Toxicity and biochemical responses induced by phosmet in rainbow trout (Oncorhynchus mykiss)

Abstract

Phosmet is a non-systemic organophosphorus insecticide exerting its toxicity by inhibiting acetylcholinesterase upon entering the body via contact, ingestion and inhalation. Data regarding its sublethal effects on fish are limited, and therefore, with this study it was aimed to investigate the effects of phosmet on liver and brain tissues of juvenile Oncorhynchus mykiss following 24, 48, 72 and 96 h of exposure to 5, 25 and 50 μg/l concentrations. Pesticide treatment caused notable decrease in the levels of serum glucose, protein and cholesterol, whereas there was prominent elevation in the activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. Anticholinesterase activity of phosmet was observed in brain tissue reaching maximum of 46%. In both tissues, increase in the activities of superoxide dismutase, catalase and glutathione peroxidase and level of glutathione was accompanied by elevated thiobarbituric acid reactive substances level. Our results clearly indicate the modulatory effect of phosmet on acetylcholinesterase activity and its potency to provoke oxidative stress condition. The determined alteration in alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities indicates hepatotoxic potential of pesticide; meanwhile, obtained hypoglycaemia and hypoproteinaemia are evaluated as adaptive responses to handle the stress to survive.

Introduction

Phosmet, O,O-dimethyl S-phthalimidomethyl phosphorodithioate, is a broad-spectrum and non-systemic organophosphorus insecticide exerting its toxic action by direct contact, inhalation and ingestion. It was first registered in the USA in 1966, and it is used in many countries in Europe, Middle East, Africa and USA against wide variety of pests in agriculture and to treat flea, tick and mite in pets [1]. In our country, it is mainly used on apple, peach and olive trees against pests. Rapid degradation in non-toxic compounds by hydrolysis and microbial action in soil and low mobility result in low persistence in the soil environment. It has low solubility in water, and reported levels were in the range of 0.0079–0.2 μg/l for groundwater and 0.3 and 0.63 μg/l for surface water [2]. Unintended consequences, however, including reproductive and teratogenic effects have been stated [3, 4]. Phosmet residues have also been reported in fruits, potato, olive oil and honey [5–7].

As an organophosphate, toxic action of phosmet originates from the anticholinesterase activity causing neurotransmitter acetylcholine (ACh) to accumulate at cholinergic synapses. The build-up of ACh leads muscarinic and nicotinic toxicity due to intensive and prolonged activation of receptor site [8]. The reports reveal the potency of pesticides in modulation of acetylcholinesterase (AChE) activity in different fish species [9, 10]. In addition to main toxic mode of action, different adverse effects on non-target organisms including genotoxicity [11], endocrine disruption [12] and oxidative stress have been evidenced [13]. The condition of oxidative stress reflects the imbalance between the oxidation and reduction reactions originated from excess production of reactive oxygen species damaging proteins, lipids and DNA. As an adaptive response to cope with the condition, alteration in the members of antioxidant defence system including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH) has been employed as biomarker of oxidative stress in fish [14, 15].

Among the non-target organisms, fish are recognized as sensitive bioindicator used in ecotoxicological and risk assessment studies. Oncorhynchus mykiss, the most frequently farmed fish species in our country, is one of the important species that has been used widely in acute and chronic toxicity studies as a sensitive test organism [16, 17]. The limited attention has been given to the toxicity of phosmet to fish. The lethal concentrations (LC₅₀) were stated as 7300 μg/l for Pimephales promelas, 150 μg/l for Oncorhynchus tshawytscha and 500 μg/l for O. mykiss and 160 μg/l for Lepomis macrochirus [18]. The sublethal effects of phosmet on Oncorhynchus kisutch and Danio rerio were studied by evaluating the endpoints of neurotoxicity and developmental toxicity in juvenile and larval life stages, respectively [4, 19].

Insufficiency of data regarding its toxicity on biological pathways in different fish species necessitates additional studies to reveal the toxic mode of action of phosmet in detail. Therefore, the current investigation was designed to assess (i) the anticholinesterase activity of phosmet by analysing AChE activity, (ii) the effects on blood biochemistry by using serum glucose, protein, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) as biomarkers and (iii) its pro-oxidant potency by evaluating SOD, CAT, GPx activities together with the levels of GSH and thiobarbituric acid reactive substances (TBARS) in liver and brain tissues of O. mykiss.

Materials and Methods

Fish

Juveniles of rainbow trout, O. mykiss, (72.80 ± 4.85 g, 16.24 ± 1.03 cm) supplied from local breeder (Firniz Alabalik), were acclimated for 14 days in 200 l aquariums containing aerated and dechlorinated tap water. The 12:12 photoperiod regime was applied, and they were fed daily with commercial trout food (Optima trout-Skretting) throughout acclimatization. The feeding were suspended 24 h before the experiments. The water quality parameters were assessed by using water testing meter (AZ 8405 Combo) on daily basis. The water quality was as follows: temperature 9.6 ± .55, pH 7.8 ± .28, dissolved oxygen 8.7 ± .71 mg/l and conductivity 745.5 ± 10.11 μS cm⁻¹.

Test chemical

The insecticide used in this study was a commercial grade formulation of phosmet (Barco 50 WP, Gowan Crop Protection Limited). The stock solution was prepared immediately before application by dissolving phosmet in acetone (final concentration was 0.002%) and desired concentrations were achieved by further dilution with aerated tap water.

Experimental design

Sublethal concentrations of 5, 25 and 50 μg/l corresponding to 1%, 5% and 10% of the 96 h LC₅₀ of phosmet for O. mykiss, 500 μg/l [18] were selected as test concentrations. Following acclimatization period, fish were divided into four groups (24 fish for each treatment). Group I was served as control and maintained in pesticide-free tap water containing 0.002% acetone. Groups II, III and IV were exposed to given sublethal concentrations of phosmet for 24, 48, 72 and 96 h. Experiments were conducted as triplicates (288 fish in total). Toxicity test procedures were performed in accordance with American Public Health Association [20]. Fish were fasted during the experiment and the medium was renewed every 24 h. Validation of nominal concentrations were performed in water taken from aquariums following each application by liquid chromatography coupled to tandem mass spectrometry (Shimadzu LC–MS/MS 8040) [21, 22]. Measurements were performed in triplicate for each sampling and average recovery rate was calculated as 95.24 ± 1.06%. All groups were observed visually for abnormal swimming as a behavioural endpoint on daily basis. At the end of each exposure period, six randomly selected fish from each aquarium were anaesthetized with MS-222 (50 mg/l, buffered with NaHCO₃) [23] and euthanized through spinal cord section. Blood samples were collected by severing caudal peduncle. Liver and brain tissues were dissected, washed with ice-cold NaCl (0.59%) and stored at −80°C until analysis. The experimental protocols were authorized by the Local Ethics Committee of Animal Experiments of Gaziantep University.

Analytical Procedure

Biochemical blood parameters

The collected blood samples were taken into anticoagulant-free tubes and centrifuged at 5000 rpm for 10 min (Hettich Universal 320R). Analyses of serum glucose, cholesterol, triglyceride, protein and ALT, AST and ALP were carried out using biochemical analyser (Siemens ADVIA 2400 Chemistry System) with kits provided by Siemens Healthcare Diagnostics Inc. (Munich, Germany).

Biochemical analysis

Liver and brain samples were homogenized (1:5 w/v) in .1 M pH 7.4 phosphate buffer saline via Ultratorrax homogenizator (Isolab) and then homogenates were centrifuged at 13 000 rpm for 30 min at 4°C (Hettich Universal 320R) to obtain supernatant. Biochemical analyses were performed spectrophotometrically (Shimadzu UV Mini-1240).

SOD activity was analysed by using INT (iodo-p-nitro tetrazolium chloride) as substrate according to the method of McCord and Fridovich [24]. GPx activity was measured by recording dismutation of t-butylhydroperoxide at 340 nm [25]. CAT activity was analysed by using the degradation rate of hydrogen peroxide at 230 nm [25]. The AChE activity was measured according to the method described by Ellman et al. [26]. Lipid peroxidation was estimated by measuring concentration of TBARS at 532 nm [27]. GSH was measured according to the method of Ellman [28]. Tissue protein levels were measured according to Lowry et al. [29], and bovine serum albumin was used as standard.

Statistical analysis

Statistical analysis of data was performed using the SPSS software (SPSS Inc., Chicago, IL, version 22). Two-way analysis of variance (ANOVA) followed by Tukey’s test were employed to compare mean values of groups and to determine the combined effects of concentration and duration on analysed parameters. The strength of association between variables was examined by Pearson’s correlation analysis. Significant difference was acknowledged when P < 0.05.

Results

Behaviour

Group I (control) showed normal behaviour during experimental period. In Group 2, control-like behaviour was observed. Group 3 exhibited frequent surfacing and faster swimming on 72 and 96 h of exposure. In Group 4, behavioural changes including localization to the corner/bottom of aquarium, loss of balance and erratic swimming were recorded on 24 h. The situation continued throughout the experimental period.

Serum biochemistry

The effect of phosmet on serum biochemistry is given on Figure 1. The activities of ALT, AST and ALP showed an ascending trend following phosmet treatment. The maximum percentage changes were calculated as 172%, 123% and 29% on 96 h at 50 μg/l concentration for ALT, AST and ALP activities, respectively (P < 0.01). A marked decrease in serum glucose level, with more pronounced change at high concentration with extended period of exposure, was observed. The calculated rise in glucose level were as 44%, 43% and 25% for 5, 25 and 50 μg/l concentrations on 96 h, respectively (P < 0.01). Statistically significant decline in serum protein level following 24- and 48-h phosmet application reaching 13% was determined (P < 0.01). Similarly, an important decrease in cholesterol level reaching maximum of 32% in groups treated with phosmet for 24, 48 and 72 h was determined (P < 0.01). The correlation coefficients calculated for the relationship among ALT, AST and ALP were significant and positive (P < 0.01).

Figure 1.

effects of sublethal phosmet exposure on serum biochemistry of O. mykiss; protein (g/l), glucose (mg/dl), cholesterol (mg/dl), ALT (U/l), AST (U/l) and ALP (U/l). Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^*P < 0.05 and ^**P < 0.01.

AChE activity

The specific activity of AChE in brain tissue is given in Figure 2. A decreasing trend was recorded in the AChE activity in brain tissue with a more marked alteration at high concentration with extended period of exposure (P < 0.01). The maximum reduction rates were calculated as 34, 44% and 46% for 5, 25 and 50 μg/l concentrations on 96 h, respectively. The correlation analyses revealed a significant and negative relationship between AChE activity and TBARS content in brain tissue (r = −0,214, P < 0.01).

Figure 2.

decrease in AChE activity following phosmet exposure in brain tissue of O. mykiss. Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^**P < 0.01.

Antioxidant enzyme activities

Alterations in specific activities of antioxidant enzymes in liver and brain tissues are presented in Figures 3 and 4. In liver tissue, SOD activity increased and the highest increase was 124% on 96 h at 50 μg/l (P < 0.01). Similarly, brain SOD activity showed an increasing trend reaching the maximum of 46% on 96 h at 50 μg/l (P < 0.01).

Figure 3.

increase in SOD, CAT and GPx activities following phosmet exposure in liver tissue of O. mykiss. Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^*P < 0.05 and ^**P < 0.01.

Figure 4.

increase in SOD, CAT and GPx activities following phosmet exposure in brain tissue of O. mykiss. Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^*P < 0.05 and ^**P < 0.01.

CAT-specific activity did not change significantly on 24 and 48 h in liver tissue (P > 0.05), whereas 12% and 21% increases were calculated on 72 and 96 h at 50 μg/l, respectively (P < 0.01). In brain tissue, CAT activity significantly increased at 25 and 50 μg/l concentrations on 96 h, 22% and 23%, respectively (P < 0.01).

GPx activity showed an increase of 17% and 18% in liver and heart tissues on 96 h at 50 μg/l (P < 0.01).

TBARS content

The effect of phosmet application on TBARS contents of liver and brain tissues of O. mykiss were given in Figures 5 and 6. In liver tissue, TBARS contents increased 56%, 55% and 78% at 5, 25 and 50 μg/l concentrations on 96 h, respectively (P < 0.01). TBARS contents showed an increase of 62%, 84% and 97% in brain tissue at the respective concentrations of 5, 25 and 50 μg/l on 96 h (Fig. 5, P < 0.01).

Figure 5.

effects of sublethal phosmet exposure on TBARS (nmol/mg protein), GSH (nmol/mg protein) and protein (mg/ml) levels in liver of O. mykiss. Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^*P < 0.05 and ^**P < 0.01.

Figure 6.

effects of sublethal phosmet exposure on TBARS (nmol/mg protein), GSH (nmol/mg protein) and protein (mg/ml) levels in brain of O. mykiss. Bars represent the mean ± standard deviation values. Asterisks on the bars indicate statistically significant difference compared with the control. ^*P < 0.05 and ^**P < 0.01.

GSH level

There was no significant alteration in GSH content following 24 and 48 h of phosmet treatment in liver tissue (Fig. 5, P > 0.05). On 72 h, 21% increase was detected at 50 μg/l concentration, whereas 42% and 49% rise in GSH level were observed in groups exposed to 25 and 50 μg/l for 96 h (P < 0.01). Phosmet application resulted in 26% and 35% increase in GSH content of brain tissue at 25 and 50 μg/l for 96 h (Fig. 6, P < 0.01). SOD, CAT, GPx activities and GSH levels were found to be positively correlated to TBARS in brain (r values were 0.362, 0.201, 0.289 and 0.322, respectively, P < 0.01) and liver tissue (r values were 0.576, 0.439, 0.350 and 0.515, respectively, P < 0.01).

Protein level

The liver protein content did not exert any significant change following 24 and 48 h of phosmet treatment; meanwhile, a concentration- and duration-dependent decrease was detected on 72 and 96 h reaching maximum of 13% and 16%, respectively (Fig. 5, P < 0.01). In brain tissue, only important change recorded was the 19% decrease in protein content at 50 μg/l concentration on 96 h of treatment (Fig. 6, P < 0.05).

The two-way ANOVA conducted to examine the effect of concentration and duration on analysed parameters revealed statistically significant interaction between the effects of these two fixed factors on activities of SOD (F_9,272: 4.913, P:0.000) and CAT (F_9,272: 2.583, P:0.007), content of GSH (F_9,272: 3.542, P:0.000) and TBARS (F_9,272: 13.043, P:0.000) in liver tissue. In brain tissue, significant interaction between the effects of concentration and duration was determined on enzymes of SOD (F_9,272: 3.063, P:0.002) and AChE (F_9,272: 4.873, P:0.000) and content of TBARS (F_9,272: 5.421, P:0.000) in brain tissue. An important interaction between the effects of concentration and duration was estimated on parameters of serum biochemistry of glucose (F_9,272: 78.143, P:0.000), protein (F_9,272: 6.936, P:0.000), cholesterol (F_9,272: 29.928, P:0.000), ALT (F_9,272: 147.112, P:0.000), AST (F_9,272: 38.587, P:0.000) and ALP (F_9,272: 13.919, P:0.000).

Discussion

The environmental pollution and toxicity risk introduced by the intensified pesticide use on a global scale have prompted research into the adverse effects of these chemicals on non-target organisms causing different adaptive responses at biochemical and cellular levels. Therefore, the anticholinesterase activity and pro-oxidant potential of organophosphorus pesticide phosmet on brain and liver tissues of O. mykiss were presented with this investigation by employing biomarker approach.

As an indication of general health status of fish, biochemical profile of blood has an important value in toxicological studies. The activities of transaminases (ALT and AST) and ALP are used as measure of tissue damage [30]. Alteration in the activities of these enzymes was reported for Clarias gariepinus and treated with carbofuran [31] and for Oreochromis niloticus applied with endosulfan [32] resulted from release of enzymes into extracellular space. Abhijith et al. [33] reported methyl parathion-induced hepatocellular damage in Cyprinus carpio evidenced with the disturbance in the activities of ALT, AST and ALP. Phosmet treatment resulted in remarkable increase in the activities of ALT, AST and ALP in concentration- and duration-dependent manner revealing toxic action of the pesticide on liver tissue.

The levels of blood cholesterol and glucose are evaluated as stress markers because of their importance in providing cellular energy to maintain the homeostasis. In this study, phosmet treatment resulted in significant decrease in the levels of both cholesterol and glucose in O. mykiss. Similar to our findings, lufenuron-induced decrease in cholesterol levels in C. carpio was reported by Ghelichpour et al. [34], and impaired absorption and synthesis of cholesterol were suggested as possible underlying mechanisms. Bharti and Rasool [35] observed decrease in serum glucose level following malathion exposure in C. carpio and explained the situation by impairment of carbohydrate metabolism and increased energy demand. The determined decrease in cholesterol level and hypoglycaemic condition reveal the involvement of metabolic pathways in the toxicity of phosmet and increased energy requirement as a response to imposed stress.

Proteins have essential role in cell metabolism, and determination of the change in the protein content was considered to be of value in screening for pesticide toxicities in fish [30]. Phosmet application caused decline in both serum and tissue protein levels in O. mykiss. The results are in agreement with the findings obtained for Labeo rohita following 96-h malathion [36] and monocrotophos applications [37]. Stress-elicited increase in proteolytic activity and the activation of physiological compensatory mechanisms are stated as possible mechanism resulting in protein depletion [38].

Similar to our findings, reduction of AChE activity under the effect of organophosphorus pesticides has been reported in different fish tissues with a focus on brain [39, 40]. Alavinia et al. [41] reported time- and concentration-dependent reduction in the AChE activity of gill, muscle, brain and liver tissues of malathion-exposed O. mykiss for 15 days emphasizing the gradual restoration of the activity following 4 weeks of recovery in clean water. The irreversible nature of the reduction depending on de novo synthesis of AChE to replenish the activity was stated as an explanation to this observation [42]. Accumulation of ACh in synaptic junctions because of reduced AChE activity disturbs normal functioning and impairs behaviour [43]. In agreement with our observations, abnormal swimming like loss of balance, hyperactivity and surfacing has been reported for Channa punctatus, Heteropneustes fossilis and Oryzias latipes treated with organophosphorus pesticides triazophos, fipronil and chlorpyrifos [9, 44, 45]. Baldissera et al. [10] suggested cerebral oxidative stress and reduced AChE activity as underlying reasons of trichlorfon-elicited abnormal swimming performance they observed in Rhamdia quelen.

Antioxidants, both enzymatic and non-enzymatic, provide defence by free radical scavenging reactions and ameliorate the toxicity caused by reactive species produced due to a stress factor like pesticide exposure. The excessive production of reactive species and inefficiency of antioxidant defence system leading to the state of oxidative stress were observed in fish following pesticide exposure [11, 46]. The vital functions of liver in detoxification and that of brain in nervous system with accompanied vulnerability to oxidative injury make them potential targets for pesticides. Phosmet application provoked increase in the activities of SOD, CAT and GPx in both liver and brain tissues pointing an evoked response of first-line defence to suppress the formation of reactive species by eliminating the superoxide radicals, hydrogen peroxides and hydroperoxides, respectively. GSH level, a non-enzymatic antioxidant conjugating electrophilic compounds, also showed an increasing tendency in both tissues providing a supportive mechanism to avoid oxidative damage by controlling the production of reactive species. Our findings are compatible with Bharti and Rasool [35] who reported increased SOD and CAT activities in the liver of malathion-treated C. punctatus and Wang et al. [15] who observed induction in SOD and CAT activities together with elevated GSH levels in brain and liver tissues of triazophos-applied Danio rerio.

Lipid peroxidation is oxidative modification of unsaturated fatty acids that generates reactive products and damages membrane by altering the permeability and fluidity [47]. Direct interaction with cellular membrane and evoked oxidative stress were suggested as mechanisms of lipid peroxidation in fish following pesticide exposure [35, 48]. Ullah et al. [36] presented the hepatotoxic potential of malathion evidenced with elevated lipid peroxidation in liver of L. rohita following 96 h of application. Similarly, endosulfan-triggered increase in the level of lipid peroxidation in liver of Oreochromis nilotus was stated [32]. Phosmet treatment resulted in increase in TBARS content in both liver and brain tissues of O. mykiss confirming the provoked oxidative stress. Also, disruption of membrane integrity could be stated as an aggravating factor for observed reduction in the activity of membrane-bound enzyme AChE [49].

Ecologically relevant biomarkers such as survival, growth and reproduction have been presented as consequential outcomes resulting from oxidative stress, reduced AChE and altered behaviour [50–53]. Thus, despite presenting organism-level responses, endpoints of this investigation could be used to predict the risks posed by phosmet at population level.

Conclusion

The research presents a clear understanding into toxic mechanisms of action of phosmet by showing evidences of oxidative stress and its anticholinesterase activity. However, limitation of our study design is that it does not represent environmentally relevant concentrations that may provide more realistic risk assessment. Therefore, integrated approaches considering nature of environment with respect to concentration, coexistence of toxicants and aquatic organisms at different life stages should be addressed in future studies.

Funding

Gaziantep University Scientific Research Projects Coordination Unit (AMYO.YLT.20.01).

Conflict of interest statement. None declared.