Ecotoxicology (2010) 19:10191026 DOI 10.

1007/s10646-010-0483-2

Assessment of biological effects of pollutants in a hyper eutrophic tropical water body, Lake Beira, Sri Lanka using multiple biomarker responses of resident sh, Nile tilapia (Oreochromis niloticus)
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 cellular level in sh have been proposed as sensitive early warning tools for biological effect measurements in environmental 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 uorescent aromatic compounds (FACs), hepatic ethoxyresorun O-deethylase (EROD) and glutathione S-transferase (GST), brain and muscle cholinesterases (ChE), serum sorbitol dehydrogenase (SDH), and liver histology of Oreochromis niloticus, the dominant sh inhabiting this tropical Lake were evaluated to assess the pollution exposure and biological effects. Some sh sampled in the dry periods demonstrated prominent structural abnormalities in the liver and concomitant increase in serum SDH and reduction in hepatic GST activities in comparison to the control sh and the sh sampled in the rainy periods. The resident sh with apparently normal liver demonstrated induction of hepatic EROD and GST activities and increase in biliary FACs irrespective of the sampling period indicating bioavailability of PAHs. Muscle ChE activities of the resident sh were depressed signicantly indicating

exposure to anticholinesterase substances. The results revealed that sh populations residing in this Lake is under threat due to the pollution stress. Hepatic abnormalities in the sh may be mainly associated with the pollution stress due to recurrent exposure to PAHs and toxigenic Microcystis blooms in the Lake. Keywords Highly eutrophic lake Tropical sh Biomarkers Liver histology

Introduction Biomarkers measured at the molecular and cellular level in sh 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 sh 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 sh 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 measurement of CYP1A dependent ethoxyresorun O-deethylase (EROD) in sh has become a promising biomarker for detecting aquatic contaminations of a variety of highly toxic pollutants such as some polycyclic aromatic hydrocarbons (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 sh (Whyte et al. 2000). Fluorescent aromatic compounds in the sh bile are reported as sensitive 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

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2000). Since PAH exposure cannot be reliably determined by measuring sh tissue levels, this parameter is a valid sh biomarker for environmental risk assessment process concerning 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 detoxication reactions within the body (George 1994). The liver is the organ mainly associated with detoxication 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 sh (Dixon et al. 1987). Histopathology of sh liver is also a sensitive and reliable monitoring tool for assessment of the effects of environmental stressors on sh 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, scientic reports of pollutant 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 sh 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 sh 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 sh, is an omnivorous feeder. Mortalities of Nile tilapia in Lake Beira have been observed in several occasions especially during dry periods. However, no studies have been carried out previously to assess the response of the resident sh 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 biochemical and histological biomarker responses of the resident sh, Nile tilapia viz. ChEs in the brain and muscle tissues, serum sorbitol dehydrogenase (SDH), hepatic EROD and GST, uorescent aromatic compounds in the bile and histological structure of the sh 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 trafc, railways, ofces, 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 cyanobacteria 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 nonrestored 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 sh which were used as Controls were obtained from a sh breeding station, National Aquaculture Development Authority, Sri Lanka and maintained at the University of Kelaniya premises (about 1215 km away from the Lake) in outdoor tanks lled 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 sh were daily fed with commercial sh food pellets (Prima Feed, Ceylon Grain Elevators Pvt Ltd., Sri Lanka) at 1% of the body weight. Control sh and the sh 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 tted to the syringe and frozen at -80C until further processing. Liver tissue from each sh was stored frozen separately at -80C until used for EROD and GST analysis. In addition liver tissues were preserved in neutral buffered formalin for histopathological studies. Fish stomach was dissected; contents were xed in 10% formalin for 24 h and examined under the microscope for identication 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 determined at 25C as described by Gerlach (1983) using D-fructose as the substrate. The ChE activities in the brain and muscle homogenates of individual sh were determined at 25C using acetylthiocholine iodide as the substrate 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 differential 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-chloro2,4-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 controlled recording 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 sh bile were determined by xed wavelength uorescence measurements as described by Aas et al. (2000) using Aminco-Bowman Series 2 Luminescence Spectrometer (Thermo Spectronic). Two microliters of bile diluted in 4 ml of 48% ethanol were used to decrease self absorption and quenching of the uorescence signal. Fluorescence at the excitation/emission wavelength pairs (FF) 290/335, 341/383 and 380/430 nm were determined for naphthalene type, pyrene type and benzo(a)pyrene type of metabolites respectively. The FF values are

expressed as arbitrary uorescence units after deducting the signal level of the solvent. Liver histology Liver tissues were xed in 10% buffered formalin at 4C and dehydrated in graded series of ethanol, cleared in xylene and embedded in parafn wax. Sections were cut in 5 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 sh collected during different sampling periods were analyzed by ANOVA (P \ 0.05). Where differences were signicant, multiple comparisons were carried out by Tukeys test (Zar 1999) as appropriate. Log transformed data were used for statistical analysis.

Results No signicant differences were obtained in relation to the body sizes of the sh collected from the Lake and the control sh (Table 1). The sampled sh included both genders. Of the 38 sh sampled from the Lake in the dry periods, livers of 16 sh (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 sh collected from the Lake during the dry period and all the sh collected during rainy periods and control sh were rm, reddish brown in colour and showed the normal appearance macroscopically. Examination of stomach contents of the sh collected from the Lake showed phytoplankton especially Microcystis as their main food items. In addition detritus were found in the stomach contents of these sh. The livers of the control Nile tilapia which showed normal appearance macroscopically had normal histological structure of the hepatopancreas. Hepatocytes are polygonal in shape, arranged in several cellular layers and surrounded by sinusoids. Each hepatocyte contained usually a centrally located single round nucleus. Pancreatic tissue was present in association with venous vessels and as isolated elements. Livers of sh 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 sh collected from the Lake in the dry periods. Large

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1022 Table 1 The body sizes of Nile tilapia used in evaluation of biomarkers at different sampling periods Sampling period Feb 2006Dry period April 2006Dry period June 2006Rainy period July 2006Rainy period
a

A. Pathiratne et al.

Source of sh Lake Beira Controls Lake Beira Controls Lake Beira Controls Lake Beira Controls

Na 16 (6) 14 (0) 22 (10) 16 (0) 20 (0) 12 (0) 18 (0) 10 (0)

Body length (cm) 20.6 3.2 19.8 2.4 19.7 1.5 18.5 2.8 20.8 2.1 19.6 3.9 20.1 2.2 20.8 1.3

Body weight (g) 188 11 162 27 150 34 161 39 175 30 159 29 174 17 188 56

Number within parentheses indicates the number of sh that demonstrated macroscopic liver abnormalities. Body sizes are presented as mean SEM of the number of sh used in each sampling period

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 sh which showed gross macroscopic abnormalities (Fig. 1c, d). The abnormalities observed in these sh were dilation of sinusoids and blood vessels and extensive vascular congestion, 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 sh and the Lake sh which had no liver abnormalities macroscopically were 014 and 228 mUnits ml-1 of serum respectively. However the serum SDH levels in the sh which had liver abnormalities had increased considerably (97160 mUnits ml-1 of serum) in comparison to those in the other sh groups. Hepatic EROD and GST activities in Nile tilapia collected from the Lake and the activities of comparable control sh are presented in Fig. 2. Of the sh sampled in the dry season, induction of EROD activity was not observed in the sh which had liver abnormalities macroscopically where as the sh 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 sh collected during the rainy period were induced by 6 to 10 folds. Of the sh sampled in the dry periods, hepatic GST activities in the sh with liver abnormalities were signicantly lower in comparison with those of the sh with apparently normal livers (Fig. 2b). Hepatic GST activities in the Lake sh were induced by nearly 23 folds during rainy periods compared to the respective controls. Fixed uorescence determinations showed (Fig. 3) that the bile samples of tilapia residing in the Lake contain signicantly higher amounts of naphthalene type, pyrene type and benzo(a)pyrene type metabolites in comparison to the control sh. The uorescence signals were higher during the rainy periods compared to those during the dry periods.

The uorescence signals in the bile samples of the sh with abnormal liver appear to be low compared to those in the Lake sh with normal livers. However the differences were not statistically signicant. Brain and muscle ChE activities in Nile tilapia collected from the Lake and the control sh are presented in Fig. 4. Muscle ChE activities in the sh sampled from the Lake during the study period were signicantly lower than those of the controls irrespective of the liver structure and sampling period. However inhibition of muscle ChE activity (3746%) was greater during the rainy periods in comparison to the extent of inhibition during the dry periods (2125%). No signicant differences were found among different groups of sh with respect to brain ChE activities.

Discussion Recent studies have reported the trophic status, gross pollution and PAH pollution in Lake Beira (Kamaladasa and Jayatunga 2007; Pathiratne et al. 2007). This is the rst study which focused on assessing the exposure and biological effects of pollutants present in this hyper eutrophic tropical Lake using a suite of biomarkers of the resident sh. Even though Nile tilapia is considered as a hardy sh, 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 environment and liver lesions are often associated with exposure to aquatic pollutants (Au 2004; Fernandes et al. 2008). In this study, the sh 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 appearance macroscopically. The most prominent histological

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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)

alterations observed in the sh that showed abnormal livers macroscopically were dilation of blood vessels and sinusoids and severe congestion in sinusoids and small blood vessels, increased vacuolation of hepatocytes, pycnotic nuclei, nuclear atrophy and focal areas of necrosis. Pycnosis of hepatocyte nuclei associated with cytoplasmic vacuolation is a non specic response of sh due to toxic conditions (Roberts 1978). The blockage of sinusoids makes the blood ow from the hepatic portal vein and hepatic artery into the central vein rather difcult. This may be responsible for the cellular degeneration and necrosis in the livers of some Nile tilapia collected from the Lake during dry periods. Observed liver abnormalities reect the biological impacts of complex mixture of pollutants present in this hypertrophic lake. Exposure of sh to anthropogenic organic contaminants with a planar conguration 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 sh 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 sh in the Lake compared to the controls. The highest EROD activities (610 fold induction) and FACs levels found in the sh captured 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 sh (Whyte et al. 2000). Hence previous exposure of sh to PAHs present in the Lake may have

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Fig. 2 Hepatic ethoxyresorun O-deethylase (EROD) and glutathione S-transferase (GST) activities in Nile tilapia collected from Lake Beira. Data are presented as mean SEM. For each enzyme, bars with different letters are signicantly 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 signicantly different from each other (P \ 0.05)

Fig. 3 Biliary uorescence 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 signicantly different from each other (P \ 0.05)

contributed to the induction of hepatic structural abnormalities in the resident sh 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 sh to PAHs in the Lake. Observed increase in serum SDH activities in these sh conrms the hepatocyte damage. GSTs are major phase II detoxication 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 sh 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 reect the natural temperature conditions in the Lake. Nevertheless, hepatic GST activities of Nile tilapia sampled from the Lake Beira in the rainy periods were signicantly higher than that of the sh sampled in the dry periods and the control sh. Even though EROD activity was induced by 610 folds, increase in detoxication capacity of the sh associated with relatively 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 sh may have lead to the hepatic damage subsequently. 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

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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 rst step of detoxication of Microcystins (Amado and Monserrat 2010). Some strains of Microcystis sp. produce the toxin microcystin-LR which is the most toxic cyanobacterial hepatotoxin. A recent study showed that both microcystin-LR and microcystinRR, can induce pathological lesions in hepatic tissues of Nile tilapia (Atencio et al. 2008). Microcystis may be toxic to sh via gastrointestinal ingestion as well as by absorption of the microcystin directly from water. Even though microcystin levels in the sh residing in the Lake were not determined in the present study, inuence of microcystins in inducing hepatic abnormalities in the sh 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 blooms present 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 nontoxic strains which co-occur and are visually indistinguishable but can be quantied 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 synthatase 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 warrants further investigations. Cholinesterases in sh have been used as a biomarker of neurotoxic contamination in aquatic environment monitoring studies (Payne et al. 1996; van der Oost et al. 2003). A recent study has shown that sh acetylcholinesterase could be used as a potential biochemical marker for fertilizer industry efuent pollution in aquatic systems (Yadav et al. 2009). In the present study, muscle ChE activities in the sh sampled from the Lake were signicantly lower (2146%) than those of control sh 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 sh 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 insecticides 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 sh (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 anticholinesterase substances present in the Lake through the brain. The muscle ChE inhibition was greater especially in the sh collected during rainy periods. It is possible that more anticholinesterase substances may have entered the Lake through surface runoff with the heavy rain which caused enhanced inhibition of muscle ChE activities of the sh in the rainy season. Although inhibition of muscle ChE activities to 2146% of the normal level may not directly induce sh mortalities it could be an additional physiological stress for the sh 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 sh which could tolerate pollution stress. 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 conrm the role of PAHs and microcystins in the Lake in inducing pollutant stress in the sh populations. It would be necessary to correlate the biomarker responses with the specic pollutant levels in abiotic components as well as in the biota including sh. The present study emphasizes the importance of multi-biomarker approach using resident sh 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 uorescence determinations in bile samples and Mr. D. D. R. U. Wanigesekera, for assistance with the histological preparations. This study was nancially supported by National Science Foundation of Sri Lanka (RG/2003/ZOO/05).

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