Lethal and sublethal effects of a methomyl-based insecticide in Hoplobatrachus rugulosus.

The aim of this study was to investigate the lethal and sublethal toxicity of a methomyl-based insecticide in Hoplobatrachus rugulosus, as methomyl-based insecticides are applied in massive amounts and agrochemicals have effects on the decline in amphibian populations. To evaluate the toxic effects of methomyl from agricultural application, a methomyl-based insecticide containing 40% methomyl was selected. The median lethal concentration of 96 hours of methomyl exposure was 8.69 ppm for H. rugulosus tadpoles. The lethal concentration also produced severe histological damage in the livers and kidneys of the exposed tadpoles. The sublethal concentration used for methomyl was 144 ppb during the metamorphosis period. It was found that the sublethal concentration of the methomyl compound could decrease growth, metamorphosis time, and size, disturb biochemical parameters, and produce histological damage. In livers, methomyl effects increased oxidative stress and dramatically decreased the glycogen level of the treated froglets. Mononuclear infiltration, blood congestion, amorphous substances, and hepatocytes vacuolization were observed throughout liver tissue. The methomyl-based insecticide also increased oxidative stress and decreased nitric oxide levels in the kidneys of the exposed froglets. Renal tissue damage including blood congestion, amorphous substances, and Bowman's capsule spaces reduction were found in the methomyl exposure group. The methomyl compound also produced vacuoles in various stages of oocytes, but no histological damage was found in testicular tissue. Our results indicated strong toxic effects of the methomyl-based insecticide on H. rugulosus, a broadly tolerant anuran.

Introduction

Methomyl, an insecticide of the carbamate type, has been classified as a potentially harmful chemical[1]. It has been applied to control many pests, such as insects, snails, and worms[2]. As a result of its broad effects, massive amounts of methomyl-based insecticides have been used without being recorded for almost half a century. Therefore, methomyl residue is frequently detected in agricultural products, soil, and natural water[3]. The primary effect of methomyl compounds is to interrupt the function of acetyl cholinesterase, causing vulnerability to damage in the nervous system[4]. Hence, contamination of the environment with methomyl-based insecticides might cause serious harm to nontarget organisms.

Pesticide contamination is considered an important factor that has been implicated for 20 years in the amphibian crisis[5]. Given the overlapping distribution of amphibians with important agricultural areas in many countries and the enormous amounts of pesticides utilized in such areas each year, most amphibians are suffering from pesticide contamination. Nowadays, population decline and species extinction have changed for the worse in anurans. Nearly 168 species have become extinct, and at least 43% of the rest have been decreasing globally[6]. In Southeast Asia, over 800 species of amphibians have been recorded[7], and large numbers of new species have continuously been discovered since 1997[8]. However, approximately 58,000 tons of pesticides are applied each year in the agricultural areas of the region[9]. For this reason, the population and diversity of amphibians in Southeast Asia have been continuously threatened by pesticide contamination.

Amphibians play roles in both terrestrial and aquatic ecosystems. Decrease of their populations could cause serious ecological problems, for example, outbreaks of insect pests and mosquitoes. In addition, freshwater fishes, small reptiles, and other amphibian predators are affected by the decline of amphibian populations.

Accordingly, pesticide toxicity in amphibian species is important. Unfortunately, study of the effects of methomyl contamination on nontarget species, especially amphibians, has been insufficient. Therefore, the present study aimed to evaluate the toxic effects of a methomyl-based insecticide on the East Asian bullfrog (Hoplobatrachus rugulosus). This species is a representative of the amphibians widely distributed throughout Eastern and Southeast Asia[10], inhabiting various types of habitats and having a broad range of tolerance. Besides, H. rugulosus is a good source of protein for local people and also an economically important animal in Thailand.

Materials and Methods

Animal and rearing condition

The present study was performed with 7-day-old H. rugulosus larvae, the mean total length of which was 2.46 ± 0.034 cm. Newly hatched tadpoles were obtained from the Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Thailand. Larvae were transferred to plastic tanks containing 4 L of dechlorinated tap water and allowed to acclimatize to laboratory conditions for a week. They were fed a commercial tadpole diet, and the accumulated residues were removed twice daily. The present study was approved by the Institutional Animal Ethics Committee of Chiang Mai University, Thailand.

Since the commercial grade of methomyl is always applied to agricultural fields, a methomyl-based insecticide with 40% active ingredient was selected in the present study. The methomyl-based insecticide (Lannate 40% SP) was purchased from Dupont Co., Ltd., Thailand. All treatment solutions were made with dechlorinated tap water. The treated concentrations were calculated from the product with 40% methomyl. Therefore, we used the term methomyl for methomyl-based insecticide (Lannate 40% SP) in this paper.

Experimental design

Lethal toxicity: Two hundred and twenty tadpoles were randomly divided into eight groups. Each group was separated into two replicates and supplied 4 L of tested solutions. The control group was kept in pesticide-free water, and seven experimental groups were exposed to 3, 5, 7, 9, 10, 15, or 20 ppm of the methomyl-based insecticide. Tested solutions were renewed every 24 hours to maintain the pesticide concentration. Dead tadpoles, if present, were immediately removed, and mortality was recorded every 24 hours for 96 hours. At the end of the experiment, all tadpoles were anesthetized for histological examination.

Sublethal toxicity: One hundred and eighty tadpoles were divided into two groups, each with three replicates. Ninety tadpoles were kept in pesticide-free water to serve as a control group. Another group was exposed to a sublethal concentration of the methomyl-based insecticide. The chosen concentration, the LC50 (0.144 ppm), to serve as the sublethal concentration was obtained from a previous study[11].

During metamorphosis, each condition was renewed every three days. Snout-vent length, developmental stage, and mortality of the tadpoles were recorded. The complete metamorphosis of H. rugulosus was recorded as days to metamorphosis, and all of the tadpoles were sampled for histological and biochemical examinations.

Histological examination: Livers, kidneys, and gonads were carefully removed and fixed in 10% formalin. Hematoxylin and eosin staining of 5-µm-thick paraffin-embedded sections was examined under a compound microscope.

Biochemical examinations

Glycogen assay: The method for estimation of glycogen in the liver was slightly modified from the method described by Seifter et al.[12]. Briefly, 0.05 g of liver was boiled in 30% (w/v) KOH for 10 min. Then 95% (v/v) ethanol was added into the cooled solution, which was subsequently boiled again. The mixture was then centrifuged at 3,000 rpm for 15 min. The pellet was redissolved in 1 mL of distilled water and mixed with 10 mL of anthrone reagent. The mixture was then boiled for 10 min and immediately placed in an ice bath. The absorbance of the solution was measured at 620 nm and calculated with reference to a glucose standard curve. Finally, the concentration of glycogen was expressed in mg/g wet weight.

MDA assay: The method for MDA measurement was slightly modified from that of Hritcu et al.[13] using tetramethoxypropane as a standard. The livers and kidneys were weighed and homogenized with phosphate buffer (pH 7.0) and centrifuged at 3,000 rpm for 10 min. Two hundred microliters of supernatant was mixed with 500 µL of 0.85% (w/v) NaCl, 1,000 µL of 100% (w/v) trichloroacetic acid in 0.06 M HCl, and 200 µL of 0.28 mM thiobarbituric acid. The mixture was boiled for 20 min and centrifuged at 3,000 rpm for 10 min. The supernatant was read at 532 nm. The results are presented in µmol/mg protein.

Nitric oxide assay: Nitric oxide was measured by its metabolites (nitrate and nitrite). The procedure was performed according to the method described by Kumar et al.[14] using NaNO3 as a standard. Two hundred microliters of homogenized tissue or standard was mixed with 20 µL of NaOH and 30 µL of ZnSO4. The sample was added into a solution containing 5×102 units of nitrate reductase, 20 µL of 0.2 M N-trimethylaminoethane sulfonic acid, and 20 µL of 0.5 M of sodium formate. The mixture was anaerobically incubated at room temperature for 20 min. After complete incubation, the mixture was added to 1 mL of distilled water and centrifuged at 260 rpm for 5 min. Supernatant was mixed with 20 µL of 1% (w/v) sulfanilamide in 15% (v/v) phosphoric acid and incubated at room temperature for 10 min. Finally, 20 µL of 0.1% (w/v) N-(1-naphthyl) ethylenediamine was added to the solution. The absorbance of each sample at 540 nm was recorded. The data were displayed in µmol/mg protein.

Protein assay: The level of protein content from each tissue was estimated following the method described by Bradford[15], and the bovine serum albumin (BSA) was used as a standard. Five milligrams of Coomassie Blue G were dissolved in 1 mL 25% (v/v) methanol containing 42.5% (v/v) H3PO4. One volume of prepared dye was diluted with four volumes of distilled water, and 0.04 mL of each sample was mixed with 2 mL of diluted dye. The absorbance was read at 590 nm.

Statistical analysis: The obtained data were statistically analyzed with IBM SPSS Statistics version 20.0 for Mac OS X (IBM Corp., Armonk, NY, USA). Lethal concentration values were calculated using probit analysis. Student’s t-test was used to analyze mortality, growth, metamorphosis traits, and biochemical values for chronic exposure.

Results

Lethal toxicity

Mortality: Mortalities were recorded only in the methomy-treated groups (Fig. 1). After 24 hours of exposure, mortalities were found in all treated groups, even the group with the lowest concentration (3.0 ppm). Mortalities increased with the increase in methomyl concentration, and 100% mortality was observed in the group exposed to 20.0 ppm methomyl.

Fig. 1.

Mean percentages of mortalities of tadpoles with exposure to different concentrations of methomyl over the course of 96 hr.

The mortality of tadpoles in each group was calculated; the LC10, LC50, and LC90 and 95% confidence limits are presented in Table 1. It was found that the value of each LC point decreased with increasing exposure time. These results indicated that the mortality of tadpoles depends on the concentration and time. Moreover, the present study found that the 96 hr LC50 value for the methomyl-based insecticide was 8.69 ppm for H. rugulosus.

Table 1.

Lethal Concentrations of Methomyl (ppm) (95% Confidence Limits) with Exposure Time for H. rugulosus

Sublethal toxicity

Survivorship: The cumulative mortalities of H. rugulosus are displayed in Fig. 2. For the methomyl-exposed group, mortality first appeared on the 21st exposure day and continuously increased up to 55% at the end of experiment. Only a minority of dead tadpoles were observed in the control group.

Fig. 2.

Cumulative mortality of H. rugulosus with exposure to 0.144 ppm methomyl during metamorphosis compared with that of the control group. *P<0.05 vs. control group (Student’s t-test). Error bars represent the standard error of the mean.

Growth: Changes in snout-vent length (SVL) of tadpoles with time in both the control and treated groups are presented in Fig. 3. It was found that the SVL of tadpoles in both the control and treated groups continuously increased until almost the end of the experiment. Obviously, the SVL of the methomyl-exposed group of tadpoles was relatively lower than that of the control group throughout the experiment. In addition, the treated group began to show slight changes in SVL (within 43 days), which was later than in the control group (within 37 days). However, at the end of the treatment period, SVL was not significantly different between the two groups.

Fig. 3.

Snout-vent length of H. rugulosus exposed to 0.144 ppm methomyl during metamorphosis compared with that of the control group. *P<0.05 vs. control group (Student’s t-test). Error bars represent the standard error of the mean.

Metamorphosis: Days to metamorphosis and SVL at metamorphosis are presented in Table 2. Methomyl-exposed tadpoles reached the complete metamorphosis stage significantly earlier than the control group. However, the treated group had a shorter SVL than the control group.

Table 2.

Metamorphosis Traits under Different Rearing Conditions

Biochemical assays: Exposure of tadpoles to 1.44 ppm methomyl during metamorphosis produced many biochemical alterations (Table 3).

Table 3.

Biochemical Analyses in Livers and Kidneys of H. rugulosus

Liver: Biochemical analysis of the livers indicated that the MDA level was significantly higher in the methomyl-exposed group (1.84 + 0.04 µmol/mg protein) compared with the control group (0.66 + 0.04 µmol/mg protein). On the other hand, the glycogen level in the liver was dramatically higher in the control group (105.98 ± 7.30 mg/g) compared with that in the methomyl-treated group (4.51 ± 1.10 mg/g).

Kidneys: For the kidney, the level of MDA in the methomyl-treated group (0.68 + 0.06 µmol/mg protein) was significantly greater than in that of the control group (0.45 µmol/mg protein). Furthermore, the nitric oxide (NO) concentrations in this tissue were also statistically lower in the treated group (0.127 µmol/mg protein) compared with the control group (0.442 + 0.02 µmol/mg protein).

Histological examination

Lethal toxicity: To evaluate the histological effects of methomyl at a lethal concentration, livers and kidneys of tadpoles from the group treated with 20 ppm were examined and are shown in Figs. 4B and 5B.

Fig. 4.

Liver tissues of H. rugulosus tadpoles exposed to 20 ppm of methomyl (B) over the course of 96 hr compared with those of the control group (A). Symbols: black arrow, vacuole; red arrow, sinusoid. H&E staining. Magnification, 400×.

Fig. 5.

Kidney tissue of H. rugulosus tadpoles exposed to 20 ppm methomyl (B) over the course of 96 hr compared with that of the control group (A). Symbols: black arrow, Bowman’s capsule space; black star, lumen of renal tubules. H&E staining. Magnification, 200×.

Liver: In the control group (Fig. 4A), liver tissues consisted of hepatocytes arranged in hepatic cords intervening with hepatic sinusoids. Hepatocytes were typically hexagonal in shape with large, round central nuclei, and the cytoplasm was regularly distributed with very fine granules of glycogen and lipid droplets. Sinusoids were hepatic capillaries carrying erythrocytes in rows. In contrast, the livers of the group treated with 20 ppm methomyl (Fig. 4B) showed severe histological damage based on the number of large vacuoles in the hepatocytes with cytoplasmic disintegration.

Kidneys: In addition to the damage in the liver, 20 ppm methomyl also produced histological damage in the kidneys (Fig. 5B). Typically, kidney tissue of amphibians consists of a renal corpuscle, renal tubules, and interstitial hematopoietic tissue. Renal corpuscles are small capillary tufts covered in a Bowman’s capsule. The renal tubules are characterized by a simple cuboidal epithelium as displayed in Fig. 5A. In the treated group, histological damage could be clearly observed in both the renal corpuscle and renal tubule of the kidney. Dilatation of the Bowman’s capsule and renal tubule was observed throughout the tissue, as shown in Fig. 5B.

Sublethal toxicity: The sublethal experiment also showed histological alterations in the liver, kidneys, and gonads (Figs. 6B, 7B, 8B).

Fig. 6.

Histological features of livers from the control (A) and group treated with 0.144 ppm methomyl (B). Symbols: black arrow, vacuole; red arrow, sinusoid; red circle, mononuclear cell infiltration; black star, amorphous substance. H&E staining. Magnification, 200×.

Fig. 7.

Kidney tissue of H. rugulosus exposed to 0.144 ppm of methomyl (B) during metamorphosis as compared with that of the control group (A). Symbols: black arrow, Bowman’s capsule space; red star, blood congestion; black star, amorphous substances. H&E staining. Magnification, 400×.

Fig. 8.

Ovarian tissue of H. rugulosus exposed to 1.44 ppm methomyl (B) during metamorphosis compared with that of the control group (A). Symbols: red arrows, vacuole. H&E staining. Magnification, 200×.

Liver: In the control group, hepatocytes were of normal shape and regularly arranged throughout tissue (Fig. 6A). In contrast, the methomyl exposed group showed various histological alterations in the livers (Fig. 6B). The hepatocytes appeared to be swollen and to have cytoplasmic disintegration accompanied by vacuolization. In addition, blood congestion was focally observed wherein amorphous substances accumulated inside sinusoids and blood vessels. Mononuclear infiltration observed throughout the tissue indicates liver inflammation.

Kidneys: Kidney tissue of control froglets (Gosner stage 46) is shown in Fig. 7A. Similar to 11-day-old tadpoles, no histological lesion was observed in the renal corpuscle, renal tubules, and interstitial hematopoietic tissue of untreated frogs. However, exposure to 144 ppb of methomyl produced histopathological evidence in the kidney tissue of tadpoles treated with methomyl (Fig. 7B). In addition, blood congestion and amorphous substances appeared in interstitial tissues. Moreover, blood congestion was also found inside the Bowman’s capsules, and there was a reduction in space in the Bowman’s capsules in the methomyl-exposed group (Fig. 7B).

Gonads: Similar to the liver and kidneys, gonadal tissue responded to the effects of methomyl. Histological examination revealed that methomyl showed toxicity in the ovarian tissue (Fig. 8B), whereas no histological damage was observed in testicular tissue (Fig. 9B). Of note, methomyl could induce histopathological changes in ovarian tissues after exposure during the metamorphosis period. A number of vacuoles were discovered in various stages of oocytes inside the ovary (Fig. 8B). On the other hand, no histopathological damage was observed in male gonadal tissue of either group (Figs. 9A and 9B).

Fig. 9.

Testicular tissue of H. rugulosus exposed to 1.44 ppm methomyl (B) during metamorphosis compared with that of the control group (A). H&E staining. Magnification, 200×.

Discussion

Mortality: The results of the present study suggest that methomyl could have a substantial effect on the mortality of H. rugulosus. This is the first report of the toxicity of methomyl in H. rugulosus. Although H. rugulosus was previously classified as a broad tolerant species[10], our experiment demonstrated that this species is sensitive to exposure to methomyl [LC50 = 10.88 (24 hr); 9.72 (48 hr)] when compared with other species of anurans. For example, the LC50 values of methomyl were 23 ppm (24 hr) in Bufo vulgaris formosus[16], 27.16 ppm (48 hr) in Rana limnocharis[17], and 40 ppm (48 hr) in Bufo bufo japonicus[18]. However, information about methomyl toxicity in amphibians is still insufficient. Most of the previous research mainly focused on the toxicity of methomyl in mammals and freshwater fishes. Furthermore, methomyl has been indicated as very highly toxic to aquatic organisms and highly toxic to terrestrial species. The LC50 values for the topmouth gudgeon (Pseudorasbora parva) and Nile tilapia (Oreochromis niloticus) were reported to be 0.425 ppm[19] and 1.81 ppm[20], which are considered to be very low and to result in high toxic effects. On the other hand, the LC50 values of other freshwater fishes were reported to be 7.7 ppm in the bluegill (Lepomis macrochirus) and 6.8 ppm in the cutthroat trout (Oncorhynchus clarki)[21]. Although the LC50 value of H. rugulosus in this study (8.69 ppm) was higher than those previously reports for freshwater fishes, the difference in pesticide formation and life stage of this bullfrog could place the LC50 values into the same range as freshwater fishes in previous experiments. In amphibians, not only species differences[22] but also differences in the ages of the tadpoles and chemical concentration affect the LC50 value. Ezemonye and Tango[23] showed that the LC50 value (96 hr) of an atrazine herbicide for the broad-banded grassland frog, Ptychadena bibroni, increased with the increase in tadpole age. In addition, the LC50 of 95% methomyl in the largemouth bass, Micropterus salmoides, was 1,250 ppb, while the LC50 of 24% methomyl was 760 ppb[24]. Due to H. rugulosus being defined as a broad tolerant species, methomyl has a high possibility to have a stronger effect against other anuran species.

Survivorship: The survival effect at the sublethal concentration confirmed the strong toxicity of methomyl in H. rugulosus. Although the tested concentration in this phase was about 60 times lower than the LC50 value, less than half of the H. rugulosus tadpoles were able to survive. In this study, the lowest observable adverse effect concentration (LOAEC) of methomyl for the early life stage of H. rugulosus was lower than 144 ppb; meanwhile, the LOAEC value of methomyl in the freshwater fathead minnow (Pimephales promelas) during the early life stage was 117 ppb[24]. Even the LC50 value of methomyl was obviously higher for H. rugulosus (8.69 ppm) than for other species of freshwater fishes, but the chronic toxicity was quite similar. Our results for the lethal and sublethal toxicities of methomyl in this study indicated strong effects on survivorship of H. rugulosus and revealed a trend regarding the methomyl toxicity concentration that could harm other amphibians as well.

Growth and metamorphosis: Our study showed that methomyl could decrease the body size at metamorphosis as well as the time to metamorphosis of H. rugulosus. This was therefore evidence of the endocrine-disrupting effect of methomyl. Clearly, metamorphosis of amphibians is directly regulated by thyroid hormones[25], [26]. Moreover, the levels of thyroid stimulating hormone (TSH), thyroxine (T4), and 3,3′,5-triiodothyronine (T3) affected the progression of metamorphosis events as well as the ratio of T3 and T4[27]. The disruption of thyroid hormone receptor gene expression and/or thyroid stimulating hormone could interrupt the whole metamorphosis event. However, the mechanism of methomyl in hormonal disruption is still unclear. The effect of methomyl on the thyroid gland was noticed earlier in mammals. For instance, Busey[28] found a significant increase in thyroid gland weight in Charles River rats treated with 200–400 ppm methomyl by oral administration. Meanwhile, Porter et al.[29] discovered that the thyroxine level increased in Sprague-Dawley rats exposed to a methomyl mixture. Besides the effects on body size at metamorphosis and time to metamorphosis, methomyl also influenced the growth of H. rugulosus. The growth rate of the anuran tadpole is normally greater in the premetamorphosis period (Gosner stages 21–30), and body length slightly increases during the premetamorphosis period (Gosner stages 31–40)[30]. Therefore, exposure to methomyl not only decreased time to metamorphosis but also reduced the premetamorphosis period, resulting in smaller sizes in the treated tadpoles throughout almost the entire experiment (Fig. 3). On the other hand, no evidence of interaction of methomyl with the thyroid pathway and no developmental endpoints or thyroid histopathological changes were observed in the amphibian metamorphosis assay[31]. Differences in species, stage of exposure, and stage of termination could affect the results. The levels of TSH, thyroid hormone (TH), corticotrophin-releasing hormone (thyrotropin-releasing hormone), and TH receptors were different in each developmental stage[32], [33]. Some TH receptors have been reported to be expressed in very early stages of life[32]. However, our results were insufficient to strongly guarantee the TH-disrupting effect of methomyl. Further studies are necessary to substantiate information about the endocrine-disrupting effect of methomyl.

Organ toxicity

Liver: In H. rugulosus, exposure to both lethal and sublethal concentrations of methomyl could produce severe liver damage. The livers of methomyl-exposed tadpoles exhibited a number of large vacuoles in the hepatocytes along with cytoplasmic disintegration. This result is in agreement with previous reports on histological lesions in livers of methomyl-treated organisms. Methomyl-treated mice previously showed cytoplasmic vacuolization[34]. Blood congestion was also reported in methomyl-exposed Sprague-Dawley rats[35]. A dilated central vein and sinusoids have been reported in some methomyl treated-rodents[34], [36], [37]. However, other histological responses to methomyl in anurans and rodents could possibly be different depending on their histology and physiology. The substance found in hepatic vacuoles of methomyl-treated tadpoles could not be identified in this study; however, abnormal tissue could alter liver function and cause failure of the homeostatic mechanism, leading to death. Crucially, all of these toxic effects at lethal concentrations were observed in the bullfrogs within 96 hours of exposure. In addition, exposure to a sublethal concentration of methomyl increased the MDA level in the liver and kidney of H. rugulosus. MDA is one of the important biochemical compounds used to indicate reactive oxygen species (ROS) generated from lipid peroxidation[38]. In general, ROS, which can be neutralized by a variety of antioxidants, are generated by cellular metabolism. However, excessive ROS would damage various chemical and biological membranes and have been suggested as a cause of toxicity in several organs[39], [40]. Furthermore, oxidative stress has also been shown to affect organ dysfunction and tissue damage[41]. In the present experiment, organ dysfunction and histological damage were observed in livers and kidneys of the methomyl-treated group. The livers of the treated frogs had dramatically low levels of glycogen, indicating failure to preserve energy for basic activities. Additionally, histological examination revealed severe damage in the liver leading to liver dysfunction. The hepatotoxicity of methomyl was previously reported to result in increasing levels of MDA together with histological damage[34], [37]. Furthermore, various liver enzymes were reported to be affected by exposure to methomyl[37]. Since the liver performs a variety of vital functions (nutrient metabolism, detoxification, and homeostasis), liver damage could lead to serious pathology and death.

Kidneys: In addition to the liver, methomyl also rapidly produced histological damage in the kidneys of H. rugulosus. The dilation of renal tubules and Bowman’s capsule space indicated the accumulation of fluid inside the kidney in treated frogs at lethal concentration. Fluid retention is related to kidney damage and acute renal failure[42], [43]. Histological damage of the kidney, in addition to the liver, has been reported in the literature. Congestion of glomerular capillaries, endothelial cell swelling, and degenerated tubules have been observed in several species of methomyl- treated rodents[36], [44], [45]. Moreover, exposure to a sublethal concentration also changed the histological features and biochemical parameters in the kidneys of the methomyl-treated frogs. The increase in MDA level was hypothesized as a cause of histological damage and NO reduction. Similar to the liver, ROS has been reported to exert histological toxicity in the kidneys[46]. Moreover, the level of NO in the kidneys of the methomyl-treated frogs was lower than that in the control group of frogs. A decreasing NO level in the kidneys has been reported to be an obvious marker for chronic kidney disease in many models[47],[48],[49], and ROS has been suggested to be involved in the progression of kidney disease through depletion of NO[47], [50]. Excessive ROS and reduction of NO have also been reported as a cause of the change in renal histopathology and biochemical reaction resulting in chronic kidney disease[51].

Gonads: For anurans, gonadal differentiation can be clearly observed in 4- to 5-week-old tadpoles (Gosner stage 27–28)[52]. Therefore, the effects of methomyl on H. rugulosus gonads could not be defined in the lethal toxicity test. However, histological damage in the ovary was found in the sublethal experiment. In the pesticide-treated group, the presence of vacuoles in oocytes could affect fertilization and/or embryonic development. Methomyl has previously been reported to have effects on reproductive toxicity in both male and female mammals. Mokhtar et al.[53] stated the toxic effects found in the ovary of female rats treated with methomyl, and Shanthalatha et al.[54] reported about the presence of vacuoles and hemorrhage in follicular cells including degenerated oocytes in female rats after treatment with sublethal doses of methomyl. The fact that the testes of froglets lacked any histological lesions in the present study is also of interest. Shalaby et al.[35] revealed that a sublethal concentration of methomyl induced histological damage in rat testes and revealed the toxic effect on sex glands and the testosterone level. Furthermore, histological and biochemical alteration in methomyl-treated male mice has also been reported[34]. In the testes of adult rodents, spermatogenesis was clearly observed, including pathological features. However, the histological findings for froglets in the present study (Gosner stage 46) showed immature testes without spermatogenesis inside. Therefore, the difference in histological effect of methomyl on testes observed in H. rugulosus might depend on the species, maturity of the testes, and/or chemical doses.

The present study clearly demonstrates the toxic effects of a methomyl-based insecticide on mortality, growth, histological and biochemical changes, and probability of endocrine-disrupting effects in H. rugulosus. The effects on histopathological changes of the ovary could be a serious problem affecting amphibian populations. Moreover, the extremely low level of liver glycogen indicates potential effects on living activities: preying, escaping predators, and mating. Our results might raise awareness about methomyl-based insecticide toxicities in amphibians and others non-tageted species.