Bioresearch Bulletin Vol 1:1

Bioresearch Bulletin (2010) 1. I I

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 ex-

pand 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

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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

Abiya Chelliah

EDITOR-IN-CHIEF

Pethuru Devathasan

EXECUTIVE EDITOR

Sam Stephen

MANAGING EDITOR

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Pharmacologist.

Mahesh

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Bioresearch Bulletin (2010) 1. II II

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Bioresearch Bulletin (2010) 1. III III

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

Assessing the ecological status of prominent medicinal herbs in central Sudan

and the challenge of sustainable management

H.H. El-Kamali

Impact of industrial effluents and sewage on river Thamirabarani and its concerns

P. Peter Baskaran, John De Britto

Effect of nutrients on in-vitro culture of Morus alba l. (white mulberry)

Ankit Pradhan, A. S. Vishwanathan, R. Basavaraju.

Antimicrobial activity of marine Bacteria associated with Polychaetes

C.V. Sunjaiy Shankar, A. Hepziba Jeba Malar, S. Mary Josephine Punitha

Laboratory culture of Microzooplankton Tintinnopsis cylindrical [Daday 1887]

S.Vijayaragavan, P.Vivek Raja

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

Bacteriological assessment of some swimming pools within Ilorin metropolis,

Kwara, Nigeria.

Sule I.O, Oyeyiola G.P

Bioresearch Bulletin (BRB) ISSN 0976-5751 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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ISSN 0976-5751

Bioresearch Bulletin (2010) 1. IV IV

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 (ISSN 0976-

5751). This new Bioindica Press journal is being intro-

duced to provide a focal point for publication of arti-

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today for Bioresearch Bulletin include advances in or

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nology, microbiology, molecular biology, neurobiol-

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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 fol-

lowing type of scientific communication,

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Bioresearch Bulletin (2010) 1: 1-6 1

ORIGINAL PAPER

Anxiolytic effect of Aridanin isolated from Tetrapleura tetraptera in mice

1A.O. ADERIBIGBE 2E.O. IWALEWA 3S.K. ADESINA

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


ABSTRACT The study was carried out to investigate the anxio-

lytic 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 en-

tries 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 ad-

ministration 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 addi-

tion it is concluded that the anxiolytic effect of aridanin

may be through interaction with GABAA- benzodi-

azepine receptor complex.

Keywords: Aridanin, Tetrapleura tetraptera, Anxiolytic, GABAA

receptor, benzodiazepine receptor.

INTRODUCTION Tetrapleura tetraptera Taub (Mimosaceae)

locally known as Aridan is a large tree growing

throughout the rain forest belt of West Africa. It is gen-

erally 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, in-

flammation 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

luteinizing hormone released by pituitary cells, sug-

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 glo-

bosus and Lymnaea natalensis (Adewunmi, 1991). The

alleopathic potential of Tetrapleura tetraptera has led

to its integration into an agro forestry system (Amoo et

al., 2008). Tetrapleura tetraptera has been shown to

improve the foaming ability of soaps (Adebayo et al.,

2000). Tetrapleura tetraptera has no influence on cell

proliferation and neither induced chromosomal aberra-

tion nor sister chromatid exchanges in Chinese hamster

ovary cells (no genotoxic effect) (Adewunmi et al.,

1991). Tetrapleura tetraptera has been shown to cause

elevation in serum AST and alteration of various me-

tabolites parameters and did not induce any marked

pathological lesion in the liver (Odesanmi et al., 2009).

The sedative, anticonvulsant and analgesic effect of

aridanin in mice have been reported (Aderibigbe et al.,

A.O. Aderibigbe

Department of Pharmacology and Toxicology,

Faculty of Pharmacy,Niger-Delta University.

Bayelsa, Bayelsa State, Nigeria.

e-mail : adebee [email protected]

E.O. Iwalewa

Department of Pharmacology,Faculty of Pharmacy

Obafemi Awolowo University,Ile-Ife, Osun State,

Nigeria.

S.K. Adesina

Drug Research and Production unit, Faculty of Pharmacy

Obafemi Awolowo University, Ile-Ife, Osun State,

Nigeria.

O.I. Agboola

Department of Pharmacognosy, Faculty of Pharmacy

Niger-Delta University, Bayelsa, Bayelsa State, Nigeria.


2007a; Aderibigbe et al., 2007b; Ojewole, 2005). The

aqueous extract of Tetrapleura tetraptera fruit have

been shown to possessed anti-inflammatory and hypo-

glycaemic 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 – acetylgly-

coside of oleanoic acid (3β- hydroxyolean-12-en-28-

oic) called Aridanin (Adesina and Reish, 1985). The

present study was carried out to investigate the anxio-

lytic 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 Phar-

macy, 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 ex-

periment 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, Flu-

mazenil 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 car-

ried out as described by Miller and Tainter (1944) and

the lethal dose was calculated by the method of Litch-

field and Wilcoxon (1949). It was carried out by in-

jecting 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

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 re-

ceived 5% Tween 80. Subsequently mice were treated

with flumazenil (2 mg/kg, i.p.) a GABA-

benzodiazepine receptor antagonist 15 min before the

administration of aridanin (10 mg/kg, i.p.). The anxi-

ety evaluation was carried out 30 min after the ad-

ministration of aridanin or vehicle. Between each trial,

the maze was wiped with 70% ethanol to prevent ol-

factory 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.

2 Bioresearch Bulletin (2010) 1: 1-6

Fig 1: The chemical structure of Aridanin

COOH

R

O

NHCOCH 3HO

R2O

OOR1

R = R1

= R2

= H = Aridanin

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 en-

tries. 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 ari-

danin (10 mg/kg, i.p.) preceded by flumazenil de-

creased 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 de-

crease 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 con-

sidered 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 anx-

iolytic effect in mice. The doses increased the en-

tries 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 simi-

lar to the one observed with diazepam, a typical

benzodiazepine drug (Rall, 1990). As expected, di-

azepam 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. Sup-

porting 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 ari-

danin. The anxiolytic effect of aridanin is similar to

that of plants such as Cissus cornifolia, Careya an-

boree, Rubus brasiliensis, Stachys lavandulifo-

lia,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 ani-

mals 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 pos-

sessed anxiolytic effect which is exerted through

interaction with the GABAA – BDZ receptor com-

plex.


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 Dose (mg/kg, i.p.) NEOA NECA PEOA

5%TW80 0.2ml/20g 4.2±0.6 7.8±1.1 29.0±1.1

Aridanin 5.0 6.2±0.4* 5.0±0.6 55.3±3.1*

Aridanin 10.0 6.4±0.5* 4.4±0.5 57.7±3.2*

Aridanin 20.0 3.0±0.5 6.8±0.4 34.1±2.1

Aridanin 30.0 1.8±0.4 7.1±0.4 23.1±1.1

Diazepam 1.0 8.4±1.2* 3.4±0.4 58.0±3.1*

Results are expressed as the mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant differ-

ence 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.


Table 2 Effect of aridanin on time spent in the open arm entries and on time spent in the close arm entries


ence between different treatment groups. TSOA: Time spent in the open arm; TSCA: Time spent in the close

arm; 5%TW80: 5% Tween 80


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


ence between different treatment groups. TSOA: Time spent in the open arm; TSCA: Time spent in the close

arm; 5%TW80: 5% Tween 80


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.).

Results are expressed as the Mean ± S.E.M, (n = 5). One way ANOVA revealed that there is a significant differ-

ence 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

Treatment Dose (mg/kg, i.p.) TSOA TSCA

5%TW80 0.2ml/20g 40.0±5.6 101.2±12.2 Aridanin 5.0 135.6±17.0* 43.2±3.3 Aridanin 10.0 140.4±20.7* 50.8.4±10.0 Aridanin 20.0 38.2±4.0 125.8±15.1 Aridanin 30.0 29.8±1.7 124.0±15.0 Diazepam 1.0 170.4±6.7* 58.4±3.4

Treatment Dose (mg/kg, i.p.) TSOA TSCA

5%TW80 0.2ml/20g 40.0±5.6 101.2±12.2 Aridanin 10.0 140.4±20.7* 50.8±10.0 FLU 2.0 200.0±24.0* 51.2±10.2 FLU+ARI 10.0 35.8±7.9* 206.8±19.8*

Treatment Dose (mg/kg, i.p.) NEOA NECA

5%TW80 0.2ml/20g 4.8±0.6 7.8±1.1 Aridanin 10.0 6.0±0.5 4.4±0.5 FLU 2.0 2.7±0.4 4.5±0.5 FLU+ARI 10.0 2.4±0.5* 6.4±0.7*

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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

G. Sugumar

Department Of Fish Processing Technology,

Fisheries College and Research Institute,

Thoothukudi, Tamilnadu,

India.

U. Ramesh, 780, Karpaganagar 3rd Street,

K.Pudur, Madurai - 625 007.

ABSTRACT The effect of crab chitosan on the susceptibil-

ity of Staphylococcus aureus (ATCC 12598) was stud-

ied in samples of chitosan prepared from shells of

whole crab, carapace, leg and claw. S. aureus main-

tained 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 expo-

sure. 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. How-

ever, chitosan prepared from whole shell of crab re-

vealed 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, Phos-

phate Buffered Saline

INTRODUCTION

Chitosan, the deacetylated form of chitin, de-

rived from the exoskeleton of shrimp and crab waste

from fish processing plants has great potential for a

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 microor-

ganisms 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; Pap-

ineau et al., 1991; Sudarshan et al., 1992; Wang,

1992; Chen et al., 1998; Tsai and Su, 1999). The anti-

bacterial activity of chitosan has been attributed to the

alteration of cell permeability causing leakage of intra-

cellular 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 bac-

teria 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.


Preparation of chitosan

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

were separated as carapace, leg and claw. The shells

were deproteinized with 5% NaOH at the ratio of shell

to solution of 1:10 (w/v) at 120-130oC for 3he. The

deproteinized shells were filtered and washed with tap

water until NaOH was washed off completely, then

dried overnight in a hot air oven at 55-60oC. The de-

proteinized shells were demineralized by continuously


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 un-

til neutral. Then deacetylation of chitosan was carrie-

dout 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.

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.

Susceptibility test Stock solution of chitosan derived from cara-

pace, leg (swimming legs) and claw sells were prepared

separately in 1% acetic acid. For susceptibility tests,

chitosan of varying concentrations namely 0.01, 0.02,

0.03, 0.04 and 0.05% (w/w) were prepared in PBS and

the final pH adjusted to 6.4. Approximately 107 cells

were inoculated into all experimental flasks containing

chitosan solution to obtain an initial cell density of 105

- 106 cfu/ml. the cell counts were estimated at 0hr, 1hr,

3hr, 6hr, 12hr, 24hr, 48hr and 72hr of incubation at

room temperature 30o ± 0.2oC to check for inhibitory of

chitosan. Inhibition of S. aureus was initially checked

with chitosan derived from carapace, leg and claw at a

concentration of 0.05%.


Effect of chitosan derived from the crab shell

waste was evaluated against Staphylococcus aureus at

a concentration of 0.05% (w/w) in PBS and the results

are presented in Table 1. In PBS without chitosan

(control), the cell density remained almost the same

with mild fluctuations, while the chitosan suspensions

inhibited the inoculated cells considerably. Chitosan

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 suspen-

sion 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 ex-

posure to chitosan suspension prepared from claw

shell also the cells of S. aureus reduced drastically.

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 chi-

tosan prepared from shells of legs and claws were

slightly more effective than that prepared from cara-

pace 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 concentra-

tion 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.

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

shrimp chitosan (Wang, 1992; Chen et al., 1998;

Sugumar et al., 2003). It was also reported that cells of S. aureus were more susceptible to chitosan than other

human pathogenic bacteria (Wang, 1992; Sugumar et

al., 2003) reported effective concentrations of chito-

san, which may probably be due to experimental mate-

rials and conditions followed. Allan et al. (1984) re-

ported that complete inhibition of S. aureus was

possible only with 1% solution of chitosan, while

0.5% was found effective by Wang (1992). However,

0.05% of shrimp chitosan was found to completely

inhibit S. aureus both 5oC and at ambient temperature

on 24hr exposure (Sugumar et al., 2003). In the recent

investigation, even a concentration of 0.02% could

inhibit S. aureus in 24hr. Complete inhibition of S. aureus with crab chitosan (leg and claw shell derived)

at 0.05% less than 6hr testified the effectiveness of

chitosan. The rapidity at which the inhibition was

achieved and the low effective concentration of crab

chitosan render the chitosan as an effective agent in

containing S. aureus. Hence, crab chitosan could be

used to inhibit S. aureus in food products involving

human handling where there is possibility of contami-

nation of S. aureus.

Table 1. Effect of chitosan derived from different parts of crab on the susceptibility of

Staphylococcus aureus at a concentration of 0.05%

Sample Cell density (cfu/ml)

0hr 1hr 2hr 3hr 6hr

Control 7.9 x 105 1.2 x 10

6 9.1 x 10

5 1.0 x 10

6 8.0 x 10

5

Carapace chitosan 7.9 x 105 1.8 x 10

4 8.5 x 10

2 1.1 x 10

2 2.0 x 10

1

Leg chitosan 7.9 x 105 3.0 x 10

4 1.1 x 10

2 3.0 x 10

1 -

Claw chitosan 7.9 x 105 2.3 x 10

4 4.0 x 10

2 1.0 x 10

1 -

Table 2. Effect of crab chitosan on S. aureus at different concentrations

Concentration

of chitosan

Cell density (cfu/ml)

0hr 1hr 2hr 3hr 6hr 12hr 24hr

Control 2.5 x 106 9.5 x 10

5 3.5 x 10

5 7.8 x 10

5 2.8 x 10

5 2.7 x 10

5 2.6 x 10

5

0.01% 2.5 x 106 1.4 x 10

5 3.0 x 10

4 1.3 x 10

3 3.9 x 10

2 3.0 x 10

1 2.0 x 10

1

0.02% 2.5 x 106 1.1 x 10

5 1.3 x 10

3 2.0 x 10

2 3.2 x 10

2 1.0 x 10

1 -

0.03% 2.5 x 106 6.8 x 10

4 8.5 x 10

3 1.4 x 10

3 2.8 x 10

2 2.0 x 10

1 -

0.04% 2.5 x 106 6.5 x 10

4 2.6 x 10

3 1.8 x 10

3 7.9 x 10

1 - -

0.05% 2.5 x 106 2.0 x 10

4 8.6 x 10

2 1.1 x 10

2 1.0 x 10

1 - -


REFERENCES

Allan, G.G., Altman, L.C., Besinger, R.E., Ghose,

D.K., Neogi, A.N. and Neogi, S. (1984). Bio-

medical effects of chitin and chitosan, in Chi-

tin, 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 Develop-

ment. Food Technol., 38(1): 85 - 97.

Madhavan, P. and Nair, K.G.R. (1974). Utilization of

prawn waste isolation of chitin and its conver-

sion 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 Biotech-

nol., 6(3): 257 - 272.

Sugumar, G., Mariappan, S., Kalaimaniarasi, P., San-

geetha, S. and Velayutham, P., (2003). Anti-

bacterial 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 chito-

san J. Food Prot., 55: 916 - 919.


ORIGINAL PAPER

Assessing the Ecological Status of Prominent Medicinal Herbs in Central

Sudan and the challenge of Sustainable Management

H.H. EL-KAMALI

H.H. EL-Kamali

Department of Botany,

Faculty of Science and Technology,

Omdurman Islamic University,

P.O. Box # 382, Omdurman, Sudan.

ABSTRACT

Central Sudan has recently attracted scientific and socio

-economic attention that stimulated active research in-

terest 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 lati-

tudes 12º 43' – 17º 34' N and longitudes 30º 14' - 36º

15' E. This study was undertaken to explore and docu-

ment quantitative and qualitative ecological setting of

the prominent herbaceous medicinal herbs that are in-

digenous 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 even-

ness (equitability), physical and chemical characteristics

of the soil, and climatic elements such as rainfall, rela-

tive humidity and temperature. Variations within distri-

bution, productivity of each of the candidate species are

seemingly related to availability of favorable environ-

mental conditions in each of the three habitats (regions).

A major objective of active research programs is to out-

line 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

ining selected areas of Central Sudan especially where

a rich and varied medicinal flora is a prominent com-

ponent of the natural vegetation. The work-plan has

been carefully designed to :

(1) evaluate the relative ecological status of each stud-

ied species for the preservation of this invaluable in-

digenous medicinal resources.

(2) an attempt to understand the natural system of the

study area by the identification of the floristic compo-

sition.

(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 hos-

pitable habitats.

METHODOLOGY The work-plan proposed for the work-field of the pre-

sent work had been carefully programmed to explore

the ecology of the most popular medicinal and aro-

matic tropical herbs in Central Sudan. Special consid-

erations, 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 tubercula-

tum (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,


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


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 eco-

logical success of any species and can be used to ar-

range 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 spe-

cies / 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 equita-

bility (E) were determined according to Shannon-

Wiener function, quoted in Peet (1974). Statistical

evaluation was carried out using one-way analysis of

variance (ANOVA) and F-ratio was computed to com-

pare 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 fruit-

ing 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 experi-

enced local research person ell; in addition, it was felt

that compilation of answers of pre-set questionnaire

addressed to collectors, traders and local knowledge-

able on indigenous herbal medicine would add impor-

tant socio-economic dimension to the knowledge gen-

erated.

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 oc-

curring 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

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 lo-

cation, the natural plant cover is dominated by Haplo-phyllum tuberculatum "Haza" was located along per-

manent irrigation canal. This sub-region includes a

number of private farms cultivated mainly with Law-sonia 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 ap-

proximately 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 catch-

ment 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 me-

dicinal plants. The region is characterized by a dry,

hot climate, typically tropicale continental with a rela-

tively 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 yel-

lowish 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 mat-

ter 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 sub-

surface 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.


Successional changes since the mid 1970s highlighted

regional degradational changes that have been related

to progressive decline of rainfall means below the nor-

mal average. However, the 1988 rainfall has been ex-

ceptional, giving an undoubted recovery. The subse-

quent years were of below average means. The econ-

omy 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. Be-

cause of its economic benefits for gum Arabic collec-

tion, 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, longi-

tude 30 39 E. The terrain is seemingly very appropri-

ate 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 sea-

sonal rainfall amounting to about to 250 mm annually.

The general landscape can generally be classified into :

Acacia steppe , Goz and sand dunes and loamy depres-

sions.

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 ap-

proximately 600 meters above sea level. The major

studied medicinal herbs include: Cymbopogon nerva-

tus "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 dis-

tinctly dominated by moderately dry savanna vegeta-

tion 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 sa-

vanna to distinctly semi- desert type.

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 Sun-

flower 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 domi-

nated by Rhynchosia minima var. memnonia , Citrul-lus 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 dis-

tance 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 ni-grum , Leonitis nepetaefolia and Xanthium strumar-

ium.

OBSERVATIONS, RESULTS AND DISCUSSIONS: Reference must be made here to the theme of the re-

search, 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 char-

acteristics in each region have been according to care-

ful selection of study sites that represent locations that

accommodate vigorous stands of medicinal herbs,

locations that are indicative of poor vitality and , loca-

tions that occupy an intermediate position.

Region I (Ed-Damer) which is located in the drier

northern region is characterized by low mean of an-

nual 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 Ed-

Damer 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 ob-

tained from Hudieba and Ed-Deheira sub-regions

showed no significant difference between the two loca-

tions. On the other hands the results of analysis of vari-

ance 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.


Such results suggest the species predominating

Hudeiba and Ed-Deheira sub-regions enjoy better con-

ditions of the soil, together with seasonally sufficient

moisture content. This is expected in view of its rela-

tive 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 soil-

moisture 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 be-

tween 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 spe-

cies still maintains its former habitats in upstream parts

(margins) of water runnels which retain relatively shal-

low 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 Moh-

moud, 1971; Tag EL-Seed and Ismail, 1982; Ismail and

Babiker, 1986 and Yahia, 1992).

In West Central Sudan (Region II), various sub-

regions within the study area: Er-Rahad, Umm Ru-

waba and Bara exhibit a wide array of vegetation

cover. Many reasons may be ecologically responsible

for these variations. The terrain in this region is con-

spicuously diverse. Although the soil is predominantly

sandy, yet it includes local variations of silty depres-

sions, hard non-cracking clays and sandy loam. This,

in addition to South –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 vege-

tation 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 spo-

radic valley habitats among steep rocky areas and on

flat hill tops around the region e.g. FAO. It must be

noted that Rufaa sub-region, has a much lower spe-

cific diversity than Abu-Kashma valley sub-region.

Despite prolonged aridity and relatively poor soil the

environmental conditions in Rufaa sub-region are pre-

sumably favorable for the development of drought-

resistant grass Cymbopogon nervatus. This is particu-

larly 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 Rhyncho-

sia minima var. memnonia folk drugs in the candidate

listing are either not recorded or only superficially

known. However, for others viz. Cassia senna, Cym-

bopogon schoenanthus ssp. proximus, Haplophyllum tuberculatum, Datura innoxia, Citrullus colocynthis

and Ambrosia maritima, the usages were found simi-

lar 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 al-

ways be guaranteed either.

The collection, trade and supply of numerous medici-

nal 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 op-

portunities for the poor people. There are many con-

strains 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 strat-

egy 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, strate-

gies and put forwards plans for its sustainability. As the

natural habitats for wild plants may suffer and indige-

nous species may be threatened, endangered or even dis-

appear completely, it may become critical to develop

alternative sources of important natural products. Tissue-

cultured cells of higher plants typically accumulate

amounts of secondary

metabolites only when subjected to specific conditions.

The leading herbs Cymbopogon schoenanthus ssp. proxi-

mus and C. nervatus plants are seasonal and the quality of

its essential oils varies according to fluctuations in eco-

logical 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, Su-

dan. Fitoterapia 57(4):263-266.

Kershaw K.A. (1979).Quantitative and Dynamic Plant

Ecology. 2nd Edition. Eduard Arnold (Publishers) Ltd.

London.

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 Univer-

sity, Sudan.

Obeid M. and Mahmoud A. (1971). Ecological Studies in

the Vegetation of the Sudan II. The Ecological Relation-

ships of the Vegetation of Khartoum Province. Vegetatio

23: 3-4, 177-198.

Peet R.K. (1974). The Measurement of Species Diver-

sity. 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 Eco-

logical 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 pro-

cera, Cassia senna and Aerva javonica in Relation to

Mineral Nutrient Factors in the Central Sudan. M.Sc.

Thesis, Khartoum University, Sudan.


Tables Contd.

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

Region / Sub-region

Plant species

Relative

frequency

Relative

density

Relative

basal

cover

Importance

Value Index

Region I

Sub-region : Hudeiba area

Francoeuria crispa 52.17 49.56 44.5 146.23

Heliotropium aegyptiacum 47.83 43.36 26.5 117.69

Sub-region : Hasaya area

Fagonia cretica 28.0 22.02 10.0 60.02

Cassia senna 28.0 36.86 32.0 96.86

Citrullus colocynths 36.0 26.61 40.0 102.61

Tribulus terrestris 4.0 4.59 1.0 9.59

Panicum turgidum 4.0 11.93 1.0 16.93

Sub-region : Ed-Deheira valley

Panicum turgidum 50.0 39.46 32.25 121.7

Cymbopogon schoenanthus ssp.

proximus

45.45 5.83 23.0 74.28

Indigofera spinosa 4.55 34.98 15.0 54.03

Region II

Sub-region : Er-Rahad area

Sporobolus pyramidatus 6.3 21.4 26.0 53.7

Geigeria alata 9.5 15.3 14.0 38.8

Alysicarpus glumaceus 6.3 11.5 11.0 28.8

Cenchrus sp. 7.9 6.9 4.0 18.8

Aerva javanica 6.3 6.9 3.0 16.2

Gueira Senegalese's 12.7 6.1 10.0 28.8

Solanum dubium 6.3 5.3 2.0 13.6

Citrullus lanatus 6.3 5.3 10.0 21.6

Aristida mutabilis 7.9 3.8 2.0 13.7

Chloris brevista 6.3 3.1 6.0 15.4

Cucumis dispaceus 6.3 3.1 3.0 12.4

Stylochiton borumensis 4.8 3.1 2.0 9.9

Echinochloa colona 4.8 2.3 3.0 10.1

Euphorbia aegyptiaca 4.8 2.3 1.0 8.1

Dactyloctenium aegyptium 1.6 0.8 1.0 3.4

Bergia suffruticosa 1.6 0.8 1.0 3.4

Ethulia conyzoides 1.6 0.8 1.0 3.4

Sub-region : Umm Ruwaba area

Indigofera oblongifolia 18.9 55.2 26.0 100.1

Datura innoxia 35.1 20.9 29.0 85.0

Abutilon figarianum 16.2 9.7 16.0 41.9

Cassia occidentalis 18.9 9.0 26.0 53.9

Aristida sp. 2.7 3.0 1.0 6.7

Calotropis procera 5.4 1.5 1.0 7.9

Cassia tora 2.7 0.7 1.0 4.4

Sub-region : Bara area

Cassia senna 46.2 46.4 55.0 147.6

Jatropha glauca 30.8 42.9 30.0 103.7

Zaleya pentandra 15.4 8.9 10.0 34.3

Blepharis ciliaris 3.8 1.8 1.0 6.6

Cenchrus sp. 3.8 1.8 1.0 6.6

Region III

Sub-region: Ab-Kashma valley

Rhynchosia minima var. memnonia 71.9 73.3 69.0 214

Setaria incrassate 21.8 20.9 16.5 59.2



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

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, dis-

solved solids and microbial study of the samples were

analysed. The tests showed that the samples of Mela-

palayam 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 bacte-

riologically contaminated. It was concluded that the

samples of river water were polluted mostly and associ-

ated 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 com-

modity. 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 in-

creasing 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

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 agri-

culture 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, hos-

pitals and hotels on the river banks and canals the wa-

ter 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, photo-

graphic 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 cellu-

losic wastes, mercaptans, mercury etc. canals also

highly polluted by sewage disposal from theater, ho-

tels, 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)

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: [email protected]


MATERIALS AND METHODS The water quality parameters decide the pota-

bility of water water samples are taken from five differ-

ent 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)


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 differ-

ences 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 al-

most 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 Mela-

palayam 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 moni-

tored regularly and allowed to discharge into the river

only after satisfactory clean up treatment.

Underground drainage system is built within munici-

pal 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 alterna-

tive source of income, treated waste water can be used

for irrigation.

Microbial disinfectants should be employed in the

water resources.


S.

No Place

Physio-chemical and Bacteriological variable

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

Table 1

0

500

1000

1500

2000

2500

3000

3500

PH Turbidity Cl Coliform

study

Physio-chemical and Bacteriological variable

Melapalayam canal Palayam canal Kottur 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. Pra-

gati 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. Environ-

mental Problems in Tirunelveli 06.

Murugesan, A.G. 2001. Environmental status of the

perennial river Thamirabarani with special

reference to domestic and industrial pollution.

Proc. Workshop and enhancing Public Aware-

ness 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.



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


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 con-

ventional propagation by stem cuttings and seeds. Ef-

fect of phytohormones, growth adjuvants, sugars and

essential minerals on the in vitro response of M. alba

explants has been described in the study. Phytohor-

mones and growth adjuvants resulted in explicit re-

sponse in terms of organogenesis. Sucrose and glucose

containing culture media elicited best response among

sugars in nodal explants. Typical symptoms were ex-

hibited 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- Dichloro-

phenoxy acetic acid; NAA: α - Naphthoxy Acetic

Acid; Indole -3- Butyric Acid; Kn: Kinetin; BAP: 6-

Benzyl Amino Purine

INTRODUCTION The mulberry plant (Morus alba L.) is chiefly ex-

ploited by the sericulture industry for its foliage, used

as feed for silk worm (Bombyx mori L.), and for feed-

ing 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

of dietary supplements such as proteins, carbohy-

drates, fats, fibers, essential minerals (Srivastava et al,

2006), ascorbic acid and β-carotene (Ercisli and Or-

han, 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, anti-

tumour 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 convention-

ally for propagation of mulberry plants which have

assumed tremendous economic importance over the

years. Genotype, environmental factors and physio-

logical state of the cuttings play a significant role in

determination of success of rooting (Lu, 2002). Propa-

gation 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 alterna-

tive, providing an effective means of rapid propaga-

tion of uniform genotypes of the plant. Application of

modern techniques for development of new high-

yielding varieties and cultivars depends on the avail-

ability of an efficient in vitro regeneration system

(Agarwal et al, 2004). This communication provides

an account of studies on the in vitro response of stem-

bit and nodal explants of M. alba to select phytohor-

mones, growth adjuvants, minerals and sugars. This

study was designed with the aim of identifying spe-

cific growth supplements which would have a positive

influence on growth of in vitro plantlets. Another ob-

jective of the study was to evaluate the occurrence of

the known in vivo physiological

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


effects of some of the selected supplements on the

plantlets in vitro.


Local varieties of M. alba were procured from local

farms in and around Puttaparthi and grown in the ex-

perimental 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 Mercu-

ric Chloride and rinsed five to six times with sterile

double distilled water.

The surface-sterilized explants were inoculated on Mu-

rashige and Skoog medium (Murashige and Skoog,

1962) under a laminar flow hood (KLENZAIDS, In-

dia). The cultures were maintained at 24 ± 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 solu-

tions 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 galac-

tose at a concentration of 3% (w/v) was used to study

the effect of different sugars. Basal MS medium with-

out addition of any supplements represented the con-

trol.

RESULTS AND DISCUSSION Studies on the in vitro response of explants of M. alba

have been undertaken to understand the effects of dif-

ferent 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 phytohor-

mones on in vitro culture of M. alba. Callus initiation

and proliferation occurred in MS medium supple-

mented 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 effec-

tive in initiating a response in terms of shoot forma-

tion and growth; the former resulting in development

of unbranched roots and the latter, profusely branched

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 com-

monly, have used MS medium culture of nodal cut-

tings 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 fre-

quency with the addition of GA3. The combination of

NAA and BAP induced shoot proliferation in mul-

berry and NAA, in particular, was found to be essen-

tial 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 re-

sults in shoot length, leaf growth and the development

of a highly branched, extensive root system. This was

followed by medium containing fructose. Quite nota-

bly, 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 carbohy-

drate metabolism, or its derivatives to enter the glyco-

lysis cycle (Brownleader et al, 1997). The varied re-

sponse to the different sugars reflects on the ability of

the plant in converting these sugars into suitable gly-

colysis 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 Pad-

maja (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 advanta-

geous for the sericulture industry.

Alteration in the mineral constitution of MS me-

dium produced pronounced physiological effects that

reflected in the morphology of the developing ex-

plants.


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 re-

sulted 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 co-

enzymes 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 chloro-

phyll 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 estab-

lished 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 gen-

erated plants to ascertain changes in phytochemical

constituents which may have a bearing on the feeding

habits of silkworms reared on mulberry. Similar stud-

ies 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

length (cm)

Average leaf

width (cm)

Root system

Monosaccharides

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

Disaccharides

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

Table 2: Effect of minerals on explants of M. alba

Alteration in MS medium

composition

Effect on growing explants

MS without Nitrogen

(NH4NO3 and KNO3)

Shoots got dried up; leaves were shriveled and gradually

turned yellow; rooting was negligible

MS without Sulphates

(MgSO4, MnSO4 ,FeSO4 and

CuSO4)

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

green to yellow in colour with black tips, roots were

profusely branched


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2004;102:359-368

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mulberry (morus alba l.) through in vitro cul-

ture of shoot tip and nodal explants. Plant Tis-sue Culture. 2003;13:47-51

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ties of various solvent extracts of mulberry

(morus indica l.) leaves. Food Chem. 2007;102:1233-1240

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355

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 monosac-

charides In: Dey P and Harborne J. Plant bio-

chemistry. Academic Press, Great Britain.

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Enkhmaa B, Shiwaku K, Katsube T, Kitajima K,

Anuurad E, Yamasaki M and Yamane Y. Mul-

berry (morus alba l.) leaves and their major

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attenuate atherosclerotic lesion development in

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berry by means of tissue culture. JARQ. 1987;21:205-210

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(morus alba), red (morus rubra) and black

(morus nigra) mulberry fruits. Food Chem.

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propagation as an aid for cloning ofmorus

laevigata wall. Plant Cell Tiss Org Cult.

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 green-

house tomato. Sci Hort. 1996;67:207-217

Lu M. Micropropagation of morus latifolia poilet us-

ing 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 tis-

sue cultures. Physiol Plant. 1962;15:473-497

Ohyama K. Tissue culture in mulberry tree. JARQ.

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602



Received: 29 May 2010 / Accepted: 12 June 2010 /Published online: 22 June 2010


ORIGINAL PAPER

Antimicrobial activity of marine bacteria associated with Polychaetes

C.V. SUNJAIY SHANKAR* A. HEPZIBA JEBA MALAR

S. MARY JOSEPHINE PUNITHA

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 tradi-

tional 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 bac-

teria 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 ; Anti-

bacterial 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, microor-

ganisms- eukaryote associations have gained signifi-

cant 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

also been illustrated by studies that have shown the

presence of unique stable communities living on geo-

graphically 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 habi-

tat for many unique microorganisms, which produce

biologically active compounds to adapt to particular

environmental conditions. For example, marine sur-

face associated microorganisms have proven to be a

rich source for novel bioactivities because of the ne-

cessity to evolve allelochemicals capable of protecting

the producer from the fierce competition that exists

between microorganisms on the surface of marine eu-

karyotes (Anahit Penesyan et al., 2010).

The number of natural products, discovered from vari-

ous 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 hu-

man diseases for millennia, and their use has been

recorded in the most ancient archaeological sources.

In contrast, the exploration of microorganisms as pro-

ducers 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% bacte-

rial 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 parapo-

dia that bear many bristles

C.V. Sunjaiy Shankar Centre for Marine Science and Technology,

Manonmaniam Sundaranar University,

Marina campus, Pannaiyoor,

Rajakamangalam-629502,

Tamil Nadu, India.

E-mail: [email protected]


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.


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 organ-

ism 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 di-

luted and appropriate dilutions were poured on zobell

marine agar. The plates were incubated at room tem-

perature 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 colo-

nies were chrecterized based on Bergey’s manual of

determinative bacteriology.

Isolation of Extracellular Polymeric Substance

(EPS) of the Bacteria associated with Polychaetes-

The amount of EPS produced by the cultures was de-

termined by estimating total carbohydrates. 100ml

culture broth was centrifuged at 5000 rpm for 15 min-

utes at 4oC. The cell pellets were discarded and the

supernatant was mixed with equal amount of cold ab-

solute ethanol. After one day incubation the precipi-

tated 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 pre-

sent 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 meas-

ured.

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

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 characteris-

tics of the isolated strains are given table-I.

Most of the strains are gram-negative, cocci and non-

motile. Strain A isolated from the polychaete was

gram-negative, cocci and showed motility. It was in-

dole 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 re-

duction 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 posi-

tive. It also utilized citrate and positive for starch hy-

drolysis and negative result for urea. In triple sugar

iron test, strain B showed positive result for acid, al-

kali. 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 poly-

chaete 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 produc-

tion and negative for hydrogen sulphide production.

Strain C also showed positive result for catalase, oxdi-

ase 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 antim-

icrobial activity against the four bacterial strains

(Galionella sp., Alteromonas sp., S.aureus and Kleb-

siella sp.). and the results are given in (Table -2).

The EPS isolated from the strain A showed inhibi-

tory 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 Kleb-

siella 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-

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 chro-

matogram 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 microor-

ganisms for the maintainance of antimicrobial de-

fenses. 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 com-

pound by bacteria when they are exposed to a different

strain of bacteria suggest that competition for space

between epibiotic bacteria may provide further anti-

fouling protection to the basibiont. In addition, some

bacteria that previously did not produce such metabo-

lites when they are exposed to other bacterial species

or extracellular products from other bacteria. As sur-

face-associated bacteria would be exposed to similar

pressures in the surface biofilm, this occurrence fur-

ther 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. Vari-

ous 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

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 poly-

chaetes.

The diversity of antibiotic producing marine bacteria

isolated in the present study suggests that polychaetes

are rich of bacteria (Hentschel et al., 2000). The bacte-

ria 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 Wein-

heimer 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 diver-

sity was not much studied from Indian marine ecosys-

tem 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 asso-

ciated 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 applica-

tion. Hence , more studies on the characterization of

the isolated strains may improve our understanding on

the chemical ecology of bacteria associated with poly-

chaetes.



Rf values (cm) Strain A Strain B Strain C

0.75 0.72 0.53

Table -3 Rf value of compounds observed in the TLC


Table-1 Biochemical and physiological characteristics of the Bacterial strains

S.No Biochemical and physio-

logical Test

Organisms

Strain A Strain B Strain C

1 Gram Staining - - +

2 Morphology cocci cocci cocci

3 Motility + - -

4 Indole production + + +

5 Methyl Red + - +

6 Voges-Prousker + + +

7 Citrate utilization + + +

8 Starch Hydrolysis - + -

9 Urea Hydrolysis - - -

10 TSI Agar test

(i)Acid

+ + +

(ii)Alkaline + + +

(iii)Gas Production + + +

(iv)H2S Production - - -

11 Catalase + + +

12 Oxidase + + +

13 Nitrate Reduction + - +

14 Casein Hydrolysis + + -

15 Gelatin Hydrolysis - + +

Table- 2 Antibacterial activity of EPS against target Bacteria

S.No

Target Bacteria

Diameter of zone of inhibition(mm)

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

REFERENCES Anahit penesyan, Staffan kjelleberg and Su-

helen Egan, 2010. Development of novel drugs from

marine surface associated microorganisms , Mar.

Drug.,8:438-459.

Berdy, J., 2005. Bioactive microbial metabo-

lites .A personal view. J. Antibiot.(Tokyo)45 : 581-

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

Bernen, V.S., M. Greenstein and W.M Maese,

1997. Marine microorganisms as a source of new

natural products. Advances in Applied Microbiology,

Burgess J.G., E.M. Jordan, M. Bregu, A.

Maerns-spragg and K.G. Boyd, 1999. Microbial an-

tagonism: a neglected avenue of natural products re-

search J. Biotechnol., 1:27-32

Correa, J.A., 1997. Infectious diseases of ma-

rine algae: Current knowledge and Approaches,

Prog.Phycol.Res.,12:149-180.

Engel S., P.R. Jensen , and W. Fenical, 2000.

Chemical ecology of marine microbial defense. J.

Chem. Ecol., 28 (10),1971-1985.

Egan, S., T. Thomas, S. Kjelleberg, 2008.

Unlocking the diversity and biotechnological potential

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 Bioac-

tive microbial products: focus upon mechanism of

action. Annu. Rev. Microbiol. , 44 : 271-301

Perez–Matos, A. E., W. Rosado, N.S. Govind ,

2007. Bacterial diversity associated with the Carib-

bean tunicate Ecteinascidia turbinate. Antonie Van

Leeuwenhoek. 92 : 155-164.

Patterson ,G.L. and C.M. Bolis, 1997. Fungal

cell-wall polysaccharides elicit an antifungal secon-

dary metabolite(phytoalexin) in the cyanobacterium

Scytonema ocellatum. J.phycol., 33:54-64.

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

Drugs from the seas-current status and microbiologi-

cal implications. Appl. Microbiol. Biol., 59: 125-134.

Taylor, M. W., R. T Hill, J. Piel, R. W.

Thacker and U. Hentschel, 2007. Soaking it up: the

complex lives of marine sponges and their microbial

associates ISME journal, 1:187-190

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

The occurrence of two new prostaglandin derivatives

(15-epi-PGA2 and acetate, Methyl ester) in the gorgo-

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ates.XV. Tetrahedron let 15:5185-5188.

Webster, N.S. and D. Bourne, 2000. Bacterial

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ORIGINAL PAPER

Laboratory culture of Microzooplankton Tintinnopsis cylindrical [Daday 1887]

S.VIJAYARAGAVANA, P.VIVEK RAJA

B



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 Tintin-

nopsis 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 microzoo-

plankton, Tintinnopsis cylindrica, under laboratory con-

dition and intensive monitoring of the water quality

(management) and species density during culture days.

Keywords : Tintinnopsis cylindrica, prawn and fish ju-

veniles.

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 emphasiz-

ing cultivation of suitable planktonic flagellates and mi-

crozooplankton. These protozoans could well meet the

essential criteria for ideal hatchery food organisms. They

have high rate of reproduction many can be mass culti-

vated, 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 de-

tergent 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 loca-

tion 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 intro-

duced 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 35oC.

The taxanomic position of Tintinnopsis cylindrica.

Phylum: Protozoa

Class : Ciliata

Order : Spirotricha

Sub order: Tintinnida

Family : Codonellidae

Genus : Tintinnopsis

Species : Cylindrica

S.VijayaragavanA, P.Vivek RajaB

A. Department of Zoology Presidency college Chennai

600005 Tamilnadu-India.

B. Department of Zoology Presidency college, Chennai

600 005, Tamilnadu


The identification characters of Tintinnopsis cylindrical

1)Almost cylindrical for about 2/3 bowl. Then some time

slightly inflated before narrowing to a stout pedicel.

2)The length is 120-240 µm, the microzoplankton were

counted (At 100 x magnification) using binocular micro-

scope and expressed as number of organism lit.

RESULTS :

During the present culture period, population density of

tintinnopsis was measured from first day to seventh day.

The Tintnnopsis cylindrica maximum concentration

reached during 5to7 days. The final day density was

40000 individuals liter (Table, 1) Fig .1

Water Quality Management

The major factor that greatly influence the rearing of mi-

crozooplankton by maintaining water quality manage-

ment. The penicillin and streptomycin (50mg/l) was

added which controlled the bacterial assemblage.

Temperature:

The temperature is important parameter in culturing sys-

tem, which ranged between 25 and 30o C, which was

favorable for the culture of microzooplankton.

Salinity:

Salinity maintenance is very important during microzoo-

plankton culture period, which was maintained between

30 and 35 ppt.

pH :

The maintenance of pH is important during the culture of

microzooplankton which was always maintained be-

tween 7.0 and 8.5.

Dissolved oxygen:

The dissolved oxygen content should be kept near the

saturation level for good health of culture organisms: the

dissolved oxygen level was maintained between 5.5 and

7.5 using an aerator vigorous aeration was provided.

Table- I

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 reproduc-

tion is of sexual activity by conjugation. ( Omarai and

Iketa, 1976; Kamamiyama and Aizawa, 1981). The

tintinnids ciliated need intensive water quality arrange-

ment and appropriate concentrations of micro flagellates

(or) dinoflagellates filtered through sterilized sea water.

(Gold, 1971; Paranjape,1980).

The major factor for the successful laboratory culture of

Tintinnids ciliates appears to be avoidance of metal and

solvent contamination and turbulence of culture medium

as reported earlier by Gifford (1985). During unfavorable

conditions it is making cyst formation and during favor-

able condition it can go for excitement as reported by

Kamiyama (1997), Paranjape (1980) Reid and John

( 1978 ).

It has been reported that the Tintinnopsis require 3 to 7

days period for the production of 1000cells/L (Gold

1968). In the present study of microzooplankton culture

which mainly depended on the diet consisted of algal

Chlorella marina and alternatively yeast (were provided)

but the algal diet stimulate much more density of micro-

zooplankton similar observations were earlier reported

by Kamiyama and Aizawa (1992) and Omari and Ikeda

( 1976).

In the present study, the population density of microzoo-

plankton increased day by day for the period of 7 days

and on the final day (7th day) attained its maximum popu-

lation density (40,000 organisms /L). because the micro-

zooplankton have a high reproduction rate in the tropical

waters and due to short regeneration period within weak

as reported earlier by Gold (1968). During the microzoo-

plankton culture, the temperature and food concentration

are the two of the main factors affecting the grazing rate

of microzooplankton a reported earlier by Hirayama and

Ogawa (1972),Dhert(1996),Nevarro (1999),Mantagnes et

al ., ( 2001).

ACKNOWLEDGEMENT :

The author (VR) is thankful to Dr..P.Vevek Raja and

Mr.T.S.Ragavan, Chennai for financial assistance.

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.

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


Gold K.(1971) Growth characteristics, of the

mass –reared tintinnid, Tintinnopsis beroidea .Mar.Biol

8:105-108.

Gold, K.(1968) Some observation on

the biology of Tintinnopsis sp.J.Prozool. 16:507-509.

Hardin, G.C(1974) .The food of deep

sea copepods J.Mar Bio.Ass U.K.54:141-155

Kamiyama,J. (1997) Growth and graz-

ing responses of tintinnids ciliates feeding on the toxic

dinoflagelate (Heterocapsa circularisquama)

Mar.Biol.128: 509-515.

Kamiyama,T and Y Aizawa (1981) Growth char-

acteristics 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, 58:877-

884.

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.

Omari, M and J.Iketa (1976) .Methods in marine

zooplankton ecology. Jhon wiley and sons publication,

Newyork. P.332.

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.

Karuppasamy, P.K.(1997).Studies on zooplank-

ton in the pichavaram mangroves and laboratory culture

of rotifer (Brachionus plicatilis). M.Phil, Theses, Anna-

malai 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 signifi-

cance of resting cysts in a hyaline tintinnid. Heli-

costomella 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.38:1207-

1214.

Navaro,N.(1999). Feeding behavior of the roti-

fers Brachionus plicatilis and Brachionus votundiformis

with two types of food: live and fresh dried micro algae

J.Exp Mar.Biol.Ecl.237.

The Population density of Tintinnopsis cylindrica during the culture period

40000

32000

26000

16000

10000

6000

3000

1000

1 2 3 4 5 6 7

GRAPH -1

Period (Lays)

Statistics (Desirevable)


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



ABSTRACT Acute toxicity experiments for Fenvalerate tech-

nical 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, one-

tenth of the 24 h LC50 was taken as sublethal concentra-

tion 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 delete-

rious than technical grade of fenvalerate. During experi-

mentation, severe respiratory distress, rapid opercular

movements leading to the higher amount of toxicant up-

take, increased mucus secretion, higher ventilation vol-

ume, 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 con-

centrate, Metabolic rate etc.

Corresponding author: [email protected]

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 immedi-

ate 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 organ-

isms to pollutants has provided essential tools for vigi-

lant contamination monitoring (Korami et al. 2000). As

pyrethroids are found to be less persistent and relatively

safe as compared to organochlorines and organophos-

phates, 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 me-

dium when pesticides, chemicals, sewage and other ef-

fluents containing organic matter are discharged into

water bodies. Pesticides in sublethal concentrations pre-

sent 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 fit-

ness of natural population. In the aquatic environment

one of the most important manifestation of the toxic ac-

tion of chemical is the over stimulation or depression of

respiratory activity. The changes in the respiratory activ-

ity 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 indica-

tor of energy expenditure in particular and metabolism in

general (Prosser and Brown, 1977).

T. ANITA SUSAN1, K. SOBHA2 and K.S. TILAK3

1 Department of Zoology, Andhra Christian College, Gun-

tur, 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

Pesticides are indicated to cause respiratory dis-

tress or even failure by affecting respiratory centres of

the brain or the tissue involved in breathing. The effect

of toxicants on the respiration of fishes and invertebrates

have received wide spread attention and were reviewed

by Hughes (1976) and Wright (1978).

As aquatic organisms have their outer bodies and

important organs such as gills almost entirely exposed to

water, the effect of toxicants on the respiration is more

pronounced. Pesticides enter into the fish mainly

through gills and with the onset of symptoms of poison-

ing, the rate of oxygen consumption increases ( Premdas

and Anderson, 1963; Ferguson et al., 1966a). Holden

(1973) observed that one of the earliest symptoms of

acute pesticide poisoning is respiratory distress. This

serves not only as a tool in evaluating the susceptibility

or resistance potentiality of the animal, but also useful to

correlate the behaviour of the animal.

By cannulating the blood system of fishes, it is possible

to measure the concentrations of oxygen, metabolites and

pollutants and hence understand more fully the mode of

action of toxic pollutants. Skidmore (1970) using cannu-

lation techniques, found that zinc reduced the oxygen

level of blood leaving the gills. It reduced the efficiency

of oxygen transport across the gill membrane, so that fish

die of hypoxia. Respiratory responses to lethal concen-

trations increase the ventilation volume and symptoms of

pyrethroid intoxication suggesting that the effect on res-

piratory surface are lethal in fish. It is known that pyre-

throids are less persistent and are effective subsituents

for organochlorine (OC) pesticides. L i k e O C

compounds, the mechanism of pyrethroid interference is

with nerve membrane function through the interaction

with the sodium channels. The symptoms of fenvalerate

intoxication suggest that, besides effect on the nervous

system, effect on respiratory surfaces and renal ion regu-

lation may be associated with the mechanism of lethality

in fish (Bradbury et al., 1987). The toxic effects of pyre-

throids on the metabolism particularly oxygen consump-

tion have been reviewed by Bradbury et al., 1986; Ku-

maraguru and Beamish, 1983; Haya and Waiwood, 1983;

Pandi baskaran, 1991.

Total oxygen consumption is one of the indicators of the

general well being of the fish. It may also be useful to

assess the physiological state of an organism, helps in

evaluating the susceptibility or resistance potentiality and

also useful to correlate the behaviour of the animal,

which ultimately serve as predictors of functional disrup-

tions of population. Hence the analysis of oxygen con-

sumption can be used as a biodetectory system to evalu-

ate the basic damage inflicted on the animal which could

either increase or decrease the oxygen uptake. Therefore

an attempt was made to study the effect of sublethal and

lethal concentrations of fenvalerate, technical grade and

20% active ingredient emulsifiable concentrate (EC) on

oxygen consumption for twelve hours to the three Indian

major carps, Labeo rohita, Catla catla and Cirrhinus

mrigala.

MATERIALS AND METHODS The fish were brought from a local fish farm and accli-

matized to the laboratory conditions in well aerated wa-

ter for one week. The water used for acclimatization and

experimentation was the same as used in the toxicity

experiments (Table 1). During this period the fish were

regularly fed, but the feeding was stopped for two days

prior to the experiment. The fish measuring 6 to 7 ± 0.5

cm in length and 6 to 8 ± 0.5 gm in weight were used in

the experiment. Toxicity experiments for fenvalerate

technical grade and 20% EC formulation were conducted

on the fingerlings of the three carps, Labeo rohita, Catla

catla and Cirrhinus mrigala, using static and continuous

flow through systems for 24h, 48h and 96h exposures.

The experiments on the oxygen consumption of the fish

Labeo rohita, Catla catla and Cirrhinus mrigala were

carried out in a respiratory apparatus developed by Job

(1955). The sublethal and lethal concentrations of the

toxicants used for the three carps for oxygen consump-

tion studies are presented in Table 2.

The respiratory chamber consists of a wide mouthed bot-

tle fitted with a rubber stopper with three holes. A glass

tube which serves as an inlet passes through one hole,

through the other hole passes a glass tube with a regula-

tor serving as an outlet. Another hole is fitted with a

glass tube and regulator which serves as atmospheric

vent.

The test fish in good condition were taken and intro-

duced into respiratory chamber filled with water. The

respiratory chamber was closed without air bubbles and

sealed with paraffin wax. The respiratory chamber is

connected to a 25 litre capacity reservoir through an

inlet. The flow rate was so adjusted, that one litre of

water flows per hour.

In each experiment, two respiratory chambers, one with

fish and another without fish (i.e. control) were taken.

The control serves to estimate initial amount of oxygen

present. After one hour of acclimatization of the fish,

desired concentration of the toxicant was added to the

reservoir and flow rate was adjusted. After fifteen min-

utes, the water of the respiratory chambers was replaced

by the test water containing the toxicant, and the experi-

ments with fenvalerate technical grade and 20% active

ingredient emulsifiable concentrate were conducted for

twelve hours. At the end of each hour, samples (50 ml)

were collected and the amount of oxygen present was

estimated by Winkler’s method (Golterman and Clymo,

1969). The difference in the rate of oxygen consumption

between the control and the test fish denotes the effect of

the toxicant on oxygen consumption.

Like wise, experiments were conducted in sublethal and

lethal concentrations of fenvalerate technical grade and

20% active ingredient EC and the data was compared

with that of control. The sublethal and lethal concentra-

tions of the two toxicants tested throughout the study are

given in Table 2.

The amount of oxygen consumption was calculated using

the formula:


O2 mg/L = 8 x 1000 x Normality of Hypo x Vol.of Hypo rundown x Vol of the respiratory chamber

Vol. of sample taken x Correction factor


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 fen-

valerate. Higher mortality rate, almost double was re-

corded in winter ( 180 C ± mean average temperature)

than in summer ( 320 C ± mean average temperature).

Thus for pyrethroids, there appears to be an inverse rela-

tionship between temperature and toxicity.

When fish were exposed to sublethal and lethal concen-

tration of fenvalerate, several behavioural changes were

observed which include swimming at the surface of wa-

ter. This surfacing phenomenon was more in fish ex-

posed to lethal concentration and sublethal concentration

over the control fish. Hyperexcitation, loss of equilib-

rium, 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 ac-

quired 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 fenvaler-

ate 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 move-

ments, the toxicant exposed fish exhibited irregular, er-

ratic 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 re-

ported erratic swimming of the test fish, their increased

surfacing, decreased rate of opercular movement, copi-

ous 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 toxi-

cants 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-

tom with the least opercular movements and die with

their mouth opened. In sublethal exposures, the fish

body becomes lean towards abdomen position as com-

pared to control owing to reduced amount of dietary pro-

tein 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, cal-

culated 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 oxy-

gen consumption, there was significant increase in oxy-

gen 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 consump-

tion decreased than that of controls.

Similarly, when a comparison is made between the ef-

fects of lethal concentrations of fenvalerate technical

grade and 20% active ingredient EC among Labeo ro-

hita, Catla catla and Cirrhinus mrigala, highest oxygen

consumption rates were attained during 2nd 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 mri-

gala as is evidenced by its death during fifth hour in le-

thal 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 con-

sumption 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 venti-

lation volume, decrease in the oxygen uptake efficiency,

laboured breathing and engulfing of air through the

mouth were observed in all the three major carps ex-

posed 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 consump-

tion 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 up-

take during first two hours of exposure followed by de-

crease in subsequent hours in Channa striatus exposed to

cypermethrin. Similar trend was reported in L. rohita

(Raju, 1991) and C. punctatus (Jeevaprada, 1990) ex-

posed to cypermethrin. Bradbury et al. (1986) stated that

the greater decrease in the rate of oxygen consumption

in the fish Cirrhinus mri-

gala may be due to inter-


sub cellular level. Similar observations were also reported

by Mushgeri and David (2003) and Jadhav and Sontakka

(1977). The decrease in oxygen consumption at sub le-

thal concentrations of the toxicant indicates lowered en-

ergy requirements which in turn indicates pronounced

haematological changes (Tilak and Satyavardhan, 2002).

Similar reduction in oxygen consumption has been re-

ported in Channa striatus exposed to organophosphate

pesticide (Natarajan, 1981), O. mossambicus due to or-

ganochlorine intoxication (Vasanthi and Ramasamy,

1987), M. cupanus following carbamide treatment

(Arunachalam and Palanichamy, 1982), and C. punctata

exposed to carbaryl (Tilak, 1979).

Changes in the gill surfaces and increased mucus produc-

tion is consistent with observed histological effects such

as hyperplasia, necrosis and lamellar aneurysms in all the

three fish exposed to sublethal concentration of technical

grade fenvalerate. Kumaraguru et al. (1982) reported

that the gill is the target organ for synthetic pyrethroid

toxicity in fish. The technical as well as commercial

formulations will pass through the gills, and interfere in

the gill movements which is directly proportional to the

respiratory activity of the fish, primarily effecting the

oxygen uptake. The respiratory metabolism was im-

paired and damage was also observed in the gill of fish

exposed to pesticides (Hughes and Perry, 1976; Rama

Murthy, 1988). In the freshwater fish, Ctenopharyn-

godon idella exposed to NuvanÒ, an organophosphate,

the depletion of the oxygen consumption is due to the

disorganization of the respiratory action caused by rup-

ture in the respiratory epithelium of the gill tissue. The

experimental data reveals that oxygen consumption de-

creases with the time of exposure to toxicant (Tilak and

Swarna kumari, 2009).

Under toxic conditions, the oxygen supply becomes defi-

cient and a number of poisons become more toxic in-

creasing the amount of poison being exposed to the ani-

mal. The fish breathe more rapidly and the amplitude of

respiratory movements will increase. Lloyd (1961) re-

ported that the toxicity of several poisons to rainbow

trout increased in direct proportion to decrease in oxygen

concentration of water. In general, it is observed that the

lack of oxygen increases the ventilation volume of fishes

and the cardiac output is reduced. This reduces the rate

of passage of blood through the gills, so allowing a

longer period of time for uptake of oxygen, and also con-

serves oxygen by reducing muscular work. The zone of

resistance is reached when the oxygen tension in the wa-

ter is so low that homeostatic mechanisms of the fish are

no longer able to maintain the oxygen tension in the af-

ferent blood and the standard metabolism begins to fall.

Changes in the architecture of gill under fenvalerate

stress would alter diffusing capacity of gill with conse-

quent hypoxic/anoxic conditions and thus respiration

may become problematic task for the fish. The results of

the present study suggest that the altered rates of respira-

tion of fresh water fish may serve as a rapid biological

monitor of the pesticide exposure to important compo-

nents of fresh water community.

ACKNOWLEDGMENT The authors sincerely thank the Heads of the Department

of Zoology, Acharya Nagarjuna University, Guntur who

have extended their fullest co-operation and provided

laboratory facilities during the period of study.

Table 1: Chemical Analysis of Water used for the present study

S. No. Water Characteristic/Parameter Quantity

1 Turbidity 8 Silica Units

2 Electrical conductivity at 280C 816 micro ohms/cm

3 pH at 280C 8.1

4a. Alkalinity: Phenophthalene Nil

4b. Alkalinity: Methyl Orange 472

5 Total Hardness (as CaCO3) 232

6 Carbonate Hardness (as CaCO3) 232

7 Non Carbonate hardness (as CaCO3) Nil

8 Calcium Hardness (as CaCO3) 52

9 Nitrite Nitrogen (as N) Nil

10 Sulphate (as SO42-) Trace

11 Chloride (as Cl-) 40

12 Fluoride (as F-) 1.8

13 Iron (Fe) Nil

14 Dissolved Oxygen (DO) 8-10 ppm

15 Temperature 28 ± 20 C


Table 2: Sublethal and Lethal Concentrations of Fenvalerate technical grade and 20% active ingredient EC

to fish Labeo rohita, Catla catla and Cirrhinus mrigala

Table 3: The amount of oxygen consumed in mg/g body weight of the fish Labeo rohita, Catla catla and Cir-

rhinus mrigala exposed to sublethal and lethal concentrations of fenvalerate technical grade.

Fish Insecticide Sublethal Concentration

(mg/L)

Lethal Concentration

(mg/L)

Labeo rohita

Fenvalerate Technical

grade

0.0014 0.014

20% EC Fenvalerate

0.0024 0.024

Catla catla


grade

0.0016 0.016

20% EC Fenvalerate

0.00203 0.0203

Cirrhinus mrigala


grade

0.0006 0.006

20% EC Fenvalerate

0.0015 0.015

Oxygen mg/g body weight

Labeo rohita Catla catla Cirrhinus mrigala

Hours

of

Expo-

Con-

trol

Sublethal

concen-

tration

Lethal

Concen-

tration

Con-

trol

Sublethal

Concen-

tration

Lethal

Concen-

tration

Con-

trol

Sublethal

concen-

tration

Lethal

Concen-

tration

2 0.452 0.750 0.684 0.531 0.874 0.782 0.874 0.485 0.883

4 0.500 0.756 0.622 0.414 0.826 0.526 0.831 0.647 1.000

6 0.480 0.748 0.685 0.417 0.957 0.654 0.788 0.681 0.883

8 0.550 0.756 0.825 0.483 0.950 0.672 0.788 0.647 0.829

10 0.500 0.700 0.658 0.480 0.900 0.600 0.790 0.500 0.700

12 0.506 0.750 0.600 0.475 0.810 0.680 0.800 0.577 0.666


Table 4: The amount of oxygen consumed in mg/g body weight of the fish Labeo rohita, Catla catla and Cir-

rhinus mrigala exposed to sublethal and lethal concentrations of 20% active ingredient EC fenvalerate .

Oxygen mg/g body weight

Labeo rohita Catla catla Cirrhinus mrigala

Hours

of

Expo-

sure

Con-

trol

Sublethal

concen-

tration

Lethal

Concen-

tration

Con-

trol

Sublethal

Concen-

tration

Lethal

Concen-

tration

Con-

trol

Sublethal

concen-

tration

Lethal

Concen-

tration

2 0.686 0.510 0.758 0.220 0.346 0.265 0.570 0.412 0.456

4 0.675 0.696 0.862 0.241 0.279 0.221 0.600 0.338 0.750

6 0.675 0.810 0.758 0.209 0.279 - 0.584 0.341 0.448

8 0.625 0.557 0.706 0.241 0.301 - 0.590 0.553 0.425

10 0.580 0.460 0.500 0.275 0.298 - 0.550 0.490 0.547

12 0.575 0.464 0.502 0.304 0.325 - 0.584 0.483 0.547

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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


ABSTRACT

Six swimming pools within Ilorin metropo-

lis were analysed after treatment prior to use by bath-

ers and after use in order to determine their physico-

chemical and microbiological parameters. These

swimming pools were Kwara hotel both adult and

children swimming pools, Bekandims, Stella, King-

stone 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 bacte-

rial 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., Mi-

crococcus 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, physico-

chemical, swimming pools.

INTRODUCTION

A swimming pool is an artificially enclosed body of

water intended for swimming or water based recrea-

tion. Bathing and swimming for pleasure has been

practiced by many land animals for unrecorded ages,

and human record of the pleasure of immersion in wa-

ter go back several thousand years ago (Perkins,

1988).

The depth of a swimming pool depends on the pur-

pose 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 re-

quire 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 be-

tween users.

Swimming pool water may become contaminated by

micro-organisms from infected swimmers, incoming

water from an unsanitary source, airborne contamina-

tion 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 sub-

merged or water swallowed.

Some potential protozoans diseases in poorly main-

tained public swimming pools include cryptosporidio-

sis (caused by Cryptosporium) and Giardiasis caused

by Giardia sp. and amoebic meningoencephalitis

caused by Naegleria fow-

eri (CDC, 2006).

Sule* I.O.

Department of Microbiology,

University of Ilorin, Ilorin, Nigeria.

E-Mail: [email protected]



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 swim-

ming 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 infec-

tions. ( Eric et al., 1982).

Strong oxidizing agents are often used es-

pecially simple chlorine compounds such as so-

dium hypochlorite, dichlor or trichlor .When any

of these pool chemicals are used, it is very impor-

tant 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; de-

termine the species of bacteria which might be

present in it and to evaluate of some of its physico-

chemical quality.


Collection of Water Samples Water samples were collected from the

following pools: Kwara hotel (Adult), Kwara hotel

(Children), Bekandims, Stella obasanjo multipur-

pose complex, Kingstone hotel, and Successxs

hotel. The water sample was collected into differ-

ent sterile sampling bottles using methods as de-

scribed 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 deter-

mined using standard plate count (SPC) as de-

scribed by APHA,(1985).The total and faecal Coli-

form 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 mor-

phology, staining reactions and biochemical char-

acteristics. The tests were carried as described by

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 differ-

ences 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 sur-

veyed public swimming pool was in full compli-

ance 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 Aus-

tralian 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 dis-

infectant (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 disin-

fection. 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).


The immediate environment of all the pools were

cleaned and tiled. Le Chevallier et al.(1996) has

reported the occurrence of coliforms in the pres-

ence of disinfectant residual. All the pools had

increment in bacterial load after use by bathers.

This is in conformity with the work of other work-

ers 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 sedi-

ments 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 re-

gime allowed (5mg/l) rather than superchlorina-

tion; the disinfectant used should be thoroughly

mixed in the pool; good water source should be

used for disinfection ; poster signs should be pro-

vided 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 mini-

mize soil contamination.

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

Swimming Pools

pH Residual chlorine ( ppm )

Before use After use Before use After use

Kwara hotel

(Adult)

6.6

7.0

9.80

9.44

Kwara hotel

(Children)

6.5

7.0

9.73

9.30

Bekandims hotel 6.9 6.4 9.94 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

Stella 2800 3000

Kingstone 23 170

Successxs 36 230


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

KEY +: Isolated - : Not isolated

Swimming

Pools

Total Coliform count ( cfu/ml) Faecal Coliform count(cfu/ml)

Before use After use Before use After use

Kwara hotel

(Adult)

0 6 0 0

Kwara hotel

(Children)

0 26 0 0

Bekandims 0 4 0 0

Stella 200 300 0 0

Kingstone 18 40 0 2

Successxs 6 20 4 6

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

Isolates Kwara

hotel

(Adult)

Kwara hotel

(Children)

Bekandi

m

Stella Kingstone Successxs

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. - + + + - +


CONCLUSION

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 su-

perchlorination.

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