International Journal of Scientific Research in Environmental Sciences, 2 (5), pp. 156-163, 2014 Available online at http://www.ijsrpub.

com/ijsres ISSN: 2322-4983; 2014 IJSRPUB http://dx.doi.org/10.12983/ijsres-2014-p0156-0163

Full Length Research Paper Effects of Almix Herbicide on Metabolic Enzymes in Different Tissues of Three Teleostean Fishes Anabas testudineus, Heteropneustes fossilis and Oreochromis niloticus
Palas Samanta1, Sandipan Pal2, Aloke Kumar Mukherjee3, Tarakeshwar Senapati4, Apurba Ratan Ghosh1*
1

Ecotoxicology Lab, Department of Environmental Science, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India 2 Department of Environmental Science, AKPC Mahavidyalaya, Bengai, Hooghly 712611, West Bengal, India 3 P.G. Department of Conservation Biology, Durgapur Govt. College, Durgapur 14, West Bengal, India 4 School of Basic and Applied Sciences, Poornima University, Jaipur 302022, Rajasthan, India *Corresponding Author: apurbaghosh2010@gmail.com
Received 03 February 2014; Accepted 05 April 2014

Abstract. Three Indian freshwater teleostean fishes Anabas testudineus, Heteropneustes fossilis and Oreochromis niloticus were exposed to almix (66.67 mg/l) under laboratory condition for a period of 30 days to investigate the activity of transaminases [alanine aminotransferase (ALT), aspartate aminotransferase (AST)], and phosphatase [alkaline phosphatase (ALP)] in the gill, intestine, heart and muscle tissues. ALT levels showed significant elevation (p< 0.05) in all the fish tissues and highest activity (183.57%) was observed in gill of O. niloticus and lowest (122.01%) in muscle of O. niloticus. AST activity was also increased significantly (p< 0.05) in all test fishes. Highest AST activity (344.05%) was observed in heart of O. niloticus, while A. testudineus showed minimum activity (114.09%) in intestine. ALP activity increased significantly (p< 0.05) in all the fish species and was highest (289.02%) in intestine of H. fossilis and lowest (142.83%) in A. testudineus. Increased activity of these metabolic enzymes resulted into tissue damage which ultimately affected the fish health; it inferred the sensitivity of these enzymes to the almix herbicide toxicity in the order of O. niloticus > H. fossilis > A. testudineus. Therefore, assessment of these metabolic enzymes in tissue systems is important for evaluation of the herbicidal contamination like almix on fish species. Keywords: Almix, Herbicide, Transaminase, Phosphatase, Tissues, Health, Teleostean fishes

1. INTRODUCTION Herbicides are recognized throughout the world as emerging means of controlling unwanted plants, at the same time considered as biological poisons due to their bioaccumulation, and non-biodegradable properties that ultimately cause the toxicity to the nontarget aquatic organisms and finally to human beings via food chain (Binelli and Provini, 2004). Contamination of aquatic bodies by these herbicides is well documented worldwide (Palus et al., 1999; Cerejeira et al., 2003). The use of fish species in toxicity based studies is now being widely used due to their greater sensitivity to toxic chemicals resulting from agricultural processes via surface run-off or indirectly through food chain (Lakra and Nagpure, 2009) and can be considered as indicators of xenobiotic contamination (van der Oost et al., 2003). Almix, based on sulfonylurea group, is one of the most commonly used modern herbicide that attacks

broad leaf weeds, and sedges in rice paddy fields both through contact and systematic pathway. It is a combination of 10% metsulfuron methyl [methyl 2-(4methoxy-6-methyl-1, 3, 5-triazin-2-ylcarbamoylsulfamoyl) benzoate], 10% chlorimuron ethyl [ethyl 2-(4-chloro-6-methoxypyrimidin 2 ylcarbamoylsulfamoyl) benzoate] and 80% adjuvant. The 96 h LC50 value of Oncorhynchus mykiss of this herbicide as active ingredient is >150 mg/l. (Safety Data Sheet, 2010). Enzymatic evaluation in the diagnosis of effects of herbicides in different tissues and/or organs of aquatic organisms is widely used as an emerging tool in early warning of herbicidal contamination and its associated remedial measures (Dutta and Arends, 2003). Alteration in the chemical composition of aquatic environment usually induces changes in the biochemical aspects of the inhabitants particularly fishes (Edwards, 1973). Alanine aminotransferase and aspartate aminotransferase activities serve as a link

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between protein and carbohydrate metabolisms and catalyze the interconversion of amino acids and -keto acids by transfer of amino groups. The AST catalyzes the transfer of this group from aspartate to ketoglutarate to form glutamate and oxaloacetate, while ALT catalyzes the transfer of the amino group from alanine to -ketoglutarate to form glutamate and pyruvate (Moss et al., 1986). Alterations in alkaline phosphatase (ALP) activities in tissues, organs and serum have been reported in fishes exposed to toxicants of varying concentrations (Jyothi and Narayan, 2000). Measurement of these enzymes in serum as well as in different tissues is frequently used as diagnostic tool in human and animals (Barse et al., 2006). Toxic responses by almix on the fishes such as Cyprinus carpio, Anabas testudineus, Heteropneustes fossilis and Oreochromis niloticus were reported recently by a limited number of authors (Jabeen et al., 2008; Samanta et al., 2013, Senapati et al., 2013). Therefore, the present study is an attempt to evaluate the effects of commercial herbicide formulation of almix on the biochemical activities of some important enzymes, viz., transaminases and alkaline phosphatase in the tissues of gill, intestine, heart and muscle of three Indian air-breathing teleosts of different trophic levels viz., Anabas testudineus (Bloch, 1792), Heteropneustes fossilis (Bloch, 1794) and Oreochromis niloticus (Linnaeus, 1758) for proper management and monitoring of almix contamination in the aquatic ecosystems at the time of application as well as post-application period. 2. MATERIALS AND METHODS 2.1. Experimental Design

a period of 30 days. Doses were applied on every alternate day. During the period of experiment both the treated and control sets of fishes were fed with live Tubifex sp. regularly. The aerator bubble was also provided in all aquaria to avoid any oxygen depletion, and also combat with the possible alteration of carbon dioxide tension. Experiments were conducted with a natural photoperiod and at an ambient water temperature. During the experimentation period, the average value of water parameters were as follows: temperature 19.670.293oC, pH 7.480.052, electrical conductivity 478.339.70 S/cm, total dissolved solids 341.446.56 mg/l, dissolved oxygen 5.820.394 mg/l, total alkalinity 317.3015.60 mg/l as CaCO3, total hardness 188.898.58 mg/l as CaCO3, ammoniacal-nitrogen 16.635.91 mg/l, nitratenitrogen 0.460.108 mg/l. 2.2. Tissue Sampling During experimentation period the quality of the aquarium water was assessed as per APHA (2005). The fishes exposed to the aforementioned test concentration of almix along with the control were sacrificed by punching the spinal cord behind the opercula and were sampled at the end of the experiment i.e., 30 days. Gill, intestine, heart, muscle samples were removed, washed in 0.75% saline solution, blotted with tissue paper, and homogenized in 2 ml of 0.5 M, pH 7.4 Tris-HCl buffer by using mortar and pestle and finally centrifuged at 8,000 rpm for 25 minutes at 0C and the supernatant was taken in teflon tubes and finally stored at -20oC for biochemical analysis. 2.3. Biochemical parameters

Three Indian carnivorous teleosts, A. testudineus (Bloch), H. fossilis (Bloch), and O. niloticus (Linnaeus) of both sexes with an average weight of 24.22 2.47 g, 44.88 5.41 g, and 49.00 5.28 g respectively and total length of 11.66 0.55 cm, 20.04 0.74 cm, and 14.32 0.54 cm respectively acquired from the local market and were acclimatized to congenial laboratory conditions for 15 days separately in aquaria of 250 L capacity. Laboratory condition was maintained by dewatering on every alternate day and supplying the live fish food viz., Tubifex sp. regularly. After 15 days fishes were divided into two groups (control and almix) and introduced in twelve aquaria, containing 10 fishes each at the laboratory: three for A. testudineus, three for H. fossilis and another three for O. niloticus including three control sets (one for A. testudineus, one for H. fossilis and one for O. niloticus). The fishes were exposed to a sublethal dose of almix i.e., 66.67 mg/l (Samanta et al., 2013) in 250 L aquaria for

2.3.1. Measurement of ALT activity Alanine aminotransferase activity was determined according to the method of Bergmeyer et al. (1976). At first 500 l of R1 reagent (mixture of L-Alanine, NADH, Lactate dehydrogenase, 2-Oxoglutarate, Tris Buffer, pH 7.50.1 at 25oC) was taken in the test tube and kept for incubation at 37C. After that 50 l of sample was added to and immediately reading was taken in the auto-analyzer for three minutes. Alanine aminotransferase activity was calculated by multiplying average absorbance difference per minute (A/minute) with the corresponding factor 1768 as described in the kit protocol. The test kit was purchased from Erba (Erba cat. # FBCEM0047).

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2.3.2. Measurement of AST activity Aspartate aminotransferase activity was determined according to the method described by Bergmeyer et al. (1976). At first 500 l of R1 reagent (mixture of 2Oxoglutarate, L-Aspartate, Malate dehydrogenase, Lactate dehydrogenase, NADH, Tris Buffer, pH 7.80.1 at 25oC, EDTA) was taken in the test tube and

kept for incubation at 37C. After that 50 l of sample was added to and immediately reading was taken in the auto-analyzer for three minutes. Aspartate aminotransferase activity was calculated by multiplying average absorbance difference per minute (A/minute) with the corresponding factor 1768 as described in the kit protocol. The test kit was purchased from Erba (Erba cat. # FBCEM0045).

Fig. 1: Alanine aminotransferase (unit/mg protein/min) activity in different tissues of three test fish species. Data are presented as mean SE (n = 9). Values with different lowercase superscripts (alphabet) differ significantly (p< 0.05) between fishes within tissue and concentration. Values with different uppercase superscripts (alphabet) differ significantly (p< 0.05) between tissues within fishes and concentration

2.3.3. Measurement of ALP activity Alkaline phosphatase activity was determined according to the method described by Bergmeyer et al. (1976). At first 400 l of R1 reagent (mixture of Diethanolamine, pH 10.2 and Magnesium chloride) was taken in the test tube and then 100 l of R2 reagent (p-Nitrophenylphosphate) added to this solution. These two solutions mixed and kept for incubation at 37C for 60 seconds. After that 10 l of sample was added to and immediately reading was taken in the auto-analyzer for four minutes. Alkaline phosphatase activity was calculated by multiplying average absorbance difference per minute (A/minute) with the corresponding factor 3397 as described in the kit protocol. The test kit was purchased from MERCK (Merck cat. # 1730PDLFT.0045).

2.4. Statistical analysis The data were statistically analyzed using SPSS package (version 16.0). Two-way analysis of variance (ANOVA) was applied to determine the effects of different parameters among different tissues, concentrations and fishes. Means were compared by Tukeys test. The values of all biochemical parameters are expressed as mean SE (n = 9). A p< 0.05 was considered statistically significant. 3. RESULTS 3.1. Alanine aminotransferase (ALT) activity Alanine aminotransferase activity, in the present study, in all the tissues of all the test fishes after exposition to the almix herbicide in the laboratory condition was significantly increased when compared to control value and is presented in the Fig 1. Muscle tissue showed highest elevation of ALT activity in H.

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fossilis 139.85% (8.00 to 11.19) followed by A. testudineus 130.29% (24.63 to 32.09), while less activity was observed in O. niloticus 122.01% (32.67 to 39.86). In case of gill tissue, maximum elevation observed in O. niloticus 183.57% (3.94 to 7.23) followed by A. testudineus 175.17% (7.45 to 13.05) and minimum activity was recorded in H. fossilis 165.41% (2.25 to 3.72). Likewise muscle tissue, similar result of increased activity was also observed in heart; highest in H. fossilis 159.32% (8.26 to 13.16)

followed by A. testudineus 134.64% (8.92 to 12.01) and lowest in O. niloticus 129.71% (13.63 to 17.68). The results clearly depicted the maximum ALT activity in gill of O. niloticus (183.57%) and minimum activity in muscle of O. niloticus (122.01%) in respect of all the tissues. But there was no definite pattern of increase in ALT activity in different tissues of test fishes as observed after almix exposure and H. fossilis was more sensitive to the changes in ALT activity than the other two species.

Fig. 2: Aspartate aminotransferase (unit/mg protein/min) activity in different tissues of three test fish species. Data are presented as mean SE (n = 9). Values with different lowercase superscripts (alphabet) differ significantly (p< 0.05) between fishes within tissue and concentration. Values with different numeric superscripts differ significantly (p< 0.05) between concentrations within tissue and fishes. Values with different uppercase superscripts (alphabet) differ significantly (p< 0.05) between tissues within fishes and concentration

3.2. Aspartate aminotransferase (AST) activity Likewise ALT activity, aspartate aminotransferase activity in gill, intestine, heart and muscle of the test fishes exposed to the almix herbicide in the laboratory condition was also increased significantly (p< 0.05) as compared to control value (Fig. 2). In case of muscle tissue, maximum elevation of AST activity was observed in O. niloticus 310.77% (117.00 to 363.60) followed by A. testudineus 287.96% (109.60 to 315.60), and H. fossilis 234.55% (178.30 to 418.20). The gill tissue showed highest elevation in A. testudineus 261.54% (116.12 to 303.70), moderate in H. fossilis 213.09% (158.80 to 338.40) and minimum in O. niloticus 187.59% (159.50 to 299.20). Likewise muscle tissue, similar pattern of enhanced activity of the enzyme was also observed in heart tissue, while intestine showed maximum activity in H. fossilis 161.55% (244.20 to 394.50) and minimum in A. testudineus 114.09% (263.30 to 300.41). Among all these tissues heart of O. niloticus showed highest elevation of AST activity (344.05%), while A.

testudineus showed minimum activity (114.09%) in intestine. This study revealed that elevation in enzyme activity in different fish species was different and O. niloticus was more sensitive than both the A. testudineus and H. fossilis. 3.3. Alkaline phosphatase (ALP) activity Alkaline phosphatase in intestine tissue of all the test fishes were also significantly increased (p< 0.05) after almix exposure in the laboratory condition (Fig. 3). ALP activity was maximally recorded in intestine of H. fossilis 289.02% (29.96 to 86.59) and minimum in A. testudineus 142.83% (109.39 to 156.39), while activity was moderate in O. niloticus 161.64% (81.07 to 131.04). The results showed that the pattern of increase in ALP activity was also different for different fish species.

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4. DISCUSSION Fish being a sentinel organism in ecotoxicological studies play a significant role in the trophic structure as well as accumulated toxic substances and respond

to the mutagens (Cavas and Ergene-Gozukara, 2005). Therefore, use of fish as indicator species to the xenobiotic compounds is of great importance and helps in early evaluation of aquatic contamination (van der Oost et al., 2003).

Fig. 3: Alkaline phosphatase (unit/mg protein/min) activity in different tissues of three test fish species. Data are presented as mean SE (n = 9). Values with different lowercase superscripts (alphabet) differ significantly (p< 0.05) between fishes within tissue and concentration. Values with different numeric superscripts differ significantly (p< 0.05) between concentrations within tissue and fishes

4.1. Transaminases activity During the toxic stress of almix under the laboratory condition, activities of transaminases: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased in gill, intestine, muscle and heart tissues of all three test fish species, these indicated tissue damage due to the increased synthesis of the enzyme in the tissues. During stress imposed condition, fishes take less amount of food, therefore, to overcome this toxic stress ALT and AST activity is enhanced by the breaking down of free amino acids to fulfill the additional energy requirement. The result is supported by Ganesh et al. (2006) and Schulman et al. (2002). These enhanced activities of transaminases (ALT and AST) in different fish tissues shown in the present study provide the oxaloacetic acid and pyruvate, ketoglutarate and glutaric acid respectively to meet the increased energy demand by the degradation of free amino acids from the amino acid pool during almix imposed stress condition. Similar results are reported by Bidigare and King (1981). The highest activity of transaminases observed in gill indicated cellular damage and this may be due to direct exposure to herbicide as well as subsequent release of these enzymes across the damaged plasma membranes

into the serum and/or blood stream. These findings were also in agreement with the observations of Sepici-Dincel et al. (2009) who found increased activity of AST and ALT in muscle and liver of common carp exposed to 10 g/l of cyfluthrin and with the results reported by Yildirim et al. (2006) in Oreochromis niloticus exposed to deltamithrin for four days and observed increase enzyme activities (AST and ALT) in the gill, liver and kidney tissues. Several other authors also reported similar findings such as Das et al. (2004), Kuester et al. (2002). On the contrary, Luskova et al. (2002) in blood plasma of carp, Cyprinus carpio L. and Begum (2004) in liver and muscle of Clarias batrachus (Linn) reported decreased aminotransferase activities in fish tissues after diazinon and carbafuran exposure respectively. Overall, the changes in transaminases activity suggested a possible change in protein metabolism in different tissues of A. testudineus, H. fossilis and O. niloticus after the exposition of almix herbicide by disrupting the various physiological and biochemical processes such as Krebs and TCA cycle. 4.2. Alkaline phosphatase activity The enhanced activity of ALP in the intestine of A. testudineus, H. fossilis and O. niloticus exposed to

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almix caused tissue damage due to disruption the permeability of cell membrane. These results were also in agreement with the results of Borges et al. (2007) who reported increased activity of ALP in tissues of Rhamdia quelen fish exposed to cypermethrin; Das and Mukherjee (2003) found similar results in Labeo rohita exposed to cypermethrin and El-Sayed and Saad (2008) reported similar findings after deltamethrin exposure in Nile tilapia. Dissimilar results were also reported by Arellano et al. (1999) who observed reduced ALP activity in fish tissues like liver, kidney and gill due to disturbance in the membrane transport system. 5. CONCLUSION The present study revealed that exposure to commercial herbicide, almix induced significant changes in physiology and metabolic state of A. testudineus, H. fossilis and O. niloticus as manifested by alterations in the enzymatic activities in different tissues of fish. The induced activity of transaminases and ALP in different fish tissues indicated perturbation in the internal integrity of biochemical and/or physiological processes by the presence of almix in the water and estimation of these enzymatic activities in the present work could be considered as indices in the biomonitoring programme as well as risk assessment. Acknowledgements The authors would like to thank Department of Science & Technology, Govt. of India for the financial assistance. The authors also would like to thank the Head, Department of Environmental Science, The University of Burdwan, Burdwan, West Bengal, India for providing the laboratory facilities and library facilities during the course of research. REFERENCES APHA (2005). Standard Methods for Examination of Water and Wastewater, 21st ed. American Public Health Association, Washington, DC, USA. Arellano JM, Storch V, Saraquete C (1999). Histological changes and copper accumulation in liver and gills of Senegales sole (Solea Senegalesis). Ecotoxicol. Environ. Saf., 44: 6272. Barse AV, Chakrabarti T, Ghosh TK, Pal AK, Jadhao SB (2006). One-tenth dose of LC50 of 4tertbutylphenol causes endocrine disruption and metabolic changes in Cyprinus carpio. Pest. Biochem. Physiol., 86(3): 172-179.

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Safety Data Sheet (2012). DuPont Almix 20 WP. Version 2.1, Revision Date 27.07.2012 (Ref. 130000029001). Samanta P, Pal S, Mukherjee AK, Senapati T, Ghosh AR (2013). Evaluation of enzymatic activities in liver of three teleostean fishes exposed to commercial herbicide, Almix 20 WP. Proc. Zool. Soc., DOI: 10.1007/s12595-013-0086-z Schulman LJ, Sargent EV, Naumann BD, Faria EC, Dolan DG, Wargo JP (2002). A human health risk assessment of pharmaceuticals in the aquatic environment. Hum. Ecol. Risk Assess., 894: 657-680. Senapati T, Samanta P, Mondal S, Ghosh AR (2013). Study on histopathological, histochemical and enzymological alterations in stomach and intestine of Anabas testudineus (Cuvier) exposed to Almix 20WP herbicide. International Journal of Food, Agriculture and Veterinary Sciences, 3(2): 100-111. Sepici-Dincel A, Karasu BC, Selvi M, Sarikaya R, Sahih D, Ozkul IA, Erkve F (2009). Sublethal cyfluthrin to carp Cyprinus carpio fingerlings: Biochemical, hematological, histopathological alterations. Ecotoxicol. Environ. Saf. 72: 14331439. van der Oost R, Beyer J, Vermeulen NPE (2003). Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol., 13: 57-149. Yildirim MZ, Benli KC, Selvi M, Ozkul A, Erko F, Kocak O (2006). Acute toxicity behavioural changes and histopathological effects of deltamethrin on tissues (gills, liver, brain, spleen, kidney, muscle, skin) of Nile Tilapia (Oreochromis niloticus) fingerlings. Environ. Toxicol., 21: 614-620.

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Mr Palas Samanta is Senior Research Fellow in DST-INSPIRE Fellowship Program at Ecotoxicology Lab, Department of Environmental Science, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India.

Dr Sandipan Pal is a Contractual Whole Time Teacher in Department of Environmental Science, Aghore Kamini Prakash Chandra Mahavidyalaya, West Bengal, India. He has done his Ph.D. on Ecological Restoration and EIA of Abandoned Opencast Coal Pits lakes in Environmental Science from The University of Burdwan, West Bengal, India. His areas of researches are ecotoxicity of different chemicals like metals, pesticides and PAHs; monitoring and assessment of different surface water quality. He has enjoyed Japanese Government (Monbukagakusho:MEXT) Scholarship as a research student in Kagoshima University, Japan.

Dr. Aloke K Mukherjee has completed his Ph.D in Environmental Science from The University of Burdwan, Burdwan-713104, West Bengal, India, in the field of opencast mine limnology and fish culture technology. His area of research includes ecotoxicology, limnology, and mine water monitoring and management. Presently he is working as Asst. Professor in PG Dept of Conservation Biology, Dugapur Govt. College, Durgapur, Burdwan- 713214. He is teaching limnoecology, resoration ecology and environmental management and in Post-Graduation classes.

Dr. Tarakeshwar Senapati has completed his PhD in Environmental Science from the University of Burdwan, Burdwan-713104, West Bengal, India in the field of ecotoxicology. His area of research includes ecotoxicology, limnology, pollution control and monitoring and biodiversity. Presently he is working as Asst. Professor in School of Basic & Applied Sciences, Poornima University, Jaipur, Rajasthan, India. He is teaching environmental engineering, environmental chemistry in B.Tech & M.Tech level.

Dr Apurba Ratan Ghosh is Associate Professor in Department of Environmental Science, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India. He has guided 47 M.Sc. students, 12 M.Phil. students, and nineteen Ph.D. students, nine of them are already awarded. His research interests are on Fish and Fishery Sciences; Ecotoxicology; EIA and Environmental Management. He has first time in India explored the possibilities of pisciculture at Khadan i.e., Open Cast Coal Pits at RCF areas, which was well documented by Nationwide. He has published so far 64 research papers in reputed international and national journals and many popular scientific articles in different magazines. A total number of three books, three contributed chapters in different edited books and review of two books of Tata McGraw Hill Publisher are in his credits.

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