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Item Heavy metal levels in Chanos chanos (Milk fish) from Negombo estuary, Sri Lanka and human health risk assessment associated with their consumption(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Bandara, P. R. C. S.; Pathiratne, A.Contamination of fish with toxic heavy metals can counteract the nutritional and health benefits of fish food on human health. Objectives of this study were to determine selected heavy metals levels viz. Aluminum (Al), Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Nickel (Ni) and Zinc (Zn) in edible fish, Chanos chanos (Milk fish) from Negombo estuary and assess potential human health risks associated with their consumption. Muscle and liver of freshly captured C. chanos (n=20) from the estuary in 2018 were used for metal analysis. Metal levels were determined by Atomic absorption spectroscopy (graphite furnace method) following microwave digestion using standard analytical chemistry techniques. Two certified reference materials for trace metals were used to check the recovery of the metals based on the analytical method used in this study and recovery percentages were within a satisfactory range. Potential human health risks associated with the dietary exposure to these heavy metals through fish consumption were assessed using multiple approaches by comparing the thresholds viz. Maximum Permissible limit (MPL), Tolerable Daily Intake (TDI) and Target Hazard Quotient (THQ) stipulated by international food standards regulatory authorities. Of the seven heavy metals measured, mean metal concentrations in C. chanos followed the decreasing order, Zn≈Al>Pb≈Cr>Cd>Ni≈Cu in muscle and Zn≈ Cu > Al≈ Cd>Cr≈ Ni> Pb in liver respectively. Concentrations of Cr and Pb in the edible muscle were significantly greater (P < 0.05) than those in the liver of the fish. Human health risk assessment indicated that the detected concentrations of the heavy metals in the edible muscle of the fish were below the MPLs stipulated by international food regulatory authorities except for Cd in 35% of the fish (range 0.06 - 0.08 mg/kg wet weight). Based on mean metal concentrations in the fish muscle, estimated daily intake values for moderate level fish consumers were below the corresponding TDI levels suggested by the food regulatory authorities. THQs for respective metals in the fish muscle were greater than the threshold value of one for Cd (1.5) and Pb (1.7) indicating potential human health risks associated with these metals in edible muscle for non-carcinogenic effects. However estimated maximum allowable fish consumption rates are high enough to safeguard the risks associated with Cd and Pb for moderate level consumers. The results revealed that it is unlikely that the measured seven heavy metals in C. chanos would pose non-carcinogenic health risks to the moderate level fish consumers.Item Assessing neurotoxic potential of titanium dioxide nanoparticles and bulk form using the fish model, Nile tilapia (Oreochromis niloticus)(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Samanthika, A.L.C.; Pathiratne, A.Titanium dioxide nanoparticles (nano TiO2) and bulk TiO2 are widely used worldwide in various sectors. Hence they can be released inevitably into the inland water bodies posing health threats to the aquatic ecosystems. Cholinesterase (ChE) activity can be used as a biomarker for screening neurotoxicity in organisms exposed to the environmental contaminants. The objective of the present study was to evaluate the neurotoxic potential of environmentally relevant concentrations of nano and bulk TiO2 using Nile tilapia as the tropical fish model. Groups of fish which had been acclimated to the laboratory conditions for two weeks were exposed continuously to relatively low concentrations (50 and 100 μg/L) of nano or bulk TiO2 for 7 or 14 days (n= 7). The control fish were exposed only to aged tap water. The experimental design also included 7 day nano or bulk TiO2 exposure groups which had been transferred to aged tap water for another 7 days to evaluate potential recovery of the toxicity. Neurotoxic potential was assessed using ChE activity measurements in the brain, gill and liver tissues by a standard spectrophotometric method. ChE activities (mean±SEM in nmol/min/mg protein) in gills were elevated significantly(P <0.05) in the fish exposed to 50 and 100 μg/L bulk TiO2 for 7 days (102±9 and 105±7 respectively) compared to those of the control fish (71 ± 5) whereas brain ChE activities were not affected. Liver ChE activities were significantly elevated at 100 μg/L continuous exposure to bulk TiO2 for 7 and 14 days (279±15 and 315±15 nmol/min/mg protein respectively) compared to those of the control fish (155±12) and were not restored to normal levels after 7 days post exposure. A consistent ChE activity stimulation pattern was not evident with respect to bulk and nanoTiO2 exposures. In the fish exposed to both concentrations of nano TiO2, ChE activities in brain and gill tissues were not significantly different from those of the control fish. However ChE activity in liver tissues of the fish exposed to 100 μg/L of nano TiO2 was elevated significantly (P < 0.05) at 7 days exposure and post exposure periods. The results revealed that environmentally relevant concentrations of nano and bulk TiO2 may pose neurotoxic risks to the fish populations inhabiting the contaminated water bodies and the bulk form of TiO2 is not ecotoxicologically inert as previously assumedItem Assessing toxicity of two industrial zone effluents reaching Kelani River,Sri Lanka(Journal of national science foundation Sri Lanka, 2018) Hemachandral, C.K.; Pathiratne, A.Evaluation of the effcaey of waste treatment technologies of industnal waste is a major challenge for sustainable industrial development world-wide. Hence, new strategies are needed to assess interactive toxic effects of all substances present in the treated waste. This study assessed potential toxic hazards of treated effluents discharged from common wastewater treatment plants of two mclustrlal zones located in the Kelani River basin using Allium cepa (common onion) test system. The results showed that the final effluents of both industrial zones under undiluted and diluted (1 IS v/v) conditions mduced cytotoxrcity on all occasions, with evidence of significant (p < 0.05) mlto-depression In the root meristem and retardation of root growth in A. cepa. Genotoxlc hazard of the effuents was evident by frequent mcrease of nuclear and chromosomal abnormalltles, and occasional development of micronuclei in the root merlstem. Dilution of the effluents to 1 reduced the genotoxic effects generated in A. cepa roots by the final effuents_ The results revealed that waste treatment technologies m these two mdustnal zones need to be upgraded m order to ehmmate cytotoxlc and genotoxrc hazards associated with the treated effuents_ The results highlight the importance of incorporatmg practically feasible bioanalytical tools such as A. cepa root based test system on a regular basis for evaluating the efficacy of waste treatment technologies.Item Biomarker responses of Nile Tilapia (Oreochromis niloticus) exposed to polluted water from Kelani river basin, Sri Lanka: Implications for biomonitoring river pollution(Sri Lanka J. Aquat. Sci, 2018) Ruvinda, K.M.S.; Pathiratne, A.Biomarkers can be considered as early warning signals for potential adverse effects on the biota. The present study examined the feasibility of using selected biomarker responses of a model fish, Oreochromis niloticus under laboratory exposure approach for identification of potential biological impacts of pollution in Kelani River. Laboratory acclimated O. niloticus were exposed under static-renewal conditions to water samples collected from selected sites of the Kelani River basin with different anthropogenic influences and biomarker responses (brain and muscle cholinesterase activities for neurotoxicity, erythrocyte micronuclei and nuclear abnormalities for genotoxicity and liver histology for hepatic damage) were evaluated at 5 and 10 days of exposure. Exposed water was physico-chemically characterized using standard analytical methods. The results revealed that exposure of O. niloticus to the water from selected polluted sites which included canals and canal confluences resulted in significant increases (p<0.05) in total erythrocyte nuclear abnormalities, evolution of erythrocyte micronuclei and induction of liver histopathological indices in comparison to the fish exposed to the water from the upper reach of the river (reference site) in most cases and the control fish exposed to the aged tap water in all cases. Brain cholinesterase activity was significantly inhibited (p<0.05) in the fish exposed to the water from the most polluted site compared to the control fish exposed to the aged tap water. Biomarker responses indicated that the fish populations inhabiting the polluted sites in the river may be under stress especially due to hepatic damage and genotoxicity. Evaluation of “effect directed biomarker responses” of the model fish, O. niloticus following laboratory exposure to the contaminated water can be a practically feasible approach for biomonitoring potential pollution impacts associated with the riverine ecosystems.Item Sensitivity of four freshwater animal species to copper and cadmium under tropical temperature exposure(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Heshani, A.L.S.; Pathiratne, A.Copper and cadmium are two common pollutants in Sri Lankan freshwater ecosystems. Water quality criteria for these metals focusing on the protection of freshwater species in Sri Lanka are yet unavailable. Formulating water quality criteria based on tropical species sensitivity distribution analysis would require credible ecotoxicity data (especially at early life/young stages) under tropical temperature exposure for a range of freshwater species belonging to different trophic levels. Thus, objective of the present study was to generate freshwater ecotoxicity data for copper and cadmium for tropical temperature exposures using four freshwater native/model test organisms: two crustaceans, Daphnia magna and Moina macrocopa, the fish Poecilia reticulata and the tadpoles of Duttaphrynus melanostictus representing primary and secondary consumers. Based on the range finding tests, the animals were exposed separately to a series of concentrations of Cu2+ (D . magna 50-300; M . macrocopa 1-150; fish 25-300; tadpole 10-300) or Cd2+ (D . magna 10-200; M . macrocopa 1-150; fish 25-300; tadpole 100-500) in μg/L along with the controls (in triplicate/quadruplicate exposure medium, n=10 animals per replicate) at 280C - 300C under static-renewal conditions (hardness ≤ 66 mg/L as CaCO3) following standard OECD toxicity testing protocols. The sensitivity of the crustaceans (neonates) for different concentrations of Cu2+ and Cd2+ exposure was assessed based on percentage neonates immobilized whereas the sensitivity of P . reticulata (fry 5-10 mm total length) and D . melanostictus (Gosner stage 25 tadpoles) was evaluated based on percent mortality. Concentrations of copper and cadmium in the exposure media were determined using atomic absorption spectrophotometry. Ecotoxicity thresholds were estimated based on concentration-toxicity response relationships using USEPA software, ‘Toxicity Relationship Analysis Program’. Estimated 48 hour median effective concentration (EC50) of copper for immobilization of the crustaceans, D . magna and M . macrocopa were 117 and 15 μg/L respectively whereas corresponding values for cadmium were 45 and 12 μg/L. Estimated 96 hour median lethal concentration (LC50) values of copper for the fish, P . reticulata and the tadpole, D . melanostictus were 63 and 72 μg/L respectively. Corresponding LC50 values for cadmium were 76 and 431 μg/L. The most sensitive species for copper and cadmium was the crustacean, M . macrocopa . The highest tolerance for cadmium was shown by the amphibian, D . melanostictus tadpole whereas D . magna showed the highest tolerance to copper. Additional ecotoxicity data for copper and cadmium focusing on more tropical freshwater species are needed for derivation of water quality criteria for tropical freshwater life protection.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 Cadmium and arsenic levels in edible fishes, Oreochromis niloticus (Nile tilapia) and Ompok bimaculatus (Pena walaya) from Padaviya reservoir of North Central Province, Sri Lanka(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Weerasekara, K.A.W.S.; Pathiratne, A.; Kithsiri, H.M.P.Oreochromis niloticus (Nile tilapia) and Ompok bimaculatus (Pena walaya) are two edible fish species captured in the reservoirs in the North Central province. Objective of this study was to assess the cadmium and arsenic levels in muscle, liver and kidney tissues of O. niloticus and O. bimaculatus inhabiting the Padaviya reservoir to assess association of metal/metalloid levels in tissues with body weights, and total lengths of the fishes. The body weight (180-300g; 110-225g) and the total length (19.0-24.1cm; 24.0-34.0cm) of Nile Tilapia (n=30), and Pena walaya (n=30) respectively were recorded during three sampling events in 2018. The tissues were analyzed for cadmium and arsenic levels using Inductively Coupled Plasma Mass Spectrometry. Cadmium and arsenic contents of the muscle tissues were below the limit of detection (LOD) (<0.05 mg/kg wet weight) except cadmium in the muscle tissues of five Nile tilapia (0.05-0.10 mg/kg). Cadmium contents in the liver tissues of both Nile tilapia and Pena walaya, ranged from <0.05 to 0.14 mg/kg (wet weight) where cadmium levels of 67-70% of the fishes were below the LOD. Arsenic levels in liver tissues of Nile tilapia and Pena walaya were <0.05–0.10 mg/kg and <0.05–0.06 mg/kg respectively where arsenic levels in 83% of Nile tilapia and 97% of Pena walaya were below the LOD. Cadmium content in the kidney tissues of Nile tilapia and Pena walaya were <0.05-0.90 mg/kg and <0.05-0.40 mg/kg respectively where cadmium levels in 30% and 33% fishes were below the LOD. Arsenic was detected only in kidney tissues of 10% Nile tilapia (<0.050.46 mg/kg). Cadmium contents in liver tissues of Nile tilapia positively correlated (p <0.05) with the total length. No other significant associations were found between the metal/metalloid content in the tissues and the body size of the fishes. Results revealed that the cadmium and arsenic contents in the edible muscle tissues of most of these fishes were within the safe limits specified by the regulatory authorities for human consumption. Bioaccumulation pattern of cadmium in the tissues of both fishes followed the order: muscle < liver < kidney, showing greater cadmium bioaccumulative capacity in the kidney tissues. Periodic monitoring of metal/metalloid levels in edible fishes in Padaviya reservoir is recommended considering human safety. The data generated in this study can serve as baseline information for future monitoring and human health risk assessment programmes.Item Screening cytotoxic potential of henna based hair dyes using human red blood cells.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Nawalage, N.M.S.K.; Pathiratne, A.Intensive usage of commercial hair dyes all over the world may lead to wide variety of health and environmental problems. Direct contact of hair dyes with the human skin may initiate toxic effects on human cells. Commercially available ‘henna based hair dyes’ are considered as less toxic but scientifically based studies on assessing toxicity of these dyes are limited. The present study was conducted to screen potential cytotoxicity of three selected henna based hair dyes on human red blood cells (RBC) in vitro using hemolysis assay. Mostly used henna based commercial hair dyes were purchased from the market. The hemolysis assay was performed by separating serum of the centrifuged blood samples and diluting the RBCs to 20% by adding phosphate buffered saline solution (pH 7.4). The diluted red blood cell suspensions were mixed with commercial hair dye solutions (final dye concentrations 0, 0.05, 0.1, 0.2, 0.4, 1.0 mg/mL) and the mixtures were incubated at 37OC. The incubated RBC samples were centrifuged and the absorbance of supernatants were measured at 540 nm to determine percentage hemolysis. The potential associations between dye concentration and hemolysis potential were analyzed using Pearson’s correlation test (P < 0.05). Triton X -100 (0.1%) and phosphate buffer solution (pH 7.4) were used as the positive control and the negative control respectively. Results showed significant positive correlations (P < 0.05) between hemolysis (%) and the hair dye concentration in all three hair dyes indicating concentration dependent cytotoxic response on red blood cells. The results may indicate potential health impacts associated with these henna based commercial hair dyes during direct applications at high doses. Further toxicity assessments especially in relation to cytogenetic effects are warranted considering human health.Item In vitro metabolism of benzene, toluene, and xylene in rat liver(North Dakota State Univ., Fargo (USA)., 1985) Pathiratne, A.A headspace gas chromatographic method was used to study the metabolism of benzene, toluene, and m-xylene in rat liver. Metabolism of benzene was lower than that of toluene, or m-xylene. Investigating metabolic rates of subcellular fractions, it was found that glutathione and glutathione S-transferase in the cytosol are involved in metabolism of benzene and toluene by microsomes. When both benzene and toluene are present in the incubation medium, they act as inhibitors of each others metabolism. Toluene and m-xylene metabolism were induced by the pretreatment of rats with phenobarbital (75 mg/kg), or 3-methylcholanthrene (25 mg/kg), or Aroclor 1254 (75 mg/kg) for 3 days, whereas benzene metabolism was not induced by all pretreatments. Another series of experiments was conducted to compare the effects of benzene, toluene, and xylene pretreatment on liver metabolism. In vivo metabolism of /sup 14/C-toluene and possible covalent binding of /sup 14/C-toluene to microsomes were also investigated. /sup 14/C-toluene when incubated with liver microsomes in the presence of a NADPH generating system formed benzylalcohol and cresols. Some of the radioactivity was covalently bound preferentially to microsomal proteins. The binding process required cytochrome P-450 dependent mixed function oxidases. This study suggests that toluene is metabolized to several reactive intermediates by liver microsomal enzymes and these metabolites are responsible for the covalent binding to macromolecules which represents a subcellular mechanism by which toluene may express its own in vivo toxicity.Item Bioassessment of the Effluents Discharged from Two Export Oriented Industrial Zones Located in Kelani River Basin, Sri Lanka Using Erythrocytic Responses of the Fish, Nile Tilapia (Oreochromis niloticus)(Springer US., 2017) Hemachandra, C.K.; Pathiratne, A.Complex effluents originating from diverse industrial processes in industrial zones could pose cytotoxic/genotoxic hazards to biota in the receiving ecosystems which cannot be revealed by conventional monitoring methods. This study assessed potential cytotoxicity/genotoxicity of treated effluents of two industrial zones which are discharged into Kelani river, Sri Lanka combining erythrocytic abnormality tests and comet assay of the tropical model fish, Nile tilapia. Exposure of fish to the effluents induced erythrocytic DNA damage and deformed erythrocytes with serrated membranes, vacuolations, nuclear buds and micronuclei showing cytotoxic/genotoxic hazards in all cases. Occasional exceedance of industrial effluent discharge regulatory limits was noted for color and lead which may have contributed to the observed cytotoxicity/genotoxicity of effluents. The results demonstrate that fish erythrocytic responses could be used as effective bioanalytical tools for cytotoxic/genotoxic hazard assessments of complex effluents of industrial zones for optimization of the waste treatment process in order to reduce biological impacts.