Histopathology of the gills, livers and kidney of Clarias gariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions

Indiscriminate use of Sniper 1000EC has become a serious problem among local fishermen in the Northern parts of Niger state. Juveniles of Clarias gariepinus (mean body weight 23.34 ±0.05 g; mean standard length, 20.00 ±0.45 cm) were subjected to 5 treatment levels of 0, 5, 10, 15 and 20 mg/l. The tissue damages were observed majorly at 15 and 20 mg/l of sniper 1000EC exposed fish species. Oedema and hyperplasia were observed in the gills of the exposed groups. Hepatocellular steatosis and vacuolations were observed in the livers. Tubular nephrosis and hyperplasias of epithelial cells were also observed in the kidney. It is concluded that alterations in gills, livers and kidney of the exposed fish species were conse- quences of exposure to the toxicant (sniper 1000EC). It is recommended that the use of Sniper 1000EC by local fishermen be banned to save the aquatic environment from destruction.


Introduction
Histopathological changes have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants in laboratory animal (Wester, Canton, 1991;Thophon et al., 2003;Abubakar, Adeshina, 2019) and wild specimen (Hinton et al., 1992;Schwaiger et al., 1997;Tech, Adams, Hinton, 1997;Drishya, Binu-Kumari, Mohan-Kumar, Ambikadevi, Aswin, 2016). One of the great advantages of using histopathological biomarkers in environmental studies is that this category of biomarkers allows the examination of the target organs (Gernhofer, Pawet, Schramm, Muller, Triebskorn, 2001). Furthermore, the alterations found in these organs are clearer than functional ones (Fanta, Rios, Romao, Vaaianna, Freiberger, 2003). It also reveals signs of damage to animal health (Hinton & Lauren 1990). The gills, being a respiratory organ of fish (Banerjee, 2007) is frequently in contact with external environment and thus vulnerable to aquatic toxicants (Abubakar, 2013). Studies have been conducted on histopathological changes in the gills, liver and kidney of fish exposed to various substances (Auta, 2001;Drishya et al., 2016;Abubakar, Adeshina, 2019) including pesticide which have been reported to cause pathological alteration in the exposed C. gariepinus (Auta, 2001). The liver ow f fish can be considered as a target organ to pollutants, alterations in its structure can be significant in the evaluation of fish health (Kolbasi, Burcu, Ucuncu, Onen, 2009).
Widespread application of various pesticides has aggravated the problem of pollution to aquatic environment. Due to these synthetic chemicals, environment has failed to keep its healthy characteristics. The insecticides of proven economic potentialities could not do well in the ecosystem when viewed on extra fronts since these revenue poisons, in a residual form or as a whole, get into the aquatic ecosystem. They cause a series of problems to aquatic organisms Mastan, Ramayya, 2010;Drishya et al., 2016;Abubakar, Adeshina, 2019).
Sniper 1000EC (2, 3-dichlorovinyl dimethyl phosphate), a brand of dichlorvos, is contact acting and fumigant insecticide (Abubakar, 2013). Like all organophosphates, it kills insects and other target organisms because of its toxicity to the nervous system. This is achieved by inhibition of enzyme acetylcholinesterase (AchE) that breaks down acetycholine at the receptor site for partial uptake into the nerve terminal (Drishya et al., 2016). Without functioning AchE, accumulation of acetylcholine results in depolarizing block of muscle membrane, producing rapid twitching of involuntary muscles, convulsions, paralysis and early death Mastan, Ramayya, 2010;Drishya et al., 2016;Abubakar, Adeshina, 2019). Indiscriminate use of Sniper 1000EC is common among local fishermen from Northern parts of Niger state.
The African catfish,Clarias gariepinus is an important food fish in Nigeria (Abubakar, 2012). Clarias gariepinus is not only the most predominant fish species raised in aquaculture in Nigeria, but has also served as an experimental model of aquatic vertebrate for two decades (Cavaco et al., 2001). Despite the indiscriminate use of Sniper 1000EC by local fishermen, there is a paucity of information on its toxicity.
The aim of the present study was to evaluate histological alterations in gills, liver and kidney of Clariasgariepinus (Burchell, 1822) exposed to sniper 1000EC under laboratory conditions.

Experimental fish and test chemical
Juveniles of Clarias gariepinus (n 150, mean body weight 23.34 ±0.05 g) were purchased from a reputable 36 fish farms in Ilorin, Kwara State, Nigeria. The samples were transported to the Central Laboratory, University of Ilorin in plastic container of 100 L capacity filled with water to two-third volume between 07:00 hours and 09:00 hours. They were held in large water baths of 160 L capacity and acclimated for 14 days to laboratory conditions. The top of water bath was covered with netted material to prevent jumping out of the fish. A slit was made at middle of the net to allow for feeding fish and cleaning of the bath. Feeding commenced two days after the arrival and stopped twenty-four hours before the commencement of the experiment. During acclimation, fish were fed twice daily (08:00 and 16:00 hours) with formulated feed (45% crude protein, Table 1) at 5% body weight. The fishwere accepted and adapted to laboratory conditions for the 14 days. The water in the bath was changed daily and uneaten food and faecal matters were siphoned out. Water parameters were monitored two times daily at the hours of 08:00 and 18:00. The water temperature, DO and pH ranges throughout the experimental period were 24.1-25.7°C, 5.6-6.6 mg/l, and 7.24-7.81, respectively which were measured with the aid of mercury-in-glass thermometer, a digital DO meter (Model AVI-660, Labtech International Ltd, Heathfield, UK) and a digital pH-meter (Model Photoic 20, Labtech International Ltd, Heathfield, UK), respectively. Dead fish were also removed to minimize contamination of water.
Test chemical (2, 3-dichlorovinyl dimethyl phosphate), a brand of Dichlorvos with the trade name Sniper 1000EC (United Phosphorous Limited, UPL) was obtained from Minna central market and was used for the study. The test concentrations were prepared with reference to the Manual of Method in Aquatic Environment Research.

Experimental design
The experimental design was a complete randomized design. A total of one hundred and fifty (150) juvenile of Clarias gariepinus were randomly distributed into the tanks (60 × 38 × 27 cm) at a stocking rate of 10 fish per tank. The fifteen (15) tanks were assigned to 5 treatments comprises 0, 5, 10, 15 and 20 mg/l. Dead fish from experimental groups were removed and dissected immediately for histological analysis while specimens from control were vivisected without anesthesia and subjected to histological analysis at the end of one-week experiment.

Histological procedures
Samples of fish from both control and experimental groups were excised, rinsed in physiological saline and fixed in Bouin's fluid for 6, 12 and 8 hours respectively. The gills, livers and kidneys were subjected to micro techniques (Bucke, 1989;Adeshina, Jenyo-Oni, Emikpe, Ajani, Abdel-Tawwab, 2019). The tissues were dehydrated in an ethyl alcohol series of ascending concentrations, embedded in paraffin and sectioned at 5 µm thickness. The tissues sectioned were stained with haematoxilin-eosin (HE). Stained slides were cleared in xylene and mounted in synthentic resin medium. The slides were examined under low power (×4) objective and high power (×10) objective with Tension Binocular microscope. Under extreme low power, the condenser was moved into a very low position to prevent the image of the lamp bulb from obscuring the image of the micrographs. Photomicrographs were then taken at low power (×40) objective with Samsung ES25 4X Zoom Lens digital Camera and downloaded into a computer. Photomicrographs of control groups were compared with those of exposed groups under the guidance of a pathologist.

Histopathology of the gill
No alterations were observed in the gills of the control ( Figure 1A). The most common pathological changes in the gills of exposed fish species were fusion of lamellae, oedema and hyperplasia of the secondary lamellae ( Figure 1B

Discussion
Gills are the primary corridor for molecular exchange between the internal milieu of fish and their external environment (Olson, 1996), such as gas transfer, acid-base regulation and ionic regulation (Eddy, 1982). The filament of gills and their secondary lamellae represent two general types of epithelium (Laurent, Dunel, 1980;Laurent, 1984) that contain three cell epithelia, the pavement, the chloride (ionocyce or mithochondria-rich) cells and the mucous cells, which are most prevalent. In some fish species, the gills contain microvillous or smooth surface lamellar pavement cells (Hossler, Harpole, King, 1986). Study of the gills in the control fish showed a typical structural organization of the lamella while gills from the fish exposed to the toxicant had several histological distortions, namely clumping/fusion, oedema and hyperplasia of the lamellae. The gill abnormalities observed in this study were similar to previous studies on fish gill morphology, which showed separation of the epithelial layers of secondary gill lamellae, deformation of secondary lamellae and degeneration of chloride cells accompanied by hyperplasia of undifferentiated cells in the primary lamellae (Daye, Garside, 1976;Chevailer, Gauthier, Moreau, 1985).
The oedema and hyperplasia in the gills of the exposed fish might be due to acute inflammatory nature of the lesion induced in such gills by the sniper 1000EC. Eller (1970) reported hyperplasia of gill lamella epithelium as findings suggestive of toxicity in several fish species following exposure to several toxicants. Oedema with epithelial separation was reported by (Eller, 1971;Van Valin, Andrews, Eller, 1968) as additional gill change in fish exposed to toxicants. Epithelia sodium pump failure might be responsible for gill oedema (Mitchel, Cotran, 2004). The hyperplasia might in addition be an attempt to increase blood supply to compromised gill in order to increase blood oxygenation. (Gabriel, Amakiri, Ezeri, 2007). This could be a compensatory maechanism in the exposed fish to counter hypoxia sequel to edema and obliterated inter-lamellae space, which impeded gaseous exchange across such gills (Gabriel et al., 2007). Oedema has been reported to increase the diffusion distance between gill capillaries and epithelial cells, thereby increasing internal diffusion resistance, which is a major factor that inhibits gaseous exchange across the affected gills (Turala, 1983).
The lamellar fusions are defense mechanisms that reduce the branchial superficial area in contact with the toxicant. These mechanisms also increase the diffusion barrier to the pollutant (Lauren, Mc Donald, 1985;Van Heerden, Vosloo, Nikinmaa, 2004). Similarly, fusion of adjacent secondary lamellae as a consequence of edema could lead to telangiectasis, which is a characteristic pathologic feature of compromised gills associated with physical or chemical damage (Robert, 2001). However, such structural changes are reported to be non-toxicant specific (Meyer, Hendricks, 1985), which could be mere stress response of fish, to toxicants exposures (Gabriel et al., 2007). The effect of sniper 1000EC that brought about edema, hyperplasia and laceration of gill lamella explains why fishermen used sniper 1000EC indiscriminately causing heavy fish kills as it destroys the gills, the major respiratory and food filtering organ in fish (Abubakar, 2013). It was recognized that liver is difficult to analyse due to its high histological variability (Kotin, 1967). However, Myers, Landahi, Krahn, Johnson, McCain (1991) and Myers, Olson, Johnson, Stehr, Varansi (1992) have described the effects of different toxicants on the livers of various fish species. They showed that the liver distortions developed gradually over the time of exposure and that this varies from individual fishes. They also showed that it is possible that liver could regenerate and recover from those distortions. In this study, the histopathologies of the liver of C. gariepinus were carried out with a view to explaining the toxic mechanism of sniper 1000EC.
The toxicant caused alterations of the liver parenchyma, such as vacuolization and necrosis. These alterations were often associated with a degenerative-necrotic condition (Myers, Rhodes, McCain, 1987). This is in agreement with the reports of Omoregie, Ufodike (1990) and Auta (2001) who separately reported reduced liver vacoulation in Oreochromisniloticus exposed to actellic 25EC, paraquat and dimethoate respectively.The reduced vacoulation of hepatocytes may be due to fatty degeneration (Thiyagarajah, Grizzle, 1985;Auta, 2001;Langiano, Martinez, 2008). Vacuolation of hepatocytes with pycknotic (condensed) nuclei in the liver was most likely due to deposition of glycogen and lipid (Myers et al., 1987) as a result of hepatotoxicity induced by the presence of the toxicant and/or reduced food intake (Khan, Kiceniuk, 1988). Ferguson (1989) observed that a common morphologic response of fish liver to toxicity is the loss of hepatic glycogen and/or lipid. This condition according to Wolfe and Wolfe (2005) might have occurred by direct intoxication or it might have occurred secondarily to deceased body condition caused by stress or concurrent disease. Furthermore, the authors noted paradoxically that toxic exposure might also result in the accumulation of fats as recorded in this study.
The steatosis and necrosis were similar to those reported for fish caught in contaminated water or those exposed to various chemicals under laboratory conditions (Brand, Fink, Bengeyfield, Birtwel, Mc Allister, 2001;Rudolph, Yanez, Troncoso, 2001;Marty, Hoffinamn, Okihiro, Hepler, Hanes, 2003;Fafioye, Adebisi, Fagade, 2004;Koehler, 2004;Olojo, Olurin, Mbaka, Oluwemimo, 2005;Wahbi, El-Greishy, 2007;Aniladevi, Philip, Smitha, Bhanu, Jose, 2008). Fatty degeneration of the liver (haemosiderosis/steatosis) recorded in the liver of the exposed fish might be suggestive of metabolic disorders and it was commonly associated with dietary deficiency in response to xenobiotic (Myers et al., 1987). These changes are normally reported in disease organisms or those exposed to toxicants (Khan, Kiceniuk, 1988;Hawkins, Walleewrr, Orerstreet, Lyte, Lyte, 1988;Ogbulie, Okpkwasili, 1999). It might have resulted from disturbances in any of the steps in the sequence of the events from fatty acid entry to lipoprotein exit (Mitchell, Cotran, 2004). The degree of fatty changes in the exposed species exceeded that of the control implicating the toxicant as being responsible for the change. Lipid or glycogen vacuolation suggested the accumulation of triglycerides usually within the hepatocytes and may be responsible for the hepatocyte enlargement/hyalination (Wolf, Wolf, 2005). Steatosis has been correlated with neoplasms; however, its role in the progression of lesions towards neoplasm formation in fish was not well understood (McCain et al., 1982). Similar changes have been reported in the liver of Astyanax sp exposed to WSFs of crude oil (Akaishi et al., 2004). Percafluviatilis and gold fish exposed to oil seed process affected water (Nero et al., 2006), centrolobular necrotic change was the main change in the species exposed to the toxicant and agrees with the observation of Popp (1991) who suggested that the distribution of the interstitial system in the liver resulting in a higher concentration of the toxicant in the centrolobular region account for the occurrence and frequency of centrolobular toxicity.
The necrosis of the liver tissue might be due to inability of the fish to regenerate new liver cells due to the effect of sniper 1000EC. Several studies have reported that chronic accumulation of some toxicant in fish livers causes hepatocyte lysis, cirrhosis and eventually death (Pourahamad, O'Brien, 2000;Varanka, Rojik, Varanka, Nemcsók, Ábrahám, 2001). The kidney has been established as one of the principal site of erythropoietin production (Gordon, Zanjani, 1970). As in higher vertebrates, the kidneys of fish perform an important function related to electrolyte and water balance and maintenance of a stable internal environment. Thurston, Russo and Smith (1978) reported mild hydrophobic degeneration in renal tubule. The hydrophobic degeneration observed in the kidneys of the exposed fish species might be due to an increase in the permeability of fish tissues to water, and increased urine out put as reported by Lloyd and Orr (1969) for rainbow trout. Following the exposure of the fish to the toxicant, histological alterations have been found at the level of tubular epithelium (Teh et al., 1997). Ortiz, de Canales and Sarasquete (2003) found kidneys of exposed fish to have received the largest proportion of post-branchial blood, and therefore renal lesions may be good indicators of environmental pollution. Any effects on the kidney are likely to lead to major problems of anaemia and a lack of new blood cells formation. In this study, there was marked diffused tubular necrosis, hyperplasia of interstitial haemopoietic tissues.
The changes recorded in the kidney section from exposed fish were similar to those caused by petroleum in English sole (McCain et al., 1978) and rats (Dede, Kagbo, 2001) and C.gariepinus exposed to plant extracts (Onusiruka, Ufodike, 2000;Fafioye et al., 2004) for various periods. Peripheral blood and cephalic kidney of turbot, Scophthalmusmaximus and Atlantic cod, Gadusmorus had micronuclei and severe nuclei abnormality such as nuclear buds, binucleated and nonylphenol (Barsiene, Dedonvte, Rybakovas, Anderson, 2006). Exposure, such as changes in kidney tubules particularly at the highest concentration recorded in this study might greatly impair the infiltration functions of the kidney with grave consequences for the exposed fish. However, the onset development and extent of these changes might be toxicant concentration and exposure time dependent.

Conclusion
In this study, alterations in gills, livers and kidney in the exposed fish species were associated with the effects of different concentrations of sniper 1000EC with LD50 of 15 mg/l. By this context, smipper at 15 mg/l causes significant damage to fish tissue and hence be prevented and prohibited for fishing.