*MEHWISH FAHEEM, ABDUAL QAYYUM KHAN SULEHRIA, MEHRAB TARIQ, IRAM KHADIJA, AYESHA FIAZ & MALEEHA SAEED

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1 BIOLOGIA (PAKISTAN) 2012, 58 (1&2), PK ISSN Effect of sub-lethal dose of Cadmium Chloride on biochemical profile and catalase activity in fresh water fish Oreochromis niloticus *MEHWISH FAHEEM, ABDUAL QAYYUM KHAN SULEHRIA, MEHRAB TARIQ, IRAM KHADIJA, AYESHA FIAZ & MALEEHA SAEED Department of Zoology, GC University, Kachery Road, Lahore, Pakistan ABSTRACT In aquatic ecosystems, heavy metals have received considerable attention due to their toxicity and accumulation. The increase in industrial activities as well as in the use of chemical fertilizers and pesticides in the agricultural practice during the past few decades led to the marked rise of heavy metals in the environment. The present study was designed to evaluate the effect of cadmium on non-commercial fish Oreochromis niloticus. Alteration in fish protein, glycogen and anti-oxidant enzyme i.e., catalase concentration was measured in tissues exposed to sublethel concentration of cadmium chloride for 10 days. LC 50 was measured and found to be mg/l. protein concentration tend to decrease in different tissues of fish in order of Liver> Heart>Gills>Muscles. concentration also showed marked decrease in order of Liver>Heart>Muscles>Gills. Catalase activity decreased in order of Liver>Heart>Muscle>Gill. Keywords: Oreochromis niloticus,lc 50, Antioxidant enzymes,,, INTRODUCTION Freshwater gets contaminated with a wide range of pollutants and it has become a matter of major concern around the globe (Voegborlo et al., 1999; Vutukuruet al., 2005; Rauf et al., 2009). Among pollutants, metals are of special concern because of their diversified effects and the range of concentrations that could cause toxic effects to fish (Rauf et al., 2009). These metals are introduced into the aquatic ecosystem through different routes such as industrial effluents and wastes, agricultural pesticide runoff, domestic garbage dumps and mining activities (Merian, 1991). In an aquatic ecosystem, fish are considered as heavy metal indicator because heavy metal readily accumulates in the fish body (Gernhofer et al., 2001). Among the aquatic organisms, these pollutants have a very adverse effect on the fishes, and because of this reason fishes are considered to be the most relevant organism for assessing the pollution in the aquatic ecosystems (Van der Oost et al., 2003). Many studies are available that demonstrate heavy metal toxicity in fish (Jagadeesan et al., 2001; Francis et al., 2002). Cadmium is considered as one of the most uncertain environmental pollutants and it is toxic for living organisms. It is mostly used in manufacturing of batteries, pigments and also in plastic industries (ATSDR, 1997). Cadmium is toxic at low level it may induce kidney, liver, gill and heart malfunctioning. It badly influences blood parameters in living organism and lead to hematological *Corresponding author:mehwish_faheem@hotmail.com

2 74 M. FAHEEM ET AL BIOLOGIA (PAKISTAN) disorders and cause oxidative stress (McCluggage, 1991; European Union, 2002; Young, 2005). Recent evidences have suggested that Labeo rohita and Catla catla can accumulate high levels of cadmium in polluted waters in Pakistan. It is interesting to note that in these waters the concentration of Cd accumulated in non-edible fishes is lesser in comparison to that of edible fishes (Nawaz et al., 2010). The aim of the present study was to determine the LC 50 value of cadmium in Oreochromis niloticusand to evaluate the effect of cadmium on oxidative enzyme (catalase) and biochemical profile. MATERIALS AND METHODS The fish, Nile tilapia (Oreochromis niloticus), were obtained from Fisheries Research and Training Institute, Manawan. Fish were acclimatized for one week in fish ponds at Zoology Department, GC University, Lahore. Following Physico-Chemical parameters were measured in pond, at the time of sampling and in laboratory glass aquaria:atmospheric temperature, water temperature with thermometer to the nearest 1 o C;pH by a portable laboratory ph meter and Dissolved Oxygen of pond water by a portable D.O. Meter (YSI- Model-57). Metal acute toxicity assays Acute cadmium chloride toxicity tests in terms of 96-hr LC 50 were conducted with Oreochromis niloticus according to APHA guidelines (APHA, 2005). These tests were performed in fifteen glass aquaria containing 70-liter water. Ten fish per group were tested against various concentrations of Cd with three replications for each test dose. Extra pure compound of Cd (CdCl 2 ) was dissolved in deionized water and stock solution prepared for required metal concentrations. The exposure concentration of Cd for each fish species was started from zero (with an increment of 10mg/l) to 50 mg/l. LC 50 (conc., in which 50% of fish die) was calculated by probit analysisusing SPSS 15 (Finny, 1971). Sub-lethal studies For sub-lethal studies, 60 fish of both sexes were randomly selected and divided into 3 groups, of 20 fishes each. Asub-lethal dose of 3.58 mg/l (approximately 4 mg/l and 18 mg/l (1/10 th, and ½ of LC 50 ) were selected and fish were exposed to these concentrations for 96 hours. At the end of 96 hours of exposure, fish were randomly selected from control and experiment aquaria for the protein, glycogen and enzyme analyses. Biochemical and anti-oxidant analysis was measured by Lowery method with folin phenol reagent (Lowery et al., 1951). was measured through calorimetric method by Kemp & Mayers (1954). activity was assayed using the method of Claiborne(1985). RESULTS AND DISCUSSION The percentage mortalities for different exposure periods at different concentrations of cadmium chloride are shown in fig 1. LC 50 value of cadmium chloride for the fish Oreochromis niloticus was determined by Probit-regression

3 Vol. 58 (1&2) EFFECT OF CADMIUM CHLORIDE ON OREOCHROMIS NILOTICUS 75 analysis (Table. 1) using SPSS 15. and LC 50 was found to be mg/l. This value obtained was lower than the LC 50 values determined in other tilapia species i.e. 200 mg/l in Tilapia mossambica (Banerjee et al., 1978) and 80mg/l as determined by Chung (1983). This shows that Oreochromis niloticus is less resistant to cadmium toxicity as compared to other tilapia species. Andaya & Gotopeng (1982) determined the LC 50 value in Oreochromis niloticus fry, they found 50 % mortality of fish fry at 1.6 mg/l. The difference may be due to size of fish as large size fish are more resistant to the toxicity as compared to fish fry. Fig., 1: % Fish Mortality Vs Exposure time of different concentration of Cadmium Table 1: Probit-regression analysis of Oreochromis niloticus against cadmium chloride at 96 hours post exposure. Concentration in mg/l Total no. of fish Observed responses Expected responses LC50 mg/l From the data presented, it is clear that at the end of 1/10 th and 1/2 exposure to sub-lethal concentration for 96 hours, content decreased in order of Liver>Heart>Gills>Muscle. This decrease in protein level may be due to metabolic utilization of keto acids to gluconeogenesis pathway (for synthesis of glucose) or for maintenance of ionic and osmo-regulation (Schmidt,. 1975). Jana & Padhay (1987) also made same observations in the muscles of C. punctatus after the treatment with heavy metals (Jana &Padhay 1987). Ravichandran (1994) observed the effect of phenol, and found decreased the protein in muscle Oreochromis mossambicus. Liver is the chief organ of carbohydrate metabolism and level of glycogen was found to be highest in liver. Muscle glycogen acts as a source of glucose for

4 76 M. FAHEEM ET AL BIOLOGIA (PAKISTAN) glycolysis while liver glycogen acts as a source of glucose to maintain the glucose level in blood. As the level of glycogen decreases in fish, when exposed to toxicant, it may be due to its rapid utilization to meet energy needs. The results of our studies are supported by the other workers (Bedii & Kenan, 2005). The maximum amount of glycogen decreased in gills, which may be due to the fact that gills rapidly utilize glycogen to meet the respiratory stress when exposed to toxicant. Table 2: Changes in the levels of biochemical constituents and Catalase activity in the fish, Oreochromis niloticus exposed to 1/10 th and ½ of lethal concentration. Organs Muscles 9.36± ± ± ±0.13 Gills 2.96± ± ± ±0.37 Liver 11.52± ± ± ±0.06 Heart 6.08± ± ± ±0.04 Exposure for 96 hours Control 1/10 th of Lc ±0.54** 6.34±0.6@ 36.0±0.28@ 6.35±0.08** 4.64±0.88** 5.34±0.31@ 6.8±0.59* 5.59±0.03* 13.52±0.66** 7.7±0.78** 23.36±0.60*** 23.52±0.02@ 8.16±0.88** 3.1±0.34@ 11.52±0.66** 0.99±0.03*** ½ of Lc ±1.15*** 5.62±0.38** 34.56±0.67** 3.491±0.03*** 7.28±0.87*** 4.3±0.82* 7.44±0.60** 6.18±0.22@ 15.12±0.87*** 6.72±0.82*** 19.16±0.67*** 22.15±0.192** 9.12±0.66*** 2.52±0.34* 9.2±1.13*** 0.55±0.10*** Biochemical constituents expressed in mg/g wet weight of the tissue. Catalase activity expressed in activity/mint/mg of Non-significant; *: Significant at P< 0.05; **: Highly significant at P<0.01; ***: Very highly significant at P< Catalase a primary antioxidant defense component protects fish from oxidative stress by converting the hydrogen peroxide to oxygen and water (Atli & Canli, 2007). It was determined that activity was maximum in liver and minimal in other tissues e.g., brain (Jena et al., 1998). Activation of activity may be due to effective oxidative defense against oxidative stress or may be to compensate the decrease in other anti-oxidant activity such as GPX and SOD. Inhibition of activity may be due to direct effect of metal. activity decreases in order Liver > Heart>Muscle>Gills. When fish is exposed to

5 Vol. 58 (1&2) EFFECT OF CADMIUM CHLORIDE ON OREOCHROMIS NILOTICUS 77 cadmium, gills are affected first. There was no significant change in activity in liver. This was also supported by work of Radii &Matkovics (1998), they suggested that no change in catalase activity may be due to increased activity of other anti-oxidant enzymes. In order to evaluate the role of cadmium as a cause of oxidative stress, there is need to estimate other oxidant and anti-oxidant enzymes. REFERENCES APHA., AWWA., WPCP Standard methods for the examination of water and wastewater. 21st ed. American Public Health Association, Washington, DC. ATSDR (Agency for Toxic Substances and Disease Registry), Toxicological profile for Cadmium. US Department of Health and Human Services, Atlanta, GA. Atli G. & Canli M.,2007. Enzymatic responses to metal exposures in a freshwater fish Oreochromis niloticus. Comp. Biochem. Physiol., 145: Andaya, A. A. & Gotopeng, E. U., Cadmium toxicity and uptake in Tilapia nilotica. Kalikasan. 11(2-3): Banerjee,.K., Dastdar, S. G., Mukhopadhya, P. K. & Dehadrai, P. V., Toxicity of cadmium: A Comparative study in the air-breathing fish, Clarias batrachus (Linn) and in the non air-breathing, Tilapia mossambica (peters). Indian J. Exp. Biol., 16(2): Bedii,C. & Kenan, E., The effects of Cadmium on levels of glucose in serum and glycogen reserves in the liver and muscle tissues of Cyprinus carpio(l.,1758); Turk. J. Vet. Anim. Sci., 29: Claiborne A., Catalase activity. In Greenwald RA ed. CRC handbook of methods in oxygen radical research. BocaRaton: CRC Press Chung, S., Lethal effects of cadmium in tropica. Bull. Jpn. Soc. Sci., 49(10): European Union., Heavy metals in wastes, European Commission on Environment. Finney, DT., Probit Analysis. 3rd ed. Cambridge University Press. London. Francis S., Mohan, K. G. & Oommen, V., Influence of steroid hormones on plasma proteins in fresh water Tilapia Oreochromis mossambicus. Indian J. Exper. Biol. 40: Gernhofer, M., Pawet, M., Schramm, M., Muller, E. & Triebskorn. R., Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams. J. Aquat. Ecosystem, Stress andrecovery, 8: Jagadeesan, G., Jebanesan, A. & Mathivanan, A., In vivo recovery of organic constituents in gill tissue of Labo rohita after exposure to sublethal concentrations of mercury. J. Exp. India., 3: Jana, S. & Padhyay., Effect of heavy metals on some biochemical parameters in the freshwater fish Channa punctatus. Environ., 5(3):

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