The organochlorine pesticide endosulfan (6,7,8,9,10,10-Hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide) is a chlorinated hydrocarbon which is among the most toxic pesticides for aquatic life, especially fish, and therefore has been registered as a priority pollutant by the US Environmental Protection Agency. Particularly in developing countries, endosulfan is in general use for pest control in jute, cotton, sugar cane and vegetables. Due to agricultural activities, endosulfan has repeatedly been reported in surface waters and soil of developing and established countries. It is more toxic to the fish model of the present study, as the fish is a mud dweller and remains in contact to the soil for longer time. Endosulfan is moderately persistent in the soil environment with a reported average field half-life of 50 days. Acute toxicity of endosulfan to fish was reported previously.
Previous histopathological studies on fishes exposed to pollutants revealed that fish organs are efficient indicators of water quality. The gills are important organs in fish to perform respiration, osmoregulation, acid base balance and nitrogenous waste excretion. Fish gills are also vulnerable to pollutants in water because of their large surface area and external location. For this reason, fish gills are considered to be most appropriate indicators of water pollution levels. Many investigators have reported the histopathological changes in the gills of different fish species exposed to various pesticides. However, there has been little information on the histopathological impact of endosulfan on fish gills.. Therefore, it was decided to determine the histopathological effects of gills in a catfish, Clarias batrachus Linn. exposed chronically to endosulfan in the present research.
MATERIALS AND METHODS:
Adult fish with an average body weight of 0.2 ± 0.03 Kg and total length of 20 ± 0.4 cm were collected from the local market. The fish were transferred in oxygenated containers to the laboratory. They were acclimated in glass aquaria at a constant temperature (24±l°C) and pH 7.5 ± 0.3 for 7 days prior to the experiment. Aged tap water was used for acclimation as well as for preparing test solutions. Water was continuously aerated. Fish were fed twice daily with Tubifex tubifex. Technical grade endosulfan (Thiodan®, 33.70% endosulfan) was provided by Hoechst Co. India Ltd.
The fish were divided into two groups and placed in separate glass aquaria. Ten fishes were used for each group. Group I was exposed to commercial formulations of endosulfan. The nominal concentrations tested were 0.5 µg/L for endosulfan, being the l/12th fractions of the 96h LC50 value. Group II was maintained in pesticide-free water to serve as control. Half the amount of test water was renewed every 24 hour. The average values for the water quality data were, temperature 24±l°C, pH 7.5 ± 0.3, dissolved oxygen 7.2 mg/L and total hardness 168 mg/L as CaCO3.
Clarias batrachus exposed to endosulfan did not show any alteration in behavioural patterns and feeding activity. Likewise, growth was not retarded following exposure to endosulfan, and apart from mucus secretion no macroscopically overt signs of pathology could be discerned during dissection.
Both the experimental and control fish were sacrificed every 15 days for 30 days. Immediately after decapitation the gills were removed and dropped into aqueous Bouin’s fluid. After fixation for 24 hours, tissues were dehydrated through a graded series of ethanol, cleared in xylene and infiltrated in the paraffin. 4-6 µm thick sections were cut on microtome and stained in Hematoxylin-Eosin. Pathological lesions were examined under optical microscope.
RESULTS AND DISCUSSION:
Endosulfan is a chlorinated hydrocarbon insecticide and acaricide of the cyclodiene subgroup which acts as a poison to a wide variety of insects and mites on contact. Although it may also be used as a wood preservative, it is used primarily on a wide variety of food crops including tea, coffee, fruits, and vegetables, as well as on rice, cereals, maize, sorghum or other grains (Extoxnet, 1996). It enters the air, water, and soil during its manufacture and use. It is often sprayed onto crops and the spray may travel long distances before it lands on crops, soil or water. Endosulfan on crops usually breaks down in a few weeks, but sticks to soil particles and may take years to completely break down. By hydrolysis, the expected half-lives for the alpha- and beta-endosulfan isomers were found to be 3.6 days and 1.7 days, respectively. Endosulfan does not dissolve easily in water. In surface water, it gets attached to soil particles floating in water or attached to soil at the bottom. It can build up in the bodies of animals that live in endosulfan-contaminated water (ATSDR, 2000). Endosulfan is moderately persistent in the soil environment with a reported average field half-life of 50 days.
The EPA recommends that the amount of endosulfan in rivers, lakes, and streams should not be more than 74 parts per billion (74 ppb). The Food and Drug Administration (FDA) allows no more than 24 parts per million (24 ppm) endosulfan on dried tea. EPA allows no more than 0.1 to 2 ppm endosulfan on other raw agricultural products. The commercial formulations of endosulfan are 1.88 times more toxic than its technical materials.
The histopathological changes were apparent in specimens exposed to endosulfan and were not observed in the control fish. After exposure, an excessive amount of mucous observed over the gills of live specimens. It has been reported that the stress caused by variations in the environment and pathologic agents induce the proliferation of mucous cells and increase secretion.
In the present study, after 15 days of exposure to 0.5 µg/L endosulfan, epithelial necrosis, hypertrophy of epithelial cells, rupture of gill epithelium, haemorrhage at primary lamellae, sloughing of the respiratory epithelium and hypertrophy of epithelial cells were noted. The lifting of the epithelium, oedema, epithelial necrosis, fusion of adjacent secondary lamellae, and haemorrhage at primary lamellae were observed in the gills of fish examined after 30 days of exposure to 0.5 µg/L.
Epithelial necrosis and rupture of gill epithelium are direct deleterious effects of the irritants. The fish’s defense responses are excessive mucus secretion. Lifting of the epithelium, lamellar fusion, and club-shaped lamellae could be protective in that it diminishes the amount of vulnerable gill surface area. The histopathological changes of gill can result in hypoxia, respiratory failure problems with ionic and acid-base balance.
Agency for Toxic Substances and Disease Registry [ATSDR]. (2000). Toxicological Profile for endosulfan . Update. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.
Extension Toxicology Network [EXTOXNET] (1996). Pesticide Information Profiles, files maintained and archived at Oregon State University.