Bioresearch Bulletin Vol 1:1

Bioresearch Bulletin (2010) 1. I

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FROM THE EDITOR “The journey of a thousand miles begins with a single step� - CHINESE PROVERB

The goal of research are as diverse as the scientists and scholars who work to expand the human knowledge and understanding. Regardless of the final destination or goal, One fact remains certain, the whole process or the quest begins with a single step. The first edition of bioresearch bulletin is one such step. Our goal is to ensure that all interested readers from around the world to access the research articles in the journal freely and to take the research motive of the authors who had their articles published with us, to the next level.

The selected manuscripts for the first issue covers from ethno-botany to animal toxicology, a perfect example of the Chinese proverb above. Many such first and second step and lots of other research journeys are highlighted in this issue of bioresearch bulletin as well. Turn the page, read on and enjoy research.

Sam Stephen Managing Editor

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BIORESEARCH BULLETIN EDITORIAL BOARD Abiya Chelliah., M.S

EDITOR-IN-CHIEF Pethuru Devathasan., M.D EXECUTIVE EDITOR Sam Stephen.,M.S MANAGING EDITOR ASSOCIATE EDITORS Harry Thomas Rodreguez Pharmacologist.

David khanna Radiologist.

Mahesh Ethno botanist.

Peter Baskaran Ecologist.

John De Britto Molecular Biologist.

Kiran Chandran Cell Biologist Sherrie Jesulyn David Plant Taxonomist.

BULLETIN STAFF

EDITORIAL OFFICE

Beneson Thilagar Language Editor

No.4, 108/A 6th Street Perumalpuram Tirunelveli- TN India - 627007

Girithara Prakash Computing Systems Manager Sam Stephen Concept and Design

Ph. No: 91-8870709052 E-Mail: info@bioresonline.com Website: www.bioresonline.com

SUBSCRIBING BIORESEARCH BULLETIN Online access at www.bioresonline.com– Free (Open access) Print Subscription- In India -Rs 750 per Year - Other countries -US $ 55 per Year

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CONTENTS Editorial Introducing Bioresearch Bulletin Anxiolytic effect of Aridanin isolated from Tetrapleura tetraptera in mice A.O. Aderibigbe, E.O. Iwalewa, S.K. Adesina & O.I. Agboola.

Susceptibility of Crab Chitosan against Staphylococcus aureus G. Sugumar, U. Ramesh & A. Selvan

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Assessing the ecological status of prominent medicinal herbs in central Sudan and the challenge of sustainable management H.H. El-Kamali

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Impact of industrial effluents and sewage on river Thamirabarani and its concerns P. Peter Baskaran, John De Britto

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Effect of nutrients on in-vitro culture of Morus alba l. (white mulberry) Ankit Pradhan, A. S. Vishwanathan, R. Basavaraju.

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Antimicrobial activity of marine Bacteria associated with Polychaetes C.V. Sunjaiy Shankar, A. Hepziba Jeba Malar, S. Mary Josephine Punitha

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Laboratory culture of Microzooplankton Tintinnopsis cylindrical [Daday 1887] S.Vijayaragavan, P.Vivek Raja

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A study on acute toxicity , oxygen consumption and behavioural changes in the three major carps, Labeo rohita (ham), Catla catla (ham) and Cirrhinus mrigala (ham) exposed to Fenvalerate T. Anita Susan K. Sobha K.S. Tilak

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Bacteriological assessment of some swimming pools within Ilorin metropolis, Kwara, Nigeria.

Sule I.O, Oyeyiola G.P

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Bioresearch Bulletin (BRB) is not responsible for the content of the individual manuscripts. The manuscripts contained in this volume were peer– reviewed. The manuscripts accepted in this issue conforms with the editorial policies.

All correspondence should be mailed to the Managing editor: Sam Stephen, No.4, 108/A 6th Street , Perumalpuram, Tirunelveli—Tamil Nadu, India - 627007

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ORIGINAL PAPER

Introducing Bioresearch Bulletin; An open access journal

SAM STEPHEN J1

ABIYA CHELLIAH D2

Published online: 22 June 2010 © Bioindica Press 2010

INTRODUCTION: Welcome to Bioresearch Bulletin (BRB). This new Bioindica Press journal is being introduced to provide a focal point for publication of articles, letters, and reviews reporting new theories based on Lifescience and its allied fields. Though the contents will evolve, some specific topics that are appropriate today for Bioresearch Bulletin include advances in or applications of Biochemistry, bioinformatics, bio-physics, bio-statistics, biotechnology, botany, cell biology, chemical biology, computational biology, developmental biology, ecology, ethno knowledge medicine, evolutionary biology, genetics, genomics, immunology, microbiology, molecular biology, neurobiology, pharmacology, pharmacology, physiology, plant biology, structural biology, systems biology, taxonomy and zoology.

TYPES OF MANUSCRIPTS: The Bioresearch Bulletin (BRB) is published every month by Bioindica. The journal accepts the following type of scientific communication, Regular Research articles: These should elaborate new and carefully confirmed findings, and experimental procedures should be given in sufficient detail for others to verify the work. The length of a full paper should be the minimum required to describe and interpret the work clearly.

Short Communications: A Short Communication is suitable for recording the results of complete small investigations or giving details of new models or hypotheses, innovative methods, techniques or apparatus.

Mini review: Submissions of mini-reviews and perspectives covering topics of current interest are welcome and encouraged. Mini-reviews are also peer-reviewed.

Scientific Images: Photographs of Lab findings, Living Organisms and their interaction with nature, 3D models 1. Managing editor Bioresearch Bulletin 2.Editor-in-Chief Bioresearch Bulletin

are published in open access image library upon acceptance.

LICENSE & COPYRIGHT: Authors publishing with Bioresearch Bulletin retain the copyright to their work, All their research articles and images are licensed under the Creative Commons Attribution License. This license allows the article / image to be downloaded from the website, copied and distributed as long as the original content is left unmodified.

Harold Urey, the editor of the Journal of Chemical Physics in 1933 observed: ―The life and interests of the individual are short and of little consequence as compared to the lives of our sciences.‖ This will always be true, and our only intention is for Bioresearch Bulletin to help enrich the life - science among researchers and scholars through improved publication and open access.

ACKNOWLEDGEMENTS: There are many people who deserve thanks for bringing Bioresearch Bulletin to fruition. The support, encouragement, and handling of innumerable details by the Board of Editors, Executive Editor Pethuru Devathasan, Editor-in-Chief Abiya Chelliah, Language Editor Benison Thilagar & Computational support Girithara Prakash (CEO of Fortab, Inc) have been essential.

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ORIGINAL PAPER

Anxiolytic effect of Aridanin isolated from Tetrapleura tetraptera in mice

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A.O. ADERIBIGBE 3 S.K. ADESINA

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E.O. IWALEWA

Received: 20 March 2010 /Accepted: 2 April 2010 /Published online: 22 June 2010 Š Bioindica Press 2010

ABSTRACT The study was carried out to investigate the anxiolytic properties of aridanin isolated from Tetrapleura tetraptera in mice. Elevated plus maze was used to investigate the effect. The possible involvement of the GABAA - benzodiazepine receptor complex was also investigated using flumazenil. Aridanin at doses of 5 and 10 mg/kg, i.p. administered 30 min prior induced anxiolytic effect expressed by increase number of entries in and time spent in the open arms and percentage of open arm entries and decrease number of entries and time spent in the closed arms. The treatment of mice with flumazenil (2.0 mg/kg, i.p.) 15 min before the administration of aridanin (10 mg/kg, i.p.) blocked the aridanin induced anxiolytic effect. It was found out that aridanin induced an anxiolytic effect in mice. In addition it is concluded that the anxiolytic effect of aridanin may be through interaction with GABAA- benzodiazepine receptor complex.

INTRODUCTION Tetrapleura tetraptera Taub (Mimosaceae) locally known as Aridan is a large tree growing throughout the rain forest belt of West Africa. It is generally found in the lowland forest of tropical Africa.

The fruit consist of a fleshy pulp with small, brownish – black seeds. The plant has many traditional uses mainly in the management of convulsion, leprosy, inflammation and rheumatic pains, schistosomiasis, asthma and hypertension (Ojewole and Adesina, 1983).

The dry fruit has a pleasant aroma (Aladesanmi, 2007).

It is used as a popular seasoning spice, a medicine and a dietary supplement rich in vitamins in Southern and Eastern Nigeria (Okwu, 2003; Essien et al., 1994). The fruit is used to prepare soup for mothers from the first day of birth to prevent post partum contraction (Nwawu and Akah, 1986). The root extract has been proven to be useful for the treatment of gastrointestinal related clinical problem (Noamesi et al., 1994). The ethanol extract and saponins from the stem bark of Tetrapleura tetraptera exerted an inhibitory effect on Keywords: Aridanin, Tetrapleura tetraptera, Anxiolytic, GABAA luteinizing hormone released by pituitary cells, sugreceptor, benzodiazepine receptor. gesting its use as contraceptive agent (El Izzi et al., 1990). Tetrapleura tetraptera is a natural molluscicides as aqueous extract of it is effective against Bulinus globosus and Lymnaea natalensis (Adewunmi, 1991). The A.O. Aderibigbe alleopathic potential of Tetrapleura tetraptera has led Department of Pharmacology and Toxicology, to its integration into an agro forestry system (Amoo et Faculty of Pharmacy,Niger-Delta University. al., 2008). Tetrapleura tetraptera has been shown to Bayelsa, Bayelsa State, Nigeria. improve the foaming ability of soaps (Adebayo et al., e-mail : adebee 738@yahoo.co.uk 2000). Tetrapleura tetraptera has no influence on cell E.O. Iwalewa proliferation and neither induced chromosomal aberraDepartment of Pharmacology,Faculty of Pharmacy tion nor sister chromatid exchanges in Chinese hamster Obafemi Awolowo University,Ile-Ife, Osun State, ovary cells (no genotoxic effect) (Adewunmi et al., Nigeria.

1991). Tetrapleura tetraptera has been shown to cause S.K. Adesina Drug Research and Production unit, Faculty of Pharmacy elevation in serum AST and alteration of various meObafemi Awolowo University, Ile-Ife, Osun State, tabolites parameters and did not induce any marked Nigeria. pathological lesion in the liver (Odesanmi et al., 2009).

O.I. Agboola The sedative, anticonvulsant and analgesic effect of Department of Pharmacognosy, Faculty of Pharmacy aridanin in mice have been reported (Aderibigbe et al., Niger-Delta University, Bayelsa, Bayelsa State, Nigeria.

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2007a; Aderibigbe et al., 2007b; Ojewole, 2005). The aqueous extract of Tetrapleura tetraptera fruit have been shown to possessed anti-inflammatory and hypoglycaemic properties (Ojewole and Adewunmi, 2004).

The ethanolic extract of Tetrapleura tetraptera fruit possessed antiplasmodial activity in mice (Okokon et al., 2007). One of the active constituents isolated from Tetrapleura tetraptera fruit is a mono – N – acetylglycoside of oleanoic acid (3β- hydroxyolean-12-en-28oic) called Aridanin (Adesina and Reish, 1985). The present study was carried out to investigate the anxiolytic effect of aridanin in mice.

MATERIALS AND METHODS Structural elucidation and characterization of Aridanin (Fig 1) from Tetrapleura tetraptera was carried out by Prof. S. K. Adesina (Adesina and Reish, 1985) of Drug Research and Product Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife. Sample used for this experiment was collected from him.

Animals Swiss albino male mice weighing between (20-25 g) were obtained from the animal house of the Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife. The animals were divided into five mice in each cage and were fed with a standard laboratory diet and tap water ad libitum. The animals were maintained at 25 +1oC under natural 12 h daylight/ night conditions. All experiment was carried out in compliance with Obafemi Awolowo University Ethics Committee on research in animals and in accordance with NIH guide for the care and use of laboratory animals.

Drugs Diazepam, Flumazenil (Sigma Chemicals Co.St. Louis, Missouri,U.S.A.), Drug dissolution Aridanin was dissolved in 5% Tween 80, Flumazenil and Diazepam was dissolved in normal saline.

The resulting solution, control vehicle or test materials were administered by intraperitoneal injection (i.p.).

Acute toxicity Acute toxicity study of aridanin in mice was carried out as described by Miller and Tainter (1944) and the lethal dose was calculated by the method of Litchfield and Wilcoxon (1949). It was carried out by injecting aridanin i.p. into 5 groups of mice containing 5 animals with the following dose levels 25, 37.5, 50, 75 and 100 mg/kg. The animals were observed for over 24 hr and the LD50 was calculated.

Elevated Plus Maze test (EPM) The elevated plus maze (EPM) test was used to evaluate the animal anxiety (Pillow and File, 1986; Lister, 1987; Nogueira and Vassilieff, 1996). The EPM for mice consisted of two open arms (30 x 5 cm) and two close arms (30 x 5 x 15 cm) that extended from a common central platform (5 x 5 cm) with an open roof, arranged such that the two arms of each

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type were opposite to each other. The floor and the walls of each arm were wooden and painted white.

The maze was elevated to a height of 38.5 cm above floor level. Testing was conducted in a quiet room that was illuminated by light. The animal’s behaviour was recorded directly by an observer sitting 2 m away in the same room.

Each animal was placed in the centre of the EPM facing one of the open arms. An entry into an arm was defined as the animals placing all four paws over the line marking that area. The number of entries and the time spent in the open and closed arms were recorded during a 5 min test period. The percentages of open arm entries (100 x Open/Total entries) were calculated for each animal.

Initially, mice were treated with aridanin at the doses of 5, 10, 20 and 30 mg/kg, i.p. 30 min before the evaluation in the EPM test. The control animals received 5% Tween 80. Subsequently mice were treated with flumazenil (2 mg/kg, i.p.) a GABAbenzodiazepine receptor antagonist 15 min before the administration of aridanin (10 mg/kg, i.p.). The anxiety evaluation was carried out 30 min after the administration of aridanin or vehicle. Between each trial, the maze was wiped with 70% ethanol to prevent olfactory cue from animals.

Statistical analysis Results are expressed as Mean ± standard error of the mean (S.E.M). All data were analysed by one way analysis of variance (ANOVA). Post hoc tests were then performed using Student Newman RESULTS AND DISCUSSION Acute Toxicity Acute toxicity of aridanin was calculated using graphical method of Litchfield and Wilcoxon, (1949). The intraperitoneal LD50 of aridanin in mice was calculated to be 60.0 mg/kg.

COOH R OR1 O O R2O HO

NHCOCH 3

R = R1 = R2 = H = Aridanin

Fig 1: The chemical structure of Aridanin

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Elevated Plus-maze Aridanin at the doses of 5 and 10 mg/kg, i.p. increased the number of open arm entries [F (5, 24) = 13.7, P < 0.001] (Table 1), decreased the number of closed arm entries [F (5, 24) = 7.8 P < 0.001] (Table 1) and increased the percentage of the open arm entries [F (5, 24) = 41.8, P < 0.001] (Table 1). Aridanin at the doses of 20 and 30 mg/ kg, i.p. decrease the percentage of open arm entries. According to the number of entries, aridanin 5 and 10 mg/kg, i.p. increased the time spent in the open arm [F (5, 24) = 29.5, P < 0.001] (Table 2) and decreased the time spent in the closed arm [F (5, 24) = 11.6, P < 0.001] (Table 2). Aridanin at the doses of 20 and 30 mg/kg, i.p. increased the time spent in the closed arm. According to the number of entries, the treatment of mice with aridanin (10 mg/kg, i.p.) preceded by flumazenil decreased the time spent in the open arms [F (3, 16) = 23.4, P < 0.001] (Table 3) and increased the time spent in the closed arm [F (3, 16) = 29.0, P < 0.001] (Table 3). and increased the time spent in the closed arm [F (3, 16) = 29.0, P < 0.001] (Table 3). Flumazenil blocked the aridanin (10 mg/kg, i.p.) induced anxiolytic effect, so there was a decrease in the number of open arm entries [F (3, 16) = 58.1, P < 0.001] (Table 4) and an increase in the number of closed arm entries [F (3, 16) = 4.8, P < 0.001] (Table 4). The elevated plus-maze is considered to be an etiologically valid animal model of anxiety because it uses natural stimuli (fear of a novel, brightly-lit open space and fear of balancing on a relatively narrow, raised platform) that can induce anxiety in human (Yellow and File, 1986; Lister, 1987). It has been extensively accepted as an ultimate test for anxiolytic drugs and their

mechanisms of action (Rodgers et al., 1997; Cole and Rodgers, 1995).

In the present study low doses of aridanin (5 and 10 mg/kg, i.p.) induce a dose dependent anxiolytic effect in mice. The doses increased the entries and time spent in the open arms and decreased entries and time spent in the closed arms in the EPM test. The anxiolytic effect of aridanin is similar to the one observed with diazepam, a typical benzodiazepine drug (Rall, 1990). As expected, diazepam produced significant increases in open arm time and in number of entries into the open arm.

Therefore it can be hypothesized that aridanin may be acting like a benzodiazepine like substance. Supporting this view, the treatment with flumazenil, a specific antagonist of the benzodiazepine site in the GABAA – BDZ receptor complex, was able to block completely the anxiolytic effect induced by aridanin. The anxiolytic effect of aridanin is similar to that of plants such as Cissus cornifolia, Careya anboree, Rubus brasiliensis, Stachys lavandulifolia,Scutellaria baicalensis which has anxiolytic properties (Musa et al., 2008; Kumar et al., 2008; Hue et al., 2002; Rabbani et al., 2003; Nogueira et al., 1998). It is noteworthy that anxiolytic doses of a drug must not affect locomotion, otherwise test animals would be sedated in one arm and the result will be an error of wrong conclusion of the effect of the drug. Reddy and kulkarni (1997) stated that; the effects of drugs seen at doses that did not markedly affect locomotor activity suggest that these changes in behaviour represent anxiolytic actions.

In conclusion, aridanin has been shown to possessed anxiolytic effect which is exerted through interaction with the GABAA – BDZ receptor complex.

Table 1 Effect of aridanin on no of entries in the open arm, no of entries in the close arm and as percentage of entries in the open arm Treatment 5%TW80 Aridanin Aridanin Aridanin Aridanin Diazepam

Dose (mg/kg, i.p.)

0.2ml/20g 5.0 10.0 20.0 30.0 1.0

NEOA 4.2±0.6 6.2±0.4* 6.4±0.5* 3.0±0.5 1.8±0.4 8.4±1.2*

NECA 7.8±1.1 5.0±0.6 4.4±0.5 6.8±0.4 7.1±0.4 3.4±0.4

PEOA 29.0±1.1 55.3±3.1* 57.7±3.2* 34.1±2.1 23.1±1.1 58.0±3.1*

Results are expressed as the mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant difference between different treatment groups. NEOA: Number of entries in the open arm; NECA: Number of entries in the closed arm; PEOA: Percentage of entries in the open arm; 5%TW80: 5% Tween 80 *Indicate significant difference from 5% Tween 80 control. P < 0.05 SNK test.

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Table 2 Effect of aridanin on time spent in the open arm entries and on time spent in the close arm entries Treatment 5%TW80 Aridanin Aridanin Aridanin Aridanin Diazepam

Dose (mg/kg, i.p.)

0.2ml/20g 5.0 10.0 20.0 30.0 1.0

TSOA 40.0±5.6 135.6±17.0* 140.4±20.7* 38.2±4.0 29.8±1.7 170.4±6.7*

TSCA 101.2±12.2 43.2±3.3 50.8.4±10.0 125.8±15.1 124.0±15.0 58.4±3.4

Results are expressed as the mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant difference between different treatment groups. TSOA: Time spent in the open arm; TSCA: Time spent in the close arm; 5%TW80: 5% Tween 80 *Indicate significant difference from 5% Tween 80 control. P < 0.05 SNK test.

Table 3 Effect of aridanin on time spent in the open arm entries and on time spent in the close arm entries in the presence of flumazenil Treatment

Dose (mg/kg, i.p.)

5%TW80 Aridanin FLU FLU+ARI

0.2ml/20g 10.0 2.0 10.0

TSOA 40.0±5.6 140.4±20.7* 200.0±24.0* 35.8±7.9*

TSCA 101.2±12.2 50.8±10.0 51.2±10.2 206.8±19.8*

Results are expressed as the mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant difference between different treatment groups. TSOA: Time spent in the open arm; TSCA: Time spent in the close arm; 5%TW80: 5% Tween 80 *Indicate significant difference from 5% Tween 80 control. P < 0.05 SNK test.

Table 4: Effect of aridanin on no of entries in the open arm and no of entries in the close arm in the presence of flumazenil (2.0 mg/kg i.p.).

Treatment

Dose (mg/kg, i.p.)

5%TW80 Aridanin FLU FLU+ARI

0.2ml/20g 10.0 2.0 10.0

NEOA 4.8±0.6 6.0±0.5 2.7±0.4 2.4±0.5*

NECA 7.8±1.1 4.4±0.5 4.5±0.5 6.4±0.7*

Results are expressed as the Mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant difference between different treatment groups. NEOA: Number of entries in the open arm; NECA: Number of entries in the closed arm. FLU = Flumazenil; ARI = Aridanin; 5% TW80 = 5% Tween 80 *Indicate significant difference form Aridanin P < 0.05 SNK test

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REFERENCES Adebayo AS, Gbadamosi I.A and Adewunmi CO. Formulation of antimicrobial dried powdered herbs in soap bases. In. Phytomedicine in Malaria and Sexually Transmitted diseases: Chalenges for rte New Millennium. Adewunmi, C.O. and S.K. Adesina (Eds.).

Obafemi Awolowo University, Ile – Ife. 2000; pp. 97.

Aderibigbe AO, Iwalewa EO, Adesina SK, Adebanjo AO and Ukponmwan OE. Neuropharmacological evaluation of Aridanin a Glycoside isolated from Tetrapleura tetraptera fruit Disc. & Inno. 2007a; 19 (3): 177 – 181.

Aderibigbe AO, Iwalewa EO, Adesina SK, Adebanjo AO and Ukponmwan OE. Anticonvulsant, Analgesic and Hypothermic effects of Aridanin isolated from Tetrapleura tetraptera fruit in mice. J Bio Sci. 2007b; 7 (8): 1520-1524.

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Hui KM, Huen MSY, Wang HY, Zheng H, Sigel E, Baur R, Ren H, Li ZW, Wong JT and Xue H. Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi.

Biochem. Pharmacol. 2002; 64: 1415 -424.

Kumar RS, Sundram RS, Sivakumar P, Nethaji R, Senthil V, Murthy NV and Kanagasabi R. CNS activity of the methanol extracts of Careya arborea in experimental animal model. Bang. J. Pharmacol. 2008; 3: 36 – 43.

Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacol. 1987; 92: 180-185.

Litchfield JT, Wilcoxon F. A simplified method of evaluation dose effects experiments. J Pharmacol Exp Therap. 1949; 95: 99

Adesina SK, Reisch J. A Triterpenoid glycoside from Tetrapleura tetraptera fruit. Phytochem. 1985; 24 (12): 30003-3006.

Miller LC, Tainter ML. Estimation of ED50 and its error by means of logarithmic probit graph paper. Proc Soc Exp Bio Med. 1944; 57: 261-269.

Adewunmi CO. Plant molluscicides: Potentual of aridanin from Tetrapleura tetraptera for schistosomiasis control in Nigeria. The Science of the Total Environment. 1991; 103: 21 – 23.

Musa AM, Yaro AH, Usman H, Magaji MG and Habu JM. Phytochemical and Some Neuropharmacological Studies on the Methanolic Leaf Extract of Cissus cornifolia [Vitaceae] in Mice. Int. J. Pharmacol. 2008; 4 (2): 145 -148.

Adewunmi CO, Anderson HC and Busk L. Potential molluscicides, aridan (Tetrapleura tetraptera), neither induces chromosomal alterations in Chinese hamster ovary cells, nor mutation in Salmonella typhimurium.

Toxicol. Envir. Chem. 1991; 30 (1&2): 69 – 74.

DOI: 10.1080/02772249109357642.

Aladesanmi JA. Molluscicidal activity and chemical constituents of Tetrapleura tetraptera.A review. Afr. J. Trad. Comp. Alter. Med. 2007; 4 (1): 23 – 36.

Amoo SO, Ojo AU and Van Staden J. Allelpathic potential of Tetrapleura tetraptera leaf extracts on early seedling of five agricultural crops. South Afr. J. Bot. 2008; 74 (1): 149 – 152.

Cole JC, Rodgers RJ. Ethological comparison of the effects of diazepam and acute/chronic imipramine on the behaviour of mice in the elevated plus-maze.

Pharmaco Biochem and Behav. 1995; 52: 473-.478.

El-Izzi A, Bennie T, Thieulant M and Dural L. Inhibitory effect of Saponins from Tetrapleura tetraptera on the luteinizing hormone release by pituitary cells. Pla. med. 1990; 56: 357- 359.

Essien EU, Izunwane BC, Aremu CY and Eka OU. Significance for human of the nutrients of the dry fruits of Tetrapleura tetraptera. Plant Foods and Human Nutrit. 1994; 45: 47 – 51.

Noamesi BK, Mensah JF, Bogale M, Dagney E and Adoley J..Antiulcerative properties and acute toxicity of some African medicinal plants extract. J. Ethnopharmacol. 1994; 1: 13 – 18.

Nogueira E, Rosa GJM and Vassilieff VS. Involvement of GABAA-benzodiazepine receptor in the anxiolytic effect induced by hexanic fraction of Rubus brasiliensis. J. Ethnopharmacol.1998; 61: 119 – 126.

Nwawu JI., Akah PA. Anticonvulsant activity of the volatile oil from the fruit of Tetrapleura tetraptera. J. Ethnopharmacol.1986; 18: 103 – 107.

Nogueira E, Vassilieff VS. Methodological evaluation of the elevated plus-maze (EPM) test for anxiety in rats. Revista de Ciencias Biomedicas. 1996; 17: 4754.

Odesanmi SO, Lawal RA and Ojokuku SA. Effects of Ethanolic Extract of Tetrapleura tetraptera on Liver Function Profile and Histopathology in Male Dutch White Rabbits. Int. J. Trop. Med. 2009; 4 (4): 136 – 139.

Ojewole JAO. Analgesic and anticonvulsant properties of Tetrapleura tetrapetra (Taub)

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[Fabaceae] fruit aqueous extract in mice. Phytothe res.

2005; 19 (12): 1023 -1029.

Ojewole JAO and Adewunmi CO. Anti-inflammatory and hypoglycaemic effect of Tetrapleura tetrapetra (Taub) [Fabaceae] fruit aqueous extract in rats. J. Ethnopharmacol. 2004; 95 (2-3),:177 -182.

Ojewole JAO, Adesina SK. Mechanism of the hypotensive effect of Scopoletin isolated from the fruit of Tetrapleura tetraptera. Pla Med. 1983; 49: 46. .Okonkon J.E, Udokpoh AE and Antia BS. Antimalaria activity of ethanolic extract of Tetrapleura tetraptera fruit. J. Ethnopharmacol.

2007; 111 (3): 537 â&#x20AC;&#x201C; 540.

Okwu OE. The potential of Ocimum gratissimum, Pergularia extensa and Tetrapleura tetra[ptera as spice and flavouring agents. Nig. Agric. J. 2003; 35: 143 â&#x20AC;&#x201C; 148.

Pellow S, File SE. Anxiolytic and anxiogenic drug

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effects on exploratory activity in an elevated plus maze: a novel test of anxiety in the rats. Pharmaco Biochem and Behav. 1986; 24: 525-529.

Rabbani M, Sajjadi SE and Zarei HR. Anxiolytic effects of Stachys lavandulifolia Vahl on the elevated plus-maze model of anxiety in mice. J. Ethnopharmacol. 2003; 89: 271 -276.

Rall TW. Hypnotics and Sedative Ethanol, In Goodman,LS, Gilman AG (eds). The Pharmacological Basis of Therapeutics: Pergamon, New York. 1990; pp 345-382.

Reddy DS, Kulkani SK. Differential anxiolytic effects of neurosteroids in the mirrored chamber behavior test in mice. Bra Res. 1997; 752 (1-2): 61 -71.

Rodgers RJ, Cao BJ, Dalvi A and Holmes A. Animal models of anxiety: an ethological perspective. Bra J Meds Bio Res. 1997; 30: 289-30.

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ORIGINAL PAPER

Susceptibility of Crab Chitosan against Staphylococcus aureus

G. SUGUMAR

U. RAMESH*

A. SELVAN Received: 2 May 2010 / Accepted: 10 May 2010 /Published online 22 June 2010 Š Bioindica Press 2010

wide range of industrial uses due to its polycationic nature. Applications include use as a flocculent in waste water treatment, a beverage clarifying agent, an immobilizing and permeabilizing matrix for microorganisms and plant cells, a biodegradable polymer for packaging materials, dietary fibre and as artificial skin, surgical sutures, anti fungal ointment etc., (Knorr, 1984; Papineau et al., 1991). The bactericidal and bacteriostatic actions of chitosan have also been reported by several authors (Allan et al., 1984; Papineau et al., 1991; Sudarshan et al., 1992; Wang, 1992; Chen et al., 1998; Tsai and Su, 1999). The antibacterial activity of chitosan has been attributed to the alteration of cell permeability causing leakage of intracellular substances (Tsai and Su, 1999), and the mechanism was investigated against Escherichia coli in most studies. A perusal of literature revealed little information on the antibacterial activity of chitosan derived from crab shell waste and therefore this study was made to evaluate the inhibitory activity of crab chitosan against S. aureus. The present study was made with Staphylococcus aureus, gram positive bacteria which is commonly associated with food product as a result of human handling, so as to use chitosan as a preservative agent against this bacteria found in food products.

INTRODUCTION Chitosan, the deacetylated form of chitin, de- MATERIALS AND METHODS rived from the exoskeleton of shrimp and crab waste Preparation of chitosan from fish processing plants has great potential for a Shell of crab (Portunus pelagicus) collected from a processing plant located at Thoothukudi were washed and dried under sun for a day. The dried shells G. Sugumar were separated as carapace, leg and claw. The shells Department Of Fish Processing Technology, were deproteinized with 5% NaOH at the ratio of shell Fisheries College and Research Institute, to solution of 1:10 (w/v) at 120-130oC for 3he. The Thoothukudi, Tamilnadu, deproteinized shells were filtered and washed with tap India. water until NaOH was washed off completely, then dried overnight in a hot air oven at 55-60oC. The deU. Ramesh, proteinized shells were demineralized by continuously 780, Karpaganagar 3rd Street, K.Pudur, Madurai - 625 007.

ABSTRACT The effect of crab chitosan on the susceptibility of Staphylococcus aureus (ATCC 12598) was studied in samples of chitosan prepared from shells of whole crab, carapace, leg and claw. S. aureus maintained at a cell density of 7.9 x 105 cfu/ml in phosphate buffered saline (0.001M and pH 6.4) were susceptible even upon 1hr exposure to chitosan at a concentration of 0.05% (w/v) and reduced considerably on 6hr exposure. Among the different chitosans tested, chitosan from crab leg shell was more effective than those from claw shell and carapace. The cell density reduced to <10 in 3hr and 6hr respectively with chitosan from leg and claw, while the cell density remained at 2.0 x 101 after 6hr exposure with chitosan from carapace. However, chitosan prepared from whole shell of crab revealed that S. aureus cells were susceptible even at a concentration of 0.01% of chitosan in about 1hr and the effect was more pronounced on exposure for 12hr or more. This study clearly indicated the effectiveness of crab chitosan against S. aureus at low concentration and on short-term exposure.

Keywords: Chitosan, Staphylococcus aureus, Phosphate Buffered Saline

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agitating with 5% HCL at the ratio of 1:10 (w/v, shell to solution) overnight at room temperature.

The shells were filtered and washed with tap water until neutral. Then deacetylation of chitosan was carriedout by hydrolyzing with 47% NaOH at the ratio of 1:20 (w/v, chitin to solvent) at 120-130oC for an hour.

This product was washed with tap water until attaining neutral and dried overnight at 55- 60oC (Madhavan and Nair, 1974). Chitosan thus prepared was used for the study.

Bioresearch Bulletin (2010) 1: 7-9

The cell density reduced by more than 4 log units 3hr and upon 6hr the density was less than 10 cfu/ml.

These clearly indicated that crab chitosan is highly effective in inhibiting S. aureus cells and that the chitosan prepared from shells of legs and claws were slightly more effective than that prepared from carapace against the tested bacteria.

Table 2 shows the inhibitory effect of chitosan (derived from whole shell crab including carapace, claws and swimming legs) at varying concentration against S. aureus. S. aureus was found susceptible even at a concentration of 0.01%. Higher concentration could inhibit the bacteria more drastically. It was observed that t five log reduction in cell density was obtained in less than 6hr at concentration of 0.04 and 0.05%, while the same effect was recorded after 12hr concentration of 0.01 to 0.03%. Similarly a complete inhibition was found after 1hr in the case of 0.04 and 0.05% concentrations while at concentrations of 0.01 to 0.03% the effect was seen at 24hr.

Bacterial culture Stock culture of Staphylococcus aureus (ATCC 12598) was subcultured on trypticase soya agar (TSA, HiMedia) and a colony was inoculated into a pair of tubes containing 10ml of TSB and incubated at 37oC for 24hr. the culture broth was centrifuged at 5000 rpm for 20 minutes to sediment the cells, washed in phosphate buffered saline (0.01 M, pH 7.0). The process was repeated twice and finally resuspended in 10ml PBS to obtain a cell density of ca. 109 cfu/ml and used for susceptibility studies.

The result clearly indicated that the S. aureus is highly susceptible to crab chitosan at ambient temperature. S. aureus was already reported to be susceptible to Susceptibility test Stock solution of chitosan derived from cara- shrimp chitosan (Wang, 1992; Chen et al., 1998; pace, leg (swimming legs) and claw sells were prepared Sugumar et al., 2003). It was also reported that cells of separately in 1% acetic acid. For susceptibility tests, S. aureus were more susceptible to chitosan than other chitosan of varying concentrations namely 0.01, 0.02, human pathogenic bacteria (Wang, 1992; Sugumar et 0.03, 0.04 and 0.05% (w/w) were prepared in PBS and al., 2003) reported effective concentrations of chitothe final pH adjusted to 6.4. Approximately 107 cells san, which may probably be due to experimental matewere inoculated into all experimental flasks containing rials and conditions followed. Allan et al. (1984) rechitosan solution to obtain an initial cell density of 105 ported that complete inhibition of S. aureus was - 106 cfu/ml. the cell counts were estimated at 0hr, 1hr, possible only with 1% solution of chitosan, while 3hr, 6hr, 12hr, 24hr, 48hr and 72hr of incubation at 0.5% was found effective by Wang (1992). However, room temperature 30o Âą 0.2oC to check for inhibitory of 0.05% of shrimp chitosan was found to completely chitosan. Inhibition of S. aureus was initially checked inhibit S. aureus both 5oC and at ambient temperature with chitosan derived from carapace, leg and claw at a on 24hr exposure (Sugumar et al., 2003). In the recent investigation, even a concentration of 0.02% could concentration of 0.05%. inhibit S. aureus in 24hr. Complete inhibition of S. RESULTS AND DISCUSSION aureus with crab chitosan (leg and claw shell derived) at 0.05% less than 6hr testified the effectiveness of Effect of chitosan derived from the crab shell chitosan. The rapidity at which the inhibition was waste was evaluated against Staphylococcus aureus at achieved and the low effective concentration of crab a concentration of 0.05% (w/w) in PBS and the results chitosan render the chitosan as an effective agent in are presented in Table 1. In PBS without chitosan containing S. aureus. Hence, crab chitosan could be (control), the cell density remained almost the same used to inhibit S. aureus in food products involving with mild fluctuations, while the chitosan suspensions human handling where there is possibility of contamiinhibited the inoculated cells considerably. Chitosan nation of S. aureus. derived from carapace showed a gruel reduction from the initial density of 7.9 x 105 cfu/ml after 2.0 x 101 after 6hr of exposure, in the case of chitosan suspension prepared with leg shells, the inhibition was more drastic that the viable cells were only 3.0 x 101 in 3hr and were less than 10 after 6hr incubation. Upon exposure to chitosan suspension prepared from claw shell also the cells of S. aureus reduced drastically.

Bioresearch Bulletin (2010) 1: 7-9

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Table 1. Effect of chitosan derived from different parts of crab on the susceptibility of Staphylococcus aureus at a concentration of 0.05% Cell density (cfu/ml)

Sample

0hr

1hr

2hr

3hr

6hr

Control

7.9 x 105

1.2 x 106

9.1 x 105

1.0 x 106

8.0 x 105

Carapace chitosan

7.9 x 105

1.8 x 104

8.5 x 102

1.1 x 102

2.0 x 101

Leg chitosan

7.9 x 105

3.0 x 104

1.1 x 102

3.0 x 101

-

Claw chitosan

7.9 x 105

2.3 x 104

4.0 x 102

1.0 x 101

-

Table 2. Effect of crab chitosan on S. aureus at different concentrations Cell density (cfu/ml)

Concentration of chitosan

0hr

1hr

2hr

3hr

6hr

12hr

24hr

Control

2.5 x 106

9.5 x 105

3.5 x 105

7.8 x 105

2.8 x 105

2.7 x 105

2.6 x 105

0.01%

2.5 x 106

1.4 x 105

3.0 x 104

1.3 x 103

3.9 x 102

3.0 x 101

2.0 x 101

0.02%

2.5 x 106

1.1 x 105

1.3 x 103

2.0 x 102

3.2 x 102

1.0 x 101

-

0.03%

2.5 x 106

6.8 x 104

8.5 x 103

1.4 x 103

2.8 x 102

2.0 x 101

-

0.04%

2.5 x 106

6.5 x 104

2.6 x 103

1.8 x 103

7.9 x 101

-

-

0.05%

2.5 x 106

2.0 x 104

8.6 x 102

1.1 x 102

1.0 x 101

-

-

REFERENCES Allan, G.G., Altman, L.C., Besinger, R.E., Ghose, D.K., Neogi, A.N. and Neogi, S. (1984). Biomedical effects of chitin and chitosan, in Chitin, Chitosan Related Enzymes, Ed by Zikakis JP, Academic Press, New York, pp 119 - 133.

Chen, C.S., Liau, W.Y. Tsai, G.J., (1998). Antibacterial effects of N-sulfonated and N- sulfobenzol chitosan and application to oyster preservation.

J. Food Prot., 61(9): 1124 - 1128.

Knorr, D. (1984). Use of chitinous polymers in food- A Challenge for Food Research and Development. Food Technol., 38(1): 85 - 97.

Madhavan, P. and Nair, K.G.R. (1974). Utilization of prawn waste isolation of chitin and its conversion to chitosan. Fish Technol., 11(1): 50 - 53.

Papineau, A.M., Hoover, D.G. Knorr, D. and Farkas, D.F. (1992). Antimicrobial effect of water soluble chitosans with high osmotic pressure.

Food Biotechnol., 5(1): 45 - 57.

Sudarshan, N.R., Hoover, D.G. and Knorr, D. (1992).

Antibacterial action of chitosan. Food Biotechnol., 6(3): 257 - 272.

Sugumar, G., Mariappan, S., Kalaimaniarasi, P., Sangeetha, S. and Velayutham, P., (2003). Antibacterial activity of chitosan. Proceeding of the National Symposium on Seafood Safety: Status and Strategies, SOFT (I) CIFT, Kochi.

Tsai, G.J. and Su, W.H. (1999). Antibacterial activity of shrimp chitosan against Escherichia coli. J. Food Prot., 62(3): 239 - 243.

Wang, G.H. (1992). Inhibition and inactivation of five species of food borne pathogens by chitosan J. Food Prot., 55: 916 - 919.

Bioresearch Bulletin (2010) 1: 10-15

10

ORIGINAL PAPER

Assessing the Ecological Status of Prominent Medicinal Herbs in Central Sudan and the challenge of Sustainable Management H.H. EL-KAMALI

Received: 19 March 2010 / Accepted: 2 April 2010 /Published online: 22 June 2010 © Bioindica Press 2010

ABSTRACT Central Sudan has recently attracted scientific and socio -economic attention that stimulated active research interest in various research organizations both local and international. Both short and long term research-plans have been launched in order to establish a database that helps developmental policies to improve human lives and apply rational approaches to deal with changes that are indicated by wide-spread ecological disturbance.

The research work in this study had been carried out in arid/semi-arid regions of Central Sudan, between latitudes 12º 43' – 17º 34' N and longitudes 30º 14' - 36º 15' E. This study was undertaken to explore and document quantitative and qualitative ecological setting of the prominent herbaceous medicinal herbs that are indigenous in North, West and East Central Sudan. The parameters measured were: frequency, density, cover and Importance Value Index, specific association of species as index of species diversity and species evenness (equitability), physical and chemical characteristics of the soil, and climatic elements such as rainfall, relative humidity and temperature. Variations within distribution, productivity of each of the candidate species are seemingly related to availability of favorable environmental conditions in each of the three habitats (regions).

A major objective of active research programs is to outline work-plans that facilitate strategies for management of natural vegetation and conserve biodiversity.

Keywords: medicinal herbs; ecological status, Central Sudan; sustainable management.

INTRODUCTION: The present ecological contribution focuses an exam H.H. EL-Kamali Department of Botany, Faculty of Science and Technology, Omdurman Islamic University, P.O. Box # 382, Omdurman, Sudan.

ining selected areas of Central Sudan especially where a rich and varied medicinal flora is a prominent component of the natural vegetation. The work-plan has been carefully designed to : (1) evaluate the relative ecological status of each studied species for the preservation of this invaluable indigenous medicinal resources.

(2) an attempt to understand the natural system of the study area by the identification of the floristic composition.

(3) to document the traditional heritage knowledge and conserve the generations long uses of medicinal plants.

(4) to stress the need for conservation of the natural habitats, and where possible extend into potential hospitable habitats.

METHODOLOGY The work-plan proposed for the work-field of the present work had been carefully programmed to explore the ecology of the most popular medicinal and aromatic tropical herbs in Central Sudan. Special considerations, based on ample literature survey, research priorities, seasonality and methodology to serve the objectives.

The field studies consisted of ecological examination of the prominent natural medicinal and aromatic herbs in each of the 3 selected regions (9 sub-regions) in Central Sudan during the period 1999-2001. The most prominent 12 herbaceous candidate species have been subjected to detailed ecological determinations. The candidate species are: Cassia senna L.; Cymbopogon schoenanthus (L.) Spreng. ssp. proximus (Hochst. Ex A. Rich.) Maire and Weiller; Haplophyllum tuberculatum (Forssk.) A. Juss. .; Ambrosia maritima L. ; Citrullus colocynthis (L.) Schrad. ; Francoeuria crispa (Forsk.) Cass.; Cassia tora L.; Cassia occidentalis L. ; Datura innoxia Mill. ; Geigeria alata (DC) Benth.

And Hook; Cymbopogon nervatus (Hochst.) Chiov. and Rhynchosia minima (L.) DC. var. memnonia (Del.) Cooke.

Measurements of relative density, relative frequency,

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relative basal cover and Importance Value Index were made in each 400 metre square quadrats randomly placed in each site. Ecological methods adopted to measure the different parameters were those described

by Phillips (1959) and Kershaw (1979). Importance Value Index (IVI) expresses the dominance and ecological success of any species and can be used to arrange a species relative to other plants of its community (Philips, 1959). It varies from place to place and from season to season. The IVI was calculated from each plant with the formula : IVI = Rbc + Rd + Rf Where: Rbc (Relative basal cover) = [Total basal area of the species / total basal area of all species]* 100 Rd (Relative density) = [Number of individual of species / number of all species] * 100 Rf (Relative frequency) = [Number of occurrences of the species / number of occurrences of all species] * 100 Species diversity (H) and Species evenness or equitability (E) were determined according to ShannonWiener function, quoted in Peet (1974). Statistical evaluation was carried out using one-way analysis of variance (ANOVA) and F-ratio was computed to compare the ecological behavior of the selected species in the different sites studied. (Hamburg, 1987).

Environmental setting of the study area: Three sites of contrasting soil types were chosen for this study within the aera of the Central Sudan.

Region I: North Central Sudan (Ed-Damer region) : This region, latitude 17º 34' N, longitude 33º 56' E, had been selected for the ecological survey, being a typical representative of production sectors for several herbs where ecological setting is most favorable. The field visit required a period of 4 weeks. The month of March signifies optimal vegetative, flowering and fruiting period for the studied species. The field visit started by collecting the relevant documented information from records at Hudeiba Research Station. The files information was confereedly discussions with experienced local research person ell; in addition, it was felt that compilation of answers of pre-set questionnaire addressed to collectors, traders and local knowledgeable on indigenous herbal medicine would add important socio-economic dimension to the knowledge generated.

Sub-region (1) : Hudeiba area This sub-region approximately lies within a radius of about 7 Km distance from the centre of Ed-Damer town. In Hudieba dense populations of naturally occurring medicinal plants are successful colonizers of the idle land along permanent canal or in fallow lands that usually fills during high floods. The silt-rich banks that receive varying deposits of Nile silt, are fairly extensive areas commercially cultivated by Lawsonia inermis "Henna". In this location, Ambrosia maritima "Damsisa", is a wild natural component in

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the fertile soils which carry cultivated Lawsonia stands.

Sub-region (2) : Gubarab area This sub-region is beyond an approximately 12 Km radius from the centre of Ed-Damer town. In this location, the natural plant cover is dominated by Haplophyllum tuberculatum "Haza" was located along permanent irrigation canal. This sub-region includes a number of private farms cultivated mainly with Lawsonia inermis. It is estimated that about 97% of Henna production of Ed-Damer region is obtained from this sub-region.

Sub-region (3) : Hasaya area The geographical location of this sub-region is at approximately 15 Km distance south of Ed-Damer town.

This sub-region is a sector of the natural cover that characterizes second terrace soils. This location is dominated by Cassia senna, Citrullus colocynthis and Fagonia cretica.

Sub-region (4) : Ed-Deheira valley This most distal sub-region lies at approximately 38 Km distance south east of Ed-Damer town. By virtue of distance from the effect of the Nile, it belongs to the high terrace soils. Its soils are semi-sterile arid/ semi-arid sandy clay soils mostly characterized by high soluble salts with a shallow calcium carbonate layer. Prolonged drought and sporadic insufficient rainfall in sub-region only permits scanty water catchment pockets supporting perennial grasses such as Cymbopogon schoenanthus ssp. proximus and the wirey tuff grass Panicum turgidum.

Region II: West Central Sudan The region lies between latitude 12º 43' – 13º 42' N and longitude 30º 14' – 31º 55' E. The distribution pattern in this region is a mosaic of stands carrying diverse assemblages of the local herbs including medicinal plants. The region is characterized by a dry, hot climate, typically tropicale continental with a relatively short rainy season. The annual rainfall totals within the region varies from about 170mm in the North to about 350 mm in the south. The soils are mostly stabilized sand dunes "Goz" consisting of yellowish red sandy loam and loam sand soils. The "Goz" land is susceptible to leaching, with low fertility and of easy nature for cultivation, is of poor organic matter and low moisture. The northern part of the region has extremely limited agricultural potential because of the low means of annual rainfall and sub-minimum soil fertility. This perhaps accounts for the fact that this part is characterized by scanty vegetation which is found only in the sporadic rain water catchment areas, drainage lines and to oasis with reasonable subsurface water e.g Bara and Kheiran. South part of this region, has the vegetation is a varying mixture of grasses and herbs with scattered bushes.

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Successional changes since the mid 1970s highlighted regional degradational changes that have been related to progressive decline of rainfall means below the normal average. However, the 1988 rainfall has been exceptional, giving an undoubted recovery. The subsequent years were of below average means. The economy of west central Sudan region is predominantly agropastorial. Crop production is mainly traditional.

Cereal and cash crops cultivated in fertile soil/ stabilized sands during good rainy years include millet as a dominant crop, Sorghum and Sesame. In areas around Umm Ruwaba, Er-Rahad and Tendelti sesame rates second to millet in terms of area. Acacia senegal (Hashab) plantation is another form of land use. Because of its economic benefits for gum Arabic collection, it is the only tree that is planted and protected by the farmers. Certain indigenous plant species in this region have prominent potential commercial attributes.

Cassia senna "Sanamakka", Grewia tenax "Godaim" Adansonia digitata "Tabaldi", Balanites aegyptiaca "Lalob" and Capparis decidua "Tundub" have local considerable consumption as fruits.

Sub-region (5): Er-Rahad area Er-Rahad is located around latitude 12 43 N, longitude 30 39 E. The terrain is seemingly very appropriate the development of rich assorted natural vegetation including a high composition of medicinal herbs.

Sub-region (6): Umm Ruwaba area This sub-region extends between altitude 12º 55' N and longitude 31º 13' E. Sub-region (7): Bara area This sub-region occupies sector around latitude 13º 42' N and longitude 30º 22' E. The area receive seasonal rainfall amounting to about to 250 mm annually.

The general landscape can generally be classified into : Acacia steppe , Goz and sand dunes and loamy depressions.

Region III: East Central Sudan This region located between latitude 13º 00' and 14º 30' N and longitude 35º 00' and 36º 15' E. This study area has been chosen to represent the ecological setting of a typical clay soil carrying dry savanna vegetation that includes production sectors for some medicinal herbs. The clay soil is contrast with the sands of Region II. It has an average altitude of approximately 600 meters above sea level. The major studied medicinal herbs include: Cymbopogon nervatus "Nal", Rhynchosia minima var. memninia "Irg el Dam" and Cymbopogon schoenanthus ssp. proximus "Mahareib". The average annual rainfall ranges from 175 mm in North to 570 mm in the centre and 650 mm in the south. The landscape of this region is distinctly dominated by moderately dry savanna vegetation which is generally characterized by medium – sized trees of Acacia senegal "Hashb" and A. seyal "Taleh". The vegetation changes gradually northwards from moderately dry savanna type, to poor dry savanna to distinctly semi- desert type.

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The clay plain of the Gedaref area is the one of the Sudanese major rain-fed agricultural sub-sectors that support the economy of the Sudan. The area potential lies mainly on growing of Sorghum, sesame, millet and the recently introduced canal – irrigated Sunflower and cotton.

Sub-region (8): Abu-Kashma valley This sub-region is at approximately 24 Km distance west of Gedaref town. It is generally a semi-arid/dry savanna grass-land on clay plain. The lands are dominated by Rhynchosia minima var. memnonia , Citrullus colocynthis and Calotropis procera.

Sub-region (9): Rufaa area This sub-region is well represented in Gedaref town. It lies to north-east and is at approximately 8 Km distance from the centre of Gedaref town. The land is a slightly sloping plain with a scattered "Jebels" and some seasonal water courses (Khors). The lands are dominated by Cymbopogon nervatus , Solanum nigrum , Leonitis nepetaefolia and Xanthium strumarium.

OBSERVATIONS, RESULTS AND DISCUSSIONS: Reference must be made here to the theme of the research, which addresses a comprehensive field survey that covers the quantitative and qualitative ecological setting of the prominent herbaceous medicinal herbs that are indigenous in North, West and East Central Sudan. Monitoring the prevalent environmental characteristics in each region have been according to careful selection of study sites that represent locations that accommodate vigorous stands of medicinal herbs, locations that are indicative of poor vitality and , locations that occupy an intermediate position.

Region I (Ed-Damer) which is located in the drier northern region is characterized by low mean of annual rainfall (39.4 mm); the rainy season is very shorter. However, seasonal Nile flood contributes an ample seasonal water supply that permit rich seasonal assemblage of a variety of medicinal herbs along the Nile banks and drainage lines.

With reference to the Plant species list in Table 1, it is clear that the three studied sub-regions in EdDamer region: Hudeiba, Hasaya and Ed-Deheira differ

markedly in their floristic composition. The results show that the soil of Hudeiba sub-region permits the development of the highest number of plant species mainly due to seasonal Nile flood which contributes an ample seasonal water supply and the soil is more fertile through occasional silt deposition.Vegetation data obtained from Hudieba and Ed-Deheira sub-regions showed no significant difference between the two locations. On the other hands the results of analysis of variance of data obtained from Hasaya sub-region showed a significant difference. On the other hands the results of analysis of variance of data obtained from Hasaya sub-region showed a significant difference.

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Such results suggest the species predominating Hudeiba and Ed-Deheira sub-regions enjoy better conditions of the soil, together with seasonally sufficient moisture content. This is expected in view of its relative nearness of Hudeiba to the Nile, where the soil is more fertile and clear heterogeneity of the Ed-Deheira landscape : soil undulations that dictate the soilmoisture balance and the nature of alluvial deposit that is transported by narrow runnels from the hilly terrain.

The findings related to medicinal herbs suggest that Haplophyllum tuberculatum , Ambrosia maritima and Francoeuria crispa are represented by a good number of individuals in Gubarab and Hudeiba sub-regions (Ed -Damer region) but its distributions are very localized.

These sub-regions which are not far from the river bank provide more evidence for the strong relationship between species abundance and relatively higher moist conditions.

The distribution of Cymbopogon schoenanthus ssp. proximus in the different habitat of Sudan has been sporadically surveyed ( Obeid and Mohmoud, 1971 and Modawi. 1975). The findings of the present study are in agreement with the above literature regarding the wide distribution of Camel Hay in Central Sudan. This species still maintains its former habitats in upstream parts (margins) of water runnels which retain relatively shallow soil and relatively less water supplies than the flat downstream water catchment areas. Water resources in margins of runnels may presumably be more effective than the actual annual means of rainfall in the region.

The distribution of Cassia senna has been recorded in a large number of habitats throughout Central Sudan (Menniger, 1952; Halwagy, 1962; Obeid and Mohmoud, 1971; Tag EL-Seed and Ismail, 1982; Ismail and Babiker, 1986 and Yahia, 1992).

In West Central Sudan (Region II), various subregions within the study area: Er-Rahad, Umm Ruwaba and Bara exhibit a wide array of vegetation cover. Many reasons may be ecologically responsible for these variations. The terrain in this region is conspicuously diverse. Although the soil is predominantly sandy, yet it includes local variations of silty depressions, hard non-cracking clays and sandy loam. This, in addition to South â&#x20AC;&#x201C;North rainfall gradient.

Geigeria alata has a distinctive geographical range restricted mainly to the Goz (stabilized sand dunes) area in West Central Sudan. Its distribution is mainly determined by amount of total annual rainfall, sandy soil and mosaic of terrain. Within its ecological range, G. alata rarely develop in habitats with heavy soils.

In East Central Sudan ( Gedaref) region, natural vegetation tend to carry a fairly limited numbers of drought -resistant forms and species rejected by migratory herds of camels, sheep and goats. Former species of trees, shrubs and perennial herbs are scattered in sporadic valley habitats among steep rocky areas and on flat hill tops around the region e.g. FAO. It must be

Bioresearch Bulletin (2010) 1: 10-15

noted that Rufaa sub-region, has a much lower specific diversity than Abu-Kashma valley sub-region.

Despite prolonged aridity and relatively poor soil the environmental conditions in Rufaa sub-region are presumably favorable for the development of droughtresistant grass Cymbopogon nervatus. This is particularly reflected in high means of to relative frequency than all other associates in their habitat.

Data on medicinal claims recorded during the survey work were checked against the available literature (Farnthworth, 1995) and it was found that usage of Geigeria alata, Cymbopogon nervatus and Rhynchosia minima var. memnonia folk drugs in the candidate listing are either not recorded or only superficially known. However, for others viz. Cassia senna, Cymbopogon schoenanthus ssp. proximus, Haplophyllum tuberculatum, Datura innoxia, Citrullus colocynthis and Ambrosia maritima, the usages were found similar with those published in the literature.

As would be expected, collection of crude medicinal herbs in the wild cannot guarantee a high and constant quality over a longer period. Very heterogeneous amounts of raw material was found on the market.

Furthermore, trained collectors are rare and, for this reason, the identify of the plant material cannot always be guaranteed either.

The collection, trade and supply of numerous medicinal herbs in Sudan is not restricted by authorized/ legislative regulations concerning endangered species and conservation of plant diversity. It is therefore vital that systematic and rationally managed cultivation of the most important medicinal herbs should be started simultaneously in rural areas in order to conserve the biodiversity and protect endangered species and also opening up of additional jobs and better income opportunities for the poor people. There are many constrains for Sudanese medicinal herbs to be competitive in the world market. Some of the problems associated with that are: (1) poor raw materials due to indiscriminate harvesting and poor post-harvest treatment and storage, (2) lack of financial resources, loans and credit facilities, and (3) difficulties in marketing (lack of access to market information and contacts).

The formulation of an appropriate national strategy should constitute an important, initial step towards the utilization of medicinal herbs. For this purpose, the governmental research organizations should establish national committees comprising experts from different related fields. These committees could help to promote the developmental programs to formulate policies, strategies and put forwards plans for its sustainability. As the natural habitats for wild plants may suffer and indigenous species may be threatened, endangered or even disappear completely, it may become critical to develop alternative sources of important natural products. Tissuecultured cells of higher plants typically accumulate amounts of secondary

Bioresearch Bulletin (2010) 1: 10-15 metabolites only when subjected to specific conditions.

The leading herbs Cymbopogon schoenanthus ssp. proximus and C. nervatus plants are seasonal and the quality of its essential oils varies according to fluctuations in ecological conditions. The production of oils by plant cell and tissue cultures could offer the possibility of better quality and availability independent of environmental changes.

REFERENCES: Farnthworth NR., ed. NAPRALERT database. Chicago, University of Illinois at Chicago. Scientific and Technical Network of Chemical Abstracts Services.

Halwagy R. (1962). The Incidence of the Biotic Factors in Northern Sudan. Oikos 13:97-117.

Hamburg M. (1987). Statistical Analysis for Decision Making. 4th Edition. Harcourt Brace Jovanovids, INC, Florida, p. 342.

Ismail A.M.A. and Babiker A.A.A. (1986). Structural Pattern of Cassia acutifolia collected in the Gezira, Sudan. Fitoterapia 57(4):263-266.

Kershaw K.A. (1979).Quantitative and Dynamic Plant Ecology. 2nd Edition. Eduard Arnold (Publishers) Ltd. London.

14 Menniger E.A. (1952). The Golden Shower and its Handsome Relatives. Garden Journal 2: 36-39.

Modawi B.M. (1975). Examination of the Terpenoids of Cymbopogon species. Ph.D. Thesis. Khartoum University, Sudan.

Obeid M. and Mahmoud A. (1971). Ecological Studies in the Vegetation of the Sudan II. The Ecological Relationships of the Vegetation of Khartoum Province. Vegetatio 23: 3-4, 177-198.

Peet R.K. (1974). The Measurement of Species Diversity. Ann. Rev. Ecol. Syst. 5: 285-307.

Phillips E.A. (1959). In: Methods of Vegetation Study.

Henry Holt and Co. INC., New York, p.70-74.

Tag El-Seed M. and Ismail A.M.A. (1982). Some Ecological Factors Controlling the Distribution of the Closely Related Species Cassia senna L. and Cassia italic Mill., around Khartoum are in the Sudan. J. Univ.

Kuwait (Si.) (9): 148.

Yahia D.A. (1992). The Distribution of Calotropis procera, Cassia senna and Aerva javonica in Relation to Mineral Nutrient Factors in the Central Sudan. M.Sc. Thesis, Khartoum University, Sudan.

Tables Contd.

15

Bioresearch Bulletin (2010) 1: 10-15

Table (1): Percentage frequency, density, cover and Importance Value Index.

Region / Sub-region Plant species Region I Sub-region : Hudeiba area Francoeuria crispa Heliotropium aegyptiacum Sub-region : Hasaya area Fagonia cretica Cassia senna Citrullus colocynths Tribulus terrestris Panicum turgidum Sub-region : Ed-Deheira valley Panicum turgidum Cymbopogon schoenanthus proximus Indigofera spinosa Region II Sub-region : Er-Rahad area Sporobolus pyramidatus Geigeria alata Alysicarpus glumaceus Cenchrus sp.

Aerva javanica Gueira Senegalese's Solanum dubium Citrullus lanatus Aristida mutabilis Chloris brevista Cucumis dispaceus Stylochiton borumensis Echinochloa colona Euphorbia aegyptiaca Dactyloctenium aegyptium Bergia suffruticosa Ethulia conyzoides Sub-region : Umm Ruwaba area Indigofera oblongifolia Datura innoxia Abutilon figarianum Cassia occidentalis Aristida sp.

Calotropis procera Cassia tora Sub-region : Bara area Cassia senna Jatropha glauca Zaleya pentandra Blepharis ciliaris Cenchrus sp.

Region III Sub-region: Ab-Kashma valley Rhynchosia minima var. memnonia Setaria incrassate

ssp.

Relative frequency

Relative density

Relative basal cover

Importance Value Index

52.17 47.83

49.56 43.36

44.5 26.5

146.23 117.69

28.0 28.0 36.0 4.0 4.0

22.02 36.86 26.61 4.59 11.93

10.0 32.0 40.0 1.0 1.0

60.02 96.86 102.61 9.59 16.93

50.0 45.45

39.46 5.83

32.25 23.0

121.7 74.28

4.55

34.98

15.0

54.03

6.3 9.5 6.3 7.9 6.3 12.7 6.3 6.3 7.9 6.3 6.3 4.8 4.8 4.8 1.6 1.6 1.6

21.4 15.3 11.5 6.9 6.9 6.1 5.3 5.3 3.8 3.1 3.1 3.1 2.3 2.3 0.8 0.8 0.8

26.0 14.0 11.0 4.0 3.0 10.0 2.0 10.0 2.0 6.0 3.0 2.0 3.0 1.0 1.0 1.0 1.0

53.7 38.8 28.8 18.8 16.2 28.8 13.6 21.6 13.7 15.4 12.4 9.9 10.1 8.1 3.4 3.4 3.4

18.9 35.1 16.2 18.9 2.7 5.4 2.7

55.2 20.9 9.7 9.0 3.0 1.5 0.7

26.0 29.0 16.0 26.0 1.0 1.0 1.0

100.1 85.0 41.9 53.9 6.7 7.9 4.4

46.2 30.8 15.4 3.8 3.8

46.4 42.9 8.9 1.8 1.8

55.0 30.0 10.0 1.0 1.0

147.6 103.7 34.3 6.6 6.6

71.9 21.8

73.3 20.9

69.0 16.5

214 59.2

Bioresearch Bulletin (2010) 1: 16-18

16

ORIGINAL PAPER

Impact of industrial effluents and sewage on river Thamirabarani and it’s concerns P. PETER BASKARAN

A. JOHN DE BRITTO*

Received: 10 April 2010 / Accepted: 20 April 2010 /Published online 22 June 2010 © Bioindica Press 2010

ABSTRACT The present study deals with the Impact of industrial effluents and sewage on river Thamirabarani.

Water samples were collected from 5 different river and canal stations such as Melapalayam canal, Palayam canal, Kottur canal, Kandiyapperi canal, Vannarpet and different chemical parameters such as colour, pH, dissolved solids and microbial study of the samples were analysed. The tests showed that the samples of Melapalayam is more polluted in all parameters. Fluoride was found normal in all sites. TDS and Turbidity in Melapalayam canal, Palayam canal and kandiyaperi canal found more. Sites I, II and III are highly bacteriologically contaminated. It was concluded that the samples of river water were polluted mostly and associated with industrial effluent and sewage discharge.

Key words: Thamirabarani, Water quality, Polluted water.

INTRODUCTION Water is essential for the survival of the world.

Good and safe drinking water is becoming a rare commodity. Rivers play a significant role because they not only serve the purpose of water supply for domestic, industrial, agricultural and power generation but also utilized for the disposal of sewage and industrial waste and therefore put under tremendous pressure due to human activities.

In the last few decades, pressure has been increasing and greater emphasis on the deterioration of the quality of Indian Rivers. Most of the rivers have been unmindfully used for the disposal of domestic and industrial effluents far beyond their assimilative A. John De Britto Plant Molecular Biology research Unit, Department of Plant Biology and Biotechnology, St. Xavier’s College (Autonomous), Palayamkottai, Tamilnadu-627 002, India., * Email: peterbaskaran@gmail.com

capacities and have been rendered grossly polluted (Agarwal & Sharma, 1982).

According to an estimate, about 80% of the total population in India is deprived of pure and safe drinking water. A recent study revealed that there were 1,53,000 villages in India, which had infected water supply. 90% of total drinking water is severely polluted. Ganga is the most polluted river in the world.

Other Indian rivers include Damodhar, Hooghly, Kulu which have almost the same story to reveal.

In South India, river ―Thamirabarani‖ serves as the principle source of water for drinking and agriculture purposes, with increasing number of industries and pollutions especially in the lower reaches, concern over the water quality of the river began to be strongly felt.

Thamirabarani, one of the perennial rivers in Tamil Nadu, originates from Pothigai hills on the Eastern slopes of the Western Ghats and drains its water into the Bay of Bengal at Punnakayal of Gulf of Mannar.

The total area is 5969 sq.km.

With the arrival of industries, workshops, hospitals and hotels on the river banks and canals the water is getting contaminated with the effluent discharges from these. Industrial waste water comprises 8-16% (Chaudhari 1982). Tamirabarani on its banks has a number of industrial units including pulp and paper, textile, state transport corporation workshops, photographic industries and other small scale industries.

The waste liquids from textile mills comprises mainly of dye stuff, sulphates, sulphide, copper, zinc, lead, phenolics and wastes from the manufacture of pulp and paper contains sulphides, chlorides, lingo cellulosic wastes, mercaptans, mercury etc. canals also highly polluted by sewage disposal from theater, hotels, houses and municipal sewage. The pollutants that are concerned to the ecosystems are those that do not degrade and those that resist to bio-degradation and are found to enter the aquatic and human systems.

(Murugesan, 1988)

17

Biores earch Bulletin (2010) 1: 16-18

MATERIALS AND METHODS The water quality parameters decide the potability of water water samples are taken from five different sites in the month November 2008. 2 litre of water sample was collected in polythene bottles and carried to the laboratory, where physio-chemical parameters were analysed as per standard methods (Manivasakam et al 1996). The parameters selected for analysis were pH, TDS, hardness, chloride, nitrate, phosphate, Co2 and DO. Bacteriological study faecal coliform study was analysed. (Papen et al 1998) RESULTS AND DISCUSSION It is evident that the water quality is altered due to the entry of sewage and other domestic wastes into the aquatic system. The pH value is measured as 8.05, 7.57, 7.35, 7.89 and 7.93 respectively. The pH value of sites I and II are more. There were great differences in the values of TDS. It was found as 422 in site I and 229 in site II and 345 in site III and 392 in site IV and 245 in site V. More amount of TDS found in site I and III. Turbidity showed great differences. In site I, it was 10 and in II 7 and in IV 9 and in VI 10 and in V 10. The turbidity was recorded maximum value in almost all sites except II. Total hardness was recorded as 178, 164, 162, 140, 120 respectively. It sites I, II, III it was recorded maximum against 200 control.

Presence of Chloride was a great significance. It was

212, 201, 112, 94 and 75 respectively. In Melapalayam and Palayam kalvai the chloride showed variations. In all five sites the presence of Fluoride was in control found below minimum level.

Bacteriological faecal coliform study showed that 3200, 860, 1400, 544 and 460 found per 100 ml of sample respectively. According to this value water samples taken from Melapalayam and Kottur canal are highly bacteriologically contaminated, which were not potable.

Control Measures The effluents from the existing units should be monitored regularly and allowed to discharge into the river only after satisfactory clean up treatment. Underground drainage system is built within municipal limits and sewage treatment plants of adequate capacity installed at chosen locations. Only treated water with permitted quality should be allowed into the river somewhere downstream after municipal limits. Domestic sewage is also treated and the waste water will be used as a bio-fertilizer which will be alternative source of income, treated waste water can be used for irrigation. Microbial disinfectants should be employed in the water resources.

Table 1

Physio-chemical and Bacteriological variable S. No

Place PH

TDS

Turbidity

TH

Cl

F

Coliform study

1

Melapalayam

8.05

422

10

178

212

0.4

3200

2

Palayam canal

7.57

229

7

164

201

0.2

860

3.

Kottur canal

7.35

345

9

162

112

0.2

1400

4.

Kandiayapperi canal

7.89

392

10

140

94

0.15

212

5.

Vannarpet

7.93

245

10

120

75

0.2

110

Bioresearch Bulletin (2010) 1: 16-18

18

Physio-chemical and Bacteriological variable 3500 3000 2500 2000 1500 1000 500 0

PH

Turbidity

Melapalayam canal

Palayam canal

Kandiyapperi canal

Vannarpet

REFERENCES Chaudhari, N. 1982. Water and air quality control. The Indian context board for the prefecture and control of water pollution, New Delhi.

Chopra, A.K and Patrick, Nirmal, J.C., 1991. Effect of domestic sewage on water quality of river Ganga at Triveni Ghat, Rishikesh, G.K.V.V. Manivasagam, N (1996) Physio-chemical Examination of water sewage and Industrial effluents. Pragati Prakashan, Meerut, India.

Murugesan, A.G and N. Sukumaran, 1999. Impact of urbanization and Industrialization on river Tamirabarani, the life line of Tirunelveli and Thoothukudi districts, Proc. Sem. Environmental Problems in Tirunelveli 06.

Cl

Coliform study Kottur canal

Murugesan, A.G. 2001. Environmental status of the perennial river Thamirabarani with special reference to domestic and industrial pollution.

Proc. Workshop and enhancing Public Awareness on ecological and Environmental Status of River Basin.

Papen, H and Von Berg, R 1998. A Most Probable Number method (MPN) for the estimation of cell number of heterotrophic nitrifying bacteria in soil Plant and Soil 199: 123-130.

Bioresearch Bulletin (2010) 1: 19-23

19

ORIGINAL PAPER

Effect of nutrients on in vitro culture of Morus alba L. (White mulberry) ANKIT PRADHAN, A. S. VISHWANATHAN* R. BASAVARAJU. Received: 3 April 2010 / Accepted: 12 April 2010 /Published online: 22 June 2010 © Bioindica Press 2010

ABSTRACT Morus alba L. is an economically important plant used extensively in sericulture, also possessesing medicinal properties. In vitro culture provides an effective means to overcome the common problems encountered in conventional propagation by stem cuttings and seeds. Effect of phytohormones, growth adjuvants, sugars and essential minerals on the in vitro response of M. alba explants has been described in the study. Phytohormones and growth adjuvants resulted in explicit response in terms of organogenesis. Sucrose and glucose containing culture media elicited best response among sugars in nodal explants. Typical symptoms were exhibited by the growing shootlets when the medium was deprived of essential minerals such as nitrogen and sulphur.

Keywords phytohormones; sugars; minerals; growth adjuvants Abbreviations MS: Murashige and Skoog; 2,4-D: 2, 4- Dichlorophenoxy acetic acid; NAA: α - Naphthoxy Acetic Acid; Indole -3- Butyric Acid; Kn: Kinetin; BAP: 6Benzyl Amino Purine INTRODUCTION The mulberry plant (Morus alba L.) is chiefly exploited by the sericulture industry for its foliage, used as feed for silk worm (Bombyx mori L.), and for feeding ruminants (Arabshahi-Delouee and Urooj, 2007).

Depending on the location where it is grown, the plant is also valued for its fruit (consumed fresh, in juice or as a preserve) and aerial parts (stem and leaves are used as vegetable) (Venkatesh Kumar and Chauhan, 2008). M. alba also contains a considerable amount

A. S. Vishwanathan Department of Biosciences, Sri Sathya Sai University, Prasanthi Nilayam, Andhra Pradesh – 515 134 (India)

of dietary supplements such as proteins, carbohydrates, fats, fibers, essential minerals (Srivastava et al, 2006), ascorbic acid and β-carotene (Ercisli and Orhan, 2007).

The plant is known to possess anti-hyperglycemic and anti-hyperpigmentation activity (Lee et al, 2002). The root bark of the plant possesses astringent and anthelmintic properties, hypotensive activity, antitumour activity and anti-microbial activity and is used in treatment of cough and cold (Venkatesh Kumar and Chauhan, 2008). Traditional reports of the application of M. alba in treatment of atherosclerosis and diabetes were experimentally validated by Enkhmaa et al.

(2005) and Shibata et al. (2007).

Stem cuttings and seeds have been used conventionally for propagation of mulberry plants which have assumed tremendous economic importance over the years. Genotype, environmental factors and physiological state of the cuttings play a significant role in determination of success of rooting (Lu, 2002). Propagation by seeds is undesirable owing to a long juvenile period (Vijaya Chitra and Padmaja, 2005) coupled with cross pollination which in turn leads to a high level of heterozygosity (Anis et al, 2003). In vitro culture or micropropagation is a sought-after alternative, providing an effective means of rapid propagation of uniform genotypes of the plant. Application of modern techniques for development of new highyielding varieties and cultivars depends on the availability of an efficient in vitro regeneration system (Agarwal et al, 2004). This communication provides an account of studies on the in vitro response of stembit and nodal explants of M. alba to select phytohormones, growth adjuvants, minerals and sugars. This study was designed with the aim of identifying specific growth supplements which would have a positive influence on growth of in vitro plantlets. Another objective of the study was to evaluate the occurrence of the known in vivo physiological

20

effects of some of the selected supplements on the plantlets in vitro.

MATERIALS AND METHODS Local varieties of M. alba were procured from local farms in and around Puttaparthi and grown in the experimental garden of the Sri Sathya Sai University, Prasanthi Nilayam campus. These constituted the mother plants for the explants used during the course of the in vitro study. The stem-bit and nodal explants were washed under running tap water for 30 minutes, surface sterilized using 0.1% (w/v) aqueous solution of Mercuric Chloride and rinsed five to six times with sterile double distilled water.

The surface-sterilized explants were inoculated on Murashige and Skoog medium (Murashige and Skoog, 1962) under a laminar flow hood (KLENZAIDS, India). The cultures were maintained at 24 ¹ 2°C under 16/8 h photoperiod at a light intensity of around 3000 lux.

MS medium fortified with auxins (2,4-D, NAA and IBA), cytokinins (Kn, BAP) and growth adjuvants (coconut milk and casein hydrolysate) as individual treatments was used in the study. MS medium without its constituent nitrogen and sulphate mineral stock solutions was used to study the effects on explant growth and development. Each of the phytohormones was used at a concentration of 2mg L-1 of MS medium and the growth adjuvants at 15% w/v or v/v. Aliquots of MS medium containing different sugars such as glucose, sucrose, fructose, mannose, maltose, lactose and galactose at a concentration of 3% (w/v) was used to study the effect of different sugars. Basal MS medium without addition of any supplements represented the control.

RESULTS AND DISCUSSION Studies on the in vitro response of explants of M. alba have been undertaken to understand the effects of different nutrients with respect to organogenesis. This study focuses on the effect of phytohormones, growth adjuvants, sugars and minerals as observed in mulberry explants in vitro.

Nodal explants showed a better response as compared to stem- bits in the study of the effect of phytohormones on in vitro culture of M. alba. Callus initiation and proliferation occurred in MS medium supplemented with 2,4-D. The callus was semi-transparent and off-white in the beginning gradually turning brownish in colour. Both NAA and IBA were effective in initiating a response in terms of shoot formation and growth; the former resulting in development of unbranched roots and the latter, profusely branched

Bi oresearch Bulletin (2010) 1: 19-23

roots. The cytokinins Kn and BAP promoted shoot growth. Kn resulted in shoots with considerably longer internodal distance compared to any other treatment and BAP treatment led to formation of leaf clusters. Treatment with casein hydrolysate in MS medium inoculated with nodal explants gave good results in shoot formation while coconut milk was more effective in callogenesis. Coconut milk showed similar response using stem-bit explants.

Micropropagation studies on mulberry, most commonly, have used MS medium culture of nodal cuttings and axillary and apical buds (Thomas, 2002).

Islam et al. (1993) observed that explants in medium containing 2,4-D showed best callogenesis and callus proliferation. Pattnaik and Chand (1997) and Bhau and Wakhlu (2003) obtained multiple shoots in BAP fortified medium which enhanced bud-break frequency with the addition of GA3. The combination of NAA and BAP induced shoot proliferation in mulberry and NAA, in particular, was found to be essential for root induction by Ponchia and Gardiman (1991). The increased inter-nodal distance in shootlets obtained in Kn containing medium, in this study, has not been reported hitherto in the available literature.

Nodal explants cultured in MS medium containing glucose and sucrose responded by producing best results in shoot length, leaf growth and the development of a highly branched, extensive root system. This was followed by medium containing fructose. Quite notably, medium containing galactose did not show any response. The results of the response of nodal explants to different sugars using MS medium are summarized in Table 1.

Sucrose is the principal product of carbon fixation in plants (Avigad and Dey, 1997). Other sugars have to be converted to glucose, the starting point of carbohydrate metabolism, or its derivatives to enter the glycolysis cycle (Brownleader et al, 1997). The varied response to the different sugars reflects on the ability of the plant in converting these sugars into suitable glycolysis intermediates. Enomoto (1987) reported that sucrose containing media elicit the best response in in vitro mulberry culture. However Ohyama (1970), Ohyama and Oka (1976) and Vijaya Chitra and Padmaja (2002) report fructose as being more favourable.

The present study revealed that sucrose is the best for morphogenic response in shoot length, leaf growth and size. These characteristics are highly advantageous for the sericulture industry.

Alteration in the mineral constitution of MS medium produced pronounced physiological effects that reflected in the morphology of the developing explants.

Bioresearch Bulletin (2010) 1: 19-23

21

Table 2 outlines the different morphological changes that occurred in the response of stem-bit explants in the absence of nitrates and sulphates in MS medium.

Absence of nitrogen compounds in MS medium resulted in very poor morphogenic response of explants.

Nitrogen supplied as ammonium (NH4+) and nitrate (NO3-) ions promotes cation anion balance within the plant (Bloom, 1994). Under severe nitrogen deficiency, leaves turn yellow and eventually fall off (Taiz and Zeiger, 2002). Sulphur is a constituent of several coenzymes and vitamins essential for plant metabolism.

Its deficiency leads to downward hooking of leaves along with stunted growth of shoots (Taiz and Zeiger, 2002). Deficiency of sulphates also decreases chlorophyll content and rate of photosynthesis (Lopez et al, 1996).

CONCLUSION The mulberry plant is reported to be the one of best

plants for silkworm rearing (Thomas, 2002). The qualitative and quantitative improvements in this plant are very essential from the aspect of enhancement of the sericulture industry. Micropropagation is an established alternative to the conventional propagation methods for obtaining improved mulberry cultivars.

The hormone treatments discussed herein provide means to obtain mulberry plants with desirable traits.

Biochemical tests could be conducted on in vitro generated plants to ascertain changes in phytochemical constituents which may have a bearing on the feeding habits of silkworms reared on mulberry. Similar studies could be designed for obtaining stress resistant varieties of mulberry which would be of benefit to the silk industry.

ACKNOWLEDGEMENTS The authors express their gratitude to the chancellor of Sri Sathya Sai University, Bhagawan Sri Sathya Sai Baba, for His constant guidance and support.

TABLES Table 1: Comparative effect of different sugars on morphogenic response of M. alba explants Sugar used

Average shoot

Average leaf

Root system

length (cm)

width (cm)

Glucose

5.7 ± 0.3

3.0 ± 0.1

Thick, long extensively branched

Fructose

4.6 ± 0.3

2.7 ± 0.2

Thin, short, few rootlets

Mannose

1.8 ± 0.1

1.8 ± 0.1

Thin, short, no rootlets

Sucrose

6.0 ± 0.4

3.9 ± 0.3

Thick, long extensively branched

Maltose

2.9 ± 0.1

1.8 ± 0.2

No response

Lactose

3.6 ± 0.1

2.6 ± 0.3

Thin, short, few rootlets

Monosaccharides

Disaccharides

Table 2: Effect of minerals on explants of M. alba Alteration in MS medium

Effect on growing explants

composition MS without Nitrogen

Shoots got dried up; leaves were shriveled and gradually

(NH4NO3 and KNO3)

turned yellow; rooting was negligible

MS without Sulphates

Shoots were very short; leaves were small, drooping, light

(MgSO4, MnSO4 ,FeSO4 and

green to yellow in colour with black tips, roots were

CuSO4)

profusely branched

22

REFERENCES Agarwal S, Kanwar K and Sharma D. Factors affecting secondary somatic embryogenesis and embryo maturation in morus alba l. Sci Hort.

2004;102:359-368 Anis M, Faisal M and Singh S. Micropropagation of mulberry (morus alba l.) through in vitro culture of shoot tip and nodal explants. Plant Tissue Culture. 2003;13:47-51 Arabshahi-Delouee S and Urooj A. Antioxidant properties of various solvent extracts of mulberry (morus indica l.) leaves. Food Chem.

2007;102:1233-1240 Avigad D and Dey P Carbohydrate metabolism: Storage carbohydrates. In: Dey P and Harborne J. Plant biochemistry. Academic Press, Great Britain. 1997.

Bhau B and Wakhlu A. Rapid micropropagation of five cultivars of mulberry. Biol Plant. 2003;46:349355 Bloom A Crop acquisition of ammonium and nitrate.

In: Boote K, Bennett J, Sinclair T and Paulsen G. Physiology and determination of crop yield.

Soil Science Society of America, Inc., Crop Science Society of America, Inc., , Madison, WI. 1994.

Brownleader M, Harborne J and Dey P Carbohydrate metabolism: Primary metabolism of monosaccharides In: Dey P and Harborne J. Plant biochemistry. Academic Press, Great Britain.

1997.

Enkhmaa B, Shiwaku K, Katsube T, Kitajima K, Anuurad E, Yamasaki M and Yamane Y. Mulberry (morus alba l.) leaves and their major flavonol quercetin 3-(6-malonylglucoside) attenuate atherosclerotic lesion development in ldl receptor-deficient mice.

J Nutr.

2005;135:729-734 Enomoto S. Preservation of genetic resources of mulberry by means of tissue culture. JARQ. 1987;21:205-210

Bioresearch Bulletin (2010) 1: 19-23

1993;33:339-341

Lee S, Choi S, Kim H, Hwang J, Lee B, Gao J and Kim S. Mulberroside f isolated from the leaves of morus alba inhibits melanin biosynthesis.

Biol Pharm Bull. 2002;25:1045-1048 Lopez J, Tremblay N, Voogt W, Dubé S and Gosselin A. Effects of varying sulphate concentrations on growth, physiology and yield of the greenhouse tomato. Sci Hort. 1996;67:207-217 Lu M. Micropropagation of morus latifolia poilet using axillary buds from mature trees. Sci Hort.

2002;96:329-341 Murashige T and Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473-497 Ohyama K. Tissue culture in mulberry tree. JARQ. 1970;5:30-34 Ohyama K and Oka S. Regeneration of whole plants from isolated shoot tips of mulberry tree. The Journal of Sericulture Science of Japan.

1976;45:115-120 Pattnaik S and Chand P. Rapid clonal propagation of three mulberries, morus cathayana hemsl, m.

Lhou koiz. And m. Serrata roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees. Plant Cell Rep. 1997;16:503-508 Ponchia G and Gardiman M. Research on in vitro propagation of mulberry. Acta Horticulturae.

1991;314:4-9 Shibata Y, Kume N, Arai H, Hayashida K, InuiHayashida A, Minami M, Mukai E, Toyohara M, Harauma A and Murayama T. Mulberry leaf aqueous fractions inhibit tnf-α-induced nuclear factor κβ (nf-κβ) activation and lectinlike oxidized ldl receptor-1 (lox-1) expression in vascular endothelial cells. Atherosclerosis.

2007;193:20-27 Srivastava S, Kapoor R, Thathola A and Srivastava R. Nutritional quality of leaves of some genotypes of mulberry (morus alba). Int J Food Sci Nutr. 2006;57:305-313

Ercisli S and Orhan E. Chemical composition of white (morus alba), red (morus rubra) and black (morus nigra) mulberry fruits. Food Chem.

2007;103:1380-1384 Taiz L and Zeiger E. Plant physiology Sinauer Associates, Sunderland. 2002.

Islam R, Zaman A, Joarder O and Barman A. In vitro propagation as an aid for cloning ofmorus Thomas T. Advances in mulberry tissue culture. J laevigata wall. Plant Cell Tiss Org Cult.

Plant Biol. 2002;45:7-21

Bioresearch Bulletin (2010) 1: 19-23

Venkatesh Kumar R and Chauhan S. Mulberry: Life enhancer. J Med Pl Res. 2008;2:271-278 Vijaya Chitra D and Padmaja G. Seasonal influence on axillary bud sprouting and micropropagation of elite cultivars of mulberry. Sci Hort.

2002;92:55-68

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Vijaya Chitra D and Padmaja G. Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6benzylaminopurine. Sci Hort. 2005;106:593602

Bioresearch Bulletin (2010) 1: 24-28

24

ORIGINAL PAPER

Antimicrobial activity of marine bacteria associated with Polychaetes C.V. SUNJAIY SHANKAR*

A. HEPZIBA JEBA MALAR

S. MARY JOSEPHINE PUNITHA Received: 29 May 2010 / Accepted: 12 June 2010 /Published online: 22 June 2010 Š Bioindica Press 2010

ABSTRACT The present study was carried out to assess the antibacterial activity of bacteria associated with Polychaetes. Polychaetes were collected from the coastal environment and the bacterial communities associated with the surfaces were isolated using traditional culture method. Four biofilm bacteria (Galionella sp., Alteromonas sp., S.aureus, Klebsiella sp.) isolated from the marine water was used as target organism for screening. Three surface associated bacteria were isolated from the surface. The EPS of the three surface associated bacteria were isolated and tested for their antimicrobial activity. The results showed that higher activity against Alteromonas sp.

The bioactive compounds were separated by thin layer chromatography. The results showed higher activity against Alteromonas sp. Based on the study, it can be concluded that bacteria associated with Polychaetes would serve as a potential source for the isolation of bioactive compounds.

Keywords Antibiotics ; Bioactive compounds ; Antibacterial activity ; Surface- associated bacteria INTRODUCTION The marine environment is a complex ecosystem with an enormous diversity of different life forms often existing in close associations. Among these, microorganisms- eukaryote associations have gained significant attention in the past decade (Egan et al., 2008).

The surface of all marine eukaryotes are covered with microbes that live attached in diverse communities often embedded in a matrix, forming a bio-film (Perez -Matos et al.,2007). Moreover, host specificity has C.V. Sunjaiy Shankar Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Marina campus, Pannaiyoor, Rajakamangalam-629502, Tamil Nadu, India.

E-mail: cvsunjaiyshankar@gmail.com

also been illustrated by studies that have shown the presence of unique stable communities living on geographically distant individuals belonging to the same species (Webster and Bourne, 2007).

Marine derived microbial natural products have been largely unexplored. The marine environment is a habitat for many unique microorganisms, which produce biologically active compounds to adapt to particular environmental conditions. For example, marine surface associated microorganisms have proven to be a rich source for novel bioactivities because of the necessity to evolve allelochemicals capable of protecting the producer from the fierce competition that exists between microorganisms on the surface of marine eukaryotes (Anahit Penesyan et al., 2010).

The number of natural products, discovered from various living organisms including plants, animals and microbes, to date exceeds one million (Berdy, 2005), with the majority (40-60%) derived from terrestrial plants. Of these natural products, 20-25% possess various bioactive properties including antibacterial, antifungal, antiprotozoal, ant nematode, anticancer, antiviral and anti-inflammatory activities, Plants and plant extracts have been used for the treatment of human diseases for millennia, and their use has been recorded in the most ancient archaeological sources.

In contrast, the exploration of microorganisms as producers of therapeutically agents only began in the 20th century (Monaghan and Tkacz., 1990).

The bacteria are found associated with all organisms.

Out standing examples from the sea are the 50% bacterial biomass in sponges of the order Verongia sp., abundant Cyanobacteria sp. and oxychlorobacteria in many sponges. Many eukaryotic microorganisms are also known as symbionts, for example dinoflagellates in sponges fungi in a variety of invertebrates, algae and sea-grass (Taylor et al., 2007). Thew polychaeta or polychaetes are a class of Annelid worms. Each body segment has a pair of fleshy protrusions called parapodia that bear many bristles

Bioresearch Bulletin (2010) 1: 24-28

called chaeta, which are made of chitin.

The microbes associated with polychaetes were not studied much. There are several advantages for using microbes as source of bioactive compounds. Hence, in the present study an attempt has been made to screen the antimocrobial activity of bacxteria associated with polychates. A study of this kind will provide more details on the bioactivity of bacteria associated with marine invertibrates.

MATERIALS AND METHODS Isolation and identification of Bacteria Polychaetes were collected from the Kanyakumari coast of west coast of India. For the collection of the polychaetes, a portion of the seaweeds Sargasum sp was detached from the surface and kept in polythene bags with seawater and brought to the laboratory .

In the laboratory, the polychaetes were removed from the seaweed by gentle agitation and collected in a jar with seawater. The polychaetes were rinsed with sterile seawater to remove the loosely attached organism and the surface was swabbed with a sterile nylon brush. The bacterial film swabbed using the brush was dispersed in 1ml filter -sterilized seawater (Millipore , 0.45µm). This bacterial suspension was serially diluted and appropriate dilutions were poured on zobell marine agar. The plates were incubated at room temperature for 48 hours and the developed colonies were purified by repeatedly streaking on zobell marine agar plates. The permanent cultures were maintained in zobell marine agar slants at 4°C. The bacterial colonies were chrecterized based on Bergey’s manual of determinative bacteriology.

Isolation of Extracellular Polymeric Substance (EPS) of the Bacteria associated with PolychaetesThe amount of EPS produced by the cultures was determined by estimating total carbohydrates. 100ml culture broth was centrifuged at 5000 rpm for 15 minutes at 4oC. The cell pellets were discarded and the supernatant was mixed with equal amount of cold absolute ethanol. After one day incubation the precipitated EPS was collected and stored at 4oC. ANTIBACTERIAL ACTIVITY OF EPS

Disc diffusion (Kirby-Bauer) method In this method sterile filter paper discs were used 50μl of the EPS was loaded on discs and placed on the sterile Zobell marine agar plates seeded with the test organisms. The test organisms used in the present study were Galionella sp., Alteromonas sp., s.aureus and Klebsiella sp. After firm placement of the discs the plates were incubated at 37oC in inverted position for 48 hours. After the period of incubation the inhibition zone if any around the discs were measured.

Characterization of Extracellular Polymers by Thin Layer Chromatography The bioactive compounds present in the EPS were partially characterized by Thin-layer chromatography.

Methanol, acetic acid and benzene (3:1:1) were used

25

as solvents for the Thin layer chromatography. The samples were loaded on silica gel plates and kept in the TLC chamber for the mobility of the compounds were detected by iodine crystals.

RESULTS Biochemical Analysis The biochemical and physiological characteristics of the isolated strains are given table-I. Most of the strains are gram-negative, cocci and nonmotile. Strain A isolated from the polychaete was gram-negative, cocci and showed motility. It was indole positive, methyl red positive and voges-proskauer positive. It also utilized citrate and showed negative results for starch hydrolysis and urea hydrolysis .In triple sugar iron test, strain A showed positive result for acid, and alkali. It also showed negative result for gas production and hydrogen sulphide production.The strain A positive for catalase, oxidase and nitrate reduction test. It also hydrolysed casein and showed negative result for gelatin hydrolysis.

Strain B isolated from surface of the polychaetes was gram-negative, cocci and non motile. It was indole positive, methyl negative and voges-proskauer positive. It also utilized citrate and positive for starch hydrolysis and negative result for urea. In triple sugar iron test, strain B showed positive result for acid, alkali. It showed positive result for gas production and negative for hydrogen sulphide production . Strain B also showed positive result for catalase, oxdiase and negative for nitrate reduction test .It hydrolysed casein and gelatin.

Strain C isolated from the surface region of polychaete was gram-positive, cocci and showed motility.

It was indole positive, voges-proskauer positive. It also utilise citrate and showed negative result for starch hydrolysis. In triple sugar iron test, strain C showed positive result for acid, alkali and gas production and negative for hydrogen sulphide production.

Strain C also showed positive result for catalase, oxdiase and positive for nitrate reduction test. It showed negative for casein hydrolysis and positive for gelatin hydrolysis.

ANTIBACTERIAL ACTIVITY OF EPS

The extracellular polymeric substance isolated from all the three bacterial strain was tested for their antimicrobial activity against the four bacterial strains (Galionella sp., Alteromonas sp., S.aureus and Klebsiella sp.). and the results are given in (Table -2).

The EPS isolated from the strain A showed inhibitory activity against all the four target bacteria and showed a maximum inhibition zone against Galionella sp.10 mm and minimum of 8 mm against S.aureus.

The zone of inhibition against Alteromonas sp. was 9 mm (Fig-1) and the zone of inhibition against Klebsiella sp. was 9 mm.

The EPS isolated from the strain B showed inhibitory activity against Alteromonas sp. and klebsiella sp. It showed a maximum inhibition zone against Alteromo-

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Alteromonas sp. 15 mm and minimum of 10mm against Klebsiella sp. The EPS of strain B did not show inhibitory activity against Galionella sp and S.aureus.

The EPS isolated from the strain C showed inhibitory activity against all the four target bacteria and showed a maximum inhibition zone against Alteromonas sp. and the diameter of zone was 13 mm. The minimum inhibition zone of 9 mm was observed against Galionella sp. and S.aureus. The zone of inhibition against klebsiella sp. was 10 mm.

THINLAYER CHROMATOGRAPHY ANALYSIS OF EPS

The EPS was loaded on silica gel glass plates for the characterization of active compounds. Thinlayer chromatogram showed the presence of a single spot in all the strains. The EPS isolated from the strain A showed a single spot with the Rf value of 0.75 cm. The Rf value of the compound present in the EPS isolated from the strain B was 0.72 cm. The EPS isolated from the strain C showed a single spot with the Rf value of 0.53 cm (Fig-3).

DISCUSSION The present study strongly revealed the ecological rationale for polychaetes and its associated microorganisms for the maintainance of antimicrobial defenses. Seawater typically contains 10-7 viruses, 10-6 bacteria, 10-3 fungi and 10-2 microalgae/ml(Engel et al.,2002), including those which have been identified as causative agents in marine infectious diseases (Correa, 1997).Given that marine invertebrates and their symbionts are continuously exposed to a broad array of potentially deleterious microorganisms, it is reasonable that the production of bioactive secondary metabolites could act as fundamental mechanisms of antimicrobial defense.

Chemical interaction between different species of bacteria can affect the production and secretion of antimicrobial secondary metabolites(Patterson and Bolis,1997).the enhancement of antimicrobial compound by bacteria when they are exposed to a different strain of bacteria suggest that competition for space between epibiotic bacteria may provide further antifouling protection to the basibiont. In addition, some bacteria that previously did not produce such metabolites when they are exposed to other bacterial species or extracellular products from other bacteria. As surface-associated bacteria would be exposed to similar pressures in the surface biofilm, this occurrence further increase the number of strains of bacteria isolated from seaweed surface that that are producing active compounds.

In the present study, of the three strains isolated from the polychaetes , two were Gram-negative. Various studies have confirmed the predominance of Gram -negative producers in the marine environment (Fenical,1993).In a study on antibiotic production in marine bacteria , (Bernen et al.,1997) have reported

26

that 36% of the strains were Gram-negative rod. In this study, Gram-positive as well as Gram-negative bacteria were isolated from the polychaetes .Since, only three strains were isolated from a polychaetes species , more rigourous culture methods may reveal the actual diversity of bacteria associated with polychaetes.

The diversity of antibiotic producing marine bacteria isolated in the present study suggests that polychaetes are rich of bacteria (Hentschel et al., 2000). The bacteria isolated in the present study may be only a fraction of the total diversity of associated bacteria, given that only a small percentage (1%) of the bacteria can be cultured using the currently available medium and fermentation techniques(Proksch et al., 2002).

Serious attempts to tap the vast potential of marine organisms as source of bioactive metabolites that may be directly utilized as drug or serve as lead structures for drug development started in the late 1960s. The discovery of sizeable quantities of prostaglandins, which had just been discovered as important mediators involved in inflammatory disease, fever and pain , from the gorgonian plexaura homomalla by Weinheimer and Spraggins in 1969 is usually considered as the ―take –off point‖ of any serious search for ―drug from the sea‖(Weinheimer and Spragginns 1969).

The discovery of new classes of antibiotics is necessary due to the increased incidence of multiple resistance among pathogenic microorganisms to drug that are currently in clinical use (Burgess et al,.1999).Peninsular India enjoys a large coastline with diverse marine ecosystems.The microbial diversity was not much studied from Indian marine ecosystem in respect to bioactive compounds search. This true in the case of polychaetes , in which only a few studies are available on the bioactive potential of associated bacteria .

In general, results of the present study suggest that bacteria associated with polychaetes are having strong antimicrobial activity and could be used as a potential source for the development of marine drugs.

The extracellular polymeric substance produced by the marine bacteria are reported to have various application. Hence , more studies on the characterization of the isolated strains may improve our understanding on the chemical ecology of bacteria associated with polychaetes.

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Table -3 Rf value of compounds observed in the TLC

Rf values (cm)

Strain A

Strain B

Strain C

0.75

0.72

0.53

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28

Table-1 Biochemical and physiological characteristics of the Bacterial strains S.No 1

Biochemical and physiological Test Gram Staining

2

Morphology

3

Organisms Strain B -

Strain A -

Strain C +

cocci

cocci

cocci

Motility

+

-

-

4

Indole production

+

+

+

5

Methyl Red

+

-

+

6 7

Voges-Prousker Citrate utilization

+ +

+ +

+ +

8

Starch Hydrolysis

-

+

-

9

Urea Hydrolysis

-

-

-

10

TSI Agar test (i)Acid (ii)Alkaline

+

+

+

+

+

+

(iii)Gas Production

+

+

+

-

-

-

11

(iv)H2S Production Catalase

+

+

+

12

Oxidase

+

+

+

13

Nitrate Reduction

+

-

+

14

Casein Hydrolysis

+

+

-

15

Gelatin Hydrolysis

-

+

+

Table- 2 Antibacterial activity of EPS against target Bacteria Diameter of zone of inhibition(mm) S.No

Target Bacteria Strain A

Strain B

Strain C

1

Galionella Sp.

10

-

9

2

Alteromonas Sp.

9

15

13

3

Staphylococcus aureus

8

-

9

4

Klebsiella Sp.

9

10

10

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29

2007. Bacterial diversity associated with the CaribREFERENCES Anahit penesyan, Staffan kjelleberg and Subean tunicate Ecteinascidia turbinate. Antonie Van helen Egan, 2010. Development of novel drugs from Leeuwenhoek. 92 : 155-164. marine surface associated microorganisms , Mar. Patterson ,G.L. and C.M. Bolis, 1997. Fungal Drug.,8:438-459. cell-wall polysaccharides elicit an antifungal seconBerdy, J., 2005. Bioactive microbial metabodary metabolite(phytoalexin) in the cyanobacterium lites .A personal view. J. Antibiot.(Tokyo)45 : 581Scytonema ocellatum. J.phycol., 33:54-64.

26. Vol.43 Academic Press, Newyork, pp. 57-90.

Proksch P, R.A. Edrada and R. Ebel, 2002.

Bernen, V.S., M. Greenstein and W.M Maese, Drugs from the seas-current status and microbiologi1997. Marine microorganisms as a source of new cal implications. Appl. Microbiol. Biol., 59: 125-134. natural products. Advances in Applied Microbiology, Taylor, M. W., R. T Hill, J. Piel, R. W. Burgess J.G., E.M. Jordan, M. Bregu, A. Thacker and U. Hentschel, 2007. Soaking it up: the Maerns-spragg and K.G. Boyd, 1999. Microbial ancomplex lives of marine sponges and their microbial tagonism: a neglected avenue of natural products reassociates ISME journal, 1:187-190 search J. Biotechnol., 1:27-32 .

Weinheimer, A.J., and R.L. Spragginns, 1969.

Correa, J.A., 1997. Infectious diseases of maThe occurrence of two new prostaglandin derivatives rine algae: Current knowledge and Approaches, (15-epi-PGA2 and acetate, Methyl ester) in the gorgoProg.Phycol.Res.,12:149-180. nian Plexaura homomalla. Chemistry of co elenterEngel S., P.R. Jensen , and W. Fenical, 2000. ates.XV. Tetrahedron let 15:5185-5188.

Chemical ecology of marine microbial defense. J. Webster, N.S. and D. Bourne, 2000. Bacterial Chem. Ecol., 28 (10),1971-1985. community structure associated with the Antarctic soft Egan, S., T. Thomas, S. Kjelleberg, 2008. coral, Alcyonium antarcticum. FEMS Microbiol.

Unlocking the diversity and biotechnological potential Ecol., 59: 81-94. of marine surface associated microbial communities.

Curr. Opin. Microbiol., 11 : 219-225.

Fenical, W., 1993.Chemical studies of marine bacteria ;developing a new resource . Chem .Rev. 93 : 1673-1683.

Hentschel U, M. Steinert, and J. Hacker, 2000.Common molecular mechanisms of symbiosis and pathogensis. Trend microbial 8:226-231.

Monaghan, R.L and J.S. Tkacz, 1990 Bioactive microbial products: focus upon mechanism of action. Annu. Rev. Microbiol. , 44 : 271-301 Perezâ&#x20AC;&#x201C;Matos, A. E., W. Rosado, N.S. Govind ,

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ORIGINAL PAPER

Laboratory culture of Microzooplankton Tintinnopsis cylindrical [Daday 1887]

S.VIJAYARAGAVANA,

P.VIVEK RAJAB

Received: 12 May 2010 / Accepted: 13 June 2010 /Published online: 22 June 2010 © Bioindica Press 2010

ABSTRACT : The culture of microzooplankton Tintinnopsis cylindrica under controlled conditions is offering within a short period. The microzooplankton is ideal food for the prawn and fish juveniles in the nursery phase and the nutrivite value of the microzooplankton depends on the such as chlorella marina, the microzooplankton Tintinnopsis cylindrica has high reproductive capacity in the tropical water. The short generation period within week and the high nutritive values and the ability to live and grow in the crowed population are the positive features of Tintinnopsis cylindrica and hence, preferred for mass culture under intensive laboratory controlled condition the present study deals with mass culture of microzooplankton, Tintinnopsis cylindrica, under laboratory condition and intensive monitoring of the water quality (management) and species density during culture days.

Keywords : Tintinnopsis cylindrica, prawn and fish juveniles.

INTRODUCTION The microzooplankton are considered to play an important role in marine food chain. The most successful rearing has involved neritic species, only few oceanic and deep water species have been reared in Mariculture, certain fish larvae generally grow better. When fed live food. Most fish larvae so far studied cannot satisfy their nutritional demands with phytoplankton as their sole food source it seems reasonable, therefore to intensive investigations of microfaunal representatives emphasizing cultivation of suitable planktonic flagellates and microzooplankton. These protozoans could well meet the

essential criteria for ideal hatchery food organisms. They have high rate of reproduction many can be mass cultivated, and they are an apparently suitable nutritional value the microzooplankton are mostly fed by copepods, salps, polychaete larvae, shrimp larvae and juvenile fishes cladocerons, pteropods, and chaetognaths. The present study deals with mass culture of Tintinnopsis cylindrica and suitable live food for fish larvae.

MATERIALS AND METHODS: The 35-liter capacity plastic tank was used for culturing the microzooplankton. All apparatus including pipettes used for the transfer of specimens from one container to another container were cleaned thoroughly before every use. Glasswares were washed first with a non-ionic detergent rinsed sequentially with an acid. Solution (5-10% HCL) in hot running water and pure distilled water, then dried completely and rearing containers were prevented the excessive evaporation, the sea water from the location where the animals acquired in the field was often used. It was filterer through a membrane filter with a pore size greater than one µm. The microzooplankton were collected from kollidom estuary using a plankton net with a cloth number.32,( mesh size 54 µm) and introduced into of 3000 organisms and during culture the feed provided was chlorella marina and yeast. The daily growth rate recorded up to a week. The water quality management was observed under laboratory condition and vigorous aeration was provided by using an aerator and the room temperature ranged between 25 and 35 oC. The taxanomic position of Tintinnopsis cylindrica.

S.VijayaragavanA,

P.Vivek RajaB

Phylum:

Protozoa

Class

Ciliata

:

A. Department of Zoology Presidency college Chennai

Order :

Spirotricha

600005 Tamilnadu-India.

Sub order:

Tintinnida

B. Department of Zoology Presidency college, Chennai

Family :

Codonellidae

600 005, Tamilnadu

Genus :

Tintinnopsis

Species :

Cylindrica

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The identification characters of Tintinnopsis cylindrical The major factor for the successful laboratory culture of 1)Almost cylindrical for about 2/3 bowl. Then some time Tintinnids ciliates appears to be avoidance of metal and slightly inflated before narrowing to a stout pedicel. solvent contamination and turbulence of culture medium 2)The length is 120-240 Âľm, the microzoplankton were as reported earlier by Gifford (1985). During unfavorable counted (At 100 x magnification) using binocular micro- conditions it is making cyst formation and during favorscope and expressed as number of organism lit. able condition it can go for excitement as reported by Kamiyama (1997), Paranjape (1980) Reid and John RESULTS : During the present culture period, population density of ( 1978 ). tintinnopsis was measured from first day to seventh day. It has been reported that the Tintinnopsis require 3 to 7 The Tintnnopsis cylindrica maximum concentration days period for the production of 1000cells/L (Gold reached during 5to7 days. The final day density was 1968). In the present study of microzooplankton culture 40000 individuals liter (Table, 1) Fig .1 which mainly depended on the diet consisted of algal Chlorella marina and alternatively yeast (were provided) Water Quality Management The major factor that greatly influence the rearing of mi- but the algal diet stimulate much more density of microcrozooplankton by maintaining water quality manage- zooplankton similar observations were earlier reported ment. The penicillin and streptomycin (50mg/l) was by Kamiyama and Aizawa (1992) and Omari and Ikeda added which controlled the bacterial assemblage. ( 1976).

In the present study, the population density of microzooTemperature: The temperature is important parameter in culturing sys- plankton increased day by day for the period of 7 days tem, which ranged between 25 and 30o C, which was and on the final day (7th day) attained its maximum popufavorable for the culture of microzooplankton. lation density (40,000 organisms /L). because the microzooplankton have a high reproduction rate in the tropical Salinity: Salinity maintenance is very important during microzoo- waters and due to short regeneration period within weak plankton culture period, which was maintained between as reported earlier by Gold (1968). During the microzoo30 and 35 ppt. plankton culture, the temperature and food concentration are the two of the main factors affecting the grazing rate pH : The maintenance of pH is important during the culture of of microzooplankton a reported earlier by Hirayama and microzooplankton which was always maintained be- Ogawa (1972),Dhert(1996),Nevarro (1999),Mantagnes et tween 7.0 and 8.5. al ., ( 2001).

Dissolved oxygen: The dissolved oxygen content should be kept near the ACKNOWLEDGEMENT : saturation level for good health of culture organisms: the The author (VR) is thankful to Dr..P.Vevek Raja and dissolved oxygen level was maintained between 5.5 and Mr.T.S.Ragavan, Chennai for financial assistance.

7.5 using an aerator vigorous aeration was provided.

Table- I Day

Organism/ L

1.

3,000

2.

6,000

3.

10,000

4.

16,000

5.

26,000

6.

32,000

7.

40,000 Fig : 1 Laboratory Culture of Microzooplankton

DISCUSSION: The culture of microzooplankton, Tintinnopsis cylindrica under controlled conditions is offering very high rate of production within a short period. (Planichamy, 1996 and Karuppasamy, 1997). The microzooplankton reproduction is of sexual activity by conjugation. ( Omarai and Iketa, 1976; Kamamiyama and Aizawa, 1981). The tintinnids ciliated need intensive water quality arrangement and appropriate concentrations of micro flagellates (or) dinoflagellates filtered through sterilized sea water.

(Gold, 1971; Paranjape,1980).

REFERENCES: Dhert P.(1996) Manual on production and use of live food for aquaculture ( FAO Technical paper ) food and agriculture organization of the united nations, Rome, PP 61-98.

Gifford, D.J.(1985). Laboratory culture of marine planktonic oligotrichy ( Ciliophora, oligotrichida ) Mar.Ecol Prog.Ser.23:257 -267.

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The Population density of Tintinnopsis cylindrica during the culture period GRAPH -1 40000 32000 26000 16000 10000 6000 3000

1000 1

2

3

4

5

6

7

Period (Lays) Statistics (Desirevable) Gold K.(1971) Growth characteristics, of the mass â&#x20AC;&#x201C;reared tintinnid, Tintinnopsis beroidea .Mar.Biol 8:105-108.

Omari, M and J.Iketa (1976) .Methods in marine zooplankton ecology. Jhon wiley and sons publication, Newyork. P.332.

Gold, K.(1968) Some observation on the biology of Tintinnopsis sp.J.Prozool. 16:507-509.

Palanichamy S, (1996). Continuous mass culture of live feed to feed different stages of prawn and fishes.

Bull. Cent. Mar. Fish.Res. Inst.48:117-119.

Hardin, G.C(1974) .The food of deep sea copepods J.Mar Bio.Ass U.K.54:141-155 Kamiyama,J. (1997) Growth and grazing responses of tintinnids ciliates feeding on the toxic dinoflagelate (Heterocapsa circularisquama) Mar.Biol.128: 509-515.

Kamiyama,T and Y Aizawa (1981) Growth characteristics of two tintinnids ciliates Tintinopsis beroidea and Amphorella quadrilineata, in laboratory culture, Bull. Plankton soc, Japan. 34:185-191.

Kamiyama,T and Aizawa,Y(1992). Effects of temperature and light on tintinnids excystment from marine sediments Nippon, Suisan, Gakkaishi, 58:877884.

Montagnes, D.J.S. S.A.Kimmmance, G.T Sounis and J.C .Gumbs (2001). Combined effect of temperature and food concentration on the grazing rate of the rotifer (Brachionus plicatilis)Marine Biology, 139:975-979.

Karuppasamy, P.K.(1997).Studies on zooplankton in the pichavaram mangroves and laboratory culture of rotifer (Brachionus plicatilis). M.Phil, Theses, Annamalai University, PP.43 Reid, P.C.and A.W.G.Jhon (1978).Tintinnid cysts, J.Mar,Biol, ASS U.K.58:551-557 Paranjape, M.A.(1980). Occurrence and significance of resting cysts in a hyaline tintinnid. Helicostomella subulata (Jurgensen) J.Exp. Man Biol, E.col 48,23.

Hirayama K.and Ogawa S. (1972) Fundamental studies on physiology of rotifer for its mass culture filter feeding of rotifer Bull. T PH .Soc. Sci.Fish.38:12071214.

Navaro,N.(1999). Feeding behavior of the rotifers Brachionus plicatilis and Brachionus votundiformis with two types of food: live and fresh dried micro algae J.Exp Mar.Biol.Ecl.237.

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ORIGINAL PAPER A study on acute toxicity , oxygen consumption and behavioural changes in the three major Carps, Labeo rohita (ham), Catla catla (ham) and Cirrhinus mrigala (ham) exposed to Fenvalerate T. ANITA SUSAN1 K. SOBHA2 K.S. TILAK3 Received: 20 May 2010 / Accepted: 14 June 2010 /Published online: 22 June 2010 Š Bioindica Press 2010

ABSTRACT Acute toxicity experiments for Fenvalerate technical grade and 20% EC formulation were conducted using static renewal bioassay and continuous flow through systems for 24h, 48h and 96 h on the three major carps, Labeo rohita, Catla catla and Cirrhinus mrigala.

Although toxicity studies were conducted on both fry and fingerling stages, further experiments were carried out with fingerling stages only. After determining the LC50 concentrations for the three fish individually, onetenth of the 24 h LC50 was taken as sublethal concentration for studies on oxygen consumption. The toxicant exposed fish showed anomalous behaviour like surfacing phenomenon, irregular, erratic and darting swimming movements, hyperexcitability, loss of equilibrium and hitting to the walls of the test tank before finally sinking to the bottom just before death. Oxygen consumption studies for a period of 12 hours, at intervals of 2 hours, in both sublethal and lethal concentrations indicated that lethal concentrations had profound effect than sublethal concentrations and 20% EC was found to be more deleterious than technical grade of fenvalerate. During experimentation, severe respiratory distress, rapid opercular movements leading to the higher amount of toxicant uptake, increased mucus secretion, higher ventilation volume, decrease in the oxygen uptake efficiency, laboured breathing and gulping of air at the surface were observed in all the three carps studied.

Keywords: Carps, Oxygen consumption, Behavioural changes, Toxicity, Fenvalerate, 20% Emulsifiable concentrate, Metabolic rate etc.

Corresponding author: sobha_kota@yahoo.co.in T. ANITA SUSAN1, K. SOBHA2 and K.S. TILAK3 1

Department of Zoology, Andhra Christian College, Guntur, Andhra Pradesh, India 2 Department of Biotechnology, RVR & JC College of Engineering, Guntur, Andhra Pradesh, India 3 Department of Zoology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India

INTRODUCTION The pollution of aquatic environment with wide array of xenobiotic compounds has become a menace to the aquatic flora and fauna and is a problem of immediate concern. These contaminants are let out into the water bodies from industrial and agricultural areas and as most of them are highly persistent, their levels fast reach to life threatening in terms of both space and time (Brack et al. 2002; Diez et al. 2002). The recent development of biomarkers based on the study of the response of organisms to pollutants has provided essential tools for vigilant contamination monitoring (Korami et al. 2000). As pyrethroids are found to be less persistent and relatively safe as compared to organochlorines and organophosphates, a variety of them are widely used to control pests, such as moth pests attacking cotton, fruit and vegetable crops, including structural pest control and/or landscape maintenance (Marigoudar et al. 2009). Fish sensitivity to pyrethroids could be explained by their relatively slow metabolism and elimination of these compounds (David et al. 2003).

Depletion in oxygen content occurs in the medium when pesticides, chemicals, sewage and other effluents containing organic matter are discharged into water bodies. Pesticides in sublethal concentrations present in the aquatic environment are too low to cause rapid death directly but may affect the functioning of the organisms, disrupt normal behaviour and reduce the fitness of natural population. In the aquatic environment one of the most important manifestation of the toxic action of chemical is the over stimulation or depression of respiratory activity. The changes in the respiratory activity of fish have been used by several investigators as indicators of response to environmental stress.

The respiratory potential or oxygen consumption of an animal are the important physiological parameters to assess the toxic stress, because it is a valuable indicator of energy expenditure in particular and metabolism in general (Prosser and Brown, 1977).

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Pesticides are indicated to cause respiratory dis- ter for one week. The water used for acclimatization and tress or even failure by affecting respiratory centres of experimentation was the same as used in the toxicity the brain or the tissue involved in breathing. The effect experiments (Table 1). During this period the fish were of toxicants on the respiration of fishes and invertebrates regularly fed, but the feeding was stopped for two days have received wide spread attention and were reviewed prior to the experiment. The fish measuring 6 to 7 ± 0.5

by Hughes (1976) and Wright (1978). cm in length and 6 to 8 ± 0.5 gm in weight were used in As aquatic organisms have their outer bodies and the experiment. Toxicity experiments for fenvalerate important organs such as gills almost entirely exposed to technical grade and 20% EC formulation were conducted water, the effect of toxicants on the respiration is more on the fingerlings of the three carps, Labeo rohita, Catla pronounced. Pesticides enter into the fish mainly catla and Cirrhinus mrigala, using static and continuous through gills and with the onset of symptoms of poison- flow through systems for 24h, 48h and 96h exposures. ing, the rate of oxygen consumption increases ( Premdas The experiments on the oxygen consumption of the fish and Anderson, 1963; Ferguson et al., 1966a). Holden Labeo rohita, Catla catla and Cirrhinus mrigala were (1973) observed that one of the earliest symptoms of carried out in a respiratory apparatus developed by Job acute pesticide poisoning is respiratory distress. This (1955). The sublethal and lethal concentrations of the serves not only as a tool in evaluating the susceptibility toxicants used for the three carps for oxygen consumpor resistance potentiality of the animal, but also useful to tion studies are presented in Table 2. correlate the behaviour of the animal.

The respiratory chamber consists of a wide mouthed botBy cannulating the blood system of fishes, it is possible tle fitted with a rubber stopper with three holes. A glass to measure the concentrations of oxygen, metabolites and tube which serves as an inlet passes through one hole, pollutants and hence understand more fully the mode of through the other hole passes a glass tube with a regulaaction of toxic pollutants. Skidmore (1970) using cannu- tor serving as an outlet. Another hole is fitted with a lation techniques, found that zinc reduced the oxygen glass tube and regulator which serves as atmospheric level of blood leaving the gills. It reduced the efficiency vent. of oxygen transport across the gill membrane, so that fish The test fish in good condition were taken and introdie of hypoxia. Respiratory responses to lethal concen- duced into respiratory chamber filled with water. The trations increase the ventilation volume and symptoms of respiratory chamber was closed without air bubbles and pyrethroid intoxication suggesting that the effect on res- sealed with paraffin wax. The respiratory chamber is piratory surface are lethal in fish. It is known that pyre- connected to a 25 litre capacity reservoir through an throids are less persistent and are effective subsituents inlet. The flow rate was so adjusted, that one litre of for organochlorine (OC) pesticides.

Like O C water flows per hour. compounds, the mechanism of pyrethroid interference is In each experiment, two respiratory chambers, one with with nerve membrane function through the interaction fish and another without fish (i.e. control) were taken. with the sodium channels. The symptoms of fenvalerate The control serves to estimate initial amount of oxygen intoxication suggest that, besides effect on the nervous present. After one hour of acclimatization of the fish, system, effect on respiratory surfaces and renal ion regu- desired concentration of the toxicant was added to the lation may be associated with the mechanism of lethality reservoir and flow rate was adjusted. After fifteen minin fish (Bradbury et al., 1987). The toxic effects of pyre- utes, the water of the respiratory chambers was replaced throids on the metabolism particularly oxygen consump- by the test water containing the toxicant, and the experition have been reviewed by Bradbury et al., 1986; Ku- ments with fenvalerate technical grade and 20% active maraguru and Beamish, 1983; Haya and Waiwood, 1983; ingredient emulsifiable concentrate were conducted for Pandi baskaran, 1991. twelve hours. At the end of each hour, samples (50 ml) Total oxygen consumption is one of the indicators of the were collected and the amount of oxygen present was general well being of the fish. It may also be useful to estimated by Winkler’s method (Golterman and Clymo, assess the physiological state of an organism, helps in 1969). The difference in the rate of oxygen consumption evaluating the susceptibility or resistance potentiality and between the control and the test fish denotes the effect of also useful to correlate the behaviour of the animal, the toxicant on oxygen consumption. which ultimately serve as predictors of functional disrup- Like wise, experiments were conducted in sublethal and tions of population. Hence the analysis of oxygen con- lethal concentrations of fenvalerate technical grade and sumption can be used as a biodetectory system to evalu- 20% active ingredient EC and the data was compared ate the basic damage inflicted on the animal which could with that of control. The sublethal and lethal concentraeither increase or decrease the oxygen uptake. Therefore tions of the two toxicants tested throughout the study are an attempt was made to study the effect of sublethal and given in Table 2. lethal concentrations of fenvalerate, technical grade and The amount of oxygen consumption was calculated using 20% active ingredient emulsifiable concentrate (EC) on the formula: oxygen consumption for twelve hours to the three Indian 8 x 1000 x Normality of Hypo x Vol.of Hypo rundown x Vol of the respiratory chamber major carps, Labeo rohita, Catla catla and Cirrhinus O2 mg/L = mrigala.

Vol. of sample taken x Correction factor

MATERIALS AND METHODS The fish were brought from a local fish farm and acclimatized to the laboratory conditions in well aerated wa-

Bioresearch Bulletin (2010) 1: 24-28 RESULTS AND DISCUSSIONS Results of the toxicity experiments revealed that 20% active ingredient EC is about 2 to 6 times more toxic than technical grade fenvalerate. Since pyrethroids are fast acting and produce mortality within 24 hours (mostly by around 8-10 hours), the static LC50 values for 24 hours are same as for 48 hours and 96 hours. The range of concentrations producing mortality is narrow for 20% EC formulation, compared to technical grade fenvalerate. Higher mortality rate, almost double was recorded in winter ( 180 C Âą mean average temperature) than in summer ( 320 C Âą mean average temperature).

Thus for pyrethroids, there appears to be an inverse relationship between temperature and toxicity.

When fish were exposed to sublethal and lethal concentration of fenvalerate, several behavioural changes were observed which include swimming at the surface of water. This surfacing phenomenon was more in fish exposed to lethal concentration and sublethal concentration over the control fish. Hyperexcitation, loss of equilibrium, increased cough rate, flaring of gills, increase in production of mucus from the gills, darting movements, hitting against the walls of test tanks and curvature of spine were also noticed in all the three major carps.

When exposed to lethal concentration, body surface acquired dark colour before their death which is one of the symptoms of toxicity. A film of mucus was observed all over the body and also on the gill. Low rates of fenvalerate elimination and metabolism seem to be contributing factors in piscicidal activity of fenvalerate (Edwards et al., 1986; Glickman et al., 1982; Bradbury et al., 1986).

Patil and David (2008) in their study on behaviour and respiratory dysfunction as an index of malathion toxicity in Labeo rohita clearly reported that while the control fish were active with controlled and co-ordinated movements, the toxicant exposed fish exhibited irregular, erratic and darting movements and loss of equilibrium due to inhibition of AChE activity leading to accumulation of acetylcholine in cholinergic synapses ending up with hyperstimulation. These findings are in corroboration with those of Murshigeri and David, 2005 and others viz., Dube and Hosetti, 2010, Rao et al., 2003 and Parma de Croux et al., 2002. Recent studies on acute toxicity and behavioural responses of common carp, Cyprinus carpio (Linn.) to an organophosphate, Dimethoate reported erratic swimming of the test fish, their increased surfacing, decreased rate of opercular movement, copious mucus secretion, reduced agility and inability to maintain normal posture and balance with increasing exposure time (Pandey et al., 2009). In gist, the various behavioral anomalies in fish exposed to different toxicants in general include initial increase in opercular movements followed by steady decrease with increased duration of exposure (Shiva kumar and David, 2004), gulping air at the surface, swimming at the water surface, disrupted shoaling behaviour and easy predation (Ural and Simsek, 2006). Gulping of air may help to avoid contact of toxic medium. Surfacing phenomenon might be a demand of higher oxygen level during the exposure period (Katja et al., 2005). Finally, fish sunk to the bot-

35 tom with the least opercular movements and die with their mouth opened. In sublethal exposures, the fish body becomes lean towards abdomen position as compared to control owing to reduced amount of dietary protein consumed by the fish at pesticide stress, which was immediately utilized and was not stored in the body weight (Kalavathy et al., 2001).

The data on the whole animal oxygen consumption, calculated per gram body weight in sublethal and lethal concentrations of fenvalerate technical grade and 20% EC for Labeo rohita, Catla catla and Cirrhinus mrigala are given in the Tables 3 and 4. When a comparison is made between the sublethal concentrations of fenvalerate technical grade and 20% active ingredient EC among Labeo rohita, Catla catla and Cirrhinus mrigala on oxygen consumption, there was significant increase in oxygen consumption as compared to the controls in Labeo rohita and Catla catla, the respiratory rate being higher throughout the experimental period. On the contrary, in toxicant exposed Cirrhinus mrigala, oxygen consumption decreased than that of controls.

Similarly, when a comparison is made between the effects of lethal concentrations of fenvalerate technical grade and 20% active ingredient EC among Labeo rohita, Catla catla and Cirrhinus mrigala, highest oxygen consumption rates were attained during 2 nd and 4th hours while in Labeo rohita, oxygen consumption reached to maximum during eighth and fourth hours in technical grade and 20% EC respectively. Catla catla proved to be more sensitive than Labeo rohita and Cirrhinus mrigala as is evidenced by its death during fifth hour in lethal concentrations of 20% active ingredient EC. From this it can be inferred that 20% EC is more toxic than technical grade fenvalerate. Lethal concentrations of the two toxicants had profound effect on the oxygen consumption than the sublethal concentrations for 12 hours during the exposure period (Tables 3 and 4).

During experimentation severe respiratory distress, rapid opercular movements, leading to the higher amount of toxicant uptake, increased mucus secretion, higher ventilation volume, decrease in the oxygen uptake efficiency, laboured breathing and engulfing of air through the mouth were observed in all the three major carps exposed to both the toxicants. However, the above said changes in the fish were more pronounced in EC than in technical grade fenvalerate.

The increased oxygen consumption in Labeo rohita and Catla catla under sublethal concentrations of both the toxicants is in corroboration with the increased consumption of oxygen in trout exposed to permethrin (Haya, 1989) and P. pugio larvae exposed to fenvalerate for twenty four hours (McKenney and Hamakar, 1984).

Reddy et al. (1977) reported an elevation in oxygen uptake during first two hours of exposure followed by decrease in subsequent hours in Channa striatus exposed to cypermethrin. Similar trend was reported in L. rohita (Raju, 1991) and C. punctatus (Jeevaprada, 1990) exposed to cypermethrin. Bradbury et al. (1986) stated that the greater decrease in the rate of oxygen consumption in the fish Cirrhinus mrigala may be due to inter-

Bioresearch Bulletin (2010) 1: 24-28

36

sub cellular level. Similar observations were also reported ACKNOWLEDGMENT

by Mushgeri and David (2003) and Jadhav and Sontakka The authors sincerely thank the Heads of the Department (1977). The decrease in oxygen consumption at sub le- of Zoology, Acharya Nagarjuna University, Guntur who thal concentrations of the toxicant indicates lowered en- have extended their fullest co-operation and provided ergy requirements which in turn indicates pronounced laboratory facilities during the period of study. haematological changes (Tilak and Satyavardhan, 2002).

Similar reduction in oxygen consumption has been re- REFERENCES ported in Channa striatus exposed to organophosphate Arunachalam S and Palanichamy S (1982). Sublethal pesticide (Natarajan, 1981), O. mossambicus due to or- effects of Carbaryl on surfacing behaviour and food utiliganochlorine intoxication (Vasanthi and Ramasamy, zation in the air breathing fish, Macropodus carpanus.

1987), M. cupanus following carbamide treatment Physiol. Behav., 29: 23-27.

(Arunachalam and Palanichamy, 1982), and C. punctata Brack W, Schirmer K, Kind T, Schrader S, Schuurmann exposed to carbaryl (Tilak, 1979).

G (2002). Effect-directed fractionation and identification Changes in the gill surfaces and increased mucus produc- of cytochrome P450A-inducing halogenated aromatic tion is consistent with observed histological effects such hydrocarbons in contaminated sediment. Environ. Toxias hyperplasia, necrosis and lamellar aneurysms in all the col. Chem., 21: 2654-2662. three fish exposed to sublethal concentration of technical Bradbury SP, Coats JR and McKim JM (1986). Toxigrade fenvalerate. Kumaraguru et al. (1982) reported cokinetics of fenvalerate in rainbow trout, Salmo gairdthat the gill is the target organ for synthetic pyrethroid neri. Environ. Toxicol. Chem., 5: 567-576. toxicity in fish. The technical as well as commercial Bradbury SP, Symonik DM, Coats JR and Atchison GJ formulations will pass through the gills, and interfere in (1987). Toxicity of fenvalerate and its constituent isothe gill movements which is directly proportional to the mers to the fat head minnow (Pimephales promelas) blue respiratory activity of the fish, primarily effecting the gill (Lepomis macrochirus). Bull. Environ. Contam. oxygen uptake. The respiratory metabolism was im- Toxicol., 38: 727-735. paired and damage was also observed in the gill of fish David M, Shiva Kumar HB, Shiva Kumar R, Mushigeri exposed to pesticides (Hughes and Perry, 1976; Rama SB and Ganti BH (2003). Toxicity evaluation of cyperMurthy, 1988). In the freshwater fish, Ctenopharyn- methrin and its effect on oxygen consumption of the godon idella exposed to NuvanĂ&#x2019;, an organophosphate, fresh water fish, Tilapia mossambica. Indian J. Environ. the depletion of the oxygen consumption is due to the Toxicol., 13: 99-102. disorganization of the respiratory action caused by rup- Diez S, Abalos M, Bayona JM (2002). Organotin conture in the respiratory epithelium of the gill tissue. The tamination in sediments from the western Mediterranean experimental data reveals that oxygen consumption de- enclosures following ten years of TBT regulation. Water creases with the time of exposure to toxicant (Tilak and Res., 36: 905-918.

Swarna kumari, 2009).

Dube BN and Hosetti BB (2010). Respiratory distress Under toxic conditions, the oxygen supply becomes defi- and Behavioral anomalies of Indian Major carp Labeo cient and a number of poisons become more toxic in- rohita (Hamilton) exposed to Sodium cyanide. Recent creasing the amount of poison being exposed to the ani- Research in Science and Technology, 2(2): 42-48. mal. The fish breathe more rapidly and the amplitude of Ferguson DE, Lude JT and Murthy GG (1966a). Dynamrespiratory movements will increase. Lloyd (1961) re- ics of endrin uptake, release by resistant and susceptible ported that the toxicity of several poisons to rainbow strain of mosquito fish. Trans. Amer. Fish Soc., 95: 335trout increased in direct proportion to decrease in oxygen 344. concentration of water. In general, it is observed that the Golterman H and Clymo C (1969). Methods for the lack of oxygen increases the ventilation volume of fishes chemical analysis of fresh water. Blackwell Scientific and the cardiac output is reduced. This reduces the rate Publications . Pp. 116. of passage of blood through the gills, so allowing a Haya K (1989). Toxicity of Pyrethroid insecticides to longer period of time for uptake of oxygen, and also con- fish. Environmental Toxicology and Chemistry, Vol. 8: serves oxygen by reducing muscular work. The zone of 381-391. resistance is reached when the oxygen tension in the wa- Haya K and Waiwood BA (1983). Adenylate energy ter is so low that homeostatic mechanisms of the fish are charge and ATPase activity: potential biochemical indino longer able to maintain the oxygen tension in the af- cators of sublethal effects caused by pollutants in aquatic ferent blood and the standard metabolism begins to fall. animals. In: Aquatic Toxicology (ed. Nriagu O.), 307Changes in the architecture of gill under fenvalerate 333. stress would alter diffusing capacity of gill with conse- Holden AV (1973). Chapter 6 in Environmental Polluquent hypoxic/anoxic conditions and thus respiration tion by Pesticides. (Ed.) CA Edwards, Plenum press. Pp may become problematic task for the fish. The results of 542. the present study suggest that the altered rates of respira- Hughes GM (1976). Polluted Fish respiratory physioltion of fresh water fish may serve as a rapid biological ogy in Lockwood. APM (ed.), Effect of Pollutants on monitor of the pesticide exposure to important compo- aquatic organisms. Cambridge University Press, Camnents of fresh water community. bridge. Pp 163-183.

Bioresearch Bulletin (2010) 1: 24-28 Hughes GM and Perry SF (1976). Morphometric studies of trout gills: A light microscopic method suitable for the evaluation of pollutant action. J. Exp. Biol., 14: 447-460.

Jadhav SM and Sontakka VB (1977). Studies on respiratory metabolism in the freshwater bivalve, Corbicula striatella exposed to carbaryl and cypermethrin.

Pollut. Res., 16: 219-221.

Jeevaprada PN (1990). Studies on Cypermethrin toxicity and changes in total protein, glycogen and oxygen consumption in the fresh water teleost, Channa punctata (Bloch). M. Phil. Thesis submitted to Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

Job SV (1955). The oxygen consumption of Salvelinas fontinalis. Pubs. Out. Fish Res. Lab., 73: 1-39.

Kalavathy K, Sivakumar AA and Chandran R (2001). Toxic effects of the pesticide dimethoate on the fish, Sarotherodon mossambicus. J. Ecol. Res. Bio., 2: 27 -32.

Katja S, Georg BOS, Stephan P, Christian EWS (2005). Impact of PCB mixture (Aroclor 1254) and TBT and a mixture of both on swimming behavior, body growth and enzymatic biotransformation activities (GST) of young carp (Cyprinus carpio). Aquat. Toxicol., 71: 49 -59.

Korami D, Eric H, Charles G (2000). Concentration effects of selected insecticides on brain acetylcholinesterases in the common carp (Cyprinus carpio L.).

Ecotoxicol. Environ.Safe, 45: 95-105.

Kumaraguru AK and Beamish FWH (1983).

Bioenergetics of acclimation to permethrin (NRDC-143) by rainbow trout. Comp. Biochem Physiol Comp. Pharmacol. Toxicol., 75(2): 247-252.

Kumaraguru AK, Beamish FWH, Ferguson HW (1982). Direct and Circulatory paths of permethrin causing histopathological changes in the gills of rainbow trout. J. Fish Biol., 29: 87-90.

Lloyd R (1961). Effect of dissolved oxygen concentrations on the toxicity of several poisons to rainbow trout (Salmo gairdneri). J. Exp. Biol., 38: 447-455.

Marigoudar SR, Ahmed RN and David M (2009). Cypermethrin induced respiratory and behavioural responses of the freshwater teleost, Labeo rohita (Hamilton). Veterinarski Archiv, 79(6): 583-590.

McKenney CL Jr. and Hamakar DB (1984).

Effect of fenvalerate on larval development of Palaeomonetes pugio (Holthuis) on larval metabolism during osmotic stress. Aquatic Toxicology, 5: 343-355.

Mushigeri SB and David M (2003). Assessment of fenvalerate toxicity on oxygen consumption and ammonia excretion in the fresh water fish, Cirrhinus mrigala. J. Ecotoxicol. Environ. Monit., 13: 191-195.

Mushigeri SB and David M (2005). Fenvalerate induced changes in the Ach and associated AChE activity in different tissues of fish, Cirrhinus mrigala (Hamilton) under lethal and sublethal exposure period.

Environ. Toxicol. Pharmacol., 20: 65-72.

Natarajan GM (1981). Effect of Lethal LC50 / 48h concentrations of metasystox on selected oxidative enzymes, tissue respiration and histology of gill of fresh water air breathing fish, Channa striatus. Curr. Sci., 50 (22): 985-991.

37 Pandey RK, Singh RN, Singh NN and Das VK (2009). Acute toxicity and Behavioral responses of common carp Cyprinus carpio (Linn.) to an organophosphate, Dimethoate. World Journal of Zoology, 4(2): 7075.

Pandi Baskaran (1991). Use of Biochemical parameters in biomonitoring of pesticide pollution in some fresh water fishes. Journal of Ecotoxicology and Environmental Monitoring, Vol. 2, 101-104.

Parma de croux MJ, Loteste A, Cazenave J (2002). Inhibition of plasma cholinesterase and acute toxicity of monocrotophos in Neotropical fish, Prochilodus lineatus (Pisces, Curimatidae). Bull. Environ.

Contam. Toxicol., 69: 356-362.

Patil VK and David M (2008). Behaviour and Respiratory dysfunction as an index of Malathion Toxicity in the freshwater fish, Labeo rohita (Hamilton).

Turkish Journal of Fisheries and Aquatic Sciences 8: 233 -237.

Premdas FH and Anderson JM (1963). The uptake and distribution of 14C labelled DDT in Atlantic salmon, Salma saleni. J. Fish Res. Board.Can., 20: 827.

Prosser CL and Brown FA (1977). Comparative Animal Physiology. 3rd Edition, W.B. Saunder Co., Philadelphia.

Raju KS (1991). Toxicity and effect of Cypermethrin to the freshwater fish Labeo rohita (Hamilton).

M. Phil. Thesis submitted to Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

Rama Murthy K (1988). Impact of heptachlor on haematological, histological and selected biochemical parameters in the fresh water edible fish, Channa punctatus. Ph. D. Thesis, S.V. University, Tirupati, Andhra Pradesh, India.

Rao JV, Rani CHS, Kavitha P, Rao RN and Madhavendra SS (2003). Toxicity of chlorpyrifos to the fish, Oreochromis mossambicus. Bull. Environ. Contam.

Toxicol., 70: 985-992.

Reddy TG, Paliappan S and Pillay KBP (1977).

Respiratory and Histopathological effects of pesticide disyston on the Colisalatia. All India Symposium on Environ. Biol., Univ. Kerala, pp 27-29.

Shivakumar R and David M (2004). Toxicity of endosulfan to the freshwater fish, Cyprinus carpio. Indian J. Ecol., 31: 27-29.

Skidmore JF(1970). Respiration and Osmoregulation in rainbow trout with gills damaged by Zinc sulphate. J. Exp. Biol., 52: 484-494.

Tilak KS (1979). Toxicity of Carbaryl and 1naphthol to the fresh water fish, Channa punctata. Ph.D. Thesis, Andhra University, Visakhapatnam, Andhra Pradesh, India.

Tilak KS and Satyavardhan K (2002). Effect of fenvalerate on oxygen consumption and haematological parameters in the fish, Channa punctatus (Bloch). J. Aquatic Biol., 17: 81-86.

Tilak KS and Swarna Kumari R (2009). Acute toxicity of Nuvan , an organophosphate to freshwater fish Ctenopharyngodon idella and its effect on oxygen consumption. J. of Environmental Biology, 30(6), 10311033.

Bioresearch Bulletin (2010) 1: 24-28 Ural MS and Simsek S ( 2006). Acute toxicity of dichlorvos on fingerling European catfish, Silurus glanis.

Bull. Environ. Contam. Toxicol., 76: 871-876.

Vasanthi M and Ramasamy M (1987). A shift in metabolic pathway of Sarotherodon mossambicus (Peters) exposed to thiodon (endosulfan). Proc. Indian Acad. Sci., (Anim.Sci.) 96: 56-69.

Wright DA (1978). Heavy Metal accumulation by aquatic invertebrates. Applied Biology, 3: 331-394.

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39

ORIGINAL PAPER

Bacteriological Assessment of some Swimming Pools within Ilorin Metropolis, Kwara, Nigeria.

SULE* I.O.

OYEYIOLA G.P.

Received 7 April 2010 /Accepted 15 April /Published Online: 22 June 2010 © Bioindica Press 2010

ABSTRACT

INTRODUCTION

Six swimming pools within Ilorin metropolis were analysed after treatment prior to use by bathers and after use in order to determine their physicochemical and microbiological parameters. These swimming pools were Kwara hotel both adult and children swimming pools, Bekandims, Stella, Kingstone and Successxs swimming pools. The pH ranged from 4.8 to 6.9 and 4.9 to 7.6 before and after use by bathers respectively. Residual chlorine also ranged from 8.95 to 11.08 ppm and 7.17 to 10.08 ppm before and after use by bathers respectively. The total bacterial count ranged from 2.0 to 2.8×103cfu/ml and1.4 ×102 cfu/ml to 3.0 ×103 cfu/ml before and after use respectively. Faecal coliform counts ranged from zero to 4 cfu/ml and zero to 6 cfu/ml before and after use by bathers while their corresponding values of total coliforms were zero to 200 cfu/ml and 4 to 300 cfu/ml respectively. T-test statistical analysis showed that there were significance between the total bacterial count and the free residual chlorine contents of the pools water before and after use by the bathers. A total of thirteen bacterial species viz: Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas sp., Bacillus sp., Streptococcus sp., Micrococcus luteus, Aeromonas aerogenes., Aerococcus sp., Lactobacillus sp., Klebsiella sp., Citrobacter freudii., Corynebacterium sp., and Escherichia coli were isolated. The possible sources of contaminations were identified and mitigation measures highlighted.

Keywords: Water supply, bacteriological, physicochemical, swimming pools.

A swimming pool is an artificially enclosed body of water intended for swimming or water based recreation. Bathing and swimming for pleasure has been practiced by many land animals for unrecorded ages, and human record of the pleasure of immersion in water go back several thousand years ago (Perkins, 1988).

The depth of a swimming pool depends on the purpose of the pool, and whether it is open to the public or strictly for private use. Many countries now have strict pool fencing laws for private pools which require pool areas to be isolated so that unauthorized children younger than six years can’t enter (EPA,2007). Public pools are often found as part of a larger leisure center or recreational complex. Public pools may belong to a hotel or holiday retort as an amenity for the recreation of their guests ( Kate and Dominck, 2008).

The water supply to a pool is usually taken from the mains of a public supply. Swimming pool sanitation refers to both visual clarity and levels of microflora such as bacteria, protozoans and viruses in swimming pools ( Totkova et al., 1994).The goal of sanitation is to prevent the spread of diseases and pathogens between users.

Swimming pool water may become contaminated by micro-organisms from infected swimmers, incoming water from an unsanitary source, airborne contamination and droppings from birds is possible ( Podewils et al., 2007).

Contaminated water can lead to a variety of disease including diarrhoea, skin, ear and upper respiratory infections particularly if the swimmer’s head is submerged or water swallowed.

Some potential protozoans diseases in poorly maintained public swimming pools include cryptosporidiosis (caused by Cryptosporium) and Giardiasis caused

by Giardia sp. and amoebic meningoencephalitis caused by Naegleria foweri (CDC, 2006).

Sule* I.O. Department of Microbiology, University of Ilorin, Ilorin, Nigeria.

E-Mail: ismailaolawales@yahoo.co.uk

Bioresearch Bulletin (2010) 1: 29-33

E. coli and Shigella are relatively sensitive to chlorine or bromine so most outbreaks have occurred in locations where no disinfectants are added. Non faecal human shedding ( from mucus, saliva, skin) in the swimming pool is a potential source of non-enteric pathogenic organisms. Other non-enetric pathogens that can be found in swimming pools are Legionella spp., Pseudomonas aeruginosa, Mycobacterium spp., Staphylococcus aureus, Leptospira interrogan, Molluscipoxvirus, Human papilloma virus, Acanthamoeba spp., Trichophyton spp., and Epidemophyton floccosum, that usually produce dermic or respiratory infections. ( Eric et al., 1982).

Strong oxidizing agents are often used especially simple chlorine compounds such as sodium hypochlorite, dichlor or trichlor .When any of these pool chemicals are used, it is very important to keep the pH of the pool in the range 7.2 to 7.6 (CDC, 2006) A pool that is in proper balance should have a pH of 7.6, calcium Hardness of 120 ppm and a residual chlorine level of 1.0 to 2.0 ppm.

The objectives of this study is to determine the level of sanitation of the swimming pools; determine the species of bacteria which might be present in it and to evaluate of some of its physicochemical quality.

MATERIALS AND METHODS Collection of Water Samples Water samples were collected from the following pools: Kwara hotel (Adult), Kwara hotel (Children), Bekandims, Stella obasanjo multipurpose complex, Kingstone hotel, and Successxs hotel. The water sample was collected into different sterile sampling bottles using methods as described by APHA (1985) and Fawole and Oso (2005). The bottles were properly tightened and taken to the laboratory immediately for analysis.

Physicochemical Analysis Residual Chlorine was determined as described by B.P.(1993) using 0.1N silver nitrate solution while the pH was determined according to standard methods (ASTM, 1985).

Bacteriological Analysis : The total bacterial count was determined using standard plate count (SPC) as described by APHA,(1985).The total and faecal Coliform count were determined using MacConkey agar and Eosin methylene blue agar respectively by pour plate technique (Salle,1993).

Characterization and identification of isolates Bacterial isolates were characterized on the basis of colonial morphology, cellular morphology, staining reactions and biochemical characteristics. The tests were carried as described by

40

Joklit et al.,(1992). Isolates were identified according to Bucchanan and Gibbon(1974).

Statistical analysis: T- test was used to determine if there is significant difference between the results obtained for each parameter before and after use by the bathers (Bello and Ajayi, 2000).

RESULTS The pH of the swimming pools water prior to use after disinfection and after use ranged from 4.8 - 6.9 and 6.4 - 7.6 respectively. Similarly, the residual chlorine level ranged from 8.95 11.08mg/l and 7.17 - 10.08mg/l before and after use respectively (Table 1).

The total bacteria count of the pools water prior to use and after use ranged from 2 – 2800 cfu/ml and 140 – 3000 cfu/ml respectively (Table 2).The total coliform count ranged from zero to 200 cfu/ml and 4 – 300 cfu/ml before and after use while their corresponding values of faecal coliform were zero to 4 cfu/ml and zero to 6 cfu/ml (Table 3).The distribution of the various bacteria species isolated were as depicted in Table 4.

The results of the T-test statistical analysis showed that there were no significant difference between the results obtained from the pools water prior to use by bathers and after use (p>0.05) for these parameters: pH, faecal coliforms and total coliforms. However, there were significant differences in the bacterial counts and the free residual chlorine levels before and after use by the bathers.

DISCUSSION It is obvious from the results that none of the swimming pool fully complied with the WHO Standards. This is similar to the observation of Attah et al.(2007) who found that none of the surveyed public swimming pool was in full compliance with the Jordanian standards for swimming pools water.

Only one of the water from the swimming pools(17%) had pH within the range of pH 7.2 – 7.8 ( WHO,2008 ). Adrian et al.(1984) found that 37% of the swimming pools in South Australian had pH outside the recommended level.

Furthermore, the residual chlorine level of the pools are well above the standard of 5mg/l ( WHO,2008 ) and this may be responsible for the low pH recorded due to excessive use of the disinfectant (hypochlorite).

Five out of the six swimming pools (83.3%) met up in terms of bacterial count of less than 100 cfu/ml before use by bathers after disinfection. The high bacteria count at Stella’s pool prior to use by bathers could probably come from contaminated water source or ineffective treatment (Table 2).

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The immediate environment of all the pools were cleaned and tiled. Le Chevallier et al.(1996) has reported the occurrence of coliforms in the presence of disinfectant residual. All the pools had increment in bacterial load after use by bathers.

This is in conformity with the work of other workers who reported that bathers tend to shed bacteria from faecal and non- faecal sources (Craun et al.,2005 ). Another perspective to increase bacteria count after use could be due to stirring up of sediments harbouring micro-organisms which chlorine did not act upon.

Fifty percent of the swimming pools had zero coliform count while only one (16.7%) had faecal coliform count of 4 cfu/ml prior to use

(Table 3). All the pools had varying number of total coliforms after use by the bathers whereas 33.3% had faecal coliforms.

It is recommended that swimming pools operators should use the required disinfection regime allowed (5mg/l) rather than superchlorination; the disinfectant used should be thoroughly mixed in the pool; good water source should be used for disinfection ; poster signs should be provided to enlightened the swimmers about good sanitary habits; Health authority should regularly monitor the pools for compliance with regulations; people should be encouraged to shower before swimming ; footbaths should be provided to minimize soil contamination.

Table 1: Some physicochemical parameters of water samples from the Swimming pools Swimming Pools

pH Before use

Kwara hotel (Adult) Kwara hotel (Children) Bekandims hotel

Residual chlorine ( ppm ) After use

Before use

After use

6.6

7.0

9.80

9.44

6.5 6.9

7.0 6.4

9.73 9.94

9.30 8.66

Stella

6.5

6.9

9.23

7.31

Kingstone hotel

5. 9

7.6

11.08

10.08

Successxs hotel

4.8

4.9

8.95

7.17

Table 2: Total Bacterial counts of the swimming pool water samples Swimming Pools

Total Bacterial count ( cfu/ml) Before use After use

Kwara hotel (Adult)

2

140

Kwara hotel (Children)

30

690

Bekandims

33

800

2800

3000

Kingstone

23

170

Successxs

36

230

Stella

Bioresearch Bulletin (2010) 1: 29-33

42

Table 3: Total coliform and faecal coliform count of water samples from the swimming pools

Swimming

Total Coliform count ( cfu/ml)

Faecal Coliform count(cfu/ml)

Pools Before use 0

After use 6

Before use 0

After use 0

0

26

0

0

Bekandims Stella

0 200

4 300

0 0

0 0

Kingstone

18

40

0

2

Successxs

6

20

4

6

Kwara hotel (Adult) Kwara hotel (Children)

Table 4: Distribution of Bacterial isolates in some swimming pools water within Ilorin Metropolis Isolates

Kwara

Kwara hotel

Bekandi

hotel

(Children)

m

Stella

Kingstone

Successxs

(Adult) Enterobacter aerogenes

-

-

+

+

-

+

Staphylococcus aureus

+

+

+

+

+

+

Pseudomonas sp.

-

-

-

+

-

+

Bacillus sp.

+

+

+

+

-

+

Micrococcus luteus

+

+

-

-

+

-

Aeromonas aerogenes

-

+

+

+

-

+

Aerococcus sp.

+

-

-

-

-

-

Lactobacillus sp.

-

-

+

+

+

+

Klebsiella sp.

-

+

-

+

-

+

Citrobacter freudii

+

+

+

-

-

-

Corynebacterium sp.

-

-

+

+

+

+

Escherichia coli

-

-

-

-

+

+

Streptococcus sp.

-

+

+

+

-

+

KEY

+: Isolated

- : Not isolated

Bioresearch Bulletin (2010) 1: 29-33

CONCLUSION

43

Eric DM, Ronel SV. Water Quality, Academic press, London. 1982; 295.

It is concluded from this study that most of the swimming pools are deficient in all or part of the standards and efforts should be geared to brace up with the challenges rather than resulting to superchlorination.

Fawole MO, Oso BA. Laboratory manual of Microbiology. Spectrum books limited, Ibadan1988; 127 pp.

REFERENCES

Joklik WK, Willett HP, Amos DB, Wilfert CM. Zinsser Microbiology 20th Edition , Appleton and lange, California. 1992; 231.

Adrian E, David MR, Cameron AS, Robinson BS, Reginald PW, Jane AL, and Peter EC. Determi- Kate R, Dominick T. Wild swim; river, lakes, lido nants of the Microbiological Charac teristics and sea: the best places to swim outdoors in Britof South Australian Swimming Pools. Applied ain, London, Guardian Books 2008; 208. and Environmental Micro biology. 1984; 47 (2): 325 - 328.

Le chevalier MW, Cawthon CD, and Lee RG. Factors promoting survival of bacteria in chlorinated American Public Health Association (APHA). water supplies. Journal of Applied Environmental Standard Methods for the Examination of water Microbiology. 1988; 54(3): 649 – 653 and waste water. 16th edition, Washington, D.C.1985 Perkins PH. Swimming pools. Applied Science, Elsevier, London and New York. 1988; 257 – American Society for Test and Measurements 296 .

(ASTM). Annual American Society for Test and Measurements Standards.1985 Podewils LJ, Zanardi B, and Hagenbuch M. Outbreak of norvirus illness associated with a swimAttah R, Yousef K, Ahmed A, and Ashraf A. Sani- ming pool. Epidemiol. Infect. 2007; 135(5): 825 – tary Conditions of Public Swimming Pools in Am- 833. man, Jordan. Int. J. Environ. Res. Public Health.

2007; 4 (4): 301 – 306.

Salle AJ. Bacteriology of Water and Fundamental Principles of Bacteriology. 7th edition. Mc Graw Bello R, and Ajayi OOS. Research Methods and Hill Book Company, Inc. Washington D.C. 1973; Statistical Analysis. Haytee Press and Publishing 687 – 710 Co. Ltd., Ilorin. 2000; 131 - 139 Totkova A, Klobusicky M, and Tirjakova A. British Pharmacopoeia. Vol. 1, International Edi- Helminth and Protozoal findings in the water of tion, HMSO publication center, London. 1993; school swimmings. Epidermol. Mikrobiol. Imunol.

604 – 605.

1994; 43(3): 130 – 136.

Bucchanan R E, Gibbons NE. Bergey’s Manual of World Health Organisation(WHO). Guidelines for Determinative Bacteriology. (8th edition). The Wil- Drinking Water Quality. 3rd. Edition, Incorporating liams and Wilkins Co., Baltimore. 1974; 1268pp. the first and second addenda,Volume 1, Recommendations. 2008; (http://www.who.int/ Centers for Disease Control and Prevention water_sanitation_health/dwq/gdwq3rev/en) (CDC). Healthy Swimming: Chlorine and pH. 2006.

Craun GF, Calderon RL, Craun MF. Outbreaks associated with recreational water in the United States. Int. J. Environ. Health Res. 2005;15(4): 243-62.

Environmental Protection Agency (EPA). Bacteria Water Quality. Standards for Recreational Waters (fresh and marine waters). 2007; 80 – 81.

Notes

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