Sc. College Journal Res.72

1
J ournal of Patna Science College ISSN 2347- 9604

IMPACT OFTWO PESTICIDES ON SERUM FREE AMINO ACID POOL OF MICE:
ACOMPARATIVE STUDY USINGTHIN LAYER CHROMATOGRAPHY.
#Bipin Bihari Mishra, *Prakriti verma , #S.R.Padmadeo and #Kumud Ranjan Thakur
#Post Graduate Department of Biochemistry Patna University, Patna- 800005
*Post graduate Department of Zoology, Patna University, Patna- 800005
drprakriti@gmail.com, bb2mishra@gmail.com
Abstract : An attempt has been made to identifyand quantify the serum free amino acids in normal and
pesticide exposed mice. The laboratory mice (Mus musculus) were exposed to Dimethoate (o,o-
dimethyl,S-methyl-carbamoyl-methyl phosphorodithioate),an organophosphatecommonly known as rogor,
witha dose of 20 mg/kg body weight and an organochlorine, Endosulfan (6,7,8,9,10,10hexachloro-
1,5,5a,6,9,9-hexahydro-6,9-Methano-2,4,3- benzodixathiepin-3-oxide) with a doseof 2 mg/kg body
weight for 21 days. Every week blood werecollected, centrifuged and serum were separated to estimate
the free amino acids by Thin layer chromatography. Amino acids were located onthe chromatogramwith
a 0.01% ninhydrin solution in acetone. Each chromatogramreveals 4-5 fractions with a wide range of
amino acid such as aspartic acid, glutamic acid, serine, tyrosine, alanine, valine. The quantity and presence
of each amino acid depends on the doses and the exposure of pesticides. Other amino acids were present
in lower concentrationand quantitative estimation was not possible. Finding indicates that the exposure of
pesticidebrings aiterationof free amino acids content in blood.
Key words : Rogor , Endosulfan, amino acid, Thin Layer Chromatography, Mus musculus
Introduction : It is well known that pesticides are beingwidelyused in agriculture, which has many
harmful effects on living organism. Animalsin the natural environment are regularly exposed to low
concentration of these xenobiotics, which are sub-lethal. Residual amounts of organochlorines and
organophosphate pesticides have been detected in thesoil, water reservoirs, vegetables, grains and other
food products (John et al., 2003). Man is the ultimateconsumer of pesticide residues. Thesepesticide
residues in animal products and other food items ultimately get accumulated in the man especially inthe
adipose tissue, blood and. lymphoid organs. When fed to man or animalsat very low dosesdailyfor
months or years, these accumulated pesticides in body, may harmthe normal functions causing various
diseases in man and animals.The widespread use of pesticides in public health and agriculture has caused
severe environmental pollution and healthhazards including cases of severe, sub- chronic and chronic
human poisoning. . Endosulfan(6,7,8,9,10,10 hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-Methano-2,4,3-
benzodixathiepin-3-oxide) is one of the organochlorine compound used extensively for the control of
agricultural pests. Its metabolites have strong tendencies to get accumulated in different organ and tissue of
the body e.g. Adipose tissue and liver (Winter and Street,1992; Thao et al.,1993).Deleterious effect of
endosulfan have been studied by many researchers (Sinha et al., 1995;Choudhary et al.,2002) but its
effect on serumamino acid pool has not been studied. Similarly, Dimethoate (O,O-dimethyl S
methylcarbomylmethyl phosphodithioate),also known as Rogor , is one of the most important
Vol. 1, 1 - 10 [2013]
2
organophosphorus insecticides used frequently in agriculture. Many studies have been carried on the
toxicity of dimethoateon non-target animal like mice(Betrsian et al., 1995; Kamathet al., 2008). Hassan
et al. (1994) have reported several changes inserumparameter and amino acid content in rats after
chronic sub lethal doseof dimethoate. In the present investigation, an attempt has been made to examine
and compare the effect of Rogor and Endosulfan on serum free amino acid pool at different oral dose.
Materials and Methods : Test animal - Mus musculus L.(Swiss albino mice):- For the present
investigation adult Swiss albino mice Mus musculus were selected. Thirty female albino mice of same
age group and average weight of 23 gmwere procured from research laboratory; MAHAVIR CANCER
SANSTHAN, Phulwarisharif, Patna. The albino mice were housed in polypropylene cagesand maintained
in controlled temperature (27degree centigrade), humidity (0.5-10%) and light cycle. They were fed
withcereal made bread and gold mohar brand animal feed manufactured by Lipton India limited company
Delhi and water ad libitum. All the experimental mice were categorized into following groups:-
Group I Normal, Group II Endosulfan(E) treatment(E7=treated for 7days,E14 =treated
for 14 days and E21=treated for 21 days), and Group III Rogor (R) treatement (R7=treated for
7days, R14 =treated for 14 days and R21=treated for 21 days).
Selection of pesticides : Two pesticides of analytical grade were used, one Endosulfan or Endocel
(EC 35%) manufactured by Excel Industries Ltd., Ruvapari Road, Bhawanagar (Gujarat), and
second Dimethoate or Rogor (EC 30%) manufactured by ANU products, old Faridabad (Haryana).
The oral LD50 value of endosulfan for mice was calculated by standard interpolation method
which was 7.36 mg/kg body weight/day(EXTOXNET, 1996). The oral LD50 value of Rogor for mice
was calculated by standard interpolation method which was 160 mg/kg body weight (EXTOXNET,
1996). After calculating the LD
50
value of endosulfan and rogor, single sublethal dose of endosulfan (2
mg/kg body weight/day ) and rogor (20 mg/kg body weight/day ) were considered ,their stock solution
were prepared in distilled water and administrated orally by gavages method for the interval of 7, 14 &
21 days (3 weeks respectively). The controlled group of mice was given only normal saline.
After scheduled interval of exposure of 7th, 14th, and 21th day; the test animal were anesthesized,
blood sample were collected in different vials, by puncturing ocular vein with the help of sterile syringe.
Blood were extracted and refrigerated at -20
o
C in sterilized paraffin covered tubes for amino acid and
biochemical analysis. Serumwas analyzed for quantitative estimation of Total protein respectively.
Methods for Amino Acids Estimation : Amino acid profile in the blood serumof Mus musculus
were assayed using Thin layer chromatography to arrive at R
f
(Retardation factor) values of standard
amino acid calculated thus:-
R = (Wilson and Walker, 2005)
Free amino acid were determined by the method of Moore and Stein (1954) using Thin Layer
Chromatographic and quantifie d on Systronic UV-Spe ctrophotome ter (UV-U75-
Spectrophotometer) at 570 nm.
Vol. 1, 1 - 10 [2013]
3
Amino acid standard preparation : Stock solution of lysine, glutamine, methionine and other amino
acid were prepared by dissolving the proper amount of each amino acid in deionised water. Each stock
solution was then used to prepare working solution fromwhich a calibration curve was constructed.
Preparation of serum for TLC : A collected blood sample were centrifuged at 3000 rpmfor 10 min
at 4C to obtain serumthat was then deproteinized with sulphosalicylic acid and centrifuged .The
supernatant was stored at low temperature pending analysis of amino acid.
Qualitative Analysis of free amino acids of the blood serum of normal and pesticide treated
Mus musculus was done using Thin Layer chromatography (TLC).
Process : The TLC sheets were activiated by heating in an oven for 30 minute at 1000- 1200 C. A line
is drawn three centimeter above the bottomand then 8-10 approximately equal small spots applied at
2cmintervals on silica gel coated 6 of TLC aluminiumsheets No-1.05554.0007 purchased from
Merck KGa A 64271 Darmstadt, Germany Tel=49(0) 615172-2440,www.merck.de. Using a
micropipette, the prepared test solution and standard is taken and lightly dotted a small amount on each
pencil marks. The sheet is then left for sometime or wafted swiftly over a blue flame to speed evaporation.
This process is repeated 25 to 30 times, applying at the same area, to build up a concentration.
Using n - butanol : acetic acid : water in the ratio of (4 : 1 : 5 ) as elutant, a 0.1% ninhydrin
in acetone as spraying reagent. The process has been done after approximate 3-6 hrs. Each amino acid
was detected through purple colour spots by heating the sheets at 1100 C for 15 minutes and then the
Rf value were calculated. Each Chromatogramreveals 4-5 fractions with a wide range of amino acid
such as aspartic acid, glutamic acid, serine, tyrosine, alanine, methionine, valine. The spots were identified
by comparing it with the Rf value of standard amino acids.
To quantify the amount of amino acid in each spot after chromatography, the sheets were
sprayed with ninhydrin to identify the spots. The position of corresponding spot in the sheet were
scrapped off and then taken in a test tube adding 5 ml of acetone to it. Then 2 ml. of 1% ninhydrin
solution were added. The tube were placed on a water bath for 20 minutes, full colour were developed
at the end of this period. The coloured solution was transferred to measuring cylinder (10 ml) made to
volume and read on Systronic UV Spectrophotometer (UV-U75-Spectrophotometer) at 570 nmwith
the help of cuvette.. Reading was compared with reading for known solution of amino acids treated in
similar manner. Using lysine (1.13mg./ml) run for comparison.Spectrophotometric reading of amino
acid present in each spot of chromatogramwere taken to know their quantity. The quantity and presence
of each amino acids depends on the doses and the exposure of pesticides. Other amino acids were
present in lower concentration and quantitative estimation was not possible.
Methods For Biochemical Analysis : All the biochemical assessments have been done for normal/
control, Rogor and Endosulfan treated mice Mus musculus, 6 independent observation have been
taken in each groups.
Vol. 1, 1 - 10 [2013]
4
Total protein level was estimated according to the method of Lowery et al (1951) using bovine serum
albumin as standard .Blood was centrifuged to separate serumat 3000 rpm.
Proteins are the polymer of amino acids. Protein are constituents of muscle , enzyme ,hormones
and several other key functional and structural entites in the body. They are involved in the maintenance
of the normal distribution of water between blood and the tissues. Consisting mainly of albumin and
globulin the fractions vary independently and widely in disease. Increased levels are found mainly in
dehydration. Decreased levels are found mainly in malnutrition, impaired synthesis, and protein losses
as in hemorrhage or excessive protein catabolism. Estimation of total protein was done to know the
impact of alteration of serumamino acid pool on serum total protein.
Biochemical analysis was done in BT- 260 plus Semi-Automatic Chemistry Analyzer,
manufactured by Nanchang Biotech A&C Biotechnical Industry Co. China.
Six observations have been taken in each case, then Mean and Standard deviation is calculated by the
formula. The t test have been applied through standard biostatistical formula by considering mean of
normal Mus musculus as standard mean and comparing individual mean of different doses and duration
of Endosulfan and Rogor exposures to their respective control mean. After applying t test the calculated
values were referred to fishers table to see level of significance at (P<0.05) and (P<0.01).
Observation
Amino Acid Analysis : For biochemical impact of pesticide, that is, Endosulphan and Rogor on
animal amino acid analysis have been done through TLC and spectrophotometer were done and
observation was done.
Mice
Rogor Treated (dose 20 mg/kg body weight) : Valine increase more than normal in R7 and then
disappear fromserumsame is the case with glutamic acid. Tyrosine was detected in control, R7 and
R21 except R14.Aspartic acid appear in control and R7 only .Methionine, alanine and serine abruptly
appear only in R21only. (Text graph 1, 2, 3)
Endosulphan Treated (dose of 2 mg/kg body weight) : Amino acid Valine, serine and Glutamic acid
which was present in control in high quantity was absent in all the treated case thus loss of amino acid
in serum. Aspartic acid was detected in high concentration in control but it decreased fromE7 TO E21.
(Text graph1, 2, 3).
There is a few serum free amino acid in case of endosulfan treated group as compared to rogor
treated group.(Table II,III,IV)
Blood Serumanalysis For biochemical impact of Endosulfan and Rogor treatment on Mus musculus,
Total protein test of blood have been done. (Table I)
Total protein test have been done for control, Endosulfan and Rogor treated (dose and duration
dependent) Mus musculus independently. After applyingt test the calculated values were referred to
Vol. 1, 1 - 10 [2013]
5
fishers table to see level of significance at (P<0.05) and (P<0.01).Where * =significant at (P<0.05)
and ** =significant at (P<0.01).
The observed serumTotal protein of control, Endosulfan and Rogor treated Mus musculus
have been shown in the following next tables
TABLE I
Showing Total Protein in blood of control, Endosulfan and Rogor treated Mus musculus.
TABLE -II
Showing amino acids in blood serumof control, Rogor and endosulfan treated Mus musculus after
7 days, [taking lysine as standard.]
O.D =Optical density at 570 nm, Rf =Retardation factor
Vol. 1, 1 - 10 [2013]
6
TABLE -III
Showing amino acids in blood serum of control, Rogor and endosulfan treated Mus musculus after
14 days, [taking lysine as standard.]
TABLE -IV
Showing amino acids in blood serumof control, Rogor and endosulfan treated Mus musculus after 21
days, [taking lysine as standard.]
S.
No
AMI N
O
ACID
CONT ROL ROGER ENDOSULPHAN
R f
VA
LU
E
O
D Mean
S.D.
R f
VAL
UE
O
D
Co
Mean
S.D.
R f
VAL
UE
O
D Mean
S.D.
1 Alanine - - - - - - - - -
2 Asparti
c aci d
0.23 0.2
18
5.9550.013 - - 0.23 0.1
29
3.5150.011
3 Glutami
c aci d
0.32 0.1
50
4.1080.004 - - - - - -
4 Glycine - - - - - - - - -
5 Methi o
nine
- - - - - - - - -
6 Serine - - - - - - - - -
7 Tyrosine 0.47 0.1
01
2.7520.009 - - - - - -
8 Val ine 0.60 0.0
94
2.5650.009 - - - - - -
S.
No
AMIN
O
ACID
CONTROL ROGER ENDOSULPHAN
R f
VA
LU
E
O
D Mean
S.D.
R f
VAL
UE
O
D Mean
S.D.
R f
VAL
UE
O
D Mean
S.D.
1 Alanine

- - - 0.39 0.0
69
1.8610.020 - - -
2 Asparti
c acid
0.2
3
0.2
18
5.9550.013 - - - 0.25 0.0
35
2.1670.017
3 Glutami
c acid
0.3
2
0.1
50
4.1080.004 - - - - - -
4 Glycine - - - - - - - - -
5 Methio
nine
- - - 0.58 0.0
80
2.1740.013 - - -
Vol. 1, 1 - 10 [2013]
7
6 Seri ne

- - - 0.27 0.1
54
4.2110.005 - - -
7 Tyrosine 0.4
7
0.1
01
2.7520.009 0.45 0.0
85
2.3150.008 - - -
8 Vali ne

0.6
0
0.0
94
2.5650.009 - - - - - -
TEXT GRAPH 1
TEXT GRAPH 2
Vol. 1, 1 - 10 [2013]
8
TEXT GRAPH 3
Discussion & Conclusion : Mus musculus is the animal model which has been included in this study
to know the effect of same pesticide in mammal as well as in human who consume fishes as well as
other pesticide affected agro-products because mice indirectly represent human. The central theme of
present investigation is to evaluate the exact comparative toxicity of endosulfan and rogor on Mus
musculus based on their serumbiochemical test exclusively serumamino acid analysis through TLC
&Total protein of serum. Various parameters have witnessed significance changes upon different doses
and duration of pesticides treatment.
In case of mice, 4 amino acid (Val,Tyr,Asp,Glu) were detected in good concentration in serum
which goes on decreasing on 7
th
day and 14
th
day while on 21 day there were increase in concentration
of amino acid in all treatment, but there is no rise of additional amino acid. The decrease is more in case
of endosulfan than rogor treated mice. The decrease in Free Amino Acid (FAA) is due to their utilization
for new protein synthesis or for production of energy to cope up with prevailing toxic condition dueto
intoxicant induced stress (Wilson and Poe,1974;James et al,1979) while increase in the concentration
of free amino acid, as seen in rogor treated group, can result froman acceleration of protein catabolism
or an inhibition of protein synthesis, or both (Chung et al.,1954) thereby indicating that pesticide can
interfere in translation process. It is clearly observed that amino acid such as valine and glutamic acid
appear in control and short day exposure but on long exposure it was absent. Aspartic acid level also
decreases significantly on long exposure to pesticide. Some amino acid which was absent in control
appear abruptly in serumon long exposure. It was also seen that endosulfan dose is more harmful. (
Text graph 1,2,3)
In case of mice Total protein start shooting up gradually from7
th
day of treatment and reaches
its peak upto 12 days treatment on endosulphan treatment. . However, on Rogor treatment, after 7
days Total protein shows slight elevation but it is not as sharp as in endosulphan. But in all cases, it is
higher than the control, signifying toxic status of mice. The decrease trend of protein content may be
due to metabolic utilization of ketoacids to gluconeogenesis pathway for synthesis of glucose or dueto
Vol. 1, 1 - 10 [2013]
9
directing FAA for synthesis of necessary protein for maintenance of osmotic and ionic regulation in
stress condition (Schmidt 1975) while increase in protein content could stimulate protein synthesis or
detoxification enzymes at the expense of glycogen to meet additional requirement in synthesis activity to
revive the body fromtoxic stress(Susan et al.,2010),(Table I), utilizing serum free amino acids.
Regarding serumfree amino acid (FAA), similar finding in increase in FAA in fish and albino
rat during fenvalerate intoxication has been reported by Radhaiah(1988) and Lakshmi(1989).
A perfect correlation among biochemical findings have clearly shown that Endosulfan and Rogor
causes biochemical abnormalities in concerned tissues but dose of Endosulfan produce more serious
biochemical abnormalities in body thus Endosulfan produce far more toxic effect as compared to rogor.
Therefore, present investigations strongly supports the reports revealing the toxicities induced
due to pesticides and helps us to be aware of the fact that we are definitely getting exposed to the
deadly chemicals and letting thempersist in our body.
Acknowledgement : Authors are thankful to Department of Biochemistry and Department of Zoology
for providing infrastructural facilities.
References:
Betrosian,A.,Balla,M.,Kafiri,G.,Kofinas,G.,Makri,R.and Kakouri,A. (1995) : Multiple systems organ
failure fromorganophosphate poisoning.,J.Toxicol.Clin.Toxicol.,33(3),257-260 .
Choudhary,N. and Joshi, S.C (2002) : Effect of short termendosulfan on hematology and serum
analysis of male rat. Ind. J. Toxicol., 9(2), 83-87.
Hassan,A.A.,Minatogawa,Y.,Hirai,T. and Kido,R. (1994): Changes of some serumparameters
and amino acids content in rats after chronic sublethal doses of dimethoate.,Arch.Environ.Contam.
Toxicol.,27(2),256-259.
J ames, J.H, Ziparo ,V., J eppsson ,B.and Fischer, J.E. (1979) : Hyperammonemia, plasma amino acid
imbalance and blood brain aminoacid transport: A unified theory of portal systemic
encephalopathy. Lancet, 2, 772-775.
J ohn,S., Kale,M., Rathore,N. and Bhatnagar,D. (2001): Protective effect of vitamin E in dimethoate
and malathion induced oxidative stress in rat erythrocytes.,J.Nutr.Biochem.,12,500-504.
Kamath,V.Joshi,A.K.R.and Rajini,.S. (2008) : Dimethoate induced biochemical perturbation in rat
pancreas and its attenuation by cashew nut skin extract.,J.Nutr.Biochem.,90,58-65.
Lakshmi RajyamC. (1989) : Evaluation of toxic effects of a synthetic pyrethroid fenvalerate in albino
rat. Ph.D. thesis, S.V. University,Tirupati, India. .
Lowry, . H.,Rosebrough, .J., Farr, A. L. and Randall,R.J. (1951) : Protein measurement with folin-
phenol reagent. J. Biol. Chem., 193, 265.
Vol. 1, 1 - 10 [2013]
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Moore, S., Stein, W.H. (1954) : A modified ninhydrin reagent for the photometric determination of
amino acids and related compounds. The J ournal of Biological Chemistry., 211, 907 913.
Radhaiah, V. (1988) : Studies on the toxic impact of a pyrethroid insecticide fenvalerate on some
metabolic aspects and histopathology of a fresh water teleost Tilapia mossambica (Peters).
Ph.D. thesis, S.V. University, Tirupati, India.
Schmidt,E., Schmidt, F.W. (1973) : Gamma glutamyl transpeptidase. Dtsch. Med. Wschr., 98, 1572-
1577.
Sinha,N.,Narayan,R.and Saxena, D.K. (1995) : Endosulfan induced biochemical changes in the testis
of rat.,Vet. Hum. Toxicol.,(37),547-549.
Susan,T.A., Sobha, K., Veeraiah, K. and Tilak, K.S (2010) : Studies on biochemical changes in the
tssue of Labeo rohita and Cirrhinus mrigala exposed to fenvalerate technical grade.,J ournal
of Toxicology and Environmental Health science.,2(5),53-62.
Thao,V.U.D., Kawano M. and Tatsukawa, R. (1993) : Persistant organochlorine residue in soils from
tropical and sub-tropical Asian countries.,Environ.Pollut.,81,61-71.
Wilson,K. and Walker,J . (2005) : Principle and Techniques of Biochemistry and Molecular biology,
Cambridge University Press, New York,6,546
Wilson RP, Poe, W.E. (1974) : Nitrogen metabolismin channel catfish Ictalurus punctatus: Relative
pool sizes of free aminoacids and related compounds in tissues of cat fish. Comp. Biochem.
Physiol., 48, 545-556.
Winter,s.and Street, B. (1992) : Organochlorine compounds in the three steps terrestrial food
chain.,Chemosphere.,24,1706-1774.
Wu, C. and Bollman, J .L. (1954) : Effect of ethionine on the free amino acids in the rat. J .Biol.Chem.,
673-680.
Vol. 1, 1 - 10 [2013]
11
MICROBIAL PRODUCTION OF CITRIC ACID
Kumar Pranay and S.R.Padmadeo
Department of Biochemistry, Patna University, Patna-800 005
Abstract : The selected fungus was cultured for the quantitative production of citric acid by using standard
and modified Doegler and Prescott broth for 7 days. In the normal Doeglers and Prescott broth the fungus
produced highest yield of citric acid (3.252g/ml) after 6 days of incubation with corresponding biomass
(3.303g/ml). It was observed that increase in the biomass was proportional to the citric acid produced. It
wasobserved that culture containing xylose as carbon source produced highest yield of citric acid (2.650g/
ml) with biomass 0.126g/ml .The lowest production was observed in culture containing sugar beet (0.396g/
ml) as carbon source having biomass 0.788g/ml. It was observed that the culture containing yeast extract
as nitrogen source produced highest yield of citric acid (2.760g/ml) with biomass 2.210g/ml.Culture
containing casein gavethe lowest yield (0.215g/ml) of citric acid with biomass 4.169g/ml. The production
of citric acid reduced drastically on increasing the concentration of MgSO
4
.7H
2
O in the broth.
Key words : Microbial production, citric acid
Introduction : Citric acid (2-hydroxy-propane-1, 2, 3-tricarboxylic acid) derives its name fromthe Latin
word citrus, a tree whose fruit is likethe lemon. Citric acid is a tricarboxylic acid with a molecular weight
of 210.14 g/mol, which contains three carboxylic functional groups with three different values of pKa (3.1,
4.7, and 6.4). It is aprimary metabolic product formed in the tricarboxylic acid (or Krebs) cycle and is
found in small quantities in virtually all plants and animals, being isolated fromlemon juice in 1784.
Wehmer was the first to demonstrate that Citromyces (now Penicillium) accumulated citric
acid in a mediumcontaining sugar and inorganic salts (Wehmer, 1893). Since then, many organisms
have been found to accumulate citric acid: A. niger, Aspergillus awamori, Aspergillus nidulans,
Aspergillus fonsecaeus, Aspergillus luchensis, Aspergillus phoenicus, Aspergillus wentii, Aspergillus
sait oi, Aspergillus flav us, Absidiasp., Acremonium sp., Botrytis sp., Eupenicillium sp.,
Mucorpiriformis, Penicillium janthinellum, Penicillium restrictum, Talaromyces sp., Trichoderma
viride and Ustulina vulgaris (Papagianni et al.,2007).
About 99% of world production of citric acid occurs via microbial processes, which can be
carried out using surface or submerged cultures. The product is sold as an anhydrous or monohydrate
acid and about 70% of total production of 1.5 million tons per year (Lancini,2008) is used in food and
beverage industry as an acidifier or antioxidant to preserve or enhance the flavors and aromas of fruit
juices, ice cream, and marmalades. 20% is used, as such, in the pharmaceutical industry as antioxidant
to preserve vitamins, effervescent, pH corrector, blood preservative, or in the form of iron citrate as a
source of iron for the body as well as in tablets, ointments and cosmetic preparations.
The accumulation of citric acid is strongly influenced by the composition of themedium, especially
in submerged fermentation processes. It was shown that the factors mainly affecting the citric fermentation
are the type and concentration of carbon source, nitrogen and phosphate limitation, pH, aeration, oligo
elements concentration, and morphology of the producing microorganism. Certain nutrients have to be
Vol. 1, 11 - 17 [2013]
12
in excess (such as sugars, protons or oxygen), other at limiting levels (such as nitrogen and phosphate)
and others below well-established threshold values (such as trace metals, particularly manganese).
The carbon source for citric fermentation has beenthesubject of many studies, especially regarding
the use ofpolysaccharides. In general, only the sugars that are quickly assimilated by the microorganism
allow high final yield of citric acid (Mattey, 1999)). In general, sucrose is preferable to glucose (Gupta et
al., 1976, Hossain et al., 1984, Kubeic et al., 1989), as A.niger has an extracellular mycelium-bound
invertase that is active at low pH. The most widely used carbon sources inindustrial fermentations are
glucose syrups fromstarch hydrolysis, sugar beet molasses and low quality-sugarcane byproducts that, in
general, are contaminated by high levels of cations fromprevious processes. Cations usually come from
insoluble residues formed by precipitation with potassiumferrocyanide. Due to the complexity of these
pretreatments, a lot of research has been conducted using refined sugars, mainly glucose or sucrose.
The concentration of carbon source is also crucial for citric fermentation. The final yield of citric
acid increases with initial sugar concentration in batch processes or glucose feeding rate in chemostat,
while the specific growth rate has an opposite behaviour (Honecker et al., 1989, Papagianni et al.,
1999, Shu et al., 1948).
Some complex media (such as molasses) arerich in nitrogen and rarely need to be supplemented
with a nitrogen source. The highly-pure media used in laboratory scale research are usually supplemented
with ammoniumsalts, particularly ammoniumnitrate and sulfate, which in turn leads to a decrease in pH
that favors fermentation (Mattey,1999). Other sources of nitrogen such as urea and yeast/malt extract
have also been employed successfully.
The pH of the mediumis important in two stages of the process. All fermentations start from spores
and their germination requirespH >5. The absorption of ammoniaby germinating sporescauses release of
protons, thus lowering the pH and improving the production of citric acid. The low pH value during the
production phase (pH >2) reducesthe risk of contamination by other microorganisms and inhibits the
production of unwanted organic acids (gluconic and oxalic acids), which makes the product recovery easier.
A. niger requires certain trace metals for growth (Mattey, 1999). However, a limitation by
other trace elements is necessary for citric acid production (Shu et al., 1948), especially in the submerged
fermentation. The metals that should be in limiting concentrations are Zn, Mn, Fe, Cu and heavy metals
Methods: The media for isolation of fungi included potato dextrose agar and Sabourouad agar
obtained from different commercial sources (Himedia and E.Merck)10g of soil obtained from4 different
herbal drug companies of India and one prepared with locally available raw was suspended in 90ml of
sterile normal saline (0.87% w/v). The samples were shaken vigorously using orbital shaker (Certomat
WR, B.Barun) for one hour followed by serial dilution to 10
-6
, 0.1ml of selected spreaded on different
agar media (in triplicate) by spread plate technique for assessment of microbial load. The plates were
incubated at 352C for 48hrs. Colony forming units (log10 CFU)/g of each sample was calculated for
assessing the microbial load (average of three plates).
Vol. 1, 11 - 17 [2013]
13
Identification of microorganisms: The parameter for identification of predominantly obtained
morphotypes of fungi in soil samples were as described in Manual of soil fungi (Gilman, 1975).
Quantitative estimation of citric acid : For citric acid production the Doegler & Prescott broths
were inoculated with fresh culture of A.foetidus and incubated at 35C for 24, 48, 72, 96, and 120
hrs.After incubation the culture containing the citric acid was filtered with the help of filter paper. The
filtrate was collected in a flask and known volume was used estimate the citric acid quantitatively. The
biomass was collected in filter paper and was dried at 37C to measure the dry mass. 50ml of the
filtrate was mixed with equal volume of Ca (OH)
2
and kept at roomtemperature for 2 to3 hours.
Precipitate of calciumcitrate was collected and equal volume of 1N H
2
SO
4
was added in it and again
kept at room temperature for 2 to 3 hours. The supernatant, containing the citric acid was collected in
another plate and dried in hot air oven at 60 to 70 C to formthe citric acid crystals. Lastly the weight
of crystal was calculated (Fig 1).
Effect of carbon sources on production: To see the effect, six different flasks of Doeglers and
Prescott broth containing lactose, dextrose, and xylose, juice of sugar beat, sugar cane and grape as
the carbon source were prepared. Fresh cultures of the isolates were inoculated in the mediumand
incubated at 35C for 144 hours.After incubation the samples were collected to estimate the citric acid
and biomass production (Fig 2).
Effect of nitrogen sources on production: In Doeglers and Prescott broths, urea, yeast extract,
casein and peptone were added in separate flasks instead of ammoniumnitrate. The modified medium
was inoculated with 48 hrs old culture and incubated for 144 hrs at 35C.Then the samples were
collected to estimate the amount of citric acid and biomass produced (Fig 3).
Effect of trace elements on production: This was doneby preparing three different flasks of Doeglers and
Prescott brothcontaining no magnesiumsulphate, 4 times of original amount and 8 times of original amount.
The medium was inoculated with 144 hours old culture and incubated at 35C for 48 hours.
Then the samples were collected to estimate the amount of citric acid and biomass produce (Fig.4).
Results: The isolated fungus was cultured for the quantitative production of citric acid by using standard
and modified Doegler and Prescott broth for 7 days. In the normal Doeglers and Prescott broth the
fungus produced highest yield of citric acid (3.252g/ml) after 6 days of incubation with corresponding
biomass (3.303g/ml). It was observed that increase in the biomass was proportional to the citric acid
produced. The isolated fungus was cultured in modified mediumcontaining lactose, dextrose, and
xylose and fruit juices like sugar cane, sugar beat and grape. It was observed that culture containing
xylose as carbon source produced highest yield of citric acid (2.650g/ml) with biomass 0.126g/ml .The
lowest production was observed in culture containing sugar beet (0.396g/ml) as carbon source having
biomass 0.788g/ml. The effect of nitrogen source was monitored on the production of citric acid. Itwas
observed that the culture containing yeast extract as nitrogen source produced highest yield of citric acid
(2.760g/ml) with biomass 2.210g/ml. culture containing casein gave the lowest yield (0.215g/ml) of citric
Vol. 1, 11 - 17 [2013]
14
acid withbiomass 4.169g/ml. The effect of MgSO
4
.7H
2
O was monitored it was observed that production
of citric acid reduced drastically on increasing the concentration of MgSO
4
.7H
2
O in the broth.
Discussion: In this work, fungus was isolated fromsoil and agro- waste. The isolated fungus was
identified on the basis of morphological characteristics. The isolate produced highest amount of citric
acid after 144 hrs of incubation. Reduction was observed in the citric acid production as the incubation
time increased, this may be due to the over growth of myceliumwhich resulted in increased viscosityof
the medium(Mattey and Allan, 1990). Here, the influence of carbon and nitrogen sources on the citric
acid by the isolates was investigated. It was found that citric acid production varies greatly among
different carbon and nitrogen sources (Ikram- ulhaqet al, 2002).The accumulation of large amounts of
citrate by the filamentous fungus A.foetidus is known to depend on Mn
2+
ion(Rohr et al., 1993).
Our work also supports the hypothesis that relief to citrate inhibition of phosphofructokinase is
a major event related to manganese deficiency stimulation of acidogenesis in A.foetidus. The citric acid
content of commercially available lemonade and other juice products vary widely (Kristina l.Penniston
et al., 2009) and similar results were obtained.
Conclusion: Citric acid production shows great variation under different growth conditions with varying
carbon and nitrogen sources. Biomass production is also variable and the production is influenced by
trace elements.
Fig.1. Citric Acid and Biomass produced at different time intervals
Vol. 1, 11 - 17 [2013]
15
Fig.2. Effect of different carbon sources on citric acid and biomass production
Fig.3. Effect of different nitrogen sources on citric acid and biomass production
Vol. 1, 11 - 17 [2013]
16
Fig.4. Effect of Magnesium Sulphate on citric acid and biomass production
References:
Gupta, JK, Heding LG and J orgensen OB (1976). Effect of sugars, hydrogen ion concentration and
ammonium nitrate on the formation of citric acid by Aspergillus niger.
ActaMicrobiologicaAcademiaeHungaricae23: 6367.
Haq I Kram, Ali Sikander, Qadeer MA and Iqbal J aved(2002).Citric acid fermentation by mutant
strain of Aspergillus niger GCHC-7 using Molasses based medium.Electronic J ournal of
Biotechnology5:121-125.
Honecker, S, Bisping B, Yang Z and RehmHJ (1989). Influence of sucrose concentration and
phosphate limitation on citric acid production by immobilized cells of Aspergillus niger. Applied
Microbiology and Biotechnology31: 1724.
Hossain M, Brooks J D and Maddox IS (1984). The effect of the sugar source on citric acid production
by Aspergillus niger. Applied Microbiology and Biotechnology19:393 397.
Kubicek C.P, Rhr M (1989).Citric acid fermentation. Critical Reviews in Biotechnology4: 331373.
Lancini, G (2008). Parte I Lusoindustrialed eimicrorganismi. Storia e campi di applicazione. In:
Donadio, S.; Marino, G. (eds.). Biotecnologie Microbiche, Casa Editrice Ambrosiana Milan,
P: 5-35.
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Mattey M and Allan A (1990).Glycogen accumulation in Aspergillus niger. Transient Biochemical
Solicitis 8:1020-22.
Mattey M (1992). The production of organic acids.Critical Reviews in Biotechnology12: 87132.
Papagianni M (2007). Advances in citric acid fermentation by Aspergillus niger: Biochemical aspects,
membrane transport and modelling. Biotechnology Advances25: 244-263.
Papagianni M, Mattey M, Berovic M and Kristiansen B (1999). Aspergillus nigermorphology and
citric acid production in submerged batch fermentation: effects of culture pH, phosphate and
manganese levels. Food Technology and Biotechnology37: 165171.
Papagianni M, Mattey M, Kristiansen B (1999). Hyphal vacuolation and fragmentation in batch and
fed-batch culture of Aspergillus niger and its relation to citric acid production. Process
Biochemistry35: 359366.
Papagianni M, Mattey M, Kristiansen B (1999). The influence ofglucose concentration on citric acid
production and morphology of Aspergillus niger in batch and fed-batch culture. Enzymeand
Microbial Technology25: 710717.
Penniston L Kristine, Nakada Y Stephen, Holmes P Assimo and G Dean(2009). Quantitative Assessment
of Lemon J uice, Lime Juice and Commercially available Fruit Juice Products.J ournal of
Endurology22:567-570.
RhrM, Kubicek CP, Zehentgruber O and Orthofer R (1993). Accumulation and partial reconsumption
of polyols during citric acid fermentation by Aspergillus niger. Applied Microbiology and
Biotechnology27:235239.
Shu, P and J ohnson MJ (1948). Citric acid production by submerged fermentation with Aspergillus
niger. Industrial & Engineering Chemistry 40:12021205.
Shu, P and Johnson MJ (1948). The interdependence of mediumconstituents in citric acid production
by submerged fermentation. J ournal of Bacteriology54: 161167.
Wehmer C (1893). Note sur la fermentation.9:728.
ATCC 9142 froma treated ethanol fermentation co-product using solidstatefermentation. Letters in
Applied Microbiology48: 639-644.
Vol. 1, 11 - 17 [2013]
18
19
J ournal of Patna Science College ISSN 2347- 9604 Vol. 1, 19 - 36 [2013]
BIOMASS AND NUTRIENT DYNAMICS OF FREE FLOATING
MACROPHYTES OF BARAILA WETLAND, VAISHALI
Shardendu*
1
, D. Sayantan
2
and S. R. Padmadeo
3
*Corresponding Author
1. Associate Professor, Laboratory of Environment and Biotechnology, Department of Botany, Patna
Science College, Patna University, Patna.
Email: shardendu77@rediffmail.com; Contact No. 9473240391
2. Junior Research Fellow (U.G.C.), Laboratory of Environment and Biotechnology, Department of
Botany, Patna University, Patna.
Email: sayantan.phd@hotmail.com
3. Professor and Head, Department of Biochemistry, Patna University, Patna.
Abstract : The present study reports the variation of biomass and nutrient concentration of
free floating aquatic macrophytes like Eichornia crassipes, Lemna minor, Azolla pinnata and
Utricularia flex uosa at monthly intervals for one year (February 2010 February 2011), and
variation their seasonal and annual net primary productivity in Baraila wetland (2576N and
8556E), Vaishali district, Bihar, India. Since, E. crassipes was present in the wetland throughout
the year, it constituted 51.8% of total annual biomass. L. minor was also reported throughout
the year with maximum biomass of 2.360.5 g m
-2
. Among the above species, E. crassipes was
recorded with the highest net primary productivity (662.07 g m
-2
season
-1
) in the rainy months
of 2010, followed by U. flexuosa (112.8 g m
-2
season
-1
) in the same month. The higher amount
of N and K were determined in L. minor and A. pinnata and the order of fall of nutrient
concentration was N >K>Ca>P>Mg>Na. There was little change in the order of nutrient
concentration of E. crassipes, and higher amount of K and Ca was measured, which gradually
decreased in the order of K>Ca>N>Mg>Na>P. In U. flexuosa, the order of decrease in the
nutrient concentration was followed as N>K>Na>Ca>Mg>P. Variation in the change of nutrients
and biomass were justified statistically by ANOVA and the relationship between accumulation
of nutrients in plant tissues with water nutrients and biomass was analysed by the two factor
Regression Analysis.
Key Words: biomass, primary productivity, nutrients, free floating aquatic macrophytes.
1. Introduction : Plants harvest solar energy in turn, involved in production of plant biomass. Aquatic
ecosystems are the best sites for studying the energy flow through primary producers (Shardendu and
Ambasht, 1991). Among primary producers, macrophytic communities are more productive per unit
area when compared with the phytoplankton communities (Westlake, 1963) but aquatic macrophytes
20
may be insignificant in the deep water lakes. They show luxuriant growth in water bodies of shallow
basin (Westlake, 1965) and belong to most productive biotopes on earth (Wetzel, 1975a). The aquatic
macrophytes may contribute to primary production by detritus formation (Adams and McCracken,
1974), actingas nutrient source (Carignan and Kalff, 1980) and serving as substrate for other organisms
such as periphytic algae, bacteria and macrofauna.
The productivity of aquatic macrophytes in turn depends on the available nutrients of
growing medium (Irfan and Shardendu, 2009; Shardendu and Ambasht, 1991). Different nutrient
elements and heavy metals often become the limiting factors affecting the aquatic ecosystem
functioning (Shardendu et al., 2003; Azaizeh et al., 2006; Sayantan and Shardendu, 2013). The
emergents differ from other aquatic macrophytes by obtaining their nutrients almost completely
from the soil. Nutrient dynamics of emergent macrophytes have been well studied by various
researchers (Westlake, 1965; Sahai and Sinha, 1970; Shardendu and Ambasht, 1991; Irfan and
Shardendu, 2009). Variation in the tissue nutrient levels have been well attributed to additive
effects of seasonal trends.
This study has been performed to assess the effect of season on the biomass accumulation,
primary production and nutrient element composition of the floating aquatic macrophytes Eichornia
crassipes, Lemna minor, Azolla pinnata and Utricularia flexuosa, growing in the Baraila wetland,
situated in Vaishali district of Bihar, India.
2. Materials and Methods
2.1 Site Description : Sampling was done fromBaraila Chaur (wetland) of Vaishali district of Bihar,
India, situated on 2576N and 8556E. Temperature varies between 30.81C and 5C with average
annual rainfall of 1168 mm. The wetland spreads over about 24 to 26 miles which remains covered with
water in the rainy season. Climatically this region comes into the Sub-humid transition climate belt.
(Misra, 2007).
2.2 Plant Sampling, Biomass Estimation and Calculation of Primary Productivity : The wetland
was divided into 5 segments and from each segment triplicates of 4 free floating aquatic macrophyte
species namely Eichhornia crassipes, Lemna minor, Azolla pinnata and Utricularia flexuosa
were sampled to obtain a total of each 15 individual species at monthly intervals between February
2009 and February 2010.
Standing crop biomass was measured by harvest method, as described by Odum(1956).
Samples were collected from25 X 25 cmarea and brought to the laboratory in acid rinsed polyethylene
bags. Plants were thoroughly washed under running tap water to remove dirt and dried in oven at 80C
Vol. 1, 19 - 36 [2013]
21
for 48 h to a constant weight. The biomass was calculated on dry weight basis. The biomass data were
utilized for the calculation of primary production. Seasonal positive monthly changes of standing crop
biomass were added to calculate seasonal net productivity.
2.3 Analysis of Plant Nutrients : The harvested plant samples were dried as described in Section
2.2, powdered in stainless steel grinder and sieved through 2 mmstainless steel mesh to obtain sub-
samples. The sub-samples (0.5 1.0 g) were divided into 3 portions for analysis of various nutrients.
First portionwas used for analysis of potassium, calcium, and sodium. The sub-samples were converted
into ash in muffle furnace at 480C for 12 h after adding 2 ml saturated solution of magnesiumnitrate (to
prevent phosphorus volatilization) and dissolved in 1 N HCl. The solution was then used to estimate K,
Ca and Na using Flame Photometer (Systronics).
The second portion of ground sample was utilized for analysis of total nitrogen and total
phosphorus. The powdered samples were subjected to Persulfate Digestion (Langer and Hendrix,
1982; Ebina et al., 1983), to convert all forms of phosphorus and nitrogen into Dissolved Inorganic
Phosphorus (DIP) and nitrate-nitrogen respectively. DIP was quantified by the Stannous Chloride
method, described by Walinga et al., (1995) and nitrate-nitrogen was determined by Phenol-di-sulfonic
Acid method (APHA, 2005).
The third portion of sub-sample was estimated for magnesiumby wet ashing method described
by Misra (1968).
2.4 Statistical Analysis : Statistical analyses were performed using ANOVA and Multiple Regression
Analysis. ANOVA was done to test for between-months variation in plant nutrients of Eichornia
crassipes, Lemna minor, Azolla pinnata and Utricularia flexuosa and Multiple Regression Analysis
was done to test the dependency of plant nutrient (PN) on nutrients in water (NW) and plant biomass
(PB). Statistics was performed using software STATISTICA v5.
3. Results
3.1 Estimation of Biomass : Free floating zone consists of four species i.e. Eichhornia crassipes,
Lemna minor, Azolla pinnata andUtricularia flexuosa. The first two were present throughout the
year, whereas Azolla pinnata was absent in April, May and J une and Utricularia flexuosa was not
recorded in May. Eichhornia crassipes constituted more than half (51.2%) of the total biomass of
the pond, and its maximumbiomass was 1190.7089.04 g m
-2
in October and minimumof
531.443.07 g m
-2
in May (Fig 1). Lemna minor was another species of this zone which had
2.360.50 g m-2 in September, afterwards there was a decrease in biomass and minimum of
Vol. 1, 19 - 36 [2013]
22
0.560.13 was recorded in May (Fig 3). Azolla pinnata showed 0.910.143 g m
-2
in August
(absent in summer) and after that there was gradual increase in biomass of the species and in February
it had 4.590.679 g m
-2
(Fig 2).
Figure 1. Mean monthly variation of biomass of Eichhornia crassipes in Baraila wetland.
Figure 2. . Mean monthly variation of biomass of Azolla pinnata in Baraila wetland.
Vol. 1, 19 - 36 [2013]
23
The fourth species of this zone was Utricularia flexuosa which was absent in May. It started
germinating in J une (4.330.56 g m
-2
). There was a gradual increase in the biomass to reach a maximum
value of 144.3324.37 g m
-2
in November (Fig 4).
Total biomass of free floating zone was 241A.38 g m
-2
. The fluctuation during different months
showed that J une (319.49 g m
-2
) and October (315.76 g m
-2
) were the most favourable period for
growth while April (173.29 g m
-2
) and J anuary (173.66 g m
-2
) were the least. The percentage contribution
of the constituent species to this zone was 91.38 for Eichhornia crassipes, 0.14 for Lemna minor,
0.21 for Azolla pinnata and 8.27 for Utricularia flexuosa.
The annual average biomass for Eichhornia crassipes was882.32 g m-2, while Lemna minor,
Azolla pinnata and Utricularia flexuosa added only 0.08%, 0.12% and 4.63% to the total biomass
respectively, having their respective annual average biomass of 1/31 g m-2, 2.04 g m-2 and 79.85 g m-2.
Figure 3. Mean monthly variation of biomass of Lemna minor in Baraila wetland.
Figure 4. Mean monthly variation of biomass of Utricularia flexuosa in Baraila wetland.
Vol. 1, 19 - 36 [2013]
24
Winter season was most suitable for the growth of all the four species of this zone while summer was the
least. So the total addition in biomass to this zone in winter was 283.14 g m-2, in rainy was 266.12 g m-
2 and in summer 174.88 g m-2 which was 39.1%, 36.75% and 24.15% respectively (Table 1).
Table 1. Variation in standing crop biomass in plants of Baraila wetland between 2010 2011.
3.2 Primary production
Primary Productivity : Free floating zone was represented by four members in the wetland; they
were Eichhornia crassipes, Lemna minor, Azolla pinnata and Utricularia flexuosa. Among them
E. crassipes was the dominant member whose rate of productivity in rainy season was 5.427 g m-2
day-1 which was the maximumrate of production in any zone or any member of the pond. This was
followed by winter season where the rate was 0.412 g m-2 day-1 (Table 2). There was no increase in
biomass in summer season. This species was responsible for approximately fifty percent of total
production in the pond. This was followed by L. minor, which was among the few species which
produced in all the three seasons of the year, though the rate of production was very low. Highest rate
of production was 0.014 g m-2 day-1 in rainy season followed by 0.0028 g m-2 day-1 in winter
season and 0.0006 g m-2 day-1 in summer season. A. pinnata was absent in summer season. Its
highest rate of production of 0.023 g m-2 day-1 was obtained in winter season followed by 0.016 g m-
2 day-1 in rainy season. The fourth species of free floating zone was Utricularia flexuosa which was
not only perennial but showed positive biomass differences in all the three seasons of the year. The
maximumrate of production of 0.925 g m-2 day-1was noted in summer season. The value (0.659 g m-
2 day-1) for winter season was intermediate between rainy and summer.
The free floating zone produced with rapid rate of 6.382 g m-2 day-1 in rainy season which
was maximumfor any zone and any season in the wetland. This was followed by winter 0.659 g m-2
day-1and the lowest rate of 0.037 g m-2 day-1was in summer season (Table 2).
The annual production of members of free floating zone was calculated by two methods in both
of themsimilar results were observed. The annual production of E. crassipes was 719.80 g m-2 day-
1which was highest addition for a species to this zone and to wetland as well. This was followed by U.
Factors Spec ies Summer Rai ny Wint er
%
contrib ution Total Standin g
% c ont ri bution
of

of species
to zone

crop bi omass of
speci es/zone

Standing crop
bi omass

Total
standi ng
1. Ei chhornia
crassi pe s 674.13 963.56 1009.28 91.88 882.32 51.2
biomass 2. Lemna mi nor 0.678 1.61 1.63 0.14 1.31 0.08

3. Azoll a
pinnata 1.29 1.31 3.53 0.21 2.04 0.12

4. Ut ricul ari a
f le xuosa 23.44 98 118.11 8.27 79.85 4.63
%
contri buti on
of the season to 24.15 36.75 39.1
thi s zone
Vol. 1, 19 - 36 [2013]
25
flexuosa 144.33 g m-2 day-1, A. pinnata 4.78 g m-2 day-1and lowest 2.14 g m-2 day-1was for L.
minor.
Table 2. Seasonal productivity of plants of Baraila wetland from2010-2011 to 2010 by positive change
method
3.3 Nutrient Composition in the Tissue of Eleocharis plantaginea : There were four species in
free floating zone, Eichhornia crassipes, Lemna minor, Azolla pinnata and Utricularia flexuosa,
in which first two were present through out the year, U. flexuosa was absent in May and A. pinnata in
April, May and J une. In E. crassipes nitrogen ranged from9.82 to 17.72 mg g
-1
, phosphorus 0.78 to
1.68 mg g
-1
, potassium20.85 to 35.51 mg g
-1
, calcium8.10 to 16.08 mg g
-1
, magnesium4.35 to 8.75
mg g
-1
and sodium1.73 to 3.28 mg g
-1
. The sequence falls in the order of potassium>calcium>
nitrogen >magnesium>sodium>phosphorus. Range of elemental content in L. minor was 18.57 to
29.57 mg g
-1
for potassium, 10.43 to 19.55 mg g
-1
for calcium2.75 to 4.11 mg g
-1
for magnesium and
1.82 to 2.93 mg g
-1
for sodium. The decrease in the mean annual content of elements were nitrogen >
potassium>calcium>phosphorus >magnesium >sodium. In A. pinnata the elemental concentration
varied from26.41 to 37.91 mg g
-1
for nitrogen, 1.22 to 2.57 mg g
-1
for phosphorus, 9.35 to 14.63 mg
g
-1
for potassium, 7.58 to 11.41 mg g
-1
for calcium, 3.59 to 6.08 mg g
-1
for magnesiumand 6.58 to 9.88
mg g
-1
for sodium. The order of fall of different elements was the same as in L. minor. U. flexuosa is the
only insectivorous species present in the study ponds in which the nitrogen ranged from 14.64 to 22.19
mg g
-1
, phosphorus from0.71 to 1.25 mg g
-1
, potassium11.55 to 18.21 mg g
-1
, calciumfrom1.51 to
2.78 mg g
-1
, magnesium0.98 to 2.67 mg g
-1
and sodium7.41 to 13.75 mg g
-1
. The order of fall of
different elements were nitrogen >potassium>sodium>calcium>magnesium>phosphorus. The
mean annual concentration of different elements in all the four species followed variable trends. The
mean annual nitrogen content were maximum30.57mg g
-1
for Azolla pinnata, 22.54 mg g
-1
for L.
minor, 18.24 mg g
-1
for U. flexuosa and 13.64 mg g
-1
for E. crassipes. The mean annual phosphorus
content decreased in the order of 6.55 mg g
-1
for L. minor, 1.78 mg g
-1
for A. pinnata, 1.11 mg g
-1
for
E. crassipes and 9.58 mg g
-1
for U. flexuosa. The potassiumcontent decreased in order 27.15 mg g
-
Summer Rainy Winter
16 Feb. - 15 J une 16 J une - 15 Oct. 16 Oct. - 15 Feb.
SPECIES Productivity Production Productivity Production Productivity Production

g m-
2
season-
1

g m
-2
season
-1
g m
-2
season
-1

g m-2
season
-1
g m
-2
season
-1
g m
-2
season
-1

1. Eichhornia
crassipes - - 662.07 5.43 50.73 0.412
2. Lemna
mi nor 0.07 0.0006 1.73 0.014 0.34 0.003
3. Azolla
pinnata - - 2.004 0.016 2.78 0.023
4. Utricularia
flexuosa 4.33 0.037 112.8 0.925 27.2 0.221

Vol. 1, 19 - 36 [2013]
26
1
for E. crassipes, 17.67 mg g
-1
for L. minor, 14.65 mg g
-1
for U. flexuosa and 11.76 mg g
-1
for A.
pinnata. The maximumannual mean for calcium was 15.58 mg g
-1
for L. minor followed by 11.66 mg
g
-1
for E. crassipes, 9.79 mg g
-1
for A. pinnata and minimum 2.15 mg g
-1
for U. flexuosa. The content
of magnesiumwhich is also the index of production was maximum6.21 mg g
-1
for E. crassipes followed
by 5.32 mg g
-1
for A. pinnata, 3.37 mg g
-1
for L. minor and minimum1.73 mg g
-1
for U. flexuosa.
Sodiumcontent varied from 10.28 mg g
-1
in U. flexuosa, 7.88 mg g
-1
for A. pinnata, 2.60 for E.
crassipes and 2.43 mg g
-1
for L. minor.
Figure 5. Mean monthly variation of Mg, Na and P in Eichhornia crassipes of Baraila
wetland.
Figure 6. Mean monthly variation of N, K and Ca in Eichhornia crassipes of Baraila wetland.
Vol. 1, 19 - 36 [2013]
27
Monthly changes in the amounts of elements of E. crassipes had a different trend.
The maximum nitrogen was a fall in the concentration and the minimum of 9.821.14 mg g
-
1
was noted in November. Seasonally, summer months April (15.591.24 mg g
-1
), May
(16.291.27 mg g
-1
) and J une (17.711.41 mg g
-1
) had higher values than the months of
rainy season, J uly (16.192.02 mg g
-1
), August (14.62 mg g
-1
) and September (13.561.75
mg g
-1
). Winter months October (12.121.87 mg g
-1
), December (11.240.71 mg g
-1
) and
J anuary (11.950.99 mg g
-1
), had lower values than both the summer and rainy months
(Fig. 6). Monthly variation in phosphorus content ranged from 1.680.12 mg g
-1
in J une to
0.780.15 mg g
-1
in October, though the very close to minimum value (0.790.15 mg g
-1
)
was observed in January 2011. Concentration of phosphorus in other months were 1.210.12
mg g
-1
for March, 1.320.18 mg g
-1
for April, 1.440.25 mg g
-1
for May and these were
higher than the contents of 1.250.14 mg g
-1
in J uly, 1.020.13 mg g
-1
in August and
0.820.11 mg g
-1
in September. In February of 1982 (1.070.12 mg g
-1
) and 2011
(1.050.09 mg g
-1
) the concentration values were very close followed by December
(1.050.15 mg g
-1
) and November (0.920.06 mg g
-1
) (Fig. 5). Maximum potassium content
(35.512.98 mg g
-1
) of E. crassipes was recorded in J une followed by fall in the content
which reached the minimum values 20.852.54 mg g
-1
in November and 20.854.40 mg g
-
1
in December. Rainy season months of J uly (32.351.96 mg g
-1
), August (30.152.11 mg
g
-1
) and September (26.672.33 mg g
-1
) had slightly lower concentration of potassium than
the summer months March (27.852.35 mg g
-1
), April (30.631.64 mg g
-1
) and May
(33.751.94 mg g
-1
). February of 1982 (24.022.02 mg g
-1
) and of 1983 (24.021.60 mg
g
-1
) had the same content followed by J anuary (21.691.75 mg g
-1
), (Fig. 6). Regarding
calciumcontent November (14.521.70 mg g
-1
), December (16.081.56 mg g
-1
) and
J anuary (14.291.85 mg g
-1
) together constituted higher content than the J uly (10.821.49
mg g
-1
), August (11.460.93 mg g
-1
) and September (12.481.24 mg g
-1
). The minimum
calcium content of 8.100.81 mg g
-1
was in May followed 9.321.17 mg g
-1
in J une (Fig.
6). Magnesium content of E. crassipes was quite low (4.620.84 mg g
-1
) in May, after
which it increased to October (8.750.97 mg g
-1
), then again decreased. Magnesiumcontent
reached the minimum (4.350.67 mg g
-1
) in J anuary 2011. The values of winter season,
6.980.87 mg g
-1
in November, 5.680.77 mg g
-1
in December and 5.380.55 mg g
-1
in
February 2011 were lower than in the months of rainy season i.e. 6.370.60 mg g
-1
in J uly,
7.750.87 mg g
-1
in August and 7.920.68 mg g
-1
in September as well as of the two
summer months, April (5.120.56 mg g
-1
) and J une (5.510.56 mg g
-1
) (Fig. 5). Sodium
content of E. crassipes had almost two equal peaks, one (3.250.38 mg g
-1
) in J une and
another (3.280.23 mg g
-1
) in J anuary. After the first peak, there was gradual fall in the
content, which reached to the minimum (1.730.20 mg g
-1
) in September, which then
enhanced. Seasonally, winter had higher content than the rainy and summer months (Fig.
Vol. 1, 19 - 36 [2013]
28
5). Monthly variation of nitrogen, phosphorus, potassium, magnesium and sodium were
highly significant (p<0.005) whereas calcium changed at p<0.025 (Table 3).
Figure 9 Mean monthly variation of Mg, Na and P in Lemna minor of Baraila wetland.
Figure10. Mean monthly variation of N, K and Ca in Azolla pinnata of Baraila wetland.
Vol. 1, 19 - 36 [2013]
29
L. minor was the another member of the free-floating zone. The maximumnitrogen content of
29.572.19 mg g
-1
was found in J une and the minimumof 18.572.77 mg g
-1
in October. April (21.022.38
mg g
-1
), May (24.631.88 mgg
-1
) and July (25.502.20 mg g
-1
) together constituted the monthsin which
higher nitrogen values were obtained than in November (19.472.26 mg g
-1
), December (20.631.95
mg g
-1
) and J anuary (21.071.98 mg g
-1
) (Fig. 10). Phosphorus content also varied in a similar way. The
highest of 10.521.10 mg g
-1
was recorded in May and the minimumof 4.520.93 mg g
-1
in October and
another lower value close to this of 4.590.75 mg g
-1
was observed in J anuary. Seasonally, higher
concentration was recorded in the summer than the rainy season (Fig. 9). In contrast to nitrogen and
phosphorus, the high contents in potassium of 21.021.84 mg g
-1
were recorded in February 2011 and in
the summer months of May (19.492.17 mgg
-1
) and June (18.462.70 mgg
-1
). The minimum14.501.62
mg g
-1
) of potassiumwas recorded for September followed by J uly (17.451.80 mg g
-1
) and J une
(16.381.81 mg g
-1
). Calciumcontent of L. minor was minimum(10.431.15 mg g
-1
) inMay, which
enhanced thereafter upto December (19.551.84 mg g
-1
). In winter calciumwas higher in L. minor than
in other seasons (Fig. 10). The trend of the monthly variation of magnesiumwas different than of other
elements. Maximumof 4.110.38 mgg
-1
was in September, which got reduced upto J anuary 2011
-1
). In April (2.850.34 mg g
-1
), May (2.950.41 mg g
-1 -
1
) values were significantly lower than in October (3.750.38 mg g
-1
), November (3.310.28 mg g
-1
) and
December(3.120.31 mgg
-1
). Variation of sodiumcontent of L. minor ranged from 2.930.32 mg g
-1
in
J uly to 1.820.30 mgg
-1
in October. Concentration in other months were2.730.38 mg g
-1
in May,
2.430.26 mg g
-1
in June, 2.670.44 mg g
-1
in August and 2.220.24 mgg
-1
in September (Fig. 9).
Phosphorus, calcium changed significantly p<0.005 between months whereasmagnesiumat p<0.025,
potassiump<0.100 and nitrogenand sodiump<0.250 (Table3).
Figure 7 Mean monthly variation of Mg, Na and P in Azolla pinnata of Baraila wetland.
Vol. 1, 19 - 36 [2013]
30
Figure8. Mean monthly variation of N, K and Ca in Azolla pinnata of Baraila wetland.
The other species of this zone was Azolla pinnata, containing an endophyte of Anabaena
azollae, a nitrogen fixing blue green algae. The nitrogen content ranged frommaximum 37.911.93 mg
g
-1
in J uly to 26.411.36 mg g
-1
in December. Among other months in August (35.282.37 mg g
-1
),
September (32.551.33 mg g
-1
) and October (30.152.62 mg g
-1
) a comparatively higher nitrogen
content was recorded than in J anuary (27.011.91 mg g
-1
) and February 2010 (29.632.12 mg g
-1
)
(Fig. 8). Phosphorus content was also highest (2.570.21 mg g
-1
) in J uly and the same was true for
potassium(14.631.79 mg g
-1
) in July. The lowest values were 1.220.09 mg g
-1
of phosphorus in
December (Fig. 7), and 9.351.24 mg g
-1
for potassiumin October (Fig. 8). For both these elements
the rainy season had higher values than winter. In contrast to N, P and K and the minimum calcium
concentration of 7.580.81 mg g
-1
was in August, followed by 8.210.63 mg g
-1
in September and
9.690.93 mg g
-1
in October. The maximum content 11.410.73 mg g
-1
was noted in January 2011
followed by February 2010 and 2011 (11.290.67 mg g
-1
and 11.021.44 mg g
-1
) (Fig. 7). The
amount of magnesium concentration was 5.780.55 mg g
-1
in November, 5.920.79 mg g
-1
inDecember
and 5.990.8 mg g
-1
in J anuary which was higher than 3.590.46 mg g
-1
in J uly, 4.110.34 mg g
-1
in
August and 4.510.49 mg g
-1
in September. The maximum of 9.880.75 mg g
-1
sodiumcontent of A.
pinnata was in July, after which it decreased to the minimumof 6.580.80 mg g
-1
in December, and it
fluctuated in January and February. In March very close (6.780.37 mg g
-1
) to the lowest value was
noted (Fig. 7). Statistically, variation of elemental concentration in A. pinnata between the months
were significant for nitrogen at p<0.025, phosphorus at p<0.005, calciumand sodiumat p<0.1 and for
magnesium at p<0.25 level, whereas the variation of potassiumwas not significant (Table 3).
Vol. 1, 19 - 36 [2013]
31
Figure 11 Mean monthly variation of Mg, Na and P in Utricularia flexuosa of Baraila wetland.
Figure12. Mean monthly variation of N, K and Ca in Utricularia flexuosa of Baraila wetland.
Vol. 1, 19 - 36 [2013]
32
The fourth and last species of this zone Utricularia flexuosa was present throughout the year
except in May. The variation of nitrogen of this insectivorous species was between 22.190.89 mg g
-
1
in July to 14.641.53 mg g
-1
in concentration of nitrogen than the rainy season months while in winter
months concentrations were the least (Fig. 11). The maximum amount of phosphorus (1.250.14 mg g
-
1
) was in J uly, after which the concentration fell down to the minimumof 0.710.17 mg g
-1
in November
and then there were ups and downs (Fig. 12). In contrast to nitrogen and phosphorus, the potassium
content was least (11.550.88 mg g
-1
) in June followed by the enhancement in the content in rainy and
winter months and maximumof 18.211.13 mg g
-1
was noted in J anuary 2011. Again it decreased. The
trend of calciumcontent of U. flexuosa was almost similar to that of potassiumhaving highest 2.780.24
mg g
-1
in J anuary 2011 and lowest 1.510.16 mg g
-1
in September. In the winter months amount of
calciumwas higher than in the other two seasons (Fig. 11). Magnesium and sodium content had almost
an opposite pattern. The minimumvalue of 0.980.14 mg g
-1
of magnesiumand maximum value of
magnesium (2.670.22 mg g
-1
) was recorded in November while lowest value of sodium (7.410.99
mg g
-1
) was found in October (Fig. 12). Statistically, calciummagnesiumand sodiumchanged between
the months highly significantly (p<0.005), whereas variation of nitrogen was significant at p<0.025,
phosphorus p<0.25 and potassium p<0.1 (Table 3).
Table 3. ANOVA for plant - nutrients plants of of free floating zone.
Nutrients Sources of Variation d.f. S.S. M.S. F P<
E. c r ass ip es
N Month 12 1.949 0.1624 4.89 0.005
Error 24 0.796 0.0332
P Month 12 0.026 0.0022 3.67 0.005
Error 24 0.0153 0.006
K Month 12 8.925 0.7438 5.48 0.005
Error 24 3.256 0.1357
Ca Month 12 2.254 0.1878 3.41 0.025
Error 24 1.321 0.055
Mg Month 12 0.649 0.054 4.50 0.005
Error 24 0.296 0.012
Na Month 12 0.081 0.007 3.50 0.005
Error 24 0.053 0.002
L. mi nor
N Month 12 3.087 0.2573 1.75 0.25
Error 24 3.533 0.1472
P Month 12 1.093 0.0911 10.47 0.005
Error 24 0.209 0.0087
K Month 12 1.029 0.0858 2.03 0.1
Error 24 1.014 0.0423
Ca Month 12 2.571 0.2143 3.86 0.005
Error 24 1.332 0.0555
Mg Month 12 0.084 0.007 3.18 0.025
Error 24 0.053 0.0022
Na Month 12 0.034 0.0028 1.57 0.25
Error 24 0.042 0.0018
Vol. 1, 19 - 36 [2013]
33
A. pi nnat a
N Month 9 3.637 0.4041 4.12 0.025
Error 18 1.768 0.0982
P Month 9 0.051 0.0057 5.18 0.0025
Error 18 0.0194 0.0011
K Month 9 0.547 0.0608 1.24 NS
Error 18 0.883 0.0491
Ca Month 9 0.533 0.0592 2.01 0.1
Error 18 0.53 0.0294
Mg Month 9 0.227 0.0252 1.75 0.25
Error 18 0.259 0.0144
Na Month 9 0.351 0.039 2.57 0.1
Error 18 0.274 0.0152
U. fl exuosa
N Month 11 1.737 0.1579 2.79 0.025
Error 22 1.244 0.0565
P Month 11 0.0088 0.0008 1.60 0.25
Error 22 0.0113 0.0005
K Month 11 1.552 0.1411 2.40 0.1
Error 22 1.297 0.589
Ca Month 11 0.051 0.0046 4.60 0.005
Error 22 0.021 0.001
Mg Month 11 0.073 0.0066 4.40 0.005
Error 22 0.034 0.0015
Na Month 11 1.144 0.104 3.77 0.005
Error 22 0.608 0.0276
Table 4 Multiple regression equations relating the plant nutrient (PN) in E. plantaginea, nutrient in water
(NW) and plant biomass (PB) of different species fromBaraila wetland; the squared multiple correlation
coefficient (R2) and F statistics are also shown/
Nutri ents Regressi on Equat ion R2 F
E. crassipes
N PN =0.27 + 0.005NW +0.001PB 0.78** 7.88***
P PN =0.02 + 0.007NW +0.00004PB

0.89*** 5.00*
K PN =1.69 - 0.681NW +0.002PB
0.10
n.s.
0.99
n.s.
Ca PN =-0.01 + 0.014NW - 0.001PB
0.43
n.s.

25.45**
Mg PN =0.04 + 0.065NW +0.0002PB 0.86*** 66.25*
Na PN =0.003+ 0.054NW +0.0001PB

0.92*** 17.50***
L. mi nor
N PN =0.73 + 0.010NW +0.511PB 0.91*** 8.24**
P PN =0.08 + 0.051NW +0.117PB

0.96*** 20.33***
Vol. 1, 19 - 36 [2013]
34
K PN =0.83 + 0.998NW - 0.105PB

0.79**. 4.81*
Ca PN =-0.01 + 0.014NW - 0.001PB

0.96*** 68.09***
Mg PN =0.04 + 0.065NW +0.0002PB 0.91*** 35.19***
Na PN =0.003+ 0.054NW +0.0001PB

0.96*** 25.00***
A. pinnata
N PN =1.76 + 0.011NW +0.154PB 0.76**
2.57
n.s.
P PN =0.07 + 0.012NW +0.005PB

0.87*** 5.00***
K PN =0.45 + 0.664NW - 0.014PB

0.85**. 3.021*
Ca PN =-0.01 + 0.014NW - 0.001PB

0.96*** 68.09***
Mg PN =0.04 + 0.065NW +0.0002PB 0.91*** 35.19***
Na PN =0.003+ 0.054NW +0.0001PB

0.96*** 25.00***
R
2
values are significant; *P<0.05; **P<0.01 with d.f. 11.
F values are significant; *P<0.05; **P<0.01; ***P<0.005; with d.f. (2,10).
n.s. Non Significant.
1. Discussion : Free-floating zone had four constituents but Eichhornia crassipes was the most
dominant species which had maximum biomass 11.70 g m
-2
in the month of October and contributed
more than 50% to the total biomass of the pond and more than 90% to the biomass of this zone. Here
also maximum biomass in October showed that these post-monsoonal months have equilibiuiumof
environmental factors, like heat, light, nutrients and other biological and geological factors, whenthe
maximumgrowth and development of aquatic plants occur. The standing crop biomass of this zone was
higher than other reports except fromWestlake (1963) (Table 1).
The free-floating zone macrophytes were intermediate in production as well as in situation
between emergent and submerged zone. A maximumrate of 5.43 g m
-2
day
-1
was recorded for E.
crassipes whereas maximumrate of production this zone was 3.19 g m
-2
day
-1
. These macrophytes
are less productive than the emergents (Westlake 1965) was true on zonal basis but rate of E.
crassipes was higher than E. plantaginea. Higher rate of net roduction was noted in rainy season
because this was the flowering and fruiting period of species. However, data of productivity on
floating macrophytes are very few. Sahai and Sinha (1970) and Srivastava (1973) have reported a
maximum rate of 3.8 g m
-2
day
-1
organic matter net production for E. crassipes which is lower than
the rate of production in present investigation while Verma (1979) observed maximumrate of 15 g
m
-2
day
-1
dry matter production for free floating zone which was quite higher than the rate of net
production in present investigation.
Vol. 1, 19 - 36 [2013]
35
In species of free-floating zone the variation in nutrients with season was well marked. In
summer, nitrogen, phosphorus and potassiumwere maximum and calciumminimum in Eichhornia
crassipes and Lemna minor while role of summer in Azolla pinnata and Utricularia flexuosa did
not arise because former was completely absent in summer season whereas latter was not noted in
May. In winter season, nitrogen, phosphorus and potassiumwere significantly lower in most of the
cases and calcium was quite high. Effect of rainy season on variation of magnesiumand sodium was
quite distinct, in most of the cases concentration of magnesiumwas higher whereas opposite was the
trend for sodium. Nitrogen, phosphorus and potassiumare the protoplasmic constituents, which
were highly needed in the early development of plants, when these are ontogenetically young and
young and also metabolically very active whereas the calcium, the cell wall constituent was enriched
with ageing of the species. Higher concentration of magnesiumin rainy season was due to higher
biomass in the same season whereas low concentration of sodium in the rainy season was due to the
dilution effect of sodiumin water. The mean annual of nutrient concentration of this zone decreased
in the order of N>K>Ca>Na>Mg>P. The result of regression analysis showed that all plant nutrients
in Eichhornia crassipes and Utricularia flexuosa strongly depended on the level of nutrient
concentration in water (Table 4). This was similar to the report of Gosset and Noris (1971) which
demonstrated positive correlation between the N and P contents of the tissues of E. crassipes and
those of the environment but opposed to this report, Boyd and Vickers (1971) were unable to
correlate the two. Concentrations of N and P in tissues of U. flexuosa were not high to justify the
third source of this elements due to its insectivorous habit as reported by Collar, Coleman and Boyd
(1971). Regression analysis showed that in Lemna minor, potassium and sodium were water
dependent whereas other four nutrients were related to variations in biomass, this might be attributed
to highly productive and relatively short life cycle of duckweed (Rajmankova 1978). In Azolla
pinnata increased in N, Ca and Mg depended on biomass whereas P, K and Na on water nutrients.
Nitrogen dependence on biomass might be assigned to occurrence of nitrogen fixing blue green alga
Anabaena azollae in Azolla. This difference may not be due to season. It may also be due to
variation in age.
References :
Adams M.S. and McCracken M.D. 1974. Seasonal production of the Myriophyllum component of
the littoral lake Wingra, Wisconsin. J . Ecol. 62: 457-65.
Boyd C.E. 1971. Further studies on productivity, nutrient and pigment relationships in Typha latifolia
populations. Bull. Torrey Bot. Club. 98: 144-50.
Boyd C.E. and Vickers D.E. 1971.Variation in elemental content of Eichhornia crassipes .
Hydrobiologia. 38: 409-14.
Carignan R and Kalff S. 1980. Phosphorus sources of aquatic weeds water or sediments? Science.
207: 987-89.
Vol. 1, 19 - 36 [2013]
36
Gosset D.R. and Norris W.E. 1971. Relationship between nutrient availability and content of nitrogen
and phosphorus in tissues of aquatic macrophytes Eichhornia crassipes Mart. Solms.
Hydrobiologia.38: 15-28.
Irfan S. and Shardendu. 2009. Dynamics of nitrogen in subtropical wetland and its uptake and storage
by Pistia stratiotes. J . Environ. Biol. 30: 977-81
Rajmankova E. 1978. Growth, production and nutrient uptake of Duckweedsin fish ponds and uin
experimental cultures. In: Pond Littoral Ecosystems (Ed.by D. Dyjykova and J. Kvet) pp.
278-84.
Sahai R. and Sinha A.B. 1970. Contribution to the ecology of Indian aquatics. I. Seasonal changes in
biomass of water hyacinth (Eichhornia crassipes Mart. Solms.). Hydrobiologia. 35: 376-82.
Sayantan D. and Shardendu. 2013. Amendment in phosphorus levels moderate the chromium toxicity
in Raphanus sativus L. as assayed by antioxidant enzymes activities. Ecotoxicol. Environ. Saf.
95: 161-70.
Shardendu and Ambasht R.S. 1991. Relationship of nutrients in water with biomass and nutrient
accumulation of submerged macrophytes of a tropical wetland. New Phytol. 117: 493-500.
Srivastava V.C. 1973. The limnology, primary production and energetic of Chilw, Gorakhpur. Ph.D.
Thesis, Gorakhpur University.
Verma K.R. 1979. Phytosociology, productivity and energetic of macropbhytes of Gujar lake
(Khetasarai) J aunpur. Ph.D. thesis, Banaras Hindu University.
Westlake D.F. 1963. Comparison of plant productivity. Biol. Rev. 38: 385-425.
Westlake D.F. 1965. Some basic data for investigations of the productivity of aquatic macrophytes.
Primary production in aquatic environment. (Ed. By C. R. Goldman). Mem. Ist Ital. Idrobiol.
18 (suppl.), pp. 229-48.
Vol. 1, 19 - 36 [2013]
37
PHYLOGENETIC STUDY OF CELLULASE PRODUCING
STREPTOMYCES SP. MTCC 7779 AND EFFECT OF NUTRITIONAL
FACTORS ON THE ENZYME ACTIVITY
Nirupa Kumari, Supriya Sharma and Birendra Prasad*
Microbial & Molecular Genetics Lab.,
Department of Botany, Patna University, Patna-800 005, India
*Corresponding author (bprasad.pu@gmail.com)
Abstract : Effect of carbon, nitrogen and metal ions on cellulase enzyme activity was investigated in
shake culture condition. Strept omyces sp. MTCC 7779 was screened out from105 strains of
actinomycetes isolated from soil of Patna, India, due to its ability to produce substantial amount of
cellulase enzyme in plate containing Carboxy Methyl Cellulose as sole carbon source. Maximum
endoglucanase and -glucosidase activity was found in 72 hours old culture filtrate at pH 6.8 and
temperature 352C.The organismalso hydrolysed different agro-wastes materials such as bagasse
and corncob, along with CMC and filter paper. To standardize the additives in the media, NaNO
3
was
found to be the best nitrogen source for the production of cellulase enzyme. Endoglucanase and
-glucosidase activities enhanced almost double when medium was supplemented with five times more
sodiumnitrate. Simultaneously, amount of reducing sugar in broth was also enhanced in same ratio.
Among the eleven different types of metal ions, Fe
+2
and Mn
+2
were found to be significant
stimulator for both cellulase activity as well as yield of reducing sugar. Ions like Zn
+2
and K
+1
were least
stimulator for enzyme production and activity. In synergistic effect, Fe
+2
and Mn
+2
greatly enhanced the
substrate enzyme affinity and showed almost triple value of V
max
. While the value of K
m
greatly enhanced
in the presence of Zn
+2
and K
+1
.
The 16S rRNA region of this strain was amplified and sequenced. The Neighbor joining and
MaximumParsimony algorithmwith topology tree of 16S rRNA was constructed. Based on observation
and phylogenetic analysis, the strain showed 98.79 % similarity with Streptomyces carpaticus, 97.95
% with S. cheonansis and 96.24% with S. xiamenensis. Sequence data has been deposited at NCBI,
Bethesda, USA having Gene Bank Accession No. GU562884.
Keywords: Streptomyces, Cellulase, Nutritional factors, Metal ions-stimulator, phylogenetic analysis.
Introduction : Microbial degradation of organic wastes, especially cellulosic agro-wastes have been
in practice for obtaining commercially useful compounds such as ethanol, glucose and single cell
protein (Solomon et al., 1999). Cellulase and hemicellulase have been evaluated for their ability to
beneficially modify pulp and paper characteristics (Kibblewhite 1996; Suurnnakki et al., 2000;
Torres et al., 2000; Roncero et al., 2000) as well as in separation of gluten fromwheat flour
(Cavaco-Paulo 1998; Bhat 2000). Economical production of cellulases has generally been considered
38
to be the main aspect for feasible production of bioethanol from lignocellulosic biomass using cellulase-
based processes (Gadgil et al., 1995; Lynd et al., 2002). Most of the cellulases for such commercial
uses have been obtained fromdifferent microorganisms, mainly thermophillic fungi (Maheshwari et
al., 2000; J atinder et al., 2006).
Actinomycetes, having fungus like morphological and nutritional characteristics, normally inhabit
the soil where they develop mycelial form. Ability of the member of this class to thrive in diverse habitat
is based on their nutritional capabilities conferred by a vast array of hydrolytic enzymes that allow them
to recycle polysaccharides, proteins and fats that formessential residues of plants and animals (Sampath
and Chandrakasan, 1988; Hodgson, 2000). Various species belonging to the genus Streptomyces are
the main representative of this group of microorganisms, all of which have a complex life cycle that
responds to different signals, among which the nutrient limitation plays a key role. Streptomyces strains
have, therefore, been commercially useful in production of a number of bioactive compounds such as
antibiotics (Champness, 1988) and enzymes degrading complex polysaccharides (Ellaiah and Srinivasulu,
1996; Jang and Chang, 2005).
As an approach to produce a more affordable enzyme, most of the researches have been
carried out using cheaper lignocellulosic biomass such as bagasse, sawdust and corncob (Ojumu et al.,
2003). Although the enzymatic degradation of cellulosic material is relatively a slow process, search for
novel microbial strains capable of producing enhanced levels of thermostable cellulase is still continuing.
This report describes the isolation of a mesophilic strain of actinomycetes, Streptomyces sp.
MTCC 7779 from decomposing agro-wastes. This strain bears some novelty as it produces a
thermostable endo-1, 4- -D-glucanase in solid-state fermentation showing a higher yield of reducing
sugar (RS) frombagasse (2450 mg/l) and corncob (2250 mg/l) as the carbon source. Successful
attempts have also been made to optimize the nutritional requirements for maximumproduction of
enzyme. The effect of nitrogen sources and different metal ions has also been assessed on the cellulase
activity and yield of RS for further commercial application of this strain. Impact of these metal ions on
the value of K
m
and V
max
has also been investigated.
Material and Methods :
Isolation of cellulase producing strains of actinomycetes
Strains of actinomycetes were isolated fromdecomposing agro-wastes using starch casein
agar (SCA) media containing per litre, 10g soluble starch, 0.3 g hydrolyzed casein, 2g KNO
3
, 2g
NaCl, 2g K
2
HPO
4,
traces of MgSO
4,
7H
2
O; CaCO
3,
FeSO
4,
7H
2
O and 1.5 % (w/v) agar. Preliminary
screening for hyper-cellulase producing strains were carried out by examining the halos formed on
solid agar plates containing carboxy methyl cellulose (CMC) as the substrate followed by congo red
staining and washing (Teather and Wood, 1982).
Vol. 1, 37 - 49 [2013]
39
Preliminary characteri zation and i dentif ication
The strain was characterized for taxonomic identification based on the parameters described in
Bergeys manual of determinative bacteriology (Williams et al., 1982). Clonal culture of the strain was
deposited in MTCC & Gene Bank, IMTECH, Chandigarh (I ndia) with Accession Number
MTCC7779. 16S rRNA gene sequencing of strain MTCC 7779 was also carried out to ascertain the
species.
Phylogeny analysis
The 16S rDNA sequences of closely related validly published taxa were retrieved from the
Genbank data base using BLASTN (Altschul et al., 1997).
Growth conditions and production of Reducing Sugar (RS)
Broth cultures were raised in 50ml media in 250ml Erlenmeyer flasks in a shaking incubator
(JeioTech, Korea) at 150rpmand 35+1
0
C. CMC of high viscosity and highest purity grade (HiMedia)
was used as the carbon source for assessment of the yield of reducing sugar (RS) as well as endo-1, 4-
-D-glucanase activity.
Test of carbon sources
A total of four carbon sources (CMC, Whatman No. 1 filter paper, bagasse and corncob)
were selected as the test substrates. The strain was grown separately in a modified basal synthetic salt
medium(BSM), pH 6.5 (containing per litre 3g NaNO
3
; 0.5g MgSO
4,
7H
2
O; 0.5g K
2
HPO
4,
1g KCl
and traces of ZnSO
4;
MnSO
4;
FeSO
4,
7H
2
O; CaCl
2,
) supplemented with 0.5% (w/v) of the respective
carbon source.
Quantitative estimation of cellulase
The enzymesamplewas prepared by passing the culture filtrates through Whatman No.1 filter
paper withthehelp of suctionpump. The filtrate was centrifuged at 1500xg for 30 minutes at 4
0
C (Beckman
J 2-21M/E). The supernatant was used as the crude enzyme in catalytic reaction. The reaction mixture
contained in a total volume of 2ml, 1.4ml sodium citrate buffer (50mM) pH 6.5, 0.5ml of CMC (1% w/v)
and 0.1ml of appropriately diluted enzyme. The mixture was incubated at 50
0
C for 20 minutes. The
reaction was terminated by adding 3ml of 3,5-dinitrosalicylic acid (DNSA) followed by heating in a water
bath at 100
0
C. The colour thus developed was read at 540nmspectrophotometrically (Hitachi U-3210)
using glucose as the standard (Miller, 1959). Thesame processwas used for FP cellulase activity except
that the substrate was replaced byfilter paper in a 50mM citrate buffer, pH 5.6.
For -glucosidase activity, p-nitrophenyl -D-galactopyranoside was used as the substrate in
50mM sodiumphosphate buffer, pH 6.5 and the reaction was stopped by adding 3ml of 1M sodium
carbonate. The amount of p-nitrophenol produced was determined at 400nmusing p-nitrophenol as
the standard (Berghemand Pettersson, 1974).
Vol. 1, 37 - 49 [2013]
40
Estimation of reducing sugar(RS)
RS was estimated by the method described by Miller (1959). The reaction mixture contained
1.5ml of crude enzyme and 3ml of DNSA. The enzyme activity was expressed in terms of amount of
RS in mmole released in ml
-1
min
-1
using glucose as the standard.
Effect of nitrogen sources on the yield of RSG42
Four different nitrogen sources (urea, peptone, NH
4
NO
3
and NaNO
3
) were tested to select
the best nitrogen source keeping the nitrogen concentration at 3g/l. The cultures were raised in a time
course study and filtrates were processed for enzyme assays.
Effect of cations on the yield of reducing sugar and endoglucanase activity
Eleven different compounds (CoCl
2
.6H
2
O, CuSO
4
.2H
2
O, NaCl, MgSO
4
.7H
2
O, KCl,
MnSO
4
.H
2
O, ZnSO
4
.7H
2
O, FeSO
4
.7H
2
O, CaCl
2
, HgCl
2
and Na
2
MoO
4
.2H
2
O) were used for testing
the effects of cations on the production of RS and enzyme activity. The yield of RS was studied by
inoculating 10
8
spores of Streptomyces sp. in 50ml of enzyme production mediumsupplemented with
5mM of respective cation. The effect of cations on enzyme activity was studied by adding themin the
reaction mixture at a concentration of 1mM.
Results : A total of 22 actinomycetes strains isolated fromdecomposing agro-wastes were screened
for cellulase production. One isolate showed significantly high level of endoglucanase activity on CMC
plates as it produced a halos of 30mmdiameter after staining with congo red. Significantly, the strain
produced a unique type of grayish black spores in chains, and reticuliniaperti arrangement of the spores
on mycelia (Fig. 1).
The endoglucanase, -glucosidase and FPase activities were studied in culture filtrates obtained
in the presence of four different carbon sources (CMC, FP, bagasse and corncob) at an interval of 24
hours upto 120 hours of incubation. Maximum endoglucanase activity was observed with corncob as
the carbon source whereas maximum -glucosidase activity in the presence of CMC. A nearly similar
level of -glucosidase activity was observed with corncob and bagasse in 72 hours old culture and
beyond which the enzyme activities decreased rapidly. FPase activity was maximal in culture grown on
filter paper (Table-1).
Carbon sources significantly influenced the productionof reducing sugar and enzymatic activities.
Table-1 shows the endoglucanase, -glucosidase and FPase activity of the partially purified enzyme
obtained after 72 hours. The maximumendoglucanase activity was found in corncob (2.500.091
U/ml). Maximum -glucosidase activity was observed in carboxy methyl cellulose (0.690.021U/ml)
and maximumFPase activity (0.1500.0004 U/ml) appeared in the culture filtrate containing filter
paper as a sole carbon source.
The nitrogen source and substrates that regulate cellulase production were also evaluated in
presence of a fixed concentration (3 gL
-1
) of urea, peptone, NH
4
NO
3
and NaNO
3.
The inorganic
Vol. 1, 37 - 49 [2013]
41
nitrogen source was found to be the most suitable in increasing the cell mass and yield of RS (data not
shown). In order to optimize the media composition for enhanced enzyme activities and yield of reducing
sugar, cultures were raised in presence of 5X-elevated level of NaNO
3
(15 gL
-1
) in the basal medium
containing different carbon sources under test. The results thus obtained revealed that endoglucanase
and FPase were secreted at 4.08 and 2.61 folds higher levels as compared to the control (Table-2).
The corresponding enzyme activities were also increased considerably in the culture broth supplemented
with extra NaNO
3.
Maximumreducing sugar (1147552.13 g/L) and their corresponding
endoglucanase (3.80.05 U/ml) activity occurred in broth containing corncob as a sole carbon source.
A slightly lower value of reducing sugar (1118065.5 g/L) and endoglucanase (3.750.024 U/ml)
were observed in case of bagasse.
The effect of addition of cations on the enzyme activity and yield of RS was tested in presence
of CMC in basal medium. A significant enhancement in the enzyme activity and corresponding yield of
reducing sugar were observed only in the presence of Fe
+2
and Mn
+2
(Fig. 2). No significant change in
the enzyme activity as compared to the control could be detected in presence of Co
+2
, Cu
+2
, Mo
+2
,
Zn
+2
and Ca
+2
whereas Na
+
, Mg
+2
and K
+
moderately stimulated the enzyme activity.
The combination of two most stimulator metal ions (Fe
+2
and Mn
+2
) and two moderate stimulator
metal ions (Zn
+2
and K
+1
) have also been tested to evaluate the values of K
m
and V
max
in the presence of
above mentioned combination of metal ions. The V
max
value for endoglucanase enzyme almost triple
(7.692 U/ml) in presence of Fe
+2
and Mn
+2
(Table-3). In contrast, the value of V
max
greatly reduced in
presence of Zn
+2
and K
+1
for endoglucanase enzyme. Similar result was observed for -glucosidase
enzyme. The V
max
value was 1.428 mole/ml/min, it was almost double in comparison to control (0.714
mole/ml/min). The V
max
value of -glucosidase enzyme was reduced to 0.500 mole/ml/min in presence
of Zn
+2
and K
+1
. K
m
value also fluctuated in combination of these metal ions. The enzyme produced by
Streptomyces sp. having good affinity with their substrate. The affinity increase significantly in presence
Fe
+2
and Mn
+2
and greatly reduced in presence of Zn
+2
and K
+1
ions for both enzymes (endoglucanase
and -glucosidase) (Fig.3).
For phylogentic analysis of this strain, a 1426 bp sequence of 16S rRNA gene was
amplified fromthe genomic DNA with the use of universal primer (Forward Primer 5 -
AGAGTTTGATCCTGGCTCAG-3 and reverse 5 TACGGCTACCTTGTTACGACTT-3 )
and its sequence was submitted to GenBank,NCBI, Bethesda, USA with accession no.
GU563884.1). The 16S rRNA gene of different Strept omyces species was obtained by
BLASTN search, however 21 strains of Strept omyces species were selected on the basis of
high identity (%) with good E value for phylogenetic analysis (Fig. 4). It showed about 98.79%
similarities with Strept omyces carpaticus , 97.95 % with S. cheonansis and 96.24% with S.
menensis. No 100% identity was observed with preexisting Streptomyces sequences
deposited in NCBI.
Vol. 1, 37 - 49 [2013]
42
Enzyme Values Control Fe
+2
& Mn
+2
Zn
+2
& K
+1

Km(mg/ml) 0.606 0.4651 1.33 Endoglucanase
Vmax(IU/ml) 2.22 7.692 2.127
Km(mM) 0.25 0.208 1.430 -gl ucosidase
Vmax (IU/ml) 0.714 1.428 0.500

Table-3. Values of K
m
and V
max
in different conditions
Table-1. Effect of different Carbon sources on the production of EG (Endoglucanase), -glucosidase
and FPase by Streptomyces. sp. MTCC 7779 under shaking condition at 35
0
+1
0
C.
*Yield of reducing sugar multiply in fold shown in bracket.
Table-2. Total yield of reducing sugar and its corresponding endoglucanase activity in control condition
as well supplemented with 5x Nitrogen sources.
Vol. 1, 37 - 49 [2013]
43
Fig.2.Cellulase production and corresponding activity in the presence of different metal ions
Fig.1. Microphotograph of Streptomyces sp. MTCC 7779 (Retinoculoperti arrangement of
spores on mycelia)
Vol. 1, 37 - 49 [2013]
44
(a) Endoglucanase activity inpresence of combination of metal ions (Fe
+2
+Mn
+2
and Zn
+2
+K
+1
) along
with control
(b) -gluosidase activity in presence of combination of metal ions (Fe
+2
+Mn
+2
and Zn
+2
+K
+1
)along
with control
Fig.3. Linewever-Burk plot of Cellulase produced by Streptomyces sp. (Effect of concentration of
substrate shown in inset
Vol. 1, 37 - 49 [2013]
45
Fig. 4. Phylogeny trees are based on the nucleotide sequence of 16S rRNA genes. The trees were
constructed by using MEGA 4.1 software. Neighbor-J oining algorithmwith topology was used
for tree construction.
Discussion : Cellulose accumulates in terrestrial environments, where a variety of cellulolytic
microorganism, existing in virtually every niche and clime, dispose it (Leschine, 1995). Maximum
production of reducing sugar and -glucosidase were obtained in 72 hours old culture filtrate by
Streptomyces sp. J ang and Chen (2004) also reported that the production of endoglucanase reached
the maximumbetween 3
rd
to 5
th
days whereas -glucosidase production occurred on the 9
th
day from
Streptomyces sp. Our strain Streptomyces sp. Has more enzymatic activities than Micromonospora
chalcae (Gallaghher et al., 2004) in which maximumendoglucanase and -glucosidase activities were
observed after 8 days and 16-18 days, respectively. After 5
th
days, a sharp decline in enzyme activities
was observed. Ojumu et al. (2004) also suggested that the depression of cellulase activity between 4-
5 days might be due to cumulative effect of cellobiose, a dimmer of glucose. According to Spiridonov
& Wilson (1998) and Gutierrez-Nova et al., (2003) the catabolic repression plays an important role in
the regulation and secretion of inducible enzymes. Such repression effect has also been observed in
other organisms.
Vol. 1, 37 - 49 [2013]
46
Maximumyield of reducing sugar was occurred in cultural filtrate containing bagasse as a sole
carbon source. Chowdhury et al. (1991) also reported that the bagasse was more easily saccharified
carbon source than any other agricultural wastes. Filter paper considered as the most resistant to
biodegradation. The highest cellulase productivity with corncob and bagasse may be due to its very
high percentage of cellulose that is the major component of the cell wall of agro-wastes (Ojumu et al.,
2003). Levels of various enzyme activities differed with different carbon sources and maximum
endoglucanase, FPase and -glucosidase activities were observed in case of corncob, filter paper and
CMC, respectively. Different levels of cellulolytic enzyme produced on these carbon source indicated
that the heterogeneous nature of the carbon source play an important role in the induction of these
enzymes (Jatinder et al., 2006). A significant enhancement in the total yield of RS in presence of
bagasse (4.5-folds), corncob (5.1-folds) and CMC (4-folds) was observed in media supplemented
with elevated level of NaNO
3
whereas in presence of FP, the yield was only 2.6-folds higher than
control sample. Similar result has also been reported by Rajoka (2004), and it has been proven that
NaNO
3
was best source of nitrogen for production of cellulase enzyme.
According to Tejirian and Xu (2010), only a few metal ions act as inhibitors of cellulases, Fe
+2
and Fe
+3
act as inhibitors of cellulase enzyme. Result of the present work was not in support of previous
result. During present investigation, maximum production of endoglucanase and -glucosidase activities
has been observed in the presence of Fe
+2
and Mn
+2
ions. Manoliu et al., (2005) showed that the
endoglucanase and -glucosidase activity tremendously increased in the presence of 80ml/L of 45%
petroleumferrofluid in broth culture on 11
th
day in stationary phase containing fungal strain Chaetomium
globosum. According to Siddiqui et al., (1997) the low concentration of Mn
+2
activate the enzymes
with apparent activation constant. The ions like Cu
+2
and Co
+2
were slightly activating the enzyme
activity under assay conditions, while Hg
+2
inhibited the activity (Ferchak and Pye, 1983). It has been
suggested that the metal inactivation of the cellulase proceeds by chelation involving carboxylates at the
active center, thereby perturbing the tryptophan residue (s) in the binding site of the enzyme (Clarke
and Admas, 1987). Acoording to Licus et al. (2001) and Yin et al. (2010) metal could interact with
the hydrophobic group of amino acids, resulting in the decreased enzyme activity.
The combination of Zn
+2
and K
+1
act as uncompetitive inhibitors [Fig-3(a) & (b)] for enzyme
endoglucanase and -glucosidase. Zn
+2
and K
+1
combine with enzyme substrate complex, and form
inhibitor complex. These complexes reduce the catalytic efficiency of the enzyme, so that the velocity of
the reaction decreased gradual (Table-3). Such inhibition does not overcome by high concentration of
substrate. In the presence of Fe
+2
and Mn
+2
ions the K
m
value reduces, therefore it is suggested that the
affinity of enzyme and substrate become high in the presence of Fe
+2
and Mn
+2
. So these two metal ions
acts as activator and greatly increases the rate of reaction.
Pernodet et al., (1989) reported that the 16S rRNA and 23S rRNA genes of various
Streptomyces species were partially sequenced and used for defining all member of the genus, groups
47
of species or individual species. As shown in Fig. 3 (NJ Algorithmwith topology) two strains belonging
to Streptomycetaceae have been relatively closely related to Streptomyces sp. MTCC 7779 has the
own branch with Streptomyces carpaticus (DQ442494.1).
Conclusion : The present study led us to conclude that the carbon and nitrogen sources play a vital
role in production of hydrolyzing enzymes. Streptomyces sp. MTCC 7779 is capable of producing
cellulase from bagasse and corncob in huge amount. Cellulase enzyme production fromthese carbon
sources could be harvested at 72 hours in shake culture, the time at which the activity is highest. This
feature may be advantageous in commercial application of the enzyme of mesophilic actinomycetes for
the saccharification of natural cellulosic substrates. On the basis of phylogenetic study of this strain no
100% identity has been observed with preexisting Streptomyces sequences deposited in NCBI.
Therefore, it may be a novel species of Streptomyces.
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50
51
ENHANCED CLONAL PROPAGATION IN RAUVOLFIATETRAPHYLLA
L. USINGADENINE SULPHATE
Rachna Kumari, M.P. Trivedi
#
and Birendra Prasad*
Microbial & Molecular Genetics Lab.,
Department of Botany, Patna University, Patna-800 005, India
#
Department of Botany, Patna Science College, Patna University, Patna-800 005, India
*Corresponding author (bprasad.pu@gmail.com)
Abstract : Nodal explant with axillary buds of Rauvolfia tetraphylla L. were inoculated on MS
mediumsupplemented with BAP (0.5-3.0 mg/l) +NAA (0.5mg/l), Kinetin (3-10mg/l) +2,4-D
(0.5-1.0mg/l), BAP (0.5-3mg/l) +2,4-D (0.1-1mg/l) and BAP (0.5mg/l) +Adenine sulphate (AS)
(3-20mg/l) +IBA (0.2mg/l). An optimal micropropagation result was achieved using BAP +AS
(3mg/l) +IBA. Maximum shoot length appeared in BAP +AS (14mg/l) +IBA and number of shoots
were also enhanced (more than 2 at all concentration after AS treatment). The highest shoot
regeneration frequency (90%) was achieved on MS mediumsupplemented with 3 mg/l AS after
eight weeks prior to transfer in rooting media. The regenerated shoots showed best rooting on MS
medium containing 2mg/l IBA. Micropropagated plantlets were hardened in mixture of soil :
vermicompost : sand in 2:1:1 proportion, aseptically. After 3 months of its survival, they were transferred
to greenhouse.
Key words: Rauvolfia tetraphylla, Benzyl amino purine (BAP), Naphthalene acetic acid (NAA),
Kinetin, 2,4-Dichlorophenoxy acetic acid (2,4-D), Adenine sulphate (AS), Indole-3-butyric acid (IBA)
Introduction: Rauvolfia tetraphylla L. is an endangered woody shrub of family Apocynaceae. The
roots often used as a substitute of R. serpentina because of the presence of alkaloid which is localized
in the roots (Patil and Jeyanthi, 1997). The plant is medicinally important in the treatment of hypertension
and used as a sedative or tranquilizing agent. Rauvolfia species is threatened in India due to its
indiscriminate collection and over exploitation of natural resources for commercial purposes to meet
the requirements of pharmaceutical industry. Hence, the conservation by in vitro propagation of these
valuable genotypes is better option (Faisal and Anis, 2002), to satisfy the growing commercial demand
of the plant.
Adenine sulphate (AS) is a potent growth regulant for in vitro propagation. Adenine sulphate
induces higher rates of adventitious shoot formation in Rauvolfia serpentina (Ilahi et al., 2007). There
are various reports on in vitro propagation of R. tetraphylla through bud, shoot and nodal cuttings as
explant using growth regulators such as IAA, IBA, NAA, BAP and kinetin by several workers (Sharma
52
et al., 1999; Ghosh and Banergee, 2003; Harisaranraj, 2009). The existing protocol gave poor plant
regeneration. The present work describes an in vitro shoot multiplication fromnodal explant of R.
tetraphylla using AS growth regulator.
Materials and Methods : Young shoots of R. teraphylla were collected fromone year old plants
growing in the green house of botanical garden of Patna Science College, Patna. The shoots were first
washed with running tap water for half an hour and then treated with detergent (Labolene 5 %, v/v for
5 minutes) followed by 0.5% savlon for 5 minutes and finally washed with sterilized tap water. The plant
material then surface sterilized with 0.1% (w/v) HgCl
2
for 3 minutes before rinsing four times with
sterilized double distilled water.
The basal mediumwas MS salt and vitamins (Murashige and Skoog, 1962) supplemented
with 3% (w/v) sucrose and gelled with 0.8% (w/v) agar. The pH of the mediumwas adjusted to 5.8
with 1N HCl or NaOH before autoclaving at 121
o
C for 20 min. After sterilization, the explants were
inoculated on MS Medium and were maintained in culture roomat 25+2
o
Cunder photoperiod of 16
-2
s
-1
provided by white fluorescent light at 50 -60% relative
humidity. For each treatment, 30 replicates were used.
For hardening, microcuttings were transferred to plastic tray in a sterile mixture of soil :
vermicompost : sand (2:1:1), covered with transparent polythene bags, irrigated with sterilized water
and maintained aseptically at temperature 25 +2
o
C and 90-100% humidity.
Result and discussion : A variety of hormonal combinations were tried to induce multiple shoot
production from nodal explant. Thenumber of shoots per explant, their length as well as growth frequency
were low in MS basal media supplemented with growth regulators BAP (0.5-3.0mg/l) +2,4-D (0.5-
1.0 mg/l) and BAP (0.5-3.0mg.l) +NAA (0.5mg/l). At higher concentration i.e. 2.0 mg/l BAP and
lower concentration of NAA (0.5mg/l), shoot proliferation was comparatively better but it did not
show more than 2 shoots/explant and also a shoot length not greater than 3.0cm (Fig.1). Harisaranraj
et al., (2009) induced multiple shoots fromnodal cutting of R. tetraphylla cultured on MS medium
containing 2.0 mg/l BAP and 0.5 mg/l NAA and got average result. Salma et al., (2008) also used this
combination on mass propagation of R. serpentina and got better result. At lower concentration of
BAP (0.5mg/l) and higher concentration of 2,4-D and NAA (1.0 and 0.5, respectively) the bud
multiplication frequency was reduced. Some of the buds appeared to be healthy and some showed
reddish brown colouration at their base.
Effect of kinetin at concentration between 3.0 to 10.0 mg/l with 2,4-D (0.5 to 1.0 mg/l)
showed poor percentage of shoot growth and decreased shoot quality because most of the shoots
were reddish brown at base and necrotic. It means lower concentration of kinetin was optimumfor
Vol. 1, 51 - 56 [2013]
53
micropropagation of R. tetraphylla. Similar result was observed by Harisranraj et al. (2009), in culture
of R. tetraphylla on liquid media.
The proliferation of shoots in AS using concentration 3.0, 5.0, 8.0, 11.0, 14.0
and 20.0 mg/l was maximal. The shoot regeneration frequency was highest (90%) in MS
medium supplemented with BAP (0.5 mg/l) +AS (3.0 mg/l) +IBA (0.2 mg/l). After eight
weeks of culture, 3-5 multiple shoots were obtained from each explant. A tremendous
increase in height of the plant was noticed (3.8 to 6.3 cm per explant) (Table- 1; Fig. 2).
Maximum shoot length appeared in BAP + AS (14m/l) + IBA (0.2mg/l). A frequent increase
in adventitious shoot in R. serpentina was also noticed by Ilahi et al. , (2007), when
incorporated AS in basal MS media with BAP. The combination of BAP with adenine sulphate
has stimulatory effect on overall growth and number of shoots production. Gabriela (2011)
has also observed the formation of callus at the base of the explant in Trifolium repens L.
in the combination of BAP and adenine sulphate. To promote maximum shoot multiplication,
higher concentration of AS i.e. 20 mg/l was incorporated with basal MS media, while the
concentration of BAP and I BA was maintained at the same level i.e. 0.5 mg/l and 0.2 mg/l,
respectively. This combination did not show much promotive response for enhancement of
higher percentage of shootlet /explant (nodal cuttings) but it was found to be promotive in
case of adventitious shoot formation from callus in many plant species (Zibbu et al., 2010;
Gabriela, 2011).
In v itro rooting of microshoots excised from proliferating cultures were carried
out in MS full strength medium supplemented with 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l Indole-
3 - butyric acid. After one week shoots were subcultured on to plain MS medium.
Roots got initiated from 0.5 to 3.0 mg/l concentration of I BA after twenty five days of
culture (Table-2). Rooting frequency was highest at 2.0 mg/l concentration of IBA. At
concentration of 3.0 mg/l rooting was also satisfactory but I BA at higher concentration
(5.0 mg/l) inhibited rooting. The suitability of I BA at concentration of 2.0mg/l and
combination of IBA and I AA (1.0 + 1.0mg/l) for rooting of microshoots of Rauv olf ia
plants has also been reported by many workers (Faisal et al., 2005; I hsan I lahi, 2007;
Salma et al., 2008). Further three months acclimatization of micropropagated plants in a
mixture of soil: vermicompost: sand in 2:1:1 proportion gave eighty percent survivability
in growth chamber. Finally the plants were transferred to greenhouse where the
survivability was observed to be about 40%.
Vol. 1, 51 - 56 [2013]
54
Fig.1. Stunted growth of
at BA P (2.0 mg/l) &
NAA (0.5 mg/l)
R. tetrap hylla
F ig. 2. regeneration of
after A S treatment
- Induction of shoots on
BAP+AS (3.0 mg/l) + IBA
- Multiplication of shoots on
BAP+AS (14.0 mg/l) + IBA
- Induction of roots in Microshoot
- Acclimatized plants
In vi tro
R. tetr aphylla
A
B
C
D
Table-1: Effect of Phytohormone on nodal cutting of R. tetraphylla for micropropagation
Growth
regulant
Regeneration
frequency
(%)
Average
No. of
shoot /
expl ant
Average
l ength of
shoot/ explant
(cm)
Cal lus
i nduction at
base
Shoot
quali ty
BAP+AS+IBA
0.5+3.0+0.2 90.0 5.0+0.58 4.0+1.0 ++ Healthy
0.5.+5.0+0.2 33.0 3.2+0.58 3.8 +0.53 + Healthy
0.5+8.0 +0.2 60.0 4.0+0.58 4.0+ 0.58 + Healthy
0.5+11.0+0.2 62.5 3.0+0.00 5.8 +0.53

+ Healthy
0.5+14.0+0.2 67.0 3.0+0.58 6.3+0.58 + Healthy
0.5+20.0+0.2 80.0 2.6+0.58 5.0+1.0 + Healthy
Values represent mean +SE of 30 replicates per treatment, recorded after two months, (-) no response,
(+) slight callusing, (++) moderate callusing
Vol. 1, 51 - 56 [2013]
55
Table -2 Effect of IBA on root induction from in vitro raised microshoot of R. tetraphylla
after two month of culture
Values represent mean +SE.
References :
Faisal, M and M Anis (2002) Rapid invitro propagation of Rauv olfia tetraphylla L. An
endangered medicinal plant. J ournal of Physiology and Molecular Biology of Plants, 8 (2):
295-299.
Faisal, M, N Ahmad and M Anis (2005) Shoot multiplication in Rauvolfia tetraphylla L. using
thidiazuron. Plant cell, Tissue and organ culture, 80: 187-190.
Gabriela Vicas (2011) Effect of adenine sulphate (ADSO4) on the invitro evolution of white clover
variety (Trifolium repens L.). Analele Universitatii din Oradea Fascicula Protectia Mediului, XVII:
203-210.
Ghosh, K C and N Banerjee (2003) Influence of plant growth regulators on invitro micropropagation
of Rauvolfia tetraphylla L. Phytomorphology , 53:11-19.
Harisaranraj, R, K Suresh and S Saravanababu (2009) Rapid clonal propagation Rauvolfia tetraphylla
L. Academic Journal of Plant Sciences, 2 (3): 195-198.
Ihsan Ilahi, Fazal Rahim and Mussarat J abeen (2007) Ehhanced clonal propagation and alkaloid
biosynthesis in cultures of Rauvolfia. Pakistan J ournal of Plant Sciences, 13 (1): 45-56.
Murashige, T and F Skoog (1962) A revised mediumfor rapid growth and bioassays with tobacco
tissue cultures. Physiologia Plantarum, 15; 473-497.
Patil, V M and J eyanthi (1997) Micropropagation of two species of Rauvolfia (Apocynaceae). Current
Sciences, 72 (12): 961-965.
Salma, U, M S M Rahman, S Islam, N Haque, M Khatun, T A Jubair and B C Paul, (2008) Mass
propagation of Rauvolfia serpentina L. Benth. Pakistan Journal of Biological Sciences, 11 (9):
1273-1277.
t
4.0 +1.0
4.0+1.0
4.3 +0.58
2.3+0.58
-
Vol. 1, 51 - 56 [2013]
56
Sharma D, S Sharma and A Baruash (1999) Micropropagation and invitro flowering of Rauvolfia
tetraphylla: a potent source of antihypertensive drug. Planta Medica, 65: 277-278.
Zibbu Garima and Amla Batra (2010) Effect of adenine sulphate on organogenesis via leaf culture in an
ornamental plant : Thevetia peruviana (Pers.) SCHUM. International Journal of Pharma and Bio
Sciences, V1 (2): 1-9.
Vol. 1, 51 - 56 [2013]
57
VEGETATION OF PATNA DISTRICT OF BIHAR
Nupur, Maheshwar Prasad Trivedi and Indrani Trivedi
Department of Botany, Patna Science College, Patna - 800005
Key words : Vegetation, Patna, Bihar.
The unique location and varied climatic conditions of Patna support luxuriant vegetation
cover with huge species diversity. The vegetation aspects of the region are being presented.
Introduction : Vegetation is actually the totality of plant growth including large or small population of
each species intermixed in a region. It is continuous though it differs fromplace to place according to
environment gradient (Whittaker, 1967; Grime, 1987). It is the mere grouping of individual plants.
Vegetations are brought about by plants modifying the habit of place in which they grow.
The present vegetation has suffered indeed fromplants, animals, soil, climate and man.
Thus, the vegetation of the region that we see around us is much interfered.
Materials and methods : The materials for the investigation are vegetation of Patna District in
Bihar. The area is predominantly urban. It is situated between 2497' 2557' N latitude and 8444'
864' E longitude at an elevation of about 60 mabove sea-level. It covers an area of 3202 Km
2
.
The town is 20 kilometer long from east to west and 5 kilometer broad fromnorth to south. The city
is situated on the land between the rivers Ganga on the north and the Punpun on the south. Adjoining
areas of Patna are Danapur, Maner, Khagaul, Bihta, Naubatpur, Masaurhi, Punpun, Fatuha, Barh
and Mokama.
Extensive field studies have been done for which several rounds of trips were arranged.
During each trip plants were collected, pressed, preserved and photographed. Listing of all the
species has been done separately. Phytosociological studies of weeds and ruderals have also been
done.
Results and Discussion : The flora of Patna has relatively different composition and characteristics
on account of variable rainfall, temperature, geology, topography and substratum of the locality which
influence the floristic and vegetation differently in various phytogeographic regions.
The only natural habitats available are a few marshy places and grass lands. The roads
traversing through the residential colonies have the trees planted on their sides for the purpose of shade
and beautification. Largest collection of species is in the Sanjay Gandhi Biological Park and Kumhrar
Park which are places of tourist interest.
With the onset of monsoon in different adjoining areas of Patna like Maner, Danapur, Barh,
Mokama, etc., green herbs make their appearance in every nook and corner. Among them Cleome
Vol. 1, 57 - 61 [2013]
58
viscosa Linn., Cleome gynandra Linn., Desmodium gangeticum D.C., Indigofera hirsuta Linn.,
Cassia occidentalis Linn., Cassia tora Linn., Dentella repens Forst., Tridax procumbens Linn.,
Xanthium strumarium Linn., Heliotropium indicum Linn., Evolvulus alsinoides Linn., Phyllanthus
fraternus, Cyperus distans Linn. f., Brachiaria ramosa Stapf. are more common.
The hedges of gardens and parks are formed by Murraya paniculata (Linn.) Jacq.,
Lawsonia inermis Linn., Tabernaemontana divaricata (Linn.) R. Br., Duranta repens Linn. and
others. The comparatively moist waste places of different area of Patna are colonized by shrubs like
Lawsonia inermis Linn., Ipomoea fistulosa Mart. ex. Choisy, Adhatoda zeylanica Medic., Lantana
camara Linn., Vitex negundo Linn. and others.
In the rainy season vegetation acquires remarkable luxuriance. The most conspicuous
climbers in the rainy season are Tinospora cordifolia (Willd.) Miers, Cayratia trifolia (Linn.) Domin.,
Clitoria ternatea Linn., Coccinia grandis (Linn.) Voigt., Zizyphus oenoplia Mill., Basella alba Linn.
and others. With the rainy season coming to an end, climbers like Cocculus hirsutus (Linn.) Deils,
Cuscuta reflexa Roxb. Antigonon leptopus Hook and Arn. become prominent. During summer,
climbers like Capparis zeylanica Linn., Zizyphys oenoplia Mill. and others are spread over bushes in
most of the areas.
The southern bank of river Ganga is lined with stone boulders with many pucca ghats; in the
crevices of rocks many amphibious and wet meadow species are seen growing, viz., Ranunculus
sceleratus Linn., Salvia plebeia R.Br., Alternanthera polygonoides (Linn.) R.Br., Polygonum
barbatum Linn., P. glabrum Willd., P. hydropiper Linn., Rumex dentatus Linn. and others. By early
summer these plants are replaced by dry meadow species such as Argemone mexicana Linn., Scoparia
dulcis Linn., etc.
The lands along roads and railway tracks are inhabited by dry-meadows comprising large
number of herbs and undershrubs like Argemone mexicana Linn., Cleome gynandra Linn., C. vicosa
Linn., Tephrosia purpurea (Linn.) Pers., Cassia occidentalis Linn., C. tora Linn., Ageratum
conyzoides Linn., Parthenium hysterophorus Linn., Lippia javanica (Burm. f.) Spreng, Chrozophera
rottleri (Geiss.) J uss. ex spreng, Croton bonplandianum Baill., Amaranthus spinosus Linn., A. viridis
Linn.,etc..
In the different areas of Patna, Mango and Guava orchards are common. The ecological
condition of these orchards favour the growth of several shade loving species, such as: Malvastrum
coromandelianum (Linn.) Gracke, Urena lobata Linn., Oxalis corniculata Linn., Desmodium
ganget icum (Linn.) DC., Ageratum conyzoides Linn., Vernonia cinerea Linn., Cynoglossum
lanceolatum Forsk and Achyranthes aspera Linn.
The lawns and parks are gradually scrapped off succulent grasses and colonized by coarser
ones. Mixed with grasses grow a number of herbaceous colonizers, e.g. Alysicarpus monilifer (Linn.)
Vol. 1, 57 - 61 [2013]
59
DC., Desmodium triflorum (Linn.) DC, Indigofera linifolia (Linn. f.) Retz., Boerhaavia diffusa
Linn., Polygonum plebeium R.Br. In sheltered spots, as under the benches and near the railings of
parks, grow Vernonia cinerea (Linn.) Less, Achyranthes apsera Linn., Amaranthus spinosus Linn.,
A. viridis Linn. and other erect forms . In many lawns and parks, the more xerophytic grasses like
Imperata cylindrica (Linn.) Beauv., Dichanthium annulatum (Forst.) Stapf., etc. are replacing the
common doob grass. The parks which have restricted entries and protected against grazing are
comparatively damper. They show a luxuriant growth of moisture loving grasses.
Due to rapid inflow of population many cultivated lands are being converted into dwelling
sites. Despite this habitational pressure, gradually the outskirts, particularly the eastern and southern
sides are still under cultivation. In the rainy season, paddy is grown on low lands and maize, millets and
pigeon peas on higher lands. In winter wheat, barley, gram, pea and oil seeds are grown. The common
vegetable crops sown in the areas are Brasscia oleraceae Linn. var. capitata Linn. (cabbage), B.
oleraceae Linn. var. botrytis Linn. (cauliflower), Raphanus sativus Linn. (radish), Abelmoschus
esculentus (Linn.) Moench., Cucumis sativus Linn., Cucurbita maxima Duch., Luffa cylindrica
Roem., Luffa cylindrica Roem., Momordica charantia Linn., Daucus carota Linn., Lycopersicon
eculentum Mill., Solanum melanogena Linn., Allium cepa Linn., Allium sativum, Amorphophallus
campanulatus Bl. and others.
Weeds growing in the crop fields compete with crop plants for various growth requirements
and deplete the soil of nutrients. They spread diseases of crop plants either as their primary carrier or as
secondary host. The weeds found with rainy season (Kharif) crops are Caesulia axillaris Roxb.,
Eclipta prostrata Linn., Bacopa monneiri (Linn.) Pennell, Scoparia dulcis Linn., Alternanthera
polygonoids (Linn.) R.Br., A. sessilis Linn., Polygonum glabrum Willd., Cyperus rotundus Linn.,
Cynodon dactylon (Linn.) Pers. etc. and the weeds of winter season (Rabi) crops are Argemone
mexicana Linn., Fumaria parviflora Lamk., Medicago lupulina Linn., Melilotus alba Desv., Caesulia
axillaris Roxb. Eclipta prostrata Linn., Anagallis arvensis Linn., Ipomoea aquatica Forsk., Solanum
nigrum Linn., S. surattense Burm f., Chenopodium album Linn., Croton bonplandianum Baill.,
Cynodon dactylon (Linn.) pers. and several others.
The walls of delapdated houses show a luxuriant growth of weeds e.g. Blumea mollis
(Don.) Merrill, Tridax procumbens Linn., Vernonia cinerea (Linn.) Less., Lindenbergia indica (Linn.)
O. kuntze, Boerhaavia diffusa Linn., Commelina benghalensis Linn., Brachiaria reptans (Linn.)
Gard et Hubb. and others. On older ruins, there are seen Ficus bengalensis Linn., F. racemosa Linn.,
F. religiosa Linn. and others. The dust heaps and garbage dumps are harboured by the common
weeds, viz. Argemone mexicana Linn., Cleome gynandra Linn., C. v iscosa Linn., Croton
bonplandianum Baill. and others. In different areas of Patna, new building construction sites are generally
low- lands having dry-meadow species like Cassia occidentalis Linn., C. tora Linn., Solanum
surattense Burm. f. and Croton bonplandianum Baill., etc.
Vol. 1, 57 - 61 [2013]
60
The aquatic floristic components of the area include species like Nymphaea nauchali
Burm. F., Trapa bispinosa Roxb., Utricularia aurea Lour., Hygrophila auriculata Heine, Eichhornia
crassipes Solms., Pistia stratioides Linn., Wolffia arrhiza and others. At certain places with excessive
organic matters, Eichhornia crassipes grows too densely to choke out all other plants in its vicinity.
Azolla pinnata R. Br. is often seen forming red carpets in some local ponds.
Newly introduced species or invasive species which are now growing successfully in the
area but were not recorded by Haines (1921-25) and Srivastava (1956) are Parthenium hyterophorus
Linn., Mecardonia procumbens (Miller) Small., Alternanthera polygonoides (Linn.) R. Br., Ageratum
houstonianum Mill. Gard. and others.
The phytosociological studies of weeds and ruderals were made at four sampling sites of
Patna-Masaurhi, Punpun, Danapur and Mokama. In Masaurhi 26 species, in Punpun 25 species, in
Danapur 24 species and in Mokama 21 species were sampled for their percentage frequency, frequency
class, density and abundance.
The most frequent species are Eclipta alba, Commelina benghalensis and Millingtonia
hortensis in Masaurhi, Cynodon dactylon and Commelina benghalensis in Punpun, Cynodon dactylon
and Vandellia crustacea inDanapur, and Vicia hirsuta and Grangea maderaspatana in Mokama. The
minimumfrequency was shown by Atylosia scarabaeoides, Cayrotia trifolia and Launaea asplenifolia
in Masaurhi, Oxalis corniculata, Blumea lacera, Desmodium gangeticum and Celosia argentea in
Punpun, Phalaris minor and Portulaca inDanapur, and Gnaphalium indicum, Chenopodium album,
Vernonia cinerea, Celosia argentea, Lathyrus aphaca and Xanthium strumarium in Mokama.
Surprisingly the histograms of almost all sampling sites are in accordance with Raunkiaers
laws of frequency (1-4). The frequency class A is approximately 40% in Masaurhi, Punpun and Danapur
while in Mokama it is 45%. The frequency class B was highest in Masaurhi but in Danapur the frequency
class C is greater than B. It is the only place where Raunkiaers law was not followed (Nupur, 2009).
No doubt, the area is wide and continuous study is needed for a wider conclusion and acceptability.
Acknowledgement : The authors are thankful to the Head, Department of Botany, Patna University
for facilities and valuable suggestions.
References :
Grime, J.P. (1987). Plant strategies and vegetation processes. Wiley Interscience, New York NY.
Haines, H.H. (1921-1925). The Botany of Bihar and Orissa 6 parts, London.
Nupur (2009). Problemand Conservational approach of angiospermic biodiversity of Patna and Vaishali
of Bihar, Ph.D. Thesis, Patna University, Bihar (Published).
Srivastava, J .G. (1956). The vegetation of Patna District (Bihar). J. Ind. Bot. Soc. 38 : 186-194.
Whittaker, R.H. (1967). Gradient analysis of vegetation. Biological Review, 42 : 207-264.
Vol. 1, 57 - 61 [2013]
61

Vol. 1, 57 - 61 [2013]
62
63
SOIL MOISTURE AND CONSERVATION POTENTIAL OF SOME
GRASSES AND WEEDS IN URBAN AREAS OF PATNA DISTRICT
Indrani Trivedi and U.K.Sinha
Department of Botany,
Patna University,Patna-800005.
Abstract: The soil binding capacity and moisture conservation potential of some grasses and weeds in
urban areas of Patna district has been worked out. An efficient grass and weedy species is one which
declines the run-off velocity, able to dissipate the rain drop energy, retains the soil particles and improves
the infiltration rate. The average root diameter was highest in Amaranthus virdis and lowest in Evolvulus
alsinoides. The Dichanthium caricosum and Trianthema monogyna were adjudged the best soil
binders.Soil moisture conservation potential is highest in Dichanthium caricosum, Sagitaria sagitifolia
and lowest in Oxalis corniculata.
Key Words : Soil binding capacity,Soil moisture, grasses,weeds, urban area, Patna district
Introduction: The explosion of population in India has exerted tremendous pressure on the land
for production of food ,fuel and fodder. On the other hand problem of soil degradation is at alarming
rate.Physical degradation is a major limitation resulting in soil erosion. In India about 5334 million
tonnes of soil is eroded every year and about 29% of it is lost permanently to seas. Also as per
estimate, about 16.35 tonnes per hectare per year soil is being eroded which is more than the
permissible limit of about 4.5. Trees are helpful in reducing the rain drop but grasses and weeds also
provide a cover on earth surface to intercept the rain water. Various weeds and grasses have their
own ability to check the erosion, improve infiltration or forma sod over ground. An efficient grass
and weedy species is one which reduces the velocity of run off, able to dissipate the rain drop
energy, retains the soil particles and improves the infiltration rate (Singh and Ratan, 2008). In the
present study an investigation has been made to work out soil moisture and conservation potential of
some grasses and weeds.
Materials and methods: The materials for the present investigation are grasses and weeds growing
naturally in diverse niches. Roots growth, their diameter and conservation efficiency were worked out.
Root growth-Root excavations of selected species were done by carefully working side ways and
down wards till the root tips were exposed (Bohm, 1979). The roots thus excavated were cleaned in
water and separated for data recording. The roots of selected species were sampled fromthe quadrats
which were clipped for estimation of above ground biomass. The soil monolith of 25x25x25cmwas
removed. The dug out monolith was carried to laboratory and flooded with water. The roots were
separated by hand.
Vol. 1, 63 - 68 [2013]
64
Root diameter-After excavations and washing, the roots of each plant were separated and divided
into four categories viz. main, primary, secondary and tertiary roots. The diameter of all the four categories
was taken at 3 points along the length with the help of a vernier caliper and average diameter of each
category of roots was worked out.
Root diameter was measured with the help of vernier caliper and root volume was measured
by placing the roots in a measuring cylinder containing known volume of water. The increase in volume
indicated the root volume. The binding capacity of the root was calculated by the formula F=V\R
where F is the binding factor, V is the volume in ml and R is the average radius in mm of the roots. The
evaluation was made in natural condition (tables 1and 2).
Moisture conservation-
Water (%) by mass:
Wet mass of soil =(wet mass of soil +box)-(mass of box)
Dry mass of soil =(dry mass of soil +box)-(mass of box)
Water (%) by mass =(wet mass - dry mass / dry mass) x 100
Results and discussion: The average root diameter was highest in Cassia tora (6.55mm) while
lowest in Evolvulus alsinoides (1.275mm).The grasses like Cynodon dactylon, Cyperus rotundus
and Dichanthium caricosum have more or less similar diameters( Table 1).
The soil binding capacity of Dichanthium caricosum was the highest (90.32) followed by
Cyperus rotundus (63.775).Cynodon dactylon showed 52.426 soil binding factor. Among the weeds
Trianthema monogyna (43.103) and then Amaranthus viridis (39.836) showed the highest value and
least (7.665) by Portulaca quadrifida( Table 2).
Soil moisture conservation potential is highest in Dichanthium caricosum (17%) Sagitaria
sagitifolia (17%) and lowest in Oxalis corniculata(1%).
Singh and Ratan (2008) have worked out the various parameters for grass ability to reduce
erosion and enhance infiltration. They have concluded that Heteropogon contortus (lumpa grass) is
best suited grass in Bundelkhand region including J alaun district (Tyagi,1997). Similarly Muthana (1981)
also recommended H. contortus,Dichanthiumannulatumand Pennisetumpurpureum most suitable for
the above mentioned region.
Singh and Soni (2009) have worked out the soil conservation value and on various parameters
for revegetation and consolidation of uranium tailings at Jaduguda in J harkhand.
Munshower,1993 emphasized that native species were less competitive and can be used
in rehabilitation and the disturbances permit the germination and development of non-seeded
species.
Vol. 1, 63 - 68 [2013]
65
Dadhwal and Singh (1993) studied the rooting behavior of five trees, two shrubs and
six grass species. The best root growth, soil binding capacity and nodulation were found in
Leucaena leucocephala, Pennisetum purpureum, Eulatiopsis binata and Cymbopogon
fulv us.
The higher values of binding capacity of species may be due to their well developed lateral
roots and higher root volume(Trivedi and Sinha,2012). Biomass is increased due to the production
of greater number of lateral roots.The absorption of water is also enhanced under moisture stress
conditions (Vasistha, 1992)Suitability of grasses, weeds and others is being judged for reclamation
of varying lands.
Aknowledgements : We are thankful to Prof. S.N. Sharma, HOD, Botany, Patna University for
providing laboratory facilities.
References:
Bohm, W.1979. Methods of studying root system: Ecological studies. Spring-Verlag, Berlin, New
York. 33
Dadhwal, K. S. and Singh, B. 1993. Rooting behavior of different plant spcies in lime stone mined
area. Indian Forester. 119(2):71-74.
Muthana, K. D.1981. Forage forest particles envisaged for the development of Bundelkhand region
(U.P.). Indian J.Range Mgmt. 2(1 and 2):73-79.
Munshower, F.F.1993. In: Practical handbook of disturbed land revegetation. Lewis publishers,
London, Tokyo.
Singh Lal and Soni Prafulla. 2009. Species selection for revegetation and consolidation of Uranium
tailings at J aduguda in Jharkhand, India , The Ecoscan 3(1&2) :19-25.
Singh U. N and Ratan Neel. 2008. Assessment of soil and water conservation of some grass
species in light Olive-Brown soils of J alaun Based on overall performance index, The
Ecoscan 2(2) :219-222.
Tyagi , R. K.1997. Grassland and fodder atlas of Bundelkhand. Indian grassland and fodder research
institute, Jhansi (India), pp. 39-40.
Vasistha ,H. B. 1992. Growth behaviour of some colonizing plant species of rock phosphate mine
spoils areas of Doon Valley. Ph.D. Thesis submitted to H. N. B. Garhwal university Springer,
(Garhwal).
Trivedi Indrani and Sinha U.K. 2012 Soil binding capacity of some grasses and weeds in urban aras
of Patna district.Int. J. Mendel,29 (1-4),23-24.
Vol. 1, 63 - 68 [2013]
66

Species

Main
roots
Primary
roots
Secondar
y roots
Tertiary
roots
Average
root
diameter
Dichanthium caricosum 5.2 2.7 1.3 0.1 2.32
Cyperus rotundus 4.5 3.7 1.8 1.2 2.80
Cynodon dactylon 3.7 2.3 1.6 1.1 2.17
Trianthema monogyna 4.4 3.5 1.8 1.2 2.72
Amaranthus viridis 10.8 7.7 4.7 1.3 6.12
Evolvulus alsinoides 2.5 1.8 o.6 0.2 1.27
Oxalis corniculata 3.8 2.6 1.3 0.2 1.97
Phyllanthus fraternus 3.6 2.4 1.7 0.9 2.15
Brachiaria ramosa 3.8 2.6 1.3 0.9 2.15
Molunga pentaphylla 5.4 4.7 2.3 0.9 3.32
Launea pinnatifida 5.4 3.6 2.3 1.4 3.17
Acalypha indica 5.6 3.7 2.5 1.8 3.40
Sagittaria sagittifolia 10.4 7.3 3.6 1.8 5.77
Ruellia tuberosa 10.9 6.4 3.9 1.8 5.75
Parthenium hysterophorus 8.2 4.5 3.7 1.4 4.45
Asplenium indicum 3.8 2.3 1.2 0.9 2.05
Lindernia spp. 6.2 4.3 2.3 1.4 3.55
Euphorbia hirta 5.6 4.5 2.7 0.9 3.42
Anisomeleus indica 6.8 5.7 4.3 1.6 4.60
Solanum nigrum 5.8 3.3 1.8 0.9 2.95
Vernonia cineraria 10.4 6.4 3.7 1.3 5.45
Poulszia indica 12.4 8.9 3.6 1.6 6.62
Brachiaria reptans 7.9 6.7 5.2 4.6 6.10
Nicotiana plumbaginifolia 9.5 4.9 1.7 0.2 4.07
Portulaca quadrifida 7.3 5.4 3.2 1.2 4.27
Table 1: Root diameter (mm) of selected plant species
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Table 2: Binding capacity (Root Conservation Value)
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Table 3 : Moisture conservation value
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69
PHYTOTHERAPEUTIC ROLE OF CENTELLA ASIATICA L.
Bimal K. Mehta
Guest faculty (Biotechnology and Env. Sc.)
Dept. of Botany,
Patna Science College, P.U. Patna
Abstract : Centella asiatica L. has been used for centuries (about 3,000 years) as a medicine in the
Ayurvedic tradition of India. The plant extracts were incorporated into the Indian Pharmacopeia,
wherein addition to being recommended for wound healing, treatment of skin conditions such as leprosy,
lupus, varicose ulcers, exzema and psoriasis and in brain stimulation, treatment of venous hypertension,
microangiopathy and in gastric ulcers. The centella asiatica L. is a rejuvenative nervine recommended
for nervous disorder, epilepsy, senility and premature aging and also as a brain tonic. The samples of the
Indian plants collected fromdifferent places showed the presence of glycosides : indocentelloside,
brahmoside, brahminoside, asiaticoside, thankuniside and isothankuniside etc. A new pligosaccharide
centellose, Kaempferol, quercetin and stigmasterol have also been reported.
With the development of science many new drugs of synthetic origin have come into existence
but times have changed and we are back to the herbs and herbal products that our ancestors used.
Key words : Phytotherapeutic role, Centella asiatica
Introduction : Human beings have to depend on Nature for sustenance and survival. The traditional
systemof medicine in indiadates back to the age of the Rigveda (2500 to 1600 B.C.). Withthe development
of science, many new drugs of synthetic origin have come into existence and with the rapid growth of the
pharmaceutical industry the value and use of the herbal medicines has come down in the recent past. Times
havechanged and we are back to the herbs and herbal products that our ancestors used.
Centella asiatica is a perennial plant native to India, China and Indonesia,. The plant commonly
knows as Brahmi belongs to the family Apiaceae (=umbelliferae.) It is found throughout our country,
more in the tarai regions of the himalayas and Bihar near marshy place or river banks. Plant is a trailing
herb, branched with soft node and internode, stem rooting at nodes.
Leaves are orbicular, reniform 1.25cmto 6.25cmin diameter, glabrous with crenate margin.
Flowers are sessile, cluster of 3 to 6, mediumsized, multicoloured. Corolla with two rows of petals and
with white petaloids intermingled with stamens in itscentre. White petals with variegations and sometimes
pink streaks . Flowering takes place during march-April. Fruits are globular nearly 8mm in diameter
with 7-9 raised ribs over which the seeds appear. The plant can be harvested at any times of the year
and is used fresh or dried. In common with most traditional phyto therapeutic agents, C. asiatica is
claimed to possess a wide range of pharmacological effects being used for wound healing capacity
(Suguna et al. 1669), Mental disorder (Apparao et al. 1973), fungicidal, antibacterial (Oyedeji et al.
2005), antioxidant and anticancer properties (J ayashree et al. 2003) Centella asiatica has also been
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reported to be useful in the treatment of inflammations, diarrhoea, asthma, tuberculosis and various skin
lesions and ailments like leprosy and psoriasis. In addition, numerous clinical reports verify the Ulcer
preventive and anti depressive, sedative effects of C. asiatica preparations, as well as their ability to
improve venous insufficiency and microangiopathy.
Popularity of C. asiatica is mostly due to its efficacy and versatility, especially referring to its
reputation as a wound healing agent and brain stimulant (promoting brain growth and improving learning
and memory.) Many scientists in the world have been conducting quite extensive experimental and
clinical investigations and focusing their interests on searching for some promising compounds with high
effectiveness and low toxicity for the benefit of human health.
The present paper deals about the recent advances in the phytochemistry and bioactivities of
C. asiatica, particularly mentioning its principally active mass-triterpenoids.
Materials and Methods : The present study is based on extensive laboratory studies on Centella
asiatica (L). The study was limited to the Urban and rural localities of Patna, (Bihar). The traditional
home remedies are still alive here. Interviews were conducted involvingsome patients, Ayurvedic doctors
and Vaidya. The diagnosis were based on clinical features. The dried powdered plant material (Leaves,
roots, aerialparts, stem, seeds) was extracted with chloroformin a Soxhlet extraction apparatus. The
solvent was removed under reduced pressure and semi solid mass was obtained (Yield 16.7%). The
extract showed positive test for alkaloids, volatile oils and saponins. The alkaloid were identified by
chromatographic comparison with reference compounds and it was further confirmed. The extract at
the different doses of 50, 100 and 200 mg/kg was suspended in aqueous between 80 solution (2%).
The dose range is usually 60-120 mg/day, although higher doses may be provided in some situations.
(Karting, T. 1986). Nausea has been reported in high level of intake. It should not be taken internally as
a supplement by children under the age 4 or breast feeding/ Pregnant mothers. People taking sedatives
should not use Centella asiatica L. as a supplement.
Results and Discussion :
Phytochemistry
Triterpenoids : Triterpene is a major and the most important component of C. asiatica, regarded as
a marker constituent in terms of quality control. The triterpenes obtained from C. asiatica are mainly
pentacyclic triterpenic acids and their respectiveglycosides, including asiatic acid, asiaticoside, madecassic
acid, madecassoside, brahmoside, brahmic acid, brahminoside, thankuniside, isothankuniside,
centelloside, madasiatic acid, centic acid, cenellic acid, betulinic acid, indocentic acid, etc. Earlier work
on this plant has led to the isolation of many triterpenoid constituents. Brinkhaus et al., (2000) has
already reviewed the chemical profile of C. asiatica before 2000, thus we predominantly collect
phytochemistry information on novel compounds isolated fromC. asiatica in recent years. Shukla et
al., (2000) separated a new ursane triterpenoid from C. asiatica and exhibited its dose-dependent
growth inhibitory activity against larvae of Spilarctia oblique. Later on, Matsuda et al.,(2001) isolated
a new olean-13-ene triterpene, Centellasapogenol A, and its Oligoglycoside fromC. asiatica.
Vol. 1, 69 - 74 [2013]
71
Continuously, in their comparative study on the genetype cultivated in Sri Lanka, however, theyobtained
two new ursane-type triterpeneoligoglycosides, Centellasaponins B and C, and an Oleanane-type
triterpene Oligoglycoside, Centellasaponin D. Kuroda et al, 2001 also seperated 5 new triterpene
glycosides fromthe aerial parts of C. asiatica and none of these saponins revealed significant cytotoxicity.
Moreover, J iang et al., (2005) identified four new triterpenoid glycosides named Asiaticoside C, D, E,
and F fromthe BuOH fraction of C. asiatica. In addition, drawing assistance fromthe technology of
biotransformation, Monti et al., (2005) prepared an array of novel derivatives of asiaticoside with
molecular diversity and functional variety.
Being the chief bioactive substances in C. asiatica, triterpenoid derivatives play an important
role in the aspect of medicinal application. Several of their traditional uses have been scientifically
validated and some of the active principles have also been reported.
Flavonoids : C. asiatica has also been reported to contain
numerous flavonoids, including quercetinand kaempferol, catechin, rutin and naringin, as a major part of
the total phenolic contents, some of which are major contributors in particular to theantioxidative activity
of C. asiatica (Zainol et al, 2003). Based on the hypothesis of free radical mediated toxicity in oxidative
stress process and depending on its antioxidant properties, C. asiatica has been recently indicated to
show anti-lipid peroxidative and free radical scavenging activities(Hussin et al. 2007 ; Wong et al. 2006).
In addition, (Matsuda et al. 2001) isolated a flavonol, petuletin, and kaempferol, 3-O- -D-glucuronide
fromthe aerial parts of C. asiatica cultivated in Vietnam, bothof which exhibited potent inhibitory activity
on aldose reductase inrats. And bioflavonoids of C. asiatica have ever been exhibited to be efficacious in
venous insufficiency, probably due to their actions on mucopolysaccharide metabolism.
Other components : Coherent researches on C. asiatica also revealed the presence of Polysaccharides,
Polyyne-alkene, amino acids, fatty acids, sesquiterpenes, alkaloids, sterols, carotenoids, tannin,
chlorophyll, pectin, inorganic salts, etc.
Phytotherapeutic Action
Wound healing properties : C. asiatica have been shown to produce different actions
on the various phases of wound repair (Suguna et al. 1996). Scientific studies proved that triterpenes
fromC. asiatica stimulated extracellular matrix accumulation in rat experimental wounds, as further
evidenced in vitro by gene-expression alternation in a human dermal fibroblast (Coldren et al. 2006).
Asiatic acid was the only component responsible for the collagen synthesis stimulation, while
madecassoside was able to increase significantly collagen secretion. Advanced studies indicated that
asiaticoside induced type I collagen synthesis via the activation of the TGF- receptor I kinase-
independent Smad pathway, which forged a basis for molecular understanding of Centellas bioactivity
on wound healing (Lee at al. 2006).
Brain stimulating effects : C. asiatica possesses various CNS effects such as stimulatory-nervine
tonic, rejuvenant, sedative, tranquilizer, especially memory improvement and intelligence promoting
property. Someof these bioactivities have been demonstrated experimentally. Scientific findings exhibited
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72
that the aqueous extract of C. asiatica has cognitive enhancing effect and an antioxidant mechanism is
involved (Rao et al. 2005 ; Veerendra et al. 2002). Additionally, C. asiatica leaf extract was not only
showed to improve spatial learning performance and enhance memory retention in neonatal rats during
growth spurt period, but also found efficient in enhancinghippocampal CA3 neuronaldendritic arborization
in rats, thus providing evidence to show the effect of this plant extract on the brain regions involved in
learning and memory (Mohan Das et al. 2005, 2006).
Treatment of venous hypertension and microangiopathy
Studies done in accordance with standardized scientific criteria have shown that triterpenic
components in C. asiatica exhibit a positive effect in the treatment of venous insufficiency and
microangiopathy and several prospective, placebo-controlled, randomized trials convinced the
effectiveness of the total triterpenic fraction of C. asiatica by improving microcirculation, edema and
decreasing capillary permeability (De Sanctis et al. 2001).
Actions on gastric ulcer : Many scientific findings suggest the potential use of C. asiatica and its
active ingredient as anti-gastric ulcers drugs. (Cheng et al. 2004) displayed the healing effects of C.
asiatica water extract and asiaticoside on acetic acid induced gastric ulcers in rats, by significantly
attenuating the myeloperoxidase activity, promoting epithelial cell proliferation and angiogenesis, and
upregulating expression of basic fibroblast growth factor in the ulcer tissues, therefore strengthening the
mucosal defensive factors. Centella extract was also reported to show anti-ulcerogenic activity against
various physical and chemical factors, such as ethanol-, aspirin-, cold-restraint stress- and pyloric
ligation induced gastric ulcers in rats (Sairam et al. 2001). In addition, (Guo et al. 2004) showed that
C. asiatica water extract and asiaticoside have an anti-inflammatory property that is brought about by
inhibition of NO synthesis and thus facilitates ulcer healing.
Anticancer activity :
light on C. asiatica, in search of potential bioactive molecules against tumor. (Babu et al. 1995) found
crude extract of C. asiatica as well as its partially purified fractions exhibited selective cytotoxicity in
vitro and anti-tumour properties in vivo.
Other effects : In addition to above-mentioned activities, triterpenoids in C. asiatica were also claimed
to be effectively applied for anti-bilharzial, antifertility, anti-herpes simplex virus, radioprotection, cosmetics,
immunomodulatoryand antagonizing liver fibrosisAdmitting of no exception, C. asiatica, despite of its
multifarious favorable uses, has been inevitable to show several adverse effects, including mutagenicity,
allergic contact dermatitis, and hepatotoxicity, perhaps mainly evoked by its triterpenoids components.
The requirement of C. asiatica in pharmaceutical industries has been sharply increasing, thus
leading to the over exploitation of this herb. It has already been listed as threatened species by the
International Union for Conservation of Nature and National Resources (IUCN) and an endangered
species. Therefore application of tissue culture approaches for rapid multiplication of elite clones and
germplasmconservation is of vital importance. However, further studies are still needed to be done for
the evaluation of the genetic resources of the plant for variation in morphological, growth, and herb and
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73
yield related characters to identify high herb and madecassol yielding populations suitable for use in
agronomical and plant breeding programs.
A great progress hasbeen made over the past decades in study of biologically active components
and bioactivities of C. asiatica, but the results are still unsatisfactory. More scientific data are required
before recommendation for increase in its utilization can be given with confidence.
References :
Apparao MVR, Srinivasan K and Rao K (1973) The effect of mandookparni (Centella asiatica) on the
general mental ability (Medhya) of mentally retarded children[J ]. J Res Indian Med, 8: 9-16.
Babu TD, Kuttan G and Padikkala (1995) J. Cytotoxic and anti-tumor propertiesof certain texa of umbelliferae
with specific reference to Centella asiatica (L.) urban[J]. J Ethnopharmacol, 48(1) :53-57.
Brinkhaus B, Lindner M and Schuppan D. (2000) Chemical, pharmacological and clinical profile of
the East Asian medical plant Centella asiatica[J]. Phytomedicine, 7(5) : 427-448.
Cheng CL, Guo JS and Luk J (2004) The healing effects of Centella extract and asiaticoside on acetic
acid induced gastric ulcers in rats[J ]. Life Sci, 74(18) : 2237-2249.
Coldren CD, HashimP and Ali J M (2003) Gene expression changes in the human fibroblast induced
by Centella asiatica triterpenoids[J ]. Planta Med, 69(8) : 725-732.
De Sanctis MT, Belcaro G and Incandela L (2001) Treatment of edema and increased capillary filtration in
venoushypertensionwith total triterpenic fraction of Centella asiatica: a clinical, prospective, placebo-
controlled, randomized, dose-rangingtrial[J]. Angiology, 52(Suppl 2) :S55-59.
Guo JS, Cheng CL and Koo MW. (2004) Inhibitory effects of Centella asiatica water extract and asiaticoside
on induciblenitric oxide synthaseduringgastric ulcer healing in rats[J]. Planta Med, 70(12) : 1150-1154.
Hussin M, Abdul-Hamid A and Mohamad S (2007) Protective effect of Centella asiatica extract and
powder on oxidative stress in rats[J ]. Food Chem, 100(2) : 535-541.
J ayashree G, Kurup MG and Sudarslal VS (2003) Anti-oxidant activity of Centella asiatica on
lymphoma-bearing mice[J]. Fitoterapia, 74(5) : 431-434.
J iang ZY, Zhang XM and Zhou (2005) J. New triterpenoid glycosides from Centella asiatica[J].
Helv ChimActa, 88(2) : 297-303.
Karting, T. (1986) Clinical application of Centella asiatica (L) urb. in herbs spices and medicinal
plants : Recent Advances in Botany, Horticulture, and Pharmacology; Vol. 3, Craker LE, Simon
J E (eds) Phoenix, AZ : Oryx Press, 145-173.
Kuroda M, Mimaki Y and Harada H (2001) Five new triterpene glycosides fromCentella asiatica[J].
Nat Med, 55(3) : 134-138.
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Lee J, Jung E andKimY (2006) Asiaticoside induces human collagen I synthesis through TGFbeta receptor
I kinase(TbetaRI Kinase)-independent smad signaling[J]. Planta Med, 72(4) :324-328.
Matsuda H, Morikawa T and Ueda H (2001) Medicinal foodstuffs. XXVI . Inhibitors of aldose
reductase and new triterpene and its oligoglycoside, centellasapogenol A and centellasaponin
A, fromCentella asiatica (Gotu Kola)[J ]. Heterocycles, 55(8) : 1499-1504.
Matsuda H, Morikawa T and Ueda H (2001) Medicinal foodstuffs. XXVII. Saponin constituents of
gotu kola (2): structures of new ursane- and oleanane-type triterpene oligoglycosides,
centellasaponins B, C, and D, from Centella asiatica cultivated in Sri Lanka[J]. ChemPharm
Bull (Tokyo), 49(10) : 1368-1371.
Mohandas Rao KG, Muddanna Rao S and Gurumadhva Rao S. (2005) Centella asiatica (linn) induced
behavioural changes during growth spurt period in neonatal rats[J ]. Neuroanatomy, 4(1) : 18-23.
Mohandas Rao KG, Muddanna Rao S and Gurumadhva Rao S. (2006) Centella asiatica (L.) leaf
extract treatment during the growth spurt period enhances hippocampal CA3 neuronal dendritic
arborization in rats[J ]. Evid Based Complement Alternat Med, 3(3) : 349-357.
Monti D, Candido A, Silva MMC. et al. (2005), Biocatalyzed generation of molecular diversity:
selective modification of the saponin asiaticoside[J]. Adv Synth Catal, 347(7-8) : 1168-1174.
Oyedeji OA and Afolayan AJ . (2005) Chemical composition and antibacterial activity of the essential
oil of Centella asiatica growing in South Africa[J]. PharmBiol, 43(3) : 249-252.
Rao SB, Chetana M and Uma Devi P. (2005) Centella asiatica treatment during postnatal period
enhances learning and memory in mice[J ]. Physiol Behav, 86(4) : 449-457.
Sairam K, Rao CV and Goel RK. (2001) Effect of Centella asiatica Linn on physical and chemical
factors induced gastric ulceration and secretion in rats[J]. Indian J Exp Biol, 39(2) : 137-142.
Shukla YN, Srivastava R,Tripathi AK, et al. (2000) Characterizationof an ursane triterpenoid fromCentella
asiatica withgrowth inhibitory activity against Spilarctia bliqua[J ]. PharmBiol, 38(4) :262-267.
Suguna L, Sivakumar P and Chandrakasan G. (1996) Effects of Centella asiatica extract on dermal
wound healing in rats[J ]. Indian J Exp Biol, 34(12) : 1208-1211.
Veerendra Kumar MH and Gupta YK. (2002) Effect of different extracts of Centella asiatica on
cognition and markers of oxidative stress in rats[J]. J Ethnopharmacol, 79(2) : 253-260.
Wong SP, Leong LP and Koh J HW. (2006) Antioxidant activities of aqueous extracts of selected
plants[J ]. Food Chem, 99(4) : 775-783.
Zainol MK, Abd-Hamid A, Yusof S, et al. (2003) Antioxidativeactivity and total phenolic compounds of leaf,
root and petiole of four accessions of Centella asiatica (L.) Urban[J ]. Food Chem, 81(4) : 575-581.
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ETHNO BOTANICAL STUDIES ON MACROPHYTES OF BHOJPUR,
(BIHAR)
Babita Singh
Department of Botany,
Patna Science College, Patna 800005.
Abstract : Bhojpur forms the district of Bihar Aquatic angiosperms of this area have been almost
ignored. This group of plants has got the potential for exploitation as animal feed, human food, medicines,
soil additives and fuel production. Present paper deals with the taxo-ethno-botanical information of
some of important macropyhtes of Bhojpur. About 10 plants have been described. The information has
been collected fromthe reliable and authentic sources by paying the periodical visit to collect aquatic
weeds for voucher specimens as well as to get the ethno-botanical informations fromthe old persons
and practioners of ayurvedic medicines.
Key words : Ethno-Botanical studies, Macrophytes, Bhojpur
Introduction : Ethno-botany is a branch of botany dealing with the utilization of plants and their parts
by the tribals or rural people fromthe times immemorial. It is a science in which the relationship between
the tribals people and the plants studied and deals with the fact that plants have close relationship with
man directly or indirectly almost in every field. Not much work has been done except S.K Jain (1963),
Bhargava (1981), Mahesh wari and Singh (1984).The plants growing around have greatly influenced
the natives fromtime to time directly or indirtectly Hindus use Ocimum sanctum(LAMIACEAE) to
bathe their idols. Flowers of Nelumbo nucifera , Nymphae stellata and Nymphea nymphoides are
offered to lord Shiva. People use aquatic weeds as fodder and also as vegetable after cooking, The
tribals use some of the aquatic weeds for burns cut etc for themselves and for their burns or cut.
Materials and Methods : Periodical visits were made to visit all places of Bhojpur for the collection
of aquatic and wetland angiosperms. Information regarding the ethno-botanical and ethno-medicinal
plants were collected from men and women of all the spheres of life. Elderly persons in the remote
areas treating tribal people by the local vaidya were also consulted. Repeated quarries were made to
get data verified and confirmed.
Observation and Result : Observations made during the course or this study are enumerated as such :-
1. Trapa natans Linn.
Family : TRAPACEAE
Common Name : Singhara
Taxonomic Notes : A much branched, annual, aquatic rooted herb, with asssimilatry root stock,
Leaves :rhomboid, with swollen petiole and purple-tinged beneath. Flower : white, solitary axillary.
Sepals : Persistent, Spinous. Nut : angled.
Commonly grown for fruits but occasionally found growing in pond as escape.
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Medicianl use : The flour of dried nuts are laxative. The unripe nut are used in the treatment of
abdominal disorders.
2. Utricularia inflex (Linn.) Cl.
Family : LENTIBULARIACEAE
Common name : Bladder wort
Taxonomic Notes : A wild annual, rooted, aquatic herb, with leaves pinnately divided into capillary
segments each with small bladder at their base. Flower : 3mmacross, yellow in aerial raceme. Calyx :
acrescent. Capsule : globose, with minute seeds
Commonly found growing in shallow water of ponds and paddy crop feild.
Medicinal use : The paste of flowers are applied externally in headache.
3. Typha ungustata Bony & chaub
Family : TYPHACEAE
Common name : Patera
Taxonomic Notes : A wild, annual, unbranched grass, with leaves exceeding the flower which are
arranged in cylindric spikes. The male and female flowers are seprated by a long interval. Female :
pale-brown Male flower :mixed with clavate toipped pistillodes.
Commonly found growing on the edges of ponds and ditches.
Medicinal uses : The infusion of floral spikes are used in the treatment of sterility among women.
4. Alternanthera philoxeroides (mart.) Griseb
Family : AMARANTHACEAE
Common name : jangli kulphi
Taxonomic Notes : A wild annual, aquatic herb with rooting at nodes. Stem: fistular, glabrous. Leaves
: oblong lanceolate, opposite decussate. Flowers :6mmlong, white, in axillary, globes head. Bracts and
bracteoles : membranous, subequal: persistent.
Commonly found growing on the slopes of pond and abundant in ditches.
Medicinal use : The paste of entire plants are used in diarrhoea of cattles.
5. Ranunculus sceleratus Tulin
Family : RANUNCULACEAE
Common Name : jaldhania
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77
Taxonomic Notes : A wild annual subaquatic herb, 40-50 cm tall with, fistula internodes, Leaves
variable, lower long petioled while uppermost sessile. Flowers : 6-9 mmacross, yellow. Achenes :
obliquely abovate on an ablong receptacles called torus.
Commonly found growing in most places on the bank of rivers pond and ditches
Medicinal use : Extract of Leaves are used in intermittent fevers
6. Nymphaea stellata Willd.
Family : NYMPHAEACEAE
Common name : Bhent
Taxonomic Notes : A wild annual, rooted aquatic herb, with rotund leaves which gets streaked with
purple beneath, Flowers : white conspicuous in solitary axillary on highly elongated peduncle.
Commonly found growing in shallow stagnant water of ponds and ditches.
Medicinal use : The decoction of roots are applied externally in sores as antiseptic
7. Potamogeton indicus Roxb.
Family : POTAMOGETONACEAE
Common Name : Wild Kumbhi
Taxonomic Notes : A wild annual, floating herb, with purple streaked internodes. Submerged Leaves
lanceolate, thin while Floating ones elliptic, thicker. Stipules : scarious. Spikes above the surface water.
Commonly found growing in shallow water of ponds .
Medicinal use : The Leaves are diuretic and used in kidney problems.
8. Ceratophyllum demersum Linn
Family : CERATOPHYLLACEAE
Common Name : Kajri
Taxonomic Notes : A wild annual rootless, submerged, aquatic herbs, with verticillate leaves. Flowers
: unisexual monoecious. Male & Female flowers : solitary. Nutlets : avoid, coriaceous.
Commonly found growing in shallow water of ponds and ditches
Medicinal use : The Paste of entire plants are used externally in cutaneous affections .
9. Nymphaea nymphoides Rox
Family : MENYANTHACEAE
Common Name : Kumudini
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Taxonomic Notes : A wild annual rooted aquatic herb, with rooting at nodes . Leaves :suborbicular,
with purple-tinged beneath. Flower : white, in clusters at the base of petiole.
Commonly found growing on the edges of ponds and canals
Medicinal use : The paste of leaves are used in jaundice.
10. Ammania baccifera Linn.
Family : LYTHRACEAE
Taxonomic Notes : A wild, annual, erect, glabrous herb, with tap-root stock. Stems : angular, with
variable length of internodes, Leaves : Opposite, lanceoiate, with cuneate and 1- nerved at base.
Commonly found growing in moist paddy field after harvesting.
Medicinal use : The paste of root are diuretic.
References :
Bhargava, N. 1981 Plants in folk life and folk love in Andaman and Nicobar Island.
J ain, S.K., 1983. Studies in Indian Ethnobotany, IRL. Bull, 1(2): 126-128.
J ain, S.K., 1980. Glimpses of Indian Ethnobotany, Oxford and IBH Publishing company, New Delhi.
Maheshwari, J.K. and J.P. Singh, 1984 Contribution of the ethno-botany of Bhora Tribe of Bijnor and
Pauri Garhwal district, U.P.J . Econ, Taxon. Bot 5:251-259.41
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FLAVONOID DIVERSITY AND SYSTEMATIC EVALUATION OF
CROTOLARIA SPECIES
Fahmida Rahman*, Naheed Ahmad**, Md. Khursheed Alam***
*Research Scholar, Department of Botany, Patna University, Patna - 5
**Associate Professor, Department of Botany, Patna University, Patna - 5
***Guest Faculty, B.Sc. Biotechnology, Patna Science College, Patna - 5
Abstract : Crotolaria belongs to family Fabaceae, comprising of both wild and cultivated species.
The genus having tremendous economic and pharmaceutical importance. It has eluded classification
till today due to presence of homoplastic characters. Various species of Crotolaria are rich in
secondary metabolites and other phytochemicals like phenols, flavonoids, anthocyanins, alkaloids
etc. They are best chemosystematic markers and are very much helpful in taxonomic characterization
of the genus Crotolaria. In the present study, distribution of flavonoids in two species of Crotolaria
viz. Crotolaria juncea L. and Crotolaria striata DC has been compared and their chemosystematic
evaluation carried out.
Key words : Flavonoid, BAW, Spectrophotometer
Introduction : The genus Crotolaria of the family Fabaceae has been withstanding adequate general
classification for more than 100 years. It can serve as a group where the distribution of some key
characters are contradicted by the presence of others.
The large amount of the homoplastic characters has often lead to extreme lumping of genera
in order to minimize contradiction in character distribution. Attempts have been made to get a homogeneric
delimitation (Van Wyk and Schutte 1995). However till today genus Crotolaria has ambiguous
classification.
The present study is an attempt to make insight into the flavonoid patterns of two species of
Crotolaria named C. juncea L. and C. striata DC.
The flavonoids are a group of polyphenolic compounds of C
3
C
6
C
3
basic skeleton,
which are widely distributed throughout the plant kingdom and consists of about 300 varieties.
Some of the important categories are Flavones, Flavonones, Flavonols, anthocyanin etc. They
are secondary metabolites and their production are genetically and physiologically controlled,
hence they are stable and reliable characters for taxonomic analysis of any taxa (Heywood V.H.
1973). These are also used as active principle against various diseases (Crawford D.J . 1978).
Multiple mechanismhave been proposed to explain the diversity of phytochemicals
between different plants (Harborne J.B. 1977). The structural variation of flavonoids in each plant
group is due to change in number and position of hydroxyl constituents of methyl and occasionally
isoprenoid group.
Vol. 1, 79 - 83 [2013]
80
Materials and Methods :Leaves of 2 species of Crotolaria namely C. juncea and C. striata were
analysed for their flavonoid compounds.
The species were collected through extensive survey of Patna and its locality. These were
studied morphologically and grown in the Botanical Garden of Deptt. of Botany Patna University. C.
juncea L. was shrub with linear oblong leaves and yellow flowers, whereas C. striata DC. was
undershrub having long petioled trifoliate leaves and yellow flowers.
For extraction of Flavonoid 1 gm. of dried leaves were extracted with methanol and
distilled water in 1 : 1 ratio, at room temperature. Extracts were Centrifuged at 8000 rpm for 5
minutes, pellets discarded and supernatant collected as Extraction sample, concentrated on
water bath for 10 minutes and chromatographed two dimensionally on whatman No. 1 paper
using 2 solvent combinations, i.e. BAW (n-Butanol; Acetic acid glacial : Water, 4:1:5) versus
15% Acetic acid and BAW versus distilled water, following standard procedure of Harborne
J .B. (1970).
Flavonoids were identified by comparing with authentic makers along with R
f
values and
colour in UV light before and after fuming with Ammonia vapours.
Compounds wererepeatedly purified bypaper chromatography, till the absorption properties
became constant. Hence and elute of a paper blank in 95% ethanol (usually about 150 cm
2
) was taken
and applied (spotted) to the paper, and run in BAW and 15% HOAc, separately. After the purification
of compounds, the spots of chromatogramwere cut and shaken in 95% ethanol for 30 minutes. The
solution was filtered and allowed to concentrate, and directly used for spectral analyses on UV-240
spectrophotometer.
Results and Discussion : A total of 21 spots were observed in the chromatographic profiles of the
two species of Crotolaria. The total numbers of spots in C. juncea were 15 and in that of C. striata
were 12. The reported R
f
and spectral values helped in identification of flavonoid compounds. Out of
21 spots, only 10 could be identified. (Table-1)
Characterisation of the compounds revealed that the occurrence of Quercitin, Fricin,
Apigenin, Genticin and Luteolin-6 glucosides were common to both the species of crotolaria. C.
juncea was distinct in its profile due to the presence of Butien, Naringin, Vixetin and Kaempferol.
Whereas, C. striata was charactersied by Isovitexin and Diadzein. Luteolin was also detected in
these two species.
The paired affinity values between species considered was 40. The group affinity was 140
and isolation values were 40% and 45% in C. juncea and C. striata respectively.
Work on flavonoid of Crotolaria has been carried out by various workers. Like
Subramanian SS and Nagarajan S. 1969, reported Luteolin-4-glucoside in C. retusa.
Dampsey, J .M. 1975, reported presence of 3,5,7- trihydroxy, flavone in aerial parts of C.
alata.
Vol. 1, 79 - 83 [2013]
81
Morris and Kays 2005, reported that most of the crotolaria species contain flavones like
Apigienin & Luteolin. The present investigation is substantiating their views.
The two species C. juncea and S. striata, although possess morphological plasticity. They
are completely distinct in their flavonoid pattern and their separate identity within the genus Crotolaria
L. is justified.
References :
Crawford, D.J . (1987) The Bot. Rev., 4431 .
Dampsey, J .M.. (1975) Fiber Crop. The University Press of Florida, Gainesvilla, Fla .
Harborne, J.B. (1977) Biochem. Syst. Evol., 5,7 .
Heywood, V.H., (1973) Pure and Appl. Chem., 34, 355.
Morris, J.B. and Kays, S.E. (2005) Total dietary fiber variability in a Cross Section of C. juncea
genetic resources Crop Sci. 45 : 1826-1879 .
Subramanian, S.S. and Nagarajan, S. (1969) Flavonoids of the Seeds of Crotalaria retusa and C.
striata. Curr. Sc. (India) 3865.
Van Wyk, B.E. and Schutte, A.L.. (1995) Phylogenetic relationship in the tribes podalyrieae, Liparieae
and Crotalarieae. In Advances in legume systematics Edited by M. Crisp and J.J . Doyle,
Royal Botanic Garden, Kew, U., pp. 283-308 .
Vol. 1, 79 - 83 [2013]
82
TABLE-1 :
Flavonoid profile and Chromatographic analysis of C. juncea L. and C. striata DC.
Fluorescence Rf 100 Spectral values
Spot
s
No.
Visible
light
UV UV
+NH
3
BA
W
15%
AcO
H
39%
AcO
H
Wat
er
Phen
ol
Forest
al
EtOH EtOH
+NaOAc
EtOH
+H3PO
4
Chemical
indentity
1. Ns Br.g
r
Br.g
r.
13.1 86.5 +
2. Br Br Br 15.4 80.2 +
3. D.Br 20.2 0.5 63.1 250;30
2,
330,40
5
301,320
380,420,5
10
200,30
1
?
4. Br.ochr
e
69.8 86.0 51 +
5. YL 70.8 224,28
5,
332
Hesperidi
n
6. Br.ochr
e
47.2 60 63.1 60.1 261 270,368 270,34
4
Luteolin
7-
gl ucoside
7. YL 56.0 227,
280,
335
Naringin
8. L. YL 58.6 253.37
0
261,265 380 Querceti
n
9. YL. Gr. 65.0 72.1 8 85.1 70.8 245,26
5,
Tricin
10. F. YL. 7.5 30 71.3 220,28
0, 345
Phlorodiz
in
11. YL. 75.4 64.2 + ?
12. Br. 76.6 0 65.0 260,38
0
Butein
Vol. 1, 79 - 83 [2013]
83
Fluorescence Rf 100 Spectral values
Spot
s No.
Visible
light
U
V
UV
+NH
3
BA
W
15%
AcO
H
39%
AcO
H
Wate
r
Phen
ol
Forest
al
EtOH EtOH
+NaOA
c
EtOH
+H3PO
3
Chemical
indentity
13. Br 77.8 56.1 0 57.0 54 265,36
6
Kaempfer
ol
14. Br.YL 83.5 +
15. F.YL 85.6 65.9 87 82 265331 Apigenin
16. D.Orch
e
92 10.2 + ?
17. F.Gr 93 23.6 + ?
18. Grey 95.9 + ?
19. Grey 90.1 + ?
20. D.Br. 27.4 65.2 + ?
21. Ns. +
Vol. 1, 79 - 83 [2013]
84
85
PHYSIOLOGICAL EFFECTS OF GIBBERELLIC ACID ON
CYTOPLASMIC-GENETIC MALE STERILE ( CMS ) LINE OF RICE.
R.K. Mandal* and Shyam Deo Mehta**
*Associate Professor in Botany, Patna Science College, Patna
**Principal, GautamBudh Evening Collage, Kumhrar, Patna.
Abstract : Gibberellic acid (GA20) on various concentrations viz-10 PPM, 20 PPM and 30 PPM
was sprayed on cytoplasmic. genetic male sterile (CMS) line of rice (V20A) to obserce its effect on
various agronomic characters viz-plant height, panicle exsertion and seed setting percentage. With
increase in GA concentrations there were increased in all asronomic characters included in the experiment
But by use of 30 PPM of GA on CMS line V20A panicle exsertion and seed setting percentage
increase were maximum. Hence 30 PPM concentration of GA should be recommended for seed
production.
Ke y words : Gibberellic acid, V20A, panicle exsertion, seed setting percentage, 30 PPM
concentration
Introduction : With the spectacular success in hybrid rice breeding in China, a newvista has emerged
in rice production. The hybrid rice varieties developed in China not only recorded an increase of 20-
30% in yield in comparison to the best commercial varieties, but were also claimed to be resistant to the
disease and pests. The tolerance of environmental stress and fertilizer response of the hybrid rice were
reported to the superion Jones (1926) This success attracted the attention of rice breeder all over the
world on account of yield advantage and superior physiological efficiency of the hybrid rice. The desirable
characters observed in the CMS lines were shorter plant height, better tillering ability, longer panicle
length, longer duration of flowering and flower opening all favouring a higher seed set. Among the
drawbacks evident in the (CMS) liner were poor panicle exsertion and lower stigma exsertion, This
causes decrease in yield.
Materials and Method : The cytoplasmic genetic male sterile line viz-V20A obtained from
International rice research Institute (IRRI) Manila, Philippines was grown in the field during kharif to
see the effect of different doses of gibberellic acid on various agronomical characters viz-Plant height,
panicle exesrtion and seed setting percentage. The experiment was conducted in randomized block
design with four replications and three concentration of gibberellic acid i.e 10 PPM,20 PPM and 30
PPM were used. these three concentration of GA were applied once on CMS line V20A at the time of
initial heading and observation was made after 20 days of GA application and seed setting percentage
was recorded after 25 days on 10 plants.
Vol. 1, 85 - 87 [2013]
86
Results and Discussion : Observation on Plant height, panicle exsertion and seed set as influenced
by the spray of gibberellic acid are given it Table 1.
It was observed that plant height in CMS line was increased in all doses of GA treatment. The
final plant height was maximumat 30 PPM concentration of GA. The increase in plant height in CMS
line was. not desirable character as it decreases seed set percentage Rutger and carnahan (1981),
Chaudhary et.al (1982), Jones Duan et.al (1997) and Ahmed et.al (1988)
In the present investigation it was recorded that the spraying of 20 PPM and 30 PPM
GA on CMS line improved the panicle exsertion. In control it was observed that 1/3 part of
panicle remained inside the sheath which results in poor seed set on CMS lines Line and Yuan,
(1980) also recommended spraying of 20 PPM GA at heading on CMS lines for improving
panicle exsertion and seed set.
Due to poor panicle exsertion seed set on CMS lines were poor. I t fact one third to half
panicle remain enclosed in the leaf sheath and can not receive any pollen and hence do not get
seed. Poor panicle exsertion in a CMS line was considered to be due the presence of sterile
cytoplasm. The sterile cytolplasm inhibited panicle exsertion (Virmani and Athwal, (1973), Lin
and Yuan, (1980), Virmani et.al (1985) and chenXionghu of al (1996).; In the present experiment
it was found that spraying of 10 PPM, 20 PPM and 30 PPM concentration of GA increased the
seed set percent 32.1, 49.5 and 57.9 respectively. This is in agreement with the result of Lin
and Yuan (1980) and Honda et.al (1996). But in present investigation it was found that 30
PPM, GA should be recommended instead of 20 PPM for general spraying on CMS lines in
seed production plots.
Table 1.
Effect of Gibberellic Acid on cytoplasmic genetic male sterile line V20A
Vol. 1, 85 - 87 [2013]
87
References :
Ahmed, M.I, Singh, S.Viraktamath. B.C., Ramesha M.S. and Vigakumar, S.S. and Khush, G.S. (1982);
Hybrid and composite breeding approach for rainfed lowland rice. IRRI Saturday seminar on
9
th
January (1982). Los Banos, Philippines.
Chen Xionghce, wan Banghce, Wu changwel and Liang kegin. (1996). Study on the flowering habit of
photo-thermo sensitive genic male sterile rice. Journal of south China Agri. Univ. 2: 1-6.
Honda, I., sado, I; Yanagiswa, T.; kato, H., ikeda, R.; Hira sawa, it and takashashi, N. (1996)
characterization of endogenous gibberelli As in dwarf rice mutants. Bioscience, Biotechnology
and Biochemistry. 6 (12) : 2073-2075.
J ones, Duan; Liang cheng ye; Huang Yuwen; Lie Hong Xian. (1997). Studies on seed setting percentage
of hybrid rice. Journal of Tropical and subtropical Botany. 5 (1) : 71-77. J un J.W. (1928)
Inheritance of earliness and other agronomic characters in rice. J our. Agric Res 36 : 581-601.
Lin, S.C. and Yuan, L.P. (1980). Hybrid rice breeding in china. In Innovative approaches to rice
breeding. PP. 36-51 Int. Rice Res. Inst-, Los benos, Philippines.
Rutger, J .N. and Carnahan, H.L. (1981). Crop Sci. 21: 373-376
Virmani, S.S. and Athwal, D.S. (1973). Genetic Variability for floral characters influence of out crossi
of in oryza sativa L. crop. Sci. 13 : 66-67.
Virmani, S.S.; Rajik Govinda, Dalmacio, R.D. and Aurin, P.A. (1985). Current Knowledge of an out
look on cytoplasmic-genetic male sterility and fertility Restoration in Rice. In Rice Genetics PP.
633-647. Proc. Int. Rice Genet. Symp., 27-31 May (1985). Int. Rice Res. Inst. P.O. Box 933.
Manila, Philippines.
Vol. 1, 85 - 87 [2013]
88
89
EFFECT OF MIXED SOLVENTS ON THERMODYNAMIC
PROPERTIES OF 4-NITROPTHALIC ACID
Dilip Kumar Verma*, Rajnish Kumar Singh and Prahlad Kumar
Department of Chemistry, Patna University, Patna-800005
Email : rajnishkumarsingh72@gmail.com.
Abstract : The study of solvent effect (water-dioxan mixture) on thermodynamic properties of 4-
nitropthalic acid was done at 25
0
C by fixing ionic strength. The PKa value increases with increase
in mole percent of dioxane which is explained with dielectric constant of medium. The free energy
change is also calculated to illustrate the effect of mixed solvent on thermodynamic properties of
organic acid.
Keywords : Nitropthalic acid, water-dioxan mixture, free energy change, ionic strength, dielectric
constant.
Introduction : The thermodynamic properties depend on many factors such as charges and size of
the constituent ions, environment of the mediumand temperature of the system. The study on
solvent on dissociation equilibria is much revealing about the structure of ions which is essential for
satisfactory understanding of the reaction. This is essential not only to confirm . The nature of ion-
solvent interaction and support the model suggested in aqueous mediumbut also to investigate, the
role of solvent in changing the model. A lot of works
1-5
have been done regarding the study of the
effect of mixed solvents. Our main purpose is to observe the effect of mixed solvents on thermodynamic
properties of organic acid.
Expe rimental Method : Potentiometric method which has been proved to be very accurate
method for determining the dissociation constant of acid in aqueous mediumcan be employed
satisfactorily for other mediums also. The dissociation constant of 4-nitropthalic acid in water
dioxan mixture composition varying (from 5% v/v to 25% v/v dioxan) at 25
0
C and at constant ionic
strength was determined. The ionic strength was made constant using sodiumchloride. The values
of dissociation constant PK
1
, G
0
, dielectric constant of o-nitropthalic acid at 25
0
C in various
compositions of dioxan- water mixture are given in table I.
Results and Discussion : When data in Table (1) was analysed, it is seen that PK increase with
increase in mole percent of dioxan. The variation of PK values with change in solvent composition
can be analysed in terms of variation of dielectric constant of medium. The dielectric constant of the
mediumwill be affected by the presence of ions. The increasing dioxan percentage in solvent
mixture is either to decrease bulk dielectric which disfavours dissolution or to increase the basicity
of the mediumwhich favours dissociation. A number of workers
6-8
have justified the former case.
This is also seen in variation of PK against 100/D in table I.
Vol. 1, 89 - 90 [2013]
90
It is seen that G
0
value is positive. This shows that unionized from will be more abundantly
populated than the ionised from.
9
The addition of dioxan increases G
0
values. This means that
ionization becomes more and more difficult with increase in the percentage of dioxan. This indicates
that proton exchange between dioxan molecule and acid molecule is less prominent than the effect of
dielectric variation. Otherwise, dioxan being more basic than water would have facilitated ionisation
resulting in the decrease of G
0
values.
References:
Rosotti F.J.C. and Rosotti H.S. (1961) : The determination of stability constant McGraw Hill, New
York, P.27.
Bag S.P. and Lahiri, S. (1975) J . Indian Chem. Soc., 52, 30.
Denison J.T. and Ramsay, J .B. (1955) J . Am. chem. Soc., 22, 2615.
Dunsmore H.S. and Speakman J.C. : (1954) Trans Farad. Soc.,20, 236.
Gilkerson, W.R. (1956) J. Chem. Phys., 25, 1199.
Kesherwani, A.K. & Khan, F. (2002) Bull. Electrochem.,18, 413.
Shabana BegumS, Siva Kumar , C.L. Mayanna S.M. and Murlidharan V.S. (2000) Electrochem.
Acta, 18, 89.
Singh, Ratan, Verma P.S. & J ain D.S. (1991) Electrochem. Soc. India, 7:, 40, 47.
Srivastava, S.B. Prakash Omand Prakash Sheo (1975) J. Chem. Thermodynamics, 7, 997.

TABLE I
Values of 100/D, Dissociation Constant, PK, G
0
at 25
0
C
Vol. 1, 89 - 90 [2013]
91
ULTRAVIOLET-VISIBLE & INFRARED SPECTRAL STUDY OF
COMPLEXES OF CO (III) WITH MIXED LIGANDS BIGUANIDE C
2
H
7
N
5
AND PYRIDINE C
5
H
5
N
Bina Rani*, Madhu Kumari Gupta** and Radhakant Prasad***
*Reader in Chemistry, Magadh Mahila College, Patna University, Patna-1
**PGT (Chemistry), Kendriya Vidyalaya, Bengdubi, Darjeeling, West Bengal.
***Professor, Dept. of Chemistry, Patna Science College, Patna University, Patna-1
Abstract : Complexes of transition metal cobalt in oxidation state III with mixed ligands biguanide
(C
2
H
7
N
5
) and pyridine (C
5
H
5
N) have been prepared and their elemental studies have been studied. In
this research paper, an effort has been made to characterize their spectroscopic characters. The blue
prints of the structure of the coordination complexes which have been prepared are illustrated with the
help of UV & IR spectroscopic data.
Key words:- UV (Ultraviolet & visible), IR (Infra-red), biguanide, pyridine, transition metal.
Introduction : Biguanide sulphate C
2
H
7
N
5
H
2
SO
4
H
2
O is a bidentate chelating molecule and its
complexes with bivalent and trivalent ions are known
1-4
.
HN C NH C NH
(1) || (3) || (5)
NH NH
(2) (4)
3 3
+ +
Biguanide : The ligand biguanide is found to coordinate with N(2) and N(4) as shown in figure given
above forming usual six membered chelate rings with metal atoms fromeach biguanide unit. The
coordination complexes of a number of biguanides with various metal ions have been investigated
extensively
5,6
.
Biguanides may be prepared by the condensation of two molecules of guanidine.
-NH
3
H2N C NH H + H2N C NH2
|| || Fusion
NH NH
Guanidine guanidine
Vol. 1, 91 - 101 [2013]
92
H2N C NH C NH2
|| ||
NH NH
Biguanide or diguanide
Actually, guanidine hydrochloride is used and fused at 180-185
0
C. Biguanide may also be
regarded as guanylguanidine. The name diguanide is not as prevalent as one finds difficulty while
naming dibiguanides for in this case one has to call di-diguanide.
The pyridine is an organic molecule which acts as a ligand. Many coordination compounds
have been prepared till now containing pyridine as a ligand. It coordinates with its nitrogen atompresent
in the ring.
pyri dine
The detailed studies on structural and biochemical aspects of coordination complexes of mixed
ligands (bidentate biguanide and pyridine) with cobalt metal are lacking. To investigate the coordinating
behaviour of bidentate biguanide C
2
H
7
N
5
and pyridine C
5
H
5
N we have prepared and characterized the
complexes of Co (III) with mixed ligands.
Results and discussion : The acidic solution of CoSO
4
, cobalt sulphate reacts with basic solution of
biguanide [BigH
2
+
] (OH
-
)
2
forming yellow silky precipitate of [Co (BigH
+
2
)
3
] (OH)
2
was obtained
which was filtered quickly to avoid oxidation on the buchner funnel and was washed with ice cold
water. The yellow coloured complex so obtained was then mixed with a little amount of distilled water
to prepare the suspension of the complex which was then transferred to an aeration flask. To this
suspension a small amount of pyridine, C
5
H
5
N (A.R.) was added and then a brisk current of air was
passed through it to oxidize Co (II) complex to Co (III) complex [Co (BigH)

2
(Py)
2
](OH)
3
(Dipyridinebisbiguanidium-cobalt(III)hydroxide). Due to aeration, the silky yellow bisbiguanidiumcobalt
[II] hydroxide [Co (BigH
+
2
)
3
] (OH)
2
gradually dissolved to a dark red solution with the separation of
a slight black oxide of cobalt due to decomposition of the complex. The mixture was then filtered
through a quantitative filter paper and the filtrate was left in cold for crystallization. The complex
sulphate[Co(BigH)
2
(py)
2
]
2
(SO
4
)
3
.12H
2
Owas obtained when red solution of the complex base
[Co(BigH
+
)
2
py
2
](OH)
3
was neutralization with dilute sulphuric acid in cold as red crystals associated
with a small amount of the trisbiguanidiumcobalt (III) sulphate. The complex nitrate, [Co(BigH)
2(
py)
2
](NO
3
)
3
, was obtained by the neutralization of the solution of the complex base
Vol. 1, 91 - 101 [2013]
93
[Co(BigH)
2
py
2
](OH)
3
with dilute HNO
3
(nitric acid)

in cold. The complex carbonate [Co(BigH)
2
(py)
2
]
2
(CO
3
)
3
was prepared by treating the complex base [Co(BigH)
2
py
2
](OH)
3
witheither ammonium
bicarbonate NH
4
(HCO
3
) or sodium bicarbonate NaHCO
3
.

The complex chloride [Co (BigH)
2
(Py)
2
]
Cl
3
.6H
2
O was prepared by dissolving the carbonate in dil. Hydrochloric acid.
Dipyridinebisbiguanidiumcobalt (III) thiosulphate[Co(BigH)
2
](S
2
O
3
)
3
6H
2
O wasprepared by neutralizing
the complex chloride with solution of sodiumthiosulphate dropwise.The complex oxalate [Co (BigH)
2
(Py)
2
]
2
(C
2
O
4
)
3
.8H
2
0 was prepared either by neutralizing the complex base with oxalic acid or by
adding sodiumoxalate to the complex chloride solution.
The trihydroxide was soluble in water and was alkaline to litmus. The sulphate was soluble in
hot water and nitrate was in alcohol. Besides this all complexes were found to be soluble in DMSO.
In case of Dipyridinebisbiguanidecobalt (III) complexes, cobalt has +3 oxidation states with
low spin type. The electronic absorption spectra of the complexes display band near 500nm(20,000cm
-
1
) and 357nm(28,000cm
-1
). This is attributed to transition of d-electrons from
1
A
1g
(ground state) to
next upper higher level
1
T
1g
and
1
T
2g
states respectively. In Co (III) complexes the spin forbidden
transition
1
A
1g
5
E
g
or
1
A
1g
5
T
2g
are seldom observed.
Here,
uv 2 =Dipyridinebisbiguanidiumcobalt (III) sulphate
uv 3 = Dipyridinebisbiguanidiumcobalt (III) thiosulphate
uv 4 =Dipyridinebisbiguanidium cobalt(III)oxalate
The appearance of strong absorption bands in the region of 4000cm
-1
to 2500cm
-1
usually
comes fromstretching vibrations between hydrogen and some other atoms with mass 19 or less. The
OH and NH stretching frequencies fall in the 3700 to 2500 cm
-1
region with various intensities.
The ligand biguanide sulphate (BigH
+
HSO
4
-
).H
2
O contains =NH, NH, NH
3
+
and SO
4
2-
groups.
The various modes of IR vibrations of =NH, NH, N
+
H
3
and SO
4
2-
groups display IR bands in
3350 to 625 cm
-1
region. The NH stretching in ammonia and alkyl derivatives of ammonia is observed
in the region 3500-3300cm
-1
. The position of absorption depends on the degree of H- bonding. The
NH and NH
2
stretches are observed between 3479.4 to 3239.7 cm
-1
.
The NH stretching in N
+
H
3
group is obtained at 3156 and 3022.9 cm
-1
. Further the peaks
2581.6 cm
-1
, 2401.6cm
-1
and 2265.3 cm
-1
are due to N H stretching in >N
+
H group. The NH and
NH
2
deformation vibration observed at 1522.3 cm
-1
and -NH
2
vibration at 1427.5 cm
-1
. NH
wagging observed at 762cm
-1.
The CN (unconjugated) stretching observed at 1025.9 cm
-1
and
928.3 cm
-1
. The C=N stretching observed at 1658.2cm
-1
.

Vol. 1, 91 - 101 [2013]
94
The ionic sulphate group displays as broad and very strong band at 1216 cm
-1
due to
3
vibration of SO
4
2-
ion. The strong and sharp band at 528cm
-1
is attributed to
4
vibration of SO
4
2-
group. The free sulphate ion has regular tetrahedral symmetry (Td). On formation of bond with metal or
hydrogen atomwith oxygen or atoms of sulphate group
[10.]
. The sulphate ion has four different modes of
vibrations
1
,
2
,
3
and
4
. The
1
and
2
vibrations of sulphate are not IR active. The
3
and
4
vibrations split into two bands while the
3
and
4
split into three bands on bidentate bonding of sulphate
group. The bidentate bonding of sulphate acts as chelating or bridging molecule
[11]
. The IR spectra of
chelating or bridging sulphate groups are differentiated clearly by position of splitting of
3
(SO
4
2-
)
stretching vibrations
[10, 12]
.
As it is well known the N H stretching decreases on complex formation with metals which is
shown in the complexes M
1
, M
2
, M
3
, M
4
, M
5
, M
6
& M
7.
All these complexes have shown the presence
of Co N & N Co N vibration near 500nm.
Here,
M
1
=Dipyridinebisbiguanidium cobalt (III) hydroxide
M
2
= Dipyridinebisbiguanidium cobalt (III) sulphate
M
3
= Dipyridinebisbiguanidium cobalt (III) nitrate
M
4 =
Dipyridinebisbiguanidium cobalt (III) chloride
M
5 =
Dipyridinebisbiguanidium cobalt (III) carbonate
M
6
= Dipyridinebisbiguanidium cobalt (III) oxalate
M
7
= Dipyridinebisbiguanidium cobalt (III) Thiosulphate
M
10
= Biguanide sulphate monohydrate
All these complexes have shown the presence of Co N & N Co N vibration near 500nm.
In the high frequency region (about 650cm
-1
), the pyridine (py) vibrations show very little shift
upon complex formation
[11]
. However, those at 604(in plane ring deformation are shifted to higher
frequencies upon co-ordination to a metal.
Clark and William
[10]
have carried out an extensive far- infrared study on metal pyridine
complexes. The C H
[11]
out of plane bendings (1000-700cm
-1
) of pyridine ring were assigned from
the analysis of combination bands at 2000-1600cm
-1
. This shows combination of pyridine with the
cobalt metal to formcomplexes. The pyridine combines with Co metal by its N atom.
Vol. 1, 91 - 101 [2013]
95
Experimental : The ligand biguanide sulphate was prepared by the reported method:
Biguanide sulphate C
2
H
7
N
5
H
2
SO
4
H
2
O was prepared according to the method described
by Smolka
1
and Friedrich with slight modification which resulted in better yield. In this method, a
mixture of ammoniumiodide (NH
4
I) and dicyandiamide, C
2
H
4
N
4
(mol.wt.-84.08) [dried at 100
0
C] in
2:1 proportion was intimately mixed in mortar and pestle and the mixture so obtained was then transferred
to a dry pyrex beaker and heated over asbestos board by means of a Bunsen burner. During this
process, the mixture was constantly stirred with the help of a thermometer and temperature was raised
gradually to 155
0
C. At this temperature, the mixture was changed to a thick liquid and was maintained
at this temperature [155+2]
0
C for ten minutes. The molten mass was then poured into a large volume of
water and then filtered fromany solid residue. The filtrate so obtained was then treated with a solution
of cuprammoniumsulphate, [Cu (NH
3
)
4
] SO
4
. Cuprammonium sulphate was prepared byadding liquor
ammonia (NH
3
) to copper (II) sulphate solution. As a result, rose coloured precipitate of copper
biguanide sulphate Cu(C
2
H
6
N
5
)
2
H
2
SO
4
was obtained at once. This was filtered in buchner funnel and
was washed thoroughly with cold water. The precipitate should be kept over the buchner funnel till
water get drained. The moist copper biguanide sulphate Cu(C
2
H
6
N
5
)
2
H
2
SO
4
was then decomposed
with cold solutions of about 10% sulphuric acid H
2
SO
4
. A blue solution was obtained which on keeping
in the cold [12
0
C] deposited large crystals of biguanide sulphate. The yield was found to be best when
a mixture of 8gms of dicyandiamide with 16gms of ammonium iodide was fused. Beside this the
temperature should also be maintained properly for good yield.
Preparation of the metal complexes :
Dipyridinebisbiguanidiumcobalt(III) hydroxide
[Co(BigH)
2
py
2
](OH)
3
It was prepared by adding calculated amount of biguanide sulphate dissolved in slight excess of
sodiumhydroxide to a solution of cobalt (II) sulphate with continuous stirring. As a result, the yellow
silky ppt. was obtained which was filtered quickly to avoid oxidation on the buchner funnel and was
washed with ice cold water. The yellow coloured complex so obtained was then mixed with a little
water to make suspension of the complex which was then transferred to an aeration flask. To this
suspension a small amount of pyridine, C
5
H
5
N (A.R.) was added and then a brisk current of air was
passed through it to oxidize Co (II) complex to Co (III) complex. Due to aeration, the silky yellow
bisbiguanidiumcobalt [II] hydroxide gradually dissolved to a dark red solution with the separation of a
slight black oxide of cobalt due to decomposition of the complex. The mixture was then filtered through
a quantitative filter paper and the filtrate was left in cold for crystallization. Dark violet permanganate
like crystals gets deposited slowly in course of a day or two. These were filtered, washed with ice cold
Vol. 1, 91 - 101 [2013]
96
water and finally with absolute alcohol .The product was then dried in a CO
2
free atmosphere. The
substance was soluble in water and was alkaline to litmus. The dried product on analysis was found to
contain
Co =12.39%
N = 35.60%
Required for [Co (BigH)
2
(Py)
2
](OH)
3
Co =12.54%
N =35.75%
Where BigH stands for one molecule of biguanide and Py for one molecule of pyridine.
The complex on heating evolved pyridine at about 95
0
C.
Dipyridinebisbiguanidiumcobalt (III) sulphate
[Co(BigH)
2
(py)
2
]
2
(SO
4
)
3
.12H
2
O
The red solution of the complex base [Co (BigH
+
)
2
(py)
2
](OH)
3
obtained as described above
on neutralization with dilute sulphuric acid in cold deposited red crystals associated with a small amount
of the trisbiguanidiumcobalt (III) sulphate .This was removed by fractional crystallization from water
.Trisbiguanidiumcobalt (III) sulphate was found to be more soluble than the dipyridinebisbiguanidium
cobalt (III) sulphate. The pure crystals were filtered & washed with cold water and alcohol and dried
in air The air dried sample on analysis was found to contain
Co =9.00%
N =24.85%
SO
4
-2
=21.59%
2(
py)
2
]
2
(SO
4
)
3
.12H
2
O
Co =8.78%
N =25.04%
SO
4
-2
=21.46%
Water could not be determined by heating as the complex evolved pyridine also when heated at 95
0
C.
Vol. 1, 91 - 101 [2013]
97
Dipyridinebisbiguanidiumcobalt(III)nitrate
[Co(BigH)
2
(py)
2
](NO
3
)
3
For the preparation of dipyridinebisbiguanidiumcobalt(III)nitrate [Co(BigH)
2(
py)
2
](NO
3
)
3
, the
solution of the complex base [Co(BigH)
2
py
2
](OH)
3
was neutralized with dilute HNO
3
(nitric acid)

in
cold. As a result deep violet crystals were separated, filtered and washed with ice cold water. Further
it was washed with alcohol & dried in air .The complex was found to be slightly soluble in alcohol.
Found
Co =9.54%
NO
3
-
=31.52%
[Co (BigH)
2(
py)
2
](NO
3
)
3
requires
Co =9.75%
NO
3
-
=30.74%
Dipyridinebisbiguanidiumcobalt(III)carbonate
[Co (BigH)
2
(py)
2
]
2
(CO
3
)
3
This was prepared by treating the complex base [Co(BigH)
2
py
2
](OH)
3
with either ammonium
bicarbonate NH
4
(HCO
3
) or sodiumbicarbonate NaHCO
3
. As a result, red precipitate of complex
carbonate was obtained which was filtered and washed.
Found
Co =12.05%
[Co (BigH)
2
(py)
2
]
2
(CO
3
)
3
requires
Co =11.59%
Dipyridinebisbiguanidiumcobalt(III)chloride
[Co (BigH)
2
(Py)
2
] Cl
3
.6H
2
O
The method of preparation as applied in case of the complex sulphate and nitrate could not be
used for the preparation of complex chloride. When the complex was neutralized with dilute hydrochloric
acid in cold, the complex chloride could not be crystallized out, but a gummy mass was obtained.
However, when a solution of complex base [Co(BigH)
2
(py)
2
]
2
(CO
3
)
3
was treated with
ammoniumcarbonate, (NH
4
)
2
CO
3
or sodium bicarbonate, NaHCO
3
red precipitate of the complex
Vol. 1, 91 - 101 [2013]
98
carbonate was obtained. The precipitate was filtered and washed. When the precipitate was just dissolve
in cold and dil HCl and cooled deposits dark red crystals. The crystals were filtered. Wash with ice
cold water and alcohol & dried in air.
Required [Co (BigH)
2
(Py)
2
] Cl
3
.6H
2
O
C =9.35%
N =26.53%
Cl =16.79%
The compound disengages pyridine when heated at 95
0
C.
Dipyridinebisbiguanidiumcobalt(III)thiosulphate
[Co(BigH)
2
(py)
2
]
2
(S
2
O
3
)
3
6H
2
O
It was prepared by neutralizing the complex chloride with solution of sodium thiosulphate drop
wise. As a result, the complex thiosulphate immediately precipitated as red crystals. The substance was
filtered, washed with cold water and alcohol. The complex was then dried in air.
Found
Co =9.25%
S
2
O
3
=27%
2
](S
2
O
3
)
3
6H
2
O
Co =9.20%
S
2
O
3
=26.23%
Dipyridinebisbiguanidiumcobalt(III)oxalate
[Co (BigH)
2
(Py)
2
]
2
(C
2
O
4
)
3
.8H
2
0
It was prepared either by neutralizing the complex base with oxalic acid or by adding sodium
oxalate to the complex chloride solution. The red crystalline precipitateDipyridinebisbiguanidiumcobalt
(III)oxalate,
[Co (BigH)(Py)
2
]
2
(C
2
O
4
)
3
.8H
2
0 so obtained was filtered washed with cold water and alcohol
and dried in air.
Found
Vol. 1, 91 - 101 [2013]
99
Co =5. 67%
[Co (BigH)
2
(Py)
2
]

(C
2
O
4
)
3
.8H
2
0 requires
Co =5.51%
On heating at 95
0
C decomposed with the loss of water and pyridine. The aqueous solution, on heating
slowly evolved pyridine and the colour changed to violet. Even incold it disengaged pyridine, but slowly.
Conclusion : From the stoichiometry and the physico-chemical properties studied about the cobalt
complex with the ligand biguanide and pyridine, the probable structures of complexes are shown below:
NH
||
C H
2
N N =C N
+
H
3

HN Co NH (OH)3

H3N
+
C =N NH2 C =NH
py py
Dipyridinebisbiguanidiumcobalt(III)hydroxide
NH
||
C H
2
N N =C N
+
H
3

HN Co NH (SO
4
)
3
12H
2
O

H
3
N
+
C =N NH
2
C =NH
py py 2
Dipyridinebisbiguanidiumcobalt(III)sulphate
NH
||
C H2N N =C N
+
H3

HN Co NH (NO3)3
H
3
N
+
C =N NH
2
C =NH
py py
Dipyridinebisbiguanidiumcobalt(III)nitrate
Vol. 1, 91 - 101 [2013]
100
NH
||
C H
2
N N =C N
+
H
3

HN Co NH (CO
3
)
3

H
3
N
+
C =N NH
2
C =NH
py py
2
Dipyridinebisbiguanidiumcobalt(III)carbonate
NH
||
C H2N N =C N
+
H3

HN Co NH (C2O4)3
H
3
N
+
C =N NH
2
C =NH
py py 2
Dipyridinebisbiguanidium cobalt(III)oxalate
NH
||
C H
2
N N = C N
+
H
3

HN Co NH Cl
3
6H
2
O
H3N
+
C =N NH2 C =NH
py py
Dipyridinebisbiguanidiumcobalt(III)chloride
Acknowl edgement : I amparticularly grateful to the sophisticated analytical instrument faculty,
Central Drug Research Institute, Lucknow, for recording UV spectra, IR spectra and elemental analysis
of my newly prepared compounds.
Vol. 1, 91 - 101 [2013]
101
I could not forget the help and the guidance by Dr. Dhananjai Singh who has helped me to
interpret the UV and IR data of the research work & tackle the obstacles faced by me. So, I amvery
grateful to himand have no words to express my gratitude.
References
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Friedrich Monatsh, (1883) : 4, 888
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Barrailough C. & Tobe M.L. (1961) : J . Chem. Soc. 1993
Earnshow A., Larkworthy L.K.& Patel K. C. (1969) : J.Chem. Soc. A, 1339.
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Sugden. J. Chem. Soc. 1932, 246
Vol. 1, 91 - 101 [2013]
102
103
ARSENIC CONTAMINATION IN GROUNDWATER OF BIHAR:
CAUSES, ISSUES AND CHALLENGES
Madhurendra Nath Sinha
1
, Ranvir Nandan
2
, Rabindra Kumar
3
1. Professor of Geology and Head of Department, Department of Geology,
Patna Science College, Patna University, Patna 800005.
email : mnsinha@gmail.com
2. Associate Professor, Department of Geology, B N College,
Patna University, Patna 800005
3. Associate Professor, Department of Geology,
Patna Science College, Patna University, Patna 800005
email : rabindra.hydrogeologist@gmail.com
Abstract: Many district of Bihar are having arsenic in its groundwater. The various causes of arsenic
contamination are mostly through geogenic channel. Human interference is not much responsible for
the problem. This paper tries to make an attempt to understand the groundwater arsenic contamination
scenario, causes, issues and challenges in Bihar. The problems on social, economic and environmental
issues are discussed in details. Almost 75-80 per cent of the rural populations rely on their drinking
water from the groundwater sources. Excess use of arsenic in drinking water leads to several disease
includes primary (black spots in the body, Keratosis) and secondary (white black spots in the body,
Hyper-Keratosis, Non-pitting edema and liver and kidney disorders) health impacts in the long run.
It has also tertiary health impacts causes gangrene of the distal organs, cancer of the skin, lungs and
urinary bladder and kidney and liver failure. It has impacts on human health, food chain nuisance and
socio-economic conditions hampers among the affected stakeholders.
Key words : Arsenic contamination, Groundwater, Bihar
The presence of Arsenic is hampering agricultural activity due to decline in soil fertility and
productivity. Social problems like depression, suicidal tendency and social ignorance are common.
Young men and women with arsenicosis problems are not getting married. Contamination in drinking
water hinders the social and economic activity to the effected person. The challenges are on the
mitigation (at macro) and adaptation (micro as well as macro) activity.
Majority of the population residing in the arsenic prone belt are from low income and are not
aware about the problems of the arsenic menace. Therefore both short termmitigation (hand pump treatment
plan or sanitarydug well) and longterm mitigation (alternative source of surface water) strategy is needed.
Introduction : Bihar along with few other states of India is facing an acute problem due to presence of
arsenic menace in its groundwater. Groundwater is the main source of drinking water and it constitutes
more than 80 per cent of drinking water source in rural Bihar. The other sources of drinking water are
fromsurface water, dug well, pond and fromnatural sources (lakes, rivers etc.). Few percentages of
rural households are using drinking water from protected dug wells. The groundwater sources were
considered safe for drinking water but over the past few years, they have reported contamination and
Vol. 1, 103 - 116 [2013]
104
pollution problems in its root due to rapid urbanisation, industrialisation and excess extraction of
groundwater for irrigation purpose. Around forty percent districts of Bihar has reported arsenic
contamination problemin its groundwater. This comprises of more than 67 blocks from15 districts
and covering more than 1500 habitations across the state where arsenic contamination in groundwater
exceeds the BIS limits for safe drinking water of 50 particle per billion (ppb) and more. I f we
consider the WHO limits of 10 ppb, the coverage area will be much more and the population which
is facing the danger of arsenic hazard will be thrice of the Bureau of Indian Standard (BIS) limit.
It has been estimated that more than 13.85 million people could be under the threat of
contamination level of above 10 ppb/l, out of which more than 6.96 million people could be above
50 ppb/l, against the total population of these area is around 50 million (MoWR, 2010). The actual
problemof arsenic menace among the population will be increasing at an alarming rate by every new
survey done by Central Ground Water Board (CGWB) and Public Health Engineering Department
(PHED), Govt. of Bihar.
Arsenic is a shiny metalloid that dissolves in water. It is a natural mineral, present in the soil
and aquifers, and the concentrations above the safe level in drinking water may cause significant
health risks. Most arsenic enters water supplies either fromnatural deposits in the earth or from
industrial and agricultural pollution. Arsenic is a natural element of the earths crust. Although surface
water are mostly considered safe for drinking water but groundwater sources are arsenic contaminated
in the range of 40 140 metre. It is used in industry and agriculture, and for other purposes. It is also
a by-product of copper smelting, mining and coal burning.
Access to safe water supply is one of the most important factors of health and socioeconomic
development (Cvjetanovic, 1986). More than 150 million people are affected worldwide by arsenic
contamination in 70 countries, out of which 50 million people in Bangladesh and 30 million people in
India are at risk (Ravenscroft et al., 2005). Arsenic is toxic in nature and the excess quantity of its
use in drinking water leads to several health hazards. Drinking arsenic contaminated water over a
long period results in various health effects including skin problems such as colour changes on the
skin, and hard patches on the palms and soles of the feet (WHO, 2010). I t also leads to skin cancer,
cancer of the bladders, kidney and lung, and diseases of the blood vessels of the legs and feet, and
also possibly diabetes, high blood pressure and reproductive disorders (ibid). Given the background,
this paper has attempted to understand the issues and challenges posed by arsenic groundwater
contamination problems and its menace of the affected population in Bihar. The paper is divided into
four sections. Followed by brief introduction and background problem, the second section is on
water pollution and arsenic scenario. In this section, water pollution and arsenic scenario have been
discussed in details. Third section deals with issues and challenges faced by arsenic in drinking water.
This section also explains about the possible solutions for the emerging challenges. The fourth section
comprises of concluding remarks.
Vol. 1, 103 - 116 [2013]
105
Figure 1. Map of Bihar showing the major rivers and physiography.
Water Pollution and Arsenic Scenario: Air and water pollution are major environmental problems
currently exist in I ndia along with depletion of non-renewable resources and degradation of
renewable resources (Sankar, 2000). Resources are economic goods while pollutants, the
degrader of resources are economic bads. Pollutants are the reverse of the resources (Dasgupta,
2010) and pollution is thus the reverse of conservation (Dasgupta, 1993 and 2007). Pollution
can be thought of as a pure public bad and hence pollution reduction as a public good (Baliga
and Maskin, 2005). Pollution is treated as negative externalities in economics literature (Pigou,
1920, Sankar, 2005). When certain actions of producers or consumers have unintended effects
on other producers or consumers externality arises. Externality is of two kinds positive and
negative. Externalities may be global public bads (emissions of greenhouse gases, climate change,
depletion of ozone layer, loss of bio-diversity and extinction of endangered species and other
are some of the examples of global public bads) which have global effect and local public bads
(problemof groundwater or surface water in a region, land degradation, air and vehicular pollutions
and others are some of the examples of the local public bads) which have local or regional
effect. Climate change problemaggravate the availability of water in the country as it threatens
the water cycle. As the population increases the demand for agriculture also grows and the
demand of water thus increase. Table 1 provides information on projected water demand in
India by different uses.
Vol. 1, 103 - 116 [2013]
106
Table 1. Projected Water Demand in India (by different uses)
Water Pollution : Water pollution is major concern in India and particular in Bihar. With around 17
percent of the worlds population but only 4 percent of its usable freshwater, India has a scarcity of
water. World oceans cover about 3/4th of earths surface. But fresh water constitutes small proportions.
About 2.7 percent of the total water available on the earth is fresh water, of which about 75.2 percent
lies frozen in Polar Regions and another 22.6 percent is present as groundwater (Ministry of Water
Resources India (MoWR), 2007; and United Nations Report (UN), 2005). A small proportion (about
2 percent) of total fresh water is available in lakes, rivers, atmosphere, moisture, soil and vegetation.
Water resources of a country constitute one of its most significant economic assets and the
different forms of water resource development differ for various uses, fluctuate fromcountry to country
depending on its climatic, physiographic, and socio-economic conditions and development (Jain, 1977).
India is rich in both surface water and groundwater resources. India has total annual replenishable
groundwater resources of 433 billion cubic meters (BCM), net annual groundwater availability of 399
BCM, annual ground water draft for irrigation, domestic and industry is around 233 BCM, and stage of
groundwater development is around 58 percent. Annual precipitation (includes snowfall) in India is
4000 cubic kilometers while average annual availability of water resources is around 1869 cubic
kilometers. Per capita water availability is 1820 cubic meters according to 2001 ministry of water
resources sources. Estimated utilized water resources is 1122 cubic kilometers in which surface water
resources share is 690 cubic kilometers and groundwater resource share is 431 cubic kilometers. Bihar
is rich in groundwater resources. In Bihar, annual replenishable groundwater resources, net annual
groundwater availability and annual groundwater draft are 29, 27.42, and 10.77 BCM. The stage of
groundwater development in Bihar is 39 percent and the annual rainfalls (in mm) are 1232. The per
capita water availability is decreasing in both Bihar and India. In 2001, per capita availability of water
(in cu. m) was 1950 and 1816 for Bihar and India. It has further decline to 1545 and 1200 (in cu. M)
in 2011. The decline in availability of groundwater in Bihar is due to the uncontrolled population growth,
excess dependence on groundwater (85 percent), over extraction of groundwater for irrigation,
uncontrolled deforestation. This leads to overall water quality problems. But water is becoming
Vol. 1, 103 - 116 [2013]
107
increasingly scarce over the years. Uncontrolled growth of population, expansion of irrigation channels
and developmental activity are responsible for the decline in water availability problems. It also leads to
problems in water quality which affects the health and other problems. Different groundwater
contamination problems in Bihar are given in table 2.
Table2. Different Groundwater Contamination in Bihar
Vol. 1, 103 - 116 [2013]
108
. Source: Authors own compilation from various sources.
Arsenic Contamination Causes and Sources : Arsenic is found in the natural environment in
plenty in the earths crust and in small magnitudes in rock, soil, water and air and is always present as
compounds with oxygen, chlorine, sulphur, carbon and hydrogen on one hand, and with lead, gold and
iron on the other (MoWR, 2010b). It can exist in both organic and inorganic formbut inorganic arsenic
is more toxic than organic arsenic. Inorganic arsenic compounds are known to be more human
carcinogens. Arsenic in element formis insoluble in water and soluble in oxidized form. Countries
including Argentina, Bangladesh, Chile, Ghana, Mexico, Mongolia, India, Taiwan, Vietnam, and United
States are exposed to arsenic problems because the sources of arsenic are primarily natural rather than
anthropogenic or geothermal. Inorganic arsenic of geological origin has been recognised as the main
formof arsenic in groundwater. Sparks (2005) suggested three source of arsenic in soil and aquatic
ecosystem. It consists of iogenic, Geogenic and anthropogenic sources of arsenic. By and large geogenic
sources are responsible for arsenic contamination but anthropogenic activities also cause contamination.
The anthropogenic sources of arsenic occur due to human activities. The main source of anthropogenic
can be further classified in three categories viz. agricultural, industrial and others. Agricultural sources of
arsenic canbe frompesticides, herbicides, seed treatment, cattle deep and fertilizer mainly, whileindustrial
sources are fromtimber treatment, tannery, electro plastic, and paints and chemicals. Other anthropogenic
sources are fromsewage and smelting.
Arsenic Scenario : Arsenic is a heavy metal and regarded as a toxic element. Excess of arsenic in
drinking water over long run is considered as a human health hazard and leads to different diseases. In
extreme cases it leads to an end of human life. Seven states of India have reported arsenic contamination
in groundwater and it is increasing at increasing rate (MoEF, 2009). Out of reported seven states, Bihar
and West Bengal have severe impact of the livelihoods of the stakeholders due to arsenic menace.
More than 70 countries are globally affected directly or indirectly with arsenic contamination in
drinking water which affects more than 150 million people across the globe. Around middle of the 20th
century, arsenic poisoning surfaced in those areas where people ingested arsenic contaminated water.
The major affected countries fromarsenic poisoning were Argentina, Chile, Mexico, Taiwan, and some
Vol. 1, 103 - 116 [2013]
109
part of the United States. In global arsenic contamination scenario, 38 countries are affected more
severely at present. At the last quarter of 20th century three Asian countries (Bangladesh, China, and
India) came to lime light due to their suffering from groundwater arsenic contamination. The major
source of arsenic contamination was contaminated hand tube-wells. As of 2010 September, 13 Asian
countries are arsenic affected and the level of arsenic contamination in Asian countries is more severe
than the rest of the world. Bangladesh is the worst affected country, as 60 of its total 64 districts have
arsenic groundwater contamination above World Health Organization (WHO), 2001 guidelines of
10mg/l for safe drinking in water. In India, flood plains of all the states in Ganga and Brahmaputrarivers
are arsenic affected.
The first case of arsenic in India was reported in 1976 from Chandigarh, where some patients
were suffering from noncirrhotic portal hypertension (NCPH) and later it was found that the water used
by patients who suffered fromNCPH came fromarsenic contaminated tube wells (MoWR, 2010b). In
1982 a patient fromNorth - 24 Parganas district of West Bengal, whose skin lesions were not like the
usual skin diseases and later similar problemfinds to many patient fromthe same village suffered from
such problems in soles of their feet, palms of their hands, ulcer in hands and bodies and found that due
to the excess availability of arsenic in tube wells in drinking water (MoWR, 2010a). Soon after the
incident four districtsof West Bengal (North 24 Parganas, South 24 Parganas, Nadia, and Murshidabad)
were found on arsenic menace in ground water. In 1983, 33 villages of 4 districts were identified,
having arsenic contamination. By the end of 2004, 3200 villages of 85 blocks from9 districts wereidentified
having arsenic contaminated water and by the end of 2008, more than 3417 villages of 111 blocks from
9 districts have reported arsenic contaminated groundwater (MoWR, 2010b).
In 2002, two villages (Barisban and Semaria Ojhapatti) from the Bhojpur district of Bihar in
the middle Ganga plain reported excess of arsenic contamination exceeding 50 mg/l (Chakraborty et
al., 2003, Sinha et al., 2010) . As of 2009, out of 38 districts of Bihar, 57 blocks from 15 districts
having total population more than 10 million have been reported to have arsenic groundwater
contamination above 50 mg/l (MoWR, 2010a and 2010b, MoEF, 2009). Due to the excess arsenic
contaminated drinking water, 18 babies were born blind in the Bhojpur district. The demographic
survey done by many organizations mainly in Bihar and West Bengal estimated that more than 13.85
million people could be under the threat of contamination level above 10 mg/l, in which more than
6.96 million people could be above 50 mg/l, against the total population of those areas of the order
of 50 million (MoWR, 2010b).
Live-stock in large number has also been exposed to arsenic contaminated groundwater. In the
arsenic affected areas, arsenic contaminated groundwater is also used for agricultural irrigation. This
leads to the possibility of arsenic exposure through food chain not only in contaminated areas but also
in areas with no contamination due to open market sale of food products.
Out of seven states, two states of India namely Bihar and West Bengal are worst affected by
arsenic contamination in their groundwater. Altogether more than 40 percent of the people fromBihar
Vol. 1, 103 - 116 [2013]
110
and West Bengal are affected by arsenic contamination in groundwater which causes serious threats to
the people of the state in health and other hazards which threats to the socio - economic status of the
affected people. Table 3 presents the arsenic contamination problemin Bihar.
Table 3. Occurrence of high Arsenic in Groundwater in Bihar (> 50 ppb/l)
Source: PHED, Govt. of Bihar (2012)
Issues and Challenges : Scarcity of safe drinking water in the rural areas of Bihar acquainted with
social and economic issues. I t also threats the environment as well as major health problems.
Contamination in drinking water hinders the social and economic activity to the affected person. The
evidence on the adverse impacts of water pollution in general and on human health in particular is well
known. High concentration of contamination in drinking water arsenic, fluoride, iron, nitrate and lead-
contribute to both human mortality and morbidity. Prolonged exposure to water contamination could
lead to different disease. Epidemiological studies show that arsenic in drinking water cause cancer
(Canter 1997, Chakraborty and Saha, 1987). Arsenic contamination (Sinha 2010) in drinking water
over long run can cause the problems in the reproductive system, birth defects and harm the central and
peripheral nervous system(Canter, 1997) and excess acquaintance of arsenic during pregnancy can
adversely affected reproductive endpoints (Mukherjee, 2006). The dose-response relation between
low arsenic concentrations in drinking water and arsenic-induced skin Keratosis and hyper pigmentation
is well characterized (Haque et al., 2003). The arsenic related skin disease may be associated with
increased risks of skin, bladder and lung cancer (NRC, 1999). Without skin lesion also cancer risks
can prevail (ibid). Such health problem has involved economic, social and environmental costs to the
affected stakeholders. Arsenic in drinking water hinders the social as well as economic costs to the
society in general and affected households in particular. Health problems caused by pollution have
Vol. 1, 103 - 116 [2013]
111
economic costs arising fromthe expenses incurred in treating the disease and loss of productivity
(Bates, 1990, Ostro, 1994, Banerjee, 2001, Adhikari, 2012). Skin lesions poses an important public
health concern in Bangladesh and West Bengal, India as advanced forms of Keratosis are painful and
if untreated can lead to social isolation among the affected villages (Haque et al., 2003, Haque and
Khan, 2011).
While most of the arsenic studies are concentrated on arsenic are epidemiological studies. In
epidemiological studies it has been tried to link exposure of arsenic in drinking water over period of
time causes acute illness. The studies are cross-section in nature and focused more on current exposure
to the illness. The long term(5 to 10 years) exposure of arsenic in drinking water forms the basis of skin
lesions (Keratosis, Melanosis), hyper pigmentation, and increased risks of lung, bladder and skin cancers,
birth defects and peripheral nervous system. Arsenic related exposure hinders the social and economic
cost of the affected person. There are very few studies available which has focused on both social and
economic factors and tried to estimate the socio-economic cost involved to the household due to
exposure of arsenic in drinking water. Arsenic in drinking water causes different types of cancer. National
Academy of Sciences (NAS) 1999 suggested that arsenic level in tap water and its total cancer risk.
Table 4 presents Arsenic in Drinking Water and Cancer.
Table 4. Arsenic in Drinking Water and Cancer
Source: National Academy of Sciences (1999)
The arsenic problem has a major effect on the socio - economic structure. The socio economic
problems can be mainly categorised into three classes as agricultural problem, health problemand
other problems. Excess presence of contaminated water leads to decrease in agricultural productivity,
soil fertility, and also enters into the food chain which createshealthproblems. Brammer (2008) suggested
that in India, Nepal and Bangladesh arsenic contaminated water used for irrigation enter into the food
chain. All these three problems lead to both social and economic problems. Skin lesions, bladder and
cancer, and mortality arefew of the health problems. Social ignorance, depression and suicidal tendency
are among few social problems. Arsenic contamination has widespread social problems among the
Vol. 1, 103 - 116 [2013]
112
affected households. Social problems are linked with the health and the economic problems. Arsenic
related diseases are not spreadable disease. It is common myth among the households in rural areas
that it is aspreadable disease. The possible solution is to initiate awareness programme by the government
at the community or grassroots level. Arsenic groundwater contamination has severe economic effect
on the people residing in the areas where the menace is found. Study found that poor people are more
prone to suffer fromsuch problem. There is dearth of studies on economic aspects of arsenic problems.
The chronic effects of inorganic arsenic exposure via drinking water include skin lesions, such as hyper
pigmentation and black foot disease, and respiratory symptoms, such as cough and bronchitis. Besides,
there is sufficient evidence to link bladder and lung cancers with ingestion of inorganic arsenic (NRC
Report, 2006).
Arsenic contaminated groundwater is used for agricultural irrigation resulting in excessive amount
of available arsenic in the crops in that area. It has been reported that second to the ingestion of arsenic,
after the direct consumption as drinking arsenic contaminated water, is through food chain, particularly
useof contaminated rice followed by vegetables. Thiseventually indicates that the effects of this occurrence
are far-reaching; sooner we search sustainable solutions to resolve the problems, lesser be its future
environmental, health, socio-economic and socio-cultural hazards (MoWR, 2010b). The fertilizers and
pesticides used for agricultural purpose also cause arsenic contamination. Rice and vegetables have
more effects on arsenic contaminated water. Brammer (2008) in his study suggested that arsenic-
polluted water used for agriculture irrigation is a health hazard for the people eating food from the crops
irrigated in the areas of India, Bangladesh and Nepal in recent times. Arsenic contaminated water used
for irrigation can adversely affect the soil quality and hence reduce food production.
Arsenic contaminated groundwater used for irrigation in the countries of south and south-east
Asia is adding arsenic to soils and rice. This poses a serious risk to sustainable agricultural production
and also the livelihoods and health of the affected population of those countries (Brammer, 2009). The
possible mitigation strategy or measures should be needed. Two possible options can be possible. The
first is to provide the alternative irrigation sources and the second will be removing the contaminated
soil by using the appropriate technology.
Concluding Remarks : Bihar was one of the least developed states of India both in terms of per
capita income and human development index. However recent developments have made Bihar a
better place for habitation. In the last few decades pollution of water level has increased due to excess
exploitation of groundwater resources for irrigation and drinking purposes, rapid increase in
industrialisation and urbanisation. Groundwater level is increasingly falling in many parts due to excess
drawls and recurrent draught like conditions leading to contamination problems with nitrate, fluoride,
arsenic and other chemicals and also contributes to contaminating potable water sources.
Accesses to safe and clean drinking water along with sanitation are basic human needs. They
are fundamentally linked to the health and wellbeing of the people. The majority of the people are facing
arsenic in their drinking water is frompoor socio-economic background. They are either not aware or
Vol. 1, 103 - 116 [2013]
113
if aware are forced to take drinking fromsame source due to lack of alternative sources of water. As
Prime minister of India Dr. Manmohan Singh rightly said in his 2012 IWW speech that With around
17% of the worlds population but only 4% of its usable freshwater, India has a scarcity of water. Rapid
economic growth and urbanisation are widening the demand supply gap. Climate change could further
aggravate the availability of water in the country as it threatens the water cycle. Our water bodies are
getting increasingly polluted by untreated industrial effluents and sewage.
Groundwater levels are falling in many parts due to excess drawls leading to contamination with
fluoride, arsenic and other chemicals. The practice of open defecation, which regrettably is all too
widespread, contributes to contaminating potable water sources. If we cannot be aware and take
action then the condition of contamination will be worse than Bangladesh which will certainly affect
sustainable health of the stakeholders in all aspects of life.
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Chakraborti, Dipankar et al. (2006): An eight-year study report on arsenic contamination in groundwater
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Chakraborti, Dipankar et al. (2003): Arsenic groundwater contamination in middle Ganga plain, Bihar
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J ain, C. K., and Ali I (2000): Arsenic: occurrence, toxicity and speciation techniques, Water Resources
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Vol. 1, 103 - 116 [2013]
117
A PLANE SYMMETRIC UNIVERSE FILLED WITH VISCOUS FLUID
IN A MODIFIED BRANS-DICKE COSMOLOGY
L. N. Rai, S. Alam and Priyanka Rai
Department of Mathematics, Patna Science College,
Patna University-800 005, Bihar, India
Abstract : A plane symmetric cosmological model filled with viscous fluid has been derived in the
Brans-Dicke theory. Some physical and geometrical properties of this model have been discussed.
Finally, this model has been transformed to the original form(1961) of BransDicke theory.
Key Words : Plane symmetric universe, Viscous fluid, Cosmological model, Brans-Dicke theory.
Introduction : Endo and Fukui [1] have studied the variable cosmological term from the point of view
of cosmology in Brans-Dicke theory [2] and elementary particle physics.
In this paper, we have considered the modified Brans-Dicke theory with the variable
cosmological termas an explicit function of a scalar field f as proposed by Bergmann [3] and Wagoner
[4] and discussed in detail by Endo and Fukui [1].
The Brans-Dicke field equations with cosmological termQ are [1] :
,k
i j ij ij , i , j ij ,k i;j ij
2
8 1 1
G g Q T g ( g )
2
(1.1)
8
(2 3)
(1.2)
(2 3) (1 ) 8 (1 )
Q . T ,
4 4
(1.3)
) deviates from that of Brans
and Dicke, w is coupling constant and
ij
T is energy-momentum tensor. Semicolons denote covariant
derivative with respect to the metric
ij
g and commas mean partial derivatives with respect to the
coordinate
i
x
. The theory can also be represented in a different form
under a unit transformation (UT) [5] in which length, time and reciprocal mass are scaled by the function
1
2 (x). Then under the conformal transformation
ij ij ij
g g g
(1.4)
equations (1.1) (1.3) have the form
Vol. 1, 117 - 123 [2013]
118
,k
ij ij
,i ,j ,k ij ij
1 1
G g Q (8 )T (2 3)( g )
2 2
(1.5)
8
(2 3)
(1.6)
Q . T,
(1.7)
where the barred quantities are defined in terms of
ij
g as their unbarred counterparts are
defined in terms of the unbarred metric
ij
g and all barred operations are performed with respect to the
barred metric and barred Christoffel symbols.
In section 2, we have studied a plane symmetric cosmological model filled with viscous fluid
in Brans-Dicke theory of gravitation. In section-3, we have discussed about some physical and
geometrical properties of this model. Lastly in section-4, we have transformed this model to the 1961
formof Brans-Dicke theory.
The Field Equations: The geometry of the universe is described by the line element.
1 a 1a
2 2 2 2 2
2 2
ds dT Tdx T dy T dz (2.1)
where a being a constant.
The energymomentumtensor for the viscous fluid distribution is given by [6]:
j j j j j l l j
j
i ;i i; i;l i ;l
i i
T p v v pg v v v vv v vv
l j j
;l i
i
2
v g v v ,
3
(2.2)
together with
i
i v v 1
(2.3)
where
p
and are the pressure and density respectively, , and are the two coefficients of
viscosity,
i
v
is the flow vector satisfying equations (2.3) and semicolons signifies covariant differentiation.
The coordinates are assumed to be comoving so that
1 2 3 4
v v v 0andv 1.
Vol. 1, 117 - 123 [2013]
119
The pressure and density in the model (2.1) are given by
1
2 2
2
2
2
(a 5 ) 1 ( 5 a ) 8
8 p 1 ta n lo g(K T ) Q
2 8( 2 3) T 16T
(2.4)
1
2 2
2
2
2
( a 5) 1 (5 a )
8 1 tan lo g(KT ) Q.
2 8(2 3) 16T
(2.5)
Also scalar field

is given by
1
2
2
(5 a )
log sec log( KT )
8( 2 3)
(2.6)
and
1
2 2
2
2
2
(1 ) (a 5) (5 a )
Q sec log(KT )
4 8(2 3) 8T
(2.7)
where K is a constant.
Physical & Geometrical Properties
The model has to satisfy the reality conditions [7] :
(i)
p 0 a nd
(ii)
3p 0
which requires that
2
3
a 5, , Q 0(i.e. 1).
2
(3.1)
and
1
2 2
2
2
2
(5 a ) (5 a )
Q sec l og(KT ) .
8(2 3 8T
(3.2)
The flow vector of the distribution for the model (2.1) is given by
1 2 3 4
v v v 0 and v 1.
Obviously
i j
;j v v 0.
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120
Therefore the flow is geodesic.
The rotation tensor

i; j j;i
ij
v v 0.
Thus, the fluid filling the universe is non-rotational.
The expansion scalar
i
;i
1
v
3
is given by
1
.
3T
(3.3)
Shear tensor i ;j j;i i j
ij ij
1
(v v ) (g v v )
2
is given by
11
1
,
6
a1
2
22
(3a1)
T ,
12
(3.4)
a1
2
33
(3a 1)
T ,
12

44
0.
Also the shear s is
2 ij 2
ij
2
1 1
[1 3a ].
2 48T
(3.5)
The volume element of the model is given by
1 1
2
2 2
V ( g) ( T ) T.
(3.6)
Here volume is directly proportional to time. If the time increases then volume increases (i.e.
the models are expanding with time) and if the time decreases then volume decreases (i.e. the models
are contracting).
Deceleration parameter

2 2
d 1
q 3
dT 3
is given by
2
4
(9T 1)
q
81T
(3.7)
The deceleration parameter acts as an indicator of the existence of inflation. If q >0, the model
decelerates in itsstandard way while q <0, the model inflates. The present model represents a decelerate
model if and inflationary model if .
2
1
T
9
and inflationary model if

2
1
T
9
.
Vol. 1, 117 - 123 [2013]
121
The surviving components of the conformal curvature
jk
hi
C tensor for the line-element (2.1) are
1 4 2 3 2
1 4 2 3 2
1
C C [1 a ],
24T
1 2 3 4 2
1 2 3 4 2
1
C C [1 6a a ],
48T
(3.8)
1 3 24 2
1 3 24 2
C C [1 6a a ].
48T
The pressure, density, scalar field and cosmological constant are singular at
1
2
2
1 2(2 3)
T exp .
K (5 a )
(3.9)
The model exists for a finite time
1
2
2
1 1 2(2 3)
T exp .
K K (5 a )
viscous fluid distribution in general relativity [8].
Transformations of the Solutions and Discussion :
Under the transformations

ij i j ij
1
g g g ,
ij ij
ij
T T T ,
2
T T T ,
2
p p p, (4.1)
2
,

e ,
Q Q Q,
1
i i i
2
v v v ,
the solutions of the field equations (1.5)-(1.7) are changed into 1961 formof Brans-Dicke
theory [2]. We now apply these transformations to the solutions obtained from field equations (1.5)-
(1.7). Thus
Vol. 1, 117 - 123 [2013]
122
1
2
2
(5 a )
se c lo g(KT )
8( 2 3)
(4.2)
1
2
2
ij ij
(5 a )
g cos log(KT) g
8(2 3)
(4.3)
i.e.
1
2 2
11
(5 a )
g cos log(KT ) T
8(2 3)
1
1 a
2 2
2
22
(5 a )
g c os log( KT ) T
8( 2 3)
1
1a
2 2
2
33
(5 a )
g cos log(K T) T
8( 2 3)
1
2
2
44
(5 a )
g co s log(KT ) .
8(2 3)
1
2 2
2
4
2
(a 5) (5 a ) 1
8 p se c log(KT ) 1
8(2 3)
32T
1
2 2 2
2
2
( a 5) (5 a )
s ec log(KT )
8(2 3)
32T
1
2 2
2
8 (5 a )
se c log(KT )
T 8(2 3)
(4.4)
1
2 2 2
4
2
(5 a ) (5 a (1 )
8 sec log(KT) 1
8(2 3) 32T
Vol. 1, 117 - 123 [2013]
123
1
2 2
2
2
2
3(a 5) (5 a )
se c lo g(KT )
8(2 3) 32T
(4.5)
1
1
2
2
4
2
(5 a )
v sec log(KT )
8(2 3)
(4.6)
1
2 2
2
3
2
(1 ) (a 5) (5 a )
Q se c lo g(KT )
4 8(2 3) 8T
(4.7)
symmetric universe filled with viscous fluid in the Brans-Dicke theory [2]. The model obtained in this
paper is new and like other models filled with viscous fluid, they may be used inthe relativistic cosmology
for the description of very early stages of the universe expansion.
References:
Endo, M., Fukui, T. (1977) : Gen. Relativ. Gravitation, 8, 833,
Brans, C.H., Dicke, R.H. (1961) : Phys. Rev., 124, 925,
Bergmann, P.G. (1968) : Int. J. Theor. Phys., 1, 25,
Wagoner, R.V. (1970) : Phys. Rev., D1, 3209,
Dicke, R.H. (1962) : Phys. Rev., 125, 2163,
Landau, L.D., Lifschitz, E.M. (1963) : Fluid Mechanics, Vol. 6, 505,
Ellis, G.F.R. (1971) : General relativity and cosmology, (ed.) R.K. Sachs, Academic Press,
New York and London, 117,
Prakash, S. (1981) : Curr. Sci., 50, 78,
Vol. 1, 117 - 123 [2013]
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125
MULTICOLLINEARITY, ITS EFFECTS AND CONSEQUENCES
L.N. Rai and Alakh Niranjan
Department of Mathematics,
Patna Science College, Patan-5, Bihar, India
Abstract :In this paper we have extensively discussed about multicollinearity its effectsand consequences.
Furthermore, exact multicollinearity versus orthogonality has been extensively studied in recent literature
where multicollinearity (
p
0) does imply non orthogonality (
p
1). Inpresence of multicollinerity
to a singular matrix
p
0, there is exact multicollinearity following the condition that defined quantity
K* as infinity.
In this context estimate of both efficient vector and dispersion matrix have been evaluated by
Farrar and Glauber (1967) related with the study of multicollinearity specifying the character of matrix
notations on assumption of parent orthogonality to the determinant |R| or conveneient transformationof
|R| for which test of significance of rejection of hypothesis
0
H : |R| =1 at specified level of significance.
In addition a light has been given on Haitovaskys Chi square, while the extent of multicollinearitycould
be assertained for measuring the departure of R-matrix fromsingularity. Also, lower bounds for
2
E( )
and
2
V( )
have been obtained in this regard and indicating the distance between b and b when eigen
values are considered in this study. We have also stressed on application of measures based on multiple
correlation implicating a test known as variance ratio test contributing matrix inversion explained with
sumof squares due to error, total and regression expressed for sampling variance and square of regression
coefficient consisted with the study of multicollinerity. Lastely, solution existence and consequences of
multicollinearity have been made on studying different approaches in the context of multicollinearity to
more extended forms.
Key words : Multicollinearity, Regression Analysis, Orthogonality, Singular Matrix, Eigen values,
Regression Coefficient.
Introduction : The present study would make an attempt to concentrate on the concept of
multicollinearity and also to visualize its effect and consequences with greater extent by means of
regression analysis study.
In this connection, the first aspect is to study relating the termmulticollinearity , which has
been brought in the case of regression analysis due to Frissh (1934) that exists for taking the
decision of exact relation. That means multicollinearity was often referred for the existence of
more than one exact linearship while the termcollinearity means the existence of single linear
relationship. Though linear regression model is also reformed for existence of all explanatory
variables.
Vol. 1, 125 - 137 [2013]
126
However, in consideration of these two studies there maintains some distinction between these
two used terms. In recent literature the termmulticollinearity is used to denote the presence of linearship
(or near - linear relationships) among the explanatory variable.
In the case of regression model
Y = X +
as we know there is one of the basic implicit assumptions of the classical linear regression that
there does not exist exact linear relationship among the observed values of the explanatory variables. In
other words, it can be said that the data matrix X which is of order (n x p) following with rank. The
reason for the assumption is that the least square estimation b =
/ 1
(X X)
/
X Y
requires the inversion
of
/
X X
which is impossible of the rank of b, and hence the rank of
/
X X
is less than p that shows
linear dependence between the explanatory variables.
This indicates the case of extreme multicollinearity which exists when some or linear all of the
explanatory variables in a relation are perfectly linearly correlated. In this situation the parameter vector
is not estimable.
Thus, the least square estimation procedure breaks down there.
In fact, an exact linear relationship is highly improbable in the case of practical work but the
general interdependence of economic phenomenon may easily result in the appearance of approximate
linear relationships for the study of regressors.
J ohnson (1963) has resorted to a asymptotic definition. Multicollinearity is the name given to
the general problemwhich arise when some or all of explanatory variables in a relation are as highly
correlated one with another that it becomes very difficult, if not impossible, to disentangle their separate
influence and obtain a reasonably precise estimate of their relative effects.
Concept Relating To Exact Multicollinearity Versus Orthogonality : The present section would
neutral to examine the exactness of multicollinearity with the concept of orthogonality. It is true that
exact multicollinearity exists when the rank of X is less than p i.e. if the columns of X are denoted by
1
X ,
2
X .........
p
X there exists non zero constants
i
a (i =1,2, .......... p) such that
i i
aX 0 (2.1)
By examining such existed relation, an assumption can be made that multicollinearity exists
when there is relationships among explanatory variables and they are perfectly linearly correlated.
The matrix is said to have orthogonal regressors when it is such that
/
X X
=1 i.e all eigenvalues
of
/
X X
are equal to unity and X consists of orthogonal regressions. Hence orthogonality of regressors
(or explanatory variables) implies that
/
X X 1
.
Vol. 1, 125 - 137 [2013]
127
In order to concentrate on near multicollinearity that exists when there are non-zero constants
such that
i i
aX 0.
When the matrix
/
X X
is singular, its inverse does not exist in the usual sense. Exact-
multicollinearity implies that
/
X X
is singular i.e. the smallest eigen values
p
near multicollinearity,,
means that
/
X X
is singular and the smallest eigen value is close to zero. In case of near orthogonality, ,
eigenvalues are different fromunity. In particular, the smallest eigen values
p
1 that does not imply
p
0
.
Thus non-orthogonality does not necessarily imply singularity or muiticollinearity. However,
muiticollinearity
p
( 0)
does imply non-orthogonality (
p
1
).
Conditioned Matrix :
Numerical analysts have mainly uncared with near singularity of matrix and hence derived the
so called conditioned number K* to index the extent of all conditioning of a matrix. It is usually
defined as
1 1
*
2 2
1 1
K
where
1 2 p
........................ .
This is a ratio of what is called the singular value of X [i.e. square - test of the eigen values of
/
X X]
. The meaning of termSingular here is not be confused with the singularity of the matrix itself.
The presence of muiticollinearity can be checked by merely looking at
p
without carrying about
1
. For a singular matrix
/
p
X X, 0 there is exact-multicollinearity, and K* is infinite.
For orthogonal data, we have
/
X X 1we have K* is equal to unity, thus the condition
number lies in the half upon interval (1,0). For any two matrices, the larger is the value of K* the
worse conditioning. All ill-conditioned matrix of available data is defined by a large condition
number. Of course, there is some ambiguity large similar to the singularity in defining elements to
zero.
Chi-Saquare (X
2
) As Measure of Multicollinearity : This study is followed by Bartletts (1950). If
the regressor variables are standardized, then
/
X X
contains elements that are the simple correlation
coefficient among the regressors. In that case
/
X X
falls in the interval (0,1).
If
/
X X
=0, one or more exact-linear dependencies exists among the columns of X. Similarly
if
/
X X =1 then column of X are orthogonal.
Vol. 1, 125 - 137 [2013]
128
In fact, most standard multiple regression computer programme have built-in checks for non-
singularity of the
/
X X
matrix. These lists commonly use that determinant of a singular matrix is zero.
Farrar and Glauber (1967) attempted to define a standard comparison for
/
X X
by defining
multicollinearity as a departure of the matrix X fromorthogonality. Estimate of both efficient vector and
its dispersion matrix viz.
/ 1 /
b (X X) X Y
and V(b) =
2 / 1
(X X)
require the operation.
The test must commonly used and rely on the property that determinant of a singular matrix is
zero. Defining a small positive test value, u >0 a solution is attempted if
/
X X u.
Computations hailed otherwise if
/
X X is based on a normalised correlation matrix (unless
stated otherwise) then 0 <
/
X X =|R| <1.
As X approaches singularity (perfect multicollinearity) then |R| approaches one.
Unfortunately the gradient between these limits is not well defined. If under in assumption of
parent orthogonality to the determinant |R| or convenient transformation of |R| could be found, the
resulting statistic could provide a useful measure of the presence and severity of multicollinearitywithin
a set of predictor variables.
However, Bartlett(1950) contributed
2 2
p(p 1)
X [n 1 (2p 5)/6] log|R|~X [ ]
2
n =no. of sample, and
p =no. of variables.
this contribution due to Bartlett on the basis of comprising the lower moments of Wilks
distribution with these
2
X
distribution.
A high value of Chi square indicates the existenceof multicollinearity. Its severity can be measured
by the level of significance at which Ho: |R| =1 is rejected.
In this connection, Monte Carle pointed out that for n =20, p =10 and a =0.05, then the null
hypothesis is rejected following with the concept that when the elements of R are larger (in absolute,
value) than 0.36.
Vol. 1, 125 - 137 [2013]
129
In the same way, when n =200, p =10, a =0.05.
One is sure in virtual sense that the null hypothesis Ho is rejected when the elements of R are
larger (in absolute value) than 0.09.
Furthermore, Williamand Watte(1978) have presented a geometric interpretation of
1
/ 2
XX
when X is in standardised form.
1
/ 2
X X represents a ratio of the volumes of the joint confidence
region based on available design relative to that of an orthogonal reference design in which the regressors
have the same variability as in the original design.
Although the Bartlett test defines multicollinearity as a departure fromorthogonality, that does
not need any solution of the least square. On the contrary, the least squares solution for multiple regression
pre supposes (in non-trivial cases) intercorrelated predictor variables, otherwise the sample correlation
coefficients may be computed for each predictor variable separately.
The only relevant requirement in the context of multicollinearity is the full rank requirement.
According to Haitovaskys Chi-square, the extent of multicollinearity can be measured as the departure
of R-matrix fromsingularity.
A Heuristic statistic which is consistent.
Which is consistent with the concept due to Haitovaskys(1969) given by
2
X
=[n1(2p +
5)/6] log (1|R|).
A small value of X
2
indicates the existence of multicollinearity, its severity can be measured by
the level of significance at which the null hypothesis
o
H ;|R | o is accepted.
Explanation For Eigen Values Mathematically As Measure Of Multicollinearity
Writing the equation as discussed in proceeding study such as
/ 1 /
b (X X) X U
(5.1)
Let D define the distance between b and b such that;
=(b ) =
/ 1 /
(X X) XU
The squared distance between b and b is obtained as;
2
=
/ 1 / / 1 /
(X X) X U[(X X) X U]
Vol. 1, 125 - 137 [2013]
130
E( ) =
/ 1 / / 1 /
E[(X X) X U] [(X X) X U]
=
/ 1 / / 1 /
E tr [(X X) XU] [(X X) XU]
=
/ 1 / / 1 / /
E tr [(X X) X U][(X X) XU]
=
/ 1 / / / 1
tr E[(X X) X U U X(X X) ]
=
2 / 1
tr[(X X) ]
Now
/
X X
is real symmetric matrix, as its inverse. Therefore,
p
2 2
i 1
i
1
( ) ( )
(5.2)
where
i
is the i th eigen value of
/
X X
. Considering the variance of
2
we have
V
2
( )
=
2 2 2
E[ E( )]
=
4 4 / 2
E[ tr (X X) ]
But,
4 / 1 / / 1 / / 1 / / 1 /
( ) [(X X) X U]' [(X X) XU] [(X X) XU] [(X X) X U]
Thus,
4 / 1 / / 1 / / 1 / / 1 /
E( ) tr E[(X X) XU]' [(X X) XU] [(X X) XU][(X X) X U]
=
/ / / 2
tr E(UU UU ) (X X)
Since U is assumed normally distributed, we can write
/ / 4
E(UU UU ) 2
4 4 / 2
V( ) 2 tr (X X)
4 / 2
2 tr (X X)
Vol. 1, 125 - 137 [2013]
131
p
4 2
i
i 1
2 (5.3)
Considering the equations (5.2) and (5.3), if
min
is the smallest value of
/
(X X)
then,
(a) A lower bound for
2
2
min
E( ) is
(b) A lower bound for
4
2
min
V( ) is
Thus, if the particular variables are related in such a manner as to result in
/
X X
matrix with one
or more small eigenvalues, the distance fromb and will tend to be greater since the eigenvalues of a
matrix are just the zeros of the characteristic polynomial, the fact that the eigen values depend continually
on the element of the matrix follows immediately if it is known that zeros of a polynomial depend
continually on its coefficients.
Then, a rule based on eigen values of the R matrix could logically suppliant the correlation
coefficient rule except that some information is lost when we deal with the characteristic polynomial,
since there are many distant matrices with a given characteristic polynomial.
But multicollinearity is essentially a problemof small |R|, and it is irrelevant what the specific
elements of R are that produces |R|.
Pointing, if
1 2 p
, ,.................................... are the eigenvalues of R (not necessarily
distinct) then
|R| =
p
i
i 1
The small eigenvalues, therefore, result in small R. In fact, a singular matrix implies the existence
of one or more zero eigenvalues. A rule can be established to constraint the smallest eigenvalue to be
greater than a specified value. Usually, 0.3 is the specified value as suggested by Daling and
Tamura(1970).
Measures Based On Multiple Correlation : Klein(1960) suggested that the multicollinearity is said
to be harmful if |
ij
r
| >
y
R

for all i =j, where
ij
r is the zero order correlation between the predictor
variables and Ry is the multiple correlation between responses and the predictor variables. In this
connection, Farrar and Glauber(1967) found same drawbacks in Kleins rules and they have developed
a set of three tests for multicollinearity, viz.
(i) test based on Chi-Square.
(ii) F-test locating which variables are multicollinear.
Vol. 1, 125 - 137 [2013]
132
(iii) t-test for finding out the pattern of multicollinearity, i.e. for determining which variables
are responsible for appearance of multicollinearity.
A particular variable
i
X would be said to be harmfully multicollinear of
i y
|R | |R |
.
Where
i
R is the multiple correlation of
i
X with other predictor variables and Ry is the multiple
correlation of dependent variable with the entire set of predictor variables. The notion of the rule can be
developed as follows.
We can define the variance of the OLS estimator as follows :
2 / 1
V(b) (X X)
/ /
1 1 1 2
/ /
1
X X X Z
Z X Z Z
(6.1)
where without loss of generality,
1
X is a vector of observations on the first predictor, and Z is a matrix
of observations of the remainder of the predictor. Applying the partitioned matrix inversion rule in
equation (6.1), we have
/ / 1 /
2 1 1 1 1
X X XZ(Z Z) Z X A
V(b)
B B
(6.2)
where A and B are vectors and C is a matrix. However,
/ / / 1 /
1 1 1 1
[X X X (Z Z) Z X ] is the
error sumof squares of the regression of the first predictor variable on the remaining particular variable.
Now fromthe equation (6.2), the variance of the first coefficient estimate is
2 / / / 1 / 1
1 1 1 1 1
V(b ) [X X X (Z Z) Z X ]
2 1
1
(SSE )
2 1 1
1 1
(SST SSR ) )
(6.3)
where SSE
1
, SST
1
. and SSR
1
are the errors, total and regression, sumof squares respectively.
Again defining SSE, SST and SSR to be the analogous sums of squares for the regression of
the criterion variable on all of the particular variables, including the first particular variable. An estimate
of s
2
is
S
2
=(SST SSR) / m-p-1) (6.4)
Vol. 1, 125 - 137 [2013]
133
Also, R
1
2
=SSR
1
/ SST
1
(6.5)
Therefore, SST
1
SSR
1
=SST
1
(1 R
1
2
) (6.6)
Analogously, SST SSR =SST (1 R
y
2
) (6.7)
Substituting equations (6.4), (6.6) and (6.7) in (6.3) we have an estimate of V(b
1
) as
2 2
1 y 1 1
1
V(b ) [ ][SST(1 R )/ SST (1 R )
(m p 1)
2 2
1 y 1
1
[ ][SST /SST ] [(1 R )/(1 R )
(m p 1)
2 2 2 2
y 1 y 1
1
[ ][S /S ] [(1 R )/(1 R )]
(m p 1)
(6.8)
Since the choice of the first predictor variable arbitrary equation (6.8) can be generalized as
2 2 2 2
1 y i y 1
1
V(b ) [ ] [S /S ] (1 R ) / (1 R )
(m p 1)
.
Thus, the magnitude of the estimated variance of such estimated coefficient, given the ratio of
2
y
S and
2
i
S , depend not only on the intercorrelation between the (i
th
) predictor variable and the rest,
but also on the relationship between the criterion variable and the predictor variables.
Concentration On Possible Solution Of Multicollinearity Problem : In the present section our
attempt is to focus on possible solution of multicollinearity which seems a vital problemrelating to
present study of research work. When multicollinearity is present in a set of multicollinearity variables,
the OLS estimates of the individual regression coefficients tend to be unstable and canmade to enumerous
inferences. After deleting its presence, some alternative methods that prove a more informative analysis
of the data than the OLS method. The possible solutions for multicollinearity are
(a) Dropping Variable(s) : It is assertained that multicollinearity arises due to lack of sufficient
information in the sample to permit reliable estimation of the individual parameters. In some situation it
may be the case that one is not interested in all the parameters. In such cases we can get estimators for
the parameters and one is interested in that have smaller mean square errors than the OLS estimators,
Considering the case
Yi =
1
X
1i
+
2
X
2i
+U
i
(7.1)
and the problemis that
1
X and X
2
are very highly correlated. In this situation one of the highly correlated
variables may be dropped. Therefore dropping X
2
the existing model becomes as
Y
i
=
1
X
1i
+V
i
(7.2)
Vol. 1, 125 - 137 [2013]
134
where Vi is said as new disturbance term.
The estimator
1
b of b
1
obtained from(7.1) is the OLS estimator, b*
1
of the same b
1
obtained
from(7.2) is called the omitted variable (OL) estimator. The estimator
*
1
b is biased estimator because
of the omitted variable X
2
, while b
1
is an unbiased estimator.
Further, it can be shown that b*
1
has a smaller variance. Since the estimate of b
1
from(7.2) is
given by
b*
1
=
2
i 1i 1i
Y X x
It can further be shown that
*
1 1 21
E(b ) b
(7.3)
where b
21
is the slope coefficient in the regression of X
2
on X
1
.
This shows that b*
1
, the estimated variable estimator is a biased estimator. If both b
21
and
1
in (7.3) are positive, then
*
1
E(b )
will be greater than
1
leading to a positive bias. Similarly, if the
product b
21
is negative, on an average
*
1
b
will under estimate
1
, leading to a negative bias.
Thus, dropping a variable fromthe model to alleviate the problemof multicollinearity may lead
to the specification bias. Hence the solution may be worse than biases in certain situations.
Existence Of First Differences In Case Of Multicollinearity : The existence of first differences is
existing in a common trend where source of multicollinearity is possible, then in such situation, the ratio
or first differences technique is often used in time series analysis. However, the transformations used
under the technique have adverse effect on the properties of the resulting residuals.
In using ratio introduction of heterosedasticity, auto correlation may be introduced with the help
of using first differences by consideration of the following model, where t is used for time
o
t . Thus we
have,
t 1 1t 2 2t t
Y X X U ......... (given for t time)
Also,
t 1 1(t1) 2 2(t1) t1
Y X X U .. (given for time t-1)
Using first differences, the following result exist in the following manner given as,
t t t1
Y Y Y
1t 1t 1,t1
X X X
2t 2t 2 t1
X X X ,
Vol. 1, 125 - 137 [2013]
135
Thus, by using first differences, there is found that for the case of regression model, there
reduces the severity of multicollinearity because
1t
X and
2t
X are existing for highly correlated
variables as there exists for
1t
X and
2t
X .
In order to concentrate on ratios, the given regression model may exist as
t 1 1t 1 2 2t 1t t 1t
Y X t X X X U X (on division of
1t
X )

1 2 2t 1t 1t t 1t
X X X U x
Obvious, resulting residuals will be heterosedastic indicating (1/
1t
X ) is used as an explanatory
variable.
Hence, a confusion may be drawn that the ratios or first differences provides for existence of
multicollinearity, unless an assumption is being made for disturbance U
t
in the original model of regression
because the transformations as made above provide independent and homosedastic residuals in the
transformed equations.
There are some special methods existing in case of multicollinearity, where traditional residual
measure, are being applied as suggested by Ranger Erisch under the conditions of more data. But the
difficulty arises for expansion or impractical due to cancellation of data that is not possible.
Concentrating on the study of multicollinearity, where some methods relating to given problems
such as (a) Chi-Square as a measure of collinearity (b) eigen values as a measure of collinearity (c)
measures based on multicorelation as they have been discussed in earlier sections of this paper.
However, possible solutions for multicollinearity, the following measures (as discussed in the
present section) have been under taken into study such as:
(i) dropping variable,
(ii) using extraneous estimates
(iii) using ratio or first differences,
(iv) using some special method.
Though, these solutions have been discussed elaborately but in conclusion a justification might
be laid there that multicollinearity as defined earlier is a statistical, rather than a mathematical condition.
As such, one thinks, one speaks, in terms of the problema severity rather than of its existence or non-
existence. It is true that the effect on estimation and specification of interdependence in X-reflected by
variances of estimated regression coefficient and a tendency towards misspecification also depends
partly on the strength of dependence between Y and X.
Consequences Of Multicollinearity : In order to concentrate on consequences of multicollinearity,
a brief concept necessiates there. The presence of multicollinearity has a number of potentially serious
effects on the least square estimates of the regression coefficients. Some of these effects may be easily
Vol. 1, 125 - 137 [2013]
136
demonstrated. It is true that collinearity does not destroy the property of minus variance. But this does
not mean, that variance of as OLS estimator will necessarily be small (in relation to the value of the
estimator) in any given sample.
Further, in consideration of high variance estimates of regresioncoefficient the following conditions
may be taken into account as;
If the intercorrelation between the explanatory is perfect then the two conditions hold there.
(i) the estimates of coefficients are indeterminate i.e. the value of b explode, and
(ii) the standard errors of these estimates become infinitely large. For near multicollinearity
Xp >0 and also Mean Square Error (MSE) tends infinitely i.e. b is subject to very large
variance (Since MSE(b)
2 1
p
).
In this connection, Marquardt(1970) remarked by consideration of correlation matrix
/
X X
indicating r
ij
for (i,j) theelement of the inverse matrix
/ 1
(X X)
asserted on variance inflation factor(VIF)
and the author contributed
ij
VIF(i) r 5
that indicates a harmful multicollinearity.
In this connection, Theil(1971) showed the following result
ij 2
i
2
i
1
r [X ]
(1 R )
where
2 /
i i i
[|X | X X
] and
2
i
R
represents the squared multiple correlation coefficient when
X
i
(i
th
col of X) is regressed on remaining (p-1) regressors.
Again, stressing on multicollinearity when it is present, there is a linear relation among regressors
as discussed in present study and
2
i
R
will be large (close to 1).
Thus, the denominator of
ii
r
will close to zero. Hence
ii
r
will be large. In otherwords,
multicollinearity leads to high variance of regression coefficients.
In addition to, giving an explanation of the difficulty with the estimated students value based on
multicollinear data is that these values are highly unstable and often change their signand relative magnitude
with minor perturbation in data.
Thus, the exact causes of wrong signs may be many, and what appears to be a wrong signs may
not even be wrong. Most regression practitioners knew about this problem, even though it is not a well-
defined problem, in a puristic sense.
Vol. 1, 125 - 137 [2013]
137
When the estimate regression coefficient bi are interpreted as practical derivatives
y i
X the
wrong sign problemis particularly serious.
Hence, it is therefore, other can be assertained that the concept of stability of bi values can be
rigorously defined by using some classical concepts in perturbation theory developed by Von
Neumann(1941), Wilkinsen(1965) and others.
References :
Frissh, R (1937) : Statistical Confluence Analysis by Means of Complete Regression Systems, Institute
of Economics, Oslo University, Publ. No. 5. cited in 1977 Maddala, G.S.
Farrar, D.E. and Glauber, R.R. (1967) : Multicollinearity In Regression Analysis : The ProblemRevisited,
Review of Economics and Statistics, 49, 92107
Feldstein, M.S. (1973) : Multicollinearity and the Mean Squared Error of Alternative Estimators,
Econometrics, 41, 337 346
Fomby, T.B. and Hill, R.C. (1978) : Multicollinearity and Minimax Conditions for the Bock Stein
Like Estimator, Econometrics, 47, 211 212
Haitovasky, Y. (1969): Multicollinearity In Regression Analysis; Comment, Review of Economics and
Statistics, 51, 486-489
Kumar, T.K. (1975) : Multicollinearity In Regression Analysis, Review of Economics and Statistics,
57, 365-366
Vol. 1, 125 - 137 [2013]
138
139
SYNCHROTRON SPECTRA OF ASTROPHYSICAL JET M87 AND THE
PROCESS OF DIFFUSIVE SHOCK ACCELERATION
Sumita Singh
Postgraduate Department of Physics, Patna University
Email :sumita.physics.pu@gmail.com
Abstract : The theory of acceleration of the synchrotron emitting electrons or positrons in the extragalactic
jets has to incorporate the fact that the spectrumis continuous fromradio to X-rays.With referenceto
the M87 jet it has been observed that the spectral shape is uniformalong the jet. And there is a constant
upper spectral cutoff of the synchrotron electrons at
6
10 * 9 . 0
c
Structure of M87 : The structure of M87 comprises of various knots which arise due to shocks in the
jet flow. The two characteristics as described above are applicable to the smallest scales of about
10pc. The synchrotron half-life at the upper cutoff is
yr T B
2 1 6
c c syn
) 1 / ( ) 10 / ( 185 ) (
(Meisenheimer et al 1996). This implies that the energy losses should affect the spectrum at a
distance fromthe acceleration site ranging from
syn
syn
42
10
pc to
pc
in the inner jet (knot F) to
2
acc
B at knot A.
This can be explained using the model of shock acceleration.
Numerical Analysis & Results : The sequential procedure for treating synchrotron losses involves
neglecting the synchrotron losseso that DSA formsa distribution that extends well beyond the synchrotron
cutoff, and then allowing synchrotron losses to modify this distribution. In order to check the validity of
this sequential procedure we treat DSA in another manner that allows one to include the acceleration
and the synchrotron losses at the same time, rather than sequentially. The numerical results show that
the two procedures produce indistinguishable results which are illustrated in the figures. In these figures
the logarithm of the distribution is plotted as a function of log N(X), so that a power law distribution
corresponds to straight line. The absolute values of f(p) and of p are unimportant. The synchrotron
cutoff momentump
c
is a free parameter and is chosen to be either three (p
c
/p
o
=10
3
) or six (p
c
/p
o
=
10
6
) orders of magnitude above the infection momentum. All the shocks have the same strength, specified
by the value of r, and the calculations are performed both for strong shocks with r =3.8 and for shocks
Vol. 1, 139 - 142 [2013]
140
with r =2.0. The adiabatic decompression after each shock moves the curve to the left [by (logr)/3],
without changing its shape, so that after N shocks the lowest energy particles in the distribution has
logp =-N(logr)/3.
Here is the compression ratio, is the power law index and a is related to the power law
index linearly and Y and X both are the ratios of p &p
o
. Log N(X) stand for log f(p).
In the theory of DSA it is usually assumed that there is injection at every shock. Hence the
cases are shown in the figures, where there is only a single initial injection with this distribution subjected
to many shocks in not realistic in practice. One expects the distribution after N shocks to consist of the
sumover the distribution injected at the first shock subjected to N-1 shocks ,and so on to the distribution
injected at the Nth shock subjected to only one shock. This sumis performed in evaluating the distribution
shown. As N is increased the slope of distribution decreases monotonically and approaches b =3 at
low p>p
o
, in accord with the theoretical predictions (White 1985; Achterberg 1990; Schneider 1993).
Nearer the synchrotron cutoff, after about 10 shocks, a peak in the slope starts to develop and becomes
increasingly prominent with increasing N. This pick may be attributed to the contribution fromthe
plateau-like portions of the distribution resulting frominjection at the earliest shocks.
The forgoing results show four notable effects of synchrotron losses on multiple DSA: (a) it
provides a high-p synchrotron cutoff (denoted p
c
) beyond which no particle can be accelerated by
DSA; (b) for a single initial injection, a plateau distribution, f(p) =const., develops at p<0.1p
c
; (c) the
cumulative effect of injection at every shock leads to distribution f(p) p
-3
for p <<p
c
; and (d) the
distribution in (c) has a slope that rises gradually to a peak (with b
min
~2 at p ~0.1p
c
).
Results and discussions : We present the results of numerical calculations that show the effect of
synchrotron losses on diffusive shock acceleration (followed by adiabatic decompression) at multiple
shocks. Our main results can be summarized as follows:
Vol. 1, 139 - 142 [2013]
141
(1) Synchrotron losses are most important during the acceleration process, when the
electrons are in thecompressed B region just downstreamfrom the shock. Synchrotron
losses imply a synchrotron cutoff, p = p
c
, to the distribution of accelerated particles;
DSA cannot cause any particle to be accelerated to p > p
c
.
(2) It is shown analytically that two procedures for treating the combination of synchrotron
losses and DSA are equivalent. In one treatment, the effects of synchrotron losses are
included in the momentumchange in each cycle of particle crossing the shock from
upstreamto downstreamand back. In the other procedure, used in our numerical
calculations, synchrotron losses are first neglected to find the distribution of electrons
resulting fromDSA alone, and then the synchrotron losses are allowed to modify this
distribution. The two procedures are equivalent provided that the time for which the
synchrotron losses are allowed to modify this distribution.
(3) J ust below the synchrotron cutoff , the distribution of particles injected at an initial
shock and subjected to DSA at many shocks without further injection tends to forma
plateau distribution [f(p) independent of p] which corresponds to an energy spectrum
N(e)
2
.
(4) The distribution below the synchrotron cutoff due to the cumulative effect of injection at
every shock tends to distribution f(p) p
-b
with 3 b at p<< p
c
, with the distribution
becoming somewhat flatter such that the slope has peak (with 2 b ) just below p
c
(at
0.1
c
p for strong shocks). Such a distribution, if the source were homogeneous (which
it is not due to the shocks), would corresponds to a flat synchrotron spectrum
[ ( 3)/ 2 0 b ] becoming a weakly inverted spectrum( 0.5) with a peak just below
a sharp cutoff due to synchrotron losses.
It can be concluded that it is possible for multiple DSA coupled with synchrotron losses to
account for a flat synchrotron spectrum. This may be a viable explanation for the flat synchrotron
spectra observed in some Galactic Centre sources.
The forgoing results apply to DSA at a single shock, and it is of interest to consider DSA at a
sequence of shocks. It is assumed that a new distribution of particles is injected at each shock and the
both these injected particles and the particles injected at earlier shocks are subjected to DSA. In
between shocks the magnetic field is decompressed to its initial value, which leads to adiabatic energy
loss by all particles between the shocks.
One other notable feature of the simple theory is the treatment in terms of test particles. In fact
the accelerated particles must contribute to the stresses; by continually reflecting off the scattering
centers embedded in the upstream and downstreamflows, the accelerated particles transfer momentum
across the shock, tending to slow down the relative flow. DSA can be very efficient under relatively
mild conditions the accelerated particles can provide the most important dissipation mechanismfor
Vol. 1, 139 - 142 [2013]
142
shocks, in the sense that a large fraction of the shock energy is ultimately transferred to accelerated
particle. In addition, if the number density of injected particles is high enough, acceleration of these
particles can lead to themproviding the dominant stress in the shock, resulting a shock structure that is
quite different fromthat in the absence of the accelerated particles. In adopting a test particle approach
it is assumed that such dynamical effects of the accelerated particles are not important.
References :
Biretta, J . A., Owen, F. N. , (1990) in Parsec scale Jets, eds. J. A. Zensus and T. J . Pearson,
(Cambridge: Cambridge Univ. Press), 125.
Melrose, Don , Crouch, Ashley, (1997) Effect of Synchrotron Losses on Multiple Diffusive
Shock Acceleration
Vol. 1, 139 - 142 [2013]
143
ON THE DETERMINATION OF REORDER LEVEL BASED ON
NEGATIVE BINOMIAL DISTRIBUTION
Arun Kumar Sinha
Department of Statistics and Patna Science College,
Patna University, Patna 800005 (Bihar) India
(arunkrsinha@yahoo.com)
Abstract : An attempt has been made in this paper to propose a method for the determination of the
reorder level or float size of a stock by extending the work done earlier in this direction. Also, a
table has been prepared for this purpose, which appears to be more useful, convenient and extensive
compared to the available tables in many respects.
Keywords: Float size, risk level, normal approximation, cumulative probabilities,
Poisson distribution, gamma distribution
Introduction : The main aimof an inventory management is to maintain an optimum level of stocks to
meet future demands. Taylor (1961) while discussing the inventory management for aircraft maintenance
pointed out two types of problems that are usually encountered. The first one focuses at the determination
of the reorder level, which is the level to which a stock is allowed to fall before an order for new items
is placed. The second problem arises in the replacement of defective parts of the aircrafts. It requires
that a store must have some extra components of each type needed. The surplus is called the float
size of each type of the component required. It means that the solution to the second problemis to
have an estimate of the float size for each component. However, if we consider the placing of a defective
component for repair as a demand and the interval between its removal from an aircraft and its entry
into the store as the lead time, the second problem reduces to the same as the first one. It is, therefore,
evident that float size is the same as reorder level. The author has used the negative binomial
distribution (NBD) for the purpose. Also, Brown (1965) has described the probability distribution for
the study of demand of replacement parts in the air force supply system. The author has prepared a
table that is divided into two parts. The first part gives the probability of n demands. The cumulative
probabilities of n or fewer demands are mentioned in the second part of the table. An attempt has
been made, in this paper, to extend the work of the earlier authors by employing the approximations
suggested by Bartok (1966) and also to prepare a table that is more extensive and convenient in many
respects. One could obtain the value of the float size or reorder level fromthe table correspondingto
the estimates of the parameters of the NBD and the desired risk level. The estimate of the parameters
is calculated on the basis of the observed demand of the component. The technique proposed in the
paper may be used in areas other than the maintenance of aircrafts. The application of the techniquehas
been illustrated in the maintenance of the inventory of the LPG (Liquefied PetroleumGas) cylinders for
cooking.
Vol. 1, 143 - 152 [2013]
144
Model : If a is the average number of demand per unit of time then the number of demand x during
a fixed interval of time t follows the Poisson distribution as given below:
P(X = x) = e
-at
(at)
x
/x! ; x = 0, 1, 2,
This assumption holds even if the demand occurs during the scheduled checks. Further, the
lead time follows the gamma distribution as mentioned below:
f(t) = (e
-bt
b
k
t
k-1
) / (k 1)! ; k> 0
This is a general assumption because it includes a wide range of lead time distribution. These
two assumptions lead to the probability P(X = x) of exactly x demands during the lead time which is
mentioned below:
where q = a / (a+b) and p = b / (a+b) ; x = 0, 1, 2,
This shows that X follows the NBD with parameters k and p. The probability of more than n
demands at the end of a lead time is given by:
Our problemis to find out n for a given risk level (P
n
), p and k. On the basis of the mean and
the variance of the observed demand, we compute the estimates of p and k as follows:
As it is difficult to obtain the value of n directly fromthe NBD, the normal approximation to
the distribution is used.
Two normal approximations out of a number of approximations proposed by Bartko (1966)
have been selected. The selection has been made after taking into consideration the maximum error E
i
(n, k, p) defined by:
Vol. 1, 143 - 152 [2013]
145
where NB
i
(n,k,p) denotes the ith approximationto the cumulative negative binomial probability.
The first approximation is given by:
We have, thus, obtained the following quadratic equation:
n
2
+ (1-kq/p)n + (k
2
q
2
/p
2
kq/p + 1/4 z
2
kq/p
2
) = 0 (1)
Bartko (1996) has calculated the maximumerrors due to this approximation which is reproduced
below :
The second approximation that we have chosen has been referred to as the Camp-Paulson
approximation by Bartko (1966). According to this:
We have derived the following equation of the sixth degree in n on the basis of the aforesaid
approximation:
531441 n
6
+ (4374 ABC 5832 AB
3
+ 1968 E) n
5
+ (A
2
C
3
+ 20412 ABC 27216 AB
3

54 ABCD + 243 E
2
+ 19683 F) n
4
+ (4 A
2
C
3
+ 38070 ABC 50760 AB
3
210 ABCD +
486 EF + E
3
) n
3
+ (6 A
2
C
3
+ 35472 ABC 47296 AB
3
306 ABCD + 243 F
2
+ 3 E
2
F) n
2
Vol. 1, 143 - 152 [2013]
146
+ (4 A
2
C
3
+ 16512 ABC 22016 AB
3
-198 ABCD + 3 EF
2
) n + (A
2
C
3
+ 3072 ABC 4096
AB
3
48 ABCD + F
3
) = 0 (2)
where A = kq/p, B = (9k 1)/k, C = B
2
9z
2
/k
2
, D = 9z
2
, E = 144 D and F = 64 D
The highest real roots of both the equations developed for different values of the parameters of
the NBD at the risk levels 0.005, 0.010, 0.025, 0.050 are shown in Table 1. The second equation
provides a better solution because of small maximum errors. The errors due to the Camp-Paulson
approximation have been computed by Bartko (1966). These are reproduced below:
In order to determine the reorder level or float size n of an item or defective component of an
aircraft, we first of all estimate the parameters of the NBD on the basis of the observed demands.
Corresponding to these estimates and the desired risk level we obtain the value of the float size or
reorder level fromthe table that we have prepared. In case the value of n is not given for a particular
set of the estimates and the desired risk level we could easily compute its value fromthe equation (2) on
the basis of the higher root obtained fromthe equation (1. Also, the value could be calculated directly
fromthe equation (2). If Table 1 does not serve the purpose of an organization then it could be modified
by taking into account the variations in the estimates of the parameters and the desired risk levels.
Describing the NBD as a tool in the study of demands for replacement parts Brown (1965) has
prepared a table of the distribution that is divided into two parts. Part 1 gives the individual probability
and Part 2 provides the cumulative probability of x demands or less for 13 different values of mean
and 10 different ratios of variance to mean. It is obvious that the table we have prepared is much
more convenient because it readily provides the values of float size or reorder level for the given values
of mean and variance of the observed demands at the desired risk level. This is also a fact that the value
of n is required for the maintenance of the optimumlevel of a stock, not the exact or the cumulative
probability of demands. The tables of Williamson and Bretherton (1963) are also not helpful for this
reason. This fact establishes the superiority of our table over those of Williamson and Bretherton (1963)
and Brown (1965).
Vol. 1, 143 - 152 [2013]
147
Numerical Example : In order to illustrate the technique let us consider the observed distribution of
the daily demands of the LPG cylinders for cooking placed at a cooking gas agency. The data are given
below:
We have computed the average demands ( ) =166.16667 and s
2
=732.966618 on the basis
of the demands placed between Sept 1 and Sept 6, 1986 and subsequently, the estimates of p and k
have been obtained as follows:
On the basis of equations (1) and (2) we have calculated the values of the reorder level or float
size (n) as 210.207 and 212.313 and 218.736 and 222.337 at the risk levels 0.05 and 0.025 respectively.
This suggests that the agency needs to maintain an inventory of only 210 or 212 cylinders at 5% risk
level.
References :
Bartko, J.J . (1966). Approximating the negative binomial. Technometrics, 8, 345-350.
Brown, B. (1965). Some tables of the negative binomial distribution and their use. Memorandum RM
4577 PR, The Rand Corporation, California, USA.
Taylor, C. J. (1961). The application of the negative binomial distribution to stock control problems.
Operations Research Quarterly, 12, 81-88.
Williamson, E. and Bretherton, M. H. (1963). Tables of the negative binomial probability distribution.
Wiley, New York.
Vol. 1, 143 - 152 [2013]
148

Table 1. The reorder level or float size n for the values of k (1 (1) 5 (5) 50 (25) 100 (50) 200) and
p (0.05 (0.1) 0.95) at the risk levels 0.005, 0.010, 0.025 and 0.050 corresponds to the standard
normal variates 2.576, 2.326, 1.960 and 1.645 respectively. For each p, the first and the second row
denote the values based on equation (1) and (2), respectively.
Vol. 1, 143 - 152 [2013]
149
k = 5 k = 10
0.05 206.785
243.127
195.888
223.490
179.934
196.663
166.204
175.339
348.295
384.867
332.884
360.910
310.322
327.630
290.905
300.650
0.15 63.237
74.685
59.801
68.503
54.771
60.054
50.442
53.352
106.235
117.766
101.376
110.218
94.262
99.728
88.140
91.222
0.25 34.454
40.918
32.517
37.436
29.683
32.678
27.242
28.881
57.719
64.237
54.980
59.982
50.971
54.066
47.520
49.267
0.35 22.054
26.380
20.766
24.060
18.881
20.886
17.259
18.359
36.836
41.204
35.015
38.368
32.349
34.426
30.054
31.227
0.45 15.104
18.238
14.183
16.571
12.834
14.289
11.673
12.471
25.147
28.318
23.844
26.280
21.937
23.446
20.295
21.148
0.55 10.616
12.989
9.934
11.743
8.936
10.038
8.077
8.681
17.617
20.023
16.653
18.501
15.241
16.387
14.026
14.673
0.65 7.435
9.275
6.926
8.330
6.181
7.036
5.540
6.007
12.299
14.170
11.579
13.018
10.526
11.417
9.619
10.121
0.75 5.007
6.451
4.634
5.735
4.088
4.758
3.619
3.981
8.264
9.739
7.737
8.870
6.965
7.666
6.301
6.693
0.85 3.007
4.137
2.752
3.613
2.379
2.920
2.058
2.335
4.976
6.138
4.616
5.508
4.089
4.638
3.635
3.938
0.95 1.119
1.971
0.987
1.751
0.795
1.178
0.629
0.822
1.944
2.829
1.758
2.434
1.485
1.895
1.251
1.467
k = 15 k = 20
0.05 478.984
515.621
460.109
488.294
432.477
450.024
408.695
418.701
604.070
640.736
582.276
610.548
550.369
568.053
522.908
533.067
0.15 145.821
157.377
139.870
148.765
131.157
136.700
123.659
126.823
183.641
195.209
176.769
185.693
166.708
172.295
158.050
161.263
0.25 79.061
85.598
75.707
803.741
70.796
73.936
66.570
68.364
99.407
105.954
95.534
100.587
89.864
93.029
84.984
86.806
0.35 50.339
54.723
48.108
51.486
44.843
46.951
42.033
43.238
63.180
67.572
60.604
63.995
56.834
58.959
53.589
54.813
0.45 34.276
37.460
32.680
35.135
30.344
31.876
28.333
29.209
42.930
46.123
41.088
43.554
38.390
39.936
36.069
36.959
0.55 23.941
26.360
22.760
24.625
21.031
21.196
19.543
20.209
29.914
32.341
28.551
30.426
26.554
27.730
24.836
25.513
0.65 16.657
18.543
15.776
17.230
14.486
15.393
13.376
13.893
20.754
22.648
19.737
21.200
18.247
19.164
16.965
17.492
0.75 11.151
12.640
10.506
11.654
9.561
10.276
8.743
9.153
13.847
15.344
13.101
14.258
12.010
12.734
11.071
11.485
0.85 6.695
7.870
6.252
7.158
5.606
6.168
5.050
5.366
8.278
9.465
7.769
8.685
7.023
7.594
6.381
6.705
0.95 2.638
3.541
2.410
3.103
2.076
2.500
1.789
2.018
3.264
4.179
3.001
3.704
2.616
3.048
2.284
2.521
Vol. 1, 143 - 152 [2013]
150
k =25 k =30
0.05 725.577
762.257
701.210
729.537
665.537
683.313
634.835
645.097
844.541
881.229
817.849
846.215
778.771
796.613
745.138
755.476
0.15 220.332
231.907
212.649
221.592
201.401
207.017
191.720
194.966
256.221
267.801
247.805
256.761
235.483
241.121
224.879
228.148
0.25 119.118
125.670
114.787
119.852
108.448
111.630
102.992
104.832
138.376
144.932
133.633
138.705
126.688
129.883
120.712
122.565
0.35 75.598
79.995
72.718
76.118
68.503
70.640
64.875
66.111
87.715
92.116
84.561
87.967
79.943
82.089
75.969
77.215
0.45 51.282
54.480
49.222
51.696
49.206
47.762
43.611
44.510
59.419
62.621
57.163
59.642
53.859
55.421
51.016
51.922
0.55 35.664
38.096
34.139
36.021
31.907
33.091
29.986
30.671
41.254
43.690
39.584
41.471
37.139
38.328
35.035
35.725
0.65 24.684
26.584
23.547
25.016
21.881
22.805
20.448
20.981
28.496
30.399
27.249
28.723
25.425
26.354
23.854
24.392
0.75 16.420
17.924
15.587
16.750
14.367
15.097
13.317
13.737
18.906
20.415
17.993
19.161
16.657
17.392
15.507
15.831
0.85 9.780
10.974
9.211
10.133
8.377
8.954
7.659
7.988
11.223
12.422
10.599
11.527
9.686
10.267
8.899
9.232
0.95 3.847
4.770
3.553
4.264
3.122
3.561
2.752
2.994
4.400
5.330
4.078
4.794
3.606
4.050
3.200
3.447

k =35 k =40
0.05 961.579
998.271
932.747
961.142
890.538
908.431
854.211
864.607
1077.091
1113.785
1046.268
1074.686
1001.145
1019.079
962.309
972.752
0.15 291.503
303.085
282.412
291.379
269.104
274.757
257.649
260.937
326.304
337.888
316.585
325.560
302.358
308.024
290.113
293.415
0.25 157.292
163.851
152.169
157.248
144.668
147.872
138.212
140.077
175.937
182.498
170.460
175.544
162.441
165.653
155.540
157.413
0.35 99.605
104.008
96.198
99.609
91.210
93.363
86.918
88.171
111.314
115.720
107.672
111.087
102.340
104.499
97.751
99.010
0.45 67.394
70.588
64.956
67.439
61.388
62.955
58.316
59.229
75.239
78.446
72.633
75.119
68.818
70.390
65.535
66.452
0.55 46.724
49.163
44.920
46.810
42.279
43.473
40.006
40.700
52.098
54.540
50.170
52.063
47.346
48.544
44.917
45.614
0.65 32.217
34.124
30.871
32.349
28.900
29.833
27.204
27.746
35.867
37.776
34.428
35.909
32.321
33.257
30.508
31.053
0.75 21.327
22.839
20.340
21.512
18.897
19.636
17.655
18.082
23.695
25.210
22.641
23.816
21.097
21.839
19.768
20.200
0.85 12.620
13.823
11.946
12.878
10.960
11.545
10.111
10.447
13.982
15.189
13.262
14.197
12.207
12.796
11.299
11.638
0.95 4.929
5.864
4.581
5.303
4.071
4.520
3.633
3.884
5.440
6.380
5.068
5.794
4.523
4.975
4.054
4.308
Vol. 1, 143 - 152 [2013]
151
k =45 k =50
0.05 1191.356
1228.052
1158.664
1187.100
1110.803
1128.770
1069.612
1080.093
1304.577
1341.274
1270.117
1298.568
1219.667
1237.662
1176.248
1186.761
0.15 360.711
372.297
350.403
359.384
335.313
340.990
322.325
325.639
394.790
406.377
383.924
392.910
368.018
373.704
354.327
357.652
0.25 194.361
200.923
188.551
193.639
180.046
183.264
172.726
174.606
212.599
219.162
206.475
211.567
197.510
200.733
189.794
191.679
0.35 122.877
127.284
119.014
122.432
113.358
115.521
108.491
109.754
134.316
138.724
130.243
133.664
124.282
126.449
119.151
120.419
0.45 82.979
86.187
80.215
82.704
76.169
77.744
72.686
73.606
90.630
93.840
87.717
90.208
83.452
85.031
79.781
80.704
0.55 57.394
59.838
55.349
57.245
52.354
53.554
49.777
50.478
62.626
65.071
60.469
62.368
57.313
58.515
54.596
55.299
0.65 39.459
41.370
37.932
39.416
35.698
36.636
33.774
34.337
43.002
44.802
41.393
42.878
39.037
39.978
37.010
37.560
0.75 26.020
27.538
24.902
26.080
23.265
24.010
21.857
22.289
28.310
29.830
27.131
28.311
25.406
26.153
23.921
26.375
0.85 15.315
16.524
14.551
15.488
13.432
14.023
12.469
12.811
16.623
17.835
15.818
16.758
14.638
15.232
13.624
13.967
0.95 5.936
6.879
5.541
6.270
4.963
5.418
4.466
4.722
6.419
7.363
6.003
6.735
5.394
5.851
4.869
5.128

k =75 k =100
0.05 1859.379
1896.075
1817.174
1845.671
1755.386
1773.473
1702.208
1712.830
2401.655
2438.349
2352.921
2381.447
2281.574
2299.716
2220.169
2230.856
0.15 561.618
573.208
548.310
557.314
528.829
534.545
512.062
515.421
724.500
736.088
709.131
718.144
686.635
692.369
667.274
670.653
0.25 301.780
308.347
294.280
299.383
283.300
286.541
273.850
275.755
388.735
395.304
380.075
385.184
367.396
370.648
356.484
358.401
0.35 190.174
194.587
185.187
188.617
177.886
180.065
171.602
172.883
244.552
248.968
238.794
242.229
230.363
232.549
223.107
224.396
0.45 127.933
131.148
124.364
126.863
119.141
120.729
114.645
115.579
164.176
167.394
160.056
162.559
154.024
155.618
148.833
149.772
0.55 88.073
90.524
85.432
87.338
81.566
82.777
78.239
78.951
112.737
115.191
109.688
111.597
105.224
106.440
101.382
102.099
0.65 60.189
62.109
58.219
59.711
55.334
56.282
52.851
53.408
76.792
78.716
74.517
76.014
71.185
72.138
68.318
68.880
0.75 39.372
40.900
37.929
39.116
35.816
36.570
33.997
34.439
50.007
51.539
48.340
49.532
45.900
46.658
43.800
44.246
0.85 22.900
24.121
21.914
22.862
20.469
21.070
19.226
19.577
28.884
30.111
27.745
28.699
26.078
26.683
24.642
24.997
0.95 8.698
9.658
8.189
8.932
7.443
7.909
6.801
7.067
10.826
11.794
10.238
10.288
9.376
9.846
8.635
8.907

Vol. 1, 143 - 152 [2013]
152
k = 150 k = 200
0.05 3464.512
3501.200
3404.825
3433.381
3317.444
3335.648
3242.238
3253.004
4509.654
4546.338
4440.733
4469.308
4339.835
4358.077
4252.995
4263.802
0.15 1043.414
1055.006
1024.595
1033.619
997.043
1002.798
973.331
976.734
1356.746
1368.338
1335.020
1344.046
1303.202
1308.968
1275.821
1279.238
0.25 558.790
565.361
548.184
553.300
532.656
535.919
519.291
521.222
725.698
732.270
713.450
718.571
695.520
698.791
680.088
682.027
0.35 350.746
355.164
343.693
347.133
333.367
335.562
324.480
325.779
454.845
459.265
446.701
450.145
434.778
436.979
424.517
425.821
0.45 234.828
238.049
229.782
232.291
222.395
223.996
216.037
216.984
303.983
307.206
298.156
300.668
289.626
291.231
282.284
283.236
0.55 160.707
163.166
156.973
158.888
151.506
152.727
146.800
147.523
207.569
210.029
203.257
205.175
196.944
198.169
191.250
192.237
0.65 108.884
110.913
106.198
107.700
102.118
103.076
98.606
99.173
140.350
142.281
137.132
138.637
132.421
133.382
128.366
128.936
0.75 70.533
72.070
68.492
69.689
65.503
66.267
62.931
63.382
90.453
91.994
88.096
89.297
84.646
85.413
81.676
82.129
0.85 40.346
41.580
38.951
39.911
36.908
37.520
35.150
35.510
51.393
52.631
49.782
50.747
47.424
48.039
45.394
45.757
0.95 14.821
15.798
14.100
14.859
13.045
13.525
12.137
14.415
18.601
19.585
17.769
18.534
16.551
17.036
15.502
15.785
Vol. 1, 143 - 152 [2013]
153
SIMULATING AVERAGE TIME COMPLEXITY OF SRSWOR:
A STATISTICAL APPROACH
Anchala Kumari
1
and Soubhik Chakraborty
2
1
Department of Statistics, Patna University, Patna-800005, India
2
Department of Applied Mathematics, BIT Mesra, Ranchi-835215, India
Abstract : Algorithmis a fundamental concept in computer science. Developing an optimal algorithm
for solving a problem depends on its complexity (which can be computational, time, space, some
weighted combination of time and space or even monetary cost) which provides a quantitative judgment
to select the best algorithmamongst the several ones.
Over the decades a lot of work has been done to measure the computational complexity but
none of the measures is realistic in nature as it merely expresses the order of complexity by computing
the minimumnumber of operations required which in turn is expressed as the function of input parameters.
In this paper attempt has been made to focus on the statistical approach to simulate the average time
complexity of an algorithmT for drawing a random sample of size (n) from a population of size(N),
sampling been done without replacement.
It has been investigated that for (i)n=N the average time complexity is of O(Nlog
2
N) and (ii)
for n arbitrarily chosen ,complexity is O(n) .While estimating the parameters of the model , emphasis
has been made on pattern recognition than on estimation since the estimates are systemdependent.
Keywords: Simple RandomSampling Without Replacement; Algorithm Complexity, Computer
Experiment.
Introduction : Simple randomsampling without replacement (srswor) is a procedure for selecting a
sample of size n from a population of size N such that the unit of the population selected at a particular
draw is not returned before the next draw and an equal probability of selection (equal to reciprocal of
number of units in the population ) is ensured to each unit of the population at the first and each
subsequent draw. In his book [5], Prof. Donald Knuth (Stanford University) has discussed about an
algorithmfor srswor .Some more algorithms related to srswor are available in W. Kennedy and J .
Gentles book [4]. These works suffer fromthe drawback that the authors have suggested the selection
procedure only, but have not focused on the order in which the observations come into the sample. In
this paper the algorithmdescribed focuses on both the aspects: the selection procedure as well as the
order in which the observations come into the sample.
The algorithmfor drawing a sample of size n froma population of size N is based on the
concept that the sample drawn is a randompermutation of the digits 1, 2,.N taking n at a time. As
the population can be labeled 1, 2,..N ,the sample consists of those units with the same labels as
those appearing in the randompermutation. The n numbers so selected not only ensure the random
selection of the units but also specifies the order in which the units come into the sample. The computer
Vol. 1, 153 - 159 [2013]
154
program for finding the execution time of the algorithmT written in Visual C++follows in the next
section.
Visual C++ code
#include<iostream.h>
#include<sys/timeb.h>
#include<time.h>
#include<math.h>
#include<stdlib.h>
void main()
{
int npop,ns,k;
int *a,*s;
clock_t start,end;
cin>>npop;
a=new int[npop];
for(int i=0;i<npop;i++)
*(a+i)=i+1;
ns=pow(npop,.5);
s=new int[ns];
start=clock();
for(int j=0;j<ns;j++)
{
k=1+rand()%npop;
*(s+j)=*(a+k);
if(k!=npop)
{
for(i=k;i<npop;i++)
a[i]=a[i+1];
}
npop=npop-1;
}
end=clock();
double elapsed=double(end-start)/CLOCKS_PER_SEC;
cout<<elapsed time=<<elapsed<<endl;
}
Vol. 1, 153 - 159 [2013]
155
For n arbitrarily chosen, the statement
ns=pow (npop,.5) is replaced by the statement
cout<<ns<<endl;
The logic here is that first an array a[] of size N with units 1,2,3,N is created such that a[i]=i,
i=1,2,3N. At first draw , a randomnumber k is selected from 1 to N. Then a[k] becomes the first
element of the sample. The elements froma[k+1] to a[N] are sifted to one place on the left to fill the gap
created by removal of the element a[k] in the array(a[]). In case the randomnumber k is equal to N ,
sifting of the elements to the left is not done. In either case, N is set equal to N-1 before drawing
another randomnumber k. The process continues till all the n units come into the sample. This means
the process has to be repeated n times.
Analysis and Results :
Case 1 n=N
The average run time is obtained for different values of population size (input parameter) or
equivalently for different values of sample size (n=N), average being taken over 100 trials for each
value of population size and is given in the table below.
Table 1 average execution time n=N
N : 10000 40000 90000 160000 250000
Avg time (sec): 0.0154 0.0532 0.206 0.4156 0.7656
N 360000 490000 640000 810000 1000000
Avg time (sec): 1.297 2.0344 3.0902 4.3084 6.006
The average timewhen plotted against different values of N usingMINITAB statistical package
agrees to the average complexity lying between O(N) and O(N
2
). Fig1 and fig 2 shows time as linear and
quadratic functions in N(population size) with 100R
2
values (coefficient of determination) equal to 97.4
and 100 respectively. For agood literature onapplied regression analysis, the reader is referred to [3].
Fig.1 N-time plot time->O(N)
Vol. 1, 153 - 159 [2013]
156
Fig.2 N-time plot time->O(N
2
)
In fig.1 we see that the some of the points lie even outside the 95%confidence bound where as
in fig. 2 confidence bounds and regression curve coincide together. The polynomial curve gives a
good fit with R
2
=100% .It may be argued that the contribution due to N
2
termis almost negligible in
the equation time=-.554417+.0000026N+.0000000N
2
.The N
2
term contributes in reducing the
distance between the confidence bounds.
Further we have O(N)<O(Nlog
2
N)<O(N
2
)
And O(Nlog
2
N) +O(N) = O(Nlog
2
N)
Thus we may experiment with the model
Y=a +bNlog
2
N
The estimated values of a,b are
^ ^
a = -.3639 b =.0000003
line of regression is time= -.3639 + .0000003 Nlog
2
N
R
2
= 98 .2%
Normal probability plot of the residuals approximates to linear function as shown below.
Fig .3. Normal probability plot of residuals
0 500000 1000000
0
1
2
3
4
5
6
N
t ime =- 0.0 5544 17 + 0. 0 00 00 26 N
+0.000 000 0 N**2
S = 0 . 041 39 92 R- Sq = 100 . 0 % R- Sq(a dj) = 100 . 0 %
Regre ssion
95%CI
Regression Plot
-1 0 1 2
-1
0
1
St andardi zed Residual
No r m a l P ro b a b i l i t y P l o t o f t he R e s id u a l s
(r esp ons e is time)
Analysis of variance table is as given below
Vol. 1, 153 - 159 [2013]
157
Table 2 : ANOVA for regression analysis
Source DF SS MS F P
Regression 1 37.396 37.396 427.42 0.000
Residual Error 8 0.700 0.087
Total 9 38.096
From the table it is clear that F is highly significant and regression sum of squares contributes
markedly to total sum of squares, implying thereby a good fit .
Case 2 n is any arbitrary value
Here no restriction is imposed on the sample size .Keeping population size fixed at 20000
samples size n is arbitrarily chosen. We get the following table of average execution time over 100
trials.
Table 3 Average execution time
Sample size n : 4000 4500 5000 5500 6000
Avg run time y (sec): 0.3126 0.3282 0.3590 0.3810 0.4064
Sample size n : 6500 7000 7500 8000
Execution time y: 0.4284 0.4560 0.4750 0.4910
Execution time when plotted against the values of n agrees to a linear pattern confirming
thereby to the fact that complexity of the algorithmis of order O(n).
The model equation is time= .125658 + .0000464 n
R
2
= 99.7%
Fig.4 n-time plot
4000 5000 6000 7000 8000
0.3
0.4
0.5
n1
time1 = 0. 125658 + 0.0000 464 n1
S = 0. 0038624 R- Sq = 99. 7 % R -S q(adj) = 99. 6 %
Regres sion
95% CI
Regression Plot
Vol. 1, 153 - 159 [2013]
158
All the points lie within 95% confidence bounds implying that the complexity can be well explained by
a linear function in n , the sample size. The very high value of F in the ANOVA table below also
supports to this fact.
Table 4 ANOVA applied to regression
Analysis of Variance
Source DF SS MS F P
Regression 1 0.0323222 0.0323222 2166.59 0.000
Error 7 0.0001044 0.0000149
Total 8 0.0324267
Vol. 1, 153 - 159 [2013]
159
The middle term follows from the fact that mean of 1,2,3-----m is (m+1)/2 where m
equals N, N-1,..N-n+1 at j=1,2,3,n respectively.
or, E(T(n))= nN/2 - n
2
/4 +3n/4
which leads to O(nN) and for n= N, the complexity is ofO(N
3/2
)
For large N, we have N< 2
N
or log2N< N or, N log2N <N
3/2
Thus the experiment
with O(N log
2
N) complexity provides an empirical bound-estimatelower than theoretical
least upper bound which is quite sensible and realistic as well.
Also for n an arbitrary value O(n)<O(nN).
Conclusion : In both the situations n = N or n is arbitrarily chosen, empirical bound estimates for the
complexity of srswor is less than the theoretical lower upper bound. As a final comment, we emphasize
that empirical O is actually a bound-estimate and itself not a bound, also when it is obtained by working
directly on time as in this case, it is estimating a weight-based statistical bound which weighs each
operation every time it comes against the corresponding time it consumes. Unlike the mathematical
bounds which are count based and operation specific, it has the provision of mixing operations of
different types conceptually which is permitted due to weighing and which is very realistic since in all
real time applications, operations perform collectively. Empirical O is written as O with a subscript
emp. So we can write for the first study T
avg
(n) =O(N log
2
N) where N=n
2
And for the second study T
avg
(n) =O(n). Empirical O is obtained by supplying numerical values
to the weights obtained by running computer experiments. A computer experiment is a series of runs of
a code for various inputs and whether the response variable will be the output or a complexity depends
on the investigators interest (e.g. we can run a sorting algorithm to get the sorted array (output) just as
we can do it to measure sorting time or measure the number of comparisons or the number of interchanges
(time/computational complexity respectively) depending on the investigators interest). For more on
empirical O, statistical bounds and the link between algorithmic complexity and computer experiments,
see [2]. If the computer experiment is designed and analyzed properly, it increases the credibilityof the
bound estimate, the so called empirical O.
References :
Aho .V, Hopcroft J , Ullman J .(2000). Data Structure and Algorithms, Pearson Education Reprint.
Chakraborty, S and Sourabh, S. K.(2010). A Computer Experiment Oriented Approach to Algorithmic
Complexity, Lambert Academic Publishing, Germany.
Draper N , Smith H.(1998). Applied regression analysis, Wiley-interscience, 3
rd
ed. 8
Kennedy J , Gentle J ames E.(1980). Statistical Computing, Marcel Dekker,
Knuth .D.E.(1998).The Art of Computer Programming,vol.2: 3
rd
ed., Addison Wesley Longman
Publishing Co,Inc.Boston, MA,USA ISBN; O-201-89684-2
Vol. 1, 153 - 159 [2013]
160
161
PESTICIDE ACCUMULATION, ALTERATION OF SERUM
VITELLOGENIN AND FSH LEVEL AND OCCURRENCE OF HEPATIC
NEOPLASM IN AIR BREATHING FISH FROM WETLANDS OF NORTH
BIHAR, INDIA
Prakriti Verma and Prabha Rani.
Department of Zoology, Patna University
Patna- 800 005, Bihar, India.
Corres.Author- drprakriti@sify.com
Abstract : In the present investigation a number of selected wetlands fromthe north Bihar particularly
Supaul and Saharsa district were surveyed. Fish soil and water sample were collected fromthe various
test zones for the assessment of organocholrine pesticide accumulation. A comparative analysis of the
toxic status of fish fromthese wetlands based on pesticide accumulation and histopathology of liver
cells were done. Various organochlorine group of pesticide incurred were HCHs, Endosulfan DDT,
DDE etc. Comparative survey of test zones showed the variation in accumulation of HCHs and Aldrin
DDTs. Fishes and water sample fromthe reference site showed almost negligible percentage of these
pesticides. Fishes collected fromdifferent test zones were screened for the presence of neoplastic and
preneoplastic liver lesion based upon histopathological and ultrastructural findings. Thehistoarchitecture
of liver of C. batrachus fromthe test reveals changes in the mitochondrial organization, reduced ER
cristae, scanty hepatoplasmand tendency of SER to transfer into glycogenated bodies, apoptic bodies,
proliferation of RER and cellular hyper activity. Blood of test fish also revealed decreased serumFSH
level & less concentration of vitellogenin.
Key words : Pesticide accumulation, SerumVitellogenin FSH level, hepatic neoplasm, air breathing
fish, wetlands, North Bihar
Introduction : Northern Bihar is lamented with a vast source of naturally occurring low lying areas,
flood plains, wet land and paddy fields inhabiting a good population of air breathing cat fishes like
Clairas batrachus (Linn) and Heteropneustes fossilis (Bloch).. The sudden death of fish indicates
heavy pollution which can be measured in terms of biochemical, physiological or histological response
(Sounders 1969, Nath Dutta, et al 2003). In aquatic environment pesticide undergo a biotic degradation
by hydrolysis, and enter in aquatic organism directly through gill or epithelial tissues. The harmful chemicals
accumulate in specific organs and then get biomagnified. Fishes take up most of the xenobiotics from
the surrounding water by passive diffusion through gills or gastro-intestinal tract. After uptake the chemical
are transmitted and deposited in the fatty portion of the tissues (Kumari et al 2001). Liver is the target
organ, which not only resists the deleterious effects of pesticides but also detoxifies it. Entering to an
organism xenobiotic bind to specific cellular structure called receptor that is localized on the cell surface
or inside the cell either in its cytoplasmor on cell organelles (Yamaguchi 2003). Several reports are
available for the study of various pesticide residues in fish and its impact on various organ have been
Vol. 1, 161 - 167 [2013]
162
well established (Muir 2003, Carla 2004 & Couch 1993, Baile 1991) but a systematic approach
regarding prevalence of a various organochlorine pesticides in soil & fish liver sample from selected
wetland test zones of North Bihar are lacking. In the present investigation a few wetland test zonesof
north Bihar have been selected to see correlation between organochlorine pesticides residues in soil
and liver of fish and occurrence of neoplasm.
Materials and Methods : Clarias batrachus were captured by local fisherman fromthe three test
zone wetlands in around Saharsa and Supaul District of Bihar viz Chitragupta Mandir Chaur (A),
Gramharnia Chaur (B) and Hardi chaur (C) and screened to find out the neoplastic and pre-neoplastic
lesions. Approximately 200 fish adult/ female were screened (Table I) and collected fromthese
wetlands. Unhealthy fishes were dissected on spot and liver tissues were sampled for the estimation of
pesticide residue (Table - III) and histopathological analysis. Among the fish haul, healthy Clarias
batrachus of 14-27" length and 50-110 gm10 gmwt were brought to the laboratory and after
disinfections with 0.1% KMNO
4
solution they were kept at roomtemperature in large plastic pool for
acclimatization. They were fed with pelleted food made up of wheat flour and egg with a pinch of starch
as binder @ 4-5% of their body weight. They were also fed with chopped goat liver on every 3
rd
day
to fulfill their dietary requirement.
Quantification of pesticide residues in soil : Collectionof soil sample Soil sediments werecollected
at the four edges of each wetlands/Chaur usinga spade. Theentire samples were sealed in polythene
bags, storing at 0
o
C and transported to the laboratorywithin two days. Freeze dried samplewere then
passed through 1.0 nm sieve to separate sample and other debris. After crushing with anhydrous sodium
sulphate powdered soil sample were further processed in n-Hexaneand send to ITRC (Industrial Toxicology
Research Institute), Lucknow for estimation of organochlorine pesticideresidues (Table II).
Collection of Blood Sample and serum analysis FSH & and Vitellogenin At autopsy, the fishes
were anesthetized with 0.1g/L of MS22, Blood sample were collected in a citrated hypodermic syringe
by resorting to cardiac puncture. The blood was centrifuged @ 15000 rpm for 15 minute and clear
supernatant fluid was stored as blood serumin appendorf at 4
o
C in deep freeze for further analysis.
SerumFSH was done on Merk minimios ELISA reader by Herichson Method. The serumFSH and
vitellogenin of both the group of fishes have been depicted in Table III & IV.
Detection of the egg yolk precursor vitellogenin (vtg) in blood, tissue sample of femalejuvenile, male
fish in a sample and sensitive biomarker for endocrine disrupt the chemicals (EDCs) with estrogenic effect.
Measurement of vtg has become an accepted routine screening test for estrogenic and anti-androgenic effect
of EDCs in fish. The lyophilized coup vtg std. was calibrated against purified cat fish vtg. Using the following
formulas (Norberg and Haux 1988) vtg concentration mg/mg =absorbance at 286 nm/0.66, range =0.24
ng/ml. The comp vtg ELISA kit werepurchased fromBioscience Laboratory, Norway.
Histopathological Analysis : The liver tissue of reference site and those of various test zones were
fixed for light and electron microscopy. For light microscopy, tissues were fixed in neutral formalin.
After dehydration through graded series of alcohol, clearing, embedding, microtomy 5msection were
Vol. 1, 161 - 167 [2013]
163
stained by Delafields haemotoxylin and eosine & mount in DPX and photographs were taken on
trinocular microscope (Labomed CXRIII) fitted with Olympus digital 14 megapixel camera. For electron
microscopic studies tissues were fixed in 2.5% gluteraldehyde in 0.1M phosohate Buffer (pH=7.4) at
4
o
C. After one hour the tissues were placed in 1% OSO
4
in 0.2M Phosphate Buffer solution (pH 7.4)
at 4
o
C for 2 hours, followed by its dehydration in graded series of alcohol and amyl acetate, cleaned in
toluene and embedded in araldite mixture. Ultrathin sections were obtained through Reichart Jung
Supersora ultra microtome stained in Uranyl acetate and lead citrate and transformed to former coated
copper grid and viewed under Philips EM-10 transmission Electron Microscopy at SIF-EM Facility
Unit, Dept. of anatomy, AIIMS, New Delhi. TheMicrographs have been shown in Text graphs. Electron
Microscopy of liver cell.
Observations
TABLE I
Resume of the test fish Clarias batrachus surveyed from different test zones after external
and internal screening during 2010-2011
(-) represent absence of lesion for the approx. 200 individual studied.
TABLE II
Concentration of organochlorine pesticide residue in soil of different test zone of North
Bihar during 2010-11
Vol. 1, 161 - 167 [2013]
164
TABLE-III
Fluctuation of Serum vitellogenin level in test fish collected from different test zones
TABLE-IV
Fluctuation of Serum Follicle Stimulating Hormone level in test fishes collected from
different test zones during the post, Pre, spawning and resting period
Values are expressed in MeanSE for No. of observations 10 in each case.
The liver of normal fish has continuous mass of hepatic cells with cord like formation. The
cells were large with more or less centrally placed nucleus and homogenous cytoplasm. Clear
division of hepatic cells into lobules has not been observed in most of the hepatic cells. Hepatocytes
were intact with dense cytoplasm. Architecture of hepatic artery was very distinct. Sinusoidal
spaces were well organized and opened into central vein. The histoarchitecture of liver of C.
batrachus from the test zone (B) wetlands had a number of necrotic changes and enlarged
perisinusoidal areas, increased eosinophilic inclusion, pyknotic and heterochromatized nuclei.
Vacuolation refers to the Initiation of pre-neoplastic changes occurring in liver. Most pronounced
abnormalities are vacuolar degeneration, Karyomegely, fibrosis of central vein, focal vacuolation
and multi-focal hemosidorosis and occurrence of appotic bodies. Few or no lesion was observed
in the tissue of fish taken fromreference site and site A. Few histological examination of liver tissue
from test zone C also revealed re-organization of liver tissue, characteristic of micro and macro-
nodular cirrhosis. Pronounced feature of ductular metaplasia of hepatocyte leading to
neocholangiolar structure. Hepatocyte tending to formrosette with the bile canaliculi located in
the center. EM Studies also reveals changes in the mitochondrial organization, reduced ER cristae,
scanty hepatoplasmand tendency of SER to transferred into glycogenated bodies, apoptic bodies,
proliferation of RER and cellular hyper activity. Fish liver were also analysed for accumulation of
organochlorine pesticides such as HCH, Aldrine, Dieldrin, Endosulphan and derivatives of DDTs.
Liver lesion and other abnormalities detected were statistically associated with sediment
contamination and water concentration of DDT & its metabolities. (Table II) Serumlevel of
FSH and vitellogenin level of test fish from these test zones also reveals decreasing trend as
compared to the reference site (Table II I & VI ).
Vol. 1, 161 - 167 [2013]
165
Discussion : A comparative analysis of pesticides in soil and fish liver shows accumulation of various
organochlorine pesticides viz. HCH HCH HCH , aldrin, endosulphan, DDE, DDT etc. the presence
fromdifferent test zone showed the variation in the accumulation of HCH HCH HCH , aldrin and
DDT whereas fishes collected fromreference site showed almost negligible percentage of organochlorine
pesecticides. Presence of pesticides in fish liver reveals the persistent use of pesticides which has also
been reported by Halden (1965). Mass mortality and behavior of Atlantic salmon on streamis polluted
by agricultural pests (Saunders, 1969). Pesticidal poisoning in fish is considered to be very serious, as
fish forms a major food resource for mankind affecting the consumers health (Dubois, 1971) and may
also adversely affect the yield of yeast. Presence of pesticide residues in liver tissues in the present
investigation can be correlated with the reports of (Yamagudi, 2003), in the muscles of fish fromUpper
Thames river. Muir et al (2003) have also observed DDT, HCH & PCBs in fishes from Barents Sea
Canadian Arctic. Occurrence of neoplasmand other degenerative changes in the present investigation,
have also well documented about the pathogenesis of liver lesion with anthropogenically introduced
contaminants Robert et al (1991). Mayers et al (1992) have shown that hepatic neoplasmas biomarkers
of contaminant exposure in fish. They have also measured fluorescent aromatic compounds in Bile and
Polychlorinated biphenyl PCBs. Carla M. et al (2004) have examined fish liver for toxicopathic lesion
and analyzed for selected chlorinated hydrocarbon such as PCBs, DDTs,and di-aldrin.George et al
(1996) have shown Rainbow trout liver as an alternative model for environment carcinogenesis research.
Parallel diagnosis of cell and tissue pathologies in c. batrachus liver showed that lysosomal per turbation
sensitivity reflected onset of progression of liver injury comprising focal to extensive necrosis and fibrosis,
as indicated by highly significant correlation between the breakdown of lysosomal stability and degree
of liver lesion. Injury of lysosomal membrane by lypopholic toxic compound may lead to leakage of the
hydrolytic lysosomal enzyme causing disturbance of cell function, resulting in degeneration and possibly
neoplasm(Moore 1985). Further (Moore et al 2007) very well illustrated about the hepatocellular
neoplasmin adult winter flounder fromBoston Harbour, as in the present investigation Mark et al
(2007) have studied the progression of hepatic neoplasia in medaka exposed to diethylnitrosamine.
Couch (1993) have very well compared about neoplastic hepatocyte with normal one under Light and
Electron microscopy. He has also well documented the hepatocellular carcinomas in teleost fish. Moore
et al (1991) in their studies have used the cellular marker of pollutant exposure and liver damage in
fish. Donald et al (1984) have shown the effect of chemical pollutants poses stress in bottomdwelling
fish and these are more prone to liver neoplasmand other diseases. Angela et al (1992) have shown
the histochemical and cytochemical indices of toxic injury in the liver of dab Limanda limanda. The
cellular death associated with this type of necrosis not only induces an inflammatory response, but also
decreases the functional no. of cells in the tissue with deleterious consequences for the organ function.
Increase in bile duct and disappearance of cellular limit suggest drastic alteration in the distribution of
organelle. Electron microscopy reveals proliferation of ER, loss of cristae of mitochondria and cellular
hypertrophy. The study confirmed the lesion described above revealed the incidence of cell death in
Vol. 1, 161 - 167 [2013]
166
individual of all tested group and reminiscent of programmed cell death. With nuclear shrinkage, irregular
shape and heterochromatization, which ultimately affect the metabolismof hepatocyte involved in important
biochemical pathway as confirmed by decrease in FSH, and Vitellogenin level. In all the wetland test
zones, test zone B seems to be the most toxic followed by test zone C and test zone A (Reference site)
as shown in the text graph (II). Organochlorine pesticide accumulation in soil and occurrence of liver
anomalies itself confirmit.It may be concluded that the bottom dwelling air breathing Clarias batrachus
fromthese wetlands is being worst sufferer by the accumulation of organochlorine pesticides and other
pollutants.
Acknowledgements : Authors are thankful to Women Scientist Scheme, SERC Division, Department
of Science & Technology New Delhi for providing fund (Project No. DSTNo:SR/WOS-A/LS-17/
2008), Vice-Chancellor, Patna University and Department of Zoology, Patna University for providing
research facility, members of EM Facility Unit, Department of Anatomy AIIMS, New Delhi for kind
cooperation for TEM.
Reference :
Baile and Oberai (1991), Rawat et al (2002) reported the histopathological changes in the fish Clarias
batrachus when subjected to endosulfan.
Donald, C., Mallins, Bruce B., mccain, Donald, W., Brown, Sin-Lam Chan, Mark S. Myers,
J ohn T. Landahl, Patty G. Prahaska, Andrew J . Friedman, Linda D. Rhodes, Douglas G.
Burrows, WilliamD. Gronlund and Harold O. Hodgins. (1984): Chemical Pollutants in
Sediments and Diseases of Bottom- Dwelling fish in Puget Sound, Washington, Environ. Sci.
Technol., 18, No.9, p. 705.
Dubois, K.P. (1971) : Acute toxicity of organophosphorus compounds to mammals. Bull. Wld. Hlth.
Org. 44: 233-240.
Dutta, et al (2003) has reported sublethal malathion induced changes in the ovary of an air breathing
fish H.fossilis.
Halden, A.V. (1965): Contamination of fresh water by persistent insecticide and their effect on fish.
Ann. Appl. Biol. 55: 332-335.
J ohn A. Couch (1993): Light and electron microscopic comparisons of normal Hepatocytes and
neoplastic Hepatocytes of well-differentiatedhepatocrellular carcinomas in ateleost fish, Dis. Aquat.
Org. 16: 1-14.
Mark, S. Ohikhiro and David E. Hinton (2007): Progression of hepatic neoplasia in Medaka (Oryizys
latipes) exposed to diethyl nitrosamine. Springer link J ournal articles pp.1-2.
Moore, M.J ., Smolowitz,R. Stegeman, J.J . (1994/2007): Cellular alteration preceding neoplasia in
Pseudopleuronectes americaus fromBoston Harpour. Marine Environ. Research. MERSDW
28, No.1/4 pp.425-429.
Vol. 1, 161 - 167 [2013]
167
Moor, M.N., Lowe. D.M., Buke. D., Dixon P. (1991): Molecular and cellular markers of pollutant
exposure and liver damage in fish. ICES. CM 1991/E: 23.
Muir, D. Savinova, T., Saninov, V., Alexeeva, L., Potelov , V., Svetochev, V. (2003): Bioaccumulation
of pcbs and chlorinated pesticides in seals, fishes and invertebrates fromthe white sea, Russia.
Sci Total Environ. 306(1-3): 111-31.
Myers, M.S., Olson, O.P., J ohson, L.l., Stehr, C.S., Hom, T., (1992) : Hepatic lesions other than
neoplasms in subadult flatfish frompuget sound, Washington: Relationship with contaminant
exposure. Response of Marine Organismto Pollutant Part I, P.45-51
Robert A., Murchelano and Richard E. Wolke (1991) Neoplasmand Nonneoplastic liver lesion in
winter flounder, Pseudopleurotectes americanus fromBosten Harbour, Massachusettes. Environ
of Health Perspective 90, pp.17-26.
Rauhani-Rankouhi, T., Van Holsteign, I., Letcher, R.J., Giery, J.P., Van den Berg, M., (2002): The
effects of pre-exposure with environmental and mnatural estrogen on vitellogenic production in
carp. (Cyprinus carpio) Hepatocytes. Toxicol. Sci. 67, 75-80.
Saunders, J.W. (1969): Mass mortalities and behavior of brook trout and atlantic salmon onstream
polluted by agriculture particles J . Fish Res. Bul. Con. 26: 695-699.
Yamaguchi N. Gazzard. D. Scholey, G. Macdonald, D.W. (2003): Concentration and hazard assessment
of pcbs, organochlorine pesticides and mercury in fish species fromthe Upper Thames: river
pollution and its potential effects on top predators. Chemosphere. 50(3): 275-73.
Vol. 1, 161 - 167 [2013]
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169
A SERO-GENETIC STUDY ON THE DISTRIBUTION OF RH-ANTIGEN
AMONG MAJOR SCHEDULED CASTE POPULATIONS
Parimal Kumar Khan and Manoj Vibhakar
Department of Zoology, Patna University, Patna
Abstract : An immune- haematological test was carried out to study the distribution of Rh- antigen
amongthe four endogamous scheduled caste populations of the district of Patna (Bihar). The populations
surveyed were the Dusadhs ,the Chamars,the Pasis and the Musahars.
All the four populations exhibited a relatively high incidence of Rh
-ve
blood group with almost
no statistically significant difference among them. It was comparatively higher amongthe Dusadhs(5.62%)
and the Chamars (5.29%) followed by a slightly lower frequency among the Musahars(4.41%) and
the Pasis(3.71%). The frequency dominant (D) and recessive (d) alleles showed a similar pattern of
their incidence in all the four populations.
Introduction : India, with its 1.2 billion people [1], has the second largest population in the world,
being represented by over 4000 endogamous group, many structured in the Hindu caste systemas
Jatis [2]. Each endogamous group, reproductively isolated fromeach other, represents a Mendelian
population [3]. The practice of endogamy , being performed for centuries, has led to the evolution of
different gene pools, one for each caste. Population geneticists use to analyze the frequencies of various
genetic markers to study the quantitative variation in differences human populations (gene pools). As
many facets of the several population groups of India are still relatively unexplored, the present study of
sample of scheduled castes (with respect to the distribution of Rh-blood group) aims to examine the
genetic polymorphismamong them.
The Rh-factor was discovered by Karl Landsteiner and A.S. Wiener [4] fromrabbits immunized
with the blood of a monkey, Rhesus macaque; the symbol Rh came from the first name of the
species of monkey. The resulting antibodies agglutinated the red corpuscles of the monkey. On the
basis of the presence or absence of Rh antigen, the whole human population has been divided into Rh
+ve
and Rh
-ve
groups. The first human blood found to lack all known antigens, Rh-null, was reported by Vos
et al.[5]. Initially the genetic mechanism of the Rh-systemseemed to be governed by a single pair of
alleles, R and r, as postulated to account for the difference between Rh
+ve
and Rh
-ve
individuals. Wiener
[6]developed a hypothesis based upon a series of multiple alleles. According to an earlier hypothesis[7],
three pairs of gene are involved in the production of Rh antigen that are not alleles but are located near
each other on the same chromosome. The dominant forms of these genes are represented by C, D and
E, and its recessive formby c, d and e. A person is classified Rh
+ve
on the basis of the presence of at
least one dominant (D) allele, whereas the dd genotype ensures the Rh
-ve
blood group. While the C,
Vol. 1, 169 - 174 [2013]
170
c, E and e alleles specify related antigens, they are less immunologically insignificant. Later, Rosenfield
et al.[8] developed a new Rh notation system to be represented by only two alleles, D (dominant) and
d(recessive).
Methodology : A test for Rh (D) incompatibility was performed by the process of slide agglutination
method[9] with the help of SpanClone anti-D (Rh
o
) monoclonal IgM antisera (Span Diagnostics Ltd.,
Surat, India). One drop of anti-D was dispensed on a clean dry slide, and a drop of blood was then
added to it, mixed well with applicator stick and the slide was tilted back and forth for 2 minutes.
Presence of D antigen resulted in agglutination of the test RBCs and the blood was assigned Rh
(+ve)
.
No agglutination with anti-D antiserumindicated the absence of D-antigen, the blood group being Rh
(-
ve)
. Agglutination of erythrocytes therefore indicated incompatibility, whereas even distribution of
erythrocytes indicated no reaction. Frequencies of dominant (D) and recessive (d) alleles were calculated
fromthe number of phenotypes scored.
Re sults and Discussion : The incidence of Rh
-ve
subjects in all the four populations was slightly
higher than the range known for I ndian populations ( Fig. 1,Table 1). It was comparatively higher
among the Dusadhs (5.62%) and the Chamars(5.29 %) with decreasing magnitude among the
Musahars (4.41%) and the Pasis (3.71%). Statistically insignificant inter-sex variations in the
frequency of Rh
-ve
persons were observed within each population (Table 1) . All the four
populations, however, did not differ in the incidence of this trait among themselves ( except
between the Pasis and the Dusadhs) (Table 3). Such a high incidence of Rh
-ve
blood group is not
unique for these populations alone, because Pingle et al. [10] have found that in Rajgonds, a tribal
group of Andhra Pradesh, it is 6.18%, and as high as 10 to 17%among the Saraswat Brahmins of
Western India [11].
The frequency of dominant (D) and recessive (d) alleles showed a similar pattern of their
incidence in all the four populations (Table 2). Consequently, the expected frequency of individuals
homozygous (DD) for the dominant allele was very high to be followed by heterozygous (Dd) and
recessive (dd) ones.
Vol. 1, 169 - 174 [2013]
171
Table1
Frequency distribution of subjects of Rh-blood group system in the different populationsz
Vol. 1, 169 - 174 [2013]
172
173
174
References :
Census of India (2011) Ministry of Information and Broadcasting, Government of India, New Delhi.
Singh K S(1998) Indias communities. People of India.National Series, vol-IV, Oxford University
press, India.
Wright S ( 1951) The genetical structure of populations. Ann Eugen,15, 325-354.
Landsteiner K and Weiner A S (1940) An agglutinable factor in human blood recognized by immune
sera for rhesus blood .Proc Soc Exper Biol,43,223.
Vos U, Gutler L and Cleve H(1961) A sample with no detectable Rh antigen.Lancet, 1,14.
Weiner A S (1970) Blood groups and disease. AmJ HumGenet, 22,476-483.
Fisher R A and Race R R(1946) Rh gene frequencies in Britain. Nature,157,48.
Rosenfield R E , Allen F H and Rubinstein P(1973) Genetic model of the Rh blood group system.Proc
Natt Acad Sci USA, 70,1303.
Bhasin M K and Chahal SMS(1996) A Laboratory Manual for Human Blood Group Analysis. Kamla
Raj Enterprises, Delhi.
Pingle U, Mukherjee B N and Das S K(1981) Blood samples belonging to five tribal groups of Andhra
Predesh . J Morphol Anthropol ,73,339-348.
Bhatia HM , Shanbhag H , Bharucha Z S , Bapat J , Sathe MS, Sharma RS , Kabeer H, Ucha ZS and
Surlacar L (1976) Genetic studies among endogamous groups of Saraswat Brahmins in Western
India.HumHered, 26,458-467.
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