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Item Characterization of tissue specific cholinesterases in native fishes, Rasbora daniconius and Dawkinsia singhala: their sensitivity to the heavy metal, copper(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Premarathna, K.I.R.; Pathiratne, A.Aquatic pollutants could pose health hazards to humans and wildlife. Cholinesterases (ChEs) have been recognized as biomarkers of exposure to a range of aquatic pollutants including organophosphate and carbamate pesticides and heavy metals. This study examined tissue specific characteristics of ChE activities in the brain, muscle and gills of two native freshwater fish species Rasbora daniconius (Sinhala; Dandiya) and Dawkinsia singhala (Sinhala; Dankola pethiya) in Sri Lanka with the aim of using ChEs of these fishes as potential biomarkers for biomonitoring aquatic pollution. The main objectives were to characterize the biochemical properties of ChEs in brain, muscle and gill tissues of R . daniconius and D . singhala, using two specific substrates and three selective inhibitors and to measure in vitro sensitivity of the ChE activities to exposure to heavy metal, Copper. Cholinesterase activity measurements with two substrates (acetyl thiocholine iodide (ATCI) and butyryl thiocholine iodide (BTCI)) showed the presence of two different cholinesterases in both fishes namely Acetyl cholinesterase and Butyryl cholinesterase. With the estimated kinetic parameters of the enzymes (Km, Vmax and Vmax/Km) for both substrates, highest catalytic efficiency was obtained for brain tissues with the two substrates for both fish species. Of the two substrates tested, the catalytic efficiencies were generally higher when ATCI was used as the substrate in crude enzyme extracts (Vmax/Km for brain ChE: 3.95 for R . daniconius; 2.04 for D . singhala). Using enzyme inhibition pattern in response to three selective inhibitors (Eserine, BW254C51 and Iso-OMPA), it was found that the brain and muscle tissues of both fishes contained mainly Acetylcholinesterase enzyme whereas the gill tissues are the sources of both Acetylcholinesterase and Butyryl cholinesterase enzymes. The results of the present study clearly proved that ChE activities of both species have a concentration depended inhibition pattern in response to in vitro exposure of Cu2+ at least in the tested concentration range (0.05-2 mM). ChEs in the gill tissues in both fish species are more sensitive to the Cu2+ than the ChEs in brain and muscle tissues. Overall results indicate that cholinesterase enzymes of R . daniconius and D . singhala may be used as biomarkers for neurotoxic contaminants such as the heavy metal, copper. Crude extracts of the ChE enzymes in brain, muscle and gill tissues of the two fish species can be used in routine procedures for screening anticholinesterase contaminations in water resources.Item Acetylcholinesterase inhibition and gill lesions in Rasbora caverii, an indigenous fish inhabiting rice field associated waterbodies in Sri Lanka(Kluwer Academic Publishers-Plenum Publishers., 2006) Wijeyaratne, W.M.D.N.; Pathiratne, A.The present study was aimed at applying condition factor (CF), brain acetylcholinesterase (AChE) and gill histology as biomarkers for detecting possible exposure/effect induced by pesticides in fish residing rice field associated waterbodies in Sri Lanka. Biomarkers of an indigenous fish, Rasbora caverii collected from five sampling sites including canals near rice fields, a river and a reservoir (the reference site) were evaluated at four sampling stages covering pesticide application periods during rice cultivation season in 2004. Results indicated that CF of the fish did not show significant alterations regardless of the sampling sites or sampling stages. Site specific differences in AChE activities of the fish were not evident either prior to application of pesticides or at 7 days after Paraquat application to the rice fields. Two days after the application of a mixture of Fenthion and Phenthoate to the rice fields, AChE activity of the fish collected from canals near rice fields was significantly depressed (65–75%) compared to the fish in the reference site. The activities remain depressed to 50–56% even at 65 days after the insecticides application. Laboratory studies showed that prior exposure of R. caverii to Paraquat (2 μg l−1, 7 days) enhanced the extent of inhibition of brain AChE activity induced by Fenthion (3 μg l−1) or a mixture of Fenthion (3 μg l−1) and Phenthoate (5 μg l−1). Gills of fish collected from canals near rice fields exhibited abnormal multiple divisions at the tips of some secondary lamellae in addition to hyperplasia, hypertrophy and club shaped deformities. Results indicate that application of pesticides in rice culture could manifest a threat to native fish populations residing rice field associated waterbodies. The response of brain AChE and histological changes in the gills of R. caverii allowed differentiating sampling sites after insecticide applications to the rice fields. Hence, R. caverii may be considered as a surrogate species in ecotoxicological risk evaluation of agrochemicals in the region.Item Body size-related differences in the inhibition of brain acetylcholinesterase activity in juvenile Nile tilapia (Oreochromis niloticus) by chlorpyrifos and carbosulfan(Academic Press., 2007) Chandrasekara, L.W.H.U.; Pathiratne, A.Influence of body size on inhibition of brain acetylcholinesterase (AChE) activity of juvenile Nile tilapia, Oreochromis niloticus by chlorpyrifos and carbosulfan was investigated concerning its potential use in the biomonitoring of anticholinesterase pesticides in tropical water bodies. Three size groups of fish (fry: 3–4 cm, fingerlings: 6–8 cm, sub-adults: 10–12 cm in total length) were exposed to a series of concentrations of chlorpyrifos (0.5–12 μg L−1) or carbosulfan (1–10 μg L−1), and concentration–response for inhibition and recovery of the AChE enzyme was evaluated in comparison to the controls at different time points, 2, 6, 10, and 14 d. The AChE activities of the control fish followed the order of decreasing activity, fry>fingerlings>sub-adults. AChE activities of the fry were nearly 2-fold higher than that of the sub-adults. Following 48 h of pesticide exposure, the AChE activity of the three size groups of fish decreased significantly in comparison to the respective controls in a concentration-dependent manner. The activity was greatly inhibited in the fry (39–85%) compared to sub-adults (18–47%) exposed to the most of the similar concentrations of the pesticides. Median effective in vivo inhibition concentrations (48 h IC50) of chlorpyrifos for fry, fingerlings, and sub-adult stages were 0.53, 0.75, and 3.86 μg L−1, respectively, whereas the corresponding values for carbosulfan were 3.37, 7.02, and 8.72 μg L−1. When fish were maintained in the initial pesticide medium for 14 days, AChE activity restored gradually depending on the initial pesticide exposure concentration and the size group of the fish. Results indicate that brain AChE of Nile tilapia is a promising biomarker for assessment of anticholinesterase pesticide contaminations in water. However, body size of Nile tilapia should be taken into account when using this biomarker in biomonitoring programmes.Item Toxicity of malathion to nile tilapia, Oreochromis niloticus and modulation by other environmental contaminants(Aquatic Toxicology, 1998) Pathiratne, A.; George, S.G.Deliberate or accidental contamination of ponds by widely utilised organophophorous (OP) insecticides such as malathion is a potential problem for aquaculture in tropical countries. The aim of the study was to investigate potential synergistic or protective effects of common environmental pollutants on malathion toxicity in the Nile tilapia (Oreochromis niloticus) and by correlation of acute toxicity (LC50) studies with biochemical parameters, identify potential enzyme systems involved in malathion toxicity. Tilapia were very sensitive to malathion (96h LC50 2ppm) and in vitro data indicated that malaoxon, formed by oxidation of malathion, was the effective toxicant. Exposure of fish to an environmentally relevant dose of the insecticide synergist and CYP inhibitor, piperonyl butoxide (PBO) markedly reduced both the sublethal and the acute toxicity of malathion by 2-fold. Correlation of toxicity data with inducer effects and biochemical analyses failed to provide any evidence for CYP1-, CYP2B- or CYP3A-mediated malathion activation or detoxication in this species, thus the effect of PBO could not be attributed to inhibition of these enzymes. Whilst interspecies comparisons implicate hepatic θ class GST and non-specific carboxylesterase in malathion detoxication there was no evidence for alterations in malathion toxicity to tilapia by inducers of these enzymes. Treatment of fish with concentrations of a prototypical polyaromatic hydrocarbon, or cadmium, exceeding those producing effects in field situations, did not alter malathion toxicity indicating a lack of interaction of other common classes of environmental pollutants with OP toxicityItem Influence of low concentrations of Trichlorfonon haematological parameters and brain acetylcholinesterase activity in common carp, (Cyprinuscarpio)(Aquaculture Research, 2005) Chandrasekara, H.U.; Pathiratne, A.Trichlorfon, an organophosphorus insecticide, is used in aquaculture to eliminate fish ectoparasites. The present study was aimed at evaluating the effects of low concentrations of Trichlorfon (0.25 and 0.50 mg L?1 for 1 and 24 h) on selected haematological parameters and brain acetylcholinesterase (AChE) of subadults of common carp, Cyprinus carpio. Results showed that Trichlorfon had no significant effect on the survival and haematocrit values of the exposed fish. However, all fish exposed to Trichlorfon exhibited leucopenia coupled with lymphocytopenia. Upon transfer to clean water, leucopenia did not return to normal in seven days. Brain AChE activity of the fish exposed to Trichlorfon for 24 h was reduced by 55?57% compared with the controls and inhibition was not restored fully within seven days. Hence, precautions should be taken when even low concentrations of Trichlorfon are used in carp culture, especially for long-term treatments.