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Assessment of biological effects of pollutants in a hyper eutrophictropical water body, Lake Beira, Sri Lanka using multiple

biomarker responses of resident fish, Nile tilapia (Oreochromisniloticus)

Asoka Pathiratne K. A. S. Pathiratne

P. K. C. De Seram

Accepted: 1 March 2010 / Published online: 13 March 2010

Springer Science+Business Media, LLC 2010

Abstract Biomarkers measured at the molecular and cel-

lular level in fish have been proposed as sensitive earlywarning tools for biological effect measurements in envi-

ronmental quality assessments. Lake Beira is a hypertrophic

urban water body with a complex mixture of pollutants

including polycyclic aromatic hydrocarbons (PAHs) and

Microcystins. In this study, a suite of biomarker responses

viz. biliary fluorescent aromatic compounds (FACs), hepatic

ethoxyresorufin O-deethylase (EROD) and glutathione

S-transferase (GST), brain and muscle cholinesterases

(ChE), serum sorbitol dehydrogenase (SDH), and liver his-

tology of Oreochromis niloticus, the dominant fish inhabit-

ing this tropical Lake were evaluated to assess the pollution

exposure and biological effects. Some fish sampled in the

dry periods demonstrated prominent structural abnormali-

ties in the liver and concomitant increase in serum SDH and

reduction in hepatic GST activities in comparison to the

control fish and the fish sampled in the rainy periods. The

resident fish with apparently normal liver demonstrated

induction of hepatic EROD and GST activities and increase

in biliary FACs irrespective of the sampling period indi-

cating bioavailability of PAHs. Muscle ChE activities of the

resident fish were depressed significantly indicating

exposure to anticholinesterase substances. The results

revealed that fish populations residing in this Lake is underthreat due to the pollution stress. Hepatic abnormalities in

the fish may be mainly associated with the pollution stress

due to recurrent exposure to PAHs and toxigenicMicrocystis

blooms in the Lake.

Keywords Highly eutrophic lake Tropical fish

Biomarkers Liver histology

Introduction

Biomarkers measured at the molecular and cellular level in

fish have been proposed as sensitive early warning tools

for biological effect measurements in environmental

quality assessments (van der Oost et al. 2003). Monitoring

of cholinesterase (ChE) enzyme inhibition in fish has been

widely used in aquatic ecosystems as an indicator of

exposure to neurotoxic pollutants and physiological effects.

Inhibition of brain and muscle ChE in fish would adversely

affect neuro-muscular transmission. ChE is sensitive to

organophosphate and carbamate pesticides, heavy metals

and complex mixture of pollutants in aquatic environments

(Payne et al. 1996; van der Oost et al. 2003). The mea-

surement of CYP1A dependent ethoxyresorufin O-deethy-

lase (EROD) in fish has become a promising biomarker for

detecting aquatic contaminations of a variety of highly

toxic pollutants such as some polycyclic aromatic hydro-

carbons (PAHs) and coplanar polychlorinated biphenyls

(PCBs). Induction of phase I biotransformation system

especially CYP1A activity has been used to infer cancer

related liver lesions in fish (Whyte et al. 2000). Fluorescent

aromatic compounds in the fish bile are reported as sensi-

tive biomarkers of recent exposure to PAH (Aas et al.

A. Pathiratne (&) P. K. C. De Seram

Department of Zoology, Faculty of Science, University

of Kelaniya, Kelaniya, Sri Lanka

e-mail: asoka@kln.ac.lk

K. A. S. Pathiratne

Department of Chemistry, Faculty of Science, University

of Kelaniya, Kelaniya, Sri Lanka

123

Ecotoxicology (2010) 19:10191026

DOI 10.1007/s10646-010-0483-2

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2000). Since PAH exposure cannot be reliably determined

by measuring fish tissue levels, this parameter is a valid fish

biomarker for environmental risk assessment process con-

cerning PAH-contaminant sites (van der Oost et al. 2003).

Glutathione S-transferases (GSTs) which catalyze the

conjugation of glutathione with xenobiotics play important

roles in xenobiotic detoxification reactions within the body

(George 1994). The liver is the organ mainly associatedwith detoxification and biotransformation of xenobiotics.

Due to its functions and rich blood supply it is also one of

the organs most affected by aquatic pollutants. Serum

sorbitol dehydrogenase (SDH) is a sensitive biochemical

indicator of chemically induced hepatic damage in fish

(Dixon et al. 1987). Histopathology of fish liver is also a

sensitive and reliable monitoring tool for assessment of the

effects of environmental stressors on fish populations in

natural water bodies (Au 2004).

Although biomarker studies in relation to water bodies

located in many temperate and sub-tropical countries have

been extensively documented, scientific reports of pollu-tant induced biomarker responses in tropical water bodies

especially under hypertrophic conditions are meager. Lake

Beira is a highly eutrophic urban water body in Sri Lanka

(Kamaladasa and Jayatunga 2007) which receives urban

and domestic wastes, automobile wastes and industrial

wastes from its catchment sources and various drain

outlets. This Lake can be considered as a model hyper

eutrophic and polluted aquatic ecosystem for assessment

of multiple biomarker responses in fish under tropical

conditions. The Lake frequently contains cyanobacteria

blooms especially Microcystis aeruginosa that are found

to be toxigenic as microcystins have been detected in

some M. aeruginosa samples collected from the Lake

(Jayatissa et al. 2006; Magana-Arachchi et al. 2008).

Occurrence of considerably high levels of PAHs in this

Lake has also been reported recently (Pathiratne et al.

2007). Nile tilapia (Oreochromis niloticus) which is the

dominant fish residing in this Lake, is used as a food

source by low income families in the area. Nile tilapia

which is considered as a hardy fish, is an omnivorous

feeder. Mortalities of Nile tilapia in Lake Beira have been

observed in several occasions especially during dry peri-

ods. However, no studies have been carried out previously

to assess the response of the resident fish exposed to

complex mixtures of pollutants present in this Lake.

Objective of the present study was to assess exposure and

biological effects of pollution in a highly eutrophic

tropical water body, Lake Beira using a suite of bio-

chemical and histological biomarker responses of the

resident fish, Nile tilapia viz. ChEs in the brain and

muscle tissues, serum sorbitol dehydrogenase (SDH),

hepatic EROD and GST, fluorescent aromatic compounds

in the bile and histological structure of the fish liver.

Materials and methods

Sampling area

Beira Lake (6450-7000 N;79300-79550 E) is located at the

commercial capital, Colombo city in the Western Province

of Sri Lanka. The Lake is surrounded by main roads with

congested motor vehicle traffic, railways, offices, hotels,food shops, ware houses, some industries, hospitals, and

residence including shanties. The extent of the Lake is about

65.4 ha and the Lake is dependent on the run off of its highly

urbanized catchments. The water is dark green colour due to

highly abundant phytoplankton biomass especially cyano-

bacteria blooms. Even though restoration activities were

carried out in 2004 in the South-West side of the Lake by

dredging the Lake bottom sediment, pumping of sea water

from the adjacent sea and closing of much of the surface

drains, restoration objectives were not fully achieved in the

Lake and the Lake is presently a hyper eutrophic stagnant

water body: mean orthophosphate levels, 0.210.52 mg l-1;mean nitrate levels, 1.31.5 mg l-1; mean chlorophyll-a

content, 0.40.59 mg l-1 (Kamaladasa and Jayatunga

2007).

Fish

Nile tilapias from Lake Beira were collected from the non-

restored East Lake during the dry periods (February 2006

and April 2006) and rainy periods associated with the south

west monsoon (June 2006 and July 2006). Rainfall is the

most seasonal climatic factor in Sri Lanka with slight

seasonal variations in temperature and day length. The

temperature in the Lake water during the sampling periods

ranged from 28 to 30C. Fish were transported live to the

laboratory with water from the same location.

The fish which were used as Controls were obtained

from a fish breeding station, National Aquaculture Devel-

opment Authority, Sri Lanka and maintained at the Uni-

versity of Kelaniya premises (about 1215 km away from

the Lake) in outdoor tanks filled with continuously aerated

aged tap water under natural photoperiod for 3 to 5 weeks

prior to their use in biomarker assays. Half of the water in

each tank was exchanged with aged tap water every 4 to

5 days. During this period, temperature, pH, and dissolved

oxygen concentration in water in the tanks ranged from

2830C, 7.37.6, and 4.25.2 mg/L respectively. Control

fish were daily fed with commercial fish food pellets

(Prima Feed, Ceylon Grain Elevators Pvt Ltd., Sri Lanka)

at 1% of the body weight.

Control fish and the fish collected from the Lake were

anesthetized with benzocaine (Treves-Brown 2000). Blood

samples were taken from the caudal vein and serum was

prepared by centrifugation and stored at -20C until

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analysis of SDH activity. Brain and a piece of muscle from

the lateral side were removed and stored frozen at -80C

until analysis of ChE activities. Bile was taken to a syringe

by puncturing the gall bladder by the needle fitted to the

syringe and frozen at -80C until further processing. Liver

tissue from each fish was stored frozen separately at -80C

until used for EROD and GST analysis. In addition liver

tissues were preserved in neutral buffered formalinfor histopathological studies. Fish stomach was dissected;

contents were fixed in 10% formalin for 24 h and exam-

ined under the microscope for identification of stomach

contents.

Analysis of biomarker enzymes

All preparation steps of the enzyme sources were carried

out on ice and/or at 4C. Serum SDH activity was deter-

mined at 25C as described by Gerlach (1983) using

D-fructose as the substrate. The ChE activities in the brainand muscle homogenates of individual fish were deter-

mined at 25C using acetylthiocholine iodide as the sub-

strate following the method of Ellman et al. (1961) as

described earlier (Pathiratne et al. 2008). Microsomal and

cytosolic fractions of liver tissues were prepared by dif-

ferential centrifugation (Pathiratne et al. 2009). GST in the

liver cytosol fraction was measured at 30C by following

the conjugation of glutathione at 340 nm using 1-chloro-

2,4-dinitrobenzene as the substrate (Habig et al. 1974). The

EROD activity in the liver microsomes was determined at

30C following the method of Klotz et al. (1984). All

enzyme assays were performed with a computer controlledrecording spectrophotometer (GBC Cintra 10e, Australia)

using a thermostated cuvette holder as kinetic assays at the

set temperature. Proteins present in the liver microsomes &

cytosol and brain & muscle homogenates were determined

according to the method of Lowry et al. (1951) with

bovine serum albumin as the standard. Chemicals used

for biochemical assays were obtained from Sigma-Aldrich

Corporation, MO, USA.

Bile analysis for PAH metabolites

PAH metabolites in fish bile were determined by fixed

wavelength fluorescence measurements as described by

Aas et al. (2000) using Aminco-Bowman Series 2 Lumi-

nescence Spectrometer (Thermo Spectronic). Two micro-

liters of bile diluted in 4 ml of 48% ethanol were used to

decrease self absorption and quenching of the fluorescence

signal. Fluorescence at the excitation/emission wavelength

pairs (FF) 290/335, 341/383 and 380/430 nm were deter-

mined for naphthalene type, pyrene type and benzo(a)-

pyrene type of metabolites respectively. The FF values are

expressed as arbitrary fluorescence units after deducting

the signal level of the solvent.

Liver histology

Liver tissues were fixed in 10% buffered formalin at 4C

and dehydrated in graded series of ethanol, cleared in

xylene and embedded in paraffin wax. Sections were cut in5 lm thickness and stained with haematoxylin and eosin

following standard procedures and examined under the

light microscope.

Statistical analysis

Body sizes and biomarker responses of the fish collected

during different sampling periods were analyzed by

ANOVA (P\ 0.05). Where differences were significant,

multiple comparisons were carried out by Tukeys test (Zar

1999) as appropriate. Log transformed data were used for

statistical analysis.

Results

No significant differences were obtained in relation to the

body sizes of the fish collected from the Lake and the

control fish (Table 1). The sampled fish included both

genders. Of the 38 fish sampled from the Lake in the dry

periods, livers of 16 fish (irrespective of the gender) were

mushy, light brown in colour and translucent showing an

extensive branching pattern of blood vessels which

appeared whitish in colour macroscopically. The livers of

the other fish collected from the Lake during the dry period

and all the fish collected during rainy periods and control

fish were firm, reddish brown in colour and showed the

normal appearance macroscopically. Examination of

stomach contents of the fish collected from the Lake

showed phytoplankton especially Microcystis as their main

food items. In addition detritus were found in the stomach

contents of these fish.

The livers of the control Nile tilapia which showed

normal appearance macroscopically had normal histologi-

cal structure of the hepatopancreas. Hepatocytes are

polygonal in shape, arranged in several cellular layers and

surrounded by sinusoids. Each hepatocyte contained usu-

ally a centrally located single round nucleus. Pancreatic

tissue was present in association with venous vessels and as

isolated elements. Livers of fish collected from the Lake in

the rainy periods also showed fairly normal appearance

macroscopically but histological structure revealed mild to

moderate vacuolation of hepatocytes in some areas

(Fig. 1a, b). Vacuolated hepatocytes were more abundant

in the fish collected from the Lake in the dry periods. Large

Biomarkers of fish in a hyper eutrophic water body 1021

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vacuoles in the cell force the nucleus to the periphery of the

hepatocytes. In addition, prominent histopathological

changes were observed in the livers of the fish which

showed gross macroscopic abnormalities (Fig. 1c, d). The

abnormalities observed in these fish were dilation ofsinusoids and blood vessels and extensive vascular con-

gestion, increased vacuolation of hepatocytes, pycnotic

nuclei, nuclear atrophy and focal areas of necrosis. No

neoplastic lesions were observed in the samples examined.

Ranges in the Serum SDH levels of the control fish and the

Lake fish which had no liver abnormalities macro-

scopically were 014 and 228 mUnits ml-1 of serum

respectively. However the serum SDH levels in the fish

which had liver abnormalities had increased considerably

(97160 mUnits ml-1 of serum) in comparison to those in

the other fish groups.

Hepatic EROD and GST activities in Nile tilapia col-lected from the Lake and the activities of comparable

control fish are presented in Fig. 2. Of the fish sampled in

the dry season, induction of EROD activity was not

observed in the fish which had liver abnormalities macro-

scopically where as the fish which had apparently normal

liver structure, demonstrated enhanced hepatic EROD

activity by nearly 2 folds in comparison to the controls

(Fig. 2a). The hepatic EROD activities in the Lake fish

collected during the rainy period were induced by 6 to 10

folds. Of the fish sampled in the dry periods, hepatic GST

activities in the fish with liver abnormalities were signifi-

cantly lower in comparison with those of the fish withapparently normal livers (Fig. 2b). Hepatic GST activities

in the Lake fish were induced by nearly 23 folds during

rainy periods compared to the respective controls. Fixed

fluorescence determinations showed (Fig. 3) that the bile

samples of tilapia residing in the Lake contain significantly

higher amounts of naphthalene type, pyrene type and

benzo(a)pyrene type metabolites in comparison to the

control fish. The fluorescence signals were higher during

the rainy periods compared to those during the dry periods.

The fluorescence signals in the bile samples of the fish with

abnormal liver appear to be low compared to those in the

Lake fish with normal livers. However the differences were

not statistically significant.

Brain and muscle ChE activities in Nile tilapia collectedfrom the Lake and the control fish are presented in Fig. 4.

Muscle ChE activities in the fish sampled from the Lake

during the study period were significantly lower than those

of the controls irrespective of the liver structure and sam-

pling period. However inhibition of muscle ChE activity

(3746%) was greater during the rainy periods in com-

parison to the extent of inhibition during the dry periods

(2125%). No significant differences were found among

different groups of fish with respect to brain ChE activities.

Discussion

Recent studies have reported the trophic status, gross pol-

lution and PAH pollution in Lake Beira (Kamaladasa and

Jayatunga 2007; Pathiratne et al. 2007). This is the first

study which focused on assessing the exposure and bio-

logical effects of pollutants present in this hyper eutrophic

tropical Lake using a suite of biomarkers of the resident

fish. Even though Nile tilapia is considered as a hardy fish,

the results indicate that population of Nile tilapia residing

in this Lake is under threat due to the pollutant impact.

Liver as the main organ of metabolism comes into

contact with xenobiotics absorbed from the aquatic envi-ronment and liver lesions are often associated with expo-

sure to aquatic pollutants (Au 2004; Fernandes et al. 2008).

In this study, the fish that were used as controls showed

typical liver structure of Nile tilapia described previously

by Vicentini et al. (2005) and Figueiredo-Fernandes et al.

(2007). In the present study vacuolation of hepatocytes

was observed in the livers of Nile tilapia collected from the

Lake even though their livers were normal in appear-

ance macroscopically. The most prominent histological

Table 1 The body sizes of Nile tilapia used in evaluation of biomarkers at different sampling periods

Sampling period Source of fish Na Body length (cm) Body weight (g)

Feb 2006Dry period Lake Beira 16 (6) 20.6 3.2 188 11

Controls 14 (0) 19.8 2.4 162 27

April 2006Dry period Lake Beira 22 (10) 19.7 1.5 150 34

Controls 16 (0) 18.5 2.8 161 39

June 2006Rainy period Lake Beira 20 (0) 20.8 2.1 175 30

Controls 12 (0) 19.6 3.9 159 29

July 2006Rainy period Lake Beira 18 (0) 20.1 2.2 174 17

Controls 10 (0) 20.8 1.3 188 56

a Number within parentheses indicates the number of fish that demonstrated macroscopic liver abnormalities. Body sizes are presented as

mean SEM of the number of fish used in each sampling period

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alterations observed in the fish that showed abnormal livers

macroscopically were dilation of blood vessels and sinu-

soids and severe congestion in sinusoids and small blood

vessels, increased vacuolation of hepatocytes, pycnotic

nuclei, nuclear atrophy and focal areas of necrosis. Pyc-

nosis of hepatocyte nuclei associated with cytoplasmic

vacuolation is a non specific response of fish due to toxic

conditions (Roberts 1978). The blockage of sinusoids

makes the blood flow from the hepatic portal vein and

hepatic artery into the central vein rather difficult. This

may be responsible for the cellular degeneration and

necrosis in the livers of some Nile tilapia collected fromthe Lake during dry periods. Observed liver abnormalities

reflect the biological impacts of complex mixture of pol-

lutants present in this hypertrophic lake.

Exposure of fish to anthropogenic organic contaminants

with a planar configuration such as PAHs and PCBs can

induce CYP1A associated EROD activity (Whyte et al.

2000; van der Oost et al. 2003). The elevated hepatic

EROD activities in Nile tilapia collected from the Lake

irrespective of the sampling period indicate that the Lake is

contaminated with CYP1A inducing chemicals such as

PAHs and/or PCBs. In a recent study, petrogenic and

pyrogenic PAHs have been detected in the water (colloid

bound) and sediments collected from different sampling

sites of this Lake (Pathiratne et al. 2007). PAHs may have

contributed partly or fully for the observed high induction

levels of hepatic EROD in the fish collected from this

Lake. This is further supported by the presence of high

levels of naphthalene type, pyrene type and benzo(a)pyrene

type FACs in the bile of the resident fish in the Lake

compared to the controls. The highest EROD activities

(610 fold induction) and FACs levels found in the fishcaptured during rainy periods indicating increase inputs of

PAHs to the Lake through surface runoff with the heavy

rain experienced during this period. This study provides

evidence that Lake Beira contains bioavailable PAHs and

other related compounds which can be associated with

induction of CYP1A dependent EROD in liver tissues.

Induction of CYP1A activity has been used to infer liver

lesions in fish (Whyte et al. 2000). Hence previous expo-

sure of fish to PAHs present in the Lake may have

Fig. 1 Histological structure of liver tissue of Nile tilapia collected

from Lake Beira: a, b liver that was normal macroscopically, showing

polygonal shape hepatocytes (H), sinusoids (S), mild vacuolation (V)

and intrahepatic pancreatic tissue (P); c, d livers which were

abnormal macroscopically showing dilation and severe congestion of

sinusoid spaces (S), increased vacuolation (V) and focal necrosis (N)

Biomarkers of fish in a hyper eutrophic water body 1023

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contributed to the induction of hepatic structural abnor-

malities in the resident fish especially hepatocyte damage

and focal necrosis which in turn could have affected the

inducing ability of CYP1A activity in the hepatocytes

following subsequent exposures of the same fish to PAHs

in the Lake. Observed increase in serum SDH activities in

these fish confirms the hepatocyte damage.

GSTs are major phase II detoxification enzymes found

mainly in the hepatic cytosol. The hepatic GST activities in

the control Nile tilapia used in the present study were

higher than the values reported for the control fish used in

our earlier study (Pathiratne et al. 2009). This may be due

to the use of higher temperature condition (30C) of the

assay medium in this study to reflect the natural tempera-

ture conditions in the Lake. Nevertheless, hepatic GST

activities of Nile tilapia sampled from the Lake Beira in the

rainy periods were significantly higher than that of the fish

sampled in the dry periods and the control fish. Even

though EROD activity was induced by 610 folds, increase

in detoxification capacity of the fish associated with rela-

tively high GST activities (increase by 23 folds) may have

provided some resistance to the pollutant stress during the

rainy periods. Depression of GST activities in the liver

tissues of some Nile tilapia collected from the Lake in the

dry season may be due to the hepatocyte damage and focal

necrosis. Alternatively, depletion of hepatic GST activities

in these fish may have lead to the hepatic damage subse-

quently. Microcystins have been detected recently in tested

M. aeruginosa samples from Lake Beira (Jayatissa et al.

2006; Magana-Arachchi et al. 2008). Microcystins can

rapidly accumulate in the liver inhibiting protein

Fig. 2 Hepatic ethoxyresorufin O-deethylase (EROD) and glutathi-

one S-transferase (GST) activities in Nile tilapia collected from LakeBeira. Data are presented as mean SEM. For each enzyme, bars

with different letters are significantly different from each other

(P\ 0.05)

Fig. 3 Biliary fluorescence levels in Nile tilapia collected from Lake

Beira. a Naphthalene type metabolites. b Pyrene type metabolites.

c Benzo(a)pyrene type metabolites. Data are presented as mean

SEM. For each PAH type, bars with different letters are significantly

different from each other (P\ 0.05)

Fig. 4 Muscle and brain cholinesterase (ChE) activities in Nile

tilapia collected from Lake Beira. Data are presented as mean

SEM. For muscle tissue, bars with different letters are significantlydifferent from each other (P\ 0.05)

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phosphatases causing hepatocellular damage followed by

intrahepatic hemorrhage that may lead to the death of the

organism (Gupta et al. 2003). Microcystins can also induce

oxidative stress. GST has been recognized as the main

enzyme which catalyzes the first step of detoxification of

Microcystins (Amado and Monserrat 2010). Some strains

ofMicrocystis sp. produce the toxin microcystin-LR which

is the most toxic cyanobacterial hepatotoxin. A recentstudy showed that both microcystin-LR and microcystin-

RR, can induce pathological lesions in hepatic tissues of

Nile tilapia (Atencio et al. 2008). Microcystis may be toxic

to fish via gastrointestinal ingestion as well as by absorp-

tion of the microcystin directly from water. Even though

microcystin levels in the fish residing in the Lake were not

determined in the present study, influence of microcystins

in inducing hepatic abnormalities in the fish collected from

Lake cannot be ruled out as Microcystis was the main food

item present in the stomachs of Nile tilapia collected from

the Lake. Hence, among the other factors, long term

exposure to the toxins produced by Microcystis bloomspresent in the Lake may have positively contributed to the

hepatic damage and concurrent GST depletion observed

during the dry period. Many common bloom forming

cyanobacteria including Microcystis have toxic and non-

toxic strains which co-occur and are visually indistin-

guishable but can be quantified effectively by molecular

methods. The study carried out by Davis et al. (2009)

suggests that elevated temperatures yield more toxic

Microcystis cells and/or cells with more microcystin syn-

thatase gene copies per cell potentially yielding more toxic

blooms. It is not known whether dry periods prevailed in

this hypertrophic tropical water body may have promoted

the growth of toxic populations of Microcystis, leading to

blooms with higher microcystin content. This aspect war-

rants further investigations.

Cholinesterases in fish have been used as a biomarker of

neurotoxic contamination in aquatic environment moni-

toring studies (Payne et al. 1996; van der Oost et al. 2003).

A recent study has shown that fish acetylcholinesterase

could be used as a potential biochemical marker for fer-

tilizer industry effluent pollution in aquatic systems (Yadav

et al. 2009). In the present study, muscle ChE activities in

the fish sampled from the Lake were significantly lower

(2146%) than those of control fish groups irrespective of

the presence of hepatic abnormalities where as brain ChE

levels were not affected. More than one form of ChE may

be present in different tissues of the fish and these different

forms have distinct sensitivities to anticholinesterase

agents (Sturm et al. 2000). The results indicate the presence

of muscle ChE sensitive anticholinesterase contaminations

in the Lake during the study period. It is unlikely that these

contaminations are organophosphate or carbamate insecti-

cides as agricultural lands are not located in the vicinity. In

addition to these insecticides, heavy metals and complex

mixtures of pollutants may also cause inhibition of ChE

levels in the fish (Payne et al. 1996; van der Oost et al.

2003; Yadav et al. 2009). Blood brain barrier may have

afforded some protection against penetration of anticho-

linesterase substances present in the Lake through the

brain. The muscle ChE inhibition was greater especially in

the fish collected during rainy periods. It is possible thatmore anticholinesterase substances may have entered the

Lake through surface runoff with the heavy rain which

caused enhanced inhibition of muscle ChE activities of the

fish in the rainy season. Although inhibition of muscle ChE

activities to 2146% of the normal level may not directly

induce fish mortalities it could be an additional physio-

logical stress for the fish populations inhabiting the Lake.

In conclusion, the biomarker responses evaluated in this

study revealed that Nile tilapia population residing in the

hyper eutrophic tropical water body, Lake Beira is under

threat due to the pollution impact even though Nile tilapias

are considered as hardy fish which could tolerate pollutionstress. Hepatic damage in the resident Nile tilapia may be

mainly associated with the pollution stress due to recurrent

exposure to PAHs and toxigenic Microcystis blooms

present in the Lake. Further studies are needed to confirm

the role of PAHs and microcystins in the Lake in inducing

pollutant stress in the fish populations. It would be neces-

sary to correlate the biomarker responses with the specific

pollutant levels in abiotic components as well as in the

biota including fish. The present study emphasizes the

importance of multi-biomarker approach using resident fish

species to assess the pollution exposure and biological

effects in hypertrophic water bodies with complex mixture

of pollutants. This approach could also be used to assess

the effectiveness of the restoration programmes which have

been implemented in order to control the pollution of

aquatic resources.

Acknowledgements We thank Prof. M. D. P. De Costa for granting

us permission to use their facilities for fluorescence determinations in

bile samples and Mr. D. D. R. U. Wanigesekera, for assistance with

the histological preparations. This study was financially supported by

National Science Foundation of Sri Lanka (RG/2003/ZOO/05).

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