Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604

IMPACT OF TWO PESTICIDES ON SERUM FREE AMINO ACID POOL OF MICE: A COMPARATIVE STUDY USING THIN 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 [email protected], [email protected]

Abstract : An attempt has been made to identify and 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 organophosphate commonly known as rogor, with a 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 dose of 2 mg/kg body weight for 21 days. Every week blood were collected, centrifuged and serum were separated to estimate the free amino acids by Thin layer chromatography. Amino acids were located on the chromatogram with a 0.01% ninhydrin solution in acetone. Each chromatogram reveals 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 concentration and quantitative estimation was not possible. Finding indicates that the exposure of pesticide brings aiteration of 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 being widely used in agriculture, which has many harmful effects on living organism. Animals in 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 the soil, water reservoirs, vegetables, grains and other food products (John et al., 2003). Man is the ultimate consumer of pesticide residues. These pesticide residues in animal products and other food items ultimately get accumulated in the man especially in the adipose tissue, blood and. lymphoid organs. When fed to man or animals at very low doses daily for months or years, these accumulated pesticides in body, may harm the 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 health hazards 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 serum amino acid pool has not been studied. Similarly, Dimethoate (O,O-dimethyl S– methylcarbomylmethyl phosphodithioate),also known as Rogor , is one of the most important

1 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 organophosphorus insecticides used frequently in agriculture. Many studies have been carried on the toxicity of dimethoate on non-target animal like mice (Betrsian et al., 1995; Kamath et al., 2008). Hassan et al. (1994) have reported several changes in serum parameter and amino acid content in rats after chronic sub lethal dose of 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 gm were procured from research laboratory; MAHAVIR CANCER SANSTHAN, Phulwarisharif, Patna. The albino mice were housed in poly propylene cages and maintained in controlled temperature (27degree centigrade), humidity (0.5-10%) and light cycle. They were fed with cereal 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 LD50 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 -20oC in sterilized paraffin covered tubes for amino acid and biochemical analysis. Serum was analyzed for quantitative estimation of Total protein respectively. Methods for Amino Acids Estimation : Amino acid profile in the blood serum of Mus musculus were assayed using Thin layer chromatography to arrive at Rf (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 ctrophotometer (UV-U75- Spectrophotometer) at 570 nm. 2 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604

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 from which a calibration curve was constructed. Preparation of serum for TLC : A collected blood sample were centrifuged at 3000 rpm for 10 min at 4°C to obtain serum that 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 bottom and then 8-10 approximately equal small spots applied at 2cm intervals on silica gel coated 6 of TLC aluminium sheets 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 Chromatogram reveals 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 nm with 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 chromatogram were 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.

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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 serum at 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 serum amino 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 fisher’s 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 from serum same 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 from E7 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 applying‘t’ test the calculated values were referred to

4 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 fisher’s 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 serum Total 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 serum of control, Rogor and endosulfan treated Mus musculus after 7 days, [taking lysine as standard.]

O.D = Optical density at 570 nm, Rf = Retardation factor

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TABLE -III Showing amino acids in blood serum of control, Rogor and endosulfan treated Mus musculus after 14 days, [taking lysine as standard.] S. AMIN C ONT ROL ROGER ENDOSULPHAN No O R f O R f O C o R f O AC ID VA D Mean± VAL D Mean± VAL D Mean± LU S .D. UE S.D. UE S .D. E 1 Alanine ------2 Asparti 0.23 0.2 5.955±0.013 - - 0.23 0.1 3.515±0.011 c a cid 18 29 3 Glutami 0.32 0.1 4.108±0.004 ------c a cid 50 4 Glycine ------5 Methio ------nine 6 Serine ------7 Tyrosine 0.47 0.1 2.752±0.009 ------01 8 Valine 0.60 0.0 2.565±0.009 ------94 O.D = Optical density at 570 nm, Rf = Retardation factor TABLE -IV Showing amino acids in blood serum of control, Rogor and endosulfan treated Mus musculus after 21 days, [taking lysine as standard.]

S. AMIN CONTROL ROGER ENDOSULPHAN No O R f O R f O R f O ACID VA D Mean± VAL D Mean± VAL D Mean± LU S.D. UE S.D. UE S.D. E 1 Alanine - - - 0.39 0.0 1.861±0.020 - - - 69 2 Asparti 0.2 0.2 5.955±0.013 - - - 0.25 0.0 2.167±0.017 c acid 3 18 35 3 Glutami 0.3 0.1 4.108±0.004 ------c acid 2 50 4 Glycine ------5 Methio - - - 0.58 0.0 2.174±0.013 - - - nine 80

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6 Serine - - - 0.27 0.1 4.211±0.005 - - - 54 7 Tyrosine 0.4 0.1 2.752±0.009 0.45 0.0 2.315±0.008 - - - 7 01 85 8 Valine 0.6 0.0 2.565±0.009 ------0 94 O.D = Optical density at 570 nm, Rf = Retardation factor TEXT GRAPH 1

TEXT GRAPH 2

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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 serum biochemical test exclusively serum amino 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 7th day and 14th 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 due to 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 from an 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 serum on 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 from 7th 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 due to

8 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 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 from toxic stress(Susan et al.,2010),(Table I), utilizing serum free amino acids. Regarding serum free 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 them persist 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 from organophosphate poisoning.,J.Toxicol.Clin.Toxicol.,33(3),257-260 . Choudhary,N. and Joshi, S.C (2002) : Effect of short term endosulfan 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 serum parameters and amino acids content in rats after chronic sublethal doses of dimethoate.,Arch.Environ.Contam. Toxicol.,27(2),256-259. James, J.H, Ziparo ,V., Jeppsson ,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. John,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 Rajyam C. (1989) : Evaluation of toxic effects of a synthetic pyrethroid fenvalerate in albino rat. Ph.D. thesis, S.V. University,Tirupati, India. . Lowry, Ï. H.,Rosebrough, Í.J., Farr, A. L. and Randall,R.J. (1951) : Protein measurement with folin- phenol reagent. J. Biol. Chem., 193, 265.

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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 Journal 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.,Journal 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 metabolism in 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.

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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 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. 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 gave the 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 MgSO4.7H2O in the broth. Key words : Microbial production, citric acid Introduction : Citric acid (2-hydroxy-propane-1, 2, 3-tricarboxylic acid) derives its name from the Latin word citrus, a tree whose fruit is like the 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 a primary metabolic product formed in the tricarboxylic acid (or Krebs) cycle and is found in small quantities in virtually all plants and animals, being isolated from lemon juice in 1784. Wehmer was the first to demonstrate that Citromyces (now Penicillium) accumulated citric acid in a medium containing 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 the medium, 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

11 Journal of Patna Science College Vol. 1, 11 - 17 [2013] ISSN 2347 - 9604 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 been the subject 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 in industrial fermentations are glucose syrups from starch hydrolysis, sugar beet molasses and low quality-sugarcane byproducts that, in general, are contaminated by high levels of cations from previous processes. Cations usually come from insoluble residues formed by precipitation with potassium ferrocyanide. 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) are rich 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 ammonium salts, particularly ammonium nitrate 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 medium is important in two stages of the process. All fermentations start from spores and their germination requires pH > 5. The absorption of ammonia by germinating spores causes release of protons, thus lowering the pH and improving the production of citric acid. The low pH value during the production phase (pH > 2) reduces the 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 from 4 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 35±2ºC for 48hrs. Colony forming units (log10 CFU)/g of each sample was calculated for assessing the microbial load (average of three plates).

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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 35°C 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 37°C to measure the dry mass. 50ml of the filtrate was mixed with equal volume of Ca (OH)2 and kept at room temperature for 2 to3 hours.

Precipitate of calcium citrate was collected and equal volume of 1N H2SO4 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 form the 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 medium and incubated at 35°C 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 ammonium nitrate. The modified medium was inoculated with 48 hrs old culture and incubated for 144 hrs at 35°C.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 done by preparing three different flasks of Doeglers and Prescott broth containing no magnesium sulphate, 4 times of original amount and 8 times of original amount. The medium was inoculated with 144 hours old culture and incubated at 35°C 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 medium containing 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. 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 gave the lowest yield (0.215g/ml) of citric

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acid with biomass 4.169g/ml. The effect of MgSO4.7H2O was monitored it was observed that production of citric acid reduced drastically on increasing the concentration of MgSO4.7H2O in the broth. Discussion: In this work, fungus was isolated from soil 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 mycelium which resulted in increased viscosity of 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- ulhaq et al, 2002).The accumulation of large amounts of citrate by the filamentous fungus A.foetidus is known to depend on Mn2+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

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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

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Fig.4. Effect of Magnesium Sulphate on citric acid and biomass production References: Gupta, JK, Heding LG and Jorgensen OB (1976). Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger. ActaMicrobiologicaAcademiaeHungaricae23: 63–67. Haq I Kram, Ali Sikander, Qadeer MA and Iqbal Javed(2002).Citric acid fermentation by mutant strain of Aspergillus niger GCHC-7 using Molasses based medium.Electronic Journal of Biotechnology5:121-125. Honecker, S, Bisping B, Yang Z and Rehm HJ (1989). Influence of sucrose concentration and phosphate limitation on citric acid production by immobilized cells of Aspergillus niger. Applied Microbiology and Biotechnology31: 17–24. Hossain M, Brooks JD 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, Röhr M (1989).Citric acid fermentation. Critical Reviews in Biotechnology4: 331–373. Lancini, G (2008). Parte I – L’usoindustrialed 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: 87–132. 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: 165–171. 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: 359–366. 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: 710–717. Penniston L Kristine, Nakada Y Stephen, Holmes P Assimo and G Dean(2009). Quantitative Assessment of Lemon Juice, Lime Juice and Commercially available Fruit Juice Products.Journal of Endurology22:567-570. RöhrM, Kubicek CP, Zehentgruber O and Orthofer R (1993). Accumulation and partial reconsumption of polyols during citric acid fermentation by Aspergillus niger. Applied Microbiology and Biotechnology27:235–239. Shu, P and Johnson MJ (1948). Citric acid production by submerged fermentation with Aspergillus niger. Industrial & Engineering Chemistry 40:1202–1205. Shu, P and Johnson MJ (1948). The interdependence of medium constituents in citric acid production by submerged fermentation. Journal of Bacteriology54: 161–167. Wehmer C (1893). Note sur la fermentation.9:728. ATCC 9142 from a treated ethanol fermentation co-product using solidstatefermentation. Letters in Applied Microbiology48: 639-644.

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BIOMAS S AND NUTRIENT DYNAMICS OF FREE FLOATING MACROPHYTES OF BARAILA WETLAND, VAISHALI Shardendu* 1, D. Sayantan2 and S. R. Padmadeo3 *Corresponding Author 1. Associate Professor, Laboratory of Environment and Biotechnology, Department of Botany, Patna Science College, Patna University, Patna. Email: [email protected]; Contact No. 9473240391 2. Junior Research Fellow (U.G.C.), Laboratory of Environment and Biotechnology, Department of Botany, Patna University, Patna. Email: [email protected] 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 (25°76’N and 85°56’E), 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 2.36±0.5 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

19 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604 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), acting as 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 from Baraila Chaur (wetland) of Vaishali district of Bihar, India, situated on 25°76’N and 85°56’E. Temperature varies between 30.81°C and 5°C 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 from 25 X 25 cm area 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 80°C

20 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604 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 mm stainless 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 portion was used for analysis of potassium, calcium, and sodium. The sub-samples were converted into ash in muffle furnace at 480°C for 12 h after adding 2 ml saturated solution of magnesium nitrate (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 magnesium by 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 and Utricularia flexuosa. The first two were present throughout the year, whereas Azolla pinnata was absent in April, May and June 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 maximum biomass was 1190.70±89.04 g m-2 in October and minimum of 531.4±43.07 g m-2 in May (Fig 1). Lemna minor was another species of this zone which had 2.36±0.50 g m-2 in September, afterwards there was a decrease in biomass and minimum of

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0.56±0.13 was recorded in May (Fig 3). Azolla pinnata showed 0.91±0.143 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 4.59±0.679 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.

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The fourth species of this zone was Utricularia flexuosa which was absent in May. It started germinating in June (4.33±0.56 g m-2). There was a gradual increase in the biomass to reach a maximum value of 144.33±24.37 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 June (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 January (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 was 882.32 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.

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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. % % cont ribution Factors Spec ies Sum mer Rainy Wint er contribution Total Standin g of of species crop biom ass of Standing crop to zone species/zone biom ass

Total 1. Eichhornia standing crassipe s 674.13 963.56 1009.28 91.88 882.32 51.2 biom ass 2. Lem na mino r 0.678 1.61 1.63 0.14 1.31 0.08 3. Azolla pinnata 1.29 1.31 3.53 0.21 2.04 0.12 4. Utricularia fle xuosa 23.44 98 118.11 8.27 79.85 4.63 % contribution of the se ason to 24.15 36.75 39.1 this z one 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 maximum rate 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 maximum rate 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 maximum for 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 them similar 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.

24 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604 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 from 2010-2011 to 2010 by positive change method Summer Rainy Winter 16 Fe b. - 15 June 16 June - 15 Oct. 16 O ct. - 15 Feb. SPECIES Productivity Production Productivity Production Productivity Produc tion g m-2 g m-2 g m-2 g m-2 g m-2 season-1 season -1 g m -2 season -1 season -1 sea son-1 season-1

1. Eichhornia crassipes - - 662.07 5.43 50.73 0.412 2. Lemna minor 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

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 June. In E. crassipes nitrogen ranged from 9.82 to 17.72 mg g-1, phosphorus 0.78 to 1.68 mg g-1, potassium 20.85 to 35.51 mg g-1, calcium 8.10 to 16.08 mg g-1, magnesium 4.35 to 8.75 mg g-1 and sodium 1.73 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 calcium 2.75 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 from 26.41 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 magnesium and 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 from 0.71 to 1.25 mg g-1, potassium 11.55 to 18.21 mg g-1, calcium from 1.51 to 2.78 mg g-1, magnesium 0.98 to 2.67 mg g-1 and sodium 7.41 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 maximum 30.57mg 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 potassium content decreased in order 27.15 mg g-

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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 maximum annual 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-1for U. flexuosa. The content of magnesium which is also the index of production was maximum 6.21 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 minimum 1.73 mg g-1 for U. flexuosa. Sodium content 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.

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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 9.82±1.14 mg g- 1 was noted in November. Seasonally, summer months April (15.59±1.24 mg g-1 ), May (16.29±1.27 mg g-1 ) and June (17.71±1.41 mg g -1) had higher values than the months of rainy season, July (16.19±2.02 mg g-1 ), August (14.62 mg g-1) and September (13.56±1.75 mg g-1). Winter months October (12.12±1.87 mg g -1 ), December (11.24±0.71 mg g-1 ) and January (11.95±0.99 mg g-1 ), had lower values than both the summer and rainy months (Fig. 6). Monthly variation in phosphorus content ranged from 1.68±0.12 mg g-1 in June to 0.78±0.15 mg g-1 in October, though the very close to minimum value (0.79±0.15 mg g-1 ) was observed in January 2011. Concentration of phosphorus in other months were 1.21±0.12 mg g-1 for March, 1.32±0.18 mg g -1 for April, 1.44±0.25 mg g-1 for May and these were higher than the contents of 1.25±0.14 mg g-1 in July, 1.02±0.13 mg g-1 in August and 0.82±0.11 mg g-1 in Septe mber. In February of 1982 (1.07±0.12 mg g-1) and 2011 (1.05±0.09 mg g-1 ) the concentration values were very close followed by December (1.05±0.15 mg g-1) and November (0.92±0.06 mg g -1 ) (Fig. 5). Maximum potassium content (35.51±2.98 mg g-1) of E. crassipes was recorded in June followed by fall in the content which reached the minimum values 20.85±2.54 mg g-1 in November and 20.85±4.40 mg g- 1 in December. Rainy season months of July (32.35±1.96 mg g-1 ), August (30.15±2.11 mg g-1) and September (26.67±2.33 mg g -1 ) had slightly lower concentration of potassium than the summer months March (27.85±2.35 mg g-1), April (30.63±1.64 mg g-1 ) and May (33.75±1.94 mg g-1 ). February of 1982 (24.02±2.02 mg g-1 ) and of 1983 (24.02±1.60 mg g-1) had the same content followed by January (21.69±1.75 mg g-1 ), (Fig. 6). Regarding calcium content November (14.52±1.70 mg g-1 ), December (16.08±1.56 mg g-1 ) and January (14.29±1.85 mg g-1 ) together constituted higher content than the July (10.82±1.49 mg g-1 ), August (11.46±0.93 mg g-1 ) and September (12.48±1.24 mg g-1 ). The minimum calcium content of 8.10±0.81 mg g-1 was in May followed 9.32±1.17 mg g-1 in June (Fig. 6). Magnesium content of E. crassipes was quite low (4.62±0.84 mg g -1) in May, after which it increased to October (8.75±0.97 mg g-1 ), then again decreased. Magnesium content reached the minimum (4.35±0.67 mg g-1 ) in January 2011. The values of winter season, 6.98±0.87 mg g-1 in November, 5.68±0.77 mg g-1 in December and 5.38±0.55 mg g -1 in February 2011 were lower than in the months of rainy season i.e. 6.37±0.60 mg g-1 in July, 7.75±0.87 mg g-1 in August and 7.92±0.68 mg g -1 in September as well as of the two summer months, April (5.12±0.56 mg g-1 ) and June (5.51±0.56 mg g-1) (Fig. 5). Sodium content of E. crassipes had almost two equal peaks, one (3.25±0.38 mg g-1 ) in June and another (3.28±0.23 mg g-1 ) in January. After the first peak, there was gradual fall in the content, which reached to the minimum (1.73±0.20 mg g-1 ) in September, which then enhanced. Seasonally, winter had higher content than the rainy and summer months (Fig.

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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.

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L. minor was the another member of the free-floating zone. The maximum nitrogen content of 29.57±2.19 mg g-1 was found in June and the minimum of 18.57±2.77 mg g-1 in October. April (21.02±2.38 mg g-1), May (24.63±1.88 mg g-1) and July (25.50±2.20 mg g-1) together constituted the months in which higher nitrogen values were obtained than in November (19.47±2.26 mg g-1), December (20.63±1.95 mg g-1) and January (21.07±1.98 mg g-1) (Fig. 10). Phosphorus content also varied in a similar way. The highest of 10.52±1.10 mg g-1 was recorded in May and the minimum of 4.52±0.93 mg g-1 in October and another lower value close to this of 4.59±0.75 mg g-1 was observed in January. 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 21.02±1.84 mg g-1 were recorded in February 2011 and in the summer months of May (19.49±2.17 mg g-1) and June (18.46±2.70 mg g-1). The minimum 14.50±1.62 mg g-1) of potassium was recorded for September followed by July (17.45±1.80 mg g-1) and June (16.38±1.81 mg g-1). Calcium content of L. minor was minimum (10.43±1.15 mg g-1) in May, which enhanced thereafter upto December (19.55±1.84 mg g-1). In winter calcium was higher in L. minor than in other seasons (Fig. 10). The trend of the monthly variation of magnesium was different than of other elements. Maximum of 4.11±0.38 mg g-1 was in September, which got reduced upto January 2011 -1). In April (2.85±0.34 mg g-1), May (2.95±0.41 mg g-1 - 1) values were significantly lower than in October (3.75±0.38 mg g-1), November (3.31±0.28 mg g-1) and December(3.12±0.31 mg g-1). Variation of sodium content of L. minor ranged from 2.93±0.32 mg g-1 in July to 1.82±0.30 mg g-1 in October. Concentration in other months were 2.73±0.38 mg g-1 in May, 2.43±0.26 mg g-1 in June, 2.67±0.44 mg g-1 in August and 2.22±0.24 mg g-1 in September (Fig. 9). Phosphorus, calcium changed significantly p<0.005 between months whereas magnesium at p<0.025, potassium p<0.100 and nitrogen and sodium p<0.250 (Table 3).

Figure 7 Mean monthly variation of Mg, Na and P in Azolla pinnata of Baraila wetland.

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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 from maximum 37.91±1.93 mg g-1 in July to 26.41±1.36 mg g-1 in December. Among other months in August (35.28±2.37 mg g-1), September (32.55±1.33 mg g-1) and October (30.15±2.62 mg g-1) a comparatively higher nitrogen content was recorded than in January (27.01±1.91 mg g-1) and February 2010 (29.63±2.12 mg g-1) (Fig. 8). Phosphorus content was also highest (2.57±0.21 mg g-1) in July and the same was true for potassium (14.63±1.79 mg g-1) in July. The lowest values were 1.22±0.09 mg g-1 of phosphorus in December (Fig. 7), and 9.35±1.24 mg g-1 for potassium in 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 7.58±0.81 mg g-1 was in August, followed by 8.21±0.63 mg g-1 in September and 9.69±0.93 mg g-1 in October. The maximum content 11.41±0.73 mg g-1 was noted in January 2011 followed by February 2010 and 2011 (11.29±0.67 mg g-1 and 11.02±1.44 mg g-1) (Fig. 7). The amount of magnesium concentration was 5.78±0.55 mg g-1 in November, 5.92±0.79 mg g-1 in December and 5.99±0.8 mg g-1 in January which was higher than 3.59±0.46 mg g-1 in July, 4.11±0.34 mg g-1 in August and 4.51±0.49 mg g-1 in September. The maximum of 9.88±0.75 mg g-1 sodium content of A. pinnata was in July, after which it decreased to the minimum of 6.58±0.80 mg g-1 in December, and it fluctuated in January and February. In March very close (6.78±0.37 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, calcium and sodium at p<0.1 and for magnesium at p<0.25 level, whereas the variation of potassium was not significant (Table 3). 30 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604

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.

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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 22.19±0.89 mg g- 1 in July to 14.64±1.53 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 (1.25±0.14 mg g- 1) was in July, after which the concentration fell down to the minimum of 0.71±0.17 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 (11.55±0.88 mg g-1) in June followed by the enhancement in the content in rainy and winter months and maximum of 18.21±1.13 mg g-1 was noted in January 2011. Again it decreased. The trend of calcium content of U. flexuosa was almost similar to that of potassium having highest 2.78±0.24 mg g-1 in January 2011 and lowest 1.51±0.16 mg g-1 in September. In the winter months amount of calcium was higher than in the other two seasons (Fig. 11). Magnesium and sodium content had almost an opposite pattern. The minimum value of 0.98±0.14 mg g-1 of magnesium and maximum value of magnesium (2.67±0.22 mg g-1) was recorded in November while lowest value of sodium (7.41±0.99 mg g-1) was found in October (Fig. 12). Statistically, calcium magnesium and sodium changed 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. crassip es N Mo nth 12 1.949 0.1624 4.89 0.005 Error 24 0.796 0.0332 P Mo nth 12 0.026 0.0022 3.67 0.005 Error 24 0.015 3 0.006 K Mo nth 12 8.925 0.7438 5.48 0.005 Error 24 3.256 0.1357 Ca Mo nth 12 2.254 0.1878 3.41 0.025 Error 24 1.321 0.055 Mg Mo nth 12 0.649 0.054 4.50 0.005 Error 24 0.296 0.012 Na Mo nth 12 0.081 0.007 3.50 0.005 Error 24 0.053 0.002 L. minor N Mo nth 12 3.087 0.2573 1.75 0.25 Error 24 3.533 0.1472 P Mo nth 12 1.093 0.0911 10.47 0.005 Error 24 0.209 0.0087 K Mo nth 12 1.029 0.0858 2.03 0.1 Error 24 1.014 0.0423 Ca Mo nth 12 2.571 0.2143 3.86 0.005 Error 24 1.332 0.0555 Mg Mo nth 12 0.084 0.007 3.18 0.025 Error 24 0.053 0.0022 Na Mo nth 12 0.034 0.0028 1.57 0.25 Error 24 0.042 0.0018 32 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604

A. pinnata 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. flexuosa 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 from Baraila wetland; the squared multiple correlation coefficient (R2) and F statistics are also shown/ Nutrients Regression Equation 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* 0.10 0.99 K PN = 1.69 - 0.681NW + 0.002PB n.s. n.s. 0.43 Ca PN = -0.01 + 0.014NW - 0.001PB 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. minor

N PN = 0.73 + 0.010NW + 0.511PB 0.91*** 8.24**

P PN = 0.08 + 0.051NW + 0.117PB 0.96*** 20.33*** 33 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604

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 2.57 N PN = 1.76 + 0.011NW + 0.154PB 0.76** 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*** R2 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 equilibiuium of environmental factors, like heat, light, nutrients and other biological and geological factors, when the maximum growth and development of aquatic plants occur. The standing crop biomass of this zone was higher than other reports except from Westlake (1963) (Table 1). The free-floating zone macrophytes were intermediate in production as well as in situation between emergent and submerged zone. A maximum rate of 5.43 g m-2 day-1 was recorded for E. crassipes whereas maximum rate 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 maximum rate 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. 34 Journal of Patna Science College Vol. 1, 19 - 36 [2013] ISSN 2347 - 9604

In species of free-floating zone the variation in nutrients with season was well marked. In summer, nitrogen, phosphorus and potassium were maximum and calcium minimum 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 potassium were significantly lower in most of the cases and calcium was quite high. Effect of rainy season on variation of magnesium and sodium was quite distinct, in most of the cases concentration of magnesium was higher whereas opposite was the trend for sodium. Nitrogen, phosphorus and potassium are 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 magnesium in 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 sodium in 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.

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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) Jaunpur. 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.

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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 ([email protected]) 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 from 105 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 35±2ºC.The organism also hydrolysed different agro-wastes materials such as bagasse and corncob, along with CMC and filter paper. To standardize the additives in the media, NaNO3 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 sodium nitrate. 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 Vmax. While the value of Km 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 Maximum Parsimony algorithm with 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 from wheat flour (Cavaco-Paulo 1998; Bhat 2000). Economical production of cellulases has generally been considered 37 Journal of Patna Science College Vol. 1, 37 - 49 [2013] ISSN 2347 - 9604 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 from different microorganisms, mainly thermophillic fungi (Maheshwari et al., 2000; Jatinder 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 form essential 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) from bagasse (2450 mg/l) and corncob (2250 mg/l) as the carbon source. Successful attempts have also been made to optimize the nutritional requirements for maximum production 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 Km and Vmax has also been investigated. Material and Methods : Isolation of cellulase producing strains of actinomycetes Strains of actinomycetes were isolated from decomposing agro-wastes using starch casein agar (SCA) media containing per litre, 10g soluble starch, 0.3 g hydrolyzed casein, 2g KNO3, 2g

NaCl, 2g K2HPO4, traces of MgSO4, 7H2O; CaCO3, FeSO4, 7H2O 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).

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Preliminary characterization and identification The strain was characterized for taxonomic identification based on the parameters described in Bergey’s manual of determinative bacteriology (Williams et al., 1982). Clonal culture of the strain was deposited in MTCC & Gene Bank, IMTECH, Chandigarh (India) 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 150rpm and 35+10C. 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 NaNO3; 0.5g MgSO4, 7H2O; 0.5g K2HPO4, 1g KCl and traces of ZnSO4; MnSO4; FeSO4, 7H2O; CaCl2,) supplemented with 0.5% (w/v) of the respective carbon source. Quantitative estimation of cellulase The enzyme sample was prepared by passing the culture filtrates through Whatman No.1 filter paper with the help of suction pump. The filtrate was centrifuged at 1500xg for 30 minutes at 40C (Beckman J2-21M/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 500C for 20 minutes. The reaction was terminated by adding 3ml of 3,5-dinitrosalicylic acid (DNSA) followed by heating in a water bath at 1000C. The colour thus developed was read at 540nm spectrophotometrically (Hitachi U-3210) using glucose as the standard (Miller, 1959). The same process was used for FP cellulase activity except that the substrate was replaced by filter paper in a 50mM citrate buffer, pH 5.6. For -glucosidase activity, p-nitrophenyl -D-galactopyranoside was used as the substrate in 50mM sodium phosphate 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 400nm using p-nitrophenol as the standard (Berghem and Pettersson, 1974).

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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, NH4NO3 and NaNO3) 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 (CoCl2.6H2O, CuSO4.2H2O, NaCl, MgSO 4.7H2 O, KCl,

MnSO4.H2O, ZnSO4.7H2O, FeSO4.7H2O, CaCl2, HgCl2and Na2MoO4.2H2O) were used for testing the effects of cations on the production of RS and enzyme activity. The yield of RS was studied by inoculating 108 spores of Streptomyces sp. in 50ml of enzyme production medium supplemented with 5mM of respective cation. The effect of cations on enzyme activity was studied by adding them in the reaction mixture at a concentration of 1mM. Results : A total of 22 actinomycetes strains isolated from decomposing 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 30mm diameter 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 production of reducing sugar and enzymatic activities. Table-1 shows the endoglucanase, -glucosidase and FPase activity of the partially purified enzyme obtained after 72 hours. The maximum endoglucanase activity was found in corncob (2.50±0.091 U/ml). Maximum -glucosidase activity was observed in carboxy methyl cellulose (0.69±0.021U/ml) and maximum FPase activity (0.150±0.0004 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 -1 presence of a fixed concentration (3 gL ) of urea, peptone, NH4NO3 and NaNO3. The inorganic 40 Journal of Patna Science College Vol. 1, 37 - 49 [2013] ISSN 2347 - 9604 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 -1 sugar, cultures were raised in presence of 5X-elevated level of NaNO3 (15 gL ) 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 NaNO3. Maximum reducing sugar (11475±52.13 g/L) and their corresponding endoglucanase (3.8±0.05 U/ml) activity occurred in broth containing corncob as a sole carbon source. A slightly lower value of reducing sugar (11180±65.5 g/L) and endoglucanase (3.75±0.024 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 +2 +1 metal ions (Zn and K ) have also been tested to evaluate the values of Km and Vmax in the presence of above mentioned combination of metal ions. The Vmax value for endoglucanase enzyme almost triple +2 +2 (7.692 U/ml) in presence of Fe and Mn (Table-3). In contrast, the value of Vmax greatly reduced in presence of Zn+2 and K+1 for endoglucanase enzyme. Similar result was observed for -glucosidase enzyme. The Vmax value was 1.428 mole/ml/min, it was almost double in comparison to control (0.714

mole/ml/min). The Vmax value of -glucosidase enzyme was reduced to 0.500 mole/ml/min in presence +2 +1 of Zn and K . Km 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 from the 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. 41 Journal of Patna Science College Vol. 1, 37 - 49 [2013] ISSN 2347 - 9604

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 350+10C.

Table-2. Total yield of reducing sugar and its corresponding endoglucanase activity in control condition as well supplemented with 5x Nitrogen sources.

*Yield of reducing sugar multiply in fold shown in bracket.

Table-3. Values of Km and Vmax in different conditions

Enzyme Values Control Fe+2 & Mn+2 Zn+2 & K+1

Endoglucanase K m(mg/ml) 0.606 0.4651 1.33

V max (IU/ml) 2.22 7.692 2.127

-glucosidase K m (mM) 0.25 0.208 1.430

V max (IU/ml) 0.714 1.428 0.500

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Fig.1. Microphotograph of Streptomyces sp. MTCC 7779 (Retinoculoperti arrangement of spores on mycelia)

Fig.2.Cellulase production and corresponding activity in the presence of different metal ions

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(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

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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-Joining algorithm with 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. Jang and Chen (2004) also reported that the production of endoglucanase reached the maximum between 3rd to 5 th days whereas -glucosidase production occurred on the 9th day from Streptomyces sp. Our strain Streptomyces sp. Has more enzymatic activities than Micromonospora chalcae (Gallaghher et al., 2004) in which maximum endoglucanase and -glucosidase activities were observed after 8 days and 16-18 days, respectively. After 5th 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.

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Maximum yield 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 NaNO3 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

NaNO3 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 80ìml/L of 45% petroleum ferrofluid in broth culture on 11th 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 +2 +2 substrate. In the presence of Fe and Mn ions the Km 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 46 Journal of Patna Science College Vol. 1, 37 - 49 [2013] ISSN 2347 - 9604 of species or individual species. As shown in Fig. 3 (NJ Algorithm with 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 from these 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. References: Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W and Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25:3389-3402. Berghem B and Pattersson G (1977) Location and formation of cellulase in Trichoderma viride. Journal of Applied Bacteriology, 42:65-75. Bhat MK (2000) Cellulase and related enzymes in biotechnology. Biotecnology Advances, 18:355-383. Cavaco- Paulo A (1998) Mechanism of cellulase action in textile processes. Carbohydrate Polymer, 37:273-277. Clarke AJ and Adams SL (1987) Irreversible inhibition of Schizophyllum commune cellulase by divalent transition metal ions. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 916:213-219. Champness WC (1988) New loci required for Streptomycces coelicolor morphological differentiation. Journal of Bacteriology, 170:1168-1174. Chowdhury NA, Oniruzzaman M, Nahar N and Choudhury N (1991) Production of Cellulases and saccharification of lignocellulosic by Micromonospora sp. World Journal of Microbiology and Biotechnology, 7:603-606. Ellaiah P and Srinivasulu B (1996) Production of extracellular protease by Streptomyces fradie. Hindustan Antibiotic Bulletin, 38:41-47. Ferchak JD and Pye EK (1983) Effect of cellobiose, glucose, ethanol, and metal ions on the Cellulase enzyme complex of Thermomonospora fusca. Biotechnology Bioengineering, 25:2865-2872.

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Gadgil NJ, Daginawala HF, Chakrabarti T and Khanna P (1995) Enhance cellulose production by a mutant of Trichoderma ressei. Enzyme and Microbial Technology, 17:942-946. Gallagher J Winters A, Barron N, Mchale L, McHale AP (1996) Production of cellulase and -glucosidase activity during growth of the actinomycetes Micromonospora chalcae on cellulose- containing media. Biotechnology Letters, 18(5):537-540. Gutierrez-Nova N, Herrera-Herrera A, Mayorga-Yeyes L, Salgado LM, and Ponce-Noyola T (2003) Expression and Characterization of the celcflB gene from Cellulomonas flavigena encoding a endo-beta-1,4- glucanase. Current Microbiology, 47:359-363. Hodgson DA (2000) Primary metabolism and its control in streptomycetes: a most unusual of Advances in Microbial Physiology, 42:47–238. Jatinder K, Chadha BS and Saini HS (2006) Optimization of medium components for of cellulases by Melanocarpus sp. MTCC 3922 under solid-state fermentation. World Journal of Microbiology and Biotechnology, 22:15-22. Jang HD and Chang KS (2005) Thermostable cellulases from Streptomyces sp: scale-up production in a 50-l fermenter. Biotechnology Letter, 27(4):239-42. Kibblewhite PR and Clark TA (1996) Enzymatic modification of radiata pine kraft fibre and hand sheet properties. Appita Journal, 49:390-396. Leschine SB (1995) Cellulose degradation in anaerobic environments. Annual Review of Microbiology, 49:399-426. Licus R, Robles A, Garcia MT, DE Cienfuegos GA and Galvez A (2001) Production, purification and properties of an endoglucanase produced by the hyphomycete Chalara (syn.Thielaviopsis) paradoxa CH32. Journal of Agricultural and Food Chemistry, 49:79-85. Lynd LR, Weimer PJ, VanZyl, WH and PetoriusIS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews, 66:506-577. Manoliu Al, Oprica L, Creanga, DE (2005) Ferrofluid and cellulolytic fungi. Journal of Magnetism and Magnetic Materials, 289:473-475. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry, 31:426-428. Ojumu TS, Soloman BO, Betiku L, Stephen K and Amigun B (2003) Cellulase production by Aspergillus flavus Linn. isolated NSPR 101 grown on baggase and corncob. African Journal of Biotechnology, 2(6):150-152.

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Pernodet JL, Alegre MT, Boccard F, Guerineau M (1989) Organization and nucleotide sequence of a ribosomal RNA gene cluster from Streptomyces ambofaciens. Gene, 79:33–46. Roncero Ma B, Torres AL, Colom JF and Vidal T (2000) Effect on xylanase treatment on fiber morphology in totally chlorine free bleaching (TCF) of Eucalyptus pulp. Process Biochemistry, 36:45-50. Rajoka MI (2004) Influence of various fermentation variables on exo – glucanase production in Cellulomonas flavigena. Electronic Journal of Biotechnology, 7:256-263. Sampath P and Chandrakasan G (1998) Physiological and nutritional factors affecting Biosynthesis of extracellular protease by Streptomyces . New Microbiology, 21:55-63. Siddiqui KS, Rashid MH and Rajoka MI (1997) Kinetic analysis of the active site of an intracellular -glucosidase from Cellulomonas biazotea. Folia Microbiologica, 42:53-58.

Thermomonospora fusca. Journal of Bacteriology, 180:3529-3532. Solomon BO, Amigum B, Betiku E, Ojumu TV and Lyokun S (1999) Optomization of Cellulase production by Aspergillus flavus Linn. isolate NSPR 101 grown on Baggase. JNSChE, 6:61-68. Suuranakki A, Tenkanen M, Siika-aho M, Nikupaavola ML, Viikari L and Buchert J (2000) Trichoderma ressei cellulases and their core domains in the hydrolysis and modification of chemical pulp. Cellulose, 7:189-209. Teather RM and Wood PJ (1982) Use of congo red-polysaccharide interaction in the enumeration and characterization of cellulolytic bacteria from rumen. Applied Environmental Microbiology, 43:777-786. Torres AL, Rocerno, Ma B, Colom JF, Pastor, F.I.J., Blanco A and Vidal T (2000) Effect of a noval enzyme on fibre morphology during ECF bleaching of oxygen delignified eucalyptus kraft pulps. Bioresources Technology, 74:35-140. Tejirian A and Xu F (2010) Inhibition of cellulase-catalyzed lignocellulosic hydrolysis by iron and oxidative metal ions and complexes. Applied Journal of Environmental Microbiology. 76:7673-7682. Williams S T and Wellington E M H (1982) Principles and problems of selective isolation of microbes in bioactive metabolic products: Search and discovery (Bu’lock JD Nisbet LJ & Winstanley DJ Ed). Academic Press London, Pp 9-26. Yin LJ, Huang PS and Lin HH (2010) Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas sp. YJ5. Journal of Agricultural and Food Chemistry, 58:9833-9837.

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ENHANCED CLONAL PROPAGATION IN RAUVOLFIA TETRAPHYLLA L. USING ADENINE 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 ([email protected]) Abstract : Nodal explant with axillary buds of Rauvolfia tetraphylla L. were inoculated on MS medium supplemented 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 medium supplemented 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

51 Journal of Patna Science College Vol. 1, 51 - 56 [2013] ISSN 2347 - 9604 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 from nodal explant of R. tetraphylla using AS growth regulator.

Materials and Methods : Young shoots of R. teraphylla were collected from one 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) HgCl2 for 3 minutes before rinsing four times with sterilized double distilled water. The basal medium was 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 medium was adjusted to 5.8 with 1N HCl or NaOH before autoclaving at 121oC for 20 min. After sterilization, the explants were inoculated on MS Medium and were maintained in culture room at 25+2oC under photoperiod of 16 -2s-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 + 2oC and 90-100% humidity. Result and discussion : A variety of hormonal combinations were tried to induce multiple shoot production from nodal explant. The number 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 from nodal 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 optimum for 52 Journal of Patna Science College Vol. 1, 51 - 56 [2013] ISSN 2347 - 9604 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 IBA 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 IBA 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 IBA at higher concentration (5.0 mg/l) inhibited root ing. The suita bility of IBA at concentrat ion of 2.0mg/l and combination of IBA and IAA (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; Ihsan Ilahi, 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 pla nts were tra nsferred to gre enhouse where t he survivability was observed to be about 40%.

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Fig.1. Stunte d growth of R. tetraphylla a t BA P (2 .0 mg/l) & NAA (0.5 mg/l)

F ig. 2. In vitro regeneration of R . tetraphylla after AS treatm ent A- Induction of shoots on BAP+AS (3.0 mg/l) + IBA B- M ultiplication of shoots on BAP+ AS (14.0 mg/l) + IBA C- Induction of roots in Mic roshoot D- Acc lim atiz ed plants

Table-1: Effect of Phytohormone on nodal cutting of R. tetraphylla for micropropagation G rowth Regeneration Average Average Callus regulant frequency No. of length of induction at Shoot (%) shoot/ shoot/explant base quality explant (cm) 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

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Table -2 Effect of IBA on root induction from in vitro raised microshoot of R. tetraphylla after two month of culture t

4.0 + 1.0 4.0+ 1.0 4.3 + 0.58 2.3 + 0.58 - Values represent mean + SE. References : Faisal, M and M Anis (2002) Rapid invitro propagation of Rauv olfia tetraphylla L. An endangered medicinal plant. Journal 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 Jabeen (2007) Ehhanced clonal propagation and alkaloid biosynthesis in cultures of Rauvolfia. Pakistan Journal of Plant Sciences, 13 (1): 45-56. Murashige, T and F Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15; 473-497. Patil, V M and Jeyanthi (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.

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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.

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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 from place 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 from plants, 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 24°97' – 25°57' N latitude and 84°44' – 86°4' E longitude at an elevation of about 60 m above sea-level. It covers an area of 3202 Km2. The town is 20 kilometer long from east to west and 5 kilometer broad from north 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

57 Journal of Patna Science College Vol. 1, 57 - 61 [2013] ISSN 2347 - 9604 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.) Juss. 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.) 58 Journal of Patna Science College Vol. 1, 57 - 61 [2013] ISSN 2347 - 9604

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. 59 Journal of Patna Science College Vol. 1, 57 - 61 [2013] ISSN 2347 - 9604

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 in Danapur, and Vicia hirsuta and Grangea maderaspatana in Mokama. The minimum frequency 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 in Danapur, 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 Raunkiaer’s 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 Raunkiaer’s 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). Problem and 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.

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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 form a 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 from the quadrats which were clipped for estimation of above ground biomass. The soil monolith of 25x25x25cm was removed. The dug out monolith was carried to laboratory and flooded with water. The roots were separated by hand. 63 Journal of Patna Science College Vol. 1, 63 - 68 [2013] ISSN 2347 - 9604

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\R² 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 Jalaun district (Tyagi,1997). Similarly Muthana (1981) also recommended H. contortus,Dichanthium annulatum and Pennisetum purpureum 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 Jharkhand. 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.

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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 Jaduguda 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 Jalaun 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.

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Table 1: Root diameter (mm) of selected plant species Secondar Tertiary Average Species Main Primary y roots roots root roots roots 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

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Table 2: Binding capacity (Root Conservation Value)

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Table 3 : Moisture conservation value

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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 from different 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 system of medicine in india dates back to the age of the Rigveda (2500 to 1600 B.C.). With the 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 have changed 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 1.25cm to 6.25cm in diameter, glabrous with crenate margin. Flowers are sessile, cluster of 3 to 6, medium sized, multicoloured. Corolla with two rows of petals and with white petaloids intermingled with stamens in its centre. 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 (Jayashree et al. 2003) Centella asiatica has also been

69 Journal of Patna Science College Vol. 1, 69 - 74 [2013] ISSN 2347 - 9604 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 involving some 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 chloroform in 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 respective glycosides, 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 from C. 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 from C. asiatica.

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Continuously, in their comparative study on the genetype cultivated in Sri Lanka, however, they obtained 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 from the aerial parts of C. asiatica and none of these saponins revealed significant cytotoxicity. Moreover, Jiang et al., (2005) identified four new triterpenoid glycosides named Asiaticoside C, D, E, and F from the BuOH fraction of C. asiatica. In addition, drawing assistance from the 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 quercetin and kaempferol, catechin, rutin and naringin, as a major part of the total phenolic contents, some of which are major contributors in particular to the antioxidative 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 from the aerial parts of C. asiatica cultivated in Vietnam, both of which exhibited potent inhibitory activity on aldose reductase in rats. 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 from C. 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 Centella’s 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. Some of these bioactivities have been demonstrated experimentally. Scientific findings exhibited 71 Journal of Patna Science College Vol. 1, 69 - 74 [2013] ISSN 2347 - 9604 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 enhancing hippocampal 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, immunomodulatory and 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 germplasm conservation 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

72 Journal of Patna Science College Vol. 1, 69 - 74 [2013] ISSN 2347 - 9604 yield related characters to identify high herb and madecassol yielding populations suitable for use in agronomical and plant breeding programs. A great progress has been 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 properties of 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, Hashim P and Ali JM (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 venous hypertension with total triterpenic fraction of Centella asiatica: a clinical, prospective, placebo- controlled, randomized, dose-ranging trial[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 inducible nitric oxide synthase during gastric 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. Jayashree G, Kurup MG and Sudarslal VS (2003) Anti-oxidant activity of Centella asiatica on lymphoma-bearing mice[J]. Fitoterapia, 74(5) : 431-434. Jiang ZY, Zhang XM and Zhou (2005) J. New triterpenoid glycosides from Centella asiatica[J]. Helv Chim Acta, 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 JE (eds) Phoenix, AZ : Oryx Press, 145-173. Kuroda M, Mimaki Y and Harada H (2001) Five new triterpene glycosides from Centella asiatica[J]. Nat Med, 55(3) : 134-138. 73 Journal of Patna Science College Vol. 1, 69 - 74 [2013] ISSN 2347 - 9604

Lee J, Jung E andKim Y (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, from Centella 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]. Chem Pharm 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]. Pharm Biol, 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) Characterization of an ursane triterpenoid from Centella asiatica with growth inhibitory activity against Spilarctia bliqua[J]. Pharm Biol, 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 JHW. (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) Antioxidative activity 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 from the 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 from the 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 from the 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 from time 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. 75 Journal of Patna Science College Vol. 1, 75 - 78 [2013] ISSN 2347 - 9604

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 : 3mm across, 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 :6mm long, 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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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 mm across, 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. Jain, S.K., 1983. Studies in Indian Ethnobotany, IRL. Bull, 1(2): 126-128. Jain, 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 C3– 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 mechanism have 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.

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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 Rf values and colour in UV light before and after fuming with Ammonia vapours. Compounds were repeatedly purified by paper chromatography, till the absorption properties became constant. Hence and elute of a paper blank in 95% ethanol (usually about 150 cm2) was taken and applied (spotted) to the paper, and run in BAW and 15% HOAc, separately. After the purification of compounds, the spots of chromatogram were 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 Rf 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.

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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 .

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TABLE-1 : Flavonoid profile and Chromatographic analysis of C. juncea L. and C. striata DC.

Fluorescence R f× 100 Spectral values

Spot Visible UV UV BA 15% 39% Wat Phen Forest EtOH EtOH EtOH Chemical s light +NH W AcO AcO er ol al +NaOAc +H PO indentity No. 3 3 H H 4

1. Ns Br.g Br.g 13.1 86.5 — — — — + — — — r r.

2. Br Br Br 15.4 80.2 — — — — + — — —

3. D.Br — — 20.2 — 0.5 63.1 — — 250;30 301,320 200,30 ? 2, 1 380,420,5 330,40 10 5

4. Br.ochr — — 69.8 — 86.0 51 — — + — — e

5. YL — — 70.8 — — — — — 224,28 — — Hesperidi 5, n 332

6. Br.ochr — — 47.2 60 63.1 60.1 — — 261 270,368 270,34 Luteolin e 4 7- glucoside

7. YL — — 56.0 — — — — — 227, — — Naringin 280, 335

8. L. YL — — 58.6 — — — — — 253.37 261,265 380 Querceti 0 n

9. YL. Gr. — — 65.0 72.1 — 8 85.1 70.8 245,26 — — Tricin 5,

10. F. YL. — — 7.5 — — — 30 71.3 220,28 — — Phlorodiz 0, 345 in

11. YL. — — 75.4 — — — 64.2 — + — — ?

12. Br. — — 76.6 — — 0 65.0 — 260,38 — — Butein 0

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Fluorescence Rf × 100 Spectral values

Spot Visible U UV BA 15% 39% Wate Phen Forest EtOH EtOH EtOH Chemical s No. light V W r ol al indentity +NH AcO AcO +NaOA +H3PO 3 H H c 3

13. Br — — 77.8 56.1 — 0 57.0 54 265,36 — — Kaempfer 6 ol

14. Br.YL — — 83.5 — — — — — + — — —

15. F.YL — — 85.6 65.9 — — 87 82 265331 — — Apigenin

16. D.Orch — — 92 10.2 — — — — + — — ? e

17. F.Gr — — 93 23.6 — — — — + — — ?

18. Grey — — 95.9 — — — — — + — — ?

19. Grey — — 90.1 — — — — — + — — ?

20. D.Br. — — 27.4 65.2 — — — — + — — ?

21. Ns. — — — — — — — — + — — —

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PHYSIOLOGICAL EFFE CTS OF GIBBERELLIC AC ID 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, Gautam Budh 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.

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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 maximum at 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. It 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

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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 9th 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. Jones, 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. Jun J.W. (1928) Inheritance of earliness and other agronomic characters in rice. Jour. 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.

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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 : [email protected].

Abstract : The study of solvent effect (water-dioxan mixture) on thermodynamic properties of 4- nitropthalic acid was done at 250C 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 medium and 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 medium but also to investigate, the role of solvent in changing the model. A lot of works1-5 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 medium can 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 250C and at constant ionic strength was determined. The ionic strength was made constant using sodium chloride. The values 0 0 of dissociation constant PK1, “G , dielectric constant of o-nitropthalic acid at 25 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 medium will 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 medium which favours dissociation. A number of workers6-8 have justified the former case. This is also seen in variation of PK against 100/D in table I.

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TABLE – I Values of 100/D, Dissociation Constant, PK, “G0 at 250C

It is seen that “G0 value is positive. This shows that unionized from will be more abundantly populated than the ionised from.9 The addition of dioxan increases “G0 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 “G0 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 Begum S, Siva Kumar , C.L. Mayanna S.M. and Murlidharan V.S. (2000) Electrochem. Acta, 18, 89. Singh, Ratan, Verma P.S. & Jain D.S. (1991) Electrochem. Soc. India, 7:, 40, 47. Srivastava, S.B. Prakash Om and Prakash Sheo (1975) J. Chem. Thermodynamics, 7, 997.

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ULTRAVIOLET-VISIBLE & INFRARED SPECTRAL STUDY OF

COMPLEXES OF CO (III) WITH MIXED LIGANDS BIGUANIDE C2H7N5 AND PYRIDINE C5H5N 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

(C2H7N5) and pyridine (C 5H5N) 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 2H7N5•H2SO4 •H2O is a bidentate chelating molecule and its complexes with bivalent and trivalent ions are known1-4.

+ + H3 N – C – NH – C – NH 3 (1) || (3) || (5) NH NH (2) (4)

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 from each biguanide unit. The coordination complexes of a number of biguanides with various metal ions have been investigated extensively5,6. Biguanides may be prepared by the condensation of two molecules of guanidine.

-NH3

H2N – C – NH H + H2N – C – NH2 || || Fusion NH NH Guanidine guanidine

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H2N – C – NH – C – NH2 || || NH NH

Biguanide or diguanide

Actually, guanidine hydrochloride is used and fused at 180-1850C. 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 atom present in the ring.

pyridine 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 C2H7N5 and pyridine C5H5N we have prepared and characterized the complexes of Co (III) with mixed ligands.

Results and discussion : The acidic solution of CoSO4, cobalt sulphate reacts with basic solution of + - + biguanide [BigH2 ] (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, C5H5N (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 bisbiguanidium cobalt + [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(SO4)3 .12H2O was obtained when red solution of the complex base + [Co(BigH )2py2](OH)3 was neutralization with dilute sulphuric acid in cold as red crystals associated with a small amount of the trisbiguanidium cobalt (III) sulphate. The complex nitrate, [Co(BigH)

2(py)2](NO3)3, was obtained by the neutralization of the solution of the complex base

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[Co(BigH)2py2](OH)3 with dilute HNO3 (nitric acid) in cold. The complex carbonate [Co(BigH)

2(py)2]2(CO3)3 was prepared by treating the complex base [Co(BigH)2py2](OH)3 with either ammonium bicarbonate NH4(HCO3) or sodium bicarbonate NaHCO3. The complex chloride [Co (BigH) 2(Py) 2]

Cl3.6H 2O was prepared by dissolving the ca rbonate in dil. Hydrochloric acid.

Dipyridinebisbiguanidiumcobalt (III) thiosulphate[Co(BigH)2](S2O3)3•6H2O was prepared by neutralizing the complex chloride with solution of sodium thiosulphate dropwise.The complex oxalate [Co (BigH)2

(Py)2]2 (C2O4)3.8H20 was prepared either by neutralizing the complex base with oxalic acid or by adding sodium oxalate 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 -1 1 ) and 357nm (28,000cm ). This is attributed to transition of d-electrons from A1g (ground state) to 1 1 next upper higher level T1g and T2g states respectively. In Co (III) complexes the spin forbidden transition 1A 5E or1A 5T are seldom observed. 1g g 1g 2g 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 from stretching vibrations between hydrogen and some other atoms with mass 19 or less. The O—H and N—H stretching frequencies fall in the 3700 to 2500 cm-1 region with various intensities. + - + 2- The ligand biguanide sulphate (BigH HSO4 ).H2O contains =NH, —NH, – NH3 and SO4 groups. + 2- The various modes of IR vibrations of =NH, N—H, —N H3 and SO4 groups display IR bands in 3350 to 625 cm-1 region. The N—H 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 -1 N—H and NH2 stretches are observed between 3479.4 to 3239.7 cm .

+ -1 The N—H stretching in – N H3 group is obtained at 3156 and 3022.9 cm . 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 N—H and -1 -1 NH2 deformation vibration observed at 1522.3 cm and ä-NH2 vibration at 1427.5 cm . N—H wagging observed at 762cm-1. The C—N (unconjugated) stretching observed at 1025.9 cm-1 and 928.3 cm-1. The C=N stretching observed at 1658.2cm-1.

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-1 The ionic sulphate group displays as broad and very strong band at 1216 cm due to õ3 2- -1 2- vibration of SO4 ion. The strong and sharp band at 528cm is attributed to õ4 vibration of SO4 group. The free sulphate ion has regular tetrahedral symmetry (Td). On formation of bond with metal or hydrogen atom with oxygen or atoms of sulphate group[10.]. The sulphate ion has four different modes of vibrations õ1, õ2, õ3 and õ4. The õ1and õ2 vibrations of sulphate are not IR active. The õ3 and õ 4 vibrations split into two bands while the õ3 and õ4 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 2- chelating or bridging sulphate groups are differentiated clearly by position of splitting of õ3 (SO4 ) 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 M1, M2, M3, M4, M5, M6 & M7. All these complexes have shown the presence of Co – N & N – Co – N vibration near 500nm. Here,

M1 =Dipyridinebisbiguanidium cobalt (III) hydroxide

M2 = Dipyridinebisbiguanidium cobalt (III) sulphate

M3 = Dipyridinebisbiguanidium cobalt (III) nitrate

M4 = Dipyridinebisbiguanidium cobalt (III) chloride

M5 = Dipyridinebisbiguanidium cobalt (III) carbonate

M6 = Dipyridinebisbiguanidium cobalt (III) oxalate

M7 = Dipyridinebisbiguanidium cobalt (III) Thiosulphate

M10= 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 form complexes. The pyridine combines with Co metal by its N atom.

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Experimental : The ligand biguanide sulphate was prepared by the reported method:

Biguanide sulphate C2H7N5•H2SO4•H2O was prepared according to the method described by Smolka1 and Friedrich with slight modification which resulted in better yield. In this method, a 0 mixture of ammonium iodide (NH4I) and dicyandiamide, C2H4N4 (mol.wt.-84.08) [dried at 100 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 1550 C. At this temperature, the mixture was changed to a thick liquid and was maintained at this temperature [155+2]0C for ten minutes. The molten mass was then poured into a large volume of water and then filtered from any solid residue. The filtrate so obtained was then treated with a solution of cuprammonium sulphate, [Cu (NH3)4] SO4. Cuprammonium sulphate was prepared by adding liquor ammonia (NH3) to copper (II) sulphate solution. As a result, rose coloured precipitate of copper biguanide sulphate Cu(C2H6N5)2•H2SO4 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(C2H6N5)2 •H2SO4 was then decomposed with cold solutions of about 10% sulphuric acid H2SO4. A blue solution was obtained which on keeping in the cold [120C] 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)2py2](OH)3 It was prepared by adding calculated amount of biguanide sulphate dissolved in slight excess of sodium hydroxide 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, C5H5N (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 bisbiguanidium cobalt [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 95 Journal of Patna Science College Vol. 1, 91 - 101 [2013] ISSN 2347 - 9604

water and finally with absolute alcohol .The product was then dried in a CO2 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 950 C. Dipyridinebisbiguanidiumcobalt (III) sulphate

[Co(BigH) 2 (py)2]2(SO4)3.12H2O

+ 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 trisbiguanidium cobalt (III) sulphate .This was removed by fractional crystallization from water .Trisbiguanidium cobalt (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%

-2 SO4 = 21.59%

Required for [Co (BigH) 2 (py) 2] 2(SO4)3.12H2O Co = 8.78%

N = 25.04%

-2 SO4 = 21.46% Water could not be determined by heating as the complex evolved pyridine also when heated at 950C.

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Dipyridinebisbiguanidiumcobalt(III)nitrate

[Co(BigH) 2(py)2](NO3)3

For the preparation of dipyridinebisbiguanidiumcobalt(III)nitrate [Co(BigH) 2(py)2](NO3)3, the solution of the complex base [Co(BigH)2py2](OH)3 was neutralized with dilute HNO3 (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%

- NO3 = 31.52%

[Co (BigH) 2(py)2](NO3)3 requires Co = 9.75%

- NO3 =30.74% Dipyridinebisbiguanidiumcobalt(III)carbonate

[Co (BigH) 2(py)2]2(CO3)3

This was prepared by treating the complex base [Co(BigH)2py2](OH)3 with either ammonium bicarbonate NH4(HCO3) or sodium bicarbonate NaHCO3. 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(CO3)3 requires Co =11.59% Dipyridinebisbiguanidiumcobalt(III)chloride

[Co (BigH) 2(Py) 2] Cl3.6H2O 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(CO3)3 was treated with ammonium carbonate, (NH4)2CO3 or sodium bicarbonate, NaHCO 3 red precipitate of the complex

97 Journal of Patna Science College Vol. 1, 91 - 101 [2013] ISSN 2347 - 9604 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] Cl3.6H2O C = 9.35% N = 26.53% Cl = 16.79%

The compound disengages pyridine when heated at 950C. Dipyridinebisbiguanidiumcobalt(III)thiosulphate

[Co(BigH)2(py)2]2(S2O3)3•6H2O 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%

S2O3 = 27%

Required for [Co (BigH)2](S2O3)3•6H2O Co = 9.20%

S2O3 = 26.23% Dipyridinebisbiguanidiumcobalt(III)oxalate

[Co (BigH)2 (Py)2]2 (C2O4)3.8H20 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 precipitate Dipyridinebisbiguanidium cobalt (III)oxalate,

[Co (BigH)(Py)2 ]2 (C2O4)3.8H20 so obtained was filtered washed with cold water and alcohol and dried in air. Found

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Co = 5. 67%

[Co (BigH) 2(Py)2] (C2O4)3.8H20 requires Co = 5.51%

On heating at 95 0C decomposed with the loss of water and pyridine. The aqueous solution, on heating slowly evolved pyridine and the colour changed to violet. Even in cold 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 – H2N N = C – N H3

HN Co NH (O H)3

+ H3N — C = N NH2 – C = NH py py

Dipyridinebisbiguanidiumcobalt(III)hydroxide

NH || + C – H2N N = C – N H3

HN Co NH (SO4 )3 • 12H2O

+ H3N — C = N NH2 – C = NH py py 2

Dipyridinebisbiguanidiumcobalt(III)sulphate

NH || + C – H 2N N = C – N H3

HN Co NH (NO 3)3

+ H3 N — C = N NH2 – C = NH py py

Dipyridinebisbiguanidiumcobalt(III)nitrate

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NH || + C – H2N N = C – N H3

HN Co NH (CO 3)3

+ H3N — C = N NH2 – C = NH py py 2

Dipyridinebisbiguanidiumcobalt(III)carbonate

NH || + C – H2N N = C – N H3

HN Co NH (C2O4)3

+ H3N — C = N NH2 – C = NH py py 2

Dipyridinebisbiguanidium cobalt(III)oxalate

NH || + C – H2N N = C – N H 3

HN Co NH Cl3• 6H2O

+ H 3N — C = N NH2 – C = NH py py

Dipyridinebisbiguanidiumcobalt(III)chloride

Acknowledgement : I am particularly 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.

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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 am very grateful to him and have no words to express my gratitude.

References Herth Ber., (1880) :13, 1358 Friedrich Monatsh, (1883) : 4, 888 Stumpf Dissertation, Berlin, (1934) : page-48 Reibenschuh Monatsh, (1883) : 4, 398 P.Ray Chem. Rev. (1961) : 61, 313 Ghosh S.P. , R.K. Prasad and Bina Rani, (2003) : J. Indian Chem. Soc. 80.912-914 Ghosh and Prasad R.K. (1987) : J. Indian Chem. Soc. 64, 765; Ghosh SP and Ghosh HM (1956) J. Indian Chem. Soc., 33,899; Ray P. and Sengupta N. R. (1959) : J. Indian Chem. Soc., 36, 201; Ghosh S.P. and Sinha A.I.P. (1961) : J. Indian Chem. Soc., 38, 179, Ghosh S.P. and Sinha A.I.P. (1964) : J. Inorg. Nucl. Chem., 41, 330, Sen D. (1975) : J. Indian Chem. Soc.(D), 52, 1741; Bera T.R. and Konar J. (1997) : J. Indian Chem. Soc., 74, 528; Ghosh S. , Mukhopadhyay C.C. De G.S. and Ghosh A.K. (1998) : J. Indian Chem. Soc., 75,219. Prasad R.K., Bina Rani and Singh Dhanajai (2006) : J. Indian Chem, 83,718 Prasad R.K. , Bina Rani and Dhanajai Singh and Kumar Prahlad (2010) : J. Indian Chem. Soc., 87, 1313. Nakamoto K. IR& Raman Spectra of Inorganic and Co-ordination compounds, Johnwiley, New York. Barrailough C. & Tobe M.L. (1961) : J. Chem. Soc. 1993 Earnshow A., Larkworthy L.K.& Patel K. C. (1969) : J.Chem. Soc. A, 1339. Clark R.J.H. and William C. S. (1965) : Inorg. Chem. ,4, 350 Kakiuchi Y., Kida S. & Quagliano J. V. (1963) : Spectrochem. Acta, 19, 201. Sugden. J. Chem. Soc. 1932, 246

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ARSENIC CONTAMINATION IN GROUNDWATER OF BIHAR: CAUSES, ISSUES AND CHALLENGES Madhurendra Nath Sinha 1, Ranvir Nandan2, Rabindra Kumar3 1. Professor of Geology and Head of Department, Department of Geology, Patna Science College, Patna University, Patna 800005. email : [email protected] 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 : [email protected]

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 term mitigation (hand pump treatment plan or sanitary dug well) and long term 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 from surface water, dug well, pond and from natural 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

103 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 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 problem in its groundwater. This comprises of more than 67 blocks from 15 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. If 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 problem of 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 from natural deposits in the earth or from industrial and agricultural pollution. Arsenic is a natural element of the earth’s 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 socio–economic 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). It 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.

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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 India 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 (problem of 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 problem aggravate 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. 105 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604

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 world’s population but only 4 percent of its usable freshwater, India has a scarcity of water. World oceans cover about 3/4th of earth’s 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 from country 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 106 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 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

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. Source: Author’s own compilation from various sources. Arsenic Contamination – Causes and Sources : Arsenic is found in the natural environment in plenty in the earth’s 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 form but inorganic arsenic is more toxic than organic arsenic. Inorganic arsenic compounds are known to be more human carcinogens. Arsenic in element form is 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 form of 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 can be from pesticides, herbicides, seed treatment, cattle deep and fertilizer mainly, while industrial sources are from timber treatment, tannery, electro plastic, and paints and chemicals. Other anthropogenic sources are from sewage 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 from arsenic poisoning were Argentina, Chile, Mexico, Taiwan, and some

108 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 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 Brahmaputra rivers 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 from NCPH came from arsenic contaminated tube wells (MoWR, 2010b). In 1982 a patient from North - 24 Parganas district of West Bengal, whose skin lesions were not like the usual skin diseases and later similar problem finds to many patient from the 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 districts of 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 from 9 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 from Bihar

109 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 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 problem in 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. It 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 110 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 economic costs arising from the 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 problem and other problems. Excess presence of contaminated water leads to decrease in agricultural productivity, soil fertility, and also enters into the food chain which creates health problems. 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 are few of the health problems. Social ignorance, depression and suicidal tendency are among few social problems. Arsenic contamination has widespread social problems among the

111 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 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 a spreadable 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 from such 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 use of contaminated rice followed by vegetables. This eventually 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 from poor socio-economic background. They are either not aware or

112 Journal of Patna Science College Vol. 1, 103 - 116 [2013] ISSN 2347 - 9604 if aware are forced to take drinking from same 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 world’s 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. References : Baliga, Sandeep and Eric Maskin (2005): Mechanism Design for Environment, in Karl-Goran Maller, and Jeffrey, R. Vincent (eds.), Handbook of Environmental Economics: Valuing Environmental Changes Vol. 1 ELSEVIER North-Holland, pp. 305-324 ISBN 0-444-50064-4 Brammer, Hugh (2008): Threat of Arsenic to Agriculture in India, Bangladesh and Nepal, Economic and Political Weekly, November 22, 2008. Brammer, Hugh (2009): Mitigation of arsenic contamination in irrigated paddy soils in south and south- east Asia, Environment International 35 pp. 856-863. Canter, Kenneth. P (1997): Drinking Water and Cancer, Cancer Causes and Control 8(3), The Harvard- Teikyo Special Issue, pp.292-308. Chakraborty, Dipankar et al. (2008): Groundwater arsenic contamination and its adverse health effects in the Ganga-Meghna-Brahmaputra plain in Kingsuk Roy (ed.) Arsenic calamity of groundwater in Bangladesh: Contamination in water, soil and plants. Nihan University Japan. Chakraborti, Dipankar et al. (2006): An eight-year study report on arsenic contamination in groundwater and health effects in Eruani village, Bangladesh and an approach for its mitigation, Journal of Health and Population Nutrition, 24 (2) pp.129-141. Chakraborti, Dipankar et al. (2003): Arsenic groundwater contamination in middle Ganga plain, Bihar India: Afuture danger, Environmental Health Perspectives 119 (9) pp. 1194-1201. Chakraborti, A. K., and Saha K C (1987): Arsenic dermatoses from tube-well water in West Bengal, Indian Journal of Medical Research 85 pp. 326–34. Conrad, Jon M (1999): Resource Economics, Cambridge University Press, New York. Cvjetanovic, B (1986): Health effects and Impacts of Water Supply and Sanitation, World Health Statistics Quarterly, 39 (1) pp. 105-117.

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Dasgupta, Partha (1993): An enquiry into well-being destitution, Oxford University Press, New York. ——(2007): Economics: A very short introduction, Oxford University Press, New York. ——(2010): The concept of natural capital, lecture delivered at royal society on inter panel academy biodiversity conference on January 12-13. Egboka, B. C. E, G. I. Nwankwor, I. P. Orajaka and A. O. Ejiofor (1989): Principles and Problems of Environmental Pollution of Groundwater Resources with Case Examples from Developing Countries, Environmental Health Perspectives, 83 pp.39-68. Ghosh, A. K, S. K. Singh, N. Bose and S. Chaudhary (2007): Arsenic Contaminated Aquifers: A study of the Ganga levee zones in Bihar, India, Symposium on Arsenic: The Geography of a global problem, Royal Geographical Society, London accessed on 12 December 2010 (Available online at) (http://www.geo.cam.ac.uk/research/projects/arsenic/symposium/S3.5_A_Ghosh.pdf ). Hotelling, H (1931): The Economics of Exhaustible Resources, Journal of Political Economy, 39(2) pp. 137-75. Jain, C. K., and Ali I (2000): Arsenic: occurrence, toxicity and speciation techniques, Water Resources 34 (17) pp. 4304- 4312. Jain, J. K (1977): India: Underground water resources and discussion, Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences 278 (962) pp. 507-524. Khan, M. Z, Hossain (2007): Managing the Disaster in Water Supply: Risk Measurement, Costs of Illness and Policy Choices for Bangladesh, SANDEE Working Paper No. 27-07 Kathmandu, Nepal. Khan, M. Z Hossain and A K E Haque (2011): Red wells, Green Wells and the Costs of Arsenic Contamination in Bangladesh in Haque, Murty and Shyamsundar (Eds.) Environmental Valuation in South Asia, Cambridge University Press New Delhi. Kondo et al., (1999): Naturally occurring arsenic in the groundwater’s in the southern region of Fukuoka prefecture, Japanese Water Research 33 (8) pp. 1967-1972. Ministry of Environment and forest, Government of India (2009): State of environment report 2009, New Delhi.—— (2010): Report to the People on Environment and Forests 2009-2010, New Delhi. Ministry of Water Resources, Government of India (2000): Annual report 1999-00, New Delhi. (2006): Dynamic groundwater resources of India, Central Ground Water Board, New Delhi. —— (2007): Annual report 2006-07, New Delhi. —— (2008): Annual report 2007-08, New Delhi.

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—— (2010a): Groundwater quality in shallow aquifers of India, Central Groundwater Board, Faridabad India. —— (2010b): Mitigation and Remedy of Groundwater Arsenic Menace in India: A Vision Document, National Institute of Hydrology, Roorkee and Central Ground Water Board, New Delhi, India. Mukherjee, A (2006): Arsenic contamination in groundwater: a global perspective with emphasis on the Asian scenario, International centre for diarrheal research centre Bangladesh. National Research Council (2003-2006): Fluoride in drinking water: A scientific review of EPA’s Standards, New Zealand. Pigou, A.C (1920): Economics of Welfare, Macmillan and Company. Rahman, M, M. Tondel, I. A. Chowdhury and O. Axelson (1999): Relations between exposure to Arsenic, Skin Lesions, and Glycosuria, Occupational and Environmental Medicine 56(4) pp. 277-281. Ravenscroft, P., W. G. Burgess, K. M. Ahmed, M. Burren, and J. Perrin (2008): Arsenic in Groundwater of the Bengal Basin Bangladesh: Distribution, field relations and hydrological setting, Hydrology Journal 13 pp.727-51. Roy, Joyshree (2007): Estimating the Economic Benefits of Arsenic Removal in India: A Case Study from West Bengal, SANDEE Working Paper No. 21-07 Kathmandu, Nepal. —— (2008): Economic benefits of arsenic removal from ground water- A case study from West Bengal, India, Science of the total Environment 397 pp. 1-12. Roy, Joyashree et al. (2004): An economic analysis of demand for water quality: A case study from Kolkata city, Economic and Political Weekly, 39(2), pp. 186-192. Saha, Dipankar (2009): Arsenic groundwater contamination in parts of middle Ganga plain, Bihar, current science, 96(6) pp. 1-3. Sankar, U (2000): Environmental Economics, Reader in Economics, Oxford University Press, Oxford India Paperback 4th impression 2004. —— (2005): Environmental Externalities, Dissemination Paper-1, Madras school of economics. Saxena et al., (2004): Occurrence, behaviour and speciation of arsenic in groundwater, Current Science 86(2) pp. 281-284. Sinha, M. N., and Mahto Ashok K : Arsenic Contamination in Groundwater affecting the Ganga Alluvial Plain, India, ANVESHIKA, 1: 75-78, 2010 Smedley, P. L., and Kinniburgh D G (2002): A review of the source, behaviour and distribution of arsenic in natural waters, Applied Geochemistry 17 (3) pp. 517-568.

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Sparks, Donald L (2005): Advances in Agronomy, Elsevier Academy, 86 pp. 440 United Nations (2009): The Millennium Development Goals Report 2009. —— (2010): The Millennium Development Goals Report 2010. UN Millennium project task force on environmental sustainability (2005): Environment and human well-being: a practical strategy, EARTHSCAN London. World Health Organization (2001): Bulletin of the WHO, 78 (9) pp. 1096-97. World Health Organization (2010): Water for health: WHO Guidelines for Drinking-water Quality

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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 Brans–Dicke 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 term as 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 term Q are [1] :

8 1 ,k 1 G i j gijQ Tij , i , j – g ij ,k ( i; j – gij ) (1.1) 2 2

8 (1.2) (2 3 )

(2 3 ) (1 – ) 8 (1 – ) Q . T , 4 4 (1.3) ) deviates from that of Brans and Dicke, w is coupling constant and Tij is energy-momentum tensor. Semicolons denote covariant derivative with respect to the metric gij and commas mean partial derivatives with respect to the coordinate xi. 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

(1.4) gij g ij g ij equations (1.1) — (1.3) have the form

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1 1 ,k Gij gQ(8)Tij (2 3)( –g ) (1.5) ij2,i ,j 2 ij ,k 8 (1.6) (2 3) (1.7) Q . T,

where the barred quantities are defined in terms of gij as their unbarred counterparts are defined in terms of the unbarred metric gij 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 form of Brans-Dicke theory. The Field Equations: The geometry of the universe is described by the line element. 1 a 1–a ds2 –dT 2 Tdx 2 T2 dy 2 T 2 dz 2 (2.1) where a being a constant. The energy–momentum tensor for the viscous fluid distribution is given by [6]:

j jj j j j l l j Ti pvvi pg–i v ;i v i; vvv i;l vvv i ;l

2 l j j (2.2) – – v;l g v i v , 3 i together with i (2.3) v vi –1

where p and are the pressure and density respectively,, and are the two coefficients of i viscosity, 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 0 and v 1.

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The pressure and density in the model (2.1) are given by

1 ( a 2 – 5 ) 1 ( 5 – a 2 ) 2 8 8 p 1 ta n 2 lo g (K T ) Q 16T 2 2 8 ( 2 3 ) T (2.4)

1 ( a 2 – 5 ) 1 (5 – a 2 ) 2 8 1 – ta n 2 lo g ( K T ) – Q . 16T 2 2 8 ( 2 3 ) (2.5)

Also scalar field is given by

1 (5 – a 2 ) 2 log s e c lo g( K T ) 8( 2 3 ) (2.6) and

1 (1 – ) (a 2 – 5) (5 – a 2 ) 2 Q s e c 2 lo g(K T ) 4 8T 2 8(2 3 ) (2.7) where K is a constant. Physical & Geometrical Properties The model has to satisfy the reality conditions [7] :

(i) p 0 a n d (ii) 3p 0 which requires that 3 a 2 5 , – , Q 0 (i.e . 1) . (3.1) 2 and

1 (5 – a2 ) (5 – a 2 ) 2 Q s e c 2 l og (K T ) . 2 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 an d v 1 .

i j Obviously v ;j v 0.

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Therefore the flow is geodesic. The rotation tensor

ij v i; j – v j; i 0. Thus, the fluid filling the universe is non-rotational.

1 i The expansion scalar v ;i is given by 3 1 . (3.3) 3T 1 Shear tensor (v i ; j v j; i ) – (g – v i v j ) is given by ij 2 ij 1 , 11 6 a–1 (3a–1) T 2 , (3.4) 22 12 –a–1 – (3a 1) T 2 , 33 12 44 0. Also the shear s is

2 1 ij 1 2 ij [1 3a ]. (3.5) 2 48T 2 The volume element of the model is given by

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

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jk The surviving components of the conformal curvature C hi tensor for the line-element (2.1) are

1 4 2 3 1 2 C 1 4 C 2 3 2 [1 a ], 24T

1 2 3 4 1 2 C 1 2 C 3 4 – [1 6 a a ], (3.8) 48T 2 C 1 3 C 2 4 – [1 – 6 a a 2 ]. 1 3 2 4 48T 2 The pressure, density, scalar field and cosmological constant are singular at

1 1 2 (2 3 ) 2 T e xp . K (5 – a 2 ) (3.9)

The model exists for a finite time

1 1 1 2(2 3 ) 2 T e x p . K K (5 – a 2 ) viscous fluid distribution in general relativity [8]. Transformations of the Solutions and Discussion : Under the transformations 1 g i j gij gij,

T ij Tij T ij ,

T T 2 T ,

p p 2 p, (4.1)

2 ,

e ,

Q Q Q ,

1 v i v i 2 v i, the solutions of the field equations (1.5)-(1.7) are changed into 1961 form of Brans-Dicke theory [2]. We now apply these transformations to the solutions obtained from field equations (1.5)- (1.7). Thus 121 Journal of Patna Science College Vol. 1, 117 - 123 [2013] ISSN 2347 - 9604

1 (5 – a 2 ) 2 se c lo g(K T ) 8( 2 3 ) (4.2)

1 (5 – a 2 ) 2 g c o s lo g (K T) g ij 8(2 3 ) ij (4.3)

1 (5 – a 2 ) 2 g c os lo g (K T ) T i.e. 11 8(2 3 )

1 1 a (5 – a 2 ) 2 g c os lo g( K T ) T 2 22 8( 2 3 )

1 (5 – a 2 ) 2 1–a g c o s lo g(K T) T 2 33 8( 2 3)

1 (5 – a 2 ) 2 g – co s lo g (K T ) . 44 8 (2 3 )

1 (a 2 – 5) (5 – a 2 ) 2 1 – 8 p se c 4 lo g (K T ) 1 32T 2 8 (2 3 )

1 ( a2 – 5) (5 – a2 ) 2 s ec 2 log (K T ) 32T 2 8 (2 3 )

1 8 (5 – a 2 ) 2 s e c2 lo g (K T ) T 8(2 3 ) (4.4)

1 (5 – a2 ) (5 – a 2 2 (1 – ) 8 s e c 4 lo g (K T) 1 32T 2 8 (2 3 )

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1 3 (a 2 – 5 ) (5 – a 2 ) 2 se c 2 lo g(K T ) 32T 2 8(2 3 ) (4.5)

1 1 (5 – a 2 ) 2 v 4 se c 2 log (K T ) 8 (2 3 ) (4.6)

1 (1 – ) (a 2 – 5) (5 – a 2 ) 2 Q se c3 lo g(K T ) 4 8T 2 8(2 3 ) (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 in the 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,

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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 effects and consequences. Furthermore, exact multicollinearity versus orthogonality has been extensively studied in recent literature where multicollinearity ( p 0 ) does imply non orthogonality ( p 1). In presence 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 transformation of |R| for which test of significance of rejection of hypothesis H0 : |R| = 1 at specified level of significance. In addition a light has been given on Haitovasky’s Chi square, while the extent of multicollinearity could be assertained for measuring the departure of R-matrix from singularity. Also, lower bounds for E()2 and V(2 ) 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 sum of 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 term ‘multicollinearity’, 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 term collinearity means the existence of single linear relationship. Though linear regression model is also reformed for existence of all explanatory variables.

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However, in consideration of these two studies there maintains some distinction between these two used terms. In recent literature the term multicollinearity 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 = (X/ X) –1 XY/ requires the inversion of XX/ which is impossible of the rank of b, and hence the rank of XX/ 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. Johnson (1963) has resorted to a asymptotic definition. Multicollinearity is the name given to the general problem which 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 X1, X2 ...... Xp there exists non zero constants ai (i = 1,2, ...... p) such that

ai X i 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 XX/ = 1 i.e all eigenvalues of XX/ are equal to unity and X consists of orthogonal regressions. Hence orthogonality of regressors (or explanatory variables) implies that X/ X 1. 126 Journal of Patna Science College Vol. 1, 125 - 137 [2013] ISSN 2347 - 9604

In order to concentrate on near ‘multicollinearity’ that exists when there are non-zero constants such that

ai X i 0. When the matrix XX/ is singular, its inverse does not exist in the usual sense. Exact- / multicollinearity implies that XX is singular i.e. the smallest eigen values p near multicollinearity,, means that XX/ is singular and the smallest eigen value is close to zero. In case of near orthogonality,, eigenvalues are different from unity. 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 K 1 1 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 XX]/ . The meaning of term “Singular” 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 X X,p 0 there is exact-multicollinearity, and K* is infinite. For orthogonal data, we have X/ X 1 we 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 (X2) As Measure of Multicollinearity : This study is followed by Bartlett’s (1950). If the regressor variables are standardized, then XX/ contains elements that are the simple correlation / coefficient among the regressors. In that case XX falls in the interval (0,1).

/ If XX = 0, one or more exact-linear dependencies exists among the columns of X. Similarly / if XX =1 then column of X are orthogonal.

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In fact, most standard multiple regression computer programme have built-in checks for non- singularity of the XX/ matrix. These lists commonly use that determinant of a singular matrix is zero. / Farrar and Glauber (1967) attempted to define a standard comparison for XX by defining multicollinearity as a departure of the matrix X from orthogonality. Estimate of both efficient vector and its dispersion matrix viz. b (X/ X) –1 X / Y and V(b) = 2(XX) / –1 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 XX is based on a normalised correlation matrix (unless / stated otherwise) then 0 < XX = |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 multicollinearity within a set of predictor variables. However, Bartlett(1950) contributed p(p – 1) X2 –[n–1–(2p 5)/6]log|R|~X 2 [ ] 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 X2 distribution. A high value of Chi square indicates the existence of 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.

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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. 1 Furthermore, William and Watte(1978) have presented a geometric interpretation of XX/ 2 1 when X is in standardised form. XX/ 2 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 from orthogonality, 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 Haitovasky’s Chi-square, the extent of multicollinearity can be measured as the departure of R-matrix from singularity.

A Heuristic statistic which is consistent.

Which is consistent with the concept due to Haitovasky’s(1969) given by X2 = [n–1–(2p + 5)/6] log (1–|R|). A small value of X2 indicates the existence of multicollinearity, its severity can be measured by the level of significance at which the null hypothesis Ho ;| R | o is accepted. Explanation For Eigen Values Mathematically As Measure Of Multicollinearity Writing the equation as discussed in proceeding study such as b – (X/ X) –1 X / U (5.1) Let D define the distance between b and b such that; = (b – ) = (XX)XU/ –1 / The squared distance between b and b is obtained as;

2 = (XX)/ –1 XU[(XX) / / –1 XU] /

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E( ) = E[(XX)/ –1 XU] / [(X / X) –1 XU] /

= Etr[(XX)/ –1 XU][(XX) / / –1 XU] /

= Etr[(XX)/ –1 XU][(XX) / / –1 XU] / /

= trE[(XX)/ –1 XUUX(X / / / X) –1 ]

= 2tr[(X / X) –1 ]

Now XX/ is real symmetric matrix, as its inverse. Therefore, p 2 2 1 ()() (5.2) i 1 i / 2 where i is the i th eigen value of XX . Considering the variance of we have

V()2 = E[2 – E( 2 )] 2

= E[4 – 4 tr (X / X) 2 ]

But, (4 ) [(XX) /–1/ XU]'[(XX) /–1/ XU][(XX) /–1/ XU][(XX) /–1/ XU]

Thus, E(4 ) trE[(XX) /–1/ XU]'[(XX) /–1/ XU][(XX) /–1/ XU][(XX) /–1/ XU]

= tr E(UU/ UU) / (X / X) –2

Since U is assumed normally distributed, we can write

E(UU/ UU / ) 2 4

V(4 ) 2 4 tr (X / X) –2

24 tr (X / X) –2

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p 4 –2 2 i (5.3) i 1 / Considering the equations (5.2) and (5.3), if min is the smallest value of (X X) then, 2 2 (a) A lower bound for E( ) is min 4 2 (b) A lower bound for V( ) is min Thus, if the particular variables are related in such a manner as to result in XX/ matrix with one or more small eigenvalues, the distance from b 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 problem of 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 p |R| = 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 r R r to be harmful if | ij | > y for all i = j, where ij 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 Klein’s 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.

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(iii) t-test for finding out the pattern of multicollinearity, i.e. for determining which variables are responsible for appearance of multicollinearity. | R | | R | A particular variable Xi would be said to be harmfully multicollinear of i y .

Where Ri is the multiple correlation of Xi 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 :

V(b)2 (X / X) –1

// 2 XXXZ1 1 1 // (6.1) ZXZZ1 where without loss of generality, X1 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 XXXZ(ZZ)ZXA1 1 1 1 V(b) (6.2) BB / / / –1 / where A and B are vectors and C is a matrix. However, [XX–X(ZZ)ZX]1 1 1 1 is the error sum of squares of the regression of the first predictor variable on the remaining particular variable. Now from the equation (6.2), the variance of the first coefficient estimate is

2 / / / –1 / –1 V(b)1 [XX–X(ZZ) 1 1 1 ZX] 1

2 –1 (SSE1 )

2 –1 –1 (SST1 – SSR 1 ) ) (6.3)

where SSE1, SST1. and SSR1 are the errors, total and regression, sum of 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 s2 is S2 = (SST – SSR) / m-p-1) (6.4)

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2 Also, R1 = SSR1 / SST1 (6.5)

2 Therefore, SST1 – SSR1 = SST1 (1 – R1 ) (6.6)

2 Analogously, SST – SSR = SST (1 – Ry ) (6.7)

Substituting equations (6.4), (6.6) and (6.7) in (6.3) we have an estimate of V(b1) as 1 V(b) [ ][SST(1–R)/SST(1–R2 2 ) 1(m p 1) y 1 1 1 [ ][SST/SST][(1–R)/(1–R)2 2 (m p 1) 1 y 1 1 2 2 2 2 [ ][S /S][(1–R)/(1–R)] (6.8) (m p 1) y 1 y 1 Since the choice of the first predictor variable arbitrary equation (6.8) can be generalized as

1 2 2 2 2 V(b) [ ][S/S](1–R)/(1–R) . 1(m p 1) y i y 1 Thus, the magnitude of the estimated variance of such estimated coefficient, given the ratio of 2 2 th Sy and Si , depend not only on the intercorrelation between the (i ) 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 problem relating 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 can made 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 X1i + 2 X2i + Ui (7.1) and the problem is that X1 and X2 are very highly correlated. In this situation one of the highly correlated variables may be dropped. Therefore dropping X2 the existing model becomes as

Yi = 1 X1i + Vi————— (7.2)

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where Vi is said as new disturbance term.

The estimator b1 of b1 obtained from (7.1) is the OLS estimator, b*1 of the same b1 obtained * from (7.2) is called the omitted variable (OL) estimator. The estimator b1 is biased estimator because of the omitted variable X2, while b1 is an unbiased estimator.

Further, it can be shown that b*1 has a smaller variance. Since the estimate of b1 from (7.2) is given by 2 b*1 = Yi X 1i x 1i It can further be shown that * E(b1 ) 1 b 21 (7.3)

where b21 is the slope coefficient in the regression of X2 on X1.

This shows that b*1, the estimated variable estimator is a biased estimator. If both b21 and 1 in (7.3) are positive, then * will be greater than leading to a positive bias. Similarly, if the E(b1 ) 1 product b is negative, on an average * will under estimate , leading to a negative bias. 21 b1 1 Thus, dropping a variable from the model to alleviate the problem of 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 to . Thus we have,

YXXUt 1 1t 2 2t t...... (given for t time)

Also, YXXUt 11(t–1) 2 2(t–1) t–1 .. (given for time t-1) Using first differences, the following result exist in the following manner given as,

YY–Yt t t–1

XX–X1t 1t 1,t–1

XX–X,2t 2t 2 t–1

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Thus, by using first differences, there is found that for the case of regression model, there reduces the severity of multicollinearity because X1t and X2t are existing for highly correlated variables as there exists for X1t and X2t . In order to concentrate on ratios, the given regression model may exist as

Yt X 1 t X 1t 1 22t1t X X U t X 1t (on division of X1t )

1 22tX X 1t X 1t U t1t x

Obvious, resulting residuals will be heterosedastic indicating (1/ X1t ) 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 Ut 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 problem a 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

135 Journal of Patna Science College Vol. 1, 125 - 137 [2013] ISSN 2347 - 9604 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 regresion coefficient 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 2 –1 variance (Since MSE(b) p ). In this connection, Marquardt(1970) remarked by consideration of correlation matrix XX/ indicating rij for (i,j) the element of the inverse matrix (XX)/ –1 asserted on variance inflation factor(VIF) and the author contributed VIF(i) rij 5 that indicates a harmful multicollinearity. In this connection, Theil(1971) showed the following result 1 rij [ X ] 2 2 i (1– Ri ) where 2 / ] and 2 represents the squared multiple correlation coefficient when [| Xi | X i X i Ri th Xi(i 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 2 as discussed in present study and Ri will be large (close to 1). Thus, the denominator of rii will close to zero. Hence rii 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 student’s value based on multicollinear data is that these values are highly unstable and often change their sign and 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. 136 Journal of Patna Science College Vol. 1, 125 - 137 [2013] ISSN 2347 - 9604

When the estimate regression coefficient bi are interpreted as practical derivatives yX i the wrong sign problem is 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 Problem Revisited, Review of Economics and Statistics, 49, 92–107 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

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SYNCHROTRON SPECTRA OF ASTROPHYSICAL JET M87 AND THE PROCESS OF DIFFUSIVE SHOCK ACCELERATION

Sumita Singh Postgraduate Department of Physics, Patna University Email :[email protected] Abstract : The theory of acceleration of the synchrotron emitting electrons or positrons in the extragalactic jets has to incorporate the fact that the spectrum is continuous from radio to X-rays.With reference to the M87 jet it has been observed that the spectral shape is uniform along the jet. And there is a constant upper spectral cutoff of the synchrotron electrons at

6 c 0.9 *10 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

6 1 2 syn ( c ) 185( c /10 ) (B / 1T ) yr (Meisenheimer et al 1996). This implies that the energy losses should affect the spectrum at a distance from the acceleration site ranging from

syn 42 pc to

syn 10 pc

2 in the inner jet (knot F) to 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 losses o that DSA forms a 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 3 cutoff momentum pc is a free parameter and is chosen to be either three (pc/po = 10 ) or six (pc/po = 106) 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 139 Journal of Patna Science College Vol. 1, 139 - 142 [2013] ISSN 2347 - 9604 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 å 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 &po. 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 sum over 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 sum is performed in evaluating the distribution shown. As N is increased the slope of distribution decreases monotonically and approaches b = 3 at low p>po, 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 from the plateau-like portions of the distribution resulting from injection 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 pc) beyond which no particle can be accelerated by

DSA; (b) for a single initial injection, a plateau distribution, f(p) = const., develops at p<0.1pc; (c) the -3 cumulative effect of injection at every shock leads to distribution f(p) á p for p <

(1) Synchrotron losses are most important during the acceleration process, when the electrons are in the compressed – B region just downstream from the shock. Synchrotron

losses imply a synchrotron cutoff, p = pc, to the distribution of accelerated particles;

DSA cannot cause any particle to be accelerated to p > pc. (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 momentum change in each cycle of particle crossing the shock from upstream to downstream and back. In the other procedure, used in our numerical calculations, synchrotron losses are first neglected to find the distribution of electrons resulting from DSA 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) Just 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 form a plateau distribution [f(p) independent of p] which corresponds to an energy spectrum N(e) á å2. (4) The distribution below the synchrotron cutoff due to the cumulative effect of injection at -b every shock tends to distribution f(p) á p with b 3 at p<< pc, with the distribution

becoming somewhat flatter such that the slope has peak (with b 2 ) just below pc (at 0.1pc 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 [ (b 3) / 2 0] 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 downstream flows, 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 mechanism for

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Vol. 1, 139 - 142 [2013] 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 them providing the dominant stress in the shock, resulting a shock structure that is quite different from that 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

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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 ([email protected])

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 aim of 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 problem is 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 from the table corresponding to 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 technique has been illustrated in the maintenance of the inventory of the LPG (Liquefied Petroleum Gas) cylinders for cooking.

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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 bk tk-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 problem is to find out ‘n’ for a given risk level (Pn), 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 from the 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 Ei (n, k, p) defined by: 144 Journal of Patna Science College Vol. 1, 143 - 152 [2013] ISSN 2347 - 9604

where NBi (n,k,p)denotes the ith approximation to the cumulative negative binomial probability. The first approximation is given by:

We have, thus, obtained the following quadratic equation:

n2 + (1-kq/p)n + (k 2q2/p2 – kq/p + 1/4 – z2kq/p2) = 0 (1)

Bartko (1996) has calculated the maximum errors 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 n6 + (4374 ABC – 5832 AB3 + 1968 E) n5 + (A2C3 + 20412 ABC – 27216 AB3 – 54 ABCD + 243 E2 + 19683 F) n4 + (4 A2C3 + 38070 ABC – 50760 AB3 – 210 ABCD + 486 EF + E3) n3 + (6 A2C3 + 35472 ABC – 47296 AB3 – 306 ABCD + 243 F2 + 3 E2F) n2 145 Journal of Patna Science College Vol. 1, 143 - 152 [2013] ISSN 2347 - 9604

+ (4 A2C3 + 16512 ABC – 22016 AB3 -198 ABCD + 3 EF2) n + (A2C3+ 3072 ABC – 4096 AB3 – 48 ABCD + F3) = 0 (2)

where A = kq/p, B = (9k – 1)/k, C = B2 – 9z2/k2, D = 9z2, 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 from the 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 from the equation (2) on the basis of the higher root obtained from the equation (1. Also, the value could be calculated directly from the 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 optimum level 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).

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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 s2 = 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.

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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.

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k = 5 k = 10 0.05 206.785 195.888 179.934 166.204 348.295 332.884 310.322 290.905 243.127 223.490 196.663 175.339 384.867 360.910 327.630 300.650 0.15 63.237 59.801 54.771 50.442 106.235 101.376 94.262 88.140 74.685 68.503 60.054 53.352 117.766 110.218 99.728 91.222 0.25 34.454 32.517 29.683 27.242 57.719 54.980 50.971 47.520 40.918 37.436 32.678 28.881 64.237 59.982 54.066 49.267 0.35 22.054 20.766 18.881 17.259 36.836 35.015 32.349 30.054 26.380 24.060 20.886 18.359 41.204 38.368 34.426 31.227 0.45 15.104 14.183 12.834 11.673 25.147 23.844 21.937 20.295 18.238 16.571 14.289 12.471 28.318 26.280 23.446 21.148 0.55 10.616 9.934 8.936 8.077 17.617 16.653 15.241 14.026 12.989 11.743 10.038 8.681 20.023 18.501 16.387 14.673 0.65 7.435 6.926 6.181 5.540 12.299 11.579 10.526 9.619 9.275 8.330 7.036 6.007 14.170 13.018 11.417 10.121 0.75 5.007 4.634 4.088 3.619 8.264 7.737 6.965 6.301 6.451 5.735 4.758 3.981 9.739 8.870 7.666 6.693 0.85 3.007 2.752 2.379 2.058 4.976 4.616 4.089 3.635 4.137 3.613 2.920 2.335 6.138 5.508 4.638 3.938 0.95 1.119 0.987 0.795 0.629 1.944 1.758 1.485 1.251 1.971 1.751 1.178 0.822 2.829 2.434 1.895 1.467

k = 15 k = 20 0.05 478.984 460.109 432.477 408.695 604.070 582.276 550.369 522.908 515.621 488.294 450.024 418.701 640.736 610.548 568.053 533.067 0.15 145.821 139.870 131.157 123.659 183.641 176.769 166.708 158.050 157.377 148.765 136.700 126.823 195.209 185.693 172.295 161.263 0.25 79.061 75.707 70.796 66.570 99.407 95.534 89.864 84.984 85.598 803.741 73.936 68.364 105.954 100.587 93.029 86.806 0.35 50.339 48.108 44.843 42.033 63.180 60.604 56.834 53.589 54.723 51.486 46.951 43.238 67.572 63.995 58.959 54.813 0.45 34.276 32.680 30.344 28.333 42.930 41.088 38.390 36.069 37.460 35.135 31.876 29.209 46.123 43.554 39.936 36.959 0.55 23.941 22.760 21.031 19.543 29.914 28.551 26.554 24.836 26.360 24.625 21.196 20.209 32.341 30.426 27.730 25.513 0.65 16.657 15.776 14.486 13.376 20.754 19.737 18.247 16.965 18.543 17.230 15.393 13.893 22.648 21.200 19.164 17.492 0.75 11.151 10.506 9.561 8.743 13.847 13.101 12.010 11.071 12.640 11.654 10.276 9.153 15.344 14.258 12.734 11.485 0.85 6.695 6.252 5.606 5.050 8.278 7.769 7.023 6.381 7.870 7.158 6.168 5.366 9.465 8.685 7.594 6.705 0.95 2.638 2.410 2.076 1.789 3.264 3.001 2.616 2.284 3.541 3.103 2.500 2.018 4.179 3.704 3.048 2.521

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k = 25 k = 30 0.05 725.577 701.210 665.537 634.835 844.541 817.849 778.771 745.138 762.257 729.537 683.313 645.097 881.229 846.215 796.613 755.476 0.15 220.332 212.649 201.401 191.720 256.221 247.805 235.483 224.879 231.907 221.592 207.017 194.966 267.801 256.761 241.121 228.148 0.25 119.118 114.787 108.448 102.992 138.376 133.633 126.688 120.712 125.670 119.852 111.630 104.832 144.932 138.705 129.883 122.565 0.35 75.598 72.718 68.503 64.875 87.715 84.561 79.943 75.969 79.995 76.118 70.640 66.111 92.116 87.967 82.089 77.215 0.45 51.282 49.222 49.206 43.611 59.419 57.163 53.859 51.016 54.480 51.696 47.762 44.510 62.621 59.642 55.421 51.922 0.55 35.664 34.139 31.907 29.986 41.254 39.584 37.139 35.035 38.096 36.021 33.091 30.671 43.690 41.471 38.328 35.725 0.65 24.684 23.547 21.881 20.448 28.496 27.249 25.425 23.854 26.584 25.016 22.805 20.981 30.399 28.723 26.354 24.392 0.75 16.420 15.587 14.367 13.317 18.906 17.993 16.657 15.507 17.924 16.750 15.097 13.737 20.415 19.161 17.392 15.831 0.85 9.780 9.211 8.377 7.659 11.223 10.599 9.686 8.899 10.974 10.133 8.954 7.988 12.422 11.527 10.267 9.232 0.95 3.847 3.553 3.122 2.752 4.400 4.078 3.606 3.200 4.770 4.264 3.561 2.994 5.330 4.794 4.050 3.447

k = 35 k = 40 0.05 961.579 932.747 890.538 854.211 1077.091 1046.268 1001.145 962.309 998.271 961.142 908.431 864.607 1113.785 1074.686 1019.079 972.752 0.15 291.503 282.412 269.104 257.649 326.304 316.585 302.358 290.113 303.085 291.379 274.757 260.937 337.888 325.560 308.024 293.415 0.25 157.292 152.169 144.668 138.212 175.937 170.460 162.441 155.540 163.851 157.248 147.872 140.077 182.498 175.544 165.653 157.413 0.35 99.605 96.198 91.210 86.918 111.314 107.672 102.340 97.751 104.008 99.609 93.363 88.171 115.720 111.087 104.499 99.010 0.45 67.394 64.956 61.388 58.316 75.239 72.633 68.818 65.535 70.588 67.439 62.955 59.229 78.446 75.119 70.390 66.452 0.55 46.724 44.920 42.279 40.006 52.098 50.170 47.346 44.917 49.163 46.810 43.473 40.700 54.540 52.063 48.544 45.614 0.65 32.217 30.871 28.900 27.204 35.867 34.428 32.321 30.508 34.124 32.349 29.833 27.746 37.776 35.909 33.257 31.053 0.75 21.327 20.340 18.897 17.655 23.695 22.641 21.097 19.768 22.839 21.512 19.636 18.082 25.210 23.816 21.839 20.200 0.85 12.620 11.946 10.960 10.111 13.982 13.262 12.207 11.299 13.823 12.878 11.545 10.447 15.189 14.197 12.796 11.638 0.95 4.929 4.581 4.071 3.633 5.440 5.068 4.523 4.054 5.864 5.303 4.520 3.884 6.380 5.794 4.975 4.308

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k = 45 k = 50 0.05 1191.356 1158.664 1110.803 1069.612 1304.577 1270.117 1219.667 1176.248 1228.052 1187.100 1128.770 1080.093 1341.274 1298.568 1237.662 1186.761 0.15 360.711 350.403 335.313 322.325 394.790 383.924 368.018 354.327 372.297 359.384 340.990 325.639 406.377 392.910 373.704 357.652 0.25 194.361 188.551 180.046 172.726 212.599 206.475 197.510 189.794 200.923 193.639 183.264 174.606 219.162 211.567 200.733 191.679 0.35 122.877 119.014 113.358 108.491 134.316 130.243 124.282 119.151 127.284 122.432 115.521 109.754 138.724 133.664 126.449 120.419 0.45 82.979 80.215 76.169 72.686 90.630 87.717 83.452 79.781 86.187 82.704 77.744 73.606 93.840 90.208 85.031 80.704 0.55 57.394 55.349 52.354 49.777 62.626 60.469 57.313 54.596 59.838 57.245 53.554 50.478 65.071 62.368 58.515 55.299 0.65 39.459 37.932 35.698 33.774 43.002 41.393 39.037 37.010 41.370 39.416 36.636 34.337 44.802 42.878 39.978 37.560 0.75 26.020 24.902 23.265 21.857 28.310 27.131 25.406 23.921 27.538 26.080 24.010 22.289 29.830 28.311 26.153 26.375 0.85 15.315 14.551 13.432 12.469 16.623 15.818 14.638 13.624 16.524 15.488 14.023 12.811 17.835 16.758 15.232 13.967 0.95 5.936 5.541 4.963 4.466 6.419 6.003 5.394 4.869 6.879 6.270 5.418 4.722 7.363 6.735 5.851 5.128

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

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k = 150 k = 200 0.05 3464.512 3404.825 3317.444 3242.238 4509.654 4440.733 4339.835 4252.995 3501.200 3433.381 3335.648 3253.004 4546.338 4469.308 4358.077 4263.802 0.15 1043.414 1024.595 997.043 973.331 1356.746 1335.020 1303.202 1275.821 1055.006 1033.619 1002.798 976.734 1368.338 1344.046 1308.968 1279.238 0.25 558.790 548.184 532.656 519.291 725.698 713.450 695.520 680.088 565.361 553.300 535.919 521.222 732.270 718.571 698.791 682.027 0.35 350.746 343.693 333.367 324.480 454.845 446.701 434.778 424.517 355.164 347.133 335.562 325.779 459.265 450.145 436.979 425.821 0.45 234.828 229.782 222.395 216.037 303.983 298.156 289.626 282.284 238.049 232.291 223.996 216.984 307.206 300.668 291.231 283.236 0.55 160.707 156.973 151.506 146.800 207.569 203.257 196.944 191.250 163.166 158.888 152.727 147.523 210.029 205.175 198.169 192.237 0.65 108.884 106.198 102.118 98.606 140.350 137.132 132.421 128.366 110.913 107.700 103.076 99.173 142.281 138.637 133.382 128.936 0.75 70.533 68.492 65.503 62.931 90.453 88.096 84.646 81.676 72.070 69.689 66.267 63.382 91.994 89.297 85.413 82.129 0.85 40.346 38.951 36.908 35.150 51.393 49.782 47.424 45.394 41.580 39.911 37.520 35.510 52.631 50.747 48.039 45.757 0.95 14.821 14.100 13.045 12.137 18.601 17.769 16.551 15.502 15.798 14.859 13.525 14.415 19.585 18.534 17.036 15.785

152 Journal of Patna Science College Vol. 1, 153 - 159 [2013] ISSN 2347 - 9604

SIMULATING AVERAGE TIME COMPLEXITY OF SRSWOR: A STATISTICAL APPROACH Anchala Kumari1 and Soubhik Chakraborty 2 1Department of Statistics, Patna University, Patna-800005, India 2Department of Applied Mathematics, BIT Mesra, Ranchi-835215, India

Abstract : Algorithm is 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 algorithm amongst 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 minimum number 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 algorithm T 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(Nlog2N) 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 system dependent. Keywords: Simple Random Sampling Without Replacement; Algorithm Complexity, Computer Experiment. Introduction : Simple random sampling 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 algorithm for srswor .Some more algorithms related to srswor are available in W. Kennedy and J. Gentle’s book [4]. These works suffer from the 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 algorithm described focuses on both the aspects: the selection procedure as well as the order in which the observations come into the sample. The algorithm for drawing a sample of size n from a population of size N is based on the concept that the sample drawn is a random permutation 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 random permutation. 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

153 Journal of Patna Science College Vol. 1, 153 - 159 [2013] ISSN 2347 - 9604 program for finding the execution time of the algorithm T written in Visual C++ follows in the next section. Visual C++ code #include #include #include #include #include void main() { int npop,ns,k; int *a,*s; clock_t start,end; cin>>npop; a=new int[npop];

for(int i=0;i

double elapsed=double(end-start)/CLOCKS_PER_SEC;

cout<<“elapsed time=”<

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For n arbitrarily chosen, the statement ns= pow (npop,.5) is replaced by the statement cout<

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 time when plotted against different values of N using MINITAB statistical package agrees to the average complexity lying between O(N) and O(N2). Fig1 and fig 2 shows time as linear and quadratic functions in N(population size) with 100R2 values (coefficient of determination) equal to 97.4 and 100 respectively. For a good literature on applied regression analysis, the reader is referred to [3]. Fig.1 N-time plot time->O(N)

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Fig.2 N-time plot time->O(N 2)

Regression Plot t ime = - 0.0 5544 17 + 0. 0 00 00 26 N + 0.000000 0 N**2 S = 0 . 041 39 92 R- Sq = 100 . 0 % R- Sq(a dj) = 100 . 0 %

6

5

4

3

2

1 Regression 95% CI

0

0 500000 1000000 N 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 R2 = 100% .It may be argued that the contribution due to N2 term is almost negligible in the equation time=-.554417+.0000026N+.0000000N2 .The N2 term contributes in reducing the distance between the confidence bounds. 2 Further we have O(N)

And O(Nlog2N) +O(N) = O(Nlog2 N) Thus we may experiment with the model

Y=a + bNlog2N The estimated values of a,b are ^ ^ a = -.3639 b =.0000003 line of regression is time= -.3639 + .0000003 Nlog2N R2 = 98 .2% Normal probability plot of the residuals approximates to linear function as shown below. Fig .3. Normal probability plot of residuals

No r m a l P ro b a b ili ty P lo t o f t he R e s id u a l s (resp ons e is time)

1

0

-1

-1 0 1 2 Standardized Residual Analysis of variance table is as given below 156 Journal of Patna Science College Vol. 1, 153 - 159 [2013] ISSN 2347 - 9604

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 algorithm is of order O(n). The model equation is time= .125658 + .0000464 n R2 = 99.7% Fig.4 n-time plot Regression Plot t im e1 = 0. 1 2 56 58 + 0.0 0 00 464 n1

S = 0. 00 3 86 24 R- Sq = 99 . 7 % R -S q( a d j) = 99 . 6 %

0.5

0.4

Regres sion

95% CI

0.3

4000 5000 6000 7000 8000 n1

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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

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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 - n2/4 +3n/4 which leads to O(nN) and for n= .O(N3/2)  3/2 For large N, we have N< 2 or log 2N< 2N

And for the second study Tavg(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 investigator’s 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 investigator’s 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 credibility of 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, 3rd ed. 8 Kennedy J , Gentle James E.(1980). Statistical Computing, Marcel Dekker, Knuth .D.E.(1998).The Art of Computer Programming,vol.2: 3rd ed., Addison Wesley Longman Publishing Co,Inc.Boston, MA,USA ISBN; O-201-89684-2

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160 Journal of Patna Science College Vol. 1, 161 - 167 [2013] ISSN 2347 - 9604

PES TIC ID E AC C UMULATION , ALTERATION O F SER UM 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- [email protected]

Abstract : In the present investigation a number of selected wetlands from the north Bihar particularly Supaul and Saharsa district were surveyed. Fish soil and water sample were collected from the various test zones for the assessment of organocholrine pesticide accumulation. A comparative analysis of the toxic status of fish from these 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 from the reference site showed almost negligible percentage of these pesticides. Fishes collected from different test zones were screened for the presence of neoplastic and preneoplastic liver lesion based upon histopathological and ultrastructural findings. The histoarchitecture of liver of C. batrachus from the test reveals changes in the mitochondrial organization, reduced ER cristae, scanty hepatoplasm and tendency of SER to transfer into glycogenated bodies, apoptic bodies, proliferation of RER and cellular hyper activity. Blood of test fish also revealed decreased serum FSH level & less concentration of vitellogenin. Key words : Pesticide accumulation, Serum Vitellogenin 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 cytoplasm or 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

161 Journal of Patna Science College Vol. 1, 161 - 167 [2013] ISSN 2347 - 9604 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 zones of 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 from the 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 from these 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 gm±10 gm wt were brought to the laboratory and after disinfections with 0.1% KMNO4 solution they were kept at room temperature 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 3rd day to fulfill their dietary requirement. Quantification of pesticide residues in soil : Collection of soil sample – Soil sediments were collected at the four edges of each wetlands/Chaur using a spade. The entire samples were sealed in polythene bags, storing at 0oC and transported to the laboratory within two days. Freeze dried sample were 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-Hexane and send to ITRC (Industrial Toxicology Research Institute), Lucknow for estimation of organochlorine pesticide residues (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 serum in appendorf at 4oC in deep freeze for further analysis. Serum FSH was done on Merk “minimios” ELISA reader by Herichson Method. The serum FSH 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 female juvenile, 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 were purchased from Bioscience 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 5m section were

162 Journal of Patna Science College Vol. 1, 161 - 167 [2013] ISSN 2347 - 9604 stained by Delafield’s 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 o 4 C. After one hour the tissues were placed in 1% OSO 4 in 0.2M Phosphate Buffer solution (pH 7.4) at 4oC 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 Philip’s EM-10 transmission Electron Microscopy at SIF-EM Facility Unit, Dept. of anatomy, AIIMS, New Delhi. The Micrographs 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

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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 Mean±SE 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 from reference 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 form rosette with the bile canaliculi located in the center. EM Studies also reveals changes in the mitochondrial organization, reduced ER cristae, scanty hepatoplasm and 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) Serum level of FSH and vitellogenin level of test fish from these test zones also reveals decreasing trend as compared to the reference site (Table – III & VI).

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Discussion : A comparative analysis of pesticides in soil and fish liver shows accumulation of various organochlorine pesticides viz. áHCH âHCH ãHCH , aldrin, endosulphan, DDE, DDT etc. the presence from different test zone showed the variation in the accumulation of áHCH âHCH ãHCH , aldrin and DDT whereas fishes collected from reference 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 stream is 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 consumer’s 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 from Upper Thames river. Muir et al (2003) have also observed DDT, HCH & PCBs in fishes from Barents Sea Canadian Arctic. Occurrence of neoplasm and 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 neoplasm as 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 neoplasm in adult winter flounder from Boston 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 bottom dwelling fish and these are more prone to liver neoplasm and 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

165 Journal of Patna Science College Vol. 1, 161 - 167 [2013] ISSN 2347 - 9604 individual of all tested group and reminiscent of programmed cell death. With nuclear shrinkage, irregular shape and heterochromatization, which ultimately affect the metabolism of 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 confirm it.It may be concluded that the bottom dwelling air breathing Clarias batrachus from these 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, John T. Landahl, Patty G. Prahaska, Andrew J. Friedman, Linda D. Rhodes, Douglas G. Burrows, William D. 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. John A. Couch (1993): Light and electron microscopic comparisons of normal Hepatocytes and neoplastic Hepatocytes of well-differentiatedhepatocrellular carcinomas in a teleost 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 Journal articles pp.1-2. Moore, M.J., Smolowitz,R. Stegeman, J.J. (1994/2007): Cellular alteration preceding neoplasia in Pseudopleuronectes americaus from Boston Harpour. Marine Environ. Research. MERSDW 28, No.1/4 pp.425-429. 166 Journal of Patna Science College Vol. 1, 161 - 167 [2013] ISSN 2347 - 9604

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 from the white sea, Russia. Sci Total Environ. 306(1-3): 111-31. Myers, M.S., Olson, O.P., Johson, L.l., Stehr, C.S., Hom, T., (1992) : Hepatic lesions other than neoplasms in subadult flatfish from puget sound, Washington: Relationship with contaminant exposure. Response of Marine Organism to Pollutant Part I, P.45-51 Robert A., Murchelano and Richard E. Wolke (1991) Neoplasm and Nonneoplastic liver lesion in winter flounder, Pseudopleurotectes americanus from Bosten 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 from the Upper Thames: river pollution and its potential effects on top predators. Chemosphere. 50(3): 275-73.

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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 among the 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 among the 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 system as ‘Jatis’ [2]. Each endogamous group, reproductively isolated from each 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 polymorphism among them. The Rh-factor was discovered by Karl Landsteiner and A.S. Wiener [4] from rabbits 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-system seemed 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 form by 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,

169 Journal of Patna Science College Vol. 1, 169 - 174 [2013] ISSN 2347 - 9604 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 (Rho) 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 antiserum indicated 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 from the number of phenotypes scored. Re sults and Discussion : The incidence of Rh-v e subjects in all the four populations was slightly higher than the range known for Indian 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-v e 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.

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Table1 Frequency distribution of subjects of Rh-blood group system in the different populationsz

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References : Census of India (2011) Ministry of Information and Broadcasting, Government of India, New Delhi. Singh K S(1998) India’s 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. Am J Hum Genet, 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.Hum Hered, 26,458-467.

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