Histopathology of Rainbow Trout Exposed to Sublethal Concentrations of Methiocarb or Endosulfan

Abstract

Liver, spleen, trunk kidney, gills, and brain of rainbow trout (Oncorhynchus mykiss) were examined histologically after exposure to different concentrations of methiocarb (2.5 and 3.75 mg/L) or endosulfan (0.6 and 1.3 μg/L) for 21 days. Histological recovery was also studied by maintaining the pesticide-exposed fish in a freshwater system for an additional 30 d. Lesions were not evident in liver, kidney, spleen, or brain of fish exposed to either concentration of methiocarb for 21 d. Lesions were observed in gills, liver, spleen, and trunk kidney (but not brain) of rainbow trout exposed to either concentration of endosulfan. There was no concentration-related effect observed on the histopathological lesions. After 30 days of recovery, fish had no histological lesions in gills, kidney, spleen, liver, or brain. Therefore all the changes observed during exposure were reversible.

Introduction

Endosulfan is a chlorinated hydrocarbon used as a broad spectrum insecticide which controls insects by contact action and by ingestion of Thiodan-treated plant material (http://www.bayercropscience.com/). Endosulfan is the only chlorinated hydrocarbon insecticide widely used in the world. Endosulfan is applied to treat a wide variety of food crops including tea, hazelnut, fruits, as well as cereals, maize and other grains (Bayer Crop Science AG, Frankfurt Germany). Methiocarb, a carbamate pesticide, has been used since the 1960s as pesticide for a variety of invertebrate pests and also used as a bird repellent on fruit crops (Hayes et al., 1991). Methiocarb is one of the most frequently used pesticides to control Balaninus nucum, Palomena prasina, Lymantria dispar, Xyleborus dispar, Agelastica alni, and Obera linearis (Bayer Crop Science AG, Frankfurt Germany).

Other than targeted pests, pesticides affect a wide range of nontarget organisms, such as invertebrates and fish inhabiting aquatic environment (Burkepile et al., 2000). Agricultural runoff of rain and irrigation water introduces pesticides into the aquatic environment, where it poses significant toxicological risks to resident organisms (Scott et al., 1990). Pesticide pollution severely affects aquatic organisms and, in turn, the entire food chain including human beings (Svensson et al. 1994). Although toxicity mechanisms of endosulfan and methiocarb were extensively studied (Davis and Wedemeyer, 1971; Hayes and Laws, 1991; Hayes et al. 1991; Naqvi and Vaishnavi, 1993; Hallenbeck and Cunningham-Burns, 1995; Ecobichon 1996; Hoffman et al. 1996; Kamijima and Casida, 2000), limited work has been done on the histopathological effects of pesticides on fishes. Histopathological studies have been conducted to help establish causal relationships between contaminant exposure and various biological responses. Histopathological investigations have proved to be a sensitive tool to detect direct effects of chemical compounds within target organs of fish in laboratory experiments (Schwaiger et al., 1996). This study was aimed to determine the histopathological effects of sublethal concentrations of endosulfan and methiocarb to rainbow trout gills, liver, spleen, brain, and kidney.

Materials and Methods

Fish

Juvenile rainbow trout Oncorhynchus mykiss (2.1 ± 0.1 g; 6.0 ± 0.3 cm; Mean ± SD) were obtained from Karadeniz Technical University, Faculty of Marine Sciences and held in 2 closed recirculating systems (200 L) for at least 15 days to acclimate to laboratory conditions prior to experiments. During the acclimation period, about 40% of the water in each recirculating system was replaced daily. Throughout the acclimation period and subsequent periods of endosulfan exposure, fish were held under a photoperiod of 12 hours of light and 12 hours of darkness. During acclimation and toxicity tests, fish were fed 5% and 2.5% body weight twice a day with commercial trout pellets, respectively.

Water Quality

During exposure to the insecticides, spring water was used and its characteristics in each treatment were measured daily. Total ammonia was measured by an indophenol method, and nitrite was measured by an azo method (Boyd and Tucker, 1992). Total hardness and total alkalinity were measured by the titration method (Boyd and Tucker, 1992). Dissolved oxygen concentration was measured by the Winkler method (Boyd and Tucker, 1992). Water temperature and pH were determined with a glass electrode (Thermo Orion, Beverly, Massachusetts, USA).

Experimental Design

Experiment was conducted as described by Altinok et al. (2006). Briefly, the fish were determined to be free of external parasites prior to the exposure (AFS-FHS, 2003). After acclimation, fish from one of the acclimation tanks were randomly transferred to one of 20 glass aquaria containing 25 L of static water. Quadruplicate random groups of 10 fish each were subjected to experimentation for 21 days in static fresh water containing 2.5 or 3.75 mg/L methiocarb or 0.6 or 1.3 μg/L endosulfan. Doses selected were based on survivability responses assessed earlier (Altinok et al., 2006; Capkin et al., 2006). Test solutions of endosulfan (applied as Thiodan) and methiocarb (applied as Mesurol) were prepared from commercial formulations containing 32.9% and 50% active ingredient (Bayer Crop Science), respectively. Quadruplicate aquaria were designated for each concentration of methiocarb and endosulfan. The pesticides were dissolved in 100 ml distilled water and then added in aquaria. Control tanks received 100 ml distilled water. This study was conducted under OECD Guideline No. 203 for static-renewal test conditions (OECD, 1992). Sixty-five percent of the test solution was renewed each day. During the toxicity experiment, water in each aquarium was aerated and had the following characteristics: dissolved oxygen 8.01 ± 0.25 mg/L, temperature 15.2 ± 0.4°C, pH 7.56 ± 0.24, total hardness 32.0 ± 1.9 mg/L as CaCO₃, alkalinity 17.2 ± 0.6 mg/L as CaCO₃, un-ionized ammonia 6.1 ± 2.2 ng/L and nitrite 2.2 ± 0.9 μg/L.

At the end of the 21 days of sublethal toxicity tests, fish were transferred to flow-through tanks to observe further effects of methiocarb and endosulfan for during 30 days. At the end of the 30 days of recovery, 20 fish were sampled for histology as described in the histopathology section.

Histopathology

After 21 days of exposure, 5 fish from the experimental and control groups were anesthetized with overdose of MS222 and then the second gill arch, liver, spleen, trunk kidney, and head were carefully removed and preserved in 10% neutral-buffered formalin (NBF) for 48 hours. The same organs were taken from rest of the fish after the 30-day recovery period. Organs were rinsed in 4 changes of 70% ethanol (ETOH), and stored in 70% ETOH until further processing. They were dehydrated in isopropanol, cleared in xylene, infiltrated in paraffin, and sectioned at a thickness of 5 μm. Sections were stained with hematoxylin and eosin (Luna, 1968), and examined with a light microscope.

Results

No fish died during the acclimation period before endosulfan and methiocarb exposure, and no fish died during toxicity tests. Fish exposed to sublethal concentrations of endosulfan and methiocarb did not show any behavioral abnormality compared to control groups.

Histopathological changes were not evident in liver, kidney, spleen or brain of fish exposed to sublethal concentration of methiocarb for 21 days. In the higher methiocarb dose group, the only lesion observed was in the gill, characterized by seperation of epithelium from gill lamellae and the space under the epithelium filled with eosinophilic material (5/10, incidence of change was 5 of 10 fish evaluated) (Figure 1).

Histological lesions were observed in gills, liver, spleen, and trunk kidney of rainbow trout exposed to endosulfan. The lesions observed in the gills of fish exposed to 0.6 or 1.3 μg/L endosulfan concentration consisted primarily of epithelial lifting (7/10), which is characterized by a lifting of the outer layer of the lamellar epithelium with the space under the epithelium filled with eosinophilic material (Figure 2a). Hyperplasia (5/10) was present as an increased number of epithelial cells at the distal or basal portions (Figure 2a). Endosulfan exposed fish gills also had hypertrophy of epithelial cells (6/10) on the lamellae (Figure 2b), fusion (Figure 2c) of two or more lamellae (6/10), and epithelial necrosis (10/10) (Figure 2d).

The trunk kidney of fish exposed to endosulfan had enlarged sinusoids within an apparently decreased amount of hematopoietic tissue (8/10) (Figure 3b). Some nephrons had occluded glomerular capillaries and separation of the renal tubular epithelium from the surrounding connective tissue (7/10) (Figure 3c). Necrosis was present in hematopoietic tissue (8/10), glomerular cells (7/10), and tubular cells (7/10). Glomeruli had eosinophilic exudate (10/10) (Figures 3b, 3c). The liver had a low number of necrotic hepatocytes and enlarged hepatic perisinusoidal areas containing eosinophilic material (8/10). Vacuolar dystrophy of hepatocytes (7/10) and hypertrophy of hepatocytes (5/10) were also observed (Figures 4b, 4c). Melanomacrophage centers (MMC) were scattered throughout spleen (9/10) (Figure 5b). Exudate (7/10) and necrosis (9/10) in the splenic white pulp were observed (Figure 5c). Histopathological lesions explained above were not seen in control fish except for MMCs, which were less common and smaller than in the endosulfan-exposed fish. There was no lesion observed in the brain and no concentration-related effect observed on the histopathological lesions.

The fish that survived the 21-day endosulfan and methiocarb toxicity tests and were then transferred to other flow-through tanks for observations did not have any abnormal behavior. After histopathological examinations of survivors, fish had no lesions in gills, kidney, spleen, liver, or brain.

Discussion

Histopathology provides a rapid method to detect effects of irritants in various organs (Johnson et al., 1993). The exposure of fish to chemical contaminants is likely to induce a number of lesions in different organs (Bucke et al., 1996). Gills (Poleksic et al., 1994), kidney (Bucher and Hofer, 1993), and liver (ICES, 1997) are suitable organs for histological examination in order to determine the effect of pollution. The exposure of aquatic organisms to very low levels or sublethal concentration of pesticides in their environment may result in various biochemical, physiological, and histological alterations in vital tissues (Anbu and Ramaswamy, 1991; Geraldine et al., 1999). We found that some of lesions were related to exposure to endosulfan and methiocarb. Lamellar lifting was observed after exposure to 3.75 mg/L methiocarb but not after exposure to 2.5 mg/L methiocarb. Furthermore, lamellar fusion was only observed in fish exposed to 1.3 μg/L endosulfan. Increasing endosulfan concentrations from sublethal to acute, melanomacrophage centers were scattered throughout the trunk kidney, head kidney, and spleen (Capkin et al., 2006). Also increasing methiocarb concentrations caused telangiectasis and necrosis between the molecular and granular layers of the cerebellum where Purkinje cells are located (Altinok et al., 2006). However, correlation of endosulfan and methiocarb concentrations and histopathological changes were weak in fish exposed to sublethal concentrations of these insecticides. Similar findings were also observed by Dalela et al. (1979). Tricklebank (2001) found the same result when damselfish, Parma microlepis, was exposed to aldrin and dieldrin.

Toxic substances can injure gills, thus reducing the oxygen consumption and disrupting the osmoregulatory function of aquatic organisms (Ghate and Mulherkar, 1979; Saravana et al., 2000). In the present study, exposure of rainbow trout to endosulfan resulted in structural alterations of the gill lamellae including edema, separation of epithelium from lamellae, lamellar fusion, and swelling of the epithelial cells. Similar findings were also observed by Sunitha and Sahai (1983), Roy and Munshi (1991), and Saravana et al. (2000). The results of these studies clearly indicate that sublethal concentration of endosulfan has diverse effects on fish gills. Gill lesions (edema with lifting of lamellar epithelium and hyperplasia of lamellar epithelium) found in the present study suggest that endosulfan causes injury to the gills and increases respiratory diffusion distance (Nowak, 1992). In the present study, gills were found to be the most seriously affected organs compared to liver, spleen, trunk kidney, and brain, perhaps because of the direct contact with the compound.

The liver has the ability to degrade toxic compounds, but its regulating mechanisms can be overwhelmed by elevated concentrations of these compounds, and could subsequently result in structural damage (Bruslé et al., 1996). In the present study endosulfan exposed fish had decreased amount of hematopoietic tissue in the trunk kidney, necrosis in hematopoietic tissue, glomerular cells, and tubular cells. Also the liver had necrosis and hypertrophy. Similar to our findings Hallenbeck and Cunningham-Burns (1995) and Choudhary et al. (2003) found that organochlorines had behavioral disturbances and hepatic and renal injury when fish were exposed to sublethal concentrations of endosulfan. Similar observations have been made in the whitefish (Coregonus clupeaformis) exposed to nickel (Ptashynski et al. 2001) and in lake trout (Salvelinus namaycush) exposed to lindane (Gill et al., 1988). In the present study, the concentrations of methiocarb tested did not cause any histological lesions in liver, spleen, brain, and trunk kidney of rainbow trout.