HAEMATO-BIOCEMICAL ALTERATIONS IN Clarias Lazara INDUCED BY DELTAMETHRIN

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1 HAEMATO-BIOCEMICAL ALTERATIONS IN Clarias Lazara INDUCED BY DELTAMETHRIN ABSTRACT Inyang 1, I. R., Pughikumo 2, D. T. and Tuesday 1, T. S. 1 Department of biological sciences, Environmental Toxicology unit, Niger Delta University, Wilberforce Island, Bayelsa State. 2 Department of Human physiology, College of Health Sciences, Niger Delta University, Wilberforce Island Bayelsa State, Nigeria. Correspondence author dr.inyang2009@gmail.com Phone: The aim of this study was to unveil the effects of deltamethrin, a type II synthetic pyrethroid on some selected haematological and biochemical parameters. Thirty African catfich (Clariaslazara, juveniles), mean length, ± 0.20cm SD and mean weight, ± 0.34gSD were acclimatized to laboratory condition for 10 days and then exposed to varying sublethal concentrations of deltamethrin (0.01, 0.02 and 0.03 mg/l) in semistatic bioassays for 14 days. Blood parameters were determined in the plasma while biochemical parameters (transferases; Alanine amino transferase, ALT and aspertate amino transferase, AST) were determined in the kidney and liver. Blood parameters were significant (P<0.05) as well as transferases (ALT & AST) in a dose dependent pattern. It is concluded that deltamethrin could be toxic at high concentation. Transferases and some haematological parameters tested could serve a good biomaker in assessment of pesticide toxicity in fishes. Further studies are required to evaluate the toxicity of deltamethrin in Clariasgariepinus fingerlings and adults and recovery response. Keywords: Clariasgariepinus, deltamethrin, transferases, haematological parameters. INTRODUCTION Pesticides in general are primarily designed to control, and eliminate pest. The wide use of chemicals to control pest has been recognized in the world. The benefit that these chemicals have brought to mankind unveiled remarkable testimonies to technological advancement in terms of lives saved, increased food production and economical gains (Adelowa, 2004). The widespread and sometimes indiscriminate uses of pesticides have led to serious ecosystem problems, especially water and soil pollution (Helfrick, et al, 1983). Pollution via pesticides has become a significant subject of discussion globally (Inyang, 2013). A sudden rise in pesticide production and utilization in agriculture have exacerbated the problem of pesticide pollution (Inyang, 2013). In recent years, there have been reports of residues of persistent organic pesticides in the aquatic environment and in the bodies of aquatic organisms. The largest amount of residues seems to occur in the tissues of animals near the top of food chain and man himself (Miller, 2004). Pesticides can reach the environment via run-off from farmlands, industrial pollution and careless disposal of pesticide containers. Careless handling of organochlorine compounds from wood workers and carpet manufacturers are also possible sources of pesticide into the environment. Alterations in water quality as a result of effects pesticide usually predispose fish to stress and diseases as a result provoke quick response in the physiology of the fish, especially the haematological and biochemical parameters (Ugwa et al., 2008). Deltamethrin (s-a-cyano-3-phenoxyloenyl ((IR)-CIS-3 (2, 2,-dibromovinyl) -2, 2 dimethyl cyclopane carboxylate) which is the active ingredient of decis, belongs to type II synthetic pyrethroid. Synthetic pyrethroids (eg. deltamethrin) are analogue of naturally occurring pyrethrins found in the flowers of chrysanthemum cinerariaefoluim. Deltamethrin was synthesized in 1974 and since then it is widely used in agriculture. In Nigeria it is sold in an open market and it is well embraced by local farmers. The presence of deltamethrin in aquatic environment as a sub lethal effect on fish such as energy metabolism and ionic regulations in the kidney and liver (Khalili et al., 2012). A contributing factor to the sensitivity of fish to deltamethrin exposure seems to be its high rate of gill adsorption due to the lipophilicity (Eells et al., 1995). The main mode of its action is neurotoxicity (Elells, et al., 1995). This xenobiotic has a great impact upon the central and peripheral nervous system and acts by modulating the opening and closing of channels that can result in synaptic discharge depolarization and ultimately death (Elells et al., 1995) although this xenobiotic is broken down via ultraviolet light and sunlight, it is quite tolerant to storage and can preserve its activity for 6 months at 40 0 c and pose risk to mammals and aquatic organisms and ecosystem as a whole. (Ozkan et al., 2012) Haematological and biochemical profiles of blood can provide important information about the internal environment of the organism (Manupust, 2000). Additionally, the evaluation of haematological and biochemical characteristics in fish has become an important means of understanding normal, pathological process and toxicological impacts of xenobiotics (Sudova et al., 2008). The objective of this work, therefore, was to study the NJAFE VOL. 11 No. 1,

2 effect of deltamethrin on haematological parameters (WBC, RBC, HGb, PCV etc) and enzyme parameters (transferases: ALT & AST) in Clariaslazara (Juveniles). MATERIALS AND METHODS Experimental Stock Thirty African catfish (Clariaslazara) juveniles, mean weight 97.00±0.34SD and mean length 14.02±0.20cmSD were obtained from a private farm at Tombia town Yenagoa, Bayelsa State. They were transported in a 20 litre trough to the wet laboratory (Ecotoxicology unit), of the Department of Biological Sciences, Niger Delta university, where the assays were conducted from April to June Fishes were acclimatized individually in rectangular aquaria for ten days during which they were fed once a day ( hr) with 35% crude protein at 1% biomass. Experimental design Completely randomized design (CRD) was used for the experiment. There were four treatment levels with three replicates. A range finding test (trial test) was carried out using the toxicant deltamethrin. Four concentrations of the toxicant were prepared from the original solution (350glL). The test solution was renewed daily. This exposure (trial test) lasted for 10days. General bioassay techniques Sub-lethal concentrations of deltamethrin (toxicant) for the assay (0.01, 0.02 and 0.03 mg/l) were determined based on the range finding test (Inyang et al., 2010). These were prepared by transferring 0.002, 0.003, ml of the original concentration of deltamethrin and making it up to 300Lwith borehole water in the test aquaria. 30L of the diluents (water) was used as control. For replications of each treatment level (concentration) and control were set up by introducing fishes individually into each aquarium. The exposure period lasted 14 days during which the exposure media were renewed daily. The physico-chemical characterization of the water used for fish bioassay was carried out using standard methods of APHA (1998) and the following values were obtained: temperature c, PH , dissolved oxygen mg/l, alkalinity mg/l, conductivity µs/cm and turbidity NTU Haemato-biochemical assay technique After 14 days exposure period, blood sample for haematological analysis were collected from each fish (behind the anal fin) with 23 G size needle and syringe. Fishes were not fed prior to blood collection. Samples were preserved in EDTA bottles and the fishes were sacrificed after blood collection and dissected for the collection of the liver and kidney. 0.5g of each organ was macerated (grounded) with pestle and mortar. Physiological saline was used for preservation and stabilization. Samples were centrifuged at the rate of 3,000rpm for 10 minutes. The supernatants were then removed and stored in plain bottles at c for analysis. The activities of aspertate amino transferase (ALT) in liver and muscle were assayed using the colimeter method of Reitman and Frankel (1957). Data analysis The data were subjected to analysis of variance (ANOVA). Where differences exists, Ducan multiple range test (DMRT) was used to test for pair wise significant differences (p<0.05) between treatment (Wahua, 1999) RESULTS Haematological parameters, Various haematological parameters tested e.g. WBC, RBC, HGB and PCV showed significant (P<0.05) deviation from the control values, indicating effects on these parameters. Nuetrophyl and lymphphocytes values were not significant compared to control, albeit experimental values slightly differ from the control (Table II). Unlike PCV and HGB, WBC values decreased as the concentration of the toxicant increases. Both were dose dependent. Enzyme parameters ALT values in the kidney and liver showed a clear elevation as the concentration of the toxicant increased akin to AST, the highest value recorded was at the highest concentration (0.03mg/l), except ALT at 0.01mg/l. the value recorded here was ±0.13SD, while the highest concentration had ± 0.98SD. DISCUSSION Fish can serve as a bio-indicator of environmental pollution and also assessment of quality of aquatic environment since they are directly exposed to xenobiotics resulting from anthropogenic, agricultural and industrial practices. Leucocytes (WBC) values decreased with increasing concentration of the toxicant. This decrease is dose dependent. The present study is contrary to the belief that since WBC functions in organisms against foreign bodies aided by phagocytosis as antibody production, values will increase as a result of lethal effect of the toxicant (Inyang, 2008). The exposure of fish to deltamethrin may have caused a depression in the established total immune response against a variety of antigenic substances. The xenobiotic may have reduce the general NJAFE VOL. 11 No. 1,

3 Table 1: Haematological profile of Clariaslazara exposed to deltamethrin Con. Of WBC RBC HGB PCV PLT MCV MCH MCHC Deltamethrin (10 3 mm -3 ) (10 6 mm -3 ) (g/l) (%) (x10-5 ) (Fl) (Pg) (g/l) (Mg/L) ±0.12 a 11.60±0.03 b 12.10±0.02 ab 19.00±0.01 b 3.00±0.00 ab ±0.30 a 67.00±0.001 a 37.00±0.06 a ±0.05 b 10.01±0.00 b 11.76±0.04 ab 20.11±0.10 b 10.80±0.02a ±0.42 ab 68.00±0.00 a 28.30±0.01 ab ±0.03 c 11.41±0.01 b 14.90±0.06a 30.00±0.03 a 7.50±0.02 a ±0.08 ab 72.30±0.20 a 40.76±0.20 a ±0.10 c 20.52±0.03 a 15.00±0.00 a 31.09±0.07 a 6.50±0.02 ab ±0.11 a 49.00±0.10 b 28.48±0.09 ab Means within column with different superscripts are significantly different (P<0.05) WBC: White Blood Cell, RBC: (Red Blood Cell), HGB (Haemoglobin), PCV (Packed Cell Volume, PLT (Platelets), MCV (Mean Cell Volume), MCH (Mean Cell haemoglobin), MCHC (Mean Cell Haemoglobin Concentration). Table 2: Haematological profile of Clariaslazara exposed to deltamethrin Con. Of Deltamethrin Neut.l( g l -1 ) Lymph (g l -1 ) Mono (g l -1 ) Eosino (g l -1 ) OCC ±0.06 a 42.00±0.03 a 20.00±0.02 a 8.00±0.06 ab ab ±0.04 a 38.00±0.21 a 7.50±0.10 b 15.01±0.05 a b ±0. 10a 30.06±0.05ab 9.00±0.01 b 10.00±0.08 ab a ±0.01 ab 35.02±0.05 a 16.50±0.02 a 15.00±0.00 a a Means within column with different superscript are significantly different (P<0.05) Neut: Neutrophyl; Lymph; Lymphocyte; Mono: Monocytes; Eosino: Eosinophyl; OCC (Oxygen carrrying Capacity) Table 3: ALT and AST in the kidney and liver of Clariaslazara exposed to deltamethrin for 14 days Conc.of ALT (µ/l) AST (µ/l) Deltamethrin (mg/l) Kidney Liver Kidney Liver ±0.02 c ±0.07 c ±0.02 c 6.00±0.00 b ±0.03 b ±0.13 a ±0.05 b 9.00±0.02 b ±0.10 a ±0.41 b ±0.03 a 7.00±0.00 b ±0.01 a ±0.08 b ±0.31 a 15.00±0.03 a Means within column with different superscript are significantly different (P<0.05) immune system leading to significant depression. This is akin to the findings of Ngodegha et al. (1999) and Inyang (2008). Changes in leucocyte system usually manifest in the form of leucocytosis and lymphocypaenia which are characteristics of leucocytic response in animals exhibiting stress. Lymphocytes and neutrophils form part of the granulocytes that make up leucocytes (WBC) in organisms. Neutrophils are very active phagocytes and constitute about 70% of the total granulocytes. In this study, a progrressive decrease (dose dependent) values was observed. The reduction may be akin to carbon tetrachloride and benzene which have been implicated in eliciting excess glucocorticoid, antibody depression, lymphatic involution and retard migration of phagocytic leucocytes which may lead to total white blood cell alteration (Ovuru, 2002). Platelets (thrombocytes) are membrane bound cell fragments, usually lacking nuclei. They are responsible for blood clotting in fish and other organisms. Values of thrombocytes increased progressively as the concentration increases. This may be because of the effect of the toxicant on tissues, hence more production to enable the organism to repair some injured tissues. Erythrocytes (RBC), Hemoglobin (HGB) and packed cell volume (PVC). Haemoglobin, packed cell volume and erythrocytes in this present study were affected by the toxicant. RBC values were not significant except at the highest concentration (0.03ppm) while haemoglobim and packed cell volume values slightly progress as the concentration of the toxicant increased. Similar result was also obtained by Atamanalp and Yanik (2003). They found a significant increase (P<0.05) in the levels of RBC, MCH, MCHC in rainbow trout (Oncorhynchusmykiss) following cypermethrin and Mancozeb acute exposure. The major function of RBC is to pick oxygen from the environment bind it haemoglobin (Oxygen carrying protein) to form oxyhaemoglobin and transport them to tissues. Fishes exposed to pesticide usually lead to tissue damage, neurological disorders, stress and behavioral abnormalities. In this present study, Clarias lazara may have been stressed in addition to haematological alterations, to cushion the effect of this toxicant fish produce enough protein and erythrocytes to pick more oxygen to tissues affected, hence deviation of values of RBC. Monocytes and eosinophils Among the granulocytes, eosinophils are phagolytic and ingest foreign proteins and immune complexes rather than bacteria (Miller and Harley, 2004). In some organism eosinophils also release chemicals that counteract the effects of certain inflammatory chemicals released during allergic reactions. A progressive increase in values was observed as the concentration increases. The sudden increase may be because of emergency of phagocytic response as a result of the toxicant effect on tissues of the probe organism. NJAFE VOL. 11 No. 1,

4 Moncytes are known to circulate in blood for a few days then migrate into the reticulo-endothelial system where they remain for a long time, known in this system as macrophages (Miller and Harley, 2004). In this study, the profound decrease was observed. The reduction is attributable to the effect of deltamethrin as well as lymphoid tissue such as spleen. Enzymology: transferases Enzyme analysis of organs such as muscles, kidney liver, heart and gills in fish can provide important information about the internal environment of the organism (Boeger et al., 2003). Activities of enzymes in fish are essential metabolic processes. We observed an increase in values of transferases tested (AST and ALT) in both Kidney and liver of the probe organism. The trend was not tissue specific but dose dependent. Elevated levels of ALT and AST indicate the enhanced transamination of amino acids, which may provide ketoacids to serve as precursor in the synthesis of essential organic elements (Prashant and Neclagund, 2008). It is likely that toxic stress imposed by deltamethrin might be one of the factors for the observed activities of ALT and AST in the present study. Similarly, sepici-dincel et al. (2009) observed that the increase in activities of AST and ALT in the muscle and liver of common carp exposed to 10.00ppm of cypermethrin may be due to a disturbance in the krebs cycle, hence a decrease in Kreb s cycle intermediate which is compensated by ALT and AST by providing α-ketoglutarate. The transferases (ALT and AST) function as a link between carbohydrate and protein metabolism by catalyzing the inter conversion of strategic compounds like α-ketoglutarate and alanine to pyruvic acid and glutamic acid respectively (Tiwari and Singh, 2004). ALT and AST were higher in the kidney and liver tissues. This suggests that both organs are very efficient in utilizing amino acid for metabolic purposes. Oxygen carrying capacity (OCC) Oxygen carrying capacity values increased slightly as the concentration of the toxicant increases. Quite unlike reports by Dhara and Gassert, (2004) and Inyang(2008). The authors reported a decrease in values of OCC as the concentration of the toxicant decreased. The authors reasoned that the toxicant causes adversity in the protein haemoglobin and possibly carbomoylation (the addition of a carbomyl group (CO-NH 2) to a protein amino acid of the haemoglobin molecule. In this present study, the xenobiotic did not alter the oxygen affinity of blood by carbomoylation of the end amino acid chains of the haemoglobin molecule. CONCLUSION This research work has unveiled the toxicity of deltamethrin on haematological and biochemical indices in Clariaslazara. WBC, RBC, HGB, AST and ALT could serve as useful biomarkers of sub-lethal effects of deltamethrin in aquatic environment. In light of the findings of this study, we therefore recommend that analysis of blood parameters should be done more than once within the exposure period in subsequent studies. It is also pertinent to ascertain the level of toxicity of the toxicant in other aquatic organisms. The monitoring of the environment for the levels of this and other pesticides should be carried out more frequently by the authorities concerned. Finally, the use of deltamethrin close to aquatic environments should be done with caution. REFERENCES Adelewa, I. 2004, Effects of pesticides on aquatic organisms. Canadian Journal of fish and aquatic science 53: Atamanalp M. and Yanik, T Alterations in haematological parameters of rainbow trout (Onchorhynchusmykiss) exposed to mancozeb. Turkish Journal of Veterinary Animal Sc. 27: Boeger, W., Ostrasky, A., Guimaraes, A. T. B. and Romao, S Histopathology as an approach to evaluate the effect of the oil spill on fishes of the rivers Saldanha Barigui and Lyvale (Brazil). Spill. Conf. 2(3) Dhara, U. R. and Gassert, H. T Acute toxicity of cyanide on common carp, Branchydaniorerio, current science vol 14, Elells, J. T., Resmusen, J. L. and Bandetini, P. A Difference in the neuroexcitatory actions of pyrethroid insecticides and sodium channel specific neurotoxins in rat and trout brain. Toxicol and Appl. Pharmacol. 123: Helfric, L. A., Weigmann, D. L., Hipkins, P. and Stinson, E. R., Pesticides and aquatic organisms a guide for reducing impacts on aquatic systems. Environ. Ecol. 11: InyangI, R., Ogamba, E. N., Frank, V. E Biochemical changes and electrolyte stabilization in Clarias gariepinus (Juveniles) induced by dichlovos. International Journal of biochemisty 108: Inyang, I. R Haematological and biochemical responses of Clarias gariepinusto diazinon. Ph.D thesis, Rivers State University of science and technology, Port Harcourt, Rivers State. NJAFE VOL. 11 No. 1,

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