1)
80
APS
1
0.5
0
20
40
Time (days)
z experiment data
1.5
60
60
80
BL
1
0.5
0
20
z experiment data
40
Time (days)
60
80
Figure 5. Estimation of experimental data with first-order kinetics model.
Table 1. The degradation rate of imidacloprid and

half-life in days.
AO
APS
ANO
ANPS
BL
kd (mg kg-1/d)
0.0208
0.0236
0.0224
0.0278
0.0141
SD
0.0045
0.0029
0.0046
0.0025
0.0033
t1/2 (days)
36
32
34
29
60
stacks (BL), concentration of imidacloprid did not reach half-life

within 60 days. Recently studies have reported degradation of
about 70% of an initial concentration of 50 ppm imidacloprid by
Pseudomonas cultures within 14 days 14.
The largest deviation of experimental data from the assumed
mathematical model emerged in pile with a randomly selected
material (BL) because of undishomogeneous of the material
collected by the probe.
Relation of physical and chemical conditions of the compost
piles and alterations in the degradation coefficient: In the
observed test containers, there was no significant difference in
the coefficient of degradation in its bearing on the physical and
chemical characteristics composting process. The degradation
rate of imidacloprid in all containers was carried out quite linearly
regardless of the change in the dynamics of physical and chemical
condition within the compost piles.
Conclusions
The degradation of imidacloprid in the composting process was
significantly faster in compost piles than in the pile with
spontaneous degradation (BL). A slightly faster half-life of
imidacloprid occurred in compost piles with anoxic conditions.

The addition of P. aeruginosa FN culture affected not significantly
but slightly the degradation, probably because of competitive
relations with microorganisms from animal manure and limited
amounts of carbon as a nutrient. The research hypothesis that
composting crop residues is an ecological and cost-effective way
to reduce accumulated imidacloprid in the agricultural soils was
confirmed. Results of this study showed significantly faster
degradation of imidacloprid in compost piles than in pile that
simulated spontaneous degradation in agricultural soil.
Composting process can be used as treatment method for crop
residues especially in a system of intensive agricultural production
where imidacloprid is used to protect plants against harmful
insects. At the same time, the compost as a final product of
composting process can be returned back in agriculture soil surface
as a fertilizer.
Acknowledgements
We wish to thank Holding Zrinjevac, Zagreb Croatia and
Association Napredak, Sarajevo, BIH for partial funding. Thanks
also to Mr Tomislav Makar for technical assistance.
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202
WFL Publisher
Helsinki, Finland
www.world-food.net
Potential methane production from manure of cattle fed diet supplemented with wet
brewery grain
Larissa Schmatz Mallmann, Maximiliane Alavarse Zambom, , Douglas Guedes Torres Batista, Leiliane Cristine
Souza, Jefferson Luiz Gonalves Silva, Silvia Renata Machado Coelho and Simone Damasceno Gomes
Research Group on Water Resources and Environmental Sanitation, Western Paran State University, Cascavel, Universitaria
Street, 2069 - CEP: 85819-110 - Cascavel, PR, Brazil. e-mail: larissasmallmann@gmail.com, mazambom@hotmail.com,
douglasgbtorres@hotmail.com, leilics@hotmail.com, j.lg11@yahoo.com.br, silvia.coelho@unioeste.br,
simone.gomes@unioeste.br
Abstract
This study aimed at evaluating the potential methane production from manure of cattle fed diets containing different levels of wet brewery grain to
replace the forage in the diet, whose levels were 0, 20, 25 and 30% dry matter. Four steers fitted with a rumen cannula were assigned in a 4 4 Latin
square design, in four 21 day-experimental periods (14 days for adaptation and 7 days for data collection). In the last week of each period, we collected
the animal manure to conduct anaerobic digestion tests for 120 days. During this period, we evaluated the removal of total and volatile solids, removal of
soluble chemical oxygen demand, daily and cumulative production of methane gas, production of volatile fatty acids (lactic, acetic, propionic and butyric),
acidity/alkalinity ratio and pH of reactors. For statistical analysis, cumulative methane production curves were fitted according to the modified Gompertz
model. Tukeys test was applied to compare mean values with significance level of 5%. The treatment with the highest level of wet brewery grain
showed higher potential methane production from manure; the inclusion of wet brewery grain did not affect the production of volatile fatty acids; the
modified Gompertz model provided a good fit to the data of methane production.
Key words: Anaerobic digestion, volatile fatty acids, modified Gompertz, biodegradability, energy, ruminants.
Introduction
Brazil has the second largest amount of cattle in the world with
over 212.8 million heads, and therefore has a crucial role in the
development of strategies to mitigate greenhouse gas emissions
in integrated production systems 1, 2.
Livestock farming is responsible for 96% of methane
emissions by the agricultural sector in Brazil. As the global
enteric CH4 is estimated at 86.109 kg year-1 and from animal
manure is in the range of 18.109 kg year-1 3, it is possible to infer
that the Brazilian cattle industry accounts for 11.3% of world
production of enteric CH4 and, on average, for 2.6% of world
production of CH4 originating from manure.
There are several techniques used to increase the efficiency
and metabolic energy of animals, one of them being the dietary
supplementation. However, the main supplemental sources are
expensive, and an alternative to reduce production costs
without compromising the balance of the diet would be the use
of agro-industrial wastes in animal feed 4-7.
Some agro-industrial wastes can be used in animal feeding but
food characteristics can be both positive and negative with
respect to the conversion of the food into animal product. The
wet brewery grain (WBG) is a by-product with high levels of
protein, neutral detergent fibre (NDF), total carbohydrates (TC)
and ether extract (EE) 5, 6, 8, 9. In this way, the use of the WBG in
nutritional supplementation for beef cattle provides a lower cost
in production because of good nutritional value of the by-product,
in addition to the correct disposal of this industrial waste.
The anaerobic digestion has proven to be an efficient method,
also called green technology, in the reduction of organic matter
in the sewage sludge, crop residues, food waste and animal

manure 10, 11. The advantages are the production of renewable
energy in form of biogas and the possibility of recycling valuable
nutrients through the biofertilizer, product obtained in the
digestion process 12.
In this way, this study aimed to evaluate the anaerobic
biodigestion process and potential methane production from
manure of cattle fed diets containing different levels of wet
brewery.
Animals and diets: Four bullocks with 400 kg average weight
fitted with a rumen cannula were assigned in a 4 4 Latin square
design with four levels of inclusion of wet brewery grain (0, 20,
25 and 30% on a dry matter basis, to replace the forage) to the
diet and four periods. Each experimental period lasted for 21
days, 14 days for adaptation and 7 days for data collection. Diet
consisted of 60% corn silage as forage and 40% concentrate
feed. Percentage and chemical composition of diets provided
to animals are listed in Table 1.
Inoculum: The inoculum used came from digester fed with cattle
manure, in the proportion of 20% of the working volume of the
reactor 13, corresponding to 0.5 litre.
Reactors: Anaerobic digestion of waste was carried out in batch
reactor at laboratory scale. The reactor was made of PVC pipe
of 10 cm diameter and 45 cm high, with a total volume of 3.0
203
Table 1. Percentage and chemical composition (% DM) of diets

provided to animals for the treatments 0, 20, 25 and
30% inclusion of wet brewery grain.
Item
0%
WBG silage
0.00
Corn silage
60.00
Concentrate fraction
40.00
Dry matter (DM)
54.87
Mineral matter (MM)
5.34
Organic matter (OM)
94.66
Crude protein (CP)
18.86
Ether extract (EE)
3.28
Neutral detergent fibre (NDF)
26.71
Acid detergent fibre (ADF)
15.77
Indigestible neutral detergent fibre (iNDF) 8.69
NDF corrected to ash and protein (NDFcp) 26.06
Lignin
2.18
Cellulose
12.61
Total carbohydrates
72.53
Non-fibre carbohydrates
24.52
Level
20% 25%
20.00 25.00
40.00 35.00
40.00 40.00
54.49 53.60
5.29 5.29
94.71 94.71
18.52 17.80
4.39 4.65
30.20 31.48
14.40 13.84
11.65 12.61
27.69 29.18
1.98 2.18
10.73 10.04
71.80 72.25
19.42 17.03
30%
30.00
30.00
40.00
53.73
5.26
94.74
18.68
5.13
33.21
13.75
12.47
30.16
2.41
9.81
70.94
14.36
litres and working volume of 2.48 litres. Twenty reactors were

made, 4 for each treatment and 1 for the inoculum, totaling 5 reactors
for each trial. The gas measurement was performed through fluid
displacement (NaOH solution) 14. The NaOH solution was used to
precipitate CO2, whose measurement was obtained corresponded
to the volume of CH4. Data obtained was transformed and
expressed in CNTP.
Reactors were kept at 35C 13, using water bath. They were
manually agitated twice daily.
The gas atmosphere was connected to a U-shaped manometer
to measure the pressure in the gasometer and in the reactor. The
volume correction was performed using equations recommended
by Motta 15 and Fernandes Jnior 16.
Conduction of the experiment: One 2.48 L reactor per treatment
was installed. Every morning and afternoon, methane production
was read. At the beginning and end of the anaerobic digestion
test, ST and SV, soluble COD were determined, and from these
parameters, we determined the removal percentage of TS, VS
and soluble COD. The anaerobic digestion lasted about 120 days,
time corresponding to stabilize the manure, i.e., until ceasing
methane production.
For each reactor of 2.48 L, six smaller reactors (400 mL)
were fitted, totaling after all collections of experimental periods,
96 minireactors. Reactors presented gas output, directly into
water layer.
The purpose of the minireactors was to monitor the larger
reactors, since these were opened only when the production of
methane ceased. The minireactors served as destructive
samples and were evaluated every twenty days, one reactor per
treatment and showed the same conditions and substrates of
reactors of greater volume. The parameters evaluated at each
sampling of minireactors were: acidity and alkalinity 17, total
and volatile solids, soluble COD and pH 18, volatile fatty acids 19,
mineral matter, organic matter, crude protein, ether extract and
neutral and acid detergent fibre 20.
Determination of volatile fatty acids (VFA): Volatile fatty acids
lactic, acetic, propionic and butyric acids were analysed by high
performance liquid chromatography (HPLC) with UV detection,
204
with the following chromatographic conditions: mobile phase

acetonitrile solution (10%) plus trifluoroacetic acid (0.025%) with
the addition of water solution (90%) plus 0.05% trifluoroacetic
acid, oven temperature of 47C, flow 0.6 mL.min-1 and wavelength of
208 nm.
The samples were prepared in a volumetric flask (5 mL) and
acidified with 200 l of 2 M sulfuric acid solution. The samples
were filtered through a membrane with 0.22 m pore size.
Statistical analysis: The responses to the replacement of corn
silage by the WBG were analysed by analysis of variance
according to the following statistical model:
Y= + Ti + Pj + Ak + eijk
(1)
where:
= general constant;
Ti = effect of treatment i (i = 0, 20, 25, 30%);
Pj = effect of period j (1, 2, 3, 4);
Ak = effect of animal k (1, 2, 3, 4);
eijk = random effect associated with each observation.
Non-linear curves were fitted to the data of cumulative methane
production for estimation of kinetic parameters of cumulative
production of methane, using the modified Gompertz model 21, 22
P(t) = P exp {-exp[
Rm
e P
( - t) + 1]}
(2)
(Eq. 2):
where:
P = potential CH4 production;
Rm = maximum production of CH4;
= time lag phase;
t = time.
A comparison of means for the variables was performed
according to the Tukeys test at 5% significance level.
Characterization of manure: Table 2 presents the average
composition of cattle manure according to WBG levels in the
diet, which were used in the test of anaerobic digestion.
In relation to the composition of manure, it can be said that the
cattle manure (Table 2) has the potential to be degraded and
produce biogas, because the levels of volatile solids accounted
for approximately 80% of the levels of total solids 23, 24. The levels
Table 2. Average composition of cattle manure
according to WBG levels in the diet,
used in the test of anaerobic digestion.
Parameters
0%
pH
6.46
TS (%)
22.02
VS (%)
17.09
Mineral Matter (%DM) 18.07
Organic Matter (%DM) 81.93
Crude Protein (%DM) 21.38
Ether extract (%DM)
3.30
NDF (%MS)
58.82
ADF (%MS)
26.57
Lignin (%MS)
2.10
WBG levels
20% 25%
6.38 6.48
22.19 23.38
17.05 19.42
19.52 18.27
80.48 81.73
19.04 19.34
3.63 3.91
56.62 58.92
29.43 25.60
2.30 2.10
30%
6.48
21.82
18.46
15.80
84.20
14.72
4.13
60.93
28.37
2.08
TS- total solids; VS- volative solids; NDF- neutral detergent fibre; ADF- acid
detergent fibre.
pH
of crude protein had a tendency to decrease with the increase of

WBV the diet. This may have occurred because the WBG can be
up to 50% of the crude protein in the form of non-degradable
protein in the rumen 6.
In Table 3, we present the initial characterisation of the
substrates (manure and inoculum) and removals of total solids
(%), volatile solids (%), chemical oxygen demand (%) and neutral
detergent fibre (NDF) and acid (ADF) of reactors in the test of
anaerobic biodigestion.
The removal of TS and VS varied from 94.67 to 95.11% and
96.57 to 97.24%, respectively. The same behaviour of total and
volatile solids can be observed in relation to soluble COD,
reaching removal of 90.78, 88.98, 89.68 and 91.00% for the
treatments 0, 20, 25 and 30, respectively. The results
characterized an efficient anaerobic system and balanced
presence of bacterial mass 25, 26.
Figure 1 shows the profile of medium pH during the test of
anaerobic digestion, according to the increase of WBG (0, 20, 25
and 30%) diet.There is a trend of increased fecal pH with the
inclusion of the WBG in the diet; however, this trend disappears
after 65 days of incubation, when the higher pH values were
observed for the manure derived from the diet without WBG. From
the values of pH, it can be stated that the inclusion of the WBG in
the diets did not cause eating disorders to animals 27. In relation to
Table 4. Production and potential methane

production from cattle manure
according to levels of inclusion of
WBG (0, 20, 25 and 30%) to the diet.
Production (L)
L.g-1 TS consumed
L.g-1 VS consumed
L.g-1 COD consumed
0
11.39
0.21
0.27
0.18
20
14.15
0.27
0.35
0.25
25
15.20
0.28
0.33
0.25
30
19.31
0.37
0.44
0.30
TS- total solids; VS- volatile solids; COD sol.- soluble chemical oxygen demand.
MM (15.8%) and higher EE content (4.13%). Higher values of

fibre and EE, which represent higher concentration of TDN (total
digestible nutrients) of the manure, tend to enhance methane
production 24, 28. The content of VS (5.24%) was higher in this
treatment (Table 3) thus providing a possible increase in methane
production. The largest reductions of fibre also correspond to the
treatment with 30% addition of WBG (Table 3).
The treatment affected (p<0.05) the cumulative production of
methane (L) (Fig. 2), which presented a high correlation with
methane production per gram of COD consumed (0.90) and
methane production per gram of VS consumed (0.92).
There was a positive effect of adding WBG on the cumulative
methane production and increased production in the third period
of evaluation (R3). This result is probably related to the different
batch of WBG which was used in animal feed.
It is possible to observe differences in digestion of fibre fraction
of barley (WBG), when compared with the food source of forage 29.
The residue is characterized as a food with high protein value of
passage, i.e. the protein that does not suffer from ruminal
Time (days)
Figure 1. Profile of medium of pH during the

test of anaerobic digestion, according to the
increase of WBG (0, 20, 25 and 30%) in the diet.
the values of VA/TA, these remained below 0.5 during the entire test
of anaerobic biodigestion, indicating the stability of the reactor 25.
Methane production from cattle manure: The production and
potential methane production are listed in Table 4.
Methane production was higher in the treatment with the
highest level of WBG (30%). This is probably due to characteristics
of the manure of this treatment (Table 2): higher concentration of
NDF (60.93%), higher content of OM (84.20%), lower values of
Cumulative CH4 production (L)
24
22
20
18
16
14
12
Period:
Period:
Period:
Period:
10
8
6
-5
10 15 20
Treatment
25
30
R1
R2
R3
R4
35
Cumulative CH4 R1 = 11.05 + 0.16x (R2= 0.93)

Figure 2. Linear regression models between levels of inclusion

of wet brewery grain (0, 20, 25 and 30%) and cumulative
methane production.
Table 3. Characterization of substrates (manure and inoculum) of the reactors at the start of the test of
anaerobic digestion for the inclusion levels of 0, 20, 25 and 30% of wet brewery grain to the diet of
cattle.
TS (%)
VS (%)
Initial COD sol.(g.L-1)
pH
VA/TA
FDN
FDA
Initial
6.46
5.03
25.26
6.47
0.13
0
Removal (%)
95.11 a
97.24 a
90.78 a
28.9 b
18.7 b
Initial
6.32
4.80
24.29
6.38
0.14
20
Removal (%)
94.67 a
96.57 a
88.98 a
31.10 ab
20.01 ab
Initial
6.14
5.06
26.61
6.48
0.09
25
Removal (%)
94.99 a
97.88 a
89.68 a
32.59 ab
20.39 ab
Initial
6.23
5.24
28.50
6.45
0.15
30
Removal (%)
94.72 a
96.79 a
91.00 a
36.04 a
24.25 a
TS- total solids; VS- volatile solids; COD sol.- soluble chemical oxygen demand; VA/TA- volatile acidity and total alkalinity; NDF- neutral detergent fibre; ADF- acid detergent
fibre. Means followed by the same letter in the row are not significantly different by Tukeys test at 5% probability.
205
degradation.
In this study, we observed an increase in the concentration of
NDF in response to an increase in additions of WBG in the diet. As
the NDF represents the fibrous fraction of diet 26 and also of faeces,
increases in their concentration may have favoured the production
of acetic acid, which is the substrate for methanogenic bacteria 30.
Manure produced from the diet with the lowest proportion of
forage (40:60) has higher contents of carbohydrates for rapid
degradation (CHOr) and lower contents of fibrous fractions, as a
result of the higher proportion of concentrate in the diet 31. It is
worth noting that the WBR had more behaviour near the food
concentrates, especially due to the lower digestibility of NDF
present in its composition 6. This explains the results obtained in
this experiment, even presenting diet with higher proportion of
forage (60:40).
Modified Gompertz model: Table 5 shows the kinetic
characteristics of the modified Gompertz model.
The coefficient of determination R2 is high, i.e., indicating good
statistical fit. With respect to the parameter P (methane production,
in litres), the treatments 0, 20 and 25 are statistically similar, but
different from the treatment 30. The maximum production (Rm)
was 0.34 L.d-1, for the treatment 30 and the lag phase in days ()
ranged from 0.78 to 2.46 d. The increased production of methane
refers to treatment with the shortest lowest lag phase (), i.e.,
treatment with the greatest increase of WBG.
Therefore, the fit of the model to the methane volume was good
and the oscillations between the expected and cumulative volumes
were associated with biological processes. The dispersion of the
points around the fitted model characterized the efficiency of the
model for the treatments 0 (Fig. 3a), 20 (Fig. 3b), 25 (Fig. 3c) and 30
(b)
Estimates production of methane
14
12
10
8
6
4
2
0
-20

T 25 (L)
(c)
20
40
60
80
100
120
Time (days)
Time (days)
(d)d)
20
18
16
14
12
10
8
6
4
2
0
-20
(L) T 20 (L)
Estimates productionTof20methane
18
16

T30 (L)

T zero (L)
20
20
40
60
Time (days)
80
100
120
P (L)
Rm (L.d-1)
(d)
R2
0
11.14 b
0.28
1.52
0.987
20
13.97 ab
0.25
1.31
0.982
25
15.47 ab
0.25
2.46
0.99
30
19.72 a
0.34
0.78
0.99
P: total methane production; Rm: maximum methane production; : lag phase. Means followed
by the same letter in the row are not significantly different by Tukeys test at 5% probability.
(Fig. 3d). In this way, the estimated gas production is good when
using the Gompertz model.
Volatile fatty acids production profile: It appears that, in the
course of the experiment, the concentrations of volatile fatty acids
showed declining trends. When we analyse the Fig. 4, we can
observe that the profile of the averages of the volatile fatty acids
originated in biodigestion of manure in function of time.
The results indicate predominance of acetic acid in every period
of evaluation and can be considered as the main precursor to the
formation of methane. The butyric acid was present in
concentrations above the propionic and lactic acid, except to acetic
acid.
Another assumption is that a low addition of WBG can stimulate
the production of these acids; however, with the increased addition
of WBG, the increase of production ceases to be expressive; the
limitation of protein in faeces can also limit the development of
micro-organisms, and this may have limited both the populations
as the productions.
The production of acetic acid was higher than that of other
20
18
16
14
12
10
8
6
4
2
0
-20
20
40
60
Time
(days)
Time (days)
80
20
40
60
80
Time (days)
100
120
20
Estimates production of methane T 30 (L)
(a)
Table 5. Kinetic parameters of the modified Gompertz

model from the production of methane from
manure in the anaerobic digestion test,
depending on the level of wet brewery grain (0,
20, 25 and 30%) in the diet.
18
16
14
12
10
8
6
4
2
0
-20
100
120
Figure 3. Estimates for fitting the modified Gompertz model, obtained from the production of
methane from manure, depending on the levels of wet brewery grain added to the diet: T zero (a),
T 20 (b), T 25 (c) and T 30 (d).
206
Concentration (mg.L-1)
Lactic
Acetic
Propionic
Butyric
Time (days)
Figure 4. Profile of the averages of the volatile fatty acids

originated in biodigestion of manure in function of time.
acids, a situation that is common in conditions of pH between 5.5

and 6. Already with pH values between 5.5 and 6.0, the production
of propionic acid is favoured 32. These values can be confirmed in
Fig. 1, in which the values of pH are along the process of anaerobic
digestion.
Conclusions
The inclusion of wet brewery grain affected the methane
production by animals.
There was effect of treatment and period for the cumulative
production of methane (L), and the treatment with greater
inclusion of wet brewery grain showed higher potential for
methane production from the manure.
The Gompertz model provided a good fit to the data of methane
production.
The wet brewery grain can be considered as an alternative food
to replace corn silage, up to 30% (DM), once it enhances
methane production from manure (energy and economic
benefits).
Acknowledgements
To CNPq National Counsel of Technological and Scientific
Development for financial support.We thank CNPQ for the
financial support during the course.
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Influence of the auxin-like activity of humic acid on bio and microbiometric

parameters of Pisum sativum L. by in vitro cultures of pea plants
Andrzej Gawlik 1*, Danuta Kulpa 2, Dorota Gobiowska 1 and Romualda Bejger
Department of Physics and Agrophysics, West Pomeranian University of Technology in Szczecin, ul. Papiea Pawa VI No3, 71459 Szczecin, Poland. 2 Department of Plant Genetic, Breeding and Biotechnology, West Pomeranian University of Technology in
Szczecin, ul. Papiea Pawa VI No3, 71-459 Szczecin, Poland. *e-mail: Andrzej.Gawlik@zut.edu.pl
1
Received 10 March 2014, accepted 20 September 2014.
Abstract
Humic acids (HA) can be found in all aquatic and land-based ecosystems. They may directly or indirectly influence the growth of plants. The aim of
the study was to determine the influence of HA extracted from peloid on the growth and morphological features of pea plants by in vitro cultures.
Sterilized pea seeds (Pisum sativum L.) of Ramdrod variety were placed on Murashige & Skoog (MS) medium with HA of the following concentration,
expressed as content of HA carbon in dm3 of medium: 0.005; 0.01 and 0.02 (gCHAdm-3). Control samples included those in which HA was not added
to MS medium. After two weeks of the experiment, morphological features were determined in young pea plants and microbiometric features
examined using the scanning electron microscope (SEM). Biometric measurements showed that HA added to the medium caused statistically
significant increase in the height of the pea plant shoot, as well as longer roots in comparison with control samples. Additionally, growth of fresh
weight of the shoot was observed, whereas in the case of the highest and the lowest concentration of HA a significant increase in fresh and dry weight
of roots. Examination using the SEM showed that humic acids had positive influence on the length of internodal shoot and root cells. At the same time,
a decrease in the number of stomas was observed. The experiment showed that HA activity is similar to auxins. No influence of HA on the green index
and dry weight of shoots were recorded.
Key words: Auxin-like activity of humic acid, biometric and microbiometric parameters of pea plants, Pisum sativum L., scanning electron microscope.
Introduction
At the moment, in both aquatic and land ecosystems, humic acid
(HA) is the most important fraction of humic substances. Despite
different origin and chemical composition, HA show a number of
common features. The core of macromolecules of HA has an
aromatic structure containing a network of aromatic rings (chiefly
derivatives of benzene) linked by various bridges and parts of
aliphatic chains. The HA structure contains numerous functional
groups, in particular COOH and OH, which contribute to its nature
of polycarboxylic organic acids. Apart from C, O and H, some of
basic elements present in the HA structure also includes nitrogen.
HA is a reservoir of nitrogen for plants. It is released gradually.
Durable forms of nitrogen in the HA structure are connected to a
large extent with aromatic chains in the form of amines or
heterocyclic structures. Due to its properties, HA plays a number
of important functions in the environment. HAs may have an
important influence on the structure and properties of soil and its
water and air properties 1. They are responsible for soil sorption
capacity as well as they may directly modify life processes in
plants 2-4.
Humic acids have the capacity of creating complexes with
various metals 5, 6. That way they may influence the concentration
of free ions in the soil solution. Ions of iron and aluminium deserve
special attention 7, 8. Moreover, humic substances show capacity
to bind heavy metals in soil and thus reducing their availability
for plants 9. The presence of sufficient quantity of humic
substances in soil and formation of stable humusenzyme
complexes is the cause of fairly continuous enzymatic activity of

soil 10.
In the early 20th century, researchers recognised influence of
humic substances on plants 11. The influence on plant cells has
been recently compared to the one exerted by auxins: indole-3
butyric acid (IBA), indole-3 acetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) 12-14. Humus substances can be both
stimulators and inhibitors for various processes in plants. Direct
influence of humic substances on plants depends on a number of
factors, such as type and dose of humic substances, manner of
application, variety and development phase of a plant 15, 16.
Examining of humic acid influence on the growth of plants is
frequently difficult due to interaction with other environmental
factors. The use of in vitro cultures enables examining the
influence of a given factor on the growth of plants in controlled
conditions better than other methods. The research aimed at
examining of the influence of various HA concentrations in soil
on the growth and development of pea plants growing in in vitro
cultures.
Humic acids: Humic acids (HA) originated from a peloid deposit
in Koobrzeg. They were extracted from peloid using Schnitzer
method 17. Samples of peloid of natural moisture content were
extracted using 0.1 moldm-3 NaOH. Extracts were acidified with
concentrated H2SO4 to pH 1.5 to obtain HA sediment. HA sediment
209
was centrifuged and purified by centrifuging and washing it with Table 1. Biometrical parameters of pea plants grown from seeds
distilled water several times. The HA gel obtained was freezeon agar base with MS medium with HA 0, 0.005, 0.01
dried in the lyophilizer. Dry HA mixture underwent elemental
and 0.02 gCHAdm-3.
analysis in the CHNS/O series II, 2400 analyser by Perkin Elmer. HA in
Fresh
Dry
Fresh
Dry
Green
Root
HA mixture used contained C (36.57 2.29%); H (38.66 1.72%); medium Height shoot
shoot
root
root
leaf
length
-3
index
N (2.18 0.42%) and O (22.59 1.70%). Atom ratios calculated (gCHAdm (cm) weight weight (cm) weight weight
medium)
(g)
(g)
(g)
(g)
(SPAD)
based on elemental analysis typical for HA mixture are as follows: Control-0
7.1a
0.639a
0.083a
7.6a
0.491a
0.054a
32a
H/C-1.06; C/N- 16.78; and O/C- 0.62.
0.005
7.9ab 0.683ab
0.089a
14.8b
0.585b
0.064b
33a
Plant material: Seeds of pea (Pisum sativum L.) Ramrod variety
were sterilized by immersing them in 70% solution of ethanol for
10 s, then in 7% solution of sodium hypochlorite for 10 min.
Sterilized seeds were placed on MS medium 18 with 0.005, 0.01 and
0.02 (gCHAdm-3) HA. Plants on an MS medium without growth
regulators were the control group. The pH of culture media was
adjusted to 5.7 using solutions of 0.1 M HCl and NaOH and
sterilized in an autoclave at 121+1C and pressure of 1 atmosphere
for 20 min. The cultures were placed in a phytotron for 14 days at
24C and relative air humidity of 70-80%. The cultures were exposed
to fluorescent light of PPFD 40 (molm-2s-1) for 16 h per day.
After two weeks of the experiment, microbiometric examination
was performed and morphological features determined, including
plant height (cm), length of roots (cm), fresh and dry weight of
shoots and roots (g) and green leaf index (SPAD). Dry weight of
plants or their parts was measured after drying at 105C to reach
constant weight.
Microscopic observations: Microbiometric measurements used
scanning electron microscope Quanta 200 by Fei. Fresh samples
of plants were taken for testing. Parts of shoots and roots were
cut longitudinally with a scalpel. Leaves sampled from the second
node from the top were placed directly on measurement tables.
After samples were placed on tables in the measurement chamber,
pumps were switched on to remove air. Measurements were made
using fresh plant material without prior irradiation and vapour
deposition.
Statistical analysis: Data were analyzed using ANOVA for a
completely randomized design. Tukey test was used to indicate
means with significant effect at P0.05. Homogenous groups
between analysed combinations were labelled with successive
letters of alphabet.
The experiment assessed influence of three concentrations of
humic acids (HA) on morphological features of two-week pea
plants. Humic acids added to MS medium at higher concentrations
resulted in the increase of pea height and fresh shoot weight
(Table 1). All concentrations of humic acids used contributed to
increased length of roots, however, statistically significant
increase in fresh and dry weight of roots appeared at the highest
and the lowest concentration of HA. No influence of HA was
recorded on the value of the green leaf index and dry weight of the
shoot. It is worth noticing that humic acids did not cause reduction
of the value of any parameters examined and HA influenced
biometrical parameters of roots rather than shoots of pea plants.
The activity of humic acids was similar to growth regulators
when added to medium in smaller quantities they had major
influence on the growth of plants. Young and Chen 19 arrived at
210
0.01
0.02
8.2b
8.7b
0.728b
0.716b
0.096a
0.095a
12.2b
14.8b
0.527ab
0.612b
0.060ab
0.063b
36a
32a
similar conclusions and explained the activity of humic substances,

similar to growth hormones, by the content of polyamines in humic
acids. Muscolo and Nardi 25 explained the same fact by the presence
of IAA in humic substances or substances capable of reacting
with markers detecting IAA 20. While examining the influence of
auxins and fine and large molecule fractions of humic substances
on pea plants, Russell concluded that the activity similar to IAA
on ion canals of plant cells is shown by fine and large molecule
fraction of humic substances 21. Research by Pinton et al. 22, 23 on
influence of HA on cucumber plants showed strong influence of
humic acids on the growth of roots. Research on influence of
humic acids on root development in plantings of strawberry by in
vitro cultures was conducted by Rzepka et al. 24. According to the
research, humic acids stimulate the root system which grow larger
than in the case HA is added to medium of 1 mgdm-3 IBA. A
reverse effect was achieved while examining the influence of SH
and auxins on the growth of roots of Lepidium sativum in aquatic
cultures. Humus substances, similarly to IAA, caused reduction
in the length and weight of roots in comparison with the control
sample. Although inhibition properties of auxins increased
proportionally to their concentration, the largest influence of humic
substances was recorded at 0.02 (gCHAdm-3). The effect was
smaller while increasing and decreasing the concentration of humic
acids 25.
Depending on its concentration, HA had various influences on
the number of stomas in plants examined (Figs. 1, 2a-b). The lowest
HA concentrations applied resulted in the reduced number of
stomas per cm2 of pea leaf blade. The larger the concentration, the
more stomas developed. It is probably caused by higher
accessibility of water due to a more developed root system in
plants examined. The reduced number of stomas is correlated with
osmotic stress or draught 26, 27. Addition of humic acids to MS
medium caused decreasing lenght of pea leaf stomas (Fig. 3).
700
Number of stomas in pea leaf per 1 cm2
600
500
400
300
200
c
b
a
100
0
MS (control)
MS + HA 0.005
MS + HA 0.01
MS + HA 0.02
Figure 1. Number of stomas per one square centimetre of pea leaf

blade grown in vitro on MS medium with HA of: 0.005; 0.01; 0.02
(gCHAdm-3) and control medium.
Figure 2. SEM picture: a) leaf blade with stomas, b) stomas length, c) shoot cross section, and d) cross section of
pea plant root exposed to HA of 0.02 (gCHAdm-3), scale: a) 400 m, b) 100 m, c) 500 m, d) 200 m.
20
Lenght of pea leaf stomas
Lenght of internodal pea cells
500
450
400
15
300
10
350
b
a
250
100
50
MS + HA 0.005
MS + HA 0.01
MS + HA 0.02
Figure 3. Length of pea leaf stomas grown in vitro on MS medium

with HA of 0.005; 0.01; 0.02 [gCHAdm-3] and control medium.
Higher concentrations of humic acids resulted in elongated

internodal cells and roots of plants growing in the case of roots
their length increased by about 30-60 % (Fig. 2c-d, 4 and 5). Similar
influence on lengthening of cells was recorded in the case of
growth regulators, especially auxins and gibberellines. Activity
of this type and influence of humic substances on enzymatic
activity in callus cells showed similarity to the activity of auxins 28.
Muscolo and Nardi 13 researched the influence of humic acids and
fulvic acids from biohumus on cells of carrot (Dacucus carota) in
the in vitro suspension culture. Different concentrations of humus
acids were tested which showed that the strongest influence can
be recorded at 0.02 (gCHAdm-3). The activity of humic substances
a
MS (control)
a
MS + HA 0.005
MS + HA 0.01
MS + HA 0.02
Figure 4. Length of internodal pea shoot cells grown in vitro on MS

medium with HA of 0.005; 0.01; 0.02 gCHAdm-3 and control medium.
Lenght of pea root cells
140
120
100
m
MS (control)
150
200
80
60
40
20
0
MS (control)
MS + HA 0.005
MS + HA 0.01
MS + HA 0.02
Figure 5. Length of pea root cells grown in vitro on MS medium

with HA of 0.005; 0.01; 0.02 (gCHAdm-3) and control medium.
211
was compared to the activity of 2,4-D and auxins: NAA; IAA.

Tests showed that humic substances acted in a similar manner to
2,4-D, and their influence was stronger on the growth of cells than
IAA and NAA. Microscopic tests enabled to establish that cells
growing in the presence of humic acids are similar in shape to
cells exposed to IAA.
Conclusions
In this experiment, humic acids present in MS medium showed
various influence on biometric and microbiometric properties of
pea plants. The influence varied depending on the HA
concentration in medium. Humic acids acted similarly to auxins
resulting in lengthening of shoots and roots, and had positive
influence on the majority of morphological properties. Elongation
of shoots and roots exposed to humic acids is closely linked with
elongation of cells.
Acknowledgements
We thank the two reviewers and the associate editor for their
comments which improved the manuscript.
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15
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Biotransformation of 2-(4-methoxybenzyl) cyclopentanone by Solanum aviculare and

Rheum palmatum plant cells
Petr Soudek 1, Zdenk Wimmer
1
and Tom Vank
1*
Laboratory of Plant Biotechnologies, Institute of Experimental Botany AS CR, v.v.i., Rozvojov 263, 165 02 - Prague 6, Czech
Republic. 2 Isotope Laboratory, Institute of Experimental Botany AS CR, v.v.i., Vdesk 1083, 142 20 - Prague 4,
Czech Republic. *e-mail: soudek@ueb.cas.cz
Abstract
The ability of plant cells cultivated in vitro synthesized and biotransformed substances of natural and synthetic origin are known for a long time. The
biotransformation of 2-(4-methoxybenzyl)-1-cycloalkanone skeleton was studied. Its knowledge is important for two reasons, partly for ease of
enzymatic synthesis juvenogens and to better understand the mechanism of action of the dicyclic juvenoids. Results obtained show that cultured
rhubarb (Rheum palmatum L.) and kangaroo apple (Solanum aviculare Forst.) plant cells were able to efficiently convert 2-(4-methoxybenzyl)-1cyclo-pentanones to its glucosides via corresponding alcohols.
Key words: Solanum aviculare, Rheum palmatum, plant cell culture, 2-(4-methoxybenzyl)-cyclopentanone, 2-(4-methoxybenzyl)cyclopentanol,
glucoside, enantiomeric purity.
Introduction
Plant cells cultivated in vitro can be used for production and
biotransformation of substances of natural and synthetic origin,
as well. Recently, we described application of this methodology
for biotransformation of plant secondary metabolites, limonene 1
and citronellal 2. To present the possibility of biotransformation
of synthetic biologically active compounds by plant cells, we
studied transformation of racemic 2-(4-methoxybenzyl)
cyclopentanone (1) towards chiral alcohols 2a and 3a. Racemic
2-(4-methoxybenzyl)cyclopentanone (1) represents an example
of a substrate unnatural for plant cells (xenobiotics). The compound
1 can serve as a model system of the dicyclic juvenile hormone
analog (JHA) skeleton. The JHAs have been investigated for many
years and reviewed recently 3- 6. In the plant tissue both enzymatic
fission of the incorporated juvenogen glucoside and eventually
the synthesis of a novel one could occur.
The juvenogens term used for compounds capable induced an
active component (juvenoids) in response to a biotic or abiotic
stress factors. Mechanism of enzymatic fission within the insect
was studied using both esters and glucosides juvenogens. They
were also tested the possibility of their practical use as a new
systemic pesticide against the herbivore insects. In the plant
tissues both pathways of enzymatic fission of incorporated
juvenogen glucoside are found 7 , 2-(4-methoxybenzyl)
cycloalkanone derivatives have been investigated intensively 3.
Presence of an alcoholic functional group in the JHAs molecule
allows their further modification in order to afford biologically
inactive complex substances (juvenogens). The juvenogens can
subsequently liberate the biologically active JHA within the insect
organism under the enzymatic action 4, 5, 8-10. The investigation of
the biotransformation of 2-(4-alkoxybenzyl)cyclopentanone
skeleton is important for studying the chemical properties of this
system under enzymatic action, the result of which could be used

for both easy enzymatic synthesis of the juvenogens and better
understanding of the mode of action of the JHAs 10, 11. The
prevailing expected reactions of the Solanum aviculare and Rheum
palmatum suspension cultures beeing observed are represented
by the enzymatic reduction of the ketone 1 to the isomeric chiral
alcohols 2a and 3a and by their enzymatic glucosylation yielding
the 2-(4-methoxybenzyl)cyclopentyl--D-glucopyranosides (4a
and 5a; Fig. 1).
Cell cultures and their cultivation: Suspension culture of
Solanum aviculare Forst 12, was obtained from callus (strain
KK1N) derived from the leaf of the plant cultivated aseptically
from seed supplied by the Royal Botanical Gardens in Kew (UK).
This culture was subcultivated in 5-days intervals in the nutrient
medium according to Murashige and Skoog 13 in the modification
of Linsmayer and Skoog, containing 10 -6 M 2,4-dichloro
phenoxyacetic acid and 10-6 M kinetin in dark on a roller (5 min-1)
at 25C.
The suspension culture of Rheum palmatum was obtained from
callus derived from the surface sterilized leaf of intact plant. The
culture was cultivated in dark on a rotary shaker (90 min-1) at 25C,
using Murashige and Skoogs medium supplemented with
naphtaleneacetic acid (10 mg l-1) and caseine hydrolyzate (2 g l-1).
The compound: The synthesis of 2-(4-methoxybenzyl)-1-cyclo
pentanone was carried out according Wimmer and Romauk 14 , 2(4-methoxybenzyl)-1-cyclopentanone ethylene acetal was
obtained by acetalisation of the starting ketone. A mixture of
isomeric cis- and trans-2-(4-methoxybenzyl)-1-cyclopentanol was
213
Figure 1. Reaction course of biotransformation of 2-(4-methoxybenzyl)1-cyclopentanone using plant cells (the starting material was racemic and
the formulae show the relative, not the absolute, configurations).
2-(4-methoxybenzyl)-1-cyclopentanone (1), cis - alcohol (2), trans - alcohol (3), cis - b - D
- glucoside (4), trans - b - D - glucoside (5).
obtained by reduction of ketone by means of lithium aluminium

hydride in ether.
mm 25 mm) packed with the SiC-18 (30 m). The column was
washed with water (100 ml), 10% methanol/water mixture (100 ml)
and the sorbed products were eluted with methanol (50 ml). The
compounds present in the reaction mixtures were separated by a
preparative HPLC, and their yields are given in Table 1. The purity
of the products was checked using analytical HPLC, and their
structure was proved by their 1H NMR spectra. 1H NMR (CDCl3):
2a: 1.44-1.88 (m, 6H), 1.97 (m, 1H), 2.63 (dd, J = 7.8, 13.8 Hz, 1H),
2.75 (dd, J = 7.8, 13.8 Hz, 1H), 3.78 (s, 3H), 4.08 (dt, J = 1.5, 4.4, 4.4
Hz, 1H), 6.96 (m, 2H), 7.14 (m, 2H). 3a: 1.20-1.88 (m, 6H), 1.99 (m,
1H), 2.49 (dd, J = 8.3, 13.7 Hz, 1H), 2.69 (dd, J = 6.8, 13.7 Hz, 1H),
3.78 (s, 3H), 3.90 (dt, J = 5.6, 5.6, 6.8 Hz, 1H), 6.96 (m, 2H), 7.11 (m,
2H). 4a: 1.45-1.90 (m, 6H), 1.96 (m, 1H), 2.64 (dd, J = 7.6, 13.7 Hz,
1H), 2.80 (dd, J = 7.5, 13.7 Hz, 1H), 3.11 (dt, J = 4.0, 4.0, 8.8 Hz, 1H),
3.22 (t, J = 8.4 Hz, 1H), 3.40 (t, J = 8.8 Hz, 1H), 3.46 (t, J = 9.1 Hz, 1H),
3.70 (dd, J = 4.2, 12.0 Hz, 1H), 3.78 (s, 3H), 3.85 (dd, J = 3.7, 12.0 Hz,
1H), 3.93 (d, J = 8.1 Hz, 1H), 4.10 (dt, J = 1.7, 4.6, 4.6 Hz, 1H), 6.96 (m,
2H), 7.13 (m, 2H). 5a: 1.18 - 1.92 (m, 6H), 2.20 (m, 1H), 2.50 (dd, J =
8.8, 13.8 Hz, 1H), 2.62 (dd, J = 6.6, 13.8 Hz, 1H), 3.06 (dt, J = 4.1, 4.1,
8.8 Hz, 1H), 3.19 (t, J = 8.3 Hz, 1H), 3.41 (t, J = 8.9 Hz, 1H), 3.50 (t, J
= 9.3 Hz, 1H), 3.73 (dd, J = 4.3, 12.0 Hz), 3.79 (s, 3H), 3.82 (dd, J = 3.9,
12.0 Hz, 1H), 3.87 (dt, J = 4.4, 4.4, 6.4 Hz, 1H), 3.95 (d, J = 7.8 Hz, 1H),
6.84 (m, 2H), 7.11 (m, 2H).
Table 1. Compounds isolated after bio transformation of
2-(4-methoxybenzyl) cyclopentanone (1) by plant cell
cultures.
Biotransformation of 2-(4-methoxybenzyl)cyclopentanone (1):

A 15 days old cell culture was filtered off and the cells (1 g dry
weight) were transferred to the nutrient medium (100 ml)
supplemented with a solution of 2-(4-methoxybenzyl)-cyclo
pentanone (1; 20 mg) in 70% ethanol/water mixture (1 ml). The
flasks were agitated in dark on a rotary shaker (90 min-1) at 25C.
The conversion rate was measured using HPLC.
Analytical methods: HPLC analysis was carried out on an
apparatus consisting of the Consta-Metric I pump (Thermoseparation Products, USA), a home-made gradient master and a
M990 PDA-detector (Waters, USA). The stainless steel column
(250
4 mm) was packed with 7 m Sepharon Si-Phenyl (Watrex,
Czech Republic). The linear gradient elution profile started with
40% of A (MeOH/H2O/MeCN = 20:67:13) and terminated with 80%
B (MeOH/H2O/MeCN = 20:27:53) within 25 min, including a 5 min
equilibration period. The flow rate was 1 ml min-1, and the
chromatograms were plotted at 268 nm wavelength, the UV
absorption maximum of 2-(4-methoxybenzyl)cyclopentanone (1).
The same conditions were used for the isolation of substances on
a preparative column (250
8 mm) filled with the same stationary
phase. The 1H and 19F NMR spectra were recorded on a Varian
UNITY 500 spectrometer (in a FT mode) in deuteriochloroform,
using either tetramethylsilane as internal reference (1H NMR
spectra, - 0.0) or hexafluorobenzene as an external reference (19F
NMR spectra, - 162.9).
Isolation and identification of the products: After the
biotransformation, the cells were filtered and homogenized in
methanol with mortar and pestle. After additional 24 h the solution
was filtered and the methanolic extract was evaporated under
vacuum. The filtrate was applied on a reverse phase column (100
214
Compound
1
2a
3a
4a
5a
Plant Cells
Solanum aviculare
Rheum palmatum
Yield (%) e.e. (%) Yield (%) e.e. (%)
10
11
25
48
10
39
35
52
24
44
5
10
6
7
16
10
Diastereoisomeric 3,3,3-trifluoro-2-methoxy-2-phenylpropanoic
acid esters 6a - 7d of the alcohols 2a - 3b: A general procedure
used for the synthesis of the 3,3,3-trifluoro-2-methoxy-2phenylpropanoic acid esters on a milligram scale starting from the
(S)-(+)- or (R)-(-)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl
chloride was already described in details 15-18. Esters 6a - 7d were
obtained in quantitative yields. Selected 1H and 19F NMR data for
esters 6a - 7d, which are important for assignment of the absolute
configuration of the parent major enantiomers 2a and 3a of the
alcohols, as well as for the minor enantiomers 2b and 3b are given
in Table 2. The enantiomeric purity of the alcohols 2a and 3a is
presented in Table 1.
The substrate 1 was fed as racemate. The reaction course was
checked using an HPLC analysis employing UV detection at 268
Table 2. Selected 1H and 19F NMR NMR data of the MTPA (3,3,3trifluoro-2-methoxy-2-phenylpropanoic acid) esters 6a 7d.
CH2Ar
CH2Ar
CF3
A. C. a
a
6a
6b
6c
6d
7a
7b
7c
7d
2.51
2.41
2.41
2.51
2.49
2.42
2.42
2.49
2.72
2.62
2.62
2.71
2.74
2.71
2.71
2.75
-67.49 -67.32 -67.31 -67.48 -67.85 -67.93 -67.94 -67.84
S,S,S R,R,S S,S,R R,R,R S,R,S R,S,S S,R,R R,S,R
A. C. = Absolute configuration given for chiral centers C(1), C(2), C(1).
analysed on the basis of their 1H and 19F NMR spectra (cf. Table 2)
and by an HPLC analysis. Using Rheum palmatum plant cells, the
2-(4-methoxybenzyl)cyclopentyl--D-glucopyranosides (4a and
5a) were found as additional products of the biotransformation
process.
The above results show that the absolute configuration of 2a
was determined as (1S, 2S), and that of 3a was determined as (1S,
2R). In addition, the Solanum aviculare and Rheum palmatum
cells transformed the substrate 1 to several main products, either
the isomeric chiral alcohols 2a and 3a or the 2-(4-methoxybenzyl)
cyclopentyl-b-D-glucopyranosides 4a and 5a. However, the
disadvantage of both these plant cell cultures is the fact that
neither of them was able to perform biotransformation process in
a way affording either of the products with more acceptable (> 90
- 95% ee) enantiomeric or diastereomeric purity. In the case of
Solanum aviculare Forst cells we found both alcohols to be the
main products in the yield of 25% cis- and 35% trans-alcohol
(Table 1). These alcohols were isolated by HPLC and their
configuration was determined using MTPA esters 18, 23, 24. Using
Rheum palmatum L. plant cells, we found both glycosides to be
the main products (Table 1, Fig. 2). This is a good example of
different activity of enzyme system in the plant cells.
25
80
20
60
15
40
10
20
Percentage of initial concentration

(Compound 2, 3, 4, 5)
100
Percentage of initial concentration
(Compound 1)
nm wavelength. Using a diode array detector, four peaks exhibiting

the same UV spectra as the starting compound 1 were found.
These peaks represented products of biotransformation of 2-(4methoxybenzyl)cyclopentanone with the unchanged skeleton, and
were subjected to a detailed analysis. The peaks corresponding
to the compounds 2a and 3a were identified by comparison of
their UV spectra and retention times with those of the reference
compounds prepared by Zarevcka 19. Their structures and
absolute configurations of the major enantiomers 2a and 3a were
checked or assigned by the NMR measurements (Tables 1 and 2).
We have shown previously 20 that the large substituent (4methoxybenzyl) always occurs as an equatorial substituent of the
cyclohexane cycle, while the small hydroxyl functionality can occur
as the axial substituent at the cis-isomer (2). Similar results were
achieved recently for cyclopentanol and cis- and trans-1,2cyclopentanediol by Abraham 21. The presence of a bulky
substituent in the axial or pseudoaxial arrangement of the six- or
five-membered ring had been found to add more energy to the
system. Therefore, the presence of this bulky substituent at such
disposals is always less favored 22. The assignment of the relative
configuration at C(1) and C(2) has to be found more complicated
due to conformational flexibility of the five-membered ring in
comparison with the six-membered one. It is evident that due to
substantially smaller difference in energies the 2-substituted
cyclopentanols do not appear to exist as single discrete
conformers. Instead, they undergo pseudorotation through more
potential minima separated by low energy barriers. Nevertheless,
the differences in chemical shifts and sum of coupling constants
of multiplets which belong to CH-O protons allowed us to
distinguish the relative configuration at the C(1)/C(2) centres. The
detailed description of our approach to assignment of relative
configuration was published in one of our previous papers 19. The
absolute configuration of major products 2a and 3a was assigned
based on characteristic differences in both 1H and 19F NMR
chemical shifts of esters starting alcohols with (S)-(+)- or (R)-(-)3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acid (MTPA). This
procedure was explained several times in literature 22 and the
principle arguments for absolute configuration assignment is
based on the steric proximity of phenyl ring resulting in different
shielding of protons in a-positions to carbon bearing the esterified
hydroxyl group. The theoretical background of changes in 19F
NMR spectra is than based on different shielding of CF3 moiety
by presence of the carbonyl group which is deviated from the
CF3-C-CO-CH plane by coexistence of a bulky group and the
phenyl moiety on the same side of this plane.
The 2-(4-methoxybenzyl)cyclopentyl--D-glucopyranosides
(4a and 5a) were identified by their 1H NMR spectra, after their
isolation using a preparative HPLC. The final results are
summarized in Table 1. The enantiomeric purity of the alcoholic
(aglycone) part of their molecule was assigned after liberation of
chiral isomers of 2-(4-methoxybenzyl)cyclopentanol from their
molecules by means of 3,3,3-trifluoro-2-methoxy-2-phenyl
propanoic acid esters 6a - 7d, and the results are given in Table 1.
When Solanum aviculare cells were employed, both isomeric
alcohols 2a and 3a were obtained in the yields of 25% and 35%,
respectively. The alcohols 2a and 3a were isolated using an HPLC
technique and their absolute configurations were assigned via
their diastereoisomeric 3,3,3-trifluoro-2-methoxy-2-phenyl
propanoic acid esters 15, 16, 18, 19. The diastereoisomeric esters were
0
0
6
Time (Days)
10
12
Figure 2. Time - course of biotransformation of of 2-(4-methoxybenzyl)1-cyclopentanone by Rheum palmatum L. suspension culture.

= starting compound (1), * = cis - alcohol (2),
glucoside (4), U = trans - b - D - glucoside (5).
= trans - alcohol (3),
= cis - b - D -
Conclusions
The transformation of 2-(4-methoxybenzyl)-l-cyclopentanone by
Solanum aviculare Forst and Rheum palmatum L. cells was
described. We obtained racemic mixture of alcohols. The plant
cells transformed the starting compounds to the different main
product (alcohols and/or glycosides) and with different yields.
This is a good example of different activity of enzyme system in
the plant cells.
Acknowledgements
This work was supported by project Ministry of Education, Youth
and Sports (KONTAKT LH 12162).
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cyclohexyl b-D-glucopyranoside enantiomers. Collection of
Czechoslovak Chemical Communications 71(10):1470-1483.
24
aman, D., Wimmerov, M. and Wimmer, Z. 2006. Synthesis and
structure assignment of 2-(4-methoxybenzyl)cyclohexyl b-Dgalactopyranoside stereoisomers. Collection of Czechoslovak Chemical
Communications 71(8):1186-1198.
21
WFL Publisher
Helsinki, Finland
www.world-food.net
Impact of nitrogen fertilisation and irrigation on water utilization efficiency, N accumulation,

growth and yields of Zea mays L.
Waleed Hassan Abou El-Hassan 1, Emad Maher Hafez 2*, Alaa A. Ahmed Ghareib 1, Mohamed Ragab Freeg
and Mahmoud Fathy Seleiman 3, 4*
Water Management Research Institute, National Water Research Center, P. O. Box 13621/5, Delta Barrage, Qalubia, Egypt.
2
Agronomy Department, Faculty of Agriculture, Kafr El-Sheikh University, P. O. BOX 33516, Kafr El-Sheikh, Egypt.
3
Department of Agricultural Sciences, P. O. Box 27, FIN-00014 University of Helsinki, Finland. 4 Department of Crop Sciences, Faculty of
Agriculture, Minufiya University, 32511 Shibin El-kom, Egypt. *e-mail: mahmoud.seleiman@helsinki.fi, emadhafez2014@gmail.com
1
Abstract
Water and N are considered the most limiting factors for the growth and yield during the grain filling stage, particularly in the arid, semi-arid zones.
The aim of the study was to investigate the effect of N applications (0, 140, 280 and 420 kg ha-1) and water regimes (I1= 100%, I2= 80% and I3= 60%
of field capacity) on nitrogen use efficiency (NUE), water utilization efficiency (WUtE) and related parameters and grain yield and its components
of Zea mays L. (cv maize hybrid Giza 10). The highest N application resulted in a significant increase in grain yield and its components, accumulated
N in grains and shoots. However, it significantly decreased apparent N fertiliser recovery (ANR) and N utilisation efficiency when all water regimes
were applied. N application of 280 kg ha-1 was the optimal in our study for such environmental conditions. Yield and its components were increased
with increasing soil field capacity from 60 to 100%. There was a significant effect of the interaction between irrigation and N treatments on N
efficiencies, growth and consequently on yields. The results showed that the yield of maize can be increased through the reasonable N application
(i.e., declining the gap between the recommended N levels and the estimated levels) and suitable irrigation regime (I2). Also, the optimal management
of N practices is highly recommended to avoid the loss of the N when there is a deficiency or excessiveness in water supplied to the soil.
Key words: Field capacity, grain yield, maize, N accumulation, N-application, NUE, WUtE.
Introduction
Globally, the cultivated area of maize (Zea mays L.) is about 177.37
M ha and the total production is about 872.06 Tg, while the
cultivated area of maize in Egypt is about 1.04 M ha and the local
production is about 8.09 Tg 1. Maize is the third most important
cereal crop after wheat and rice in the world 2 and also in Egypt. It
has an exceptional importance because the Egyptian national
production is not sufficient. This is imputable to the growth in the
gap between the internal output of maize and the demands 3. Thus,
an important aim of the Egyptian government is consequently to
reduce the dependence on imported maize by enhancing grain
yield and production. Maize is usually used for a human food and
livestock feed, as well as it can be used in the production of
alcohol and medical materials 2-6. Recently, it has been used as a
biomass for bioenergy purposes 7, 8.
Irrigation is considered one of the most important factors that
can influence the yield and the quality of the crop 6, 9, 10. The water
deficiency is considered a serious issue that can limit maize growth
due to its effect on the most important process, such as
physiological and morphological processes 11. Water management
and N-fertiliser application are considered the most significant
elements to increase crop productivity 11. For example, about 30%
of the gap between potential and actual maize grain yields in the
farms of developing countries is attributable to the drought and N
deficiency. Many studies have been conducted to examine the
only effects of irrigation or N applications on the grain yield and
its parts as well as the N-related parameters of maize. Nevertheless,
few investigations have been made out along the interaction
effects of both of such these factors, in particular with maize.

Globally, drought stress is considered the main key of the
challenge for the stability of the field crop yield. The water and N
availability during the grain filling stage of the crop in temperate
regions consider the most environmental limiting issues. In such
areas, the primary region of N, which demands for the grains is
provided via vegetative organs. Roughly 70% to 80% of needed
N for grain filling in cereals is acquired from vegetative organs
before flowering stage 12. Thus, this process is called
demobilisation. The productivity of the dry matter was increased
when N uptake was increased. However, grain yield was declined
due to the relative water deficit during the growing season 13. In
dried zones, the N translocation is a critical and important key to
yield production of grains. Only, the high temperature following
flowering stage could cause drought stress, which could limit the
N translocation among plant parts 14. This indicates that grain
yield as well as protein content are dependent on the N assimilation
and translocation from vegetative parts before flowering into the
reproductive parts 15.
The N absorption, for instance by cereals, plays a main role in
plant growth 5. As a result of this, N is considered a strong tool for
high crop yield 5, 16. The worldwide output of chemical fertilisers
has steadily raises from 33 Tg in 1961 to 180 Tg in 2007 1. N was
accounted for about 58%, while P was accounted for 24% and K
18% of the total chemical fertiliser production 1. N is an important
component and the key element of proteins, nucleic acids,
secondary compounds, chlorophyll and cell walls of the plants 17.
217
Commonly, the period of vegetative growth is reduced and plants

mature earlier when there is a deficiency in the supplied or soil N 18.
However, the extra application of synthetic fertilisers on cropland
has increased the concern regarding the sustainability in agricultural
production and environmental security, since a part of it can be
leached to the groundwater 19. Thus, the excessive application of
such inorganic form of fertilisers should be controlled 19.
N use efficiency (NUE), which is considered an important factor
in the management of N applications in crop productivity, is
expressed as the ratio between the grain yield and the total N
accumulation 5. In cereals, the NUE should be improved through
the optimal management for the N applications as well as through
the different methods of plant breeding to increase the crop yield 20.
Since, unsuitable irrigation and N applications are the most
significant factors that can limit maize productivity. The estimation
of the suitable N applications is a vital issue for the increase of N
uptake efficiency 21.
The objectives of the current investigation were to examine the
effects of different N applications and water regimes on water
utilization efficiency, NUE and yield and its components of maize
crop. Specific attention was paid to the harvest index (NHI) and
its remobilisation as well as the uptake and usage efficiency of N
in maize under such these environmental conditions.
Plant material and experimental design: Two field experiments
were conducted in 2012 and 2013 at El Karada located at 4 km
south of Kafr El-Sheikh, Water Requirements Research Station,
Kafr El-Sheikh Governorate, North Delta, Water Management
Research Institute, National Water Research Centre, Egypt, to
investigate the effect of different N applications and water regimes
on NUE as well as the growth and yield of maize [Zea mays L.,
hybrid single cross (Giza 10)]. The experiments were conducted in
split-plot design with four replicates. Irrigation treatments (i.e.,
60%, 80% and 100% of field capacity) were contributed in the
main plot, while N applications (i.e., 0, 140, 280 and 420 kg N ha-1)
were put in sub-plots. Sub-plot size was 10.5 m2 consisting of 5
rows (3.0 m 0.7 m). Seeding rate was 35 kg ha-1 and seeds were
sown on 25 cm distance. Seeds of maize were sown on May 18th
during 2012 and in June 2th during 2013.
Maize plants were thinned to one plant per hill prior to the first
irrigation. The preceding crop was white clover (Trifolium
alexandrinum L.) in both of the growing seasons. N applications
were applied in the form of urea (46.5% N) at sowing date and at
reproductive stage. The first irrigation was added directly after
sowing of the seeds. About 0.2 g of each soil sample was used to
analyse N content by Kjeldahl method 22. Phosphorus (P) fertiliser
(70 kg P2O5 ha-1) was added during tillage practice as supplementary
for the crops based on the soil analysis and also the crop
requirements from P. All recommended agricultural practices were
done during both of the growing seasons. The soil was clayey in
texture during both of the growing seasons. The chemical
properties of the soil are presented in Table 1. The electrical
conductivity (EC) of soil (0-60 cm layer) was 2.03 dS m-1 and soil
pH was 8.1. Field capacity, permanent wilting point, bulk density
and soil moisture content were measured in the soil from 15 to 60
cm depth according to Klute 23 (Table 2).
Table 1. Chemical properties of soil used in growing seasons

2012 and 2013.
Zn
S
Fe
Cu
P
K
N
(mg L-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1)
2012 27.0
14.1
255.0
1.2
12.2
13.1
6.9
2013 26.1
12.2
230.4
1.1
10.5
11.5
6.2
Year
Table 2. Soil moisture constants for the experimental site.

Soil depth Field capacity Wilting point Bulk density Available soil water
(%)
(cm)
(%)
(%)
(g cm-3)
0-15
24.55
1.11
45.17
20.62
15-30
23.92
1.17
44.01
20.09
30-45
21.71
1.25
39.96
18.04
45-60
21.69
1.34
39.91
18.22
Average
42.26
22.97
1.22
19.29
measured and recorded using Parshall flume (20 cm90 cm). After
measuring the water discharge and the required time, the amount
of water applied per unit area was calculated. The water utilization
efficiency (WUtE) (kg m-3) was estimated as the weight of
marketable crops produced (kg) per unit volume of applied water
(m3).
Plants were harvested at 120 days after sowing (DAS) in both
of the growing seasons. Ten plants at harvest, were randomly
chosen from the fourth inner rows for measuring the yield
components, i.e. ear length (cm), number of rows per ear, number
of grains per row and 100-grain weight (g) as well as the plant
height (cm). From the central area of each plot 1 m2 was harvested
to measure the shoots (i.e., whole biomass above soil except grains)
and grain yield to get the shoot and grain yield per ha. Grain yield
of maize was then adjusted to 15.5% moisture content.
To determine N in shoots and grains, ten maize plants were
chosen randomly and harvested at the soil surface and shoots
and grains were separated. Different parts were dried in oven for
72 h at 70C and ground in a mill to produce a fine powder, which
is needed for the N analysis. About 0.2 g of each sample was used
to analyse N content using the standard procedure of Kjeldahl
method. Then, N harvest index (NHI) at maturity was calculated 24
and also N accumulation (kg N ha-1) in the shoots or grains was
calculated 8, 25 as follows:
N harvest index =
Grain N accumulation (kg ha-1)

Total N accumulation (kg ha-1)
(1)
where, the total N accumulation includes all N that accumulated in

leaves, stem, shank, cobs and husk organs in addition to the grain
N.
Shoots N accumulation =
(kg ha -1)
Shoots N content
Shoots DM
(g kg-1)
(kg ha-1)
1000
Grains DM
Grains N content
(kg ha-1)
(g kg-1)
Grains N accumulation =
1000
(kg ha-1)
(2)
(3)
N use efficiency (NUE) and apparent N fertiliser recovery (ANR)

was calculated as it is described by Azizian and Sepaskhah 5. In
addition, N uptake efficiency and N utilisation efficiency were
calculated. N utilisation efficiency was calculated as described by
Haegele 26.
NUE (kg kg-1) =
GYt - GYc (kg ha-1)

NFt - NFc (kg ha-1)
(4)
Measurements and analysis: Amounts of water applied (cm) were

218
where GYt and GYc express the grain yield at different N treatments
and control, respectively. While NFt and NFc express the N
applications for different N treatments and control, respectively.
Total N accumulationt - Total N accumulationc (kg ha-1)
ANR
-1 =
(kg kgN )
NFt - NFc (kg ha-1)
(5)
where total N accumulation either for different treatments or for

the control expresses the total N accumulation in both of grain
and stover.
NUtE (kg kg-1 planN) =
GYt - GYc (kg ha-1)

N uptaket - N uptakec (kg ha-1)
(6)
where GYt and GYc express the grain yield at different N treatments
and control, respectively. While N uptaket and N uptakec express
the total N accumulation in whole plant biomass above ground
(grains and stover) for different N treatments and control,
respectively.
Statistical analysis: Data obtained from the current investigation
was subjected to analysis of variance (ANOVA) using PASW
statistics 20.0 (IBM Inc., Chicago, IL, USA). Different means were
compared using Duncans multiple range test, when the ANOVA
showed significant differences (P<0.05).
The total amounts of irrigation water delivered to different
treatments were measured and statistically analyzed. In general,
the amounts of water applied and water utilization efficiency in
different treatments increased with the increase in N application
for maize crop. In addition, with decreasing soil moisture content,
both amount of applied water as well as the WUtE were decreased.
The highest value recorded for the WUtE, when N 420 kg ha-1 and
80% of field capacity were applied. In general, the highest WUtE
was obtained with 80% of field capacity (Fig. 1). The management
of the irrigation process has an important role not only for the
increasing production of plant species via the better availability
of the water resources, but also for supplying water with reducing
the potential risk for water-borne diseases 27. Generally, the WUtE
is more associated to the grain yield, and contrariwise in the
relationship with the quantity of water applied to plant species 28.
N fertiliser application significantly increased grain yield and
its components, but the N effect depended on the availability of
water for maize (Tables 3 and 4). There was an improvement in the
plant growth, when N 280 kg ha-1 and 80% of field capacity were
applied. This might be due to the well utilisation of the supplied N
in the metabolism and the meristemic activity, which consequently
improve growth characters and yield components. The marked
increase in growth characteristics and yield components
contributed to the significant increase in maize grain yield 29.
There was not a significant difference in plant height and number
of grains per row when maize was fertilised either with N 280 or
420 kg ha-1 under all of the studied water regimes (Table 3). However,
the lowest field capacity (I3) and the lowest N application (zero N)
resulted in the lowest plant height, ear weight and number of
grains per row during both of growing seasons (Table 3). This
reduction might be caused by the low field capacity of the soil.
The data of the current study showed also that some of yield
components (i.e., ear weight and number of grains per row) was
significantly affected by the interaction between both of the
studied factors. These results are in agreement with Iqbal et al. 30,
who reported the significant interaction effect between the
fertilisation and irrigation treatments on the plant growth. Uptake
of N during silking influences the number of grains 37. This might
be due to the high demand of embryos for the N following the
fertilisation 40. Therefore, the number of grains is very sensitive to
the N deficiency compared with grain weight.
On the contrary, 1000-grain weight was decreased when N
application level was increased, regardless of the different
treatments of water regime (Table 3). The 1000-grain weight of
plants grown with 60% of field capacity was noticeably lower
than in those grown with either 80% or 100% of field capacity
(Table 3). This was shown when this parameter was reduced due
to decrease in field capacity by 9% in 2012 and by 3% in 2013.
Generally, the impact of soil moisture stress, in the case of
increasing or reducing soil moisture, on the plant growth could be
associated with unbalanced water-air relations in the soil.
Consequently, this can cause a reduction in the photosynthesis
activities as well as can affect the balance in the relations between
plant hormones and different biological processes in the different
organs of the plant 31. These undesirable conditions in the treated
soils are certainly critical, since they can affect the growth and
accumulation of dry matter in maize.
In the current study, there was an interaction effect between
the N applications and the capacity on the dry weight productivity
(i.e., shoots and grain yield) of the maize crop. The shoot and
grain yields were significantly increased when both of N
application and field capacity were increased (Table 4). There was
no significant difference between the effect of highest two N
application under the different field capacity treatments, in
Figure 1. Amount of water applied (cm) and water utilization

efficiency (kg m-3) in maize grown with different soil moisture contents
(SMC, I1, I2, I3) and N applications (0, 140, 208, 420 kg N ha-1).
219
particular on the dry weight of maize shoots. These results are

similar to those of Arun Kumar et al. 32, who reported that the
increase in yield of maize is generally linked to the level of the N
applied to crops. The optimum combination of N fertilisation and
field capacity to the crop, could result in the highest maize yield.
The results of the current investigation indicate obviously the
important role of N in the plant growth as well as its role in the
increase of the total shoot and grain yields. The results also prove
that N is considered the key element of the plant cell division and
elongation in addition to its role for the root growth 33.
The N content in grains and shoots was significantly increased
when the N application was increased in both of growing seasons
(Table 4). The N applications of 140, 280 and 420 kg ha-1 resulted
in an increase in the N content of grains by about 35, 76 and 104%,
respectively, in comparison to the grains of non-fertilised plants
in both of growing seasons. In addition, N applications at same
levels (i.e., 140, 280 and 420 kg ha-1) resulted in an increase in the
shoots N content by around 15, 26 and 33%, respectively, in
comparison to the shoots N content of non-fertilised plants as
average of both growing seasons. There were significant

differences in the interaction effect of N applications and field
capacity on N content in plants. Generally, the N content in grain
and shoot of maize (g N kg-1) was increased with the reduction in
field capacity (Table 4).
N harvest index (NHI) estimates the efficiency of plants in
concern of the N assimilation 34 and it is described as the ratio of
N accumulation in biomass and grains 35. The highest NHI was
obtained when the highest field capacity was applied in comparison
to 60% or 80% of the field capacity (Table 4). It seems that the
moisture content in soil is the main effect on the NHI. Embryos,
which considered the main part of grains, contain high N. This
means the higher ratio between embryo and endosperm would
explain the increase in NHI under water deficient treatments.
Generally, the highest NHI was found when the highest N
application and field capacity were applied (Table 4). Different
plant species in cereals varied in the NHI according to the genetic
variability 34. For examples, NHI ranges from 57-74% in spring wheat,
Triticum aestivum L.35, 58-86% in durum wheat, Triticum durum L.36
and 42-68% in oat, Avena sativa L. 37.
The grains and shoots N accumulations were
Table 3. Yield components of maize grown with different soil moisture
significantly increased when N applications and field
contents (SMC) and N applications.
capacity were increased (Figs. 2 and 3). This might be
Number of
1000-grain
Plant height
Ear weight
grains
weight
N-fertiliser
attributed to the high quantity as well as to the quality of
(cm)
(g)
SMC
per row
(g)
(kg N ha-1)
the plant root system, since there is a relationship
2012 2013 2012 2013 2012 2013 2012 2013
between the N uptake and the plant root system 38. At
0
300.3 302.6 265.1 266.7 46.1 46.2
371
370
maturity, the low N uptake is a result of the low N
140
310.4 313.4 270.4 271.8 46.3 46.5
360
360
I1
availability in the soil as a consequence of the low ability
280
325.4 323.2 281.6 280.6 50.7 50.8
351
350
420
328.6 327.7 286.4 285.4 52.8 52.8
341
341
of the plant to absorb N, when N is applied in low
0
290.9 293.5 260.8 257.9 37.2 37.3
360
361
applications 16. On the contrary of low N application,
140
305.7 309.4 266.5 264.6 39.5 39.4
351
350
I2
there is a large difference in the uptake of the N when it is
280
319.4 319.4 273.2 272.3 41.8 41.6
331
340
applied in high applications without a negative
420
324.3 325.8 278.2 277.1 42.9 42.8
323
319
0
270.1 271.7 252.1 250.7 32.4 32.5
311
310
association between the N content and crop yield 16. The
140
280.6 280.9 255.4 255.5 33.7 33.8
301
301
relationship was positive and significant between the
I3
280
298.8 299.4 265.7 265.6 35.9 35.9
281
281
grain N uptake and N application in combination with the
420
301.6 301.7 272.8 270.2 36.9 36.9
270
279
field capacity treatments.
S.E.M
0.21
0.19
0.15
0.18
0.24 0.21 0.39 0.35
*
*
**
**
**
**
**
**
SMC
The highest ARR, agronomical NUE and NUtE were
*
*
**
**
**
**
**
**
N-application
obtained when 140 and 280 kg N ha-1 applications were
ns
ns
*
*
*
*
**
**
SMC N
applied (Table 4). This was similar to Tilman et al. 41, who
I =100% of field capacity, I = 80% of field capacity, I = 60% of field capacity. S.E.M.=Standard error of means.
*
= P0.05 and **= P0.01.
Table 4. Grains and shoots N contents (g kg-1), grains and shoots yield (Mg ha-1), N harvest index (NHI), apparent N recovery
(ANR), N utilisation efficiency (NUtE) and agronomical NUE of maize grown with different soil moisture content
(SMC) and N applications.
SMC
I1
I2
I3
N-fertiliser
(kg ha-1)
0
140
280
420
0
140
280
420
0
140
280
420
S.E.M.
SMC
N- fertilizer
SMC N
Grain N
(g kg-1)
2012 2013
11.9 12.1
13.5 13.7
14.6 14.9
15.9 15.9
12.7 12.8
13.9 14.0
15.2 15.5
16.2 16.4
13.1 13.2
14.1 14.2
15.9 16.3
16.8 16.7
0.11 0.13
Shoot N
(g kg-1)
2012 2013
8.9
8.4
9.6
9.1
10.4
9.8
11.1 10.5
9.4
9.5
10.4
9.9
11.3 10.7
11.8 11.5
10.1
9.7
11.0 10.5
11.8 11.2
12.5 12.1
0.10 0.09
Shoot DM
(Mg ha-1)
2012 2013
12.2 11.8
13.9 13.5
15.5 15.1
15.9 15.4
11.9 11.6
13.3 13.1
15.2 14.9
15.6 15.1
10.2
9.7
12.2 11.8
14.1 13.5
14.3 13.7
0.13 0.15
Grain yield
(Mg ha-1)
2012 2013
5.86 6.21
6.90 7.24
8.28 7.93
8.97 8.62
5.65 5.84
6.75 6.91
8.02 7.75
8.75 8.52
5.27 5.33
6.20 6.32
7.00 7.48
8.36 8.19
0.05 0.07
NHI
2012
0.32
0.35
0.39
0.42
0.26
0.34
0.38
0.42
0.26
0.28
0.33
0.34
0.01
2013
0.34
0.37
0.42
0.45
0.29
0.36
0.40
0.44
0.28
0.31
0.36
0.39
0.01
ANR
(kg kg-1)
2012 2013
-
0.30
0.31
0.27
0.34
0.33
0.24
0.27
0.24
0.24
0.28
0.27
0.25
0.16
0.20
0.16
0.005
0.16
0.21
0.18
0.007
NUtE
(kg kg-1)
2012 2013
24.6 21.6
27.7 18.7
26.8 31.8
29.8 27.6
34.3 24.9
30.5 25.0
40.2 44.0
38.2 35.2
43.8 35.9
0.12 0.14
ANUE
(kg kg-1)
2012 2013
-
7.37
8.61
7.38
7.39
6.16
5.74
7.85
8.46
7.38
7.64
6.82
6.37
6.64
7.96
7.35
0.06
7.07
7.67
7.21
0.09
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
ns
ns
ns
ns
**
**
**
**
ns
ns
I1= 100% of field capacity, I2= 80% of field capacity, I3= 60% of field capacity. S.E.M.=Standard error of means. *= P0.05 and **= P 0.01.
220
Season 2012
175 I
1
150
125
100
75
50
25
0
75
50
25
0
Shoots N accumulation (kg ha-1)
Season 2013
175 I
1
150
125
100
175
I2
150
175
125
100
125
100
75
150
75
50
I2
50
25
25
0
0
175
175
I3
150
150
125
125
100
I3
100
75
75
50
50
25
25
0
140
280
420
140
280
420
N application (kg ha-1)
Figure 2. N accumulation in shoots (kg ha-1) of maize grown with different soil
moisture content (SMC) and N applications during two growing seasons. Data
are meanSE and r = 4.
120
100
Season 2012
I1
100
80
Grain N accumulation (kg ha-1)
120
60
40
40
20
20
100
120
I2
100
80
60
40
40
20
20
100
120
I3
100
I3
80
80
60
60
40
40
20
20
I2
80
60
120
I1
80
60
120
Season 2013
140
280
420
140
280
420
Figure 3. N accumulation in grains (kg ha-1) of maize grown with different soil
moisture content (SMC) and N applications during two growing seasons. Data
are mean SE and r = 4.
reported that ARF was highest with the lowest N application.

Improving the management practices during plant growth could
improve ARF and ultimately improve the productivity when low
level or alternate N is applied 42. ARF was reduced when the field
capacity was reduced from 100 to 60% (Table 4). The

highest N application and the lowest field capacity resulted
in the lowest ARR (Table 4). This might be due to increased
grain N uptake when the N applications were increased, as
well as N uptake in grains was closely associated with
grain yield and N content in grains 33.
In the current study, the interaction effect of N application
and field capacity treatments on agronomic N efficiency in
maize was significant (Table 4). The highest field capacity
resulted in the highest agronomic N efficiency compared
to the other field capacity treatments (Table 4). Agronomic
N efficiency shows the efficiency of the plants to take up
the N from the soil 43. Usually, the sufficient water supply
increases the fertilizer use efficiency to the plants. However,
the high N application could cause an unbalance between
the available water and used fertiliser, which could decrease
the NUE. Generally, genetic variation in agronomic NUE,
when N is applied in high application, might be due to the
N uptake differences. At low N applications, the variation
in agronomic NUE could be primarily caused by variation
in NUtE 16. This indicates that the limited factors for N
assimilation can vary in the plants depending on the N
input level 16. In the current study, the results indicated
that the optimal management of N practices is highly
recommended to avoid the loss of the N when there is a
deficiency or excessiveness in the irrigation.
Conclusions
The different irrigation treatments significantly affected
the shoot and grain yields of maize. The optimal N
application was a function of field capacity, which means
that there was an optimal application of N for each field
capacity treatment. Generally, the optimal N application
was increased as the field capacity was increased. In the
arid region, the irrigation with either 80% of field capacity
with the optimal total N supplied is recommended for the
highest biomass production in maize. The total N
application should include soil N content beside the
fertiliser. The significant effect of interaction between soil
moisture content and N applications was obtained for the
N efficiencies as well as the growth and yields of maize,
which implies that 80% of field capacity improved the
response of maize plants to the N application. It is
concluded that the increase in the yields of maize [cv. hybrid
single cross (Giza 10)] in the current study can be achieved
through the reasonable increase in the N application
(decreasing gap between the recommended N application
and estimated level) and the suitable irrigation regime (80%
of field capacity). Also, the results indicated that the optimal
management of N practices is highly recommended to avoid
the loss of N when there is deficient or excessive irrigation.
Acknowledgements
The Water Management Research Institute, National
Water Research Centre, Egypt, is gratefully acknowledged
for providing us the experimental farm and all other
materials needed to finish the investigation. Technicians
are acknowledged for their invaluable technical assistance.
221
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WFL Publisher
Helsinki, Finland
www.world-food.net
Effect of water stress on yield components of sorghum (Sorghum bicolor)

Cndido Ferreira de Oliveira Neto, Ricardo Shigueru Okumura, Ismael de Jesus Matos Vigas, Herclito
Eugnio Oliveira da Conceio, Lucila Elizabeth Fragoso Monfort, Raimundo Thiago Lima da Silva, Jackeline
Arajo Mota Siqueira, Luma Castro de Souza, Roberto Cezar Lobo da Costa and Daiane de Cinque Mariano
Core Research and Plant Production, Federal University Rural of Amaznia, Belm city, 66077-830, State of Par, Brazil.
e-mail: candido.neto@ufra.edu.br, ricardo.okumura@ufra.edu.b
Abstract
The aim of the study was to evaluate the influence of water stress on yield components of hybrid of sorghum BR-700. The experiment was conducted
in a greenhouse at the Federal University of Rural of Amaznia, Belm city, State of Par, Brazil. The experiment was carried out in the completely
randomized design in a 2 x 3 factorial scheme, consisting of two water conditions (control and stress), and initiation of water stress at three growth
stages of sorghum (vegetative, reproductive and physiological maturity), with 5 repetitions. The evaluations were performed after each period of
stress in the plant, measuring variables: dry mass of stem, dry mass of leaves, dry mass of panicle, dry mass of shoots, total dry mass, number of
grains plant-1, mass of 100 grains and dry mass of grains plant-1. Water stress promoted a reduction in the number of leaves and stem diameter,
regardless of the phenological stage of culture, except plant height, which was statistically equal to the control. Concerning the variable dry mass of
stem, dry mass of leaves, dry mass of panicle, dry mass of shoots and the total dry mass were reduced dramatically with fifteen days of water stress.
Water stress promoted low production of plant, evidenced by the low number of grains plant-1, 100 grain mass and grain yield plant-1, showing that
the hybrid sorghum BR-700 was sensitive to water stress, independent of phenological stage of water stress occurrence.
Key words: Grain yield, drought tolerance, plant biometrics.
Introduction
Cultivated plants are constantly exposed to abiotic or biotic
stresses, and interactions that cause changes in growth,
metabolism and yield. The main abiotic factors limiting crop
productivity are: water stress, salinity, low and high temperatures
and excess water, pollution and radiation 1, emphasizing water
stress to be considered the main obstacle of global agricultural
productivity 2-4.
The water, besides being the major component of the cell, is
essential for the maintenance of cellular turgor and allows the
continuation of the process of plant growth, expansion,
photosynthesis and cell division 5. According to Lawlor and
Cornic 6, the leaf water potential, and also the relative water content
are reduced with the declining availability of soil water, resulting
in the loss of turgor and stomatal closure 7.
Water stress is a common situation to occur during the
development of many cultures, and may cause negative impact
on growth and development of crop plants 8. Thus, there is a need
to classify species, biotypes and cultivars in drought tolerant or
sensitive plants, in which each of the possible methods of
classification is by allometric data of the plants. Camacho and
Caraballo 9, studying maize cultivars exposed to water stress in
soil, determined that the dry mass of root can be used as an
indicator parameter for drought tolerant plants.
Studies about the distribution of the root system are considered
essential to the proper management of cultivated plants. The water
stress stimulates root growth in depth leading to areas with higher

moisture in the soil profile 10 and this trait may confer adaptation
to drought 11.
Exposed to water stress plants initially reduce the growth of
shoots to affect the development of the root system, resulting in
increased root/shoot ratio, promoting decrease in leaf growth and
reducing the photosynthetic rate, resulting in excess
carbohydrates in the roots 2.
The physiological indices of evaluation of plant development
are widely studied with the purpose of evaluating the performance
of plants grown under natural stresses or induced by human
action. Despite the importance in knowing the vegetative growth,
there is little research in the northern Brazil, more specifically in
State of Par, involving the development of sorghum under
unfavourable environmental conditions. Thus, the aim of this study
was to evaluate the yield components of sorghum hybrid BR-700
subjected to water stress at three phenological stage of the crop.
Experimental site: The experiment was conducted from May 11
to 2007 to August 11, 2007 in a greenhouse located at the Institute
of Agricultural Sciences, Federal University Rural of Amaznia, in
Belm city, State of Par, Brazil (0127 S and 4826 W). The
minimum/maximum air temperature, relative humidity, photoperiod
and maximum photosynthetically active radiation during the
223
experimental period were: 24.5/39.7, 40/89%, 12 h and 968 mol-2 s-1,

respectively.
250
(a)
Conducting the experiment: Initially, the seeds were put to

germinate in the pots, the population density of six seeds pot-1.
After emergence, held the roughing operation, keeping one plant
pot-1. Then, the plants were subjected to two water regimes:
irrigated, with plants irrigated daily, taking into account the crop
evapotranspiration; and water stress, in which the suspension of
irrigation occurred at 15, 45 and 75 days after germination. The
suspension of irrigation was performed for 15 days before each
period, simulating a severe water stress at three phenological
stages of sorghum 12, 13: vegetative stage, reproductive stage and
physiological maturity.
Data collection: For assessments of vegetative development,
plant height, leaf number and stem diameter were measured.
Plants were separated into root, stem, leaf and panicle and weighed
on an analytical balance to determine fresh weight and forwarded
to the forced movement of air at 70C (5C) until the constant
mass was obtained. By this method, the dry mass of root, stem,
leaf, panicle and grainst were determined.
At physiological maturity of sorghum measurements of yield
components (number of grains per panicle, mass of 100 grains
and grain yield of the crop) were conducted, and the data corrected
to 13% moisture.
Statistical analysis: Initially, the experimental data submitted to
test the normality and homogeneity of variance. The treatment
means were compared by Tukey test, at 5% probability, by using
the statistical software Sisvar 14.
Plant height, number of leaves plant-1 and stem diameter: The
height of the sorghum plant showed statistical differences between
the growth stages, except the physiological maturation of the
grains, which despite having the maximum values of 206 and 199
cm for the control plants and water stress, respectively, did not
differ statistically between treatments (Fig. 1a), probably, because
sorghum plants obtained their maximum height. The plant height
is fundamental for being a characteristic that usually correlates
with the characteristics of grain production 11.
224
Plant height (cm)
Ba
Ba
150
Bb
100
50
Ca*
Ca*
Cb
Number of leaves plant-1
10
(b)
E
Aa
Aa
8
6
4
Ba
Ba
*
Ca
Ca*
Ab
Ab
Ab
Bb
Bb
2
0
25
(c)
Aa
20
Stem diameter (mm)
Experimental design: The experimental design was completely

randomized in a 2 x 3 factorial, two moisture conditions (control
and water stress) and three periods of water stress according to
the phenological stage of sorghum (vegetative stage, reproductive
stage and physiological maturity observed at 30, 60 and 90 days,
respectively), with 5 replications.
Stress
Aa
Aa
Aa
200
Substrate and materials: The substrate consisted of a 3:1:1 mixture

(v: v: v) [(3) black earth, consisting Oxisol soil, medium texture (1)
and chicken manure (1) earthworm humus]. The pots filled with
substrate had the following dimensions: 0.30 m high and 0.30 m in
diameter, with a capacity of 28 kg substrate.
The seeds of sorghum (Sorghum bicolor (L.) Moench.) used
was the hybrid of sorghum BR-700, which features forage sorghum
yield potential of 30 to 40 Mg ha-1 of green biomass and 4-5 Mg
ha-1 grain, as well as being tolerant to soil acidity.
Control
Ba
Ba
15
10
5
0
*
Ca
Ca*
Ab
Ab
Bb
Bb
Cb
Cb
R
Phenological state
PM
Figure 1. Plant height (a), number of leaves plant-1 (b) and

stem diameter (c) in Sorghum bicolor plants cv BR-700
submitted to water stress.
* Averages followed by the same uppercase letter within of the stages
(vegetative, reproductive and maturation) and lowercase letter among the
conditions (control and stress), do not differ among themselves by the
Tukey test at 5% of probability. The bars represent the mean standard error.
Height of control plants was 40, 170 and 206 cm at vegetative

growth and reproductive stages and physiological maturity,
respectively. In the treatment subjected to water stress plant height
was 25, 125 and 199 cm, initiating water stress on vegetative, and
reproductive period and physiological maturity of sorghum,
respectively (Fig. 1a). Similar results were described by Tomich et
al. 15 and Amaral et al. 16, assessing 23 and 11 sorghum genotypes,
respectively, verifying that the reduction in water availability had
a negative influence on plant height.
Water availability significantly affected the plants at vegetative
and reproductive growth stages, possibly, water stress have
promoted severe inhibition of photosynthesis by stomatal closure,
as well as the deleterious effects in chloroplasts. According to
Taiz and Zeiger 17, the stomatal closure contributes significantly
to reduce water losses during limited availability of water and/or
high evaporative demand; however, provides the entry limit of
carbon dioxide (CO2) and, consequently, a decrease in the
intracellular concentration of CO2, resulting in a decrease in plant
height, provided by the lower dry matter production.
The interpretation of plant height in different types of abiotic
stresses can be very complex, since at the time the system has
Dry mass of stem (g plant-1)
70
Stress
Aa
Aa
60
Ba
Ba
50
40
30
20
Ab
Ab
Ab
Ab
Ca*
Ca*
Bb
Bb
Dry mass of leaves ( g plant-1)
25
(b)
Aa
Aa
20
15
Ba
Ab
Ab
Ca
Ca**
Bb
Bb
10
Cb
Cb
5
0
R
Phenological state
PM
60
(c)
Aa
50
40
30
20
10
0
Dry mass of stem, leaf and panicle: There was a significant

reduction in the dry mass of stems and leaves in sorghum plants
subjected to water stress. The effect of water stress caused
variation in the values of the dry mass of the stem from 13.2 to 4.34
g, from 56.57 to 21.75 g and from 44.66 to 25.1 g for the control and
water stress at the vegetative, reproductive and physiological
maturity growth stages, respectively (Fig. 2a). For dry mass of
sorghum leaf, there was also a variation in the values of the control
and water stress treatments from 11.1 to 5.2 g in the vegetative
growth stage, from 18.8 to 11.36 g in the reproductive stage and
from 13.19 to 8.7 g for the physiological maturity of the grain (Fig.
2b).
With respect to dry mass of panicle there were reduction ranging
from 8.48 and 2.66 g for control plants and subjected to water
stress at reproductive stage. At the physiological maturity the
application of water stress negatively influenced the values, in
control plants 47.26 g and in stressed plants of 12.08 g (Fig. 2c).
The change in metabolism promoted by water stress provides
difficulty in translocations of assimilates from leaves and stems
to panicles, resulting in lower dry matter accumulation compared
to control plants. Similar results are described by Costa 23 and
Leite et al. 24, there was negative effect of water stress on dry
matter production of the panicle in sorghum.
The water stress caused a drastic decrease in photosynthetic
Control
(a)
10
Dry mass of panicle ( g plant-1)
water available, overheating of the leaves can be prevented by

cooling transpiration 18. The effect of water loss from the leaves
increases the resistance of photosystem II (PSII) caused by
temperature optimum above, which induces a decrease in plant
height 19.
By decreased availability of water, the number of leaves
decreased by 40, 44 and 29% at vegetative, reproductive and
physiological maturity stages, respectively (Fig. 1b). According
to Carlesso and Santos 20, the water stress influences the division
and cell expansion, by being involved with the turgidity and cell
division, resulting in the reduction of elongation and curling of
the leaves.
The stem diameter was significantly influenced with the
application of water stress on sorghum (Fig. 1c). The plants in
physiological maturity of the grain showed greater stem diameter,
compared to vegetative and reproductive growth stages,
regardless of the treatment control or water stress. The values
showed a decrease in the percentage of 47, 31 and 25% for the
vegetative, reproductive and physiological maturity growth stages,
respectively.
The decrease in values, probably, occurred as a result of water
stress have affected directly the vegetative growth, in the plant
height and stem diameter, reducing cell expansion and cell wall
formation and, indirectly, by decreasing the availability of
carbohydrates and/or influencing the production of plant growth
regulators 21. Another possibility for the reduction in stem diameter
was due to greater stomatal closure, which negatively influenced
the production and accumulation of assimilates. The stomatal
closure, usually observed in plants that prevent dehydration, has
several side effects, such as: decrease in the production of
assimilates and increase in oxidative enzyme activity, as a result
of increase in temperature of the plant that elevates breathing and
spending assimilates and, consequently, reduce vegetative
growth 22.
Ab
Ab
Ba*
Ba*
Bb
Bb
R
Phenological state
PM
Figure 2. Dry mass of the stem (a), leaf (b) and panicle (c) in
Sorghum bicolor plants cv BR-700 submitted to water stress.
* Averages followed by the same uppercase letter within of the stages
(vegetative, reproductive and maturation) and lowercase letter among the
conditions (control and stress), do not differ among themselves by the Tukey
test at 5% of probability. The bars represent the mean standard error.
rate, promoting a decrease in leaf area and number of leaves

plant-1 (Fig. 1b), thus, the dry mass of leaves decreased (Fig. 2b),
reducing the production of assimilates in leaf tissues, causing the
imbalance between the source/sink relations, as well as provided
a greater inactivation of hormones responsible for the growth of
plant height and stem diameter, significantly affecting the dry
mass of shoots (Fig. 3a). Leite 25 observed a decrease in
photosynthetic efficiency promoted by water stress, causing a
reduction in dry mass of shoots.
Dry mass of shoot, root and total: The results showed a
significant decrease in shoot and total dry matter (Fig. 3a, c), and
an increase in the root dry mass (Fig. 3b) in plants subjected to
water stress. The dry mass of shoots ranged from 24.7 to 9.55 g,
from 83.85 to 35.77 g and from 95.2 to 45.88 g for the control
treatment and water stress at the vegetative, reproductive and
physiological grain maturity growth stages, respectively (Fig. 3a).
225
Dry mass of shoot (g plant-1)
120
(a)
Control
Stress
of abscisic acid (ABA), which has been accumulated in the root,

by inhibiting the production of ethylene 27, changing the elasticity
of the wall of the root cells, and allowing root growth 28.
In the physiological maturity, no statistical difference was
observed between control and water stress plants for the dry
mass of root (Fig. 3b), due to root growth, in general, stop its
development after sorghum reaches the reproductive stage,
because, in this period the plant begins to prioritize reproductive
parts (panicles), which have great nutritional requirement 29.
For total dry mass of sorghum observed variation from 32.7 to
22.55 g, from 101.25 to 61.57 g, and from 113.6 to 75.65 g for the
control and water stress plants at the vegetative, reproductive
and physiological grain maturity growth stages, respectively (Fig.
3c). The decrease observed for the variables of shoot and total
dry mass occurred due to lack of water promoted the reduction in
photosynthetic rate of the plants, affecting their physiological
and metabolic conditions. Therefore, the reduction of substances
of reserves and an increase in the inefficiency of transportation of
assimilates promoted a reduction in shoot growth of sorghum
plants 11.
Aa
Aa
100
Ba
Ba
80
60
40
Ab
Ab
Bb
Bb
*
Ca
Ca*
20
Cb
Cb
Dry mass of root (g plant-1)
30
(b)
Aa
Aa
25
20
Bb
Ba
Ba
Ca
Ca
15
10
Aa
Aa
Cb*
Cb*
5
0
Dry mass of the total plant (g palnt-1)
140
(c)
Aa
120
Ba
Ba
100
Ab
Ab
80
Bb
Bb
60
40
*
Ca
Ca*
Cb
Cb
20
0
R
Phenological state
PM
Figure 3. Dry mass of shoot (a), root (b) and total (c) in
Sorghum bicolor plants cv BR-700 submitted to water stress.
* Averages followed by the same uppercase letter within of the stages (vegetative,
reproductive and maturation) and lowercase letter among the conditions (control
and stress), do not differ among themselves by the Tukey test at 5% of probability.
The bars represent the mean standard error.
The dry mass of root was the only variable studied, which
showed higher values for plants subjected to water stress compared
to control plants, verifying the superiority of 4.61, 8.40 and 1.37 g
compared with control plant (Fig. 3b). The significant increase in
root dry mass in plants subjected to water stress was promoted
by more developed root system, thus, characterizing a more deep
and extensive root system, promoting a greater efficiency in
capturing water in the soil. Thus, more specific researches,
biochemical and molecular levels, to elucidate the detailed
mechanism of tolerance to drought, must be made, since
physiological measures such as water potential and osmotic
adjustment do not correlate with differences in productivity of
grain sorghum under water stress 26.
The increase in root growth can be explained that water stress
have been little severe, affecting a greater proportion of shoot
growth of the plant, which promoted an increase in the availability
of assimilates to the roots 11. Another possibility to increase the
dry matter of the roots in plants under water stress can be
correlated with the increase and/or cell expansion by increasing
226
Number of grains plant-1, mass of 100 grains and grain yield:

The water stress negatively affected the number of grains plant-1
resulting in a decrease in the values from 1427 to 710 grains plant-1
in the control and water stress treatment, respectively (Fig. 4a).
The mass of 100 grains was influenced negatively, observing
values of 2.96 in control plants and 1.80 g for plants subjected to
water stress (Fig. 4b).
The grain yield of sorghum was directly influenced by the
application of water stress, with decreasing values of 42.23 g
plant-1 in control plants to 8.25 g plant-1 in plants subjected to
water stress (Fig. 4c). Thus, it was found that the application of
water stress had a negative influence on the number of grains
plant-1, mass of 100 grains and grain yield, reinforcing the results
obtained for plant height, number of leaves plant-1, stem diameter
(Fig. 1) and dry mass of stem, leaf, panicle (Fig. 2), shoot and total
dry mass (Fig. 3a, c). Similar results on corn were presented by
Bergamaschi et al. 30, who observed a decrease in grain yield in
plants subjected to water stress.
The water stress caused a reduction in grain yield of culture, in
the reproductive stage and physiological maturity, the water stress
was extremely limiting, due to the dry mass of grain be dependent
on water, thereby, the stress in this period decreased translocation
of assimilates in the leaves and stem to the grains 29, 31, 32, reducing
the flow of assimilates, causing marked decrease in the number of
grains plant-1 and dry matter accumulation in the grain 33. Another
possible explanation is the extreme sensitivity of sorghum BR-700
to water stress at reproductive stage and physiological maturity,
and physiological processes associated with the formation of the
zygote and the early development of the grains.
From the results obtained, it appears that during the vegetative
stage, the water stress had a negative influence on the growth of
sorghum, due to the decrease in the number of leaves plant-1 and
dry matter accumulation by shoots. In the reproductive stage the
water stress resulted in a negative effect. According to
Bergamaschi et al. 30 this period the culture is highly sensitive to
water limitation, because changes in the metabolism of plant in
reproductive stage were faster than those observed in the
vegetative stage.
Control
Number of grains plant-1
1600
(a)
Stress
a*
a*
1400
1200
1000
bb
800
650
References
400
Mass of 100 grains (g)
(b)
a*
a*
b
b
0
50
45
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Camacho, R. and Caraballo, D. 1994. Evaluation of morphological
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Santos, F. R. and Carlesso, R. 1998. Water deficit and morphologic and
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11
Pimentel, C. 2004. The relationship of the plant with water. Seropdica,
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Oliveira Neto, C. F., Lobato, A. K. S., Costa, R. C. L., Maia, W. J. M.
S., Santos Filho, B. G., Alves, G. A. R., Brinez, B., Neves, H. K. B.,
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13
Oliveira Neto, C. F., Lobato, A. K. S., Gonalves-Vidigal, M. C., Costa,
R. C. L., Santos Filho, B. G., Alves, G. A. R., Maia, W. J. M. S., Cruz,
F. J. R., Neves, H. K. B. and Lopes, M. J. S. 2009b. Carbon compounds
and chlorophyll contents in sorghum induced to water deficit during
three stages. International Journal of Food, Agriculture & Environment
7(3&4):588-593.
14
Ferreira, D. F. 2011. Sisvar: A computer statistical analysis system.
Cincia e Agrotecnologia 35:1039-1042.
15
Tomich, T. R., Rodrigues, J. A. S., Tomich, R.G., Gonalves, L. C. and
Borges, I. 2004. Forage potential of sorghum sudangrass hybrids.
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16
Amaral, S. R., Lira, M. A., Tabosa, J. N., Santos, M. V. F., Mello, A. C.
L. and Santos, V. F. 2003. Forage sorghum lines submitted to water
deficit stress under controlled conditions. Pesquisa Agropecuria
Brasileira 38:973-979.
17
Taiz, L. and Zeiger, E. 2009. Plant Physiology. 4th edn. Artmed, Porto
Alegre, 848 p.
18
Larcher, W. 2000. Ecofisiologia vegetal. RIMA, So Carlos, 531 p.
19
Havaux, M. 1992. Stress tolerance of photosystem II in vivo.
Antagonistics effects of water, heat and photoinhibition stresses. Plant
Physiology 100:424-432.
20
Carlesso, R. and Santos, R. F. 1999. Soil water availability to maize
plants cultivated in soils with different textures. Revista Brasileira de
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1
200
Grain yield (g plant-1)
Acknowledgements
The authors are grateful to the Federal University Rural of
Amaznia for the financial support of this work and collaborations
of researchers participating in the Core Research and Plant
Production, Belm city, State of Par, Brazil, the granting of the
area study and scientific contribution of researchers.
(c)
a*
a*
40
35
30
25
20
15
10
bb
5
0
Physiological maturity
Figure 4. Number of grains plant-1 (a), mass of 100 grains

(b) and grain yield (c) in Sorghum bicolor plants cv BR700 submitted to water stress.
* Averages followed by the same lowercase letter among the conditions
(control and stress), do not differ among themselves by the Tukey test at 5%
of probability. The bars represent the mean standard error.
In general, the water stress had a direct influence on the

reduction of growth and development, therefore, the decrease in
grain yield , demonstrates that the hybrid of sorghum BR-700 is
susceptible to water stress. It is not recommended to regions of
water stress and/or cultivation during the year with limited periods
of rain.
Conclusions
The water stress negatively influences on plant height, number of
leaves plant-1, stem diameter and dry mass of stem, leaf, panicle,
shoot and total plant, regardless of the period of water stress
occurence at the phenological stage of vegetation, reproductive
and physiological maturity of sorghum, with the exception of the
dry mass of root which presents an increase in values in plants
subjected to water stress.
The hybrid of sorghum BR-700 is sensitive to water stress at
the vegetative, reproductive and physiological maturity stages,
resulting in a decrease in the values of number of grains plant-1,
mass of 100 grains and grain yield.
227
Gonalves, M. R. and Passos, C. A. M. 2000. Growth of five species

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peanut (Arachis hypogaea L.) subjected to water deficit. Revista de
Biologia e Cincias da Terra 4:1-7.
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in Plants Nodulated Bean-to-String [Vigna unguiculata (L.) (Walp)]
Subjected to Water Stress. Ph.D. thesis, Plant Biochemistry, Federal
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Leite, M. L., Rodrigues, J. D., Mischan, M. M. and Virgens Filho, J. S.
1999. Effects of water deficit on the culture of cowpea (Vigna
unguiculata (L.) Walp), cv. EMAPA-821. II Analysis of growth.
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sudanense (Piper) Stapf), Depending on the Availability of Water and
Phosphorus Sources. Dissertation (Master of Animal Science), Federal
University of Paraiba, Areia, 85 p.
26
Costa, R. C. L., Oliveira-Neto, C. F. and Freitas, J. M. 2004.
Physiological parameters of the sorghum plant used in the production
of silage. 1 Workshop sobre Produo de silagem na Amaznia. Federal
University Rural of Amaznia, Belm, pp. 9-31.
27
Sharp, R. E. and Lenoble, M. E. 2002. ABA, ethylene and the control
of shoot and root growth under water stress. Journal of Experimental
Botany 53:33-37.
28
Hsiao, T. C. and Xu, L. K. 2000. Sensitivity of growth of roots versus
leaves to water stress: Biophysical analysis and relation to water
transport. Journal of Experimental Botany 51:1595-1616.
29
Magalhes, P. C., Dures, F. O. and Schaffert, R. E. 2000. Physiology
of Sorghum Plant. Embrapa Milho e Sorgo, Sete Lagoas, 46 p.
30
Bergamaschi, H., Dalmago, G. A., Comiran, F., Bergonci, J. I., Mller,
A. G., Frana, S., Santos, A. O., Radin, B., Bianchi, C. A. M. and
Pereira, P. G. 2006. Water deficit and yield in maize crop. Pesquisa
Agropecuria Brasileira 41:243-249.
31
Magalhes, P. C. and Jones, R. 1990. Increase of assimilates in growth
rate and final weight of corn. Pesquisa Agropecuria Brasileira 25:17471754.
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Magalhes, P. C., Dures, F. O. M., Carneiro, N. P. and Paiva, E. 2002.
Physiology of Corn. Embrapa Milho e Sorgo, Sete Lagoas, 23 p.
33
Matzenauer, R., Bergamaschi, H., Berlato, M. A. and Riboldi, J. 1995.
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water availability in the state of Rio Grande do Sul. Pesquisa
Agropecuria Gacha 1:225-241.
21
228
WFL Publisher
Helsinki, Finland
www.world-food.net
Linseed response to treatment with swine wastewater as biofertilizer

Jhonatas Antonelli *, Cleber Antonio Lindino, Reginaldo Ferreira Santos, Samuel Nelson Melegari de
Souza, Willian Czar Nadaletti, Paulo Cremonez, Eduardo Rossi and Flvio Gurgacz
Department of Agricultural Energy, University of West Paran, Paran, Brazil. e-mail: jonatas-a@hotmail.com,
cleberlindino@yahoo.com.br, reginaldo.santos@unioeste.br, samuel.souza@unioeste.br, williancezarnadaletti@gmail.com,
pa.cremonez@gmail.com, eduderossi@gmail.com, flavio.gurgacz@unioeste.br
Abstract
This study aimed to verify the linseed responses when subjected to different doses of swine wastewater (SWW). The experiment was conducted in
the city of Cascavel, Paran state, Brazil. The treatments considered were: T1 control sample; T2 5; T3 10; T4 20; T5 30 and T6 40 m3ha-1 SWW.
The sowing was performed in a 12 m2 area. After 180 days of cultivation stem diameter, plant height, number of capsules, stem branch number and
fresh and dry weight of the aerial part were measured. The SWW application proved to have a positive effect on the linseed culture with an application
of 10 m3ha-1. The application of 40 m3ha-1 inhibited the linseed development. The immobilization of nutrients and the shortage of oxygen in the soil
may have been the main cause of the linseed development decrease in applications exceeding 10 m3ha-1 of SWW.
Key words: Macronutrients, linseed, biofertilizer.
Introduction
The linseed (Linum usitatissimum L.), belonging to the Linaceae
family, is native of Asia. A fiber of flax can be extracted from bark,
which is used as raw material in fabrics manufacturing; from
capsule is possible to extract a seed rich in oil, which can be used
for human consumption, oils industrialization for painting or in
animal feeding 4.
The linseed is cultivated as a commercial or subsistence culture
in over than 30 countries. The world production of linseed in
2004 was 1,902,688 tons in 2,620,396 ha. Canada and China are
the main producers, representing 27 and 24%, respectively, of the
linseed worldwide production in 2004 (FAO, 2005).
The cultivation of linseed can be used as an alternative in
agriculture, which allows an increase in diversification of the
production systems in temperate environments. However, in many
regions where the traditional planting system is predominant, it is
necessary to have an agro-economic incentive, in order to
encourage the farmers to work with the linseed culture 5.
For an adequate plant development, it is necessary that a soil
can supply its nutritional needs. This soil should provide adequate
amounts of nutrients and ideal conditions for plants to absorb them2.
According to Prado 12, the factors that affect the availability of
nutrients in soil are: pH, aeration, humidity, organic matter,
temperature and presence of other ions.
The aeration is an important aspect of physical soil quality,
because part of biological activities needs oxygen for good
development. For good aeration, it is necessary to be gas
exchanges between soil and atmosphere 17. The soil aeration is an
important limiting factor for the root system development, growth
and culture productions 3.
The use of bio-fertilizers in organic agriculture has been widely
used to supply the plants nutritional demand and the trophobiotic
management of pests and diseases. In general, the biofertilizers
when applied in the cultures, act as a source of micronutrients

and can contribute to increase the natural resistance of plants
from attack of pests and pathogens, besides exerting a direct effect
on phytoparasites due to the presence of toxic substances in the
mixture.
The application of organic biofertilizer provides a better
displacement of the nutrients needed for the plants, offering
nutrients in a readily available form, improving the nutritional
property of soil due to the high concentration of nitrogen,
phosphorus, calcium and magnesium.
The organic matter in soil can be a natural source of nutrients,
however, its availability does not occur immediately, but after
decomposition and mineralization. Thus, plants can absorb the
nutrients. The organic nutrients mineralization depends of several
factors, including edaphoclimatic characteristics, handling
practices and quality of residues 16.
Sampaio et al. 15 observed that the application of cattle manure
in the first month caused immobilization of soil nutrients, but in
the following months there was liberation of these nutrients until
reaching the maximum point in the sixth month. The organic matter
mineralization occurs simultaneously with the immobilization of
the nutrients, which is also mediated by the soil microbial biomass.
Thus, depending on the magnitude of each process, either a
positive result (mineralization) or negative one (immobilization)
can be obtained within a certain period of time, making the exclusive
use of organic matter in the soil insufficient to meet the crop
needs 14.
The swine culture in Brazil is increasing in the recent decades,
undergoing great technological changes in order to raise the
productivity and decrease production costs. Therefore, the
centralized pig production had a great growth, creating massive
problems regarding the correct disposal of effluents. Thus, the
229
use of SWW is an excellent biofertilizer option to be used in

agriculture, aiming the increase of agricultural production.
In view of these facts, this study aimed to evaluate the culture
of linseed under different concentrations of SWW. The variables
evaluated were: stem diameter, plant height, fresh and dry weight
of aerial parts, number of branches in stem and number of capsules.
The study lasted 180 days between April and October of 2013.
60
55
A
Plant height in cm
50
45
40
35
30
25
20
y = -0.0428x + 1.7532x + 30.255

R2 = 0.8433
2
10
20
30
m3.ha-1 of swine wastewater
C
5
4
3
y = -0.008x2 + 0.3097x + 2.428

R2 = 0.9455
2
1
0
0
10
20
30
40
70
65
60
55
y = -0.0225x2 + 0.6898x + 60.105

R2 = 0.7317
50
45
40
40

Figure 1 shows that the treatment with SWW influenced
significantly (p < 0.05) the plant height, stem diameter and fresh
and dry mass of the aerial part, in a quadratic form. The number of
branches on stem and capsules had no significant influence.
According to Fig. 1, the culture of linseed had a better
development until the T2 treatment with 10 m3ha-1 of SWW. After
this, the culture had a lower development with the increase of
SWW dose. The T5 treatment with 40 m3ha-1 of SWW gave some
results lower than those found in the control sample, showing
that the excess of SWW hindered the development of culture.
One of the factors that contributed to the results in Fig. 1 might
have been the immobilization of the nutrients by the
microorganisms present in the soil. This occurs because they
consume nutrients to produce energy and multiply themselves,
releasing nutrients in an immobilized form when they die, which
after some time becomes mineralized nutrients.
Another factor that might have contributed to the results was
consumption of oxygen by soil microorganisms. To multiply and
produce energy, the microorganisms consume oxygen, the greater
the amount of organic matter in the soil, the higher the rates of
microorganisms multiplication, leaving the soil poor in oxygen.
The lack of oxygen can inhibit the development of the plants.
Analysing the stem diameter variable (Fig. 1A), it can be
observed that it received a positive influence in its development
up to the application of 10 m3ha-1 of SWW, having a development
reduction from the application of 20 m3ha-1. According to a derived
75
Dry mass from aerial part in g
Fresh mass from aerial part in g
Stem diameter in mm

The experiment was carried out at the State University of West of
Paran (UNIOESTE), campus Cascavel, with a geographic location
245920.5 South, 532658.7 West. The local climate is
considered super humid, subtropical and mesothermal, with an
annual precipitation of 1640 mm and an average temperature of
19C 10. The soil of the region is classified as dystrofic Red Latosol.
The design used was randomized blocks with 6 treatments, with
dimensions of 0.80 m x 2.5 m, totaling 2.00 m2 each treatment, and
the total area used in the experiment was 12.00 m2.
The treatments were: T1 control; T2 5 m3ha-1; T3 10 m3ha-1; T4
20 m3ha-1; T5 30 m3ha-1 and T6 40 m3ha-1 of SWW. The analysed
factors were plant height, stem diameter, number of capsules,
number of branches in stem, and fresh and dry mass of the aerial
part.
The sowing was performed at 1 cm depth of the soil, 20 days
after the sowing a distance between the plants of 1 cm between
plants was delimited and the treatment with SWW was initiated
30 days after sowing .
The SWW was collected fom an output of biodigestor and
allocated in plastic barrels of 30 L each. The collection took place
at a rural property located in the city of Marechal Cndido Rondon,
85 km far from Cascavel. The treatments with SWW were done
weekly, with support of a 10 L water can. The treatments began in
April 2013 and finished in October 2013, totalling 180 days of
treatment.
The analyses were conducted when the plant were still in
reproductive phase with the seeds in formation. To harvest the
plants, a cut close to the ground was performed, after this
procedure a measurement of height, stem diameter, number of
branches and number of capsules per plant was carried out. To

measure the height, a millimetre tape was used and for the stem
diameter, a pachymeter. To determine the fresh and dry mass of
the aerial part it was weighed on an analytical scale and placed at
a temperature of 105C until constant weight and then weighed
again on analytical scale.
For analysis of the data the Tukey test was applied at 1 and 5%
significance level in order to determine the statistical difference
between means. To support the tests the software ASSISTAT 7.5
beta was used.
10
20
30
40
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
y = -0.0051x2 + 0.1964x + 1.898

R2 = 0.8896
10
20
30
40
Figure 1. Stem diameter (A); Plant height (B); Fresh mass from aerial part (C); Dry mass from aerial part (D).
230
mathematical equation, the maximum point of the stem diameter

development occurs at 20.5 m3ha-1 of SWW, in which the plant
diameter can reach up to 48 mm of thickness.
The results agree with El-Nagdy et al. 7, that biofertilizers and
mineral fertilizers had a positive influence on the stem diameter,
having a development around 14% greater than the control sample.
At the plant height variable (Fig. 1B), there was a positive influence
on development up to the application of 10 m3ha-1 SWW, having
an accentuated reduction on its development with applications
above 20 m3ha-1. According to a derived mathematical equation,
the maximum point of plant height development occurs at 15.5
m3ha-1 of SWW, and can reach up to 65 cm height.
Rahimi et al. 13, showed that the plant height variable had a
positive effect, having a development according to the amount of
nitrogen fertilizer applied up to 100 kgha-1. According to El-Nagdy
et al. 7, the development of linseed culture was positive with the
application of nitrogen fertilizer and biofertilizers, when compared
to the control.
The plant development and its productivity can be restricted
by the amount of nutrients in the soil and by the period of the year
it has been planted. Therefore, it is necessary to perform a soil
analysis in order to determine the nutrients needed for the plant
to reach its maximum productivity using minimal amount of
fertilizer 9, 11. The seeds productivity is directly related to the plant
height, number of capsules and seed weight 1.
The fresh mass of the aerial part (Fig. 1C) has a positive effect in
applications of 10 m3ha-1 of SWW, having an accentuated
reduction with applications above 10 m3ha-1. Using a derived
mathematical equation, the maximum point of dry mass would be
with application of 19.5 m3ha-1 of SWW, reaching 5.5 g of dry
mass of aerial part.
According to Flnet et al. 8, the fresh mass of the aerial part had
a significantly increase when 40 and 80 kgha-1 doses of urea were
applied, the results being similar to those found in this work.
According to Dordas 5, the greater the plant dry mass relation,
the greater its seed production rate will be, and larger its amount
of oil per seed. The biomass production is directly related with the
seed generation, seed weight per plant, number of capsules per
plant and absorption rate of nutrients.
Analysing the dry mass variable of aerial part (Fig. 1D), the
results showed a similar effect to that found in Fig. 1C, in which
the best results were found with 10 m3ha-1 of SWW. According to
the derived mathematical equation, the best production results
would be with the application of 19 m3ha-1 of SWW, reaching a
dry mass of the aerial part of 3.8 g.
Dordas 6 reported that the linseed responded positively to the
application of nitrogen fertilizer, having a relatively high increase
in its mass of 33% and 44% in the dry mass of leaves, 43% and
51% in the dry mass of stem, with the application of 40 and 80
kgha-1 of nitrogen fertilizer, when compared to the control sample.
These results are similat to those found in this work.
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biochemical compositions under various planting dates and nitrogen.
African Journal of Agricultural 6:31673175.
14
Siqueira Neto, M., Piccolo, M. C., Venzke Filho, S. P., Feigl, B. J.,
Cerri, C. C. 2010. Mineralizao e desnitrificao do nitrognio no
solo sob sistema plantio direto. Bragantia 69:923-936.
15
Sampaio, E. V. S. B., Oliveira, N. M. B. and Nascimento, P. R. F. 2007.
Eficincia da adubao orgnica com esterco bovino e com Egeria densa.
Revista Brasileira de Cincia do Solo 31:995-1002.
16
Santos, G. A., Silva, L. S., Canellas, L. P. and Camargo, F. A. O. 2008.
Fundamentos da Matria Orgnica do Solo: Ecossistemas Tropicais &
Subtropicais. Metropole, Porto Alegre, 654 p.
17
Warkentin, B. P. 1995. The changing concept of soil quality. Journal of
Soil and Water Conservation 50:226-228.
1
Conclusions
The SWW application proved to have a positive effect on the
linseed culture with an application of 10 m3ha-1. The application
of 40 m3ha-1 inhibited the linseed development. The immobilization
of nutrients and the shortage of oxygen in the soil may have been
the main cause of the linseed development decrease in applications
exceeding 10 m3ha-1 of SWW.
231
WFL Publisher
Helsinki, Finland
Journal of Food, Agriculture & Environment Vol.12 (3&4):232-236. 2014
www.world-food.net
Thermic sum and crop coefficient of canola (Brassica napus L.) for the region of
Tangar da Serra, Mato Grosso State, Brazil
Kssio De Marco 1, Rivanildo Dallacort 1*, Adalberto Santi 1, Ricardo Shigueru Okumura 2, Mirian Hiroko Inoue 1,
Joo Danilo Barbieri 1, Dejnia Vieira de Araujo 1, Roberto Antnio Savelli Martinez 1 and Willian Fenner 1
Campus de Tangar da Serra, Universidade do Estado de Mato Grosso, 78300-00, Tangar da Serra, Brasil. 2 Campus de
Capito Poo, Universidade Federal Rural da Amaznia Par, 66650-000, Capito Poo, Brasil. *e-mail: rivanildo@unemat.br
1
Abstract
The canola cultivation is highly dependent on water resources, which makes it necessary to conduct studies to determine the crop coefficient (Kc),
with the purpose of improving the efficiency of water use. Thus, the objective of this study was to determine the Kc and calculate the thermic sum
required to complete its development cycle. The experiment was conducted at the University of the State of Mato Grosso - UNEMAT Campus of
Tangar da Serra, Brazil, in the period of February 20th 2013 to May 25th 2013, in which the hybrids Hyola-433 and Hyola-61 were sown. To
determine the reference evapotranspiration (ETo), the Penmam - Monteith method was used, while the crop evapotranspiration (ETc) was determined
using drainage lysimeters, and the Kc obtained by the relation between ETc and ETo. In calculating the thermic sum, base temperatures were used,
determined in the literature for each cultivar. The ETc had a mean of 4.50 mm day-1 for the hybrid Hyola-433 and 3.38 mm day-1 for the hybrid Hyola61, the ETo had an average of 3.14 mm day-1 and the Kc of the hybrid Hyola-433 was 1.09; 2.15 and 1.43, while the hybrid Hyola-61 showed 0.79;
1.65 and 1.33 in the vegetative growth, flowering and maturity stages, respectively. During its growing cycle the hybrids and Hyola-61 and Hyola433 required a thermic sum of 1,778 and 1,816 degree days, respectively.
Key words: Degree day, evapotranspiration, plant development.
Introduction
The decrease of fossil fuel reserves, increase of oil prices and the
concern with the environment caused by the greenhouse gas
emission have stimulated research on alternative sources for
biofuels 1, 2, such as canola (Brassica napus L.).
The canola is oilseed species which has high oil content (about
38% 3). Moreover, the crop has shown a great potential for
incorporation in grain production systems in Brazil due to the
possibility of production of alternative fuel 4, 5 as well as the oil
extraction for human and animal nutrition 6.
According to FAO statistics 7, canola is the third most widely
grown oilseed in the world, and in Brazil its cultivation has
increased significantly in recent years, with a range of
approximately 45,000 hectares of cultivated crop area in 2012. From
that amount, the Brazilian South region accounts for 95% of the
total 8.
Recently, the crop was introduced in the Midwest region,
cultivated as off-season between soybean harvests in rotation
and/or succession in crop systems 4; however, being an extremely
demanding plant in temperatures and water has its vegetative
growth and grain yield affected in extreme conditions 9.
Thus, the plant is still little explored in the central region of the
country and one of the first steps to be defined before the
domestication of a species is the determination of the agroclimatic
adaptability of the crop 10, the correct quantification of the crop
evapotranspiration being extremely important for the specific
region 11.
232
Knowing the evapotranspiration of a crop (ETc) throughout its

cycle and its crop coefficient (Kc) is critical for the design and
management of irrigation systems 12, since it can get a better
management of irrigation 13, which contributes to a sustainable
and rational agriculture 14.
In this regard the objective of this study was to perform the
thermic sum and determine the values of evapotranspiration and
Kc of two canola hybrids (Hyola-433 and Hyola-61) in its
developmental stages, in the climatic conditions of the municipality
of Tangar da Serra, State of Mato Grosso, Brazil.
Experimental site: The study was conducted at the experimental
field of the State University of Mato Grosso (UNEMAT)
University Campus of Tangar da Serra, Mato Grosso, Brazil, which
is located at the geographical coordinates of 1439' S and 5725'
W, with average altitude of 321.5 m. The region has a humid tropical
megathermic (Aw) climate, with annual average temperature of
24.4C, precipitation of 1,500 mm and a relative humidity between
70 and 80% 15. The soil is classified as Dystroferric Red Latosol
(Oxisol) 16, with a very clayey texture (664 g kg-1).
Cultivar choice: The genotypes used were the hybrids Hyola433 with an early maturity cycle, installed in the lysimeters 1, 2, 3
and 4, and the hybrid Hyola-61 with a medium maturity cycle,
installed in the lysimeters 5, 6, 7 and 8. The hybrids choice occurred
Table 1. Soil chemical characteristics in the plow layer 0-0.20 m.

prior to the experiment installation.
pH
M.O.
g
dm-3
41.0
CaCl2
4.7
P
mg
dm-3
6.0
K+
Ca2+
Mg2+
Al3+
H+Al
CTC
-3
-----------------mmolc dm ----------------0.8
14.0
7.0
2.0
46.0
68.0
V
%
33.0
Tmed
30
100
25
80
20
60
15
40
10
20
0
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101
DAS
Temperature (C)
Soil correction, fertilization, seeding, pest and disease control:

Soil amendment, sowing fertilization and coverage were performed
according to Tomm et al. 17, taking into account the results of the
chemical analysis of the soil in the plow layer of 0-0.20 m and
expected grain yield of 1,500 kg ha-1 (Table 1). The seeding of the
experiment was manually held on February 20th, 2013, as
recommended by Tomm 18 for the Midwest region of Brazil,
distributing 15-20 seeds m-1 at a spacing of 0.45 m between rows,
resulting in a population density of 40 plants m-2. At the time of
sowing 200 kg ha-1 of mineral fertilizer with 10-25-15 formulation
was applied in the furrows, and complementation of nitrogen
fertilization in the phenologic stage of four developed leaves at a
dose of 60 kg ha-1 of N, using the source of urea (45% N) according
to technical indications for the canola crop 17. The weed control
was done by hand weeding, so that the area was maintained
without the presence of weeds during the period of the experiment.
In regards to the prevention and control of major pests and
diseases of canola, pulverizations with pesticides recommended
for the crop through manual spraying were held 18.
Prec
120
Precipitation (mm)
due to the good grain yields in southern Brazil region and to

present the polygenic disease resistance to the principal culture
disease blackleg (Leptosphaeria maculans, Phoma lingam) 17.
Figure 2. Average precipitation and temperature in the days after

sowing (DAS) of canola.
UNEMAT Campus of Tangar da Serra. Mato Grosso State. Brazil. 2013.
least one open flower; end of flowering (FF) when the plants had
no more flowers except atypical plants; and finally the physiological
maturity (MF) when 50% of the seeds switched to the dark colour
silique located in about the middle of the main raceme of plants.
Determination of thermic sum: The thermic sum was determined
for each stage of crop development, considering the base
temperature (Tb) of: -0.8C (E-IF), 10C (IF-FF) and 7.2C (FF-MF)
for the hybrid Hyola-61 and 0.3C (E-IF), 9.9C (IF-FF) and 7.9C
(FF-MF) for hybrid Hyola-433 as determined by Luz et al. 19, using
the equation:
GD = (Tmd - Tb)
where GD = degree days; = summatory; Tmd = daily average

temperature and Tb = base temperature according to the different
stages of the crop.
UNEMAT Campus of Tangar da Serra. Mato Grosso State, Brazil, 2013.
Gravimetric moisture (kg kg-1)
Soil moisture and meteorological variables: To monitor soil

moisture, reflectometry probes in the time domain (TDR) were
installed vertically (0.30 m) in each lysimeter, CS -616 type, duly
calibrated by the quadratic equation in the local soil (Fig. 1).
Meteorological data (pluviometric precipitation and average
temperature) used were collected by a conventional automatic
weather station, Campbell Scientific, model UT 30, installed next
to the experimental area, which belongs to the Agrometeorology
Laboratory of the University of the State of Mato Grosso UNEMAT (Fig. 2).
0.6
Soil moisture
Field capacity
Wilting point
0.45
0.3
y = -4.0918x2 + 2.4307x - 0.0102
R2 = 0.8137
0.15
0
11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86
DAE
Figure 1. Gravimetric soil moisture behavior as a function of days

after emergence (DAE) canola and sensors calibration equation.
UNEMAT Campus of Tangar da Serra, Mato Grosso State. Brazil, 2013.
Crop phenological stages: Phenological stages were defined by

the methodology described by Tomm et al. 17, which proposed
the date of emergence (E) at the time when 50% of the seedlings
emerged; early flowering (IF) at which 50% of the plants had at
Determination of the crop evapotranpiration (ETc):

Determination of the crop evapotranpiration (Etc) was performed
by the use of drainage lysimeters with the soil maintained near
field capacity. The replenishment of water to the soil occurred by
the pluviometric precipitation and manual irrigation by sprinklers.
Irrigation were performed according to the methodology proposed
by Bernard et al. 20, in which the plants contained in the lysimeters
were irrigated daily with the same amount of water sufficient to
provide a small amount of drainage water, about 10% of the applied
irrigation. Eight lysimeters were used in the experimental area,
spaced by 3m x 3 m, constructed in fiber boxes with a capacity of
1000 litres and diameter of 1.5 m. The daily ETc was determined by
the difference between the amount of water applied and the volume
drained in 1 litre for each lysimeter, divided by the useful area of
the lysimeters in m2 21.
Reference evapotranspiration determination (ETo): ETo was
performed using the CLIMA software, developed by the
Agronomic Institute of Paran (IAPAR) 22, using the PenmanMonteith equation, considered the standard for this type of study.
D etermination of crop coefficient (Kc): The daily values of the
single Kc were determined after 10 days of sowing, when the crop
was well established in the field, by the division ratio of the crop
evapotranspiration (ETc) and ETo using the equation given by
Doorenbos and Pruitt 23. Subsequently, the daily Kc values were
grouped so that for each phenological stage a crop coefficient
was determined.
233
Evaluation of yield components: Yield components were

determined at the end of the crop cycle. 1) Plant height (PH) was
measured from the base of the plant to the top of the main stem,
expressing the average values in cm. 2) Height of insertion of the
first silique (HIFS) was measured with a graduated ruler the
distance of the base of plant to the point of insertion of the first
silique. 3) Stem diameter (SD) was measured using a caliper, and
the value expressed in mm. 4) Weight of 1000 grains (M1000) was
determined by weighing 250 grains, multiplied by four and
registered in g. 5) Grain yield (Yield) was obtained by harvest of
the whole area of the lysimeters, determining the grain moisture
content and correcting it to 10%, and subsequent conversion to
kg ha-1 6.
Statistical analysis: The data of yield components of canola were
subjected to variance analysis and the treatment means were
compared by Tukey test at 5% probability using SISVAR 24
statistical software.
Crop cycle: Canola presented a total development cycle of 90
days for the hybrid Hyola-433 and 95 days for the hybrid Hyola61, in the climatic conditions of the city of Tangar da Serra, Mato
Grosso State, Brazil. Similar results to those were found by Tomm et
al. 25 in the northeastern state of Paraiba, where the culture had an
average cycle of 92 and 94 days for Hyola-433 and Hyola-61
hybrids, respectively (Table 2). Despite the development cycles
were similar even being grown in different locations, Dalmago et
al. 26 report that there is great heterogeneity in the responses of
canola crop to cultivation environments, a result of both the
agricultural management as well as adaptations to environmental
variables and water availability, which highlights the influence of
temperature on the duration of the culture of canola cycle 19. Luz
et al. 19 found that there is a negative linear relationship between
the air temperature and the sub periods duration (in days) for two
canola hybrids, wherein the total crop cycle ranged from 100 to
162 days for Hyola-433 and from 104 to 162 for Hyola-61, according
to the sowing dates. The variation of the canola cycle occurs
because higher temperatures decrease the number of days between
emergence and flowering initiation, and the more sensitive sub
period to the culture of canola is from the beginning of flowering
to the end of flowering 27.
The emergence of canola occurred on February 25th, 2013, five
days after sowing for both cultivars, flowering initiation was
observed on April 10th for the hybrid Hyola-433 and April 13th for
the hybrid Hyola-61, finalizing with 30 days, which features a
short flowering period, probably attributed to the high temperature
average 20, 27. Finally, grain maturity occurred on May 21th and 25th
for the Hyola-433 and Hyola-61, respectively (Table 2).
For the environmental conditions during the experimental period,

it is verified that the air average temperature after the canola seeding
varied between 17.2 and 28.2C with a mean of 24.6C, slightly
higher values than the recommended as appropriate for the
development of the crop. According to Thomas 28 and Tomm et al.
27
, the optimum average temperature is 20C throughout the cycle
of canola with amplitude of variation from 12 to 30C, where air
temperatures above 27C promote flower abortion.
During the crop cycle, the pluviometric index was 456.9 mm,
distributed so that by the full flowering totalled up precipitation
of 437.2 mm and 19.7 mm after flowering, remaining within the
optimum range established by Thomas 28.
Regarding canola water consumption after its full establishment
in the lysimeters, with soil condition of field capacity was 363 mm
for the hybrid Hyola-433 and 290 mm for the hybrid Hyola-61, in
which the phases between the emergence and full flowering
showed the largest water consumption (Table 2). According to
Thomas 28, canola flowering is the most sensitive period to water
deficit and when this occurs a reduction of the components of
grain yield and oil content can be verified.
Productivity: The grain productivity was 2,332.55 and 1,772.11
kg ha-1 for Hyola-433 and Hyola-61 hybrids, respectively (Table
3), similar results to those obtained in other regions of Brazil for
the same genotypes 18, 27, which is higher than the national average
of 1,400 kg ha-1 8.
Plant height (AP), height of insertion of the first silique (AIPS)
and stem diameter (DC) did not differ between the two cultivars,
possibly because both are commercial hybrids that exhibit the
defined characteristics in the breeding program of the company
to obtaining high grain yield. The mass of 1000 grains (M1000)
was higher in hybrid Hyola-61 (3.19 g), but had no direct effect on
grain yield, since grain productivity was less than Hyola-433
(Table 3).
Table 3. Average plant height (AP), height of insertion of
the first sliquoa (AIPS), stem diameter (DC), 1000
grain weight and yield of two canola hybrids.
Cultivars
Hyola-433
Hyola-61
CV (%)
AP
(cm)
145.64 a
147.08 a
2.63
AIPS
(cm)
74.08 a
72.52 a
3.34
DC
(mm)
10.25 a
10.98 a
6.94
P 1000
(g)
2.84 b
3.19 a
6.42
PROD
(kg ha-1)
2,332.55 a
1,772.11 b
15.48
UNEMAT Campus of Tangar da Serra. Mato Grosso State. Brazil. 2013. *Means followed by
the same letter in the column do not differ by the Tukey test at 5% probability.
Thermic sum: For the municipality of Tangar da Serra, the canola

cycle completed with a thermic sum of 1,778 degree days (GD) for
the hybrid Hyola-433 and 1,816 GD for the hybrid Hyola-61, wherein
the thermic sum for the sub periods E-IF, IFFF and FF-MF were 1,179, 414 and 185 GD
Table 2. Initiation and duration (Dur) of the phenological phases: Emergency (EMER);
for the hybrid Hyola-433 and 1,203, 436, and
beginning of flowering (IF); flowering finalization (FF) and maturation (MAT);
177 GD for the hybrid Hyola-61, respectively
and water consumption (mm) (Cons.) of hybrids Hyola 433 (H433) and Hyola
(Fig. 3).
61 (H61).
Cycle
Hyola
Start
Dur.
Cons. (mm)
EMER
H433
H61
25/02 25/02
5
5
-
IF
H433
10/04
45
139
FF
H61
13/04
48
106
H433
10/05
30
185
H61
15/05
32
153
MAT
H433
H61
21/05 25/05
10
10
39
31
Total
H433 H61
90
363
95
290
234
40
Hyola-61*
FF-MF = 177 GD*

FF-MF = 185 GD**
30
Degree days
Hyola-433**
20
E-IF = 1.203 GD*
E-IF = 1.179 GD**
10
0
1
IF-FF = 436 GD*

IF-FF = 414 GD**
11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91
DAE
Figure 3. Degree days in relation to days after emergence of two

canola hybrids, using base temperature defined by Luz et al. 19.
Starting from the same basal temperature, Luz et al. 19 found a

thermic sum for the hybrid Hyola-61 of 1644 GD and for the hybrid
Hyola-433 of 1,515 GD, showing an average cycle of 134 and 136
days for Hyola-433 and Hyola-61 hybrids, respectively. Battisti et
al. 29 working with base temperature of 5C, found the thermic
sum during the canola cycle inferior to 1,223 GD for hybrid Hyola433 and 1,306 GD for the hybrid Hyola-61. The accumulated thermic
sum can vary within the same crop, according to the studied region,
cultivar, irrigation use, among other factors 30 .
To determine the crop coefficient (Kc), data of crop evapotranspiration (ETc) and reference evapotranspiration (ETo) were
used (Fig. 4). In the first 45 days after emergence, the values of
reference evapotranspiration and of the crop were found proximate,
with some minimal peaks of crop evapotranspiration due to be
days with high pluviometric indexes. Between 45 and 80 days
after emergence, comprising the period of full bloom, the values
of crop evapotranspiration were always higher than the reference
evapotranspiration, which characterizes this period of maximum
water consumption, and subsequently to physiological maturity
values were close to each other, as occurred in the first phenological
stage.
The hybrid Hyola-433 showed higher values of evapotranspiration that the hybrid Hyola-61 throughout the crop cycle.
According to Tomm et al. 17, this occurs because the hybrid Hyola12
mm
9
6
3
0
1 4 7
61 has a very high stability of grain yield and wide adaptation,

even under conditions of water stress, while the hybrid Hyola433 needs high requirement of favourable environmental
conditions, especially on soil fertility and water availability to
express their full productive potential.
The crop coefficient values were daily calculated and grouped
according to different phenological stages (Table 4), which shows
the evolution of the crop coefficient in different phenological
stages. The observed values between sub periods E-IF, IF-FF and
FF-MF showed a variance of 0.4 10-2, 0.2 10-2 and 0.3 10-2 and
standard deviation of 0.6 10-1;, 0.4 10-1 and 0.5 10-1, respectively,
for each sub period in the hybrid Hyola-433 and the hybrid Hyola61 variance values were of 0.9 10-2, 0.1 10-1 and 0.2 10-2 and standard
deviation of 0.9 10-1, 0.9 10-1 and 0.4 10-1, respectively.
The Kc values had similar behaviour to those described by
Allen et al. 31, where the intermediate Kc value (1.15) is higher
than the initial values and maturation (0.35), in which these values
are lower than those found for the region of Tangar da Serra,
Mato Grosso, Brazil. The phase which has the highest water
requirement is from start to the end of flowering, which is
considered the critical stage of development for the crop 28.
Conclusions
Based on the results the following conclusions were obtained:
a) The average high temperature during the crop cycle (24.6C)
resulted in a rapid development, ending its cycle with 95 days
for the Hyola-61 hybrid and 90 days for the Hyola-433 hybrid;
b) Thermic sum for the hybrid Hyola-433 was 1,778 GD and 1,816
GD for the hybrid Hyola-61, wherein the thermic sum for the
sub periods E-IF, IF- FF and FF-MF are 1,179, 414 and 185 GD
for hybrid Hyola-433 and 1,203, 436 and 177 GD for the hybrid
Hyola-61, respectively;
c) The period of greatest water consumption of canola was
comprised by the phenological stages of flowering initiation to
the final flowering period, with a consumption of 187 mm for
Hyola-433 and 166 mm for Hyola-61 in suitable water conditions;
d) The hybrid Hyola-433 presented evapo-transpiration values
higher than the hybrid Hyola-61, with water
consumption during the whole cycle of 363 mm,
as well as higher grain yield, with an average of
Eto
Etc Hyolla 433
Etc Hyolla 61
2,332.55 kg ha-1;
e) The average crop coefficients (Kc) for the hybrid
Hyola-433 in the initial phase was 1.09, the
flowering phase was 2.15 and 1.43 for the
maturation and for the hybrid Hyola-61 was 0.76
10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85
in the initial stage, 1.65 in the flowering stage and
DAE
1.33 at maturity.
Figure 4. Crop evapotranspiration (ETc) and reference evapotranspiration (ETo)

as a function of days after the establishment of two canola hybrids.
Table 4. Crop coefficient (Kc) as a function of phenological phases, E =

Emergency, IF = Beginning of flowering, FF = End of flowering, MF =
Physiological maturation of two canola hybrids.
Phenologic
phases
E-IF
IF-FF
FF-MF
L1*
1.02
2.08
1.41
Hyola-433
L2
L3
1.12 1.18
2.18 2.13
1.42 1.38
Kc observed at the lysimeters

Hyola-61
Mean
Kc
L4
L5
L6
L7
1.04 1.09 0.86 0.80 0.61
2.20 2.15 1.76 1.72 1.50
1.52 1.43 1.37 1.35 1.34
L8
0.79
1.61
1.25
Mean
Kc
0.76
1.65
1.33
UNEMAT. Tangar da Serra - MT. 2013. *Lysimeters from 1 to 4 (Hyola 433) and lysimeters from 5 to 8 (Hyola 61).
Acknowledgements
The authors would like to express their
acknowledgement to the National Council for
Scientific and Technological Development - CNPq
for granting a Scientific Initiation scholarship to the
first author and to the Foundation for Research
Support of the State of Mato Grosso - FAPEMAT
for the financial support for the research project
UNIVERSAL EDITAL/ FAPEMAT - No. 009/2011
Process No. 749427/2011, as well as to Celena
Alimentos S/A for the technical support.
235
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1
WFL Publisher
Helsinki, Finland
www.world-food.net
Soil health sustainability and organic farming: A review

Sudarsan Biswas, Md. Nasim Ali*, Rupak Goswami and Somsubhra Chakraborty
IRDM Faculty Centre, RKM Vivekananda University, Ramakrishna Mission Ashrama, Narendrapur, Kolkata-700103, West Bengal,
India. e-mail: sudarsanbiswas@yahoo.com, nasimali2007@gmail.com, goswamirupak@rediffmail.com, som_pau@yahoo.com
Abstract
The need of sustainable agriculture is universal and way to achieving it has been defined through intensive empirical research. Several indicators for
the sustainability of agricultural systems have also been developed. Use of synthetic fertilizers and their effects on crop production, soil health,
environmental quality, biodiversity conservation and self-reliance of farming system have been discussed in the context of agricultural sustainability.
Degradation of soil fertility due to use of synthetic agro-inputs is considered as one of the most important factors affecting sustainability of
agricultural systems. Presence of soil organic matter and soil microbial population are primarily useful indicators of soil health and productivity of
both crops and livestock. A long-term integrated approach will be an appropriate solution for standardizing fertility management in organic farming
considering the complex interactions among different components of this system. A comprehensive and systematic review on different qualitative and
quantitative changes of soil health parameters for improved nutrient management supports these observations.
Key words: Organic farming, soil fertility, soil health, sustainable agriculture.
Introduction
According to the International Federation of Organic Agriculture
Movement 112, the primary objective of organic farming is the
sustainable crop production for maintaining long-term soil fertility
in harmony with natural systems. Therefore, to sustain the
agricultural productivity and environmental quality soil health
management should be the primary concern of all stakeholders of
agricultural development 87. According to Larson and Pierce 59,
apart from nourishing plants, mother earth creates a congenial
atmosphere for the survival of the soil organisms. Therefore, better
soil health is inevitable for better growth and development of
crop leading to higher production 29. Soil health is defined as the
continued capacity of soil to function as a vital living system. The
biological elements are key to ecosystem function within landuse boundaries 23, 50 and are able to sustain not only biological
productivity of soil but also maintain the quality of surrounding
environment. These ultimately promote plant, animal, and human
health. Conversely, Oldeman 71 defined soil degradation as the
process, which lowers the current and/or future capacity of soil to
produce goods or services. Application of excessive chemical
fertilizers, imbalanced nutrient management, and soil pollution are
the main causes of soil degradation 42, 117. Noticeably, the United
Nations Environment Programme (UNEP) sponsored project,
Global Assessment of Soil Degradation estimated more than
two decades ago 38% of degraded agricultural land globally
due to anthropogenic reason 71.
The context is no exception to a country like India, which has a
glorious agricultural background since the Vedic era 9, 85. In Vedic
era, the cultivation procedure was solely based on local resources,
which were completely free from the application of synthetic
compounds 9. The uses of animal manure, oil cakes, green manures
etc. were emphasized in the Vedic Era to maintain the soil fertility82.
The Indian epic Ramayana described the importance of organic
matter in soil management as all dead things rotting corpse or
stinking garbage returned to earth are transformed into
wholesome things that nourish life. Such is the alchemy of mother
earth. Farmers treated the soil as living entity, which was one of
the five elements of life along with air, water, fire, and sky,
collectively known as Panchabhuta 51. In Vedas, soil was
considered as Mother and the human beings were treated as her
Sons: Mata bhumi putro aham prthvya (Atharva Veda
12.1.12) 86. In the modern context, Doran et al. 24, in similar vein,
has described soil as the mother of any agricultural activity.
With the advent of industrial agriculture or so called green
revolution technologies agriculture became dependent on external
inputs, environment became polluted, natural resources depleted,
human health deteriorated, and agriculture itself experienced the
challenge of sustainability 104. There are several evidences, which
prove that agrochemical based, external input intensive agriculture
is not sustainable in long run due to gradual decline in productivity
factor and adverse impact on soil health and quality including soil
organic carbon 90, 102, 104, 110. Thus, the Europeanization of Indian
agriculture, i.e. the introduction of green revolution has threatened
long-term soil health, and at the same time farmers have become
frustrated because of declining agricultural productivity and surged
cost of cultivation.
Organic farming system is based on the management of soil
organic matter, which in turn maintains the physical, chemical,
and biological properties of soil 84, 101. It is now a well-established
fact that organically managed soil exhibits greater soil organic
carbon and total nitrogen, lower nitrate leaching 26 and biological
237
soil quality 61, 116 than conventionally managed soil. Long-term

studies have shown higher soil microbial biomass carbon under
organic than conventional management 39. From the environmental
perspective also, organic practices were claimed to have improved
soil ecosystem quality 28 and long-term farm-level sustainability 46.
objectives of the review were set to study the impact of organic

farming on soil health in terms of physical, chemical and biological
properties of soil, and to compare the outcomes of organic and
conventional soil health management. Second, we established a
search protocol including selection of key words, bibliographic
databases, establishing selection criteria for search engine hit
etc. We used a combination of key words involving organic
farming/organic agriculture and all the soil quality parameters
conceptualized in Fig. 1. Sciencedirect, Google Scholar and DOAJ
were used as the bibliographic database and the first 50 hits
were considered for screening literature. Third, we screened the
results as per the screening criteria, i.e. publication in the last 20
years, article published in English, key word match etc. Fourth,
analysis of the literature was performed through the principle of
qualitative analysis of literature. This employed iterative coding
of themes related to soil quality parameters and their relationship
with organic agricultural practices 17.
Conceptualization of the Impact of Organic Farming

on Soil Quality
Soil quality is often argued as an ecosystem concept that integrates
diverse soil functions, including nutrient supply, which leads to
crop productivity (instead of seeing it as the continued ability of
soil to supply nutrients to crops) 106. This understanding suites
well to organic farming, since there are complex relationships
between different system components and the system
sustainability depends heavily on functioning of the whole
system6. It is then crucial to conceptualize the relationship of
organic farming with soil quality for a sound appreciation of organic
farmings impact on soil fertility. The integration of different
components of organic practices enhancing soil properties to
sustain soil health is shown in Fig. 1.
Impact of Organic Farming on Soil Health

Impact on physical properties of soil: The physical properties of
soil denote structure, texture, bulk density, porosity, water-holding
capacity etc. 43 and positive effects of organic farming on soil
physical properties viz. soil structure, water holding capacity, soil
aeration and soil temperature are well-reported 74, 81, 82, 89.
Papadopoulos et al. 73 notice that organic management can
improve soil structure, organic matter content, and porosity in
soil. Crop rotation is an important component under organic
farming which directly and indirectly influences the physical
structure of soil. Accumulation of organic matter in soil during the
lean phase has a direct influence in the modification of soil
structure 16, 40. The architectural form of different root systems of
several crops included in the crop rotation also helps to modify
the soil structure 13. Mulching of soil surface with organic materials
renders the soil soft, pulverized, and humid that ultimately creates
a congenial environment for beneficial microbes to maintain bulk
density and porosity in the soil 58, 67, 77. Organic farming adds more
organic matter to the soil, which is the basic requirement for
improving soil health 4, 7. Presence of this organic matter in soil
Review Methodology
Although case study or single source of information on a given
issue may generate rich insight, inferring relationships or
developing theory on the issue require information from a wider
universe 78. Literature survey is no exception to this, since it is
also laden with the biasness of social surveys and questions may
well be raised on the validity of making conclusions based on a
single review 76. Thorne et al. 107 propose synthesis-based
methodologies to address this limitation and calls for building
new knowledge from rigorous analysis of existing research
findings, which is clearly distinguishable from single reviews in
terms of collection and treatment of data and detection of literature
omissions 78. Although systematic reviews have widely been
quantitative in nature, recently, qualitative systematic reviews
have been used as useful methodology 68. Following Plummer et
al. 78, a four-step approach for the systematic review and analysis
of literature was undertaken for the present paper. First, the
G. Nutrient Management
Manure (FYM, Vermicompost, Compost etc.),
Bio-fertilizer,
Organic Liquid Manure
Soil Organic
Matter
B. Crop
Rotation
Provides Macro and

micro nutrients
Improves soil texture
Improves soil Structure
Increases soil water
holding capacity
Improves soil micro.
Meso and macro fauna
Soil moisture
conservation
Check soil erosion
HEALTHY
FERTILE
&
a) Healthy
Crop
b) Healthy
Livestock
LIVING
SOIL
c) Healthy
Human
d) Food
Security
e) Nutrition
al
security
C. Mulching
Biomass
D. Live
Mulching
E. Live Fencing
A. Pest
Management
Bio-control
Botanical control
Microbial control
Protect crop from

grazing
Crop protection
No residual toxicity
Figure 1. Integration of different of organic practices for sustainability of soil health.

238
increases its moisture retention capacity 5. A combination of crop

residue mulching and no-tillage increases soil fertility, crop
production, and control soil erosion. Further, residue
decomposition adds organic matter to the soil, which contributes
to reduce the soil hydrological response, increase soil water
repellency that reduces infiltration rates 36. Application of organic
fertilizer not only provides nutrient to the standing crop but also
to the succeeding crop 49. The improvement of soil physical
properties due to organic farming has spatio-temporal dimension
also. Lotter et al. 62 reported that organic farming is better in areas
having extreme rainfall because of the higher absorption and less
run-off of water in the field.
Impact on chemical properties of soil: Unlike conventional
agriculture, organic agriculture follows the natural cycle to add
essential nutrients for quality improvement. Organic farming has
potential to maintain soil fertility and increase organic carbon in
soil 72. Application of different organic inputs like FYM, vermicompost, green manuring etc. ensures both the sustainability of
soil organic carbon and supply of nutrients to the plants 107.
Application of good quality FYM improves the total nitrogen 10
and organic matter in the soil, which is an important substrate of
cationic exchange and the warehouse of most of the available
nitrogen, phosphorus, and sulphur; the main energy source for
microorganisms; and is a key determinant of soil structure 30.
Significant differences and higher values of soil organic carbon,
carbon stocks, and carbon sequestration rate were observed in
organically managed plots compared to non-organic plots 37. It is
undoubtedly an important controlling factor for C:N ratio, total
and available N, N mineralization, soil moisture, microbial activity,
and soil texture 3, 12, 27. Strikingly, several studies have reported
that organically amended soil holds more available N than the soil
receiving inorganic fertilization 14, 52, 91, 95, 105, mainly due to relatively
slower and constant mineralization rates, ultimately decreasing
nitrogen leaching. Organic acids and humus fraction of
decomposing matter are more efficient in releasing phosphorus
and reducing its fixation in soil 48. Nutrient supply through organic
sources also ensures micronutrient availability to the plant 74.
Impact of organic inputs on biological properties of soil:
Although many researchers confine the concept of soil quality to
physical and chemical properties, others value biological parameter
as an important aspect, which should be incorporated in soil
quality assessment process 108. These biological properties are
very important while assessing soil quality 1, 25, 72 since soil quality
is strongly influenced by the flora and fauna present in the soil.
Soil micro-organisms are the living part of soil organic matter
present in the soil 18, 35, 96. The microbial biomass and microbial
activities in soil are crucial to sustain the productivity of soil. For
ensuring consistent release of nutrients to the plants, there is a
need to have balanced ratio of microbial biomass and activity in
soil 72. Organic farming is reported to have enhanced both microbial
biomass and microbial activity by 20-30% and 30-100%,
respectively 103. The soil having high organic matter content
ensures greater microbial activity and greater soil N supplying
power than the soil having less organic matter (which is managed
inorganically) 41, 54. In addition to this, soil organic matter has a
capacity to sink the atmospheric CO2 56 and thereby increasing
the carbon content in the soil, which further enhances the microbial
biomass and respiration 99. It has also been well documented that
the organically managed soil enriched with several beneficial
microorganisms like arbuscular mycorrizal fungi for ensuring
improved crop nutrition and decreasing soilborne diseases 8, 64.
Arbuscular mycorrizal fungi is a special fungal group, which makes
symbiotic association with the plants root system 93 enhancing
plant nutrient uptake and water absorption 32, 97, 98. This mutualistic
relationship primarily helps plant to take more P from the soil and
also protects plants from several diseases 97. As organic farming
increases the microbial activity, leads to increased competition,
parasitism and predation in the rhizosphere, it collectively reduces
the chances of plant disease infestation 53, 115. Application of
quality organic inputs enhances the microbial population in the
soil 15, 33, 75. Organic fertilizer application improved nodule dry
weight, photosynthetic rates, N2 fixation, and N accumulation as
well as N concentration in several crops 49. However, it was also
found that organic agroecosystem management strongly
influences the soil nutrients and enzyme activity while it has lesser
influence on soil microbial communities 11. Several composts like
vermin-compost, farmyard manure etc. are generally used for
nutrient management in organic farming, which ultimately promote
the beneficial macro and micro flora in the soil 94. Application of
organic inputs like human urine, sewage sludge, municipal waste,
deep litter, cattle slurry, cattle manure etc. ensures higher soil
microbial biomass. Hence, household waste and sewage sludge
help to maintain the highest number of colony forming
heterotrophic bacteria in the soil 66, 79.
Organic farming and biodiversity: Biodiversity in soil refers to a
variety of taxonomic groups including bacteria, fungi, protozoa,
nematodes, earthworms, and arthropods present in the soil 108.
Intensification and expansion of modern agriculture have created
a threat to biodiversity worldwide 22, 55 and several studies have
shown that it has been reducing the abundance and diversity of a
host of plant and invertebrate taxa over the past four decades 21, 80,
114
. On the contrary, organic farming helps to maintain biodiversity 20,
44, 63, 69
. After a study of 21-years on agro-economic and agroecological performance of biodynamic, bioorganic, and
conventional farming systems in Central Europe, organic farming
resulted higher biodiversity and enhanced soil fertility than
conventional farming 65. However, recent studies found no effect
of landscape heterogeneity, and differences in any of the measured
soil and microbial variables between conventional and organic
farms; but N mineralization was higher in organic farms 113.
Outcome of Soil Management through Organic
and Inorganic Means
At the end of 40-47 years of dairy farm management in Denmark,
organically managed soil had greater fragment size, aggregate
stability in water, and microbial biomass carbon than
conventionally managed soil 92. Moreover, at the end of 21 years
of long-term crop rotation management in Switzerland, soil organic
carbon and total N were greater under biodynamic than
conventional management, but organic management and
integrated management (combination of manures, inorganic
fertilizers, and herbicides) were moderate 34. Soil microbial biomass
carbon and dehydrogenase activity were greater under organic
than that of conventional management, but basal soil respiration
did not vary between systems. In North Dakota and Nebraska,
239
total and microbial C and N, and mineralizable C and N were greater

under organic than that of conventional management 60. In
Washington State, a comparative study of organic, conventional
and integrated apple production systems from 1994 to 1999
indicated that the organic and integrated systems had higher soil
quality and potentially lower negative environmental impact than
the conventional system 88. Limited studies of intensive organic
farming systems in Australia have generally shown an increase in
soil health compared to conventional practice 45, 100, 111. Lampkin 58
reported that nitrate leaching may be less under organic than
conventional systems.
It is reported that the bulk density of organic soil is less than
the soil which was managed chemically, indicating better soil
aggregations and soil physical conditions owing to increased
soil organic matter 109. There is a 29.7% increase in organic carbon
under organically managed farm (1.22%) as compared to
conventionally managed farm (0.94%) 83. Dehydrogenase, alkaline
phosphate, and microbial biomass carbon were higher in organic
soils by 52.3%, 28.4%, and 34.4%, respectively, as compared to
conventional farms 83.
The multidimensional effects of organic soil management
approaches on soil health are summarized in Table 1.
Conclusions
To achieve sustainable crop and livestock production, the primary
requirement is the maintenance of soil fertility and soil health.
Organic farming systems being highly complex and integrated
biological systems could be the potential technology option to
maintain good soil heath. Organic practice has both direct and
indirect effect to soil properties as it affects more than one
component of the system simultaneously. The previous studies
on the impact of organic practices on different aspects of crop
production, soil health and environment envisage the potentiality
of the organic farming in maintaining the soil health and soil fertility.
Acknowledgements
The financial assistance of Department of Science and
Technologys INSPRIRE Fellowship, Govt. of India, New Delhi, is
gratefully acknowledged.
Table 1. Effects of selected organic farming practices on soil health properties.

Organic Components /
Management
Soil Properties
Physical
Effects on Soil Properties
Chemical
FYM, Vermi-compost,
Green Manuring,
Household waste and
sewage sludge and Soil
Organic Matter
Biological
Physical
Crop Rotation
Chemical
Biological
Physical
Mulching
Chemical
Biological
240
Improve soil structure, porosity, moisture retention capacity etc. in

the soil.
Supply several macro and micro nutrients to the plants.
Increase total nitrogen, organic matter in the soil which is an
important substrate of cationic exchange, is the warehouse of most
of the nitrogen, phosphorus, and sulphur potentially available to
plants
Soil Organic Matter is the main energy source for microorganisms
and it increases the microbial population in the soil.
Soil micro-organisms are the living part of the soil organic matter.
Soil organic matter has a capacity to sink the atmospheric CO2 and
thereby increase in the carbon content in the soil which further
enhance the microbial biomass and elevate respiration
In general, organic fertilizer application improved nodule dry
weight (DW), photosynthetic rates, N2 fixation, and N accumulation
as well as N concentration in several crops.
Household waste and sewage sludge helps to have the highest
number of colony forming heterotrophic bacteria in the soil.
Architectural form of different root systems of several crops
included in crop rotation and which influences the physical
structure of soil.
Crop rotations significantly increased soil pH, available phosphate,
exchangeable K and Ca in soil.
Crop rotation decreases the incidence of soil-born pathogen by
increasing soil chemical properties and soil microbial biomass.
It makes the soil softer, pulverized and humid that ultimately helps
to maintain bulk density and porosity in the soil.
It increases soil fertility, crop production and control soil erosion;
residues become decomposed and add organic matter to the soil.
Better absorption and less run off-of water in the field.
Mulch materials improve soil physicochemical properties, suppress
soil temperature, reduce evaporation and increase the soil moisture.
The mulching materials become decomposed and add organic
matter and other nutrients to the soil.
Mulching helps to increase the population, species diversity and
activity of macro fauna in the soil.
It improves biological activities in the soil and after decomposition
it adds nutrients to the soil.
Citation
Altieri and Nicholls 5, Papadopoulos e
al. 73 and Jannoura et al. 49.
Bharadwaj and Guar 10 and
Parthasarathy et al. 74.
Ewel 30, Smith and Paul 96,

Lal et al. 56, Dalal 18,
Chowdhury et al. 15, Friedel et al. 35;
Peacock et al. 75, Sparling et al. 99,
Poulsen et al. 79 and Mattana et al. 66.
Clement and Williams 16, Chan and

Heenan 13 and Grace et al. 40.
FAO 31.
Dick 19 and FAO 31.
Lampkin 58, Pinamonti 77, Naeini and

Cook 67, Lotter et al. 62, GarcaMorenoa et al. 36, Inyang 47 and
Gbadebor 38.
Agbede et al. 2.
Lal 57, Ojeniyi and Adetoro 70,
Awodun and Ojeniyi 6.
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243
WFL Publisher
Helsinki, Finland
www.world-food.net
Ergonomic assessment of traditional and improved methods of paddy threshing for

drudgery reduction of hill region
Divya Singh 1 and Deepa Vinay 2*
Family Resource Management, College of Home Science, G. B. Pant Agriculture University and Technology, Pantnagar,
Uttarakhand 263145, India. *e-mail: singhdivya546@gmail.com
Abstract
To compare paddy threshing activity undertaken by farm women, an ergonomics study using four methods, viz. traditional method, thresher cum
winnower, VL paddy thresher and motorized thresher, was taken up. Although paddy threshing by using wooden plank is popular among small and
marginal farmers of hill region and about nine percent of paddy is lost due to use of this outdated method therefore time saving motorized thresher
was developed. Split plot method was used to ascertain total number of experiments and response surface methodology (RSM) technique required
for standardization of design was selected. The results revealed that improved thresher was best among all threshing methods, and it reduces the
drudgery and gives maximum production with minimum energy expenditure. New motorized paddy threshing gives maximum production, i.e. 16.69
kg with 9.6 kJ/min energy expenditure, and 299 beats TCCW, 127 beats/min, heart rate 20.94% MSD and 20.86% RPE.
Key words: Agriculture, threshing methods, paddy, drudgery, women, musculo-skeletal disorder.
Introduction
The women are backbone of agriculture workforce and are a vital
part of Indian economy. Over the years, there is a gradual realization
of the key role of women in agricultural development and their
contribution in the field of agriculture, food security, horticulture,
dairy, nutrition, sericulture, fisheries, and other allied sectors. The
role of women in agricultural production in India can never be
overemphasized. They perform crucial roles in the domestic and
economic life of the society. They contribute a higher proportion
of labour in agricultural sector than men. However, they are not
active in decision making. Swaminathan, the famous agricultural
scientist, describes that it was woman who first domesticated
crop plants and thereby initiated the art and science of farming.
The nature and extent of womens involvement in agriculture, no
doubt, varies greatly from region to region 8. Even within a region,
their involvement varies widely among different ecological
subzones, farming systems, castes, classes and stages in the family
cycle, but regardless of these variations, there is hardly any activity
involved. Many studies have shown that the Indian women carry
out the most arduous activity on farm. Modernization of
agriculture is taking place at a faster pace. However, job attended
by women, more or less, remained the same. Though, considerable
work has been done to develop agriculture with major emphasis
on technical and economic achievement, very little attention has
been given to gender issues.
The importance of developing farming technologies relevant to
farm women has only recently been recognized as an extensive
participation of farm women in the field of agriculture and other
allied sectors has been gradually realized in coming years. For
assessment of equipment suitable of farm women, ergonomic
consideration is better option than other as it covers all aspects
244
that deal with anthropometry, assessment of workload, working

environment and safety features/mechanism. For human machine
interface, the shape, size and position, angle (to minimize radial
and lunar deviation of the wrist) need to be considered for fitting
to the capabilities and limit of human operator/workers, so that
their working efficiency could be increased with reduced drudgery.
The equipment or tools designed with proper application of human
factors, improve safety, have flexibility, increase comfort and also
have consumer acceptance. The purpose of women friendly
improved farm tools and implements is to help in reducing drudgery,
increasing utilization efficiency of inputs, ensuring timeliness in
field operations, increasing productivity of women, improve work
efficiency so that they can also get leisure time, conserve energy,
and improve quality of work. The purpose can be fulfilled if one or
more of the above mentioned parameters are met.
Agriculture has changed significantly with advances in science
and technology. Traditional agriculture was mostly dependent on
human labour and draught animal. During the manual operation
by human beings, the load on the operator as well as occupational
and health hazards are found to be increased which lead to impair
the performance of the operator.
Wooden planks are used extensively by Indian farmers for
threshing grain like rice. To develop a cost effective, improved
design for safe operation of threshers based on ergonomic
principles, a motorized paddy thresher for small-scale farmers was
designed by college of Home Science, G. B. Pant University of
Agriculture & Technology, Patnagar, Uttarakhand, India. This
thresher was tested for its threshing capacity, and energy
expenditure, postural discomfort and perceived exertion of the
respondents during threshing activity on one variety, i.e. PR-113
through RSM (Response Surface Methodology). Studies carried

out on comparative performance on manual beating (Traditional
method), thresher cum winnower, VL peddle thresher and new
developed motorized thresher. For comparative performance of all
threshing methods, the present study was planned with the
following objectives:
To study the knowledge and awareness of respondents regarding
available models of paddy thresher.
To assess the practice followed for threshing of paddy to ascertain
the physiological cost of work in compliance to the standards.
Assessment of problem perceived and constraints faced in
various practices followed by small holding families.
The ergonomic evaluation of paddy threshing activity was
conducted with farm women in Tarai and hill region of Uttarakhand
using four methods. All the paddy threshers were operated for 20
min for threshing in the month of December including traditional
method, where women were asked to perform the activity for 20
min. At the end of each experiment, the subjects were given 30 min
rest so that all the physiological parameters regained to their resting
level. The detailed specification of the threshers is given in Table
1.
Selection of subjects: The selected thirty subjects were in the age
group of 20-45 years, because they usually attain their highest
strength level between 20-45 years 9. All the subjects were right
handed, physically fit and were not suffering from any physical
abnormalities to perform the selected activity.
Measurement of muscular stresses: Muscular stresses during

the performance of the activity were measured by recording the
incidences of pain perceived by the subjects from the body map
indicating different parts of the body. Intensity of pain in the
various parts of the body was measured on five-point scale. Score
intensity of pain was: 4 very severe, 3 severe, 2 moderate, 1 mild,
1 very mild.
Measurement and classification of data:
TCCW = CCW + CCR; where
Height 2 (m)
Split-plot design (Fig. 1):

Details of the experiments:
Design: Split plot
No. of subjects: 15
Replication: 3
Trial duration: 15 min
No. of treatments: 4 (Three existing methods and one improved
method)
Parameters for comparison: Heart rate beats/min.
R1
S1
S15
D1
D2
D4
D3
D2
D3
D1
D4
R2
D3
D4
D2
D1
D4
D1
D3
D2
S1
D1
D2
D2
D3
S15
D3
D3
D1
D4
D4
D1
D4
D2
D2
D4
D3
D1
R3
S1
Measurement of physiological cost of work: Circulatory stress

was evaluated from the cardiac cost of work and cardiac cost of
recovery. The cardiac cost of recovery is the total number of heart
beats above the resting level occurring between the end of work
and return to the resting state 15. Heart rate was measured with
polar heart rate monitor and recorded as HR = beat/min. The
following equation was used to calculate the total cardiac cost of
work (TCCW) and physiological cost of work (PCW) 16.
Total cardiac cost of work = cardiac cost of work + cardiac cost of
recovery
Weight (kg)
BMI (kg/m2) =
S15
D1
D3
D2
D1
Replication
I.
Replication: R1
II. Replication: R2
III. Replication: R3
D3
D1
D3
D4
D4
D1
D4
D3
D2
D4
D1
D2
Sub treatments subject

S1: Respondents name
-: Respondents name
S15: Respondents name
Main treatment threshing method

D1: Traditional method
D2: GBPUAT method
D3: VPKAS method
D4: Improved method
Figure 1. Split plot design for the experiments.

Knowledge and awareness of paddy thresher: In the era of
technical advancement, it becomes very essential to make the
agriculture and the farmers technically sound in order to increase
the productivity and further for food security.
AHR = Avg. working HR - Average resting HR

CCR = (Avg. recovery HR - Average resting HR) Duration
Energy expenditure was calculated by using the equation:
Awareness: Data pertaining to awareness of farm families on

traditional method (t-1), thresher cum winnower (t-2) and VL peddle
thresher (t-3), in Dogara village revealed that 93.33% of
EE (KJ/min) = 0.159 HR (beats/min) - 8.72
Table 1. Design specification of all existing threshing methods.

Design specification
Mode of operation
Production/Operation
Height (mm)
Width (mm)
Length (mm)
Weight (kg)
Conventional
method
Manual beating
50 kg
-
Thresher cum
winnower
Manually operated
25 kg
1300
1000
600
40
VL peddle
thresher
Peddle operated
50-70 kg
970
1030
630
35
Motorized
paddy thresher
Motor operated
200.28
1120
560
93
55
245
respondents aware about traditional manual beating method,

whereas 46.66% aware about thresher cum winnower method and
only 26.66% for Vl peddle thresher, respectively (Fig. 2).
Figure 2. Knowledge and awareness of paddy thresher.
Knowledge: Data regarding to knowledge of farm families on

traditional method (t-1), thresher cum winnower (t-2) and VL
peddle thresher (t-3), revealed that 86.66% of respondents were
having knowledge about traditional manual beating method
whereas 33.33 and 20% were having knowledge regarding thresher
cum winnower and VL peddle thresher, respectively (Fig. 2).
Ergonomic assessments of various threshing methods performed
by respondents:
Physiological characteristics: The physiological characteristics
of the respondents selected for ergonomic experiments to carry
out the identified drudgery prone activities of threshing with mean
has been presented in the Table 2. The mean age of the
respondents was 25.53 years and mean height 153 cm. The mean
body weight was 52.4 kg. The computation of Body Mass Index
(BMI), revealed that the average BMI was 21.10% and almost all
the respondents fell in the normal range (Table 2). The mean blood
pressure was 80/115 and pulse rate (72 beats/min) and the body
temperature was 97.13 F, which were in the normal range 3.
Table 2. Physiological characteristic of the respondents.
Physiological
characteristics
Age
Height
Weight
BMI
Mean SD
25.53 3.75
153 3.02
52.4 4.67
21.10 5.54
Physiological
characteristics
Blood pressure
Pulse pressure
Body temperature
Mean SD
80 / 115 7.31 / 6.22
72 7.189
97.13 0.83
Energy expenditure: The average energy expenditure rate of the

selected respondents for threshing activity in the present
investigation was estimated from the heart beat responses using
the equation given by Varghese et al. 18.
Physiological cost of work: The analysis of physiological cost of
work plays a pivotal role in the process of carrying out ergonomic
evaluations of any job. The objective of applying ergonomic
principles in work analysis is to maintain a balance between the

work and the physical capacity of the worker. The physiological
cost of threshing task performed by selecting respondents was
calculated by the equation given by Singh et al. 16.
The physiological classification of different activities has been
proposed by different researchers in the field of work physiology
and ergonomics from time to time. They are either based on the
monitoring of some principle parameters such as heart rate both
during work and recovery, energy cost or on some derived
parameters, i.e. net cardiac cost. The severity of the workload as
per the physiological responses of heart rate and energy
expenditure rate were analysed for the threshing activity of
respondents against the classification given by Astrnad and
Rodahl 1, Huskisson 5, ICMR 6, NIOSH 13 and Varghese et al. 18
(Table 3). The finding of the experiments evident that the average
energy expenditure rate by traditional method was 14.209 kJ/min.
The task of threshing by traditional method fell under the category
of moderately heavy work as per the classification given by
Astrnad and Rodahl 1 and Grandjean 4. When compared with the
classification given by Varghese et al.18, it was found to be heavy
work. Extremely heavy and very heavy classification of workload
based on the energy expenditure rates was given by ICMR 6 and
NIOSH 13, respectively. However, the average energy expenditure
rate by using thresher cum winnower and VL peddle thresher was
10.80 and 10.45 kJ/min, respectively. The task of threshing by
thresher cum winnower and VL peddle thresher also fell under the
category of moderately heavy work as per the classification given
by Astrnad and Rodahl 1. When compared with the classification
given by Grandjean 4, it was found to be light work, whereas it was
found extremely heavy and heavy on the basis of the energy
expenditure rates given by ICMR 6, NIOSH 13 and Varghese et al. 18,
respectively.
The average energy expenditure rate by improvised thresher is
8.542 kJ/min. The task of threshing by improved thresher fell under
the category of light work as per the classification given by
Astrnad and Rodahl 1. When compared with the classification
given by Grandjean 4 and Varghese et al. 18, it was also found as
light work, whereas it falls in the category of heavy workload
based on the energy expenditure rates given by ICMR 6 and
NIOSH 13, respectively (Table 3).
Based on the above observations, it can be concluded that,
among threshing activities, traditional manual beating was found
to be maximum drudgery prone and improved threshing method
was found to be very light work based on the workload
classification given in Table 3.
Perceived exertion as rated by respondents: It is evident from
the results that the perceived exertion is expressed by respondents
during the threshing while performing the task in traditional method
and it was perceived as very heavy (73.33%) followed by 26.66%
Table 3. Physiological workload of respondents by different threshing methods.

Sl. no.
1
2
3
4
5
6
246
Physiological parameters
Average working heart rate (Beats/min)
Average energy expenditure (kJ/min)
Peak heart rate (Beats/min)
Peak energy expenditure (kJ/min)
Average TCCW (Beats)
Average physiological cost of work (Beats/min)
Traditional
method
144.21
14.20
158.57
14.50
1036.16
69.07
Thresher cum
winnower
122.50
10.80
134.60
12.68
780.40
52.03
VL peddle
thresher
120.63
10.45
130.00
11.56
592.30
39.84
Improvised
thresher
108.57
8.54
113.33
8.77
452.16
30.14
of respondents who expressed their exertion as heavy, followed

by 73.33% of respondents who found the task as heavy and 26.6%
of samples who reported their exertion as moderately heavy by
thresher cum winnower. Of respondents 73.33% found the task
moderately heavy by the VL peddle thresher, followed by only
26.66% of respondents who found their exertion heavy. Of
respondents 86.66 % found an improved method of threshing in
the moderately level of exertion followed by 13.33% who reported
as heavy. Similar results were also reported by Kwatra et al. 7 (Fig.
3).
d
s>W /
d
d
d
D
Treshing method
s>
>
D,
s,
Figure 3. Perceived rate of exertion while performing

threshing by different methods.
Assessment of musculo-skeletal disorders: Musculo-skeletal

disorder is the name given to a variety of physical conditions,
which affects joints, limbs and muscles. These are also known as
RSI (repetitive strain injury), ULDs (upper limb disorders) and
WRULDs (work-related upper limb disorders). Specific conditions
include tenosynovitis, carpal tunnel syndrome, writers cramp,
tendonitis and tennis elbow. Symptoms include numbness, pins
and needles, pain or aching, muscle weakness, loss of grip strength
and stiffness. Here, musculo-skeletal disorders are identified as a
principle risk associated with display screen equipment work. The
musculo-skeletal problems and the body pain perceived during
threshing activity with different methods were determined by
administering of Standardized Nordic Questionnaire. All the
selected respondents had given their responses, which were
analysed. In a study, an appreciable number also reported musculoskeletal problems, such as backache and pain in upper limbs and
hand. Risk factors for upper-extremity of musculo-skeletal
disorders include biomechanical factors (force, repetition, posture
and psychosocial factors (job stress) as stated by Morse et al. 11.
Menzel 10 also has brought out the role of psychosocial factors
(job strain, social support at work, and job dissatisfaction) in
musculo-skeletal disorders. He suggested measures for reducing
the incidence of musculo-skeletal disorders and addressing
psychosocial risk factors to prevent delayed recovery. Williams
et al. 19 have carried out a systematic review of psychometric
evaluation of health related work outcome measures for musculoskeletal disorders. Toomingas et al. 17 conducted a study which
revealed that six percent female and sixty eight percent male
reported for musculo-skeletal problems, especially pain in neck
and shoulder regions.
Postural discomfort: The postural discomfort analysis

questionnaire was used to find out the discomforts of different
body parts during the threshing of paddy by different methods.
The questionnaire was given to each subject who were asked to
put the mark on the line, which was then analysed. Mean and SD
values were determined for VAS (Visual Analogue Scale) validated
by Huskisson 5. Similar work was reported by Newell 12. She
conducted studies to find out the comparison of instantaneous
and cumulative loads of the low, back and neck in orthodontists.
She applied the VAS scale ranging from 0-10, with 0 meaning no
discomfort and 10 meaning severe discomfort. Her individual
values ranged between 0.6-9. The neck had the highest mean
value 2.6, with shoulder and lower back closest behind at the 2.3
and 1.6, respectively. The mean and SD results of the postural
discomfort questionnaire were analysed and found among
respondents.
The mean score of the pain felt by the respondents in threshing
of paddy by traditional method was highest in the upper arm
(7.26) followed by lower back (7.2), mid back (6.13) and shoulder
(5.73) among the respondents. The pain and discomfort in legs
was highest, whereas discomfort in buttock thighs and upper
back where comparatively low (Table 4). This revealed that the
workers suffered from pain and discomforts more in upper arm
due to manual beating for a long duration, which also caused
musculo-skeletal disorders. The pain in lower arm was noticed
due to respective motion of lower arms during beating. The
discomfort in shoulder and upper back were due to blending
postures adopted by them at work place.
Table 4. Postural discomfort of the respondents by using different
threshing methods.
Traditional Thresher cum
methods
winnower
MeanSD
MeanSD
Neck
5.2 1.74
1.730.79
Shoulder
5.731.09
4.21.01
Upper back 3.931.09
1.20.41
Upper arm 7.261.03
3.80.86
Mid back
6.131.24
1.530.63
Lower arm
3.40.50
2.660.48
Lower back 7.20.94
2.80.67
Buttock
3.570.75
0.260.45
Thighs
3.640.84
1.10.63
Legs
5.281.38
2.260.79
Body parts
VL peddle
thresher
MeanSD
2.250.44
2.810.75
1.180.40
3.750.68
2.180.54
1.180.40
2.750.85
6.430.89
4.181.16
3.931.12
Motorized
paddy thresher
MeanSD
0.730.45
1.20.41
0.60.50
2.460.83
1.40.50
1.260.45
1.930.79
0.20.41
0.530.51
1.80.67
The mean and SD results of the postural discomfort

questionnaire were analysed. It can be evidenced from the mean
value that the discomfort was highest in the shoulder (4.2) followed
by the upper arm (3.8) among the respondents. The pain and
discomfort in lower back (2.8) was highest, while it was slightly
less in legs (2.26), lower arm (2.66), neck (1.73) and mid back (1.53),
upper back (1.2), thighs (1.1) and buttock (0.26), respectively, which
revealed that the continuous maintaining of static posture and
repetitive movements were the main reason for discomfort in these
body parts.
Table 4 shows that the mean value of postural discomfort was
highest in the buttock (6.43) followed by the thighs (4.18) among
the respondents. The pain and discomfort in upper arms (3.75)
was highest, while it was slightly less in lower back (2.75), neck
(2.25), shoulder and mid back (2.81), respectively, which revealed
that the continuous maintaining of static posture and repetitive
movements were the main reason for discomfort in these parts.
Singh et al. 16 revealed that 86% of female and 68% of male reported
for musculo-skeletal problems, especially pain in neck and
shoulder regions.
247
The mean score of the pain experienced by the respondents in

threshing by improved method is depicted in Table 4, the mean
score of pain felt by the respondents in threshing of paddy crop
with improvised thresher was reported as very slightly moderate
when compared with all other threshing methods. The upper arm
had the highest mean value of discomfort, i.e. 2.46, followed with
shoulder, mid back, lower back and leg as calculated means for all
respondents were 1.2, 1.4, 1.93 and 1.8, respectively. The mean
values of neck and buttock discomfort was comparatively lower,
the values were 0.73 and 0.53, respectively. Deakin 2 studied the
sensitiveness of the Standardized Nordic Questionnaire. It was
administered by both a physiotherapist and an ergonomist to two
similar but separate workstations of a manufacturing plant.
Analysis of the injury-reporting frequencies results that indicating
increased reporting to the ergonomist in two of five upper limb
and trunk body regions considered at one workstation and in one
region at the other. The differences in reporting frequencies
between workstations were also found. Physical demand analyses
performed in each area confirmed that differing job requirements
existed in the two workstations under consideration. Early
identification of potential job-specific injuries by an in-house
ergonomist can initiate appropriate intervention strategies prior
to the onset of chronic injuries. Hence, Nordic questionnaire was
found sensitive, therefore used in the present study.
Technology and user satisfaction matrix: The matrix has three
dimensions, each of which is to be rated at 5-point scale as per
degree of satisfaction experienced, time cost, pace of work and
human power used.
The first section of technology and user satisfaction matrix
defined the manual threshing activity with no technology, which
is performed by farm workers (Table 5). Regarding satisfaction
with time of cost majority of respondents were dissatisfied (86.6%)
and only 13.4% of respondents were fairly satisfied. It indicates
that traditional method was time consuming. Maximum farm
workers (80%) were dissatisfied with the pace of work and only
20% fairly satisfied, which denotes that output to ratio of time is
comparatively very less, whereas posture at work 93.3% were
dissatisfied and only 6.7% were fairly satisfied, since they have
reported pain and discomfort in various body parts. Satisfaction

regarding human power used in manual threshing activity, 86.66%
of farm workers were dissatisfied and 13.4% were very dissatisfied,
hence need of introduction of some other alternatives was felt on
the basis of available matrix.
First available option, i.e. thresher cum winower, shows that
73.33% of workers were dissatisfied regarding cost of time and
26.7% were satisfied with cost of time (Table 6). Whereas
satisfaction regarding pace of work 80% of workers felt dissatisfied
and 20% were satisfied. Maximum farm workers (20%) were fairly
satisfied and 80% were dissatisfied regarding use of human power
used in thresher cum winnower machine (Table 6).
The second available option, i.e. VL peddle thresher, shows
that as far as time cost was concerned, 93.33% of workers were
dissatisfied and 6.7% were satisfied. Of workers 73.33% were fairly
satisfied and 26.7% were satisfied with the pace of work of
machine. Satisfaction regarding use of human power used in VL
peddle thresher machine, 13.4% were fairly satisfied and 86.6%
were found to be dissatisfied (Table 6).
Data on satisfaction with use of time cost with power operated
technology, i.e motorized improvised thresher revealed that
maximum people (86.66%) were satisfied, 13.4% were very satisfied.
When inquired about satisfaction regarding pace of work, 73.33%
of people reported satisfaction and 26.7% were very satisfied.
New improvised thresher is motorized and power operated and it
reduced the human power during threshing activity, therefore
93.33% of people were satisfied and 6.7% of people were very
satisfied (Table 7).
Users acceptability of new improved threshing machine: The
users acceptability of new improved threshing machine was
evaluated on the basis of four parameters: relative advantage with
the use of machine, compatibility of the machine with the workers,
simplicity of the machine and satisfaction obtained from the use
of machine (Fig. 4). Workers felt that machine was time saving
(93.33%), reduces drudgery (86.6%), was advantageous (86.6%)
and labour saving (73.33%) as well.
Regarding compatibility with the use of machine majority of
workers (86.66%) felt that there was no problem in storage of
Table 5. Activities manually perform with no technologies/implements.

Activity
Performed by
Threshing
Farm Workers
Time of cost
Dissatisfied (86.66)
Fairly satisfied (13.4)
Satisfaction
Pace of work
Posture at work
Dissatisfied (80)
Dissatisfied (93.3)
Fairly satisfied (20) Fairly satisfied (6.7)
Human power used

Very dissatisfied (13.4)
Note: Very satisfied: 5, satisfied: 4, fairly satisfied: 3, dissatisfied: 2, very dissatisfied: 1. **Percentage in paranthesis.
Table 6. Manually operated mechanical technology/hand implements.

Name of the technology/implement
User
Thresher cum winower
Farm workers
VL peddle thresher
Farm workers
Time of cost
Satisfied (26.7)
Satisfied (6.7)
Satisfaction with use

Pace of work
Human power used
Dissatisfied (80)
Dissatisfied (80)
Satisfied (20)
Fairly satisfied (20)
Dissatisfied (73.33) Fairly satisfied (13.4)
Satisfied (26.7)
Table 7. Power operated technologies/implements.

Name of the technology/implement
New improvised thresher
User
Farm workers
Time of cost
Satisfied (86.66)
Very satisfied (13.4)
Satisfaction with use

Pace of work
Satisfied (73.33)
Very satisfied (26.7)
Human power used

Satisfied (93.33)
Very Satisfied (6.7)
248
93.33
100
80
60
40
20
0
86.66
86.66
73.33
Respondents
Time saving
Reduces
Advantageous Labour saving
drudgery
Relative advantage of machine
Figure 4. Relative advantage with use of machine.
machine and machine could easily be used (93.33%), 90% felt that
it was easy to operate device and 73.33% of workers agreed for
passed the information regarding usefulness of the machine. The
majority of workers (80%) was satisfied with the working of
machine (Fig. 5). All the workers were very satisfied and they
believed that the improved threshing machine was very convenient
and comfortable for hill region as compared to other threshing
machines. Similar results were reported by Rai 14.
E^
W

W
^
h
K /
Z
D
h

Figure 5. Compatibility and simplicity of machine.
Statistical analysis by split plot method: Heart rate for different

subjects at different methods of paddy threshing: The experiment
was organized as a split plot design where fifteen subjects were
the whole plots and four threshing methods were the subplot
(Table 8). The purpose of this experiment was to test the relationship
of heart rate with the different threshing methods, such as
traditional method, thresher cum winnower, VL paddle thresher
and improved thresher.
All the selected methods of threshing were done for a particular

trial duration, i.e. 15 min, and same crop, i.e. PR-113, were taken.
Three replicates under every treatment combination of different
subjects and different threshing methods were selected. The
experiments were completely randomized by randomly selecting
the subjects for threshing.
In split plot design, grand mean of various threshing methods
was taken as a main plot whereas subjects heart rate was taken as
sub plot. All four designs were found to be significant and the
results showed that the maximum heart rate decreased in following
order: traditional method > thresher cum winnower > VL paddle
thresher> improved thresher, respectively, during threshing. The
minimum heart rate was found in improved threshing method, i.e.
D4, which shows that this thresher was best among all threshers.
Conclusions
Manual beating is common in all paddy growing areas across the
country particularly in cases of marginal land holdings. The
process of paddy threshing by footy is mostly carried out by farm
women and sharp edges of paddy kernels often wound their feet.
In under to minimize drudgery and energy expenditure of farm
women, need to develop user friendly thresher for small
landholding farm families. Response surface methodology was
used for optimization of improved thresher. RSM results obtained
gave a threshing capacity 200.28 kg/hour with 9.6 kJ/min energy
expenditure, and 299 beats TCCW, 127 beats/min heart rate, 20.94%
MSD and 20.86% RPE, respectively.
Acknowledgements
The authors are thankful to All India Coordinated Research project
(AICRP) for providing financial assistance for carrying out the
work under study. Thanks are also extended to the respondents,
without their cooperation this work could not be undertaken.
Table 8. Heart rate for different subject at different method of paddy threshing.
Sl. no.
Number of
subject
Traditional
method
146.07
143.57
143.17
142.07
143.83
143.67
144.37
144.43
145.13
144.10
144.37
144.37
143.43
145.70
145.00
144.22
1
S-1
2
S-2
3
S-3
4
S-4
5
S-5
6
S-6
7
S-7
8
S-8
9
S-9
10
S-10
11
S-11
12
S-12
13
S-13
14
S-14
15
S-15
Mean
Factors
Method (D)
Subject (S)
Factor S at Same level of D
Factor D at Same level of S
Heart rate
Thresher cum
VL peddle
winnower
thresher
126.01
116.57
129.69
123.63
127.17
123.77
132.13
110.53
130.00
122.37
135.67
115.70
124.67
110.07
124.00
127.57
126.67
123.23
134.00
124.07
129.33
121.53
133.67
122.73
128.33
123.23
128.67
121.10
126.33
123.47
129.09
120.64
C.D. at 5% LOS
2.63
1.93
4.09
4.55
Improvised
method
107.60
109.40
110.07
109.83
109.63
108.77
107.93
107.43
106.43
105.77
108.87
109.87
110.07
108.60
108.33
108.57
SE (d)
1.08
0.98
1.95
2.17
Mean
124.06
126.57
126.04
123.64
126.46
125.95
121.76
125.86
125.37
126.98
126.03
127.66
126.27
126.02
125.78
SE (m)
0.76
0.69
2.96
1.54
249
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Varghese, M. A., Saha, P. N. and Atreya, N. 1994. A rapid appraisal of
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Williams, R. M., Schmuck, G., Allwood, S., Sanchez, M., Shea, R. and
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1
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Phenology and green leaf yield of coriander at different sowing dates and harvesting
times
Sagarika Guha 1, Amit Baran Sharangi 2* and Sandip Debnath 3
Department of Spices and Plantation Crops, Faculty of Horticulture, BCKV (Agricultural University), Mohanpur
741252, Nadia, West Bengal, India. 3 Department of Genetics and Plant Breeding, Faculty of Agriculture, BCKV
(Agricultural University), Mohanpur- 741252, Nadia, West Bengal, India. *e-mail: dr_absharangi@yahoo.co.in
1,2
Abstract
Coriander (Coriandrum sativum L.), one of the major seed spices, is valued both for leaves as well as seeds that are often used for culinary and
medicinal purposes worldwide. However, there is limited research on management practices for efficient utilization of this crop for both leaf and seed
production. The principle objective of this study was to optimise sowing time of the crop and harvest times of leaves. The experiment comprised of
seven dates of sowing and three harvest treatments, no harvest, 1 harvest and 2 harvests, at 30 - 63 DAS when each harvest was made on coriander
plants cultivated during months of September through March. The first and second harvests were made at about 15 days interval when leaves were
without any signs of serration and in ideal marketable condition and preferred by the consumers. The time of sowing influenced significantly the days
taken for seedling emergence and the production of the 1st, 2nd and 3rd leaf. Thereafter, November sown seeds were earliest to emerge and produce
leaves among different sowing dates. Both, sowing date and harvest time had significant effect on flowering time, leaf serration, seed setting and
physiological maturity of coriander. Sowing in October resulted in superior leaf yield compared to other sowing dates. Two harvests may be
recommended as a crucial agronomic practice for better green leaf yield.
Key words: Coriander, Coriandrum sativum, sowing dates, leaf harvesting, phenology, green leaf yield.
Introduction
Coriander (Coriandrum sativum. L.), one of the major seed spices,
is grown over 531,000 ha with an annual production of 482,000
tons of fresh leaf at the rate of 0.9 tons ha-1 1. It is a dual purpose
crop, grown for fresh leaf as well as for seed for use as a spice.
The plant has regenerative capacity and hence 2 - 3 harvests can
be made very easily. It was suggested that leaf plucking of
coriander seed crop at early stages can provide an extra income to
its growers 2, 3. For leaf purpose, coriander is grown all the year
round. Harvesting at leafy stage has also been found to improve
the seed yield in coriander. A single harvest at 30 DAS provides
higher yield of seeds 4. The harvested leaves apart from being
used raw as such could also be used for extraction of essential oil.
India being the largest producer as well as consumer of fresh
leaves, there is a persistent demand for fresh leaves in the market.
It has been documented that, seed and green leaf yield had
increased with increase in the levels of nitrogen from 0 to 60 kg
ha-1, whereas seed yield had decreased with the increase in
frequencies of leaf cutting 5. Coriander sown on 1st November and
having treated with nitrogen @ 60 kg ha-1 and cut once (at 30 DAS)
had the highest seed yield and good green leaf yield 6. Several
studies have been reported on the response of coriander to sowing
dates 7-11 as well as to cutting management 5, 12. However, the
information available on leaf production of coriander at different
sowing dates in multiple harvest system is very much limited
especially of coriander crops grown on Gangetic alluvial soils of
West Bengal. Therefore, a field experiment was conducted to study
the phenology and green leaf yield of coriander at different sowing
dates and multiple harvests.

The experiment was conducted at the Horticultural Research
Station, Mondouri, Bidhan Chandra Krishi Viswavidyalaya
(Agricultural University), India. The experiment was laid out in a
Factorial Randomized Block Design with three replications. The
treatments comprised of seven sowing dates: September (D1),
October (D2), November (D3), December (D4), January (D5),
February (D6) and March (D7); and three times of leaf harvests: i)
no harvest (C0), ii) one harvest (C1), and iii) two harvests (C2) over
a period of 7 months between September and March. The
respective dates (DAS) of each harvest are summarized in Table 1.
The seed of coriander variety X-47 (Leafy type) was sown in the
third week of each month in plots 2 m 1.5 m and at a 30 cm 15
cm between and within row spacing, respectively. Standard
management practices were followed throughout the growing
period. In addition to 20 tons of organic manure (FYM) and
inorganic fertilizer, nitrogen, phosphorus and potassium at the
rate of 60:40:20 kg/ha was applied. The crop was irrigated when
required to maintain moisture-stress-free conditions. Inorganic
fertilizer nitrogen was applied @ 20 kg/ha along with full dose of
P and K as basal at the time of sowing. The remaining 40 kg/ha of
N was applied at the time of harvest in treatment C1 and in two
equal doses after harvests in treatment C2. In treatments C1 and
C2, leaves were harvested when they were in ideal marketable
condition without any signs of serration, preferred by the
consumers at the market.
251
Table 1. Sowing times and times of fresh green

leaf harvesting days after sowing
(DAS) of coriander variety X-47 in
Mondouri, WB.
Month
September
October
November
December
January
February
March
Treatment
One cut
Two cut
One cut
Two cut
One cut
Two cut
One cut
Two cut
One cut
Two cut
One cut
Two cut
One cut
Two cut
first leaf (Tables 2 and 3). The first leaf emerged at about 10 DAS
in November sown seeds, which was the earliest one among
different dates of sowing. However, March sown seedlings took
the longest period of time of about 15 DAS. Emergence of 1st leaf
was earlier in early sown crops and was progressively delayed
with delay in sowing time.
Time of cutting
(Days after sowing, DAS)
30
45
42
56
45
63
45
63
42
56
43
56
35
49
Emergence of second leaf: Time of sowing had a significant effect

on the time taken for emergence of second leaf (Tables 2 and 3).
Second leaf emerged at about 12 DAS in November sown plants
while it took 17 days to emerge in plants sown in March. Emergence
of second leaf was earlier in earlier sown crop and later in late
sown crop and was similar to the trend observed for the emergence
of first leaf.
Results
Time of emergence: When 50% of the seed in each plot was
visually seen above the ground level it is called as emergence of
seedling. Time taken for emergence (Tables 2 and 3) was
significantly influenced by the sowing time of coriander seeds.
November sown seeds emerged at about 8 DAS which was earliest
among different sowing dates. However, March sown seeds
emerged in the longest time period of about 13 DAS. No significant
effect of cutting was found in germination on coriander. The most
interestingly, emergence of seedlings had taken shorter time period
where early sowing was done. On the other hand, late sown seeds
were found to take more time for emergence.
Emergence of first leaf: Different time of sowing of coriander
seeds significantly influenced the time taken for emergence of
Emergence of third leaf: Different time of sowing of coriander

seeds influenced significantly the time taken for emergence of
third leaf (Tables 2 and 3). Third leaf emerged at about 14 DAS in
November sown plants and 19 DAS in March sown plants. Time
of emergence of the third leaf was directly related to time of sowing
with emergence of third leaf being delayed with sowing time.
Time of flowering: Both time of sowing and harvest significantly
affected time of flowering(Tables 2 and 3). Flowering started at
about 79 DAS for October sown seeds, which was observed to be
most delayed among the treatments. Nevertheless, early flowering
was noticed for March sown seeds with the shortest time period
of about 64 DAS. Hence, in case of October sown seeds, farmers
may get prolonged vegetative period with ample green leaf
production. Early flowering is not desirable for farmers as green
leaf yield decreases. Two harvests may be recommended as a
crucial agronomic practice for avoiding early flowering and better
green leaf harvest. Flowering was later in early sown plants
compared to that in later sown plants.
Table 2. Mean comparison of effects of sowing dates on phenological variables under study.
Sowing
Date
September
October
November
December
January
February
March
S.Em (r)
CD (0.05)
Germination
(DAS)
First
leaf
10.11c
9.44c
7.89d
9.11dc
10.44c
12.22b
13.11a
0.21
0.59
12.00c
11.56d
9.56e
11.11d
12.44c
14.11b
15.56a
0.25
0.72
Emergence of
Second
Third
leaf
leaf
DAS
14.00d
16.11ba
13.67c
15.56bc
11.56c
13.67d
13.78c
15.11c
13.78c
15.67ba
15.89b
17.89a
17.11a
18.56a
0.29
0.25
0.83
0.71
Flowering
Leaf
serration
71.67ab
78.67a
78.00a
75.67ab
73.33ab
68.67bc
64.00c
2.35
6.72
72.56c
82.56a
78.22ab
75.78bc
75.89bc
79.89ab
75.56bc
1.58
4.52
Time of
Seed
setting
DAS
82.56ab
84.89a
84.56a
83.56ab
82.89ab
79.78ab
78.56b
1.20
3.43
Physiological
maturity
114.22a
117.00a
116.89a
116.78a
115.78a
110.56ab
107.33b
1.99
5.68
Means within a column followed by the same letter are not significantly different at the P 0.05 probability level. Means within a column followed by a different letter
denotes significant differences (P 0.05) as determined by Fishers protected least significant differences.
Table 3. Mean comparison of effects of level of cutting on phenological variables under study.
Harvest
levels
No cut
One cut
Two cut
S.Em (r)
CD (0.05)
Germination
(DAS)
10.14q
10.29pq
10.57p
0.13
NS
Emergence of
Second
leaf
DAS
12.10q
14.10p
12.62p
14.57p
12.29pq
14.10p
0.17
0.19
NS
NS
First
leaf
Third
leaf
Flowering
16.14p
16.00p
16.10p
0.16
NS
66.29r
73.33q
78.95p
1.54
4.40
Time of
Seed
setting
DAS
70.05r
76.33r
78.71q
82.76q
82.86p
88.10p
1.03
0.79
2.96
2.25
Leaf
serration
Physiological
maturity
105.29r
115.71q
121.24p
1.30
3.72
Means within a column followed by the same letter are not significantly different at the P 0.05 probability level. Means within a column followed by a different letter
denotes significant differences (P 0.05) as determined by Fishers protected least significant differences. # NS, not significant at P 0.05.
252
Time of initiation of serrated leaf: Serration occurs more towards

maturity of leaves and are often having lesser consumer demand.
Time of sowing and harvest times significantly influenced the
time taken for initiation of serrated leaf (Tables 2 and 3). Initiation
of serration of leaf was most delayed (82 DAS) for October sown
plants, but it was earliest (72 DAS) in September sown plants.
Two harvests of leaves delayed initiation of serration of leaves by
12 days. Thus, two harvests may be considered as crucial
agronomic practice for avoiding serration and enable better green
leaf harvest. Initiation of serrated leaves had taken longer in early
sown plants. On the other hand, late sown seeds took less time
for initiation of serrated leaves. Early serration is not desirable for
farmers as it reduces marketability of leaves.
Time of seed setting: Sowing time and harvests had significant
effect on time of seed setting (Tables 2 and 3). For green leaf
production purpose early seed setting is not favourable for farmers
as green leaf yield is hampered. Seed setting occurred at about 85
DAS for October sown plants and this was most delayed among
the treatments. However, early seed setting (78 DAS) was observed
for March sown plants. In October sown plants, the vegetative
period is longer which promotes better green leaf production.
Then again two harvests were found to promote prolonged
vegetative growth and result in better green leaf harvest.
Time to physiological maturity: Physiological maturity is that
stage of growth in which the plant has completed all of its
development, including seeds which, when planted, can survive
on their own. It was significantly influenced by different dates of
sowing and different levels of cutting (Tables 2 and 3). Plants
sown in October achieved physiological maturity in 117 DAS which
was relatively later than that for other sowing dates. In contrast,
March sown plants took only 107 DAS for achieving physiological
maturity. In case of October sown plants, late physiological
maturity indicates a prolonged vegetative phase and better seed
yield. Then again two harvests prolonged vegetative phase.
Green leaf yield: Among different dates of sowing (Fig. 1), October
sown plants produced the highest green leaf yield (577.27 g/3 m2).
Among different levels of cutting, the highest leaf yield was found
in two cuttings (775.68 g/3 m2). October sowing and two cuttings
recorded highest leaf yield (788.09 g/3 m2).
Green leaf yield (g/3 sq.m)
One cut
Two cuts
900
800
700
600
500
400
300
200
100
0
Discussion
Coriander cultivation starts with direct sowing of seeds to the
open field during winter. If the environmental conditions, such as
temperature, humidity and sunshine hours in particular, are
unfavourable during this time, emergence could be delayed and
result in uneven plant stand. This, in turn, affects negatively
further growth and development of plants and their yield. There
may be some macro and micro agro-climatic factors responsible
for early or delayed germination. Low temperature seems to favour
germination by promoting the breakdown of reserve proteins in
the seed to the particular amino acids which are necessary for
growth of the embryo 13. The effects of unfavourable temperature
and light on seed germination of celery were discussed.
Germination of seeds was affected by mean maximum and mean
minimum temperatures and minimum relative humidity. It was
further reported that seeds sown on 4th November at 20 20 cm
spacing took minimum time for germination 14. The earliness or
delay in first, second and third leaf emergence are also governed
by several or more environmental factors. Among the factors,
temperature is the main determinant of the rate of leaf appearance
in plants 15-17.
Flowering in coriander was earlier with delay in sowing time 18.
It was reported that flowering was delayed when sown early 19. So
far as green leaf yield is concerned, the result is in accordance
with the earlier reports 20 with regards to sowing dates. The highest
green leaf yield was recorded with 60 kg N ha-1 and two cuttings 5,
21
. It was also opined that lower yield in delayed sowing was due
to insufficient time for vegetative growth as the plant entered the
reproductive phase at a faster rate 22. It has been reported that,
with 2C increase in temperature resulted in 15 to 17% decrease in
grain yield in rice under low temperature conditions 22, 23.
Conclusions
The results showed that time of sowing of coriander seeds
influenced significantly the time taken for germination and
emergence of 1st, 2nd, and 3rd leaves. November sown plants were
earliest in leaf emergence. Sowing dates and different levels of
cutting had significant effect on time of flowering, time taken for
serration, for seed setting and for physiological maturity of
coriander. October sown plants were most superior among the
treatments as all reproductive stages were delayed and plants
with prolonged vegetative phase also gave higher fresh green
leaf yield. Two cuttings may be recommended as crucial agronomic
practice for better green leaf yield.
Acknowledgements
The authors greatfully acknowledge the help and assistance of
Dr M. K. Nanda in calculating protected situation data and of Ms
Samima Sultana during various stages of manuscript preparation.
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Sowing time of coriander
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Figure 1. Green leaf yield of coriander at differential sowing dates and

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Applications of xerophytophysiology in plant production: Potato yield increase

induced by drying the cut trace of seed tuber blocks
Feifei Su
, Hui-lian Xu 2*, Fenglan Li 3, Feifei Qin 4, Yili Chen 1*, Dianqiu L 5, Linshuang Hu 5, Yong Li 5,
Shaopeng Wang 5 and Ying Shi 1
1, 5
College of Agriculture, Northeast Agricultural University, 59 Mucai Road, Xiangfang, Harbin, Heilongjiang 150030, China.
International Nature Farming Research Center, 5632-1 Hata, Matsumoto, Nagano 390-1401, Japan. 3 College of Life Science,
Northeast Agricultural University, Harbin, Heilongjiang 150030, China. 4 Peanut Institute, Shandong Academy of Agricultural
Sciences, 126 Wannianquan Road, Qingdao 266100, China. 5 Virus-Free Seedling Research Institute, Heilongjiang Academy of
Agricultural Sciences, 360 Xuefu Road, Nangang, Harbin 150086, China. e-mail: xuhuilian@163.com, potato@mail.neau.edu.cn
1
Abstract
Seed tubers of potato are usually cut into blocks to reduce seed cost, break dormancy and induce dominance while desiccation of the tuber blocks is
prevented. In the present research, the cut trace of the seed tuber blocks was intentionally dried as a treatment to induce positive xerophytophysiological
regulations. Drying the seed tuber block cut trace induced more production of O2-, which might act as a stimulation signal, and consequently activate
the antioxidant enzyme, SOD. The stimulation of drying the cut trace was not strong enough to activate POD and CAT and showed also no damage
to cells, which was confirmed by unchanged MDA concentration. Properly drying the cut trace induced osmotic adjustment, leaf turgor improvement,
disease resistance and yield increase in the potato crop although severely drying dramatically decreased plant growth and tuber yield. These results
were confirmed similar under all the three different organic material application regimes although the surface soil layer application was more favourite
to the potato crop. Analysis of pressure-volume curve showed that active solute accumulation was higher, symplastic water fraction was larger and
osmotic potential at plasmolysis was lower in plots of drying cut trace compared with the non-dry controls. Leaf turgor potential at both midday and
at full turgid status was maintained higher in plots of drying cut trace. High maintenance of leaf turgor and symplastic water fraction was accountable
for the improved tuber yield. Higher osmotic potential and relative water content at incipient plasmolysis suggested higher stress tolerance in the
potato plants treated by drying cut trace of the seed tuber blocks. Higher water retention ability analyzed by using excised leaf transpiration declining
curve suggested higher resistance to water stress and disease. In conclusion, properly drying cut trace of seed tuber blocks was feasible to improve
potato crops as one of applications of xerophytophysiology in plant production.
Key words: Antioxidant enzyme, osmotic adjustment, potato (Solanum tuberosum), cut seed tuber, signal, turgor, xerophytophysiology.
Introduction
When the adverse environment approaches, the animal can move
to evade, but the plant cannot and must come up to adapt the
changed environment in the original position. The adverse
environment includes windblown and sunburn, drought and flood,
winter cold and summer heat, shading and radiation, salinity and
alkalinity, and disease and insect pests. In the long duration of
adaptation and evolution, the plant has formed a series of
sensation and intrinsic adjustment mechanisms to adapt adverse
environment. In other words, the plant can perceive changes of
its environment, produces signal substances in response to the
stimulation by the environmental changes, and transduces crisis
signals to the gene system, where the corresponding genes are
activated, expressed and transcripted, leading to the corresponding
adjustments or regulations for resistance to the adverse
environment. Even if the plant is intelligent in this sense, its
intelligence is limited. In other words, the plant cannot clearly
distinguish whether the change is true or false, slight or severe,
and long time or temporary. Therefore, one can use the virtual,
mild, short-term and partial stimulations to induce expected
positive regulations in plant. The theories of plant environmental
perception and signal transduction have been well studied using
the model plant of Arabidopsis and detailed mechanisms have
been clarified 1, 2. However, the theories have not been sufficiently
used to food or cash plant production. According to the
abovementioned theories and situations, Xu et al. 3 have proposed
and started a research field in applications of xerophytophysiology
and signal transduction in plant production. Some silent or
sleeping genes can be activated to make the plant more vigorous
and healthier than usual if a drought stimulus is imposed to certain
organ or part of a plant in guarantee without real water stress. The
related techniques include PRD (partial root drying), which means
that the crisis signals and responsive regulations are induced by
placing partial root zone in dry soil while another part of root zone
is in moist soil to ensure a sufficient water supply to the plant 4, 5.
255
Another is the exposure of an underground part, such as

hypocotyl of peanut 6, mesocotyl of sorghum 7, and clove of garlic8,
tulip or gladiolus to light and dry air. In addition, restricted
irrigations, such as sub-irrigation 9 and horizontal infiltration
irrigation 10, can also produce mild drought stimulation to the
plant and induce xerophytophysiological regulations. As a
hardening practice, repeat of soaking and drying of wheat seeds
can also induce drought resistance and health of the wheat crop 11.
As for the potato seed tubers, cutting into blocks is adopted but
drying is usually intentionally avoided 12, 13. In the present study,
the cut seed tubers were intentionally dried before sown into the
soil. The hardening effects were examined in terms of osmotic
adjustment, plant growth, photosynthetic activities, disease
resistance and the final tuber yield.
Experimental site, climate and soil properties: The experimental
field is located in Matsumoto highland area, Nagano Prefecture,
Japan, with an altitude of 700 m above sea. The climate is humid
continental with a precipitation less than the coastal area. The
weather factors are shown in Table 1. Experiments were conducted
in organic field, where chemical fertilizers and pesticides have not
been used for 20 years. The experimental plots were designed in
three rainout shelters, each with an area of 250 m2. As shown in
Table 2, the original fertilization regime was kept unchanged with
three treatments of organic material application, the surface layer
(top 3 cm), the whole soil layer (25 cm) and no application as
control. In this way, the three organic application treatments acted
actually as three different experimental sides and could better
confirm the effect of seed tuber cutting treatment. The fertility
shown by the nitrogen contents and the chemical properties were
different between treatments (Table 2).
Plant materials and cutting treatment of the seed tubers: The
plant material used in the present experiment was potato (Solanum
tuberosum L. cv. Danshaku). Seed tubers of about 80 g were
selected and cut into two blocks. The lower part of 1/4 of each cut
tuber was cut off again to keep the apical dominance. The cut
seed tubers were dried outdoors in shading for 8 h in Experiment
I and for 4 h in Experiment II. Then the treated seed tubers were
sown into soil. In Experiment II, the cut trace was pasted with
bamboo charcoal for fast drying and protection of the cut trace.
Experiment I - design and field management: Exp. I was

conducted in 2011, treatment of organic material application was
taken as the main plot and the cut seed tuber drying as the subplot in a 23 factorial split design. The main plots, soil surface
application, whole soil layer application and non-application as
control, were arranged in a 33 Latin square, and each line with
three plots was arranged in one of the three rainout shelters. One
main plot included three sub-plot treatments: 1) cut tubers dried
properly, 2) cut tubers dried severely, and 3) cut tubers not dried.
Each sub-plot was arranged in three 12 m 0.75 m ridges. Space
between plants was 20 cm. Severely dried cut seed tubers were
characterized by dehydrated and curled edge and black cut trace
(Fig. 1).
Experiment II - design and field management: In Exp. II,
arrangement of the main plot was the same as in Exp. I. One main
plot of organic application included two sub-plot cutting
treatments, 1) cut tubers dried properly, 2) cut tubers not dried, 3)
cut tubers dried properly with charcoal powder pasted, and 4) cut
tubers not dried but with charcoal powder pasted, and each subplot was arranged in four 12 m 0.8 m ridges. Space between
plants was 20 cm.
Measurements of soluble proteins, O2-, activities of SOD, POD
and CAT as well as MDA in Exp II.
Soluble protein: Soluble protein was extracted in 50 mM
phosphate buffer solution (pH 7.5) and determined according to
the Bradford method using bovine serum albumin (BSA) as the
standard 14. Standards were prepared in a range of 0 to 150 g
protein using BSA. Absorbance at 595 nm was measured using
spectrophotometry (Hitachi U-2000). The content of soluble
proteins was expressed as g per g of fresh weight.
Superoxide radical: The free superoxide radical (O2-) was
measured as described by Bissenbaev et al. 15 with some
Figure 1. Drying the cut trace of the seed tuber blocks.

A: Drying (Exp I). B: Properly dried (Exp I). C: Severely dried (Exp I). C: With charcoal pasted
(Exp I).
Table 1. Climate data in the Matsumoto highland plain area, Nagano Prefecture, Japan,
Climate variable
Record high (C)
Average high (C)
Average low (C)
Record low (C)
Precipitation (mm)
Snowfall (mm)
Jan
18.8
4.9
-5.5
-24.8
31.1
230
Feb
21.1
5.6
-5.3
-20.4
42.5
260
Mar
25.9
10
-2
-17.9
73.5
180
Apr
30.9
17.5
3.9
-10.1
86.8
20
May
32.3
22.6
9.4
-2.7
92.5
0
Jun
35.9
25.5
14.8
2.3
135.9
0
Jul
37.9
29
18.9
10.2
132.6
0
Aug
38.5
30.5
19.8
8.0
95.8
0
Sep
35.3
25
15.3
3.0
162.3
0
Oct
31.8
19
7.9
-3.6
89.4
0
Nov
25.6
13.3
1.7
-8.4
52.9
0
Dec
21.5
7.9
-3.1
-19.2
23.3
110
Year
38.5
17.6
6.3
-24.8
1018.8
800
Table 2. Improvements in soil fertility and chemical properties by application of half-decomposed

organic materials.
Plot
Surface
Whole
CK
256
pH
5.84a
5.83a
5.89a
EC
(mS cm-1)
0.05a
0.05a
0.04a
T-C
T-N
g kg-1
38.6a 3.3a
37.7b 2.9b
37.1b 2.7b
NH4-N
NO3-N
0.58a
0.57a
0.65a
1.17a
0.80b
0.57c
P2O5
K2O
mg g-1
19.6a 36.2a
19.3a 36.8a
20a
34.7b
CaO
MgO
283.3a
292.7a
286.5a
45.1a
43.4b
41.1c
CEC
(meq 100 g-1)
18.5a
18.1a
18.3a
modifications by monitoring the nitrite formation from

hydroxylamine in the presence of O2-. Enzyme extract as that used
for soluble protein measurement, 0.5 ml, was mixed with 0.5 ml of
50 mM phosphate buffer (pH 7.5) and 1 ml of 1 mM hydroxylamine
hydrochloride to form the incubation solution. After incubation
at 25C for 1 h, 1 ml of 17 mM sulfanilamide and 1 ml of 7 mM naphthylamine were added to the incubation mixture. The reaction
mixture was incubated again at 25C for 20 min. The absorbance
was measured at 530 nm using the same spectrometer as mentioned.
A standard curve with NO2- was used to calculate the production
rate of O2-. The generating rate of O2- was expressed as nmol
mg-1 protein min-1 and O2- content was expressed as mol kg-1 FW.
SOD activity: The activity of superoxide dismutase (SOD) was
measured using riboflavin-nitroblue tetrazolium (NBT) method
according to Beyer and Fridovich 16 and Chakrabarty et al. 17. The
enzyme extract (100 l) was first mixed with 2.9 ml of 50 mM
phosphate buffer (pH 7.5) containing 10 M EDTA, 13 mM Met,
and 75 M NBT, and then 2.0 M riboflavin was added. The
reaction mixture was illuminated under 135 mol m-2 s-1 for 5 min
and the absorbance was read at 560 nm. One unit of activity was
defined as the amount of enzyme necessary to produce a 50%
inhibition of the riboflavin-mediated reduction of NBT.
CAT activity: Activity of catalase (CAT) was measured by deletion
of H2O2 at 240 nm 1. One unit of CAT gives a H2O2 decomposition
rate of 1 mol min-1 at 25C.
POD activity: Activity of peroxidase (POD) was measured by
oxidation with guaiacol as substrate 19. The reaction mixture
contained 100 l of enzyme extract, 2.9 ml of 50 mM phosphate
buffer (pH 5.5), 1 ml of 50 mM guaiacol, and 1 ml of 2% H2O2.
Spectrophotometrically, the absorption was read at 470 nm for the
product formation. One unit of POD activity was defined as the
quantity of the enzyme that oxidizes 1 M guaiacol per min at
25C.
Malondialdehyde: Malondialdehyde (MDA) was measured using
the thiobarbituric acid method 20, 21. The enzyme extract 1 ml was
mixed with 2 ml TBA reagent (0.6%). The reaction mixture was
heated at 100C for 30 min and terminated in an ice bath and
centrifuged at 4500 rpm for 10 min. The absorbance of the coloured
supernatant was read at 532 nm and corrected for non-specific
absorbance at 450 and 600 nm. The concentration of MDA (mol
L-1) was calculated as 6.45(OD532-OD600)-0.56 OD450. All
assays were conducted at 25C, and enzyme activity was expressed
as unit per mg protein.
Analysis of plant growth dynamics: The dynamics of plant growth
was analysed as changes in dry mass production during the whole
growth period and modelled by G = GM [1-GO (1-t)]-1 [1+(1-t)e(t-) -1
] -GO(1-t) 5. In the equation GM is the maximum increment of
biomass; GO the original biomass of the plant at the beginning;
the constant related with the steep part of the curve; the
constant related with sloping part of the curve before and after
the fast growth; the time point when the biomass increment
reached half amount of GM; and t the growth time (day).
Measurement and analysis of leaf photosynthesis: At the

flowering stage, the second expanded leaf from top of the main
stem was used to measure photosynthesis with LI-6400 (LI-COR
Co. Ltd., Lincoln, NE, USA) at different photosynthetic photon
flux (PPF) from 0 through 250, 600, 800, 1200 and 1600 to 2000 mol
m-2 s-1. The light response curve of net photosynthesis (PN) was
modeled as PN = PC (1 - e-KI) RD, where PC is photosynthetic
capacity; K, time constant, I, PPF, RD, dark respiration 5.
Construction and analysis of pressure-volume curve for
estimation of osmotic adjustment: Analysis of pressure-volume
(P-V) curve was made only in Exp. I but not in Exp. 2. The leaf after
photosynthetic measurement was used for construction of P-V
curve. According to Xu et al. 5, P-V curve was modeled as --1 =
{FT-1-s+a-1[o-(1-)-ap]}e-(1-)+s+a-1[o-(1-)-ap], where, is
leaf water potential; , osmotic potential; , leaf relative water
content; and the subscripts, FT, s+a, IP, sym and apo, mean those
at full turgid status, symplastic water + apoplastic water, incipient
plasmolysis, symplastic water fraction and apoplastic water
fraction, respectively. Calculation of the active ability of osmotic
adjustment shown by CFT and the details of P-V curve analysis
were described by Xu et al. 5.
Estimation of leaf water retention ability by constructing and
analyzing excided leaf transpiration declining curve:
Construction and analysis of the excised leaf transpiration curve
were conducted according to Xu et al. 5. The curve was modeled
as =[sat-SC(1-t)] e-t+SC(1-t)), where, is relative water content
of the excised leaf; the subscripts, sat and SC mean those at
saturated status and at the time point of stomatal closure; and
are constants, respectively, related with the slope of the first part
and the slope of the second part in the curve; and t is the time
from beginning of the transpiration course. The construction and
analysis of the excided leaf transpiration declining curve were
described by Xu et al. 5.
Estimation of disease index: The disease index (DI, %) was defined
as DI = (Number of infected leaves to a certain degree Degree
coefficient)/(total sample leaf number highest degree coefficient)
as described by Xu et al. 5. The main disease was brown leaf spot
caused by Mycosphaerella arachidis.
Statistical analysis: The data from both experiments were
subjected to statistical analysis based on Tukeys multiple
comparisons using the software of DPS Data Processing System 22.
Results
Increased O2- production and antioxidant enzyme activation
were induced but cell damage was not caused by drying cut
trace of the seed tuber blocks: Content of O2-, activities of
superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)
as well as the contents of malondialdehyde (MDA) and soluble
protein were examined and compared between treatment of drying
cut tuber blocks and no-dry control both with and without
charcoal powder pasting in Exp II. Both O2- producing rate and O2content were higher in treatment of drying cut trace of the tuber
blocks than in the no-dry control (Fig. 2). As a consequence, SOD
was activated in treatment of drying cut trace of the tuber blocks
compared to the no-dry control (Fig. 3). However, as shown in
257
400
Dry
Dry
0.10
POD (Unit g-1 FW min-1)
O2- rate (nmol mg-1 protein min-1)
0.12
No-Dry
No-Dry
0.08
0.06
0.04
0.02
0.00
0
40
80
Time (h)
120
200
150
100
50
0
CAT (Unit g-1 FW)
Dry
Dry
No-Dry
No-dry
20
160
No-Dry
No-Dry
25
20
15
10
0
80
Time (h)
120
Fig. 4, activity of POD or CAT showed no difference between

treatment and control. It was suggested that the stimulation was
not strong enough to induce activation of POD or CAT, which
was usually activated by strong stress 23, 24. Although drying cut
trace of the seed tubers increased production of O2-, which was
supposed being damageable to cells, concentration of MDA was
not increased by cutting cut trace of the seed tubers (Fig. 5). The
level of lipid peroxidation can be indicated by MDA concentration 25,
a decomposition product of peroxidized polyunsaturated fatty
acid component of membrane lipid (Fig. 5). Activated SOD might
delete the extra O2- produced by the stimulation of drying cut
trace of seed tuber blocks and consequently avoid the damage to
30
Dry
No-Dry
Do-Dry
20
15
10
5
40
80
Time (h)
120
160
8
Dry
Dry
No-Dry
No-Dry
6
4
2
0
40
80
Time (h)
120
160
Figure 3. Activities of SOD in the cut seed tuber blocks

after sowing (Left: on fresh weight; right: on protein).
80
Time (h)
120
160
2.0
Dry
Dry
No-Dry
No-Dry
1.6
1.2
0.8
0.4
0.0
40
80
120
Time (h)
160
8
7
6
5
4
Dry
Dry
No-Dry
Np-Dry
3
2
1
0
0
40
Figure 4. Activities of POD and CAT in the cut

seed tuber blocks after sowing.
Figure 2. Generating rates and contents of O2- in the

cut seed tuber blocks after sowing.
25
160
MDA (mmol kg-1 FW)
40
Solube protein (g kg-1 FW)
SOD (Unit g-1 FW)
120
Dry
Dry
30
SOD (Unit mg-1 protein)
80
Time (h)
10
258
40
40
30
35
40
Dry
Dry
No-Dry
No-Dry
160
50
O2- content (mmol kg-1 FW)
350
300
250
40
80
Time (h)
120
160
Figure 5. MDA and soluble protein content in the cut

seed tuber blocks after sowing.
cells. Soluble protein content, which was proportional to enzyme

content, was also higher in treatment of drying cut trace of the
tuber blocks than in the no-dry control (Fig. 5).
Tuber yield: The treatment of properly drying cut trace of the
seed tubers significantly increased tuber yield under all the organic
application regimes in both Exp I (Table 3) and Exp II (Table 4).
The increased tuber yield was attributed to both the tuber number
per plant and tuber size. The tuber yield increase by properly
drying the cut seed tuber reached 17.8, 19.0 and 16.3%, respectively,
in surface application, whole soil layer application and nonapplication plots in Exp I. In Exp II, the yield increase by drying
the cut seed tuber reached 12.7% (with charcoal powder) and
19.1% (with charcoal powder), 12.0% and 18.3%, and 15.2% and
16.8% in the abovementioned three organic application plots,
respectively. However, in Exp I, severely drying the cut seed tubers
caused rotting of the seed tubers in soil and dramatically decreased
plant growth and the final tuber yield. In Exp II, pasting bamboo
charcoal onto the cut surface of the seed tuber also significantly
increased the tuber yield with percentage increments of 7.5, 8.2
and 10.7% in the three organic regimes when the cut seed tubers
were not dried but the increments reached 13.6, 14.2 and 11.5% in
the three organic regimes when the cut seed tubers were properly
dried. There were positive additive interaction effects on tuber
yield between seed tuber cut trace drying treatment and cut trace
charcoal powder pasting treatments.
Photosynthetic activities: The parameters obtained from the

photosynthesis-light response curve include photosynthetic
capacity (PC), the maximum quantum use efficiency (YQ) and the
dark respiration rate (RD). Generally in the two experiments in the
present study, PC, YQ and RD were proportional to each other. In
both experiments, PC, YQ and RD were increased under all the three
organic application regimes by the treatment of drying the seed
tuber cut trace. However, PC, YQ and RD were not changed by the
treatment of pasting charcoal powder to the seed tuber cut trace
in Exp II. Leaf colour was proportional to PC and YQ and significantly
increased by drying seed tuber cut trace in both experiments but
not changed by pasting charcoal powder to the seed tuber cut
trace in Exp II (Tables 3 and 4).
Disease incidence: The extent of disease incidence of brown leaf

spot caused by Mycosphaerella arachidis is shown by disease
index (DI, %) (Tables 3 and 4). In Exp I, DI was significantly
decreased in both plots with seed tuber trace properly and severely
dried under all the three organic application regimes. In Exp. II,
both drying the cut trace of seed tubers and pasting charcoal
powder onto the cut trace of the seed tubers significantly decreased
DI. There were positive additive interactions of disease lightening
effect between seed tuber cut trace drying treatment and cut trace
charcoal powder pasting treatments.
Plant growth dynamics: Plant growth dynamics was analysed in

both experiments. In Exp I, the analysis was based on the whole
plant dry mass without separating the shoot and tubers. In Exp II,
dry mass of shoot and tubers was separated for the dynamic
analysis. From the dynamic curves (Figs 6-8) can only be seen in
the differences at the late growth stages, which are consistent
with differences in tuber yield. Nevertheless the detailed
differences in plant growth dynamics can be understood from the
parameters shown in Tables 5 and 6. In Exp I (Table 5), GM, the
maximum dry mass, was higher in drying treatment than in control
and also higher in organic application plots than in non-organic
Table 3. Effects of drying cut trace of the seed tubers on tuber yield, disease incidence and photosynthetic activities
(Exp I).
Organic
Drying
Surface
No dry
Dry
Over-dry
No dry
Dry
Over-dry
No dry
Dry
Over-dry
Whole
No org
Organic
Drying
DryOrg
(g pl-1)
461
543
73
343
408
67
320
372
59
**
**
*
Yield
(kg m-2)
3.8
4.48
0.6
2.83
3.37
0.55
2.64
3.07
0.49
**
**
*
%
100
117.8
15.8
100
119
19.5
100
116.3
18.4
**
**
*
Tuber no
(pl-1)
8.4
9.6
3.7
6.6
7.2
3.3
5.4
6.4
2.6
**
*
*
Size
(g)
54.9
56.8
19.6
52
56.7
20.5
59.3
57.8
22.4
*
*
ns
DI
(%)
13.4
7.3
8.9
16.7
8.4
7.6
17.6
9.2
8.7
*
**
*
PC
RD
(mol m-2 s-1)
25.2
3.3
26.4
3.7
22.1
3.1
23.8
3.8
24.9
4.1
24.9
4.1
21.2
3.5
23.2
3.9
21.4
3.1
*
*
*
*
*
*
YQ
(mol mol-1)
0.077
0.089
0.072
0.069
0.081
0.081
0.066
0.075
0.061
**
**
*
Leaf colour
(SPDA)
65.6
69.7
68.2
62.4
67.3
67.3
60.3
65.6
64.7
*
**
*
PC, photosynthetic capacity; RD, dark respiration; YQ, the maximum photon use efficiency. DI, disease index.
Table 4. Effects of drying cut trace and pasting charcoal powder onto cut trace of the seed tubers on tuber yield,
disease incidence and photosynthetic activities (Exp II).
Organic
Drying
Charcoal
Surface
No dry
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
Dry
Whole
No dry
Dry
CK
No dry
Dry
Organic
Drying
DryOrg
DryCha
DryChaOrg
(g pl-1)
351.9
411.1
396.2
449.2
310.5
337
348.5
397.5
253.5
277.9
293.2
324.4
**
**
*
**
*
Yield
(kg m-2)
2.92
3.14
3.29
3.74
2.58
2.79
2.89
3.3
2.11
2.32
2.43
2.71
**
**
*
**
*
%
100
107.5
112.7
128.1
100
108.2
112
12.9
100
110.7
112
128.2
**
**
*
**
*
Tuber
(pl-1)
7.1
7.8
7.6
7.9
6.4
7
6.6
7.2
5.6
6.0
6.7
7.2
**
*
*
ns
ns
Size
(g)
49.7
52.9
52.1
57.1
48.4
52.1
53.6
5
45.4
48.7
43.8
46.9
*
*
ns
ns
ns
DI
(%)
15.4
10.3
8.3
6.6
19.6
17
17.9
8.3
23.4
20.3
19.9
17.4
*
**
*
*
*
PC
RD
(mol m-2 s-1)
22.8
2.7
22.6
2.9
23.4
3.1
23.8
3.2
21.6
2.8
21.9
2.6
22.8
2.7
22.6
2.9
20.3
2.7
20.1
2.5
20.9
2.8
21.2
2.9
*
*
*
*
*
*
*
**
*
ns
YQ
(mol mol-1)
0.058
0.059
0.066
0.068
0.052
0.053
0.061
0.062
0.052
0.051
0.059
0.061
**
**
*
ns
*
Leaf colour
(SPDA)
59.3
60.6
61.1
62.7
58.2
57.4
60.5
60.3
55.1
55.3
58.1
58.6
*
**
*
ns
*
259
Dry mass per plant (g)
350
C-No-Dry
C-No-Dry-Model
C-Dry
C-Dry-Model
W-No-Dry
W-No-Dry-Model
W-Dry
W-Dry-Model
S-No-Dry
S-No-Dry-Model
S-Dry
S-Dry-Model
C-No-Dry
C-No-Dry-Model
300
C-Dry
C-Dry-Model
W-No-Dry
W-No-Dry-Model
W-Dry
W-Dry-Model
250
200
150
S-No-Dry
S-No-Dry-Model
S-Dry
100
50
0
S-Dry-Model
20
40
60
Time (day)
80
Table 6. Parameters from growth dynamics

analysis (Exp II).
Treat
No-Dry
Dry
Ch-No-Dry
Ch-Dry
Charcoal
Drying
DryCh
Figure 6. Dynamic of total dry mass per plants.
Shoot dry mass per plant (g)
Exp I, C, no organic application; W, whole soil layer organic application; S, surface soil layer
organic application.
60
No-Dry
No-Dry-Model
Dry
Dry-Model
EM-No-Dry
EM-No-Dry-Model
EM-Dry
EM-Dry-Model
No-dry
50
No-Dry-Model
Dry
40
Dry-Model
EM-No-Dry
30
EM-No-Dry-Model
EM-Dry
EM-Dry-Model
20
10
0
10 20
30 40 50 60 70
Time (day)
80 90
Tuber dry mass per plant (g)
Figure 7. Dynamic of shoot dry mass per plants (Exp II).

120
No-Dry
No-Dry-Model
Dry
Dry-Model
EM-No-Dry
EM-No-Dry-Model
EM-Dry
EM-Dry-Model
No-dry
No-Dry-Model
100
80
Dry
Dry-Model
60
EM-No-Dry
EM-No-Dry-Model
EM-Dry
EM-Dry-Model
40
20
0
10
20 30
40 50 60 70
Time (day)
80 90
Table 5. Parameters from growth dynamics

analysis (Exp I).
GM
192.2
225.4
214.2
243.6
278.4
306.6
**
**
*
Go
11.7
11.5
11.9
11.8
11.9
11.6
ns
ns
ns
0.117
0.128
0.126
0.141
0.126
0.146
*
**
**
0.0087
0.0092
0.0087
0.0093
0.0082
0.0094
ns
*
*
63.5
64.5
61.5
64.5
62.5
64.5
ns
*
*
C, no organic application; W, whole soil layer organic application; S, surface

soil layer organic application. GM, the maximum dry mass; Go, base dry
mass; and are constants related with the slope of the steep part and the
sloping parts respectively; , time when the dry mass reached half of GM.
plots with a significant synergistic interaction. GM is consistent

with the tuber yield. Go is the base dry mass of the seed tuber and
not different between treatments. The constant is related with
the slope of the steep part and showed consistence with GM. The
constant is related with the slope of the second sloping part of
the curve and was higher in drying treatment but not different
between organic regimes although there was a synergistic
interaction between organic and drying treatment. The constant
260
0.0072
0.0082
0.0069
0.0075
ns
*
*
50.2
46.8
52.7
51.3
ns
*
*
0.0079
0.0091
0.0082
0.0094
ns
*
ns
61.6
63.3
60.7
62.3
ns
*
ns
is the time when the dry mass reached half of GM and higher in
drying treatment than in no-dry control but showed no difference
among organic application regimes although there was a
synergistic interaction between drying treatment and organic
application.
In Exp II, dry mass was separated into shoot and tuber part.
Interestingly, the change trends of all GM, and for shoot part
dry mass were not consistent but opposite to those of tuber dry
mass. Although GM of tuber dry mass was consistent with the
tuber yield, GM of shoot part was smaller in the treatment of drying
cut tubers than in no-dry control. The leaf area index was not
measured in this experiment, but it could be suggested from the
shoot dry mass that leaf area index was not higher in drying
treatment than in no-dry control. It is also suggested that higher
tuber yield in drying treatment was attributed to its higher
photosynthetic activities as shown in Table 4.
Osmotic adjustment ability: Osmotic adjustment ability was
estimated by analyzing the P-V curves. The parameters are shown
in Table 7 for Exp I and Table 8 for Exp II.
Figure 8. Dynamic of tuber dry mass per plants (Exp II).
Treat
C-No-Dry
C-Dry
W-No-Dry
W-Dry
S-No-Dry
S-Dry
Organic
Drying
DryOrg
No-Dry
Dry
Ch-No-Dry
Ch-Dry
Charcoal
Drying
DryCh
GM
Go
Shoot dry mass

40.4
0.1 0.147
33.5
0.1 0.138
46.1
0.1 0.146
39.4
0.1 0.116
**
ns
*
**
ns
**
*
ns
**
Tuber dry mass
92.2 0.01 0.128
106.2 0.01 0.143
101.4 0.01 0.146
115.6 0.01 0.161
**
ns
*
**
ns
**
ns
ns
**
Turgor maintenance: In both experiments, leaf water potential at

full turgid status (FT) showed no difference between treatments.
However, at the same level of FT, osmotic potential at full turgor
(FT) was lower, which was caused by higher solute concentration
(CFT) in treatments of drying the seed tuber cut trace than in the
no-dry controls. Therefore, leaf turgor potential (PFT) was higher
in treatments of drying the seed tuber cut trace than in the no-dry
controls. The osmotic potential when the symplastic water was
diluted by apoplastic water (s+a) showed the same trends as FT.
The plant water relations including , and P at midday were
similar as those at full turgid status (Tables 7 and 8). On other
words, leaf turgor potentials both at full turgor and at midday
were maintained higher in treatments of drying the seed tuber cut
trace than in the non-dry controls. In Exp I, leaf turgor was also
slightly higher in the surface organic material application treatment
than in other two organic treatments. The mechanisms might be
different from those involved in the treatment of drying seed tuber
cut trace. In Exp II, the effect of charcoal powder pasting was
small but there was a significant synergistic interaction between
cut tuber drying and charcoal powder pasting.
Cell desiccation tolerance: In both experiments, at the point of

incipient plasmolysis, both osmotic potential (IP) and relative
water content (IP) were lower and, as suggested, the desiccation
tolerance was higher in treatments of drying the seed tuber cut
trace than in the no-dry controls. IP was slightly lower but IP did
not change much in treatments of drying the seed tuber cut trace
compared with the non-dry controls (Tables 9 and 10). There was
also a synergistic interaction between drying cut tubers and
applying organic materials or pasting charcoal powder.
Symplastic and apoplastic water fractions: The relative water
fraction in symplast (sym) was higher or that in apoplast (apo) was
lower in treatments of drying the seed tuber cut trace than in the
no-dry controls. This suggested that the cell water was
recompartmented and part of the water in apoplast moved into the
symplast where all biochemical metabolisms occurred. sym was
slightly higher or apo was only slightly lower in surface organic
application plots than in other two organic plots.
Active net increment of cell solute concentration: In the present
research, the active net increment of cell solute concentration at
full turgor or in water-saturated conditions (CFT) in comparison
with the corresponding control was used to the ability of osmotic
adjustment. The values of CFT showed that osmotic adjustment
really induced by drying the seed tuber cut trace. Osmotic
adjustment showed by CFT was higher in plots applied with
organic materials than in non-organic control.
The constants, and : The constant is related with the slope
at the first part and higher in treatments of drying the seed tuber
cut trace than in the non-dry controls although there was not
much difference among organic application regimes. The constant
is the second part and related to the cross of the P-V curve with
the abscissa or the fraction of symplastic water. The value showed
not much difference among treatments (Tables 7 and 8).
Leaf water retention ability estimated by analyzing the excised
leaf transpiration declining curve: The excided leaf transpiration
declining curves in both experiments are shown in Fig. 9. The
curve is clearly different in shape between treatment and control.

In the initial phase of the curve, the relative water content () in
the excised leaf decreases more sharply because of higher stomatal
conductance in treatment compared with control. Then the
decrease in slows down and shows a less deep slope in treatment
compared with control. This might be attributed to thicker cuticular
layer and more wax deposit on the leaf surface in treatment than in
control. The quantified data are shown in Tables 9 and 10.
Relative water content at stomatal closure point (SC): As shown
in Tables 7 and 8, the relative water content at stomatal closure
point (SC) was lower in treatment of drying cut tubers than in nodry control under all regimes of organic material application (Exp.
I) and with or without charcoal powder pasted onto the cut surface
(Exp II). This suggested that stomatal conductance, as one of
plant physiological activities, could performed under and sustain
to lower leaf water conditions, showing higher water stress
tolerance in treatment of drying cut tubers than in control. Both
application of organic materials to the soil and pasting charcoal
powder onto the cut surface did not much affect (SC), but the
synergistic interaction between drying cut tubers and organic
application or charcoal powder pasting was significant.
Constants and showing stomatal and cuticular transpiration:
The constant shows the slope of the initial part of the curve and
is proportional to stomatal transpiration rate. The value of was
larger in treatment of drying cut tubers than in control, and not
much affected by organic application and charcoal powder
pasting, but showed a significant synergistic interaction between
drying cut tubers and organic application of charcoal pasting.
The constant shows the slope of the second part of the curve
and is proportional to cuticular transpiration rate. Opposite to ,
the value of was smaller in treatment of drying cut tubers than in
control. The value of was not much affected by organic
application and charcoal powder pasting, but showed a significant
synergistic interaction between drying cut tubers and organic
application of charcoal pasting. The lower cuticular transpiration
shown by the small value of might be attributed to thicker
cuticular layer and more wax deposit on the leaf surface.
Table 7. Parameters obtained from the P-V curve analysis (Exp I).
Plot
No dry-Surface
No dry-Whole
No dry-No org
Dry-Surface
Dry-Whole
Dry-No Org
Organic
Drying
OrgDry
FT
-0.20
-0.21
-0.21
-0.2
-0.22
-0.21
ns
ns
ns
FT
-0.83
-0.79
-0.81
-0.99
-0.9
-0.86
*
**
*
PFT
0.63
0.58
0.60
0.79
0.68
0.65
*
**
*
s+a
-0.51
-0.47
-0.53
-0.65
-0.6
-0.59
*
**
*
MD
-0.77
-0.76
-0.77
-0.78
-0.74
-0.75
ns
ns
ns
MD
-0.96
-0.94
-0.93
-1.05
-1.01
-1.02
*
**
*
PMD
0.19
0.18
0.16
0.27
0.26
0.26
*
**
*
IP
-1.1
-1.02
-1.05
-1.2
-1.1
-1.13
*
*
*
sym
0.3
0.28
0.28
0.37
0.35
0.34
*
**
*
apo
0.7
0.72
0.72
0.63
0.65
0.66
*
**
*
35
37.9
42.1
51.9
45.1
52
ns
*
*
0.99
0.99
0.98
0.98
0.98
0.99
ns
ns
ns
IP
0.842
0.845
0.842
0.831
0.827
0.821
ns
*
*
CFT
340.3
323.9
332.1
405.9
369
352.6
*
**
*
CFT
65.6
45.1
20.5
*
**
*
-, leaf water potential. , osmotic potential. , leaf relative water content. Subscripts, FT, s+a, IP, sym and apo, mean those at full turgid status, symplastic water + apoplastic water, incipient
plasmolysis, symplastic water fraction and apoplastic water fraction, respectively. and are constants related with the slope at the first part and that at the second part, respectively. CFT,
concentration of osmotic substances at full turgor. CFT, the active increment of CFT compared with control.
Table 8. Parameters obtained from the P-V curve analysis (Exp II).
Plot
No dry
Ch-No-dry
Dry
Ch-Dry
Dry
Charcoal
DryCharcoal
FT
-0.21
-0.21
-0.22
-0.21
ns
ns
ns
FT
-0.8
-0.81
-0.88
-0.945
ns
**
*
PFT
0.59
0.6
0.66
0.735
ns
**
*
s+a
-0.5
-0.49
-0.61
-0.63
ns
**
*
MD
-0.76
-0.77
-0.74
-0.73
ns
ns
ns
MD
-0.93
-0.95
-1.02
-1.05
ns
**
*
PMD
0.17
0.18
0.28
0.32
ns
**
*
IP
-1.04
-1.05
-1.11
-1.16
ns
*
*
sym
0.28
0.27
0.31
0.34
ns
**
*
apo
0.72
0.73
0.69
0.66
ns
**
*
40.1
36.5
48.4
48.5
ns
*
*
0.985
0.991
0.985
0.983
ns
ns
ns
IP
0.844
0.843
0.824
0.829
ns
*
*
CFT
328
332.1
360.8
387.45
ns
**
*
CFT
0
4.1
32.8
59.45
**
**
**
261
Relative water content
1.00
0.96
No-Day
No-Day-Model
Dry
Dry-Model
Exp. I
Exp I
0.92
0.88
0.84
0.80
20
40
60
80 100
Time (min)
120
140
160
180
Relative water content
1.00
0.96
No-Day
No-Day-Model
Dry
Dry-Model
Exp. II
Exp II
0.92
0.88
0.84
0.80
20
40
60
80
Time (min)
100
120
140
Figure 9. Excised leaf transpiration declining curves for

treatment of drying cut tuber blocks and the do-dry control
(Exp I and Exp. II).
Table 9. Parameters from analysis of the excised

leaf transpiration declining curves
(Exp I).
Treat
No-Dry-S
No-Dry-W
No-Dry-C
Dry-S
Dry-W
Dry-C
Drying
Charcoal
DryCh
SC
0.856
0.859
0.864
0.826
0.834
0.842
**
ns
*
0.117
0.123
0.121
0.141
0.134
0.129
**
ns
*
0.000404
0.000408
0.000417
0.000229
0.000236
0.000244
**
ns
**
10 (min)
2187
2165
2120
3944
3728
3610
**
ns
**
Table 10. Parameters from analysis of the excised

leaf transpiration declining curves (Exp II).
Treatment
No-Dry
Dry
Ch-No-Dry
Ch-Dry
Drying
Charcoal
DryCh
SC
0.881
0.858
0.89
0.849
**
ns
*
0.102
0.107
0.101
0.111
**
ns
*
0.000471
0.000235
0.000479
0.000214
**
ns
**
10 (min)
1832
3761
1852
4121
**
ns
**
The time used to dry the excised leaf to 10% of the leaf water
content (10): The value of 10, showing the time used to dry the
excised leaf to 10% of the leaf water content, was larger in treatment
of drying cut tubers than in control.
Discussion
The main objective of this experiment was to confirm the positive
physiological regulations, such as osmotic adjustment, leaf turgor
maintenance, photosynthetic improvement and yield increase in
the potato crop induced by drying the cut trace of seed tubers
according to the principle of xerophytophysiology. Reproduction
of potato is mainly through vegetative or clonal propagation, while
sowing seeds is only used for breeding purpose and for producing
virus-free seed tubers. Compared with the grain seeds, the tuber
262
seed reproduction can make the crops homogeneous and vigorous.

The tubers used as seeds are usually cut into blocks in order to
low the seed cost, break the dormancy and induce the dominance.
In general, the cut trace of tuber blocks need to be healed and if
so, the margin forms a cork layer to prevent from infections. Cutting
seed tubers of potato results in increased respiration during the
healing process. The cut seed tubers are still living organisms
and require oxygen. Therefore, adequate air movement through
the pile of cut seed tubers during storage is essential. Good
aeration helps cut wound healing and prevent seed tuber decay.
Too much air circulation or drying promotes dehydration. Usually,
the cut seed tubers are stored in good aeration but prevented
from being dried. There has been no one who tries to dry cut seed
tubers intentionally. In the present research, we dried the cut seed
tubers intentionally. This is based on the theory of applications
of xerophytophysiology in plant production proposed by Xu 3.
When a short-term stimulation caused by some drought factors,
such as dry soil, high radiation, low humidity or salinity, is imposed
to a part or organ of the plant, the plant perceives the stimulation
and sends the signal to its internal gene system, where the
corresponding gene or genes are activated, and expressed for a
series of positive physiological regulations. A cascade of signal
transduction might be involved in these regulations 15, 26. The
precondition is that the stimulation must not cause real water
stress to the plant. In Exp. I, plant growth and the final tuber yield
were dramatically decreased in the plots with severely dried cut
seed tubers because the drying damaged the seed tubers and
also caused seed tuber rotting as the secondary damage. Properly
drying cut trace of the seed tuber in both experiments induced
osmotic adjustment and the consequent leaf turgor improvement.
Leaf turgor potential is the drying force for cell enlargement in
plant growth and for stomatal opening in photosynthesis
processes. Another consequence of osmotic adjustment caused
by drying seed tubers was cell water recompartmentation between
symplast and apoplast, i.e. part of the apoplastic water moved
into the symplast, where most biochemical metabolisms occurred 27.
Physiological activities are totally improved and consequently
the final tuber yield was increased by the treatment of properly
drying cut trace of the seed tubers. The potato plant might
remember the drought stimulation perceived by the seed tuber
and did not experience any real water stress during its growth
period. Although the cascade of signal transduction and the
responsive gene expression were not examined in the present
experiment, it was clearly found that production of O2-, which
might act as one of the signal chemicals in response to the
stimulation, was increased and, as a consequence, SOD was
activated by drying cut trace of the tuber. However, the drying
stimulation was only a mild stress but not strong enough to
activate POD and CAT, which was confirmed by the unchanged
MDA (Fig. 5). As one of the damages to the cell by stresses, lipid
peroxidation can be indicated by concentration of MDA, a
decomposition product of peroxidized polyunsaturated fatty acid
component of membrane lipid (Fig. 5). Activated SOD might delete
the extra O2- produced by the stimulation of drying cut trace of
seed tuber blocks and consequently avoid the damage to cells.
Without real stress and damage, the stimulation of drying cut
seed tubers resulted in a series of responsive xerophytophysiological regulations such as osmotic adjustment and the
consequent physiological improvement and tuber yield increase.
The results were confirmed in three different regimes of organic

material application in both Exp I and Exp II, which were equivalent
to three different experiments in addition to the repeated
experiments in two years. Although the effect of organic material
application was not the research objective, it was found that
surface layer application of half-decomposed plant residues
improved plant growth and tuber yield of the potato crop. As
shown by the plant growth analysis in Exp II, the interesting result
was that aboveground part dry mass was slightly decreased but
the tuber dry mass was significantly increased by properly drying
the cut trace of seed tubers. It was suggested that the stimulation
as crisis promoted the translocation of carbohydrates to the tubers
in addition to improved photosynthetic activities. Similar
treatments of xerophytophysiological application include properly
drying the wheat seedlings used for transplanting cultivation 28
and repeated soaking and drying of wheat seeds before sowing 11.
In similar ways, the wheat plant has remembered its experience
with the seedling properly dried before transplanting or with the
seed repeatedly soaked and dried before sowing, and the plant
grows in well watered conditions without any water stresses during
its whole growth period. These treatments all belong to
applications of signal transduction and xerophytophysiology in
plant production. Other techniques based on xerophytophysiology include the PRD technique 25, i.e. drying a part of root
zone while keeping the whole plant with good water supply. Part
of the plant root grows in dry soil and perceives the adverse
environment and sends the crisis signal to the gene system with
positive regulations induced. The preconditions are not only the
ensured sufficient water supply to the whole plant but also the
severity of drying treatment or the treatment timing. If PRD is
applied to a cherry tomato crop, pollination and fruit setting are
easily negatively affected with the final fruit yield reduced because
of reduced fruit number 4. As found in an experiment of our research,
partial root zone drying in late growth periods of the potato crop
reduced tuber yield since the growing tubers might be restricted
in the drying soil 5. In addition to PRD, other restricted irrigations
to induce xerophytophysiological regulations include subirrigation 9 and horizontal infiltration irrigation 10, which are all
effective in tomato crops. Other treatments used as applications
of signal transduction and xerophytophysiology in plant
production include sudden exposure of a plant part or organ,
which usually grows underground in soil, such as hypocotyl of
peanut 6, mesocotyl of sorghum 7, and clove or bulb of garlic, tulip
or gladiolus 8 . The sudden stimulations by exposing the
underground plant parts to light and dry air all induce
xerophytophysiological regulations and make the plant grow better
and be healthier and resistant to stresses and diseases. The
precondition is also that no real water stress was caused since the
principal root grows in moist soil although a plant part is exposed
receiving light and dry air as drought factors. Irradiation from
blue LED lamps to the canopy of tomato in the evening also induces
xerophytophysiological regulation and makes the tomato crop
healthier with both yield and fruit quality improved 29. Even in
case of hydroponic cultivation, xerophytophysiological
regulations in greenhouse tomato crops can also be induced with
fruit quality improved by suddenly flushing the root with salt
water or by irrigating the crop with variable solution
concentrations higher in evening and lower in midday 30, or by
maintaining low air humidity in the greenhouse 31. Of course, there
is no real or severe water stress in conditions of hydroponic

greenhouse culture and the stimulations are caused by shortterm rhizosphere salinity as a factor of physiological drought or
by low air humidity as the atmosphere drought with the whole
root system in nutrient solution, where sufficient water is supplied
to the plants. Properly drying cut trace of seed tubers of potato is
among the abovementioned applications of xerophytophysiology
in plant production and many positive effects can be expected
from its use in potato production.
Conclusions
As a mild stimulation without real stress and damage to the plants,
intentionally drying cut seed tubers induced xerophytophysiological regulations such as osmotic adjustment and turgor
maintenance and the consequent improvements in plant growth,
photosynthesis and the final tuber yield. The significant
consequences of the regulations included active increases in
solutes and re-compartmentation of cell water to the symplast. As
one of the signal chemicals, O2- was increased and consequently
the antioxidant enzyme SOD was activated. Thus, cells were
protected without damages, which were confirmed by the
unchanged MDA concentration. High maintenance of leaf turgor
and increase in symplastic water fraction were accountable for
the improved photosynthesis, plant growth and tuber yield. Higher
osmotic potential and relative water content at incipient plasmolysis
suggested higher stress tolerance in the tomato plants treated by
drying cut trace of the seed tuber blocks. Higher water retention
ability suggested higher resistance to water stress and disease. It
was concluded that properly drying cut trace of seed tuber blocks
was feasible to improve potato crops as one of applications of
xerophytophysiology in plant production.
Acknowledgements
This research was supported by National Science and Technology
Support Program (Grant no. 2012BAD06B02), International
Cooperation Project (Grant no. 2013DFR30270), National Natural
Science Foundation for Distinguished Young Scholars of China
(Grant no. 31201470) and National Natural Science Foundation for
Fostering Talents in Basic Research (Grant no. J10106).
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26
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Hydric and physiological monitoring of soybean in oxisol and oxisol incorporated with
biodegradable waste residue
Alexandre C. Salvestro*, Paulo Sergio Loureno de Freitas, Roberto Rezende, Erci Marcos Del Quiqui,
Cludia Regina Dias Arieira, Jos Carlini Junior, Magnun Rodrigo Silva, Vinicius Hicaro Frederico Abe and
Matheus A. Mendes
Universidade Estadual de Maring/CCA/DCA Agronomia, Umuarama - P. R, Brazil. *e-mail: alexandresalvestro@gmail.com
Abstract
The objective of this work was to determine and compare the evapotranspiration of soybean cultivation at vegetative stage in two soils, oxisol and
oxisol incorporated with biodegradable organic waste, for environmental incorporation and water retention, as well as to determine the physiological
conditions of the culture in relation to moisture.The experiment was conducted in Umuarama, PR, at the State University of Maring in the
greenhouse. Sowing was carried out in October 2011 in pots with two hydraulic systems (field capacity and 80% of field capacity). The physiological
parameters, net photosynthetic rate (A) and stomatal conductance (gs), were assessed using IRGA (Infrared Gas Analyze). From A and gs the
efficiency of water use (A/gs) was determined. During the physiological assessments, mass measurements of the pots were carried out through
gravity balance, which together with the data from the meteorological station site determined the evapotranspiration of the cultivation. The
conclusion was that the average evapotranspiration of the culture at the vegetative phase was 3.0 mm day-1 for both moisture and soil conditions and
culture coefficient of 0.86, where oxisol had lower water use efficiency than the soil incorporated with biodegradable waste.
Key words: Evapotranspiration, soil moisture, soy.
Introduction
Studying water dynamic in soil refers to include contradictory
and evolutionary concepts and definitions of several works;
highlighting this dynamic in a research about water availability in
soil disposed as an energy state (potential) for the plant and match
it to a volumetric limit of minimum disposal, deduces a reference
of relative significance to establish criteria of ideal water
replacement for agricultural productivity 7.
Water is the most important and limiting factor to crop
productivity, it has a great influence over several physiological
and chemical processes of the plants, a soil reserve conditioned
to the plant in a disposal potential. According to Ritchie et al. 16,
this water disposal potential is related to the available water
concept, which is a result between field capacity (FC) and
permanent wilting point (PWP). Appropriate water management
must be established through the connexion between available
water in concise parameters and total water availability in the soil,
crop water necessity, and evapotranspiration levels 8.
Water restriction is the main factor that delimits the growing
phenomena, development and production, however, resulting the
minimum amount of water that the soil provides to the plant without
causing irreparable damage in this phenomenon is fundamental.
According to Lima et al. 6, quantitative information about
evapotranspiration and evaporation is necessary in the several
scientific fields that deal with numerous problems concerning water
management.
There are several physiological factors that are affected by the
the available water fraction, among them photosynthesis stands
out. Machado et al. 10 measured photosynthesis rates for three
varieties of citrus, they were from 9.8, 13.0 and 12.8 mol m-2 s-1 in
Valence, Murcott and Tahiti, respectively, indicating different
capacities of electron transport and CO2 assimilation efficiency
according to moisture conditioning.
However, as the soil water potential changes, these
physiological factors will be reflected in plant turgidity, that serves
as an indicator for the permanent wilting point, the preservation
of turgidity 3 demonstrates an effective mechanism of tolerance
to drought when the water potential decreases, emphasizing water
relation of minimum availability to the plant with its physiology
and water reposition quantity appropriate to its productivity.
The identification of a threshold value of water potential, in
which the photosynthetic process would start to decay, would be
greatly relevant, for it would determine a scale of the soil and
plant water status, which would probably result in massive
biological productivity reductions 2.
Reassuring this concept of hydric and physiological relation
Machado et al. 9 adds that weather factors are like photosynthesis
intervenors as a consequence of water stress, and the
photosynthesis rate increases with the irradiance, whose most
elevated values happened at times around 1 pm more than in
other periods, when the temperatures were higher and air relative
humidity was lower, consequently having higher plant
transpiration demand, but, under water deficiency, transpiratory
demand is not answered and therefore, stomatal closure and a
decrease in photosynthesis occur.
In this context, this work aimed to determine soybean culture
evapotranspiration in developing stages and the culture
265
physiological conditions for oxisol and oxisol incorporated into

biodegradable waste residue in an attempt to improve water
disposal to the plant in the respective soil, under two humidity
conditions in vegetative phase (field capacity and 80% of field
capacity) until it reaches the permanent wilting point.
The experiment was carried out in a greenhouse, under natural
conditions day/night (minimum and maximum air temperature, and
relative humidity 13.4, 38.8C and 60%, respectively, inspected
during the pediod of experiment), located in Advanced Campus of
Umuarma (ACU), that belongs to the Agronomic Science
Department, from the Agricultural Sciences Center, in the State
University of Maring, located in Umuarama, PR, in the period
from November 2011 to January 2012. Testing location is 232531
latitude south, 515619 longitude west of Greenwich and 542 m
altitude.
The soils which were used to install the experiment in pots were
oxisol and oxisol incorporated into biodegradable organic waste
residue (25% of pot volume). Physical and chemical characterization
of the soil samples (0-20 cm), macronutrients (P, H+Al, K, Ca,
Mg) and the granulometric composition are presented in Tables 1
and 2 for oxisol, and chemical caracterization for the residue is
The soils in the pots were kept at two moisture levels, around
80% of field capacity and in the field capacity until pre-flowering
stage, when the permanent wilting point was determined. Field
capacity was found after the time in which the refering soils
drainage became insignificant. The plants were irrigated twice a
day, even in periods pre-determined for the beginning of the water
stress in pre-flowering, complying evapotranspirometric demand,
measured in a greenhouse.
During permanent wilting point analysis, in clear days with few
clouds, CO2 assimilation rates as well as transpiration and stomatal
conductance were measured with a photosynthesis measurement
portable system (IRGA, Infra-red Gas Analyzer), these variables
were measured in a period between 10 a.m. and 12 p.m. in all the
plants. Concomitant to physiological assessments at developing
stages, measurements in the post mass were carried out through
gravimetric scale, that together with data from the local weather
station, gave the crop evapotranspiration.
In the measurements, completely expanded leaves were used,
exposed to the sun during all the measurement period 9.
The experiment was randomized blocks in factorial design and
factors were two hydric conditions (field capacity and 80% of
field capacity) and two soil conditions (oxisol with and without
incorporation of biodegradable urban residue) with 8 repetitions.
Table 2. Physical characteristics of oxisol (0- 20 cm).

Sample
identification
0-20 cm
Grit
36
%
Fine sand
36
Silt
03
Clay
25
Table 3. Chemical analysis of biodegradable

urban residue.
Parameter
Calcium (mg kg-1)
Total organic matter (mg kg-1)
Magnesium (mg kg-1)
Total nitrogen (mg kg-1)
Nitrate (mg kg-1)
Nitrite (mg kg-1)
Ammonia (mg kg-1)
Ammoniacal N (mg kg-1)
Total nitrogen Kjeldahl (mg kg-1)
pH
pH (CaCl2)
Potassium (mg kg-1)
Sodium (mg kg-1)
Sulfur (mg kg-1)
Total phosphorus (mg kg-1)
Volatile suspended solids (mg kg-1)
Total moisture (%)
Value
6,152.91
399,798.00
2,501.72
1,166.06
21.85
391.27
339.80
413.13
752.93
8.07
7.91
4,809.57
1,914.01
1,380.10
2,846.49
358,460.00
43.53
The values of Etc varied from 1.6 a 4.7 mm day-1, with average
value 3.0 mm day-1 for both soil conditions, in developing stage
(vegetative stage) until pre-flowering, point of greatest water
demand, where parallely to the work the permanent wilting point
(PWP) was assessed, which results for oxisol 7% weight-based
moisture 7.
This humidity is similar to the one in studies that highlighted
permanent wilting point, for sandy texture soils, according to
Fooladmand 14, Puckett et al. 15, mentioned by Ghanbarian et al. 11,
the values for the relative soil texture class were 0.089 (m3 m-3) and
0.062 (m3 m-3), totalizing in base weight 6.2 and 8.9%, respectively.
The lamina evapotranspiration did not differ statistically in
relation to the humidity variation kept in soil in vegetative stage,
until it reaches PWP in the two soils studied. With three humidity
layers in relation to the field capacity for the wheat in 40-60%, 6080% and 80-100%, Freitas et al. 5 concluded that the change in
humidity layer from 40-60 to 60-80% provided a greater variation
in averages of crop evapotranspiration, than from 60-80 to 80100% maximum water capacity stored in soil.
Still according to the mentioned authors, this fact that ETc
variation is smaller in 80-100% humidity in relation to the other
humidities studied, regardless of the plant development stage,
corroborates with the idea of the work not having different
evapotranspiration in relation to humidities kept until pre-flowering
and the soil conditions.
According to Fig. 1A-B, ETc monitoring in relation to different
water conditionings for oxisol showed the same behavior with a
tendency to maximum evapotranspirometric demand in the
Evapotranspiration values of the crop (ETc) and crop coefficient
beginning of data collection until a decrease in permanent wilting
(kc) for oxisol with field capacity moisture (CC) and 80% of CC, are
point evaluation (7% weight-based volume), this same analysis
presented in Fig. 1 for oxisol with and without incorporation of
was carried out for oxisol incorporated, presenting the same
residue, demonstrating some experimental units averages in
evapotranspirometric monitoring (Fig. 1C-D).
relation to replications.
Between the two respective soil conditions,
evapotranspiration showed the same average whereas
Table 1. Chemical analysis of oxisol.
the weather conditions are the same, it was expected
DPT pH
mg dm-3 g dm-3
cmolc/dm3
only a difference according to oxisol humidity
V%
(cm) CaCl2 Al H + Al Ca Mg
K
SB CTC
P
C
incorporated into biodegradable waste residue, which
0-20
266
4,14
0,15
6,21
1,25 0,88 0,18 3,30 8,51
7,6
6,62
27,1
Evapotranspiration (mm d-1)
Humidity (%)
Humidity (%)
C
Humidity (%)
Humidity (%)
Figure 1. Evapotranspiration behavior of soybean for oxisol with and without residue incorporation at vegetative
stage in relation to humidity: (A) humidity to 80% of field capacity kept at development stage for oxisol, (B)
humidity in field capacity kept at development stage for oxisol, (C) humidity to 80% of field capacity kept at
development stage for oxisol incorporated, (D) humidity in field capacity kept at development stage for oxisol
incorporated.
would increase soil moisture, thus conditioning a greater

evapotranspiration rate, since ETc is directly connected to a Ks
fact that depends on soil humidity, however, such fact was not
statistically proved.
Crop coefficient (Kc) showed average value of 0.81 for both
situations. This Kc value corroborates with Mendes 4, that for the
development stage (vegetative stage) of soybean, Kc values
variated between 0.45 and 1.02 at pre-flowering, the stage with
greatest water needs for the crop. A necessary factor for an
appropriate water management for irrigation projects is knowing
the amount of water to be applied in each culture, and the crop
coefficient (Kc) is an important parameter to make this decision 12.
Kc variation (ETo/ETc) (Fig. 2) has similar behavior for both soil
humidity conditions, this factor happens according to growing
stages of the culture. In the beginning, after plant emergency and
while vegetation is not complete, the values of this coefficient are
small. At the end of vegetative stage, the crop has a full vegetation
and the growing of the root system is stabilized, thus, Kc reaches
maximum values. On the other hand, there is a decrease of values
when reaching maturation phase 13. According to Allen et al. 1, Kc
is also affected by the predominant weather conditions and water
availability in soil.
Concerning the physiological assessment, this study refers that
the intrinsic efficiency of water use for oxisol is smaller than for
the one incorporated into biodegradable waste residue under both
humidity conditions. This result corroborates the idea that content
incorporation in soil provides a greater condition of water
availability, wich does not mean that more water is stored for the
plant, pointing the same evapotranspiration rate for the soil
conditions faced.
Conclusions
Considering the experimental conditions and the response to the
variables studied:
Average reference evapotranspiration at development stage
(vegetative) of soybean was 3.0 mm day-1.
Average crop coefficient for the vegetative stage was 0.81.
There was no significant difference in the evapotranspiration
rate for oxisol with or without incorpotation of biodegradable waste
residue and humidities assessed.
Intrinsic efficiency of water use for oxisol is smaller than for
oxisol incorporated into biodegradable waste residue.
Acknowledgements
Acknowledgements awarded to State University of Maring.
References
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Ghanbarian, A. and Milln, H. 2009. The relationship between surface
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12
Costa, G. F. and Marenco, R. A. 2007. Fotossntese, condutncia
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Humidity (%)
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ETo/ETc
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Figure 2. Crop coefficient (Kc) of soybean for oxisol oxisol with and without biodegradable waste residue
incorporation at vegetative stage in relation to humidity: (A) soybean Kc for humidity to 80% of field capacity
for oxisol, (B) soybean Kc for humidity in field capacity for oxisol, (C) soybean Kc for humidity to 80% of
field capacity for oxisol incorporated, (D) soybean Kc for humidity in field capacity for oxisol incorporated.
268
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Sowing dates and plant density of peanut cultivars in different soil and climatic
conditions of Mato Grosso state, Brazil
Joo Danilo Barbieri 1, Rivanildo Dallacort 1*, Adalberto Santi 1, Kssio De Marco 1, Alcir Jos Modolo 2,
Santino Seabra Jnior 3, Ronicely Pereira Rocha 1 and Rafael Cesar Tieppo 1
1
Campus de Tangar da Serra, Universidade do Estado de Mato Grosso, Brazil. 2 Campus de Pato Branco, Universidade
Tecnolgica Federal do Paran, Brasil. 3 Campus de Nova Mutum, Universidade do Estado de Mato Grosso, Brazil.
*e-mail: rivanildo@unemat.br
Abstract
The aim of this study was to evaluate the influence of plant density on the development, yield components (plant height, number of pods per plant,
thousand grain weight) and yield of peanut (Arachis hypogaea L.) cultivars IAC Runner 886 and IAC Tatu ST in two regions under different soil and
climatic conditions. The experimental design was a randomized block design in a factorial arrangement of 2 x 2 x 2 x 3, with 4 replications, located in
Barra dos Bugres and Tangar da Serra cities, both in Mato Grosso. For each location, the sowing of cultivars (IAC Tatu ST and IAC Runner 886)
occurred on two dates, 12 and 24 of February of 2013, and at three plant densities (5, 10 and 15 plants m-1) with 4 replications. For the Barra do
Bugres city, the highest total weight, yield and productivity of peanut was obtained at density of 10 pl m-1, regardless of the sowing date. However,
in Tangar da Serra city, sowing on February 12 limited the yield due to excessive rainfall during the harvest season, thus sowing held on February 24
provided better agronomic performance for the two peanut cultivars in density of 10 pl m-1. Due to the different cultivar characteristics, the choice
of cultivar depends on the commercial interest of the region and for what purposes the production will be allocated.
Key words: Arachis hypogaea L., plant density, yield.
Introduction
The peanut (Arachis hypogea L.) is a species of the Fabaceae
family, is a crop of great importance in human nutrition, because
of characteristics of oil production (40 - 45%) and protein (20 28%) 1, 2. O il can be used directly in human nutrition and in industry
of paints, preserves and pharmaceutical products, besides the
potential for the biodiesel production 3.
The Mato Grosso state, a large arable territory, focuses its
production in some crops with higher commercial value such as
soybean, corn, cotton, sugarcane, among others, however, the
peanut crop has established itself in the region. In 2012 the crop
productivity was approximately 1630 kg ha-1, however, for 2013
the productivity has reached 2460 kg ha-1, due to the application
of agricultural technology resources 4. This increase becomes
significant in relation to the largest producer, Sao Paulo state,
with cultivated area of 79,000 ha and achieved production of 3400
kg ha-1. The expression is related to the fact that Mato Grosso has
more arable land, but not yet invested in peanuts as a secondary
crop.
The yield potential of peanut is genetically determined and
depends on limiting factors such as soil and climatic conditions,
plant arrangement and incidence of pests or diseases. Among the
factors affecting the productivity of a culture, plant density affects
directly the components production 5. For the peanut crop, plant
density may vary depending on cultivar, sowing date, fertilization
and cultivation region.
Nakagawa et al. 5 also mention that the plant density is one of

the factors that most affect the yield, to exercise direct influence
on production components; thus planting configuration,
characterized by the spacing between and within rows, should
also significantly influence the behaviour of these variables, since
it is a determinant of population density. In general, productivity
grows as plant density increases, until reach a point where
competition for light, nutrients and water begins to limit the plant
development.
Based on the hypothesis that a combination of optimal density
within favourable sowing date gives the highest productivity of
pods and grains, this study aimed to evaluate the agronomic
characteristics and productivity of two peanut cultivars IAC 886
and IAC Runner Tatu ST in different spatial arrangements, in two
sowing dates in the adverse conditions in Tangar da Serra and
Barra do Bugres cities, Mato Grosso.
Experimental site: The study was conducted in two areas under
different soil and climatic conditions, on the rainy season in the
2013 harvest, the experimental area of the Universidade do Estado
de Mato Grosso (UNEMAT), University Campus of Tangar da
Serra - MT, located at 1439' south latitude and 5725' West
longitude, with an average altitude of 310.5 m, according to the
INMET. The another experiment was located in the So Joo
269
farm, in Barra do Bugres city, MT, located at 1504' south latitude

and 5710' west longitude, with an average altitude of 171 m. The
climate of the region is tropical moist megathermic (Aw) according
Kopen. Soil is Rhodic Dystroferric with 689 g of clay per kg of soil
for Tangar da Serra and quartz sand with 121 g of clay per kg of
soil for Barra do Bugres.
Crop management: The planting was done in two sowing dates,

the first on 02.12.2013 and the second day being 02.24.2013, for
both locations. Cultural practices were performed according to
the daily monitoring and the incidence of pathogens or undesirable
plants following recommendations given in the literature of culture,
not to compromise the yield and research.
Meteorological data: The annual average of temperature,

precipitation and relative humidity are respectively 24.4C, 1500
mm and 70 - 80% for Tangar da Serra and 25.3C, 1550 mm and 70
- 80% for Barra do Bugres. The rains are practically concentrated
from October to March, and the dry season occurs between April
and September, with a dry season of six months. The
meteorological data were obtained from stations installed at both
locations.
Phytosanitary control of pest: It was conducted through

preventive and curative sprays, thus for all treatments were applied
the same dosage and the same products, indicating the period
after emergence, so for sowing the treatment of seed with fipronil
(0.3L cp/100 kg of seed) at 8 days after emergence (DAE)
application of imidacloprid (30 g cp/100 L); imidacloprid at 28 DAE
(30 g cp/100 L) + strobilurin + triazole (0.01 L cp/100 L) + foliar
fertilizer (0.4 L cp/100 L) + methomyl (0.02 L L cp/100 ); 35 DAE
pyrethroid (0.02 L cp/100 L) + strobilurin + triazole (0.01 L cp/100
L); at 43 DAE anthranilamide + lambda-cialorina (0.01 L cp/100 L)
+ foliar fertilizer (0.4 L cp/100 L); 55 DAE strobilurin + triazole (0.01
L cp/100 L) + pyrethroid (0.02 L cp/100 L) + foliar fertilizer (0.4 L
cp/100 L).
Treatments and experimental design: The experimental design

was randomized blocks, arranged in factorial design 2 x 2 x 2 x 3,
located in Barra do Bugres and Tangar da Serra cities, both in
Mato Grosso. For each location, the sowing of the two peanut
cultivars (IAC and IAC Runner Tatu ST 886) was conducted in
two sowing dates, 12 and 24 of February, respectively, using three
plant densities (5, 10 and 15 plants m-1) with 4 replications (Table
1). The plots consisted of 4 rows of 4.0 m, spaced 0.45 m, totaling
7.2 m2. To evaluate and data collection the area of two rows 2.0
m long was used.
Table 1. Description of the structure of treatments in different
spatial arrangements of plants per linear meter.
Treatments
D1
D2
D3
Spatial arrangements
(Plants m-)
5
10
15
Explored area
per plant (m)
0.090
0.045
0.022
Plants m
Plants ha-1
11.11
22.22
33.33
111.100
222.200
333.300
Soil management: Before sowing, with adequate soil moisture

conditions, conventional tillage was performed with two heavy
disking and one levelling harrow. Soil analysis (Table 2) was
performed. The amount of lime and fertilizer needs was applied as
recommended by Quaggio and Godoy 6. Liming was performed 60
days before sowing for the two sites studied in doses of 932 kg
ha-1 for Barra do Bugres and 1360 kg ha-1 for Tangar da Serra, of
dolomitic limestone with PRNT of 80%. This was applied entirety
and incorporated with a leveling harrow depth of 15 cm. Fertilization
was based on interpretation of soil chemical analysis, followed by
recommendations for the peanut crop for the state of Mato Grosso 25.
A week before sowing 30 and 60 kg ha -1 of P 2O5 (simple
superphosphate) 60 and 30 kg ha-1 of K2O (potassium chloride),
10 and 10 kg ha-1 of N (urea), respectively, were applied for Barra
do Bugres and Tangar da Serra.
Evaluated agronomic characteristics: The following parameters

were evaluated at vegetative growth of the plants: Emergency
(E); first tetrafoliadas leaf (FT); first branches (PR); flowering (FL);
appearance of gynophore (AG); stretching of gynophore (ALG);
training pod (FV); end of flowering (FF) and full maturation of the
pod (MCV). Table 3 shows the description of the phenological
stages of peanut cultivars IAC Runner 886 and IAC Tatu ST,
sown at two dates and growing in two areas of Mato Grosso, in
the agricultural year of 2013. According Gonalves 7, at
phenological aspect, the stages of growth and development among
the Valencia genotypes are particularly defined, but may vary
depending on the location and climatic conditions, especially
temperature, where it is grown. Production parameters, such as
plant height (AP); fresh weight of 1000 grains (P1000), pod weight
(PVAG); income (REND); number of pods per plant (NVP); total
weight of pods (PTOT); productivity in the bark ( PRODC ) and
productivity of grain (PRODG ), were determined at the end of the
crop cycle.
Statistical analysis: The data were subjected to analysis of
variance by F test using the program ASSISTAT 7.7 beta. When
the F value was significant at 1 or 5% of probability, the Tukey
test for comparison of means and the factorial for developments
of the variables wre applied.

Variable climate: The climate elements are the key to enhance the
productivity of a species 8, therefore the mean values of temperature,
relative humidity, solar radiation and precipitation from seeding to
harvest of the first and second sowing date
are shown in Fig. 1A-B.
Table 2. Chemical analysis* of soil in the depth of 0 - 0.2 m from the experimental area of
It was observed strait distinction to the
Unemat in Tangara da Serra and from the farm called So Joo in Barra do Bugres.
mean values of temperature, humidity and
pH
P
K
Ca+Mg
Ca
Mg
Al
H
H+Al
S
CTC
V
M.O
solar radiation between the two regions,
H2O
Mg dm3
cmolc dm3
cmolc dm3
%
g dm3
but for precipitation there was a significant
Barra do Bugres
5.20 2.90 35.00
1.07
0.66 0.41 0.13 1.87 2.00 1.20 3.20 36.70 13.00
difference between one and another ten
Tangar da Serra
days time, it directly interferes on the
6.30 1.30 50.00
3.90
2.83 1.07 0.00 4.50 4.50 4.00 8.50 47.20 32.00
determination
of sowing date in relation to
*PLANTE CERTO - Analysis of: Soil, Limestone, Water, Nematode, Fertilizer, Feed, Salt and Fabric Leaf LTDA, Varzea Grande - MT.
(November/2012 and January/2013).
270
Table 3. Description of phenological stages of the peanut cultivars IAC Runner 886 and
IAC Tatu ST (days after sowing, DAS), grown at different sowing dates in the
Tangara da Serra and Barra do Bugres areas, MT (2013).
Some cultivars begin flowering between

30 and 35 days after planting, and
continue to produce flowers until
almost 85 days 11.
Tangar da Serra
Barra do Bugres
The reduction of the duration of
Runner
Tatu
Runner
Tatu
Symbol Description
flowering without compromising the
12
24
12
24
12
24
12
24
Feb.
Feb.
Feb. Feb.
Feb.
Feb.
Feb.
Feb.
yield has been an important
E
Emergency
9
13
8
11
8
9
6
7
characteristic sought in the breeding of
FT
Trifoliate leaves
18
18
16
17
13
15
12
13
peanuts. This is because, with an
PR
First branches
25
25
24
23
20
24
19
21
indeterminate plant growth, and the
FL
Flowering
28
36
26
34
29
34
25
27
fruits with hypogeal nature, there is no
AG
Appearance of gynophore
38
45
32
43
36
42
31
33
ALG
Stretching gynophore
52
53
46
52
47
53
45
46
way to delay the harvest, for a greater
FV
Training pod
68
75
63
74
69
75
58
61
yield of immature pods without incurring
FF
End of flowering
83
91
77
85
88
93
75
78
losses or sprouting or rotting fruits that
MCV
Full maturation of the pod
110
115
98
110
113
116
97
102
CO
Harvest
122
137
110
126
127
129
117
121
are already at stage of full maturity. The
ideal in a peanut plant is that it has
the growing site. Considering that the water requirement can interfere concentrated production of flowers, with flowering lasting around
in the crop establishment, reducing the plant population, the six weeks. This would provide greater uniformity in the number of
thousand grain weight and yield 9, the sowing date has been studied mature pods at the end of the cycle, as well as reduction of the
by many authors, as this may influence the accumulation of dry losses in production 12.
matter of shoots, the harvest index and the mass of pods, number of
As the plant density rises there is an exponential increase over
pods per plant on seed quality and final yield of peanut 7, 9.
plant height and weight of pod and yield, however, for number of
pods per plant, total weight and productivity there has been a
Phenological stage: Assumption and Escobedo 10, estimating positive linear growth. This behaviour is probably due to the
the water requirement of the peanut crop in Eutrustox on the lower competition among individuals and greater availability of
UNESP campus in Botucatu, noticed that after emergence (7 DAS) factors of production in the lower plant density. These results are
until the beginning of flowering (27 DAS), the crop consumed 67 similar to those found by Gonalves 7, Romanini 13 and Bulgarelli 14
mm. During the flowering of peanut (28 - 62 DAS), judiciously the that the highest number of pods per plant was in lower plant density.
most demanding phase in water demand, there was a consumption
of 166 mm. After flowering (63 DAS) until the end of the grain
Analyzed variables: The results of the analysis of variance for
filling stage (90 DAS), the water demand was 124 mm. Between plant height (Table 4) show that there are no statistically significant
physiological maturity (91 DAS) until harvest (109 DAS), there differences (p < 0.05) for the effect of different sowing date. The
was a consumption of 46 mm, totaling 403 mm for the entire cycle. mean of plant height was 60.31 cm considering the two cultivars,
It was observed that the flowering cultivar Runner in Tangar approximate values observed by Assuno et al. 15. For the sowing
da Serra in E1 occurred at 28 DAS, for E2 has occurred at 36 DAS, date variable there was increase in plant growth due to satisfactory
the difference between sowing dates repeats for Barra do Bugres hydro and thermal conditions, however, for the plant density there
(Table 3), but follow the same periods, which leads us to understand were larger plants in D2 that differ statistically for other densities.
that the location for this cultivar did not affect the flowering date. These results corroborate with data obtained by Belletini and
The same can be observed for all phenological stages in relation Endo 16, spacing and plant density influenced the plant height
to sowing date where E1 is closer to the ideal for the crop, in and in higher densities branches were shorter due to competition
relation to the location, Tangar da Serra showed more favourable between plants. Regarding cultivars, the Tatu by presenting erect
precocity of cultivars.
type was statistically different from Runner with prostrate growth17.
Among the characters of the peanut plant flowering, the period
For the site variable there was no significant difference for
of flowering is one of the most important in the effective production plant height and yield. Bolonhezi et al. 18 observed that vegetative
of pods, since the shorter the period that starts the beginning to the and genetic or quantitative characteristics of the plants were not
end of flowering, the higher the recovery in the phase filling of the changed by different types and soil management, however, for
pods as a result of reduction in the number of immature pods. the qualitative characteristics as productivity, number of pods
A
Figure 1. (A) Decennial (ten days time) averages of precipitation and relative humidity; (B) temperature and solar
radiation between 10 of February and 21 of July of 2013 for the Tangar da Serra (TGA) and Barra do Bugres (BB).
271
Table 4. Mean values for plant height (AP); number of pods per plant (NVP); total weight (PTOT);
thousand grain weight (P1000); bark productivity per ha-1 (PRODC); grain productivity
per ha-1 (PRODG); yield (REND); coefficient of variation (CV) and F value.
Evaluated characteristic
Barra do Bugres
Tangar da Serra
F value
AP
60.46a
60.16a
0.27ns
12/02/2013
24/02/2013
F value
66.72a
53.90b
489.43**
IAC Tatu ST
IAC Runner 886
F value
68.43a
52.18b
787.22**
D1 (5 pl m-1)
D2 (10 pl m-1)
D3 (15 pl m-1)
F value
CV (%)
59.87b
62.33a
58.73b
13.47**
4.70
NVP
PTOT
P1000
Sites
14.67a
183.43a
485.65b
6.89b
123.14b
562.06a
781.91** 128.49** 175.30**
Sowing dates
11.36a
176.51a
556.61a
10.20b
130.06b
491.10b
17.19**
76.22**
128.82**
Cultivars
10.25b
139.81b
404.33b
11.30a
166.76a
643.37a
14.23**
25.67** 1715.54**
Densities
7.57c
85.73c
523.65a
11.72b
178.48b
531.22a
13.05a
195.64a
516.68a
140.41** 164.76**
2.11ns
12.65
17.00
5.40
PRODC
PRODG
REND
2292.96a
1539.26b
128.49**
1579.42a
1106.31b
83.50**
68.98a
68.01a
2.93ns
2206.38a
1625.85b
76.22**
1611.78a
1073.95b
107.92**
72.83a
64.16b
230.45**
1747.67b
2084.55a
25.67**
1158.76b
1526.98a
50.58**
64.60b
72.39a
186.14**
1071.66c
2231.09b
2445.58a
164.76**
17.00
755.86b
1583.60a
1689.14a
129.94**
18.89
69.59a
67.98ab
67.91b
3.68*
4.08
Means followed by the same letter do not differ significantly by the Tukey test ** p < 0.01; * P < 0.05; ns (not significant).
colour for Runner 3. The Runner cultivar had higher productivity

and yield than Tatu, which encouraged the producers to deploy
such cultivar in large-scale farming 13.
As the average plant density, factor very researched for
productivity of any crop, Nakagawa et al. 5 affirm that this
factor is predominant for plant height variable, determining
the D2 as the most satisfactory, but for production variables
despite the competition reduced the income, an increase in
grain productivity and total weight results are similar to
those obtained by Leonel et al. 21, who observed higher
productivity in density until 17 plants m -1.
With regard to the interaction between the factors of production
and the proposed treatments, the influence of variables within
each level of site x sowing date x cultivar x density (Tables 5 -11),
represented developments.
For the variable plant height (Table 5), there was no significant
difference in the different sowing dates in relation to the sites,
however, Ep1obtained higher cultivars and densities, these results
are similar to Centurion 22. Due to cultivar Tatu being erect, it was
better in relation to Runner in this aspect. As expected and
compared with Leonel et al. 21, the best density observed for
locations, sowing dates and cultivars was the D2 that simultaneously
shows the best result.
In studies assessing plant height with the interference of plant
density, Peixoto et al. 9 concluded that at lower densities there is
a smaller plant height, this happens while the
Table 5. Split of plant height (cm) at two sites with two peanut cultivars, two sowing
plants compete for light, but from a certain
dates and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar density the competition for nutrients is
da Serra; Sowing date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) greater, which leads to a nutritional deficiency
- IAC Tatu ST; Cultivar 2 (C2) - IAC Runner 886; Density 1 (D1) - 5 pl m-1;
and it becomes impossible to achieve
Density 2 (D2) - 10 pl m-1; Density 3 (D3) - 15 pl m-1.
maximum plant height. Compared to this study
Plant height
it was observed that D2 had higher plant
Factor
E1
E2
C1
C2
D1
D2
D3
DMS
height for both cultivars and sowing dates.
62.28aA
57.85aB 1.6335
L1
66.39ns 54.53ns 70.20aA 50.72bB 61.25aA
The number of pods per plant (Table 6) was
ns
ns
L2
67.04
53.27
66.67bA 53.64aB 58.49bB
62.38aA
59.61aB 1.6335
higher
in L1 for both sowing dates, this due
1
------------- 77.22aA 56.22aB 64.48aB
70.62aA
65.05aB 2.4026
to differences in soil texture, because L1 is
2
------------- 59.65bA 48.15bB 55.26bA 54.04bAB 52.40bB 2.4026
C1
------------------------67.26aB
71.95aA
66.10aB 2.4026
sandy and soils with this characteristic,
C2
------------------------52.48bA
52.71bA
51.36bA 2.4026
according to Godoy et al. 24, are best for
DMS
1.6335
1.6335
2.0006
2.0006
2.0006
growing of peanuts crop, because it favours
Means followed by the same lowercase letter in the column and uppercase on the line do not differ statistically by Tukey test (p < 0.05) ( ) not significant.
per plant, weight of thousand grains and weight of pod are
significantly influenced by soil type. In other hand Gerin et al. 19
evaluated the productivity of peanut in clayey and sandy soils
and observed that the texture of the soil interferes in some features,
but is irrelevant because it does not indicate the best treatment.
The sowing date is defined by a set of environmental factors,
besides to affect the productivity, also interferes in the architecture
and development of the plant. Gonalves 7 and Peixoto et al. 9
affirm that sowing date in inappropriate season can cause drastic
reductions in yield of pods and grains, due to changes in plant
height, number of branches, stem diameter and lodging in soybean
and peanut. Similar results were obtained in this study considering
the dry period in 27 February to 15 March, causing a water deficit
for the emergence and vegetative development, thus characterizing
the E2 as unfit for sowing, due to the crop did not express their
potential of production significantly compatible to E1.
It was observed that peanut deployed in the first sowing date
showed better agronomic performance for most characteristics
evaluated, except for the pod weight that obtained more weight in
E2, which means a higher weight of bark and lower weight of
grains, which can be proven by the income which is the percentage
of bark in relation to grain production.
Cultivars from different botanical groups have different growth
habit. The cultivar Tatu is erect sized and the Runner prostrate
sized 20, besides the pods and grains are larger and have beige
ns
272
Table 6. Split of number of pods per plant at two sites with two peanut cultivars, two
sowing dates and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) Tangar da Serra; Sowing date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02;
Cultivar 1 (C1) - IAC Tatu ST; Cultivar 2 (C2) - IAC Runner 886; Density 1 (D1)
- 5 pl m-1; Density 2 (D2) - 10 pl m-1; Density 3 (D3) - 15 pl m-1.
Silveira 20 concluded that the thousand

grain weight is strongly influenced by the
density, the higher the density the lower
the weight of grain, which explains the
relationship of sowing density in
competition for nutrients. Therefore, there
Number of pod per plant
is greater weight in D2, both in different
Factor
E1
E2
C1
C2
D1
D2
D3
DMS
places and in the related sowing dates.
15.27ns 6.97bC 16.85aB 20.20aA 0.7849
L1
15.36aA 14.67aA 14.07ns
L2
8.04bA
5.73bB
6.44ns
7.34ns
8.17aA 6.60bB
5.90bB 0.7849
The productivity of peanuts in bark (Table
1
------------11.55aA 11.16aA 8.35ns
12.46ns
13.26ns
0.7849
9) showed higher means in L1 and did not
ns
ns
ns
10.98
12.83
0.7849
E2
------------8.96bB 11.45aA 6.79
differ between sowing dates, the C2 showed
C1
------------------------8.00aC 10.41bB 12.35bA 1.1545
the best productivity for both sites with D3,
C2
------------------------7.14aB 13.03aA 13.75aA 1.1545
DMS
0.7849
0.7849
0.7849
0.7849
0.9613
0.9613
0.9613
similar to the results of Silveira 20, that the
Means followed by the same lowercase letter in the column and uppercase on the line do not differ statistically by Tukey test (p < 0.05) ( ) not significant.
density of 15 pl m-1 increases the yield of
pods per hectare, but the yield per plant is
the penetration of gynophores and facilitates harvesting. Bolonhezi reduced which become more expensive the planting and prevents
et al. 18 in experiments in the region of Ribeiro Preto observed the the use of this density.
same behaviour for peanut cultivars and reported that in more porous
Peixoto et al. 9, in studies of peanut productivity in different
soils there is increase of production of pods per plant. However, the spacing and sowing dates in the Reconcavo Baiano, observed
C1 suffered a stress in E2 and showed no potential compared to C2, that productivity in grain begin to be reduced from a density
which according to Carneiro 23, occurs due to shorter crop cycle above 15 pl m-1, which is stated to this study where D3 did not
and requires a more concentrated rainy season.
differ from D2 to both sites and sowing dates (Table 10).
According to Romanini 13 and Bulgarelli 14, the highest number
The lowest plant density leads to lower productivity due to the
of pods per plant was in lower plant density, and it can be observed small number of plants. The lowest productivity of 608 kg ha-1
that the L2 fits, but for L1 to D3 is best for the variable number of was in D1 against the D3 which showed highest productivity of
pods per plant, this difference is similar to Bolonhezi et al. 18
2141 kg ha-1. Similar results were found by Romanini 13 that the
For the total weight (Table 7), the sowing E1 was highest in the productivity is related to the optimal density for sowing which is
two planting sites and the C2 was higher for both locations. In D3 between 12 and 18 pl m-1.
the total weight of pods was greater, which according to Silva and
The weight of the pod confirms the reduction of productivity in
Beltro 11, more plants per metre the higher the gross production grains per hectare with the increasing of plant density per metre.
of pod and grain, but the yield is reduced, so the best treatment According to Romanini 13, the higher the density is less the weight
on the total weight is the L1 in the E2 with the C2 in the D3.
of the pod due to competition among plants and lower is the
According to Romanini 13, these results are directly related to weight of the grain within the pod. This relationship is called the
the plant population, in other words, occurring compensation in yield, which according to Centurion et al. 22, is influenced by plant
individual production of plants, that develop in low-level density and corroborating with their results the best yield is in the
competition, providing the same productivity of peanuts lowest density. The results indicate C2 with higher yield and E1
compared to lower densities where there is no level of as the best sowing date, however, with relation to the site there is
competition.
no restrictions, but must observe the best sowing date as described
The results of the analysis of variance for thousand grain weight by Romanini 13, who studied two different soils for cultivation.
(Table 8) indicate that the L2 in E1 is the best treatment presenting
Mean values of yield were affected by the density only in E2, in
632.58 g, but for the L1 in E2 is more appropriate with 490.66 g which the lower plant density (5 pl m-1) showed the highest yield
what fits to the results found by Romanini 13, the cultivars showed (66%), and higher plant density (10 pl m-1), lower to the other, with
no significant differences between location and sowing date, the the lowest yield of pods (62.70%). The cultivars in both sowing
D2 showed the best weight for all locations and cultivars and did dates did not differ significantly. These results support those
not differ between sowing dates.
found by Silva and Beltro 11, Gonalves 7, Peixoto et al. 9 and
Bulgarelli 14, where higher productivity
-1
were obtained in higher plant
Table 7. Split of total weight (g pl ) at two sites with two peanut cultivars, two sowing
dates and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar da populations. However, like the fresh pods,
the cultivars in both sowing dates did not
Serra; Sowing date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) IAC Tatu ST; Cultivar 2 (C2) - IAC Runner 886; Density 1 (D1) - 5 pl m-1; Density differ significantly. Regarding to the yield
(%) the analysis of variance revealed
2 (D2) - 10 pl m-1; Density 3 (D3) - 15 pl m-1.
significant effect for volume of fresh pods
Total weight
only for the factor plant density in sowing
Factor
E1
E2
C1
C2
D1
D2
D3
DMS
L1
189.63aA 183.43aA 175.93aA 190.93aA 89.59aC 150.93bB 178.91bA 15.00
date 1 (Table 11), with the treatment of
L2
169.58aA 76.69bB 103.69bB 142.59bA 81.86aB 206.04aA 212.38aA 15.00
higher plant density (15 pl m-1). There were
ns
ns
186.80
100.09aB 210.68aA 218.75aA 22.06
1
------------166.21
no statistical differences among cultivars.
146.72ns
71.37bC 146.28bB 172.54bA 22.06
E2
------------113.40ns
Also related to the yield in volume of
169.99ns
192.97ns
ns
C1
------------------------78.97ns
186.98ns
198.31ns
ns
C2
------------------------92.49ns
pods, densities and cultivars evaluated
DMS
15.00
15.00
15.00
15.00
18.37
18.37
18.37
18.37
did not differ significantly in both sowing
ns
Means followed by the same lowercase letter in the column and uppercase on the line do not differ statistically by Tukey test (p < 0.05) (ns) not significant.
273
Table 8. Split of thousand grain weight (g) at two sites with two peanut cultivars, two sowing
dates and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar da
Serra; Sowing date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) - IAC
Tatu ST; Cultivar 2 (C2) - IAC Runner 886; Density 1 (D1) - 5 pl m-1; Density 2 (D2)
- 10 pl m-1; Density 3 (D3) - 15 pl m-1.
Thousand grain weight
Factor
E1
E2
C1
C2
D1
D2
D3
DMS
L1
480.63bA
490.66aA
370.78ns
600.52ns
505.66bA
495.38bA
455.90bB
23.93
L2
1
E2
632.58aA
-------------
491.54aB
-------------
437.89ns
431.88ns
376.79ns
686.23ns
681.33ns
605.42ns
541.65aB
548.41ns
498.90ns
567.06aA
569.72ns
492.73ns
577.47aA
551.69ns
481.68ns
23.93
ns
ns
C1
-------
-------
-------
-------
402.75bA
402.09bA
408.16bA
23.93
C2
-------
-------
-------
-------
644.56aAB
660.36aA
625.20aB
23.93
DMS
16.27
16.27
ns
ns
19.93
19.93
19.93
Means followed by the same lowercase letter in the column and uppercase on the line do not differ statistically by Tukey test (p < 0.05) (ns) not
significant.
Table 9. Split of productivity in bark (kg ha-1) at two sites with two peanut cultivars, two sowing dates
and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar da Serra; Sowing date
1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) - IAC Tatu ST; Cultivar 2 (C2) - IAC
Runner 886; Density 1 (D1) - 5 pl m-1; Density 2 (D2) - 10 pl m-1; Density 3 (D3) - 15 pl m-1.
Factor
L1
L2
E1
E2
C1
C2
DMS
E1
2292.96aA
2119.79aA
------------------------187.50
E2
2271.52aA
958.73bB
------------------------187.50
Productivity in bark
C1
C2
2199.21aA 2386.71aA
1296.13bB 1782.39bA
2335.02ns
2077.73ns
1417.61ns
1834.08ns
------------------------187.50
187.50
D1
1089.84aC
1053.49aB
1251.17aB
892.16bC
1119.97aC
1023.35aB
229.64
D2
2632.81aB
1829.37bA
2633.59aA
1828.59bB
1886.64bB
2575.54aA
229.64
D3
3156.25aA
1734.92bA
2734.37aA
2156.79bA
2236.40bA
2654.76aA
229.64
DMS
187.50
187.50
275.79
275.79
275.79
275.79
Means followed by the same lowercase letter in the column and uppercase on the line do not differ statistically by Tukey test (p <0.05) (ns) not significant.
Table 10. Split of productivity in grain (kg ha-1) at two sites with two peanut cultivars in two sowing
dates and three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar da Serra; Sowing
date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) - IAC Tatu ST; Cultivar 2 (C2) IAC Runner 886; Density 1 (D1) - 5 pl m-1; Density 2 (D2) - 10 pl m-1; Density 3 (D3) - 15 pl m-1.
Factor
L1
L2
1
E2
C1
C2
DMS
E1
1613.14aA
1610.42aA
------------------------145.99
E2
1545.70aA
602.21bB
------------------------145.99
Productivity in grain
C1
C2
1447.51aB 1711.33aA
870.01bB 1342.62bA
1784.32ns
1439.24ns
ns
878.28
1269.63ns
------------------------145.99
145.99
D1
756.30aC
755.42aB
903.02aB
608.71bB
766.39aB
745.33aB
178.80
D2
1840.82aB
1326.39bA
1942.32aA
1224.88bA
1258.38bA
1908.83aA
178.80
D3
2141.14aA
1237.13bA
1990.00aA
1388.27bA
1451.51bA
1926.77aA
178.80
DMS
145.99
145.99
214.73
214.73
214.73
214.73
Table 11. Split of yield (%) at two sites with two peanuts cultivars, two sowing dates and
three densities. Site 1 (L1) Barra do Bugres; Site 2 (L2) - Tangar da Serra;
Sowing date 1 (E1) - 12/02; Sowing date 2 (E2) - 24/02; Cultivar 1 (C1) - IAC Tatu
ST; Cultivar 2 (C2) - IAC Runner 886; Density 1 (D1) - 5 pl m-1; Density 2 (D2) - 10
pl m-1; Density 3 (D3) - 15 pl m-1.
Factor
L1
L2
1
E2
C1
C2
DMS
E1
70.21bA
75.44aA
------------------------1.60
E2
67.76aB
60.57bB
------------------------1.60
C1
66.47aB
62.73bB
69.61aB
59.60bB
------------1.60
Yield
C2
71.49bA
73.28aA
76.04aA
68.73bA
------------1.60
D1
69.68aA
69.50aA
72.45aA
66.74bA
66.578bA
72.61aA
1.97
D2
69.62aA
66.34bB
73.26aA
62.70bB
63.07bB
72.89aA
1.97
D3
67.65aA
68.17aAB
72.77aA
63.06bB
64.17bB
71.66aA
1.97
DMS
1.60
1.60
2.36
2.36
2.36
2.36
274
dates. According to Silva and Beltro 11 the intraspecific

competition, determines in each cultivar the plant population that
provides higher yield and better utilization of available resources.
It is noted that the density of 15 pl m-1, although of the plant
exploit the smallest area of soil compared to the other treatments,
this effect is compensated by the higher plant population, resulting
in a higher yield in pods. These results are consistent with those
found by Gonalves 7 who recommends to a higher yield of pods
(%) the arrangement of 15 pl m-1 x 0.80 m, more efficient for volume
of pods.
Conclusions
Based on the results the following conclusions were obtained:
a) The site, sowing date, density and the cultivar influenced the
agronomic characteristics such as plant height, number of pods
per plant, total weight, thousand grain weight, productivity on
bark; productivity in grain; weight of pod and yield of peanut
grown under conditions of Mato Grosso state.
b) The choice of plant density that expresses higher yields of
pods and grains depends on the sowing date, cultivar used and
the commercial purpose of the producer (volume or mass).
Therefore, it is recommended density of 15 pl m-1 to the total
weight and productivity in bark and the density of 10 pl m-1 to
yield, weight of the pod, and the a thousand grain weight and
total weight of grain.
c) The best site for growing peanuts was Barra do Bugres due
to soil type and climatic conditions occurred, considering the
density of 15 pl m-1 for C1 and 10 pl m-1 for C2, in the sowing
conducted in E1.
Acknowledgements
Production linked to the research project, Application and transfer
of technologies in optimizing sustainable agricultural systems,
linked to the sub-network of social, environmental and
technologies studies for the production system in the southwest
region of Mato Grosso state - REDE ASA, funded under the official
announcement MCT/CNPq/FNDC /FAP/ MEC/CAPES/PROCENTRO-OESTE No. 031/2010. And the project funded by the
Foundation for Research Support of the State of Mato Grosso,
OFFICIAL ANNOUNCEMENT UNIVERSAL/ FAPEMAT No.
009/2011 process No. 749427/2011.
The authors also thank the Universidade do Estado de Mato
Grosso - UNEMAT by the scholarship to the first author.
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6
275
WFL Publisher
Helsinki, Finland
www.world-food.net
Effect of composted sewage sludge on durum wheat: Productivity, phenolic

compounds, antioxidant activity, and technological quality
Antonella Pasqualone 1, Laura Nunzia Delvecchio 1, Giovanni Lacolla 2, Luciana Piarulli 1,
Rosanna Simeone 1 and Giovanna Cucci 2*
1
2
University of Bari Aldo Moro, Dept. of Soil, Plant, and Food Science (Di.S.S.P.A.), Via Amendola, 165/A, I 70126, Bari, Italy.
University of Bari Aldo Moro, Dept. of Agricultural and Environmental Science (Di.S.A.A.T.), Via Amendola, 165/A, I 70126,
Bari, Italy. e-mail: antonella.pasqualone@uniba.it, lauranunziadelvecchio@yahoo.it, giovanni.lacolla@uniba.it,
luciana.piarulli@libero.it, rosanna.simeone@uniba.it, giovanna.cucci@uniba.it
Abstract
The beneficial effects of whole meal wheat products are mainly attributed to dietary fibre and secondary metabolites related to it, such as phenolic
compounds. To date, no studies have investigated the effect of fertilization with sewage sludge on the levels of wheat phenolic compounds. The aim
of this study was to point out the effects of increasing doses of composted sewage sludge, also in combination with mineral fertilization, on phenolic
compounds and antioxidant activity of durum wheat. Moreover, the effects on productivity and technological quality were verified. A randomized
block experimental system with six replicates was adopted and seven treatments were compared: unfertilized control (absence of any fertilization or
composted sewage sludge application); four doses of composted sewage sludge (3, 6, 9, and 12 Mg ha-1); mineral fertilization (MF) consisting of 120,
100 and 100 kg ha-1 of N, P2O5 and K2O, respectively; combined fertilization with 6 Mg ha-1 of composted sewage sludge and 60 kg ha-1 of N. There
was a significantly positive effect of sewage sludge application on productivity as well as on phenolic compounds, antioxidant activity, and
technological quality of durum wheat whole meal. Regarding the productivity, the use of 12 Mg ha-1 of composted sewage sludge can effectively
substitute mineral fertilization. Regarding phenolics and antioxidant activity, a further increase can be achieved by employing a combination of
composted sewage sludge (at the level of 6 Mg ha-1) and mineral fertilization. The phenolic compounds of whole meal raised from 1.31 mg ferulic acid
equivalents (FAE) g-1 (unfertilized control) to 1.93 mg FAE g-1 (combined application of 6 Mg ha-1 of sewage sludge and 60 kg ha-1 N). The antioxidant
activity range was 1.89-2.02 mol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) g-1 d.m., corresponding to a level of scavenging
capacity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical ranging from 56.26% (unfertilized control) to 62.29% (6 Mg ha-1 of sewage sludge plus
60 kg ha-1 N).
Key words: Triticum durum, fertilization, yield.
Introduction
Among cereals, durum wheat is one of the most used for human
nutrition and is a fundamental element of Mediterranean diet.
Moreover, the interest towards the healthy features of food is
increasingly growing and many studies have demonstrated the
association between the reduction of degenerative diseases and
the regular consumption of whole grains 1, 2. The beneficial effects
are mainly attributed to dietary fibre and secondary metabolites
related to it, such as phenolic compounds 3. In fact, several
researches assessed the antioxidant properties of wheat phenolic
compounds 4-6. Moreover, the phenolic compounds are thought
to be part of the chemical defence of the plant, where exert
physiological functions such as stabilizing cell walls, screening
UV radiation and protecting against herbivore attacks and insect
damages 7.
Doses and modality of nitrogen fertilization are known to have
a significant effect on the overall wheat grain quality 8. At this
purpose, the use of composted sewage sludge of municipal origin
to substitute the commonly used fertilizers has been proposed to
preserve or restore soil fertility, due to its ability to increase the
organic substance 9. According to the Council Directive 91/271/
EEC of 21 May 1991 10, concerning urban waste-water treatment,
276
an increase of the amount of sewage sludge is expected, and the

agronomic use of a part of it would be advisable to convert this
waste into a resource. On the other hand, it has to be taken into
account the risk related to the possible presence of heavy metals
or other undesired elements, eventually subjected to plant
uptake 11. This risk could be minimized by a rational managing of
sewage sludge, opportunely composted, in a soil quality
perspective 9, 12. In particular, doses, time and technique of
application should be planned and established only after an
accurate characterization of both soil and sewage sludge, without
excluding the eventuality of combining the application of
composted sewage sludge and mineral fertilization 13, 14.
Various studies have been carried out to evaluate the effects of
composted sewage sludge on wheat 15-17. By comparing mineral
fertilization and sewage sludge application, positive relationships
between nutrients of sewage sludge and photosynthetic activity
were observed by Lakhdar et al. 15, while a positive effect on yield
and yield components was pointed out by zyazc 16 and ElGhany et al. 17. Also, the biomass of wheat increased by increasing
doses of sewage sludge compost 18. Regarding the effect of
different agricultural practices on phenolic compounds of cereals,
few studies reported about them, mainly discussing the differences

between conventional and organic systems in spring and winter
wheat 19, 20, as well as in oat 21. To date, however, no studies have
investigated the effect of sewage sludge application on the levels
of wheat phenolic compounds.
The aim of this study was to point out the effects of increasing
doses of composted sewage sludge, also in combination with
mineral fertilization, on phenolic compounds and antioxidant
activity of durum wheat. Moreover, the effects on productivity
and technological quality were also verified. Unfertilized control
and mineral fertilization alone were considered for comparisons.
Table 2. Amounts of N, P, and K (kg ha-1)

corresponding to the various treatments.

Plant cultivation: Durum wheat (Triticum durum Desf.) cv. Iride
was grown at the experimental field of DISAAT of University of
Bari Aldo Moro in a three-year rotational crop system potato/
durum wheat/barley. The cultivation was carried out in 240 dm3
pots filled with sandy-loam soil having the characteristics reported
in Table 1.
Parameter
pH
Humidity (g 100 g-1)
Organic carbon (g 100 g-1 d.m.)
Total nitrogen (g 100 g-1 d.m.)
Organic nitrogen (% of total N)
C/N
Total phosphorus (g 100 g-1 d.m.)
Total potassium (g 100 g-1 d.m.)
Humic and fulvic acids (g 100 g-1 d.m.)
Pb (mg kg-1)
Cd (mg kg-1)
Ni (mg kg-1)
Zn (mg kg-1)
Cu (mg kg-1)
Hg (mg kg-1)
Cr (mg kg-1)
Salinity (meq 100 g-1 d.m.)
Particle size (mm)
Specific weight (kg m-3)
Table 1. Main properties of the soils where the trials

were carried out.
Parameter
Chemical properties:
Total nitrogen (Kjeldahl method) (g kg-1)
Available phosphorus (Olsen method) (mg kg-1)
Exchangeable potassium (BaCl2 method) (mg kg-1)
Organic matter (Walkley Black method) (g 100 g-1)
Total limestone (g 100 g-1)
Active limestone (g 100 g-1)
pH
ECe (dS m-1)
ESP
CEC (BaCl2 method) (meq 100 g-1 of soil d.m.)
Particle-size distribution:
Total sand (2 > > 0.02 mm) (g 100 g-1)
Silt (%) (0.02 > > 0.002 mm) (g 100 g-1)
Clay (%) ( < 0.002 mm) (g 100 g-1)
Hydrologic properties:
Field capacity (g 100 g-1 of soil d.m.)
Wilting point (-1.5 MPa) (g 100 g-1 of soil d.m.)
Bulk density (t m-3)
Value
0.9
22.5
252
1.6
2.6
1.4
7.3
0.4
0.8
20.2
60.5
20.0
19.5
23.6
12.5
1.4
ECe = saturation extract electrical conductivity; ESP = exchangeable sodium percentage;

CEC = cation exchange capacity.
The pots, equipped with a bottom valve to enable water

percolation, were kept open air. A randomized block experimental
system with six replicates was adopted and seven treatments were
compared: unfertilized control (C, carried out without any
fertilization or composted sewage sludge application); four doses
of composted sewage sludge (3, 6, 9, and 12 Mg ha -1 ,
corresponding to the treatments SS3, SS6, SS9 and SS12,
respectively); mineral fertilization (MF); combined fertilization with
6 Mg ha-1 of composted sewage sludge and 60 kg ha-1 of N (SS6N).
The amounts of N, P, and K (kg ha-1) corresponding to the various
treatments are reported in Table 2.
The composted sewage sludge, of municipal origin, was
produced by ASECO (Ginosa Marina, Taranto, Italy) and fulfilled
the heavy metals and bacteriological limits imposed by the Italian
Legislative Decree no. 217/06 22. The main physico-chemical
characteristics of the composted sewage sludge are shown in
Table 3.
The composted sewage sludge was incorporated 20 cm below
soil surface about 2 months before sowing, which was effected
Treatment
C
SS3
SS6
SS9
SS12
SS6N
MF
N
0
27
55
82
110
115
120
P2O5
0
38
77
115
153
77
100
K2O
0
20
41
61
81
41
100
Table 3. Main physico-chemical characteristics of the

composted sewage sludge used in the trials.
Value
7.7
23
> 22
1.1
> 80
< 20
2.3
1.2
>7
< 140
< 1.5
< 100
< 500
< 230
< 1.5
< 0.5
21.00
15
600
December 5th, 2012. Supplemental irrigations were carried out, from

the plantlet emergence to the end of crop cycle, when 50% of the
maximum available water was lost by evapotranspiration. The
applied volume of water was calculated to restore field capacity in
the whole soil mass contained in each pot. Harvesting was carried
out at the stage of full ripening (June 28 th, 2013).
Morpho-physiological and yield parameters: Plant height, culms,
ears, shoot dry biomass, seed yield, 1000-seed weight, test weight,
and non-vitreous kernels were determined at the stage of full
ripening. Moreover, at late flowering (May 14th, 2013) the flag leaf
was tested for the chlorophyll index by means of Chlorophyll
Meter SPAD-502 (Spectrum Technologies Inc., Aurora, IL, USA)
to characterise the crop nitrogen status.
Sample production and basic analyses: Harvested grain samples
from each plot were separately milled to whole meal by means of a
laboratory mill equipped with 1-mm sieve (Cyclotec Sample Mill,
Tecator Foss, Hillerd, Denmark). Moisture content was
determined at 105C by means of an automatic moisture analyser
(Radwag Wagi Elektroniczne, Radom, Poland). Colorimetric
evaluations were carried out by means of the reflectance
colorimeter Chroma Meter CR-300 (Minolta, Osaka, Japan). Protein
content was determined by using a dual beam near infrared
reflectance spectrophotometer (Zeutec Spectra Alyzer Premium,
Zeutec Bchi, Rendsburg, Germany).
Quantification of phenolic compounds: Soluble (free and soluble
conjugated) phenolic compounds were extracted from 0.1 g whole
meal flour by using 50:50 (v/v) acetone/water at 1:10 sample to
277
solvent ratio. After shaking at 200 rev/min for 2 h, and

Table 4. Effect of different doses of composted sewage sludge and mineral
subsequent centrifugation at 7,000 g for 5 min, the
fertilization on the morpho-physiological parameters of durum
supernatant was subjected to Folin-Ciocalteu reaction. The
wheat cv. Iride (mean SD).
reaction mixture contained 100 l supernatant, 500 l FolinShoot dry
Chlorophyll Plant height
Culms
Ears
Treatment
biomass
Ciocalteu reagent (Sigma-Aldrich Chemical Co., St. Louis,
index
(cm)
(n per pot) (n per pot)
(Mg ha-1)
MO, USA) and 2 ml of 15% (w/v) sodium carbonate. The
C
26.91.50E
62.52.1D
1448F
1347E
5.70.6F
final volume was made up to 10 ml with distilled water.
SS3
29.81.20E
69.81.4C
1647E
1516D
6.60.7E
After 1 h in the dark, and centrifugation at 12,000 g for 3
SS6
33.63.46D
71.22.0C
1756D
1625C
8.20.9D
min to precipitate any particles, the absorbance of the
SS9
36.94.51C
74.01.2B
1807D
1704C
9.51.0C
SS12
48.62.45B
76.70.8A
2097C
19810B 12.40.7B
solution was measured at 765 nm (Cary 60 UV-Vis
SS6N
52.52.06A 76.81.6A
2188B
20511B 12.61.1B
spectrophotometer, Agilent Technologies Inc., Santa Clara,
MF
54.72.00A 77.71.4A
23912A
22811A 14.50.3A
CA, USA). A calibration curve was built by using 50:50 (v/
Different letters in column indicate significant differences according to the SNK test at P 0.01.
v) acetone/water solutions of ferulic acid (Sigma-Aldrich
Chemical Co., St. Louis, MO, USA). The results were expressed as Table 5. Effects of different doses of composted sewage sludge
ferulic acid equivalents (FAE). All tests were carried out in triplicate.
and mineral fertilization on the yield parameters of durum
wheat, cv. Iride (mean standard deviation).
Determination of antioxidant activity: Antioxidant activity (AA)
1000-seed
Non-vitreous
Test
Seed yield
was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
Treatment
weight
kernels
weight
(Mg ha-1)
-1
(g)
(%)
(kg hL )
scavenging capacity assay. Whole meal flour (0.1 g) was mixed
C
3.030.35E
42.31.3E
79.91.1D
34.33.2A
with 1 ml of methanol and kept on orbital shaker at 200 rev/min for
SS3
3.500.25D
43.50.9E
81.10.7C
31.63.0B
2 h. After centrifugation at 7,000 g for 5 min, 500 l of supernatant
SS6
3.710.29CD
45.31.6D
82.20.5B
30.00.9B
were added to 1.5 ml of freshly-prepared 60 M methanol solution
SS9
3.970.17C
46.81.6C
82.50.5B
24.21.7C
of DPPH radical (Sigma-Aldrich Chemical Co., St. Louis, MO, USA).
SS12
4.500.14B
49.70.7B
83.70.3A
20.21.2D
SS6N
4.810.32AB 50.51.2AB 83.90.6A
19.32.2D
After 30 min of reaction in the dark, the absorbance was measured
MF
4.970.53A
51.81.3A
84.00.3A
18.61.5D
at 515 nm (Cary 60 UV-Vis spectrophotometer, Agilent Technologies Different letters in column indicate significant differences according to the SNK test at P 0.01.
Inc., Santa Clara, CA, USA). At the same wavelength, but at t = 0
min, it was also read the absorbance of the methanol solution of relationships between nutrient levels of composted sewage sludge
DPPH radical without sample (control). Methanol was the blank. and photosynthetic activity of wheat have been observed by
The AA of the samples was expressed as percent capacity of Lakhdar et al. 15.
Plant height maximum value was recorded at the highest compost
scavenging the DPPH radical (SC%) according to the following
equation: SC% = (1 Abs of samplet=30/Abs of controlt=0) 100, dose, with no significant difference with the treatment involving
where Abs of samplet=30 is the absorbance of DPPH radical solution the use of mineral fertilization (Table 4). Moreover, the use of
+ sample at t = 30 min; Abs of controlt=0 is the absorbance of the composted sewage sludge induced an increase of the number of
DPPH radical solution at t = 0 min. All tests were carried out in culms and ears per pot, and improved the level of shoot dry
triplicate. Besides, a standard curve was built by using methanol biomass. In particular, increases of 45%, 48% and 118% respect to
solutions of the synthetic antioxidant 6-hydroxy-2,5,7,8- control were observed at 12 Mg ha-1 of compost, with further
tetramethylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich increases with mineral fertilization.
Consequently, seed yield and its components were positively
Chemical Co., St. Louis, MO, USA), to measure the AA activity
also as mol/g Trolox, as suggested by Liu et al. 23. All tests were influenced by the application of composted sewage sludge (Table
5), in agreement with the results of zyazc 16 and El-Ghany et al. 17.
carried out in duplicate.
The values reached at the highest dose (4.50 Mg ha-1 for seed
Statistical analysis: Data were submitted to statistical analysis yield, 49.7 g for 1000-seed weight and 83.7 kg hL-1 of test weight)
by using MSTAT-C software (Michigan State University, East were not statistically different to those obtained by applying 6
Lansing, MI). One-way analysis of variance (ANOVA) was Mg ha-1 of sewage sludge and 60 kg ha-1 N or by employing
performed, followed by the Student-Newman-Keuls (SNK) test mineral fertilization only.
The amount of non-vitreous kernels, also, was positively
for multiple comparisons.
influenced by the application of high doses of sewage sludge: a
41% decrease was observed at 12 Mg ha-1 of compost respect to
Effect on morpho-physiological and yield parameters: The control, without significant differences with the combined
experimental results show that the morpho-physiological and yield application of sewage sludge and mineral fertilization or with
parameters of durum wheat Iride were significantly influenced mineral fertilization only (Table 5).
by increasing doses of composted sewage sludge of municipal
Effect on phenolic compounds: Whole meal of samples grown in
origin (Tables 4 and 5).
In particular, the chlorophyll index increased significantly and soils amended with different levels of sewage sludge showed
roughly doubled its value respect to control (from 26.9 to 48.6) at levels of phenolic compounds ranging between 1.31 mg FAE g-1
the highest composted sewage sludge dose (12 Mg ha-1). Further (unfertilized control) and 1.93 mg FAE g-1 (combined application
increases were observed when the combined application of 6 Mg of 6 Mg ha-1 of sewage sludge and 60 kg ha-1 N) (Table 6). These
ha-1 of sewage sludge and 60 kg ha-1 N was considered, without levels were in the range of those reported in durum wheat by
statistical difference with the results of mineral fertilization. Positive other authors: Lempereur et al. 24 assessed 0.69-2.44 mg FAE g-1,
278
Table 6. Effect of different doses of composted sewage sludge and mineral

fertilization on the antioxidant activity, soluble phenolic
compounds, and protein content of durum wheat whole meal,
cv. Iride (mean SD).
sewage sludge alone was applied, at any of the doses

considered (Table 6). A significant difference was observed
between control and the combined application of 6 Mg
ha-1 of sewage sludge plus 60 kg ha-1 N. Moreover, grains
from the latter trial showed the highest value of antioxidant
Antioxidant activity
Protein
Soluble phenolic
Treatment (mol Trolox
content
compounds
activity among all the treatments considered, not
(DPPH SC %)
(g/100 g)
(mg FAE g-1 d.m.)
g-1 d.m.)
statistically different from the chemically fertilized trial.
C
1.890.07b
56.261.59c
1.310.04c
8.350.44c
The antioxidant activity of whole meal was comprised
SS3
1.920.06ab
57.312.12bc
1.660.09b
8.810.60c
between
1.89-2.02 mol Trolox g-1 d.m., corresponding to a
SS6
1.960.06ab 57.722.18abc
1.660.10b
9.210.33c
DPPH radical scavenging capacity ranging from 56.26%
SS9
2.000.07ab 59.291.64abc
1.700.14b
11.231.78b
SS12
1.920.09ab 58.302.24abc
1.710.16b
12.470.89ab (unfertilized control) and 62.29% (6 Mg ha-1 of sewage
SS6N
2.020.06a
60.851.21a
1.930.08a
13.451.28a
sludge plus 60 kg ha-1 N). These values agreed with those
MF
1.980.03ab
59.711.15ab
1.740.10b
13.010.63a
reported by other authors in similar products: Yu and
Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; DPPH = 2,2-diphenyl-1-picrylhydrazyl radical;
FAE = ferulic acid equivalents. Different letters in column indicate significant differences according to the SNK test at Zhou27 observed levels close to 75% in bran, and Yu et
P 0.05.
al.28 determined DPPH radical remaining percentages as
low as 36%, corresponding to SC values up to 64%, in whole grain
and Menga et al. 5 ascertained 0.78-0.95 mg FAE g-1.
The amount of phenolic compounds was positively influenced wheat-based breakfast cereals.
by the application of composted sewage sludge: all the treatments
with the composted sewage sludge application showed levels of
phenolics significantly higher than control, with the maximum level
in the treatment involving the combined application of 6 Mg ha-1
of sewage sludge and 60 kg ha-1 N. The latter led to levels of
phenolic compounds that overcome those observed when mineral
fertilization only was applied. This is an interesting result,
indicating a synergic effect of sewage sludge and mineral
fertilization. Hence, the use of sewage sludge as a partial substitute
of mineral fertilizers could be effectively implemented in durum
wheat cultivation.
Although impossible to compare our results with other studies,
due to the absence of reports on the effect of sewage sludge on
phenolics, we can consider that significant differences in the
concentration of particular phenolic compounds, as well as in the
total phenolics content, were observed by uchowski et al. 19, 20
as a consequence of different agricultural practices. In particular,
organically produced spring and winter wheat had significantly
higher concentrations of ferulic and p-coumaric acid, and higher
total phenolics content, than conventional wheat, though the
differences were not large 19, 20. However, since phenolics are
localized in the peripheral part of caryopsis 24, the authors comment
that these differences were probably caused mainly by smaller
size of organic wheat kernels (lower 1000-seed weight). Our results
unexpectedly coupled high phenolics levels and high 1000-seed
weight as a consequence of sewage sludge application: probably
the raise of the overall phenolic concentration overcome the
increase of kernel weight. On the other hand, N deficiency has
been shown to strongly affect the synthesis of polyphenols 25
and phenol accumulation. In sorghum, phenolics concentration
of vegetative parts was found to be higher when N nutrition
improved 26. Grain yield and the phenol pool of aerial parts were
also positively correlated, indicating that the environmental factors
that promote growth and grain yield also enhance the total phenol
synthesis in vegetative parts 26. Hence, further studies should be
carried out to separate and analyse specifically the peripheral part
of wheat caryopsis collected after sewage sludge application.
Effect on antioxidant activity: In spite of the significant influence
of composted sewage sludge application on phenolic compounds,
the antioxidant activity assessed by DPPH radical scavenging
capacity assay showed an increase, but not significant, when
Effect on protein content: Protein content was positively

influenced by sewage sludge application starting from the dose
of 9 Mg ha-1 (Table 6). The highest dose of sewage sludge led to
results not statistically different from those obtained applying 6
Mg ha-1 of sewage sludge plus 60 kg ha-1 N or mineral fertilization
alone. These data confirm the positive effect of composted sewage
sludge on protein content of wheat observed by Lakhdar et al. 15
and zyazc et al. 16, and could be due to an increase of nitrogen
availability, known to be positively correlated with protein content 29.
Effect on colour: The colorimetric indices were significantly
influenced by sewage sludge application (Table 7). Soils amended
with sewage sludge led to browner whole meals than control, with
progressively higher values of brown index at increasing levels of
composted sewage sludge application, and with significant
differences starting from doses of 3 Mg ha-1. A similar behaviour
was observed for both red index and yellow index, but with
significant differences starting from doses of 6 Mg ha-1 and 9 Mg
ha-1, respectively.
Table 7. Effect of different doses of composted sewage sludge
and mineral fertilization on color indices of durum wheat
whole meal, cv. Iride (mean SD).
Treatment
C
SS3
SS6
SS9
SS12
SS6N
MF
Brown index (100-L*)

11.180.28d
12.330.76c
13.090.32c
14.321.08b
15.200.71ab
15.621.28a
15.800.48a
Red index (a*)

0.320.09c
0.500.14bc
0.710.07b
1.010.28a
1.090.16a
1.270.32a
1.100.18a
Yellow index (b*)

13.670.05c
13.850.56c
14.250.13c
15.300.78b
15.570.50ab
16.250.48a
16.070.79ab
Different letters in column correspond to significant differences according to the SNK test at P 0.05.
Brown index was significantly correlated with phenolic

compounds (R = 0.7678, P < 0.001). This result confirms the role
of phenolics in enzymatic browning, prompted by the action of
polyphenol oxidase 30, 31. Significant correlations were observed
also between phenolic compounds and both red index (R = 0.7677,
P < 0.001), and yellow index (R = 0.7011, P < 0.001).
The observed increase of brown tone of whole meal, able to
give a brown colour to the end-products, should be taken into
account because sometimes it is not well accepted by consumers.
Eventually, an accurate varietal choice to select durum wheat
cultivars with low polyphenol oxidase activity could be affected
279
in view of keep lower the brown level 31.

Conclusions
The study demonstrated a significant effect of sewage sludge
application on productivity as well as on phenolic compounds,
antioxidant activity, and technological quality of durum wheat
whole meal. Regarding the productivity, the use of 12 Mg ha-1 of
composted sewage sludge proved to be an efficient soil
conditioner, and could be a possible alternative to mineral
fertilization. Regarding phenolics and antioxidant activity, a further
increase can be achieved by employing a combination of composted
sewage sludge (at the level of 6 Mg ha-1) and mineral fertilization.
Although these results have been obtained after a single year
of trials, and need to be confirmed in subsequent years and by
testing other durum wheat cultivars, they seem promising.
Moreover, these results are particularly interesting for areas poor
in organic matter, which could effectively take advantage from the
use of composted sewage sludge as a cheap and environmentally
friendly alternative to organic and mineral fertilization. Therefore,
recycling of sludge for agricultural purpose seems to be an
appealing solution for its sustainable management.
Acknowledgements
The authors acknowledge the financial support of MIUR in the
framework of the Project PON/01_01145/8 ISCOCEM.
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12
WFL Publisher
Helsinki, Finland
www.world-food.net
Genetic diversity and presence of DREB gene in watermelon cultivars and wild type
of watermelon based on molecular markers
Abdullah S. Alsohim * and Mohamed I. Motawei
Plant Prod. & Prot. Dept., College of Agri. & Vet. Medicine, Al-Qassim Univ., Saudi Arabia. *e-mail: a.alsohim@qu.edu.sa
Abstract
Watermelon (Citrullus lanatus) is commonly grown in traditional agrosystems throughout the drought-prone Saudi Arabia. There has been little work
on investigating the relationships between wild and cultivated forms, and to study amount and partitioning of genetic variation, to allow for better
conservation strategies. Previous studies have reported relatively low levels of genetic diversity in cultivated watermelon, but these have been based
mainly on US plant introductions and modern watermelon cultivars linked to breeding programmes for disease resistance. The genetic relationships
among six cultivars from different countries of origin and with different horticultural characteristics and one related wild-type species (Citrullus
colocynthis) were assessed using inter-simple sequence repeats (ISSR) markers. Also, the presence of the resistance stress gene (DREB) as a marker
of drought tolerance in watermelon cultivars and wild-type species was investigated. The cluster analysis results demonstrated low genetic diversity
among commercial cultivars and high genetic diversity between wild species and commercial cultivars. Dendrograms produced two major clusters; one
with all the watermelon cultivars and the other with the wild-type species. The low level of marker polymorphism among the adapted cultivars
implies that a severe bottleneck in genetic diversity existed in watermelon during the initial breeding practices. There were no significant differences
in chlorophyll content and plant height of wild-type species either in three-day intervals or watered daily. All watermelon cultivars gave lower
chlorophyll content and plant height at three- and five-day intervals compared to watered daily. Specific PCR assays using AP2 primers (designed
on the basis of the AP2/EREBP sequence of gene DREB) represent a sensitive tool for screening watermelon genotypes for the DREB gene. The
DREB gene was present only in wild-type species. The amplifying DREB gene could be valuable in watermelon breeding programs for selection of
desirable alleles under drought stress.
Key words: Watermelon cultivars, wild type of watermelon, genetic diversity, DREB gene, water stress.
Introduction
Citrullus lanatus, commonly known as watermelon and belonging
to Cucurbitaceae, is an important food crop in many countries.
However, watermelon is less drought tolerant as Citrullus
colocynthis, which is closely related to watermelon (Citrullus
lanatus (Thunb.) Matsum. & Nakai.), a member of the
Cucurbitaceae family. This plant is a drought tolerant species with
a deep root system, widely distributed in the Sahara-Arabian
deserts in Saudi Arabia.
Drought is the major abiotic stress that has adverse effects on
growth and productivity of crop plants. During the last century,
watermelon production has increased steadily. Today, watermelon
accounts for 2% of the world area devoted to vegetable production.
Although many watermelon cultivars were developed throughout
the world during the last century, there is an ongoing need for
watermelon improvement, especially for increased drought
tolerance 9. Citrullus colocynthis (L.) Schrad is closely related to
domesticated watermelon (C. lanatus (Thunb)) Matsum & Nakai
var. lanatus) and wild watermelon (C. lanatus var. citroides). The
species, commonly known as the bitter apple or bitter gourd, is a
non-hardy drought-resistant herbaceous perennial vine.
Domesticated watermelons have been selected for their
productivity and quality. Domestication of crop plants and plant
breeding has dramatically eroded allelic variations of crop species,
which has contributed to an increasing susceptibility of crop
plants to environmental stresses, diseases and pests 17. Wild

germplasm has been used with great success in breeding for simply
inherited resistance to diseases and insects. For effective
conservation of watermelon, it is important to obtain information
about genetic diversity within and between accessions 14. Few
such studies have, however, been conducted in Saudi Arabia,
except for an investigation of morphological diversity in landraces
of Citrullus. Lee et al. 8 used RAPD markers to estimate genetic
diversity among watermelon cultivars, and to construct an initial
genetic linkage map for watermelon. Jarret et al. 6 used SSR markers
to determine genetic variation among PI accessions of C. lanatus
var. lanatus, C. lanatus var. citroides and the wild species C.
colocynthis, and delineated 4 groups at the 25% level of genetic
similarity. Citrullus colocynthis (L.) Schrad. grows in northern
Africa, southwestern Asia and the Mediterranean, and is drought
resistant 2. Many genes that respond to drought, high-salt and
low-temperature conditions were discovered to have DRE or CRT
(C-repeat element) in their promoter regions 19. The dehydration
responsive element binding (DREB) transcription factors, which
specifically interact with C-repeat/DRE (A/GCCGAC), play an
important role in plant environmental stress tolerance by
controlling the expression of many stress related genes 15.
In the present study, the aim was to assess genetic relatedness
among watermelon cultivars and wild-type species (Citrullus
281
colocynthis). In addition, our study seeks to screen watermelon

genotypes for the DREB gene of desirable alleles for drought
stress.
Plant materials and greenhouse experiment: This research was
carried out at the Experimental Research Station, at the Agriculture
and Veterinary College, Qassim University. Six watermelon
cultivars, Sun shade, Diamond, Charleston, Charleston gray,
Crimson sweet and Fashion, and wild type species (Citrullus
colocynthis) have been used in this study (Table 1). Each cultivar
was irrigated in three- and five-day intervals, including a control
(watered daily). A split-plot in a randomized complete block design
with three replicates was used. The three irrigation treatments
were considered as main plots, and the six watermelon cultivars
and wild type species (Citrullus colocynthis) were regarded as
sub-plots. At harvest, five plants were randomly chosen for
measuring plant height. Chlorophyll pigment was measured on
the fifth upper leaf using a SPAD-501 chlorophyll meter (Konica
Minolta, Co. Ltd., Japan). Data were statistically analysed by using
a split-plot design with three replicates according to Snedecor
and Cochran 16. The Costat computer program (CoHort Software,
Monterey, CA) was used to perform the analysis of ANOVA. The
least significant difference (LSD) test was used to compare means
at the 5% level.
Table 1. Original for watermelon genotypes.
Watermelon genotype
Sun shade
Black diamond
Charleston
Fashion
Charleston gray
Crimson sweet
Wild-type species
Origin
USA
USA
Niagara
Holland
USA
USA
Saudi Arabia
DNA extraction: Total genomic DNA was extracted from young

leaves with the CTAB-DNA extraction protocol 4 and Promega
Wizard Genomic DNA Purification Kit. The DNAs were quantified
on the Nanodrop ND-1000 spectrophotometer.
ISSR analysis: The ISSR-PCR method was conducted according
to Negaoka and Ogihara 11. Amplification was performed in 25 l
reaction volumes, containing 1X Taq polymerase buffer (50 mM
KCl; 10 mM Tris, pH 7.5; 1.5 mM MgCl2) and 1 unit of Taq
polymerase (Pharmacia Biotech, Germany) supplemented with
0.01% gelatin, 0.2 mM of each dNTP (Pharmacia Biotech, Germany),
50 pmol of ISSR primers (Table 2), and 50 ng of total genomic
DNA. Amplification was performed in a thermal cycler (Thermolyne
Amplitron) programmed for 1 cycle of 2 min at 94C; 35 cycles of
30 s at 94C, 45 s at 44C, 1.3 min at 72C, and 20 min at 72C. After
the completion of PCR, the samples were cooled immediately to
10C and stored at 4C until gel separation. A gel-loading solution
(5 l) was added, and 10 l of the total product volume was resolved
in 1.5% agarose in 1X TAE buffer for 2 h with a 100-bp ladder
(Pharmacia, Germany) as the size standard. The gels were stained
with ethidium bromide, and the images were recorded.
Simple sequence repeats (SSR) marker for DREB gene: SSR
primers for amplification of DREB gene was AP2 primer. This primer
was designed on the basis of the AP2/EREBP sequence of DREB
282
gene 20. Amplification was carried out in 25 l reaction volumes,

containing 1X Taq polymerase buffer (50 mM KCl, 10 mM Tris, pH
7.5, 1.5 mM MgCl2) and 1 unit of Taq polymerase (Pharmacia
Biotech, Germany) supplemented with 0.01% gelatin, 0.2 mM of
each dNTPs (Pharmacia Biotech, Germany), 25 pmol of forward
and reverse of each primer, and 50 ng of total genomic DNA.
Amplification was performed in a thermal cycler (Thermolyne
Amplitron) programmed for 1 cycle of 30 s at 94C; 40 cycles of 1
min at 94C, 1 min at 55C and 1 min at 72C; followed by 5 min at
72C.
Cluster analysis: Data from the ISSR analysis were scored for
cluster analysis on the basis of the presence or absence of the
amplified products for each ISSR primer. If a product was present
in a cultivar, then it was designated 1; if absent, then the product
was designated 0. Pairwise comparisons of genotypes, based
on the presence or absence of unique and shared polymorphic
products, were used to generate similarity coefficients based on
the SIMQUAL module. The similarity coefficients were used to
construct a dendrogram by UPGMA (Unweighted Pair-Group
Method with Arithmetical Averages) using NTSYS-PC software
version 2.0 (Exeter Software, New York) 13.
Genetic diversity among watermelon genotypes: The results of
ISSR-PCR are presented in Table 2. The ten primers amplified 42
band positions (loci). In 19 of the 42 loci amplified, the watermelon
genotypes were polymorphic. The number of amplification bands
per primer varied between 2 and 5. The dinucleotide repeat (AC)n
primer had more bands than the other primers (Fig. 1, Table 2),
likely because of its greater abundance in watermelon genome.
The repeats (GA)n and (CA)n were the most abundant in rice 12,
jojoba 1, and date palm 18 genomes. Fig. 1 shows the amplification
profiles generated by the HB 13 and UBC 827 primers. In the
current study, 8 out of 10 ISSR primers exhibited polymorphism
among watermelon genotypes. Therefore, the ISSR technique can
play an important role in watermelon improvement programs as it
is effective in polymorphism identification between very closely
related lines.
Table 2. ISSR primers used in this study and a summary of
the ISSR markers from 7 watermelon genotypes.
Primer
UBC807
UBC810
UBC823
UBC825
UBC826
UBC827
UBC864
HB13
HB14
HB15
Primer
sequence
(AG)8T
(GA)8T
(TC)8C
(AC)8T
(AC)8C
(AC)8G
(ATG)6
(GAG)3GC
(CTC)3GC
(GTG)3GC
Annealing
temperature
(C)
50
50
50
50
50
50
50
42
42
42
Amplified
products
4
5
2
5
4
5
4
4
5
4
Fraction
polymorphic
fragments
3/4
1/5
0/2
1/5
3/4
5/5
3/4
1/4
0/5
2/4
Determined empirically. Number of polymorphic fragments/number of fragments amplified.
The ISSR-derived data were analysed to calculate the genetic

similarity (ISSR-GS) (Table 3). The genetic similarity coefficient
varied between 0.53 and 0.95. The minimum GS value was between
wild-type genotype and cultivar Diamond, while the maximum GS
value was between cv. Crimson and cv. Charleston gray. Wild-
Table 3. Simple matching coefficients of similarity determined from the analysis using 42 ISSR loci.
Watermelon genotypes
Sun shade
Diamond
Charleston
Fashion
Charleston gray
Crimson
Wild genotype
Sun shade
1.0
0.83
0.95
0.85
0.95
0.90
0.65
Diamond
Charleston
Fashion
Charleston gray
Crimson
Wild genotype
1.0
0.78
0.83
0.83
0.78
0.53
1.0
0.85
0.90
0.85
0.60
1.0
0.90
0.85
0.65
1.0
0.95
0.70
1.0
0.75
1.0
HB13
M
C1
C2
C3
C4
C5
C6
Sun shade
C7
Charleston
Char. gray
Crimson
Fashion
Diamond
Wild
0.65
0.72
0.80
Coefficient
0.87
0.95
Figure 2. Dendrogram constructed from similarity coefficients and

showing the clustering of the tested watermelon genotypes.
UBC827
M
C1
C2
C3
C4
C5
C6
C7

Figure 1. Polymorphism revealed using primer ISSR primers (HB 13,
UBC827) to amplify genomic DNA purified from the tested watermelon
genotypes (C1 = Sun shade, C2 = Diamond, C3 = Charleston, C4 =
Fashion, C5 = Charleston gray, C6 = Crimson, and C7 = Wild genotype).
type genotype was quite distinct from watermelon cultivars. A

cluster analysis was conducted to generate a dendrogram showing
the relationships between watermelon genotypes. The
dendrogram generated using the pooled ISSR data divided the
watermelon genotypes into two main clusters (Fig. 2). Cluster I
included all watermelon cultivars (C. lanatus) that originated from
countries in Holland and USA. Cluster II was composed of wildtype species. The genetic distance between the nodes in these 2
clusters was approximately 0.65, indicating that a high level of
genetic dissimilarity exists between adapted watermelon species
and related other species 9. The first cluster was divided into three
sub-clusters that included the following: (i) cultivars Sun shade,
Charleston, Charleston gray, and Crimson sweet, (ii) cultivar
Fashion, and (iii) cultivar Diamond. The similarity values among
the cultivars in Cluster I were very high (0.83-0.95), demonstrating
cross relationships and a narrow genetic background 6-8, 10.
Previous studies by Levi et al. 9 indicated that among Citrullus
species, the wild C. colocynthis species, which has the widest

geographic distribution, also has the highest genetic diversity.
These studies also demonstrated higher genetic diversity within
the wild subspecies C. lanatus var. citroides, than in C. lanatus
var. lanatus.
SSR marker for DREB gene: Irrigation treatments of three- and
five-day intervals decreased chlorophyll content and plant height
as compared with control watered daily. There were no significant
differences in chlorophyll content and plant height of wild-type
species either in three-day intervals or watered daily. All watermelon
cultivars gave lower chlorophyll content and plant height at threeand five-day intervals compared to watered daily (Table 4).
Drought induces the expression of a number of plant genes
that encode proteins to enhance tolerance of plants to water stress.
One group of these genes is to regulate gene expression and
signal transduction. They include transcription factors, for example
CBF/DRE-binding protein or CBF/DREB 15. Specific PCR assays
using AP2 primers (designed on the basis of the AP2/EREBP
sequence of gene BeDREB) represent a sensitive tool for screening
watermelon genotypes for the BeDREB gene. The amplification
of the BeDREB gene of watermelon genotypes produced one
fragment of approximately 800 bp. The BeDREB gene was present
only in wild-type C. colocynthis (Fig. 3). This species, also known
as bitter gourd, is a drought-resistant perennial and is used as a
M
C1
C2
C3
C4
C5
C6
C7
Figure 3. Detection of DREB gene in watermelon genotypes (C1 = Sun

shade, C2 = Diamond, C3 = Charleston, C4 = Fashion, C5 = Charleson
gray, C6 = Crimson, and C7 = wild genotype) using AP2 primer. M is a
ladder marker (100 bp). Arrow shows the one polymorphic band for
DREB gene.
283
Table 4. Effect of interaction between irrigation treatments and cultivars on chlorophyll content
(SPAD) and plant height (cm) of seven watermelon genotypes.
Cultivars
Sun shade
Diamond
Charleston
Charleston gray
Crimson sweet
Fashion
Wild type
Chlorophyll content (SPAD)

Every 5 days Every 3 days Every day
31.9g
32.8g
37.73f
39.83f
45.33cd
47.9b
32.2g
37.37f
43.4de
32.1g
37.3f
43.2de
33.0g
38.0f
44.0de
40.73ef
44.17de
49.3a
31.07g
39.3f
38.67f
Plant height (cm)

Every 5 days Every 3 days Every day
100f
125d
155b
108e
140c
165a
63hi
67h
73g
60hi
66h
74g
62hi
67h
75g
48k
57j
61ij
26m
32l
34l
medicinal plant in the pharmaceutical industry 3. Therefore, the

BeDREB gene is important for breeding watermelon cultivars to
drought tolerance.
Conclusions
ISSR-PCR was efficient for distinguishing among cultivated
watermelons and wide-type C. colocynthis. Also, BeDREB gene
was present only in wild-type C. colocynthis. Therefore,
interspecific crosses between wild-type species and adapted
cultigens will enable breeders to develop new breeding sources
with diverse characteristics, including resistance to drought stress.
This process can be facilitated by a backcross that applies
molecular markers in both the foreground and background
selection. The abundance in polymorphisms shown in this study
ensures readiness for molecular marker-based introgression of
useful traits from wild-type species 5.
Acknowledgements
The authors gratefully acknowledge the Deanship of Scientific
Research at Qassim University for funding this project. The
authors thank Prof. H. Abidallah for his assistance in experimental
design of greenhouse experiment and collecting the data of
chlorophyll content and plant height.
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9
WFL Publisher
Helsinki, Finland
www.world-food.net
Impact of PAR interception at different time points on total dry matter production in
rice (Oryza sativa L.) crop transplanted on different dates
Shrabani Basu 1*, Srijani Maji 1, Swaraj Kumar Dutta 2, Sarika Jena 3, Rajib Nath 1 and Prodip Kumar Chakraborty 4
Department of Agronomy, Faculty of Agriculture,Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal741252, India. 2 Department of Agronomy, Faculty of Agriculture, Bihar Agriculture University, Sabour, Bihar-813210, India.
3
AINP Jute Research Station, Kendrapara, Odisha University of Agriculture and Technology, Odisha -754250, India.
4
Department of Agric. Meteorology and Physics, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur,
Nadia, West Bengal-741252, India. *e-mail: shrabanibasu808@gmail.com
1
Abstract
The main rice-growing season in Eastern India spans from July-October. The average productivity is low because of cloudiness during the season.
Interception of photosynthetically active radiation (PAR) by the crop at the different time points of a day plays a significant role in biomass
accumulation, which is ultimately translated into yield. The effect of radiation has been investigated but the impact of different time-point has not
been discussed in the literatures. To identify the congenial time point for PAR interception, an experiment was carried out for two years (2007 and
2008) on rice [variety IET-4786 (Satabdi)], which was transplanted during a period of 1st to 29th July having one week interval. The experiment was
conducted at the B.C.K.V Research Farm (2256'N and 8832'E) in a randomised block design with four replications having a plot size of 30 m2. The
PAR interception was measured at 7.30, 9.30, 11.30, 13.30 and 15.30 h during tillering, panicle initiation, flower emergence and 100% flowering
stages. The total dry matter accumulation and leaf area index (LAI) were measured at these same growth stages. The mean interception was maximum
at 100% flowering and minimum at the tillering stage; interception by the rice crop declined gradually after 15th July transplanting. Maximum dry
matter accumulation was observed at 100% flowering. About 88, 92,73 and 97% variations in dry matter accumulation were explained by PAR
interception at 13.30 h under 1st, 8th, 22nd and 29th July transplanted rice; whereas, 73% variation in dry matter accumulation could be explained
through the variation in interception of PAR under 15th July transplanted crop. It was observed that the interception during early morning and
afternoon played a significant role in biomass production. In case of late-planted crop, interception almost throughout the day played a regulatory
role in dry matter production. The LAI increased gradually and the maximum LAI was recorded at 100% flowering. Delay in transplanting reduced
the LAI. PAR interception increased significantly with the increment in LAI; the strength of relationship declined beyond 15th July transplanting. The
results suggested that the rice should not be transplanted beyond 15th of July depending on PAR interception by the crop.
Key words: Dry matter, interception, tillering, panicle initiation, flower emergence, 100% flowering, PAR, planting dates, leaf area index, rice.
Introduction
Rice yield in Eastern India enhanced when the high yielding
varieties replaced the traditional ones. Modern research helped
to evolve a large number of high yielding varieties in rice for yield
improvement. However, these varieties could not reach the
potential yield level of rice crop. The rice yield in the main rice
growing rainy season is comparatively lower than the summer
season when irrigation is given to this crop for high productivity.
Use of groundwater causes a number of problems such as arsenic
contamination, depletion of groundwater level and fluoride
contamination. The concentration of arsenic in groundwater has
crossed the critical limit (>0.01 ppm) and has caused a human
disaster, affecting more than nine million people 1, 2. In spite of
these difficulties, farmers are reluctant to grow other alternative
crops having low water requirement. Therefore, it is highly
important to improve the rice yield by transplanting the crop in an
ideal weather situation, so that the crop may use the weather
input in a better aspect. Rice yield increases with the increase in
total solar radiation under unstressed condition 3. Solar radiation
in a rice canopy plays an important role in energy balance on both
the plant leaves and soil/water surface. Absorption coefficients
of rice leaves throughout the growing period were almost constant,
but the leaf inclination factor increased with the growth of the
crop 4. Absorption or interception of solar radiation changes with
the solar elevation angle during the apparent diurnal journey of
the sun. Therefore, it is logical to measure the interception or
absorption of solar radiation by the crop through a diurnal course.
The photosynthetic efficiency of a crop enhances with the diurnal
variation in interception of solar radiation. Utilisation of solar
radiation during morning and afternoon hours in peanut and
mustard were better than other time points in a diurnal course 5-7.
However, no such studies have been reported in rice crop so far.
The main rice-growing season (July-October) very often suffers
from cloudiness, which reduces the crop yield. With an objective
to indicate an ideal time-point for interception of PAR and its
relationship with the total biomass production and leaf area index
in rice crop, this experiment has been framed.
Experimental site: The experiment was conducted at the B.C.K.V
research farm (C-Block, 2256' N Latitude, 8832' E Longitude and
9.75 m above sea level), Kalyani, West Bengal.
285
Climate: The experimental site falls under tropical humid climate

and experiences three distinct seasons - March to June as summer,
June to September as rainy season and October to February as
winter; the summer season is humid and receives rainfall with
thunderstorm occasionally. The mercury reaches at its maximum
during the month of May, whereas the coldest day is usually
observed during January. The South West Monsoon season starts
during the first week of June and withdraws from the area during
1st week of October. Kalyani receives an average annual rainfall of
1,600 mm out of which 1,300 mm occurs during monsoon. NorWester shower comes during March, April or May and the area
experiences dry spell for long period with concomitant high air
temperature.
Determination of dry matter accumulation: Total dry matter

accumulation was measured at tillering, panicle initiation,
emergence of flower and 100% flowering. Five plants were selected
from the 2nd row and plants were cut from the ground level. The
leaves, stems and roots were separated and dried in hot air oven
at 75C temperature for 48 h. The summation of the dry weight of
stem leaves and roots gave total dry matter accumulation, which
was then calculated in terms of g m-2.
Determination of LAI: The leaf area index (LAI) was estimated
by area weight relationship method 9. Leaf area index was
calculated as follows:
LAI =
Experimental details: Twenty-five days old seedlings of rice (var.

Satabdi i.e. IET-4786) was transplanted within a span of 1st and
29th July having a weekly interval. The experimental design was
completely randomised block design. Each treatment is allotted to
a plot of 30 m2 having four replications. The soil is Entisol having
the pH of 7.45, N 0.07%, organic carbon 0.67%, available P and K
are 22.02 and 126 kg P2O5 and K2O ha-1. Before transplanting, each
plot was puddled, fertilised with N, P2O5 and K2O at the rate of
80:60:60 kg ha -1, respectively, through urea, single super
phosphate (SSP) and muriate of potash (MOP). Half of the N was
applied at the time of transplanting and the rest half was applied
at the time of tillering stage. Twenty-five days old seedlings were
transplanted giving an inter and intra-row spacing of 20 and 15
cm, respectively.
Measurements: The photosynthetically active radiation (PAR)
was measured with the help of line quantum sensor (APOGEE
Logan UK, Model no. MQ-301), placing across the row 50 cm
above the crop to measure the incident radiation. The instrument
was horizontally lowered down the canopy and placed 50 cm above
the water level to measure the PAR at the bottom. The reflected
PAR from the canopy was measured at the same position by simply
inverting the sensor. The observations were recorded at 7.30, 9.30,
11.30, 13.30 and 15.30 h on each phenophase to have a diurnal
variation.
Intercepted PAR (IPAR): Intercepted PAR was calculated with
the help of the following equation 8:
IPAR = PAR(o) T PAR R PAR(c)
where PAR(o) is the portion of the incident PAR above the canopy,
T PAR is transmitted portion of the PAR through the canopy to
the soil surface (incident PAR value at the bottom of the rice
canopy) and R PAR(c) is reflected PAR from crop (reflected PAR
value at the uppermost layer of the rice canopy).
Total leaf area for a given land area

Land area considered
Statistical analysis: The total dry matter and LAI were analysed
and the results were given separately for two years with a twoyear pooled mean values. The radiation data could not be
statistically analysed. Impact of PAR interception during different
hours of a day on dry matter accumulation was computed through
regression analysis. The statistical calculation was done by SPSS
version 7.5 (SPSS 7.5, 1997, copyright by SPSS Inc., USA Base 7.5
Application guide).
Interception: The mean interception of PAR in rice canopy ranged
from 71.8 to 72.4% at the tillering stage in the first year, whereas, in
the second year, it ranged from 69.8 to 83.7% (Table 1). Interception
increased with the progress of growth because of increased LAI.
Flowering stage recorded maximum interception under all dates of
transplanting. Among the five different transplanting dates, the
8 th July planted crop recorded the maximum interception.
Interception reduced with the delay in transplanting, which might
be due to the low LAI under delayed planted crop.
Total dry matter production: The total dry matter accumulation
was maximum under D1 planting irrespective of phenophase and
year of experimentation. The two-year pooled mean values recorded
the similar trend. Dry matter accumulation significantly reduced
with the delay in transplanting. The reduction in dry matter
accumulations were 6.9, 9.0, 25.9 and 1.7%, respectively, for each
seven days delay during the span of 1st July and 29th July (Table
2). The interception at different time points significantly affected
the total biomass in rice crop. The interception at 7.30, 9.30 and
13.30 h significantly affected the biomass production. The
relationship was highly significant at 9.30 and 13.30 h under D1
planting (Fig. 1). Under D2 planting, the interception at 13.30 and
15.30 h was found to be highly significant. About 92% variation in
total biomass could be explained through the variation in
intercepted PAR at these two time points (Fig. 2). In case of D3
Table 1. Mean interception (%) of PAR by rice at the different phenophases under different dates of transplanting.
Mean interception of PAR (%)
Treatments
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
286
Tillering
PI
72.4
77.6
71.8
73.1
73.4
83.7
86.2
78.7
81.0
76.7
2007
Flower
emergence
86.4
88.0
86.0
85.3
85.6
100%
flowering
89.8
91.7
87.6
86.4
87.2
Mean
Tillering
PI
83.1
85.9
81.0
81.5
80.7
83.7
80.0
74.9
77.3
69.8
80.7
85.4
78.5
83.3
75.8
2008
Flower
emergence
85.0
88.9
84.3
87.8
87.4
100%
flowering
88.6
91.7
85.7
87.4
87.3
Mean
Year
mean
84.5
86.5
80.9
84.0
80.1
83.8
86.2
80.9
82.7
80.4
Total biomass (g m-2)
1500
1000
500
0
0
y = -0.3612x2 + 65.813x - 875.19

R2 = 0.6506 *
(7:30)
10
D1
2000
20
30
40
Intercepted PAR (W m-2)
50
1500
1000
500
0
80
100
D1
2000
120
140
160
y = -0.1255x2 + 33.133x - 689.65

R2 = 0.8422 **
(9:30)
1500
1000
500
0
0
y = -0.506x2 + 161.16x - 11531

R2 = 0.5028
(11:30)
D1
2000
60
D1
2000
20
D1
2000
40
(13:30)
60
80
100 120
140
160
y = 0.2586x2 - 34.148x + 1421.5

R2 = 0.8827 **
1500
1000
180
500
0
40
60
80
100
120
140
160
y = -1.497x2 + 186.5x - 4434

R2 = 0.215
(15:30)
1500
1000
500
0
20
40
60
80
100
Figure 1. Impact of PAR interception on dry matter accumulation in rice at different time points in D1 (1st July) transplanted crop
(averaged over phenophases and year).
Table 2. Total dry matter accumulation in rice at the different

phenophases under different dates of transplanting.
2007
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
2008
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
Pooled
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
Tillering
Panicle
initiation
Flower
emergence
100%
Flowering
333.6
300.7
240.9
195.1
161.0
8.0
23.8
652.5
504.1
427.8
486.4
354.0
8.1
24.2
1329.6
1091.0
981.7
893.0
636.1
17.2
51.5
1557.3
1376.7
1131.6
784.9
852.3
14.4
43.2
283.3
249.2
227.1
217.1
209.6
8.9
26.8
671.7
713.8
617.2
492.8
303.7
17.9
53.8
1336.3
1138.6
1387.7
1002.3
783.1
13.8
41.4
1473.1
1443.4
1433.7
1116.7
1016.5
17.5
52.6
308.5
274.9
234.0
206.1
185.3
6.7
20.1
662.1
608.9
522.5
489.6
328.8
9.3
28.0
1332.9
1114.8
1184.7
947.6
709.6
10.6
31.8
1515.2
1410.0
1282.7
950.8
934.4
11.6
34.7
planting, the interception at 9.30 and 11.30 h was found to affect

significantly the biomass production (Fig. 3). Under D4 planting,
the interception at 9.30, 11.30, 13.30 and 15.30 h significantly
affected the biomass production (Fig. 4). Under D5 planting, the
interception of PAR at 7.30, 9.30 and at 13.30 h significantly affected
the biomass production (Fig. 5). Interception of PAR at different
time points in a day significantly affected the total biomass in rice

crop. Among the five time points, the interception at 13.30 h had
the maximum effect (Fig. 6). The capture of radiation at different
time points plays a significant role in dry matter accumulation 10.
The development of leaf area and the pattern of intercepted radiation
determine the carbon and nitrogen capture by the crop 11. The
radiation in the early morning and late afternoon hours
significantly affected the photosynthetic processes in groundnut
and mustard, respectively 6, 7. However, in rice crop, the effect of
interception of PAR on dry matter accumulation at different
phenophases was different at different time points of a day. When
the crop was transplanted within 15th July, the intercepted PAR in
the morning or afternoon hours played a significant role, whereas,
in case of delayed planting, the interception almost throughout
the day played a regulatory role in biomass production. The dry
matter production in rice was significantly affected by the solar
radiation absorbed by the crop 12, 13. However, the present
experiment showed that the regulatory role of PAR interception
for dry matter production had been altered at different time points.
This variation has been initiated due to the cloudy weather present
during the monsoon season. The mean interception was also
declined under the late-planted crop because of low LAI.
Leaf area index (LAI): The leaf area index recorded a continuous
increase from tillering to flower emergence (Table 3). In 2007, LAI
increased up to flowering, but in 2008, it recorded a marginal decline
during flowering. The 1st July sown crop recorded the maximum
LAI, whereas, a gradual reduction in LAI was recorded with the
late-transplanted crops. During tillering stage, the magnitude of
reductions in LAI due to late transplanting were 10, 17.7, 10 and
22.5%, respectively, for D2, D3, D4 and D5 transplanting compare
287
1500
1000
500
0
20
40
D2
2000
60
80
100
1000
500
0
120
D2
2000
140
160
180
200
2000
D2
220
y = 0.119x2 - 18.36x + 1370

R2 = 0.360
(9:30)
1500
1000
500
0
20
y = -0.017x2 + 18.95x - 1761

R2 = 0.477
(11:30)
1500
100
y = -0.111x2 + 2.433x + 346.9

R2 = 0.303
(7:30)
D2
2000
2000
40
D2
60
(13:30)
1500
140
80
100 120
160
180
y = -0.2431x2 + 59.709x - 2321

R2 = 0.9273 **
1000
500
0
20
40
60
100
120
80
140
160
y = -0.2409x2 + 43.782x - 538.34

R2 = 0.9268 **
(15:30)
1000
0
40
60
20
80
100
D3
2000
y = -1.991x2 + 194.7x - 3676

R2 = 0.433
(7:30)
Figure 2. Impact of PAR interception on dry matter accumulation in rice at different time points in D2 (8th July) transplanted crop
(averaged over phenophases and year).
1500
1000
500
0
20
40
D3
2000
(9:30)
1500
1000
500
0
60
20
40
60
D3
1500
1000
500
0
100
y = 0.1394x2 - 25.69x + 1356.2

R2 = 0.7307 **
(11:30)
2000
120
D3
2000
140
160
180
200
y = -1.6749x2 + 214.83x - 5641.9

R2 = 0.6656 *
2000
D3
(13:30)
1500
80
y = 0.053x2 + 1.282x + 265.0

R2 = 0.463
1000
500
0
20
40
60
80
100
120
140
y = 0.124x2 + 13.30x + 55.16

R2 = 0.389
(15:30)
1500
1000
500
0
0
20
40
60
80
Figure 3. Impact of PAR interception on dry matter accumulation in rice at different time points in D3 (15th July) transplanted
crop (averaged over phenophases and year).
288
(7:30)
y = 0.818x - 25.49x + 385.7

R2 = 0.421
2
200
0
1200
1000
800
600
400
20
40
D4
y = -0.022x2 + 21.698x - 2212.9

R2 = 0.651 *
200
0
120
140
160
180
D4
(9:30)
y = 0.00007x2 + 10.301x - 129.11

R2 = 0.575 *
200
0
0
(11:30)
100
1200
1000
800
600
400
60

D4
1200
1000
800
600
400
1200
20
D4
1000
800
600
400
60
100
40
80
D4
140
140
160
(13:30)
y = 0.056x2 - 0.2711x - 121.13

R2 = 0.7361 **
200
0
0
200
20
40
1500
120
60
80
100
120
y = -0.467x2 + 53.498x - 635.31

R2 = 0.7314 *
(15:30)
1000
500
0
20
40
60
80
D5
800
600
(7:30)
y = 1.9873x2 - 93.089x + 1178.5

R2 = 0.9642 **
400
200
0
20
40
D5
1500
(11:30)
500
0
100
D5
1500
(15:30)
120
140
160
180
D5
200
0
1500
60
40
80
20
D5
1000
100
120
(13:30)
y = 0.1303x2 - 5.2915x - 49.38

R2 = 0.9736 **
500
0
40
200
(9:30)
y = 0.0209x2 + 9.8248x - 309.18

R2 = 0.8449 **
y = 0.007x + 5.515x - 363.5

R2 = 0.393
1000
1200
1000
800
600
400
60
80
1200
1000
Figure 4. Impact of PAR interception on dry matter accumulation in rice at different time points in D4 (22nd July) transplanted
60
80
100
120
y = 0.956x2 - 48.62x + 1079

R2 = 0.131
1000
500
0
0
40
20
60
Figure 5. Impact of PAR interception on dry matter accumulation in rice at different time points in D5 (29th July) transplanted
289
1500
1000
500
0
0
20
40
60
500
0
15:30
2000
y = 0.0071x2 + 6.9544x + 176.3

R2 = 0.3663 *
1500
1000
500
0
50
150
100
200
1000
100
150
9:30
1500
50
2000
100
80
y = 0.0283x2 + 3.2881x - 396.81

R2 = 0.4204 **
11:30
2000
y = -0.2899x2 + 44.325x - 508.67

R2 = 0.3558 **
7:30
2000
200
250
y = 0.0371x2 + 3.6882x + 9.0773

R2 = 0.5022 **
13:30
2000
1500
1000
500
0
50
100
150
200
y = -0.2109x2 + 34.412x - 239.85

R2 = 0.4394 **
1500
1000
500
0
0
20
40
60
100
80
Figure 6. Impact of PAR interception on dry matter accumulation in rice at different time points (averaged over phenophases, dates
of transplanting and year).
Table 3. Effect of dates of transplanting on LAI at the different

phenophases of rice.
2007
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
2008
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
Pooled
D1 (1st July)
D2 (8th July)
D3 (15th July)
D4 (22nd July)
D5 (29th July)
S.Em ()
LSD at 5%
290
Tillering
Panicle
initiation
Flower
emergence
100%
Flowering
4.2
3.7
3.2
3.3
2.9
0.11
0.33
6.5
6.1
5.6
5.2
4.8
0.10
0.31
7.8
6.7
6.9
6.8
5.6
0.19
0.58
8.7
8.0
8.0
7.7
7.6
0.13
0.39
6.8
6.1
5.9
5.8
5.2
3.7
3.6
3.5
3.9
3.4
0.11
0.34
6.3
5.9
6.2
5.2
5.1
0.51
1.53
8.6
8.6
8.2
7.9
7.9
0.22
0.65
8.0
8.0
8.1
7.8
7.9
0.15
0.46
6.7
6.5
6.5
6.2
6.1
4.0
3.6
3.3
3.6
3.1
0.09
0.26
6.4
6.0
5.9
5.2
4.9
0.24
0.73
8.2
7.7
7.5
7.3
6.8
0.17
0.52
8.4
8.0
8.1
7.8
7.7
0.09
0.26
6.8
6.3
6.2
6.0
5.6
Mean
with the D1 transplanting. The extent of reductions in LAI during

panicle initiation stage was 6.7, 7.8, 18.7 and 23.4%, respectively,
for D2, D3, D4 and D5 transplanting. The rate of LAI development
was maximum during transplanting to tillering in case of D1
planting. The rate of LAI development declined from panicle
initiation to flowering because of the increment of the maximum
temperature and reduction of the afternoon humidity. Increment
in LAI in rice might be observed until the anthesis 14. The late
transplanted crop tried to cope up the situation by accelerating
the rate of development during tillering to panicle initiation, but
the total light harnessing by the late planted crop was severely
affected leading to the lower productivity.
Interception of PAR increased with the increase in LAI
irrespective of dates of transplanting. The strength of association
became insignificant under D4 and D5 transplanting (Fig. 7) as the
LAI increased, the interception of PAR increased, which may have
significant influence on the yield of the crop 15.
Conclusions
PAR interception during early morning (7.30-9.30 h) and in the
afternoon (13.30 15.30 h) played a significant role in biomass
production in rice crop, when the crop was transplanted within
15th July. However, for late transplanted crop, PAR interception
throughout the day significantly contributed to the biomass
production, although, biomass accumulation was found to be
reduced. The results suggested that the rice should not be
transplanted beyond 15th of July in eastern Gangetic plains of
West Bengal for better productivity based on interception pattern
of PAR by the rice canopy.
Intercepted PAR (%)
92
90
88
86
84
82
80
D1
Intercepted PAR (%)
D2
95
Intercepted PAR (%)

Intercepted PAR (%)
10
10
y = 0.0701x2 + 0.5982x + 81.326

R2 = 0.7625**
90
85
80
0
Intercepted PAR (%)
4
6
Leaf area index (LAI)
95
D3
4
6
y = 0.1171x2 + 0.3806x + 77.673
R2 = 0.8529**
90
85
80
75
D4
84
4
6
10
y = 0.0002x2 + 0.672x + 76.582

R2 = 0.5100*
82
80
78
76
0
100
4
6
Pallas, J. E. Jr. and Samish, Y. B. 1974. Photosynthetic response of

peanut. Crop Sci. 14:478-482.
6
Nayyar, H., Malik, C. P., Singh, P., Parmar, U., Grewal, M. and Kaur, S.
1990. Diurnal variation in photosynthetic parameters in peanut.
Photosynthetica 24:276-279.
7
Chakraborty, P. K. 1994. Effect of date of sowing and irrigation on the
diurnal variation in physiological process in the leaf of Indian mustard
(Brassica juncea). J. Oilseeds Res. 11(2):210-216.
8
Dhaliwal, L. K., Hundal, S. S., Kular, J. S., Chahal, S. K. and Aneja, A.
2007. Radiation interception, growth dynamics and agroclimatic indices
in raya (Brassica juncea). J. Agromet 9:242-246.
9
Radford, P. J. 1967. Growth analyses formulae their use and abuse.
Crop Sci. 7:171-175.
10
Hay, R. and Porter, J. 2006. The Physiology of Crop Yield. 2nd edn.
Blackwell Publishing, New York, USA, 330 p.
11
Lemaire, G., van Oosterom, E., Sheehy, J., Jeuffroy, M. H., Massignam,
A. and Rossato, L. 2007. Is crop nitrogen demand more closely related
to dry matter accumulation or leaf area expansion during vegetative
growth? Field Crops Res. 100:91-106.
12
Ishikawa, T., Fujimoto, H., Kabaki, N., Maruyama, S. and Akita, S.
2003. Effect of temperature and solar radiation on dry matter production
and translocation during the ripening period in rice cv. Takanari. Japanese
J. Crop Sci. 72(3):339-344.
13
Al-Khaffaf, A., Marzah, T. K. and Hujairi, A. O. 2003. Effect of
temperatures and light accumulations on some vegetative growth
characteristics and dry weight of flowering and harvest of three rice
cultivars (Oryza sativa, L.). University of Aden J. Natural and Applied
Sci. 7(1):1-13.
14
Takai, T., Matsuura, S., Nishio, T., Ohsumi, A., Shiraiwa, T. and Horie,
T. 2006. Rice yield potential is closely related to crop growth rate
during late reproductive period. Field Crops Res. 96:328-335.
15
Campbell, C. S., Heliman, J. L., McInnes, K. J., Wilson, L. T., Medley,
J. C., Wu, G. and Cobos, D. R. 2001. Seasonal variation in radiation
use efficiency of irrigated rice. Agric. Forest Meteorol. 110:45-54.
5
y = -0.2438x2 + 4.0272x + 71.344

R2 = 0.7378**
10
D5
50
y = 1.0442x2 - 10.086x + 104.23

R2 = 0.4776*
0
0
4
6
10
Figure 7. Relationship between LAI and intercepted PAR under different

dates of transplanting.
Acknowledgements
The first author acknowledges the financial help to the
Department of Science and Technology, Govt. of India for
providing INSPIRE Fellowship.
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Yang, C. M. 1994. Response of rice yield in relation to solar radiation
and air temperature under soil water deficits. Chinese J. Agromet.
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1
291
WFL Publisher
Helsinki, Finland
www.world-food.net
Growth and visual symptoms of nutrient deficiencies in young Mentha piperita plants
Diocla Almeida Seabra Silva 1*, Mrio Lopes da Silva Jnior 1, Ismael de Jesus Matos Vigas 2*,
Allan Klynger da Silva Lobato 3, Vnia Silva de Melo 1, Snia Maria Arajo Botelho 1, George Rodrigues da
Silva 1, Joze Melisa Nunes de Freitas 1, Cndido Ferreira de Oliveira Neto 1, Milton Leite Alves da Cunha 4
and Ana Regina da Rocha Araujo 1
Instituto de Cincias Agrrias, Universidade Federal Rural da Amaznia, Avenida Tancredo Neves, n 2501, Bairro: Montese, CEP.
66.077-830, Belm, Par, Brasil. Campus de Capanema, Universidade Federal Rural da Amaznia, Par, Brasil. Ncleo de Pesquisa
Vegetal Bsica e Aplicada, Universidade Federal Rural da Amaznia, Paragominas, Brasil. 4 Ministrio da Agricultura Pecuria e
Abastecimento, Par, Brasil. e-mail: dioclea@ibest.com.br, ismael.viegas@ufra.edu.br
Abstract
The characterization of the deficiency symptoms of macronutrients and micronutrients on the growth of young plants of Mentha piperita were
evaluated by the missing element technique. The experiment was carried out under greenhouse conditions at Universidade Federal Rural da Amaznia.
The experimental design was completely randomized blocks with 12 treatments and four replicates and a total of 48 plots. The following treatments
were tested: (C) Complete (macro-and micro-nutrients) and omissions of N, P, K, Ca, Mg, S, B ,Cu, Fe, Mn and Zn. The variables analyzed were:
height, stem diameter, leaf area, and dry matter production. The omissions of nutrients induced morphological changes translated into deficiency
symptoms. The omission of N and Ca showed the first visual symptoms of deficiency, followed by P, K, Mg, S, B, Cu, Fe and Mn=Zn; omissions
of N, P, K, Mg, S, B, Cu, Fe, Zn and Mn = compromise the plant growth. The most limiting nutrients for the plant growth based on the total dry
matter and foliar area was N, followed by Ca; boron is the micronutrient that most affects the plant growth.
Key words: Mentha piperita, mineral nutrition, nutritional deficiency.
Introduction
Mentha piperita L., a native plant to tropical regions, belongs to
the family Lamiaceae, perfectly adapted to the climatic conditions
of Amazon region. This plant has been used for decades by
industries in the manufacture of perfumes, essences and cosmetics.
It is common tourists visit the state of Para in search for those
fragrances to introduce it in other countries, making the demand
for mint great attraction, moving the trade and allowing opening
of new markets.
Despite having high economic importance because of the role
it plays in the manufacture of perfumes, peppermint plants also
have medicinal, antiseptic, cardiotonic, bleaching hair, collagen,
digestive, skin stiffener, stimulant and purgative properties 1, 2.
The availability of raw materials is not sufficient to meet local
demand so the products from the peppermint are made by artisans
to increase family income. M. piperita plays an important role in
the economy of the State of Para, precisely in the perfume and
cosmetic industries 3, which had increases of about 66.5% due to
a demand for aromatic products, and a strong industrial demand
of about 2,065 tons.
Despite the intense search for M. piperita, there is little
information about the nutritional limitations that the plant may
show. Blank et al. 4 report that there is little information in the
literature, with no specific work related to the best nutrient solution
that provides the best plant growth. As it is not known which are
the most nutrients that could limit M. piperita production, it has
been used hydroponics to obtain a better response in a short
292
period of time 5, 6. Hydroponics is a technique where nutrients are

supplied by a nutrient solution, which has been widespread in the
forage production.
Therefore, one of the major limitations of market expansion is a
nutritional issue, and poor investment in research to the needs of
the species for nutrients that limit its growth. This culture is very
important by its ability of producing essential oil, a major
constituent of menthol, used as flavouring, additives, food and
oral hygiene products7. The objective of this study was to evaluate
the growth, symptomatology and dry matter production of M.
piperita, grown in nutrient solution.
Material and Methods
Experimental conditions: Experiment was carried out in an
experimental area of Instituto de Cincias Agrrias ICA) of
Universidade Federal Rural da Amazonia, in Belm State of Par.
Recipients and plant obtaining: Seedlings of Mentha piperita
L., from the Department of Plant Science were transplanted to
plastic pots containing 3 kg of ground quartz substrate (zero
coarse, type 4).
Plant material and acclimation: Peppermint plants were daily
watered with a complete solution Hoagland et al. 8, in a rate 1:1,
for a period of 15 days for adaptation (Table 1).
Table 1. Chemical composition of nutrient solutions (mL-1) used in the experiment, according
to Hoagland and Arnon 8, modified.
Stock solution
NH4H2PO4
KNO3
Ca(NO3)2
MgSO4
KH2PO4
KCl
CaCl2
NH4NO3
NaNO3
K2SO4
MgCl2
Solution a*
Solution a - B
Solution a - Cu
Solution - Fe
Solution - Mn
Solution - Zn
Solution de -Fe EDTA**
Conc.
1M
1M
1M
1M
1M
1M
1M
1M
1M
1M
1M
C
2
6
4
1
1
1
-N
1
2
4
4
-
-P
8
4
1
2
-
-K
2
4
1
6
-
-Ca
2
6
1
4
-
-Mg
2
6
4
1
-
-S
2
6
4
1
-
-B
1
-
-Cu
1
-
-Fe
1
-
-Mn
1
-
-Zn
1
-
*Composition of solution: 2.86 g of H3BO3; 1.81 g of MnCl2.4H2O and 0.22 g of ZnSO4.7H2O; 0.88 g CuSO4.5H2O; 0.02 g of H2MO3;
**Composition of solution of Fe-EDTA, 26.1 g of Fe-EDTA; 286 ml of 1N KOH and 24.9 g of FeSO4.7H2O, per litre of solution.
Note: The omission of micronutrient treatments had similar composition to the full treatment and solution, with the exception of solutions -B (B omitted),
the -Cu (Cu omitted); a -Mn (Mn omitted) a -zinc (Zn omitted) and in the treatment- Fe omitted solution of Fe-EDTA.
Experimental design: The experimental design was completely

randomized with 12 treatments (N, P, K, Ca, Mg, S, B, Cu, Fe, Mn
and Zn deficiencies, and control), and 5 replicates. These
treatments composed of complete nutrient solution for control
and omissions of individual nutrients for nitrogen, phosphorus,
potassium, calcium, magnesium, sulphur, boron, copper, iron,
manganese and zinc.
Qualitative parameters and harvest: The symptoms of
deficiencies in macronutrients and micronutrients with the
evolution of symptoms and descriptions were made from the first
signs of discoloration until symptoms become well established,
when plant samples were harvested.
Determination of growth parameters: Plants were sorted out in
old leaves, young leaves, stems and roots and washed in distilled
water, and dried in an oven with forced air circulation at 70C, until
constant weight. Immediately after getting dry matter, plant material
was ground in a Wiley mill for subsequent chemical analysis.
Plant height was defined as the distance between the stem, at the
substrate level (5 cm) to the apex. The measurement of diameter at
5 cm from substrate was made with a caliper. Leaf area was
determined at the end of the experiment, measurements were made
with graduated scale. Six leaves were taken at random from two
plant strata (old leaves and young leaves), and total twelve samples
per plant. In each leaf, length and the greatest width of the blade
were measured.
from leaf dry weight and nutrient contents were calculated results
of accumulation of nutrients. Low concentration of nutrient
induced deficiency symptoms in peppermint tissues were
compared to control treatment (C) which contains appropriate
amount of nutrients to promote optimal plant growth.
Data analysis: Data were analyzed by computer software Stat for
analysis of variance and F-test, and Tukey test at 5% probability,
comparing means between treatments on each variable.
Visual symptoms and consequences of macronutrient
deficiencies:
Nitrogen: The first symptoms of nitrogen deficiency appeared 12
days after initiation of treatment. Initially, peppermint plants showed
intense purple colour in old leaves, turning light purple (Fig. 1). It
was also observed that the plants had reduced height (34.95 cm)
and foliage with small leaf area (113.13 cm2), and thin stem (0.30 cm
diameter), as compared to control treatment. Root system showed
long and fine roots. According to Taiz et al. 10, symptoms of nitrogen
deficiency first appear because it is the most important element
Leaf area (AF) = C L
where, C is length and L is the greatest width of the blade.

Nutrient determinations: Macronutrients such as nitrogen (N),
phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg),
sulphur (S) and micronutrients such as boron (B), copper (Cu),
iron (Fe), manganese (Mn) and zinc (Zn) were determined using
methods described by Malavolta et al. 9. Considering data obtained
Figure 1. Visual symptoms linked to restriction of nitrogen (B),

phosphorus (C), potassium (D), calcium (E), magnesium (F), sulphur
(G), boron (H), copper (I), iron (J), manganese ( K ) and zinc (L) in plants
of Mentha piperita compared with the full treatment (A) with no deficiency.
293
required by the plant. The carbohydrate unutilized in the nitrogen

metabolism can be used in the synthesis of anthocyanin, leading
to accumulation of this pigment which causes the purple colour
of leaves and stems of plants deficient in nitrogen. Yellowish
colour is a consequence of loss of chlorophyll, and plants with
nitrogen deficiency have smaller cells. Batista et al. 11 attributed
the symptoms of chlorosis in plants with nitrogen deficiency in
older leaves firstly, due to its high mobility. Silva et al. 12, studying
the omission of nitrogen in peach plants found that nitrogen is
the first element to induce symptoms of deficiency.
Phosphorus: The omission of phosphorus in plants of M. piperita
grown in nutrient solution caused the appearance of deficiency
symptoms of phosphorus 23 days after initiation of treatment in
older leaves, characterized by intense purple color and rough
leaves. The stem was rough and coarse, leading to overturning
(Fig. 1). The leaves showed necrosis (burning), progressing to
the fall and reduced growth in height (67.30 cm), and small leaf
area (116.43 cm2). Plants with thick stem presented 0.65 cm diameter
(Table 2). The root system had normal growth and shape. Symptoms
similar to those described in this study were also observed by
Marques et al. 13 on paric (Schizolobium amazonicum, Herb.)
cultivated under omission of phosphorus and by Sobral 14 on
coconut. Salvador et al. 15 attributed the appearance of purplish
spots in between veins due to phosphorus deficiency, caused by
the accumulation of photoassimilates in plant tissues, promoting
synthesis of anthocyanin.
Potassium: Symptoms of potassium deficiency in plants of M.
piperita L. grown in nutrient solution with potassium omission
appeared 25 days after the initiation of treatment and were
characterized by marginal chlorosis on older leaves, stiffening of
leaves from the edges to inside; and buds with necrosis and
whitening of the stem, leading to necrosis in most affected areas
(Fig. 1). Potassium deficiency also affected significantly both the
height (79.75 cm) and leaf area (59.36 cm2), but did not affect the
diameter (0.63 cm) of peppermint plants (Table 2). Vigas et al. 16
observed that the omission of potassium in Alpinia purpurata
causes the appearance of small orange spots in the old leaves,
which coalesces to form larger patches and necrosis followed by
blight. Those symptoms are results of accumulation of putrescine
which is highly phytotoxic due to non-deployment of nitrogen
compounds of the urea cycle caused by lack of potassium. In Fig.
1, a comparison between peppermint plants grown in pots
containing full nutrients and with omission of potassium is made.
Calcium: Peppermint plants grown in nutrient solution with the

omission of calcium showed deficiency symptoms after 15 days,
which is characterized by reduced growth, mainly in aerial part
(Fig. 1). The evolution of calcium deficiency gave rise to
widespread necrosis and fall of young leaves, bud blight and
stem rot. Omission of calcium caused a significant reduction of
aerial part (37.02 cm) and leaf area (116.11 cm2), but did not affect
the diameter (0.51 cm) (Table 2). Also root rot was observed
especially in younger ones. Leaf area of the plants was smaller
than that found by Fazio et al. 17 on peppermint. Those results
can be explained because in that study different calcium
concentrations were supplied in nutrient solution. Freitas et al. 18
attributed the reduction of leaves, hence leaf area, to distortions
and necrosis that appear when there is lack of calcium.
Symptoms of calcium deficiency observed in peppermint plants
are similar to those described by several authors to different
cultures. In young plants of teak (Tectona grandis), Barroso et
al. 19 describe the presence of yellowish colour between veins,
curling, necrosis of leaves and root rot. Those symptoms were
also described by Santos et al. 6 in castor bean seedlings, in
which hardening of terminal leaves was due to calcium deficiency.
Magnesium: Symptoms of magnesium deficiency in peppermint
plants grown in nutrient solution with the omission of magnesium
appeared 27 days after initiation of treatment (Fig. 1), characterized
by yellowish colour between veins with deep green colour on the
tips and margins of the leaves and yellowing between and within
veins. In the absence of magnesium significant reduction in plant
height (65.17 cm), the leaf area (141.52 cm2) and the diameter of the
stem (0.52 cm) were observed as compared to the complete
treatment (Table 2). Santos et al. 6 considered the lodging of
plants on the stem and reduction in dry matter production as
magnesium deficiency.
Sulphur: Peppermint plants grown in nutrient solution with the
omission of sulphur presented at 35 days after initiation of
treatment, chlorosis and burning in old leaves, with purple
coloration between the veins (Fig. 1). Later spots and curving
stem appeared and new leaves became with pale green tones and
other chlorotic areas. The omission of sulphur significantly
affected the development of the aerial part (72.42 cm) and leaf area
(163.41 cm2), but did not affect the diameter (0.62 cm) (Table 2).
Symptoms of sulphur deficiency are similar to nitrogen because
both nutrients are essential components of proteins, resulting in
slower plant growth and stunt 20.
Table 2. Production of dry matter (g) in different organs of Mentha piperita plants in different treatments.
Treatment
Control (C)
N restriction
P restriction
K restriction
Ca restriction
Mg restriction
S restriction
B restriction
Cu restriction
Fe restriction
Mn restriction
Zn restriction
Young leaf (g)

9.64 b
5.99 d
8.26 c
7.65 c
5.57 d
8.44 c
12.40 a
24.49 d
26.16 d
42.27 b
28.57 c
29.30 c
Old leaf (g)

59.77 a
8.63 e
19.80 d
27.03c
9.47 e
19.27 d
30.62 b
8.24 de
10.46 b
7.9 e
9.14 cd
12.80 a
Plant organs (g planta-1)

Stem (g) Aerial part (g)
45.29 a
114.71 a
15.15 e
29.77 e
30.48 b
56.05 c
31.20 b
65.89 b
22.34 d
37.39 d
31.09 b
58.80 c
26.79 c
69.82 b
22.98 d
55.72 e
32.82 c
69.45 d
53.01 a
102.48 b
31.47 c
69.18 d
52.19 a
94.30 c
Root (g)
14.29 b
10.26 c
7.27 e
8.67 d
8.80 d
8.59 d
16.22 a
9.58 d
12.75 b
10.63 c
12.50 b
12.69 b
Total dry matter (g)

129.00 a
40.04 f
63.33 d
74.57 c
46.20 e
67.39 d
86.05 b
65.30 e
82.20 d
113.12 b
81.68 d
106.99 c
Means followed by the same letters in columns are not significantly different at 5% probability by Tukey test.
294
Visual symptoms and consequences of micronutrient

deficiencies:
Boron:The symptoms of boron deficiency appeared 40 days after
starting treatment. It is characterized by chlorosis with burning at
the margins of younger leaves (Fig. 1). Plants showed reduced
height (35.80 cm), very small leaves and small leaf area (163.73
cm2). Boron deficiency caused a significant increase in the diameter
of plant stems (0.54 cm) as compared to the control treatment
(Table 2). Siebeneichler et al. 21 studied the pineapple culture and
found that the symptoms of boron deficiency are characteristics
of young leaves, which present leathery and wrinkled probably
due to failures in the structure of the cell wall. The symptoms are
similar to those observed in the peppermint culture, which had
small and wrinkled leaves. The same has happened in seedlings
of jatropha (Jatropha curcas), which presented wrinkle at the
edges of young leaves with reddish ribs on the underside of older
leaves 22.
Copper: Peppermint plants grown in nutrient solution with the
omission of copper deficiency manifested symptoms 42 days after
initiation of treatment. Initially, the plants showed slight chlorosis
between the veins, progressing to necrosis (Fig. 1). We observed
a whitening of the stem and the plants showed reduced size when
compared with the full treatment (91.93 cm), whose leaves had
reduced leaf area (128.51 cm2). The plants showed thick stem (0.59
cm diameter) in relation to the control (Table 2), and such symptoms
are similar to those described by Neves et al. 23 on ombe plants
and Camargo et al. 24 on Bertholletia excelsa plants.
Iron: Symptoms of iron deficiency were observed 46 days after
starting treatment. Young leaves became chlorotic, and later red
in colour with reddish spots between veins (Fig. 1). At more
advanced stages of symptoms, the old leaves also showed
chlorosis. The plant height (78.93 cm) and leaf area (129.24 cm2)
were reduced significantly compared to the complete treatment,
but there was no effect of the lack of iron in stem diameter (Table
2). Similar symptoms were observed on ombe (Spondias
tuberosa), whose iron deficiency leads to alterations in young
leaves, stem purple in colour and decrease in chlorophyll level,
turning leaves chlorotic and whitish, and causing slow growth 16,
23
.
Manganese: The symptoms of manganese deficiency in
peppermint plants appeared 47 days after initiation of treatment
by means of chlorosis in between ribs and black spots on young
leaves (Fig. 1). It was also observed that the plants had small size
(59.98 cm), reduced foliage with small leaf area (153.40 cm2), plant
with very thick stem (0.75 cm diameter), as compared with the
complete treatment (Table 2). There was thickening of the stem,
bending down and cracks. The manifestation of manganese
symptoms in peppermint was very similar to the symptoms
observed on ombes, which occurred thickening of the basal
portion of the stem, and leaves become chlorotic 23. Omissions of
manganese and zinc did not affect the diameter of the plants 25.
Zinc: The first symptoms of zinc deficiency occurred 47 days of
initiation of treatment showed initially chlorosis between veins
and fall of the younger leaves. The leaves were well spaced from
each other on the stem (Fig. 1). Plants had small size (72.00 cm),
reduced foliage with small leaf area (130.74 cm2) thick stem (0.63
cm diameter), as compared to the complete treatment (Table 2).
These plants have been subject to bend, with fissures. There
were bending and cracking of the plant stems. Zinc deficiency
caused morphological changes in young leaves, characterized by
small and narrow leaves, sometimes distorted with short internodes
and chlorotic areas in between veins 23, which were observed in
this study with peppermint.
Deficiency effects on plant growth and dry matter production:
The largest reductions in plant growth were observed in plants
grown under omission of nitrogen, calcium, boron and manganese
as compared with the full treatment (Table 2). The omission of
manganese and zinc caused an increase in stem diameter compared
to the control treatment diameter was (0.43 cm). Batista et al. 11
and Lange et al. 26 observed a reduction in growth of soursop and
castor bean, with the omission of zinc and manganese. The smaller
leaf area were observed in plants under omission of copper, iron
and zinc. Leaf area was the most affected with the omission of
potassium, nitrogen and calcium with values of 59 cm2, 113.13 cm2
and 116.11 cm2. Similar results were shown by Fasabi 27 with
mallow plants, variety BR-01.
The values of total dry matter present in different organs of M.
piperita according to the treatments in Table 2 show that the total
dry matter regarding the omission of sulphur in young leaves
(12.40 g kg-1 of plant) and roots (16.22 g kg-1 of plant) was superior
to control treatment, except nitrogen and calcium that limited more
the production of dry matter in the plant, consistent with the results
of Silva Jnior et al. 25 and Freitas et al. 18.
Conclusions
Nitrogen and calcium are the first to express nutrient deficiency
symptoms, followed by phosphorus, potassium, magnesium,
sulphur, boron, copper, iron and manganese = zinc. For omission
of nitrogen, phosphorus, potassium, magnesium, sulphur, boron,
copper, iron, manganese and zinc compromise the growth of M.
piperita inducing morphological changes with characteristic visual
symptoms of deficiencies of these nutrients. The most limiting
nutrient for growth of M. piperita based on total dry matter and
leaf area is nitrogen, followed by calcium, as well as the nutrient
that most affects the growth of M. piperita is boron. In relation to
stem, aerial part and total dry matter the omissions of nitrogen,
calcium, boron and copper were most limiting, and in the root the
omission of phosphorus and boron was the most outstanding.
Acknowledgements
We are very grateful to CAPES by the offered opportunity, to
Universidade Federal Rural da Amaznia and Embrapa Amaznia
Oriental for supports during the research.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Decrease in photosynthetic pigments promotes negative consequences on carbon

compounds in young Euterpe oleracea plants submitted to progressive water deficiency
Emilly dos Santos Pereira 1, Allan Klynger da Silva Lobato 1*, Odyone Nascimento da Silva 1, Argemiro Pereira
Martins Filho 1, Carla Leticia Figueredo de Carvalho Souza 1, Tiago Rodrigues Ferreira 1, Gustavo Antonio
Ruffeil Alves 1, Elaine Maria Silva Guedes 1, Ismael de Jesus Matos Vigas 1, Ricardo Shigueru Okumura 1,
Augusto Jos Silva Pedroso 1, Roberto Cezar Lobo da Costa 1 and Benedito Gomes dos Santos Filho 1
Ncleo de Pesquisa Vegetal Bsica e Aplicada, Universidade Federal Rural da Amaznia. Paragominas, Par, Brazil.
*e-mail: emilly_2009_sp@hotmail.com
Abstract
This research aimed to (i) evaluate the interference of the water supply on photosynthetic pigments and to (ii) determine if there are changes in carbon
compounds of young Euterpe oleracea plants subjected to progressive water deficit. The experimental design was in factorial randomized entirely with
two water conditions (water deficit and control) and four evaluation points (0, 6, 12 and 18 days). The parameters evaluated were leaf relative water
content and hydrogen peroxide, chlorophylls and carotenoids, as well as total soluble carbohydrates, reducing carbohydrates, starch and sucrose. The
water deficit caused reduction in leaf relative water content and increase in levels of hydrogen peroxide, and it also provoked decrease in all
photosynthetic pigments, being the changes more intense and significant after 18 days. In relation to carbon metabolism, the water deficit induced
oscillation in levels of total soluble carbohydrates and reducing carbohydrates, as well as significant increases in starch during all evaluated points and
in sucrose until the 12th day. Therefore, this study reveals that progressive water deficit provoked reductions in chlorophylls and carotenoids, and
these modifications induced negative consequences on translocation and utilization of carbon compounds in young Euterpe oleracea plants.
Key words: Euterpe oleracea, water deficit, chlorophylls, sucrose, starch, carbon metabolism.
Introduction
Euterpe oleracea (Mart.) is a palm that grows in the Amazon
forest, and this plant has nutritional and economical significance
due to the commercialisation of energy drinks and the heart of
the palm, which is produced from the fruit and stem 1, 2. Regarding
its nutritional properties, the fruit contains higher amounts of
lipids and minerals such potassium, calcium, and magnesium 3, 4.
Additionally, a study conducted by Bobbio et al. 5 revealed that
the fruit is a source of anthocyanins. Brazil is the main producer
of E. oleracea and is a significant consumer and exporter of
products derived from this palm 6.
Water deficiency represents an abiotic stress and is considered
as one of the most important agricultural limitations due to its
negative effects on growth and development 7 and the
corresponding yield losses 8. Water supplies are essential in
several metabolic and physiological processes such as the
assimilation of nutrients 9, gas exchange 10, and translocation of
organic solutes 11.
The photosynthetic pigments are substances responsible for
the light absorption necessary for carrying out the
photosynthesis12, and the concentration can be modified by
several factors such as water deficit, light intensity and temperature
variation, in which combination or isolation can affect the molecule
integrity 13. The severe water deficiency normally promotes the
overproduction and consequently accumulation of reactive
oxygen species 14, which will cause damages in structures due to
oxidation of photosynthetic pigments, promoting decrease in
chlorophyll levels 15.
The water deficiency is one of the main abiotic factors that

influence the biosynthesis and distribution of carbon compounds
in several plant species 16. Higher plants can storage the
carbohydrates to utilization as reserve, as well as being used
during osmoregulation process aiming to increase the tolerance,
when subjected to stress periods 17. The drought affects the CO2
assimilation, and frequently there are negative consequences on
photosynthetic activity in plants exposed to deficit deprivation 18,
19
, and it will limit the carbohydrates synthesis to supply the organ
exigencies. In agreement with McCormick et al. 20, the complexity
to study carbon metabolism is related to diverse pathways that
carbon skeletons can be directed.
Based in this overview, this research aimed to (i) evaluate the
interference of the water supply on photosynthetic pigments and
to (ii) determine if there are changes in carbon compounds of
young Euterpe oleracea plants subjected to progressive water
deficit.
Localization and growth conditions: The experiment was
performed in Campus of Paragominas of the Universidade
Federal Rural da Amaznia, Paragominas city, Brazil (255' S
and 4734' W). This study was conducted in greenhouse without
environment control, and minimum, maximum and median
temperatures were 33, 26, and 22C, respectively. The relative
humidity during the experimental period oscillated between 65
and 93%, and the photoperiod was set to 12 h of light. During
297
measurements the photosynthetic active radiation oscillated

between 451 and 1453 mol m-2 s-1 (at 12 h).
Plants, containers, and substrate: Seeds of Euterpe oleracea
(Mart.) were germinated in pots with dimensions of 0.25 and
0.20 m for height and diameter, respectively, presenting capacity
of 8 L, and holes for water drainage. Pots were filled with
substrate Plantmax, and irrigation was implemented daily with
1 L of distilled water. After 100 days, seedlings with similar
aspect and size were selected. Subsequently, seedlings with 120
old-days received 0.2 L of nutrient solution 21 modified for this
species, and this nutritional supplementation was applied at regular
intervals of 30 days until the 12th month.
Experimental design: The experimental design was in factorial
randomized entirely with two water conditions (water deficit and
control) and four evaluation points (0, 6, 12 and 18 days),
resulting in a total of 8 measurements. This experiment was
assembled with 5 replicates for a total of 40 experimental units,
with one plant in each unit.
Water deficit application and harvest: All the plants were grown
until the 12th month as described above. Subsequently, the plants
under the water deficit treatment were submitted to 18
consecutive days without irrigation. The plants under the control
treatment were watered daily with distilled water. Measurements
linked to gas exchange and leaf relative water content were
carried out to each evaluation period. Additionally, the plants
were harvested, and the leaves present in middle region of the
plant were removed. Afterwards, the leaves were frozen in liquid
nitrogen and kept at -20C for subsequent biochemical
determinations.
Determination of leaf relative water content: The leaf relative
water content (LRWC) was evaluated in leaf disks of 10 mm diameter.
For each plant, 40 disks were removed and this parameter was
calculated using the equation proposed by Slavick 22:
LRWC = [(FM-DM)/(TM-DM)] 100
where FM is fresh matter, TM is turgid matter evaluated after 24 h

and saturated in deionized water at 4C in the dark, and DM is the
dry matter determined after 48 h in an oven with forced air
circulation at 80C.
Determination of total soluble carbohydrates and starch: For
determination of total soluble carbohydrates 50 mg of leaf
powder was incubated with 5 mL of ultrapure water at 100C for
30 min, centrifuged at 2,000 g for 5 min at 20C and the supernatant
was removed. For determination of starch 50 mg of powder was
incubated with 5 mL of ethanol at 80C for 30 min, centrifuged at
2,000 g for 10 min at 25C, and the supernatant was removed. In
addition, the second extraction was carried out with the same
powder incubated with 5 mL of 30% HClO4 at 25C for 30 min and
centrifuged in conditions previously described. The supernatants
of the two extractions were mixed. The quantifications of the total
soluble carbohydrates and starch were carried out at 490 nm using
the method of Dubois et al. 23, who used glucose (Sigma
Chemicals) as standard.
298
Determination of sucrose: The determination of sucrose was

carried out with 50 mg of leaf powder incubated with 1.5 mL of
solution MCW (methanol, chloroform and water) in the
proportion of 12:5:3 (v/v) at 20C by 30 min under agitation,
centrifuged at 10,000 g for 10 min at 20C and the supernatant was
removed. The sucrose quantification was carried out at 620 nm, in
agreement with Van Handel 54 using sucrose (Sigma Chemicals)
as standard.
Hydrogen peroxide quantification: In extraction of hydrogen
peroxide (H2O2), a mixture was prepared by homogenising 500 mg
of fresh leaf matter in 5 mL of 5% (w/v) trichloroacetic acid.
Subsequently, the samples were centrifuged at 15,000 x g for 15
min at 3C, and the supernatants were collected, being this
procedure described by Wu et al. 24. For H2O2 detection, 200 L of
supernatant and 1,800 L of reaction mixture were mixed, with
reaction mix containing 2.5 mM potassium phosphate buffer (pH
7.0) and 500 mM potassium iodide, and the absorbance was
measured at 390 nm 25.
Data analysis: The data were subjected to variance analysis and
significant differences between means were determined by F
test at 5% level of error probability 26. The standard deviations
were calculated to each treatment in all evaluation points. The
correlation analysis was performed by the Pearson parametric
method, and the statistical procedures were carried out with the
SAS software.
Impact of water deficit on leaf relative water content and
hydrogen peroxide: The water deficit caused reduction in leaf
relative water content, being significant from the 12th day after
experiment implementation (Fig. 1A). The most intense decrease
in the 18th day was approximately 60% when compared to control
treatment. The water deficiency promoted an increase in hydrogen
peroxide (Fig. 1B), being significant in the 12th and 18th day after
the application of water restriction.
The increase obtained was about 20%, in relation to control.
The reduction in leaf relative water content was promoted by
water deficit and it is related to lower amount of water in tissue.
These results can be explained by contribution of transpiration
rate combined to lower water availability in substrate 27, during
several consecutive days 28. Similar results were found by
Nogueira et al. 29 who studied water relations of Malpighia
emarginata submitted to water deficiency.
The increase in hydrogen peroxide is an indicator of stress in
oxidative system, resulting of inadequate work into photosynthetic
apparatus, and this effect is promoted by damages caused by the
water stress that plants were submitted 30. When a plant is exposed
to water deficiency, normally intensification in photorespiration
and limitation in photosynthesis rate occur, resulting in increase
linked to production of hydrogen peroxide in leaves, produced by
peroxisomes 31 . Similar results were reported by Deuner et al. 32 on
Coffea arabica plants submitted to water restriction, corroborating
with results obtained in this research.
Negative influence induced by water deficit on photosynthetic
pigments: The water limitation provoked reduction in total
chlorophyll, with significant results only in the 18th day, and most
Leaf relative water content

(%)
100
80
60
40
20
Control
Water deficit
Hydrogen peroxide
(nmol g-1 FM)
99
8
7
a
a
a
a
a
B
3
0
12
18
Time (days)
Figure 1. Leaf relative water content (A) and

hydrogen peroxide (B) in young Euterpe
oleracea plants subjected to water deficit.
Same letters do not show significant differences at F test (P < 0.05) to
each time. Squares represent the mean values of 5 repetitions, and bars
represent the standard deviations.
intense decrease was 47% , if compared to control treatment (Fig.

2A). In carotenoids, reduction was also obverved, being significant
in the 18th day (Fig. 2B). This decrease was approximately 77%, in
comparison with control. The water stress promoted decreases in
levels of chlorophyll a (Fig. 2C), and reductions verified were of
23, 26, and 45% in periods of 6, 12, and 18 days, respectively, in
which it was significant in the 18th day. The plants exposed to
water limitation presented decreases in levels of chlorophyll b
(Fig. 2D). In addition, this effect was significant in the 18th day,
with decrease of approximately 51%, if compared to control plants.
The reduction in chlorophyll a content was provoked by the
reduced water absorption, and this decrease can be associated
with action of agents linked to oxidative degradation of pigments
increased during the period on water restriction 33. Cardoso et

al.34, who investigated the pigments during the initial growth of
Myracrodruon urundeuva fr. Allemo under water restriction, also
showed reductions in chlorophyll a level.
The fall accentuated linked to chlorophyll b in plants under
water deficiency, mainly in last evaluated point, can be primarily
related to large leaf number already indicates senescence, and
secondarily by water restriction in soil to promote indirectly the
lower production of pigments in leaves 35. Silva et al. 36, who studied
the biochemical behaviour of Piper hispidinervum exposed to
water restriction, showed decrease in levels of chlorophyll b.
The reduction in levels of total chlorophylls was entirely related
to stress that plants were subjected, in which the water deficit
induced the production of hydrogen peroxide that is associated
with chlorophyll degradation in thylakoid membrane 37. Carvalho
et al. 38 showed reduction in total chlorophyll and evaluated the
physiological responses in Hyptis pectinata plants under water
stress.
The carotenoids are accessory pigments in absorption and
transference of energy; besides, it acts as defensive of
chlorophyll molecules during photo-oxidation 13. The degradation
of pigments frequently is related with the decrease in synthesis or
degradation of carotenoids 39. The water stress induced the
degradation of -carotene and the reduction in levels of zeaxanthin
and carotenoid pigments involved in protection against the
photoinhibition 40 . Similar results were reported by Cha-um et
al.41 who evaluated Oryza sativa plants under water deficiency,
and corroborated with results of this study.
Carotenoids (mg g-1 FM)
Chlorophyll b
(mg g-1 FM)
Chlorophyll a
(mg g-1 FM)
Total chlorophyll
(mg g-1 FM)
Consequences provoked by water deficit on carbon compounds:

The water deficit induced oscillation in levels of total soluble
carbohydrates, and increases are shown during the 6th and 12th
day, and these increases were of 3 and 28% (Fig. 3A). However, in
the 18th day, significant reduction of 26% was verified, when
compared to control. The water deficiency promoted an oscillation
in results found to reducing carbohydrates (Fig. 3B), presenting a
non-significant increase until the 12th day, and
14
4.0
decreases in the 18th day, when compared to
A
B
Control
Water deficit
3.5
control plants. The difference, which was more
12
a
a
a
a
3.0
expressive in starch, was of approximately 16%
10
a
a
in the 12 th day. The drought promoted
a 2.5
8
a
2.0
significant increases from the 12th to 18th day in
b
a
6
a
1.5
starch levels (Fig. 3C). The most intense
a
a
4
1.0
increase being observed in the 12th day, was of
b
2
0.5
approximately 74% in comparison with control
0
0.0
plants. The results reveal increases in sucrose
9
9
D
C
levels of plants exposed to drought during the
8
8
6th and 12th day (Fig. 3D). However, significant
a
a
7
7
a
reduction of 71% when compared to control
a
a
6
6
a
a
was obtained in the 18th day. In plants under
a
5
5
water restriction, the increase in the 12th day in
4
4
a
a
b
total soluble carbohydrates is related to
b
3
3
a
a
osmotic adjustment in plant, in which the first
2
2
moment of the stress will modify the cell
0
0
composition and induce the production of
0
6
12
18
0
6
12
18
Time (days)
osmotically active substances, such as
sucrose42, 40. The stimulation linked to osmotic
Figure 2. Total chlorophyll (A), carotenoids (B), chlorophyll a (C), and chlorophyll b (D) in
substances in plants exposed to water deficit
young Euterpe oleracea plants subjected to water deficit.
Same letters do not show significant differences at F test (P < 0.05) to each time. Squares represent the mean values of 5 repetitions, and
occurs aiming to keep the plant with adequate
bars represent the standard deviations.
299
33
Control
Water deficit
30
27
27
24
21
18
15
24
18
15
a
a
12
12
a
9
0
10
9
8
7
6
5
4
3
2
1
21
C
b
b
a
a
a
a
9
0
10
9
8
7
6
5
4
3
2
1
Reducing carbohydrates
(mg g-1 DM)
30
Sucrose
(mg g-1 DM)
Total soluble carbohydrates

(mg g-1 DM)
Starch
(mg g-1 DM)
33
progressive water deficit provoked reductions

in chlorophylls and carotenoids, and these
modifications induced negative consequences
on translocation and utilization of carbon
compounds in young Euterpe oleracea plants.
Acknowledgements
This research had financial support from
Conselho Nacional de Pesquisa (CNPq/Brazil)
and Universidade Federal Rural da Amaznia
(UFRA/Brazil) for Lobato, A.K.S., as well as
from Fundao Amaznia Paraense (FAPESPA/
Brazil) for Pereira, E.S., Souza, C.L.F.C.
0
0
12
18
12
18
Time (days)
Figure 3. Total soluble carbohydrates (A), reducing carbohydrates (B), starch (C), and sucrose
(D) in young Euterpe oleracea plants subjected to water deficit.
Same letters do not show significant differences at F test (P < 0.05) to each time. Squares represent the mean values of 5 repetitions, and
bars represent the standard deviations.
water levels, and consequently to avoid the dehydration of

tissues43. Similar results were obtained by Lobato et al. 44 studying
Vigna unguiculata plants in response to water deficiency.
The reduced carbohydrates increased due to water limitation,
this effect being related to osmotic adjustment. Normally, the
decrease of the water potential is by the accumulation of solutes
in symplast 45, and the reduction in translocation of carbohydrates
is directly proportional to accumulation in synthesis sites, such
as leaf. Similar results are related to Lobato et al. 11 when evaluated
Glycine max plants submitted to water deficit.
The water restriction caused an increase in starch levels, which
is probably related to reduction in enzyme activity of -amylase,
that is the main enzyme responsible for the starch degradation in
leaves 46. The starch degradation process occurs by hydrolytic
pathway, which uses the water as substrate 47. In plants exposed
to water deficit the water content is reduced 48, decreasing the amylase efficiency, and it will promote accumulation of the starch
in leaf. Similar results were reported by Silva et al. 49 on three
grass species submitted to water deficiency, and this fact is
corroborated with results found in this research.
The increase in sucrose levels in plants under water deficit
occurred probably in function of lower invertase activity. This
enzyme is responsible by the sucrose degradation 50. The
breakdown of sugars such as fructose and glucose by hydrolysis
is combinated with action of invertase enzyme, which is necessary
in carbon metabolism and energy production 51. However, this
research showed decrease linked to sucrose in last evaluated
period. This fact reveals the starch consumption aiming to the
plant survival in conditions of severe deficit 52. Similar results are
described by Pastorini et al. 53 on Solanum tuberosum, resulting
also in increase of sucrose.
Conclusions
The water deficit caused reduction in all photosynthetic pigments.
The water deficit induced significant increases in starch and
sucrose until the 12th day. Therefore, this study reveals that
300
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302
WFL Publisher
Helsinki, Finland
www.world-food.net
Microbes and dietary values of some major fish sources in Nigeria

Olajide Adedayo Ajayi
*, Emmanuel I. Adeyeye 2 and Anthony I. Okoh
1, 3
Applied & Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology,
University of Fort Hare, Private Bag X1314, Alice 5700, South Africa. 2 Department of Chemistry, Ekiti State University, AdoEkiti, Ekiti State, PMB 5363, Nigeria. 3 Department of Microbiology, Adekunle Ajasin University, Akungba-Akoko, Ondo State,
PMB 001, 23434, Nigeria. *e-mail: AAjayi@ufh.ac.za
1
Abstract
This study shows some nutritional values and metal components of health concern in fish sources such as mackerel (Titus) Scomber scombrus, shawa
Sardinella eba and kotwe Carangoides armatus meant for public consumption in Nigeria. The moisture content, ash content, crude fat, crude protein,
crude fibre, vitamin A and vitamin C were determined. Titus Scomber scombrus showed highest value of vitamin A (96.7 i.u), while kotwe recorded
relatively high value of vitamin C (0.60 mg/100 g) compared with other fish sources. Variations in the levels of some metal components can serve as
measure of pollution, especially the amount of lead (P) (1.09%) and iron (Fe) (2.0%) in Titus Scomber scombrus could be an indication of pollution
for this fish sources or possibly result of biomagnifications. The moisture and ash content of the sampled fish sources during the study ranged from
66.7% to 77.5% and 3.79% to 4.03%, respectively. Total bacterial count estimated for fish samples studied were 113 x 104 cfu/mL for Titus, 40 x 104
cfu/mL for Sardinella eba (shawa) while coliform count gave value of 9.5 x 103 cfu/mL and 9.65 x 104 cfu/mL for both fish sources, respectively.
Generally, this study shows that mackerel (Titus) Scomber scombrus has more nutritional quality than other two fish sources analyzed, that is,
shawa Sardinella eba and kotwe Carangoides armatus. Complementary to this, the level of microbial and chemical contaminants including heavy
metals such as lead and chromium that could be inimical to human health from these sources were determined. Thus, the data obtained during the
study, can serve as benchmark for environmental monitoring and aquaculture protection services in the country, Nigeria, and other parts of the world.
Key words: Aquaculture, assessment, environment, fishes, health benefits, microbiological quality, mineral composition, Nigeria, proximate analysis,
protection, public.
Introduction
Many aquatic organisms served as major food sources for people
in different parts of the world. Fish is an aquatic organism that
serves as a good protein and nutrient source for people in both
rural and urban areas of the world. Fish feed on nearly all types of
marine organisms from bacteria to crustaceans, whereby the
organisms reside in fishes as their home before other animals feed
on them in the food chain systems 1. In the context or this study
mackerel (Titus) Scomber scombrus, shawa (Sardinella eba) and
kotwe Carangoides armatus which is a longfin trevally 2, are three
major species of fish that are widely consumed in Nigeria. Naturally,
these sources of food supplement vary in their expenses based
on their nutritional quality assumed by people. Fishes need food
for growth, body maintenance and survival. The food supplies
available for fishes determine such important matters as
distribution, abundance, rate of growth, condition, movement,
fertility and even the taste of fish 3. Fishes play vital role as sources
of protein and some vitamins food supplements to human beings.
Thus it helps to protect the body immune system.
The microbiota of marine fish from temperate waters is usually
composed of Gram-negative psychrotrophic bacteria, whose
growth is possible at 0C and optimal at around 25C. The majority
belongs to the class of proteobacteria: Pseudomonas, Shewanella,
Acinetobacter, Aeromonas, Vibrio, Moraxella, Psychrobacter,
Photobacterium, etc., and to a lesser extent to the CFB group

(Cytophaga-Flavobacter-Bacteroides). Nevertheless, Grampositive bacteria, such as Micrococcus, Corynebacterium,
Bacillus, Lactobacillus and Clostridium may also be present in
variable proportions 3-10. The same bacterial genus can be found
in tropical marine fish but Gram-positive bacteria of
Enterobacteriaceae and Vibrionacea are often dominant 10, 11.
Bacteria play diversified role in association with fishes.
According to Austin 12, the role of the bacteria includes the ability
to degrade complex molecules (therefore exercising a potential
benefit in nutrition), to produce vitamins and polymers, and to be
responsible for the emission of light by the light-emitting organs
of deep-sea fish. Taxa, including Pseudomonas, may contribute
to spoilage by the production of histamines in fish tissue. Bacteria
play a significant role inside our aquaculture environments 13.
Without it, we would not be able to maintain sufficient water quality
to possess healthy fish. Hence, this will adversely affect the
quantity and quality of fish supplied for human consumption.
The fish samples were purchased locally from the Oja-oba market,
Akure, Nigeria, and wrapped in sterile nylon bags. They were
immediately transported to the laboratory and stored in the
303
refrigerator prior to analyses. The microbiological quality of the

samples were determined by estimating the total bacterial and
coliform counts.
The proximate analysis done to determine the nutritional value
of the fish sources included the moisture content, ash content,
crude fat, crude protein, crude fibre, vitamin A and vitamin C.
Similarly the fish samples were analyzed for some mineral
components and heavy metals such as copper (Cu), iron (Fe),
manganese (Mn), zinc (Zn), chromium (Cr), nickel (Ni), cadmium
(Cd), and lead (Pb).
Moisture content determination: The moisture content was
determined and recorded as a percentage of the wet weight of
food (i.e. on a wet basis). Known weight of the samples was
measured into crucible and dried to constant weight in the oven
at 105C for about 3 hours, and calculated as:
% Moisture = W1-W2 x100
(Wi-a) or W
where a = initial weight of crucible, W = weight of sample,W1 =

initial weight of crucible + sample, W2 = final weight of crucible +
sample after drying
The most rapid method usually adopted for moisture
determination is to heat a thin layer of sample in the bottom of a
beaker at 105C. Foods that are heat labile, especially those
containing sugar, may decompose at this temperature and so they
are dried at 70C in vacuum. True moisture content is determined
by Karl Fisher method 14.
Ash content determination: Ash content was obtained by igniting
a known amount of dried material (moisture face) in a muffle
furnace15. The dried sample obtained from moisture determination
was kept in the muffle furnace maintained at 550C for 1 hour, cooled
in desiccators, weighed, and calculated as:
wt of ash = wt of sample after burning in furnace wt of crucible.
% ash = (wt of ash x 100)/wt of sample
Determination of crude fat: The fish samples were well wrapped
and tied with thread inside a known wt of filter paper and placed in
a continuous extractor (Soxhlet) and subjected to extraction with
petroleum ether. This is capable of extracting the triglyceride
phospholipids, sterols, essential oils, pigments and waxes as the
crude fat components 15.
Wt of fat =initial wt of sample final wt after extraction. Hence, %
Fat = (loss in wt x 100)/ Wt of sample
Determination of crude protein: The usual method employed for
this purpose is based on Kjeldahl method of nitrogen
determination. The percentage of nitrogen in the sample food is
multiplied by 6.25 to get the protein value, and this is based on the
assumption that plant proteins contain an average, 16 percent
nitrogen. The factor for the test was prepared. A catalyst selenite
tablet was used in conjunction with conc. H2SO4 for digestion of
sample. Boric acid (4%) with bromocresol green/methyl red
indicator solution was prepared by dissolving 4 g of boric acid in
about 12 cm3 of boiled but still very hot deionizer water, mixed,
304
boiled water (90 cm3) added, cooled and 1 cm3 of bromocresol

green solution (l mg in l cm3 of alcohol) was added. This was made
up to 100 ml with distilled H2O, and 25 cm3 of the boric solution
was transferred to a receiver flask and distilled a blank (no
sample, only chemicals). This was titrated with 0.1M sodium
hydroxide solution until a neutral grey colour was obtained.
Determination of crude fibre: One g of fat free residue of the fish
sample obtained after crude fat determination (1.25%) was
transferred into 1-litre conical flask. The sample was boiled in
dilute H2SO4 for 30 min maintaining the liquid level during boiling
by adding boiling water from time to time. Contents of the flask
were filtered though fine linen (18 threads to a cm) held in a funnel
and washed with boiling water until the washings were no longer
acid to a stage of dilution to extinction. All residues were scrapped
with spatula and transferred back to original digestion flask, and
200 cm3 of dil. NaOH was boiled and used to wash the residue of
the linen into digestion flask and boiled for 30 min. This content
of the sample was rotated and filtered through the same linen
cloth. Residue was thoroughly washed with boiling water till free
from alkali. This was transferred into crucible and dried to constant
weight at 105C. The weight of the sample was noted and
transferred in the crucible which was eventually placed in furnace
at 550C (600C) until all carbonaceous matter was burnt. The test
sample was cooled 14.
The weight of the residue dried at 105C was subtracted to
obtain crude fibre content. Percentage can be calculated on
moisturefree basis.
Determination of vitamin A: Four g estimated to contain approx.
20,000 U.S.P units vitamin A was transferred to saponification
flask. It was boiled under reflux condenser in all glass apparatus
with 50 mL isopropyl alc and 5 ml KOH solution for at least 30 min
until saponification was completed. This was cooled and 500 mL
water added. All these are incoherent.
The sample was transferred to separation funnel and extracted
with four successive 30 mL portions of ether. The combined ether
extracts were washed by swirling gently with 50 mL water. Washing
was repeated until the last gives no colour of phenolphthalein.
The washed ether extract was transferred into 250 mL volume
flask and to volume 10 mL of aliquot was transferred with pipet
into volumetric flask, without application of heat. The ether was
removed in stream of nitrogen.
Residue was dissolved in sufficient isopropyl alcohol to give
expected concentration of 8-12 units vitamin A per mL such that
absorbance is in range of 0.4-0.8 at 325 mm.
One of the matched cells was filled with isopropyl alcohol placed
in instrument and adjusted to zero absorbance at 325 mm. The test
absorbance was determined at this wavelength and also repeated
at 310 and 334 mm 14, 15. Estimation of vitamin A content was as
follows:
Vitamin A USP. Units/g = A (corr) x diln. Factor x 18.30
1
sample wt g
Determination of vitamin C (ascorbic acid): Vitamin C content

of the sample was determined by pipetting 5 mL of the working
standard solution into a 100 mL conical flask. This was followed
by adding 10 mL of 4% oxalic acid and titrated against the dye (V,
mL). End point is the appearance of pink colour which persists for
a few min. The amount of dye consumed is equivalent to the
amount of ascorbic acid. The sample was extracted (2 g) in 4%
oxalic acid, made up to known volume (100 mL) and centrifuged.
Consequently, 5 mL of this supernant was obtained using a pipette
and 10 mL of 4% oxalic acid was added. It was then titrated against
the dye (v 2 mL) 15. Vitamin C was estimated as follows:
Table 2. Vitamin A (I. U. ) and vitamin C (mg/100 g) in fish

sources.
Fish sample
Vitamin A (I.U)
A- Titus -Scomber scombrus

B- Kotwe
C- Sardinella eba
Vitamin C
(mg) content
0.40
0.60
0.50
96.7
65.0
56.1
Table 3. Mineral components of fish samples.

Vitamin C mg/100 g = 0.5mg x v2 x 100 mL x 100
V1mL 5mL
Fish samples
The result vit C. can also be expressed in mg/100 mL. Sample

weight will be substituted with sample volume.
This study shows the microbiological quality, nutritional value
and heavy metal components of fish sources commonly consumed
by people especially in the South-West, Nigeria. In Table 1, the
moisture and ash contents show a range of 66.7 - 77.5% and
3.79% to 4.03%, respectively. Mackerel (Titus) Scomber scombrus
showed relatively higher content of crude fat and protein at values
of 16.2 and 18.90%, respectively, compared with lower values
recorded in other two fish sources (shawa Sardinella eba and
kotwe Carangoides armatus.). Kotwe and shawa, however, had
higher moisture and ash contents than Titus fish. The crude fibre
for all fish sources studied was at zero level (Table 3).
Titus showed highest value of vitamin A while kotwe recorded
relatively high value of vitamin C compared with other fish sources
(Table 2). Some toxic contaminants that can constitute health
hazard were also determined. In this regard, there are variations in
the level of metal components which can serve as measure of
pollution, particularly the amount of lead (P) (2.0%) and iron (Fe)
(1.09%) in Titus could be an indication of pollution for this fish
sources. It can possibly be result of biomagnifications which can
be carefully investigated in subsequent researches (Table 3). Table
4 shows the microbial load of the fish samples studied. In this
context, the total bacterial count estimated for fish samples are
113 x 104 cfu/mL for Titus, 40 x 104 cfu/mL for Sardinella eba and
coliform count was 9.5 x 103 and 9.65 x 104 cfu/mL for both fish
sources, respectively.
This study shows that there are valuable nutritional contents
that can be derived from major sources of fishes viz. mackerel
(Titus) Scomber scombrus, shawa Sardinella eba and kotwe
Carangoides armatus. During the study, it was shown that Titus
had the highest crude fat (16.2%), crude protein (18.90%) and
vitamin A (96.7 I.U) which are among the major nutritional
components analyzed during this study compared with other fish
sources khote and Sardinella eba with lower values (Tables 1
and 2). This kind of variation was described by Franoise 10 who
explained the variation in nutritional quality of some fish sources
A - Titus
B - Kotwe
C - Sardinella eba
Zn
0.48
0.35
0.5
Fe
1.09
1.07
0.65
Mineral components (%)

Cr
Pb
Cd
Ni
0.31 2.0 0.00 0.91
0.22 1.1 0.00 0.76
0.07 1.2 0.00 0.38
Cu
0.02
0.25
0.19
Mn
0.13
0.15
0.22
based on ecological attributes and species. He emphasized that,

changing in consumers habit has led to an increase of ready-toeat and convenient food, concept that includes both the easy-touse aspect and an extended shelf-life of the products. The
nutritional aspects are also more and more taken into consideration
by the consumers who want natural products, with technological
treatment and level of preservatives as low as possible. Thus,
nutritional quality assurance is taken to be of importance by some
previous investigators 9, 10, 16.
Relatively high levels of lead (P) (2.0%) and iron (Fe) (1.09%) in
Titus fish coupled with some observable variation in other fish
sources (Table 3) can serve as measure of pollution of this metal
components for this fish sources. These metals detected can
possibly be result of biomagnifications which can be carefully
investigated in subsequent researches. However, the maximum
allowable level of lead is 0.1mg/L 17. It is important to know their
level of contamination before being finally consumed and possible
resistant pattern to diseases. Level of coliforms up to 9.5 x 103 as
intensified in this study is not healthy enough for human
consumption. The presence of these coliforms is an index of
pollution 18. Thus, there is need to protect our aquaculture
products from causing disease spread. In correlation to this, Burr
et al. 16 described aquaculture as one of the fastest growing
industries in the world, hence their substantial need for enhanced
disease resistance, feed efficiency, and growth performance of
cultured organisms. If growth performance and feed efficiency
are increased in commercial aquaculture, then the costs of
production are likely to be reduced. Similarly, the ability of fish to
resist disease and survive until they are of marketable size depends
on favourable ecological conditions. Thus, the subsequent cost
of medication and overall production becomes more managable.
It has been documented in a number of food animals that
gastrointestinal microbiota play important roles in affecting the
nutrition and health of the host organism. Dietary supplementation
of prebiotics, which are classified as non-digestible food
ingredients that beneficially affect the host by stimulating growth
Table 1. Moisture content, ash content, crude fat, crude protein and crude fibre values.
Sample
A- TitusB- Kotwe
C- Sardinella
eba
Wt of
crucible (g)
19.4832
19.2766
W1
Wt of crucible
+ sample (g)
21.6537
21.3117
W
Wt of
sample (g)
2.1705
2.0351
W2
crucible + sample
after drying (g)
20.2059
19.8440
W1-W2
Loss in
wt (g)
1.4478
1.4677
moisture
(%)
66.7
72.1
Ash content
(%)
3.79
4.35
18.4614
20.6265
2.1615
18.9475
1.6790
77.5
4.03
Crude
fat
16.2
13.4
6.5
Crude
protein (%)
18.90
16.63
Crude
fibre
0
0
14.88
Note: % Moisture = W1-W2 100

W
305
Table 4. Total bacterial count of fish samples on solid culture medium (cfu/mL).
Sample
Titus - Scomber scombrus
Sardinella eba
Total viable count

113 x 104
40 x 104
Coliform count
9.5 x 103
9.65 x 104
Anaerobic count
17 x 103
1.07 x 104
and/or activity of a limited number of health-promoting bacteria

such as Lactobacillus and Bifidobacter spp. in the intestine, while
limiting potentially pathogenic bacteria such as Salmonella,
Listeria and Escherichia coli, have been reported to favorably
affect various terrestrial species; however, such information is
extremely limited to date for aquatic organisms. In general, the
microbiota of fishes studied in this context has been determined,
including the anaerobic microbiota. Table 4 shows that fish can
be colonized by various forms of microorganisms, especially
bacteria. This is in correlation with the reports of Austin 12 and
Wilson et al. 19 that fish possess bacterial populations on or in the
skin, gills, digestive tract, and light-emitting organs. In addition,
the internal organs (kidney, liver, and spleen) of healthy fish may
contain bacteria, but there is debate on whether or not muscle is
actually sterile. The number and taxonomic composition of the
bacterial populations often reflect those of the surrounding water.
Similarly, the ecology of some fish sources influence their
nutritional components and metabolites produced. Some of this
fish products are of industrial importance 20, 21.
Conclusions
This study shows that mackerel (Titus) Scomber scombrus has
higher nutritional quality than other two fish sources analyzed,
shawa Sardinella eba and kotwe Carangoides armatus.
Furthermore, the health benefits of various classes of fish widely
consumed in South-west Nigeria, their level of microbial
contaminants and possible control of some substances such as
some observable levels of lead and chromium that could be inimical
to human health from these traceable sources were determined.
Hence, the data obtained during the study, can serve as benchmark
for environmental monitoring and aquaculture protection services
in this part of the country, Nigeria. It can also serve as a guide for
environmental amelioration and food protection in other parts of
the world.
Acknowledgements
We appreciate the support of the Institute of Public Analyst,
Nigeria (IPAN) and University of Fort Hare, Alice, South Africa,
for granting the opportunity to conclude this research work.
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Impact of micro credit and training on efficiency of small-scale entrepreneurs:

Evidence from National Directorate of Employment (NDE) loan/training programmes
in Nigeria
Olumide Oyewole Akinrinola 1*, M. M. Fasoranti
and Oluyede Adeleke Aturamu3
Department of Agricultural & Resource Economics, Federal University of Technology, P. M. B. 704, Akure, Nigeria.
2
Department of Economics, Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria. 3Ekiti State
College of Education, Directorate of External Degree Programme, Ikere, Ekiti, Nigeria. *e-mail: akinrinola01@yahoo.com
1
Abstract
This study analyses the impact of micro-credit and training on efficiency of small-scale entrepreneurs, using the National Directorate of Employment
(NDE) programme as a case study. National Directorate of Employment (NDE) has been found to be functioning effectively in its training services.
However, in the area of credit provision and utilization, there is no empirical evidence to support its efficiency. In order to evaluate the link between
the loan/training programmes of the Directorate and the level of efficiency of micro-entrepreneurs in the state, we estimate technical efficiencies and
identify significant policy variables influencing efficiency of selected micro enterprises, using the stochastic frontier production function. Results
indicate that there are intra and inter group variations in the efficiency of bakers, furniture makers and burnt-brick makers, showing that there is
possibility of improving the level efficiency of these entrepreneurs. The most significant determinants of technical efficiencies of bakers, furniture
makers and burnt-brick makers are the level of education, business experience, age of operators, credit access, training experience and level of working
capital and initial capital outlay.
Key words: Micro credit, small scale, entrepreneurs, employment, loan.
Introduction
The dynamic role of small and micro enterprises as an engine for
industrial growth and development of any nation cannot be over
stressed. These enterprises create employments and contribute
significantly to output growth of any nation of the world, of which
Nigeria is a part. Any nation that neglects the small/micro enterprise
sector will always have unemployment problem, together with its
attendant vices and remain a dependent economy. It is in
recognition of this that successive governments have evolved
policies, programmes to develop the small and micro enterprises.
Employment generation in the small/micro-business is hinged
on adequacy of investment outlay and working capital. Such
capital is difficult to raise in a low-income and poverty stricken
society. Hence, loan advancement in such circumstance can assist
micro entrepreneur to invest in income and employment generating
activities 7.
The formal credit institutions have not been supportive in
meeting credit need of self-employed persons due to their stringent
loan terms and conditions, and cumbersome loan application
procedures. These formal institutional arrangements have
constituted a major barrier to the growth of small scale and since
they cannot meet the knife-edge conditions, they are often
considered not credit-worthy 1. Cooperative societies have helped
in meeting the credit need of the small/micro business owner in
Nigeria, but the extent of their support cannot satisfy the credit
demand of the small-scale entrepreneur due to their limited
resources 4, 10. This is because as small-scale/micro-enterprises

grow, their credit requirement become increasingly large for this
indigenous credit source to meet, yet too small for the commercial
banks 5.
Consequently, successive Nigeria governments have evolved
strategies to address the diverse needs of this sector. Such efforts
culminated into policies and programmes like establishment
National Directorate of Employment, restructuring of National
Industrial Development Bank to Bank of Commerce and Industry.
The National Directorate of Employment is saddled with the
responsibility of offering training programme to develop the
entrepreneurial, technical and management capabilities of smallscale business owners and support them with the required startup and working capitals. The Small-scale Enterprise Development
Department of the Directorate has provided credit to over 62,013
beneficiaries in Ondo State since inception with a total loan
package of =
N 506,000,000.00 until 2006 when the restructuring
policy of the present administration streamlined the Directorates
mandate to training only.
Problem Statement/Justification
Increase productivity of small-scale businesses in terms of output
growth could emanate from improvement in technical and allocative
efficiency, use of adaptable improved technology and increase in
input consumption. This could be made possible with adequate
307
provision of easily accessible credit to effect their input demand

and adoption of relevant technologies and tailor-made training
scheme that will keep small-scale business investors abreast of
relevant technical and managerial skills. The credit constraint and
high cost of capital confronting small-scale enterprises in Nigeria
in general and Ondo state in particular, constrict output growth.
With the restructuring policies of the present administration, which
make provision of credit and entrepreneurial training a priority,
one of the challenges to policy makers is how to improve technical
efficiency of these small-scale business entrepreneurs to increase
productivity. This can only be done when we know the critical
factors influencing efficiencies of the small-scale enterprises.
Vijay and Wisdom 13 posited that technical efficiency is
influenced by human capital variables, which control the decisionmaking process of the entrepreneurs, and socio-economic and
institutional variables that could influence an entrepreneurs
capacity to apply his/her decisions at the enterprise level without
any constraints. The human capital variables include level of
education, business experience and age; socio-economic and
institutional variables are loan-interest, loan size, contact with
lender, and training programmes, training experience, receptively).
Pertinent to determine the technical efficiency of micro-business
enterprise are certain questions, such as: are small-scale business
enterprises technically efficient or inefficient? What are the factor
contributing to their efficiency or otherwise? Can the level of their
technical efficiency be improved upon? What is the contribution
of micro-finance and training services provided by NDE to the
technical efficiency or otherwise of these micro-enterprises?
This study therefore is premised on determining the link between
credit access, training and technical efficiency and highlight other
significant factors influencing the level of efficiency in
confectionery, furniture making and burnt-brick making microenterprises.
Literature is replete with studies on the measurement of technical
arid allocative efficiency in the primary, manufacturing and service
sectors of sub-saharan African countries. Ajibefun and Daramola3
have shown that the significant determinants of technical efficiency
of block-makers, metal fabrication and saw-millers in Nigeria are
age of operator, level of education, business experience and
number of employed and level of investment. The significant
determinant of technical efficiency of tobacco growers in Uganda
have been estimated by Obwona 12 to be family size, level of
education, health status, hired labour, credit accessibility,
fragmentation of land and extension services. Njikam 11 examined
the impact of trade liberalisation on the technical efficiency of
electrical industry of Cameroon and found a positive effect of the
trade policy on the level of efficiency in the industry.
This study was carried out in Akure South Local Government
Area (LGA) of Ondo State, Nigeria. The LGA is located in the
state capital with relative infrastructure advantage suitable for
micro-enterprises. Like other state capital, most government
programmes on small-scale enterprises are better implemented in
the LGA owing to its comparative and competitive advantages in
terms of infrastructure, population and market.
The NDE headquarters are situated in the LGA where credit and
training activities are carried out. The Small-scale Enterprise
Department handles the Directorates Entrepreneurship Training/
308
credit service. The department organises entrepreneurship

awareness seminars, management development workshop,
working capital management, record keeping and management
and improve-your-own business seminars at the state capital.
Between 2005 and 2011, the NDE organised three hundred and
twenty-eight training programmes. These included seventy
entrepreneur seminars, forty-seven management development
workshops, twenty-five record keeping and managements, one
hundred and three feasibility report preparatory seminars, fiftyseven working capital managements and twenty-six seminars on
internal control over cash. During the period a total loan package
of ninety-six million (N
=596,000,000 equivalent of $3,701,863.36 at
exchange rate of =
N 161 to $1) naira was advanced to 3840 smallscale entrepreneurs who engaged in manufacturing, service,
agriculture and trading 9.
Data collection and sampling procedure: Data used for this study
consist of age, business experiences and level of education of
respondents. Training programmes and training experiences credit
intervention by NDE, value of output and physical quantity of
input, equipment, and man-hours. These data were generated
through structured questionnaire administered by trained
enumerated after correction for validity and reliability test.
Collection process involved purposive and random sampling
techniques. The lists of loan beneficiaries who are bakers, burntbrick makers and furniture manufacturers were obtained from the
NDE and sixty-five respondents were randomly selected from each
group.
Analytical technique: The study used the stochastic frontier also
called the composed error model of Aigner et al. 2 and Meeusen
and Van den Broeck 8. Conceptually, a firm producing a single
output Q using 2 inputs efficiently transforms such inputs into
output through a production function. This shows the functional
relationship between output obtainable form input vectors.
The stochastic production function is given as:
Qi = zi + (Vi - Ui),i = 1, 2 ... ... ... ... ... ... N
(1)
where Qi is the production of the i-th firm, zi is a mxl vector of input

quantities of the i-th firm. is a vector of unknown parameter ,Vi
random variable; and Ui non-negative random variable presumed
to account for technical inefficiency in production.
The random errors of Vi are said to be independently and
identically distributed - N (0, 2) independent of Ui. The Ui is
assumed to be independently and identically distributed as, for
instance, exponential and half normal 2. In this study, Uis are
assumed to follow half normal distribution, and a mixed chi-square
distribution (Likelihood Ratio Test) to test for one-sided error.
The stochastic frontier production functions technical efficiency
measure for an individual firm defined in equation is given as:
TEi = B ( Q */Ui, Zi)/E (Qi */Ui = 0., Zi)
(2)
where Q* is the output of the i-th firm, which will be equal to Qi

when the dependent variable is expressed in original units and
will be equal to exp (Qi) when the regressor is in logs. The non-log
version of equation was used for all the selected micro-enterprises,
the technical efficiency is given as ((zi-Ui)/zi). The computer
programme Frontier 4.1 6, was used to obtain the maximum

likelihood estimate for the parameters of the stochastic frontier
production functions and the predicted technical efficiency. The
variance parameters are expressed in terms of:
indicates that capital outlay and labour were important factors

affecting the value output. The marginal effect of expenditure on
capital outlay and labour on the value of output of burnt-brick
makers was 0.22 and 0.84, respectively.
3 = (u2 + v2) and
Estimates of technical efficiency: Table 2 presents the technical

efficiency distribution of bakers, furniture makers and burnt-brick
makers. The average technical efficiency for bakers was 62.3%,
this was on the lower side when compared with the mean technical
efficiency obtained for furniture makers (78.2%) and burnt-brick
makers (73.42%). Forty-three bakers (66%) were operating at
technical efficiency over 60%, maximum efficiency being 86.28%,
while only 3% (2 bakers) were found to be operating at below
40%. Only 3 furniture makers (5%) were operating at efficiency
level not more than 40 and 89% of them were at most 80%
technically efficient. The two extreme levels of technical efficiency
of furniture maker were 32.3 and 88.34% efficiency level.
Majority (85%) of the burnt-brick makers were operating at
efficiency level greater than 50%, while the least and most efficient
burnt-brick makers were operating at 62.25 and 87.13%,
respectively. Implicit in the fore goings is that there exists some
level of inter and intra group variations of technical efficiencies.
This signals that there is room for improvement of technical
efficiencies of bakers, furniture makers and burnt-brick makers in
Ondo state.
The findings of this study conform to similar findings for
technical efficiencies of other previous studies 3, 11-13. Ajibefun
and Daramola 3 found that the technical efficiency of block makers
varied between 19.5 and 85% with a mean efficiency of 72%. They
also observed that the technical efficiency of metal fabricators
and saw millers in Nigeria lie between 2.7% and 92%, 30% and
90% with mean technical efficiency of 80% and 78%, respectively.
Obwona 12 has observed that tobacco growers technical efficiency
in Uganda varied between 44.8% and 97.3% with mean technical
efficiency level of 76.2%. Vijay and Wisdom 13 has observed that
the technical efficiency of wood processors, dressmakers and
hairdressers varied from 39.3% to 94.4%, 41.9% to 99% and 69.7%
to 100% with mean technical efficiency of 75.7%, 83.4% and 89,1%,
respectively. Also the technical efficiency of electrical firms in
Cameroon was observed to vary from to 94.39% with a mean
efficiency level of 81.9% before trade liberalisation and remained
between 38.85 and 95.76% with mean efficiency level of 72% after
trade liberalisation 11.
= u / (u + v )
2
(3)
In order to estimate the technical inefficiency model, the

predicted technical efficiency was regressed on a vector of socioeconomic variables and, human capital variables (age of
entrepreneur, educational level, and business experience), and
institutional variables (training programmes, training experiences
arid credit access). The technical inefficiency model is given as u3
where:
(4)
where Ku is a vector of independent variables. The parameters of
the above equation were estimated by OLS. The statistical
significance of s help to identify variables causing technical
inefficiencies in the chosen micro-enterprises. As a result of product
differential associated with the selected micro-enterprise, value
of output was used instead of physical output as a depend variable
in the empirical estimation of production functions. The inputs
used in the production functions are expenditure on equipment,
man-hour worked and raw material values.
Estimates from the maximum likelihood production function frontier
are presented in Table 1.
Results of the Cobb-Douglas frontier for bakers showed that
capital outlay and man-hour were significant factors influencing
the value of output with elasticity coefficient of 0.20 and 0.23,
respectively, among bakers. The Return to Scale (RTS) is less
than I, signifying a decreasing return to scale. The estimated
production function frontier for furniture makers indicates that
capital outlay, man-day worked, and equipment in that order were
statistically significant determinant of value of output. The
elasticity of expenditure on capital outlay labour, equipment and
on the furniture makers output was 0.37, 0.36 and 0.21, respectively.
In the burnt-brick firms, production function frontier estimate
Table 1. Maximum likelihood estimates of production
functions.
Enterprise varialbe
Capital outlay
Labour
Equipment
Intercept
Gamma ()
Total variance (2)
Long-likelihood function
Likelilhood ratio test
Bakers
0.2009*
(0.0494)
0.2311*
(0.0321)
0.1757
(0.2751)
5.3432*
(2.0532)
0.3724*
(0.0545)
0.0773
-2.8694
1.75*
Furniture
makers
0.3691
(0.1143)
0.2098*
(0.0795)
0.3455**
(0.1345)
23.543*
(4.2412)
0.4042*
(0.1678)
123.032
-27.52
9.34*
Bunt-brick
makers
0.2179**
(0.0974)
0.8485**
(0.4012)
0.2179
(0.1556)
-28.1342*
(7.0052)
0.4348*
(0.1321)
85.345
31.75
11.34*
Source: Data analysis, * Statistical significance at 1%, ** Statistical significance at 5% level.
Estimate of technical efficiency determinants: The computed

technical efficiencies for the selected enterprises were modelled
on certain policy variables (age, level of education, training
experience/training programmes, business experience; credit
access and working capital and investment outlay) to determine
their influence on the efficiency of these small-scale entrepreneurs.
Age is expected to negatively affect technical efficiency, while all
other policy variables listed above were expected to positively
influence the technical efficiency of these small-scale business
operators 13. Estimated coefficients for selected enterprises are
Among the baking firms, the significant policy variables were
business experience, credit access, age, level of education and
training experience. Experienced, educated, young bakers with
credit access were relatively more efficient. The significance of
309
Table 2. Distribution of technical efficiencies.

Efficiency
21-30
31-40
41-50
51-60
61-70
71-80
91-100
Total
Bakers
No of firms
2
5
7
8
11
24
65
Mean
S.D.
Min
Max
(%)
3
8
11
12
17
37
100
62.37%
10.25
23.45%
86.28%
Furniture
No of firms
1
2
15
19
13
11
1
65
Mean
S.D.
Min
Max
(%)
2
3
23
29
20
17
2
100
78.2%
14.11
2.18%
88.34%
Burnt-brick makers
No of firms
10
10
15
17
13
65
Mean
S.D.
Min
Max
Source: Survey data.
Table 3. Determinats of technical efficiencies.

Enterprise variables
Age of operator
Level of education
Training experience
Business experience
Credit access
Working capital
Invesment outlay
Intercept
R2
F-Statistics
Bakers
-0.1123*
(0.0351)
0.2846*
(0.1124)
0.0312
(0.1712)
0.5816*
(0.1326)
0.0528*
(0.1999)
0.4872**
(0.0041)
0.0077***
(0.0041)
0.7723
(0.2467)
79.3%
83.31*
Funiture
makers
-0.3215*
(0.0542)
0.1111*
(0.0296)
0.3769**
(0.1896)
0.4215
(0.5896)
0.0746*
(0.0245)
0.6734**
(0.0091)
0.0162***
(0.0091)
0.5539
(0.2356)
82.3%
77.5*
15
15
23
26
20
100
73.23%
9.24%
44.15%
87.45%
Conclusions
In the study, we have estimated the technical efficiency and
significant policy variables influencing the technical efficiencies
of bakers, furniture makers and burnt-brick makers in Ondo state.
Our findings show that there exist some level of inter and intra
group variations of technical efficiencies. This signals that there
is room for improvement of technical efficiencies of bakers, furniture
makers and burnt-brick makers in Ondo state. The significant
determinants of technical efficiencies of bakers, furniture makers
and burnt-brick makers were identified as age of operator, business
experience, level of education, training experience, credit access,
working capital and initial capital outlay. Implicit in these findings
is that well structured entrepreneurship training programmes
complemented with easy credit access can facilitate the desired
improvement in the efficiencies of small-scale business people in
the state.
Burnt-brick
makers
-0.2114*
(0.0475)
0.0159*
(0.0045)
0.4012*
(0.1996)
0.0115
(0.1150)
0.0356*
(0.0102)
0.1678**
(0.2848)
0.1884
(0.2848)
0.4972
(0.1896)
69.4%
25.7*
Notes: The figures in paretheses are the standard errors, b. * means that statistic are significant
at 1% level of significance, c. ** means that statistic are significance at 5% level of
significance, d. *** means that statistic are significance at 10% level of significance.
credit at 1% level might not be unconnected with its enhancement

of adequacy of initial capital outlay and working capital. Thus,
increasing inflow of fund through both formal and informal credit
institutions may probably improve efficiency of these microentrepreneurs.
Age had a negative impact on efficiency of bakers. Exposing
unemployed youths to vocational training may enhance the
propensity of bringing forth the desired improvement in the bakers
efficiency. The R2 shows that 79.3% of the total variability in the
efficiency of bakers is jointly explained by the relevant policy
variables.
In furniture making, the significant variables were education,
credit access, initial capital investment, adequate working capital
and training experience. The significance of education and training
is hinged on the fact that they enhance the stock of human
knowledge, and management skills, this consequently improves
efficiencies. Furniture makers who have accessed NDE loan (both
in kind and cash) showed greater efficiency, as this guaranteed
adequacy of working capital and initial investment outlay. Since
credit access, adequate working capital and initial capital outlay
matter for efficiency of furniture makers, provision of adequate
credit at favourable terms will ultimately raises the level of
efficiency of this category of entrepreneurs. The R2 value implies
that about 82.3% of total variability in the efficiency of furniture
maker is jointly explained by the policy variables.
310
(%)
In burnt-brick making the significant

variables were ages of operator, level of
education, credit access, working capital and
training experience. In line with a priori
expectation, age had negative impact on
efficiency of burnt-brick makers. The old
burnt-brick makers are expected to be less
efficient in comparison to the young ones.
Training programmes, working capital and
credit access are positively related with the
level of efficiency of burnt-brick makers. With
best practice, training programmes for the
burnt-brick makers, can significantly improve
their efficiencies.
Acknowledgements
We sincerely thank the management of National Directorate of
Employment for making their data available for this study, Also,
on record is the unalloyed cooperation of our enumerators and
their undaunted spirit in spite of numerous challenges during
data collection. We cannot but appreciate the spirited efforts of
chairperson of trade associations who provided us with list of
their members and guided us on the operation of their union. We
say a big thank you all.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Modulation of micronutrients and antioxidants defenses in Conocarpus lancifolius

under abiotic stress
Amina Redha, Redha Al-Hasan and Mohammad Afzal *
Department of Biological Science, Faculty of Science, Kuwait University, P. O. Box 5969, Safat-10360, Kuwait.
*e-mail: afzalq8@gmail.com
Abstract
Plant adaptation, in response to biotic/abiotic stress, may take place by adjusting nutrient requirements, metabolic enzymes, cellular membrane
composition or an increase/decrease of metabolites such as amino acids, carbohydrates, lipids etc. The present study focused on the adjustment of
antioxidant defenses in Conocarpus lancifolius, subjected to four abiotic oxidative stresses including temperature shock, salinity, water deficit and
polyethylene glycol - 800. C. lancifolius exposure to elevated temperature at 40C significantly increased the antioxidant defenses while the plant
resistance to oxidative stress decreased by other abiotic stressors. This was confirmed by measuring the antioxidant capacity of C. lancifolius by
employing four different assays with two distinctive mechanisms. These assays confirmed that C. lancifolius developed thermo tolerance on
exposure to increased temperature by generating antioxidant defenses against free radicals. This may be an important factor that contributes to
adaptation of C. lancifolius to harsh desert environment. In addition, C. lancifolius showed high accumulation of potassium and boron ions to keep
its osmotic balance that could help it survive under severe abiotic stress.
Key words: Abiotic stress, Conocarpus lancifolius, heat-shock, oxidative stress.
Introduction
Drought, temperature and salinity are common modifiers that
introduce stress tolerance in plants 1-3. These abiotic pressures
cause oxidative stress (OS) generated through reactive oxygen
species (ROS) 4. It is recognized that radical damaging species are
generated within minutes in response to salt stress 5 initiating
ROS in mitochondria or chloroplast affecting multiple signaling
pathways in plants 6. Thus, formation of ROS may initiate a chain
reaction challenging membrane integrity and making them leaky
with pathological consequences 7-10. The rapid generation of ROS
such as a damaging hydroxyl radical (HO) and others may be a
result of salt stress imposed by K+ ion efflux 11-13. The resulting
ion imbalance and the subsequent oxidative burst may adversely
affect growth and photosynthetic apparatus in plants by
compromising potassium channels that are sensitive to ROS thus
affecting cellular transport and energy metabolism 13-15. In plants,
ROS may also be provoked with a variety of stressors including
heat shock 16, 17 and the damage caused by ROS may be alleviated
by a subtle balance and accumulation of natural antioxidants 18.
Many preventive antioxidant defenses such as phenols,
flavonoids, tocopherols, ascorbate, reduced glutathione etc. are
naturally present or may be induced in plants protecting
biosystems from ROS 19, 20. In addition, antioxidant enzymes such
as superoxide dismutase (SOD), glutathione reductase (GR) and
ascorbate peroxidase (APX) play an important role in plant
defenses against ROS 21.
Numerous methods have been described for the measurement
of antioxidant activity in plants 22, 23. Diverse types of free radicals
are generated by different methods of measurement with a range
of end-points. This dictates to accumulate and compare data
generated by idiosyncratic methods to reach a consistent
312
deduction. This approach was adopted in the present study.

Abiotic stress can also result in the accumulation or loss of
metabolites, enzyme activities and protein synthesis. One such
example is the accumulation of carbohydrates and amino acids
such as proline that serve as nitrogen storage materials to balance
osmotic potential. Thus accumulation of amino acids in sorghum
plants, under moderate water deficit increased from 32 to 39 mM
and under severe water deficit amino acids increased from 29 to 45
mM. The main increase was observed in aspartate, glutamate,
proline, alanine, and valine 24.
Conocarpus lancifolius Engl. (Combretaceae) is an imported
plant used for horticulture drives in the State of Kuwait. It has
developed resistance to high temperature that occasionally goes
beyond 50C during summer. The dry climatic conditions in Kuwait,
with elevated summer temperature may be damaging for a
sustainable growth of this plant critical for the greenery projects
in the State of Kuwait. The present study describes endurance
and sustainability of this plant under stark free radical producing
heat shock and other abiotic stressors.
Plant material and growth conditions: Single shoots of
vegetatively propagated C. lancifolius were obtained from the
Public Authority for Agriculture Affairs and Fish Resources
(PAAFR) in the State of Kuwait and maintained under greenhouse
conditions. Shoots at the 13 - 15 leaf growth stage were randomly
selected and transferred to plastic pots (19.0 cm diameter and 16.3
cm deep) containing local sandy soil and peat moss (3:1 v/v).
Ninety plants were randomly grouped into five groups (a - e) with
eighteen plants in each group. Each group of plants was exposed
to a singular stress regime as follows: drought (water deficit),

polyethylene glycol (PEG), salinity and temperature. The plants
in group a (control) were watered daily. Eighteen plants in group
b were subdivided into six groups (b1- b6), three plants in each
group and each subgroup was irrigated with 100 ml of 10 - 60%
polyethylene glycol (PEG) (MWt 6000) in 10% increments on daily
basis for 6 days. Plants in group c were subdivided into five
groups (c1 - c5), three plants in each group and each subgroup
was irrigated with 2 - 10% NaCl solution for 6 days. Group d
plants were exposed to drought by water withdrawal (water deficit)
for a period of 6 days. The plants in group e were subdivided
into five subgroups (e1 - e5) and were subjected to temperature
regime between 10 - 50C for 6 days. All plant groups (a - e) were
placed in growth chambers at 45% relative humidity and
fluorescent light (95 E m-2 s-1). Plant leaf tissues were harvested
following drought, PEG, NaCl and temperature treatments after
completion of the stress period. Tissues were either stored (-20C),
air dried or used fresh for further analyses.
Free amino acids determination in the plant samples: Free amino
acids dissolved in cell sap offering buffering capacity. These are
products of protein hydrolysis and a source of stored soluble
nitrogen to be utilized in later synthesis of peptides and proteins.
Fresh plant mature leaves (3 g) were macerated in 80% ethanol (5
ml), warmed to 50C for 15 min and centrifuged. The pallet was
extracted three times with the same solvent and the total extract
was pooled and membrane filtered and brought to a 20 ml with 80%
ethanol. Free amino acids were determined by PICOTAG method
as described in the literature 25. The amino acids were resolved by
HPLC using a Waters PicoTag column with PicoTag eluent solvent
A while the solvent B acetonitrile:water (3:2 v/v) was used with a
flow rate of 1 ml /min The chromatogram showed 26 well resolved
peaks and 16 were identified as protein free amino acids (Fig. 1,
Table 1).
Mineral nutrients in C. lancifolius: Mineral accumulation was
determined by inductively coupled plasma-optical emission
spectrometry (ICP-OES). Using optimal experimental conditions, 1 g
of the plant material was used. Digestion was carried out in 20 ml 1 M
HNO3. The extraction method described by Santos et al. 26 was
used for mineral analysis.
Table 1. Peak table for the amino acid

analysis of Conocarpus lancifolius.

Preparation of plant extracts for antioxidant activities: Leaf

samples were collected from different plants in the same group to
avoid injury response of the plant. At the specified time, 2 g of leaf
tissue was taken, homogenized (Polytrone, Kinematica AG,
Switzerland) in 2 ml 80% methanol and the homogenate was
centrifuged at 2000 rpm for 15 min. The clear supernatant was
collected and the pellet was re-extracted twice with the same
solvent. The organic solvent from the pooled extracts was removed
on a rotavapor at 30C. Freeze-drying the samples eliminated water
left behind after the evaporation of organic solvent. A known
weight of the freeze-dried sample was re-dissolved in methanol (2
ml), warmed and charcoaled to remove pigments. The solution
was syringe filtered (0.45 m membrane filter) and the yellowish
filtrate was brought to a fixed volume. Each extract was run in
triplicate for antioxidant assay.
DPPH radical scavenging assay: 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH+ ) is a stable purple coloured cation radical, soluble
in methanol and in its oxidized form has an absorbance 517 - 520
0.20
0.18
0.16
AU
0.14
16
0.12
0.10
0.08
0.06
15
12
0.04
78
33 4
0.02
10 11 12
14
13
0.00
2
10
12
14
16
18
20
Minutes
Figure 1. HPLC separation of free amino acids in Conocarpus lancifolius on WATERS - PicoTag Column.
313
nm. The cation radical is reduced to a yellow coloured non-radical

diphenyl-picryldydrazine (DPPH-H) in the presence of H-donating
antioxidant. A methanolic solution of 0.2 mM DPPH was daily
prepared before use. DPPH solution (3.9 ml) was thoroughly mixed
with freeze-dried plant extract, re-dissolved in methanol (1.1 ml),
at different concentrations. The mixture was vortexed and left in
the dark for 30 min. For linearity of the curve, different
concentrations of DPPH were tried to find out the best
concentration for taking the absorbance within the rage. A solution
of the same concentration of DPPH in methanol was used as a
blank and the absorbance was measured at 517 nm. The reduction
scavenging activity (RSA) was calculated from the equation as
described in the literature and the IC50 values were determined
from the graph obtained from standards by using the y = mx+c
equation from the slope of the graph as described by Tang et al. 27.
ABTS radical scavenging assay: This assay measures the total
antioxidant activity where a blue-green colour stable radical is
generated prior to a reaction with antioxidants and its discoloration
measured photometrically. The assay was carried out using a
freshly prepared solution of 2,2-azinobis-3-ethylbenzothiazoline-6sulphonic acid (ABTS+) radical cation, as described by Tang et al.27
A stock of 7 mM ABTS aqueous solution was vortexed with 2.45
mM potassium persulphate and left for 12 - 24 h at room temperature
25C. The concentration of the solution was adjusted to an
absorbance of 0.70 0.02 at 734 nm. Two ml of the ABTS
solution was pipetted into 200 l of the plant extract or Trolox
(standard) in ethanol and the absorbance was immediately taken
at 734 nm. The percentage antioxidant activity was calculated as
described by Tang et al. 28:
Scavenging rate (%) = [1- (As - AB)/ Ao] 100
Ao is the absorbance of ABTS solution without sample; As =

absorbance of the test sample + ABTS solution; AB = absorbance
of the sample without ABTS solution. The results were expressed
as Trolox equivalent antioxidant capacity (TEAC). IC50 values were
determined as described for DPPH.
Ferric reducing/antioxidant activity (FRA): This assay measures
the total reductive capability of the sample using potassium
hexacyanoferraate reduction of Fe3+(CN)6 to Fe2+(CN)6 by an
electron abstraction from the sample antioxidant. Different
concentrations of the freeze-dried plant extract (FDE) 0.01- 0.1 mg
ml-1 were prepared in 0.2 M phosphate buffer pH 6.6 and mixed
with 0.1% of the ferrate reduction solution. The solution was
incubated at 50C for 20 min. The solution was acidified with 1.25
ml of 10% trichloroacetic acid (TCA) solution and vortexed with 0.5
ml of 0.1% FeCl3 solution and after 10 min the absorbance was read at
700 nm. Absorbance value of the blank was subtracted from the
sample and the results were expressed as reducing power of the
plant extract by ascorbic acid 29 and quercetin (AsAE; QrE) 30, 31.
The results were interpreted as higher the OD at 700 nm, the higher
the FRA value.
Cupric ions (Cu++) reducing-cuprac assay: Cupric reducing
antioxidant capacity (cupric) is also an electron transfer based
method which is much more sensitive and superior compared to
FRAP assay. It can account for antioxidant values in complex
314
samples including thiols where total antioxidant capacity (TAC)

assay gives negligible absorbance 32, 33. This method measures
the cupric ions (Cu2+) reduction ability of the plant extract. A 0.01
M, CuCl2 solution (0.25 ml) was vortexed with 0.25 ml ethanolic
solution of 7.510-3 M neocuproine and 0.25 ml, 1 M ammonium
acetate buffer (pH 7). This solution was mixed with different
concentrations of the freeze-dried plant extract dissolved in
ethanol:ammonium acetate buffer (1:1 v/v) to a final concentration
of 20 g ml-1. After 30 min at room temperature, the final volume
was adjusted to 1.5 ml, vortexed and the absorbance was read at
450 nm against reagent blank. The increased absorbance indicated
bigger reduction capability expressed as AsAE and QrE 34, 35.
A total of sixteen free amino acids, including five major free amino
acids were detected (Fig. 1, Table 1) in C. lancifolius extracts.
These five amino acids were identified as aspartic acid, glutamic
acid, argenine, proline, and lysine. Accumulation of free amino
acids in response to drought stress has been reported in sorghum
and sunflower plants 24. The free amino acids work as source of
soluble nitrogen as well offer buffering capacity in the cellular
environment. Free amino acids and micronutrients present in C.
lancifolius suggest that plant thrives under harsh environmental
conditions in Kuwait by accumulating amino acids and minerals
such as potassium that assists in maintaining plant cellular osmotic
pressure 36. Potassium is accumulated at high concentration of
11.273 g kg-1 while boron is accumulated at a concentration of
162.25 mg kg-1. Boron is known to contain fertilizer like properties
that help plants in their growth and development 37.
Free radical damage caused by drought stress: In water deficit
plants FRA values decreased non-significantly from 0.617 0.0018
(control) to 0.596 0.0017 (3.5% decrease) on the 6th day. The nonsignificant decrease may be due to an improved drought tolerance
and/or adaptation characteristics of C. lancifolius under applied
experimental conditions. When plants were exposed to stronger
drought settings induced by a treatment with 10, 30 or 60% PEG
for 2, 4 or 6 days, the decrease in FRA values were PEG
concentration and time dependent. The FRA values decreased
from 0.617 0.0018 (control) to 0.515 0.0026 (16.5% decrease) on
the 6th day after treatment with 60% PEG (Fig. 2a). This indicated
that drought conditions created by PEG were further detrimental
than simple water deficit. The decrease in FRA values are
statistically significant (p = 0.001), demonstrating that C.
lancifolius does not retain resistance to high drought conditions
caused by PEG. In water stress physiology, effect of drought has
been studied under many conditions in various plants and drought
is known to reduce stomatal conductance (gs), photosynthetic
rate (A) and photosynthetic potential (Apot). Thus, drought can
bring about many changes in plant physiology including cell
signaling through generation of ROS 38. Relative water content
(RWC) is known to drop in plants irrigated by PEG solution with a
decrease in antioxidant enzymes such as catalase (CAT),
peroxidase (POD) and superoxide dismutase 39. Therefore, in PEG
induced drought, ROS may be generated at an increased rate
reducing FRA values in the plant. Diminution of FRA, DPPH and
ABTS % inhibition rates are known to decrease under drought
conditions 23.
Measurement of radical scavenging activity (RSA % inhibition)
0.60
0.55
0.50
10
20
40
30
56
6.2
53
6.0
5.8
50
5.6
47
5.4
44
5.2
0
60
50
10
20
EC50 Day-2
18
EC50
40
10
20
0
50
50
50
-6
10
(D) Effect of drought on DPPH reduction
l
6
2
4
0
t r o E C 50 ay- E C 50 ay- E C 50 ay- E C 5
on
D
D
D
Exposure time and EC50 values

(C) Effect of drought on % reduction of ABTS
Day-6
EC50 Day-4
30
16
14
20
12
10
10
6
10
EC
20
10
60
EC 50 Day-6
50
E C Day E C Day E C Day

C
Exposure time and EC50 values
t
on
50
30
00
EC50
20
Reduction ABTS %
60
-4
40
Day-4
Day-2
30
80
-2
30
40
(B) Effect of PEG on % reduction of DPPH
(A) Effect of PEG on reduction potential
50
20
PEG %
PEG %
ol
30
6.4
20
30
40
EC50
EC50 Day-6
Day-6
Reduction ABTS %
Day-4
Day-4
EC50 Day-4
CUPARAC g QrE/ 10 g/ml of Fde
Day-6
Day-2
Day-2
Reduction DPPH %
Reduction potential g ml-1 Fde
0.65
Reduction DPPH %
Day-6
Day-6
Day-4
EC50 Day-2
EC50
Day-2
Day-4
Day-2
50 60
PEG %
(E) Effect of PEG on % Reduction of ABTS
Co
nt
ro
l
%
10
PE
G
%
30
PE
G
%
60
PE
G
Dr
ou
gh
PEG %
(F) Effect pf PEG on Cuparac value
Figure 2. Effect of drought stress on antioxidant parameters.
was determined using DPPH, in water deficit and PEG exposed

plants. The plants exposed to water deficit showed a consistent
decrease in RSA % in the plants. Thus RSA % inhibition values
significantly decreased (p 0.05) from control 66.40 0.6 [EC50 =
4.67 0.03; ascorbic acid equivalent 3.77 0.4; quercetin equivalent
(QrE) 4.22 0.08 g g-1 FDE] leading to 55.58 0.55 (16.6% decrease)
[EC50 = 5.52 0.04; AsAE 3.1 0.02; QrE 2.86 0.03 g g-1 FDE], on
the 6th day of the exposure to water deficit (Table 2). This percentage
decrease is the same as measured for FRA induced by more
powerful water depleting agent PEG indicating better sensitivity
for a sample electron donation assay (FRA) by the antioxidant.
An increase in EC50, and decrease in AsAE and QrE were 15.3%,
17.7% and 32.2%, respectively. This data indicated a loss in
antioxidant activity of the plant under water deficit, generating
ROS. The significant decrease (p 0.05) in RSA indicated enhanced
production of ROS under water deficit, diminishing the antioxidant
defenses including polyphenols and antioxidant enzymes in plant.
Table 2. %Decrease/increase in RSA % inhibition, EC50, ascorbate
equivalent (AsAE) and quercitin equivalent (QrE)
values with different stresses on C. lancifolius.
Treatment
DPPH/H2O deficit
DPPH/PEG
ABTS/H2O deficit
ABTS/PEG
DPPH/10% salt
ABTS/10% salt
DPPH/Temperature
ABTS/Temperature
AsAE
RSA Inhibition
EC50
%Increase %Decrease
%Decrease
16.0
15.30
17.7
35.0
25.80
39.0
47.0
55.00
52.0
64.0
61.00
71.7
24.4
22.44
25.7
59.3
55.75
65.7
19.0
17.50
20.6
18.0
12.80
17.3
QrE
%Decrease
32.20
60.70
50.00
69.00
48.00
63.00
35.00
16.70
The RSA % inhibition values also decreased significantly (p

0.05), in plants exposed to incremental PEG % and with increasing
exposure time (Fig. 2b). The RSA % inhibition value was 46.12 0.46
(EC50 6.29; AsAE 2.53 0.04; QrE 1.66 0.08 g mg-1 FDE), compared
with control RSA values given above, indicating a highly
significant decrease (p 0.001) in antioxidant defenses of the
plants under PEG drought. Thus plants exposed to 60% PEG on
the 6th day presented 35.5%, a two-fold decrease in RSA values
compared with water deficit plants. In addition, the EC50 increased
by 25.8% associated with a decrease in AsAE and QrE values by 32.9
and 60.7%, respectively (Table 2). This reflected on the fact that the
non-permanent drought induced by PEG was much more damaging
to the plants compared with drought created by water deficit (Fig.
2c). The data indicated Stern oxidative stress induced by PEG
which may have led to oxidative membrane and chloroplast damage
with an inhibition of Rubisco supply. In response to PEG, inhibition
of stomatal conductance and photosynthesis are well established
in the literature 40.
In water deficit plants, ABTS % reduction decreased
significantly (p 0.05) in a time dependent manner. A 47% decrease
in ABTS value was observed in plants exposed to water deficit for
six days. Thus, in control plants, the ABTS % scavenging activity
significantly decreased (p 0.001) from control values 19.76 0.21,
(EC50 19.74 0.26; AsAE 4.6 0.05; QrE 5.8 0.06 g g-1 FDE) to 10.48
0.30, a 47% decrease (EC50 43.95, AsAE 2.200.8 and QrE 2.91 0.09
g g-1 FDE) (Fig. 2d). These figures corresponded to an increase
in EC50 by 55% and a decrease in AsAE and QrE by 52% and 50%,
respectively. A highly positive correlation (Tukey test) in EC50
values indicated extensive oxidation of the antioxidants as a result
315
of increased rate of production in ROS under water deficit 41, 42.

A similar pattern was observed in plants exposed to incremental
PEG stress where the ABTS % scavenging activity was time and
PEG concentration dependent. Compared with water deficit, plants
experienced at least 30% more ABTS % scavenging activity when
exposed to 60% PEG treatment for six days. Thus, a 64% decrease
in ABTS % scavenging value 7.07 0.61 (EC50 50.62; AsAE 1.3 0.15;
QrE 1.8 0.19 g g-1 FDE), compared with control (19.74 0.21),
was obtained after 6 days of plants exposure to 60% PEG (Fig. 2e).
This 64% decrease in ABTS % value along with a highly
significant 61% increase in EC50 and associated decrease in AsAE
and QrE by 71.7 and 69%, respectively, indeed indicated the extreme
stress experienced by plants by PEG treatment. Comparing the
PEG induced increase in EC50 and decrease in AsAE and QrE with
water deficit values, again supported our view that PEG created
drought was more detrimental than water deficit. The data also
suggests that ABTS assay was more sensitive than DPPH for
measuring antioxidant defenses in plants. Contrary to our results,
Rautenbach et al. 43 have observed an increase in antioxidant
capacity in response to drought in sweet potato varieties.
Cupric assay also showed that drought induced by PEG was
considerably more damaging to the free radical defenses of the
plants. Thus, cupric values significantly decreased (p 0.05)
from 7.01 0.01 (control) to 3.99 0.07 g g-1 (QrE), which
Day-2
Reduction potential g ml-1 fd plant extract
Day-4
Free radical damage by salt stress: Salinized plants are known

for stomatal closure, stomatal conductance, osmotic potential,
transpiration rate, RWC, and many other biochemical changes
including accumulation of competitive solutes that help in
protecting (osmoprotectants) the plants from oxidative burst 44.
Salinity plays a dual negative role in plants. First, salinity creates
drought situation in the roots and second it creates ion imbalance.
Thus salinity is more deadly than drought since it creates osmotic
pressure difference, ion toxicity and oxidative damage due to
drought 45, 46. In our studies, salt stress was time and concentration
dependent. The FRA non-significantly (ns) dropped from 0.68
0.0038 (control) to 0.551 0.0012 (FDE sample concentration;
concentration100 g ml-1) on the 6th day of treatment with 10%
NaCl treatment of the plants (Fig. 3a).
The RSA % inhibition (DPPH) significantly decreased (p 0.01)
from control 66.4 0.6 (EC50 4.670.03; AsAE 3.770.04; QrE 4.22
0.08 g g-1 FDE) to 50.15 0.20 (EC50 6.06 0.06; AsAE 2.8 0.01;
QrE 2.2 0.03 g g-1 FDE). This represents 24.4% decrease, in
Day-4
EC 50 Day-2
Day-2
accounts for 43% decrease in antioxidant defenses in plants on

the 6th day of the exposure to 60% PEG (Fig. 2f). This confirmed
that PEG induce drought was much more damaging to the plant
compared with water deficit. This is the first report to measure
drought induced free radical damage by cupric assay.
Day-6
EC 50 Day-4
EC 50 Day-6
Day-6
65
0.62
6.0
0.58
0.56
60
5.5
EC50
Reduction DPPH %
0.60
55
5.0
50
0.54
4.5
4
6
8
NaCl %
(A) Effect of salt on reduction potential
2
Day-2
Day-4
EC 50 Day-2
10
10
Day-6
45
40
30
10
EC50
35
15
25
20
15
4
10
NaCl %
(C) Effect of salt on % reduction of ABTS and EC50 values
CUPRAC g quercetin equivalent/

10 g/ml of fd sample
20
Reduction ABTS %
6
NaCl %
EC 50 Day-4
25
(B) Effect of salt on % reduction of DPPH and EC50 values
EC 50 Day-6
Cl
Cl
Cl
Na
Na
Na
%
%
%
2
6
10
Salt concentration
(D) Effect of salt on Cuprac values
Co
nt
ro
Figure 3. Effect of salt stress on antioxidant parameters and EC50 values.

316
DPPH % inhibition in plants, after 6 days of exposure to 10% NaCl

(Fig. 3b). In addition, the highly significant increase (p 0.05)
22.44% in EC50 with the associated decrease in AsAE and QrE
values 25.7% and 48% are informative of the reduction status of
the plant.
ABTS % reduction also significantly decreased (p 0.003) with
increasing time and NaCl concentration. 59% decrease in ABTS
value was observed in plants exposed to 10% salt solution for six
days. Thus, in control plants ABTS % reduction diminished
from 19.76 0.21 (EC50 19.74; AsAE 4.55 0.05; QrE 5.8 0.07),
to 8.04 0.52; (EC50 44.77; AsAE 1.58 0.13; QrE 2.15 0.16 g g-1
FDE), after six days of exposure to 10% NaCl exposure (Fig. 3c).
This represented 59.3% decrease in ABTS radical % inhibition
and the associated increase in EC50 was 55.75% with a decrease in
AsAE and QrE as 65.7% and 63%, respectively. The ABTS values
from salt stressed plants are comparable with the values obtained
for plants exposed to PEG drought. This suggested that salt and
PEG were both equally damaging to the plants by increasing ROS
in the plants.
The cupric assay also confirmed that the antioxidant defenses
of the plants decreased with increasing concentration of salt stress.
Thus, cupric value decreased significantly (p 0.01) from 7.01 0.01
(control) to 4.39 0.11 QrE in plants exposed to 10% NaCl for six
days, accounting for a 37% decrease in cupric value (Fig. 3d). To
measure the oxidative consequence of salt stress in plants, cupric
assay has been used for the first time.

There are conflicting reports about the salt stress on plants.
Thus, an increase in antioxidant polyphenolics has been reported
in Salvia officinalis but a decrease has been reported in Nigella
sativa 47-49.
Temperature stress: High temperature is considered to be a major
abiotic stress that deleteriously affects growth and development
of plants through free radical production 50, 51. The % RSA values
in plants exposed to higher temperature were indicative of plant
protection from free radicals generated at higher temperature. The
FRA values like drought and salt stress showed a non-significant
increase in temperature stress. For the plants exposed to
temperature stress, FRA values negligibly altered from 0.62 0.0018
(control) to 0.66 0.0029 in plants exposed to 20C after six days.
While FRA values were logged as 0.67 0.0015 after 6 days of
exposure to 40C (Fig. 4a).
The RSA % inhibition (DPPH) value for control was 57.72 0.59;
(EC50, 5.15; AsAE 3.23; QrE 3.13 g g-1 FDE) and the plants exposed
to 40C for six days resulted in % RSA values 71.33 0.35; (EC50, 4.25;
AsAE 4.07; QrE 4.86 g g-1 FDE). The increase in % RSA was 19%
greater which was significantly higher (p 0.05) in plants exposed to
higher temperature (Fig. 4b). In addition, EC50 decreased by 17.5%
while the AsAE and QrE content increased by 20.6% and 35%,
respectively, indicating a highly significant increase in the
Day-2
Day-4
EC 50 Day-2
Day-4
Day-6
0.68
0.66
0.64
70
5.0
65
EC50
0.70
4.5
60
0.62
55
20
30
Temperature C
4.0
40
20
(A) Reduction potential Vs Temperature

Day-4
EC 50 Day-2
10
21
19
22
18
20
17
18
16
30
35
Temperature C
(B) ABTS % reduction Vs temperature
40
EC50
20
25
40
Day-6
24
20
30
35
Temperature C
EC 50 Day-4
EC 50 Day-6
26
25
(B) DPPH % reduction Vs temperature
CUPRAC g equivalent/ 10 g/ml-1 of

fd sample
Day-2
Reduction ABTS %
5.5
EC 50 Day-6
0.72
Reduction DPPH %
Reduction potential g/ml-1 FD

plant extract
Day-2
Day-6
EC 50 Day-4
6
4
2
0
Co
nt
ro
C
C
20
30
Temperature
40
(D) Cuprac values Vs temperature
Figure 4. Effect of temperature on antioxidant parameters and EC50 values.

317
antioxidant potential of the plants at higher temperature. An

increase in ascorbic acid and phenolic content and consequently
detoxification of ROS in response to higher temperature has been
extensively reported 17, 52, 53.
ABTS % reduction values tracked % RSA pattern confirming
that increased temperature had an advantageous effect on the
growth and development of the plants by combating free radical
stress. Thus, ABTS % reduction values significantly increased (p
0.05) from 20.29 0.44 (EC50 19.62;AsAE 4.82 0.14; QrE 6.15 0.17)
in plants exposed to 20C compared with 24.80 0.55, (EC50 17.1;
AsAE 5.83 0.13; QrE 7.38 0.17 g g-1 FDE). This presented an
increase of 18% in ABTS % reduction. The EC50 reduced by 12.8%
while the AsAE and QrE increase was 17.3% and 16.7%,
respectively again indicating improved antioxidant defenses and
decreased ROS formation in C. lancifolius at higher temperatures
(Fig. 4c).
Cupric assay of antioxidants in plants exposed to variable stress
also resulted in increased cupric values for QrE from 6.58 0.15 in
plants exposed at 20C to 8.19 0.06 g g-1 FDE in plants exposed
at 40C for six days accounting for 9.6% increase in cupric values
(Fig. 4d).
Our data suggest that C. lancifolius is well adapted to Kuwait
environment where summer temperature can reach 50C which is
appropriate for the growth and development of the plant. Higher
temperature may also off set the other environmental stresses like
drought and salinity that may be detrimental to the growth of the
plant. Our results also reflect on the sensitivity of the four
antioxidant assays employed in this work. Thus, ferric reducing
potential is the least sensitive assay for evaluating antioxidant
activity of plant extracts. DPPH, ABTS and cupric assays
demonstrate that ABTS was more sensitive and performed better
than DPPH or cupric assays.
Conclusions
C. lancifolius (CL) exposure to elevated temperature at 40C,
significantly increased the antioxidant defenses while the plant
resistance to oxidative stress decreased by other abiotic stressors.
These results confirmed that CL developed thermo-tolerance on
exposure to increased temperature by generating antioxidant
defenses against free radicals. This may be an important factor
that contributes to the adaptation of CL to harsh desert
environment. In addition, CL showed high accumulation of
potassium and boron ions to keep its osmotic balance that could
help it survive under severe environmental stress.
Acknowledgements
The authors gratefully acknowledge research grant # 2011/1207/
08 from Kuwait Foundation for the Advancement of Science
(KFAS). The authors are also thankful to Public Authority of
Agriculture and Fish Resources (PAAF), Kuwait for providing us
Conocarpus plantlets. Technical help of Mrs. Jacquilion Jose and
Mrs. Divya Saju is thankfully acknowledged.
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319
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Heavy metal distribution in Fagonia indica and Cenchrus ciliaris

native vegetation plant species
Adel M. Ghoneim *, Soud M. Al-Zahrani, Salem E. El-Maghraby and Abdullah S. Al-Farraj
Department of Soil Sciences, College of Food and Agricultural Sciences, King Saud University, P. O. Box 2460,
Riyadh 11451, Saudi Arabia. *e-mail: aghoneim@ksu.edu.sa
Abstract
In recent years, phytoremediation of polluted soil and water resource have gained growing interest. This study address the issue of Cr, Ni, Cu, Zn,
Cd and Pb metals accumulation in Fagonia indica and Cenchrus ciliaris native plant species in the context of their possible use for the sanitation of
wastewater of Riyadh city, Saudi Arabia. Represented soil and plant samples were analysed for their total heavy metal concentrations. The results
indicated that, Cr and Zn are the most abundant heavy metals in soil samples and the total concentrations of Cd and Pb in soil exceed the average values
proposed for common soil. The concentrations of Cr, Zn and Pb metals increased with soil depth, while Ni, Cu and Cd concentrations did not show
a systematic pattern. The results indicated that enrichment factor values ranged from 1 to 5. The enrichment factor values for Cr, Ni and Cu elements
fall into the normal range while medium enrichments of soil with Zn, Pb and Cd. The accumulation coefficients of the studied heavy metals in the
roots of Fagonia indica and Cenchrus ciliaris plants ranged from 0.71-7.50 and 0.31-2.30, respectively. The order of accumulation coefficients in the
roots of of Fagonia indica and Cenchrus ciliaris as follows: Ni > Cu > Zn > Cr > Cd > Fe and Cd > Ni > Cu > Zn > Fe > Cr, respectively. The
translocation factors varied between the two plant species and highest for Ni and Cu in both Fagonia indica and Cenchrus ciliaris plants. The highest
concentrations were found in roots of Fagonia indica, with 75 mg kg-1 Ni and 65 mg kg-1 Cr.
Key words: Heavy metals, accumulation capacity, Fagonia indica, Cenchrus ciliaris.
Introduction
Industrialized activities in recent decades resulted in aggravation
of a lot of environmental problems, especially contamination with
heavy metals. The problem is that these heavy metals when
available in higher concentrations will be toxic to humans and
other organisms 1. In addition, metals may seep into the
groundwater and surface water, making them absorbed by the
plant, and thus enters the food chain in a manner directly or
indirectly. Heavy metals are becoming increasingly prevalent in
soil environments as a result of wastewater irrigation, sludge
application, solid waste disposal, automobiles exhaust and
industrial activities 2, 3. Heavy metals are highly toxic elements
that can concentrate and accumulate in live tissues. Lead, Cu and
especially Cd can become a sanitary and ecological threat to
drinking water resources, even at very low concentrations. Also,
Cd and Zn are common industrial pollutants. Both Cd and Zn are
harmful to plant at relatively low concentrations 4. Thus, clean
alternatives must be developed in order to remove heavy metals
from effluents 5.
In Saudi Arabia, which is facing freshwater shortage, sewage
water and industrial effluents are useful water sources that are
commonly used for agricultural production. The domestic
wastewater and industrial effluents are biologically treated and
subsequently used for irrigation, however, the wastewater contains
substantial amount of nutrients and organic and inorganic
pollutants, which are creating problems for the farming sector 6.
Phytoremediation is a method of environmental treatment that
320
makes use of the ability of some plant species to accumulate certain

heavy metals, in amounts exceeding the nutrition requirements of
plants. Phytoextraction is one of the elements of phytoremediation
in which heavy metals from the contaminated site are taken up by
plants and then transported from roots to shoots and removed
with crops from a specified area of nature 7. It is difficult to state
definitely which plant species could be particularly useful in
environmental phytoremediation. Plants spontaneously growing
on natural and anthropogenic grounds reflect their adaptation to
the given conditions of growth.
Knowledge on the biomass element accumulation capacity,
which would enable the development of a treatment method for
the obtained plant mass, seems to be essential, as well as the
assessment of the possibility of ground sanitation by plants. Plants
of particular ability for the uptake of elements from the air, water
and soil and their accumulation are considered to be indicative
and therefore they happen to be called bioaccumulators 9.
Considering the possibilities for phytoremediation of waste
landfill sites and areas surrounding industrial plants, both the
ability of plant species to accumulate high amounts of heavy
metals per biomass unit, as well as the possibility of high biomass
production over a given time and area are important 10-12.
Therefore, despite numerous studies on the sources,
accumulation and transfer of heavy metals of wastewater
contaminated soils, however, information regarding the combined
uptake, translocation and accumulation of heavy metals present
in wastewater-contaminated soil are few. The main purpose of

this study was to determine soil-plant relationship for the Fagonia
indica and Cenchrus ciliaris native plant species that were
naturally present at the second industrial effluent site, Southeast
of Riyadh city, Saudi Arabia.
Area description and soil sampling: The second industrial area
located in southeast of Riyadh city (2439.8 N; 4657 E), is kind
of industrial waste water canal covering an area of about 300 m2.
This canal receives most of the liquid industrial waste water.
Composite soil sample was collected from the upper horizon (0-5
and 0-30 cm) depth by 30 m 30 m. Soil samples were air- dried,
sieved through a 2-mm mesh, and then used to measure the
chemical and physical properties according to standard
procedures. The pH and EC values of soil samples were measured
using 1:5 ratio of w/v with distilled water by pH-meter and the
electrical conductivity meter, respectively. Particle size distribution
was determined according to method of Gee and Bauder 13. Calcium
carbonate content was determined using a calcimeter 14. Some soil
properties are presented in Table 1. Total soil heavy metal
concentrations were extracted by digestion with HNO3-HClO4HF mixture 15 and metal concentrations were determined using
ICP (Perkin Elmer, Model 4300DV). Results of total heavy metal
concentrations in soil (mg kg-1) are presented in Table 2. Also,
Enrichment Factor (EF) was calculated for soil using Fe
concentrations as a as a background element, which is generally
considered as mainly originated from natural lithogenic source
(rock weathering). The EF is the relative abundance of a chemical
element in a soil sample compared to the earth crust concentration.
EF = ([M] / [Fe]) soil / ([M] / [Fe]) Earth crust
where [M] is the total heavy metal concentrations measured in

soil sample and [Fe] = total concentration of Fe.
Plant sample preparation and heavy metal analysis: The Cr, Fe,
Ni, Cu, Zn and Cd concentrations in plants grown on the industrial
waste water of Riyadh city were the subject of this study. Cenchrus
ciliaris and Fagonia indica plant species based on their coverage
at the site, were collected, separated into roots, and shoot, washed
gently with deionized water for approximately 3 min to remove soil
particles adhered to the plant, dried at 60C and finally ground to
powder using a Wiley mill. The plant samples were acid digested
with concentrated mixture of HNO3-HClO4 16. After digestion, the

cooled solutions were diluted to 50 ml with ultrapure water and
filtered into plastic bottles pre washed with acid. Finally, the
concentrations of heavy metals in acid digests were measured as
mentioned before. Reagent blanks and internal standards were
used where appropriate to ensure accuracy and precision in heavy
metal analysis.
Quality control: Reagent blank standard, soil and plant samples
reference were included in the sample batch to verify the accuracy
and precision of the digestion methods and subsequent chemical
analysis. The results indicated satisfactory recovery ranged from
91-115% and 90-102% for soil and plant samples, respectively.
Root and shoot transfer factors: Heavy metal concentrations in
the roots, shoots and soil extracts were calculated on the dry
weight basis. Roots and shoots concentrations are often used for
contaminant concentration in plants because soil to plant tranfer
is one of the major pathways for pollutants to enter the food
chain 17.
Metal
TF =
Metal
AC =
Metal
Metal
shoot
roots
shoot
soil
where TF and AC are the translocation factor and accumulation

coefficient of heavy metals, respectively. Metal shoot, root and
soil represent the contaminant content in shoot, root and soil on
the dry basis, respectively.
Soil characteristics and heavy metal contents: As shown in Table
1, soil in the surrounding area of industrial wastewater was slightly
alkaline with pH average of approximately 7.70. The average of
EC and CaCO3 were 6.30 dS m-1 and 15.2%, respectively, and the
texture of soil was sandy. A wide range of soil heavy metal
concentrations was observed and ranged from 0.10 to 40 mg kg-1
depending on soil depth and heavy metal type. According to
Lindsay 18, 100 mg kg-1 Cr, 40 mg kg-1 Ni, 30 mg kg-1 Cu, 50 mg kg-1
Zn, 0.06 mg kg-1 and 10 mg kg-1 Pb would be considered the average
range of heavy metal concentrations in common soils (Table 2).
The concentration of Cr, Ni, Cu and Zn in surrounding area of
Table 1. Soil characteristics of the surrounding area of industrial wastewater of Riyadh city (n = 4).
CaCO3%
15.2
Particle size distribution %

Sand
Silt
Clay
85
6
9
EC
dS m-1
6.30
CO30
Anions (meq L-1)

SO4- HCO3Cl0.09
1.40
3.50
Cations (meq L-1)

Ca2+ Mg2+ Na+ K+
0.80 0.30 0.80 4.0
pH
7.70
Table 2. Evolution of total heavy metal concentrations and enrichment factors for Cr, Ni, Cu, Zn, Cd and
Pb in soil.
Metal
type
Cr
Ni
Cu
Zn
Cd
Pb
Concentration of heavy metals (mg kg-1)

Soil depth (0-5 cm) Enrichment factor (EF) Soil depth (0-3 cm)
40
1
30
10
1
10
8
1
9
30
3
20
0.1
4
0.1
10
5
6
Enrichment
factor (EF)
1
1
1
2
4
3
Standard values of heavy

metals in soil (mg kg-1)a
100
40
30
50
0.06
10
a: Adapted from Lindsay 18. EF is calculated against earth crust values.
321
Wastewater samples characterization: Wastewater samples were

collected from the experimental site and processed for heavy metal
determination. Table 3 indicates that, most heavy metals in
wastewater effluent were above permissible limits set by King
Saudi Arabia and USEPA 22 standard limits for agriculture reuse.
Soil contamination with heavy metals in this area is caused mainly
by mining activity, wastewater irrigation and solid waste disposal23.
Table 3. Wastewater heavy metal concentrations in
comparison with recommended maximum
concentration of KSA and USEPA standards limits
for agriculture reuse.
Element
Fe
Cd
As
Cu
Pb
Zn
Co
Concentration (mg L-1)

15.8
3.90
5.01
3.01
0.30
5.60
0.02
KSA Standard
0.02
0.01
0.20
2.0
1.00
-
USEPA 22
5.0
0.01
0.10
0.20
5.0
2.00
0.05
Heavy metal concentrations in plant species: The differentiation

in heavy metal concentrations in plants is shown in Fig. 1. The
heavy metal concentrations of roots were higher than that in
shoots. Plant uptake of heavy metals from soil occurs either
passively with the mass flow of water into the roots, or through
active transport crosses of the plasma membrane of root epidermal
cells. Under normal growing conditions, plants can potentially
accumulate greater amounts of certain heavy metal ions than are
in the surrounding medium 24. Plant species vary in their capacity
for heavy metals accumulation. Uptake and accumulation of heavy
metals by shoots and roots varied with heavy metal type and
322
Heavy metal concentrations

(mg kg-1) dry weight
Enrichment factor: Enrichment factor (EF) values ranging between

0.5 and 2 can be considered in the range of natural variability,
whereas ratios higher than 2 indicate some enrichment
corresponding mainly to anthropogenic activity. The results of
EF calculations indicated that EF values ranged from 1 to 5 (Table
2). The EF values for Cr, Ni and Cu heavy metals fall into the
normal range while medium enrichments of soil with Zn, Pb and
Cd occur. Pb is taken as example because Pb is known as partly
coming from anthropogenic input. Table 2 indicates that the soil
was enriched in Pb when compared to average range of soil
abundance. For other heavy metals (Cr, Ni and Cu), the soil is not
heavy metal enriched (EF = 1) with reference to the deep soil layer.
The variations can be attributed to Fe concentration differences
between the earth crust and the deep soil layer. Cr and Zn are the
most abundant heavy metals in soil samples. The high Cr
concentration in deep soil layer could be explained by the fact
that Cr is like Zn, Ni and Cu that are strongly bound to clay
minerals 20. Cu concentration shows relatively little variation in
the soil profile, and it is mainly related to Fe and Al oxides and soil
pH 21.
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Fagonia indica
Roots
Shoots
Cr
Ni
Cu
Heavy metal
Zn
Cd
Cenchrus ciliaris
30
Heavy metal concentrations
(mg kg-1) dry weight
industrial wastewater site was less than the standard values

proposed for common soil 18 and European norms 19. Except for
Cd, the concentrations of Cr, Zn and Pb increased with soil depth,
while Ni, Cu and Cd concentrations did not show systematic
patterns. Zn and Pb are enriched in the surface soil layer and then
decreased in the deep horizon.
Roots
25
Shoots
20
15
10
5
0
Cr
Ni
Cu
Zn
Cd
Heavy metal
Figure 1. Heavy metal concentrations in the root and shoot

tissues of Fagonia indica and Cenchrus ciliaris native plants
(mg kg-1 dry weight). Vertical bars represent Standard Error (SE, n = 4).
plant species. Heavy metal concentrations in the shoot and roots

of studied plant species were compared with the standard set by
State Environmental Protection Administration (SEPA) China, the
maximum limits of Cr, Ni, Cu and Cd elements are 0.5, 9, 20 and 0.2
mg kg-1 on a dry weight basis, respectively. In this study, the Cr,
Ni and Zn concentrations in roots and shoot samples exceeded
the SEPA limits, especially in Fagonia indica plant. However, our
data indicated that heavy metal concentrations in Fagonia indica
and Cenchrus ciliaris were markedly higher than the heavy metal
concentration in Rhazya stricta grown on industrial wastewater
of Riyadh city 25. This study shows that elevated concentrations
of some heavy metals were accumulated in Fagonia indica and
Cenchrus ciliaris grown in contaminated soil. In the study area,
soil contamination with some heavy metals is mainly due to the
industrial wastewater and mining activity 23, 26.
Accumulation coefficient: The accumulation coefficient of the
heavy metals in the roots of Fagonia indica and Cenchrus ciliaris
were 0.717.50 and 0.312.30, respectively (Table 4). These high
values, especially in Fagonia indica, indicated that these species
are suitable for phytoextraction but are not hyperaccumulators.
These plants may be more suitable for soil stabilization. The order
of accumulation coefficients in the roots of Fagonia indica is Ni
> Cu > Zn > Cr > Cd > Fe, while in roots of Cenchrus ciliaris Cd
> Ni > Cu > Zn > Fe > Cr. Melendo et al. 27 studied some plant
species that grew in Southeast Spain and reported higher Pbaccumulated levels in P. miliaceum (981 mg kg-1) than in Z. fabago
(50 mg kg-1).
Table 4. Heavy metal accumulation factors in plants grown

in the industrial wastewater-contaminated area.
Metal type
Cr
Fe
Ni
Cu
Zn
Cd
Accumulation Factor (AF)

Fagonia indica
Cenchrus ciliaris
Root/Soil
Shoot/Soil
Root/Soil
Shoot/Soil
1.88
0.64
0.31
0.20
0.71
0.18
0.14
0.10
7.50
1.25
2.25
0.88
3.23
0.82
1.18
0.79
2.20
1.10
0.70
0.60
1.30
ND
2.30
1.30
AF = Heavy metals in plant tissues (mg kg-1)/total heavy metal content in soil (mg kg-1); All data
are means of 4 replications. ND = not detected.
Transfer factor: Soil to plant transfer factor is often used to

estimate heavy metals uptake ability of plants and it is one of the
key components of human exposure risk associated with industrial
wastewater-contaminated soil. The translocation factor (TF)
values are presented in Table 5. The TF values varied between the
two plant species and were highest for Ni and Cu in both Fagonia
indica and Cenchrus ciliaris plants. The high transfer values for
Ni and Cu elements from soil to plant indicated a strong
accumulation of those metals (Table 5). The TF for heavy metals
was found in the order of Ni > Cu > Zn > Cd > Cr > Fe. The ability
of the Fagonia indica plant for accumulation of heavy metals
was higher than that for Cenchrus ciliaris.
Table 5. Heavy metal translocation factors on dry weight basis
for Fagonia indica and Cenchrus ciliaris plants grown
in industrial wastewater-contaminated soil.
Metal type
Cr
Fe
Ni
Cu
Zn
Cd
Translocation Factor (TF)a

Fagonia indica
Cenchrus ciliaris
Roots/Soil Shoots/Roots Roots/Soil Shoots/Roots
1.88
0.35
0.31
0.65
0.71
0.25
0.14
0.73
7.50
0.17
2.25
0.39
3.23
0.26
1.18
0.68
2.20
0.50
0.70
0.86
1.30
ND
2.30
0.56
a: Translocation Factor (Ratio of metals in above-ground biomass to roots).
Conclusions
This study was conducted to screen Fagonia indica and Cenchrus
ciliaris plant species growing on a contaminated site for their
potential for heavy metal accumulation. In plant species, heavy
metal translocation into shoots appears to be very restricted.
Therefore harvesting plants will not be an effective source of
metals removal in this site. However, plants with low shoot
accumulation should be used in order to stabilize the heavy metals
and reducing the metals dispersion through grazing animals.
Acknowledgements
Authors wish to thank College of Food and Agricultural Research
Center and Deanship of Scientific Research, King Saud University,
Saudi Arabia, for supporting this work.
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Performance of lateral move type sprinkler irrigation system

Jonathan Dieter 1, Silvio C. Sampaio 2, Gisele Vogel 3, Mrcio A. Vilas Boas 2, Elisandro P. Frigo
and Alvaro Mari Junior 4
UFPR, Department of Engineering and Exact Sciences, Federal University of Paran (Palotina Sector), Pioneiro R. Street,
2153, Jd Dallas, Palotina (85950-000), Pr, Brazil. 2UNIOESTE University of Western Paran, 3 Agricultural Engineer,
4
Environmental Engineer. e-mail: jdieter@ufpr.br, silvio.sampaio@unioeste.br, vogelgisa@hotmail.com,
marcio.vilasboas@unioeste.br, epfrigo@gmail.com, professor.alvaro.mari@gmail.com
Abstract
This trial aimed at evaluating the performance of a lateral move type sprinkler irrigation system. Data collection was obtained according to ABNT and
the precipitation assay was carried out at relay speeds of 100, 80, 60 and 40%, collectors were distributed in 4.5 m distance and arranged in three rows
separated by 10 m between rows. It was verified that the mobile showed good performance at all speeds. The best uniformity coefficients, CUC
(88.54%) and CUD (80.79%) were for 60% speed, however, the irrigation suitability was lowest (26.34%).
Key words: Water resources, uniformity, efficiency.
Introduction
Farmers have sought several techniques of yield management in
order to find a high yield; including irrigation systems, which
were chosen in accordance with the characteristics of soil, climate,
topography and crops to be irrigated 12, 19, availability, energy
cost 15 and economic feasibility of each irrigation system 10.
Irrigation is not only to apply water without control or planning,
but it aims at applying the correct water amount at the right time,
especially with uniformity during the irrigation process 12, 13, 26.
Thus, in order to achieve water optimization in agriculture, it is
important to frequently evaluate the performance of installed
irrigation system, using some parameters that express and quantify
the operation quality. The water distribution uniformity coefficient
is often used as an indicator of problems concerning irrigation
distribution 4.
Christiansen 7 was the first researcher to study the uniformity
of spray distribution and to quantify it by using a statistical index
called as Christiansen uniformity coefficient (CUC), which uses
the average absolute deviation to express water depth dispersion.
Criddle et al. 8 have already proposed a distribution uniformity
coefficient (CUD), which relates the fourth part of the total irrigated
area that receives less water, with the applied average depth. In
this case, the whole area receives at least the required real water
depth and the CUD low value indicates excessive loss by leaching.
In high economic yield crops, besides the fact that water makes
part of yield cost, the sprinkler irrigation should show high
distribution uniformity.
According to Merriam and Keller 17, in crops with shallow root
systems, the CUD should be greater than 80%, while CUC should
be above 88%. In crops with an average root system, CUD should
range from 70 to 80%, while CUC ranges from 80 to 88%. In crops
with a deep root system, the distribution uniformity may vary
from 50 to 70% and CUC from 70 to 80%.
In recent years, several studies have been carried out in Brazil

on irrigation performance by central pivot to identify the main
problems of irrigation efficiency 3, 9, 11, 12, 21-23. However, studies
concerning the evaluation of sprinkler irrigation system
performance with lateral move type were not found, thus, this trial
achievement is characterized and justified. In Brazil, the sprinkler
lateral move type was introduced in 1986, with an increasing use
in recent years due to technological innovations, management
convenience, economics and water. Linear systems, also known
as mobile side, have their structures similar to central pivot.
However, the circular movement is replaced by the linear one, so
that the water application rate is constant along the entire length
of lateral move type 6.
In recent years, some papers regarding uniformity and water
efficiency application have addressed the development of new
methods and equipments 1, 14, 24, localized irrigation 18, 25, gravity
irrigation 22, conventional sprinkler irrigation 14, 16, 25 and central
pivot irrigation 3, 5, 9, 11, 12, 21- 23. Therefore, there is a gap on uniformity
and efficiency application in sprinkler of lateral move types. In
this sense, this study aimed at evaluating the performance of
lateral move type sprinklers with relay at 100%, 80%, 60% and
40% speeds.
The trial was carried out at Pioneer Seeds Ltd Research Station, in
the municipality of Coxilha (RS), Brazil, geographically located at
latitude 2815' S, longitude 5224' W and 684 m altitude.
This research applied sprinkler irrigation of lateral move type
manufactured by FOCKINK company and S2 AF3000L model,
composed of two towers. The total length of lateral line is 110.5 m,
including a 14.5 m oscillation. The Super Spray type sprinklers
were set at 4.2 m height. The displacement was performed by 1.5
325
hp geared motor, with distance to be covered of 1,393 m total area

to irrigate 15,392 m.
The assays for water depth and uniformity distribution followed
NBR 14244:1998 recommendations 2. Groups of collectors were
arranged along 250 m, separated by 50 m, in order to evaluate
relay speeds at 100%, 80%, 60% and 40%. Each group consisted
of three line collectors, separated by 4.5 m, and 10 m between
rows. Collectors of water showed the following dimensions: 0.08
m diameter collectorneck, 0.10 m height.
Water depth in each collector was measured in a beaker with 0.1
mm precision. The wind speed was measured by manual
anemometer, with a 10% error probability. The evaluated
parameters at each speed (100%, 80%, 60% and 40%) were water
depth (WD), Christiansen uniformity coefficient (CUC) and
distribution (CUD). The CUC was calculated by applying the
equation proposed by ABNT (Equation 1) and for CUD the
equation by Bernardo et al. 4 (Equation 3).
n
Vi VMA
CUC = 100 1 i =1 n
Vi
i =1
(1)
where CUC - Christiansen uniformity coefficient (%); Vi - water

volume collected in the i-th collector (mm); n - number of rain
pluviometers; VMA - weighted average volume of collected water
(mm) (Equation 2).
VMA =
V
i =1
(2)
where CUD - coefficient uniformity distribution (%) and lq - average

of 25% water depths with the lowest values (mm); Lm - average
depth of all observations (mm).
CUD =
lq
(3)
Lm
A descriptive statistical analysis was carried out of all data. In

this analysis, it can be highlighted the Anderson-Darling normality
test, which evaluated the hypothesis of the applied water
distribution to be normal at 5% significance level. CUC and CUD
classifications were taken using the table from ABNT 2 (Table 1).
Table 1. CUC and CUD qualification 2.
CUC (%)
<80
80- 84
85-89
>90
CUD (%)
<70
70-74
75-81
>82
Classification
Bad
Regular
Good
Very good

Table 2 shows the values for the water depth descriptive analysis
that was already observed for the four relay speeds. The proximity
among the average and the median values were observed for all
tests, indicating symmetry among the sampled data. Data variability
in relation to the average may be classified 20. This indicates low
variability when CV is lower than 10%, whose average is between
10% and 20%; it shows high variability from 20% to 30% and very
high for values greater than 30%. In this study, it was found out
326
Table 2. Descriptive statistics of water depths obtained in

assays of water uniformity distribution in lateral
move at speeds of 100%, 80%, 60% and 40%.
Relay- Relay- Relay- Relay100% 80%
60%
40%
Number of samples
75
75
75
75
Minimum (mm)
0.00
1.20
0.50
2.00
Maximum (mm)
3.30
4.00
4.00
7.70
Average (mm)
2.11
2.71
3.12
5.29
Median (mm)
2.00
2.60
3.20
5.50
Standard deviation (mm)
0.63
0.52
0.59
0.99
CV (%)
29.80 19.33 22.58 18.61
Asymmetry
-0.88 -0.09 -2.83 -0.44
kurtosis
2.41
0.23 10.61 0.97
Kolmogorov-Smirnov* (p-value) 0.05
0.15
0.01
0.15
Parameters
CV: Coefficient of variation; * Test at 5% significance
that variability of data was classified as averages (relay at 60%

and 80%) and high (relay at 40% and 100%).
It is observed in the results for Kolmogorov-Smirnov test (Table
2) that assays with relay speeds at 100% (p-value 0.05), 80% (pvalue 0.15) and 40% (p-value 0.15) were normal. The assay to
speed at 60% (p-value 0.01) did not show any normal distribution.
It can be highlighted that to calculate CUC and CUD, there was no
data processing technique, but in Fig. 1, WDP, WDE and DP can
be seen along the lateral line to the four moving speeds.
In Fig. 1, it can be seen similar behaviour in DP along the profile
for all tested speeds. Water distribution remained around WDE
until 100 m from moving side. The maximum variations can be
positive or negative and they occurred within 0 to 100 m of the
moving side, between WDE and DP, which were from 18% to 40.5
m (Fig. 2A), 17.47% to 85.5 m (Fig. 2B), 18.33% to 90.0 m (Fig. 1C),
and from 12.97% to 94.5 m (Fig. 1D) for relay speeds at 100, 80, 60
and 40%, respectively. After 100 m of lateral moving side, variation
at the end of lateral side and WDE and DP were negative at 108 m
in the following ratios: -92.10% (Fig. 1A); -20.46% (Fig. 1B); 57.35% (Fig. 1C), and -29.94% (Fig. 1D) to speeds at 100 to 80, 60
and 40%, respectively.
A similar behaviour was found in many studies 5, 12, 21. Other
researchers 9, 21, 22 observed that with central pivot, the pressure at
the end of the wing was lower than the recommended one and the
applied water depth was below the average. Folegatti et al. 12
realized that the lack of pressure, caused by malfunctioning of
regulators pressure at the end of lines, induced water depth
reduction at the end of the line.
The obtained values of Ws, DP, CUC and CUD on equipment
evaluation for different percentage and classification of relay
settings according to ABNT 2 are presented on Table 3.
The DP was below WDE (Fig. 1A, 1B and 1D), except for the
assay at 60% speed (Fig. 2C). The main cause of these differences
between DP and WDE is the relay percentage speed 5, 22. According
to ABNT 2, in those places where WDE differs by 10% (negatively
or positively) in relation to WDD, the reasons for such variation
should be investigated. For the four tested relay speeds,
variations were lower than 10% recommended by ABNT 2 (Table
2). However, preventive measures must be taken, since the values
are close to the recommended limit. Cainelli et al. 5 have already
evaluated the central pivot at different relay speeds and observed
negative variations (-1.67, -2.74, -2.81 and 2.81%) between WDE
and LP, while for the speeds, the answers were 100%, 75%, 50%
and 25%.
DP
WDE
WDD
A
0
Water depth (mm)
Water depth (mm)
3
2
1
0
3
2
1
0
0
Water depth (mm)
6
5
4
10 20 30 40 50 60 70 80 90 100 110
Distance between the collectors (m)
DP
WDE
WDD
1
0
0 10 20 30 40 50 60 70 80 90 100 110
Water depth (mm)
4
3
DP
WDE
WDD
0
0 10 20 30 40 50 60 70 80 90 100 110
Figure 1. Water distribution profile (DP), water depth

assay (WDE) and water depth design (WDD) to speeds
at 100% (2A), 80% (2B), 60% (2C) and 40% (2D).
25
Irrigated area (%)

50
75
100
2
3
4
IS
WDE
WDD
25
B
Irrigated area (%)
50
75
100
1
2
3
4
IS
WDE
WDD
5
6
IS
WDE
WDD
Water depth (mm)
100
Water depth (mm)
Irrigated area (%)

50
75
Water depth (mm)
Water depth (mm)
DP
WDE
WDD
25

6
1
2
25
IS
WDE
WDD
C
Irrigated area (%)
50
75
100
D
3
4
5
6
Figure 2. Irrigation suitability (IS), water depth design

(WDD) and water depth assay (WDE) to speeds at
100% (2A), 80% (2B), 60% (2C) and 40% (2D).
During the assays, it was noted some variation in Ws to 40%,

60 to 80% speeds (Table 3). However, for 100% speed, there was a
higher Ws value. ABNT 2 has indicated that the uniformity assay
should not be valid when Ws is greater than 3 m s-1 and, if it is
greater than 1 m s-1, the test accuracy begins to decrease. All Ws
values for the four assays were lower than 3 m s-1, but for 100 and
40% speeds, the values were higher than 1 m s-1.
Ratings for CUC are inverse to moving side speeds, while for
100% speed, it was obtained the lowest CUC (78.57%), thus, it
was classified as bad and regular at 80% speed (CUC =
84.32%) 2.
The best CUC ratings and values were found for speeds at 60%
(CUC = 88.54%) and 40% (CUC = 85.30%), so they were rated as
good. CUD values for the three speeds that were less than
100% showed a good (<70%) rating. At 100% speed, CUD was
rated as bad, so, it did not meet the minimum acceptable levels
by ABNT 2. Sandri et al. 24 have worked with sixteen central pivots
and observed that four of them showed CUD answers lower than
70%. Cainelli et al. 5 recorded that average CUD answers were
greater than 80%, while, Folegatti et al. 12 studied the performance
of a large central pivot with low pressure and obtained 85.9%
values for CUD.
If it is considered the use of the moving side
Table 3. Wind speed (Ws), water depth variation of the experiment in relation to
in crops of high commercial value, the results at
water depth design (WDD), Christiansen uniformity coefficient (CUC),
all speeds are not feasible because all CUC
coefficient uniformity distribution (CUD) and classification according to
values were lower than 90% 4. In extensive crops
NBR 14244 2.
and average root system, the relay speeds at
Speeds (%) Ws (m s-1) DP (%) CUC (%) Classification CUD (%) Classification
60% and 40% showed an appropriate CUC (85%
100
1.80
6.06
78.57
Bad
65.08
Bad
to 90%). During the application of deep root
80
0.80
8.96
84.32
Regular
76.23
Good
crop system, the relay speed at 80% showed
60
0.90
-7.01
88.54
Good
80.79
Good
appropriate levels (80% to 85%). The
40
1.30
5.78
85.30
Good
75.66
Good
327
application on relay speed at 100% does not meet any of these

criteria (<80%) 4.
Based on the sixteen central pivots evaluated by Sandri et al. 24,
only three of them showed acceptable levels for the use on crops
of high commercial value. Five pivots showed appropriate indexes
to be applied in extensive crops and medium root system; while
for deep root system, four pivots showed acceptable levels for
the application and four pivots did not fit the criteria.
Fig. 2 shows irrigation suitability (IS), water depth assay (WDE)
and water depth design (WDD) according to each side moving
relay speed. The moving lateral irrigated 26.34% of the area with
IS larger than WDE (Fig. 2C). This was probably due to a deficiency
on flow, between what was designed and what was applied,
causing an extensive area under water deficit 11.
Irrigation suitability was above 80% with speeds at 100 (83.57%),
80 (91.23%) and 40 (85%). According to Bernardo et al. 4, the
values of a correct irrigated crop of high economic value should
usually be above 90% and when a crop has less economic value,
this answer should be above 80%. In this case, crops with high
economic value can be irrigated only with 80% speed.
Conclusions
Based on the obtained results and the conditions in which this
trial was carried out in the field, it can be concluded that:
1-When water distribution profile was similar to the water depths
of assay and design along the moving side, and after 100 m depth,
the collected water depth reduced intensively.
2-In all tested speeds, the water depth did not differ at 10% water
depth design.
3-The increased speed of moving side provided some decrease
on water application uniformity.
Acknowledgements
To the Pioneer Seeds Ltd at Coxilha Research Station (RS), since
it allowed me to use the lateral move and carry out the assays.
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9
WFL Publisher
Helsinki, Finland
www.world-food.net
Improving water quality in the Nile Delta irrigation network by regulating

reuse of agricultural drainage water
Abd Elhamed Khater 1*, Yoshinobu Kitamura 2, Katsuyuki Shimizu 2, Hiroaki Somura
and Waleed H. Abou El Hassan 4
The United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8553, Japan. 2 Faculty of Agriculture,
Tottori University, Tottori 680-8553, Japan. 3 Faculty of Life and Environmental Science, Shimane University, 1060
Nishikawatsu, Matsue, Shimane 690-8504, Japan. 4 National Water Research Center (WMRI), Delta Barrage NWRC Building,
13621/5, Egypt. e-mail: abdo_khatter@yahoo.com, yoshikita1949@gmail.com, shimizu@muses.tottori-u.ac.jp,
som-hiroaki@life.shimane-u.ac.jp, waleed-hassan@live.com
1
Abstract
In Egypt, the reuse of agricultural drainage water provides an integral supplement to the fresh water supply. Government pumping stations (official)
and farmers small diesel pumps (unofficial) lift water up from drainage canals and direct it back into the irrigation canals for reuse in agriculture,
thereby increasing the countrys available water resources by 12.6%. However, as water passes through the soil and drainage network, it sorbs salts,
agricultural chemicals and other pollutants, leading to differences in the quality of drainage and irrigation water. Therefore, mixing the two water types
deteriorates the overall quality. The common practice in Egypt is to mix drainage into fresh water up to the point, where the salinity of the mixed water
approach 1000 mg L-1. Some canals in the Kafr El-Sheikh Governorate have deficits of fresh water in some months. Therefore, agricultural drainage
channeled back in culverts connect the canal ends with the main drain (Bhr Nashrat) to provide supplemental water. However, this return is not
controlled and the flows are based only on differences in the hydraulic head. Current study evaluated the efficiency of using backflow to supplement
the fresh water. Water supply ratio (WSR) indicator was employed in the analysis within two water supply conditions: (1) Fresh water supply only
and (2) Fresh water supply plus backflow. During the summer 2008, WSR showed an average value of 0.93 and adding the backflow increased it to
1.27. During the following winter, WSR showed an average value of 1.50 and adding the backflow increased it to 1.82. Salinity measurements of water
were taken during the study period at four locations - head, middle, tail and drain. Salinity significantly increased toward the end of the canals.
Excessive backflow is the most serious constraint. The effect of backflow on the salinity was calculated and improved salinity values were obtained
by regulating the backflow. During the summer, backflow significantly deteriorated water quality, but only part of this backflow was actually required
in June and July, just to make up the shortage and not to exceed the requirements. During the following winter, fresh water availability was generally
sufficient. However, backflow still occurred, leading to unnecessary deterioration of the water quality. If backflow is controlled according to the actual
requirements, the water quality would be improved. An improvement in water salinity of over 30% was realised in June and July and by 100% in
May, August, September and all winter months.
Key words: Agricultural drainage reuse, water quality, non-conventional water, irrigation management, water supply indicator, Nile Delta.
Introduction
Egypt water resources: Egypt has a negative water balance. This
negative water balance arises from the use of non-conventional
water resources, which represent 21.3% more water than is
available as fresh water. Annual fresh water amounts to 59.6 billion
cubic metres (bcm) and is derived from Nile river (56.8 bcm), rainfall
along the Mediterranean coast (1.8 bcm) and the non-renewable
Nubian Sandstone Aquifer (1 bcm) 1. Egyptians use 72.3 bcm each
year, where 12.7 bcm represents the negative water balance arising
from agricultural drainage reuse (7.5 bcm), municipal wastewater
reuse (2.9 bcm) and renewable groundwater extraction (2.3 bcm) 2.
Current study focused on reuse of agricultural drainage water,
which represents 12.6% of Egypts annual supply. Egyptians use
Nile water multiple times on its journey through the country
towards the tail end of the system. The main drains discharge a
total of approximately 12.7 bcm of water per year, either directly
into the Mediterranean Sea or into the coastal lakes of Mariut,
Edku, Burullus and Manzala 3. Egyptians do not consider drainage
water as waste, but as a resource in the face of water scarcity.
However, its diversion back into irrigation system for reuse

deteriorates the quality of the water in the canals.
History of agricultural drainage reuse in the Nile Delta: Projects
for the reuse of drainage water date back to the 1870s, when
authorities started to divert two million cubic metres a day from
one of the main drains in the delta into a canal to boost irrigation
supplies during the period of low water prior to the annual Nile
flood 4. In the 1930s, the administration completed a pumping
station on a main drain in the Eastern Delta. This station was
designed to propel water along the drain to its outlet point in the
coastal Manzala Lake. However, officials found that the water
being lifted at the pumping station was of a reasonable quality.
Therefore, they decided to channel it back into one of the branches
of the Nile instead 5. In 1964, after construction of the high Aswan
dam, the introduction of perennial irrigation in the Nile River Delta
and Valley of Egypt led to increased water demand. In the 1970s,
the Ministry of Water Resources and Irrigation (MWRI) developed
329
a specific policy for drainage water reuse. The increasing pressure

on the nations water resources from agricultural expansion and
intensification, as well as agricultural expansion through desert
reclamation, led to mounting claims on irrigation supplies, leaving
some farmers with no option but to use drainage water 2. In the
1980s, the reuse of agricultural drainage water became a formal
Egyptian policy 6. The Ministry of Water Resources and Irrigation
(MWRI) officials recognised that drainage water offered a good,
short-term solution for enhancing the countrys water supply, so
reuse of drainage water will be a key solution for meeting increasing
regional demands for water in the next decades 7. Agricultural
drainage water is a relatively inexpensive and costs less than a US
penny. Other measures for developing new sources of water, such
as desalinating seawater, for example, costs almost one US dollar
per cubic metre 3. By 1984, 2.9 bcm of drainage water was pumped
back into the main canals and the Niles branches for reuse. Since
then, the ministry has further expanded its network of pumping
stations, so that by 2011 the official reuse had reached 7.5 bcm 8.
Types of agricultural drainage irrigation reuse in Egypt: MWRI
has traditionally focused on recycling water from the main drains,
which hold the most water. This is called main reuse. Pumping
stations lift water from these drains and discharge it into the main
canals or branches of the Nile. A major problem results from the
deteriorating water quality in many drains, which are polluted
from municipal and industrial sources. Mixing of this water with
canal water in a number of cases has threatened other water users
located downstream of the mixing points. For this reason, a number
of main drain reuse stations have been closed in the past. This
also poses a substantial problem for the Egyptian company
responsible for provision of drinking water, since the water
treatment plants also draw water from the canals 9. Bringing these
pumping stations into operation again would require great efforts
to reduce the pollution loads. Therefore, alternatives for this type
of reuse have to be developed.
One solution to the reuse of drainage water from larger drains
would be to shift the reuse to smaller, less polluted drains in the
upper part of the system. This intermediate reuse would pump
drainage water to lower order irrigation canals, where it would not
have harmful impacts on downstream domestic water intakes 1.
The branch drains are shallower. Thus, this method of reuse does
not require huge pumping stations and is therefore less costly
and the infrastructure less prone to malfunction. However, MWRI
officials are concerned that intermediate reuse will adversely
affect their main reuse program by reducing the quantity and
quality of water flowing down the branch drains into the main
drains.
The amount of drainage water farmers are recycling back into
the irrigation system is difficult to gauge. When farmers do not
have the option of mixing the drainage water with fresh water
before reuse, they instead apply the drainage water directly to
their fields. This water moves straight from the drain to field, so it
does not directly affect water quality in the canals. However, it
does pass through the soil and thus directly affects soil salinity.
Farmers turn to the drains to irrigate only if their irrigation canals
are empty. This is the practice of what MWRI terms unofficial
reuse and is a strategy for survival 10. The amount of drainage
water each farmer uses is relatively small, but the aggregate impact
is large. The ministry estimates unofficial reuse 2 - 3 bcm a year 8.
330
Salinity measurements: The average level of salinity in the main

drains is 565 mg L-1, but can reach up to 6,000 mg L-1 in northern
parts of the delta 11. With increasing distance downstream, water
quality deteriorates in the river, canals and drains. The drains in
the northern parts of the delta are wide and full of dark, polluted
water with high salinity - on average, over 2800 mg L-1 3. The Niles
large discharge means that salts and pollutants are highly diluted,
leading to salt concentration levels well below those found in the
drains. Therefore, the Niles salinity remains within the bounds of
what is considered to be fresh water (typically under 500 mg L-1),
increasing from 110 mg L-1 at Aswan to just 280 mg L-1 at Cairo, 650
km to the north 11.
The Ministry of Water Resources and Irrigation (MWRI) officials
judge the drainage reuse policy based on good versus bad water.
However, water quality is not an absolute concept, but has
connotations of both scientific understanding and cultural beliefs 12.
In the mid-1990s, a team of international consultants, working
with MWRI conducted an evaluation of Egypts maximum reuse
potential. The teams target salinity level of 1,500 mg L-1 led to a
reuse potential of 8.1 bcm. However, the ministry raised the target
salinity level to 3,000 mg L-1, so the team calculated a significantly
higher reuse potential of 13.3 bcm, almost two times its current
level. The team ultimately recommended that the ministry should
adopt an intermediate threshold value of salinity of 2,250 mg L-1
for drainage water reuse. This would mean that Egypt could
achieve a maximum reuse of 9.6 bcm, almost 30% higher than its
current official reuse 3.
The main objectives of the present study were to check the
efficiency of using backflow to counteract the shortage of fresh
water supply and to find out its effect on water quality
deterioration. Unutilised backflow were calculated and computed
the improved water quality based on utilized backflow only.
Farmers face water shortages and depend partially on backflow
from the main drain, Bhr Nashrat. Previous studies 7, 13, 14 have
indicated a water supply shortage in the Irrigation Improvement
Project areas of El-Wasat (31,500 ha) and El-Manaifa (19,740 ha) in
the Kafr El-Sheikh Governorate of northern Egypt (Fig. 1). In the
study area, MWRI has proposed the use of available drainage
water within the project areas to supplement the fresh water supply
during periods of shortage 1. However, backflow exceeds the
amounts required to make up the shortages. All crops grown in
the area suffer from the increased irrigation water salinity, although
the concentrations of common metals and salts do not cause
problems for soils and plants 13. An environmental management
plan is also needed that will ensure safe reuse of drainage water
after operation of the appropriate mixing locations 13. However,
the ministry is able to track water quality parameters at over 300
sites. These data shows that the salinity of the reused drainage
water has increased from an average of 945 mg L-1 in 1984 to over
1,200 mg L-1 by 1997 15. For project planning and management,
water delivery systems are often evaluated in terms of the desire
to best meet the water requirements of the systems users. As the
ability to access a resource is often a marker of privilege, but with
drainage water reuse, this is not necessarily the case. The most
privileged are those, who have no need for this recycled water 10.
The study to evaluate the efficiency of using backflow to
supplement the water provided by the fresh water supply and to
examine the improvement in water quality when only the utilised
volume of backflow was channeled back into the canals, would
Lake Burullus
Mediterranean Sea
Kafr
El-Sheikh
Gaza
Meet
Yazeed
Cairo
El-Masharka canal
r
ive
eR
Nil
Libya
Bhr Nashrat Drain
M
ee
tY
az
ee
d
Eliwa Canal
Red
Sea
Aswan
High
Dam
Ca
na
l
100
200
N
Sudan
Road
Saafan Canal
Drain
0
N
km
Culverts
Head regulatiors
Culvert
10
Canal
Figure 1. Detailed map of the study area.
help planning and management of irrigation delivery system in

the area.
Study area: The study area is within the Kafr El-Sheikh
Governorate in northern Egypt (Fig. 1), in the western part of the
Nile River Delta (31.21N 30.78E, 31.27N 30.85E) at a mean
elevation of 20.0 m above sea level. One area located in the
downstream regions of the study area is a part of the Nile Delta
with saline groundwater. The salinity of the drainage water is
2,688 mg L-1, so there is limited opportunity to reuse this water
and it is channeled into Burullus Lake through open drains 16. The
study area has a Mediterranean-type climate with hot, dry summers
and cool, wet winters. The maximum monthly average temperature
is about 33.3C in July. The minimum is about 6.6C in January and
the mean annual temperature is 20.2C. Evapotranspiration is
relatively high during summer at 6.1 mm d1 in June and is a
minimum of 1.6 mm d1 in December. The maximum relative humidity
is 74% in November and the minimum is 53% in May. Mean wind
speeds range from 1.0 m s1 in October to 1.7 m s1 in March. The
soil originated from Holocene alluvial deposits, mainly dark
greyish-brown sediments from the suspended materials in the
Nile River 17. The average annual rainfall is low (about 63 mm y1),
occurring for twelve days a year, mostly as occasional
thunderstorms, mainly in winter from November to March. The
main source of irrigation water is from the tail of the main canal,
Meet Yazeed. Three branch canals (Saafan, Eliwa and El-Masharka)
branch from the Meet Yazeed. These canals suffer from inadequate
fresh water supply from head regulators. Therefore, the main drain
(Bhr Nashrat) is used to supply the study area during a shortage.
For our study, we obtained salinity measurements during the study
period from the Water Management Research Institute (WMRI)
of the National Water Research Center (NWRC). These

measurements were taken once every month at four locations:
Head, middle, tail and drain. The quality deteriorated from 400 mg
L-1 at the head and reached a value less than 1,000 mg L-1 at the
drain. Canal water quality deterioration by backflow was within
the acceptable level.
Water supply: As mentioned earlier, the study area suffers from
inadequate fresh water supply owing to its position at the tail of
the Meet Yazeed feeder canal. Managers have proposed
compensating for shortages by using the water from the Bhr
Nashrat drain. The potential water supply for the study area, thus
comprises a fresh water supply and agricultural drainage backflow.
Continuous discharge records are not collected and fresh water
supply data is collected only four times per month. However, gate
openings and water levels upstream and downstream of the head
regulators were continuously monitored with automatic recorders
(OTT Thalimedes, Hydromet-Germany) during the study period
by staff of the WMRI of the NWRC, so it was possible to establish
a relationship between water levels in the canals, gate openings,
and discharges. We converted flow heights into discharges using
individual rating curves for each canal (Table 1, Fig. 2). These
curves were verified at each measurement point by using the flow
velocities and the cross-sectional areas of the canals. The curves
were then used to obtain the discharges, which were summed to
give the monthly fresh water supply. No discharge records are
available for agricultural drainage backflow at the culverts
connecting the canals with the drain and there are no control
gates or valves. However, we measured culvert diameter and
length, and continuously monitored water levels in the canal tail
and the drain with automatic recorders during the study period.
Daily discharges were calculated using HEC-RAS model.
331
Table 1. Equations for discharge from the head regulators.

Canal
Season
Summer
Saafan
Winter
Summer
Eliwa
Winter
Summer
El-Masharka
Winter
Flow status
Submerged
Submerged
Submerged
Submerged
Free flow
Free flow
Equation
y=0.91 GO+0.449
y=2.2143 GO0.441
y=3.1676 GO+0.1074
y=1.4768 GO+0.3322
y=0.0863e1.521GO
y=0.0228e2.3607GO
the following equation:
R2
0.72*
0.79*
0.73*
0.83*
0.76*
0.78*
HL = hen + hf + hex
(1)
where: hen = Entrance loss (m)

hf = Friction loss (m)
hex = Exit loss (m)
The friction loss in the culvert is computed using Mannings
equation, which is expressed as follows:
y=Q h1, where Q=Discharge (m3/s), h=Difference in the hydraulic head (m), e = 2.72, GO=Gate
opening (m) and R2=Coefficient of determination (*: Significant at P<0.05).
hf = L
Qn
AR2/3
(2)
2.5
Qh-0.5
2
1.5
1
+ 0.1074
yy=3.1676*GO
= 3.1676*
GO + 0.1074
R =0.7347
R = 0.7347
2
0.5
0
0
0.2
0.4
0.6
Gate opening (m)
0.8
Figure 2. Discharge calibration for the example of summer irrigation in

the Eliwa canal. Explanation of the parameters and the equations for the
other canals and seasons are shown in Table 1.
The model developed by the United States Army Corps of

Engineers has been applied extensively in calculating the hydraulic
characteristics of rivers. It is designed to perform one-dimensional
hydraulic calculations for a full network of natural and constructed
channels (HEC-RAS 2008). The model requires details of cross
sections of the canal and upstream flow rate for execution. Use of
the energy conservation equation allows calculation of the
velocity and water depth of the given cross section. HEC-RAS
computes energy losses caused by structures, such as culverts,
in three parts. The first part consists of losses that occur in the
reach immediately downstream from the structure, where an
expansion of flow takes place. The second part consists of losses
that occur as flow travels into, through and out of the culvert. The
last part consists of losses that occur in the reach immediately
upstream from the structure, where the flow contracts towards
the opening of the culvert. Culverts connect canal ends with the
main drain (Bhr Nashrat) for the three studied canals were modeled
with HEC-RAS. If the water level at the canal tail was higher than
that at the drain, we assumed that water flowed into the drain.
When the water level in the drain exceeded that in the canal tail,
we assumed that backflow occurred. Data required for each
simulation were culvert length, material, diameter, entrance and
exit loss coefficients, river cross sections, Mannings roughness
coefficient, downstream water level and discharge. The culvert
used in the study area is circular in cross section with a 0.8 m
diameter and 30 m length. For each simulation, discharge was
assumed, actual downstream water level was used in the simulation
and the corresponding upstream water level was calculated from
HEC-RAS model and then compared to the actual upstream water
level. Discharge was repetitively assumed until we obtained
sufficient agreement between the actual and calculated upstream
water levels. Various coefficients were given based on the HECRAS model (HEC-RAS 2008). The head loss, HL is computed using
332
where: hf = Friction loss (m)

L = Culvert length (m)
Q = Flow rate in the culvert (m3 s1)
n = Mannings roughness coefficient
A = Area of flow (m2)
R = Hydraulic radius (m)
The exit energy loss is computed as a coefficient times the
change in velocity head from just inside the culvert at the
downstream end, to outside of the culvert at the downstream end.
The entrance loss is computed as a coefficient times the absolute
velocity head of the flow inside the culvert at the upstream end
(HEC-RAS 2008).
We summed the daily discharges to obtain the monthly
agricultural drainage backflow for each canal.
Water requirements: Data on cropping patterns in the study area
was collected from the agricultural directorate of each canals
command area. Rice, cotton, maize and a variety of summer
vegetables were grown during the summer. Wheat, alfalfa, sugar
beet and a variety of winter vegetables were the main winter crops
(Table 2). Calculation of water demand was based on the
CROPWAT model 18. Water application efficiency (by surface
irrigation) was assumed to be 70% and conveyance efficiency to
be 80% 19. CROPWAT for Windows V.4.3 is application software
for irrigation planning and management, developed by several
scientists 20-22. This model can be considered a useful method to
support decision making to calculate the reference crop
evapotranspiration and crop water requirements. CROPWAT
model requires climatic, crop and soil data as inputs for the model.
The crop water requirements (CWR) are calculated as:
CWR = ET0 Kc
(3)
Table 2. Irrigated cropping pattern during the summer 2008 and

the following winter.
Summer
2008
Winter
2008-2009
Crops
(%)
Rice
Cotton
Maize
Other
Total
Area (ha)
Wheat
Alfalfa
Sugar beet
Other
Total
Area (ha)
Saafan
Canal
55
33
5
7
100
661.5
38
32
23
7
100
661.5
Eliwa
Canal
57
36
6
1
100
656.5
37
28
29
6
100
656.5
El-Masharka
Canal
58
35
5
2
100
628.7
34
32
26
8
100
628.7
where ET0 = Reference crop evapotranspiration, which represents

the potential evapotranspiration of a well-watered grass crop. The
water needs of other crops are directly linked to this climatic
parameter. Values were calculated using the FAO Penman-Monteith
equation based on geographic and geologic information of latitude,
longitude and altitude and monthly climatic average data of the
minimum and maximum air temperature (C), relative humidity (%),
sunshine duration (h) and wind speed (m s1). Climatic data was
obtained from records at the Sakha Station of the Agricultural
Research Center (ARC), Egypt.
A cropping pattern consisting of the crops and their planting
date, crop coefficient data files (including Kc values, stage days,
root depth and depletion fraction) and the area planted (0-100%
of the total area). A set of typical crop coefficient data files are
provided in the program. Planting dates were obtained from the
agricultural cooperatives in the canals command area. Irrigation
requirements were then calculated monthly for the entire study
period (Table 3).
The theoretical crop water requirements in the areas around the
monitored canals were calculated for comparison with the water
supply (Table 4).
Water supply ratio: A water supply indicator was used to measure
the water delivery performance at the study area. The indicator of
water supply rate (WSR) was calculated by the following equation 23-26:
WSR =
QD
QR
(4)
where QD is the amount of water delivered (m3) and QR is the total

irrigation water requirements for the area served (m3). A value of
WSR=1 shows that enough water is being supplied to meet
requirement, a value of WSR<1 shows that supply is less than
requirement, and a value of WSR>1 shows that more water is
being supplied than is required.
where fws is the fresh water supply (m3) and QR is the total irrigation
water requirements for the area served (m3). A value of Percent of
unutilised backflow = 100% shows that enough water is being
supplied to meet requirement, a value of Percent of unutilized
backflow>100% shows that backflow exceeds the shortage
occurring.
When 100% of the backflow was unutilised, we considered the
improved salinity values along the canal are equal to the first
salinity value measured at the entrance of the canal. When no
backflow occurred, we considered the improved salinity values
exactly the same as the actual measurements. When part of the
backflow were utilised, we calculated improved salinity values
based on the utilised backflow using the following equation:
Improved salinity = Actual salinity % Percent of unutilised backflow (6)
The accuracy of these estimates is a matter of debate among
international water experts, but they provide at least some
indication of the improved salinity values.
Salinity: Salinity is a measure of the concentration of salts in
water. Excessive salinity adversely affects crop production and
yields specially for a number of crops such as most vegetables,
maize, alfalfa, flax and a number of fruit trees 1. Therefore, Egypt
must reuse agriculture drainage more sensitively and accurately.
Salinity measurement data was obtained during the study period
from the WMRI of the NWRC. As mentioned before, these
measurements were made once a month at four locations (head,
middle, tail and drain) and are shown in Fig. 3. These values were
evaluated against the performance standards proposed in
guidelines by the Food and Agriculture Organization (FAO):
Salinity of up to 450 mg L-1 in irrigation water will not cause any
problems, 450-2,000 mg L-1 may cause slight to moderate problems
and over 2,000 mg L-1 will cause severe problems for use in irrigation 27.

Crop pattern: The total study area is 1946.7 ha out of which 661.5
ha, 656.5 ha and 628.7 ha were irrigated by Saafan, Eliwa and ElMasharka canals, respectively, for both summer and winter
seasons. Table 2 shows the crop distribution for each canal during
the study period. The cultivation pattern did not differ greatly
among the three branch canals. Rice and cotton were the main
fws + backflow - QR
(5)
100
Percent of unutilised backflow =
crops cultivated during the summer season, collectively, 93% of
backflow
the area is cultivated with these water-intensive
Table 3. Crop water requirements during the summer of 2008 and the winter of
crops. Water consumption by these and other crops
2008-2009.
represent almost twice the water consumption for
Water requirements (m3 ha-1 month-1)
maize (Table 3). This explains the high water
Summer
Winter
requirements in the summer season compared to
Cotton
Rice
Maize Other Alfalfa Wheat Sugar beet Other
the winter season, as shown in Table 4. Farmers are
May 2008
1636
1575
478
1110
free to decide the crops to cultivate in their fields

June 2008
1999
2884
1039
1577
and rice is considered a high value crop, so they

July 2008
1914
2624
1765
1890
Aug. 2008
1714
2290
1648
1928
frequently exceed the governments stipulated

Sept. 2008
1248
1021
1690
maximum limit of 50% area under rice (as a water

Oct. 2008
464
252
591
conservation measure due to high water
Nov. 2008
663
387
344
598
consumption by paddy rice 28). Stricter monitoring
Dec. 2008
370
204
294
365
of farmers and more effective crop management
Jan. 2009
441
379
498
416
Feb. 2009
561
633
683
policies are required 29.

Mar. 2009
866
1017
1011
The main crops in winter were wheat, alfalfa and

Apr. 2009
1117
902
494
sugar
beet, collectively accounting for 94% of the
Total
8511
10 394 4930
8195
4482
3522
3576
1970
Unutilised backflow and improved salinity values: Unutilised
backflow was used to calculate the improved salinity values and
to check the effect of regulating the backflow on improving the
salinity. Unutilised backflow was calculated by the following
equation:
333
Table 4. Fresh water supply, backflow and requirements during the summer 2008 and the following winter.
Season
Summer
2008
Winter
20082009
Month
Fresh
water
supply
May
June
July
Aug.
Sept.
Total
Oct.
Nov.
Dec.
Jan.
Feb.
Mar.
Apr.
Total
1681
1538
1654
1766
1068
7707
870
1199
1086
1086
919
942
1051
7155
Saafan Canal
Fresh
water
Water
supply +
requirement
Backflow
(m3 ha-1)
2209
1602
2180
1970
2354
2035
1766
1804
1068
973
9577
8384
1162
551
1546
583
1119
277
1296
417
1162
564
1394
830
1132
774
8811
3996
El-Mashaka
1546
1394
1564
1693
1255
7451
809
890
803
838
641
735
1038
5755
Eliwa Canal
Fresh
Water
water
requirement
supply +
Backflow
(m3 ha-1)
2443
1492
2173
1863
2544
1913
1669
1932
907
1328
10 419
7843
521
828
906
363
268
803
419
905
575
641
892
893
825
1290
3862
6265
Fresh
water
supply
1499
1324
1491
1737
1175
7225
706
722
603
576
627
754
580
4568
El-Masharka Canal
Fresh
water
Water
supply +
requirement
Backflow
(m3 ha-1)
2340
1520
2136
1853
2224
1900
2121
1656
1486
768
10 306
7697
734
486
840
340
989
254
929
381
832
580
1032
901
811
817
6167
3759
Water salinity based on utilised backflow
Actual water salinity
1500
1000
500
0
1500
Eliwa
EC (mg L-1)
Fresh
water
supply
1000
500
0
Saafan
1500
1000
500
Nov-08
Dec-08
Head
Mid
Tail
Drain
Head
Mid
Tail
Drain
Oct-08
Jan-09
Feb-09
Head
Mid
Tail
Drain
Sep-08
Head
Mid
Tail
Drain
Head
Mid
Tail
Drain
Head
Mid
Tail
Drain
Aug-08
Head
Mid
Tail
Drain
July-08
Summer 2008
Head
Mid
Tail
Drain
Jun-08
Head
Mid
Tail
Drain
May-08
Head
Mid
Tail
Drain
Head
Mid
Tail
Drain
Head
Mid
Tail
Drain
Mar-09
Apr-09
Winter 2008
Figure 3. Actual water salinity and those based on utilised backflow (mg L-1) during the summer of 2008 and the winter of 20082009.
area cultivated. Water consumption by the main winter season

crops was almost half that of cotton and rice. Therefore, water
requirements in the winter season were low compared to
requirements in the summer season (Table 4). Water consumption
by other crops was much lower than that of the three main crops.
Crops composition are random along the canals. However, the
quality of water along these canals is deteriorating towards their
ends. Studies conducted by MWRI Irrigation with saline water
(1,000 mg L-1) showed a reduction in yields of up to 29% compared
to fresh water irrigation and yield declines were most marked for
sensitive crops like maize and alfalfa in comparison to more tolerant
crops like cotton and wheat, which can cope with a higher salinity30.
Alfalfa is considered salt-sensitive crop and it represents 30% of
the area cultivated in the winter season. Hence, significant amount
of alfalfa crop is subjected to saline water and not only in the
study area but also over a million acres in the delta depend either
partially or entirely on drainage water for irrigation 31. Stricter
measures should be taken to redistribute the crops along the canals
334
based on their sensitivity to salinity, as this would increase the

yield and profit from the crops cultivated.
Water supply and requirements: Fresh water supply, agricultural
drainage backflow and irrigation water requirements during the
summer of 2008 and the following winter are listed in Table 4 for
the three branch canals. This table permits two important
comparisons: fresh water supply versus irrigation water
requirements and fresh water supply plus backflow versus
irrigation water requirements. Water supplied was higher in
summer than in winter, as water requirements for summer crops
were more than those for winter crops (Table 3). Farmers have
been free to decide what crops they wish to plant and many farmers
have turned towards profitable but water-intensive crops like rice
as shown in Table 2. In the summer season, some farmers cannot
irrigate their fields because not enough water flows down their
irrigation canals. A national survey indicated that 71% of farmers
find the water in their irrigation canals to be insufficient during
summer months, when pressure on water supplies Table 5. WSR values during the summer of 2008 and the following winter.
is most intense 32. Rice was considered the most
Saafan Canal
Eliwa Canal
El-Masharka Canal
Month
WSR
WSR
WSR
WSR
WSR
WSR
water-intensive crop in the study area and the Season
(cond.
1)
(cond.
2)
(cond.
1)
(cond.
2)
(cond.
1)
(cond.
2)
higher water requirements occurred in June and
1.04
1.64
0.99
1.54
May
1.05
1.38
July as shown in Table 3.
June
0.78
1.11
0.75
1.17
0.71
1.15
During the summer season, if only the fresh Summer
July
0.81
1.16
0.82
1.33
0.78
1.17
water supply was used, water excess occurred 2008
Aug.
0.98
0.98
1.01
1.16
1.05
1.28
Sept.
1.10
1.10
1.38
1.46
1.53
1.93
in September and deficiency occurred in June
Average
0.92
1.14
0.95
1.33
0.94
1.34
and July. The additional water supplied by
1.55
1.59
1.45
1.51
Oct.
1.58
2.11
backflow, occurring at all months, boosted the
Nov.
2.06
2.65
2.45
2.50
2.13
2.47
water supply but was required to augment the
Dec.
3.92
4.04
3.00
3.00
2.37
3.89
shortage of fresh water supply only in June and Winter
Jan.
2.60
3.11
2.00
2.16
1.51
2.44
Feb.
1.63
2.06
1.11
1.11
1.08
1.43
July. The fresh water supply in June and July 2008-2009
Mar.
1.13
1.68
0.82
1.00
0.84
1.15
for the three branch canals was less than that in
Apr.
1.36
1.46
1.26
1.56
0.71
0.99
May and August, while requirements for
Average
1.79
2.20
1.49
1.62
1.22
1.64
irrigation water was higher (Table 4). The
shortage was due to an ineffective water
delivery plan and intensive water use by upstream users to meet June and July. The second is that fresh water contamination by
the high water consumption at these months. The total fresh water agricultural drainage water occurred to a significant degree in the
supply for each of the three branch canals was somewhat lower system, as backflow was mixed with the fresh water supply and
than the requirements. However, backflow was more than sufficient channeled out again in the culvert to the drainage canal. During
to counteract the shortage of fresh water supply.
the winter season, when only the fresh water supply was used,
During the winter season, an excess of fresh water was supplied the WSR values were greater than 1 (1.79, 1.49 and 1.22) for the
to the three branch canals at most times, this was to secure a three branch canals, while additional water supplied by backflow
hydraulic head required for gravity irrigation for winter crops under raised the values (2.2, 1.62 and 1.64). These values indicate that
canal systems with large conveyance capacity designed to provide backflow increased water supply by more than 100%, even though
maximum water requirements of the water-consuming summer there was no need of the backflow. Also, fresh water waste occurred
crops in the extremely flat topography. This indicates a lack of to a significant degree in the system, as the fresh water supply
utility of any backflow supply except for the Eliwa canal during was mixed with backflow and channeled out again unused into
March and for the El-Masharka canal during March and April. the pipe to the drainage canal.
However, backflow occurred in most months. The total fresh water
supply for each of the three branch canals was approximately Impact on salinity: Salinity measurements of water are shown in
60% greater than the requirements and backflow raised the water Fig. 3 for the three branch canals during the summer season of
supply to approximately twice of the requirements. This situation 2008 and the following winter season. This figure shows two
deteriorates water quality.
salinity values along the studied canals: One curve shows salinity
Farmers channel the vast majority of Nile water (90%) to cultivate values as measured at the field representing the use of the fresh
their fields and any water not taken up by growing crops infiltrates water supply and the total backflow. The second curve shows
the soil and passes into the drainage system. A recent assessment calculated salinity values representing the use of the fresh water
at the main canal level showed that 53% of the annual irrigation supply and the actual utilized backflow (Fig. 3). Due to the actual
water supply entered drainage systems and saline groundwater field conditions and existing culverts connecting the canal end
sinks, indicating an oversupply 14. This waste of water can only with the drainage canal, water salinity is increasing towards the
be reduced if farmers are motivated not to demand more water end of the canal. The most fortunate farmers are those, who have
than necessary 33. In the study area, 27% of water was channeled no need for drainage water in the first place, as they have enough
out in the culvert to the drainage canal during the summer and good quality irrigation water. However, those at the end of the
82% during the winter. Water excess over water requirements canals have access to enough water, but of bad quality caused by
means that backflow channeled in the pipe as a supplement is mixing of drainage water, as drainage water is different in quality
simply drained back again into the drainage canal after mixing to irrigation water. When water passes through the soil and
with the canal fresh water, leading to an unnecessary loss of the drainage network, it picks up salts, agricultural chemicals and
fresh water and deterioration of the canal water quality.
other pollutants. Drainage water is not only a substandard source
but a symbol of marginality 10.
Water supply ratio: Calculated values for the water supply ratio
During the summer season, backflow occurred during all summer
(WSR) are listed in Table 5. During the summer season, when only months and exceeded the requirements. Based on the actual field
the fresh water supply was used, the average WSR values were measurements the salinity level increased from 400 to 1,000 mg
less than 1 (0.92, 0.95 and 0.94) for the three branch canals, while L-1 that could affect plants with moderate and high salinity
additional water supplied by backflow raised values to greater sensitivity, such as maize and vegetables. Backflow deteriorated
than 1 (1.14, 1.33 and 1.34), respectively. These values indicate the quality unnecessarily. Control of the backflow according to
two important points: The first is that backflow increased the the actual requirements would improve the water quality. We
water supply by 40% while only 9% was required and backflow calculated the amount of unutilised backflow, as shown in Table
accrued during all summer months while it was required only in 6. During May, August and September, 100% of the backflow was
335
Table 6. Unutilised backflow as a percentage of total backflow

during the summer of 2008 and the following winter.
Season
Summer
2008
Winter
2008-2009
Saafan Canal
Unutilised
Month
backflow
(%)
May
100
June
33
July
46
Aug.
0
Sept.
0
Average
64
Oct.
100
Nov.
100
Dec.
100
Jan.
100
Feb.
100
Mar.
100
Apr.
100
Average
100
Eliwa Canal
Unutilised
backflow
(%)
100
40
64
100
100
87
100
100
100
100
100
0
100
100
El-Masharka Canal
Unutilised
backflow
(%)
100
35
44
100
100
85
100
100
100
100
100
47
0
100
unutilised, while in June and July, some of the backflow was still
unutilised. We calculated the salinity values based on the utilised
backflow while omitting any effect of unutilized flows, as shown
in Fig. 3. Water salinity values compared to actual measurements
at the end of the canal showed an improvement in salinity by 30%
in June and July and 100% in May, August and September.
During the winter season, backflow was not required for any
winter months. However, it still occurred. Based on the actual
field measurements, the salinity deteriorated from 400 mg L-1 and
reached values of 1,000 mg L-1 that could cause slight to moderate
problems to crops, especially the sensitive ones like alfalfa and
most vegetables. Backflow, therefore, also deteriorated the quality
unnecessarily in the winter season. Again, control of the backflow
would improve the water quality. Unutilised backflow was
calculated, as shown on in Table 6. During all winter months,
100% of the backflow was unutilised, except in March for the ElMasharka canal, where only some of the actual backflow was
unutilised (Table 6). We calculated the salinity values based on
utilised backflow while omitting any effect of unutilised flows, as
shown in Fig. 3. Salinity values compared to actual measurements
at the end of the canal reduced by 100%. Using utilised backflow
decreased the water salinity to values under 500 mg L-1, except at
June and July were little more than 500 mg L-1. The recycling of the
saline and often polluted drainage water impacts the quality of
water flowing through Egypts irrigation network. A farmer, who
can access a better quality of water for irrigation will achieve
higher yields and profits. Irrigation with saline water is possible
while still maintaining good levels of production (so-called
biosaline agriculture), but this requires careful land management
practices, which although already piloted have yet to become
widespread throughout the country 34, 35.
Conclusions
Current study investigated the efficiency of using agricultural
drainage backflow to supplement the fresh water of the Nile Delta
irrigation network and its effect on the water quality in Kafr ElSheikh Governorate. We calculated the water supply ratio (WSR)
as an indicator to compare the results based on fresh water use
with those based on fresh water plus backflow. During the summer
season (May-September) of 2008, backflow significantly
336
deteriorated water quality, while it was actually required only in

June and July and only to make up the shortage, not to exceed the
requirements. During the following winter season (October-April),
water availability was generally sufficient from the fresh water
supply. However, drainage backflow occurred and unnecessarily
deteriorated the water quality. The use of fresh water supply plus
backflow perfectly supplemented all fresh water supply shortages.
However, the backflow exceeded the requirements. Therefore,
control of the backflow according to the actual requirements would
improve the water quality. An improvement of over 30% in June
and July was realised and 100% improvement was realised in May,
August, September and all winter months.
Excessive backflow and high water levels in the drainage canal
indicate that upstream users withdraw more water than they need,
exceeding the crop consumption and unused water was drained
out to the drainage canals. Therefore, our study area faced
freshwater supply shortages and excessive saline backflow. The
freshwater supply approach used in the area should therefore be
modified to improve the management of water distribution. The
farmers must also be motivated not to demand more than
necessary. The canal systems were designed to provide the
maximum water requirements of the water-consuming summer
crops, but this has led to an excess freshwater supply in the winter
season. Further measures should be considered to improve the
management of water distribution.
Cropping pattern along the canals indicates random cultivation
of crops, as farmers are free to decide which crops to cultivate.
The mixing ratio of saline backflow water is higher at the end of
the branch canals. Here, a better organised cropping pattern should
be adopted based on the salt tolerance of each crop (i.e., sensitive
crops should be planted at the canals head and tolerant crops at
the end).
The calibrated equations for fresh water supply and HEC-RAS
calculations for backflow provided a generally good fit to the
observed data. However, direct measurements would improve the
accuracy of the calculations.
Acknowledgements
The authors are grateful for a post GCOE project (Dryland Sciences)
funded by Tottori University to support this research. This study
was partly carried out under the Science and Technology Research
Partnership for Sustainable Development (SATREPS) project,
Sustainable Systems for Food and Bioenergy Production with
Water-Saving Irrigation in the Egyptian Nile basin. The Japanese
Government (Ministry of Education, Culture, Sports, Science and
Technology) supported the first author by providing him a
scholarship (20112014) for the first author. The authors thank
the Water Management Research Institute (WMRI) of the
National Water Research Center (NWRC) in Egypt for providing
required data.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Antioxidant potential and secondary metabolite content of grape berries influenced by

microclimate
Mustafa Ozden
Department of Horticulture, Agriculture Faculty, Harran University, 63040 Sanliurfa, Turkey. e-mail: mozden70@gmail.com
Abstract
In this study, the effects of the microclimatic changes around the grapes on quality parameters in Shiraz berries were investigated. Variations in
microclimate condition of grapes were created by the four different shading treatments: fully exposed (Exp), natural shading (Nsh), 40% of shading
(V40), and 60% of shading (V60). During the experiment, illuminance (lux), ultraviolet light (W/cm2), temperature (C), and relative humidity (%) of
each microclimate was continuously recorded by data loggers placed in adjacent grapes perceiving the same microclimate conditions. Shading
treatments were set from the initial stage of ripening, and maintained to harvest. Subsequently, influence of the shading treatments on berry quality
characteristics was measured by determining physicochemical composition, phenolic accumulation, and antioxidant potential of berries at harvest. A
quantitative increase in berry weight (g), pH, and titratable acidity (%) of its juice were observed in shaded grapes, but they decreased in Exp grapes,
particularly for total soluble solids (24.3 0.5 Brix). Similarly, total phenolic (1795.5 40.1 mg GAE/kg fwt), and flavonoid (310.9 8.7 mg CE/
kg fwt) contents of berries exposed to solar radiation were higher than grapes grown under shading (V40, V60) conditions. The response of total
antioxidant potential to shading treatments was variable and depended on shading level. Fully exposed grapes may not be desirable for some reasons
including low anthocyanin accumulation, fast ripening process, berry desiccation, and crop loss due to sunburn. Even if exposed berries reached the
highest phytochemical content and antioxidant potential, they are not enough for grape berry quality standards normally accepted. Under the study
conditions, 40% of shading may meet the expected grape berry quality. Microclimatic changes in fruit zone of grapevines significantly affected berry
quality characteristics, secondary metabolite content and antioxidant potential of grape tissues. Therefore, modifications in microclimate conditions
of fruit zone may receive considerable research interest in the future studies under harsh climate conditions.
Key words: Vitis vinifera L., solar UV radiation, shading, phytochemical content, antioxidant potential.
Introduction
Sunlight and temperature at ambient levels are essential for the
process of photosynthesis. However, high light, high temperature,
and high ultraviolet (UV) radiation have been regarded as elements
of environmental stress variables caused to solar injury in plants
or fruits 1. To cope with adverse effects of these environmental
stresses, plants have evolved morphological, physiological, and
biochemically adaptive responses such as thicker epidermal cell
layers, excessive biosynthesis of phenolics and flavonoids. These
natural bioactive compounds are important because of their
contribution to the grape quality characteristics and ultimately to
human health 2, 3. Epidemiological studies have shown a positive
correlation between fruit and vegetable consumption and reduced
incidence of chronic and degenerative diseases 4, 5. These
antioxidants have scavenging properties against oxygen free
radicals, offering protection to lipids, proteins and nucleic acids 6.
Therefore, beneficial human health effects of grape natural
bioactive compounds have provided incentive to many
researchers to study composition of active components and their
contents in grape.
In the past two decades, there had been significant advances in
analytical chemistry and secondary metabolites of grapes that are
affected by variety, climatic conditions, agricultural and
environmental factors. Researchers began to understand the
importance of grapevine or grape shading 7. It was reported that
anthocyanin accumulation was more depended on temperature
338
rather than light exposure 8-10. Up to now, many studies have been
reported related to microclimate of grapes affected the degree of
exposure to solar radiation. It has been concluded that changing
in the light and temperature of the microclimate around grapes
caused differences in quality, quantity, and content of secondary
metabolites of berries. Relatively, low light intensity and moderate
daytime grape temperature gave full berry color and best
anthocyanin accumulation 11-14. Although we have now better
understanding of the impact of fully sun exposure on grape quality,
little is known about influence of microclimate changes in fruit
zone of grapevine on grape berry quality characteristics, secondary
metabolite accumulation, and antioxidant activities.
This study was undertaken to determine how shading treatments
affect physicochemical composition, secondary metabolite
accumulation as bioactive compounds, and antioxidant potential
of grapes growing in hot and sunny climate of Turkey. When
quality characteristics of berries was determined by measuring
berry weight, total soluble solids, pH, titratable acidity, and hue
angle, total bioactive content of samples was detected with total
phenolics, flavonoids, and anthocyanins. In addition, antioxidant
potential of berry extracts was assessed by the 2, 2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity, the ferric
reducing capacity (FRAP) and phosphomolybdenum reducing
capacity.
Data collection of microclimates: Measurements of the microclimate data were made from the beginning of the experiment until
to the harvest continuously. Data loggers (TR-74Ui Data Loggers,
Japan) were programmed to simultaneously measure and record
of illuminance (lux), solar UV radiation (W/cm2), temperature (C)
and relative humidity (%) of each microclimate condition at fruit
zone. Readings were made in a 30 - minute interval and the hourly
average was calculated. To do that, the probes of the data loggers
were fastened and positioned vertically on the trellising wires
next to the clusters to measure microclimate conditions around
the grapes. Hourly average readings of the daytime during the
experiment are given in Fig. 1. Each data point represents the
mean of forty independent readings in a forty day of experimental
shading period.
Grape sampling and extraction: When berries of Nsh grapes
reached at commercial maturity level, grapes grown under different
microclimate conditions were harvested on the same day. Prior to
analysis, whole grape clusters or 100 - berry weight of the samples
representing shading treatments were determined. Subsequently,
about 10 g of berry tissue homogenized in 50 ml of ddH2O and the
liquid level was adjusted to 100 ml for measuring pH and titratable
acidity. Total soluble solids (Brix) was measured by using a digital
Nsh
Exp
A
A
40
Temperature (C)
V60
38
36
34
32
30
27
Relative humidity (%)
25
23
21
19
17
15
13
11
Solar illuminance (1000 lux)
Shading treatments: When berries reached ~5 mm in diameter,

experimental shading treatments were initiated with shoot
positioning, leaf orientation or polyethylene shading nets in order
to create shaded and fully exposure. In artificial shading
treatments, grapevines were shaded from the initial stage of
veraison, onset of ripening (June 20) to harvest (July 30) with a
green polyethylene net with two different mesh sizes holding 40%
and 60% of the direct sunlight. Polyethylene shading nets placed
over grapevines were supported by the shade frames, so that
there was no contact between the cluster and shading nets. Briefly,
to characterize berry exposure level under natural light conditions,
four shade treatments were used as follows: 1 - exposed (Exp),
(berries exposed to direct sunlight); 2 - natural shading (Nsh),
(control - berries sheltered by the foliage); 3 - 40% vine shading
(V40), (vines shaded by a 40% shading net); and 4 - 60% vine
shading (V60), (vines shaded by a 60% shading net). Fully exposed
treatments were shoot positioned or leaf oriented weekly in order
to maintain sunlight exposure. The experiment was arranged in a
randomized complete block design, consisting of three replications
with twelve vines per replication, was used for four shading
treatments. Each treatment was replicated at least three times on
three adjacent rows.
V40
42
140
120
100
80
60
40
20
0
3.0
Solar UV intensity (mW/cm2)

Plant material and experimental conditions: This study was
conducted in a 10 year old vineyard of Vitis vinifera L. cv. Shiraz
grown on SO4 (V. berlandieri x V. riparia) rootstock at Harran
University, Turkey (3710N, 3859E) in 2012/2013. Row and vine
spacing were 3.0 and 2.5 m, respectively, with the rows orientated
in a north - south direction. Vines were trained on bilateral cordon
system (0.4 m above ground), and the foliage was supported by
two wires. Plants were irrigated with a drip system. The climate of
the experimental site is classified as Mediterranean type and
experiences dry, hot, and sunny conditions without any significant
precipitation during the summer months.
2.5
2.0
1.5
1.0
0.5
0.0
10
12
14
16
Local standard time (h)
18
20
Figure 1. Daily changes in relative humidity (%) - A,

temperature (oC) - B, illuminance (lux) - C, and solar
UV intensity (mW/cm2) - D in microclimatic fruit zone
of Shiraz created by shading treatments, namely, SunExposed (Exp), Natural shading (Nsh), 40% vine
shading (V40), and 60% vine shading (V60) from prior
to veraison to harvest.
refractometer, 0.1 N sodium hydroxide (NaOH) solution was added

to the homogenized berry juice until to the endpoint of pH 8.2.
Titratable acidity results were expressed as the percentage of a tartaric
acid reference. For sample extraction, berries were homogenized
in an ice cold blender after removal of seeds and a 25 g of the
homogenate were macerated in 100 ml of ethanol containing 0.1%
HCl and set aside overnight in darkness. Then the extract was
filtered over Whatman No. 1 paper under vacuum and the residue
was repeatedly extracted with the same solvent until it was
colourless. One part of the extracts was separated for determining
anthocyanin content and the other remaining portions concentrated
by a rotary evaporator at 50C and were used for determining total
339
phenolic, flavonoid contents, and antioxidant potential of samples.

Determination of total phenolics, flavonoids and anthocyanins:
Total anthocyanin content of samples was determined using the
pH differential method 18. Anthocyanin content was estimated in
a spectrophotometer (UV - visible spectrophotometer, Shimadzu 1601)
at 510 and 700 nm buffers at pH 1.0 and 4.5 17, 18. All values were
estimated as malvidin-3-O-glucoside using a molar extinction
coefficient of 28,000. Total phenolic concentrations in the ethanolic
berry extracts were determined by using a modified Folin-Ciocalteu
colorimetric method 15. Total phenolic content were obtained from
the calibration curve prepared with gallic acid (GAE) (20 - 250 mg/l)
and expressed as mg GAE/kg of fresh weight. Total flavonoid
concentration of the extracts was determined by the modified
colorimetric method 16. Total flavonoid content obtained from the
calibration curve prepared with catechin (20 - 250 mg/l) and
expressed as mg of catechin equivalents (CE) per kg g fresh weight.
Determination of antioxidant potential: Total antioxidant activity
of different ethanolic berry extracts was measured by DPPH free
radical scavenging assay 19 and expressed as percentage of
inhibition. IC50 values of the extracts, i.e. concentration of extract
necessary to decrease the initial concentration of DPPH by 50%
was calculated. However, total antioxidant capacity of samples
was determined by the ferric reducing power assay (FRAP) and
phosphomolybdenum antioxidant assay. The total antioxidant
capacity of samples was determined by the ferric reducing
antioxidant power (FRAP) assay 20 and phosphomolybdenum assay21.
FRAP assay was a measurement in reduction of Fe3+(CN-)6 to
Fe2+(CN-)6, resulting in the formation of Perls Prussian Blue
complex following the addition of excess ferric ions (Fe3+). One ml
of grape extract (20 g/ml) was mixed with 2.5 mlL of 0.2 M (pH 6.6)
phosphate buffer and 2.5 ml of 1% potassium ferricyanide [K3Fe(CN6)].
The mixture was incubated at 50C for 20 min, then rapidly cooled
and mixed with 2.5 ml of 10% trichloroacetic acid and centrifuged
at 1500 rpm for 15 min. An aliquot of the supernatant (2.5 ml) was
diluted with distilled water (2.5 ml) and then freshly prepared 0.5
ml of 0.1% FeCl3 was added and allowed to stand for 10 min. The
absorbance was measured at 700 nm. Butylhydroxytoluene (BHT)
in a concentration of 20 - 250 mg/ml was used as standard for
construction of the calibration curve and reducing power was
reported as BHT equivalent per mg/ml of extract. The
phosphomolybdenum assay 2 is based on the reduction of Mo
(VI) to Mo (V) by the extract and subsequent formation of a green
phosphate - Mo (V) complex. A 0.3 ml of extract (20 g/ml) was
mixed with 3 ml of reagent solution (0.6 M H2SO4, 28 mM sodium
phosphate, 4 mM ammoniummolybdate) in Falcon tubes. The tubes
were incubated for 90 min at 95C. Thereafter, the mixture was
cooled to room temperature and the absorbance was read at 695
nm against blank. Ascorbic acid (20 - 250 mg/ml) was used as
standard for construction of the calibration curve and antioxidant
capacity was expressed as mg of ascorbic acid equivalents (AE)/

ml extract.
Statistical analysis: All data were analyzed by analysis of variance
(ANOVA). Shading treatment effects were compared using mean
separation by LSD test. All analyses were performed using the
Proc Glm function of SAS (SAS Institute, Cary, NC).
Cluster microclimate: Shading treatments changed the
temperature, relative humidity, solar light intensity, and UV
intensity in microclimate conditions of grapes (Fig. 1A-D). The
mean values of the readings at solar noon, temperature around
the exposed clusters were 4.5%, 6.8%, and 10% higher than
temperatures measured around the clusters grown in Nsh, V40,
and V60 shading treatments, respectively (Fig. 1A). As expected,
increases in shading degree lead to an apparent increase in relative
humidity at microclimate conditions of fruit zone.
The highest and lowest relative humidity readings were
recorded as 18% and 14% around the grapes grown under V60
and Exp shading treatments, respectively, in the middle of day
time (Fig. 1B). On the contrary to the relation between temperature
and relative humidity, illuminance and solar UV radiation were
reduced at microclimate fruit zones, when shading degree increased
(Fig. 1C-D). The highest solar UV radiation value was recorded
around the exposed clusters as 2.55 mW/cm2 which was equal to
10.2 UV index regarded as highly dangerous level for living
organisms 22. Based on the data set recorded, it is anticipated that
shading treatments alter microclimate conditions considerably
around grapes by reducing evapotranspirational or transpirational
water loss, and by blocking solar UV light transmission through
the shading nets.
Physicochemical characteristics of grape berries: Impact of
microclimatic changes on fresh berry weight and phytochemical
composition of berries are given in Table 1. Shading treatments
had significant influence on cluster or berry weight, soluble solids,
titratable acidity, and hue angle. The heaviest clusters or berries
were weighed in V40 shading treatment (251.4 7.7 g), followed
by V60 (227.5 6.4 g), Nsh (183.1 10.2 g), and Exp (113.0 7.3 g).
The V40 shaded grapes were 45% heavier than exposed grapes in
comparison with Nsh (27%) and Exp (10%) grapes (Table 1). Despite
these differences in cluster weight, berries of exposed grapes
reached the highest level of total soluble solids (24.3 0.5 Brix),
followed by a descending order Nsh (22.1 0.5 Brix), V40 (20.8
0.4 Brix), and V60 (18.8 0.5 Brix) shaded berries harvested on
the same day time. The pH of samples showed considerably less
response to shading treatments than did Brix and titratable acidity
as shown in Table 1. Except for V60 shading treatment, no
significant (p > 0.05) changes were found in the pH values of any
of the shade-treated berries. While percentage of titratable acidity
Table 1. Physicochemical characteristics of grape berries from different fruit zone microclimates.
Shading
Treatments
Exp
Nsh
V40
V60
Cluster
weight (g)
113.0 7.3d
183.1 10.2c
251.4 7.7a
227.5 6.4b
100-Berry
weight (g)
120.0 5.8d
137.0 5.5c
159.5 2.1a
141.1 5.6b
Soluble
Solids (oBrix)
24.3 0.5a
22.1 0.5b
20.8 0.4c
18.8 0.5d
pH
3.80 0.02a
3.89 0.02a
3.83 0.01a
3.70 0.04b
Ti tra table
Acidity (%)
0.33 0.03c
0.37 0.02c
0.51 0.05b
0.59 0.01a
Hue angle
( h o)
21.98 0.2c
23.47 0.2b
20.55 0.2d
24.95 0.2a
Values representing means SE (n = 3) with lowercase letters on the same row indicate significant differences (P 0.05).
340
of samples was highest (0.59 0.01%) in V60-treated berries, it

was lowest in exposed berries (0.33 0.03 %) (Table1).
Hue angle of samples, indicating colour appearance of berry
extracts, was determined as the physical characteristic. The berry
juice colour of V40-treated berry extracts had dark red and the
lowest hue angle (h) value as 20.55 0.2. Berry juice colour of
exposed grapes had red and 21.98 0.2 h, Nsh had light red and
23.47 0.2 h, and V60-treated berries had lighter red and 24.95
0.2 h (Table 1). Previous reports indicate that microclimate change
around the grapes is an important factor determining the growing
condition of berries. Therefore, some degree of grape exposure to
solar UV radiation may change light and thermal conditions around
grapes with consequence berry phytochemical characteristics 11, 13, 14.
Our studies indicated that artificially shaded grapes (V40 and V60)
had significantly higher berry weights, titratable acidity, and pH
values than fully exposed or naturally shaded grapes as control.
Also, heavily shade treatment (V60) delayed berry ripening period
based on commercially harvest criterion, but it was an opposite
situation in berries of exposed clusters (Table 1). These results
agree with previous work examining the influence of microclimate
changes in fruit zone of vines on berrys physical characteristics 14, 23, 24.
Grapes grown under excessively sunlight exposure might be
undesirable for some reasons such as berry desiccation and sun
burn (Fig. 2A). These negative influences of excessive sunlight
exposure could be attributed to heat stress resulting from high
light and solar radiation 7, 8. Therefore, modification of microclimate around grapes appeared to be important to reduce adverse
effects of fruit exposure that could be recommended for hot, sunny,
and dry climates where fruit quality is a major priority.
Figure 2. Shiraz grapes grown under various microclimate

conditions created by shading treatments.
A; sun-exposed grape, B; naturally shaded grape, C; 40% shaded grape,
D; 60% shaded grape (Photograph was taken on the same day of harvest).
Phytochemical contents of berries: Some phytochemical contents

of berries grown under different shading treatments are shown in
Table 2. The results confirmed that influence of the shading
treatments on phytochemical content of grape berries was highly
variable with each sample ranging from 903.3 to 1261.9 mg/kg for
total anthocyanin, from1182.3 to 1795.5 mg/kg for total phenolic,
and from 211.8 to 310.9 mg/kg for total flavonoids. Grape Exp
berries exhibited the highest anthocyanin (1261.9 mg malvidin-3O-glucoside/kg fwt), phenolic (1795.5 mg GAE/kg fwt), and
flavonoid (310.9 mg KE/kg fwt) contents among the assayed
samples (Table 1). On the contrary, grapes grown under V60
treatment comprised the least amount of phytochemicals among
the samples tested (Table 2). Total anthocyanin, phenolic, and
flavonoid concentrations of berries shaded with V60 were 903.3,
1297.7 and 250.1 mg/kg, respectively. Although natural shaded
berries had the second highest anthocyanin (1352.7 mg/kg),
phenolic (1636.8 mg/kg), and flavonoid (235.3 mg/kg) contents,
V40-shaded berries contained relatively lower amounts of
anthocyanin, phenolic, and flavonoid as 1064.5, 1182.3 and 211.8
mg/kg, respectively (Table 2).
Grape berries of the shaded vines (V40 - V60) tended to have
lower phytochemical content than natural shaded or sun-exposed
berries. Artificial shading treatments (V40, V60) caused a reduction
in polyphenol synthesis particularly in total anthocyanin content
of berries grown under V60 treatment. However, grapes exposed
to direct sunlight would suffer from some type of degradation;
desiccation or sunburn caused the loss of yield as shown at Fig.
2A. Except for the total anthocyanins, total amounts of phenolics,
and flavonoids were clearly lower in the berries of shaded grapes
(V40, 460) than in exposed berries (Exp) (Table 2). Total anthocyanin
concentration decreased significantly in berries exposed to direct
sunlight in comparison with naturally shaded berries. As it was
reported in literature, high degree of grape exposure caused to
high berry temperatures in hot and sunny climates. Due to inhibition
of anthocyanin metabolism, this type of harsh environmental
condition was not conducive to optimal anthocyanin
accumulation25.
Depending on density of shading, concentrations of bioactive
compounds decreased in artificially shaded grape berries 25-27.
Especially, V60-shaded grapes had significantly lower amount of
bioactive compounds than V40-shaded grapes. It is clearly stated
in the literature that some enzymes of the shikimic acid pathway
being mostly responsible for synthesizing of phenolic compounds
were light-stimulated. That might be one of the reasons of low
phytochemical accumulation in grape berries grown under artificial
shading conditions 27. Secondly low light intensity might cause
the reduction in photosynthetic rates, leading to less sugar
accumulation and possibly delayed ripening. In our climatic region
pressurized by highly stressful solar radiation, shading of
grapevines might be beneficial because the photosynthesis is
more efficient under diffuse light conditions 28. In our case, the
question arisen was how much shading is really necessary for
good berry quality characteristics rather than be concerned about
a lack of light intensity. The data set obtained implicates a 40% of
grapevine shading may meet the grape berry quality expectations
without negative influences of excessive grape exposure under
hot and sunny climates.
Table 2. Physicochemical composition of grape berries from different

fruit zone microclimates.
Shading
Treatments
Exp
Nsh
V40
V60
Total Anthocyanins
(mg Malvidin-3-Oglucoside/kg fwt)
1261.9 28.0a
1352.7 18.9b
1064.5 38.0c
903.3 20.0d
Total Phenolics
(mg GAE/kg
fwt)
1795.5 40.1a
1636.8 25.1b
1182.3 44.9d
1297.7 14.9c
Total Flavonoids
(mg CE/kg
fwt)
310.9 8.7a
235.3 5.2b
211.8 4.0c
250.1 3.0d
Values representing meansSE (n=3) with lowercase letters on the same row indicate significant differences at P 0.05.
Antioxidant potential of berry extracts: Total antioxidant

activity of grape berry extracts is shown in Fig. 3A-B, and
expressed as the median effective dose IC50 values (Fig.
3B). The lowest IC50 value was found in berry samples taken
from Exp shading (59.06 1.6 mg/l), followed by V40 shading
(67.65 1.3 mg/l), Nsh shading (76.84 1.6 mg/l), and V60
shading treatments (83.5 1.3 mg/l). There is an inverse
proportion between antioxidant activity and IC50 value.
341
60
Nsh
Exp
V40
V60
50
40
30
20
10
0
0
50
100
150
200
IC50 (mg/ml)
80
BB
a
b
c
70
d
60
50
40
30
20
Nsh
Exp
V40
Shading treatment
200
550
180
AA
Antioxidant capacity by
FRAP assay
160
c
d
140
120
100
80
60
250
90
Reducing power mg BHT/ml extract
Reducing power mg AE/ml extract
DPPH inhibition (%)
70
V60
500
BB
a
b
Antioxidant capacity by
phosphomolibdenum assay
450
c
400
d
350
300
250
h
Nsh
40
Exp
V40
Shading treatment
60
V60
Figure 3. Antioxidant activity of Shiraz berry extracts. A-DPPH

antiradical scavenging activity (%) of berry extracts of grapes
grown under Nsh, Exp, V40, and V60 treatments. B-IC50 (mg/
ml) value of grapes grown under the shading treatments. Data
presented here are means of three replicates with SE. The
letters on the top of the bars, which are the same, indicate that
there is no statistically significant difference at P 0.05 level
based on LSD test.
Figure 4. Antioxidant capacity of Shiraz berry extracts. AFerric reducing antioxidant power (FRAP) of ethanolic berry
extracts (mg BHT/ml extract) of grapes grown under Nsh,
Exp, V40, and V60 treatments. B- Phosphomolybdenum
reducing antioxidant power of samples (mg AE/ml extract)
taken from different shading conditions. Data presented here
are means of three replicates with SE. The letters on the top
of the bars, which are the same, indicate that there is no
statistically significant difference at P 0.05.
According to our results, the highest total antioxidant activity

was detected in berry extracts of exposed grapes (Fig. 3A) and
reflected by the lower IC50 value (Fig. 3B). On the contrary, the
lowest antioxidant activity was found in berries grown under V60
shading treatment. The antioxidant activity differences among
the shading treatments were statistically significant (P 0.05).
Antioxidant activity of Exp samples exerts nearly twofold higher
antioxidant capacity in comparison with that of V60 shading
samples (Fig. 3B). Therefore, berry extract of the exposed grapes
was in the first place, when samples of V40 shaded grapes was
ranked to second place in the antioxidant activity power for
inhibition of DPPH stable free radical. The lowest antioxidant
activity of samples was detected in samples obtained from V60
shading conditions. These findings were parallel with previous
reports, indicating that higher anthocyanin, phenolic, and
flavonoid contents attributed to higher antioxidant activity
property 29, 30.
FRAP and Mo (VI) reducing power of grape berry extracts are
shown in Fig. 4A-B. The highest FRAP reducing power was
observed for sun exposed berries as 176.6 2.0 mg BHT/ml,
followed by Nsh shading (162.9 3.0 mg BHT/ml), V60 shading
(149.4 1.5 mg BHT/ml), and V40 shading treatments (139.2 5.2
mg BHT/ml).
Similarly that of FRAP assay, the highest Mo (VI) reducing
power was accounted for the samples extracted from exposed
grapes as 531.2 9.9 mg AE/ml, followed by Nsh shading (492
3.3 mg AE/ml), V60 shading (390 2.6 mg AE/ml), and V40 shading
treatments (358.04 6.4 mg AE/ml) (Fig. 4B). Higher FRAP and
Mo (VI) reducing power values translates into higher antioxidant
capacity. The findings showed that microclimatic conditions

around the grapes were effective on the biosynthesis of secondary
metabolites, and total antioxidant capacities of Shiraz berries.
Overall, most plants may synthesize anthocyanins, phenolics
and flavonoids to protect them against solar UV radiation,
temperature above ambient levels. These metabolites, responsible
for antioxidant activities in fruits and vegetables, also play an
important role in human health 29, 31. Because epidemiological
studies have shown that these natural antioxidants have
scavenging properties against free radicals, offering protection
nucleic acids, proteins and lipids 6.
Correlations among the total bioactive compounds and total
antioxidant potential were calculated using Pearsons correlation
coefficient (Table 3). According to these results, there was a
positive correlation between antioxidant potential determined by
DPPH, FRAP, and phosphomolybdenum assay with total phenolic
and flavonoid content. Correlation between total anthocyanin and
antioxidant potential designated by DPPH, FRAP was not
statistically significant at P 0.05 (Table 3). This finding was
supported by many studies reporting weakly correlation between
anthocyanin content and antioxidant activity of different fruit
species 32, 33. Taking the data obtained in this study into
consideration, it could be concluded that phenolic compounds
and flavonoids are the major contributors of antioxidant potential
of Shiraz grapes. It was previously reported that vitamin C exerted
only 0.4% of total antioxidant activity of the apple skin, and majority
of the antioxidant activity may come from phenolics and flavonoids34.
The data obtained in the study represented that the higher the
phenolic and flavonoid contents of samples, the higher DPPH
342
Table 3. Correlations (R) between antioxidant potential determined by the DPPH, FRAP,
phosphomolybdenum assays and total bioactive compounds of grapes grown
under different microclimatic conditions.
Total anthocyanins (mg/kg)

Total phenolics (mg/kg)
Total flavonoids (mg/kg)
DPPH inhibition
(mg/ml extract)
0.55
0.70*
0.76**
Reducing power (FRAP)

(mg BHT/ml extract)
0.47
0.87**
0.84**
radical scavenging ability, the higher FRAP and Mo (VI) reducing

power of grape berry extracts.
Conclusions
In the past two decades, there have been significant advances in
analytical chemistry of grapes. Recently, secondary metabolites
have driven significant attention for their ability to scavenge free
radicals generated within cell of living organisms normally.
Currently, we have the ability of monitoring more of the secondary
metabolites than before. Therefore, we studied the effects of
microclimatic changes on biosynthesis of bioactive compounds
which are the main responsible compounds for the antioxidant
potential of berries. The shading treatments used in the study
altered significantly the microclimatic conditions significantly of
grapes. A quantitative decrease in some physicochemical
characteristics was observed in exposed berries, but they inversed
in shaded berries. Grape exposure to solar UV radiation might be
undesirable for berry desiccation and sunburn. This negative
influence of excessive sun exposure could be attributed to heat
stress resulting from high light and solar radiation. However, direct
sun exposure stimulated synthesizing of secondary metabolites
in berries compared to shaded berries.
The highest total antioxidant activity was detected in berry
extracts of exposed grapes, and the lowest in V60-shaded berry
extracts. The data set obtained points out 40% of grapevine
shading may meet the grape berry quality expectations without
negative influences of excessive grape exposure. Our results
showed that excessive grape exposure may not be desirable for
the reasons such as low anthocyanin accumulation, fast ripening,
desiccated berries, and yield loss due to sunburn, and also
reaching at the highest level possible for the phytochemical
compounds is not necessary to grape quality standards accepted.
Based on the results, microclimatic changes in fruit zone of
grapevines significantly affected berry quality characteristics,
secondary metabolite contents and antioxidant potential of grape
tissues. Therefore, modifications in microclimate conditions
around the grapes may receive considerable research interest in
the future studies under harsh climate conditions.
Acknowledgements
This study was supported by a research grant from Scientific
Research Coordinator of Harran University (HUBAK Project no:
13028).
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Tourists perception on local economy of Terengganu state in Malaysia

Md. Anowar Hossain Bhuiyan 1, 4, Chamhuri Siwar 2, Shaharuddin Mohamad Ismail 2 and Aini Aman 3
Center for Entrepreneurship and SMEs Development (CESMED), 2Institute for Environment and Development (LESTARI), 3Faculty
of Economics and Management (FEP), Universiti Kebangsaan Malaysia (UKM), 43600 UKM, Bangi, Selangor, Malaysia. 4 Lecturer
(Management), National University-1704, Bangladesh. *e-mail: anowaranu@yahoo.com.
1
Abstract
Tourists give support to the tourism places, local economy and conservation programs by their spending, entrance fees and voluntary donations.
These initiatives can affect local economic structures as well as tourist perception towards local economy. Terengganu state is well endowed with
natural and mineral resources, unspools beauties, islands, beaches, Islamic heritages and cultural attractions. Lake Kenyir and Sekayu Recreational
Forest have seen identified as prospective ecotourism sites in Terengganu for their natural beauties, recreational facilities and tourism activities. The
present study examined the tourists intention for enhancing local economy in Lake Kenyir and Sekayu Recreational Forest from their perception.
The study follows non-probability convenience sampling design and purposive sampling technique to collect the primary data from the respondents.
A structured questionnaire has been used to collect the data. This study uses descriptive analysis to measure the domestic and foreign tourists
intention towards local economy according to their opinions. Most of respondents from domestic tourists (>87%) agreed to help the local economy
by accommodations, shopping, buying food and spending more income. Moreover, highest number of respondents from foreign tourists (>81%)
agreed to help the local economy by accommodations, shopping, buying food, boat service and spending more income. The study shows that foreign
tourists have less positive intention towards local economy compared to domestic tourists. The destination authorities can motivate the tourists to
buy local food and products, shopping from local stores, stay in local accommodation and enjoy local tourism products. Moreover, local products
and service qualities also improved according to the demand of tourists.
Key words: Intention, Kenyir, local economy, Sekayu, tourist, Terengganu.
Introduction
Tourist exploitation in a destination depends on social, cultural,
economic and environmental factors. From this point of view,
tourist destinations change their initiatives connecting with
natural attractions and tourism activities to ensure tourist
satisfaction. These initiatives can affect local economic structures
as well as tourist intention towards local economy 1. Visitors also
give support to the tourism places, local economy and
conservation programs by their entrance fees and voluntary
donations 2.
In the 21st century, tourists are interested to alternative forms of
tourism, such as ecotourism and nature-based tourism due to
change their tastes. They give focus on local customs, cultural
diversity and history for searching a destination. Moreover,
tourism is linked to natural and cultural resources, productive and
social activities of local communities 1. Accommodation facilities
are one of the main components for tourist satisfaction towards a
destination. Krippendorf 3 found that environment friendly
facilities are influencing tourist to choice their accommodation in
a destination. In ecotourism destinations of rural areas, tourists
like alternative accommodations, which are different from traditional
hotels, hostels and boarding houses 4. Hearne and Salinas 5 pointed
that tourist preferences and perceptions towards ecotourism
destination promote nature conservation, rural development and
economic potential. Taiwan Ecotourism Association 6 defines that
tourist behaviour and perception can influence culture and lifestyle of local communities, improve communities welfare,
conserving natural resources and environment.
Journal of Food, Agriculture & Environment, Vol.12 (3&42), April 2014
Thrane 7 addressed that the main focus of tourism development

in rural areas is to attract the tourists and motivate them to
contribute in local economy. Yusof et al. 8 identified through a
study on Lake Kenyir in Malaysia that environment friendly
practice and sufficient facilities of local accommodation increase
tourist satisfaction in a tourism destination. Barogh 9 identified
tourist intention towards local economy in Taman Negara,
Malaysia. The study used tourist perception on local food,
products, shopping from local stores and boat service. The study
pointed that tourists intention towards local economy is not so
positive in study area. The tourists feel that some tourism events
especially boat-rides and guiding, accommodation and food in
restaurant are relatively expensive. They were less interested for
shopping from local stores and spending more income for local
people. The study identified tourist intention towards creating
employment opportunity not so positive for local people through
tourism development. The above discussion revealed that tourist
intention on local economy and local accommodation can influence
sustainable livelihood of local communities.
Tourism is a mentionable earning industry of Malaysia.
Malaysian government has played a significant role in instituting
legal and institutional framework for ensuring sustainable tourism 10.
Terengganu is one of the states of East Coast Economic Region
(ECER) of Malaysia. There are some socio-economic
backwardness situations remaining in Terengganu state rather
than in other states of Malaysia. Among them poor incomes,
unemployment, poverty, less urbanisation, limited investment and
345
poor infrastructure development are mentionable. Terengganu is

full of tourism resources, which have huge natural diversity for
suitable tourism development. Terengganu state is well endowed
with natural and mineral resources, unspools beauties, islands,
beaches, Islamic heritages and cultural attractions. Terengganu
state government has identified three elements, culture, heritage
and environment, to develop and promote tourism industry in this
state 11. The ecotourism sites of this state are attractive to the
tourists for their activities. The main activities of ecotourism sites
are boating, jungle trekking, nature observation, camping, fishing,
canoeing and water sports. According to ECER Master Plan, Lake
Kenyir and Sekayu Recreational Forest of Terengganu have seen
identified as prospective ecotourism sites for their natural beauties,
recreational facilities and tourism activities. Both of these
ecotourism destinations are situated in the district of Hulu
Terengganu. This district is relatively backward in Terengganu in
terms of poverty rate, monthly income and number of poor
households 12. Ecotourism development in Lake Kenyir and Sekayu
Recreational Forest can be ensured socio-economic development
in this area as well as well-being of local people. The present
study examines the tourists intention for enhancing local economy
in Lake Kenyir and Sekayu Recreational Forest from their
perception.
Study sites: Sekayu is the largest and popular recreational forest
of Terengganu, which is well facilitated with shelters, changing
room, chalets and rest house for visitors convenience. Seven
cascades of waterfalls, which become the natural swimming pools
make it a famous retreat for visitors. Lake Kenyir is the largest
man-made lake in Malaysia. The lake area covers 340 islands
including hilltops and highlands, more than 14 waterfalls,
numerous rapids and rivers. This site is one of the famous and
lucrative fresh water ecotourism destinations for natural beauties,
recreational facilities and tourists attractions.
Sampling design: The study follows non-probability convenience
sampling technique for the sample selection. Within the sample
design, purposive sampling technique has been used to collect
the primary data from the respondents.
Data attainment: A total of 230 respondents perception from
tourists was used in this study. Among the respondents, 180 were
selected from domestic tourists and 50 from foreign tourists. From
domestic tourists 110 were selected from Sekayu and 70
respondents from Kenyir. On the other hand, 30 and 20 foreign
tourists were selected as respondents from Kenyir and Sekayu,
respectively. In Sekayu Recreational Forest, field survey was
conducted in January, 2011. The field survey in Tasik Kenyir was
completed on second half of September, 2011 and first half of
April, 2012. Statistical Package for Social Science (SPSS) software
was used to measure the tourists perception on local economy.
Methodology: A structured questionnaire was used to collect the
data. The questionnaire was divided in two sections. Section-A
included the socio-demographic profile of tourists. Section-B
consisted of 6 statements (for Sekayu 5 items) to examine the
tourists intention on local economy. The statements were buying
local products, staying in local accommodations, shopping from
346
local stores, using boat service (for Lake Kenyir), spending more
income for local people and creating employment opportunities.
This section used 5-point Likert-type scale ranging from 1 =
strongly disagree, 2 = disagree, 3 = neither disagree nor agree, 4 =
agree and 5 = strongly agree. Barogh 9 used descriptive analysis
to measure contribution of tourists towards local economy in
Taman Negara. This section uses descriptive analysis to measure
the domestic and foreign tourists intention towards local economy
according to their opinions.
Tourism in Terengganu: The main tourism resources of this state
are beaches, islands, highlands, hills, mountains, lakes and
waterfalls, parks, forest reserves, arts, crafts, culture and heritage 13.
Some beaches and islands are world-class and not crowded, which
are appropriate for tourist attraction. This state has lush tropical
landscape and nature, marine parks and wide variety of tree and
food crops. This state has good mix of royal, religious, historic
and cultural heritage. The people of this state are friendly and
welcoming hosts. All of these are suitable for potential tourism
development in this state 12.
Most of the respondents of the two study sites came from
Terengganu, are of Malay ethnicity and students. Maximum
respondents were female, single and with college/diploma and
university level education. This demographic characteristic shows
that these two destinations are favorites to the local tourists,
especially to the students. These two sites have good facilities
for group visits and for female tourists (Table 1).
Most of the respondents to the two ecotourism destinations
came from three neighboring countries (Thailand, Indonesia and
Singapore) of Malaysia, whereas Kenyir also attracts tourists from
Europe. That means Kenyir is well known to the foreign tourists
as an ecotourism destination. Most of respondents were job
holders, male, married and educated (College and university level).
This indicates that they come to visit these ecotourism destinations
during their vacation period (Table 2).
Table 1. Demographic profile of domestic tourists.
Variable
State
Ethnicity
Occupation
Sex
Marital status
Education
Source: Survey data
Item
Terengganu
Pahang
Kelantan
Johor
Melaka
Selangor
Kedah
Perak
Negri Sembilan
Malay
Chinese
Indian
Student
Job (govt. and non-govt.)
Business
Male
Female
Single
Married
Primary
Secondary
College/Diploma
University
Frequency and percentage (%)

Sekayu
Kenyir
61 (55.5%)
22 (31.4%)
8 (7.3%)
10 (14.3%)
9 (8.2%)
15 (21.4%)
6 (5.5%)
6 (8.6%)
5 (4.5%)
-11 (10%)
6 (8.6%)
10 (9.1%)
-----4 (5.7%)
--7 (10%)
90 (81.8%)
39 (55.7%)
18 (16.4%)
20 (28.6%)
2 (1.8%)
11 (15.7%)
66 (60%)
39 (55.7%)
27 (24.5%)
24 (34.3%)
17 (15.5%)
7 (10%)
50 (45.5%)
34 (48.6%)
60 (54.5%)
36 (51.4%)
78 (71%)
45 (64.3%)
32 (29%)
25 (35.7%)
7 (6.4%)
---22 (20%)
16 (22.9%)
53 (48.2%)
16 (22.9%)
28 (25.5%)
38 (54.3%)
14, 15.
Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014
Table 2. Demographic profile of foreign tourists.

Variable
Item
Thailand
Indonesia
Singapore
Europe
Student
Job
Business
Male
Female
Married
Single
Secondary
College/Diploma
University
Country
Occupation
Sex
Marital status
Education
Source: Survey data
14, 15
Frequency and percentage (%)

Sekayu
Kenyir
4 (20%)
3 (10%)
12 (60%)
20 (66.7%)
4 (20%)
2 (6.7%)
--5 (16.7%)
2 (10%)
8 (26.7%)
14 (70%)
18 (60%)
4 (20%)
4 (13.3%)
16 (80%)
19 (63.3%)
4 (20%)
11 (36.7%)
18 (90%)
23 (76.7%)
2 (10%)
7 (23.3%)
---2 (6.7%)
10 (50%)
6 (20%)
10 (50%)
22 (73.3%)
Tourists visit in ecotourism destination are contributing in local

economy. Their contributions give benefits to local communities
in destination. A total of 6 items have been used in the
questionnaire to examine the tourists intention on local economy
through their perception. Table 3 represents domestic tourists
perception on local economy of those, who visit the study areas.
Most of the respondents agreed that they would like to help the
local economy through their several activities in the study areas.
They show a positive perception towards local food and products
and shopping from local stores. Again, most of the respondents
were willing to give opportunity for employment and more income
to the local communities. Most of the respondents in Kenyir
agreed to use the boat service by the local people. This scenario
shows that domestic tourists had a positive perception towards
economic activities of local communities for tourists. Moreover,
about 13% of the respondents disagreed about staying in local
accommodations. This means local accommodations have a lack
of facilities, which should be improved. Table 4 represents foreign
tourists perception on local economy of those, who visit the
study areas. Most of the respondents agreed that they would like
to help the local economy by staying in local accommodations,
shopping from local stores and creating opportunity for
employment and more income for the local communities. This means
foreign tourists are attracted to the local communities commercial
activities in the study areas. Moreover, a mentionable number of
respondents disagreed to buying local food and products (14%)
and using the boat service (10%) of local people. This indicates
that local food, products and boat service do not highly attract
the foreign tourists.
The descriptive analysis of tourists intention of enhancing

local economy has been measured by the mean scores and
standard deviations acquired from the six statements. Most of the
statements concerning this topic yielded a mean score in the high
range from the intention of domestic tourists. These statements
are Help the local economy by shopping, Help the local
economy by spending more income, Help the local economy by
creating employment opportunity and Help the local economy
by using their boat service (in Kenyir). This indicates the positive
intention of domestic tourists on local economy. They are
interested in shopping from local stores and shops. The domestic
tourists have good intention towards employment and income
opportunities for the local people. They would like to help the
local economy by creating employment opportunities and
spending more income for local communities. In Lake Kenyir, boat
service is a famous and traditional event for the tourists. Domestic
tourists want to help the boat operators as well as the local
economy by using their boat service. Again, two statements Help
the local economy by buying food and local products and Help
the local economy by staying in local accommodation yielded a
mean score in the minimum range. This indicates that domestic
tourists are not highly attracted to local food and accommodations.
They choose alternative foods in place of the local food. On the
other hand, domestic tourists prefer the non-local accommodations
for their stay during the visit periods. Moreover, most of
statements concerning the above topic yielded a mean score in
the minimum range from the intention of foreign tourists. All
statements of this topic from foreign tourists scored between 3.76
and 3.86. This indicates that foreign tourists intention is not high
for enhancing the local economy. This means local communities,
tour operators and related authorities do not have specific
programs and activities for creating positive responses from
foreign tourists for enhancing the local economy in the study
areas (Table 5).
Conclusions
The study reveals both domestic and foreign tourists perception
on local economy in the study areas. Most of the respondents
from domestic tourists (>87%) agreed to help the local economy
by accommodations, shopping, buying food and spending more
income. Domestic tourists showed the highest positive perception
for shopping from local stores (mean = 4.22) and taking the local
boat service in Kenyir (mean = 4.14). Moreover, they posed the
lowest positive perception towards buying food from local people
Table 3. Domestic tourists perception on local economy.

Statements
I would like to help the local economy by buying food and other local products from local people
I would like to help the local economy by staying in their accommodation
I would like to help the local economy by VKRSSLQJIURPORFDOVVWRUH
I would like to help the local economy by using boat service of local people (for Lake Kenyir )
I would like to help the local economy by spending more income
I would like to help the local economy by creating employment opportunity
Source: Survey data
14, 15
Agreed
90%
87.2%
95.6%
97%
96.1%
97.3%
Disagreed
10%
12.8%
4.4%
3%
3.3%
2.8%
Neutral
0.6%
Table 4. Foreign tourists perception on local economy.

Statements
,ZRXOGOLNHWRKHOSWKHORFDOHFRQRP\E\VKRSSLQJIURPORFDOVVWRUH
I would like to help the local economy by using boat service of local people (for Lake Kenyir )
Source: Survey data
14, 15
Agreed
82%
86%
83.3%
88%
86%
Disagreed
8%
6%
10%
6%
6%
Neutral
10%
8%
6.7%
6%
8%
Journal of Food, Agriculture & Environment, Vol.12 (3&42), April 2014
347
Table 5. Tourists intention of enhancing local economy.

Statements
I would like to help the local economy by buying food and other local products from local people
,ZRXOGOLNHWRKHOSWKHORFDOHFRQRP\E\VKRSSLQJIURPORFDOVVWRUH
I would like to help the local economy by using their boat service (for Lake Kenyir )
Source: Calculated from survey data
14, 15
Domestic tourist
Mean
S.D.
3.86
0.671
3.82
0.748
4.22
3.02
4.14
0.546
4.02
0.483
4.05
0.464
Foreign tourist
Mean
S.D.
3.76
0.796
3.84
0.710
3.88
0.627
3.83
0.746
3.88
0.593
3.86
0.606
(mean = 3.86) and staying in local accommodations (mean = 3.82).

On the other hand, a highest number of respondents from foreign
tourists (>81%) agreed to help the local economy by
accommodations, shopping, buying food, boat service and
spending more income. Foreign tourists expressed minimum
positive perception (mean>3.75) towards the local economy. This
finding is supported by Carrier and Maclead 2, which showed that
tourists give support to local economy by their voluntary
donations. The study shows that foreign tourists have less
positive intention towards local economy compared to domestic
tourists. The destination authorities can motivate the tourists to
buy local food and products, shopping from local stores, stay in
local accommodation and enjoy local tourism products. Moreover,
local products and service qualities also improved according to
the demand of tourists.
Development Council, Kuala Lumpur, Malaysia, pp. 15-16. http://

www.ecerdc.com.my/ecerdc/ENG/ECERAR_ENG.pdf
12
ECER 2007. East Coast Economic Region Master Plan. East Coast
Economic Region Development Council, Kuala Lumpur, Malaysia,
pp. 1-3. http://www.ecerdc.com/ecerdc/dc.htm
13
Bhuiyan, M. A. H., Siwar, C. and Ismail, S. M. 2013a. Socio-economic
impacts of home stay accommodations in Malaysia: A study on home
stay operators in Terengganu state. Asian Social Science 9(9):11-18.
14
Survey data 2011.Survey data from tourists in Sekayu Recreational
Forest, Terengganu, Malaysia in January, 2011.
15
Survey data 2012. Survey data from tourists in Lake Kenyir,
Terengganu, Malaysia in April, 2012.
Acknowledgements
Financial assistance provided from the Research Grants (ERGS/1/
2013/SS07/UKM/01/1 & FRGS/1/2013/STWN01/UKM/03/1),
Institute for Environment and Development (LESTARI), Universiti
Kebangsaan Malaysia (UKM) are gratefully acknowledged.
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Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014
WFL Publisher
Helsinki, Finland
www.world-food.net
Identification of brownfields in China: Concept, procedure and practice

Hongbin Xie 1*, Mingshui Lin 1, Changchun Zhou 2, Yang Zhang 3 and Liangjin Zhou 4
1
College of Geographic Science, Fujian Normal University, Fuzhou 350007, Fujian, China. 2 College of Geography and Tourism,
Qufu Normal University, Rizhao 276826, Shandong, China. 3 College of Resource and Environment, Fujian Agriculture and
Forestry University, Fuzhou 350002, Fujian, China. 4 Fuzhou Academy of Environmental Sciences, Fuzhou 350001, Fujian,
China. *e-mail: xiehongbin933@sina.com
Abstract
Based on the definition of brownfield, this article proposes a methodology for identifying and classifying contaminated brownfields in the Chinese
situation. The methodology developed consists of a two-stage procedure: 1) selecting the sites suspected to be contaminated by environmental
information for the site collected from existing data sources and 2) inspecting the suspected contaminated sites by the use of sampling and testing. A
case study of 37 land sites in Fuzhou, capital of Fujian Province in southeast of China, is used to illustrate the application and the suitability of this
methodology. Following this two-stage procedure, 37 sites have been subdivided into the following three categories: 27 sites found to be without
suspected contaminants are identified as type I; another 8 sites exhibiting contaminants concentration less than the soil clean-up standards are
classified as type II; and the remaining 2 sites with higher contaminants concentration than the soil clean-up standards were identified as type III. The
above described identification procedure is designed to be beneficial to the local Chinese government in particular because it reduces the time it takes
to identify brownfields and thus the associated costs.
Key words: Brownfields, contaminated sites, identification, classification, China.
Introduction
Since the second half of the 20th century, due to fierce international
competition and technological progress, traditional industries in
urban areas have been in decline and some factories have been
shut down or relocated out of the central parts of urban areas. As
a result, this industrial transformation has left some land sites
unused. These abandoned land sites, which might bring about
environmental risks, are named brownfield sites or simply
brownfields 1-3. As brownfields redevelopment can promote the
intensive use of urban land, such development has come under
close scrutiny by many countries, local governments, enterprises
and non-government organizations (NGOs). Similarly, with the
acceleration of industrialization and economic reforms in China
since the 1990s, many state-owned traditional industrial
enterprises, which previously occupied inner-city sites, have been
relocated to the urban periphery. Accordingly, obsolete industrial
sites, which might potentially also be contaminated sites, have
been replaced by non-industrial purposes such as high-density
residential or commercial areas. This land transformation is a wellknown part of Chinas industrial policy of suppressing secondary
industries and developing tertiary industries 1. However, these
obsolete or derelict industrial sites exhibiting soil contamination
have the capability to inflict an ecological shadow on sustainable
urban development 4, with clearly serious consequences. As a
result of this, the redevelopment of these contaminated sites has
received increasingly close attention in China. However, a method
for identifying what is brownfield and how to define it are initial
bases.
Brownfield definition: The definition of brownfields has received

an increasing degree of attention in many countries over recent
decades, but there is still no commonly agreed denition of this
term. Brownfields have different meaning in various countries
and contexts 5-10. The first official definition of brownfields was
defined by US Environmental Protection Agency (USEPA) as
abandoned, idled, or under-used industrial and commercial facilities
where expansion or redevelopment is complicated by real or
perceived environmental contamination. The US Department of
Housing and Urban Development defines the term in a similar
way, except that real property has been substituted for
facilities, and industrial and commercial specifications have
been omitted. Similarly, in Europe, the Concerted Action on
Brownfield and Economic Regeneration Network (CABERNET)
has defined brownfields as currently derelict or underused sites
which have been affected by former uses of the site or surrounding
land; they are mainly located in fully or partly developed urban
areas and may have real or perceived contamination problems
thus require intervention to bring them back to beneficial use.
Drawing on these various definitions, several common
characteristics of brownfields can be identified as follows 11:
Previously developed land
Locating in urban area
Currently abandoned
Requiring redevelopment
May have real or perceived contamination problems.
349
At present, an official definition of what constitute brownfields

in China does not exist, although a number of Chinese scholars
have attempted to formulate basic definition of brownfields by
adopting it from European and American countries, and in
particular from the US. However, land redevelopment processes
in China are quite different from those developed countries, and
the pace of urban construction and transformation in China is
extremely rapid. Following the brownfield definitions of domestic
and foreign scholars, and incorporating the Chinese context into
the general understanding, the concept is defined as follows:
previously developed sites located in urban areas, which have
lost their former activities and which might be contaminated and
therefore redevelopment for other purposes be required within a
specified period of time.
Overview of brownfield identification:There is no unified and
universally applicable method for identifying contaminated
brownfield sites. A review of the relevant literature reveals that
existing researches on identifying contaminated brownfields
identification are relatively limited. In general, in western developed
countries, such researches can be classified into two groups. The
first group of researches focus on using different highly advanced
techniques and technical equipment to identify contaminated
brownfields. For example, Ferrara 12 proposed techniques for
identifying and monitoring brownfields based on hyper spectral
remote sensing, during which the airborne Multispectral Infrared
and Visible Imaging Spectrometer (MIVIS) sensor is used to detect
dangerous materials or contaminants. Kadioglu and Daniels 13
illustrated a new approach of integrating ground penetrating radar
and EM-61 data in a 3D image for the monitoring and interpretation
of buried remains and their characteristics. Morio and Finkel 14
presented a flow guided interpolation method (FGI) to detect the
distribution of contaminants in groundwater during the early
stages of brownfield assessments. Hayek et al. 15 used Geographic
Information Systems (GIS) to build a comprehensive brownfield
database with a series of historical fire insurance plans and city
directories for successive eras of development, thereby providing
extensive documentation about the location and condition of
brownfield. All the aforementioned identification methods are
relatively convenient, efficient and accurate, but in most cases,
their practice is restricted by lack of equipment or difficulties in
using the equipment.
The second group of researches focus on using traditional
frameworks and historical data sources to identify contaminated
brownfields. For example, Coffin 16 identified such brownfields by
linking currently available and easily accessible information held
by official and unofficial organizations as well as by websites.
Bezama et al. 17 presented a method of identifying and evaluating
contaminated sites which consists of developing a list of sites
potentially contaminated in any particular region, identifying any
vulnerable areas in that region, and then prioritizing and classifying
the sites identified as potentially contaminated in those vulnerable
areas. Cheng et al. 11 identified potential brownfield sites using
four steps: defining input sites; verifying environmental liability;
confirming tax delinquency; and cross-checking with the relevant
industrial classification codes. Frickell and Elliot 18 outlined the
need to identify brownfield sites and presented a method of
identifying current and past hazardous sites. They began by listing
polluting industries and then cross-referenced this list with the
350
manufacturing directories of a particular city. Using this approach,

they were able to identify firms which had been operating as
polluting industries.
In general, such kind of identification procedure for brownfields
is facilitated by comparing the characteristics of sites with the
predefined criteria of brownfield identified in European and
American countries. The sites, which characteristics are accordant
with these predefined criteria, are defined as contaminated
brownfield sites 11, 19, 20. Consequently, this identification process
needs abundant sources of information such as an accurate
inventory of old industrial sites, which are currently abandoned;
environmental records of polluting activities; tax records, which
help identify a current declining business requiring redevelopment;
and redevelopment registrations 11, etc. This information is
primarily drawn from national records and local governments as
well as city communities. Given that the data and information
required for identification process are easily acquired, the
aforementioned methods are simple and feasible, as well as suitable,
for identifying a wide range of brownfields. However, brownfield
contaminants are complex to identify, so it is difficult to spot them
by qualitative screening methods only. Therefore, further
inspections and analyses of the degree of contamination of each
typical site, especially a seriously contaminated one, should be
carried out.
This article outlines a procedure which has been developed to
provide support in identifying contaminated brownfield sites in
the Chinese situation. A case study from Fuzhou in Fujian Province
is used to illustrate the application of this methodology. The article
addresses designing an identification procedure; applying the
identification procedure to a case study from Fuzhou.
Methods and Materials
Methods: Brownfield identification is a process of screening
potentially contaminated sites by means of the pre-defined
characteristics of brownfield; for this purpose, site information is
more fundamental and critical. Here we propose an efficient and
effective identification method by using site information from land
transactions list of the city. In general terms, the methodology
consists of the following stages (see Fig. 1).
The first stage is to narrow down the number of sites potentially
contaminated by focusing only on old industrial sites, which would
be the sources for heavy contamination in most cases. Such sites
are selected and based on the knowledge of their former industrial
activities, and the potential contaminants are determined by crosschecking the industries with the official standards of polluted
industry classification in China, including consultation of the First
Technical Census Report of National Pollution Sources in Fuzhou.
The second stage involves inspection of the seriously
suspected contaminated sites by means of field investigation,
sample analysis of soil and groundwater contaminants, and by
comparison between analytical results and the corresponding
executive standards. Thus, brownfields are classified and ranked
according to the contamination degrees.
An important aspect of this identification procedure is its
consideration of preliminary information and its establishment of
a hierarchy among the sites suspected to be contaminated. Such
hierarchical ranking can help authorities to use their (often limited)
budgets for remediation of seriously contaminated sites.
Moreover, this procedure has some other advantages, above all,
Selecting identification period
Selecting sites from the transaction list
Collecting sites information and cross-checking with industrial contamination sources

First stage
No
Was the site used for industry

with contamination?
Brownfield type I
Yes
Further screening
Field investigation and sampling
Soil assessment
Groundwater assessment
Second stage
Is there any contaminant

in the sites?
No
Brownfield type I
No
Brownfield type II
Yes
Is contaminant over environmental
standard?
Yes
Brownfield type III
Figure 1. The framework and procedure for brownfield identification.
it selects suspected contamination by information easily acquired;

furthermore, the staffs involved in this selection do not need
special knowledge and training; last but not least, only the
suspected contamination sites undergo a more detailed
investigation. Thus, the procedure is time saving and cost reducing
through minimizing the use of technical equipment and experts,
both of which are often not available.
Case study area: Fuzhou is the capital city of Fujian Province,
which is situated on the southeast Chinese mainland, close to
Taiwan. From the late 1950s to the 1990s, with the rapid
industrialization in Fuzhou, this city attracted and set up many
industries, such as chemical and food-processing, timber-working,
engineering, paper making, printing, and textile factories. Since
the 1990s, Fuzhou has experienced rapid economic transformation,
while the industrial sites in central parts of the city began to be
replaced due to the special land administration system and market
mechanisms, and the factories, which could not afford to stay in
the central areas, have been closed down or moved out to the
industrial parks or technological zones, such as Fuzhou Economic
& Technological Development Zone, Fuzhou Export Processing
Zone, Fuzhou Free Trade Zone, Fuzhou Hi-Tech Industrial
Development Zone, Fuzhou Science and Technology Park and
Fuzhou Taiwan Merchant Investment Area. Therefore, many
industrial sites in the urban centre from which the

enterprises have moved away have been transferred to
commercial and residential purpose by bid, auction and
list for sale.
According to the current land transaction system in
China, the land sites abandoned by the former occupants
and awaiting re-use are reserved in a land bank by the
local Land and Resources Bureau before any transaction.
Thereafter, the land use rights will be sold by auction,
bid or list for sale in the given period. Normally, in
accordance with environmental protection regulations,
these sites will have to be conducted an environmental
impact assessment by the local environmental protection
departments before or after the transaction. Hence, the
environmental condition of the sites will be confirmed,
and soil contaminants will be sampled and analysed.
As a result, identified cases can be selected from the
transaction list on the website of Fuzhou Land and
Resources Bureau.
Selection of suspected contaminated sites: There are
37 sites (see Fig. 2) which land use rights were
transacted from January to December in 2011. Detailed
information such as location, area size, original and
planned use of these sites is acquired from the official
website of Fuzhou Land and Resources Bureau. The
sites, which were used for manufacturing industries in
the past, are screened as suspected contaminated sites,
while the others are considered to be non-contaminated
sites. Thus, 10 out of 37 sites are screened as suspected
contaminated, while they are transformed to the other
purposes, such as the commercial and service industries,
residence and public green space (Table 1).
Brownfiled type I
Brownfiled type II
Brownfiled type III
Figure 2. Distribution of types of the simple sites in the city of Fuzhou.
Inspection of suspected contaminated sites based on sampling:

In accordance with the characteristic of the former industries and
the official classification criterion of industrial contamination
sources, the main suspected contaminants of soil and groundwater
on the potentially contaminated sites are specified. This
classification criterion of industrial contamination sources is based
on the First Technical Census Report of National Pollution Sources
in Fuzhou, which was issued by the State Council of China in
351
Table 1. Landuse information of sites transacted in Fuzhou (2011).

Land parcel number
Original land use
Planned land use
03, 04, 05, 07, 08, 09, 10,

11, 12, 13, 17, 19, 20, 21,
22, 23, 24, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35
02
06-1
16
15
18
14
06-2
06-3
Business&Service,Residence,
Warehouse, Public green space
Business & Service, Residence, Logistic,

Public green space
Wood industry
Wood industry
Electro-mechanical industry
Beer production
Electronics industry
Printing industry
01
Chemical production
25
Chemical production
Business, Residence, Public green space

Residence
Residence
Residence, Business & Service
Residence
Residence
Residence
Residence
Residence, Business & Service, Public
green space
Residence, Business & Service, Public
green space
order to cover the basic environmental information of all kinds of

enterprises and institutions. The report involves the classification
of contamination sources according to different industrial sectors.
In doing so, the main suspected contaminants and their potential
degrees in soil and groundwater for each site can be deduced in a
preliminary way. Based on this screening, further environmental
investigation and assessment on the suspected sites will be carried
out.
In order to investigate the environmental conditions of soil and
groundwater in suspected contaminated sites, Fujian Academy
of Environmental Science carried out an inspection from August
to September in 2011. This inspection consists of three steps:
The first step is field environmental investigation. It is necessary
to acquire the sites historical environmental information with the
purpose of estimating the main contaminants. Environmental
information of a suspected contaminated site includes: (1) land
use history; (2) raw materials and production technology used in
the process of manufacture and pollution discharge; (3) storage
of dangerous and poisonous goods; (4) soil environmental and
hydrological conditions; (5) environmental accident reports. All
of this information is collected from media and interviewees. Media
information is primarily acquired from the websites of local
environmental protection departments, newspapers and other
news reports, etc. Interviewees include staff in the environmental
protection departments, urban planning and land administration,
former users of the sites, land developers as well as residents
around the sites, among which the workers on the original sites
and the nearby residents are main part of interviewees. Based on
inquiries about the former industrial procedures on suspected
contaminated sites, the used chemicals, toxic and harmful
substances, as well as possible leakage of contaminants in the
process of industrial production can be further inferred.
The second step is soil sampling and analysis. This procedure
is based on Chinas Technical Specification for Soil Environmental
Monitoring (HJ/T166-2004). For each suspected contaminated
site, 1 to 3 sample locations are selected on which a sampling
depth in the range of 0 to 20 cm will be carried out. This can be
done without any disturbance, since the sampling depth is
determined by the characteristics of vertical migration in the soil
profile and, in general, the heavy metal contaminants are mainly
concentrated in the soil surface. Other contaminants should be
352
Area
(hm2)
306.6
14.3
2.8
3.1
4.3
2.2
7.0
4.6
2.2
14.3
16.8
considered in the context of the characteristics of vertical migration

in soils.
The third step is direct comparison between analytical results
and the corresponding executive standards. These standards are
as follows: (1) Standard of Groundwater Quality (GB/T1484893): III; (2) Standard of Soil Quality Assessment for Exhibition
Sites in China; (3) Generic Soil Screening Values of USEPA. In
the matter of above standards selection, China has Environmental
Quality Standard for Soil (GB15618-1995), Environmental
Quality Risk Assessment Criteria for Soil at Manufacturing
Facilities (HJ/T25-1999), and Standard of Soil Quality
Assessment for Exhibition Sites (HJ350-2007). However, there
are certain limitations in these standards, which are not completely
applicable to brownfields identification. For these standards, the
first one is set up according to soil background values and the
eco-environmental effects, which is mainly applicable to estimate
agricultural and forestry land but not suitable for residential,
industrial and commercial sites. The second one is designed to
protect the workers who might absorb dust or contact soil in the
process of work and to protect underground drinking water sources
located beneath industrial enterprises. The third one is instituted
to protect soil environment of exhibition sites, which is not only
suitable for stadiums, green spaces, commercial and public
municipal sites, but also for residential, industrial and commercial
sites. As there are no specific assessment standards for residential
sites, this study referred to the Standard of Soil Quality
Assessment for Exhibition Sites (HJ350-2007), which has been
widely used in soil contaminants assessment in China. In addition,
the values which cannot be found in the Standard of Soil Quality
Assessment for Exhibition Sites (HJ350-2007) are referred to the
Generic Soil Screening Values of USEPA. In this way, excessive
contaminants of the 10 sites were identified.
Identification of the suspected contaminated sites: By checking
the list of sites transacted in 2011 from the official website of
Fuzhou Land and Resources Bureau, 10 suspected contaminated
sites were selected from the 37 sites. The results are shown in
Table 2. These 10 sites have been used for industrial activities in
the past, such as chemical, mechanical and electrical, timber, beer,
electronics and printing industries, and might create or at least
pose a risk of damage to soils and groundwater in Fuzhou.

The identification result is that 10 out of the 37 sites were
suspected contaminated, and only 2 sites out of these 10
suspected contaminated sites, which contaminants concentration
exceed standards. Since the 2 sites with serious soil contamination
are located in the city centre of Fuzhou, they might bring risks to
the local environment or/and citizens. For that reason, it is
imperative to carry out further inspection and detailed
investigation on these seriously suspected contaminated sites.
Classifying and ranking suspected contaminated sites:
According to the First Technical Census Report of National
Pollution Sources in Fuzhou, and by field investigation and
interviews, the suspected contaminants of each site have been
specified. Then the 10 sites have undergone further inspection
and assessment based on the specified contaminants in order to
estimate the exact degree of contamination of soil and groundwater.
Results are shown in Table 2.
From sample analysis, it was found that for 8 sites, specifically
numbers 02, 14, 15, 16, 18, 06-1, 06-2 and 06-3, the contaminants
concentration is less than the corresponding standard values and
these sites are identified as brownfield type II. However, site
numbers 01 and 25 are identified as brownfield type III, since the
contaminants of number 01 (Cu, phenol, sulfide, PH, NH3-N) are
higher than the corresponding standard values, and the ones of
number 25 (Hg, Pb, hexachloro, cyclohexane, chloride, PH, NH3-N)
also exceed the corresponding standard values.
From the obtained results, it is clear that contaminant
concentration found in the chemical industry exceeds the standard
values, corresponding to former serious industrial contaminant
sources; in contrast, the contaminant concentration in the

mechanical and electrical, timber, beer, electronics and the printing
industries do not exceed the standard values, corresponding to
the normal industrial contaminant sources, according to the First
Technical Census Report of National Pollution Sources, which
contains the official classification criteria of industrial contamination
sources.
Conclusions and Final Remarks
Before the re-use of a brownfield site, environmental investigations,
risk assessments and any necessary rehabilitation of the site
should be carried out. Of these processes, classifying the sites
according to contamination and identifying contaminated sites
are a priority for the management and rehabilitation of brownfields.
At present, studies of brownfield re-use in China have only just
begun and there are no specific studies on brownfield identification
and classification.
Based on the definition of what constitutes a brownfield,
identification is determined by selecting suspected contaminated
sites in the first stage and by screening and classifying the
potentially contaminated sites in the second stage. Finally, each
site is identified as one of three types: non-suspected contaminated
sites are ranged into type I; slightly contaminated sites which
contaminant concentrations are less than their standard values
are ranged into type II and seriously contaminated sites which
contaminant concentrations exceed their standard values are
ranged into type III.
In total, 37 land sites in the central parts of Fuzhou were publicly
transacted in 2011 by the Fuzhou Land and Resources Bureau.
These land sites were selected to undergo the identification
Table 2. Process and results of brownfields identification in Fuzhou.

Land parcel number
Have the land

sites usage
been changed?
03, 04, 05, 07, 08, 09, 10, 11, 12,

13, 17, 19, 20, 21, 22, 23, 24, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35
No
02
Yes
06-1
Yes
16
Yes
15
Yes
18
Yes
14
Yes
06-2
Yes
06-3
Yes
01*
Yes
25*
Yes
Do suspected soil and

groundwater contaminants exist?
No
Formaldehyde, benzene heavy
metal
Formaldehyde, xylene, heavy
metal
Waste paint, waste acid and
alkali, heavy metal, asbestos,
cyanide
Waste paint, waste acid and
alkali, heavy metal, cyanide,
asbestos
Waste acid and alkali, heavy
metal, asbestos, cyanide
Diatomite, waste yeast, waste
vinasse, cold and hot waste
sludge
Waste acid and alkali, petroleum,
heavy metal, benzene
Heavy metals, printing ink,
toluene, aromatic hydrocarbons
Heavy metals, phenol, cyanide,
sulfide, ammonia, petroleum,
waste acid and alkali
Heavy metal, sulfide, petroleum,
polyethylene, dipterex, chloride,
hexachloro-cyclohexane, PH,
ammonia
Do the sites
contaminants exceed
the standard values?
Do the sites need

to be cleaned or
remedied?
Brownfield
type
No
No
No
No
No
No
No
No
No
No
II
No
No
No
No
No
No
No
No
Yes (Cu, phenol,

sulfide, ammonia)
Yes
Yes (Hg, Pb,

hexachlorocyclohexane, chloride,
PH, ammonia,)
III
Yes
*These two sites were cleaned up by soil excavation, solidification and stabilization for heavy metals and thermal desorption for organic compounds before they were developed and approved for residential use.
353
procedure. Of these 37 land sites, 27 were identified as brownfield

type I; another 8 land sites with contaminant concentrations less
than their standard values were identified as brownfield type II;
and only land sites number 01 and 25, with contaminant
concentrations in excess of their standard values, were identified
as brownfield type III. Accordingly, these land sites have to be
cleaned up and remedied before they are fit for a new occupation.
This has been carried out after the investigation.
An important aspect of this procedure is combining qualitative
and quantitative analyses. The application of the two-step
assessment analysis for identifying and classifying suspected
contaminated sites not only can reduce time and invested
resources in the first stage, but also can make the identification
reliable by undergoing further investigation and sampling
procedures in the second stage. Moreover, data sources of this
identification procedure not only come from official environment
notices and reports on environmental impact assessment, but also
depend on sampling analysis; besides, information also comes
from interviews with the public, the original enterprise employees,
government officers, and the community around the sites. Thus,
collecting data from different departments and individuals can
overcome the limits of previous methods based only by secondhand reports and information.
Yet this identification procedure is not perfect, because it is
only a first sketchy screening. For seriously contaminated sites,
further inspection and identification should be carried out. In
addition, the identification method based on the Chinese domestic
standards is not as universal as the international standards.
It is important to emphasize that identification of contaminated
brownfields allows a citys administration to determine the extent
of the contemporary brownfields situation and to improve the
management of these sites for the future. The two-step
identification makes it helpful for Fuzhou Land and Resources
Bureau to determine land value. Moreover, it can be used as the
basis of different types of brownfield risk assessment and soil
remediation.
Based on the information about land transaction in Fuzhou,
this study narrowed the scope of suspected contaminated sites
by means of rough screening. On the basis of sketchy screening,
further sampling and analysis were carried out, thus reducing the
cost of identification. This being so, this procedure may well have
relevance to other cities in China. Finally, this study has the
potential to generate further studies about brownfield site re-use
in China.
Acknowledgements
The source of financial support of study: China National Natural
Science Foundation (grant number: 41171119).
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4
WFL Publisher
Helsinki, Finland
www.world-food.net
Evaluation of the microbiological and physicochemical quality of artesian well water

used for irrigation in ArRiyadh
Sulaiman Ali Alharbi 1*, M. E. Zayed 1, Arunachalam Chinnathambi 1, Naiyf S. Alharbi 1
and Milton Wainwright 1, 2
1
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh-11451, Kingdom of Saudi Arabia.
2
Department of Molecular Biology and Biotechnology, University of Sheffield, S102TN, UK. *e-mail: sharbi@ksu.edu.sa
Abstract
The quality of water from artesian wells used for irrigation was analysed. Water samples were collected from 12 wells from different farms along a
8.5 km transect of the Hayer, which is an area located approximately 35 km south of ArRiyadh. The major parameters for assessment of the
groundwater quality used here were analysis of the major cations (K+, Na+ and NH4 +) and the major anions (Cl-, SO42-, NO3- and PO43-). A total
dissolved solid (TDS) is a summation of all major constituents. The pH, temperature and electrical conductivity (EC) were also measured as
important indicators of groundwater quality. The samples were also tested for the presence of total and fecal coliform bacteria. All the samples were
free from contamination by coliforms; the physicochemical parameters of all the samples were not, however, within the acceptable limits prescribed
by WHO and FAO.
Key words: Physicochemical quality, artesian well water quality, irrigation, coliforms, cations.
Introduction
Water from rivers, lakes, reservoirs, and groundwater aquifers is an
essential human resource and is needed for direct consumption as
well as for recreational purposes 1. Groundwater is a vital source for
fresh water in Saudi Arabia and the surrounding Gulf states 2;
groundwater being the major source of both potable and irrigation
waters in Saudi Arabia. As the population of Saudi continues to
increase, especially in the big cities such as ArRiyadh, the demand
for adequate and high-quality groundwater resources continues to
increase. The Kingdom of Saudi Arabia (about 2.25 million km2) is
one of the hottest and most arid countries in the world, with an
average summer temperatures of 46C and an average rainfall of 120
mm per year over most of the country 2. The available surface water
and groundwater resources are limited, precipitation rates are low,
and evaporation is high. With increasing population and agricultural
use, there is an increasing need for high quality water in Saudi
Arabia 3.
The total population of Saudi Arabia has increased from about
7.7 million in 1970 to 11.8 million in 1990 and is expected to reach 19
million in 2010, if the present growth rate of 3% per annum continues.
Consequently, domestic water demand has increased from about
446 MCM in 1980 to about 1,563 MCM in 1997, and is expected to
reach 2,800 MCM in 2010 4, 5. Agriculture accounts for some 88% of
water use, while industry consumes only around 3% 6. Saudi Arabia
faces severe water problems and as a result, is in need of new water
policies to achieve sustainable development in its harsh environment.
Problems include balancing supply and demand while facing aridity
and water scarcity, non-renewable supplies, poor quality of ground
water, poor distribution of supplies, salt water intrusion, and the
overuse and contamination of aquifers 7.
Available water resources in Saudi Arabia are a) conventional,

i.e. groundwater and surface water, and b) non-conventional such
as desalinated seawater and treated waste water. About 88% of
the water consumption in Saudi Arabia is met from groundwater
supplies 2. Groundwater is generally presumed to be ideal for
human consumption and is used as a potential source of drinking
water, agricultural development, urbanization and industrialization 8.
Around 47% of the water supplied in ArRiyadh is groundwater pumped
from local aquifers 9.
It is estimated that 18% of worldwide cropland is irrigated, producing
40% of all food. Irrigation water and any foliar applied water, in intimate
contact to the developing or mature edible portions of fresh produce,
is likely to lead to contamination with human waste, although irrigation
using surface water is likely to pose a greater risk to human health
than irrigation water obtained from deep aquifers drawn from properly
constructed and protected wells 10.
Water-borne pathogens infect around 250 million people and
result in 10 to 20 million deaths worldwide each year. An estimated
80% of all illness in developing countries is related to water and
sanitation, with some 5% of all child deaths under the age of five
years occurring in developing countries resulting from diarrheal
diseases 11, 12. Pathogens pose a risk to human health as a result
of the various uses of water. For example, it was suggested that
contaminated irrigation water was a possible source of a recent
outbreak of E. coli across USA 13. Fruit and vegetables are
frequently contaminated impacted by fecallypolluted irrigation
water 14. As a general rule, surface water resources are more
susceptible to microbial contamination than are groundwater
supplies. Microbial contamination introduced through sprinkler
355
irrigation systems may also affect the surface of a crop for varying
periods of time, and the risk is increased when the irrigated crop is
consumed raw and sometimes unwashed 15.
Pathogen contamination of fresh, ready-to-eat fruits and
vegetables is a significant issue in agriculture. In many cases,
fecal-oral pathogens such as toxin-producing E. coli, Salmonella
spp., and norovirus are the causative agents 16. Fecally
contaminated irrigation water is frequently a possible or likely
source of contamination of fresh, ready-to-eat fruits and
vegetables 17. According to the Centre for Disease Control and
Prevention (CDC) 18, at least 12% of food-borne outbreaks during
the 1990s were attributable to fresh produce, and the economic
cost of food-borne illnesses is estimated at around $10 to $83
billion per year 19.
Water is subject to varying degrees of fecal pollution, and
consequently fresh waters are a transmission vector of many
pathogenic bacteria, viruses, and protozoa. Fecal pollution can
reach water resources as the result of human activities, such as
sewage treatment plants and communities where sewage treatment
is not available. Many diseases are related to fecal polluted water,
but the majority is caused by enteropathogenic microorganisms,
and not surprisingly therefore, the presence of enteric pathogens
in waters is of considerable concern. For this reason, maintaining
the microbiological safety of water is very important issue relating
to the protection of public health 1. Washing and disinfection
practices are less effective against pathogens which penetrate
the plant interior 20, and for this reason the prevention of water
borne contamination is considered to be an important primary
means of controlling health risk associated with food-borne
pathogens 19.
The quality and safety of farm irrigation water determines the
quality and safety of the resultant crop, and the safety of water
depends on its source. Human pathogens can be introduced into
irrigation water via run-off of manure from animal production
facilities, from domestic/urban sewage systems or directly from
wildlife. Extreme rainfalls (which lead to storm overflows), spills of
manure, or human waste can all increase the probability of the
occurrence of contamination 21. The quality of water needed for
various uses is determined by its physical characteristics, chemical
composition, biological parameters and the conditions of use and
all surface or sub-surface waters contain varying amounts of salts
which increase in irrigated soil due to evaporation.
The aim of the work reported here was to determine the
microbiological and physicochemical quality of waters obtained
from artesian wells used for irrigation near the city of Riyadh.
Description of the artesian wells: The samples were taken from
wells of depth ranging from (60-100 m); some wells were open
while others were closed.
Sampling collection: All groundwater samplings (chemical or
microbial) were conducted with the existing well pumps, which
were run for a sufficient time (10-15 min) in order to replace the old
water in the pipes with fresh water and thereby obtaining reliably
stable readings of pH, specific conductance and temperature. Well
water depths were measured with a graduated (l/l00th foot) steel
tape.
A total of three water samples were collected from 12 different
356
wells located in different farms along a 8.5 km transect of the

Hayer, which is an area located some 35 km south of Riyadh,
during November 2010. The water samples were collected in plastic
bottles, pH, EC and TDS were measured on site; samples were
subsequently transported to the laboratory in an ice box. Each
sample was divided into three portions; one for cation analysis,
one for anion determinations and the third for coliform analysis.
The concentration of total dissolved ions, Na, K, P, Cl, SO4, NH4
and NO3 were determined. The analytical procedures used for
these determinations were those described in standard methods
or the examination of water and wastewater.
The evaluation of the suitability of groundwater for irrigation
purpose is based here on the irrigation water specification provided
by the Saudi Arabian Standards organization (SASO), irrigation
water standards 1993, and water quality for use in agriculture by
the Food and Agriculture Organization (FAO)11. Table 1 shows
the concentration (mg/l) of individual constituents, groundwater,
hardness, electrical conductance and pH of the groundwater.
Table 1. Laboratory determinations used to evaluate
common irrigation water quality problems.
Water parameter
SALINITY
Salt content
Electrical conductivity
(or)
Total dissolved solids
Cations and anions
Calcium
Magnesium
Sodium
Carbonate
Bicarbonate
Chloride
Sulphate
NUTRIENTS
Nitrate-nitrogen
Ammonium-nitrogen
Phosphate-phosphorus
Potassium
MISCELLANEOUS
Boron
Acid/basicity
Sodium adsorption ratio
Usual range in
irrigation water
Symbol
Unit
ECw
dS/m
03
dS/m
TDS
mg/l
0 2000
mg/l
Ca++
Mg++
Na+
CO--3
HCO3ClSO4--
me/l
me/l
me/l
me/l
me/l
me/l
me/l
0 20
05
0 40
0 0.1
0 10
0 30
0 20
me/l
me/l
me/l
me/l
me/l
me/l
me/l
NO3-N
NH4-N
PO4-P
K+
mg/l
mg/l
mg/l
mg/l
0 10
05
02
02
mg/l
mg/l
mg/l
mg/l
B
pH
SAR
mg/l
114
(me/l) 1, 2
02
6.0 8.5
0 15
mg/l
dS/m = deciSiemens/metre in S.I. units (equivalent to 1 mmho/cm = 1 millimmho/centimetre).

mg/l = milligram per litre parts per million (ppm). me/l = milliequivalent per litre (mg/l
equivalent weight = me/l); in SI units, 1 me/l= 1 millimol/litre adjusted for electron charge.
Coliform determination: The samples were diluted in the range

of 10-1 to 10-6 and the original water sample was aseptically diluted
into 9 ml buffered peptone prepared in three series. The number of
total and fecal coliforms was determined using the MPN method
and statistical tables were used to interpret the results. From each
dilution, 1 ml was removed and added aseptically to triplicate
tubes containing 5 ml of lauryl tryptose broth (LSB). The tubes
were incubated at 37C for 48 hours. Tubes showing colour change
or gas production were recorded as positive, and the number of
positive tubes at each dilution was referred to MPN tables to
obtain the number of bacteria present in the original sample.

Microbiological analysis: None of the water samples obtained
from any of the wells contained coliforms, a fact which shows that
the general sanitary conditions around the wells are excellent.
Analysis of physicochemical parameters: Table 1 shows the
laboratory determinations used, together with the acceptable range
to evaluate common irrigation water quality, as prepared by FAO11.
Table 2 shows the physical parameters of the groundwater samples.
pH: The pH values of all the groundwater samples tested were
alkaline (around 8); a pH which is generally not conducive to
optimal crop plant growth.
Total dissolved solids (TDS): Suspended solids and total
dissolved solids (TDS) are indicators of polluted water. The value
for TDS of the samples ranged from 2393 to 7123 mg/l. Most of
these values are outside the standard values generally considered
to be suitable for irrigation purposes. TDS values exceeding 3000
mg/l are high for irrigation of some crop types. The high TDS
values found in groundwater sampled from the study area are
likely to be due to high concentrations of sodium, chloride, sulfate
and nitrate.
Conductivity: Electrical conductivity gives a measure of all of the
dissolved ions in solution. Electrical conductivity values measured
in this study varied from 3.74 to 11.13 mS/cm with sample K
exhibiting the highest conductivity (11.13) and sample D the lowest
one (3.74). The acceptable limit of conductivity is 1.5 mS/cm 22.
Generally, the conductivity of clean water is lower but as water

moves down the soil profile it leaches and dissolves ions and also
picks up organic matter from the biota and detritus 23. Generally,
the conductivity values recorded for wells sampled here were not
within the acceptable limit prescribed by WHO and FAO limits11.
Total water hardness: Water hardness is primarily a measure of
the amount of calcium and magnesium, and to a lesser extent, iron
in a water sample. Water hardness is measured by summing the
concentrations of calcium, magnesium and converting this value
to an equivalent concentration of calcium carbonate (CaCO3); a
value which is expressed in mg/l of water. Water with hardness
greater than 200 mg/l is considered to be of poor quality and
water with hardness greater than 500 mg/l is normally considered
to be unacceptable for domestic purposes. If the hardness
concentration range is between 1200 and 3800 mg/l, the samples
would be assessed as belonging to the fourth category with very
hard water and unacceptable for domestic purpose without
treatment.
Cations and anion loads of the groundwater samples: Table 3
shows the cations and anions loads of groundwater samples.
Sodium: An infiltration problem related to water quality occurs
when the normal infiltration rate for the applied water or rainfall is
appreciably reduced and water remains on the soil surface for
long periods, or infiltrates too slowly to supply the crop with
sufficient water to maintain acceptable yields. The infiltration rate
of water into soil varies widely and can be greatly influenced by
Table 2. Physical parameters of analysed groundwater samples.

Sample
ID
A
B
C
D
E
F
G
H
I
J
K
L
Temperature
(Degree Celsius)
25.0
25.5
24.5
25.5
23.5
24.5
28.0
25.0
26.5
26.0
27.0
27.0
pH
8.15
8.13
8.17
7.98
8.19
8.05
8.02
7.84
8.29
8.07
8.06
8.11
Parameters
E.C*
T.D.S**
(mS/cm)
(mg/l)
3.87
2476
8.89
5689
4.48
2867
3.74
2393
5.49
3513
9.41
6022
9.19
5881
10.78
6899
9.41
6022
10.29
6585
11.13
7123
10.16
6502
Turbidity
(NTU)
11.30
28.70
20.50
18.00
6.24
2.98
0.73
21.90
0.94
5.78
12.30
5.63
Total hardness
(mg/l as CaCO3)
1800
3000
1200
1400
1000
2600
2800
3600
3200
3200
3800
3600
* E.C = Electrical conductivity; ** T.D.S = Total dissolved xolids.
Table 3. Cations and anion loads of the groundwater samples.

Sample
ID
A
B
C
D
E
F
G
H
I
J
K
L
Sodium
Na (mg/l)
500
1375
750
500
750
1500
1375
1375
1125
1375
1375
1125
Potassium
K (mg/l)
17.0
28.0
15.0
15.0
23.0
27.0
26.0
27.0
30.0
27.0
31.0
30.0
Phosphorus
P (mg/l)
0.53
0.37
0.15
0.11
0.10
0.00
0.33
0.25
0.81
0.00
0.00
0.25
Parameters
Sulphate
SO4 (mg/l)
1437
3275
1302
1380
1607
3675
3275
2587
1737
2987
3075
1595
Ammonia
NH3 (mg/l)
0
0
0
0
0
0
0
0
0
0
0
0
Nitrate
NO3 (mg/l)
2.0
10.0
5.5
2.0
4.0
3.5
13.5
49.5
138.0
35.0
142.0
158.0
Chloride
Cl (mg/l)
1250
2500
1500
1250
1500
2850
2500
3000
2750
2750
3250
3000
357
the quality of the irrigation water. The two most common water
quality factors, which influence the normal infiltration rate, are
water salinity (total quantity of salts in the water) and sodium
content relative to the content of calcium and magnesium. Water
which is highly saline will increase infiltration, while a low salinity
water, or a sample with high sodium to calcium ratio will decrease
infiltration; both of these factors may operate simultaneously.
One serious side effect of an infiltration problem is the potential
to develop plant disease and vector (mosquito) problems.
An infiltration problem related to water quality in most cases
occurs in the surface few centimetres of soil and is linked to the
structural stability of this surface soil and its low calcium content
relative to that of sodium. When a soil is irrigated with sodiumrich water, a high sodium surface soil develops which weakens
soil structure. The surface soil aggregates disperse into much
smaller particles which clog soil pores. The problem may also be
caused by an extremely low calcium content of the surface soil. In
some cases, water low in salt can cause a similar problem but this
is related to the corrosive nature of the low salt water and not to
the sodium content of the water or soil. In the case of the low salt
water, the water dissolves and leaches most of the soluble minerals,
including calcium, from the surface soil. Analyses of the
groundwater samples tested here shows that all have sodium
ranges over 500 (mg/l); sodium contents greater than 500 mg/l are
normally considered unacceptable for irrigation according to water
quality standards used by the FAO 11for agricultural use.
Nitrate: The nitrate content of the analysed groundwater samples
ranges between 2 mg/l in well A and D and reaches a maximum of
158 mg/l in well L. Many of the sampled groundwater wells contain
nitrate exceeding the guideline values for irrigation water prescribed
by FAO (0-10 mg/l), with most of the nitrogen present being
probably derived from the biosphere. The nitrogen originally fixed
from the atmosphere, is mineralized by soil bacteria into ammonium,
which is converted into nitrate by nitrifying bacteria under aerobic
conditions 24.
The main sources of nitrate result from either natural or
anthropogenic activities - rainfall and dry fall out, soil nitrogen,
nitrate deposit, sewage, septic tank and animal waste, manure or
compost, green manure and plant residues, atmospheric nitrogen
fixation, fertilizer nitrogen from irrigated overflow water outlets
and industrial effluent 25. Nitrate is the end product of the oxidation
of nitrogen in the environment. Particularly high nitrate
concentrations indicate pollution from either sewage or agricultural
fertilizer waste. Nitrate is without doubt an essential plant nutrient,
but is equally a potential threat to human health when present in
excess concentrations in the drinking water 26. The data obtained
from the samples tested here shows that the groundwaters
examined contain high level of nitrate, concentrations which exceed
the permissible limits for drinking purposes (Table 3).
Ammonia: The term ammonia includes the non-ionized (NH3) and
ionized (NH4+) species. Ammonia originates in the environment
from metabolic, agricultural and industrial processes and from
disinfection with chloramines. Natural levels in groundwater and
surface water are usually below 0.2 mg/l 27. Anaerobic groundwaters
may contain up to 3 mg/l. Intensive rearing of farm animals can
give rise to much higher levels in surface water. Ammonia
contamination can also arise from cement mortar pipe linings.
358
Ammonia in water samples is an indicator of possible bacterial,

sewage and animal waste pollution 27. The samples analysed here
showed that all of the well waters were ammonia-free (Table 3).
Phosphate: Slight increases may cause numerous undesirable
effects, such as accelerated plant growth, algal blooms and low
dissolved oxygen levels. Phosphate levels below 0.03 mg/l are
generally considered to be unpolluted. The concentration of
phosphate encountered in the natural water environment is
normally not sufficient to cause any detrimental health effect on
humans or animals. Phosphate like any other nutrient is harmless
in low concentrations but becomes harmful only in higher doses.
Higher doses of phosphate are known to interfere with digestion
in both humans and animals 28. Levels between 0.03 and 0.1 mg/l
are sufficient to stimulate plant growth. However, the PO4
concentration of analysed groundwater samples was within
permissible limit according to water quality for agriculture
guidelines by FAO (0-2 mg/l) 11.
Chloride: The chloride content of the water sampled here ranged
from 1250 to 3250 mg/l. The FAO standard 11 for chloride in irrigation
water is 030 mg/l so all of the samples exceeded the acceptable
limits prescribed by FAO (Table 3). Chlorine is an active chemical
which has disinfecting capabilities. Chlorine in natural water may
originate from mine drainage waste and from dissolving rocks.
The closeness of the water source to the sea can also influence
the chloride content since the sea is characteristically saline.
Chloride in water may react with sodium to form sodium chloride.
A salinity problem exists if salt accumulates in the crop root zone
to a concentration that causes a loss in yield. In irrigated areas,
these salts often originate from a saline, high water table or from
salts in the applied water. Yield reductions occur when the salts
accumulate in the root zone to such an extent that the crop is no
longer able to extract sufficient water from the salty soil solution,
resulting in a water stress for a significant period of time. If water
uptake is appreciably reduced, the plant slows its rate of growth.
Sulphate: Sulphate salts affect sensitive crops by limiting the
uptake of calcium and increasing the adsorption of sodium and
potassium, resulting in a disturbance in the cationic balance within
the plant. The sulphate content of the water samples tested here
ranged from 1302 to 3675 mg/l (Table 3). The FAO standard 11 for
the concentration of sulphate in irrigation water is 0-20 mg/l, so
the samples tested here all exceeded the acceptable limits
prescribed by FAO.
Potassium: High concentration of potassium may introduce
magnesium deficiency and iron chlorosis into a soil. An imbalance
of magnesium and potassium may be toxic to plants, but the effects
of both can be reduced by high calcium levels. The potassium
content of the water sampled here ranged from 15 to 31 mg/l (Table
3). The FAO standard for potassium in irrigation water is 0-2 mg/
l, so the samples tested here were all within the acceptable limits
prescribed by FAO11.
Conclusions and Recommendations
Based on the outcome of the study the following is recommended:
1- The high physicochemical values of all samples make them,
under class 5 (unsuitable for irrigation waters).
2- It is vital to create the necessary awareness of irrigation

water quality among the involved agencies, such as the Ministries
of Agriculture and Water, Health, and Environment, as well as
other stakeholders. It has proven efficient and successful to initiate
national interdisciplinary working groups with specialists of the
involved authorities for the elaboration of guidelines and
monitoring programs. Furthermore it is not sufficient to intervene
in only one field of activities, such as guidelines for irrigation
water quality. Other relevant issues including agricultural use of
groundwater, crop quality monitoring and awareness campaigns
have to be included and to complement each other.
3-A careful review should be made of the geology and geothermal
nature of the study area because the geology may reveal unique
or explanatory features.
4- It is recommended that water quality analysis be carried out
on all the wells in the district at least once every two years. This
will ensure that incidences of contamination are noticed earlier for
remedial action to be taken.
5- Communities should be educated on the need to keep their
surroundings clean, especially in the region of groundwater
sources.
Acknowledgements
Authors extend their appreciation to the Deanship of Scientific
Research at King Saud University for funding the work through
the research group project No. RGP-VPP-332.
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WFL Publisher
Helsinki, Finland
www.world-food.net
X-ray diffraction (XRD) and x-ray fluorescence (XRF) analysis of ancient bricks from
Sungai Batu Temple (site SB1), Bujang Valley, Kedah
Zuliskandar Ramli 1*, Nik Hassan Shuhami Nik Abdul Rahman 1, Abdul Latif Samian 1, Muhammad Rizal
Razman 2, Sharifah Zarina Syed Zakaria 3 and Hossein Sarhaddi Dadian 4
Institute of the Malay World and Civilisation (ATMA), 2 Research Centre for Sustainability Science and Governance (SGK),
Institute for Environment and Development (LESTARI), 3 Research Centre for Environment, Social and Economics Sustainability
(KASES), Institute for Environment & Development (LESTARI), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia. 4 Department of Archaeology, University of Zabol, Iran. *e-mail: ziskandar2109@gmail.com
1
Abstract
The aim of this study was to determine whether the ancient bricks from Sungai Batu Temple (SB1) are made from local materials or not.
Chronometric and relative dating have given different results, the absolute dating gave the date between 2nd to 3rd century AD while the relative dating
gave the date between 6th to 7th century AD. The structure which was fully built using bricks is a relatively large stupa structure and not a structure
associated with animism practised. In order to determine whether the bricks that were used to build the structure of Site SB1 used local raw materials
or otherwise, thus material composition analysis needs to be carried out which involves X-Ray Diffraction (XRD) and X-Ray Fluorescence (XRF)
analysis. Analysis shows that the mineral content present in the ancient brick samples consists of quartz, muscovite, microcline, mullite and albite.
The mullite mineral shows that some of the bricks were baked at sufficient temperature of more than 550C. This shows that the open burning
technique was used in the production of the temples bricks because some of the bricks have an indication of low firing burning. The dry weight
percentage graph of silica and aluminium, and magnesium and titanium, as well as the lead and copper concentration graph indicate that the raw
materials used to produce the ancient bricks are local raw materials and these raw materials were obtained from the surrounding area of Bujang
District.
Key words: Sungai Batu Temple, x-ray diffraction (XRD), x-ray fluorescence (XRF), Bujang Valley.
Introduction
Sungai Batu Temple was found by a group of archaeologists from
Science University of Malaysia in 2006 where a structure which is
believed to function as a stupa was discovered. The structure is
entirely made of bricks and its roof made of tiles. It is the first time
that a temple of this architecture (Photos 1 and 2) was found in
Bujang Valley. The rectangular shaped structure with an empty
circle was built on top of a circular floor structure that was also
built using bricks and it is proposed that this structure is a stupa
and not an animistic structure because there is no evidence to
show that the local people that practised animism built a structure
Photo 2. Overall structure of Sungai Batu Temple.
i The1main
h structure
i
f S BatuiTemple.
Photo 1.
of Sungai
360
or monument for their practices. The size of the site is approximately

900 m2. The bricks used are relatively smaller and of uniformed
size if compared to the bricks found in Sungai Mas. Dating that
was given to this site is between the 3rd to the 7th century AD 1.
There was also the finding of an inscription that contained
Buddhist credo verses in Sanskrit and of Pallava script and based
on the script, it can be dated to the 6th or 7th century AD and this is
a crucial evidence which indicated that the structure is a stupa.
The structure of Sungai Batu Temple is unique and the size of the
bricks used to build the structure is also relatively uniform and
standard compared to the temples of the early stage that were

found along Sungai Muda and Sungai Bujang. In order to
determine whether the bricks used are bricks that were produced
by the local people or imported, material composition analysis of
the bricks needs to be performed. The analysis involves X-Ray
Diffraction (XRD) and X-Ray Fluorescence (XRF) Analysis where
the content of the mineral, major elements and trace elements will
be able to be identified. The research carried out based on the
brick material composition in the Bujang Valley has indicated that
local clay was used in producing the bricks used to build the
temples in Bujang Valley. Among them are Sungai Mas Temple
(Site 32/34), Pengkalan Bujang Temple (Site 23), Bukit Pendiat
Temple (Site 17), Pengkalan Bujang Temple (Site 19, Site 21, 22, 23)
Pengkalan Bujang Temple (Site 2211) and Bukit Kechil Temple 2-9.
In addition to the bricks, analysis was also applied on other
artefacts such as pottery 10-12, votive tablets 13, monochrome glass
beads 14, 15 and bronze drum 16.
Use of local raw materials will prove that the local people were
the ones involved in the production of the bricks and the
construction of Temple Sungai Batu (SB1), similar to what took
place in other sites in Bujang Valley. Therefore the objective of
this research was to determine whether ancient bricks from Sungai
Batu Temple (Site SB1) are local materials or imported from other
places. This can be done by comparing bricks composition with
clay composition taken from Bujang Valley, Kedah17.

Mineral content analysis of the ancient bricks of Sungai Batu
Temple showed that the ancient bricks in this site contained major
elements such as quartz, muscovite, microcline and other minerals
such as mullite and albite (Table 1). Sample SB(i) only contained
quartz mineral content and showed that this sample was exposed
to a very high temperature, namely exceeding 950C. Samples SB(ix),
SB(xi) and SB(xix) contained mullite mineral content while sample
SB(xvi) has albite mineral content. Open burning technique was
used in the production of bricks at this site and it is proposed that
the firing temperature was between 750C and 850C or even higher,
exceeding 1000C. The x-ray diffraction pattern of the brick samples
of Sungai Batu Temple are in Figs 1-3.
Major element contents in the ancient brick samples of Sungai
Batu Temple are in Table 2. Analysis indicated that these brick
samples contained dry weight percentage of silica of between
66.53% and 80.09%. Dry weight percentage for titanium element
was between 0.40% and 1.13%. Iron element contained dry weight
percentage of between 1.56% and 3.46%. Dry weight percentage
for aluminium element was between 16.75% and 27.46%.
Table 1. Mineral content of ancient bricks from Sungai
Batu Temple.
Location
Sungai Batu
Methodology
A total of 19 brick samples were taken from the excavation site of
Sungai Batu (Site SB1) and taken to the lab for cleaning and labelled
with the names SB1, SB2, SB3, SB4, SB5, SB6, SB7, SB8, KSGC9,
SB10, SB 11, SB 12, SB 13, SB 14, SB 15, SB 16, SB 17, SB 18 and SB
19. Samples weighing 0.4 g were refined and heated up for one
hour at a temperature of 105C and mixed until homogenous with
the flux powder of a type of Spectroflux 110 (product of Johnson
and Mathey). These mixtures were baked for one hour in a furnace
with a temperature of 1100C. The homogenous molten was
moulded in a container and cooled gradually into pieces of fused
glass with a thickness of 2 mm and a diameter of 32 mm. The
samples were of 1:10 dilution. Samples in the form of fused glass
were prepared for analysis of major elements such as Si, Ti, Fe, Al,
Mn, Ca, Mg, Na, K and P2O2. Pressed pallet samples were prepared
for analysis of trace elements such as As, Ba, Ce, La, Nb, Ni, Pb,
Rb, Th, V, Y, Zn, U, Cr, Sr, Ga, Cu, Hf, Co and Zr. These samples
were prepared by mixing 1.0 g of samples together with 6.0 g of
boric acid powder and then pressure of 20 psi was applied by
using hydraulic pressure equipment. The samples of fused pallets
and pressed pallets were analysed using Philips PW 1480
equipment. Samples in the form of very fine powder were put into
the pellets (sample holder) and analysed using the X-ray diffraction
instrument (D500 Diffractometer SIEMEN). A scatter plot diagram
of TiO2 and MgO; Zr and V was performed to demonstrate if there
were any differences among the samples and analysed using
Microsoft Excel software. The applicability of the analytical
methods for the multi elemental analysis by XRF of the votive
tablets is evaluated by the analysis of certified reference material,
315 Fire Brick (Calibration: G_FBVac 28 mm) for major elements
and certified reference material, SY-2 (Calibration: Trace Element
P_20) for trace elements. The CRM was also used as the quality
control material of the analytical procedure.
Sample
SB (i)
SB (ii)
SB (iii)
SB (iv)
SB (v)
SB (vi)
SB (vii)
SB (viii)
SB (ix)
SB (x)
SB (xi)
SB (xii)
SB (xiii)
SB (xiv)
SB (xv)
SB (xvi)
SB (xvii)
SB (xviii)
SB (xix)
Mineral Content
SiO2Quartz
SiO2Quartz
KAl2Si3AlO10(OH)2 Muscovite 1M
SiO2Quartz
SiO2Quartz
KAlSi3O8 Microcline
SiO2Quartz
SiO2Quartz
K2O.3Al2O3.6SiO2.2H2O Muscovite
SiO2Quartz
KAl2Si3A1O10(OH)2 Muscovite 1M
SiO2Quartz
KAl2Si3A1O10(OH)2 Muscovite 1M
SiO2Quartz
Al3Si3K(OH)2O10 Muscovite 3T
Al5.65Si0.35O9.175Mullite
SiO2Quartz
KAlSi3O8 Microcline
SiO2Quartz
KAl2Si3AlO10(OH)2 Muscovite 2M1
Al2(Al2.8Si1.2)O9.6Mullite
SiO2Quartz
SiO2Quartz
SiO2Quartz
SiO2Quartz
SiO2Quartz
NaAISi3O8Albite
SiO2Quartz
SiO2Quartz
SiO2Quartz
(Al4SiO8)1.2Mullite
361
q,m
q,m
q
m
SB7
mq
SB13
q,m
q,m
q
m
SB11
SB6
q,m
m
q,m
q
m
mc SB4
q,m
SB3
ml
q
ml ml
q,m
q
m
SB11
SB5
Intensity(arbitrary
(Arbitraryunits)
Units)
Intensity
Intensity
(arbitrary
units)
Intensity
(Arbitrary
Units)
SB12
mc
SB10
ml ml
SB8
q,m
q,m
SB9
SB2
q
m
ml
q,m
SB1
20
Legend: q = Quartz, m = muscovite.
40
60
Figure 1. XRD diffraction pattern of bricks from Sungai Batu Temple.
q
m
2
20
40
2
Legend: q = Quartz, m = muscovite, mc = microcline, ml = mullite.
60
Figure 2.XRD diffraction pattern of bricks from Sungai Batu Temple.
Manganese has dry weight percentage of 0.01% to 0.03% while

calcium element contained dry weight percentage of between
0.30% and 0.10%. Dry weight percentage for magnesium and
sodium elements were 0.14-0.81% and 0.01- 0.10%, respectively.
Potassium and phosphorus elements contained dry weight
percentage of 0.38-1.05% and 0.02-0.10%, respectively.
Silica and aluminium content showed that these bricks have

higher sand content compared to clay. Dry weight percentage
graph of SiO2 and Al2O3 (Fig. 4) as well as dry weight percentage
graph of MgO and TiO2 (Fig. 5) for the brick samples in Sungai
Meriam Temple and clay in Bujang Valley were plotted to see the
result of the comparison between the brick samples and clay
samples according to major elements. Based on the graph, it
Table 2. Major element contents of ancient bricks from Sungai Batu
was found that the major element composition of Sungai Batu
Temple.
Temples brick samples has a slight difference based on its
Dry Weight (%)
Sample
Si
Ti
Al
Fe
Mn
Ca
Mg
Na
K
P2O3 silica and aluminium content while the composition content
SB 1
75.59 0.69 19.97 1.78 0.01 0.03 0.57 0.10 0.65 0.10 of magnesium and titanium showed similarities with the raw
SB 2
75.69 0.71 19.05 2.69 0.01 0.08 0.61 0.10 0.63 0.10 material in Sungai Baru, Sungai Bujang and Sungai Terus. It
SB 3
77.39 0.87 18.54 1.92 0.01 0.05 0.22 0.05 0.78 0.10
is clear that the raw material used is local raw material.
SB 4
69.42 1.10 23.10 2.23 0.02 0.04 0.18 0.05 0.45 0.03
Trace element contents for the brick samples of Sungai
SB 5
70.75 0.82 22.53 3.46 0.01 0.07 0.81 0.10 1.05 0.02
SB 6
72.04 0.95 21.11 2.90 0.01 0.04 0.18 0.06 0.74 0.05 Batu Temple (Tables 3-5) showed content exceeding 100 ppm
SB 7
76.10 0.84 20.22 2.41 0.01 0.04 0.17 0.05 0.82 0.06 for barium, cerium, chromium, vanadium and zircon. Other
SB 8
67.03 1.12 27.46 3.24 0.02 0.05 0.57 0.03 0.70 0.04
SB 9
70.03 1.09 24.8 2.50 0.01 0.05 0.48 0.10 0.86 0.04 elements were at a slightly lower concentrations, less than
SB 10
76.52 0.79 20.29 2.12 0.01 0.03 0.22 0.05 0.64 0.04 100 ppm. Barium element content was between 684 and 837
SB 11
66.53 1.02 23.48 3.01 0.02 0.03 0.17 0.06 0.76 0.10 ppm and for cerium element between 544 and 633 ppm.
SB 12
80.09 0.80 16.75 1.56 0.01 0.03 0.14 0.40 0.04 0.10
Chromium and vanadium element contents were between 8
SB 13
68.94 1.03 24.11 2.50 0.03 0.05 0.42 0.01 0.80 0.10
SB 14
69.47 1.13 25.14 3.12 0.01 0.03 0.49 0.05 0.38 0.02 and 104 ppm and 90 and133 ppm, respectively, while zircon
SB 15
70.18 0.87 25.53 1.66 0.01 0.04 0.31 0.05 0.63 0.03 element has concentration of 231-500 ppm. Fig. 6 shows the
SB 16
74.72 0.95 21.20 1.95 0.01 0.04 0.25 0.10 0.58 0.03 graph that was plotted to see the distribution of copper
SB 17
77.85 0.77 17.91 2.11 0.01 0.10 0.44 0.05 0.43 0.10
SB 18
76.79 0.80 19.86 1.72 0.03 0.03 0.18 0.05 0.62 0.10 element against lead for the brick samples of Sungai Batu
SB 19
74.15 0.40 21.64 2.03 0.01 0.06 0.47 0.05 0.38 0.10 Temple where the concentration of both elements is between
362
Table 3. Trace element content of ancient bricks

from Sungai Batu Temple.
q,m
Element
(ppm)
As
Ba
Ce
Co
Cr
Cu
Ga
Hf
La
Nb
Ni
Pb
Rb
Sr
U
Th
V
Y
Zn
Zr
SB19
q
ml ml
q,m
Intensity
(arbitrary
units)
Intensity
(Arbitrary
Units)
SB18 q
SB17
SB16
SB15
q,m
q
q,m
SB 1
10
712
596
8
74
12
24
8
30
36
29
44
63
16
9
18
90
5
52
320
SB 2
10
689
602
10
83
10
22
7
29
36
26
41
51
16
9
18
98
0
45
246
Sample
SB 3 SB 4
11
13
837
694
633
570
8
8
79
89
12
9
24
28
8
8
31
29
40
35
30
29
49
50
58
46
10
6
9
9
27
26
112
127
8
12
48
53
388
500
SB 5
16
685
567
21
103
12
28
7
29
33
28
45
109
31
9
24
111
13
61
231
SB 6
14
769
618
10
88
13
24
8
30
38
28
49
54
11
9
22
115
2
68
317
q,m
q
m
Table 4. Trace element content of ancient bricks from

Sungai Batu Temple.
q,m
SB14
2
20
2
Legend: q = Quartz, m = muscovite, ml = mullite.
40
60
Figure 3. XRD diffraction pattern of bricks from Sungai Batu Temple.

35
30
Al2O3 (%)
25
20
15
Sg. Batu
10
Lempung
5
0
20
40
60
SiO2 (%)
80
100
120
Figure 4. Dry weight percentage (%) of SiO2 and Al2O3 element for
the brick samples of Sungai Batu Temple and clay in Bujang Valley.
1.2
1
TiO2 (%)
0.8
0.6
Sg. Batu
0.4
Lempung
0.2
0
0.5
1
MgO (%)
1.5
Figure 5. Dry weight percentage (%) of MgO and TiO2 element for
the brick samples of Sungai Batu Temple and clay in Bujang Valley.
Element
(ppm)
As
Ba
Ce
Co
Cr
Cu
Ga
Hf
La
Nb
Ni
Pb
Rb
Sr
U
Th
V
Y
Zn
Zr
SB 7
13
729
600
7
80
11
27
7
28
40
27
48
68
7
8
23
102
4
46
353
SB 8
14
684
544
10
104
10
32
8
29
33
29
45
52
15
9
29
129
10
56
397
Sample
SB 9 SB 10
16
11
710
733
590
602
118
77
9
8
12
12
39
25
8
8
30
30
34
37
32
30
47
47
88
56
31
7
9
9
38
16
133
99
19
1
50
55
344
276
SB 11
13
725
590
14
98
10
23
6
27
40
21
43
83
21
8
20
102
6
60
280
SB 12
8
770
603
7
66
9
20
8
31
38
26
45
32
6
9
15
98
1
54
447
12 and 17 ppm and 48 and 62 ppm, respectively. The results showed

that there is one major source of raw material that was used and
based on lead and copper element concentrations, slight
differences were found with the clay in Bujang Valley, Kedah.
Although the trace element composition of the bricks found in
Sungai Batu Temple is slightly different from the trace element
composition of the clay available, based on the trace composition
content of the bricks in this site, it is more of local clay composition.
Data of clay composition around the Sungai Batu basin has yet to
be performed and most likely this raw material was sourced from
the areas in the vicinity of the site.
Conclusions
The study on material composition of the ancient bricks of Sungai
Batu Temple (Site SB1) shows that the bricks have almost similar
material composition as the clay in Bujang Valley, Kedah, which is
363
Table 5. Trace element content of ancient bricks from Sungai

Batu Temple.
Element
(ppm)
As
Ba
Ce
Co
Cr
Cu
Ga
Hf
La
Nb
Ni
Pb
Rb
Sr
U
Th
V
Y
Zn
Zr
SB 13
16
777
583
9
103
12
32
8
30
38
31
52
113
19
9
30
127
19
47
320
SB 14
14
715
586
6
93
10
28
9
32
38
28
50
82
10
7
24
110
3
50
282
SB 15
10
744
616
7
79
13
27
8
30
37
33
46
46
14
9
18
104
4
53
312
Sample
SB 16 SB 17
12
14
733
730
589
572
8
7
83
96
12
10
25
26
8
4
30
26
38
28
31
30
48
48
55
72
9
14
9
7
26
28
116
124
6
11
50
51
425
380
SB 18
9
753
607
7
75
11
22
8
30
40
29
47
53
7
9
16
105
2
42
307
SB 19
12
773
60
8
95
10
26
4
28
25
29
50
71
13
20
22
121
13
52
314
25
Cu (ppm)
20
15
Sg. Batu
10
Lempung
5
0
50
100
Pb (ppm)
150
200
Figure 6. Graph of lead and copper element concentration for the

brick samples of Sungai Batu Temple and clay in Bujang Valley.
based on major and trace elements which is similar to composition

of clay samples. The mineral content present in the ancient brick
samples consists of quartz, muscovite, microcline, mullite and
albite. The mullite mineral shows that some of the bricks were
baked at sufficient temperature of more than 550C. This shows
that the open burning technique was used in the production of
the temples bricks because some of the bricks have an indication
of low firing burning. The dry weight percentage graph of silica
and aluminium and magnesium and titanium as well as the lead
and copper concentration graph indicate that the raw materials
used to produce the ancient bricks are local raw materials and
these raw materials were obtained from the surrounding area of
Bujang District.
Acknowledgements
This study was conducted using the GGPM-2013-070 and II/004/
2012; and therefore, we would like to express our gratitude to
National University of Malaysia and Ministry of Higher Education
for the research grant awarded.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Estimating residents willing to pay using contingent valuation for ecological

restoration and recreational benefits of AL-Prespa protected area in Albania
Dorina Grazhdani
Agricultural University of Tirana, Faculty of Economy and Agribusiness, Kamz, Tirana, Albania.
e-mail: d.grazhdani@yahoo.com
Abstract
Protected areas (PAs) provide some important goods and services, thus making them an important contributor to social well-being. They are widely
regarded as the cornerstones of biodiversity conservation strategies worlwide, providing the main foundation for in situ conservation of flora and
fauna, being an important element of any sustainable development. The double impacts of climate change and biodiversity loss are major threats to
achieving the Millennium Development Goals, especially those relating to environmental sustainability, poverty alleviation and food and water
security. Protected undeveloped areas are an important tool for land conservation in developing nations. Efficient land allocation decisions and
resource management requires knowledge of non-market benefits. The contingent valuation method is a stated preference method of non-market
valuation where respondents are asked to state their preferences for an environmental good or service that is not bought and sold in traditional
markets. This study used contingent valuation method (CVM) to derive estimates of economic value for recreational benefits use of Albanian part of
Prespa Park (AL-Prespa), in southeastern Albania. The contingent valuation survey used a dichotomous choice (DC) format followed by an openended question asking for their maximum WTP for restoration. Park visitors were surveyed regarding their willingness-to-pay (WTP) for ecological
restoration and recreational benefits of area. The WTP responses were analysed using a probit procedure. The results of the study indicated that
visitors are willing to pay increased entrance fees to finance ecological restoration and recreational benefits of areas damaged by human activities.
Consumer surplus for ecological restoration and recreational benefits of the AL-Prespa elicited through the close-ended DC exercise was found to be
1.4 per person, and elicited through the open-ended exercise was found to be about 1.6. The factors and personal characteristics of the visitors that
affect respondents WTP for ecological restoration and recreational benefits damaged by human activities in AL-Prespa could be important inputs in
order to ensure successful environment protection. The results of this study also should be helpful to assist the authority for the fee system and how
much they can increase the new entrance fee charged to the visitors.
Key words: AL-Prespa, contingent valuation, willing to pay, non-market value, protected area, ecological restoration, recreational benefits.
Introduction
Protected Areas (PAs) are widely regarded as the cornerstones of
biodiversity conservation strategies worldwide, providing the main
foundation for in situ conservation of flora and fauna, and being
an important element of any sustainable development plan.
Furthermore, national parks and other PAs contribute to human
well-being in a variety of ways 26. However, economically efficient
resource management requires knowledge of the flow of park
benefits and costs, including non-market benefits 25. Public
benefits derived from a national park in the form of environmental
amenities and ecosystem services implies that the park contributes
to public welfare, and loss of the park or decline in park quality
could result in a loss in welfare 33. Information on the welfare
contribution or economic value of protected areas in developing
countries, however, is scarce.
Non-market valuation techniques, such as the Contingent
Valuation method (CVM) can be used to measure the economic
value of recreation in PAs. The CVM applications in the literature
range from examining WTP for public projects in urban areas 5 to
the protection of vast and isolated areas of tropical forest 19.
Recently, the use of CVM for improving PA management or
developing sustainable ecotourism in national parks has received
considerable attention 32, 34, 36. When it comes to the question of
restoration of ecological goods or services, the majority of previous
studies involve valuing the restoration of river flows and riparian

ecosystems 21, 30. The restoration of native land ecosystems has
also been investigated in the United Kingdom. It has been
suggested that contingent valuation can be used as way to gauge
peoples WTP for entrance fees to PAs 32, 34. Similar studies have
also estimated values for improving the conditions or infrastructure
of a park 20, 25. There is evidence that synergies between biodiversity
conservation and economic and social development exist, and
such synergies can be developed in protected area management9.
A few studies have combined the use of Travel Cost method
(TCM) and CVM to measure the economic benefits of protected
areas in the developing world 24, 29. This study employs the CVM,
as recommended by Cameron 6.
The purpose of this study was to estimate the recreational use
value accrued to visitors to AL-Prespa Park in southeastern
Albania, and the visitors WTP for restoration of areas affected
by human activities. In addition, valuations were tested for
sensitivity to information about the ecosystem services provided
by the park, as well as respondent characteristics.
Study area: Prespa Park is the first trans-border protected area in
the Balkans established in February 2nd 2000. It is situated in the
365
Balkan Peninsula at the cross-border area shared between Albania,

Macedonia and Greece (Fig. 1). It is a high-altitude basin, which
includes two inter-linked lakes, the Macro Prespa (259.4 km2) and
the Micro Prespa (47.4 km2) at approximately 850 m above sea
level. AL-Prespa enjoys a wide array of assets to attract visitors,
mostly due to its rich natural and cultural capital, which create the
opportunities for the development of the ecotourism during the
whole year. These assets include biodiversity (both emblematic
and endemic), landscapes and aesthetic values including lakes,
clean air, quiet and peaceful environment, mountains and wetlands,
cultural and high quality local food tradition, skilled craftsmen,
architecture and archeological sites. In the five villages round the
shoreline of Prespa, tourism is small-scale rural and family tourism.
At present, tourism to the area is mostly limited to seasonal visits
by tourists.
LEGEND
Political borders
Park borders
Kilometers
0 10 20 30
Figure 1. Prespa park area.
Data collection: Data used for the analysis were obtained from
personal interviews conducted at the AL-Prespa during July 2013.
A total of 134 visitors were interviewed. The contingent valuation
of the interview began by asking respondents how familiar, if at
all, they were with the human activities which damage the
environmental quality. Then, they were informed about the
magnitude and location of these activities and were confronted
by a scenario in which the park authorities are considering an
increase in the park admission fee to cover restoration expenses.
A card with the proposed fee increase (one of four values) was
then shown to the respondent, followed by the question: Please
consider how much you spend on recreation each year, including
the cost of this trip. Would you have been willing to pay this
extra amount per person during this visit?
The four price levels were 25%, 50%, 75%, and 100% increases
of the existing entrance fee of 1. Regardless of their response,
respondents were then asked to identify their maximum willingness
to pay an increased fee as an open-ended question.
The contingent valuation method: The contingent valuation
method (CVM) is a stated preference approach to the measurement
of the value of changes in the allocation of non-market
environmental and natural resources 27. The development of CMV
as a survey method over the last four decades has been one of the
major theoretical innovations in the field of resource and
environmental economics 1, 15, 16. The CMV is widely applied to the
problem of estimating economic values of goods and services
that are not traded in markets and for which no economic behaviour
is observable. These non-market characteristics are present when
366
the good in question is in the form of an environmental amenity.

As a result, contingent valuation is receiving increasing
application for estimating the economic value of environmental
goods.
The main assumption underlying CVM is that people have true,
but hidden, preferences for all kinds of environmental goods.
Furthermore, people are assumed to be capable of transforming
these preferences into monetary units 11. Cummings et al. 10
provided a high-quality state of the art assessment of the
technique with commentary from a range of interested parties
representing a variety of opinions.
Several problems with the CVM have been identified throughout
the literature and most of them result from the hypothetical nature
of the approach 17. CVM and its various components have been
studied extensively in the literature with numerous papers on its
strengths, weaknesses, applications and misapplication.
Meanwhile, there was an intensive debate over the CVM. The
debate included different CVM results with different framing of
the issue. A major strength of the CV method is that it is designed
to directly ask the respondent in a simulated market to respond to
monetary costs as they relates to their individual situation,
household, or spending habits. The 1980s experienced an explosion
in the number of CVM studies in the US and worldwide. In the
1990s, the CVM has become the dominant technique for the
valuation of non-market environmental costs and benefits. To
date, the importance of CVM remains undimmed 2, 8.
Typical weaknesses and criticism of CVM relate to its
consistency with economic theory and the utility function 13, 17, 31.
The second set of identified weaknesses of CVM surrounds the
dependence of CVM on surveys and the various weaknesses of
survey methodology. Another commonly cited weakness of CVM
process is that the monetary values assigned by the respondent
are determined or created by the survey process. Yet another
weakness of the CVM process is that respondents may not have
the background knowledge to make informed decisions typically
requested in CVM and thus may be prone to surveyor or other
influences. Finally, a major weakness of CVM is that the survey
itself cannot be verified by a second, follow-up effort, or following
the scientific method replicated by other parties 12-14, 17, 27.
To address these concerns and mitigate potential weaknesses,
recommended protocols have been developed for the CVM
process. A major step to this end was achieved through an
evaluative process and report of the National Oceanic and
Atmospheric Administration (NOAA) Panel on Contingent
Valuation 4. To ensure the integrity of CVM the NOAA panel
recommends that all CVM survey efforts meet prescribed
requirements 4 pertaining to sampling, response rates, a preference
for interviews over mail or telephone surveys, pre-testing,
preference for referendum-style CV, follow-up and related
questions to aid interpretation, background on substitutes, and
economic trade-offs and constraints.
In recent CVM studies, a single bound dichotomous choice
(DC) format has been used to address some of these concerns.
Respondents are asked whether they would be willing to pay a
specified amount for the good or service in question, followed by
an open-ended survey in which they are asked to provide their
maximum WTP 30. In the closed-ended DC portion, different
respondents are confronted with different prices. Conceptually,
this process should result in a downward sloping demand curve,
as more respondents are expected to respond positively to low

prices and vice versa for a normal good.
In this study, the single bound DC format with the follow-up
maximum WTP is used to reduce the potential for starting-point
bias. Each model is described in turn below. Hypothetical bias
and payment vehicle bias are not expected since the interviews
were conducted in the park entrance. It is possible to test for
information bias through survey design, but warm glow bias will
only be evident ex post if the positive responses are overwhelming.
Close-ended dichotomous choice WTP model: Many CVM
studies, including the one presented here, apply the DC elicitation
format as recommended by Carson et al. 7. The DC CVM involves
sampling respondents and asking if they would vote in favour of
a referenda and pay a particular randomly assigned dollar amount.
The decision process of a respondent to the DC survey can be
modelled in a simple utility framework following 18. Let the utility
or satisfaction of a visitor to AL-Prespa be given by:
u = u (y, x, q)
where utility (u) is a function of income (y), personal characteristics

(x) and the quality of the park (q) as perceived by the visitor.
Furthermore, let there be two states of the world corresponding to
different levels of environmental quality: q as the quality after
restoration of the park and q as the quality before restoration or
if restoration is not pursued. If A is the amount the respondent is
asked to pay to finance restoration of the park, the respondent
will choose to pay (i.e., give a yes response) if:
u (y - A, x, q) u (y, x, q)
In essence, the individual is willing to pay the specified amount

(A) if more satisfaction is received by paying the amount (income
becomes y - A) and having the park restored than in the status
quo where income is unchanged and the park is not restored.
Thus, the researcher observes a binomial random variable that
takes a value of 1 if the individual is willing to pay A or 0 if they are
not. This variable is assumed to reflect the underlying and
unobserved response variable y*i that is described by:
y*i = xi + i
where xi is a vector of respondent characteristics and i is an error

term. While y*i is unobservable, we observe the binomial variable
that takes a value of 1 if y*i > 0, or a value of 0 otherwise. The
probit model is obtained by modeling the probability that yi takes
a value of 1:
Pr (yi = 1) = Pr (i xi) = 1 F ( xi)
where F is the standard normal distribution 23. A logit procedure

could also have been used as in previous studies 34, 36, but both
procedures are likely to yield similar results.
Open-ended follow-up WTP model: Respondents who were
willing to pay the predetermined amount in the DC portion of the
survey were expected to have a maximum WTP that was at least
as high as the predetermined amount. Respondents who were not
willing to pay the predetermined amount could be willing to pay a
lower amount than they were initially asked. Some respondents,

when asked about their WTP above the entrance fee, said the fee
was already too high.
These respondents have a negative WTP for restoration given
the existing entrance fee. The questionnaire did not accommodate
negative WTP values so the minimum value was zero, which means
the dependent variable is censored at zero. A censored regression
model is known as the Tobit model, which also assumes that there
is an underlying response variable (i.e. the true WTP), defined by
the regression relationship:
y* = X + ,
y = y* if y* 0,
y = 0 if y* < 0,
where X is a vector of respondent characteristics and is a normally

distributed error term 28.
Exploration of the factors and personal characteristics that affect
respondents WTP for restoration of areas damaged by human
activities in AL-Prespa requires inclusion of these characteristics
in the empirical equations. Summary statistics and descriptions of
the included variables are shown in Table 1.
Dichotomous choice results: The WTP responses were analysed
using two probit models. The models were estimated to account
for two possible functional forms assumed by the price variable.
Given the small number of observations and the relatively small
variation among prices, there was no a priori information
regarding the correct functional specification for these models.
The results of the analysis are summarized in Table 2.
While not all the variables included in the analysis were
statistically significant, there were several variables that were
statistically significant in both models. Price was statistically
significant at the 5% level in both models. Both the parameter
estimate and the marginal effect have a negative sign. This implies
that as the proposed entrance fee shown to respondents increased,
they were less likely to agree to pay the extra money for ecological
restoration. Marginal changes are shown along with the base
values with which they were calculated.
Respondents who expressed that they are regular visitors to
other National Parks or other natural areas (RVisit) exhibited a
higher probability of being willing to pay for restoration than
those who did not. Regular visits to natural areas may indicate a
pro-environment attitude, which can be expressed as WTP for
restoration of damaged ecosystems. Regular users of natural
amenities also have higher direct-use values that result from more
frequent use and enjoyment of these amenities.
Survey respondents traveling in tour buses (TourBus), most of
whom were traveling in large groups, exhibited a higher probability
of responding yes to the DC question. It was hypothesized that
respondents traveling in large groups would be less likely to
respond yes, as they were told that the additional fee would
apply to everyone in their group. However, tour travel may also
reflect an individuals desire to be part of a group that is interested
and concerned with nature, thereby resulting in a higher WTP for
restoration. On the other hand, tour bus visitors may perceive
entrance fees as sunk costs, and they may see the increased
367
Table 1. Summary statistics of variables in the contingent valuation analysis (N = 134).

Variable
WTP_Yes
Description
1 if WTP specified fee,
0 otherwise
Max_WTP
Maximum WTP
Ln (Max_WTP)
Natural logarithm of Max_WTP
Price
Specified fee increase
Ln (Price)
Natural logarithm of Price
Interaction
Product of WTP_Yes x Price
FirstVis
RVisit
PrimPurpose
TourBus
FireAware
EcoAware
Version
Child
Older
Income
Member
Gender
1 if first visit park,

0 otherwise
1 if visit regularly park,
0 otherwise
1 if visit is primary purpose of trip,
0 otherwise
1 if used a tour bus,
0 otherwise
1 if aware of human activities,
0 otherwise
1 if aware ecological services
0 otherwise
1 if CV before awareness questions,
0 otherwise
1 if minor present in group,
0 otherwise
1 if older than 40,
0 otherwise
Residential strata
(1 - 6)
1 if member to environmental organization,
0 otherwise
1 if male,
0 otherwise
Mean (St. Dev.)

0.813
(0.393)
6.742
(5.080)
7.783
(2.8220
0.503
(0.225)
8.402
(0.523)
3.968
(2.794)
0.703
(0.460)
0.594
(0.495)
0.813
(0.393)
0.281
(0.453)
0.234
(0.427)
0.531
(0.503)
0.516
(0.504)
0.469
(0.503)
0.375
(0.488)
3.578
(1.166)
0.141
(90.350)
0.625
(0.488)
entrance fee in relation to the total cost of the package, which is

relatively small.
Respondents were queried on their awareness of three different
ecosystem services provided by AL-Prespa: hydrologic regulation,
habitat for threatened or endangered species, and carbon
sequestration, and were given an EcoAware value of 1, if they
had prior knowledge of all three services. Contrary to expectations,
those respondents who knew about these services had a lower
probability of responding yes. This seems counterintuitive, as
individuals more familiar with the services provided by an
ecosystem are expected to value that ecosystem more than those
who are not. However, more knowledge regarding the services
provided by an ecosystem may also be an indication of more
knowledge about this particular ecosystem or about ecology in
general.
Open-ended WTP results: Responses were analysed using a
censored Tobit model. Three different models were regressed in
order to accommodate different functional forms of the WTP_Yes
and Price variables and to test for starting-point bias. The results
are summarized in Table 3.
The Interaction variable, which was included to account for
the anchoring effect, was found to be positive and statistically
significant in all models. Respondents who were showed higher
bids (A) in the closed ended exercise were more likely to have
higher maximum WTP amounts. There are no statistically
368
Min.
Max.
30.0
10.3
2.0
8.0
7.6
9.0
significant coefficients for the Price and WTP_Yes variables in

models 2 and 3. This may imply that the anchoring effect is
accounted for with the interaction of the price shown to the
respondent and their likelihood of responding yes to the exercise.
The variable indicating if the respondent was visiting the park
for the first time (FirstVis) was associated with positive and
statistically significant coefficients in models 1 and 2, indicating
that repeat visitors experience diminishing marginal utility.
The parameter estimate associated with the Version variable
was positive and statistically significant in all three models. This
indicates that respondents who went through the CV exercise
before they were asked about their awareness of the ecological
services provided by the park showed a higher WTP. It was
expected that respondents who were asked about their awareness
of the ecological services provided by the park, and thus informed
or reminded about these services, before going through the CV
exercise would show a higher WTP, as they had more information
about these services. The results, however, did not support this
hypothesis. It is possible that respondents perceived the extra
information about ecological services as a blatant attempt to
increase their WTP, or they resisted paying for a public good.
Respondents who were traveling with children showed a higher
WTP than those who did not, as indicated by the positive and
statistically significant parameter estimate associated with the
Child variable in models 1 and 3. This result could be an indicator
of bequest values.
Table 2. Probit results of the dichotomous choice CV survey.

Variable
Model 1
Intercept
Ln (Price)
FirstVisit
RVisit
PrimPurp
TourBus
FireAware
EcoAware
Version
Child
Older
Income
Member
Gender
Model 2
Intercept
Ln (Price)
FirstVisit
RVisit
PrimPurp
TourBus
FireAware
EcoAware
Version
Child
Older
Income
Member
Gender
Parameter
Estimate p-value
Marginal Effect
(dy/dx)
p-value
Table 3. Tobit results of the open-ended willingness to

pay models.
Variable
0.375
-2.397**
0.671
1.352**
1.008*
0.861*
0.709
-1.307**
0.508
-0.485
0.657
0.381
0.803
-0.962
0.981
0.048
0.347
0.031
0.074
0.074
0.307
0.026
0.345
0.534
0.342
0.266
0.435
0.205
-0.041**
0.1108
0.254**
0.257
0.085
0.093
-0.217**
0.0891
-0.0789
0.164
0.075
0.089
-0.134*
0.034
0.456
0.024
0.169
0.087
0.209
0.033
0.317
0.561
0.187
0.364
0.208
0.078
4.853
0.682
0.604
0.982
0.307
0.295
0.605
0.601
0.576
0.463
3.671
0.165
0.652
6.974*
-0.987**
0.658
1.265**
0.965*
0.907*
0.587
-1.165**
0.4074
-0.425
0.678
0.212
0.682
-1.018**
0.078
0.048
0.402
0.028
0.056
0.078
0.207
0.031
0.277
0.643
0.334
0.264
0.408
0.043
-0.187**
0.123
0.0253**
0.321
0.132*
0.056
-0.1945**
0.084
-0.045
0.056
0.023
0.083
-0.153**
0.038
0.487
0.028
0.231
0.075
0.172
0.046
0.321
0.582
0.201
0.321
0.252
0.047
7.899
0.789
0.602
0.768
0.341
0.234
0.342
0.444
0.488
0.405
3.758
0.132
0.652
Intercept
WTP_Yes
Price
Interaction
FirstVis
RVisit
PrimPurp
TourBus
FireAware
EcoAware
Version
Child
Older
Income
**, * statistical significance at the 0.01, 0.05, and 0.10 level, respectively.
Younger survey respondents were found to have higher WTP

values for restoration than respondents who were at least 40
(Older). Environmentalism is a recent phenomenon in the
developing world, and pro-environment attitudes may be more
common among the young. Older individuals may also be more
sceptical that the additional fees collected would be used for
restoration of AL-Prespa.
Income, as proxied by the tax stratum of the respondents home 3,
was positively related to maximum WTP for restoration. This supports
the theory higher incomes result in larger demand for goods and
services. Individuals with higher incomes have more disposable
income that can be spent in recreation or ecological restoration.
The coefficient associated with the Member variable indicates
that individuals who are members of, or donate money to,
environmental organizations have a higher WTP for ecological
restoration than those who do not. This is not a surprising result,
as these individuals were expressing their non-market environmental
preferences through the survey.
The results obtained in the analysis highlight several important
issues concerning protected area management in the developing
world. At the forefront of these issues is the role of non-market
valuation in the improvement of park management. Non-market
valuation methods are particularly suitable for answering
questions concerning visitors WTP for improvements in park
management or park quality. Moreover, non-market valuation can
help answer questions like what are parks worth to society?
Such questioning of the worth or value of parks to society is
incomplete without an examination of the role and purpose of PAs
in the 21st century. In this section, the results obtained from this
study are used to better understand these issues, particularly as
Member
Gender
Model Statistics:
R2
Adj. R2
Model 1
Model 2
Model 3
Parameter Estimate (p-value)
-4.632*
-3.656
-5.4362*
(0.087)
(0.228)
(0.072)
N/A
N/A
2.7641
N/A
N/A
(0.196)
N/A
-0.4224
N/A
N/A
(0.142)
N/A
0.9665***
1.8087***
0.8157***
(< 0.0001)
(< 0.0001)
(0.005)
2.1467*
2.0789*
1.8761
(0.087)
(0.077)
(0.207)
-0.7451
-1.0543
-1.0876
(0.654)
(0.432)
(0.409)
0.3025
-0.0357
-0.1987
(0.866)
(0.879)
(0.988)
0.3087
0.3098
0.4351
(0.833)
(0.843)
(0.841)
-0.0352
-0.0609
-0.1277
(0.895)
(0.619)
(0.944)
0.1873
0.3421
0.3832
(0.823)
(0.796)
(0.765)
2.7863**
2.7654**
2.4953**
(0.021)
(0.033)
(0.046)
2.0423**
1.8533
1.9874*
(0.047)
(0.143)
(0.082)
-2.9156**
-2.9398**
-2.9235**
(0.034)
(0.033)
(0.031)
1.3877***
1.4532***
1.3654***
(0.008)
(0.005)
(0.007)
3.9087**
3.8765**
3.7926**
(0.024)
(0.032)
(0.027)
-0.3856
-0.2059
-0.2065
(0.666)
(0.694)
(0.588)
0.68
0.56
0.69
0.57
0.66
0.55
***, **, * statistical significance at the 0.01, 0.05, and 0.10 level, respectively.
they relate to AL-Prespa. However, the discussion is extended to

other PAs in the developing world.
The average WTP for restoration elicited through the closeended DC exercise was found to be about 1.4 per person. The
average WTP for restoration elicited through the open-ended
exercise was found to be about 1.6.
Conclusions
Economic valuation of protected areas, in general, and of parks
and reserves in the developing world, in particular, is an area
where little research exists but much more is needed. Most parks
in the developed and developing world alike are highly valuable
but are extremely undervalued under the existing institutional
framework, opening the way for other non-conservation land uses.
Research on the total economic value of these parks and reserves
can fill an important gap and allow decision-makers and the general
public to make more informed choices regarding these important
social assets.
Furthermore, visitors are willing to pay increased entrance fees
to finance restoration of areas damaged by human activities.
However, park authorities are cautioned in regards to the use of
increased entrance fees as a way to protect PAs from overcrowding
and overuse, as high fees may prevent large segments of the
population from the enjoyment of these natural assets, thereby
369
decreasing their value to society. Consumer surplus measures of

the economic value created by parks would, for example, decline if
access to PAs was curtailed. Economic valuation of protected
areas is an important component of sustainable park development,
where increased access and enjoyment of parks does not diminish
the provision of ecosystem services and the protection of
ecological processes.
Acknowledgements
The author would like to acknowledge the Agricultural University
of Tirana for the financial support of this study and the colleagues
of Agribusiness Department of this University for outstanding
support to improve the consistency and quality of the work.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Jatropha cake as a fertilizer for the growth of Blc. Amy Wakasugi Yamanashi orchid
Roberto A. Ribeiro *, Maria Flvia R. Starling and Luiza A. R. Rossi-Barbosa
State University of Montes Claros, Bocaiuva Campus, Professora Augusta Ribeiro Drumond Street, 441 39390-000,
Bocaiuva/MG, Brazil. *e-mail: roberto.ribeiro@unimontes.br, flapth@hotmail.com, luiza.rossi@unimontes.br
Abstract
Jatropha curcas is oil plant which has been used to obtain biodiesel once produces oil with potential as raw material to this application. Jatropha cake
is a waste product of the process of oil extraction from seeds that can be utilized as a fertilizer. The objective of this work was to investigate the
potential of Jatropha cake as a fertilizer for the growth of Blc. Amy Wakasugi Yamanashi orchid. Sixty seedlings were treated with Jatropha cake,
enriched castor cake, calcium nitrate, chemical fertilizer and their mixtures. Chemical analyses were done to verify nitrogen, phosphorus and
potassium contents in these fertilizers. The length, the width and the largest part of the aerial unit were measured, and the number of leaves was
determined to evaluate the development of the orchids. Compositions (g kg -1) of Jatropha cake and enriched castor cake were, respectively, nitrogen
38.5 0.4 and 29.3 0.7, phosphorus 5.60 0.23 and 36.81 0.43, potassium 25.6 0.8 and 21.0 0.7. Slight differences in the length and width
were observed among the orchids subjected to the different treatments over nine months. For the number of leaves, the treatments with Jatropha cake
were found to exhibit the highest values over nine months, whereas the measurements of the largest part of the aerial unit revealed that the use of
enriched castor cake was slightly better than the use of the other fertilizers. The results indicated that Jatropha cake exhibits potential for application
as a fertilizer for this orchid.
Key words: Jatropha cake, fertilizer, orchids.
Introduction
Fossil fuel reserves are known to be decreasing around the world.
This fact has demanded research into the development of
alternative fuels, such as biodiesel, that can be produced through
the transesterification reaction of vegetable oils 1. Oil plants, such
as soybean, sunflower and rapeseed, have been used to
commercially produce biodiesels. Studies have demonstrated the
availability of using non-edible oil extracted from the feedstock,
for example, Jatropha curcas 2.
There are different possibilities of using the Jatropha plant in
addition to the production of oil for biodiesel production 3, 4. The
plant has been used as a living fence and for soil erosion control.
Biogas and fertilizers are produced from the seed cake, and soap
that exhibits biocidal and medicinal properties is prepared from
the oil. Toxic substances (phorbol esters and curcin) in Jatropha
seeds prohibit its use as food for animals and humans. However,
a non-toxic variety of the plant was identified in Mexico, and the
toxicity level can be reduced via chemical treatments.
After oil extraction, a high amount of Jatropha cake is obtained.
Economic and environmental issues have led researchers to
investigate the potential use of this waste in industrial applications.
Acid pre-treated Jatropha cake exhibited efficient Cr(VI) biosorption
from wastewater 5. The maximum adsorption capacity was 22.727
mg of Cr(VI)/g of biosorbent at 30C, and the thermodynamic
properties indicated that the Cr(VI) sorption process is
spontaneous and endothermic.
The extracted fibres from the Jatropha plant are used as
reinforcement materials in composites 6. Materials composed of
Jatropha cake combined with either glass fabric-epoxy 7 or styrene-
butadiene rubber have exhibited appropriate chemical and

physical properties for the production of biocomposites. Through
the incomplete combustion of the extracted fibres, the biomass is
converted to carbon black, which can be used as a polymer filler7,
as well as activated carbon 8, which is obtained from thermic or
acid treatment of the carbon black.
The nitrogen, phosphorus and potassium contents make
Jatropha cake attractive for use as a fertilizer. Kumar and Patra 9
found that Jatropha and mustard cake supplements increased the
yield and modified the chemical composition of the essential oil of
Mentha piperita L. These organic wastes were used in earthen
pots filled with garden soil and fly ash at different compositions.
Composting of de-oiled Jatropha cake with rice straw and animal
dung was tested in mustard, sorghum, vigna and wheat plants,
and the resulting germination percentage was assessed 10. The
use of horse dung exhibited the best results of the average
germination percentage: 94.2%, 73%, 60.1% and 79.5% for wheat,
sorghum, vigna and mustard, respectively. Moreover, a low
concentration of phorbol ester (0.12 mg/g) was observed in the
composted material with the highest proportion of de-oiled cake.
Orchids are used as ornamental plants due to their beauty; of
course, they require nutrients to grow 11. According to the Brazilian
Institute of Florists, from January to May (2011), Brazil imported
US$ 2.34 million of orchid seedlings from Holland, Thailand and
Japan12. The Brazilian production of orchids is low, and investments
are necessary to promote such production. In general, the
consumption of orchids and other flowers tends to increase,
requiring the use of appropriate agricultural techniques and
371
manures. Jatropha cake can be a feasible option as a fertilizer for

orchids. The present work investigated the potential use of
Jatropha cake as a fertilizer for the growth of Blc. Amy Wakasugi
Yamanashi orchid.
Preparation and chemical characterization of the fertilizers:
Jatropha seeds were acquired in the Biojan NNE Minas
Agroindstria Limitada of Janauba city. The cake was obtained
through hot extraction, triturated in the knife mill and stored in
dark packing. Organic manure used by the Norte Mineira
Orchidophiles Society, which was composed of castor cake,
vegetable ashes, bone flour and chicken dung (enriched castor
cake) 13, was also investigated. This organic manure was processed
using the same procedure used for the Jatropha cake. Calcium
nitrate and chemical fertilizers were used as purchased.
The nitrogen content was determined using the micro-Kjeldahl
method, and spectrophotometry and flame photometry were used
to determine the potassium and phosphorus contents,
respectively14. Triplicate determinations were performed in this
study.
Preparation of the orchids in the study: Seedlings of Blc. Amy
Wakasugi Yamanashi orchids were acquired from Binot of
Petrpolis/RJ. The plants, which were approximately 2 years old
and exhibited four pseudobulbs each, were placed in 0.5-L pots
filled with gneiss rock and Macauba coconut husk. The orchids
were kept under a screen that exposed them to a 70% level of
sunlight for one and a half months before beginning the
experiment.
Evaluation of the development of the orchids: The first three

leaves of each pot were identified, and both the length and the
width of each of the leaves were measured using a digital
pachymeter. The width measurement was performed at the half
length of each leaf. The number of leaves of each plant was
obtained by counting the leaves for each plant. These measurements
were performed prior to the first application of the manures and
over nine months of growth. After nine months, the largest part of
the aerial unit of a new leaf per pot was measured.
Statistical analysis: A comparison of the mean values was
performed using one-way ANOVA, and the Tukey test was used
to identify the treatments that resulted in a significant difference
in the plant growth at the 0.05 level of significance.
Physical and chemical characterizations of the fertilizers: The
distribution of the grain sizes of the Jatropha cake and the enriched
castor cake are presented in Fig. 1. The grain sizes of the enriched
castor cake exhibited a heterogeneous distribution, with 24.1% of
the largest grains being smaller than 0.500 mm, whereas a higher
amount (56.7%) of the Jatropha cake exhibited a homogeneous
distribution, with grain sizes smaller than 0.840 mm. The enriched
castor cake is composed of materials with different textures, which
were submitted to the same grid for grinding. Thus, the mixture
resulted in a heterogeneous material relative to the grain sizes.
The Jatropha cake is merely vegetable material that contributes to
the production of a more homogeneous material.
Jatropha
castor
cake
Jatrophacake
cake Enriched
Enriched
castor
cake
Treatments to grow the orchids: Sixty seedlings were organized

into 12 groups, with five plants in each group. For each group, a
different composition of fertilizer was prepared, as presented in
Table 1.
The irrigation of the plants was performed daily using wellwater at pH 8.0. Jatropha cake and enriched castor cake were
applied at intervals of 2 months, whereas calcium nitrate and
chemical fertilizer were applied at intervals of 15 days. Before
application of the Jatropha cake and the enriched castor cake, the
plants were watered with deionized water for approximately 16 h
and 40 min, and depending on the treatment, they received calcium
nitrate and chemical fertilizer as well. The fertilizer solutions were
sprayed in the superior and inferior parts of the leaf.
Granulometric distribution (%)
60
50
40
30
20
10
0
0.2
Concentrations
0
25 mL.pot-1
25 mL.pot-1
25 mL.pot-1
8 g.pot-1
8 g.pot-1 + 25 mL.pot-1
8 g.pot-1 + 25 mL.pot-1
8 g.pot-1 + 25 mL.pot-1
8 g.pot-1
8 g.pot-1 + 25 mL.pot-1
8 g.pot-1 + 25 mL.pot-1
8 g.pot-1 + 25 mL.pot-1
*1 g.L-1 concentrations for the calcium nitrate and chemical fertilizer solutions. The use of both in the same treatment, with
a 0.5 g.L-1 concentration used for each fertilizer.
372
0.6
0.8
Sieve size (mm)
1.0
Figure 1. Distribution of the grain sizes of organic fertilizers.
Table 1. Treatments used to grow the orchids.

Fertilizer types
Control (T1)
Calcium nitrate (T2)
Chemical fertilizer (T3)
Calcium nitrate + chemical fertilizer (T4)*
Enriched castor cake (T5)
Enriched castor cake + calcium nitrate (T6)
Enriched castor cake + chemical fertilizer (T7)
Enriched castor cake + calcium nitrate + chemical fertilizer (T8)
Jatropha cake (T9)
Jatropha cake + calcium nitrate (T10)
Jatropha cake + chemical fertilizer (T11)
Jatropha cake + calcium nitrate + chemical fertilizer (T12)
0.4
The chemical composition of the fertilizers is

presented in Table 2. A comparison of the Jatropha
cake and the enriched castor cake indicated that the
highest difference in the compositions of these
materials is the amount of phosphorus, which is
justified by the composition of the enriched castor cake
that has bone flour rich in phosphate. The nitrogen
and potassium contents present in the Jatropha cake
are higher than the values obtained by Chaturvedi and
Kumar 15 of 2.95 and 1.0 g/100 g, respectively, whereas
the P content is smaller (0.83 g/100 g).
Evaluation of the development of the orchids: The
initial and final measurements of the mean length of
Table 2. Macronutrients of the manures used in the growth of the Table 3. D values of the orchid leaves calculated for the cases of
orchids.
before the application of fertilizer (Di) and over 9 months
(Df) of treatment.
Nitrogen Phosphorus Potassium Proportion
Fertilizer
g N.kg-1
29.30.7
38.50.4
250.98.7
Enriched castor cake

Jatropha cake
Chemical
g P.kg-1
36.810.43
5.600.23
39.416.56
g K.kg-1
21.00.7
25.60.8
133.44.6
N: P: K*
3:8:3
4:1:3
25:9:16
Treatments
T1
*Quantities per 100 g of sample for N, P as P2O5 and K as K2O.
Initial After
After99months
months
Initial
16
T2
Length of leaves (cm)
14
12
10
T3
8
6
4
T4*
2
0
2
6 7
8
Treatments
10 11 12
Figure 2. Mean length of the leaves for each treatment of

the Blc. Amy Wakasugi Yamanashi orchid.
the leaves are shown in Fig. 2. There is no significant difference

among the various treatments over nine months according to the
ANOVA test at the 0.05 level (F = 1.98016 and p = 0.05180). Similar
behaviour was observed in the analysis of the mean width (F =
1.79270 and p = 0.08175).
Table 3 presents the product of the length and width
measurements (D) for one leaf of each pot (the results of the other
two leaves are not presented). These results indicated that some
of the final values were lower than the initial values. Once the
length and the width of the first leaves were measured, it is likely
that some of them had their growth disturbed. Thus, the final
values of the length and the width are lower than the initial values,
leading to low values for the product (D).
Fig. 3 presents the number of leaves determined for each of the
treatments, which increases over the nine months. From the T9
(Jatropha cake) to T12 (Jatropha cake + calcium nitrate) treatments,
the highest differences in the number of leaves during the
experiment were obtained. Nevertheless, the measurements of the
largest part of the aerial unit revealed that the use of enriched
castor cake (the T5, T6, T7 and T8 treatments) was slightly better
than the use of the other fertilizers (Fig. 4). New leaves were not
observed for the orchids of the control treatment (T1).
Jatropha cake, or its association with either the chemical fertilizer
or the amount of calcium nitrate, was found to have the appropriate
composition of macronutrients to grow the orchids. Unfortunately,
the plants submitted to treatments based on enriched castor cake,
which is used by orchidophiles, did not exhibit suitable conditions
for the development of the orchids, even though this manure
exhibited smaller-sized grains (Fig. 1) that contribute to the
absorption of macronutrients. According to these results, the
macronutrient proportion present in the manure likely is a relevant
aspect in the growth of this type of orchid. Wang 16 investigated
the effects of various concentrations of soluble fertilizer on the
growth of the phalaenopsis orchid and found that the mean leaf
T5
T6
Df/ cm2
24.956
25.014
14.36805
25.86105
12.41625
43.66526
41.19311
23.07582
24.6756
20.1522
39.32
45.792
47.4
18.879
17.00475
105.77357
54.24725
96.22714
99.83122
72.8717
37.485
52.857
69.92
16.7167
33.2556
10.5493
15.9327
36.03597
34.13093
90.82368
Treatments
T7
T8*
T9
T10*
T11
T12
Di / cm2
24.905
57.13
21.144
23.2001
51.7762
41.4232
28.2576
20.46465
39.66405
28.43165
26.7618
40.62165
53.41245
33.47872
32.51395
37.8708
51.48
60.3603
22.4612
73.16588
55.5528
15.10604
15.5496
46.754
39.32195
42.661
11.79524
24.3903
7.35318
36.72
Df/ cm2
29.0719
103.00125
32.16975
46.9718
21.9773
42.53779
50.6482
20.76588
66.2642
79.86062
33.23852
18.8445
40.80113
21.243
15.50174
50.7585
51.3604
61.55095
5.3636
53.8593
57.96067
14.92302
15.62252
48.63098
39.82804
43.94039
10.86492
22.45456
6.842
37.87251
*Mean values of Df exhibited a significant difference at the level 0.05 using the Tukey test in relation to the
control treatment. T4: F = 41.77952 and p = 0.00020; T8: F = 8.94072 and p = 0.01733; T10: F = 5.33798
and p = 0.04966.
Initial After
After99months
months
Initial
amonginitial
inital and
and after
after 99months
months
Difference
Difference among
55
50
45
40
Leaf number
Di / cm2
29.6682
25.2925
15.74955
23.72055
16.276
11.3364
52.4667
22.81838
25.0355
21,27375
25.0355
46.276
45.98825
14.03825
17.2724
102.62763
52.8066
89.7974
91.2912
70.1807
37.7349
51.4402
71.8716
19.7736
32.984
11.09395
15.65078
33.7644
33.20488
83.752
35
30
25
20
15
10
5
0
6 7
8
Treatment
10 11 12
Figure 3. Total number of leaves for each treatment.
number was greater when the following fertilizer types were used:
10N: 13.1P: 16.6K and 15N: 8.7P: 20.8K. Parameters such as leaf
size, total leaf area, shoot and root fresh weight did not exhibit
significant differences among the fertilizer types studied.
Jatropha cake has demonstrated attractive properties for its
use as a fertilizer for various plants. Lycopercicum esculatum
(tomato) was grown in soil with different proportions of Jatropha
cake 15. The use of 3% cake in plants during 60 days of the stages
of growth enhanced the levels of total soluble solids, vitamin C,
proteins, reducing sugars and tomato fruit yield. Studies
performed by Srinophakun et al. 17 indicated that the application
of Jatropha cake associated with chemical fertilizer or farm manure
373
Length of largest aerial part unit (cm)
30
27
24
21
18
15
12
9
6
3
0
2
6
7
8
Treatment
10 11 12
Figure 4. Measurements of the largest part of the aerial

unit of the orchids for each treatment.
resulted in the appropriate conditions for the growth of Chinese

kale, tomato and sweet potato. The best yields were obtained for
the use of the half rate of chemical fertilizer mixed with 10 and 2.5
t/ha of Jatropha cake for Chinese kale and sweet potato,
respectively, whereas all of the rates studied exhibited good results
for tomato crops. Moreover, phorbol ester residues were not found
in the leaves, fruit or soil.
In this work, the potential of Jatropha cake as a fertilizer to grow
Blc. Amy Wakasugi Yamanashi orchid was verified. The results
indicated that the use of Jatropha cake to grow this orchid was
not significantly higher than the use of a chemical fertilizer. Thus,
the use of Jatropha cake should not entirely replace the use of
chemical fertilizer; however, the use of Jatropha cake is associated
with the decreased amounts of chemical compounds in the soil
and the plants in addition to the reduced costs of planting.
Studies regarding orchids are scarce in the literature, and
important aspects concerning the growth and nutrition of these
plants require scientific investigations to improve the conditions
of growth and consumption of orchids.
Conclusions
Jatropha cake exhibits the potential to be used as a fertilizer for
the Blc. Amy Wakasugi Yamanashi orchid. The application of
Jatropha cake to offset the use of chemical fertilizer is
recommended to decrease the amount of chemical compounds
present in the environment and to reduce the costs of planting.
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Bose, A., Kavita, B. and Keharia, H. 2011. The suitability of Jatropha
seed press cake as a biosorbent for removal of hexavalent chromium
from aqueous solutions. Bioremediat J. 15:218229.
6
Abdul Khalil, H. P. S., Sri Aprilia, N. A., Bhat, A. H., Jawaid, M.,
Paridah, M. T. and Rudi, D. 2013. A Jatropha biomass as renewable
materials for biocomposites and its applications. Renew. Sust. Energy
Rev. 22:667685.
7
Mohan, N., Natarajan, S., Kumaresh Babu, S. P. and Siddaramaiah 2011.
Investigation on sliding wear behaviour and mechanical properties of
Jatropha oil cake-filled glass-epoxy composites. J. Am. Oil Chem.
Soc. 88:111117.
8
Kurniawan, A. and Ismadji, S. 2011. Potential utilization of Jatropha
curcas L. press-cake residue as new precursor for activated carbon
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composition of essential oil of Mentha piperita L. plant: Effect of fly
ash amendments and organic wastes. Ecol. Eng. 47:237241.
10
Das, M., Uppal, H. S., Singh, R., Beri, S., Mohan, K. S., Gupta, V. C.
and Adholeya, A. 2011. Co-composting of physic nut (Jatropha
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Hew, S., Lim, L. Y. and Low, C. M. 1993. Nitrogen uptake by tropical
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flowering of Phalaenopsis orchid. Sci. Hortic. 65:191-197.
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Acknowledgements
We thank Fundao de Amparo Pesquisa do Estado de Minas
Gerais (FAPEMIG) and Rede Mineira de Qumica and Biojan NNE
Minas Agroindstria Limitada for their support.
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WFL Publisher
Helsinki, Finland
www.world-food.net
In vitro regeneration of Acacia crassicarpa A. Cunn Ex Benth through organogenesis

from juvenile sources
Griffin Akeng 1, Sures Kumar Muniandi 2 and Nor Aini Ab Shukor 2, 3*
Forest Department of Peninsular, Terengganu Forestry Training Centre, Malaysia Headquarters, Jalan Sultan Salahuddin,
50660, Kuala Lumpur, Malaysia. 2 Department of Forest Management, Faculty of Forestry, 3 Institute of Tropical Forestry and
Forest Product, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. e-mail: anishukor@yahoo.com,
smallmammal2006@yahoo.com, griffin@forestry.gov.my.
1
Abstract
Micropropagation through tissue culture technique offers an alternative to traditional vegetative propagation to mass propagate selected trees for largescale forest plantation. Therefore, this study aimed to develop a protocol for the micropropagation of A. crassicarpa. Nodal stem segment and leaf
obtained from 2 month-old aseptically germinated seedlings were used as explant in this study. Nodal stem segment was found to be the most appropriate
explant for shoot formation when cultured on a MS medium supplemented with 6-benzylaminopurine (BAP). The highest mean number of shoots (5)
and the longest mean shoot elongation (8 mm) occurred on a medium supplemented with 0.5 mg l-1 BAP. The longest mean shoot length (8 mm) and the
highest mean number of explants per culture (7) were obtained on medium without any plant growth regulator. When cultured on a medium supplemented
with 2,4-dichlorophenoxy acetic acid (2,4-D), nodal stem segment explant developed roots and callus (after 14 days). The highest mean number of roots
(8.3 = 8) and the longest mean root length (12.0 = 12 mm) were obtained from the medium supplemented with 10.0 and 2.0 mg l-1 2,4-D, respectively.
The highest mean number of roots (20.6 = 21) and the longest mean root length (10.4 = 10 mm) were obtained from the medium supplemented with 10.0
and 2.0 mgl-1 2,4-D, respectively, while the highest intensity of callus was produced on a medium supplemented with 8.0 and 10.0 mg l-1 2,4-D, and which
was only able to produce root without any shoot formation. The calli produced were compact, watery and white in colour. Survival rate of plantlets was
higher (100%) when transferred into the autoclaved mixture of soil, sand and peat (3:3:1) than those transplanted in an unautoclaved soil mixture (6.6%).
Survival percentages of the plantlets in the culture room and greenhouse condition were 85 and 100%, respectively.
Key words: A. crassicarpa, micropropagation, organogenesis, plant growth regulator, cytokinin, auxin, leaf segment, nodal segment, Murashige and
Skoogs medium (MS), in vitro rooting, in vitro shooting, acclimatisation.
Introduction
The planting of exotic tree species in Malaysia has started long
time ago since as early as 1893 in peninsular Malaysia with the
introduction of Eucalyptus species 1. The planting of these tree
species was actually aimed at rehabilitating vast areas that suffered
from shifting cultivation within the permanent forest estates/
reserve, as a fire-break and as boundary markers in the plantation
plots 2. One of the main species that has been chosen for these
purposes is Acacia with the introduction of Acacia auriculiformis
back in 1932 and Acacia mangium in 1966. The genus Acacia is
a member of the Leguminous family, sub family Mimosoidae. It
encompasses about 1100 species, in which over 850 occurs in
Australia and its neighbouring countries, i.e. Papua New Guinea
and Indonesia. The remainder are endemic largely to Africa and
Tropical America. Acacia species are fast growing multipurpose
trees, suitable for timber, pulp and paper production and various
end products such as furniture 3.
Since the introduction of A. mangium in Sabah back in 1966, it
has become an important plantation species in Malaysia, where
most of the forest plantation development was based mainly on
this species. Because of the successful introduction of A. mangium,
keen interest was generated to test the suitability of other related
Acacia species such as A. crassicarpa, A. aulococarpa and A.
auriculiformis. These species also provide alternative to
A.mangium when there is a need to alleviate the underlying
dangers of monoculture 4. Among these species, A.crassicarpa is
becoming increasingly popular among commercial planters. It has

shown good growth performance, possesses good wood quality
and adapts well to a wide range of environments 5. The estimation
of adaptability and stability suggested that A. crassicarpa was
best for various sites tested 4. Growth trials of this species also
showed better growth than A. mangium and A. auriculiformis
on certain site 6. It ranked the third best of the Acacia species
according to the Consultative Group for Research and
Development of Acacia in Southeast Asia 7. Since then, it has
been widely planted in Indonesia as well as in the southern
provinces of China for reforestation, reclamation of wastelands,
and industrial material production 7-9. However, it produced very
small amounts of seeds, which might be due to its small crown
and few branches. Its flowering period and duration are also
uncertain where flower development occurs once every 2 - 3 years.
Therefore, regeneration of this species for large-scale plantation
establishment is still a problem.
Despite the ease by which most Acacia species can be
propagated by seed, there is a need for vegetative propagation if
rapid breeding is to take place 10. Micropropagation technique
can facilitate such effort by allowing mass propagation of
individual selected for desired characteristic and the potential to
overcome the above problem. Micropropagation system employed
in plantation forestry has been used for large-scale production of
commercially important tree species with desired characteristic to
375
achieve maximum and faster genetic gain 11. There have been
reports stating that some Acacia species, such as A. mangium1214
, A. auriculiformis 15, 16, A. nilotica 17-19 and A. sinuata 20, 21, have
been successfully micropropagated via tissue culture techniques.
Apart from the callus induction using leaf and stem segments of
plant 22, regeneration using phyllode segment also has been
reported for A. crassicarpa 23, 24. The present study was undertaken
to determine suitable explants, appropriate plant growth regulators
and medium required for culture initiation and maintenance. In
this paper, we report regeneration of A. crassicarpa through
organogenesis in calli cultures from leaf and nodal segment of
aseptically germinated seedlings
Plant materials and disinfection of explants: Mature and healthy
seeds of A. crassicarpa were collected from a 5 year-old
provenance trial at Universiti Putra Malaysia (UPM), Serdang,
Malaysia. Prior to germination, the seeds were pre-treated by
soaking them in boiling water at 120C for 2 min to soften the seed
coat. Treated seeds were sterilized with 70% (v/v) ethanol for 2
min with three rinsing in sterile distilled water. Seeds were further
sterilized in 15% commercial Clorox (containing 5.25% sodium
hypochlorite) for 15 min and rinsed thoroughly three times with
sterile distilled water. The seeds were germinated in test tubes
containing half strength hormone-free MS medium. When the
seedlings were 2 months old, two types of explants were excised;
namely leaves and nodal stem segments. Leaf and nodal segment
explants were cut into 5.0 mm 5.0 mm and 3.0 - 5.0 mm long in
size, respectively.
Media preparation and culture conditions: Three types of media
tested in this study were Murashige and Skoogs basic medium
(MS) 25, B5 medium (B5) 26 and Woody Medium (WPM) 27. MS
and B5 media were obtained from FLOWLABTM (Sydney, Australia)
while the WPM was supplied by DUCEFA (Haarlem, the
Netherlands). MS, B5 and WPM media were prepared by weighing
their powder of 4.41, 3.75 and 2.46 g, respectively. The powder
was mixed together with other additives such as agar and plant
growth regulators and dissolved in ultra-pure water to make up to
1 litre. Amount of 30.0 g l-1 sucrose was also added during the
preparation of the medium. The pH of the medium was adjusted to
5.5-5.8 before dissolving Difco-Bacto agar (7.5 g) into the medium.
This medium was poured into borosilicate test tubes and the mouth
was closed with a plastic cap. They were autoclaved at 121C for
15 min. Plant growth regulators were filter sterilised with 0.02 m
membrane filter and added to the media after autoclaving under
sterile condition. All cultures were maintained under cool-white
fluorescent light with a 16-h photoperiod and incubated at 25
2C.
Effects of basal medium, plant growth regulators and their
interaction on morphogenesis of leaf and nodal stem segment
explants: Following surface disinfection and pre-treatment, each
explant was trimmed and placed individually in culture tubes
containing 15 ml of three types of medium (MS, B5 and WPM)
supplemented with various concentration (0, 0.5, 1.0, 2.0, 4.0, 6.0,
8.0 and 10.0 mg l-1) of cytokinins (BAP) and auxin (2,4-D). The
experiment, utilized a factorial design followed with 20 explants
per treatment. Initial morphogenesis was assessed weekly and
376
photographs of the culture were taken after 8 weeks. Data for

shoot formation including mean number of shoots, mean shoot
length, shoot elongation, number of explants obtained per culture,
mean number of roots, mean root length and callus formation
were collected after 8 weeks of culture incubation. All cultures
were maintained as condition described above.
Effects of sub-culturing on the number of shoots produced: The
shoots utilised in this experiment were obtained from the ones
produced on MS medium supplemented with 1.0 and 2.0 mg l-1
BAP. Shoots from the previous culture were used for the following
subculture. Sub-culturing on a fresh medium was done every 30
days (once a month) and assessment of shoots produced was
assessed monthly. These shoots were cultured in 25 mm 85 mm
borosilicate test tubes, closed with plastic caps and sealed with
parafilm.
In vitro rooting: Shoots initiated using 2.0 mg l-1 BAP from the
initial culture and the fourth subculture where shoot produced
were 5 - 6 cm long, were transferred to half strength MS basal
medium and B5 medium containing various concentrations (0, 0.5,
1.0, 2.0 and 5.0 mgl-1) of auxin 1-naphthaleneacetic acid (NAA)
and indole-3-butyric acid (IBA). The control comprised of shoots
transferred to auxin-free half strength MS medium. The experiment
followed a complete randomised design (CRD) with 20 replicates
per treatment. Root ability was monitored for percentage of shoots
rooted, mean root number and mean length of root per explant for
30 days. Root ability between shoots obtained from the initial
culture and fourth culture were also made. A total of 20 explants
from each culture were used and cultured on the MS medium
supplemented with 2.0 mg l-1 NAA.
Acclimatisation: Partial plantlet autotrophy was stimulated as
early as the in vitro stage through the gradual reduction in the
relative humidity in the culture environment. Ten week-old plantlets
were subjected to 3 stages of experiment. The first stage involved
cultures, which were placed under the room temperature of 25
2C for a period of 3 - 5 days. At the second stage, the cultures
were placed near the window to expose them to a higher
temperature, a lower relative humidity and longer light period for
14 days. Finally, rooted plantlets were rinsed thoroughly in distilled
water and transplanted into polyethylene bags containing a
mixture of autoclaved soil, sand and compost (3:3:1) and covered
with plastic sheet. The newly transplanted plantlets were placed
in the culture room under a control condition and watered regularly.
During the last three days, the plastic sheet was removed and
plantlets were exposed to room environment (Fig. 1E). The plantlets
were left in culture room for 5 periods (1, 2, 3, 4 and 5 weeks) for
hardening and each period consisted of 20 replications. The plants
were taken out and grown in polyethylene bags containing a
mixture of soil:peat:quartz sand (1:1:1) after one month in the
greenhouse.
Statistical analysis: The data was analysed for their variance
using Statistical Analysis System (SAS) computer package. The
Duncan Multiple Range Test (DMRT) comparison was used to
determine significant differences. Differences found at the
probability level of P < 0.05 were considered to be significant.

Type of explants for shoot initiation: Nodal segment explants
responded very well in terms of shoot formation when cultured
on three types of medium supplemented with various concentration
of BAP. Axillary buds and adventitious shoots started to
proliferate from nodal segment explants after 14 days in culture
incubation. The shoots were healthy, green in colour and produced
juvenile compound leaves. Leaf explants on the other hand, failed
to undergo organogenesis when cultured on a medium with BAP
and were swollen and died after 6 - 7 weeks in culture. Nodal
segment has been reported to be appropriate explant choice for
propagation of other Acacia species, such as A. auriculiformis 15,
16
, A. nilotica 19, A. sinuata 20, A. mearnsii 28 and A. mangium 12, 29.
Nodal stem segment explants were found to undergo rhizogenesis,
callus induction and true-to-type plantlet formation when cultured
on a medium containing 2,4-D. It is likely that cell ability to develop
rhizogenesis somehow acts as a trigger for organogenesis or the
other way round, especially under appropriate plant growth
regulator conditions. Formation of true-to-type plantlet directly
from nodal explants was earlier reported for Eucalyptus alba 30.
This phenomenon was also reported by Hsia and Korban 31 on
internodal stem segment of Rosa hybrida.
Effects of different type of medium on shoot initiation and root
formation: All of the parameter tested was not significantly
affected by different types of basal media, with the exception of
the mean number of shoots. The appearance of shoot was similar
in all media. These results were consistent with those obtained by
Juddy 32 where she reported that there was no significant effect of
different types of media (MS, B5 and WPM) on the proliferation
of shoot of Azadirachta excelsa. However, among three types of
media tested, MS medium was found to produce the highest mean
number of shoots (4.0) and the longest mean shoot length (5.4
mm) while WPM and B5 media were found to produce the longest
mean shoot elongation (6.5 mm) and the highest mean number of
explant per culture, respectively, (3.5) (Table 1). In another study,
Table 1. Effects of different types of basal medium on the
morphogenesis of nodal stem segment explants.
Medium
MS
B5
WPM
Mean
number of
shoots
4.0 (1.9a)
3.6 (1.9a)
1.7 (1.2b)
Mean shoot
length
(mm)
5.4 (2.2ab)
4.7 (2.1b)
5.1 (2.2a)
Mean shoot
elongation
(mm)
6.4 (2.5a)
6.5 (2.5a)
6.5 (2.5a)
Mean number of
explants obtained
per culture
3.4 (1.8a)
3.4 (1.8a)
3.5 (1.8a)
Values expressed in parenthesis indicate square root transformation. The values with same superscripts
are not significantly different at P < 0.05 based on Duncans Multiple range test.
MS media also proved to be more appropriate than B5 medium

and produced a similar number (4.23) of shoots with 43.2% shoot
regeneration frequency 19. This difference might be due to the
higher ratio of NO3-/NH4+ (nitrate/ammonium) in MS compared to
B5 and WPM, which may favour good growth of buds, although
other differences in the media cannot be excluded as contributing
to this response. This confirmed postulation made by Bonga 33
where lower NO3-/NH4+ and higher KNO3 in the medium were
believed to be factors that could enhance shoot formation.
Effects of BAP on morphogenesis of nodal stem segment and
leaf explants: Cytokinins were believed to control shoot formation
through their direct action upon enzyme activities, which among
others could control processes in cell division 34. In this study, it
was found that BAP alone when supplemented with 0.5 - 10.0
mg l-1 in each type of media (MS, B5 and WPM) was effective to
stimulate axillary shoot bud development in A. crassicarpa. A
similar range of BAP was utilised to initiate axillary shoots in A.
koa 35. Rajadurai et al. 36 also reported a successful attempt to
initiate shoot proliferation in A. mangium but utilizing a wider
range of BAP, i.e. from 0 to 20.0 mg l-1. BAP concentrations showed
significant effects on all of the parameters tested.
The highest percentage of shoot formation (71.7%) from nodal
segment explants was obtained in the medium supplemented with
1.0 mg l-1 BAP (Table 2). The highest mean number of shoots
(4.5) was obtained on medium supplemented with 2.0 mg l-1 BAP
(Fig. 1A). This was concurrent with the result obtained by Khayri
and Gbur 37 on A. mearnsii, where the optimum multiple shoot
bud formation (2.6 = 3) was obtained with 2.0 mg l-1 BAP. Galiana
et al. 38 also reported that 2.0 mg l-1 BAP was optimum for shoot
formation in A. mangium where 14 to 18 axillary shoots were
produced per explant. A similar amount of BAP concentration
required in multiple shoot induction of mature nodal segments of
A. auriculiformis but addition of 0.1 mg l-1 of NAA further enhanced
the percentage of shoot multiplication 16.
These results, however, were slightly different from those
obtained by Rajadurai et al. 36 that a higher concentration of BAP
(5.0 mg l-1) was better for the proliferation of shoot in A. mangium,
which produced an average of 4 shoot buds per explant. In some
cases, BAP in combination with other cytokinins required much
lower concentration to induce maximum shoot induction. Haliza
et al. 15 found that nodal segment of 14 month-old seedling
produced the highest shoot regeneration (67%) with 6.8 mm mean
shoot length when sub-cultured with combination of 0.5 mg l-1
BAP and 0.1 mg l-1 kinetin. Moreover, they also found that more
Table 2. Effects of BAP on the morphogenesis of nodal stem explant after 8 weeks in culture.
BAP
(mgl-1)
0
0.5
1.0
2.0
4.0
6.0
8.0
10.0
Percentage of
explants
producing shoots
41.7
60
71.7
46.7
55
50
50
48.3
Mean
number
of shoots
3.7 (1.8)bc
3.1 (1.7)c
4.1 (1.9)ab
4.5 (2.0)a
3.3 (1.8)bc
2.1 (1.5)d
2.3 (1.5)d
1.6 (1.3)e
Mean
shoot
length (mm)
8.5 (2.7)a
5.7 (2.3)b
5.5 (2.3)b
5.2 (2.2)bc
3.9 (2.0)d
3.2 (1.7)e
4.6 (2.1)cd
3.9 (2.0)d
Mean
elongation
of shoots (mm)
4.8 (2.2)d
8.1 (2.8)a
7.9 (2.8)a
6.8 (2.6)b
7.5 (2.7)ab
5.9 (2.4)c
5.2 (2.3)cd
5.4 (2.3)cd
Mean number
of explants
obtained per culture
6.7 (2.6)a
3.3 (1.8)c
3.2 (1.8)c
4.5 (2.1)b
2.0 (1.4)f
2.2 (1.5)ef
3.0 (1.7)cd
2.6 (1.6)de
Intensity
of callus
formation 1
+
+
++
++
Morphological
appearance
of shoot 2
+
+
+
++
++
++
++
Values expressed in parenthesis indicate square root transformation. The means with same superscripts are not significantly different at P < 0.05 based on Duncan Multiple Range Test.
1: Number of (+) sign indicates the extent of callusing, single plus (+) represent minimum and four pluses (++++) indicates maximum amount of callusing. 2: The (-) sign indicates shoots
showing sign of necrosis, single plus (+) indicates less than 10% of the cultures producing curly leaves shoot with stunted growth, two pluses (++) indicates 11 to 30% of cultures producing
curly leaves shoot with stunted growth, three pluses (+++) indicates 31 to 50% of the cultures producing curly leaves and stunted growth and Four pluses (++++) indicates > 50% of
the culture producing curly leaves with stunted growth.
377
in the medium supplemented with lower concentration or without

BAP. Shoots showing apical necrosis were very severe in the
medium without BAP where they produced yellowish long and
tiny internodes with curly leaves. Upon longer culture of more
than 4 weeks, the leaves slowly detached, which subsequently
led to death.
The inhibitory effects of cytokinins on the elongation of axillary
shoots were reported by Sukartiningsih et al. 40 on Gmelina
arborea, where BAP exceeding 10.0 mg l-1 was found to cause
fewer shoot elongation. The inhibitory effects of BAP were also
reported by Juddy 32 on micropropagation of A. excelsa. However,
the inhibitory effect of BAP on A. excelsa was only apparent at a
concentration level of 6.0 mg l-1, but for this study the inhibitory
effects of BAP were observed even at a lower concentration, i.e,
4.0 mg l-1.
The longest elongation of shoots (8.1 mm) was obtained from
those cultured in medium supplemented with 0.5 mg l-1 BAP.
However, the longest mean shoot length (8.5 mm) and the highest
mean number of explants produced per culture (6.7) were obtained
from those cultured in medium without any plant growth regulators.
The interaction of different types of basal medium and BAP caused
significant effects in terms of mean shoot number and length but
not on shoot elongation and number obtained per culture.
Figure 1. A) The highest mean number of shoots (4.5) was obtained on

medium supplemented with 2.0 mg l-1 BAP; B) Dwarf-like shoot formed
from nodal stem segment when cultured on the B5 medium supplemented
with 8.0 mg l-1 BAP after 3 weeks in culture incubation; C) Microshoot
cultured on MS medium supplemented with 5.0 mg l-1 IBA showing the
highest mean root number (11.0 mm) after 30 days in culture incubation;
D) Microshoot cultured on MS medium supplemented with 1.0 mg l-1
NAA showing longest mean root length (25.5 mm) after 30 days in
culture incubation; E) Plastic covers were removed after 3 days in
greenhouse; and F) Established plantlets of A.crassicarpa after 1 month
in soil mixture. * (white bar = 3.0 cm).
mature explants (72 month old) required only 0.1 mg l-1 BAP in
combination with 0.1 mg l-1 kinetin to produce the highest shoot
regeneration (50%) with much shorter mean shoot length (2.0 mm).
These differences might be mediated by the differences in the
endogenous levels of growth hormones in the species 39.
There is a tendency for shoot growth with increased BAP
concentration in term of mean number of shoot produced.
However, further increase in BAP concentration (exceeding 2.0
mg l-1) has resulted in a decrease in the number of shoots produced
per explant, and also formation of more abnormal shoots (Fig. 1B).
The abnormal dwarf like shoots had shorter internodes and
thicker leaves and they formed compact clumps, which were
difficult to separate. Shoots of this type tend to remain stunted
and produced more bud-like structures around the base of the
explants. Based on the fact that BAP acts on nucleus-based events
or on membranes in the cytoplasm, it is expected to produce toxic
effects beyond a certain levels. So far, no information whether
cytokinins possess inhibitory effects as most of these negative
results with other species was undoubtedly been left unreported 34.
Apical necrosis was observed to occur on the shoots produced
378
Effects of 2,4-D on morphogenesis of nodal stem segment and

leaf explants: Roots and callus started to develop from leaf and
nodal stem segment explants, while complete plantlets only
developed from nodal stem segment explants cultured on media
(MS, B5 and WPM media) supplemented with 2,4-D. Thin long
roots developed either individually or in clusters at the edge of
the leaf explants. Among the three types of media tested, the
highest mean number of roots produced from leaf (8) and nodal
stem segment (3.8) were achieved in the WPM and B5 media,
respectively, while the longest mean root length for leaf (8.0 mm)
and nodal stem segment (6.3 mm) were both achieved in the WPM
medium (Table 3).
Table 3. Effects of different types of basal medium on the
morphogenesis of leaf and nodal stem segment explants.
Medium
MS
B5
WPM
Leaf
Mean number
Mean root
of roots
length (mm)
6.5 (2.2b)
4.6 (2.2a)
6.9 (2.4a)
3.9 (2.0b)
8.0 (2.1a)
8.0 (2.2b)
Nodal stem segment

Mean number
Mean root
of roots
length (mm)
3.1 (2.2a)
3.0 (2.2b)
3.8 (2.0b)
4.4 (2.3b)
3.4 (1.8c)
6.3 (2.9a)
Values expressed in parenthesis indicate square root transformation. The means with same superscripts
are not significantly different at P < 0.05 based on Duncans Multiple Range Test.
The highest mean number of roots for leaf (20.6) and nodal stem
segment explants (8.3) were both achieved in the medium
supplemented with 10.0 mg l-1 2,4-D while the longest mean root
for leaf explants (10.4) and nodal stem segment (12.0 mm) were
achieved in the medium supplemented with 2.0 mg l-1 2,4-D (Table
4). Callus started to develop from the cut end for leaf segment
after one week in culture incubation and produced compact white
and watery callus in all concentration tested. Callus turned
yellowish and started to produce roots after 5 weeks. The highest
intensity of callus produced (+++), from both leaf and nodal stem
segment explants were obtained in the medium supplemented with
10.0 mg l-1 2,4-D.
The pattern of rhizogenesis in this study was similar to that
Table 4. Effects of various concentration of 2,4-D on the morphogenesis of leaf and nodal segment explants
after 30 days in culture incubation.
2, 4-D
(mgl-1)
0
0.5
1.0
2.0
4.0
6.0
8.0
10.0
Mean number
of roots
0.2 (0.5e)
1.4 (0.3e)
1.2 (0.3e)
2.6 (1.1d)
2.9 (2.2c)
9.3 (3.8b)
18.7 (4.2a)
20.6 (4.4a)
Leaf segment
Mean length
Intensity of
of roots (mm)
callus formation 1
0.4 (1.5d)
+
5.0 (2.0bc)
+
9.5 (2.9a)
+
10.4 (3.1a)
++
2.7 (2.1b)
++
2.0 (1.5d)
++
3.4 (1.5d)
+++
4.1 (1.7cd)
+++
Mean number
of roots
0.8 (1.3d)
1.3 (0.9e)
1.4 (1.0e)
2.0 (1.4d)
3.3 (2.1c)
3.7 (2.3c)
6.6 (2.6b)
8.3 (4.0a)
Nodal stem segment

Mean length
Intensity of
of roots (mm)
callus formation 1
1.1 (1.9de)
+
2.9 (1.9de)
+
3.9 (2.1cd)
++
12.0 (4.2a)
++
2.7 (1.9de)
++
4.3 (2.4c)
+++
3.9 (1.8e)
+++
5.7 (3.2b)
+++
Values having the same superscripts are not significantly different at P < 0.05 based on Duncans Multiple range Test. Values expressed in parenthesis indicate square root
transformation. 1: Number of (+) sign indicates the extent of callusing, single plus (+) represent minimum and four pluses (++++) indicates maximum amount of callusing.
described for Acacia lebbeck 41. Rajadurai et al. 36 also reported

similar observation on leaf and nodal stem explants of A. mangium
and Pithecellobium jiringa cultured on the medium supplemented
with 2,4-D (0 - 10.0 mg l-1). However, the exact concentration of
2,4-D that proved to be most effective was different in each case.
This could be possibly due to the different requirement of auxin
for a different species. There was no clear distinction in the
appearance of roots produced from nodal stem segment from those
produced from leaf explants. Both of them showed similar
characteristics such as having rudimentary roots with the
secondary roots attached to them. The roots produced from leaf
explants were believed to originate from the rachis part of the leaf
while in the nodal stem segment roots were believed to develop
from the phloem parenchyma, similar to that demonstrated in P.
jiringa 36.
Besides root formation, the presence of 2,4-D in all the media
tested was found to induce callus proliferation in both leaf and
nodal stem segment explants. It produced compact, white and
watery callus. This type of callus was considered as nonembryogenic, which is unable to produce somatic embryos
through induction of direct somatic embryogenesis. On the other
hand, an embryogenic callus was compact and nodular in
appearance and is often surrounded by friable and translucent
non-embryogenic callus 42. The formation of callus in this study is
believed to originate from the cell patches or nodules, which
exist in the hypocotyls and nodal stem explants.
After the eighth week, the callus turned yellowish and roots
started to emerge from its surface. A histological study on callus
of Malus revealed that the friable callus was embryogenic, which
was rich in starch and having thick cell walls 43. The induction of
callus from leaf and stem segment explants of the same species
has been reported earlier 22 where the callus produced was friable
and light green in colour. However, this contradicts with the results
obtained by Rout et al. 44 on A. catechu that the callus produced
from cotyledon explants cultured on medium containing 2,4-D
was friable and yellowish in colour.
Despite the fact that 2,4-D has long been associated with root
formation and callus induction, its presence in tissue media was
found to stimulate axillary buds proliferation. As observed in
this study, nodal stem segment explants of A. crassicarpa have
the tendency of developing complete plantlets when cultured in
medium with 2,4-D. The proliferation of axillary buds occurred
simultaneously with the formation of roots from the implanted
part of the nodal stem segment. The plantlets produced from the
implanted part of nodal stem explants are physiologically
connected to the axillary shoots or just attached to the basal end
of explants. The roots developed mainly from the implanted part

of the explants or attached to the surface of callus produced at the
implanted part of the nodal stem segment explants. A similar
observation has been reported for Eucalyptus alba 30 where the
emergence of shoots occurred simultaneously with root formation.
Brown and Sommer 45 also reported that formation of both shoots
and roots from callus cultures of Populus tremuloides, indicated
association in the plantlet formation.
Formation of callus at the cut end of the nodal stem segment
explants in this study did not affect axillary buds initiation and
root formation, even though it is believed that its presence at the
cut end of the explants could suppress axillary buds proliferation.
Rajadurai et al. 36 reported that the presence of callus at the cut
end of hypocotyl explants of A. mangium suppressed axillary
buds initiation where they remained dormant for some time or
failed to develop at all. The presence of callus in a small amount,
however, was believed to increase the multiplication rate of
plantlets. This happens only when adventitious buds regenerated
simultaneously or earlier than the initiation of callus.
Effects of sub-culturing on the number of shoots and multiplication
rate: The ability of shoots to multiply rapidly in vitro is essential
for the establishment of economically feasible micropropagation
systems. Usually, once proliferating shoot cultures of particular
tree species have been stabilized, they can provide a continuous
source of microshoots. In this study, multiplication of shoots has
been achieved by repeated sub-culturing of shoot tips at a 30-day
interval for 5 months where the mean number of shoots produced
and multiplication rate were assessed. It was observed that the tip
portion of explants stopped to elongate while shoot buds were
differentiated from the sides and developed prolific buds,
indicating the easy release of the axillary buds. The cut ends of
the cultures showed marginal callusing activity in every subculture
and its presence did not seem to affect the multiplication of shoot.
It was found that the multiplication rate of shoots increased
with subsequence sub-cultures. This occurred only after the fourth
sub-culture of shoots cultured on medium supplemented with 2.0
mg l-1 BAP, where the highest multiplication rate (4.2 = 4) was
achieved in the 4th culture (third subculture) (Table 5). The
multiplication rate of shoots started to decrease from the fourth to
the subsequent subcultures. Explants cultured on medium
supplemented with 1.0 mg l-1 BAP, showed no clear trend of the
multiplication rate at each subculture where the highest
multiplication rate (2.3 = 2) was produced in the second and fourth
subcultures. Repeated sub-culturing caused production of white
and spongy callus at the basal end of the shoot. The morphological
379
Table 5. Number of shoots produced and multiplication rates

from every subculture for a period of 5 month (30
days per subculture).
Number
of
culture
1st
2nd
3rd
4th
5th
6th
1.0 mgl-1 BAP

Number
Multiplication
of shoots
rate
produced
20
43
2.2
97
2.3
137
1.4
315
2.3
420
1.3
2.0 mgl-1 BAP

Number
Multiplication
of shoots
rate
produced
20
54
2.7
151
2.8
635
4.2
1651
2.6
3467
2.1
feature of callus was similar for each subculture. This finding is

similar to those obtained by Aziah et al. 46 on A. mangium A.
auriculiformis hybrids, that the multiplication rates for the six
hybrid clones tested varied between 1.6 and 2.5.
In vitro rooting and acclimatisation: Rooting of shoots is an
essential step in tissue culture before they can be established in
the field. It can be carried out either in vitro or in vivo. In most of
the tissue culture work on Acacia, exogenous auxin was required
to initiate adventitious rooting in vitro. However, Williams et al. 47
reported that adventitious rooting of shoots of A. ligulata occurred
when cultured in auxin-free medium. Auxin IBA and NAA used in
this study were found to be effective for initiation of adventitious
rooting of A.crassicarpa, especially when they were cultured in
MS media supplemented with 0.5 - 5.0 mg l-1. Such inclusion in the
rooting medium was believed to promote rooting, thus producing
a higher percentage of rooting than those cultured without auxin.
For example, the percentages of root formation on medium
supplemented with 0.5 and 2.0 mg l-1 of IBA were 55 and 65%,
respectively, compared to only 25% for those cultured on an auxinfree medium. A similar observation was made in the experiment
with NAA where the percentages of rooting on the medium
supplemented with 0.5 and 2.0 mg l-1 NAA were 40 and 60%,
respectively, compared to only 10% on medium without NAA.
Mittal et al. 48 who conducted similar study on A. auriculiformis
reported that 24% of shoots rooted when cultured on medium
supplemented with 1.0 M NAA, but none of them produced
root when no hormone was included. Galiana et al. 38 also reported
a higher percentage of rooting (60%) with in vitro shoots of A.
mangium were cultured on medium containing 0.05 mg l-1 IBA
than those cultured on a hormone-free medium (10%). These results
were in agreement with those obtained by Sukartiningsih et al. 40
on G. arborea that the inclusion of IBA in rooting medium has
increased the frequency of rooting and shortened the time required
for root initiation. Auxin as a rooting promoter is believed to
influence the initiation of adventitious rooting by its direct effect
on processes like gene activation, protein synthesis and membrane
functions 49. On the other hand, the initiation of adventitious root
on auxin-free medium was mainly dependent on the endogenous
auxin. This type of auxin is produced in young leaves and shoot
buds and was translocated downward within the phloem stream
to the rooting zone at the base of shoots, which also forms a sink
for other compounds like nitrogen, carbohydrates and other
synergists necessary for rooting process.
Similar range of concentrations of auxin (0.5 - 5.0 mg l-1) were
also reported to be effective for adventitious root formation in
other Acacia species including A. mangium 36, A. koa 10 and
380
A.mernsii 28. Rajadurai et al. 36 reported that both IBA and NAA
when added at 0.02 - 2.0 mg l-1 gave the best result for root formation
in A.mangium while the rooting of shoots in A. koa 10 and A.
mearnsii 28 were reported to be successful on medium containing
0.2 and 1.0 mg l-1 IBA, respectively. Other forest species like
Eucalyptus citriodora 50 and A. heterophyllus 51 have been
reported to require similar concentrations of auxin, 2.0 mg l-1 NAA
and 2.0 - 5.0 mg l-1 IBA, respectively, for optimal adventitious root
formation.
The highest percentage of rooting (30%) and the highest mean
number of shoots (3.3) occurred on the MS medium supplemented
with 5.0 mg l-1 NAA. The longest mean root length of 25.5 mm was
obtained from those cultured on MS medium supplemented with
1.0 mg l-1 NAA (Table 6, Fig. 1D). Shoots cultured on B5 medium
failed to develop any root and died after 20 days in incubation in
all concentrations. Shoot from fourth subculture responded better
in IBA compared to NAA where the highest percentage of roots
produced (70%) was achieved in medium supplemented with 1.0
and 5.0 mg l-1, respectively (Table 7, Fig. 1C). Better performance
of rooting was observed on the shoots obtained from the fourth
culture where 60% of the shoots formed roots, compared to those
obtained from the initial culture with only 21% of the shoots rooted.
Juddy 32 also reported similar finding on A. excelsa where shoots
from the fourth subculture rooted better (100%) than those
obtained from the initial culture (70%). This could be due to the
rejuvenation process that took place during each subculture.
According to Harry and Thorpe 52, rejuvenation of shoots can be
achieved by repeated subculture on a cytokinin supplemented
medium, which is believed to increase the auxin:abscisic acid ratio
and subsequently improve the rooting ability of shoots.
Table 6. Effects of concentration of NAA on the
formation of roots after 30 days in MS medium
(shoots from first sub-culture).
NAA
(mgl-1)
0
0.5
1.0
2.0
5.0
Percentage of
rooted shoots (%)
10
20
20
21
30
Mean number
of roots
1.8b
1.3bc
1.0c
2.8a
3.3a
Mean length
of roots
23.3a
24.0a
25.5a
24.5a
18.0b
Values having the same superscripts are not significantly different at P < 0.05 based on
Duncans Multiple Rank Test.
Table 7. Effects of concentration of NAA and IBA on the

formation of roots after 30 days in MS medium (shoots
from fourth subculture).
Plant growth
regulators
(Auxin)
IBA
NAA
Concentration
(mgl-1)
0.0
0.5
1.0
2.0
5.0
0.0
0.5
1.0
2.0
5.0
Percentage
of rooted
shoots (%)
25
55
70
65
70
10
40
20
60
30
Mean
number of
roots
2.6c
3.4bc
3.3c
9.2b
10.5a
1.8b
1.6b
2.0ab
2.4a
1.7b
Mean
length of
roots
23.2a
19.1b
18.0b
13.8c
12.0d
23.3b
21.4c
31.1a
16.5d
15.3d
Values having the same superscripts are not significantly different at P < 0.05 based on Duncans
Multiple Rank Test.
The effect of the rejuvenation process on the rooting ability of

shoots has been comprehensively documented by Gupta et al. 53
on E. citriodora, that shoots obtained from 20 year-old tree did
not produce root when cultured on medium with 2.0 mg l-1 NAA,
but upon repeated sub-culturing, the rooting percentage increased
in the fourth and subsequent subcultures. On the other hand, the
present study produced opposite result with those obtained on
A. auriculiformis and A. excelsa, respectively 32, 48. NAA performed
better than other auxins like IAA and IBA for root formation. The
highest survival rate (100%) obtained for the plantlets incubated
for 2 weeks in the culture room and 95 - 100% survival rate was
recorded when the plantlets were transferred to the greenhouse
(Fig. 1F).
Conclusions
Considering the fact that A.crassicarpa is a multipurpose tree
species with having a great economical potential, the
micropropagation techniques developed and elucidated in this
study, are viable ways of mass producing large number of planting
stock for clonal plantation program. Appropriate sterilization
technique, explant type, media and plant growth regulators for
culture initiation and culture maintenance were obtained and it
was demonstrated that these trees can be clonally multiplied and
can be differentiated from tissue culture.
Acknowledgements
This study is part of a M.Sc thesis funded by Intensification of
Research in Priority Area (IRPA) grant provided by Ministry of
Science, Technology and the Innovative (MOSTI). Authors also
thank The Department of Forest Management, Faculty of Forestry,
Faculty of Agriculture and Institute of Tropical Forestry and Forest
Product of Universiti Putra Malaysia for providing support and
facilities.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Assessing salt-affected degraded soils using remote sensing

Case study: Al-Qassim region, Saudi Arabia
Abdulla S. Modaihsh, Abdelazeem Sh. Sallam, Adel M. Ghoneim * and Mohamed O. Mahjoub
Department of Soil Sciences, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460,
Riyadh 11451, Saudi Arabia. *e-mail: aghoneim@ksu.edu.sa
Abstract
Continued unplanned and unscientific exploitation of land resources could result in land degradation. Mis-management regarding land and land use
practices are aggravated by over-exploitation of water and land resources, over cultivation of marginal lands, and the use of inappropriate technologies.
Identification of land degradation is essential to check the problem and to implement the remedial measures needed. The study area falls under parts
of Al-Qassim province, Saudi Arabia, that is an arid region in climate. In recent years, the region has witnessed an intensive agriculture and unplanned
utilization of natural resources, which may result in land degradation. This study was carried out in 2006-2007 as part of a project aimed to study
features and causes of land degradation in Al-Qassim area, Saudi Arabia. Satellite imagery in addition to the field and laboratory studies to identify
salinity-induced soil degradation was adopted in this study. Identification of different land degradation types revealed that calcareous and saltaffected area covers about 4335.86 km2. The major degradation process is chemical degradation, particularly by salinization, which affects vast area
of the cultivated soils in the region. Morphological, chemical and physical characteristics of soils in salinity-induced degraded sites in Al-Qassim,
Saudi Arabia, were depicted. The main results of a thorough evaluation of salinity-induced soil degradation in Al-Qassim, Saudi Arabia, are presented.
The data revealed that extent of salinity-induced degradation was generally related to some physical properties of soil, quality of irrigation water and
previous soil management practices. These results are useful as the basis for designing soil conservation and restoration programs, as a base line for
evaluating the performance of conservation programs and for assessing the impact of other soil-related activities (e.g. agriculture and livestock rising).
Key words: Soil degradation, S-induced land degradation, remote sensing.
Introduction
Land degradation is a great threat to the world, not merely as an
environmental issue, but also a social and economic problem.
Land degradation can be defined as a decrease in either or both
the biological productivity and usefulness of a particular place,
due to human interference 1. Salt-prone land degradation is a major
environmental constraint with severe negative impacts on
agricultural productivity and sustainability, particularly in arid and
semi-arid regions of the world 2. Salt-affected soils exist in more
than 100 countries, extending to 76 million hectare 3. It is reported
recently that about 10% of presently arable lands of the world are
affected by salinity 4. Salinity and sodicity affect an estimated 952
M ha-1 of land 5. In several large-scale irrigation schemes, salinityinduced soil degradation has increased steadily over the last few
decades with concurrent reductions in agricultural productivity
and sustainability.
In studying areas expected to face desertification in Saudi
Arabia 5, it was concluded that about 97% of Saudi Arabia is
extremely arid while the remaining 3%, which is located in the
elevated areas of the Southwestern corner of the country, is subject
to desertification. Desertification indicators that can be used in
monitoring desertification are changes in both groundwater and
surface water as resources and the consequence changes in natural
vegetation density and extend of agricultural areas. Classification,
evaluation, and mapping of degraded land are a major issue
throughout the world. Some methods to evaluate degraded land

have been proposed 6. The capability of wide spatial coverage of
remote-sensing data is its advantageous feature. In semi-arid
environments, there is a serious problem for quantitative
assessment of land degradation with remote sensing data and
techniques. To overcome the problems related to sub-pixel mixture,
different approaches have been developed for mapping soil surface
condition using mixture modelling technique 7.
Several researchers have demonstrated that the use of
multispectral data Landsat, in mapping of soils and land
degradations at reconnaissance level. Two degrees of salinity
can be differentiated using computer-aided multispectral data
analysis system 8. Describing potential use of hyper-spectral data
in mapping of desert like features in semi-arid areas 9 was reported.
Desert soil surface feature, like desert pavements, surface
accumulation of salts, CaCO3 accumulation and surface exposure
of gypsum materials are manifestations of some kind of land
deterioration in semi-arid regions. Mapping of spatial distribution
and extent of these features would be relevant in different aspects
of environmental studies especially in areas vulnerable to land
degradation in arid regions.
Remote sensing techniques have been widely applied to identify
and characterize degraded land and to monitor the trends of
degraded land and desertification. However, due to the lack of
383
perception and information about the environmental state, different

physical and social background, no satisfactory evaluation system
of degraded land has been adapted to the specific characteristics
of each ecosystem. This research attempts to provide a synthesis
and analysis of the state of land degradation in Al-Qassim region,
Saudi Arabia.
field work and previous work in the region. This approach can be
summarized in the following steps:

Study area: Al-Qassim region is considered as one of the most
important agricultural areas in the Kingdom of Saudi Arabia that
offers valuable groundwater potential. The study area lies between
2425 to 2710 N and 4130 to 4454 E covering an area of about
14,142 km2 (Fig. 1). The major geologic formations are the Arabian
shelf including most of cultivated areas, except some areas at the
north-western boundaries (Ar-Rass, Ad Dulaymiyah), which fall
in the Arabian shield. Al-Qassim province is divided by the Wadi
Al-Rumma (Rumma Valley). The valley crosses the entire region
from the west to the northeast. It is the longest valley in the whole
Arabian Peninsula; it stretches for about 600 km from near Medina,
to the Thuayrat dunes in the east, and northeast of the region.
The land height in Al-Qassim is about 600-750 m above sea level,
and it is gliding from west to east in general. The study area has
an arid climate, characterized by hot summer with and cold winter.
The maximum summer temperature ranges between 36C and 40C
and a maximum winter temperature ranges from 18.2 to 24.9C with
a minimum of 5.30 to 10.9C. Mean annual rainfall is 110 mm, major
portion of which is received during January and March. Agriculture
is still the cornerstone of the region economy, although the region
has been famous for its agricultural assets for a long time, it wasnt
until recently that wheat production has been introduced to the
local agricultural industry. The region also produces dates, grapes,
lemons, grapefruits, mandarin oranges, oranges, pomegranates,
and a large group of vegetables.
Second step: Identification of the changes in the cultivated area

by comparing the dates of the successive Landsat images through
the analysis of Normalize Density Vegetation Index (NDVI) and
vector generation.
First step: Use of relevant research conducted previously in the

region 1, 10. This is mainly done to identify potential indicators
from the scientific literature for study area.
Third step: To identify surface features associated with the land

degradation (e.g., salinization, erosion, and sand creep).
Different techniques for image processing have been applied.
These techniques include both principal component analysis
(PCA) and unsupervised classification. Images of satellite
Landsat-5 for the years between 1993 and 2001 of the study area
were used as a basis for the digital processing. Tables 1 and 2
show the specifications of satellites used; note that all the original
satellite images with the following specifications:
Orientation: Satellite
Resampling: NN.
Forma: Eosat Fast Format for Landsat-TM or Spim for Spot image.
Product type: level - 2a.
Organization: BSQ for TM, BIL for Spot.
After delineation the studied area, a number of Landsat-5 and
Spot-4 for 2004 were selected. Table 3 shows paths, (rows), and
the dates of satellite images of the study area of Landsat-5 and
Spot-4. Radiometric correction for Landsat images of the selected
study area was done using improved dark object subtraction
Table 1. Landsat-5 specifications.
Satellite
Landsat-5
Methods: An integrated approach for evaluating salinity induced

soil degradation in Al-Qassim area was adopted in this study,
through the combined analysis of satellite imagery, supported by
Sensor
TM
300'0"N
300'0"N
270'0"N
180'0"N
180'0"N
210'0"N
210'0"N
240'0"N
240'0"N
270'0"N
330'0"E 360'0"E 390'0"E 420'0"E 450'0"E 480'0"E 510'0"E 540'0"E 570'0"E
360'0"E
390'0"E
Figure 1. Study area.

384
420'0"E
450'0"E
480'0"E
510'0"E
540'0"E
Technical specifications
The coverage frequency: every 16 days.
The area coverage was: 185 185 km
IFOV: 28.5 meters.
Radiation accuracy: 8 bit
The degree of gray scale: 0-255
The number of TM bands: (7)
Bands range is:
Range 1 (0.45- 0.52 m) Blue
Range 2 (0.52- 0.60 m) Green
Range 3 (0.63- 0.69 m) Red
Range 4 (0.76- 0.90 m) Reflective infrared
Range 5 (1.55- 1.75 m) Mid-infrared
Range 6 (10.40- 12.5 m) Thermal-infrared
Range 7 (2.08- 2.35 m) Mid-infrared
Table 2. Spot-4 specifications.

Satellite
Spot-4
Sensor
XS
Pan
Technical specifications
Coverage frequency: every 26 days.
Area of coverage: 60 60 km
IFOV: XS 28.5 meters. Pan = 10 meters
Radiation accuracy: 8 bit
The degree of gray scale: 0-255
The number of XS bands:
Seven spectral bands and a range is:
Range 1 (0.50- 0.59 m) Green
Range 2 (0.61- 0.68 m) Red
Range 3 (0.79- 0.89 m) Reflective infrared
The number of Pan bands:
Range 1 (0.51- 0.73 m)
167
168
168
167
168
168
167
168
168
1
2
3
4
5
6
7
8
9
296
297
296
138
138
139
1
2
3
technique. As for the images from Spot-4 was carried out by the
radiometric correction of simple type. Geometric correction for the
Orthro rectification of spot image was generated by using image
technique. Digital processing of the images was carried after
making primary corrections in accordance with the primary
objective of the research as follows:
1. Mosaic work of the selected images covering the study area.
2. The identification of the study areas.
3. Colour enhancement of the images of the bands 7, 4, 2 (RGB) of
Landsat-5, bands 3, 2, 1 (RGB) in Spot-4 by using non-linear
extension.
To identify the characteristics of ground features, and how it
spread through the analysis of satellite images principal component
analysis and unsupervised classification have been made. Several
attempts were made for the classification and the latest satellite
images used for the study area in a way that helps to identify
areas with degraded soils, which can show features of specific
type of degradation such as surface salinity.
Fourth step (field investigations): The ground truth was done
through several field trips to the study area, which identified a
number of sites experienced a decline in productivity. The total
number of these sites amounted to 49 locations representing all
surface features related to land degradation. Nine sites were
chosen to represent salinity induced land degradation. In these
sites, profiles were dug, described and sampled according to Soil
Survey Staff 11.
Analysis of soil samples: A representative soil samples collected
during fieldwork were air-dried, sieved using 2 mm sieve and used
for analysis. The pH and EC values of soil samples were measured
using 1:5 ratio of w/v with distilled water by pH-meter and the
electrical conductivity meter, respectively. Particle size distribution
and soil organic matter was determined according to Gee and
Bauder 12 and Klute 13, respectively.
Previous research: Al-Mashhady et al. 10 conducted reconnaissance soil survey in Al-Qassim area. Their results revealed
that soils of the area under investigation were highly variable in
topography and surface cover with poor to moderate drainage.
Dominant part of the area is composed of shallow to moderately
deep soils. The land suitability map based on the degrees of land
Identification of degraded areas by remote sensing: Technique

used for surface features reflected by remotely sensed imagery
and mapping soil surface conditions, such as salinity, sand sheet
and sand dune are presence or absence of spectral absorption
features. Simple identification and mapping of degradation
features was performed by digital processing and analysed using
a principal component (PC) and unsupervised classifications.
These techniques were used to identify the ground features, which
could be related to land degradation. Fig. 2 shows the map obtained
from Spot-4 satellite after improvement for the year 2004, for bands
3, 2 and 1 RGB of the Al-Qassim region. It was noticed that the red
colour showed the existing vegetation. Fig. 3 shows a map obtained
by Landsat-5 for 2001, for bands 7, 4 and 2 RGB of Al-Qassim
regions after improvement. It also was noticed that that the green
color shows the existing vegetation. To identify the land
characteristics (e.g., vegetation and land forms) that reflect aspects
of the surface and how it spread through the analysis of satellite
images, and to see some of the aspects of the surface function for
the possibility of a deterioration of the soil, especially what is
arising from salinization, principal component (PC) and
430'0"E
4330'0"E
440'0"E
270'0"N
42
41
42
42
41
42
42
41
42
2630'0"N
Number
260'0"N
Path
270'0"N
March-May 2004
Row
2630'0"N
Dates
Landsat-5 Thematic Mapper
22-03-1993
13-03-1993
130-3-1993
02-04-1997
09-04-1997
09-04-1997
31-05-2001
06-05-2001
06-05-2001
Spot-4
limitation salinity and texture showed these areas as marginally

suitable or not-suitable for agriculture. Nonetheless, in recent years
most of these soils, which were classified as unsuitable, were
largely introduced for crop production. Generous subsidies from
the government plus the use of modern technology for water
drilling and sophisticated irrigation system enabled the farmers to
turn these soils to productive soils.
260'0"N
Table 3. Orbits of Landsat-5 Thematic Mapper for the years

1993, 1997, 2001 and Spot-4 2004 in Al-Qassim Region.
SPOT image, April - May 2004

430'0"E
4330'0"E
20
10
km
440'0"E
40
Figure 2. Satellite image map Spot-4, RGB 3, 2, 1 band for year 2004.
Red colour shows cultivated areas.
385
430'0"E
270'0"N
PCA image
430'0"E
440'0"E
Figure 3. Satellite image map Landsat-5, RGB 7, 4, 2 bands for year

2001. Red colour shows cultivated areas.
430'0"E
Unsupervised classification: The unsupervised classification was

conducted for Landsat-5 2001, for some spectral bands, to identify
the differences in ground units in the Al-Qassim region. It is
concluded that number of 15 units is the most suitable in terms of
showing the agricultural coverage. It should be noted that the
unsupervised image does not gives accurate estimation of the
areas of the land units however; the normalize density vegetation
index (NDVI) is more accurate in assessment of the vegetation
cover (Fig. 5).
Verification by ground truth for the unsupervised image (Fig. 5)
20
10
km
440'0"E
40
440'0"E
270'0"N
270'0"N
4330'0"E
2630'0"N
Legend
260'0"N
Principal component: To demonstrate the variation in the studied

area, analysis of basic components was done for Landsat-5 images
in 2001, using 7, 4, 3, 2, 1 bands (Band 5 was excluded due to of
the high reflectivity high gain). Fig. 4 showed clearly some ground
units, such as zones of central pivots (dark green), areas of sand
dunes (light brown and dark), and areas of lime and salt sediments
(green yellowish). Also, the river course of Wadi Al-Rumma was
shown with some resemblance to lime and salts sediments.
Characteristics of the rest of the area were difficult to trace
accurately by this technique. To identify the various landforms in
the studied area, unsupervised classification for Landsat-5 (2001)
was applied. Several attempts were performed to identify the
suitable number of taxonomic units. It was concluded that 15 units
was more appropriate to show the cultivated area clearly with no
overlapping with other units.
4330'0"E
Figure 4. Satellite image map Landsat-5 obtained by PC for Al-Qassim

region.
unsupervised classification have been made.
386
2630'0"N
40
2630'0"N
20
km
260'0"N
4330'0"E
10
260'0"N
260'0"N
430'0"E
440'0"E
2630'0"N
270'0"N
2630'0"N
2630'0"N
260'0"N
Vegetation cover May 2001

Background image: LANDSAT image, May 2001
4330'0"E
270'0"N
440'0"E
260'0"N
4330'0"E
270'0"N
430'0"E
???
Classified image
430'0"E
4330'0"E
10
20
km
40
440'0"E
Figure 5. Satellite image map (Landsat-5) obtained by unsupervised

classification for Al-Qassim region (2001).
and with reference to interpretation of satellite images 14 revealed

that there are three basic units and several sub-units in the studied
area. The basic units are sand sheets and sand dunes (pink, yellow,
and orange colours), which covered an area that is nearly about
2905 km2. The second unit represents lime and high saline land
forms (dark brown, and navy blue), which covered an area of
about 4335 km2. The third unit represents the cultivated areas
(dark and light green) and covered an area of about 1405.3 km2.
These findings agree somehow with land forms presented in the
atlas of land resources 15.
The logic behind the concept of choosing these three units can
be summarized as follows:
1) After studying the remotely sensed data using interpretation
skill, field check, literature survey and soil sample analysis, three
main land degradation units were identified based on the data.
2) Careful analysis of land degradation units showed that the
whole area suffered from different degree of salinity this is the
main type of degradation in the area.
3) Salt affected crop could not be selected as a pure indication of
salinity because there spectral response varies according to the
degree of damage and different kind of crops even its pixel purity
index cannot detect the pure pixel in salinity suffered crop.
4) Bare non-vegetated sand dune can be seen clearly in the image
Table 4. Description of salt-affected profile.
Location: 60 (Zayab Farms, Al-Bukairiyah)
Features of degradation: Soil salinization
Coordinates: Geographical:4346'10.3" E, 2615'24.8" N
Elevation: 651 m
Parent material: Alluvial & eolian deposits
Surface cover: A thin salt crust
Natural vegetation: The land was planted, few of
natural weeds resistance to salinity
Physiography: Plain
Topography and slope: Flat
Erosion: None
Drainage: Moderately drained
Classification: Typic Aquisalids
Horizon
1Cz
Depth / cm
0 20
20 40
2Cz
40 75
Cz
75 95
95+
including its large extent and surrounded by poor or no vegetation.

This area is considered as the pure pixels for sand end members.
5) Crops are in abundance in large tract of the area, but they are
quite modified by the presence of different degree of land
degradation. EC values showed salinity and alkalinity.
Morphological, physical and chemical characteristics:
According to the unsupervised classification, monitoring of the
change in cultivated area, vector generation and field verifications,
nine sites representing the salt-affected soils within the studied
area were chosen. The morphological study of these sites showed
that some of these soils were left without cultivation for either a
very long or medium periods. Some of them are currently planted
with palm trees or barley. The spread of small and sparse herbs at
sites that were left without cultivation were also observed. A
reprehensive soil profile description of one of the salt-affected
site is given in Table 4.
Soils of these sites comprised of deposits that have been
transported by wind or water, was reflected clearly by the soil
texture that ranged from sand to clay, and corresponds to the
estimated texture of the soil in the laboratory. Depth of the soil
ranged between very shallow and very deep, while the structure
varied between single grain and loose structure. Some of the
morphological features emerged in some layers
of the profile is the appearance of salts in the
form of amorphous or non-amorphous
crystallization. Gypsum was also seen in the
form of very thin amorphous layer. Results of
soil analysis indicated that a high level of
soluble salts in the first soil layer (100.7 dS m1
), however, the salinity dropped in the lower
layers but remained high (Table 5).
Description
Reddish yellow (7.5YR 6/6, dry), brown (7.5YR 5/4, moist);
loamy sand; single grain; loose, slightly sticky, non-plastic;
very few fine roots; few very fine salt crystals; abrupt smooth
boundary.
Very pale brown (10YR 7/4, dry), yellowish brown (10YR
5/6, moist); loamy sand; single grain; loose, slightly sticky,
non-plastic; few fine roots; few very fine salt crystals; very
few fine soft decomposed organic matter; clear smooth
boundary.
Light yellowish brown (10YR 6/4, dry), yellowish brown
(10YR 5/6, moist); sand; single grain; loose, non-sticky, nonplastic; few very fine salt crystals; very few fine soft
decomposed organic matter; clear wavy boundary.
White (10YR 8/2, dry), yellowish brown (10YR 5/6, moist);
sandy loam; massive; slightly hard, slightly sticky, slightly
plastic; few very fine salt crystals; some fine soft distinct
yellow (2.5Y 7/8) mottles.
Water table
Table 5. Some physical and chemical properties of the soil profile.

Depth (cm)
020
2040
4075
7595
Particle size distribution (%)

Sand
Silt
Clay
84.3
6.02
9.68
88.3
4.02
7.68
88.3
6.02
5.68
72.3
20.0
7.68
Texture class
Loamy sandy
Loamy sandy
Sand
Sandy loam
O.M
%
1.20
0.50
ND
ND
pH
8.91
8.76
8.62
8.39
EC
dS m-1
100.7
38.5
34.4
45.0
O.M, Organic matter; EC, Electrical conductivity, ND; not detected.
CaCO3
%
35.5
39.4
23.9
23.3
Conclusions
Repeated coverage of satellite imageries could
provide an efficient tool for monitoring land
degradation in Saudi Arabia. Implementation of
remote sensing technology in desertification
monitoring can be done through various means:
the direct approach is by measuring the area of
reduction in plant cover. However, social and
economic factors may also have an effect. The
indirect mean is by studying indicators of
desertification that can be measured on satellite
imageries. Monitoring saline degraded lands
has always been a primary issue for efficient
irrigation systems management and
rehabilitation policies. The solutions to reduce
salinization in the study area could be one of
the following: reduce irrigation, switch to salttolerant crops, use humic acids to fix anions
and cations and eliminate them from the root
region of the plants. It is suggested that, the
salinization in Al-Qassim region occurred by
one of the following: high level of salts in the
soils, over cultivation, irrigation
mismanagement and climate trends that favour
accumulation of salts. Salinization
consequences in Al-Qassim region resulted in
387
stunts crop growth, lowers crop yields, decreased soil and fertility,
damage to infrastructure and reduction of water quality.
Acknowledgements
Authors wish to thank College of Food and Agricultural Research
Center and Deanship of Scientific Research, King Saud University,
Saudi Arabia, for supporting this work. The authors extend their
thanks to King AbduIaziz City for Science and Technology
(KACST) for financial support of project # AT-25 43 under the title
Assessment of land degradation in some irrigated soils (Causes
and Features) in Saudi Arabia.
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(ed.). Methods of soil Analysis. Part 1: Physical and Mineralogical
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13
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1
388
WFL Publisher
Helsinki, Finland
www.world-food.net
Determination of pond water quality for aquaculture and ecosystem management

Umme Shahina Khanom 1, Sabrina Sharmeen 2, Jannatul Ferdouse 1, Wahhida Shumi
Hazandy Abdul Hamid 4, 5 and Md. Aktar Hossain 4, 6*
1, 3*
, Arifin Abdu
4, 5
Department of Microbiology, 2 Department of Soil Science, University of Chittagong, Chittagong - 4331, Bangladesh.
Department of Bioprocessing Technology, Faculty of Biotechnology and Biomolecular Science, 4 Faculty of Forestry,
5
Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
6
Institute of Forestry and Environmental Sciences, University of Chittagong, Chittagong - 4331, Bangladesh.
*e-mail: wahhida_shumi@hotmail.com, aktar_forestry@hotmail.com
1
Abstract
Fish is the main source of animal protein for the common people of Bangladesh. About 37% of the inland fishes are obtained from the closed water
fish culture where ponds play vital roles in the production system. The present study was carried out to determine the water quality (physicochemical and biological factors) of five uncultivated or under cultivated ponds in the Chittagong University campus, Bangladesh, for aquaculture and
ecosystem management. Temperature, pH, EC, BOD, COD, TSS, turbidity, NO2-, PO4-, free CO2, CO3 -, HCO3-, Cl-, total viable bacterial count and
total coliform were determined. Isolation and identification of Escherichia coli and Salmonella sp. from the water samples were performed to
investigate the antibiotic sensitivity. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against ciprofloxacin
was also investigated. The findings reveal that there was no significant difference in temperature (19C to 20C), pH (6.7 to 7.4), electrical
conductivity and Cl- values in the water among the ponds and that was within the range of the requirements for the fish cultivation. Both turbidity
and total dissolved solids was highest in the uncultivated botanical garden pond 2 (BGP2). BOD, COD, NO2-, PO4-2, HCO3-, and free CO2
concentration was beyond the permissible limits for aquaculture. Total viable count and total coliform ranged from 1.3 10 4 to 6.6 10 5 cfu ml-1 and
from 0.020510 2 to 1.1010 2 ml-1 respectively, among the ponds where both these parameters were highest in the naturally polluted BGP2. Most
of the isolated E. coli and Salmonella sp. were sensitive but few showed resistance against the antibiotics, especially in BGP1, where fishes were
cultivated commercially.
Key words: Aquaculture, ecosystem management, water quality, physico-chemical property, total bacterial count.
Introduction
Water is essential for maintaining healthy ecosystems and socioeconomic development. Properly managed water resources are
the critical component of growth, poverty alleviation and equity.
Livelihoods of the people are intimately associated with the access
to water services. Managing water resources using integrated
approach will be critical to mitigate social, economic and
environmental impacts. Rice and fish constitute the principle diet
where fish is the main source of animal protein for the common
people of Bangladesh. Bangladesh is one of the leading inland
fisheries producer in the world with an estimated production of
1,646,819 tons out of 9,14,752 tons from aquaculture and 2.1 million
tons in the country during the year 2003 - 2004 1. Aquaculture
accounted for about 43.5 percent of the total fish production in
the country where inland open and closed water fisheries
contributed 34.8 and 37 percent, respectively 1. Among the closed
water culture fisheries, pond fish culture plays vital roles in the
production system.
The estimated number of ponds in the country is 1.3 million,
covering an area of 0.151 million ha, of which 55.3 percent is
cultured, 28.52 percent is culturable and 16.18 percent is unused.
The percentage of fish produced from these three types of ponds
was 72.09, 20.01 and 7.90, respectively, in 2002 2. The size of these
ponds varied from 0.020 ha to 20 ha with an average of 0.30 ha. In
the country, the highest number of ponds exist in Barisal district

(12.11%), followed by Comilla (9.36%), Sylhet (9.10%), Chittagong
(8.02%; 14,306 ha) and Noakhali (7.75 %) 2. However, most of the
ponds in Chittagong district are either uncultivated or under
cultivated.
Fisheries and aquaculture play inevitable roles in nutrition,
employment and foreign exchange earnings in the country. About
12 million people are associated with the fisheries sector, of which
1.4 million people rely exclusively on fisheries related activities 3.
An estimated 9.5 million people are involved in subsistence
fisheries on the countrys flood plains 4. The number of fishermen
increases dramatically to 11 million between June to October each
year. There are 3.08 million fish farmers, 1.28 million inland fishermen
and 0.45 million fry collectors (fish and shrimp) in the country 5. It
has also been estimated that fisheries and related activities support
more than 7 percent of the countrys total population. Aquaculture
is the high-density production of fish, shellfish and plant forms in
a controlled environment.
At present per capita annual fish consumption in Bangladesh
is 14 kg year -1 against a recommended minimum requirement of
18 kg year -1 indicates there is still need to improve fish
production and consumption in the country. The Ministry of
Fisheries and Livestock (MoFL), Department of Fisheries (DoF),
389
Bangladesh Fisheries Development Corporation (BFDC) and the

Bangladesh Fisheries Research Institute (BFRI) are the main
organisations responsible for aquaculture and its development.
Universities, organisations within other ministries and local and
international NGOs are also involved in this area. Integrated efforts
from all this agencies/groups must be united together in the
production system. However, Bangladesh is among the most
densely populated countries in the world and very difficult to
expand the fish cultivation area. Therefore, extensive cultivation
in the under cultivated and uncultivated water body might be one
of the important options for the sustainable aquaculture
production system in the country. However, pond water quality
largely determines the fish production in the closed water
aquaculture. Water pollution is one of the common obstacles to
achieve the stated objective. Water body is being polluted rapidly
due to increasing agricultural, industrial and municipal activities.
The effects of water pollution on aquatic systems largely depend
on whether polluted waters are standing (lakes and ponds) or
flowing (rivers). Standing water in the ponds are generally more
susceptible for the pollution because of slow turnover. The major
water pollutants are organic and inorganic nutrients, infectious
agents, toxic organic and inorganic pollutants, sediment and heat.
Quality water should therefore be ensured for the growth and
development of fishes 6. Several physicochemical and biological
parameters of water are of great concern in this regard. Biological
oxygen demand (BOD), chemical oxygen demand (COD), pH,
temperature, conductivity, total dissolved solids (TDS), salinity,
total suspended solids (TSS), turbidity, concentration of different
ions like nitrate, nitrite, ammonia, phosphate, sulfate, chloride,
carbonate, bi-carbonate, total bacterial count, etc. are the most
important water quality parameters.
BOD is the amount of oxygen required by the micro-organisms
to decompose the organic substances aerobically in water. A high
BOD is an indication of water pollution. When industrial,
municipal, and domestic sewage enter a surface water body, microorganisms begin to decompose the organic materials by
consuming the dissolved oxygen (DO) of water. This can quickly
deplete the available oxygen and subsequently affect aquatic life
forms 7. In aquatic ecosystem, the COD is often used as an index
of the total organic carbon in the samples 8. Water turbidity
interferes with light penetration in the water column, thereby
limiting photosynthesis in the bottom layer. High turbidity can
cause clogging of gills or direct injury to tissues of prawns. The
total dissolved solids and total suspended solids are very useful
parameters describing the chemical constituents of the water and
can be considered as general edaphic relations that contribute to
productivity within the water body 9. Electrical conductivity is
usually used for indicating the total concentration of the ionized
constituents of water. Most fish species survive well within the
pH range of 6.5 to 9.5. Chronic pH levels below 6.5 may reduce
fish reproduction and are associated with fish die-offs that
sometimes occur in the late winter. Newly hatched fish (fry) are
often sensitive to pH levels above 9.0 to 9.5 10. Sulphate is a
common compound found in water as a result of the dissolution
of minerals from soil and rocks. Typical levels are between 0 and
1000 mg L-l. Fish tolerate a wide range of sulphate concentrations,
and levels of sulphate above 500 mg L-1 are a concern only if the
water is used for other purposes, such as watering cattle 11. Almost
all of the phosphorus in water is in the form of phosphate ion. The
390
principle concerns associated with phosphorus in freshwater

aquatic systems, however, are algal blooms and increased
eutrophication 12, which is an increase in levels of production in a
water body 13. Eutrophication may result in decreased DO levels
as bacteria decompose dead algae, consuming oxygen in the
process. When DO concentrations fall below the critical levels for
metabolic requirements of aquatic biota, both lethal (e.g., fish kills)
and sublethal effects can occur. Chloride is beneficial to fish in
maintaining their osmotic balance. High chloride levels (above
100 mg L-1) are a concern only if the water is also used to irrigate
sensitive land-based crops 11. Under the current circumstances,
the present research has been undertaken to evaluate the water
quality of five uncultivated or under cultivated ponds in Chittagong
University campus for aquaculture and other ecosystem
management.
Study area and sample collection: Water samples were collected
from the surface of five different ponds in the Chittagong
University campus, Bangladesh (Fig. 1) during the winter in 2011.
The selected ponds were 1) Botanical Garden Pond 1 (BGP 1)
(where fishes were cultivated), 2) Botanical Garden Pond 2 (BGP 2)
(uncultivated), 3) Science Faculty Pond (SFP) (under cultivated), 4)
South Campus Pond (SCP) (under cultivated) and 4) Biological
Science Faculty (BSF) (uncultivated). These ponds are the main
source of drinking water for the wildlife of university forest.
Samples were collected in sterile reagent bottles and screw cap
tubes, and transported immediately to the laboratory for analysis.
Whenever necessary, samples were preserved in refrigerator until
analyzed.
Figure 1. Map of Bangladesh showing the study area

in Chittagong University campus.
Determination of physicochemical parameters of water:

Important physical, chemical, and biological parameters of the
water samples collected from several ponds of Chittagong
University campus were determined in laboratory. Temperature

was determined by dipping a thermometer into the water at the
sampling spot. Turbidity was determined by spectrophotometer
at 660 nm. The pH was determined with an electric pH meter (pH
Hanna Instrument Ltd. & 3310, pH meter Janway, UK). Electrical
conductivity was determined with a conductivity meter (Hanna I
nstrument Ltd., EC 214, UK). Total dissolved solid (TSS) was
measured by drying the water samples.
Assessment of biological oxygen demand (BOD) and chemical
oxygen demand (COD) of the samples: Biological oxygen demand
(BOD) was determined by a modification of Winkler method.
Collected water samples from each sampling point of the selected
ponds were poured in two bottles. The dissolved oxygen (DO) for
one BOD bottle was determined immediately and other bottle was
kept in the dark for five days. After five days, the DO for water
sample in the dark bottle was calculated. The BOD level of the
sample was determined from the difference between the initial and
final (dark) DO and calculated in mg L-1 of water sample. Chemical
oxygen demand (COD) was determined by titrating re-flasked
sample in ferrous ammonium sulphate using ferroin indicator 14.
To determine COD, 25 mL water sample was poured in a 250 mL
conical flask. Similarly 25 mLdistilled water was taken in a conical
flask as control. Ten mL of 0.25 N K2Cr2O7 in 18 N H2SO4 solution
was poured in both of the flasks. Then each pinch of salt of Ag2SO4
and HgSO4 was added separately into the flask and incubated at
100C for 2 h in water bath. Flasks were cooled for 10 min and
added with 8 - 10 drops of ferroin indicator. Excess K2Cr2O7 with 0.1
N Mohrs salt solution in 8 N H2SO4 from the burette was titrated. The
end point of the titration (colour change to bottle green) was recoded
and expressed as mg L-1 sample.
Enumeration of total viable bacteria and total coliforms: Tenfold serial dilution and pour plate technique was followed for the
enumeration of total viable bacteria. The total bacterial load of the
samples was determined by using nutrient agar (NA) media. The
calculated result was expressed as colony forming unit per mL
(cfu mL-1) of water. Enumeration of total coliform was done by
MPN (Most Probable Number) method. Results were computed
using MPN (Most Probable Number) chart as total coliform number
per mL.
Isolation and identification of Salmonella sp. and E. coli: BSA
(Bismuth Sulfate Agar) media was used for Salmonella sp. and
EMB (Eosin Methylene Blue) and MacConkey agar media were
used for isolation of E. coli from the collected water sample by
serial dilution and plating with pour plate and spread plate
techniques. The selected bacterial colonies were isolated on the
basis of the colony morphology and identified on the basis of
their morphological, cultural, physiological and biochemical
characteristics etc. All the characteristics were compared with the
standard description of Bergeys Manual of Determinative

Bacteriology 15.
Determination of antibiotic sensitivity of the micro-organisms:
Antibiotic sensitivity of the micro-organisms was determined
following the method described by Bauer et al. 16 using MllerHinton agar media, 0.85% saline suspensions of test bacterial
isolates were prepared. Aseptically, sterile Mller-Hinton agar
medium was poured in the sterile Petri plates so that the depth of
the medium filled two-third of the plate. The plates were stand for
several min for solidification and isolated inoculums were spread
over the medium. This process was repeated three times for each
test culture. Then all the culture plates were allowed to dry for 5
min in clean bench. Antibiotic discs against each test organism
were placed by using sterile forceps. A control disc was placed at
the centre of each plate. The plates were then chilled for 4 h to
diffuse the antibiotics properly and incubated (invertedly) for 24
h at 37C. After incubation, the plates were observed and diameter
of inhibition zone was measured in mm.
Physical properties of water samples: The temperature, pH,
turbidity, total suspended solid (TSS) and electrical conductivity
(EC) of the water samples are presented in Table 1. The temperature
of all water samples was almost similar and recorded as 19C or
20C during the winter. The pH of water varied from 6.72 to 7.40
but there was no significant difference among the ponds.
Turbidity varied from 0.05 to 0.20 among the ponds where
remarkably higher (0.203) turbidity was in BGP2 than the other
ponds. The highest electrical conductivity was recorded 7933.3
S L-1 in the SFC pond water followed by BSF and lowest was in
BGP1. Total suspended solid (TSS) ranged from 160 to 360 mg
L-1 with maximum (360 mg L-1) in BGP2 and minimum (160 mg L-1)
value in SFC pond.
Since the samples were collected in the winter season, the
temperature of the water samples remained above 20C rest of the
year, which was favourable for the growth of aquatic organisms.
The pH values of studied water were neutral to slightly alkaline,
which was also suitable for the fish population. According to
Environmental Quality Standard for Bangladesh 17, the standard
pH value for fish culture is 6.5 to 8.5. Chronic pH levels below 6.5
and above 9.5 may reduce fish production and sometimes kill the
fish population in late winter. However, the increased concentration
of alkali helps to buffer against pH changes 18. Total suspended
solids of all samples were beyond the permissible limits of 80 mg
L-1 for fish culture, which might turn the water unsuitable for
domestic, agricultural and fisheries utilization 19-21. Higher amounts
of suspended solids in water contributed to a higher turbidity
that restricts light penetration (Table 1). The conductivity values
of pond water were within the permissible limits of 30 to 5000 S
cm-1 for fisheries 18.
Table 1. Physical parameters of the collected water samples.

Sample
BGP 1
BGP 2
SFC
SCP
BSF
Sites
Botanical garden pond 1
Botanical garden pond 2
Science faculty pond
South campus pond
Biological science faculty
Temperature
20C*
19C
20C*
19C
19C
pH
6.88
6.84
7.40*
7.05
6.72
Turbidity
0.094
0.203*
0.050
0.067
0.088
TSS (mg L-1)

280
360*
160
200
240
EC (S L-1)
653.3
1033.3
7933.3*
1286.7
7866.7
Here * indicates the highest value.
391
Biochemical components of water samples: BOD, COD and total

bacterial count of the water samples are shown in Table 2. BOD
and COD ranged from 40.2 to 122.6 mg L-1 and from 119.68 to
401.28 mg L-1 respectively, among the ponds. Total bacterial count
of water was found to vary from 1.3 10 4 to 6.6483 10 5 cfu mL-1.
The BGP2 was characterized by significantly higher biochemical
components including BOD (122.6 mg L-1), COD (401.28 mg L-1)
and total bacterial count (6.64810 5 cfu mL-1) indicates highly
polluted water in the pond 22. The optimum value of BOD for
drinking, irrigation and fisheries is less than 6 mg L-1 19, 22.
Table 2. BOD, COD, and total bacterial count of the water samples.
Sample
BGP 1
BGP 2
SFC
SCP
BSF
COD
(mg L-1)
387.2
401.28*
264
119.68
281.6
BOD
(mg L-1)
92.4
122.6*
40.2
48.6
80.6
Total bacterial count

(cfu ml-1)
3.998 105
6.648 105*
1.3 104
2.3 104
4.3 104
Ions and free CO2: The concentration of nitrite in the present

study ranged from 1.333 to 2.6784 mg L-1. Phosphate concentration
of water varied from 0.6889 to 17.591 mg L-1 which was significantly
different among the ponds. Chloride content was from 3.19 to 12
meq L-1 in the water samples with a highest value in BGP1 and
PGP2. The concentration of HCO3- ranged from 1464 to 5856 mg
L-1 with the maximum value (5856) in SFC and BSF ponds followed
by BGP1 and BGP2 and lowest in SCP. All the values were beyond
the permissible limit for drinking, irrigation and aquaculture. The
permissible limit of HCO3- content is up to 158.6 mg L-1 for drinking

and irrigation 23 and 50 to 300 mg L-1 for aquaculture purpose 18.
Under this consideration, the water reported herein was not safe
enough for domestic, agricultural and aquacultural uses. However,
CO3-1 could not be recorded in the water sample of the studied
ponds.
The phosphate content in the samples ranged from 0.6889 to
17.591 mg L-1 among the ponds with highest value in SCP (Table
3). The permissible limit of phosphate 0.1-0.5 mg L-1 18 indicates
the concentrations of nitrite and phosphate are much higher than
the acceptable limit. All the chloride values were within the
permissible limits of 100 mg L-1 for drinking, 250 mg L-1 for irrigation
and 1 - 100 for aquaculture purpose 18, 19, 24. The values of pH and
electrical conductivity of the present study were almost similar
with the results found 25. Again, results of COD and TSS were
very close to those (TSS 85 - 206 mg L-1 and COD 162 - 397 mg
L -1) found by Ehiagbonare and Ogunrinde 26, where they
determined the water samples from concrete and earthen fish ponds
in different locations in Okada and its environs, Edo State, Nigeria.
Isolation and identification of Salmonella sp. and E. coli: During
study, Salmonella sp. and E. coli (2 strains from each of the pond
water) were isolated from the collected samples on the basis of
their growth characteristics on selective media. Total five strains
of Salmonella sp. and five strains of E. coli were tested for their
morphological, biochemical and physiological characteristics and
compared with standard description 15 (Table 4).
Sensitivity of antibiotic against the E. coli and Salmonella sp.:

Table 3. Some important chemical parameters of the water samples All the strains of E. coli and Salmonella sp. were tested for their
antibiotics sensitivity against commercially available disc of
collected from various ponds in the study area.
PO4-P
Cl
HCO3
CO3
Free CO2 amoxicillin, erythromycin, tetracycline and cefixime and results
NO2-N
Samples
are shown in Table 5 and Fig. 2. In this study, all the strains of E.
(mg L-1) (mg L-1) (meq L-1) (mg L-1) (mg L-1) (mg L-1)
BGP 1
2.0336
0.6889
12*
2928
Absent
0.1936
coli and Salmonella sp. were sensitive to cefixime and amoxicillin
BGP 2
2.6784*
6.122
12*
2928
Absent
0.5808*
but exhibited resistance against erythromycin. On the other hand,
SFC
1.333
5.989
3.47
5856*
Absent
0.5808*
most of the strains of isolates were sensitive to tetracyclin but
SCP
1.488
17.591*
4.80
1464
Absent
0.1936
some also showed resistance. Onyuka et al. 27 reported that E.
BSF
1.0416
5.373
3.19
5856*
Absent
0.3872
coli isolates were resistant to ampicillin, tetracycline, cotrimoxazole,
Table 4. Biochemical reaction of the isolates at a glance.
Test
Gram Staining
Shape
Size (m)
Length
Width
GFT
SFT
LFT
MFT
Indol
H2S
Motility
Urease
Oxidase
Citrate
Catalase
MR
VP
Gelatin hydrolysis
Nitrate reduction test
392
BGP1
Rod
1.54
0.98
+
+
+
+
+
+
+
+
+
Salmonella sp.
BGP2 SFC SCP
Rod
Rod Rod
1.14
0.82
+
+
+
+
+
+
+
0.98
0.7
+
+
+
+
+
+
+
+
+
1.26
1.12
+
+
+
+
+
+
+
+
+
BSF
Rod
BGP1
Rod
BGP2
Rod
0.84
0.42
+
+
+
+
+
+
+
+
1.26
0.7
+
+
+
+
+
+
+
+
+
1.12
0.74
+
+
+
+
+
+
+
+
+
E. coli
SFC
Rod
SCP
Rod
BSF
Rod
1.4
0.84
+
+
+
+
+
+
+
+
+
+
1.54
0.98
+
+
+
+
+
+
+
+
+
+
1.12
0.98
+
+
+
+
+
+
+
+
+
+
Table 5. Antibiotic sensitivity of E.coli and Salmonella sp. isolated from different
ponds against various antibiotics.
Pond
BGP1
BGP2
SFC
SCP
BSF
Antibiotics
Erythromycin
Tetracyclin
Cefixime
Amoxicillin
Erythromycin
Tetracyclin
Cefixime
Amoxicillin
Erythromycin
Tetracyclin
Cefixime
Amoxicillin
Erythromycin
Tetracyclin
Cefixime
Amoxicillin
Erythromycin
Tetracyclin
Cefixime
Amoxicillin
Antibiotic
g disc-1
15
30
5
30
15
30
5
30
15
30
5
30
15
30
5
30
15
30
5
30
Zone of inhibition(cm)
E. coli Salmonella sp.
Nil
Nil
Nil
Nil
1.7
2.3
2.4
1.7
Nil
Nil
1.8
1.8
2.5
2.5
2.5
2.5
Nil
Nil
1.5
1.7
3
2
1.7
1.5
Nil
Nil
1.7
1.5
3.1
1.7
1.7
1. 3
Nil
Nil
1.5
1.7
2.9
2.5
1.5
1.5
Comment
E. coli
Salmonella sp.
Resistance Resistence
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
sensitive
Sensitive
Sensitive
Sensitive
Sensitive
Sensitive
sensitive
Sensitive
were beyond the permissible limits for aquaculture. The

bacteriological quality of each pond water also exceeded the
permissible limits. The highest number of bacterial population
was observed in Botanical garden pond 2. BOD and COD were
sufficiently high in all water samples, which is not desirable.
Salmonella sp. and E. coli of Botanical garden pond 1 and Science
Faculty pond showed more resistance than the other ponds, which
might be due to use of vaccinated poultry waste, medicine and
also other chemical contamination with the fertilizer used in the
crop field around the ponds.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Impact of pre-sowing treatment on seed germination and seedlings growth attributes

of Calamus longisetus Griff. at nursery and field conditions
M. Rafiqul Haider 1*, Md. Sah Alam 1, Md. Aktar Hossain
1
2, 3
* and Nor Aini Ab. Shukor
3, 4
Bangladesh Forest Research Institute, P. O. Box 273, Chittagong - 4000, Bangladesh. Institute of Forestry and Environmental
Sciences, Chittagong University, Chittagong - 4331, Bangladesh. 3 Faculty of Forestry, 4 Institute of Tropical Forestry and
Forest Products, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
*e-mail: haider_bfri@yahoo.com, aktar_forestry@hotmail.com
2
Abstract
The paper describes the germination behaviour of Calamus longisetus Griff. seeds under three different pre-sowing treatments and seedlings growth
performance in nursery and field condition. Whole fruits, seeds with pulp and cleaned seeds were sown in germination trays filled with soils mixed with
decomposed cowdung at a ratio of 3:1. The growth performance of the seedlings were determined by transferring the young seedlings (having 2 - 3 leaves)
from germination bed to the polybags filled with soil mix followed by outplanting (at 1-year old) in the field. Germination percentage was significantly
(p 0.001) enhanced by the pre-sowing treatment where the highest germination percentage was in cleaned seeds followed by the seeds with pulp and
lowest in whole fruits. The survival percentage was over 91% in the field after one year and the average height 126.2 cm after two years of planting. Clean
seeds for nursery raising and one-year old seedlings for outplanting were found suitable for raising successful plantations for the species.
Key words: Calamus longisetus, cleaned seeds, germination potential, survival percentage, pre-sowing treatment, seedling growth.
Introduction
Rattan is a climbing spiny palm belonging to the family Arecaceae
(Palmeae) and constitutes an integral part of the tropical forest
ecosystem 1. Rattans are the most important non timber forest
products (after bamboo) in the tropical and sub-tropical counties
of Asia and Africa. It has gained additional interest among the
people because of increased awareness as well as their vital roles
in socioeconomic development and socio-ecological research
issues 2. Rattans are important sources of income and employment
for millions of people all over the world directly or indirectly. In
India, rattan industries alone provides jobs for 200,000 people 3 and
its contribution is about 25 - 35% of the total household income of
the tribal communities in North Eastern India. Rattan furnitures
are much valued in many countries, and are being exported abroad
from the producing countries 4.
Six hundred species of rattans under 13 genera were recorded in
the world in early nineties 4 but recent report shows that only 558
species exist 5. Of these, 15 species were recorded in Bangladesh 6-11
but currently there are only 10 species of rattans under two genera
growing in the country 12, 13. These rattan resources have depleted
fast in recent years due to over exploitations and poor
management 14 including almost complete removal of stems from
the forest during harvesting. Consequently, steady loss of forest
habitat due to urbanization and industrialization is also posing a
serious threat to rattan supply. High demand for these resources,
coupled with uncontrolled harvesting and deforestation, has led
the resources towards the exhaustion in many rattan-producing
areas 15. To cope with the increasing global demand for rattan,
there is an urgent need for sustainable management of rattan
resources. There are two different approaches to achieve the goals,
by establishing rattan plantations and managing existing rattans
properly in their natural habitats 15. However, establishing the

rattan plantation requires appropriate knowledge about the nursery
development, plantations raising and stand management for
sustainable development 14.
Calamus longisetus locally known as udum bet, occurs naturally
in the dry hill slopes, sandy loam soils of mixed evergreen forests
of Chittagong, Chittagong Hill Tract and Coxs Bazaar district in
Bangladesh 7, 12. The species is found in robust clustering to form
dense clumps, erect for climbing where support tree is available.
Stems covered with sheath and internode ranges from 18 - 25 cm
long. Leaf is ecirrate, 3 - 4 cm long with petiole. Leaf sheath is
greenish-yellow, densely armed with 3 - 5 cm long, flat, erect,
spreading and reflexed spines (Fig. 1). The species is dioecious;
flowering occurs in October to December, fruiting in March to
Figure 1. Leading shoot and spiny stem of Calamus longisetus.
395
June and is pollinated mainly by insects. It is mainly used for

making furniture frame, umbrella handles, walking sticks, binding
materials and stem water for drinking by the tribal people. With
depletion of rattan resources C. longisetus is also depleted all
over the country and hardly found in the natural condition in
forests. To ensure sustainable development and continuous supply
of rattans, several initiatives have already been taken to increase
the rattan plantations and the overall production by involving the
government, non-government and other agencies in the country.
For the purpose sound knowledge on nursery raising and
plantation development, updated techniques and management
systems are required for the species.
Rattan research in Bangladesh was initiated about four decades
back and considerable information relating to germination and
nursery techniques for selected rattan species are available 16-21.
Rattans cannot be grafted or rooted by cuttings, therefore their
propagation largely depends on seed germination, wildings,
suckers and tissue cultured materials for plantation. Among them,
seeds are the most important propagating materials for large-scale
plantations program for rattans 22, 23. There are considerable
variations among the rattan species in respect to germination
period and percentage. The seed viability after collection may
affect the germination period and percentage. Moreover, because
of hard seed coat in association with possible seed dormancy,
most of the species of cane require longer period to germinate 24.
Pre-sowing treatments, especially, removal of fleshy pulp or scales
from the intact fruits has been reported to influence the seed
germination and seedling growth performance in many tropical
tree species. For instance, depulping of fruits and soaking of the
seeds in water for 48 h enhanced the seed germination and seedling
growth of T. belerica 25-27, T. chebula 28-30 and Grevillea robusta 31.
Nainar et al. 28 showed that seed pre-treatments including
mechanical scarification offered 60% germination in T. chebula
seeds. Although there are some reports on pre-sowing treatment
and germination of some rattan species, for example, pre-treatment
of Daemonorops jenkensiana clean seeds with 10% H2SO4 and
HCl enhanced seed germination (68.14% with both the
treatments) 21, the information about the regeneration of Calamus
longisetus with seed treatments is very scare. Further research
related to the nursery raising techniques including seed
germination, pre-sowing treatments and initial seedling growth
performance of this species is therefore required with an ultimate
objective of successful rattan plantation. Considering the facts
the present study was undertaken to investigate the effects of
pre-sowing treatment on seed germination and seedling growth
performance of C. longisetus in nursery and field condition.
Present research was carried out in the nursery of Bangladesh
Forest Research Institute (BFRI), Chittagong, Bangladesh, over a
period of three and half years from June 2010 to November 2013.
The study area was located between 222227 and 22290 North
latitude and 914630 and 914630 East longitude enjoys a
tropical climate, characterised by hot humid summer and cool dry
winter. The maximum and minimum temperature in the area are
28.3 - 31.9C and 15.2 - 25.2C 32. Relative humidity is usually low
in winter (November - February) and high in summer (June September). Mean annual rainfall is around 300 cm, mainly
occurring from June to September.
396
Seed collection and pre-sowing treatments: Ripe fruits of Calamus

longisetus were collected from natural forest of Ukhia, under Coxs
Bazaar district of Bangladesh in the first week of June 2010. To
determine the effect of pre-sowing treatment on seed germination
and seedling growth attributes, three different treatments were
applied namely, i) whole fruit ( seed + sarcotesta + scale), ii) seed
with pulp (seed + sarcotesta without scale) and iii) clean seed
(seed without sarcotesta and scale) (Fig. 2). Clean seeds were
obtained by removing the scale and pulp through rubbing. The
numbers of whole fruits and cleaned seeds per kg were 200 - 220
and 600 - 650, respectively. Seeds of C. longisetus were sown in
the germination trays filled up with soil mixed with decomposed
cowdung at a ratio of 3:1 by volume. Complete Randomized Design
(CRD) was adopted for the experiment with three replications.
Fifty seeds were sown in each replication and total of 450 seeds
were subjected to three treatments for the germination trial. The
trays were kept under nursery shade for one week and then
exposed to partial sunlight.
A)
(A)
Fi
B)
2 Wh l f it (A)
d l
(B)
d (B) f C l
i t
d f
Figure 2. Whole fruits (A) and cleaned seeds (B) of C. longisetus are
ready for sowing in the germination trials.
Assessment of seed germination and seedling growth

performance: The effects of pre-sowing treatments on seed
germination and seedling growth were explored periodically
through counting the germinated seeds and assessing initial
growth performance of seedlings. Cumulative germination was
recorded in every third day from the day of sowing and continued
till ending the germination (106 days after sowing the seeds).
Germination phase like imbibition period was determined by
counting the number of days required for the commencement of
germination from the day of sowing and germination period was
the number of days required for completion of germination from
sowing the seeds. For assessing the growth performance, all
seedlings were measured for above ground height (form base to
leaf tip) and number of leaves four months after sowing the seeds.
Besides these ten seedlings from each replicate (30 from each
treatment) were randomly uprooted and measured for total length
(root length and shoot length separately) for the assessment.
Seedling vigor index (VI) was calculated according to Abdul-Baki
and Anderson 33 as the germination percent multiplied by total
length of seedling (i.e. sum of shoot and root length).
Assessment of seedling growth performance in the nursery and
the field: To determine the seedling growth performance in the
nursery and the field, clean seeds were first sown in the nursery
bed. When the seedlings were about two months old (with 2 - 3
leaves), they were transferred in to the polybags (23 cm x 15 cm in
size) filled with soil mixed with cowdung. The polybags were kept
under full shade for one week and then placed under direct sunlight
where they were allowed to grow. When the seedlings were about
one-year old, 1000 seedlings were out planted in five plots in the
field at the beginning of the monsoon, e.g. June. Rest of the
seedlings (1000), were allowed to grow in the nursery for one
more year. Data on shoot and root length and leaf number of
these seedlings were also recorded at three, six, twelve and twenty
four months after transferring them in polybags. Seedlings in the
field were planted at 2 m 2 m spacing at the foot hills of Hinguli
Research Station, Chittagong, Bangladesh. The soil was sandyloam with a pH 5.7 - 6.0. Average rainfall of the area was about
3200 mm and average maximum and minimum temperature was
34.7C and 20.7C, respectively, indicating the ecophysiological
conditions of the planting site were ideal for the rattan plantations34.
Weeding was done at every six months in the field but no fertilizer
or water was added after planting. One hundred seedlings from
each of the five selected plots were measured to determine the
height growth of the species in the field. Data on the height of
each plant were recorded at six, twelve and twenty four months
after planting. Survival percentage of the planted seedling in the
field was determined one year after planting the seedlings. All
data were analyzed with computer software IBM SPSS ver. 21 to
determine the significant (p 0.001) variations among the
treatments.
400
200
00
Whole
fruit
80
80
800
600
Cummulative
germination(%)(%)
Cummulative germination
Germination percentage

Seed germination and initial growth performance of the
seedlings: Pre-sowing treatments significantly affected the
germination period and germination percentage of C. longisetus
seeds. Seed germination started 56 days after sowing (DAS) the
seeds and continued until 106 DAS. The fastest germination was
noticed 56 DAS in cleaned seeds followed by seeds with pulp (62
DAS) and slowest seed germination (64 DAS) was in whole fruits.
Clean seeds showed 60% germination between 70 and 80 DAS
days and germination continued up to 100 days. In the seeds with
pulp and whole fruits 40% and 35% germination occurred in
between 70 and 80 DAS and germination continued up to 106
DAS (Fig. 2). Finally, the germination percentage reached to 73.3
in clean seeds, 45.7 in seeds with pulp and 35.7 in whole fruits
(Fig. 3). Although the germination percentage in the cleaned seeds
was significantly (F = 56.083; p < 0.001) higher than in the other
treatments, there was no variation between whole fruits and seeds
with pulp.
Like other members of the family Palmeae, the species Calamus
required long time to germinate. Generalao 35 reported that cane
seeds take weeks to six months to germinate depending on the
(A)
60
60
Whole fruit
(B)
Seed with
pulp
Seed with pulp
species and method of treatment. Sumantakul 36 reported that C.

longisetus seed in different media starts to germinate from 30
days and continues till 60 days. Banik and Nabi 16 mentioned that
the seeds sown with intact sarcotesta require two or three months
to start germination and give poor germination percentage (10 - 26
only). Manokaran and Wong 37 reported that removal of the
sarcotesta is a necessary pre-treatment for cane to shorten the
germination period and obtain good levels of success. The findings
of the present research are also unidirectional to those reported in
the previous studies mentioned here.
The initial growth performance of the C. longisetus seedlings
was also significantly affected by the pre-sowing treatment of the
seeds. The shoot length (from base to leaf tip), root length, leaf
number and vigor index are shown in Table 1. The highest length
of shoot and root and vigor index was noticed in the cleaned
seeds which values were much higher than those in the whole
fruits and seeds with pulp. However, there was no significant
variation in the leaf number among the treatments. Similar results
were reported by several authors and mentioned that depulping
or removing the fleshy outer parts of the fruits enhance the seed
germination and seedling growth performance in the nursery
conditions. Hossain et al. 29, 30 mentioned that the seedling growth
including root, shoot and total length of T. chebula was
significantly increased with pre-sowing treatment specially by
depulping the fruits. Again, in separate experiments, Hossain et
al. 27, 29 showed maximum growth including shoot length, root
length, total length and vigor index in T. belerica seedlings when
fruits were depulped and soaked in cold water. However, similar to
this experiment, there was no remarkable difference in average
number of leaves per seedling of T. belerica seedlings 26.
Table 1. Initial growth performance of the seedlings germinated
from various treatments one month after germination.
Growth parameter
Shoot length (cm)
Root length (cm)
Leaf number
Vigor index
Cleaned seeds
12.53 1.6 a
6.7 0.87 a
2.1 0.52 a
1409.56 a
The vigor index of the seedlings in this experiment was

dramatically increased from 489.8 in the whole fruits to 1409.56 in
the cleaned seeds (Table 2). The vigor index usually depends on
the germination percentage and the seedling length. In this study
we noticed that the seedling length was marginally significantly
different among the treatments but the germination percentage in
the cleaned seeds was almost doubled of that in
the whole seeds and the seeds with pulp for the
Clean
seed
Clean
seed
species which led the vigor index substantially
higher in the cleaned seeds than in the other
treatments.
20
20
00
Seeds with pulp

9.04 1.3 b
5.32 0.36 ab
1.4 0.51 a
647.11 b
Note: The same different letter(s) in each row are significantly different at p 0.05, according to
Duncans Multiple Range Test (DMRT). indicates the standard error of mean.
40
40
Whole fruit Seed with pulp Clean seed

Treatment
Whole fruits
9.1 1.03 b
4.62 0.42 b
1.3 0.3 a
489.80 b
60 64 67 70 73 76 79 82 85 88 91 94 97 100 103 106 109

Day after sowing
Figure 3. Germination percentage (A) and the germination pattern (B) of C. longisetus for
whole fruits, seeds with pulp and cleaned seeds over time.
Seedlings growth performance in nursery

condition: In our preliminary experiment (Fig.
3), it was noticed that the germination percentage
of the cleaned seed was almost double of that in
the whole fruits and seeds with pulp. The initial
growth performance of these seedlings including
seedling length and vigor index were also
397
Table 2. Seedling growth of C. longisetus at different age (up to

24 months) in the nursery.
Age of seedlings
(month)
3
6
12
24
Average height
(cm)
17.5 2.5
22.8 2.8
30.2 3.7
82.7 6.2
Average length of
Roots (cm)
10.1 2.2
13.4 2.8
16.6 1.7
27.2 3.8
The figure in each column mean followed by standard error (SE) of means.
No. of leaves
Per seedlings
3.1 0.9
5.0 1.2
7.6 1.4
11.3 2.1
significantly higher than those in the other treatments (Table 1). We,
therefore, sowed only the cleaned seeds in the seed beds for
assessing the seedling growth performances in the nursery and
the field. Three thousand cleaned seeds were sown in three blocks
(considered as replications) of the nursery bed for the purpose.
The germination percentage (71.6 2.3) was almost similar of the
previous experiment (Fig. 3). Two-months old (after germination)
seedlings having 2 - 3 leaves were transferred in to the polybags
filled with soil mixed with cowdung and allowed them for growing
there. After one year of transferring the seedlings in the
polybags, 1000 seedlings were outplanted in the field. Rest of
the seedlings (1000) were grown in the nursery for one more year.
The seedling mortality in the nursery bed (less than three percent)
and during and after transplanting the seedlings to the polybags
(around 1%) was very negligible. Growth performance of seedlings
in the nursery determined at different age is shown in Table 2.
The seedlings attained 17.5 cm height (above ground) with
average length of root 10.1 cm and 3.1 leaves in three months. The
seedlings became quite tough and attained a height of 22.8 cm
with 13.4 cm root and 5.0 leaves at six months. The average height
30.2 cm with 16.6 cm root and 7.6 leaves was recorded at twelve
months. The seedlings attained a height of 82.7 cm with 27.2 cm
long root and 11.3 leaves at twenty four month (Table 3 and Fig.
3).
Table 3. Survival percentage and seedling growth of Calamus

longisetus after out planting.
Plots
Plot - 1
Plot - 2
Plot - 3
Plot - 4
Plot - 5
Survival (%) at
12 months
90.0
88.5
92.5
91.0
94.5
Average height (cm) at

6 months
12 months
24 months
52.8 2.9 c 70.9 2.4.7 b 117.3 3.8 b
67.6 3.7 a
82.4 3.7 ab 128.5 2.8 ab
61.7 2.2 ab
88.6 4.4 a
134.0 6.6 a
69.4 1.8 b
122.9 3.8 b
58.8 2.0 b
63.5 1.0 ab 80.2 3.5 ab
128.3 4.7 ab
Means followed by the same letter (s) are not significantly different at p 0.05, according to Duncans
Multiple Range Test (DMRT). indicates the standard error of the mean.
(A)A)
(B)
B)
Seedling survival and growth performance in the field: One year old seedlings of C. longisetus developed from cleaned seeds
grown in polybags were planted in the field. Survival and seedlings
growth performances were determined at 6, 12 and 24 months
after planting in the field (Table 3). Survival percentage varied
from 88.5 to 94.5 with an average of 91.3 among the plots. The
seedling height varied from 52.8 to 67.6 cm at six months, from
69.4 to 88.6 cm in one year and from 117.3 to 134.0 cm in two years
of planting (Figs 4 and 5).
Figure 5. Growth performance of C. longisetus 18

months after transplanting in the field.
The height growth of seedlings was always significantly higher

in plot 3 than in the other plots and lowest in plot 1. This variation
of the height growth in the seedlings was probably due to the
microclimate of the plots or some other unknown reasons. Since
the survival percentage of the seedlings in the field was quite
satisfactory, out planting of one-year old seedlings instead of
older seedlings may be considered for the planting the species in
the field. Similar report was also made by the Kerala Forest
Research Institute, India, and mentioned that rattan seedlings
were out planted at one-year old 38. Another interesting finding in
this study was that the plant height in the nursery condition at
two years (82.7) after transferring them into polybags was almost
similar to that in the field (69.4 - 88.6 cm) after one year of planting
(two years after transferring in to the polybags). However, it was
not possible to maintain the seedlings in the nursery after two
years of transferring them in polybags due to some unavoidable
circumstances.
(C)
C)
Figure 4. Different stages of seedlings of C. longisetus. One-month old seedlings of

C. longisetus in nursery bed (A), an individual seedling prior to transfer into the
polybag (B) and two-year old seedlings in the nursery (C).
398
Conclusions
Germination behaviour of C. longisetus is similar to
other members of the family Palmeae and needs longer
time to germinate. C. longisetus seeds start germination
after 56 days of sowing and complete within 106 days.
Maximum seed germination and highest initial growth
performance of the seedlings was observed in clean
seeds which was much higher than those in the other
two treatments. Delayed pricking at 100 - 120 days after
sowing the seeds from nursery bed to polybags
ensures least or no mortality of the seedling. Survival
of seedlings (91%) and growth performance in the field
was satisfactory when one year old seedlings were
outplanted. Therefore, clean seeds for the nursery
raising and one-year old seedlings for outplanting the species

may be recommended for large-scale plantation programs.
Acknowledgements
We are thankful to Mr. S. M. Zahirul Islam, Research Officer for
his support in data analysis. Thanks are due to Mr. Abul Bashar,
Forest Ranger, Mr. Abdus Salam, Nursery Supervisor and other
staff members of Minor Forest Products Division, BFRI for their
help in the execution of the study.
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Trophic State Index (TSI) applied in the assessment of anthropic impacts on the
surface water of a watershed
Adir Otto Schmidt *, Slvio Cesar Sampaio, Ralpho Rinaldo dos Reis, Camila Jussara Schmidt,
Edison Barbosa da Cunha and Lisdefferson Hamann Andrade
Western Paran State University, UNIOESTE/CCET/PGEAGRI /RHESA, Rua Universitria, 2069 CEP 85819-110, Cascavel,
PR, Brazil. *e-mail: adir.schmidt@unioeste.br
Abstract
The consolidation of the urbanization process in the cities and the intensification of agriculture and breeding in rural areas have caused impacts on
water resources, particularly the eutrophication of rivers. In order to contribute to the monitoring and preservation of water quality, this study aimed
to employ the Trophic State Index (TSI) to assess impacts of human actions on the surface water of a watershed of So Francisco Verdadeiro river.
In the period from 2011 to 2013, 52 monitoring campaigns were performed at intervals of 15 days. Water samples were collected from nine strategic
locations of the watershed and from a control location. The collection sites were defined by taking into account the possibility of different impacts
from each activity developed along the watershed. Discharge readings and analyses of total phosphorus and chlorophyll-a concentrations were
performed in order to calculate the TSI. Based on the results obtained, the trophic classification was mesotrophic in six locations, and eutrophic,
supereutrophic and hypereutrophic in one location each. It was concluded that different forms of use and occupation cause impact on the eutrophication
process, which varied in space and time. The highest concentrations of total phosphorus for sites with influence of urban activities occurred during
periods of low discharge, whereas for places with influence of agricultural activities they occurred in high discharge events. Sites with influence of
urban activities presented higher trophic classification than those influenced by agricultural activities. TSI, TSI(Chl), and TSI(TP) classifications may
vary for a same location, depending on environmental conditions and activities developed on site.
Key words: Water pollution, eutrophication, monitoring of water quality.
Introduction
Human activities are developed from natural resources and exert
influence on these resources by modifying the environment, which
affects such activities in a way that in some cases it is not possible
for people to inhabit those locations anymore. In China, the
eutrophication threatens water quality with indirect impact on
economic development and social stability 1.
The occupation and land use for agricultural activities and the
process of urbanization alter the physical, chemical and biological
processes of natural ecosystems with direct impact on water
quality 2-5. Chemical parameters of water bodies are modified due
to the supply of nutrients, particularly phosphorus and nitrogen,
which induce the process of eutrophication in rivers and lakes
and a consequent inadequate utilization for domestic, industrial,
agriculture and livestock consumption. The eutrophication of
rivers and lakes is worsening in tropical regions, which offer more
favourable environmental conditions for the growth of algae and
macrophytes. Apart from natural factors, the introduction of
elements derived from the use and occupation of land by man in
their watershed enhances the process of eutrophication. Studies
in western Paran indicate increased level of nutrients in high
rainfall events that occurred during the plantation of corn and
soybean, respectively 6-8.
The Trophic State Index (TSI) evaluates water quality regarding
nutrient enrichment. Its effect is related to excessive algal growth
or increased infestation of aquatic weeds. The TSI has been
consolidated as an indicator of eutrophication in aquatic
400
environments based on the quantification of total phosphorus

and chlorophyll-a. The result for the TSI-phosphorus is a measure
of the potential for eutrophication. The result for the TSIchlorophyll-a is considered as a measure of response of the water
body to the agent. Thus, the average index comprises the cause
and effect of the process 9.
The characterization of this process occurs by monitoring water
quality and is one of the main instruments to sustain a policy of
planning and management of water resources 10. The objective of
this study was to apply the trophic state index for the assessment
of human impacts on the rivers in the watershed of So Francisco
Verdadeiro, as well as to identify the impact of different human
activities on the eutrophication process.
The research was conducted in the watershed of So Francisco
Verdadeiro river, located at the western end of the Paran state, in
southern Brazil, with systematic monitoring every two weeks
between July 2011 and June 2013, totalling 52 samples for each
studied site. Monitoring was conducted in nine monitoring sites
and one control site, totaling 520 samples throughout the study.
Characterization of the studied area: The watershed of So
Francisco Verdadeiro river is located on the third plateau of Paran,
formed in the upper Jurassic and lower Cretaceous with
predominant soil classified as clayey oxisol. It presents drainage
area of 2,219.1 km2, with an average discharge of 46.9 m3 s-1, which

is the result of average rainfall of 1,800 mm yr-1, and altitude
between 729 and 221 m. The predominant activity is agriculture
with 69% occupancy in relation to the total area 11.
The definition of the sampling sites (Fig. 1) was performed in
order that the prevalence of different existing human actions and
their impact on water quality could be separately characterized.
Strategically, the nine sampling sites were defined in sections
of So Francisco Verdadeiro river and its major tributaries that
divided the watershed into nine subwatersheds that have
characteristic use and occupation. This division, whose
characteristics are presented in Table 1, allowed a study of the
influence of different human activities on the watershed
eutrophication process.
In general, the watershed can be divided into four important
human actions that contribute in different ways to the process of
eutrophication, namely: urban activity (sediment from construction
of buildings and nutrients from industrial and domestic sewage);
agricultural activity (surface runoff with presence of fertilizers);
extensive cattle ranching (surface runoff originating from
pastures); and poultry and swine intensive activity (application
and disposal of animal waste). The sub-watersheds in the
riverhead influence the following sub-watersheds according to

the diagram shown in Fig. 2.
Collection and preservation of samples: In each of the nine
monitoring sites, a limnimetric scale was installed in order to obtain
values for the hydrological regime of the river at the time of
sampling. Water sampling was performed with the aid of a glass
container with a manual bar sampler. The volume collected formed
an independent composite sample for each of the nine sites
studied. The containers with the samples were prepared so that
the content did not receive light and were placed in an isothermal
box with ice in order to be cooled at a temperature that could not
exceed 4C until arrival at the laboratory. All analyses were initiated
on the same day of collection so that there was no need for sample
preservation longer than the time required for transportation.
Discharge determination: For the control site, the discharge rate
was calculated for each sample based on the record of the time
required for filling a container of a known volume. At least seven
discharge measurements were performed at each site at all nine
monitoring sites, noting that each one was in a different regime
from the other, and discharge rating curves were built. Discharge
measurements were performed with a propeller
flowmeter, a coupled pulse counter and happened
on different days from those of sample collections.
Figure 1. Watershed location, urban perimeters, control site and collection sites along So
Francisco Verdadeiro river and its tributaries.
Evaluated parameters: For each location studied

in each campaign, water regime record for
discharge estimate and water collection for
analysis of chlorophyll-a and total phosphorus
concentrations were performed. The determination
of chlorophyll-a concentratrions was carried out
according to technical standard L5306 12 with 90%
acetone extraction after filtration on cellulose
acetate membrane with a porosity of 0.45 m. Total
phosphorus was determined by means of ascorbic
acid applied to the samples subjected to
autoclaving with potassium persulfate in acid
media (H2SO4), in accordance with methods
4500PB and 4500PE 13.
Table 1. Location and characterization of the control site and the nine collection sites along So Francisco Verdadeiro river
and its tributaries.
Site
C00
River
Geographic Coordinates
Fonte dos Mosaicos
S 2457; W 5328
S02
So Francisco Verdadeiro
after the urban perimeter
So Francisco Verdadeiro/Lope
S 2447; W 5343
S03
Lope (river mouth)
S 2447; W 5343
S04
So Francisco Verdadeiro/Toledo
S 2445; W 5347
S05
Toledo (river mouth)
S 2445; W 5347
S06
So Francisco Verdadeiro/ Santa Quitria
S 2446; W 5404
S07
Santa Quitria (river mouth)
S 2446; W 54 04
S08
So Francisco Verdadeiro (river mouth)
S 2444; W 5407
S09
Marrecos (river mouth)
S 2441; W 5409
S01
S 2455; W 5330
Characteristics
One of the several springs of So Francisco Verdadeiro river
in the urban perimeters of Cascavel.
After the urban perimeter of Cascavel and discharge of a
sewage treatment station.
Rural area.
Tributary of a rural area, tanks for fish farming and intensive
swine production.
Rural area with influence on the urban perimeter of Toledo.
Tributary of a rural area that receives domestic and industrial
sewage from the urban perimeter of Toledo.
Area with higher land declivity (building of a small
hydropower plant) with agriculture and pastures.
Tributary of an area with extensive production of cattle.
Close to the reservoir of Itaipu hydropower plant: rural area
and pastures with higher land declivity.
Close to the reservoir of Itaipu hydropower plant: rural area,
with intensive production of swine and poultry.
401
sub-watershed, since phosphorus concentrations

do not justify the difference in relation to other
places. Low concentrations of chlorophyll-a could
be due to environmental conditions that are
unsuitable for the development of phytoplankton
or to phosphorus restriction, for its adsorption to
clay, what makes it unavailable in the water
column 14. Mean values of chlorophyll-a below
5.0 mg L-1, considering all sampling points from
the source to the mouth, were found in a study in
Pariquera-Au river, with higher concentrations
recorded just below the release of the effluent
treatment station 15.
Figure 2. Schematic diagram of the control site and collection sites in the watershed of
Values below the minimum detection limit (0.01
So Francisco Verdadeiro river and their influences. A - Area of contribution of the
mg L-1) were obtained for the concentrations of
watershed (km2); A - Area of total contribution (km2); C contributory subwatersheds.
total phosphorus in C00. S01 and S05 stood out
Trophic State Index TSI: The TSI for rivers consists of the for presenting concentrations approximately 6 times higher than
trophic state index for phosphorus TSI(TP), shown in Equation the average concentrations of other sites. The concentrations of
1, and the trophic state index for chlorophyll-a TSI(Chl-a), in total phosphorus showed discrepant values (outliers) for the nine
studied sites. Reduction in phosphorus concentrations with
Equation 2 14.
increasing rainfall in places subject to the presence of domestic
sewage were found in rivers of So Paulo state 16. The high
TSI(TP) = 10 (6 ((0.42 0.36 (lnTP)) / ln2)) 20
(1)
concentration of phosphorus in rivers can be attributed to the
release of untreated sewage and fertilizers used in agriculture 17.
TSI(Chl-a) = 10 (6 ((-0.7 0.6 lnChl-a)) / ln2)) 20
(2)
In places subject to the release of manure, there may be a
in which TP Total phosphorus concentration, in mg L-1; Chl-a decrease of total phosphorus and orthophosphate in the rainy
season due to the dilution effect 18. Higher phosphorus levels are
chlorophyll-a concentration, in g L-1.
To calculate the TSI, the geometric means of the results of the common in rivers in rural areas, in a period of higher rainfall 16. A
concentrations of total phosphorus and chlorophyll-a from each study on the components present in the water of a micro-watershed
collection site within the study period were used. The final index of Piracicaba river - So Paulo found average phosphate
was the result of simple arithmetic means of the indices for total concentrations ranging from 0.08 to 0.49 mg L-1. The conclusion is
phosphorus and chlorophyll-a. To interpret the results, the points that phosphorus concentrations showed significant changes
were classified according to the results obtained for the TSI, using between dry and wet periods 19. The phosphorus present in the
surface runoff is an important component of diffuse pollution
the categories of trophic state, shown in Table 2 9.
sources in rural watersheds 2.
Table 2. Trophic State Index classification for rivers, according to
Higher concentrations of phosphorus in rainy periods, possibly
modified Carlsons index 14.
due to sediment load in also higher suspension under those
conditions, have been recorded in Tiet and Pinheiros rivers in
Chlorophyll-a
Total phosphorus
Classification
Range
(g.m-1)
(mg.L-1)
So Paulo state 20. A research conducted in 35 sampling sites in
Ultraoligotrophic (U)
TSI47
P0.013
Chl-a0.74
rivers in the So Paulo state found 64% of the average
Oligotrophic (O)
47<TSI52
0.013<P0.035
0.74<Chl-a1.31
concentrations of total phosphorus above 0.1 mg L-1, with 24 of
Mesotrophic (M)
52<TSI59
0.035<P0.137
1.31<Chl-a2.96
Eutrophic (E)
59<TSI63
0.137<P0.296
2.96<Chl-a4.70
the 35 areas surveyed being located in watersheds with population
Supereutrophic (S)
63<TSI67
0.296<P0.640
4.70<Chl-a7.46
density greater than 100 inhabitants km-2 14. Another study
Hypereutrophic (H)
TSI>67
0.640<P
7.46<Chl-a
reported average concentrations of total phosphorus above 0.15
TSI = Trophic State Index; P = Total Phosphorus; Chl-a = Chlorophyll-a.
mg L-1 for all points assessed in Queimados river, with results that
were three times greater for the stretch after the city limits of
Table 3 shows average values of each parameter assessed at all Concrdia - Santa Catarina state 21.
The TSI found for each site studied is presented in Table 4 and
nine collection sites and at the control site. S01 stands out from
the other sites for presenting concentrations of chlorophyll-a allows the division of points researched into five different
above average, possibly due to the presence of stabilization ponds categories. C00, where there is no eutrophication, is a protected
in the wastewater treatment station that is upstream of the spring in the urban perimeter of the city of Cascavel.
S01 is classified as hypereutrophic and located downstream of
collection site. The chlorophyll-a found in S03 is possibly
originating from the establishment of fish tanks present in the the release of effluents from the sewage treatment station of
Cascavel city. Facultative treatment ponds
Table 3. Average values found for the parameters assessed at the control site and all have high concentrations of nutrients and
nine collection sites.
favour algae development. The release in a
water body with little dilution capacity
Parameters
C00 S01 S02 S03 S04 S05 S06 S07 S08 S09
Chlorophyll-a (g L-1)
0.0
73.9
3.3
8.6
3.5
5.3
2.6
2.1
2.3
2.9
explains the TSI found at this point. TSI
Total Phosphorus (mg L-1) 0.001 0.764 0.147 0.129 0.138 0.819 0.121 0.071 0.108 0.135 results are associated with pollution along
3 -1
Discharge (m s )
402
0.001
0.56
8.95
2.78
13.28
3.05
26.75 19.80 49.85
9.80
Table 4. Trophic state indices and trophic state classification for the
control treatment and nine collection sites.
watershed discharges with such turbulence that its strength

disaggregates and carries particles of soil with nutrients and
pollutants that accumulate on the inside of rivers, lakes and
TSI
TSI
Site Chl-a TSI(a) TP TSI(b)
TSI(c)
TSI(d) TSI TSI(e)
(Chl-a)
(TP)
reservoirs. Low discharges, caused by lack of water storage in
C00 0.01
U
0.00
U
10.24
U
10.02
U
10.13
U
the system, decrease the dilution capacity for releases of
S01 63.44
H
0.69
H
86.02
H
67.92
H
76.97
H
pollutant loads that have no other place to deposit 24. Areas
S02 2.44
M 0.12 M
57.81
M 58.73 M 58.27 M
occupied by forests promote the reduction of the intensity of
S03 7.91
H
0.10 M
68.00
H
58.00 M 63.00
E
S04 2.80
M 0.11 M
59.01
E
58.53 M 58.77 M
surface runoff and also contribute to the improvement of water
S05 4.04
E
0.77
H
62.19
E
68.45
H
65.32
S
quality by promoting the settlement of discharge because of
S06 2.15
M 0.12 M
56.72
M 58.63 M 57.67 M
increased coverage, stability and water infiltration into the
S07 1.82
M 0.04 M
55.27
M 53.47 M 54.37 M
soil 25. Overall, inhabited areas (urban and rural residences),
S08 1.96
M 0.09 M
55.93
M 57.55 M 56.74 M
S09 2.15
M 0.10 M
56.72
M 58.09 M 57.40 M
farmed areas (perennial and annual crops) and degraded forests
Chl-a (g.m ) Chlorophyll-a; TSI Trophic state classification (Chl-a); TP (mg.L ) Total phosphorus;
contribute to the reduction of the regularity of discharge and
TSI Trophic state classification (TP); TSI(Chl-a) - Trophic state index for chlorophyll-a; TSI Trophic
state classification (TSI(Chl-a)); TSI(TP) - Trophic state index for total phosphorus; TSI Trophic state
water quality due to temporary changes in the intensity of
classification (TSI(TP)); TSI - Trophic state index (average) ; TSI(e) Trophic state classification (TSI); U
runoff.
Ultraoligotrophic; M Mesotrophic; E Eutrophic; S Supereutrophic; H - Hypereutrophic.
Each river is influenced by the characteristics of its
the watershed, due to land use in riverside properties 23.
surroundings and has capacity to recycle nutrients that increase
S03, classified as eutrophic by the TSI, results from the mean of with distance. An overload leads the problem to be transferred
a high TSI(Chl-a) and a low TSI(TP), what can be explained by the downstream. If there are new overloads downstream then loss of
absence of urban activities and a large number of fish tanks, which water quality occurs. The growth of population, urbanization
are conducive environments to algae growth, because their waters process and intensification of agriculture and animal farming lead
are free of turbulence, exposed to the sun and also enriched by to increased demand for water for domestic, agricultural and
phosphorus present in the feed given to fish.
industrial uses and create increasing pressure on the water quality
S05, classified as supereutrophic by the TSI, results from the of rivers. Therefore, there is a need for greater control in the origin
mean of a low TSI(Chl-a) and a high TSI(TP) and is located of nutrients and improvement of ecosystem management 3.
downstream of the release of effluent from sewage treatment plants
of Toledo city, which do not have stabilization ponds. The release
Conclusions
of treated effluent water enriches the water body with phosphorus; Considering the result of 52 campaigns performed during 24
however, the environmental conditions are unfavourable to the months in nine different locations and a control site along the
development of algae.
watershed of So Francisco Verdadeiro river, one can conclude
S02, S04, S06, S07, S08 and S09, all classified as mesotrophic by that the presence of total phosphorus impacts on water quality,
the TSI, are sites with discharges that provide them large dilution increasing its TSI. Different forms of land use and occupation
capacity or are located far from point sources, thus potential caused impact on the eutrophication process that varied in time
impacts are mitigated by their natural ability of self-purification. and space. The highest concentrations of total phosphorus for
Due to the self-purification process and the influx of rivers with sites with influence of urban activities occurred in periods of low
better conditions, downstream sites can provide improvements in discharge. In contrast, the highest concentrations in sites with
water quality 22.
influence of agricultural activities occurred in high discharge
The TSI of Queimados river in the Santa Catarina state recorded events.
a sharp rise in the stretch where the river has urban influence,
Sites with urban influence and do not have an environment,
which declines as it receives other tributaries of rural origin and which is favourable for the development of algae prior to discharge
approaches the mouth 21. A study conducted in the watershed of into the water body, were classified into different classes by the
upper Uruguay river in western Santa Catarina state concludes TSI(Chl-a) and the TSI(TP). Sites with agricultural influence and
that for most rivers, from source to mouth, there is a gradual present an environment, which is favourable for algae
increase of phosphorus, especially the rivers flowing through development prior to discharge into the water body, were also
urban areas, where there is a deviation from this pattern, caused classified into different classes by the TSI(Chl-a) and the TSI(TP).
by high input of organic material 23.
Sites with influence of urban activities showed trophic state
S01 and S05, strongly influenced by urban activities, have classification rating higher than those found in sites influenced
increased concentrations of phosphorus with decreasing by agricultural activities.
discharge. S07, mouth of a sub-watershed with predominantly
extensive production of cattle and agricultural, has high
Acknowledgements
concentrations of total phosphorus at higher discharge events. The authors would like to thank Fundao Araucria de Apoio ao
Its lower concentrations occurred at times of lower discharges. In Desenvolvimento Cientfico e Tecnolgico do Paran [Araucria
S02, S03, S04, S06, S08 and S09 there is junction of urban and Foundation in Support of the Scientific and Technological
agricultural impacts, what resulted in total phosphorus Development of Paran] for financially supporting this research.
concentrations above 0.1 mg L-1 at the extremes of high and low
discharge.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Seasonal elemental variations of Fe, Mn, Cu and Zn and conservational management

of Rastrelliger kanagurta fish from Karachi fish harbour, Pakistan
Quratulan Ahmed 1, Farzana Yousuf 2, Maliha Sarfraz 3, Nor Kartini Abu Bakar 4, Mansour A. Balkhour 5
and Muhammad Aqeel Ashraf 6*
The Marine Reference Collection and Resource Centre, 2 Department of Zoology, University of Karachi, Karachi, 45320 Pakistan.
Department Physiology & Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan. 4Department of Chemistry,
University of Malaya, 50603 Kuala Lumpur, Malaysia. 5Department of Environmental Sciences, King Abdulaziz University, Jeddah
21589, Saudi Arabia. 6 Department of Geology, University of Malaya 50603 Kuala Lumpur, Malaysia.
*e-mail: aqeelashraf@um.edu.my
1
3
Abstract
Fish samples of Rastrelliger kanagurta of various lengths (cm) and weight (g) were collected from the coast of Karachi fish Harbour during August,
2006 to December, 2011. Samples were analysed as seasonal variation for Fe, Mn, Cu and Zn by using Atomic Absorption Spectroscopy. The highest
(24.01.75) and the lowest mean (23.51.65) lengths of fish were measured in 2009-2010 and 2010-2011. The maximum (13222.76) and minimum
(12618.65) weights were also measured in 2009-2010 and 2010-2011. The highest (498.21161.37 g/g) and lowest (16.7414.07 g/g) mean
concentrations of Fe were recorded in liver and gonads of fish during 2007-2008. Maximum (18.239.46 g/g) and minimum (0.510.45 g/g)
concentrations of Mn were determined in liver and gonads during 2006-2011. Fishing operations can cause ecological impacts of different types: by
catches, damage of the habitat, mortalities caused by lost or discarded gear, pollution, generation of marine debris, etc. Periodic reassessment of the
R. kanagurta potential is also required with adequate inputs from exploratory surveys as well as commercial landings and this may prevent any
unsustainable trends in the development of the tuna fishing industry in the Arabian Sea.
Key words: Heavy metals, Rastrelliger kanagurta, fish harbour, Karachi, Pakistan.
Introduction
For so many years, preventive measures have been taken to reduce
the input of trace metals into oceans, rivers and estuaries, but
accumulation in the different aquatic systems have been reported
even today 19, 22, 23, 26, 29, 30. The anthropogenic activity generating
the cause for environmental concern may have divesting effects
on the ecological balance of the aquatic environment and diversity
of biota 8, 9. Sea is one of the precious gifts awarded by nature to
the human beings. Trace elements occur in minute concentrations
in biological systems. They may exert beneficial or harmful effects
on plant, animal and human life depending upon the concentration.
Heavy metals have the tendency to accumulate in the various
aquatic animals, and the accumulation depends upon the intake
and the elimination from the body 17. Metal pollution of the sea is
less visible and direct than other types of marine pollution but its
effects on marine ecosystems and humans can be intensive and
extensive.
Fish being a best nutritional source is a component of a balanced
diet that is a vital part of a healthy lifestyle. It is far healthier than
many alternative foods. Marine fishes exposed to these heavy
metals have been consumed as sea foods and hence, a connecting
pathway for the transfer of toxic heavy metals in human beings.
Fishes are major part of the human diet because they have high
protein content, low saturated fat and omega fatty acids, which
are known to support good health 6, 15. Fish is a major source for
iron in adults and children. Iron deficiency causes anemia. Iron is

the most abundant element examined in the muscle tissue of the
various fishes.
The Karachi coastline growing pollution level, tied to the
increasing of the shipping industry through the Karachi port,
severely contaminating the mangrove forests and marine life.
Industries produce large amount of industrial waste 33. Untreated
waste and sewage from factories drained into the sea as well as oil
spills from ships and fishing trawlers transiting the port 25. Marine
food, especially fish, are an important part of the human diet across
the world. It is generally known that marine fish have seasonal
variations in chemical composition, especially in fatty acids. Study
was done on a fish, Rastrelliger kanagurta commonly called
surmai in Pakistan, a shallow warm water fish in Karachi coast.
The main objective of this study was to determine the heavy
metal (Fe, Mn, Cu, Zn) concentrations in the liver, muscles, kidney,
gills and gonads of fish Rastrelliger kanagurta from Karachi
coast. Thus the abundance and bioavailability of metals in the
marine environment, assessed as the bioaccumulation of metals
by the tissues of marine organisms, is of great concern. Mapping
area is shown in Fig. 1.
Fishes of various lengths (cm) and weights (g) were collected in
405
Figure 1. Map of Karachi coast.
August, 2006 to December, 2011 from the coast of Karachi. The

fishes were collected seasonally for four seasons of Pakistan
(autumn, winter, spring, summer) for analysis of heavy metals.
Sampling point and sample collection: Samples were collected
seasonally (autumn, winter, spring, summer) from Karachi coast
and Fish Harbour West Wharf Karachi as follows: autumn
(September and October), winter (November, December and
January), spring (February and March) and summer (April, May,
June and July) between August 2006 and December 2011. Eighty
six Rastrelliger kanagurta samples were collected in 2006-2007,
91 samples in 2007-2008, 92 samples in 2008-2009, 84 samples in
2009-2010 and 81 samples in 2010-2011.
Measurement of length (cm) and weight (g) of fishes: The length
(L) of the fishes collected during the seasonally surveys from
Fish Harbour of Karachi was measured from the tip of the anterior
part of the mouth to the caudal fin in (cm). Fish weight was
measured after blot drying with a piece of clean towel. From the
fresh samples total length (TL) in cm and body weight (W) in g,
measured to the nearest 0.1 cm and 0.01 g, respectively.
Sample preservation: Samples were immediately transported to
the laboratory, thawed and rinsed in distilled water to remove
foreign particles. After that length in cm and weight in g was
determined. Fish were tagged for identification and stored at 4C
until time for analysis.
Sample preparation: Samples collected for the analysis of heavy
metals were dissected using steel scissors and scalpels to remove
approximately 5 g dorsal muscles, entire liver, 2 rakers of gills,
entire kidney, and entire gonads, washed with deionized water
and weighed 17, 27, 32. Samples were ground and ashed at 500C for
3 hours until turned to white or grey ash. The ashes were dissolved
406
in 0.1 M HCl according to the method described by Gutierrez et

al.12. The ashes were dissolved in 10 ml HCl in a beaker and the
dissolved ash residue filtered with Whatman filter paper, and 1.0
ml of filtered solution was diluted with 25 ml distilled water for
elemental analysis.
Analysis of samples on atomic absorption spectrophotometer:
One ml of prepared sample was diluted in 25 ml of distilled water.
The instrument (Analyst 700) was started and programme a Win
lab 32 software prepared. Three standards from 1000 ppm stock
solution to 2, 4 and 6 ppm were prepared and the equipment was
calibrated with the standards. The samples were aspirated one by
one to detect the required metal. Finally, results were collected
from monitor.
Statistical analysis: Descriptive statistics (mean, standard
deviation, range) and three-way analysis of variance (ANOVA)
were conducted using Excel software. The data transformations
allowed for adjusting of all the zero values in the analytical results
prior to the ANOVA test. A three-way ANOVA statistical procedure
was employed in the assessment of variation in metal
concentrations. Three-way analysis of variance (ANOVA) 31, in
which the within sample variances and between the sample
variances were considered, was utilized to test for the significance
of differences between organs and metals. The three way analysis
of variances method was used to consider the first order
interactions between the three factors, namely organs, year and
seasons. The Bonferroni test was used for correction to counteract
the problem of multiple comparisons.
Length and weight of fishes from Karachi coast (2006-2011):
The maximum mean length of Rastrelliger kanagurta in cm was
recorded (25.51.39) in winter (2010-2011) and second highest
(25.51.259) was also measured in winter (2009-2010). The minimum
Manganese (Mn): The highest mean concentration of Mn

(20.835.83 g/g d.w) in liver was estimated in autumn 2010-2011
and the second highest mean (19.366.23 g/g d.w) in winter 20092010. The lowest mean concentration in liver (5.531.95 g/g d.w)
was in autumn 2006-2007 and second lowest mean (6.112.44 g/
g d.w) in autumn 2007-2008.
Highest mean Mn concentration of muscles (6.154.44 g/g d.w)
was recorded in summer 2010-2011 and second highest (4.222.86
g/g d.w) in summer 2008-2009. The lowest mean concentration
of muscles (1.421.20 g/g d.w) was recorded in spring 2006-2007
and second lowest (1.641.22 g/g d.w) in autumn 2007-2008.
The highest mean Mn concentration of kidney (3.522.05 g/g
d.w) was recorded in summer 2010-2011 and second highest
(2.801.25 g/g d.w) in spring 2009-2010. Lowest mean
concentration of kidney (1.450.35 g/g d.w) was recorded in
winter 2008-2009.
Maximum mean concentration of gills was recorded (8.014.20
g/g d.w) in summer 2009-2010 and the second highest
2006-2007
Seasons
Summer
Spring
Muscles
Winter
Liver
Kidney
Autumn
Gills
0
100
200
300
400
500
600
Gonads
700
Concentration of Fe (
g/g)
B
2007-2008
Seasons
Summer
Spring
Muscles
Liver
Winter
Kidney
Autumn
Gills
0
200
400
600
800
Gonads
g/g)
C
2008-2009
Summer
Seasons
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
100
200
300
400
500
600
700
800
g/g)
D
2009-2010
Summer
Spring
Seasons
Metal concentrations in fish samples:

Iron (Fe): The highest mean concentration of Fe (603.28 140.94
g/g d.w) was recorded in liver in summer 2010-2011, second
highest (595.45115.15 g/g d.w) was also recorded for liver in
summer 2009-2010. The lowest mean concentration in liver
(297.0772.30 g/g d.w) was determined in autumn 2006-2007,
second lowest mean (322.32124.34 g/g d.w) was in autumn 20102011.
The maximum mean Fe concentration of muscles (56.5724.23
g/g d.w) was recorded in spring 2007-2008, (51.9921.79 g/g
d.w) in winter 2007-2008 and (50.8123.45 g/g d.w) in spring 20092010. The minimum mean concentration of muscles (18.9213.12
g/g d.w) was recorded in spring 2006-2007 and second lowest
(19.3510.14 g/g d.w) was in autumn 2010-2011.
Highest mean Fe concentration of kidney was found (57.5220.19
g/g d.w) in in spring 2010-2011 and second highest (53.7816.53
g/g d.w) in summer 2010-2011. Lowest mean concentration of
kidney was found (17.929.60 g/g d.w) in autumn 2007-2008 and
second lowest (18.2424.80 g/g d.w) in winter 2007-2008.
The highest mean Fe concentration of gills was recorded
(49.7320.30 g/g d.w) in autumn 2010-2011 and the second highest
(43.1918.09 g/g d.w) in autumn 2009-2010. The lowest mean
concentration of gills was recorded (18.467.57 g/g d.w) in autumn
2007-2008 and second (19.7611.78 g/g d.w) in winter 2007-2008.
The highest mean Fe concentration of gonads (93.6319.89 g/
g d.w) was recorded in spring 2010-2011 and second highest
(54.8923.11 g/g d.w) in summer 2010-2011. The lowest mean
concentrations of gonads was (15.016.37 g/g d.w) in autumn
2006-2007 and second lowest mean concentration (15.619.06 g/
g d.w) in spring 2006-2007 as shown in Fig. 2.
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
200
400
600
800
g/g)
E
2010-2011
Summer
Seasons
mean length in cm was obtained (221.003), (22.30.908),

(22.51.024) in spring (2007-2008), (2008-2009), (2010-2011),
respectively, and (22.81.23) in summer (2006-2007).
The highest mean weight in g was obtained (14828.63) and
(14615.05) in winter (2006-2007), (2008-2009), respectively, and
(14618.40) in autumn (2010-2011). The lowest mean weight was
observed (11111.11), (11210.20), (11210.20), (11613.58) in
spring (2007-2008), (2008-2009), (2009-2010), (2010-2011),
respectively, and (11622.96) in autumn (2007-2008).
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
200
400
600
800
1000
g/g)
Figure 2. Concentration of Fe (g/g) in different sample of fish

Rastrelliger kanagurta (2006-2011) from Karachi coast.
(7.682.16 g/g d.w) in spring 2009-2010. The lowest mean

2007-2008 and second (2.512.28 g/g d.w) in spring 2008-2009.
Highest mean concentration of gonads was recorded (2.581.26
g/g d.w) in summer 2010-2011 and second highest (2.130.83 g/
g d.w) in summer 2008-2009.
407
2006-2007
Seasons
Summer
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
10
15
20
Concentration of Mn (
g/g)
B
2007-2008
Summer
Seasons
Copper (Cu): Highest mean Cu of muscle was recorded (8.213.37

g/g d.w) in summer 2009-2010 and second highest (5.894.55 g/
g d.w) in the spring 2009-2010. The lowest mean concentration of
muscles was recorded (2.032.23 g/g d.w) in spring 2010-2011
and second lowest (2.251.18 g/g d.w) in winter 2010-2011.
In liver, the highest mean Cu was recorded (87.8336.01 g/g
d.w) in summer 2009-2010 and lowest (19.5213.29 g/g d.w) in
spring 2007-2008.
The maximum mean Cu concentration of kidney was recorded
(7.613.17 g/g d.w) in summer 2008-2009 and second maximum
(6.232.72 g/g d.w) in spring 2008-2009. Minimum mean
concentration of kidney was recorded (2.031.78 g/g d.w) in
autumn 2007-2008 and second minimum (2.171.48 g/g d.w) in
winter 2006-2007.
The highest mean concentration of gills was recorded (6.802.05
g/g d.w) in summer 2009-2010 and second highest (6.652.16
g/g d.w) in summer 2010-2011. The lowest mean concentration
of gills was recorded (1.950.85 g/g d.w) in autumn 2006-2007
and second (2.430.92 g/g d.w) in winter 2006-2007.
The highest mean concentration of gonads was recorded
(4.631.82 g/g d.w) in summer 2010-2011 and second highest
(3.591.16 g/g d.w) in spring 2010-2011. The lowest mean
concentrations of gonads was recorded (0.820.54 g/g d.w) in
autumn 2006-2007 as shown in Fig. 4.
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
10
15
20
25
g/g)
2008-2009
Summer
Seasons
The lowest mean concentrations of gonads (0.440.30 g/g

d.w) was recorded in winter 2006-2007 and second lowest mean
concentration about (0.460.38 g/g d.w) was recorded in winter
2007-2008 as shown in Fig. 3.
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
408
Gonads
0
10
15
20
25
30
35
g/g)
D
2009-2010
Seasons
Summer
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
10
15
20
25
30
35
40
g/g)
E
2010-2011
Summer
Seasons
Zinc (Zn): The highest mean concentration of Zn in liver was

estimated (68.8126.59 g/g d.w) in autumn 2010-2011 and the
second highest mean (61.7220.91 g/g d.w) in spring 2009-2010.
The lowest mean concentration in liver (26.5416.39 g/g d.w)
was estimated in winter 2007-2008.
The highest mean Zn concentration of muscles (9.413.14 g/g
d.w) was recorded in spring 2009-2010 and second highest
(7.163.67 g/g d.w) in summer 2009-2010. The lowest mean
concentration of muscles (2.961.22 g/g d.w) was recorded in
autumn 2006-2007 and second lowest (3.021.24 g/g d.w) in
winter 2006-2007.
The highest mean Zn concentration of kidney (6.462.82 g/g
d.w) was recorded in summer 2009-2010 and second highest
(6.042.52 g/g d.w) in autumn 2007-2008. Lowest mean Zn
concentration of kidney was recorded (2.151.86 g/g d.w) in
autumn 2006-2007 and second lowest mean concentration
(2.161.91 g/g d.w) in autumn 2010-2011.
The highest mean Zn concentration of gills was recorded
(8.144.99 g/g d.w) in summer 2009-2010 and the second highest
2006-2007 and second lowest (1.310.41 g/g d.w) in winter 20062007).
The maximum mean Zn concentration of gonads was recorded
(7.643.37 g/g d.w) in summer 2010-2011 and second maximum
concentration of gonads was recorded (1.550.75 g/g d.w) in
winter 2007-2008 and second lowest mean concentration (1.581.36
g/g d.w) in autumn 2007-2008 as shown in Fig. 5.
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Gonads
0
10
15
20
25
30
35
40
g/g)
Figure 3. Concentration of Mn (g/g) in different sample of fish

2006-2007
Spring
Muscles
Liver
Winter
Seasons
Summer
Seasons
2006-2007
Summer
Spring
Muscles
Liver
Winter
Kidney
Gills
Autumn
Kidney
Gills
Autumn
Gonads
Gonads
0
10
20
30
g/g)
Concentration of Cu (
40
50
2007-2008
Muscles
Liver
Seasons
Seasons
Spring
Kidney
2007-2008
Spring
10
20
30
g/g)
40
Kidney
Gills
Gonads
0
Gonads
0
Muscles
Liver
Winter
Autumn
Gills
Autumn
10
50
Seasons
Muscles
Liver
Winter
2008-2009
Spring
Muscles
Gonads
Gills
Autumn
Liver
Kidney
Gills
Winter
Autumn
Kidney
10
20
Gonads
20
40
60
g/g)
60
Summer
Spring
20
30
40
50
g/g)
Concentration of Zn (
2008-2009
Summer
80
100
30
40
50
60
70
g/g)
D
2009-2010
Summer
Seasons
2009-2010
Summer
Seasons
60
50
Summer
Winter
Spring
Muscles
Spring
Muscles
Liver
Kidney
Gills
Winter
Autumn
Liver
Winter
Gonads
0
Kidney
10
20
Gonads
0
20
40
60
g/g)
80
100
30
40
50
60
70
g/g)
Gills
Autumn
2010-2011
Summer
Seasons
2010-2011
Summer
Seasons
20
30
40
g/g)
Summer
Seasons
10
Spring
Muscles
Liver
Kidney
Gills
Gonads
Winter
Spring
Muscles
Winter
Autumn
Liver
Kidney
Gills
Autumn
Gonads
0
20
40
60
80
g/g)
100
120
Figure 4. Concentration of Cu (g/g) in different sample of fish

20
40
60
80
100
g/g)
Figure 5. Concentration of Zn (g/g) in different sample of fish

A three-way analysis of variance (ANOVA) 31, in which the within

sample variances and between sample variances were considered,
utilized for testing the significance of differences between organs,
between seasons and between metals. The advantage of this
409
method compared with the two-way ANOVA is that three-way

method considers the first-order interactions between the three
factors, namely organs, seasons, and metals. The effects of these
interactions are indicators of the species specificity as well as the
organ specificity for metals to accumulate. Three-way ANOVA
tests the significance of the single factor effects as well as the
first-order interaction effects using the statistics called Snedecors
F statistics. The significance of the statistics F is always associated
with a certain prefixed level of significance or probability of type
1 error, denoted by P. The Bonferroni method is a simple method
that allows many comparison statements to be made (or confidence
intervals to be constructed) while still assuring an overall
confidence coefficient is maintained. This method applies to an
ANOVA situation when the analyst has picked out a particular set
of pairwise comparison contrasts or linear combination in advance.
The metal interaction with tissues, season and year observation
were significant in R. kanagurta fish in the case of Fe, Mn, Cu
and Zn. The explanation is given in Appendices I and II.
Aquatic organisms such as fish and shell fish accumulate metals
to concentrations many times higher than present in water or
sediment 11. They can take up metals concentrated at different
levels in their different body organs 18 under certain environmental
conditions such as salinity and pH. Metal accumulation in the
living organisms up to toxic concentrations can cause ecological
damage 13. Thus, heavy metals are acquired through the food
chain as a result of pollution and potential chemical hazards are
threatening consumers. At low levels, some heavy metals such as
copper, cobalt, zinc, iron and manganese are essential for enzymatic
activity and many biological processes. Other metals, such as
cadmium, mercury and lead have not known to have essential role
in living organisms and are toxic even at low concentrations. The
essential metals also become toxic at high concentrations 3, 4.
Studies carried out on fish have shown that heavy metals may
have toxic effects, altering physiological activities and biochemical
parameters both in tissues and in blood of fish 20.
Conservation and management: Rastrelliger kanagurta is like
any other renewable living resource; the rate of harvest affects
the abundance and ability to sustain various levels of exploitation.
As fishing pressure for Rastrelliger kanagurta increases on a
global scale, management and conservation measures are essential
if the populations of Rastrelliger kanagurta are to remain at
desired levels of abundance. However, the management is
complicated by their migratory nature, and calls for special
cooperation among nations, since a single nation cannot manage
Rastrelliger kanagurta effectively. This is reflected in Article 64
of the United Nations Convention on the Law of the Sea, which
calls on states to cooperate directly or through appropriate
international organizations to ensure the conservation of highlymigratory species. The socio-economic importance of Rastrelliger
kanagurta coupled with the global spread of industrialized fishing
has led to the reduction of global stocks to dangerously low
levels28. Recent analyses suggest that large, predatory fishes have
declined more than 90% globally in the past 50 years 24, raising
concerns regarding the future of many species. This is particularly
troubling, as any ecosystem-wide effect is bound to be widespread
and likely irreversible due to the global nature of the decline 24.
Maritime countries engaged in fishing Rastrelliger kanagurta
and like species cooperate regarding conservation and fisheries
410
management within several international frameworks 21. Currently,

there are many regional fisheries management organizations
(RFMOs) dedicated to the conservation and management of
Rastrelliger kanagurta whose common objective is to maintain
the populations at or above levels of abundance that can support
the maximum sustainable yield (MSY). However, as demand for
Rastrelliger kanagurta continues to rise, and with it the levels of
exploitation, these organizations find it ever more difficult to reach
agreement on the implementation of effective management
measures. Cooperation must also extend beyond the scale of single
oceans. Industrial Rastrelliger kanagurta fleets are highly mobile
and in the principle market are intensively traded on a global scale.
In addition many Rastrelliger kanagurta research, conservation
and management problems are similar in all oceans. Therefore,
there is a need for exchange of information and collaboration on a
global scale regarding fisheries for Rastrelliger kanagurta and
other species with wide global distribution.
Major issues in Rastrelliger kanagurta fishery in Arabian Sea:
Rastrelliger kanagurta is an important but not well managed
fishery in the Arabian Sea. The evolution of Rastrelliger kanagurta
longline fisheries in all oceans has changed fishing strategies as
different species have been targeted. These tactics increase the
use of longliners, simultaneously making the stock seem bigger
but damaging the fishs breeding capacity 2. The present study
represents an attempt to generate the main fishery interactions
issues in the Arabian Ocean as they appear on the basis of the
present knowledge we have of the fisheries for Rastrelliger
kanagurta and other similar species. Severe overfishing leads to
species ecological extinction because overfished populations no
longer interact significantly with other species in the community16.
Periodic reassessment of the tuna potential is also required with
adequate inputs from exploratory surveys as well as commercial
landings and this may prevent any unsustainable trends in the
development of the Rastrelliger kanagurta fishing industry in
the Indian Ocean.
As many fisheries in the region continue to be open access, i.e.
no effective controls are in place to limit the growth of fishing
capacity and fishing efforts or to limit catches through a quota
regime, the high resource rent potential manifests itself initially in
high returns to the owners of fishing vessels. This high profitability
attracts new entrants into the fisheries as well inciting current
operators to invest in technological improvements of fishing craft
and gear, causing the fishing power to augment. The capacity and
effort of expanding investments commonly continue to take place
until the time when the fishery becomes unprofitable and crew
incomes have dropped to a low level.
The economic incentive regarding fisheries resources to be
found in these deeper waters, combined with insufficient
monitoring, control and surveillance of fishing activities has led
to a proliferation of illegal, unregulated and unreported fishing
(IUU). IUU fishing activities are now one of the biggest threats to
Indian Ocean resources and ecosystems. However, IUU fishing is
a pervasive problem in many of the worlds oceans 7. Whereas
IUU fishing occurs, or has the potential to occur, in all captured
fisheries, both in marine and inland waters, it has raised particular
concern with regard to fisheries on the high seas for highly
migratory and straddling fish stocks as well as pure highseas
stocks, i.e. fishery resources whose entire life cycle is within waters
outside of national jurisdictions 5. The IOTC estimated that, in

1996, IUU fishing amounted to nearly 100,000 t in the Arabian
Ocean, i.e., 10% of all reported landings of tuna and tuna-like
species. It is estimated that the lower and upper estimates of the
total value of current IUU losses worldwide are between $10 billion
and $23.5 billion annually, representing between 11 and 26 million
tonnes in fish catch.
The present study represents an attempt to examine the main
fishery interaction concerned with the Rastrelliger kanagurta
fisheries issues in the Karachi coast regarding heavy metals. These
include, (i) lack of reliable information on the status of stock
position of Rastrelliger kanagurta, (ii) lack of information on
Rastrelliger kanagurta migration, (iii) lack of trained man power
in manning Rastrelliger kanagurta longliners and high sea
purseiners, (iv) investors are reluctant to invest in high sea
Rastrelliger kanagurta fishing as they are still not convinced
whether it is a profitable venture, (v) permitting foreign Rastrelliger
kanagurta fishing vessels to operate in the Arabian Ocean
countries, (vi) fishermen are not familiar with the modern postharvest handling procedures for high priced Rastrelliger
kanagurta, (vii) countries yet to develop satellite based PFZ
advisories for oceanic Rastrelliger kanagurta to help Rastrelliger
kanagurta vessel operators to save energy and scouting time,
(viii) no proper deep sea fishing policy/guidelines, (ix) The El
Nino Southern Oscillation (ENSO) phenomenon is considered the
main cause for inter annual climatic variability around the globe.
There is a lack of studies on oceanographic environmental
parameters variability during extreme climatic events and its
relationship with Rastrelliger kanagurta catch in Arabian Ocean.
We have presented major issues responsible for declines in many
coastal and oceanic Rastrelliger kanagurta over a short period.
Our results indicate that Rastrelliger kanagurta should be given
serious attention similar to that given to other threatened large
marine predators.
Fishing capacity: Studies have indicated overcapacity exists in
the purse-seine fishery in the Karachi Coast. In an effort to address
this problem, several measures have been approved calling on
states to not exceed the number of vessels they had fishing at
earlier times, but they have had limited success in controlling fleet
growth.
Compliance: There is strong need to implement a number of
measures to improve compliance with conservation measures.
However, many of the nations do not provide the information
needed to monitor compliance.
Database: There is lack of catch-statistical data for some of the
important fisheries in the Karachi Coast, which hinders its ability
to make comprehensive stock assessments, timely conservation
and management recommendations, and to monitor compliance.
Data on discards and by catches are also lacking.
IUU fishing: There is strong need to introduce a number of
measures to control IUU fishing, but few of these have been
implemented by Arabian Sea nations.
Insufficient financial resources: Field research is needed to
improve estimates of vital parameters for Rastrelliger kanagurta,
such as the rates of natural mortality, fishing mortality, migration,

and mixing. Large-scale tagging programs are useful for this, but
they are costly. The budget is insufficient for such programs, so
there is a strong need to seek funding from other sources.
Bycatches: The very complex issue of bycatches in fishery is of
great concern to governments and stakeholders. Any measures
to deal with this issue must be based on sound science if they are
to be effective in reducing bycatches and maintaining sustainable
fisheries. Obtaining the data needed to quantify the impact of
bycatches on the various species will entail expanding observer
programs to longline fleets, and require scientific studies of the
ecosystem to which the bycatch species and Rastrelliger
kanagurta belong and research into the development of fishing
gear and technology that will allow the target species to be caught
without harming the bycatch species. All of this will require
considerable resources, both human and financial. There are
several questions that need to be considered under this heading:
a) What are the levels of populations? Are the populations
increasing, remaining stable, or decreasing?
b) Are any of the populations in danger of extinction?
Rastrelliger kanagurta fishery management approaches:
Virtually all human activities have some impact on the ecosystem
in which they take place, and fishing is no exception. Management
strategies must be adopted for Karachi Coast stocks.
These strategies should include the following elements:
a. Species-specific management objectives; procedures for data
collection, verification and analysis.
b. Precautionary limit and target reference points reflecting
international best practice; where necessary,
rebuilding programmes aimed at returning stocks to
sustainable levels within biologically reasonable time-frames.
c. The harvest is managed in a way that avoids, or at least
minimizes, the loss of genetic diversity;
d. The waste of resources is kept at a minimum low [bycatch
of target species/catch] ratio;
e. The use of the energy by the vessels is minimized low
[energy use/catch] ratio;
f. The gear used is the best to harvest the resource with the
least impact on the habitat low [habitat damage/catch] ratio.
From the ecological point of view, a fishery should operate in
such a way that it meets or approaches the conditions stated
before. This view does not include economic or social
considerations, which may also be important to humans. For
example, yields less than the maximum possible may be preferable
if the value of this fish caught or the employment of fishers
increases, but larger catches increase employment in the
processing plants 14.
Bycatch management: Bycatch of juvenile Rastrelliger kanagurta
and unmarketable species and size groups of other fish in purse
seine fisheries, and juvenile swordfish in longline fisheries,
contributes to the overexploitation of some stocks, and is an
allocation issue. There has been substantial progress in identifying
gear technology solutions to seabird and sea turtle bycatch on
longlines and to direct dolphin mortality in purse seines 10. The
magnitude of incidentally caught species (by-catch), their discards
as well as catch of small individuals of target species and the
411
status of stocks of the bycatch species have been another area of

concern 1, 14. Generally, bycatch of Rastrelliger kanagurta fisheries
are relatively low. However, they include species of dolphins,
turtles, seabirds and sharks, which receive particularly high
attention from the international community. In future, a greater
utilization of bycatch species may be expected. (1) Fishing may
become more selective through gear modifications and changes
in fishing areas and seasons. Also, more research is likely to be
undertaken to determine the status of stocks of species incidentally
caught. (2) Conservation and management measures for sharks
must be reviewed to ensure that they are comprehensive; that
they provide specific protection to the most vulnerable species
and that the ratios of fins to carcass weight are meaningful. (3)
Bycatch mitigation measures for seabirds should be based on the
current best practice approaches.
Structure and process: The structure and charter of advisory
bodies must reflect the adoption of an ecosystem approach to
management. Decision-making processes of the Commissions must
be reviewed to maximize the likelihood that appropriate
conservation and management measures will be agreed and
adhered to. The basis upon which fishing rights will be allocated
amongst members and cooperating non-members and a process
for accommodating the interests of new members must be
developed as a priority. Mechanisms to maximize opportunities
for sharing of data and research and for harmonization of
conservation and management measures must be formalized and
improved. Documentation schemes for Rastrelliger kanagurta
must cover all components of the catch rather than only product
entering international trade.
Rastrelliger kanagurta fisheries management to date has often
been ineffective, it focus on maximizing the catch of a single target
species and often ignores habitat, predators and prey of the target
species and other ecosystem components and interaction. To
address the critical need for more effective and holistic management
approach a variety of advisory panels have recommended
ecosystem considerations be considered broadly and consistently
in managing sustainable fisheries. The overall objective of
conservation and management approach is to sustain healthy
marine ecosystem and the fisheries. In particular, Rastrelliger
kanagurta conservation and fishery management should include
the following (a) early warning signs of pressure on the stock of
Rastrelliger kanagurta catches should not exceed the MSY level,
(b) high seas drift gillnetting should be banned and FAD
associatedfishing should be regulated, (c) annual species wise
stock assessment to be made, (d) the number of hooks and length
of line to be regulated in longline fishing, (e) introduction of large
meshed purse-seine for high sea tuna fishing, (f) capture based
culture of Rastrelliger kanagurta should be regulated / banned,
(g) countries operating their vessels under letter of permission
(LOP) agreement for Rastrelliger kanagurta fishing should report
their catches to the native country, (h) illegal unreported
unregulated (IUU) fishing in Arabian Ocean waters by other
countries should be banned, (i) tagging experiments for
Rastrelliger kanagurta in the Arabian Ocean waters should be
undertaken, (j) ensure constant supply of high quality raw material
and enable optimum utilization of the capacity of Indian processing
plants, (k) provide essential support for training and research,
The development of new deep sea fishing policy will be a major
412
step forward in efforts to halt the global decline of Rastrelliger

kanagurta species.
What can be done to improve stock assessment inputs to
management?
i. Catch per unit effort series and other data
Increased scientific observer coverage will improve accuracy
Need to account for changes in species, size targeting
Need more scientific sampling to obtain more accurate estimates
of growth and aging and expand tag release and recapture
experiments should be increased
Need to explore interactions between seasonal migrations of
Rastrelliger kanagurta, stock structure and historic shifts in effort
distribution
ii. Need further research on
Decision rules for annual quota and other regulations
Improved spatial resolution in stock assessment models to
account for seasonal migrations and stock mixing
Provide essential support for training and research of
Rastrelliger kanagurta scientists, more closely follow scientific
advice.
Put more resources into enforcement and find ways to give
industry incentives to harvest sustainably.
iii. Conservation, protection and sustainable management
Poverty alleviation and the provision of supplementary
livelihood among municipal fisher folk, improvement of
productivity of aquaculture within ecological limits;
Optimal utilization of off-shore, and deep-sea resources and
upgrading of postharvest and value addition technology.
Good networks and strong relationships with sellers and
importers also play an important role.
Lack of experienced labor for distantwater Rastrelliger
kanagurta vessel operation, high cost of production, increasing
influence of imports on pricing of Rastrelliger kanagurta in
domestic markets, and a sharp decline in landings by its own feet.
Without adequate fisheries management, future catches of some
species may decline in the long term due to over fishing. With the
present status of stocks, the catches of principal market
Rastrelliger kanagurta should not increase on a global scale in
the near future unless future technological developments will
allow increasing catches of Rastrelliger kanagurta. Furthermore,
previous studies 34 have indicated that species richness, when
considered in conjunction with species density, fine scale
information regarding habitat use, spawning areas, migration
patterns, and fishing mortality could be useful in identifying priority
areas for marine conservation. We believe the fish are there and
they will not stay permanently down, so when the preferred climatic
conditions and temperatures improve they will move higher up
where fisherman can catch them.
Conclusions
This study provides an overview of the seasonal elemental
variations and conservational management of Rastrelliger
kanagurta fish in the Arabian Ocean. The maximum sustainable
yield estimated was higher than the average annual catch
indicating scope for further exploitation. The level of four heavy
metals iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) in
organs of Rastrelliger kanagurta fish. Numerous fish samples
collected seasonally from fish harbour of Karachi clearly revealed
significant differences (p<0.05) in organs of fishes. Fishing
operations can cause ecological impacts of different types:

bycatches, damage of the habitat, mortalities caused by lost or
discarded gear, pollution, generation of marine debris, etc. Periodic
reassessment of Rastrelliger kanagurta fish potential is also
required with adequate inputs from exploratory surveys as well
as commercial landings and this may prevent any unsustainable
trends in the development of the Rastrelliger kanagurta fishing
industry in the Arabian Sea.
Acknowledgements
The research was financially supported by the University Malaya
PPP Grant (PG008-2013B), (RG257-13AFR) and FRGS (FP0382013B).
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413
414
F
2833.390
36.665
11.573
7.434
6.227
4.492
5.485
822.834
28.059
125.768
14.629
39.032
15.460
20.720
1267.365
50.486
34.616
26.397
19.020
4.910
4.234
21.330
21.330
113.835
45.17
18.282
43.478
132.632
21.773
Mean square
1.5487
200316.436
63227.547
40616.048
34020.327
24543.949
29965.326
5463.481
8812.057
300.494
1346.905
156.663
418.004
165.569
221.895
10.709
130652.302
5204.580
3568.558
2721.309
1960.784
506.137
436.524
103.090
2777.626
14823.368
5882.512
2380.673
5661.685
17271.113
2835.230
130.219
* = significant at P < 0.05 ** = significant at P < 0.01 *** = significant at P < 0.001. F statistics, df degree of freedom.
Zn
Cu
Mn
Fe
df
4
3
4
12
16
12
48
2070
2170
1269
4
3
4
12
16
12
448
2070
2170
1269
4
3
4
12
16
12
48
2070
2170
1269
4
3
4
12
16
12
48
2070
2170
1269
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
P
0.000
0.000
0.000
0.000
0.000
0.000
0.000
Zn
Cu
Mn
Fe
Winter
Summer
Spring
Autumn
Winter
Summer
Spring
Autumn
Winter
Summer
Spring
Autumn
Winter
Summer
Spring
Autumn
Season (I)
Season (J)
Spring
Summer
Winter
Autumn
Summer
Winter
Autumn
Spring
Winter
Autumn
Spring
Summer
Spring
Summer
Winter
Autumn
Summer
Winter
Autumn
Spring
Winter
Autumn
Spring
Summer
Spring
Summer
Winter
Autumn
Summer
Winter
Autumn
Spring
Winter
Autumn
Spring
Summer
Spring
Summer
Winter
Autumn
Summer
Winter
Autumn
Spring
Winter
Autumn
Spring
Summer
Mean difference (I-J)

-22.80936
-43.43027
-9.44522
22.80936
-20.62091
13.36414
43.43027
20.62091
33.98505
9.44522
-13.36414
-33.98505
0.22594
-1.28320
-0.74812
-0.22594
-1.50914
-0.97406
1.28320
1.50914
0.53508
0.74812
0.97406
-0.53508
-1.35325
-6.30987
0.23945
1.35325
-4.95662
1.59271
6.30987
4.95662
6.54933
-0.23945
-1.59271
-6.54933
-6.27678
-5.54715
-12.14118
6.27678
0.72963
-5.86440
5.54715
-0.72963
-6.59403
12.14118
5.86440
6.59403
Std. Error
4.683256
4.450861
4.414836
4.683256
4.596317
4.561441
4.450861
4.596317
4.322502
4.414836
4.561441
4.322502
0.207463
0.197057
0.195462
0.207463
0.203616
0.202073
0.197057
0.203616
0.191374
0.195462
0.202073
0.191374
0.643311
0.611388
0.606440
0.643311
0.631369
0.626578
0.611388
0.631369
0.593756
0.606440
0.626578
0.59.756
0.723019
0.687141
0.681849
0.723019
0.709597
0.704474
0.687141
0.709597
0.667600
0.681849
0.704474
0.667600
Metal
Sum of square
6.1927
600949.308
252910.190
487392.580
544325.233
294527.384
1438335.649
1.1317
1.1118
7.7717
35248.229
901.481
5387.619
1879.960
6688.064
1986.829
10650.940
22157.750
147290.070
86054.529
522609.207
15613.740
14274.232
32655.703
31372.536
6073.641
20953.171
213395.741
1169657.543
871289.316
11110.504
44470.104
23530.047
28568.078
90586.965
207253.360
136091.043
269422.185
1175701.157
818354.910
Metal
Effect
Organs
Season
Year
Organs*Season
Organs*Year
Season*Year
Organs*Season*Year
Error
Total
Corrected Total
Organs
Season
Year
Organs*Season
Organs*Year
Season*Year
Organs*Season*Year
Error
Total
Corrected Total
Organs
Season
Year
Organs*Season
Organs*Year
Season*Year
Organs*Season*Year
Error
Total
Corrected Total
Organs
Season
Year
Organs*Season
Organs*Year
Season*Year
Organs*Season*Year
Error
Total
Corrected Total
Appendices II (i). Bonferroni test fish Rastrelliger kanagurta

collected from Karachi coast.
Appendices I (i). Analysis of variance (ANOVA) in Rastrelliger kanagurta fish

from the coast of Karachi.
P
0.000
0.000
0.195
0.000
0.000
0.021
0.000
0.000
0.000
0.195
0.021
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.031
0.001
0.000
0.031
0.213
0.000
0.000
0.213
0.000
0.067
0.000
0.000
0.000
0.000
0.067
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
WFL Publisher
Helsinki, Finland
www.world-food.net
The use of urea molasses multinutrient block on pica symptom of cattle

Haili Li *, Keling Wang, Limin Lang, Yindi Xu, Qinxian Zhang, Wenhao Zhu, Lixian Zhang, Yi You,
Feng Xu and Wan Lu
Animal Husbandry and Veterinary Research Institute, Henan Academy of Agricultural Sciences, 116 Huayuan Road,
Zhengzhou, Henan, 450002, China. *e-mail: haili8693@sina.com
Abstract
This study discusses the effect of feeding urea molasses multinutrient block (UMMB) as an alternative supplement on the pica symptom of
Limousin cattle. Thirty indigenous pica cattle were chosen from Ningnan mountainous district Guyuan City on the basis of similar bodyweight. The
Ningnan mountainous district cattle produce was limited by harsh environments and nutritional factors. Especially the forage quality and availability
are affecting nutrient intake. Forage was reduced during winter and early spring of the year. A background of these blocks manufacturing process and
their effect on pica symptom of cattle are reported. Sixty cattle were randomly divided equally into control and UMMB treatment supplemental
groups. Both groups had the same forage except the treatment group had free access to supplement with UMMB. The experiment lasted for 30 days.
The contents of mineral elements (Se, Zn, Cu, Co, I, Ca, P) in blood in the experiment group were significantly increased compared with control group
(P<0.05) after feeding the UMMB. The activity of ceruloplasmin (CP), superoxide dismutase (SOD) and lactate dehydrogenate (LDH) in the serum
of experimental group were significantly increased in contrast with control group (P<0.05). The contents of mineral elements Se, Zn, Cu, Co on
feather in the experiment group were significantly increased in contrast with control group (P<0.05). According to field observation and stock owner
reflect, the pica symptom was disappeared in the treatment group after feeding UMMB three days, and the intake and drinking have significantly
increased. Cattle have the symptom of pica and the hair was coarse and had lack of luster before experiment in the experiment group. The symptom
of pica was gradually disappeared and the appearances of cattle were dramatically changed with feeding the UMMB. Furthermore, the colour of hair
was bright and bushy, and the cattle was in good condition in the treatment group after feeding UMMB, but the hair of the cattle in the control group
was sparse and dirty. The symptoms of pica all disappeared, which means the UMMB was effective in treated cattle pica. Hence, the UMMB can
be used as an effective way mineral supplement and treatment in cattle pica.
Key words: Urea molasses mltinutrient block, pica, cattle, Ningnan mountainous area.
Introduction
Nutrient deficiency is a commonly problem in the world, especially
the mineral deficiencies and imbalances for cattle 1. In arid and
semi-arid regions, feed shortage is the main constraint to their
productivity. During the hot and dry seasons, the available feed
resources are not enough in energy and digestion proteins, which
are insufficient to ensure maintenance requirements and reducing
productivity throughout the year. In drought conditions,
ruminants need enough nutritious forage to improve animal
performance. Earlier studies have assessed the nutritional quality
in Ningnan mountainous district forage. The nutrition consent in
this mountain was deficiencies and required to supplement the
desired level of production. In the past decades, UMMB was
chosen as a supplementation to deficient diets in cattle 2-4, sheep57
, and buffaloes 8-11. However, there are many kinds of UMMB in
the market. Since these UMMB are too soft or hygroscopic due to
humidity resulted in gobbled rapidly by the animals, which lead to
extensive supplement to animals. In order to study the UMMB of
local cattle in Guyuan, the present study was carried out on UMMB
formulas, raw material, technical parameters and feeding effect.

Ingredient composition of UMMB: The UMMB was prepared in
this study using the process as described by Li et al. 12. Different
ingredients of UMMB licks were chosen from local place with the
objective of supplying minerals nutrition. The UMMB consisted
of mineral mixture, common salt, urea, sodium bentonite, molasses,
wheat bran, calx and calcium hydrogen phosphate at the level of
8.5, 20, 10, 20, 20, 4, 5 and 12.5%, respectively.
Animals feeding and management: The study was done at the
Ningnan mountainous district, Guyan City, Ningxia province,
China. Sixty indigenous cattle with symptom of pica were chosen
from Ningnan mountainous district Guyuan City on the basis of
similar bodyweight, divided randomly into two groups of thirty
animals in each group. Two groups had the same formula except
the treatment group was supplemented with UMMB to meet the
mineral requirement.
The cattle animals were kept in sheds during the experimental
conduction. Clean and fresh drinking water was provided in the
morning and afternoon. During the 30 days of study, hematology
415
index, serum enzymology index, mineral concentrations of the

blood, mineral contents of the hair, the consumption of UMMB
and the pica animal clinical manifestation and disease development
were tested at the beginning and end of the experiment.
Sample preparing: Blood sample of 30 ml was taken in the neck
venous before the experiment and at the end of the experiment
with 1% heparin anticoagulation and stored at -25C. Ten ml was
used for the mineral element test, 10 ml for the determination of
hematology index and 10 ml for the biochemical index test. One g
of clothing hair was taken before and at the end of the experiment
for mineral test.
Routine hematological examination: Routine hematological test
was done by automatic animal blood analyser. The routine
hematological test indexes included number of red blood cells
(RBC) and white blood cells (WBC), hematocrit (HCT), hemoglobin
(HGB), neutrophils (GR), lymphocyte (LY), median cells (MO),
platelet (PLT), lymphocyte ratio (LY%), mid-value cell ratio (MO
%), neutrophils ratio (GR %), mean corpuscular volume (MCV),
mean content of hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), mean platelet volume (MPV) and platelet
cubic thrombocytocrit (PCT).
ml of strong HCl was put in cook stove digestion 15 to 16 h (130C

temperature), cooled and added to 10 ml and subjected to hydride
generation atomic fluorescence spectrometric determination
(HGAFC), 200~290 nm wavelength, standard curve equation I
(fluorescence intensity value) = 40.4049 C(density) - 3.9259. (2)
Calcium, phosphorus, copper, zinc, cobalt, selenium and iodine
were determined by plasma atomic emission spectrometer
(American Varian company VISTA-AES). The former was dealing
similar with selenium determination, determination conditions
temperature was -35C, Water temperature 22C, Power 1.2 kW;
Gas flow for argon was 2.25 L/min, nitrogen 1.5 L/min. (3) Fluorine
and chlorine were determined by ion chromatography (Dianne
Company). The eluent and leachate were 12% of chromatographic
pure NaOH; temperature 30C, and the sample volume 20 l, the
volume of leachate 250 l/time, each sample elution time was 20
min.
Statistical analysis: The SPSS 13.00 software on one-way ANOVA
analysis of variance and Duncans multiple comparison, the result
in average standard deviation.
Results
Routine blood index: Routine blood test results are in Table 1.
The number of red blood cells (RBC), hematocrit (HCT),
Blood biochemical index examination: Blood biochemical index hemoglobin (HGB) and the number of white blood cells (WBC) of
was examined by chemical colorimetric method (Test Kit). The pica cattle were below normal index. The number of RBC, WBC,
evaluation indexes included ceruloplasmin (CP), glutathione HCT, HGB, neutrophils (NP), mean corpuscular volume (MCV),
peroxidase (GSHPX), superoxide dismutase (SOD), lactate and medial plaque volume (MPV) in the experiment group were
dehydrogenase (LDH) and alkaline phosphatase (AKP).
increased by 49% (P>0.05), 4.19% (P>0.05), 12.88% (P<0.05), 3.64%
(P>0.05), 6% (P>0.05), 7.67% (P<0.05), 2.54% (P<0.05), 11.68%
Blood element analysis: The blood element analysis included (1) (P>0.05) compared with control group. Lymphocytes (LY), median
selenium determination. Instrument was AFS-930 double ways cells (MC), platelets (PLT), lymphocyte rate (LY%), median cells
for atomic fluorescence photometric (Beijing auspicious day rate (MC%), mean content of hemoglobin (MCH), mean
Instrument Company.). Blood 2.0 g with 15 ml of nitric acid and 1 corpuscular hemoglobin concentration (MCHC) and platelet cubic
thrombocytocrit (PCT) in the experiment
Table 1. Blood routine tests in sick cattle.
group were reduced by 9.2% (P>0.05),
Blood
At the beginning of the experiment
At the end of the experiment
23.38% (P>0.05), 16.18% (P<0.05), 2.04%
Unit
routine item Experiment group Control group Experiment group Control group
(P>0.05), 18.97% (P>0.05), 9.16% (P>0.05),
12
RBC
4.640.99
4.560.92
9.180.99*
4.740.94*
10 /L
11.65% (P<0.05), 5.26% (P<0.05) compared
WBC
7.882.39
7.932.32
8.462.33*
7.552.03*
109/L
with control group.
HCT
22.725.54
21.465.23
26.086.14*
23.215.52*
%
HGB
NP
LY
MC
PLT
LY%
MC%
NP%
MCV
MCH
MCHC
MPV
PCT
109.412.86
2.980.8
4.131.76
0.770.41
413.5216.37
51.058.4
9.492.94
39.468.08
48.672.52
24.667.32
515.9185.81
91.78
0.380.26
108.6412.41
2.760.73
3.941.48
0.710.34
415.57217.3
50.837.48
9.132.51
40.128.53
47.862.17
25.677.85
513.1183.65
9.561.92
0.350.17
113.5311.58
3.170.92
3.751.38*
0.590.17*
346.6146.09*
50.018.34*
7.691.09*
42.748.03*
49.943.18
22.42.51*
455.8102.56*
10.191.86
0.360.21
112.1312.45
3.150.86
4.441.72*
0.780.36*
417.8193.17*
52.348.86*
8.992.61*
38.678.32*
48.732.61
24.546.16*
511.8158.01*
9.331.67
0.40.24
g/L
109/L
109/L
109/L
109/L
%
%
%
fL
Pg
g/L
fL
%
Experiment group compared with control group. *Notice P< 0.05, Significant difference.
Table 2. Enzyme activity in blood serum.

Item
CP
GSHPX
SOD
LDH
AKP

Experiment group
Control group
1.380.99
1.290.91
208.718.98
205.978.53
105.986.02
104.565.87
2653.29171.67
2651.11169.88
20.698.08
21.518.11

Experiment group
Control group
8.452.56*
1.410.77*
246.1575.63*
211.1615.09*
130.812.55*
106.896.32*
4544.51347.7*
2643.99137.37*
28.0811.68*
19.136.82*
416
Unit
1012/L
109/L
%
g/L
109/L
Serum enzyme activity: Enzymatic activity

in blood serum is shown in Table 2. There
was no significant difference (P>0.05) in
the enzyme activity between the
experimental and control groups at the
beginning of the experiment. At the end
of the experiment, the CP, GSHPX, SOD,
AKP and LDH activities were higher
(significant difference, P<0.05) in
experimental group.
The mineral element contents in blood:
There was significant difference (P<0.05)
between experimental and control groups
in calcium, but the other indexes were not
significantly (P>0.05) different at the
beginning of the experiment (Table 3). At
the end of the trial, experiment group and
Table 3. Mineral contents in blood (mg/L).

Item
Se
Zn
Cu
Co
I
Ca
P

Experiment group Control group
0.0110.005
0.0120.005
1.470.41
1.350.4
1.660.16
1.670.148
0.0250.004
0.0270.004
0.010.003
0.0130.004
81.991.16*
83.440.72*
150.130.48
150.160.53

0.0840.015*
0.0260.011*
8.20.28*
3.150.26*
5.090.25*
2.410.16*
0.080.002*
0.030.003*
0.030.002*
0.0110.002*
97.913.46*
84.330.38*
250.87.52*
153.361.3*
Reference
0.13
9
5
0.1
-102
260.15
Table 4. Mineral contents in hair (mg/kg).

Item
Cu
Zn
Co
Se

6.190.17*
6.330.14*
92.440.99
92.511.32
0.210.026
0.210.03
0.1730.02
0.1610.014

9.350.13*
6.420.09*
208.150.78*
105.750.88*
0.420.036*
0.240.02*
1.30.15*
0.180.01*
Reference
10
1~5
control group were significantly different (P<0.05), and the

concentrations of the indexes were close or higher than the normal
index.
The mineral elements contents in hair: The concentration of
copper was significantly different (P<0.05), but the other indexes
were not significantly (P>0.05) different between experimental
and control groups at the beginning of the experiment (Table 4) .
At the end of the trial, experiment group and control group were
significantly different (P<0.05), and the concentration of the
indexes were close or higher than the normal index.
The block consumption:The consumption of the block was about
126.41 g/d during 1-5 d , 76.20 g/d during 6-15 d and 76.00 g/d
during 16-30 d (Table 5).
Table 5. The consumption of the block (g/d).
Day
Consumption
1-5 d
126.418.43
6-15 d
76.25.14
16-30 d
766.73
Experiment group compared with control group. *Notice P< 0.05, Significant
difference.
The consumption of the block: The pica cattle liked to lick block
and the block palatability was better, especially at the first stage,
some cattle not only suck the block but also bite it. It means that
the mineral deficiencies of cattle are pretty serious in the
countryside. In the first days of the test, the time of lick was
controlled in order to prevent the excessive suck. After four or
five days, the average intake was normal (76.2 g/d). According to
the lick intake each day, each block was available for every cattle
suck 20 ~ 30 days or so. There were differences among individuals,
in cattle feed block intake, but urea poisoning did not appear.
The effect of the block on the hair and the symptom of pica:
Every cattle had the symptom of pica before experiment. In the
pica cattle symptoms were gradually reduced since entering the
experiment period in the experimental group. According to the
client, the cattle with pica symptoms in the experiment group
gradually disappeared after licking the block 3 days, and the feed
and water intake were increased. The pica symptom was
disappeared after 15 days, but the control group still had pica
symptom.
The hair was harsh and lusterless etc. before the experiment,
the hair was improved greatly and hair removal time neatly and
with shining fur after the experiment. The results showed that
UMMB has a good curative effect on cattle pica. It can improve
hair nutrition, promote the hair take off and growth as early as
possible.
The appetite, spirit and different body status: All the cattle feed
and excrement was normal during the experiment. The feed and
water intake was increased according to the field observation and
reflect from the client. The feed intake still could not be measured
although a lot of effort on feed intake, because this experiment
was conducted in production conditions. The body status and
fur condition in experiment group were better than in control group.
The above statement showed that UMMB can improve the cattle
nutrition and health conditions, and can improve the dry matter of
feed intake, especially straw feed intake.
Discussion
Routine blood index is a comprehensive index, which reflects the
cattle nutrition and metabolism condition, the environmental
balance in the body, body health, growth speed and production
performance 13. The present results showed that the index of RBC,
WBC and HGB were below normal in pica cattle. The blood routine
index was normal after feeding UMMB. It means that the
physiological and biochemical indexes had relatively
comprehensive improvement when feeding UMMB.
The most important function of mineral elements is the
composition of enzymes and maintaining of the enzyme activity.
The active enzyme in the blood and tissue can be used to diagnose
certain mineral elements. The results showed that the enzyme
activity was significantly higher in the experiment group than in
the control group. AKP is a kind of metal enzyme containing zinc,
the AKP activity dropped significantly when animals lack zinc or
vice versa, the activity of AKP increasing significantly with
supplementary zinc 14. A lack of selenium resulted in the GSHPX
activity change. The relationship between blood GSHPX activity
and food selenium levels is confirmed in mouse, chicken, lamb,
calves and pig. The activity of GSHPX can be used as an index in
the early diagnosis of selenium deficiency 15. Siddons and Mills 16
reported that GSHPX changes provide an objective evidences for
417
the ruminant animals and clinical diagnosis of selenium deficiency

index of this diseases. The RBC GSHPX can well reflect the
selenium condition 17. The advantages of mineral nutrition
evaluation through enzyme activity not only can be early
monitoring of mineral nutrition, but also can avoid sample pollution
of the elements, but this approach also has some shortcomings
because the activity of GSHPX may reduce especially the activity
of GSHPX 18.
Hair is part of the animal organization. Hair roots cells, blood,
lymphatic and extracellular fluid are in close contact to get fully
nutrition and the mineral elements deposit on the base of the hair
in the short term. So the concentration of element in hair can
reflect the body metabolism. Animal hair plays a tape, which
reflects the elements intake and metabolism in different periods.
Use of hair in monitoring of animal nutritional status has
advantages of easy sampling without any damage to the animal,
long-term preservation, analysis convenience etc. The element
concentrations of hair are changed with the breed of livestock
and poultry, age, body parts, body colour, hair period and the
different parts of the season.
The levels of Se, Zn, Cu, Co, I, Ca and P in the experiment group
reached or exceeded the reference requirements (Table 3). The
main biological functions of elements regulated the bodys
physiological activities through proteins and enzymes involved
in metabolism of the body or form hormones 19. The lack of trace
elements will lead to metabolism disorders, which affect livestock
production performance 20-22. Mineral nutrition imbalance has
become recognized as one of the main factors that restrict livestock
production, affecting the growth of livestock, and causing more
serious consequences than infectious diseases 3. Reports on
adding trace elements to improve ruminant livestock production
performance (weight and lactation) are mainly concentrated in
weight gain effects after addition of a single element selenium
(Se) and iodine (I) and filling zinc nutritional status 2, 4-7, 11. There
are also a large number of literature on zinc supplementation, which
promoted the growth of cattle and sheep, and increased feed intake
of the stress calf and promoted weight on cattle 6. For the bulls,
zinc deficiency will result in seminiferous tubule degeneration,
abnormal mesenchymal cells, sperm dysplasia, reduction of
testosterone production, impact on shape of testis and delay of
the estrus onset 9. Zinc is also associated with animals taste and
appetite, its deficiency may result in corneal cornification and
cover or block the small taste buds and lead to taste loss 10. Adding
Co can increase the hemoglobin and weight gain in the process of
production 8. The influence of Co deficiency on cattle immune will
lead to lowered immunity 23. Cu, Zn, Co, Se, Fe and I are necessary
mineral elements for livestock. Co and Fe are mainly involved in
hematopoiesis; Co can also boost ruminants rumen digestion. Se
is involved in antioxidant effect and regulation of thyroid function
by forming glutathione peroxidase and iodine enzyme,
respectively24, 25. Se deficiency (white myopathy) and anemia are
worldwide widespread diseases.
Urea molasses multinutrient block treatment can help in
prevention of many diseases. Parasites were significantly reduced
by giving male lamb urea molasses multinutrient blocks 26, 27. The
incidence of intestinal parasitic diseases were effectively reduced
by adding licking block (drug urea molasses lick block, molasses
lick block with 7% urea and 21% urea molasses lick block) 28. Urea
molasses multinutrient block can also used for treatment the water
418
buffalos lack of mood disorder, which is the most common summer

buffalo reproductive disorders 29, 30. Supplementary feeding urea
molasses to 5 month old east African goat not only improved the
body weight gain, but also significantly reduced gastrointestinal
nematode parasites 31.
Conclusions
It could be concluded that UMMB could be used in treatment of
the Ningna Mountains cattle pica. Supply of UMMB in experiment
increased the intake and the performance of cattle. The results
showed that UMMB supplementation is an effective strategy to
increase the production, while maintaining animal performance
and feed efficiency.
Acknowledgements
The authors are grateful to Xishan Li, Guanglin He, Guoping
Zhang, Yaofa Du, Xiaomei Yang, Lizhen Yan, Yafeng Zhao who
participated in this experiment. This study was financed by Henan
academy of agricultural sciences outstanding youth fund of
science and technology (Project number: 2013YQ22), and Henan
Academy of Agricultural Sciences Dr. Start-up fund of scientific
research.
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419
WFL Publisher
Helsinki, Finland
www.world-food.net
Views and attitudes of mulberry cultivators according to the Regulation (EC) No

1257/99: The case of Evros prefecture
S. Ch. Tsiantikoudis 1*, A. Parissi 2, Z. Papadopoulou 3, A-M. Fidani-Mantzoula 4, G. Kourtelis 5 and Z. Andreopoulou 6
Department of Forestry and Management of the Environment and Natural Resources, Democritus University of Thrace, Pantazidou 193,
68200, Orestiada, Greece. 2 Direction of Primary Education, Rthnikis Antistasis 56, Skydra, 58500, Greece.
3
Department of Accounting and Finance, Eastern Macedonia and Thrace Institute of Technology, Agios Loukas, 65404, Kavala,
Greece. 4 National and Kapodistrian University of Athens, Faculty of Economics and Political Sciences, Department of Economics,
Pesmazoglou 8, 10509, Athens, Greece. 5 Department of Food Technology, School of Agricultural Technology, Technological
Educational Institute of Kalamata, Antikalamos, 24100, Kalamata, Greece. 6 Department of Forestry and Natural Environment,
Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece. e-mail: stsianti@fmenr.duth.gr,
garamp@fmenr.duth.gr, zoepap@yahoo.com, anniemary@live.com, 77cyanide77@gmail.com, randreop@for.auth.gr
1
Abstract
Nowadays, the future of sericulture is prosperous, since the European Union adopted sound economic reforms to support the mulberry cultivators.
This study aims at investigating the individual and societal characteristics of mulberry cultivation investors and farmers, in compliance to the 1257/
99, article 31 of EU legislation regarding the afforestation of agricultural lands and its specific application at the prefecture of Evros (North Greece).
The adopted methodology of the study was implemented via the structure and distribution of a questionnaire. The data was gathered and manipulated
with the use of the SPSS software. According to the study outcomes, the mulberry cultivation meets the social approval and landowners acceptability,
since it is considered a prosperous and economically safe investment.
Key words: Prefecture of Evros, mulberry cultivation, Regulation (EC) No 1257/99, individual and societal characteristics.
Introduction
During the last decades, European Union under the Framework of
CAP has directed its policy into the integrated rural development
and the agri-environmental measures 1, 2. In particular, European
Union has adopted and initiated regulations and directives in
relation to transition of agricultural areas from food production
towards forest production 3-5.
In the relevant literature, many of published studies have
addressed the forest production and particular the fuelwood
production for energy in Greece 6, 8-11.
Also, many studies investigated the forest management and its
impact to local societies, in according to issues of safety and
optimum exploitation 7, 8, 12-15 under the framework of the Greek
rural development.
Particularly, the Regulation (EC) No 1257/99 manipulates issues
of rural development 16. The article 31 of the Regulation 1257/99
mention the forestry areas development under policies that
address the forest costs: Those are allocated in the annual
economic subsidiary for the operation and development of these
new forest areas, as well as in balancing out the revenue losses
for investments 17. One of forest species, that were selected to
implement these forestry management reforms, is the mulberry.
Mulberry is a native species that is endemic in China and the
Far East. Its introduction to Greece was noted during the 12th
century to rearing silkworms as well as an edible fruit 18. In
420
particular, in the Greek prefecture of Evros (North Greece) the

main utilization of mulberry is its cultivation for leaf production,
that are subsequently gathered to feed the silkworms and
eventually produce silk for the clothing industry. At the early 20th
century, sericulture was one of the most valuable and profitable
entrepreneurial sector of the prefecture of Evros and especially in
the area of Soufli town. Nevertheless, after the Asia Minor disaster
and the exchange of Greek and Turkish population in effect to the
relevant Treaty of Lausanne, the national borders of two countries
changed and a significant portion of agricultural area that was
devoted to the mulberry cultivation was transferred to the Turkey
ownership 19, 20.
Also, the outbreak of economic crisis in 1929 and the 2nd world
war followed have contributed to the change in the textile industry
and the innovation of the synthetic fibres, thus the traditional
silkworm-based method of silk production faced a severe economic
recession. During the 1970s, the land redistribution of the Soufli
area contributed to fundamental changes in land use. These
changes included the introduction of new and more productive
mulberry species, comparing to the former mulberry species. The
economic recession for the mulberry cultivation was apparent
throughout the 1980s, by the time of the introduction of the
aforementioned Regulation 1257/99 and its accompanying
economic initiatives to the renaissance of the stagnant mulberry
industry to economic growth and national profitability.

This study aimed at investigating the personal and social
characteristics of the mulberry investors/cultivators in the
prefecture of Evros, who invested their cultivations in compliance
with the Regulation 1257/99 (article 31) of the European Union
Council regarding with the afforestation of agricultural lands.
The study was realized in the prefecture of Evros, which is the
northernmost prefecture of Greece. The Evros prefecture is 4,242
km2 and its population is 144,023 inhabitants, according to the
Greek census at 2001. The lowland accounts of 60.7% and the
agricultural area 35.5%, of the total area of the prefecture,
respectively 21. The prefecture of Evros supports habitants of
abundant flora and fauna, including the Dadia forest 22, 23.
The reasoning of the Evros prefecture, mainly its region of
Soufli, selection is that Soufli is an area of the mulberry cultivation
and a major producer of mulberry leaves and silkworms feeding.
This silkworm production is estimated at about two thirds of the
total national production 21.
The adopted methodology was materialized using a specific
questionnaire that was structured mainly by closed type
responses. The questions involved the personal and social
characteristics of the investors, the structural characteristics of
their cultivations, as well as their attributes, preferences or dislikes,
regarding the adopted cultivations. For the smooth manipulation
of the data gathering a catalogue of 107 accredited-to-subsidiary
beneficiaries in according to the Regulation 1257/99 and (article
31) in the Forest Service of Alexandroupolis, Soufli, Didymoticho,
was used 24-26.
A number of 94 valid questionnaires were returned, mainly from
land owners, mainly farmers. The latter group of investors
accounted of 87.7% of the total number of beneficiaries that
correspond to the aforementioned European policy. A small portion
of the remaining beneficiaries group was difficult to be identified.
The data manipulation was made with the support of the SPSS
package, as well as in accordance to descriptive statistics.
Results
According to the data of the three Forest Services of
Alexandroupolis, Soufli, Didymoticho, the cultivators/investors
who were engaged to mulberry cultivation in compliance to the
Regulation 1257/99 (article 31) were 107 out of the total of 905
investors, which is equivalent to 11.8%. The afforestation area
covered was 355.5 ha out of the total of 2,225.84 ha, which is
equivalent to 16%. The regional allocation of these 107 mulberry
farmers is: 52 are located in the Soufli, 50 are located in
Didymoticho, and 5 are located in Alexandroupolis. All the three
aforementioned Forestry Departments of the prefecture of Evros
were totally funded by 1,279,922 for these mulberry cultivations.
The data processing showed that the majority of cultivators/
investors , 53.2%, were male and 46.8% were female. The average
age of participants was 48 years old. The profile of the age of
cultivators/investors was such that 9.6% were under 35 years old,
28.7% between 35 and 45 years old, 28.7% between 45 and 55
years old, and 33% above 55 years old. The youngest participant
was a male of 25 years old and the oldest participant was a male of
67 years old.
The average working engagement with the agriculture was 18.7
years, while the average gross annual income was 13,350. Above
average income are positioned 26 participants (27.6%) and below
average income 72.4%. Farming has been declared as a secondary
occupation for 13.8% with an annual revenue of 3,250, while
from other professional activities their annual revenue is 7,680.
Farming has been declared as the primary occupation from 81
participants, which is equivalent to 72.4%. The professional
allocation was 3 public servants, 5 private servants and 5
freelancers; their percentage allocation corresponds to 3.2%, 5.3%
and 5.3%, respectively. The educational allocation was such that
51 (54.3%) participants had primary education, 20.2% had
secondary education, and 13.8% completed their gymnasium
studies without completing their secondary education, 4.3% had
vocational education and 4.3% were bachelor graduates from
universities and 3.2% from technical foundations.
In parallel, 49% of the cultivators/investors had participated in
agricultural-oriented seminars, while 24.5% and 28.7% of the
cultivators/investors were informed frequently and most frequently
by agricultural-oriented broadcasts, respectively, and 37.2% and
9.6% of them were informed sometimes or seldom, respectively.
Even though farming is mainly an outdoors profession an overall
53.2% have been informed by agricultural-oriented broadcasts.
One fourth (26.6%) of the participants have read articles and
news of agricultural interest, and 23.3% have expressed occasional
engagement. Of the cultivators/investors 19.1%, 14.9% and 17%
were frequent, less frequent or not at all readers of similar
information.
On average, 55.4% declared that the value of irrigated land has
been decreased in the last 5 years, showing prospects to decrease
more, as some of the respondents annotated. Of the respondents
10.6% answered that the value of irrigated land has been increased
during the last 5 years, especially in lowland areas, while a
significant percentage of the respondents (34%) declared that the
respective value has been remained stable during the same period.
The privately owned land was from 0.38 ha to 45 ha. The average
land was 7.8 ha from which 3.4 ha was irrigated and 4.4 ha non
irrigated. The average agricultural farm measures 8.21 holdings
and finally the average fallow agricultural farm is 0.52 ha.
Only 21.3% of the respondents were satisfied by the selling
prices of agricultural products, while the majority (78.7%) was not
satisfied by the respective selling prices. Among respondents
67% considered the cotton cultivation more profitable, while a
small proportion (10.6% and 6.4%) considered sugar beet and
maize cultivations, respectively, as more profitable.
As far as the reason of why investors/cultivators prefer to utilize
a part of their farms by planting with forest trees, 38.3% considered
this action as more profitable than other kinds of cultivation. Of
the respondents 24.5% prefer forest planting as an easiest way of
utilizing their farms, and 23.4% declared that forest plantations
are having better subsidies than others. Finally, 6.4% of the
respondents choose this kind of plantations and especially
mulberry plantations as they have the proportional knowledge in
order to manage it efficiently. A small proportion of the respondents
(5.3%) were affected by other local cultivators in their decision to
plant their land with forest trees. The easy disposal of the product
in the free market was one of the reasons that the rest 2.1% establish
mulberry plantations in their farms, as the foliage can be available
for sericulture and the wood for selling after 20 years which is the
maximum period of regulation subsidy.
421
Half of the respondents (50%) learned about forest plantations

and the respective regulation from their neighbours. Forest service
has informed 25.5% of the investors/cultivators, while 13.8% was
informed by freelance foresters. A small percentage of the
investors/cultivators (5.3%) learned for the first time about
afforestation of agricultural land from agricultural associations
and another 4.3% from the Agricultural Administration Office. Only
1.1% heard about forest plantations by their family.
The attitude of investors/cultivators has been influenced by
their family by 26.6% in their decision to invest in forest plantations
using the regulation 1257/99 and especially mulberry, and 24.5%
said to be affected by no one in this decision. Neighbours affected
investors by 19.1%. At the same percentage (13.8%) was the
influence by the freelance foresters and Forest Service.
Cumulatively the two previous factors had the most significant
role (27.6%). Finally, only 2.1% was influenced by agricultural
associations.
In the decision to establish forest plantation in the farms,
neighbours reacted positively by 36.2%, neutral by 35.1% and
19.1% unfavourable as they thought that this kind of action would
have negative impacts in their farms. On the other hand, 9.6%
declared that their neighbours were very favourable and supported
them.
About their family, the attitude in the decision of mulberry
investors/cultivators was positive for the 55.3% of them and
absolutely positive for 36.2%. Family was neutral for 5.3% of the
respondents, while for 2.1% and 1.1% was against and completely
against their decision, respectively.
More than half of the investors/cultivators (51.1%) stated that
forest plantations are a non-risky investment for them and for
their farms, 24.5% stated that they took small and moderate risk
about this investment. On the other hand, 17% of the respondents
said that the risk of establishing forest plantations in their farm
was high and the rest 1.1% considered this investment as high
risky.
In the question if their investment is compatible with current
forest law and the requests of the regulation that subsidies the
implementation of forest plantations, the majority of the investors
(73.4%) stated that they dont have any fear about implementing
it following all the rules and respective requests. A moderate
percentage (16%) stated that they have little fear about
implementing the requests of the forest law and the respective
regulation, while the rest 10.6% did not have any reason to fear
about implementing forest law.
The change in agricultural farms brought also changes in family
occupation. Of the respondents, 38.3% stated that family
occupation increased with this new cultivation, while for 18.1%
decreased. The rest 43.6% did not notice any change in the duration
of the family occupation.
Further goal of the investors/cultivators is the direct or indirect
occupation with sericulture (70.3%). Only 7.4% stated that is in
place to harvest and exploit the produced timber of the mulberry
cultivations as the case to deal with sericulture is not a reliable
scenario and therefore quite risky. Finally, 22.3% of the respondents
stated that they are indecisive about their plans of future utilizing
of their mulberry plantation.
More than half of the investors/cultivators (52.1%) were not in
place to express satisfaction about their plantation; 45.7% were
satisfied about their plantation, while only 2.1% were not satisfied
422
from the revenues it earns from it. The investors/cultivators that

have not been regretted about their choice of establishing a
mulberry plantation in their farm were at the same level (52.2%).
Only 1.1% of them regretted about occupying with this kind of
plantations. The rest 11.7% was undecided.
The role of yearly economic incentives of the program was a
very strong reason of the investors/cultivators to deal with the
certain investment. Otherwise they would not be able to realize
such an investment without any incentive, as 2.1% stated.
Conclusions and Proposals
The Regulation 1257/99 boosted the governmental initiatives in
compliance to mulberry cultivation management, in comparison
to former national regulations. The study revealed that the
prosperity of mulberry cultivation, under the new governmental
reforms, is very promising.
In particular, the majority of the investors/cultivators was above
the age of 35 years old. The half of them had participated in
agricultural-oriented seminars, while many of the participants were
informed by agricultural-oriented broadcasts. The majority of them
were farmers with a considerable little or substantial experience in
agricultural tasks. The half of the participants was graduates of
elementary education.
In parallel, many participants had received valuable support
from neighbourhoods, who were foresters or freelancers, while
there was a significant portion of participants who were selfmotivated in order to be involved in the relevant policy. Their
decision was mainly made in line to their belief of affirmation that
the mulberry cultivation would be proven highly profitable.
Besides, the implementation of relevant programs that foster
the sericulture competitiveness in regional and national level of
analysis, and the appropriate funding, would attract many other
farmers to be affiliated with the specification of the mulberry
cultivation as a credible alternative solution of profitability,
regarding the existing cultivations.
Finally, the enhancement of economic subsidiaries and the
accompanying motives should familiarize the majority of the
agricultural population and the landholders to be directed the
replacement of agricultural areas into the forestry management of
sericulture and livestock (cattle, sheep and goats).
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WFL Publisher
Helsinki, Finland
www.world-food.net
A proposal to standardize herbicide sorption coefficients in Brazilian tropical soils

compared to temperate soils
Kassio Ferreira Mendes 1*, Marcelo Rodrigues Dos Reis 2, Ana Carolina Ribeiro Dias 3, Jos Ari Formiga 4,
Pedro Jacob Christoffoleti 4 and Valdemar Luiz Tornisielo 1
Laboratory of Ecotoxicology, Center for Nuclear Energy in Agriculture, University of So Paulo, Piracicaba - So Paulo Brazil, CEP: 13416-000. 2 Institute of Agricultural Sciences, Federal University of Viosa, Rio Paranaba - Mina Gerais, Brazil,
CEP: 38810-000. 3 Department of Agronomy, State University of Mato Grosso, Alta Floresta - Mato Grosso, Brazil, CEP: 78580000. 4 Department of Crop Sciences, College of Agriculture Luiz de Queiroz, University of So Paulo, Piracicaba , So Paulo,
Brazil, CEP: 13416-000. *e-mail: kassio_mendes_06@hotmail.com
1
Abstract
Current mathematical models for predicting herbicide behaviour in tropical soils use sorption coefficients (Kd) and organic carbon affinity (Koc)
obtained from temperate soils, indicating that the information obtained may be distorted. Moreover, organic matter content in tropical soils varies
greatly when methods and laboratories are compared; consequently, those conditions cause Koc values to vary. Thus, the objectives of this paper were
to review the scientific literature and discuss problems associated with the determination, calculation and interpretation of Kd and Koc values for
Brazilian tropical soils, evaluate Kd and Koc values of tropical soils using the values described in the databases for temperate soils, correlate Kd values
with tropical soil properties and suggest a method for calculating standardized Kd values applicable to soils with properties similar to those reported
for tropical soils. Koc values were calculated based on an OC/OM (organic carbon/organic matter) index of 0.54 for 22 herbicides. Pearson correlation
was used to compare Kd values and soil properties (organic matter, OM; clay mineral, CM; potential hydrogen, pH and cation exchange capacity,
CEC) for each herbicide in tropical soils. The results indicate that Kd and Koc values for herbicides in tropical soils presented an OC/OM index ranging
from 0.395 to 1.275, with some similarities and differences compared to temperate soil values. The Kd values of the majority of the herbicides
correlate with the variation of OM and CEC contents in tropical soils. Standardized values confer more precision and accuracy to the mathematical
models when used to represent herbicide behaviour in soils with similar properties to topical soils.
Key words: Databases, environmental behaviour, mathematical models, soil properties.
Introduction
Herbicides play an important role in modern agriculture, but
concerns about food safety and environmental impacts of herbicide
residues have increased 1, 2. Surface and subterranean waters have
become contaminated because of agricultural and non-agricultural
applications of herbicides, due to the processes of runoff and soil
leaching 3.
Herbicide registration processes focus on requirements such
as environmental impacts and animal toxicology. Additionally, after
being available on the market, most products go through a
revalidation procedure to meet newer country-specific legislation
to comply with current guidelines and national security profiles.
Recently, global conservation programs have been developed to
protect all countries from environmental contaminants. Studies of
herbicide applications and production system efficiency are tools
to monitor environmental impacts and support food safety 1.
It should be noted that applied pre-emergent herbicides are
more problematic compared to post-emergent herbicides, because
they are deposited directly into the soil and therefore more subject
to leaching and runoff, thus leading to further environmental
contamination 4, 5.
Soil herbicide behaviour is controlled by a set of complex
processes, such as degradation (chemical, microbiological and
photodegradation) 6, retention (sorption from soil colloids) 7,
424
leaching, volatilization, runoff and absorption by plants.

There is evidence that herbicides applied to soil tend to be
more easily transported by groundwater. The hypotheses
proposed to explain this transport include preferential flow and
colloidal material transport, as well as a combination of processes.
The rate and magnitude of rapid transport are affected by many
parameters, such as the physico-chemical properties of the
herbicide, hydrology and soil use 8. Therefore, non-volatile and
water-soluble molecules are moved through the soil profile,
following the flow of water by the difference in water potential
between two points 9.
Leaching refers to the entrainment of the herbicides in the soil
matrix or the groundwater. Intensity is dependent on herbicide
physico-chemical properties and soil and climate characteristics.
Understanding these processes is important for predicting
herbicide behaviour in distinct soil types and selecting appropriate
dosages, with the goal of avoiding harmful environment effects
and residual effects on succeeding crops 10, 11. The sorption
process is comprised of solute movement (an herbicide molecule)
from the aqueous phase to a solid surface (soil) connected by
means of physico-chemical interactions 8. Sorption is influenced
by soil conditions (texture, organic matter content, nutrients,
cations and pH), environment (temperature, humidity and light)
and herbicide characteristics (water solubility, sorption coefficient,

vapour pressure and chemical composition).
Factors that influence herbicide sorption, such as organic matter
(OM), pH and temperature, were evaluated 12-14. The OM,
associated to the soil and sediment, is known to play an important
role in herbicide sorption 15-17, which could result in hydrogen
bond formation 18.
As a function of its high specific surface area and functional
group diversity, OM can interact with organic molecules in
different ways. The forces responsible for the sorption reactions
of herbicides in soil include physical forces (Van der Waals),
hydrogen bonds, hydrophobic bonds, electrostatic bonds,
reactions of coordination and exchange bonds, among others 19.
Soil OM consists of partially humidified organic waste at various
decomposition stages and is considered a key indicator of soil
quality. It acts as a source of nutrients, increases cation retention,
acts on metal complexation and is a source of carbon and energy
to soil microorganisms, in addition to aiding water infiltration and
retention and functioning as an essential component of soil
sustainability maintenance 20.
OM is considered heterogeneous, consisting of hard and soft
components and may be sequestered by a combination of both
mechanisms (Fig. 1) that exhibit different herbicide sorption
behaviours. Most accessible components are denominated
rubbery OM (soft) and the less accessible components, which
exhibit different sorption behaviour, are denominated rigid or glassy
(hard) 21.
Diffusion in pores
Surface sorption
Water-soluble fraction
Organic
matter
Mineral
fraction
Surface
sorption
Organic
matter
Diffusion into rubbery organic

matter (soft)
Diffusion into glassy organic
matter (hard)
Figure 1. Summary of the physical behaviour (surface sorption) of an

herbicide in the soil.
Source: Adapted from Semple et al. 48; DAgostinho and Flues. 21.
Therefore, herbicide can be superficially sorbed by OM adhered

to the mineral fraction or the soluble organic fraction. In a second
phase, herbicide can diffuse into micropores and mesopores and
interact with soft OM (more accessible micropores) or hard OM
(less accessible micropores). Most adsorbents are highly porous
bodies with a very large internal surface area and a smaller external
surface comprising only a small fraction of total surface adsorbent.
Nonlinear sorption is generally the result of the sorption process
in hard fraction OM soil. Moreover, temperature and pH have
different effects on herbicide sorption 22.
Herbicide bioavailability can decrease depending on the
sorption process, but can also increase detoxification problems
depending on soil properties and the molecules themselves 23.
Sorption behaviour is fundamental for determining the negative
effects of herbicide sorbed in soil, in contact with the succession

culture.
The equilibrium sorption is calculated as the difference in
concentration between the initial solution and the solution in
equilibrium with the soil, in a single solution concentration for
each soil. Assuming the sorption to be an instantaneous, reversible
and a linear process, the sorption coefficient (Kd) value in L kg-1,
which is commonly used to determine herbicide sorption intensity
on soil, was estimated.
Kd is calculated for the initial herbicide concentration, as
represented by Equation (1):
Kd = Cs/Ce
(1)
Cs is the concentration of herbicide sorbed to soil (g g-1) and Ce

is the concentration of herbicide in solution at equilibrium (g mL-1).
This coefficient is normalized to the organic carbon (OC) content
of soil evaluated by Koc, calculated with Equation (2):
Koc = Kd 100/OC(%)
(2)
The value calculated for Kd is a very important parameter for

mathematical models used for assessing environmental
contamination. Therefore, when starting a risk analysis and
defining the Kd value to be applied to the model, the pesticide
being used must first be evaluated.
In the second phase, the percentage of OM present in the soil
being studied and the soil particle size must be evaluated;
especially the percentage of clay, which can contribute to the
increase of area-specific OM attached to the clay mineral and
allowing greater interaction of the compound to the soil 21. In the
OM composition, OC content prevails (from 50 to 58%), with soil
test laboratories traditionally using a conversion factor ranging
from 1.724 for OM (equal to 58% OC) to 2.0 for OM (equal to 50%
OC) when reporting the OM content of soil. Therefore, the
determination of total OC has been used to quantitatively estimate
the organic fraction of the soil 24.
Different analytical procedures have been used to determine
the carbon content of the soil, from those based on the oxidant
agent dichromate method and its variants to those employing
automated dry combustion, which may underestimate or
overestimate actual soil OC values 25. The quantitative scaling of
carbon stored by the different systems of soil use and management
requires the application of methodologies that have been verified
and shown to be representative for the region.
Many mathematical models are described in the literature, such
as the screening criteria of the U.S. Environmental Protection
Agency (EPA) 26, Groundwater Ubiquity Score (GUS) index 27,
Attenuation Factor (AF), Retardation Factor (RF) 28, Attenuation
Factors for multilayered soils (AFi) 29, LIX index (Screening
Leachability) 30 and TLPI (Temperature Leaching Potenctial Index) 31,
to evaluate the leaching potential of herbicides in soil. The absence
of Kd and Koc values in herbicides used on tropical soils, typically
Brazilian in these models, is pointed out as a limitation by the fact
that the essential values have been derived from databases of
temperate soils, meaning that the information may be distorted.
Considering the importance and lack of information on herbicide
behaviour in soil under varied environmental conditions, the
objectives of this paper were to review the scientific literature and
425
discuss problems associated with the determination, calculation

and interpretation of Kd and Koc values for Brazilian tropical soils;
evaluate Kd and Koc values of tropical soils with the values
described in databases for temperate soils; and to correlate Kd
values with the tropical soils properties, suggesting a method for
calculating standardized Kd values applicable to soils with
properties similar to those reported in tropical soils.

The methodology adopted in this paper was in form of research
literature reviews in scientific journal articles by nationally
recognized Brazilian authors. The respective sources are reported
in Table 1, demonstrating the values of sorption coefficients (Kd)
and OC affinity (Koc) for tropical soil conditions in Brazil.
After obtaining the data, a database was prepared for soil
Table 1. Values reported in the literature of herbicide sorption coefficients (Kd), OC affinity coefficients (Koc) in Brazilian
tropical soils, selected soil property values, calculated OC and OC/OM indices, and calculated OC and Koc values,
assuming an OC/OM index of 0.54 as proposed by Weber et al. 32.
Reported valuesa
Calculated values
Herbicide
Acetochlor
Alachlor
Ametryn
Aminocyclopyrachlor
Atrazine
Dicamba
Diclosulam
426
Kd
(L kg-1)
0.76
1.22
1.67
2.75
0.53
0.54
1.48
2.15
2.19
5.51
4.57
5.49
6.35
13.44
33.50
0.05
0.06
0.09
0.14
0.23
0.29
0.34
0.48
0.50
0.50
0.62
0.85
1.07
1.17
1.34
2.16
2.86
3.03
4.87
5.96
0.51
0.85
1.69
3.81
4.06
10.53
1.60
1.72
1.93
1.95
2.35
2.42
28.58
0.07
0.12
0.13
0.15
0.22
0.52
1.95
2.03
Koc
(L kg-1)
86.00
116.00
126.00
166.00
150.00
94.00
102.00
77.00
126.00
74.00
n/a
n/a
n/a
n/a
318.44
10.00
9.00
16.00
22.00
32.00
32.00
33.00
27.00
27.00
47.00
39.00
39.00
66.00
57.00
67.00
81.86
105.15
135.27
225.46
248.33
145.00
146.00
61.00
219.00
280.00
140.00
233.90
247.50
143.10
170.00
148.80
194.50
271.67
11.00
34.00
9.00
9.00
8.00
7.00
98.00
159.00
OM
(%)
1.63
1.94
2.46
3.07
0.65
1.07
2.68
5.15
3.22
13.80
1.70
1.70
1.70
2.55
18.89
0.92
1.20
1.13
1.18
1.26
1.67
1.89
3.31
3.39
1.96
2.96
4.02
3.02
3.80
3.44
4.55
4.68
3.86
3.72
4.13
0.65
1.07
5.15
3.22
2.68
13.80
1.67
1.67
3.00
2.72
3.28
2.58
18.89
1.07
0.65
2.68
3.22
5.15
13.80
3.68
2.37
CM
(%)
22.0
22.0
22.0
22.0
10.0
6.0
42.0
75.0
34.0
14.0
44.0
44.0
44.0
25.0
59.8
10.0
5.0
7.0
11.0
7.0
17.0
11.0
50.0
56.0
17.0
65.0
59.0
57.0
57.0
23.3
58.3
32.7
33.7
34.0
53.0
10.0
6.0
75.0
34.0
42.0
14.0
27.0
29.0
46.0
39.0
56.0
64.0
59.8
6.0
10.0
42.0
34.0
75.0
14.0
35.5
34.1
pH
5.50
5.40
4.70
4.70
4.30
4.50
4.80
6.30
4.50
4.60
5.80
4.90
4.40
5.90
4.10
5.40
7.20
6.50
6.50
5.10
5.70
4.90
6.10
5.70
6.00
6.00
6.20
5.20
5.90
5.80
5.10
6.00
5.40
5.20
5.10
4.30
4.50
6.30
4.50
4.80
4.60
6.10
6.30
5.60
6.00
6.00
6.20
4.10
4.50
4.30
4.80
4.50
6.30
4.60
5.0
4.4
CEC
(cmolc kg-1)
9.00
9.00
9.00
9.00
3.00
4.40
14.50
26.10
14.40
46.80
11.87
2.37
2.29
4.47
50.00
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
9.00
8.90
7.60
11.70
7.50
11.70
3.00
4.40
26.10
14.40
14.50
46.80
9.03
9.03
16.19
14.68
17.69
13.93
50.00
4.40
3.00
14.50
14.40
26.10
46.80
n/a
n/a
OC (%)
0.884
1.052
1.325
1.657
0.353
0.574
1.451
2.792
1.738
7.446
n/a
n/a
n/a
n/a
10.520
0.500
0.667
0.562
0.636
0.719
0.906
1.030
1.778
1.852
1.064
1.590
2.179
1.621
2.053
2.000
2.639
2.720
2.240
2.160
2.400
0.352
0.582
2.770
1.740
1.450
7.521
0.684
0.695
1.349
1.147
1.579
1.244
10.520
0.636
0.353
1.444
1.667
2.750
7.428
1.990
1.277
OC/OM
indexc
0.542
0.542
0.539
0.540
0.543
0.536
0.541
0.542
0.540
0.539
n/a
n/a
n/a
n/a
0.557
0.543
0.556
0.497
0.539
0.571
0.542
0.545
0.537
0.546
0.543
0.537
0.542
0.537
0.540
0.581
0.580
0.581
0.580
0.581
0.581
0.541
0.544
0.538
0.540
0.541
0.545
0.409
0.416
0.450
0.422
0.481
0.482
0.557
0.594
0.543
0.539
0.518
0.534
0.538
0.541
0.539
Calculated values
assuming
OC/OM = 0.54
OCd
Koce
(%)
(L kg-1)
0.880
86.364
1.048 116.412
1.328 125.753
1.658 165.862
0.351 150.997
0.578
93.426
1.447 102.280
2.781
77.310
1.739 125.934
7.452
73.940
0.918 497.821
0.918 598.039
0.918 691.721
1.377 976.035
10.201 328.399
0.497
10.060
0.648
9.529
0.610
14.754
0.637
21.978
0.680
33.823
0.902
32.151
1.021
33.301
1.787
26.861
1.831
27.307
1.058
47.259
1.598
38.798
2.171
39.152
1.631
65.604
2.052
57.018
1.858
72.120
2.457
87.912
2.527 113.178
2.084 145.393
2.009 242.409
2.230 267.264
0.351 145.299
0.578 147.059
2.781
60.770
1.739 219.091
1.447 280.580
7.452 141.304
0.902 177.383
0.902 190.687
1.620 119.136
1.469 132.743
1.771 132.693
1.393 173.726
10.201 280.169
0.578
12.111
0.351
34.188
1.447
8.984
1.739
8.626
2.781
7.911
7.452
6.978
1.987
98.138
1.280 158.594
Source
Ferri et al. 49
Ferri et al. 50
Oliveira Jr. et al. 40
Andrade et al. 38
Matallo et al. 51
Arantes et al. 42
Inoue et al. 43
Matallo et al. 51
Lavorenti et al. 52
Table 1. Continued.
Reported valuesa
Calculated values
Herbicide
Diuron
Hexazinone
Imazaquin
Imazethapyr
Indaziflam
Metamitron
Metolachlor
Metsulfuron-methyl
Nicosulfuron
Picloram
Prometryn
Simazine
Kd
(L kg-1)
1.44
2.65
4.34
4.58
8.42
10.99
14.31
2.70
3.10
14.70
5.07
10.14
12.98
13.40
15.77
17.05
0.13
0.18
0.71
0.74
0.75
1.58
0.33
0.58
1.63
0.08
0.10
0.29
0.42
0.54
0.76
4.86
5.22
9.42
12.44
21.14
27.44
23.05
1.80
3.10
3.90
0.09
0.12
0.13
0.15
0.16
0.27
0.14
0.14
0.31
0.32
0.35
0.38
0.81
0.92
2.40
86.28
0.34
0.54
0.76
1.09
2.02
6.45
3.60
4.00
36.10
Koc
(L kg-1)
145.00
180.00
310.00
627.00
2,631.00
2,290.00
917.00
145.00
n/a
917.00
741.60
1,482.50
1,131.60
966.60
1,267.70
1,079.80
36.00
30.00
25.00
42.00
52.00
21.00
66.31
52.77
108.82
22.00
17.00
17.00
6.00
19.00
53.00
972.00
855.00
434.00
1,173.00
1,321.00
1,339.00
219.11
179.00
261.00
n/a
16.00
33.00
5.00
10.00
9.00
4.00
39.00
24.00
18.00
11.00
24.00
5.00
82.7
n/a
n/a
820.15
96.00
93.00
27.00
75.00
117.00
86.00
367.00
299.00
343.16
OM
(%)
1.83
2.72
2.59
1.35
0.59
0.89
2.89
1.46
0.87
2.87
1.67
1.67
2.72
3.00
2.58
3.28
0.65
1.07
5.15
3.22
2.68
13.80
0.92
2.04
2.78
0.65
1.07
3.22
13.80
5.15
2.68
0.92
1.13
4.02
1.96
2.96
3.80
18.89
0.87
2.87
1.46
1.07
0.65
5.15
2.68
3.22
13.80
0.65
1.07
3.22
5.15
2.68
13.80
1.81
1.70
3.10
18.89
0.65
1.07
5.15
2.68
3.22
13.80
1.81
2.48
18.89
CM
(%)
35.0
27.0
32.0
40.0
35.0
37.0
33.0
45.0
48.4
31.6
29.0
27.0
39.0
46.0
64.0
56.0
10.0
6.0
75.0
34.0
42.0
14.0
8.0
20.0
70.0
10.0
6.0
34.0
14.0
75.0
42.0
10.0
7.0
59.0
17.0
65.0
57.0
59.8
48.4
31.6
45.0
6.0
10.0
75.0
42.0
34.0
14.0
10.0
6.0
34.0
75.0
42.0
14.0
22.0
35.0
26.0
59.8
10.0
6.0
75.0
42.0
34.0
14.0
22.0
40.0
59.8
pH
4.90
4.60
4.30
4.50
5.10
4.20
4.70
5.00
5.10
5.10
6.30
6.10
6.00
5.60
6.20
6.00
4.30
4.50
6.30
4.50
4.80
4.60
5.90
6.10
5.50
4.30
4.50
4.50
4.60
6.30
4.80
5.40
6.50
6.20
6.00
6.00
5.90
4.10
5.10
5.10
5.00
4.50
4.30
6.30
4.80
4.50
4.60
4.30
4.50
4.50
6.30
4.80
4.60
5.60
6.33
4.96
4.10
4.30
4.50
6.30
4.80
4.50
4.60
5.60
5.50
4.10
CEC
(cmolc kg-1)
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
9.03
9.03
14.68
16.19
13.93
17.69
3.00
4.40
26.10
14.40
14.50
46.80
n/a
n/a
n/a
3.00
4.40
14.40
46.80
26.10
14.50
n/a
n/a
n/a
n/a
n/a
n/a
50.00
n/a
n/a
n/a
4.40
3.00
26.10
14.50
14.40
46.80
3.00
4.40
14.40
26.10
14.50
46.80
n/a
18.75
4.96
50.00
3.00
4.40
26.10
14.50
14.40
46.80
n/a
n/a
50.00
OCb (%)
0.993
1.472
1.400
0.730
0.320
0.489
1.560
1.862
n/a
1.603
0.684
0.684
1.147
1.184
1.244
1.579
0.361
0.600
2.840
1.762
1.442
7.524
0.498
1.099
1.498
0.364
0.588
1.706
7.000
2.842
1.434
0.500
0.610
2.170
1.060
1.600
2.049
10.520
1.005
1.188
n/a
0.562
0.364
2.600
1.500
1.778
6.750
0.359
0.583
1.722
2.909
1.458
7.600
0.979
n/a
n/a
10.520
0.354
0.581
2.815
1.453
1.726
7.500
0.981
1.338
10.520
OC/OM
indexc
0.543
0.541
0.540
0.541
0.542
0.550
0.540
1.275
n/a
0.558
0.409
0.409
0.422
0.395
0.482
0.481
0.555
0.561
0.551
0.547
0.538
0.545
0.541
0.539
0.539
0.560
0.550
0.530
0.507
0.552
0.535
0.543
0.540
0.540
0.541
0.540
0.539
0.557
1.155
0.414
n/a
0.525
0.560
0.505
0.560
0.552
0.489
0.552
0.545
0.535
0.565
0.544
0.551
0.541
n/a
n/a
0.557
0.545
0.543
0.547
0.542
0.536
0.543
0.542
0.539
0.557
Calculated values
assuming
OC/OM = 0.54
Koce
OCd
(%)
(L kg-1)
0.988
145.749
1.467
180.641
1.399
310.221
0.729
628.258
0.319 2,639.498
0.481 2,284.823
1.561
916.720
0.788
342.639
0.470
659.574
1.550
948.387
0.902
562.084
0.902 1,124.168
1.620
801.234
1.469
912.185
1.771
890.457
1.393 1,223.977
0.351
37.037
0.578
31.142
2.781
25.530
1.739
42.553
1.447
51.831
7.452
21.202
0.497
66.398
1.102
52.632
1.501
108.594
0.351
22.792
0.578
17.301
1.739
16.676
7.452
5.636
2.781
19.417
1.447
52.522
0.497
977.867
0.610
855.738
2.171
433.901
1.058 1,175.803
1.598 1,322.904
2.052 1,337.232
10.201
225.958
0.470
382.978
1.550
200.00
0.788
494.924
0.578
15.571
0.351
34.188
2.781
4.674
1.447
10.366
1.739
9.201
7.452
3.623
0.351
39.886
0.578
24.221
1.739
17.826
2.781
11.507
1.447
24.188
7.452
5.099
0.977
82.907
0.918
100.218
1.674
143.370
10.201
845.799
0.351
96.866
0.578
93.426
2.781
27.328
1.447
75.328
1.739
116.159
7.452
86.554
0.977
368.475
1.339
298.730
10.201
353.887
Source
Carbo et al. 44
Dores et al. 53
Inoue et al. 43
Barizon et al. 54
Alonso et al. 7
Matallo et al. 51
Dores et al. 53
Regitano and Koskinen 55

Assis et al. 56
Matallo et al. 51
Regitano et al. 57
Matallo et al. 51
427
Table 1. Continued.
Reported valuesa
Calculated values
Herbicide
Sulfometuron-methyl
Tebuthiuron
2,4-D
Kd
(L kg-1)
0.14
0.21
0.46
0.73
0.77
1.18
0.72
0.79
1.58
1.61
2.50
2.57
0.13
0.23
0.31
0.36
0.40
0.43
0.49
0.81
0.90
0.91
0.94
0.97
1.02
1.07
1.17
1.29
1.60
2.10
4.63
Koc
(L kg-1)
38.00
37.00
17.00
50.00
44.00
16.00
258.21
374.30
135.40
159.40
138.20
151.40
27.80
21.30
26.70
42.90
31.10
40.90
32.20
53.70
70.70
60.30
82.00
98.80
70.00
89.10
80.60
99.40
102.30
131.30
306.50
OM
(%)
0.65
1.07
5.15
2.68
3.22
13.80
0.48
0.36
2.02
1.74
3.12
2.93
0.92
2.04
2.22
1.48
2.41
2.04
2.78
2.78
2.41
2.78
2.04
1.80
2.59
2.22
2.59
2.41
2.96
2.96
2.78
CM
(%)
10.0
6.0
75.0
42.0
34.0
14.0
8.0
8.0
62.0
62.0
36.0
36.0
10.0
16.0
28.0
13.0
41.0
27.0
54.0
41.0
24.0
51.0
50.0
5.0
39.0
19.0
61.0
42.0
52.0
64.0
2.0
pH
4.30
4.50
6.30
4.80
4.50
4.60
7.30
7.30
5.70
5.90
5.40
5.90
5.00
5.60
5.60
6.30
5.50
6.60
5.80
5.70
6.50
5.80
5.00
6.40
6.00
4.60
5.70
6.10
5.30
4.90
4.80
CEC
(cmolc kg-1)
3.00
4.40
26.10
14.50
14.40
46.80
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
OCb (%)
0.368
0.568
2.706
1.460
1.750
7.375
0.279
0.211
1.167
1.010
1.809
1.697
0.468
1.080
1.161
0.839
1.286
1.051
1.522
1.508
1.273
1.509
1.146
0.982
1.457
1.201
1.452
1.298
1.564
1.599
1.512
OC/OM
indexc
0.566
0.531
0.525
0.545
0.543
0.534
0.581
0.586
0.578
0.580
0.580
0.579
0.509
0.529
0.523
0.567
0.534
0.515
0.547
0.542
0.528
0.543
0.562
0.546
0.562
0.541
0.561
0.538
0.528
0.540
0.544
Calculated values
assuming
OC/OM = 0.54
Koce
OCd
(%)
(L kg-1)
0.351
39.886
0.578
36.332
2.781
16.541
1.447
50.449
1.739
44.278
7.452
15.835
0.259 277.992
0.194 407.216
1.091 144.821
0.940 171.276
1.685 148.368
1.582 162.452
0.497
26.157
1.102
20.871
1.199
25.855
0.799
45.056
1.301
30.746
1.102
39.020
1.501
32.645
1.501
53.964
1.301
69.178
1.501
60.626
1.102
85.299
0.972
99.794
1.399
72.909
1.199
89.241
1.399
83.631
1.301
99.154
1.598 100.125
1.598 131.414
1.501 308.461
Source
Souza et al. 46
Spadotto et al. 47
OM, organic matter; OC, organic carbon; CM, clay mineral; pH, potential hydrogen (H2O or CaCl2); CEC, cation exchange capacity; n/a, not available. b (%OC) = Kd/Koc 100. c (%OC)/(%OM)
ratio. d (%OC) = (%OM) 0.54. e Koc = Kd/(%OC) 100.
a
conditions in tropical climate, with the maximum number

of herbicides found in Brazilian scientific literature, where
possible reporting the soil chemical properties (organic
matter, OM; clay mineral, CM; potential hydrogen, pH
and cation exchange capacity, CEC) wherein the sorption
experiments were performed for each herbicide.
In addition, the OC percentage and Koc values were
calculated based on the OC/OM index equal to 0.54
proposed by Weber et al. 32. This is a mean value used
to provide a more accurate comparison of Koc values
among herbicides with the limited information on tropical
soil properties currently available 32.
A comparative analysis with Students t-test at the
0.01 and 0.05 levels was done using Kd and Koc values
from tropical soils, typically Brazilian, with Kd and Koc
values referring to temperate soils described in the
FOOTPRINT 33 and EXTOXNET 34 databases, as shown
in Table 2.
Herbicide Kd values were calculated for hypothetical
tropical soils with OM contents of 1.0, 2.5, and 5.0%
(0.54, 1.35, and 2.70% OC, respectively), based on the
OC/OM index of 0.54 of Weber et al. 32 described in Table
3.
For herbicides used on Brazilian tropical soils reported
in the literature, linear equations were described to
calculate Kd values from the analysis of significant
Pearson correlation at the 0.01 and 0.05 level among each
428
Table 2. Reported mean values of herbicide sorption coefficients (Kd) and

OC affinity coefficients (Koc) in Brazilian literature on tropical soils
compared to temperate soils from reference database 33, 34.
Herbicide
Acetochlor
Alachlor
Ametryn
Aminocyclopyrachlor
Atrazine
Dicamba
Diclosulam
Diuron
Hexazinone
Imazaquin
Imazethapyr
Indaziflam
Metamitron
Metolachlor
Metsulfuron-methyl
Nicosulfuron
Picloram
Prometryn
Simazine
Sulfometuron-methyl
Tebuthiuron
2,4-D
Reported mean
Kd (L kg-1)a
Trsb
Tes
1.600 0.84*
3.210c
2.067 1.83ns
2.890d
12.670 12.16
n/a
0.460 0.36** 0.390c
4.327 6.29** 2.340d
0.202 0.16* -0.530d
1.990 0.05
n/a
8.852 5.41** 8.300c
0.682 0.52* -4.400d
0.847 0.68
n/a
0.365 0.26
n/a
13.420 9.09
n/a
23.050
0.890c
2.933 1.05*
0.670c
0.153 0.06
n/a
0.350 0.22** 0.440d
0.140d
1.660 1.04ns
86.280
3.340d
6.100 10.81ns 1.960d
0.582 0.39** -0.500d
1.628 0.79** 1.780d
1.040 1.00** 1.240c
Reported mean
Koc (L kg-1)a
Trsb
Tes
123.500 33.04**
156.000c
103.833 29.43**
335.000c
318.440
316.000c
32.571 16.45**
24.000c
171.765 68.15**
100.000c
13.000 10.37*
2.000d
90.000d
128.500 43.13ns
988.787 731.04**
813.000c
34.333 11.46**
54.000c
75.967 29.24
n/a
22.333 15.97*
52.000c
1,015.667 343.09** 1,000.000c
219.110
77.700c
220.000 57.98ns
120.000c
12.833 10.75
n/a
29.100 25.99*
30.000c
n/a
13.000c
820.150
400.000c
167.018 130.43**
130.000c
33.667 14.09*
85.000c
202.818 95.62**
80.000c
77.242 63.66**
88.400c
Trs, tropical soils; Tes, temperate soils; n/a, not available. b Mean reported from Brazilian literature as described in Table
1. Kd and Koc values associated with standard deviation (SD) of the mean. Significant at the * 0.05 and ** 0.01 level by
Students t-test; ns, nonsignificant. c Source: FOOTPRINT 33. d Source: EXTOXNET 34.
a
Table 3. Reported mean of herbicide OC affinity coefficients (Koc)

and calculated herbicide sorption coefficient (Kd) values
from Brazilian literature on tropical soils, assuming soils
with 1.0, 2.5 and 5.0% OM contents and OC/OM index
of 0.54 proposed by Weber et al. 32.
Calculated Kd values (L kg-1)a,b
Assuming
Assuming
Assuming
Herbicide
1.0% OM
2.5% OM
5.0% OM
(0.54% OC) (1.35% OC) (2.70% OC)
Acetochlor
123.500
0.667
1.667
3.334
Alachlor
103.833
0.561
1.402
2.804
Ametryn
318.440
1.720
4.299
8.598
Aminocyclopyrachlor
32.571
0.176
0.440
0.879
Atrazine
171.765
0.928
2.319
4.638
Dicamba
13.000
0.070
0.176
0.351
Diclosulam
128.500
0.694
1.735
3.470
Diuron
988.787
5.339
13.349
26.697
Hexazinone
34.333
0.185
0.464
0.927
Imazaquin
75.967
0.410
1.026
2.051
Imazethapyr
22.333
0.121
0.302
0.603
Indaziflam
1,015.667
5.485
13.712
27.423
Metamitron
219.110
1.183
2.958
5.916
Metolachlor
220.000
1.188
2.970
5.940
Metsulfuron-methyl
12.833
0.069
0.173
0.346
Nicosulfuron
29.100
0.157
0.393
0.786
Prometryn
820.150
4.429
11.072
22.144
Simazine
167.018
0.902
2.255
4.509
Sulfometuron-methyl
33.667
0.182
0.454
0.909
Tebuthiuron
202.818
1.095
2.738
5.476
2,4-D
77.242
0.417
1.043
2.086
Reported
mean Koc
(L kg-1)c
OM, organic matter; OC, organic carbon. b Kd = Koc (%OC)/100. c Reported mean from Brazilian
literature described in Table 1.
a
of the tropical soil properties (OM, CM, pH and CEC) of Brazil

available in the scientific literature (Table 4).
Table 1 contains reported sorption coefficients (Kd), OC affinity
(Koc), selected tropical soil property values and calculated OC
and OC/OM indices for 22 herbicides. The computed OC/OM
indices, with reported values ranging from 0.395 to 1.275, emphasize
larger discrepancies for the soils tested with diuron and metolachlor.
Corroborating with the data, Weber et al. 32 found OC/OM
indices ranging from 0.100 to 1.538 for 20 herbicides, reflecting the
inaccuracy of the values reported in the literature (i.e., the Koc and
Kd values determined from %OM values are inaccurate).
In general, the OC/OM index of 0.54 used to calculate %OC
from the reported %OM values were close to 0.54 for most studied
herbicides (Table 1). This fact can be justified by calculations
already proposed by Weber et al. 32 with herbicide sorption
coefficients evaluated in temperate soils listed in the Weed Science
Society of America Herbicide Handbook and Supplement.
In many cases, the newly calculated Koc values, which assume
the OC/OM index equal to 0.54 using Equation (2), do not vary
greatly from the Koc values reported in literature. For example, for
acetochlor, where OM = 1.63%, an OC/OM index of 0.54 resulted
in OC = 0.880% (1.63 0.54 = 0.880), was similar to the values
reported in the literature (OC = 0.884%), providing Koc = 86.364 L
kg-1 (0.76/0.880 100), similar to Koc of 86 L kg-1 (Table 1). In some
cases, however, the values vary greatly, as in the case of atrazine,
which shows variation of 24% in Koc (233.90 vs. 177.383 L kg-1),
diuron with 36% (145.00 vs. 342.639 L kg-1) and metolachlor with
114% (179.00 vs. 382.978 L kg-1).
In addition, all of the newly calculated Koc values are based on
the same OC/OM index described in Table 1. For selected
herbicides, linear equations are provided later in this paper to

allow more accurate computation of Kd values for tropical soils
with the reported properties (Table 4).
The differences of mean Kd values among herbicides reported
in the literature on tropical soils and databases on temperate
soils 33, 34 are usually evident for acetochlor, aminocyclopyrachlor,
atrazine, dicamba, diuron, hexazinone, metolachlor, nicosulfuron,
sulfometuron-methyl, tebuthiuron and 2,4-D. For example,
metamitron and prometryn showed variation of 96% (23.050 vs.
0.890 L kg-1 and 86.280 vs. 3.340 L kg-1) between tropical and
temperate soils (Table 2). The similarities of mean Kd values
reviewed in the literature are expressive for alachlor, picloram and
simazine.
For mean Koc values, alachlor in temperate soils showed a value
of 335.000 L kg-1, differing from tropical soils where the mean value
was 103.833 L kg-1. These differences were also observed for
acetochlor, aminocyclopyrachlor, atrazine, dicamba, diuron,
hexazinone, imazethapyr, indaziflam, nicosulfuron, simazine,
sulfometuron-methyl, tebuthiuron and 2,4-D.
By comparison, similar mean Koc values were observed for
diclosulam and metolachlor across temperate and tropical soils
(Table 2). It should be emphasized that the missing information
(n/a, not available) in databases with temperate soils corresponded
to 27 and 9% for mean Kd and mean Koc values, demonstrating the
difficulty in comparing sorption coefficients.
Usually in sorption herbicide studies, K d is calculated
considering the herbicide-soil interactions as not only a surface
phenomenon, but one that occurs homogeneously in the entire
soil volume with Kd or Koc 35. Higher and lower values of these
coefficients are indicative of greater and lesser retention of
herbicides by soil 36.
For hydrophobic herbicides such as aromatics, halogenates,
phenols and bisphenols, which have low water solubility, their
mobility and the risk of leaching to groundwater are related to low
sorption in soil matrices and lower quantified values of Koc.
Therefore, herbicides with high K oc values are found in
groundwater and drainage, presumably as a result of leaching
from a combination of factors with emphasis on rapid and torrential
rain after herbicide application and the presence of preferred
channels, which occurs more easily in the tropics 8.
Given the above and considering the mean Kd and Koc values
reported in databases using temperate soils (Table 2), it is not
recommended to estimate herbicide behaviour in tropical soils
using mathematical models based on temperate soils as this can
distort the compiled information.
Silva et al. 19 report that in Brazil, Koc has been widely used to
predict the sorption capacity of several herbicides in soil and it is
also used, along with the texture, to recommend herbicide dosages.
Standardizing Kd in relation to soil OC, however, provides no
consensus among researchers in this area, since the sorption of
herbicides to soil OM occurs in heterogeneous form, as a function
of the mechanisms and organic fraction involved in the sorption
process, the indices of which may not represent reality. At the
same time, Kd and Koc are not always sufficiently accurate to
describe the sorption of an herbicide in the concentration range
considered.
Table 3 contains the mean Koc values of 21 herbicides in tropical
soils, with the exception of picloram, which was listed and
referenced in Table 1. Assuming an OC/OM index of 0.54, Equation
429
Table 4. Pearson correlation of herbicide sorption coefficient (Kd) and OC affinity coefficients (Koc) values vs. selected
soil properties and equations for calculating Kd values when soil property values were available (values taken
from Brazilian literature).
Herbicide
Chemical
family
Mean Kd
(L kg-1)b
Acetochlor
Nonionizable
1.600
Alachlor
Nonionizable
2.067
Ametryn
Base
12.670
Aminocyclopyrachlor
Acid
0.460
Atrazine
Base
4.327
Dicamba
Acid
0.202
Diuron
Hexazinone
Nonionizable
Base
1.600
0.682
Imazaquin
Acid
0.847
Imazethapyr
Acid
0.365
Indaziflam
Acid
13.420
Metolachlor
Nonionizable
2.933
Metsulfuron-methyl
Nicosulfuron
Simazine
Sulfometuron-methyl
Tebuthiuron
2,4-D
Acid
Acid
Base
Acid
Nonionizable
Acid
0.153
0.350
6.100
0.582
1.628
1.040
Reported soil property valuesa, d

OM
CM
CEC
pH
(%)
(%)
(cmolc kg-1)
0.990*
-0.844
0.986**
0.039
0.092
0.969**
0.970**
0.524
-0.508
0.925*
0.866**
0.833**
-0.233
0.923**
0.226
-0.501*
0.809**
0.990**
-0.025
0.099
0.958**
0.535*
0.385
0.434
0.855*
0.943*
0.161
0.141
0.954**
0.897
0.999**
-0.870
0.313
0.683
0.511
0.455
0.677
0.733
-0.071
0.415
-0.323
-0.790
0.934**
-0.092
-0.108
0.891*
0.098
0.110
0.503
0.793
0.851**
0.384
-0.403
0.730
0.836*
0.103
-0.018
0.839*
0.986**
0.490
-0.864*
0.486*
-0.075
-0.414
Equationc
R2
Kd = - 1.440 + 1.336 (OM)

Kd = ns
Kd = 0.416 + 0.373 (OM)
Kd = ns
Kd = ns
Kd = 0.079 + 0.109 (CEC)
Kd = 4.432 + 1.552 (OM)
Kd = ns
Kd = ns
Kd = 4.840 + 0.551 (CEC)
Kd = - 0.190 + 0.285 (OM)
Kd = 4.432 + 1.552 (CM)
Kd = ns
Kd = - 1.517 + 1.310 (OM)
Kd = ns
Kd = 27.338 - 4.290 (pH)
Kd = - 1.931 + 0.402 (CEC)
Kd = 0.054 + 0.033 (OM)
Kd = ns
Kd = ns
Kd = 0.027 + 0.010 (CEC)
Kd = 1.968 + 3.340 (OM)
Kd = ns
Kd = ns
Kd = - 1.201 + 1.013 (CEC)
Kd = 0.232 + 0.101 (OM)
Kd = ns
Kd = ns
Kd = 0.124 + 0.031 (CEC)
Kd = ns
Kd = 0.161 + 0.021 (CM)
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = 0.100 + 0.012 (OM)
Kd = ns
Kd = ns
Kd = 0.092 + 0.003 (CEC)
Kd = ns
Kd = ns
Kd = ns
Kd = ns
Kd = - 2.311 + 1.522 (OM)
Kd = ns
Kd = ns
Kd = ns
Kd = 0.284 + 0.067 (OM)
Kd = ns
Kd = ns
Kd = 0.216 + 0.020 (CEC)
Kd = 0.439 + 0.670 (OM)
Kd = ns
Kd = 6.793 0.826 (pH)
Kd = - 1.103 + 0.921 (OM)
Kd = ns
Kd = ns
0.99
ne
4
0.99
6
0.97
0.97
5
0.92
0.87
0.83
14
0.92
0.50
0.81
0.99
19
6
0.96
0.54
16
0.86
0.94
6
6
0.95
0.99
3
0.93
6
0.89
7
6
0.85
9
7
0.84
6
0.84
0.99
6
0.87
0.49
19
OM, organic matter; OC, organic carbon; CM, clay mineral; pH, potential hydrogen (H2O or CaCl2); CEC, cation exchange capacity. b Reported mean from Brazilian literature as described
in Table 1. c Most significant linear equation; ns, nonsignificant. d Significant at the * 0.05 and ** 0.01 levels. e Number of values correlated.
a
430
(2) was used to calculate the hypothetical Kd values of herbicides

in tropical soils with OM contents of 1.0, 2.5, and 5.0% (0.54, 1.35,
and 2.70% OC), as proposed by Weber et al. 32.
For example, acetochlor with a mean Koc value of 123.5 L kg-1
and 1% OM content (0.54% OC) in tropical soil showed a Kd of
0.667 L kg-1 (123.5 0.54/100); and with OM content of 2.5 and
5.0%, the Kd values were 1.667 and 3.334 L kg-1 (Table 3). For 2,4D with Koc equal to 77.242 L kg-1, the resultant Kd values were
0.417, 1.043 and 2.086 L kg-1, as a function of increased OM content.
Aminocyclopyrachlor and indaziflam herbicides are two new
molecules in the development stage in Brazil, according to Guerra
et al. 37 with Koc values of 32.571 and 1,015.667 L kg-1, respectively.
Aminocyclopyrachlor presents Kd values of 0.176, 0.440 and 0.879
L kg-1, while indaziflam presents Kd values of 5.485, 13.712 and
27.423 L kg-1, as a function of increased OM content (Table 3).
It is important to report that works developed by Weber et al. 32
using mean Koc values to calculate Kd values may be quite
inaccurate. Nevertheless, the calculation of Kd values at levels
close to the OM contents of the soils being modeled are much
more accurate than the mean Kd value for all soils.
For herbicides with Kd values between 1 and 10 L kg-1, small
changes in sorption may result in large variations in the quantity
of product in the solution of the soil and consequently on its
leaching 38.
The 2,4-D herbicide presents Koc in the form of acid salt and
ester; however, only values for the acid salt form are listed in
Table 3. This is because, as proposed by Weber et al. 32, only the
acid forms remain for significant periods of time in soil. Ester
formulations are inactive until they are readily hydrolyzed in soil,
changing from the ester to the acid form, with the acid forms being
much more mobile 39.
Several additional problems reported by Weber et al. 32 are
apparent in using mean Koc values for estimating Kd values in soils
with different OM contents, as is the case of herbicides with arsenic
acid moieties (cacodylic acid and DSMA) and phosphoric acid
moieties (fosamine and glufosinate), which bind and react with
CM and metallic hydrous oxides but not with OM, rendering Koc
values meaningless. The authors also report that strongly basic
herbicides (difenzoquat2+, diquat2+ and paraquat2+) that ionize to
cationic species in solution react primarily with CM in soils, again
resulting in Koc values with little significance. It is an important
report in this work that herbicides belonging to these groups
described above were not listed (Table 3).
Kd values of 18 herbicides reported in literature were correlated
with four properties (OM, CM, pH and CEC) of tropical soils and
described in Table 4. Diclosulam (n = 2), metamitron (n = 1),
picloram (n = 2) and prometryn (n = 1) were not correlated and the
differences may be attributed to the small number of values
reported for each herbicide from the Brazilian literature. The
significant correlation coefficients and linear equations to calculate
Kd values using the described soil properties were found for 14
herbicides, with the exceptions being imazethapyr, indaziflam,
metolachlor and nicosulfuron.
OM content was correlated with Kd values of five weakly acidic
herbicides (aminocyclopyrachlor, dicamba, metsulfuron-methyl,
sulfometuron-methyl and 2,4-D), four weakly basic herbicides
(ametryn, atrazine, hexazinone and simazine) and four nonionizable
herbicides (acetochlor, alachlor, diuron and tebuthiuron) (Table
4). CM content was correlated only with the Kd value for
aminocyclopyrachlor and imazaquin, both of which are weakly

acidic. Soil solution pH was inversely correlated with Kd values of
atrazine (weakly basic) and tebuthiuron (nonionizable). CEC of
tropical soils was correlated with Kd values of three weakly acidic
herbicides (dicamba, metsulfuron-methyl and sulfometuronmethyl), three weakly basic herbicides (ametryn, atrazine and
hexazinone) and two nonionizable herbicides (alachlor and diuron)
(Table 4).
In the case of highly weathered tropical soils, where the clay
fraction is predominantly composed by oxides and hydroxides of
Fe and Al silicate clays 1:1, of low reactivity (kaolinite), most of
the CEC part is due to soil OM, which explains the concurrence of
significance for the correlations 40, 41. In general, Brazilian
recommendations for herbicide rates applied to the soil are based
on the texture rather than the OM content as the main parameter
of initial reference, generating doubt about the adequacy of this
criterion 40.
These data are in general agreement with Silva et al. 19 where
OM and CEC are the soil constituents most highly correlated with
binding of most organic herbicides registered for use in tropical
soils. CM is correlated with cationic herbicide retention and the
pH is inversely correlated with the retention of many herbicides,
being strictly related to the capacity of electrolytic dissociation
- pKa. The differences in correlation between Kd values and soil
properties may be attributed to the small number of values
(minimum n = 3) reported for each herbicide in tropical soils (Table
4), corroborating with the data of Weber et al. 32.
Oliveira Jr. et al. 40 studied the correlation between soil properties
and found that sorption coefficients (Kd and Koc) of certain
herbicides in Brazilian soils were significantly correlated with the
OC and CEC content of the soil for most studied herbicides.
Generally, weakly acidic herbicides (imazethapyr, metsulfuronmethyl, nicosulfuron and sulfometuron-methyl) showed less
sorption, while weakly basic herbicides (atrazine, hexazinone and
simazine) and nonionizable (alachlor) were more sorbed by the
OC and CEC content of tropical soils. Since most of the CEC in our
soils is related to OM, this characteristic can be considered the
most important for these herbicides.
Linear equations for estimating the calculation of Kd values
(Table 4), as also reported in the literature for alachlor, atrazine,
dicamba, diuron, hexazinone, imazethapyr, metsulfuron-methyl,
nicosulfuron, simazine, sulfometuron-methyl 40, 42-44,
aminocyclopyrachlor 45, indaziflam, 7 tebuthiuron 46 and 2,4-D 47
using tropical soil properties, may be useful for calculating the
optimum rates for herbicide applications and should improve the
predictability of herbicide sorption using mathematical models in
soils with similar properties.
In addition to the soil properties evaluated and correlated with
Kd values by the authors cited in this work, more works are expected
to be developed on herbicide sorption in tropical soils correlated
with climatic factors, such as temperature and soil humidity; and
edaphic factors, such as the humified part of OM (composed of
fulvic, humic and humin acids) and the characteristics of clay
minerals, iron oxide and aluminum.
Conclusions
The OC/OM index values proposed in this paper were on average
close to 0.54 for most herbicides found in literature using tropical
soils, but ranged from 0.395 to 1.275.
431
Kd and Koc values of herbicides in tropical soils do not indicate

inferiority or superiority when compared to values found in
temperate soils, with similarities and differences among the values
reported in the scientific literature. The Kd and Koc values of
temperate soils are still used in research on herbicide behaviour in
tropical soils, however, being a resource for estimating
environmental contamination.
Research in Brazil is already generating information such as
sorption coefficients, however, they are not tabulated in a database
as has been done for temperate soils. Thus, it was possible to
group the information related to Kd and Koc values of herbicides in
tropical soil conditions.
The Kd values of most of herbicides were linearly correlated
with OM content and CEC variation in tropical soils, generating
standardized values to more accurately estimate the behaviour of
herbicides in soils with similar properties and to offer better
accuracy when using mathematical models.
Acknowledgements
The authors thank the Fundao de Amparo Pesquisa do Estado
de So Paulo/FAPESP for financial support for the completion of
this research.
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433
WFL Publisher
Helsinki, Finland
www.world-food.net
The relocation of undisturbed soil in long-term experiment impacts organo-mineral

complex degree and combined humus of black soil
Fengqin Chi *, Enjun Kuang, Baoku Zhou, Jiiuming Zhang, Qingrui Su and Shanshan Cai
Institute of Soil Fertilizer and Environment Resource, Heilongjiang Academy of Agricultural Sciences, The Key Laboratory of
Soil Environment and Plant Nutrition of Heilongjiang Province, Harbin 150086, Heilongjiang, China.
*e-mail: zouwenxiu@hotmail.com
Abstract
The effect of soil relocation on organo-mineral complex status and combined humus was considered to be based on the relocation of undisturbed soil
in a long-term experiment in Agricultural Soil Ecological Environment Key Field Scientific Observation and Experiment Station (Harbin) of the
Ministry in Northeast China. Four fertilizer treatments were selected in this study including CK (control, no fertilizer), M (organic fertilizer, horse
manure), NPK (chemical fertilizer), MNPK (horse manure plus chemical fertilizer). Tested soils were relocated at December 18, 2010 to March 15,
2011 by the relocation of undisturbed soil columns under freezing conditions. There were no effect of soil relocation on soil organo-mineral complex
degree, quantity of combined humus and the ratio of loosely and tightly combined humus. The youngest humus, the highest complex degree of organomineral complex and the highest soil fertility were observed in the combination of chemical plus organic matter (NPKM) in all tested soils. Soil organic
carbon, heavy fraction of organic carbon and organo-mineral complexes contents did not change much in all tested soils. However, the soil relocation
impacted significantly the content of additional organo-mineral complexes and the degree of additional organo-mineral complexes which showed the
trend of increase-decrease order after two years of soil relocation. Soil relocation did not impact significantly the quantity of combined humus and the
ratio of loosely and tightly combined humus in 0 - 20 cm soil layer in all tested soils.
Key words: Long-term experiment, black soil, combined humus, organo-mineral complex degree.
Introduction
Long-term located fertilization experiment is the most basic and
effective way to study the soil science, because it can
systematically explain the evolution of soil fertility, provide a
comprehensive evaluation of fertilization effect and the scientific
basis for fertilization 1. Long-term experiment has 170 years
history since Rothamsted Experimental Station in UK was
established in 1843. From the early 1980s, more than 100 longterm experiments related to soil and fertilizer were established in
typical agricultural regions in China, but finally only about 30
long-term experiments related to soil and fertilizer were preserved
due to various reasons. Because the limitations of the initial trial
design of long-term experiments, especially with the accelerated
urbanization process in China, some long-term experiment sites
must be relocated in order to protect the long-term experiment
resources. Soil relocation in long-term experiment site has been
recorded in a volume of literatures, but the information about the
effect of soil relocation in the long-term experiment on soil humus
is limited.
Humus is the main resource of soil nutrients, affects the soil
physical, chemical and biological properties, and controls the soil
fertility. Soil humus is composed of complex components, there is
much difference between humus which determines soil properties
and soil fertility. Combined humus played an important role in
soil fertility 2. Combined soil humus and minerals include 3 forms:
loosely combined humus, stably combined humus and tightly
combined humus 3. The contribution of soil humus to soil fertility
depended on the degree of the combination of humus and
minerals 4. The effect of soil relocation on the organo-mineral
434
complexes and combined humus of black soil were studied on the

basis of undisturbed soil relocation of long-term located
experiment5. The objectives of this study were: (1) to reveal the
relationship and variation of soil humus after black soil relocation,
(2) to provide feasible basis for undisturbed soil relocation of longterm located experiment.
Study site: Agricultural Soil Ecological Environment Key Field
Scientific Observation and Experiment Station (Harbin) of the
Ministry built in 1979, is the longest running long-term fertilization
monitoring experiment station in the black soil region of Northeast
China. Due to the expansion of urban development, the experiment
station has to be relocated. The overall relocation method of the
undisturbed permafrost was used. Harbin Black Soil Ecology
Environment Key Field Observation Station had achieved a
successful implementation of the overall relocation at December 18,
2010 to March 15, 2011 by the relocation of undisturbed soil columns
under freezing conditions in order to keep soil original performance
and structure. The new study site locates in Harbin Democracy
Township which is away 40 km from old study site. There are
similar climate conditions, soil parent material, groundwater levels,
soil physical and chemistry properties between two locations.
Soil sampling: The selected experiment includes four fertilizer
treatments: CK (control, no fertilizer), M (organic fertilizer, horse
manure), NPK (chemical fertilizer), MNPK (horse manure plus
chemical fertilizer). Soil samples were collected in 2010 (before
relocation) and 2012 and 2013 (after relocation), respectively. The

sampling depth is 0 ~ 20 cm and 20 ~ 40 cm soil depths, respectively.
Soil samples collected from Harbin Democracy Township was used
as a control treatment. The basic properties of the tested soil and
application amount of fertilizer were shown in Tables 1 and 2.
Chemical analysis: Soil heavy fraction was measured using
relative density fractionation method, combined humus was
measured by the improved Xiongyi-Fujiping method, solid organic
carbon and extracted organic carbon were measured by TOC
instrument (Multi N/C 2100S) 6. Soil organic matter was measured
by the potassium dichromate external heating method, alkaline
hydrolysis N was measured by the alkaline hydrolysis diffusion
method, available P was measured by the NaHCO3 extraction Mo-Sb colorimetry method, available K was measured by the
NH4OAC extraction - flame photometry 7.
The calculation of organo-mineral complex degree:
Soil complexes (SQC) = HC HW / SW
Soil complex degree (SCD) = SQC / SC 100%
Quantity of additional complex (QAC) = MQC - SQC
Degree of additional complex (DAC) = QAC / (MC - SC) 100%
where, HC is heavy fraction of soil organic carbon (g kg-1), HW is
heavy fraction carbon (g), SW is soil weight (g), SC is soil organic
carbon content (g kg-1), MQC is soil added organic manure
complex, MC is organic carbon content of soil added organic
manure (g kg-1).
Statistical analysis: Statistics were processed with Microsoft
Excel 2003 software. A correlation analysis was carried out with
SPSS software.
Results
The effect of soil relocation on soil organic carbon and heavy
fraction organic carbon contents: Heavy fraction organic carbon
fractionated from soil organic carbon includes organo-mineral
complex which is hard to be decomposed, thus it can be called as
the real soil humus with lower C/N and longer turnover.
Soil organic carbon content in 2010 fluctuated from 12.87 to
16.20 g kg-1 (Table 3), and higher values were found in 0 ~ 20 cm
soil layer compared with 20 ~ 40 cm soil layer, the highest values
were observed in MNPK treatment. Soil organic carbon content
Table 1. The fertilization amount of different treatments for
maize, soybean and wheat from 1979 to 2013.
Crop
Wheat
Maize
Soybean
N
(kg hm-2)
150
150
75
P2O5
(kg hm-2)
75
75
150
K2O
(kg hm-2)
75
75
75
M (house
manure) (t hm-2)
18.75
decreased and changed from 12.28 to 15.07g kg-1 after two years
of soil relocation (2012 and 2013). Soil organic carbon content
in 2012 was decreased by 6.5% and 7.7% for CK and MNPK
treatments, increased 4.9% and 3.6% for NPK and M treatments,
respectively, compared with that in 2010. Soil organic carbon
content in 2013 were increased 7.1%, 1.0% and 2.3% for CK, M
and MNPK, decreased 4.6% for NPK treatment, respectively,
compared with that in 2012. The variation of soil organic carbon
in 20-40 cm soil layer was more than that in 0 - 20 cm soil layer.
There was a significantly positive relationship between soil
organic carbon content and heavy fraction organic carbon (y = 0.579x
+ 4.258, r0.01 = 0.996, n = 8) in 2010, indicating that the process of soil
organic matter transformation involved gradually the formation
of organo-mineral complex. This significantly positive relationship
also was recorded in two years of soil relocation (2012: y = 0.672x +
3.067, r0.01 = 0.975, n = 8 , 2013: y = 0.753x + 2.072, r0.01 = 0.978, n = 8),
heavy fraction organic carbon showed the similar change trend with
soil organic carbon in different soil layers and treatments. Heavy
fraction organic carbon content in 2010 was decreased 3.5%
and 3.4% for CK and MNPK, increased by 2.9% and 3.8% for
NPK and M, respectively, compared with that in 2012. Heavy
fraction organic carbon content in 2012 was increased by 7.6%,
1.7% and 3.2% for CK, M and MNPK, decreased by 3.6% for NPK,
respectively, compared with that in 2013. The range of change of
heavy fraction organic carbon content in 20 - 40 cm soil layer was
more than that in 0 - 20 cm soil layer.
The effect of soil relocation on organo-mineral complex degree:
The degree of organo-mineral complex and quantity of additional
organo-mineral complex are used to represent complex degree of
soil organic matter and minerals, and reflect the status of soil
fertility, it is an important quantitative indicator to characterize
complex of soil organic matter and inorganic minerals. The quantity
of additional organo-mineral complex of the organic manure is the
ratio of the composite organic carbon from organic manure and
soil mass during the process of improving soil fertility 8. The degree
of additional organo-mineral complex is the percentage of the
composite organic carbon from organic manure and the increased
value of total soil organic carbon.
The quantity of original soil organo-mineral complex of M and
MNPK treatments was higher than CK and NPK, and was higher
in 0 ~ 20 cm soil layer than 20 ~ 40 cm soil layer in 2010 (Table 4).
The similar trends were found in 2012 and 2013. The quantity of
original soil organo-mineral complex in 2010 was decreased by 4.3%
and 2.3% for CK and MNPK, increased by 2.3% and 3.9% for M
and NPK, respectively, compared with that in 2012. The quantity
of original soil organo-mineral complex in 2013 was increased
by 10.9%, 5.7% and 6.3% for CK, M and MNPK, decreased by 1.1%
for NPK, respectively, compared with that in 2012. The degree of
original soil organo-mineral complex was in the increasing order of
CK > NPK > M > MNPK, and was more in 20 - 40 cm soil layer than
that in 0 - 20 cm soil layer, indicating that organic manure application
Table 2. Basic properties of the tested soils in 2010.

Treatments
CK
NPK
M
MNPK
Organic carbon
(g kg-1)
26.62
28.96
29.14
30.48
Total N
(g kg-1)
1.39
1.52
1.49
1.60
Total P
(g kg-1)
0.94
1.50
1.19
1.42
Total K
(g kg-1)
21.75
19.40
18.22
20.11
Available
(mg kg-1)
103.56
116.41
119.63
114.85
Available
P2O5 (mg kg-1)
14.49
57.18
17.22
66.78
Available
K2O (mg kg-1)
155.75
205.03
167.64
213.77
pH
6.9
6.5
7.2
7.0
435
Table 3. The difference of soil organic carbon and heavy fraction organic carbon
contents in 2010, 2012 and 2013 under different treatments.
Soil depth
cm
0 - 20
20 - 40
Treatments
CK
NPK
M
MNPK
CK
NPK
M
MNPK
Soil organic carbon (g kg-1)

2010
2012
2013
13.56
12.41
13.17
14.29
14.38
14.39
14.67
14.48
14.63
16.20
14.82
15.07
12.87
12.28
13.27
11.79
12.86
11.68
13.08
14.18
14.32
15.41
14.36
14.79
Heavy fraction organic carbon (g kg-1)

2010
2012
2013
12.11
11.40
12.26
12.49
12.59
12.67
12.67
12.77
12.87
13.66
13.01
13.46
11.58
11.45
12.33
11.14
11.71
10.78
11.93
12.73
13.05
13.24
12.98
13.36
Table 4. The effect of soil relocation on the organo-mineral complex degree.

Soil depth
(cm)
0 - 20
20 - 40
Treatments
CK
NPK
M
MNPK
CK
NPK
M
MNPK
2010
11.14
11.27
11.61
12.24
10.77
10.85
10.88
11.94
SQC
(g kg-1)
2012
10.53
11.81
11.77
11.94
10.44
10.82
11.58
11.68
2013
11.48
12.11
12.3
12.52
11.76
10.31
12.35
12.59
2010
82.15
78.87
79.14
75.56
83.68
92.03
83.18
77.48
SDC
(%)
2012
84.85
82.13
81.28
80.57
85.02
84.14
81.66
81.34
could improve soil organo-mineral complex and more significant

effect was observed in 0 - 20 cm soil layer. The increase of original
soil organo-mineral complex degree meant the decrease of fresh
soil organic carbon, soil tended to age 9, 10. There was an increasing
trend for original soil organo-mineral complex degree from 2010 to
2013, but generally soil relocation did not affect on the original
soil organo-mineral complex degree. The quantity and degree of
organo-mineral complex changed much, and were increased in 2012
and decreased in 2013 due to that organic manure was more quickly
decomposed by more active soil microbiology resulted from the
change of environment conditions after soil relocation.
The quantity of additional organo-mineral complex in 0 ~ 20 cm
soil layer was higher than that in 20 ~ 40 cm soil layer, the degree of
additional organo-mineral complex was inverse in 2010. The quantity
of additional organo-mineral complex of MNPK was higher than
that in M, but the degree of additional organo-mineral complex
was lower than that in M. Soil relocation significantly impacted
the quantity and degree of additional organo-mineral complex,
and were increased in 2012 and decreased in 2013. The carbon in
out of organo-mineral complex was more quickly decomposed
than that in organo-mineral complex, which resulted in the degree
of additional organo-mineral complex was increased with the
increase of plantation period 17.
The quantity of organo-mineral complex of black soil tented to
be increased by short-term application of chemical fertilizers, and
decreased by long-term application of chemical fertilizer 11, longterm application of organic manure and chemical fertilizers could
improve quantity of organo-mineral complex. The quantity of
organo-mineral complex was decreased in 2012 then increased
in 2013 for M and MNPK treatments, and was increased in 2012
and then decreased in 2013 for NPK treatment, which may be due
to the fluctuations of the quantity of organo-mineral complex under
the influence of the surrounding environment after soil relocation.
The quantity of organo-mineral complex was increased in short
time, but decreased in long-term for NPK treatment. The longterm application of organic manure could increase the quantity of
organo-mineral complex.
436
2013
87.17
84.16
84.07
83.08
88.62
88.27
86.24
85.13
QAC
(g kg-1)
2010 2012 2013
2010
DAC
(%)
2012
2013
0.47
1.10
1.24
1.41
0.82
1.04
42.34
41.67
59.90
58.51
56.16
54.74
0.11
1.17
1.14
1.24
0.59
0.83
52.38
46.06
60.00
59.62
56.19
54.61
The effect of soil relocation on combined humus: Most of the

soil humus was bound with the minerals to form organo-mineral
complexes, the amount of the free humus is tiny. The combination
of soil humus and minerals include three statuses: loosely
combined humus, stably combined humus and tightly combined
humus. Different combined humus forms had different impact on
soil fertility. Loosely combined humus had the most contribution
for soil fertility due to that was fresh humus and had the largest
activity. Tightly combined humus was the most stable because it
was tightly combined with mineral. The stability of stably
combined humus was between two other forms.
The content of the loosely combined humus was higher in 0 ~ 20
cm soil layer than 20 ~ 40 cm soil layer in 2010 (before soil relocation),
the content of the loosely combined humus of NPK, MNPK and
M was decreased in 20 ~ 40 cm soil layer, and was lower than that
in 0 ~ 20 cm in 2012 (the first year of soil relocation), the same
change trend of loosely combined humus was found in 2010 and
2013 (Table 5).
The content of loosely combined humus was increased in 0 20 cm soil layer, decreased in 20 - 40 cm soil layer in 2012, decreased
by 7.1% for CK, increased by 6.2 - 11.6% for NPK, MNPK and M,
compared with that in 2010. The content of loosely combined
humus was decreased by 2.5% for CK, and increased by 1 - 18.9%
for NPK, MNPK and M in 2013, compared with that in 2012. There
was no obvious change law of stable combined humus in both 0
~ 20 cm and 20 ~ 40 cm soil layers. Compared with the content of
stable combined humus in 2010, the content of stable combined
humus was increased by 92.2% in 2012, and decreased 45.9% in
2013 for M treatment, decreased by 27.8% in 2012, increased by
62.8% in 2013 for MNPK treatment. The content of tightly combined
humus was higher in 0 ~ 20 cm soil layer than 20 ~ 40 cm soil layer in
experimental three years. The content of tightly combined humus
showed an order of increase-decrease in 2012 and 2013 for NPK,
MNPK and M treatments, and an order of decrease-increase in
2012 and 2013 for CK treatment.
Table 5. The change of combined humus after soil relocation.

Soil depth
(cm)
0 - 20 cm
20 - 40 cm
Treatment
CK
NPK
M
MNPK
CK
NPK
M
MNPK
Loosely combined humus Stably combined humus

(g kg-1)
(g kg-1)
2010
2012
2013
2010
2012
2013
5.18
5.95
5.46
0.64
0.70
0.62
5.99
6.38
6.69
0.86
0.9
0.7
7.33
7.54
7.59
0.94
1.91
0.97
9.01
8.49
8.98
1.36
1.00
1.54
6.05
6.01
6.20
0.67
0.94
0.64
7.44
6.04
5.87
1.01
0.92
0.59
8.97
6.64
9.10
1.01
1.83
1.05
9.61
7.98
9.81
1.34
0.95
1.63
Loosely combined humus has the largest activity in combined

humus as fresh humus, thus it has significant contribution to soil
fertility 12, 16. The effect of soil relocation on loosely combined
humus was more than stable and tightly combined humus due to
that loosely combined humus was more active than stable and
tightly combined humus. The contents of soil nutrients and humus
were higher in 0 ~ 20 cm soil layer than in 20 - 40 cm soil layer
because soil fertility in 0 ~ 20 cm could be replenished by fertilizer
application 13. The content of loosely combined humus of NPK,
MNPK and M treatments was increased gradually for 0 ~ 20 cm
soil layer and showed decrease-increase trend for 20 ~ 40 cm soil
layer in 2012 and 2013.
The effect of soil relocation on the ratio of loosely and tightly
combined humus: Loosely combined humus was more active as
fresh humus; tightly humus was hard to be uptake because of
slow decomposition and low activity. So higher the ratio of loosely
and tightly combined humus was corresponding to higher soil
fertility 14. The highest ratio of loosely and tightly combined humus
was observed in MNPK treatment, there was no obvious change
of the ratio of loosely and tightly combined humus in 0 ~ 20 cm
soil layer among 2010, 2012 and 2013 (Fig. 1). The ratio of loosely
and tightly combined humus in 20 ~ 40 cm soil layer was decreased
53.1 ~ 66.1% in 2012, and then increased in 2013 compared with
that in 2010, but the ratio of loosely and tightly combined humus
in 20 - 40 cm soil layer was still lower in 2013 than 2010.
66
55
CK
CK
NPK
NPK
M
M
MNPK
MNPK
0-20 cm 0 - 20 cm
44
33
22
11
00
2010
2010
2012
2012
Experimental year
2013
2013
The
ratio of
combined
humus
he ratio
of loosely
looselytototightly
tightly
combined
hum
The ratio of loosely to tightly combined humus
Conclusions
The contents of soil organic carbon and heavy fraction of organic
carbon were in an increasing order of CK < NPK < M < MNPK in
all tested soils. The effect of soil relocation on soil organic carbon
and heavy fraction of organic carbon did not be found. The
quantity of organo-mineral complex showed an increasing order
Tightly combined humus

(g kg-1)
2010
2012
2013
6.29
4.75
6.18
5.64
5.31
5.28
4.40
3.32
4.31
3.29
3.52
2.94
4.86
4.50
5.49
2.69
4.75
4.32
1.95
4.26
2.90
2.29
4.05
1.92
of CK < NPK < M < MNPK in 2010 (before soil relocation), 2012 and
2013 (after soil relocation), the organo-mineral complex degree
showed an inverse trend. The organo-mineral complex degree
showed a decreasing order of CK > NPK > M > MNPK, indicating
that organic manure application could improve the status of soil
organo-mineral complex. The organo-mineral complex degree
increased gradually in experimental three years, indicating that
soil organic carbon aged with the increase of fertilization and
tillage periods. The quantity of additional organo-mineral complex
in MNPK was higher than in M, but the degree of additional
organo-mineral complex in MNPK was less than in M. The ratio of
loosely and tightly combined humus showed a higher range in 20
~ 40 cm soil layer than in 0 ~ 20 cm soil layer, and was decreased in
2012 and increased in 2013 compared with that in 2010. There was
no significant difference of the ratio of loosely and stable combined
humus, the higher change was observed in M treatment, which
needs more research in the future. Generally, the properties of soil
fluctuated after soil relocation, and showed that activity of soil
fertility supply was decreased. However, most properties did not
have significant change. Relocated soil would be suitable for new
environmental conditions with the increase of experimental years.
The properties of soil would be recovered to the status before soil
relocation, but this needs continually observation in the future.
Acknowledgements
This work was supported by National Natural Science Foundation
of China (41171244), and Key Program of the Heilongjiang Province
Natural Science Fund Project, China (ZD201113), the National
Science and Technology Support Program of China (2013BAD07B01),
the Special Fund for Agro-scientific Research in the Public Interest
of China (201303126), Soil restoration and high efficient utilization
science team of Northeast Agricultural University.
66
55
CK
C
K
20-40 cm20 - 40 cm
NPK
NPK
M
M
MNPK
MNPK
44
33
22
11
00
2010
2010
2012
2012
Experimental year
2013
2013
Figure 1. The effect of soil relocation on ratio of loosely to tightly combined humus.
437
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1
438
WFL Publisher
Helsinki, Finland
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Environmental guidelines of So Francisco Verdadeiro river according to

Brazilian standards
Kayla W. Garmus*, Silvio C. Sampaio, Maria Hermnia F. Tavares, Adir Otto Schmidt and Marcelo Remor
Research Group of Water Resources and Environmental Sanitation, Western Paran State University, Cascavel, Brazil.
e-mail: kaylawr@gmail.com, ssampaio@unioeste.br, mhstavar@gmail.com, adir.schmidt@unicoeste.br, remor_hotmail.com
Abstract
The So Francisco Verdadeiro River watershed reaches 11 cities in Western Paran. It is the largest tributary of Paran River and essential to the economy
and public supply of this region, where human activities such as agriculture, swine and poultry farming, industries and urban supply are held. The 9.433/
97 Legislation - National Water Resources Policy has foreseen the watershed as a management tool for water resources and its 5th article deals with water
bodies classification according to the predominant uses. Thus, this study aimed at classifying the So Francisco Verdadeiro River according to the limits
of 357/05 CONAMA Resolution, based on the following physicochemical parameters: turbidity, dissolved oxygen, pH, cadmium, lead, cobalt, copper,
chromium, manganese, nickel and zinc. Ten points were selected along the river, where fortnightly collections of water samples were taken over a year.
It was concluded that So Francisco Verdadeiro River has been undergone by several environmental impacts due to human activities, which have
contributed to water quality changes. According to 357/05 CONAMA Resolution, the studied river was classified as Class III.
Key words: Anthropogenic actions, 357/05 CONAMA, water quality.
Introduction
Brazil is worldwide known for its abundance in water; however, its
demand is increasing due to the development of industries,
agriculture and cities, possibly jeopardizing water quality. Many
studies have pointed out that there is a trend of water resources
to become increasingly scarce in both quantitative and qualitative
features 1-4. Therefore, urgent measures must be taken in order to
improve the water supply and demand management for different
uses. Thus, maintenance of water supply, not only in amount but
also in quality, has been the greatest challenge to be overcome by
society.
Water quality evaluation is an important tool to manage its
quality and resources, so, it must be included the trends monitoring
in space and time. This allows to identify some anthropogenic
and natural factors that determine surface waters quality 5-7. The
evaluation of water quality as well as its temporal and spatial
evolution will only be possible based on the implementation of
systematic monitoring via programs. This results in several
historical data that, in the future, can be analyzed to establish
standards for seasonal and spatial distribution 8.
Monitoring and evaluating of surface and groundwater quality
are major factors for the correct management of water resources.
They allow features and analyses of trends in river basins, essential
for several management activities, such as planning, granting,
levy taxing and classification of watercourses.
Developing countries have also experienced a growing problem
of water or soil pollution due to, for example, heavy metals toxicity
with industries discharges, agricultural waste and urban sewage 9.
In Brazil, the National Water Resources Policy, Law 9.433/97,
establishes as one of its warranties that water management should
always provide the multiple uses of water, are associated to its
quality. Waters with higher quality allow the existence of more

demanding uses, while waters with low quality allow only the
least demanding uses. Law 9.433/97, in its Article 5, foresees the
guidelines on water bodies in classes, according to the
predominant uses of water.
Resolution 357/05 CONAMA 10 - defines that the Class II
waters may be used to: a) supply human consumption, after its
conventional treatment; b) protect aquatic communities; c) primary
contact recreation such as swimming, water skiing and scuba
diving; d) irrigate vegetable crops, fruit and park trees, gardens,
sports and leisure fields, with which the public might have direct
contact; e) aquaculture and fishing activity.
The So Francisco Verdadeiro River, object of this study, is the
largest tributary of the Paran River and has essential importance
for the economy and public supply of the region, where activities
such as cropping, swine and poultry farming, industries and urban
supply are held. Therefore, despite the studied region does not
have problems with the water amount, there is some concern
regarding the water quality changes. The So Francisco Verdadeiro
River, due to its water regime, is classified according to 357/05
CONAMA Resolution as a Class II river. Thus, this study aimed
at carrying out the monitoring, according to the physicochemical
parameters determination as well as recording the environmental
impacts caused by human actions.
Experimental location: The So Francisco Verdadeiro basin is in
the third plateau of Paran, structured between the superior
Jurassic and the inferior Cretaceous periods (Fig. 1). It has a 2219.1
km2 drainage area and reaches 11 municipalities, with 46.9 m3 s-1
439
-5410
-5400
-5350
-5340
-5330
-5400
-5350
-5340
-5330
-2450
-2450
-2440
-2440
-5420
10
20 km
-5420
-2500
-2500
-5410
Figure 1. Locations of watershed, urban perimeters and collection sites in So Francisco Verdadeiro River and its
tributaries 12.
average flow, 1,800 mm yr-1 average rainfall as well as altitude

between 729 and 221 m. Its most important activity is agriculture,
with 69% occupancy in relation to the total area 11.
The response variables quantification was performed in 10
points, described on Table 1, selected along the So Francisco
Verdadeiro watershed. The selection of sampling points was carried
out aiming at showing the different anthropogenic activities in
adjacent areas.
Treatments and parameters: The collections of water samples
were carried out with a DH-48 sampler and a wading rod. The
samples were stored in 2.5 L amber glass containers, transported
in cold boxes at 4C to the laboratory. The fortnightly collections
were made from August, 1st 2012 to July, 17th 2013. The values of
temperature (T) and electrical conductivity (EC) were quantified
with the multiparameter probe Instrutherm PH1500/SC100. The
pH values were measured by multiparameter probe Instrutherm
PH1500/EPC70 (T). The dissolved oxygen (DO) values were
obtained with OM Instrutherm measurer and turbidity (Turb)
values were determined at the laboratory with Hach 2100P
turbidimeter.
The extraction of Cu, Cr, Mn, Ni and Zn followed the procedure

described by NBR 13809:1997 ABNT (Brazilian Technical
Standards Association). According to this standard, the samples
undergo acid digestion to be treated with nitric acid with pH below 2.
Thus, 250 mL of sample was transferred to a glass beaker and 5 mL of
concentrated nitric acid was added. The beakers were placed in a hot
plate at 90C (without boiling) for evaporation until the sample
volume was reduced to 15 mL. After cooling, 5 mL of concentrated
hydrochloric acid was added. The beakers were covered with a
watch glass and heated to keep a mild reflux on the beaker walls.
Then, for a complete digestion, more hydrochloric acid was added
to obtain a clear solution. After cooling, the solution was filtered
to separate insoluble compounds and avoid clogging the nebulizer.
The supernatant was transferred to a 50 ml volumetric flask and
its volume was completed with deionised water. Finally, the treated
samples were quantified by atomic absorption spectrometry.
The average values of the parameters are shown on Table 2 and
compared with 357/205 CONAMA Resolution for Class II rivers.
The Turb averages showed values that were below the maximum
Table 1. Location and characterization of the collection sites in So Francisco Verdadeiro River and its tributaries.
Location
River
S01
Mosaics Source
S02
S03
S04
S05
S06
S07
S08
S09
S10
440
So Francisco Verdadeiro
after urban perimeter
Lope (estuary)
So Francisco
Verdadeiro /Lope
Toledo (estuary)
So Francisco
Verdadeiro/Toledo
Santa Quitria (estuary)
So Francisco
Verdadeiro/Santa Quitria
So Francisco
Verdadeiro (estuary)
Marrecos (estuary)
Geographic Coordinates
S 24 57 W 53 28
S 24 55 W 53 30
S 24 47 W 53 43
S 24 47 W 53 43
S 24 45 W 53 47
S 24 45 W 53 47
S 24 46 W 54 04
S 24 46 W 54 04
S 24 44 W 54 07
S 24 41 W 54 09
Characteristics
One of several springs of San Francisco Verdadeiro River
within urban area of Cascavel city.
After urban perimeter of Cascavel and discharge of sewage
treatment plant.
Tributary area of agriculture and ponds for fish production.
Agricultural Region.
Farming region tributary that receives domestic and
industrial sewage from the urban perimeter of Toledo.
Agricultural region with influence of the urban perimeter of
Toledo.
Regional tributary of extensive farming.
Region with the highest slope of the land (construction of a
small dam plant) with agriculture and grasslands.
Next to the reservoir of Itaipu dam: agricultural and grazing
region with the steepest ground.
Next to the reservoir of Itaipu Dam: agricultural region, with
intensive swine and poultry production.
Table 2. Averaged values of hydric and physicochemical parameters.

Parameters
Turb.
DO
pH
Cr
Mn
Ni
Zn
(a)
CONAMA
357/05 class II
100 NTU(a)
5.0 mg/L(b)
6 a 9(c)
0.05 mg/L
0.1 mg/L
0.025 mg/L
0.18 mg/L
S01
S02
S03
S04
S05
S06
S07
S08
S09
S10
0.35
6.23
5.47
0.08
0.07
0.02
0.13
20.77
7.08
7.19
0.09
0.05
0.02
0.15
69.70
7.90
7.38
0.11
0.08
0.02
0.21
83.70
7.96
7.26
0.10
0.09
0.03
0.21
78.60
7.08
7.33
0.11
0.09
0.02
0.26
81.60
8.19
7.49
0.11
0.08
0,02
0.19
85.50
8.28
7.36
0.01
0.12
0.01
0.25
48.02
8.30
7.41
0.07
0.08
0.01
0.31
71.30
8.24
7.47
0.10
0.10
0.02
0.36
92.20
7.84
7.49
0.08
0.16
0.01
0.32
Maximum acceptable; (b) Minimum acceptable; (c) acceptable range.
limit, determined by 357/05 CONAMA Resolution - Class II rivers.

The DO averages at each collection site showed values above the
minimum limit already determined by 357/05 CONAMA Resolution
- Class II rivers, except for the S01 site. It is implied that S01 site
can be a natural source since it showed the lowest DO average. It
was recorded that DO was close to 4.0 mg L-1 throughout the year
on a spring 13. The pH averages at each collection site, except at
S01 site, showed values between the minimum and maximum limits
established by 357/0 CONAMA Resolution - class II rivers. S01
site can be a natural spring, since it showed the lowest average
for pH.
The averages concentration of Cr showed values above the
upper limit established by 357/05 CONAMA Resolution - class II
rivers. The average concentration of this element in natural waters
is 0.00043 mg L-1 14, which indicates anthropogenic influence that
has come from industries, chroming products, steel welding,
metalworking, leather tanning. The averages of Mn concentration
at S07, S09 and S10 collection sites presented values above the
maximum limit established by 357/05 CONAMA Resolution - Class
II rivers. It should be highlighted that livestock is the main activity
at S07 and S10 sites and both livestock and agriculture are
developed at S09. Manganese is combined with other elements to
produce batteries, oxidizing agents, pyrotechnics, catalysts,
disinfectants, deodorants, agricultural products and medicines
prescribed for human and animal 15.
The average concentration of Ni at S04 collection site showed
values above the upper limit determined by 357/05 CONAMA
Resolution - Class II rivers and it is important to inform that
agriculture is the main activity developed there. The use of
pesticides and the increasing number of industries have
contributed to Cr and Ni discharges at a very superior acceptable
level concerning the environment in recent years 16.
The averages concentration of Zn at the studied collection sites,
except for S01 and S02, showed higher values when compared to
those ones from 357/05 CONAMA - Class II rivers, because these
points are predominantly agricultural.
Conclusions
Despite the So Francisco Verdadeiro River has been commonly
classified due to its high values of Cr, the results are consistent
with data from rivers that matches class II.
The S07, S09 and S10 sites have registered Mn concentration
values above the limit already established by 3657/05CONAMA
Resolution - Class II rivers.
The S04 site showed Ni concentrations values above the limits
established by 3657/05CONAMA Resolution - class II rivers.
Except for S01 and S02 sites, all the other sites presented Zn
concentration values above the limit determined by 357/05
CONAMA Resolution for Class II rivers.

The So Francisco Verdadeiro River has been suffering
environmental impacts due to human activities and this has
contributed to water quality changes and, according to 357/05
CONAMA Resolution, it is classified as a class III river.
Acknowledgements
The authors express their gratitude to the Post Graduate
Agricultural Engineering Program of the Western Parana State
University, for the support.
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J. 2008. Trend study and assessment of surface water quality in the
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Valdman, E., Erijman, L., Pessoa, F. L. P. and Leite, S. G. F. 2001.
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sets forth other provisions. Union Official Journal, 18 March 2005.
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Schmidt, A. O. 2014. Impacts of the Soil Use and Occupation at the
Physical and Chemical Qualities of the Sao Francisco Verdadeiro
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442
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Helsinki, Finland
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Isolation and identification of microorganisms from soil in a young oil palm plantation
Nur Masirah Mohd Zain 1, Rosli B. Mohamad 1*, Kamaruzaman Sijam 2 and Yahya Awang 1
1
Department of Crop Science, 2 Department of Plant Protection, Faculty of Agriculture, University Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia. *e-mail: rosli@agri.upm.edu.my, yahya_awg@putra.upm.edu.my
Received 28 January 2014, accepted 20 September 2014.
Abstract
Microorganisms in soil could be beneficial to the ecosystem or pathogenic to crop plants. Their distribution depends on the environmental habitat
favoring each species. This study was intended to isolate and identify the types of microorganisms in soil habitat of a young oil palm plantation, and
associate them to their role as being beneficial or pathogenic to the ecosystem. The isolation process was done by soil sampling and soil dilution
technique. The bacterial population were identified through BIOLOG, whereas the fungal and actinomycetes were identified through conventional
methods of microscopic observations of the isolates. Five of the isolates were bacterial species, identified as Malikia spinosa, Bacillus humi, Bacillus
cereus/thuringiensis, Bacillus pseudomycoides and Bacillus sp. Other isolates were three fungi and two actinomycetes of Mucor sp., Penicillium sp.
and Aspergillus sp., and Streptomyces spp. isolate B1 and C2, respectively. None of the isolated species were reported to be pathogenic on oil palm,
but Aspergillus sp. could be pathogenic to other crop plants. Bacillus cereus/thuringiensis is commonly used commercially as biological control agent
mainly for Lepidopterous insects.
Key words: Soil bacteria, soil fungi, soil actinomycete, oil palm soil.
Introduction
Soil provides diverse habitats for a wide range of organisms. It
serves as a complex habitat of soil microorganisms, in which the
populations are crucial for sustainable agriculture 1. Soils are made
up of different components of solid fractions including sand, silt,
clay and organic matter, which provide a wide range of
microhabitats for various type of microorganisms 2, 3. Major groups
of microorganisms in soil include bacteria, fungi and
actinomycetes, and their population varies according to the soil
microhabitats.
Each fertile soil could contain high bacterial number, indicating
that the microorganisms can be an important indicator of soil
quality and ecologically important parameter 4-6 . Soil
microorganisms are also crucial in primary production through
decomposition of organic matter and nutrient cycling 7. Soil
microorganisms have essential roles in elemental cycles including
carbon, nitrogen and sulfur cycle. These organisms also transform
phosphorus, iron and potassium to more soluble form which assists
the uptake of the compounds by plants 8.
Despite of the importance of microorganisms in soil processes,
several soil-borne microbial species are known to cause plant
diseases. In oil palm plantation, at certain stages of the crop
growth, Ganoderma boninense is widely known to cause basal
stem rot of oil palm in Malaysia 9. Fusarium oxysporum f. sp.
elaeidis has been reported to cause vascular wilt in African oil
palm 10. Other pathogenic soil-borne microorganisms include
Colletotrichum spp. (antrachnose disease), Phytophthora spp.
(root rot disease) and Rhizoctonia solani (damping-off disease) 11.
This study was aimed to isolate and identify the microorganisms
present in soil of young oil palm plantation, and grouped them as
beneficial or pathogenic to the ecosystem.

Soil samples: Soils were randomly sampled using auger from 0-15
cm layer in young oil palm area at University Putra Malaysia (UPM),
Selangor, Malaysia. Soil composition was determined for organic
carbon, analyzed by the Walkley-Black procedure: total N was
extracted using micro-Kjeldahl method; total P was extracted using
6 M HCl; K, Mg and Ca were extracted by leaching the soil sample
with 1 M ammonium acetate solution (pH 7.0), and K was
determined using flame photometer, and Mg and Ca were
determined using atomic absorption spectrophotometer. The
composition was as follows: 1.94% C, 0.32% N; 219 ppm P, 104
ppm K, 119 ppm Ca and 32 ppm Mg, and were classified as sandy
clay (40% clay, 10% silt and 50% sand); pH 4.1 0.01.
Isolation of microbial colony: Microbial extracts were prepared
from the soil samples by serial dilution in sterile distilled water.
Ten g of soil was transferred into 90 mL sterile distilled water
followed by serial dilutions to 10-5. The dilutions were plated using
the spread plate technique on the specific culture media
accordingly: Potato Dextrose Agar (PDA) amended with 30 mg/L
streptomycin sulphate for fungi; Nutrient Agar (NA) amended
with 0.1 g/L cyclohexamide for bacteria; Actinomycete Isolation
Agar (AIA) amended with 0.5 g/L cyclohexamide for actinomycete.
Additions of streptomycin sulphate to PDA and cyclohexamide
to NA and AIA were, respectively, for the suppression of bacterial
and fungal growth. The plates were inverted and incubated at
25C in darkness. The plates were examined for microbial colonies
within 24 hours to 10 days after incubation. Visible microbial
colonies on the respective media were counted after 24 hours for
bacteria, 5 days for actinomycetes and 7 days for fungi. The
colonies were isolated by subculturing accordingly in the specific
443
media in order to obtain pure culture of single colony of the

microbial species. The pure cultures were maintained in the
respective media accordingly for identification purpose.
Identification of microbial isolates:
Bacteria: Bacterial identification was done using Biolog Gen III
Technology, which needs no Gram staining, no pre-tests and no
following tests. The technology is claimed to be much more simple
and user-friendly compared to the previous Gen II Technology 12.
The Gen III Microplate test panel involves a standardized
micromethod using 94 biochemical tests for profiling and
identification of both Gram-positive and Gram-negative bacteria.
This procedure requires sterile condition during bacterial
inoculation steps. Two to three single colonies of unknown bacteria
grown on NA for 24 hours were suspended in a special gelling
Inoculation Fluid A (IF-A) at the recommended cell density (9098%) using the Biolog turbidometer. After a slight upside down
conversion of the IF-A tube, the cell suspension was inoculated
into the Gen III Microplate at 100 L per well. The microplate
was then placed in the Omnilog incubator at 33C to allow the
phenotypic fingerprint to form. The Omnilog software identifies
the bacterium from the phenotypic fingerprint in the Gen III
Microplate by performing all reading and interpretation of
results. ID results (microbial species) were obtained within 24
hours. Gram staining was conducted later, prior to viewing under
light microscope for visual recording.
Actinomycetes: Classical approach was chosen in identifying the
actinomycetes 13, 14. The identification process was done
macroscopically and microscopically based on morphological
appearances. Macroscopic observations involved the examination
of colonial appearance grown on AIA and Blood Agar (BA).
Microscopic appearances were observed using light microscope
upon Gram staining and acid fast staining using modified Kinyoun
method. Biochemical tests were also conducted for confirmation
of genus. The genus of actinomycete was determined based on
the UK National Standard Method: Identification of Aerobic
Actinomycetes 14 and Bergeys Manual of Determinative
Bacteriology 13.
Fungi: The fungal identification involved morphological
examinations of the fungal mycelium (radial growth, colour, and
pigmentation) on PDA and Malt Extract Agar (MEA) 8, 15, 16. The
spores and other structures were examined under light microscope.
The genus was identified based on A Manual of Soil Fungi 16
and Compendium of Soil Fungi 15.
Results
Microbial counts of total population of bacteria, actinomycetes
and fungi showed that bacteria recorded the highest (3.9x106)
colony-forming unit per gram of dry weight soil (CFU/g of dry
weight soil) among the three microbial groups in the oil palm soil
(Table 1). The colony count was not significantly different to that
of the actinomycete population with a 3.8x106 CFU/g of dry weight
soil, but significantly higher than the fungal population count of
3.9x105 CFU/g of dry weight soil.
Ten isolates were obtained from the isolation process, which
consisted of three fungi, five bacteria, and two actinomycete
strains. Specific identification of the isolates revealed that the
444
Table 1. General count of microorganisms in soil of young

oil palm plantation.
Type of microorganisms
Bacteria
Actinomycetes
Fungi
LSD
CFU/g of dry weight soil (mean SEM)

3.9a 0.9 x106
3.8ab 1.4 x106
3.9b 0.7 x105
3.5 x106
Note: Values in the same column followed by a similar letter are not significantly different by
LSD (P<0.05).
fungal species were Mucor sp., Penicillium sp. and Aspergillus

sp.; and actinomycete species were Streptomyces sp. isolate B1
and Streptomyces sp. isolate C2, as listed in Table 2. Bacterial
species identified by BIOLOG (Table 3) were Malikia spinosa,
Bacillus humi, Bacillus cereus/thuringiensis, Bacillus
pseudomycoides, and Bacillus sp.
Table 2. Specific identification of fungal and
actinomycete isolates from oil palm soil.
Microbial group
Fungi
Actinomycete
Microbial isolate
Mucor sp.
Penicillium sp.
Aspergillus sp.
Streptomyces sp. isolate B1
Streptomyces sp. isolate C2
Table 3. BIOLOG identification of bacterial isolates from oil palm

soil.
Bacterial ID
Malikia spinosa
Bacillus humi
Bacillus cereus/thuringiensis
Bacillus pseudomycoides
Bacillus sp.
Probability Index (%)

96.8
64.5
86.1
76.2
-
Similarity Index
0.7
0.5
0.6
0.5
0.2
Mucor sp. (Fig. 1) was a fast growing organism, which was

observed to be cottony white turning grayish on PDA with radial
growth of 8 cm within 3 days. The sporangiophores could grow
up to more than 10 mm high and unbranched. The sporangia would
turn from yellowish to dark brown after several days with ellipsoidal
and smooth-walled spores.
The vegetative hyphae of Penicillium sp. were observed as
septate and branched (Fig. 2). The spores were formed in chains
from verticillate phialides, and the conidiophores branched to form
a brush-like head structure. The conidial areas appeared on PDA
were yellow-green and reverse side were yellow with some red
spots. The radial growth was between 2 and 3 cm within 7 days.
The vegetative mycelium of Aspergillus sp. consisted of
branching hyphae (Fig. 3). The conidiophores were nonseptate
and smooth with blackish brown conidial heads. The conidial
areas appeared on PDA were blackish brown, and reverse side
were yellowish white. The radial growth was between 2 and 5 cm
within 7 days.
Streptomyces spp. produced extensively branched vegetative
hyphae which did not fragment easily. Both isolates, B1 (Fig. 4)
and C2 (Fig. 5) grew well on AIA, and the colonies were small,
discrete, and creamy white, similar to the reverse-side. Later, aerial
mycelia developed which appeared powdery. On BA, the colonies
appeared glisten. The colonies adhered to both agar, and could
be hardly removed by loop. The isolates were not acid fast when
stained using modified Kinyoun method, catalase-positive and
reduced nitrate to nitrite. They also produced strong earthy smell.
Malikia spinosa was only Gram-negative among the bacterial
445
Figure 4. Macroscopic appearances of Streptomyces

sp. isolate B1 on AIA (a) and BA (b), and Streptomyces
sp. isolate B1 (c) under 1000x magnification.
Figure 1. Macroscopic appearances of Mucor sp. on PDA

(a and b), and Mucor sp. (c) under 1000x magnification.
Figure 5. Macroscopic appearances of Streptomyces sp.

isolate C2 on AIA (a), and BA (b), and Streptomyces sp.
isolate C2 (c) under 1000x magnification.
Figure 2. Macroscopic appearances of Penicillium sp. on PDA

(a and b) and Penicillium sp. (c) under 1000x magnification.
Figure 3. Macroscopic appearances of Aspergillus sp. on PDA

(a and b) and Aspergillus sp. (c) under 400x magnification.
isolates. The cells were straight rods with rounded ends which
occurred singly, in pairs or in short chains. The colonies on NA
appeared to be circular, raised, smooth, creamy white and
translucent. After few days, the colonies appeared mucoid.
Bacillus humi was observed to be a rod shaped Gram-positive
organism. The cells were slightly curved with rounded end,
occurred singly or in pairs. On NA, the colonies were convex and
appeared to be glossy and white with watery texture.
Bacillus cereus/thuringiensis appeared Gram-positive, and
when grown on NA, the colonies were white, large, raised and
dull surface, irregular margins and opaque. The cells were rod
shaped and occurred singly or in pairs.
Bacillus pseudomycoides was also observed as Gram-positive.
The appearance of colonies on NA was whitish cream and opaque.
Microscopic observation showed that the cells were rod shaped
and occurred in short chains.
Another species, only identified as Bacillus sp. appeared to be
convex, white and opaque on NA. The cells were stained Gram- p
ositive, and rod shaped.
Discussion
Fungi, bacteria and actinomycetes were recorded to be present in
soils from young oil palm plantation. Bacteria and actinomycetes
recorded significantly higher population counts compared with
that of the fungi. Bacteria, however, were observed to have slightly
higher CFU/g of dry weight soil than actinomycetes. Bacteria were
reported to replicate rapidly in favourable conditions 17. There
was indication in this study that actinomycetes had slower growth
rate compared with bacteria. This could be explained by the visible
and distinct colonies of actinomycetes that could only be seen
after 5 to 10 days, compared with bacterial colonies which normally
appeared after 12 to 24 hours. Fungi recorded the lowest CFU/g of
dry weight soil compared with bacteria and actinomycetes. Despite
of the low population count, the presence of fungi in the soil of
young oil palm plantation remains important as they can still
dominate the soil through their extensive mycelia and reproductive
spores, which ensures the survival of most fungal species 18.
The fungal species identified were all considered as common
soil fungi. Mucor sp. can be regarded as one of the commonest
soil fungi 15. Certain Mucor spp. are beneficial, capable of
producing large quantities of gamma-linolenic acid (GLA) which
is effective in curing certain human diseases 19.
Penicillium species occurred with particular frequency in soil
dilution plate technique; differences in soil washing number will
not alter the number of colonies 15. Penicillium sp. is a fungus of
major importance due to its ability to produce penicillin, used as
antibiotics to control human pathogens 20. Penicillium sp. and
Aspergillus sp. are strong phosphate solubilizing microorganisms
in soil, which convert phosphate for plant uptake 21. Aspergillus
sp., unlike Penicillium sp., is found predominantly in warmer
regions 15. The species are known to cause serious infections in
humans and animals by producing aflatoxin 22. In agriculture, certain
Aspergillus spp. were recorded as plant pathogens by producing
mycotoxins 23.
Streptomyces spp., which represented the actinomycete group,
were considered non-pathogenic. Streptomyces spp. are known
as antibiotic producers, particularly Streptomyces griseus, which
is widely recognized to produce streptomycin, essential antibiotic
in medicine 8. Streptomyces has been observed to produce
446
antibiotic depending on the growth phase 24. Candicidin (antifungal antibiotic) was produced after vegetative growth in liquidgrown culture of S. griseus, while Streptomyces antibioticus
grown on agar surface only produces oleandomycin at the end of
the vegetative growth state 25.
The BIOLOG Gen III Identification System was not able to
differentiate between B. cereus and B. thuringiensis. These species
are closely related, and can only be distinguished through
molecular methods such as polymerase chain reaction combined
with a restriction endonuclease 26. Many literatures have reported
B. cereus to be responsible of minor food-borne illnesses, causing
nausea, vomiting and diarrhea 27, 28. This opportunistic pathogen
has endospore which can survive and stimulate bacterial growth,
thus producing enterotoxin. This enterotoxin can survive high
temperatures and wide range of pH, which finally will cause
poisoning in humans 29. B. thuringiensis is also known to produce
toxins, which are termed Cry proteins which specifically targets
insects including Hymenoptera, Diptera, Lepidoptera and others30.
B. pseudomycoides was also reported to be closely related to B.
cereus, but can easily be separated through fatty acid composition 31.
It was even proposed as a new species, after previous studies
showed that this species was genetically distinct but
phenotypically similar to B. mycoides 31. Therefore, no previous
studies were found on the pathogenicity of this species. B.
mycoides, a non-pathogenic bacterium, which has distinctive
rhizoidal colony formations, was reported to induce systemic
resistance in sugar bit, thus acting as biological control agent 32, 33.
Little is known of B. humi and M. spinosa, as both species were
proposed in 2005, and therefore, no report was found on the
pathogenicity of both species. B. humi was first isolated in
agricultural research area in Netherlands 34, whereas, M. spinosa
has been misclassified as Pseudomonas spinosa and was proposed
to the new genus Malikia in 2005 35.
Conclusions
Fungi, bacteria and actinomycetes made up the major groups of
microorganisms present in soil of young oil palm plantation from
UPM. Ten microbial isolates were obtained from the soil, consisting
of three fungi, two actinomycetes and five bacteria. The fungal
isolates were identified as Mucor sp., Penicillium sp. and
Aspergillus sp. Two actinomycete isolates were identified as
Streptomyces spp. isolate B1 and C2. The bacterial isolates were
identified as M. spinosa, B. humi, B. cereus/thuringiensis, B.
pseudomycoides and Bacillus sp. No previous report was found
on the pathogenicity of all the identified isolates towards oil palm.
However, Aspergillus sp. could be pathogenic to other plant crops.
Acknowledgements
The authors wish to thank University Putra Malaysia for providing
the research fund under Research University Grant Scheme
(RUGS) vot. 91329.
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WFL Publisher
Helsinki, Finland
www.world-food.net
Monitoring larval populations of Aedes aegypti in different residential districts of

Jeddah governorate, Saudi Arabia
Khalid M. Al-Ghamdi 1, Abbas M. Al-Azab1, Hassan M. Khormi
*, Lalit Kumar 3 and Jazem A. Mahyoub
2, 4
Department of Biological Sciences - King Abdulaziz University, P.O. Box 80203, Jeddah, 21989, Saudi Arabia. Department of
Geography, Umm Al-Qura University, P.O. Box 715, Makkah, 21955, Saudi Arabia. 3 Ecosystem Management, University of New
England, Armidale NSW 2351, Australia. 4 KACST GIS Technology Innovation Centre, Umm Al-Qura University, P.O. Box 715,
Makkah, 21955, Saudi Arabia. *e-mail: hmkhormi@uqu.edu.sa
Abstract
House-to-house surveys of larval population of Aedes aegypti were conducted to determine the importance of house index for each habitat in Jeddah
governorate. In this study, we aimed to survey and monitor mosquito population and potential breeding sites by using House index (HI), Container
index (CI), and Breteau index (BI). The statistical analysis showed that the presence of larval stages of Ae. aegypti reported throughout the year inside
houses in the studied locations (Ghuleel, Al-Balad, Al-Jameiah, Al-Nazlah Al-Yamaneyyah, and Al-Safa) with some significant differences among
investigated areas showed that Ghuleel had highest and Al-Safa lowest in density of larvae, respectively. House indices of each study area compared
with the highest ratio of standard WHO (5-10%) were as follows: 8.7, 7.6, 6.6, 6.22 and 4%, respectively, for the above sites (P<0.05). The results
showed that there were significant differences among types of containers of water in the inspected houses. Large containers were most significant
compared with medium and small containers. Container index (CI) was 12% (Ghuleel), 13% (Al-Balad) and 14% (Al-Jameiah), 12% (Al-Nazlah AlYamaneyyah) and 9% (Al-Safa), whereas Breteau index (BI) was 8, 6.6, 4.7, 4.5 and 1.43%, respectively. Significant increase in the density of larvae
was found in November, March, June and January due to the effect of the environmental factors including temperature and humidity.
Key words: Dengue fever, populations, monitoring, Aedes aegypti, residential areas, house index.
Introduction
Mosquitoes are considered Public Enemy Number One for
humans because of their biting and blood feeding habits and their
role as vectors of dengue fever 1. Dengue is the most common
arthropod-borne viral (Arboviral) illness in humans. Globally, 2.5 3 billion individuals, in more than one hundred countries, have
experienced dengue transmission. Approximately 50-100 million
individuals are infected annually with 30,000 deaths 2-5.
Dengue fever outbreaks can cause high morbidity and mortality
in a short span of time, and may create panic among the people,
who expect urgent action from the government. Distribution of
mosquitoes (Diptera: Culicidae) in Saudi Arabia has been
investigated by many researchers. Mattingly and Knight 6 studied
the distribution of mosquito larvae in the Arabian Peninsula and
recorded 46 species and subspecies. The commonly-used larval
indices (house, container and Breteau) are useful for determining
general distribution, seasonal abundance and principal larval
habitats, as well as for evaluating environmental sanitation
programmes 7. Ae. aegypti, widely distributed in tropical and
subtropical regions, is highly adapted to the urban environment
and beyond the Arctic Circle. Ae. aegypti plays an important role
in the transmission of arboviruses such as dengue and urban
yellow fever 8.
One of the fundamental requirements in the epidemiology and
control of vector-borne diseases is information on the biology
and ecology of the vectors 9. Understanding the spatiotemporal
distribution of risk for mosquito-borne infections is an important
step in planning and implementing effective control measures.
448
Numerous approaches have been adopted during dengue

surveillance and dengue case investigation studies including the
use of traditional mosquito indices that take into account the
density of Ae. aegypti eggs 10, larvae 11, 12, and pupae 13, 14.
Environmental heterogeneity has been recognized as an important
factor affecting the abundance and quality of mosquito larval
habitats and hence the production of adult mosquitoes 15.
Several indices have been described and used to monitor Ae.
aegypti populations for dengue virus transmission. Those related
to immature populations include the HI, i.e. the percentage of
houses infested with larvae or pupae; the CI, the percentage of
water-holding containers infested with larvae or pupae, and the
BI, the number of positive containers per 100 houses inspected.
Using the HI or the BI, the definition of a house should be one
unit of accommodation and the surrounding premises, irrespective
of the number of people residing therein. The basic sampling unit
is the house or premise, which is searched for water-holding
containers 16. The surveillance of Ae. aegypti is important in
determining the distribution, population density, major larval
habitats, spatial and temporal risk factors related to dengue
transmission, and levels of insecticide susceptibility or resistance.
HI has been most widely used for monitoring infestation levels,
but it does not take into account the number of positive containers
or the productivity of those containers 1. In this study, we aimed
to survey and monitor mosquito population and potential breeding
sites by using house index, container and Breteau index of larvae.

Study sites: HI, CI and BI surveillance was studied in Jeddah
governorate that is located in 2132 '36" N latitude and 3910 '22"
E longitude on the west of the Kingdom of Saudi Arabia (Fig. 1)
in five residential districts. These districts were Ghuleel, AlSafa, Al-Jameiah, Al-Balad and Al-Nazlaah Al-Yamaneyyah.
They were selected according to the frequent reports of density
of mosquitoes in the studied localities (Fig. 2).
40E
45E
50E
55E
60E
30N
30N
33N
35E
im
lQ
A
35E
600 KM
40E
21N
Ash Sharqiyah
18N
`Asir
300
Ar Riyad
Najran
45E
50E
55E
15N
JEDDAH Makkah
24N
ua
ss
27N
24N
Al
Madinah
21N
18N
Hail
27N
Al Jawf
Tabuk
Figure 1. The study area location in the west of Saudi Arabia.
Mosquito collection and larvae identification: Larvae were

collected monthly from different areas of Jeddah from January
to December 2010. A similar number of houses (100 houses)
from around 20 locations or areas in five residential districts
were surveyed for the presence or absence of Aedes larvae
(Fig. 2). Moreover, survey of water-holding containers for
mosquito larvae was undertaken in each of the five regions. All
potential domestic breeding sites associated with that
household were inspected. Potential breeding sites were
classified into three of container types according to the volume
of water held, large, medium and small containers held >200 L,
50-200 L and <50 L of water, respectively. Containers with
immature mosquitoes were recorded, and samples were collected
for identification of the species. Larval and adult mosquitoes were
identified using the taxonomic keys of Rueda 17 and Huang 18 and
followed the Walter Read Biosystematics Unit (WRBU) 19.
Survey was conducted by searching for containers containing
water and recording their types and site locations. Larval habitats
were first identified as indoor and examined for all accessible water
containers inside the house. The collected immature stages were
brought to the laboratory and numbers of larvae were recorded
individually for each positive site. Larvae were subsequently
counted and identified at the late 3rd instars. Fresh water was
added into the container and the larvae mosquito were reared in
the plastic trays and provided with a ground fish food. Sugar
solution (10%) was provided to the newly emerging adults. The
houses, which were confirmed or suspected, were used as a house
index for this study as described by Chadee 20. The indices were
calculated using the followed equation:
House index (HI) =
Number of infested houses

100
Number of inspected houses
(1)
Container index (CI): percentage of water-holding containers

infested with larvae or pupae per all houses.
Figure 2. The study sites and Ae. aegypti larval collection locations
in five residential districts of Jeddah governorate.
CI =
Number of positive containers

100
Number of inspected containers
(2)
Breteau index (BI): number of positive containers per 100 houses

inspected.
BI =
Number of positive containers

100
Number of inspected containers
(3)
Statistical analysis: This study was conducted in completely

randomized design (CRD) in a factorial experiment. The collected
data were statistically analysed using analysis of variance (ANOVA)
and means were compared by LSD at P 0.05 according to ElNakhlawy 21 and SAS software program SAS Institute 22 version 9.3.
A total of 2000 houses were inspected for Ae. aegypti breeding in
five residential districts. Fig. 3 shows continued presence of larvae
over the study period. Also we have noticed differences on the
infestation degree between the areas under investigation as
shown in Fig. 4 and Table 1. Significant increase in the density of
larvae was observed in March, November, June and January,
respectively, in Figs 3 and 5.
449
studied locations were 8.7, 7.6, 6.6, 6.2 and 4% of Ghuleel, AlBalad, Al-Jameiah, Al-Nazlah Al-Yamaneyyah and Al-Safa,
respectively, while the values of container indices (CI) were 12,
13, 14, 12 and 9%, respectively, whereas (BI) were 8, 6.6, 4.7, 4.5
and 1.43%, respectively. These results, in some studied locations
(Al-Jameiah, Al-Nazlah Al-Yamaneyyah and Al-Safa), are in
agreement with Al Thabiany et al. 23, who studied the abundance
of Ae. aegypti in container breeding mosquitoes indoors and
nearby homes in Makkah City. They reported that the values of
HI, CI and BI indices during dry and wet season in Makkah City
were 4.44/12.73, 2.5/6.13 and 1.66/4.83, respectively.
The results as shown in Table 2 and Fig. 4 revealed that there is
significant difference among locations with mean values are 0.91,
0.86, 0.81, 0.77 and 0.65 of Ghuleel, Al-Balad, Al- Nazlah AlYamaneyyah, Al-Jameiah and Al Safa, respectively.
1.2
0.8
0.6
0.4
0.2
0.0
Jan
Feb Mar Apr May June Jul

Month
Aug Sept
Oct Nov Dec
Figure 3. Degree of infestation throughout the study period.
No. of infested houses (log)
1
0.9
Table 2. Mean infested houses in five residential

locations in Jeddah.
0.8
0.7
Location
Guleel
Al-Balad
Al-Jameiah
Al-Nuzlaah Al-Yamaniah
Al-Safa
0.6
0.5
0.4
0.3
0.2
Guleel
Al-Balad
Al-Jameiah
Al-Nuzlaah
Al-Yamaniah
The containers of water in the surveyed houses showed

significant differences between the large containers as compared
to medium and small containers. Significant differences between
containers of water in the surveyed houses showed that the large
containers were the most significant compared to medium and
small containers (Table 3). This could be interpreted by the
shortage of water abundance of these sites and the use of
containers as an alternative source of regular water. Water in these
containers were consumed in different periods according to their
size. The medium containers may be consumed during short
periods. Therefore, the life cycle of mosquitoes will not be
completed (Figs 5 and 6).
Al-Safa
Locations
Figure 4. Infested houses in all studied locations.
Table 1. Larval indices of Ae. aegypti mosquito in

the selected areas of Jeddah.
Location
Guleel
Al-Balad
Al-Jameiah
Al-Nuzlaah Al-Yamaniah
Al-Safa
HI (%)
8.70
7.60
6.60
6.22
4.00
CI (%)
12.00
13.00
14.00
12.00
9.00
BI (%)
8.00
6.60
4.70
4.50
1.43
House index (HI): Percentage of positive houses presence of Ae. aegypti larvae.
Container index (CI): Percentage of positive containers per 100 houses. Breteau
index (BI): Number of positive containers of Ae. aegypti larvae per 100 houses.
Table 3. Mean of infested containers at

different sizes.
Total infested containers (log)
Size of containers
Large containers
Small containers
Medium containers
Means of infested containers

0.86168 a*
0.43915 b
0.27573 c
* Means followed by the same letter are not significant different according
to LSD at (0.05).
1
0.9
Month
Figure 5. Relationship between infested containers and months.
Based on the density of larvae, the order of the infested areas

were Ghuleel, Al-Balad, Al-Jameiah, Al- Nazlah Al-Yamaneyyah,
and Al-Safa, respectively. Ghuleel was the highest in the density,
while Al- Safa location had the lowest density in larval abundance
by comparing house indices in the studied areas with the highest
ratio of standard WHO (HI) 5-10% as shown in Table 1 and Fig. 4.
Table 1 shows that demonstrated the values of (HI) of five
450
Means of infested house

0.91759 a*
0.86315 b
0.81964 c b
0.77178 c
0.65572 d
* Means followed by the same letter are not significant different according
to LSD at (0.05).
0.1
0
Infested containers (log)
No. of infested houses (log)
1.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Large containers
Small containers
Medium containers
Size of container
Figure 6. Mean infested containers based on their size.
Total infested containers
2.5
1.5
0.5
Guleel
Al-Balad
Al-Jameiah
Al-Yamaniah
Al-Safa
Location
Figure 7. Total infested containers in the five residential locations.
Entomological indices, BI in particular, allows the identification

of geographic units at high risk for dengue transmission. The
results showed the presence of high risk in some study areas
such as Ghuleel and Al-Balad compared with the described 3 levels
of risk for dengue transmission as follow: low (HI<0.1%), medium
(HI 0.1%-5%) and high (HI>5%). Dengue outbreaks occurred in
Singapore when the overall national HI was <1% 11, while in
Salvador, Brazil sentinel surveillance in 30 areas detected a
significant of 1.4 higher when the HI was >3% 24.
Fig. 8 shows a decrease on larvae abundance from March to
October correlated to an increase on average temperature for the
same period. Also it shows that abundance of larvae has a
significant correlation with average temperature comparing to
other climatic factors.
70
Climatic factors
60
HI mean
50
40
Mean temp.
(C)
30
Mean RH
(%)
20
10
Rain total
(mm)
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Month
Figure 8. Relationship between house index of Ae. aegypti larvae and

climatic factors.
Fig. 8 shows that the effect of climatic factors on larval

abundance of Ae. aegypti is consistent with Tun-Lin et al. 25 and
Espinoza et al. 26, who reported that the density of Aedes larvae
are influenced by many factors, such as mosquito-human
interaction and climate, which have a major effect on mosquito
biology.
Furthermore, warmer temperatures may allow arthropod vectors to
survive and reach maturity much faster than at lower temperature 27.
It may reduce the size of mosquito larvae resulting in smaller adults
that have a high metabolism rates and need to lay eggs more often 28,
but the effect of temperature on mosquito survival and abundance is
difficult to consider as a constant factor according to Reiter 29 and
Al-Ghamdi et al. 30, because temperature at daytime is usually
higher than at nights 31. Rainfall is one of the important elements

for the breeding and development of mosquitoes and significantly
influences them 32. Many studies have shown that rain plays an
important role in dengue epidemiology 33. In Jeddah, mosquito
population increases after rain, which creates suitable habitats 30.
It may prove beneficial to mosquito breeding if moderate, but it
may destroy or wash out existing breeding sites and interrupt the
development of mosquito eggs when the rain is excessive 34.
Increasing rain may increase suitable larval habitats and vector
population size by creating new breeding sites or increase adult
survival 33. The results of this study are in agreement with those
observations, because water not only provides the medium for
the aquatic stages of the mosquitos life cycle but also increases
the relative humidity and hence longevity of adult mosquitoes.
Our data shows that the indices dont correlate with the mean of
rain, but with the increase in rain in the prior 1-2 months (Fig. 8).
Conclusions
We conclude that the indices for larval habitats (HI, CI and BI) are
good indicators of heavy infestation of houses, which is necessary
for controlling the dengue fever vector. This conclusion is in
agreement with previous study of Tun-Lin et al. 35 and Mazine et
al. 36, where they suggested that routine larval controls should
not be uniformly applied in the whole environment. Particular
attention should be given to specific containers, which represent
the most productive breeding sites, either because they are more
likely than others to contain immature Ae. aegypti (containers
with a high risk factor), or because they are very common though
having a moderate risk factor.
The results showed the presence of Ae. aegypti larvae
throughout the year with significant increase in population density
during March, January, April, November and June, respectively.
There is a relationship between temperature and the abundance
of dengue fever vector density. The spread of Ae.aegypti in the
studied areas relates to human behaviours especially when the
containers are left uncovered an important reason for a breeding
habitat, due to a non-continuous water supply. Therefore,
awareness in water storage practices and removal of stagnant water
could reduce the population density of larval habitats 37. This
suggestion is also in agreement with Chen et al. 38, who reported
that water-holding containers produced by humans are very
important larval habitats for Aedes mosquito. The installation of a
water supply system in residential areas may lead to a reduction
in number of water holding containers, which are used as an
alternative due to the lack of continuous water supply. Hence,
this vector surveillance should be conducted continuously for
evaluation of the progress of the control programs and further
pertinent planning
Acknowledgements
We deeply thankful King Abdulaziz City for Science and
Technology (KACST) for financial support under grant no. A-S10-0013 & GISTIC-13-04. We gratefully acknowledge the house to
house control program teams in the Municipality of Jeddah for
helping us during this study. Special thanks to the Ministry of
Health for providing data of dengue infested areas in Jeddah.
451
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1
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