Fenthion induced toxicity and histopathological changes in gill tissue of freshwater African catfish, Clarias gariepinus (Burchell, 1822)

1 Department of Biological Sciences, Federal University Lokoja, Kogi State, Nigeria. 2 Department of Zoology and Environmental Biology, University of Nigeria Nsukka, Enugu State, Nigeria. 3 Department of Plant Science and Biotechnology, University of Nigeria Nsukka, Enugu State, Nigeria. 4 Department of Crop Science and Land Scape Management, Ebonyi State University Abakaliki, Ebonyi State, Nigeria. 5 Department of Vocational Teacher Education, University of Nigeria Nsukka, Enugu State, Nigeria. 6 Department of Agricultural Economics/CDR, University of Nigeria Nsukka, Enugu State, Nigeria.


INTRODUCTION
Organic insecticide poisoning remains one of the major health issues in both developing and developed communities (Peter and Cherian, 2000). A great propor-tion of acute poisoning cases are caused by exposure to pesticides, especially organophosphate (OP) compounds. The primary mechanism of action of OP pesticides is *Corresponding author. E-mail: didigwunwani@yahoo.com, chris.nwani@unn.edu.ng. Tel: +2348037509910.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License based on inhibition of the acetylcholinesterase (AChE) enzyme (Eto, 1979). Once AChE has been inactivated, acetylcholine (ACh) accumulates throughout the nervous system, resulting in overstimulation of muscarinic and nicotinic receptors. Fenthion (O, phenyl] phosphorothioate) is a systemic OP insecticide widely used in both agricultural and urban areas for controlling many sucking and biting pests (Cong et al., 2009;Sevgiler and Uner, 2010). Like all organophosphate insecticides, fenthion acts on the nervous system as inhibitor of acetylcholinesterase (AchE), an enzyme that hydrolyzes acetylcholine (Ach). Acetylcholine is a molecule that is involved in the transmission of nervous signals from nerves to muscles and between neurons in the brain (Jungera, 2014). Due to fenthion toxicity, the Environmental Protection Agency (EPA) has classified it as restricted use pesticide (RUP) that warrants special handling (ATSDR, 2005). Large amounts of pesticides find their ways into water bodies due to repeated application for the control of pest. The indiscriminate use of pesticides, careless handling, accidental spillage or discharge into natural water ways have harmful effects on fish population and other aquatic organisms and may contribute to long term effects (Nwani et al., 2013b).
Histopathological studies help to establish causal relations between contaminant exposure and various biological responses (Boran et al., 2012) and have proven to be a sensitive tool used in detecting direct effects of chemical compounds within target organs of fish in laboratory experiments (Altinok and Capkin, 2007;Capkin et al., 2009). A study on histopathology provides very important and useful data concerning changes in cellular or sub cellular structure of an organ much earlier than external notification. One of the advantages of using histopathological biomarkers in environmental studies is that it allows the examination of target organs (Gernhofer et al., 2001) and the alterations found in these organs are easier to identify than functional ones . These alterations serve as warning signs of damage to the well-being of an organism.
Research has demonstrated that fenthion induced DNA damage and should be considered potentially hazardous to humans (Wu et al., 2011). Recent research by Kanter and Celik (2012) revealed that exposure of frogs to fenthion induced an increase in melondialdehyde (MDA) and antioxidant defense systems. The effect of fenthion on biochemical changes in Cirrhina mrigala (Israel and Sam, 2012), liver function and pigmentation (Muralidharan and Pillai, 2012) in Cyprinus carpio have been reported. Studies have demonstrated that exposure of fish to different pesticides resulted to histopathological changes in the gill, liver and kidney tissues (Auta, 2001;Maduenho and Martinez, 2008;Capkin and Altinok, 2013). The fish species Clarias gariepinus is an attractive model for ecotoxicological studies because of its hardiness in culture, omnivorous feeding habits and availability throughout the year. In spite of the wide use of fenthion in Nigeria, there is however paucity of information on its effects on indigenous fish species. The present study was thus aimed at determination of the lethal concentration, behavioral effects and histopathological changes in gill tissues of freshwater African catfish, C. gariepinus exposed to fenthion.

Experimental fish specimen and chemical
Freshwater African catfish C. gariepinus (Burchell 1822) (Family: Clariidae, Order: Siluriformes), mean standard length of 27.36 ± 0.23 cm and weight of 197.39 ± 2.34 g were procured from Freedom Fisheries Ltd Nsukka, Enugu, Nigeria and transported to our wet laboratory where they were treated with 0.05% potassium permanganate (KMnO4) for 2 min to avoid any dermal infections. The fish were acclimatized for three weeks in plastic tanks of 300 L capacity and fed ad libitum daily with commercial available food (Coppens International Helmond Netherlands) containing 35% crude protein. To maintain hygienic condition and prevent pollution caused by food and feces, fecal matter and other waste materials were siphoned off daily. Dead fish were removed with plastic forceps to avoid possible deterioration of water quality. During the period of acclimation, the water in the tanks was renewed daily with well aerated tap water. The feeding was terminated 24 h prior to the range finding and acute toxicity test to avoid interference of feces.
The mean water quality of the test water analyzed daily during the experimental period following the standard methods (APHA, AWWA, WPCE, 2005) had the following physicochemical characteristics: temperature (24.40°C; SE 0.56), dissolved oxygen (6.77 mg/L; SE 0.80), conductivity (285 Μm/cm; SE 6.50), pH (7.22; SE 0.60), and total hardness (as CaCO3 -240 mg/L; SE 6.60). For the present study, commercial formulation of fenthion manufactured by Yufull Industry Co, Ltd., China containing 600 g/L fenthion was purchased from the local market and used.

Acute toxicity bioassay and behavioral responses
Acute toxicity bioassay to determine the 96 h LC50 values of fenthion was conducted using standard methods (APHA, AWWA, WPCF, 2005). The range finding test was carried out before determining the concentrations of the test chemical for the definitive test. For the definitive test, a set of 10 fish were randomly exposed to fenthion concentrations of 12, 24, 36, 48, 60 and 72 mg/L derived from a range finding test using plastic aquaria (60 × 30 × 30 cm size) containing 25 L aerated tap water. Another set of 10 fish was simultaneously maintained in equal amount of tap water but without the test insecticide and considered as the control. The experiments were set in triplicates to obtain the LC50 values of the test chemical. Fish were not fed throughout the experiment and lethality was the toxicity end point. Fish were visually examined daily and considered dead when no sudden swimming in response to gentle touch was observed. Dead fish were removed with plastic forceps and the mortality was recorded at intervals of 24, 48, 72 and 96 h. The LC10-90 values of the test insecticide for the fish at 24, 48, 72 and 96 h was determined by probit analysis (Finney, 1971). During the acute toxicity test, the no observed effect concentration (NOEC) was determined as the highest concentration in which no mortality was observed while lowest observed effect concentration (LOEC) was also determined using acute toxicity results. The safe levels of the test pesticide was estimated by multiplying the 96 h LC50 with  different application factors (AF) and were based on the methods of Hart et al., (1948), Sprague (1971), CWQC (1972), NAS/NAE (1973), IJC (1977), and CCREM (1991). The behavioral responses of Clarias gariepinus at different concentrations of fenthion were observed from 24 to 96 h of the exposure.

Sublethal concentrations and tissue preparation
The 96 h LC50 value of fenthion in the present study was determined to be 39.97 mg/L. Based on this value, three sublethal concentrations of 2.0, 4.0 and 8.0 mg/L corresponding to 1/20, 1/10 and 1/5 th of fenthion, respectively were prepared by serial dilution of the stock solution. A total of 30 acclimatized fish were exposed to each of the aforementioned sublethal test concentrations in triplicates of 10 fish per replicate. Control fish specimens were maintained in dechlorinated tap water without fenthion. The exposure lasted for 21 days after which the fish were withdrawn from the insecticide and kept for seven days in dechlorinated tap water for possible recovery. To avoid catabolism due to starvation during the chronic exposure, the fish were fed daily small quantity of food (approximately 1% of total body weight) an hour before renewal of the test solution. On day 1, 7, 14, 21 and 7 days recovery, one fish from each replicate treatment group and control was sacrificed after anesthetizing with tricaine methanesulfonate (MS 222) to minimize stress. The fish were dissected and gill tissues were removed, preserved in 10% phosphate buffered formalin for 24 h, dehydrated by a series of upgraded ethanol solution, embedded in paraffin, and sectioned at 5 μm thick. A total of three tissue sections per fish for each replicate concentration were routinely processed and stained with Hematoxylin and Eosin (H & E) and examined by light microscopy according to Roberts (2001). Photomicrographs were then taken at (x100) objective and the fish of the control groups were compared with that of exposed groups under the guidance of a pathologist.

Statistical analysis
The data obtained were statistically analyzed using statistical package for social sciences (SPSS) version 21.0 software (SPSS, Inc., Chicago, Illinois, USA) and Duncan's multiple range test was used to determine the significant difference at 5% probability level.

Median lethal concentration (LC 50 ) and safe level
The mortality of C. gariepinus exposed to different concentrations of fenthion is presented in    (Table 3).

Behavioural responses of C. gariepinus exposed to fenthion
The behavioural responses of the test fish were observed at 24 to 96 h of the exposure (Table 4). In the control group, normal behavioural responses such as nonhyperactivity, normal swimming patterns and fin movements were observed. Fish exposed to different concentrations of fenthion displayed behavioural abnormalities in response to the test chemical. At the initial exposure, fish were alert, stopped swimming and remained static in position in response to the sudden changes in the surrounding environment. After some few seconds, fish in the experimental group tried to avoid the test water by swimming rapidly and trying to jump out of the aquaria. Fish in aquaria that had higher concentrations of the pesticide showed abnormal behaviour and tried to avoid the test water by swimming very fast, jumping and displaying erratic with vigorous jerky movements, faster opercula movement, and gulping of air. Skin discoloration was mostly observed in fish that were exposed to the highest concentration of fenthion while it was least in the lowest concentration. After sometime, the fish lost their balance and became exhausted owing to respiratory difficulty. They settled down passively at the bottom of the aquaria and died.

Histopathological observations of gills in Clarias gariepinus exposed to fenthion
Histopathological changes in C. gariepinus were observed during the experiment (Figures 2 to 4). No alterations were observed in the gills of the control. The Table 4. Effects of fenthion on the behavioural characteristics of Clarias gariepinus at different exposure durations. severity and frequency of gill lesions were found to be more pronounced in fish exposed to the highest concentration of fenthion. Histopathological alterations were concentration and duration dependent. The most common pathological changes in the gills of the exposed fish species were fusion of the tip of lamella, deformed lamella, congestion of blood spaces, epithelial lifting, intercellular space variation and hypertrophy of lamella (Table 5).

DISCUSSION
Acute and chronic toxicity tests are mostly used to assess the toxicity of chemicals on non-target organisms (Santos et al., 2010). The 96 h LC 50 is one of the most important parameters for evaluating the toxic effects of pollutants (Nwani et al., 2015). In the present study, the 96 h LC 50 values of fenthion for the African catfish, C. gariepinus was found to be 39.97 mg/L. This suggests that fenthion is toxic to fish. The observed mortality was dose and time dependent such that as the exposure time increased from 24 to 96 h, the median lethal concentration required to kill the fish was reduced. The 100% mortality observed in fish exposed to 72.0 mg/L of fenthion indicates that the insecticide was very toxic. The 96 h LC 50 value obtained in the present investigation was higher than the 1.5 mg/L 96 h LC 50 value of fenthion previously reported in C. carpio (Muralidharan, 2012). The disparity in the toxic potential of the pesticide may be attributed to the difference in age and hardiness of the test species and water quality parameters. According to Johnson and Toledo (1993), the difference in the toxic potential of fenthion can be related to its accumulation, biotransformation and excretion. In general, toxicity of chemicals to aquatic organisms has been reported to be affected by temperature, pH, dissolved oxygen, size and age, strain of species, water quality, concentration and formulation of test chemicals (Nwani et al., 2010;Boran et al., 2012;Rauf and Arain, 2013). The magnitude of toxic effects of pesticides also depends on the length and weight, corporal surface/body weight ratio and breathing rate (Murty, 1986). Oh et al. (1991) also reported that varied inhibition of acetylcholinesterase, detoxification and absorption are factors causing the selective toxicity of pesticides for various species of fish. The safe levels obtained for fenthion in the present study varied from 3.99 × 10 -4 to 3.99. However, the large variation in safe levels determined by various methods has resulted in controversy over its acceptability as extrapolation of laboratory data to field data was not always meaningful hence, the difficulty in deciding the acceptable concentration that may be considered 'safe' based on laboratory experiments (Buikema et al., 1982;Pandey et al., 2005). Behavioural changes in fish are the most sensitive indicators of potential toxic effects of pesticide exposure (Banaee et al., 2011;Rauf and Arain, 2013). Clinical symptoms observed during acute exposure of C. gariepinus to fenthion in the present study are consistent with the findings of other authors and may indicate fenthioninduced suppressed activity of acetylcholinesterase. The abnormal behaviour exhibited by fish in the experimental groups such as abnormal swimming behaviour are due to inhibition of AChE activity leading to accumulation of acetylcholine in choligenic synapses thus causing hyper  Table 5. Summarized histopathological effects in the gill tissues of Clarias gariepinus exposed to fenthion and the control. stimulation. Fenthion may inhibit AChE activity due to its accumulation in the brain tissues. This may lead to impairment of the normal functioning of nerve impulse causing physiological and behavioural modifications that can lead to reduction in survival ability (Muralidharan, 2014a). The behavioural symptoms observed during C.
gariepinus exposure to fenthion in this study is similar to observations by other authors on the responses of different fishes exposed to organophosphate pesticides. It has been reported that under stress condition, fish become hyperactive, perhaps to get out of the stressful medium and would require an increased amount of oxygen to meet their energy demand (Alkahem et al., 2011). Muralidharan (2012) reported that hyperactivity of fish exposed to pollutants could be attributed to impaired gill function and the secretion of increased amount of mucus to coat the body and gills may be attempt to get relief from the irritating pollutants. The secretion of copious mucus by fish could also be a defence mechanism to neutralize the effect of pesticides which gradually cover the body, gills, and buccal cavity. Repeated opening and closing of the mouth and opercula covering accompanied by partially extended fin (coughing) as observed in the present study could be due to clearance of the accumulated mucus debris in the gill region for proper breathing. Similar behavioural changes in C. gariepinus exposed to atrazine and chlorpyrifos (Nwani et al., 2013a) and C. mrigala exposed to diazinon (Rauf and Arain, 2013) have been reported.
Exposure of organisms to sub-lethal concentrations of pesticides in the environment has been reported to result in biochemical, physiological and histopathological alterations in vital tissues (Anbu and Ramaswamy, 1999). The histological results observed in gill tissues of C. gariepinus in the present study indicate that sub-lethal concentrations of fenthion caused moderate to severe alteration in gill architecture which are important organs performing vital functions. Histopathological lesions observed in gill tissues of C. gariepinus exposed to fenthion in the present study are similar to reports in C. carpio exposed to (Muralidharan, 2014b). Similar pathological lesions in the gill architecture were observed in Oreochromis niloticus exposed to dimethoate (Elezaby et al., 2001), Puntius gonionotus exposed to paraquat (Cengiz and Unlu, 2006), Oncorhynchus mykiss exposed to the fungicide captan (Boran et al., 2012) and in Gobiocypris rarus (Yang et al., 2010), Gnathonemus petersii (Alazemi et al., 2012) and C. carpio (Blahova et al., 2014) exposed to atrazine. Epithelial hypertrophy could be as a result of epithelial detachment as stated by Machado and Fanta (2003). Epithelial lifting increases the distance through which the toxicant reach the blood stream thereby causing impaired oxygen uptake  and could result in dysfunction or even nonfunctional gills and eventually suffocate the fish. Lamella fusion could be a protective mechanism as it reduces the amount of vulnerable gill surface area. According to Olurin et al. (2006) these pathological changes may be a reaction to toxicant intake or an adaptive response to prevent the entry of the pollutants through the gill surface and probably increase capillary permeability. The oedema observed in fish gills exposed to fenthion could be due to acute inflammatory nature of the lesion induced by fenthion. Oedema with epithelial separation was reported by Eller (1971) as additional change in fish gills exposed to toxicants.
Though histological changes observed in the present study and that of Muralidharan (2014b) revealed that the changes were concentration dependent, some authors reported that such structural changes are non-toxicant specific (Meyers and Hendricks, 1985), which could be mere stress response of fish to toxicant exposures (Gabriel et al., 2007). The structural changes observed in tissues throughout the duration of exposure were expected to normalize as reported by Myers et al. (1992) who stated that it is possible for the gill to regenerate and recover from the alterations but no such regeneration and recovery were observed at the post exposure test which shows that the changes observed were non reversible.

Conclusion
Fenthion is toxic to fish and possibly other aquatic organisms. It has the potential to damage the physiology of fish leading to observed changes in behavioural pattern with resultant dose dependent mortality. Histopathological results clearly indicate that sublethal concentrations of fenthion had destructive effects in the gill tissues of C. gariepinus. The study shows the significance of behavioural parameters in assessing the risk associated with pesticide exposure to fish. Environmental monitoring programs for the pesticide is necessary in view of the hazard and side effects that might be associated with excessive use on non-target organisms especially fish.

Conflict of interests
The author(s) did not declare any conflict of interest.