Sabrina Hossain Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh-2202.

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1 International Journal of Environment, 5(1): 8 13 (2015) ORIGINAL ARTICLE Impacts of Sumithion on Blood Glucose and Some Hematological Parameters in Common Carp Sabrina Hossain, Mst. Halima Khatun, Md. Khalilur Rahman, Zahid Parvez Sukhan, Md. Shahjahan,* ISSN: (print) ISSN: (online) ARTICLE HISTORY Received: 12 May 2015 Revised: 17 November 2015 Accepted: 8 December 2015 Published online: 13 December 2015 Abstract An experiment was conducted to assess the effects of an organophosphorous pesticide sumithion on blood glucose and some hematological parameters in common carp, Cyprinus carpio. The experiment was conducted with three treatments, each having three replications. Treatment one (T1) was used as control (0 ppm) and two concentrations, such as 0.85 ppm and 1.70 ppm were used as Treatment two (T2) and Treatment three (T3), respectively. Fish were sacrificed at 7, 14, and 21 days after start of exposure. The sexual dimorphic changes in blood glucose levels were observed in the present study. In male, the blood glucose levels were significantly (P<0.05) increased with the toxicity of sumithion at 7, 14, and 21 days of exposure periods in higher concentrations (T2 & T3) compared to control (T1), while only tended to increases were observed in female. The Hb (g/dl) tended to decrease with toxicity of sumithion though significantly decreased in males at 7 and 14 days of exposure in T3. The RBCs ( 10 6 /mm 3 ) were decreased significantly (P<0.05) during the toxicity of sumithion at 7, 14, and 21 days of exposure periods in higher concentrations (T3), whereas Hct (%) showed no significant changes in both concentrations (T2 & T3). On the other hand, WBC were significantly (P<0.05) increased in higher concentrations both in male and female. The present investigation highlighted that sumithion has adverse effects on the metabolism of macromolecules of fish. Key words: Organophosphorous; Environment; Pesticide; Fish; Toxicology. 2015, International Journal of Environment. All rights reserved. I. INTRODUCTION 1 The use of pesticides for the safety of humans and to increase agricultural production is alarming for their harmful effects on other non-target animals like fish. Contamination of water by pesticides, either directly or indirectly can lead to fish kills, reduced fish productivity. Several hundred types of pesticides and insecticides are used for crop protection in agriculture in Bangladesh. Among them, sumithion, the O, O Dimethyl O-(3- AUTHORS INFO Sabrina Hossain Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh Mst. Halima Khatun Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh Md. Khalilur Rahman Bangladesh Fisheries Research Institute, Freshwater Station, Mymensingh, Bangladesh. Zahid Parvez Sukhan Department of Marine Fisheries & Oceanography Patuakhali Science & Technology University Dumki, Patuakhali 8602, Bangladesh Md. Shahjahan,* Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh *Corresponding author mdshahjahan@bau.edu.bd Tel: ; Fax: methyl-4-nitrophenyl) is important one which falls under the organophosphate groups. It is effective to control wide range of important insects and certain other arthropod pests. It is mainly used to control brittle in paddy field. It is also used in larval rearing fish pond to control tiger bug. Since sumithion is widely used for crop protection and for eradication of aquatic insects in aqua-ponds, it is very important to know the extent of damage made by this chemical to fish. Sumithion is considered somewhat toxic to fish [1]. The pesticides affect the aquatic ecosystem by interrupting the aquatic food chain resulting in the loss/shift in abundance of natural species in the aquatic environments [2 and 3]. Fish appear to possess the same biochemical pathways to deal with the toxic effects of endogenous and exogenous agents as mammalian species does [4]. Hematological parameters like hemoglobin, hematocrit, and blood cell counts can be used to find physiological response of contaminated environment [5]. Therefore, the blood parameters are often measured when a clinical diagnosis of fish physiology is applied to determine the sub-chronic effects of pollutants [5]. In the present study, the changes in blood glucose, hemoglobin, RBCs, WBCs and hematocrit values were monitored after sub-lethal exposure of this pesticide to the common carp (Cyprinus carpio), an economically important freshwater fish. II. MATERIALS AND METHODS A. Animals and chemicals The fish was collected from Bangladesh Catfish Limited, Valuka, Mymensingh and maintained in aquaria at the wet laboratory of the Faculty of Fisheries,

2 Impacts of sumithion on haematological parameters Bangladesh Agricultural University, Mymensingh at 25 ± 0.5 C under a controlled natural photo-regimen (14/10 h, light/dark) for a period of 21 days before the experiments. The fish were fed twice a day. The length and weight of fishes ranged from 17 to 19 cm and 75 to 90 g, respectively. To conduct the present experiment, sumithion (60E/C) was collected from retail pesticide shop from local market. B. Experimental design and sampling To conduct the experiment, nine aquaria (75 cm 45 cm 45 cm) were first cleaned and washed thoroughly by tap water. Then the aquaria were filled with 100 L of tap water. The experiment was conducted with three treatments, each with three replications. Treatment one (T1) was used as control (0 ppm) and two concentrations, such as 0.85 ppm (10% of 96 h LC50) and 1.70 ppm (20% of 96 h LC50) were used as Treatment two (T2) and Treatment three (T3), respectively. Ten fish were transferred in each aquarium. Fish were sacrificed at 7, 14 and 21 days after start of exposure and blood was collected from the caudal peduncle into citrated tuberculin syringes. C. Measurement of haematological parameters Blood glucose (mg/dl) and hemoglobin (Hb; g/dl) were measured by EasyMate GHb, blood glucose/haemoglobin dual-function monitoring system using glucose and hemoglobin strips. The red blood cell (RBC; x10 6 /mm 3 ) and white blood cell (WBC; x10 3 /mm 3 ) count was made using Neubauerhemocytometer. Hematocrit (Hct, %) values were determined by using a micro-hematocrit centrifuge. To measure haemoglobin, SAHLI s hemometer was used. At first 90µl 0.1N HCL was taken in an eppendorf tube using micropipette. Then 10 µl blood was added and the tube was shacked thoroughly for proper mixing. After 2-3 minutes the mixture was transferred to the tube. Then distelled water was added in drops until color was adjusted with colorimeter of haemometer. When the color was adjusted then the reading was taken upto the level of the mixture specified on the body of tube. To count RBC and WBC 990 µl 2% EDTA was taken in an appendorf tube. Then 10µl blood was added with it. During counting, 10 µl of RBC solution and small amount of gimsa stain was taken on haemocytometer. After covering by a cover slip, it was observed under microscope. During counting, 5 large square units (each large square contains 16 small square units) were selected randomly. Numbers of RBC within a large square unit not touching any line were counted. Using this procedure, number of RBC was counted from randomly selected 5 large square units. Then total number of RBC was counted using the following formula. Number of red blood cells sumof RBC* 4000*100 = ( ) cells/mm 3 5 *16 In case of WBC, total number of WBC found within large squares of four corners were counted. Then total number of wbc was counted using following formula. Number of white blood cells D. Statistical analysis ( sumtoalof W BC/ 4)*100 = ( ) cells/mm Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey s post hoc test to assess statistically significant differences among different treatments (P < 0.05). Statistical analyses were performed using PASW Statistics 18.0 software (IBM SPSS Statistics, IBM, Chicago, USA). Fig. 1. Effects of sub-lethal exposure of sumithion on blood glucose levels (Means ± SD) at different time intervals in common carp. The blood glucose levels of T2, and T3 were compared to the control group (T1). Asterisk (*) indicates the statistically significantly different (P<0.05, n=4). 9

3 Hossain et al. III. RESULTS A. Effects of sumithion on blood glucose The blood glucose levels of experimental fish were examined after exposure of fish to sumithion. The sexual dimorphic changes in blood glucose levels were observed in the present study. In male, the blood glucose levels were significantly (P<0.05) increased with the toxicity of sumithion at 7, 14, and 21 days of exposure period in higher concentrations (T2 & T3) compared to control (T1), while only tended to increases were observed in female. The blood glucose levels were higher in female compare to male (Fig. 1). B. Effects of sumithion on Hemoglobin (Hb) The blood hemoglobin (Hb) was examined after exposure of fish to sumithion (Fig. 2). The Hb (g/dl) tended to decrease with toxicity of sumithion though significantly decreased in male at 7 and 14 days of exposure in T3. C. Effects of sumithion on RBCs In the RBC count, it is found that the RBCs count ( 10 6 /mm 3 ) were decreased significantly (P<0.05) in higher concentrations (T3) during the toxicity of sumithion at 7, 14, and 21 days of exposure periods compared to control (T1) in both male and female (Fig. 3). D. Effects of sumithion on Hematocrit (Hct) In the present study, the value of Hct (%) though tended to decrease in higher concentrations of sumithion (Fig. 4) but showed no significant changes in both concentrations (T2 and T3). Fig. 2. Effects of sub-lethal exposure of sumithion on Hb (g/dl) at different time intervals in common carp. The Hb of T2 and T3 were compared to the control group (T1). Asterisk (*) indicates the statistically significantly different (P<0.05, n=4). Fig. 3. Effects of sub-lethal exposure of sumithion on RBC ( 10 6 /mm 3 ) at different time intervals in common carp. The Hct of T2 and T3 were compared to the control group (T1). Asterisk (*) indicates the statistically significantly different (P<0.05, n=4). 10

4 Impacts of sumithion on haematological parameters Exposure time (weeks) Fig. 4. Effects of sub-lethal exposure of sumithion on Hct (%) at different time intervals in common carp. The Hct of T2 and T3 were compared to the control group (T1). Asterisk (*) indicates the statistically significantly different (P<0.05, n=4). WBCs ( 10 3 /mm 3 ) Fig. 5. Effects of sub-lethal exposure of sumithion on WBCs ( 10 3 /mm 3 ) at different time intervals in common carp. The WBCs of T2 and T3 were compared to the control group (T1). Asterisk (*) indicates the statistically significantly different (P<0.05, n=4). E. Effects of sumithion on WBCs The results of WBCs ( 10 3 /mm 3 ) values are presented in Fig. 5. The WBCs ( 10 3 /mm 3 ) were increased significantly in both concentrations (T2 and T3) compared to control (T1) in both male and female. IV. DISCUSSIONS Studies of blood glucose and hematological parameters are effective physiological tools to know the toxicological effects of pollutants and chemicals. In the present study, differential changes were observed in blood glucose and hematological parameters exposed to sumithion indicating the toxicological impacts of sumithion on fish. Blood glucose is regulated by complex interactions of hormones such as glucagon and cortisol. After analyzing the hematological parameters in both controlled and in treatment conditions and in both male and female of common carp, it was found that blood glucose level was high 11 in treatment than the control. Blood glucose level was increased due to exposure to sumithion indicating the stressed condition of fish in pesticide exposure. Mamta [7] observed that organophosphate pesticide have effect on blood glucose level of H. fossilis. Increased glucose levels in the present study can cause hypoglycemic condition. Hypoglycemia in fish may reflect stress induced hormone mediated response. Such elevation may be due to enhanced gluconeogenesis response of stressed fish in their attempt to satisfy their new energy demands [8]. Alterations in blood glucose levels have been reported in H. fossilis exposed to sub-lethal concentration of testosterone [9]. Hypoglycemia in H. fossilis exposed to various concentration of pesticides viz. rogor and aldrin has been noticed [10, 11]. This is probably due to the rapid utilization of blood glucose during hyper excitability, shocks and tremors, which are characteristic behavior of organophosphate pesticide toxicity in fish [12]. Interestingly, sexual dimorphic changes in blood glucose

5 Hossain et al. levels were observed in the present study indicating that male common carps are more sensitive to pesticides. In the present study, main hematological response of common carp to the sub-lethal exposure of sumithion were a significant decrease (P<0.05) of RBC, Hb and Hct values compared to the control group. Reductions of RBC and Hb contents were reported in common carpafter acute exposure to another organophosphorous pesticide, diazinon [13, 14, and 15]. Similarly, changes in erythrocyte profile induced by acute effect of organophosphorous pesticides, dichlorvos in Clarias batrachus [16], formothion in H. Fossilis [17], malathion in Cyprinion watsoni [18], and trichlorphon in Piaractus mesopotamicus [19] provides evidence for decreased hematopoiesis followed by anemia induction in fish. Considering the previous findings, it is revealed that the decrease in RBCs count and Hb content observed in this study may be due to the disruptive action of the pesticides on the erythropoietic tissue as a result of which the viability of the cells might be affected. Changes in the hematological parameters were observed by diazinon as an anaemic condition due to decreased synthesis of RBCs and erythrocyte in bone marrow [20]. Moreover, decreases in hemoglobin concentration may be due to either an increase in the rate at which hemoglobin is destroyed or decrease in the rate of hemoglobin synthesis [21]. Several morphological changes were observed in liver and kidney of common carp exposed to malathion [22] and sumithion [23], indicates that the disruptive action of the pesticides on the erythropoietic tissue as a result of which the viability of the cells might be affected. Increased WBC count established leucocytosis, which is considered to be of an adaptive value for the tissue under chemical stress. This also helps in the removal of cellular debris of necrosed tissue at a faster rate [24]. In the presence of foreign substances or under pathological conditions leucocytosis in fish may be the consequence of direct stimulation of immunological defense [24]. The increase in WBC count can be correlated with an increase in antibody production which helps in survival and recovery of the fish exposed to organophosphorous pesticide malathion [25, 26 and 27]. V. CONCLUSIONS The current research was conducted to evaluate the effects of sumithion, an organophosphorous pesticide, on some hematological parameters in common carp. Sumithion enhanced the blood glucose level suggesting that glycogen might be breakdown to glucose due to toxicant of sumithion. Conversely, reduction of Hb, RBCs and Hct might be because of failing of hematopoietic system.the increase in WBC count can be correlated with an increase in antibody production which helps in survival and recovery of the fish exposed to pesticide. Therefore, the present study revealed that the sumithion has adverse effects on various blood parameters in common carp. ACKNOWLEDGMENTS This work was supported by the grants from Aquadrugs project of Bangladesh Fisheries Research Institute. REFERENCES [1] Thomson WT Agricultural Chemicals. Book I: Insecticides. Thomson Publications, Fresno, CA. 120 pp. [2] Ventura BC, AngelisDF, Maria AM and Morales M Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus Perciformes, Cichlidae) detected by the micronuclei test and thecomet assay. Pestic. Biochem. Physiol., 90: [3] Rahman MS, Shahjahan M, Haque MM and Khan S Control of euglenophyte bloom and fish production enhancement using duckweed and lime. Iranian J. Fish. 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