Abstract

Endosulfan is an important environmental pollutant that is a pesticide of organochlorine. The 24, 48, 72 and 96h median lethal concentrations of endosulfan (35 EC) were assessed by an acute static renewal test. The sublethal concentration taken for the present study is one-tenth of the 96h LC₅₀ and fish were exposed to that concentration (1.69 ppb) for 30 days. The histopathological observations revealed the presence of catecholamine neurons in the optic tectum and severe depletion of the layer stratum grisum periventricular. An altered pattern of neurons as an “interrupted string of pearls” was noticed in the posterior tuberal nucleus. From the present study, we could conclude that endosulfan is a serious toxicant that even affects the vital organs like the brain of vertebrates. Histology of the spleen showed necrosis in red pulp, hyperplasia and exudate in white pulp and an increased number of melanomacrophage centers (MMC) of the exposed fish species. Based on the results of this study, it is concluded that endosulfan is highly toxic to edible freshwater fish Anabas testudineus. Subsequently, damage to the structures of cells and tissues and histopathology of target organs comprise a significant parameter to be considered in evaluating the toxic possibility of contaminations on living organisms in various environmental conditions and represent the time exposure to which organisms are subjected.

1. Introduction

Large scale application of pesticides, insecticides and other chemicals are drastically deteriorating the quality of our aquatic environment. The greater use of pesticides for high agricultural production and pesticide residues may constitute a significant source of contamination of environmental factors such as air, water and soil. Environmental health and ecosystem services are adversely affected by the misuse /overuse of pesticides. Aquatic microorganisms, invertebrates, plants and fish are badly affected by pesticides ^{1, 2, 3, 4}. Pesticide exposure is the primary occupational hazard among agriculturalists of developing countries, this leads to various health issues and environmental contamination associated with pesticide use ^{5, 6, 7, 8, 9}.

Organochlorines (OC) are a group of chlorinated compounds and persistent organic pollutants (POPs) with high persistence in the environment and are widely used as pesticides. For controlling, malaria and typhus Organochlorine pesticides were used in earlier days, but presently many countries banned the usage of OC compounds ¹⁰. The organochlorine pesticides pose a constant threat to the non-target organisms and are known to alter the behavior, growth, nutritional value, cellular functions and physiology of aquatic organisms ^{11, 12, 13}. In developing countries of Asia organochlorine insecticides such as DDT, hexachlorocyclohexane (HCH), endosulfan and dieldrin are among the most widely used pesticides due to their low cost and the need against various pests, ^{14, 15, 16}. Organochlorine toxicity is mainly due to stimulation of the central nervous system. Cyclodines, such as the GABA antagonists endosulfan and lindane, inhibit the calcium ion influx and Ca- and Mg-ATPase causing the release of neurotransmitters ¹⁷.

Pesticide residue from the agricultural field enters the nearby aquatic ecosystem through water runoff, where they seriously affect nontarget species in the aquatic environment. The bioaccumulation of pesticide residues in various trophic levels of the food chain, in turn, affects biodiversity and homeostasis ¹⁸. Changes in the surrounding environment such as an increase in pollution severely affect fishes since they are very sensitive to changes. The direct effect of chemical compounds within target organs of fish in laboratory experiments is well explained by histopathological investigations ¹⁹ and it provides valuable pieces of information about the health status of the organisms and also environment and period of exposure. This will also help us to access the potential for human exposure to the same environmental pollutants, and to predict human health risks. Hence, the present study was undertaken to determine the histopathological effects of sublethal concentration of endosulfan (35EC) on the brain and spleen of Anabas testudineus

2. Materials and Methods

Anabas testudineus ²⁰ are found mostly in canals, lakes, ponds, swamps and estuaries and also in areas with dense vegetation. The climbing perch is native to . They are considered as hardy fishes that can tide over extremely unfavorable conditions and are associated mainly with turbid, stagnant waters. During the dry season, they remain buried under mud. Anabas testudineus feed on macrophytic vegetation, shrimps and fish fry. This fish can tolerate low oxygen levels due to the presence of accessory respiratory organs. Using long robust spines on their gill covers they move over land (usually at night) between pools. Anabas testudineus is an edible fish of Kerala.

Irrespective of sex, healthy specimens of adult freshwater fish Anabas testudineus were collected from Akkulam lake, Thiruvananthapuram, Kerala, India. The fish weighing 45-50 g with a total length of 14-15 cm were brought to the laboratory and acclimatized to the laboratory conditions for 15 days in large glass aquaria with seasoned tap water. The average water quality parameters for the experiment were temperature 26.93 ± 2.16 PH 7.18 ± 0.05 and dissolved oxygen 7.92 ± 0.51 mg/L. The fish were fed daily 2-3 times with laboratory prepared food comprising rice bran, tapioca, fish meal, groundnut, oil cakes and an adequate amount of vitamins during acclimatization. To remove food debris and excretory waste routine cleaning of aquaria was performed every 48 h.

Before the experiment, all experimental glass aquaria were cleaned and disinfected with potassium permanganate. All glass aquaria were filled with 40 L of seasoned (chlorine-free) tap water. Three aquaria were maintained as control and three as experimental tanks for each concentration in the determination of LC₅₀ concentration. The average water quality parameters for the experiment were temperature 26.93 ± 2.16 PH 7.18 ±0.05 and dissolved oxygen 7.92 ± 0.51 mg/L. The fish were starved 24 h before the test and throughout the test. An acute static renewal test was conducted to determine the 24, 48, 72 and 96h LC₅₀ concentrations of endosulfan. Three replicates of each containing eight fish were exposed for each concentration (3, 5, 7, 9, 14, 16, 18, 20, 25, 28, 31, 35, 38 and 40 ppb) of endosulfan. A blank solution (00 ppb) in three replicates was used as a control. The control and test solution were renewed daily ²¹. One-tenth of 96h LC₅₀ was taken as sublethal concentration. For histopathological studies, fishes were exposed to sublethal concentrations for 30 days

Commercial grade endosulfan (35 EC) was used in the present study purchased from the local Agrochemical market, Madurai, Tamil Nadu. The required experimental concentration of pesticide was prepared by serial dilution of Commercial grade endosulfan (35 EC)

After the exposure period fishes were sacrificed (control and experiment) and tissue of choice (brain and spleen) were dissected pooled and fixed in neutral buffered formalin (NBF) for two days. This will harden and preserve the tissue by slowly penetrating the tissue. This will also protect it against subsequent processing steps. Fixed tissues were then taken out for dehydration by immersing specimens in a series of alcohol solutions of increasing concentration until pure, water-free alcohol is reached (70%, 90% and 100%) so that the water in the specimen is progressively replaced by the alcohol. The dehydrated tissues were cleared in methyl benzoate. Infiltration was done in two changes of wax for 1h each at 60°C (melting point of paraffin wax 58 - 60°C). Blocks were prepared and sections of 5µm thickness were cut on a rotary microtome. The sections were stained in hematoxylin-eosin following standard histological procedure. Stained sections were studied under a compound research microscope and photomicrographs were made using a photomicrography system.

LC₅₀ values of endosulfan at different exposure periods were determined by probit analysis by following a computerized statistical package namely SPSS 24. Upper and lower confidence limit and slope functions were also calculated.

3. Results

LC₅₀ values of endosulfan (35 EC) for Anabas testudineus at different exposure periods were determined and is presented in Figure 1 with the upper and lower confidence level.

The fish were exposed to a sublethal concentration of endosulfan for 30 days showed marked histological variations from the normal structure. The effect of endosulfan is predominantly seen on the midbrain of A. testudineus. The midbrain roof of the teleost contains structures involved in the sensory relay and integrative functions, and the midbrain tegmentum (optic tectum) contains a variety of cranial nerves and other nuclei. When A. testudineus was exposed to sublethal concentration for 30 days, the layer stratum grisum periventriculare and catecholamine neurons in the optic tectum were severely depleted (Figure 3). In the control group brain, histology was normal without any architectural variation (Figure 2). In the posterior tuberal nucleus, endosulfan caused an altered pattern of neurons defined as an “interrupted string of pearls” (Figure 5). Histology of control fishes show no alterations in cell structure (Figure 4).

The spleen is an important lymphomyeloid organ in teleosts and it is the only lymph node-like organ in teleosts. The splenic mass is composed of red and white pulp with very little connective tissue. The white pulp mainly consists of lymphocytes of different sizes and mature and developing plasma cells scattered around reticular cell processes. White pulp is also seen around scattered melanomacrophage centers. Macrophage centers are arrogated of closely packed histoenzymaticaly heterogenous macrophages that contain diverse inclusions, the most frequent being lipofuscin, melanin and haemosiderin. The red pulp is composed of sinusoids and it contains mature erythrocytes, reticular cells and macrophage. Sinusoids are the region where erythropoiesis and thrombopoiesis occur. Anabas testudineus exposed to sublethal concentration (1.691 ppb) of endosulfan (35 EC) for 30 days showed necrosis in the red pulp, hyperplasia and exudate in white pulp and an increased number of melanomacrophage centers (MMC) which were scattered throughout the spleen (Figure 7). In control groups, the spleen exhibited a typical histological structure except for melanomacrophage centers, which were less common and smaller than in pesticide treated fish (Figure 6).

4. Discussions

Toxicity of endosulfan (35 EC) on A. testudineus ²⁰ increased with increasing concentration and exposure periods. The test result of the LC₅₀ for 24, 48, 72 and 96h were 27.58, 23.35, 18.79 and 16.91 ppb respectively. These values show that endosulfan (35 EC) is a potent environment toxicant. Several workers have estimated the median lethal concentration (LC₅₀) of endosulfan for fish. In some of the other fish species already reported LC₅₀ values of endosulfan for 96 h were 1.5µg/L in rainbow trout, 1.4 µg/L fathead minnow ²² 7.75 ppb in Channa punctatus ²³ 41µg/L in Anguilla anguilla ²⁴ These findings indicate that the LC₅₀ values obtained in the present study are not exactly similar to some of earlier observations. These variations in toxicity may be due to species specificity of test chemical, age and stage in the life cycle of the species, the capacity of individual species to detoxify the compound, differences in test condition and factors of the medium such as temperature, dissolved oxygen, hardness, turbidity, P^H. Various studies indicate that hydrolyzation of endosulfan increases with a decrease in P^H, of less than 5. Hydrolyzation product endosulfan sulfate is more toxic than endosulfan ²⁵. According to ²⁶ LC₅₀ values of Rainbow trout are influenced not only by the size of the fish and temperature but also water qualities like hardness, alkalinity, temperature and P^H. Anabas testudineus is considered a hardy fish of the aquatic ecosystem. From the present study, it is interesting to find that this fish is one of the most sensitive freshwater fish to endosulfan (35 EC).

A. testudineus that were exposed to sublethal concentration of endosulfan for 30 days showed marked variations in the histology of the brain and spleen. In the present study, the brain histology of control fish was normal without any architectural alterations but the toxicity of endosulfan (35 EC) is very predominant in the histology of the brain of treated fish with depletion of catecholamine neurons in the optic tectum and layer stratum grisum periventriculare and caused an altered pattern of neurons in the posterior tuberal nucleus. Brain histology of spotted murrel, Channa punctatus after exposure to endosulfan for 96 hours showed mild necrosis and vacuolar changes ²⁷. Similar observations were also made by ²⁸ in the brain of toad Buffo regularis exposed to endosulfan and diazinon. Many studies with organochlorine pesticides reveal alteration in the reproductive behaviors in many teleost ^{29, 30} and it is mediated through cerebral neuromediating systems like histaminergic systems ³¹. Catla catla exposed to heavy metals observed degeneration of the nerve cells, cellular damage in the anterior and posterior regions of the brain, vacuolization and necrosis of the brain cells ^{32, 33}. Clarias gariepinus exposed to glyphosate herbicide showed degeneration of Purkinje neurons, the proliferation of glial cells, oedema and vacuolar changes in brain histology ³⁴. In Labeo rohita massive cerebral tissue degeneration was noticed when exposed to 0.001 mg/L endosulfan ³⁵. Reference ³⁶) opinioned that vacuolar changes in the parenchymal tissue at the lower dose and severe necrosis of neuronal cells of the cerebrum at higher dose on L. rohita were due to the neurotoxic effect of hexachlorocyclohexane.

In teleost, the spleen is primarily hemopoietic in function. Exposure of A. testudineus to a sublethal dose of endosulfan (35 EC) for 30 days caused serious alterations in the histology of the spleen such as, an increased number of melanomacrophage centers, necrosis in the red pulp, hyperplasia along with exudate in the white pulp. Earlier reports of toxicity associated with environmental contamination in the spleen of fish indicated an increase in the size of the spleen, haemosiderosis, white pulp proliferation, lymphocyte depletion and increase in melanomacrophage centers ³⁷. Oedema, cell necrosis and degeneration are examined in rainbow trout (Oncorhynchus mykiss) after exposure to different sublethal concentrations of carbosulfan, propineb and benomyl for 14 days ³⁸. In the spleen of rainbow trout, Oncorhynchus mykiss was exposed to endosulfan, necrosis and exudates in the white pulp and an increase in the number of melanomacrophage centers were observed ³⁹, which is in agreement with this study. As envisaged in the present research stressful conditions to the animal often result in the increased number of its splenic MMC’s ⁴⁰.

5. Conclusion

Histology is the direct, effective, economic and reliable method for studying ecotoxicology. Histopathological investigations on vital organs of fish are useful tools for toxicological studies and monitoring water pollutions. The histopathological studies performed in the brain and spleen of Anabas testudineus (Bloch 1792) exposed to a sublethal concentration of endosulfan (35 EC) for 30 days showed significant alterations from normal histological architecture. Thus histological investigation is a useful methodology for monitoring the long term effects of pesticides on fish. Damages to organs and tissue injury will affect the fitness, growth and survivability of fish. From the present study, it is clear that endosulfan (35 EC) is highly toxic to A. testudineus (Bloch, 1792) even at sublethal concentrations and the absence of mortality cannot guarantee the physiological health of the fish population. A screening program of pesticides based on their performance on impairing tissues of various organs is of credible necessity. When pesticides are applied in agriculture fields surrounding water sources these facts also should be taken into consideration.

References