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© by PSP Volume 25 ± No. 4/2016 Fresenius Environmental Bulletin
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© by PSP Volume 25 ± No. 4/2016, pages 957-958 Fresenius Environmental Bulletin
CONTENTS
ORIGINAL PAPERS
REDUCTION OF Cr(VI) TO Cr(III): A DETOXIFICATION MECHANISM IN HYPERACCUMULATOR LEERSIA 959
HEXANDRA SWARTZ
Shimin Chen, Xuehong Zhang, Jie Liu, Shaohong You, Hui Zhang and Hua Lin
MODELING NUTRIENT AND HEAVY METAL TRANSPORT AT SELECTED CATCHMENTS IN THE MARMARA 969
REGION
=H\QHS$NGR÷DQ%DúDN*YHQ,úÕO%DOFÕR÷OX
ENHANCED HEAVY METAL IMMOBILIZATION IN POULTRY LITTER USING SODIUM DIETHYL 981
DITHIOCARBAMATE
Wen-gang Liu, De-zhou Wei, Hui-liang Guo, Bao-yu Cui and Yan-bai Shen
SYNTHESIS, CHARACTERIZATION, ELECTROKINETIC AND ADSORPTION PROPERTIES OF AN ORGANO- 991

MODIFIED ADSORBENT FOR SELECTIVE REMOVING OF METAL IONS
3ÕQDU7XUDQ%H\OL0HKPHW'RJDQ0DKLU$ONDQ$\GÕQ7UN\ÕOPD]<DVHPLQ7XUKDQg]NDQ
Demirbas, Hilmi Namli
SETTLEMENT OF BLUE CRAB (Callinectes sapidus) MEGALOPAE IN THE NORTH SHORE OF YUMURTALIK COVE 1007
(TURKEY)
Irem Nur Yesilyurt and Canan Tureli
CONTAMINATION STATUS OF ERZENI RIVER, ALBANIA DUE TO HEAVY METALS SPATIAL AND TEMPORAL 1019
DISTRIBUTION
Alma Shehu, Majlinda Vasjari, Edlira Baraj, Rajmonda Lilo, Roza Allabashi
ASSESSING CONTAMINATION IN STURGEONS GROWN IN RECIRCULATING AQUACULTURE SYSTEM BY LUX- 1028

BIOSENSORS AND METAL ACCUMULATION
Adem Yavuz Sönmez, Marina Sazykina, Soner Bilen, Nejdet Gültepe, Ivan Sazykin, Ludmila E.
Khmelevtsova, Natalia V. Kostina
THE EFFECT OF SOILS HAVING DIFFERENT SALT CONTENT ON MINERAL ACCUMULATIONS OF SOME 1038
FORAGE LEGUME SPECIES
Süleyman Temel, Bilal Keskin8÷XUùLPúHNøEUDKLP+DNNÕ<ÕOPD]
EFFECT OF THE IRON CONCENTRATION ON THE PROPERTIES OF AN ALUMINOSILICATE GLASS USED FOR 1051
STORAGE OF RADIOACTIVE WASTE
D. Moudir, S. Ikhaddalene, N. Kamel, A. Benmounah, F. Zibouche, Y. Mouheb, F. Aouchiche
ROLE OF SOME ANTIOXIDANTS IN SUPPRESSING OF FLAX POWDERY MILDEW 1059

Abd El-Rahim M.A. El-Samawaty, Mohamed A. Yassin, Mohamed A. Moslem and Moawad R.
Omar
ELEMENTAL COMPOSITIONS OF SOME COSMETIC PRODUCTS MARKETED IN TURKEY 1068

g6|÷W+5H\KDQOÕR÷OX0(]HU+%DOWDú
SUSTAINABLE SECOND-CROP SILAGE CORN PRODUCTION UNDER CENTRAL ANATOLIAN HIGHLANDS 1078
Taner Akar, Mahmut Kaplan, Seyma Havva Yilmaz, Nami Sagir, Nedim Tabak, Ahmet Gelebur
EVALUATION OF HEAVY METALS IN POTENTIAL FODDERS AND FORAGES GROWING IN KHUSHAB, 1089
PAKISTAN
Zafar Iqbal Khan, Kafeel Ahmad, Hafiz Abdalla Muhammad Ali, Abrar Hussain, Humayun
Bashir, Muhammad Sohail, Ameer Khan, Asma Zafar, Zahara Bibi, Zill-e-Huma, Muhammad
Fahad Ullah, Saif Ur Rehman, Nudrat Aisha Akram, Muhammad Sher, Ijaz Rasool Noorka,
Fahim Ashrad, Aima Iram Batool and Irfan Mustafa
ANAEROBIC COMPOSTING OF ORGANIC FRACTION OF MUNICIPAL SOLID WASTE 1097
<DVHPLQdDOÕúNDQ1LKDO%HNWDú0HVXW7HNEDú
957
COMPARISON OF SELECTED METHODS FOR PREDICTION OF LOW-FLOW IN YILDIZ RIVER BASIN, TURKEY 1105
2VPDQ(PUH<ÕOGÕ]
EVALUATION OF POTASSIUM AND SODIUM SILICATES AGAINST FUSARIUM SPP. CAUSING DAMPING-OFF 1117
DISEASE OF COTTON SEEDLING
Mohamed A. Yassin, Abd El-Rahim M. A. El-Samawaty, Mohamed A. Moslem and Shaban R.
M. Sayed
INFLUENCE OF LEAD STRESS ON GROWTH, ANTIOXIDATIVE ENZYME ACTIVITIES AND ION CHANGE IN 1125
ROOT AND LEAF OF STRAWBERRY
)HUKDG0XUDGR÷OX, 7DUÕN(QFX0XWWDOLS*QGR÷GX, Sibel Boysan Canal
THE EFFECTS OF HEAVY METALS ON THE SEED GERMINATION AND SEEDLING GROWTH OF TWO ENDEMIC 1134
VERBASCUM SPECIES
Gürcan Güleryüz6HUDS.ÕUPÕ]Õ+O\D$UVODQ6HYJL'HU\D
EVALUATION OF COMMON BEAN (PHASEOLUS VULGARIS L.) COLLECTION FOR AGROMORPHOLOGICAL 1143
AND SEED MINERAL CONCENTRATIONS
Faruk Toklu, Clarice J. Coyne, Safiye Asikli2÷X]KDQ$\GLQ, Tolga Karaköy, Hakan Özkan
HAZARDOUS WASTE CHEMICAL STABILIZED BY TRICALCIUM SILICATE: KINETIC AND ISOTHERM STUDIES 1153
Ionela Carazeanu Popovici, Alina Ioana Badanoiu, Georgeta Voicu and Ichinur Omer
CASPASE-3 ACTIVATION IN CYTOTOXICITY OF ISOLATED RAINBOW TROUT (ONCORHYNCUS MYKISS) 1167

HEPATOCYTES INDUCED BY BISPHENOL A
Burak .DSWDQHUDQG(UWX÷UXO.DQND\D
SEASONAL CHARACTERISATION OF THE LOCAL SEWAGE SLUDGE 1175

Fouzia Benoudjit, Mohamed Amine Benoudjit, Messaoud Hachemi
SEASONAL-VARIED CHEMICAL CHARACTERISTICS AND POLLUTION SOURCES OF PM2.5 IN HARBIN, CHINA 1183
Likun Huang, Guangzhi Wang, Deying Mu, Zhi Xia, Wei Wang
DELAYED EFFECTS OF CAPTOPRIL ON THE MEDITERRANEAN FLOUR MOTH: REPRODUCTIVE EVENTS, 1199
BIOCHEMICAL COMPOSITION AND MOLTING HORMONE CONTENTS OF OVARIES
Samira Yezli-Touiker, Nadia Soltani-Mazouni, Leïla Kirane-Amrani, Noureddine Soltani
EFFECT OF PRE-OZONATION ON ANAEROBIC TREATABILITY OF MEMBRANE CONCENTRATE 1206

Mehmet Cakmakci, F. Busra Yaman, Kaan Yetilmezsoy, Dogan Karadag, Bestamin Ozkaya,
Hande Turkay, Tuba Genc
INTEGRATED HEALTH ASSESSMENT OF A MULTI-TYPE RIVER TO OPTIMIZE REMEDIAL WORKS IN THE 1221
DIANCHI LAKE BASIN
Zhu-jing Jin, Jin-hua Li, Feng-le Yang, Ke Huang, Bao-xue Zhou
ECO-TOXIC EFFICACY OF NANO-SIZED MAGNESIUM OXIDE IN FRESHWATER SNAIL RADIX LEUTEOLA L 1234
Daoud Ali, Huma Ali, Saud Alarifi, Agha P Masih, S. Manohardas, S. A. Hussain
FACTORS AFFECTING TH( 678'(176¶ (19,5210ENTAL AWARENESS, ATTITUDES AND BEHAVIORS IN 1243
ONDOKUZ MAYIS UNIVERSITY, TURKEY
Mehmet Bozoglu, Abdulbaki Bilgic, Bakiye Kilic Topuz, Yuksel Ardali
EVALUATION OF SPAD CHLOROPHYLL IN SPRING WHEAT GENOTYPES UNDER DIFFERENT ENVIRONMENTS 1258
&HODOHGGLQ%DUXWoXODU0HKPHW<ÕOGÕUÕP0XMGH.Ro&XPD$NÕQFÕ,UHP7RSWDú2QGHU
$OED\UDN$EGXONDGLU7DQUÕNXOX, Ayman El Sabagh
BI2S3 FLOWER-LIKE NANOSTRUCTURES COMPOSED OF NANOBELTS: SYNTHESIS, GROWTH PROCESS AND 1267
THERMOELECTRIC PROPERTIES
Kuili Liu, Jinghua He, Wei Gu, Lisong Xiao
958
REDUCTION OF Cr(VI) TO Cr(III): A DETOXIFICATION

MECHANISM IN HYPERACCUMULATOR LEERSIA
HEXANDRA SWARTZ
Shimin Chen1, Xuehong Zhang1,2, Jie Liu2, Shaohong You2, Hui Zhang2 and Hua Lin2
1
Institute of Light Industry and Food Engineering, Guangxi University, Nanning, P. R. China
2
Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guilin University of Technology, Guilin, P. R.
China
ABSTRACT metal phytoextraction is only a fraction of that

associated with conventional engineering
The mechanisms underlying the detoxification of technologies [1, 2].
Cr(VI) by Leersia hexandra Swartz, a Cr- L. hexandra has great potential for use in the
hyperaccumulator, remain unclear. To determine phytoremediation of Cr-contaminated soil and
whether L. hexandra was able to detoxify Cr(VI) by water. This species has high tolerance of Cr(III) and
reduction, we studied the oxidation state and Cr(VI) and can accumulate high concentrations of
possible complexation mode of Cr in plant tissues these contaminants. L. hexandra shows good
using non-destructive techniques, namely, XANES adaptability, wide distribution, and rapid growth
and EPR spectroscopy. In L. hexandra, treated with and can easily be grown by artificial propagation
Cr(VI) solution, XANES and EPR indicated that [3]. Thus, L. hexandra is an ideal experimental
highly toxic Cr(VI) was reduced to the intermediate material for studying the mechanisms underlying
Cr(V) and accumulated as less toxic Cr(III), and detoxification of Cr in a Cr hyperaccumulator. We
reduction of Cr(VI) mainly depended on the aimed to determine the potential applications of L.
chemical reduction of Fe(III) and the complexation hexandra in the phytoremediation of Cr-
of organic acid. We also aimed to analyze the contaminated water and soil.
possible complexation mode of Cr in Cr(III)- and Cr is not an essential element, but rather a
Cr(VI)-treated L. hexandra. In roots, Cr was metal of high toxicity for plants [4]. Accumulation
accumulated in the form of Cr(OH)3. A portion of of high Cr concentrations in plants inhibit various
Cr in roots was possibly bound to organic acid activities, such as seed germination and seedling
ligands, which promoted the translocation of Cr. growth, and may seriously damage nutrients and
However, The Cr in leaves was was primarily water balance, interfere with synthesis of pigment,
Cr(III)-trioxalate, with small amounts of Cr(III)- and decrease antioxidant enzymes activities [5-8].
dioxalate and Cr(III)-citrate㸪 and a small amount Plants have mechanisms of Cr tolerance and
of Cr(OH)3 were also found. Therefore, oxalate was detoxification, which eliminate the damage from Cr
the final receptor of Cr in the vacuole. under normal conditions; such mechanisms include
the distribution of metals to apoplast tissues (i.e.,
KEYWORDS: trichome and cell walls), the variation of valence of
Leersia hexandra Swartz, Chromium, Hyperaccumulator, metals, the chelation of the metals by a ligand, and
XANES, EPR the sequestration of metals in vacuoles [9-11].
However, the mechanism underlying Cr
detoxification in a hyperaccumulator is still unclear.
INTRODUCTION The mechanism of Cr detoxification needs to be
studied to improve the Cr tolerance of
Unregulated application of Cr has led to hyperaccumulators and the efficiency of
serious environmental pollution and has caused phytoremediation of Cr-contaminated water and
great harm to human health. An efficient, cost- soil. We previously reported the subcellular
effective, and environmentally sound method for distribution of chromium in L. hexandra [12]. Cr
removing Cr from contaminated sites is required. mainly existed as oxalic-integrated Cr in the leaves
Such methods can be used in the remediation of of L. hexandra [13]. In the current work, we
environments contaminated with low levels of determined the oxidation state and the possible
Cr(VI). Phytoremediation is a cost-effective, complexation mode of Cr in intact plant tissues by
environment-friendly, and sustainable method for using nondestructive techniques, such as XANES
removing toxic chemicals from the environment. and EPR spectroscopy. We also explored the
Previous studies have demonstrated that the cost of
959
mechanism of Cr detoxification in a with ultrapure water for three replicates. The roots
hyperaccumulator (L. hexandra). and leaves of the washed plants were separated.
Roots and leaves were initially dried at 105 °C for
30 min and then at 70 °C for 48 h until constant
MATERIALS AND METHODS weight was achieved. The biomass (dry weight,
DW) was determined. Furthermore, the dried plant
Plants Culture and Cr Treatment. We used tissues were ground with an agate mortar and
a method that was described in our previous work passed through a 40-mesh screen. The triturated
[3] with little modification. Seedlings of L. plant tissues (approximately 0.5 g) were digested
hexandra were collected from an unpolluted region with a mixture of HNO3 and HClO4 (5:3, v/v) and
of Taohua River in Guilin, China. From this parent heated in an oven. After cooling, the extracts were
population, replicate daughter plants were grown diluted to 50 mL 0.2% HNO3. Extract Cr
under greenhouse conditions (12 h photoperiod, concentration was determined by AAS.
25 °C day/20 °C night and relative humidity 55% to
65%) in half-strength Hoagland nutrient solution. XANES Analyses. The XANES experiments
Antimicrobial agents, rifampicin (20 ȝg mL-1) and were performed at Shanghai Synchrotron Radiation
amphotericin B (5 ȝg mL-1) (Sigma Chemical Facility (SSRF), Shanghai Institute of Applied
Company), were added to nutrient solutions as a Physics, Shanghai, on BL14W. The source of
pretreatment prior to each Cr(VI) treatment [14]. BL14W is a 38-pole wiggler device with a
The antibiotic treatments eliminated possible maximum magnetic field of 1.2T and magnetic
interference caused by microbial reduction of period of 80 mm. The main optical components
Cr(VI) in the nutrient solution or on root surfaces. included the following: a collimation mirror; a
After 10 d, young plants with good and liquid nitrogen-cooled double crystal
uniform growth were selected. Cr treatments monochromator (DCM) with two sets of Si crystals,
included 0.4 and 1.0 mmol L-1 Cr(III) solutions (as namely , Si(111) and Si(311); a focusing mirror;
CrCl3) and 0.05 and 0.6 mmol L-1 Cr(VI) solutions and a harmonic rejection mirror. Our study used
(as K2Cr2O7). The treatments were added to the Si(111) DCM with an energy range of 4.0 KeV to
modified half-strength Hoagland nutrient solution 22.0 KeV and an unfocused mode with a spot size
(replacing FeSO4 with Fe2(SO4)3 to prevent the of >1 mm × 1 mm. To prepare samples for XANES
reaction between Fe (II) and Cr(VI)). An untreated analysis, leaf and root tissues of L. hexandra were
group served as the blank group. Each treatment rinsed thrice in deionized water, frozen in liquid
had three replicates, comprising 25 to 30 plants per nitrogen, ground into a fine powder with an agate
replicate. The pH values of the nutrient solutions mortar and pestle, and stored at -80 °C. The
and Cr treatments were buffered to 6.3 ± 0.2. The reference compounds, including K2Cr2O7, CrCl3,
solutions were renewed every 3 d to maintain the and Cr foil, were prepared and frozen in liquid
Cr concentrations and species during the 30 d nitrogen before the XANES spectra were obtained.
culture period. Several milliliters of the nutrient The XAFS analysis data were analyzed using
solution in each treatment were sampled daily Athena of IFEFFIT Version 1.2.11.
during the first 3 d of culture to inspect the possible
transformation of the valence state of Cr. The EPR Analysis. Cr standards, including
concentrations of total Cr and Cr(VI) in the nutrient K2Cr(VI)2O7, CrCl3, Cr(OH)3, and Cr(III)-
solution were determined by a flame atomic trioxalate, were prepared in solution or suspension
absorption spectrophotometer (AAS; model PE- and frozen in liquid nitrogen before the EPR spectra
AA700) and a UV-Vis light spectrophotometer were obtained. Cr(OH)3 was prepared by
(Model UV-9100), respectively. The concentration precipitation method with CrCl3 and NH3·H2O as
of Cr(III) was calculated by subtracting Cr(VI) raw materials. Cr(III)-trioxalate was prepared
from the total Cr. Cr in the nutrient solution of the according to the method of Tian et al. [15]. EPR
CrCl3 treatment was maintained as Cr(III), and 84% analyses of plant material and standards were
to 96% of the Cr in the nutrient solution of K2Cr2O7 performed at the Shanghai Institute of Applied
treatment was maintained as Cr(VI) during the 3 d Physics, Shanghai. Spectra were obtained at
period, thereby indicating that the transformation of approximately 10 or 293 K at a microwave power
Cr species was not significant. of 0.2 mW to 20 mW with a fixed X-band
frequency of 9.4 GHz. The data were analyzed with
Cr Concentration Measurement. This the ESR data processing program Version 3.1.2.
method was conducted as described in our previous
work [3] with little modification. At 30 d after Cr
treatment, the plants were harvested and washed
960

RESULTS leaves of Cr(III)- and Cr(VI)-treated L. hexandra

was very high (>1,000 mg kg-1 DW), except for the
Cr Accumulation by L. hexandra under roots and leaves of plants treated with 0.05 mmol L-
1
Different Treatments. After 30 d, all the Cr- Cr(III) (Table 1). The Cr concentration in the
treated plants grew well and did not show roots was much higher than in the leaves; Cr was
symptoms of Cr toxicity. Cr concentrations in tissue poorly translocated and was largely retained in the
of L. hexandra under different treatments are listed roots [13].
in Table 1. The Cr concentration in the roots and
TABLE 1
Chromium concentrations in tissue of L. hexandra of different treatments
Treatment Cr concentration (mg kg-1 DW) Bioaccumulation coefficient a

-1
(m mol L ) Root Leaf Root Leaf
blank 0 ND b ND 㸫㸫
Cr (III) 0. 4 8632±1093 4680±276 415±52.5 225±13.3
Cr (III) 1 19455±752 5892±2436 374.1±14.5 113.3±46.8
Cr (VI) 0.05 620±36 248±78 238.5±13.8 95.3±30
Cr (VI) 0.6 3354±1320 2246±465 107.5±42.3 72.0±14.9
Ă
ZĂƚŝŽŽĨƌĐŽŶĐĞŶƚƌĂƚĞŝŽŶŝŶƉůĂŶƚƚŝƐƐƵĞƐƚŽƚŚĂƚŝŶŶƵƚƌŝĞŶƚƐŽůƵƚŝŽŶ͘
ď
EсŶŽƚĚĞƚĞĐƚĂďůĞ͘

Chemical Speciation of Cr by XANES tissues was similar to that of the Cr(III) in the
Spectra. &U.Į;$1(6VSHFWUD RI&UIRLO &U9, reference solution.
(K2Cr2O7), and Cr(III) (CrCl3) standards (Figures 1
A, 1 B, and 1 C, respectively) were compared. 7KH &U .Į ;$1(6 VSHFWUD RI WKH OHDI DQG
Comparison results revealed the potential utility of root tissues from L. hexandra treated with 0.05 and
XANES to determine the coordination structure and 0.6 mmol L-1 Cr(VI) (Figures 2 B, 2 C, 2 D, and 2
valence of Cr in the plant because the structures E) were similar to those of plants treated with 1.0
showed different shapes and absorption peak mmol L-1 Cr(III) (Figures 1 D and 1 E). The sample
energies. The Cr(VI) standard has a pre-edge peak spectra were compared with the reference materials
attributable to the 1s to 3d electron transition, of Cr foil, Cr(VI) (K2Cr2O7), and Cr(III) (CrCl3).
which showed characteristics of orbital mixing in Figures 2 clearly illustrates that the oxidation state
four-coordinated Cr(VI). With Cr(III), this peak of accumulated Cr in the plants treated with Cr(VI)
showed much lower intensity and was at a slightly was primarily Cr(III).These data provided clear
lower energy compared with Cr(VI). evidence that L. hexandra efficiently and
thoroughly reduced Cr(VI) to Cr(III) in roots and
Under the 1.0 mmol L-1 Cr(III) treatment translocated Cr(III) to the aboveground parts, as
)LJXUHV'DQG(WKH&U.Į;$1(6VSHFWUDRI shown by Lytle et al. [14] and Howe et al. [16].
leaf and root tissue from L. hexandra failed to show However, the pre-edge observed in the spectrum of
a Cr(VI) pre-edge peak, but the spectra had two the samples (Figures 2 B, 2 C, 2 D, and 2 E)
small pre-edge peaks attributable to the 1s to eg* and indicated that other Cr oxidation states were present
t2g electron transition. Such transition is a in the samples㸪which is very similar to the spectra
characteristic of the octahedral coordination and has of the leaves of Gynura pseudochina (L.) DC.
the same energy and position of absorption peak as treated with Cr(VI) [17]. So we speculated that a
Cr(III). Thus, the valence of Cr in leaf and root few of the accumulated Cr in L. hexandra treated
with Cr(VI)was Cr(V) or Cr(VI).
961

FIGURE 1
XANES spectra of standards, leaves and roots of L. hexandra grown with 1.0 mmol Cr(III).
FIGURE 2
XANES spectra of Cr(III) standard, leaves and roots of L. hexandra grown with 0.05 and 0.6 mmol Cr(VI).
962
Chemical speciation of Cr by EPR spectra. produced broad signals. We were able to

EPR spectra of Cr standards. EPR spectra of Cr distinguish between Cr(VI) and Cr(III) by EPR, and
standards (K2Cr2O7, CrCl3, Cr(OH)3, and Cr(III)- JLVDVSOLWWLQJIDFWRURIWKHVSHFWUDLH³J-IDFWRUV´
trioxalate) revealed distinctly different patterns RU³J-YDOXH³6XFKIDFWRULVDQLPSRUWDQWSDUDPHWHU
(Figures 3 A, 3 B, 3 C, and 3 D). Because Cr(VI) is because it provides information on molecular
nonparamagnetic, it did not produce an EPR signal structure and environment.
(Figure 3 A). CrCl3, Cr(OH)3, and Cr(III)-trioxalate
FIGURE 3
EPR spectra of standards, leaves and roots of L. hexandra grown with 0.4 and 1.0 mmol Cr(III).
CrCl3 produced broad signals in the g = 1.98 ligands affected the unpaired electrons differently,
region of the spectrum (Figure 3 B), whereas the complexed Cr(III) EPR signal might be used as
Cr(OH)3 produced broad signals in the g = 2.0 a fingerprint to differentiate complexes.
region of the spectrum (Figure 3 C), and their
spectra had the same line shape and width. Cr(III)- EPR spectra of plants grown in CrCl3. The leaf
trioxalate produced broad signals in the g = 2.2 and tissues of L. hexandra treated with 0.4 mmol L-1
g = 4.0 regions of the spectrum (Figure 3 D). EPR Cr(III) exhibited signals in the g = 2.0 and g = 4.0
signals in the g = 2.2 region was attributed to regions of the spectrum (Figure 3 E). The weak
Cr(OH)3 impurity. Another signal in the g = 4.0 peak in the g = 4.0 region showed that the Cr in leaf
region was the characteristic peak of Cr(III)- tissues of L. hexandra was primarily Cr(III)-
trioxalate. Organically complexed Cr(III) [Cr(III)- trioxalate, with small amounts of Cr(III)-dioxalate
trioxalate, g = 4.0; Cr(III)-dioxalate, g = 4.0 and g = and Cr(III)-citrate. Plants detoxify Cr by combining
5.3; Cr(III)-citrate, g = 4.0 and g = 4.7; and Cr(III)- Cr and organic acids into chelates, which are then
EDTA, g = 4.7] produced signals in the g = 4 to g = transported to the shoot [ 18]. Other signals in the
5.5 region [16]. Differences in the electronic g = 2.0 region were detected with hyperfine
environment of the unpaired electrons resulted in splitting. Such signals were attributed to Cu(II) and
changes in the Cr EPR signal. Because different Mn(II). We also detected Cu and Mn in the leaf
963
tissues of L. hexandra by synchrotron X-ray cationic Cr(III) species further formed the
fluorescence microscopy (SXRF). However, the precipitation of Cr(OH)3, which may have reduced
unusual size and shape of these signals suggested the uptake and translocation of Cr.
the underlying presence of Cr(III). The peak shape EPR spectra of leaves from 1.0 mmol L-1
suggested a precipitated phase, possibly Cr(III) Cr(III)-treated plants (Figure 3 F) were consistent
hydroxide (Figure 3 C), because soluble Cr3+ would with those of leaves from 0.4 mmol L-1 Cr(III)-
not be retained in the presence of a hydrolysis treated plants (Figure 3 E). A broader signal shape
reaction in the plant cell cytoplasm (pH =7.3-7.6) was observed in the g = 4.0 region for 1.0 mmol L-1
[16]. Cr(III)-treated plants, thereby indicating that leaves
EPR spectra of roots of 0.4 mmol L-1 Cr(III)- of plants treated with 1.0 mmol L-1 Cr(III) have
treated plants revealed strong broad signals more Cr(III)-trioxalate than those treated with 0.4
centered at the g = 2.0 region (Figure 3 G). Such mmol L-1 Cr(III). EPR signals of roots of plants
signals were consistent with those of the Cr(OH)3 treated with 1.0 mmol L-1 Cr(III) (Figure 3 H) were
(Figure 3 C). This result indicated that Cr(III) had consistent with those of the Cr(III)-organic complex
precipitated in the root because we failed to observe (Figure 3 D), thereby indicating that the roots of
precipitation on the root surface or in the nutrient plants treated with 1.0 mmol L-1 Cr(III) contained
solution. Cationic Cr(III) species in the nutrient not only Cr(OH)3 but also massive Cr(III)-organic
solution were attracted to negatively charged sites complexes. Such complexes promoted the
on the root surface and in the apoplast. These translocation of Cr.
FIGURE 4
EPR spectra of standards, leaves and roots of L. hexandra grown with 0.05 and 0.6 mmol Cr(VI).
EPR spectra of plants grown in K2Cr2O7. For F). Thus, Cr(III) was present in the leaves of plants
comparison, EPR spectra of leaves of plants treated treated with both low and high concentrations of
with both low and high concentrations of Cr(VI) Cr(VI). Cr(III) mainly existed as Cr(III)-trioxalate,
(Figures 4 E and 4 F, respectively) were similar to but may also exist as Cr(OH)3, Cr(III)-dioxalate,
EPR signals of leaves of plants treated with and Cr(III)-citrate.
different concentration of Cr(III) (Figures 3 E and 3
964
EPR spectra of roots of plants treated with efficiently reduce Cr(VI) to Cr(III) and absorb Cr
both low and high concentrations of Cr(VI) from nutrient solution constantly. Thus, the
exhibited broad signals in the g = 2.0 and g = 1.97 intermediate state Cr(V) was detected in roots of L.
spectral regions (Figures 5 and 6). The signal at g = hexandra. In addition, the spectrum of the roots of
2.0 was attributed to Cr(OH)3. The additional signal plants treated with a high concentration (0.6 mmol
at g = 1.97 with a ppw of approximately 2 mT to L-1) of Cr(VI) had an additional weak broad peak
4 mT corresponded closely to the Cr(V) signals, near g = 4.0 (Figure 4 H), thereby indicating the
which typically exhibited g-values near two and presence of low amounts of organically complexed
one ppw of less than 6 mT [19]. Howe et al. [16] Cr(III). However, this peak was absent at the
observed Cr(V) signals at g = 1.97 with a ppw of 2 spectrum of the roots of plants treated with a low
mT to 3 mT using subterranean clover. Micera and concentration (0.05 mmol L-1) of Cr(VI).
Dessi [20] observed an EPR signal at g = 1.970 for The EPR spectra of L. hexandra tissues under
plant roots and shoots of 4- to 5-day-old garlic Cr(VI) treatments produced broad signals in the g =
(Allium sativum) seedlings subjected to Cr(VI) 4.3 spectral region (Figure 4 G, H). Such signals are
treatments at 0.15 mmol L-1 to 4.8 mmol L-1. characteristics of Fe(III), thereby indicating that
Differences in g-value between these studies both the leaves and roots of plants contained
suggest the presence of different predominant Fe(III). We also detected abundant Fe in both the
Cr(V)-complexes. The roots of L. hexandra can leaves and roots of L. hexandra by SXRF.
FIGURE 5
Details of EPR spectra of /KH[DQGUD¶V roots grown with 0.05 mmol Cr(VI).
FIGURE 6
Details of EPR spectra of /KH[DQGUD¶V roots grown with 0.6 mmol Cr(VI).
965
DISCUSSION AND CONCLUSIONS Cr(III) in roots by Fe(III)-reductase enzymes. Cary

et al. [25] observed that Fe hyperaccumulators more
In our study, although the Cr concentration effectively translocate Cr to aerial tissues than
was very high (Table 1), Cr-treated L. hexandra plants that do not accumulate Fe. We also observed
grew well and did not develop symptoms of Cr by TEM that Fe and Cr coexisted in electron-dense
toxicity. All results demonstrated that L. hexandra precipitates of L. hexandral tissues, and such
had an extraordinary Cr tolerance and accumulation coexistence indicated that low biotoxic Cr might
capacity, and a powerful detoxification system. Our accumulate in leaf vacuoles as a part of an Fe±Cr
previous study showed that the accumulated Cr in complex [12]. In the current study, the EPR spectra
L. hexandra was isolated to the cell walls in roots of Cr(VI)-treated L. hexandra tissues produced
and to the vacuoles in leaves, thereby reducing Cr broad signals in the g = 4.3 spectral region (Figures
toxicity in the plant cell [12]. Such distribution of 4 G and 4 H), and such signals were characteristics
Cr in the plant cell explained the tolerance and of Fe(III). L. hexandra can detoxify Cr(VI) through
detoxification of Cr at the subcellular distribution the absorption of Fe and can accumulate Fe(III) in
level. Howe et al. [16] reported that to detoxify leaves and roots. In conclusion, Fe may be serves
Cr(VI), plants must first convert the highly toxic an important function in the Cr(VI) reduction [17,
Cr(VI) to the considerably less toxic Cr(III), which 26] of L. hexandra. Metallothionein synthase,
in turn is either immobilized or compartmented. phytochelatin synthase, and organic and amino
Similarly, the XANES and EPR spectroscopy acids are important plant chelates that alleviate
results (Figures 1, 2, 3 and 4) provided clear heavy metal toxicity by forming a low or non-toxic
evidence that the accumulated Cr was primarily complex with the heavy metal [27-30]. L. hexandra
Cr(III) with small amounts of Cr(V) in Cr(VI)- cells only contained carbon, oxygen, and other
treated L. hexandra. Antimicrobial agents were major elements and did not detect sulfur in
added to experimental nutrient solutions as a electron-dense precipitates of Cr, as shown by
pretreatment to eliminate the interference of TEM; thus, the complexation of Cr in L. hexandra
bacteria or fungi in the system. So the result with metallothionein synthase, phytochelatin
demonstrated that L. hexandra absorbed Cr(VI), synthase, or glutathione is unlikely [12]. Cr mainly
efficiently and thoroughly reduced Cr(VI) to Cr(III) contained oxalic-integrated Cr in leaves of L.
in roots, and subsequently translocated Cr(III) to hexandra, as shown by chemical analysis [13]. In
the aboveground parts. In addition, the presence or the current study, we presented sufficient proof by
absence of Cr(VI), intermediate Cr(V) and Cr(III) nondestructive techniques. All EPR spectra of
and their translocation from the root to the shoots leaves of Cr(III)-treated and Cr(VI)-treated L.
may depend on plant species and its reduction hexandra plants (Figures 3 and 4) revealed broad
efficiency. Zayed et al. [21] found only Cr(III) in 9 signals at the g = 4.0 region, suggesting that Cr(III)-
vegetable plant species tissues treated with Cr(VI). organic complex [mostly Cr(III)-trioxalate with few
Arteaga et al. [22] and Aldrich et al. [23] reported Cr(III)-dioxalate and Cr(III)-citrate] is the main Cr
the transportation and accumulation of Cr(VI) and compound in tissues of L. hexandra.
Cr(III) in the shoot parts creosote bush (Larrea Previous studies have shown the existence of
tridentata) and mesquite (Prosopis spp.) treated two combined forms of Cr(III) in plants for
with Cr(VI), respectively. Cr(VI), Cr(V) and a immobilization and compartmentation, as follows:
significant amount of Cr(III) were found in the (i) the precipitation of a Cr(III) hydroxide phase at
leaves of G. pseudochina (L.) DC treated by a high the root, and (ii) the complexation of Cr(III) and
concentration of Cr(VI) [17]. However, Howe et al. probable storage as Cr(III)-organic complexes, such
[16] found that Cr(VI), Cr(V) and Cr(III) were as Cr(III)-EDTA, Cr(III)-trioxalate, and sulfurated
observed in Subterranean clover (Trifolium ligands [16,17, 21, 31]. In this paper, we analyzed
brachycalycium) treated with a high concentration the combined form of Cr in roots and leaves of L.
of Cr(VI) but only Cr(III) under a low hexandra under Cr(III) and Cr(VI) treatments. In
concentration of Cr(VI), So the presence or absence roots, all the EPR spectra of roots of Cr(III)- and
of Cr(VI), intermediate Cr(V) and Cr(III) may also Cr(VI)-treated plants showed that the accumulated
effected by the concentration of Cr(VI) in the Cr mainly existed as Cr(OH)3. The EPR signals of
experimental system. roots of 1.0 mmol L-1 Cr(III)-treated plants (Figure
In plants, the decrease the chemical valence of 3 H) were consistent with those of the Cr(III)-
heavy metal mainly depends on the chemical organic complex (Figure 3 D), which indicated that
reduction of reductase or on the complexation of a portion of the Cr(III) in root may be bound to
chelates. Hexavalent Cr has powerful oxidization organic acid ligands. Such ligands can promote the
capacity. Zayed et al. [21] and Mallick et al. [24] translocation of Cr. The intermediate state, Cr(V),
reported that Cr(VI) is immediately converted into was detected in roots of Cr(VI)-treated L. hexandra
966

plants (Figure 2, 5 and Figure 6). All the EPR [9] Salt, D.E., Blaylock, M., Kumar-Nanda,
spectra of leaves from Cr(III)- and Cr(VI)-treated P.B.A., Dushenkov V, Ensley BD and Chet I,
plants were similar. In leaf, Cr existed mainly as Raskin I. (1995) Phytoremediation: a novel
Cr(III)-organic complex [mostly Cr(III)-trioxalate strategy for the removal of toxic metals from
with few Cr(III)-dioxalate and Cr(III)-citrate] and the environment using plants, Bio Technology,
as Cr(OH)3 in small amounts. This finding is in 13(5), 468-474.
agreement with results obtained by chemical [10] Shah, K. and Nongkynrih, J.M. (2007) Metal
analysis [5]. The Cr that accumulated in the leaf of hyperaccumulation and bioremediation, Biol
L. hexandra was mainly located in the vacuole of Plantarum, 51, 618-634.
the mesophylls [4]. Therefore, oxalate is the final [11] Yang, X.E., Feng, Y., He, Z.L. and Stoffella,
receptor of Cr in the vacuole. P.J. (2005) Molecular mechanisms of heavy
metal hyperaccumulation and
phytoremediation, J Trace Elements Med Biol,
ACKNOWLEDGEMENTS 18(4), 339-353.
[12] Liu, J., Duan, C., Zhang, X., Zhu, Y.N. and Hu,
The authors thank the financial supports from C. (2009) Subcellular distribution of chromium
the Special Funding for Guangxi 'BaGui scholars' in accumulating plant Leersia hexandra Swartz,
Construction Projects and Innovative Project of Plant Soil, 322, 187-195.
Guangxi Graduate Education (No. YCBZ2014004). [13] Zhang, X.H., Liu, J., Wang, D.Q., Zhu, Y.N.,
The authors gratefully acknowledge the Shanghai Cheng, H. and Sun, J.J. (2009)
Institute of Applied Physics which provided beam Bioaccumulation and chemical form of
time and facilities for EPR analysis. chromium in Leersia hexandra Swartz, Bull
Environ Contam Toxicol, 82, 358-362.
[14] Lytle, C.M., Lytle, F.W., Yang, N., Qian, J.H.,
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968
MODELING NUTRIENT AND HEAVY METAL TRANSPORT

AT SELECTED CATCHMENTS IN THE MARMARA REGION
=H\QHS$NGR÷DQ1 ā%DúDN*YHQ1, ā,úÕO%DOFÕR÷OX1
1
Institute of Environmental Sciences, %R÷D]LoL8QLYHUVLW\,VWDQEXO7XUNH\
ABSTRACT INTRODUCTION
Increasing human activities and land use Urbanization, industrial and agricultural
changes in recent years have contributed to water practices, fertilizer applications and other human
quality degradation by affecting the transport of activities has led to an increased accumulation of
contaminants to receiving waters by overland flow. contaminants in natural resources. These
This necessitates the development of modeling contaminants affect water quality and human health
approaches for planning and management of by being transported to surface and ground waters
catchments. The aim of the study presented here is as a result of runoff, which is highly affected by
to develop a GIS-based catchment hydrology and land use and cover, soil structure, climate and
contaminant transport model for selected topography [1, 2, 3, 4]. Agricultural activities have
catchments in the Marmara Region and to predict been identified as a dominant factor in generating
total loads of nutrients and heavy metals to large amounts of nonpoint source (NPS) pollution
receiving waters from those catchments. The [5]. Previous studies have also indicated a close
contaminant transport module of the model includes relationship between water quality and urbanization.
a kinetic equation for nutrient loss and Urban expansion leads to an increase of the
mathematical equations for distribution of heavy impervious area percentage significantly and a
metals in environmental compartments. Storm decrease of natural sinks for storm flows [6]. This
Water Management Model (SWMM) is used as the results in contribution to flood events and water
modeling environment for the conversion of quality impairment. Research revealed that storm
predicted concentrations to catchment runoff water runoff from urban areas is a critical nonpoint
contaminant loads. Total nitrogen (TN), total source of pollution, which transports some of the
phosphorus (TP), Copper (Cu), Nickel (Ni), and same pollutants found in rural and agricultural
Zinc (Zn) concentrations in runoff are estimated runoff, such as sediment, nutrients, oxygen-
using previously analyzed soil samples collected demanding organic materials, pesticides, and also
from the different lands of the Marmara Region. toxic contaminants, such as heavy metals [7]. In
Spatial data and concentrations obtained are addition to land use information, soil characteristics
transferred to SWMM to develop the catchment are also effective in runoff production and
hydrological model and describe the influence of contaminant transport, which are governed by
land use and precipitation on contaminant loads. infiltration and sorption processes, respectively. For
An integrated modeling procedure is developed by example, Sharpley et al. [8] investigated the
combining kinetic equations both for nutrient loss kinetics of P desorption for several soils at different
and heavy metal transport, and applied to the water/soil ratios using an empirical model, and
relatively urbanized catchments of the Marmara concluded that empirical equations were successful
Region. Results revealed the validity of the in estimating P release from soil to runoff water for
proposed modeling method, indicating the positive relatively short periods of time.
correlation between runoff production and Physically-based catchment hydrology models
contaminant loads in catchments (R2 > 0.9). The have been widely used, particularly in the last few
initial soil contaminant concentrations are found to decades, in order to better understand and interpret
be more effective in simulating runoff the behavior of surface and subsurface processes, as
concentrations, when compared to the heavy metal well as the mobility of water and contaminants.
partition coefficients. According to Jamieson et al. [9], analysis tools and
simulation models have been developed to properly
evaluate alternate management practices and to
KEYWORDS: predict water quality improvements at the
Catchment hydrology; Contaminant transport; Modeling; catchment scale. During the hydrological modeling
Nutrient loss; Heavy metal distribution; SWMM
studies, water quality should be considered with
runoff quantity due to the unequal distribution of
969
water and its spatio-temporal variability. For this determined by a kinetic model [8], whereas heavy
reason, it is important to have realistic models metal concentrations in soil pore water and runoff is
including hydrologic or hydraulic component, predicted via an exposure model [18]. SWMM is
which have the appropriate spatial and temporal used as the major modelling environment for
resolution required for the problem [10]. In hydrological modelling and required input variables
reference to catchment models, Environmental for SWMM are determined via GIS-based maps. 30
Protection Agency (EPA) Storm Water soil samples are collected and analyzed for the
Management Model (SWMM) is the one model modelled catchments of the Marmara Region, and
developed primarily for urban areas [11], which can nitrogen, phosphorus and heavy metal
clearly simulate the catchment hydrology and water concentrations are determined.
quality.
All basic units such as water, soil and
pollutants in environmental modeling have spatial MATERIALS AND METHODS
distributions that affect the processes and their
dynamics considerably [12]. In the last few decades, The methodology adopted in this study can be
Geographical Information Systems (GIS) has classified under 3 main categories, including (i)
become a very helpful tool in environmental determination of soluble nutrient runoff
modeling, since it has the ability to identify NPS concentrations are estimated by a kinetic model [8],
pollution and serves as a common data and analysis (ii) determination of heavy metal concentrations in
framework for hydrological and transport models soil pore water and runoff is estimated by an
[13]. GIS technology provides boundary conditions exposure model developed by RIVM (National
as a practical solution, as it is designed to collect, Institute of Public Health and the Environment in
store, manipulate and display geographic The Netherlands) [18], (iii) development of a semi-
information. Multivariate statistical analyses and distributed, physically-based catchment hydrology
transport models integrated with GIS facilitate to model for selected catchments in the Marmara
identify the contaminants and their spatial patterns Region, to investigate the relationships between
and distributions in soil and in catchments by annual nutrient and heavy metal loads, basin
spatial data management combined with temporal topography, land use and meteorological variables.
variable simulation [1, 14, 15]. In the literature, A schematized representation of the modeling
different modeling approaches integrated with GIS procedure and information flow together with basic
have been developed to investigate the transport of data requirements are outlined in Fig. 1.
nutrients [5, 16, 17]. While several studies are
encountered concerned with nutrients, the modeling Study area. Marmara Region is situated in the
studies with GIS are scarce for metals at catchment northwest of the country and occupies 8.5 % of
scale. Bhaduri et al. [7] investigated the impact of Turkey with an area of 67000 km2 approximately.
urban/impervious areas and land use change on There is a dense stream network throughout the
annual runoff volume and NPS pollution at region. The climatic characteristics of the Black Sea
catchment scale using a long-term hydrologic influences the north of the region, while the typical
impact assessment (L-THIA) model with GIS. The Mediterranean climate prevailing in its south.
study stated the variation of annual nutrient and Precipitation averages about 600-700 mm annually
heavy metal (Cu, Pb, and Zn) loads as a and mainly falls between November and January.
consequence of the land use change. Average annual temperature is between 14oC and
This paper aims to investigate overland 16oC and the mean monthly temperatures range
transport and total loads of total nitrogen (TN), total from 5oC in the coldest months to 25oC in warm
phosphorus (TP), Copper (Cu), Nickel (Ni), and seasons [19]. The Marmara Region generally has
Zinc (Zn) at selected catchments in the Marmara the slightly undulating lands. Land use activities in
Region by undertaking an integrated, semi- the region include residential, industrial and
distributed modeling approach. Input agricultural practices. Currently about 30% of the
concentrations of contaminants are obtained from region is arable land with intense agricultural
previously analyzed 30 soil samples taken from the activities and 11.5% is woodland. Forests are found
modelled catchments of the Marmara Region. particularly in Trakya region at high elevations.
Concentrations of soluble nutrients in runoff are
970
FIGURE 1
A schemati ed representation of the modeling procedure and associated data re uirements.
The region consists of three major catchments: involves 7 selected catchments in the Marmara
Meriç-Ergene, Sakarya, and Susurluk. This study Region (Fig. 2).
FIGURE 2
Selected cachments in the region and soil sample collection stations.
971
Meriç-Ergene Catchment (catchment 1) is construction of the backdrop image, and basin tool
found in the northwest of the region. 72 % of the of the program is used to delineate the 51
catchment is arable land and 65 % of the basin area catchments in the Marmara Region. The
involves agricultural practices. Industrialization has topographical data and discretizated catchments
also been developed rapidly for many years, (Fig. 2) are transferred to SWMM for the
especially in the southern part of the catchment [20]. construction of the hydrological model. SWMM
While a portion of Sakarya (catchment 2) is located model is preferred because of its suitability for
in the eastern side of the Marmara Region, Susurluk hydrological modeling procedure in urban areas [11]
(catchment 3) covers the great part of the southern and convenience to operate with spatial interface.
side. The percentages of agricultural areas are 52 The catchment map with 51 discretized catcthments
and 42 for Sakarya and Susurluk Catchments, (Fig. 2) is used as a backdrop image for SWMM
respectively [21, 22]. Catchment 7 represents application, and the map is divided into manageable
Ömerli Basin which provides 48% of daily drinking HRUs (Hydrological Response Unit). Input
water (872000m3) for the city of Istanbul and parameters including topographical data and land
includes the biggest reservoir in northern part of the use information, time-series precipitation, and other
Marmara Region. The prominent land use activities required data produced by the SWMM are entered
and environmental issues in such catchments are to the model. Meteorological data is obtained from
also intense agricultural and livestock activities, the Turkish Meteorological Service, and 26
growing industrialization and urbanization, and precipitation stations within the study area
discharge of domestic and industrial wastewaters boundary, generally located in the city centers of
[21, 22]. Catchments 5 and 6 include Lake Iznik the Marmara Region, are chosen to provide daily
DQG $÷YD &UHHN UHVSHFWLYHO\ 7KH LQWHQVH precipitation data between years 1950 and 2005.
residential areas around these water sources It is possible to classify the variables and
indicate that water quality of those are of special parameters required for SWMM in two main
interests to the local people. categories as the quantities describing catchments
and hydraulic characteristics. While the
Modeling catchment hydrology. In this study, topographical data describing the catchment
GIS-based digital elevation maps are used to obtain characteristics and hydraulic parameters regarding
topographical data, such as elevation, slope, area, rivers are determined via GIS, values of parameters
and width, and to prepare the background map to be VXFK DV 0DQQLQJ¶V FRHIILFLHQW GHSUHVVLRQ VWRUDJH
used for the hydrological model. Spatial analyst depths, and channel roughness are obtained from
tool of the ArcGIS 10 program is combined with the literature. Values and corresponding sources of
precipitation and land cover data for the such data are given in Table 1.
TABLE 1
Input data for HRUs and conduits
Parameter Value Description Source

Manning's n for overland flow over the
N-imperv 0.011
impervious portion of the HRU
[23]
Manning's n for overland flow over the
N-perv 0.13
pervious portion of the HRU
Depth of depression storage on the
DǦstoreǦimperv 1.27 mm
impervious portion of the HRU
[24]
Depth of depression storage on the
D-storeǦperv 3.8 mm
pervious portion of the HRU
Conduit Roughness 0.03 Manning's roughness coefficient [25]
The percent imperviousness (% I) values used GIS based maps, obtained from Republic of Turkey
in the model are determined by the help of land use Ministry of Food, Agriculture and Livestock. The %
and cover data regarding the provinces located I values for all HRUs are estimated using runoff
within the study area, which are available for the coefficients that are corresponding to three main
year 2005. This information is derived from the
972
land use classes; commercial, residential and rural, and subsequent transport of those to receiving
are obtained from the literature [26]. waters are dependent upon soil type and
Obtaining an average runoff volume is characteristics. Soil data are obtained from
required in order to assess the mean load of the extensive field and laboratory work carried out by
pollutants in surface runoff. In this respect runoff %DOFÕR÷OX HW DO >@ VRLO VDPSOHV DUH FROOHFWHG
coefficients, produced by the model, are used to and analyzed for the modelled catchments of the
help to characterize the amount of runoff in each Marmara Region, and nitrogen, phosphorus and
HRU. Here, we used runoff quantities produced heavy metal concentrations are determined. Table 2
from SWMM (Equation (1)), for the estimation of provides the contaminants detected in soil samples,
TN, TP, Cu, Ni, Zn at selected catchments of the as well as soil characterizations such as; texture, pH
Marmara Region. value, percent of water content of for residential
and rural land use classes [27]. Partition
Runoff=HRU Area ×Annual Rainfall ×Runoff coefficients of each heavy metal for partitioning
Coefficient (1) between soil and water are obtained from the
literature [28], and are determined by considering
Modeling contaminant transport and data soil characteristics, such as pH and concentration of
re uirements. Sorption behavior of contaminants dissolved organic carbon (DOC).
TABLE 2
Data re uirements for water uality modeling.
Soil Data Contaminant Data
Input Source Input Source Modelled
Soil texture (% clay, sand, silt), [27] TN and TP [27] TN and TP

pH, organic carbon content, concentrations in soil concentrations in runoff
water content
Soil texture type Calculated Cu, Ni, and Zn [27] Cu, Ni, and Zn
concentrations in soil concentrations in
porewater
Fresh bulk density of soil Calculated Partition coefficients [28] Cu, Ni, and Zn
for Cu, Ni, and Zn concentrations in runoff
In the model, 30 soil samples that were ୏ൈ୔౥ ൈ஡౩౥౟ౢൈୈൈ୲ಉ ൈ୛ಊ

୰ ൌ (2)
analyzed for contaminant concentration and soil ୚
characteristics (soil texture, pH, organic carbon
content, and water content) are used. The data are where; Pr is the average phosphorus
split into two sets to be used for calibration and concentration of runoff (mg L-1), P0 is the
validation steps. Half of the data are used in model phosphorus content of the soil (g kg-1), ȡsoil is the
equations used for prediction of nutrient and heavy fresh bulk density of the soil (kg m-3), D is the
metal concentrations in runoff, particularly for effective depth of interaction between surface soil
calibration of partition coefficients of contaminants, and runoff (mm), t is the storm duration (min), W is
and fraction of those in different environmental runoff water/soil (suspended sediment) ratio, V is
compartments (soil, water), which are used in total runoff during the event (mm) and K, Į, and ȕ
model equations, described later in this section. are constants for a given soil. Equation (2) is also
Other half is used for model evaluation. applied for determination of soluble nitrogen
concentrations in runoff. Since actual storm
Prediction of nutrient concentrations in duration data are not available, in the model
runoff. Nutrient concentrations in runoff are calculations, t is set to 30 min. The mean monthly
predicted using Equation (2), which describes the rainfall intensities corresponding to sampling dates
kinetics of soil soluble phosphorus desorption are entered and total runoff values (V) for each
process [8]: corresponding event are calculated via SWMM.
973
Average values of D and W are obtained from the and ȡsolid is density of soil solids (kg m-3). Predicted
literature [29, 30] and constants of the Equation (2) Environmental Concentrations of heavy metals in
are estimated from surface soil clay/organic carbon pore water (PECporewater) (mg L-1) transported from
content using Equations (3) to (5) [29]: contaminated soil are found by the below equation:
ൌ ͲǤ͸͵ ൈ ሺΤ ୭ୡ ሻି଴Ǥ଺ଽ଼

୑୉େ౩౥౟ౢ ൈ஡౩౥౟ౢ
(3) ୮୭୰ୣ୵ୟ୲ୣ୰ ൌ
୏౩౥౟ౢష౭౗౪౛౨ ൈଵ଴଴଴
(7)
ି଴Ǥହଶ଺
Ƚ ൌ ͲǤ͹͹ͻ ൈ ሺΤ ୭ୡ ሻ (4)
଴Ǥସଵଽ where MECsoil (Measured Environmental
Ⱦ ൌ ͲǤͳͶ͵ ൈ ሺΤ ୭ୡ ሻ (5)
Concentration) is metal concentration in the
where, Foc is fraction organic carbon in soil (%). analyzed soil samples (mg kg-1), and ȡsoil is fresh
bulk density of soil (kg m-3).
Prediction of heavy metal concentrations in Fraction of metals not adsorbed to soil
runoff. Exposure model equations developed by particles, but existing in the soil water is
RIVM are used in the determination of sorption of transported to runoff water governed by
Cu, Ni, and Zn to soil, and in estimation of these precipitation. It is assumed that predicted metal
concentrations in pore water and runoff. The model concentration in runoff (PECr) (mg L-1) is equal to
adopted here [18] was primarily developed for PECporewater diluted by one order of magnitude:
veterinary products, but subsequent studies revealed
୔୉େ౦౥౨౛౭౗౪౛౨
WKHPRGHO¶VDSSOLFDELOLW\IRULQYHVWLJDWLRQRIKHDY\ ୰ ൌ (8)
ଵ଴
metals and their capacity to adsorb to the soil [31,
32]. Each type of metal has a specific partition Runoff water uality prediction. In SWMM
coefficient that varies according to soil runoff water quality is modelled with the help of
characteristics. The previous studies state that the pollutant and land use characteristics
soil/sediment-water partition coefficients of metals (Residential/Rural), using the washoff function.
are critical for exposure models [32], and the Tsihrintzis and Hamid [34] described this as the
relationship with standard soil characteristics, such process through which storm water washes off all
as pH, clay content and organic carbon, must be the accumulated contaminants into receiving water
considered for metal Kp values [33]. In this study, in a catchment. Appropriate values of predicted TN,
partition coefficients of metals in soil are obtained TP, and heavy metal concentrations in runoff are
from the literature [28] by taking into account the entered to SWMM as Event Mean Concentrations
similarities of pH values and soil textures between (EMCs). The EMC for an individual storm event
the soil samples collected from the Marmara can be defined as the total contaminant load divided
Region, and those studied in literature. Since metals by total runoff volume (Equation (9)) [35], hence
have low vapor pressures, the partitioning to air loads for TN, TP, Cu, Ni, and Zn can be estimated
phase is neglected by the assumption partitioning by Equation (10):
occurs only between soil solids and pore water.
After Kpsoil values are determined, Ksoil-water σ ௤೔ ൈେ౟
partition coefficients for each heavy metal are ൌ σ ௤೔
(9)
calculated by Equation (6):
where, qi is the time variable flow (m3 s-1) and
Ksoil-water = Fwatersoil+(Fsolidsoil x Kpsoil Ci is the time variable concentration (mg L-1).
x ȡsolid)/1000 (6)
ൌ ൈ (10)
where; Ksoil-water is partition coefficient of
metals between solids and water in soil (m3 m-3), The modeling method for predicting contaminant
Fwatersoil is fraction water in soil (m3 m-3), Fsolidsoil is concentrations in runoff and associated loads in
fraction solids in soil (m3 m-3), Kpsoil is partition catchments is schematized in Fig. 3.
coefficients in solids and water in soil (dm3 kg-1),
974
FIGURE 3
Contaminant transport modeling flowchart.
RESULTS AND DISCUSSION initial check of model verification. For this reason,
first, EMC of each contaminant is calculated using
This section presents the predicted nutrient Equations (2)±(5) for TN and TP, and Equations
and heavy metal mass loadings within selected (6)±(8) for Cu, Ni, and Zn, respectively. Predicted
catchments. Prior to SWMM application, it is EMCs are qualitatively verified by comparison with
ZRUWKZKLOH WR DVVHVV WKH PRGHO HTXDWLRQV¶ a similar study carried out for Maryland, U.S. [36],
reliability by comparison with other studies, as an and provided in Table 3.
TABLE 3
Predicted EMCs vs. EMC ranges in the literature.
Catchment number
EMC Range Land use
1 2 3 4 5 6 7
class
1.307 1.485 0.98 4.366 0.754 3.46 4.815 1.5-2.69 Residential
TN (mg L-1)
11.973 13.198 12.333 8.012 5.774 6.268 6.234 2-7.84 Rural
0.642 0.136 0.11 0.928 0.523 0.307 0.529 0.2-0.39 Residential
TP (mg L-1)
14.191 6.484 4.29 4.338 3.287 2.007 1.39 0.2-0.82 Rural
0.0106 0.0104 0.0194 0.0124 0.0207 0.0248 0.0152 0.0125-0.0826 Residential
Cu (mg L-1)
0.0023 0.0068 0.0051 0.0063 0.0091 0.0137 0.0088 0.1166-0.1774 Rural
0.0086 0.0144 0.0129 0.0123 0.0177 0.0395 0.0342 NA Residential
Ni (mg L-1)
0.0002 0.0129 0.0123 0.0113 0.0111 0.0148 0.0177 NA Rural
0.015 0.0192 0.0354 0.0206 0.0399 0.0497 0.0192 0.0449-0.1814 Residential
Zn (mg L-1)
0.0072 0.0066 0.0101 0.0053 0.0174 0.0217 0.0136 0.0239-0.0383 Rural
36
Contaminant mass loadings. Following the land use and % I values of selected catchments for
prediction of EMCs, catchment contaminant loads the year 2005, together with their HRUs.
are determined via SWMM. Catchments 6 and 7 Fig. 4 provides an overall representation of
(Fig. 2) located in the north cost of the Marmara nutrient and heavy metal loads for each modelled
Region within the boundaries of Istanbul and catchment for the wettest and driest years observed
Kocaeli are chosen to present load and runoff in the past 55 years. The results presented below
graphs to visualize the influence of changing land indicate the positive linear relationship (R2 > 0.9)
use and % I on runoff quality. Table 4 summarizes between contaminant load and runoff production.
975
TABLE 4
Land use data of selected catchments in 2005.
Land Use ( )
Catchments HRUs I
Residential Rural
1 31.8 68.2 26.8
2 7.4 92.6 7.4
6 3 48.5 34.1 51.5 65.9 37.4 27.1
4 48.7 51.3 37.5
5 27.4 72.6 22.0
1 8.1 91.9 8.3
2 30.5 69.5 25.8
7 3 4.6 10 95.4 90 5.6 9.8
4 6.5 93.5 7.1
5 4.5 95.5 5.5
According to the figure, as an expected CONCLUSION AND

outcome of soil nutrient concentrations, runoff TN RECOMMENDATIONS
load is higher than that of TP. Figure also underpins
the importance of % I on pollutant loading, by The aim of the study presented here is to
suggesting, although the EMCs of TN and TP are develop a catchment hydrology and contaminant
higher in rural areas than those in residential, load transport model for selected catchments in the
values of these contaminants are found to be higher Marmara Region and to observe the total loads of
in catchment 6 that has a higher % I, which can be nutrients and heavy metals to receiving waters from
attributed to greater volume of runoff production. those catchments. The contaminant transport
Previous studies also indicates that pollutant module of the model includes a kinetic equation for
loading to runoff is positively correlated with % I nutrient loss and mathematical equations for
[6]. It can be suggested that Kpsoil values are distribution of heavy metals in environmental
effective in simulating runoff concentrations, when compartments. SWMM is used as the modeling
measured soil contaminant concentrations are close environment for the conversion of predicted
to each other. In Fig. 4c and 4d, Cu has the lowest concentrations to catchment runoff contaminant
mass loading, despite its lowest Kpsoil value loads.
amongst other metals (316.23 dm3 kg-1) [28] These Pollutant mass loading results reveal that TN
results can be attributed to differences between load in runoff are higher than the other
metal concentrations in soil samples, which seems contaminants significantly and Zn has the largest
to be a more effective factor than soil partition loading values amongst other heavy metals.
coefficients, in the transport of heavy metals to Although soil partition coefficient value of Cu is
runoff water. lower than the other metals, it has the lowest
Half of the data, which were not used before loading values as a result of the low concentrations
for calibration model equations including partition in soil. The influence of different land use
coefficients of contaminants, and fraction of those percentages on pollutant loading are examined by
in different environmental compartments (soil, selecting catchments 6 and 7, which have 34.1%
water), is used for model evaluation. Fig. 5 and 10% residential areas, and 27.1% and 9.8%
represents the measured concentrations in soil and imperviousness values, respectively. The results
predicted catchment loads for each type of indicate a significant correlation between
contaminant. precipitation, land use and pollutant loading;
therefore, it is possible to conclude that the
modeling method proposed and schematized in Fig.
3 is appropriate for predicting contaminant
concentrations in runoff and associated loads in
catchments.
976
FIGURE 4
Total load and runoff values in catchments in the wettest and driest years (a) nutrient load for the
year 1997 (b) nutrient load for the year 1977 (c) heavy metal load for the year 1997 (d) heavy metal load
for the year 1977.
Data gathering and harmonization are one of water quality. Results, suggest that when supported
the most challenging steps towards developing with appropriate kinetic equations, SWMM can be
distributed catchment models due to the spatial used for establishing the relationship between
variability of data. This study suggests that GIS runoff concentration and catchment contaminant
incorporated SWMM modeling approach is an load.
efficient tool in catchment modeling and has the Further research may focus on developing
potential to evaluate the storm rainfall and pollution catchment hydrology and runoff quality models in a
prevention planning. SWMM, itself has not been a more distributed manner to account for the rather
common modelling environment for predicting high spatio-temporal variability of catchments.
977
FIGURE 5
Measured concentrations in soil and predicted catchment loads of (a) TN (b) TP (c) Cu (d) Ni (e) Zn
for the year 1997.
ACKNOWLEDGEMENTS AGNPS integrated with a Geographic

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University Scientific Research Projects (Bogazici (2007). Rainfall intensity and phosphorus
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surface runoff from soil trays. Sci. Total
Environ. 373, 334.
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Received: 26.06.2015
Accepted: 13.01.2016
CORRESPONDING AUTHOR
%DúDN*YHQ
,QVWLWXWH RI (QYLURQPHQWDO 6FLHQFHV %R÷D]LoL
University, 34342
e-mail: guven.basak@gmail.com
980
ENHANCED HEAVY METAL IMMOBILIZATION IN

POULTRY LITTER USING SODIUM DIETHYL
DITHIOCARBAMATE
Wen-gang Liu, De- hou Wei, Hui-liang Guo, Bao-yu Cui and Yan-bai Shen
College of Resources and Civil Engineering, Northeastern University, Shenyang 110819, Liaoning, China
ABSTRACT desirable methods are expected to treat poultry litter

before its disposal to avoid secondary pollution.
Sequential extraction procedure was applied to Accordingly, the remediation of heavy metal
investigate the immobilization of heavy metals (Cu, wastes through manipulating their availability using
Mn and Zn) in poultry litter when sodium diethyl a range of soil amendments has been presented,
dithiocarbamate (DDTCNa) was used as including physical means, electro±kinetic
immobilizing agent by column leaching tests. The remediation, biological remediation, and combined
results indicated that DDTCNa showed high remediation technologies [5,6]. In contrast to
immobilization effect for converting Exchangeable classical remediation techniques (electro±kinetic
and Carbonate Bound fractions of heavy metals into remediation or soil flushing), immobilizing (or
more stable fraction, i.e. Organic Matter Bound stabilization) technique has been strongly
fraction. Moreover, better immobilization effect on recommended and used to treat heavy metal wastes
Cu and Zn was obtained than on Mn. When for its environmental safety and cost effective [6,7].
''7&1DGRVDJHZDVKLJKHUWKDQPJNJíQR Some additives, such as silicates [8], cement,
Exchangeable fractions of Zn could be detected. phosphates [9], organic materials [10,11], and
pH-dependent leaching experiment showed that inorganic sulphides [12], are frequently used to
percentage of Organic Matter Bound fraction decrease heavy metal availability. Demir and Bin-
increased with Exchangeable and Carbonate Bound Shafique [13] has demonstrated the decrease of
fractions decreasing when the leaching pH was heavy metal leaching in bioretention when
increased from 4.50 to 7.00, and better phosphate was added. Meanwhile, NovoGro (NG),
immobilization effect was obtained at neutral pH. chicken manure compost (CMC), bacillus subtilis
XRD analysis indicated that DDTCNa could and their mixtures were also used to increase the
immobilize heavy metals by the formation of stable residual fraction of cadmium (Cd) in farmland [14].
sulphur±coordinated four±membered chelates. Dithiocarbamates, which are a versatile class
of mono-anionic 1,1±dithio ligands, can coordinate
KEYWORDS: with transaction metals strongly. Thus, their
Heavy metals, immobilization, poultry litter, sodium stabilization abilities on heavy metals have been
diethyl dithiocarbamate, sulphur chelates studied by several researchers. Hou et al. [15]
indicated that dithiocarbamates (DTCs) can
precipitate Hg2+ well, and heavy metal ions (Cu2+,
INTRODUCTION Pb2+, Ni2+ and Zn2+) inhibited Hg2+
precipitation. Jiang et al. [16] synthesized one
Poultry litter is a mixture of manure, bedding chelating agent by the reaction of polyamine (or
material, wasted feed, and feathers. Currently, a polyethylene imine) and CS2, and discussed its
large quantity of poultry waste generated as by± stabilization effect on Pb, Cr, Zn, and Cu. Fu et al.
products of intensive poultry production has been [17] demonstrated the precipitation ability of one
disposed by adding to agricultural land as organic new dithiocarbamate±type compound (HTDC) on
fertilizer [1,2]. Cu2+.
However, in addition to nutrients, poultry Sodium diethyl dithiocarbamate (DDTCNa),
litter also contains high levels of potentially toxic which has been widely used in sulfide mineral
metals, such as Cu, Mn, Zn, Cd, or As, in labile flotation as a collector [18], has shown strong
forms and is viewed as a major source of soil metal chelating actions with some transition metal ions,
contamination. Thus, continued disposal of large such as Cu, Pb, Zn, and Cr, to form four±membered
amounts of raw manure on limited areas may pose a ring chelates [19]. Therefore, it may present
significant threat to soil or groundwater quality via potential stabilization effect on mobile heavy
leaching of toxic elements [3,4]. Thus, some metals via generation of sulfide compounds.
981
Compared with the existing dithiocarbamate highest concentration in the sample. However, it
immobilization agents, DDTCNa is much cheaper was not usually being viewed as harmful heavy
and more accessible. However, fewer studies have metal. Cr content in the sample was relatively
focused on the immobilization effect of DDTCNa lower. Thus, Mn, Zn, and Cu were the main
on heavy metals. Thus, the purpose of this work is immobilizing targets that must be paid more
to evaluate the immobilization effect of DDTCNa attention to.
on heavy metals in polluted poultry litter by column DDTCNa and Na2S, which were analytical
leaching experiments in laboratory, and to pure, were purchased from Sinopharm and used as
investigate the subsequent chemical speciation of immobilizing agents at 0, 0.2, 0.8, 1.4, 2.0, and 2.6
Cu, Zn and Mn through sequential extraction J/íRIDTXHRXVVROXWLRQ
procedure. The immobilization mechanism of Column leaching tests. Immobilizing effects
DDTCNa on heavy metals was also discussed. of DDTCNa and Na2S on heavy metals in poultry
litter were conducted by column leaching tests. A
portion of 500 g poultry litter was packed into a
MATERIALS AND METHODS PVC column with 6.0 cm in inner diameter to reach
approximately 15 cm high as shown in Fig. 1. The
Samples and characterization. Poultry litter bulk density of the packed poultry column was
was collected randomly from one poultry house HYDOXDWHG WR J FPí 7KH ERWWRP RI WKH
located in Qingyuan District, Liaoning Province, column consisted of a Plexiglas plate containing
China. The sample was air±dried at room several 5 mm±wide holes. The plate was covered
temperature, homogenized, and ground to pass with 6 layers of nylon cloth to prevent poultry litter
through a 2 mm nylon sieve. And then, the sample loss during leaching process. Two pieces of filter
was subjected to X±Ray Fluorescence analysis. The paper (Whatman #42) were placed on top of the
organic matter in the sample was determined by column to avoid surface disturbance of the sample
dichromate oxidation method. Heavy metal by influent droplets. The column was tightly closed
contents were determined by Inductively Coupled to prevent the evaporation of immobilizing agent
Plasma Atomic Emission Spectroscopy (ICP±AES aqueous solution when leaching was performed.
2000) after being dissolved in acid. Selected Before leaching, the poultry columns were
physicochemical properties of the investigated saturated with deionized water from bottom for
sample were measured and given in Table 1. As three hours. And then, the poultry column was
shown in the table, the organic matter in the poultry leached by immobilizing agent aqueous solution
was as high as 71.23%. And such heavy metals as (approximately 500 ml) from the top at an influx
Mn, Zn, Cu, Fe, and Cr were existed. According to UDWH RI PO PLQí )RU HDFK OHDFKLQJ WHVW
the result, the order of total metal content in poultry leachates were collected into a 1000 ml plastic
litter was Fe > Zn > Mn > Cu > Cr. Fe had the beaker below the poultry columns.
TABLE 1
Physicochemical properties of the sample.
Moisture/ Heavy metals background values/mg kgí1 Organic Oxides/%

% Mn Zn Cu Fe Cr matter/% Fe2O3 Al2O3 SiO2
3.28 328.43±2.1 634.15±3.2 199.97±1.5 5943.75±25.1 24.91±0.8 71.23±0.1 0.91 2.13 12.54
Finally, poultry was removed from the PVC and experimental conditions were given in Table 2.
column and mixed thoroughly. Each 2.0 g After each extraction, the suspension was
subsample of the poultry was air±dried and ground centrifuged for 20 min at 12000 g with a TG16-WS
to pass through a 60±mesh sieve for heavy metal high speed table-top centrifuge. The solution was
speciation determination. separated, while the precipitate was washed with 10
Chemical speciation determination of heavy ml high purity water and centrifuged again for 5
metals. In this study, five±step sequential extraction min. The washing water was added to the
technique developed by Tessier et al. [20] was used supernatant, while the precipitate was used for
to analyse the chemical fractions of Mn, Zn and Cu. subsequent extractions. The extracts were diluted to
The extraction steps were implemented based on 25 ml for soluble ion analysis. Concentrations of
references [21], and the details of different steps heavy metals in solution were measured by ICP±
982
AES. The sum of EXCH, CAR, Fe-MnOx, OMB, of heavy metals. And percentage of each fraction to
DQG 5(6 FRQFHQWUDWLRQV LQ PJ NJí GU\ PDWWHU total heavy metal concentration was calculated.
was defined as total concentration of the three types
FIGURE 1
Schematic diagram of leaching apparatus in column leaching study.
Analysis of immobili ing products. The the total amount. The different fractions of Cu were
crystallographic structure of the immobilizing in the order: OMB (79.44%) > RES (8.75%) >
product generated by adding immobilizing agent FeMnOx (7.13%) > CAR (3.19%) > EXCH (1.5%).
into the leachate was investigated by means of
glancing angle X±ray diffractometer (GAXRD) Immobili ing effect of sulphuring agent on
(Shimadzu XRD±ZLWK &X.ĮUDGLDWLRQ7KH heavy metals. The immobilization of heavy metals
incident angle was 0.5°. in poultry litter using DDTCNa as immobilizing
agent was investigated and the corresponding
results were shown in Table 3.
RESULTS AND DISCUSSION Accordingly, heavy metal stabilization in
poultry litter treated with Na2S was conducted as
Heavy metal speciation in poultry litter. The comparison. The dosage of immobilizing agent was
percent contributions of EXCH, CAR, FeMnOx, J NJí DJHQWSRXOWU\ OLWWHU ,W FRXOG EH
OMB, and RES fractions of Mn, Zn and Cu in their concluded from Table 3 that EXCH fractions of
total metal contents were shown in Fig. 2, Mn, Zn and Cu were reduced when Na2S and
respectively. DDTCNa were used as immobilizing agent.
As shown in the figure, the order of different Moreover, it was evident that better immobilization
fractions of Mn were: FeMnOx (34.89%) > CAR effect could be achieved when DDTCNa was used
(34.77%) > RES (11.76%) > OMB (10.92%) > for reducing EXCH fractions of heavy metals
EXCH (4.07%). And the percentage of EXCH significantly. In addition, DDTCNa and Na2S had
fraction of Mn was higher than that of other three similar immobilization effect on CAR fractions of
heavy metals. Among these three heavy metals, the heavy metals. And the fact that DDTCNa and Na2S
concentration of Zn was highest while its could immobilize heavy metals by the formation of
percentage of EXCH fraction was lowest. The order sulphide precipitates should be concluded from the
of different fractions of Zn was: FeMnOx (46.31%) results for OMB fractions of the heavy metals were
> OMB (22.07%) > CAR (18.17%) > RES (13.2%) increased obviously corresponding with the
> EXCH (0.24%). Cu mainly existed in OMB decrease of EXCH and CAR fractions.
fraction, whose percentage was more than 79% of
983
FIGURE 2
Percent contributions of heavy metal speciation in poultry litter. (a) Mn (b) Zn (c) Cu.
TABLE 2
Speciation scheme for poultry litter sample.
Fraction Reagents Experimental conditions

Exchangeable (EXCH) 1 mol/L MgCl2 (pH=7.0) Stirred for 1 h at 25Ԩ
Carbonate bound (CAR) 1 mol/L NaAc adjusted to pH=5.0 with Stirred for 8 h at 25Ԩ
HOAc
Fe±Mn oxide bound 0.04 mol/L NH2OH·HCl in 25% (v/v) Stirred for 4 h at 96Ԩ
(FeMnOx) HOAc
Organic matter bound 30% H2O2+3.2 mol/L NH4OAc in 20% Heated at 85Ԩ for 4h and stirred
(OMB) HNO3, adjusted to pH=2.0 with HNO3 for 1 h at 25Ԩ
Residual (RES) HNO3:HF:HClO4:HCl=4:1:1:1 Heated at 85Ԩ for 3h
984
TABLE 3
Comparison of heavy metal speciation in poultry litter when Na2S and DDTC was 2.0 g kgí1
(agent poultry litter).
Immobili ing agent Heavy metal speciation

EXCH CAR FeMnOx OMB RES
Mn mg kgí1
None 13.37±0.13 114.25±1.2 126.50±1.1 35.87±0.4 38.63±0.3
Na2S 9.13±0.08 109.75±0.7 130.93±1.7 35.87±0.4 38.75±0.3
DDTC 7.88±0.03 111.00±0.8 126.50±1.2 44.38±0.5 38.63±0.3
Cu/mg kgí1
None 3.00±0.2 6.38±0.05 14.25±0.8 158.87±1.0 17.50±0.1
Na2S 1.12±0.03 5.38±0.04 14.30±0.8 160.83±0.9 17.50±0.1
DDTC 0.75±0.01 5.25±0.04 14.25±0.8 161.88±0.9 17.50±0.1
Zn/mg kgí1
None 1.52±0.02 115.25±0.6 293.75±1.5 140.00±1.2 83.75±0.4
Na2S 0.72±0.01 110.46±0.4 293.98±1.5 145.98±1.2 83.87±0.4
DDTC 0.13±0.01 111.00±1.0 293.75±1.5 145.75±1.2 83.75±0.4
Effect of DDTCNa dosage on heavy metal again in acidic soils with breakdown of organic
immobili ation. Effect of DDTCNa dosage on matter which binds heavy metals [1,6]. Thus, pH±
heavy metal speciation variations were described in dependent leaching tests were conducted to
Fig.3. As shown in Fig.3, different immobilization investigate the sensibility of immobilized heavy
results were obtained when DDTCNa dosage varied metals to leaching pH. Before leaching, the
from 0 to 2.6g kgí1. (1) In the case of Mn, immobilization agent solution was adjusted to the
percentages of EXCH and CAR fractions tent to desired value by HCl or NaOH solutions.
decrease from 4.07% and 34.77% to 2.36% and
33.63%, respectively; the percentage of OMB Fig. 4 illustrated different speciation
fraction was clearly increased from 10.92% to percentages of heavy metals in poultry litter when
13.71%, while the percentages of FeMnOx and leaching pH changes from 4.50 to 7.00 at DDTC
RES fractions kept constant. (2) With agent dosage dosage of 2.0 mg kgí1. It was found that the
increasing, percentages of EXCH and CAR percentage of EXCH and CAR fractions increased
fractions of Zn decreased gradually, while the with leaching pH decreasing. For Mn, the
percentage of OMB fraction was rising. When the percentages of EXCH and CAR fractions were
GRVDJH ZDV KLJKHU WKDQ PJ NJí (;&+ decreased from 4.07% and 34.77% to 2.10% and
fraction of Zn could not be detected. (3) The 29.13%, when the leaching pH was increased from
increasing of agent dosage resulted in continuous 4.50 to 7.00, while the OMB fraction was increased
decrease of EXCH and CAR fractions of Cu and from 10.92% to 18.33%. The FeMnOx and RES
increase of OMB fraction. Especially, when the fractions were changeless with the variation of
GRVDJH ZDV DERYH PJ NJí QR REYLRXV leaching pH. For Zn, the increase of EXCH and
change was found for percentages of EXCH and CAR fractions were also negatively correlated with
CAR fractions of Cu. EXCH, associated with CAR the increase of leaching pH. When the leaching pH
fractions, are more labile than other fractions [22]. was increased from 4.50 to 7.00, the percentages of
According to Fig. 3, it was found that the EXCH and CAR fractions were decreased from
percentage of less stable fractions (EXCH and 0.24% and 18.17% to 0 and 16.97%, while the
CAR) was decreased with the increasing of less OMB fraction was increased from 22.07% to
mobility fraction (OMB) with DDTCNa addition. 23.47%. This revealed that EXCH fraction of Zn
Thus, low heavy metal risk to environment was has changed into more stable fraction entirely when
achieved. Moreover, weaker immobilization effect the leaching pH was higher than 7.0. When Cu was
of DDTCNa on Mn than on other two heavy metals discussed, the fact that leaching pH increase was
due to feeble metal chelate ring strain between responsible for the transformation of Cu from
DDTCNa and Mn [19] was also concluded. mobile fraction to stable organic fraction could be
concluded. And the percentages of EXCH and CAR
Effect of leaching pH on heavy metal fractions were decreased from 1.50% and 3.19% to
immobili ation. Previous reports indicated that 0.38% and 2.75%, respectively, with OMB fraction
immobilized heavy metals may be remobilized increased from 79.44% to 80.90%.
985
FIGURE 3
Heavy metal speciation in poultry litter with DDTC dosage.
Immobili ation mechanism analysis. LQWUDOLJDQGʌ±ʌ WUDQVLWLRQVDVVRFLDWHGZLWK1±C=S

According to immobilization results, OMB and S±C=S. Hence, stable sulphur±coordinated
fractions of the heavy metals were increased when four±membered ring of Me(DDTC)n predominated
DDTCNa was used as immobilizing agent. Thus, LQ FRPSOH[HV ZLWK WKH H[LVWHQFH RI VWURQJ ʌ-
organic sulphides were generated during coordinated bonds [15,18]. Thus, strong
immobilizing process. In fact, DDTC± was a stabilization effect on heavy metals could be
versatile class of anionic dithio ligand that could achieved.
coordinate around transition metals strongly due to
986
FIGURE 4
Percentage of heavy metal speciation in poultry litter at various leaching pH.
In order to demonstrate the immobilizing according to JCPDS±ICDD numbers 19±1634, 44±

mechanism of DDTCNa on heavy metals, 1815 and 44±1765, and no other compound peaks
immobilized products, generated by adding were identified. This revealed such four±membered
DDTCNa into heavy metal solutions, were chelating sulfide precipitates were generated when
subjected to an XRD analysis and the results were heavy metals were mixed with DDTCNa. And the
shown in Fig.5. Compared with the standard immobilizing mechanism could be described as Eq.
images, characteristic peaks of C10H20MnN2S4, (1). In the equation, Me represented such heavy
C10H20ZnN2S4 and C10H20CuN2S4 were found, metals as Mn, Zn or Cu.
987
Intensity (a.u.)
Mn
Zn
Cu
5 10 15 20 25 30 35 40 45
2 theta (Deg)
FIGURE 5
XRD patterns of precipitates generated by mixing DDTCNa with heavy metal solutions.
Generally, the electronic configuration of the reducing of leaching pH may result in

Mn(II), Zn(II) and Cu(II) were [Ar] 3d5, [Ar] 3d10 immobilized heavy metal leakage, so the optimal
and [Ar] 3d9. According to the ligand field immobilization effects can be estimated based on
stabilization energy (LFSE) theory, the lattice leaching pH of 6.0-7.0 for significant reduction of
energy for first row d±block metals was the order available heavy metals in the poultry litter. The
d5 < d6 < d7 < d8 < d9 > d10. As Mn(II), Cu(II) immobilization of DDTCNa on Mn, Zn and Cu was
and Zn(II) were d5, d9 and d10 ions in the first d- achieved for the formation of such stable sulphide-
block row, the stability order for the sulfide coordinated four-membered chelates of
precipitates were C10H20CuN2S4 > Me(DDTC)n.
C10H20ZnN2S4 and C10H20CuN2S4 >
C10H20MnN2S4, which was consistent with Yang
et al. [19] report. Thus, highest reduction rate can ACKNOWLEDGEMENTS
be obtained to Cu(II) when DDTCNa was used as
immobilizing agent. This work was financially supported by Major
Science and Technology Program for Water
Pollution Control and Treatment of China
CONCLUSIONS (2012ZX07202003).
Column leaching tests were carried out to

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989
CORRESPONDING AUTHORS
Wen-gang Liu
Associate professor
College of Resources and Civil Engineering
Northeastern University
Wenhua Road 3-11, Heping District
Shenyang 110819, Liaoning ± P. R. CHINA
e-mail: liuwengang@mail.neu.edu.cn
De- hou Wei

Professor
College of Resources and Civil Engineering
Northeastern University
Wenhua Road 3-11, Heping District
Shenyang 110819, Liaoning ± P. R. CHINA
e-mail: dzwei@mail.neu.edu.cn
990
E\3639ROXPH²1RSDJHV )UHVHQLXV(QYLURQPHQWDO%XOOHWLQ

SYNTHESIS, CHARACTERIZATION, ELECTROKINETIC AND

ADSORPTION PROPERTIES OF AN ORGANO-MODIFIED
ADSORBENT FOR SELECTIVE REMOVING OF METAL
IONS
3ÕQDU7XUDQBeyli, Mehmet Dogan, Mahir Alkan1$\GÕQ7UN\ÕOPD]2, Yasemin Turhan1, Özkan
Demirbas1, Hilmi Namli1
1
%DOÕNHVLU8QLYHUVLW\)DFXOW\RI6FLHQFHDQG/LWHUWXUH'HSDUWPHQWRI&KHPLVWU\dD÷Õú%DOÕNHVLU-TURKEY
1
Kastamonu University Faculty of Engineering and Architecture, Department of Enviromental Engineering Kastamonu-TURKEY
Firstly, sepiolite was modifed using N1-[3- The release of heavy metals such as Ni(II),
(trimethoxysiliylpropyl)]. In order to support the Cu(II), Zn(II), Pb(II), Co(II), etc. into the
modification of sepiolite surface, FTIR, XRD, environment in aqueous waste streams is a potential
DTA/TG and SEM analyses were carried out. It threat to water and soil quality. Unlike organic
was found that the chemical bonding took place pollutants, heavy metal ions are not biodegradable
between the hydroxyl groups and/or oxygen atoms and tend to accumulate in living organisms, leading
within the structure of sepiolite and methoxy to several types of disseases and disturbances
groups of N1-[3-(trimethoxysiliylpropyl)] (Coruh and Geyikçi 2012). The removal of heavy
diethyltriamine. Then, the removal of heavy metal metal ions from industrial wastewaters is a major
ions was systematically investigated as a function problem. Numerous processes exist for removing
of pH, ionic strength and temperature using AAS. It dissolved heavy metals, including ion exchange,
was found that the amount of adsorbed metal ions adsorption, precipitation, phytoextraction,
increased with increase in solution equilibrium pH ultrafiltration, reverse osmosis and electrodialysis.
and temperature, whereas it generally decreased One of the major methods for the removal of
with the ionic strength. The experimental data were pollutants from aqueous effluent is adsorption,
analyzed using Langmuir and Freundlich isotherms especially using low-cost natural sorbets such as
to identify the adsorption mechanism of metal ions agricultural wastes, clay materials, zeolite, biomass,
on organo-modified sepiolite, and found that the and seafood processing wastes. Activated carbon
isotherm data were reasonably well correlated by has been tested for the removal of inorganic ions
Langmuir isotherm. The Langmuir monolayer from aqueous solutions. Despite its prolific use in
adsorption capacity of modified sepiolite was water and wastewater industries, commercial
estimated as 118, 118, 110, 80, 37 and 30 mol g -1 activated carbon is not suitable in developing
for Cu(II), Zn(II), Fe(III), Mn(II), Cd(II) and countries due to the high costs associated with
Co(II), respectively. The affinity order of production and regeneration of spent carbon. This
adsorption was Cu(II)=Zn(II)>Fe(III) >Mn(II)> has led to search for low-cost materials to remove
Cd(II)> Co(II). The experiments demonstrated that pollutants (Coruh and Geyikçi 2012, 8÷XUOX
Cu(II), Zn(II) and Fe(III) ions were more efficiently Clays and clay minerals are used for a large
adsorbed than Cd(II) and Co(II) ions by modified variety of environmental applications such as water
sepiolite. The results show that modified sepiolite purification, waste treatment mineral barries for
can be used as an alternative low-cost selective- waste deposits and slurry walls for the
adsorbent in adsorption process, in encapsulation of contaminated areas (Bradl 2002).
chromatographic purification process and in The use of chemically modified clays and clays
nanocomposite synthesis. Finally, the zeta potential minerals containing a variety of functional groups
of the modified sepiolite suspensions was measured on a pendant organic chain can be designed for the
and it was found that the zeta potential of the adsorption and preconcentration of metal ions from
modified sepiolite in studied concentrations and pH aqueous and non-aqueous solutions. The
values was negative. preparation of solid adsorbents, to be applied for
removal of these contaminants from waste effluent
KEYWORDS: streams, is one of the major goals of green
Metal ions, modified-sepiolite, zeta potential, selective- chemistry (Prado et al. 2002). There are five types
adsorption, characterization of surface modifications that have received
991

attention: (1) adsorbed organic cations or sepiolite with quaternary amines; Alkan et al.
polycations, (2) adsorbed polyvalent metal ions (2005) the electrokinetic properties of sepiolite
with attached organic acid groups (metal soaps), (3) treated with some organosilanes; Torro-Palau et al.
surface silanol groups esterified with alcohols to (1997) the structural modification of sepiolite by
cover the surface with SiOR groups, (4) polymeric thermal treatment; Akyuz et al. (2001) the
organic coatings of various types, and (5) surface properties of adsorption of 3-aminopyridine on
silanol groups reacted with organosilicon sepiolite and montmorillonite; but no studies about
intermediates to produce a surface of Si-O-SiR3 or modification, adsorption and electrikinetic
similar multiply bonded (SiO)2SiR2 JURXSV'R÷DQ properties by N1-[3-(trimethoxysiliylpropyl)]
et al. 2008). One of the most important schemes of diethyltriamine compound of sepiolite have been
chemically surface modification involves silylation found. It is therefore the objective of this study; i. to
reaction reacted with organosilicon intermediates, modify the sepiolite surface by N1-[3-
and in general, reactions with MXm where M is a (trimethoxysiliylpropyl)] diethyltriamine, ii. to
metal and X is a halid, alkoxide, alkyl, alyl, characterize the modified sepiolite using FTIR,
carbonyl, etc. Silylation is the displacement of an XRD, DTA/TG and SEM, iii. to investigate the
active hydroxyl group by an organosilyl group. A adsorption behavior and electrokinetic properties of
major application for silylated surfaces has been in heavy metal ions on modified sepiolite and iv. to
modified glass fibers and fillers for preparation of describe the equilibrium isotherm. The effects of
organic polymer composites and chromatographic various parameters such as pH, ionic strength and
materials (Monde 2002). At the same time, temperature on metal ion adsorption and such as
chemically modified adsorbents have been heavy metal ion concentrations and equilibrium pH
considered for the selective removal processes of on the electrokinetic properties have been
metals from aqueous solutions. investigated. Langmuir and Freundlich adsorption
Sepiolite is a natural clay mineral with a unit models were applied to describe the equilibrium
cell formula of magnesium hydrosilicate isotherms and the isotherm constants were also
Si12Mg8O30(OH)4(OH2)48H2O. It is structurally determined.
formed by blocks and channels extending in the
fibre direction. Each structural block is composed
of two inverted tetrahedral silica sheets and a MATERIAL AND METHODS
central magnesium octahedral sheet. In the inner
blocks, all corners of the silica tetrahedral are Materials. Sepiolite sample used in this study
connected to adjacent blocks, but in the outer ZDV REWDLQHG IURP $NWDú /OHWDúÕ &R (VNLúHKLU
blocks, some of the corners are Si atoms bound to Turkey). The chemical composition of sepiolite has
hydroxyls (Si±OH) (Kuncek and Sener 2010). been given in Table 1. The cation exchange
These Si-OH groups play an important role in capacity of sepiolite is 25 meq 100g-1; the density
modification and adsorption processes. There are 2.55 g cm-3; the specifice surface area 342 m2 g-1;
limited studies related to the modification of particle size in the range of 0 to 50 μm. All
sepiolite, which may be used in the adsorption chemicals were obtained from Merck, Aldrich,
process, in chromatographic purification process Sigma and Fluka, were of analytical grade and used
and in nanocomposite synthesis due to increasing without further purification (Alkan et al. 2005).
the mechanical properties. For example, Lemic et
al. (2005) investigated the surface modification of
TABLE 1
The chemical composition of sepiolite
Composition Weight (%)

SiO2 53.47
MgO 23.55
CaO 0.71
Al2O3 0.19
Fe2O3 0.16
NiO 0.43
Loss of ignition 21.49
992
Methods. Modification of sepiolite with N1- agitation for 24 h in an incubator, the suspension
[3-(trimethoxysiliylpropyl)]diethyltriamine. was allowed to stand for 15 min to let the larger
Sepiolite samples were treated before using in the particles settle. An aliquot taken out from the
experiments in order to obtain a uniform size suspension was slowly poured into the
sample of adsorbent as follows: the suspension electrophoresis cell. Molybdenum anode and
containing 10 g L-1 sepiolite was mechanically platinum cathode electrodes were inserted into the
stirred for 24 h, after waiting for about two minutes cell. After a desired voltage applied between the
the supernatant suspension was filtered through electrodes, the movement of particles was observed
filter paper. The solid sample was dried at 105 oC via a microscope nodule. Minimum 10
for 24 h, ground, and then sieved by 50 μm sieve. measurements were carried out to present the
The particles under 50 μm were used in further measured potential. The applied voltage during the
experiments 'R÷DQ HW DO 6HSLROLWH J measurements was varied in the range 50±150 mV.
suspended in toluene (100 mL) was refluxed and The pH that was made the zeta potential
mechanically stirred for 1 h under dry nitrogen. To measurement was recorded as equilibrium pH. The
this suspension N1-[3-(trimethoxysiliylpropyl)] instrument was tested prior to each test using the
diethyltriamine (5.0 mL) was added dropwise. The test colloid, as recommended by the supplier
mixture was refluxed for another 24h, filtered and 'R÷DQHWDO
washed with water, followed by methanol and
acetone. Modified sepiolite was dried at 110 0C Adsorption procedures. Batch sorption
(Menezes et al. 1996). experiments were performed at room temperature
on an incubator at 150 rpm using 100 mL capped
Zeta potential. To determine the zeta polyethylene bottles containing metal ion solutions
potential of organo-modified sepiolite, the and the adsorbent. 0.1 g of modified sepiolite was
electrokinetic measurements were conducted using added into 50 mL of metal solutions and shaken
a Zeta Meter Model 3.0+ (Zeta Meter Inc., USA) continuously at room temperature. A thermostated
equipped with a microprocessor unit to directly orbital shaker incubator was used to keep the
calculate the zeta potential. The instrument temperature constant. Stock solutions were
determines the electrophoretic mobility of the prepared in deionized water for each metal ion and
particles automatically and converts it to the zeta further experimental solutions were prepared from
SRWHQWLDO XVLQJ 6PROXFKRZVNL¶V HTXDWLRQ The the stock solution by successive dilution. The pH of
6PROXFKRZVNL¶V HTXDWLRQ WKH PRVW HOHPHQWDU\ the solutions was adjusted with NaOH or HNO3
expression for zeta potential, gives a direct relation solution by using an Orion 920A pH meter with a
between zeta potential and electrophoretic mobility, combined pH electrode. The pH meter was
standardized with NBS buffers before every
4ʌȘ measurement. A preliminary experiment revealed
ȗ uU that about 24 h is required for metal ions to reach
İ the equilibrium concentration. The effect of pH,
ionic strength and temperature on the adsorption of
(1) heavy metals such as Cu(II), Zn(II), Fe(III), Mn(II),
Cd(II) and Co(II)) was also studied. It was used the
where U is electrophoretic mobility at actual nitrate salts of metal ions in the experiments. At the
temperature, K is viscosity of the suspending liquid, end of the adsorption period, the solution was
H LVGLHOHFWULFFRQVWDQWʌLVFRQVWDQWDQG ] is zeta centrifuged for 15 min at 5000 rpm. Five standard
potential. The pH measurements were performed solutions for each metal ion were prepared in 100
using a Orion 920A pH meter with a combined pH mL flasks. Calibration graph for each batch of
electrode. Double-distilled water was used to experiments were re-constructed by using the
prepare aqueous solutions for all experiments. The standard solutions. The blank experiments were
samples were conditioned under the adsorption test simultaneously carried out without the adsorbent.
conditions. For the electrophoretic mobility Filter paper was not used in any part of the
measurements, the suspensions were prepared in a experiments, since filter paper itself adsorbs about
polyethylene container by mixing 0.1 g of the § RI WKH SUHVHQW PHWDO LRQV SDUWLFXODUO\ DW ORZ
modified sepiolite with 100 mL of double-distilled concentration levels. Each run was repeated at least
water, after the desired amounts of nitrate salts of twice. The final concentration of metal ions in the
heavy metals were added according to the solutions was determined by Unicam 929 flame
experimental conditions. The zeta potential atomic absorption spectrometer operating with an
measurements were carried out as a function of the air-acetylene flame (AAS). The amount of metal
metal ion concentration and equilibrium pH. After ions adsorbed onto modified sepiolite was
993
calculated from the difference between initial and transmission mode. The interaction between the
residual concentration in solution as follows: sepiolite surface and the modifier N1-[3-
(trimethoxysiliylpropyl)]diethyltriamine during the
V modification was investigated by a series of FTIR
qe (C0 - C e ) (2) spectral analysis: 1. modifier sample, 2. sepiolite
W sample and 3. modified sepiolite sample. X-ray
diffraction measurements of natural and modified
where qe is the amount adsorbed (mol g-1), C0 and sepiolite samples were performed using a
Ce are the initial and equilibrium liquid-phase $QDO\WLFDO 3KLOLSV ;¶3HUW-Pro X-ray diffractometer
concentrations of metal ion solution (mol L-1), equipped with a back monochromator operating at
respectively, V is the volume of metal ion solution 40 kV and a copper cathode as the X-ray source (Ȝ
(L), and W is the mass of the modified-sepiolite = 1.54 Å). A simultaneous DTA/TG system was
VDPSOHJ$ONDQDQG'R÷DQ used for thermogravimetric (TG) analysis
(PerkinElmer Diamond DTA/TG). To observe the
Characterization. Infrared spectra of samples surface topography of sepiolite and modified
were obtained using a PerkinElmer BX 1600 FTIR. sepiolite, scanning electron micrographs (SEM)
About 0.01 g of clay was mixed with 1 g of were performed using a Zeiss EVO LS 10. Most of
potassium bromide (KBr) and pelletized in the the measurements were carried out using an
hydraulic press at 10 kPa. FTIR spectra were taken accelerating voltage of 20 kV.
in the range from 4000 to 400 cmí1 in the
FIGURE 1
FTIR spectra of (a) N1-[3-(trimethoxysiliylpropyl)]diethyltriamine, (b) sepiolite and (c) modified structure
in the KBr disks.
RESULTS AND DISCUSSION treatments or modification processes may be fully

characterized. Infrared spectra of adsorbed probe
Characterization of Modified-Sepiolite. molecules are widely used to provide additional
FTIR Analysis. The existence of different types of characterization not only of surface hydroxyl
surface hydroxyl groups on an oxide is easily groups but also of other types of exposed site
recognized from infrared spectra, and the behavior (Anderson and Rochester 2002). Figure 1 has
of the hydroxyl groups when subjected to thermal shown FTIR spectra of sepiolite, modifier and
modified sepiolite. As can be seen from spectra,
994
there are important interactions between modifier process, and the peak of modifier at 1307 cm-1
and sepiolite. The peak at 2939 cm-1 of modifier shifted at 1314 cm-1. According to the explanations
due to alkyl groups was observed at 2938 cm-1 in above, the reaction between modifier and sepiolite
modified sepiolite. Moreover, C-N peak of modifier can be written as following:
at 1470 cm-1 shifted at 1478 cm-1 after modification
6(3,2/,7(685)$&(
OH
OH
OH H3CO Reflux
+ H3CO Si (CH2)3 NH (CH2)2 NH (CH2)2 NH2
OH Toluene
H3CO
OH
OH
Sepiolite N1-[3-trimethoxysilylpropyl)]diethyltriamine
6(3,2/,7(685)$&(
O
O Si (CH2)3 NH (CH2)2 NH (CH2)2 NH2
O
O
O
Modified Sepiolite (3)
TG analysis. The variation of percent weight ଼ ଵଶ ଷ଴ ሺሻସ ሺଶ ሻସ ՜ ଼ ଵଶ ଷ଴ ሺሻସ ሺଶ ሻଶ ൅ ʹଶ
loss with temperature (TG curves) and differential (5)
weight loss with temperature (d[TG] curves) for
sepiolite and modified sepiolite under nitrogen and The bound water and structural water leave the
oxygen atmospheres have been given in Figure 2. mineral at higher temperatures. The bound water is
Sepiolite mineral is known to contain four types of found in magnesium coordination in the crystal
water molecules in different chemical states; i. structure. It is known that dehydration of it occurres
hydroscopic water, ii. zeolitic water, iii. bound in two steps, accompanied by structural changes.
water and iv. structural water (Alkan et al. 2005; Literature studies have shown that the first part of
Balci 1996) which are removed from the mineral by the bound water leaves the structure between 250°C
thermal dehydration at different temperatures. The and 450 0C. The following equation represents the
amount of hydroscopic water depends on the explanations above:
humidity of the environment. It leaves the mineral
at lower temperatures. In this case, the reaction may ଼ ଵଶ ଷ଴ ሺሻସ ሺଶ ሻଶ ՜ ଼ ଵଶ ଷ଴ ሺሻସ ൅ ʹଶ (6)
be written as following:
By the removal of some parts of the bound
଼ ଵଶ ଷ଴ ሺሻସ ሺଶ ሻସ ͺଶ ՜ ଼ ଵଶ ଷ଴ ሺሻସ ሺଶ ሻସ ൅ water, rearrangements within the crystal structure
ͺଶ (4) may have occurred. The breakage of water
molecules and the transportation of them from the
The zeolitic water is found in the channel-type small pores to the channel voids, become difficult
voids and starts to leave the structure above room due to the crystal folding. The remaining part of the
temperature. The removal from the structure of bound water begins to leave the structure at higher
zeolitic water can be given in following reaction: temperatures.
଼ ଵଶ ଷ଴ ሺሻସ ՜ ͺଷ ൅ Ͷܱܵ݅ଶ ൅ ʹଶ (7)
995
FIGURE 2
(A) TG and (B) d[TG] spectra of natural and modified sepiolite samples under different conditions: a.
modified sepiolite under oxygen atmosphere, b. modified sepiolite under nitrogen atmosphere, and c.
sepiolite under nitrogen atmosphere.
The removal of bound water is completed oxygen were calculated as 20.5 and 20.1,
before 750°C. The removal of structural water respectively. Again, percent weight losses for
molecules (hydroxyl groups) is completed up to modified sepiolite under nitrogen and oxygen were
850°C (Balci 1996;) As can be seen from Figure 2, determined as 4.5 and 11.8 in the temperature range
the removal of hydroscopic water from the external of 0 to 200 0C; 19.6 and 24.0 in the temperature
surface and zeolitic water from the voids of the range of 200 to 600 0C; and 5.0 and 4.1 in the
structure was completed by 200 °C. The bound temperature range of 600 to 900 0C, respectively.
water molecules left the structure higher Total percent weight losses for modified sepiolite
temperatures (200-600 0C) than the hydroscopic under nitrogen and oxygen were calculated as 29.1
and zeolitic water molecules. The removal of and 39.9, respectively. In addition, any weight loss
structural water molecules and hydroxyl groups was was not observed by heating of the samples up to
completed until 850 0C. Percent weight losses for 1100 0C. Results showed that the percent weight
sepiolite under nitrogen and oxygen were loss of modified sample increased. In this case, it
determined as 9.6 and 9.4 in the temperature range can be said that sepiolite was modified by
of 0 to 200 0C; 7.4 and 7.5 in the temperature range organosilane compound and formed a chemical
of 200 to 600 0C; and 3.5 and 3.2 in the temperature bond between the hydroxyl groups on the sepiolite
range of 600 to 900 0C, respectively. Total percent and modifier substance.
weight losses for sepiolite under nitrogen and
996
Ă͘
ď͘
FIGURE 3
XRD spectra of (a) natural and (b) modified sepiolite samples.
997

Ă͘
ď͘ FIGURE 4
SEM images of (a) natural and (b) modified sepiolite samples.
XRD. Figure 3 shows XRD patterns of modified by N1-[3-

sepiolite and modified sepiolite. The XRD pattern (trimethoxysiliylpropyl)]diethyltriamine.
of sepiolite is clearly affected by the incorporation
of N1-[3-(trimethoxysiliylpropyl)]diethyltriamine. Electrokinetic properties. The zeta potential
Covalent bonding is formed because, as seen in of modified sepiolite in single aqueous solutions of
Figure 3, the some peak intensities both increased Cu(II), Zn(II), Fe(III), Mn(II), Cd(II) and Co(II)
and decreased by modification. When the covalent was investigated. Figure 5 illustrates the zeta
bonding is formed by the interaction between potential of modified sepiolite as a function of
hydroxyl groups on sepiolite and N1- [3- different metal ion concentrations at natural pH.
(trimethoxysiliylpropyl)] diethyltriamine, some Since there is no experimental method for
structural distortions occur at the plane atoms in the determining both the surface potential and Stern
sepiolite. As a result of these distortions, the peak layer potential, the zeta potential is the measurable
intensity is changed. surface potential at the shear plane between the
particle and the surrounding liquid (Kara et al.
SEM. Sepiolite and modified sepiolite were 2003). Although the zeta potential values with
examined by SEM and representative micrographs heavy metal cations are quite different, their
are shown in Figure 4. The modification process tendency to change with zeta potential is similar
produced a reduction in size of aggregates that (Figure 5). The values on the curves obtained for
showed flake type morphology. SEM image of each heavy metal did not change the sign of surface
modified sepiolite showed that sepiolite losed its negative charge. The results indicated that
fibrous morphology after modification process. increasing metal ion concentration in solution
This result has shown that sepiolite surface was decreased the negative charge of surface (becomes
less negative) without changing the sign of the
998


surface charge. This case can be explained the then the negative charge decreases as the
compression of the electrical double layer at the concentration of metal ion increases. Note that, at
modified sepiolite surface. For a cation to be a the heavy metal ion concentrations studied,
potential-determining ion (pdi), it should render the modified sepiolite does not exhibit a charge
surface more positive with increasing concentration reversal. Indeed, charge reversal requires, in
of the cation. As can be seen from Figure 5, the addition to ion exchange, specific adsorption of
surface is, at first, more negatively charged, and counterions in the electrical double layer,
ż : Fe
Ŷ : Mn
Ɣ : Co
' : Cd
Ƒ : Cu
Ÿ : Zn
FIGURE 5
Effect of heavy metal ion concentrations on zeta potential of modified sepiolite.
that is, in the inner Helmhotz plane of the Stern range. This is another sign that the sepiolite surface
layer. As the heavy metal cations did not reverse was modified by N1-[3-
the sign of the surface, these cations adsorb as (trimethoxysiliylpropyl)]diethyltriamine. For all
indifferent electrolytes onto modified sepiolite and metal ions, the zeta potential became more negative
are not capable of causing a charge reversal. with increasing equilibrium pH. There is no change
Because of Coulombic attraction, they accumulate in the sign of the surface charge of modified
only as counterions in the electrical double layer on sepiolite for any of the metal ions. The modified-
the negatively charged modified sepiolite. sepiolite surface is negatively charged, and metal
Consequently, they compress the electrical double ions cannot specifically adsorb onto the modified-
layer and change the magnitude of the zeta potential sepiolite surface and cannot alter the sign of the
of modified sepiolite (Turhan et al. 2008). zeta potential. At low pH values, the adsorption of
The zeta potential curves as a function of pH H3O+ hinders the specific adsorption of metal ions.
for modified sepiolite in the presence of different For this reason, as the pH increases, the negative
metal ions are displayed in Figure 6. Previous sign of the surface increases because of the
investigations on the electrokinetic properties of releasing of H+ ions on two ±NH2+- or one ±NH3+
different types of clay minerals indicated groups of modifier.
differences between various minerals in their pH
dependence (Sondi and Pravdic 1996; 'R÷DQHWDO Adsorption properties. There are some
2000). The pH-dependent changes are due to physical and chemical parameters that influence the
reactive hydroxyl groups at the edge surfaces. adsorption of heavy metal ions in aqueous
Indeed, the fraction of the edge surfaces and, thus, solutions. The most important ones are pH, the
of reactive hydroxyl groups in the total surface area presence of other cations in solution, and
varies depending on the type of clay minerals. As temperature. All these variables may change the
seen from Figure 6, the most important metal adsorption isotherms (Bradl 2002). We have
observations are that modified sepiolite does not discussed the experimetal data obtained for
has isoelectrical point over the pH range of 1 to 7 adsorption of metal ions from aqueous solutions in
and has negative zeta potential in the studied pH this section.
999


-50
=HWDSRWHQWLDOP9
-100
ż : Fe
-150 Ŷ : Mn
Ɣ : Co
-200 ' : Cd
Ƒ : Cu
-250 Ÿ : Zn
-300
1 2 3 4 5 6 7
(TXLOLEULXPS+
FIGURE 6
Effect of pH on zeta potential of modified sepiolite
ż : Fe
Ŷ : Mn
Ɣ : Co
' : Cd
Ƒ : Cu
Ÿ : Zn
FIGURE 7
Isothern curves for adsorption data
After incorporating the silylating agent into changing of adsorbed metal ions versus the
clay structure, the pendant groups disposed on the concentration of solution under equilibrium
structure enable this material to interact with a wide conditions has been given in Figure 7, which shows
range of cations due to the presence of three basic that the amount adsorbed of metal ions increased
centres; three characteristically hard basic centres. with increasing metal ion concentration. The higher
This fact improves the process of interaction with the initial metal ion concentration, the larger the
various metal ions (Prado et al. 2002). The metal ion sorbed per unit weight of adsorbent at
1000


equilibrium. The selectivity of different adsorbents for Cu(II), Zn(II) and Fe (III) ions than other ions.
for metal ions cannot be given according to When Cu(II), Zn(II) and Fe (III) have hard and
universally consistent rules as it depends on a borderline acid character, the nitrogen atom of
number of factors such as i. the chemical nature of modifier has hard basic character (Yantasee et al.
the reactive surface groups, ii. the level of 2004). Based on the structural features presented by
adsorption, iii. the pH at which adsorption is the pendant groups attached to the sepiolite surface,
measured, iv. the ionic strength of the solution in the hard basic atoms of modifier can easily bond to
which adsorption is measured and v. the presence the hard or borderline cations. Thus, metal ion
of soluble ligands that could complex the free metal adsorption should be via nitrogen in the case of
[Bradl, 2002]. As seen from Figure 7, the hard and borderline metal cations, as proposed in
adsorption capacity of modified sepiolite is higher reaction (8).
6(3,2/,7(685)$&(
O
O Mn+
+
O
O
6(3,2/,7(685)$&(
O
O
Mn+ Mn+
O
O
(8)
The most important factor which controls the of adsorption for metal ions was investigated in
adsorption capacity of metal ions on different detail. The pH dependence of metal ion uptake onto
adsorbents is pH, which affects the surface structure modified sepiolite is shown in Figure 8. The
of sorbents, the formation of metal hydroxides, and removal efficiency of metal ions generally
the interaction between sorbents and metal ions. increased with increasing pH in the pH range of 1
Change of pH affects the adsorptive process to 7. This can be explained by considering the
because of the protonation and deprotonation of the surface charge of modified sepiolite. For clay
acidic and basic groups on the adsorbent surface minerals, the potential determining ions are H+ and
active sites (Kuncek and Sener 2010; Duru et al. OH- and complex ions formed by bonding with H +
2001). Therefore, in this study, the pH dependence and OH-.
1001


ż : Fe
Ŷ : Mn
Ɣ : Co
' : Cd
Ƒ : Cu
Ÿ : Zn
FIGURE 8
Variation of the adsorption of metal ions with pH on modified sepiolite
As the pH is lowered, the hydrogen ions Figure 9 shows the adsorbed amount of metal
compete with the metal ions for the sorption sites ions on modified sepiolite from aqueous solutions
on the sorbent and may hinder the binding of in different NaCl concentrations. As seen in Figure
positively charged metal ions. In this case, the 9, increasing the ionic strength of the solution
adsorption of metal ions decreases. Increasing pH slightly causes a decrease in the adsorption of metal
means decreasing H+ concentration and more metal ions onto the modified sepiolite surface. This
ions are able to occupy adsorption sites at the indicates that the negative charge of the surface of
surface. Therefore, the adsorption of metal ions modified sepiolite, which has no pHpzc in the pH
increases. As a result, as the pH of the system range of 1 to 7, decreases with increasing ionic
increases, the number of negatively charged sites strength, resulting in reducing the adsorption
increases due to deprotonation of the surface. A capacity (Eren et al. 2010).
negatively charged surface site on the modified In order to study the effect of temperature to
sepiolite favors the adsorption of metal cations. the adsorption of metal ions on modified sepiolite a
Lower adsorption of metal ions at acidic pH is series of experiment was conducted in the
probably due to the presence of excess H+ ions temperature range of 30 to 60 0C. The results are
competing with the metal cations for the adsorption presented in Figure 10. There is an increase in the
sites. In low and high pH, the surface of modified adsorbed amount of metal ions with a rise in
sepiolite can be shown as following: temperature which may be due to higher adsorption
caused by an increase in the thermal energy of the
adsorbates. This indicates that the adsorption
6(3,2/,7(
2
process is endothermic. The effect of temperature is
2 6L &+ 1+ &+ 1+ &+ 1+
fairly common and increasing temperature results in
2
/RZS+ an increase in the rate of approach to equilibrium.
In addition, the temperature coefficient for the
2
reverse reaction is lower than for the forward
6(3,2/,7(
reaction and consequently the equilibrium capacity

2 6L &+ 1+ &+ 1+ &+ 1+
increases with increased temperature (Kuncek and
2
+LJKS+
Sener 2010).
(9)
1002


ż : Fe
Ŷ : Mn
Ɣ : Co
' : Cd
Ƒ : Cu
Ÿ : Zn
FIGURE 9
Variation of the adsorption of metal ions with ionic strength on modified sepiolite
ż )H
Ŷ 0Q
Ɣ &R
' &G
Ƒ &X
Ÿ =Q

FIGURE 10
Variation of the adsorption of metal ions with temperature on modified sepiolite.
1003


Adsorption isotherms. The analysis of the comparison of adsorption performance. q m and K

isotherm data is important to develop an equation are calculated from the slopes and intercepts of the
which accurately represents the results and which straight lines of plot of Ce/qe vs. Ce.
could be used for design purposes. The adsorption
curves were applied to both the Langmuir and The Freundlich isotherm is given as,
Freundlich equations. The widely used Langmuir
isotherm has found successful application to many qe K FC1e n
real sorption processes and is expressed as in the
linear form:
in logarithmic form,
Ce 1 C
e 1
qe qmK qm lnqe lnKF lnCe
n
(10)
(11)
-1
where qe is the amount adsorbed (mol g ), Ce is the
where KF is roughly an indicator of the adsorption
equilibrium concentration of the adsorbate ions capacity and (1/n) of the adsorption intensity. KF
(mol L-1), and qm and K are Langmuir constants and (1/n) can be determined from the linear plot of
related to maximum adsorption capacity lnqe vs. lnCe.
(monolayer capacity) and energy of adsorption,
The parameters of Freundlich and Langmuir
respectively. qm represents a practical limiting
isotherms are given in Table 2.
adsorption capacity when the surface is fully
covered with metal ions and assists in the
TABLE 2
Isotherm parameters for the adsorption of metal ions on the modified sepiolite
Langmuir isotherm Freundlich isotherm

Metals t (0C) pH
qmx105 (mol g-1) Kx10-4 (L mol-1) R2 n KF x103 R2
Mn 30 4.0 80 4.34 0.981 1.67 49.29 0.965
Cu 30 4.0 118 2.05 0.998 3.71 8.31 0.918
Fe 30 3.0 110 1.39 0.998 2.06 41.17 0.928
Zn 30 4.0 118 1.77 0.996 2.47 23.75 0.968
Co 30 4.0 31 1.19 0.992 2.42 6.15 0.946
Cd 30 4.0 37 1.18 0.984 1.60 53.39 0.945
Langmuir isotherm fits quite well with the Theoretically, sepiolite has a finite capacity
experimental data (correlation coefficient R2>0.98), for adsorbing metal ions (Kocaoba 2009; 8÷XUOX
whereas the low correlation coefficients (R2<0.96) 2009).
show poor agreement of Freundlich isotherm with According to the equilibrium isotherms, the
the experimental data. selectivity series is
The Langmuir isotherm models the monolayer Cu(II)=Zn(II)>Fe(III)>Mn(II)>Cd(II)>Co(II). An
coverage of the adsorption surface. Once metal ion inspection of the qm values in Table 2 demonstrate
occupies a site on the modified sepiolite surface, no that Cu(II), Zn(II) and Fe(III) are easily the most
further adsorption of metal ions can take place at effective in binding to the pendant groups. This
that site. This model assumes that adsorption occurs behaviour reflects the high affinity of the nitrogen
at specific homogeneous adsorption sites within the basic centres for Cu(II), Zn(II) and Fe(III). This fact
adsorbent and intermolecular forces decrease suggests that this surface is more favourable in
rapidly with the distance from the adsorption reacting with hard and borderline cations. The
surface. The Langmuir adsorption model further selectivity of different clay minerals for heavy
based on the assumption that all the adsorption sites metal ions has changed. For example, it was found
are energetically identical and adsorption occurs on that the selectivity series for montmorrillonite was
a structurally homogeneous adsorbent. Ca>Pb>Cu>Mg>Cd>Zn, for illite
Pb>Cu>Zn>Ca>Cd>Mg, for kaolinite
1004


Pb>Ca>Cu>Mg>Zn>Cd, and for smectite [2] $ONDQ 0 dHOLNoDSD 6 'HPLUEDú g 'R÷DQ 0
Zn>Mn>Cd>Hg (Bradl 2002). In light of these (2005) Removal of reactive blue 221 and acid
observations, this material could be potentially blue 62 anionic dyes from aqueous solutions
applied as a selective electrode when the objective by sepiolite. Dyes and Pigments 65: 251-259.
is to determine or identify hard and borderline [3] $ONDQ 0 'R÷DQ 0 $GVRUSWLRQ RI
acids. Another feature connected to the use of this copper(II) onto perlite. Journal of Colloid and
surface is its good ability to separate these cations Interface Science 243: 280±291.
from a water solution. [4] Alkan M, Tekin G, Namli H (2005) Zeta
potentials of perlite modified with dimethyl-
dichlorosilane, 3-aminopropyltriethoxysilane
CONCLUSIONS and dimethyloctadecylchlorosilane.
Microporous and Mesoporous Materials 84(1-
Sepiolite surface was modified with N1-[3- 3): 75-83.
(trimethoxysiliylpropyl)]diethyltriamine. This [5] Anderson JA, Rochester CH (2002) Infrared
surface presents a high affinity for Cu(II), Zn(II) spectroscopy of silica, alumina, and related
and Fe(III) due to the presence of three basic surfaces. Encyclopedia of Surface and Colloid
nitrogen centres. This anchored surface also Science. Marcel Dekker, Inc..
presents a good adsorption capability for other [6] Balci S (1996) Thermal decomposition of
divalent cations. Modified sepiolite surface has a sepiolite and variations in pore structure with
negative charge in solutions in the pH range of 1 to and without acid pre-treatment, J. Chem.
7. The results of this study indicate that modified Technol. Biotechnol 66: 72±78.
sepiolite can be successfully used for the removal [7] Beene GM, Bryant R, Williams DJA (1991)
of heavy metal ions from aqueous solutions. Electrochemical properties of illites. J. Colloid
Correlation coefficients for the isotherm models Interface Sci. 147: 358±369.
indicate that the Langmuir model best describes the [8] Bradl H (2002) Adsorption of heavy metal ions
metal adsorption process, experimental data are on clays. Encyclopedia of Surface and Colloid
better fitted to the Langmuir isotherm and Science. Marcel Dekker, Inc..
adsorption capacities order was found as [9] Coruh S, Geyikçi F (2012) Adsorption of
Cu(II)=Zn(II)>Fe(III)>Mn(II)>Co(II)>Cd(II). In copper (II) ions on montmorillonite and
summary, the surfaces of modified sepiolite present sepiolite clays: equilibrium and kinetic
a high affinity for Cu(II), Zn(II) and Fe(III), and studies. Desalination and Water Treatment 45:
low affinity for Co(II) and Cd(II). This fact permits 351±360. [@'R÷DQ0$ONDQ02QJDQHU
an excellent separation of Cu(II), Zn(II) and Fe(III) Y (2000) Adsorption of methylene blue from
from other cations, and these surfaces may be aqueous solution onto perlite. Water, Air and
useful in separating these cations. These abilities Soil Pollution 120: 229-248.
can be explored for applications in clean [12] 'R÷DQ 0 7XUKDQ < $ONDQ 0 Namli H,
technologies reinforcing the importance of Turan P, Demirbas O (2008) Functionalized
developments in basic sciences for green chemistry, sepiolite for heavy metal ions adsorption.
in chromatographic applications and in the Desalination 230: 248±268.
manufacture of a chemical sensor. [13] 'XUX3(%HNWDV6*HQog3DWÕU6'HQL]OL$
(2001) Adsorption of heavy-metal ions on
poly(ethylene imine)-immobilized
ACKNOWLEDGEMENT poly(methyl methacrylate) microspheres.
Journal of Applied Polymer Science 81:. 197±
205.
The authors gratefully acknowledge the
[14] Eren E, Cubuk O, Ciftci H, Eren B, Caglar B
ILQDQFLDO VXSSRUW RI 78%ø7$. 7%$*-2455
(104T067)). (2010) Adsorption of basic dye from aqueous
solutions by modified sepiolite: Equilibrium,
kinetics and thermodynamics study.
Desalination 252: 88±96.
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thermal treatment: effect on the properties of
1006


SETTLEMENT OF BLUE CRAB (Callinectes sapidus)

MEGALOPAE IN THE NORTH SHORE OF YUMURTALIK
COVE (TURKEY)
Irem Nur Yesilyurt and Canan Tureli
Cukurova University, Faculty of Fisheries, Department of Basic Sciences 01330 Adana, Turkey.
ABSTRACT Turkey [1]. Besides, the Blue Crabs are

economically the most important and useful species
There is not any information on the time in the Eastern Mediterranean of Turkey among
series, type and density of the Blue Crab post- other crab species [2,3]. The Blue Crabs mate in the
larvae (megalopae) settlement in the Mediterranean areas of low salinity in the lagoons and mated
Basin in Turkey. This study aims to uncover the female blue crabs migrate from areas of low salinity
settlement time, area and amount of the Blue Crab to areas of high salinity in order for the eggs to
post-larvae in the north shoUH RI <XPXUWDOÕN &RYH hatch and grow up and the eggs are hatched in that
(northwestern of Iskenderun Bay) and to define the area [4, 5]. Newly hatched planktonic zoeal larvae
relations between post-larvae settlement and are transported to inshore waters of the continental
ecological factors like temperature, salinity, moon shelf via surface currents. The larvae remain on the
SKDVHV ,Q WKH <XPXUWDOÕN &RYH SRVW-larvae water surface in their approximately 50-day
samples were collected every other day on standard development process [6, 7, 8]. In this period, the
artificial collectors during June-November 2011 at larvae metamorphose to post- larvae through
the 4 stations which have different bottom completing 7 or 8 complicated zoeal stages [9].
structures. At the end of study 28 a i After these stages are completed, post- larvae
a i , 218 sp. and 8 from other species (megalopae) or juveniles are transported back to the
of the Portunidae family post-larvae were caught. lagoon [10]. The post-larvae stage occurs in
54% of the total number of blue crab post-larvae shallow waters and concentrated in neuston layer.
settled on sea grass beds, 25% on marhes and The metamorphosis from planktonic stage to
muddy bottoms, 14% on sandy and grassy areas benthic stage is the most important phase in the life
and 7% on sandy bottom areas. Settlement occurred cycle of many benthic invertebrates with planktonic
between mid-June and mid-October. The densest larvae. This period in the life cycle of many benthic
settlement was recorded in August and September organisms substantially affects the population
(86%). Largest settlement peak occurred at the time density. It has been known that the settlement of the
of the full moon and major settlement peak on a post-larvae is affected by biotic (fecundity,
single sampling date was detected on the 19th of behavior, predation) and abiotic factors (currents,
July (full moon) and 29th of August (new moon). In temperature, salinity, phases of the moon) [9]. In
this study, at least some very important information early stages of crabs are more sensitive to
on the post-larval stage ± one of the most important pathogens and environmental stressors [11].
life stages- of the Blue Crab population in our Diseases have been established causing mortality
region has been obtained for the first time. and decreasing fecundity of blue crabs and the other
crab species [12, 13].
KEYWORDS The information on the settlement time, type
Blue Crab, Callinectes sapidus, Post-larvae (Megalopae), and density of the Blue Crab post-larvae in South-
Settlement, Iskenderun Bay, Mediterranean and Mid-Atlantic and the Gulf of Mexico has been
acquired from the data obtained from the passive
collectors [14, 15, 16, 17]. For the Mediterranean
INTRODUCTION Basin, on the other hand, there is not any
information on the settlement time, type and density
The Blue Crab, Callinectes sapidus Rathbun, of the Blue Crab post-larvae. By conducting this
1896 is an endemic species of the Atlantic basin. study, it has been aimed to obtain information about
Outside its endemic borders, it has been reported the settlement period, area and abundance of the
that they have densely populated in 15 lagoon Blue Crab post-larvae in the north shore of
systems found in the Mediterranean shores of <XPXUWDOÕN&RYHDQGWRGHILQHWKHUHODWLRQEHWZHHQ
1007


ecological factors like temperature, salinity, lunar were designated as four different stations with
phase that affect the settlement of the post-larvae. It different bottom structures on the same line starting
is of essential importance to know the life cycle of from the inner side of the cove on the northwestern
the Blue Crab population in the cove in terms of part of the cove to the Bay (Figure 1). The 1 st
determining the supply density and sustainability of Station was set on the marshes and muddy areas in
the Blue Crabs in our region. the inner right side of the cove; the 2nd Station was
on a grassy area in the middle of the cove; the 3 rd
and the 4th Station was located near mouth of the
MATERIALS AND METHODS Bay; the 3rd Station was set on a sandy area and the
4th Station in a sandy and grassy area. Each station
<XPXUWDOÕN Cove is located on the had three artificial collectors placed in the form of
northwestern part of the Bay of Iskenderun, where an equilateral triangle.
the study was conducted. Our sampling stations
FIGURE 1
<XPXUWDOÕNCove and Stations.
The sampling was conducted every other day (www.tug.tubitak.gov.tr). Besides the special
between the 7th of June and the 30th of November. phases of the moon as the new moon (I) (1-7 days),
A 37.5 cm long, 16.3 cm wide artificial settlement the first quarter (II) (8-14 days), the full moon (III)
collectors which has a 0.26 m2 surface area and is (15-22 days) and the last quarter (IV) (23-30 days)
made up of glass-fiber wound cylindrical PVC pipe were taken into account.
was used in the sampling [18]. The standard The samples in the Petri dish that brought in
procedure published by Metcalf et al. [18] was used the laboratory were examined, classified different
in the sampling, cleaning and sorting of the species and enumerated with the help of an
megalopae from the artificial collectors. The Olympus SZX-16 stereoscopic microscope under
samples were preserved in 70% ethyl alcohol and favorable conditions. First, crab post-larvae were
then brought in the laboratory. Temperature and distinguished from other zooplankton samples.
salinity values taken from each station were Then, the post-larvae under the Portunidae family
recorded in the protocol after being measured by were sorted from crab post-larvae [19,20] and
YSI 30 salinity, conductivity and temperature meter lastly, Blue Crab post-larvae were defined and
every other day. Lunar information was taken from enumerated. The studies conducted by Costlow and
the Calendar of Celestial Events prepared by Bookhout, Ingle, Kennedy and Ogburn were highly
TUBITAK National Observatory
1008


used Blue Crab post-larvae identification RESULTS AND DISCUSSION

[21,22,23,24].
The analysis of variance was conducted for As a result of this study, 28 a i (Blue
WHPSHUDWXUH DQG VDOLQLW\ GDWD DQG 'XQFDQ¶V Crab) post-larvae, 218 sp. post-larvae and
Multiple Comparison Test was applied for those 8 post-larvae from other species of the Portunidae
attributed significant based on the results of the family were collected from the four stations on a
analysis of variance [25]. monthly basis (Table 1).
SPSS 10.0 software package was used for
statistical analysis [26].
TABLE 1
The Number of Individuals of Post-larvae of Crab Species Monthly
The first settlement of the Blue Crab post- 7DEOH 7KHQ 'XQFDQ¶V 0XOWLSOH &RPSDULVRQ
ODUYDHLQ<XPXUWDOÕN&RYHZDVUHFRUGHGRQWKH th Test was conducted on the data and it was revealed
of June and the last one was on the 14th of October that the temperature average of each month during
(Figure 2). The densest settlement period was the course of the study was different from each
appointed as August and September (Table 1). The other. In terms of the stations, the temperature
highest number of Blue Crab post-larvae (10) was difference between the 2nd-4th stations and the 1st-3rd
achieved in the full moon (III) during study period stations was found insignificant while the 2nd-4th
(Figure 3). The highest number of Blue Crab post- stations had different temperature values than the
larvae in the collectors was sampled on the full 1st-3rd stations (Table 3).
moon (19th of July) and the new moon (29th of The highest average salinity measured in the
August) on a single sampling date (4 on each date) study field was recorded in November as
(Figure 2). Å ZKLOH WKH ORZHVW RQH ZDV UHFRUGHG LQ
When the sampling stations are considered, -XQHDVÅ (Figure 6). The average salinity
the highest post-larvae population (54%) was in the ZDVFDOFXODWHGDVÅ7DEOH,WKDVEHHQ
2nd station the bottom of which was a grassy area found important that the salinity varied in different
and the lowest one was in the 3rd station the bottom months and in different stations (p<0.01) (Table 6).
of which was a sandy area (Table 2, Figure 4). 7KHQ 'XQFDQ¶V 0XOWLSOH &RPSDULVRQ 7HVW ZDV
The average bottom water temperature in conducted on the data and it was revealed that the
<XPXUWDOÕN &RYH EHWZHHQ -XQH-November 2011 salinity difference between the 2nd and 4th stations
was recorded as 25.1±0.9ºC (Table 3, Figure 5). with the 1st and 3rd stations was found insignificant.
The highest temperature was recorded in August However, it was determined that the salinity
and measured as 30.9ºC while the lowest one was difference between the 1st-2nd stations and the 3rd-4th
recorded in November and measured as 9.8ºC It has stations was found significant, as can be seen in
been found important that the temperature varied in Table 5.
different months and in different stations (p<0.01)
1009


FIGURE 2
The Number of Individuals of C. sapidus Post-larvae
FIGURE 3
The Settlement of C. sapidus Post-ODUYDHLQ<XPXUWDOÕNCove Based on the Phase of the Moon (I: New
Moon, II: First Quarter, III: Full Moon, IV: Last Quarter)
1010


TABLE 2
The Number of Monthly Caught Blue Crab Post-larvae Based on the Stations
FIGURE 4
The Percentage Distribution of C. sapidus Post-larvae Based on the Stations (%)
CONCLUSION study on the crab post-larval settlement in our

shores. For this reason the data obtained from this
In Turkey, the number of the studies study have been discussed in the light of the studies
conducted on Decapod Brachyuran crab larvae is on the post larval settlement in the Atlantic shores
highly limited [27,28,29,30] and there is not any of the USA where the Blue Crab is endemic.
1011


TABLE 3
The Water Temperature Averages (Ԩሻ with Standard Error on a Monthly Basis and Based on the
ഥ ±ࡿ࢞ഥ ). a,b: Common letters show no statistical difference (p<0.05).
Stations (࢞
FIGURE 5
7KH0RQWKO\$YHUDJH:DWHU7HPSHUDWXUHV&LQ<XPXUWDOÕNCove.
TABLE 4
The Table of Analysis of Variance of Temperature (ºC)
Variation Source df Mean Square Sig

Station 3 8.899 .01**
Time (months) 5 1.145.025 .00**
Station-Time 15 3.987 .04*
Error 218 2.232
S S
1012


TABLE 5
ഥ ±ࡿ࢞ഥ )
7KH6DOLQLW\$YHUDJHVÅZLWK6WDQGDUG(UURURQD0RQWKO\%DVLVDQGBased on the Stations (࢞
a,b: Common letters show no statistical difference (p<0.05).
FIGURE 6
7KH0RQWKO\$YHUDJH6DOLQLW\LQ<XPXUWDOÕNCove Å
1013


TABLE 6
7KH7DEOHRI$QDO\VLVRI9DULDQFHRI6DOLQLW\Å
Variation Source df Mean Square Sig

Station 3 18.151 .00**
Time (months) 5 15.095 .00**
Station-Time 15 1.185 .05*
Error 218 0.68
S S
The studies conducted in the same area for a larvae chose those areas for food resources and
few years indicate that the most densely populated disguise from the predators.
areas by the post-larvae in different years and the There was a significant statistical difference
dates they were the most sampled in one day varied between the stations in the cove in terms of
(38, 35). It was suggested that the effect of the temperature and salinity (p<0.05) (Table 4 and 6).
environmental factors like raining, wind, tides on The number of the post larvae (22 in total) in the 1 st
the larva transportation [39, 40] and the consequent and 2nd station where the salinity was lower was
shift in post larval entrance time in the cove might higher than the number (6 in total) in the 3 rd and 4th
have caused this variance. stations (Table 2 and 5). Metcalf and Lipcius [45]
Many studies indicated that the settlement of reported that the post-larvae were usually found in
the Blue Crab post-larvae peaked on different dates low-salinity areas of the lagoon.
between July and November. Chenery [41] reported The salinity of the four stations was measured
that the peak settlement occurred in mid-August in Å DV WKH ORZHVW YDOXH Å DV WKH KLJKHVW
Hudson River, New York (USA) and Bishop et al. YDOXH DQG Å DV WKH DYHUDJH YDOXH LQ
[16] reported the major settlement period between <XPXUWDOÕN &RYH RQ WKH GDWHV WKH VDPSOLQJ ZDV
the end of August and the beginning of September. conducted (Table 5). Several studies showed that
The highest settlement of post-larvae occurred the salinity was between 10-ÅLQ$WODQWLF2FHDQ
between August and October in Charleston Harbor where the Blue Crab had an endemic distribution
[31], York River, Virginia [18, 42], Mississippi [38, 43], between 1-ÅLQWKHVKRUHRIWKH*XOIRI
Cove [32] and the Gulf of Mexico [33]. The 0H[LFR DQG Å LQ DYHUDJH >14]. It seems the
postlarval settlement peaked in October in Duplin salinity of the sampling site was much higher than
River, Georgia and North Carolina [43, 15]. Van the one in Atlantic shores and lagoons. Wrona et al.
Montfrans et al. [34] observed the highest [43] indicated that there was a lot more barely
settlement at the end of September and at the settlement than expected in Duplin River where the
beginning of November in York River, Virginia. salinity was high (64 in 1990). At the end of this
Morgan et al. [44] conducted a two-year study and study, 28 Blue Crab post-larvae in total were caught
reported that the number of the post-larvae peaked in the collectors. It is possible that this situation
in between July and August in 1990 and in mid- resulted from the low fresh water flow and high
September and in mid-October in the other year. In VDOLQLW\LQ<XPXUWDOÕN&RYH
this study, some peak points in the number of Several studies were conducted on the
a i post-larvae occurred on the 19th of July, interaction between temperature and salinity and
29 of August and 4th of September (Figure 2).
th
the settlement of the Blue Crab post-larvae in the
Depending on these data, the highest settlement of lagoon and different results came out. Mense et al.
the Blue Crab post-larvae occurred in one day in [35] and Rabalais et al. [33] found the relation
mid-July, at the end of August and at the beginning between water temperature and salinity and
RI6HSWHPEHULQ<XPXUWDOÕN&RYH abundance in post-larvae insignificant in North
When the stations are considered in terms of Carolina and in Galveston Cove (the Gulf of
the abundance of a i post-larvae sampled in Mexico) respectively (ANCOVA Test) (r=0.002,
the study, the highest number of the post-larvae was r=0.056). However, there are other researches that
found in grassy areas (2nd Station) (Table 2, Figure attributed statistical importance in this relation.
4). Morgan et al. [44] indicated that the a i Boylan and Wenner [31] indicated that there was a
post-larvae chose a vegetated habitat over a non- positive correlation between the number of the
vegetated habitat. It has been assumed that post- post-larvae and the bottom temperature (r=0.277
1014


p<0.0001) and the salinity (r=0.132, p<0.05). )LJXUH LQ WKH FROOHFWRUV LQ <XPXUWDOÕN &RYH
Bishop et al. [16] found out that there was a The tide occurs in the new moon and the full moon
positive correlation between the settlement and phases at the most, as a result of which those phases
temperature (r=0.3324) whereas there was a DUH HIIHFWLYH LQ WKH ODUYDH¶V HQWUDQFH LQ WKH ODJRRQ
negative correlation with the salinity (r=-0.2900). A [46, 38]. For this reason it has been thought that the
study conducted in Louisiana (USA) for two years highest number of the post-larvae was achieved in
showed that there was a relation between the those phases on a daily basis at the mouth of the
number of the post-larvae and temperature in one cove.
year (p<0.05); there was no relation between the It has been known that abiotic factors like
two in the other year (p>0.05) and there was no wind regimes, currents and tides affect the post-
difference between the years in terms of average larval ingress into the lagoon [47,17]. Ocean
temperature [14]. The same study indicated that currents and especially wind force are highly
there was a positive correlation with the salinity in important in post-larval movement in the Atlantic
the first year whereas there was a negative shores where the Blue Crab is endemic. However,
correlation in the second year. Our study, on the the wind force and the current speed could not be
other hand, could not clearly reveal the relation measured because of lack of proper equipment;
between the settlement and temperature and salinity therefore, their effect on the settlement could not be
as the number of the Blue Crab post-larvae was examined. Besides as the tidal range in the cove
considerably low. was quite low, the tidal effect could not be clearly
There are various studies indicating that the defined, either. Despite all these deficits, the study
settlement of the post-larvae are significantly results have been considered important as they are
affected by the phases of the moon. Boylan and the first data on the post-larvae and the settlement
Wenner [31] suggested that the settlement of the of the Blue Crab outside its endemic distribution
post-larvae considerably varied depending on the area.
phases of the moon (p<0.0001, X2=96.01, Kruskal- Environmental factors, movement
Wallis test) and the highest abundance was detected mechanisms, spawning models of the adults and
in the last quarter and the least one was in the full post larval behavior affect post-larvae migration,
moon in South Carolina (USA). 42% of the samples their time of the entrance in the lagoon, their
were gathered on the 26th lunar day (the last quantity in the lagoon and their recruitment.
quarter) in August. Forward et al. [15] reported that Moreover, total number of post-larvae and their
the first and the last quarter±3 days caused peak number vary annually and from region to
significantly higher settlement of the post-larvae region [43]. Therefore, it is of very importance - at
than the new and the full moon±3 days (z=4.162, least for our region- that long-term observational
p<0.001, Z test) and the highest amount of the post- studies should be conducted in order to explain the
larvae was caught in the last quarter in North relation between post-larvae migration and
Carolina. environmental factors. It is necessary that long-term
Apart from the above mentioned studies, Jones post-larvae settlement studies should be conducted
and Epifanio [38] asserted that there was no relation and the annual data obtained from these studies
between the lunar phases and the maximum should be compared in order to establish the
settlement. In their study conducted in four development model of the blue crab in the study
different coves in the Gulf of Mexico (USA), field, to comprehend their settlement parameters, to
Rabalais et al. [33] found out that the settlement of estimate supply amounts and to provide a fisheries
the post-larvae occurred in the new moon in three management.
coves whereas there was no apparent relation
between the number and the settlement of the post-
larvae in one cove. The maximum settlement was ACKNOWLEDGEMENTS
reported to have occurred right after the full moon
in Chesapeake Bay [34]. Besides, the maximum This study was supported by Cukurova
settlement of the Blue Crab post-larvae in North University Scientific Research Projects
Carolina and Virginia was reported to have Coordination Unit (Project No: SÜF2010YL22).
occurred after the new moon and the full moon [36, We would like to thank Dr. Matthew B. Ogburn
18]. In this study period, the highest settlement of from Smithsonian Environmental Research Center
the Blue Crab post-larvae occurred in the full moon (USA) and Dr. Allen Place from Institute of Marine
of the month (Figure 3) whereas the greatest and Environmental Technology, University of
numbers of megalopae were found highest in the Maryland (USA) for their contribution to the
full moon and the new moon on a daily basis diagnosis of blue crab megalopae.
1015


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1017


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875
CORRESPONDING AUTHOR:
Irem Nur Yesilyurt

Cukurova University
Faculty of Fisheries
Department of Basic Sciences
%DOFDOÕ-Adana ± TURKEY
e-mail: iyesilyurt@cu.edu.tr
1018


CONTAMINATION STATUS OF ERZENI RIVER, ALBANIA

DUE TO HEAVY METALS SPATIAL AND TEMPORAL
DISTRIBUTION
Alma Shehu1, Majlinda Vasjari1, Edlira Baraj2, Rajmonda Lilo2, Roza Allabashi3
1
Department of Chemistry, Faculty of Natural Sciences, University of Tirana
2
Departmentof Chemistry, Faculty of Mathematical Engineering and Physical Engineering, Polytechnic University of Tirana
3
University of Natural Resources and Life Sciences, Vienna; Department of Water, Atmosphere and Environment, Institute of Sanitary
Engineering and Water Pollution Control
ABSTRACT the aquatic ecosystem have recently become subject

of widespread concern; since beyond the tolerable
Heavy metals are considered as serious limits they may become very toxic [1]. Sediments
pollutants of aquatic ecosystems because of their and biota are generally metal reservoirs in aquatic
environmental persistence, toxic potential and environment and may assume several orders of
ability to be incorporated into the food chain. In the magnitude greater than the amount found in water
present investigation, the contamination status of [2]. Contamination of surface water systems by
one section of Erzeni River was assessed in regard heavy metals may be either due to natural or
to occurrence and distribution of some heavy anthropogenic activities. Naturally occurring metals
metals. Due to concerns about potential pollution move through aquatic environments independently
resulting from watershed sources and adjacent of human activities, usually without any detrimental
increased human activities, the study was effects [3, 4]. Some of these metals are essential for
performed at a representative section of this river, proper metabolism in all living organisms yet toxic
during the period December 2013-April 2014. The at high concentrations; other metals currently
filterable fraction of heavy metals such as Pb, Cu, thought of as non-essential are toxic even at
Cd, Zn and Ni was determined using the GF-AAS relatively low concentrations [5]. Major sources of
technique. BGBI.II, Nr. 96/2006 as well as toxic metals arising from human activities are
2008/105/EC Directives were used for the domestic and industrial wastewaters and their
estimation of the contamination status of the river associated solid wastes. Metal contamination can
water. Distribution of heavy metals in the river also originate from industrial activities as well as
water was also evaluated by means of the from urban storm water runoff from roads, parking
Contamination Index (CI) and the Mean areas and other impervious surfaces, ending up
Contamination Index (MCI), values. The relative either in waste water treatment plants, or directly in
abundance of heavy metals followed the order: the river [6].
Zn>Ni>Pb>Cu>Cd, while probability plots (D = Metals that are naturally introduced into the
0.05) confirm that external pressures can contribute river come primarily from such sources as rock
to heavy metal levels in waters of Erzeni River. weathering, soil erosion, or the dissolution of
Although CI varied evidently in different sites, water-soluble salts [7]. All heavy metals exist in
assuming that the levels of heavy metals are very surface waters in colloidal, particulate and
dependent with location, the MCI values prove that dissolved phases, although dissolved concentrations
there exist a high degree of spatial and temporal are generally low. The solubility of trace metals in
homogeneity at all scales due to a high rate of water surface waters is predominately controlled by the
mixing in the river. water pH, the type and concentration of ligands on
which the metal could adsorb and the redox
KEYWORDS: environment of the system [8, 9].
Heavy Metals, Water Contamination, Erzeni River. Albania is rich in water resources, including
rivers, groundwater, lakes, lagoons and seas.
INTRODUCTION Although its resources exceed by far its
consumption, locally water shortage and conflicts
The fate of heavy metals introduced by human among users may occur in the dry season [10].
activities, chemical and geochemical processes into
1019


General assessment of the state of HM depth in each site using PET bottles, according to
pollution of rivers is difficult, primarily because the standard method Nr 105, described in the
measurements of metals are rarely included in ³6WDQGDUG 0HWKRGV IRU WKe Examination of Water
monitoring programmes, but also because DQG :DVWH :DWHU´ FDVH RI KHDY\ PHWDOV >@
concentration levels are usually so low that Samples were transported to the laboratory within
problems arise with sample preparation and the the same day, using cooling boxes, were
precision of analytical results [11, 12]. Comparison immediately filtered with 0.45μm glass filters and
and assessment of the state of HM in rivers is the filtrate was acidified to pH=2 with concentrated
therefore more difficult than for most of the other HNO3.
water quality variables. Acidified samples were stored at temperature
Erzeni River is located in the western part of +4oC till the analyses. The analyzed metals were
Albania. It springs from the east mountains of Copper, Cadmium, Lead, Nickel, Zinc and their
Tirana city, near the Shëngjergji village and flows content in water samples was determined according
west through Bërzhitë, Petrelë, Vaqarr, Ndroq, WR WKH VWDQGDUG PHWKRG 1U RI WKH ³6WDQGDUG
Shijak and Sukth into the Adriatic Sea, north of Methods for the Examination of Water and Waste
Durrësi [10]. The catchment of this river is about :DWHU´ >@ 129$$ $WRPLF $EVRUSWLRQ
759 km2 while its annual discharge volume is about Spectrometer, equipped with a graphite furnace
1,439 million m3, with a specific discharge of about system, was used to carry out water analyses.
31 L/sec*km2. The water quality is poor as the Analytical blanks were run in the same way as
river flows through one of the most developed the samples and concentration was determined
urban areas of the central part of Albania. In recent using standard solutions prepared in the same acid
years, human activities such as abusive PDWUL[5HVXOWVDUHH[SUHVVHGLQȝJ/-1, as well as
deforestation, the plugging up of new agricultural standard deviation was calculated for three repeated
lands and their intensive cultivation, extraction of analyses of each sample
gravel and sand from the riverbed have been
intensified [10]. Sampling sites description. The water quality
Environmental situation of the main rivers in of Erzeni River was monitored in four selected sites
Albania has been monitored in the framework of along the river, namely S1- located at Ibe area; S2-
several national and international projects [12, 13, located at Peshkatari Bridge ; S3- located at Domje
14]. Previous studies on Erzeni River have been Bridge; S4- located at Sharra area.
focused mainly on physical-chemical parameters The sampling site S1 is located in the
while there are no reliable studies regarding heavy upstream area of the river, being suggested as the
metals. The aim of the present study was the reference point. At the area of S2, a considerable
evaluation of the spatial and temporal number of restaurants and hotels develop their
contamination of this section of Erzeni River due to activities, while in S3 extraction of gravels is
heavy metals levels in the water. evident in several points. Station S4 is located close
Evaluation of the contamination status of to the Sharra landfill, classified by the UNEP, 2000,
waters of Erzeni River was carried out based on two DV D ³KRW VSRW´ DUHD LQ $OEDQLD >@ %HIRUH WKH
important standards, which determine the allowable landfill construction, Sharra dump was the most
levels of heavy metals in surface waters, such as potential source of pollutants of Erzeni River, for
%*%O,, 1U ³4XDOLWlWV]LHOYHURUGQXQJ both inorganic and organic pollutants [19].
&KHPLH 2EHUIOlFKHQJHZlVVHU´ DQG WKH Location of all selected sampling sites for the
2008/105/EC Directive [15, 16]. present study is illustrated in figure 1.
Quality control of measurements and

MATERIALS AND METHODS statistical analysis. Quality control of the results
was conducted by analyzing a certified reference
Estimation of the environmental situation of material (QC-TM#1), provided by GEMS/Water
Erzeni River regarding heavy metals content in (PE) Study No 8, 2013. Obtained results were in
water was accomplished by determining the metals good agreement with the certified values given in
content in water samples of four sites, selected in a respective certificates of the CRM (z-score ranging
representative section of the river. The study was between -0.03 to-0.6). The concentration of each
performed in different months, during the period metal in water samples in Erzeni River was
December 2013-April 2014. calculated using linear regression and expressed in
μg.L-1 as the mean value of three sample replicates.
Sampling procedure for water samples. Limit of quantification (LOQ) and working range
Water samples were manually collected at surface
1020


for each element studied in this work are presented statistics for the variability of metals
in table 1. concentrations, including Anderson-Darling (AD)
Descriptive statistics including mean, standard test statistics. The Anderson-Darling (AD) test
deviation, min, max, value and median were compares the observed distribution with a normal
calculated for each of the variables. All data were distribution based on the calculated p-value. If the
tested for normal distribution by means of the p-value is <0.05 the data do not fit to normal
probability plots, using the MINITAB 15 Statistical distribution.
Software. The Minitab plots summarize the
FIGURE 1
Map of sampling sites
TABLE 1
Performance parameters of the analytical method
Cu Cd Pb Zn Ni
LOQ (μg.L-1) 0.250 0.015 0.250 0.010 3.0
Working range (μg.L-1) 10-50 1-10 20-100 0.5-4 5-50
RESULTS AND DISCUSSIONS elements while Cd resulted to be the metal with the
lowest content. The metals concentration in Erzeni
Descriptive statistics. Some statistical water followed the order: Zn>Ni>Pb>Cu>Cd while
parameters of the obtained results regarding metals the variations relating their content between the
concentration in water samples are presented in sampling time and sites, estimated as relative
table 2. As it can be seen, Zn and Ni were found to standard deviation were within 55% (Cu) to 64%
be in higher concentration, compare to other (Ni).
TABLE 2
6WDWLVWLFDOSDUDPHWHUVRIKHDY\PHWDOVLQ(U]HQL5LYHUZDWHUVȝJ/-1)
Cd Pb Ni Cu Zn
Parameter n=17 n=16 n=18 n=20 n=18
mean 0.30 4.76 19.41 4.58 23.86
min 0.02 0.60 5.13 1.71 8.58
max 0.72 9.67 51.20 9.59 50.00
median 0.25 5.22 14.05 3.78 18.00
St.dev. 0.18 3.10 12.58 2.55 13.57
Distribution pattern of the elements. Results River are presented in Figures 2 and 3 and are
on heavy metals distribution in waters of Erzeni discussed in detail in the paragraphs below. Interval
1021


Plots were used aiming to evaluate the temporal and 2 and Fig. 3) while the distribution pattern was
spatial variation of each element concentration (Fig. evaluated by means of the probability plots (Fig. 4).
Interval Plot of concentration of elements in different months

95% CI for the Mean
60
50
Concentration (ppb)
40
30
20
10
0
r
r
c.
n.
b.
r.
c.
n.
b.
r.
r.
c.
n.
b.
r.
r.
c.
n.
b.
r.
r.
c.
n.
b.
r.
Ap
Ap
Ap
Ap
Ap
Ma
Ma
Ma
Ma
Ma
De
De
De
De
De
Fe
Fe
Fe
Fe
Fe
Ja
Ja
Ja
Ja
Ja
Ni Cu Pb Zn Cd

FIGURE 2
Variation of heavy metals concentration during the studied period.
Interval Plot of Concentration of Elements in Different Stations

95% CI for the Mean
50
Concentration (ppb)
40
30
20
10
0
S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4 S1 S2 S3 S4
Ni Cu Pb Zn Cd

FIGURE 3
Variation of metals concentration in each sampling site.
Cadmium. In the present study, Cd stations as well as between months. Even though
concentration varied from 0.02- ȝJ/-1, while the Maximal Allowable Concentration value
the highest values were recorded during December recommended for Cd content in surface waters by
2013 and April 2014 (Fig. 2). Mean values of Cd the EC, 2008, DireFWLYH >@ 0$& ȝJ/-1),
concentration varied evidently between different was not exceeded, it was observed that the content
1022


of Cd during the monitoring period exceeded the from natural origin, may also derive from
annual average concentration (AA-(46 ȝJ/- anthropogenic sources.
1
) recommended by the above mentioned
regulations. Copper. Concentration of copper in waters of
Probability plots of Cd give relevant Erzeni River varied between 1.71- ȝJ/-1
information regarding Cd distribution pattern in (Table 1). Levels of Cu varied significantly in
waters of Erzeni River, as well as to the natural or samples collected in different months of
anthropogenic sources of this element. The monitoring, where the highest concentrations were
concentration values of Cd (Fig. 4), in most recorded in January and April. The mean
analyzed samples fall between the confidence limits concentration values of Cu, recorded in samples
area, set for p<0.05, but obviously obtained values collected from different sites resulted to be very
do not fulfill the condition to fit the normal FORVH DERXW ȝJ/-1), which assumes for a high
distribution pattern. This is also confirmed by the homogeneity scale of the river. The environmental
AD test, which has generated a value of p<0.05, quality standard of copper in river waters is closely
concluding that the distribution of Cd related to water hardness [15]. If the hardness of
concentrations in (U]HQL 5LYHU ZDWHU GRHVQ¶W water exceeds 100 mg CaCO3/L the allowed
comply with normal distribution. additional concentration of copper is set to 8.8
ȝJ/-1. In the present study total hardness exceeded
Lead. The concentrations of Pb in the water the limit of 100 mg CaCO3/L in all analyzed water
samples of Erzeni River varied between 0.60-9.67 samples [14]. Referring the above, even though the
ȝJ/-1. Especially during the winter period, the PD[LPXP YDOXH ȝJ/-1), recorded during
highest values were recorded (Fig. 2) and in three January at S3 is higher than the AAC, the mean
cases, respectively during December, February and values of Cu concentration, either referring to the
March, the mean concentration of lead exceeds the sites or to the monitoring months (Fig. 2, 3) do not
annual average value (AA-(46 ȝJ/-1) of the H[FHHGWKHUHFRPPHQGHGYDOXHȝJ/-1. Relating
environmental quality standards recommended in to the distribution pattern of the recorded values, it
[15] and [16], for lead content in surface waters. was observed a similar situation as in the case of Ni
Mean values of Pb concentration varied more and Cd. Statistical parameters obtained from
evidently in different periods of monitoring, while probability plots (Fig. 4), (AD=1.030 and p<0.05),
no significant difference was observed for the mean mean that the distribution of Cu concentration in
content of this metal in different sampling sites ZDWHU VDPSOHV GRHVQ¶W FRPSO\ ZLWK QRUPDO
(Fig. 3). Probability plots show that the values of distribution which is a fact that possible
Pb content in almost all analyzed samples fall anthropogenic contamination sources can contribute
within the confidence limits area, set for p<0.05 and to Cu content in the river.
the AD test generating a p-value 0.38, confirms that
the distribution of Pb concentrations in Erzeni Zinc. The allowed additional Zn concentration
River waters comply with normal distribution (Fig. is related to water hardness. According to [15] if the
4). hardness of water exceeds 100 mg CaCO3/L the
Nickel. In the present investigation Ni allowed additional concentration of Zn is set to
concentration in the river water varied between ȝJ/-1 The content of Zn in water of Erzeni
5.13- ȝJ/-1 (Table 2). It was also observed River, expressed as the mean value of results
that the highest levels of Ni were recorded during obtained in different sites and in different
December and April (Fig. 2) as well as in station S3 monitoring periods, varied evidently within sites
(Fig. 3), exceeding the annual average value (AA- DQG H[SHGLWLRQV DOVR ,W¶V VSDWLDO DQG WHPSRUDO
(46 ȝJ/-1), recommended by EC, 2006, [15]. distribution range of concentration fall below the
Referring to the probability plot (Fig. 4), even allowed additional concentration value, AAC. As it
though the recorded values of Ni mostly fall inside is confirmed by the AD test (Fig. 4), the
WKH FRQILGHQFH OLPLWV DUHD WKH\ GRQ¶W ILW WR D GLVWULEXWLRQ RI =Q LQ (U]HQL 5LYHU ZDWHUV GRHVQ¶W
random distribution. Moreover, the AD test, which comply with normal distribution pattern (p<0.05),
has generated a p-value <0.05, confirms that the concluding that the presence of zinc in waters of
GLVWULEXWLRQ RI 1L LQ (U]HQL 5LYHU ZDWHU GRHVQ¶W Erzeni River is mainly derived by anthropogenic
comply with normal distribution. This assumes that sources.
the content of Ni in waters of Erzeni River, except
1023


Probability Plot
Normal - 95% CI Cu
Mean 4.583
&X &G 3E StDev 2.615

N 20
AD 1.030
P-Value 0.008
Cd
Mean 0.3012
StDev 0.1847
N 17
AD 1.225
P-Value <0.005
3HUFHQW
Pb
Mean 4.761

StDev 3.102

N 16
AD 0.380
1L =Q
P-Value 0.360
Ni
Mean 19.41
StDev 12.94
N 18
AD 0.976

P-Value 0.011
Zn
Mean 23.86
StDev 13.97
N 18
AD 0.939
P-Value 0.013
FIGURE 4
Probability plot of metals distribution in waters of Erzeni River.
Contamination Index. In the absence of calculated for each element at all sites and obtained
³EDFNJURXQG OHYHOV´ RI KHDY\ PHWDOV LQ D ZDWHU values are presented in Figure 5.
media without anthropogenic inputs, several
authors propose the calculation of the Accordingly, major changes of Contamination
Contamination Index (CI) and the Mean Index are evident for Cd and Ni. Although the CI
Contamination Index (MCI), to evaluate the degree varied in different stations, assuming that the levels
of contamination of a selected site compared to the of the studied metals are dependent with location,
overall pollution in the whole region according to the MCI values prove that there exist a degree of
the formula: spatial and temporal homogeneity at all scales due
MX to a high rate of water mixing in the river.
CI X ( metal )
M The comparison of results with reported heavy
where Mx is the metal mean value at station x
metal levels in Erzeni River is rather difficult,
ȝJ/-1) and M is the mean concentration of
because of the lack of existing data. To our
selected metal in all sites [20]. The Mean
knowledge, heavy metal content in surface and
Contamination Index (MCI) aiming to classify the
ground water is not yet included in the water
overall environmental status of the river selected
quality monitoring program in Albania and
section was determined using the formula:
n
therefore not periodically reported [21].
Nevertheless, the comparison with a study
¦ CIi 1
x
performed in 2002-2004 [12] for other rivers in
MCI Albania shows that Cd and Cu concentration ranges
n at Erzeni River are at the same level as reported
ZKHUH³Q´LVWKHQXPEHURIPHWDOVDQDO\]HGIRUWKH there, but higher concentrations are found for Pb,
present study, n = 5). CI and MCI values were Ni and Zn in our study.
1024


FIGURE 5
CI and MCI values for each element in all sites.
Due to the geology of the zone, large areas of ȝg.L-1). To our opinion, this situation has
central Albania are covered by serpentine soils changed drastically due to the negative impact in
which are highly enriched in heavy metals such as development of traffic situation in Albania. As a
Cr, Cu, Ni, Fe, and Zn [10]. According to the consequence, Pb concentrations up to nearly 10
Geochemical Atlas of Europe Albania belong to the ȝJ/-1 are measured in Erzeni River in this study,
region with the highest Ni soil content in Europe. showing an continuously increasing of Pb
The subsoil Ni map shows a strong anomaly over concentration in river water in Albania, also
most of Greece and Albania, caused by mineralized compared to the study performed in 2002-2004
ophiolitic rocks, and sedimentary rocks derived >@ ZKHUH FRQFHQWUDWLRQV XS WR ȝJ/-1 are
from them [22]. Through enhanced interventions in reported.
the area of Erzeni River during the last decade for Zn concentrations measured in this study in
road construction, excavation for tunnel opening, or (U]HQL5LYHUXSWRȝJ/-1) are rather higher than
quarrying operation the Ni concentration in Erzeni those reported before [12, 23] for Albanian rivers
River is found to be much higher than reported 10 (20- ȝJ/-1). They are comparable to typical
years earlier for most of Albanian Rivers (up to 17 concentrations in river water from the literature,
ȝJ/-1) [12]. On the other hand some known which are about 10- ȝJ/-1 [22]. These high
anthropogenic sources of nickel including concentrations in stream water can be explained
fertilizers, fuel combustion and detergents are with the high content of Zn in topsoil of the region
strongly represented in this newly developed which is reported to be among the highest in
touristic area. In the presence of some organic Europe [22]. The enhanced interventions in the area
complexing agents, Ni is capable of forming neutral of Erzeni River during the last decade, especially
or negatively charged complexes, making the metal excavations or quarrying operations can be one of
highly mobile in relation to other trace elements. the reasons for the change of water quality
Consequently, Ni concentrations maybe high in regarding Zn content in Erzeni River.
stream water contaminated by sewage and leachate
from waste tips [23]. This can be an additional
reason for the nickel pollution of Erzeni River, as it CONCLUSIONS
serves actually as receiving water course for all
economic and touristic activities in this area. In the present study, the contamination status
Lead tends to low concentrations in the of Erzeni River was assessed in regard to
alkaline environments of central Spain, Greece, occurrence and distribution of Pb, Cu, Cd, Zn and
Albania, and throughout the Alps [22]. The lowest Ni in one section of the river, represented by four
values in Europe are reported for Pb concentrations selected sites. The concentrations of studied heavy
in stream water in Albania between 1998 and 2000 metals are determined in the filterable sample
1025


fraction. The European Water Frame Directive [3] Qarri F., Kane S., Lazo P., 2014. Environmental
(2008/105/EC) and the respective Austrian assessment of dissolved heavy metals in Vlora
regulation for surface water (BGBI.II, Nr. 96/2006) Bay, Albania. Pranvera Lazo. Fresenius
were used to classify the contamination status of the Environmental Bulletin. Vol. 23 No. 7, 1539-
river water. 1546.
Except Cd, the means concentrations of other [4] Qi Y., Ma J., Fan G., 2015. Assessment of
metals were found to be lower than the values heavy metals contamination in fen river
recommended in those standards while the channels. Fresenius Environmental Bulletin.
variations relating the sampling time and sites were Vol. 24, No. 5. 1230-1242.
within 55% (Cu) to 64% (Ni). The metal contents [5] Baker, Lawrence A., Ed., 1994. Environmental
were found to be in the order: Zn> Ni> Pb>Cu> Cd. Chemistry of Lakes and Reservoirs, American
Comparative studies with other publications Chemical Society, Washington, D.C.
on river water quality based on heavy metals [6] Nicolau, R., Galera, C. A., and Lucas, Y., 2006.
contents confirm that Cd and Cu concentration Transfer of nutrients and labile metals from
ranges at Erzeni River are at the same level as the continent to the sea by a small
reported there, but higher concentrations are found Mediterranean river, Chemosphere, 63 (3),
for Pb, Ni and Zn in our study. The enhanced 469 - 476.
interventions in the area of Erzeni River during the [7] Hirsch, R.M., Hamilton, P.A., and Miller, T.L.,
last decade for road construction, excavation for 2006. U.S. Geological Survey perspective on
tunnel opening, or quarrying operation have caused water-quality monitoring and assessment.
the enrichment of water samples of Erzeni River Journal of Environmental Monitoring 8(5),
with heavy metals originated from the geology of 512-518.
the zone. On the other hand, some known [8] Forstner U, Wittmann G.T.W., 1981. Metal
anthropogenic sources including fertilizers, fuel Speciation in the Aquatic Environment, 2nd
combustion and detergents are strongly represented ed., 26, Springer-Verlag.
in this newly developed touristic area.Although the [9] Huang Y., Zhang D., Li Y., Xu Zh., Yuan Sh.,
CI values varied significantly for some elements Wang L., 2015. Heavy metals sorption on
such as Cd and Ni, assuming that the levels of river sediments: kinetics, equilibria and
studied metals are dependent with location, the relative selectivities. Fresenius Environmental
MCI values prove that there exist a degree of spatial Bulletin, Vol. 24, No. 09.
and temporal homogeneity at all scales due to a [10] Selenica A., Ardicioglu M.,Kuriqi A. Risk
high rate of water mixing in the river. assessment from floodings in the rivers of
Albania. International Balkans Conference on
Challenges of Civil Engineering, BCCCE, 19-
ACKNOWLEDGEMENTS 21 May 2011, EPOKA University, Tirana,
ALBANIA.
The authors would like to thank the team of [11] Gashi F., Troni N., Hoti R., Faiku F., Ibrahimi
WKH SURMHFW ³$VVHVVPHQW RI WKH ZDWHU TXDOLW\ RI R., Laha F., Kurteshi K., Osmani S., Hoti F.,
Erzeni River in Albania and the impact of economic 2014. Chemical determination of some
GHYHORSPHQWVWKHUHLQ´5HI1R$-13, co- elements in water of Sitnica river (Kosovo) by
financed by the Central European Initiative (CEI) ICP-MS technique. Fresenius Environmental
with funding of the Austrian Development Bulletin, Vol. 23, No. 01.
Cooperation (ADC) for providing the water [12] Miho A., Cullaj A., Hasko A., Lazo P., Kupe
samples for this study. L., Bachofen R., Brandl H., Schanz F., Baraj
%³(QYLURQPHQWDOVWDWHRIVRPHULYHUV
RI $OEDQLDQ $GULDWLF /RZODQG´ (YDOXDWLRQ
REFERENCES Report/ Joint research project 7ALJ065583
(SCOPES).
[13] Flanagan M., Paukstys B., 2012, Report on:
[1] Manahan S. E., 2001. "Front matter
"Fundamentals of Environmental Chemistry. ³&(06$:DWHU0RQLWRULQJ)LHOG6XUYH\DQG
Boca Raton: CRC Press LLC. 7UDLQLQJ´
[14@ 3URMHFW 5HSRUW ³$VVHVVPHQW RI WKH ZDWHU
[2] Zhang Z., Wang J., Delaune R., 2015. Trace
quality of Erzeni River in Albania and the
elements contamination of Tangipahoa river
LPSDFW RI HFRQRPLF GHYHORSPHQWV WKHUHLQ´
water and sediment in Louisiana. Fresenius
Environmental Bulletin, Vol. 24, No. 3, 910- Ref. No. 1206.002A-13, co-financed by the
919. Central European Initiative (CEI) with
1026


funding of the Austrian Development

Cooperation (ADC).
[15] BGBl.II, Nr. 96/2006,
³4XDOLWlWV]LHOYHURUGQXQJ &KHPLH
2EHUIOlFKHQJHZlVVHU´
[16] Directive 2008/105/EC of the European
Parliament and of the Council of 16 December
2008 on environmental quality standards in
the field of water policy.
[17] APHA, 1985. American Public Health
Association.Standard methods for the
examination of water and wastewater. 18th
Edition APHA Washington, DC.
[18] Post ±conflict Environmental Assessment-
Albania UNEP 2000
[19] Billing K.,Wenborn M.,Selfo L., 2011.Sharra
Waste Dump Site, Albania-Feasibility Study
and Urgent Rehabilitation
Measures.http://www.sida.se/publications.
[20] Chafika, A., M.Cheggourb, D. Cossac and
Siffedine, S.B.M., 2001. Quality of Morocoan
Atlantic coastal waters: Water monitoring and
mussel watching. Aquatic Living Resour.,
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[21] State of the Environmental Report, Albania
2011, 2012, 2013, 2014
[22] Geological Atlas of Europe, Part1, ISBN 951-
690-913 2 (electronic version)
http://weppi.gtk.fi/publ/foregsatlas/index.php
[23] Cullaj A., BarDM % ³$VVHVVPHQW RI WKH
environmental situation of Albanian rivers
based on physico-FKHPLFDO DQDO\VHV´
http://siba-ese.unisalento.it/.
Majlinda Vasjari
Department of Chemistry
Faculty of Natural Sciences
University of Tirana ± ALBANIA
e-mail: majlinda.vasjari@fshn.edu.al
1027


ASSESSING CONTAMINATION IN STURGEONS GROWN IN

RECIRCULATING A UACULTURE SYSTEM BY LU -
BIOSENSORS AND METAL ACCUMULATION
Adem Yavuz Sönmez1, Marina Sazykina2, Soner Bilen1, Nejdet Gültepe3, Ivan Sazykin2, Ludmila E.
Khmelevtsova2, Natalia V. Kostina2
1
Department of Basic Sciences, Faculty of Fisheries, Kastamonu University,37100 Kastamonu, Turkey
2
Research Institute of Biology, South Federal University, Rostov-on-Don, 344006 Russia
3
Kastamonu University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, Kastamonu, Turkey
ABSTRACTS much more prevalent. This approach is used both

for commercial fish breeding and for cultivation of
In the present study, it was aimed to assess of fry of different types of fishes for the purpose of
the quality of fish grown in a recirculation replenishment of natural populations. Taking into
aquaculture system (RAS). For this purpose, three account that demand for fish grows every year, it is
sturgeon species especially i en e possible to assume that aquaculture growth rates
el en tae tii, i en e ae ii, and i en e will overtake fisheries soon (Bruge're and Ridler,
tellat were investigated. Concentrations of 2004). However, problems such as contamination
certain metals (Cu, Cd, Pb, Zn, Fe, Ni) in gills, liver of surrounding waters by solid waste, antibiotics,
and muscle samples were examined. Metal and pathogenic strains of microorganisms resulting
concentrations in i en e el en tae tii were; from aquaculture operations are significant and
Fe:172.43-196.52 μg g-1, Cd: 0.21-0.33 μg g-1, Cu: require attention (Seymour and Bergheim, 1991;
29.65-45.11 μg g-1, Ni: 0.52-0.86 μg g-1, Pb: 14.24- Wu, 1995; Katranitsas et al., 2003; Matos et al.,
19.63 μg g-1, and Zn: 60.13-88.15 μg g-1. In 2006; Fernandes et al., 2008; Cömert et al., 2008;
i en e ae ii, metal concentrations were; Fe: Kapetanovic et al., 2013).
151.47-182.32 μg g-1, Cd: 0.18-0.36 μg g-1, Cu: Actions aimed at reduction of pollution from
27.17-38.52 μg g-1, Ni: 0.72-0.98 μg g-1, Pb:16.45- aquaculture farms have led to rapid development of
25.16 μg g-1, and Zn: 72.14-95.41 μg g-1. recirculating aquaculture systems (RAS). RAS
Concentrations in i en e tellat were; Fe: enables more intensive fish breeding operations.
206.39-262.38 μg g-1, Cd: 0.26-0.39 μg g-1, They occupy a small area and recirculation of water
Cu:30.26-55.63 μg g-1, Ni: 0.54-0.87 μg g-1, provides stable aquaculture production (Devillera et
Pb:14.36-21.51 μg g-1, and Zn: 53.52-82.15 μg g-1. al., 2005). Nevertheless, dissolved substances
In general, the highest metal levels were found in which cannot be removed in the course of RAS
i en e ae ii. Negative biological effects caused water processing can gradually accumulate. The
by the presence of various toxicants in fish tissues process of their accumulation depends on feeding
were studied with bacterial lux-biosensors. These and water renewal rates (Leonard et al., 2002;
assays revealed that the accumulation of Pagand et al., 2000).
genotoxicants, oxidative stressors, and substances Chemicals used in aquaculture can negatively
causing damage to proteins and membranes mainly affect the health of fishes and, respectively, their
occurred in gills and to a smaller extent in liver. consumers. In this respect, there are uncertainties
i en e ae ii showed the highest accumulation regarding the quality of cultured fishes. It is
of toxic substances according to the bacterial lux- necessary to consider the fact that among all aquatic
biosensor assays. organisms, fish are most subject to the action of
WR[LF VXEVWDQFHV SUHVHQW LQ ZDWHU $OLEDELü HW DO
KEYWORDS: -DULüHWDO
i en e el en tae tii i en e ae ii, i en e Sturgeon is considered to be especially
tellat , Resirculation Systems, Lux-biosensors, Metal susceptible to bioaccumulation of recalcitrant
Accumulation environment pollutants. Pollution by toxicants is
one of the main threats for sturgeon (Kruse and
Scarnecchia, 2002; Stanic et al., 2006; Webb et al.,
INTRODUCTION 2011).
Chemical analysis is not adequate to describe
In recent decades, artificial reproduction of the adverse effects of the complicated chemicals
different types of fish in breeding farms has become mixtures which can accumulate in fish tissues.
1028


Chemical analysis does not consider integrated In the present work, six different recombinant
toxicological effects on biological systems. Solving bioluminescent li strains containing the
the problem requires application of biological promoters recA (DNA damage), katG and soxS
analysis methods. A combination of chemical (responsive to oxidative damage), grpE (protein
analytical methods and biological testing into a damage), fabA (membrane damage) and lac
uniform complex monitoring platform will increase (constitutive expression) fused to the bacterial
the efficiency of cultured fish quality assessment. operon luxCDABE from t a
Metals are of greatest concern among the l mine en (Kotova et al., 2010; Manukhov et al.,
pollutants that can be accumulated in fish. Metals 2008; Zavilgelsky et al., 2007) were used to
have strong impacts on the stability and describe the mechanism and degree of toxicity of
development of aquatic ecosystems as they are fish extracts on the bacteria. It was aimed that to
highly toxic (Has-Schon et al., 2006; Omara et al., evaluate toxicity by using bioluminescent sensors
2012). They can cause oxidative stress and and to assess the accumulation of heavy metals in
negatively affect growth and development of fish sturgeons cultured in fish-breeding farms with
(Baker et al., 1997, 1998). The concentrations of recirculating aquaculture systems and also to
some metals in fish intended for human analyze their distribution among different tissues.
consumption are routinely monitored. Their
concentrations can be defined precisely in trace
quantities (Pe´rez Cid et al., 2001). MATERIALS AND METHODS
Among modern biotesting methods,
biosensors based on luminescent microorganisms The present research was carried out on
are the optimal tool for toxicity control of cultured cultured fish species in Russia: i en e
fish. There are two types of lux-biosensors, el en tae tii, i en e ae ii, i en e
biosensors with constitutive expression and tellat .
biosensors with inducible luminescence expression.
The biosensors of the first type ("lights off") Site of collection and sampling. The studied
usually have a high level of luminescence fish species were collected from a fish farm in the
expression which decreases in the presence of toxic south of Russia in the Rostov region. This farm is a
substances. As a rule, "lights-off" strains are used recirculating aquaculture system. Fish samples were
when testing toxicity of pure chemicals, and also of collected from the studied site during the spring
various natural samples. Microtox®, BioTox, season of 2013. A total of 9 fish (3 fish of each
ToxAlert and others are among the more widely species) were collected.
used test systems based on "lights-off" bacterial
strains (Parvez et al., 2006). Bacterial strains and culture conditions.
Low baseline luminescence is characteristic Strains li MG1655 (pRecA-lux), li
for inducible strains. In these strains, luminescence MG1655 (pKatGlux), li MG1655 (pSoxS-lux),
increases in the presence of specific compounds li MG1655 (pGrpElux), li MG1655
("lights on"). These sensors have been designed (pFabA-lux), li MG1655 (pXenA-lux) were
using genetic engineering methods. Usually, used in this study. The strains kindly furnished by
promoter and reporter genes are introduced from Manukhov I.V., Federal State Unitary Enterprise,
other bacteria. Reporter genes in this case refer to a "GosNIIGenetika". The bacterial strains were
bacterial gene cassette (luxCDABE) which is cultivated in Luria±Bertani (LB) medium (Maniatis
necessary for bioluminescence formation. These et al., 1982) containing 100 μg of ampicillin/ml.
strains respond to intoxication in a graded dose- The cultures were grown under constant shaking to
response manner (Woutersen et al., 2011). early exponential phase at 37ƕC. Cells were used
Use of bioluminescent bacteria provides immediately for stress-induction tests.
opportunity to carry out rapid and efficient
assessment of ecotoxicological potential of various Biosensors assay system. The fish samples
ecosystem components and allows simultaneous were extracted and homogenized using 1% Tween-
evaluation the presence of various types of toxic 80 solution in 96% ethanol in a 1:1 ratio.
substances: Mutagenic compounds, substances Homogenate was incubated at room temperature for
causing oxidative stress, heavy metals, etc. 1 hour than centrifuged (3000 rpm, 20 min).
Research of toxic chemicals by means of biological Supernatant was used for toxicity test. To determine
analysis methods will help to estimate potential the luminescence, cells of bacterial strains were
consequences for the health of present and future grown overnight in the presence of ampicillin (100
populations of fishes (Omar et al., 2012; Kligerman, mg.l-1), diluted to a concentration of approximately
1982; Da Silva Souza et al., 2006). 107 cells.ml-1 in fresh LB medium (Maniatis et al.,
1029


1982), and allowed to grow at 37°C for 2 h. 72800 Enien Germany) (Mertens, 2005a) followed
Extracts were added in 10-μl portions to wells by two parallel readings with an ICP OES
containing 190 μl of the culture. In the control, 10 spectrophotometer (inductively coupled plasma
μl of 1% Tween-80 solution in 96% ethanol was spectrophotometer) (Perkin-Elmer, Optima 2100
DGGHG ȝO RI WR[LFDQW VROution was introduced DV, ICP/OES, Shelton, CT 06484-4794, USA)
into other wells. Methylviologen was used to (Mertens, 2005b).
control activation of the PsoxS promoter, hydrogen
peroxide for PkatG, MNNG for PrecA, high Statistical Analyses. Difference reliability of
temperature (50ɨɋ PLQ IRU 3JUS( bioluminescence in samples versus control value
pentachlorophenol for PfabA, and ZnSO4 for Plac. was estimated by t-criterion using Excel with p
To determine genotoxicity of a sample with <0.05. All experiments were carried out three times
metabolic activation, 180 μl of culture, 10 μl of the independently.
test extract (in the control variant - 10 μl of a 1%
solution of TWEEN-80 in 96% ethanol) and 10 μl
of activating mixture comprised of the S9 fraction RESULTS AND DISCUSSIONS
of rat liver microsomal enzymes (Moltox) were
added to the wells. Luminescence measurements Results of fish extract testing using li
were carried out on a microtablet luminometer LM± MG1655 (pRecA-lux) biosensor without metabolic
01T (Immunotech, Czech Republic). Numerical activation are presented in Fig. 1. The highest
values of bioluminescence were expressed in bioluminescence response of E. coli MG1655
relative luminescence units (Sazykina et al., 2002). (pRecA-lux) was registered in gill extracts from all
three studied fishes. The induction factor values
Determination of the concentration of were 1,82, 2,25 and 2,62 from el en tae tii,
heavy metals in fish. The heavy metal content of ae ii, and tellat , respectively. The values of
the samples was determined with nitric induction factors registered for liver extracts were
acidhydrogen peroxide (2:3) in three different steps lower (1.7, 1.57 and 1.80). The average effect
(1; at 145 ºC, 75 % microwave power for 5 minutes, (I=2.62) indicates that the highest concentration of
2; at 180 ºC 90 % microwave power for 10 minutes genotoxicant was in gill extracts of tellat . The
and 3; at 100 ºC 40 % microwave power for 10 minimum response of li MG1655 (pRecA-lux)
minutes) exposing samples to 40 bar pressure was found against muscle extracts from all three
microwave wet incineration units (speedwave studied fishes.
MWS-2 Berghof products + Instruments Harresstr.1.
FIGURE 1
Induction of E. coli MG1655 (pRecA-lux) luminescence by fish extracts without metabolic activation.
The chemicals used for prevention and formaldehyde, oxytetracycline, and CuSO4 are
treatment of diseases may be the sources of intensively used in aquaculture. These substances
genotoxicity in fish cultivated in recirculating can promote emergence of high levels of mutation
aquaculture systems. Chemicals such as (Chairi et al., 2010; Jerbi et al., 2011). High
1030


concentrations of heavy metals are also potential the purpose of identification of pro-mutagenic
sources of fish genotoxicity (Talapatra et al., 2007; substances, a similar picture was obtained (Fig. 2).
Omar et al., 2012). The magnitudes of the induction factors were 1.59,
Genotoxicity can also be caused by 2.02 and 2.27 for el en tae tii, ae ii, and
ozonization, which is often used in aquaculture for tellat , respectively. For liver extracts, the
stabilization of water quality and disease control. factors were lower (1.59, 1.56 and 1.59). This
Genotoxicity effects caused by ozone can lead to demonstrates the simultaneous presence of both
negative consequences in a population of fishes and direct mutagens and pro-mutagenic substances in
reduce aquaculture productivity (Silva et al., 2011). all of the studied fish tissues.
When testing genotoxicity with li
MG1655 (pRecA-lux) with metabolic activation for
FIGURE 2
Induction of E. coli MG1655 (pRecA-lux) luminescence by fish extracts with metabolic activation.
The results of testing with li MG1655 registered by means of li MG1655 (pKatG-lux)

(pKatG-lux) biosensor confirm the presence of and li MG1655 (pSoxS-lux) biosensors could
substances causing oxidative stress (peroxides) in also promote emergence of the above-mentioned
the gills of fishes (Fig. 3). Almost identical weak genotoxic effects.
pro-oxidant effects were obtained for the Siberian
sturgeon ( el en tae tii) and for stellate Substances causing protein damage were
sturgeon ( tellat ) (induction factors were 1.64 registered by means of biosensor li MG1655
and 1.61, respectively), while for the Russian (pGrpE-lux) in gill extracts (fig. 5). The maximum
sturgeon an average induction effect of 2.58 was average induction factor of 2.69 was recorded in
registered. In muscles and liver of all fishes, gills of the Siberian sturgeon ( ae ii). In
slightly different induction factor values were tellat gills, the induction factor was 2.35 and in
registered. el en tae tii ZDV 2.08. In liver, the
bioluminescent response of li MG1655
Induction factors of 1.7, 1.89 and 2.27 were (pGrpE-lux) was 1.6, 1.5 and 1.4 times lower,
registered for gill extracts with the li MG1655 respectively. For tests with muscles, the induction
(pSoxS-lux) biosensor which detects those oxidants factor values were minimal. The concentration of
in the medium that form superoxide anion radical membrane-damaging substances was maximum in
intracellularly (fig. 4). The maximum average gills of el en tae tii and ae ii (Fig. 6),
factor of induction (2,27) was registered in gills of with average induction factors of 2.54 and 2.43,
the Russian sturgeon ( ae ii). In respectively. Induction factors for liver of the same
el en tae tii and tellat liver, the species of fish were 1.53 and 1.49 times lower (1.66
bioluminescent response of li MG1655 and 1.63, respectively). For muscles, the
(pSoxS-lux) was practically at the same level (1.62 bioluminescence induction factors were
and 1.57). Additionally, these pro-oxidant effects insignificant.
1031


FIGURE 3
Induction of E. coli MG1655 (pKatG-lux) luminescence by fish extracts.
FIGURE 4
Induction of E. coli MG1655 (pSoxS-lux) luminescence by fish extracts.
FIGURE 5
Induction of E. coli MG1655 (pGrpE-lux) luminescence by fish extracts in the presence of metabolic
activation.
1032


FIGURE 6
Induction of E. coli MG1655 (pFabA-lux) luminescence by fish extracts in the presence of metabolic
activation.
The data obtained using lux-biosensors 1993) and habitat (Canli and Atli, 2003). Although
indicates that gills and liver are more subject to there were similar results between the species in
toxin accumulation. The most negative biological terms of accumulation, these inter-species
effects were revealed in these tissues. differences make it difficult to compare the
FRQFHQWUDWLRQVEHWZHHQWKHVSHFLHVVWXGLHG<ÕOPD]
Similar results were also found in regards to et al., 2007). It is possible that pollutant
metal accumulation trends amongst the different accumulation in certain tissues depends on the
tissues of the species. habitat of fishes. Accordingly, in the work of
Kalantzi et al. (2013), it was shown that wild fish
According to Table 1, the average heavy metal from sites with anoxic substrata accumulate metals
concentrations in the muscles, gills and livers in all from the ambient habitat in their gills. At the same
three fish species can be sorted in descending order time, fishes of from sites with toxic substrata
as; Fe>Zn>Cu>Pb>Ni>Cd. All metal accumulate metals and other chemicals by means of
concentrations in gills and livers were higher food via their intestine.
compared to those in muscles in all three fish
species. Accumulation of genotoxicants, substances
causing oxidizing stress, and damagers of proteins
Results of many studies confirm the pattern of and membranes mainly takes place in gills and to a
variation of toxicant (particularly heavy metals) smaller extent in liver according to the data
accumulation in different organs. Accumulation obtained by means of bacterial lux-biosensors. The
takes place mainly in gills and liver, kidney and bioaccumulation of toxicants in liver and gills is
bones and to a smaller extent in muscles and skin most likely to be connected with the fact that they
(Coetzee et al., 2002; Farkas et al., 2002; Yilmaz et belong to the main metabolizing organs. It is known
DO3ROHNVLüHWDO9LãQMLü--HIWLüHWDO that liver and gills are the main accumulators of
2010; Sönmez et al., 2012). metals and trace elements (Adhikari et al., 2009;
Saha et al., 2006). Additionally, the liver contains
It has been reported that the differences in metal-binding proteins, metallothioneins.
heavy metal levels between fish species are Accordingly, significantly higher metal
associated with dietary habits (Romeoa et al., 1999), concentrations accumulate in liver in comparison
ecological needs, metabolism conditions (Canli and with muscle tissue (Has-Schön et al., 2006; Ploetz
Furness, 1993), age, body dimensions (Linde et al., et al., 2007; Verep et al., 2007).
1033


Cu Cd Pb Zn Fe Ni
A.G gill 43.573±1.37 0.247±0.015 18.34±1.08 83.93±4.97 190.43±6.18 0.65±0.04
(42.45-45.11) (0.23-0.26) (17.14-19.24) (78.45-88.15) (184.15-196.52) (0.61-0.69)
liver 38.433±1.68 0.300±0.020 16.66±2.73 79.94±5.94 189.65±8.49 0.82±0.04
(36.52-39.67) (0.28-0.32) (14.24-19.63) (75.12-86.59) (180.14-196.47) (0.78-0.86)
muscle 30.636±1.32 0.223±0.015 17.29±0.87 65.90±6.11 177.40±7.17 0.57±0.06
(29.65-32.14) (0.21-0.24) (16.52-18.24) (60.13-72.31) (172.43-185.62) (0.52-0.65)
A.B gill 34.040±3.31 0.227±0.025 23.80±1.53 92.25±.87 169.01±6.91 0.84±0.026
(30.26-36.45) (0.2-0.25) (22.14-25.16) (90.12-95.41) (162.35-176.15) (0.81-0.86)
liver 36.276±2.21 0.337±0.020 18.18±1.56 86.53±5.66 176.56±6.11 0.95±0.04
(34.1-38.52) (0.32-0.36) (16.47-19.53) (80.24-91.24) (170.14-182.32) (0.90-0.98)
muscle 28.980±1.58 0.190±0.017 17.35±0.85 77.47±5.03 156.51±5.59 0.76±0.05
(27.17-30.12) (0.18-0.21) (16.45-18.15) (72.14-82.14) (151.47-162.53) (0.72-0.82)
A.S gill 52.320±3.48 0.340±0.034 18.95±2.46 79.01±2.89 247.20±8.33 0.67±0.03
(48.69-55.63) (0.3-0.36) (16.60-21.51) (76.45-82.15) (240.11-256.39) (0.65-0.72)
liver 49.686±1.37 0.373±0.015 16.36±2.36 71.21±2.57 255.06±8.93 0.84±0.037
(48.63-51.24) (0.36-0.39) (14.36-18.49) (69.35-74.15) (245.11-262.38) (0.80-0.87)
muscle 34.340±3.66 0.270±0.010 16.55±1.52 56.93±.60 215.44±9.97 0.58±0.04
(30.26-37.35) (0.26-0.28) (15.12-18.15) (53.52-62.18) (206.39-226.14) (0.54-0.62)
TABLE 1
Heavy metal concentrations on the various tissues of fish A. gueldenstaedtii, A. baerii, and A. stellatus.
A.G: A. gueldenstaedtii, A.B: A. baerii, A.S: A.stellatus. All values are mg.kg-1 wet wt.
The degree of accumulation of various atmosphere as a metal or a compound and both

elements in fish tissues also depends on the situations have toxic properties, it is one of the most
function of the tissue in question and on gender and important heavy metals in terms of environmental
physiology of fishes. Important roles are played by SROOXWLRQ .DKYHFLR÷OX HW DO, 2002). Its
habitat, its pollution, and feeding type of fishes accumulation from the water cycled by the system
(Alam et al., 2002). A significant concentration of is possible in cases in which filtration is not
pro-mutagenic substances in muscles of the studied adequate. Factors including the source of water
fishes was registered only in el en tae tii. As circulated to the RAS system, the levels in these
a rule, the majority of studies on toxicant water sources, metal load that the source is exposed
accumulation is carried out on muscular tissue. to and the performance level of the system may
Muscles are the main part of fish which is used for have all have effects on the metal levels in water
food. However, our results show that muscles are used for aquaculture. This information will help to
not the best indicator of fish pollution. ae ii direct efforts towards effective prevention.
was the leader among the studied species of fish in Currently, information about the levels of pollution
terms of toxicant accumulation. In 50% of cases, to which fish in recirculating aquaculture systems
the induction factor was the highest in tissues of are exposed and about the accumulation and
this sturgeon species. It is necessary to point out toxicological consequences of this pollution is
that sturgeon fishes in general are considered limited.
especially susceptible to bioaccumulation of Pollution levels in recirculating aquaculture
recalcitrant pollutants, which is one of the main systems mostly depend on the degree of
threats for them. Generally, the high level of recirculation and the system design. Poor quality of
toxicant accumulation in sturgeon is connected with mechanical or biological purification can lead to
the high concentration of lipids in their body, long incomplete removal of organic matter (waste feed
maturation period, and long lifespan (Kruse and and faeces), nutrients and pharmaceutical agents
Scarnecchia, 2002; Stanic et al., 2006). and cause serious ecological problems (Maul et al.,
Metal levels analyzed for the three species 2006; Hahn and Schulz, 2007; Schreiber and
were within the limits of consumption permitted by Szewzyk, 2008). Monitoring with the use of
international standards, except for Pb and Cd. Pb bioluminescent sensors is a useful tool. It has the
and Cd especially in muscle tissues were above the advantage of providing rapid, mechanism-specific
limits set by EU (2005) and FAO/WHO (1987). information on negative adverse effects caused by
Cadmium has the highest water-solubility among water pollution.
the other heavy metals. It has a high velocity of
propagation in nature. As lead is spread to the
1034


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402 CORRESPONDING AUTHOR
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Hisar, O., (2012) Metals in Two Species of Soner Bilen
Fish in Karasu River. Bull Environ Contam Kastamonu University
Toxicol 89:1190-1195 Fisheries Faculty
[49] Talapatra S.N., Banerjee S.K. (2007) Department of Basic Sciences,
Detection of micronucleus and abnormal Kastamonu ± TURKEY
nucleus in erythrocytes from the gill and e-mail: sbilen@kastamonu.edu.tr
1037


THE EFFECT OF SOILS HAVING DIFFERENT SALT

CONTENT ON MINERAL ACCUMULATIONS OF SOME
FORAGE LEGUME SPECIES
Süleyman Temel1, Bilal Keskin18÷XUùLPúHN2øEUDKLP+DNNÕ<ÕOPD]1
1
Department RI)LHOG&URSV)DFXOW\RI$JULFXOWXUH,÷GÕU8QLYHUVLW\,÷GÕU7XUNH\
2
'HSDUWPHQWRI6RLO6FLHQFHDQG3ODQW1XWULWLRQ)DFXOW\RI$JULFXOWXUH,÷GÕU8QLYHUVLW\,÷GÕU7XUNH\
ABSTRACTS INTRODUCTION
Determining the mineral contents and Mineral contents of the plants are significantly
cultivation potentials of forage species with affected by species, varieties, development stages
different degrees of salt tolerance in soil having of the plants, parts of the plant, applied agricultural
different chemical properties and salt contents can techniques (fertilization, irrigation, harvesting,
provide information about the fodder quality of silage), regions, seasons, soil characteristics
forage species and the importance about animal including pH and different interaction types
feed. In the evaluation of saline soils, growing of between different mineral elements [1-4]. In
salt tolerant or resistant plants is recently a particular, the mineral ions in different classes and
widespread implementation. For this purpose, this amounts dissolved in the soil have a significant
study was planned to determine the effect of impact on productivity of the plant, nutrient and
salinity stress on macro (P, K, Ca, Mg and Na) and mineral contents [5-7]. It has been recorded that salt
micro (B, Mn, Cu, Fe and Zn) mineral stress negatively affect some nutrient element
accumulations of alfalfa (Medicago sativa L.), amounts (except for Na) removed from the soil by
ELUG¶V IRRW WUHIRLO Lotus corniculatus L.) and plants [8] and some nutrients such as K+ and NO3-
sainfoin (Onobrychis sativa Lam.) species cause a nutritional imbalance in plants due to Na+
cultivated under non-saline soil (0.43 EC dS m-1) and Cl- competition [9]. Furthermore, it has been
and saline soils (9.80 EC dS m-1) and to reveal reported that excess Na+ concentration in plant
whether or not the existing forage is adequate for tissues prevents the nutrient element and osmotic
mineral requirements of ruminants. Research was balance and so, it causes to specific ion toxicity
conducted in randomized blocks design with three [10]. However, it is known that some forage species
UHSOLFDWLRQV RQ WKH ,÷GÕU 3ODLQ ORFDWHG LQ HDVWHUQ can be easily cultivated in some marginal areas
Turkey, between the years of 2011-2013. The affected by salt without causing a lot of yield and
mineral concentrations of the fodders were detected quality loss and it can meet nutrient and mineral
by ICP-OES. As a result of the study, significant substance needs of the animals [11].
differences were found among species in respect of As it is known, there is a close relationship
the other minerals examined except for K and Na, between mineral nutrient content of the soils and
and L.corniculatus was identified to be the richest mineral composition of the plants cultivated in
species in terms of mineral content. Mineral these lands, and the mineral needs of the animals
compositions of the plants differed significantly and the mineral composition of the produced forage
among the year, and generally significant increases [1]. For example, since pasture areas have species
were recorded in mineral contents of the plants in diversity in different compositions, mineral content
years following the establishment year. In terms of of the produced herbage is higher than the mineral
soil types, only K, B, Mn and Cu contents were composition of monoculture produced forage
found statistically significant. The results of this species. However, it has been reported that macro
study showed that soil salinity had no significant and micro mineral contents of the herbs produced in
effect on especially the macro mineral contents of especially disturbed rangelands which soil fertility
the plants. Besides, it was revealed that forage is low are less than the levels required by the
species cultivated in both soil types are an grazing ruminants [2]. Therefore, since grazed
important mineral source (except for K) for animals cannot meet mineral requirements except
ruminants. for salt, this gap is required to close by making
additional feeding [12]. On the other hand, the
KEYWORDS: excess of some minerals have toxic effect on the
Forage crops, mineral contents, ruminants, tolerance to animals while some of the minerals in fodders is
salt
1038


enough for the animals in very little amount [13]. MATERIALS AND METHODS
Generally, mineral needs of the animals are high;
especially the amounts of P, Ca2+, Na+ and Cl- in Climate and soil characteristics of trial
fodders are not enough for the animals. However, it areas. Research was carried out irrigable lands of
is required to meet their mineral requirements for WKH,÷GÕUSODLQORFDWHGLQHDVWSDUWRI7XUNH\ZKLFK
the balanced nutrition of the ruminants. Otherwise, have arid climatic conditions. Based on long-term
excessive and insufficient mineral intakes may climatic date, the annual precipitation, relative
cause a bad effect on the production of animals and humidity and temperature are 264.0 mm, 51.2% and
animal health, and the feeding disorders in animals 12.5 oC, respectively [17]. In the years of 2011,
[14]. 2012 and 2013 when the experimental was
In marginal areas due to salinity, cultivating conducted, total annual precipitation amounts
fodder species with high mineral content is an realized as 340.0 mm, 237.2 mm and 226.9 mm,
important potential in terms of providing mineral respectively; mean temperatures were recorded as
necessities of the animals because many plants that 12.6 oC, 13.5 oC and 14.1 oC, respectively and
can be grown under saline conditions have been relative humidity was measured as 56.5%, 53.6%
used as fodder for the animals within historical and 51.4%, respectively [17]. 7KH WULDO DUHD¶V
process [15]. Therefore, comprehension of mineral altitude was 825 m.
contents of fodder species cultivated in the soils Soil samples were taken at 0-30 cm deep of
having different chemical features is important to experiment areas and air dried. Then initial physical
estimate the mineral necessities of the animals [16]. and chemical properties of the soils were
The aim of this study is to determine the mineral determined by using the standart methods after
concentrations of some forage legume species passing a 2 mm screen, and soil characteristics of
cultivated on the normal and salt-affected soils and research site were given in Table 1.
to reveal whether or not the produced fodder meet
the mineral needs of the animals.
TABLE 1
Some physical and chemical characteristics of the experimental sites.
Soil types Texture EC pH O.M. P Ca Mg Na K B ESP

dS/m ½. 5 % Mg/kg %
Non-saline soil clayed loam 0.43 8.2 4.4 27.9 3640 537 552 1251 4.3 8.9
Saline soil loamy sand 9.80 8.5 2.1 33.8 3680 549 759 1329 12.4 11.9
Texture analysis of the soil was determined by different soil properties; saline soil (coordinates 39 o
Bouyoucus Hydrometer method [18]. The soil pH ¶´1o ¶¶¶((&HG6P(63
was measured in 1:2.5 (soil:water) extracts 11.9%) and non-saline soil (coordinates 39o
according to Rhoades [19]. The organic matter ¶¶¶ 1 o ¶¶¶ ( (&H G6P
(OM) of the soil was performed by using the Smith- ESP 8.9%) with an altitude of 820 m. As the test
Weldon method [20]. Available phosphorus (P) materials, three perennial legumes forage species
content was determined by sodium bicarbonate with different degrees of salt tolerance were used
(NaHCO3) extraction and subsequent for this study. Alfalfa (Medicago sativa L.) and
spectrophotometry [21]. Exchangeable potassium ELUG¶V IRRW WUHIRLO Lotus corniculatus L.) have
(K), sodium (Na), calcium (Ca) and magnesium moderate salinity tolerance. Sainfoin (Onobrychis
(Mg) were determined using an ammonium acetate sativa Lam.) has low salinity tolerance. Seed
extraction followed by the atomic absorption amounts for each species were determined by
method [22]. Boron (B) was determined as considering the cultivations in the ideal conditions
described by John et al [23]. Electrical conductivity of plants. According to this, 30, 20 and 120 kg of
(EC) was determined by a conductivity meter in VHHGV SHU KHFWDUH ZHUH XVHG IRU DOIDOID ELUG¶V IRRW
saturation extract [24]. trefoil and sainfoin species, respectively [25]. The
fodder crops were manually sown in 30 cm row
Experimental design. Experiment was carried spacing with a seeding depth of 2 cm for alfalfa and
out in randomized blocks design with three birG¶V IRRW WUHIRLO DQG FP IRU VDLQIRLQ [25].
replications between the years of 2011-2013. The Sowings were done trial plots prepared in 3 m x 4
research was established at two locations with
1039


m dimensions on 20th of April 2011. In each year, harvested plants were first washed with tap water,
weeds were controlled with hand hoeing as needed. and later with pure water. All the forage samples
For the fertilization, the amount of nutrient were first air dried and then dried at 70 oC in an
elements in soil were neglected. For the oven for 48 h. After measured dry weight, samples
fertilization, 40 kg/ha N (ammonium sulphate) and were ground in a Wiley mill to pass through a 1
80 kg/ha P2O5 (triple superphosphate) were given to mm screen and made prepared for mineral analyses.
legume forages at sowing in the first year of the Then the ground plant samples were prepared by
study. For the subsequent years, 150 kg/ha P2O5 wet digestion method using H2SO4 and H2O2 [26].
(triple superphosphate) for DOIDOID DQG ELUG¶V IRRW Mineral element (Ca, K, Mg, Na, P, S, B, Cu, Fe,
trefoil and 50 kg/ha P2O5 (triple superphosphate) for Mn and Zn) contents of each sample solution were
sainfoin were applied between row after the last determined by inductively coupled plasma optical
harvest in season of autumn for maintenance years, emission spectrometry (ICP-OES, Thermo
but N fertilizer was not applied. Irrigation periods Scientific, Cambridge, United Kingdom). All
of the plants were determLQHG ZLWK µµ6RLO :DWHU analyses were carried out on duplicate samples.
3RWHQWLDO 0HDVXUHPHQW 'HYLFH¶¶ :DWHU6FRXW 60
100 Sensor), taking soil texture classes into Statistical analysis. In the present study,
account. Irrigation was started when the available multiple harvests were made from the examined
moisture level in soil dropped to 50 %. Plants were species during the year, and in each harvest mineral
irrigated five times within one year and in every content of the species differed. Thus, the mineral
irrigation period, about 75 mm of water was given contents of the plants were analyzed by calculating
by means of surface irrigation method which is according to the weighted average. The data were
widely and practically used in the region. analyzed using general linear models with SPSS
(version 20). The measurements of treatments were
Data collection. The examined plants in the FRPSDUHG DQG JURXSHG XVLQJ 'XQFDQ¶V PXOWLSOH
study were harvested at the convenient growth range tests at the 0.01 significance level.
stages. Alfalfa (M. sativa), ELUG¶V IRRW WUHIRLO (L.
corniculatus) and sainfoin (O. sativa)were cut at
early flowering, full flowering stages and between RESULTS AND DISCUSSIONS
early flowering and 50 % flowering stages,
respectively according to the procedure described Macro-minerals. Macro-mineral contents of
by Tan and Serin [25]. In both soil types, alfalfa Medicago sativa L., Onobrychis sativa Lam. and
DQGELUG¶VIRRWWUHIRLOZHUHKDUYHVWHGWKUHHWLmes in Lotus corniculatus L. species cultivated in different
2011, but four times in the maintenance years. On soil conditions between the years of 2011 and 2013
the other hand, sainfoin was cut three times in are given in Table 2.
2012, but twice in 2011 and 2013 years. The
TABLE 2
Mean macro-mineral compositions (%) of forage legume species grown on the two different soils during
the three years.
P K Ca Mg Na
In terms of species
Medicago sativa 1.28b 0.38 0.57A 0.32B 0.52
Lotus corniculatus 1.37a 0.37 0.58A 0.36A 0.53
Onobrychis sativa 1.39a 0.41 0.46B 0.31B 0.52
In terms of soil types
Non-saline soil 1.34 0.41a 0.52 0.33 0.55
Saline soil 1.36 0.37b 0.55 0.33 0.50
In terms of years
2011 1.59A 0.33B 0.29C 0.12C 1.13A
2012 1.64A 0.62A 0.87A 0.33B 0.40B
2013 0.82B 0.21C 0.45B 0.54A 0.04C
A,B,C,. Means indicated with different letters in the same column are significantly different by Duncan test at p<0.01. a,b,c,.
Means with different letters in the same column are significantly different by Duncan test at p<0.05
1040


Significant differences (p<0.05) were recorded increase in salinity, while increases in P content of
among forage species in terms of phosphate M. sativa and L. corniculatus species, some
content. According to these results, O. sativa had decreases are seen in P content of O. sativa plant.
the highest phosphorus content followed by L. On the other hand, while P content of M. sativa
corniculatus and M. sativa (Table 2). Similar plant is showing a continuous decrease following
results were revealed by also different researchers the establishing year, P content of L. corniculatus
and that phosphorus composition of the fodders was and O. sativa plant increased in the second year
reported to change according to the species and the (2012) according to establishing year but in the
varieties [2]. In current study, P content of the third year (2013) it decreased again (Figure 1).
fodders has a significant difference between the Many researchers mentioned that increased salinity
years and the highest P contents were recorded in in the soil and especially Na decreased taking
2012. Generally, temperature, light, airing, soil pH, phosphate by the plants [28-29]. However, Kopittke
the mutual effects of the ions, plant species and the et al. [30] reported that increased salinity levels
growth are the main effective factors on uptake the cause significant increases in phosphate content of
elements of plant nutrients [27]. Therefore, one or fodder species. As a result, while salinity is
more factors that are effective in uptake plant decreasing P concentration in plant tissues in many
nutrient element may cause different mineral situations, it increases P concentration in some
content of the plants between the years. situations or it has no effect [31]. Therefore, there is
According to soil types and years, the changes no mechanical explanation of the matters of salinity
in P content of the species were found to be and P interaction [32].
significant (p<0.05) (Figure 1). Accordingly with
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FIGURE 1
The effect of species x soil type (a) and species x year interaction (b) on P accumulations of the fodders.
NSS: Non-saline soil, SS: Saline soil, M.s: Medicago sativa, L.c: Lotus corniculatus and O.s: Onobrychis
sativa.
In respect of the potassium content, no other ions in the content of soil decreased taking K+
significant difference was found between species, by the plants. Besides, Na+ accumulated in the plant
but with the increase in salt concentration in the in salt stress prevents the potassium intake [35]. In
soil, decreases were seen in K+ contents of the also our current study, the K+ content of fodder
fodders (Table 2). Similar results have also been differed significantly (p<0.01) among years. When
revealed by different researchers and it has been Figure 2-a is examined, the highest K+ contents in
reported that salinity stress had a preventive effect three species were determined in 2012 and K+
on potassium accumulation in the plants [33, 29]. content of the species examined in the establishing
For example, Teakle et al. [34] stated that 200 mM year was found to be higher compared to the year of
NaCl application in Lotus corniculatus and Lotus 2013. This may be caused by high salt content of
tenuis plants reduced the K+ rate in plants. the soil and low air temperature in the establishing
Especially in saline soils, that Na+, Mg2+ and Ca2+ year according to the maintenance year. In a study
ions are more compared to control soil conditions conducted, in M. sativa and in many other fodders,
may cause this (Table 1). About the subject, Kacar it has been stated that the increased air temperature
and Katkat [27] stated that being high of Na and during growth period increased K+ accumulation
1041


[36]. Besides, it is well known that salinity stress that high Na+ content and drought conditions
causes a decrease in K+ accumulation in plant body caused low potassium concentration in the fodders.
[37]. About the matter, Abdullah et al. [2] stated
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й
й
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Ϭ͕ϱϬ
Ϭ͕ϯϬ K͘Ɛ K͘Ɛ
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FIGURE 2
The effect of species x year interaction on K+ (a) and Ca2+ (b) accumulations of the fodders. M.s: Medicago
sativa, L.c: Lotus corniculatus and O.s: Onobrychis sativa.
In our current research, Ca2+ concentrations results [42, 38]. Mg2+ content of L. corniculatus
between the species and years were found plant was found higher than the other species. This
significantly different (p<0.01) (Table 2). The may be caused by the fact that L. corniculatus is
highest calcium concentration between the species more tolerable to salinity than the other two
was found in L. corniculatus and M. sativa, and the species. Similar results have also been found by
lowest one was found in O. sativa plant. Similar Boga et al. [43] and they reported that Mg2+ content
UHVXOWV ZHUH DOVR IRXQG E\ (OPDOÕ DQG .D\D [38] of L. corniculatus was higher than M. sativa plant.
and they reported that Ca2+ content of O. sativa was When Table 2 was examined, the highest Mg2+
lower than the plant of M. sativa. Besides, content was determined in 2013, and the lowest
Khorshidi et al. [39] stated that salt-tolerant values were detected in 2011. It is thought to be
varieties of M. sativa may absorb more Ca2+ ions. resulted from temperature differences. In a study
The difference in the amount of Ca2+ taken by the conducted, it was reported that Mg2+ concentration
plants can be described with genetic and root reduced in M. sativa plant with the increase in air
structures. Generally, the plants having high cation temperature [36]. Besides Mg2+ content of the
exchange capacity take more Ca2+ compared to the species taken to the examination showed a
lower ones. Again, the plants having enough root continuous increase in the following years after the
growth and improvement provide more ion establishing year (Figure 3-a). It is thought that this
absorption [40]. According to the years when Ca2+ situation causes an improvement in soil structure
content of the plants are examined, in all three and a reduction in salinity degree of the soil in the
species, the highest Ca2+ content was detected in years following the establishing year as a result of
2012, and the lowest values were detected in 2011. applied agricultural activities (fertilizing, hoeing,
Also, the species examined had more Ca2+ content irrigation). In a study conducted with this aim, it
in 2013 when compared to the year of 2011 (Figure was stated that Mg2+ contents of the plants reduced
2-b). This may be caused by the fact that Na+ in the with the increase in salt concentration in the soil
soil in establishing year causes Ca2+ deficiency in while Mg accumulation increased with the decrease
plants. On the subject, Grattan and Grieve [41] in salinity degree in the soil [33].
stated that being high Na+ concentration in root Sodium content was found to be very
environment prevents the intake and transportation important (p<0.01) between the years and Na +
of Ca2+. content of the obtained fodders continually
In present study, the content of Mg2+ has decreased in years following the establishing year
shown significant differences (p<0.01) between the (Table 2). Especially in establishing year (2011),
species and years (Table 2). That there are different Na+ accumulation in fodders was found to be higher
Mg2+ concentrations in most of the fodder species in saline soil conditions (Figure 3-b). The studies
by the studies conducted beforehand supports our conducted beforehand reported that with the
1042


increase in salinity in most of the plant species, Na+ Micro-minerals. Mean micro-mineral
content increased in both root and the shoots [44- contents of Medicago sativa L., Onobrychis sativa
45]. Therefore, that Na+ content is high in saline Lam. and Lotus corniculatus L. species cultivated
soils in the establishing year may have caused in different soil conditions between the years of
higher Na+ accumulation in plant body. 2011 and 2013 are presented in Table 3.
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й
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FIGURE 3
The effect of species x year interaction (a) on Mg 2+ and soil type x year interaction (b) on Na +
accumulations of the fodders. NSS: Non-saline soil, SS: Saline soil, M.s: Medicago sativa, L.c: Lotus
corniculatus and O.s: Onobrychis sativa.
TABLE 3
Mean micro-mineral compositions (ppm) of forage legume species grown on the two different soils during
the three years.
B Cu Fe Mn Zn
In terms of species
Medicago sativa 20.08B 18.18A 40.61B 28.93B 39.78A
Lotus corniculatus 23.09A 17.72A 44.96AB 35.92A 33.79B
Onobrychis sativa 19.96B 15.82B 50.26A 29.46B 33.24B
In terms of soil types
Non-saline soil 19.31B 17.68A 45.28 29.28B 36.35
Saline soil 22.78A 16.81B 45.27 33.60A 34.86
In terms of years
2011 9.93C 2.85C 46.88A 12.31C 18.16C
2012 16.06B 8.27B 51.04A 20.17B 28.39B
2013 37.15A 40.61A 37.90B 61.83A 60.26A
A,B,C,. Means indicated with different letters in the same column are significantly different by Duncan test at p<0.01.
Boron content in plant tissues differed saline soils had higher boron content compared to
significantly (p<0.01) depending on species, soil control soils where the experiment was conducted
types and the years (Table 3). B content of L. 7DEOH 2Q WKH VXEMHFW .Õ]ÕOJ|] [47] reported
corniculatus plant was found higher than M. sativa that there was more boron accumulation in the body
and O. sativa plants, and B values of both species of barley plant while boron concentration is getting
were under the same statistical group. In a study increased in the soil. Similar results were also
conducted previously, it was revealed that boron revealed by different researchers and they stated
accumulation had differences among fodder species that B contents of the plants increased depending on
and M. sativa had higher boron content than O. increased soil salt concentration [48, 28]. The
sativa [46]. In saline soil conditions, plants had changes in B content of the plants due to the years
more B content compared to control soil conditions were found to be significant and the highest B
(Table 2). This may be caused by the fact that accumulation between the years was found in 2013
1043


and the lowest values were detected in 2011 (Table transpiration increase in the plants. Being high of
3). Accordingly, while B contents of the plants transpiration rate may increase boron transportation
taken to be examined was at the lowest level in and accordingly boron accumulation in plants,
establishing year, it increased a little in the second because, it is known that increase in transpiration
year and reached to the highest in the third year rate has positive and significant effect on the plants
(Figure 4-a). According to establishing year, being [49]. Besides, B contents of the soil types according
higher of boron contents in the plants in to the years were found significant (Figure 4-b). In
maintenance years may be thought to be resulted 2011 and 2012, saline soil conditions had higher B
from different air temperature during the years content than control soil conditions and the highest
(2011: 12.6 oC, 2012: 13.5 oC and 2013: 14.1 oC) boron content was determined in both soil types in
when the experiment was conducted. As it is 2013.
known, temperature increase may cause
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FIGURE 4
The effect of species x year (a) and soil type x year interaction (b) on the B accumulations of fodder. NSS:
Non-saline soil, SS: Saline soil, M.s: Medicago sativa, L.c: Lotus corniculatus and O.s: Onobrychis sativa.
Copper content differed significantly (p<0.01) stated that soil salinity reduced Cu2+ content in the
between species and the highest Cu2+ content was stems and leaves of corn plants. Cu2+ content was
measured in M. sativa L. corniculatus plants (Table found extremely important among the years and the
5HJDUGLQJWKLVLVVXHùHQJODQG+DOLOR÷OX>@ highest Cu2+ accumulation was determined in 2013,
reported that there are big differences between the while the lowest values were determined in 2011.
varieties and genotypes belonging to M. sativa in When evaluated in respect of plant x year
terms of Cu2+ content. Moreover, the studies interaction, Cu2+ content was found to be
conducted revealed that there were significant significant. According to these results, while Cu2+
variations even in the mineral levels of the different contents of M. sativa, L. corniculatus and O. sativa
forage species grown in the same soil [51]. Cu2+ species were at the lowest level in the establishing
content of the forage species were significantly year, it increased slightly in the second year and
affected by the soil types and the levels of Cu2+ reached to the highest level in the third year (Figure
were significantly found to be higher under non- 5-b). In our present study, in the region chosen as
saline soil conditions than the saline soils. Similar trial area, no crop production had been conducted
results were also revealed by Uddin et al. [52] and for a long time. Therefore, the activities such as
they stated that in saline soil conditions, the fertilizing, weeding, irrigating applied following the
solubility of micro elements such as Cu2+ and being establishing year is thought to cause improvements
taken of them by the plants decreased. Besides, in soil structure in the next years. As a result, intake
with increased salinity, while Cu2+ content of M. by the plants and the solubility of Cu2+ elements
sativa and O. sativa plant was reduced, Cu2+ content may increase depending on the improvement in soil
of L. corniculatus plant did not differ significantly characteristics. Similar results have been reported
in addition to the soil types (Figure 5-a). This may also by Turan et al. [35], and these results are in
resulted from being different of the ability to agreement with our findings.
tolerate and prevent the excessive salt Significant differences (p<0.01) were found in
concentrations. For example, Hasan et al. [53] terms of iron accumulation among the species and
1044


the highest Fe2+ content was obtained from O. content of the soil types was found significant
sativa plant (Table 3). About the subject, Jalali- between the years and the highest Fe2+
Honarmand et al. [29] stated that Fe2+ ion amounts accumulation was detected under Non-saline soil
removed by the plants differed even among the conditions in 2012 (Figure 6). However, in saline
varieties. Fe2+ content was found highly significant soil conditions, it was seen that Fe2+ content
among the years and in 2013, Fe2+ accumulation continually decreased in years following the
was lower compared to the previous years. Fe2+ establishing year.
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FIGURE 5
The effect of species x soil type (a) and species x year interaction (b) on the Cu accumulations of fodder.
NSS: Non-saline soil, SS: Saline soil, M.s: Medicago sativa, L.c: Lotus corniculatus and O.s: Onobrychis
sativa.
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FIGURE 6
The effect of soil type x year on the Fe accumulations of fodder. NSS: Non-saline soil, SS: Saline soil.
In terms of manganese content, the effects of in terms of Mn2+ content (p<0.01) and the highest
year, soil type and species were found to be very Mn2+ concentration (33.60 ppm) was obtained from
significant (Table 3). L. corniculatus plant had high saline soil conditions (Table 3). This may
statistically more Mn2+ content than M. sativa and resulted from weak drainage that is effective in the
O. sativa plant. About the subject, Grzegorczyk et formation of saline soils. Because the weak
al. [54] stated that 12 forage species grown in drainage increase Mn2+ content of fodders [55].
different soil conditions had different mineral These results are in agreement with reports of
compositions and Lotus corniculatus species had Martinez et al. [48], who stated that soil salinity
higher Mn2+ content than many other species. In increased Mn2+ accumulation in the body of plant.
another study conducted, they reported that alfalfa Among the years, maximum Mn2+ accumulation
had lower Mn2+ content than white clover [55]. Soil was measured in 2013 while minimum values were
types included under the different statistical groups measured in 2011. According to these results, while
1045


Mn2+ contents of M. sativa, L. corniculatus and O. of root surface width and root growth. Generally,
sativa species were at the lowest level in the the plants having higher root surface width can take
establishing year, it increased a little in the second zinc from the soil more easily. So, zinc can be used
year and reached to the highest level in the third relatively more effectively by the plants [56].
year (Figure 7-a). Mn2+ contents of soil types were Among the years, the lowest Zn2+ content was
found to be significant due to the years. When determined in the establishing year and in the years
Figure 7-b was examined, the highest Mn2+ content following the establishing year; increases were seen
were determined under saline soil conditions in in Zn2+ accumulation of the fodders (Table 3). As it
2013. Whereas Mn2+ contents of non-saline soil and is known, the usefulness of zinc and intake by the
saline soil conditions in 2011 and 2012 were found plants are in close relationship with temperature
to be close to each other. degree. During the periods when the temperature is
In this study, Zn2+ content of the fodders low, zinc intake slows down and vice versa it
differed significantly depending on the species and increases [57]. In our present study, it is also seen
years (Table 3). Zn2+ content of M. sativa plant was that temperature degree was lower in the
significantly found to be higher compared to the establishing year when compared to the
other two species. This may resulted from the effect maintenance years.
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FIGURE 7
The effect of species x year (a) and soil type x year interaction (b) on the Mn accumulations of fodder. NSS:
Non-saline soil, SS: Saline soil, M.s: Medicago sativa, L.c: Lotus corniculatus and O.s: Onobrychis sativa.
Moreover, in our present study, it was tried to recommended for ruminants by NRC [58] (0.12 to
be revealed whether or not legume fodder crops 0.48%). However, when the content of phosphate
grown in different soil conditions meet the mineral was found higher than 1.0% according to dry matter
needs of the animals. The recommended ranges of principle, the symptoms of poisoning can be seen.
K, Ca, Mg, Na, Cu, Fe, Zn and Mn, for all classes Therefore, the fodders must be fed with caution in
of ruminants as suggested by NRC [58] were 0.5 to terms of phosphate content.
1.0%, 0.19 to 0.82%, 0.12 to 0.20%, 0.06 to 0.18%,
6 to 12 ppm, 30 to 60 ppm, 7.0 to 100.0 ppm and 18
to 36 ppm, respectively. In our present study, the K, CONCLUSION
Ca, Mg, Na, Cu, Fe, Zn and Mn of the fodders
varied from 0.38 to 0.41%, 0.46 to 0.58%, 0.31 to % In general, it was determined that the salinity
0.36%, 0.52 to 0.53%, 19.96 to 23.09 ppm, 15.82 to of soil had not a significant effect on mineral
18.18 ppm, 40.61 to 50.26 ppm, 28.93 to 35.92 ppm contents of forage legume species, only the content
and 33.24 to 39.78 ppm, respectively. According to of K decreased with increased salinity. Besides,
these results, it was determined that mineral saline soil conditions caused an increase in B and
contents of forage species cultivated in both saline Mn contents in the plant body, and a decrease in Cu
and non-saline soils were DGHTXDWH IRU DQLPDOV¶ content. The fact that the forage legume species
demands except for K. On the other hand, examined response different against salinity
phosphate contents of the plants ranged from 1.26 degrees also lead to differ the mineral contents
to 1.44% according to the soil types and years and among the species. Accordingly, L. corniculatus
were found to be higher than the levels was determined to be the richest species in terms of
1046


all the examined mineral contents except for Zn. in urban environment. Fresenius Environ. Bull
Mineral contents of the obtained fodders differed 22: 987-994.
significantly depending on the years and in the [6] Tong, H.Y., Gu, C.S., Yuan, H.Y., Ma, J.J.
years following the establishing year, significant
and Huang, S.Z. (2014). Effects of salt stress
increases were observed especially in micro mineral
contents of the plants. Fodder species grown in on the growth, photosynthetic pigments and
both soil types were revealed to be a significant structure of two halophytes, Iris halophila and
mineral resource for ruminants. Moreover, it was I. lactea var. Chinensis. Fresenius Environ.
revealed that the forage legume species cultivated Bull 23: 84-90.
in both soil types were an important mineral source [7] Abari, A.K., Nasr, M.H., Hojjati, M. and
for ruminants. However, Potassium content of the Bayat, D. (2011). Salt effects on seed
fodders obtained was found to be low, and
germination and seedling emergence of two
phosphate content was found to be extremely high.
Therefore, it was foreseen that K needs of the Acacia species. African J Plant Sci 5: 52-56.
animals should be met by additional mineral [8] 7XUDQ0$$úÕN%%dHOLN+DQG.DWNDW
supplement in order to obtain satisfactory A.V. (2012). Effect of foliar applied humic
productivity, and the fodders obtained in terms of acid on growth and some nutrient elements
phosphate content should also be given with uptake of maize plant under salinity
caution to the animals. conditions. J Arts and Sci Sakarya U 14: 529-
539.
ACKNOWLEDGEMENT [9] Jouyban, Z. (2012). The effects of salt stress
on plant growth. Technical J Eng Appl Sci. 2:
This research was supported by the Scientific 7-10.
and Technological Research Council of Turkey [10] Arzani, A. (2008). Improving salinity
with Project No. 110O259. The authors thank tolerance in crop plants: a biotechnological
7h%ø7$.IRUÕW¶VIXQGLQJ view. Vitro Cell Dev Biol Plant 44: 373-383.
[11] $FDU=6DEDQFÕ&27DQ06DQFDN&
.Õ]ÕOúLPúHN 0 %LOJLOL 8 $\DQ ø
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.DUDJ|]$0XW+$úFÕgg%DúDUDQ8
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1049


[58] N.R.C. (1985). Nutrient requirements of CORRESPONDING AUTHOR

domestic animals. Nutrient Requirements of
Beef Cattle. 6th edition. National Academy of Assist. Prof. Dr. Süleyman Temel
Sciences, Washington, DC. USA. ,÷GÕU8QLYHUVLW\Faculty of Agriculture
Department of Field Crops
,÷GÕU ± TURKEY
Accepted: 13.12.2015 e-mail: stemel33@hotmail.com
1050


EFFECT OF THE IRON CONCENTRATION ON THE

PROPERTIES OF AN ALUMINOSILICATE GLASS USED
FOR STORAGE OF RADIOACTIVE WASTE
D. Moudir1, S. Ikhaddalene2, N. Kamel1, A. Benmounah3, F. Zibouche2, Y. Mouheb1, F. Aouchiche1
1
Nuclear Research Center of Algiers, Nuclear Technology Division, 2. Bd Frantz Fanon, BP 399, RP-Algiers, Algiers, Algeria
2
Faculty of Science. Department of Chemistry, Railway Station Road - 35000 Boumerdes, Algeria
3
Research Unit, Process Materials and Environment, University M'Hamed Bougurra, Boumerdes, 35000, Algeria
ABSTRACTS environment for extended period of time. A variety

of matrix materials is available for the
Aluminosilicate glasses doped with iron of the immobilization of HLW. The choice of the
valence state III have satisfactory properties, and immobilization technology depends on the physical
may be suitable as nuclear glasses, dedicated for and chemical nature of the waste and the
storage of radioactive waste solutions, cause of the acceptance criteria for the long term storage and
good reception capacities of actinides and / or disposal facility to which the waste will be
lanthanides, and excellent long-term performance consigned. The main immobilization technologies
properties. In this work, a simplified that are available commercially are cementation,
aluminosilicate glass composition: SiO2-Al2O3- bituminization, and vitrification [2] .
Fe2O3-MgO-Li2O-Na2O-K2O-Y2O3-ZrO2-La2O3- Vitrification is a particularly attractive
MoO3 is synthesized by a batch method of double immobilization route because of the high chemical
melting of oxides at 1,450 ° C during 1h30min. The durability (remain in a corrosive
influence of the content of Fe2O3 in the glass in the environment). Furthermore, glasses have a flexible
interval: 0.50 to 7.00% was studied. Most of the microstructure which permits waste compositions.
basic properties increase with the glass Fe2O3 Nuclear glasses must be durable against both self-
content. The Archimedes density of the materials is irradiation and water corrosion [3-5].
in the range 2.55-2.63 g / cm3, the molar volume in At an industrial scale, many glass systems are
the range 22.92 to 23.20 cm3/mol, Both X-ray employed in nuclear industries, mainly alkali
diffraction and scanning electron microscopy borosilicates and aluminoborosilicates glasses [6],
analyses reveal a pure amorphous structure. each kind will exhibit a complex behavior
Infrared Spectroscopy Fourier Transform gave depending on its basic structure and the dopents
spectra representative of the chemical composition elements [7-9]
of the glasses studied. The values of Vickers With regard to their physico-chemical
indentations are from 516 to 724 HV and Young properties, it has been demonstrated that these kinds
modulus around 0.330-1.840 GPa. The electrical of glasses may be suitable candidates for minor
resistivity show a spring in the concentration of actinides and (REE elements or fission products)
3.0% Fe2O3, display both the combined effect of Fe [6]. Aluminosilicate glasses are widely used as
valence change and the mixed alkali effect (MAE) glasses with good mechanical, thermo-mechanical
in the glass. and high chemical durability [10, 11], especially
when added with specific metals, like iron.
Many physical properties of oxide glasses
KEYWORDS: show non-linear behavior as a function of alkali
nuclear Glass, Fe, storage, radioactive waste, XRD, SEM. content, if one alkali type is gradually changed for
another alkali ion type [12-14], Which is referred
to the mixed alkali effect (MAE) [12].
INTRODUCTION In this paper we study the effect of iron
concentration ranging between 0.50 to 7.00 wt.%
If one considers a typical nuclear fuel cycle, on the properties of an aluminosilicate glass.
majority of the radioactivity of the entire cycle is Lanthanum (La) and Yttrium (Y) was employed as
concentrated in the so called high level waste a lanthanide/actinide surrogate.
(HLW) [1]. Management of this waste is a major The materials are characterized by their
challenge for nuclear scientists. They require physical, microstructural, mechanical and electrical
special treatment to ensure its isolation from human properties. The glasses densities are measured for
calculating the molar volume, the amorphous
1051


structure was confirmed by X-ray diffraction mixtures are melted and refined at 1,450 °C in air,
(XRD) and a scanning electron microscopy (SEM), for 1h30min, in a platinum crucible using a
samples structure was characterized by the FTIR Carbolite BLF 18/3 1800.The heating step was of
VSHFWUD 9LFNHUV PLFURKDUGHQHVVHV \RXQJ¶V 5°/min. The melted glasses are casted in steel
modulus and the electrical characteristic using the cylindrical moulds.
adequate equipment.
RESULTS AND DISCUSSIONS

MATERIALS AND METHODS
GLASSES CHARACTERIZATIONS
The glasses were prepared using the method
described by M. Chavoutier, and al. [15] which The synthesized glasses have been
consisted in a two melting/casting steps. The characterized by their physical, microstructural,
following commercial reagents were employed: mechanical and electrical properties.
ZrO2 $/'5,&+ 3XULW\ $O2O3
)/8.$3XULW\0J2)/8.$3XULW\ PHYSICAL CHARACTERIZATION
97%), Na22 0(5&. 3XULW\ )H2O3
0(5&.3XULW\.220(5&.3XULW\ Density and molar volume Measurements.
99%), Li22 0(5&. 3XULW\ /D2O3 The glasses density was measured by the
)/8.$3XULW\<2O3 (MERCK, Purity Archimedes method ( ), the samples was carried
0R23 0(5&. 3XULW\ 6L22 out by a hydrostatic balance using distilled water as
352/$%23XULW\5((HOHPHQWVR[LGHV an immersion liquid.
are dried over night at 1,000 °C, and the other Archimedes density gives the specific gravity
oxides at 400 °C, for the same time. This step of the several glasses. For each sample, three
ensures a good homogeneity of the products, and consecutive measures were made
confers them isotropic properties. La2O3 is prepared This last result we served for calculating
by calcination at 450 °C of La (NO3)3,6H2O another parameter which is the molar volume, using
(FLUKA, Purity 99,99%). Six powder mixtures the formula,
were prepared with Fe contents ranging between
0.50 and 7.00 wt.%. Fe content of 0.50, 1.00, 2.00, σ௜ ‫ݔ‬௜ ‫ܯ‬௜
3.00, 6.00 and 7.00 wt.% respectively. A batch of ܸ௠ ൌ
25g was prepared for each glass, having the ݀୅ ‫ͲͲͳ כ‬
chemical compositions gathered in Table 1. The With:
mixtures are homogenized in an Automatic Sieve Mi: are the molar masses (g / mol);
Shaker D403 during 7 h, to achieve a good particles xi: the molar compositions oxides ith (%),
dispersion. For each composition, the powders : the glass density (g / cm3).
TABLE 1
Chemical composition of the studied glasses.
Oxyde Contents (wt.%)

ZrO2 4.50 4.50 4.50 4.50 4.50 4.50
Al2O3 15.00 15.00 15.00 15.00 15.00 15.00
MgO 5.00 5.00 5.00 5.00 5.00 5.00
Na2o 6.00 6.00 6.00 6.00 6.00 6.00
Fe2O3 2.00 0.50 1.00 3.00 6.00 7.00
K2O 0.50 0.50 0.50 0.50 0.50 0.50
Li2O 12.00 12.00 12.00 12.00 12.00 12.00
La2O3 0.50 0.50 0.50 0.50 0.50 0.50
Y 2O 3 0.50 0.50 0.50 0.50 0.50 0.50
MoO3 3.00 3.00 3.00 3.00 3.00 3.00
SiO2 51.00 52.50 52.00 53.00 47.00 46.00
Total 100 100 100 100 100 100
1052


As can be seen, the Archimedes density of the synthesized between1000 and 1150 °C for 2 h.
glasses, increases on the average [2.55 to 2.63] These values are comparable to our values.
g/cm3with an increase in iron concentration except We can say that the atomic mass of each element
for the case of 7wt.%, were we noticed a slight added into the glass influences its density.
decrease in density. The increase in density is S.Ibrahim [16] showed an increase in density
primarily due to the increased atomic weights of the in the range 4.2632 -4.4566g / cm3 with increasing
modifying cation. levels of Fe2O3, for glasses in the system(25-x)
Overall, iron reinforces the glass structure and Na2O-x Fe2O3-25PbO-50 SiO2 where x varied from
densifies it .This result is consistent with the work 0.5 to 15 mol.%. This increase is due to the
of the literature [16]. substitution of Na2O oxides by Fe2O3 that provides
R. Laopaiboon and C. Bootjomchai [17] good durability of the material.
reported density values comprised between 2.839 The molar volume is of higher interest since it
and 2.863g /cm3 for a series of glasses (LGS) in the relates directly to the special distribution of the
system xCeO2-20Na2O-Al2O3-13B2O3-6.5CaO- oxygen in the glass network. The effect of Fe2O3
1.5PbO-(58-x) SiO2. These values are slightly content on density and molar volume system is
higher than our values, because lead is a heavier represented on the fig1. As the concentration of
element which slightly increases the density of the Fe2O3 (x) increases in the glass system, a
glasses. monotonic increase of the molar volume is seen.
On the other side N.Srinivasa Rao and al. [18] The results of the calculated molar volume are
have reported density values between 2.24 and schematized on the Fig1 which represents the
2.74g / cm3for a series of boroarsenates glasses, by change in molar volume and density depending on
varying the K2O amount in the composition system the content of Fe2O3 in the glass.
K2O-x(40-x) Na2O-50B2O3-10As2O3, (x =0-40)

Vm (cm / mol )

dA (g / cm )
3

%(Fe2O3)
FIGURE 1
Variation of the Archimedes density and molar volume of the glass depending on the content of Fe2O3.
The molar volume varies nonlinearly with the MICROSTRUCTURAL CHARACTERIZATION

content of Fe2O3 in the glass. Overall and for
contents of Fe2O3 greater than 3.0 wt.%, The molar X-Ray Diffraction. For the six studied
volume increases with the content of Fe2O3 in compositions of the glasses, X-ray diffraction
accordance with the density of the material. the (XRD) analysis was conducted by Philips ;¶3HUW
nonlinearly variation of the molar volume of the PRO equipment. The crystalline phase (s)
glass is due to the mixed alkali effect (MAE) which LGHQWLILFDWLRQ ZDV GRQH XVLQJ WKH 3KLOLSV ;¶3HUW
modifies the structure, by growing the amount of plus 2004 software [21]. The result confirm the
non-bridging oxygen (NBO) following the amorphous structure of the glasses, with no residual
polymerisation of the glass network, which crystalline phase.
increases the space in the network and thus the
volume increases [19,20].
1053


Scanning Electron Microscopy (SEM) visualized, at least with these spectroscopic

Analysis. The glasses microstructure was revealed methods.
by both the bulk and surface SEM observations.
The SEM analysis was conducted on a Philips Infrared Spectroscopy Fourier Transform
XL30 microscope. A typical micrograph of the (FTIR). Fourier Transformed IR (FTIR) spectra of
glasses is shown on Fig. 2.The micrograph all samples were recorded using a spectrometer
confirms the homogeneity of the glasses for the with the following equipment (NICOLET 380). The
different Fe contents. The SEM confirms the XRD FTIR spectra of the samples were measured from
results, and thus the amorphous nature of the 400 to 4000 cm-1 (wave number) at room
products. No crystalline micro-grains can be temperature.
FIGURE 2
SEM micrograph of typical glass with 6 % Fe2O3
Taking this into consideration and combining The absorption bands of Fe-O in an octahedral
it to the literature, the experimental FTIR data are structure of structural units FeOOH appear around
interpreted as follows: (458-470) cm-1. [28,29].
The band between (457 and 467) cm-1 [22] Absorption bands (580-582) Cm-1 and ‫׾‬538 Cm-1
corresponds to the vibration Si-O-Si. Additionally, occur primarily Because of vibrations Fe-O
another band occurs at around 452 cm-1 is assigned assimilated to the group Fe3O4 and Fe-O for the
to O-Si-O stretching [23]. group Fe2O3, respectively. [22]
Another band occurs at around 470 cm-1 due to The absorption bands between 412 cm -1 correspond
the asymmetric stretching of Si-O-Si units and Si- to the antisymmetric vibration
O. This band reflects the bridging oxygen atoms Fe-OH bond. [30]
(BO) [17]. The absorption bands between 416 and 488
The absorption peak of SiO2 spectra, from 470 cm-1 correspond to the harmonic vibration of the
to 480cm-1 corresponds to the vibration of Si-O group ZrO6 and FeO4 [31]
bond. [24] The interval 440-530 cm-1 is attributed to The absorption bands between (460-480) cm-1
the deformation vibration of Si-O bond containing correspond to the Zr-O vibration [32].
the SiO4 tetrahedral group [25]. Na-O, Na interference stretching vibration Na+ -
The displacement of these bands towards OSiO is observed around 460 cm-1. [33]
lower values indicating the formation of the Si-O-
Me (Me = metal) in a crystalline structure. Me = MECHANICAL CHARACTERIZATION
can be replaced by Mg or Al. [26]
The absorption bands in the region of 600 to MicroHardness and Young modulus
800 cm-1can be related to the Stretching bonds Al-O Measurement. The micro-hardness measurements
[27]. Low absorptions between (674-685) cm-1 are made on a polished surfaces with Vickers
correspond to the vibration of elongation octahedral indenter applying a load of 300g for 10 seconds. A
(AlO8) [26]. total of 10 indents were conducted on each sample.
The equipment used was a Zwick microdurometer.
1054


On the other side the Young modulus can be The results of Vickers microhardness and
calculated by RFDA basic software for its elastic Young modulus for the prepared samples are shown
properties. in Table 2.
In our case the vibration mode used is the
bending mode for a rectangular bar [34].
TABLE 2
Vickers hardness and Young modulus for glasses of different contents of Fe 2O3.
% Fe2O3 Young modulus

La micro-dureté Vickers (HV) E (GPa) ઢE
0.5 516.4 1.240 0.040
1.0 630.6 0.390 0.030
2.0 667.9 0.980 0.980
3.0 683.0 0.330 0.030
6.0 724.7 1.780 0.080
7.0 722.0 1.840 0.060
As we see for density, the microhardness polymerization of the glassy network grow the
increases with the content of Fe2O3 in the glass. For quantity of nonbridging oxygen, which increases
all samples, it is between 516.4 and 724.7 HV. the molar volume, and the resistivity of the glass.
These results are in agreement with those of
literature which give an increase of the hardness in According to AC. Hannon [36] when a second
the average [431-508] Hv with increasing the Fe2O3 alkali oxide is added in varying proportions, it is
content from 0.25 to 10 m.%, for a silicate glass in observed that the resistivity goes through a
the system Na2O-SiO2-Fe2O3. [35] pronounced maximum when the two oxides are
present in approximately equal proportions. The
The Young modulus reflecting the elasticity of electric current in the vitreous silica is provided by
the materials, showing the ability of the material to monovalent cations, which are the most mobile
recover its structure in response of the system to an ions, such as, sodium and lithium, which are
external stress. responsible for this conductivity. The conductive
nature of the material depends directly on the
The Young modulus of glass studied is concentration of the introduced ion [35, 37].
between 0.33 and 1.84 GPa. The highest values
correspond to the higher value of Fe2O3 in the glass. These values are in agreement with those of
The amelioration of these results goes in the same literature.
road as the density and molar volume.
R. Hakim et al. [38] reported a conductivity of
3.57 x 10-8 (ȍ.cm)-1 measured at 350 °C in a silicate
ELECTRICAL CHARACTERIZATION glass system 7.5 Li2O - 7.5 Cs2O - 85 SiO2.
S. Sindhu et al. [139] reported conductivity of 4.40
The resistivity and the electrical conductivity x 10-7 (ȍ.cm) -1; for a glass composition xV2O5.
of glasses for different contents of Fe2O3 are (60-x) B2O3. 40 BaO (x = 12) tested at 200 °C.
represented by the histograms on the fig 3. These results are close to our values.
Pavic L. et al. [37] have synthesized a sodium
The electrical resistivity of the glass depend to sulfo-phosphate glass doped Fe2O3, from three
the concentration of Fe2O3, decreasing from 0.5 to modifier oxides MgO, CaO, BaO. They find a
3.0 wt.%, then increases on the average of 3.0 to conductivity of 4.57 x 10-10 (ȍ.cm)-1 for the system
7.0 wt%. these results varies on the same way as the 19.0Na2SO4-20.0BaO-60.0P2O5 with 1.0 Fe2O3; and
molar volume, which translate directly the oxygen a value of 3.81 x 10-10 (ȍ.cm)-1 for the system
bonds within the material, this behavior is 19.0Na2SO4-20.0MgO-60.0P2O5 with 1.0 Fe2O3.
explained by the mixed alkali effect, the
1055


FIGURE 3
Electrical resistivity of glass for different contents of Fe2O3.
CONCLUSION REFERENCES
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1058


ROLE OF SOME ANTIOXIDANTS IN SUPPRESSING OF

FLAX POWDERY MILDEW
Abd El-Rahim M.A. El-Samawaty1,2, Mohamed A. Yassin1,2, Mohamed A. Moslem1 and Moawad R.
Omar2
1
Botany and Microbiology Department, Faculty of Science, King Saud University, Riyadh, Saudi Arabia.
2
Agricultural Research Center, Plant Pathology Research Institute, Giza, Egypt.
ABSTRACT the world as well as it is a significant problem for

Egyptian flax growers. It is occurs annually in all
Five antioxidants; ascorbic acid, benzoic flax-production areas and infects all the
acid, citric acid, salicylic acid, and thiourea along aboveground organs including stems, leaves,
with Punch C fungicide were evaluated against flax flowers and capsules [7]. The lack of resistance in
powdery mildew disease. Efficacy of foliar sprays the nowadays commercial flax cultivars grown in
of these compounds on disease incidence, disease Egypt [8], make the search for alternatives
severity, seed weight/plant, straw weight/plant and ecofriendly and not risky methods, instead of
number of capsules/plant were investigated in an synthetic fungicides are necessary [9, 10]. Among
outdoor pot experiments over two seasons. the various alternatives are the antioxidant
Significant reductions in disease incidence and compounds; which are known for their broad
severity were achieved by those antioxidants spectrum activity against plant pathogens and as
compared with the control, particularly after two resistance inducers [4]. Some antioxidant
and three sprays. Punch C fungicide and thiourea compounds such as salicylic acid could indirectly
were the best compounds in reducing disease enhance plant resistance to different pathogens and
incidence and severity followed by benzoic and successfully used as resistance inducers [11, 12]. In
salicylic acid. Two and three sprays of the tested addition, various promising compounds have been
compounds increased the seed weight, straw discovered seemed to act as abiotic inducers
weight, and number of capsules per plant compared protecting many crop plants against many
with the control over seasons. These results may be phytopathogens [13-16]. Therefore, the objective of
due to the potential of the tested antioxidants to this study was to evaluate some compounds for
promote plant productivity and profitability. inducing the resistance of flax against powdery
Therefore, these antioxidants have potential in mildew disease and to determine the response of
suppressing flax Powdery mildew and may be flax plants to foliar application of such compounds.
alternatives to synthetic fungicides.

KEYWORDS:
Punch® C, control, in m itati im m L., i i m lini Plant Material. The experiments were
±Skoric, thiourea. carried out in the cotton Pathology department,
Plant Pathology Research Institute, Agricultural
Research Center, Giza, Egypt during two seasons.
INTRODUCTION Seeds of Sakha 1 cultivar were sown in No. 25 clay
pots; filled with autoclaved soil to eliminate the
Flax ( in m itati im m L.) is considered pathogenic soil-borne fungi which may attack the
a historically important fiber crop; widely grown in plants. Planted pots were received normal irrigation
some countries for its high-quality fibers, and and fertilization and kept under outdoor conditions
primarily grown for its seed oil in the others [1, 2]. from December to April.
It is the second most important and economic crop
following cotton in Egypt, whereas it is cultivated Antioxidant compounds. Five antioxidant
for both fiber and oily seed [3, 4]. compounds, i e , Ascorbic acid (C6H8O6), benzoic
One of the common, and easily recognized acid (C6H5COOH), citric acid (C6H8O7.H2O),
foliar diseases attack flax is powdery mildew salicylic acid (C7H6O3), and thiourea (CH4N2S),
caused by i i m lini Skoric [5, 6]. Flax powdery along with Punch® C fungicide (11.8%
mildew is widespread in most flax-growing areas of
1059


carbendazim & 23.6% flusilazole) were used in this thiourea on the disease severity. On the other hand
study. significant difference was found between the effect
of salicylic acid and thoiurea on the disease severity
Treatments. Flax powdery mildew was after two sprays, while they insignificantly differed
allowed to develop naturally, and the initial after one and three sprays (Table 2).
application of treatments to plants coincided with
the first signs of the disease. Tested compounds Regarding the effect of treatments on the
were dissolved in deionized distilled water (DDW) seed and straw weights; obtained results (Table 3)
at 200 ppm concentrations. DDW was applied as a indicated that significant difference was found in
control of foliar spray. Three foliar sprays with the the seed weight between the number of sprays and
tested compounds were applied to run-off on 7 control except after one spray treatment.
March, 22 March and 5 April in the first season and
on 1, 15 & 30 March in the second season. All trials Significant difference was also found in the
were conducted in a complete randomized block straw weight between the number of sprays and
design; with four and three replicates for the first control. Significant difference was also found in the
and second seasons respectively. Ten days after the seed and straw weights between sprays themselves.
last spray, disease incidence and severity were
recorded to evaluate the effect of treatments on In respect of the effect of treatments on the
disease development. Disease incidence was number of capsules/plant; Table 4 indicated that
measured as percentage of infected plants / pot. significant difference was found between number of
Disease severity was measured as percentage of the sprays and the control except after one spray.
infected leaves/plant in a random sample of 10 Significant difference was also found among sprays
plants/pot [1]. At harvest, straw and seed weights themselves.
were recorded for each plant to evaluate the effect
of treatments on the plant growth. All collected data Results of the second season (Table 5)
were subjected to statistical analysis. indicated that Treatment x number of sprays
interaction was the only significant source of
Statistical analysis. Analysis of variance variation in disease incidence and severity of
(ANOVA) of the data was performed with powdery mildew but number of sprays was the only
MSTAT-C statistical package (A Microcomputer significant source of variation in seed, straw weight
program for the Design, Management and Analysis and number of capsules per plant.
of Agronomic Research Experiments, Michiagan
State Unvi., USA). Least significant difference The significant of the interaction indicated
(LSD) was used to compare treatment means. that the reduction in disease incidence and severity
was dependent on the material and number of
sprays. For example no significant difference was
RESULTS found between thiourea and citric acid in their
effects on the reduction of disease incidence and
ANOVA (Table 1) of the first season severity after one spray. Meanwhile, significant
indicated that Treatment, number of sprays and differences were found between those materials
their interaction were highly significant sources of after two and three sprays. Significant difference
variation in the disease incidence and severity of was also found between benzoic and citric acid in
powdery mildew, but number of sprays was the their effects on the reduction of disease incidence
only significant source of variation in weight of and severity after three and two sprays respectively
seed, weight of straw and number of capsules per (Table 6).
plant.
Regarding the seed and straw weight per
The significance of the interaction indicated plant; no significant difference was found between
that disease incidence and severity are depending treatments and control in increasing of the seed
on applied material and number of sprays. For weight after one spray. On the other hand
example, no significant difference was found significant differences were found between
between the effects of thiourea and Punch C treatments and control and among sprays
fungicide on the reduction of disease incidence themselves after two and three sprays. Straw
except after two sprays. Similar result was obtained weights were significantly increased after all
for disease severity in all treatments except after treatments compared with the control. Significant
three sprays. Meanwhile, significant difference was differences were also found between numbers of
found between the effect of two and three sprays of sprays (Table 7).
1060


TABLE 1
Analysis of variance of the effect of treatments (Antioxidants), number of sprays and their interactions on
the disease incidence, disease severity, number of capsules, weight of seed and weight of straw
(First season).
Experiment Source of variation D.F M.S F. value P>F

Replication 3 108.854 1.587 00.199
Treatment (T) 6 1579.830 23.037 0.000
Disease
incidence No. of sprays (S) 3 5207.345 75.934 0.000
TxS 18 303.841 4.431 0.000
Error 81 68.577
Replication 3 57.33 0.620 0.604
Treatment (T) 6 2253.521 24.376 0.000
Disease
severity No. of sprays (S) 3 7758.558 83.922 0.000
TxS 18 328.58 3.55 0.000
Error 81 92.450
Replication 3 9.166 8.912 0.000
Treatment (T) 6 1.563 1.519 0.182
Number of
capsules No. of sprays (S) 3 14.555 4.15 0.000
TxS 18 0.977 0.950 0.524
Error 81 1.029
Replication 3 0.0222 10.443 0.0000
Treatment (T) 6 0.003 1.248 0.291
Seed No. of sprays (S) 3 0.036 17.184 0.0000
weight TxS 18 0.002 1.068 0.399
Error 81 0.002
Replication 3 0.089 7.452 0.000
Treatment (T) 6 0.007 0.559 0.761
Straw No. of sprays (S) 3 0.444 37.024 0.000
weight TxS 18 0.010 0.843 0.646
Error 81 0.012
TABLE 2
Effect of treatments (Antioxidants) and number of sprays on disease incidence and disease severity under
greenhouse conditions (First season).
Disease Incidence % Disease Severity %

Treatments Control b 1st Spray 2nd Spray 3rd Spray Control b 1st Spray 2nd Spray 3rd Spray
Ascorbic acid 44.77 37.47 31.42 21.17 71.30 55.90 45.95 37.80
Benzoic acid 50.75 42.00 28.17 18.32 64.40 54.32 39.97 33.40
Citric acid 51.92 43.15 23.77 16.60 69.50 52.45 44.77 37.57
Salicylic acid 49.05 34.70 28.22 13.45 70.15 60.32 54.35 36.57
Thiourea 52.32 35.05 21.15 10.07 75.02 49.90 41.52 26.77
Punch C 53.17 36.22 00.00 00.00 61.15 40.07 00.00 00.00
Control a 54.15 54.15 57.10 53.10 74.35 65.35 62.80 62.25
Control a = Deionized water were used LSD for (T x S) interaction=11.340
Control b = Without any sprays
LSD for (T x S) interaction=11.59
1061


TABLE 3
Effect of treatments (Antioxidants) and number of sprays on weight of seed (gm) and weight of straw (gm)
per plant under greenhouse conditions (First season).
Weight of seed (gm) per plant Weight of straw (gm) per plant
st nd rd
Treatments Control b 1 2 3 Mean Control b 1st 2nd 3rd Mean
Spray Spray Spray Spray Spray Spray
Ascorbic acid 0.14 0.15 0.18 0.22 0.17 0.42 0.46 0.59 0.72 0.55
Benzoic acid 0.13 0.16 0.19 0.22 0.18 0.41 0.53 0.67 0.73 0.59
Citric acid 0.10 0.17 0.21 0.20 0.17 0.43 0.50 0.58 0.76 0.57
Salicylic acid 0.11 0.14 0.17 0.21 0.16 0.37 0.47 0.61 0.74 0.55
Thiourea 0.14 0.12 0.19 0.23 0.17 0.40 0.44 0.59 0.68 0.53
Punch C 0.12 0.16 0.20 0.26 0.19 0.41 0.55 0.65 0.72 0.58
Control a 0.14 0.15 0.15 0.13 0.14 0.46 0.61 0.60 0.58 0.56
Mean 0.13 0.15 0.18 0.21 0.41 0.51 0.61 0.70
Control a = Deionized water were used
Control b = Without any sprays LSD for (T) =NS
LSD for (T) =NS LSD for (S) = 0.06
LSD for (S) = 0.023 LSD for (T x S) interaction=NS
LSD for (T x S) interaction=NS
TABLE 4
Effect of treatments (Antioxidant) and number of sprays on number of capsule/plant under greenhouse
conditions (First season).
Number of capsule/plant
Treatment Control b 1st Spray 2nd Spray 3rd Spray Mean
Ascorbic acid 6.26 6.67 7.71 7.84 7.12
Benzoic acid 6.48 6.72 7.36 7.98 7.14
Citric acid 6.38 7.08 7.91 8.03 7.35
Salicylic acid 6.30 6.37 7.12 8.19 7.00
Thiourea 6.26 6.45 7.28 9.22 7.30
Punch C 5.92 7.20 7.31 7.99 7.11
Control a 6.19 6.57 6.78 6.09 6.41
Mean 6.26 6.72 7.35 7.91
LSD for (T) =NS
LSD for (S) = 0.54
In respect of the number of capsules/plant; between number of sprays and control and among
significantly different increasing were found the number of sprays themselves (Table 8).
1062


TABLE 5
Analysis of variance of the effect of treatments (Antioxidant), number of sprays and their interactions on
the disease incidence, disease severity, number of capsules/plant, weight of seed and weight of straw.
Experiment Source of variation D.F M.S F.value P>F

Replication 2 17.58 0.340 0.713
Treatment (T) 6 1374.878 26.592 0.000
Disease
incidence number of sprays (S) 3 3801.527 73.528 0.000
TxS 18 244.532 4.730 0.000
Error 54 51.702
Replication 2 350.584 3.231 0.047
Treatment (T) 6 1423.058 13.117 0.000
Disease
severity number of sprays (S) 3 6185.288 57.011 0.000
TxS 18 45.969 4.166 0.000
Error 54 108.493
Replication 2 2.35 0.911 0.408
Treatment (T) 6 4.48 1.733 0.131
Number of
capsules number of sprays (S) 3 32.58 12.58 0.000
TxS 18 0.831 0.321 0.995
Error 54 2.58
Replication 2 0.030 4.358 0.180
Treatment (T) 6 0.013 1.984 0.840
Seed number of sprays (S) 3 0.019 2.868 0.045
weight TxS 18 0.007 0.964 0.512
Error 54 0.007
Replication 2 0.280 11.86 0.000
Treatment (T) 6 0.042 1.760 0.125
Straw number of sprays (S) 3 0.340 14.417 0.000
weight TxS 18 0.25 1.063 0.412
Error 54 0.24
TABLE 6
Effect of treatments (Antioxidant) and number of sprays on disease incidence and disease severity under
greenhouse conditions (Second season).
Disease Incidence Disease Severity

Treatments Control b 1st Spray 2nd Spray 3rd Spray Control b 1st Spray 2nd Spray 3rd Spray
Ascorbic acid 27.37 23.59 21.93 18.81 50.05 38.14 36.97 24.66
Benzoic acid 44.34 33.49 21.63 7.11 63.86 48.62 31.26 29.39
Citric acid 48.77 34.53 26.77 18.50 66.49 55.43 42.44 31.01
Salicylic acid 49.84 32.18 22.85 16.51 71.02 54.20 39.40 26.56
Thiourea 48.73 34.03 9.60 3.03 65.31 54.66 23.20 7.03
Punch C 54.00 14.58 0.00 0.00 64.56 44.25 0.00 0.00
Control a 48.97 54.04 45.96 42.26 54.86 68.83 60.78 63.65
Control b = Without any sprays LSD for (T) =4.24
LSD for (T) =2.93 LSD for (S) = 3.20
LSD for (S) = 2.21 LSD for (T x S) interaction=8.48
LSD for (T x S) interaction=5.85
1063


TABLE 7
Effect of treatments (Antioxidant) and number of sprays on weight of seed (gm) and weight of straw (gm)
per plant under greenhouse conditions (Second season).
Weight of seed (gm) per plant Weight of straw (gm) per plant
Treatments Control b 1st 2nd 3rd Mean Control b 1st 2nd 3rd Mean
Spray Spray Spray Spray Spray Spray
Ascorbic acid 0.10 0.11 0.13 0.18 0.13 0.64 0.87 0.84 0.85 0.80
Benzoic acid 0.11 0.12 0.15 0.14 0.13 0.64 0.73 0.88 0.91 0.79
Citric acid 0.11 0.15 0.15 0.15 0.14 0.74 0.75 0.80 0.92 0.80
Salicylic acid 0.09 0.12 0.14 0.17 0.13 0.61 0.84 1.04 1.15 0.91
Thiourea 0.15 0.15 0.40 0.16 0.22 0.58 0.83 0.84 1.05 0.83
Punch C 0.12 0.16 0.18 0.19 0.16 0.69 0.80 0.80 1.12 0.85
Control a 0.11 0.12 0.14 0.12 0.12 0.70 0.70 0.73 0.74 0.72
Mean 0.11 0.13 0.18 0.16 0.66 0.79 0.85 0.96
Control b = Without any sprays LSD for (T) =NS
LSD for (T) =NS LSD for (S) = 0.48
LSD for (S) = 0.025 LSD for (T x S) interaction=NS
TABLE 8
Effect of treatments (Antioxidant) and number of sprays on number of capsule/plant under greenhouse
conditions (second season).
Number of capsule/plant
Treatment Control b 1st Spray 2nd Spray 3rd Spray Mean
Ascorbic acid 3.76 4.40 5.51 6.75 5.11
Benzoic acid 3.80 4.13 6.01 7.49 5.36
Citric acid 5.14 5.02 6.38 7.47 6.00
Salicylic acid 4.08 5.01 5.91 6.03 5.26
Thiourea 5.26 5.84 6.56 8.60 6.57
Punch C 4.60 5.76 8.16 7.68 6.55
Control a 4.63 5.25 5.44 6.71 5.51
Mean 4.47 5.06 6.28 7.25
LSD for (T) =NS
LSD for (S) = 0.49
DISCUSSION 17, 18]. Their effectiveness have attributed to

triggering of several morphological and/or
Ascorbic acid, benzoic acid, citric acid, SK\VLRORJLFDO FKDQJHV LQ WKH KRVW¶V GHIHQVH-related
salicylic acid, thiourea and Punch C fungicide were compounds, which in turn increase the systemic
tested in this study against flax powdery mildew. resistance [4, 5]. Thiourea was the best in reducing
Significant reductions in disease incidence and disease incidence and severity at all, however,
severity were achieved by these antioxidants benzoic and salicylic acid were promising. No
compared with the control and Punch C fungicide, significant differences were found in the current
particularly after two and three sprays. Therefore, study between the effects of thiourea and Punch C
these antioxidants have potential in suppressing flax fungicide in reducing the disease incidence in the
Powdery mildew and may be alternatives to two seasons when three sprays were applied.
synthetic fungicides. These compounds have Similar result was observed for disease severity in
effectively been used to induce systemic resistance the second season. This funding may be due to the
in different plants against several plant diseases [13, induction of some morphological and/or
1064


SK\VLRORJLFDO FKDQJHV LQ WKH KRVW¶V GHIHQVH-related ACKNOWLEDGEMENTS
compounds such as phenolic compounds and
proteins [4, 19, 20]. The flax resistance to powdery The authors would like to extend their
mildew on the other hand; has been suggested to be sincere appreciation to the Deanship of Scientific
related to the oxidative potential of phenolic Research at King Saud University for its funding of
compounds of leaves [9, 21]. The phenolic this research through the Research Group Project
compounds have fungi-toxic effects mainly due to No. RG- 298.
their interaction with fungal lipids or phospholipids,
causing an increase in fungal membrane
permeability, leakage of cell contents, and REFERENCES
cytoplasm aggregation [22]. The role of phenolic
compounds in the susceptibility of many plants to
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some biotic and abiotic inducers on root rot of Agriculture and Forestry í
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(2011) Pathogenesis-related protein and pp 239- 241.
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powdery mildew by elicitors. International Ascorbic acid, thiamine or salicylic acid
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and Wali, A.M. (2013) Effect of humic and Botany and Microbiology Department
ascorbic acids foliar application on yield and Faculty of Science
yield components of two Wheat cultivars King Saud University
grown under newly reclaimed sandy soil. P.O. Box 2455, Riyadh 11451 ± SAUDI ARABIA
International Journal of Agronomy and Plant
Production 4 (6):1125-1133. e-mail: myassin@ksu.edu.sa
[30] Talaat, N.B. (2003) Physiological studies on
the effect of salinity, Ascorbic acid and
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Thesis, Fac Agric, Cairo Univ Egypt.
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Acid and Salicylic Acid on Growth, Yield,
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of Soybean Plant. World Journal of
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a calcareous soil: sorption reactions and
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[33] Talebi, M., Hadawi, E. and Jaafari, N. (2014)
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[35] Anitha, S., Sreenivasan, E. and
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[36] Balai, L.R. and Keshwa, G.L. (2011) Effect of
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Journal of Species and Aromatic Crops. 20
(1): 34±37.
1067


ELEMENTAL COMPOSITIONS OF SOME COSMETIC

PRODUCTS MARKETED IN TURKEY
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5HFHS7D\\LS(UGR÷DQ8QLYHUVLW\)DFXOW\RI6FLHQFHDQG$UW'HSDUWPHQWRI3K\VLFV53100 Rize-Turkey.
ABSTRACTS Romans were using lead and mercury to look

beautiful but had no idea about the toxic effects of
The aim of this study was to determine heavy metals. For example, HgS was used for many
elemental composition of various cosmetic products years by the Romans as a red colour pigment. This
marketed in Turkey by X-Ray Fluorescence pigment was commonly used as wall decoration,
Spectrometry (XRFS). These cosmetic products VFXOSWXUH G\H JODGLDWRU¶V ERG\ SDLQW DQG DV WKH
ranged from blush, eye shadow, lipstick, nail lipstick of Roman women in the Roman [3].
polish, shampoo, hand cream, cellulite cream, baby Although there is no specific definition of heavy
powder, soap, to toothpaste (50 samples). Elements metals, it is often used as a group name for metals
that were determined in these samples included V, and semimetals (metalloids) that have been
Cr, Mn, Ni, Cu, As, Br, Mo, Ag, Sn, I, Hg, Pb and associated with contamination and potential toxicity
Bi. Metal concentration levels of Cr, As, Hg and Pb or ecotoxicity [4]. Many of these products are in
in certain cosmetic products are very important our homes and actually add to our quality of life
since these elements are toxic and dangerous for when properly used. Heavy metals may enter the
human health at high concentrations. In a few human body through food, water, air, or absorption
sample brands of cosmetic products As and Hg through the skin when they come in contact with
were detected at higher concentrations than limits humans in agriculture and in manufacturing,
defined by both German federal government and pharmaceutical, industrial, or residential settings
Health Canada regulations. In the majority of the [5]. Although several adverse health effects of
samples, the concentrations of lead are higher than heavy metals have been known for a long time,
limits defined by both German federal government exposure to heavy metals continues, and is even
and Health Canada regulations. In lipsticks alone, 8 increasing in some parts of the world, in particular
out of 11 brands contain Pb at concentrations higher in less developed countries, though emissions have
than that of the maximum permissible concentration declined in most developed countries over the last
of 10 μg g-1 in cosmetics. 100 years [6].
As certain heavy metals such as lead,
cadmium and mercury have been recognised to be
KEYWORDS: potentially toxic, a considerable potential hazard
Cosmetic products, toxic element, XRF, health hazard. exists for human exposure through consumer
products. Therefore, the contents of the heavy or
toxic metal of these products, including cosmetic
INTRODUCTION products, should be investigated on a regular basis.
Several important factors, for selection of analytical
Cosmetic products can be used on various methods used for the elemental analysis, are
parts of the human body such as the epidermis, reliability, good precision and accuracy (better than
nails, hairs and lips to look good or to change the 10%), low detection limit and cost. The
appearance. Although cosmetic products are used determination of heavy and toxic elements permits
by many people including children, young and the study of their distributions, the pollution level
elderly people, studies related to cosmetic products as well as the risk assessment in the investigated
in the literature have been limited [1, 2]. The use of ecosystems. Undoubtedly, the most popular
cosmetics is as old as human history, and almost all methods for this purpose nowadays are X-Ray
societies on earth have been used in many cosmetic Fluorescence Spectrometry (XRFS), Atomic
products since very early on. The first Absorption Spectrometry (AAS) and Inductively
archaeological data corresponding to the use of Coupled Plasma Atomic Emission Spectrometry
cosmetic products were found in the ancient (ICP-AES). However, the need for exhaustive the
Egyptians in BC 4000 years. Ancient Egyptians and sample preparation for AAS and ICP-AES, has led
1068


to increasing interest towards energy dispersive X- Spectrometer (Epsilon 5, PANalytical, Almelo, the
ray Spectrometry (EDXRF) in environmental Netherlands) featuring a three-dimensional
investigations [7]. X-ray Spectrometry, in its polarizing optical geometry. All the samples were
various forms is now a powerful, well established measured at least three times and the averages of
and mature technique for environmental analysis. It these results were obtained to minimize statistical
is also one of the Atomic Spectrometric techniques errors. The measurement parameters of sample
that can be adapted for true field portable use [8]. It pellets were set up by using the Epsilon 5 EDXRF
offers multi-element capability, low cost, high V\VWHP¶V LQEXLOW VRIWZDUH 7KH FRPELQDWLRQ RI
speed and easy operation, and its advantages and features including a-three dimensional, polarizing
limitations are now well understood [9-11]. More geometry, together with a 600 W Gd-anode, X-ray
specifically EDXRF has increasingly been applied, tube and 100 kV generator, up to 15 polarizing and
in the last 20 years, to the analysis of aerosols, secondary targets and a high resolution PAN 32
waters, sediments, soils, solid waste and other GHWHFWRUW\SLFDOO\H9EXWH9FSV
environmental samples. EDXRF is a non- 0Q.Į 6HFRQGDU\ WDUJHWV ZHUH LUUDGLDWHG E\ ;-
destructive and multi-elemental technique and can rays from Gd tube under a vacuum equipped with a
be applied to any kind of sample: liquid or solid liquid nitrogen cooled PAN-32 Ge X-ray detector
(thick, intermediate or thin) and in many cases with KDYLQJ D %H ZLQGRZ WKLFNQHVV RI ȝP WRJHWKHU
sufficient sensitivity for the determination of many with crystal thickness of 5mm and area of 30 mm2.
trace elements [12,13]. Element analysis and Then, the sample pellets were irradiated by
biological studies on ten oriental spices using XRF characteristic X-rays of the secondary targets. The
and ames test have reported [1] that the spices power, current, and high voltage of the Gd X-ray
contained the following elements: Mg, Al, Si, P, S, tube was 600W, 6mA and 100 kV, respectively.
C, K, Ca, Ti, Mn, Fe, Cu and Zn, with varying 7KH V\VWHP¶V VRIWZDUH (SVLORQ VRIWZDUH
concentrations. automatically analyzed the sample spectrum and
The aim of this study has been the determined the net intensities of element peaks as
determination of elements and heavy metal soon as the measurement was completed. A set of
concentrations in various brands in products of Auto Quantify, available from PANalytical, was
cosmetic such as lipstick, nail polish, blush, make- used for the calibration of this application. Auto
up materials, hand and cellulite cream, shampoo, Quantify offers quantitative analysis without
soap, toothpaste and cleaning products. The standards for the Epsilon 5 spectrometer. Epsilon
EDXRF spectrometer (Epsilon 5, PANalytical, 5's optional Auto Quantify routine easily handles
Almelo, the Netherlands) was used to determine the the analysis of completely unknown samples. Based
heavy metal concentrations in these samples. on Fundamental Parameter calibrations, it can
handle concentrations from % to μg g-1 levels, in a
variety of sample types including solids, powders
MATERIALS AND METHODS and liquids (https://www.panalytical.com).
Sampling and sample preparation. The

cosmetic samples were taken from different stores RESULTS AND DISCUSSIONS
LQ .DKUDPDQPDUDú 7XUNH\ All the samples
were separately put into a box that was made of Results of present measurements for trace
aluminium foil to protect from external effects. All metal contents in 50 cosmetic samples are listed in
the samples were dried at 105 to 350 0C for 10 h. Tables 1-9. As presented in Tables 1-9, each
Dried samples were homogenized using a mixer cosmetic sample type is coded according to its
and stored in polyethylene bottles prior to analysis. group. The samples of blush, lipstick, eye shadow,
All polyethylene bottles were cleaned by soaking nail polish, shampoo, hand cream, cellulite cream,
overnight in 10% nitric acid solution and then were soap and baby powder have been presented with the
rinsed with water before the samples were placed. symbols of B, L, E, NP, SH, HC, CC, SO, and BP,
After that the prepared samples were placed in respectively. The investigated samples consist of
polyethylene bottles. Samples were compressed for cosmetic products used for cleaning and make-up.
20 sec with 7 ton hydraulic press and reduced to 40 All the samples examined contained various
mm in diameter and the pellets at the mass of concentrations of V, Cr, Mn, Ni, Cu, As, Br, Mo,
500±3mg were prepared prior to analysis [14]. Ag, Sn, I, Ba, Hg, Pb and Bi.
The most important (most widely spread and
Measurement of elemental compositions in toxic) heavy metals are known to be lead, mercury,
cosmetic samples. Measurements of cosmetic nickel, manganese, chromium and cadmium. Some
samples were performed with the EDXRF heavy metal limits in cosmetic are defined by
1069


Health Canada regulation [15]. According to these its compounds, thallium and its compounds,
regulations, some heavy metal limits are limited to zirconium and its compounds, cobalt sulphate,
10 ppm for lead, 3 ppm for arsenic, 3 ppm for nickel monoxide, dioxide nickel, nickel and zinc
cadmium, 3 ppm for mercury and 5 ppm for salts. However, metals in cosmetic products are not
antimony. The limit values for heavy metals in currently regulated by the U.S. Food and Drug
cosmetic by the German federal government are: 20 Administration (FDA). Acceptable limits for heavy
ppm for lead, 5 ppm for arsenic, 5 ppm for metals vary according to the subpopulation of
cadmium, 1 ppm for mercury and 10 ppm for interest (for example, children are more susceptible
antimony [16]. In addition to this, cosmetic to heavy metal toxicity than adults and have greater
regulation established by the government of the exposure potential due to hand-to-mouth activity),
Republic of Turkey have been prepared in parallel the amount of product used and the site of
with 96/335/EC Commission Decision with application (for example arms versus lips) [15, 16].
76/768/EEC council directive of cosmetic As seen from Table 1, the blush samples of the
regulations of the European Union. By this nine different brands were analyzed and V was
regulation the presence of some metals and their determined in samples coded as B1, B2, B6, B7 and
compounds mentioned below in cosmetic products B9; Cr was determined in samples coded as B3,
are prohibited as follows: Antimony and its B4, B5, B8 and B9; Mn, Ni and Zn were
compounds, arsenic and its compounds, barium determined in all samples; Cu was determined in
salts except for barium sulphate, beryllium and its all samples except for B6 ; Ba was determined in
compounds, bromine, cadmium and its compounds, all samples except for B5; Br was determined only
chlorine, chromium, chromic acid and its salts, in B1 and B4; Sn was determined in all other
iodine, mercury and its compounds, lead and its samples except for B4 and B6 samples; I was
compounds, radioactive materials, gold salts, determined in B3, B7 and B8 ; Pb was determined
selenium and its compounds, tellurium and its in all samples except for B1, B3 and B4, and As
compounds, neodymium and its salts, tellurium and was determined in B2, B7 and B8.
TABLE 1
ഥrV, number of measurements n=3)
The results of elemental analysis of blush samples (ppm), (࢞
B1 B2 B3 B4 B5 B6 B7 B8 B9
V N.D. N.D. 149.114 41.956 89.007 N.D. N.D. 61.057 N.D.
±5.779 ±6.318 ±35.795 ±19.243
Cr N.D. N.D. 21.822 149.346 22.060 N.D. N.D. 12.500 N.D.
±1.946 ±2.794 ±4.316 ±1.261
Mn 89.180 88.298 325.455 205.547 35.483 129.307 108.008 278.319 110.380
±6.454 ±2.550 ±3.882 ±4.220 ±2.97 ±5.685 ±2.405 ±3.143 ±6.378
Ni 14.889 5.192 18.718 15.791 13.276 66.337 9.437 17.248 N.D.
±0.279 ±1.031 ±1.720 ±1.673 ±0.0977 ±1.890 ±1.267 ±1.800
Cu 24.293 60.462 10.891 23.580 N.D. N.D. 9.716 91.783 13.469
±0.991 ±1.311 ±1.376 ±0.986 ±0.715 ±2.112 ±0.503
Zn 144.888 50.360 3200 8190 8980 68.578 35.618 8630 9380
±0.507 ±1.011 ±0.001 ±0003 ±0.006 ±1.628 ±1.824 ±0.001 ±0.002
As N.D. 0.369 N.D. N.D. N.D. N.D. 2.694 0.199 N.D.
±0.045 ±0.577 ±0.010
Br 128.739 N.D. N.D. 3.766 N.D. N.D. N.D. N.D. N.D.
±1.251 ±0.512
Sn 35.546 32.680 185.908 N.D. 7.122 N.D. 35.919 8.622 12.420
±3.024 ±3.189 ±4.283 ±1.868 ±0.724 ±0.543 ±1.303
I 58.162 57.577 N.D. 60.486 35.874 61.691 N.D. N.D. 47.299
±4.782 ±0.517 ±0.501 ±5.278 ±2.312 ±2.066
Ba 629.733 229.762 88.438 114.767 N.D. 85.764 87.062 401.356 81.089
±5.454 ±1.048 ±3.907 ±1.410 ±0.911 ±0.849 ±2.003 ±3.255
Pb N.D. 24.769 N.D. N.D. 24.156 6.364 11.482 59.042 9.929
±0.179 ±0.387 ±0.319 ±1.220 ±0.468 ±0.096
Not detected=N.D.
1070


The average Pb concentration in blush for lead impurities is 10 ppm, which is considerably
samples was determined to be 22.62 ppm which is high by comparison [21]. The analyses of the trace
higher than the limit defined by Health Canada metal of facial (eyeliners, eye pencils and lipstick)
regulations. Arsenic concentration in the B7 sample cosmetics used extensively in Nigeria were reported
(a blush sample) was also determined to be higher [22] and who found Pb, Cd, Cr, Ni, Zn and Fe at
than the limit defined by the German federal various concentrations. They reported that
government. V, Cr, Mn, Ni, Cu, Zn, Br, Sn, I and continuous use of these cosmetics could result in an
Ba concentrations found in most of the blush increase in the trace metal levels in the body
samples are greater than that which can be tolerated beyond acceptable limits [22]. It has been reported
by the body. As an example, Cr was found in 4 out that investigated trace metals such as Cr, Ni, As,
of 9 blush samples, at concentrations higher than 10 Cd, Sb, Hg and Pb in lipsticks produced in various
ppm. countries and they reported levels of Pb, Cd, Ni, Cr,
Presented in Table 2, are the results of eleven Hg, Sb, and As to be 13, 15, 10, 10, 0, 11, and 23
different brands of lipstick samples marketed in ppm, respectively [17]. The heavy metals such as
.DKUDPDQPDUDú7XUNH\,QWKHVHOLSVWLFNVDPSOHV Al, Ti, Cr, Mn, Co, Ni, Cu, Cd and Pb
V was determined in L3, L8 and L9; Mn was concentrations in 8 lipsticks and in 24 lip glosses
determined in L3, L6, L8, L9 and L10; Cr was were measured previously [18]. In addition, a
determined in L3, L9 and L10; Ni was determined research related to presence of lead (Pb) in lipsticks
in L1, L2, L8, L9 and L10; Cu, was determined in sold in the United States has been done using
all samples except for L6; Zn was determined in all inductively coupled plasma±mass spectrometry
samples except for L1 and L2 ; Br was determined (ICP±MS) and it was reported that Pb levels found
only in L3, L5 and L9; Sn was determined in by the FDA are within the range that might be
samples L1, L8, L9, L10 and L11; I was expected from lipsticks formulated with permitted
determined in samples L1, L2, L7, L8, L9, L10 and colour additives and other ingredients prepared
L11; Ba was determined in samples L3, L4, L6, L7, under GMP conditions [23]. Concentration
L8, L9 and L10; As was determined only in L1, L7 determination of heavy metals such as Pb, As, Cr,
and L10; and Pb was determined in all samples Co, Cd, Ni and Cu in lipstick samples of the various
except for L2, L5 and L6. brands were performed and it was reported that
It can be inferred from Table 2, that the almost all brands contained safe levels of Pb, As,
average Pb concentration in lipstick samples was Cr, Co, Cd, Ni and Cu but in several cases the
20.35 ppm which is higher than the limits defined presence of Pb and As exceeded permissible limits
by both the German federal government and Health rendering the product unsafe for human
Canada regulations. Arsenic was found only in 3 consumption and cosmetic application [19].
lipstick samples but all of these samples had As seen in Table 3, six different brands of eye
concentrations higher than 1 ppm which is higher shadow samples were analyzed. The investigated
than the limit defined by Health Canada samples contained V, Mn, Ni, Zn, As, Br, Sn, I, Ba,
regulations. In general, most of the lipstick samples Hg, Pb and Bi in various concentrations. V was
contained V, Cr, Mn, Cu, Zn, B, Sn, In and B in determined in E3 and E6; Mn was determined in
concentrations higher than 10 ppm. These findings all samples; Ni was determined in all samples
are in agreement with most of the previous reports except for E4; Cu was determined in all samples
[17-19]. In a previously reported study, the except for E3 and E6; Br was determined only in
measurement of Pb in 26 brands and in 72 lipstick E4; Sn was determined in all samples except for E2
samples from China, Germany, Thailand, USA and and E4; I was determined in all samples except for
Taiwan was performed[17] and it was reported that E2; As was determined only in E1; Hg was
the mean value of Pb in the lipstick samples as an determined only in E2; Pb was determined in all
average of two batches with the same LOT number samples except for E2 and E4, and Bi was
was 117.40 ppm wet wt. Lipstick samples in four determined only in E1. Four out of six eye shadow
different brands have been investigated in another samples contained Pb and the average concentration
study and Al (10.98- 694.5ppm), Cr (1.30- of Pb in four eye shadow samples was 19.65
81.60ppm), Mn (0.89- 48.89ppm), Ni (4.94ppm), (4.548-45.859) ppm which is higher than the limits
Cu (118.6ppm), Zn (10.62- 345.8ppm), Cd (0.14 defined by both the German federal government
ppm), Ba (10.37- 1895 ppm), Pb (1.32- 15.92 ppm) and Health Canada regulations. Most of the
and Bi(21.3- 698.1ppm) metals in the lipstick concentrations (27 out of 28) determined for V, Mn,
samples were reported[20]. A study of 20 lipsticks Cu, Br, Sn, I and Ba in eye shadow samples are too
conducted by the United States Food and Drug great to be tolerated by human body. The
Administration showed Pb impurity levels averaged measurement of lead in the eye shadows in the
SSP ZKHUH &DQDGD¶V FXUUHQW GUDIW JXLGHOLQH eight different brands were done [17] and it has
1071


been reported an average 1.38 (0.944-1.854) ppm powders. Nine brands of mascara and eyeshade
Pb in the eye shadows samples. It has been reported purchased from the Saudi market were analysed by
[20] heavy metal contents of various types of Inductively Coupled Plasma Mass Spectrometer
cosmetics such as lipsticks, Kohl (eyeliner), henna (ICP-MS) and a flow injection mercury system
(hair dye or temporary tattoo), eye shadows, cream; (FIMS) and twenty eight elements in the mascara
freckles, moisturizing and foundation, and face and eyeshade samples were determined [24].
TABLE 2
The results of the elemental analysis of the lipstick samples (ppm) (࢞
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11
17.352 835.355 559.611
V N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±5.731 ±118.133 ±26.823
30.287 12.446 71.197
Cr N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±1.030 ±0.859 ±3.183
362.625 170.058 193.978 254.757 272.807 131.429 49.500
Mn N.D. N.D. N.D. N.D.
±12.511 ±3.433 ±8.572 ±7.956 ±12.051 ±8.236 ±2.735
32.795 34.755 46.315 51.734 19.597
Ni N.D. N.D. N.D. N.D. N.D. N.D.
±2.813 ±2.541 ±4.526 ±6.150 ±1.887
164.458 100.242 143.158 35.250 39.244 36.688 41.553 28.862 36.308 22.853
Cu N.D.
±3.902 ±3.343 ±2.463 ±0.891 ±5.480 ±4.095 ±4.166 ±2.711 ±2.449 ±2.362
203.174 560.342 813.484 1372.130 1812.604 575.365 160.534 254.693 513.975
Zn N.D. N.D.
±3.468 ±7.919 ±0.095 ±17.816 ±68.081 ±4.878 ±4.835 ±8.968 ±1.912
3.228 2.200 4.591
As N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±0.317 ±1.689 ±0.933
334.886 33.074 11.705
Br N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±1.861 ±8.123 ±0.829
1.295
Ag N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±0.014
115.928 457.244 24.381 20.325
Sn N.D. N.D. N.D. N.D. N.D. N.D. N.D.
±17.029 ±21.620 ±3.752 ±2.348
397.668 119.816 156.605 177.072 55.760 136.336 82.699 123.710 86.610
I N.D. N.D.
±2.080 ±0.543 ±1.008 ±45.113 ±48.30 ±118.077 ±1.567 ±8.584 ±0.368
180.316 278.458 297.078 1007.107 144.576 90.589 133.244
Ba N.D. N.D. N.D. N.D.
±4.624 ±4.650 ±5.377 ±30.629 ±4.380 ±0.523 ±8.304
22.091 31.487 23.367 13.533 25.530 20.228 16.161 10.397
Pb N.D. N.D. N.D.
±0.675 ±5.474 ±1.314 ±0.537 ±0.503 ±1.446 ±3.014 ±0.531
TABLE 3
ഥrV, number of
The results of the elemental analysis of the eye shadow samples (ppm), (࢞
measurements n=3).
E1 E2 E3 E4 E5 E6
V N.D. N.D. 353.978±5.201 N.D. N.D. 190.678±0.587
Mn 192.389±4.023 316.55±3.668 205.071±3.547 33.336±4.561 206.626±4.335 94.477±7.145
Ni 13.709±2.957 9.494±0.904 10.841±0.717 N.D. 7.319±0.277 12.540±1.056
Cu 8.936±0.285 15.215±0.336 N.D. 35.076±4.002 102.099±3.540 N.D.
As 0.058±0.003 N.D. N.D. N.D. N.D. N.D.
Br N.D. N.D. N.D. 160.377±1.383 N.D. N.D.
Sn 53.684±4.298 N.D. 720.904±2.282 N.D. 182.330±0.750 193.242±1.031
I 60.150±0.646 N.D. 84.629±1.876 67.542±0.505 21.783±0.396 51.253±1.192
Ba 878.230±4.544 425.921±2.436 108.185±1.454 N.D. 156.993±1.110 97.957±2.941
Hg N.D. 1967.704±37.835 N.D. N.D. N.D. N.D.
Pb 19.544±1.095 4.548±0.923 N.D. N.D. 45.859±0.837 8.638±0.212
Bi 1.186±0.080 N.D. N.D. N.D. N.D. N.D.
1072


As seen from Table 4, four different brands of been analyzed and the various concentrations Al
hand creams samples were found to contain various (15.31ppm), Cr (4.30ppm), Mn (0.70ppm), Ni
concentrations of V, Cr, Mn, Ni, Cu, As, I, Hg and (2.89ppm), Cu (0.84ppm), Zn (996.3ppm), Mo
Pb. Hg was determined in all samples except for (0.51ppm) and Ag (0.07ppm) were found [20]. The
HC4; Pb and As were determined in HC2 and HC3. elemental analysis of epidermal creams by using X-
In addition, I was determined only in HC3; Ni and Ray Fluorescence Spectrometer (XRF) have been
Cu were determined in all samples; Cr was done and in these creams Ca, Ti, Cr, Mn, Fe,
determined in all samples except HC3; V was Co,Ni, Cu, Zn, Br, Rb, Sr, Y, Zr, Nb, Mo and Pb
determined in all samples except HC1; Mn was were determined [2].
determined only in HC2 and HC4. As seen from As shown in Table 5, the samples of the
Table 4, Hg and Pb concentrations in hand cream cellulite creams of the four different brands were
samples are detected to be higher than the limits investigated and in the investigated samples V, Cr,
defined by Health Canada regulations. As Mn, Ni, Cu, As, Br, I, Ba, Pb and Bi elements were
concentrations in hand cream samples are found to determined at various concentrations. In all samples
be lower than the limits defined by both the except for the CC2 sample, As and Pb were found
German federal government and Health Canada which are accepted to be toxic to human body. As
regulations. shown in Table 5, Hg was not detected in any
Since As, Hg and Pb are toxic elements for cellulite cream samples. However the Pb
human health, they should not be present even in concentration in one cellulite cream sample (CC4)
trace levels in cosmetic products. In addition to was found to be higher than the limit defined by
these, V, Cr, Mn, Ni, Cu and I concentrations are Health Canada regulations. V, Cr, Mn, Ni, Cu, I,
also found to be large enough to cause toxic effects Br, I and Ba concentrations were found high
on the body. In addition to these results in a enough to be harmful to human health.
previously reported study, a moisturizing cream has
TABLE 4
ഥrV, number of measurements n=3).
The results of elemental analysis of hand cream samples (ppm), (࢞
HC1 HC2 HC3 HC4

V N.D. 817.717±58.968 4.651±0.638 34.353±0.561
Cr 49.677±2.141 234.483±26.953 N.D. 10.436±0.512
Mn N.D. 26.140±3.248 N.D. 28.419±1.814
Ni 12.265±2.773 64.446±4.231 13.134±1.632 4.955±0.628
Cu 61.330±44.965 235.304±6.231 112.016±5.256 366.962±4.422
As N.D. 2.644±0.204 0.654±0.051 N.D.
I N.D. N.D. 101.269±1.103 N.D.
Hg 8.427±0.559 5.360±0.555 7.388±1.297 N.D.
Pb N.D. 20.210±2.145 10.586±1.648 N.D.
TABLE 5
The results of elemental analysis of the cellulite cream samples (ppm), (࢞
CC1 CC2 CC3 CC4

V N.D. 708.540±16.611 88.164±14.451 3.478±0.501
Cr 30.557±0.509 N.D. 107.221±6.802 211.180±17.407
Mn N.D. 54.314±0.344 20.123±3.481 18.375±0.545
Ni N.D. 27.999±2.891 N.D. 45.140±2.199
Cu 92.191±2.683 156.459±2.086 85.945±2.704 32.668±3.606
As 2.417±0.121 N.D. 2.905±0.145 1.044±0.155
Br 8.199±0.491 24.871±1.742 N.D. N.D.
I 75.358±0.557 91.634±0.551 98.436±1.005 94.696±2.570
Ba N.D. N.D. 105.053±3.486 N.D.
Hg N.D. N.D. N.D. N.D.
Pb 8.090±1.889 N.D. 9.983±2.241 14.374±1.445
Bi 8.035±0.498 N.D. N.D. N.D.
Not detected=N.D.
1073


As shown in Table 6, three different brands of reported studies contain similar results to this study
baby powder samples were analyzed for their V, [22, 25].
Cr, Mn, Ni, Cu, As, I, Hg and Pb contents. In a few As shown in Table 7, five different brands of
of these baby powder samples toxic elements such nail polish samples were analyzed and V, Mn, Ni,
as As in BP3 , Hg in BP2 and BP3, and Pb in BP3 Cu, Zn, As, Br, Ag, I, Ba, Hg, Pb and Bi were
were found. Pb and Hg concentrations in baby observed in various concentrations. In a few of
powder samples are lower than the limits defined these nail polish samples, toxic elements such as As
by both the German federal government and Health (NP1, NP4 and NP5), Hg (NP4) and Pb (NP1, NP3,
Canada regulations. However, the As concentration NP4 and NP5) were found. V was found in all
in BP3 is higher than the limit defined by Health samples except for NP3; Mn, Cu and Zn in all
Canada regulations. In most of the analyzed samples; Ni only in a NP1; Br in all samples except
samples Cr, Mn, Cu and I concentrations are found for NP3 and NP5; Ag only in NP3; I and Ba in only
high enough to be harmful to human health. In a two of the samples (NP2 and NP3) and Bi only in
previous study, elemental contents of talcum baby NP2 were determined. Pb concentrations in a
powder have been analyzed by using X-ray majority of the nail polish samples were found to be
florescence and fast neutron activation techniques higher than the limits defined by both the German
[25] and it was reported that the samples were federal government and Health Canada regulations.
found to contain Mg, Si, Al, Fe, Zn, and Ba. In In addition to this, V, Mn, Cu, Zn, Br, I, and Ba
another study, analysis of the trace metals (such as concentration were also found to be large enough to
Pb, Cd, Co, and Cr) in cosmetic facial talcum cause toxic effects to human health. Ten lipsticks
powders marketed in Nigeria were performed and and ten nail polishes sold at a cosmetic store were
it has been reported that the mean concentrations of investigated using Atomic Absorption
the trace metals to be 5.0-1.0 mg/kg for Pb, 2.1-0.3 Spectrophotometer (AAS) by Ouremi and Ayodele
mg/kg for Cd, 0.7-0.1 mg/kg for Co, and 0.2-0.1 and the concentrations of six heavy metals,
mg/ kg for Cr [22]. Both of these previously including Pb, Ni, Cd, Mn, Cr and As were reported
[26].
TABLE 6
The results of elemental analysis of baby powder (ppm),(࢞
BP1 BP2 BP3

V 7.853±0.300 21.144±4.677 N.D.
Cr 14.665±2.208 N.D. N.D.
Mn 25.697±2.116 35.687±0.497 42.0022±0.303
Ni 13.329±3.805 N.D. 3.101±0.155
Cu 25.493±0.500 17.036±1.144 11.614±0.431
As N.D. N.D. 3.194±0.160
I 37.144±1.031 49.286±0.623 N.D.
Hg N.D. 1.221±0.018 2.276±0.477
Pb N.D. N.D. 6.477±0.879
As seen from Table 8, two different brands of analysed. However, Cu and Zn were detected in all
shampoos (SH1 and SH2) and three different shampoo samples. In addition Pb was detected in
brands of baby shampoos (BSH1, BSH2, and SH2, BSH1 and BSH3. As seen from Table 8, Hg
BSH3) were analyzed for their metal content and concentrations in S1 and BSH3 were higher than
V, Cr, Ni, Cu, Zn, Br, I, Hg, Pb and Bi were found the limits defined by both the German federal
in various concentrations. In the three different government and Health Canada regulations. Most
brands of baby shampoos V, Ni, Cu, Zn, Br, I, Hg, of the determined concentrations (19 out of 22) for
Pb and Bi elements were measured. Arsenic was V, Cr, Ni, Mn, Cu, Zn, Br, Ag, I, Bi and Ba are also
not detected in any shampoo samples that were found to be too high to be tolerated by the body.
1074


TABLE 7
The results of elemental analysis of nail polish samples (ppm), (࢞
NP1 NP2 NP3 NP4 NP5

V 47.988±9.791 849.787±49.683 N.D. 13.959±4.027 207.171±0.154
Mn 71.296±4.593 140.625±0.545 59.266±1.101 22.267±1.387 51.322±2.715
Ni 18.270±1.388 N.D. N.D. N.D. N.D.
Cu 34.120±4.627 24.342±4.053 27.825±3.285 49.448±2.004 40.663±2.873
Zn 2010±9.849 182.178±6.383 67.284±1.146 43.008±2.384 298.350±6.115
As 7.965±0.461 N.D. N.D. 7.858±0.891 3.216±0.189
Br 11.013±0.071 1544.268±2.967 N.D. 182.504±1.932 N.D.
Ag N.D. N.D. 11.403±0.363 N.D. N.D.
I N.D. 184.953±4.929 157.397±1.028 N.D. N.D.
Ba N.D. 384.976±9.544 2040±18.788 N.D. N.D.
Hg N.D. N.D. N.D. 0.998±0.004 N.D.
Pb 26.878±3.117 N.D. 11.677±0.586 9.927±0.064 14.826±0.283
Bi N.D. 9.736±3.281 N.D. N.D. N.D.
As shown in Table 9, three different brands of was determined in SO2 and SO3. Concentration
soap samples were investigated for their metal values of As, Hg and Pb in soap samples were
content. After completing the analysis of these soap found to be less than the limits defined by the
samples only V, Cu, Zn, As, I, Hg and Pb were German federal government and by Health Canada
detected. V was determined in SO1; while Cu and regulations. However, it is important to note that all
Zn were determined in all soap samples; As was the concentrations for Cu, Zn and I are higher than
determined in SO2; I was determined in SO1 and 10 ppm.
SO3; while Hg was determined only in SO1, and Pb
TABLE 8
ഥrV, number of measurements
The results of elemental analysis of shampoos and baby shampoos (ppm) (࢞
n=3).
SH1 SH2 BSH1 BSH2 BSH3

V N.D. N.D. N.D. 13.435±0.513 14.473±3.913
Cr 19.500±1.356 N.D. N.D. N.D. N.D.
Ni 4.465±0.399 N.D. 7.736±1.646 N.D. N.D.
Cu 27.782±1.305 56.679±0.327 27.226±2.024 33.364±1.849 50.020±1.881
Zn 803.312±2.535 131.787±1.325 1073.982±13.393 11160±20.000 676.872±3.305
Br 278.867±3.416 N.D. 3.416±0.104 10.445±0.374 12.553±0.514
I N.D. 48.654±1.166 71.457±0.505 N.D. 36.432±1.689
Hg 3.376±0.464 0.410±0.014 2.121±0.626 N.D. N.D.
Pb N.D. 5.597±0.611 9.339±0.573 N.D. 9.595±1.204

Bi N.D. N.D. N.D. 51.080±2.599 N.D.
CONCLUSION contained As and 11 samples contained Hg out of

50 samples analyzed. These results suggest that
This study has investigated the levels of trace toxic metals in cosmetics should be regulated and
metals in nine different types of cosmetic and good manufacturing practice must also be followed
cleaning products (50 samples) marketed in Turkey. WRSURWHFWZRPHQ¶VKHDOWKLQ Turkey, as has already
A total of 20 samples contained Pb at elevated undertaken by German federal government and by
concentrations (more than 10 ppm) of the 33 Health Canada regulations.
samples that contained Pb. In addition, 17 samples
1075


TABLE 9
The results of elemental analysis of soap samples (ppm), (࢞
SO1 SO2 SO3

V 19.401r0.529 N.D. N.D.
Cu 36.299r2.404 15.668r1.754 14.964r0.413
Zn 28.243r0.670 53.511r0598 23.049r0.228
As N.D. 0.385r0.022 N.D.
I 48.380r0.451 N.D. 12.476r1.610
Hg 0.136r0.005 N.D. N.D.
Pb N.D. 4.598r0.528 4.638r0.119
Not detected=N.D.
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Prof. Dr. Ömer 6|÷W
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KahraPDQPDUDú6Wo,PDP8QLYHUVLW\
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.DKUDPDQPDUDú± TURKEY
(2009) Determination of total lead in
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e-mail:omersogut@gmail.com
microwave-assisted digestion, inductively
1077

E\363 9ROXPH²1RSDJHV )UHVHQLXV(QYLURQPHQWDO%XOOHWLQ
SUSTAINABLE SECOND-CROP SILAGE CORN

PRODUCTION UNDER CENTRAL ANATOLIAN HIGHLANDS
Taner A ar1, Mahmut Kaplan2, Seyma Havva Yilma 3, Nami Sagir4, Nedim Taba 4, Ahmet Gelebur4
1
Department of Field Crops, Faculty of Agriculture, University of Akdeniz, Antalya,, Turkey
2
Department of Field Crops, Faculty of Agriculture, University of Erciyes, Kayseri, Turkey
3
Department of Field Crops, Faculty of Agriculture, University of Bingol, Bingol, Turkey
4
ILCI Cicekdagi Agricultural Farm, Cicekdagi-Kirsehir, Turkey
Objective of the study is to determine the The feeds supplied as hay and silage provide
effects of different liquid-manure doses and about 40-50% dry matter intake of dairy cows [1,
manure-chemical fertilizer combinations on yield 2]. Corn with high quality silage characteristics has
and quality parameters of silage corn grown as a great potential to play a key role in livestock
second crop after vetch + barley combination. feeding with high yields [3], therefore, it has been
Experiments were carried out in randomized block widespread all around the world [4]. Its popularity
design with 4 replications during the years 2010 as a second crop is also increasing day by day [5].
and 2011 in Turkey. Totally, 12 treatments Nitrogen fertilization directly effects yield and
consisting of different doses of liquid manure alone quality of forage crops. Excessive and unconscious
and combinations of liquid manure with chemical chemical fertilizer-use on the other hand negatively
fertilizers were tested in the experiment. Two year effects soil microbial population, organic carbon
average indicated that green herbage yield, plant content and structure. Increased nitrogenous
height, plant diameter, cob ratio, stem ratio, crude fertilizer-use also threatens the water resources and
protein ratio, NDF and ADF ratios varied between consequently the health of human and animals [6].
35396.7-44610.0 kg ha-1; 193.13-237.07 cm; So that reason, crop nitrogen status can be
13.25-17.30 mm; 30.52-33.75 %; 20.20-24.61 %; monitored by unmanned aerial vehicles to timely
42.45-49.05 %; 3.96-8.46 %; 65.01-69.77 % and provide adequate nitrogen for optimization of
33.51-35.85 %, respectively. Research results nitrogen fertilizer input [48]. Manure is not only a
showed that 15 ton liquid manure + 30 kg ha-1 quality plant nutrient [7] but also a significant
chemical nitrogen combination (L1.5N3) had regulator for soil physical, chemical and biological
higher green herbage yield (44610.0 kg ha-1) than characteristics [8]. Combining manure with
120 kg ha-1 chemical nitrogen treatment (N12). chemical fertilizers can decrease the input rates in
However, crude protein ratio of N12 treatment agricultural activities, increase the current yields
(8.46 %) was higher than L1.5N3 (5.55 %) and the and provide healthier environments to future
difference between them was found to be generations [9]. Environment-friendly manure
significant. The first study on sustainable second storage, handling and application will both increase
crop silage maize production under Central soil fertility and reduce chemical fertilizer costs.
Anatolian highlands conditions demonstrated that Cattle manure contains average 1.67 % nitrogen,
chemical nitrogen use can significantly be reduced 1.07 % phosphorus and 2.73 % potassium [10] and
by using liquid manure as an alternative to chemical may provide significant savings from commercial
fertilizers without any negative effects on soil fertilizers [11]. Another factor reducing chemical
chemical characteristics. fertilizer use is use of legumes before maize
cultivation in the rotation. In this rotation system,
the second greatest enzyme activity and the highest
KEYWORDS: soil total N content were determined in barley
Vetch+barley mixture, liquid manure, second-crop silage +Hungarian vetch mix as pre-crops application
corn, green herbage yield, chemical composition,
sustainability.
under silage maize [47].
This is the first attempt to cultivate sustainable
second crop silage corn under Central Anatolian
highland conditions safely escaping from early frost
damage due to the fact that second crop silage corn
is generally cultivated under lowland conditions. In
addition to this, main aim of the research was to
1078
determine the potential use of liquid manure either different combinations of liquid manure and
alone or in combination with chemical fertilizers chemical fertilizers were used in present study and
and the amounts to be used in second-crop silage these doses of liquid manure were applied to
corn cultivation under traditional soil tillage experimental plots before traditional tillage.
conditions of Central Anatolian highlands without Following the harvest of vetch + barley, soils were
any loss in green herbage yield and quality while plowed, disc-harrowed and then harrowed with 20-
not creating any negative side effect to the soil. 25 cm 10-15 cm with 5 cm depth, respectively
before corn plantation. Finally corn sowing was
performed just after traditional soil preparation and
MATERIAL AND METHODS experiments were carried out in randomized block
design with 4 replications. Treatments were
Experimental design, climate and soil arranged as follows: 1) Control; 2) 10 tons ha-1
conditions. Experiments were carried out over Liquid Cattle Manure (L1); 3) 10 tons ha-1 Liquid
experimental fields of ILCI Private Agricultural Cattle Manure +40 kg ha-1 N (L1N4); 4) 15 tons
Farm which is above 900 m sea level during the ha-1 Liquid Cattle Manure (L1.5); 5) 15 tons ha-1
years 2010 and 2011. In both years, 75 % vetch and Liquid Cattle Manure +30 kg ha-1 N(L1.5 N3); 6)
25 % barley combination was cultivated as previous 20 tons ha-1 Liquid Cattle Manure (L2); 7) 20 tons
crop of second crop of silage corn. TarP %H\D]Õ ha-1 Liquid Cattle Manure +20 kg ha-1 N(L2N2);
+XQJDULDQ YHWFK JHQRW\SHDQG$\GDQKDQÕPEDUOH\ 8) 25 tons ha-1 Liquid Cattle Manure (L2.5); 9) 25
genotype were used in vetch/barley combination. tons ha-1 Liquid Cattle Manure +10 kg ha-1 N
Combined sowing was carried out in winter, row (L2.5 N1); 10) 30 tons ha-1 Liquid Cattle Manure
spacing was 36 cm and sowing depth was 5 cm. (L3); 11) 30 ton ha-1 Liquid Cattle Manure +5 kg
Vetch was harvested in spring for forage at 25 % ha-1 N (L3 N0.5) and 12) 120 kg ha-1 P205 and N
flowering stage. A total of 12 treatments, consisting (L12). Macro and micro nutrient contents of liquid
of different doses of liquid manure alone and manure are provided in Table 1.
TABLE 1
Macro and micro nutrient content of li uid manure.
Years N P K Fe Zn Mg Ca
(%) (%) (%) (%) (%) (%) (%)
2010 2.7 0.2 0.8 0.14 0.02 0.5 2.2
2011 2.4 0.4 0.9 0.13 0.04 0.3 2.5
Medium-early corn cultivar of OSSK 644 was applications. Soil texture was sandy-loam with
used as the plant material of this study. Liquid slight alkalinity, low organic matter, phosphorus,
manure was applied over the 8.4 x 30 m plots just nitrogen and medium lime (Table 2).
before sowing and 2.8 m strips were left between
the plots to prevent interferences. Chemical Temperatures of the first year were higher
fertilizer combinations were applied all in one as than both the temperatures of the second year and
upper fertilization practice. Row spacing, on-row long-term averages. Temperatures of the second
seed spacing, and sowing depth were 70 cm, 15 cm year were close to long-term averages. Distribution
and 6 cm respectively. In chemical fertilizer of precipitations varied with months and total
treatment 12 (L12), all of 120 kg ha-1 phosphorus precipitations during experimental years were
and half of nitrogen were applied at sowing and higher than long-term averages (Table 3).
remaining half of nitrogen was applied before the
second hoeing. Weed control was performed twice Effects of treatments on silage corn green
with mechanical hoeing. Irrigations were performed herbage yield and quality were investigated over
after the first hoeing in every 15 days and a total of plant samples harvested from a 5.2 (3x1.4) m2 area
600 mm irrigation water was applied. from each plot. Beside green herbage yield, plant
height, shoot diameter, cob weight like
Soil samples were taken to determine the soil morphological characteristics were also determined
characteristics (texture, pH, organic matter, salinity, [12].
N-P-K contents, lime content) before liquid manure
1079
TABLE 2
Pre-sowing soil characteristics of experimental site.
Treatments Texture pH Organic Salt K P N CaCO3

Matter ( %) (kg/da) (kg/da) (%) (%)
The First Year
Control Sandy-Loam 8.18 0.79 0.085 117.97 4.21 0.04 13.78
L1 Sandy-Loam 8.15 1.07 0.105 106.17 3.69 0.05 13.02
L1N4 Sandy-Loam 8.20 0.97 0.085 106.17 3.72 0.05 13.02
L1.5 Sandy-Loam 8.11 0.96 0.107 106.17 3.60 0.05 14.55
L1.5N3 Sandy-Loam 8.17 0.89 0.075 106.17 2.83 0.04 14.55
L2 Sandy-Loam 8.19 0.89 0.073 106.17 2.40 0.04 16.47
L2N2 Sandy-Loam 8.18 0.76 0.076 70.78 1.72 0.04 21.06
L2.5 Sandy-Loam 8.15 0.90 0.067 94.38 3.78 0.05 14.17
L2.5N1 Sandy-Loam 8.15 1.07 0.086 106.18 3.69 0.05 9.57
L3 Sandy-Loam 8.13 1.01 0.083 129.77 2.97 0.05 13.40
L3N0.5 Sandy-Loam 8.19 1.03 0.048 106.18 3.43 0.05 13.78
N12 Sandy-Loam 8.26 0.94 0.096 94.38 2.32 0.05 15.32
The Second Year
Control Sandy-Loam 8.17 1.04 0.015 117.97 3.77 0.05 12.89
L1 Sandy-Loam 8.14 1.07 0.012 106.17 3.03 0.05 17.44
L1N4 Sandy-Loam 8.19 0.94 0.014 176.96 3.61 0.05 14.78
L1.5 Sandy-Loam 8.17 0.74 0.006 129.77 3.53 0.04 15.09
L1.5N3 Sandy-Loam 8.20 0.85 0.014 141.57 3.69 0.04 14.78
L2 Sandy-Loam 8.10 0.68 0.015 153.36 3.77 0.03 13.65
L2N2 Sandy-Loam 8.10 1.07 0.017 153.36 4.02 0.05 14.78
L2.5 Sandy-Loam 8.09 1.07 0.017 153.36 5.25 0.05 13.27
L2.5N1 Sandy-Loam 8.14 1.08 0.015 165.16 3.12 0.05 13.65
L3 Sandy-Loam 8.05 0.94 0.016 188.76 3.69 0.05 14.33
L3N0.5 Sandy-Loam 8.08 1.14 0.016 188.76 4.02 0.06 12.89
N12 Sandy-Loam 8.18 1.13 0.012 200.55 4.26 0.06 11.37
TABLE 3
Climate data for experimental years and long-term averages (1975-2011).
Temperature Precipitation
(oC) (mm)
Months 2010 2011 Long-Term 2010 2011 Long-Term
May 16.6 13.9 15.2 24.3 30.6 42.1
June 20.9 18.6 19.6 82.5 84.6 34.4
July 25.6 24.3 23.2 12.9 5.9 7.4
Agust 26.7 22.6 22.9 0 1.0 5.6
September 21.4 18.8 18.4 0.4 4.0 12.1
Measurements and statistical analyses. NY, USA). Experimental results were subjected to
Dried samples were ground in a grinder with 1 mm variance analysis by using SAS [16] software.
sieve and analyses were performed over these Significance of differences between treatments
samples. Kjeldahl method was used to determine PHDQVZHUHWHVWHGE\'XQFDQ¶VPXOWLSOHUDQJHWHVW
nitrogen (N) content. Crude protein was calculated
with Nx6.25 formula [13] NDF [14] and ADF
[15] were determined by using ANKOM 200 Fiber
Analyzer (ANKOM Technology Corp. Fairport,
1080
RESULTS L2.5 and L2N2 were placed in the same group. The
lowest value of the first year was observed in L1.5
Both single liquid manure treatments and with 29850.0 kg ha-1 and control, L1, L1N4, L2
combinations with chemical fertilizers had and N12 treatments were placed into the lowest
significant effects on silage corn green herbage group. While the lowest value of the second year
yields and chemical compositions of both years. was seen in control treatment, the highest value was
Effects of liquid manure treatments on green seen in L1.5 treatment and L1.5N3 was also placed
herbage yield, plant height and plant diameter of into the highest group. As the average of years, the
second crop silage corn in both years (p<0.01). highest values were observed in L1.5N3 (44610.0
Treatments, years and year x treatment interaction kg ha-1) and L2N2 (43913.3 kg ha-1) treatments
were found to be significant with regard to average and the lowest value in control treatment with
yields of the years 2010 and 2011 (p<0.01) (Table 35396.7 kg ha-1.
4). Plant heights varied between 170.00-220.33
Green herbage yields varied between 29850.- cm in 2010 and between 216.26-254.13 cm in 2011
36580.0 kg ha-1 in the year 2010 and between and average plant height of the experimental years
40943.3-55083.3 kg ha-1 in the year 2011. The changed between193.13-237.07 cm. The highest
highest yield was obtained from L3N0.5 treatment plant height was observed in L2 treatment of the
in the first year and the treatments L3, L2.5N1,
TABLE 4
Effects of single and combined li uid manure treatments on green herbage yield, plant height and plant
diameter of second crop silage corn
Green Herbage Yield Plant Height Plant Diameter

Treatments (kg/ha) (cm) (mm)
2010 2011 Mean 2010 2011 Mean 2010 2011 Mean
Control 29850.0 d 40943Õ 35396.7 e 170.00 f 216.26 c 193.13 f 13.50 e 13.00 d 13.25 g
L1 32860.0 bcd 47613.3 ef 40236.7 d 205.00 abcd 232.15 b 218.57 cd 14.57 cd 16.11 c 15.34 f
L1N4 31510.0 cd 53446.7 b 42478.3 bc 193.00 cd 222.04 c 207.52 e 14.33 d 18.07 b 16.20 cde
L1.5 29850.0 d 55083.3 a 42466.7 bc 175.33 ef 254.13 a 214.73 cde 14.21 d 18.02 b 16.11 de
L1.5N3 34710.0 abc 54510.0 a 44610.0 a 212.67 ab 252.68 a 232.68 a 14.03 de 18.11 b 16.07 de
L2 32860.3 bcd 46926.7 f 39895.0 d 220.33 a 253.81 a 237.07 a 14.72 bcd 19.03 a 16.88 ab
L2N2 35400.0 ab 52436.7 c 43913.3 ab 212.00 ab 234.72 b 223.36 bc 14.41 d 17.17 b 15.79 def
L2.5 35400.0 ab 44163.3 h 39781.7 d 192.00 cd 231.14 b 211.57 de 14.60 cd 18.06 b 16.33 cd
L2.5N1 34700.0 abc 48293.3 de 41496.7 cd 189.67 de 254.07 a 221.87 bc 15.60 a 19.00 a 17.30 a
L3 36580.0 a 45306.7 g 40943.3 cd 202.33 bcd 240.24 b 221.29 bc 14.67 cd 16.06 c 15.37 f
L3N0.5 36580.0 a 44623.3 gh 40601.7 d 199.67 bcd 236.34 b 218.00 cd 15.29 abc 16.10 c 15.70 ef
N12 31510.0 cd 48926.7 d 40213.3 d 206.67 abc 251.76 a 229.22 ab 15.40 ab 18.00 b 16.70 bc
Mean 3348.4 b 4852.3 a 198.22 b 239.94 a 14.61 b 17.23 a
Years ** ** **
Treatments ** ** ** ** ** ** ** ** **
Year*treatment ** ** **
S
first year and L1, L2N2 and N12 treatments were treatment and L1.5N3, L2, L2.5N1 and N12
also placed into the same group. The lowest value treatments were also placed into the highest group.
of the first year was observed in control treatment. With regard to average of years, while the
The highest value of the year was observed in L1.5 lowest value was seen in control treatment, the
highest values were observed in L2 and N12
1081
treatments. While the lowest plant diameter of the ratio was found to be significant at 0.05 level in the
first year was seen in control treatment with 13.50 year 2010 while the level was 0.01 in average of
cm, the highest values were observed in L2 (15.60 years and year*treatment interaction of the year
cm), N12 (15.40 cm) and L3N0.5 (15.29 cm) 2011.
treatments. The lowest plant diameter of the year
2011 was again observed in control treatment with Cob ratios varied between 29.08-33.98 % in
13.00 cm and the highest value was seen in L2 the first year, between 22.76-34.12 % in the second
treatment with 19.03 cm and L2.5N1 was year and average of years was between 30.52-33.75
statistically placed into the same group. With %. While the loest value of the first year was
regard to average of years, while the lowest value observed in control treatment, the highest value was
(13.25 cm) was seen in control treatment, highest seen N12 treatment and the treatment L2N0.5,
values were observed in L2.5N1 (17.30 cm) and L2 L2.5, L1N4 and L1 were placed into the highest
(16.88 cm) treatments. group. During the second year, the lowest cob ratio
was again obtained from control treatment and the
Cob, leaf and stem ratios of second-crop silage treatment L2.5 was also placed into the lowest
corn and effects of treatments on these parameters group. The highest value of the second year was
are provided in Table 5. While the effects of observed in L2.5N1 treatment and the entire
treatments on cob ratio was found to be statistically treatments except control and L2.5 were placed into
significant at 0.01 level in the first year, in average the highest group. With regard to average of years,
of years and year*treatment interaction were also the lowest value was seen in L2 treatment and the
significant at levels of 0.05 in the second year. treatments control; L1, L1.5, L1.5N3, L2N2, L2.5
Effects of treatments on leaf ratios were found to be and L3 were placed into the lowest group. The
significant at 0.05 level in both years, the level for highest values were seen in N12, L3N0.5, L3,
difference between years and for average of years L2.5N1, L1N4 and L1 treatments.
was the level of 0.01. Effects of treatments on stem
TABLE 5
Effects of single and combined li uid manure treatments on cob, leaf and stem ratios.
Cob Ratio Leaf Ratio Stem Ratio

Treatments (%) (%) (%)
2010 2011 Mean 2010 2011 Mean 2010 2011 Mean
Control 29.08 d 22.76 c 30.75 dc 22.03 abc 19.70 c 20.20 c 48.89 a 52.57 a 49.05 a
L1 34.11 abc 31.75 ab 31.93 abcd 21.57 bc 20.69 bc 21.13 bc 46.31 ab 47.56 b 46.93 b
L1N4 32.18 abc 33.56 ab 32.87 ab 21.16 cd 20.91 bc 21.04 bc 46.66 ab 45.53 bc 46.09 b
L1.5 31.22 bcd 31.19 ab 31.21 bcd 22.48 abc 23.26 abc 22.87 ab 46.64 ab 45.54 bc 46.09 b
L1.5N3 29.30 d 31.76 ab 30.53 d 24.00 a 21.84 abc 22.92 ab 46.70 ab 46.40 bc 46.55 b
L2 29.67 d 31.36 ab 30.52 d 23.67 ab 25.54 a 24.61 a 46.67 ab 43.10 cd 44.88 bc
L2N2 30.14 cd 32.47 ab 31.31 bcd 22.15 abc 24.07 ab 23.11 ab 47.70 ab 43.46 cd 45.58 bc
L2.5 32.27 abc 30.23 bc 31.25bcd 22.07 abc 25.63 a 23.85 a 45.67 bc 44.14 bcd 44.90 bc
L2.5N1 30.96 bcd 34.12 a 32.54 abc 22.32 abc 25.30 a 23.81 a 46.72 ab 40.59 d 43.65 cd
L3 30.89 bcd 32.95 ab 31.92 abcd 21.52 bc 23.50 abc 22.51 ab 47.59 ab 43.55 cd 45.57 bc
L3N0.5 32.69 ab 33.19 ab 32.94 ab 19.29 d 23.53 abc 21.41 bc 48.01 ab 43.27 cd 45.64 bc
N12 33.98 a 33.51 ab 33.75 a 22.27 ab 25.34 a 23.80 a 43.74 c 41.14 d 42.45 d
b a a b
Mean 31.60 31.99 21.93 23.28 46.50 44.74
Year NS ** **
Treatments ** * ** * * ** * ** **
Year*treatment ** NS **
S S; NS; Not significant
1082
While the lowest leaf ratio of the first year was L1.5 treatments and the treatments of control and
observed in L3N0.5 treatment with 19.29 %, the L1 were also included in the lowest group.
highest value was observed in L1.5N3 treatment
with 24.00 % and the treatments such as control, NDF ratios varied between 63.09-69.40 % in
L1.5, L2, L2N2, L2.5, L2.5N1 and N12 were also 2010, between 65.16-71.46 % in 2011 and between
included in the highest group. In the year 2011, the 65.01-69.77 % as the average of years. The lowest
lowest value was seen in control treatment with NDF ratio was observed in N12 in the first year,
19.70 % and highest value was seen in L2.5 L2.5 in the second year and again in N12 in average
treatment. As an average of the years, the lowest of years. The highest NDF ratio was observed in L3
value was obtained from control treatment with in the first year, control in the second year and L2
20.20 %, the highest value from L2 treatment with in average of years.
24.61 %.
While lowest ADF ratio of the first year was
Stem ratios of the first year varied between observed in N12 with 32.90 the highest value was
43.74-48.89 %. The lowest value was observed in seen in L2.5 treatment with 36.70 %. The lowest
N12 and the highest in control treatment. The ratios value of the second year was obtained from L1.5
of the second year varied between 40.59-52.57 % treatment with 33.62 % and the highest from L1
with the lowest value in control and the highest treatment with 36.79 %. Average of the years
value in L2.5N1 treatment. With regard to average revealed the lowest value for N12 treatment with
of years, the lowest value was obtained from 33.50 % and the highest value for L2.5 treatment
L2.5N1 treatment with 42.45 % and the highest with 35.85 %.
value was obtained from control treatment with
49.05 %.
DISCUSSION
Chemical composition parameters of silage
corn under different liquid manure doses and
combinations are provided in Table 6. Treatment The differences between green herbage yields,
had significant effects on crude protein, ADF and agro-morphological characteristics and protein
NDF ratios of both years (p<0.01). Effects of years ratios of the years were mainly due to higher
and year x treatment interaction were also found the temperatures of the second year above long term-
significant at 0.01 level. While the differences averages (Table 3). Similar green herbage yield of
between the crude protein ratios of the years were control plot of especially the first year without any
not significant, the differences in NDF and ADF fertilization implementation with L15 treatment and
ratios were significant. similar ADF and NDF ratios were basically resulted
from soil residual nitrogen due to fixated nitrogen
Crude protein ratios varied between 3.80-7.93 of preceding Hungarian vetch cultivation. Such
% in the first year, between 4.03-9.00 % in the findings point out the significance of legumes to be
second year and 3.96-8.46 % as the average of the used as preceding cropping in silage corn culture
years. The highest value of the first year and [47]. In this way, [17] reported that forage quality
average of years was observed in N12 treatment of silage corn after Hungarian vetch + barley
and the lowest value of the year 2010 and average combination was higher than that of silage corn
of years was observed in control treatment. The after wheat cultivation.
lowest value of the years 2011 was obtained from
1083
TABLE 6
Effects of single and combined li uid manure treatments on chemical compostion of silage corn.
Crude protein (%) NDF (%) ADF (%)

Treatments 2010 2011 Mean 2010 2011 Mean 2010 2011 Mean
Control
3.80 4.12 fg 3.96 f Õ 71.41 a 68.70 b Õ 36.72 b 35.19 d
L1 5.46 b 4.49 efg 4.98 c 67.03 g 69.50 c 68.27 c 33.98 h 36.79 a 35.39 c
L1N4 5.24 c 4.92 cde 5.08 c 67.61 e 66.31 j 66.96 g 34.76 g 34.94 e 34.84 f
L1.5 4.68 g 4.03 g 4.36 e 67.40 f 66.81 h 67.10 f 35.79 c 33.62 34.70 g
L1.5N3 5.39 b 5.71 b 5.55 b 66.55 h 68.89 d 67.72 d 35.08 e 34.81 f 34.95 e
L2 4Õ 4.69 de 4.39 e 69.04 c 70.50 b 69.77 a 34.97 ef 36.32 c 35.65 b
L2N2 4.81 f 4.61 def 4.71 d 64.35 j 68.42 e Õ Õ 34.42 h 34.04 h
L2.5 4.95 e 5.05 cd 5.00 c 68.24 d 65.16 l 66.70 h 36.70 a 35.00 d 35.85 a
L2.5N1 5.07 d 5.3133 bc 5.19 c 68.51 c 67.90 f 68.20 c 35.58 d Õ 34.89 ef
L3 4.43 h 4.90 cde 4.67 d 69.40 a 65.95 k 67.67 d 36.54 b Õ 35.38 c
L3N0.5 5.42 b 5.03 cde 5.23 c 68.11 d Õ 67.31 e 34.85 fg 34.50 g 34.67 g
N12 7.93 a 9.00 a 8.46 a 63.09 66.94 g 65.01 32.90 34.12 j Õ
Mean 5.11 5.16 67.11 b 67.86 a 34.87 b 34.97 a
Year ** **
Treatments ** ** ** ** ** ** ** ** **
Year*treatment ** ** **
S161RWVLJQLILFDQW
Nitrogen has a vital role in protein and diameter, [31] indicated insignificant effects on
enzyme synthesis in plants and enzymes control the nitrogen treatments on plant diameter but [32],
entire metabolic processes of plants. Nitrogen is [26] reported increased diameters with nitrogen
also included in synthesis of chlorophyll, the basic treatments. Nitrogen is one of the an essential
molecule absorbing the energy required for elements for growth and development of plant
photosynthesis [18]. Previous researches reported height, leaf ratios, plant diameter and finally green
elevated growth and development but decreased herbage yield of corn but all these parameters are
nitrogen-use efficiency and consequent green also controlled by genetics and also environmental
herbage yield decreases in corn with increasing factors so that reason there is always a balance
nitrogen doses [19, 20]. So that reason nitrogen among these parameters and green herbage yield.
deficiency or sufficiency should be monitored by Nitrogen deficiency negatively affects the
visually or technologically such as unmanned aerial vegetative development of corn and result in weak
vehicles suggested [48]. Results of present study plant development. When the plant is not able to get
reflected the impacts of organic nitrogen in liquid sufficient nitrogen, it is forced to blossom earlier
manure on silage yield. Similar findings were also and growth period is shortened. In this case, upper
reported by [18, 21, 22]. Nitrogenous fertilizer tassel and cob tassel development are slowed down
treatments generally increase the plant heights of and consequently very few kernels are formed over
corn but such an increase is not continuous [23]. the tip of cob [33]. Normal growth, cob and kernel
[24] reported nitrogen as the most significant development are only possible with sufficient
nutrient effecting plant heights of corn. Similarly, nitrogen. Increased cob ratios normally indicate
[25] reported decreasing leaf ratios with increasing relatively decreased leaf and stem ratios. However,
nitrogen doses but [26] indicated insignificant [18] reported increased plant heights with nitrogen
effects of different nitrogen doses on leaf ratios. treatments. Plant height is mostly affected by plant
Some others also reported increased leaf area index genetic characteristics, soil nutrients, climate
values and consequent leaf ratios [27,28] and conditions, light intensity and quality and growth
increased green herbage yields (29, 21, 22] with seasons. [26] and [34] reported insignificant
increasing nitrogen doses. [30] reported excessive effects of increased nitrogen doses on stem ratios of
impacts of environmental conditions on plant corn. [35] indicated that plant height may
1084
significantly be affected by environmental with the findings of [36] and [40], [39] and
conditions. Compared to control treatment, in [18].
present study, significant relationships were Nitrogenous fertilization decreases ADF and
observed between increasing liquid manure doses NDF ratios of plants. [44] reported significant
and plant height, diameter and leaf ratios. effects of liquid manure treatments on ADF and
Increasing liquid manure doses increased these NDF ratios. Yolcu et al. (2010) carried out a two-
parameters until the dose of L2, and then a decrease year study to investigate the effects of liquid
was observed after this dose. This situation can be manure treatments and reported insignificant effects
explained by optimum use of nitrogen and after this on ADF and NDF ratios in the first year but
level excessive doses of nitrogen sometimes can significant effects in the second year of
result in toxic effects on plant growth and experiments. [45] observed increasing ADF and
development. Treatments of present study had also decreasing NDF ratios in Bermuda grass with liquid
significant effects on plant diameter and cob ratio lagoon effluent treatments. Although significant
but increases were not regular in any treatments effects of liquid manure doses on ADF and NDF
except for control treatment. With regard to plant ratios were observed in present study, the
height, current findings relatively comply with the differences between different liquid manure
findings of [18] but fully comply with the results treatments with regard to these two parameters
of [26] (2002) and [34]. were not significant except for pure nitrogen (N12)
Nitrogenous fertilizers generally increase cob treatment. Current findings were higher than the
ratios but such an increase is again not continuous. results observed by [46, 18] but comply with
Although, leaf and stem ratios decreases, cob ratio results of [8]. As indicated by many researches,
and kernel formation increases with nitrogenous quality parameters such as protein, NDF and ADF
fertilizers. Formation of cobs and kernels occurs in are mainly determined by environment and
the generative stage of corn development and in cultivation practices. So, this situation can be
order to stimulate high cob and kernel formation explained both highland conditions and second crop
halves of the nitrogen is generally supplied just cultivation of corn which are the major effects to
before this level. Additionally, all reserve nitrogen high NDF and ADF ratios together with manure
in different parts of the plant is also transferred to application time.
generative organs, so these two factors can
contribute to increase of the cobs and kernels.
Current findings comply with the results of CONCLUSION
previous studies [19, 36, 37, 38]. However, [26]
indicated insignificant effects of increasing nitrogen
doses on cob ratios. This can be result of different In first study of second crop silage corn
corn genotype used in the trial, cultivation practices production under Central Anatolian highlands,
such as first and second cropping and experimental liquid manure treatments had generally positive
conditions. effects on primarily the green herbage yield, plant
[39] reported increasing crude protein ratios height, cob ratio like morphological characteristics
with nitrogenous fertilizer treatments. [36] and and crude protein, ADF and NDF like quality
[40] also indicated positive impacts of increasing parameters. It was observed that 15 tons ha-1
nitrogen doses on crude protein ratios. Increased liquid and 30 kg ha-1 chemical nitrogen
crude protein ratios were also reported with liquid combination was able to meet all of the phosphorus
manure treatments in barley + vetch combination need and large portion of nitrogen need of corn
[8], oat [41], corn [42] and grass [43]. Results without any adverse impacts. This can contribute to
of variance analysis revealed significant effects of sustainable second crop silage maize production
different liquid manure doses on crude protein under highland conditions among especially
ratios of present study. More importantly, resource poor farmers. Additional chemical
additional nitrogen doses to liquid manure yielded nitrogen fertilization as upper fertilizer resulted in
more stable increases in crude protein ratios than in significant increases in green herbage yield and
single liquid manure treatments. Such a case quality parameters of silage corn. Such a case may
indicates that liquid manure application should be be due to application of liquid manure in at once
repeated one more time after the second hoing. In just before the sowing and consequent nitrogen
this stage protein accumulation starts at dough deficiency or nitrogen leaching through irrigations.
stage of kernel formation of all cereals including In case of ecological and economical sustainable
corn so that reason plants urgently need of available second-crop silage corn cultivation with only liquid
nitrogen to maximize protein ration in the grain. manure application, liquid manure should be
The results obtained from the research fully comply applied twice, one just before the sowing and
another one just before upper tassel formation
1085
period to increase also silage quality parameters growth increase in West Africa. Soil Science
under highland conditions. Society of America Journal, 64, 346-358.
[10] Mkhabela, T.S. and Materechera, S.A. (2003).
ACKNOWLEDGEMENTS Factors influencing the utilization of cattle and
chicken manure for soil fertility management
We kindly acknowledge the partial financial by emergent farmers in moist Midlands of
contribution of TAGEM (General Directorate of Kwazulu-natal province, South Africa.
Agricultural Research and Policy) for the project. Nutrient cycling in Agrosystem, 65, 151:162.
[11] Chadwick, D. and Laws, J. (2002). The impact
of novel slurry application techniques on
amonnia emission , silage quality and sward
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Taner A ar
Faculty of Agriculture
University of Akdeniz
07100 Antalya ± TURKEY
email: yezakar66@yahoo.com
1088

EVALUATION OF HEAVY METALS IN POTENTIAL

FODDERS AND FORAGES GROWING IN KHUSHAB,
PAKISTAN
Zafar Iqbal Khan1, Kafeel Ahmad1, Hafiz Abdalla Muhammad Ali2, Abrar Hussain4, Humayun Bashir1,
Muhammad Sohail1, Ameer Khan1, Asma Zafar1, Zahara Bibi1, Zill-e Huma1, Muhammad Fahad Ullah7,
Saif Ur Rehman7, Nudrat Aisha Akram5, Muhammad Sher3, Ijaz Rasool Noorka6, Fahim Ashrad8, Aima
Iram Batool and Irfan Mustafa9
1
Department of Botany, University of Sargodha, Sargodha, Pakistan
2
Graduate College, University of Bahri, Khartoum-Sudan
3
Department of Chemistry, University of Sargodha, Pakistan
4
Science & Technological Division, University of Education, Township, Lahore, Pakistan
5
Department of Botany, Govt. College University, Faisalabad, Pakistan
6
University College of Agriculture, University of Sargodha, Pakistan
7
Department of Earth Sciences, University of Sargodha, Pakistan
8
Department of Botany, University of Education Okara Campus, Okara, Pakistan
9
Department of Zoology,University of Sargodha,Pakistan
ABSTRACT reason, a strict monthly observation of such

contaminants is advisable [1]. The main
Heavy metals posed poisonous effects on the contributors of heavy metals are heavy-duty electric
health of ruminants and hence it is very important power generators, use of pesticides and crop
to analyze the heavy metal profile of plants residues burning in agriculture, industrial activity,
consumed by these species. The present study was municipal wastes and automobile exhausts which
conducted to explore the heavy metal profile of concentrates them in to the atmosphere [2].
wild plants of Khushab, Soon Valley, Pakistan. The livestock grazing depends upon the
Heavy metal studied were Cadmium (Cd), composition of nutrients in wild forage vegetation
Chromium (Cr), Lead (Pb) and Nickel (Ni). The during various seasons of year. Microminerals i
results showed that except for Cd, all heavy metal Fe, Mn and Zn play significant role in the
contents exhibited significant (P < 0.05) variations biological mechanisms of living bodies. Copper as
among studied plant species. The highest Cd a trace metal is required in very small quantity. The
content was observed in C n n a t l n amount of copper differs in various parts of the
(0.059%). til n t e a tii (6.85%) presented plant however the mechanism of action of copper
maximum value of Pb and the highest Cr ions slowed down with the gradual increase in the
concentration was observed in e i i m lati li m soil pH [3]. Such inconsistent accumulation of
(5.39%). ali m a a ine showed the highest Ni heavy metal in plants depends on developmental
value (4.72). stages and ecological factors i.e., supply of nitrogen
and soil chemical properties. Excessive amount of
KEYWORDS: Cu concentration is required if the nitrogen supply
Wild Plants, Heavy Metals, Soon Valley, Pakistan. is higher [4].
Due to the high oxidation potential, mobility
and solubility across the membrane of living
INTRODUCTION organism Chromium hexavalent is much more
toxic. It is impossible to dissolve the trivalent
As a result of environmental pollution and chromium in water. Trivalent chromium is oxidized
climatic changes on the earth, heavy metals have into hexavalent in the presence of high
spread widely in reservoirs. These heavy metals concentration of oxygen. Presence of hexavalent
could be absorbed by plants and the accumulation chromium becomes the cause of bronchitis, cancer,
of these metals in plants may have short as well as asthma and destruction of genetic material such as
long term risks for the health of both animals and DNA.
humans. For example, accumulation of lead could Sometimes the dietary nutrients can be up
cause breakdown of liver and brain cells and also taken by human through consuming the
reduced the functioning of the kidney. For that contaminated meat which was due to grazing on
pastures containing lead thus having the possibility
1089


of causing lead toxicity in humans [5]. The main collected from Soon Valley, Khushab (Pakistan) to
contributor of lead toxicity comes from confidential evaluate their nutritional status and safety.
centers discharging lead [6-7-8].
Zinc and Iron are important components of
insulin and hemoglobin respectively [9]. Both MATERIALS AND METHODS
Insulin and hemoglobin are necessary in the regular
performance of heart, nerves and muscles that are Study Site. This study was conducted in Soon
vital for the continuation of the composition of Valley, Pakistan. This valley is located in the
body fluid along with others [10]. Punjab Province (Fig. 1). It is situated in the north
Cadmium is used for the processing of food west of Khushab and is surrounded from the South
preparation, germination of seed, metabolism and by Punjab Plains, North by Potohar plateau, East by
growth in forages and for the formation of proteins Kallar Kahar and West by Indus River at some
and phosphorous. Cadmium along with its distance and the main town of the Soon Valley is
compounds in excess is toxic for the human health Naushehra. Geographically, the valley is located
[11]. between Long.: 71o 50' 33" - 72o 30' 07" East and
This present study was designed to investigate Lat.: 32o 26' 11" ± 32o 41' 18" North.
the levels of four heavy metals (Cd, Cr, Pb and Ni)
in leaf and stem samples of twenty wild plants
oon a esar
FIGURE 1
Index map of Soon Sakesar (Western Salt Range, Pakistan).
Plant Samples Collection. Twenty plants leaves and other parts) were collected without
having potential of forage were collected randomly damaging them and stored in paper bags. The
from various localities across Soon Valley, Punjab, forages were cleaned of contaminants, dried (oven
Pakistan. Each plant has three replicates from at 70 0C) and stored for analysis.
different sites. Various plant parts (viz.; stems,
1090


Plant Samples Identification. This was For all plant species, Cr concentrations were
carried out in the Department of Biological lower compared to those of Ahmad et al [19] and
Sciences, University of Sargodha using published Cr values of emi e m in i as found by
taxonomic manuals [12]. Kulhari et al [17].
Mineral Analysis. Forages and soil were Livestock health is badly affected by high
analyzed for their minerals concentration using concentration of Cr. Nose irritation, lungs cancer,
Atomic Absorption Spectrophotometer Perkin- liver damage, kidneys upset, bleeds and skin rash
Elmer AAS-5000 following Perkin-Elmer Corp are the indications of Cr toxicity. Proteins, lipids
[13] and Lindsay and Norvell [14] respectively. and glucose metabolism are significantly affected
by deficiency of Cr [20]. The recommendation of
Statistical Analysis. Data were subjected to daily Cr intake of young people as reported by US
statistical analysis (ANOVA) using SPSS software National Academy of Sciences is up to 50-ȝJ
version (17). The significance test was carried out [21].
at 0.001, 0.01 and 0.05 probability levels [15].
Lead (Pb). Lead contents showed significant
(P < 0.05) differences in all plant species. It ranged
RESULTS AND DISCUSSION from 4.85mg/kg to 7.66mg/kg. The maximum Lead
content was observed in a a m ntane m
Analysis of variance for heavy metals of while the minimum in a i a a a.
different plant species was shown in table 1. The
analysis indicated that except for Cd, all heavy High concentration of lead was observed in
metal contents exhibited significant (P < 0.05) the current study as compared to the lead
variations among the twenty plant species studied. concentration in ate i i e plant as reported by
The concentration heavy metals in different plant Lasisi et al. [22] $OOHQ¶V [23] work presented
species is presented in table 2 and figures 1-4. higher concentrations of Pb in various plant species
as compared to Pb values obtained in the present
Cadmium (Cd). Cadmium showed non- study. Lead has tremendous toxic effects for
significant (P > 0.05) variations among plant microorganism, animals and plants. Accumulation
species studied. The cadmium contents ranged from of Pb is caused by regular fertilizers consumption,
0.044 mg/kg in ali m a a ine L. to 0.059 mg/kg sewage sludge and fuel combustion. WHO [24]
in C n n a t l n. Similar Cd values were suggested high amount of Pb (10ppm) as compared
reported by Kloke [16]. Current results were also to the amount of Pb observed in the present study.
comparable with those of Kulhari et al [17]. Headache, brain damage, anemia, colic are among
However, results reported by Aksoy et al [18] were the symptoms of Pb toxicity [25].
higher than the present findings.
Nickel (Ni). Nickel exhibited significant (P <
Cadmium is a very poisonous heavy metal 0.05) variation among plants. It varied from
which is present in plants. Plants yield is badly 3.61mg/Kg ( e m le in m) to 4.72 mg/Kg
affected at a range of 5-30 mg/kg of Cadmium ( ali m a a ine ).
which usually come from the combustion of fossil
fuels, lead and zinc mines, phosphate fertilizers, Nickel contents obtained in the current study
non-ferrous smelters and sewage sludge were similar to those reported by Adriano [26],
applications. These are the known sources of Cd in higher than the values of Kulhari et al [17] and
plants and soil [17]. lower than those found by Tokalioglu and Kartal
[27].
Chromium (Cr). Chromium contents showed
significant (P < 0.05) differences between species. Anthropogenic activities are the main reason
The range was 3.55mg/kg to 5.39mg/kg. The behind of high Ni concentration in plants. It also
maximum Cr concentration was reported for plays a pivotal role in the production of insulin and
e i i m lati li m while til n t e a tii mainly present in pancreas. Nickel produces
Me i showed the lowest. allergic reactions on moist skin as well as nasal
allergies and lungs infection. Nickel deficiency is
the cause of most of the liver disorders [28].
1091


TABLE 1
Analysis of variance for Heavy Metals of Different Plant species.
Source of Degree of Mean Squares (in mg/kg)

Variation freedom (df)
S.O.V. Cd Cr Pb Ni
Plant Spp. 19 .001ns .823*** 1.455*** .248***
Error 40 .001 .011 .002 .001
*** = significant at 0.001 level
Ns = non-significant
TABLE 2
Heavy Metal mean concentration of different plant species.
Plant Species Mean + S.D

No. Cd Cr Pb Ni
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
1 i ita ia an inali (L.) Scop. 0.049±0.007 3.75±0.68 5.28±0.69 4.23±0.45
2 nit m l m ian m Nutt. 0.052±0.006 4.04±0.56 5.33±0.54 4.32±0.48
3 naea i a Jacq. 0.053±0.005 5.16±0.54 6.17±0.62 3.66±0.43
4 a i a a a L. 0.055±0.006 4.28±0.58 4.85±0.61 3.83±0.45
5 temi ia t ii Gray. 0.047±0.007 4.04±0.56 5.12±0.52 3.76±0.51
6 M na a n tata L. 0.049±0.006 5.04±0.61 5.93±0.61 4.11±0.41
7 Cean t el tin Douglas ex 0.046±0.005 4.12±0.54 5.78±0.63 4.22±0.43
Hook.
8 til n t e a tii Medik 0.051±0.006 3.55±0.58 6.85±0.68 4.42±0.42
9 ali m a a ine L. 0.044±0.005 3.94±0.64 6.31±0.66 4.72±0.39
10 nat e m men i e 0.046±0.005 5.04±0.53 6.59±0.71 4.04±0.52
(Roem. & Schult.) Barkworth.
11 a a m ntane m L. 0.057±0.006 4.21±0.56 7.66±0.62 3.93±0.41
12 C n n a t l n (L.) Pers. 0.059±0.005 4.02±0.67 6.13±0.61 4.14±0.47
13 e m le in m (Link) 0.056±0.006 4.92±0.62 5.39±0.64 3.61±0.46
Arcang.
14 e i i m lati li m L. 0.046±0.005 5.39±0.65 5.84±0.59 4.24±0.43
15 en a l n i l Hook. 0.052±0.006 4.43±0.58 5.62±0.57 3.74±0.52
16 Ca ella a a t i (L.) 0.051±0.006 4.25±0.52 6.74±0.61 3.87±0.45
Medik
17 e i la a (Pursh) Raf. 0.053±0.007 4.39±0.61 6.63±0.62 3.93±0.52
18 i e n i e en Torrey & A. 0.045±0.006 4.93±0.62 5.85±0.61 3.99±0.44
Gray.
19 C n a ana en i (L.) 0.052±0.005 4.08±0.57 5.77±0.56 4.06±0.52
Cronquist.
20 C ll mia linea i Nutt. 0.045±0.005 3.88±0.58 6.75±0.63 3.64±0.41
CONCLUSIONS AND RECOMMENDATIONS contaminated meat. Hence it is recommended that a

close observation of the heavy metals in potential
The present study showed that the twenty wild forage plants is required and also investigations
plants species studied contain appreciable quantities should look for the presence of other heavy metals
of heavy metals, Cd, Cr, Pb and Ni, which could be such as Copper (Cu), Cobalt (Co), Iron (Fe),
harmful to animals foraging on them and ultimately Manganese (Mn) and Zinc (Zn).
to humans through consumption of the
1092


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Ϭ͕Ϭϰ
Ϭ͕Ϭϯ
Ϭ͕ϬϮ
Ϭ͕Ϭϭ
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WůĂŶƚƐ
FIGURE 1
Cadmium concentrations in plants studied
ŚƌŽŵŝƵŵ
ƌĐŽŶĐĞŶƚƌĂƚŝŽŶŝŶWůĂŵƚƐ;ŵŐͬŬŐͿ
ϳ
ϲ
ϱ
ϰ
ϯ
Ϯ
ϭ
Ϭ
WůĂŶƚƐ
FIGURE 2
Chromium concentrations in plants studied.
1093


>ĞĂĚ
WďĐŽŶĐĞŶƚƌĂƚŝŽŶŝŶƉůĂŶƚƐ;ŵŐͬŬŐͿ
ϵ
ϴ
ϳ
ϲ
ϱ
ϰ
ϯ
Ϯ
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WůĂŶƚ
FIGURE 3
Lead concentrations in plants studied.
EŝĐŬĞů
ϲ
EŝĐŽŶĐĞŶƚƌĂƚŝŽŶŝŵƉůĂŶƚƐ;ŵŐͬŬŐͿ
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ϰ
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ϭ
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WůĂŶƚƐ
FIGURE 4
Nickel concentrations in plants studied.
ACKNOWLEDGEMENT 1721/RGD/104624 to the first and second authors.

The authors thank all the supporters of this project
The Higher Education Commission, Pakistan and the referees for their constructive comments.
is acknowledged for providing the financial support
through the research project # 20-
1094


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Development of DTPA soil test for Zn, Fe,
[1] A.A.K. Abou-arab, M.A. Aboudonia (2001) Mn and Cu. Soil Science Society of American
Pesticide residues in some Egyptian spices Journal. 42, 421-428.
and medicinal plants. Food Chemistry. 72, [15] R.G.D. Steel, J.H. Torrie (1980) in i le
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[2] L. Jarup (2003) Hazards of heavy metal a (2nd Ed.). New York: McGraw Hill
contamination. Journal of Book Co. 1, pp.1.
Ethnopharmacology. 68, 167-182. [16] $ .ORNH 5LFKWZHUHWH¶
[3] S. Sauve, M.B. McBride, W.A. Norvell, W.H. orientierungsdaten fur tolerierbare
Hendershot (1997) Copper solubility and gesamtgehalte einiger elemente in
speciation of in situ contaminated soils: Kulturboden, Mih, vdlufa, h. 2, 9-1.
effects of copper level, pH and organic matter. [17] A. Kulhari, A. Sheorayan, S. Bajar, S. Sarkar,
Water, Air and Soil Pollution. 100, 133-149. A. Chaudhury, R.K. Kalia (2013)
[4] H. Marschner (1995) Mineral nutrition of higher Investigation of heavy metals in frequently
plants gulf. Professional Publishing Nature. 1, utilized medicinal plants collected from
pp. 889. environmentally diverse locations of north
[5] J.D. McEvoy, M. McCoy (1993) Acute lead western India. Springer Plus 2, 676.
poisoning in a beef herd associated with [18] A. Aksoy, W.H.G. Hale, J.M. Dixon (1999)
contaminated silage. Veterinary Record. 132, Capsella bursa-pastoris (L.) Medic. as a
89±90. biomonitor of heavy metals. The Science of
[6] Z.P. Liu (2003) Lead poisoning combined with the Total Environment. 226, 117-186.
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non-ferrous metal smelters. Science of the Valeem, Z.A. Shah, L.R. McDowell (2009)
Total Environment. 309, 117±126. Determination of forage concentration of lead,
[7] %6 'ZLYHGL 'HYHORSPHQW RI IDUPHUV¶ nickel and chromium in relation to the
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supply systems: experience of a FAO-ICAR- Range. Pakistan Journal of Botany. 41, 61-65.
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soil test crop response correlation. Bhopal: Nickel in heavy metals in soils. In: B.J.
Indian Institute of Soil Science. 1, pp. 50-75. Alloway (Ed.), Blackie, Glasgow. 1, pp.125.
[8] D. Swarup, R.C. Patra, R. Naresh, P. Kumar, P. [21] D. Waston (1993) Safety of chemicals in food,
Shekhar (2005) Blood lead levels in lactating chemical contaminants Published by Ellis,
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Environment. 347: 106±110. Nwosu, M.A. Olayiwola (2005) Heavy metals
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Reeves (1989) The possibility of in it heavy medicines with reference to contaminants and
metal decontamination of polluted soils using residues. World Health Organization, Geneva.
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[13] Perkin-Elmer Corp (1980) Analytical Weed Science and Research. 14, 101-110.
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[27] S. Tokalioglu, S. Kartal (2005) Determination

of heavy metalin soil extracts and plant tissues
at around of zinc smelter. International Journal
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Raton F1: CRC Press. 1, pp. 365.
Zafar Iqbal Khan

Department of Botany
University of Sargodha
Sargodha ± PAKISTAN
1096


ANAEROBIC COMPOSTING OF ORGANIC FRACTION OF

MUNICIPAL SOLID WASTE
<DVHPLQdDOÕúNDQ1LKDO%HNWDú0HVXW7HNEDú
Gebze Technical University, Environmental Engineering Department, 41400, Gebze/Kocaeli, Turkey
ABSTRACT solid wastes were sent composting facilities [6].

This number is not satisfactory in the perspective of
The amount of solid waste has been increasing the integrated MSW management hierarchy.
by rapid population growth, improved technology, Besides, according to the European Union Landfill
and economic development and changing Directive (1999/31/EC), it is envisaged that
consumption habits. This increase leads to find biodegradable parts of solid wastes send to landfills
different municipal solid waste (MSW) are needed to be reduced at certain rate until the
management alternatives. Anaerobic composting year 2020 [7, 8]. Therefore there is a need for the
offers a cost-effective treatment strategy to MSW alternative disposal methods of solid wastes which
management alternatives. Therefore the aim of this content high organic matter. Organic matters in
study was selected to investigate anaerobic MSW include food, leaves and grass, branches and
composting process for food waste from catering stumps and manure. Organic matter constitutes
company since large amount of organic wastes are around % 68 of the MSW [9]. The important part of
generated from this type of sources. Anaerobic organic solid wastes comes from food wastes,
bioreactor was filled with mixed organic solid generated from supermarkets, restaurants, hotels,
waste and monitored throughout 24 weeks. sport centres, schools and cafeterias. Also catering
Leachate was re-circulated instead of mechanic companies usually generate very high organic solid
mixing. Some parameters of leachate such as pH, wastes.
COD, BOD, TKN, NH3-N, alkalinity, VFAs were Composting is a useful alternative disposal
analysed to gain information about biodegradability method of organic solid wastes and application of
and stabilization of the anaerobic compost process. compost product improves soil fertility [10].
Compost which is the final product of our process Although there are many studies related to
also analysed at the end of the study and it was seen composting of MSW with aerobic conditions in the
that the compost is stable and can be used for literature, there are limited number studies on
agricultural purposes. anaerobic composting of MSW. The anaerobic
composting of the biodegradable organic fraction of
municipal solid waste was deeply discussed in the
KEYWORDS: early work published in 1993 by Kayhanian and
Municipal solid waste (MSW), Organic waste, Anaerobic Tchobanoglous [11]. The physical, chemical, and
composting biological characteristics of humus produced from
anaerobic composting process were evaluated and
based on this results the anaerobic composting
INTRODUCTION process could be an effective alternative technology
to aerobic composting with the added advantage of
the recovery of energy in the form of methane [12].
The population growth, improved technology, The study undertaken by Murphy and Power (2006)
economic development and unplanned urbanization was investigated technical, economic and
in many cities of the developing world bring along environmental comparison between composting and
many environmental problems particularly in terms anaerobic digestion processes [13]. The results of
of solid waste management. Sustainable the research suggest that composting is
management of solid wastes has become a major economically preferable to anaerobic digestion for
social and environmental concern [1, 2, 3]. less than 50 kton/year of biowaste treated.
Landfilling is the oldest and most used disposal However, in terms of greenhouse-gas production,
method for MSW. However there are some anaerobic digestion is more beneficial than
important issues with landfilling such as lack of composting due to the substitution of fossil fuel.
space and leachate problem [4,5]. Therefore the aim of this study was selected to
About 25 million ton solid waste were investigate anaerobic composting process for food
collected in 2010 in Turkey and only 0.8 % of total waste from catering company. Process parameters
1097


such as pH, COD (Chemical Oxygen Demand), reactor made of plexiglas material. The dimensions
BOD (Biological Oxygen Demand), TKN (Total of the reactor were the height of 1m and the
Kjeldahl Nitrogen), NH3-N (Ammonia), alkalinity, diameter of 40 cm. A jacket was covered the reactor
VFAs (Volatile Fatty Acids) were monitoring with continuous water circulation (the heat of water
during the composting process to gain information was set to 40±2 oC) in the jacket to provide to
about biodegradability and stabilization of waste. control the heat various valves were placed to rotate
leachate from bottom to top of the reactor and to
give biogas outside (Figure 1). Perforated plaque
EXPERIMENTAL placed on the leachate input to distribute leachate
homogeneously. A peristaltic pump was used to
Design of lab-scale anaerobic digester. The recycle of leachate. pH, temperature values and gas
experimental set up was carried out in a cylindrical volume were monitored using on-line probes [14].
FIGURE 1
Schema of the lab-scale anaerobic bioreactor
Experimental Procedure. Mixed and crushed purged by nitrogen gas to create anaerobic
vegetable wastes (pepper, squash, aubergine, conditions. Anaerobic composting usually takes
cabbage and lettuce) provided from a catering place in mesophilic conditions. Hence, temperature
company were used in the experiments. Sewage of the process was set to 40±2 oC to provide
sludge was added into waste mixture to set optimal microbial activity formation. The leachate
C/N ratio, moisture content and also provide initial accumulated in the bottom of the reactor was
biological activity as inoculum. 10% of waste recirculated by sprinkler system for two hours
cardboard was also included to the waste mixture to every morning. pH and temperature values of
enhance carbon content of mixture. The final leachate were monitored daily and some important
mixture was consisted of 30% organic waste, 60% process parameters such as COD, BOD, NH3-N,
sewage sludge and 10% cardboard. After placing TKN, alkalinity were analysed weekly. Also, pH,
waste mixture to residual air inside the reactor was moisture content, organic content, volatile solids,
1098


total nitrogen, and TKN analyses were performed were performed according to the Standard Methods
on the sewage sludge and waste mixture at [15].
beginning of the study (Table 1). The analyses
TABLE 1
Characteristic of waste mixture
Parameter Waste Mixture Sewage sludge

Water content,% 78 75
Organic material,% 87 50
C,% 50.4 27.9
N,% 1.93 2.7
C/N, ratio 26 10.3
RESULTS AND DISCUSSIONS as hydrolysis and acidification phases that

occurring consecutively in anaerobic process. In
pH measurements. The variation of weekly this stage, organic acids and carbonic acid were
average pH values in leachate over time is shown in occurred resulting low pH values. When the
Figure 2. As can be seen in Figure 2, pH value of methanation stage took place at the anaerobic
leachate was measured as 5.5 at the beginning of process, pH values increased and remained in the
the experiment. After that, pH value was increased region neutral pH until the end of study. This
slightly and then raised and finally stabilized attitude of pH was agreed with the studies reported
around 7.5. This changing of pH can be explained by various studies [16-18].
FIGURE 2
The change of pH values in time.
COD and BOD measurements. Chemical 28750 mg/L at the initial stage of composting. Then
oxygen demand (COD) and biochemical oxygen it rose to 32.940 mg/L very rapidly due to of the
demand (BOD) in leachate were monitored during complex organics in the waste mixture. Then the
experiments to follow biological activity in the COD values were regularly decreased following 12
reactor (Figure 3). COD value was measured as weeks. In these phases, organic acids were
1099


decomposed and formed volatile fatty acids causing of usage of organic materials by microorganisms
COD and conductivity values rapid decline [1,19]. and it was analysed as 300 mg/L.
At the end of the study, COD value was calculated BOD/COD ratio generally used to show the
as 1124 mg/L. degree of biodegradability of waste mixture. In this
As can be seen from the results, BOD study, the initial and final BOD/COD ratios were
concentrations showed similar trend with the COD. calculated as 0.8 and 0.26, respectively (Figure 3).
BOD initial concentration was found as 23000 The ratio of between 0.02-0.13 shows very low
mg/L and the second week it increased to 25400 biodegradability of organic waste [1,18]. Therefore
mg/L due to hydrolysis of organic material in the the lower ratio shows that the biological treatment
waste mixture. After reach to maximum BOD of organic waste was very slowed at the end of
concentration, decreasing was observed as a result study.
FIGURE 3
The change of COD and BOD values and BOD/COD ratio in time.
The decrease in BOD/COD ratio was in the monitored and presented in Figure 4, respectively.
agreement with other reports in the literature. TKN and NH3-N parameters in leachate were
$÷GD÷ HW al. explained this decrease with analysed every week. TKN concentrations was
degradation of organic wastes through fermentation initially measured as 1106 than it raised up to 1474
phase which demonstrated a decreasing mg/L in four weeks then it was measured as 969
biodegradability due to methane formation [20]. mg/L at the end of the study. NH3-N concentrations
Yazdani et al. reported that this ratio was initially were measured as 628, 1160 and 857 mg/L for
calculated as 0.31 than as the solid was initially, after eight weeks and at the end of the
decomposed it was measured as 0.02 at end of 78 study, respectively. Increasing of the total nitrogen
days digestion time [14]. concentrations in the six weeks can be explained
with decomposing of organic compounds into
TKN and NH3-N Measurements. High nitrogen and proteins. Then NH3-N concentrations
ammonia values cause inhibition in the biological rose due to decomposing of proteins and amino
systems and are in main cause for delaying the acids in organic waste into NH3-N. The slight
system to run. Therefore the variations in leachate decreasing of NH3-N and TKN concentrations
TKN and NH3±N concentrations in the reactor were continued until end of the study.
1100


FIGURE 4
The change of TKN and NH3-N concentrations in time.
The results of the present study are similar to Volatile fatty acids (VFAs) are important
the findings of Romero et al. who reported TKN intermediates and metabolites in biological
concentrations of (970-1105 mg/L) and NH4-N processes. Therefore monitoring of leachate for
concentrations of (887-970 mg/L) for initial and VFAs is very important to understand stability of
final stage of operation [20]. Similar trends for the anaerobic digestion systems. As it seen from
NH3-N concentrations were also reported in co- Figure 5, initial VFAs concentration of this study
digestion of industrial sludge with municipal solid was measured as 10956 mg/L. This value increased
waste in a laboratory scale simulated anaerobic due to hydrolysis and fermentation of organic
landfilling reactors [22]. material and it was analysed as 15391 mg/L at the
end of the seventh week. Then it was started to
Al alinity and olatile Fatty Acids decrease after six weeks due to degradation of
Measurements. Alkalinity can be described as acid organic materials quickly. After that VFAs can be
buffering capacity effects when the pH is decreased converted to methane by methanogenic bacteria. At
to below the desired level. Sufficient and the end of the study, VFA value was measured as
appropriate buffering capacity is necessary for 400 mg/L.
optimum biological activity and stable condition of VFA/Alkalinity ratio describes the
pH value in the bioreactor. Alkalinity in the relationship between volatile fatty acids and the
bioreactor can result from carbonates, bicarbonates, UHDFWRU¶V EXIIHULQJ FDSDFLW\ 7KLV UDWLR JLYHV
borates, ammonia, organic compounds, sulphites knowledge about the stability of anaerobic process.
and phosphates in leachate occurred from anaerobic If this ratio is the lower than 0.4 the process is
conditions [9]. stable, if it is higher than 0.8, it can be said that
The initial alkalinity value was analysed as process is instable. Between these values it is
7176 mg CaCO3/L. After three month the alkalinity assumed that some disability can be occur in the
value was measured as 11397 mg CaCO3/L. After process. Because microbial activity can be inhibited
that, as it is seen from the Figure 5, the alkalinity by acidic conditions not buffered by alkalinity in
were decreased in the system. This problem was the reactor. In our study, Total volatile fatty acids
kept under control via adding sodium bicarbonate (TVFAs)/Alkalinity ratio was increased to 1 from
as a buffer solution. In the end of the study, the 0.8 due to effect of decreasing alkalinity in first
alkalinity value was measured as 6157 mg CaCO3/L four weeks. The ratio was started to drop after
(Figure 5). Alkalinity which analysed in leachate in addition of sodium bicarbonate as a buffer solution.
this study corresponds to literature [23] so it can be At the end of the study this ratio decreases to 0.06
said that there was enough alkalinity in our system (Figure 5).
during the process.
1101


FIGURE 5
The change of FA and Al alinity values in time.
Characteristics of compost product. were under the limit values given in the regulations
Compost samples were mixed homogenously and [24]. Also (C/N)final/(C/N)o ratio was calculated as
dried in the drying-oven at 105°C. Then it was an indication for waste stabilization [25]. The rate
shattered in a house-type food processor and was of 0.43 and 0.31 showed that compost is stable and
analysed to control the possibility of different usage can be used directly for agricultural purposes. In
such as growing plant. Table 2 shows the this study the ratio was calculated as 0.42 showing
characteristic properties of compost product. The stability of compost product.
results showed that the compost analysis results
TABLE 2
Analysis of compost
Parameter Compost Product Regulation

Water content,% 25 <%30
Organic material,% 48.9 >%35
C,% 25 -
N,% 1.87 -
C/N, ratio 13.36 10-30
TKN, (g/kg) 21.2 -
pH 8.02 5.5-8.5
Zn, (mg/kg) 108 1100
Cu (mg/kg) 114 450
Ni (mg/kg) 16 120
Cr (mg/kg) 98 350
Cd (mg/kg) 0 3
Pb (mg/kg) 0 150
Hg (mg/kg) 0 5
E.coli (CFU/g wet sample) 0 0
1102


CONCLUSIONS human health risks. Environment
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ACKNOWLEDGEMENT solid waste activated sludge with low organic
content at different organic loading rates by
pretreatment. Fresenius Environmental
The authors would like to express appreciation
Bulletin, 24(2), 510-518.
for the support of the TUBITAK [Project Number =
[13] Jerry D. M. and Power N. M. (2006) A
111Y011].
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Digestion of Biodegradable Municipal Waste.
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FRPSRVW SURGXFWLRQ 0DVWHU¶V 7KHVLV
Department of Environmental Engineering,
Graduate School of Natural and Applied
Sciences, Yildiz Technical University,
Turkey.
<DVHPLQdDOÕúNDQ
Gebze Technical University
Environmental Engineering Department
41400, Gebze/Kocaeli ± TURKEY
e-mail: ycaliskan@gtu.edu.tr
1104

COMPARISON OF SELECTED METHODS FOR

PREDICTION OF LOW-FLOW IN YILDI RI ER BASIN,
TURKEY
2VPDQ(PUH<ÕOGÕ]
Department of Civil Engineering, Cumhuriyet University, Sivas, 58140, Turkey
ABSTRACT problem from the tail-behavior of the parent

distribution via the asymptotic theory of extreme
In this study, low-flow frequency analyzes for value distributions. Hamilton et al. (2008)
<ÕOGÕ] 5LYer Basin in the middle of Turkey with investigated a regression model for computing
various predict methods. The study area was index flows describing the median flow for the
identified as measurement low-flow characteristics summer month of lowest flow in Michigan.
RI WKH <ÕOGÕ] 5LYHU %DVLQ LQ .Õ]ÕOÕUPDN 'UDLQDJH Low-flow mechanism ensures fish and other
Basin. Low-flow forecast methods are used with alive survive, protect aquatic life and ecological
concurrent flows measurement at a continuous systems in stream and river channel. Low-flow and
stream flow-JDJLQJ 6WDWLRQ 6*6 <ÕOGÕ] 5LYHU regionalization characteristics are identified for
Basin SGSs depend on the State Hydraulic Works selected streams in Arkansas (Funkhouser et al.,
(SHW). SHW is gaged daily average flow metric 2008). The relationship between low flows and
Q7,10 at each index and partial sites. Correlation large-scale climate variability is explored in France
methods are used to daily concurrent low-flow (Giuntoli et al. 2013). They were set up a national
records at four index-partial sites for estimate to low flow reference network of near-natural
low-flow characteristics of partial-record sites. catchments, consisting of 236 gauging stations.
Error and bias are calculated with three methods They were detected trends in a number of severity
such as Q-ratio, MOVE.1, and Base Flow and timing drought indices with a subset of 220
Correlation. The Base Flow Correlation method has daily stream flow records for the period 1968±2008.
the lowest error and the least amount of bias for Another potential application of the hydro-climatic
scenarios. relationships highlighted in this study pertains to
projecting the impact of climate change on low
flows. The regionalization of low-flow
KEYWORDS: characteristics are studied for selected streams in
Low-flows, Base Flow, MOVE. 1, Q-ratio, Base-Flow Arkansas by Funkhouser et al. (2008).
Correlation, low-flow frequency analyzes methods Low-flow prediction is the difficult problem
still at ungagged watersheds. The applicability of
physiographical space-based interpolation
INTRODUCTION techniques are investigated for the prediction of
low-flow indices in ungagged basins (Castiglioni et
Low-flow estimation is very significance to al., 2009). The study considers 51 catchments
evaluate the availability of water resources. Low- located in a wide region of central Italy, for which
flow forecasting is vital for various hydrologic several geomorphological and climatic descriptors
studies such as water quality management, are available. Four catchment grouping methods
determination of minimum flows requirement for were compared by Laaha et al. (2006). The
hydropower, irrigation system design. Based on the grouping methods are the residual pattern approach,
minimum frequent-flows and levels program was weighted cluster analysis, regression trees and an
developed in northeast Florida, USA (Ouyang, approach based on seasonality regions in terms of
2012). A new approach is namely the frequent-low their performance in predicting specific low flow
(FL) approach (or frequent-low index). A new discharges q95, i.e. the specific discharge that is
theoretical framework is developed for minimum exceeded on 95% of all days. Low-flow frequency
stream flow (Gottschalk et al., 2013). This statistics is estimated for unregulated streams in
framework was derived distribution approach Idaho and Oregon by Hortness et al. (2006), and
allowing a physical interpretation of its parameters. Risley et al. (2008)
It was grasped over the whole spectrum of the
1105

Meteorological and climatic data lacking in diagram (MRD) is expressed an easy way to
terms of low flow forecast cannot be expressed identify regional homogeneity of a region. The
exactly the region can cause. In addition, the preliminary process of data (annual minimum flow)
amount of time the dry season flow deficiency is achieved by carrying out the test for randomness
makes it difficult to forecast future low flow. A and homogeneity. The homogeneity of low flow
range of efficiency criteria is advised for low flows time-VHULHV ZDVGHWHUPLQHGE\7KRP¶VWHVWRUUXQ
analyzes. (Pushpalatha et al., 2012). The analysis test) at the 95% confidence level.
mainly concentrates on criteria computed on Effects of hydrodynamic conditions on the
continuous simulations that include all model diatom communities were investigated in the
errors. The criteria were evaluated using two reservoir has a fluctuant water level of 30 m
rainfall±runoff models and a set of 940 catchments annually in China. Variations of water flow, level,
located throughout France. The recession functions temperature and velocity are used to indicate the
are used for derivation of low-flow distribution (Yu spatio-temporal patterns of diatoms heterogeneity
et al. (2014). In this study without taking into during the flood and dry seasons (Zhou et al.,
consideration the statistical dependence structure 2015). An EFR (Environmental flow requirement)
between the characteristics of low-flow event. model was advanced based on the dominant eco-
Temporal evolution of annual and seasonal low- function and structure of the river ecosystem in the
flow regimes are investigated by Fiala et al. (2010), Haihe River Basin such that semiarid area where
for the 144 Czech river basins in 1961±2005 water resources are scarce China (Yang et al.,
periods. They were identified and delimited to 2014). A computational intelligence approach so
summer and winter low-flow seasons. They were Genetic Programming (GP), based on evolutionary
used to classify two categories of gauging stations. algorithm was used to estimate the response of
According to their low-flow regime, these are stream-flow to hydro-climatic variables in the upper
mountain and lowland stations. The standard and Mkomazi River in South Africa using limited
modified versions of the Mann±Kendall trend test amount of datasets (1994-2012). Individual
were used in the study to detect trends in several monthly models that evaluated using three
low-flow variables. Dukic (2006) found statistical measures of accuracy were used for
hydrologic±hydraulically model for determining the stream-flow forecasting for each month of the year
base flow of the catchment by analyzing the water (Oyebode et al., 2014). The streams were analyzed
balance of the basin. And vertical exchange of in the Western Mediterranean Basin of Turkey
water between the atmosphere, unsaturated media, monthly and annually. Mann-Kendall trend test was
and ground water, basing on the known physical used to observe whether a trend changed or not, in
characteristics of the basin in the Kolubara River the safety range of 95%. Scatter diagrams are
basin, in Serbia. created to determine the slopes of variations on the
Some of the low flow estimates to start with trend-line of flow-rates. The Mann-Kendall test is
the statistical behavior to determine a homogeneity evaluated to specify plotting diagrams of each
test. Then this estimates is expressed with the most trends (Saplioglu et al., 2014).
appropriate of probability density function. The main objective of this article is to analyze
According to the most appropriate function of the the extent to low-IORZLQ<ÕOGÕ]ULYHUEDVLQ7KUHH
probability density, the parameters of low-flow low flow predicting methods were used in this
distribution are found. Low-flow frequency analysis. These methods were compared to
statistics and hydrological parameters were statistically. Root Mean Square Error (RMSE),
estimated for selected stream flow-gaging stations Error of Arithmetic Average (BIAS), Mean
at Nooksack River basin in northwestern Absolute Deviation (MAD) and Square of
Washington and Canada (Curran, et al., 2009). A Correlation Number (R2) values are calculated and
number of commercial software packages are used used for this comparison. This study estimate of
by Joshi and Hilaire, (2013) for fitting statistical low flow in the region for the first time, these three
distributions to the flow data sample to low-flow methods is important in terms of use.
frequency analysis of three rivers in Eastern
Canada. A regional low flow frequency analysis
were carried out in the north of Iran using L- MATERIALS AND METHODS
moments technique by Modarres (2008). Low flow
events have been represented by the 7-day annual STUDY AREA
minimum series, and the L-moments approach was
used to assign these data to homogenous regions. A <ÕOGÕ]5LYHU%DVLQ is located in the KÕ]ÕOÕUPDN
high degree of heterogeneity can be identified from Drainage Basin and covering a total geographical
the L-moment ratio diagrams. The moment ratio area of 1285 km2. The regions are surrounded by
1106

<ÕOGÕ]HOL City Hall in the west, Sivas City in the nourished with mostly melt water from snow cover
east, Kabasakal Municipality in the south and Tokat and fields especially during spring and winter
City in the north (see Figure 1). Most of the months in <ÕOGÕ] 5LYHU %DVLQ /RZ-flow typically
precipitation falls during late autumn, winter and occur during extended dry periods in the spring,
spring. The climate of <ÕOGÕ] 5LYHU %DVLQ is wet summer and early autumn, but also can occur
winters and generally cool, dry summers. The during cold, dry periods in winter in here. Low-
topography ranges in elevation from sea level to flow can be changed by many factors such as sub-
about 2150 m. Most precipitation in the mountains surface water trend, return flows, groundwater
and lowlands occurs as snow. Land cover in the pumping, reservoir operation studies and land use.
region is mostly unfrosted areas, with developed People and ecosystems influence from this reasons
and agricultural lands leading the lowland areas. in terms of water availability.
Stream, base flow and groundwater discharge is
FIGURE 1
.Õ]ÕOÕUPDN'UDLQDJHBasin in Tur ey
MATERIALS once every ten years. Four stream gauging stations

GLVWULEXWHGDOORYHU<ÕOGÕ]5LYHU5HJLRQ,W¶V<ÕOGÕ]
The recorded daily concurrent average low- LV WKH QXPEHU RI .Õ]ÕOÕUPDN 'UDLQDJH
flow data are obtained from stream gauging stations Basin, 185 is the number of station, in Turkey)
(SHW). SHW network to measure the daily average *QH\ND\D .D\DOÕJ|O DQG
flow metric Q7,10 at each gaging site. Q7,10 is Çeltek stations. Stations are included at least ten
defined as the lowest average stream flow for a years recorded data. <ÕOGÕ] 5LYHU %DVLQ DQG
sequential 7-day period that returns on average locations of these stations are shown in Figure 2.
1107

FIGURE 2
<ÕOGÕ]5LYHU%DVLQLQ.Õ]ÕOÕUPDN'UDLQDJH%DVLQ
METHODS
(2)
-ratio Method. Q-ratio method is advanced
by Potter at 2001. When the number of base-flow Where is the number of pairs of concurrent base-
measurements at the partial-record site is fewer flow observations, Qp,k is the measured stream flow
than eight measurements, the method is suggested. of the kth pair at the partial-record site, and Qi,k is
In this method, concurrent flow pairs (all stream the measured stream flow of the kth pair at the
flow measured at the partial-index record site on the index site. Pushpalatha et al. (2012), a relative
same day) are arranged, and the ratio of flows is efficiency criterion is obtained by calculating the
determined for each pair. The average flow ratio is ratio between the simulated and observed values.
then computed and magnified by the flow statistic The weighted cumulative frequency for the kth
at the index site to predict the statistic at the partial- flow is therefore given by the ratio between the
record site. The Q-ratio methods predict discharge accumulated error for the k smallest flows and the
is defined as: total error (SSE) over the N time steps. The daily-
value correlation, variable space, and flow-duration
(1)
ratios, and other factors are related to individual
Where Qp is the estimated low-flow statistic at the sub-basins using methods of hydrograph
partial-record site, Qi is the low-flow statistic at the comparison (Curran et al., 2009).
index site (in volume per time). The dimensionless
average flow ratio, is defined as:
1108

MO E.1 Method. The Maintenance of and a simple linear regression between the log-
Variance Extension, Type 1 (MOVE.1) method
transformed concurrent flows at the partial-record
developed by Hirsch, at 1982. MOVE.1 is generally
site. Index site is achieved to get the regression
used for spreading the length and completing
FRHIILFLHQWV ȕ1 VORSH DQG ȕ0 (intercept). These
lacking and scarce periods of the flow record at a
continuously recording SGS. The method can be coefficients are further used to estimate and
used to predict low-flow frequency or duration expressed as:
statistics at a partial-record site. MOVE.1 is
recommended over more general linear regression (5)
techniques for ordinary least squares are used. and
Because it maintains the characteristic variance of
stream flow data at the partial-record site and
(6)
results in predict of flow that is less biased. This
method is recommended referred when at least
where mi and i2 are the mean and variance of the
eight base-flow measurements are suitable for a
annual Q7 at the index site, 2 is the variance of the
partial-record site and requires concurrent flows
concurrent flows at the index site, n is the number
from a hydrological parallel index site. The
of concurrent base-flow measurement pairs, and e2
MOVE.1 method equation is defined:
is the square of the standard error of the regression
(3) among log-transformed concurrent flows at the
partial-record site and index site.
where is the predicted logarithmic (log) low-flow The standard deviate for the partial-record
statistic at the partial-record site, is the average site, , is assumed equal to the standard deviate for
the index site, i, and, if not already known, is
of the log base-flow measurements at the partial-
calculated as:
record site, and x are the standard deviations of
the log base flows at the partial-record site and
index site, respectively, X is the log low-flow (7)
statistic at the index site, and is the average of
Where is known at the index site, mi is
log concurrent flows at the index site. The
coefficient of determination (R2) of the model i.e. described previously, and i is the standard
the ratio of the variance explained by the deviation of the at the index site.
classification and the total variance of low flows, is
a measure of the goodness-of-fit of this simple The Stedinger and Thomas (1985) method, or
model in Analysis of variance (Blöschl et al., base-flow correlation method, estimates low-flow
2006). The least squares method were used by Chen characteristics by using the mean and standard
et al. (2006), for calculated correlation coefficient. deviation of the time series of 7-day low flows at
the partial-record station. The example is worked
Base-Flow Correlation Method. The Base- shown in the report applies some of the low flow
Flow Correlation method was improved Stedinger frequency analysis techniques to flow data from
and Thomas (1985). Method is an un-biased river Ouelle in Eastern Canada. Joshi et al. (2013).
technique for forecasting low-flow frequency For each discharge station correlation coefficients
statistics at partial-record sites. The method were computed for the low flow indices and the
presumes an equal skew coefficient Log-Pearson yearly, seasonal and monthly series of every
Type III distribution for the annual Q7 low-flow at climate variable. A linear relationship is found
both sites. When using this method, a minimum of between the low flow and the 10-year increment
10 base-flow measurements at a partial-record site with very good correlation coefficients by Ouyang
is suggested for consistent results. The Base-Flow (2012).
Correlation methods estimate discharge is given as:
(4)
RESULTS AND DISCUSSION
where is the predicted mean of the annual 7 at
the partial-record site, is the log-Pearson Type The low-flow prediction techniques are
III standard deviate for a return interval of 10 years, applied to 4 stream-JDXJLQJVWDWLRQVLQ<ÕOGÕ]5LYHU
and is the forecasted standard deviation of the Region, whose locations are shown in Figure 2. Q-
ratio, MOVE.1 and Base-Flow Correlation methods
annual 7 at the partial-record site. Predicted are used to annual low-flow series (Q7,10) records
1109

DW6*6LQ<ÕOGÕ]5LYHU%DVLQ for forecasting low- the given equation (1). Low-flow measuring and
flows. The first low-flow estimation method such as predicting hydrograph are given in Figure 3. Low-
Q-ratio method is given by equation (1). The flow predicting results are expressed for Q-ratio
dimensionless average flow ratio (Z) is computed Method in Table 1. Q-ratio Method gauging and
with equation (2) and the low-flow statistic at the estimating low-flows are showed according to most
index and partial sites (Qi, Qp) are obtained from high R2 values and linear equation in Figure 4.
SGS Q7,10 records. Qp has been calculated with
FIGURE 3
Low-flow measuring and predicting hydrograph values calculated from SGS 7,10 records.
TABLE 1
Low-flow prediction results are calculated with -ratio Method.
Qp,k Qi,k
Date Q-ratio Z Qp (m3/s) MAD BIAS RMSE
(m3/s) (m3/s)
0,037 0,008 0,045
13/10/2005 0,227 2,72 11,982 0,0698 0,19
for n=8 for n=8 for n=8
0,02 0,002 0,026
27/10/2006 0,191 2,31 12,094 0,0698 0,191
0,014 0,0013 0,016
16/09/2007 0,175 2,28 13,029 0,0698 0,188
0,001 0,00008 0,001
13/10/2008 0,138 2,09 15,145 0,0698 0,172
R2=0,922
25/10/2009 0,202 2,66 13,168 0,0698 0,219
for n=8
R2=0,856
23/10/2010 0,283 2,85 10,071 0,0698 0,235
for n=6
R2=0,923
03/09/2011 0,08 1,95 24,375 0,0698 0,161
for n=4
R2=0,999
19/10/2012 0,048 1,44 30 0,0698 0,119
for n=2
* Qp is the estimated low-flow statistic at the partial-record site; Qi is the low-flow statistic at
the index site (in volume per time), Zi is the dimensionless average flow ratio.
According to Table 1 and Figure 4; MAD, For n=2, MAD, BIAS and RMSE values are most
BIAS, RMSE and R2 are valued respectively 0.037, low and R2 value is most high. But for n=6, R2
0.008, 0.045 and 0.999 to low-flow frequency value is most low while MAD, BIAS and RMSE
DQDO\]HLQ<ÕOGÕ]5LYHU%DVLQZLWK4-ratio method. values are third low.
1110

FIGURE 4
-ratio Method predict and measured low flows for n 8 and n 4.
ThH VHFRQG ORZ-IORZ HVWLPDWLRQ PHWKRG VXFK flow statistics for Log-Pearson Type III and linear
DV 029( PHWKRG LV H[SUHVVHG E\ HTXDWLRQ distribution values between index and partial sites.
:KHUH <ࡄ 6\ 6[ ; DQG ;ࡄ DUH WKH QXPHULFDO ,W¶V GHULYHG IURP FRPPRQ VWDWLVWLFDO IRUPXODV DQG
characteristic values for the index and partial sites. equation (5), (6) and (7). Q7,10 is obtained from
,W¶V FDOFXODWHG ZLWK FRPPRQ PHDQ DQG VWDQGDUG equation (4). Base-Flow Correlation Method results
deviation equations. Y is determined by equation are showed in Table 3. Base-Flow Correlation
(3). Low-flow estimation results are showed for Method gauging and estimating low-flows are
MOVE.1 Method in Table 2. MOVE.1 Method showed according to most high R2 values and linear
Low-flow estimating and gauging values is showed equation in Figure 6.
according to most high R2 values and linear According to Table 3 and Figure 6; MAD,
equation in Figure 5. BIAS, RMSE and R2 values are altered respectively
MAD, BIAS, RMSE and R2 values are altered 0.0019-0.0002, 0.0019-0.0002, 0.0095-0.0002 and
respectively 0.016-0.0008, 0.0015-0.000001, 0.7521-0.999 to low-flow prediction with Base
0.0195-0.00008 and 0.8635-0.999 in Table 2 and Flow Correlation method. For n=4, MAD, BIAS
Figure 5. For n=2, MAD, BIAS and RMSE values and RMSE values are most low and R2 value is
are most low and R2 value is most high. But for most high. For n=10, MAD, BIAS and RMSE
n=6; when RMSE is third high value, MAD and values are most high when R2 is the worst value.
BIAS is second high and R2 is the lowest value to Root Mean Square Error (RMSE), Error of
low-flow estimation with MOVE 1 method in Arithmetic Average (BIAS), Mean Absolute
<ÕOGÕ]5LYHU%DVLQ Deviation (MAD) and Square of Correlation
In this study, Base-Flow Correlation Method Number (R2) values are calculated with common
is to be given to third low-flow prediction method statistical equations. Calculated RMSE, BIAS and
with equation (4), (5), (6) and (7). Where Vp, Kp MAD values showed for every Low-flow methods
and Pp are the known mean and standard deviation in Figure 7.
or namely first, second statistical moments of low-
FIGURE 5
MO E.1 Method predict and measured low flows for n 8 and n 4.
1111

© by PSP
TABLE 2
Low-flow estimation results are computed with MO E.1 Method.
Date log Yi A log Yi A log Xi S log Yi S log Xi log YPredct Predict
Yi (m3/s) Xi (m3/s) log Xi MAD BIAS RMSE
13/10/2005 0,227 2,72 0,016

-0,644 0,435 -0,83 0,3506 0,257 0,097 -0,606 0,248 0,0015 0,0195
for n = 8
27/10/2006 0,191 2,31 0,0147 0,017
-0,719 0,364 -0,795 0,16 0,00007
for n = 6
16/09/2007 0,175 2,28 0,0096
-0,757 0,358 -0,81 0,155 -0,00015 0,0106
for n = 4
13/10/2008 0,138 2,09 0,00008
Volume 25 ± No. 4/2016, pages 1105-1116
-0,86 0,32 -0,911 0,123 0,00001 0,00008

for n = 2
1112
25/10/2009 0,202 2,66 R2=0,9364
-0,695 0,425 -0,632 0,233
for n = 8
23/10/2010 0,283 2,85 R2=0,8635
-0,548 0,455 -0,553 0,28
for n = 6
03/09/2011 0,08 1,95 R2=0,9059
-1,097 0,29 -1,06 0,087
for n = 4
19/10/2012 0,048 1,44 R2=0,999
-1,319 0,158 -1,341 0,046
for n = 2
<LVWKHSUHGLFWHGORJDULWKPLFORJORZ-IORZVWDWLVWLFDWWKHSDUWLDO-UHFRUGVLWH<ࡄLVWKHDYHUDJHRIWKHORJEDVH-IORZPHDVXUHPHQWVDWWKHSDUWLDO-
Fresenius Environmental Bulletin
UHFRUGVLWH6\DQG6[DUHWKHVWDQGDUGGHYLDWLRQVRIWKHORJEDVHIORZVDWWKHSDUWLDO-UHFRUGVLWHDQGLQGH[VLWHUHVSHFWLYHO\;LVWKHORJORZ-IORZ
VWDWLVWLFDWWKHLQGH[VLWHDQG;ࡄLs the average of log concurrent flows at the index site.

FIGURE 6
Base-Flow Correlation Method predict and measured low flows for n 8 and n 6.
FIGURE 7
Calculated RMSE, BIAS, MAD and R2 values are related with all low-flow methods.
CONCLUSIONS prediction techniques from Table 1, 2, 3 and Figure

6.
In this study, correlation methods such as Q- Q-ratio, MOVE.1, and Base Flow Correlation
ratio, MOVE.1, and Base Flow Correlation are used low flow prediction method using, measurement
to low-IORZ IUHTXHQF\ DQDO\]H LQ <ÕOGÕ] 5LYHU values with estimated values are quite consistent in
Basin in the middle of Turkey. <ÕOGÕ]5LYHU%DVLQ
The aforementioned low flow prediction Figure 7 is showed to best method is Base
methods have been very successful when flow Correlation Method according to calculated
observations and prediction results are compared to MAD and RMSE values to low-flow frequency
use to low-IORZIRUHFDVWLQJLQ<ÕOGÕ]5LYHU%DVLQLQ DQDO\]HLQ<ÕOGÕ]5LYHU%DVLQ
.Õ]ÕOÕUPDN'UDLQDJH%DVLQ7XUNH\ Q-ratio method is the best method among the
It is understood that Base Flow Correlation other methods according to computed R2 values.
low flow estimation method is the most consist and MOVE: 1 method is the best method among
reliable method through in other low flow the other methods according to calculated BIAS
values.
1113

TABLE 3
Low-flow forecasting results are derived with Base-Flow Correlation Method.
© by PSP
Date log Yi E0 Si2 Sc2 Vp mi

Yi (m3/s) E1 Se2 Pp Kp A E 7 7 pred MAD BIAS RMSE
18/109/2003 0,112 0,1641 0,166

0,0019
-0,9508 -1,5764 1,9787 0,0098 0,0125 0,2718 0,2258 0,3761 -0,8322 0,2321 0,0019 0,00195
for n=10
07/10/2004 0,185 0,2566 0,168 0,1709

0,0029
-0,7328 0,0029 0,0028
for n = 8
13/10/2005 0,227 0,1052 0,2027 0,2025

0,00014
-0,644 -0,00014 0,00014
for n = 6
27/10/2006 0,191 0,09 0,1828 0,183

0,0002
-0,719 0,0002 0,0002
for n = 4
16/09/2007 0,175
R2=0,752
Volume 25 ± No. 4/2016, pages 1105-1116
-0,757
for n = 10
1114
13/10/2008 0,138 R2=0,934
-0,86
for n = 10
25/10/2009 0,202 R2=0,948

-0,695
for n = 10
23/10/2010 0,283 R2=0,923

-0,548
for n = 10
03/09/2011 0,08
-1,097
Fresenius Environmental Bulletin
19/10/2012 0,048 -1,319
* is the predicted mean of the annual 7 at the partial-record site, is the log-Pearson Type III standard deviate for a return interval of 10 years, is the forecasted standard deviation of the annual 7 at the
partial-UHFRUGVLWHȕ1 VORSHDQGȕ0 (intercept) are the regression coefficients. mi and i2 are the mean and variance of the annual Q7 at the index site, 2 is the variance of the concurrent flows at the index site, n is the
number of concurrent base-flow measurement pairs, and e2 is the square of the standard error of the regression among log-transformed concurrent flows at the partial-record site and index site. is the standard deviate
for the partial-record site,, i is the standard deviate for the index site.

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1115

2VPDQ(PUH<ÕOGÕ]
Department of Civil Engineering
Cumhuriyet University
Sivas, 58140 ± TURKEY
e-mail: oemrey@gmail.com,
oeyildiz@cumhuriyet.edu.tr
1116

E ALUATION OF POTASSIUM AND SODIUM SILICATES

AGAINST usariu spp. CAUSING DAMPING-OFF DISEASE
OF COTTON SEEDLING
Mohamed A. Yassin1,2, Abd El-Rahim M. A. El-Samawaty1,2, Mohamed A. Moslem1, and Shaban R. M.
Sayed3
1
Plant Pathology Research Institute, Agricultural Research Center. Giza, Egypt.
2
Botany and Microbiology Department, College of Science, King Saud University. Riyadh, Suadi Arabia.
3
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
ABSTRACT to the a i m isolates and cultivated germplasm

[6].
Sodium and potassium silicates were in it Fungicidal application in controlling
evaluated against seven a i m species; causing a i m fungi has negative effects on the human
cotton seedling damping-off. Scanning electron and animal health as well as the biological balance.
microscopy-SEM was used to investigate the However, alternative control methods against
morphological changes in the fungal hyphae. All pathogenic fungi should be developed [7,8].
collected data were statistically analyzed, and the Several alternative control methods had
least significant difference was used to compare successfully been used against phytopathogenic
means. Mycelial growth of the tested isolates was a i m species [9,10]. It has been known that
strongly inhibited by both silicates. Varied silicon adequate application in most soils, and at the
morphological changes in a i m mycelia proper time can significantly decrease fungal
following silicate treatments were observed infection and increase the plant yield [11,12]. Such
including; asymmetric mycelium compared with major micronutrient was documented as effective
the control. Silicate-treated cultures of ai m suppressor of phytopathogenic fungi [13-15].
species were generally characterized by wizened The efficiency of silicon against some
mycelia, curling, twisting, and distorted hyphae. phytopathogenic fungi had been in it and/or in
Diameter of silicate treated hyphae of ai m i investigated [16,17]. ai m l e m
species was generally smaller than the control. and eni illi m i itat m [18,19], and
Promising antifungal activity of potassium and Ma mina a e lina [20] have greatly
sodium silicates in it ; suggest that inhibited by sodium silicate in it treatment.
phytopathogenic species of a i m could be t t a root rot of avocado [21],
controlled by both materials. Further researches are t t a blight in Pepper [22], rice brown spot
needed in order to confirm the in i effects of [23] and tomato powdery mildew [24] have
such materials and identify the optimal effective successfully controlled by silicon applications in
doses. i .
This study was conducted to investigate the in
it effect of potassium and sodium silicates on
KEYWORDS the growth of seven a i m species; causing
Disease control, Phytopathogens, SEM, Silicon cotton seedling damping-off disease.
compounds.

INTRODUCTION
usariu species. Seven isolates of a i m
a i m is a widespread filamentous fungal species; ant il m i i i
genus threatens cotton ( i m a a en e L.) m nili me x m emite t m
as well as many plant species [1-3]. Pathogenicity lani and t i i i e were used in this
of a i m on the Egyptian cotton throughout study. The pathogenicity of these isolates have been
much of the growing areas is well documented confirmed in two cotton ( i m a a en e
[4,5]. a i m is one of the major causes of cotton L.) cultivars i Giza-86 and Giza-90, under
seedling damping-off, and the disease incidence in greenhouse conditions [9].
the commercial cotton cultivars is varied according
1117

Effect of the silicates on the mycelia tetroxide for 2 h at 4°C and then air-dried. The
growth. The methods described by Li et al., 2009 treated samples were placed on aluminum stubs
[19] was used to assay the effects of potassium and using carbon tape coated with gold, and examined
sodium silicates on the growth of a i m species. by SEM (using a JEOL JSM-6380LA microscope)
Sodium and potassium silicate solutions (12% SiO2, operated at 10 kV. The hyphae diameter of all
molar ratio [MR] = 2) were incorporated into the treated a i m species were measured (in μm).
Potato Dextrose Agar media to obtain Five replicates per isolate were measured and the
concentrations of 0.5%, 1%, 2% and 4% of data were subjected to statistical analysis.
silicates. Supplemented media were then poured
into petri plates and inoculated with 5-mm-diameter Statistical analysis. All statistical analyses
plugs of 7-days-old a i m cultures. Four were performed with SPSS software package
replicate plates were used for each treatment and version, 16 (SPSS Inc., Chicago, Ill., U.S.A.). Data
untreated plates served as controls. The colony were analyzed using the one-way analysis of
diameters of the developing fungi at 25 ± 2°C was variance (ANOVA). The least significant
measured daily until the fungi in the control plates differences 3 ZHUH XVHG WR LGHQWLI\
reached the full growth. Inhibition of fungal growth differences and compare mean values.
was measured using comparisons with the control
plates. Collected data were subjected to statistical
analysis. RESULTS
Effect of the silicates on the hyphal Effect of the silicates on the mycelia
morphology. The scanning electron microscopy growth. ANOVA (Table 1) showed that the
(SEM) procedure described by Li et al , 2009 [19] a ia, silicate concentration (C), and their
was used in this investigation. Fungal samples were interaction (F × C) were significant sources of
excised from 7-day-old cultures of ai m variation in the growth of a i m species.
species grown in 0.5% and 1% silicates. Samples
were vapor-fixed with 2% (w/v) aqueous osmium
TABLE 1
ANO A of the effect of Fusaria (F), silicate concentration (C) and their interaction on the growth of
usariu species.
Source of
D. f M.S. F Sig.
variance
Potassium Rep. 3 21.410 3.10 0.030
silicate F 6 684.724 99.169 0.000
C 4 10837.632 1569.620 0.000
FxC 24 237.140 34.345 0.000
Error 102 6.905
sodium Rep. 3 9.550 1.441 0.235
silicate F 6 813.357 122.707 0.000
C 4 6161.918 929.619 0.000
FxC 24 219.935 33.180 0.000
Error 102 6.628
The significance of the F × C interaction between i i i and emite t m in 1% and

indicates that the inhibitory effects of silicates 4%, while, significant differences were found
varied depending on the concentration and between the same fungi in 0.5% and 2% potassium
a i m species. For example, no significant silicate. No significant differences were found
differences were found between the growth of between m nili me and lani for 0.5% and
i i i and ant il m in 1% and 2% 1% potassium silicate; but, significant differences
potassium silicate; however, significant differences were found between the same species in 2% and
were found between the same species in 0.5% and 4% potassium silicate (Table 2).
4%. No significant differences were also found
1118

TABLE 2
Effect of potassium silicate on the growth of usariu species in itro.
Linear growth
Fusaria Control 0.5% 1.00% 2.00% 4.00% Mean
a i m emite t m 90.00 80.00 70.00 59.00 32.00 66.30
ti i i e 90.00 90.00 90.00 73.25 24.50 73.75
ant il m 90.00 82.75 69.50 53.50 25.75 64.30
x m 90.00 86.50 81.00 77.50 61.75 79.45
m nili me 90.00 78.75 78.00 63.75 59.00 73.90
i i i 90.00 73.25 68.50 53.50 33.50 63.75
lani 90.00 81.75 78.25 67.50 41.25 71.75
Mean 90.00 81.85 76.46 64.00 39.67
LSD for (FxC) interaction (3
No significant differences were found between between the same species in 2% and 4%
ai m x m and t i i i e in all concentrations. No significant differences were
concentrations except 2% of sodium silicate. No found between m nili me and lani in all
significant differences were found between concentrations except 0.5% of sodium silicate
ant il m and m nili me in 0.5% and 1%; (Table 3).
however, significant differences were found
TABLE 3
Effect of sodium silicate on the growth of usariu species in itro.
Linear growth
Fusaria Control 0.5% 1.00% 2.00% 4.00% Mean
a i m emite t m 90.00 80.25 63.50 64.50 59.25 71.60
ti i i e 90.00 82.00 81.25 77.75 70.00 80.40
ant il m 90.00 87.75 70.25 51.50 42.25 66.75
x m 90.00 82.75 80.75 71.50 68.25 78.75
m nili me 90.00 84.50 72.00 62.50 53.25 72.45
i i i 90.00 79.25 74.00 37.25 30.00 62.10
lani 90.00 78.75 69.75 63.25 50.75 70.50
Mean 90.00 81.04 73.07 61.17 53.39
LSD for (FxC) interaction 3
The highest efficiency of potassium and Varied morphological changes in a i m

sodium silicates was against a i i e e ie mycelia following silicate treatments were
however; the minimum efficiency of potassium observed including; asymmetric mycelium
silicate was against x m and the minimum compared with the control. Silicate-treated cultures
one of sodium silicate was against of a i m species were generally characterized
t i i i e (Figure 1). by wizened mycelia, curling, twisting, and distorted
hyphae.
Effect of the silicates on the hyphal ANOVA (Table 4) showed that a i m
morphology. Figures 2 & 3 illustrated the SEM species, treatment (T), and their interaction (AG ×
observations of silicate-treated and -untreated T) were significant sources of variation in the
mycelia of a i m species cultures. diameter of a i m species hyphae.
1119

FIGURE 1
Percentages of the efficacy of potassium and sodium silicates on the growth of usariu species in
itro
FIGURE 2
SEM observations of silicate treated and untreated cultures of usariu species. Asymmetric, curling,
twisting wi ened mycelium and distorted hyphae shown in silicate treatments.
1120

FIGURE 3
SEM observations of silicate treated and untreated cultures of usariu species. Asymmetric, curling,
twisting wi ened mycelium and distorted hyphae shown in silicate treatments.
TABLE 4
ANO A of the effect of usaria (F), treatment (T) and their interaction on the diameter of usariu
species hyphae.
Source of
D. f M.S. F Sig.
variance
a ia (F) 6 2.137 57.936 0.000
Treatment (T) 2 2.533 68.675 0.000
F xT 12 0.326 8.836 0.000
Error 105 0.037
This result indicates that the effectiveness of of emite t m x m and i i i

applied silicate depends on the a i m species was significantly different from the control.
and treatment (tested compound). Diameter of However, significant differences were only found
a i m species hyphae was lower in the silicate- between the effect of potassium and sodium silicate
treated cultures than in the control for all tested treatments on the hyphae diameter of x m
isolates. Hyphae diameter in the silicate treatments and i i i (Table 5).
1121

TABLE 5
Effect of potassium and sodium silicates on the diameters of usariu hyphae.
Hyphae diameter (μm)

Fusaria Control Potassium silicate Sodium silicate
emite t m 2.18 1.49 1.70
ti i i e 1.27 1.05 1.06
ant il m 1.06 1.01 1.03
x m 2.16 1.10 1.84
m nili me 1.18 0.96 1.05
i i i 2.20 1.15 1.91
lani 1.35 1.19 1.21
LSD for (F x T) LQWHUDFWLRQ3
DISCUSSION materials or cavities within the cells, collapsing the

cells that abnormally contracted, and complete
This study indicated that the growth of hyphal damages in the higher concentrations [19].
a i m species were significantly inhibited by The plasma membrane damage was suggested to
treatment with potassium and sodium silicates. This play a crucial role in the antifungal effects since the
result was in agreement with Smith et al., 2005 [25] leakage of protein and sugar from Si-treated spores
who stated that ai m x m f. sp. was significantly higher than it was in the control
a in e t m growth was significantly inhibited by treatment due to fungal plasma membrane damage
potassium silicate treatment. Sodium silicate has [18].
also greatly suppressed the growth and spore Potassium and sodium silicates were
germination of ai m l e m in it [19]. promising as antifungal agents since they
Bi et al. (2006) found that mycelial growth of suppressed the growth of some a i m species in
lte na ia alte nata, emite t m and this study. Further research confirming the
i t e i m e m is completely inhibited by antifungal property and determining the optimal
100 mM sodium silicate [26]. The effectiveness of doses of potassium and sodium silicates is needed.
sodium silicate against the in it growth of M
a e lina has also been documented [20]. Similar
observations have also been recorded for ACKNOWLEDGEMENTS
t ae ia t i ea [27], ex an m and
M nilinia ti la and i itat m [18]. The authors would like to extend their sincere
Several morphological changes in silicate- appreciation to the Deanship of Scientific Research
treated hyphae of a i m species were recorded at King Saud University for its funding of this
according to the SEM observations in this study. research through the Research Group Project No.
This result was in agreement with the observations RGP- 298.
of Li et al., 2009 [19] who found that sodium
silicate 100mM treatment resulted in great
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Dr. Mohamed Abdallah Yassin

Botany and Microbiology Department
Faculty of Science
King Saud University
P.O. Box 2455, Riyadh 11451 ± SAUDI ARABIA
e-mail: myassin@ksu.edu.sa
mohamdyassin@gmail.com
1124


INFLUENCE OF LEAD STRESS ON GROWTH,

ANTIOXIDATI E EN YME ACTI ITIES AND ION CHANGE
IN ROOT AND LEAF OF STRAWBERRY
)HUKDG0XUDGR÷OX17DUÕN(QFX20XWWDOLS*QGR÷GX1, Sibel Boysan Canal3
1
Department of Horticulture, Faculty of Agriculture and Natural Sciences, Abant Izzet Baysal University, Bolu, Turkey.
2
Department of Horticulture, Faculty of Agriculture, YuzuncuYil University, Van, Turkey
3
Department of Soil Science and Plant Nutrition, Faculty of Agriculture, YuzuncuYil University, Van, Turkey
ABSTRACTS of Pb ores, metal plating and finishing activities,

pesticides, fertilizers, pigments additives and
Heavy metals are of widespread occurrence as gasoline [1]. Pb is accessible to plants from soil and
consequences of human, agricultural and industrial aerosol source. Pb can be accumulated into plants
activities. Among heavy metals, lead is a potential though it is not necessary for plant. Previous studies
pollutant that can accumulate in different tissues show that roots usually accumulate significant
organs and thus restrict plant growth. In this study, quantities of Pb and roots greatly restricting its
mechanisms of plant tolerances to lead stress were translocation to aerial parts but certain species have
studied on strawberry (Camarosa cv.) using pot ability to transferring large quantities of Pb to their
experiment with different Lead(PbNO3)2 shoot and leaves [2]. Many research demonstrated
applications ranging from 0 to 80 mg kg-1 using soil that excess Pb causes toxic effect which influence
culture. The accumulation of Pb2+ and its influence various morphological and biochemical processes
on chlorophyll and malondialdehyde (MDA) in plant. Pb toxicity inhibits cell division and
contents, activities of superoxide dismutase (SOD), transpiration [3], chlorophyll production [4], leaf
catalase (CAT), ascorbate peroxidase (APX) and rolls, chlorosis and inhibition of root and stem
ion concentrations were investigated in both root growth [5], Lead to changes in plant nutrient upset
and leaves of strawberry plant. Despite severally [6,7], changes hormonal status [1], water balance,
decrease in the chlorophyll a and b contents, Pb2+ and nitrate assimilation [8]. Heavy metals cause
accumulation, MDA, SOD, and CAT activities oxidative damage to plant either directly or
were increased with the increase in the indirectly. ROS (reactive oxygen species) can
Pb2+concentrations both in roots and leaves. efficiently eliminated by non-enzymatic
However APX activity was shown unsteady components such as glutathione, ascorbic acid, a-
increase in roots and leaves. In the roots and leaves tocopherol and carotenoids as well as enzymatic
macro and micro ion concentrations were shown components superoxide dismutase (SOD),
altered. While micro element concentrations ascorbate peroxidase (APX), catalase (CAT),
decreased in contrary to macro element peroxidase (POD) and glutathione reductase,
concentrations which were increased compare with ascorbic acid peroxidase [9, 10, 11]. Pb can cause
control. These results indicate that lead stress cause oxidative stress in plant with accompanying
reduction on growth and oxidative damage as increased ROS production. When exceeding the
shown by the decline in chlorophyll, increase in capacity of cell defense mechanisms, these ROS
lipid peroxidase and antioxidant enzyme and alter can induce oxidative stress and damage the
levels of macro-micro ion concentrations. proteins, lipids and eventually causing the death of
plant [12, 13]. Turkey is one of the most important
countries in world with 353.173 tones Strawberry
KEYWORDS: productions. Turkey comes after United States of
Lead stress, antioxidant enzyme activity, lipid America (1366.850) and Mexico (360.426) [14].
peroxidase, macro-micro element, strawberry The strawberry is a grown area of around 134.23
decares in Turkey [1@ 6R ÕW LV YHU\ LPSRUWDQW WR
study on Strawberry and understand the effect of Pb
INTRODUCTION toxicity in root and leaf of strawberry.
Up to the present, there is not study in the
Lead is a toxic and residual environmental literature dealing with Pb tolerance on strawberry
pollution. The major source of Pb in environmental cultivars. Although, Pb toxicity in plant is depend
are automobiles exhaust fumes , chimneys of on Pb range, soil types and plant species. Thus, the
factories, wastes of the storage battery, processing objectives of the this study were study Pb
1125


accumulation and influence of Pb toxicity on Preparation of extracts and determination of

Photosynthetic performance, ion alter in root and antioxidant enzymes: For the analysis of
leaf and also help to clarify Pb effect on antioxidant antioxidant enzyme, 1 g fresh tissue from fourth
enzymes in tolerances mechanisms in root and leaf leaves and the roots was homojenized in 5mL cold
of Camarosa cultivars (Strawberry) in increasing Pb 0.1M Na-phosphate, 0.5 mM Na-EDTA and 1mM
applications. ascorbic acid (pH7.5). Samples were centrifuged
(18.000 g; 4ºC for 30 min). Then Catalase activity
was determined immediately and the supernatant
MATERIALS AND METHODS was stored at -20ºC until determined for SOD.
SOD activity was measured by monitoring the
Plant materials and pot experiment: The inhibition of nitroblue tetrazolioum (NBT)
experiment was carried out in the green house of reduction at 560 nm as reported by Giannopolitis
<]QF<ÕO University during growing period and Ries [19]. Briefly, the reaction mixture
(from middle May to end of July). The experiment contained phosphate buffer (50 mM; pH 7), Na-
was conducted frigo plant of Strawberry (Fragaria x ('7$ P0 ULERIODYLQ ȝ0 PHWKLRQLQH
ananassa cv. Camarosa) and pot experiment. Four (13 mM) and enzyme extract (0.1-0.2 ml).Reaction
frigo plant was planted into every pots was carried out in test tubes at 25°C under
(72x20x17cm) that were filled peat (4 kg). Initial fluorescent lamp (40 W) with irradiance of 75
stage of grown, plants were fed by adding nutrient ȝPR/ P-2 s-1. The reaction was allowed to run for
solution to the pots. The nutrition solutions 10 min and stopped by switching the light off.
contained N 200, K 208, P 37, Ca 167, Mg 49, Fe Blanks and controls were run similarly but without
1.53, Mn 1.16, B 0.46 Zn 0.09, Cu 0.03 and Mo irradiation and enzyme, respectively. Under the
0.02 mg/L [16]. Flower buds were cut of early stage experimental condition, the initial rate of reaction,
RISODQW¶VJURZWK$IWHUWKHSODQWVKDGIRXURUILYH as measured by the difference in increase of
leaves about 4 weeks, Lead applications were absorbance at 560 nm in the presence and absence
started. Lead was added to pots at concentration of of extract, was proportional to the amount of
0, 20, 60 and 80 mg kg-1 in the form of Pb(NO3)2 enzyme.
four equal times with watering during growth CAT activity was determined using the
period. In harvest, 12 plants were harvested to modified Aebi [20] method, by measurement the
every application, plants were sectioned into leaves decrease in absorbance at 240 nm for 2 min, in a
and roots and then this sections were stored -80ºC solution containing H2O2 (10 mM) in phosphate
until antioxidant analysis, also for macro-micro buffer (50 mM; pH 7.0). Enzyme activity was
analysis, fresh root and leafs dried in a oven (80ºC) defined as the consumption of 1 μmoL H2O2 per
and dried parts were ground and stored. min and mL using a molar absorptivity of 39.4mM-1
Chlorophyll determination: Chlorophyll a and cm-1.
chlorophyll b were extracted by using Lichtentaler APX activity was assayed according to the
formula Fresh strawberry leaves (0.5g) were method of Nakano and Asada [21] by recording the
extracted in 80% acetone and determined by decrease in ascorbate content at 290 nm as
spectrophotometrically [17]. ascorbate was oxidized. . The reaction mixture
Lipid peroxidation content: The level of lipid contained potassium phosphate buffer (pH 7.0; 50
peroxidation was determined as a malondialdehyde mM), ascorbic acid (5mM), EDTA (0.1 mM), H 2O2
(MDA) content, following the method of Heath and (0.1mM) and diluted enzyme (0.1 ml) in a total 3.0
Packer [18]. A leaf sample (0.5g) was homogenized ml. The reaction was started with the addition of
in trichloro acetic acid, TCA (10 ml; 0.1%). The H2O2 and absorbance was recorded at 290 nm
homogenate was centrifuged (15 000 g; 5 min) and spectrophotometer for 1 min.
supernatant was collected. To aliquot (1.0 ml) of Macronutrient and micronutrient
the supernatant 4 ml of 0.5% thiobarbituricacid determination: In dried leaves and roots, Pb 2+ and
(TBA) in TCA (20%) was added. The mixture was others nutrient elements concentrations were
heated at 95ºC for half an hour and then quickly analyzed by an atomic absorption
cooled in an ice bath. After centrifugation (10.000 spectrophotometer (Varian Techtron Model AAS
g; 10 min), the absorbance of the supernatant was 1000, Varian Associates, Palo Alto, CA). The
recorded at 532 nm. The value for non specific samples which were digested in an acid solution
absorption at 600 nm was substracted. The MDA (HCL 3%) were passed through the ASS system
content was calculated by its extinction coefficient using different lamps and calibrated with related
of 155 mM-1cm-1 and expressed as nmol MDA per minerals in different concentrations for different
gram fresh weight. macronutrients.
1126


Statistical analysis. The experiment was (strawberry) cultivars. Pb applications reduced

designed as a complete random block design and all chlorophyll a to 7, 14 and 23% and Chlorophyll b
measurements were replicated four times. The to 2, 12 and 16% in comparison to control
statistical analysis of the data was performed using respectively (Figure 1). Chlorophyll a content was
SPSS (22.0) program. The results were statistically found higher than chlorophyll b content at control
evaluated by ANOVA using the Duncan test for and end of Pb applications. At higher applications
significant differences between means (p<0.05). (80 mg kg-1), decrease in chlorophyll a was found
Error bars in graphs shows ±standard error. higher than chlorophyll b.
Several researchers found decrease in
chlorophyll a, b and total chlorophyll content under
RESULTS AND DISCUSSIONS 3E DSSOLFDWLRQV <ÕOPD] HW DO >@ UHSRUWHG WKDW
chlorophyll a and b in eggplant seedlings decreased
Effect of Pb on chlorophyll and Pb by about 20.8-21.9% at 75 mg L-1 Pb application.
accumulation in Strawberry. The chlorophyll a Significant chlorophyll decrease under Pb stress
and chlorophyll b contents showed significant was reported in other plant species including Wheat
decrease in strawberry plant under Pb stress and 7ULWLFXPDHVWLYÕP DQG 6SLQDFK 6SLQDFLDROHUDFHD
chlorophyll a and b contents indicated regularly a [8], Salviniacuculatta [23], peanut seedling [11] and
decrease. Higher levels of Pb application severely maize (Zea mays L) [24].
decreased chlorophyll a and b contents of Camarosa
30 Chlorphyll a Chlorphyll b
a
25 ab
bc
c
20
mg g-1 FW
a a
15 b b
10
0
Control 20 60 80
Pb (mg kg-1 )

FIGURE 1
Changes of Chlorophyll contents exposed to different Pb applications in leaf. Same letters are not
VLJQLILFDQWO\GLIIHUHQWDFFRUGLQJWR'XQFDQWHVWS
Pb content in roots and leaves of Camarosa accumulation in roots and leaves increased with
cultivars increased with increasing Pb applications. exposure to excess lead in tomato plant. Other
Figure 2 Shows changes in the ratio of Pb studies have also determined that Pb accumulation
accumulation in roots and leaves. Results showed in root higher than in shoot and leaf. Our results are
Pb accumulated mainly in root approximately two LQ DFFRUGDQFH ZLWK WKRVH <ÕOPD] HW DO >@ ZKR
times higher than in leaf and somewhat of Pb found the highest Pb accumulation in roots of
transported from root to leaf. Excessive Pb eggplant 6.3 fold higher than shoots and 4.4. fold
applications significant accumulation noted in Pb higher than leaf. Similary, Dahmani et al., [26]
level. Pb applications caused to increase from 0.79, reported that the highest Pb concentration was
0.93 to 1.09 mg kg-1 Pb accumulation in root and measured in roots of Armeria maritime ssp. 10 fold
0.62, 0.70 to 0.78 mg kg-1 Pb accumulation in leaf higher than Pb concentration in shoots and 88 times
at Pb applications respectively (Figure 2). fold than leaves.
This data are in agreement with the findings of
previous study. Akinci et al., [25], observed that Pb
1127


1,4
Root Leaf a
1,2
Pb content (mg kg-1 DW)

ab
1
b
0,8
0,6 ab a
c ab
0,4
0,2 b
0
Control 20 60 80
Pb (mg kg-1 )

FIGURE 2
Lead accumulation in strawberry plant exposed to different Pb applications. Same letters are not
VLJQLILFDQWO\GLIIHUHQWDFFRUGLQJWR'XQFDQWHVWS
Effects of Pb on MDA content. Increasing MDA content in leaves was found higher about 2
MDA content is an indicator of free radicals times than root. When compared to control, MDA
generation, when plants expose to excess heavy content was increased gradually from 16, 18 to 27%
metals. It was suggested that Pb was responsible for in roots and in leaves from 2, 8 to 21% with
the generation of free radicals which leading to increasing Pb applications from control to 80 mg
membrane damage and lipid peroxidation. kg-1 (Figure 3). Reddy et al., [28] observed similar
Generally, first response of plants to heavy metal changes in both horsegram and bengalgram that
stress is to generation of free radicals. MDA MDA content was increased in both roots and
increasing was due to more lipid peroxidation that leaves of horsegram and bengalgram the rising ratio
was provoked by heavy metal stress. When plant from 2.3 to 3.5 times at 800 ppm Pb in both plants
exposed to stress condition, plants produce compared to control. Similar, increases in MDA
malondialdehyde (MDA) and MDA production is content have been reported by Wang et al., [29] in
an indicator which demonstrate the degree of the leaf of vallisnerianatans plant when exposed to
injuries in plant occurred [27]. Pb stress. Britto et al., [13] also declared that lead
This study shows that an increase in lead increased MDA content in capsicum annum and
applications lead to generation of lipid peroxides MDA content increased from 009 to 2.46 mg g-1
content in both roots and leaves of strawberry plant. FW on 30th day at 500 ppm Pb applications.
20 Leaf Root a
18 ab
16 b
MDA (nmol g-1) FW)
b
14
12
10
8 a a
6 a a
4
2
0
Control 20 60 80
Pb (mg kg-1)
FIGURE 3
Changes in malondialdehyde content in strawberry plant exposed to different Pb applications. Same
letters are not significantly different according to Duncan test S
1128


Effect of Pb on antioxidant en yme activity. in the activity of SOD in both roots and leaves of
Superoxide dismutase (SOD) activity was strawberry. SOD activity in both roots and leaves
determined to be statistically significant different in by stages increased from 13, 44 to 60% in roots and
booth roots and leaves of strawberry (Figure 4). Pb from 28, 55 to 64% in leaves respectively compare
applications caused significant and regular increase with control.
180 a
160 Leaf Root a
SOD (U mL-1 g-1 FW)
140 b
120 c a
ab
100
ab
80 b
60
40
20
0
Control 20 60 80
Pb (mg kg-1)
FIGURE 4
SOD activity in strawberry plant exposed to different Pb applications. Same letters are not
VLJQLILFDQWO\GLIIHUHQWDFFRUGLQJWR'XQFDQWHVWS.
Roots and leaves catalase activities increased applications enhanced CAT activity about 13, 44
LQ UHVSRQVH WR 3E DSSOLFDWLRQV øQFUHDVLQJ 3E and 60% in root and 28, 55 and 64% in leaves
applications were ensure to regularly increase CAT respectively as compared with control (Figure 5).
activity in roots and leaves. Increasing Pb
0,6
Leaf Root a
0,5
a
CAT (mmol g-1 FW)
0,4 a
a
0,3 ab
b b
0,2
c
0,1
0
Control 20 60 80
Pb (mg kg-1)
FIGURE 5
CAT activity in strawberry plant exposed to different Pb applications. Same letters are not
significantly different DFFRUGLQJWR'XQFDQWHVWS
APX results are shown in figure 6. For respectively. But APX activity in leaves was
Camarosa (Strawberry) cultivars exposed to monitored decline about 86% at 20 mg kg-1Pb
different Pb application. Increasing in APX activity application, whereas APX activity was increased to
belongs to Pb application was found significant. about 68 and 166% respectively treatment with 60
APX activity was monitored regularly increased by and 80 mg kg-1Pb as compare with control.
2.7, 4.4 and 2.9 times in root compared with control
1129


10 b a
Leaf Root
APX (nmol min-1g-1 FW)

8
a
6
b b
4 b
b
2 c
0
Control 20 60 80
Pb (mg kg-1)
FIGURE 6
APX activity in strawberry plant exposed to different Pb applications. Same letters are not
VLJQLILFDQWO\GLIIHUHQWDFFRUGLQJWR'XQFDQWHVWS.
Biotic and abiotic stresses stimulate Camarosa cultivars. Macro ion accumulation was
production of reactive oxygen species (ROS), that found higher than micro ion accumulation in both
cause severe oxidative stress to DNA, proteins and roots and leaves. Contrarily, Ca, Fe, Cu and Zn
lipids damage and eventually causing the death of contents in roots was determined higher than in
plant. Toxicity of ROS can be alleviated by leaves, K, Mg and Mn contents in leaves was
antioxidant defense system through ROS determined higher than in roots of strawberry. Ca
scavenging enzymes (SOD; CAT, APX) and non- and Mg contents in both roots and leaves showed
enzymatic (ascorbic acid, glutathione, phenolic similar increasing pattern with increasing Pb
compounds) antioxidant enzyme. Our results show applications. Ca and Mg concentrations increased
that higher Pb applications cause significant up to 10.8% (3291.5 to 4757.4 mg kg-1) and 32.3%
increase of antioxidant enzymes activity (SOD; (1744.0 to 18986.9 mg kg-1) from 0 to 80 mg kg-1 in
CAT, APX) in both roots and leaves of strawberry. root and 46.6% (11842.5 to 17359.2 mg kg-1) and
These results were in agreement in with early 40.7% (4779.5 to 6728.4 mg kg-1) from 0 to 80 mg
reports on other plants like, Capsicum annum [13], kg-1 in leaf respectively. Zn contents in roots and
Vallisnerianatans [29]. Reddy et al., [28] who leaves of Camarosa cultivars was decreased
observed serious increasing in antioxidant enzyme depending on Pb applications. This decrease in
activity with increasing concentration and duration roots was clearer and it decreased 78.9% (206.6 to
of Pb stress that in both roots and leaves of 43.86 mg kg-1), but in leaves decrease was 9.7%
horsegram and Bengal gram. They reported that (9.34 to 2.19 mg kg-1) when compared with control.
SOD and CAT enzymes activities were increased The concentrations of K, Fe, Mn and Cu in roots
more than two times and APX enzyme activity was were increased with increasing Pb applications.
increased 2-3 fold in roots and leaves of both the Unlike the roots, K, Fe, Mn and Cu concentrations
plants at 800 ppm Pb applications compare to in leaves were decreased compare to control. When
control. Wang et all., [29] also who reported that a Pb applications were increased from 0 to 80 mg kg-1
significant increasing of SOD, CAT and APX in study, concentrations of K, Fe, Mn and Cu
activity was observed Vallisnerianatansin leaves increased by 44.5, 164.8, 45.5 and 5.9 % in roots
treatments with 75 μM Pb. Similarly, Malecka, et and the concentrations were decreased up to 10.7,
al., [12] reported that Pb increased by about 17% 17.3, 9.1 and 12.4% in leaves.
SOD enzyme activity and about 33% CAT enzyme Some elements such as K, P, Ca, Mg, Mn, Cu,
activity in roots of pea compare to control. Liang, et Zn and Fe are necessary for plants and these
al., [30] also reported a negative effect about 62% elements contribute to biosynthesis of many
(SOD) and 67% (CAT) in leaves of Jatropha enzymatic activities [31, 32]. According to Sengar
seedlings at 500 mg kg-1 Pb applications compared et al., [33] there is a antagonistic effect among
to control. nutrient uptake and heavy metal stress in plants and
changes in mineral nutrition may occur from
Effects of Pb on ion accumulation in blockage of roots absorption, a decrease in
Strawberry. As shown in Table 1. Increasing of Pb translocation processes, or by competition between
application affected the accumulation of mineral ions, impairing critical physiological processes [4].
ions in both roots and leaves of strawberry Many researchers decelerated that Pb have a
1130


negative effect of nutrition uptake and these effect and calcium concentration (in root) in spinach.
were shown widely in different plant species and Lamhamdi et al., [8] also reported that Pb decreased
cultivars. Kibria et al., [34] found similar results in the uptake of Na, K, Ca, P, Mg, Cu and Zn,
spinach plant. Who reported that Pb decreased Although Mn concentration is increased in wheat
potassium and magnesium concentration in both and spinach. Similar study reported in various plant
roots and shoots but Zinc and iron concentration species increasing Pb applications generally
decreased only in roots with increasing Pb level. On inhibited the uptake of all mineral elements in
the contrary increasing Pb level increased tomato [25], eggplant seedling [22], canola [35] and
manganese concentration (in both roots and shoots), azolacaroliniana- Anabaena [36].
nitrogen and magnesium concentrations (in shoots)
TABLE 1
Effects of Pb applications on macro-micro nutrient elements concentrations in roots and leaves of
strawberry Camarosa cultivar (mg g-1DW).
Pb Applications
Macro Nutrient Element Control 20 60 80
Root 3291.50 b 4521.60 a 3902.98ab 4757.43 a
K Leaf 18655.50 a 18744.09 a 14235.44b 16672.24ab
Root/Leaf 0.17 0.24 0.27 0.28
Root 17144.00a 20610.18a 19299.83a 18986.99a
Ca Leaf 11842.50b 18603.19a 18067.76a 17359.24a
Root/Leaf 1,44 1.10 1.06 1,09
Root 4038.86b 4926.53ab 4325.66b 5344.16a
Mg Leaf 4779.57b 7243.34a 5550.96b 6728.45a
Root/Leaf 0.85 0.69 0.77 0.79
Pb Applications
Micro Nutrient Element Control 20 60 80
Root 980.00c 1739.42b 2416.17ab 2594.69a
Fe Leaf 203.50a 175.99a 174.98a 168.24a
Root/Leaf 4.82 9.88 13.80 15.42
Root 38.75b 57.92a 56.93a 56.41a
Mn Leaf 105.15b 149.47a 96.81b 95.57b
Root/Leaf 0.37 0.38 0.58 0.59
Root 15.49 a 17.71 a 16.61 a 16.40 a
Cu Leaf 10.99a 8.89b 5.75c 9.62ab
Root/Leaf 1.41 1.99 2.88 1.70
Root 206.62a 93.75b 35.49c 43.86c
Zn Leaf 22.10a 19.49a 15.24b 19.96a
Root/Leaf 9.34 4.81 2.32 2.19
Same letters are not significantly GLIIHUHQWDFFRUGLQJWR'XQFDQWHVWS
In conclusion, our research indicated that this enzyme has a key role for chlorophyll
higher levels of Pb could be accumulated in both biosynthesis [37]. Treatments with Pb caused
roots and leaves of strawberry plant. The oxidative stress as namely increased lipid peroxide
accumulation of Pb in roots was higher than leaves content induces the production free radicals. The
because root is the first organ exposed to Pb and defense mechanism of strawberry against Pb
could be expanded to prevent translocation of applications triggered the production of some
higher amount of Pb in shoots. Pb accumulation protective mechanism (antioxidant enzyme) such as
reduced chlorophyll a and chlorophyll b and increased SOD, CAT and APX activities were
proportional decrease of chlorophyll a was found increased with increasing Pb concentrations. In
higher than chlorophyll b with increasing Pb addition, excess Pb applications were seriously
applications from 20 mg kg-1 to 80 mg kg-1. The blocked to nutrition uptake. These results ensure an
decreased chlorophyll content caused reduction of important mark for understanding the change of
JURZWK EHFDXVH RI Į-amino laevulinate chlorophyll content in leaves, accumulation of Pb
dehydrogenase is strongly inhibited by Pb ions and
1131


and defense mechanisms of strawberry plant in both and mineral nutrient accumulation in
roots and leaves. strawberry. Biological Research 48:1-7.
[11] Dogan, M. Akgul, H. Tozlu, I. 2013. Lead
accumulation and toxicity in peanut (Arachis
ACKNOWLEDGMENT hypogaea L.) seedlings. Fresenius Environ.
%XOOí
This study was supported by <X]XQFX <ÕO [12] Malecka, A. Jarmuszkiewicz, W. and
Universty of the head of scientific research (BAP, Tomaszewska, B. 2001. Antioxidative defense
2010-ZF-B015) Van, Turkey to lead stress in subcellular compartments of
pea root cells. Acta Bochimica Polonica,
48:687-698.
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Van-Themaat, EVL. Koornneef, M. and Aarts, CORRESPONDING AUTHOR
MGM. 2006. Large expression differences in
genes for iron and zinc homeostasis, stress
)HUKDG0XUDGR÷OX
response, and lignin biosynthesis distinguish Department of Horticulture
roots of Arabidopsis thaliana and the related Faculty of Agriculture and Natural Sciences
metal hyperaccumulator Thlaspicaerulescens.
Abant Izzet Baysal University
Plant Physiol., 142:1127±1147.
Bolu ± TURKEY
[32] Ali, M.B. Singh, N. Shohael, A.M., Hahn, E.J.
and Paek, K.Y. 2006. Phenolics metabolism
e-mail: muradogluf@ibu.edu.tr
and lignin synthesis in root suspension
1133


THE EFFECTS OF HEAVY METALS ON THE SEED GERMINATION

AND SEEDLING GROWTH OF TWO ENDEMIC VERBASCUM
SPECIES
Gürcan Güleryüz16HUDS.ÕUPÕ]Õ2, Hülya Arslan1, Sevgi Derya1
1
University RI8OXGD÷)DFXOW\RI6FLHQFHDQG$UWV'HSDUWPHQWRI%LRORJ\*|UNOH 16059, Bursa, Turkey
2
8QLYHUVLW\RI8OXGD÷*HPOLN$VÕP.RFDEÕ\ÕN*UDGXDWH9RFDWLRQDO6FKRRO*HPOLN%XUVD7XUNH\
ABSTRACT They can also remediate these habitats by

accumulating heavy metals in their aboveground
In this study, we aimed to investigate the biomass. This process is called phytoremediation
effects of heavy metals (Cd, Cu, Cr, Ni and Zn) on [2] and is based on the use of plants to clean up
seed germination and seedling growth of two heavy metals from the environment. Using native
endemic e a m species, m ie m plant species for heavy metal-contaminated sites
Boiss. and l m i m Boiss. We determined the provides some advantages because they are adapted
root and shoot elongation, seed germination to the local soil conditions [3]. For this reason, it
percentage, mean germination time (MGT), seed is also important to know how the germination of
vigour index (SVI) and metal tolerance index % such plant species is affected by heavy metals.
(MTI). These species have been defined as a However, there is more literature on the effects of
pioneer and dominant species of ruderal plant heavy metal on the germination and seedling
communities in disturbed areas, such as roadsides, growth parameters of crop species, such as en
developed building areas, rubber hips, and mining lina i [4], a l nia t nei [5], a an
DUHDV DW GLIIHUHQW DOWLWXGHV RQ 8OXGD÷ 0RXQWDLQ ati [6], and C mi ati [7]. Reports on
Seed germination was reduced significantly in monitoring the effects of heavy metals on the
l m i m with increased metal concentrations (P germination and seedling growth of native plants
< 0.05). However, only Cd and Cr resulted in are comparatively scarce, although there are some
significantly decreased seed germination in examples such as reports on ni m
m i e m (P < 0.05). MGT was also altered by enta l m [8], native Australian tree species
metal treatments in both species. Although the [3], ae a al a [9], and Brazilian savannah tree
metal treatments resulted in root growth inhibition, species [10].
shoot growth was not inhibited. SVI and MTI were Most of the heavy metals are essential
also negatively affected by treatments. microelements but become toxic at higher
concentrations [11]. Metals are known to affect
seed germination through both toxicity and the
KEYWORDS: inhibition of water uptake [12]. For example, Cd
Heavy metal, e a m l m i m, e a m inhibits water uptake in in m enta l m
m i e m, Seed germination seeds [8]. Copper has been found to allow
germination in eliant ann [13] but inhibits
seedling development and reserve mobilization at
INTRODUCTION higher concentrations in i ia ati a seeds [14].
e a m is the second largest genus among
Environmental pollution is among the high- Turkish flora and includes numerous endemic
priority problems of the world. Many pollutants species. e a m m i e m Boiss. and
such as heavy metals are accumulated in many e a m l m i m Boiss. are two endemic
types of habitats. Land degradation can be caused VSHFLHVJURZLQJRQ8OXGD÷0RXQWDLQ%RWKVSHFLHV
by both human and climate factors. Mining, were observed growing in destroyed areas such as
agricultural activities, and industrial organizations roadsides, picnic areas, and rubbish dumps as one
frequently cause soil contamination by heavy of the dominant pioneer species [15]. Having the
metals [1] and lead to land degradation. advantages of rapid growth, l m i m and
The effects of heavy metals on plants are m i e m are also prominent for their high
specific to plant species [12], and some plant biomass production [16]. This species produces
species can survive in metal-contaminated sites. high biomass, increases the soil organic material,
1134


and contributes to the subsequent re-vegetation Differences among treatment means were tested by
processes in destroyed areas [17]. In previous TurNH\¶V +RQHVW 6LJQLILFDQFH 'LIIHUHQFH +6'
studies, it was reported that l m i m and test. Germination percentages were arcsine
m i e m can be considered bio-indicators and transformed before statistical comparison. All tests
accumulators for certain heavy metals in heavy ZHUHSHUIRUPHGDWWKHVLJQLILFDQFHOHYHORIĮ
metal-contaminated soils [18, 19]. using the Statistica 6.0 [22] software package.
In this study, our aim was to understand the
effects of heavy metals (Cu, Cr, Ni, Cd, Zn) on the
seed germination and early seedling growth RESULTS
characteristics such as germination (%), mean
germination time, root and shoot elongation (mm), In this study, we assessed the effects of a wide
seed vigour index and metal tolerance index (%) of range of heavy metal concentrations on the seed
m i e m and l m i m germination and seedling growth of l m i m
and m i e m. The germination percentage
of m i e m seeds was decreased by Cd
MATERIALS AND METHODS treatment. The value was reduced by 22 %
compared to the control at 250 μM Cd (Table 1).
Seed collection. Seeds of two e a m MGT was increased significantly with increasing
species were collected on September 2011 from Cd concentration. Root length was decreased
8OXGD÷0RXQWDLQ´ N, 13 º 23´ 26´É). fourfold compared to the control, but shoot lengths
The seeds were collected from a single plant of were increased at 250 μM. The germination
each species to reduce the genetic variations. Seeds percentage of l m i m was decreased by 20 %
were maintained for a week at room temperature compared to the control at 250 μM, and MGT
for drying and stored in paper bags. increased significantly with increasing Cd
concentration (Table 2). Root length decreased
Seed germination. The seeds were surface fourfold at 250 μM Cd concentration. Shoot length
sterilized with 1 % NaOCl for 5 minutes and then was increased at higher concentrations and
rinsed with tap water. Standard 9 cm plastic sterile remained consistently higher than the control shoot
Petri dishes were used. The seeds were maintained length. Cd concentrations above 500 μM caused
in Petri dishes (30 seeds per Petri dish and 5 Petri atypical germination for both e a m species.
dishes per treatment) on two layers of sterile filter SVI and MTI (%) were decreased significantly in
paper moistened with 5 ml of test solution or both species upon Cd application (Table, 3, 4). The
distilled water as control, under a 12 h photoperiod lowest SVI and MTI (%) values were found at the
DWDWHPSHUDWXUHRIÛ&LQDJURZWKFKDPEHUIRU highest Cd concentrations.
21 days. The heavy metals were selected based on Zn treatment did not affect the seed
our previous study on the heavy metal germination percentage of m i e m seeds,
contamination of the soil and the plant growth but MGT was increased significantly with Zn
around DPLQHZRUNRQ8OXGD÷0RXQWDLQ[18]. Test concentration (Table 1). Root length decreased
solutions were applied as Heavy metal salts tenfold compared to the control, and shoot length
(CdSO4.8H2O, K2Cr2O7, CuSO4, NiSO4, was slightly increased with Zn application (P<
ZnSO4.7H2O). The seeds were considered 0.05). Zn reduced the germination percentage
germinated once the radicle emerged by significantly by 10 %, at 1000 μM compared to the
approximately 2 mm from the seeds. The numbers control in l m i m seeds (Table 2). The
of germinated seeds were recorded daily. At the end germination percentage of l m i m was 67.2 ±
of the incubation, root and shoot lengths were 2.9 in 100 μM Zn treated seeds and was higher than
measured, and means were calculated. Seed in the control. The lowest MGT (2.00 ± 0.5) was
germination percentage and Mean Germination also found at 100 μM Zn concentration. While the
Time (MGT) were also calculated. Seed highest root length was measured in 100 μM Zn
germination percentages were arcsine transformed treated seedlings, the highest shoot length was
before statistical comparisons. The Seedling Vigor measured at 500 μM Zn. Atypical germination was
Index (SVI) was calculated according to Abdul- observed in seedlings treated with Zn above 1000
Baki and Anderson (1973) [20]. The metal μM for both e a m species.
tolerance index was calculated using the formula of
Turner and Marshal (1972) [21].
Statistical Analyses. The data were analysed

using one-way analysis of variance (ANOVA).
1135


TABLE 1
Root lengths (mm), shoot lengths (mm), final germination percentage and mean germination times (MGT)
of 21 days incubated seedlings of . bo b ci eru under heavy metal concentrations. [Values are means
followed by standard error (n = 5); the values followed by the same letter were not statistically significant
DWWKHĮ OHYHO@na: not applicable.
Shoot length MGT

Element Metal Concentration Root length (mm) Germination (%)
(mm) (day)
ȝ0FRQWURO 13.0a ± 2.6 2.5ab ± 0.1 84.0a ± 0.0 3.3c ± 0.3
ȝ0 11.2a ± 3.9 2.3ab ± 0.1 71.4ab ± 9.8 6.2ab ± 0.7
ȝ0 8.4ab ± 3.2 2.2b ± 0.3 73.6ab ± 8.6 4.1b ± 0.4
ȝ0 9.6a ± 1.2 2.8ab ± 0.3 79.6a ± 4.3 6.8a ± 0.2
Cd2+ ȝ0 4.2b ± 1.4 3.0a ± 0.4 66.0b ± 7.8 6.2ab ± 0.4
ȝ0 na na atypical na
ȝ0FRQWURO 13.0a ± 2.6 2.5b ± 0.1 84.0a ± 0.0 3.3ab ± 0.3
ȝ0 8.7b ± 1.4 3.2ab ± 0.4 84.0a ± 15.2 3.3ab ± 0.3
ȝ0 2.4c ± 0.6 2.9b ± 0.8 82.0a ± 4.5 3.6a ± 0.3
Cu2+
ȝ0 1.9c ± 0.1 4.4a ± 1.8 84.0a ± 0.0 3.0b ± 0.2
ȝ0FRQWURO 13.0a ± 2.6 2.5b ± 0.1 84.0a ± 0.0 3.3ab ± 0.2
ȝ0 12.6a ± 2.3 2.5b ± 0.2 80.0ab ± 2.9 4.6b ± 0.4
ȝ0 10.7ab ± 0.0 2.8b ± 0.2 80.0ab ± 14.5 4.5b ± 0.1
ȝ0 12.5a ± 1.6 2.5b ± 0.2 84.0a ± 0.0 5.0ab ± 0.4
Cr2+
ȝ0 7.8 b ± 1.4 2.8b ± 0.1 80.0ab ± 6.4 5.6a ± 0.5
ȝ0 6.2bc± 1.4 2.7b ± 0.3 81.0ab ± 4.5 5.6a ± 0.3
ȝ0 3.3c ± 0.8 3.6a ± 0.6 80.0ab ± 9.6 5.3ab ± 0.7
ȝ0FRQWURO 13.0a ± 2.6 2.5a ± 0.1 84.0a ± 0.0 3.3b± 0.3
ȝ0 8.7b ± 1.4 2.3a ± 0.5 82.8a ± 2.7 5.4a ± 0.4
ȝ0 4.2c ± 0.3 2.5a ± 0.6 84.0a ± 0.0 3.6b ± 0.9
Ni2+
ȝ0FRQWURO 13.0ab ± 2.6 2.5a ± 0.1 84.0a ± 0.0 3.3b ± 0.3
ȝ0 16.7a ± 5.1 1.4b ± 0.7 84.0a ± 6.4 4.1ab ± 0.6
ȝ0 8.9b ± 4.3 2.5a ± 0.8 84.0a ± 0.0 5.1a ± 1.1
Zn2+
ȝ0 1.2c ± 0.2 2.9a ± 1.4 80.8a ± 7.2 5.3a ± 1.0
Zn caused significant decreases in the SVI and MTI length was not affected by Ni (P >0.05). However,
(%) of both species, and 1000 μM Zn caused more 250 μM Ni treatment decreased the germination
than a 90 % decrease in SVI and MTI (%) values percentage of l m i m seeds slightly (7 %)
in both species, a greater effect than was shown by compared to the control. MGT was also slightly
the other metals (Table 3, 4). decreased by this treatment (Table 2). Root length
Similarly to the Zn treatments, Ni treatment decreased sharply and significantly with increasing
did not affect seed germination percentage, but Ni concentrations, and shoot length increased. Ni
MGT was increased significantly in treatment caused significant decreases in the SVI
m i e m seeds (Table 1). The root length and MTI (%) values in both e a m species in a
decreased by more than threefold, whereas shoot dose-dependent manner (Table 3, 4).
1136


TABLE 2
Root lengths (mm), shoot lengths (mm), final germination percentage and mean germination times (MGT)
of 21 days incubated seedlings of . ol icu under heavy metal concentrations. [Values are means
followed by standard error (n = 5); the values followed by the same letter were not statistically significant
DWWKHĮ OHYHO@ na: not applicable.
Shoot length MGT

Element Metal Concentration Root length (mm) Germination (%)
(mm) (day)
ȝ0FRQWURO 13.5a ± 1.7 2.7c ± 0.6 66.0a ± 2.8 3.1c ± 0.1
ȝ0 3.6b ± 1.4 4.6a ± 0.5 59.8b ± 3.5 2.8d ± 0.3
ȝ0 4.5b ± 0.7 4.0b ± 0.3 61.8ab ± 5.1 3.4c ± 0.3
ȝ0 3.4b ± 1.3 3.0c ± 0.4 54.8c ± 6.4 3.6b ± 0.3
Cd2+
ȝ0 3.4b ± 1.2 2.9c ± 0.3 53.2c ± 6.7 4.0a ± 0.4
ȝ0FRQWURO 13.5a ± 1.7 2.7b ± 0.6 66.0a ± 2.8 3.1b ± 0.1
ȝ0 5.8b ± 1.3 6.0a ± 0.8 58.4b ± 4.9 3.2b ± 0.4
ȝ0 2.0c ± 0.8 6.0a ± 1.4 57.2b ± 5.1 4.0a ± 0.6
Cu2+
ȝ0FRQWURO 13.5a ± 1.7 2.7c ± 0.6 66.0a ± 2.8 3.1b ± 0.1
ȝ0 4.9b ± 1.6 5.9a ± 0.8 62.0b ± 2.0 2.7c ± 0.2
ȝ0 3.8b ± 0.5 5.3b ± 0.3 63.6ab ± 3.3 3.2b ± 0.3
ȝ0 2.4c ± 0.8 4.9b ± 0.7 63.6ab ± 3.3 3.5a ± 0.3
Cr2+
ȝ0FRQWURO 13.5a ± 1.7 2.7b ± 0.6 66.0a ± 2.8 3.1a ± 0.1
ȝ0 3.6b ± 0.7 3.2a ± 0.3 64.6a ± 2.2 2.6c ± 0.2
ȝ0 1.7c ± 0.6 2.8ab ± 0.2 61.8b ± 1.6 2.9b ± 0.3
Ni2+
ȝ0FRQWURO 13.5a ± 1.7 2.7b ± 0.6 66.0a ± 2.8 3.1a ± 0.1
ȝ0 5.0b ± 1.6 4.6b ± 0.5 67.2a ± 2.9 2.0b ± 0.5
500 ȝ0 3.1c ± 0.4 5.5a ± 0.3 58.4b ± 4.5 3.1a ± 0.4
Zn2+
ȝ0 1.1d ± 0.2 4.2b ± 0.8 59.0b ± 3.1 2.9a ± 0.1
1137


Cu did not significantly affect germination shoot length was increased by Cu treatments. Cu
percentage, but MGT was increased slightly in concentrations above 250 μM caused atypical
m i e m seeds (Table 1). Root length germination in l m i m. Atypical germination
decreased by more than sixfold, while shoot length was also observed in Cu treatments above 500 μM
increased. The germination percentage of in m i e m. SVI and MTI (%) decreased
l m i m was significantly decreased by the Cu significantly with increased Cu concentrations in
treatments (Table 2). The highest germination both Verbascum species (Table 3, 4).
percentage was observed in the control seeds (66.00 Seed germination percentage was decreased in
± 2.8), while the lowest was observed in 250 μM 750 μM Cr treated seeds compared to control seeds,
Cu treated seeds (57.2 ± 5.1). MGT was increased and MGT increased with Cr treatment in
by Cu treatment in this species. In contrast to roots, m i e m seeds (Table 1).
TABLE 3
SVI and MTI (%) values of 21 days incubated seedlings of . bo b ci eru under heavy metal
concentrations.
[Values are means followed by standard error (n = 5); the values followed by the same letter were not
VWDWLVWLFDOO\VLJQLILFDQWDWWKHĮ OHYHO@.
MTI
Element Metal Concentration SVI
%
ȝ0FRQWURO 1178.2a ± 70.3 100.0a ± 0.0
ȝ0 932.6ab ± 58.8 78.8ab ± 2.1
Cd2+ ȝ0 660.2b ± 84.1 64.0b ± 7.8
ȝ0 869.2ab ± 140.9 87.5ab ± 14.4

ȝ0 277.8c ± 51.6 31.8c ± 3.7
ȝ0FRQWURO 1178.2a ± 70.3 100.0a ± 0.0
ȝ0 708.4b ± 8.63 63.5b ± 1.3
Cu2+
ȝ0 188.5c ± 29.1 18.8c ± 3.4
ȝ0 165.1c ± 4.5 16.3c ± 2.0
ȝ0FRQWURO 1178.2a ± 70.3 100.0ab ± 0.0
50 ȝ0 1064.5ab ± 75.8 111.5a ± 18.6
ȝ0 828.8bc ± 45.2 78.3ab ± 4.11
Cr2+ ȝ0 1010.4ab ± 61.7 81.0ab ± 8.1
ȝ0 623.1cd ± 67.4 65.8bc ± 11.5
ȝ0 534.6d ± 59.1 55.5bc ± 8.8
ȝ0 289.8d ± 14.2 29.8c ± 5.5
ȝ0FRQWURO 1178.2a ± 70.3 100.0a ± 0.0
Ni2+ ȝ0 764.4b ± 53.2 72b ± 4.6
ȝ0 361.2 c± 11.4 34.8c ± 4.6
ȝ0FRQWURO 1178.2a ± 70.3 100.0a ± 0.0
ȝ0 1543.5a ± 82.9 112.8a ± 23.3
Zn2+
504 504.0b ± 35.4 51.5b ± 12.4
ȝ0 105.0b ± 4.4 8.2c ± 0.65
1138


TABLE 4
SVI and MTI (%) values of 21 days incubated seedlings of . ol icu under heavy metal
concentrations. [Values are means followed by standard error (n = 5); the values followed by the same
letter were not statistically significant at the Į OHYHO@
MTI
Element Metal Concentration SVI
%
ȝ0FRQWURO 890.8a ± 18.1 100.0a ± 0.0

ȝ0 217.7bc ± 19.5 27.2bc ± 2.7
Cd2+ ȝ0 280.6b ± 13.1 32.2b ± 1.2
ȝ0 187.8c ± 15.6 24.4c ± 1.6
2ȝ0 177.1c ± 11.1 24.6c ± 2.2
ȝ0FRQWURO 890.8a ± 18.1 100.0a ± 0.0
Cu2+ ȝ0 339.8b ± 19.3 42.4b ± 2.9
ȝ0 157.4c ± 29. 4 13.3c ± 0.9
ȝ0FRQWURO 890.8a ± 18.1 100.0a ± 0.0
ȝ0 304.9b ± 22.2 36.4b ± 2.9
Cr2+
ȝ0 282.6b ±8.61 28.2c ± 1.4
ȝ0 150.8c ±11.8 16.8d ± 1.4
0 ȝ0FRQWURO 890.8a ± 18.1 100.0a ± 0.0
Ni2+ ȝ0 239.5b ±12.4 27.0b ± 1.9
ȝ0 104.1c ± 8.2 12.4c ± 1.2
ȝ0FRQWURO 890.8a ± 18.1 100.0a ± 0.0
ȝ0 328.6b ± 29.4 37.6b ± 3.5
Zn2+
ȝ0 180.9c ±4.3 22.6c ± 0.6
ȝ0 62.4d ± 19.1 7.6d ± 2.4

Root length was decreased by threefold, and Cu is an essential element in plant nutrition,
shoot length was unchanged. The Cr treatments but it can be toxic at higher concentrations
resulted in a decreased germination percentage in produced by anthropogenic and industrial inputs
l m i m seeds (Table 2). However, MGT [23]; Under high Cu concentrations in the growing
increased with increasing doses. Root length medium, Cu in the roots can increase
decreased sharply with doses, and shoot length proportionally, but the content in shoots may not
increased (P < 0.005). Treatment with 100 μM Cr [23]. The germination percentage was not affected
decreased SVI and MTI (%) by 83 % in by Cu treatments in m i e m but was
l m i m, and 750 μM Cr reduced SVI and MTI decreased significantly in l m i m (Table 2).
(%) by 75 % and 71 %, respectively, in This result suggests the sensitivity of l m i m
m i e m (P< 0.05). (Table 3, 4). to Cu. The increased MGT of V. olympicum seeds
also support this conclusion (Table 3). Cu has been
previously reported to affect the germination
DISCUSSION percentage of certain species. In alfalfa, Cu reduced
germination at concentrations higher than 300 μM
In this study, we examined the germination [24], in wheat at concentrations higher than 80 μM
and seedling growth responses of two e a m [25] and in rice at concentrations higher than 1000
species to different heavy metals (Cd, Cr, Cu, Ni μM [26]. As reported by Marquez-Garcia et al.
and Zn). Except for Cr and Zn, metal (2013) [27], in the two salt marsh species t i lex
concentrations above 500 μM resulted in atypical alim and ali nia am i ima, germination
(abnormal) germination in l m i m and was not affected by Cu, but root elongation was
m i e m plants. We observed delayed affected. Root length was reduced in both species,
germination rather than germination inhibition as a while shoot length was not affected. Ouzouniudou
result of heavy metal treatments in general. et al. (1994) [28] investigated the Cu tolerance in
resistant and sensitive populations of Min a tia
i ta seedlings, finding that the resistant
1139


population showed increased root growth up to 80 certain concentrations of heavy metals, seed
μM Cu, but in the sensitive population, root growth germination was not normal and
stopped above 0.5 μM Cu. hypocotyls/radicles did not grow. They also
Zinc is the second most abundant transition suggested that these heavy metal concentrations
metal in living organisms after Fe [29]. The could be considered as indicators of the plant
application of Zn to Brazilian native savannah tree sensitivity threshold. However, the threshold
species seeds decreased germination and increased toxicity level is higher in hyperaccumulator or
germination time at all levels in tree species [10]. metal-tolerant species than in non-tolerant ones.
Zn treatments above 1000 μM resulted in atypical Seed germination and seedling growth are
germination in both species. The seed germination very important stages in plant life that are affected
percentage was not significantly affected in by environmental changes. There is a general trend
m i e m, while MGT was significantly in the literature indicating that the effects of heavy
changed by Zn application in both species. metals depend on intra- and interspecific variations
Cadmium is not an essential element, and [12, 30]. In this study, e a m species differed
some species are known to keep Cd at the testa and in their germination response to metals, and
not permit it to enter the endosperm and embryonic l m i m seemed to be more sensitive to applied
tissues [18, 31]. This situation was not valid for heavy metals. MGT increased in proportion to
the examined e a m species, as the increasing metal concentrations. While root growth
germination percentage was significantly decreased was more susceptible, shoot growth was not
in both species upon Cd treatment (Table 1, 2). affected in most cases. The inhibition of root
Rice was found to be affected by Cu and Cd mostly JURZWK LV WKH ILUVW VLJQ RI D SODQW¶V UHVSRQVH WR
in terms of the root growth, followed by shoot heavy metals, and some plants have resistance
growth and germination, which was affected only at mechanisms to exclude heavy metals from shoots
higher concentrations [28]. MGT was found to [12, 29, 37]. We suggest that heavy metal
increase by Cd applications in both species. Cd was concentrations over 500 μM could be a threshold
found to reduce the germination speed in Ce ia value for effects on the seed germination and
l e a and na enant e a e e ina [10]. seedling growth of l m i m and
Ni is an essential element for the activities of m i e m. Our results could constitute basic
certain enzymes, such as urease, and is involved in knowledge to support more advanced studies on the
the functions of some other proteins [29]. Similar heavy metal tolerance of these two endemic
to Cu and Zn, Ni treatments did not affect seed e a m species.
germination in m i e m but did affect Sevgi Derya presented e e e t ea
l m i m. Ni has been reported to be toxic for metal n ee e minati n an ee lin t
germination at over 30 μM in Lolium perenne l m i m as her MSc Thesis in the Institute of
[32], 100 μM in radish [33], 300 μM in alfalfa Science, University RI 8OXGD÷ %XUVD 7XUNH\ in
[24], and 250 μM in ali nia am i ima [27]. 2012.
Only root growth was reduced in both e a m
species (Table 2, 3).
Chromium is not an essential element and has REFERENCES
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1140


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156.
1142


EVALUATION OF COMMON BEAN (PHASEOLUS

VULGARIS L.) COLLECTION FOR AGROMORPHOLOGICAL
AND SEED MINERAL CONCENTRATIONS
Faruk Toklu1, Clarice J. Coyne2, Safiye Asikli32÷X]KDQ$\GLQ3, Tolga Karaköy4, Hakan Özkan1
1
Department of Field Crops, Faculty of Agriculture, University of Cukurova, 01330, Balcali-Adana, Turkey
2
Washington State University, Department of Crop and Soil Science/USDA lab Johnson Hall 35, Pullman, WA
3
Department of Soil Science and Plant Nutrition, Faculty of Agriculture, University of Cukurova, 01330, Balcali-Adana, Turkey
4
Vocational School of Sivas, Cumhuriyet University, Sivas, Turkey
A collection of common bean comprising Common bean (Phaseolus vulgaris L.) is a

totally 223 accessions of which 176 genotypes from self-pollinating, diploid (2n=2x=22) food legume
the USDA, 37 common bean landraces and 10 crop originated in Latin America and has two
commercial cultivars from Turkey, evaluated for primary centers of origin in the Mesoamerican and
several agromorphological plant characters and Andean regions [1-3]. The two major gene pools
mineral concentrations in seeds. There were wide (Andean and Mesoamerican) have been identified
range of variations for the investigated based on morphological and agronomical traits,
agromorphological characters and mineral phaseolin seed protein, isozymes, molecular
concentrations. The majority of the common bean markers, and adaptation traits [4-13,3,14]. The
genotypes had indeterminate climber growth habit genus, Phaseolus contains five domesticated
(35% and 69% for collection and cultivars species, common bean (P. vulgaris L.), lima bean
respectively), with white flower color (46% for (P. lunatus L.), tepary bean (P. acutifolius A. Gray)
collection 100% for cultivars). The majority of the and year bean (P. polyanthus Greenman) with
USDA collection and Turkish landraces had very distinct adaptations and reproductive systems [15].
small seed size (52%) while common bean cultivars Currently common bean is the most widely
had medium and average seed size (38% cultivated grain legume in the world with its 29
respectively). The results showed that several million hectares growing area and 22 million tonnes
genotypes such as Pinck, Feijao Pico de Oro, production. In Turkey common bean is the third
Estrada Rosado (Colombian genotypes), most important food legume after chickpea and
08KMYOF25, 12KMYOF8 and 12KMYOF17 lentil with 84.7 thousand hectares sowings and 195
(Turkish landraces) found to be promising thousands tons production [16]. Common bean is
genotypes for grain yield and yield components. also a major legume crop with significant
The genotypes G878, G16837, Frijol canario, nutritional importance [17, 48] that provides a good
P8474-A-D (from USDA collection), 12KMYOF25 source of protein in the diet of many people around
landrace and Göynük cultivar (from Turkey) were the world [13]. Common bean plays an important
found to contain high Fe and Oaxana 5-1, role in the nutrition of low-income people in many
Queretaro 7-5, Frijol canario and Negro for high Zn developing countries due to its dietary source of
seed concentrations. The first two principal protein, dietary fiber, carbohydrate and minerals
components (PC1 and PC2) which are the most [18, 19, 14]. The crop is consumed principally for
important components accounteds for 29.78% and its dry (mature) beans, shell beans (seeds at
18.70% of total variation respectively. PC1 is physiological maturity) and green pods [15].
positively correlated to yield components such as Humans require at least 49 nutrients to meet their
number of pods per plant, weight of pods per plant, daily metabolic need and today over three billion
number of seeds per plant, weight of seeds per plant people are faced with both vitamins and essential
and grain yield per plot while PC2 is positively micronutrient elements deficiencies [20, 21]. It is
correlated to the seed Zn, Fe, Cu and Mn reported that 60-RIWKHZRUOG¶VSRSXODWLRQIDFH
concentrations. iron (Fe) deficiency, >30% zinc (Zn) deficiency
[22, 23]. In general, common bean have high Zn
and Fe concentrations as well as a good source of
KEYWORDS: vitamin B complex such as niacin, riboflavin, folic
Common bean, collection, characterization, mineral acid and thiamin [24]. Additionally, it is
concentration
characterized by low glycaemic index (GI) which is
1143


associated with several long term health benefits and 10 commercial cultivars commonly grown in
such as reducing heart disease, cardiovascular Turkey. Collection and registration informations of
disease, diabetes, obesity, some forms of cancer etc these genotypes indicated in supplemental Table 1
[25-27, 19, 14]. Several researchers reported that (Table S1, available online). Each genotype was
mineral malnutrition could be solved by increasing sown by hand in a 3 m long row with a 70 cm row
the consumption of mineral-rich foods called spacing at a 15 cm seed spacing in the row. Several
biofortification [28, 23]. Recently, biofortification agromorphological plant characters were collected
by for the enrichment of staple crops with minerals five randomly selected plants in each row such as
(iron, zinc, vitamin A, etc.) [29, 47], using plant plant height, the number of pods per plant, the
breeding strategies and/or mineral fertilization [23]. weight of pods per plant, the number of seeds per
There is considerable genetic variations reported for plant, the weight of seeds per plant, the 100 seed
mineral concentrations and contents in staple crops weight (computed by the dividing the weight of
such as common bean [49, 50]. The use of plant seeds per plant to number of seeds per plant), grain
genetic resources is one of the most effective way yield per plot (g plot-1), days to flowering (days
to increase grain yield and quality in staple crops from sowing to appearance of 50% flowers), flower
such as common bean. Utilization of the genetic color (1: white, 2: greenish, 3: lilac, 4: white with
resources for achievement of continued genetic lilac edge, 5: white with lilac stripes, 6: dark lilac
variability in plant breeding studies is indicated as with purple outer edge, 7: dark lilac with purplish
key factor for successful crop improvement. Almost spots, 8: carmine red, 9: purple) and growth habit
1 M samples of grain legume genetic resources are (I: determinate bush, II: indeterminate bush, III:
preserved ex-situ genebanks globally and managing indeterminate prostrate, IV: indeterminate climber)
and utilizing such large diversity in germplasm [31].
collections are great challenges to germplasm For the mineral nutrient analysis, 0.3 g seed
curators and crop breeders [19]. Common bean is samples were ground and then digested in
an imortant grain legume also in Turkey for its microwave digester using 2 ml of 35% H2O2 and 5
nutritional value directly human consumption and ml of 65% HNO3. Following the digestions, Zn, Fe,
recently, several research projects have been Cu and Mn were analyzed by an inductively
conducted in Turkey, for collection, evaluation and coupled plasma optical emission spectrometer
utilization of common bean genetic resources [30]. (ICP-OES; Varian-Vista Pro). Reference leaf
Although improved cultivars of common bean are samples from National Institute of Standards and
available, many producers still grow landraces for Technology (Gaithersburg, MD, USA) were used to
marketing in many parts of the world and also in check the related elemental measurements.
Turkey. The objectives of this study were to Simple statistics of the mean, minimum and
characterize of common bean genetic resources for maximum values and the coefficient of variation
adaptation ability, grain yield and yield components were computed using the recorded data with Excel
and trace mineral concentrations in seed so software (Microsoft Office). For the principal
facilitate to more efficient use of common bean component analysis, pairwise correlation
genetic resources for crop improvement. coefficients and scatter diagram were calculated
using JMP statistical software [32].

RESULTS AND DISCUSSIONS
This research was conducted in 2013-14
cropping season at the research field of Vocational The mean values of considered
School of Kozan, Çukurova University at Adana, agromorphological plant characters and mineral
Turkey. The research materials were comprised content of common bean collections and cultivars
totally 223 common bean genotypes of which 176 presented in supplemental Table S1 (Table S1,
genotypes kindly provided from USDA, ARS, available online). The means, ranges and CVs of
WRPIS, Washington State University (WSU), the quantitative and qualitative plant traits and
Regional Plant Introduction Station, 37 were mineral and seed mineral nutrient concentrations
landraces from Turkey (around Erzincan, Sivas, are indicated in Table 1 and Table 2 respectively.
Kozan, Feke, Saimbeyli and Tufanbeyli provinces)
1144


TABLE 1
Mean, range and coefficient of variation (CVs) for agromorphological plant characters and
micronutrient concentration of common bean collection and varieties
Common bean collection Common bean cultivars

Plant characteristic
Mean Range CV(%) Mean Range CV(%)
Days to flowering 50.3±6.0 35.0-60.0 12 43.00±5.0 40.0-54.0 12
Plant height (cm) 61.3±18.7 23.2-137.8 31 48.3±12.3 35.6-75.0 25
Number of pods per plant 19.4±9.1 5.7-60.2 47 20.9±5.9 11.6-32.8 29
Weight of pods per plant (g) 25.4±13.1 4.7-92.0 51 35.4±8.6 24.0-53.0 24
Number of seeds per plant 81.3±44.7 14.7-239.8 55 74.5±44.4 33.4-184.4 60
Weight of seeds per plant (g) 17.2±9.2 2.1-49.1 54 23.6±7.7 12.6-37.2 32
100-seed weight (g) 22.9±10.6 5.7-56.7 46 35.6±8.8 15.8-46.3 25
Grain yield per plot 104.3±77.5 0.8-372.0 74 157.9±69.5 28.4-261.4 44
Zn (mg kg-1) 36.7±6.0 13.3-72.4 16 38.2±4.2 30.5-44.8 11
Fe (mg kg-1) 65.7±14.2 26.1-146.03 22 71.0±16.8 52.1±114.2 24
Cu (mg kg-1) 9.1±1.4 4.3-13.4 16 10.5±1.9 7.5-13.2 19
Mn (mg kg-1) 15.7±2.5 7.1-25.1 16 18.2±3.6 12.4-23.9 20
TABLE 2
Frequency distribution of morphological plant characteristics of common bean collection and cultivars.
Plant Frequencies (%)

Classes
characteristic Collection Cultivars
1: Determinate bush 26 31
2: Indeterminate bush, with erect stems 10 0
Growth habit
3: Indeterminate prostrate, with many lateral guides 29 0
4: Indeterminate climber 35 69
1: White 46 100
3: Lilac 20 0
Standard flower colour
8: Carmine red 6 0
9: Purple 28 0
Very small (<20 g) 52 8

Small (20-30 g) 22.6 8
Medium (30-40 g) 16 38
Seed size
Normal (40-50 g) 9 38
Large (50-60 g) 0.4 8
Very large (>60 g) 0 0
1145


A valuable phenotypic diversity has been respectively. Lower levels of diversity for Fe, Zn,
detected in the common bean genotypes for Cu and Mn were obtained for the common bean
agromorphological plant characters and seed cultivars compared to the collection genotypes
mineral concentrations (Table 1). High CVs were (Table 1).
obtained for the plant height, number of pods per
plant, weight of pods per plant, weight of seeds per Table 2 shows that the frequency distribution
plant, 100 seed weight, grain yield per plot, and Zn of several morphological plant characters of
and Fe concentrations compared to common bean common bean genotypes and cultivars with USDA
cultivars. Greater diversity for the days to flowering core collection and Turkish landraces and cultivars
ranged from 35.0 to 60.0 days with a mean of 50.3 mainly composed of indeterminate climber growth
days. Cauca 38 genotype from Colombia has the habit respectively 35% and 69%. The remaining
longest days to flower whereas Frijol canario and USDA core collection and Turkish landraces
G2595 from Mexico has the shortest days to showed 29% indeterminate prostrate growth with
flowers. Plant height ranged from 23.2 to 137.8 cm many lateral branches, 26% determinate bush habit
with a mean of 61.3 cm. The tallest plant height and 10% indeterminate bush with erect stems. 31%
was recorded for Col.No.23,sel.#10 from Costa of the common bean cultivars showed determinate
Rica and the shortest one for Canario 102 from bush growth habit. USA core collection and
Mexico (Table S1). The number of pods per plant Turkish landraces showed 46% white flower color,
varied from 5.7 to 60.2 from the lowest in genotype 28% purple, 20% lilac and 6% carmine red. All of
Narino 47 from Colombia and the highest in the Turkish common bean cultivars (100%) showed
12KMYOF8 from Turkey, respectively. The weight white flower color. Diversity of the seed size
of pods per plant ranged from 4.7 to 92.0 g with a frequency differed between the USDA core
mean of 25.4 g. The highest weight of pods per collection and Turkish landraces compared to
plant was observed for 08KMYOF13 landrace from Turkish common bean cultivars. While most of the
Turkey. The average number of seeds per plant USDA core collections and landraces (52%) exhibit
ranged from 14.7 to 239.8 for Narino 47 and Pinck very small seed size, most of the common bean
from Colombia respectively. Weight of seeds per cultivars showed medium and normal seed size (38
plant ranged from 2.1 to 49.1 g with a mean of 17.2 and 38% respectively) (Table 2).
g. Feijao Pico de Oro from Colombia has the
highest weight of seeds per plant while P8474-A-D The contribution of the agromorphological
from Peru has the lowest. The highest and lowest characteristics in the principle components (PC)
100 seed weights were recorded for F421 numbered and scatter diagram are shown in Table 3 and
landrace (56.7 g) from Turkey and G2494 (5.7 g) Figure 1. The first five PCs contribute the 81.18%
from Mexico. Grain yield per plot ranged from 0.8 of total cumulative variance. The first two principal
g for G2676 from Mexico to 372.0 g for Feijao Pico components (PC1 and PC2) are the most important
de Oro from Colombia. components accountings for 29.78% and 18.70% of
total variation respectively. PC1 is positively
A wide range of variability of seed Zn, Fe, Cu related to yield components such as number of pods
and Mn concentrations was found among the per plant, weight of pods per plant, number of seeds
common bean genotypes and cultivars (Table 1). per plant, weight of seeds per plant and grain yield
The seed Zn concentration was ranged from 13.3 to per plot. PC2 is positively correlated to the garin
72.4 mg kg-1 with a mean of 36.7 mg kg-1. The Zn, Fe, Cu and Mn concentrations and negatively
highest Zn concentration was recorded for Qeretaro correlated to 100 seed weight. PC3 is accounted for
7-5 from Colombia and lowest for Canario 102 17.25% of the total variance and positively
from Mexico. Seed Fe concentration ranged from correlated to 100 seed weight and grain Zn, Fe and
26.1 to 146.0 mg kg-1 with a mean of 65.7 mg kg-1 Cu concentrations but, negatively correlated to days
(Table 1). The highest Fe concentration was to flowering. PC4 and PC5 are accounted 9.54%
observed for 12 KMYO F25 landrace (146.0 mg kg- and 5.90% of the total variation respectively and
1
) and lowest for G1989 from Guatemala (Table PC4 is positively correlated to plant height but
S1). Copper concentration ranged from 4.3 to 13.4 negatively correlated to grain Mn concentration.
mg kg-1. The Mexican genotype M7323-3-1-3 has PC5 is positively related to days to flowering, grain
the highest seed Cu concentration (13.4 mg kg-1) yield per plot, and seed Zn cocentration but
and the G1989 has the lowest one (4.3 mg kg-1) negatively correlated to seed Fe concentration. The
(Table S1). The Mn concentration of common bean scatter diagram of common bean genotypes (Figure
genotypes has a wide diversity ranged from 7.1 to 1) showed that the first and second PCA
25.1 mg kg-1 for the Guatemala genotype G1989 components allow the common bean genotypes for
and Honduras genotype Frijol Blanco de Verdura the investigated traits. The scatter diagram of
1146


common bean genotypes showed that all genotypes was positively correlated with number of seeds per
grouped under 6-7 different groups (Figure1). plant, weight of seeds per plant, 100 seed weight
and grain yield per plant. Number of seeds per plant
The correlation coefficients between the was positively correlated to weight of seeds per
agromorphological characters and micronutrient plant and grain yield per plant but negatively
concentration of common bean genotypes are correlated with 100 seed weight. Weight of seeds
indicated in Table 4. Days to flowering was per plant was positively correlated to grain yield per
positively correlated to plant height but negatively plot but negatively correlated with 100 seed weight.
correlated with weight of pods per plant, weight of 100 seed weight was positively correlated with
seeds per plant, 100 seed weight and grain yield per grain yield per plot. Seed Zn concentration was
plot. Also there was a significant negative positively correlated with Fe, Cu and Mn grain
correlation in the plant height with 100 seed weight. concentrations. Grain Fe concentration was
The number of pods per plant was positively positively correlated to Cu and Mn concentrations
correlated to weight of pods per plant, number of and seed Cu concentration was positively related
seeds per plant, weight of seeds per plant, and grain with grain Mn concentration (Table 4).
yield per plot (Table 4). Weight of pods per plant
TABLE 3
Eigenvalues, proportion and cumulative variance and correlation coefficients between the first five
principle components for agromorphological and seed micronutrient concentrations in common bean
genotypes
Plant characteristic PC1 PC2 PC3 PC4 PC5

Eigen value 3.57 2.24 2.07 1.14 0.70
Proportion of variance (%) 29.78 18.70 17.25 9.54 5.90
Cumulative variance (%) 29.78 48.48 65.73 75.28 81.18
Days to flowering -0.100 0.373 -0.409 0.175 0.273
Plant height (cm) 0.098 0.251 -0.069 0.714 -0.300
Number of pods per plant 0.464 0.178 -0.160 -0.092 -0.030
Weight of pods per plant (g) 0.465 -0.155 0.084 0.115 -0.095
Number of seeds per plant 0.443 0.227 -0.230 -0.129 -0.002
Weight of seeds per plant (g) 0.483 -0.149 0.071 0.074 -0.105
100-seed weight (g) 0.033 -0.494 0.378 0.205 -0.102
Grain yield per plot (g plot-1) 0.328 -0.167 0.023 -0.064 0.598
Zn (mg kg-1) -0.010 0.285 0.429 0.040 0.416
Fe (mg kg-1) 0.032 0.345 0.402 0.276 -0.076
Cu (mg kg-1) 0.060 0.310 0.448 -0.064 0.180
Mn (mg kg-1) 0.060 0.310 0.212 -0.534 -0.483
1147


FIGURE 1
Scatter diagram of common bean genotypes.
TABLE 4
Pearson correlation coefficients between the agromorphological characters and micronutrient
concentrations of common bean genotypes.
Traits 1 2 3 4 5 6 7 8 9 10 11 12
1-Days to flowering 1.00
2-Plant height 0.27** 1.00
3-Number of pods per plant 0.07 0.07 1.00
4-Weight of pods per plant (g) -0.27** -0.07 0.65** 1.00
5-Number of seeds per plant 0.19 0.08 0.94** 0.58** 1.00
6-Weight of seeds per plant (g) -0.26** -0.10 0.69** 0.92** 0.65** 1.00
7-100-seed weight (g) -0.59** -0.29** -0.17 0.42** -0.33** -0.33** 1.00
8-Grain yield per plot (g plot-1) -0.23** -0.18 0.39** 0.55** 0.35** 0.55** 0.28** 1.00
9- Zn (mg kg-1) 0.01 0.09 -0.04 -0.03 -0.08 -0.07 -0.02 -0.06 1.00
-1
10- Fe (mg kg ) -0.06 0.16 -0.02 -0.04 -0.09 -0.10 -0.04 -0.09 0.38** 1.00
-1
11- Cu (mg kg ) -0.04 0.03 0.08 0.01 0.07 0.05 -0.01 0.06 0.49** 0.48** 1.00
12- Mn (mg kg-1) 0.02 0.04 0.11 -0.03 0.12 -0.03 -0.19 -0.04 0.30** 0.35** 0.38** 1.00
**P<0.01
The use of legume genetic resources in valuable way to increase agricultural production
breeding and crop improvement is one of the most and food security. It is reported as approximately
1148


~1M samples of grain legume genetic resources are productivity of common bean [41,39]. In our study
preserved in ex-situ genebanks globally and flowering duration found to be negatively
management and utilization of such large diversity correlated to yield component and grain yield and
in germplasm collection are great challenges to this is an indication of common bean genotypes
germplasm curators and crop breeders [19]. A which with early flowering provide advantages for
common bean collection with 223 genotypes of breeding programs. G2595 and Frijol canario from
which 176 genotypes kindly provided from USDA, Mexico and Col. No. 23 from Peru scored under 40
ARS, WRPIS, Washington State University days for flowering time and could be used to
(WSU), Regional Plant Introduction Station, 37 introgress earliness into common bean breeding
were landraces from Turkey (around Erzincan, programs.
Sivas, Kozan, Feke, Saimbeyli and Tufanbeyli Common bean is an important source of
provinces) and 10 commercial cultivars commonly nutritional components including proteins, vitamins
grown in Turkey was investigated for important and minerals [42]. Humans require several
yield and quality traits. Common bean (Phaseolus micronutrient such as Fe, Zn, Cu, I and Se for
vulgaris L.) is the third most important grain healthly diets [23]. Recently a term
legume after the chickpea and lentil in Turkey. µELRIRUWLILFDWLRQ¶KDVEHHQSURSRVHGDQGGHILQHGDV
Common bean seeds are commonly used by people the process of increasing the bioavailable
for a highly nutritional consumption as dry bean concentrations of essential elements in edible
and common bean landraces commonly grown still portions of several crops [23]. Food security and
many parts of the cropping regions in Turkey. nutritional quality is another important aspect
Identifying new variation and genetic resources are associated with increased grain legume production.
needed to improve and introduce new cultivars in So, genetic resources with useful variation
common bean breeding programs. In this research discovered in this research should be considered
agromorphologically traits especially ralated to and promising genotypes with higher nutritional
grain yield and and mineral nutrient concentration quality are under development. Zn and Fe are
of seeds were studied. Many investigations have reported as essential micronutrients for human
covered agromorphological traits but there is growth, development and immune system [43] and
limited data on grain quality available [19]. Great common bean is reported as an important source of
variation and exotic genotypes were observed for Zn and Fe and genetic differences have been
grain yield components compared to commercial reported for seed mineral concentrations among
cultivars. Distinct variation for grain yield and yield genotypes [44,45,42,46]. In our investigation we
components as plant height, number of pods and suggest that the genotypes G878, G16837, Frijol
seeds per plant and 100 seed weight in this canario, P8474-A-D from USDA collection, 12
collection may facilitate introgressing alleles for KMYO F25 landrace and Göynük variety from
these traits into new cultivar breeding programs. Turkey could be evaluated for high Fe and Oaxana
Many researchers have reported utilization of grain 5-1, Frijol canario, Queretaro 7-5 and Negro for
legumes genetic resources in crop improvement as high seed Zn cocentration.
an important tool [33-38]. It is reported that growth As a conclusion, we have considered a large
habit is an important trait for bean adaptation. For collection of common bean and we identified
example, indeterminate type is predominantly promising genotypes both for yield and yield
grown in the mountain regions because with air components and seed mineral concentration. These
humidity and low temperatures and determinate common bean genotypes could be useful as new
type is grown mainly under dry environments genetic sources of variation for common bean
[39,40] reported that the most important breeding objectives.
characteristics related to grain yield are the number
of pods per plant and number of seeds per plant. In
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Blair, M.W., Osorno, J., Dilawari, M.,
1151


CORRESPONDING AUTHOR University of Cukurova

01330, Balcali-Adana ± TURKEY
Faruk Toklu
Department of Field Crops e-mail: fapet@cu.edu.tr
1152


HAZARDOUS WASTE CHEMICAL STABILIZED BY

TRICALCIUM SILICATE KINETIC AND ISOTHERM STUDIES
Ionela Cara eanu Popovici1 , Alina Ioana Badanoiu2, Georgeta oicu2 and Ichinur Omer3
1*
Ovidius University of Constanta, Faculty of Applied Science and Engineering, 124 Mamaia, 900527 Constanta, Romania
2
Politehnica University of Bucharest, Faculty of Applied Chemistry and Material Science, 1-7 Gh Polizu Str., 011061, Bucharest, Romania
3
Ovidius University of Constanta, Faculty of Civil Engineering, 124 Mamaia, 900527 Constanta, Romania
ABSTRACTS metal ions, binding them irreversibly under ambient

conditions, and simultaneously possessing the
The present work gives new insights regarding ability to release the same under different
the kinetics of chemical immobilization of heavy conditions such that it can be regenerated for
metals by low-cost material like tricalcium silicate further use [5-7]. Several different solid phases
(Ca3SiO5 or C3S). The effects of pH, metal ion have been found useful in taking up of heavy
concentration and contact time on the binding metals from solution such as clay minerals,
ability of tricalcium silicate were investigated by activated carbons, metals oxides, biosorbents and
dynamic leachability test. The immobilization rate natural or synthetic zeolites [8].
of Cu(II), Ni(II) and Pb(II) in Ca3SiO5 and the Portland cement is the most popular
leaching characteristics of the Ca3SiO5 hydrated solidification agents for inorganic wastes [9-14].
matrixes containing heavy metals were investigated The high pH of Portland cements tends to keep
by X-ray diffraction, electron microscopy and metals in their insoluble forms (i.e. carbonates,
atomic absorption spectrometry. hydroxides or double hydroxides), hence reduced
The feasibility of transforming heavy metals subsequent leaching. The development of hydration
into stable compounds in tricalcium silicate hydrate products provided an interlocking framework that
and the successful reduction of heavy metals physically encapsulates waste particles and is also
mobility from the matrix of hydrated C 3S was responsible for the strength development. Moreover
demonstrated. Kinetics study results revealed that the adsorption of heavy metals ions is possible on
the sorption data on heavy metals with correlation calcium silicates hydrates formed during Portland
coefficient of 0.999 can best represent by pseudo cement hydration [15].
second±order. The sorption data fitted with various Tricalcium silicate (C3S) is the major phase in
isotherm models and Langmuir model was the best Portland cement (40-65%). This phase reacts with
one. The sorption capacity of tricalcium silicate for water to produce calcium silicate hydrate gel (C-S-
Pb(II), Ni(II) and Cu(II) was found to be 80 mg/g, H) and portlandite (Ca(OH)2) [16-18]. Calcium
17.889 mg/g and 22.173 mg/g, respectively. silicate hydrate gel (C-S-H), is a mixture of poorly
crystallized particles with different morphologies
KEYWORDS: and contains the bulk of micro-porosity. The gel
Tricalcium silicate; Heavy metals; Stabilization; pores give rise to a high surface area and largely
Leaching; Sorption; Kinetics control the sorption properties [16,17]. Adsorption
of heavy metals inhibits homogeneous or
heterogeneous nucleation and growth of hydration
INTRODUCTION products and in some cases enhances silicate
polymerization [18-20].
In the removal of hazardous contaminants This paper is focuses on the influence of some
from industrial waste waters from, among others, heavy metals ± Cu(II), Ni(II) and Pb(II) on early
metal finishing, electroplating, plastics, pigments hydration of C3S in order to their immobilization
and mining industries, there is the distinct tendency inside the matrix of hydrated C3S. The controlling
to replace the costly activated carbons and ion mechanisms of the sorption of Cu(II), Ni(II) and
exchangers with various low cost adsorbents, which Pb(II) on C3S were examined by comparing the
have metal binding capacities [1-4]. Minimization properties of the hydrated C3S to their sorption
and removal of heavy metals from aqueous systems characteristics including sorption kinetic and
is therefore an important area of study. This can be isotherm model parameters. In order to achieve aim
achieved by transfer of the metal ions from the of this work the effect of metal ion concentration,
aqueous phase to a solid phase by adsorption. The pH and contact time on binding ability of tricalcium
solid phase should have strong affinity for the target silicate was investigated. Three kinetic models
1153


including a PF-order, PS-order and intraparticle dynamic leachability test. 50 mL of metal ion
diffusion equation were selected to follow the solutions with the initial concentration ranging from
sorption process in C3S - heavy metals systems. 110-2 M to 510-2M were placed in 100 mL flasks
The sorption data were also analyzed by the with a required dose of C3S samples (0.5 g) and
Langmuir, Freundlich, Halsey and Redlich-Paterson shaken at a constant speed (200 rpm) for a specific
models of adsorption. period of contact time (5 ± 360 minutes). After that
the samples were separated by centrifugation at
4000 rpm for 4 minutes and the liquid phase was
MATERIALS AND METHODS filtered through a 0.22 Pm Millipore filter. The
resulting solid phase of selected samples was
MATERIALS treated with ethanol and acetone and dried in oven
Tricalcium silicate (3CaOSiO2 or C3S) had at 105oC, for 2 hours in order to be examined by X-
been synthesized from a 3:1 molar ratio mixture of ray diffraction.
CaCO3 and SiO2 (aerosol) with a mineralizer The concentrations of Cu(II), Ni(II) and
addition (Na2SiF6) of 0.3%. The materials were Pb(II), before and after equilibrium, were analyzed
grinded to a fine powder in a ceramic ball mill, by Analytik Jena ContrAA 700 Atomic Absorption
pelletized and the then subjected to a double Spectrometer at 324.75 nm for Cu(II), 232 nm for
sintering at 1400oC for 4 hours. After the initial Ni(II) and 217 nm for Pb(II). The experiments were
firing, the cycle of grinding, pelletizing and carried out in duplicate and the average values were
sintering was repeated until no free lime content obtained (± 1±2% for the percentage removal and ±
was detected [21]. The 3CaOSiO2 powder was 0.005 ± 0.01 mg/g for the amount adsorbed). Also,
checked for phase purity by XRD and milled to a the pH of suspensions was measured with a 720
Blaine specific surface of 0.2416 m2.g-1 [22]. The Inolab Multiparameter using a combined glass
peaks present on XRD spectra of synthesized electrode.
material are attributed to 3CaOSiO2 (JCPDS 42- The adsorption amount of heavy metals on
0551). This material was used for batch adsorption C3S is given by Eq.(1):
experiments. Heavy metals were introduced in the C0 Ct (1)
t
water added for 3CaOSiO2 hydration (water/solid m
ratio was 100). Three single-component metal ion where t is the adsorption amount of metal ion at
solutions containing Cu (II), Ni (II) or Pb (II), with time t (mg/g), m is the weight of C3S sample (g),
different concentrations (110-2 to 510-2 M), were is the total volume of solution (L); C and Ct are the
used in this work. The stock solutions (1M) were initial and equilibrium concentrations of M(II) ions
prepared from soluble salts (CuSO4·5H2O, in solution, respectively the concentrations of M(II)
Ni(NO3)2·6H2O and Pb(NO3)2 ± Merck Chemical ions in solution (mol/L) at time t.
Company, purity >98%) and deionized water. The immobilization efficiency, Ef (%) is
express as:
METHODS (C0 Ct )
X-ray powder diffraction (XRD). A 100 (2)
C0
Shimadzu XRD 6000 diffractometer (CuK D
radiation, O = 1.5418Å) was used for crystalline When t is equal to the equilibrium contact
phases identification in anhydrous and hydrated time, Ct = Ce, t = e and the amount of M(II)
C3S in presence of heavy metals ions, for 6 hours adsorbed at equilibrium ( e), is calculated by Eq.
and 12 hours. The finely ground samples were (1).
examined between 5o (15o) and 50o 2ș at scanning Kinetic studies of heavy metals adsorption on
rate of 2 deg/min. C3S were carried out at various concentrations of
the metal ion solution (110-2M, 310-2M, 510-2M)
Electron microscopy (SEM and BSE). where in the extent of adsorption was investigated
Scanning electron microscopy (SEM) Hitachi S as a function of time. The kinetics and adsorption
2600 N was used to evaluate the morphology and dynamics of Cu(II), Ni(II) and Pb(II) on C3S have
microstructures of anhydrous and hydrated C3S in been investigated by applying three models: the
presence of heavy metals ions. SEM and BSE (back pseudo first order (PF-order) kinetics model,
scattered electron) analyses were performed on pseudosecond order (PS-order) kinetics model and
selected pastes specimens coated with silver. intraparticle diffusion model (IPD): Sorption
equilibrium isotherms. The adsorption data of the
Sorption capacity studies. Sorption metal ions on C3S were analyzed using four
experiments, in the single component systems, were models: Langmuir, Freundlich, Halsey and Redlich-
carried out at room temperature (20 oC) using the Paterson (Table 2).
1154


TABLE 1
Kinetic parameters for adsorption process of Cu(II), Ni(II) and Pb(II) on hydrated C 3S
1t
PF-order model ( log( e t) log( e ) )
2.303
C0 e,exp 1 1 R2 Chi s uare
(mol/L) (mg/g) (mg/g) (1/min) Ȥ2
Cu (II)
1x10-2 M 1.480 1.399 0.024 0.980 0.0045
3x10-2 M 4.118 4.016 0.031 0.990 0.0025
5x10-2 M 6.356 6.258 0.035 0.993 0.0015
Ni (II)
1x10-2 M 1.747 1.575 0.019 0.958 0.0168
3x10-2 M 4.702 4.500 0.026 0.980 0.0086
5x10-2 M 7.415 7.167 0.028 0.985 0.0083
Pb (II)
1x10-2 M 7.491 7.031 0.023 0.980 0.0282
3x10-2 M 20.682 20.186 0.031 0.987 0.0119
5x10-2 M 33.666 32.789 0.030 0.978 0.0229
t t t
PS-order model ( 2
)
t 2 t
e
C0 e,exp 2 2 100
R2 &KLVTXDUHȤ2
(mol/L) (mg/g) (mg/g) (g/mg.min)
Cu (II)
1x10-2 M 1.480 1.431 6.659 0.994 0.0017
3x10-2 M 4.118 4.054 2.553 0.997 0.0010
5x10-2 M 6.356 6.311 1.326 0.996 0.0003
Ni (II)
1x10-2 M 1.747 1.643 3.803 0.986 0.0061
3x10-2 M 4.702 4.591 1.745 0.996 0.0026
5x10-2 M 7.415 7.291 1.056 0.997 0.0021
Pb (II)
1x10-2 M 7.491 7.165 0.831 0.990 0.0142
3x10-2 M 20.682 20.397 0.353 0.997 0.0039
5x10-2 M 33.666 33.090 0.203 0.997 0.0099
1 2
Intraparticle diffusion model (IPD) ( t it I )
C0 e, exp m Chi s uare
I R2
(mol/L) (mg/g) (mg/g) i (mg/g.min0.5) Ȥ2
Cu (II)
1x10-2 M 1.480 1.505 0.0812 0.6159 0.979 0.0004
3x10-2 M 4.118 4.294 0.2354 1.715 0.965 0.0075
5x10-2 M 6.356 6.726 0.421 2.113 0.955 0.0215
Ni (II)
1x10-2 M 1.747 1.747 0.117 0.4649 0.961 0.0000
3x10-2 M 4.702 4.919 0.3165 1.4521 0.932 0.0101
5x10-2 M 7.415 7.844 0.5251 2.0914 0.930 0.0248
Pb (II)
1x10-2 M 7.491 7.625 0.5358 1.756 0.982 0.0024
3x10-2 M 20.682 21.895 1.5063 5.3941 0.950 0.0711
5x10-2 M 33.666 35.555 2.5139 8.0168 0.947 0.1060
1155


TABLE 2
Isotherm parameters and correlation coefficients for the adsorption
of Cu(II), Ni(II) and Pb(II) on C3S
Model Cu (II) Ni (II) Pb (II)

Langmuir q0 22.173 17.889 80
Ce KL 0.004 0.009 0.004
0
e RL 0.599 0.431 0.326
1 Ce
R2 1 0.998 0.999
KF 0.135 0.242 0.490
Freundlich n 1.176 1.251 1.234
e Ce1 / n 1/n 0.850 0.799 0.810
R2 0.999 1 0.999
Halsey KH 10.585 5.914 2.410
1 nH -1.176 -1.251 -1.234
§ ·n
e ¨¨ ¸¸
© Ce ¹ R2 0.999 1 0.999
Redlich-Paterson KRP 0.093 0.163 0.287
Ce aRP 0.004 0.009 0.004
e ȕ 1 1 1
1 a CeE
R2 1 0.998 0.999
RESULTS AND DISCUSSIONS Scanning electron microscopy (SEM) images

presented in fig. 2 provide more information
X-ray diffraction patterns of anhydrous C 3S regarding the influence of heavy metals on the
and hydrated C3S with heavy metals (2.5 mmol/L as microstructure on hardened C3S paste. The SEM
Me(II)), after 6 h and 12 h of hydration are shown images of C3S paste hardened for 28 days (Fig.2
in Fig. 1. The results obtained in XRD analyses a,b) shows the presence of calcium silicate hydrates
reveal that the presence of heavy metals affects the (C-S-H) gel identified as short needle-like bundles
evolution process of C3S hydration. This influence and platy formations, at the surface of prismatic
can be attributed to the uptake of heavy metals in C3S particles [23]. When the Cu(II) ions are
C-S-H gel [7,10,16], which would be expected to presented in the C3S ±water system (Fig. 2c) there
be more slowly mobilized during leaching. The are visible mainly the prismatic C3S particles and
inhibition of portlandite formation or/and scarce C-S-H gel phase at their surface. The low
precipitation in the systems with Cu(II) and Pb(II) amount of calcium silicate hydrates suggests an
can be also due to the consumption of Ca2+ ions in important delay of C3S hydration process; these
formation of different compounds such as data are in good correlation with the results
CaCu4(SO4)(OH)6.3H2O (Fig.1a) or Ca2Pb2O5(OH)2 obtained by XRD i.e. minor changes of intensity of
and Ca(NO3)2(H2O)1.235 (Fig. 1b). For the system C3S XRD peaks vs. time. The micrograph of
with Ni(II) ions no crystalline hydrates are detected hydrated C3S pastes in the presence of Ni(II) ions
by XRD (Fig. 1c); nevertheless the decrease of the (Fig. 2d) shows the presence of a high amount of C-
X-ray reflection peaks specific for C3S, after 6h and S-H gel phase; these data confirms the information
12h of contact with the solution, correlated with the obtained by XRD i.e. Ni(II) do not exert a delaying
board peak (halo) assessed between 30-35 degree effect upon the C3S hydration opposite to Cu(II)
suggests the development of C3S hydration with ions. The microstructure of C3S hydrated in the
formation of phases with a low crystalline degree presence of Pb(II) ions (Fig. 2 e,f) shows also the
(calcium silicate hydrates C-S-H). presence of a high amount of low crystallinity
phase (most probably calcium silicate hydrates).
1156


FIGURE 1
X-ray diffraction patterns of anhydrous C3S and hydrated for 6h and 12h (initial solution concentration of
2.5 mmol/L as Me(II)): a) Cu(II) b) Pb(II)) c) Ni(II))
1157


(a) (b)
(c) (d)
(e) (f)
FIGURE 2
SEM micrographs of C3S hydrated for 28 days without heavy metals (a-b) C3S hydrated for 28 days in
the presence of: Cu (II) ions (c) Ni (II) ions (d) Pb (II) ions (e ± f)
To understand the effects of heavy metals on (Fig. 3). This value is close to the pH of the
portlandite precipitation and C3S hydration, the pH saturated solution of pure portlandite. Additions of
of C3S suspensions was measured. In the absence of heavy metal salts decreased pH due to hydrolysis of
heavy metals, C3S reacts with water rapidly and in heavy metal cations at an initial concentration of
no more than 10 minutes a pH of 11.43 is recorded 2.5 mmol/L of heavy metal, the initial pH values of
1158


C3S suspensions with Cu(II), Ni(II) and Pb(II) were 60 minutes. This value confirming that Ni(II)
5.26, 7.30 and 5.63, respectively. The pH of the C3S severely retarded the hydration of C3S. It is known
suspensions increases vs. time due to the hydration that in low pH media (<12.4), the precipitation of
of C3S at different rates, which reflected portlandite is inhibited. As a result, no X-ray
interactions between C3S (or species formed by C3S reflection peaks of portlandite are detected in X-ray
hydration) and heavy metals ions. In the presence diffraction patterns obtained from these systems
of Ni(II), the pH value was below 7.63 even after after 6 and 12 hours of hydration.
FIGURE 3
Evolution of pH vs. time in C3S - M(II) - H2O systems
The effect of initial concentration of Cu(II), the sorption process, it was observed that the
Ni(II) and Pb(II) on the extent of their removal by increasing of concentration of heavy metals
C3S, in terms of immobilization efficiency (Ef, %) solution (from 110-2 M to 510-2 M) determined the
and amount adsorbed ( t, mg/g) at different time increasing of adsorbed amount of heavy metals on
intervals was also studied (Fig. 4 a and b). Heavy C3S and also a decreasing of the removal efficiency.
metals sorption increases with contact time and an This indicates that if the metal ions concentration in
equilibrium is established when the concentration solution increases, the difference of concentration
of metal in a bulk solution is in a dynamic balance between the bulk solution and solid surface
with the concentration on the surfaces of the C3S increases too, intensifying the mass transfer
particles. Equilibrium conditions were established processes. It was found that removal efficiency
within 120 min for all investigated systems. As it decreases with the increasing of initial
can be seen in Fig. 4a, the highest values of heavy concentration of M(II) ions from 110-2 M to 510-2
metals sorption on C3S were achieved for Pb(II). M. For Cu(II) adsorption in the C3S sample these
These data support the information brought by the values are equal to 56.86%, 53.26% and 50.11%,
XRD spectra presented in Fig. 1. For the system for Ni(II) removal efficiency values were equal to
with Pb(II), even after 6 hours of hydration, the C3S 69.82%, 63.46% and 60.98% and for Pb(II)
peaks intensity has an important decrease, 82.46%, 76.92% and 75.12%. It is obviously that
suggesting that C3S is consumed in hydration the sorption rate is rather fast at the initial stage and
process. There are detected also new diffraction then it slows down gradually.
peaks, specific for compounds with Ca and Pb. Kolodynska et al. [24] considers that the
Therefore we can assume that Pb(II) sorption process rate and dynamic behavior of the
immobilization in this system is determined both by system are very important factors for the process
sorption on C-S-H phase (detected in high amount design and operation control. Sorption of an
in SEM analysis, Fig. 2 e, I DQG DOVR E\ LW¶V adsorbent from the aqueous phase involves three
consumption in reaction with Ca2+ ions formed by steps: (1) the transport of the adsorbate from the
C3S hydration. bulk phase to the exterior surface of the adsorbent
When initial Me(II) concentration was (film diffusion), (2) the transport into the adsorbent
considered as a parameter which would influence by pore diffusion and/or surface diffusion
1159


(intraparticle diffusion) and (3) the adsorption on Sorption of metal ions increases with time, until the
the surface of the adsorbent. The slowest of these equilibrium between the amounts of adsorbates
steps determines the overall rate of the sorption adsorbed on the adsorbents was achieved and the
process. A strong dependence seems to be between amounts of adsorbates remained in solution.
the sorption kinetics and the physical and/or Generally, sorption reactions took place rapidly at
chemical characteristics of the adsorbed material initial stages and gradually slowed down in
which also influences the sorption mechanism. reaching its equilibrium state [25,26].
FIGURE 4
Adsorption inetics of heavy metals on hydrated C 3S (suspensions with initial concentration of 2.5
mmol/L as Me(II)): a) Effect on the phase contact time of Cu(II) Ni(II) and Pb(II) adsorption on hydrated
C3S b) removal efficiency of Cu(II) Ni(II) and Pb(II) on hydrated C 3S
In order to investigate the controlling equilibrium sorption capacities, the rate constants
mechanism of sorption processes such as mass and the related correlation coefficients, for all
transfer and chemical reaction, a proper kinetic kinetic models. Fig. 5 ± 7 shows the plots of
model is required to analyze the data. Three linearized form of PF-order model, PF-order model
simplified kinetic models including a PF-order and IPD model at all concentrations studied. To
equation, PS-order equation and intraparticle determine the kinetics parameters ( i,
diffusion equation were selected to follow the I), the slopes and intercepts of plots of l e ±
adsorption process in C3S - heavy metals systems. vs. t, t t vs t and t vs. t were used. The PF-order
Table 1 shows the kinetic parameters, the model did not adequately describe the sorption
1160


results of Cu(II), Ni(II) and Pb(II) ions onto the concentration increases. For example, for Pb (II)
hydrated C3S due to slightly low correlation the rate of sorption was highest at the initial
coefficients ( ) between the predicted and the concentration of 110-2 M ( = 0.831 g/mg.min)
experimental values ( and e ex ). Taking into and lowest for a concentration of 510-2 M ( =
account the values, a better fit was achieved 0.203 g/mg.min). The mean value of (0.353
when the PS-order model has been used (Fig. 6). It g/mg.min) was obtained for the concentration of
can be noticed that with the increasing of the initial 310-2 M. The analogous values were obtained in
concentration the values increase and we can the case of Cu(II) and Ni(II) ions adsorption.
considered that the process is controlled by
diffusion. Moreover, the values decrease as the
FIGURE 5
PF-order plots of adsorption of Cu(II), Ni(II) and Pb(II) on hydrated C 3S.
1161


FIGURE 6
PS-order plots of adsorption of Cu(II), Ni(II) and Pb(II) on hydrated C3S.
1162


FIGURE 7
Intraparticle diffusion model plots of adsorption of Cu(II), Ni(II) and Pb(II) on hydrated C3S.
1163


The possibility to use the IPD model on the Isotherm model was determined to be 22.173 mg/g
heavy metals sorption process was also evaluated. for Cu(II), 17.889 mg/g for Ni(II) and 80 mg/g for
The metal ion sorption is controlled usually by Pb(II), KL (Langmuir isotherm constant) is 0.004
either the intraparticle (pore diffusion) or the liquid- L/mg for Cu(II) and Pb(II) and 0.009 L/mg for
phase mass transport rates (film diffusion). The Ni(II), RL is 0.599 for Cu(II), 0.431 for Ni(II) and
experiment was made in a batch system with rapid 0.326 for Pb(II) indicating that the equilibrium
stirring. Therefore, it is a possibility that sorption was favorable and the value is 1 for
intraparticle diffusion to be the rate determining Cu(II) and Pb(II) and 0.998 for Ni(II), proving that
step. In Fig. 7 was plotted the relationship between the sorption data fitted well to Langmuir Isotherm
e and t , in order to establish whether the model.
transport of M(II) ions from the solution into the The constant is an approximate indicator of
pores of the hydrated C3S is the rate controlling adsorption capacity, whereas n is a function of the
step. Fig. 7 confirms that the straight lines do not strength of adsorption in the sorption process. If n =
pass through the origin. The deviation of the 1 then the partition between the two phases are
straight lines from the origin can be attributed to the independent of the concentration. A normal
difference between the rate of mass transfer in the adsorption take place when the value of n is
initial and the final steps of the sorption process. below one [27,28]. However, and n are
Also, the deviation of straight line from the origin characteristic parameters of the sorbent - sorbate
indicates that the pore diffusion is the sole rate- system. These parameters should be determined by
controlling step in this process [25]. Moreover, data fitting, whereas linear regression is usually
comparing the intercept (I) values, which give us an used to determine the parameters of kinetic and
idea about the boundary layer thickness one can isotherm models. Specifically, the linear least-
state that if the intercept is larger the boundary squares method and the linearly transformed
layer effect is greater. It was observed that the equations have been widely applied to correlate
values of the intercept increase with the increasing sorption results. The n is the heterogeneity
of initial concentration of the heavy metals. parameter and for a smaller value of n a greater
The kinetic data indicate that the mechanism heterogeneity is expected. When n = 1 a linear
of metal ions sorption by the hydrated C3S is isotherm adsorption takes place. If n value lies
complex and probably is a combination of external between 1 and 10, this indicates a favorable
mass transfer, intraparticle diffusion through the sorption process [26]. All the data from the Table 2
macropores and micropores of hydrated C3S and (the value of n is 0.850 for Cu(II), 0.799 for
sorption processes. It should be also noted that the Ni(II) and 0.810 for Pb(II) while n lies between
kinetic process fitted by the PS-order model 1.176 and 1.251) indicate that the sorption of
assumes chemisorption. In the case of the rate Cu(II), Ni(II) and Pb(II) onto C3S is favorable;
limited chemisorption the inner-sphere moreover, the value is 0.999 for Cu(II) and
complexation and precipitation involves the metal Pb(II) and 1 for Ni(II). These results also suggest
ions sorption whereas the role of electrostatic ion that the surface heterogeneity of the composite
exchange can be negligible. These assertions are adsorbent is very significant, indicating that all the
supported by the results obtained by XRD analysis. ³PLQHUDO´ FRPSRQHQWV VLOLFD JHO FDOFLXP VLOLFDWH
The sorption data of the metal ions on C3S hydrate gel and portlandite) take part in the sorption
were analyzed using the Langmuir, Freundlich reaction. The high percentage adsorption, the
isotherm, Halsey isotherm and Redlich-Paterson values of the Langmuir, Freundlich, Redlich-
isotherm models. The isotherm constants were Paterson and Halsey isotherms as well as the low
obtained from the intercepts and slopes of the values of the separation factors ( ), show that the
respective plots and are shown in Table 2. The data C3S is a good adsorbent for investigated heavy
for sorption of the above-mentioned metals on the metals.
C3S fit closely to the Langmuir adsorption mode.
The values of the separation factor,
( C ) indicating the nature of the CONCLUSION
sorption process are also presented in Table 2.
value indicates the sorption nature to be either Based on the obtained results, we conclude
unfavorable if >1, linear if =1, favorable if 0< that tricalcium silicate can be suitable and
<1 and irreversible if =0. From the calculated economically feasible for heavy metals
data, the RL is greater than 0 but less than 1 immobilization such Cu(II), Ni(II) and Pb(II). The
indicating that Langmuir isotherm is favorable. effects of metal ion concentration and contact time
In our research work, the maximum on binding ability of hydrated tricalcium silicate
monolayer coverage capacity (q0) from Langmuir
1164


were investigated by kinetic and equilibrium [3] Fenglian F. and Wang Q. (2011) Removal of
adsorption isotherm studies. heavy metal ions from wastewaters: A review.
Heavy metals immobilization mechanism is Journal of Environmental Management, 92,
complex and relies on competition of the following 407-418.
processes: chemisorption, substitution and [4] Jin F. and Al-Tabbaa A. (2014) Evaluation of
precipitation of the mixed salt. The XRD and SEM novel reactive MgO activated slag binder for
data suggests that the inhibition of portlandite the immobilisation of lead and zinc.
formation and precipitation in the systems with Chemosphere 117, 285±294.
Cu(II) and Pb(II) can be attributed to the [5] Bullard J.W., Jennings H.M. et al. (2011)
consumption of Ca2+ ions in formation of different Mechanisms of cement hydration. Cem.
compounds such as CaCu4(SO4)(OH)63H2O or Concr. Res. 41, 1208±1223.
Ca2Pb2O5(OH)2 and Ca(NO3)2(H2O)1.235. [6] Chen Q.Y., Tyrer M., Hills C.D., Yang X.M.
This study demonstrates the feasibility of and Carey P. (2009) Immobilisation of heavy
transforming heavy metals into stable compounds metal in cement-based solidification/
in tricalcium silicate hydrate and the successful stabilisation: A review. Waste Management
reduction of heavy metals mobility from the matrix 29: 390±403
of hydrated C3S. Examination of the sorption [7] Gineys N., Aouad G. and Damidot D. (2010)
capacity of the C3S adsorbent at saturation for Managing trace elements in Portland cement ±
investigated heavy metals indicates that: (1) the Part I: Interactions between cement paste and
binding capacities were 80 mg/g for Pb(II), 17.889 heavy metals added during mixing as soluble
mg/g for Ni(II) and 22.173 mg/g for Cu(II); (2) salts. Cem. Concr. Composites 32, 563±570
equilibrium conditions were established within 120 [8] Mallampati S.R., Mitoma Y., Okuda T.,
min. Adsorption data were strongly correlated with Sakita S. and Kakeda M (2012) Enhanced
the PS-order kinetic equation and the Langmuir heavy metal immobilization in soil by
adsorption isotherm. grinding with addition of nanometallic
The kinetic data indicates that the mechanism Ca/CaO dispersion mixture Chemosphere 89,
of metal ions sorption by the C3S is complex and 717±723.
probably is a combination of external mass transfer, [9] Badanoiu A., Georgescu M. and Zahanagiu A.
intraparticle diffusion through the macropores and (2008) Properties of blended cements with
micropores of C3S (or of the formed hydrates) and hazardous waste content. Rev. Roum. Chim.
sorption processes. Moreover, a high degree of 53, 229-237.
immobilization of heavy metals ions inside the [10] Chen Q.Y., Hills C.D., Tyrer M. et al. (2007)
matrix of hydrated C3S also suggests that the Characterisation of products of tricalcium
surface heterogeneity of the composite adsorbent is silicate hydration in the presence of heavy
YHU\ VLJQLILFDQW LQGLFDWLQJ WKDW DOO WKH ³PLQHUDO´ metals. J. Hazard. Mater. 147, 817±825.
components (silica gel, calcium silicate hydrate gel [11] Giergiczny Z. and Krol A. (2008)
and portlandite) take part in the sorption reaction. Immobilization of heavy metals (Pb, Cu, Cr,
Zn, Cd, Mn) in the mineral additions
containing concrete composites. J. Hazard.
ACKNOWLEDGMENT Mater. 160, 247±255.
[12] Sobiecka E. (2013) Investigating the chemical
Authors are thankful to Mrs. Adriana Puhacel stabilization of hazardous waste material (fly
(chemist) for atomic absorption spectroscopy ash) encapsulated in Portland cement Int. J.
analysis. Environ. Sci. Technol. 10, 1219±1224.
[13] Song F., Gu L., Zhu N., Yuan H. (2013)
Leaching behavior of heavy metals from
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[2] Bhattacharyya K.G. and Gupta S.S. (2011)
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of some sulfonated naphthalene formaldehyde
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[23] Stutzman P. (2001) Scanning electron Ovidius University of Constanta
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e-mail: icarazeanu@univ-ovidius.ro
1166


CASPASE-3 ACTIVATION IN CYTOTOXICITY OF

ISOLATED RAINBOW TROUT (ONCORHYNCUS MYKISS)
HEPATOCYTES INDUCED BY BISPHENOL A
Burak Kaptaner1 and (UWX÷UXO.DQND\D2
1
Department of Biology, Faculty of Science, Yuzuncu Yil University, 65080 7XúEDVan, Turkey
2
Faculty of Fisheries, Yuzuncu Yil University, 65080 7XúED9an, Turkey
ABSTRACT including fish, are a major target of BPA and

environmentally relevant concentrations of this
The present study was carried out to examine substance have been reported to cause altered sex
caspase-3 activity in cytotoxicity of isolated differentiation, gonadal abnormalities, and hepatic
rainbow trout ( n n m i ) hepatocytes vitellogenin induction [2].
induced by bisphenol A. For this purpose, cultured Apoptosis is an early indicator of acute and
hepatocytes were exposed to various concentrations chronic chemical stress and serves as a warning
of BPA (10, 50, 100, 250, 500, and 1000 μM) for regarding the loss of cellular function and structure
24 and 48 h. Cell viability was detected by [3]. Caspases are the primary drivers of apoptotic
measuring lactate dehydrogenase release into the cell death and are regulated by a variety factor
medium. BPA demonstrated a cytotoxic effect on under physiological and pathological settings.
hepatocytes in a dose- and time-dependent manner, Initiator caspases (caspase-2, -8, -9, and -10)
and was found to be severely toxic for hepatocytes cleavage the inactive precursor forms of effector
with concentrations of 250 μM BPA and higher at caspases (caspase-3, -6, and -7) and activates them
both 24 and 48 h. Caspase-3 activity was and caspase-3 in an effector caspase expressed in
significantly induced by BPA with 100 and 250 μM cells undergoing apoptosis and plays a central role
concentrations at 24 h, and 50 and 100 μM in mediating nuclear apoptosis, including chromatin
concentrations at 48 h. The results suggest that condensation, DNA fragmentation, and cell
cytotoxicity of rainbow trout hepatocytes induced blebbing [4].
by BPA is mediated by caspase-3 activation. Fish cells used in toxicological studies are
practical and advantageous tools allowing widely
KEYWORDS: different species to be evaluated for their sensitivity
n n m i , bisphenol A, cytotoxicity, to environmental contaminants at the cellular level
caspase-3, isolated fish hepatocytes [5]. Thus, they constitute an attractive model to
understand time- and dose-dependent toxicant
induction processes under defined experimental
INTRODUCTION conditions [6]. Primary hepatocyte cultures from
fish, such as rainbow trout, n n m i ,
Bisphenol A (BPA) is one of the important are well characterized screening tools for the
monomers used in the production of determination of the impacts of a single chemical or
polycarbonates, epoxy resins, and other plastic chemical mixtures [7].
products. It has received particular attention Previous studies have mostly focused on the
because of its large-scale production and estrogenic effect of BPA on fish hepatocytes [8, 9,
widespread usage in the manufacturing of many 10, 11]. However data on the responses of
consumer products, such as adhesives, baby bottles, cytotoxicity in fish exposed to BPA is limited,
dental sealants, compact discs, thermal papers, especially in fish primary hepatocytes. In this study
powder paints, cans, electronic parts, etc., on rainbow trout, which frequently selected in
encountered in daily life [1]. BPA has been reported toxicological studies for testing chemicals and
to be estrogenic and possess endocrine-mediated recommended by the Organisation for Economic
adverse effects on wildlife and humans due to its Co-operation and Development as a test animal
weak binding to estrogen receptors. It is released [12], the hepatocytes were used to determine the
into the environment through sewage treatment dose- and time-dependent toxic effect of BPA on
effluents, landfill leachate, or the natural primary cultured hepatocytes of rainbow trout using
degradation of polycarbonate plastics, and collects lactate dehydrogenase release, and the role of
in rivers, lakes, and estuaries. Aquatic organisms, caspase-3 in BPA-induced cytotoxicity.
1167


MATERIALS AND METHODS softened liver tissue was triturated by pipetting with
different pipette tips. At the final stage, the obtained
Fish. Gonadally immature rainbow trout cell suspension was gently passed through the
( n n m i ) weighing 180200 g were needle of a sterile injector for dissociation of the
purchased from a local supplier in Van, Turkey. cells. The resulting cell suspension was transferred
The fish were anesthetized with 2-phenoxyethanol to sterilized centrifuge tubes and then centrifuged at
for 3 min by placing them in dechlorinated water. 30 × for 3 min. Cells were washed two times with
The ethic regulations were followed in accordance L-15 medium containing antibiotic/antimycotic 1%
with national and institutional guidelines for the (v/v) and NaHCO3 (0.38 g/L). After the last wash,
protection of animal welfare during experiments. the cell pellet was resuspended in L-15 medium.
All of the procedures were approved by the The cells were counted using a Thoma slide and the
<]QF <ÕO 8QLYHUVLW\ $QLPDO ([SHULPHQWV cell viability was assessed using a trypan blue
Ethics Committee for the ethical concerns of the exclusion test. The cells were observed to possess
study. more than 90% viability for cell culture studies.
Cell culture medium and chemicals. The cell Hepatocyte culture and exposure. The
FXOWXUH PHGLXP /HLERYLW]¶V / /-15) with L- isolated hepatocytes were seeded at a density of 1 ×
glutamine, was purchased from Sigma-Aldrich (St. 106/mL (200 μL: 2 × 105 per well) in 48 well
Louis, MO, USA). The antibiotic/antimycotic culture plates (Greiner Bio-One, USA). The cells
solution and collagenase (crude type IV) were also were cultured at 18 °C in a sterile incubator
purchased from Sigma. The BPA [(2,2-bis (4- (Binder, Germany) without additional O2/CO2 for
hydroxyphenyl) propane), purity: t99%] was 24 h prior to chemical exposure. A stock solution of
purchased from Sigma-Aldrich (St. Louis, MO, BPA was made in absolute ethanol and the final
USA) and dissolved in absolute ethanol (Merck, concentration of ethanol in the media never exceed
Darmstadt, Germany). The kit for the lactate 0.1%. After 24 h of preculture, the old medium was
dehydrogenase (LDH) release analysis was removed, and the attached monolayer hepatocytes
purchased from Abcam (Cambridge, UK) and the were exposed the BPA at concentrations of 10, 50,
kit for the caspase-3 activity measurement was 100, 250, 500, and 1000 μM for 24 and 48 h.
purchased from Sigma (Jerusalem, Israel). All of Control cells received only L-15 medium
the other chemicals used in this study were containing 0.1% (v/v) absolute ethanol. Three
analytical grade and obtained from Merck replicates were used in each experiment. The cell
(Darmstadt, Germany). cultures were routinely checked by morphological
changes and contamination using an inverted
Hepatocyte isolation. The hepatocytes were microscope (Leica DM 6000, Germany) during
isolated in a two-step perfusion method as experiment. After the exposures were completed
described by Berry and Friend [13] and modified by photos of cultures were taken.
Mortensen et al. [14]. Prior to the cell culture
studies, all of the glassware and instruments were Lactate dehydrogenase release. Cellular
sterilized and the solutions were sterilized by damage was monitored by measuring the LDH
filtering using a 0.22-μm filter (Millipore, Ireland) release into the culture medium. After the 24 and 48
before use. The hepatocytes were prepared from h BPA exposures, the culture medium was removed
four individual fish. Briefly, the liver of the from 48 wells by gentle pipetting and transferred to
anesthetized fish with 2-phenoxyethanol was eppendorf tubes. The tubes were centrifuged at 600
carefully excised and transferred onto a glass petri × for 10 min to precipitate the cells. Clear
dish, and rinsed with a cold calcium-free solution medium was removed and used for the LDH
containing NaCl (7.14 g/L), KCl (0.36 g/L), MgSO 4 analysis. LDH activity in the medium was
(0.15 g/L), Na2HPO4 (1.6 g/L), NaH2HPO4 (0.4 performed using a LDH-cytotoxicity assay kit
g/L), NaHCO3 (0.31 g/L), and EGTA (20 mg/L) DFFRUGLQJWRWKHPDQXIDFWXUHU¶VLQVWUXFWLRQV All of
under sterile conditions. The liver was dissected the experiments were carried out in triplicate.
into small pieces in this solution and agitated by Samples were loaded onto 96-well plates and their
pipetting. Next, tissue pieces were rinsed with a absorbance values were measured with an ELISA
clear solution and this process was repeated until all plate reader (DAS Instruments, model A3, Rome,
of the blood was flushed out. The whitened tissue Italy) at 450 nm. All of the measurements were
pieces were then transferred into the same buffer taken in duplicate and the LDH release was
with 0.22 g/L CaCl2, instead of EGTA, and expressed as the percentage of cytotoxicity.
collagenase (0.53 mg/mL), and the tissue was
digested for 15 min in this solution. Thereafter, the
1168


Caspase-3 activity. Caspase-3 activity was cells were observed with concentrations of 250 μM
determined by a colorimetric assay based on the BPA and higher (Figure 2(eíg)).
hydrolysis of the peptide substrate acetyl-Asp-Glu-
Val-Asp p-nitroaniline (pNA) by caspase-3, Lactate dehydrogenase release. LDH
resulting in the release of the pNA moiety. An correlates with cell viability, thus being a useful
increase in the absorbance was used to quantify the indicator of cellular damage. Parallel to
activation of the caspase-3 activity. For the morphological observations, time- and dose-
determination of caspase-3 activity, a commercial dependent increases in the LDH release into the
caspase-3 assay kit was used and all of the media were observed after BPA exposures at 24
processes were performed according to the and 48 h with all of the concentrations ( 0.05).
manufactXUHU¶V LQVWUXFWLRQV. After exposure, The LDH release was at the highest levels with
hepatocytes from 48 wells were collected and the concentrations of 250 μM BPA and higher in both
medium was removed by a low speed 24 and 48 h (Figure 3).
centrifugation (600 × , 5 min), and the cells were
then washed with cold phosphate buffered saline Caspase-3 activity. BPA caused significant
(PBS). After removal of the PBS by a second increases in the caspase-3 activity with 100 and 250
centrifugation, the cells were lysed in lysis buffer μM BPA at 24 h, while the caspase-3 activity
(20 μL) for 20 min on ice. All of the experiments remained unchanged in the other exposure groups.
were carried out in triplicate. The cell lysates were At 48 h exposure to BPA, significant increases
centrifuged at 20,000 × for 15 min at 4 °C. The were observed with 50 and 100 μM BPA. The
samples were loaded onto 96-well plates and their caspase-3 activity returned to that of the control
absorbance values were measured with an ELISA levels with 250 μM BPA. Significant differences in
plate reader (DAS Instruments, model A3, Rome, the caspase-3 activity were not observed in the
Italy) at 412 nm. All of the measurements were other exposure groups at 48 h (Figure 4).
taken in duplicate and activity was expressed as
μmol pNA/min/mL.
DISCUSSION
Statistical analyses. Statistical analyses were
performed using the Statistical Package for the In this study, we demonstrated that the
Social Sciences software, version 16.0. Differences exposure of rainbow trout cultured hepatocytes to
among the data were analyzed using a one-way BPA caused morphological changes and significant
analysis of variance (ANOVA) with a post hoc decreases in the cell viability in a dose- and time-
'XQQHWW¶V WHVW 7KH UHVXOWV ZHUH H[SUHVVHG DV WKH dependent manner. It has been generally accepted
mean ± standard error of the mean (SEM). Values that the release of the cytosolic LDH correlates with
of < 0.001, < 0.01 and < 0.05 were cellular viability, thus being an important indicator
considered to be statistically significant. of plasma membrane damage [15]. BPA
prominently decreased cell viability at a
concentration of 100 μM and it was found to be
RESULTS highly cytotoxic at concentrations of 250 μM and
higher at 24 and 48 h. Similar to our observations,
Hepatocyte morphology and viability. After Iida et al. [16] reported apoptotic changes and
24 h incubation of the cells, morphological changes decreased cell viability in rat Sertoli cells at
were not observed at lower concentrations (100 150300 μM BPA. It has been reported that BPA
μM) of BPA (Figure 1(aíc)). The cells started to significantly reduced the viability of TM4 cells, a
degenerate and shrink at 100 μM BPA (Figure Sertoli cell line, by elevating of intracellular
1(d)). Cytotoxicity was marked with concentrations calcium level at between 100 and 600 μM BPA
of 250 μM BPA and higher. Mostly shrunken and [17]. BPA was reported to be relatively nontoxic at
dead cells were observed at these concentrations concentrations below 250 μM and toxic at
(Figure 1(eíg)). After 48 h exposure, the concentrations above 500 μM in isolated rat
hepatocytes demonstrated a cord-like arrangement hepatocytes after 3 h exposure [18]. On the other
in the control groups, and in the 10 μM (Figure hand, BPA-induced cytotoxicity was observed at
2(a,b)) and slow cord-like arrangements 50 μM concentrations of 50100 μM in isolated rat
BPA-exposed cells (Figure 2(c)). An increased hepatocytes at 18 h [19]. Our results demonstrated
amount of the degenerated and shrunken cells were WKDW%3$FRQFHQWUDWLRQVRI0VHHPWRUHVXOW
observed with 100 μM BPA (Figure 2d)). Similar to in slight toxic effects at 24 h, while clear evidence
that of the 24 h exposure, mostly shrunken and dead of cytotoxicity of BPA at concentration of 100 μM
at 48 h. BPA was severely cytotoxic at higher
1169


FRQFHQWUDWLRQV 0 RQ UDLQERZ WURXW BPA between the present study and other studies
hepatocytes at both 24 and 48 h. The different may be attributed to the design of the experiment,
results observed in the cytotoxic concentrations of cell type, and species.
FIGURE 1
Effects of BPA on hepatocyte morphology after 24 h. Intact cells in the control cells (a). Similar views to
control cells at the 10 (b) and 50 μM (c) BPA exposed cells. Cell degenerations (arrows) and shrunkens
(arrowheads) in the 100 μM concentration (d). Mostly degenerated and shrunken cells in the 250 (e), 500
(f) and 1000 (g) μM BPA exposed cells (bars = 40 μm).
1170


FIGURE 2
Effects of BPA on hepatocyte morphology after 48 h. Cord-like arrangement in the control (a) and 10 μM
exposed (b) cells. Slow cord-like arrangements in the 50 μM BPA exposed cells (c). Increased number of
degenerated (arrows) and shrunkens (arrowheads) cells in the 100 μM BPA exposed cells (d). Mostly
degenerated and shrunken cells in the 250 (e), 500 (f) and 1000 (g) μM BPA exposed cells (bars = 40 μm).
Caspase-3 is an effector caspase expressed in the stage when commitment to loss of cell viability
cells undergoing apoptosis. It can process is made [20]. Therefore, we measured caspase-3
procaspase -2, -6, -7, and -9, and plays a central activity in the hepatocytes treated with BPA in
role in mediating nuclear apoptosis, including here. The results showed significantly increased
chromatin condensation, DNA fragmentation, and caspase-
cell blebbing, but it may also function before or at
1171


FIGURE 3
Dose- and time-dependent effects of selected BPA concentrations on cytotoxicity of primary cultured
hepatocytes at 24 and 48 h. Cytotoxicity was judged by lactate dehydrogenase release into the culture
media. Results were expressed as % cytotoxicity. Data are the mean ± SEM of three experiments each
were performed in duplicate. Symbols obscure SEM bars.
FIGURE 4
Dose- and time-dependent effects of selected BPA concentrations on caspase-3 activities of primary
cultured hepatocytes at 24 and 48 h. Results were expressed as μM pNA/min/mL. Data are the mean ±
SEM of three experiments each were performed in duplicate. Data are the mean ± SEM of three
experiments each were performed in duplicate. Significance at : 0.05; : 0.01; : 0.001
compared to control.
3 activity with 100 and 250 μM BPA at 24 h, and induced apoptosis occurred by caspase-3 activation
with 50 and 100 μM BPA at 48 h. These results in HL-60 cells treated with BPA and the active
demonstrate that caspase-3 activation occurs in a form of caspase-3 showed a peak at 3h and declined
dose- and time-dependent manner. On the other at 5 h. An explanation for the unchanged activities
hand, caspase-3 activity declined with 250 μM at of caspase-3 in this study with 500 and 1000 μM
48 h, and remained unchanged at both 24 and 48 h BPA at 24 and 48 h might be that caspase-3
at higher concentrations. In accordance with our activation occurred in the cells in the early stages of
results, Terasaka et al. [21] reported that BPA- the incubation and declined before 24 h with 500
1172


and 1000 μM BPA, in a similar fashion to that [6] Segner, H. (1998). Isolation and primary culture
observed with 250 μM BPA. The results suggest of teleost hepatocytes. Comp. Biochem.
that BPA-induced hepatocyte apoptosis in fish Physiol. A 120, 71±81.
occurs by caspase-3 activation and caspase-3 is an [7] Tollefsen, K. E., Blikstad, C., Eikvar, S., Finne,
early signal of BPA-induced cytotoxicity. E. F., Gregersen, I. K. (2008). Cytotoxicity of
alkylphenols and alkylated non-phenolics in a
primary culture of rainbow trout
CONCLUSION ( n n m i ) hepatocytes.
Ecotoxicol. Environ. Saf., 69, 64±73.
In conclusion, the current study demonstrates [8] Rankhouhi, T. R, van Holsteijn, I., Letcher, R.,
that BPA exhibited cytotoxic effect on rainbow Giesy, J. P., van den Berg, M. (2002). Effects of
hepatocytes. It is suggested that cytotoxicity of primary exposure to environmental and natural
rainbow trout hepatocytes induced by BPA is estrogens on vitellogenin production in carp
mediated by caspase-3 activation. (C in a i ) hepatocytes. Toxicol. Sci.,
67, 75±80
[9] Park, C. B., Kim, B. H., Na, O. S., Choi, Y. C.,
ACKNOWLEDGEMENTS Lee, Y. D., Baek, H. J., Kim, H. B., Takemura,
A. (2003). Induction of in it vitellogenin
synthesis by bisphenol, nonylphenol and
This work was supported by the Yuzuncu Yil
octylphenol in Chinese minnow ( xin
University, Directorate of Scientific Research
Projects, under project number: 2013-FEN-B045. x e al ) hepatocytes. Korean J. Biol. Sci.,
The authors wish to thank the Directorate of 7, 227±235.
[10] Petersen, K., Tollefsen, K. (2011). Assessing
Scientific Research Projects for providing financial
combined toxicity of estrogen receptor agonists
assistance.
in a primary culture of rainbow trout
( n n m i ) hepatocytes. Aquat.
e a t e la e t at t e a e n n li t
inte e t Toxicol., 101,186±195.
[11] Turker, H., Takemura, A. (2011). Effects of
environmental contaminants and natural
substances on vitellogenesis in tilapia primary
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2¶%ORFN67+DUULV/5$UHYLHZ chemicals. Test no. 203: acute fish test.
of the environmental fate, effects, and [13] Berry, M. N., Friend, D. S. (1969). High yield
exposures of bisphenol A. Chemosphere 36, preparation of isolated rat liver parenchymal
2149±2173. cells. J. Cell Biol., 43, 506±520.
[2] Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., [14] Mortensen, A. S., Tolfsen, C. C., Arukwe, A.
Laufer, H., LeBlanc, G.A., Guilette, Jr L. J. (2006). Gene expression patterns in estrogen
(2007). An ecological assessment of bisphenol- (nonylphenol) and aryl hydrocarbon receptor
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Toxicol., 24, 225±239. trout ( n n m i ) primary
[3] Franco, R., Sánchez-Olea, R., Reyes-Reyes, E. hepatocyte culture. J. Toxicol. Environ. Health
M., Panayiotidis, M. I. (2009). Environmental A, 69, 1±19.
toxicity, oxidative stress and apoptosis: ménage [15] El-Shenawy, N. S. (2010). Effects of
á trois. Mutat. Res., 674, 322. insecticides fenitrothion, endosulfan and
[4] Parish, A. B., Freel, C. D., Kornbluth, S. (2013). abamectin on antioxidant parameters of
Cellular mechanisms controlling caspase isolated rat hepatocytes. Toxicol. In Vitro, 24,
activation and function. Cold Spring Harb. 1148±1157.
Perspect. Biol., 5(6). doi: [16] Iida, H., Maehara, K., Doiguchi. M., Mori, T.,
10.1101/cshperspect.a008672 Yamada, F. (2003). Bisphenol A-induced
[5] Bols, N. C., Dayeh, V. R., Lee, L.E.J., apoptosis of cultured rat Sertoli cells. Reprod.
Schirmer, K. (2005). Use of fish cell lines in the Toxicol., 17, 457±464.
toxicology and ecotoxicology of fish. Piscine [17] Hughes, P. J., McLellan, H., Lowes, D. A.,
cell lines in environmental toxicology. Zafar Khan, S., Bilmen, J. G., Tovey, S. C.,
Biochemistry and Molecular Biology of Fishes, Godfrey, R. E., Michell, R. H., Kirk, C. J.,
vol. 6. Elsevier Science, Amsterdam, pp 43±84. Michelangeli, F. (2000). Estrogenic
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alkylphenols induce cell death by inhibiting

testis endoplasmic reticulum Ca2+ pumps.
Biochem. Biophys. Res. Commun., 277, 568±
574.
[18] Nakagawa, Y., Tayama, S. (2000). Metabolism
and cytotoxicity of bisphenol A and other
bisphenols in isolated rat hepatocytes. Arch.
Toxicol., 74, 99±105.
[19] Pritchett, J. J., Kuester, R. K., Sipes, I. G.
(2002). Metabolism of bisphenol A in primary
cultured hepatocytes from mice, rats, and
humans. Drug Metab. Dispos., 30, 1180±1185.
[20] Porter, A. G., Janicke, R. U. (1999). Emerging
role of caspase-3 in apoptosis. Cell Death
Differ., 6, 99±104.
[21] Terasaka, H., Kadoma, Y., Sakagami, H.,
Fujisawa, S. (2005). Cytotoxicity and
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hydroquinone in HL-60 cells. Anticancer Res.,
25, 2241±2248.
Received: September 23, 2015

Accepted: January 04, 2016
Burak Kaptaner
Department of Biology
Faculty of Science
Yuzuncu Yil University
65080 7XúED Van ± TURKEY
e-mail: bkaptaner@yahoo.com,
bkaptaner@yyu.edu.tr
1174


SEASONAL CHARACTERISATION OF THE LOCAL

SEWAGE SLUDGE
Fou ia Benoud it1, Mohamed Amine Benoud it2, Messaoud Hachemi1
1
Research Unit of Materials, Processes, Environment (URMPE), 0¶KDPHG%RXJDUD8QLYHUVLW\%RXPHUGqV$OJHULD
2
The School of Architecture, Computing and Engineering (ACE), University of East London, United Kingdom
ABSTRACT inevitable by-product from wastewater treatment

plants (WWTP) [1-3], and its quantities are
7KH VHZDJH VOXGJH¶V FKDUDFWHULVDWLRQ LV growing dramatically on a yearly basis, and thus
necessary to reuse it in a safe way or to dispose of it owe to be seriously considered. Activated sludge
in an environmental friendly manner. The present processing technique is one of the largely adopted
paper focuses on the seasonal characterisation of systems to treat urban and industrial WW, but it
the municipal sewage sludge from wastewater generates a huge amount of SS of which the
treatment plant located in the town of Boumerdès, inadequate disposal can be dangerous. At present,
Algeria, and on the comparison of the obtained the main methods of disposal and reuse are:
results with literature. This work describes the incineration, landfill and land application.
VHZDJH VOXGJH¶V SK\VLFDO FKHPLFDO Pineralogical Recent researches concluded the possibility to
and morphological features during the four seasons use municipal SS as a fertiliser [4-7], for
of year 2013 and identifies their seasonal changes. composting or energy production by incineration
The analysis showed a small variation in the [8] and for the development of building materials
organic matter and heavy metals content. [2, 9, 10].
$SSUR[LPDWHO\ KDOI VHZDJH VOXGJH¶V ZHLJKW LV SS is a heterogeneous solid whose volume and
organic matter which is composed of several composition vary which depend on the origin of to
functional groups that were identified by Fourier be treated effluent and on the technology used to
transform infrared spectroscopy. The atomic treat WW [2]. Therefore, it is essential to
absorption spectrometer analysis revealed a low characterise the sludge over period of time in order
concentration of heavy metals, which is much lower WR HYDOXDWH LWV FRPSRVLWLRQ¶V VWDELOLW\ DQG LGHQWLI\
WKDQ WKH $OJHULDQ¶V VWDQGDUG. The X-ray alternative methods for its safe reuse or dispose.
fluorescence analysis showed the presence of some Algeria has 72 WWTPs that use the activated
oxides especially SiO2, Al2O3, Fe2O3 and CaO. In sludge technique producing over 400000
order to deepen the understanding of sewage tonnes/year of SS whose majority is disposed of in
VOXGJH¶V PLFURVWUXFWXUH DQG FU\VWDOOLQH SKDVH landfills which could negatively affect the
respectively, it was analysed with an X-ray environment and underground water. This paper
diffraction and a scanning electron microscopy. The studies the physicochemical characterisation during
analyses have revealed the presence of the same the four seasons of the year 2013 of the municipal
major cryVWDOOLQH SKDVHV LQ DOO IRXU VHDVRQV¶ SS of WWTP which is located in Boumerdès,
samples. The analysis have also confirmed the low Algeria, and has processing capacity of 15000 m3
density of the sewage sludge, its high porosity, its and uses a biological treatment through an activated
SRURXV VKDSH LWV GLIIHUHQW SDUWLFOHV¶ VL]H DQG LWV SS with low loading mass and an extended aeration
irregular morphology. process. The aim is to identify alternative ways to
UHXVH %RXPHUGqV¶ DQG $OJHULD¶V 66 ZKLFK LV E\-
product of domestic WW which channels to the
KEYWORDS: station through a unitary sewerage system.
Sewage sludge, seasonal characterisation, physico-
chemical analysis, organic matter, heavy metals
INTRODUCTION Sewage sludge samples collection. The SS

samples used for the characterisation were collected
Several sewerage systems and treatment plants from the filter press outlet. They were sampled and
have been planted worldwide to minimise the preserved respectively according to the
environmental pollution of discharged untreated international standards ISO 5667-13 [11] and ISO
wastewater (WW). Sewage sludge (SS) is an 5667-15 [12].
1175


The approximate flow of WW and the quantity flow of WW is probably due to an increase in the
of dehydrated SS discharged by the plant for each water consumption when the weather is warmer.
season of the year 2013 are listed in Table 1 below. From the data in Table 1, it seems that although the
The WW flow and SS quantity increase between water flow in autumn is more than its flow in
the seasons. They are at their lowest amount in spring, the quantity of dehydrated SS is higher in
winter, and their highest amount in summer. The spring than in autumn. The quantity of discharges
flow and the quantity for the spring and the autumn SS decreases when it rains because the access to the
seasons are less than the summer season but much fill during this period is difficult and the excess
more than the winter season. The variation in the sludge is simply kept inside the thickener.
TABLE 1
Seasonal WW flow and dehydrated SS uantity.
Seasons Winter Spring Summer Autumn

3
WW flow (m ) 833606 1112549 1335453 1214357
Dehydrated SS quantity (t of dry solids) 44 160 272 153
Experimental. The experimental process VOXGJH¶V PRUSKRORJ\ DQG VWUXFWXUH DUH VWXGLHG RQ
VWDUWV E\ RYHQ GU\LQJ WKH 66¶ VDPSOHV JULQGLQJ the dried samples with a PHILIPS FEI Quanta 250
them with a disc mill and then storing them for the scanning electron microscope (SEM). The bulk and
physico-chemical analyses listed below. The SS the real density of each sample are calculated
VDPSOHV¶S+DQGHOHFWULFDOFRQGXFWLYLW\(&YDOXHV following the standards ISO 11272 [18] and ISO
are measured before and after drying the samples 11508 [19], respectively, and the porosity is
following the standards NF EN12176 [13] and deduced from them.
,62 >@ UHVSHFWLYHO\ 66¶ PRLVWXUHV LV
calculated according to the mass difference before
and after thH66EHLQJGULHGLQDQRYHQDWÛ&IRU RESULTS AND DISCUSSIONS
K 1) (1 >@ 7KH 66¶V YRODWLOH
suspended solid (VSS) content is determined by pH and EC. The results of the pH and EC
FDOFLQDWLRQDWÛ&IRUKIROORZLQJ1)(1 analyses before and after drying the SS are in Table
standard [16], while the loss on ignition (LOI) is 2.
estimated by calcination at Û& 7RWDO RUJDQLF The tested dehydrated SS was from slightly
carbon (TOC) content is also determined using the acidic to neutral between 6.69 and 7.02. This is
ISO 10694 [17] international standard and a LECO typical of the sludge produced during the biological
WR-112 carbon analyser. The concentration of treatment of municipal sewage [5]. The pH values
major elements in the sludge is performed by X-ray decrease and become more acidic [5.85-6.61] when
fluorescence (XRF) with an AXIOS minerals SS is dried, since drying SS decreases the
PANALYTICAL X-ray spectrometer (working liquid/solid ratio and thus increases the
conditions: 50 kV and 60 mA). The soluble salts concentration of free acidic ions. Changes in the pH
(SO42- and Cl-) and carbonates (CaCO3) are values during the four seasons are not really
determined by means of analytical methods. The significant for either dry SS or dehydrated SS.
concentration of minor elements in the sludge is The tests completed in this section show that
determined using an Atomic Absorption the EC of the dehydrated SS samples is relatively
Spectrometer (AAS) VARIAN AA 240. The low and varies between 0.350 and 0.618 mS/cm,
mineralogical composition of the SS is determined which means the presence of soluble salts is low. At
by employing the X-ray diffractometry (XRD) this stage, a comparison between the EC values of
analysis, which is performed in a PANALYTICAL the four seasons cannot be completed since the
XPERT-PRO X-ray diffractometer (working water flow is not constant. Table 2 shows that the
conditions: 45 kV and 40 mA). Fourier transform EC values of the dried SS are way above those
infrared spectroscopy (FTIR) characterisation is recorded for dehydrated SS since their values
performed on KBr pellet (4-6 mg of sample/200 mg oscillated between 3.7 and 4.4 mS/cm. The rise in
KBr) at various wavenumber infrared rays with a the EC values of the dried SS compared to the
range of frequencies from 4000 to 400 cm-1 with an hydrated SS is also due to the decrease of the
ALPHA-T BRUKER FTIR spectrophotometer. The liquid/solid ratio. The highest EC values of the dry
1176


and the dehydrated SS are recorded in the summer weather is cool or cold. The percentage of
which is possibly due to the increase in the water carbonates (CaCO3) is between 3.98% and 5.34%.
consumption during this period of the year. Due to the unitary sewer system and soil erosion
caused by rainfall, CaCO3 is at its highest in winter.
Moisture, SS, TOC and LOI. The values of According to the international standard NF ISO
moisture, VSS, TOC and LOI are in Table 2. The 10693 [22] for soil classification, the carbonate
amount of moisture in SS is between 83.98% and FRQWHQW RI %RXPHUGHV¶ ::73¶V 66 PDNHV LW
86.72% which is high. The weight percentage of similar to from a non-calcareous to a low
VSS is also relatively high since it varying between calcareous soil.
DQG 7KLV 966¶ FRQVLGHUDEOH
SHUFHQWDJH FRQILUPVWKHVOXGJH¶V XUEDQRULJLQDQG The nature and the concentration of
shows that this sludge is rich in organic matter. The contaminants in the sludge, such as heavy metals,
amount of TOC amount recorded varies between are dependent on the origin of the raw water
7.32% and 12.05%. TOC and VSS are linked since arriving to a WWTP. The dewatered SS studied
they both increase and decrease together. Loss on contains heavy metals but their concentrations
ignition (LOI) during the period of study varies (Table 2) are lower than the maximum values
between 50.89% and 61.77 %. For each season, the accepted by the Algerian standard NA 17671 [23]
difference between VSS and LOI is not significant, for farmland application. These low concentrations
which means that LOI is principally due to the confirm the urban origin of the water from which
decomposition of organic matter. the studied SS is extracted. The fact that the
concentrations of heavy metals are in line with the
Chemical composition. The major and the Algerian standard makes this sludge a good
minor elements and the soluble salts are expressed candidate for agricultural reuse.
in in percentage by weight (%) and in mg/kg of dry
matter. The results listed in Table 2 show that 7KHUHDUHVOLJKWYDULDWLRQVLQWKHKHDY\PHWDOV¶
sulphate (SO42- LRQV¶ DQG FKORULGH LRQV¶ &O-) concentrations between the four seasons of year
percentages in the sludge vary, respectively, 2013. The sum of the heavy PHWDOV¶FRQFHQWUDWLRQV
between 0.72% and 1.46%, and 0.24% and 1.07%. in the autumn and winter seasons is slightly higher
These results also show that the sludge contains than their sum in the spring and summer seasons.
several oxides. The four majors are: silica (SiO2), The difference in this sum between the seasons is
alumina (Al2O3), calcium oxide (CaO) and iron possibly caused by the rainfall which transfers
oxide (Fe2O3). The silica percentage is higher than heavy metals from the pavements and road
the other oxides by a significant margin. The sum infrastructures into the unitary sewer system. It is
RIWKHDOXPLQRVLOLFDWHV¶DPRXQWZKLFKcompose the evident that the total concentration of heavy metals
reactive part of pozzolanic materials [20] for each in sludge indicates the extent of contamination, but
of the four seasons is between 19.19% and 28.69%. provides only a little insight about the heavy
These oxides are in the same range as in [2] and PHWDOV¶ IRUP WKHLU PRELOLW\ DQG WKHLU
[10] who studied the valorisation of SS in ceramic bioavailability when used in farmland applications
material and lightweight aggregate. [24].
The amount of phosphates (P2O5) in the SS Mineralogy. The crystalline phases in the
oscillated between 1.54% and 3.21%, and its seasonal SS were composed of quartz, calcite and
presence is likely due to the discharge of domestic illite (Table 3). The following differences in it
detergents in WW [21]. P2O5 is at its lowest in crystalline composition were observed. First, albite
winter, but it increases in spring to reach its highest was not detected in summer, but traces of calcium
is summer. P2O5 in autumn is lower than in summer sulphate (anhydrite) were found. Second, dolomite
but higher than the two other seasons. As a only appeared in winter, and kaolinite was
conclusion, the amount of P2O5 seems to be high presented in both winter and spring. Last,
when the weather is warm or hot and low when the clinochlore was noticed in summer and autumn.
1177


TABLE 2
Physical and chemical composition of SS during the year 2013.
Parameters Units Winter Spring Summer Autumn NA 17671 [23]

pH (dehydrated SS) - 7.02 6.81 6.90 6.69 -
pH (dry SS) - 6.61 5.99 6.31 5.85 -
EC (dehydrated SS) mS/cm 0.364 0.472 0.618 0.350 -
EC (dry SS) mS/cm 3.7 4.3 4.4 4.2 -
Moisture % 84.27 83.98 86.72 85.49 -
VSS % 60.99 47.04 44.79 55.73 -
LOI % 61.28 50.89 50.89 61.77 -
TOC % 12.05 7.32 7.08 9.54 -
SiO2 % 22.02 15.02 19.97 19.88 -
Al2O3 % 6.67 4.17 5.97 5.69 -
Fe2O3 % 6.73 6.4 6.45 7 -
CaO % 7.45 6.38 6.53 6.62 -
MgO % 1.03 0.22 1.02 1.03 -
Na2O % 0.24 0.12 0.44 0.27 -
K2O % 1.74 1.43 1.64 1.58 -
TiO2 % 0.58 0.54 0.55 0.51 -
MnO % 0.29 0.3 0.28 0.38 -
P2O5 % 1.54 2.06 3.21 2.82 -
BaO % 0.17 0.19 <LOD <LOD -
SrO % 0.05 0.05 <LOD 0.06 -
Cl- % 0.24 0.26 1.07 0.84 -
SO42- % 0.72 1.02 0.84 1.46 -
CaCO3 % 5.34 4.21 4.64 3.98 -
Co mg/kg <LOD <LOD <LOD <LOD -
Mo mg/kg <LOD <LOD <LOD <LOD -
Cd mg/kg <LOD <LOD <LOD <LOD 20
Cu mg/kg 130 150 90 150 1000
Cr mg/kg 90 80 80 230 1000
Pb mg/kg 80 90 100 90 800
Zn mg/kg 770 480 50 360 3000
Ni mg/kg <LOD 50 <LOD 50 200
Cr+Cu+Ni+Zn mg/kg 990 760 220 790 4000
TABLE 3
&U\VWDOOLQHSKDVHVRI66¶VDPSOHVRIWKHIRXUVHDVRQVRI\HDU
Seasons Winter Spring Summer Autumn

Crystalline Quartz Quartz Quartz Quartz
phases Calcite Calcite Calcite Calcite
Illite Illite Illite Illite
Albite Albite Clinochlore Albite
Kaolinite Kaolinite Anhydrite Clinochlore
Dolomite
FTIR spectra. Figure 1 contains the FTIR corresponds to O±H stretching vibrations present in
spectra of the dried SS for all four seasons. The the acids and alcohols groups, and the broad bands
IXQFWLRQDO FKDUDFWHULVWLFV RI WKH VOXGJH¶V VDPSOHV centred between 3300-3400 cm-1 correspond to the
were qualitatively similar but with different H-bonded of two functional groups OH and NH.
amplitudes. In the spectra, the peak at 3423 cm-1 These two functional groups could indicate the
1178


presence of components such as alcohols, around 1033 cm-1 can be attributed to the C±O
carboxylic acids, amides or amines. The weak stretching attributed to polysaccharides and
peaks around the wavenumber 2900 cm-1 could be aromatics ethers. These peaks could also be due to
attributed to the asymmetrical stretching of C±H silicates (Si±O stretching). The bands centred at
bonds from methyl and methylene groups, and the 778-797 cm-1 are attributed to the symmetric
bands at 2853 cm-1 could represent the symmetrical stretching of Si±O±Si which confirms the presence
stretching of C±H from the methylene groups. of silicates. The bands around 469 cm-1 are due to
Pronounced peaks between 1638 and 1654 cm-1 the bending frequency of O±Si±O, and the bands
correspond to the aromatic C=C stretching around 600-900 cm-1 might be attributed to Si±O±
vibrations, H-bonded C=O and NH2 deformation in Al vibrations which results from the substitution of
the amide I. Absorption at around 1560 cm-1 and Al by Si in the Si±O vibrations. Finally, peaks
1542 cm-1 correspond to the N±H bending in the below 900 cm-1 could also be due to aromatic
amide II structures. C±C aromatic skeletal structures or skeletal vibrations, or to one of the
vibrations which might correspond to the lignin following functional groups: amine, amide,
structures are found between wavenumbers 1600 phosphate or sulphur.
and 1500 cm-1. The main absorption bands and peaks for
The peaks between 1420 and 1440 cm-1 and %RXPHUGHV¶ ::73¶V VOXGJH DUH VLPLODU WR WKRVH
bands that spread between 875 and 876 cm-1 reported by Abrego et al. [25], Guibaud et al. [26],
represent the presence of calcite (CaCO3). The band Thipkhunthod et al. [27], Filip et al. [28], and
around 1238 cm-1 is assigned to C=O stretching in Laurent et al. [29].
carboxylic acids. The strong peaks appearing at
FIGURE 1
)7,5VSHFWUDRI66¶VDPSOHVRIWKHIRXUVHDVRQVRI\HDU
Morphological characterisation. Figure 2 Density and porosity. The apparent density

VKRZVPLFURJUDSKVRIWKHGULHG66¶VDPSOHGGXULQJ (i.e. bulk density), the real density and the porosity
the four seasons of year 2013 which taken by a values of the studied SS are summarised in the
scanning electron microscope (SEM). These Table 4. The bulk density values of the dry SS
micrographs show that the dried SS has a porous during the four seasons oscillates between 0.822
VKDSH DQ LUUHJXODU SDUWLFOHV¶ PRUSKRORJ\ DQG and 1.057 g/cm3. The real density for the same
YDULDWLRQV LV WKH SDUWLFOHV¶ VL]H 7KHVH PLFURJUDSKV period varies between 1.723 and 1.983 g/cm3.
are similar to those in Bouzid et al. [30] who These relative low density values are due to the
studied SS sample which was collected from presence of organic matter. The slight difference in
municipal WWTP of the city of Sfax, south of density between the four seasons may be caused by
Tunisia. the slight difference in the proportion between
1179


PLQHUDO DQG RUJDQLF PDWWHUV 7DEOH 7KH 66¶ and can be attributed to the high amount of the
density values are close to the density of the clay, organic matter. These results are in accordance with
DQG WKXV %RXPHUGHV¶ ::73¶V VOXGJH FDQ EH porous shape in the SEM micrographs in Figure 2.
considered as a lightweight aggregate. The high
porosity of SS varies between 43.60% and 52.30%
FIGURE 2
6(0PLFURJUDSKVRIGULHG66¶VVDPSOHVRIWKH\HDUE\VHDVRQV
TABLE 4
%XONDQGUHDOGHQVLWLHVDQGSRURVLW\RI66¶VDPSOHVRIWKHIRXUVHDVRQVRI\HDU
Parameters Units Winter Spring Summer Autumn

Bulk density g/cm3 1.015 1.057 0.822 0.908
Real density g/cm3 1.866 1.983 1.723 1.911
Porosity % 43.6 46.7 52.3 52.2
CONCLUSION PDWWHU WKHUH LV RQO\ D VPDOO YDULDWLRQ LQ WKH 66¶
composition between the samples. All the
This study was concerned with the seasonal variations including those of the organic matter
characterisation of the sewage sludge of the have been mostly noticed between the samples
wastewater treatment plant located in the city of extracted in the cold and rainy seasons and those
Boumerdès, Algeria. The aims were to learn about extracted in the warm and dry seasons, and are
WKH66¶FRPSRVLWLRQDQGTXDOLW\DQGWRLGHQWLI\WKH principally due to an increase in water consumption
most appropriate ways to reuse it. The results of the when the weather is warm and the rainfall in the
analyses described in the previous section shows cold weather.
that the studied SS is composed of salts, mineral The low concentration in heavy metals, the
matter, half weighted organic matter and a low richness in organic matter, and the aluminosilicates¶
concentration of heavy metals. The heavy metals content which are in line with the literature makes
concentration is well below the maximum threshold the studied SS ideal as fertiliser or as a components
DOORZHGE\$OJHULDQ¶VVWDQGDUG$VH[SODLQHGDWWKH for building materials, or simply incinerated to
EHJLQQLQJ RI WKLV SDSHU WKH DQDO\VHG 66¶ VDPSOHV produce energy. At this stage of the
were sampled during all four seasons of year 2013. characterisation, the reuse of excess sludge seems
The results of the analyses have also shown that to be a good alternative.
overall and with the exception of the organic
1180


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- Part 15: Guidance on preservation and
1 Chen, J.C. (2004) Emission characteristics of handling of sludge and sediment samples.
industrial sludge incineration in different [13] NF EN 12176: 1998 : Caractérisation des
operating conditions. Environ Technol., 25, boues - Détermination de la valeur du pH.
1285-1292. [14] ISO 11265: 1994: Soil quality - Determination
[2] Jordán, M.M., Almendro-Candel, M.B., of the specific electrical conductivity.
Romero, M. and Rincón, J.M. (2005) [15] NF EN 12880: 2000 : Caractérisation des
Application of sewage sludge in the boues - Détermination de la teneur en matière
manufacturing of ceramic tile bodies. Appl sèche et de la teneur en eau
Clay Sci., 30, 219-224. [16] NF EN 12879: 2000 : Caractérisation des
[3] Liang, Z., Peng, X., Wang, J., Luan, Z., Liu, boues - Détermination de la perte au feu de la
Z. and Wang, Y. (2011) Immobilization of matière sèche
phosphorus in sewage sludge using inorganic [17] ISO 10694: 1995: Soil quality - Determination
amendments. Environ Earth Sci., 63, 221-228. of organic and total carbon after dry
[4] Tamoutsidis, E., Papadopoulos, I., Tokatlidis, combustion (elementary analysis)
I., Zotis, S. and Mavropoulos, T. (2002) Wet [18] ISO 11272: 1998: Soil quality - Determination
sewage sludge application effect on soil of dry bulk density.
properties and element content of leaf and root [19] ISO 11508: 1998: Soil quality - Determination
vegetables. J Plant Nutr., 25(9), 1941-1955. of particle density.
[5] Dusza, E., Zablocki, Z. and [20] Cyr, M., Coutand, M. and Clastres, P. (2007)
Mieszczerykowska-Wójcikowska, B. (2009) Technological and environmental behavior of
Content of magnesium and other fertilizer sewage sludge ash (SSA) in cement-based
compounds in stabilized and dewatered materials. Cement Concrete Res., 37, 1278-
sewage sludge from the municipal sewage 1289.
treatment plant in Recz. J Elementol., 14(1), [21] Merino, I., Arévalo, L.F. and Romero, F.
63-70. (2007) Preparation and characterization of
[6] Karef, S., Kettab, A. and Nakib, M. (2013) ceramic products by thermal treatment of
Characterization of byproducts from sewage sludge ashes mixed with different
wastewater treatment of Medea (Algeria) with additives. Waste Manage. 27, 1829-1844.
a view to agricultural reuse. Desalin Water [22] ISO 10693: 1995, Soil quality - Determination
Treat., 52, 2201-2207. of carbonate content -- Volumetric method.
[7] :RáHMNR ( %XWDUHZLF] $ :\GUR 8 DQG [23] NA 17671: 2010: Matières fertilisantes -
àRERGD 7 $GYDQWDJHV DQG SRWHQWLDO Boues des ouvrages de traitement des eaux
risks of municipal sewage sludge application usées urbaines - Dénominations et
to urban soil. Desalin Water Treat., 52, 3732- spécifications.
3742. [24] Lombardi, A.T. and Garcia, O.JR. (2002)
[8] Zorpas, A.A., Coumi, C., Drtil, M. and Biological leaching of Mn, Al, Zn, Cu and Ti
Voukalli, I. (2011) Municipal sewage sludge in an anaerobic sewage sludge effectuated by
characteristics and waste water treatment plant thiobacillus ferrooxidansand its effect on
effectiveness under warm climate conditions. metal partitioning. Water Res. 36, 3193-3202.
Desalin Water Treat., 36, 319-333. [25] Ábrego, J., Arauzo, J., Sánchez, J.L., Gonzalo,
[9] Sales, A. and De Souza, F.R. (2009) A., Cordero, T. and Rodríguez-Mirasol, J.
Concretes and mortars recycled with water (2009) Structural changes of sewage sludge
treatment sludge and construction and char during fixed-bed pyrolysis. Ind. Eng.
demolition rubble. Constr Build Mater., 23, Chem. Res., 48, 3211-3221.
2362-2370. [26] Guibaud, G., Tixier, N., Bouju, A. and Baudu,
[10] Latosinska, J. and Zygadlo, M. (2011). The M. (2003) Relation between extracellular
application of sewage sludge as an expanding SRO\PHUV¶ FRPSRVLWLRQ DQG LWV DELOLW\ WR
agent in the production of lightweight complex Cd, Cu and Pb. Chemosphere. 52,
expanded clay aggregate mass. Environ 1701-1710.
Technol., 32(13), 1471-1478. [27] Thipkhunthod, P., Meeyoo, V., Rangsunvigit,
[11] ISO 5667-13: 1997: Water quality - Sampling P. and Rirksomboon, T. (2007) Describing
- Part 13: Guidance on sampling of sludges sewage sludge pyrolysis kinetics by a
from sewage and water treatment works. combination of biomass fractions
decomposition. J Anal Appl Pyrolysis., 79,
78-85.
1181


[28] Filip, Z., Pecher, W. and Berthelin, J. (2000)

Microbial utilization and transformation of
humic acid-like substances extracted from a Received: 18.09.2015
mixture of municipal refuse and sewage Accepted: 26.12.2015
sludge disposed of in a landfill. Environ
Pollut. 109, 83-89.
[29] Laurent, J., Casellas, M., Pons, M.N. and CORRESPONDING AUTHOR
Dagot, C. (2009) Flocs surface functionality
assessment of sonicated activated sludge in
Fou ia Benoud it
relation with physico-chemical properties.
Research Unit of Materials, Processes,
Ultrason Sonochem. 16, 488-494.
Environment (URMPE)
[30] Bouzid, J., Elouear, Z., Ksibi. M., Feki, M.
0¶KDPHGBougara University
and Montiel, A. (2008) A study on removal Boumerdès ± ALGERIA
characteristics of copper from aqueous
solution by sewage sludge and pomace ashes.
e-mail: ffouzzia@yahoo.fr
J Hazard Mater., 152, 838-845.
1182

SEASONAL-VARIED CHEMICAL CHARACTERISTICS AND

POLLUTION SOURCES OF PM IN HARBIN, CHINA
Li un Huang1,2, Guang hi Wang2 , Deying Mu1, hi Xia1, Wei Wang1,
1
School of Food Engineering, Harbin University of Commerce, Harbin 150076, China
2
School of municipal and environmental engineering, Harbin Institute of Technology, Harbin 150090, China
Chemical characteristics and pollution sources With the rapid economic and industrial
of PM2.5 were analyzed during four seasons, development in the past few decades, energy
including 3 carbonaceous compounds, 9 ionic consumption has an escalating increase, thus
species and 20 metallic elements. In addition, 9 influences the ambient air quality to a great extent
sources were identified by principal component and air pollution has become ubiquitous (Li et al.,
analysis (PCA) and 11 sources by chemical mass 2009; Li et al., 2010; Ozelkan et al., 2015; Wang et
balance (CMB). Results indicated that the al., 2015; Zhang et al., 2011). According to the
distribution variations of PM were mainly caused statistics of the Chinese central and local
by PM2.5. It had the lowest value in July and the governments, atmospheric particulate matter are
highest value in January, which was 2~5 times considered to be one of the most serious air
larger of the EU standards. Therefore, controlling pollution problems, particularly airborne fine
fine particles was an effective method to reduce the particulate matter (PM2.5), and nearly 70% of urban
particulate matter concentrations. Affected by areas in China do not meet national ambient air
emission sources properties and specific weather quality standards, which are even much laxer than
conditions, chemical characteristics in particulate the air quality exposure standards/guide lines of the
matter presented obvious seasonal differences. World Health Organization (WHO, 2005) (Lu et al.,
OC/EC in four seasons was greater than 2, which 2015; Zhang et al., 2013). Especially in recent
meant that secondary organic carbon was generated years, haze incidents occurred frequently, while
to different extent. NO3- and SO42- were the highest PM2.5 is the main reason which has been frequently
ions in winter which were related to coal reported (Yin et al., 2012). PM2.5 pollution was
combustion. NH4+ was the highest ion in summer controlled to improve the ambient air quality to
mainly from natural decomposition process. Ca, Na some extent, while it still remains a nationwide
and Mg were the highest in summer. Al and Fe problem despite considerable efforts for its
mainly came from industrial and natural emissions. removal.
Pb was comparatively high in winter. Coal
combustion, secondary sulfate and petrochemical It is important to understand which emission
industry had contributed higher ratio in spring than sources contribute to the elevated daily PM2.5 levels
other seasons. Incinerator contributed more in for developing effective control strategies (Zosima
summer. Soil was the lowest (1%) while the cement et al., 2015). This places increased emphasis on
industry was the highest (4%) in summer. Coal correct daily source apportionments to such high
combustion and secondary sulfate had higher levels vary by location and time, and epidemiologic
contribution rate (14% and 17%) in autumn than studies suggest that such sources have different
other seasons which was similar to spring. Both health outcomes (Laden et al., 2000; Sarnat et al.,
dust and traffic led to more particulate matter in 2006; Zheng et al., 2007).
autumn. In wintertime, coal combustion maintained
the highest contribution rate (24%). Secondary The complexity in emission sources of PM2.5
organic carbon and secondary sulfate also reached hinders effective air quality control measures and
the highest rate (5% and 20%). In general, the has become a world-wide problem. Understanding
contribution of coal combustion, secondary sulfate, of each source contribution to PM2.5 and the extent
and petrochemical industry varied violently, while of accuracy of the apportionment results is a
the effects of steel industry, soil and secondary prerequisite for the formulation of effective control
nitrate were relatively stable. strategies for PM2.5 emissions (Shrivastava et al.,
2007). Source apportionment can be achieved by
KEYWORDS: using a variety of air quality models. Amongst
Source Apportionment; Seasonal Variation; PCA; CMB; these, receptor-oriented modeling is one of the
PM2.5 conventional techniques and has been in broad use
1183
for source apportionment studies in the past wind speed and relatively stable atmospheric
decades. The most commonly used receptor models structure (Tolis et al., 2014).
include element-based or molecular marker-based
chemical mass balance (CMB).Applications of Several major cities in China have carried out
CMB using traditional speciation data (i.e.,organic the investigations on the characteristics of PM2.5
carbon (OC), elemental carbon (EC), major ions, (Dai et al., 2013; Li et al., 2013; Li et al., 2012; Lu
and metals) have been reported around the world et al., 2012; Zhang et al., 2012), however, there was
(Andriani et al., 2011; Belis et al., 2011; Belis et al., only limited studies on PM2.5 in Harbin, especially
2013; El Haddad et al., 2011; Lin et al., 2008; chemical characteristics and source apportionment.
Perrone et al., 2012; Yin et al., 2010). Therefore, in order to improve atmospheric quality,
protect human health, and reduce ecological
Principal component analysis (PCA) is one of damage, it is very much crucial to investigate the
the oldest and most widely used multivariate characteristics and sources of PM2.5 for developing
statistical techniques in the atmospheric sciences effective air pollution control measures in Harbin.
(Hopke et al., 1976). Usually the data for Herein, we emphasized the chemical characteristics
atmospheric aerosols exhibit many large and the source apportionment of PM2.5 during four
correlations among parameters and PCA results in a seasons in Harbin by using chemical analysis and
much more compact representation of their varia- source apportionment techniques.
tions (Ho et al., 2006). By using PCA, Saucy
(1991) was able to identify three major sources that
contributed to the atmospheric aerosol (near MATERIAL AND METHODS
Phoenix, Arizona), namely crustal material, copper
smelters and marine air (Saucy et al., 1991). Fung SAMPLE COLLECTION
and Wong (1995) sampled total suspended
particulates (TSP) in the western part of the New Prevailing wind in Harbin comes from the
Territories in Hong Kong between 1986 and 1987 southwest direction. Considering the effect of
and analyzed various trace metals (e.g. Se, As, Sr, atmospheric transmission and diffusion, we set up
V, etc) as markers (Fung et al., 1995). Then PCA two sampling sites respectively on upwind and
was applied to identify the sources and the mass downwind direction of the city, as shown in Fig. 1.
contributions of each source obtained. In many The background sampling site was upwind and
recent source apportionment studies, quantitative located in the suburb of Harbin, about 40 miles
aerosol source apportionment was performed using away from downtown. Air quality around the
absolute principal component analysis (Astel, 2010; sampling site was not influenced by anthropogenic
Bruno et al., 2008; Callén et al., 2009; Contini et pollution sources and the monitoring data were
al., 2010; Maenhaut et al., 2002; Manoli et al., taken as the background value to analyze the effects
2002; Negral et al. , 2008; Salvador et al., 2007). of the pollution sources from city on atmospheric
environment. The monitoring sampling site was
As a typical industrialized urban city in downwind and located on the south campus of
northeastern China, Harbin has aroused much more Harbin University of Commerce. This area is the
attention domestically and internationally. Due to center of residential and commercial district which
its high latitude and cold climate, there might own need to satisfy higher air quality standard (the
different air quality characteristics from the other second level of GB3095-2012). Therefore,
cities. In addition, seasonal features of Harbin are continuous air quality monitoring was very
more obvious than other cities (Xu et al., 2012; Tan necessary.
et al., 2009). Especially in winter, PM2.5 has
different mass concentration, chemical
characteristics and pollution sources due to low
1184
FIGURE 1
Locations of sampling sites in Harbin, China
In order to investigate seasonal variation and consistent with the requirements of national
relative content of particulate matter, TSP, PM10 standard.
and PM2.5 had been collected in parallel for two
years from September, 2010 to August, 2012. The CHEMICAL ANALYSIS
data of TSP and PM10 were just applied to analyze
relative content of PM2.5. According to the season Total and elemental carbon content (TC and
division of Harbin, sample data were divided into EC) of each quartz filter were determined with an
four seasons including spring from March to May, elemental analyzer (EA, Fisson, Model CHNS/O
summer from June to August, autumn from 1108). The first one eighth of each filter sample
September to November, and winter from was heated in advance in a 340 Ԩ oven for 100 min
December to next February. Samples were to expel the organic carbon (OC), and then fed into
collected every day except raining, snowing, dust the elemental analyzer to obtain the elemental
and other special weather. At least 10 samples of carbon (EC) content. The second one eighth of each
each particulate matter were selected every month filter was fed directly into the elemental analyzer
for the further mass concentration and chemical without pre-heating to obtain the total carbon (TC)
analysis. 6~8 samples were used for data processing content (Lin, 2002). Organic carbon could then be
every month. Total 250 samples per year including determined by subtracting the elemental carbon
TSP, PM10, and PM2.5 are counted and analyzed. from the total carbon.
TSP and PM10 were just analyzed in mass The third eighth of the quartz filter for ionic
concentration. The sampling height is about 10m species analysis was put into a 50-mL PE bottle for
and not influenced by the surrounding buildings. each sample. Distilled-de-ionized water was added
The distance between the sampler and the main into each bottle for ultrasonic vibration for
obstacles is more than 10 times the height of the approximately 120 minutes. Ion chromatography
obstacles. There is no barrier in the range of 45 (Dionex, Model 100) was used to analyze the
degrees, so as not to affect the collection of concentration of major anions (F±, Cl±, NO3±, and
particulate matter. The filters used in this study SO42±) and cations (Na+, NH4+, K+, Mg2+, and Ca2+)
were 80 mm quartz filters which were initially (Huang et al., 2011).
heated at 900 Ԩ before sampling in order to The forth one eighth of the quartz filter was
eliminate the interference of residual organic soaked in 15 mL mixed acid (HNO3:HClO4 = 3:7)
compounds. Three medium-flow air samplers and placed on electrical heating panel at 150~200Ԩ
(Wuhan Tianhong Intelligent Instrument, Model for at least 2h until the solution boiled and clarified.
TH-150) were applied to collect TSP, PM10 and During the digestion period, distilled-de-ionized
PM2.5 with the same flow rate of 100 L/min and water was added into the residual solution twice or
sampling time of 24 h every time. Around the more in order to completely dissolve the metallic
sampling site, there were no large pollution sources elements. The residual solution was then diluted to
and obvious obstacles. Sampling approach was 50 mL with 0.5 N HNO3(aq) and stored in a
1185
polyethylene (PE) bottle. After the above- effective variance and the least squares, which
mentioned steps, metallic content was further results in the contribution ratio of each source. The
measured with inductively coupled plasma-atomic model needs the data from the chemical profile of
emission spectrometry (ICP-AES, Perkins Elmer, emission sources and the elements concentrations
Model 400) (Yuan et al., 2004). of receptor samples. Based on these data the model
can calculate the apportionment of each source
SOURCE APPORTIONMENT (Huang et al., 2011).
Principal component analysis (PCA). PCA

is frequently used in data reduction to identify a RESULTS AND DISCUSSION
small group of factors which can explain most of
the variance observed in a larger number of CONCENTRATION AND RELATI E
variables. In this study, principal component CONTENT ARIATION OF ATMOSPHERIC
analysis (PCA) was applied to identify the potential PARTICULATES
sources. The experiment was performed by utilizing
the orthogonal transformation method with varimax Seasonal and monthly variation of
rotation to determine the eigenvalues of variance atmospheric particulates concentration. Monthly
matrix of original variables. Usually, factors with and seasonal variations of atmospheric particulates
eigenvalues > 1 were chosen (Wang et al., 2010). are shown in Fig. 2, which are the average mass
Once a factor is determined by PCA, it will consist concentrations from the analysis results in two
of patterns of variation of the factor loadings of years.
input parameters. The factor loadings indicate the
correlation of each species with each component The analysis of seasonal mass concentration of
and a species was said to load on a given particulate matter was aimed at showing the PM
component if the factor loading was 0.5 or greater pollution degree of the city center area.
for that component. The characteristics of a factor
can then be inferred from the dominant pollutants. As seen from the figure, the variations of
The variances of individual factors in PCA indicate particle concentration from two sampling sites were
the relative magnitudes among dominant potential similar, which showed the characteristics of higher
sources (Huang et al., 2012). value on both sides and lower value in the middle.
In monitoring area of the commercial and
Chemical mass balance (CMB) receptor residential center, the lowest values of TSP, PM10
model. Based on CMB, the software CMB8.2 was and PM2.5 appeared in July with the mass
used to estimate the contribution of emission FRQFHQWUDWLRQ RI DQG ȝJP3.
sources by determining the best-fit combination of The highest values of them appeared in January
emission source and chemical composition profiles with the average mass concentration of 227.57,
through reconstructing the chemical composition of DQGȝJP3. In background area, the
ambient samples. The basic calculation principle of lowest values of TSP, PM10 and PM2.5 appeared in
the model is as follow: summer with the average mass concentration of
ȝJP3. The highest values of
&L )L6)L6«)LM6M«)L- them appeared in winter with the mass
6-L «,M «- (1) FRQFHQWUDWLRQ RI ȝJP3.
Because January was the middle period of heating
where Ci is the concentration of element i by coal burning, coal combustion resulted in a
measured at the receptor site of atmospheric significant increase of particulate concentrations.
particulate matter (μg/m3); Fij is the concentration Compared with the Ambient Air Quality Standard
of elements i measured at the source j of emitted of National Standard of China (GB3095-2012),
particulate matter (μg/m3); Sj is the contribution TSP and PM10 all exceeded the second level of this
ratio of particulate matter emitted from source j; I is standard in January, February, March, April,
the number of elements; J is the number of November and December. PM2.5 concentration was
emission sources. The contribution ratio of the 2~5 times higher than Environmental Air Quality
source j is defined as Sj/C×100%. Standard of European Union (2008/50/EC, 25
ȝJP3) and had a higher value all the year, so we
:KHQ³,´LVJUHDWHUWKDQ³-´DJURXSRIUHVXOWV should endevour to control PM2.5.
can be obtained by using the equation based on the
1186
250
225 TSP
Mass concentration (ȝJP3)
TSP
200
PM10
PM10
175
150 PM2.5
PM2.5
125
100
75
50
25
0
May
Aug
Spring
Nov
Autumn
Mar
Apr
Jun
Winter
Jul
Oct
Summer
Dec
Sep
Feb
Jan
Spring Summer Autumn Winter Background value
FIGURE 2
Seasonal and monthly distribution of atmospheric particles in monitoring area and bac ground area.
The average concentrations of TSP, PM10 and relative content and distribution of atmospheric
PM2.5 ZHUH ȝJP3 in spring, particles in different periods. Mass concentration
ȝJP3 in summer, 156.44, percentage of different size particulates is shown in
ȝJP3 in autumn and 218.87, 171.56, Fig. 3.
ȝJP3 in winter. The PM concentrations in
monitoring area were 2~3 times larger of that in the The proportion variation of PM2.5 was more
background area with the highest multiple in winter obvious with higher percent content of more than
and the lowest multiple in summer. Because of 50% in January, February, March, October,
high pressure and low dispersal ability, atmospheric November, and December. At the same time,
particles were easy to gather during wintertime, PM2.5/PM10 also reached the higher value. This
resulting in increased particles concentration. showed that the fine particle pollution was serious
Because of better air diffusion capacity and less during these periods and could cause great harm to
pollution sources, TSP and PM10 could meet the our environment. However, the variation range of
national standard of level two in summer, but PM2.5 PM10/TSP was only from 0.71 to 0.79. It could be
still exceeded the EU standards during all the concluded that the distribution variation of
seasons. Therefore, limiting fine particles was an particulate matter was mainly caused by PM2.5 and
effective method to reduce the particulate matter PM2.5-10, especially PM2.5. The concentration of
concentrations and improve the air quality. PM2.5 was mainly determined by the physical and
chemical processes between particles and other
Relative content variation of atmospheric pollutants, the properties of pollution source and
particulates. Atmospheric particles with different weather conditions, when these conditions changed,
size have different hazards to environment and particle distribution caused by PM2.5 also changed
human health. It is very necessary to investigate the accordingly.
1187
100%
90%
80%
Cumulative percentage
70% ˘PM2.5
PM with diameter > 10 μm
60% PM2.5-10
PM2.5-10
50%
PM2.5
˚PM10
40%
30%
20%
10%
0%
Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb
Spring Summer Autumn Winter
FIGURE 3
Relative content of different si e scales particulates.
CHEMICAL CHARACTERISTICS OF PM2.5 Seasonal distribution of OC, EC and OC/EC

DURING FOUR SEASONS in PM2.5 was shown in Fig.4.
Seasonal variation of carbonaceous content Due to different times and intensities of coal
in PM2.5. Affected by the properties of emission combustion heating in spring, autumn and winter,
sources and specific weather conditions, carbon OC was higher in the three seasons, particularly in
component in particulate matter presented obvious winter; the seasonal variation of EC was not
seasonal differences, especially secondary organic obvious; OC/EC at four seasons was greater than 2,
carbon. Carbon components in atmospheric which indicated that four seasons had the
particulates were usually formed by three ways: generation of secondary organic carbon to some
first, when the concentrations of volatile organic different extent. During the summer, OC/EC was
compounds exceeded the saturation vapor pressure, the lowest with the rate of more than 2. Although
organic matter with low saturated vapor pressure high temperature in this season was beneficial to
would condense on particulate matter and form strengthen the atmospheric photochemical
secondary organic carbon; second, gaseous organic activities, gaseous pollutants from less pollution
compounds in the particle surface could enter the source had low concentration and could not be
interior of particulate matter by physical or absorbed by particulate matter, so the generation of
chemical process, adsorption or absorption; third, secondary organic carbon was limited, mainly
gaseous organic compounds formed low volatile through the first and the third way. OC/EC was the
substances through oxidation reaction in the highest in winter and 5~6 times larger of that in
atmospheric environment, thereby generating summer. Low temperature was not conductive to
secondary particles. Generally, when OC and EC photochemical reactions, but with a favorable
are from direct emissions of pollution sources, the condition of high pressure and low wind speed in
rate of OC/EC is a relatively stable value which is winter, gaseous compounds had a higher
related to the types of pollution sources. When concentration and were easily absorbed by
OC/EC exceeds the threshold, it means the particulate matter, which made the absorption
formation of secondary organic carbon. Chow et al processes smooth.
thought when OC/EC >2, the secondary reaction
occurs (Chow et al, 2001).
1188
40 16
Mass concentration (ȝJP3) 35 OC 14
30 12
EC
25 10
OC/EC
OC/EC
20 8
15 6
10 4
5 2
0 0
Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb
FIGURE 4
Seasonal variation of carbon component in PM 2.5
Seasonal variation of ionic species in PM 2.5. distribution, which accounted for the relatively
Because of different seasonal characteristics, the stable pollution sources and not affected by the
distribution of ions in PM2.5 had a significant seasons. Strong air diffusion capacity and outside
distinction between four seasons. The seasonal sources led to the increase of Cl-, so the highest
distributions of 9 ions were shown in Fig. 5. percentage of Cl- occurred in summer. K+ and F-
had the lowest percentage in summer, associated
As the main ions in PM2.5, SO42+, NO3- and with biomass burning .In a word, the percentage of
+
NH4 occupied the highest levels of 90% in total DOO WKHVH LRQV KDG GLIIHUHQW GHJUHHV¶ LQFUHDVH LQ
ions in winter,, and the lowest of 85% in fall. In spring, autumn and winter.
addition, there was a gas-solid balance reaction on
NH4NO3 in the atmosphere: Seasonal variation of metallic elements in
PM2.5. Different environmental factors and
NH4NO3(s) NH3(g)+ HNO3(g) (2) pollution emissions during the different seasons
would lead to the different composition of the
The reaction was endothermic and low chemical elements in particulate matter. Fig.6
temperature was conducive to generate NH4NO3- showed the seasonal variations of elements in
(s), so the content of NO3- was the highest in winter PM2.5.
and the lowest in summer. SO42- had direct
relationship with coal combustion during the Part of the elements in PM2.5 had the same
winter. With the increase of the heating intensity, trend in different seasons, such as the ratio of Ca,
the concentration of SO42- was increasing, which Na and Mg which were the highest in summer and
showed that coal combustion had a greater the lowest in winter. It indicated that the flue
contribution to the generation of SO42-. NH4+ was combustion in winter increased the ratio of other
mainly from natural decomposition process, but had elements. S was marked as a trace element of coal
obviously different with NO3- and SO42-. NH4+ combustion. From the figure, it was lower in
showed a higher level with the percentage of 44% summer and higher in autumn and winter, so coal
in spring and 49% in summer because of the high combustion caused the releasing of S in winter and
air temperature. Some NH4+ coming from the autumn. Si was high in winter and low in autumn,
natural release of plant and other part was generated mainly from natural source input and partly from
by the nitrogen organic matter decomposition. It the flying ash caused by coal combustion in winter.
indicated that NH3 was easier to generate smaller
particle diameter by secondary reactions. Na+,
Mg2+and Ca2+ had not an obvious seasonal
1189
н
<н DŐ EĂ
Ϯн
<н DŐ EĂн
Ϯн
Ϯй Ϭй ϭй Ϯнϭй
Ϭй ϭй
ĂϮн Ă
ϭй E, ϰн Ϯй
ϰϰй EKϯͲ E, ϰн
EKϯͲ
ϭϵй ϰϵй
Ϯϰй
^KϰϮͲ ^KϰϮͲ
ϮϮй Ϯϰй
&Ͳ
ů ϭй Ͳ
&Ͳ
ůͲ
ϱй Ϭй
ϰй
(a) Spring (b) Summer
Ϯн
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DŐ Ϯн н
<н DŐ EĂ
Ϯн
ϯй Ϯй Ϭй EĂн &Ͳ
ůͲ
Ϭй Ϭй Ϯй
EKϯͲ ϭй ĂϮнϮй ϱй
E, ϰн
Ϯϱй E, ϰн ϭй
ϮϬй
Ϯϴй
^KϰϮͲ EKϯͲ
ϯϯй ^KϰϮͲ
ϯϮй
ϯϳй
&Ͳ
ůͲ ϭй
ϴй
(c) Autumn (d) Winter
FIGURE 5
Seasonal variation of water-soluble ions in PM2.5
Zn and K had more obviously seasonal between 2% to 3%, no substantial changes. Pb was
variations. Because of low temperature in spring from vehicle exhaust, steel industry, coal dust, soil
and winter, fuel burning is used to heating which dust, diesel exhaust, moped exhaust and cement
released a lot of Zn. Autumn was the harvest time, dust in major cities. Since the ban of using leaded
and straw burning released more K. In winter, straw gasoline, the dust from iron and steel industry was
burning was also used to keep warm in the city the major source of Pb. Pb was high in winter,
suburbs and countryside which resulted in high which indicated that coal burning had a great
concentration of K. Al and Fe were mainly from contribution to Pb and more fine particles were
industrial and natural emissions. Al was high in from coal combustion emissions.
summer and low in winter, while Fe was high in
winter and low in autumn. Due to the low SEASONAL SOURCE ANALYSIS OF PM2.5
concentration of Cu, Cr, Cd, Ni, Mn, Sr, V, As and BY PCA
Ba, these elements had no obvious changes in
concentration ratio. It also illustrated that the Seasonal sources of PM2.5 were analyzed by
sources of these elements emissions was stable and PCA and shown in Table 1 (Xu, 2008).
less affected by seasonal factors. Ti was mainly
from natural sources, and the content of Ti was
1190
Mg Mn Mg Mn
6% 1% Ni
K 6% 0%
Fe 0%
Pb Fe 6%
6% K Na
Cu 10%
1% Cu 6% Na Ni
1% 7% 17% 0%
1%
Cr Cr Pb
S
1% 1% 0%
18%
Cd Cd S
0% 0% 15%
Si Si
7% 2%
Ca
30% Sr Ca Sr
Ti 0% 36% Ti 0%
3% 3%
Ba Zn V
As Al Zn V Ba As
Al 2% 0%
2% 0% 3% 4% 0% 0% 0%
4%
Ni Pb
Mn Pb Ni
0% 3%
Mg 0% 2% 0%
5% Na Na
15% Mn 9% S
Fe 1% 23%
4% K Mg
Cu 10% S 3%
1% 24% K
Cr 8% Si
0% Si 18%
2% Fe Sr
Cd
0% Sr 8% 0%
Ca Ca Ti
0% Cu
29% V Ti 16% V 3%
0% 2%
1% Cd Zn 0%
Cr
Ba As Al Zn Ba As Al 5%
0% 0%
0% 0% 3% 2% 1% 0% 2%
FIGURE 6
Seasonal variation of water-soluble ions in PM2.5
1191
TABLE 1
Principle component analysis of PM 2.5 during four seasons

Principal
1 2 3 1 2 3 1 2 3 1 2 3
components
Eigenvalue 19.69 7.43 2.88 14.72 8.60 6.68 17.30 8.70 4.00 14.59 10.18 5.23
Contribution˄
62.64 22.38 7.59 46.08 24.52 20.26 53.73 26.01 11.34 44.31 31.72 14.45
%˅
Mn 0.98 -0.19 0.01 0.82 -0.52 0.26 -0.57 0.76 0.30 0.95 0.29 0.10
Na 0.05 -0.96 -0.26 0.95 0.24 0.18 0.99 -0.13 -0.03 -0.79 -0.50 0.35
Ni 0.86 -0.36 0.37 0.39 0.92 -0.05 0.47 -0.33 -0.82 0.41 0.90 0.18
Pb 1.00 0.09 0.00 0.48 -0.88 -0.08 0.99 -0.13 0.11 0.12 -0.96 0.26
S 0.88 0.47 0.07 0.23 0.77 0.60 0.97 0.25 0.02 0.12 -0.35 -0.93
Si 0.95 -0.30 -0.06 0.95 0.09 0.31 0.39 0.92 -0.05 0.96 0.28 0.07
Sr 0.94 -0.35 -0.05 0.27 0.91 -0.32 0.92 0.39 0.09 0.81 0.57 0.17
Ti 0.85 0.41 -0.33 0.75 -0.51 0.42 -0.67 0.74 0.07 0.33 0.94 0.11
Zn 0.98 -0.17 -0.07 -0.89 0.43 0.12 1.00 -0.01 -0.05 -0.14 0.90 0.42
Al -0.01 0.99 0.16 0.80 0.58 -0.18 0.70 0.66 0.29 0.71 -0.62 -0.34
As 0.88 -0.48 -0.02 -0.61 0.36 0.70 0.94 0.26 -0.22 -0.82 0.57 0.07
Ba 0.95 -0.32 -0.05 0.95 -0.13 0.28 -0.40 0.90 0.19 1.00 0.06 0.05
Ca 0.64 0.74 0.19 0.58 -0.32 -0.75 -0.35 -0.80 0.48 -0.77 0.64 0.06
Cr 0.88 0.48 0.02 -0.96 0.24 0.11 0.99 -0.01 -0.17 -0.39 0.52 -0.76
Cu 0.28 0.94 0.18 0.06 -0.15 0.99 -0.40 0.41 -0.82 0.93 -0.35 -0.12
Fe 0.99 -0.09 0.15 0.64 0.72 0.25 -0.41 0.90 -0.17 0.45 0.81 0.37
K 0.86 0.50 0.08 0.61 -0.35 0.71 0.91 -0.36 -0.19 -0.72 -0.02 0.69
Mg 0.52 0.85 0.10 0.55 0.80 0.27 0.77 0.38 -0.51 -0.88 0.32 0.36
TC 0.97 0.23 0.02 0.89 0.34 -0.31 1.00 0.05 0.08 1.00 0.04 -0.09
EC -0.47 0.85 -0.25 0.52 -0.22 -0.83 -0.24 0.19 0.95 0.68 0.16 -0.71
OC 0.98 0.18 0.03 0.86 0.51 -0.01 1.00 0.03 0.01 1.00 0.04 -0.08
Ca2+ -0.79 0.18 0.59 0.98 0.18 -0.07 -0.35 -0.93 -0.08 0.19 -0.86 0.47
K+ 1.00 -0.08 0.01 0.83 -0.53 0.19 0.88 0.44 0.17 -0.46 -0.60 -0.65
Mg2+ -0.25 -0.28 0.93 0.64 0.71 -0.29 0.48 -0.88 0.06 0.48 -0.28 0.84
Na+ 0.24 -0.45 0.86 0.68 0.39 -0.63 0.72 -0.44 0.53 -0.97 0.14 0.18
NH4+ -0.88 0.39 0.26 -0.41 0.75 -0.52 0.31 -0.94 0.15 -0.54 0.72 -0.45
F- 0.93 -0.36 -0.03 0.78 -0.37 0.50 0.94 0.33 0.06 0.10 0.93 -0.36
Cl- 0.85 0.38 0.37 0.54 -0.27 -0.80 0.90 0.26 0.35 -0.94 -0.20 -0.27
SO42+ 0.88 -0.37 0.28 0.61 0.38 0.70 0.96 -0.09 -0.26 0.93 -0.63 0.34
NO3- 0.92 0.21 -0.33 0.58 -0.74 -0.33 0.79 0.21 0.57 0.86 0.24 0.18
CC
CC
SA SD DS CC
SA PI
TD AD SA SI SA ID
Sources* - ID DS SD TD ID -
PI DS TD PI TD SI PI
SD SI
SI ID AD AC
AD
AC
*CC²²Coal combustion SA²²Secondary aerosols TD²²Traffic dust DS²²Dust
PI²²Petrochemical industry ID²²Incinerator SI²²Steel industry
AC²²Agricultural burning SD²²Soil dust AD²²Construction dust
The contribution rate of extracted cumulative and winter, which expressed that the sources of
variance were 92.61%, 90.86%, 91.08% and PM2.5 were relatively stable. Extracted factor 1 had
90.48% corresponding to spring, summer, autumn a high load in spring, which meant the sources of
1192
this factor had an obvious characteristic. The industry. K and K+ were from agriculture
sources of Factor 1 were coal combustion, combustion. Crustal materials had a high load in
secondary aerosols, traffic dust, petrochemical Factor 2, which considered that the pollution was
industry, steel industry and agricultural burning. from soil or dust. EC had a high load in Factor 3,
Factor 2 showed the sources of soil dust, which considered that the pollution was from
construction dust, dust and incinerator. Factor 3 incinerator or chemical industry. In winter, TC and
showed that Mg2+ and Na+ had a high load, which 2&¶V KLJK ORDG LQ )DFWRU PHDQW WKDW VHFRQGDU\
considered the release of natural sources. In aerosol pollution was severe. Other ions and
summer, crustal elements and carbon had a high elements might be from coal combustion, dust, soil
load in Factor 1, which considered the pollution and construction dust. Factor 2 was judged from
was from soil, dust, traffic dust and construction incinerators, chemical and metal industries.
dust. Some ions and metal elements had a high load
in Factor 2, which showed the pollution were from SOURCE APPORTIONMENT OF PM2.5 BY
steel industry and petrochemical industry. In CMB
DXWXPQ 2& DQG 7&¶V ORDGV ZHUH XS WR ZKLFK
indicated that secondary aerosol pollution was very Contributions of identified sources to PM 2.5.
serious. The loads of some heavy metals and SO42- By using the CMB receptor model, the contribution
were also high, which inferred the pollution were characteristics of pollution sources were analyzed
from coal combustion, traffic dust, or metal and shown in Table 2.
TABLE 2
Contributions of identified sources to PM 2.5 during four seasons by CMB
DF 11 11 13 12
R Square 0.84 ±0.04 0.89±0.05 0.85±0.05 0.88±0.04
Chi-square 1.89±1.24 1.62 ±1.15 1.86±1.29 1.45±1.35
% mass explained㸦%㸧 88.57 90.82 84.96 91.90
3
Soil (ȝJP ) 1.20 0.66 1.34 2.25
Dust (ȝJP3) 6.35 4.06 7.35 6.25
Traffic (ȝJP3) 7.35 5.18 8.85 10.65
Incinerator(ȝJP3) 4.35 5.26 4.18 8.09
Coal combustion (ȝJP ) 3 15.30 6.21 11.25 29.25
Cement industry (ȝJP3) 1.25 2.16 1.24 1.06
Steel industry (ȝJP3) 3.54 3.06 2.89 4.11
Petrochemical industry (ȝJP3) 9.36 8.20 8.85 10.36
Secondary sulfate (ȝJP ) 3 10.84 5.03 13.31 24.06
Secondary nitrate (ȝJP3) 5.52 4.31 6.28 9.06
Secondary organic carbon (ȝJP3) 2.35 1.03 1.97 5.79
Others (ȝJP3) 8.70 4.56 11.95 9.78
3
Sum of identified sources (ȝJP ) 67.41 45.17 67.52 110.93
Source profile was from Europe, the United of categories and characteristics of pollution
States and Taiwan to (Lee et al., 2007; Lee et al., sources including soil type, traffic equipment, fuel
2008; Manoli et al., 2002; Xu, 2008). The properties, coal quality and combustion equipment,
selection of source profile focused on the consistent
1193
in order to ensure the accuracy of source steel industry, and petrochemical industry were not
apportionment results. obvious with relatively stable contribution. Soil and
As shown in Table 2, R Square was between incinerator had higher contribution in winter than
0.84 and 0.89, DF was greater than 10, and other seasons. The release amount of OC was
PERCENT MASS was between 84.96% and relatively high from coal combustion, incinerator,
91.90%. These parameters could ensure the high and traffic, so the seasonal variation of secondary
simulation accuracy and similarity with the actual organic carbon was consistent with these sources.
situation. The identified sources by CMB included Because of good weather conditions, particles could
soil, dust, traffic, incinerator, coal combustion, easily transmit horizontal and vertical. Therefore,
cement industry, steel industry, petrochemical all pollution sources had lower contributions in
industry, secondary sulfate, secondary nitrate, and summer.
secondary organic carbon. Because of major human
behaviors, traffics, coal combustion, petrochemical Seasonal contribution rate of pollution
industry, secondary sulfate and secondary nitrate sources to PM2.5. Each identified source of PM2.5
were major sources with higher concentration in four seasons was illustrated in Fig. 7 and their
DQG ȝJP3) in average contribution rate showed obviously
winter. The seasonal feature of cement industry, different during these periods.
Others Soil Secondary

Soil
Secondary 11% 2% Dust organic Others Dust
organic carbon 8% carbon 9% 1% 8%
3% 2%
Traffic Secondary Traffic
Secondary 10% nitrate 10%
nitrate 9%
7% Incinerator
Incinerator
6% Secondary
Secondary 11%
sulfate
sulfate Coal 10%
14% Coal
combustion combustion
20% Petrochemical 13%
Petrochemical industry
Cement Steel industry Cement Steel industry
industry 17%
industry 5% industry 6%
12%
2% 4%
Others Soil Dust

Others Soil Dust Secondary 8% 2% 5%
Secondary 15% 2% 9% organic carbon Traffic
organic carbon 5% 9%
2% Traffic
11% Secondary
nitrate Incinerator
Secondary
7% 7%
nitrate Incinerator
8% 5%
Secondary Coal
Coal
Secondary sulfate combustion
combustion
sulfate 20% 24%
14%
17% Steel industry
4% Petrochemical Steel industry
Petrochemical Cement industry industry Cement 3%
industry 9% industry
2%
11% 1%
FIGURE 7
Contribution rate of sources to PM 2.5 during four seasons by CMB
1194
Coal combustion, secondary sulfate and source profiles, though atmospheric processing and
petrochemical industry had higher contribution limitations in the CMB method also affect profiles.
ratio in spring. Early spring was the final stage of
heating, thus PM2.5 mainly came from primary
pollutants and secondary aerosols emitted from coal CONCLUSIONS
combustion. With the arrival of summer, particle
concentration decreased and the contribution rates A seasonal comparison of PM concentrations
of coal combustion and secondary sulfate were showed that the lowest values appeared in summer
greatly reduced. Petrochemical industry was still in and the highest values emerged in winter. The PM
high contribution rate (17%) due to the increasing concentrations in monitoring area were 2~3 times
factory workload in summer. Because of frequent larger of that in the background area. The
human activities and enormous waste volumes, the distribution variations of PM were mainly caused
contribution rate of incinerator increased. There by PM2.5 and PM2.5-10, and the latter played more
was no difference for traffic and dust in spring and important role. A monthly comparison of PM 2.5
summer. Compared with the other seasons, the concentrations showed that PM2.5 had the lowest
contribution of soil was the lowest with the rate of value in July and the highest value in January,
1% and that of cement industry was the highest which was 2~5 times larger of the EU standards.
with the rate of 4% in summer. Therefore, controlling fine particles was an
During autumn, coal combustion and effective method to reduce the particulate matter
secondary sulfate had higher contribution rate concentrations and improve the air quality.
which shared similar reason to that of spring. Both Affected by emission sources properties and
dust and traffic contributed more particulate matter specific weather conditions, Chemical
in autumn than other seasons. Because these two characteristics in particulate matter presented
sources had similar characteristic and strong obvious seasonal differences. OC/EC in four
correlation, the variations were consistent. In seasons was greater than 2, which meant that
wintertime, coal combustion had the highest secondary organic carbon was generated to
contribution rate of 24%, followed by secondary different extent with the highest value in winter and
sulfate (20%), petrochemical industry (9%), and the lowest value in summer. NO3- and SO42- were
traffics (9%). At the same time, secondary organic the highest in winter due to coal combustion; NH4+
carbon also reached the highest contribution rate was mainly from natural decomposition process;
(5%), which was mainly due to the formation of the highest percentage of Cl- occurred in summer;
secondary aerosols. In other words, part of primary Ca, Na and Mg kept the highest in summer; Al and
pollutants emitted from these sources could evolve Fe came mainly from industrial and natural
into secondary aerosols through chemical reactions emissions. Pb was higher in winter and Ti was
at low wind speed and in stable atmospheric mainly from natural sources.
condition in winter, which resulted in the Two different receptor models (PCA and
contribution of these sources. CMB) were applied to estimate PM2.5 the impacts
of PM2.5 sources. 9 sources were identified by PCA
THE COMPARISON OF PCA AND CMB and 11 sources by CMB, which showed a close
correspondence. The source profiles of primary
Two different receptor models (PCA and emission sources in PCA may not be representative
CMB) were applied to estimate the impacts of due to a lack of available source profiles, while
PM2.5 source. 9 sources were identified by PCA and CMB may provide more information for developing
11 sources by CMB, which showed a close more representative source profiles, though
correspondence between the two methods. In atmospheric processing and limitations in the CMB
general, the contribution changes of coal method also affect profiles. From the result of
combustion, secondary sulfate, and petrochemical CMB, coal combustion, secondary sulfate and
industry were larger, while steel industry, soil, and petrochemical industry had higher contribution
secondary nitrate were relatively stable. For some ratio in spring, among which coal combustion and
other sources/factors, even for those highly secondary organic carbon had the highest
correlated, there are differences in estimated source contribution rate of 24% and 5% in winter. The
contributions. Several factors may contribute to the contribution rate of incinerator increased in
differences in the source apportionment results. The summer. Compared with the other seasons, the
source profiles of primary emission sources in PCA contribution of soil was the lowest and that of
may not be representative due to a lack of available cement industry was the highest in summer. In
source profiles, while CMB may provide more autumn, coal combustion and secondary sulfate had
information for developing more representative higher contribution rate which was similar to that of
1195
spring. Dust and traffic both contributed more Comparison of receptor models for source
particulate matter in autumn. In general, the apportionment of the PM10 in Zaragoza
contribution of coal combustion, secondary sulfate, (Spain). Chemosphere, 76:1120-1129.
and petrochemical industry varied violently, while [7] Chow J C, Waston J G, Lu Z, 1996.
the effects of steel industry, soil and secondary Descriptive Analysis of PM2.5 and PM10 at
nitrate were relatively stable. Regionally Representative Locations during
SJVAQS/AUSPEX. Atmospheric
Environment, 30: 2079-2112.
ACKNOWLEDGMENTS [8] Contini D, Genga A, Cesari D, Siciliano M,
Donateo A, Bove M C, Guascito M R, 2010.
This study was supported by the National Characterisation and source apportionment of
Natural Science Foundation of China (No. PM10 in an urban background site in Lecce.
51408168) and the Open Project of the State Key Atmospheric Research, 95:40-54.
Laboratory of Urban Water Resource and [9] Dai W, Gao J Q, Cao G, 2013. Chemical
Environment, Harbin Institute of Technology (No. composition and source identification of
ES201417). The authors would like to express their PM2.5 in the suburb of Shenzhen, China.
sincere appreciation for the financial support to Atmospheric Research, 122: 391-400.
accomplish this study. Special thanks go to Mr. [10] El Haddad I, Marchand N, Temime-Roussel
Hsieh-Hung Tsai and Mr. Tsung-Chang Li from the B, Wortham H, Piot C, Besombes J L, Baduel
Institute of Environmental Engineering, National C, Voisin D, Armengaud A, Jaffrezo J L,
Sun Yat-sen University,Taiwan, for their kind 2011. Insights into the secondary fraction of
assistance in chemical analyses. the organic aerosol in a Mediterranean urban
area: Marseille. Atmospheric Chemistry and
Physics, 11:2059-2079.
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1198

DELAYED EFFECTS OF CAPTOPRIL ON THE

MEDITERRANEAN FLOUR MOTH REPRODUCTIVE
EVENTS, BIOCHEMICAL COMPOSITION AND MOLTING
HORMONE CONTENTS OF OVARIES
Samira Yezli-Touiker, Nadia Soltani-Mazouni, Leïla Kirane-Amrani and
Noureddine Soltani
Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences, Badji Mokhtar University 23000-Annaba, Algeria
ABSTRACT important pest in stored products worldwide. This

species has been used as a model target
Captopril is an inhibitor of angiotensin lepidopteran to investigate normal developmental
converting enzyme. We evaluate its effects on and reproduction-related events [7-10]. As a
reproduction of e tia e niella (Lepidoptera), consequence, these data obtained provide a strong
an important pest in stored products worldwide. basis to examine the potential effect of insect
The drug was applied topically (10 Pg/pupa) on growth regulators on development and reproduction
newly molted pupae. In follow±up experiment, the of this laboratory model. The molting hormone
adults that survived from treated pupae were (ecdysteroids) synthesis can be performed by
investigated for different reproductive event several tissues besides prothoracic glands and is not
parameters. Captopril was found to decrease the restricted to larvae [11]. Moreover, it now well
duration of the oviposition period and the established that in adult females, ecdysteroids are
morphometric measurements of ovaries, the synthesized by the follicle cells in the ovaries and
fecundity and the egg viability. Moreover, play a major role in several aspects of reproduction
biochemical analyzes revealed that treatment in insects [12]. Then the ovarian ecdysteroids, in
reduced total protein, lipid and carbohydrate both free and conjugated forms, are almost entirely
amounts of ovaries, respectively. Lastly, enzyme taken up by and stored/concentrated in the maturing
immunoassay measurements of ovarian eggs and may serve as hormonal substrate for
ecdysteroids indicated that captopril increased the embryonic molts during embryogenesis [13].
amounts of both total and free ecdysteroids, and Captopril, a strong and specific inhibitor of
decreased that of conjugated. The reduction of ACE, is often used in the treatment of hypertension
reproductive capacity was probably due to an [14]. It enhances both trypsin and vitellogenin titers
interference of captopril with the vitellogenesis in the grey fleshfly Neobellieria bullata [15] while
process and/or ecdysteroid biosynthesis. in Tenebrio molitor, it affects the morphometric
measurements of ovary and no significant effect
was observed on both thickness and fine structure
KEYWORDS: of chorion [16]. To date, few authors have
e tia e niella, Captopril, Reproduction, Ovaries, examined the delayed effects of captoril during the
Biochemistry, Ecdysteroids adult stage. Therefore, the current study was
designed to evaluate the toxicity of captopril,
applied topically on newly molted pupae of
INTRODUCTION e niella, on the morphometric measurements, the
biochemical composition (carbohydrates, proteins
Angiotensin-converting enzyme (ACE) is a and lipids) and the ecdysteroids (free, conjugated
dipeptidyl carboxypeptidase and a key player in the and total) of ovaries from newly emerged adult
renin±angiotensin system, responsible for the females. The reproductive events (duration of pre-
removal of the C-terminal dipeptide of angiotensin oviposition and oviposition periods, fecundity and
I to generate the powerful vasoconstrictor, egg viability) were also examined. The attempt
angiotensin II [1, 2]. Little is known about the role results should help in better understanding of the
of the invertebrate angiotensin converting enzyme mode of action of this drug.
(ECA) [3]. In several insects, a peptidyl dipeptidase
similar to mammalian ACE has been found [4- 6].
The Mediterranean flour moth, e tia
e niella Zeller (Lepidoptera: Pyralidae), is an
1199


MATERIALS AND METHODS Germany) as a reagent and glucose as a standard.

Lipids were measured by the sulfo-phospho-
Insects. e tia e niella (Zeller, 1879) vanillin method [20] using a lipid solution (Lyotrol,
was reared on wheat flour at 27°C and 80% relative BioMérieux, France) as a standard. Proteins were
humidity in almost continuous darkness as reported quantified by the Coomassie Brilliant Blue G-250
before [10]. Pupae were collected from a stock dye-binding method [21] with bovine serum
colony and sexed. albumin (Sigma) as a standard.
Chemical and treatment. Captopril (D- Extraction and enzyme immunoassay of

3mercapto-2-methyl-propionyl-L-proline) was ecdysteroids. Ovaries were dissected from newly
purchased from Sigma Co. (98%, Bornem, emerged adult females in control and treated series.
Belgium). A stock solution of ACE inhibitor and submitted to extraction with methanol by
captopril (5 mg/ml) was prepared in acetone. Newly sonication. After centrifugation at 5000 g for 10
molted pupae (<8 h old) were topically treated (10 min. [22]. For determination of the total ecdysteroid
μg/pupae). The drug is easily diluted in acetone, amounts (free and conjugated), the samples were
allowing a better diffusion throughout the cuticle further submitted to an enzymatic hydrolysis with
[10, 17]. In the control groups, pupae were treated porcine liver esterase (EC3.1.1.1. Sigma Co., St.
with 2 ȝl of solvent (acetone). Louis, IL, USA) as previously described [13]. Then
each individual sample of free and total
Morphometric measurements of ovaries. As ecdysteroids was analyzed in duplicate by an
above, newly molted E. kuehniella pupae were enzyme immunoassay (EIA) [23] using a conjugate
treated topically with captopril. Subsequently, the of 20E coupled to peroxidase as the enzymatic
surviving adults were collected and the fresh weight tracer and tetramethyl benzidine as the color
of ovaries, the number of oocytes, and the size reagent. The rabbit polyclonal B antibody and the
(length, width) and the volume of basal oocytes tracer were kindly supplied by Dr J.P. Delbecque
were respectively determined in the newly emerged (University of Bordeaux I, France) and Dr. C. Blais
adult females. Six females per series were used for (UMPC, Paris, France), respectively. The
the morphometric measurements of ovaries. polyclonal antibody used in the present study is
more sensitive to E (6 times more sensitive to
Reproductive events. To determine potential ecdysone than to 20-hydroxyecdysone) [24].
effects of captopril on the duration of pre- Absorbances were red in a multiplate reader
oviposition and oviposition periods, an extra series (Labystem, Finland) at 630 nm. The amounts of
of newly molted pupae were treated (10μg/pupa) as conjugated ecdysteroids were deduced by
above. Untreated adult males were used to mate subtracting the amounts of free ecdysteroids from
with surviving adult females from treated pupae. that of total ecdysteroids. Data are expressed as pg
Six pairs per series were investigated. The time of ecdysteroids per mg tissue.
first oviposition and the fecundity (number of eggs
per female throughout its lifespan) were recorded. Statistical analysis. Results are expressed as
The egg hatchability (percentage of neonates that mean ± standard deviation (SD). Comparison of
emerged from eggs) was determined on four groups PHDQ YDOXHV ZDV PDGH E\ 6WXGHQW¶V t-test. All
of 10 eggs. For each series, freshly laid eggs were statistical analyses were performed using the
randomly collected at the beginning of the MINITAB Software (Version 16, PA State College,
oviposition period from different tested pairs and USA) and p< 0.05 was considered to be a
the number of first-instar larvae was scored. statistically significant difference.
Ovarian biochemical components. The

surviving adults from treated pupae were collected. RESULTS AND DISCUSSIONS
Paired ovaries from the newly emerged adult
females were dissected from control and treated The effect of captopril applied topically on
series. Each pair of ovaries was individually newly molted pupae was examined on some
extracted [18]. In brief, each sample consisting of morphometric measurements of ovaries sampled
one pair of ovaries was homogenized in 1mL of from newly emerged adult females. Data showed
trichloroacetic acid (20%). Then the quantification that the compound caused a significant reduction in
of the amounts of the main components both the weight (p= 0.004) of ovaries, the number
(carbohydrates, lipids, proteins) determined as of oocytes per paired ovaries (p= 0.000), the width
described previously [10]. Carbohydrates were (p= 0.000), the length (p= 0.003) and the volume of
determined [19] using the anthrone (Merck,
1200


basal oocytes (p= 0.000) as compared to control series (Table 1).
TABLE 1
Effects of captopril (10 μg/pupa) applied topically on newly molted pupae of E. kuehniella on the
weight of ovaries, the number of oocytes per paired ovaries and the size of basal oocytes (mean ± SD,
n= 6 females). Values followed by different letters are significantly different at 5% level.
Treatment Weight Oocyte number Length Width Volume

(mg) (μm) (μm) (mm3)
Control 5.16 ± 0.40a 189.00 ± 13.60a 431.44 ± 6.39a 297.15 ± 6.37a 0.015 ± 0.005a
Captopril 2.13 ± 0.55b 73.00 ± 12.20b 317.10 ± 7.00b 237.10 ± 3.40b 0.007 ± 0.0001b
The ecdysteroid contents were determined 2). Captopril increased significantly (p= 0.001) the
before and after hydrolysis using EIA. In ovaries of rates of total (p=0.001) and free ecdysteroids.
e niella the presence of free ecdysteroids was Treatment also resulted in a slight but significant
the predominant form as compared to conjugated reduction in conjugated ecdysteroids as compared
ecdystroids in both control and treated series (Table to control series.
TABLE 2
Effect of effect of captoril (10 μg/pupae), applied topically on newly molted pupae of E. ue niella, on the
amounts of free, conjugated and total ecdysteroids (pg/mg tissue) (mean ± SD, n= 6 females). Values
followed by different letters are significantly different at 5% level.
Treatment Free Conjugated Total

(pg/mg tissue) (pg/mg tissue) (pg/mg tissue)
Control 15.66 ± 0.94 a 1.87 ± 0.04 a 17.12 ± 0.82 a
Captopril 32.89 ± 2.70 b 1.43 ± 0.01 b 34.56 ± 2.85 b
The amounts of main biochemical components 0.001) reduction in the ovarian amounts of
were performed in each ovaries collected from carbohydrates, lipids and proteins as compared to
newly emerged adult females after treatment of control series.
pupae (Table 3). Captopril caused a significant (p<
TABLE 3
Effect of captoril (10 μg/pupa), applied topically on newly molted pupae of E. ue niella on amounts of
carbohydrates, lipids and proteins (μg/mg tissue) (mean ± SD, n= 6 females). For each components, values
followed by different letters are significantly different at 5% level.
Treatment Carbohydrates Lipids Proteins

(μg/mg tissue) (μg/mg tissue) (μg/mg tissue)
Control 30.61 ± 0.38 a 71.09 ± 4.21 a 28.72 ± 1.06 a
Captopril 18.88 ± 1.10 b 47.21 ± 4.19 b 10.02 ± 0.66 b
Captopril was administered topically on newly pre-and oviposition periods, fecundity and egg
molted pupae and its effects on the duration of the hatchability of each adult female from treated
1201


pupae were also investigated (Table 4, Fig. 1). The period (p= 0.01). Moreover, captopril applied
compound had no significant (p= 0.09) effect on the topically on newly molted pupae of e niella
duration of the pre-oviposition period. In contrast, it reduced significantly (p= 0.001) the fecundity and
reduces significantly the duration of the oviposition the egg hatchability (Table 4).
FIGURE 1
Effect of captoril (10 μg/pupa), when applied topically on newly molted female pupae of E. ue niella, on
the duration (days) of pre-oviposition and oviposition periods (mean ± SD, n= 6 females). For each
parameter values followed by different letters are significantly different at 5% level.
TABLE 4
Effect of captopril (10μg/pupa) applied topically on newly molted pupae of E. ue niella on the fecundity
(number of eggs laid females; mean ± SD; n=6 females) and eggs hatchability (%, mean ± SD, 4 replicate
each of 10 eggs). For each parameter values followed by the same letter are not significantly different at p
> 0.05).
Treatment Fecundity Egg hatchability

(eggs/female) (%)
Control 165.00 ± 2.16 a 70.18 ± 0.92 a

Captopril 155.70 ± 1.10 b 61.04 ± 0.45 b
In insects, the steroid molting hormone develop embryos and pre-hatching larvae [12].
(ecdysteroids), and the sesquiterpenoid juvenile Recently, it has been reported that ovarian follicle
hormone play a central role in the regulation of the cells are sites of biosynthesis of ecdysteroids and
growth, development and reproductive processes proteins for egg shell formation [25, 26]. The
[12]. The effect of captopril applied topically on quantitative analysis of ecdysteroids was tested by
newly molted pupae was examined on some EIA. In ovaries of e niella the presence of free
morphometric measurements of E. kuehniella ecdysteroids was the predominant form as
ovaries sampled from newly emerged adult compared to conjugated ecdystroids in both control
females. Results revealed that captopril caused a and treated series. In addition, captopril increased
significant reduction in both the weight of ovaries, significantly the rates of total and free ecdysteroids.
the number of oocytes per paired ovaries, the width, Free and conjugated forms of ecdysteroids are
the length and the volume of basal oocytes as confirmed in ovaries of many insects [12, 13, 27].
compared to controls. Our results agree with data The regulation of ovarian ecdysteroidogenesis
obtained after topical treatment of captopril on T. involves a range of different factors, which exert
molitor adults [16]. their effects through various pathways. Captopril
In adults, ecdysteroids are synthesized by the was reported to decrease the ovarian ecdysteroid
follicle cells in the ovaries and are available to amounts in females of m lit [16] and S.
1202


littoralis [28]. Lisinopril, another ACE inhibitor, mode of action of captopril is still unknown, our
induced a significant decrease in the whole body finding suggests an interference with the
content of ecdysteroids in e niella pupae [29]. reproductive events probably via the vitellogenesis
Methoxyfenozide, an ecdysteroid agonist, was process and its endocrine control. However, further
found to increase the amounts of free ecdysteroids research, for instance on the composition of
in ovaries of e niella [10]. Similarly, ecdysteroids (ecdysone and 20-hydroxyecdysone)
treatment of newly emerged adult females of in the embryo, is required to better understand the
mealworms with halofenozide, an ecdysteroid mechanism underlying the depressive effects of this
agonist, increased the amounts of free ecdysteroids drug on reproduction.
in eggs, and decreased that of conjugated
ecdysteroids but without any significant effect on
total ecdysteroids [13]. ACKNOWLEDGEMENT
In Lepidoptera such as e niella, the
process of vitellogenesis take place during the The authors are grateful respectively to Dr.
pupal stage and the oocyte accumulates and J.P. Delbecque (University of Bordeaux I, France)
organizes yolk from precursors imported from for the antibodies, Dr. C. Blais (UMPC, Paris,
hemolymph, while the cells of follicular epithelium France) for the tracer and Pr. G. Smagghe (Ghent
synthesize additional components of the yolk as University, Belgium) for the ACE inhibitors. This
well as the vitellin envelope and chorion [30]. research was supported by the National Fund for
Biochemical analyses showed that captopril applied Scientific Research of Algeria to Pr. N. Soltani and
on newly molted pupae exhibited an inhibitory the Ministry of Higher Education and Scientific
effect on both carbohydrate, protein and lipid Research of Algeria (CNEPRU and PNR projects to
amounts of ovaries from adult females that survive Pr. N. Soltani-Mazouni).
to treatment of pupae. It has been reported that
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SK (eds). Comprehensive Molecular Insect protein-dye binding. Analytical Biochemistry
Science, Vol. 3. Elsevier, Oxford, pp125±195. 72: 248-254.
[13] Berghiche, H., Houamria, M., Van De Vielde, [22] Soltani, N., Aribi, N., Berghiche, H., Lakbar,
S., Soltani, N., Smagghe, G. (2008). Effect of S., Smagghe, G. (2002). Activity of RH-0345
two insect growth regulators on the on ecdysteroid production and cuticle
secretion in ene i m lit pupae in vivo
1204


and in vitro. Pesticide Biochemistry and angiotensin converting enzyme on growth,

Physiology 72: 83±90. development, and ecdysteroid contents of the
[23] Kirane-Amrani L, Yezli Touiker S, Soltani- mediterranean flour moth, e tia e niella
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mealworms. Fresenius Environmental Bulletin Lepidoptera. Journal of Insect Science, 9(50):
24(5A): 1856 -1861 1-21.
[24] De Reggi, ML., Pitoizet, N., Gharib, B., [31] Hami, M., Taibi, F., Smagghe, G., Soltani-
Delbecque, J.P. (1992). New enzyme Mazouni, N. (2005). Comparative toxicity of
immunoassay for ecdysteroids using three ecdysteroid agonist insecticides against
peroxydase enzyme and polyclonal or the Mediterranean flour moth.
monoclonal antibodies. Xth Ecdysone Communications in Applied Biological
Workshop Leverpool 6-7th April, Abstract, pp Sciences, Ghent University 70(4): 767±772.
91. [32] Dhadialla, T.S., Retnakaran, A., Smagghe, G.
[25] Soltani-Mazouni, N. and Soltani, N. (1995) (2005). Insect growth and development
Protein synthesis in the fat body of ene i disruptingin insecticide. In: Gilbert, L.I.,
Kostas, I., Gill, S. (eds.). Comprehensive
m lit (L) during oocyte maturation: effect of
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[26] Soltani-Mazouni, N., Khebbeb, M.E.H.,
Soltani, N. (1999). 3URGXFWLRQG¶HFG\VWpURwGHV
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[27] De Loof, A. (2008). Ecdysteroids, juvenile
hormone and insect neuropeptides: recent
successes and remaining major challenges.
Pr. Dr. Noureddine Soltani
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3-13. Faculty of Sciences, Department of Biology
[28] Vercruysse, L., Gelman, D., Raes, E., Hooghe, University Badji Mokhtar of Annaba
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G. (2004). The angiotensin converting enzyme
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Physiol 57: 123±132.
[29] Kirane-Amrani L, Soltani-Mazouni N (2012)
Comparative activity of three inhibitors of the
1205

EFFECT OF PRE-O ONATION ON ANAEROBIC TREATABILITY OF

MEMBRANE CONCENTRATE
Mehmet Ca ma ci, F. Busra Yaman, Kaan Yetilme soy ,
Dogan Karadag, Bestamin O aya, Hande Tur ay, Tuba Genc
Department of Environmental Engineering, Faculty of Civil Engineering, Yildiz Technical University, 34220, Davutpasa, Esenler, Istanbul, Turkey
ABSTRACT treated physically, chemically and biologically,

however, in some cases existing treatment units
An integrated process composed of of 5 μm may not be sufficient to meet the desired effluent
cartridge filter, ultrafiltration and nanofiltration discharge standards. Particularly, it is sometimes
membranes has been used for the pre-treatment of very difficult to accomplish the removal of colour
cotton dyeing textile wastewater. Treatability of and turbidity from textile wastewaters by a single-
membrane concentrate has been further investigated step treatment, due to complex nature of these
by means of the anaerobic treatment with and wastewaters. Therefore, integration of existing
without pre-ozonation. Two identical fermentors treatment units and effective treatment processes
have been simultaneously operated to compare the has become inevitable when the improved effluent
effect of pre-ozonation on removals of organic will be reused in the main process. In recent
carbon, total Kjeldalh nitrogen, total phosphorus decades, the membrane technology has become a
and sulfate from the concentrate. During the study, promising alternative to conventional treatment
average removal efficiencies of COD and SO42- technologies due to its profound treatment
have been determined as 54% and 59% for non- efficiency, easiness and cost-effectiveness [2]. It
ozonated and 45% and 52% for ozonated has been reported that nanofiltration (NF) can be
concentrate, respectively. Results demonstrate that successfully applied to wastewaters from various
ozonation has a slight positive effect on the textile industrial processes to obtain high quality
biodegradability of membrane concentrate and the effluent by completely removal of organics and
removal of possible hazardous pollutants. Low salts [3-5]. Moreover, NF enables to reuse of
methane content of the produced biogas is wastewater and also decreases the consumption of
attributed to high sulfate concentration in the textile freshwater in textile production.
wastewater. Findings of this study indicates that the Distinctive constituents are condensed in the
combination of ozonation and anaerobic treatment concentrate stream during the membrane treatment
can be a suitable alternative for the post-treatment of the wastewater. The volume and composition of
of the concentrate from the membrane treatment of membrane concentrate depend on the quality of the
the textile wastewater. feed water, quality of the produced water, and the
pre-treatment method and cleaning procedures
used. The amount of constituent in the concentrate
KEYWORDS: may arise double or higher than that in the feed
Anaerobic Treatment; Membrane Concentrate; Ozone; water [6,7]. Concentrate from the membrane
Textile Wastewater treatment of textile industry is a colored waste
stream with high concentrations of organic and
inorganic compounds [8]. Further treatments of
INTRODUCTION concentrate with membrane fouling are the major
drawbacks of membrane processes [8].
Textile industry represents an important
environmental problem due to considerable water Environment. For this reason, there is an
consumption and highly polluted wastewater urgent need to develop efficient and cost effective
generation from dyeing, rinsing, bleaching and techniques for treating the textile wastewater to
finishing processes. Compared to many other improve the quality of the final discharge in terms
industrial effluents, textile wastewater is of residual pollutant contents. Recently, various
characterized by high color, COD and inorganic advanced chemical technologies have been
loads [1]. Various treatment methods have been proposed for the treatment of membrane
proposed for the post-treatment of textile concentrate such as electrochemical [6,9], H2O2
wastewater. Although textile wastewaters can be >@ )HQWRQ¶V R[LGDWLRQ >@ DQG R]RQDWLRQ >@
1206
*Corresponding author
Single application of advanced chemical process is digestion process. It is considered that all these
effective for the treatment of concentrated problems might be reduced by means of ozonation
pollutants while their main drawback is high process.
running cost. To overcome this problem, Considering the above-mentioned facts, the
physicochemical technologies have been specific objectives of this study were: (1) to
successfully combined with biological treatment investigate the effect of pre-ozonation on
methods [13-15]. Biological treatment technologies treatability of membrane concentrate in anaerobic
have been proven for the degradation of organic completely stirring fermentor by means of COD,
pollutants at relatively low costs. In particular, BOD5, TP, TKN, sulfate and TOC removals, (2) to
anaerobic degradation is an environmental friendly analyse of gas production and gaseous components
technology due to its high efficiency and rich in anaerobic fermentor fed with pre-ozonized and
energy content of biogas [16]. Various non-ozonized membrane concentrate, (3) to
configurations of anaerobic treatment have been measure the changes in volatile fatty acids and
successfully conducted for the treatment of textile components and (4) to evaluate the applicability of
wastewater [17-20]. Although numerous studies the proposed system in real-scale.
regarding the reuse of wastewaters by membrane
processes have been reported so far, however, a few
reports have been released for successfully E PERIMENTAL
application of anaerobic technology on singular or
combined treatment of membrane concentrate [21]. Experimental set-up and operation.
In the textile sector, where water consumption Continuous anaerobic treatment studies were
is quite high, the re-use of water in order to conducted by using two identical lab-scale-
minimize the water supply and wastewater fermentor systems (Fermac 320, Electrolab) for one
treatment costs is of utmost importance. While the year by feeding ozonated and non-ozonated
use of membranes allows wastewater to be re-used, membrane concentrate. Both fermentors were fed
they also create a concentrated solution, which by various amount of synthetic wastewater at start-
necessitates further treatment. This solution, where up period while Fermentor 1 and Fermentor 2 were
contaminants become more intense, is not only an fed by ozone-treated and raw concentrate,
important environmental problem, but because of respectively. Each fermentor had a total volume of
intensified trace contaminants, also a threat to the 6 L and a working volume of 5 L. To obtain a
health of the people who are in close contact. uniform environment, fermentors were
Moreover, the concentration of the pollutants in the simultaneously operated under continuous mixing
membrane concentrate is expected to be more 3 to 6 mode at 220 rpm. Fermentors had ports for
times than raw textile wastewater. Therefore, there sampling, feeding, effluent and gas collecting. Gas
will be expectedly differences between the was collected from the headspace on the top of the
anaerobic treatments of textile wastewater and reactor and gas production was measured by the
textile membrane concentrate. Since organic liquid displacement method. Wastewater samples
substances are biologically treated by anaerobic were taken from a cotton dyeing textile factory in
treatment, therefore, potential harmful Istanbul. Membrane system was consisted of a
environmental impacts of a waste will also be cartridge filter (5 μm), a ultrafiltration membrane
minimized. Besides, at the end of the anaerobic (UP150) and a nanofiltration membrane (NF270).
digestion of the concentrated solution with high UP150 and NF270 membranes were operated under
chemical oxygen demand (COD) values, methane TMP of 2.5 and 5 bars, respectively. Details on
gas that can be used as a source of energy gets membrane and concentrate characteristics can be
liberated. found in the previous work conducted by Cakmakci
There is a substantial gap in the relevant et al. [22]. Experimental scheme of ozonation and
literature about the elimination of this concentrated anaerobic treatment are depicted in Fig. 1. The
solution. In this study, the anaerobic digestibility of concentrate produced in the membrane system was
the concentrated solution formed at the end of the kept at 4°C to minimize substrate decomposition
direct treatment or ozone-based pre-treatment of before subsequent experimental studies. Some of
textile wastewater was investigated. Acetic acid, membrane concentrate was firstly subjected to
peroxide, salt, direct dyes, detergents, sodium ozone treatment. Ozone generator (Erwin Sander
bisulfite, fabric conditioners, bleaching agents, and Apparatebau, Sander Laboratory Ozonizer 300.5)
caustic chemicals are used, especially in textile was operated with air flow of 20 L/h for 15 min. pH
processes such as cotton fabric washing and dyeing. and ORP values were online measured and pH was
High concentrations of some of these chemicals in maintained at pH 7.1 by using 3 N NaOH and 5 N
the solution might adversely affect the anaerobic H3PO4 solutions. Temperature within the
1207
fermentors was kept at 37°C with electric blanket. fermentors was attempted to be kept at 7.0 by
The inoculum was anaerobic granular sludge from a means of online measurements and an automatic
full-scale UASB fermentor treating wastewater control system. Apart from very small deviations
from food industry at 37°C. TS content of the due to short-term mechanical problems, the pH
granular sludge was about 326.6 g TS/L. The VS level of the fermentors has always been stable.
content of the sludge was found to be about 36% of
TS. During the adaptation studies, the pH level of
FIGURE 1
Photograph of the experimental scheme of o onation and anaerobic treatment (a) o one set-up and
(b) anaerobic fermentor.
Analytical measurements. The Absorbance values were recorded at 420 nm and at

characterization of concentrate and the performance 690 nm using a spectrophotometer (UV-1800) for
of anaerobic fermentors were monitored by several sulfate and TP analyses, respectively. Deionized
chemical measurements. All experimental analyses water used in the experiments were supplied from a
were performed by the procedures described in the purification system (Meck Millipore Direct-Q 3, 5,
Standard Methods of APHA [23]. COD was 8 Ultrapure Water Systems). TOC was measured as
measured by closed reflux-titrimetric method (5220 described in the method of 5310 by using a HACH
C) while BOD was determined in 5 day test (5210 IL 550 TOC-TN analyser. The volatile fatty acid
B). Total phosphorus (TP), total Khjeldahl nitrogen (VFA) fractions were analyzed using a GC-2010
(TKN) and sulfate concentrations were determined device. CH4 and CO2 that was produced in the
by the methods of 4500-P C reactors was measured using SHIMADZU brand
(vanadomolybdophosphoric acid colorimetric GC-2014 gas chromatograph in accordance with the
method for total phosphorus), 4500-Norg B (macro- method number of 6211, and using a Thermal
Kjeldahl method for total Kjeldahl nitrogen) and Conductivity Detector (TCD). Stability of the
4500-SO4-2 E, respectively. Other parameters were treatment process and components of wastewater
determined by the procedures described in method samples were monitored in Environmental
numbers of 2320 B (titration method for alkalinity), Engineering Laboratory at Yildiz Technical
4500 NH3-N E (titrimetric method for ammonia), University in Istanbul, Turkey.
4500-Nr-L (titrimetric method for NH4+-N). 2540 B
(total solids dried at 103±105°C), 2540 D (total
suspended solids dried at 103±105°C) and 2540 E
(fixed and volatile solids ignited at 550°C).
1208
RESULTS AND DISCUSSION and vitamins was prepared for an optimum

anaerobic microbial growth is summarized in Table
Start-up period. Start-up periods of both 1.
fermentors lasted for 75 days in 5 stages. At start- At the fourth stage, ratio of synthetic
up, fermentors were initially fed by fully synthetic wastewater was reduced to 50% and the remaining
wastewater then amount of real textile wastewater half was of real textile wastewater while fermentors
was gradually increased to acclimate the were finally fed by 75% of membrane concentrate
microorganisms. At first three stages, synthetic with 25% of synthetic wastewater. Fermentors were
wastewater was supplied with glucose as a carbon operated simultaneously at identical hydraulic
source with COD equivalent of 5000 mg/L. The retention time (HRT) whereas it was increased from
composition of synthetic wastewater [24] 5 days in the first stage to 10 days at subsequent
containing all necessary micro- and macro-nutrients operation duration.
TABLE 1
Composition of synthetic wastewater 24 .
Chemical Concentration Chemical Concentration

NaH2PO4*1H2O 10.7 g/L EDTA (C10H16N2O8*2H2O) 50 μg/L
Na2HPO4 3.2 g/L Piridocsamine (C8H12N2O2*2HCl) 250 μg/L
NaHCO3 4 g/L Nikotinic Acid (C6H5NO2) 100 μg/L
NH4Cl 0.6 g/L Nikotinamide (C6H5N2O) 100 μg/L
KH2PO4 0.125 g/L DL-Pantotenic Acid (C9H16NO5*1/2Ca) 50 μg/L
CaCl2*2H2O 0.11 g/L Vitamine B12 (C63H88CoN14O14P) 50 μg/L
MgCl2*6H2O 0.1 g/L p-Aminonezoic Acid (C7H7NO2) 50 μg/L
FeCl2*4H2O 2 mg/L Piridocsine-HCl (B6: C8H11NO3*HCl) 100 μg/L
H3BO3 50 μg/L D Biyotine (C10H16N2O3S) 20 μg/L
ZnCl2 50 μg/L Tioctic Acid (Lipoic Acid; C8H14O2S2) 50 μg/L
CuCl2*2 H2O 38 μg/L Folic Acid (C19H19N7O6) 20 μg/L
MnCl2*2 H2O 41 μg/L Riboflavine (B2;C17H20N4O6) 50 μg/L
(NH4)6Mo7O24*4H2O 50 μg/L Tiamine-HCl (B1;C12H17ClN4OS*HCl) 100 μg/L
AlCl3 50 μg/L Na2SeO3*5H2O 26.3 μg/L
CoCl2* 6H2O 50 μg/L NaWO4*2H2O 32.9 μg/L
NiCl2* 6H2O 50 μg/L
Both fermentors had closer COD removal 10 days. Performances of fermentors were
efficiencies during the operation with synthetic evaluated mainly based on total COD (tCOD)
wastewater (Fig. 2). COD removal efficiencies of removal efficiencies and changes in soluble COD
both fermentors gradually increased from 40% in and BOD5 were monitored as well. Changes in total
first stage to 50% in the second stage. COD COD and soluble removals for both fermentors are
removal in Fermentor 2 reached to around 60% at given in Fig. 3a and b.
the end of third stage while Fermentor 1 has During the operation period, the influent total
slightly lower performance due to short-term COD values in the Fermentor 1 ranged between
mechanical problems. COD removal in both 2080 and 5835 mg/L, while the average COD
fermentors continued to increase during the concentration of the substrate fed to the Fermentor
following operation days and reached to 70% in 1 was detected as 3400 mg/L. The COD values of
Fermentor 2 and 58% in Fermentor 1. The the samples taken from the effluent of the
significant reduction in COD removal between Day Fermentor 1 varied between 421 and 3840 mg/L
60 and Day 65 were related with the due to short- and the average effluent COD value for the
term mechanical problems. Fermentor 1 was found to be 1420 mg/L.
According to these values, the COD removal
Comparison of treatment performances of efficiency in the Fermentor 1 was found to be 8-
fermenters. Following the start-up period, 82% for the operation period and the average COD
Fermentor 1 was fed by ozone-treated concentrated removal efficiency for the whole process was
while Fermentor 2 was fed by non-ozone-treated determined as 59% (Fig. 3a). The experimental
concentrated and both fermentors were operated in results indicated that the influent COD values in the
identical environmental conditions with the HRT of Fermentor 2 were measured as 1977-5810 mg/L
1209
and the average COD concentration of the substrate results indicated that the influent COD values in the
fed to the Fermentor 2 was found to be 3538 mg/L. Fermentor 2 were measured as 1977-5810 mg/L
The COD values of the wastewater samples taken and the average COD concentration of the substrate
2080 and 5835 mg/L, while the average COD fed to the Fermentor 2 was found to be 3538 mg/L.
concentration of the substrate fed to the Fermentor The COD values of the wastewater samples taken
1 was detected as 3400 mg/L. The COD values of from the effluent of the Fermentor 2 was
the samples taken from the effluent of the determined as 650-3443 mg/L, while the average
Fermentor 1 varied between 421 and 3840 mg/L effluent COD value for the Fermentor 2 was
and the average effluent COD value for the measured as 1684 mg/L. According to these values,
Fermentor 1 was found to be 1420 mg/L. the COD removal efficiency in the Fermentor 2,
According to these values, the COD removal which was fed by the non-ozonized membrane
efficiency in the Fermentor 1 was found to be 8- concentrate, changed between 4 and 74% and the
82% for the operation period and the average COD average COD removal efficiency for the whole
removal efficiency for the whole process was process was determined as 54% (Fig. 3a).
determined as 59% (Fig. 3a). The experimental
FIGURE 2
COD removal performances of Fermentor 1 and Fermentor 2 at start-u period
The influent soluble COD (sCOD) values in The sCOD values of the wastewater samples taken
the Fermentor 1 changed between 561 and 4610 from the effluent of the Fermentor 2 was
mg/L, while the average sCOD concentration of the determined as 420-2793 mg/L, while the average
substrate fed to the Fermentor 1 was detected as effluent sCOD value for the Fermentor 2 was
2441 mg/L. The sCOD values of the samples taken measured as 1114 mg/L. According to these values,
from the effluent of the Fermentor 1 varied between the sCOD removal efficiency in the Fermentor 2,
191 and 3074 mg/L and the average effluent sCOD which was fed by the non-ozonized membrane
value for the Fermentor 1was found to be 905 concentrate, changed between 12 and 88% and the
mg/L. Based on these values, the sCOD removal average sCOD removal efficiency for the whole
efficiency in the Fermentor 1 was found to be 12- process was determined as 58% (Fig. 3b). When the
87% for the operation period and the average sCOD average sCOD removal efficiencies for fermentor
removal efficiency for the whole process was systems are considered, it was seen that the sCOD
determined as 64% (Fig. 3b). During the removal efficiency of the Fermentor 1, which was
experiment, the influent sCOD values in the fed by the ozone-treated membrane concentrate was
Fermentor 2 were measured as 970-4560 mg/L and approximately 16% higher than that of the
the average sCOD concentration of the substrate Fermentor 2 fed by the non-ozonized membrane
fed to the Fermentor 2 was found to be 2524 mg/L. concentrate. The average ratios of sCOD/tCOD in
1210
influents were 71.7% and 71.3% for Fermentor 1 higher than that of the Fermentor 2 fed by the non-
and Fermentor 2, respectively, which indicated that ozonized membrane concentrate. Biodegradability
total COD in membrane concentrate is mainly ratios (BOD5/COD) of both fermentors were 0.39
comprised of soluble COD. and 0.33 in the influent and changed to 0.32 and
At first 20 days, COD removal efficiencies of 0.31 in the effluent for Fermentor 1 and Fermentor
both fermentors decreased sharply due to due to a 2, respectively. Those values indicated that both
short-term mechanical problem at day 2. After Day raw and ozone-treated concentrates had lower
21, both fermentors recovered the performance and biodegradability and pre-ozone treatment had a
COD removal efficiencies simultaneously increased slight effect on biodegradability of the membrane
up to 65% (Fig. 3a). Both fermentors performed concentrate. The changes in phosphorus
similar COD removal at subsequent operational concentration for the influent and effluent of two
days and the average COD removal were 59% and fermentors are as given in Fig. 4a and b. Total
54% for Fermentor 1 and Fermentor 2, respectively. phosphorus in the influent of Fermentor 1 was
Both fermentors had closer removal efficiencies for between 15.3-170 mg/L while phosphorus
soluble COD with 64% and 56% for Fermentor 1 concentration in the influent of Fermentor 2 was
and Fermentor 2, respectively. The difference measured as 20.1-192 mg/L. As can be clearly seen
between total and sCOD removal efficiencies of from Fig. 4a and b, phosphorus concentration in the
two fermentors were 5% and 8% which indicates effluent was higher in both fermentors. Average
that ozone treatment has very slight effect on COD increase ratio of phosphorus in the effluent was
removal by anaerobic bacteria. Similarly, Carballa 131% in Fermentor 1 and 109% in Fermentor 2.
et al. [25] found that pre-ozone treatment had only Similar results were reported for varied anaerobic
5% improvement on anaerobically treatment of fermentor systems which is the result of phosphorus
sludge. Andreozzi et al. [26] stated that ozone by- release by phosphate accumulating organisms
products have stronger inhibitory effect on [27,28]. Between Day 90 to 135, the highest
methanogenic bacteria rather than acidogenic phosphorus release was accompanied with the
bacteria. highest BOD5 removal efficiencies as well. It has
The influent BOD5 values in the Fermentor 1 been reported that phosphate accumulating bacteria
changed between 460 and 2380 mg/L, while the release phosphorus to gain energy which is used to
average BOD5 concentration of the substrate fed to take up organic carbon, primarily short-chain
the Fermentor 1 was measured as 1354 mg/L. The volatile fatty acids, and stored as intracellular
BOD5 values of the samples taken from the effluent polyhydroxyalakanoates within the cell [29,30].
of the Fermentor 1 varied between 70 and 1930 Changes in TKN values during the operational
mg/L and the average effluent BOD5 value for the period are given in Fig. 4c and d. Influent TKN
Fermentor 1 was found to be 447 mg/L. According values ranged from 95 to 213 mg/L and from 95 to
to these values, the BOD5 removal efficiency in the 196 mg/L for Fermentor 1 and Fermentor 2,
Fermentor 1 was found to be 2-90% for the respectively. On the other hand, average TKN
operation period and the average BOD5 removal concentrations in the effluent streams were 132
efficiency for the whole process was determined as mg/L for Fermentor 1 and 134 mg/L for Fermentor
67% (Fig. 3c). During the experiment, the influent 2. These figures show that very little ammonia
BOD5 values in the Fermentor 2 were measured as removal can be obtained during anaerobic
390-2185 mg/L and the average BOD5 treatment. Demirer and Chen [31] indicated that
concentration of the substrate fed to the Fermentor anaerobic treatment suplies so little nutrient
2 was found to be 1177 mg/L. The BOD5 values of removal and most of removed nitrogen and
the wastewater samples taken from the effluent of phosprous are used for new cell sysnthesis and
the Fermentor 2 system was determined as 25-1465 precipitation in the fermentor [32]. In anaerobic
mg/L, while the average effluent BOD5 value for systems fed with sulfate-rich wastewater,
the Fermentor 2 was measured as 514 mg/L. methanogenic bacteria and sulfate reducing bacteria
According to these values, the BOD5 removal survive in the similar environmental conditions and
efficiency in the Fermentor 2, which was fed by the compete on the same organic carbon. Because of
non-ozonized membrane concentrate, changed that, small lower methane production is
between 5 and 97% and the average BOD5 removal accompanied with high amount of hydrogen sulfide
efficiency for the whole process was determined as in the biogas despite higher organic carbon
59% (Fig. 3c). When the average BOD5 removal removal. In our system, raw concentrate had higher
efficiencies for fermentor systems are considered, it sulfate concentration and ozone treatment had no
was seen that the BOD5 removal efficiency of the effect on sulfate amount. Influent sulfate
Fermentor 1, which was fed by the ozone-treated concentration in Fermentor 1 was measured as 533
membrane concentrate was approximately 8% to 1876 mg/L while it was 548 to 1972 mg/L in
1211
Fermentor 2. Sulfate removal efficiencies for both different fermentor systems are considered, it was
fermentors are shown in Fig. 5a. Both fermentors seen that the gas production performance of the
had similar patterns like COD removal efficiencies Fermentor 1, which is fed by the ozonized solution,
with average values around 50%. The average is approximately 16.1% higher than that of the
sulfate removal efficiency in Fermentor 1 was 52% Fermentor 2, which is fed by a non-ozonized
while Fermentor 2 had a slight lower removal membrane concentrate The gas composition
efficiency with 45%. analyses that were carried out at different stages of
During the operation period, the total organic this study are shown in Fig. 6b and c. During the
carbon (TOC) values in the Fermentor 1 fermentor study, it was observed that the methane content in
changed between 508 and 1551 mg/L, while the the Fermentor 1 (which was fed by ozonized
TOC concentration of the substrate fed to the membrane concentrate) varied between 1.9 and
Fermentor 1 was detected as 1172 mg/L. The TOC 66.4%, while the average methane value in the
values of the samples taken from the effluent of the biogas obtained from the Fermentor 1 was 27.6%.
Fermentor 1 varied between 171 and 978 mg/L and Similarly, the methane content in the Fermentor 2
the average effluent TOC value for the Fermentor 1 (which was fed by non-ozonized membrane
was found to be 375 mg/L. According to these concentrate) changed between 0.8 and 70.9% and
values, the TOC removal efficiency in the the average methane content in this fermentor was
Fermentor 1 was found to be 4-85% for the found to be 23.1%. In addition, the carbon dioxide
operation period and the average TOC removal content in the biogas obtained from the Fermentor 1
efficiency for the whole process was determined as changed between 31.4 and 98.1% and the average
66% (Fig. 5b). During the experiment, the influent carbon dioxide value was 72.4% for this fermentor.
TOC values in the Fermentor 2 were measured as The carbon dioxide content analyzed in the
508-1570 mg/L and the average TOC concentration Fermentor 2 was 2-99.2% and the average methane
of the substrate fed to the Fermentor 2 was found to percentage in the Fermentor 2 was 76.9% (Fig. 6b
be 1189 mg/L. The TOC values of the wastewater and c).
samples taken from the effluent of the Fermentor 2 In terms of energy values, when the average
was determined as 194-895 mg/L, while the methane content in the biogas obtained from these
average effluent TOC value for the Fermentor 2 fermentor systems are considered, it was seen that
was measured as 443 mg/L. According to these the methane production performance of the
values, the TOC removal efficiency in the Fermentor 1, which was fed by the ozonized
Fermentor 2, which was fed by the non-ozonized membrane concentrate, was approximately 19.5%
membrane concentrate, changed between 6 and higher than that of the Fermentor 2, which was fed
83% and the average TOC removal efficiency for by a non-ozonized membrane concentrate.
the whole process was determined as 61% (Fig. 5b). During the experimental study, based on the
When the average TOC removal efficiencies influent (S0) and effluent (Se) COD concentrations,
for fermentor systems are considered, it was seen the COD removal efficiency values, active
that the TOC removal efficiency of the Fermentor fermentor volume (5 L), and the applied hydraulic
1, which was fed by the ozone-treated membrane retention time (10 days), the removed COD loads
concentrate was approximately 6% higher than that (Q × [S0 ± Se]) changed between 165 and 1625 mg
of the Fermentor 2 fed by the non-ozonized (COD)removed/day, and the average COD load
membrane concentrate. removal for the Fermentor 1 was determined as 978
mg (COD)removed/day. Similarly, during the
Gas production and composition. The operation, the COD loads in the Fermentor 2 (fed
volumes of the gas produced in the reactors are by the non-ozonized membrane concentrate)
shown in Fig. 6a. During the operation period, the changed between 64 and 1625 mg
gas volume produced in the Fermentor 1 (which (COD)removed/day and the average COD load
was fed by ozonized membrane concentrate) removal in the Fermentor 2 was found to be 915 mg
changed between 5.1 and 1887 mL, and the average (COD)removed/day.
gas production value was determined as 656 mL for
this system. Similarly, the gas volume values at the
Fermentor 2 (which was fed by non-ozonized
membrane concentrate) were found to change
between 4.25 and 1905 mL and it was seen that the
average value for this system was 565 mL. When
the average gas production values for these
1212
100
D
80
COD removal efficiency (%)

60
40
20
Fermentor 1
Fermentor 2
0
0 100 200 300 400
Time (day)
100
E
Soluble COD removal efficiency (%)
80
60
40
20
Fermentor 1
Fermentor 2
0
0 100 200 300 400
Time (day)
100
F
80
BOD5 removal efficiency (%)
60
40
20
Fermentor 1
Fermentor 2
0
0 100 200 300 400
Time (day)
FIGURE 3
Comparison of sCOD(a), COD(b) and BOD5(c) removal efficiencies of Fermentor1 and Fermentor2.
1213
300 250
Fermentor 1 (Influent)
D F
Total Kjeldahl nitrogen (TKN, mg/L)

250 Fermentor 1 (Effluent)
200
Total phosphorus (TP, mg/L)
200
150
150
100
100
50 50
Fermentor 1 (Effluent)
0 0
0 100 200 300 400 0 100 200 300 400
Time (day) Time (day)
300 250
E G
Total Kjeldahl nitrogen (TKN, mg/L)

250 Fermentor 2 (Effluent) 200
Total phosphorus (TP, mg/L)
200
150
150
100
100
50
50 Fermentor 2 (Influent)
Fermentor 2 (Effluent)
0 0
0 100 200 300 400 0 100 200 300 400
Time (day) Time (day)
FIGURE 4
Changes in total phosphorus and TKN values in Fermentor 1 (a and b) and Fermentor 2 (b and c).
The CH4 production rate per COD removed consequently the amount of the volatile fatty acids
within the framework of this study was determined in the environment increases. This situation can be
based on the average CH4 contents analyzed in the evaluated as an indication for the imbalance in
produced gas compounds and the average COD terms of methane production inside the reactor. In
load removal values and were found to be 0.205 L anaerobic conditions, where the volatile fatty acid
CH4/g (COD)removed and 0.181 L CH4/g (COD)removed concentration is relatively high, the fractional
for the Fermentor 1 and Fermentor 2, respectively. balance between the acid-producing
When these values are compared to 0.397 L CH 4/g microorganisms (acitogen and acetogen
(COD)removed, which is equal to the theoretical CH 4 population), as well as the acid-consuming
production for the stable operation temperature for microorganisms (methanogens, sulfate-reducing
the fermentors, it was seen that 51.7% and 45.6% of bacteria and nitrogen-reducing bacteria) is
the theoretical maximum CH4 production are disturbed and the carbon dioxide content in the
obtained for the Fermentor 1 and Fermentor 2, biogas increases [33]. As a result of this
respectively. Based on the values obtained from the characteristic change in the metabolic steps, the
biogas composition, it was seen that the carbon sulfate-reducing bacteria begin competing against
dioxide content was higher than the methane methanogens and homoacitogens. During the
content from day 220 onwards. When the relevant anaerobic decomposition the sulfate ions in the
literature is surveyed, the following conclusions can environment result in an increase in the sulfate-
be drawn: reducing bacteria (SRB). The sulfate-reducing
bacteria compete with the methane-reducing
(1) As a result of the changes in organic and bacteria for the same substrate (hydrogen and
hydraulic loading rates in certain periods, the acetate) [22].
microbial equilibrium between acitogens and
methanogens has become unbalanced, and
1214
100
D Fermentor 1
Fermentor 2
80
SO4 removal efficiency (%)
60
40
2-
20
0
0 100 200 300 400
Time (day)
100
E
80
TOC removal efficiency (%)
60
40
20
Fermentor 1
Fermentor 2
0
0 100 200 300 400
Time (day)
FIGURE 5
Comparison of sulfate (a) and TOC (b) removal and efficiencies of Fermentor 1 and Fermentor 2.
Within the framework of this study, the possible reasons for the carbon dioxide content
average influent total volatile fatty acid being higher than the methanecontent is thought to
concentrations measured after day 220, where the be the microbial balance in the fermentors and the
carbon dioxide content was higher than the methane biogas characteristics changing in favor of carbon
content for both fermentor systems, were 529 mg/L dioxide due to the increase in the volatile fatty acid
and 445 mg/L for the Fermentor 1 and Fermentor 2, concentrations in the membrane concentrate fed to
respectively. On the other hand, the average the fermentor after day 220. (2) In the case of an
influent total volatile fatty acid concentrations increase in carbon dioxide composition, it is known
measured before day 220 were 267 mg/L and 193 that most acitogenic bacteria demonstrate a more
mg/L in the Fermentor 1 and Fermentor 2, active bio-reaction in comparison to methanogenic
respectively. Based on both the experimental results bacteria [34]. In addition, the high carbon dioxide
and the above-mentioned studies, one of the content in the biogas might be due to the fact that
1215
the hydrogen consumption in the environment is isovaleric acid, corresponding to 11.5% and 10.6%
much higher than the carbon dioxide consumption of the total acid concentration, respectively.
as a result of the acetic acid produced by According to the average values obtained at
homoacetogenic reactions of homoacetogens [35]. the end of a series of analyses made on the volatile
Accordingly, another possible reason for carbon fatty acid compounds of the samples taken from the
dioxide contents being higher than methane effluent of the Fermentor 1 (between days 18 and
concentrations is considered to be the bio-reactions 343), the highest concentration among the effluent
of the acitogenic bacteria more active than those of samples was 182 mg/L acetic acid, which was equal
methanogenic bacteria due to the changing to approximately 39% of the total effluent acid
conditions after day 200. concentration. According to the average values
obtained for the Fermentor 1, the next highest
ariation of volatile fatty acids ( FA) in volatile fatty acid compounds (after acetic acid)
fermentors. A considerable number of analyses were isocaproic acid (48 mg/L) and hexanoic acid
showed that the volatile fatty acid compounds were (40 mg/L), corresponding to the 10.2% and 8.5% of
below the detection limit. During the study, the the total volatile fatty acid concentrations (Table 2).
volatile fatty acids (acetic acid, propionic acid, Details regarding analyses of the volatile fatty acid
isobutyric acid, butyric acid, isovaleric acid, valeric compounds (acetic acid, propionic acid, isobutyric
acid, isocaproic acid, hexanoic acid, and heptonic acid, butyric acid, isovaleric acid, valeric acid,
acid) in the samples taken from the influent of the isocaproic acid, hexanoic acid, and heptonic acid)
Fermentor 1 (which was fed by ozonized membrane in the samples taken from the influent of the
concentrate) between days 46 and 343 of the Fermentor 2, which was fed by the non-ozonized
operation were analyzed (Table 2). According to membrane concentrate, between days 98 and 351 of
the average values determined by means of these the operation are given in Table 3. According to the
analyses, the highest concentration among the average values determined by means of these
samples taken from the influent belonged to acetic analyses, the highest concentration among the
acid and was equal to 129 mg/L, which was samples taken from the influent belonged to acetic
approximately 31.6% of the total acid concentration acid and was equal to 101 mg/L, which was
in the influent. For the Fermentor 1, this value was approximately 30.3% of the total acid concentration
followed by 47 mg/L isocaproic acid and 43 mg/L in the influent.
TABLE 2
Average concentrations of volatile fatty acid compounds in the influent and the effluent of
Fermentor1.
Volatile fatty acids (VFA)a
Time (day)
AA PA IBA BA IVA VA ICA HXA HPA TA
(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
46-343 Influent VFA concentrations

Average (mg/L) 129.00 28.25 33.30 31.31 43.07 38.34 46.95 37.86 19.71 401.54
Percentages (%) 31.6 6.9 8.2 7.7 10.6 9.4 11.5 9.3 4.8 100
18-343 Effluent VFA concentrations
Average (mg/L) 181.80 32.06 28.99 33.41 39.33 39.19 47.67 39.85 27.33 452.17
Percentages (%) 38.7 6.8 6.2 7.1 8.4 8.3 10.2 8.5 5.8 100
aAA: acetic acid, PA: propionic acid, IBA: isobutyric acid, BA: butyric acid, IVA: isovaleric acid, VA: valeric acid, ICA:
isocaproic acid, HXA: hexanoic acid, HPA: heptonic acid, TA: total acid.
For the Fermentor 2, similar to the Fermentor corresponding to 11.8% and 10% of the total acid
1, this value was followed by isocaproic acid (39.3 concentration, respectively.
mg/L) and isovaleric acid (33.2 mg/L),
1216
2000
Fermentor 1 D
Fermentor 2
1500
Gas production (mL/day)
1000
500
0
0 100 200 300 400
Time (day)
100
Fermentor 1 (CH4) E
Fermentor 1 (CO2)
80
Gas composition (%)
60
40
20
0
100 150 200 250 300 350 400
Time (day)
100
Fermentor 2 (CH4) F
Fermentor 2 (CO2)
80
Gas composition (%)
60
40
20
0
100 150 200 250 300 350 400
Time (day)
FIGURE 6
Changes in gas production values (a) and comparison of gas compositions (CH 4 and CO2) in Fermentor 1
(b) and Fermentor 2 (c).
1217
According to the average values obtained at bacteria (MPB) and sulfate-reducing bacteria (SRB)
the end of a series of analyses made on the volatile inside the fermentors and increasing organic
fatty acid compounds of the samples taken from the loading conditions in the anaerobic conditions, not
effluent of the Fermentor 2 (between Days 18 and all of the intermediate products can be converted to
351), similar to the Fermentor 1 reactor, the highest methane and consequently, the methane percentage
concentration among the effluent samples was in gas composition is less than the carbon dioxide
acetic acid, again, with a concentration equal to 177 percentage.
mg/L, corresponding to approximately 42.8% of the
total effluent acid concentration. According to the
average values obtained for the Fermentor 2, the CONCLUSIONS
next highest volatile fatty acid compounds (after
acetic acid) were 40.1 mg/L isocaproic acid and Although the removal of membrane
34.2 mg/L hexanoic acid, corresponding to 9.7% concentrates is a considerable problem today, the
and 8.3% of the total volatile fatty acid related studies are quite few in number. Results of
concentrations (Table 3). this study indicated that wastewaters containing
Based on the average values obtained from the resistive organic matters such as textile industry
volatile fatty acid compound analyses carried out wastewaters, can be actually suitable for real-scale
for the Fermentor 1 and Fermentor 2, it was seen concentrate management. The conclusions drawn
that the compound with the highest concentration at based on the present study are considered to
the influent and effluent was acetic acid and there provide an important data-base for scale-enlarging
were acid compounds in the fermentors effluent pilot facilities. Furthermore, operation of anaerobic
that were not able to be converted to methane, systems with the high inert heat produced by
resulting in an increase in the total acid dyeing, rinsing, bleaching units might also make
concentration in the effluent. When the average the usability of these processes for large-scale
acid concentration percentages at the effluent are textile facilities attractive for the concentrate
considered, it can be concluded that the acetic acid management.
concentration found in the samples taken from the
effluent of the Fermentor 1 (which was fed by the
ozonized concentrate) was approximately 39% of
the total acid concentration. Similarly, the acetic ACKNOWLEDGEMENTS
acid concentration found in the samples taken from
the effluent of the Fermentor 2 (which was fed by This research has been supported by The
the non-ozonized concentrate) was approximately Scientific and Technological Research Council of
42.8% of the total acid concentration. Furthermore, Turkey (TUBITAK-CAYDAG) (Project Number:
these results support the fact that depending on the 110Y222) Ankara-Turkey. The authors have
bacterial competition between methane-producing declared no conflict of interest.
TABLE 3
Average concentrations of volatile fatty acid compounds in the influent and the effluent of
Fermentor2.
Volatile fatty acids (VFA)a
Time (day)
AA PA IBA BA IVA VA ICA HXA HPA TA
(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)
98-351 Influent VFA concentrations

Average (mg/L) 100.63 26.97 23.87 25.41 33.18 31.81 39.30 31.29 20.12 332.59
Percentages (%) 30.3 8.1 7.2 7.6 10.0 9.6 11.8 9.4 6.0 100
18-351 Effluent VFA concentrations
Average (mg/L) 176.48 25.80 24.08 27.15 31.21 32.61 40.07 34.27 20.47 435.61
Percentages (%) 42.8 6.3 5.8 6.6 7.6 7.9 9.7 8.3 5.0 100
aAA: acetic acid, PA: propionic acid, IBA: isobutyric acid, BA: butyric acid, IVA: isovaleric acid, VA: valeric acid, ICA:
isocaproic acid, HXA: hexanoic acid, HPA: heptonic acid, TA: total acid.
1218
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1220

INTEGRATED HEALTH ASSESSMENT OF A MULTI-TYPE

RIVER TO OPTIMIZE REMEDIAL WORKS IN THE DIANCHI
LAKE BASIN
hu- ing in1, 2, in-hua Li1, Feng-le Yang2, Ke Huang1, Bao-xue hou 1
1
School RI(QYLURQPHQWDO6FLHQFHDQG(QJLQHHULQJ6KDQJKDL-LDR7RQJ8QLYHUVLW\1R'RQJFKXDQ5G6KDQJKDL3HRSOH¶V
Republic of China.
2
<XQQDQ,QVWLWXWHRI(QYLURQPHQWDO6FLHQFH.XQPLQJ3HRSOH¶V5HSXEOLFRI&KLQD
ABSTRACT suggest that an estuarine wetland could be built to

further decrease pollutant loading.
The Xinbaoxiang River is a typical multi-type
river that flows through both suburban and urban
areas in the Dianchi Lake Basin. In this paper, we KEYWORDS:
determined the health of the river by applying a Dianchi Lake, river health assessment, multi-type river,
remedial works
river health assessment index system to Dianchi
Lake. Our results show that the comprehensive
river health assessment index for the upper reach is
INTRODUCTION
2.695, indicating a basically healthy status despite
the low natural water supply. The middle reach is
sub-healthy, with an assessment index of 2.014, Dianchi Lake is a typical plateau lake located
because of the direct inflow of suburban wastewater in the arid Yunnan-Guizhou Plateau in Southwest
directly flowing into the river with no pollutant China. It is one of the largest and most
interception and a low natural water supply. The contaminated lakes in China. Rapid industrial
river is also sub-healthy in its lower reach, with an development in the region in recent decades has
assessment index of 2.376, because of the high caused environmental deterioration, especially
nitrogen loading and a low capacity for self- because of surface water contamination. In total, 35
purification. Based on this health assessment and rivers flow through densely populated urban and
the characteristics of the Xinbaoxiang River, we suburban areas in the Dianchi Lake watershed
propose the following remedies: in the upper and before entering the lake. These rivers contribute an
middle reaches, the problem of insufficient natural annual average water volume of 900 million m3,
water supply could be solved by artificial water and the contaminants they carry account for 72% of
replenishment, in which water could be injected the chemical oxygen demand (CODCr), 78% of the
back into the river from the Airport Wastewater total nitrogen loading (TN), and 80% of the total
Treatment Plant and the No. 12 Wastewater phosphorous (TP) loading of Dianchi Lake [1].
Treatment Plant (which are currently under Therefore, treatment of these polluted rivers is
construction). In the middle reach, the sewage FUXFLDO WR WKH UHPHGLDWLRQ RI 'LDQFKL /DNH¶V
interception system could be enhanced in several ecological system.
ways to decrease the amount of pollutants flowing Various river management approaches have
into the river; potential approaches include a traditionally been used to enhance riverbank and
sewage interception project along both sides of the riverbed security, including cutting off meander
riverbank, a dispersal treatment project utilizing the bends, dredging and constructing vertical
existing ponds and wetlands and a retention pond riverbanks. However, these approaches often do not
project to regulate overflow storm water with high work well in polluted rivers because their
SROOXWDQW ORDGLQJ ,Q WKH ORZHU UHDFK WKH ULYHU¶V ecological health and capacity for self-purification
self-purification capacity could be increased by are impaired [2]. In the case of Dianchi Lake,
implementing in situ treatment projects that use many preventive actions and remedial works have
ecological concrete to repair the hardened riverbed. been implemented in an attempt to solve these
In addition, fill biological stuffing could help problems, such as blocking wastewater pipes,
increase the biomass in the river. Finally, we intercepting sewage for further treatment and
implementing ecological recovery measures. These
1221


measures have improved the water quality of many MATERIALS AND METHODS
rivers in the Dianchi watershed, including the
Xinbaoxiang River, which has the highest flow rate. Sampling sites. The 292-km2 Xinbaoxiang
However, because each river has its own River catchment lies 45.8 km northeast of Dianchi
unique ecosystem, treatment should be individually Lake. The Xinbaoxiang River is one of the most
WDLORUHGEDVHGRQDWKRURXJKDQDO\VLVRIHDFKULYHU¶V important rivers to the city of Kunming in Yunnan
hydrology, water quality, riparian zone, SURYLQFH )LJXUH 7KH ULYHU¶V KHDGZDWHUV DUH LQ
morphology and aquatic life. Use of a river health the Xinbaoxiang River Reservoir, which extends
assessment index system can help identify the for 14.4 km. The ecological environment of the
primary problems facing these rivers and ensure reservoir is not heavily polluted; its water quality is
that their ecological systems are improved to the rated as class II, which meets the standard of
greatest degree possible using the relevant remedial drinking water. The upstream portion of the river
works. extends for 10.54 km before passing under the
The river health assessment index system is an Dahua Bridge, where the natural riverbank and
advanced approach to river management that was ecological environment are well maintained. The
developed to guide river environmental midstream reach stretches 12.05 km from the
management and ecological restoration. It has Dahua Bridge to the Yangfu Flood Diversion Gate
proved to be effective for the study of river and passes through suburban areas. The
conditions and key indices of river health [3]. The downstream reach extends from the Yangfu Flood
assessment results can help identify cost-effective Diversion Gate to the estuary for 8.81 km and
remedial works for river restoration and investment passes through urban areas.
management. Numerous studies have reported the There are three wastewater treatment plants in
successful use of river health assessments in the Xinbaoxiang River watershed. The No. 6
various areas, including the Hawkesbury-Nepean Wastewater Treatment Plant has a processing
River in Australia [4], the Keum-Ho River in capacity of 130,000 m3/d and is located in the
Korea [5], and the Brantas River in Indonesia [6]. downstream region; the No. 12 Wastewater
The method has also been applied to some of the Treatment Plant has a processing capacity of
largest watersheds in China, such as the Liao River 100,000 m3/d and is located in the midstream
[7, 8] the Hai River [9,10], the Dongjiang River region; and the Airport Wastewater Treatment
[11] and Taihu Lake [12-14]. Plant, which is currently under construction, is
The Xinbaoxiang River, a typical multi-river located in the upstream region and will have a
in the Dianchi Lake watershed, was selected as a processing capacity is 70,000 m3/d.
model river for this study. This river flows through During the period of the 11th Five-Year Plan,
a large variety of urban and suburban areas and has several remedial works were implemented in the
diverse types of riverbanks in different sections, lower reach including the construction of a sewage
includingnatural habitats with few human impacts interception trunk, the blockage of wastewater
and imitation natural reaches constructed with pipes, riverbed dredging and the renovation of the
gabions and straight solidified reaches. Although No.6 Wastewater Treatment Plant. After the
several remedial works have been implemented implementation of those remedial works, the water
along the river, its reaches have displayed different environment had been obviously improved in the
capacities for self-purification. Moreover, because lower reach.
different regions have unique ecological Seven sampling sites were selected along the
characteristics, they also face various health river to assess its health condition. Sites 1 and 2 are
problems. located in the lower reach; Sites 3, 4 and 5 are
This paper establishes a river health located in the middle reach; and Sites 6 and 7 are
assessment index system designed for Dianchi Lake located in the upper reach.
and applies it to the Xinbaoxiang River to study
river health. The index system provides scores for Sampling and chemical analysis. Sampling
ILYH FRPSRQHQWV WKDW GHILQH D ULYHU¶V KHDOWK frequency. Surface water was collected from the
condition: hydrology, water quality, riparian zone, sampling sites twice every month during the period
morphology and aquatic life. We demonstrate that from January 2013 to November 2014.
WKHULYHU¶VKHDOWKFRQGLWLRQFDQEHGHVFULEHGE\WKH Zooplankton, phytoplankton and benthic organisms
health index and its overall score. We also suggest were sampled in October 2013.
that the assessment result is fundamental to the
design of effective restoration projects, the Water uality. Assessment of water quality
optimization of strategies, and the improvement of included both chemical and physical analyses and
river health conditions. involved eleven parameters. The chemical
1222


parameters included dissolved oxygen (DO), and suspended solids (SS). Among these
demand (CODCr), five-day biological oxygen parameters, DO and pH were measured in the field
demand (BOD5), total nitrogen (TN), total using portable instruments, and the remainder were
phosphorous (TP), ammonia-nitrogen (NH4+-N), measured in the laboratory following the
nitrate-nitrogen (NO3--N), and nitrite-nitrogen Monitoring and Analysis Methods for Water and
(NO2--N). Physical parameters included pH value Wastewater (SEPA, GB3838-2002).
FIGURE 1
Sampling sites on the Xinbaoxiang River.
A uatic animal collection. For the zooplankton. The density of aquatic animals in the
zooplankton collection, a No. 25 mesh net was used samples was then calculated.
to sample protozoa and a No. 13 mesh net was used The benthic samples were collected using a
to sample cladocerans and copepods. Samples for mud dredger (29 cm×17 cm) and then filtered
qualitative studies were collected using the No. 13 through a No. 40 mesh net. The benthic animals
mesh net, which was GUDJJHGLQD³´VKDSHIRU were placed in 50 mL sampling bottles and
min at a depth of 0.5 m, after which the samples preserved in a 10% formaldehyde solution. The
were preserved in a formaldehyde solution in a 100 animals were identified to the species level, counted
mL specimen bottle. and weighed, and then, the density of aquatic
Samples for quantitative studies filtered animals per square meter of water was calculated.
through the No. 25 mesh net were concentrated
from 10 L to 20 mL and preserved in a Plants. We used the classic sampling method
formaldehyde solution in a 100 mL specimen involving a 20 m×20 m quadrat. For each quadrat,
bottle. To determine the density of aquatic animals, we recorded data, including the name, GPS site,
the samples were diluted to 100 mL and shaken. elevation, gradient, slope, exposure, slope position,
Next, 1 mL was extracted into a counting box to human influence, and coverage rate for the entire
determine the quantity of different types of plant community and for each layer of the plant
community. The plant community was divided into
1223


layers based on height: the upper 5 m was classified concentration could be attributed to the increase in
as the arborous layer, and the lower 5 m was the NO3--N concentration.
classified as the shrub layer. Herbaceous plants Figure 3 shows a cluster analysis for the seven
formed the herb layer, and vines and epiphytes sampling sites. The sites fall into two groups: S6
formed the interlayer. and S7 (in the upper reach) and S1-S5 (in the
middle and lower reaches). Pollutant monitoring at
Other indicators. Other indicators, such as each site suggests that the upper reach is clean,
hydrology and morphology, were determined from whereas the middle and lower reaches are
previously published literature, expert consultation, contaminated.
field investigation and comparison.
A uatic life.
Data analyses. The SPSS 22.0 software was a. Zooplankton: In total, 42 species of
used for the analysis of mean values and cluster zooplankton were detected from sampling in the
analysis. An analytical hierarchy process (AHP) estuary, downstream, midstream and upstream.
was used to calculate the weights of the indicators Among these, 10 were from Protozoa 17 from
in the river health assessment index system Rotifera 4 from Cladocera and 5 from Copepoda.
designed for Dianchi Lake. To fully assess the diversity of zooplankton in the
Xinbaoxiang River, three biodiversity indices were
calculated for all sampling sites: the Shannon-
RESULTS AND DISCUSSION Wiener index, the Margalef index and the Pielou
index (Table 1).
Water uality and ecology. Water uality. b. Phytoplankton: In total, 77 species of
Figure 2 shows the six main water pollution indices phytoplankton were detected in the estuary,
(CODcr, BOD5, TN, TP, NH4+-N and NO3--N) at downstream, midstream and upstream sampling
the seven sampling sites in the Xinbaoxiang River. sites. They belong to 5 phyla: Cyanophyta,
The organic pollutant loads and concentrations of Cryptophyta, Bacillariophyta, Euglenophyta and
nitrogen and phosphorus are lower in the upper Chlorophyta The Shannon-Wiener, Margalef and
reach, at S6 and S7, than in the middle or lower Pielou indices were also calculated for
reaches; the levels of CODcr, BOD5, TP and NH4+- phytoplankton to examine their distributions along
N are similar at all five sampling sites. A the river (Table 2).
comparison of S6 (in the upper reach) and S5 (in c. Benthic invertebrates: For benthics, 17
the middle reach) illustrates that the average species were detected in the estuary, downstream,
concentrations of CODcr and BOD5 in the upper midstream and upstream sampling sites. They
reach are 12.1 mg/L and 3.53 mg/L, respectively, belong to 3 phyla, Arthropoda Annelida and
which is less than half of those (26.5 mg/L and 9.74 Mollusca. Table 3 shows the density, biomass,
mg/L, respectively) in the middle reach. Similarly, Margalef index and Pielou index for the benthic
the average concentrations of TP and NH4+-N are invertebrates at all sampling sites. Different species
0.109 mg/L and 0.687 mg/L in the upper reach, have vastly different pollution tolerances, but only
respectively, which is a third of the concentrations the pollution tolerant species are able to reproduce
in the middle reach (0.316 mg/L and 2.177 mg/L, in contaminated rivers. Thus, the Margalef index is
respectively). most suitable for assessing the biodiversity of
The average concentrations of TN and NO3--N benthic invertebrates.
are significantly higher at S1 and S2 in the lower
reach than at the other sampling sites. A Water uantity. The distribution of rainfall in
comparison of S3 (in the middle reach) and S2 (in the Dianchi Lake Basin is uneven due to the
the lower reach) illustrates that the average complex local topography. The Xinbaoxiang River
concentrations of TN and NO3--N are 2.790 mg/L is located east of the lake and has an annual rainfall
and 6.72 mg/L, respectively, in the middle reach of 820-890 mm. The natural water supply for the
and 4.625 mg/L and 9.28 mg/L, respectively, in the Xinbaoxiang River is 0.557 m3/s, the majority of
lower reach. The concentrations of NH4+-N are which comes from the Xinbaoxiang Reservoir and
highly similar at all sites in the middle and lower the Cao River (its main tributary), which provide
reaches, which suggests that the high TN water supplies of 0.057 and 0.5 m3/s, respectively.
1224


FIGURE 2
Chemical analysis of water uality at the seven sampling sites.
7KH FRQFHSW RI ³ULYHU HFRORJLFDO ZDWHU method) for self-purification and 0.0084 m3/s for
GHPDQG´KDVEHHQSURSRVHGWRPDLQWDLQWKHULYHU¶V evaporation. However, the actual natural water
ecological health. For the Xinbaoxiang River, the supply is only 0.557 m3/s, which is less than two
ecological water demand (0.8944 m3/s) can be thirds of the ecological water demand (0.8944
divided into three categories: 0.489 m3/s (Tennant m3/s).
method) for the basic flow, 0.397 m3/s (7Q10
1225


FIGURE 3
Dendrogram showing the clustering of water uality indices at the seven sample sites.
TABLE 1
Biodiversity indices for ooplan ton from upstream to downstream.
Site Estuary (S1) Downstream (S2) Midstream (S4) Upstream (S7)

Species number, S 16 15 14 13
Shannon-Wiener LQGH[+¶ 1.826 1.468 1.734 1.294
Margalef index, DM 1.870 1.899 1.716 1.391
Pielou index, Je 0.658 0.542 0.657 0.505
TABLE 2
Biodiversity indices for phytoplan ton from upstream to downstream.
Downstream
Site Estuary (S1) Midstream (S4) Upstream (S7)
(S2)
Species number, S 20 24 16 10
Shannon-:LHQHULQGH[+¶ 2.667 2.466 2.331 2.158
Margalef index, DM 1.367 1.618 1.106 0.719
Pielou index, Je 0.890 0.776 0.841 0.937
Riparian one. The riparian zone has Morphology. In the upper reach of the
significantly different morphologies in the various Xinbaoxiang River, the natural curves of the river
reaches. In the upper reach, the natural ecology is are still present, the riverbed is stable, and there is
undisturbed, and the river has rich vegetation, broad sound connectivity. Similarly, in the middle reach,
buffering riparian areas and stable riverbanks. In the river still follows its natural path and its
the middle reach, some parts of the riverbank have meanders have not been cut off, maintaining the
been reinforced with rock-filled gabion walls along stability and ecology of the river. In contrast, the
WKH ULYHU¶V ZLQGLQJ SDWK E\ WKH UHVWRUDWLRQ SURMHFW lower reach has been overhauled and now has a
team. The lower reach is a straight channel that was straight channel without any natural morphology.
excavated in 1977. The downstream riverbed was Consequently, the return period of floods in the
channelized and hardened in 2006 for flood control river has lengthened to approximately 50 years.
purposes.
1226


a. Riparian zone of the upstream reach b. Riparian zone of the midstream reach
c. Riparian zone of the downstream reach
FIGURE 4
Riparian ones from upstream to downstream.
River health assessment index system system for river health around Dianchi Lake. We
designed for Dianchi La e. Assessment of chose 20 indicators to assess five aspects of the
indicator species assessment and integrated indices rivers: the hydrology, water quality, riparian zone,
are the two methods that are most often used for river morphology and aquatic life. These indicators
river health assessment. Due to the rapid fully cover the complex characteristics of river
development of the cities around Dianchi Lake, the functioning and can thus accurately reflect the
aquatic ecosystems of the main rivers flowing into ULYHU¶V HFRV\VWHP ZDWHU ODQGVFDSH DQG IORRG
the lake are being degraded. In many of the rivers, control capacity.
severe water pollution and low precipitation have River flow is one of the most basic indicators
led to ecological problems such as flow cut-off and RI D ULYHU¶V K\GURORJLFDO Fondition [24]. Fresh
fish extinction [15]. Therefore, indicator species water supply is a particularly important indicator
assessment cannot provide a sufficient amount of for the Dianchi Basin because of its dry climate and
information for river health assessment, and abundant river cut-offs. A sufficient water supply
assessment of integrated indices is more suitable FDQ LPSURYH WKH ULYHU¶V HFRV\VWHP DQG KHDOWK
[16]. condition. In terms of water quality, TN, NH4+-N
There are several methods to assess and TP are key indicators of water pollution for the
integrated indices, such as the Rapid Bioassessment rivers that enter the lake [25]. CODcr is also an
Protocols (RBPs) [17], the Riparian, Channel and important indicator for total quantity control.
Environmental (RCE) inventory [18], the Index of Similarly, DO can directly reflect the general status
Stream Condition (ISC) [19] and the River Habitat of the aquatic environment. The riparian zone
Survey (RHS) [20]. Some methods were indicators include the coverage rate of riparian
specifically developed for Chinese rivers, including vegetation, the width of the riparian area, riverside
the assessment methods for the Yangtze River stability and the types of riverbanks reported in
[21], Yellow River [22] and Pearl River [23]. previous literature [26]. In addition, a new
However, some specific issues must be considered waterside landscape comfort level indicator is used
for the rivers around Dianchi Lake, such as the to measure the landscape function of the
insufficient water sources and dry climate. Xinbaoxiang River. The river morphology
Therefore, we established an integrated index indicators include riverbed stability, natural
1227


FRQQHFWLYLW\WKHULYHU¶VGHJUHHRIFXUYDWXUHDQGLWV Weighting of the indicators (Table 4) was

capacity for flood control (a significant function of calculated using an analytical hierarchy process
the Xinbaoxiang River). The majority of rivers (AHP) [28], in which each indicator was rated by
around Dianchi Lake have severely damaged river restoration specialists in Yunnan province.
ecological systems; thus, biodiversity indicators, The indicators were rated based on a five-point
such as benthic organisms, zooplankton and VFDOH7DEOH7KHILYHOHYHOVDUH³KHDOWK\´VFRUH
phytoplankton, are more useful as indicators of the 3.2- ³EDVLFDOO\ KHDOWK\´ VFRUH - ³VXE-
ULYHU¶V KHDOWK FRQGLWLRQ ,Q VRPe cases, the KHDOWK\´ VFRUH - ³VXE-siFN´ VFRUH -1.6)
Shannon-Wiener index was found to be more DQG³VLFN´VFRUH-0.8).
suitable than other indices for water quality
monitoring and assessment [27].
TABLE 4
Components of the multi-type river health index system and indicator weighting.
(Weight) Total
System (Weight) Indicators
Elements weight
(0.3333)Water quantity, C11 0.0837
(0.2511)
(0.6667) Supplementary quantity of ecological water
Hydrology, B1 0.1674
demand/ ecological water demand, C12
(0.2881) CODcr, C21 0.1097
(0.1425) TN, C22 0.0543
(0.3806) Water
(0.4471) NH4+-N, C23 0.1702
quality, B2
(0.0725) TP, C24 0.0276
(0.0498) DO, C25 0.0190
(0.0736) Vegetation species, C31 0.0068
River health (0.1560) Riparian width/river width, C32 0.0145
(0.0929)
assessment index (0.4034) Riverside stability, C33 0.0375
Riparian zone,
system (0.2288) Type of riverbank, C34 0.0213
B3
(0.0852) Comfort level of water landscape, C35 0.0079
(0.0530) Gradient of riverbank, C36 0.0049
(0.4904) Riverbed stability, C41 0.0601
(0.1226)
(0.1304) Natural connectivity, C42 0.0160
Morphology,
(0.0740) Degree of curvature, C43 0.0091
B4
(0.3052) Flood control, C44 0.0374
(0.3333) Margalef index of benthic organisms, C51 0.0509
(0.1528)Aquatic
(0.3333) Shannon-Wiener index of zooplankton, C52 0.0509
life, B5
(0.3334) Shannon-Wiener index of phytoplankton, C53 0.0509
Assessment of river health. Table 6 shows The water quality and aquatic life indices
the assessment results based on the index weight received scores of 1.798 and 2, indicating sub-
and score in the assessment system. In the upper healthy conditions. The hydrology index received a
reach, the water quality, riparian zone and score of 1, indicating sub-sick conditions. The
morphology indices received scores of 3.572, 4 and overall score was 2.014, indicating sub-healthy
4, respectively, which indicates healthy conditions. conditions in the middle reach. In the lower reach,
The aquatic life index scored 2, a sub-healthy level. the hydrology and morphology indices received
Finally, the hydrology index received a score of scores of 3 and 3.443, respectively, which are
0.667, which falls into the sick category. The healthy. The water quality, riparian zone and
overall score was 2.695, indicating a basically aquatic life indices received scores of 1.656, 2.302
healthy condition in the upper reach. In the middle and 2.333, respectively, indicating sub-healthy
reach, the riparian zone and morphology indices conditions. The overall score is 2.376, meaning that
indicate healthy conditions, with scores of 3.236 the lower reach is sub-healthy.
and 3.852, respectively.
1228


TABLE 5
River health assessment rating standard.
Rating
Indicators Basically healthy,
Healthy, 4 Sub-healthy, 2 Sub-sick, 1 Sick, 0
3
Without
< 25% riverbed <50% riverbed >50% riverbed
C11 riverbed Dried up
exposed exposed exposed
exposed
C12 >2 1-2 0.6-1 0.2-0.6 < 0.2
C21 PJ/ PJ/ PJ/ PJ/ > 60 mg/L
C22 PJ/ PJ/ PJ/ PJ/ > 10.0 mg/L
C23 PJ/ PJ/ PJ/ PJ/ > 8.0 mg/L
C24 PJ/ PJ/ PJ/ PJ/ > 0.8 mg/L
C25 PJ/ PJ/ PJ/ - < 2 mg/L
Without
C31 >3 3-2 2-1 1
vegetation
C32 >1 0.5-1 0.25-0.5 0.1-0.25 < 0.1
80%-100%
C33 without erosion < 20% eroded 20%-50% eroded 50%-80% eroded
eroded
Natural soil Ecological slope Hydrophilic Artificial stepped Vertical concrete
C34
riverbank riverbank platform riverbank riverbank riverbank
C35 Excellent Good Medium Poor Very poor
C36 0-15° 15°-45° 45°-60° 60°-80° η80°
Minor erosion and Moderate erosion
C41 stable unstable severely unstable
deposition and deposition
Excellent Moderately good Poor Very poor
C42 Good connectivity
connectivity connectivity connectivity connectivity
Curve partially cut
Natural curved Curve cut off,
C43 - off, otherwise -
shape river channelized
unchanged
C44 50-100a 30-50a 20-30a 10-20a < 10a
C51 >3 2-3 1-2 0-1 0
C52 >3 2-3 1-2 0-1 0
C53 >3 2-3 1-2 0-1 0
The upper reach is in fairly pristine condition, mainly affected by hydrology and water quality.
with healthy ecology and few contaminants 7KH ULYHU¶V K\GURORJ\ KDV D QHJDWLYH LPSDFW
entering the river. Therefore, the river has a strong because the natural water supply cannot meet the
capacity for self-purification, which maintains the ULYHU¶V HFRORJLFDO GHPDQG OHDGLQJ WR D ZDWHU
water quality, riparian zone and morphology in a shortage of 30,000 m3 per day. The water supply
healthy state. However, the hydrology is sub- VKRUWDJH FDQ DOVR EH DWWULEXWHG WR WKH UHJLRQ¶V DULG
healthy, mainly because the natural water supply climate. Low water quality in the area is caused by
RQO\ PHHWV WZR WKLUGV RI WKH ULYHU¶V HFRORJLFDO the direct discharge of polluted water to the river
demand. The dry climate also reduces the water from nearby villages. Figure 2 shows the
flow. Thus, the upper reach was deemed to be concentrations of contaminants at the sampling
basically healthy. sites. The organic loads and nitrogen/phosphorus
Figure 5b shows the health assessment for the loads are significantly higher at the middle reach
middle reach, which is located in a suburban area. sampling sites (S3, S4 and S5) than at the upper
7KH KHDOWK VWDWXV RI WKH ULYHU¶V PLGGOH UHDFK LV reach sampling sites (S6 and S7).
1229


TABLE 6
Scores for the river health condition in the upper, middle and lower reaches.
Upper reach Middle reach Lower reach

Indicators Indicator Element Health Indicator Element Health Indicator Element Health
score score level score score level score score level
C11 2 3 Sub- 3 Basically
0.667 Sick 1 3
C12 0 0 sick 3 healthy
C21 4 3 3
C22 1 1 0
Sub- Sub-
C23 4 3.572 Healthy 1 1.798 1 1.656
healthy healthy
C24 4 2 2
C25 4 4 4
C31 4 4 2
C32 4 2 2
C33 4 4 4 Sub-
4 Healthy 3.236 Healthy 2.302
C34 4 3 1 healthy
C35 4 2 0
C36 4 3 0
C41 4 4 4
C42 4 4 2
4 Healthy 3.852 Healthy 3.443 Healthy
C43 4 2 0
C44 4 4 4
C51 1 1 2
Sub- Sub- Sub-
C52 2 2 2 2 2 2.333
healthy healthy healthy
C53 3 3 3
Total Basically Sub- Sub-
2.695 2.014 2.376
score healthy healthy healthy
The lower reach sites (S1 and S2) are located Figure 2 shows that the concentrations of NO3-
in an urban area where the river has been artificially -N and TN at the lower reach sampling sites (S1
channelized. As shown in Figure 5c, the hydrology and S2) are 4.625 and 9.28 mg/L, respectively,
index of the lower reach indicates healthy which are significantly higher than at other
conditions, but its water quality index received a sampling sites and nearly two times higher than the
ORZ VFRUH 7KLV LV WKH SULPDU\ WKUHDW WR WKH ULYHU¶V concentrations in the middle reach. These high
health. In the lower reach, there is sufficient water values occur because the effluent from the No. 6
supply due to the discharge of treated wastewater Wastewater Treatment Plant contributes significant
from the No. 6 Wastewater Treatment Plant, which NO3--N to the lower reach. Furthermore, the lower
has a daily processing capacity of 130,000 m3. This reach has a lower capacity for self-purification
addition of water is sufficient to elevate the because it is channelized and hardened with
hydrology index to a healthy level. The low water concrete. As a result, the pollutants cannot be
quality in the lower reach is mainly due to pollutant eliminated in the lower reach and are transferred to
effluent from the middle reach and treated water Dianchi Lake.
from the No. 6 Wastewater Treatment Plant. In
Kunming city, the wastewater discharged by the Options for remedial wor s. According to
No. 6 Wastewater Treatment Plant is rated as Class the river health assessment index system designed
1-A based on the criteria from e ll ti n for Dianchi Lake, indices with a score below 2
i a e tan a M ni i al a te ate indicate the primary problems for the Xinbaoxiang
eatment lant (GB18918-2002). The nitrogen River. These include a low hydrology index in the
and phosphorus loading of the treated water is upper reach, a low hydrology index and a low
higher than from natural water sources; thus, the water-quality index in the middle reach, and a low
treated wastewater is identified as a minor source of water-quality index in the lower reach.
pollution.
1230


FIGURE 5
River health assessment for the upper, middle and lower reaches.
The hydrology problem in the upper and pond project to control the overflow of storm water
middle reaches can be solved by artificial water with high pollutant loading.
supplementation. Two wastewater treatment In the lower reach, the river has high TN and
projects are currently under construction: one is the NO3--N loading because of treated water from the
Airport Wastewater Treatment Plant located in the No. 6 Wastewater Treatment Plant. Several
upstream region, with a processing capacity of methods could feasibly help solve this problem:
70,000 m3/d, and the other is the No. 12 first, the water quality from the No. 6 Wastewater
Wastewater Treatment Plant located in the Treatment Plant could be improved by decreasing
midstream region, with a processing capacity of the nitrogen loading. Second, in situ treatment
100,000 m3/d. Upon completion, the two projects could be implemented to increase the
wastewater treatment plants will provide a ULYHU¶V VHOI-purification capacity. These in situ
sufficient amount of treated water to meet the projects include the use of ecological concrete to
30,000 m3/d ecological water demand of the repair the hardened riverbed and stocking to
Xinbaoxiang River. This additional water will increase the biomass in the river. Finally, an
LPSURYH WKH ULYHU¶V K\GURG\QDPLF VLWXDWLRQ DQG estuarine wetland could be built to further decrease
ecological system. pollutant loading.
The low water quality in the middle reach can
be improved by preventing the direct inflow of
polluted water from suburban areas by sewage CONCLUSIONS
interception. Proposed methods are a sewage
interception project along the riverbanks, a The objective of this study was to evaluate the
dispersal treatment project that makes full use of health condition of different reaches of the
the existing ponds and wetlands and a retention Xinbaoxiang River. We used a river health
assessment index system designed for Dianchi Lake
1231


that includes five aspects, hydrology, water quality, [7] Meng, W., Zhang, N., Zhang, Y., Zheng, B. H.
riparian zone, morphology and aquatic life. The (2009). Integrated assessment of river health
results indicate that the upper reach is basically based on water quality, aquatic life and
healthy, and that the middle and lower reaches are physical habitat. Journal of Environmental
sub-healthy. The primary problems were identified Sciences, 21(8), 1017-1027.
for each reach, and remedial works were [8] Zhang, N., Meng, W., Zhang, Y., Zheng, B. H.
recommended based on the health assessment (2009). Multi-Variable Assessment of River
results and the characteristics of each reach of the Ecosystem Health in Liao River Basin.
Xinbaoxiang River. Research of Environmental Sciences, 22(2),
It is imperative to identify the primary 162-170.
problems of river health to ensure that any [9] Hao, L. X., Sun, R. H., Chen, L. D. (2014).
investment in remedial works results in the greatest Health Assessment of River Ecosystem in
possible improvements. The assessment method Haihe River Basin, China. Environmental
and results of this study are valuable for river health Sciences, 35(10), 3692-3701.
management and the implementation of effective [10] Yang, T., Liu, J. L., Chen, Q. Y., Zhang, J.,
remedial works for other plateau lakes with low Yang, Y. (2014). Environmental flow
natural water resources. assessment for improvement of ecological
integrity in the Haihe River Basin, China.
Ecotoxicology, 23(4), 506-517.
ACKNOWLEDGEMENTS [11] Jia, Y. T., Chen, Y. F. (2013). River health
assessment in a large river: Bioindicators of
This study is supported by the National Water fish population. Ecological Indicators, 26, 24-
Pollution Control and Treatment Science and 32.
Technology Major Project (2012ZX07102-002). [12] Guan, B. H., An, S. Q., Gu, B. H. (2010).
Assessment of ecosystem health during the
past 40 years for Lake Taihu in the Yangtze
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1233


ECO-TOXIC EFFICACY OF NANO-SIZED MAGNESIUM

OXIDE IN FRESHWATER SNAIL RADIX LEUTEOLA L
Daoud Ali1, Huma Ali2,Saud Alarifi1, Agha P Masih3, S. Manohardas4͕S. A. Hussain1

1
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia
2
Department of Chemistry, Maulana Azad National Institute of Technology, Bhopal, MP, India
3
Department of Zoology, Shia Post Graduate College, Lucknow, UP, India
4
Department of Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
ABSTRACT this is an important to find out the danger related

with engineered nano level particles in water body
This study aimed to estimate the ecotoxic [3]. Many investigators reported that metal based
properties of magnesium oxide nanoparticles (MgO nanomaterials widely used in many applications
nanoparticles), in freshwater pond snail a ix such as cosmetics, sunscreens, drug delivery, gene-
le te la L. ( l te la). Before observations of therapy and textiles [4]. Gajjar et al., [5]
MgO nanoparticles toxic potential, average size and demonstrated that metallic nanoparticles are toxic
shape of MgO nanoparticles was measured by but some examinations had been performed to
dynamic light scattering and field transmission measure ecological toxicity of nonmetal oxide in
electron microscope. Exposure of MgO aquatic organisms [6].
nanoparticles encouraged an elevation in levels of Quantum dots, magnetic materials and
malondialdehyde, glutathione peroxidase and inorganic /organic luminescent molecules have
coincidentally decreases in glutathione reduced manufactured and they are useful in various fields
after 1 and 4 days, suggesting pro-oxidant effects of [7]. The solubility of metallic particles, in turn,
the nanoparticles in l te la. Other anti-oxidant appears to be dependent upon alkalinity of water.
enzyme, i.e., catalase and glutathione-s-transferase Many researchers reported that MgO nanoparticles
were declined at 34 μg/ml of MgO nanoparticles caused mortality in adult zebrafish [8]. Ecotoxic
and increased at 51 μg/ml of MgO nanoparticles ability of carbon nanotubes, silver nanoparticles,
during exposure periods. Single strand DNA silica nanowires and fullerenes has been
disintegration happened in l te la due to lethal investigated by using zebrafish model [9]. Current
effect of MgO nanoparticles was confirmed by study we used l te la to find out toxic effects of
comet test and exhibited in the form of olive tail MgO nanoparticles. However observations of MgO
moment and per cent tail DNA. DNA damage and nanoparticles toxicity on l te la have been very
enzyme increased more effectively at the higher rare. Ge et al., [10] have reported that MgO
concentration of MgO nanoparticles at 4 days. This nanoparticles induce cytotoxicity and disturbed
approach provides crucial new information enzyme activity in HUVECs cells at high
regarding the unknown effects of MgO concentration.
nanoparticles in freshwater snail. The evaluation of l te la is a sensitive model for
DNA integrity in l te la reveals quick indication ecotoxicology examination, and it has been
of pollution of the aquatic ecosystem by MgO suggested as an appropriate organism for
nanoparticles. nanotoxicity. However, there are no data yet about
MgO nanoparticles toxic effects on l te la. In
KEYWORDS: sight of widely existence and eco-toxicological
Oxidative stress; DNA fragmentation; MgO importance of MgO nanoparticles in water
nanoparticles; Snail ecosystem, an examination of the impact of MgO
nanoparticles in digestive glands of freshwater
pulmonate l te la was performed in this study.
INTRODUCTION
The fast production and exploitation of nano- MATERIALS AND METHODS

materials have elevated alarms that discharges of
nano-scale particles may cause a severe ecological Chemicals. Magnesium oxide nanoparticles
hazard [1]. Nano scale ecotoxicology research is (MgO nanoparticles) (mean size QP), SSB,
presently in the prominence because advancement EDTA, DMSO, EtBr, were bought from M/s.
of nanotechnology based industrial products [2]. So Sigma (St. Louis, MO).
1234


Collection of snail and acclimation. Fresh stored in deep fridge (-80oC). Digestive glands was
water snail l te la with average wet weight 491 homogenized in chilled buffer (50 mM, 150 mM
mg and size 16.5 mm were collected from fish NaCl,1mM EDTA, 1mM DTH, pH 7.5) by using
culture ponds. Test specimens were maintained and tissue homogenizer. Tissue lysate was centrifuged
acclimatised in aquarium under aeration with (12000 x g for 20 minute) at 4ºC and supernatant
photoperiod (12 hour dark: 12 hour light) at used for quantification of biochemical analysis.
temperature 22ºC and were provided to feed green
leaves of aquatic plants. LPO. Thiobarbituric acid-reactive substances
were quantified to assess concentration of digestive
Nano magnesium oxide suspension. Stock gland malondialdehyde by using Ohkawa et al.,
suspension MgO nanoparticles were made in milli- method [12].
Q water using sonication at 45 kHz in a sonictor for
25 minutes before exposure. Distribution of MgO Reduced GSH. GSH was evaluated as per
nanoparticles size in water was evaluated by DLS Owens and Belcher [13] method.
(Malvern, Zetasizer nano ZS) to see agglomeration
in exposure concentrations. MgO nanoparticles Estimation of glutathione peroxidase
were characterized by TEM (JEOL Inc.) at 200 kV. activity. Glutathione peroxidase activity was
estimated by using Flohe and Gunzler [14] method.
Evaluation of sub lethal dosages. The
primary range of dosage of MgO nanoparticles was Determination of catalase activity. Catalase
examined with a range finding tests. After that level was quantified by Beers and Sizer, method
confirmed acute toxicity tests was carried out by [15].
treating snails to various concentration of MgO
nanoparticles. Determination of GST activity. Activity of
Stock solution (1 mg/ml) of MgO GST was estimated by Vessey and Boyer [16]
nanoparticles was prepared in distilled water. The method.
quality of test water such as total hardness, DO, pH,
temperature, and conductivity were examined [11]. Determination of DNA strand breakage.
Ten fresh water snails were randomly treated to six DNA breakage was done according Ali et al [17]
MgO nanoparticles concentrations (0, 10, 20, 40, method.
80, 120 and 200 μg/ml) in 200 ml beakers and test
was done in duplicate to find out EC50 -96 hour of Protein. Amount of protein in digestive glands was
MgO nanoparticles for snail. The 96 hour EC50 determined by Bradford, [18] method.
(66.8 μg/ml) of MgO nanoparticles was measured
by using the probit analysis method (Finney, 1971). Statistical Analysis. Data are presented as average
We have selected three sub lethal concentrations of ± standard errors. Statistical analysis was done
MgO nanoparticles such as sub lethal 1 (1/4th of using one-way analysis of variance followed by
EC50 = 17 μg/ml), sub lethal 2 (1/2nd of EC50= 34 'XQFDQ¶V WHVW 6WDWLVWLFDO VLJQLILFDQFH ZDV
μg/ml) and sub lethal 3 (3/4th of EC50= 51 μg/ml) considered at -value < 0.05.
for further study.
In vivo MgO nanoparticles exposure and RESULTS

collection of digestive glands. Specimens were
treated with different concentrations (0, 17, 34, 51 MgO nanoparticles characterization. Result
μg/l) of MgO nanoparticles over four days. of DLS indicated that mean size of MgO
Untreated specimens were retained in tap water. nanoparticles was 154±6.0 nm and zeta potential
Five snails were sacrificed at day 1 and 4 as per 19 ± mV (Table 1).The mean size of MgO
exposure durations. nanoparticles was determined by TEM was 35±4.0
Digestive glands was removed quickly and nm (Fig. 1).
washed in snail saline buffer solution and
immediately proceed for DNA damage and rest part
1235


TABLE 1
Dynamic light scattering (DLS) measurements of MgO nanoparticles in water
DLS measurements Value

Hydrodynamic size 154±6.0 nm
Zeta potential 19 - mV
FIGURE 1
TEM images of MgO nanoparticles (a) TEM image (b) their particle size distribution. Scale bar ( --- ) is
100 nm.
Analysis of the test water. Physiochemical mg/l. The conductivity of test water varied 247 to
properties of test water were illustrated in Table 2. 298.6 μM/cm and Cl- was 45.8 to 57.2 μg/ml. Total
Test water temperature (between 22.5 to 24°C) and alkalinity of water were found 260.4 to 291 μg/ml
pH of test water (between 6.9 to 7.94) were found as CaCO3 and hardness of water ranged 159 to 180
in experiment duration. DO level was 6.8 to 8.20 μg/ml.
TABLE 2
Physiochemical characteristics of test water
Parameters Values
Temperature 22.5 to 24°C
pH 6.9 to 7.94
Dissolved oxygen (mg/l) 6.8 to 8.20
Total hardness (as CaCo3) μg/ml 260.4 to 291
Chloride (μg/ml) 45.8 to 57.2
Conductivity (μM/cm) 247 to 298.6
Mortality of snails. Fig. 2 indicates the during experimental durations in control. It is clear
correlation between dosage of MgO nanoparticles from Fig. 2 that snail mortality was increased as
and % mortality of snail. No snail death was seen dosages of MgO nanoparticles increased.
1236


FIGURE 2
Mortality (%) of snail after 4 days exposure to different concentration of MgO nanoparticles.
Levels of oxidative biomarkers. The hepato- malondialdehyde level was observed in unexposed
pancreatic level of MDA, that indicate the amount specimen. On the other hand, the level of
of lipid peroxidation of cell, is explained in Fig. 3. malondialdehyde was increased significantly
During whole exposure duration no alteration according dose and period basis (Fig. 3).
FIGURE 3
Levels of lipid peroxides (LPO) in digestive glands after exposure of MgO nanoparticles to . luteola.
Each value represents the mean ± SE of three experiments. *p < 0.05 vs. control.
1237


Fig. 4a represents reduced glutathione for four concentrations of MgO nanoparticles exposure,
days of MgO nanoparticles treatment. Glutathione however, a significant increase was noted (Fig. 4b).
was constant during experimental period in control. Fig. 4c illustrates changes in GPx activity. It
There was a strong trend for a decline glutathione increased significantly as dose and time dependent
with MgO nanoparticles (34 and 51 μg/ml) manner. GST activity was decreased at lower
exposure. concentration but increased significantly as at
Activity of catalase did not alter in unexposed higher dose (Fig. 4d).
throughout the four-day study period. At all
4a
4b
1238


4c
4d
FIGURE 4
a. GSH b. Catalase activity c. GPx activity d. GST level
in digestive glands of R. luteola after treatment of MgO nanoparticles. Each value represents the mean ±
SE of three experiments. *p < 0.05 vs. control.
DNA fragmentation. It was observed as % DNA was found in digestive gland tissue as dose of
tail DNA and olive tail moment in the untreated and MgO nanoparticles and time increased. Maximum
MgO nanoparticles treated snail digestive gland fragmentation of DNA was found at MgO
cells. The uneven proliferation in fragmentation of nanoparticles (51μg/ml) at fourth day (Fig. 5).
1239


FIGURE 5
Fragmentation of DNA in digestive glands cells of . luteola after treatment of MgO nanoparticles.
a. % Tail DNA b. Olive tail moment (OTM) c. Control d. Exposed.
Data is showed as average ± SE of triplicate tests. *p < 0.05 vs. control. Scale bar ( --- ) is 50 μm.
DISCUSSION was affected when treated with high dose of MgO

nanoparticles during acute toxicity assay. There is
Anthropogenic activity is an important source no reported data for MgO nanoparticles
of contaminations of heavy metal to aquatic body concentration in aquatic ecosystem. Therefore, the
[19]. In contaminated areas, heavy metalloids observed high lethal dose of MgO nanoparticles
produced hazards to snail due to containing cannot be unnoticed.
electrons (unpaire). Amphibious creatures have The purpose of this work was to scrutinize the
developed antioxidant defence mechanism which level of antioxidant enzymes in snail after treatment
inhibit and interacts between pollutants and of MgO nanoparticles. Our results showed change
organism causing in enzymatic turbulences. of antioxidant enzymes in snail during the exposure
Oxidative stress is an adverse effect from of MgO nanoparticles for 4 days, which indicating
treatments of pollutants of cells or tissues to surplus that oxidative stress was produced in snail digestive
levels of free radicals oxidants such reactive glands due to the exposure of MgO nanoparticles.
oxygen species [20]. Production of reactive oxygen species may be
Lethal observations are initial significance in induce oxidation of nuclear material, promoting to
ecotoxicology to measure susceptibility, acute cell death. Reactive oxygen species are main source
stress and viability response of living organisms for of DNA impairment [23]. Repetto et al., [24]
detecting the effects of environmental pollutants reported that reactive oxygen species degrades
such as metal nanoparticles to ecosystem cytoplasmic membrane by oxidizing double bonds
functioning. Few observations on toxicity of metal of membrane phospholipids. The quantification of
oxide nanoparticles to aquatic organisms are MDA level, final products of lipid peroxide delivers
existing [21]. Ali and Ali [22] had been reported a effect of MgO nanoparticles to induce oxidative
acute toxicity of nano-copper to freshwater stress. In this study, LPO levels were increased in
gastropods. In the present study the lethal test -96 h digestive glands. Elevated LPO levels indicate
on l te la indicated that fresh water gastropods tissue damage and let-down mechanism of
was unable survive up to 100 μg/ml of MgO antioxidant system to inhibit production of too
nanoparticles. Although survivability of l te la much free radicals [25]. Glutathione reduced
reflected as a major inhibitor against ROS
1240


metabolites in tissue by aiding as a substrate for and L. Sigg. (2008) Environmental behavior
glutathione peroxidase [26].Our observation was and ecotoxicity of engineered nanoparticles to
according the finding of Ali et al. [27] in which algae, plants, and fungi. Ecotoxicology
glutathione declined in digestive gland in response 17:372-386.
of metal nanoparticles. Catalase induces the [3] MacCormack, T.J., and Goss, G.G., (2008)
transformation of H2O2 to H2O in the presence of Identifying and predicting biological risks
cofactor Fe or Mn [28]. In the present study activity associated with manufactured nanoparticles in
of catalase reduced at low concentration but it aquatic ecosystems. J. Ind. Ecol. 12: 286-296.
increased at higher dose in soft tissue of snail. The [4] Nel, A., Xia, T, Madler, L, and Li, N. (2006)
inhibition of catalase at lower may be due to Toxic potential of materials at the nanolevel.
development of MDA which is known to prevent Science 311:622-627.
synthesis of protein and some enzymes. GST is an [5] Gajjar, P., Pettee, B., Britt, D.W., Huang, W.,
enzyme that participates in the detoxification Johnson, W.P., and Anderson, A.J. (2009)
process due to conjugation reaction between GSH Antimicrobial activities of commercial
and xenobiotics [29]. The present investigation nanoparticles against an environmental soil
showed a significant decrease in GST in MgONP- microbe, Pseudomonas putida KT2440. J.
exposed snails compared with the control group. Biol. Eng. 3: 13.
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concentration and time±response relationship 2883.
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[9] Cheng, J., Flahaut, E., and Cheng, S.H. (2007)
CONFLICT OF INTEREST STATEMENT Effect of carbon nanotubes on developing
zebrafish (Danio rerio) embryos. Environ
There are no conflicts of interest. Toxicol Chem. 26(4):708-16.
[10] Ge, S., Wang, G., Shen, Y., Zhang, Q., Jia,
ACKNOWLEDGEMENT D., Wang, H., Dong, Q., and Yin, T. (2011)
Cytotoxic effects of MgO nanoparticles on
The authors would like to extend their sincere human umbilical vein endothelial cells in
vitro. IET Nanobiotechnol. 5(2):36.
appreciation to the Deanship of Scientific Research
at King Saud University for its funding this
[11] A.P.H.A., A.W.W.A., and W.P.C.F., (2005).
Research group NO (RG -1435-076).
Standard Methods for the Examination of
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1242


)$&7256$))(&7,1*7+(678'(176¶(19,5210(17$/
AWARENESS, ATTITUDES AND BEHAVIORS IN ONDOKUZ
MAYIS UNIVERSITY, TURKEY
Mehmet Bo oglu1 , Abdulba i Bilgic2, Ba iye Kilic Topu 1
and, Yu sel Ardali3
1
Ondokuz Mayis University, Faculty of Agriculture, Department of Agricultural Economics, 55200 Samsun, Turkey
2
Ataturk University, Faculty of Agriculture, Department of Agricultural Economics, 25240 Erzurum, Turkey
3
Ondokuz Mayis University, Faculty of Engineering, Department of Environmental Engineering, 55200 Samsun, Turkey
ABSTRACT environmental sources resulted in environmental

problems such as deforestation, loss of biodiversity,
7KLV VWXG\ DLPHG WR GHWHUPLQH WKH VWXGHQWV¶ pollution, ozone depletion, global climate change
environmental awareness, attitude and behavior and over-consumption of natural resources [2, 3].
levels and factors influencing their environmental Environmental problems are the most vital
awareness, attitude and behavior. A face-to-face problems we face today and all are mostly due to
survey with 621 candidate graduate students in the the human behavior [4, 5], or they are
Ondokuz Mayis University was used to collect the anthropogenic of origin [6]. Such threats to the
data. The environmental awareness, attitude and environment are generally listed as industrialization,
EHKDYLRU ZHUH GHWHUPLQHG E\ WKH VWXGHQWV¶ over population, developments in science and
assessments based on a five-point Likert scale. The technology and increasing needs and globalization
ordered probit procedure was used to determine [7].
IDFWRUV DIIHFWLQJ WKH VWXGHQWV¶ HQYLURQPHQWDO Environmental issues are being eroded with
awareness, attitudes and behaviors. The study more university education in the long run, since
UHYHDOV WKDW WKH FDQGLGDWH JUDGXDWH VWXGHQWV¶ universities educate young generations expected to
environmental awareness, attitude and behavior are be the future leaders in many different areas in the
found to be high. The probit models showed that society, and who will be the decision-makers and
both environmental attitude and behavior were therefore their graduates are expected to deal with
influenced by the environmental education and VXVWDLQDELOLW\ LVVXHV LQ ERWK WKHLU¶ SHUVRQDO DQG
information factors, while the socio-demographic professional lives [8]. Students should acquire an
factors generally have important impacts on the appropriate range of awareness, understanding, and
VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV DV H[SHFWHG concepts about the environment while they were in
While the variables of gender had the highest school, so that critical judgment can be achieved [9].
LPSDFWV RQ WKH VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV Environmental education can help create positive
both awareness and curiosity levels towards awareness and attitudes about environmental issues,
environmental news had the highest influences on whilst curbing the negative role of human actions
WKH VWXGHQWV¶ HQYLURQPHQWDO DWWLWXGH DQG EHKDYLRU on the environment [10, 11]. Therefore, universities
UHVSHFWLYHO\ 7R LQFUHDVH WKH VWXGHQWV¶ are sole responsible for increasing their students¶
environmental literacy levels, the departments environmental awareness, attitudes and behaviors to
should put sufficient compulsory environment create an environmentally sustainable future [12].
courses on their programs, and they should focus on Environment is defined as all factors affecting
providing their graduates with environmental the physical, biological, socio-psychological, socio-
attitudes and behaviors. economic and cultural life of an individual or
society [13]. Environmental awareness is defined as
an understanding of natural systems combined with
KEYWORDS: how they interact with human social systems [14],
Environment, awareness, attitudes, behavior, ordered while many consider this as the ultimate driving
probit, Turkey force that stimulates knowledge [15]. Contrarily,
environmental attitude is defined as learned
tendencies in the form of consistent behaviors
INTRODUCTION against environment either positive or negative [16],
providing a good understanding of the set of beliefs,
Natural resources are being consumed at a interests or rules that influence environmentalism or
faster rate than they can be restored [1]. The pro-environmental action [17]. On the other hand,
uncontrolled and uncoordinated usage of behavior is what people do, whether it is
1243


environmentally appropriate or inappropriate [18]. conducted among science teacher candidates in

Since environmental behavior is considered as the thirteen universities in the country to obtain their
basis of the environmental crisis, social scientists environmental attitudes found very high level of
have long been interested in the causation of this environmental attitudes of candidates [42]. Similar
behavior [1]. results were also echoed in other studies [43-49].
The rise of environmental issues has produced However, to our knowledge there was no specific
many studies with students on explaining the study to investigate awareness, attitudes and
variation in individual knowledge, awareness, behavior levels of the candidate graduates towards
attitudes and behaviors toward the environment. environmental issues and how their determinants
Aklin et al. [19] attempted to identify the main affect perceptions of these issues. Therefore, this
determinants which predict a person's awareness of study was to identify this relationship and levels of
environmental issues, while many other researches awareness, attitudes and behavior among the
revealed the relation between environmental students of the Ondokuz Mayis University in
awareness, concern, knowledge and behavior [20- Turkey who would soon graduate.
27]. In parallel, some other researchers have Universities have the responsibility to prepare
investigated whether schools [1] and infusions of their graduates for preserving the environment.
environmental education [28] make a difference in Every department is related with the environment
environmental awareness and attitudes among DQG WKHLU JUDGXDWHV¶ SURIHVVLRQDO activities can
students. Ogunbiyi and Ajiboye [29] examined pre- somehow effect the environment. Therefore, every
VHUYLFH WHDFKHUV¶ NQRZOHGJH DQG DWWLWXGHV WR VRPH department should prepare responsible graduates
environmental education concepts. Grob [30] for a more livable environment. The emerging
estimated a structural model for environmental interest of universities to participate in the increase
attitudes and behaviors, whilst Bradley et al. [10] in the consciousness of environmental values makes
and Kaiser et al. [31] overemphasized that attitude LW QHFHVVDU\ WR H[SORUH WKH FDQGLGDWH JUDGXDWHV¶
is the most important factor affecting individual environmental awareness, attitude and behavior.
behavior. While Klöckner [32] and Masud and Kari However, it is very important to explore if the time
>@ H[DPLQHG WKH GHWHUPLQDQWV RI LQGLYLGXDOV¶ students spend at the university actually improves
environmentally relevant behaviors, Tanner [34] their environmental awareness, attitude and
identified prevalent constraints inhibiting behavior. Determining environmental awareness,
LQGLYLGXDOV¶ HQYLURQPHQWDO EHKDYLRUV attitude and behavior of the candidate graduates is
Environmental awareness, attitudes and behaviors also crucial in establishing the sustainability of a
may vary with gender, age, education, income, community.
family, residence, country, political tendency, Based on the logical approach described in the
knowledge/awareness of the environment, school previous section, it is an important task to analyze
and etc. [35-38]. the levels of awareness, attitudes and behavior
In Turkey, researchers have investigated attributed to environmental issues by university
XQLYHUVLW\ VWXGHQWV¶ HQYLURQPHQWDO NQRZOHGJH students, since it can present significant results to
responsibility, risk perceptions, attitude, and draw appropriate decisions about the effective
behaviors. For example, Beyhun et al. [39] educational programs for students who are currently
GHWHUPLQHG WKH VWXGHQWV¶ HQYLURQPHQWDO ULVN enrolled and future generations on environmental
perception levels and their determinants in one of issues. The aim of this study was to determine the
WKHFRXQWU\¶V0HGLFDO6FKRROVDQGVWDWHGWKDWVWUHVV environmental awareness, attitude and behavior
damage of ozone layer and motor vehicle accidents levels of the students and understand how socio-
were found as high or very high risk factors demographic, economic, and environmental-related
towards environment. On the other hand, Yilmaz et factors influence their environmental awareness,
DO >@ H[DPLQHG WKH XQLYHUVLW\ VWXGHQWV¶ attitude and behavior. The research results can
environmental knowledge levels and determined contribute to the program curricula and shed light
that the environmental education was not sufficient on penetrative protection of the environment those
enough so that students generally tended to obtain policy makers of local in particular and central
information about environmental issues from the government in general seek for.
media. Contrarily, Sadik and Sadik [41]
investigated environmental knowledge and attitudes
of those who are teacher candidates at Cukurova MATERIALS AND METHODS
University and found that they had a moderate
environmental knowledge level and positive Ondokuz Mayis University is a state
environmental attitude, but with a low level of the university and has totally 52,301 undergraduate and
environmental behavior. In parallel, a study graduate students at 5 graduate institutions, 16
1244


faculties, 2 colleges, 1 conservatory and 12 average response score for the questions. The
vocational schools [50]. The population of this student who scored less than 2.5 was classified
study includes all candidate students for graduation under the low environmental awareness, attitude
at the undergraduate programs of the departments, and behavior group, 2.5 to 3.5 was classified under
which are somehow related with environment, the moderate environmental awareness, attitude and
under the body of the faculties of Ondokuz Mayis behavior group, and more than 3.5 was classified
University. Totally 621 students participated in the under the high environmental awareness, attitude
study, and the number of participant students from and behavior group.
the departments were as follows: Agricultural 7KH VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV
Economics (26), Agricultural Construction and attitude and behavior scores were given in Table 1
Irrigation (20), Horticulture (27), Plant Protection based on their statements. To calculate and interpret
(31), Field Crops (24), Agricultural Biotechnology the mean values of the level of agreement for each
(32), Agricultural Machinery (20), Soil Science and item of awareness, attitude and behavior, an
Plant Nutrition (30) and Animal Science (18) all interpretative scale was developed as follows: If the
from the College of Agriculture, and Environmental mean value was less than 2.5, it meant the
Engineering (43), Food Engineering (55) and participant strongly disagreed or disagreed. If the
Industrial Engineering (42) from the College of mean value was from 2.5 to 3.5, it was neutral, and
Engineering, while Biology (37), Chemistry (33) if the mean value was higher than 3.5, it meant that
and Geography (89) from the College of Science the participant agreed or strongly agreed. Based on
and Education, and lastly Science Teacher (72) and this scale, of 26 environmental awareness
Biology Teacher (22) from the College of statements, while 21 statements were agree or
Education. strongly agree, 3 awareness statements were neutral
This study used a survey technique to collect and 2 awareness statements were disagree or
the data. The technique aims to reveal a past or strongly disagree. The three items of environmental
FXUUHQWSKHQRPHQRQ>@RUGHWHUPLQHSDUWLFLSDQWV¶ awareness with the highest means weUH ³&UHDWLQJ
views or characteristics such as interest, skill and/or environmentally conscious individuals is
attitude [52]. The survey consisted of totally 64 compulsory for future generations to live in a
questions and four parts: The first part contained 12 KHDOWK\ DQG VDIH HQYLURQPHQW PHDQ ´
items to measure socio-demographic and economic ³8VLQJLQVHFWLFLGHVDQGKHUELFLGHVIRUDJULFXOWXUHLV
characteristics of the students such as age, gender, QRWKDUPIXOIRUWKHHQYLURQPHQWPHDQ ´DQG
family size, education of mother and father, ³(QYLURQPental activities help raise awareness of
residence, monthly income of family, information HQYLURQPHQWDO LVVXHV PHDQ ´ 7KH WKUHH
sources on environment, frequency of following items of environmental awareness with the lowest
environmental news, research and developments, PHDQV ZHUH ³7KH PDOQXWULWLRQ LQ XQGHUGHYHORSHG
the most important institution about environment, countries is a consequence of environmental
membership to environmental organizations, and SUREOHPV PHDQ ´ ³7KH XQiversity is not
whether the student has taken courses about the sensitive enough towards environmental problems
environment. The second part included 26 items to PHDQ ´DQG³(QYLURQPHQWDOHGXFDWLRQLVQRW
PHDVXUH WKH VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV HQRXJKLQ7XUNH\PHDQ ´
The third part contained 11 items to measure the Of 11 environmental attitude statements, while
VWXGHQWV¶ HQYLURQPHQWDO DWWLWXGH DQG WKH ODVW SDUW 10 statements were agree or strongly agree, only 1
LQFOXGHG LWHPV WR PHDVXUH WKH VWXGHQWV¶ statement was neutral. The three items of
environmental behaviors. The Likert scale environmental attitude with the highest means were
measurement was used for every statement of ³3HRSOH ZKR VSLW RQ RU OLWWHUWKH JURXQG VKRXOG EH
environmental awareness, attitude and behavior on LQWHUIHUHGPHDQ ´³:HVKRXOGWU\WRSURWHFW
a 5 point scale. Each alternative item is assigned (DUWK¶V SODQWV DQG DQLPDOV HYHQ WKRXJK LW LV
from 5 (strongly agree) to 1 (strongly disagree) for H[SHQVLYH PHDQ ´ DQG ³&RXUVHV RI
favorable items. In case of unfavorable items (2, 5, HQYLURQPHQW PXVW EH SUDFWLFDO PHDQ ´ 7KH
7, 9, 10, 11, 13, 16, 18, 20, 21, 23, 36, 37 and 40) in three items of environmental attitude with the
Table 3, the scoring is reversed from 1 (strongly ORZHVW PHDQV ZHUH ³, WKLQN , DP YHU\ VHQVLWLYH
agree) to 5 (strongly disagree). The students were DERXWHQYLURQPHQWDOLVVXHVPHDQ ´³,SUHIHU
asked to indicate whether they agree or disagree the option of highway construction to the protection
with each item, as well as the magnitude of their RISODQWVSHFLHVPHDQ ´DQG³)RUP\FRXQWU\
agreement or disagreement. The students were then I prefer technological development to noise
categorized in three groups according to their SROOXWLRQPHDQ ´
1245


TABLE 1.
Average awareness, attitude and behavior scores
Statements Level
Moderate
General
High
Low
1.Creating environmentally conscious individuals is compulsory for future generations to live in a healthy
and safe environment*** 1.33 3.77 4.60 4.41
2.Using insecticides and herbicides for agriculture is not harmful for the environment *** 1.33 3.38 4.55 4.29
3.Environmental activities help raise awareness of environmental issues*** 1.50 3.73 4.42 4.25
4.Environmental protection is a constitutional obligation** 2.00 3.63 4.37 4.21
5.Field activities related to the environment are a waste of time, within class activities are important *** 1.83 3.53 4.39 4.20
6.If universities carry out more activities on environment, it will help better understand environmental
issues*** 1.50 3.57 4.38 4.19
7.Protecting the environment is a duty of the state, not people** 2.17 3.65 4.24 4.10
8.Human mistreat the environment ** 2.00 3.45 4.23 4.05
9.Environmental education activities are useful only for children*** 1.17 3.57 4.19 4.04
10.A squatter is not an environmental problem** 2.00 3.28 4.23 4.02
11.I believe that environmental problems are exaggerated. 2.67 3.38 4.19 4.02
Awareness
12.Rapid population growth is a serious environmental problem** 2.00 3.32 4.15 3.97
13.Air, water and soil are inexhaustible resources*** 1.17 3.20 4.19 3.96
14.Necessity of using water filters at homes is an indicator of water pollution *** 1.50 3.52 4.08 3.94
15.Being sensitive to the environmental problems is not an obstacle for the development of a country*** 1.83 3.38 4.09 3.93
16.The idea of environmental protection was invented by western people to prevent the development of
developing countries 2.67 3.45 4.03 3.90
17.I increasingly need more information about the effects of our activities on the environment*** 1.83 3.42 4.00 3.86
18.There is no desertification problem in Turkey*** 1.83 3.28 3.96 3.80
19.Immigration affects negatively environmental problems 2.50 3.17 3.95 3.79
20.Delivering environmental education does not help solving environmental problems** 1.83 3.09 3.89 3.71
21.No international institution or organization should intervene on using the natural resources. 2.33 3.02 3.81 3.64
22.There have not been enough protest meetings for protecting the environment in Turkey. 2.50 2.97 3.55 3.42
23.The increase of natural gas usage in Turkey did not contribute to the solution of the air pollution problem.
2.67 3.05 3.44 3.36
24.The malnutrition in underdeveloped countries is a consequence of environmental problems. 2.67 2.65 3.23 3.11
25.The university is not sensitive enough towards environmental problems*** 4.00 2.52 2.32 2.37
26.Environmental education is not enough in Turkey*** 4.67 2.16 1.80 1.89
27.People who spit on or litter the ground should be interfered. ** 2.50 3.89 4.58 4.43
:HVKRXOGWU\WRSURWHFW(DUWK¶VSODQWVDQGDQLPDOVHYHQWKRXJKLWLVH[SHQVLYH ** 2.50 4.00 4.50 4.38
29.Courses of environment must be practical.** 2.33 3.92 4.51 4.37
30.People who pollute the environment should be fined.** 2.50 3.91 4.50 4.36
Attitude
31.Newspapers, magazines and televisions should make more programs about environmental issues.** 2.67 3.95 4.48 4.36
32.I love to visit recreation areas outside of the city.** 2.33 3.99 4.47 4.36
33.Courses about environment should be compulsory. 2.83 3.36 4.23 4.05
34.Regardless of their position, any country engaging in nuclear testing should be protested. 2.67 3.45 3.77 3.70
35.I think I am very sensitive about environmental issues.** 2.50 3.39 3.73 3.65
36.I prefer the option of highway construction to the protection of plant species.
2.83 3.13 3.74 3.61
37.For my country, I prefer technological development to noise pollution. 2.50 3.08 3.38 3.32
38.I take care of using both sides of papers when I write something. ** 2.67 3.91 4.32 4.22
39.I usually economically use resources such as water and electricity. 2.17 3.77 4.13 4.04
40.When I am outside of the room, I do not turn the lights off, because I think it does not consume energy
very much. 3.83 3.62 3.97 3.90
41.I like participating in the environmental protection activities, because it is the best way to understand the
environment.** 2.50 3.37 3.94 3.82
42.I often talk about environmental issues with people around me. * 2.33 3.29 3.82 3.71
43.I often watch television programs about environmental issues. 2.67 3.22 3.72 3.61
44.I usually use public transports such as the tram in order to avoid air pollution. *
2.50 3.34 3.57 3.52
45.I usually read publications on the environmental issues. 3.00 3.17 3.59 3.50
46.I take care of buying recycling packaged products in my daily life. 3.00 3.08 3.57 3.46
47.I take care of collecting household waste separately in my daily life. 3.00 2.89 3.38 3.28
Behavior
48.I take care of consuming products which do not contain harmful substances to the ozone layer. 3.33 2.99 3.35 3.28
49.I choose courses about environment as elective courses at the university. 3.33 2.66 3.36 3.23
50.I vote politicians who are concerned about environmental protection. 2.50 2.97 3.29 3.22
51.When I get a chance, I plant seedling. 2.83 3.03 3.14 3.11
52.I join actively in activities of environmental issues. 3.33 2.78 2.85 2.84
***, **, * denote that there are statistical differences among the groups at 1%, 5% and 10% significance
levels, respectively.
1246


Of 15 environmental behavior statements, 8 Descriptive statistics were used to identify

statements were agree or strongly agree, 7 variables reflecting socio-demographic and
statements were neutral. The three items of economic characteristics and environmental
environmental behavior with the highest means
awareness, attitude and behaviors of the students.
ZHUH ³, WDNH FDUH RI XVLQJ ERWK VLGHV RI SDSHUV
when I write something (mean=4.2´ ³, XVXDOO\ To compare the socio-demographic and economic,
economically use resources such as water and and environmental variables in which students were
HOHFWULFLW\PHDQ ´DQG³:KHQ,DPRXWVLGHRI identified low, moderate and high for subsequent
the room, I do not turn the lights off, because I awareness, attitude and behavior groups, ANOVA
think it does not consume energy very much and Kruskal Wallis tests were used for parametric
PHDQ ´ 7KH WKUHH LWHPV RI HQYLURQPHQWDO and non-parametric variables, respectively. The
behaYLRU ZLWK WKH ORZHVW PHDQV ZHUH ³, YRWH
ordered probit model was used to determine the
politicians who are concerned about environmental
SURWHFWLRQ PHDQ ´ ³:KHQ , JHW D FKDQFH , environmental awareness, attitude and behaviors of
SODQWVHHGOLQJPHDQ ´DQG³,MRLQDFWLYHO\LQ the students by means of socio-demographic,
DFWLYLWLHVRIHQYLURQPHQWDOLVVXHVPHDQ ´ economic and environmental variables.
The questionnaire was pre-tested and modified Definitions of variables were presented in
to improve its reliability. The questionnaires were Table 2. The dependent variable had three response
conducted with students at the beginning of classes categories for which students have stated their
by lecturers in May 2015. The students answered understanding levels of environmental awareness,
the survey questions in about 15 minutes. SPSS attitude and behavior. Since the dependent variable
17.0 and Limdep 10 software programs were used takes discrete values and these values are inherent
for data analysis. ordinal ranking, the ordered model fits the data best
[53-56].
TABLE 2.
Definitions of the model variables
Variable name Variable definition

GENDER Female=0, Male=1
AGE Age (year old)
HOUSHSIZE Household size (people)
MOTHEDUC Mother education (year)
FATHEDUC Father education (year)
RESIDENCE If student lives in rural residences =1, other = 0
FAMINCOM Household monthly income (༲, Turkish Lira)
ENVCOUR If the student takes two or more courses about environment =1, other = 0
INFSMEDIA If the main information source of environmental awareness is the media =1, others=0
INFSUNIV If the main information source of environmental awareness is university =1, others=0
INFSINST If the main information source of environmental awareness is the state institutions =1,
others=0
FENVNEWS If student follows usually or always environmental news =1, other = 0
MEMENVO If student is a member of voluntary environmental organizations=1, other=0
ENVAWARL /HYHO RI HQYLURQPHQWDO DZDUHQHVV ‫ ޒ‬ORZ DZDUHQHVV OHYHO -3.5= moderate
DZDUHQHVVOHYHODQG‫ޓ‬KLJKDZDUHQHVVOHYHO
ENVATTL /HYHO RI HQYLURQPHQWDO DWWLWXGH ‫ ޒ‬ORZ VHQVH OHYHO -3.5= moderate sense level
DQG‫ޓ‬KLJKVHQVHOHYHO
ENVBEHL /HYHORIHQYLURQPHQWDOEHKDYLRU‫ ޒ‬ORZEHKDYLRUOHYHO-3.5= moderate behavior
OHYHODQG‫ޓ‬KLJKEHKDYLRUOHYHO
1247


The ordered model, for which the dependent as high each for awareness, attitude or behavior, is
variable was coded 0 as low, 1 as moderate, and 2 expressed as
(1)
*
i ȕc[i H i , H i H i | T , H i | [i 0, an a H i | [i 1
where LV WKH XQREVHUYHG ³ODWHQW´ GHSHQGHQW distribution as İ a1[0,1]) and F stands for any
variable, ȕ is a vector of coefficients to be distribution that a researcher might consider.
estimated, i a vector of explanatory variables, İa The above observation mechanism results from
vector of error terms (e.g., we assume normal a complete censoring of the latent limited
dependent variable as follows:
i 0 i i d P0 ,
i 1 i P0 i d P1 , (2)
i 2 i P1 i d P2 ,
where is the observed counterpart to , have similar cutoff points, resulting with the cutoff
while ȝ represents the threshold values or the cutoff points being normally distributed [57, 58].
points. The cutoff points vary with the individual If we assume that the İis normally distributed
respondents. We expect students with similar socio- across observations, then the probability of
demographic and economic characteristics, and choosing a specific ranking by a respondent can be
environmental awareness, attitude or behavior to expressed as [54, 57, 59].
Pr > i @ Pr ª¬ *
i i in t e t an e º¼ ) P ȕc[i ) P 1 ȕc[i (3)
0,1, 2.
where ) is the normal cumulative density Note that μ -1 = - DQG 0 = 0.These probabilities
function (cdf), ȝ and ȝ represent the upper and are specifically as follows:
lower threshold values for category , respectively.
Pr > i 0@ ) ȕc[i
Pr > i 1@ ) P1 ȕc[i ) ȕc[i (4)
Pr > i 2@ ) P2 ȕc[i ) P1 ȕc[i
The log likelihood function is:

2
log ¦¦
i 1 0
i
log ) P ȕc[i ) P 1 ȕc[i
(5)
¦ 0
log ) ȕc[i ¦ 1
log ) P1 ȕc[i ) ȕc[i
i i
¦ 2
log ) P2 ȕc[i ) P1 ȕc[i
i
Marginal effects were calculated to determine a The marginal effect of a continuous variable for the
unitary effect of each exogenous variable on each ordered probit model for three categories can be
of the three categories of the dependent variable. calculated as [57, 60].
1248


w Pr 0
wx
i

I ȕÖ c[i EÖ ,
w Pr 1
wx
i
¬
ªI ȕÖ c[i I PÖ1 ȕÖ c[i º EÖ ,
¼ (6)
w Pr 2
wx
i
¬
ªI PÖ1 ȕÖ c[i º EÖ
¼
where I is the normal probability density between ) of the corresponding probability with
function. Marginal effects for a dummy variable, on and without the presence of the variable in question.
the other hand, can be calculated as the difference For example:
0
w Pr
wxm
i

) ȕÖ c[i
xm 1 ) ȕÖ c[
i
xm 0
(7)
The standard errors of these marginal effects main information source, respectively. There were a
can be obtained by utilizing the delta method. statistical difference among the environmental
attitude and behavior groups in terms of the
variable of university information source (p<0.05
RESULTS AND DISCUSSIONS and p<0.01, respectively). Fifty eight percent of the
students follow usually or always environmental
Descriptive statistics of the variables used in news, and there were statistical differences among
the models were presented in Table 3. Regarding the environmental awareness, attitude and behavior
socio-demographic and economic variables, while groups in terms of the following frequency of
57% of the students were male, 43% of the students environmental news (p<0.01). Only 10.5% of the
were female. There were statistical gender students were members of an environmental
differences among the groups in terms of awareness organization, and there were statistical differences
and behavior (p<0.00 and p<0.05). The average age among the environmental awareness and behavior
of the students was 23 years old, and there were groups in terms of membership (p<0.05 and
statistical differences among the groups in terms of p<0.01). The average environmental awareness
age (p<0.10). The average household size was 5.04, score was 3.80, and this meant that the students had
and there were not statistical differences among the a high level of environmental awareness. There
groups in terms of household size. While the were statistical differences among the
average education duration of mothers and fathers environmental attitude and behavior groups in
were about 7 and 9, respectively, there were terms of environmental awareness (p<0.01). The
statistical differences among the groups in terms of average environmental attitude score was 4.05,
the mother education duration (p<0.01). indicating that the students had a high level of
While 16.1% of the students were from the environmental attitude. There were statistical
rural residences, 83.9% of the students were from differences among the environmental behavior
the urban residences. There were statistical groups in terms of environmental attitude (p<0.01).
differences among the environmental awareness The average environmental behavior score was 3.52,
groups in terms of residence (p<0.05). The average and this indicates that the students had a high level
family monthly income was ༲ 2.5 thousand, and of environmental behavior. There were statistical
there was no statistical difference among the groups differences among the groups in terms of
in terms of income. environmental awareness, attitude and behavior.
Regarding environmental variables, 64.7% of the Our results echoed with previous findings. For
students took two or more courses about example, Ozsoy [42] indicated that pre-service
environment and there were statistical differences science teachers had a high level of environmental
among the environmental attitude and behavior attitudes, whilst Altinoz [61] concluded that the
groups in terms of the number of courses taken VFLHQFH HGXFDWLRQ FDQGLGDWH WHDFKHUV¶
(p<0.05 and p<0.10). While 81% of the students environmental attitudes were found to be at a high
use the media as a main information source about level and contrarily their environmental knowledge
environmental issues, 22.5% and 18.4% of the and behavior occurred at low levels. However,
students use university and other institutions as a (UNDO HW DO >@ IRXQG WKH XQLYHUVLW\ VWXGHQWV¶
1249


environmental attitudes as above the medium level, [63] found that the environmental knowledge and
while Karatekin [62] determined social science behavior levels of science education candidate
FDQGLGDWH WHDFKHUV¶ HQYLURQPHQtal knowledge, teachers as intermediate, and their level of
behavior and literacy levels as intermediate. Timur environmental attitude were high.
TABLE 3
Descriptive statistics of the variables used in the environmental awareness, attitude and behavior models.
Variables General Environmental awareness level Environmental attitude level Environmental behavior level
(N=621)
Low Moderate High Low Moderate High Low Moderate High
(n=6) (n=122) (n=493) (n=16) (n=166) (n=539) (n=27) (n=252) (n=342)
GENDER 0.433 0.833*** 0.549 0.400 0.438 0.500 0.425 0.444** 0.500 0.383
(0.496) (0.408) (0.500) (0.490) (0.512) (0.504) (0.495) (0.506) (0.501) (0.487)
AGE 23.068 22.167* 22.598 23.195 23.688* 22.485 23.193 23.259* 22.683 23.336
(2.815) (0.753) (1.689) (3.033) (3.995) (1.491) (2.799) (2.105) (2.352) (3.133)
HOUSHSIZE 5.040 4.667 4.779 5.110 5.250 5.106 5.026 5.407 4.929 5.094
(1.776) (1.033) (1.561) (1.828) (1.844) (1.781) (1.776) (2.325) (1.603) (1.847)
MOTHEDUC 6.628 3.167*** 7.975 6.337 6.438 7.478 6.529 5.852 6.743 6.604
(3.761) (2.137) (4.204) (3.571) (3.705) (4.178) (3.703) (3.890) (3.832) (3.702)
FATHEDUC 8.940 7.167 9.298 8.873 8.500 9.035 8.942 8.222 9.030 8.931
(3.911) (5.193) (3.848) (3.910) (3.983) (4.199) (3.879) (3.588) (4.034) (3.848)
RESIDANCE 0.161 0.333** 0.082 0.178 0.188 0.212 0.154 0.222 0.143 0.170
(0.368) (0.516) (0.275) (0.383) (0.403) (0.412) (0.361) (0.424) (0.351) (0.3769)
FAMINCOM 2467.021 1750.000 2331.230 2509.351 2368.750 2356.515 2483.469 2218.148 2513.135 2452.690
(2064.585) (1036.822) (1131.474) (2243.444) (1285.156) (1254.041) (2161.963) (1015.480) (1984.762) (2182.917)
ENVCOUR 0.647 0.667 0.574 0.665 0.750** 0.455 0.668 0.630** 0.587 0.693
(0.478) (0.516) (0.497) (0.472) (0.447) (0.502) (0.471) (0.492) (0.493) (0.462)
INFSMEDIA 0.810 0.667 0.803 0.813 0.750 0.803 0.813 0.815 0.845 0.784
(0.393) (0.516) (0.399) (0.390) (0.447) (0.401) (0.391) (0.396) (0.362) (0.412)
INFSUNIV 0.225 0.167 0.172 0.239 0.188** 0.106 0.241 0.185*** 0.167 0.272
(0.418) (0.408) (0.379) (0.427) 0.403 0.310 0.428 (0.396) (0.373) (0.446)
INFSINST 0.184 0.167 0.164 0.189 0.063 0.212 0.184 0.185 0.151 0.208
(0.387) (0.408) (0.372) (0.392) 0.250 0.412 0.388 (0.396) (0.359) (0.406)
FENVNEWS 0.580 0.667*** 0.418 0.619 0.500*** 0.258 0.622 0.296*** 0.349 0.772
(0.494) (0.516) (0.495) (0.486) 0.516 0.441 0.485 (0.465) (0.478) (0.420)
MEMENVO 0.105 0.000** 0.041 0.122 0.125 0.030 0.114 0.037*** 0.060 0.144
(0.307) (0.000) (0.199) (0.328) 0.342 0.173 0.318 (0.192) (0.238) (0.351)
ENVAWARL 3.797 2.135 3.274 3.934 3.124*** 3.446 3.849 3.459*** 3.682 3.890
(0.401) (0.180) (0.232) (0.262) 0.738 0.421 0.343 (0.661) (0.411) (0.327)
ENVATTL 4.053 2.561 3.644 4.172 2.801 2.985 3.602 3.064*** 3.875 4.262
(0.580) (1.156) (0.624) (0.481) (0.742) (0.426) (0.511) (0.874) (0.534) (0.445)
ENVBEHL 3.516 2.867*** 3.206 3.601 2.801*** 2.985 3.602 2.193*** 3.119 3.913
(0.556) (0.481) (0.586) (0.517) (0.742) (0.426) (0.511) (0.274) (0.264) (0.304)
Note: The numbers in parentheses indicate standard deviations. ***, **, * denote that there are statistical
differences among the groups at 1%, 5% and 10% significance levels, respectively
Estimates of the ordered probit for the significant socio-demographic and economic
probabilities of the environmental awareness, variables were gender, age, mother education,
attitude and behavior of the students were presented father education, residence and family income.
in Table 4. The chi-square value for the Fernandez-Manzanal et al. [17] underlined also that
environmental awareness (69.83 with 12 degrees of socio-demographic development had an important
freedom) was statistically significant at the 0.01 role in the formation and growth of environmental
level of probability. The estimated threshold value awareness.
was positive and statistically significant (μ=1.655) However, Fransson and Garling [21] found
at the same level of probability. This indicated that that residential area had influences on
there was a natural ordering among the three environmental concern. The significant
response categories of environmental awareness of environmental variables were environmental
the students. Thirteen independent variables were courses taken, information source of media and
included in the ordered probit model, and the usually or always following environmental news.
estimated coefficients of these variables were tested This is because as the students are more likely from
using t-test statistics. Of the 13 independent rural areas and generally follow environmental
variables, 7 socio-demographic and economic news from media. Some studies [40, 64, 65] stated
characteristics and, 6 environmental education that university students in Turkey had usually got
characteristics were statistically significant at the their environmental knowledge from the mass
0.10 level of probability or better. The statistically media. However, Sadik and Sadik [41] determined
1250


that the internet and television as the most If the information source of the student was
LPSRUWDQWIDFWRUVLQUDLVLQJWKHXQLYHUVLW\VWXGHQWV¶ the media, his likelihood of having a high
environmental awareness. However, the variables environmental awareness increased by 9.29%
of household size, university and other institutions points. However, the likelihood of having a
as main information sources and membership to moderate and low environmental awareness
environmental clubs or organizations had no decreased by 8.7 and 0.6% points, respectively. If
statistical influence on the environmental awareness. the student usually or always followed
The results indicate that the environmental environmental news, his likelihood of having a high
DZDUHQHVV LQFUHDVHV DV WKH VWXGHQW¶V DJH IDWKHU environmental awareness increased by 10.4%
education, family income and number of points. However, the likelihood of having a
environmental courses taken boost. moderate and low environmental awareness
Timur et al. [49] found that pre-service decreased by 9.8 and 0.6% points, respectively.
WHDFKHUV¶ WDNLQJ HQYLURQPHQW FODVVHV VLJQLILFDQWO\ The chi-square value for the environmental
increased their environment awareness levels attitudes (61.26 with 13 degrees of freedom) was
which did not statistically differ their behaviors statistically significant at the 0.01 level of
towards environment. On the other hand, the probability. The estimated threshold value was
likelihood of the environmental awareness of the positive and statistically significant (μ=0.947) at the
student would be higher with younger and less same level of probability. This indicated that there
educated mother. Of the socioeconomic variables, was a natural ordering among the three response
the marginal effect for gender indicated that if the categories of the environmental attitudes of students.
student was male, his likelihood of having a high Fourteen independent variables were included
environmental awareness decreased by 16.8% in the ordered probit model and the estimated
points. However, the likelihood of having a coefficients of these variables were tested using t-
moderate and low environmental awareness test statistics. The statistically significant socio-
increased by 1.84 and 0.11% points, respectively. demographic variables were age and father
This is because women tended to display a higher education. The significant environmental variables
level of commitment and responsibility than men in were environmental awareness and following
social aspects and collective actions [17], and they environmental news. However, the variables of
were more environmental sensitive [22, 41, 66]. household size, university and other institutions as a
Previous studies [17, 44, 67-70] echoed with our main information sources and membership to
findings. The marginal effect for age indicated that environmental clubs or organization had no
if the student had a higher age, his likelihood of statistical influence on the environmental attitude.
having a high environmental awareness increased The results verified the likelihood for
by 1.95% points. However, the likelihood of having environmental attitudes increases with a decrease in
a moderate and low environmental awareness DJH RI WKH VWXGHQW .D÷ÕWoÕEDúÕ >@ VWDWed that
decreased by 1.84 and 0.11% points, respectively. environmental attitude started at a very early age,
If the mother had a longer education duration, his and if there was no important experience or
likelihood of having a high environmental circumstances, it did not change easily. However,
awareness decreased by 1.8% points. However, the Masud and Kari [33] concluded that age had a
likelihood of having a moderate and low positive influence on environmental attitudes. Ercan
environmental awareness increased by 1.7% and and Bilen [70] determined that education levels of
0.0015% points, respectively. If the father had a both mother and father did not play a role on their
longer education duration, his likelihood of having environmental attitudes, whilst Ozsevgec and Artun
a high environmental awareness increased by 1.0% [72] underlined that educated parents with
points. However, the likelihood of having a sufficient knowledge about the environment would
moderate and low environmental awareness transfer these information and experiences to their
decreased by 1.0 and 0.1% points, respectively. If children, and they could develop positive
the student was from rural areas, his likelihood of environmental attitudes. As a student had a higher
having a high environmental awareness increased environmental awareness score and usually or
by 11.0% points. However, the likelihood of having always followed environmental news, it was likely
a moderate and low environmental awareness that his environmental attitudes would be higher.
decreased by 10.5 and 0.5% points, respectively. If Masud and Kari [33] found also that environmental
the student took more than two courses about DZDUHQHVV KDG D JUHDW LQIOXHQFH RQ FRPPXQLWLHV¶
environment, his likelihood of having a moderate perception and attitudes. Fernandez-Manzanal et al.
environmental awareness increased by 5.8% points. [17] underlined also that it was essential to increase
However, the likelihood of having a low the awareness of the actions that made
environmental awareness decreased by 5.5 percent. environmental problems worse and to define the
1251


behavior that helped prevent and mitigate these moderate and low environmental awareness
problems. The marginal effect for age indicated that increased by 1.01 and 0.25% points, respectively. If
if the student had an older age, the likelihood of WKHVWXGHQW¶VIDWKHUKDGDORQJHUHGXFDWLRQGXUDWLRQ
having high environmental attitudes decreased by the likelihood of having high environmental
1.25% points. However, the likelihood of having a attitudes increased by 1.08% points.
TABLE 4.
2UGHUHG3URELWHVWLPDWHVIRUWKHSUREDELOLW\RIVWXGHQWV¶HQYLURQPHQWDODZDUHQHVVDWWLWXGHDQGEHKDvior.
Environmental awareness Environmental attitude Environmental behavior

Marginal effect Marginal effect Marginal effect
Coeff. Y=0 Y=1 Y=2 Coeff. Y=0 Y=1 Y=2 Coeff. Y=0 Y=1 Y=2
*** **
GENDER -0.626 0.010 0.156*** -0.167*** 0.107 -0.003 -0.015 0.019 -0.237** 0.007 *
0.086** -0.093**
AGE 0.075*** -0.001** - 0.018*** 0.019*** -0.068*** 0.002*** 0.010*** -0.012*** -0.025 0.000 0.009 -0.010
HOUSHSIZE 0.040 -0.000 -0.009 0.010 -0.033 0.001 0.004 -0.006 -0.030 0.000 0.011 -0.011
MOTHEDUC -0.069*** 0.000** 0.017*** -0.018*** -0.012 0.000 0.001 -0.002 -0.002 0.000 0.001 -0.001
FATHEDUC 0.038* -0.000 -0.009* 0.010* 0.058*** -0.002** -0.008*** 0.010** -0.001 0.000 0.000 -0.000
RESIDANCE 0.505*** -0.004** - 0.105*** 0.110*** 0.026 -0.000 -0.003 0.004 0.149 -0.004 -0.054 0.058
FAMINCOM 0.000** 0.000 0.000** 0.000* 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
ENVCOUR 0.220* -0.055* 0.058* 0.000 -0.011 0.001 -0.002 0.000 0.113 -0.003 -0.041 0.044
INFSMEDIA 0.328** -0.006 -0.086* 0.092* 0.209 -0.008 -0.032 0.041 -0.306** 0.007** 0.110** -0.118**
INFSUNIV 0.200 -0.002 -0.046 0.049 0.307 -0.009* -0.041* 0.050* 0.150 -0.004 -0.054 0.058
INFSINST 0.188 -0.002 -0.043 0.045 0.013 -0.000 -0.001 0.002 -0.053 0.001 0.019 -0.021
FENVNEWS 0.392*** -0.006* - 0.098*** 0.104*** 0.437*** -0.017** -0.066*** 0.083*** 1.002*** -0.04*** -0.340*** 0.383***
MEMENVO -0.001 0.000 0.000 -0.000 -0.001 0.000 0.000 -0.000 0.001 0.000 -0.000 0.000
ENVAWARL 0.799*** -0.028*** -0.117*** 0.146*** -0.155 0.056 -0.061 0.000
ENVATTL 0.802** -0.02*** -0.291*** 0.316***
Mu : 1.65509*** 0.94684*** 2.11599***
Log-Likelihood : -305.25056 -252.19175 -405.23229
Chi squared : (12) 69.82779 (13) 61.25905 (14) 221.43063
McFadden R2 : 0.1026383 0.1082999 0.2145864
N : 621 621 621
***, **, * denote significance levels at 1%, 5% and, 10%, respectively.
However, the likelihood of having moderate variable was only gender. The significant
and low environmental attitudes decreased by 0.87 environmental variables were the environmental
and 0.21% points, respectively. If the student had a attitude, information source of media and usually or
higher environmental awareness, the likelihood of always following environmental news. However,
having high environmental attitudes increased by the variables of household size, university and other
14.6% points. However, the likelihood of having institutions as a main information sources,
moderate and low environmental attitudes membership to environmental clubs or
decreased by 11.77 and 2.87% points, respectively. organizations and taking environmental courses had
If the student usually or always followed QR VWDWLVWLFDO LQIOXHQFH RQ WKH VWXGHQWV¶
environmental news, his likelihood of having a high environmental behavior. The results verified that if
environmental attitude increased by 8.38% points. the student was male and used the media as the
However, the likelihood of having a moderate and information source, so the likelihood of the student
low environmental attitude decreased 6.64 and 1.74 to behave environmental friendly would be less.
percentage points, respectively. Some previous studies [33, 73] contradicted with
The chi-square value for the environmental this phenomenon by indicating that men were more
behavior (221.43 with 14 degrees of freedom) was concerned about environmental issues, whilst some
statistically significant at the 0.01 level of stuGLHV FRQFOXGHG WKDW WKH VWXGHQWV¶ OHYHOV RI
probability. The estimated threshold value was environment knowledge and behavior did not
positive and statistically significant (μ=2.116) at the significantly differ on gender [62, 63, 74]. On the
same level of probability. This indicated that there other hand, as the student had a higher
was a natural ordering among the three response environmental attitude and usually or always
cDWHJRULHV RI VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV followed environmental news, the likelihood for the
Fifteen independent variables were included in the student to behave environmental friendly would be
ordered probit model, and the estimated coefficients higher. Altinoz [61] concluded that taking
of these variables were tested using t-test statistics. environment classes increased the science and
The statistically significant socio-demographic technology pre-VHUYLFH WHDFKHUV¶ HQYLURQPHQWDO
1252


knowledge and behavior. Karatekin [62] found that students aged, their environmental awareness
while taking environment courses at the university LQFUHDVHG+RZHYHUDVWKHVWXGHQWV¶DJHLQFUHDVHG
VLJQLILFDQWO\ GLIIHUHG WKH VWXGHQWV¶ HQYLURQPHQWDO their environmental attitudes decreased. The
behaviors. Timur et al. [48] concluded also that the students form the rural residences had higher
level of environmental literacy of teacher positive environmental awareness than the urban
candidates, who took courses on environment, was residence students. This might be that the rural
higher than the candidate teachers who did not. residence students were more practically involved
Monroe et al. [75] underlined also that behavior, in with environment, so that they are obliged to
general, was supported by knowledge and attitude, protect their surrounding resources. While high
but there was no direct cause-and-effect progression level of mother education had negative influence on
from knowledge to attitude and/or behavior..The WKH VWXGHQWV¶ HQYLURQPHQWDO DZDUHQHVV KLJK OHYHO
marginal effect for gender indicated that if the of father education had positive influence on the
student was male, his likelihood of having a high VWXGHQWV¶HQYLURQPHQWDODZDUHQHVVDQGDWWLWXGH
environmental behavior decreased by 9.36% points. As the curiosity levels of the students towards
However, the likelihood of a having moderate and environmental news increased, their environmental
low environmental behavior increased by 8.60 and awareness, attitude and behavior levels increased as
0.76% points, respectively. However, if the main well. While the mass media as a main information
information source of student was the media, the source had an important positive influence on the
likelihood of having a high environmental behavior studenWV¶ HQYLURQPHQWDO DZDUHQHVV LW KDG D
decreased by 11.84% points. However, the negative influence on their environmental behaviors.
likelihood of having a moderate and low While the students who took two or more
environmental behavior increased by 11.07 and environment courses had significantly higher
0.SRLQWVUHVSHFWLYHO\,IWKHVWXGHQW¶VDWWLWXGHV environmental awareness, this had no influence on
were more environmental friendly, the likelihood of their environmental attitudes and behaviors. Since
having a high environmental level increased by the students had taken about 3.4 environment
31.64% points. However, the likelihood of having courses, this number might not be enough to show
moderate and low environmental levels decreased their environmental attitude or behavior in their
by 29.19 and 2.46% points, respectively. If the JUDGXDWLRQ WLPH:KHUHDVVRPHGHSDUWPHQWVGRQ¶W
student usually or always followed environmental have any course or enough courses related to
news, his likelihood of behaving more environment in their curricula of education
environmental friendly increased by 38.31% points. programs, they may have a direct or indirect impact
However, the likelihood of having moderate and RQ HQYLURQPHQW E\ WKHLU DFWLYLWLHV 7KH VWXGHQWV¶
low environmental levels decreased by 34.07 and environmental awareness had the biggest positive
4.23% points, respectively. influence on their environmental attitudes. Thus, if
the students had higher environmental awareness,
their attitudes would be more environmentally-
CONCLUSIONS conscious. However, this study concluded that the
students who had higher environmental awareness
In this study, the average levels of would not behave more environmentally-conscious.
environmental awareness, attitudes and behaviors 7KHVWXGHQWV¶HQYLronmental attitudes had very high
of students were found to be high. Control variables positive influence on their environmental behaviors.
as socio-demographic and economic factors Thus, if the students had higher environmental
JHQHUDOO\ KDG LPSRUWDQW LPSDFWV RQ WKH VWXGHQWV¶ attitudes, they would behave more environmentally-
environmental awareness. Our findings verified conscious. However, our study concluded that
DOVR WKDW WKH VWXGHQWV¶ HQYLURQPHQWDO DWWLWXGH DQG environmental awareness of the students was not
behavior were generally affected by explanatory sufficient for them to put this awareness into
variables of environmental education or practice in their daily life.
information. Gender had very high impacts on the This study provided a clear picture about the
environmental awareness and behaviors of the environmental awareness, attitude and behavior
students in question. Thus, female students were levels of candidate graduates from undergraduate
more interested in environmental issues and their programs and the determinants on their
likelihood of high environmental awareness and environmental sensitivities. Therefore, this study
behaviors were higher by 16.8% and 9.4% than would help decision makers to improve the
male students, respectively. Age had an important curricula of undergraduate programs and to
impact on both thH VWXGHQWV¶ HQYLURQPHQWDO formulate better strategies for conserving
awareness and attitudes, whereas, age had no environment. Considering the results of the current
LPSDFWRQWKHVWXGHQWV¶HQYLURQPHQWDOEHKDYLRU$V
1253


study, the following suggestions could be put The research subject should be also conducted in
forward: other departments and universities to compare the
7R LQFUHDVH WKH JUDGXDWHV¶ HQYLURQPHQWDO levels of environmental literacy.
awareness, the departments should put sufficient
environment courses as compulsory on their
SURJUDPV 7R LQFUHDVH WKH VWXGHQWV¶ HQYLURQPHQWDO ACKNOWLEDGEMENT
DZDUHQHVV¶V DQG DWtitudes, the departments should
focus especially on male students in the classes by The authors thank all students participating in
encouraging them to participate in environmental the surveys and the lecturers for their help in
conservation activities such as nature and applying the surveys in Ondokuz Mayis University.
environmental trips. However, it should be The authors are grateful to the editor and
benefited actively from the leadership of female anonymous referees for their very useful comments
students on environmental education and and suggestions. The authors gratefully
conservation activities. The departments should try acknowledge the help of Birol Kurt and Emine Bol
WR LPSURYH WKH \RXQJHU VWXGHQWV¶ HQYLURQPHQWDO Yazici (International Relations Office of Ondokuz
awareness by interference with older age students. Mayis University) in the English editing of this
The departments should enable the students to share article.
their environmental knowledge and experiences in
their classes and environmental activities. Since
mass media was the main information source of the REFERENCES
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1257


EVALUATION OF SPAD CHLOROPHYLL IN SPRING

WHEAT GENOTYPES UNDER DIFFERENT
ENVIRONMENTS
Celaleddin Barutçular10HKPHW<ÕOGÕUÕP2, Mujde Koç1&XPD$NÕQFÕ2,UHP7RSWDú1, Onder Albayrak2,
$EGXONDGLU7DQUÕNXOX3, Ayman El Sabagh4
1
Department of Field Crops, Faculty of Agriculture, University of Çukurova, 01330 Adana, Turkey
2
'HSDUWPHQWRI)LHOG&URSV)DFXOW\RI$JULFXOWXUH8QLYHUVLW\RI'LFOH'L\DUEDNÕU7XUNH\
3
Department of Field Crops, Faculty of Agriculture, University of Kahramanmaras Sutcu Imam, Kahramanmaras, Turkey
4
Department of Agronomy, Faculty of Agriculture, University of Kafrelsheikh, Egypt
ABSTRACTS INTRODUCTION
Wheat is an important food crop. Its Wheat iti m ae ti m L.) is the most
production is limited by multiple environmental important cereal crop in Turkey and around the
stresses, especially terminal heat stress. High world. The grain yield of wheat is usually
temperature and drought are the main stresses determined by genetic and environmental factors.
affecting wheat production in the Mediterranean The physiological characteristics of wheat are
region. Hence, the chlorophyll content in leaves controlled by genetic factors that are of great
reflects photosynthetic activity and the yield interest to researchers and breeders. It is important
potential of wheat plants. So, the aim of this study to characterize the water relations of species or
was to evaluate SPAD (Soil Plant Analysis cultivars at the whole-plant or cellular level to
Development) chlorophyll meter readings and their evaluate their resistance to environmental stresses
relationships with the grain yield of spring wheat such as drought, heat, or salinity [1].
growing in different environments, and to examine Although global wheat production has
the physiological responses to environmental stress increased, it is still insufficient to meet the demands
at different growth stages. Sixteen spring wheat of the growing population. Wheat production in
genotypes were grown under two temperature (cool many regions of the world is below average
and warm) and two moisture (rain-fed and irrigated) because of adverse environmental conditions, and
regimes during the 2011/2012 growing season in wheat cultivation is often restricted in zones with
WZR ORFDWLRQV LQ 7XUNH\ $GDQD DQG 'L\DUEDNÕU scarce water resources [2]. Drought has affected
There were significant genotypic variations in wheat productivity worldwide, especially in arid,
SPAD values under all conditions. There was a semi-arid, and Mediterranean climates owing to the
significant positive correlation between third-leaf unpredictable and erratic rainfall in these regions
SPAD values and grain yield in the cool-climate [3]. The increase in wheat yield has been
conditions at Adana. There was a significant characteristically slow in the Mediterranean region,
positive relationship between flag-leaf SPAD as in other stressful environments. For example, the
values at the heading stage and grain yield under annual genetic yield gain for durum wheat over the
cool-FOLPDWH FRQGLWLRQV LQ 'L\DUEDNÕU ZKLOH WKH last century was about 20 kg haí1 yearí1 in Italy [4].
relationship between SPAD values and grain yield In wheat breeding, the selection efficiency can
was negative under warm-environment conditions. be increased if specific physiological and/or
The SPAD values were more strongly affected by morphological traits related to yield under specific
location and heat stress than by drought stress. environments can be identified and used as
Regarding to the range of SPAD values within each selection criteria to complement traditional plant
genotype was relatively stable. Together, these breeding [5]. Total chlorophyll content per leaf area
results showed that SPAD values can be used as an can be estimated in a fast, single, and
indicator of grain yield in spring wheat, providing nondestructive way using a portable chlorophyll
that the measurements are taken at appropriate meter such as the SPAD-502 (Soil-Plant Analysis
stages at each location. Development Section, Minolta Camera Co., Ltd.,
Osaka, Japan). The chlorophyll meter (or SPAD
meter) is a simple, portable diagnostic tool that
KEYWORDS: measures leaf greenness, i.e., the relative
Bread wheat, drought, heat stress, irrigation, SPAD chlorophyll concentration in leaves. Compared with
reading
traditional destructive methods, this method
1258


provides substantial savings in time, space, and MATERIALS AND METHODS

resources [6]. The SPAD meter measures
transmittance by leaves at two wavelengths (650 Plant materials and growth conditions.
nm and 940 nm) that are differentially absorbed by Sixteen spring bread-wheat ( iti m ae ti m L.)
chlorophyll, and estimates leaf chlorophyll and genotypes with similar developmental patterns were
nitrogen contents. Chlorophyll meter readings have used in these experiments. The widely adapted
been extensively used in the last few years, genotypes comprised commercial cultivars from
especially to control nitrogen nutrition in cereals. Turkey (Adana-99, Balattila, Cemre, Cumakalesi,
Researchers have used chlorophyll meters to Genç- .DUDFDGD÷-98, Meta-2002 and Özkan),
optimize the timing of nitrogen application to wheat ICARDA (Cham-6 and Siete Cerros), Israel (Dariel
and rice [7, 8], to determine the nitrogen and Galil), Italy (Colfiorito), Spain (Mané-Nick),
requirements of wheat and barley [9, 10], to and Pakistan (Inqilab-91 and V-3010).
estimate the nitrogen status of plants [11] and to The experiments were conducted during the
estimate nitrogen deficiency [12]. 2011±2012 growing season at two locations in
In previous studies, significant relationships Turkey; Adana (Experimental area of Cukurova
between SPAD values and grain yield were 8QLYHUVLW\ DQG 'L\DUEDNÕU ([SHULPHQWDO DUHD RI
detected at the heading stage [13] and the middle of Dicle University). In each location, the 16
the grain-filling stage [14] in winter wheat. genotypes were sown at two sowing times, normal
<ÕOGÕUÕP HW DO 3) found positive (cool environment) and late (warm environment), to
correlations between SPAD values and grain yield represent the two temperature regimes, and two
at the heading and mid-milk grain development irrigation conditions (rain-fed and irrigated).
stages [15, 16]. The strength of the relationship The soil type at Adana is a slightly alkaline
changed depending on the location and (pH 7.1±7.6), fine loamy, montmorillonitic typic
phenological stage [17]. xerofluvent, with a low organic matter content. The
SPAD-validation studies on materials with VRLO W\SH DW 'L\DUEDNÕU LV DQ DONDOLQH S+
wide genetic backgrounds are useful to improve loamy incepticol, with a low organic matter content.
selection efficiency in durum wheat breeding. 'L\DUEDNÕU DQG $GDQD KDYH D W\SLFDOO\
Strong genotype±environment interactions in leaf Mediterranean climate. In the experimental season,
chlorophyll contents can result in lower selection 'L\DUEDNÕU ZDV GULHU DQG ZDUPHU WKDQ $GDQD ,Q
accuracy in conventional breeding programs. 'L\DUEDNÕUWKHWHPSHUDWXUHUDQJHZDVJUHDWHULQWKH
Recently, environmental conditions were shown to cool environment (early planting) than in the warm
affect the relationships between grain yield and environment (late planting) (Table 1). In rain-fed
chlorophyll retention in populations of spring and conditions, Adana was wetter at the early stage
winter wheat [18,19]. Therefore, it is necessary to (235.2 mm in the cool environment and 129.2 mm
analyze SPAD values of plants in target in the warm environment; Table 2). The seeding
environments to verify the relationship between rate was adjusted to a density of 450 seeds mí,
SPAD values and grain yield. If there is a strong according to standard practice. The plot size was
relationship between SPAD chlorophyll at a 8.4 m2 (six rows, 20 cm apart). The drilled plot
particular stage and grain yield, then this may serve sizes were six rows, each 7 m long, and 6 m of each
as an indirect selection tool to differentiate high- row was harvested. Phosphorus (40 kg haí P2O5)
yielding genotypes. was applied before sowing in the form of triple
The relationships between chlorophyll content super phosphate. Nitrogen was applied as
and yield in spring wheat under moisture stress are ammonium nitrate in three split doses (40 + 80 + 40
not well documented. Therefore, the objectives of kg N haíDW=DGRN¶V>@JURZWKVWDJHV=*6
this work were as follows: a) to evaluate variation 20, and 30, respectively. Sowing was conducted on
in chlorophyll contents in a set of phenologically the same date in both locations at the beginning of
similar spring wheat cultivars at different December (cool environment/early planting) and
developmental stages; and b) to analyze the April (warm environment/late planting). The
relationships between SPAD chlorophyll readings drought and heat stress were mild on the first
and grain yield under rain-fed and irrigated cool sowing date, and stronger on the second sowing
and warm climatic conditions. Because of date. The plants were subjected to two moisture
increasing drought and heat damage to crops- conditions: irrigated or rain-fed. No supplementary
especially in Turkey with its arid and semi-arid irrigation was applied in rain-fed conditions, except
climate zones- it is important to select varieties that during emergence. Irrigation was adjusted to
are well adapted to water shortage. maintain plants at field capacity (Table 2)
1259


TABLE 1
Mean temperature at each growth stage and temperature regime in Adana DQG'L\DUEDNÕU
Adana 'L\DUEDNÕU
Min. Max. Mean Min. Max. Mean
Growth Stage Temp.°C Temp.°C Temp.°C Temp.°C Temp.°C Temp.°C
Cool environment (early sowing date)
ZGS: 00-13 4.09 16.50 9.58 -0.3 9.1 4.0
ZGS: 13-15 4.43 12.49 7.97 8.6 23.2 16.2
ZGS: 15-55 4.89 17.42 10.93 11.6 26.1 19.1
ZGS: 55-75 11.44 24.86 17.94 12.7 27.8 20.3
Warm environment (late sowing date)
ZGS: 00-13 5.88 19.45 12.63 9.2 24.1 17.1
ZGS: 13-15 10.67 26.79 18.16 12.6 26.8 19.6
ZGS: 15-55 13.20 25.48 19.07 14.9 29.1 22.0
ZGS: 55-75 14.80 26.54 20.60 17.9 33.8 26.4
TABLE 2
Amount of water (mm) at each JURZWKVWDJHDQGWHPSHUDWXUHUHJLPHLQ$GDQDDQG'L\DUEDNÕU
Adana 'L\DUEDNÕU
Growth stage Rain-fed Irrigated Rain-fed Irrigated
Cool environment (early sowing date)
ZGS: 00-13 227.55 227.55 201.6 246.6
ZGS: 13-15 247.20 247.20 12.0 27.0
ZGS:15-55 205.10 226.54 12.2 111.2
ZGS: 55-75 9.60 45.68 9.4 64.4
Warm environment (late sowing date)
ZGS: 00-13 94.93 94.93 53.2 60.2
ZGS: 13-15 30.71 30.71 0.0 44.2
ZGS:15-55 129.15 129.15 28.4 121.0
ZGS: 55-75 25.80 71.10 0.0 123.0
Crop sampling and measurements. The Experimental design and statistical analysis.
chlorophyll content, hereafter referred to as SPAD The experiment was designed as a completely
value, was measured at four developmental stages randomized block with a spilt-split plot
(ZGS13: three leaves folded; ZGS15: five leaves arrangement with four replications. Temperature
folded; ZGS55: heading stage; and ZGS75: medium regime (sowing time), irrigation, and genotypes
milk stage) at the midpoint of the flag leaves of 10 were arranged as main factor, sub factor, and sub-
randomly selected plants in each plot, using a sub factors, respectively.
portable chlorophyll meter (Minolta SPAD-502, The data were subjected to analysis of
Osaka, Japan). Grain yield was determined on the variance (ANOVA) and least significant difference
basis of the harvested plot in all trials. Grain yields (LSD) values were calculated. Genotypic
were obtained after physiological ripening from one correlations were calculated between grain yield
square meter in the two middle rows of each plot. and SPAD measurements at the four different
The grain weight includes a moisture content of stages at both locations. The effect of location was
12%. also tested. Significant differences among
The drought and heat resistance indices (DRI genotypes, irrigation, and temperature (sowing date)
and HRI, respectively) were calculated from the WUHDWPHQWV ZHUH GHWHUPLQHG XVLQJ )LVKHU¶V
grain yield and SPAD values according to [21]. protected LSD at a probability level of 0.05.
Each RI is the ratio of grain yield and SPAD (total
chlorophyll) reduction due to stress in a given
genotype as compared with the mean reduction
over all genotypes in the given conditions.
1260


RESULTS AND DISCUSSIONS same plant. The SPAD values among leaves in a
single plant had significant relationships (data not
Chlorophyll loss is associated with shown).
environmental stress, and changes in the There were significant temperature × irrigation
chlorophyll/carotenoids ratio may be a good interactions for SPAD values measured at ZGS55 at
indicator of stress in plants [22]. The SPAD both locations (Table 3). This result indicated that
measurements, which estimated leaf chlorophyll the changes in SPAD values in response to
content per unit area, were taken from different irrigation conditions depended on the temperature
points on a leaf and from different leaves of the regime (i.e., early vs. late sowing date).
TABLE 3
ANOVA table for SPAD values of wheat genotypes at four different growth stages in two locations.
Adana 'L\DUEDNÕU
Source Df ZGS12 ZGS15 ZGS55 ZGS75 ZGS12 ZGS15 ZGS55 ZGS75
Replication 3 62.1 30.2 186.6 92.3 2.1 1.2 39.9 48.1
Temp. Regime
(T) 1 1046.2 * 677.7 ** 1154.9 * 104.3 ns 451.0 ** 90.0 ** 0.0 ns
Error 3 62.7 16.1 64.9 191.7 1.5 1.9 18.0
Irrigation (I) 1 19.8 ns 0.0 ns 159.6 ns 318.0 ns 10.8 ns 11.1 * 6.7 ns 48.5
TxI 1 82.1 ns 5.1 ns 278.2 * 152.1 ns 1.9 ns 0.0 ns 582.3 **
Error 31.4 7.3 41.7 125.5 4.5 1.8 19.6 30.1
Genotype (G) 15 76.9 ** 70.9 ** 90.1 ** 45.4 * 39.8 * 84.2 ** 86.0 ** 56.7 **
TxG 15 12.5 ns 29.9 ** 17.0 ns 65.5 ** 21.0 ** 1.1 ns 5.7 ns
IxG 15 7.2 ns 12.6 ns 24.7 * 22.6 ns 1.6 ns 0.0 ns 8.1 ns 13.5 ns
TxIxG 15 14.4 ns 12.6 ns 8.0 ns 19.0 ns 0.0 ns 1.1 ns 3.3 ns
Error 18 11.0 11.8 13.1 21.8 1.7 1.2 5.9
CV, % 7.49 8.36 8.17 10.99 3.04 2.44 5.17 6.88
'IIRU'L\DUEDNÕU=*6; * and **, significant at 0.05 and 0.01 probability level, respectively; ns, not significant.
Genotype × irrigation interactions were not (Table 3). Cham-6, Colfiorito, and Dariel had
significant at either of the locations (Table 3). This higher SPAD values than those of other cultivars at
result indicated that the genotypes performed all measurement times at Adana. Cham-6,
independently of the moisture conditions, and thus, Colfiorito, Dariel, and Galil had higher SPAD
genotypes suitable for growth in drought and values than those of all of the other cultivars at
irrigated conditions could determined based on their =*6 DQG =*6 LQ 'L\DUEDNÕU $W $GDQD PRVW
SPAD values. Genotype × temperature interactions genotypes showed higher SPAD values at younger
were significant at ZGS15 and ZGS75 at Adana and growth stages. Cham-6 had high SPAD values at all
DW =*6 DW 'L\DUEDNÕU EXW QRW VLJQLILFDQW DW measurement times in both locations, suggesting
ZGS55 at either location (Table 3). This result that a high chlorophyll content might be a heritable
indicated that the studied genotypes responded trait in this variety.
differently in the cool and warm environments (i.e., The SPAD values varied among the 16
at early and late sowing dates). Based on these genotypes. From a plant selection perspective, the
results, genotypes with a high yield potential were average value of a series of SPAD measurements
reliably determined by using SPAD readings at on a single leaf provides an adequate ranking
different growth stages under drought and irrigated assessment of genotypes in terms of leaf
conditions. Similar results were reported by chlorophyll content per unit area. Averaging several
<ÕOGÕUÕPHWDOIRUGXUXPZKHDW[15]. measurements to obtain a mean value improves the
The mean SPAD values for all of the selection efficiency and reduces measurement and
genotypes at four growth stages in both locations recording times and costs [23]. Mean SPAD values
are shown in Table 4. There were significant have been used successfully by many researchers in
differences in SPAD values among genotypes the screening and selection of drought-tolerant
1261


wheat cultivars [24]. Additionally, the assessment the SPAD values first decreased at ZGS15,
of photosynthetic pigments and their relationships increased at ZGS55, and finally decreased at
is an important indicator of senescence [25]. ZGS75 at Adana (Tables 4 and 5).
The SPAD values fluctuated depending on the
growth stage. Averaged across all of the genotypes,
TABLE 4
SPAD values for 16 VSULQJZKHDWJHQRW\SHVDWIRXUJURZWKVWDJHVLQ$GDQDDQG'L\DUEDNÕU
Adana 'L\DUEDNÕU
Genotypes ZGS13 ZGS15 ZGS55 ZGS75 ZGS13 ZGS15 ZGS55 ZGS75
Adana-99 44.9 40.3 43.5 42.9 44.1 44.9 46.1 45.8
Balattila 43.8 42.3 42.9 41.9 42.7 46.2 44.2 46.7
Cemre 43.4 40.6 41.6 41.4 43.2 45.3 45.3 45.7
Cham-6 49.7 46.3 49.2 44.7 46.9 50.0 51.0 52.2
Colfiorito 47.9 45.4 49.2 45.0 43.5 44.2 51.4 52.8
Cumakalesi 42.8 39.2 42.7 40.3 43.6 47.1 46.2 46.8
Dariel 46.1 42.2 46.5 44.7 41.2 42.3 50.2 51.7
Galil 45.8 40.9 46.5 42.6 41.4 43.3 49.1 51.1
Genç- 99 41.7 39.0 45.2 43.1 41.1 42.7 48.4 49.4
Inqlab-91 43.6 40.4 44.6 44.1 43.0 46.2 46.3 49.5
.DUDFDGD÷-98 44.0 40.0 45.2 43.5 45.1 49.4 44.9 44.8
Manenick 41.4 41.2 43.3 40.4 41.4 41.9 45.7 46.3
Meta-2002 44.6 39.7 42.2 40.9 44.1 45.0 44.4 46.9
Ozkan 43.4 41.1 42.3 41.6 43.7 46.3 46.7 45.3
Siete Cerros 44.4 39.9 43.0 43.1 41.9 44.0 46.1 46.1
V-3010 42.4 39.0 42.8 39.6 44.1 45.6 45.7 47.9
Mean 44.4 41.1 44.4 42.5 43.2 45.3 47.0 48.1
LSD0.05 2.31 2.4 2.52 3.25 0.92 0.77 1.69 3.28
TABLE 5
Mean SPAD values of 16 spring wheat genotypes in different moisture and temperature conditions at
$GDQDDQG'L\DUEDNÕU
Adana 'L\DUEDNÕU
ZGS13 ZGS15 ZGS55 ZGS75 ZGS13 ZGS15 ZGS55 ZGS75
Cool environment
Rain-fed 42.6 39.6 44.1 45.0 44.4 45.7 45.6 n.d.
Irrigated 42.1 39.3 40.5 41.2 44.6 46.0 48.3 n.d.
Warm environment
Rain-fed 45.5 42.6 46.3 42.2 41.6 44.4 48.6 48.7
Irrigated 47.2 42.8 46.8 41.5 42.2 44.9 45.3 47.4
LSD0.05 0.82 0.85 0.89 1.15 0.32 0.27 0.6 1.16
n.d., not determined
In contrast, the SPAD values continuously barley under different temperature and nitrogen
LQFUHDVHG GXULQJ JURZWK DW 'L\DUEDNÕU $W $GDQD fertilizer regimes [10]. In this study, the SPAD
the SPAD values decreased from ZGS55 to ZGS75 values at ZGS55 and 75 were generally higher
in the warm environment (late sowing date), but under rain-fed conditions than under irrigated
increased in the cool environment (early sowing conditions. This can result from increased density
date). The decline in the chlorophyll content was of cells, organelles, and membranes under non-
indicative of the start of senescence. Spaner et al. irrigated conditions.
(2005) reported a significant decrease in SPAD The flag leaf width, area, and weight
values from ZGS59 to ZGS 75±79 in wheat and decreased similarly at both locations under rain-fed
1262


conditions, except for leaf area in the cool wheat genotypes, SPAD values declined in a cool
environment at Adana (data not shown). The environment, but increased in a warm environment
decrease in relative water content was accompanied [16]. In other studies, SPAD values decreased
by increased irradiance, and both factors can affect under high temperature and drought conditions [27].
SPAD values without affecting the chlorophyll In this study, the SPAD values increased from
content [26]. The decreased relative water content ZGS55 to ZGS75 in some conditions (Table 5).
after the heading stage (ZGS55) resulted in Previous studies have also reported that SPAD
increased SPAD values under rain-fed conditions in values increased under heat stress [28] and drought
Adana. <ÕOGÕUÕPHWDOUHSRUWHGWKDWLQEUHDG stress [29].
TABLE 6
Correlation analysis between grain yield and SPAD values at four growth stages, two temperature
regimes, and two irrigation conditions in two locations.
Adana 'L\DUEDNÕU
Cool environment
Rain-fed 0.298* 0.106 0.030 0.182 -0.088 0.366** n.d.
Irrigated 0.338** -0.135 0.012 -0.115 -0.081 0.346** n.d.
Warm environment
Rain-fed -0.236 -0.139 -0.003 -0.127 -0.059 0.033 -0.436** -0.053
Irrigated -0.159 -0.106 -0.007 -0.085 -0.11 0.047 0.039 -0.376**
and **, significant at 0.10, 0.05 and 0.01 probability levels, respectively; n.d., not determined.
Sikuku et al. (2010) reported that chlorophyll the growth stage advanced [34]. There were
synthesis was inhibited under water deficit in negative relationships between SPAD values and
drought-sensitive wheat lines [30]. High JUDLQ\LHOGLQWKHZDUPHQYLURQPHQWDW'L\DUEDNÕU
chlorophyll content is a desirable characteristic In this case, the genotypes with high SPAD values
because it indicates a low degree of photoinhibition had low yield potential. Therefore, researchers and
of the photosynthetic apparatus; therefore, more plant breeders should interpret SPAD data
carbohydrates are available for grain growth in cautiously [35].
lines with a higher chlorophyll content [31]. In most cases, the relationships between grain
Except for normal sowing, SPAD values and yield and SPAD stress resistance indices were not
grain yield were not strongly related in all of related to drought and heat stress (Table 7). The
growth stages of late conditions (Table 6). In DRI was only significant at the heading stage (ZGS
previous studies, the changes in SPAD values of 55) in the cool environment at Adana. This result
cultivars differed depending on the temperature and could be related to the narrow range of RIs obtained
drought conditions [32, 33]. In the cool in these experiments (data not shown).
environments, there were significant relationships The results of this study showed that the
between SPAD values at ZGS13 and grain yield SPAD values were affected by location, sowing
under rain-fed and irrigated conditions at Adana, time, irrigation, and developmental stage. However,
and between SPAD values at ZGS55 and grain the ranking of genotypes based on their SPAD
\LHOGLQ'L\DUEDNÕU<ÕOGÕUÕP HWDOUHSRUWHG values was generally stable throughout all of the
significant positive correlations between SPAD growth stages and in both of the stress conditions.
values at ZGS55 and ZGS75 and grain yield in a Some genotypes showed differences in SPAD
warm environment [16]. Babar et al. (2006) values at different locations. The SPAD values at
reported a significant positive phenotypic the heading stage were related to grain yield in the
correlation between SPAD values and biomass at FRRO HQYLURQPHQW DW 'L\DUEDNÕU UHJDUGOHVV RI WKH
the grain filling stage in wheat under irrigated drought conditions.
conditions, and the correlation became stronger as
1263


TABLE 7
Correlation analysis between grain yield and chlorophyll stress resistance indices (based on SPAD values)
of wheat at four different growth stages and two temperature regimes in two locations.
Adana 'L\DUEDNÕU
DRI cool environment
0.391 -0.326 0.544* 0.171 -0.068 -0.112 0.218 n.d.
DRI warm environment
0.425 -0.201 -0.021 -0.078 -0.138 0.329 0.060 0.145
HRI
0.266 -0.258 0.105 -0.265 -0.228 -0.019 -0.141 n.d.
DQG, drought and heat resistance indices, respectively; *, significant at 0.05 probability level; n.d., not determined.
CONCLUSION Since AD 1500, Routledge, London, pp. 246±

268.
Based on the results of the study, the SPAD [4] De Vita, P. Nicosia, O.L.D. Nigro, F. Platani,
values of the 16 genotypes showed different C. Riefolo, C. Fonzo, N.D. and Cattivelli, L.
responses to drought and heat stresses. The values (2007) Breeding progress in morpho-
generally decreased under environment stresses. physiological, agronomical and qualitative
Temperature had the strongest effect on SPAD traits of durum wheat cultivars released in
values. The relationship between SPAD values and Italy during the 20th century. Europ. J.
grain yield was stronger in the cool environments. Agronomy 26: 39-53.
This relationship was significant at the early growth [5] Acevedo, E. (1991) Improvement of winter
stage (ZGS15) under cool environment at Adana, as cereal crops in Mediterranean environments:
well as at the later growth stage (ZGS55) under use yield, morphological and physiological
FRRO HQYLURQPHQW DW 'L\DUEDNÕU 7KXV WKH FRRO traits. In: E. Acevedo, A.P. Conesa, P.
environment (early sowing date) was more suitable Monneveux and P. Srivastava (eds),
for detecting the relationship between SPAD values Physiology breeding of winter cereals for
and grain yield. From above-mentioned, to screen stressed Mediterranean environments.
for drought-tolerant genotypes, the SPAD-based Montpellier, France, INRA, pp. 273-305.
DRI should be evaluated in rain-fed and warm [6] Netto, A.T. Campostrini, E. Oliveira, J.G. and
environments. In the same way, use of the SPAD- Bressan-Smith, R.E. (2005) Photosynthetic
based HRI to screen for heat-tolerant genotypes pigments, nitrogen, chlorophyll a florescence
should be evaluated in further studies. and SPAD readings in coffee leaves. Scientia
Horticulturae 104: 199-209.
[7] Singh, B. Singh, Y. Ladha, J.K. Bronson, K.F.
ACKNOWLEDGMENTS Balasubramanian, V. Singh, J. and Khind, C.S.
(2002) Chlorophyll meter and leaf color chart-
based nitrogen management for rice and wheat
This research was supported by the Scientific
in northern India. Agronomy Journal 94: 821-
and Technological Research Council of Turkey
829.
(TUBITAK) Project no. 110O345.
[8] Shukla, A.K. Ladha, J.K. Singh, V.K.
Dwivedi, B.S. Balasubramanian, V. Gupta,
R.K. Sharma, S.K. Singh, S.Y. Pathak, H.
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Celaleddin Barutçular
University of Çukurova
01330 Adana ± TURKEY
e-mail: cebar@cu.edu.tr
1266


BI S FLOWER-LIKE NANOSTRUCTURES COMPOSED

OF NANOBELTS SYNTHESIS, GROWTH PROCESS AND
THERMOELECTRIC PROPERTIES
<Ƶŝůŝ>ŝƵϭ͕Ϯ͕:ŝŶŐŚƵĂ,Ğϭ͕tĞŝ'Ƶϭ͕>ŝƐŽŶŐyŝĂŽϭ͕Ύ
1
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, Jiangsu, P. R. China
2
Department of Physics and Electronic Engineering, Zhoukou Normal University Zhoukou, 466001, Henan, P. R. China
ABSTRACT including our previous works, [5-6] have suggested

WKDW ³QDQRVWUXFWXULQJ´ RI WKH 7( PDWHULDOV is a
Bi2S3 flower-like nanostructures composed of promising strategy for the enhancements in TE
single-crystalline nanobelts have been fabricated property owing to a high density of states and an
via a facile and low-cost hydrothermal approach. increased phonon scattering or reduced lattice
[7-12]
The structures and morphologies of the samples thermal conductivity in nanosystems.
were characterized via X-ray diffraction (XRD), X- Moreover, compared with simple nanostructures of
ray photoelectron spectroscopy (XPS), Field- Bi2Te3 and PbTe, the 3D hierarchical micro-
emission scanning electron microscopy (FE-SEM) /nanostructured counterparts show obviously
and transmission electron microscopy (TEM). On enhanced TE properties. [13-16]
the basis of the effects of kinetic parameters such as In the past several years, diverse methods
the concentrations of reactant, time and reducing such as chemical vapour deposition technique, [17-20]
agent type on the morphologies of the resultant hydrothermal and solvothermal routes, [21-28]
products, a stepwise growth process is rationally solution-phase approach [29-34] have been developed
proposed including the initial formation of Bi to fabricate various shapes of Bi2S3 nanostructures.
nanospheres by aggregation of nuclei and the Among them, the hydrothermal approaches are
subsequent site-specific oriented growth of powerful, simple, high-efficient and cheap, and
nanobelts. The as-obtained Bi2S3 flower-like have been used to successfully obtain some 3D
nanostructures show enhanced electrical transport Bi2S3 nanostructures. [35-37] But they all require
properties and power factor compared with other organic molecules or bio-molecules as the S source,
reports. This work also provides a facile and green such as thiourea and L-cysteine, which are
strategy to fabricate 3D hierarchical nanostructures relatively expensive, highly toxic and easily
based on Bi2S3 nanobelts. decomposed. Therefore, it still remains a major
challenge to develop an environmentally friendly,
organic S source-free and low-cost hydrothermal
KEYWORDS: synthetic route for the fabrication of 3D Bi2S3
Flower-like / Nanostructure / Thermoelectric / hierarchical nanostructures.
Hydrothermal / Growth process Herein, we develop a novel hydrothermal
approach for 3D Bi2S3 flower-like nanostructures
composed of single-crystalline nanobelts through
INTRODUCTION
using inorganic salts (Bi(NO3)3·5H2O and Na2SO3)
as raw materials. The structures and shapes of the
Thermoelectric (TE) materials have attracted as-obtained Bi2S3 products are sensitive to the
tremendous attention because they can generate synthetic conditions including the reactant
electricity by recovering waste heat or be used as concentration, reaction time and the types of
solid-state cooling devices in various application reducing agent. A possible stepwise growth process
fields such as military and civilian. [1] Recently, of 3D Bi2S3 nanoflowers is proposed. The product
Bi2S3 has received much interest as a promising powders after consolidated into bulk flakes by the
candidate to replace the traditional materials such as spark plasma sintering (SPS) treatment show an
Sb2Te3 and Bi2Te3 because of the non-toxicity and H[FHOOHQWSRZHUIDFWRU3)YDOXHRIȝ:P.2
low-cost. [2-4] But the so far obtained Bi2S3 peaked at 470 K.
generally show low TE efficiency, which has
hindered the real application. Both theoretical
predictions and experimental investigations,
1267


Experimental Section. Bismuth nitrate RESULTS AND DISCUSSION

pentahydrate (Bi(NO3)3·5H2O, AR), exsiccated
sodium sulfite (Na2SO3,AR), ethylenediamine- In the synthetic process, typical Bi2S3 flower-
teraacetic acid disodium salt dihydrate (EDTA, like nanostructures products were synthesized at
C10H14N2O8Na2·2H2O, AR) and sodium 140 oC under hydrothermal treatment for 12 h. The
hypophosphite monnhydrate (NaH2PO2·H2O, AR) phase and composition of the sample are
were purchased from Sinopharm Chemical Reagent characterized by X-ray diffraction (XRD), as shown
Co. Ltd. All the reagents were used for the in Figure 1. The peaks can be indexed to
synthesis without further purification. orthorhombic Bi2S3 (space group: Pbnm (62)) with
In a typical experiment, 0.1 mmol lattice constants a=11.137 Å, b=11.297 Å and c=
Bi(NO3)3·5H2O and 1 mmol ethylene- 3.981 Å (Joint Committee on Powder Diffraction
diamineteraacetic acid disodium salt dihydrate Standards (JCPDS) card number 89-8964). No
(EDTA) was firstly added into 35 ml deionized other crystalline impurities were detected,
water, and then formed the dissolvable demonstrating the high purity of obtained Bi2S3
Bi(III)(EDTA) complex solution at 40 oC under products. The strong intensity of the XRD pattern
stirring for 10 mins. 0.15 mmol Na2SO3 and 1.8 g suggests the high crystallinity of the product. The
NaH2PO2·H2O was introduced and further stirred quality and composition of the as-prepared samples
for 5 mins. The mixed solution was then transferred were further studied by X-ray photoelectron
into a Teflon-lined stainless steel autoclave (50 ml spectroscopy (XPS) analysis. The Bi 4f7/2, Bi 4f5/2
capacity), sealed, maintained at 140 oC for 12 h, and S 2s features shown in Figure 2a and b have a
and cooled to room temperature naturally. The binding energy (BE) of 537.9, 158.0, 163.2 and
resultant black solid product was washed with 224.8 eV, respectively. The results are in agreement
deionized water and absolute alcohol several times. with the reported Bi2S3. [35], [38] In addition, taking
Then the sample was dried in a vacuum at 60 oC for into account the atomic sensitivity factors of Bi and
5 h. The powders were charged into column S, the atomic ratio of Bi/S is approximately 2 : 3
graphite dies with internal diameters of 10 mm, according to quantification of the peak areas of Bi
respectively, and then sintered in a vacuum using 4f and S 2s. No peaks of other elements are
an SPS system (DR.SINTER SPS-752, Japan) observed in the wide XPS spectrum, indicating the
under 60 MPa at 623 K for the 5 mins. The sintered high purity of the Bi2S3 product.
bulk samples were disks with a dimension ø10 The morphology and structure of Bi2S3
mm×3 mm. flower-like nanostructures were characterized by
X-ray diffraction (XRD) measurements were field emission scanning electron microscope (FE-
conducted with a D8 Advance X-ray diffractometer SEM), transmission electron microscopy (TEM)
(Bruker, Germany) using Cu-.Į UDGLDWLRQ Ȝ and selected area electron diffraction (SAED).
1.5406Å) with a scan rate of 0.2 o sí1. The Figure 3a is a typical low-magnification SEM
morphologies of the powders were observed with a image of the as-synthesized products, which clearly
Hitachi S-4800 field-emission scanning electron reveals uniform flower-like nanostructures with the
microscope (FE-SEM) at an accelerating voltage of diameter of 7-ȝP)XUWKHUREVHUYDWLRQIURPKLJK
10 kV. Transmission electron microscopy (TEM) magnification SEM image (Figure 3b) indicates that
and selected-area electron diffraction (SAED) the flower-like nanostructures is built of nanobelts
patterns were performed on an JEM-2100F (JEOL, with a thickness of ca. 100 nm and length of ca. 3.5
Japan) electron microscope with an accelerating ȝP $OO QDQREHOWV LQ D VLQJOH IORZHU-like
voltage of 200 kV. For TEM characterization, the nanostructure seem to grow from a centre.
products were ultrasonically dispersed in ethanol, Interestingly, the flower-like structure cannot be
and then the resulting suspension was dropped on a destroyed to become some discrete nanobelts even
Cu grid coated with carbon film. The Seebeck under a long time of ultrasonication, indicating that
coefficient and electrical resistivity were measured the complex architectures are actually integrated
at 300-470 K in a helium atmosphere using a but not made up of loosely aggregated nanobelts.
Seebeck coefficient /electrical resistance measuring This indicates that the Bi2S3 flower-like
system (ZEM-3 Ulvac-Riko, Japan). nanostructures were synthesized successfully by
this simple hydrothermal method.
1268


FIGURE 1
XRD pattern of Bi2S3 flower-li e nanostructures composed of nanobelts synthesi ed at 140 oC for 12 h.
FIGURE 2
XPS analysis of the Bi2S3 flower-li e nanostructures. (a) Bi 4f spectra, and (b) S 2s spectra.
1269


FIGURE 3
(a) SEM image of Bi2S3 flower-li e nanostructures. (b) high-magnification SEM image of an
individual flower-li e nanostructure. (c) TEM image of a Bi2S3 flower-li e nanostructure. (d)
SAED pattern ta en from the single nanobelt in (c).
Further insight into the microstructural details shows XRD pattern and FE-SEM images of sample
of the Bi2S3 flower-like nanostructures is provided obtained with corresponding hydrothermal
by the TEM images (Figure 3c). It clearly indicates durations of 2, 4 and 8 h. XRD results (Figure 4a)
that the flower-like nanostructures consists of some exhibit that the crystallized element Bi (JCPDS 85-
nanobelts growing out of the centre, consistent with 1330) is obtained accompanied by a minority of
the SEM observations. The SAED pattern (Figure Bi2S3. After 2 h, the obtained products are many
3d) taken from the edge of single nanobelt of this VSKHULFDO SDUWLFOHV ZLWK WKH GLDPHWHU RI FD ȝP
flower-like nanostructure shows that the nanobelt is (Figure 4b) under supersaturated solutions. Some
a well-crystallized single crystal. Those diffraction spheres even have belt-like protuberances
spots can be easily indexed to orthorhombic Bi2S3 perpendicular to the surfaces. When the reaction
crystal recorded. The calculations suggest that the time prolonged into 4 h, the obtained intermediates
lattice spacings of 3.75 Å and 7.93 Å corresponding are many undeveloped flower-like structures
to the distance space of the (101) and (110) planes, (Figure 4c). It seems that several nanobelts grow
respectively. The nanobelt is oriented with its [- out of the surface of spherical interior core. The
111] zone axis perpendicular to the substrate. nanobelts are a width of ca. 500 nm and length of
Therefore, the {110} reflections appear across the FDȝP The diffraction peak intensities of Bi2S3
width of the belt and the {211} and {101} gradually increase with time, suggesting its
reflections appear at 53.73o and 76.13o from the continuously improving crystallinity. Based on the
{110} reflections. Note that no lattice reflections above analysis, we may confirm these nanocores
appear in the SAED pattern parallel to the long axis are element Bi. As time prolonged into 8 h, more
because of the particular crystallographic zone axis nanobelts with different lengths appear (Figure 4d),
parallel to the incident electron beam. Such a and eventually the 3D Bi2S3 flower-like
similar crystal structural has also been reported for nanostructures are obtained through the Ostwald
other literature. [39] ripening process. This suggests that element Bi is
To illustrate the morphology evolution of the continuously transformed into Bi2S3 with increasing
Bi2S3 flower-like nanostructures, a series of time- time.
depended experiments were carried out. Figure 4
1270


FIGURE 4
(a)XRD patterns showing the transformation of the products obtained at different times. (b-d) SEM
images of samples obtained at various times of (b) 2 h, (c) 4 h, (d) 8 h.
Recently, the element Bi nanoparticles have But the hydrolysis rates of by-product HPO32-
been successfully prepared through using the groups in the reducing reaciton of NaH2PO2·H2O
reducing reaction between the Bi-based complex are much higher than their ionization rates, which
ions ( [Bi2(C4H2O6)2]2-) and NaH2PO2·H2O. [40] In leads to the production of a certain amount of OH -
our protocol, we propose that the Bi(III)(EDTA)3+ groups in the reaction system. These OH- groups
ions should be reduced into element Bi under the can easily neutralise a certain amount of newly-
effect of reducing agent NaH2PO2·H2O. Meanwhile, born H+ ions, and so the reducing rate of element Bi
the SO32- groups in the aqueous solution are also are decreased. This is the important reason that
reduced into the anion S2-. Lots of H+ ions as the element Bi can appear in the obtained products in
by-products are also continuously produced in these the beginning step. With further increasing the
reduction processes. These H+ ions in the presence reaction time, the OH- groups could be eliminated
of NO3- ions can further reduced elemental Bi into completely owing to the limited amount of HPO 32-
Bi3+ ions. To further clarify this reaction process in groups, and relatively low-concentration residual
our case, two contrast experiments were performed. H+ ions are dominant in reaction solution, which
)LUVWO\ WKH REWDLQHG SURGXFWV DUHQ¶W LQGH[HG LQWR leads to the slow growth process of the Bi2S3
Bi2S3 under the hydrothermal conditions of the nanoflowers in our case. All possible reactions
element Bi and Na2S are raw materials, which can process in the aqueous solution could be formulated.
indicate the element Bi cannot directly react with S
ions to form Bi2S3. Therefore, the possible route 2Bi(III)(EDTA)3+ + 3H2PO2- + 3H22 ĺ %LĻ
can only take place in the reaction between the Bi3+ 3HPO32- + 9H+
ions and S2- ions. Secondly, elemental Bi cannot be 2SO32- + 3H2PO2- ĺ62- + 3PO43- + 6H+
dissolved in the aqueous solution of other acids HPO32- + H22ĺ+2PO3- + OH-
(such as dilute HCl, dilute H2SO4) besides dilute Bi + 4H+ + NO3- ĺ%L3+ + NOĹ+ 2H2O
HNO3, which shows the NO3- ions play an 2Bi3+ + 3S2- ĺ%L2S3Ļ
important role in reduction process of element Bi.
1271


FIGURE 5
SEM images of Bi2S3 synthesi ed at (a) absence of EDTA (b) hydra ine hydrate as reducing agent.
To decipher the role of EDTA, a contrast suggest that the additives EDTA plays a key role
experiment was carried out. In the process of the via chelate action in the formation of the flower-
synthesis, the introduction of EDTA can make like nanostructures. In addition, the mild reducing
Bi(NO3)3·5H2O completely dissolve in water ability of NaH2PO2 has strong effect on the
because they can form the diffluent complex formation of 3D flower-like nanostructures. When
Bi(III)(EDTA). In the absence of EDTA, the hydrazine hydrate (N2H4·H2O) were used as the
obtained product was the element Bi and Bi2S3 reducing agent under keeping the other
nanoparticles (Figure 5a and Figure 6a). When experimental parameters, many monodisperse
Bi(NO3)3·5H2O and H2O were mixed together, Bi3+ nanobelts were obtained (Figure 5b). The XRD
reacted rapidly to form BiONO3. The BiONO3 pattern shows that the Bi2S3 (JCPDS 43-1471) is
cannot easily be reduced to element Bi with obtained in Figure 6b. These above results indicate
NaH2PO2·H2O as reductant. Meanwhile, it was the significant influence of the reducing agent on
difficult reduced into Bi3+ ions. Therefore, the the morphology of the products. N2H4 has stronger
product is gradually formed to element Bi and Bi2S3 reducing ability than NaH2PO2, which results in
nanoparticles because the reaction rate was not well faster reducing rate from SO32- ions into S2- ions. So
controlled in this process. When EDTA was added the Bi(III)(EDTA) in solution would react with
in a proper content (1 mmol), the high quality of these S2- ions quickly but not have a chance to form
flower-like nanostructures composed of nanobelts the interim Bi particles as the cores for the growth
were formed, as shown in Figure 3a. Further studies of 3D flower-like Bi2S3 nanostructures.
FIGURE 6
XRD patterns of the samples prepared at (a) absence of EDTA (b) hydra ine hydrate as reducing agent.
On the basis of the above-mentioned analysis, nucleation and aggregation step followed by a slow
the possible stepwise growth process is illustrated crystallization of primary Bi particles under
in Figure 7. Such a process involved a fast reducing agent conditions. The subsequent growth
1272


was the rapid formation of small Bi2S3 nanostructures composed of single-crystalline

nanoparticles as the crystal nuclei on the surface of nanobelts (Figure 3a) is obtained at the reaction
Bi particles. In this process, the Bi3+ ions gradually temperature of 140 oC for 12 h sintered by SPS,
generated and the crystal grew to form nanobelts which is a pressure-assisted rapid sintering process
from the crystal nuclei according to the inherent by using a pulsed DC to produce spark discharges
crystal structure. These Bi2S3 nanobelts in the Bi to heat samples under high pressure and to press the
particles surface further gradually turn into Bi2S3 powders into graphite dies. Instead of taking the
nanoflowers. This process is easy deduced if the risk of forming larger crystal grains in the long-time
reaction rate increases, the equilibrium growth conventional thermal press, the process is a much
condition is broken and difficult to form 3D flower- shorter. Figure 8a shows electrical resistivity and
like nanostructures. Using this equilibrium growth Seebeck coefficient of obtained Bi2S3 flower-like
to control the reaction rate for the formation of 3D nanostructures at different temperature (300 to 470
hierarchical nanostructures has been reported by K). The temperature dependence of the electrical
our groups. [41-43] Further work to better understand UHVLVWLYLW\ȡREWDLQHGIURPDIRXU-probe resistance
the formation of these 3D Bi2S3 flower-like PHDVXUHPHQW JLYHV D ȡ YDOXH GHFUHDVHV IURP
nanostructures is still in progress. 3.7×10-4 ȍP DW . WR î-4 ȍP DW .
The thermoelectric property of the Bi2S3 The Seebeck coefficient measurement shows an
flower-like nanostructures is shown in Figure 8. absolute value increasing trend from 1ȝ9.DW
The inset in Figure 8a shows the SEM image of . WR ȝ9. DW . 7KH QHJDWLYH
fractured surfaces for the Bi2S3 bulk samples. It is Seebeck coefficient value indicates the n-type
clear that the nanobelts structure is maintained in conduction, which was the same as that of other
the bulk body after SPS. The Bi2S3 flower-like literature result. [2]
FIGURE 7
Schematic illustration for the stepwise growth process of Bi2S3 flower-li e nanostructures composed of
nanobelts.
The calculated power factor of the Bi2S3 This trend probably due to the introduction of larger
flower-OLNH QDQRVWUXFWXUHV LV ȝ:P. 2 peaked grain boudary potential barriers that has been
at 470 K (Figure 8b), which is about higher than shown to alter electron transport drastically. This
that of the Bi2S3 nanorods to the perpendicular performance comparison further supports the
press direction for applying to the measurement or research demand for fabricating Bi2S3 flower-like
the mechanical alloying (MA)-bulk sample. [2], [44] nanostructures.
1273


FIGURE 8
(a) Seebec coefficient and thermal conductivity. Inset: SEM image of fractured surfaces for the
Bi2S3 bul obtained by SPS (b) power factors of the bul composites made of the Bi 2S3 flower-
li e nanostructures measured at a temperature range from 300 to 470 K.
CONCLUSION ACKNOWLEDGEMENTS
In summary, 3D Bi2S3 flower-like This work was supported by the National

nanostructures have been synthesized by a Natural Science Foundation of China (21301076),
convenient and environmentally friendly the Key Technologies R & D Program of Henan
hydrothermal treatment. Based on a series of Province of China (142102210642).
contrast experimental results, we propose a possible
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1275

AUTHOR INDEX
A
Ahmad, K. 1089 Ali, H. A. M. 1089
Akar, T. 1078 Allabashi, R. 1019
Akdogan, Z. 969 Aouchiche, F. 1051
Akinci, C. 1258 Ardali, Y. 1243
Akram, N. A. 1089 Arslan, H. 1134
Alarifi, S. 1234 Ashrad, F. 1089
Albayrak, O. 1258 Asikli, S. 1143
Alkan, M. 991 Aydin, O. 1143
Ali, D. 1234
Ali, H. 1234
B
Badanoiu, A. I. 1153 Benoudjit, F. 1175
Balcioglu, I. 969 Benoudjit, M. A. 1175
Baltas, H. 1068 Beyli, P. T. 991
Baraj, E. 1019 Bibi, Z. 1089
Barutcular, C. 1258 Bilen, S. 1028
Batool, A. I. 1089 Bilgic, A. 1243
Bektas, N. 1097 Bozoglu, M. 1243
Benmounah, A. 1051
C
Cakmakci, M. 1206 Coyne, C. J. 1143
Caliskan, Y. 1097 Chen, S. 959
Canal, S. B. 1125 Cui, B.-y. 981
D
Demirbas, Ö. 991 Dogan, M. 991
Derya, S. 1134
E
El Sabagh, A. 1258 Encu, T. 1125
El-Samawaty, A. E.-R. M. A. 1059, 1117 Ezer, M. 1068
G
Gelebur, A. 1078 Gündogdu, M. 1125
Genc, T. 1206 Güven, B. 969
Güleryüz 1134 Gu, W. 1267
Gültepe, N. 1028 Guo, H.-l. 981
H
Hachemi, M. 1175 Huma, Z.-e. 1089
He, J. 1267 Humayun, B. 1089
Huang, K. 1221 Hussain, A. 1089
Huang, L. 1183 Hussain, S. A. 1234
I
Ikhaddalene, S. 1051
J
Jin, Z.-j. 1221
1276
K
Kamel, N. 1051 Khan, Z. I. 1089
Kankaya, E. 1167 Khmelevtsova, L. E. 1028
Kaplan, M. 1078 Keskin, B. 1038
Kaptaner, B. 1167 Kirane-Amrani, L. 1199
Karadag, D. 1206 Kirmizi, S. 1134
Karaköy, T. 1143 Koc, M. 1258
Khan, A. 1089 Kostina, N. V. 1028
L
Li, J.-h. 1221 Liu, J. 959
Lilo, R. 1019 Liu, K. 1267
Lin, H. 959 Liu, W.-g. 981
M
Manohardas, S. 1234 Mouheb, Y. 1051
Masih, A. P 1234 Mu, D. 1183
Moslem, M. A. 1059, 1117 Muradoglu, F. 1125
Moudir, D. 1051 Mustafa, I. 1089
N
Namli, H. 991
Noorka, I. R. 1089
O
Omar, M. R. 1059 Özkan, H. 1143
Omer, I. 1153 Ozkaya, B. 1206
P
Popovici, I. C. 1153
R
Rehman, S. U. 1089 Reyhanhoglu, H. 1068
S
Sagir, N. 1078 Simsek, U. 1038
Sayed, S. R. M. 1117 Sögüt, Ö. 1068
Sazykin, I. 1028 Sönmez, A. Y. 1028
Sazykina, M. 1028 Sohail, M. 1089
Shehu, A. 1019 Soltani, N. 1199
Shen, Y.-b. 981 Soltani-Mazouni, N. 1199
Sher, M. 1089
T
Tabak, N. 1078 Topuz, B. K. 1243
Tanrikulu, A. 1258 Türkyilmaz, A. 991
Tekbas, M. 1097 Tureli, C. 1007
Temel, S. 1038 Turhan, Y. 991
Toklu, F. 1143 Turkay, H. 1206
Toptas, I. 1258
U
Ullah, M. F. 1089
V
Vasjari, M. 1019 Voicu, G. 1153
1277
W
Wang, G. 1183 Wei, D.-z. 981
Wang, W. 1183
X
Xia, Z. 1183 Xiao, L. 1267
Y
Yaman, F. B. 1206 Yildirim, M. 1258
Yang, F.-l. 1221 Yildiz, O. E. 1105
Yassin, M. A. 1059, 1117 Yilmaz, I. H. 1038
Yesilyurt, I. N. 1007 Yilmaz, S. H. 1078
Yezli-Touiker, S. 1199 You, S. 959
Yetilmezsoy, K. 1206
Z
Zafar, A. 1089 Zhou, B.-x. 1221
Zhang, H. 959 Zibouche, F. 1051
Zhang, X. 959
1278
by PSP olume 25 ± No 4/2016, pages 12 -1281 Fresenius Environmental Bulletin

SUB ECT INDEX
A
i en e ae ii 1028 Anaerobic treatment 1206
i en e el en tae tii 1028 Antioxidant enzyme activity 1125
i en e tellat 1028 Attitudes 1243
Anaerobic composting 1097 Awareness 1243
B
Base flow 1105 Bisphenol A 1167
Base-flow correlation 1105 Blue Crab 1007
Behavior 1243 Bread wheat 1258
Biochemistry 1199
C
Calline te a i 1007 CMB 1183
Captopril 1199 Collection 1143
Ca a e 1167 Common bean 1143
Catchment hydrology 969 Contaminant transport 969
Characterization 991, 1143 Control 1059
Chemical composition 1078 Cosmetic products 1068
Chromium 959 Cytotoxicity 1167
D
Disease control 1117 DNA fragmentation 1234
Drought 1258
E
Ecdysteroids 1199 e tia e niella 1199
Environment 1234 EPR 959
F
Fe 1051 Forage crops 1038
Flower-like 1267
G
Green herbage yield 1078 Growth process 1267
H
Health hazard 1068 Heavy metals 981, 1019, 1089, 1134, 1153, 1175
Heat stress 1258 Hydrothermal 1267
Heavy metal distribution 969 Hyperaccumulator 959
I
Immobilization 981 Isolated fish hepatocytes 1167
Irrigation 1258
K
Kinetics 1153
L
Leaching 1153 Liquid manure 1078
Lead stress 1125 Low-flow frequency analyzes methods 1105
Leersia hexandra Swartz 959 Low-flows 1105
in m itati im m L. 1059 Lux-biosensors 1028
Lipid peroxidase 1125
1279


M
Macro-micro element 1125 Mineral contents 1038
Membrane concentrate 1206 Modeling 969
Metal accumulation 1028 Modified-sepiolite 991
Metal ions 991 MOVE. 1 1105
MgO nanoparticles 1234 Multi-type river 1221
Mineral concentration 1143 Municipal solid waste (MSW) 1097
N
Nanostructure 1267 Nutrient loss 969
Nuclear glass 1051
O
i i m lini ±Skoric 1059 Organic waste 1097
n n m i 1167 Ovaries 1199
Ordered probit 1243 Oxidative stress 1234
Organic matter 1175 Ozone 1206
P
PCA 1183 Post-larvae (Megalopae) 1007
Physico-chemical analysis 1175 Poultry litter 981
Phytopathogens 1117 Punch® C 1059
PM2.5 1183
Q-ratio 1105
R
Radioactive waste 1051 Resirculation systems 1028
Remedial works 1221 River health assessment 1221
Reproduction 1199 Ruminants 1038
S
Seasonal characterization 1175 Sorption 1153
Seasonal variation 1183 Source apportionment 1183
Second-crop silage corn 1078 SPAD reading 1258
Seed germination 1134 Stabilization 1153
SEM 1051, 1117 Storage 1051
Selective-adsorption 991 Strawberry 1125
Settlement 1007 Sulphur chelates 981
Sewage sludge 1175 Sustainability 1078
Silicon compounds 1117 SWMM 969
Snail 1234
T
Textile wastewater 1206 Tolerance to salt 1038
Thermoelectric 1267 Toxic element 1068
Thiourea 1059 Tricalcium silicate 1153
Verbascum bombyciferum 1134

Verbascum olympicum 1134 Vetch+barley mixture 1078
W
Water contamination 1019 Wild plants 1089
1280


X
XANES 959 XRF 1068
XRD 1051
Zeta potential 991
1281


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Fresenius Environmental Bulletin is abstracted/indexed in:

Biology & Environmental Sciences, BIOSIS, CAB International, Cambridge Scientific

SYNTHESIS, CHARACTERIZATION, ELECTROKINETIC AND ADSORPTION PROPERTIES OF AN ORGANO- 991

ASSESSING CONTAMINATION IN STURGEONS GROWN IN RECIRCULATING AQUACULTURE SYSTEM BY LUX- 1028

ROLE OF SOME ANTIOXIDANTS IN SUPPRESSING OF FLAX POWDERY MILDEW 1059

ELEMENTAL COMPOSITIONS OF SOME COSMETIC PRODUCTS MARKETED IN TURKEY 1068

CASPASE-3 ACTIVATION IN CYTOTOXICITY OF ISOLATED RAINBOW TROUT (ONCORHYNCUS MYKISS) 1167

SEASONAL CHARACTERISATION OF THE LOCAL SEWAGE SLUDGE 1175

EFFECT OF PRE-OZONATION ON ANAEROBIC TREATABILITY OF MEMBRANE CONCENTRATE 1206

REDUCTION OF Cr(VI) TO Cr(III): A DETOXIFICATION

ABSTRACT metal phytoextraction is only a fraction of that

RESULTS leaves of Cr(III)- and Cr(VI)-treated L. hexandra

Treatment Cr concentration (mg kg-1 DW) Bioaccumulation coefficient a

Chemical speciation of Cr by EPR spectra. produced broad signals. We were able to

DISCUSSION AND CONCLUSIONS Cr(III) in roots by Fe(III)-reductase enzymes. Cary

MODELING NUTRIENT AND HEAVY METAL TRANSPORT

Parameter Value Description Source

Soil Data Contaminant Data

Input Source Input Source Modelled

Soil texture (% clay, sand, silt), [27] TN and TP [27] TN and TP

In the model, 30 soil samples that were ୏ൈ୔౥ ൈ஡౩౥౟ౢൈୈൈ୲ಉ ൈ୛ಊ

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According to the figure, as an expected CONCLUSION AND

ACKNOWLEDGEMENTS AGNPS integrated with a Geographic

[30] Sharpley, A.N., Smith, S.J., Berg, W.A., and

ENHANCED HEAVY METAL IMMOBILIZATION IN

ABSTRACT desirable methods are expected to treat poultry litter

Moisture/ Heavy metals background values/mg kgí1 Organic Oxides/%

Fraction Reagents Experimental conditions

Immobili ing agent Heavy metal speciation

Immobili ation mechanism analysis. LQWUDOLJDQGʌ±ʌ WUDQVLWLRQVDVVRFLDWHGZLWK1±C=S

In order to demonstrate the immobilizing according to JCPDS±ICDD numbers 19±1634, 44±

Generally, the electronic configuration of the reducing of leaching pH may result in

Column leaching tests were carried out to

[4] Guerra-Rodríguez E, Alonso J, Melgar MJ, farmland in Tianjin, China. Fresenius

De- hou Wei

SYNTHESIS, CHARACTERIZATION, ELECTROKINETIC AND

Composition Weight (%)

RESULTS AND DISCUSSION treatments or modification processes may be fully

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XRD. Figure 3 shows XRD patterns of modified by N1-[3-

reaction and consequently the equilibrium capacity

Adsorption isotherms. The analysis of the comparison of adsorption performance. q m and K

Langmuir isotherm Freundlich isotherm

[17] Kocaoba S (2009) Adsorption of Cd(II), polyurethane adhesives. Intern. J. Adhesion

SETTLEMENT OF BLUE CRAB (Callinectes sapidus)

ABSTRACT Turkey [1]. Besides, the Blue Crabs are

used Blue Crab post-larvae identification RESULTS AND DISCUSSION

CONCLUSION study on the crab post-larval settlement in our

Variation Source df Mean Square Sig

Variation Source df Mean Square Sig

REFERENCES [12] Butler IV, M. J., Tiggelaar II, J. M., Shields, J.

[47] Tankersley, R. A., Welch, J. M., Forward, R.B.

Irem Nur Yesilyurt

CONTAMINATION STATUS OF ERZENI RIVER, ALBANIA

ABSTRACT the aquatic ecosystem have recently become subject

Quality control of measurements and

Interval Plot of concentration of elements in different months

Interval Plot of Concentration of Elements in Different Stations

funding of the Austrian Development

ASSESSING CONTAMINATION IN STURGEONS GROWN IN

ABSTRACTS much more prevalent. This approach is used both

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