Background

Although pesticide is beneficial for diseases and pest control but it also poses a harmful effect to our environment such as the pollution of pond water. Pesticides have been mentioned as the contributor of gradual degradation of the aquatic ecosystem (Konar, 1975).It is assumed that about 25% of the used pesticides drain off into open water bodies through rainfall and floods and the aquatic environment obviously gets polluted. The pollution hazards for aquatic life are increasing pointedly. Sometimes this pollution may cause sudden death of fishes and other aquatic organisms (Rahman and Alam, 1997). Due to natural and human induced phenomena, day by day a number of fish species is going to vulnerable, endangered and critically endangered situation. Among the indigenous fish species, 12 have been categorized as critically endangered, 28 as endangered and 14 as vulnerable (IUCN, 2015). Trichogaster fasciata is considered as least concern by IUCN (2015). One of the major causes of this situation is the indiscriminate use of various insecticides and pesticides in the agricultural sectors.

The organochlorines pesticides are DDT, Eldrin, Chloroden, Dieldren, Heptachlore etc. In general organochlorine compounds are more toxic than other groups of 3 pesticides to fish. Durability of its action in water is about 3-15 years. Activities of nervous system are paralyzed by the effects of these types of pesticides. Convulsion of muscle occurs and animals loss their balance of Na and K in neuron (Sachsen and Sultana, 1999). Carbamates viz. Carbaryl, Marshal are moderately toxic to fish but highly toxic to other aquatic invertebrates. The biological activities of these pesticides are very similar to organophosphorus. Pyrethroids pesticides are similar to organochlorine but degraded promptly, within 7-30 days Organophosphorus insecticides viz. Diazinon, Nogos, Sumithion, Basudin, Malathion, Darsban etc. inhibit the enzymatic (Cholinesterase) activity of blood (Amin et al., 2013). Convulsion of muscles, paralysis and damage of respiratory system also occur due to the effects of organophosphorus pesticides.

Pesticides at high concentrations are known to reduce the survival, growth and reproduction of fish (Rathore and Nollet, 2012). Due to the residual effects of pesticides, important organs like gonads, kidney, liver, gills, stomach, brain and muscle are damaged. In Bangladesh, more than 300 types of insectides and pesticides are used for crop protection in agriculture. Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including non target species, air, water, bottom sediments, and food (Miller and Miller, 2004).

Chlorpyrifos (IUPAC name: O,O-diethyl O-3,5,6-trichloropyridin-2yl phosphorothioate) is a crystalline organophosphate insecticide, acaracide and miticide (Figure 1). It was introduced in 1965 by Dow Chemical Company and is known by many trade names including Dursban and Lorsban. It acts on the nervous system of insects by inhibiting acetylcholinesterase.

Chlorpyrifos is moderately toxic to humans, and exposure has been linked to neurological effects, persistent developmental disorders and autoimmune disorders. According to Dow, chlorpyrifos is registered for use in nearly 100 countries and is annually applied to approximately 8.5 million acres. The crops with the most use are cotton, corn, almonds and fruit trees including oranges, bananas and apples. In agriculture, chlorpyrifos is commonly used as a foliar spray, or applied directly to soil and incorporated into it before planting. Exposure to sub-lethal concentrations of chlorpyrifos has caused the severe effects in species of freshwater and marine fauna: ataxia, delayed maturation, growth and reproduction impairment, deformities and depressed populations (Marshall and Roberts, 1978).

Trichogaster fasciata, the stripped gourami, is a tropical labyrinth fish native to Bangladesh (Figure 2). It is bentho pelagic and prefers weedy environments such as ponds, large rivers, ditches, lakes and rice fields. Size of a fully grown male can be up to 12 centimeters (4.7 inches). Females are usually a little smaller and are used for food in its natural habitat. The natural resources of this species are declining fast, especially in Bangladesh, due to drastic reduction of its natural feeding and breeding ground as a result of human intervention, climate change and habitats modification. At present, T. fasciata under ‘lower risk near threatened’ as least concern category although is not listed in the IUCN Red Data Book 2015. To date, so far no study has been carried out describing the potential toxic effects of chlorpyrifos on the reproduction of striped gourami. Therefore, the present study was conducted to evaluate the physiological toxicity of striped gourami induced by chlorpyrifos through the histopathological alterations of gills.

The major objectives of the present research work was to investigate the effect of chloropyrifos on the histo-architecture of gills of stripped gourami as well as to find out the histopathological responses of the gills in stripped gourami exposed to various concentrations of chlorpyrifos. Further, the potential health risks to consumer due to the intake of this chloropyrifos contaminated fishes.

1 Materials and Methods

1.1 Experimental site

Experiments were conducted at the Wet Laboratory and Fish Conservation Laboratory under the Department of Fisheries Management of the Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh. Active and healthy specimens of striped gourami fish (Trichogaster fasciata) were randomly collected and stocked in the cisterns of Mini hatchery and Breeding complex located at the southern side of the Fisheries Faculty building. The size of each cistern was 250 cm × 195 cm × 70 cm. Aeration was maintained and the fishes were kept under natural photoperiod. Commercial fish feed were fed twice daily to satiation.

1.2 Experimental design for gills histology

After acclimatization in the cisterns the fishes were exposed to different concentrations of chlorpyrifos 20 EC for different times to find the LC₅₀ value and to identify the histopathological changes of gills. Using Probit-Analysis LC₅₀ of chlorpyrifos for striped gourami was determined. There are five sub-lethal doses, such as 0, 15, 50, 150 and 500 µg/L of chlorpyrifos which were denoted as T_c (control), T₁₅, T₅₀, T₁₅₀, and T₅₀₀ respectively. There are five treatments and 3 replications in each treatment. Each of the concentrations and control group was maintained in triplicates. To conduct this experiment, 15 PVC tanks (86 cm diameter, 81 cm depth) were used and cleaned with disinfectant, washed thoroughly with ground water. After that each of the tanks was filled with 300 L of dechlorinated ground water. Then 10 female (length 7.97 ± 0.72 cm and weight 9.76 ± 2.31 g) fishes were kept in each of the 15 tanks which were identified by their silvery body color and swollen abdomen. Sub-lethal doses were applied according to LC₅₀ of the chlorpyrifos which was estimated for adult striped gourami. After 21 days of conditioning, the sub-lethal concentrations were exposed to 15 tanks for 15, 30, 45, 60 and 75 days. About 90% of tank water was exchanged every alternate day and fresh chlorpyrifos was used. Fishes were feed twice daily and excess food and excretion were removed through siphoning every day. After 15 days interval, female fishes were sampled regularly. Total length and body weight of each fish was determined using a specialized scale.

1.3 Histology of gills

The collected fish were taken on a tray and operculum was cut carefully by scissor and gills were exposed. Gills were removed with the help of scissor and placed on a petridish. Then the samples were preserved in 10% neutral buffered formalin for further analysis. The fixed gills were passed through graded alcohol series to dehydrate them. The dehydrated gills samples were taken for clearing with 100% benzene for 2 times with an interval of 1 hr at each step. The samples were embedded into paraffin. Sectioning was done using microtome. The gill sections were then stained for 2 times with haematoxylene and eosin stains. Finally the gills sections were observed under microscope.

1.4 Microscopic observations

The slides were observed under electric microscope (Olympus) which was connected to computer with a viewer (Magnus viewer). The viewer was also equipped with a camera. By the help of this mechanism numerous photographs were snapped at different magnifications.

2 Results

Histopathological results indicated that gill was the primary target tissue affected by chlorpyrifos. The gills are key organs involved in nutrient uptake, ingestion and respiration. The gill tissue of Trichogaster fasciata in general consists of well structured primary and secondary gill lamellae. This is evident in gill tissues not exposed to chloropyrifos (control). Continuous exposure to the chlorpyrifos caused architectural distortion of the gill tissues of the exposed fish as shown in figures (sub lethal concentrations). No histological changes were observed in the gill of the control fish (0 μg/L). The most common changes occurred in a range of 50 μg/L-150 μg/L concentrations of chlorpyrifos were hypertrophy, necrosis, missing of gill lamellae, hemorrhage, vacuum, pyknotic cells and splitted of gill lamellae.

The gills exhibited chloropyrifos induced changes on the 15^th day exposure (Figure 3). Almost normal structure appeared when exposed to 15 μg/L. But significant structural change like hypertrophy was observed at 50 μg/L concentration. Notable structural changes occurred at a concentration of chlorpyrifos 150 μg/L like necrosis, missing of gill lamellae. Chlorpyrifos induced alterations at 45 days in histoarchitecture of the gills were very significant (Figure 4). Control showed normal structure but changes like hypertrophy, hemorrhage, vacuums and necrosis were observed after exposure at different concentration of chlorpyrifos (15 μg/L, 50 μg/L, 150 μg/L). The 96-h LC₅₀ of chlorpyrifos for this species was found to be 880 µg/L.

Greater damage of gills was observed after 60 days exposure of chlorpyrifos like the cells of gills got thickness and were reduced into a dense solid mass (pyknosis), increased in size of cells (hypertrophy), escaped of blood from blood vessels and occurred in the membrane, skin and vessels (hemorrhage), circumscribed death of cells with black structural evidence (necrosis) at different concentrations of chlorpyrifos (Figure 5).

A notable observation with respect to gills damage was observed after 75 days exposure of chlorpyrifos was that, the gills exposed to the 15 µg/L, 50 µg/L, 150 µg/L concentrations exhibited greater distortion of gills architechtecture than the 15, 45 and 60 days exposure. Concentration 500 μg/L of chlorpyrifos showed lethal effect to Trichogaster fasciata (Figure 6).

All the histopathogical observations indicated that exposure to sublethal concentrations of chlorpyrifos caused destructive effect in the gills of Trichogaster fasciata. Gill histopathogical alterations, such as those observed in this study could result in severe physiological problems which ultimately leads to the death of fish. The findings of the present histological investigations demonstrated a direct correlation between chlorpyrifos exposure and histopathological disorders observed in gills.

3 Discussion

Histopathological observation is a sensitive bio-monitoring tool in toxicant impact assessment to indicate the effect of toxicants on fish in pesticides polluted aquatic ecosystems (Marchand et al., 2009). Pesticides in polluted aquatic ecosystem are accumulated mainly in the metabolically active tissues of fish such as liver, kidney, gonads and gill (Oruce and Usta, 2007) and cause histopathological damage of those organs. Trichogaster fasciata was exposed to different sub-lethal concentrations. Four sub-lethal concentrations (15, 50, 150 and 500 μg/L) of chlorpyrifos based on previously estimated LC₅₀ of 880 μg/L were used in this study for 75 days.

The histopathological changes in gills of common carp (C. carpio) exposed to organophosphate pesticide, malathion at 1.5 and 3.0 ppm was observed by Sharmin (2014). Several morphological changes were seen in the gills of fish exposed to malathion. The gills of fish exposed to low dose (1.5 ppm) showed Telangiectasia and Blood Lamellar congestion while Telangiectasia, Blood Lamellar Congestion, Hypertrophy of filaments, Lamellar Fusion observed in the gills of fish exposed to high concentration (3.0 ppm).

However, at the higher tested concentrations of chlorpyrifos 20 EC viz., 50 and 150 μg/L marked degenerative changes, severe necrosis, pyknosis, haemorrhage and vacuolation were observed in all the tested fish species which agreed with the finding of Rahman et al. (2002) and Omitoyin et al. (1999). All of the fishes died within 5 days when exposed to 500 μg/L.

Navaraj and Yasmin (2012) investigated the impact of tannery industry waste water on O. mossambicus. The histopathological changes observed in gills, liver, kidney and brain of the studied fish have demonstrated the quality status of industrial wastewater. In the vital organs, the following marked changes were observed: filament cell proliferation, cellular infiltration, haemorrhage and epithelial lifting in gills, vaculation of hepatocytes and necrosis in liver, exfoliation and swollen with pyknotic nuclei in kidney and enlarged pyramidal cells, binucleated nuclei, vaculation, and necrosis in brain.

To determine the histopathological effects of gammalin 20 on African catfish (C. gariepinus) an experiment was conducted by Lawrence and Tamiotan (2010). The 96 hrs lethal concentration (LC₅₀) value was 30 ppm. Histopathological changes of the gill, liver, and intestinal tissues of fish treated with sub lethal concentrations of gammalin 20 for twelve weeks showed gill distortion and fusion of adjacent secondary lamella as a result of hyperplasia and excessive mucus accumulation. The liver showed swelling of hepatocytes with mild necrosis, pyknosis, and vacuolation, while the intestine showed yellow bodies of the lamina propria at the tip of the mucosal fold.

Degenerative changes in gill, such as intraepithelial edema in the secondary lamellae, thick coating of mucus covering the entire gill filaments and lamellae, erosion of secondary lamellae, thickening of lamellae, inflammation of epithelial cells, breakages in primary lamellae, degeneration of secondary lamellae, necrosis, rupture of epithelium were noticed during exposure of sublethal concentrations of monocrotophos by Rao et al. (2005). Histopathological changes observed were hemorrhage in the primary and secondary gill lamellae, degeneration and necrosis of epithelial cells, distortion of the secondary lamellae, disruption of epithelial cells from pillar cells, shorter gill lamellae, fusion, complete destruction of lamella, increased vacuolation, irregular appearance of gill lamellae were observed in guppy Poecilia reticulate exposed to chlorpyrifos (De Silva and Samayawardhena, 2002).

However, the result of the present study revealed that chlorpyrifos 20 EC is toxic to fish and causes histopathological changes in fish organs. The LC₅₀ values recorded in this study were very lower which indicated that T. fasciata was quite susceptible to chlorpyrifos and its mortality increased proportionally at higher doses. In case of histopathological study, it was observed that at higher doses of chlorpyrifos 20 EC, gills of T. fasciata were severely affected which was responsible for the death of the fish. Therefore, indiscriminate use of chlorpyrifos 20 EC by farmers should be discouraged particularly in an area closed to aquatic environment.

4 Conclusion

The research findings provided a significant understanding of the pesticidal toxicity of crop insecticide chlorpyrifos 20 EC to the development and physiology of T. fasciata. It is lend a hand the policy makers to make people conscious about the impact of insecticides used in crop production indiscriminately on normal physiological development of fish and other aquatic organisms. The research findings are very much effective to find a safety level of using the pesticide in crop lands in Bangladesh. Therefore, illegal use of pesticide in agricultural land should be reduced to prevent exposure of pesticide in commercial aquatic resources.

Authors’ contributions

Mukti S.S. has conducted this research as a part of her Master of Science Degree, Ahmed G.U., Ahmed Z.F., Sumon K.A. and Fatema M.K jointly facilitated the histopathological study in Diseases Laboratory under the Department of Aquaculture and Aquatic Conservation Laboratory under the Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh. Fatema M.K. and Ahmed G.U. are Supervisor and Co-Supervisor of this student, respectively. All authors read and approved the final manuscript.

Acknowledgements

We thank all staffs of Diseases Laboratory and Aquatic Conservation Laboratory of Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh for technical support.

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