ISSN Original Article Histological changes induced by monocrotophos in the kidney of Ctenopharyngodon idellus (Cuvier and Valenciennes)

Transcription

1 Available online at International Journal of Research in Fisheries and Aquaculture Universal Research Publications. All rights reserved ISSN Original Article Histological changes induced by monocrotophos in the kidney of Ctenopharyngodon idellus (Cuvier and Valenciennes) NanditaSingla Department of Zoology, Panjab University, Chandigarh, India Current Address: Department of Zoology and Environmental Sciences, Punjabi University, Patiala, India Received 08 April ; accepted 19 April 2015 ABSTRACT Fresh water fish, grass carp, Ctenopharyngodon idellus (Cuvier and Valenciennes) was exposed to sublethal concentration of monocrotophos and fishes were acclimatized to the laboratory conditions for several days in the dechlorinated tap water. The live specimens of fish were exposed to monocrotophos for 45 days and they were studied on the basis of exposure periods i.e. after 15th, 30th and 45th day. The median lethal concentration (LC50) of monocrotophos to Ctenopharyngodon idellus (Cuvier and Valenciennes) was calculated by Probit analysis method which was found to be 0.27ml/L. The histological changes in the kidney revealed the change in glomerulus, increase in exposure period exhibited drastic damaging effects showing damage of the glomerulus and vacuolisation of cytoplasm. The changes in the damage tubules with pyknotic nuclei were also observed Universal Research Publications. All rights reserved Key words: Ctenopharyngodon idellus, monocrotophos, LC50, Kidney INTRODUCTION With the onset of industrial revolution, there is significant change in water quality. Increase in population and proliferation of industries saw the emergence of civilization that began to have much greater impact on its surroundings. Water pollution in various water bodies like lakes, rivers, oceans and ground water is caused by various anthropogenic activities which in turn have a bearing on the health of other aquatic organisms. Therefore, water pollution is a major problem in the global context. The contamination results in instability, discomfort, disorder or harm to ecosystem and is called pollution. Pollution in fact is one of man s greatest crimes against himself. The major water pollutants can be categorized as inorganic and organic. Inorganic impurities include some suspended particles which may consist of fine sand, clay, soil and precipitated salts. These impurities cause turbidity in water which interferes with various life processes of aquatic animals. There are several reports which point to the increasing load of certain heavy metals in the drinking water. These metals are directly associated with nerve damage, birth defects, mental retardation, certain cancers and increased susceptibility to disease. Pesticides are one of the major water pollutants. In 1940 s, synthetic pesticides were manufactured in large quantities and their use became widespread. Their use has been increasing since 1950 and about 2.3 million tons of industrial pesticides are now used every year. 75% of the pesticides in the world are used in developed countries, however, their demand is increasing drastically in the developing countries. These pesticides are not selective and are generally toxic to non-target species. World Health Organisation (WHO) in 1973 suggested that 500,000 cases of pesticide poisoning occurred annually (Jeyaratnam, 1990). Organophosphate compounds work by inhibiting important enzymes of the nervous system which play a vital role in transmission of nerve impulses. Chronic effects also occur due to exposure of organophosphates. Behavioral and psychological changes occur after exposure of humans or experimental animals to them. Organophosphates don t tend to persist or bioaccumulate in the environment but they are highly toxic to aquatic environment. The use of these chemicals must be reduced in order to combat environmental pollution. Generally, these pesticides enter the water bodies and directly affect the growth, 54

2 reproduction and other metabolic activities of fish besides other aquatic fauna. An ordinarily pollutant harms a fish either directly or indirectly. Indirect damage occurs largely through destruction of habitat. Direct effects of pollution on the fish life is either catastrophic or depends on the amount and kind of the undesirable effluent. Sudden discharge of large volume of boiling water or heavy salts or other chemicals may instantly kill fishes. Some substances when absorbed are directly toxic to fishes, even if present in small quantities. Though monocrotophos is banned for control of vegetable pests, but it is still being used widely because it is non-specific systemic insecticide and acaricide which is used to control a wide spectrum of chewing and sucking insects, ticks, spiders and also common mites (Burkhard and Drabek, 1994). It is widely used against cotton pests and on various crops like rice, bajra, cotton, wheat, citrus, sugarcane, sugarbeet, ornamentals, tobacco, potato, soyabeans etc. Grass carp is a very hardy fish which can tolerate a wide range of temperatures and is adaptable to the varying environment. Keeping the above facts in view, the present investigation has been carried on the toxicity of organophosphate pesticide, monocrotophos on the kidney of grass carp, Ctenopharyngodon idellus (Cuvier and Valenciennes). The major aspects of these studies are to determine the LC50 value of monocrotophos for grass carp and histological changes in kidney of control and treated fish. MATERIAL AND METHODS Procurement of fish The live specimens of fish, Ctenopharyngodon idellus (Cuvier and Valenciennes) were collected from Neelamber Fish Farm, Nanoke village, District Patiala, Punjab. Fishes were acclimatized under laboratory conditions for 15 days in dechlorinated water and the tank was provided with an aerators and filters. The average length and weight of the fishes under investigations varied from cm (± 15.5) and gm (± 7.65) respectively at the start of experiment. The fishes were fed with fresh Hydrilla during the experiment so that fish tissues does not show any changes due to starvation. The experiment was carried out by using the organophosphate Hilcron 36SL (Monocrotophos 36SL), manufactured by Hindustan Insecticides Ltd., having 36% W/W of monocrotophos as an active ingredient. Acute toxicity tests Toxicity tests were performed according to the standard methods given in APHA (1998). To calculate the LC50 value of pesticide for grass carp, a group of five fishes each were exposed to the pesticide with varying concentration in plastic tanks of 25 litres capacity. These concentrations were 0.22ml/L, 0.23ml/L, 0.24ml/L, 0.25ml/L and 0.26ml/L. An appropriate control was set along with the test. Dechlorinated water was used for making various concentrations. Water was changed after every 24 hours, so as to remove the faecal matter and to maintain the required concentration of pesticide. On the basis of this test, 96 hrs LC50 was calculated by Probit analysis (Finney, 1980). Chronic toxicity tests To carry out the chronic toxicity test, 15 fishes were exposed to pesticide in an aquarium of 175L water capacity. 50L of dechlorinated water was used to for the experiment with concentration of 0.10ml/L of the pesticide. Control was set up along with the test so as to compare the results. Five fishes each were sacrificed after 15 days, 30 days and 45 days and were dissected immediately to remove the kidney for further processing. Behavioral and morphological changes The exposed fishes were observed for any behavioral and morphological changes during acute and chronic toxicity tests. They were observed from time to time for colour change, jerky movements, feeding habits, equilibrium etc. Histological studies - Kidney After completion of exposure periods, fishes were sacrificed and the kidneys were removed for light microscopic investigations. It was washed in 10% saline for 5 minutes so as to make it free from blood.the procedure for light microscopic studies using Bouin s fixative (Pearse, 1968) RESULTS AND DISCUSSION The organic insecticides are one of the major health issues both in developing and developed countries and many of the acute poisoning cases are caused by exposure to these pesticides especially the organophosphate compounds. The accumulation of these pesticides is known to adversely affect the liver, muscle, kidney and other important tissues of the fish. The kidney of the fish receives a vast majority of post branchial blood and the renal lesions are expected to be good indicators of environmental stress (Hinton and Lauren, 1990). It is very unlikely that the use of these very pesticides would be replaced by other means of pest control such as integrated pest management (IPM) in near future because of the cost and the effectiveness involved. Very limited work has been done on the histopathological effect of these pesticides on fishes and fewer histological studies have been done with organic pollutants than with metals. The aim of this present research is to study the effect of an important organophosphate, monocrotophos on the kidney of an exotic carp, Ctenopharyngodon idellus, which is a widely cultured fish, which is a great food source and is relished for its excellent flavor and high protein value. The study has been undertaken to determine the median lethal concentration (LC50) values, behavioral changes coupled with the histological changes in the kidney of the tested fish species at a sub-lethal concentration with exposure periods i.e. 15 days, 30 days and 45 days. In the present studies, both acute and chronic toxicity experiments were performed to test the effect of monocrotophos on Ctenopharyngodon idellus and the LC50 value was calculated. Estimation of LC50: The LC50 value for monocrotophos in the experimental fish i.e. Ctenopharyngo don idellus was found to be 0.27 ml/l and this is based on the mortalities of 55

3 fish which were exposed to a series of known concentrations of the toxicant, monocrotophos in this case along with a control in parallel which received no pesticide treatment. The concentrations of the toxicant were selected on the basis of the LC50 values of monocrotophos for Cyprinus carpio as there were no available references for LC50 value of grass carp. Acute and Chronic Toxicity tests of Monocrotophos on the Kidney of Ctenopharyngodon idellus The fishes are very sensitive to any change in aquatic environment particularly by the changes in physical and chemical characteristics of water which affect the homeostatic equilibrium resulting in altered behavioral and physiological responses (Wanderlaar Bonga, 1997). Both acute and the chronic treatment of the fish with 0.10 ml/l of the monocrotophos resulted in substantial changes in the fish behavior. The initial pesticide exposure lead to the abnormal behavior such as sudden quick movements and jerks. The rolling movements were observed at higher doses of pesticide i.e. monocrotophos. Occasional jumping over the walls of aquaria was also noted. On prolonged exposure with the pesticide, the fish became weak and settled on the bottom and were almost stationary most of the time in one corner of the experimental tank. These observations are in line with the observations of Rahman et al. (2002) who also observed sudden quick movements of fish when the media i.e. Diazinon 60 EC, an organophosphate started to act on the test species i.e. Anabas testudineus, Channa punctatus and Barbodes gonionotus. The general conditions of the fish deteriorated with the increase of the pesticide exposure as e.g. the colour of the fish became pale with higher doses in 96 hours experiment as well as with increase of the duration of the pesticide exposure. Moreover, loosening of scales occurred after pesticide exposure but more importantly, there were no cases of scale loss from the fish during the course of the experiment. Iqbal et al. (2004) observed rapid scale loss especially from the head region in a common carp, Cyprinus carpio following acute treatment of the nitrate and the changes were more pronounced during initial hours of exposure of fish to nitrate. Another important observation is the increased rate of defecation by almost all the fishes in the treated group (15th day, 30th day and 45th day) and it was particularly more marked in the fish exposed to the higher dosage of the pesticide in acute toxicity assay. Excessive secretion mucous was also observed particularly when the aquarium water became dirty/cloudy which is in line with our observations on grass carp. On exposure to pesticide, several other workers have observed similar results in different species of fish (Hoque et al., 1993; Lovely, 1998 and Veiga et al., 2002). HISTOLOGICAL STUDIES ON KIDNEY OF GRASS CARP Kidney is a vital excretory organ and proper kidney functioning is important to maintain the homeostasis (Iqbal et al., 2004). The gross anatomy of the kidney varies in different species from distinctly bilobed kidneys to the kidneys that are fused and intimately embedded between vertebrae. The kidney occupies dorsal position in the body cavity and is ventral to the vertebral column. Kidney is divisible into anterior kidney (also known as head kidney) and posterior kidney (also known as trunk kidney). Embryologically, anterior kidney derives from pronephros and posterior kidney derives from mesonephros as studied by Takashima and Hibiya (1995). It not only is involved in removal of metabolic wastes but is also responsible for selective reabsorption, thereby, helping in maintaining volume and ph of the body fluids. Therefore, any change in the gross structure of the kidney leads to a corresponding change in the physiological and behavioral parameters. The damage may be in the form of necrosis, vacuolization of the tubule cells, breakage of the tubules and also because of degeneration of the epithelial cell lining. There have been many studies on fish kidney showing effects of various toxins by many researchers but there are no studies about the effect of monocrotophos on the histology of kidney of grass carp (Kumar and Pant, 1981; Saleh, 1982; Kumari and Banerjee, 1986; Bhuiyan et al., 2001; Veiga et al., 2002, Velmurugan et al., 2007; Staicu et al., 2007). The kidney of the fish is made up of numerous renal tubules. At the light microscopic level, the renal corpuscle is composed of glomerulus and bowman s capsule, connected further to the coiled uriniferous tubule. The proximal tubule which is nearer to the glomerulus is composed of cuboidal / columnar cells with a welldeveloped brush border containing vacuoles whereas the distal tubule lacks the brush border and is connected to the collecting duct and the space between these (Proximal and distal tubules) is filled with parenchymatous lymphoid tissue with rich blood supply. The light microscopic studies were carried out by staining kidney sections with haematoxylin-eosin. In the control group, no recognizable changes were observed. The kidney consisted of the renal tubules with lumens, certain glomerulus along with rich supply of blood vessels (Plate 1, Figs. a and b). After the fishes were exposed to monocrotophos, at a sub-lethal concentration of 0.10 ml/l of the pesticide for varying duration i.e. 15 days, 30 days and 45 days; the kidney showed varying degrees of damage in glomerulus, renal tubules and necrosis. The histological analysis of grass carp kidney exposed to monocrotophos for 15 days showed gross morphological changes at many places which include damage to the renal tubules, shrinkage/ contraction of the glomerulus/ disintegration of the glomeruli, vacuolization, narrowing of the tubular lumen, disorganization of the tubules with pyknotic nuclei coupled with hemorrhagic areas (Plate 1, Figs. c, d, e, f). Upon exposure of pesticide for 30 days, the corresponding damage to the renal tubules increased coupled with increase in the vacuolization (Plate 2, Figs. a and b). Moreover, there was alteration of the shape of the renal tubules and the necrosis of the tissue was more evident (Plate 2, Figs. c and d). Further exposure of the pesticide led to far greater vacuolization, increase of the pyknotic nuclei vacuolization and tubular cells were in 56

4 complete disarray. Similar changes have been reported by many other investigators but with a different toxicant in a different fish (Dubale and Shah, 1981; Rashatwar and Llyas, 1984; Sharma et al., 1985; Srivastava and Pandey, 1986; Gill and Pant, 1987; Gill et al., 1988; Bais et al., 1990; Singhal and Jain, 1997; Tilak et al., 2001). Rahman et al. (2002) showed necrosis, degenerated kidney tubules and pyknosis with hemorrhagic areas in the kidney tissue of Anabas testudineus, Channa punctatus and Barbodes gonionotus upon exposure to Diazinon 60 EC and the impact of the toxicant increased with increase of dosage. Iqbal et al. (2004) observed similar changes in the kidney of common carp, Cyprinus carpio, following exposure of the fish to nitrate even for 1 to 4 days and these again showed increased damage in the glomerulus coupled with vacuolization with increased duration of the nitrate. Similar results have been observed in the kidney tissue of many other fishes using different toxicants (Velmurugan et al., 2007; Staicu et al., 2007; Ayoola, 2008; Mishra and Mohanty, 2009; Anthony Reddy et al., 2015). Thus, it can be said that the sub-lethal dosages of monocrotophos results in significant histological changes in the grass carp kidney and these changes increase with increase of the exposure period of the pesticide. The type of changes found in our investigations may also serve as biological markers of environmental stress. It becomes more important when the pesticide does not cause the death of the organism but instead causes cellular damage which can be observed with light microscopy. These include degeneration, accumulation of cytoplasmic inclusions and other changes in the cells. CONCLUSION The results of present study on the histological changes observed in the kidney of fish exposed to monocrotophos have been correlated with the previous findings of various workers in various toxicological studies conducted with other toxicants and it has been found that the monocrotophos also induces degenerative changes as induced by other toxicants. Hence, viewing the deleterious effect of these insecticides on the non-target species, the indiscriminate use of monocrotophos should be prohibited through legislation. Acknowledgements We acknowledge Dr. Y. K. Rawal and Dr. M.S. Johal, Department of Zoology, Panjab University, Chandigarh for their much needed guidance in the work. I would also like to acknowledge UGC-CAS and Department of Zoology, Panjab University, Chandigarh for financial support and required laboratory facilities. Reference 1. ANTHONY REDDY, P., VEERAIAH, K., TATA RAO, S. and VIVEK, C The effect 57

5 of confidor on histology of the gill, liver and kidney of fish LabeoRohita (Hamilton). Int. J. Bioassays 4(2): APHA Standard methods for the examination of water and waste water; American Public Health Association; 20 th edition; New York. 3. AYOOLA, S.O Toxicity of glyphosate herbicide on Nile tilapia (Oreochromisniloticus) juvenile. African J. Agric. Res. 3(12): BAIS, V.S., BHARGAVA, A. and BHARGAVA, S Toxic effects of DDT on the kidney of freshwater fish, Channa punctatus. Trend. Ecotoxicol. Pp BHUIYAN, A.S., NESA, B. and NESSA, Q Effect of sumithion on the histological changes of spotted murrel, Channa punctatus (Bloch). Pak. J. Biol. Sci. 4(10): BURKHARD, N. and DRABEK, J Monocrotophos- chemical and physical properties. Rev. Environ. Contam. Toxicol. 139: DUBALE, M.S. and SHAH, P Histopathology of kidney of fish Channa punctatus exposed to cadmium in India. J. Ani. Morph. Physiol. 28(1-2): FINNEY, D.J Probit Analysis, 3 rd Ed., Cambridge University Press, London and New York. 9. GILL, T. S. and PANT, J. C Hematological and pathological effects of chromium toxicosis in the freshwater fish, Barbusconchonius (Ham.). Water, Air and Soil Polln. 35: GILL, T. S., PANT, J. C. and PANT, J Gill, liver and kidney lesions associated with experimental exposures to carbaryl and dimethoate in the fish (Puntius conchonius ham.). Bull. Environ. Contam. Toxicol. 41(1): HINTON, D.E. and LAUREN, D.J Integrative histopathological approaches to detect the effect of environmental stressors on fishes. pp In: Biological indicators of stress in fish (ed. S.M. Adams.). 12. HOQUE, M.M., MIRJA, M.J.A. and MIAH, M.S Toxicity of diazinon and sumithion to Puntius gonionotus. Bangladesh.J. Tran. Dev. 6(1): IQBAL, F., QURESHI, I.Z. and ALI, M Histopathological changes in the kidney of common carp, Cyprinus carpio, following nitrate exposure. J. Res.Sci.15 (4): JEYARATNAM, J Acute pesticide poisoning: A major global health problem. WldHlth. Stat. Quart.43 (3): KUMAR, S. and PANT, S.C Histopathological effects of acutely toxic effects of copper and zinc on gills, liver and kidney of Puntius conchonius (Ham.). Indian J. Exp. Biol. 19: KUMARI, K. and BANERJEE, V Effect of sublethal toxicity of zinc, mercury and cadmium on gills, liver and kidney of Puntius conchonius (Ham.). Indian J. Exp. Biol. 19: LOVELY, F Toxicity of three commonly used organophosphorus insecticides to Thai sharputi (Barbodesgonionotus) and African catfish (Clariasgariepinus) fry. M.Sc. Thesis. Department of Fisheries Biology and genetics, Bangladesh University, Mymensingh, Bangladesh. 83 p. 18. MISHRA, A.K. and MOHANTY, B Chronic exposure to sublethal hexavalent chromium affects organ histopathology and serum cortisol profile of a teleost, Channa punctatus (Bloch). Science of the Total Environment 407: PEARSE, A.G.E Histochemistry: Theoretical and Applied (revised edition of A.G.E. Pearse) J. and A. Churchill. London. 20. RAHMAN, M.Z., HOSSAIN, Z., MOLLAH, M.F.A. and AHMED, G.U Effect of Diazinon 60EC on Anabas testudineus, Channa punctatus and Barbodes gonionotus. The ICLARM Quarterly 25(2): RASHATWAR, S. S. and LLYAS, R Effect of phosphamidon in a freshwater teleost fish Nemacheliusdensoni (Day): Histopathological and biochemical studies; J. Environ. Biol. 5(1): SALEH, H Fish liver as indicator of aquatic environmental pollution. Bull. Inst. Oceanographic Fisher.8: SHARMA, R.K., QUARESHI, A. and QUARESHI, N.A Toxic effects of lead on kidney of a freshwater catfish, Clariasbatrachus. Ind. J. Zool. 13: SINGHAL, R.N. and JAIN, M Cadmium induced changes in histology of kidney in common carp, Cyprinus carpio. Bull. Environ. Contam. Toxicol. 58(3): SRIVASTAVA, A. and PaNDEY, A.K Effects of cadmium chloride on kidney of Puntius sophore, a freshwater fish. Com. Physiol. Ecol. 11: STAICU, A.C., MUNTEANU, M.C., COSTACHE, D., COSTACHE, M. and DINISCHIOTU, A Histological changes in Deltamethrin induced intoxication in Carassiusauratusgibelio (Pisces Cyprindae). Biotechnol. Anim. Husb. 23(5-6): TAKASHIMA, F. and HIBIYA, T An atlas of fish pathology: normal pathological features; second edition; Kondansha Ltd., Tokyo, Japan, 195 pp. 28. TILAK, K.S., VEERAIAH, K. and YACOBU, K Studies on histopathological changes in the gill, liver and kidney Ctenopharyngodon idella (Valenciennes) exposed to technical fenvalerate and EC 20%. Poll. Res. 20(3): VEIGA, M., RODRIGUES, E.de L., PACHECO, F.J. and RANZANI-PAIVA, M.J.T Histopathological changes in the kidney tissue of Prochiloduslineatus Valenciennes, 1836 (Characiformes, Prochilodontidae) induced by sublethal concentration of trichlorfon exposure. Brazilian Arch. Biol. Tech. 45(2):

6 30. VELMURUGAN, B., SELVANAYAGAM, M., CENGIZ, E.I. and UNLU, E The effects of monocrotophos to different tissues of fresh water fish Cirrhinusmrigala. Bull. Environ. Contam. Toxicol. 78(6): WANDERLAAR BONGA, S.E The stress response in fish. Physiological Reviews, 77(3): Source of support: Nil; Conflict of interest: None declared 59