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Bioresearch Bulletin Vol 1:1ISSN 0976-576XTRANSCRIPT
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-
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fact remains certain, the whole process or the quest begins with a single step. The first edition of
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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
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Pethuru Devathasan
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Sam Stephen
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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-
cles, letters, and reviews reporting new theories based
on Lifescience and its allied fields. Though the contents
will evolve, some specific topics that are appropriate
today for Bioresearch Bulletin include advances in or
applications of Biochemistry, bioinformatics, bio-
physics, bio-statistics, biotechnology, botany, cell biol-
ogy, chemical biology, computational biology, devel-
opmental biology, ecology, ethno knowledge medi-
cine, evolutionary biology, genetics, genomics, immu-
nology, microbiology, molecular biology, neurobiol-
ogy, pharmacology, pharmacology, physiology, plant
biology, structural biology, systems biology, taxonomy
and zoology.
TYPES OF MANUSCRIPTS: The Bioresearch Bulletin (BRB) is published
every month by Bioindica. The journal accepts the fol-
lowing type of scientific communication,
Regular Research articles: These should elaborate
new and carefully confirmed findings, and experimen-
tal procedures should be given in sufficient detail for
others to verify the work. The length of a full paper
should be the minimum required to describe and inter-
pret the work clearly.
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vestigations or giving details of new models or hy-
potheses, innovative methods, techniques or apparatus.
Mini review: Submissions of mini-reviews and per-
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reviewed.
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upon acceptance.
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Authors publishing with Bioresearch Bulletin
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unmodified.
Harold Urey, the editor of the Journal of
Chemical Physics in 1933 observed: ―The life and in-
terests of the individual are short and of little conse-
quence as compared to the lives of our sciences.‖ This
will always be true, and our only intention is for Biore-search Bulletin to help enrich the life - science among
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ACKNOWLEDGEMENTS: There are many people who deserve thanks for
bringing Bioresearch Bulletin to fruition. The support,
encouragement, and handling of innumerable details by
the Board of Editors, Executive Editor Pethuru
Devathasan, Editor-in-Chief Abiya Chelliah, Language
Editor Benison Thilagar & Computational support
Girithara Prakash (CEO of Fortab, Inc) have been es-
sential.
1. Managing editor
Bioresearch Bulletin
2.Editor-in-Chief
Bioresearch Bulletin
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
© Bioindica Press 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.
Bioresearch Bulletin (2010) 1: 1-6 3
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.
4 Bioresearch Bulletin (2010) 1: 1-6
Table 2 Effect of aridanin on time spent in the open arm entries and on time spent in the close arm entries
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. TSOA: Time spent in the open arm; TSCA: Time spent in the close
arm; 5%TW80: 5% Tween 80
*Indicate significant difference from 5% Tween 80 control. P < 0.05 SNK test.
Table 3 Effect of aridanin on time spent in the open arm entries and on time spent in the close arm entries
in the presence of flumazenil
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. TSOA: Time spent in the open arm; TSCA: Time spent in the close
arm; 5%TW80: 5% Tween 80
*Indicate significant difference from 5% Tween 80 control. P < 0.05 SNK test.
Table 4: Effect of aridanin on no of entries in the open arm and no of entries in the close arm in the
presence of flumazenil (2.0 mg/kg i.p.).
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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6 Bioresearch Bulletin (2010) 1: 1-6
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Bioresearch Bulletin (2010) 1: 7-9 7
ORIGINAL PAPER
Susceptibility of Crab Chitosan against Staphylococcus aureus
G. SUGUMAR U. RAMESH*
A. SELVAN
© Bioindica Press 2010
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.
MATERIALS AND METHODS
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
8 Bioresearch Bulletin (2010) 1: 7-9
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%.
RESULTS AND DISCUSSION
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 - -
Bioresearch Bulletin (2010) 1: 7-9 9
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.
Bioresearch Bulletin (2010) 1: 10-15 10
ORIGINAL PAPER
Assessing the Ecological Status of Prominent Medicinal Herbs in Central
Sudan and the challenge of Sustainable Management
H.H. EL-KAMALI
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,
© Bioindica Press 2010
Received: 19 March 2010 / Accepted: 2 April 2010 /Published online: 22 June 2010
11 Bioresearch Bulletin (2010) 1: 10-15
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.
Bioresearch Bulletin (2010) 1: 10-15 12
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.
13 Bioresearch Bulletin (2010) 1: 10-15
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.
Bioresearch Bulletin (2010) 1: 10-15 14
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
15 Bioresearch Bulletin (2010) 1: 10-15
Bioresearch Bulletin (2010) 1: 16-18 16
ORIGINAL PAPER
Impact of industrial effluents and sewage on river Thamirabarani
and it’s concerns
P. PETER BASKARAN A. JOHN DE BRITTO*
© Bioindica Press 2010
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)
RESULTS AND DISCUSSION
It is evident that the water quality is altered
due to the entry of sewage and other domestic wastes
into the aquatic system. The pH value is measured as
8.05, 7.57, 7.35, 7.89 and 7.93 respectively. The pH
value of sites I and II are more. There were great 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.
17 Bioresearch Bulletin (2010) 1: 16-18
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.
Bioresearch Bulletin (2010) 1: 16-18 18
Bioresearch Bulletin (2010) 1: 19-23 19
ORIGINAL PAPER
Effect of nutrients on in vitro culture of Morus alba L. (White mulberry)
ANKIT PRADHAN, A. S. VISHWANATHAN*
R. BASAVARAJU.
Received: 3 April 2010 / Accepted: 12 April 2010 /Published online: 22 June 2010
© Bioindica Press 2010
ABSTRACT Morus alba L. is an economically important plant used
extensively in sericulture, also possessesing medicinal
properties. In vitro culture provides an effective means
to overcome the common problems encountered in 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)
20 Bioresearch Bulletin (2010) 1: 19-23
effects of some of the selected supplements on the
plantlets in vitro.
MATERIALS AND METHODS
Local varieties of M. alba were procured from local
farms in and around Puttaparthi and grown in the 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.
Bioresearch Bulletin (2010) 1: 19-23 21
Table 2 outlines the different morphological changes
that occurred in the response of stem-bit explants in the
absence of nitrates and sulphates in MS medium.
Absence of nitrogen compounds in MS medium 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
22 Bioresearch Bulletin (2010) 1: 19-23
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secondary somatic embryogenesis and embryo
maturation in morus alba l. Sci Hort.
2004;102:359-368
Anis M, Faisal M and Singh S. Micropropagation of
mulberry (morus alba l.) through in vitro cul-
ture of shoot tip and nodal explants. Plant Tis-sue Culture. 2003;13:47-51
Arabshahi-Delouee S and Urooj A. Antioxidant proper-
ties of various solvent extracts of mulberry
(morus indica l.) leaves. Food Chem. 2007;102:1233-1240
Avigad D and Dey P Carbohydrate metabolism: Stor-
age carbohydrates. In: Dey P and Harborne J.
Plant biochemistry. Academic Press, Great
Britain. 1997.
Bhau B and Wakhlu A. Rapid micropropagation of five
cultivars of mulberry. Biol Plant. 2003;46:349-
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.
1997.
Enkhmaa B, Shiwaku K, Katsube T, Kitajima K,
Anuurad E, Yamasaki M and Yamane Y. Mul-
berry (morus alba l.) leaves and their major
flavonol quercetin 3-(6-malonylglucoside)
attenuate atherosclerotic lesion development in
ldl receptor-deficient mice. J Nutr.
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Enomoto S. Preservation of genetic resources of mul-
berry by means of tissue culture. JARQ. 1987;21:205-210
Ercisli S and Orhan E. Chemical composition of white
(morus alba), red (morus rubra) and black
(morus nigra) mulberry fruits. Food Chem.
2007;103:1380-1384
Islam R, Zaman A, Joarder O and Barman A. In vitro
propagation as an aid for cloning ofmorus
laevigata wall. Plant Cell Tiss Org Cult.
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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.
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Lopez J, Tremblay N, Voogt W, Dubé S and Gosselin
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on growth, physiology and yield of the green-
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Lu M. Micropropagation of morus latifolia poilet us-
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Murashige T and Skoog F. A revised medium for
rapid growth and bio assays with tobacco tis-
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Ohyama K. Tissue culture in mulberry tree. JARQ.
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Ohyama K and Oka S. Regeneration of whole plants
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Bioresearch Bulletin (2010) 1: 19-23 23
Bioresearch Bulletin (2010) 1: 24-29 24
Received: 29 May 2010 / Accepted: 12 June 2010 /Published online: 22 June 2010
© Bioindica Press 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]
Bioresearch Bulletin (2010) 1: 24-29 25
called chaeta, which are made of chitin.
The microbes associated with polychaetes were not
studied much. There are several advantages for using
microbes as source of bioactive compounds. Hence, in
the present study an attempt has been made to screen
the antimocrobial activity of bacxteria associated with
polychates. A study of this kind will provide more
details on the bioactivity of bacteria associated with
marine invertibrates.
MATERIALS AND METHODS
Isolation and identification of Bacteria Polychaetes were collected from the Kanyakumari
coast of west coast of India. For the collection of the
polychaetes, a portion of the seaweeds Sargasum sp
was detached from the surface and kept in polythene
bags with seawater and brought to the laboratory .
In the laboratory, the polychaetes were removed
from the seaweed by gentle agitation and collected in
a jar with seawater. The polychaetes were rinsed with
sterile seawater to remove the loosely attached 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.
Bioresearch Bulletin (2010) 1: 24-29 26
Bioresearch Bulletin (2010) 1: 24-29 27
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
Bioresearch Bulletin (2010) 1: 24-29 28
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
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Berdy, J., 2005. Bioactive microbial metabo-
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Bioresearch Bulletin (2010) 1: 30-32 30
ORIGINAL PAPER
Laboratory culture of Microzooplankton Tintinnopsis cylindrical [Daday 1887]
S.VIJAYARAGAVANA, P.VIVEK RAJA
B
Received: 12 May 2010 / Accepted: 13 June 2010 /Published online: 22 June 2010
© Bioindica Press 2010
ABSTRACT :
The culture of microzooplankton Tintinnopsis
cylindrica under controlled conditions is offering within
a short period. The microzooplankton is ideal food for
the prawn and fish juveniles in the nursery phase and the
nutrivite value of the microzooplankton depends on the
such as chlorella marina, the microzooplankton 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
Bioresearch Bulletin (2010) 1: 30-32 31
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
Bioresearch Bulletin (2010) 1: 30-32 32
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)
Bioresearch Bulletin (2010) 1: 33-38 33
ORIGINAL PAPER
A study on acute toxicity , oxygen consumption and behavioural changes in the three major
Carps, Labeo rohita (ham), Catla catla (ham) and Cirrhinus mrigala (ham) exposed to
Fenvalerate
T. ANITA SUSAN1 K. SOBHA2
K.S. TILAK3
Received: 20 May 2010 / Accepted: 14 June 2010 /Published online: 22 June 2010
© Bioindica Press 2010
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:
Bioresearch Bulletin (2010) 1: 33-38 34
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
Bioresearch Bulletin (2010) 1: 33-38 35
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-
Bioresearch Bulletin (2010) 1: 33-38 36
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
Bioresearch Bulletin (2010) 1: 33-38 37
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
Fenvalerate Technical
grade
0.0016 0.016
20% EC Fenvalerate
0.00203 0.0203
Cirrhinus mrigala
Fenvalerate Technical
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
Bioresearch Bulletin (2010) 1: 33-38 38
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
© Bioindica Press 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]
Bioresearch Bulletin (2010) 1: 39-43 42
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.
MATERIALS AND METHODS
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).
Bioresearch Bulletin (2010) 1: 39-43 43
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
Bioresearch Bulletin (2010) 1: 39-43 44
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. - + + + - +
Bioresearch Bulletin (2010) 1: 39-43 45
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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Notes
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