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Richard CallaghanUniversity of Calgary, AB, CanadaDavid T CrambUniversity of Calgary, AB, CanadaMatthew CooperGrand Valley State University, AWRI, Muskegon, MI, USAAnatoly S BorisovKazan State University, Tatarstan, RussiaRon ColeyColey Water Resource & Environment Consultants, MB, CanadaChia-Chu ChiangUniversity of Arkansas at Little Rock, Arkansas, USAMichael J DreslikIllinois Natural History, Champaign, IL, USADavid FederUniversity of Calgary, AB, CanadaDavid M GardinerUniversity of California, Irvine, CA, USAGeoffrey J HayUniversity of Calgary, AB, CanadaChen HaoanGuangdong Institute for drug control, Guangzhou, ChinaHiroyoshi ArigaHokkaido University, JapanGongzhu HuCentral Michigan University, Mount Pleasant, MI, USAMoshe InbarUniversity of Haifa at Qranim, Tivon, IsraelSA IsiorhoIndiana University - Purdue University, (IPFW), IN, USABor-Luh LinUniversity of Iowa, IA, USAJinfei LiGuangdong Coastal Institute for Drug Control, Guangzhou, ChinaCollen KellyVictoria University of Wellington, New ZealandHamid M.K.AL-NaimiyUniversity of Sharjah, UAEAQ KhanUniversity of Illinois at Chicago, IL, USAEric L PetersChicago State University, Chicago, IL, USARoustam LatypovKazan State University, Kazan, RussiaFrances CP LawSimon Fraser University, Burnaby, BC, CanadaGuangchun LeiRamsar Convention Secretariat, SwitzerlandAtif M MemonUniversity of Maryland, MD, USASR NasyrovKazan State University,Kazan, RussiaRussell A NicholsonSimon Fraser University, Burnaby, BC, CanadaShakeel A KhanUniversity of Karachi, Karachi, Pakistan

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Asian Institute of Technology, Thailand

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Indian Institute of Technology Kharagpur, India

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University of Catania, Italy

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Chemistry & Environment College, Normal University, China

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Asian Institute of Technology, Thailand

Indraneil Das

Universiti Malaysia, Sarawak, Malaysia

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Indian Institute of Technology , Guwahati, India

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American Museum of Natural History, New York, NY, USA

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Bio-Sciences Research Institute, University College Cork, Ireland.

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Volume 2, Number 2May 2008

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Volume 2, Number 2 May, 2008

CONTENTS NATURAL SCIENCES Falodun A, Owolabi OJ and Aigbogun OO Chromatographic Characterization, Proximate Analysis and In vitro Inhibitory Evaluation of Pyrenacantha staudtii Hutch and Dalz (Icacacinancae) on the Guinea Pig Ileum ..................................................................................317 Joydev Maity and Bidhan C Patra Effect of Replacement of Fishmeal By Azolla Leaf Mael on Growth, Food Utilization, Pancreatic Protease Activity and RNA/DNA Ratio in the Fingerlings of Labeo rohita (Ham.) .....................................................................323 ABM Sharif Hossain and Fusao Mizutani Dwarf Peach Trees by using Abscisic Acid Hormone, Maleic Hydrazide and Cycocel as Growth Inhibitors in Phloemic Stress Condition Grafted on Vigorous Rootstock.............................................................................................335 Samar Kumar Pal, Syam Sundar Konar, Abhishek Mukherjee and Tapan Kumar Das Removal of cadmium ion by cadmium resistant mutant of Aspergillus niger from cadmium contaminated aqua – environment ..........................................................................................................................................................343 M Zaheer Khan, Ghazala Yasmeen , SNH Naqvi and Aisha Perveen Activity of Cholinesterase and Alkaline Phosphatase in Liver, Kidney and Brain of Euphlyctis cyanophlyctis under the Effect of Chlorpyrifos and Dathrin ..................................................................................................................349 Chinedu, Nwodo S, Nwinyi, Obinna C and Okochi, Veronica I Growth and Cellulase Activity of Wild-Type Aspergillus niger ANL301 in Different Carbon Sources .........................357 Pratap B Singh and Vandana Singh Exposure and Recovery Response of Pesticides on Tissue Bioconcentration and Plasma Sex Steroid Hormones in Heteropneustes fossilis................................................................................................................................363 Hala M Rifaat and Osama Hamed El-Sayed Rare Actinomycetes from Egyptian Habitats: Isolation and Screening for Antimicrobial Activities...............................373 Gabriel Rosangkima and Surya Bali Prasad Inhibitory Effect of Dillenia Pentagyna Stem Bark Extract on Cisplatin and Benzo[A]Pyrene-induced Mutagenicity ...........................................................................................................................381

Fahrul Huyop, Ng Hong Jing and Ronald A Cooper Overexpression, Purification and Analysis of Dehalogenase D of Rhizobium sp.............................................................389 Okafor, PN, Anoruo, K, Bonire AO and Maduagwu EN The Role of Low-Protein And Cassava-Cyanide Intake in the Aetiology of Tropical Pancreatitis..................................393 Ghulam Sarwar, MA Haq, Akbar Ali Cheema and MB Chaudhary Induced Polygenic Variation Study in Sesame (Sesamum indicum L.) and its Implication in Selection..........................399 Omonkhelin J Owolabi, Eric Ki Omogbai, Anthony B Eledan And Abiodun Falodun Smooth Muscle Relaxant Activity and Proximate Evaluation of Kigelia africana ..........................................................405

Muhammad Siddique Sadiq, Sajjad Haidar, Muhammad Ahsanul Haq, Ghulam Abbas, Munir Ahmad Chaudhary and Noor Muhammd A high Yielding and Disease Resistant Mutant of Lentil Developed through Seed Irradiation of an Exotic Germplasm .......................................................................................................................................................411 PHYSICAL SCIENCES Greg Gaston Use of Electrical Resistivity Imaging to Investigate Depth and Concentration of Subsurface Ice in a Suspected ‘Ice Cored’ Moraine .................................................................................................................................417 Arjunudu, K., Srinivasa Rao, M and Kasipathi, C A New Gemstone Province of Eastern Ghats Mobile Belt, India: Results of Geological and Mineralogical Investigations....................................................................................................................................................................423 Salem Issa An Integrated Geo- Database for Land Management and Greening Assessment of an Arid Island at the Fringes of Abu Dhabi City ...............................................................................................................................................431 K Muni Krishna and S Ramalingeswara Rao Study of Indian Summer Monsoon Rainfall on Decadal Scales vis-à-vis Circulation Patterns........................................441 Suprakash Patra, Goutam Sutradhar, Amitava Mandal FEM Simulation with Experimental Verification of Sintered Forged Components .........................................................451 B Ahmad and S Hussain γ0-Compact , γs-regular and γs-Normal Spaces ................................................................................................................. 459 Muhammad Asif Khan and Saleh Al Turki Evaluation of Software Development Controls in Information Systems Organizations...................................................463

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 317-321, 2008 ISSN: 1715-9997

1

CHROMATOGRAPHIC CHARACTERIZATION, PROXIMATE ANALYSIS AND IN VITRO INHIBITORY EVALUATION OF PYRENACANTHA STAUDTII HUTCH

AND DALZ (ICACACINANCAE) ON THE GUINEA PIG ILEUM

*Falodun A1, Owolabi O J2 and Aigbogun OO1 Departments of 1Pharmaceutical Chemistry and 2Pharmacology and Toxicology

Faculty of Pharmacy University of Benin, Benin City, Nigeria

ABSTRACT

Pyrenacantha staudtii (Hutch and Dalz), Icacinaceae is a medicinal plant used in trado – medical practice for the treatment of dysmenorrheal and gastrointestinal disorders. The leaf extract is also used in ethno - medicine for the treatment of threatened abortion. The present study aimed at assessing the scientific evaluation of the ethnomedicinal claim on isolated guinea pig ileum suspended in an organ bath containing a physiological salt solution of Tyrode solution at a pH of 7.35, and also to investigate the phytochemical constituents and quantitative parameters like the moisture content, ash value, acid insoluble ash, water soluble ash and extractive values such as alcohol and water extractives which can be used in the identification of the leaf of P. staudtii. The crude extract of the plant was subjected to thin layer and vacuum liquid chromatography with different solvent systems. The phytochemical evaluation revealed the presence of alkaloids, tannins and saponins in the leaves of P. staudtii. The proximate analysis gave 5.40 ± 0.02 % as the moisture content, 5.25 ± 0.01 % as ash value, 2.58 ± 0.13 % as acid insoluble ash and 3.25 ± 0.08 % as water soluble ash value. The alcohol and water extractives of 7.20 ± 0.04 % and 8.10 ± 0.13 % respectively were obtained. The thin layer chromatography profile of the fractionated crude extract indicated the presence of compounds with different Rf values. The pharmacological evaluation revealed a very significant (P<0.05) inhibitory effect of the extract on histamine and acetylcholine induced ileum contractions. The relaxation of the guinea ileum by the extract, has justified the claims for which the plant is known and used.

Keywords: Pyrenacantha staudtii, extract, ileum, chromatography.

INTRODUCTION Despite the amazing progress in the fields of genomics and genetics, and the importance of synthetic pharmaceutical Chemistry and microbial fermentation, plants remain an essential source of new lead compounds (Hosettmann and Marston, 2001). Natural products are an important source of new structures leading to drugs in all major diseases areas. Traditional medicine practice in the treatment of diseases and infections have assumed a more scientific and wider dimension as the emphasis on ethnomedicine is on the increase especially in the developing countries where the primary health care needs of the populace are not easily affordable. In the developed countries, the use of herbal medicine is attracting attention as a result of the increased resistance posed by orthodox drugs which are expensive and with numerous adverse side effects. The support for the use of medicinal plants by the World Health organization (WHO) is quite encouraging due to the numerous therapeutic benefits (WHO, 1995). For example, acetylsalicylic acid was obtained from Flipendula umaria, the antihypertensive and antipsychotic agent, reserpine from Rawolfia

vomitoria is still clinically in use today. This support for traditional medicine still needs the support and cooperation of the regulatory agents in various countries to discourage the abuse and misuse of these herbal medicines. This attraction for herbal medicine led to the present scientific investigation of the local medicinal plant Pyrenacantha staudtii used in the treatment of spastic conditions of the gastrointestinal tract disorders. The aqueous extract is used for the treatment of dysmenorrheal and of threatened abortion. Previous phytochemical studies reported the isolation and characterization of 3-Carbomethoxylpyridine and Hexahydroxylcyclohexane (Falodun and Usifoh, 2006; 2007). The present study aimed at validating the ethnnomedicinal use as anti - spamolytic agent via an in vitro experimental model. MATERIALS AND METHODS Collection and preparation of plant material The fresh leaves of the plant were collected from Ikpoba river axis of the University of Benin, Ugbowo campus, identified by Mr. Sunny of the Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Benin City, Nigeria. Botanical authentification *Corresponding author email: faloabi25@yahoo.com

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was done by Dr. BA Ayinde of the same Department. [A voucher specimen was kept in the herbarium of Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Nigeria]. The plant sample was dried at room temperature and powdered with the aid of a mechanical mil. Quantitative analysis of the crude powdered sample Proximate composition of the sample was determined according to standard methods (African Pharmacopoeia, 1986, British Pharmacopoeia, 1988, and AOAC, 1990) with Analytical Codex Number14.062, 14.063, 14.064 and 14.065. Extraction and Partitioning of extract The powdered sample (400g) was extracted exhaustively by maceration at room temperature with ethanol for 72hrs. Removal of the solvent under pressure at 40˚C yielded a greenish residue (7.86g). The concentrated extract was stored in air tight containers, labeled and refrigerated at - 4˚C prior to use. The crude ethanolic extract was partitioned with petroleum ether 40-60OC, chloroform and then ethyl acetate. Phytochmemical screening of crude powdered and biologically active extract The crude powdered and the solvent free petroleum ether and ethanolic extracts of the plant was subjected to phytochemical examination using chemical and chromatographic methods. The extracts were separately screened for following constituents, carbohydrates alkaloids and / or nitrogenous bases, tannins, saponins and flavonoids, using standard procedures (Trease and Evans, 1989, Sofowora 1980 and Harborne, 1984). Fractionation of crude extracts using open column chromatography with Sephadex The bioactive extract of P. staudtii was subjected to chromatographic investigation using thin layer, column and vacuum liquid chromatography. The ethylacetate, ethanolic and petroleum ether fractions were subjected to thin layer chromatographic analysis with different solvent systems of chloroform, methanol and petroleum ether ratio. The Rf values recorded. Ethylacetate fraction (305mg) was applied on a column of sephadex, elution was done with 100 % toluene followed by a mixture of toluene/methanol of increasing polarity (1:9; 2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; and 9:1 v/v) and then 100 % methanol followed by a mixture of MeOH/H20 (75:25 and 50:50 v/v) each 50ml. The spots were visualized with sulphuric acid and heating at 105oC for 10 minutes. The spots were also characterized with UV lamp at 254nm and 366nm.

Pharmacological evaluation Drugs and Chemicals The following drugs were used: Acetylcholine (Roche), Histamine (Roche), DMSO (Sigma Aldrich). Stock solutions of the various drugs, ethanolic and petroleum ether of Pyrenacantha staudtii extracts were prepared fresh for each experiment. Animals Guinea pigs of either sex weighing 250 -300g were obtained from the Animal house of the College of Medicine, Ambrose Alli University, Ekpoma, Edo State, Nigeria. Animals were acclimatized for two weeks before being subjected to the experimental protocol. The animals were maintained on a standard diet (Ladokun feeds, Ibadan, Oyo State, Nigeria) and had free access to food and water ad libitum. Animals were housed in a cage with a twelve hour light-dark cycle. [Approval for use of animals in this work was obtained from the Ethical Committee on the use of Animals for Experiments of the Faculty of Pharmacy, University of Benin, Benin City]. Animal preparation The guinea pigs were killed by cervical dislocation and exanguinations. The abdomen was opened and the ileum carefully isolated, freed of mesenteric fat and about 3 - 4cm piece in length was mounted in a 50 ml organ bath containing Tyrode physiological salt solution having the following chemical composition: NaCL, 8 g/l, NaHCO3, 1.0 g/l, D-glucose, 1.0 g/l NaH2PO4, 0.05g/l, MgCl2, 0.1g/l, KCL, 0.2 g/l, CaCL2.2H20, 0.26 g/l. Each intestinal strip was suspended vertically with the lower end fastened to the tissue holder and the upper end attached to an isometric or isotonic transducer connected to a chart recorder. The tissue was aerated with 95 % O2 and 5 % CO2 and temperature maintained at 37oC with a pH of 7.4. The contractions of the ileum which occurs both circularly and longitudinally were recorded with FT 03 transducer connected to an Ugo Basile recorder (7075). The transducer was previously calibrated to establish a relationship between the force applied to the transducer and the gauge deflection (800mg). The tissue was allowed to equilibrate for 30 minutes before the commencement of the experiment. A dose response for acetylcholine obtained using 0.004ug to 4ug in geometric doses with a dose cycle of 120seconds (30sec contact period and 90sec washout period) was maintained. The dose response then repeated in the presence of 400ug of the crude extract after a contact time of 120sec. Similarly, a dose response for histamine was obtained using 0.032ug to 4ug in geometric doses with a dose cycle of 120seconds (30sec contact period and 90sec washout period) was maintained. The dose response then repeated in the presence of 400ug of

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the crude extract and its fractions after a contact time of 120sec. Statistical analysis All results are expressed as the mean of 5 experiments ± SEM (standard error of mean) and continuous line graph. The data were analyzed statistically by student’s t-test using Graphpad instant version 2.05a. The level of significance was P<0.05. RESULTS AND DISCUSSION

Determination of the proximate composition is used to further establish and differentiate crude drug identity particularly where similar macroscopic and microscopic features are observed. The results of the quantitative parameters are shown in (Table 1). The moisture content shows the susceptibility of crude drug samples to microbial attack especially fungi, and also to degradation due to hydrolysis of the crude powdered drug. A moisture content of 5.55 ± 0.02 % obtained from this study is indicative of the storage quality for some time without microbial degradation or hydrolytic break down of the chemical constituents. The maximum range is between 6-8 % in African Pharmacopoeia (1986). Table 1. Percentage (%) values of quantitative parameters examined on the leaf of Pyrenacantha staudtii

Parameter Values ± SEM (%) Moisture content 5.40 ± 0.02 Total ash 5.25 ± 0.01 Acid insoluble ash 2.58 ± 0.13 Water soluble ash 3.25 ± 0.08 Alcohol extractive 7.20 ± 0.20 Water extractive 8.10 ± 0.18

The total ash is a measure of the non-volatile inorganic constituents remaining after ashing. It is made up of two parts, the physiological and the non-physiological ash. The physiological ash consists of carbonates, phosphates, nitrates, sulphates, chlorides and silicates of metals which the plant took up when it was growing. The non-physiological ash represents ash from extraneous matter. The acid insoluble ash is residue obtained when total ash is boiled with 10 % HCl. It is a measure of the sandy matter in the crude drug samples. The values of 5.25 ± 0.01 and 2.58 ± 0.13 were obtained. The results of the phytochemical test (Table 2) as well as the chromatographic profile (Table 3) of Pyrenacantha staudtii leaf revealed the presence of phyto constituents which include simple and complex sugars, tannins, saponins and alkaloids. The column chromatography with sephadex (Table 3) afforded one broad spot Rf 0.84 which tested positive for reducing sugar. Because of the intensively bitter taste of the leaf, locals usually add common salt (NaCl) to the aqueous decoction before ingestion. This has important implications since there is a co-transport of Na+ and glucose across the epithelial cells of the ileum and glucose enhances sodium absorption and thus water uptake, these locals are actually rehydrating themselves – an important first step in diarrhea treatment. The pharmacological evaluation as revealed in figure 1 and figure 2, showed that graded doses of acetylcholine at 0.064ug/ml, 0.128ug/ml, 0.256ug/ml, and 0.0512ug/ml elicited dose-dependent contractions of the ileum producing 77.63, 79.12, 78.73%, and 88.33% mean responses respectively. The crude ethanolic extract inhibits acetylcholine and histamine induced contractions of the isolated guinea pig ileum. In the presence of 400ug/ml of the crude ethanolic extract, there was

Table 2. Results of phytochemical screening of the crude extract and fractions of Pyrenacantha staudtii leaf.

Secondary metabolite

Powdered leaf

Crude ethanolic extract

Petroleum ether fraction

Chloroform fraction

Ethanolic fraction

Ethylacetate fraction

Carbohydrate present present present present Present present Reducing sugar present present present present Present present Saponins present present absent absent present absent Tannins present present present present absent absent Alkaloids present present present present present absent

Table 3. Resolutions of vacuum liquid chromatography.

Codes Fractions Rf Values UV reaction (nm) A 7 0.91, 0.82 254, 366 B 8 0.93, 0.82, 0.27 254, 366 C 10 0.93, 0.82, 0.64, 0.27 254, 366 D 11 0.94, 0.27 254, 366 E 29 0.84 254, 366

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significant reduction of the mean percentage responses (P<0.0001). Similarly, as shown in figure 2, histamine at 0.512ug/ml, 1.04ug/ml, 2ug/ml, and 4ug/ml elicited dose-dependent contraction of the ileum producing 86.89 %, 91.19 %, 97.53 %, and 96.99 % mean responses respectively. In the presence of 400ug/ml of the crude ethanolic extract, there was significant (P<0.0001)

reduction of the contractile responses. Except for the dose of 0.064ug/ml of histamine (P>0.05), there was significant reduction in the contractile responses (P<0.05). It therefore suggests strongly that (Fig. 1 and 2), as the concentration of the drugs (acetylcholine and histamine) are increased in the presence of the extract, there is a gradual increase in the contractile responses suggesting

Fig. 1. Effect of acetylcholine and the crude ethanolic extract of Pyrenacantha staudtii on isolated guinea pig ileum.

Fig. 2. Effect of histamine and the ethanolic extract of Pyrenacantha staudtii on isolated guinea pig ileum.

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that the extract act through competitive antagonism. Acetylcholine acting through muscarinic receptor (M3-subtype) and histamine acting through histamine (H1-subtype) both activates G-protein resulting in the cleavage of phospholipase C and formation of IP3 (inositol triphosphate) and DAG (diacyl glycerol) both of which cause increase in intracellular calcium. The extract may work through interaction with the second messenger (Ca2+) rather than by specific receptor antagonism. However, since the extract also inhibits H2-receptor mediated ulcer in the gastrointestinal tract (Aguwa and Okonji, 1986; Aguwa and Mittal, 1983; which act through increase in cAMP (cyclic adenine monophosphate) Rang and Dale, 2003. It therefore suggests that the specific mechanism of action is unclear. More work need to be done in order to establish the precise mechanism of action of the extract. Further characterization of the extract is necessary for the structure elucidation of the bioactive chemical constituent(s) and its structure-activity relationship. CONCLUSION The study was carried out with the objective of investigating the ethno medical claim of the leaves of Pyrenacantha staudtii in folk as a remedy against diarrhoea and intestinal colic and to authenticate its use. The present work confirmed and justified its use in traditional medicine, and the active constituents when isolated and purified would be a potential anti diarrhea agent. ACKNOWLEDGEMENT The authors acknowledge Sunny A of the Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Benin City, Nigeria for the technical assistance. REFERENCES African Pharmacopoeia. 1986. Vol. 2. 1st ed. OAU/ STRC Publications. pp.128-144.

Aguwa, CN. and Mittal, GC. 1983. Study of the Antiulcer Activities of the Aqueous Extract of the Leaves of

Pyrenacantha Staudtii (Icacinaceae) Using Various Models of Experimental Ulcer in Rats. European Journal Pharmacology. 74: 215-219.

Aguwa, CN. and Okunji, CO. 1986. Gastrointestinal Studies Of Pyrenacantha Staudtii Leaf Extracts. European Journal Pharmacology. 15: 45-55.

AOAC. 1990. Ref. No. 968.08, 946.06, Official Method of Analysis Association of Official Analytical Chemists (13th ed.) Washington, DC, USA.

British Pharmacopoeia BP. 1988. Vol.II. Her Majesty’s Stationary Office, London. p.1022.

Falodun, A. and Usifoh, CO. 2006. Isolation and Characterization Of 3-Carboxymethypyridine from the Leaves of Pyrenacantha Staudtii (Hutch and Dalz) (Icacinaceae). Acta Poloniae Pharmaceutica-Drug Research. 63 (3): 235-237.

Falodun, A. and Usifoh, CO. 2007. Isolation and Characterization of hexahydroxylcyclohexane from the leaves of Pyrenacantha staudtii (Hutch and Dalz). J. Pharm. Sci. & Pharm. Pract. 8(3): 60-63.

Harborne, JB. 1984. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 2nd Ed. Chapman and Hall, USA, p.46.

Hosettmann, K. and Marston, A. 2001. The Search for New Drugs From Higher Plants; CHIMIAD. 61(6): 322-324.

Rang, HP, Dale RT. and Ritter, JM. and Moore, PK. 2003. The Gastrointestinal Tract. Pharmacology 5th Ed. 24: 367.

Sofowora, A. 1980. Screening Plants for Bioactive Agents. In: Medicinal plants and traditional Medicine in Africa. Spectrum Books Limited, 1st Ed. 128-161.

Trease, GE. and Evans, WC. 1989. Pharmacognosy (13th Ed.). English Language Book Society, Bailliere Tindall, Britain. 378: 386-480.

World Health Organisation (WHO). 1995. The World Health Report. Bridging the gap WHO Geneva 1. p.118.

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EFFECT OF REPLACEMENT OF FISHMEAL BY AZOLLA LEAF MAEL ON GROWTH, FOOD UTILIZATION, PANCREATIC PROTEASE ACTIVITY AND

RNA/DNA RATIO IN THE FINGERLINGS OF LABEO ROHITA (HAM.)

*Joydev Maity1 and Bidhan C Patra2

1Department of Aquaculture Management and Technology, Vidyasagar University Midnapore-721102, West Bengal

2Aquaculture Research Unit, Department of Zoology, Vidyasagar University Midnapore-721102, West Bengal, India

ABSTRACT

Dried water fern (Azolla pinnata) leaf meal was substituted for fish meal in practical diets of Labeo rohita fingerlings. Five isonitrogenous diets (38% crude protein) were formulated in which fish meal (68.9% crude protein) was replaced at varying levels (D1 = Control, 0% replacement; D2 = 25%; D3 = 50%; D4 = 75% and D5 = 100%) with protein from Azolla meal (25.81% crude protein). Carp fed a control diet had significantly (p < 0.05) better growth response and nutrient utilization. Growth depression of fish increased with increasing dietary levels of Azolla meal. Diet D5 containing 100% replacement of Azolla meal gave the poorest feed conversion ratio as it contain higher amount of trypsin inhibitor. There was marked reduction in cost the diets incorporated with Azolla meal. With the gradual inclusion of Azolla meal in the diets, the pancreatic enzyme activity was increased significantly up to 50% replacement and then decreasing trend was noticed during 60 days feeding trial. Growth rate of Labeo rohita and RNA/DNA ratio were reduced by insufficiently heated meals but properly heated Azolla meal improve nutritional quality as well as improvement of growth rate. Dry processing methods other than sun drying are suggested for the improvement of nutritional quality of Azolla leaf meal in carp diets. It was concluded that the antinutritional or limiting factor present in Azolla leaf meal are antitryptic and reduces the growth rate, if do not take proper care for the nutritional security. Keywords: Non-conventional fish feed, Azolla pinnata, Labeo rohita, antinutritional factor, pancreatic protease, RNA/DNA ratio.

INTRODUCTION The increasing cost and scarcity of feed ingredients has created an urgent demand for cheaper and more abundant substitutes. There is an increasing research effort taken to assess the nutritive value of different non-conventional resource including terrestrial and aquatic macrophytes (Edwards et al., 1985; Wee and Wang, 1987; Patra, 2000). Because of the high cost of fish meal in carp diets, most studies of their nutrition have been concerned with substitution of this component with lower cost protein sources and by-product materials (Hanley, 1991). Most of the studies have included materials of plant origin such as soybean, groundnut, rapeseed, sunflower, cottonseed, alfalfa, Leucaena leaves and other macrophytes (Olli and Krogdahl, 1994; Jackson et al., 1982; Davies et al., 1990; Ray and Das, 1992; Mirnova, 1975; Olvera-Novoa et al., 1990; Sadiku and Jauncey, 1995; Hasan et al., 1997; Patra and Ray, 1988; Patra, 2000; Patra et al., 2002, Patra, 2003) for aquaculture practices. Most of the feed ingredients used in animal feed manufacture are believed have some potential for

inclusion as ingredients for aquaculture feeds. The International Network of Feed formulation has described over 18,000 feed ingredients (Harris, 1980). Martyshev (1983) also gave several combinations of feedstuffs used in Northern Asia for feeding carps. There are several reports on the use of various supplementary feeds for culture of carps in ponds (Lakshmanan et al., 1971; Chakraborty et al., 1973; Patra et al., 1999, 2001; Hajra, 1987; Hajra and Tripathi, 1985). Recently, formulated and pelleted diets have been used in the experimental culture of carp (Das et al., 1994; Sehgal and Sharma, 1991). Edwards et al. (1985) tried culture of Tilapia sp. in outdoor tank by using compost and dried water hyacinth in pelleted forms. Feed based on Salvinia (Murty and Devraj, 1991) and other aquatic weeds (Chiayvareesajja et al., 1989; Patra et al., 1999; Patra, 2001) have been used as feed in fish culture. Studies on green plant leaves as dietary sources for fish have focused on the use of leaf protein concentrates such as rye grass and alfalfa leaf protein concentrate (Ogino et al., 1978; Olvera-Novoa et al., 1990). From a nutritional perspective such protein concentrates have proven beneficial for only a limited number of cultured fish species. However, their potential usefulness may still depend upon the cost of *Corresponding author email: jmaity2003@yahoo.co.in

Canadian Journal of Pure and Applied Sciences 324

their extraction and preparation (Olvera-Novoa et al., 1990). To identify economical and locally available feed stuffs, this study was designed to evaluate the use of Azolla leaves in formulated diets for carp. Water fern (Azolla pinnata) is a free-floating, tiny aquatic plant which grows as a weed, thereby covering the surface of ponds, especially during the rainy season. It represents a “free food” source, being rapidly propagating (Ayinla and Adindu, 1992). Azolla has attracted attention as a nitrogenous fertilizer (Anon, 1985; Peters et al., 1982) and as a source of dietary nitrogen for herbivorous fish and livestock (Singh and Subudhi, 1978; Edwards, 1980). Its popularity has stirred a rush among fish farmers for its propagation as livestock-fish-integration culture system (Gavina, 1994; Rav and Shanmugasundaram, 1992). The Indian major carp, Labeo rohita is endemic in Indian subcontinent. The fish enjoys high consumer preference in West Bengal as well as in other Asian countries and its price increases over the years. This is due to the growth and survival which is not so far been satisfactory, one reason possibly is due to the non-availability of suitable artificial feed at a cheaper rate. In the present study, therefore, aimed to evaluate the nutritive value and nutritional efficiency, feed utilization, growth performance, protease enzyme activity and RNA/DNA ratios of Labeo rohita fingerlings fed diets containing varying levels of dehydrated Azolla leaf meal and identify the limitations to their use arising from antinutritional factors. MATERIALS AND METHODS Collection and Processing of Azolla Fresh colonies of Azolla pinnata were harvested from the water bodies of Midnapore district, India and thoroughly washed to remove dirt and mud debris. They were then dried in a hot-air oven at 30-32oC for 72h. The dried Azolla was milled and packed in a polythene bag and kept in a freezer at –2oC prior to use. The proximate composition of both fresh and dried Azolla were performed following AOAC’s (Association of Official Agricultural Chemist) (1990) standard analysis procedure (Table 1). Table 1. Proximate composition of dried and fresh Azolla pinnata.

Constituents Sundried Azolla (% content)

Fresh Azolla (% content)

Moisture 13.12 83.17 Crude protein 25.92 3.19 Crude lipid 4.79 2.04

Formulated Experimental diets Five isonitrogenous (≅ 38% crude protein) diets (D1-D5) were formulated, containing increased levels of dried Azolla meal as replacement of fish meal at 0% (control), 25%, 50%, 75% and 100% (Table 2) and another four isonitrogenous (CP = 38%) diets (D6 – D9) processed with heat treatment (90oC for 10 minutes) using the same ingredients for experimental treatments. All dietary ingredients were hand mixed and produced pellets in pelletilizer to form noodle like strands which were mechanically broken into pellets of suitable size (2 mm diameter) for Labeo rohita fingerlings. The dry pieces of feed were then stored in a freezer at –20oC in sealed plastic bags until fed. Experimental Design The feeding trial was conducted in specially designed glass aquaria of 130 liter capacity in the Aquaculture Research Unit, Dept. of Zoology, Vidyasagar University, Midnapore, India. In order to avoid metabolic accumulation, the aquaria’s water was changed daily. Each aquaria was also supplied with air by aerator. Water temperature, dissolve oxygen, alkalinity and pH were monitored everyday alternative day. The photoperiod was set on 12 hour light and dark cycle using fluorescent lamp at the light source. Labeo rohita fingerlings (mean weight = 10.82 + 0.60g) were obtained from rearing pond of Aquaculture Research Unit, Vidyasagar University Campus and acclimatize for two weeks in the laboratory condition with standard diet (30.0% crude protein) containing the mixture of fish meal, mustard oil cake, rice bran. The fingerlings were randomly distributed between the aquaria at a stocking density of 10 fish/aquaria. Fish were fed once daily to satiation (6% body weight) at 15.00 to 16.00 hour daily feeding allowance was adjusted each week on the basis of the average weight of the fish. The study was conducted for 60 days. Estimation of Protease activity Protease activity was estimated following the method of Bernfeld (1955) and which was modified by Snell and Snell (1971). 500mg hepatopancreas tissue was taken in a glass homogenizer and the tissue was homogenized in cold temperature (4 - 6°C) with phosphate buffer (0.01M, pH 7) and then centrifuged the homogenate at low speed (13,000 rpm) followed by high speed (20,000 rpm) in 4 - 6°C, continued for 10 minutes. The supernatant was used as enzyme source. 2.4ml phosphate buffer, 1.2ml enzyme extract, 1.8ml distilled water and 0.6ml 0.1% BSA were taken in a test tube and incubate it for 1 hour at 37°C. Then added 6.0ml 10% TCA, shaken thoroughly and centrifuged it at 5000 rpm for 10 minutes in cold condition. 1.0ml supernatant and 1.0 ml ninhydrin were taken in separate test tube and placed it in boiling water bath for 15 minutes. After cooling 8 ml distilled water

Maity and Patra 325

was taken in the same test tube and read the OD in UV Spectrophotometer (Hitachi, Japan) at 570nm. The OD compared with standard curve of Glycine for calculation of protease activity. Protein was estimated following the methods of Lowry et al. (1951) using BSA (Bovine Serum Albumin) as a standard. Estimation of DNA and RNA The DNA and RNA content of fish muscle were estimated according to the method of Munro and Fleck (1969) with some modifications. 200 mg liver (Hepatopancreas) tissue was taken from L. rohita and homogenised with 0.25M sucrose solution. 250 µl homogenate and 500 µl 5% TCA (Tri-chloro aceticacid) mixed thoroughly, centrifuge and wait for 15 minutes and then supernatant was discarded. The precipitate was dissolved in 500 µl of 0.6N PCA (Per-chloric acid) and 0.3N PCA each and wait for 15 minutes and then centrifuged for 5 minutes at 5000 rpm at 5°C. The supernatant was discarded and precipitate was again dissolved in 2 ml cold 0.3N PCA and wait for 10 minutes and then centrifuged at 5000 rpm for 5 minutes at 5°C. Now, precipitate was dissolved in 0.3M KOH, and incubated in water bath at 37°C for 2 hours with occasional shaking. Cooled at room temperature. With this solution 1.0ml cold 0.6M PCA and 250 µl cold 0.6N PCA were mixed and wait for 10 minutes. The supernatant was used for RNA estimation (read at 290 nm in UV spectro, Hitachi). The precipitate was dissolved in 1.5ml cold 0.6N PCA and heated at 70°C for 40 – 60 minutes in water bath and cooled at room temperature and then kept at 4°C for 10 minute Then with it 400µl of 0.6N PCA was added and centrifuged for 10 minutes at 5000 rpm in cold condition. The supernatant was read at 270 nm in UV Spectro for DNA estimation. For RNA the blank was prepared by 0.3N PCA and for DNA it was with 0.6N PCA. The calculation was done by the method of Strove and Makaravo, 1989. Protease inhibitor activity assay Trypsin inhibitor activity was measured by the methods of Smith et al. (1980) and this is the modified method of Kakade et al. (1974) using BAPNA (Benzoyl-DL-Arginine-Paranitroanilide) as substrate. A solution containing 100 µl of inhibitor solution, 200 µl (20 µg ml-1) bovine pancreatic trypsin and 100 µl of distilled water was pre-incubated at 370C for 20 minutes. Then, 500 µl (0.4 mg ml-1) of BAPNA (pre-warmed at 37oC) was added and vortex immediately to start the reaction. After incubation for 20 minutes, 100 µl of 30% glacial acetic acid (v/v) was added to terminate the reaction. The reaction mixture was centrifuged at 8000 rpm for 5 minutes. Residual activity of trypsin was measured the absorbance at 410 nm in UV-spectro photometer (Hitachi, Japan) and calculated the protease or trypsin inhibitor activity according to the methods of Hamerstrand et al., 1981.

Analytical Procedure The chemical analysis of dietary ingredients, diets were performed according to the procedures of the AOAC (1990). Diet performance was evaluated on experimental fish according to Olvera-Novoa et al. (1990) as follows:

100 (%)gain Weight =

⎥⎦

⎤⎢⎣

⎡tbody weigh Initial

tbody weigh Initial -t body weigh Final

100 )day (SGR% RateGrowth Specific -1 =

⎥⎦

⎤⎢⎣

⎡in trial days

tbody weigh initial log -t body weigh final (log ee

= (FCR) Ratio Conversion Feed

⎥⎦

⎤⎢⎣

⎡(g)gain ht Fish weig

(g) fed feed of Dry weight

= (PER) Ratio EfficiencyProtein

⎥⎦

⎤⎢⎣

⎡basis) dry weight (g, intakeProtein

(g)gain ht Fish weig

Water Quality Analysis The water quality was monitored periodically (every alternative day) following the methods of APHA (1989) (American Public Health Association). The water temperature during the experimental period determined by thermometer ranging from 29-30oC. The pH (pH meter, Systronics 302), dissolve oxygen (Winkler’s method) content and alkalinity (titrimetric method) of the water ranged between 6.65-7.19, 9.12-9.59 mg liter-1 and 102.27 – 105.75 mg liter-1 respectively. Statistical analysis All calculations and statistical analysis were done on IBM P-III using statistical packages STATISTICA and ASP. The level of significance was chosen at p < 0.05, results are means of three separate determinations and presented as means ± SEM (Standard Error of Mean). Analysis of variance was performed and mean comparisons were accomplished using Duncan’s multiple range test. RESULTS Fish in all the experimental units become accustomed to the general diets within the first week of acclimation. Low mortality (<5%) occurred during this period which may be due to initial stress and they were subsequently replaced with fish of similar size prior to the feeding trial.

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However, fish fed on 100% Azolla meal (D5) dietary inclusion has lowest percentage of survivality (Table 3). There was a decreasing rate of survival with an increasing inclusion of Azolla meal in the diets during the growth trial. Mortality ranged from 0% in D1 diet and 65% in D5 diet. The summary of growth responses (Table 3 and Fig. 1) showed that Labeo rohita fed the control diet (D1) had significantly (P<0.05) better weight gain, specific growth rate as compared to fish fed diets (D2 - D5) supplemented at various levels with dried Azolla meal. The lowest results were observed for fish fed 100% Azolla dietary inclusion. The control diet gave the best FCR (2.08) but diets containing Azolla meal at the 100% level (D5) showed the poorest FCR (7.58) and PER value for control diets (D1) have best performance (1.18) but poorest D5 diets (0.33) (Table 3).

10121416182022242628303234

0 15 30 45 60

Days

Wei

ght g

ain

(g)

D1 D2 D3 D4 D5

Fig. 1. Growth study of L. rohita fingerlings of different experimental diets (D1 – D5) during 60 days feeding trial. The response of fingerling Labeo rohita fed the different diets (D2 - D5) containing Azolla meal with varying trypsin inhibitor activity is presented in Table 3. Fish fed the diets containing the unheated Azolla meal showed poor growth performance and had a low PER value. Both growth rates and PER values increases in fish fed diets containing less trypsin inhibitor activity. No significant difference ( p < 0.05) in growth rate or PER was observed in fish fed diets (D2) containing 11.8 mg TI g-1 diet or less. The best growth and PER were observed in fish fed diet (D6) containing 1.8 mg TI g-1 diet prepared with Azolla meal in which 90% of the trypsin inhibitor activity has been destroyed (Table 4). Azolla meal heated with

90oC for 10 minutes reduces the trypsin inhibitor activity in the diets D6 - D9 (Table 4). A comparative growth study was presented in Fig. 2 during 60 days feeding trial, containing control (D1), unheated Azolla meal (D2 – D5) and heated Azolla meal (D6 – D9).

0

20

40

60

80

100

120

D1 D2 D6 D3 D7 D4 D8 D5 D9

Experimental diets

Wei

ght g

ain

(%)

Fig. 2. Comparative study of growth of L rohita in control (D1), unheated (D2-D5) and heat treated (D6-D9) experimental diets during the experimental period. The protease activity in hepatopancreas was also recorded during 60 days growth study and presented in Table 5. It was observed that protease activity (µg glycine liberated h-1 mg-1 tissue protein) in the hepatopancreas gradually increased as compared to control with the inclusion of different level of Azolla meal with different T1 activity in the diets (D2 - D5) but later decreasing trends was followed with the higher levels of T1 inclusion. During the 60 days feeding trial protease activity in control (D1) was 92.75 µg glycine liberated h-1 mg-1 tissue protein and gradually increased in D2, D3 (100.34, 102.94 µg glycine liberated h-1 mg-1 tissue protein respectively but decreased in diets D4, D5 (99.32, 94.62 µg glycine liberated h-1 mg-1 tissue protein respectively). It was also noted that during 60 days feeding trial protease activity increased upto 15 days fed the diet (D5) and later decreased during 30, 45 and 60 days fed. Muscle RNA/DNA ratio estimated on 15th, 30th, 45th and 60th day was presented in the Table 6. RNA/DNA ratio gradually decreased with the inclusion of high percentage of unheated Azolla meal containing high level of T1 as compared to control. RNA/DNA ratio in control (D1) was 2.89 but decreased fed diets D2, D3, D4, D5 (1.91, 1.39, 1.34, 1.27 respectively) containing high inclusion of Azolla meal during 60 days feeding trial. This may also be explained and concomittented with the growth and conversion of fingerlings of L. rohita during 60 days feeding trial.

Maity and Patra 327

DISCUSSION The performance of the fish fed the diets supplemented with different levels of dehydrated Azolla meal was inferior (P<0.05) to that of the fish fed the control diet. The growth depression of fish tends to be more pronounced with increasing dietary level of Azolla meal. This study reveals the inadequacy of total replacment fishmeal with dried Azolla meal in diet L. rohita fingerlings. Almazan et al. (1986) reported similar growth depression and worsening FCR value with increasing dried Azolla meal incorporated in the diet for Nile tilapia (Oreochromis niloticus). Yousif et al. (1994) also supported similar growth depression and decreasing PER value with increasing level dehydrated alfalfa and salt bush (Atriplex sp.) leaves in the diets for tilapia

(Oreochromis aureus). The poor growth responses and feed utilization values of the fish at increasing inclusion levels of dried Azolla meal in the diets indicates its nutrient inferiority to fish meal. A possible reason for this may be due to relatively high level of antinutritional factor specially the trypsin inhibitor in 100% Azolla dietary inclusion (Yousif et al., 1994; Ensminger et al., 1990; Olvera Novea et al., 1990). High inclusion level of Azolla meal in the diets has been resumed results poor digestibility and lowered availability of energy content of plant feeds as well as reduction in fish growth responses (Buddington, 1979; Appler and Jauncey, 1983; Patra et al., 1999; Birk, 1989; Maity, 2003). The growth depression of fish fed diets containing Azolla meal incorporation could also be attributed to the

Table 2. Ingredients and proximate composition of experimental diets.

Diets (% fishmeal replacement) Ingredients D1 (0%,

control) D2 (25%) D3 (50%) D4 (75%) D5 (100%)

Fish meal 34.86 26.07 22.14 17 - Azolla powder - 6.36 13.14 22.07 39.50 α-cellulose 5.00 9.68 9.68 9.62 10.28 Wheat flour 38.14 35.89 33.04 29.31 28.22 Mustard oil cake 12.50 12.50 12.50 12.50 12.50 Rice bran 5.00 5.00 5.00 5.00 5.00 Cod liver oil 1.50 1.50 1.50 1.50 1.50 Vitamin + Mineral 1.00 1.00 1.00 1.00 1.00 Binder (gelatin) 2.00 2.00 2.00 2.00 2.00 Proximate composition (% Dry matter basis) Moisture 8.09 8.30 8.20 8.45 8.35 Crude protein 38.82 37.95 39.21 37.75 37.97 Crude lipid 6.43 5.50 5.98 6.02 5.71

Table 3. Cumulative growth performance and feed utilization of fish feed the experimental diets, during 60 days feeding trial.

Experimental Diets Parameters D1 (0%,

control) D2 D3 D4 D5

mg T1 g-1 diet 0 11.8 24.4 38.9 48.4 Average initial weight (g) 14.86 + 0.94 14.78 + 0.92 14.80 + 0.62 14.83 + 0.91 14.80 + 0.84 Average final weight (g) 31.38 + 0.38a 29.65 + 0.99a 25.67 + 0.54b 24.88 + 0.75b 18.18 + 0.69c

Weight gain (%) 111.23a 100.84b 74.01c 67.94d 22.83e

SGR (% day-1) 0.89a 0.83a 0.66b 0.62b 0.24c

Feed intake (g day-1) 0.41a 0.39a 0.37a 0.29b 0.28b

FCR 2.08a 2.20a 2.85a 2.44a 7.58b

PER 1.18a 1.10a 0.87a 1.00a 0.33b

Survival (%) 100a 95a 80b 75b 65c

Results are means of three separate determinations (Mean ± SEM), Values with the same superscript in the same row are not significantly different (p < 0.05) from each other.

Canadian Journal of Pure and Applied Sciences 328

presence of possible growth inhibitors such as trypsin inhibitor, which are reported to occur in vegetative tissues of several aquatic plants (Gleen et al., 1882; Yousif et al., 1994; Fasakin and Balogun, 1998; Soto and Mitchell, 1960; Humphries, 1980; Liener and Kakade, 1980). Trypsin inhibitor impairs the digestion and absorption of protein (Jauncey and Ross, 1982; Maity and Patra, 2003). It is possible from this study that antinutritional factor i.e. trypsin inhibitor might have impaired the absorption of essential components of the diets containing Azolla meal and causing depression of growth responses in L. rohita fingerlings. The possible effect of these inhibitory factors

was more pronounced in 100% Azolla dietary substitution for fishmeal. The present observation demonstrates that high dietary level of trypsin inhibitors may cause negative effect on L. rohita either in growth or conversion. In a similar study with rainbow trout (O. mykiss) in fresh water, Krogdahl et al. (1994) found protein digestibility significantly reduced by increasing dietary inclusion of the same inhibitor. Soybean trypsin inhibitors have been reported to be important when using untreated Soybean products in diets for the carp (Viola et al., 1983), Channel Catfish (Wilson

Table 4. Growth performance of L. rohita fed with heated diets (D6 - D9) Azolla meals in the experimental period.

Experimental diets D1 D6 D7 D8 D9 mg T1 g-1 diet 0 1.8 8.4 16.5 24.4 %T1 destroyed 100 90 70 60 52 Initial body weight (g) 14.86 ± 0.94 14.80 ± 0.75 14.84 ±0.82 14.42 ± 0.42 14.49 ± 0.39 Final body weight (g) 31.38 ± 0.38a 30.80 ± 0.31a 27.65 ± 0.44b 25.60 ± 0.16b 20.20 ± 0.69c

Weight gain (%) 111.23a 108.10a 86.32b 77.53c 39.40d

SGR (% day-1) 0.89a 0.79b 0.56c 0.48d 0.41e

FCR 2.08a 2.24a 3.04b 4.19c 4.24c

PER 1.18a 1.08a 0.81b 0.57c 0.58c

Results are means of three separate determinations (Mean ± SEM), Values with the same superscript in the same row are not significantly different (p < 0.05) from each other. D1 = Control : without Azolla meal. D6 - D9 Heat treated (90oC for 10 minutes) Azolla meal.

Table 5. Protease activity (µg glycine liberated hour-1 mg-1 of protein) in the hepatopancras of L. rohita fingerlings fed with different levels of Azolla meal containing different level of TI during 60 days feeding trial.

Feeding days Experimental diets 0 15 30 45 60

D1 Control 92.51 ± 1.52a 93.32 ± 0.79 92.15 ± 0.94 93.17 ± 0.39 92.75 ± 0.27a

D2 93.01 ± 0.15a 98.52 ± 0.57 101.32 ± 0.61 102.74 ± 0.50 100.34 ± 0.91b

D3 92.98 ± 0.72a 103.02 ± 0.94 109.17 ± 0.29 108.10 ± 0.97 102.94 ± 0.20b

D4 92.21 ± 0.94a 108.36 ± 0.71 109.27 ± 0.54 104.27 ± 0.72 99.32 ± 0.78b

D5 93.27 ± 0.19a 114.79 ± 0.57 110.38 ± 1.02 103.50 ± 0.74 94.62 ± 0.59c

Results are means of three separate determinations (Mean ± SEM), Values with the same superscript in the same column are not significantly different (p < 0.05) from each other. Table 6. Muscle RNA/DNA ratio in L. rohita fingerlings fed with Azolla meal containing different level of T1 during the experimental period.

Feeding days Experimental diets 0 15 30 45 60 D1 Control 2.02 ± 0.12a 2.44 ± 0.09 2.69 ± 0.29 2.77 ± 0.51 2.89 ± 0.42a

D2 2.15 ± 0.43a 2.05 ± 0.29 2.02 ± 0.09 1.97 ± 0.12 1.91 ± 0.42b

D3 1.99 ± 0.99a 1.74 ± 0.31 1.68 ± 0.29 1.52 ± 0.14 1.39 ± 0.14c

D4 1.95 ± 0.37a 1.72 ± 0.18 1.62 ± 0.11 1.42 ± 0.19 1.34 ± 0.32c

D5 2.10 ± 0.41a 1.64 ± 0.14 1.58 ± 0.50 1.40 ± 0.09 1.27 ± 0.13d

Results are means of three separate determinations (Mean ± SEM), Values with the same superscript in the same column are not significantly different (p < 0.05) from each other.

Maity and Patra 329

and Poe, 1985), rainbow trout (Olli and Krogdahl, 1994), Atlantic Salmon (Olli et al., 1994) or in mammals (Krogdahl and Holm, 1983; Kakade et al., 1973). Olvera-Novoa et al. (1990) evaluated the use of purified alfalfa leaf protein concentrate (LPC) in diets for O. mossambicus. They reported reduced performance at higher inclusion levels of LPC (>45%). To explain this reduction in growth, they suggested that at high dietary inclusion levels, and apparent digestibility was adversely affected by the presence of high trypsin inhibitory activity in alfalfa LPC. It appears that processing of Azolla meal heating in 90oC for 10 minutes to enrich the nutritive quality of the feedstuff as a replace of fishmeal in Labeo rohita diet. Growth rates and PER values improved as the trypsin inhibitor activity of the Azolla meal decreased to tolerable levels. Viola et al. (1982, 1983), Maity and Patra (2002) and Sadiku and Jauncey (1998) reported that the growth rates of carp were reduced when fed diets containing insufficiently heated Soybean meal. They observed equal growth rates in carp fed diets containing properly heated and slightly over heated (100% destruction of trypsin inhibitors) Soybean meal. The properly heated and slightly overheated Soybean meals used has 90-100% of the trypsin inhibitor activity destroyed. During this investigation, the protease activity gradually increased as compared to fish receiving the control diet and later decreased with the higher inclusion of Azolla meal containing high level of T1. Similar observations were recorded by Olli et al. (1994), Vidal Valverde et al. (1997) in the Atlantic Salmon. They suggested that the synthesis of trypsin increases in total trypsin in the pyloric caeca homogenate seemed to be peak and then fall off, suggesting an exhaustion of the pancreatic storage of enzyme and synthesizing capacity. Diets with raw Soybean have been reported to increase pancreatic enzyme secretion in the rat (Crass et al., 1987; Green and Lyman, 1972), the pig (Corring et al., 1985; Zebrowska et al., 1985) and Man (Calam et al., 1987; Holm et al., 1991, 1992). Again a negative impact has been observed with the incorporation of Azolla meal in the diet containing high level of TI on the feed intake, appetite, RNA/DNA level in the muscle. There are several studies in which tissue RNA concentration and RNA/DNA ratios have been used as indicators of recent growth or nutritional status in fish (Bulow et al., 1978; Mitra and Mukhopadhyay, 2002, 2003). The usual interpretation is that RNA/DNA ratios are sensitive to nutritional status of fish. The amount of DNA per cell is stable under changing nutritional conditions, whereas cellular RNA, which is directly involved in protein synthesis, fluctuates with nutritional status of the fish (Foster et al., 1992). Thus the RNA/DNA ratio indicates metabolic intensity which is

deeply influenced by the nutritional status of the diet (Clemmesen, 1987). Similar observations has also been reported previously by Lone and Matty (1980a, b; 1982 a, b; 1983) and Patra et al. (2002). In fish, growth can be termed as accretion of protein (Love, 1970, 1980) and it depends on food availability and state of starvation has been shown to effect on the nucleic acid levels in different body tissue (Bulow, 1970, 1971; Bulow et al., 1978, 1981; Haines, 1973; Bouche, et al., 1977). Furthermore, the quality of food has profound effects on cellular growth response in different fishes. So, the present studies definitely establish a direct relationship between the growth and RNA/DNA level. CONCLUSION The present investigation indicated that heat treated Azolla meal was partially substituted for fish meal resulted a significant growth of L. rohita. It may therefore, be incorporated as a non-conventional sources of protein in the diet of IMC on one hand and reduces the cost of feed on the other and gave the nutritional security to improve better aquaculture practices. ACKNOWLEDGEMENTS This study was financially supported by All India Council for Technical Education (AICTE), New Delhi, India. The authors are deeply grateful to the members of Aquaculture Research Unit, Department of Zoology, Vidyasagar University, Midnapore, West Bengal, India for their untiring help. REFERENCES Almazan, GJ., Pullin, RSV., Angeles, AF., Nanalo, TA., Agbayani, RR. and Trono, MTB. 1986. Assessment of Azolla pinnata as dietary component for Nile Tilapia (O. niloticus). In: Median, JL., Dizon, LB., Hosillos, LV (eds.). First Asian Fisheries Forum, Asian Fisheries Society, Manila, Philippines. 523-528.

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SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 335-342, 2008 ISSN: 1715-9997

1

DWARF PEACH TREES BY USING ABSCISIC ACID HORMONE, MALEIC HYDRAZIDE AND CYCOCEL AS GROWTH INHIBITORS IN PHLOEMIC

STRESS CONDITION GRAFTED ON VIGOROUS ROOTSTOCK

*ABM Sharif Hossain and Fusao Mizutani Department of Bioresource production Science, Faculty of Agriculture, Ehime University

Hattanji, Matsuyama 799-2424, Japan

ABSTRACT

The study was undertaken to evaluate the dwarfing effects of phloemic stress (represented by bark ringing) and growth inhibitors [abscisic acid hormone (ABA), Maleic hydrazide (MH) and cycocel (CCC)] applied to a connecting bark strip of partially ringed trunk by using two-year-old peach trees. A 2 cm length of bark was removed from the trunk leaving a 2 mm width connecting bark band to which the aqueous chemical solutions (ABA, MH and CCC) were applied. Positive correlations were found between the bark regeneration and tree growth. Higher concentrations of ABA, MH and CCC retarded both bark regeneration and shoot growth. ABA and MH showed the greater effect than CCC. ABA 2000 showed the greater effects than ABA 1000 ppm which completely inhibited the bark regeneration even resulting in tree death like complete ringing. Root weight was also reduced in accordance with the decline in bark regeneration. Thus these results indicate that the tree growth can be controlled by chemically modifying bark regeneration of partially ringed trees in which a 2 mm connecting strip (97% ringing) was left. The use of chemicals will be greatly reduced compared with the whole tree spray for tree dwarfing. Keywords: Dwarfing, ringing, growth inhibitor, shoot growth.

INTRODUCTION Small, compact, dwarfed or size controlled fruit trees provide easier pruning, thinning, spraying and harvesting, high production of high-grade fruit and lower cost of production (Tukey, 1964). The primary factor limiting the use of size controlling rootstocks in stone fruit production is the lack of suitable rootstocks with a wide range of compatibility among cultivars (De Jong et al., 2001). Dwarfing rootstocks or genetically dwarfed cultivars that produce high quality fruit are not yet available for peaches as they are for apple (Erez, 1984). Dwarfing techniques other than utilizing dwarfing rootstocks are to be developed. The effect of bark ringing (cut once) was not long lasting because new bark phloem was regenerated after few years that permit normal phloem transport downward (Tukey, 1964; Hossain et al., 2005). Hossain et al. (2006) observed that partial ringing (cut once) without using growth inhibitors retained the tree dwarfing until four years in three-year-old peach trees. Johnson (1998) reported that complete ringing was almost fatal, even though plant might die within months to 2-3 years (Hossain et al., 2005 and 2006). Arakawa et

al. (1997) reported that bark ringing reduced vegetative growth in apple. They also stated that flowering in apple was significantly increased by bark ringing. Sole use of growth inhibitors as spraying is costly, laborous and much more polluted the environment than use to the bark band (bridge) of trunk by swabbing cotton. That is why partial ringing combined with growth inhibitors can be more effective than sole use of either partial ringing or growth inhibitors. ABA has been known as a natural growth inhibitor. It was reported that lignin content increased in one-year-old peach shoot sprayed with ABA and CCC resulting dwarf the trees (Khamis and Holubowicz, 1978). They also reported that foliar application of cycocel (CCC) at 1000 ppm led to growth inhibition. Ito et al. (2000) observed that foliar application of MH increased the number of laterally born flower buds on Japanese pear shoots. The present research was attempted to innovate a new technique to use growth inhibitors on bark band by avoiding the spray of whole tree. Therefore, we determined the influences of different concentrations of ABA, MH and CCC on tree growth and the relationship between bark thickness (regeneration) and shoot growth as well as root growth.

[A part of this manuscript was presented in the Spring meeting of Japanese Society for Horticultural Sciences, 2003 and 2004]*Present address: Laboratory of Plant Physiology and Biotechnology, Institute of Biological Sciences, Faculty ofScience, University of Malaya, 50603 Kuala Lumpur, Malaysia *Corresponding author email: sharif@um.edu.my

Canadian Journal of Pure and Applied Sciences 336

MATERIALS AND METHODS Experiment 1 Site: The experiment was carried out in an orchard in the Ehime University Farm located in southern Japan. Plant materials: Two-year-old peach (Prunus persica Batsch cv. ‘Hikawahakuho’) trees grafted on wild peach seedling rootstocks were used in this experiment starting from April 2002 to December 2002. The trees were planted in mid April 2002. The trees were spaced at 0.60 m x 1.0 m. Intercultural operations Weeding and irrigation were done at 7 days intervals and insecticides were applied when necessary. Fertilizers were applied in mid May at the rate of N, P and K (10%, 10% and 10%) 10 g per tree respectively. Five shoots were maintained per tree to ensure proper growth. Treatment setting The treatments were control (unringed), partially ringed (PR) + water, PR + ABA 500 ppm, PR + 1000 ppm, PR + MH 1000 ppm, PR + MH 2500 ppm, PR + CCC 500 ppm and PR + CCC 1000 ppm. A partial ring was made by using a knife (thin razor blade type) on 6 June 2002. The partial ringing was consisted of removing a 2 cm length (vertically) bark (from trunk) leaving a 2 mm width (horizontal thickness) connecting bark band (strip) in the trunk, 10 cm above from the ground (Fig. 1). The aqueous solutions (ABA, MH and CCC) were applied to the bark strip (2 cm length and 2 mm width) swabbing 2-3 times by using cotton with the point of forceps (Fig. 1). They were applied at 15 days intervals and were continued until 3 months from 6 June 2002-15 August 2002). Data collection Shoot and regenerated bark growth were measured at 7 days intervals from 6 June 2002 – 22 August 2002. Regenerated bark growth was measured horizontally with vernier caliper. Total shoot length were measured in late November 2002 after tree growth was stopped completely. The percentage of flower bud and tree circumference were measured in late December 2002. Experiment Design The experimental design was completely randomized design. There were 5 replications and 8 treatments (including control) used in the experiment. A total of 40 trees used in the experiment. Mean seperation was done by Duncan`s multiple range test (DMRT). Standard errors were calculated for some data. Experiment 2 Site: The experiment was carried out in the same location as mentioned in experiment 1.

Plant materials Two-year-old peach (Prunus persica Batsch cv. ‘Hikawahakuho’) trees grafted on wild peach seedling rootstocks were used in this experiment starting from April 2003 to February 2004. The trees were planted in mid April 2003. The trees were spaced at 0.60 m x 1.0 m. Intercultural operations Weeding was done at 15 days intervals. Irrigation and insecticides were applied as needed. Fertilizers were applied in mid May at the rate of N, P and K (10%, 10% and 10%) 10 g per tree respectively. Heading back was done for all trees after one month of transplantation (in mid May). Five shoots were maintained per tree to ensure proper growth. Treatment setting The treatments were control (unringed), patially ringed (PR) + water, PR + ABA 1000ppm and PR + 2000ppm. A partial ringing was made by using a knife (thin razor blade type) in mid May 2003. The partial ringing was consisted of removing a 2 cm length (vertically) bark (from trunk) leaving a 2 mm width (horizontal thickness) connecting bark band (strip) in the trunk, 10 cm above from the ground. The aqueous solutions (ABA) were applied to the bark band (2 cm length and 2 mm width) swabbing 2-3 times by using cotton with the point of forceps. Growth inhibitors were applied at weekly intervals from the week of treatment setting and continued until 6 weeks (June 19 2003). Data collection Per Shoot growth and final regenerated bark growth were measured on 28 August 2003 after 3.5 months (15 weeks) of treatment setting. Regenerated bark growth was measured horizontally with vernier caliper. Total shoot length were measured in late December 2003 after tree growth was stopped completely. Percent flower bud and trunk circumference were measured in late December 2003. Flower bud was measured per 10 cm of shoot then it was converted into percent. Finally trees were uprooted and the root weights were determined in late February 2004. Experiment Design The experimental design was completely randomized design. There were 4 replications and a total of 16 trees used in the experiment. Mean seperations were done by Duncan`s multiple range test (DMRT). Standard errors were calculated in case of some data. Measurement of leaf chlorophyll in vivo: The chlorophyll meter SPAD-502 (Minolta Co. Japan) was used for determination of chlorophyll in leaves. SPAD value was measured in late July 2003. Three leaves were selected from the middle part of shoot and a total of 15 leaves per tree were measured.

Hossain et al. 337

RESULTS AND DISCUSSION Experiment 1 Figure 1 shows the ringing structure and position where aqueous solutions of the chemicals (growth inhibitors) were used. Shoot growth was lower in partial ringing (PR) + growth inhibitors treated trees than control (unringed) and PR + water treated trees (Fig. 2). A similar trend in shoot growth was observed in case of all treatments from 1-12 weeks. Similar trend of bark growth was found to shoot growth (Fig. 3). Total shoot length was lower in higher concentration of growth inhibitors (ABA 1000ppm, MH 2500ppm, CCC 1000ppm) treated trees than lower concentration (ABA 500ppm, MH 1000ppm, CCC 500ppm) (Table 1). The greater bark thickness (width) was observed in PR + water treated trees than PR + growth inhibitors treated trees (Table 1). The higher inhibition of trees was found in lower concentration and the lower inhibition of trees was found in higher concentration of growth inhibitors. The percentage of flower bud was recorded higher in PR + growth inhibitors treated trees than control (unringed) and PR + water treated trees. Trunk circumference was higher above ring of PR + growth inhibitor treated trees than PR

+ water treated trees and was lower below ring in PR + growth inhibitor treated trees than PR + water treated. There was a little difference in case of trunk circumference among treatments (Table 1). Figure 4 shows relationship between shoot growth and regenerated bark thickness. A positive correlation was found between shoot growth and bark thickness. When bark thickness was higher then shoot growth was also higher. On the contrary, when bark thickness was lower then shoot growth was also lower. Plant architectures have been shown in Figure 5 as affected by PR and different concentrations of growth inhibitors. It was shown that shoot growth was influenced by bark thickness (width/regeneration) which affected by partial ringing and growth inhibitors. Experiment 2 Shoot growth was lowest in partial ringing (PR) + ABA 2000ppm treated trees and was highest in un-ringed (control) trees (Table 2). Among all PR and ABA treatments, shoot length was lower in PR + ABA 2000 and ABA 1000 ppm treated trees than PR + water treated trees (Table 2). The highest final bark thickness was observed in PR + water treated trees and the lowest was in PR + ABA 1000ppm treated trees (Table 2). The bark

Table 1. Effect of growth inhibitors on shoot length, flower bud and trunk circumference of peach trees.

Trunk circumference (cm) Treatments Total shoot

length (cm) Flower bud

(%) Above ring Below ring Control (unringed) 525.4±8.8a 68.76±2.7a 6.0±0.24c 6.1±0.25a Water 397.2±5.6b 65.52±5.9a 6.7±0.25a 6.3±0.25a ABA 500ppm 290.0±5.3c 61.05±2.2a 6.1±0.26c 5.8±0.26b ABA 1000ppm 245.2±8.0c 39.12±1.8b 6.2±0.25bc 5.7±0.25b MH 1000ppm 261.1±6.2c 47.40±3.2b 6.1±0.28c 5.7±0.28b MH 2500ppm 254.2±3.7c 44.84±1.4b 6.2±0.23bc 5.6±0.23b CCC 500ppm 300.6±4.8c 53.44±5.1ab 6.3±0.27b 6.1±0.26a CCC 1000ppm 275.5±5.4c 42.02±2.0b 6.4±0.26ab 5.9±0.26b

Means followed by the common letters in column are not significantly different at the 5%level by Duncan`s multiple range test (DMRT). Mean±SE (n = 5). In case of control trees, ring position were measured to take an idea about trunk circumference without ringing. Table 2. Peach shoot growth, flower bud and root weight as affected by different treatments of PR and ABA.

RBT TC

Initial Final Above ring

below ring Treatment PSG

(cm) TSL/ tree

(cm) (mm)

SPAD unit

Bud Flower

(%) (cm)

Root weight

(g)

Control (Unringed) Water ABA 1000ppm ABA 2000ppm

47.4a 34.2b 25.5c 18.4d

237.0a 177.0b 127.5c 92.0d

--- 2.0 2.0 2.0

--- 10.9a 5.3b 2.0c

41.8a 38.6a 27.8b 20.0c

58.0b 63.0a 64.8a

---

5.4b 5.5b 5.9a 5.2b

5.6a 5.2b 5.5ab 4.8c

111.7a 109.5a 77.4b 41.5c

Means followed by the common letters are not significantly differenence at the 5%level by Duncan`s multiple range test (DMRT). PR = Partial ringing, ABA = Abscsic acid, PSG = Per shoot growth, TSL = Total shoot length, RBT = Regenerated bark thickness, SPAD unit = From chlorophyll meter, TC = Trunk circumference.

Canadian Journal of Pure and Applied Sciences 338

growth was higher in the lower concentrations (PR + ABA 1000ppm) compared with higher concentration (PR + ABA 2000 ppm). Trunk circumference was higher above ring and lower below ring in PR + ABA 1000 ppm than PR + water treated trees (Table 2). The minimum SPAD value was found in PR + ABA 2000 ppm treated trees and was a maximum in un-ringed trees (Table 2). The percent flower bud was greater in PR + growth inhibitors treated trees than PR + water and un-ringed trees (Table 2). Root weight was lower in PR + ABA 2000ppm than PR + ABA 1000 ppm and PR + water treated trees (Table 2). It seems that partial ringing and

growth inhibitors, not only affected shoot growth but also root system. A positive correlation between bark width (thickness/regeneration) and shoot length was shown in Figure 6. The higher bark thickness the higher shoot length (Fig. 6) and root growth. The lower bark thickness the lower shoot length and root weight (Fig. 7). In Figure 8 photos show the shoot and root architecture as affected by PR and different concentrations of ABA. It was shown that shoot growth was influenced by bark thickness (width/regeneration) which affected by partial ringing and ABA.

Xylem (wood)

Growth inhibitors (ABA, CCC, MH solutions) were applied to the 2cm length x 2mm width of bark band surface.

Fig.1. Show the partial ringing structure (bark regeneration) after treatment.

Fig. 2. Effect of partial ringing and growth inhibitors on new shoot growth in peach trees. Vertical bars indicate SE. Weeks are (0: June 6, 11:August 22). Means followed by the common letters in column are not significantly different at the 5%level by Duncan`s multiple range test (DMRT).

Hossain et al. 339

The results show that growth inhibitors are effective as dwarfing components in peach trees when used together with ringing. Shoot growth was greater in control (unringed) and partial ringing + water treated trees compared with other treatments where growth inhibitors

were used. Khamis and Holubowicz (1978) stated that foliar application of cycocel (CCC) at 1000 ppm led to growth inhibition and acceleration of leaf fall. It was found that ABA 2000ppm was toxic effect. When it was used weekly, tree growth were stopped after a certain

Fig. 3. Effect of growth inhibitors on bark width in peach trees. Vertical bats indicate SE (n=5). Weeks are (0: June 6; 11: August 22). Means followed by the common letters in column are not significantly different at the 5%level by Duncan`s multiple range test (DMRT).

Fig. 4. Relationship between shoot growth and bark width (thickness) of peach trees in different concentrations of ABA, MH and CCC.

Canadian Journal of Pure and Applied Sciences 340

period, withered and finally died. One possible explanation of the growth inhibition by growth inhibitor applied to bark is that growth inhibitors applied to the bark (cambial layer) might be translocated to the shoot through the phloem. Another explanation is indirect effect of the growth inhibitors through the growth inhibition of bark. It was observed that effectiveness of inhibitors on shoot and bark growth was higher in lower concentration and lower in higher concentration. It might be due to an increase of concentration of inhibitors. The inhibitors may inhibit shoot elongation by interfering with cell division as reported by Khamis and Holubowicz (1978). It was reported that when bark band (strip) of partial ringing in

peach trees was cut (weekly and continuous) by razor blade which attached to mid portion of bark band, after few weeks peach trees were withered and finally died in case of continuous treated trees and for weekly treated trees after bark inhibition again bark started to regenerate (unpublished data). In our result we have found CCC 1000 ppm was more effective than CCC 500ppm. Smith (1994) observed that higher (CCC 500 ppm) concentration was more effective than lower (250 ppm) concentration. He also explained that cytokinin and other plant growth hormones stimulate cell division (cytokinesis) and influence the path of

Tree structure after treatment

Fig. 5. Photos show the partial ringing and plant structure after treatment. A partial ring (arrow) of bark, 2cm in length was removed leaving a 2mm connecting strip. A: Control; B: PR + water; C: PR + ABA (500 ppm); D: PR + ABA (1000 ppm); E: PR + MH (1000 ppm); F: PR + MH (2500 ppm); G: PR + CCC (500 ppm); H: PR + CCC (1000 ppm). ABA: abscissic acid; MH: maleic hydrazide; CCC: cycocel.

Fig. 6. Relationship between shoot length and bark thickness of peach trees in different concentration of growth inhibitor (ABA). In case of ABA 2000ppm there was no bark regeneration that is why after a certain period shoot growth was stopped and finally trees were died due to higher (toxic) effect.

E F G H

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differentiation by stimulating RNA and protein synthesis. When growth inhibitors are used, they may interfere with cell division, elongation and differentiation. They may accumulate in leaves and cause the stomata to close, reducing transpiration and preventing further water loss. This way they may affect the plant physiological process (Smith, 1994). Kamuro (1995) reported that maleic hydrazide (MH) was a growth regulator that was used as an inhibitor of sucker development in tobacco. We have observed that percent flower bud was higher in PR + water and PR + growth inhibitors than control (unringed) trees. Ito et al. (2000) observed that foliar application of MH increased the number of laterally born flower buds on Japanese pear shoots. In our study Hossain et al. (2006) also found the similar result to our result. Trunk circumference was greater above ring and lower below ring in PR + growth inhibitors trees than PR + water trees.

It might be due to more carbohydrate deposition above the ring by the suppression of bark band. Onguso et al. (2004) also found similar result to our results. Root growth was inhibited by ringing and ringing plus growth inhibitors. Ringing caused a significant decrease in gibberellin level in the root system (Wallerstein et al., 1974). Onguso et al. (2004) stated that ringing blocks the translocation of sucrose from leaves to the root zone through phloem bundles. The block decreases starch content in root system and accumulation of sucrose in the leaves. It might be that the reduced level of gibberellins that prevent the hydrolysis of starch. A positive correlation was found between regenerated bark width and new shoot growth. From positive correlation we can understand the degree of effectiveness of PR + water and PR + growth inhibitors.

Fig. 7. Relationship between root weight and bark thickness of peach trees in different concentration of growth inhibitor (ABA). A= Partial ringing (PR), B = PR + ABA 1000 ppm, C = PR + ABA 2000 ppm.

Plant Structure

Root structure

Fig. 8. Photos show plant and root structure after treatment. A partial ring (arrow) of bark, 2 cm in length was removed leaving a 2mm connecting strip. A: Control, B: PR + water; C: PR + ABA 1000 ppm; D: PR + ABA 2000ppm, ABA: abscissic acid.

Canadian Journal of Pure and Applied Sciences 342

CONCLUSION This study has shown that it is possible to make peach tree greatly dwarfed by using PR and PR + ABA, MH and CCC applied to the bark strips of partially ringed trees. PR + growth inhibitors are more effective than PR + water (sole use of PR). In our research technique, we used 97% ringing + growth inhibitors (ABA, MH and CCC) by swabbing method with cotton to the bark band (strip) surface only. We have found that they had dwarfing effect on vigorous peach trees grafted on vigorous rootstocks. However, the method might greatly reduce the use of amount (volume) of chemicals compared with the whole tree spray for tree dwarfing. Furthermore, this method is applicable for all fruit species even if they have no adequate dwarfing rootstocks. ACKNOWLEDGEMENTS The authors are grateful to the Ministry of Education, Culture, Sports, Science and Technology, Japan for providing fund in this research. REFERENCES Arakawa, O., Kanno, K., Kanetsuka, A. and Shiozaka, Y. 1997. Effect of girdling and bark inversion on tree growth and fruit quality of apple, Proc. 6. International Symposium on Integrating Canopy, Acta Horticultuare. 451: 579-586.

De Jong, TM., Weibel, A., Tsuji, W., Doyle, JF., Johnson, RS. and Ramming, D. 2001. Evaluation of size controlling rootstocksfor California peach production, Acta Horticultuare. 557: 103-110.

Erez, A. 1984. Dwarfing peaches by pruning and by paclobutrazol, Acta Horticultuare. 146: 235-241.

Hossain, ABMS. 2003. Maintence of slender spindle bush type of peach trees grafted on vigorous rootstocks by summer pruning and partial ringing. M.S Thesis. Department of Bioresource Production Science, Faculty of Agriculture, Ehime University, Japan (unpublished).

Hossain ABMS., Mizutani, F., Onguso, JM., El-Shereif, AR. and Yamada, H. 2005. Effect of partial ringing on the shoot growth, fruit yield and quality of peach. Journal of Applied Horticulture. 7: 117-120.

Hossain ABMS., Mizutani, F., Onguso, JM., El-Shereif, AR. and Yamada, H. 2006. Dwarfing peach trees by bark ringing, Scientia Horticultuare. 110: 38-43.

Ito, A., Hayama, H., Kashimura, Y. and Yoshioka, H. 2000. Effect of Maleic hydrazide on endogenous cytokinin contents in lateral buds and its possible role in flower bud formation on the Japanese pear shoot, Scientia Horticulturae. 87: 199-205.

Johnson, G. 1998. Plant health care update. A Newsletter. Minnesota University Extension Service, Glenwood Avenue, Minneapolis. pp 2.

Kamuro, Y. 1995. Review of PGRs and their development in the future, Chemical Regulation of Plants. 30: 197-216 (in Japanese).

Khamis, M. and Holubowicz, T. 1978. Effect of foliar application of alar, ethrel, ABA and CCC on the frost resistance of peach trees, Acta Horticultuare. 8: 67-72.

Onguso JM., Mizutani, F. and Hossain, ABMS. 2004. Effects of partial ringing and heating of trunk on shoot growth and fruit quality of peach trees. Botanical Bulletin of Academia Sinica. 45: 301-306.

Smith, M. 1994. A comparison of growth retardant application in Euphorbia pulcherrima. Byng Junior High School, Byng, Oklahoma. pp. 3.

Tukey, HB. 1964. Tree structure, physiology and dwarfing. Dwarf Fruit Trees. pp. 562.

Wallerstein, I., Goren, R. and Monselise, SP. 1974. The effect of girdling on starch accumulation in sour orange seedlings. Canadian Journal of Botany. 52: 935-937.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 343-348, 2008 ISSN: 1715-9997

1

REMOVAL OF CADMIUM ION BY CADMIUM RESISTANT MUTANT OF ASPERGILLUS NIGER FROM CADMIUM CONTAMINATED

AQUA - ENVIRONMENT

Samar Kumar Pal1, Syam Sundar Konar2, Abhishek Mukherjee1 and *Tapan Kumar Das1

Department of Biochemistry and Biophysics University of Kalyani, Kalyani, West Bengal, Kalyani-741235 2Department of Microbiology, University of Kalyani, Kalyani, West Bengal, Kalyani-741235

ABSTRACT

N-methyl N’-nitro N-nitroso guanidine induced cadmium resistant mutant (Cd2) of Aspergillus niger was selected for the study of removal of cadmium ions from cadmium contaminated aqueous systems like sewage, river and pond water. Growth studies of the Cd2 mutant in plane and glucose supplemented sewage, river and pond water indicated that Cd2 mutant could grow well both in plane and glucose supplemented river and sewage water but not as growth in glucose supplemented aqueous systems. The intracellular as well as extracellular Cd+2 concentration of the mutant grown in cadmium contaminated river water reflected the capability of the mutant for removal of cadmium ions both by the mycelial absorption and adsorption methods from an aqueous system. The enhancement of the mutant strengthened the support for high tolerance of the mutant to cadmium ions which favors the removal of cadmium ions. Keywords: Cadmium resistant mutant, Aspergillus sp, mycelial biosorption & adsorption, reduced glutathione.

INTRODUCTION The effect of heavy metals on global pollution through mining operation, industrialization and urbanization has gradually enhanced the potential of heavy metals in ecosphere. The industries of electronics, plating, battery and ammunition manufacture excrete heavy metal like cadmium, lead and zinc as waste materials in streams and contaminate the environment. Cadmium, one of the heavy metals widely used in industry affects human heart and liver through occupational (Ossola and Tomaro, 1995) and environmental exposure and also involved in cell proliferation, differentiation, apoptosis and other cellular activities and it may be considered as carcinogen due to its direct or indirect interaction with adenine and guanine in DNA (Hossain and Huq, 2002). People around Jinstsu river were attacked by painful Itai-Itai disease in eating rice cultivated in cadmium, zinc and lead contaminated soil with polluted water of the river. Agricultural soils were primarily contaminated with Cd2+ due to the excessive use of phosphate fertilizers, dispersal of sewage sludge and atmospheric deposition. Cd2+ was readily taken up by numerous crops including cereals, potatoes, rice and fruits (Ingwersen and Streck, 2005). Consumption of rice grown in paddy soils contaminated with Cd2+, Cr6+ or Zn2+ may pose a serious risk to human health, because 22–24% of the total metal content in rice biomass concentrated in the rice grains (Wang et al., 2003). Thus, contamination by Cd2+ is increasing in both human food and overall in the agricultural environment. Plants readily take up Cd2+ from the soil. Microorganisms

are of increasing importance in biotechnological processes applied for removal of heavy metal ions like biosorption (Kapoor and Viraraghavan, 1995), intracellular absorption (Pal and Das, 2005). Bioremediation of cadmium can be partially fulfilled by producing various mutants of different microorganisms and using the same to study biochemistry and molecular genetics of cadmium toxicity as reported in Pseudomonus fluorescens (Rossbach et al., 2000), Paxillus involtus (Courbot et al., 2004) and Aspergillus niger (Pal and Das, 2005), unicellular algae (Yoshida et al., 2006). The assay of intracellular uptake of cadmium ion, study of metallothionein and cadmium sensitive enzymes of cadmium resistant mutants of Aspergillus niger (Cd1 and Cd2) were described in our earlier paper (Pal and Das, 2005). In the present study the cadmium resistant mutant of Aspergillus niger has been used to verify the growth of the same in plane river , sewage and pond water with or without glucose. An attempt was also taken to investigate its cadmium tolerance and uptake (both intracellular and extracellular) capacity by using sewage and river water contaminated with cadmium ion and also to assay the content of reduced glutathione in cell-free extract of the mutant. MATERIALS AND METHODS Source of organism and composition of growth media Aspergillus niger culture was obtained from the Department of Botany, University of Kalyani and N-methyl N’-nitro-N-nitroso guanidine induced cadmium resistant mutant (Cd2) as reported earlier (Pal and Das, 2005) was selected for the present study. River, pond and *Corresponding author email: tapndas@hotmail.com

Pal et al. 344

sewage water were collected from Kalyani region. Czapex-Dox (CD) as broth (Raper and Thom, 1949) was used. Growth of mutant in river water, pond water and sewage water: Spore suspension of Cd2 mutant (1010 conidia in one liter) of A. niger was separately inoculated to plane pond, sewage and river water (Ganges; Kalyani, W.B. India) and also supplemented only with variable concentration of glucose as a source of carbon and allowed to incubate at 30o C for 96h in a shaker incubator. Harvested mycelia from each sample were washed repeatedly with sterile distilled water and dried completely in a vacuum drier.. The dried mycelia of each sample were weighed. Spore suspension of Cd2 mutant (107conidia/l) was separately inoculated to plane River and sewage water containing variable concentration of CdCl2 and incubated at 300C for 96h in shaker incubator. The harvested mycelia collected from respective water source were washed repeatedly with sterile distilled water. The excess water was removed from mycelia and was placed in drier to obtain dried mycelia and weighed. Estimation of biosorped/ mycelial adsorbed and absorbed cadmium: Spore suspension of Cd2 (1010 conidia in one litre) mutant was inoculated separately to river water containing varying concentration of CdCl2, and incubated at 30oC for 96 h in a shaker incubator. Harvested mycelia were washed repeatedly by sterile distilled water. The excess water was removed from mycelia and preserved at –20oC. Squeezed water also was preserved for estimation of cadmium ion concentration. The repeatedly washed mycelia were ground with alumina (1:1) for the preparation of cell free-extract. Spectrophotometric method (dithiozone method) was followed as described earlier (Pal and Das, 2005) to estimate concentration of cadmium ion present in squeezed water and also in cell free-extract. Measurement of reduced glutathione Spore suspensions of wild type and the test mutant (Cd2) were inoculated to CD broth medium and CD broth with variable concentration of CdCl2 at a final concentration of 1010 conidia per liter and allowed to incubate in a shaker at 300C for 48 h. The harvested mycelia were washed with sterile distilled water under aseptic conditions. The excess liquid was removed and mycelia were preserved at –20oC for the preparation of cell-free extract. The frozen mycelia were ground with neutral alumina (1:1) and extracted with 0.05M potassium phosphate buffer (pH 7.0) and 0.001M EDTA for the assay of reduced glutathione.

GSH content was assayed according to the method of Akerboom and Sies (1981). The reaction mixture (1.0ml) contained 50 µl Cell free extract, 50µl 5,5’-Dithiobis (2-benzoic acid) (DTNB) (1.5mg/100ml), 50µl gluthione oxidoreductase solution (5units/ml). 50 µl of NADPH (3.5mg/ml)and 0.8ml potassium buffer (0.05M, PH 7.0). The mixture was allowed to incubate for 5 to 10 minutes at room temperature. The absorbance was measured after one minute and after 5 minutes at 412 nm.. RESULTS Reduced GSH content in cell free extract of wild type and Cd2 mutant The reduced GSH content in cell free extract of the wild type grown in liquid CD medium. was found to be very low but the content sharply increased when the same strain was grown in liquid CD medium containing 0.5mM CdCl2. Since then the rate of increment of GSH content was found to be infinitesimally small with further increase of CdCl2 up to the tolerable concentration (1.5mM) of the same to the wild type (Fig. 1). The GSH content in Cd2 mutant was found to be increased by 47.5% as compared to that of the wild type when the cell free extract was prepared from the mycelia grown in CD medium only. Whereas the GSH content of the Cd2 increased linearly up to the concentration of 1.5mM CdCl2, after that the increment rate of GSH content in the mutant was found to be almost the same as the wild type up to the tolerable concentration of CdCl2 (3.0mM) to the mutant type.

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Fig. 1. Reduced Glutathione Content in cell free extract of Wildtype and Cd2 in presence of CdCl2. Growth of Wild type and Cd2 mutants in River’s water, pond water and sewage water Both the wild type and the Cd2 mutant could grow in river/sewage water and solid agar medium prepared with

Pal et al. 345

river/sewage water only (without addition of any nutrients exogenously) (Fig. 2a, 2b and 3a); but more growth for the both, the wild type and the Cd2 mutant was found to be at 40 gram/ L glucose in river /sewage water without addition of any other nutrients (Fig. 2 and 3). There was no significant growth difference between the wild type and the mutant in plane pond and even in pond water supplemented with glucose only and also in solid agar medium prepared with pond water only. (Fig. 4 ,4a and 4b).

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Fig. 2. Growth of Wild type and Cd2 mutant in River water/ River water in presence of glucose only.

Fig. 2a. Growth of the Cd2 mutant in plane river water and river water with supplemented with CdCl2.

Fig. 2b. Growth of the Cd1 and Cd2 mutants in plane solid agar medium prepared with river water only.

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Fig. 3a. Growth of Wild type and Cd2 mutant in sewage water / supplemented with glucose only.

Fig. 3b. Growth of the Cd1 and Cd2 mutants in plane solid agar medium prepared with sewage water only.

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Fig. 4. Growth of Wild type and Cd2 mutant in pond water/ supplemented with glucose only.

Pal et al. 346

Fig. 4a: Growth of the Cd2 mutant in plane pond water and pond water supplemented with CdCl2.

Fig. 4b. Growth of the Cd1 and Cd2 mutants in plane solid agar medium prepared with pond water only.

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Fig. 5. Growth of Wild type and Cd2 mutant in River water in presence of CdCl2. Growth of Wild type and Cd2 mutant in River/ sewage water in presence of different concentration of CdCl2 The Cd2 mutant was found to grow both in River / sewage water in presence of CdCl2 and also in solid agar medium prepared with river/ sewage water only (Fig. 2a, 2b, 3a, 5 and 6). Wild type produces very reduced amount of mycelia as compared to that of the mutant in presence of CdCl2. But the mutant could grow even at 3.5mM CdCl2; whereas the mutant showed about 10% less amount of dry

mycelia with increase in CdCl2 concentration from 0.5 to 1.0 mM. The wild type showed about 43 to 46% less amount of dry mycelia with the increase of CdCl2 concentration from 0.5 to 1.0 mM and could not produce any mycelia at 2mM CdCl2.

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Fig. 6. Growth of Wild type and Cd2 mutant in sewage water supplemented with CdCl2. Biosorped/ mycelial adsorbed (B.M) and absorbed (cell free extract) cadmium(A.M) Growing mycelia / cell free extract prepared from the mycelia of the wild type absorbed Cd+2 maximally from river water containing 0.5mM CdCl2 but the absorption capacity of the same strain declined gradually from the liquid media containing higher concentration of CdCl2, whereas both of growing mycelia / cell free extract prepared from the mycelia of the Cd2 mutant showed gradually increasing absorption capacity of Cd+2 till the tolerable concentration (Fig. 7).

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The results presented in Fig. 7 indicates that the Cd2 mutant could biosorb extracellularly about 1.5 times more cadmium ion than that of the wild type, whereas absorbed cadmium ion prepared for cell free-extract of the Cd2 was found to be about 1.7 times more compared with that of the wild type.

Fig. 7. Cd+2 content in biosorbed (squeezed water) andabsorbed (cell free extract) mycelia of Wild type and Cd2mutant.

Pal et al. 347

DISCUSSION The wild type A. niger and its Cd2 mutants could not grow in pond water as ponds usually accumulates water with limited micronutrients and carbon source from a specific area whereas the same strains could grow in sewage water, because the same water assimilates all nutrients from different geographical regions which are appropriate to the growth of all types of microorganisms (Bartsch and Mllum, 1997; Okun, 2002) and remarkable growth of the mutants has also been found in river water without addition of any sugar exogenously. Growth of the mutant in river water supplemented with CdCl2 indicates that the mutants can tolerate CdCl2 up to 3.5mM. Investigation of growth of the mutants in sewage water indicated that, cadmium resistant mutant (Cd2) can grow well in sewage rich in cadmium ion. It signifies that resistance mechanism of cadmium ion has been developed in the same mutant of Aspergillus niger as reported in S.cerevisiae (Joho et al., 1987). The Cd2 mutant had the capacity to take up cadmium ion from plane river water containing CdCl2 only in both process of biosorption (extracellular) and absorption (intracellular) without inhibition of growth of the mutant as it showed increased metallothionein activity with partially defective cadmium-sensitive enzymes as reported earlier in the same mutant strain of Aspergillus niger grown in liquid CD medium containing CdCl2 (Pal and Das, 2005). Similarly Talaromyces helicus (Romero et al., 2006) showed activity of biosorption of heavy metals. . The enhanced content of reduced GSH in the cell free extract of Cd2 mutant indicated that it could bind more cadmium and allow the growth of the mutant type even at higher concentration of cadmium ion compared to the wild type as presented by Xiang and Oliver (1998) and strengthened the support for intake of cadmium ion by the Cd2 mutant of A. niger. The results presented in Figure 1 indicated that CdCl2 induces the wild strain to synthesize more GSH for its survival under stress condition of cadmium ion as reported by Xiang and Oliver (1998). Since the Cd2 mutant strain possessed high content of GSH in normal and Cd+2 stress condition compared to that of the wild type, the same mutant strain could tolerate higher concentration of CdCl2 as reported by Dessislava et al. (2007). As presented in the earlier paper the cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger (Dessislava et al., 2007; Pal and Das, 2005) showing increased metallothionein activity along with partially defective cadmium sensitive enzymes and having more GSH content as presented here, have the capacity to take up Cd+2 intracellulary in different concentrations without

inhibition of its growth, the mutant may be considered as a unique tool to be used for construction of a water purifying system by which cadmium can be removed from cadmium rich aqua-environment or grown mycelia or growing mutant cells can be used roughly in a perforated bag to be placed at the outlet of a sewage drain for remediation of cadmium ions from the waste water. ACKNOWLEDGEMENTS Authors are thankful to Prof. KR Samaddar (Retd.), Dept. of Botany, University of Kalyani for his valuable suggestion and critical discussion in the preparation of this manuscript. REFERENCES Akerboom, TP. and Sies, H. 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77: 373-382.

Bartsch, AP. and Mllum, MO. 1997. Biological factors in treatment of raw sewage in artificial ponds. Limonology and Oceanography. 2: 77-84.

Courbot. M., Diez, L., Ruotolo, R., Chalot, M. and Leropy, P. 2004. Cadmium responsive thiols in the Ectomycorrhizal fungus Paxillus involutus. Applied and Environmental Microbiology. 70: 7413-7417.

Dessislava, T., Lyuba, M., Sergei, I., Alexieva, V. and Tsekova, K. 2007. Role of glutathione and sulfhydryl groups for cadmium tolerance of Aspergillus niger B77. Abstract ID: 1111, No.:P05019 Plant Biology & Botany (Joint Congress) Hilton Chicago Chicago, Illinois.

Joho, M., Imada, Y. and Murayama, T. 1987. The isolation and characterization of Ni+2 resistant mutants of Saccharomyces cerevisiae. Microbios. 51: 183-90.

Hossain, Z. and Huq, F. 2002. Studies on the interaction between Cd+2 ions and nucleobases and nucleotides. Inorganic Biochemistry. 90: 85-86.

Ingwersen, J. and Streck, T. 2005. A regional-scale study on the crop uptake of cadmium from sandy soils: measurement and modeling. Journal of Environmental Quality. 34: 1026–1035.

Kapoor, A. and Viraraghavan, T. 1995. Fungal biosorption an alternative treatment option for heavy metal cleaning waste waters: a review. Bioresource Technology. 53: 195-206.

Okun, OA. 2002. Water reuse introducer the need to integrate both water supply and waste water management at local and regulatory levels. Water Science Technology. 46: 273-280.

Ossola, JO. and Tomaro, ML. 1995. Heme oxygenase induction by cadmium chloride:

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evidence for oxidative stress involvement. Toxicology. 104: 141-147.

Pal, SK. and Das, TK. 2005. Biochemical characterization of N-methyl N’-nitro-N-nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger. Journal of Biosci ences. 30: 639-646.

Raper, KB. and Thom, C. 1949. Manual of the penicillia (Baltimore: The Williams and Winkins).

Romero, MC., Reinoso, EH., Urrutia, MI. and Kiernan, AM. 2006. Biosorption of heavy metals by Talaromyces helicus: a trained fungus for copper and biphenyl detoxification. Biotechnology and Environment. 9: 221- 226.

Rossbach, S., Kukuk, ML., Wilson, TL., Feng, SF., Pearson, MM. and Fisher, MA. 2000. Cadmium-regulated gene fusions in Pseudomonas fluorescens. Environmental Microbiology. 2: 373-82.

Wang, QR., Cui, YS., Liu, XM., Dong, YT. and Christie, P. 2003. Soil contamination and plant uptake of heavy metals at polluted sites in China. Journal of Environmental Science and Health, Part-A. 38: 823–838.

Xiang, C. and Oliver, DJ. 1998. Glutathione Metabolic Genes Coordinately Respond to Heavy Metals and Jasmonic Acid in Arabidopsis. Plant Cell. 10: 1539-1550.

Yoshida, N., Ikeda, R. and Okuno, T. 2006. Identification and characterization of heavy metal-resistant unicellular alga isolated from soil and its potential for phytoremediation Bioresource Technology. 97: 1843-1849.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 349-356, 2008 ISSN: 1715-9997

1

ACTIVITY OF CHOLINESTERASE AND ALKALINE PHOSPHATASE IN LIVER, KIDNEY AND BRAIN OF EUPHLYCTIS CYANOPHLYCTIS

UNDER THE EFFECT OF CHLORPYRIFOS AND DATHRIN

*M Zaheer Khan1, Ghazala Yasmeen2, SNH Naqvi2 and Aisha Perveen2 1Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia Canada V5A 1S6

2Department of Zoology – Wildlife and Fisheries, University of Karachi, Karachi-75270

ABSTRACT

Globally many scientists believe that several common pesticides already exist at levels capable of killing amphibians in the earth. The aquatic biota may be harmed by pesticide-contaminated water. The present study was done to investigate the effects of two pesticide groups organophosphate and pyrethroid, on the activity of cholinesterase (ChE) and alkaline phosphatase (ALP) in the liver, kidney and brain tissue of Euphlyctis cyanophlyctis. LD50 of each pesticide was determined before the selection of final concentrations of both pesticides. The frogs were treated by two concentrations of chlorpyrifos i.e. 2 and 4%. The effect of these two concentrations on ChE activity in the liver, kidney and brain was estimated. According to results it was decreased upto 30.0 and 45.0% in liver (F2,6=116.90, P=0.001), 20.0 and 50.0% (F2,6=8.99, P=0.016) in kidney and 33.33 and 55.55 % (F2,6=63.96, P=0.001) in the brain, respectively. Under the effect of two concentrations of Dathrin i.e. 0.04 and 0.08% the ChE activity in the liver was decreased upto 15.0 and 45.0% (F2,6=14.90, P=0.005), in the kidney it was decreased upto 20.0 and 40.0 % (F2,6=7.30, P=0.025), while in the brain the activity decreased upto 22.0 and 44.0 %, respectively (F2,6=6.80, P=0.029). The effects of same concentrations of both pesticides were also observed on alkaline phosphatase (ALP) activity in liver, kidney and brain of E. cyanophlyctis. In the case of chlorpyrifos it was decreased upto 25.0 and 50.0 % (F2,6=7.00, P=0.027), in kidney ALP activity decreased upto 33.33 and 50.0 % (F2,6=1.98, P=0.219), while in the brain it decreased upto 22.22 and 44.44 %, respectively (F2,6=1.89, P= 0.231). The effect of Dathrin on ALP in liver it was decreased upto 37.50 and 50.0 % (F2,6=73.0, P= 0.001), in the kidney the activity was decreased upto 16.66 and 50.0% (F2,6=2.02, P=0.214), while in the brain it was decreased upto 44.44 and 55.55%, respectively (F2,6=1.98, P=0.219). Keywords: Euphlyctis cyanophlyctis, cholinesterase, alkaline phosphatase, chlorpyrifos, dathrin.

INTRODUCTION

The widespread application of agricultural pesticides has attracted the attention of ecologists to understand the impacts of these chemicals on natural communities (Relyea et al., 2005). Amphibians are important to the overall ecosystem balance. The large biomass of amphibians makes them significant prey for other animals (Khan et al., 2007a). Direct contact of sprays of some pesticides can be highly lethal to amphibians (Relyea, 2005). The loss of amphibian populations was first recognized in 1989 as a phenomenon that deserved worldwide attention (Barinaga, 1990; Wake, 1991; Blaustein, 1994; Alford and Richards, 1999). The obvious factor contributing to amphibian population declines are habitat destruction and alteration (Alford and Richards, 1999). A wide array of contaminants may affect amphibian populations which include pesticides, herbicides, fungicides, fertilizers and numerous pollutants (Sparling et al., 2000; Boone and Bridges, 2003). A diversity of pesticides and their residues are present in a

wide variety of aquatic habitats (Harris et al., 1998; McConnell et al., 1998; Le Noir et al., 1999; Kolpin et al., 2002). While pesticides have the potential to affect many aquatic species, the impacts on amphibians are of particular concern in the past decade because of the apparent global decline of many species (Blaustein and Wake, 1990; Alford and Richards, 1999; Houlahan et al., 2000; Kiesecker et al., 2001) and the amphibians living in these habitats exhibit physiological signatures of pesticides (i.e. reduced acetylcholine esterase activity; Sparling et al., 2001) and declining populations are correlated with greater amounts of upwind agriculture where pesticide use is common (Davidson et al., 2001, 2002). Pesticides can severely affect amphibians in a variety of ways, as they destroy the natural biotic balance in agricultural soils and reduce the diversity and abundance of biodiversity with cascading effects at higher trophic levels (Larson et al., 1997). They can kill amphibians directly, affect their behaviour, reduce their growth rates, act as endocrine disrupters or induce immunosuppression (Bishop, 1992; Carey and Bryant, 1995; Alford and *Corresponding author email: zaheer_khan@sfu.ca

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Richards, 1999). The causes of these declines include human involvement in an effort to increase agricultural products and indiscriminate use of pesticides. The effects of these toxic materials remain to be studied on non-target biodiversity in many regions of the world. (Khan et al., 2002b). The pesticides, organophosphate and carbamate are widely used and have a variety of lethal and sublethal effects on non-target wildlife species (Parsons et al., 2000; Khan et al., 2003a). Pyrethroids appear to effect voltage-dependent neuromuscular sodium channels producing tremors, hyperexcitation and convulsions (Van den Bercken, 1977; Vijverberg et al., 1982; Ruigt and Van den Bercken, 1986). In Pakistan pesticides have been reported to have reduced enzyme activity of cholinesterase in frog Rana tigrina (Khan et al., 2002a,b, Khan et al., 2003a,b) in skittering frog Rana cyanophlyctis (Khan et al., 2003b, c,d; Khan and Yasmeen, 2005; Khan et al., 2007a). Khan et al. (2007b) determined the induced effect of chlorpyrifos (organophosphate) on skin of E. cyanophlyctis. In the present study the effects of Chlorpyrifos (organophosphate) and Dathrin (pyrethroid) were observed on the activity of two enzymes cholinesterase and alkaline phosphatase in the liver, kidney and brain of skittering frog E. cyanophlyctis. MATERIALS AND METHODS The experimental work was carried out on adults skittering frog Euphlyctis cyanophlyctis, collected from selected areas of Province of Sindh. Collected frogs were brought in laboratory and kept in glass aquarium in the Wildlife Lab, Department of Zoology, University of Karachi. Frogs were fed with prawns and insects. Two concentrations of both pesticides were applied, i.e. 2 and 4% of Chlorpyrifos, while 0.04 and 0.08% of Dathrin were injected in the sub-cutaneous abdominal region of frog by using insulin syringe. The effects of pesticide were observed in liver, kidney and brain tissue. The liver, kidney and brain were taken as per Shakoori and Ahmad’s (1973) techniques. Organs (liver, kidney and brain) were crushed in 2 ml bidistilled water and homogenized. The homogenates were centrifuged in Labofuge 15000 at 5000 rpm for 20 minutes and placed in cold chamber. Supernatents were taken in separate glass tubes to use in estimation of enzyme activity. The activity of cholinesterase was estimated by Randox Kit No. CE-190. In colorimetric method (Knedel and Boetteger, 1967) the reagent composition was consisting of Buffer (Phosphate buffer=50 mmol/l having pH 7.7 and DTNB=0.25 mmol/l) and Substrate (Butyrylthiocholine iodide=6 mmol/l). The method based upon the hydrolysis of butyrylthiocholine by the action of enzyme butyryl cholinesterase. The reaction between thiocholine and dithiobis (nitrobenzoate) gave 2-nitro-5-

mercaptobenzoate, a yellow compound which was measured at 405 ηm. Principle: Cholinesterase Butyrylthiocholine + H2O → thiocholine + butyrate Thiocholine + DTNB → 2-nitro-5-mercaptobenzoate DTNB = Dithiobis (nitrobenzoate) The activity of alkaline phosphatase was estimated by Randox Kit No.AP-307. In colorimetric method (Rec.GSCC, 1972) the reagent composition was Buffer (Diethanolamine buffer=1 mol/l, pH=9.8 and MgCl2= 0.5 mmol/l) and Substrate (p-nitrophenylphosphate=10 mmol/l). The reaction is based upon the hydrolysis of p-nitrophenylphosphate by the action of alkaline phosphatase. Principle: ALP p-nitrophenylphosphate + H2O → phosphate + p-nitrophenol All statistical analyses were conducted by Micro Soft Excel and Minitab (Minitab Inc,1996). Data presented as percentages were arcsine-square-root transformed before analyses. One way analysis of variance (ANOVA) was used to compare the effect of two concentrations of each pesticide on activity of enzymes cholinesterase and alkaline phosphatase. RESULTS

In this study, effect of two concentrations of chlorpyrifos on cholinesterase in the liver, kidney and brain was determinate. The enzyme level decreased up to 30.0 and 45.0% in liver (F2,6=116.90, P=0.001) (Table 1), 20.0 and 50.0% (F2,6=8.99, P=0.016) (Table 2) in kidney and 33.33 and 55.55 %, respectively (F2,6=63.96, P=0.001) (Table 3) in the brain. Under the effect of two concentrations of Dathrin the cholinesterase activity in the liver was decreased up to 15.0 and 45.0% respectively (F2,6=14.90, P=0.005) (Table 4), in the kidney it decreased up to 20.0 and 40.0 % (F2,6=7.30, P=0.025) (Table 5), while in the brain the activity decreased up to 22.0 and 44.0 %, respectively (F2,6=6.80, P=0.029) (Table 6). The effects of same concentrations of both pesticides were also observed on alkaline phosphatase in liver, kidney and brain. In the case of chlorpyrifos it was decreased up to 25.0 and 50.0 % (F2,6=7.00, P=0.027) (Table 7), in kidney ALP activity decreased up to 33.33 and 50.0 % (F2,6=1.98, P=0.219) (Table 8) and in the brain it decreased up to 22.22 and 44.44 % respectively (F2,6=1.89, P= 0.231) (Table 9).

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The effect of Dathrin on alkaline phosphatase in the liver was evident by a decrease upto 37.50 and 50.0 % (F2,6=73.0, P= 0.001) (Table 10), in kidney the activity decreased upto 16.66 and 50.0% (F2,6=2.02, P=0.214) (Table 11), while in the brain it was decreased up to 44.44 and 55.55% respectively (F2,6=1.98, P=0.219) (Table 12).

DISCUSSION On the basis of lab experiments, amphibians are known to be vulnerable to pesticides and they are cholinesterase inhibitors (Wang and Murphy, 1982). There is some indication, that field application of these chemicals may be deleterious to amphibians (Jolly et al., 1978; Thybaud, 1990; Berril et al., 1993; Materna et al., 1995). A number

of non-target species can be affected when pesticides are used because of their affect on cholinesterase activity. The enzyme inhibition occurs in a number of species and reduction can result in sub-lethal toxicity and death (Cooper, 1991). Anticholinesterase pesticides function by binding with this enzyme in animals and disrupting nervous system activity, usually causing death by respiratory failure. Decreased cholinesterase activity can indicate exposure to some commonly used pesticides and can be harmful to wild animals (Catherine and Gloria, 2000). In the present study, the estimation of two enzymes i.e. cholinesterase and alkaline phosphatase were carried out in the liver, kidney and brain of E. cyanophlyctis under

Table 1. Activity of cholinesterase in liver of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 18.40 10.449 6.039 6.561 - 30.238 00 2% 12.88 5.745 3.321 6.370 - 19.389 30.0 4% 10.12 5.745 3.321 3.610 - 16.629 45.0

F 2,6 = 116.90, P = 0.001

Table 2. Activity of cholinesterase in kidney of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 9.20 6.373 3.684 1.978 - 16.421 00 2% 7.36 5.745 3.321 0.850 - 13.869 20.0 4% 4.60 3.186 1.842 0.989 - 8.210 50.0

F 2,6 = 8.99, P = 0.016

Table 3. Activity of cholinesterase in brain of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 8.28 2.76 1.595 5.153 -11.406 00 2% 5.52 2.76 1.595 2.393 - 8.646 33.33 4% 3.68 1.593 0.921 1.874 - 5.485 55.55

F 2, 6 = 63.96, P = 0.001

Table 4. Activity of cholinesterase in liver of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 18.4 10.449 6.039 6.561 - 30.238 00 0.04% 15.64 10.449 6.039 3.801 - 27.478 15.0 0.08% 10.12 4.215 2.436 5.343 - 14.896 45.0

F 2, 6 = 14.90, P = 0.005

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the effects of Chlorpyrifos and Dathrin as compared with lab standard (control). The organophosphate can affect cholinesterase activity in both red blood cells and in blood plasma, and can act directly, or in combination with other enzymes, on cholinesterase in the body. The first notable studies to examine the effects of organophosphates on amphibians were reported in the early sixtees (Edery and Schatzberg-Porath, 1960; Mulla, 1962; Mulla et al., 1963). More recently several studies indicated that standard field application rates of organophosphates insecticides may have a deleterious effect on amphibian population (Anguiano et al., 1994; Berril et al., 1993 and1994;

Schuytema et al., 1995; Sparling et al., 1997). While pyrethroid have gained a reputation as “safe insecticides” and are widely used in agricultural, aquatic and house hold products (Elliot et al., 1978; Smith and Stratton 1986). During this study, the effect of 2 and 4% concentration of chlorpyrifos (organophosphate) on cholinesterase activity in the liver, kidney and brain showed significantly decrease i.e. upto 30.0 and 45.0% (P<0.001) in liver, 20.0 and 50.0% (P<0.016) in kidney and 33.33 and 55.55 % (P<0.001) in the brain, respectively while in the case of Dathrin the effect of 0.04 and 0.08% concentrations on cholinesterase activity in the liver, kidney and brain were found significantly decreased upto 15.0 and 45.0% (P<0.005) in the liver, 20.0 and

Table 5. Activity of Cholinesterase in kidney of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 9.2 6.373 3.684 1.978 - 16.421 00 0.04% 5.745 3.321 6.509 0.850 - 13.869 20.0 0.08% 5.52 4.780 2.763 0.103 - 10.936 40.0

F 2, 6 = 7.30, P = 0.025

Table 6. Activity of cholinesterase in brain of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 8.28 2.76 1.595 5.153 - 11.406 00 0.04% 6.44 4.215 2.436 1.663 - 11.216 22.00 0.08% 4.60 1.593 0.921 2.794 - 6.405 44.00

F 2, 6 = 6.80, P = 0.029 Table 7. Activity of alkaline phosphatase in liver of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 7.36 3.186 1.842 3.749 - 10.970 00 2% 5.52 4.780 2.763 0.103 - 10.936 25.00 4% 3.68 1.593 0.921 1.874 - 5.485 50.00

F2, 6 = 7.00, P = 0.027

Table 8. Activity of alkaline phosphatase in kidney of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 5.52 2.76 1.595 2.393 - 8.646 00 2% 3.68 1.593 0.921 1.874 - 5.485 33.33 4% 2.76 00 00 - 50.00

F2, 6 = 1.98, P = 0.219

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40.0% (P<0.025) in kidney, 22.0 and 44.0% (P<0.029) in the brain, respectively. Tilak et al. (2003) investigated the effect of fenvalerate in Indian bullfrog to sublethal dose (1/3 of LC50 I.E. 1.166 mg/kg) and the effect was studied on the specific activity of acetyl-cholinesterase in the different tissues of frog liver, kidney, brain, muscle, and testis at different time periods viz., 3,6, 12, 24, 48 and 72 hours. It was found that the inhibition in activity of acetyl-cholinesterase was in the order of kidney > brain > muscle > liver > testis. A significant inhibition was noticed in kidney at 12 hours (-64.33%) and no effect was noticed at 3 hours in testis (+0.67%). It was suggested that the AChE activity was inhibited in first three hours of administration of

fenvalerate in all the tissues tested. The inhibition continued upto 6 hours or 2 hours in different tissues but the recovery started by 24 hours and almost completed by 72 hours. Another study, Khan et al. (2003a) investigated that the effect of cypermethrin and permethrin on cholinesterase in Rana tigrina and reported that the ChE activity decreased in the treated frogs. The present findings are generally in accordance with the previous reports. Khan et al. (2003b) compared the effect of two pyrethroids lambda cyhalothrin with permethrin on cholinesterase in R. cyanophlyctis and R. tigrina and reported that the amphibian in general are sensitive and R. cyanophlyctis is more sensitive to R. tigrina, and lambda cyhalothrin is

Table 9. Activity of alkaline phosphatase in brain of E. cyanophlyctis treated with Chlorpyrifos.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 8.28 7.302 4.220 0.006 - 16.553 00 2% 6.44 4.215 2.436 1.663 - 11.216 22.22 4% 4.6 3.186 1.842 0.989 - 8.210 44.44

F2, 6 = 1.89, P = 0.231 Table 10. Activity of alkaline phosphatase in liver of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 7.36 3.186 1.842 3.749 - 10.970 00 0.04% 4.6 3.186 1.842 0.989 - 8.210 37.50 0.08% 3.68 1.593 0.921 1.874 - 5.485 50.00

F2, 6 = 73.0, P = 0.001

Table 11. Activity of alkaline phosphatase in kidney of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 5.52 2.76 1.595 2.393 - 8.646 00 0.04% 4.6 1.593 0.921 2.794 - 6.405 16.66 0.08% 2.76 00 00 - 50.00

F2, 6 = 2.02, P = 0.214

Table 12. Activity of alkaline phosphatase in brain of E. cyanophlyctis treated with Dathrin.

Treatment Mean (U/l)

S.D. +

S.E. +

Range at 95% Confidence limit

Inhibition %

Control 8.28 7.302 4.220 0.006 - 16.553 00 0.04% 4.6 3.186 1.842 0.989 - 8.210 44.44 0.08% 3.68 1.593 0.921 1.874 - 5.485 55.55

F2, 6 = 1.98, P = 0.219

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more toxic among the pesticides tested. In the present finding also, it was observed that Dathrin inhibited the ChE activity in the liver, kidney and brain. Khan et al., 2003c determined the effect of two pesticides lambda cyhalothrin and monocrotophos on ChE in the liver, kidney and brain of R. cyanophlyctis, two different concentrations were used and ChE activity was observed. It was decreased up to 34.6 and 46.3 % in the liver, 25.08 and 57.1% in the kidney and 31.64 and 50.7% in the brain under the effect of lambda cyhalothrin. In the case of monocrotophos treatment, cholinesterase decreased up to 37.7 and 57.7% in liver, 57.5 and 67.5% in kidney and 47.6 and 65.9% in brain, respectively. The brain cholinesterase activity of R. cyanophlyctis was decreased up to 4.10 and 13.84 % under the effect of sandaphos and 5.16 and 23.28% under the effect of β-cypermethrin, respectively (Khan and Yasmeen, 2005). Khan et al. (2006) reported that the effects of sandaphos and β-cypermethrin on ChE activity in liver and kidney of E. cyanophlyctis, under the effect of sandaphos, ChE activity decreased upto 41.28 and 51.46% in the liver and 4.43 and 22.85% in the kidney, respectively. Under the effect of β-cypermethrin, ChE activity decreased upto 24.46 and 26.34 in the liver and 21.46 and 26.63% in the kidney, respectively. The alkaline phosphatase is a unique enzyme that is usually present in the tissues involved in transport function and regeneration (McGomb et al., 1979). This enzyme has been extensively studied in liver, bone, placenta, intestine and serum. The localization of this enzyme in plasma membrane strongly suggests its involvement in membrane functions (Fishman, 1974). Yora and Sakagishi (1986) studied the activity of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals. The alkaline phosphatases from the liver, kidney and intestine in various vertebrates were strongly inhibited by beryllium, 2-mercaptoethanol, potassium cyanide and EDTA. The enzymes showed various sensitivities to the inhibition by zinc and to heat denaturation at 56 degrees Celsius for 5 min at pH 7.0. The liver and kidney enzymes showed higher sensitivity to the inhibition by L - homoarginine than by L - phenylalanine. The intestinal enzymes in higher vertebrates were more sensitive to the inhibition by L - phenylalanine than by L - homoarginine, whereas the intestinal ones in lower vertebrates showed quite similar sensitivities to both amino acid. Goseki et al. (1990) examined the enzymatic and immunological properties of alkaline phosphatase (ALP) in several tissues of bullfrog Rana catesbeiana. The inhibition and thermal inactivation studies showed that bullfrog ALP in kidney, liver and intestine had similar enzymatic properties. In addition, mouse antiserum against bullfrog liver ALP cross-reacted with kidney and intestine enzymes as well as with liver enzyme.

The present study with reference to Pakistan is the first attempt to determine the effects of pesticides on ALP activity in E. cyanophlyctis and findings have showed the effect of 2 and 4% concentration of chlorpyrifos on alkaline phosphatase in the liver, kidney and brain decreased upto 25.0 and 50.0% (P<0.027) in the liver, 33.33 and 50.0% (P<0.219) in kidney, 22.22 and 44.44% (P<0.231) in the brain, respectively. Under the effect of Dathrin the effect of lower 0.04 and 0.08% concentrations on alkaline phosphatase in the liver, kidney and brain was found significantly decreased upto 37.50 and 50.0% (P<0.001) in the liver, 16.66 and 50.0% (P<0.214) in kidney, 44.44 and 55.55% (P<0.219) in the brain, respectively. In general the present results are in agreement with earlier reports. CONCLUSION On the basis of present findings it is concluded that the selected pesticides Chlorpyrifos (organophosphate) and Dathrin (pyrethroid) decreased the cholinesterase and alkaline phosphatase activity in the liver, brain and kidney of E. cyanophlyctis. The organophosphate group is more harmful to E. cyanophlyctis as compared to pyrethroid group. It is therefore, suggested that pyrethroid group could be a better pesticide if used at lower doses in agricultural fields and other places. ACKNOWLEDGEMENTS We gratefully acknowledge the technical assistance of Prof. Dr. Nikhat Yasmeen Siddiqui, Department of Zoology, and Dr. S. Hamid, Faculty of Pharmacy, University of Karachi REFERENCES Alford, RA. and Richards, SJ. 1999. Global amphibian declines: a problem in applied ecology. Ann. Rev. Ecol. Syst. 30: 133-165. Anguiano, OL., Montagna, CM., Chifflet De Llamas, M. 1994. Comparative toxicity of parathion in early embryos and larvae of toad, Bufo arenarum Hensel. Bull. Environ. Contam. Toxicol. 52: 649-655. Barinaga, M. 1990. Where have all the froggies gone? Science. 247: 1033-34. Berril, M., Bertram, S., Wilson, A., Louis, S., Brigham, D. and Stromberg, C. 1993. Lethal and sub-lethal impacts of pyrethroid insecticides on amphibian embryos and tadpoles. Environ. Toxicol. Chem. 12: 525-539. Berril, M., Bertram, S., McGillivary, L., Kolohhon, M. and Pauli, B. 1994. Effects of low concentrations of forest use pesticides on frog embryos and tadpoles. Environ. Toxicol. Chem. 13: 657-664. Bishop, CA. 1992. The effects of pesticides on amphibians and the implications for determining causes

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of declines in amphibian populations. In Declines in Canadian Amphibian Populations: Designing a National monitoring Strategy, (eds. Bishop, CA. and Pettit, KE.) pp. 67-70. Occas. Pap. No. 76, Can. Wildlife Serv. Blaustein AR. and Wake, DB. 1990. Declining amphibian populations: a global phenomenon? Trends Ecol. Evol. 5: 203-4. Blaustein, AR. 1994. Chicken Little or Nero’s fiddle? A perspective on declining amphibian populations. Herpetologica. 50: 85-97. Boone, MD. and Bridges, CM. 2003. The problem of pesticides: Implications for amphibian populations. In: Amphibian Conservation. (eds. Semlitsch, RD.). Smithsonian Institution Press, Washington, DC, USA. 152-167. Carey, C. and Bryant, CJ. 1995. Possible interrelationships among environmental toxicants, amphibian development, and decline of amphibian populations. Environ. Health Perspec. 103(4): 13-17. Catherine, H. and Gloria, M. 2000. USGS Research Finds that contaminants may play an important Role in California Amphibian Declines. US. Geological Survey, MS119 National Center, Reston, VA. www.usgs.gov/public/press/public_affairs/press_releases/pr1338m.html. Cooper, K. 1991. Effects of pesticides on wildlife, Handbook of Pesticides Toxicology, Vol. 1. General Principles. Academic Press, New York, USA. 463-496. Davidson, C., Shafer, HB. and Jennings, MR. 2001. Declines of the California red-legged frog: climate, UV-B, habitat, and pesticides hypotheses. Ecological Applications. 11: 464-479. Davidson, C., Shafer, HB. and Jennings, MR. 2002. Spatial tests of the pesticide drift, habitat destruction, UV-B, and climate-change hypotheses for California amphibian declines. Conservation Biology. 16: 1588-1601. Edery, H. and Schatzberg-Porath, G. 1960. Studies of the effects of organophosphorus insecticides on amphibians. Arch. Int. Pharmacodyn. 124: 212-224. Elliot, M., Janes, NF. and Potter, C. 1978. The future of pyrethroids in insect control. Ann. Rev. Entomol. 23: 443-469. Fishman, W. H. 1974. Perspectives of alkaline phosphatase isoenzymes. Am. J. Med. 56(5): 617-650. Goseki, M., Oida, S. and Sasaki, S.1990. Enzymatic and immunological properties of alkaline phosphatase of bullfrog. J. Exp. Zool. 254 (1): 1-5. Harris, ML., Bishop, CA., Struger, J., Ripley, B. and Bogart, JP. 1998. The functional integrity of northern leopard frog (Rana pipiens) and green frog (Rana clamitans) populations in orchard wetlands. II. Genetics, physiology, and biochemistry of breeding adults and

young of the year. Environ. Toxicol. and Chemistry. 17: 1338-1350. Houlahan, JE., Findlay, CS., Schmidt, BR., Myer, AH. and Kuzmin, SL. 2000. Quantitative evidence for global amphibian population declines. Nature. 404: 752-755. Jolly, AL. Jr., Avault, JW. Jr., Koonce, KL. and Graves, JB. 1978. Acute toxicity of Permethrin to several aquatic animals. Trans. Am. Fish Soc. 107: 825-827. Khan, MZ., Fatima, F. and Ahmad, I. 2002a. Effect of Cypermethrin on Protein Contents in Lizard Calotes versicolor in comparison to that in Frog Rana tigrina. Online Journal of Biological Sciences. 2 (12): 780-781. Khan, MZ., Shah, EZ., Ahmad, I. and Fatima, F. 2002b. Effects of agricultural pesticides permethrin (pyrethroid) on protein contents in kidney and liver of lizard species Calotes versicolor in comparison to that in frog Rana tigrina. Bull. of Pure and Appl. Sci. 21A (2): 93-97. Khan, MZ., Tabassum, R. Naqvi, SNH., Shah, EZ., Tabassum, F., Ahmad, I. Fatima, F. and Khan, MF. 2003a. Effect of Cypermethrin and Permethrin on Cholinesterase Activity and Protein Contents in Rana tigrina (Amphibia).Turk. J. Zool. 27: 243-246. Khan, MZ., Nazia, M., Fatima, F., Rahila, T. and Gabol, K. 2003b. Comparison of the effect of Lamda cyhalothrin with permethrin on cholinesterase activity in Rana cyanophlyctis and Rana tigrina (Ranidae: amphibian). Bull. of Pure and Appl. Sci. 22 A (1): 43-49. Khan, MZ., Zaheer, M. and Fatima, F. 2003c. Effect of Lamda Cyhalothrin (Pyrethroid) and Monocrotophos (Organophosphate) on Cholinesterase activity in liver, kidney and brain of Rana cyanophlyctis. Korean J. Biol. Sci. 7: 165-168. Khan, MZ., Fatima, F., Mahmood, N. and Yasmeen, G. 2003d. Comparison of Cholinesterase activity in the brain tissue of lizard Calotes versicolor with that of frog Rana cyanophlyctis under the effect of Cypermethrin, Lamda Cyhalothrin, Malathion and Monocrotophos. Bulletin of Pure and Applied Sciences. 22 A (2): 105-112. Khan, MZ. and Yasmeen, G. 2005. Pesticide dependent cholinesterase activity in brain of Rana cyanophlyctis (amphibian) J. Exp. Zool. India. 8(1): 135-140. Khan, MZ., Yasmeen, G. and Hamid, S. 2006. Effect of Sandaphos (Organophosphate) and ß-cypermethrin (Synthetic Pyrethroid) on cholinesterase activity in liver and kidney of Euphlyctis cyanophlyctis. Hamadryad. 30 (1 & 2): 176-180. Khan, MZ., Rais, M. and Yasmeen, G. 2007a. Inhibitory effects on cholinesterase activity produced by the two different pesticides on brain, liver and kidney of Euphlyctis cyanophlyctis. J. Exp. Zool. India. 10 (1): 89-93 Khan, MZ., Yasmeen, G., Akbar, A. and Khan, MF. 2007b. Determination of induced effect of pesticide

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chlorpyrifos (organophosphate) on skin of Euphlyctis cyanophlyctis. J. Basic Appl. Sci. 3(2): 101-105. Kiesecker, JM., Blaustein, AR. and Belden. LK. 2001. Complex causes of amphibian population declines. Nature. 410: 681-684. Knedel, M. and Boetteger, R. 1967. Kinetic method for determination of pseudocholinesterase (acylcholine acylhydrolase) activity. Klin. Wochenschr. 45(6): 325-327. Kolpin, DW., Furlong, ET., Meyer, MT., Thurman, EM., Zaugg, SD., Barber, LB. and Buxton, HT. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U. S. streams, 1999-2000: a national reconnaissance. Environmental Science and Technology. 36: 1202-1211. Larson, SJ., Capel, PD. and Majewski, MS. 1997. Pesticides in surface waters: Distribution, Trends, and Governing Factors. Ann Arbor, Inc., USA. LeNoir, JS., McConnell, LL., Fellers, GM., Cahill, TM., Seiber, JN. 1999. Summertime transport of current-use pesticides from California’s central valley to the Sierra Nevada mountain range, USA. Environ Toxicol Chem. 18: 2715-2722. Materna, EJ., Rabeni, CF. and La Point, TW. 1995. Effects of synthetic pyrethroid insecticides, esfenvalerate, on larval leopard frogs( Rana spp.) Environmental, Toxicol. Chem. 14: 613-622. McConnell, L.L., LeNoir, JS., Datta, S. and Seiber, JN. 1998. Wet deposition of current-use pesticides in the Sierra Nevada Mountain range, California, USA. Environmental Toxicology and Chemistry. 17: 1908-1916. McGomb, RB., Bowers, GN. and Posen, S. 1979. Alkaline phosphatase. Plenum Press, New York, USA. Minitab Inc.1996. Minitab statistical software, version 11.12. Mulla, MS. 1962. Frog and toad control with insecticides! Pest Control. 30: 64. Mulla, MS., Isaak, LW. and Axelrod, H. 1963. Field studies on the effects of insecticides on some aquatic wildlife species. J. Econ. Ent. 59: 1085-1090. Parsons, KC., Matz, AC., Hooper, MJ. and Pokras, MA., 2000. Monitorinh eading bird exposure to agricultural chemicals using serum cholinesterase activity. Environ. Toxicol. Chem. 19(5): 1317-1323. Rec. GSCC (Deutsche Gesellschaft für Klinische Chemie). 1972. Optimised standard colorimetric methods. J. Clin. Chem. and Clin. Biochem. 10: 182. Ruigt, GSF. and Van den Bercken, J. 1986. Action of pyrethroids on a nerve muscle preparation of the clawed frog Xenopus leavis. Pestic. Biochem Physiol. 25: 176-187.

Relyea, RA. 2005. The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecological Applications. 15(2): 618-627. Relyea, RA., Schoeppner, NM. and Hoverman, JT. 2005. Pesticides and Amphibians: The Importance of Community Context. Ecological Applications. 15(4): 1125-1134. Schuytema, GS., Nebeker, AV. and Griffis, WL. 1995. Comparative toxicity of Guthion and Guthion 2S to Xenopus laevis and Pseudacaris regilla tadpoles. Bull. Environ.Contam.Toxicol. 54: 382-388. Shakoori, AR. and Ahmad, MS. 1973. Studies on the liver of chicken Gallus domasticus Liver growth and nucleic acid content. Pakistan J. Zool. 5: 111-117. Smith, TM. and Stratton, GW. 1986. Effect of synthetic pyrethroid insecticides on non target organisms. Residue Rev. 97: 93-120. Sparling, DW., Lowe, TP. and Pinkney, AE. 1997. Toxicity of Abate to green frog tadpoles. Bull. Environ. Contam.Toxicol. 58: 475-481. Sparling, DW., Linder, G. and Bishop, CA. eds. 2000. Ecotoxicology of amphibians and Reptiles. SETAC Press, Pensacola, FL, USA. Sparling, DW., Fellers, GM. and McConnell, LS. 2001. Pesticides and amphibian population declines in California, USA. Environ. Toxicol. and Chem. 20: 1591-1595. Thybaud, E. 1990. Acute toxicity and bioconcentration of lindane and deltamethrin in tadpoles of Rana temporaria and the mosquito fish Gambusia affinis. Hydrobiologia. 190: 137-146. Tilak, KS., Veeraiah, K. and Sastry, LV. 2003. Effect of fenvalerate technical grade on acetyl cholinesterase activity in Indian bull frog Haplobatrachus tigerinus (Daudin). J. Environ. Biol. 24(4): 445-8. Van den Bercken, J. 1977. The action of allethrin on the peripheral nervous system of the frog. Pestic. Sci. 8: 692-699. Vijverberg, HPM., Zalm, JM. and Bercken, JVD. 1982. Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves. Nature. 259: 601-603. Wake, DB. 1991. Declining amphibian populations. Science. 253. pp 860. Wang, C. and Murphy, SD. 1982. Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides. Toxicol. Appl. Pharmacol. 66: 409-419. Yora, T. and Sakagishi, Y. 1986. Comparative biochemical study of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals. Comp. Biochem. Physiol. 85(3): 649-58.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 357-362, 2008 ISSN: 1715-9997

1

GROWTH AND CELLULASE ACTIVITY OF WILD-TYPE ASPERGILLUS NIGER ANL301 IN DIFFERENT CARBON SOURCES

*Chinedu, Nwodo S1, Nwinyi, Obinna C1 and Okochi, Veronica I2

1Department of Biological Sciences, College of Science and Technology Covenant University, KM 10 Idiroko Road, Canaan Land, PMB 1023 Ota, Ogun State, Nigeria 2Department of Biochemistry, College of Medicine, University of Lagos, PMB 12003 Idiaraba

ABSTRACT

A wild-type Aspergillus niger (ANL301) isolated from wood-waste in Lagos, Nigeria, produces extracellular proteins with cellulase (EC 3. 2. 1. 4) activity. Three different carbon sources (Glucose, Cellulose and Sawdust) influenced the organism’s growth and the production of extracellular cellulase enzymes. Best growth was obtained with glucose at 72 hours of incubation. The peak mycelia weight of 1.56 mg/ mL obtained with glucose was about 3 times the maximum weight of 0.58 and 0.49 mg/ mL respectively obtained with cellulose and sawdust at 96 hours. The peak protein contents of the culture filtrates were 0.02, 0.15 and 0.69 mg/ mL respectively in the media containing glucose, cellulose and sawdust. There was no significant cellulase activity in the filtrates from glucose-containing media. The culture filtrates of the organism from cellulose- and sawdust-containing media yielded significant cellulase activities with maximum values of 105.6 Units /L (at 72 hours for cellulose) and 101.9 Units /L (at 144 hours for sawdust). There is a correlation between the protein content and cellulase activity of the culture filtrates. Sawdust can serve as a low-cost substrate for cellulase production by the organism. Keywords: Aspergillus niger ANL301, growth, cellulase activity, cellulosic materials.

INTRODUCTION There is a growing interest in the conversion of lignocelluloses into bulk chemicals and biofuels as a means of alleviating energy shortages and reducing pollution-load (Howard et al., 2003). One method being intensively studied is the hydrolysis of cellulosic materials into simple sugars and subsequent fermentation into ethanol (Fan et al., 1987; Spano et al., 1978). Cellulase is the generic name for the group of enzymes which catalyze the hydrolysis of cellulose and related cellooligosaccharide derivatives. The enzyme is potentially useful for industrial saccharification of cellulosic biomass. An economic process for its production is thought to be critical for successful utilization of cellulosic materials (Solomon et al., 1999; Wu and Lee, 1997). Cellulase is adaptive in most fungi; substances such as cellulose and sophorose are known to induce the enzyme production (Berry and Paterson, 1990; Ryu and Mandels, 1980). Major limiting factors on the commercial use of the enzyme are low activity and high cost of the available enzyme preparations (Spano et al., 1978). This has necessitated a renewed search for cellulolytic organisms with novel cellulase properties and strategies for low-cost enzyme production. In search of viable cellulolytic organisms, we isolated

different cellulolytic microfungi from decomposing wood-wastes in Lagos, Nigeria, which included Aspergillus niger AN301 (Nwodo-Chinedu et al., 2005). Aspergillus niger group is particularly noted for the secretion of extracellular enzymes which can hydrolyze the β-glycosidic bonds of native cellulose and associated hemicelluloses (de Vries and Visser, 2001). Aspergillus niger AN301grows rapidly on media containing sugarcane pulp and sawdust as sole carbon sources (Nwodo-Chinedu et al., 2007) and produces celulase (Nwodo-Chinedu et al., 2005) and xylanase enzymes (Okafor et al., 2007) in such media. In the present study, growth and cellulase production by the wild strain of Aspergillus niger AN301 cultured in submerged liquid media containing glucose, cellulose and sawdust were investigated. Our data shows rapid growth but no significant cellulase activity in glucose-containing media. In contrast there was low growth and very significant cellulase production in the media containing cellulose and sawdust. There appears to be a correlation between the protein content and cellulase activity of the cell-free filtrates. MATERIALS AND METHODS Chemicals All chemicals and reagents were of analytical grade. Potato Dextrose agar and crystalline cellulose (Avicel) were obtained from Merck, Germany. Carboxymethyl-Cellulose (CM52) was obtained from Whatman Ltd, *Corresponding author email: sncresearch@yahoo.com

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England. All other chemicals and reagents were obtained from Sigma Chemicals Co. Ltd, England. Sawdust Sawdust of Abora wood (Mitragyna ciliata) was collected from Okobaba Saw-mills, Ebute-Metta, Lagos, Nigeria. The sample was dried in the oven at 80 OC for 2 hours, ground with Marlex Exceller Grinder (Mumbai, India) and passed through a sieve (about 0.5 mm pore size) to obtain the fine powder used for the study. Media preparations The liquid media contained (per liter of distilled water): NaNO3, 3.0 g; KCl, 0.5 g; KH2PO4, 1.0 g; MnSO4. 7H2O4, 0.5 g; FeSO4. 7H2O, 0.01 g; and 10.0 g of the carbon source (Glucose, Crystalline cellulose or sawdust). One liter (1 L) of the media was supplemented with 1.0 mL of trace solution containing (per liter of distilled water) ZnSO4, 1.0 g and CuSO4. 5H2O, 0.5 g. The pH of each media was adjusted to 5.6. Organism The strain of Aspergillus niger (ANL301) was isolated from wood-wastes in Lagos, Nigeria and identified as described previously (Nwodo-Chinedu et al., 2005). The organism was maintained at 4oC on Potato Dextrose Agar (PDA) slants. Growth Studies Fresh sub-culture of the organism was made on sterile PDA plates and incubated at 30oC for 72-120 hours. The colonies on PDA plates were covered with 10 mL of 1.0% Tween 80 [Polyoxyethylene (20) Sorbitan Monooleate]. Conidia were harvested using sterile cotton swab and transferred into a sterile test tube. Serial dilutions of the suspension were made using 1.0% Tween 80 to obtain the spore suspension of 2.0-4.0 X 106 spores/ mL used as inoculum for the growth and other analyses. Two (2.0) mL of the spore suspension was inoculated into 100 mL of the respective sterile liquid medium placed in 250 mL Erlenmeyer flask. The flask was covered with sterile cotton wool and incubated at 30oC for 24 – 168 hours with continuous agitation at 100 osc min-1 using the Griffin flask shaker. Cultures were harvested at 24 hour intervals by filtration over a 168-hour period. The mycelium was washed and dried in the oven at 80 OC for 2 hours. The cell-free filtrates were used as crude protein and enzyme source. Protein assay Protein content of the culture filtrates was determined by the folin ciocalteau method described Lowry et al. (1951) using Bovine Serum Albumin (BSA) as standard. Cellulase (Endo-1, 4-β-Glucanase; EC 3. 2. 1. 4) assay A modification of the reducing sugar method described by Khan (1980) was used to for the assay of Endo-1, 4-β-

Glucanase (EC 3. 2. 1. 4) activity. Carboxylmethyl-cellulose (CMC) was used as enzyme substrate. The reaction mixture contained 2.0 mL of 0.1% (w/v) substrate in 0.1M sodium acetate buffer (pH 5.0) and 2.0 mL of cell-free culture supernatant (or 0.5 mL of partially purified enzyme). The mixture was incubated at 37oC in water bath with shaking for 30 minutes. The reducing sugar released was measured using 3, 5-dinitrosalicylic acid and read at 540nm using a spectrophotometer (Miller, 1959). The released reducing sugar was expressed in glucose equivalent and expressed in Units mL-1. A unit of activity was defined as amount of enzyme required to liberate 1µmol of Glucose per minute under the assay conditions. RESULTS The growth of Aspergillus niger ANL301in liquid media containing glucose, cellulose and sawdust is shown in figure 1. Very rapid growth was obtained in the media containing glucose where the mycelia weight reached a peak of 1.56 mg/mL after 96 hours. In cellulose containing media, the mycelia weight attained a peak of 0.58 mg/ mL after 72 hours. A maximum weight of 0.49 mg/mL was obtained after 96 hours in sawdust-containing medium. A sharp decline in mycelial weight was noted in all the media after the organism attained the peak growth. Figure 2 shows the cellulase activities of A. niger ANL301 in media containing cellulose and sawdust respectively. The cellulase activity of the organism cultured in cellulose-containing medium gave a peak of 105.6 Units/mL (X103) after 96 hours while a peak of 101.9 Units/mL (X 103) was obtained after 144 hours in sawdust-containing medium. The Protein yield of the organism cultured in the media containing cellulose and sawdust respectively is shown in figure 3. The organism produced much more protein in medium containing sawdust compared to that containing cellulose. The protein content of culture filtrates in cellulose-containing medium was highest (1.5 mg/mL) after 96 hours and maximum (6.9 mg/mL) in sawdust-containing medium after 72 hours. Plots of the protein contents and cellulase activities of culture filtrates of A. niger ANL301 cultivated in modified Czapek-Dox Broth containing cellulose and sawdust respectively are shown in figure 4. The plots show a direct relationship between the protein content and cellulase activity of the culture filtrates. In cellulose-containing medium, the protein content and cellulase activity of the cell-free filtrates peaked at the same period and declined afterwards (Fig. 4 a). In the medium containing sawdust, the protein content of the culture filtrates peaked before the cellulase activity. The highest protein content was obtained at 72 hours while the

Chinedu et al. 359

maximum cellulase activity was attained at 144 hours of incubation (Fig. 4b). DISCUSSION The liquid media containing glucose yielded higher amounts of mycelia compared to the modified media

containing cellulose or sawdust as the respective carbon source (Fig. 1). The peak growth period varied for the different carbon sources. It was 72 hours with cellulose as sole carbon source whereas the peak mycelial weight was at 96 hours for the media containing glucose or sawdust as sole carbon source. The differences in the carbon sources of the media could account for the disparity in the

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growth of the organism in the different media. Since glucose is more readily assimilated and metabolized by cells, there is greater tendency for organisms to grow very rapidly in media containing the simple sugar compared to that which contain cellulose or sawdust. Cellulose is a polymer of β-D-glucose while sawdust (wood) is composed of complex plant cell wall components which include cellulose, hemicelluloses and lignin (Grant and Long, 1981). In order to obtain simple sugars from cellulose or sawdust, the organism have to synthesize the enzymes required for the hydrolysis of the macromolecules. This may account for the much slower growth in media containing cellulose and sawdust. The protein content and cellulase activity of filtrates from glucose containing media were extremely low and thus considered insignificant. This is expected because the organism already has the simple sugar, glucose, in its media and hence do not need to produce the hydrolytic enzymes (proteins). Cellulases of most fungi are inducible and are also regulated by catabolite repression (Berry and Paterson, 1990). Absence of cellulose or other inducers as well as the high concentration of glucose in the medium will thus turn off the production of the enzymes. This may account for the low protein content and insignificant cellulase activity recorded in glucose-containing media, and goes on to suggest that the production of cellulase enzyme by the wild-type A. niger ANL301 is induced by cellulose and sawdust in the absence of glucose.

There were different periods for peak cellulase activity for the two carbon sources (cellulose and sawdust). The time was shorter in cellulose compared to sawdust. Cellulose is a homo-polysaccharide containing only β-D-glucose monomers whereas sawdust contains other polymers such as hemicelluloses, in addition to cellulose. When cellulose is the sole carbon source, the production of cellulases will be more rapid since glucose molecules needed for the organism’s metabolism must come from cellulose hydrolysis. This may not be the case when sawdust is the sole carbon source. Most microorganisms generally have far greater ability to depolymerize hemicelluloses compared to cellulose due to the greater solubility of the former (Grant and Long, 1981). Aspergillus niger ANL301 was found to produce high levels of xylanases when cultured on media containing sawdust and other agro-wastes (Okafor et al., 2007). In terms of protein yield, higher values were obtained in media containing sawdust compared to that containing crystalline cellulose (Fig. 3). The high protein released in the sawdust suggests the presence of other proteins (beside the cellulase enzyme) which may include other cell-wall hydrolyzing enzymes. Fungi such as Aspergillus species are known to produce many plant cell-wall hydrolyzing enzymes (de Vries and Visser, 2001). Hemicellulases particularly xylanases are also required for the hydrolysis of natural cellulose (Khan, 1980).

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Chinedu et al. 361

There appears to be a correlation between the protein content and cellulase activity of the crude enzyme obtained at the different period of incubation (Fig. 4). The organism seems to secrete the hydrolytic enzymes for the breakdown of the polymers into the growth media which largely accounts for the protein contents of the cell-free filtrates. The amounts of protein released appear to be a

function of the complexity of the carbon sources. The more complex the carbon source, the greater the amount of proteins secreted. In conclusion, the strain of Aspergillus niger ANL301 produces extracellular proteins with significant cellulase activity when cultured on media containing cellulose and

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sawdust as sole carbon sources. On the other hand, Aspergillus niger ANL301did not yield significant extracellular protein or cellulase activity when cultured on media containing glucose as sole carbon source. Much more protein was produced on sawdust compared to cellulose, but the maximum cellulase activities of the filtrates from both culture media were about the same. Sawdust is indicated as a good inducer of cellulase activity in Aspergillus niger ANL301. The waste cellulosic material is available in abundance and can be used as low-cost carbon source for the production of commercial cellulases. Such use could help reduce the pollution due to wood-wastes. REFERENCES Berry, DR. and Paterson, A. 1990. Enzymes in Food Industry. In: Enzyme Chemistry, Impact and applications, 2nd Edition. eds. Suckling C.J. 306-351.

De Vries, RP. and Visser, J. 2001. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiology and Molecular Biology Review. 65: 497-552.

Fan, LT., Gharpuray, MM. and Lee, YN. 1987. Cellulose Hydrolysis Berlin, Germany: Springer-Verlag. 3: 1-68.

Grant, WD. and Long, PE. 1981. The carbon cycle. In: Environmental Microbiology, Tertiary level Biology. Thomas Litho Ltd., Scotland. 91-116.

Howard, RLI, Abotsi, E., Jansen Van Rensburg, ELI. and Howard, S. 2003. Lignocellulose biotechnology: issues of bioconversion and enzyme production. Review. African Journal of Biotechnology. 2 (12): 602-619.

Khan, AW. 1980. Cellulolytic enzyme system of Activibrio cellulolyticus, a newly isolated Anaerobe Journal of General Microbiology. 121: 499-502.

Lowry, OH., Rosebrough, N.J., Farr, AL. and Randall, RJ. 1951. Protein measurement with the folin-phenol reagent. Journal of Biological Chemistry. 193: 265-275.

Miller, GL. 1959. Use of dinitrosalicyclic reagent for the determination of reducing sugars. Analytical Chemistry. 31: 426-428.

Nwodo-Chinedu, S. Okochi, VI., Smith, HA. and Omidiji, O. 2005. Isolation of cellulolytic microfungi involved in wood-waste decomposition: Prospect for enzymatic hydrolysis of cellulosic wastes. International Journal of Biomedical and Health Sciences. 1(2): 41-51.

Nwodo-Chinedu, S., Okochi, VI., Omidiji, O., Omowaye, O. O., Adeniji, B. R., Olukoju, D. and Chidozie, F. 2007. Potentials of cellulosic wastes in media formulation. African Journal of Biotechnology. 6 (3): 243-246.

Okafor, UA., Okochi, VI., Onyegeme-Okerenta, MB. and Nwodo-Chinedu, S. 2007. Xylanase production by

Aspergillus niger ANL301using agro-wastes. African Journal of Biotechnology. 6 (14): 1710-1714.

Ryu, DD. and Mandels, M. 1980. Cellulases: Biosynthesis and Applications. Enzyme Microbiology and Technology. 2: 92-102.

Solomon, BO., Amigun, B., Betiku, E., Ojumu, TV. and Layokun, SK. 1999. Optimization of cellulase production by Aspergillus flavus Linn Isolate NSPR101 Grown on Bagasse. JNSCHE. 16: 61-68

Spano, L., Alien, A., Tarssinane, T., Mandels, M. and Ryu, DD. 1978. Reassessment of economics of Technology for production of ethanol Proceedings in Industrial fuel from biomass symposium. Troy, New York, USA. 671-674.

Wu, Z. and Lee, YY. 1997. Inhibition of the enzymatic hydrolysis of cellulose by ethanol. Biotechnology Letters. 19: 977-979.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 363-371, 2008 ISSN: 1715-9997

1

EXPOSURE AND RECOVERY RESPONSE OF PESTICIDES ON TISSUE BIOCONCENTRATION AND PLASMA SEX STEROID HORMONES

IN HETEROPNEUSTES FOSSILIS

*Pratap B Singh and Vandana Singh Department of Zoology, Tilak Dhari College, Jaunpur-222002, India

ABSTRACT

Effects of 40 days exposure and 20 days recovery response at sublethal concentration of technical grades of gamma isomer of hexachlorocyclohexane (γ-HCH-0.025 ppm), dichlorodiphenyltrichloroethane (DDT-5.0 ppm) and chlorpyrifos (0.5 ppm) on the percentage rate of bioconcentrations in blood, brain, liver, muscles, ovary, gonadosomatic index (GSI) and plasma levels of testosterone and estradiol-17β were studied during prespawning phase in catfish, Heteropneustes fossilis (Bloch) during its annual breeding cycle. All pesticides caused maximum bioconcentration either in liver or ovary. Recoveries of these pesticides have also been recorded in aforesaid tissues except DDT in blood, brain, muscles and ovary when kept the fishes in pesticide free water. The percentage of bioconcentrations were maximum in all tissues but recoveries were maximum for blood (γ-HCH), muscles (DDT) and liver (chlorpyrifos). The GSI, testosterone and estradiol-17β were decreased at all doses of pesticides. The exposed catfish kept in pesticide free water caused recoveries in GSI, testosterone and estradiol-17β. Our results indicate that pesticides have preferential order of percentage of bioconcentration in different tissues and have very selective effects on sex hormones thereby affecting reproductive physiology. Restoration of normal reproductive physiology during recovery phase might be due to dissipations of pesticides. Keywords: Pesticide residues in fish; recoveries; testosterone; estradiol-17β; Heteropneustes fossilis.

INTRODUCTION Tissue concentrations of pesticides in wild captured fish have been reported by several workers (Letherland and Sonstegard, 1982; Von Westernhagen et al., 1987; Kannan et al., 1995; Singh et al., 1997; Sahagun et al., 1998; Ferreira et al., 2004; Antunes and Gil, 2004; Saqib et al., 2005; Amado et al., 2006; Antunes et al., 2007a, b). It has been reported that pesticides even very low doses inhibits lipid metabolism (Singh, 1992) and steroidogenesis (Singh et al., 1994; Kime and Singh, 1996; Singh and Canario, 2004). Hilmy et al. (1983) have reported when exposure of DDT was done to Anguilla vulgaris and Mugil cephalus the bioconcentration of DDT in tissue recorded highest in liver than gonads, gills and muscles. Organochlorine pesticide show very low accumulation in the muscles, but concentrations in liver are in general 10-100 fold higher. Recovery response for different pesticides have also been reported (Aditya and Chattopadhyay, 2000; Kumari et al., 2001; Sarkar et al., 2005). Very few literatures are available which indicate the exposure study of pesticides under laboratory condition and their bioconcentration in different tissues during pre-

spawning phase to monitor percentage rate of bioconcentration in different tissues and recovery response in pesticide free water during reproductive growth. This prompted us to explore 1- Exposure for 40 days at sublethal concentration of γ-HCH, DDT and chlorpyrifos on percentage of tissues bioconcentration of total hexachlorocyclohexane (ΣHCH), dichlorodiphenyl-trichloroethane (ΣDDT), gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and estradiol-17β) during prespawning phase (when the vitellogenesis was continuing and have maximum steroidogenic activity) and 2. Percentage recovery studies by keeping the exposed fish in pesticide free water for 20 days on the above parameters. MATERIALS AND METHODS Experimental fish The original research reported herein was conducted under ethical guidelines for the treatment of animals in behavioral research and teaching (Anonymous, 1998) established for animal usage by Tilak Dhari College, Jaunpur (UP). The experimental fish, Heteropneustes fossilis (65-70 g and length 21-22 cm) were collected from a pond of the same brood stock during prespawning

*Corresponding author email: pratap_b_singh@rediffmail.com

Canadian Journal of Pure and Applied Sciences 364

phase season and maintained in cemented tank of size 2 x 1 x 1-m supplied with circulating constant flow of dechlorinated tap water and enjoyed natural photoperiod (13.0L : 11.OD) and temperature (30 ± 2oC). They were fed ad libitum with minced goat liver comprising 20% protein, 5% lipid, 15% carbohydrate the remaining 60% being water, minerals and vitamins etc. Chemicals Analytical grade chemicals were obtained from E. Merck, Hi Media, India and Sigma Chemicals Co., USA. After a week of acclimation, experiments were performed in glass aquaria. In each glass aquaria, 15 fish were kept in 20 l water separately for individual pesticides. The total fish used in this study was 40. Experiment 1- Toxicity test of γ-HCH, DDT and chlorpyrifos The toxicity tests have been done as per method described by Singh et al. (1994; Singh and Singh, 2007). The LC50 for 96 hr of technical grade for γ-HCH (99.8%), DDT (72-74%) and chlorpyrifos (94%) was 0.125, 25 and 2.5 ppm respectively. The dose 1/5th of LC50 96 h was taken for each pesticide as sublethal concentration for 40 days exposure. The details of treatments for exposure and recoveries study have been given (Tables 1, 2). Experiment 2- Estimation of percentage of bioconcentration of pesticide residue in fish after 40 days exposure during prespawning phase of the annual reproductive cycle During experimentation fish were fed every fourth day when the aquarium water was changed with fresh water having similar concentration of pesticides. At the end of exposure fish were bled by caudal puncture and blood was collected to required volume in heparinized (1% heparin sodium salt activity 1,00,000 units 140.3 U/mg)

glass tubes. After decapitation brain, liver, abdominal muscles and gonad were dissected out, washed in saline (0.6% NaCl) blotted and kept at -20oC for extraction and analysis of percentage of bioconcentration of total individual pesticide residues. The half of the blood was centrifuged at 10,000 rpm for 15 minutes at 4oC for plasma sex steroid hormone analysis by radioimmunoassay. The GSI was calculated (gonad weight x 100/ body weight). Experiment 3- Recovery studies in pesticide free water of exposed fish tissue concentrations and sex steroid hormones Five fish from exposed fish of each batch were kept in pesticide free water for 20 days to observe the recoveries of percentage of tissue concentration of pesticide, GSI and plasma sex steroid hormones.

Extraction procedure and cleanup of organochlorines insecticides from blood and tissues Extraction and cleanup procedure was followed as per method described (Dale et al., 1965) with modifications. Briefly, the fatty tissues from 5 individual of different groups of each in duplicate (0.5 g-brain and ovary or 1.0 g non-fatty tissues-liver, muscles and 1ml blood in heparinized tubes) were homogenized in a homogenizer cup by using 7 ml of formic acid and 5ml n-hexane (glc grade only). The homogenate was transferred in the conical flask by rinsing the cup and teflan pestle twice with 10 ml of n-hexane each time. The homogenate was shaken on shaker for 1 h at 37oC. The upper layer of n-hexane was separated in another conical flask and the aqueous layer was again extracted with 10 ml of n-hexane 2 times (20 ml) by similar fashion. The organic layer was collected together. The resididue (formic acid) from the extract was removed by shaking the organic phase with distilled water (50 ml) in separating funnel. This extract

Table 1. Details of treatments of Heteropneustus fossilis for 40 days exposure at sublethal concentrations of pesticides*.

Batches Treatments of technical grade (γ-HCH- 99.8%, DDT-72.74%, chlorpyrifos-94%) of pesticides

1 2 3

γ-HCH treated with 0.025 ppm DDT treated with 5.0 ppm

Chlorpyrifos treated with 0.5 ppm

* The dose 1/5th of LC (50) 96 hour was used (0.125, 25, 2.5 ppm for HCH, DDT and chlorpyrifos respectively). Table 2. Details of treatments of exposed fish for 20 day recovery during pre-monsoon.

Batches Treatments

1 2 3

Control HCH treated fish kept for 20 days in pesticide free water* DDT treated fish kept for 20 days in pesticide free water

Chlorpyrifos treated fish kept for 20 days in pesticide free water

* dechlorinated water

Singh and Singh 365

was demoisturised by passing through anhydrous sodium sulfate bed. The demoisturised extract was concentrated on rota evaporator up to 1 ml and finally volume was made up to 2 ml in volumetric flask with help of n-hexane by rinsing the rota evaporator flask. The cleanup procedure for required pesticide was done by adding 2 ml

concentrate sulfuric acid in the above extracted 2 ml extract. This mixture was vortexed and centrifuzed to separate the aqueous and non-aqueous layer. The upper n-hexane layer was separated in 2 ml clean volumetric flask with the help of Pasteur pipette individual samples. This concentrated sample was applied on Gas Liquid

*

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Blood Brain Liver Muscles Ovary

40 days exposure of γ-HCH

Tiss

ue b

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f ΣH

CH

(Mea

n +

SEM

, n =

5)

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Fig. 1. Tissue bioconcentrations of γ-hexachlorocyclohexane (γ-HCH-0.025 ppm technical grade at sublethal concentration) in different tissues of the catfish, H. fossilis after 40 days exposure during prespawning phase of the annual reproductive cycle. Control vs γ-HCH exposed were compared by Students t -test. The level of significance (P)- *P< 0.001.

**

NS

* *0

500

1000

1500

2000

2500

3000

Blood Brain Liver Muscles Ovary

20 days recovery in γ-HCH free water

γ-HCH exposed controlΣHCH recovered in fresh water

Fig. 2. Tissue recoveries of ΣHCH after 20 days treatments wγ-HCH free water of 40 days γ-HCH exposed catfish, H. fossilγ-HCH exposed control vs recovered values were compared Students t-test. The level of significance (P)- *P< 0.001. NS- nsignificant.

*

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Blood Brain Liver Muscles Ovary

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ue b

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ns o

f ΣD

DT

(Mea

n +

SEM

, n =

5)

Control DDT exposed

Fig. 3. Tissue bioconcentrations of dichlorodiphenyl-trichloroethane (DDT-5.0 ppm technical grade at sublethal concentration) in different tissues of the catfish, H. fossilis after 40 days exposure during prespawning phase of the annual reproductive cycle. Control vs DDT exposed were compared by Students t-test. The level of significance (P)- *P< 0.001.

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Blood Brain Liver Muscles Ovary

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DT

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CH

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ean

+ SE

M, n

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)

Canadian Journal of Pure and Applied Sciences 366

Chromatography (GLC) for identification and quanti-fication of isomers of HCH and metabolites of DDT from which ΣHCH and ΣDDT calculated. Extraction procedure and cleanup of chlorpyrifos from blood and tissues The method for extraction was used with modification as have been described earlier (Lino et al., 1994). Known amount of each tissue from 5 individual of different groups in duplicate (0.5 g fatty tissue- brain and ovary, 1 g non-fatty tissue- liver and 1 ml blood in heparinized tubes) were collected separately and then homogenized with 5 ml of n-hexane. The content was then transferred into conical flask by adding the wash (10 ml) of teflon pestle twice with n-hexane (20 ml). Now, each flask was shaken 1 h for the extraction of pesticides at 37oC. Decanted the n-hexane layer and residue was re-extracted with 10 ml of n-hexane twice after 30 min shaking each. Filtered the extracted content with Whatman filter paper No. 1. Now contents were passed through the anhydrous sodium sulfate bed and concentrated on rota evaporator up to 5 ml. This concentrated extract was passed through the bed of anhydrous sodium sulfate (1g), activated Florisil (4g) and anhydrous sodium sulfate (1g) prepared in column 400 mm x 20 mm i.d. for pesticides cleanup. The contents was eluted with 200 ml of diethylether : n-hexane mixture (6 : 94) for the extraction of chlorpyrifos. Now concentrated the contents into 1 ml on evaporator and finally made up the volume to 2 ml by rinsing the evaporatory flask with n-hexane for the analysis and quantification of chlorpyrifos by GLC.

Quantitative analysis The quantitative analysis of organochlorines (isomers of HCH and metabolites of DDT) and organophosphorus (chlorpyrifos) were performed by Gas Chromatography (Nucon 5765) equipped with 63Ni electran capture detecter (ECD). The GC column (6 inch x 1/8 inch i.d.) filled with 80-100 mesh, Gas Chrome coated with a mixture of 1.5 / SP-2250 and 1.95% SP-2401. Oven temperature was 190oC. The injector and detector temperature were kept at 250oC. Nitrogen IOL-AR grade I was kept as carrier gas (flow rate 60 ml/min). The volume of injection for each unknown samples and standard were 2-5µl depending upon concentration of pesticides in samples. Pesticides were estimated from individually resolved peak of samples with corresponding peaks of standards. The confirmation of chlorpyrifos was done on NPD detector in same column and conditions. Recovery studies for pesticide extraction and elution Representative samples of each matrix were spiked with known concentrations of the isomers of HCH and DDT metabolites as well as chloripyrifos and kept at least for 3 h. The samples were extracted and cleaned up for analysis using GLC equipped with ECD system. The commodities are calculated and the recovery was approximate 94% and recovery factor 1.0638. Detection limit was 0.1ng/g (ppb) for HCH, DDT and chlorpyrifos. Extraction and radioimmunoassay (RIA) of sex steroids Sex steroids were extracted twice from plasma (400 µl or less as appropriate) with 5 ml of distilled dichloro-

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Blood Brain Liver Muscles Ovary

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Control Chlorpyrifos treated

7

Fig. 5. Tissue bioconcentrations of chlorpyrifos (0.5 ppm technical grade at sublethal concentration) in different tissues of the catfish, H. fossilis after 40 days exposure during prespawning phase of the annual reproductive cycle. Control vs treated were compared by Students t-test. The level of significance (P)- *P< 0.001.

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Blood Brain Liver Muscles Ovary

20 days recovery in chlorpyrifos free water

Chlorpyrifos treated control

Chlorpyrifos recovered in fresh water

Fig. 6. Tissue recoveries of chlorpyrifos after 20 days treatments with pesticide free water of 40 days chlorpyrifos exposed catfish, H. fossilis. Chlorpyrifos exposed control vs recovered values were compared by Students t-test. The level of significance (P)- *P< 0.001.

Tiss

ue re

cove

red

of c

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pyrif

os (M

ean

+ SE

M, n

= 5

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orpy

rifos

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Singh and Singh 367

methane. The details of RIA have been described elsewhere (Singh and Singh, 1992). The precision of the method as expressed in the percentage coefficient of variation were 4.2 and 3.9% intra-assay and 5.1 and 6.1% inter-assay for testosterone (T) and estradiol-17β (E2), respectively. Detection limits in the present assay were 6 pg for T and E2. Steroid antiserum had a cross reactivity of 100% with the respective hormones. The % recovery of double extraction of T and E2 were 97.7 and 97.3% respectively. The concentration coefficient between observed and expected values was 0.99 for each of sex steroids. Statistical analysis Values were expressed as ng / ml or ng / g of tissue (ppb) for pesticides and ng / ml plasma for sex steroid hormones (Mean ± SEM, n = 5). Data were analyzed by Students t-test for the level of significance (Bruning and Kintz, 1977). RESULTS Effect of γ-HCH exposure for 40 days on tissue bioconcentrations at sublethal concentrations and recoveries in γ-HCH free water for 20 days during prespawning phase in H. fossilis under laboratory conditions The effect of γ-HCH exposure for 40 days have indicated that the bioconcentrations was maximum in liver and minimum in blood. The preferential order of bioconcentration for total HCH were recorded in tissues (liver > ovary > brain > muscles > blood) (Fig. 1). After 40 days of γ-HCH exposure when fish were treated with γ-HCH free water for 20 days, the bioconcentrations were dissipated in preferential order of tissues (ovary > liver > brain > muscles > blood) (Fig. 2). Effect of DDT exposure for 40 days on tissue bioconcentrations at sublethal concentration and depuration in DDT free water for 20 days during prespawning phase in H. fossilis The effect of DDT exposure for 40 days has indicated that the bioconcentrations was maximum in liver and minimum in muscles. The preferential order of bioconcentrations for ΣDDT were recorded in tissues (liver > ovary > brain > blood > muscles) (Fig. 3). After 40 days of DDT exposed fish when treated with 20 days of DDT free water the bioconcentration of DDT was raised in preferential order of tissues (brain > ovary > muscles > blood) whereas declined in liver (Fig. 4).

Effect of chlorpyrifos exposure for 40 days on tissue bioconcentrations at sublethal concentration and depuration in chlorpyrifos free water for 20 days during prespawning phase in H. fossilis The effect of chlorpyrifos exposure for 40 days has showed that the bioconcentrations was maximum in ovary

and minimum in blood. The preferential order of bioconcentrations for chlorpyrifos were recorded in tissues (ovary > liver > brain > muscles > blood) (Fig. 5). After 40 days of chlorpyrifos exposed fish when treated with 20 days of chlorpyrifos free water the bioconcentration were dissipated in the preferential order (blood > ovary > liver > brain > muscles) of tissues (Fig. 6). Percentage of bioconcentrations of ΣHCH, ΣDDT and chlorpyrifos exposure for 40 days under laboratory conditions in different tissues during prespawning phase in H. fossilis After 40 days of γ-HCH and chlorpyrifos exposure the tissue bioconcentrations for ΣHCH were maximum for liver and minimum in brain whereas ΣDDT showed high percentage of incorporation in liver but minimum in muscles (Fig. 7).

γ-HCH exposed

Liver 1,530.28 (56%)

Ovary 391.44 (14%)

Muscles 405.52 (15%)

Brain 182.35(7%)

Blood 215.81 (8%)

DDT exposed

Liver 2,734.27 (43%)

Muscles 476.56 (7%)

Ovary 1,288.3 (20%) Blood 802.69

(13%)

Brain 1,074.32(17%)

Chlorpyrifos exposed

Liver 30,292.76 (63%)

Muscles 147.07(7%)

Ovary 11,656.26(24%)

Blood 2,364.45(5%)

Brain 684.62 (1%)

Fig. 7. Pie diagram showing percentage of bioconcentrations of ΣHCH, ΣDDT and chlorpyrifos exposure for 40 days under laboratory conditions in different tissues during prespawning phase in H. fossilis.

Canadian Journal of Pure and Applied Sciences 368

Percentage of depuration in tissue bioconcentrations of γ-HCH, DDT and chlorpyrifos treated fish when kept for 20 days in pesticide free water Treatments of exposed fish with 20 days in pesticide free water the tissues dissipations of ΣHCH were maximum in blood and minimum in brain. The preferential order of depuration were recorded (blood > muscles > liver > ovary > brain) in tissues. The ΣDDT has shown the increased bioconcentrations (muscles > brain > blood > ovary) in tissues whereas only liver showed dissipations by 69.78%. The chlorpyrifos exposed fish when kept in chlorpyrifos free water for 20 days showed depuration maximum in ovary and minimum in blood and were recorded in preferential order of depuration in tissues (ovary > liver > muscles > brain > blood) (Fig. 8).

ΣHCH recoveries

Muscles 51.01 (26%)

Liver 44.71(23%)

Brain 2.22(1%)

Blood 72.45(37%)

Ovary 24.72(13%)

ΣDDT recoveries

Blood 260.58(17%)

Ovary 112.56(7%)

Brain 429.39(28%)

Liver 69.78(5%)

Muscles 650.03 43%

Chlorpyrifos recoveries

Liver 95.6(22%)

Brain 93.66(23%)

Blood 46.74(11%)Ovary 95.71

(22%)

Muscles 95.48 (22%)

Fig. 8. Pie diagram showing percentage of recoveries of ΣHCH, ΣDDT and chlorpyrifos exposed fish when treated with pesticide free water for 20 days in H. fossilis. Effect of γ-HCH, DDT and chlorpyrifos exposure for 40 days on gonadosomatic index (GSI) and plasma levels of testosterone (T) and estradiol-17β (E2) in H.

fossilis during prespawning phase of the annual reproductive cycle Exposure of pesticide caused significant decline in GSI and was maximum by γ-HCH than DDT and chlorpyrifos tested. The plasma levels of T was maximum in chlorpyrifos than the γ-HCH and DDT whereas the levels of E2 was suppressed maximum by DDT than the γ-HCH and chlorpyrifos exposure (Fig. 9, 10).

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SI, M

ean

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7̀

Fig. 9. Gonadosomatic index (GSI) after 40 days exposure at sublethal concentration of technical grade of γ-HCH, DDT and chlorpyrifos during pre-spawning phase (May and June) of the annual reproductive cycle in the female catfish, H. fossilis. Control vs pesticide treated were compared by Students t-test. The levels of significance (P)- *P< 0.001; **P< 0.005.

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DDT Chlorpyrifos

Fig.10. Plasma levels of testosterone and estradiol-17β after 40 days exposure at sublethal concentration of technical grade of γ-HCH, DDT and chlorpyrifos during pre-spawning phase of the annual reproductive cycle in the female catfish, H. fossilis. Control vs pesticide treated were compared by Students t-test. The level of significance (P) *P< 0.001.

Singh and Singh 369

Recoveries of GSI, plasma levels of T and E2 in pesticide free water for 20 days The γ-HCH, DDT and chlorpyrifos exposed fish when kept in pesticide free water showed varied effects. In γ-HCH and DDT groups the GSI has tendency to recover but it was not significant up to 20 days of the recovery in pesticide free water whereas chlorpyrifos treated fish recovered significantly. Interestingly, the plasma levels of T recovered significantly but more recovery was noticed by DDT. The recoveries for E2 was recorded only by γ-HCH and chlorpyrifos but remained insignificant by DDT treated fish kept in DDT free water (Fig. 11, 12). DISCUSSION Our results have clearly demonstrated that the exposure of technical grades of γ-HCH, DDT and chlorpyrifos at sublethal concentrations during pre-monsoon caused bioconcentration of these pesticides in different tissues (Fig 1,3,5) and also diminished the plasma levels of sex steroids (Fig.10) thereby affected the reproductive physiology of this species. Tissue bioconcentrations of pesticide in different tissues has varied effects in preferential order of bioconcentration. Although ΣHCH and ΣDDT has similar trends of bioconcentrations in tissues (liver > ovary > brain) and gonad has intermediate in position. The chlorpyrifos bioconcentration in liver was in the intermediate (ovary > liver > brain) position in H.

fossils. It is suggested that prespawning ovary appears to be the important organs of γ-HCH, DDT and chlorpyrifos bioconcentrations. Aditya and Chattopadhyaya (2000) have reported the accumulation of pesticide residue occurred in the order muscle > testes > ovary when adult Labeo rohita were sub lethally (1/5th 96h LC 50) exposed to methyl parathion during prespawning, spawning and post-spawning phase. Among the pesticide DDT was maximum in its percentage bioconcentration in liver compared to γ-HCH and chlorpyrifos which may be due to more persistent than the other insecticides. The decreased levels of sex steroids have been reported by Singh and Singh (1992) in this species for malathion and γ-BHC exposure. Results have shown that when exposed fish kept in 20 days in pesticide free water pesticides dissipation occurred for ΣHCH and chlorpyrifos in different tissues. The DDT level was increased in all the studied tissues but declined in liver when exposed fish were kept in DDT free water for 20 days. Here it may be interpreted that the loss or dissipation in liver may be due to metabolism of these pesticides by microsomal enzymes involved in detoxification but reason for increase in bioconcentrations by DDT only is unclear at this stage. The percentage of bioconcentrations was highest in the liver by all pesticide tested. However, the percentage of bioconcentrations varied for γ-HCH, DDT and chlorpyrifos exposure in

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GSI

, Mea

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5γ-HCH-exposed controlΣHCH-recoveryDDT exposed controlΣDDT recoveryChlorpyrifos exposed controlChlorpyrifos recovery

Fig. 11. Recoveries of gonadosomatic index (GSI) in pesticide free water for 20 days of exposed fish in the H. fossilis. Pesticide exposed (Control) vs recovered values were compared by Students t-test. Level of significance (P)- *P< 0.05. NS- not significant.

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γ-HCH exposed controlΣHCH recoveryDDT exposed controlΣDDT recoveryChlorpyrifos exposed controlChlorpyrifos recovery

Fig. 12. Recovery of plasma levels of testosterone and estradiol-17β hormones in pesticide free water for 20 days of exposed fish in the H. fossilis. Pesticide exposed (control) vs recovered values were compared by Students t-test. The levels of significance (P)- *P< 0.01; **P< 0.02; ***P< 0.05. NS- not significant.

Canadian Journal of Pure and Applied Sciences 370

different tissues among which chlorpyrifos was maximal. This result is supported by the work of Hilmy et al. (1983) who have demonstrated 10-100 folds higher for organochlorines in freshwater fishes. The recovery percentages have also been recorded for above pesticides when kept in free water for 20 days. The recovery responses have also been reported by Chandra et al. (2004) in Cyprinus carpio after exposure to carbofuran which also supports result obtained in our observations. Another report of Begum (2005) has also demonstrated the recovery response in Clarias batrachus when transferred to cypermethrin free water for protein, glycogen and enzymes. In the present observations, GSI was found to be depressed after pesticide exposure but recovery was noticed in GSI by chlorpyrifos. The decrease in GSI, T and E2 in exposed fish when recovered in pesticide free water indicates the significant correlation between GSI and steroidogenesis. Here it can also be suggested that withdrawal of pesticide may be due to recovery in aromatase enzyme responsible for steroidogenesis in this species. These results get support from the result of Sarkar et al. (2005) who has also recorded recovery response in histological changes in liver of Labeo rohita when kept in carbofuran and cypermethrin free water. Smita et al. (2003) has reported gonadal recrudescence and recovery response in Cyprins carpio after long term exposure to carbamate pesticide. Above findings supports our result. Interestingly in H. fossilis on exposure of three pesticide caused decline in plasma T and E2, and exposed fish when kept in pesticide free water for 20 days there was recovery in plasma T levels. The levels of E2 was elevated only by γ-HCH and chlorpyrifos free water but not by DDT which suggested that enzyme involved for the conversion of T into E2 is disrupted by DDT during prespawning phase. These findings get support form the results of Mills et al. (2001) who have reported that flounder treated with DDT showed in reduction in T and E2. In conclusion, pesticide on exposure get bioconcentrated in tissues in the preferential order in which gonad is an important organ for its bioconcentrations causing disturbance in metabolism and inhibits steroidogenesis thereby affecting reproductive physiology of this species. Recovery studies have indicated that exposed fish has tendency to restore the hormonal profile in due course required for normal reproductive physiology, information which may be of use to pisciculturists. ACKNOWLEDGEMENTS A grant-in-aid from Department of Science and Technology (DST Ref. No. SR/SO/AS-07/2004 dated 12.7.05), Government of India, New Delhi-110 016 to

PBS is greatly appreciated. The authors are grateful to Dr. B. Jayaraman, Hindustan Insecticide Ltd., Gurgaon (Haryana) for the gift of technical grades of DDT (70-72%), γ-HCH (lindane-99.8%) and chlorpyrifos (94.0%) for my experiments. We are also grateful to Industrial Toxicology Research Centre (ITRC), Lucknow for GLC facilities. REFERENCES Aditya, AK. and Chattopadhyaya, S. 2000. Accumulation of methyl parathion in the muscle and gonad of Labeo rohita. Journal of Environmental Biology. 21(1): 55-57.

Amado, J., Antunes, P., Gil, O. and Vale, C. 2006. Mobility of organochlorines in muscles of sardine (Sardina pilchardus) during spawning in the Portugues coast. Ciencias Marinus. 32 (2B): 369-377.

Antunes, P. and Gil, O. 2004. PCB and DDT contamination in cultivated and wild sea bass from Ria de Aveiro, Portugal. Chemosphere. 54 (10): 1503-1507.

Antunes, P., Gil, O., Ferreira, M., Vale, C. and Reis-Henriques, MA. 2007a. Depuration of PCBs and DDTs in mullet under captivity clean conditions. Chemosphere. 67 (9): 558-564.

Antunes, P., Amado, J., Vale, C. and Gil, O. 2007b. Influence of the chemical structure on mobility of PCB cogeners in female and male sardine (Sardina pilchardus) from Portuguese coast. Chemosphere 69: 395-402.

Anonymous, 1998. Guidelines for the treatment of animals in behavioral research and teaching. Animal Behavior. 55: 251-257.

Begum, G. 2005. In vivo biochemical changes in liver and gill of Clarias batrachus during cypermethrin exposure and following cessation of exposure. Pesticide Biochemistry and Physiology. 82(3): 185-196.

Bruning, JL. and Kintz, BL. 1977. Computational Handbook of Statistics, Second Edition, Freeman, Chicago, IL, USA.

Chandra, S., Ram, RN. and Singh, IJ. 2004. First ovarian maturity and recovery response in common carp Cyprinus carpio after exposure to carbofuran. Journal of Environmental Biology. 25(3): 239-249.

Dale, WE., Copeland, MF. and Hayes, WJ Jr. 1965. Chlorinated insecticides in the bodies of peoples in India. Bulletin of World Health Organisation. 33: 471-477.

Ferreira, M., Antunes, P., Gil, O., Vale, C. and Reis-Henriques, MA. 2004. Organochlorine contaminants in flounder (Platichthys flexux) and mullet (Mugil cephalus) from Douro estuary, and their use as sentinel species for environmental monitoring. Aquatic Toxicology. 69 (4): 347-357.

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Hilmy, AM., Badawi, HK. and Shabana, MB. 1983. Organochlorine pesticide residues in 12 freshwater Egyptian fish species with special emphasis on Anguilla vulgaris and Mugil cephalus. Comparative Biochemistry and Physiology. 76C: 163-171.

Kannan, K., Tanabe, S. and Tatsukawa, K. 1995. Geographical distribution and accumulation features of Organochlorine residues in fish in tropical Asia and Oceania. Environmental Science and Technology. 29: 2673-2683.

Kime, DE. and Singh, PB. 1996. In vitro effects of γ-hexachlorocyclohexane on in vitro biosynthesis and metabolism of steroids in goldfish, Carassius auratus. Ecotoxicology and Environmental Safety. 34(2): 165-173.

Kumari, A., Sinha, RK. and Gopal, K. 2001. Quantitative estimation of DDT residues in some freshwater fishes of river Ganga at Patna, Bihar. Environment and Ecology. 19(2): 396-399.

Letherland, JF. and Sonstegard, RA. 1982. Bioaccumulation of organochlorines by yearling coho salmon (Oncorhychus kisutch Walbaum) fed diets containing Great Lakes coho salmon, and the pathophysiological responses of the recipients. Comparative Biochemistry and Physiology. 72C: 91-99.

Mills, LJ., Gutjahr-Gobell, RE., Haebler, RA., Horowitz, DJB., Jayaraman, S., Pruell, RJ., McKinney, RA., Gardner, GR. and Zaroogian, GE. 2001. Effect of estrogenic (o,p′-DDT; octylphenol) and antiandrogenic (p,p′-DDE) chemicals on indicators of endocrine status in juvenile male summer flounder (Paralichthys dentatus). Aquatic Toxicology. 52 (2): 157-176.

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Saqib, TA., Naqvi, SNH., Siddiqui, PA. and Azmi, MA. 2005. Detection of pesticide residues in muscles, liver and fat of 3 species of Labeo found in Kalri and Haleji lakes. Journal of Environmental Biology. 26 (2): 433-438.

Sahagun, AM. Tern, MT., Garcia, JJ., Sierra, M., Fernandes, N. and Fiz, MS. 1998. Organochlorines pesticide residues in muscles tissues of rainbow trout Onchorhynchus nukiss taken from four fish farms in Leon, Spain. Food Additive and Contamination. 15 (5): 501-505.

Sarkar, B., Chatterjee, A., Adhikari, S. and Ayyappan, S. 2005. Carbofuran-and cypermethrin-induced histo-pathological alterations in the liver of Labeo rohita

(Hamilton) and its recovery. Journal of Applied Ichthyology. 21(2): 131-135.

Singh, PB. 1992. Impact of malathion and γ-BHC on lipid metabolism in the freshwater female catfish, Heteropneustes fossilis, Ecotoxicology and Environmental Safety. 23: 22-32.

Singh, PB. and Singh, TP. 1992. Impact of malathion and γ-BHC on steroidogenesis in the freshwater catfish, Heteropneustes fossilis. Aquatic Toxicology. 22: 69-80.

Singh, PB. and Singh V. 2007. Exposure and recovery response of isomers of HCH, metabolites of DDT and estradiol-17β in the female catfish, Heteropneustes fossilis. Environmental Toxicology and Pharmacology, 24: 245-251

Singh, PB., Kime, DE., Epler, P. and Chyb, J. 1994. Impact of γ-hexachlorocyclohexane exposure on plasma gonadotropin levels and in vitro stimulation of gonadal steroid production by carp hypophyseal hemogonate in Carassius auratus. Journal of Fish Biology. 44(2): 195-204.

Singh, N., Shukla, MM., Chand, SK. and Sharma, VP. 1997. Outbreak of falciparum malaria in submerged villages of Narayanganj PHC, district Mandla due to Narmada irrigation Project, Central India (Madhya Pradesh). Current Science. 73: 689-691.

Singh, PB. and Canario, AVM. 2004. Reproductive endocrine disruption in the freshwater catfish, Heteropneustes fossilis, in response to the pesticide γ−hexachlorocyclohexane. Ecotoxicology and Environmental Safety. 58(1): 77-83.

Smita, C., Ram, RN. and Singh, IJ. 2003. Testicular recrudescence and recovery response in Cyprinus carpio after long term exposure to carbamate pesticide. Journal of Ecophysiology and Occupational Health. 3(1&2): 15-36.

Von Westernhagen, H., Dethlefsen, V., Cameron, P. and Janssen, D. 1987. Chlorinated hydrocarbon residues in gonads of marine fish and effects on reproduction. Sarsia. 72: 419-422.

Singh, PB., Kime, DE. Epler, P. and Chyb, J. 1994. Impact of γ-hexachlorocyclohexane exposure on plasma gonadotropin levels and in vitro stimulation of gonadal steroid production by carp hypophyseal homogenate in Carassius auratus. Journal of Fish Biology. 44(2): 195-204.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 373-380, 2008 ISSN: 1715-9997

349

RARE ACTINOMYCETES FROM EGYPTIAN HABITATS: ISOLATION AND SCREENING FOR ANTIMICROBIAL ACTIVITIES

*Hala M Rifaat1 and Osama Hamed El-Sayed2

1Microbial Chemistry Department, National Research Centre, Cairo 2Microbial Biotechnology Department, National Research Centre, Cairo, Egypt

ABSTRACT

Sixty-six of rare actinomycetes were isolated from samples of soil, fresh water and decayed plants collected from different Egyptian localities. They were recovered on humic-vitamen B agar medium using dilution, several antibiotics as selective agents and mild heat techniques. They assessed for their antimicrobial activity using diffusion assay methods against seven Gram-positive bacteria, seven Gram-negative bacteria, two yeasts and two filamentous fungi. Among the 66 isolates, 35 (53%) strains showed an activity against some of the test organisms. The polyphasic identification of the active isolates revealed that they are commonly Micromonospora (23 isolate, 65.71%), less commonly Actinoplanes (11 isolates, 31.43%) and rarely Dactylosporangium (1 strain, 2.86%) genera. Keywords: Antimicrobial activity, decayed plants, rare actinomycetes, soil, water.

INTRODUCTION Screening of microorganisms for the production of novel antibiotics has been intensively pursued for many years by scientists. Actinomycetes have the capability to synthesise many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic and enzymes like cellulase and xylanase (Waksman, 1961; Lacey, 1973; Ouhdouch et al., 2001, Saadoun and Gharaibeh, 2003; Rifaat et al., 2007). The term rare actinomycete was introduced by many workers to denote actinomycetes less frequently found in soil. Several genera of rare actinomycetes such as Actinomadura, Actinoplanes, Amycolatopsis, Dactylo-sporangium, Microbispora and Micromonospora have been described from which many enzymes and antibiotics have been discovered (Hacene et al., 1994; Lazzarini et al., 2000; Ouhdouch et al., 2001; Mazza et al., 2003). The present investigation carried out for isolation of rare actinomycetes from Egyptian habitats as well as the determination of antagonistic spectra of whole isolates. The active rare actinomycetes isolates were also identified. MATERIALS AND METHODS 1- Samples and bacterial strain isolation Five cultivated rhizosphere soil samples from Qalubiya (S.Q), Sharkiya (S.S), Ismaelliya (S.I), Dakahliya (S.D) and Gharbiya (S.G); 5 fresh water samples from River Nile at Cairo (W.C1 & W.C2), Giza (W.G1 & W.G2) and

El-Fayoum (W.F) as well as 5 different decayed plant samples from papyrus of the Gold Island (D.P1 & D.P2), Ward El-Neel at Sharkiya (D.W1 & D.W2) and maize at Kalubiya (D.M) were collected under aseptic conditions. Bacterial strains were isolated from the soil samples by dilution techniques on humic-vitamin B agar medium which was recommended for isolation of rare actinomycetes (Hayakawa and Nonomura, 1984). For water samples, mild heat was applied for pre-treatment steps to reduce the dominance of fast growing actinomycetes and to facilitate the recovery of slow growing and relatively less competitive types (Cross, 1981). 0.2 ml of the pre-treatment sample was spread on the same previous medium. In case of decayed plant samples, small pieces of approximately 3 cm2 are cut out and each piece transferred to a 100 ml conical flask containing 25 ml of sterile water. After 2 min. of agitation on rotary shaker, 0.1 ml of the leaf washing liquid was spread on the surface of the previous agar medium. The antifungal cycloheximide (50 µg/ml) was used to inhibit the development of invasive fungi. Also, one of the following antibacterial agents was added to the isolation media: cycloserine, gentamycin and strepto-mycin (10 µg/ml). These antibiotics were chosen on the basis of good results obtained previously during the selective isolation of rare actinomycetes (Sabaon et al., 1998). The plates were incubated at 30oC for 3-4 weak and all colonies were examined directly by light microscopy to detect the rare actinomycetes isolates. 2- Antimicrobial assay For determining the antimicrobial spectrum of the isolated rare actinomycetes, the diffusion assay method was used Bauer et al., 1966). Inhibition zones were measured after *Corresponding author email: halamohamed6@hotmail.com

Canadian Journal of Pure and Applied Sciences 374

incubation at 300 C for 24 hours for bacteria and yeast and 48 hours for filamentous fungi. The used target organisms were: (i) Gram-positive bacteria as Staphylococcus aureus, Streptococcus thermophilus, Streptococcus pneumoniae, Micrococcus luteus, Enterococcus faecalis, Bacillus cereus and Bacillus subtilis, (ii) Gram-negative bacteria as Escherichia coli, Pseudomonas aeruginosae, Klebsiella pneumonia, Proteus sp., Shigella dysenteriae, Serratia marcescens and Acinetobacter calcoaceticus, (iii) yeast as Candida albicans and Saccharomyces cerevisiae and (iv) fungi as Aspergillus niger and Mucor racemosus. 3- Morphological and cultural characteristics of the isolates Morphological and cultural characteristics of isolated rare actinomycetes were examined according to the method described by Shiriling and Gottleib (1966) and Holt et al. (1994). 4- Chemotaxonomical analysis Isolates were grown on yeast -malt extract broth for seven days at 300 C. Mycelia were harvested by centrifugation and washed by distilled water. These were used for chemical analysis of diaminopimelic acid (DAP) isomer and whole cell sugars according to the method of Hasegawa et al. (1983).

RESULTS AND DISCUSSION 1. Isolation of rare actinomycetes Rare actinomycetes are present in small quantity in the various habitats and cannot be isolated by the current methods used in microbiology. Their selection must pass through elimination of other microorganisms which obstruct the growth of actinomycetes. Actinomycetes/ microorganisms ratio of a sample increases by (i) the use of certain sources of carbon and nitrogen making the cultures media less favourable to the growth of bacteria (Hayakawa and Nonomura, 1984; Cavalla and Eberlin, 1994), (ii) by the use of antibiotic substances which inhibit the development of bacteria and fungi (Larpent and Larpent-Gouragand, 1990) and (iii) by physical pre-treatment such as mild heat (Cross, 1981). In the present work, results presented in Table 1 reveal a considerable variation of the numbers of rare actinomycetes strains isolated from each sample on the selective medium. Among the 66 isolated strains of rare actinomycetes, 26 were isolated from soil sample (39.5%), 18 from water sample (27%) and 22 from decayed plants (33.5%). The presence of a high number of rare actinomycetes in the soil and decayed plants is in agreement with the bibliographical data which let appear the soil as the principle reservoir of actinomycetes (Lemriss et al., 2003).

Table 1. Initial screening of rare actinomycetes strains.

Sample No. Origin Number of actinomycetes isolates

Number of active actinomycetes

S.Q Soil of Qalubiya 8 5 S.S Soil of Sharkiya 5 3 S.I Soil of Ismaelliya 4 4 S.D Soil of Dakahliya 4 2 S.G Soil of Gharbiya 5 2 W.C1 Fresh water from Cairo 4 2 W.C2 Fresh water from Cairo 4 2 W.G3 Fresh water from Giza 3 1 W.G4 Fresh water from Giza 5 3 W.F Fresh water from El-Fayoum 2 0 D.P1 Decayed plant of Papyrus 8 5 D.P2 Decayed plant of Papyrus 4 2 D.W1 Decayed plant of Ward El-Neel 3 3 D.W2 Decayed plant of Ward El-Neel 4 1 D.M Decayed plant of Maize 3 0 Total 66 35

Rifaat and El-Sayed 375

2. Antibiotic production Among the rare actinomycetes isolates, only 35 (53%) were active against at least one of the tested microorganisms (Table 1). No active rare actinomycetes were isolated from fresh water sample of El-Fayoum (W.F) and sample from decayed plant of maize (D.M). Lemriss et al. (2003) indicated that the isolation of actinomycetes with antimicrobial activity is higher than 40% while Jiang and Xu (1996) mentioned that it is less

than 10%. Figure 1 showed the percentage of the active strains of rare actinomycetes against the target test organisms. The percentage of isolates showed anti Gram-negative and anti yeast of 32.71%, while 23.37% showed anti fungal activity. In addition, only 11.21% showed anti Gram-positive bacteria. Rare actinomycetes with antibacterial and antifungal activities were 43.9 and 56.1% respectively.

Table 2. Antimicrobial activity of isolated rare actinomycetes.

Sample No.

Staphy-lococcus aureus

Strepto-coccus

thermophilus

Strepto-coccus

pneumoniae

Microco-ccus

luteus

Enteroco-ccus

faecalis

Bacillus cereus

Bacillus subtilis

Escherichia coli

Pseudomonas

aeruginosae

S.Q1 - - - - - - - + ++

S.Q2 - - - - - - - + ++

S.Q3 - - - - - - - +++ +++

S.Q6 - - - - - - - +++ +++

S.Q8 - - - - - - - +++ +++

S.S2 - - - - - - - + ++

S.S3 ++ + + - + + + + +++

S.S4 ++ + + - + + + + ++

S.I1 ++ + + - + + + + ++

S.I2 ++ + + - + + + ± ++

S.I3 - - - - - - - + ++

S.I4 - - - - - - - +++ +++

S.D1 - - - - - - - +++ +++

S.D3 - - - - - - - +++ +++

S.G3 - - - - - - - +++ +++

S.G5 - - - - - - - + ++

W.C1.2 - - - - - - - +++ +++

W.C1.3 ++ + + - + + + ± ++

W.C2.1 - - - - - - - ++ ++

W.C2.2 + + + - ± + + + +

W.G1 ++ + + - + + + + +++

W.G2.1 - - - - - - - + +++

W.G2.2 - - - - - - - +++ ++

W.G2.5 - - - - - - - ++ ++

D.P1.1 - - - - - - - ++ +++

D.P1.2 - - - - - - - ++ ++

D.P1.4 - + + - + + ++ ++ -

D.P1.6 - - - - - - - + +++

D.P1.7 - - - - - - - ++ +

D.P2.3 - + + - ± + + + -

D.P2.4 - - - - - - - - ++

D.W1.1 - + ± - + + ++ + -

D.W1.2 - ± + - + + ++ ++ -

D.W1.3 - - - - - - - - +

D.W2.4 ++ ++ + ++ + + + ++++ ++++

Canadian Journal of Pure and Applied Sciences 376

The activity of the isolated strains against the tested organisms is illustrated in Table 2. All strains isolated from soil, water together with the majority of decayed plants reveal high to moderate activity towards Escherichia coli, Pseudomonas aeruginosae, Klebsiella pneumonia, Proteus sp., Shigella dysenteriae, Serratia marcescens, Acinetobacter calcoaceticus and Saccharo-myces cerevisiae. In addition, all strains isolated from soil and water samples showing moderate activity towards Aspergillus niger and Mucor racemosus. Some strains from the different studied habitats are moderately active

against Staphylococcus aureus, Streptococcus thermophilus, Streptococcus pneumoniae, Micrococcus luteus, Enterococcus faecalis, Bacillus cereus and Bacillus subtilis. All isolates from the different samples (except one from decayed plants) have no activity against Micrococcus luteus and Candida albicans. The strain isolated from the decayed plant of Ward El-Neel (sample D.W2.4) is active all over the tested organisms and showing strong activity towards Escherichia coli, Pseudomonas aeruginosae, Serratia marcescens and Acinetobacter calcoaceticus.

Table. 2. Cont.

Sample No.

Klebsiella pneumonia

Proteus sp.

Shigella dysenteriae

Serratia marcescens

Acinetobacter calcoaceticus

Candida albicans

Saccharomyces cerevisiae

Aspergillusniger

Mucor racemosus

S.Q1 ++ + + + ++ - + + +

S.Q2 ++ + + + ++ - + + +

S.Q3 ++ ++ ++ ++ ++ - + + +

S.Q6 ++ ++ ++ ++ ++ - + + +

S.Q8 ++ ++ ++ ++ ++ - + + +

S.S2 ++ + + + ++ - + + +

S.S3 +++ +++ + ++ ++ - + + +

S.S4 ++ + ± + ++ - + ± ±

S.I1 + ++ + + ++ ± + + ±

S.I2 ++ ++ + + + - + + +

S.I3 ++ + + + ++ - + + +

S.I4 ++ ++ ++ ++ ++ - + + +

S.D1 ++ ++ ++ ++ ++ - + + +

S.D3 ++ ++ ++ ++ ++ - + + +

S.G3 ++ ++ ++ ++ ++ - + + +

S.G5 ++ + + + ++ - + + +

W.C1.2 ++ ++ ++ ++ ++ - + + +

W.C1.3 +++ +++ + ++ ++ - + + +

W.C2.1 ++ + ++ + ++ - + + +

W.C2.2 ++ +++ ± ++ ++ - + ± +

W.G1 +++ ++ ++ + + - + + ±

W.G2.1 ++ ++ ++ ++ + - + + +

W.G2.2 ++ + ++ ++ + - + + +

W.G2.5 + + + + ++ - + ± +

D.P1.1 ++ + + ++ - - + - -

D.P1.2 + + + + - - + - -

D.P1.4 + + ± ++ ++ - ++ - -

DP.1.6 ++ + + ++ - - + - -

DP.1.7 ++ + + ++ - - + - -

D.P2.3 ± + + ++ ++ - ++ - -

D.P2.4 ++ + + + - - + - -

D.W1.1 + ± + + + - + - -

D.W1.2 + + + ++ ++ - ++ - -

D.W1.3 + + + ++ - - + - -

D.W2.4 + + + ++++ ++++ + + + +

Rifaat and El-Sayed 377

3. Morphological characteristics The morphological characteristics of the active strains are presented in Table 3 from which the following criteria can be indicated: a. Half of the strains have no diagnostic mycelial

pigment, while the rest have orange or purple colour. b. Fourteen strains have no diffusible pigment, while

the others have brown to dark brown and rarely olive or green colours.

c. All isolates grow on the Czapek-sucrose agar and none of them grow on potato slice.

d. The utilization with different carbon sources show unlike patterns.

e. Approximately all isolates can not reduce nitrate. f. Half of the isolates can tolerate the presence of NaCl

till 3%, while others tolerate till 1.5%.

Table. 3. Morphological and physiological characteristics of the active rare actinomycetes isolates.

Growth on Growth utilisation Sample No.

Diagnos-tic

mycelial Pigment

Diffusible pigment

Czabek- sucrose

agar

Potatoslice

α- Meli-biose

Raffi-nose

D- Mannitol

L- Rha-mnose

Gly-cerol

Inosi-tol

D-Ri-bose

Nit-rate

reduc-tion

Maximum NaCl

tolerance (% w/v)

S.Q1 - Olive + - - - - - - - + - 3

S.Q2 - Olive + - - - - - - - + - 3

S.Q3 - Green + - - - - - - - + - 3

S.Q6 - Olive + - - - - - - - + - 3

S.Q8 - Green + - - - - - - - + - 3

S.S2 - Dark brown + - + + - - + - - - 1.5

S.S3 Orange - + - + - + - - - + - 3

S.S4 Orange - + - + - + - - - + - 3

S.I1 Orange - + - + - + - - - + - 3

S.I2 Orange - + - + - + - - - + - 3

S.I3 - Dark brown + - + + - - + - - - 1.5

S.I4 - Brown + - + + - - + - - - 1.5

S.D1 - Dark brown + - + + - - + - - - 1.5

S.D3 - Dark brown + - + + - - + - - - 1.5

S.G3 - Dark brown + - + + - - + - - - 1.5

S.G5 - Brown + - + + - - + - - - 1.5

W.C1.2 - Dark brown + - + + - - + - - - 1.5

W.C1.3 Orange - + - + - + - - - + - 3

W.C2.1 - Dark brown + - + + - - + - - - 1.5

W.C2.2 Orange - + - + - + - - - + - 3

W.G1 Orange - + - + - + - - - + - 3

W.G2.1 - Dark brown + - + + - - + - - - 1.5

W.G2.2 - Dark brown + - + + - - + - - - 1.5

W.G2.5 - Dark brown + - + + - - + - - - 1.5

D.P1.1 Purple - + - - - - + - - - + 3

D.P1.2 Purple - + - - - - + - - - + 3

D.P1.4 Orange Brown + - + + - - - - + - 1.5

D.P1.6 Purple - + - - - - + - - - + 3

D.P1.7 Purple - + - - - - + - - - + 3

D.P2.3 Orange Brown + - + + - - - - + - 1.5

D.P2.4 Purple - + - - - - + - - - + 3

D.W1.1 Orange Brown + - + + - - - - + - 1.5

D.W1.2 Orange Brown + - + + - - - - + - 1.5

D.W1.3 Purple - + - - - - + - - - + 3

D.W2.4 - - + - + + + + - - - - 3

Canadian Journal of Pure and Applied Sciences 378

4. Chemotaxonomical analysis All of the studied isolates showed meso DAP in their cell wall as well as xylose and arabinose as a characteristic sugars in cell hydrolysates. 5. Identification of rare actinomycetes isolates Results obtained after the polyphasic identification of isolated rare actinomycetes are presented in Table 4 from which, it is clear that the active actinomycetes belong generally to Micromonospora (65.7%), less commonly

Actinoplanes (31.3%) and rarely to Dactylosporangium (3%) genera. The soil isolates revealed the presence of three different phena. The first was identified as Micromonospora olivasterospora, while the second belongs to Micromonospora purpurea. The third phena was identified as Actinoplanes teichomyceticeus. In addition, rare actinomycetes in water samples are commonly

Table.4. Taxonomic groups of active rare actinomycetes isolates

Sample No. Locality Identified rare actinomycetes S.Q1 Soil of Qalubiya Micromonospora olivasterospora S.Q2 Soil of Qalubiya Micromonospora olivasterospora S.Q3 Soil of Qalubiya Micromonospora olivasterospora S.Q6 Soil of Qalubiya Micromonospora olivasterospora S.Q8 Soil of Qalubiya Micromonospora olivasterospora S.S2 Soil of Sharkiya Micromonospora purpurea S.S3 Soil of Sharkiya Actinoplanes tecichomyceticus S.S4 Soil of Sharkiya Actinoplanes tecichomyceticus S.I1 Soil of Ismaelliya Actinoplanes tecichomyceticus S.I2 Soil of Ismaelliya Actinoplanes tecichomyceticus S.I3 Soil of Ismaelliya Micromonospora purpurea S.I4 Soil of Ismaelliya Micromonospora purpurea S.D1 Soil of Dakahliya Micromonospora purpurea S.D3 Soil of Dakahliya Micromonospora purpurea S.G3 Soil of Gharbiya Micromonospora purpurea S.G5 Soil of Gharbiya Micromonospora purpurea W.C1.2 Fresh water from Cairo Micromonospora purpurea W.C1.3 Fresh water from Cairo Actinoplanes tecichomyceticus W.C2.1 Fresh water from Cairo Micromonospora purpurea W.C2.2 Fresh water from Cairo Actinoplanes tecichomyceticus W.G1 Fresh water from Giza Actinoplanes tecichomyceticus W.G2.1 Fresh water from Giza Micromonospora purpurea W.G2.2 Fresh water from Giza Micromonospora purpurea W.G2.5 Fresh water from Giza Micromonospora purpurea D.P1.1 Decayed plant of Papyrus Micromonospora echinospora D.P1.2 Decayed plant of Papyrus Micromonospora echinospora D.P1.4 Decayed plant of Papyrus Actinoplanes capillaceus D.P1.6 Decayed plant of Papyrus Micromonospora echinospora D.P1.7 Decayed plant of Papyrus Micromonospora echinospora D.P2.3 Decayed plant of Papyrus Actinoplanes capillaceus D.P2.4 Decayed plant of Papyrus Micromonospora echinospora D.W1.1 Decayed plant of Ward El-Neel Actinoplanes capillaceus D.W1.2 Decayed plant of Ward El-Neel Actinoplanes capillaceus D.W1.3 Decayed plant of Ward El-Neel Micromonospora echinospora D.W2.4 Decayed plant of Ward El-Neel Dactylosporangium aurantiacum

Rifaat and El-Sayed 379

Micromonospora purpurea followed by Actinoplanes teichomyceticeus. Several workers emphasised that Micromonospora olivasterospora, Micromonospora purpurea and Actinoplanes teichomyceticeus showed antimicrobial activity (e. g. Bardone et al., 1978; Wagman and Weinstein, 1980; Borghi et al., 1984; Lazzarini et al., 2000). In the case of decayed plants samples, the dominant group (6 strains) were identified as Micromonospora echinospora, followed by the presence of Actinoplanes capillaceus (4 isolates). These species reported as potential producers of new antibiotic (Lazzarini et al., 2000). One strain could be identified as Dactylosporangium aurantiacum.

Anti Gram-positivebacteria 11.21%

Anti Gram-negative bacteria 32.71%

Anti yeast32.71%

Anti fungi23.37%

CONCLUSIONS In the course of screening for new antibiotics, sixty six rare actinomycetes strains were isolated from different Egyptian habitats using humic-vitamin B agar medium. Antibiotic production of the isolates has been tested against Gram-positive, Gram-negative, yeast and fungi. Thirty five isolates showed activity against the tested organisms. Approximately all the isolates were active against Gram-negative bacteria. Isolates from soil and fresh water samples were active towards fungi and one tested yeast. The isolates showed different pattern of activity against Gram-positive bacteria. The taxonomic study revealed that the active isolates belonging to Micromonospora, Actinoplanes and Dactylosporangium genera. Screening of rare actinomycetes will hopefully generate new leads. REFERENCES Bardone, MR., Paternoster, M. and Coronelli, C. 1978. Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. II. Extraction and chemical characterization. Journal of Antibiotic. 31: 170-177.

Bauer, AW., Kirby, WM., Sherries, JC. and Turk, M. 1966. Antibiotic susceptibility testing by standard single disk method. American Journal of Clinical Pathology. 45: 493-496.

Borghi, A., Coronelli, C., Faniuolo, L., Allievi, G., Pallanza, R. and Gallo, GG. 1984. Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. IV. Separation and characterization of the components of teichomycin (teicoplanin). Journal of Antibiotic. 37: 615-620.

Cavalla, M. and Eberlin, T. 1994. Isolement des streptomycetes du sol. L’operon. 19: 13-17.

Cross, T. 1981. Aquatic actinomycetes: A critical survey of the occurrence, growth and role of actinomycetes in aquatic habitats. Journal of Applied Bacteriology. 50: 397-423.

Hacene, H., Sabaou, N., Cavaletti, l., Sosio, M. and Donadis, S. 1994. Diversity of Actinoplanes and related genera isolated from an Italian soil. Microbial Ecology. 45: 362-372.

Hasegawa, T., Takizawa, M. and Tanida, S. 1983. A rapid analysis for chemical grouping of aerobic actinomycetes. Journal of Genetic and Applied Microbiology. 29: 319.

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Holt, JG., Williams, ST. and Sharp, ME. 1994. In: Bergey’s Manual of Determinative Bacteriology. The Williams and Wilkins Com., Baltimore, London.

Jiang, CL. and Xu, LH. 1996. Diversity of aquatic actinomycetes in lakes of the middle plateau, Yunnan, China. Applied Environmental Microbiology. 62: 249-253.

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Larpent, JP. and Larpent-Gouragand, M. 1990. Memento technique de microbiologie. Technique et documentation lavoisier.

Lazzarini, A., Cavaletti, L., Toppo, G. and Marinelli, F. 2000. Rare genera of actinomycetes as potential producers of new antibiotics. Antonie van Leeuwenhoek. 78: 399-405.

Lemriss, S., Laurent, F., Couble, A., Casoli, F., Lancelin, JM. and Saintpierre-Bonaccio, D. 2003. Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes. Canadian Journal of Microbiology. 49: 669-674.

Fig. 1. Percentage of the active strains of rareactinomycetes against the target organisms.

Canadian Journal of Pure and Applied Sciences 380

Mazza, P., Monciardini, P., Cavaletti, L., Sosio, M. and Donadio, S. 2003. Diversity of Actinoplanes and related genera isolated from an Italian soil. Microbial Ecology. 45: 362-372.

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Rifaat, HM., El-Sayed, OH., Hassanein, SM. and Selim, MSM. 2007. Protease activity of some mesophilic streptomycetes isolated from Egyptian habitats. Journal of Culture Collections. 5: 16-24.

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Sabaon, N., Boudjella, H., Bennadjj, A., Mostefaoui, A., Zitouni, L., Lamari, H., Bennadji, G., Lefebvre, G. and Germain, P. 1998. Les sols des oasis du Sahara Algerian, source d'actinomycetes rares producteurs d'antibiotiques. Secheresse. 9: 147-153.

Shiriling, EB. and Gottleib, D. 1966. Methods for characterisation of Streptomyces species. International Journal of Systematic Bacteriology. 16: 313.

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SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 381-387, 2008 ISSN: 1715-9997

1

INHIBITORY EFFECT OF DILLENIA PENTAGYNA STEM BARK EXTRACT ON CISPLATIN AND BENZO[A]PYRENE-INDUCED MUTAGENICITY

Gabriel Rosangkima and *Surya Bali Prasad

Cell and Tumor Biology Lab., Department of Zoology North-Eastern Hill University, Shillong- 793022, India

ABSTRACT

Modulatory effect of methanol extract of stem bark of Dillenia pentagyna (DPE) was evaluated against mutagenicity induced by cisplatin (CIS) and benzo[a]pyrene (B[a]P) in the mouse using bone marrow chromosomal aberration, micronucleus and sperm abnormality as mutagenicity parameters. In one group of experiment, animals received a single dose (20mg/kg body weight) of DPE through intraperitoneal injection (i.p) followed by cisplatin or benzo[a]pyrene treatment. In the other groups, three doses of DPE (20, 50 and 100mg/kg b. wt/day) were given through diet for seven consecutive days prior to a single treatment with cisplatin or benzo[a]pyrene. The animals from different treatment groups were used for the study of chromosomal aberration (CA), micronucleus (MN) and sperm abnormality assays. The result of present study shows that a single treatment with DPE did not show significant changes in the incidence of chromosomal aberration, micronucleus and sperm abnormality induced by CIS and B[a]P. However, pretreatment with DPE for seven consecutive days dose dependently reduced CIS and B[a]P-induced mutagenicity suggesting the protective role of D. pentagyna on CIS and B[a]P mutagenic potentials. Keywords: Dillenia pentagyna – antimutagenicity - benzo[a]pyrene – cisplatin.

INTRODUCTION Cis-diamminedichloroplatinum-II, commonly known as cisplatin (CIS) is widely used as a chemotherapeutic agent alone or in combination with other agents against a variety of cancers (Carter, 1984). However, its therapeutic efficacy has been limited due to the side effect and also its mutagenic potential (Krakoff, 1979; Khynriam and Prasad, 2001; Overbeck et al., 1996). An increased carcinogenic risk with the development of secondary tumors in patients/animals treated with cisplatin has also been reported (Cross et al., 1996; Greene, 1992). It has been shown to cause genotoxic effects in cultured mammalian cells (Zwelling et al., 1979) and bone marrow cells (Giri et al., 1998). Benzo[a]pyrene (CAS Reg. No. 50-32-8), also known as 1,4-benzo[a]pyrene (B[a]P), is a polycyclic aromatic hydrocarbon (PAH) generated from the combustion of fossil fuels and tobacco and is both inhaled and consumed (Phillips, 1983). It has shown various toxicological effects, such as haematological effects, reproductive and developmental toxicity and immunotoxicity. It is the carcinogenic and genotoxic potential of these compounds that has attracted most attention. A large number of dietary agents have the inhibitory potentials against genotoxicity and carcinogenicity (Ames, 1983; Ferguson, 1994). Phenolic compounds,

fibre, chlorophyll, b-carotene, and vitamins such as C and E, a component of fresh fruits and vegetables were suggested to have antimutagenic and/or anticarcinogenic properties (Stavric, 1994; Ho, 1992; Kuo et al., 1992), and a negative association between the incidence of cancer and consumption of diet rich in fibres, fresh vegetables, vitamins and minerals was also reported (Archer, 1988; Stainmetz and Potter, 1991). Some of the food ingredients including vitamins, flavonoids and organosulphur compounds possess antimutagenic and anticarcinogenic activities (Stavric, 1994), and extracts of certain plants were reported to have the ability to inhibit the mutagenic activity of well established genotoxins (Ito et al., 1986; Khanduja and Majid, 1993; Abraham et al., 1986; Mejia et al., 1999). Dillenia pentagyna Roxb. (Dilleneaceae) is a deciduous tree, distributed in Indo-Malaysian areas extending to tropical Australia and throughout India particularly in sub-tropical Himalayas. It is found in most places of Mizoram state in India. Our preliminary investigation through literature review and personal interview with local herbal practitioners revealed that the stem bark of this plant has been used by the people of Mizoram for the treatment of gastric cancers, diarrhea and other human ailments. The stem bark and fruit has also been used by some of the Indian ethnic communities as cure for blood dysentery, stomach pain and fistula (Pal and Jain, 2000). We have noticed the promising antitumor activity of methanol extract of stem bark of this plant against murine ascites Dalton’s lymphoma (Rosangkima and Prasad, *Corresponding author email: sbpnehu@hotmail.com

Canadian Journal of Pure and Applied Sciences 382

2004). We have also reported the inhibitory effect of this plant extract in the level of sialic acid and lipid peroxidation in the tissues of Dalton’s lymphoma-bearing mice (Rosangkima and Prasad, 2007a), and a significant inhibitory effect in the level of total reduced glutathione and glutathione reductase activity in Dalton’s lymphoma cells was also noted (Rosangkima and Prasad, 2007b). All these findings, thus, generated an interest to investigate the antimutagenic activity of this plant in murine model. Therefore, the present investigation was undertaken to evaluate the antimutagenic potential of Dillenia pentagyna against CIS and B[a]P induced mutagenicity in mice. MATERIALS AND METHODS Chemicals Colchicine, cisplatin, corn oil, benzo[a]pyrene and Giemsa stain were obtained from Sigma Chemicals Co. Ltd., USA. All other chemicals of analytical grade were purchased from SRL Co. Mumbai, India. Animals Inbred Swiss albino mice colony is being maintained under laboratory conditions keeping 5-6 animals in a propylene cage at 23-250C. The animals were fed with commercially available food pellets and water ad libitum. For some experiment in involving micronucleus assay, tumor-bearing mice were used. For this Dalton’s lymphoma cells (1x 107 cells) in PBS , 0.25 ml vol. were transplanted. Tumor transplanted animals generally survived for 19-21 days. Plant material and preparation of test sample The stem bark of D. pentagyna was collected from Kawlkulh village, Mizoram state, India, in July 2006. The methanol extract of stem bark of D. pentagyna (DPE) was prepared as described previously (Rosangkima and Prasad, 2004). For the treatment through intraperitoneal (i.p) injection, the plant extract was dissolved in 0.05% NaOH solution. For the treatment through the diet, plant extract was dissolved in 50% alcohol (14, 35, and 70mg/100 ml) and mixed thoroughly with the feed powder in the ratio of 1:2 (vol:wt) which was then dried in an oven at 35oC to 40oC. Since the daily intake of feed per animal was approximately 7.5g, different doses of extract treatment come to 20, 50 and 100mg/kg b. wt/day. Antimutagenic activity In the antimutagenic activity studies, experimental animals were divided into 3 groups. Group I (CIS/B[a]P control) animals received a single dose of mutagen (CIS/B[a]P). Group II animals received a single i.p injection of DPE (20mg/kg b. wt) and CIS/B[a]P. Group III animals were given DPE pretreatment through the diet at the dose of 20, 50 and 100mg/kg b. wt/day for 7 consecutive days prior to a single dose of mutagen

(CIS/B[a]P). CIS (8mg/kg b. wt) and B[a]P (125mg/kg b. wt) were administered through i.p. injection and gavages respectively. Chromosomal analysis Mice were sacrificed by cervical dislocation 24 h after mutagen treatment. The animals were subjected to mitotic arrest by injecting colchicine (i.p 4mg/kg b. wt.) 2 h prior to sacrifice. Bone marrow cells were collected from humerous and femur by flushing in PBS. The cells were washed with PBS and collected by centrifugation (1000 rpm, for 5 min at 4°C). The cell pellet was subjected to treatment with hypotonic solution (0.075M KCl) for 25 min at 37oC. The cells were separated and fixed in acetic acid: methanol (1:3; v/v), repeated again with a 30 min interval. Two drops of cell suspension were dropped on a clean and chilled slide, subjected to flame drying and stained for 5 to 7 min with working Giemsa (1ml of stock giemsa + 0.25ml methanol + 2.8ml Sorensen’s buffer, pH 6.8), washed and mounted in DPX. Five hundred good metaphase spreads were examined per animal under 100x oil immersion. The observed chromosomal aberrations (CA) included chromatid break (CB), chromosomal fragment (CF), exchange (Exch) and sister chromatid union (SCU). Micronucleus assay Micronucleus (MN) was assayed following the method of Fenech et al. (2003). Briefly, 24 h after mutagen treatment, bone marrow cells from both the femurs were collected by flushing in PBS. The cell suspension was centrifuged at 1000 rpm for 5 minutes at 4oC. The cell pellet was treated with a weak hypotonic solution (0.075M KCl:saline, 1:9 v/v) for 5 min. After centrifugation, the cells were fixed in fresh fixative (acetic acid:methanol, 1:3 v/v) for 15 min at room temperature and repeated twice. Two drops of cell suspension were dropped onto clean and wet chilled slide. The slides were air-dried and stained with May Grunwald-Giemsa stain. A total of 2500 polychromatic erythrocytes (PECs) were scored per animal to determine the frequency of micronucleated polychromatic erythrocytes (MnPCEs). Only rounded bodies approxi-mately one-fifth to one-sixteenth the size of the main nucleus, lying within three nuclear diameter distance from the main nucleus, and possessing a staining intensity similar to that of the main nucleus, were scored as micronucleus. All the slides were scored by the same observer. Sperm abnormality assay The male mice in different treatment groups (Group I, II and III) were sacrificed on the 10th day of mutagen treatment. The cauda epididymis were removed and placed in physiological saline. It was then minced into pieces and kept undisturbed for 20 min. The spermatozoa were spread on a clean slide, air-dried, fixed in absolute

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methanol for 15 min and then stained with 1% aqueous eosin-Y on the following day. Five hundred sperms from each mouse were examined for the abnormalities in sperm head and tail shapes following the criteria as close as possible to those established by Wyrobeck and Bruce (1975). Statistical analysis Results were expressed as mean ± S.D of number of experiments. Significance was evaluated by Student’s t-test. p values less than 0.05 were regarded as significant. RESULTS

Chromosomal aberrations CIS and B[a]P caused the development of various chromosomal aberrations in bone marrow cells of mice.

Chromatid break, chromosomal fragment, exchange and sister chromatid union were observed after treatment with CIS and B[a]P (Fig. 1), with CB and Exch occurring more frequently (Table 1). Treatment with a single dose of DPE (24 h) did not show significant changes in the frequency of CA induced by CIS and B[a]P. However, pretreatment of animals with DPE (20, 50 and 100mg/kg b. wt./day) significantly decreased the frequency of CA induced by both CIS and B[a]P (Table 2). Out of three different doses used, 100mg/kg b. wt./day showed maximum inhibitory effect against CIS and B[a]P-induced chromosomal aberrations. Micronucleus The development of MN was observed in bone marrow cells of tumor-bearing mice after CIS and B[a]P treatment. Treatment with a single dose of DPE (20

Fig. 1. Representative of different types of chromosomal aberrations (chromatid break - CB, chromosomal fragment - CF, exchanges - Exch and sister chromatid union - SCU) induced by CIS or B[a]P in the bone marrow cells of mice.

Fig. 2. Representative of micronuclei induced by CIS and B[a]P in the bone marrow cells of mice.

0.00.20.40.60.81.01.21.41.61.82.0

DPE(20mg)+B[a]P

Control(B[a]P)

DPE(20mg)+CIS

Control(CIS)

% M

nPC

E (m

ean

± S.

D)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Control(B[a]P)

Control(CIS)

DPE(100mg)+B[a]P

DPE(50mg)+B[a]P

DPE(20mg)+B[a]P

DPE(100mg)

+CIS

DPE(50mg)+CIS

DPE(20mg)+CIS

**

*

% M

nPC

E(m

ean

± S.

D)

Fig. 3. Frequency of micronucleus induced by CIS and B[a]P in the bone marrow cells of mice after a single treatment with DPE (20 mg/kg b. wt.). Results were mean ± S.D. Student’s t- test, n=6 as compared to the corresponding mutagen control. *P<0.05.

Fig. 4. Frequency of micronucleus induced by CIS and B[a]P in the bone marrow cells of mice after pretreatment with different doses of DPE through the diet. Results were mean ± S.D. Student’s t- test, n=6 as compared to the corresponding mutagen control. *P<0.05.

Canadian Journal of Pure and Applied Sciences 384

mg/kg b. wt) did not show significant changes in the development of MN induced by CIS and B[a]P (Fig. 3), while pretreatment with DPE through the diet in a dose depend manner decreased the incidence of MN induced by both CIS and B[a]P (Fig. 4). Sperm abnormality Various form of sperm abnormalities were induced after CIS and B[a]P treatment (Table 3). A single treatment

with DPE did not show significant changes in the frequency of sperm abnormality induced by CIS and B[a]P (Table 3). Amorphous, hookless, banana, microhead and doubled tail were among different types of abnormality observed in sperms(Fig. 5A-F). Out of different types of abnormal sperms analyzed, amorphous heads were noted to be more than the others. However, pretreatment with DPE at different doses (20, 50 and 100mg/kg b. wt/day) for 7 consecutive days prior to

Table 1. Changes in the frequency of chromosomal aberrations induced by CIS and BaP in bone marrow cells of mice following single co-treatment with DPE (20 mg/kg b. wt).

Chromosomal aberrations Treatment

CB CF Exch SCU Totala Total CA (mean±SD) Per 100 cells

Control (CIS) 425 55 242 59 909 181.8 ± 19.13 DPE+CIS 348 201 179 75 809 161.8 ± 12.27 Control (B[a]P) 47 2 - - 49 9.8 ± 0.83 DPE+ B[a]P 38 4 - - 42 8.6 ± 1.14

aFive hundred metaphase plates were scored per group (n = 5 animals) for chromosomal aberrations. Student’s t-test, as compared to the respective control values, *p<0.05. CB = Chromatid break, CF = Chromosomal fragment, Exch = Exchange, SCU = Sister chromatid union. Table 2. Effect of different doses of pretreatment with DPE on the frequency of chromosomal aberrations induced by CIS and BaP in bone marrow cells of tumor-bearing mice.

Chromosomal aberrations Mutagensa DPE

(mg/kg/day)b CB CF Exch SCU Totalc Total CA (mean±SD) Per 100 cells

CIS

20 50 100

398 329 292

78 72 57

218 176 156

87 65 55

781 642 560

156.2 ± 5.31* 128.4 ± 13.22* 112.0 ± 7.31*

B[a]P 20 50 100

31 25 20

2 1 1

- - -

- - -

33 26 21

6.6 ±1.14* 5.2 ± 0.83* 4.6 ± 0.54*

aAnimals were administered with a single dose of CIS or BaP. bAnimals were treated with DPE through the diet for 7 consecutive days prior to CIS or BaP treatment. cFive hundred metaphase plates were scored per group (n = 5 animals) for chromosomal aberrations. Student’s t-test, as compared to the respective control values, *p<0.05. CB = Chromatid break, CF = Chromosomal fragment, Exch = Exchange, SCU = Sister chromatid union. Table 3. Changes in the frequency of sperm abnormalities induced by CIS and BaP in mice following single co-treatment with DPE (20 mg/kg b. wt).

Treatment Amorphous Banana Hookless Microhead Double tail Mean % of abnormal sperms ± SD

Control (CIS) 132 28 83 11 6 8.66±0.89 DPE+CIS 123 23 80 9 3 7.93±0.38 Control (B[a]P) 77 19 18 14 2 4.33±0.35 DPE+B[a]P 70 18 22 13 4 4.23±0.19

A total number of 3000 sperms were observed in each treatment group. Results are expressed as Mean ± S.D. Student’s t- test, n=6 as compared to the corresponding control. A single dose of DPE and CIS or B[a]P were administered on the same day.

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mutagen(s) treatment significantly decreased the frequency of sperm abnormalities induced by both CIS and B[a]P (Table 4). Out of three different doses of DPE studied, maximum inhibition of sperm abnormality was observed with 100mg/kg b. wt/day (Table 4).

Fig. 5. Representative of normal (A) and different types of abnormal sperms (amorphous - B, banana - C, hookless - D, microhead - E and doubled tail - F) induced by CIS or B[a]P in mice. DISCUSSION The development of chromosomal aberration, micronucleus and sperm abnormality has been used as reliable biological indicators in the mutagenic bioassay of different drugs (Tandon and Sodhi, 1985; Giri et al., 2002). Micronucleus is chromatin masses that arise from chromosome fragments of intact whole chromosomes lagging behind at the anaphase (Czyzewska and Mazur, 1995). Micronucleus can be easily accredited in the cytoplasm of immature polychromatic erythrocytes (Schmid, 1976). Since blood cells originate in the bone marrow cells, the incidence of chromosomal aberration and micronucleus were analyzed in the bone marrow cells. The therapeutic efficacy of CIS has been limited by its dose dependent side effects (Krakoff, 1979) and its genotoxic properties have also been reported (Overbeck et

al., 1996; Cross et al., 1996; Giri et al., 1998). B[a]P can bind to the aryl hydrocarbon receptor (AHR), which then induces the expression of many genes, including members of the cytochrome P450 family of enzymes. B[a]P is then metabolized to an array of reactive species that form covalent bonds with nucleic acids and proteins within target cells, generate reactive oxygen species (Xie and Herschman, 1995; Balinsky and Jaiswal, 1993), and cause genetic mutations and cancer (Conney, 1982; Shields et al., 1993). It is clear that B[a]P also induced mutagenicity in the strain YG1024 (Watanabe et al., 1990). Carcinogens bind to the cell macromolecules resulting in mutagenic events leading to cell transformation and neoplastic changes. Some phytochemicals prevent these changes from occurring either by directly binding to the carcinogens/their metabolites or by metabolising and eliminating toxic xenobiotics. The results of present studies show that CIS and B[a]P treatment of mice causes significant elevation of chromosomal aberration, micronucleus and sperm abnormality depicting their mutagenic and genotoxic potentials (Table 1 and 3, Fig. 3). A single treatment with DPE did not show significant changes in the frequency of chromosomal aberration, micronucleus and sperm abnormality induced by CIS and B[a]P, while pretreatment for 7 consecutive days dose dependently decreased the frequency of chromosomal aberration, micronucleus and sperm abnormality. Since the micronuclei in young erythrocytes arise mainly from chromosomal fragments, the observed decrease in the incidence of micronuclei can be considered to indicate an inhibitory effect of DPE on the in vivo chromosomal damage induced by CIS and B[a]P. In conclusion, the result of present study showing significant reduction in CIS and B[a]P induced mutagenicity in presence of DPE suggests the protective role of this plant on CIS and B[a]P mutagenic/genotoxic potentials. Further, based on our earlier findings on changes in reduced glutathione etc, it is proposed that D. pentagyna may exert its antimutagenic potential by inducing some of the antioxidant enzymes that detoxify

Table 4. Effect of different doses of pretreatment with DPE on the frequency of sperm abnormalities induced by CIS and BaP in mice.

Mutagens DPE (mg/kg)

Amorphous Banana Hookless Micro-

head Double

tail Mean % of abnormal

sperms ± SD CIS 20

50 100

84 78 77

20 19 9

74 74 70

6 5 2

2 1 1

6.19±0.32* 5.85±0.55* 5.30±0.24*

B[a]P 20 50 100

72 66 59

15 7 11

13 10 10

8 3 5

1 1 0

3.62±0.18* 2.89±0.15* 2.83±0.31*

A total number of 3000 sperms were observed in each treatment group. Results are expressed as Mean ± S.D. Student’s t- test, n=6 as compared to the corresponding control, *P≤0.05. DPE was administered through the diet for 7 consecutive days prior to mutagen(s).

Canadian Journal of Pure and Applied Sciences 386

mutagens, or by acting as a free radical scavenger. However, other contributory steps may also be involved in its antimutagenic potential. Therefore, further investigation is needed in this direction. ACKNOWLEDGMENT The financial assistance rendered by University Grants Commission, Department of Science and Technology, New Delhi, and North-Eastern Hill University is gratefully acknowledged. REFERENCES Abraham, SK., Mahajan, S. and Kesavan, PC. 1986. Inhibitory effect of dietary vegetables on the in vivo clastogenicity of cyclophosphamide. Mutation Research. 172: 51-54.

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SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 389-392, 2008 ISSN: 1715-9997

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OVEREXPRESSION, PURIFICATION AND ANALYSIS OF DEHALOGENASE D OF RHIZOBIUM SP.

*Fahrul Huyop1, Ng Hong Jing1 and Ronald A Cooper2

1Industrial Biotechnology Department, Faculty of Bioscience and Bioengineering University Technology Malaysia, 81310 Skudai, Johor, Malaysia

2Biochemistry Department, University of Leicester, LE1 7RH Leicester, United Kingdom

ABSTRACT

Halogenated organic compounds are found widely throughout the environment and microbial catabolism can lead to their biodegradation. The action generally involves enzyme-catalysed carbon-halogen bond cleavage. Dehalogenase D enzyme act only on D-2-chloropropionic acid and D,L-2-chloropropionic acid. Other substrate for the enzyme included monochloroacetic acid and monobromoacetic acid but not 2,2-dichloropropionic acid. From 4 g (wet weight) of cells, 2.8 mg protein (4.3 U enzyme) was efficiently purified using Fast Protein Liquid Chromatography (FPLC). The cell-free extract was prepared in 0.01M Tris-acetate buffer pH7.6 and was applied to a FPLC Mono Q HR 5/5 anion-exchange column equilibrated with 5 mM sodium phosphate, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10%(mass/vol.) glycerol buffer, pH7.6 and eluted with sodium phosphate gradient to 100 mM. The dehD gene consisted of 798 bp which encoded a 266 amino acid protein with a predicted molecular mass of 29 386 Da. This value corresponds to the value of 29 000 Da estimated by SDS/PAGE. DehD protein has some similarity to the previously published sequence of several stereospecific dehalogenases with 59% identity to Rhizobium sp. NHG3 and 23% identity to Pseudomonas putida strain AJ1. Keywords: Dehalogenase D, D,L-2-chloropropionic acid, Rhizobium sp.

INTRODUCTION Halogenated organic compounds are found widely throughout the environment and microbial catabolism can lead to their biodegradation. The action generally involves enzyme-catalysed carbon-halogen bond cleavage. This process is known as ‘dehalogenation reaction’. A bacterium isolated from soil by elective culture on 2,2-dichloropropionic acid and identified as Rhizobium sp. was found to produce three dehalogenases (Allison, et al., 1983). All three enzymes acted on 2-chloropropionic acid, with dehalogenase L (DehL) being stereospecific for L-2-chloropropionic acid, dehalogenase D (DehD) being stereospecific for D-2-chloropropionic acid and dehalogenase E shows non-stereospecific and can act on both isomers of D- and L-2-chloropropionic acid (Leigh et al., 1986). The use of dehalogenases is important for industrial biocatalysis, for example in the manufacture of chiral intermediates. D-2-haloacid dehalogenase has found commercial application in the production of L-2-chloropropionic as a chiral feedstock chemical for the production of herbicides (ICI patent no. 179603) and anti-inflammatory agents (Swanson, 1999). A dehalogenase from the D-2-haloacid dehalogenase was studied in detail for use in industry for developing a bioreactor system where D-2-haloacid dehalogenase from Pseudomonas

putida AJI/23 was immobilised. This resulted in increased temperature stability and ability to withstand mildly alkaline conditions (Parker and Colby, 1995). In the present paper, we describe the overexpression of the gene of a DehD enzyme of Rhizobium sp. and the purification of enzyme produced by the overexpression system. This overexpression system may shed light into the properties, structure and function of the enzyme in the future. MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions E.coli K-12 strain NM522 was used for plasmid pUC18. Cells were grown aerobically at 30oC in PJC minimal medium containing 20 mM D,L-2-chloropropionic acid as carbon source. Ampicillin (100 µg/ml) and isopropyl thio-β-D-galactoside (IPTG) were incorporated as appropriate. Growth was followed by measurement of the absorbance at A680nm. Enzyme purification E.coli K-12 strain NM522 carrying plasmid pSC3 containing dehD+ gene, was grown in a 10 ml LB/amp culture overnight at 37oC. Cells from 2 ml of the culture were washed with sterile PJC before inoculating into 20 mM D,L-2-chloropropionic minimal medium containing 0.05% yeast extract plus 0.3 mM IPTG. Cells were grown at 30oC and harvested at late logarithmic phase (A680nm 0.7 to 0.8). The A680nm readings were taken every two hours. The cells 4 g (wet weight) were harvested and *Corresponding author email: fahrul@bio.fs.utm.my

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rinsed in 0.01 M Tris-acetate buffer pH7.6. The cell pellets resuspended in 20 ml of the same buffer was centrifuged at 10,000 g for 10 minutes at 4oC. The cells were then resuspended in 4 ml of 0.01 M Tris-acetate buffer pH7.6 and maintained at 0oC for ultrasonication in an MSE Soniprep 150W ultrasonic disintegrator at a peak amplitude λ=10 microns for 30 seconds. Unbroken cells and cell wall material were removed by centrifugation at 20,000 g for 15 minutes at 4oC. The latter was centrifuged at 140,000 g for 60 minutes to give a soluble preparation. To further purify the enzyme, the 2.8 mg protein (4.3 U enzyme) was applied to a Mono Q HR 5/5 anion-exchange column equilibrated with 5 mM sodium phosphate, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10% (mass/vol.) glycerol buffer, pH 7.6 and eluted with sodium phosphate gradient to 100 mM. Assay of dehalogenase activity Cells were harvested at late log phase and extracts were assayed for dehalogenase activity using D-2-chloropropionic as substrate for DehD. Cell-free extract was prepared in 0.01 M Tris-acetate buffer pH7.6. The enzyme reaction was carried out at 30oC in a mixture of 5 ml 0.09 M Tris-acetate (pH 7.5), substrate and enzyme. Samples were removed at 5 min intervals, and the free halide was determined colorimetrically (Bergman and Sanik, 1957). The colour was allowed to develop for 10 mins at room temperature and measured at A460nm. Enzyme activity (1U) was defined as the amount of enzyme that catalyses the formation of 1 µmol halide ion/min. The assay was carried in duplicate. RESULTS AND DISCUSSION Growth analysis E.coli NM522[pSC3] (dehD+) were inoculated into minimal medium flasks containing (i) 20mM D,L-2-

chloropropionic + 0.05% yeast extract. IPTG (final concentration 0.3 mM) was added to the growth medium before incubating at 30oC. The A680nm readings were taken every two hours. The growth curves and the maximum growth measured at A680nm are shown in Fig. 1. From the curve, the doubling time, Td = 3 hours. SDS-Polyacrylamide Gel Electrophoresis (PAGE) analysis of DehD The specific activity was calculated as in Table 1. SDS-PAGE analysis of the crude cell-free extracts that was carried out shown how much dehalogenase protein had been produced. SDS-PAGE for DehD showed the expected 29 kDa protein was expressed (Fig. 2). The amount of DehD was estimated visually to be about 20 – 30% of the total cellular proteins indicating that only 5-fold purification would be needed to obtain the pure enzymes. Purification of the DehD enzyme Cell-free extract was prepared in 0.01 M Tris-acetate buffer pH7.6 and 2.8 mg protein (4.3U enzyme) was applied to a Mono Q HR 5/5 anion-exchange column equilibrated with 5 mM sodium phosphate, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10%(mass/vol.) glycerol buffer, pH7.6 and eluted with sodium phosphate gradient to 100 mM. DehD eluted in four fractions between approximately 30 mM and 40 mM sodium phosphate. Each fraction had 1.3 U, 1.0 U, 1.15 U and 0.5 U enzyme, respectively and the enzyme recovered was estimated to be 92%. Analysis of the fractions by SDS-PAGE is shown in Fig. 3 In Fraction 11, there was slight contamination from host cellular protein. Specific activity of one of the fractions was measured (Fraction 10) which gave 14.4U/mg with D-2-chloropropionic as substrate. Since more than one fraction had DehD, possibly there are different forms of DehD protein. The Km value for each

0.1

1

0 2 4 6 8 10 12 14 16 18 20 22

Time(Hours)

Log

A68

0nm

20mM D,L2-CP+0.05%Yeast

Fig. 1. Growth of E.coli NM522 [pSC3] (dehD+) in D,L-2-chloropropionic minimal medium. Table 1. Specific activity for DehD, using D-2-chloropropionic acid as substrates.

Growth condition Specific activity (µmolCl-/min/mg protein) (20 mM D,L-2-chloropropionic acid + 0.05% yeast extract) 1.61

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fraction was determined using D-2-chloropropionic as substrate. All showed similar Km values of approximately 0.064 mM, suggesting that they are the same DehD protein. A possible reason for the protein being eluted at different ionic strengths is that a host protease may act on the dehalogenase protein. This problem may be avoided by expressing the gene in a protease-negative host cell.

Fig. 2. SDS-PAGE analysis of crude extracts from E.coli NM522 [pSC3] (dehD+) grown on D,L-2-chloropropionic acid minimal medium. Lane 1: NM522[pUC18](8µg protein) Lane 2: Crude extract from NM522[pSC3] (dehD+) (8µg

protein) Lane 3: SDS VII marker proteins Determination of Km values for dehalogenase enzymes with various substrates A range of substrates was used to obtain Km values for DehD enzyme. The overall results are shown in Table 2. Compounds that are not a substrate for an enzyme are indicated by a dash. Using DehD, the Km for D-2-chloropropionic was 0.067 mM. When D,L-2-chloropropionic with equimolar amounts of the two isomers was used the Km values were very similar to those with the single enantiomer. This indicates that under these conditions, the presence of the non-substrate enantiomer has little or no effect on Km. From all this information on Km values, the actual substrate concentration to use for enzyme assay to ensure that the enzyme was saturated with substrate could be determined. Molecular analysis From the complete dehalogenase D gene, the amino acid sequence was determined. The initiation codon was ATG (methionine). The deduced amino acid sequence of this

open-reading frame corresponded to a 29 kDa protein (consisted of 798 bp, encoded by a 266 amino acids protein with a calculated subunit molecular weight of approximately of 29 386 Da (Table 3). The calculated protein size agreed to the SDS-PAGE analysis that was carried out as shown in Fig. 3.

Fig. 3. SDS-PAGE analysis of the purification of DehD

Lane 1: Protein markers. Lane 2: MonoQ fraction 9 Lane 3: MonoQ fraction 10 (4µg protein) Lane 4: MonoQ fraction 11 (4µg protein) Lane 5: MonoQ fraction 12 (3µg protein) Lane 6: MonoQ fraction 13 (1µg protein) Lane 7: MonoQ fraction 14 Lane 8: Protein markers Lane 9: Crude extract of DehD

Bioinformatic analysis The Rhizobial DehL and DehD were completely different with each other by amino acid sequence comparison (18% identity). The deduced amino acid sequence of DehE also showed little identity to DehD (14%) and DehL (16%). It is of interest to compare DehD amino acid sequence by Blast search (NCBI) programme to see if there is any other protein with a similar amino acid sequence now present in the databases. Sequence from DehD showed slight identity to D,L-DEX enzyme (18% identity) derived from Pseudomonas sp. strain 113 (Nardi-Dei et al., 1997). However, the DehD protein has higher sequence similarity to the previously published sequence of several stereospecific dehalogenases for example 59% identity to Rhizobium sp. NHG3 (Higgins et al., 2005) and 23% to Pseudomonas putida strain AJ1 (Barth et al., 1992). CONCLUSIONS Dehalogenase D gene was very efficiently expressed in the recombinant cells. The amount of the enzyme produced correspond to about 20 – 30% of the total cellular proteins judging from the specific activities of the crude extract of 1.61 µmolCl-/min/mg protein. The purified enzyme from each fraction had 1.3, 1.0, 1.15 and 0.5 U/mg enzyme, respectively and the enzyme recovered was estimated to be 92%. The dehD gene encoded a 266

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amino acid protein with a predicted molecular mass of 29 386 Da. This value corresponds to the value of 29 kDa estimated by SDS/PAGE. This procedure is very useful for purification of a large amount of enzyme for characterization and application. Table 3. Predicted amino acid composition of DehD from Rhizobium sp.

Amino Acids DehD (Residue Per–Subunit)

Alanine (A) Cysteine (C) Aspartic acid (D) Glutamic acid ((E) Phenyalanine (F) Glycine (G) Histidine (H) Isoleucine (I) Lysine (K) Leucine (L) Methionine (M) Asparagine (N) Proline (P) Glutamine (Q) Arginine (R) Serine (S) Threonine(T) Valine(V) Tryptophan (W) Tyrosine (Y) Stop (Z)

27 05 12 11 10 16 09 19 03 26 05 01 18 10 30 26 17 12 04 04 1

Number of amino acids 266 Calculated molecular weight

29 386 Da

ACKNOWLEDEGEMENTS The author thanks the Malaysian Government Ministry of Science Technology and Innovation (Research Grant: 71925) for their generous support.

REFERENCES Allison, N., Skinner, AJ. and Cooper, RA. 1983. The dehalogenases of a 2,2 dichloropropionate degrading bacterium. Journal of General Microbiology. 129:1283-1293.

Barth, PT., Bolton, L. and Thomson, JC. 1992. Cloning and partial sequencing of an operon encoding two Pseudomonas putida haloalkanoate dehalogenases of opposite stereospecificity. Journal of Bacteriology. 174(8): 2612–2619.

Bergman, JG. and Sanik, J. 1957. Determination of trace amounts of chlorine in naptha. Analytical Chemistry. 29: 241-243.

Higgins, TP., Hope, SJ., Effendi, AJ., Dawson, S. and Dancer, BN. 2005. Biochemical and molecular characterization of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile Agrobacterium sp. Biodegradation. 16(5): 485-492.

Leigh, JA., Skinner, AJ. and Cooper, RA. 1986. Isolation and partial characterisation of dehalogenase - deficient mutants of a Rhizobium sp. FEMS Microbiology Letters. 36: 163-166

Nardi-Dei, V., Kurihara, T., Chung P., Esaki, N. and Soda, K. 1997. Bacterial D,L-2-haloacid dehalogenase from Pseudomonas sp. strain 113: Gene cloning and structural comparison with D and L-2haloacid dehalogenases. Journal of Bacteriology. 179(3): 4232-4238.

Parker, K. and Colby, J. 1995. Immobilisation of the D-2haloacid dehalogenase from Pseudomonas putida strain AJ1/23. Biodegradation. 6: 191-201

Swanson, PE. 1999. Dehalogenases applied to industrial-scale biocatalysis. Current Opinion in Biotechnology. 10: 365-369.

Table 2. Km values (mM) for different substrates using crude dehalogenase D (DehD) preparations.

Substrates Km values (mM) for DehD D-2-CP (D-2-chloropropionic acid) 0.067±0.014 L-2-CP (L-2-chloropropionic acid) - D,L-2-CP (D,L-2-chloropropionic acid) 0.089±0.015 2,2DCP (2,2-dichloropropionic acid) - D,L-2,3DCP (D,L-2,3-dichloropropionic acid) 0.53±0.063 MCA (monochloroacetic acid) 1.02±0.34 DCA (dichloroacetic acid) - TCA (trichloroacetic acid) - MBA (monobromoacetic acid) 0.62±0.11 DBA (dibromoacetic acid) - TBA (tribromoacetic acid) -

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 393-397, 2008 ISSN: 1715-9997

1

THE ROLE OF LOW-PROTEIN AND CASSAVA-CYANIDE INTAKE IN THE AETIOLOGY OF TROPICAL PANCREATITIS

*Okafor, PN, Anoruo, K, Bonire AO and Maduagwu EN2

Department of Biochemistry, Michael Okpara University of Agriculture Umudike, Abia State, Nigeria 2. Department of Biochemistry, University of Malawi

ABSTRACT

The contribution of low-protein and cassava-cyanide intake in the aetiology of tropical pancreatitis was investigated in male albino Wistar rats fed for 28 days with cassava diet containing 50mgCN-kg-1DM and 3% protein supplement, using acceptable biochemical methods. Assay for pancreatic amylase activity (indicator for pancreatic dysfunction) in blood of both control and cassava-cyanide fed groups indicated 9.43% and13.57% rise in activity of this enzyme in the latter above the former after 14 and 28 days respectively. Increases activity of some hepatic enzymes in the blood were also measured. Depletion of whole blood glutathione of the test animals by 57.33% and 84.38% after 14 and 28 days respectively above that of control was also observed. There was non-significant increase (p> 0.05) in plasma malonaldehyde (lipid peroxidation status) of the cassava-cyanide fed group when compared to the control. Significant decreases in plasma albumin and elation in blood and urine thiocyanate levels were also measured. The results are discussed from toxicological and mechanism of action points of views. Keywords: Cassava-cyanide, low-protein, tropical pancreatitis, rats.

INTRODUCTION Chronic pancreatitis is a relatively progressive disease that causes untold misery in its victims. Cyanogenic glycosides in dietary staples have been postulated as an aetiologic factor in this disease (McMillan and Geevarghese, 1979; Pitchmunoni, 1988) The good geographical match between the occurrence of tropical chronic pancreatitis and consumption of cassava (Manihot esculenta Crantz) a plant rich in cyanogenic glycosides, linamarin and lotaustralin formed the basis for the cassava cyanide toxicity theory (McMillan and Geevarghese, 1979) This theory has been discredited by the lack of cassava connection in some tropical areas (Narendranathan and Cheriyan, 1994), the apparently innocus nature of cassava in other zones (Abubakare et al., 1986) and the declining incidence of chronic pancreatitis in the province of Kerala, South India in the past decades (Geervarghese, 1986) although dietary pattern has not changed in this region where the cassava connection was first postulated (McMillan and Geevarghese, 1979). That long term feeding of cassava itself can produce chronic pancreatitis-like morphological changes in rat and rabbit exocrine pancreas has been demonstrated (Geldof et al., 1992: Shenoy et al., 1996). However no direct link between chronic cyanide exposure per se and pancreatitis has been made in humans or in animal studies. This paradox suggests that either the non-cyanide moiety

is the true pancreatic toxin, or that additional factor most likely low sulfur-amino acid content in diet or tissue contribute to the damage. The present study is aimed at investigating the latter, ‘the possible role of low-protein and cassava cyanide intake in the aetiology of tropical pancreatitis’. This will be carried out by feeding rats on cassava diet containing cyanide and low protein (i.e. 3% protein supplement) and then monitor indices of cyanide toxicity, destruction of exocrine pancreas and pancreatitis. This will be achieved by determining the activity of pancreatic amylase, whole blood glutathione (GSH) and malonaldehyde (lipid peroxidation) product along with cyanide and thiocyanate in blood and urine as well as plasma albumin. Blood test for amylase is used to diagnose pancreatitis, and glutathione (GSH) plays vital role in determining organ toxicity from plant glycosides in general, cyanogenic and non-cyanogenic (Wallig et al., 1988). Increased lipid peroxidation products (such as malonaldehyde) resulting from oxidant stress has been associated with pancreatitis (Sanfey et al., 1985) MATERIALS AND METHODS Animals and their diet Twenty male albino rats of the wistar strain (weighing 150-165g), bred in animal house of University of Nigeria Nsukka were used. All animals were kept at room temperature and had free access to drinking water and their diets. The animals were acclimatized to their environment and their diet for 7days before experiments *Corresponding author email: pnokafor@yahoo.com

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were commenced. The test rats were fed before and throughout the period of investigation on a high cassava (Manihot esculenta Crantz) starch diet containing 50mgCN-equivalentKg-1DM(see table 1). Control diet prepared as above with cornstarch in the place of cassava starch was fed to the control animals. Treatment of animals The rats were grouped into three. Groups 1 and 2 comprised 5animals each while group 3 of 10 rats served as the control. Groups 1 and 2 were fed cassava diet containing 50mgCN-kg-1 DM for 14 and 28 days respectively. Group 3 were fed control diet prepared with corn starch in place of cassava (see Table 1 and 2). Sacrificing of the animals After 14days, Group 1 animals together with five from control group were sacrificed and their blood collected with 5.0ml syringe intravenously from the heart. The group 2 animals and the remaining five in control were sacrificed at the end of 28days and their blood collected as above Determination of whole blood glutathione (GSH) Glutathione in blood is well established as an accurate indicator of whole body glutathione status (10). Whole blood glutathione was determined spectrophotometrically as described by Duron and Kelly (1963). Malonaldehyde determination Malonaldehyde was determined by the modified thiobarbituric acid (TBA) method of Gutteridge and Whilkin (1982). Cyanide and thiocyanate Cyanide was determined by the method of Esser, et al. (1993) and thiocyanate by ferric nitrate reagent (Sorbo, 1953). Serum protein and albumin Total protein was determined by the Biuret method as described by Layne (1975) and albumin by the dye-binding (Bromo-cresol green) method (Doumas, 1971) Determination of serum amylase activity and some other serum enzymes The assay of some serum enzymes as indicator of damages to some organs such as the pancreas, liver and kidney was carried out. The pancreatic amylase activity was determined using a kit from Quimca Clinica Aplicada S.A. Amylase-BPS. Kinetic enzymatic test with benzylidene-G7 PNP as substrate for in vitro determination of amylase in serum or plasma. Aspartate aminotransferases (AST) and alanine aminotransferases (ALT) were determined as recommended by Reitman and Frankel (1957) and alkaline phosphatase (ALP) as described by Klein et al (1960)

STATISTICS Students’ t-test was used for statistical analysis. Table 1. Composition of cassava diet for the feeding experiment.

Bassir (Personnel communication, 1979). Table 2. Some chemical components of the test and control diets.

Cyanide, mgCN-kg-1equivalent. Diet Free Bound Total Cassava based diet 3.45±0.11 46.55±0.21 50.00±0.3

Control diet ND ND ND The values given are the mean of three determinations. ND = not detected by the method of assay. RESULTS Physical Examination The physical activities of the rats during the period of the experiment were closely monitored. It was observed that all the rats were very active. The rate of feed consumption before and during the feeding experiment did not change in any of the groups. However, there was decrease in body weight of the cassava diet rats and the decrease was more pronounced at the end of the experiment (Table 5). On the other hand there was weight gain in the control rats. Increase in relative organ weight was also observed among the cassava diet animals compared to the control (Table 5). Table 3 shows the blood and urine cyanide levels, whole blood reduced glutathione (GSH) and serum malondialdehyde. There was statistically significant (p< 0.05) elevation in blood and urine thiocyanate of the test rats compared to the control. Depletion of whole blood glutathione (GSH) of the test rats by 57.33% and 84.38% after 14 and 28 days respectively. There was statistically non-significant (p> 0.05) rise in serum malondialdehyde of the cassava diet animals compared to the control. Table 4 shows the levels total protein, serum albumin, and activities of some hepatic enzymes and pancreatic

Ingredients Quantity gKg-1 Cassava flour 830 Fat-free soybean 30 Vitamin mixture 40 Salt mixture 20 Banana flavour 40 Groundnut oil 40

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amylase in the blood of both the control and test animals. There was 60% decrease in the serum albumin of the cassava diet rats compared to the control. Some hepatic enzymes and pancreatic amylase were elevated in the blood of the test groups. DISCUSSION In this study, we cold not establish conclusively a direct link between cassava cyanide exposure and low-protein intake in the aetiology of tropical pancreatitis. However, our results strongly indicate diverse manifestations of dietary cyanide toxicity in rats. The toxic manifestations strongly suggest that high dose of cyanide in diet in association with low protein intake could precipitate pancreatic dysfunction akin to tropical pancreatitis.

The appearance of significant amounts of both cyanide and thiocyanate in the blood of the test animals clearly indicate exposure to cyanide through the diet. That the concentrations of blood cyanide of the test animals (2.61± 0.42 and 3.2± 0.31after 14 and 28 days respectively) were higher than that reported to cause toxic manifestations in rat (Egekege et al., 1979) lends support to the exposure of the animals to cassava cyanide and the resultant toxic manifestations. Among the toxic manifestations were decreased body weights and increases in relative organ weight of the cassava diet groups (Table 5). The deceased body weights is as a result of the animals using the sulfur-containing amino acids of their body to detoxify the cyanide, since their dietary sulfur-containing

Table 3. Concentrations of total cyanide, thiocyanate, glutathione, and malonaldehyde in blood and urine of rats red the cassava cyanide diet.

Blood (mg mL-1) Urine (mg mL-1) CN SCN GSH MDA CN SCN

Group 1 (after 14 days) 2.61±0.42 14.41±1.03 23.00±2.11 10.31±0.42 6.73±0.82 17.82±2.03

Group 2 (after 28 days) 3.21±0.33 19.66±1.14 8.42±0.53 10.98±0.37 6.33±0.45 30.63±1.51

Group 3 (Control) ND 4.03±0.32 53.91±4.01 9.97±0.17 0.79±0.11 6.60±0.32

ND: Non detectable. Each is an average of three determinations. Table 4. Levels of some serum enzymes, protein and albumin in rats fed cassava diet.

Total protein Albumin ALP AST ALT Pancreatic amylase Groups

gdL-1 gdL-1 UL-1 UL-1 UL-1 UL-1 Group 1 3.04 2.21 120 14.00 12.00 101.00 Group 2 2.61 1.32 147 18.00 15.00 105.00 Group 3 4.82 3.30 101 10.00 8.00 92.00

ALP…. Alkaline phosphatase; AST…..Aspartate aminotransferases; ALT…..Alanine aminotransferases. Table 5. Relative organ weight and % weight gain/loss of the various animal groups.

Groups Liver Kidney Pancreas % weight gain/loss

1. After 14days 4.42 x 10-2 7.41 x 10-3 5.92 x 10-3 11.66

2. After 28days 4.89 x 10-2 7.50 x 10-3 6.55 x 10-3 23.26

3. Control 3.40 x 10-2 7.26 x 10-3 5.17 x 10-3 9.01 Relative organ weight was determined as the ratio the organ to that of the body weight of the animal. Increase Decrease

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amino acids, being insufficient could not support the detoxification of such high cyanide dose. Statistically significant (p<0.05) elevation in blood and urine thiocyanate is an indication of the attempt by the animals to detoxify the ingested cyanide. Thiocyanate (SCN) remains the most useful biomarker for cyanide exposure since it is a stable metabolite (Haque and Bradbury, 1999). One of the obvious implications of utilization of the sulfur amino acids for detoxification of dietary cyanide under low protein intake is that protein synthesis is compromised. This is clearly seen in decreased body weight (Table 5) and very low serum albumin level (Table 4) of the cassava diet group compared to the control. Depletion of whole blood glutathione (an important antioxidant of the body) of the rats fed cassava diet by 57.33% and 84.36% after 14 and 28 days respectively (Table 3) is a significant finding in this work. The decreased concentrations of whole blood glutathione could be attributed in part to reduced synthesis of this important biological compound as a result of cyanide detoxification as well as its consumption in the course of scavenging for reactive intermediates generated from metabolism of glucosidic and non-glucosidic cyanide as well other chemical species from the diet. In this connection, cysteine, a sulfur-containing amino acid needed for cyanide detoxification in the body (Nagahara et al., 1999) is the limiting amino acid in glutathione synthesis. Depletion in glutathione (GSH) levels to the extent observed in this study could further lead to drastic decrease in the total antioxidant status of the body of the animals. This is because glutathione (GSH) helps to recycle vitamins C and E (cellular antioxidants), blocks free radical damage, enhances the antioxidant activity of vitamin C, and plays a critical role in the detoxification of harmful compounds (Meister and Anderson, 1983). It is important to note at this point that both ‘oxidant stress’ and depletion of antioxidant system has been reported in pancreatitis (Dabrowski et al., 1991; Luthen and Greendell, 1994). Drastic decrease in antioxidant status of the body could then precipitate “oxidant stress” with concomitant attack of reactive intermediates or free radicals on cells of some target organs and tissues of the body. Our findings also include increases in the serum aminotransferases (aspartate and alanine aminotrans-ferases) and alkaline phosphatase as well as serum pancreatic amylase activity of the cassava diet animals above that of the control (see table 4). Similar findings have been reported in humans exposed to cassava cyanide through large scale cassava processing (Okafor et al., 2002). Increases in the serum concentrations of these

enzymes indicate damage to cell membrane of some organs (Joan et al., 1987; Bogusz, 1975) such as the liver, kidneys and exocrine pancreas. That exposure to cyanide either in its organic form or as dietary cyanide induces damage to some organs and tissues of animals have been demonstrated (Okolie and Osagie, 1999; Okafor and Maduagwu, 1999). The rise in pancreatic amylase activity in blood of the test animals by 9.43% and 13.57% above that of control after 14 and 28 days respectively is a suggestive evidence of destruction of the exocrine pancreas as elevation in serum amylase activity above normal range is an indication of leakage of this enzyme from exocrine pancreas. The damaging effects of cyanide on these tissues and organs could be mediated either by cyanide ion (being a nucleophile) or free radicals generated in the course of its metabolism or the metabolism of other chemical species within the system. The free radical damaging effect is further suggested by the increase (though not statistically significant, p> 0.05) in the blood malondialdehyde (a by product of lipid peroxidation) concentration of the cassava diet rats above the control (Table 3). Free radicals are known to induce lipid peroxidation and to generate of malonaldehyde and other lipid peroxidation products. Pancreatitis is a condition in which there is premature activation of the pancreatic enzymes resulting in self-destruction of the gland. It appears from our findings that the destruction of the pancreas could be attributed more to the attack of free radicals or reactive intermediates on the organ than from premature activation of the pancreatic enzymes. This is because other organs such as the liver and the kidney were affected as indicated in rises in aminotransferases and alkaline phosphatase as earlier discussed. Thus, the divers effects of cyanide on the organs and tissues including exocrine pancreas of experimental animals as reported in this work and by other authors are more of direct attack of reactive intermediates or free radicals on these tissues and organs. This holds true and more pronounced when the antioxidant status is affected as in the case of high cassava-cyanide and low protein intake over a considerable period of time. The body then tries to detoxify the ingested cyanide using sulfur-containing amino acids of the body. Protein synthesis is compromised and as well as the synthesis of glutathione that has cysteine (a sulfur-containing amino acid) as the limiting amino acid for its synthesis. This important biological compound (glutathione) plays central role in the antioxidant activities of the body. REFERENCES Abubakare, A., Taylor, R., Gill, GV. and Alberti, KGMM. 1986. Tropical or malnutrition-related diabetes. A real syndrome. Lancet. 1135-1138.

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Bogusz, M. 1975. The usefulness of the enzymatic tests in acute poisoning. Archives of Toxicology. 34: 159-167.

Dabrowski, AA., Gabrylewiez, A. and Chwieko, M. 1991. Products of lipid peroxidation and changes in sulfuhydryl compounds in pancreatic tissues of rats with caerulein-induced acute pancreatitis. Biochem. Metab. Biol. 46:10-16.

Doumas, BT., Watson, WA. and Biggs, HG. 1971. Albumin standards and measurements of serum albumin with Bromo-cresol Green. Clin. Chim. Acta. 31:87.

Duron, O. and B.Kelly, B. 1963. Improved method for determination of blood Glutathione. J. Lab. Clin. Med. 61: 882-887.

Esser, AJA., Bosveld, M., Van der Grift, RM. and Voragem, AG. 1993. Studies on cassava products and introduction of a new chromogen. J. Sci. Food Agric. 63: 287-296.

Egekege, JO. and Oehme, FW. 1979. Blood and liver cyanide concentrationa in rats poisoned oral doses of potassium cyanide. Toxicol. Lett. 31: 243-247.

Geevarghese, PG. 1986. Calcific pancreatitis. Bombay Varghese Publishing House.

Geldof, AA., Beching JLF., De Vries, CD. and Van Der Ven, E. 1992. Histopathological changes in rat pancreas after fasting and cassava feeding. In Vivo. 6: 545-552.

Gutterridge, JMC. and Wilkins, C. 1982. Copper-dependent hydroxyl radical damage to ascorbic acid. Formation of thiobarbituric acid reactive product. FEBS letl. 137: 327-340.

Haque, R. and Bradbuny, JH. 1999. Simple kit method for determination of thiocyanate in urine. Clin. Chem. 45: 1459-1464.

Joan, FZ., Peter, RP. and Philip, DM. 1987. Biochemical test for liver disease. In: Clinical Chemistry in Diagnosis and Treatment. 5th Edn. pp 291-292.

Klein, B., Read, PA. and Babson, LA. 1960. Rapid method for the quantitative determination of serum alkaline phosphatase. Clinical Chemistry. 6: 269-275.

Layne, E. 1975. Spectrophotometric and Turbidimetric methods for measuring proteins. In: Colowick, S. P and Kaplan, (eds). Methods in Erizymology. vol.3 New York Academic Press.

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Meister, A. and Anderson. 1983. Glutathione. Ann. Rev. Biochem-52:711-760.

McMillan, DE. and Geervarghese, PH. 1979. Dietary cyanide and tropical malnutrition diabetes. Diabetes Care. 2: 202-208.

Nagahara, N, Ito, T. and Minami, M. 1999. Mercaptopyruvate sulphur transferase as a defense against cyanide toxication . Molecular properties and mode of toxication. Histol. Histopathol. 14: 1277-1286.

Narendranathan, M. and Cheriyan, A. 1994. Lack of association between cassava consumption and tropical pancreatitis syndrome. J. Gastroenterol Hepatol. 9: 282-285.

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Shenoy, KT., Leena, KB. and Nair, RB. 1996. Pancreatic changes (enzymes and histological) in experimental model fed with cassava based diet. in: Tropical Tuber Crops: Problems, Prospects and Future strategies. Lebanon Science Publisher. 458-466.

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INDUCED POLYGENIC VARIATION STUDY IN SESAME (SESAMUM INDICUM L.) AND ITS IMPLICATION IN SELECTION

*Ghulam Sarwar, MA Haq, Akbar Ali Cheema and MB Chaudhary

Nuclear Institute for Agriculture and Biology (NIAB) Faisalabad, Pakistan

ABSTRACT

In this study, ninety four single plants selected from M2 generation were evaluated during summer 2006 in M3 generation. Single plant data pertaining to different morphological traits viz. plant height, number of branches, capsule per plant, capsule length, capsule breadth, seeds per capsule and seed yield were recorded and analyzed for genetic parameters, correlation coefficients and path coefficient. Number of branches and capsule per plant showed highly significant and positive correlation with seed yield. Capsule length exhibited maximum direct positive effect (0.8791) followed by capsule/plant (0.755) and plant height. Except capsule length other two attributes showing positive direct effects also exhibited highly significant and positive genotypic correlation which indicated that these characters may be selected directly for the improvement of seed yield. Highest heritability in broad sense was estimated in case of capsule length (74.5%) followed by plant height (63%), capsule breadth (60.8%) number of branches (51.9%) and seed per capsule (50.3%). Seed yield indicated the highest value of genetic advance (41.72%) followed by number of branches (34.32%), capsule length 17.84%) and capsule breadth (17.77%). High heritability in case of capsule length, number of branches, capsule length and capsule breadth combined with corresponding higher values of genetic advance indicated that these characters are controlled by additive type of genes and may be selected directly for seed yield improvement. From these results, it may be concluded that for making selection directly in this type of population, the main emphasis should be placed on plant height, number of capsule and to some extent number of branches. Secondarily to a minor extent capsule length may be considered alongwith seed yield and other yield components. Keywords: Induced polygenic variation, Sesamum indicum L, selection.

INTRODUCTION Pakistan is facing an acute shortage of edible oil and an amount of Pak Rupees 59,187/- million is being spent on its import. The country is producing only 30% of its requirement while 70% is being met through import (anonymous, 2005). Sesame is an important edible oil seed crop containing about 50 – 58% oil and 25% protein Sesame oil and foods fried in sesame oil have a long shelf life because the oil contains two very powerful antioxidant called sesamole and sesamin together with tocopherols. In addition to this it has tremendous food, industrial and medicinal value (Yosida and Tagaki, 1997; Morris, 2002). The use of sesame oil reduces the blood pressure due to the presence of polyunsaturated fatty acid (PUFAS), the good kind of fat that cuts cholesterol. The seed of sesame contain a protein with eight amino acids and vitamin of B complex which are important for cell oxygenation and thus have a favorable influence on the liver cells. So sesame seeds are recommended specially to those who suffer from some type of liver disorder. Sesame is an import substitute/export item of Pakistan,

earning an amount of 19.3 million dollars (Anonymous, 2005) and is cultivated on an area of 82.0 thousand hectare with an annual production of 35.1 thousand tons having an average seed yield of 42.8 kgha-1 (Anonymous, 2006) which is very low as compared to 1267 and 1063 kgha-1 of world’s leading countries, like Hondrus and Egypt respectively (Anonymous, 2000). Presently two cultivars viz. TS3 and Til 89 are available for cultivation which are simply the collection from locally available germplasm from the farmer’s field (Khan and Din, 1999). The major constraints to low yield are poor yielding varieties susceptible to various biotic and abiotic stresses alongwith improper management practices. Breeding for crop improvement, genetic variability is a prerequisite. Induced mutation has proved to be an efficient and supplemented tool for creation of genetic variability. Furthermore, the induced mutation is also helpful in breaking of unwanted/negative linkages present among different qualitative and quantitative attributes (Ashri, 1994; Kang et al., 1994; Rajput and Sarwar, 1994; Rehman and Das, 1994). Keeping in view these facts mutation breeding work on sesame improvement was initiated for creation of genetic variability and selection of desirable mutants in the succeeding generations. Genetic parameters such as genotypic and phenotypic correlations, path analysis, *Corresponding author email: sarwar_niab@yahoo.com

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heritability and genetic advances are helpful in identifying the characters which are important for selection directly or indirectly. Furthermore, it also tells us that to what extent weightage should be given to certain trait and on which characters the selection should be based for a particular population and environment. Heritability and genetic advance combination tells about the nature and type of genes whether they are of additive or non additive. Keeping in view all these facts, genetic parameters were worked out in induced population of sesame and based on this genetic information, selection criteria was formulated for further improvement. MATERIALS AND METHODS Sesame seed of four different genotypes viz. TS3, Til 89, Gp 11504, Gp 117 04 were got irradiated with gamma rays ranging from 100 – 800 Grey and M1 generation was grown during Kharif 2004. The M2 was raised during Kharif 2005 and single plant selection based on visual field observation regarding high bearing and disease resistance in respect of phyllody and virus were selected. Ninety four single plants selected from M2 generation were grown as plant progeny rows of 3 M length, keeping row to row and plant to plant distance of 45 and 15 cm respectively and studied during Kharif 2006 in M3

generation in three repeats in RCBD. Single plant data pertaining to different morphological traits viz. plant height, no. of branches, capsule per plant, capsule length, capsule breadth, seed per capsule and seed yield were recorded. The data thus collected were then subjected to analysis of variance (Steel and Torrie, 1980). The genetic parameters, genotypic/ phenotypic correlations and path coefficient were estimated following the procedures as described by Singh and chaudhry (1985). RESULTS AND DISCUSSION Plant height showed positive and highly significant genotypic correlation with number of branches, capsules per plant and seed yield (Table 1). Negative and highly significant correlation of plant height was observed in the capsule length and seeds/capsule. Number of branches showed highly significant and positive correlation with capsule per plant and seed yield. Capsules per plant showed positive and highly significant correlation with seed yield and highly significant but negative with capsule length and seed/capsule. Capsule length exhibited positive and highly significant correlation with capsule breadth and seed per capsule, while negative and highly significant with seed yield. Capsule breadth and seed/capsule showed negative and highly significant correlation with seed yield

Table 1. Genotypic correlation between different traits in M3 generation of sesame 2006.

Variables Plant Height(cm)

No. of Branches

Capsule/ plant

Capsule Length(cm)

Capsule Breadth (cm)

Seed/ Capsule

Plant Height (cm) No. of Branches .6379* Capsule/ plant .4275** .6449** Capsule Length(cm) -.5970** -.7317** -.3715**

Capsule Breadth(cm) .0770 -.3677** .0782 .4781**

Seed/ Capsule -.5177** -.6740** -.4289** .8259** .2011 Seed Yield (g) .3166** .6600** .6900** -.4050** -.4137** -.4400**

Table 2. Phenotypic correlation between different traits in M3 generation of sesame 2006.

Variables Plant Height (cm)

No. of Branches

Capsule/ plant

Capsule Length (cm)

Capsule Breadth (cm)

Seed/ Capsule

Plant Height(cm) No. of Branches -.3530** Capsule/ plant .1979 .4555** Capsule Length(cm) -.53553** -.4189** -.0321 Capsule Breadth(cm) .0860 -.1563 .1015 .4129** Seed/ Capsule -.2234* -.3196** -.0496 .6578** .2524* Seed Yield (g) .2395* .4926** .4988** -.1120 -.0908** -.1241

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In the case of phenotypic correlation (Table 2), plant height showed positive and significant correlation with number of branches and seed yield. Number of branches showed highly significant and positive correlation with capsule per plant and seed yield. Capsule per plant showed positive and highly significant correlation with seed yield. Capsule length exhibited positive and highly significant correlation with capsule breadth and seed per capsule. Capsule breadth showed significant and positive correlation with seed per capsule. Capsule length exhibited maximum direct positive effect (0.8791) followed by capsule/plant (0.755) and plant height (Table 3). Except capsule length other two attributes showing positive direct effects, also accompanied highly significant and positive genotypic correlation which indicated that these characters may be selected directly for the improvement of seed yield. Capsule length showed highest positive indirect effects (0.726) via seed per capsule followed by capsule per plant via number of branches (0.487) and capsule length via capsule breadth (0.4202). Genotypic coefficient of variation (Table 4) was highest (37.846%) in case of seed yield followed by number of branches (26.662%) and capsule per plant (14.949%). Phenotypic coefficient of variation (Table 4) was also highest in case of seed yield per plant (61.107%) but followed by capsules per plant (40.682%) and number of branches per plant (37.004%).

Highest heritability in broad sense (Table 4) was estimated in case of capsule length (74.5%) followed by plant height (63%), capsule breadth (60.8%), number of branches (51.9%) and seed per capsule (50.3%). The highest value of genetic advance as percent of mean was computed in seed yield per plant (41.72%) followed by number of branches (34.32%), capsule length 17.84%) and capsule breadth (17.77%). High heritability in case of capsule length, number of branches, capsule length and capsule breadth combined with corresponding higher values of genetic advance indicated that these characters are controlled by additive type of genes and may be selected directly for seed yield improvement (Johnson et al., 1955; Panse, 1957; Sarwar et al., 2004). Morphological data of elite selected mutants (Table 5) based on high bearing and seed yield indicated that maximum plant height (200 cm) was noted in mutant NS4/3 as compared to check variety TS3 (192.7 cm). Other high yielding mutants had less plant height than TS3. The minimum plant height (147 cm) was observed in mutant NS288/1 followed by mutant NS202/3 (164 cm). Branches per plant were highest in case of NS 14/3 (15) followed by NS 146/1 (13) and NS 60/1 (12). As compared to this, TS3 had 7 branches per plant. Almost all the selected high yielding mutants had more number of capsules per plant than the overall mean (103.22) and check variety TS3 (131.3). Maximum capsule per plant were noted in NS 4/3 (257) followed by NS 321/1 (228) and NS 14/3 (222). The capsule length was highest in

Table 3. Direct (in parenthises) and indirect effect of different traits in M3 generation of sesame.

Variables Plant

Height (cm)

No. of Branches

Capsule/ plant

Capsule Length (cm)

Capsule Breadth

(cm)

Seed/ Capsule

Genotypic correlation (rg) with

Seed Yield (rg) Plant Height (cm) (.3602) -.0616 .3228 -.5248 -.0651 .2851 .3166** No. of Branches .2304 (-.0963) .4870 -.6432 .3109 .3712 .6600** Capsule/plant .1540 -.0621 (.7551) -.3266 -.0661 .2362 .6905** Capsule Length (cm) -.2150 .0705 -.2805 (.8791) -.4042 -.4248 -.4050** Capsule Breadth (cm) .0278 .0354 -.0591 .4202 (-.8454) -.1108 -.4137** Seed/Capsule -.1865 .0649 -.3238 .7260 -.1700 (-.5507) -.4401**

Table 4. Genetic parameters of different traits in M3 generation of sesame 2006.

Variables Mean G.VAR. G.COV. (%) P.VAR. P.COV.

(%) h2 (%) GA (% of Mean)

Plant Height (cm) 174.10 239.558 8.784 380.007 11.064 63.00 12.41 No. of Branches 607 3.285 26.662 6.382 37.004 51.90 34.32 Capsule/plant 103.22 239.108 14.949 1770.812 40.682 13.50 9.68 Capsule Length (cm) 2.69 0.100 11.731 0.134 13.587 74.50 17.84 Capsule Breadth (cm) 0.63 0.007 12.665 0.011 16.425 60.80 17.77 Seed/Capsule 70.80 35.346 8.405 70.389 11.840 50.30 10.48 Seed Yield (g) 11.58 26.961 37.846 70.289 61.107 38.40 41.72

G. VAR.= Genotypic variance, G. COV. = Genotypic coefficient of variation P.VAR. = Phenotypic variance, P. COV. = Phenotypic Coefficient of variation h2 = heritability in broad sense, GA = Genetic advance

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case of mutant NS 288/1 (3.9 cm) and NS 202/3 (3.8 cm). These two mutants had also the highest number of seeds per capsule i.e. 96 and 88 respectively. The highest seed yield (53 g) was estimated in mutant NS 60/1 followed by NS 321/1 (50 g) and NS 14/3 (49 g). As compared to this over all mean seed yield per plant was 13.58 g and that of check variety TS3 18.6 g/plant. Plant height, number of branches and capsule per plant showed highly significant and positive correlation with seed yield, hence following these results more stress should be given to these traits while selecting sesame genotypes for seed yield improvement. Except number of branches, plant height and capsule per plant also showed positive direct effects alongwith positive and significant genotypic correlation with seed yield which indicated that seed yield can be exploited directly by selection genotypes based on these attributes. In case of branches per plant where correlation coefficient was positive, but the direct effect was negative, the indirect effects seem to be cause of correlation. In such situation, the indirect casual factors are to be considered simultaneously. Highest positive direct effect was noted in case of capsule length but the correlation was negative. Under these circumstances, a restricted simultaneous selection model is to be followed, i.e. restrictions are to be imposed to multiply the undesirable indirect effects in order to make use of the direct effect (Singh and Kakar, 1977). Govindarsu and Ramamoorthi (1998) observed strong correlation of seed yield with number of branches and capsule number in irradiated and non irradiated segregating population of sesame. These attributes had also high direct effects in both types of populations, revealing that they had significant effects on determining seed yield.

In another study Govindarsu et al. (1998), observed consistently high strong positive genotypic correlation and high positive direct effect of branches per plant and capsule number with seed yield in M3, F3 and F2M2 generations. The other component characters fluctuated in their relationship with seed yield. They suggested that in determining seed yield, branch number and capsule number are the most important traits; hence selection for these traits would ensure the improvement of seed yield in sesame. Studying 60 diverse types of sesame, Nimbalkar et al. (1999) estimated highly significant and positive correlation of seed yield with number of capsule, plant height, 1000 seed weight and number of branches. Backiyarani et al. (1999) found positive correlation of seed yield with capsule number, 1000 seed weight and plant height and negative with percentage of seed oil in F2 and F3 populations of sesame. In both the generations, path coefficient analysis revealed the importance of number of branches as selection criteria for seed yield improvement. Intermating in the F2 or a later generation was suggested for improvement of yield and oil content simultaneously. Gandhara (2005) observed high heritability and high genetic advance in case of plant height, capsule number and 100 seed weight in 72 genotypes of sesame but combination of high heritability and low genetic advance in case of branches per plant and seed yield. He has explained that in the first case, simply selection at this stage may be benefited and workable for seed yield improvement, but for the 2nd case to select superior lines, recurrent selection would be effective in the succeeding generations. High heritability indicates that the character is controlled by such type of genes which are less influenced by environmental factors and vice versa. It was

Table 5. Morphological attribures of sessame mutants in M3 generation 2006.

S. No. Genotype

Plant height (cm)

Branche/ plant

Capsule/ plant

Capsule length (cm)

Capsule breadth

(cm)

Seed/ capsule

Seed yield/

plant (g)

Phyllody Reaction

(%) 1. NS 4/3 200 11 257 2.9 0.9 68 33 0 2. NS 14/3 185 15 222 3.0 0.8 72 49 0 3. Ns 32/2 150 10 201 2.6 .06 68 43 0 4. NS 44/1 190 8 190 2.6 0.5 72 30 0 5. NS 60/1 187 12 214 2.4 0.6 48 53 0 6. NS 120/1 130 11 184 2.6 0.6 72 39 0 7. NS 146/1 184 13 150 2.7 0.6 80 33 0 8. NS 146/2 179 10 183 2.7 0.6 76 35 0 9. NS 202/3 164 3 102 3.8 0.8 88 22 0 10. NS 288/1 147 4 126 3.9 0.8 96 24 0 11. NS 311/1 185 9 200 2.5 0.6 76 33 0 12. NS 321/1 180 8 228 2.5 0.6 72 50 0 13. TS3 (Check) 192.7 7 131.3 2.56 0.76 64 18.6 0

Mean 174.10 6.7 103.22 2.69 0.63 70.80 13.58 1.07 CV(%) 11.06 37.00 40.68 13.58 16.24 11.84 61.107 -

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further reported that in case of high heritability combined with a low genetic advance, the character is mainly under the control of non additive type of genes (epistasis, dominance or their interaction). Saravanan et al. (2003) evaluating sesame lines also emphasized that if a character is influenced less by the environment and controlled by additive type of genes, selection can be made directly for the improvement of this particular character at an early stage, and if the character is controlled by non additive type of genes, then the selection for this trait should be postponed and performed safely in the advanced/ succeeding generations. From the results of foregoing study, it may be summarized and concluded that through nuclear techniques, first the variability can be created in seed yield and its components and secondly positive variability may be exploited through continuous selection based on certain scientific knowledge. For making selection directly in this type of population, the main emphasis should be placed on plant height, number of capsule and to some extent number of branches. Secondarily to a minor extent capsule length may be considered alongwith seed yield and other yield components. REFERENCES Anonymous. 2000. FAO production year book. 54:123.

Anonymous 2005. Government of Pakistan. Federal Bureau of Statistics, External trade section, Karachi, Pakistan.

Anonymous. 2006. Agri. Statistic of Pakistan. Ministry of Food and Agri. Pakistan.

Ashri, A. 1994. Modification and adaptation of the induced determinate sesame mutant by cross breeding and its evaluation. Mutation breeding of oil seed crops. IAEA- TEC DOC- 781. pp: 111-114.

Backiyarani, S., Subramanian, M. and Shanthi, S. 1999. Character association and path coefficient analysis in segregating generation of sesame (Sesamum indicum L.). Crop Research. 18: 251-255.

Gandhara Rao, SVS. 2005. Genetic variability in sesame (Sesamum indicum L.). Sesame and Safflower Newsletter. 20: 26-28.

Govindarsu, R., Subramaniam, M., Nadarajan, M. and Ramamoorthi, N. 1998. Selection criteria for yield improvement in sesame. Annals of Agriculture Research. 19: 433-436.

Govindarsu, R. and Ramamoorthi, N. 1998. Character association in irradiated and non irradiated segregating population in sesame. Crop Improvement. 25: 83-87.

Johnson, HW., Robinson, HE. and Comstock, RE. 1955. Estimate of genetic and environmental variability in soybean. Agron. J. 47:314-318.

Kang, CW., Lee, JI. and Choi, BH. 1994. Mutation Breeding for disease resistance and high yield of sesame (Sesamum indicum L.) in the Republic of Korea. Mutation breeding of oil seed crops. IAEA – TECDOC-781.pp: 69-82.

Khan, NI. and Din, F. 1999. TS3 a new sesame white cultivar. J. Agric. Res. 37: 123-128.

Morris, JB. 2002. Food, industrial, nutraceutical, and pharmaceutical uses of sesame genetic resources. P. 153-156. In: J. Janik and A. Whipkey (eds.), Trends in new crops and new uses. ASHS press, Alexandria VA.

Nimbalkar, CA., Navale, PA. and Uplap, DD. 1999. Relative contribution of component characters on yield of sesamum. Journal of Maharashtra Agricultural University. 24: 260-262.

Panse, VG. 1957. Genetics of quantitative characters in selection plant breading. Int. J. Genet.17: 318-32.

Rehman, A. and Das, ML. 1994. Evolution of improved varieties of sesame through induced mutation. Mutation breeding of oil seed crops. IAEA-TEC DOC – 781: 115-118.

Rajput, MA., Sarwar, G., Siddiquei, KA. and Siddiqui, MA. 1994. Genetic improvement of sesame for plant architecture and grain yield through nuclear techniques. Mutation breeding of oil seed crops. IAEA – TEC DOC – 781: 89-96.

Saravanan, S., Nadarajan, N. and Kumari, TV. 2003. Variability Study in sesame. Crop Research. 25: 325-327.

Sarwar, G., Sadiq, MS., Saleem, M. and Abbas, G . 2004. Selection criteria in F3 and F4 population of mungbean (Vigna radiate L. wilczek). Pak. J. Bot. 36: 297-31.

Singh, RK. and Kakar, SN. 1977. Control on individual trait means during index selection. Proc. Third congr. SABRAO (Canberra), 3(d): 22-25.

Singh, RK. and Chaudhry, BD. 1985. Biometrical methods in quantitative genetic analysis. Kalyan’s publisher New Delhi. 1-303.

Steel, RGD. and Torrie, JH. 1980. Principles and procedures of statistics. A biometrical approach. 2nd ed. Mc. Graw Hill Book Company.

Yermanos, DM., S. Hemstreet, S., Salleb, W. and Huskar, CK. 1972. Oil content and composition of the seed in the world collection of sesame introductions. J. Amer. Oil Chem. Soc. 49: 20-23.

Yoshida, H. and Tagaki, S. 1997. Effect of seed roasting temperature and time on quality characteristics of sesame (Sesamum indicum L.) Oil J. Sci. Food Agric. 75: 19-26.

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SMOOTH MUSCLE RELAXANT ACTIVITY AND PROXIMATE EVALUATION OF KIGELIA AFRICANA

*Omonkhelin J Owolabi1, Eric KI Omogbai1, Anthony B Eledan1 and Abiodun Falodun2

1Departments of Pharmacology and Toxicology and 2Pharmaceutical Chemistry, Faculty of Pharmacy University of Benin, Benin City, Edo State, Nigeria

ABSTRACT

The ethanolic extract of Kigelia africana at 400.0 and 800.0 µg /ml concentrations were tested on the isolated rat uterus. Both concentrations significantly (p<0.0001) exerted high smooth muscle relaxant activity on the uterus (a reduction of oxytocin and acetylcholine induced contractions as well as inhibition of the spontaneous contractions of the uterus were observed). Evaluation of the data also indicated that the relaxant effect was dose-dependent. Its relaxant activity was 80 and 90% of the inhibitory effects produced by salbutamol (0.002µg/ml) and atropine (0.02µg/ml) on oxytocin and acetylcholine induced contractions respectively. Proximate analysis of the powdered crude stem bark was also carried out. The results indicate the presence of active principles in the bark extracts of Kigelia africana which may be responsible for some of the applications in traditional medicines as remedy against threatened abortion and retained placenta. The proximate analysis carried out in this study is used to establish the identity of the crude drug sample. A moisture content of 5.55 ± 0.02 % was obtained. The total ash is a measure of the non-volatile inorganic constituents remaining after ashing. The values of 3.26 ± 0.13 were obtained. Keywords: Kigelia africana; rat uterus; tocolytic activity; bark extract.

INTRODUCTION The use of medicinal herbs is as old as mankind and throughout ages, traditional medicinal plants have long contributed tremendously to alleviate the sufferings of the human race (WHO, 1995). Today there is no doubt that complementary medicines have gained wide acceptability and is increasingly attracting attention from scientific community world wide (Newal et al., 1986). Kigelia africana, a medium sized tree, 30 feet high or more is classified under the Bignoniaceae species. It’s commonly referred to as the sausage tree and known locally by Cucasians as the cucumber tree. The sausage tree draws its name from its large sausage- shaped fruit, suspended from lengthy stalks (Cox and Balick, 1994). It is abundant in the tropics and is widely used in Southern, Central and West Africa as a herbal remedy for various ailments such as eczema and psoriasis, diarrhoea, malaria, rheumatism, retained placenta and dizziness (Gill, 1992). The stem bark in particular has a wide reputation in folk medicine for the treatment of malaria, rheumatism, wounds, ulcers, retained placenta, venereal diseases, diarrhoea and to combat infections (Burkill, 1985). As hollow organs, uterus shares a number of similarities with the stomach, both belong to the group of smooth muscles that are spontaneously active and both

maintain force as their volume increases and to expel its content, but even more differences exist between them (Bulletti et al., 2000). Data on the effect of Kigelia africana on the contraction or relaxation of the uterine smooth muscle is lacking. Recent reports indicate the antibacterial activity of the fruits (Grace et al., 2002), antimicrobial activities of the stem bark (Akunyili, 1991) and the antidiarrhoeal activity of the aqueous leaves extract (Akah, 1996). In the present study, we evaluated the ethanolic extract of the stem bark for possible activity on the isolated uterus of non-pregnant rats based on its use as a remedy for retained placenta. MATERIALS AND METHODS Plant material The barks of Kigelia africana were collected in Okhoro Village, Egor Local Government Area of Edo State, Nigeria, between February and July 2005. The botanical identity of the plant and its bark were authenticated by Alhaji Alasa Abubakar (of blessed memory), a hebarium curator of the Department of Pharmacognosy, Faculty of Pharmacy, University of Benin, Nigeria. Botanical authentication was confirmed at the Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria where a voucher specimen (No.FHI107654) was deposited for future reference. Immediately after collection, barks were cut into small pieces and dried under sunlight. The dried barks were pulverized into a fine powder using impact mill, weighed and kept for further analysis. *Corresponding author email: josphineomo@yahoo.com

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Extraction The powdered material (500g) was macerated with absolute alcohol (2.5 litres) and left for 72 hours. The mixture was stirred at six-hourly intervals using a sterile glass rod. The extract was filtered and the filtrate evaporated to dryness with the aid of a rotary evaporator attached to a vacuum pump at 40oC. The concentrated extract was stored in air tight containers, labelled and refrigerated at 4oC prior to use. Drugs and Chemicals The following drugs were used: diethylstilbestrol (Merck), Oxytocin (Rotex Medica), Salbutamol (Glaxosmithkline), Acetylcholine (Sigma Aldrich), and Atropine (Sisbu Xierkang Pharm Co.Ltd), Absolute ethanol (Sigma Aldrich). Animals Female non-pregnant Wister rats weighing 120-150g and swiss albino mice (20-40g) of either sex were obtained from the Animal house of the College of Medicine, Ambrose Alli University, Ekpoma, Edo State, Nigeria. Animals were given two weeks acclimatization time before being subjected to the experimental protocol. The animals were maintained on a standard diet (Ladokun feeds, Ibadan, Oyo State, Nigeria) and had access to food and water ad libitum. Animals were housed in a cage with a twelve hour light-dark cycle. [The ethical committee on the use of animals, Faculty of Pharmacy, University of Benin, Edo State. Nigeria, approved all animal experiments]. Proximate analysis of the powdered crude bark The following quantitative parameters were carried out using standard methods (African Pharmacopoeia, 1986, British Pharmacopoeia 1988, and AOAC, 2003). Moisture content /water loss on drying The powdered drug (2.0 g) was weighed into a clean crucible of known weight. After oven drying at 105oC for 5 hours and cooled. The crucible was weighed to determine weight loss in the powdered drug. The average percentage weight loss, with reference to the air dried powdered drug was determined for four replicates. Total Ash Determination The crucibles were washed thoroughly, dried in hot oven at 100OC, cooled in desiccators and weighed. A 2.0 g portion of each of the samples were weighed into the crucible and put in the furnace. Heating was started gradually until temperature of 600oC was reached. This temperature was maintained for 6 hours. The crucible was then put inside the desiccators and cooled. After cooling the sample was reweighed and the percentage ash calculated.

N

100Z-W Ash % ×=

W=Weight of the crucible and ash Z=Weight of empty crucible N=Weight of Sample. Acid Insoluble Ash Value Determination The crucible with the ash of the stem bark from the experiment above was transferred into a beaker containing 25 ml of dilute HCl. The beaker and its contents were boiled for 5 minutes and the boiled contents filtered through an ashless filter paper. The washings were then passed through the filter paper in a manner as to allow the collection of the residue at the tip of the cone of the filter paper. The weight of the clean and heated porcelain crucible was accurately determined. The filter paper with the residue was folded with a small cone and transferred into the crucible. The crucible was gently heated until the filter paper was completely ashed, and then heated strongly for few minutes. The crucible and its contents were cooled, weighed and the final weight was noted. The weight of the residue (ash) was then calculated. This was done by subtracting the constant weight of the crucible and ash. The weight of the ash was divided by the initial weight of the drug multiplied by a hundred was taken as the acid insoluble ash value. Water Soluble Ash Value Determination The crucible with the total ash as in acid insoluble ash was transferred into a beaker containing 25 ml of distilled water. The beaker and its contents were boiled for 5 minutes and filtered through an ashless filter paper. The filter paper containing the residue was folded and placed in a weighed porcelain crucible. The crucible was then heated in the muffle furnace, until the filter paper was completely ashed. The crucible and its content were cooled and weighed and the final weight noted. The weight of the residue was then calculated by subtracting the constant weight of the second crucible and its ash. This is the water insoluble ash. The weight of the water soluble ash was obtained by subtracting the weight of the water insoluble ash from the total ash. The weight of the water soluble ash divided by the initial weight of the crude drug was multiplied by 100 and was taken as the water soluble ash value. Determination of Extractives (a) Alcohol Soluble Extractive Value Powdered leaf drug (5.0 g) was weighed into a 250 ml stopper conical flask. Ethanol 90 % (100 ml) was added and the conical flask and stoppered. The flask was shaken in a mechanical shaker for 6 hours and then allowed to stand for 18 hours. The extract was filtered by suction filtration using a Buckner funnel. The weight of a heated cooled flat bottom porcelain crucible was accurately determined. The filtrate (20 ml) was poured into weighed crucible and evaporated to dryness at 100oC. The residue

Owolabi et al. 407

was dried to constant weight and the final weight noted. The weight of the residue obtained from the extract (20 ml) was determined by subtracting the constant weight of crucible from the residue. The alcohol extractive was then calculated with reference to the initial weight of the powdered drug and expressed as percentage. (b) Water Soluble Extractive Value The above experiment was repeated using chloroform: water 400:1. The water soluble extractive value was done for the powdered drug. Animal preparation Female non-pregnant Wister rats were pretreated intraperitoneally with 1 mg/kg of Diethylstilbestrol 24 hours prior to the actual experiment (Veale, 1989). The rats were killed by cervical dislocation and exanguinations. The abdomen was opened and the two horns of the uterus carefully isolated, freed of mesenteric fat and a 1 cm piece was mounted in a 50 ml organ bath containing De Jalon physiological salt solution having the following chemical composition: NaCL, 9 g/l, NaHCO3, 0.5 g/l, D-glucose, 0.5 g/l, KCL, 0.402 g/l, CaCL2.2H20, 0.08 g/l. Each uterine strip was suspended vertically between two parallel stainless steel hooks for measurement of isometric tension. The tissue was aerated with 95% oxygen 5% carbon (IV) oxide and temperature maintained at 37oC, with a PH of 7.4. The spontaneous contraction of the uterus was recorded with FT 03 transducer connected to an Ugo Basile recorder (7075). The transducer was previously calibrated to establish a relationship between the force applied to the transducer and the gauge deflection (500 mg). The tissue was allowed to equilibrate for 30 minutes before the commencement of the experiment. The dose- response curves of oxytocin and acetylcholine induced contractions were first obtained, the effect of the ethanolic extract (400.0 and 800.0 µg/ml) and that of two positive controls (salbutamol and atropine) were also determined. Statistical analysis All results are expressed as the mean of five experiments ± SEM. The statistical package used was SAS, 1994 Users guide, Version 8.2. SAS Institute Inc., Cary, NC, USA. The statistical significance (p <0.0001) of differences between means was assessed by an analysis of variance (ANOVA) followed by Duncan’s multiple range test. RESULTS The plant extraction gave a yield of 3.78%. On the proximate analysis shown in table 1, a moisture content of

5.55 ± 0.02 % was obtained. The total ash which is a measure of the non-volatile inorganic constituents remaining after ashing was 3.26 ± 0.13. The results showed that various concentrations of oxytocin (0.2 to 0.8ml of 0.1 and 1.0 I.U) produced a significant contraction of the rat uterus, with maximum response been produced at 0.8 ml of 1.0 I.U. This was also noted for acetylcholine (0.2 to 0.8 ml of 1.0, 10.0 and 100.0µg/ml) which also produced significant contraction of the uterus with the maximum been produced by 0.8 ml of 100.0 µg/ml. Table 1. Percentage (%) values of proximate analysis of the stem bark of Kigelia Africana.

Parameter Values ± SEM (%) Moisture content 5.55 ± 0.02 Total ash 6.42 ± 0.01 Acid insoluble ash 3.26 ± 0.13 Water soluble ash 2.88 ± 0.18 Alcohol extractive 8.68 ± 0.50 Water extractive 9.42 ± 0.18

Evaluation of the data indicates that there was a significant (p<0.0001) dose-dependent reduction in oxytocin and acetylcholine induced contractions by the ethanolic extract at 400.0 and 800.0µg/ml concentrations tested (Figs. 1 and 2). It is noteworthy that a complete blockade of acetylcholine induced contraction was observed by both doses of the extract against the first four doses of acetylcholine, this was also observed for atropine that completely blocked the response to the first three doses of acetylcholine (Fig. 2). The results in figure 1 also shows a comparative inhibitory activity produced by the extract and salbutamol which is used clinically in the treatment of threatened abortion in gravid uterus, while figure 2 shows a comparative inhibitory activity produced by the extract and atropine on acetylcholine induced contraction. The activity of the two concentrations of the extract compares well with salbutamol and atropine, two positive controls that significantly (p<0.0001) relaxed the uterus (Figs. 1 and 2 respectively), producing about 80% and 90% of the inhibitory effects of salbutamol and atropine. DISCUSSION The proximate analysis carried out in this study is used to establish the identity of the crude drug sample. The moisture content shows the susceptibility of crude drug samples to microbial attack especially fungi, and also to degradation due to hydrolysis of the crude powdered drug. A moisture content of 5.55 ± 0.02 % (Table 1)

Canadian Journal of Pure and Applied Sciences 408

obtained from this study is indicative of the storage quality for some time without microbial degradation or hydrolytic break down of the chemical constituents. The maximum range is between 6-8% in African Pharmacopoeia (1986). The total ash is a measure of the non-volatile inorganic constituents remaining after ashing. It is made up of two parts, the physiological and the non physiological ash. The physiological ash consists of carbonates, phosphates, nitrates, sulphates, chlorides and silicates of metals which the plant took up when it was growing. The non-physiological ash represents ash from extraneous matter. The acid insoluble ash is residue obtained when total ash is boiled with 10% HCl. It is a measure of the sandy matter in the crude drug samples. The values of 3.26 ± 0.13 were obtained. The uterus is spontaneously active, which means that, without any nervous or hormonal stimulation, a piece of isolated, pregnant or non-pregnant, uterus will produce regular spontaneous contractions (Brenninkmeijer et al., 1999). Our results showed that the ethanolic extract at concentrations of 400.0 and 800.0 µg/ml dose-dependently produced significant inhibition of oxytocin and acetylcholine induced contractions of the uterine

smooth muscle in non-pregnant rats (Figs. 1 and 2). The 800.0 µg/ml gave the higher percentage inhibition. A complete blockade of acetylcholine induced contraction was also observed by both doses of the extract against the first four doses of acetylcholine. It can thus be inferred that the extract might act via muscarinic receptors since there was complete blockade at lower doses and significant reduction in acetylcholine induced contractions at higher doses of acetylcholine. In addition, interestingly the extract was found to inhibit the normal spontaneous contraction of the uterus. The results suggest that the ethanolic extract of Kigelia africana has a potential tocolytic effect that can be explored for therapeutic advantage as an alternative treatment for threatened abortion and dysmenorrhoea. On the basis of its acclaimed folkloric use in the treatment of retained placenta, a contractile or oxytocin like effect on the uterus was expected. However the results points to a relaxant rather than a contractile effect. CONCLUSIONS The findings of our study indicate that the ethanolic extract of Kigelia africana stem bark possess inhibitory

Fig. 1. Inhibitory activity of the ethanolic stem bark extract of Kigelia africana on oxytocin induced contraction ofthe isolated rat uterus. Values are mean percentage responses + SEM; (n = 5 animals); K.A stands for the ethanolic extract of Kigelia africana. The extract significantly (P<0.0001) reduced the percentage response due to oxytocin.

Owolabi et al. 409

activity on the uterine smooth muscles in non-pregnant rats, which is inconsistent with the literature report (of its use in the treatment of retained placenta). Rather it’s been envisaged that the active ingredients (compounds) will have a potential for being added to the present list of tocolytic agents used clinically. Further work is in progress to determine the mechanism of action at the receptor site. ACKNOWLEDGEMENTS We gratefully acknowledge the technical assistance of Dr Z.A.M Nworgu and Pharm F.C Amaechina. REFERENCES African Pharmacopoeia. 1986. Vol. 2. 1st Ed. OAU/STRC Publications. pp.128-144.

Akah, PA. 1996. Antidiarrhoeal activity of the aqueous leaf extract of Kigelia africana in experimental animal. Journal of Herbs, spices and medicinal plants. 4(2): 31-38.

Akunyili, DN. 1991. Antimicrobial Activities of the Stembark of Kigelia Pinnata. Journal of Ethno-pharmacology. 35: 173-177.

AOAC. 2003. 14th Ed. Official Method of Analysis Association of Official Analytical Chemists. Washinghton DC, USA. 1112 -1114.

Brenninkmeijer, CB., Price, SA., Lopez, BA. and Phaneuf. 1999. Expression of G-protein-coupled receptor kinases in pregnant term and non-pregnant human myometrium. Journal of Endocrinology. 162: 401-408.

British Pharmacopoeia BP. 1988. Vol.II. Her Majesty’s Stationary Office, London. P.1022.

Bulletti, C., De Ziegler, D., Polli V., Diotallevi L., Del Ferro E. and Flamigni, C. 2000. Uterine contractility during the menstrual cycle. Human Reproduction, 15: 81-89.

Burkill, HM. 1985. The useful plants of West Tropical Africa. Planta West Africa. 1: 254-257.

Cox, PA. and Balick, MJ. 1994. The ethnobotanical approach to drug discovery. Sci Am. 270(6):82-87.

Gill, LS. 1992. Ethnomedical uses of plants in Nigeria. Uniben Press, Benin City. pp 143.

Grace, OM., Light, ME., Lindsey, KL., Moholland, DA., Staden, JV. and Jager, AK. 2002. Antibacterial activity and isolation of antibacterial compounds from the fruit of

Fig. 2. Inhibitory activity of the ethanolic stem bark extract of Kigelia africana on acetylcholine induced contraction of the isolated rat uterus. Values are mean percentage responses + SEM; (n = 5 animals). K.A stands for the ethanolic extract of Kigelia africana. The extract significantly (P<0.0001) reduced the percentage response dueto acetylcholine. The first four doses of acetylcholine were completely blocked by both doses of the extract.

Canadian Journal of Pure and Applied Sciences 410

the traditional African medicinal plant Kigelia africana. South African journal of Botany. 68: 220-222.

Newal, CA., Anderson, LA. and Phillipson, JD. 1986. Herbal medicine, a guide for health care professionals. Pharmaceutical press. 291-296.

Veale, DJH., Oliver, DW., Arangics, NS. and Furman, KJ. 1989. Preliminary isolated organ studies using an aqueous extract of Clivia miniata leaves. Journal of Ethnopharmacology. 37: 341-346.

World Health Organization (WHO). 1995. The World Health Report, Bridging the gap. Geneva 1, p.118.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 411-416, 2008 ISSN: 1715-9997

1

A HIGH YIELDING AND DISEASE RESISTANT MUTANT OF LENTIL DEVELOPED THROUGH SEED IRRADIATION OF AN EXOTIC GERMPLASM

Muhammad Siddique Sadiq, *Sajjad Haidar, Muhammad Ahsanul Haq, Ghulam Abbas,

1Munir Ahmad Chaudhary and Noor Muhammd1 Mutation Breeding Division, Nuclear Institute for Agriculture and Biology, Faisalabad

1 Pulses Research Institute, Ayub Agriculture Research Institute, Faisalabad

ABSTRACT

A high yielding mutant derived after seed irradiation of an exotic ICARDA (International Center for Agriculture Research in the Dry Areas) accession ILL 2580, was evaluated under the name of NL 20-9-4 for seed yield and wide adaptation in different yield trials during 1993-2006. It has shown superb yield performance in various yield trials i.e. yield screening nursery, intermediate, advanced and adaptation, conducted during 1993-2000 by producing 20% to 60% higher seed yield as compared to standard varieties; Masoor 85 and Masoor 93. In Lentil National uniform yield trials; NL 20-9-4 was tested at eleven locations including five locations in Punjab province of Pakistan during 2001-02 and twelve locations including five locations in Punjab during 2002-03. NL 20-9-4 ranked second by producing 1121 kg ha–1 during 2001-2002 and secured fourth position by producing seed yield of 1763 kg ha–1 during 2002-03 in the country. NL 20-9-4 showed an increase of 15% and 12% in seed yield over Masoor 93 during the year 2001-02 and 2002-03 respectively. NL 20-9-4 produced significantly the highest yield of 1983 kg ha-1 and 1685 kg ha-1 when planted on November 10, 2004 and November 22, 2005 respectively in agronomic trials. This line has shown resistance against Ascochyta blight, rust, and Botrytis. It has distinctness of thick stem with profuse pubescence, erect growth habit, dark green leaf, synchronous pod maturity and higher number of pods. Based on high seed yield potential along with other desirable traits, NL 20-9-4 has been approved with the name of “NIAB MASOOR 2006’’ for general cultivation in the lentil growing areas of the Punjab province. Its release is expected to increase lentil production and will curtail import bill of the country. Keywords: Seed irradiation, exotic germplasm, lentil, mutant, high yielding, disease resistant.

INTRODUCTION Lentil is an important traditional winter pulse crop in the world having about 25% protein. In cereal-based diet of common man in Pakistan, it plays an important role to meet the protein requirements. It is planted on an area of 43.4 thousand hectares with an annual production of 25.9 thousand tonnes having an average seed yield of 597 kg ha-1 which is significantly low as compared to the other lentil growing countries of the world having similar acreage (Anon, 2005a). The low seed yield may partially be attributed to meager genetic diversity in lentil germplasm for desirable seed yield traits (Malik and Malik, 1988). Punjab contributes 67% in respect of area as well as production of the country. Its cultivation is mainly concentrated in the districts of Narowal, Sialkot, Jhelum, Rawalpindi, Chakwal and Gujrat (Anon, 2005b). Lentil acreage has reduced significantly to 28.0 thousand hectares during the previous one decade, which was 43.9 thousand hectares during 1995-96. In this period, the area and production decreased by 36 and 24% respectively which may partly be attributed to the non-suitability of existing lentil germplasm to this particular ecological

niche and the cultivation of one variety in this growing environment will prove risky (Sadiq et al., 2004). Nuclear Institute for Agriculture and Biology, Faisalabad has been engaged in lentil improvement through conventional breeding and induced mutations techniques. Intraspecific hybridization has resulted in the develop-ment of MASOOR 2002, a short duration variety suitable for growing in cotton based cropping system and for late planting (Sadiq et al., 2003). Whereas, a series of improved mutant lines of lentil with high seed yield potential and resistance against biotic and abiotic stresses developed through induced mutations are in hand. Out of these, an elite line i.e. NL 20-9-4 has shown superb yield performance in different yield trials. MATERIALS AND METHODS High Yielding Elite Line Nursery from ICARDA, Syria was planted at NIAB, Faisalabad during 1985-86 to evaluate seed yield potential. The accession ILL 2580 produced high seed yield (in the absence of diseases in the field), and has spreading growth habit with tendrils, and reddish seed coat color. *Corresponding author email: sajjadhaidar_pk@yahoo.com

Canadian Journal of Pure and Applied Sciences 412

To induce desirable genetic variability for seed yield, yield related traits and disease resistance, the seed of ILL 2580 was treated with 200 Gy gamma rays from 60Co source (Sharma and Kant, 1975; Tripathy and Dubey, 1992). M1 generation was raised during 1986-87 and at maturity; all the surviving plants were individually harvested/threshed in each treatment. To raise M2 generation, individual plants were grown in single row of 5m length with row-to-row distance of 0.25m during 1987-88. Based on desirable morph-physiological traits, (Solanki and Sharma, 1999) selections were made from M2 mutated population. Single plant progenies were raised from the selected M2 plants as M3 during 1988-89 for their evaluation/confirmation of their true to type behavior and resistance against diseases. Susceptible checks i.e. ILL 4401 (for rust), ILL 6301 (for wilt), JL 171 (for Ascochyta blight), and AARIL-505 (for botrytis) were grown after every 10th rows to create natural epiphytotic environment in all the segregating generations for field screening (Nene et al., 1975; Agrawal et al., 1976; Pandey et al., 1983; Mishra et al., 1985; Ahmad and Morrall, 1996). These selections remained under evaluation for desirable agronomic traits during 1988-92 in the succeeding generations.

True breeding lines/mutants of lentil were screened under tunnel condition at Pulses Research Institute AARI, Faisalabad during fours years (2000-01, 2001-02, 2004-05 and 2005-06). Each test entry was planted in one m. long and 0.3m apart rows. Highly susceptible check 94548 was planted as spreader after every two test entries. Diseases were produced artificially in the tunnel by spraying spore suspension (30000 spores/ml) of blight and rust at three days interval till the initiation of diseases. The temperature in the tunnel was maintained at 20 + 2 Co and humidity at more than 80%, being congenial for disease development. Disease severity was recorded on scale; 1 with no visible lesion and 9 with complete death of the plant. The entries rated 1-3, were considered resistant, 5 moderate and those rated 7-9 were considered susceptible. Accordingly susceptible variety had more than 10% bud infection, more than 40% foliar infection on all branches with lesions, more than 40% of the branches girdled and leaf lesions with large number of pycnidia (Mishra et al., 1985). Selection for desirable mutants continued in M 4 and M 5 generations of ILL 2580 during the years 1989-1990 and 1990-1991. The progenies of M5 selected plant were grown during 1991-1992 and true to type progenies were

Table 1. Seed yield performance of NL 20-9-4 in different Yield Trials.

Mean Seed yield (Kg ha-1)

% Increase/decrease over Trial Year

*NL 20-9-4 ILL 2580 (Parent)

Masoor 85

Masoor 93

ILL 2580

Masoor 85

Masoor 93

Rank

Yield Screening Nursery (15 entries) 1992-93 1643A 1295 E 1177 F - 27 40 - 1st

Intermediate Yield Trial (15 entries) 1993-94 2009A 1653DE 1259 I - 21 60 - 1st

Advanced Yield Trial (12 entries) 1994-95 1647A 997 D 867 H - 65 90 - 1st

Adaptations yield trials**

(8 entries) ARS, Sheikhupura Adap. Farm.Gujranwala ARS, Pasroor ARS, Sahowali NIAB, Faisalabad Mean NIAB, Faisalabad A.R.S Narowal ARS, Siailkot PRSS, Sahowali Mean NIAB, Faisalabad A.R.S Sheikhupura PRSS, Sahowali ARS, Narowal Mean Over all Mean

1997-98

1998-99

1999-00

* * 1799a 1306a 1114a 1083e 1750a 1410

2118 C 1870 A 601 N.S 1744 A

1583

2069A

1639N.S

1802 A

1545 A

1764 1586

- - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - -

* * 959 g 1118 e 1125de 1153 a 1800 a 1231 2454 A 1348 C 597 N.S 1561C 1490 1740E 1621NS 1509 D 1070BC 1485 1402

- - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - -

87 17 -1 -6 -3 15

-15 38 1 12 6

19 1 20 44 19

13

1st

2 nd

1st

** Data of other entries is not shown *Approved by Punjab Seed Council as NIAB MASOOR 2006.

Sadiq et al. 413

bulked. To evaluate their yield potential, different sets of trials; yield screening nursery (1992-1993), intermediate yield trial (1993-94), advanced yield trial (1994 -1995), adaptation trials (1997-2000) and National Uniform Yield trials (2001-2003) were laid out by following appropriate experimental design along with recommended agronomic practices. Lentil National Uniform Yield Trials (LNUYT) were organized in Pakistan by National Coordinator (Pulses) NARC, Islamabad in which different research organizations working on lentil contributed elite lines for testing their seed yield potential. During 2001-2002, NIAB contributed NL 20-9-4, NL 20-27-2 NL 768-2-1, NL 20-39, NL 20-11-1 and TCM 85, Pulses Research Institute AARI, Faisalabad; 94505, 94517, 94550, 95511 and Masoor 93, NARC; Markaz 2000, Arid Zone Research Institute, Bhakhar; 93 L 026, 93 L 055, AZRC Quetta; ILL 8076 and ILL 8081, ARI D.I. Khan; DL-1 and DL-3 and NIA Tandojam; AEL 49/20. During 2002-2003, NIAB contributed NL 20-9-4, NL 20-27-2, NL 768-2-1 and NL 20-39, Pulses Research Institute AARI, Faisalabad; 94550, 95511, 95520, 98505 & Masoor 93, ARI D.I. Khan; DL-1, DL-3 and DL-5, NARC, Islamabad; NARC-02-1, NARC-02-2 and NARC-02-3, NIA Tandojam; AEL 23/49/91 & 49/20/91, AZRC, Quetta; ILL 8076 and ILL 8081, BARI, Chakwal; 98CL-008 and 98CL-007, AZRI, Bhakhar; 93L 055, and BARS, Kohat; KTM-1. Masoor 93 and Markaz 2000 were used

as standard varieties. To determine optimum sowing time to realize yield potential, sowing date trials were conducted during 2004 -05 and 2005-06 at NIAB, Faisalabad. Data on seed yield and yield related traits were recorded and statistically analysed (Steel and Torrie, 1984). RESULTS AND DISCUSSION A. Seed yield Seed yield performance of NL 20-9-4 in comparison with standard varieties/parent-ILL 2580 in different yield trials; yield screening nursery, intermediate and advanced is shown in Table 1. NL 20-9-4 produced significantly the highest seed yield of 1643 kg ha–1, 2009 kg ha –1 and 1647 kg ha –1 respectively as compared to the seed yield of parent ILL 2580 and standard variety Masoor 85. In adaptation yield trial, NL 20-9-4 produced significantly the highest yield of 1410 kg. ha–1 in 1997-98, 1583 kg ha–

1 in 1998-99 kg. ha–1 and 1764 kg ha–1 in 1999-2000. On overall mean basis NL 20-9-4 showed 13% increase in seed yield over Masoor 93 and secured first position in 1997-1998, & 1999-2000 and second position during the year 1998-1999. Non-significant differences for highest seed yield were observed between DL-3 and NL 20-9-4 to TCM 85 and

Table 2. Seed yield (Kg ha –1 ) of different genotypes in Lentil NUYT during 2001-2003.

Genotype Seed yield (2001-2002) Genotype Seed yield

(2002-2003) DL-3 1127 A NARC-02-2 2117 A NL 20-9-4* 1121 A 95511 1833 AB 93 L 055 1090 AB KTM 1 1764 BC NL 20-39 1065 AB NL 20-9-4* 1763 BC 95511 1063 AB 94550 1688 BC DL-1 1049 AB AEL 49/20/91 1679 BC Markaz 2000 1038 AB 98505 1664 BC NL 20-27-2 1034 AB NL 768-2-1 1659 BC NL 20-11-1 1031 AB DL-1 1653 BC 94550 1015 AB NARC-02-1 1633 BC 94517 1009 AB NL 20-27-2 1631 BC EL 49/20 1002 AB 93 L 055 1597 BC 94505 935 AB Masoor 93 1582 BC Masoor 93 925 AB NL 20-39 1578 BC 93 L 026 910 AB AEL 23/40/91 1576 BC TCM 85 848 AB DL-3 1554 BC NL 20-768-2-1 794 BC 98 CL-008 1546 BC ILL-8076 549 CD 95520 1516 BC

ILL-8081 529 D NARC-02-3 1474 BC 99 CL-007 1435 CD DL-5 1152 D ILL-8081 628 E ILL-8076 611 E

*Approved by Punjab Seed Council as NIAB MASOOR 2006.

Canadian Journal of Pure and Applied Sciences 414

the lowest between ILL 8076 and ILL 8081 in Lentil NUYT during 2001-02 (Table 2). NARC– 02-2 and 95511 showed non-significant differences for seed yield during 2002-2003. However 95511, KTM-1, NL 20-9-4, 94550 to NARC-02-3 showed non-significant differences in seed yield. The lowest seed yield was observed between ILL 8081 and ILL 8076. NL 20-9-4 exhibited 15% and 12% increase over Masoor 93 in seed yield on mean basis during the year 2001-2002 and 2002-2003 respectively (Table 3). Non-significant differences in seed yield were recorded between the sowing of November 10 and November 25 and December 10 & December 25 (Table 4). NL 20-9-4 produced significantly the highest seed yield of 1983 kg ha-1 when planted on November 10, 2004. Number of pods per plant and biomass yield contributed for this high seed yield. Non-significant differences in high seed yield were observed between November 12 and November 22

sowing and significantly the lowest yield was produced by December 12 planting (Table 5). NL 20-9-4 produced significantly the highest seed yield of 1685 kg ha-1 when planted on November 22, 2005. Number of pods and biomass yield were the yield contributing factors. These results indicated that November 10-25 is the optimum-sowing time for getting the highest seed yield. B. Disease resistance NL 20-9-4 exhibited desirable level of resistance to Ascochyta blight, whereas susceptible variety 94548 (spreader) has shown high degree of susceptibility during the four years. Other elite lines also showed similar disease resistance reaction (Table 6). As regard to rust and Botrytis, NL 20-9-4 showed resistance response (Table 7). Masoor 85 and Masoor 93 also showed resistance reaction to rust and BGM. MASOOR 2002 showed highly resistant reaction to these diseases (Table 7). Susceptible check 94548 showed highly susceptible reaction.

Table 3. Seed yield (Kg ha-1) of NL 20-9-4 in comparison with Masoor 93 in Lentil National Uniform Yield Trials during 2001-2002 and 2002-2003.

2001-2002 2002-2003 Location NL 20-9-4 Masoor 93 % Increase/

Decrease *NL 20-9-4 Masoor 93 % Increase/ Decrease

AZRI, Bhakkar 675 1025 - 34.0 542 867 - 37.0 NIA, Tandojam 727 477 + 52.0 2361 2375 = AZRI, Bahawalpur 65 81 - 20.0 2083 1563 + 33.0 AARU, Fausakabad 2708 2097 + 29.0 2825 2976 - 5.0 ARSM, Swat 309 198 + 56.0 1025 1296 - 21.0 BARI, Chajwak 535 694 - 23.0 1492 1484 = AZRI, Quetta 1943 1343 + 45.0 1984 1084 + 84.0 ARI D. I. Khan 1618 1262 + 28.0 2500 2222 + 13.0 NARC, Islamabad 1768 1171 + 51.0 2819 2075 + 36.0 NIAB, Faisalabad 1347 1478 - 9.0 1507 1364 + 10.0 RRI, Dhokri 632 345 + 83.0 BARS, Kohat 903 510 + 77.0 ARI, Sariab 1118 1173 = Sariab Quetta Average 1083 933 + 15.0 1764 1582 + 12.0

Table 4. Performance of NL 20-9-4 at different Sowing dates at NIAB, Faisalabad during 2004 -05.

Nov. 10, 2004 Nov. 25, 2004 Dec. 10, 2004 Dec. 25, 2004 Trait/

Variety/

Sowing date

Mas

2002

Mas 93 NL* 20-

9-4

Mas

2002

Mas 93 NL 20-

9-4

Mas

2002

Mas 93 NL

20-9-4

Mas

2002

Mas 93 NL

20-9-4

Days to flowering 66 C 109 A 105 B 60 C 101A 97 B 59 C 98 A 93 B 60 C 95 A 90 B

Days to maturity 110 C 149 A 140 B 115C 142A 136 B 90 C 136 A 131B 102 C 129 A 123 B

Plant height (cm) 43.9B 46.7B 57.7A 42.3B 46.1A 47.5A 40.1AB 42.6A 37.3B 37.3A 34.9AB 33.6B

P. Branches/plant 2.4B 2.9A 2.9A 2.1B 2.7A 2.5A 2.0B 2.7A 2.7A 2.1B 2.5A 2.3AB Pods/plant 234C 269B 344A 222B 247B 282A 156AB 146B 180A 150AB 132B 178A Pod length 1.13A 1.03C 1.10B 1.13A 1.05B 1.06C 1.11A 1.07B 1.07C 1.11A 1.03C 1.06B Seeds/pod 2.0A 1.70B 2.0A 2.0A 1.7B 1.9A 2.0A 1.7B 1.7B 2.0A 1.6B 1.7B 1000 seed weight 23.2A 22.2B 21.7B 22.4B 24.4A 21.7B 23.5B 24.0A 21.7B 23.7A 24.2A 21.8B Biom. Yield (kg ha-1) 4715C 6379B 7280A 4776C 5616B 6517A 3924C 4507B 4997A 2981B 2704B 4090A Harvest Index (%) 35A 27C 28B 31A 27A 29A 36A 16C 30B 47A 25C 35B Seed Yield (kg ha-1) 1636B 1352C 1983A 1407B 1498B 1885A 1407A 721B 1525A 1421A 679B 1421A Date of sowing 1657 A 1596 A 1218 B 1174 B

Sadiq et al. 415

Genetic variability is one of the important pre-requisites for advancement of crop breeding program. The magnitude of genetic variability existing in lentil germplasm and generated through mutation breeding had earlier been reported (Dixit and Dubey, 1986; Sarkar and Sharma 1988, Badiya et al., 1988; Ramgiry et al., 1989). In the present investigation, mutation has resulted in an

increase in variance in quantitative characters, which has been utilized by selection. The larger response to selection for seed coat colour, number of pods, lodging resistance, biomass yield and seed yield was achieved. Economically important mutations were induced earlier (Sharma and Sharma 1977, 1979). One of the major factor to low production in lentil is vulnerability to diseases i.e.

Table 5. Performance of NL 20-9-4 at different Sowing dates at NIAB, Faisalabad during 2005-06. Nov. 12, 2005 Nov. 22, 2005 Dec. 2, 2005 Dec. 12, 2005 Trait/

Variety/ Sowing date

Mas 2002

Mas 93 NL* 20-9-4

Mas 2002

Mas 93 NL 20-9-4

Mas 2002

Mas 93

NL 20-9-4

Mas 2002

Mas 93 NL 20-9-4

Days to flowering 60E 106A 105A 55F 98B 97B 55F 98B 97B 60E 94C 92D

Days to maturity 108H 145A 140BC 113G 139B 141C 86I 132D 132D 109H 127E 124F

Plant height (cm) 36.3CD 41.8A 40.4AB 38.3BC 42.7A 38.5BC 33.3EF 40.9A 34.6DE 32.0F 34.7DE 29.7G

P. Branches/plant 1.7E 2.3AB 2.0BCDE 1.3F 2.3ABC 2.5A 1.13F 2.07BCD 1.93CDE 1.2F 2.0BCDE 1.8D

Pods/plant 68F 159C 184B 63F 180B 227A 37G 103E 157C 36G 113E 137D

Pod length 1.04B 1.00D 1.01CD 1.07A 1.00D 1.00D 1.07A 1.01G 1.00D 1.03BC 1.00D 1.00D

Seeds/pod 2.0A 1.7B 2.0A 2.0A 1.4C 2.0A 2.00A 1.40C 1.93A 1.9AB 1.3C 1.9AB

1000 seed weight 25.9AB 24.3BC 23.9CD 26.8A 23.9CD 23.8CD 25.6AB 22.9CD 21.0EF 24.3BC 22.4DE 20.8E

Bio. Yield (kg ha-1) 1944FG 4021B 4437A 2427DE 4645A 3883B 2357DE 3953B 3189C 1656G 2691D 2247EF

Harvest Index (%) 50.6A 31.9D 39.3C 48.0AB 30.9D 41.0C 45.0BC 28.8D 40.6C 32.3D 22.1E 29.4D

Seed Yield

(kg ha-1)

985D 1283BC 1671A 1165CD 1359B 1685A 1060D 1283BC 1293BC 531E 614E 657E

Date of sowing 1313 A 1403 A 1212 B 601 C

Table 6. Screening of NIAB Lentil Elite Lines against Ascochyta lentis under artificial epiphytotic environment (Tunnel) at Pulses Research Institute, Faisalabad.

S. No. Genotype 2000-01 2001-02 2004 -05 2005-06

1 NL 20-9-4* 3 3 3 3

2 NL 20-11-1 5 3 - 3

3 NL 20-27-2 5 3 - 5

4 NL 20-39 3 3 - 3

5 NL 768-2-1 5 3 - 3

6 94548 (Spreader) 7 7 9 9

Table 7. Screening of NIAB Lentil Elite Lines against Uromyces fabae and Botrytis cinerea Under Tunnel Conditions during 2005-06 at Pulses Research Institute AARI, Faisalabad.

Sr. No. Genotype BGM Rust 1 NL 20-9-4* 3 1 2 NL 56-1 3 1 3 Masoor 85 3 3 4 Masoor 93 1 1 5 Masoor 2002 1 1 6 94548 (Spreader) 9 7

Disease Scoring

Disease reaction Disease Score Disease reaction Disease Score Highly resistant 1 Susceptible 6-7 Resistant 2-3 Highly susceptible 8-9

Canadian Journal of Pure and Applied Sciences 416

lentil blight, rust and botrytis. These biological stresses cause appreciable yield losses. Induced mutant variety NIAB MASOOR 2006 has shown field resistance under natural conditions, which was confirmed by artificially inducing epiphytotic environment (under tunnel). Similar results for induction of disease resistance were earlier reported (Haq et al., 2002). In sequel, mutation breeding has played a significant role in augmenting the existing genetic variability in lentil. A series of mutants with desirable traits has been developed. Out of this one mutant NL 20-9-4 with the name of NIAB MASOOR 2006 has been approved by Provincial Seed Council in its 32nd meeting held on 07-11-2006 at Lahore for general cultivation in the major lentil growing areas of the Punjab province. REFERENCES Agrawal, SC., Khare, MN. and Agrawal, PS. 1976. Field screening of lentil lines for resistance to rust. Indian journal of Phytopathology. (29): 208.

Ahmad, S. and Morrall, RA. 1996. Field reaction of lentil lines and cultivars to isolates of Ascochyta fabae f. sp. lentis. Canadian Journal of Plant Pathology. 18: 362-369.

Anonymous. 2005a. Agricultural Statistics of Pakistan. Government of Pakistan, Ministry of Food, Agriculture and Livestock (Economic Wing), Islamabad.

Anonymous. 2005b. Pakistan Statistical Year Book. Statistical Division, Federal Bureau of Statistics. Government of Pakistan.

Badiya, BN., Eunus, AN. and Sen, S. 1988. Estimation of variability and correlation in yield and yield contributing characters in lentil (Lens culinaris Medik). Environment and Ecology. 6 (3): 694-697.

Dixit, P. and Dubey, DK. 1986. Three interesting mutants in lentil. LENS Newsletter. 13(2): 5-7.

Haq, MA., Sadiq MS., Hassan, M., Shah, TM. and Ali, H. 2002. CM-2000: A new Kabuli chickpea variety. Pakistan Journal of seed technology. 1(1): 45-50.

Malik, B.A. and Malik, IA. 1988. Yield potential of lentil cultivars in Pakistan. LENS Newsletter. 10 (1): 989-990.

Mishra, RP., Kotasthane, SR. and Khare, MN. 1985. Reaction of lentil varieties and exotic germplasm to rust (Uromyces fabae). LENS Newsletter. 12:25-26.

Nene, YL., Kannaiyan, J. and Saxena, GC. 1975. Note on performance of lentil varieties and germplasm culture

against Uromyces fabae (Pres). De Bary. Indian Journal of Agricultural Sciences. 45:177-178.

Pandey, GP., Sharkar, S. and Gupta, PC. 1983. Occurrence of pulse beetle in the field in Gaziabad district (U.P). Bulletin of Grain Technology. 21:160-162.

Ramgiry, SR., Paliwal, KK. and Tomar, SK. 1989. Variability and correlations of grain yield and other quantitative characters in lentil. LENS Newsletter. 16(1): 129-210.

Sadiq, MS., Saleem, M., Haidar, S. and Abbas, G. 2004. Genetic diversity in different exotic and indigenous genotypes of mungbean (Vigna radiata (L.) Wilczek. Journal of Agriculture Research. 42 (3-4): 235-243.

Sadiq, MS., Sarwar, G, Saleem, M. and Abbas, G. 2003. NIAB Masoor 2002: An early maturing, high yielding and disease resistant variety of Lentil. Journal of Agriculture Research. 40(3-4): 187-192.

Sarkar, A. and Sharma, B. 1988. Efficacy of early generation selections for induced polygenic mutations in lentil. Indian Journal of Genetics and Plant Breeding. 48(2): 14 -15.

Sharma, B. and Kant, K. 1975. Mutation studies in lentil (Lens culinaris Medik). LENS. 2: 17-20.

Sharma, SK. and Sharma, B. 1977. Description of various morphological mutations in lentil. LENS Newsletter. 2: 2-14.

Sharma, SK. and Sharma, B. 1979. Pattern of induced mutability in different genotypes of lentil (Lens culinaris Medik). Zeitschrift fuer Pflanzenzuchtung. 83: 315-320.

Solanki, SI. and Sharma, B. 1999. Differentiate behavior of polygenic character to mutagenic treatments and selection in macroscopic lentil (Lens culinaris Medik). Indian Journal of Genetics and Plant Breeding. 61(3): 238-241.

Steel, RGD. and Torrie, JH. 1984. Principles and Procedures of Statistics. A Biometrical Approach, McGraw Hill Book Company, New York, USA.

Tripathy, A. and Dubey, DK. 1992. Frequency and spectrum of mutation induced by separate and simultaneous application of gamma rays and EMS in two microsperma varieties of lentil. LENS Newsletter. 19(1): 3-8.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 417-421, 2008 ISSN: 1715-9997

1

USE OF ELECTRICAL RESISTIVITY IMAGING TO INVESTIGATE DEPTH AND CONCENTRATION OF SUBSURFACE ICE IN A SUSPECTED

‘ICE CORED’ MORAINE

Greg Gaston Geography Department, University of North Alabama, USA

ABSTRACT

“Ice Cored” moraines are common features near many alpine glaciers. The presence of subsurface ice in both recessional and lateral moraines is believed to be indicated by slopes steeper than would be expected from the normal angle of repose of unconsolidated glacial debris. The ‘ice core’ probably takes the form of discontinuous ice lenses that serve as structure holding the otherwise unconsolidated material in an oversteepened state. The work reported herein tested electrical resistance image tools to confirm the presence of ice in the lateral moraine of the Elliot glacier, Mt. Hood Oregon. The presence of what are believed to be discontinuous ice lenses in the lateral moraine is supported by the electrical resistance images. In addition, this tool was found to be potentially very useful in assessing the depth of debris cover for many ‘rock glaciers’. Keywords: Electrical resistance imaging, ice cored moraines, rock glaciers, debris covered glaciers.

INTRODUCTION Alpine glaciers are sensitive indicators of climatic conditions. From the time of the most recent glacial maximum (~ 11,000 BP), through the glacial retreat and ice free conditions of the 13th and 14th centuries, through the Little Ice Age of the 1600’s when glaciers globally show evidence of advance, to the current evidence of warming and melting, alpine glaciers record very clearly changes in climatic conditions. Globally, it appears that glacial retreat occurring as part of the overall warming trend that started after the retreat of the most recent continental glaciation (~11,000 BP) ceased during the Little Ice Age (mid 1600’s). Glacial retreat (melting) began again during recent time, as the globe warmed (Lonne and Laesa, 2005). Abundant evidence for fluctuations in alpine glaciers is readily visible in the mountainous areas of the globe. In the United States, the stratovolcanoes of the High Cascades of the Pacific Northwest have a long record of advances and retreats and the formation/destruction of supraglacial features. Glaciers in the Cascades of Oregon have been shown to be in continuous retreat since at least 1900 with the greatest retreat occurring in the first decades of the 20th century (1900-1924) (McDonald, 1994). These observations fit with many other published reports suggesting that the global climate has re-entered a warming period. However, in many places in the Cascades glacial ice is still present and active glacial processes can be directly observed at relatively low elevations (6000 to 10,000 feet).

During retreat of an alpine glacier, ice continues to flow down slope, but the rate of ablation (loss due to melting and sublimation) exceeds advance and sediments cover the ice front forming what is called a debris-covered glacier. Glaciers of this type form a significant population of alpine glaciers worldwide. Ice left in the core of moraines usually forms steep sided features with the ice core covered by 1-3 meters of glacial debris (Kruger and Kjaer, 2000). There is well-accepted support for an assumption of an ‘ice core’ when the slopes of a moraine are extremely steep and the relief is higher than would be expected under the simple limits of gravity and the angle of repose of glacial debris [Photo 1 shows the steep slopes of the lateral moraines of the Elliot glacier].

Photo 1 (1990): Steep slopes and high local relief of this moraine provides strong evidence of an ice core.

*Corresponding author email: gggaston@una.edu

Canadian Journal of Pure and Applied Sciences 418

Rapid melting of ice-cored moraines forms a significant potential natural hazard. Ice cored moraines often act as dams, holding back melt-water lakes at high altitudes. Failure of these dams has the potential to cause great damage to communities and environments on the lower slopes (Richardson and Reynolds, 2000). In recently deglaciated areas in the Cascade Range, at least 30 lakes are dammed by unconsolidated moraines that are susceptible to breaching (O’Connor et al., 2001) [Photo 2 is a vertical air photo of a meltwater lake (Carver Lake on South Sister, Oregon) with what appear to be ice cored moraines acting as the dam containing the lake].

Photo 2. 2006: Vertical Air Photo of Carver Lake, South Sister. This lake is formed by steep moraines that are believed to be ice cored. Anecdotal evidence suggests that not only is glacial ice retreating, but supraglacial features such as ice-cored moraines are rapidly retreating (melting) (Rosenfeld, 2005 Personal Communication) [Photos 3 and 4 clearly show the retreat of the debris covered Elliot glacier between 1990 and 2006].

Photo 3 (1990): Lateral Moraine (ice cored) and debris covered Elliot glacier, Mt. Hood again, the steep slope of the lateral moraine suggests an ice core and the hummocky irregular surface indicates debris covered glacial ice.

Photo 4. 2006: The retreat of Elliott glacier is obvious when compared with the 1990 photograph (Photo 3). Saturated sediments (‘weeping’) on both sides of the main valley and at the base of the lateral moraines indicate melting ice. Electrical resistance imaging tools are fairly common in geophysical investigations. Electrical current is injected into the ground from a line of electrodes. Resistance to the flow of electrical current is measured at other electrodes along the array line. Image processing software integrates the measured resistance and models a cross section of electrical resistance in the sub-surface. While an extremely useful tool, the results are very open to interpretation. In spite of any limitations, used alone or integrated into an array of other geophysical archeological tools, electrical resistivity imaging equipment has been widely used at archeological sites in Great Britain, Europe, the Middle East and the United States. Electrical resistance imaging allows location of a variety of underground features including building sites, resource production sites, and graves. Because data is gathered quickly and non-invasively, electrical resistance imaging is ideal for preliminary evaluations of archeological impact at modern building and development sites and for location of unmarked graves at established cemeteries. See for example; Powell 2004, Iowa Office of the State Archaeologist 2003, and Johnson 2003. Confirmation of the surface expression of the ice cored features should be possible by examining an electrical resistivity cross section of the subsurface. Elvin et al. (1997), briefly reported success in identifying and mapping ice lenses in rock glaciers of the Yukon using electrical resistance tools. Water or saturated subsoil are good conductors of electrical current. Saturated glacial debris should show low resistance to electrical current. Dry, unconsolidated glacial materials should show moderate resistance to the

Gaston 419

passage of electrical current and ice, which is very poor conductor of electricity, should be highly resistant to electrical current flow. MATERIALS AND METHODS Glaciers of the Cascades of Oregon are well suited for this study. Found at relatively low elevations with comparatively easy access the Elliott glacier on Mt Hood was chosen for this study. A SuperSting R1/IP, a single channel automatic resistivity instrument developed by Advanced Geosciences of Austin TX was packed to the lateral moraines of the Elliott Glacier in July 2006. A

survey line was established down the slope of the lateral moraine just below the current debris covered terminus of the glacier. Fourteen electrodes were placed 3 meters apart down the slope. A second line was established across the surface of the debris-covered Elliott glacier. Melting ice indicated by the presence of melt water saturating the sediments was visible in the cut below the location of the electrical resistance line. Again, fourteen electrodes were placed at 3 meter spacing. Current injections and resistance measurements were performed at both sites using three different patterns (Schlumberger, Wenner and Dipole-Dipole).

Fig. 1. Electrical Resistance cross-section from site 2 which is the debris field covering known glacial ice. Theextremely high resistance can be anticipated, as glacial ice is an extremely poor conductor of electrical energy.

Fig. 2. Electrical resistance cross section from lateral moraine. Upslope is to the right of the figure. Moderateresistance near on the upper slopes indicates dry, unconsolidated glacial materials. Low resistance near the surfaceon the downslope end of the section (the left side of the figure) indicates water in the glacial material. The highresistance area that occurs at approximately 1.5 meters beneath the surface indicates discontinuous ice lenses.

Canadian Journal of Pure and Applied Sciences 420

Slopes at the moraine site and both up valley and down valley were measured using the clinometer of a Brunton pocket transit. An average value was calculated for the morainal slope. The accepted penetration and accurate assessment of sub surface conditions by these methods is 10-20% of the total length of the electrode array (Advanced Geosceinces 2006). Fourteen electrodes at 3 meter spacing should provide an accurate picture of conditions 4 to 8 meters beneath the surface. It was expected that this number and spacing of electrodes would be sufficient to locate and identify ice lenses in the moraines. RESULTS AND DISCUSSION Figure 1 shows the electrical resistance cross section produced by the array over known glacial ice. Clearly, at depths slightly more than one meter this environment shows an extremely high electrical resistance ~ 10,000 micro ohms, suggesting solid ice. There is little doubt that this environment produces results as expected. Figure 2 shows the electrical resistance cross section produced from the electrode array down the slope of the moraine. The electrode array was started near the top of the moraine (the left side of the cross section) and run directly down slope for approximately 50 meters. Moderately low resistance materials to the right side of the section suggest dry unconsolidated glacial materials at the drier top of the moraine. Near the lower end of the run, blue colors show low resistance indicating the presence of saturated sediments beneath the surface. Observations made when driving the electrodes support this interpretation, as it was possible to remove 8-10 inches of dry material and exposing visibly moist conditions. Higher resistance beneath this layer of moisture is logically interpreted as ice. The resistance values ~2000 micro ohms are significantly lower than found on the solid glacial ice, lending support to an interpretation of discontinuous ice lenses that begin about 1.5 meters below the surface. Slope measurements at the location of the electrode run and immediately up and down valley were very consistent (79% to 82%) with an average of 80% slope; this converts to a slope of 40-42 degrees. The expected slope of unconsolidated earth materials, the angle of repose, is expected to be approximately 35 degrees. The electrical resistance results lend support to a theory that includes ice structures within the moraine that provides structure to the otherwise loose moraine materials. CONCLUSIONS These results provide powerful support of the utility of electrical resistance imaging equipment as a tool to

evaluate the presence and characteristics of subsurface ice in many glacial environments. The glaciers of the Cascades produce very heterogeneous sediments, from silt to boulders with good contact between materials in the moraine. In a glacial environment of hard rock, the moraine materials can be large rocks with very little particle-to-particle contact and no opportunity to obtain good electrical contact. Glacial moraines in the Snowy Range of Wyoming between Laramie and Saratoga are perfect examples of an environment where electrical resistance imaging equipment would be of very limited utility. The dense quartzite of the mountains has shattered into very large boulders, which make up the moraines. In the Cascade glaciers with well mixed sediments and good electrical contact, electrical resistance imaging is projected to be superior to other subsurface imaging equipment such as ground penetrating RADAR (GPR). GPR functions best in a dry environment, the saturated conditions of the glacial moraine may make GPR problematic in this environment. The Electrical Resistance imaging equipment has no limitations from saturated materials and results actually improve with the presence of some moisture. While not conclusive, these results tend to support the long held tenet that steep slopes on a moraine indicate support from sub-surface ice. If current warming trends continue, not only will the surface area of alpine glaciers continue to decrease, but the ice supporting many moraines will also melt causing the moraine to collapse, reaching a new equilibrium. In the case of melt-water lakes that are contained by these ice reinforced moraines, there is an increasing likelihood that these dams will fail as the supporting ice melts. REFERENCES Advanced Geosceinces. 2006. Resistivity Imaging Seminar. Austin TX, April 19-21 2006.

Elvin, M., Denis, F. and Peter, GJ. 1997. Electrical Resistivity Measurements on the Rock Glaciers of Grizzley Creek, St. Elias Mountains, Yukon, Permafrost and Periglacial Processes. 8: 179-189.

Iowa office of the state Archeologist, University of Iowa. 2003. Locating Unmarked Cemetery Burials.

Kruger, J. and Kurt, K. 2000. De-icing progression of ice-cored moraines in a humid, subpolar climate. Kotlujokull, Iceland, Holocene. 10 (6): 737-747.

Lønne, I. and Astrid, L. 2005. Deglaciation dynamics following the Little Ice Age on Svalbard: Implications for shaping of landscapes at high latitudes. Geomorphology. 72 (1-4): 300-319.

McDonald, GD. 1994. Changes in mass of Collier Glacier, Oregon, 1910-1994.

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Unpublished MS Thesis, Department of Geosceinces, Oregon State University, USA.

O'Connor, J., Jasper, HHardison III. and John, EC. 2001. Debris Flows from Failures of Neoglacial-Age Moraine Dams in the Three Sisters and Mount Jefferson Wilderness Areas, Oregon: US. Geological Survey Professional Paper 1606.

Johnson, WJ. 2003. Geophysical Detection of Graves – Basic Background and Case Histories from Historic Cemeteries, Council for West Virginia Archeology Spring Workshop.

Powell, K. 2004, Detecting buried human remains using near-surface geophysical instruments Exploration Geophysics. 35: 88-92.

Richardson, S. and John, MR. 2000. Degradation of ice-cored moraine dams: implications for hazard development. Proceedings of a workshop, Debris-Covered Glaciers, held September 2000, Seattle Washington, USA.

Rosenfeld, Charles, Professor Emeritus, Oregon State University Department of Geosciences, 2005 Personal Communication regarding geomorphic changes in the High Cascades.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 423-430, 2008 ISSN: 1715-9997

1

A NEW GEMSTONE PROVINCE OF EASTERN GHATS MOBILE BELT, INDIA: RESULTS OF GEOLOGICAL AND MINERALOGICAL INVESTIGATIONS

Arjunudu, K., Srinivasa Rao, M and *Kasipathi, C

Mineral Exploration Division, Department of Geology Andhra University, Visakhapatnam-530 003, Andhra Pradesh, India

ABSTRACT

A new province of invaluable precious and semi-precious gemstone resources has been explored from parts of Eastern Ghats Mobile Belt (EGMB) in the states of Andhra Pradesh and Orissa, India for the first time. The chief precious minerals are alexandrite, chrysoberyl, chrysoberyl cat’s eye, aquamarine, ruby and the semi-precious stones are moonstone, zircon, sillimanite, garnet, tourmaline, and a variety of silica minerals viz. rock crystal, amethyst, rose quartz, smoky quartz, citrine and green quartz. These gem variety stones have their hostage in pegmatites, and potential when criss-crossed by the basic and ultrabasic rock types. The pegmatites are the immediate hostages and intruded through the gneissose bands of khondalite and leptynite; thus made the EGMB, India as a special precious province of gemstones. The exploration carried out in the pegmatitic bodies of the EGMB region, the potentiality of the gemstones in various pegmatitic colluvium bodies, their nature of characterization, their reserve positions, the activities of the government, the outcome of the mining activities, the illegal practices of mining, the beneficiaries of the precious stone scenario and the mineral dressing activities carried out by the mining organizations are briefly presented in this paper. Keywords: India, eastern ghats, precious stones, geology and reserves.

INTRODUCTION

Eastern Ghats Mobile Belt (EGMB) rock formations of India are well known for a variety of mineral deposits viz. apatite, bauxite, beach placers, feldspars, building stones, chromite, decorative stones, graphite, manganese ores, mica, quartz / quartzite, radioactive minerals, and sulphide minerals. The principal lithological members of the Eastern Ghats Mobile Belt comprise khondalite, charnockite, leptynite, granite, calc-granulite and quartzite. Pegmatite is one of the important rock types in the present context, which is a treasure-house of many rare, resistive and durable gemstones (Kasipathi, 1993). Pegmatite is an intrusive vein rock body that is intruded in the EGMB members, through khondalite and leptynite gneissose bands. The pegmatite when found criss-crossed even by thin veins of basic and ultrabasic rocks types, potential gemstone bodies were traced out. The details are presented in the following lines. DISCUSSION Geology of Eastern Ghats Mobile Belt Khondalite, charnockite, granite, quartzite, leptynite and calc-granulite are the important members of EGMB (Fig.1), in which a number of thin intrusive bodies of acidic to basic pegmatitic types are delineated. Pegmatite has formed to be an important association of the entire EGMB, which can be considered as a target for mineral exploration of gemstones. It is well known that micas, apatite, feldspars, tourmaline, magnetite and vermiculite

occur in abundance in the major part of the EGMB. These minerals are the products of liquid immiscible injections (Kasipathi, 1995). Weathered colluvial bodies after pegmatite, at the midst of the hill ranges in the valley portions show concentrated bodies of gemstones. Pegmatite shows variable lengths and widths, but continues for long distances. The thickness of the pegmatite veins is variable from about 3 cm to 120 cms in a zigzag pattern. It is very difficult to trace the extensions of the pegmatite vein bands in the field due to their variable thinning and bulging habit, but they follow the strike direction of the host rocks, i.e. khondalite and leptynite along their gneissose bands. Three parallel gemstone zones were recognized in Visakhpatnam and East Godavari Districts (Kasipathi, 1996a,b) (Fig. 2), viz. Zone-I - Shallow in-situ gemstone zone in the plains

and low-lying EGMB ridges parallel to the east coast with moderate concentrations of precious gemstones to a depth below 50 m. without any weathering zones and formation of colluvium.

Zone-II - Moderate hill ranges parallel to the Zone –I in the same strike direction of EGMB comprising moderate to high concentrations of precious gemstones with rich secondary colluvium bodies even to a depth beyond 100 m.; and,

Zone-III - Highly elevated hill ranges parallel to the strike direction of the other two zones, with rich concentrations of the precious stones and semi-precious stones in the hard unweathered in-situ EGMB rock-types and intensely in the *Corresponding author email: kasigeolau@rediff.com

Canadian Journal of Pure and Applied Sciences 424

deep colluvial bodies to an estimated depth beyond 300 m.

Precious and Semi-Precious Stones of the region The identified precious and semi-precious stone resources of the region are alexandrite, chrysoberyl, chrysoberyl cat’s eye, aquamarine, ruby (Plate-I), moonstone, garnet, tourmaline, sillimanite, zircon, epidote and a variety of silica minerals viz. rock crystal, citrine, rose-quartz, smoky quartz, and green quartz (Plate-II). Minor amounts of precious minerals viz. emerald and sapphire are also identified at places in tiny sizes. These gemstone minerals have shown close association of silicate minerals in the form of plates and books of phlogopite, vermiculite and chlorite, within the pegmatite bodies. The first four are the abundant precious minerals and the next two are rare precious minerals and the remaining minerals are semi-precious stones. The chemistry of the precious minerals is presented in Table 1 for a general understanding of the minerals (Lakshminarayana et al., 2006). The following are the characteristic features of these minerals (Kasipathi, 2007). Alexandrite is a variety of chrysoberyl with the chemical composition BeO.Al2O3.n.Cr2O3. It is light green to deep green colored, silky, chatoyant to opalescent, indistinct in cleavage, uneven to conchoidal in fracture, vitreous, about 8.5 hardness and about 3.6 to 3.8 specific gravity. The crystals are medium to coarse in size, dominantly less than one carat and often show pseudo-hexagonal forms. Crystals are tabular, vertically striated and twinned. Colour transformation to violet and red with artificial / incandescent light is it’s valuable character (Kasipathi, 1997). It is a valuable gemstone. It is distributed in parts of Visakhapatnam and East Godavari districts of Andhra Pradesh and in parts of Sambalpur district of Orissa.

Chrysoberyl and Chrysoberyl Cat’s Eye: Their chemical composition is BeO.Al2O3. Chrysoberyl is crystalline, mostly tabular, columnar, short prismatic with distinct prismatic striation. It varies in length from 5 mm to 30 mm and width from 2 mm to 35 mm. It is light yellow to yellowish-green coloured, green and deep yellow, transparent to translucent, hardness 8.2 to 8.5 and specific gravity is 3.7 to 3.8. It produces beautiful cut stones after processing. Chrysoberyl Cat’s Eye is a chatoyant chrysoberyl, massive, variable in dimension, translucent, yellowish, yellowish-green, lemon yellow and golden yellow in colour. Different fine layers of transparent and translucent chrysoberyl bands result a cat’s eye effect. Chrysoberyl cat’s eye is suitable for processing to yield cabachons. These stones are distributed in the north coastal districts of Andhra Pradesh and southern coastal districts of Orissa (Bhaskara Rao et al., 2002). Aquamarine: It is Be3(Al,Fe,Cr)2 Si6 O18. It belongs to hexagonal system and is a member of beryl group with sky blue colour, light blue-green or even light green. It is pleochroic. They are free from inclusions. Lustre is vitreous. It is found so far only in parts of Orissa (Badmal-Mursundi area of Subarnapur district) as per Das (1993), but not in Andhra Pradesh. Ruby: It is a gem variety of corundum with chemical composition Al2 O3 and belongs to hexagonal system. It occurs as barrel-shaped / pyramidal crystals / hexagonal bipyramids and also as massive and granular bodies. There is no cleavage. It’s luster is vitreous and fracture is uneven or conchoidal. It is reddish colored, sometimes violet-red and pleochroic. It shows fluorescence under ultraviolet light. They are mostly crystalline cylindrical prisms with a width of 3 mm to 12 mm and height of 25 mm to 40 mm. It has hardness of 9 and specific gravity of

Table 1. Chemistry of precious stones (Neutron Activation studies).

Wt.% 1 2 3 4 5 6 7 8 Be O 18.70 19.10 17.82 17.66 19.16 19.15 --- --- Al2O3 75.32 76.88 78.50 78.54 76.32 76.34 99.68 98.68 Fe2O3 0.28 0.34 2.45 1.76 2.41 --- 0.08 Tr. Cr2O3 0.38 0.35 0.08 0.06 Tr. --- 0.01 0.82 FeO 0.42 0.86 Tr. Tr. 0.42 3.60 Nil Tr. MnO --- --- --- --- --- --- 0.01 Tr. MgO 0.46 0.38 0.27 0.40 0.30 --- 0.01 0.06 CaO 1.24 1.05 0.22 0.31 0.22 --- Nil 0.02 TiO2 0.47 0.21 0.18 0.26 0.24 0.55 0.02 0.01 SiO2 2.65 0.92 0.31 0.81 0.52 --- 0.05 0.12

Tr. : Traces; --- = Not determined / Under detection limits; Nil = Absent. Sample details : (1) and (2) Alexandrite from Chintapaka, (3) Chrysoberyl from Chintapaka, (4) Chrysoberyl from Pappusettipalem, (5) Chrysoberyl Cat’s Eye from Pappusettipalem, (6) Dana (1992), (7) Grey corundum from Pallalabhavi area, and (8) Red transparent ruby from Mekalakunta area. [Samples numbers 1 to 5 are collections from Visakhapatnam district and 7 to 8 are from Khammam district, Andhra Pradesh].

Arjunudu and Kasipathi 425

4. It is available only in parts of Southern parts of Khammam district of Andhra Pradesh adjoining the supracrustal rocks and near Bhawanipatna (Orissa). Zircon: It is Zr.SiO4, colourless, pale-yellow, grey, brownish-yellow and reddish-brown, transparent to opaque, conchoidal in fracture, brittle and adamantine in luster. It’s hardness is 7.5 and specific gravity is variable between 4.35 and 4.82. It is found in the association of Chrysoberyl and Chrysoberyl Cat’s Eye in parts of EGMB. Garnets: Due to isomorphism, they show variable chemical composition as 3(Ca,Mg,Feii)3O. (Al,Feiii,Cr,Tiiii)2 Si3O12. These are light to deep pink, red, rose, yellow and brown coloured, crystalline, transparent to translucent, and relatively coarse grained among the gemstones family. They show mostly dodecahedral and

trapezohedral crystalline forms and massive also. They are sub-conchoidal to uneven in fracture. Hardness is 6.5 to 7.5 and specific gravity is 3.15 to 4.30. It is brittle and parting is distinct. It is vitreous and resinous. The transparent and coloured garnets are used as gemstones. Huge concentration of variety of garnets are found independently without any sympathetic and antipathetic relation with the other gemstone members; but associated with the rubies of Khammam district (Andhra Pradesh) and near Bhawanipatna (Orissa). Moonstone: It is Na Al Si3O8. It is a variety of orthoclase feldspar. It is vitreous and often pearly. It is colorless, white, pale yellow, red, grey and green. It is transparent to translucent and usually crystalline and shows opalescent play of colors, when polished. It is distributed rarely in the association of chrysoberyl and chrysoberyl cat’s eye, but not much with alexandrite.

Table 2. Reserve Estimations of gemstone deposits in different locations of Andhra Pradesh, India.

Reserve Estimations in tonnes District Name of the location

of the gem tract Chryso-beryl* Alexandrite Ruby Moon-

stone Garnet

1. Asakapalli 2.95 --- --- 8.69 4.53 2. Pappusettipalem 815 --- --- 27 46 3. Chintapaka 8.58 51.26 --- --- 72.10 4. Paderu 0.16 --- --- 0.23 --- 5. Pedabayalu 0.05 --- --- 1.18 ---

Visakhapatnam

6. Araku Valley 0.01 --- --- 0.01 --- 1. Mekalakunta --- --- 7.22 --- 10.18 2. Lakshmipuram --- --- 18.30 --- 53.34 3. Singaraipalem --- --- 3.10 --- 9.97 4. Pallipadu --- --- 7.69 --- 23.66

Khammam

5. Kodavatimetta --- --- 2.10 --- 2.88 (* Including Chrysoberyl Cat’s Eye Reserves)

Fig 1. Geological map of the Eastern Ghats mobile belt (after Mishra,1998).

Canadian Journal of Pure and Applied Sciences 426

Sillimanite: It is Al2 SiO5 . It shows a columnar structure and also a cat’s eye (Rao and Basu, 1994). Colour is deep brown, hair-brown, greyish-brown, pale-yellow, green and black. It is transparent to translucent and pleochroic. It is distributed rarely only in the association of chrysoberyl and chrysoberyl cat’s eye only in the EGMB. Tourmaline: It is (H9Al3(B.OH)2 Si4O)19. Crystals are usually prismatic, acicular and flattened. Prismatic faces strongly striated vertically and barrel in shape. Cleavage is indistinct and fracture is uneven. Hardness varies from 7.0 to 7.5 and specific gravity from 2.98 to 3.20. Lustre is vitreous. Color is variable viz. black, brownish-black, bluish-black, marine blue, green, red, colorless, deep blue and intermixed colors. It is mostly opaque (Schorl). The transparent varieties are the semi-precious stones. This is

a common associate in almost of all the pegmatite bodies of this region. It is commonly distributed with all the pegmatitic bodies of EGMB in association with the precious minerals, except the ruby-bearing pegmatitic bodies of Andhra Pradesh and Orissa. Varieties of Silica: It is SiO2. It is colorless (Rock crystal), yellow (citrine), rose (rose quartz), violet (amethyst), smoky (smoky quartz), green and red. Its hardness is 7 and specific gravity is 2.65. It is vitreous and transparent. These are used as common semi-precious stones and in sculpturing. These minerals are omni-present with chrysoberyl, chrysoberyl cat’s eye and alexandrite and distributed in the gemstone province of EGMB, both in Andhra Pradesh and Orissa.

Fig 2. Location of gemstone occurrences in parts of Visakhapatnam district, Andhra Pradesh, India. 1. Pappusettipalem, 2. Paderu, 3. Arakuy, 4. Turaiguda, 5. Pittagedda, 6. Matsyapuram, 7. Venkataramannapeta, 8. Eruwada hill,9. Lothugedda, 10. Goppulapa1em, 11. Khutikonda, 12. Pinapadu, 13. Gee1ugumetta, 14. Pedakonda, 15. Pedapittagedda, 16. Ravipalem, 17. Korramamidipalem, 18. Goppuru, 19. Siripuram, 20. Yeduvokala, 21. Chintapaka, 22. Pedda Madina, 23. ChinnaMadina, 24. K. Vallapuram, 25. Turakalapudi, 26. Asakagalli, 27. Rolugunta, 28. Gunnempudi, 29. Karaka, 30. Gurralagondi, 31. Salika Mallavaram.

Arjunudu and Kasipathi 427

The following significant mineral affinities and departures are noted in the gemtracts of EGMB (Mishra et al., 1993; Kasipathi et al., 1999): (a) Rock crystal and beryl are often found together, (b) Topaz and beryl seems antipathic with each other, (c) Tourmaline and aquamarine are generally sympa-

thetic, and (d) Corundum (Ruby) and muscovite usually occur in

each other’s company, but the pair show antipathic relation with biotites.

(e) Chrysoberyl / Chrysoberyl Cat’s Eye / Alexandrite / Aquamarine sympathetic with (i) zircon and (ii) hydroxyl micaceous minerals viz. biotite, lepidolite,

phlogopite and (iii) hydrous micaceous silicates of chlorite family, including vermiculite.

Gem mineral localization in EGMB The described gemstones are found present in the EGMB along the gneissose bands of khondalite and leptynite. The miraculous rock, ‘pegmatite’ is found to be the important rock type that holds the gemstones in close affinity with the micaceous minerals viz. phlogopite, biotite, vermiculite and muscovite in the lower order of abundance. If the pegmatite is found in close interference with the mafic and ultramafic rock types, the pegmatite shows potential pockets of the gemstones. Primary pegmatite shows variable dimensions, thinning and

Plate I

Canadian Journal of Pure and Applied Sciences 428

thickening and bulging structures in the unaltered rocks. The secondary plastic clayey mass, after pegmatite is described as colluvium (Kasipathi, 1997). The secondaries after pegmatite are the rich exploration targets for gemstones in EGMB. The deposits so far observed indicates mono-mineral potentials of any one of the precious minerals viz. Chrysoberyl / Chrysoberyl cat’s eye / alexandrite / aquamarine with the remaining semi-precious stones. It implies that the pegmatitic body favours only one precious mineral, as per the geochemical affinities and the intensity of pneumatolysis. The remote plain areas within the vicinity of pegmatite dominated EGMB hilly terrains were thoroughly checked for the potential gem pockets in the colluvium. Many secondary bodies were identified in Andhra Pradesh and Orissa in this perspective. Mining for gemstones in the hard pegmatitic terrains is difficult, as separation of the valuable gemstones from the adjoining hard mineral zone

is not really possible, unless we cut the stone. Even to cut the stone, the hilly rocks have to be blasted and the net result is the powdered gemstone material. So, mining in the colluvium is suggested, where every gem mineral can be separated, picked, and washed easily. The mineral ‘alexandrite’ must have formed at a very high temperature, when compared to the other precious minerals. The chemical composition of alexandrite is more or less the same as that of chrysoberyl, but constitutes a little amount of chromium impingment in its chemical structure, which results in green colored mineral. Gemstone Resource Estimations Gemstones are not new to Indians, where India was exclaimed as a ‘Ratnagarbha’ in the ancestral times and the present ‘Golconda’ (Hyderabad) was the centre for

Plate II

Arjunudu and Kasipathi 429

gemstone trade in this country. After a few centuries, the same tradition has been continued in the country, where the present annual turnover of the gem and jewellery trade of India crosses about US $ 20 Billion. Considering the geology, field relations, structural features and lithological controls, geoscientific exploration was carried out in some of the important gem-bearing locations of Andhra Pradesh. Geological mapping, physiographic contouring, structural para-meters, pitting, trenching, drilling and sampling were carried out, in addition to Electrical Resistivity surveys. The collected samples were categorized in the laboratory and characterized with their physical and optical parameters. The samples were also graded and the ratio of gemstone resource and the gangue was also determined. Reserves were estimated using cross-section methods. The gemstone concentration levels with different lithological units were also determined. The results are shown in Table. 2. Government’s Policies – Illegal Mining Activities It is very well understood that a few precious stone varieties and many a semi-precious stones are available with the EGMB and there piled a history of ‘geology and mining of gemstones’ of 15 years. The first known gemstone incidence to the government was at Sambalpur (Orissa) during 1992 and Araku (Andhra Pradesh) during 1992-93. Mining of corundum, ruby and garnet was already active by that time at Khammam. It became a major issue to the governments of Andhra Pradesh and Orissa to control the un-lawful mining activities of the rural and tribal sections all along the EGMB and the news reached the headlines of all the leading national and international newspapers and magazines. Many illiterate men and women have been died in these illegal mining activities (Kasipathi and Rao, 2006), even though the government missionary comprising the revenue, police, forest, mines & geology and mineral development corporations have been vigilant day and night to stop the illiterate labour mobs and the government could not really control these activities and it is humanly impossible for any agency to stop it, as these workers have been maintained by some of the top gem businessmen. Much of the vegetation of the Reserve Forests is vanished with the illegal prospecting operations of the illiterate rural and tribal folks and many of the labour were died in these activities. The green vegetation was completely scraped out by the labour for the sake of the precious gemstones. And the hilly regions lost their beautiful soil cover and made ugly, resulting rat holes everywhere in the EGMB. The iron and steel implements used in the illegal mining activities were made use of killing other workers of the same mining sphere. Many a common men became multi-billionaires and could earn a lot of properties. In nutshell, there is a lot of change in the life

style of many rural and tribal families and villages of EGMB, viz. Paderu, Araku, Dumbriguda, Narsipatnam, Eleswaram, Bhawanipatna, Elamanchili, Payakaraopeta, Addateegela, Karaka and Krishnadevipeta. After a clear observation of the situation, the Government of Andhra Pradesh has sanctioned prospecting licences in parts of Visakhapatnam district to seven parties through Andhra Pradesh Mineral Development Corporation by public auction. The government could collect about Rs. 3.5 Crores through the auction and the royalties of the mined-out gemstone material in a span of 6-months period of prospecting and mining. In other words, the government has given a chance to the prospective agents and agencies to purchase the adjoining and surrounding gemstone-bearing prospective lands of the declared gemstone prospects and, there by, there is no free land available with the government and the rural locals for furtherance of legalized gemstone mining activities in these areas. Now, the gemstone-bearing areas are being given mining leases for systematic mining, which is very much welcoming for the prosperity of the gemstone mining and the associated small-scale lapidary industries. The government may immediately take up detailed gemstone exploration programmes in the entire EGMB, so that the correct potential locations can be recognized and the mining can be initiated by the government or by the private agencies. If it is done, a good amount of employment can be generated and the minimum infrastructural facilities will be resulted in the tribal areas. Outcome of the Gemstone Province 1. Authorised systematic mining and ore dressing

activities will be opened. 2. The government can collect good amount of revenue. 3. Required infrastructural facilities can be provided by

the mining organizations. 4. Employment can be provided to the locals. 5. Major, minor and small-scale lapidary units will be

established in the nearby cities. 6. A new gemstone (raw and cut) marketing and trade

will be initiated in the nearby towns and cities. CONCLUSION To conclude the paper, the gemstone-bearing parts of Eastern Ghats Mobile Belt of Andhra Pradesh and Orissa, India forms a separate ‘gemstone province’ and their time and space relations have to be worked out in detail. The total gemstone reserves may be estimated, good amount of mining can be taken up, there by can provide a lot of employment, infra-structural development and mineral-based industrialization in the rural and tribal areas of this remote region.

Canadian Journal of Pure and Applied Sciences 430

REFERENCES Bhaskara Rao, A., Divakara Rao, V. and Kasipathi, C. 2002. Chrysoberyl (Alexandrite-Cat’s Eye) in the Eastern Ghats and it’s genetic and exploration aspects. J. Applied Geochemistry. 4(2): 355-358.

Das, SK. 1993. Gem-bearing pegmatites and the gem beryls of Badmal-Mursundi area of Subarnapur district, Orissa. Proc. Natl. Sem. Gemstones., Organised by the Society of Geoscientists and Allied Technologists, Bhubaneswar, India, Dec. 11-12: 91-92.

Kasipathi, C. 1993. Exploration for gemstones in parts of Eastern Ghats, North Coastal Andhra Pradesh. Proc. Natl. Sem. Gemstones., Organized by Soc. Geosci. All. Tech., Dec.11-12, 36.

Kasipathi, C. 1995. Exploration for gemstones in parts of tribal tracts, Paderu Division, Visakhapatnam, Andhra Pradesh. Invited Review paper submitted to the Chairman, A.P. State Committee on gemstones, Hyderabad, India. 48.

Kasipathi, C. 1996a. Chrysoberyl from Visakhapatnam and East Godavari Districts, Andhra Pradesh. J. Geol. Soc. India. 48 (10): 463-465.

Kasipathi, C. 1996b. A tribal welfare measure – Utility of gemstone resources from parts of Andhra Pradesh. Ind. J. Geology. 68 (3):178-184.

Kasipathi, C. 1997. Mineral Resources of Visakhapatnam District, Andhra Pradesh. Visakha Science Journal – A publ. of A.P. Akademi of Sciences. 1: 39-42.

Kasipathi, C. 2007. Minerallogeny of Precious and Semi-precious stones of north coastal Andhra Pradesh, India. MoST/DST Group Discussion on ‘Metallogeny, Crustal Evolution and Advanced Laboratory Techniques of Ore Genesis’, organized by the Dept. of Geology and Geophysics, IIT, Kharagpur during 07-09 March: 23.

Kasipathi, C. and Janardhana Rao, KV. 2006. Illegal mining activities for gemstones in parts of north coastal Andhra Pradesh. Ind. Min. & Engg. J., Bhubaneswar (In Press).

Kasipathi, C., Padmavathi, MVL., Srinivasa Rao, K., Arjunudu, K., Ravi, C., Krishna, C., Trinadha Rao, P. and Janardhana Rao, KV 1999. Precious and Semi-Precious stone occurrences with Eastern Ghat formations, Visakhapatnam District, Andhra Pradesh. J. Ind. Mineralogist. 33 (2): 23-33.

Lakshminarayana, S., Krishnarjuna Rao, I., Thirumala Rao, BV., Naidu, TY., Kasipathi, C, Jagannadha Rao, M., Newton Nathaniel, T., Acharya, R. and Reddy, AVR. 2006. Multi-Element Analysis of Sediments, Coal Samples and Gemstones by INAA. Proc. DAE-BRNS Discussion meet on current trends and future perspectives

of neutron activation analysis, BRNS/DAE, Mumbai, India.109-110.

Mishra, R.N. 1998.Metallogenetic frame of the Eastern Ghats Mobile Belt : Conceptual approach. Geol. Surv. Ind. Spl. Pub. No.44, pp.128-136.

Mishra, RN., Sarangi, BB. and Das, JN. 1993. Gem tracts of Orissa. Proc. Natl. Sem. Gemstones, Organised by the Soc. Geosci. All. Tech., Dec. 11-12: 37-45.

Rao, KN. and Basu, PK. 1994. Occurrences of unusual sillimanite cat’s eye from Visakhapatnam and Vizianagaram districts, Andhra Pradesh. Abs. Workshop on Eastern Ghats Mobile Belt., Visakhapatnam. 15-16 June : 93.

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363

AN INTEGRATED GEO- DATABASE FOR LAND MANAGEMENT AND GREENING ASSESSMENT OF AN ARID ISLAND

AT THE FRINGES OF ABU DHABI CITY

Salem Issa Department of Geology, College of Science, United Arab Emirates University

P O Box 17551, AL AIN, UAE

ABSTRACT

The objective of this study is to build a spatial GIS database for land management on the AL Sammalyah Island. Twenty GIS layers were created and inserted into a spatial database. GIS overlay analysis was applied to five major land cover types between 1999 and 2005 to estimate vigor of land cover change. The study drew the attention on the fact that real increase took place in most important land cover types namely mangrove, palm trees, and buildings registering more than 336%, 130%, and 300% increase respectively in six years. Hence, highlighting the success of planting salt tolerant vegetation to protect the environment and enhance the landscape in arid lands. Finally, building the GIS database was a real success; managers of the island started to use it as a source of information and generate statistics for land management, consequently reliability and flexibility of the remote sensing and GIS products were demonstrated. Keywords: Geo-database, sustaining management, Abu Dhabi city, arid island, GIS overlay analysis.

INTRODUCTION Arid lands are the results of various influences which prevent moisture-bearing weather systems reaching certain areas of the land surface; such influences are climatic, topographic and oceanographic (Beaumont, 1989; Cook et al., 1993; Thomas, 1989a; Thomas, 1989b; Thompson, 1975). Although deserts or drylands typically do not have a large number of inhabitants, they are often the loci of economic and cultural activity. For example, the oil-producing nations of the Middle East are all found within a single arid region. Furthermore, deserts tend to be fragile ecosystems, requiring little in the way of perturbations in order to cause tremendous changes in the landscape (Okin et al., 2001a; Schlesinger et al., 1990; Starbuck and Tamayo, 2006). The size, remoteness, and harsh nature of many of the world’s deserts make it difficult and expensive to map or monitor these landscapes or to aid planning for and management of, renewable natural resources. The situation exacerbates in developing countries where lack of accurate maps and the need for rapid and relatively accurate mapping techniques are urgent. This is becoming challenging if we know the dimension of large scale engineering projects being implemented, particularly in the wealthy Gulf States (Alhameli and Alshehhi, 2004; Essa et al., 2005; Sohl, 1999). Remote sensing and GIS emerge as promising new technologies potentially considered as a time and cost effective techniques to defy these challenges. Because of the nature of the information on land use and

cover change offered by these data, remote sensing has become central to many natural resource planning programmes that utilize geographical information systems (GIS) (Mulders and Girard, 1993; Ustin and Xiao, 2001). Moreover, with the use of ancillary field data and the calibration of remote sensing inputs, data integration within a GIS can enhance the extraction of information from satellite imagery and improve the accuracy of a variety of outputs (Jenssen et al., 1990; Salami et al., 1999). This has led to a synergistic approach in spatial data handling (Jenesen, 2000; Suga et al., 1994). Studying land resources management using spatial-related technology starts by assessing the amount of changes occurred during certain period (Deer, 1995; Mertens and Lambin, 2000; Mouat and Lancaster, 1996; Roy and Tomar, 2001; Singh, 1989). This assessment can be both qualitative (visualizing the extent) and/or quantitative (rate). Change detection is defined as the process of identifying differences in the state of an object or phenomenon by observing it at different times. The basic principle in using remotely sensed data for change detection is that: changes in the objects of interest will result in changes in reflectance values or local textures that are separable from changes caused by other factors such as differences in atmospheric conditions, illumination and viewing angles, and soil moistures (Larsson, 2002; Luque, 2000; Sohl, 1999; Zhang et al., 2002; Weng, 2002). Numerous works have been reported in these fields. For landuse change detection, imagery data from various sensors such as aerial photographs, Landsat MSS, TM, ETM, SPOT HRV, IRS and IKONOS *Corresponding author email: salem.essa@uaeu.ac.ae

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are often used, and it is common that images with various scales and from two or more sensors are used (Essa et al., 2005; Lu et al., 2004; Maldanado et al., 2002; Miller et al., 1998; Petit and Lambin, 2001; Petit et al., 2001; Prakash and Gupta, 1998; Salami, 1999; Yang and Lo, 2002; Zhou et al., 2004). Furthermore, all large operational GIS are built on the foundation of a geographic database. After people, the database is arguably the most important part of a GIS because of the costs of collection and maintenance, and because the database forms the basis of all queries, analysis, and decision making (Ayeni and Ikwuemesi, 2002; Chen, 2002; Periera et al., 2002; Suga et al., 1994; Travaglia et al., 2001). A database can be thought of as an integrated set of data on a particular subject. Geographic databases are simply databases containing geographic data for a particular area and subject. In recent years geographic databases have become increasingly large and complex. For example, AirPhoto USA's US National Image Mosaic is 25 terabytes (TB) in size, EarthSat's global Landsat mosaic at 15 m resolution is 6.5 TB, and Ordnance Survey of Great Britain has approximately 450 million vector features in its MasterMap database covering all of Britain (Longley et al., 2005). In the United Arab Emirates (UAE), where deserts compose more than 97 percent of its land, extensive development works have been and continue to be undertaken in the country. Thanks to investments generated from oil exports (Alhameli and Alshehhi, 2004). Transformation of islands into high value land indicates that islands in the UAE will become a focus for many investors. This development is manifested in the rapid change in landscape, settlements, and infrastructure (CER, 2000; DER, 2004), hence generating opportunities and challenges and there is a need to create spatial databases that can be used to address problems associated with the reality. During the last decade, many studies were conducted to highlight the rates and extent of change in the UAE, including change analyses studies using satellite and archived data but non of these studies were focusing on creating and managing spatial databases (Essa et al., 2005; Starbuck and Tamayo, 2006; Sohl, 1999; Yagoub, 2004). To assess development vigor, capture its footprint, and evaluate the wise exploitation of its natural resources, remote sensing and geographical information systems (GIS) are applied to a study area at the fringes of Abu Dhabi capital city with the main objective of building a spatial GIS database for optimal land resources management on AL Sammalyah Island.

MATERIALS AND METHODS Study area AL Sammalyah is located at approximately 24° 26′10″N - 24° 28′56″N and 54° 29′22″E - 54° 34′12″E (Fig.1). Situated in the Arabian Gulf, about 12 km north east of Abu Dhabi Island near (Um al-Nar Island) area and just opposite Shati’-Al Raha beach, is characterized by its rich ecosystems and marine life. The non-populated Island covers an area about 14.7 square kilometers. Its geomorphology is characterized by a flat desert surface with small artificial sandy hills and dispersed coastal Sabkhat especially in the low lands along the shoreline. The island is characterized by its hyper arid climate; the mean annual rainfall is just below 50mm, while the mean monthly temperature exceeds 30ºC. Soil texture is dominated by sand, with high salt content reaching 31.5% Total Soluble Salts (TSS) on the surface, being mostly Chloride soluble salts giving a white color to the soils of the island, which produces high brightness levels on satellite imagery operating in the visible portion of the EMR, however the very narrow mangrove soils surrounding the island have dark color because of high content of organic carbon content. The Island is a natural reserve containing high biodiversity, particularly mangroves (Avicenna marina) -local name: Al qarm, which grow throughout the coastal areas of Abu Dhabi Emirate and is associated with varieties of plant communities including: Al Qasaba plant (Arthrocnemum macrostachyum), Al shinnan plant (Seidlitzia rosmarinus), Al Suaed plant (Suaeda vermiculata), and Al Rasha plant (Cyperus conglomerates) (CER, 2000), (DER, 2004).

Fig. 1. Location of the study area. MATERIALS A multi-temporal dataset of remote sensing data was acquired for the study area. It was composed of aerial photographs from 1985, 1999, 2005, and 2006 (Table 1). Most aerial photographs were procured at the Military Survey Department (MSD), and Abu-Dhabi Municipality (AD). The software used includes ESRI ArcGIS v9.1 software for vector processing, and ERDAS Imagine 8.4 for image processing. The hardware used includes PC

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Pentium IV 3.2 GH speed, and HP color LaserJet printers. The selection of data used in this study was largely governed by availability and accessibility of archived data especially at the MSD archives; the selection of hardware, and software was governed by the UAEU geology department's facilities. In addition to the raster datasets, an elevation layer was available from the AD municipality. METHODOLOGY 1. Aerial photographs are scanned at a resolution of

1200dpi. The nearest neighbour method of grey-level interpolation is applied. An image-to-image registration is used to register other datasets using the corrected 2000-aerial photograph as a master image.

2. Five land cover types representing the years 1999 and 2005 are created namely: Roads; Water constructions; Vegetation; Buildings; and Barren land, using heads up on-screen digitizing. The years 1999 and 2005 were chosen because changes were visible on available aerial photographs only from the 1990s; no major activities were noticed from the mid-1980s aerial photographs. On the other hand, there was a delay in acquiring the 2006 aerial photograph which led to the adoption of the 2005 as a second date.

3. Evaluation and visualization of the change between past and present to assess the vigour of human footprint manifested by construction and rehabilitation activities on the island is achieved

Table 1. Imagery dataset produced for the GIS database.

Type Date Resolution Projection B/W Aerial photograph 1985 2meters UTM, zone40N Color Aerial photograph 1999 2 meters UTM, zone40N Color Aerial photograph 2005 2 meters UTM, zone40N Color Aerial photograph 2006 2 meters UTM, zone40N

)a(

)b(

)c(

)d(

Fig. 2. Snap shots for a specific area on the Island: (a) 1985; (b) 1999; (c) 2005; and (d) 2006. Simple visual inspection of the different dates shows evidences of human footprints and engineering developments occurring on the Island.

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using traditional change detection techniques and GIS overlay analysis.

4. TIN is generated using Geoprocessing tools available in ArcGIS 9.1. Data used to generate the TIN are spot heights with vertical accuracy of 2 meters.

5. Designing, building and populating the GIS database with respect to the following general steps:

i. Data sources selected for entities and attributes are user requirements identified through field visits, discussions and scientific reports.

ii. ArcGIS version 9.1 geodatabase structure is adopted for the design and building of the database

iii. Shapefiles created by digitizing using ArcGIS 9.1 under Edit session, and then converted to the database using geoprocessing tools available in ArcGIS.

iv. Metadata is created using ArcGIS metadata standards.

v. All data are projected to UTM, zone 40N, Nahrawan Datum.

vi. Integration of different data types including: aerial photographs, vector and ancillary data, and TIN.

RESULTS Imageries dataset A total of four large scale aerial photographs spanning the period from the mid-1980s to include the most recent aerial photos of 2005 and 2006 (Table 1, Fig. 2) were processed and integrated into the database. The dataset was registered to Nahrwan_1967 coordinate system, Universal Transverse Mercator projection, zone 40. A minimum of 10 ground control points (GCPs) were collected for each photograph and a second degree polynomial transformation was used to project them. The total RMS was less than 0.4 pixels. The resulting aerial photographs were resampled using ERDAS Imagine Map Interpreter Function and a unique pixel resolution of 2 meters was achieved for the whole dataset. The 1999 and 2005 aerial photographs were used to create GIS layers for main land cover types. The layers were then used to evaluate the change during this active period; furthermore they were used to populate the spatial database produced for the island (Fig. 2). GIS layers Ten GIS vector layers (feature classes in the database) representing five land cover types for each of 1999 and 2005 were created (Table 2). These feature classes were

chosen as they reflect most of the engineering and rehabilitation and greening works carried out on the island. Thus analyzing those parameters is a key factor to evaluate the level of development and to assess the vigour of human footprint on the study area during the research period. Field evidences confirmed the level of development and demonstrated the presence of human footprint on the study area (Essa et al., 2005). Table 2. Vector GIS layers considered for representing and studying land cover classes for 1999 and 2005.

Land cover classes (visual Interpretation of large scale aerial photographs) of 1999 and 2005

1. Transportation: Roads / Footpaths/ tracks and Roundabouts

2. Water constructions: bodies / Water channels

3. Vegetation: Shrubs & grass / Palm trees / Mangroves

4. Buildings

5. Barren land

A Triangulated Irregular Network (TIN) was generated from available spot heights with vertical accuracy of 2 meters (Fig. 3). The TIN was created to be used for 3D modeling and visualization as will as to estimate the volume of soils brought into the island during the study period.

Fig. 3. AL Sammalyah Triangulated Irregular Network (TIN). Geo-database As a result, the Al Sammalyah database integrates 20 GIS vector and raster layers encompassing various types of features on the surface of the Island (Table 3). The building of the actual GIS database is essential for sustainable development and management of the island in the long term.

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Table 3. AL Sammalyah GIS Database layers

No. GIS Layer The image dataset 1. B/W Aerial photograph, 1985 2. Color Aerial photograph, 1999 3. Color Aerial photograph, 2005 4. Color Aerial photograph, 2006 The 1999 change analysis input layers 5. Roads Network –first date 6. Water constructions –first date 7. Vegetation –first date 8. Buildings –first date 9. Barren Land –first date The 2005 change analysis input layers 10. Roads Network –second date 11. Water constructions –second date 12. Vegetation –second date 13. Buildings –second date 14. Barren Land –second date The Overlay output layers 15. Roads change analysis 16. Water change analysis 17. Vegetation analysis 18. Buildings change analysis 19. Barren land change analysis The TIN layer 20. Al Sammalyah TIN layer

DISCUSSION The main activity of the research was the assembling of a complete set of multi temporal remotely sensed dataset for the Al Sammalyah Island. GIS overlay analysis method for detecting and visualizing changes between the two dates was applied. Roads buffers; Water constructions; Vegetation; Buildings; and Barren land layers were mapped, also areas and lengths were measured hence producing a qualitative (Fig. 4) and a quantitative (Table 4) estimation of the change. Evaluation and visualization of the change Change maps shown in Figure 4 together with statistical analysis presented in Table 4 and Table 5 confirm the following:

• An increase in the buildings surface of more than 300% in six years, totaling an area of about 10 hectares in 2005, representing 0.7% of the total island area.

• Results of the change analysis indicate good progress in the level of greening of the island, especially in the increase of the salt-tolerant mangrove plantation during the study period: o Mangrove testifies the most significant land

cover type area increase. An increase of more than 336% in six years, totaling an area of about 165 hectares in 2005, representing 11.2% of the total island area. This indicates the amplitude of greening and reclamation efforts occurring on the island particularly when we know that this very adapted salt-tolerant plant is irrigated using sea water during high tide-water time.

o Palm trees show an increase of more than 130% in six years, totaling an area of about 21 hectares in 2005, representing 1.4% of the total island area.

o Grass is a new land cover type introduced at a later stage. An estimated area of about 2.4 hectares in 2005, representing 0.2% of the total island area. Indicating that the island has reached an advanced stage in its urbanization and development.

• In 1999 barren land alone occupied about 71% of the total area of the island. This percentage has only slightly changed in six years period approaching around 69% of the island area in 2005. This low decrease of barren land percentage shown on aerial photographs in the second period is attributed to the removal of natural vegetation cover during reclamation works.

A close examination of the above items points out many remarks, e.g., the low percentage decrease in barren land during the study period, despite good advancements in land reclamation and greening. This is attributed to the huge engineering works undertaken on the island in order to replace original high salt content soils by a new layer of soil brought from the main land. This has destroyed almost all existing natural vegetation cover mainly bushes, from 151.6 to 23.1 ha and mixed forest, from 153.9 to 144.4 ha in 1999; resulting in a more vegetation-void land appearing on the aerial photographs of the second date. However, if we look at the thematic distribution of land cover types on the island, significant achievements were acquired demonstrated in terms of land reclamation, urbanization and engineering works (Table 5). Engineering works and urbanization were demonstrated by the increase in built up areas and roads network infrastructure. Whereas, rehabilitation and human fingerprints were demonstrated by the increase in water

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constructions, manifested by either artificial water bodies for hosting migrant birds or by the construction of water channels for land reclamation and irrigation. This is confirmed by the high expansion rate of Mangrove vegetated areas which increased from near 2% in 1999 to reach more than 11% of the total area of the island in 2005. One last interesting observation is the chronological development of the island. The researcher found that

first, roads were constructed, then water channels, then vegetation, then water bodies, then buildings were constructed the last, reflecting ideal chronological sequence of human settlement in a relatively very short time (six years). This proves the rate and level of urbanization at which the island in particular and the UAE in general are being transformed from a once deserted area, into one of the most urbanized countries on Earth at an unprecedented scale in human history.

(a) Roads Network: Main roads / Footpaths/ Tracks and Roundabouts dynamics.

( b ) Water construction dynamics

(c) Vegetation: Shrubs & grass / Palm trees / Mangroves dynamics

(d) Buildings construction dynamics

(e) Barren land dynamics

Fig. 4. Overlay analysis mapping results of the change analysis between 1999 and 2005 for five major land cover classes: (a) Roads buffers dynamics; (b) Water construction dynamics; (c) Vegetation dynamics; (d) Buildings construction dynamics and; (e) Barren lands dynamics. The vigor of human footprints and development magnitude (rehabilitation and engineering works) on the ground was demonstrated.

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Table 5. Land cover types percentage distribution in 1999 and 2005.

Land cover types

% of Island area (1999)

% of Island area (2005)

Buildings 0.2 0.7 Mangrove 2.6 11.2 Palm trees 0.6 1.4 Grass 0 0.2

TIN building and 3D Visualization Derived contour lines layer and a Triangulated Irregular Network (TIN) were created from spot heights; the later is used in the database for visualization purposes. A 3D surface model of the island was produced permitting the creation of a fly-through traversing the island following a predefined path. Likewise, the idea of producing and comparing two TIN models for the island from 1999 and 2005 was innovative. This is true in the sense that it would have allowed for the estimation of the volume of soils brought into the island during the study period, therefore, demonstrating the capability of remote sensing and GIS to assist even in large scale engineering construction works. Unfortunately, the researcher was unable to achieve this challenging idea, as huge amounts of new soils continued to be added to the island during the study period hence hindering the possibility of producing the second 3D model!

GIS layers creation and Database building The building of the actual Al Sammalyah database is essential for sustainable development and management of the island in the long term. The GIS database integrates raster, vector and TIN data. Large scale aerial photographs were scanned, corrected, processed, interpreted and converted to ArcGIS geodatabase format. An integrated geodatabase of 20 GIS vector and raster layers encompassing various types of features on the surface of the Island (Table 3), and spanning four different dates is now in the hands of decision makers of the island for the best management of its land resources. The history of each unit of the total 1475 hectares of the island can be studied and analyzed back to the mid eighties. The database helps in planning the land of the Island for any future development. Information can be extracted about the dimensions or extent of any of the main land cover types e.g. extent of mangrove, or building of new site seeing constructions for tourists and visitors, engineering works, etc. Another important point to learn about the database of the island is its open structure nature, in the sense that it is possible to add new data or GIS layers for any feature class of the island at any time (e.g. soil layer, water table layer, salt distribution layer, land suitability layer, etc.). Land suitability maps are of particular importance, as they will help in assisting and directing managers in their greening and construction efforts for sustainable development of the island in the long run.

Table 4. Land cover change analysis statistics in the study area (1999 - 2005).

N o Class name 1999 2005 Change % Net Change

1. Roads Network - Main Roads (km) - Footpath/ tracks (km) Total Lengths (km) - Roundabout (number)

17.43 36.06 53.49 2

19.92 43.90 63.82 8

2.49 7.84 10.33 6

+14.27 +21.8 +19.31 +300

2. Water bodies (ha) Water Channels (km)

0.09 21.1

1.94 23.2

1.85 2.1

+2000 +10

3. Vegetation - Bushes (ha) - Mixed Forest (ha) - Mangrove (ha) - Palm trees (ha) - Grass (ha) Total Areas (ha)

151.6 153.9 37.7 9.1 - 352.3

23.1 144.4 164.5 21.3 2.4 355.7

128.5 9.5 126.8 12.2 2.4 3.4

-84.8 -6.2 +336.3 +134.1 new +1%

4. Buildings (ha) 2.31 9.66 7.35 +318

5. Barren land (ha) 1045 1025 20 -1.4

Note: * Decrease carries negative sign while increase carries positive sign. * Total island area = 14.75 km2 = 1475 ha

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The importance of building GIS spatial databases gains its importance in the region especially with growing attention given to islands that resulted in building artificial islands such as Palm and World islands built in Dubai. Definitely, the associated high cost of these artificial islands greatly justifies the implementation of GIS spatial databases to manage and sustain the development on these islands. Flexibility and reliability of Remote Sensing and GIS Demonstrating the flexibility and accuracy of remote sensing and GIS technologies in providing essential and updated information for resources mapping and management is one of the objectives of this study. This is of paramount importance undertaken to convince high ranking administrators of the importance of using RS/GIS technologies in management and decision making process. Since most developing countries’ governments have a preference towards rapid solutions with low maintenance cost in the long run. These governments are welling to provide a one-time don with the expectation to see quick and concrete results. Investing this one-time money don to build a digital, accurate and comprehensive geo-database to manage land resources is a well thought-out and a great achievement. This geo-database has the potentiality for archiving, retrieval, querying and processing, visualization and disseminating of results amongst decision makers and the public. Furthermore, building the GIS database was a real success. Managers of the island started to use it to do measurements and generate statistics about main land cover types like mangrove and palm trees plantation. Visualization is another product being used to print maps and generate reports for important meetings to justify funding and persuade superiors. The open structure nature of the GIS database makes it possible to expand and add more layers deemed necessary to the database in the future. The reliability and flexibility of the remote sensing and GIS products are demonstrated by assisting and directing managers in their efforts for the sustainable development of the island in the long run. Significance of the study This is probably the first time that an integrated spatial GIS database for land management of a specific protected island is built in the UAE. The uniqueness of such database resides in the following elements: i. First of its kind in the UAE. Other examples can

follow for other islands especially, those artificially constructed to manage tourism and sustainable development of these high costly islands.

ii. Inclusion of historical large scale aerial photographs spanning more than 20 years period.

iii. Inclusion of the change detection analysis results integrated with the rest of the basic GIS database layers.

iv. Provides evidence of environmental conservation and urban development being carried out on the island. Indicators include increase in vegetation cover extent, especially salt-tolerant plants, and increase in buildings, roads and water constructions.

v. Opportunities to undertake future GIS-based research to conduct environmental impact studies of oil or marine pollution on the ecosystems of the island.

vi. Creation of 3D simulation for the island to assess the important engineering works undertaken and visualize the artificial landscape created on the island.

The building and maintenance of a geodatabase for the AL Sammalyah Island is another step in the construction and publication of a Spatial Decision Support System (SDSS). Such a system is considered as a priority for the actual research project forthcoming period. Once built, published and maintained the SDSS will serve three types of customers: i) Decision makers who will use the system to make the decision making process quicker and more efficient; ii) Tourists and eco-tourists who will be able to search the database and get answers to their queries, and iii) Students and researchers who can retrieve data and extract information about the island. Correlation between the amount of money invested and the level of engineering works achieved is now possible and open for planners. Future work should invite developers and GIS researchers to use RS and GIS technologies to calculate and model the relation between the amount of “money spent” and volume of “engineering work” undertaken. CONCLUSION The present study is the first conducted primarily to, first; quantify rates of change and levels of development using GIS and remote sensing; second build a spatial geodatabase to integrate and store all spatial data available. The geodatabase shows the evolution of the island landscape in time, in addition to the actual status of the island as of 2005, 2006. The geodatabase provides opportunities for quick and timeless maintenance, and provide a basis for the construction and publication of the Island SDSS. On the island, large-scale reclamation started in the early 1990s and has increased very rapidly since then. Urbanization and the spread of water bodies was testimony to the development of the island for enhancing scientific research and developing the ecosystem. Further, results provided convincing evidences of modernism, but also conservation, greening, and desert watering which were successfully achieved. The successful engineering and reclamation works conducted proved that dedication and wise decisions can make differences in enhancing local environmental

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conditions. The production of an integrated geospatial database for land management was the first in the UAE. It is believed that other institutes will follow as past experiences proved that such study example was usually followed by other public institutes in the country as well as in other Gulf States. The spread of artificially built islands in the UAE as well as in the region such as Palm and World islands in Dubai justifies the implementation of such GIS spatial databases to help in the management of tourism and economic development on these islands. ACKNOWLEDGMENT The author would like to express his thanks to all those who participated in the accomplishment of this study inside or outside the UAEU. Many thanks are due to all colleagues from the Emirates Heritage Club (EHC) for allocating the necessary money to achieve this study. Thanks should be directed to Dr. Maithaa Al Shamsi, director of the UAEU research affaires sector for her assistance and continuous support. Efforts exercised by Dr. Mohammed Al Ghali, from the EHC, for acquiring the data are highly appreciated. REFERENCES Alhameli S. and Alshehhi, M. 2004. Images are an outstanding evidence of rapid development “a perfect example from United Arab Emirates (UAE)”. Proceedings of the ISPRS XXth Congress, Commission PS IC, Working Group II/4, XXXV, part B4, p. 505 ff. Istanbul, Turkey.

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STUDY OF INDIAN SUMMER MONSOON RAINFALL ON DECADAL SCALES VIS-À-VIS CIRCULATION PATTERNS

*K Muni Krishna and S Ramalingeswara Rao

Department of Meteorology and Oceanography, Andhra University Visakhapatnam-530003, India

ABSTRACT

The decadal variations of all India summer monsoon seasonal rainfall during 1871-2000 are studied. Two extreme decades, one positive (1951-60) and another negative (1981- 1990) in the recent decades are considered depending upon the values of all-India monsoon seasonal rainfall. For these two decades, the synoptic features are studied for the parameters like mean seasonal values of air temperatures, zonal wind and meridional wind at two levels, 850hPa and 150hPa and latent heat flux in domain of 30oE-120oE and 30oS-40oN. The frequency of Bay of Bengal cyclonic systems during June through September is also studied. During good (poor) monsoon decade there is an enhancement (a reduction) in the intensity of easterly jet and frequency of Bay of Bengal cyclonic systems during June through September. It is also observed that the strength of the monsoon trough is pronounced/reduced during active/poor monsoon situations. Bay of Bengal and Arabian Sea play an important role in transporting moisture towards Indian main land and deciding the behaviour of Indian summer monsoon. Keywords: Summer monsoon, rainfall, circulation, latent heat flux.

INTRODUCTION The Indian summer monsoon is very important in all its aspects namely onset, withdrawal and variability because it is a rain giving monsoon. Quite many studies have been carried out on all these aspects in the past, but far more is yet to be done because we lack in correct understanding of their details. The Indian summer monsoon is a part of global circulation, which gives abundant amount of rainfall, which is crucial not only for agriculture and drinking purposes, but also for generation of hydroelectric power. The rainfall is not homogenous over the entire country. The seasonal summer monsoon rainfall is maximum over the west coast (200-290 cm) and northeastern parts (153-222 cm) of India, moderate over the central parts (70-120 cm) of India and minimum over northwestern (25-45 cm) parts (Shukla, 1987). If one part receives good amount of rainfall, another may suffer from drought conditions. The rainfall is not uniform with respect to the time also. There are some wet spells and dry spells during the monsoon season. Proper management of the abundant amounts of the rainfall occurred during the active monsoon periods or wet spells is very much essential to overcome the problems arising during the dry spells. The spatial and temporal variability of the rainfall play an important role on the economy of India. A strong cross equatorial low level jet stream is one among the monsoon elements. It exits with its core close to 850hPa level over Indian Ocean and south Asia (Joseph

and Raman, 1966; Findlater, 1969) and brings the moisture generated by trade winds over the south Indian Ocean and the evaporative flux from Arabian Sea to the areas of rainfall production over south Asia. The cyclonic vorticity north of this jet in the atmospheric boundary layer is a dynamic forcing for the generation of vertical upward air motion and rainfall and for the genesis of depressions in north Bay of Bengal (Joseph and Simon, 2005). During active monsoon period the core of the Jet passes eastward through peninsular India in between 12.5oN and 17.5oN. But in the break monsoon this moves southeastward from the central Arabian Sea and by-passing India passes eastward between latitudes 2.5oN and 7.5oN (Joseph and Sijikumar, 2004). The presence of a strong jet over peninsular India favors the formation of monsoon depressions in the north Bay of Bengal (Sikka, 1977). Generally the monsoon depressions form over the north Bay of Bengal north of 180N during the period June through September and move in west-north-westerly direction along the Ganges valley up to the central parts of the country before weakening generally (Rao, 1976). Rainfall is quite high to the left of their tracks. If they take more northern track over northern India, the Ganges plains receive good rains with corresponding deficiency over central India and northern peninsula. When they follow more southern course over central India, the central parts of India and Gujarat get good rain with the corresponding deficiency over the north. The wind convergence is strong in the southwestern sector of depression area (Subbaramayya, 1961). The absence of these depressions in July and August causes the rainfall deficit upto 40% over different parts of India (Raghavan, 1967; Dhar and Rakhecha, 1976).

*Corresponding author email: kailasam15@yahoo.co.in

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Several authors suggested that baroclinic instability is responsible for the generation of the monsoon depressions (Shukla, 1977, 1978; Mishra and Salvekar, 1980; Moorti and Arakawa, 1985; Aravequia et al., 1995). They considered the baroclinic instability of the zonal wind with easterly shear associated with tropical easterly jet. They showed that higher (lower) wind shear leads to higher (lower) growth rates with or without the cumulus heating. Thus an increase or decrease of the easterly shear in the earlier / later period leads to monsoon depressions with higher/lower growth rates. Disturbances with weak growth rates may not sustain the frictional dissipation and may not develop. This may explain the decrease in the number of monsoon cyclonic systems when the shears are weak and increase when the shears are strong (Srinivas Rao et al., 2004). According to the results of Bhalme and Mooley (1980) India experienced frequent large-scale droughts in summer monsoon during 1891-1920 and 1961-1975 and only a few in the intervening period. They found the following conditions for large-scale droughts: (1) Weak meridional pressure gradients, (2) Larger northward seasonal shifts of the monsoon trough, (3) Larger number of days in the breaks of the monsoon, (4) Smaller frequency of depressions and shorter westward extent of depression tracks, and (5) Abnormally low 200-hpa surface in May in the latitude belt 150-300N along 700E. Opposite features were reported in large-scale floods. Awade et al. (1985) reported larger northward transport of momentum in the upper troposphere across 300N in good monsoon years than in poor monsoon years in both pre-monsoon and monsoon months. In good monsoon years there is large divergence in the momentum transport in sub-tropics, while there is large convergence in middle latitudes. In poor monsoon year, there is large divergence of sensible heat in sub-tropics and large convergence in the middle latitudes in mid troposphere. After approaching the Indian coastline as a southwesterly current the monsoon air is deflected to northeastern region of India and northern parts of Burma. Subsequently, it flows along the plains of northern India and southern periphery of the Himalayas as an easterly current. The flow of air is around a quasi-permanent zone of low pressure over the plains of northern India. This is referred to as the monsoon trough. Its axis is oriented in northwest-southeast direction and runs parallel to the southern edge of Himalayas representing a line of symmetry between westerly or southwesterly winds to the south and easterly or southeasterly winds to its north. It passes through Delhi, Allahabad and Kolkatta. Active phases of monsoon occur when its axis is to the south of its normal position and its eastern end dips into Bay of Bengal. In such a situation there is heavy rain over plains of northern India, the central parts of India, and along the west coast. An extension of the axis into the Bay of

Bengal is often the precursor of a Bay depression. Once a depression is formed, it is followed by an increase in rainfall intensity over those parts of central India that lie to south of the axis (Das, 1987). The monsoon current picks up copious moisture by evaporation from the Arabian Sea and the Indian Ocean. This moisture is essential not only for the monsoonal rainfall but also important as a driving force for monsoon as the latent heat release in the monsoon trough strengthens the monsoon circulation. More moisture transport leads to active monsoon conditions which are indicated by dense multilayered and convective clouds over central parts of the country, over the eastern Arabian Sea and Bay of Bengal and strong low level (westerly/southwesterly) flow over the Arabian Sea often with a low level jet of strength, 25-30 m/s below 850 mb level (Keshavamurthy and Sankara Rao, 1992). The summer monsoon climate exhibits variability in a variety of time scales, intraseasonal variability, interannual variability, intradecadal variability etc. Various components of the Asian summer monsoon also exhibit significant interdecadal variability (Joseph, 1976; Parthasarathy and Mooley, 1978; Bhalme and Mooley, 1980; Mooley and Parthasarathy, 1984; Kripalani et al., 1997; Mehta and Lau, 1997; Chang et al., 2001, 2000; Parthasarathy et al., 1991; Wu and Wang, 2002). Folland et al. (1986) have found low frequency variability of global sea surface temperature (SST) on this time scale. The gradient in SST between northern and southern hemispheres also seems to vary on this time scale. The Indian monsoon is known to have gone through alternating epochs of above normal and below normal conditions, each lasting about three decades (Krishna Murthy and Goswami, 2000). The interdecadal variability is evident in various monsoon parameters such as all India monsoon rainfall (Partharasathy et al., 1994; Kripalani and Kulkarni, 1997; Kripalani et al., 1997; Webster et al., 1998), the frequency of cyclones in Indian monsoon region (Joseph, 1976), homogeneous monsoon rainfall covering the northwestern and central parts of India (Partharasathy et al., 1993) and circulation features such as the April position of the 500 hPa ridge (Kripalani et al., 1997). The behaviour of the interdecadal epochs of the monsoon may have great socioeconomic impact in South Asia region. A strong monsoon is associated with anomalous ascent around 25°N and weak monsoon is associated with anomalous ascent near the equator (Goswami et al., 1999). Monsoon rainfall fluctuates around mean causing runs of wet and dry years. The drought and wet decades occur in runs rather than scatter randomly. In the same manner the

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decadal rainfall amounts also fluctuate around mean causing runs of wet monsoon decades and dry monsoon decades. These fluctuations were persistent. In view of the importance of rainfall the authors examined the decadal variability of all-India summer monsoon rainfall in terms of circulation patterns over India and neighbourhood and cyclonic activity over the Bay of Bengal, which are connected well with the rainfall activity. MATERIALS AND METHODS Data and Methodology The all-Indian summer monsoon rainfall (June-September) is the area-weighted average of the rainfall of 29 meteorological sub-divisions (Parthasarathy et al., 1994). They have not included the hilly regions due to sparse rain gauge network. Later this data was updated by Indian Institute of Tropical Meteorology and kept in their web site (www.tropmet.res.in). This data set during 1871 to 2000 is considered. Another data set used in this paper, including air temperatures, zonal wind and meridional wind at two standard levels, 850 hPa and 150 hPa and latent heat flux covering a 50-year (1951-2000) period, for the domain 30oE-120oE and 30oS-40oN are taken from the National Centers for Environment Prediction-National Center for Atmospheric Research (NCEP/NCAR) re-analysis datasets (Kalnay et al., 1996). The frequencies of the cyclonic systems generated over the Bay of Bengal during June through September for the period 1881-2000 are obtained from the published report of the India Meteorological Department (1979) and from the weather reports published in Mausam journals, India Meteorological Department. The average seasonal monsoon rainfall amounts in different decades starting from 1871 to 2000 are

evaluated. From these values the extreme events are obtained. If any value exceeds or equal to mean+one standard deviation, the corresponding decade is considered as extreme positive decade. If any value precedes or equal to mean-one standard deviation, the corresponding decade is considered as extreme negative decade. One positive and another negative extreme decades are considered. For these two extreme decades, the mean monsoonal thermal patterns, circulation patterns, zonal and meridional wind anomalies in the lower (850 hPa) and upper (150 hPa) troposphere are studied. The frequency of cyclonic systems generated in the Bay of Bengal is also studied. RESULTS AND DISCUSSION Figure 1 clearly indicates that the decadal variation of summer monsoon rainfall during the period 1871-2000. Rainfall fluctuates around the mean value of 85.1cm. However, decadal mean values are showing changing dry/wet monsoons. In the beginning the values showed a positive trend from 1871-1880 to 1881-1890. After that there was a sharp decrease up to 1900-1910. Then the rainfall increased gradually with time and reached peak value in the decade 1940-1950. After that there was a decreasing tendency up to 1980-1990. Monsoon was very active during 1871-1900 and 1930-1960 and poor during 1900-1920 and 1980-1990. During the period of study 1871-2000, India experienced very heavy rainfall amounts during 1880-1890 and 1940-1950 and very less amounts during 1900-1920. From this it was observed that wet/dry monsoon conditions were prevailed for three consecutive decades. In the recent decades 1980-1990 experienced less amount of rainfall (about mean-1 standard deviation). NCEP/NCAR data is available only from 1950 onwards and so the authors wish to study the thermal as well as circulation features which are responsible for the good monsoon activity during 1951-60 and for the poor monsoon activity (1981-1990) (Fig.1).

Fig. 1. Decadal variation of All-India Monsoon Rainfall during 1871-2000. Brown line indicates mean+SD, blue lineindicates mean-SD green, orange bars indicate good and poor monsoon decades respectively.

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Thermal patterns 850hPa: The thermal features in good and poor monsoon decades resemble some important differences. Over the Bay of Bengal, Arabian Sea and India the north-south temperature gradient is more during good monsoon decade (1951-1960) when compared to the poor monsoon decade (Fig. 2a and b). 150hPa: Thermal distribution at 150 -hPa over India and neighboring areas for 1951-1960 and 1981-90 are depicted in figures 2c and d. There are few differences between these two patterns over southern India; the upper troposphere is relatively cool during good monsoon (1951-1960) when compared to poor monsoon (1981-1990) decade. Temperatures over southern India during good monsoon decade are relatively less by 1° to 1.5°C. The feature is clearer over tropical Indian Ocean with a temperature difference of 2°C-2.5°C. Circulation patterns 850hPa: The authors wish to note differences between the circulation features associated with active and poor monsoon decades. In active monsoon decades the cross equatorial flow and the low level jet across the western

coast of India exhibited maximum intensity (Fig. 3a and b). In good monsoon decade the cross-equatorial flow is strongly spreading over a wide area. The intensities are about 10 m/s. The low level jet is also strengthened. The speed is about 16 m/s., which is present in between 50°E to 65°E and 5-12°N over the Arabian Sea, the speed gradually decreases towards north. In the Bay of Bengal, the wind speeds are in between 6-10 m/s. While in the case of poor monsoon decade the cross equatorial flow is weaker, wind speeds are less when compared to the good. The strength of the low level jet is also decreased. 16 m/s contour is concentrated over a small area only when compared to the good monsoon. Over the Bay of Bengal there is no appreciable change in the wind speeds. Over land area there is a difference in the speeds. During good monsoon decade, the wind speeds are ranging from 11 m/s in the south to 2 m/s in the north. During poor monsoon, winds with relatively low intensity exist over southern peninsula (Fig. 3a and b). 150hPa: The circulation patterns in the upper troposphere (150-hPa) infer the differences between the poor monsoon

Fig. 2. Monsoon seasonal (JJAS) thermal patterns of the atmosphere at 850 hPa and 150 hPa for good (a, c) /poor (b, d) monsoon decades (Temperatures in oC).

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and good monsoon decades. Generally the easterlies are dominant in the upper troposphere. The maximum in the easterlies is concentrated in the southern latitudes. The core of maximum easterlies is known as easterly Jet stream. First time Koteswaram (1958) discovered the existence of easterly Jet. In the good monsoon case the wind speeds over the southern tip and adjoining ocean areas are exceeding 30 m/s covering wider area 40°E-90°E and equator to 10°N. Over the land area (India) the wind speeds are less when compared to oceanic regions, they are varying from 30 m/s over southern tip to 5 m/s at 25°N. In the poor monsoon the area under 30 m/s contour is very much reduced and limited to the region 55°E to 85°E and equator to 10°N. There is no appreciable changes over India when compared to good monsoon (Fig. 3c and d). Anomalies: The wind anomalies are obtained by subtracting the long term (50 year) means from the corresponding actual values. Anomaly U wind at 850-hPa: The U wind anomaly in good monsoon decade showed positive values over south

and central Arabian Sea and the major part of the India, south of 200N (Fig. 4a and b). This indicates that a strong westerly belt is perceived during good monsoon decade, which helps to enhance monsoon activity by picking up the moisture from south and central Arabian Sea. Over northern India anomaly easterly belt is seen. Generally over the north India the deflected monsoon (southeasterly belt) persists. The presence of easterly anomalies indicates that the deflected monsoon also has greater strength during good monsoon years. In the poor monsoon situation the reverse is true. The presence of easterly belt in the most parts of the Arabian Sea (except a small region over the north) and south of 25oN indicates the existence of weak westerlies over these regions. This indicates that the moisture transport from the Arabian Sea will be diminished due to the presence of weak westerlies. At the same time over the north India, north of 25oN the anomalies are positive, which indicates that the deflected monsoon is also weak. From the above anomalies and their patterns we may come to conclusion that the strength of (1) the westerlies over the Arabian Sea and the India south of 20oN and (2) deflected monsoon over northern India will be a key factor for deciding the monsoon behaviour. If the strength of these two branches is reduced, it will result in poor moisture transport causing

Fig. 3. Monsoon seasonal (JJAS) circulation patterns of the atmosphere at 850 hPa and 150 hPa for good (a, c)/poor (b, d) monsoon decades (wind speed in m/s).

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the poor monsoon. If the two branches are intensified one can expect good monsoon situation due to the transport of more moisture (Fig. 4a and b). Anomaly U wind at 150-hPa: Anomaly picture (Fig. 4c and d) indicates differences clearly between the poor and good monsoon situations. Easterly anomalies are dominant over equatorial Indian Ocean in good monsoon

situation. This indicates that the easterly Jet is intensified in good monsoon situation. In the case of poor monsoon, over the equatorial Indian Ocean the anomalies are westerlies, which indicate the weakening of easterly Jet. From these observations one can say that the easterly jet plays an important role in deciding the activity of monsoon over the Indian subcontinent (Fig. 4c and d).

Fig. 4. Monsoon seasonal (JJAS) zonal wind anomaly of the atmosphere at 850 hPa and 150 hPa for good (a, c) /poor (b, d) monsoon decades (anomalies are in m/s).

Fig. 5. Monsoon seasonal (JJAS) meridional wind anomaly of the atmosphere at 850 hPa for good (a) /poor (b) monsoon decades (anomalies in m/s).

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Anomaly V wind at 850- hPa: There are some differences between the anomalies of poor and good monsoons over the Arabian Sea. In good monsoon case, over Arabian Sea and adjoining northern parts of equatorial Indian Ocean, the anomalies are positive. Further the positive anomalies are extended from Sumatra to central parts of North India in SE-NW direction. In the case of poor monsoon, northerly anomaly is present over southeastern Arabian Sea, south and central Bay of Bengal, adjoining Indian mainland (Fig. 5a and b). This pattern indicates the strengthening/weakening of southerlies during good/poor monsoon. Northward transport of moisture from equatorial Indian Ocean is enhanced/reduced during good/poor monsoon. Anomaly latent heat flux The anomaly latent heat flux values are calculated by subtracting the long term mean (50 Year) mean values from the corresponding actual values. In good monsoon decade, the latent heat flux anomaly is positive over Eastern Arabian Sea and adjoining parts. The anomaly is high over southeastern Arabian Sea and the values range from 10 to 20 W/m². The anomaly is positive over the

entire Bay of Bengal and the values lie in between 10 to 20 W/m² (Fig. 6a). In poor monsoon decade, the latent heat flux anomaly is negative over southeastern parts of the Arabian Sea and adjoining area. The values lie in between -10 to -20 W/m². Over the Bay of Bengal also, negative values are present, particularly they are pronounced over south central parts of the Bay of Bengal (Fig. 6b). Cyclonic systems The cyclonic activity over the Bay of Bengal is more during good monsoon decade. The number of cyclonic systems (of all categories of systems, depressions, storms, severe storms) formed over Bay of Bengal is 33 during poor monsoon conditions. The number is doubled in the case of good monsoon (Fig. 7). The time series of frequency of cyclonic systems and rainfall indicates that they are positively related (Fig. 1). There is a decreasing trend in the frequency of cyclones in the recent four decades, which is closely connected with the tendency of rainfall.

Fig. 6. Monsoon seasonal (JJAS) latent heat flux anomalies in good (a) and poor monsoon (b) decades (values are in w/m2).

Fig. 7. Decadal variation of cyclonic systems formed in the Bay of Bengal during monsoon season. Brown line indicates mean.

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CONCLUSIONS In good monsoon decades, the westerly belt in the latitudes to south of monsoon trough and easterly belt in the latitudes north of the trough are quite pronounced at 850-hPa during June through September. This indicates that the trough is strengthened and inflow of air due to these two branches coming from the Arabian Sea and the Bay of Bengal enhances the transport of moisture to the monsoon trough. Ultimately this situation leads to formation of more rainfall over India during monsoon months. In poor monsoon decade opposite features are observed; weak westerlies/easterlies to the south/north of trough reduce the inflow of moisture from the regions of the Arabian Sea /the Bay of Bengal. In good monsoon decade, the intensity of tropical easterly jet stream is enhanced and elongated in east-west direction occupying more area (covering southern tip of India and tropical equatorial Indian Ocean) during June through September. While in poor monsoon decade, it is observed that the strength of easterly jet stream is reduced and squeezed in east-west direction occupying less area. In good monsoon decade, the cyclogenetic activity is enhanced over Bay of Bengal, while in the poor monsoon decade, the cyclogenetic activity is reduced. Bhaskar Rao et al. (2001) reported that total cyclonic systems indicated decreasing tendency after the 1970’s which may be coinciding with the observation of global warming and climate change during last three decades. Bengtsson et al. (1996), reported that doubling of CO2 reduces the number of cyclonic storms. The reduced cyclonic activity in the decade 1980- 90 is agreeing with the above two studies. Srinivasa Rao et al. (2004) also reported a decreasing trend in tropical cyclonic systems of the Bay of Bengal during monsoon and a significant negative correlation between the tropical easterly Jet strength and the number of tropical cyclonic systems. From the results on the latent heat flux it can be concluded that both the Arabian Sea and the Bay of Bengal play an important role for the enhancement of evaporation in good monsoon decade. The enhanced water vapour is imported to Indian main land through both the branches, (a branch of monsoon current from the Arabian Sea and another from the Bay of Bengal). The latent heat is the hidden heat supplied by the water vapour and after copious rains are produced over northeast India, monsoon trough area and central India, the large amounts of latent heat will be released and it will be given to atmosphere. This heat enhances the north-south temperature gradient and hence monsoon meridional circulation and the zonal winds. So the winds get more strengthened in the upper troposphere and it may be responsible for the strengthening of tropical easterly jet stream at upper troposphere during good monsoon decade opposite features are observed for poor monsoon decade.

ACKNOWLEDGEMENTS The authors are thankful to NCEP/NCAR Reanalysis data team and Indian Institute of Tropical Meteorology, Pune, India for free supply of their data for this study. REFERENCES Aravequia, JA., Rao, VB. and Bonathi, JP. 1995. The role of moist baroclinic instability in the growth and structure of monsoon depressions. J. Atmos. Sci. 52: 4393-4401.

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SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 451-458, 2008 ISSN: 1715-9997

1

FEM SIMULATION WITH EXPERIMENTAL VERIFICATION OF SINTERED FORGED COMPONENTS

*Suprakash Patra1, Goutam Sutradhar2, Amitava Mandal3

1AWS, CWISS, Indian Institute of Technology, Kharagpur 2Department of Mechanical Engineering, Jadavpur University, Kolkata

3National Institute of Foundry and Forge Technology, Hatia, India

ABSTRACT Powder metallurgy (P/M) is one of the most diverse approaches in metal forming. The main attraction of this process is the ability to fabricate high quality complex parts to close tolerances in an economical manner. Extensive research work has been done on this field. It has been reported by so many authors that if this process is properly performed the P/M products approaches to full density which belongs better mechanical response compare to their equivalent wrought materials, largely because of the microstructural homogeneity. In this present work P/M steel components were forged under cold condition and the predicted results were verified with the help of DEFORM-3D software package. The theoretical analysis and the FEM models agree the experimental results. Keywords: Cold forging, iron powder, metal powder preform, simulation, FEM.

INTRODUCTION Forging of Powder metallurgy (P/M) components deliver net shape parts having reliable dimensional tolerances and accurate chemical composition etc. several advantages over ordinary P/M components eliminating the machining operation increases material utilization. It was reported that this process leads to a net cost saving compared to the conventional approach under mass production and the P/M products approaches to full density which belongs better mechanical response compare to their equivalent wrought materials, largely because of the microstructural homogeneity, Ramakrishna (1980). Powder forged parts are used in connecting rods in US automobiles since 1987, which are superior than conventional products due to uniform microstructure and better material distribution. Now-a-days development of super plastic ceramic materials from ceramic perform has been developed for high bearing and seal applications. The plastic deformation of powder particles during compaction promotes the mechanical interlocking which ultimately control the density and hardness (Haglund and Agren ,1998; German, 1994). The particle bonding is dependent on interatomic and electrostatic force at surface. The small size powder with clear surface has stronger force and irregular particle shape, enhance better mechanical interlocking between the particles (David et al., 1998; German, 1990). A substantial work has been done by the various workers Vedis and Geiling (1981), Sutradhar et al. (1994, 1995) on cold forging of sintered iron powder performs. Work has also been done on

modeling on prediction of shrinkage Shama et al. (2002). It is needless to mention that there is an immense potential in this particular field. A lot of authors (Oozo and Yang, 1992; Ibhadode, 1989; Mori et al., 1980; Im and Kobayashi, 1986; Oh et al., 1987) have been reported the P/M forging by FEM analysis and simulation technique with the following assumptions. Elastic portion of deformation is neglected because,

practical forging process involves very large amount of plastic deformation.

The normality of the plastic strain rates to the yield surface holds.

An-isotropic behavior, which occurs during deformation, is negligible.

Thermal properties of porous material are independent of temperature.

Following observations have been made by the above authors, for small reduction, almost no change in relative density at the central region, as the reduction increases the relative density near the bulged free surfaces increases and the equatorial free surface is possible fracture site which was indeed by experience also. Substantial work has been reported by various authors on cold forging of sintered powder performs during last two decades. But no work has been published regarding the verification of Upper Bound method with the Simulation Software like Ausys or Deform-3D. For analysis the preform is divided into 1500 elements (Fig.1). Therefore, an attempt has been made to access the die load in two methods and which have been verified by experimental results.

*Corresponding author email: suprakashpatra@lycos.com

Canadian Journal of Pure and Applied Sciences 452

Fig. 1. Preform divided into 1500 elements. Analysis by Upper Bound Method In metal forming process friction condition between work piece and deforming tool is of most important and a number of factors such as force and mode of deformation, properties of the finished specimen and resulting surface roughness are the most deciding factors for the successfulness of the process. The relative velocity between the work piece material and the die surface together with high interfacial pressure and deformation modes will create the conditions essential for adhesion in addition to sliding. Due to application of compressive hydrostatic stress in cold forging i, e, plastic deformation the pores will close and the relative density will increase, but in case of tensile hydrostatic stress the pores will grow and relative density will decrease. The density distribution is also not uniform. It is high at the central region and low at edges. The density will be more uniform for smaller coefficient of friction µ and for a greater initial density Tabata et al. (1980). During cold forging of metal powder preforms the compressive force gradually increases the relative density that is directly proportional to the real area of contact. The relative density gradually approaches the apparent relative density and this approach is asymptotic. During the sinter cold forging processes it is very important to keep special consideration on interfacial friction, as this will determine the success or failure of the operation. The relative velocity between the workpiece material and the die surface, together with high interfacial pressure and deformation modes, create a condition of composite friction, which is due to the adhesion and sliding Deryagin et al. (1952). The sheer equation becomes τ=µ (p+ρ0 φ0), the first term µp is due to the sliding and the second term µρ0 φ0 being due to adhesion, which latter arises from chance of relative density of the pre-form during forging process. The pattern of metal flow during the cold forging of a metal powder pre-form is such that there exists two zones, an inner where no relative movements between work piece and die occurs (the sticking zone) and an outer zone where sliding occurs. Therefore the appropriate friction laws for different conditions are:

Axisymmetric Conditions τ = µ [ p + ρ ϕ{ 1- (

0nrrrm − )}] (1)

Plain strain conditions

)}](1{[ 00 nbxxmp −−+= ϕρµτ (2)

rm and xm denote the radius of the sticking zone axisymmetric and plane strain conditions respectively, which may be approximated by the relationship given by Rooks (1974) and n>>1. As it is a case of axisymmetric condition. Velocity field and strain rates

ro

-U

Xh

Fig.2. Forging of disc.

The velocity of the upper die is taken as - .

U as shown in

Fig.2. The radial and axial velocity components .

U r and .

U z are assumed as .

U r = hU)1(2)21(

.

ηη+

− r (3) .

U z = - hUz

.

(4) .

U θ = 0 The corresponding strain rates are

.ε r =

rU r

δδ

.

= hU)1(2)21(

.

ηη+

− (5)

.ε θ =

rU r

.

= hU)1(2)21(

.

ηη+

− =

.ε r (6)

.ε z =

zUzδδ

.

= - hU

.

.

(7)

The above normal-strain components satisfy the compressibility equation for the axisymetric condition for porous material.

.ε +

.

)1(2)21( εη

η±

± = 0 (Refer to Appendix) (8)

Patra et al. 453

For plastic deformation of a metal powder, the external power J* supplied by the die is given as

J* = 3

2σ0 e∫ 2

1ijeij dV + ∫s IvIDsτ + dVUa i

Vi∫ ρ -

dsVT ist

i∫ (9)

The first term on the right hand side denotes the rate of internal energy dissipation Wi the second term denotes the frictional shear energy losses Wf, the third term denotes the energy dissipation due to inertia forces Wo, and the last term covers power supplied by predetermined body tractions Wt. In this case forces due to inertia are negligibly small and no external surface traction is stipulated. Moreover forces due to inertia are negligibly small. Therefore Wa = Wt = 0. The external power J* supplied by the press through the piston is

J* = Wi + Wf = P.

U (10) The rate of energy dissipation per unit volume is given by DWi = σ1 dε1+σ2 dε2 + σ3 dε3 (11) The total internal energy dissipation in the disc is

Wi = 2

.

0

)1(b

Uk

πη

σρ+

(12)

The rate of energy dissipation due to the friction Wf is given by Wf = dsV

s∫ ∆τ (13)

Considering the magnitude of the relative velocity ∆V

∆V = hrhU

Zr )1(2

)21(,0 0.

ηη

+−

== − (14)

ds =4πr dr

Wf = 32

⎥⎦

⎤⎢⎣

⎡

⎭⎬⎫

⎩⎨⎧

−+++−

000

.3

0

431

)1()21(

nrr

nhUr m

av φρρηηπµ (15)

Die Load Using the equations

PU = ++

2.

00

)1( ηπσρ rUk

32

⎥⎦

⎤⎢⎣

⎡

⎭⎬⎫

⎩⎨⎧

−+++−

000

.3

0

431

)1()21(

nrr

nhUr m

av φρρηηπµ (16)

Pav = 20r

Pπ

, for ρ0ϕ0 = 0.3 pav (17)

PU = ++

2.

00

)1( ηπσρ rUk

32

⎥⎦

⎤⎢⎣

⎡

⎭⎬⎫

⎩⎨⎧

−+++−

02

02

0

.3

0

4313.0

)1()21(

nrr

nrP

rP

hUr m

ππηηπµ

(18)

P =

)1(431

)1()21(2.0

)1(3)21(2

12

00

1

0

00

ηπσρ

ηηµ

ηµµ

+⎥⎦

⎤⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛−+

+−

−+−

−−

rnrr

nhr

hr k

m

(19) For 0<x<1 where ρ0ϕ0 = xpav. and n>>1. Experimental Work Automised iron powder of purity 98% and finer than 150 µm was used. The sieve analysis of iron powder is as follows: Screen Size (micron) -150 -106 -75 -63 -45 +150 +106 + 75 +63 +45 Weight of the 2 38 12.5 8.5 21.5 17.5 powder (%) Apparent Density = 3.9 gm/cc. Tap Density = 4.3 gm/cc. Chemical Analysis of Iron Powder used. C Si Mn S P Fe 0.12 0.35 0.15 0.43 0.03 Balance Compacting Pressure = 25 – 30 Kg./mm2 Platen Speed = 1 mm/Sec. Iron powder was compacted in a closed circular die. The die wall was lubricated with graphite. The compaction pressure of 25-30 Kg./mm2 was maintained at 1000 psi (6.89 k Pa) and fifteen green compacts were prepared. These compacts were sintered in an argon atmosphere at about 1050oC temperature for 2 hours in a horizontal Tubular furnace of maximum temperature capacity 1450 ± 1oC. In order to minimize the non-uniformity of density distribution, the sintered compacts were re-pressed at the same compaction pressure in the same die and the specimens re-sintered. The average relative density of the re-sintered performs was found to be 0.70 to 0.80. The pre-forms were machined to the dimensions of diameter 12.7mm and 27.2 mm and different heights as shown in the tables. The surfaces of the specimens were polished with fine emery paper.

Canadian Journal of Pure and Applied Sciences 454

Following this the specimen was placed between lower and upper platens in both dry and lubricated conditions. Silicon grease is used as lubricant. The top diameter, middle diameter, and lower diameter and reduction in height were measured at various stages of compression and the loads were recorded. The upper platen moved at a speed of 1 mm/sec. The lower platen was held stationary. The results obtained are shown in the tables. Simulation of Cold Forging of Metal Powder Preform The simulations are carried out using software package DEFORM 3D. The pre-form is considered as porous and dies are considered as rigid. The elastic deformation is neglected. For analysis the per-forms are divided into 15000 elements. The ambient temperature is selected as 20oC. The bottom die is fixed and top die is moving with the speed of 1 mm/sec. The heat exchange between work piece and dies and between the dies and ambient surrounding is incorporated in FEM calculation. The friction factor for pre-form and top die is calculated as 0.25 and for pre-form and bottom die as 0.30 in lubricated condition. The friction factor for un-lubricated case is calculated as 0.50 for top die and 0.55 for bottom die. The values of friction factors are assumed initially and then for one case these values are adjusted to give the observed dimensions of the pressed cylinder. Using these values for all the other cases gave good results. The stress-strain curve for the powder material is shown in figure 3.

Fig. 3. Stress-strain curve of the iron powder used in the experiment.

The experimental results and simulation results are given in a tabular form. TD(f) = the coefficient of friction value between top

die and work-piece BD(f) = the coefficient of friction value between

bottom die and work-piece RD = the relative density of pre-form. Red. % = reduction in percentage of the height of

original preform Sim. Means results obtain after FEM simulation.

Table 1. Diameter 27.2, Height 11.4, RD 0.8, TD (f) 0.25, BD (f) 0.30

Change in Hit. mm. Load (N) Top Dia(mm) Middle Dia.(mm) Bottom Dia.mm Red. % Actual Step Sim. Actual Sim. Actual Sim. Actual Sim. Actual Sim. 12 1.368 17 1.36 224525 299058 27.70 28.013 28.80 28.291 28.00 27.90 24 2.888 36 2.88 343350 428177 28.54 29.382 30.40 29.986 28.80 29.12 32 3.65 46 3.68 490500 516592 30.00 30.373 31.11 31.152 29.95 30.03

Table 2. Diameter 12.7, Height 13.2, RD 0.7, TD (f) 0.50, BD (f) 0.55

Change in Hit.(mm) Load(N) Top Dia. mm Mid. Dia.(mm) Bottom Dia.mm Red. % Actual Step Sim. Actual Sim. Actual Sim. Actual Sim. Actual Sim. 30 3.96 49 3.92 96138 69957 13.54 13.62 14.52 14.38 13.40 14.32 42 5.54 69 5.52 100062 100331 13.92 13.89 15.70 15.90 13.90 13.78 55 6.52 83 6.64 117720 125232 14.50 14.054 16.92 16.40 14.65 14.043

Table 3. Diameter 12.7, Height 12.6, RD 0.8, TD(f) 0.25, BD(f) 0.3

Change in Hit.(mm) Load(N) Top Dia.mm Mid. Dia.(mm) BottomDia.(mm.) Red. % Actual Step Sim. Actual Sim. Actual Sim. Actual Sim. Actual Sim. 21.36 2.69 33 2.64 343350 378108 28.7 28.813 29.6 29.415 28.45 28.483 22.70 2.86 36 2.88 343350 396558 28.12 29.052 29.4 29.681 28.25 28.666 30.76 3.875 49 3.92 490500 490992 30.10 30.079 31.1 31.004 29.60 29.683

Patra et al. 455

% Reduction in height Fig. 4. Actual height reduction & Sim. Height reducation against % height reduction for Table 1.

Fig. 5. Actual load & Sim. Load against % reduction height for Table 1.

Fig. 6. Actual bottom dia. & Sim. dia against % reduction in height for Table 1.

Fig. 7. Actual top dia. & sim. top dia. against % reduction in height for Table 1.

Fig. 8. Actual middle dia. & sim. middle dia. Against & reduction in height for Table 1.

Fig. 9. Actual change in height against sim. change in height for particular % reduction in height for Table 1.

Canadian Journal of Pure and Applied Sciences 456

Fig. 10. Dense powder particles at center.

Fig.11. Powder Particles at the middle of sample.

Fig. 12. Cracks formed at the bulging edge of sample

Fig. 13. Single crack (Enlarged view)

Fig. 14. Powder Particles at the edge of the sample. RESULTS AND DISCUSSION The results obtained from the experiments and from finite element based simulation are shown in tables 1-3. From the tables it is observed that at any percentage reduction in height the calculated load is close to the actual load recorded taking into consideration the low sensitivity of the machine used. The diameters of the cold forged performs at top, middle and bottom obtained through simulation at various stages are also quite close to the actual dimensions obtained from experiments. Which are shown in Figures 4-9. A few cold pressed specimens were cut vertically and photographs were taken. The trends of relative density variation obtained from the photographs after analyzing them using SEM (Fig.10-14). It is clearly visible from the figure that the density is highest at the center and it gradually decreases towards the edges. Figure15 shows the variation of relative density with reduction in height for three points as obtained from FE simulation. The

Patra et al. 457

calculated relative density is also more at the center for lubricated case than for un-lubricated case. Two samples were pressed till the crack appeared (Fig.12 and 13). The cracks are located at the middle of bulged portion. The simulation results (Fig.16) show that the relative density is minimum at those locations where cracks appeared. CONCLUSIONS FEM modal is a convenient tool for the prediction of density distribution and change in dimensions with

reduction in height of the specimens at a particular time and boundary conditions. The friction factor calculated is 0.25 for preform and top die and 0.30 with bottom die in lubricated condition. The friction factors for un-lubricated case are calculated as 0.50 for top die and 0.55 for bottom die. The difference of 0.05 in the friction factors between top and bottom is found to be effective in predicting the actual dimensions of the pressed cylindrical performs. Though the machine used is not very sensitive still the load recorded for height reduction matched well with the calculated load. From the trends of relative density

Fig.15. Density distribution of Cold Forged Preform obtained through Simulation.

Fig.16. Probable damage area (with the rate of more susceptible to damage) of the Cold Forged Prefrom.

Canadian Journal of Pure and Applied Sciences 458

variation at different points in the pressed cylinder the crack prone areas can be effectively identified and necessary precautions can be taken accordingly. ACKNOWLEDGEMENT Authors thankfully acknowledge the Vice-Chancellor of Jadavpur University for Financial assistance under ‘Seed Support’ of Potential for Excellence Scheme in carrying out the experimental part of this work and the Director, NIFFT, Ranchi, Jharkhand for providing the necessary infractural facilities in carrying out the Simulation of the above experiments, otherwise the work could not be given the present shape. Notations b: perpendicular distance from center of the disc to

the side .ε x ,

.ε y ,

.ε z : principal strain increments

h: thickness of the specimen k: constant equals to 2 n: constant quantity L: die load p: average pressure at the die/work piece interface S: surface of velocity discontinuity V: volume of the zone of plastic deformation η : constant, a function of ρ only ρ0: dimensionless, ρr/ρ*

ρr: real density of real contact area ρ*: apparent density of the apparent contact area τ: shear stress ϕ0: specific cohesion of the apparent contact area µ: co-efficient of friction σ0 : yield stress of the non-work hardening matrix

metal J* : External power supplied REFERENCES David, Dr., Zener, C. and Cai, Haimain. 1998. Common Causes of Cracks in Powder Metallurgy Compacts. The International Journal of Powder Metallurgy. 34 (4): 33-52.

Deryagin, BV., Izd. Akad. and Nauk. 1952. USSR.

German, RM. 1994. Powder Metallurgy Science, 2nd edition, Powder Metallurgy. Industries Federation, Princeton, NJ, USA.

German, RM. 1990. Powder Packing Characteristics, Metal Powder Industries Federation, Princeton, NJ, USA.

Haglund, S. and Agren, J. 1988. W content in Co binder during sintering of WC-Co. Acta Metall.46: 2801.

Ibhadode, AOA. 1989. Simulation and experimental verification of completely closed cavity die forging on a mechanical press. Journal of Engineering Manufacturing. 30: 17-32.

Im, YT. and Kobyashi, S. 1985. FE analysis of plastic deformation of porous Materials in Metal Forming and Impact Mechanism’ Pergamon Press, Oxford, UK. pp 103.

Im YT. and Kobayashi. S. 1986. Analysis of Axisymetric Forging of porous materials by FEM’. Adv. Manufacturing Processes, Vol.1, pp.473.

Mori, K., Shima.S. and Sakada, KO. 1980. Finite Element Method for the analysis of Plastic Deformation in Porous Metals’ Bulletin of JSME. 23 (178): 516.

Oh, SI., Wu, WT. and Park, JJ. 1987. Application of the Finite Element Method to P/M forging processes, Proceedings of 2nd ICPT, Stuttgart, West Germany. pp 961.

Oozo, K. and Yang, G. 1992. Application of Networks to Expert System for Cold Forging. Machine Tool Manufacturing. 577- 587.

Ramakrishna, P. 1980. Forging of Metal Powder Perform. Proceedings of the International Seminar on Metal Working Technology Today and Tomorrow. Ranchi, India.

Rooks, BW. 1974. The effect of die temperature on metal flow and die wear during high-speed hot forging, 15th Int. MTDR Conf., Birmingham, UK. Macmillan. London. pp 487.

Shama, S., Mahesha, S, Pavanachand, C., Rengarajan, R., Ramesh Rao S. 2002. Modeling Liquid Phase Sintering of Hard Metal Powder Compacts. PM2TEC World Congress on Powder Metallurgy and Particulate Materials.

Sutradhar, G., Jha, AK. and Kumar, S. 1994. Production of sinter-forged components. Journal of Materials Processing Technology. 41: 143-169.

Sutradhar, G., Jha, AK. and Kumar, S. 1995. Cold Forging of Sintered Polygonal Discs. Journal of Institute of Engineers (India), Nov. 76: 148-152.

Tabata, T., Masaki, S. and Hosokawa, K. 1980. Int. J. Powder Metallurgy Powder Tech. 16, p.149.

Vedis, WV. and Geiling, KR. 1981. Relationship between Mechanical Properties and Particle Size on Iron Powder Compacts. The International Journal of Powder Technology. 17(2): 135.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 459-462, 2008 ISSN: 1715-9997

345

γ0-COMPACT , γs-REGULAR AND γs-NORMAL SPACES

B Ahmad1 and *S Hussain2

1Centre for Advanced studies in Pure and Applied Mathematics, Bahauddin Zakariya University, Multan

2 Department of Mathematics, Islamia University, Bahawalpur, Pakistan

ABSTRACT

We define and study the properties of γ s -regular and γ s -normal spaces. We also continue studying γ0-compact spaces defined in Ahmad and Hussain (2006). Keywords: γ-closed(open), γ-closure, γ-regular (open), (γ,β)-continuous (closed, open) functions, γ0-compact, γs-regular spaces and γs-normal spaces. AMS (2000) subject classification. Primary 54A05, 54A10, 54D10.

INTRODUCTION Kasahara (1979) defined an operation α on topological spaces. He introduced and studied α-closed graphs of a function. Jankovic (1983) defined α- closed setss and further worked on functions with α- closed graphs. Ogata (1991) introduced the notions of γ-Ti ,i = 0,1/2,1,2; and studied some topological properties. Rehman and Ahmad (1992) [respt. Ahmad and Rehman 1993] defined and investigated several properties of γ-interior, γ-exterior, γ-closure and γ-boundary points in topological spaces (respt. in product spaces), and studied the characterizations of (γ,β)-continuous mappings initiated by Ogata (1991). Ahmad and Hussain (2003) continued studying the properties of γ-operations on topological spaces introduced by Kasahara (1979). Ahmad and Hussain (2005) defined γ-nbd, γ-nbd base at x, γ-closed nbd, γ-limit point, γ-isolated point, γ-convergent point and γ*-regular spaces and discussed their several properties. They further established the properties of (γ,β)- continuous, (γ,β)- open functions and γ-T2 spaces. In this paper, we continue studying γ0-compact spaces defined in Ahmad and Hussain (2006) and study its properties. We also define and study some properties of γs-regular and γs-normal spaces. First, we recall some definitions and results used in this paper. Hereafter we shall write spaces in place of topological spaces. Definition (Ogata, 1991). Let (X,τ) be a space. An operation γ : τ → P(X) is a function from τ to the power set of X such that V ⊆ Vγ , for each V ∈τ, where Vγ

denotes the value of γ at V. The operations defined by γ(G) = G, γ(G) = cl(G) and γ(G) = intcl(G) are examples of operation γ.

Definition (Ogata, 1991). Let A ⊆ X. A point a∈A is said to be γ-interior point of A iff there exists an open nbd N of a such that Nγ ⊆ A and we denote the set of all such points by intγ (A). Thus intγ (A) = { x ∈A : x ∈N ∈τ and Nγ ⊆ A } ⊆ A. Note that A is γ-open ( Ogata, 1991) iff A = intγ (A). A set A is called γ- closed (Ogata, 1991) iff X−A is γ-open. Definition (Rehman and Ahmad, 1992). A point x∈X is called a γ-closure point of A ⊆ X, if Uγ ∩ A ≠ ∅, for each open nbd U of x. The set of all γ-closure points of A is called γ-closure of A and is denoted by clγ(A). A subset A of X is called γ-closed, if clγ(A) ⊆ A. Note that clγ(A) is contained in every γ-closed superset of A. Definition 1. An operation γ : τ → P(X) is said to be strictly regular, if for any open nbds U , V of x ∈X, there exists an open nbd W of x such that Uγ ∩Vγ = Wγ. Definition 2. An operation γ : τ → P(X) is said to be γ-open, if Vγ is γ-open for each V∈ τ. Example 1. Let X={a,b,c}, τ ={ ∅, X, {a}, {b},{a,b}}. Define an operation γ : τ→ P(X) by γ(A) = intcl(A). Clearly the γ-open sets are only ∅, X, {a}, {b},{a,b}. It is easy to see that γ is strictly regular and γ-open on X. Example 2. Let X={a,b,c}, τ ={ ∅, X, {a}, {b},{a,b}}. Define an operation γ : τ→ P(X) by γ(A) = cl(A).

*Corresponding author email: sabiriub@yahoo.com Present Address: Department of Mathematics, King Abdul Aziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

Canadian Journal of Pure and Applied Sciences 460

Clearly the γ-open sets are only ∅, X. It is easy to see that γ is strictly regular but not γ-open on X. 1. γ0-compact spaces Definition (Ahmad and Hussain, 2006). A space X is said to be γ0-compact, if for every cover {Vi : i ∈ I} of X by γ-open sets of X, there exists a finite subset I0 of I such that X= ∪ clγ ( Vi). i∈I0

Then the following characterization of a γ0-compact space is immediate: Theorem (Ahmad and Hussain, 2006). A space X is γ0-compact iff every class of γ-open and γ- closed sets with empty intersection has a finite subclass with empty intersection. Definition (Ogata, 1991). A space X is said to be γ-T2 space. If for each pair of distinct points x, y in X, there exist open sets U , V such that x ∈ U, y ∈ V and Uγ ∩ Vγ = ∅. Definition (Ogata, 1991). An operation γ is said to be regular, if for any open nbds U ,V of x ∈X, there exists an open nbd W of x such that Uγ ∩Vγ ⊇ Wγ. Thorem 1. Let X be a γ-T2 space and suppose that C be a γ0-compact subset of X and x ∈ X − C, then there are open sets Ux and Vx in X such that x ∈ Uγ

x and C ⊆ Vγ

x

and Uγx ∩ Vγ

x = ∅, where γ is regular and γ-open.

Proof. Suppose that C is γ0-compact subset of X and x ∈ X − C. For each y ∈ C , y ≠ x. Since X is γ-T2 , there are open sets Uxy and Vy containing x and y respectively such that Uγ

xy ∩ Vγy = ∅. Now, let { Vγ

y ∩ C : y ∈ C } be γ-open cover of C. Since C is γ0-compact , then γ-open cover has a finite subset {Vγy1 ∩ C ,Vγy2 ∩ C, … ,Vγyn ∩ C } such that n

C = ∪ clγ ( Vγyi ∩ C ). i = 1

Let Uγy1 ,Uγy2 , … ,Uγ yn be the corresponding γ-open sets containing x. Take n

Uγx = ( ∩ clγ (Uγxyi))

i = 1

n and Vγ

x = ( ∪ clγ (Vγxyi)), i = 1

then x ∈ Uγx and C ⊆ Vγ

x . Where Uγx and Vγ

x are γ-closed , since γ is regular.

n

n

Also Uγx ∩ Vγ

x = ( ∩ clγ (Uγxyi )) ∩ ( ∪ clγ (Vγyi )) i = 1 i = 1 n n = ∩ ( ∪ clγ (Uγxyi) ∩ clγ (Vγyi )) i = 1 i = 1

n n = ∩ ( ∪ clγ (Uγxyi ∩ Vγyi )) i = 1 i = 1 [ γ is regular ( Rehman and Ahmad, 1992) ]

n n = ∩ ( ∪ clγ ( ∅ )) = ∅. i = 1 i = 1

Theorem 2. Let X be a γ-T2 space. Then every γ0-compact subset A of X is γ-closed, where γ is regular and γ-open. Proof. Let X be a γ-T2 space and A be a γ0-compact subset of X. We show that X − A is γ-open. For this, let x ∈ X − A. Then y ∈ A gives x ≠ y. Since X is γ-T2 , there are open sets Uxy and Uy in X containing x and y respectively such that Uγ

xy ∩ Uγy = ∅.

Let the collection { Uγ

y ∩ A : y ∈ A } is a cover of A by γ-open and γ-closed sets of A. Since A is γ0-compact, there is a finite subset { Uγyi ∩ A : i = 1,2, … ,n} such that n n A = ∪ clγ (Uγyi ∩ A) = ∪ (Uγyi ∩ A ). i=1 i=1 Now corresponding to each yi , let Uγxyi be the γ-open set containing x, then Uγ

x = ∩ Uγxyi is γ-open containing x, since γ is regular. Also n n Uγ

x ∩ A = Uγx ∩ (∪ (Uγyi∩ A )) ⊆ Uγ

x ∩ (∪ Uγyi ) i=1 i=1 n ⊆ ∪ (Uγ

x ∩ Uγyi ) = ∅ i=1

or Uγx ∩ A = ∅. Hence Uγ

x ⊆ X − A implies x∈ intγ (X −A). Consequently, X −A = intγ (X −A). That is, X −A is γ-open. So, A is γ-closed. This completes the proof. Definition 3. Let X be a space and A ⊆ X. Then the class of γ-open sets in A is defined in a natural way as : τγA = { A ∩ O : O ∈ τγ } , where τγ is the class of γ-open sets of X. That is, G is γ-open in A iff G = A ∩ O, where O is a γ-open set in X.

Ahmad and Hussain 461

2. γs-regular spaces Definition 4. A space X is said to be γs-regular space, if for any closed set A and x∉A, there exist open sets U , V such that x∈U, A ⊆ V and Uγ ∩Vγ = ∅. Example. Let X= {a,b,c}, τ ={ ∅, X, {a}, {b,c}}. For b∈X, define an operation γ : τ→ P(X) by

⎩⎨⎧

∉∈

=AA

Ab if cl(A),b if ,A

)(γ

Then easy calculations show that X is a γs-regular space. Theorem 3. Every subspace of γs-regular space X is γs-regular, where γ is regular. Proof . Let Y be a subspace of a γs-regular space X. Suppose A is γ-closed set in Y and y∈Y such that y∉A. Then A = B ∩ Y, where B is γ-closed in X. Then y∉B. Since X is γs-regular, there exist open sets U , V in X such that y∈U, B ⊆ V and Uγ ∩ Vγ = ∅. Then U ∩ Y and V ∩ Y are open sets in Y containing y and A respectively, also (U ∩Y)γ ∩ (V∩ Y)γ ⊆ ( Uγ ∩ Yγ ) ∩ ( Vγ ∩ Yγ )

( γ is regular ) = ( Uγ ∩ Vγ ) ∩ Yγ

= ∅ ∩ Yγ = ∅. This completes the proof. 3. γs-normal spaces Definition 5. A space X is said to be γs -normal space, if for any disjoint closed sets A , B of X, there exist open sets U, V such that A ⊆ U, B ⊆ V and Uγ ∩Vγ = ∅. Example . Let X= {a,b,c,d}, τ ={ ∅, X, {a}, {b}, {a,b}, {b,d}, {a,b,d}, {b,c}, {b,c,d}, {a,b,c}}. For b∈X, define an operation γ : τ→ P(X) by

⎩⎨⎧

∉∈

= )Α(AA

b if ,clintcl(A)b if cl(A),

γ

Then X is γs-normal. Next, we characterize γs -normal space as : Theorem 4. A space X is γs -normal if for any closed set A and open set U containing A, there is an open set V containing A such that A ⊆ V ⊆ clγ (Vγ) ⊆ Uγ, where γ is γ-open and strictly regular.

Proof . Let A , B be disjoint closed sets in X. Then A ⊆ X−B, where X−B is open in X. By hypothesis, there is a open set V such that clγ (Vγ) ⊆ ( X− B)γ … (1) (1) gives Bγ ⊆ (X−clγ (V))γ and V∩ (X− clγ (Vγ)) = ∅. Consequently, A ⊆ V, B ⊆ X− clγ (Vγ) and Vγ ∩ ((X− clγ (Vγ)))γ = ∅. This proves that X is γ-normal. This completes the proof. Theorem 5. A γs -normal γ-T1 space is γs –regular, where γ is strictly regular. Proof . Suppose A is a closed set and x∉A. Since X is a γ-T1 space, therefore by Proposition 4.9 ( Ogata, 1991) each {x} is γ-closed in X. Since X is γs-normal, therefore there exist open sets U , V such that {x} ⊆ U, A ⊆ V and U ∩ V = ∅, or x ∈U, A ⊆ V and U ∩ V = ∅ implies that Uγ ∩ Vγ = ∅, since γ is strictly regular. Thus X is γs -regular.This completes the proof. Theorem 6. A closed subspace of a γs -normal space X is γs -normal, where γ is regular. Proof . Let A be a closed subspace of γs -normal space X. Let A1, A2 be disjoint closed sets of A. Then there are closed sets B1, B2 in X such that A1= B1 ∩ A , A2 = B2 ∩ A. Since A is closed in X, therefore A1, A2 are closed in X. Since X is γs-normal, there exist open sets U1, U2 in X such that A1 ⊆ U1, A2 ⊆ U2 and Uγ

1 ∩ Uγ2= ∅. But then

A1 ⊆ A ∩ U1, A2 ⊆ A ∩ U2 , Where A ∩ U1, A ∩ U2 are open in A and (A ∩ U1) γ ∩ (A ∩ U2) γ ⊆ (Aγ ∩ Uγ

1 ) ∩ (Aγ ∩ Uγ2 )

(since γ is regular) = Aγ ∩ ( Uγ

1 ∩ Uγ2 )

= Aγ ∩ ∅ = ∅. This proves that A is γs -normal. Hence the proof. Theorem 7. Every γ0-compact and γ-T2 space is γs-normal, where γ is regular and γ-open. Proof. Let X be γ0-compact and γ-T2 space and C1 , C2 be ant two disjoint γ-closed subsets of X. Then being γ-closed subset of γ0-compact space, C1 is γ0-compact. By Theorem 1, for γ0-compact C2 and x ∉ C2, there are open sets Ux , Vx such that x∈Uγ

x , C2 ⊆ Vγx and Uγ

x ∩ Vγx = ∅. … (2)

Let the set { Uγ

x : x∈ C } be a cover of C1 by γ-open and γ-closed sets of C1 . Since C1 is γ0-compact, so there are finite number of elements x1, x2, … , xn such that

Canadian Journal of Pure and Applied Sciences 462

n n

C1 ⊆ ∪ clγ (Uγxi ) = ∪ (Uγxi ). i=1 i=1

n n

Let U = ∪ ( Uγxi ) , V = ∩ ( Vγxi ). i=1 i=1

Then C1 ⊆ U, C2 ⊆ V and

n n

( U ∩ V ) γ = ( (∪ (Uγxi) ∩ ( ∩ ( Vγxi ))γ i=1 i=1 = ( ∅ ) γ = ∅. Hence X is γs-normal. This completes the proof. REFERENCES Ahmad, B. and Rehman, FU. 1993. Operations on topological spaces II, Math. Today. 11: 13-20.

Ahmad, B. and Hussain, S. 2003. Properties of γ-operations on topological spaces, Aligarh Bull. Math. 22 (1): 45-51.

Ahmad, B. and Hussain, S. 2005. γ-Convergence in topological spaces, Southeast Asian Bull. Math. 29(5): 835-842.

Ahmad, B. and Hussain, S. 2006. γ*- regular and γ- normal spaces, Math. Today. 22 (1): 37- 44 .

Jankovic, DS. 1983. On functions with closed graphs, Glasnik Mat. 18: 141-148.

Kasahara, S. 1979. Operation-compact spaces, Math. Japon. 24: 97-105.

Ogata, H. 1991. Operations on topological spaces and associated topology, Math. Japon. 36 (1): 175-184.

Rehman, FU. and Ahmad, B. 1992. Operations on topological spaces I, Math.Today. 10: 29-36.

Willard, S. 1970. General Topology, University of Alberta, Volume A, Addison Wesely., MR 41# 9173.

SENRA Academic Publishers, Burnaby, British Columbia Vol. 2, No. 2, pp. 463-468, 2008 ISSN: 1715-9997

1

EVALUATION OF SOFTWARE DEVELOPMENT CONTROLS IN INFORMATION SYSTEMS ORGANIZATIONS

*Muhammad Asif Khan and Saleh Al Turki

Department of Information Systems, College of Computer Sciences and Information Technology King Faisal University, Kingdom of Saudi Arabia

ABSTRACT

Information Systems organizations have become more vigilant in identifying risks to their infrastructures. In fact, organizations have recognized the significance of IS audit and controls to remove or mitigate the risks for their infrastructures by implementing appropriate measures. The aim of this study is to analyze, explain and demonstrate that how Information Systems organizations implement and ensure that business applications are developed under a controlled environment, thus preventing and/or mitigating the risks involved in development. Also, the study focuses on whether organizations are careful in carrying out the acquisition process as efficiently and effectively possible. To complete our work we have collected and analyzed data from different large organizations in Saudi Arabia, which have an existing IS audit function in order to compare between the approach used by these organizations and the industry standards of IS audit and control set by organizations. Keywords: Information systems, software development and acquisition, audit controls.

INTRODUCTION The study of information systems deals with deployment of information technology in organizations, institutions and society at large. Information systems are becoming essentials for businesses to be more productive and efficient, and since the internet has taken a leading edge in business growth, control weaknesses and system vulnerabilities have become the top issues in organizations (Ciborra, 2002). In early 1970s IS organizations had not realized the extent of risks and losses of various business and technology sectors but thereafter, professionals from various sectors such as technology, security, business, manufacturing, government and general public joined efforts in order to confront these issues. Consequently, organizations, associations and institutions were established to lay down standards, guidelines and procedures, which were designed and developed by these professionals to address the increasing control weakness and security concerns. Initially, security threats, system vulnerability and control weaknesses were given much consideration in applications and network infrastructure, but soon it was determined that the way the technology was developed and managed had a significant impact on organizations. Consequently, appropriate action was taken to further develop and evolve the control procedures to comprehend most of the major processes, which included the development, design, management and implementation of the information systems facility. Since the focus started to comprise the business processes

and the governing procedures, the concept of auditing evolved. Altar (2003) said that with the advent of computer systems, the scope of auditing expanded to encompass both general controls over computer installations and application controls for assuring that recording, processing and reporting of data are performed properly. CISA (2007) described that information systems auditing is a process that collects and evaluates evidence to determine whether the information systems and related resources adequately safeguard assets, maintain data and system integrity, provide relevant and reliable information, achieve organizational goals effectively, consume resources efficiently and have in effect internal that provide reasonable assurance that business, operational and control objectives will be met and that undesired events will be prevented or detected and corrected in timely manner. Globally every organization should undergo a periodic security audit. A security audit is a systematic assessment of security level of a system and the effectiveness of controls. It is important to obtain an understanding of the audit area before a risk assessment can be accomplished, prioritized and categorized. Data regarding the existing controls of the audit area is collected, compiled and analyzed to evaluate the controls’ appropriateness, adequacy, effectiveness and efficiency (Solomon, 2005). METERIALS AND METHODS The study was carried out with the aim to demonstrate the effectiveness of IS Audit and Control and analyze the effectiveness and efficiency of IS Audit in reducing and/or mitigating risks, vulnerabilities, security issues and weaknesses within IS organizations. We started our study *Corresponding author email: asifkhan@kfu.edu.sa

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by collecting data regarding the IS Audit approach through surveys and interviews with the companies identified [to maintain the privacy of the organizations we will denote the two financial organizations as F1, F2 and other public services organization as G]. Data about different technical infrastructure and operational practices in use, security techniques available, most commonly used approach for software development and acquisition, business continuity and disaster recovery planning were collected through interviews and surveys. Our study started with a questionnaire prepared for the F1, F2 and G companies where IS audit function in place. The main aim of the questionnaire was to allow the companies under research to rate the importance, effectiveness before IS audit, the effectiveness after IS audit, risk rating before IS audit and the risk rating after IS audit. We have used following indicators in the questionnaire:

Importance – level of importance of the control.

Effectiveness before IS audit – effectiveness of the control before IS audit function in the company.

Effectiveness after IS audit - effectiveness of the control after it has been reviewed by the IS audit function in the company.

Risk rating before IS audit - risk level the IT function is exposed to with regard to the corresponding rated control area and the governed IS processes before an IS Audit review was conducted on that process.

Risk rating after IS audit - risk level after the corresponding control area and the governed processes was reviewed by the IS audit function.

We used the ratings from scale One to Five, one being the minimum and five is the maximum for all above stated indicators (i.e. 1 = Minimum, 2 = Low, 3 = Medium, 4 = High and 5= Maximum). For example if the “Importance” was rated as 5 (i.e. Maximum) it implies that the control is of maximum and/or extreme importance to the management in order to govern the subsequent IT process. Likewise, if the “Effectiveness before/after IS Audit” was rated as 1 (i.e. Minimum) it means that the control is of poor effectiveness. RESULTS AND DISCUSSION In any software development environment operating systems have their significance and data was gathered to know operating systems in the companies. Following table 1 describes the available operating systems in the companies under research:

Table 1. Operating Systems used in the companies under research

Operating System F1 F2 G Windows ● ● ● OS/400 ● ● Unix ● ● ● Linux Novell ● ● Sun Solaris ●

It is observed from the table 1 that the company F1 uses OS/400 for their core banking system due to its high security, reliability, scalability and efficiency. Unix is used for their other critical applications due to its reliability and Novell is used for their front-end banking solution. Windows is used as the network operating system. The company F2 uses OS/400, Unix, Windows and Sun Solaris for their infrastructure. However, they use OS/400 and Unix for their critical applications. Only one application is hosted on Windows. According to them Windows is an excellent network operating system but not a secure application server. The company G uses Windows, Unix and Novell operating systems. Unix is used for the core banking system due to its higher reliability, stability and security compared to Windows and Novell. Windows is used as their network operating system. According to them Windows provides the best networking service out of all operating systems. Novell is used for their legacy systems. From the above, it is observed that operating systems such as OS/400 and Unix are more reliable and secure application operating systems than Windows. On the other hand Windows is a more reliable network operating system. Since databases are the backbone of software development, therefore, we carried out a research on available databases in the respective companies and following table 2 shows the databases in use in the companies: Table 2. Databases used in the companies under research

Databases in use F1 F2 G Oracle ● ● SQL ● DB2 ● ● MS Access ● Sybase Other(s) ●

Both companies F1 and F2 use Oracle and DB2 for their core applications systems due to their integrity, reliability

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and security. The company F1 uses SQL and MS Access due to their business requirements, which demand some applications that utilize these databases. Audit analysis We carried out a comprehensive study with regard to the audit controls implemented in the organizations. Company F1 Despite that Policies and Procedures was rated as “Maximum” Importance, it was completely ineffective before the IS Audit function and therefore the risk was at its maximum as well. The IS Audit function recommended to have policies and procedures in place to govern the Software Development function of the IT Division. Once this was done the risk rating reduced drastically. Although Development Methodology and Project Management were also rated as “High” Importance, they were of “None” effectiveness before the IS Audit function. This means that no Development Methodology and Project Management controls were in place before IS Audit function. When the IS Audit function made its recommendations a Development Methodology and a Project Management approach were implemented subsequently increasing the effectiveness. As a result the risk, of unstructured software development approaches which might result in poor design software, expensive developments, inefficient software development and bad project management approaches was reduced. User training was effective from the beginning at the F1

Company. The IT Audit function could only make small recommendations in order to improve the effectiveness of the user training thereby further reducing the risk of unqualified staff handling critical processes. Software change management was not at the desired level of effectiveness until appropriate recommendations were made by the IS Audit function to bring it at the desired level of effectiveness to reduce and/or mitigate the risk of implementing unauthorized changes onto the application systems developed. Protection over source code was not as sufficient as required before the IS Audit function, though its “Maximum” importance. The IS Audit function was able to identify weaknesses with a potential of losing the source code or it destruction. However, with appropriate recommendations this risk was reduced and/or mitigated and the effectiveness of the control was increase. As shown in the figure 1, it is observed that Policies and Procedures, Software Change Management and Protection of Source Code were rated “Maximum” (score 5) for Importance. Development Methodology, Project Management and User Training were rated “High” (score 4). Effectiveness before IS Audit for Policies and Procedures, Development Methodology and Project Management were at “None” (score 0). User Training and Software Change Management were rated at “Medium” (score 3). As far as Protection of source code is concerned, it was rated as “Low” (score 2). As a direct impact to the above the risk rating before IS

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Fig. 1. Software Development Controls – Company F1.

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Audit for Policies and Procedures was at “Maximum” (score 5). The risk rating before IS Audit for Development Methodology and Project Management was “High” (score 4). User Training it was at “Minimum” (score 1). For Software Change Management it was at “Low” (score 2) and for Protection of Source Code it was as at “Medium” (score 3). Effectiveness after IS audit for most of the controls increased to “High/Maximum” (score 4.5) except for Development Methodology and Project Management where it increased to “Medium/High” (score 3.5) with a difference of 1 compared to the other controls. As a consequence the risk rating after IS Audit for all the controls dropped to “None/Minimum” (score 0.5) except for User Training where it dropped to “None” (score -0.5). Company F2 There were only some policies and procedures governing software development. The IS Audit function recommended to have these policies and procedures complete to adequately govern the Software Development function of the IT Division. Once this was done the risk rating reduced significantly. A development methodology existed before the IS Audit function. However, its effectiveness improved after the IS Audit Methodology and the risk of inadequate developments reduced. A Project Management structure did exist before the IS Audit function as well. Its effectiveness improved after the IS Audit Methodology and the risk of inadequate developments reduced.

User training was effective from the beginning at F2 Company. The IT Audit function could only make small recommendations in order to improve the effectiveness of the user training thereby further reducing the risk of unqualified staff handling critical processes. Software Change Management and Protection of Source Code was not at the desired level of effectiveness until appropriate recommendations were made by the IS Audit function to bring it at the desired level of effectiveness to reduce and/or mitigate the risk of implementing unauthorized changes onto the application systems developed and losing the source code. As illustrated in the figure 2, it is observed that most of the controls were of “Maximum” (score 5) Importance except for User Training where it was rated as “High” (score 4). Effectiveness before IS Audit for Policies and Procedures was rated as “Minimum/Low” (score 1.5). Development Methodology and Project Management were rated as “Low/Medium” (score 2.5). User Training was rated as “Medium” (score 3). Effectiveness of Software Change Management and Protection of Source Code before IS Audit were rated as “Medium/High” (score 3.5). As a result the risk rating before IS Audit for Policies and Procedures was “Medium/High” (score 3.5). Development Methodology and Project Management were rated as “Low/Medium” (score 2.5). User Training was rated as “Minimum” (score 1). Software Change Management and Protection of Source Code were rated as “Minimum/Low” (score 1.5). Effectiveness after IS audit for nearly all the controls increased to “High/Maximum”

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Fig. 2. Software Development Controls – Company F2.

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(score 4.5) except for Development Methodology and Project Management where it increased to “Medium/High” (score 3.5) and “High” (score 4) respectively. Therefore, the risk rating after IS Audit for Policies and Procedures, Software Change Management and Protection of Source Code dropped to “None/Minimum” (score 0.5). Risk Rating after IS Audit for Development Methodology dropped to “Minimum/Low” (score 1.5), for Project Management to “Minimum” (score 1) and for User Training to “None” (score -0.5). Company G There were sufficient policies and procedures governing software development. The IS Audit function recommended to have these policies and procedures refined to further reduce the risk rating. User training was also effective from the beginning. The IT Audit function could only make small recommendations in order to improve the effectiveness of the user training thereby further reducing the risk of unqualified staff handling critical processes. Source Code was also protected well enough. The effectiveness of the above controls was increased by (0.5 points) and the risk rating was reduced by (0.5 points), thereby placing the risk rating after IS Audit to “None”, which was much acceptable by the Company’s management. Development Methodology and Project Management existed before the IS Audit function. However, their effectiveness improved after the IS Audit function and the risk of inadequate developments reduced.

Software Change Management was not at the desired level of effectiveness until appropriate recommendations were made by the IS Audit function to bring it at the desired level of effectiveness to reduce and/or mitigate the risk of implementing unauthorized changes onto the application systems developed and losing the source code. As pointed out in the figure 3 below, the Importance of Policies and Procedures, User Training and Protection of Source Code were rated as “High” (score 4). The effectiveness of these controls before IS Audit was given a rating of “Medium/High” (score 3.5) as a result their risk rating before IS Audit was “None/Minimum” (score 0.5). The effectiveness of these controls after IS Audit increased to be as “High” (score 4), subsequently the risk rating after IS Audit dropped to “None/Minimum” (score 0.5). The Importance of Development Methodology was rated as “High” (score 4). The effectiveness before IS Audit was given a rating of “Medium” (score 3) as a result the risk rating before IS Audit was “Minimum” (score 1). The effectiveness of this control after IS Audit increased to be at “Medium/High” (score 3.5), subsequently dropping the risk rating after IS Audit to “None/Minimum” (score 0.5). Project Management’s Importance was rated as “Maximum” (score 5). The effectiveness before IS Audit was given a rating of “Medium/High” (score 3.5) as a result the risk rating before IS Audit was “Minimum/Low” (score 1.5). The effectiveness of this control increased to be at “High/Maximum” (score 4.5) after IS Audit, subsequently the risk rating after IS Audit dropped to “None/Minimum” (score 0.5). The Importance of Software Change Management was rated as “Maximum”

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Fig. 3. Software Development Controls – Company G.

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(score 5). The effectiveness before IS Audit was given a rating of “Medium” (score 3) as a result the risk rating before IS Audit was “Low” (score 2). The effectiveness of this control after IS Audit increased to be at “High/Maximum” (score 4.5), subsequently dropping the risk rating after IS Audit to “None/Minimum” (score 0.5). We noticed from the technological infrastructure of the companies (i.e. F1, F2 and G) have almost similar infrastructure and methodologies due to the same standards that these organizations adopt. It is clearly evident that the effectiveness of the controls improved significantly after the IS Audit function, which one of its responsibilities is to ensure that the organizations adhere to best practices and international standards. The recommendations made by the IS Audit function to rectify the controls weaknesses also enhanced the control effectiveness. The standards put in place by international bodies such as ISACA, ISACF, ITGI and ISC2, proved to be effective and efficient in improving the control structure over the IT processes and management by stipulating the required controls. By adhering to the standards put in place by ISACA, ISACF, ITGI and ISC2, the organizations had a similar level of control effectiveness and compliance level after the IS Audit was conducted. At the company F1 it was observed that, the effectiveness of the controls increased and, the risk rating dropped, by an average of 2.1 points after IS Audit. For company F2 it was observed that, the effectiveness of the controls increased and, the risk rating dropped, by an average of 1.5 points after IS Audit. With regard to company G it was observed that, the effectiveness of the controls increased and, the risk rating dropped, by an average of 1.1 points after IS Audit. CONCLUSION It was observed that the organizations under the research did not reveal further information about their weaknesses, which somewhat affected the analysis of the control and risk evaluation. However, enough information was provided to conduct the research effectively. Furthermore, it was observed that some controls were not implemented despite the repeat recommendations of the IS Audit function. When further inquired, it was found that the higher management and/or the board of directors of that particular company were not applying enough force on the line management including IT to implement these controls. Based on the findings and analysis of the data gathered it is proved that, by implying the control objectives and standards stipulated by industry leaders such as ISACA, ISACF, ITGI and ISC2 using effective, efficient and adequate IS Audit methodologies, the IS Audit function was successfully able to identify and address control weaknesses, security, systems vulnerability and threat concerns of the information systems and supported business processed.

REFERENCES Ciborra, C. 2002. Labyrinths of Information. Oxford University Press. 15-25.

Altar, S. 2003. Information Systems: a management perspective. Pearson Inc. USA. 480-486.

Solomon, GM. and Chapple, M. 2005. Information Security Illuminated. Jones and Bartlett Publishers, MA, USA. 340-343.

CISA Review Manual. 2007, ISACA, USA. 20-24.