| Methomyl (C5H10N2O2S), S-memy1-1-N-(methylcarbamoy1)-oxy]-thioacetimidate, is an insecticide belonging to the family of carbamate pesticides. And it was first introduced by E.I. du Pont de Nemours in 1968 and was regarded as one of the environmental estrogens by World Wild Fund for Nature in 1997. Because of its broad biological activity, relatively rapid disappearance and high efficiency against insects, methomyl is widely used in many agricultural countries for crop protection and soil or plant treatment. Methomyl has high water solubility (57.9 g·L-1 at 25℃), long half life and a weak-to-moderate adsorption to soils, and therefore poses a contamination risk to surface and groundwater, especially the methomyl applied in the agricultural area are expected to infiltrate into the groundwater and threatens the safety of the resource for drinking water. Methomyl residue level ranging from 0 to 97 μg·L-1 in environmental water has been reported, and even as high as 0.65 mg-L"1 has been detected in canal beside the field in which the methomyl had been sprayed. Methomyl has showed its harms to water ecology system and even drinking water resource of human being, for its large usage, high toxicity, long persistence, high detection ratio in natural water-bodies, high residual in environment and endocrine disruption to aquatic organisms. Although, there were some reports about endocrine disrupting effects and antioxidant system impairment of methomyl to mammal, such as rats, however, information on the endocrine disruption capacity and antioxidant system impairment of sublethal concentration of methomyl to aquatic orgnanisms is still generally limited and even no information has been available on fish. Therefore, in order to assess the security of methomyl to water ecology system scientifically and provide basic data for drawing and revising the standards of the maximum residue limit of methomyl in fisheries water bodies, it is very important and necessary to research the sublethal toxicological effects of methomyl to aquatic organisms, especially fish.Male Nile tilapia, Oreochromis niloticus, was chosen for this study because methomyl is one of the environmental estrogens having endocrine disrupting effects and tilapia is commonly available in most fish farms worldwide. The acute toxicity of methomyl to tilapia was done by the method of static water contacting toxicity tests. And based on the results of acute toxicity test, other three chronic toxicity tests of methoyl to male tilapia were done. That is, endocrine disruption effects of different concentrations (0.2 μg·L-1,2 μg·L-1,20 μg·L-1,200 μg·L-1) and different exposure time (0 d,6 d,12 d,18 d,24 d,30 d) of methomyl to male tilapia Oreochromis niloticus were investigated by analyzing the responses of the fish gonadosomatic index (GSI) and serum 173-estradiol (E2), testosterone (T),11-ketotestosterone (11-KT), vitellogenin (VTG); endocrine disruption mechanism of methomyl to male tilapia Oreochromis niloticus were investigated by analyzing the responses of mRNA expression levels of gonadotropin-releasing hormone (GnRH), estrogen receptor (ER), cytochrome P450 aromatase (CYP19), gonadotropin-releasing hormone receptor (GnRHR), follicle stimulating hormone (FSH), luteinizing hormone (LH), androgen receptor (AR), follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), steroidogenic acute regulatory protein (StAR),3 β-hydroxysteroid dehydrogenase (3β-HSD), vitellogenin (VTG) in hypothalamus-pituitary-gonadal axis; and antioxidant defense system impairment effects of different concentrations (0.2 μg·L-1,2 μg·L-1,20 μg·L-1,200 μg·L-1) and different exposure time (0 d,6 d,12 d,18 d,24 d,30 d) of methomyl to male tilapia Oreochromis niloticus were investigated by analyzing the responses of superoxide dismutase (SOD), catalase (CAT), glutathions-s-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione (GSH) and reduced glutathione (GSSG) in serum, kidney and liver. And the recovery test lasting 18 d was carried out simultaneously to assess whether the impairments caused by methomyl could recover or not.The results of acute toxicity of methomyl to tilapia showed that 24 h,48 h and 96 h IC50 of methomyl to tilapia were 1.25 mg·L-1,0.75 mg·L-1 and 0.43 mg·L-1 respectively. And the safety concentration of methomyl to tilapia was 0.043 mg·L-1. Methomyl is of very high toxicity to fish according to the standard on the grade of acute toxicity of pesticide. Therefore, it is very easy for methomyl to make fish die.The results of the endocrine disruption effects of methomyl to male tilapia showed that there were no significant changes in E2, T,11-KT, VTG levels in serum of male tilapia exposed to 0.2 μg·L-1 and 2 μg·L-1 methomyl compared to controls. Thus it would appear the 2 μg·L-1 methomyl might be considered as the no observed adverse endocrine disruption effect level. However, the significant changes in E2, T,11-KT, VTG levels in serum of male tilapia exposed to 20 μg·L-1 and 200 μg·L-1 were observed. Serum E2 and VTG levels were induced to increase significantly (p<0.05). The highest levels of serum E2 and VTG were reached at 24 d and 30 d respectively in the male tilapia exposed to 200 μg·L-1, and the highest levels were 261.81% and 2156.95% of the control respectively. Serum T and 11-KT levels were inhibited to decrease significantly (p<0.05). The lowest levels of serum T and 11-KT were both reached at 30 d in the male tilapia exposed to 200 μg·L-1, and the lowest levels were 43.78% and 41.22% of the control respectively. The results of effects of methomyl on male tilapia GSI showed that there were no significant changes in GSI of male tilapia exposed to 0.2 μg·L-1,2 μg·L-1 and 20 μg·L-1 methomyl compared to controls. However, GSI was inhibited to decrease significantly (p<0.05) in male tilapia exposed to 200 μg·L-1, and lowest value was reached at 30 d and the lowest value was 76.64% of the control. The significant changes in E2, T,11-KT, VTG levels in serum of male tilapia exposed to 20 μg·L-1 and 200 μg·L-1 in the present test indicated methomyl with concentrations no less than 20 μg·L-1 could affect the synthesis and metabolism of sex steroid hormones and VTG in male tilapia and therefore might result in its reproduction dysfunction. With respect to the sensitivity of GSI, E2, T,11-KT and VTG to methomyl, it could be concluded from our results that they followed the order:VTG> E2> 11-KT> T> GSI, suggesting VTG is the most sensitive biomarker to indicate methomyl pollution.The results of the endocrine disruption mechanism of methomyl to male tilapia showed that there were no significant changes in all the genes mRNA levels in HPG axis of male tilapia exposed to 0.2 μg·L-1 and 2 μg·L-1 methomyl compared to controls. Thus it would appear the 2 μg·L-1 methomyl might be considered as the no observed adverse endocrine disruption effect level, which is consistent with the conclusion drew from the effects of methomyl on E2, T,11-KT, VTG levels in serum of male tilapia exposed to 0.2 μg·L-1 and 2 μg·L-1. However, the significant changes in some genes mRNA levels in HPG axis of male tilapia exposed to 20 μg·L-1 and 200 μg·L-1 were observed. The mRNA levels of GnRH2, GnRH3, ERa, ERβ genes in hypothalamus, GnRHR and FSH(3 genes in pituitary, CYP19a、FSHR、ERα genes in testis and VTG and ERa genes in liver were induced to increase significantly (p<0.05). The highest mRNA levels of GnRH2, GnRH3, ERa, ERβ genes in hypothalamus, GnRHR and FSHβ genes in pituitary, CYP19a、FSFH、ERa genes in testis and VTG and ERa genes in liver were reached at 200 μg·L-1,20 μg·L-1,200 μg·L-1, 20 μg·L-1,20 μg·L-1,20 μg·L-1,200 μg·L-1,200 μg·L-1,20 μg·L-1,200 μg·L-1 and 200 μg·L-1 respectively, and the highest levels were 1.84 fold,2.35 fold,1.75 fold,1.56 fold,2.23 fold, 2.57 fold,3.42 fold,2.59 fold,2.03 fold,123.26 fold and 3.01 fold of the control respectively. The mRNA levels of LHR, StAR,3β-HSD, ARa genes in testis and LHβ gene in pituitary were inhibited to decrease significantly (p<0.05). The lowest mRNA levels of LHR, StAR,3β-HSD, ARa genes in testis and LHp gene in pituitary were reached at 20 μg·L-1,200 μg·L-1,200 μg·L-1,200 μg·L-1 and 200 μg·L-1 respectively, and the lowest levels were 0.46 fold,0.42 fold,0.39 fold,0.38 fold and 0.42 fold of the control respectively. However, there were no significant changes in mRNA levels of CYP19a gene in hypothalamus, ERP gene in testis and ARa and ERP genes in liver of male tilapia exposed to 20 μg·L-1 and 200 μg·L-1 methomyl compared to controls. This study provided convincing evidence for endocrine disruption of methomyl by disrupting multiple sites in HPG axis, leading to the changes of GSI and E2, T,11-KT, VTG levels in serum of male tilapia and resulting in reproduction dysfunction to fish.The results of the antioxidant defense system impairment effects of methomyl to male tilapia showed that there were no significant changes in the activities and levels of all the antioxidant parameters in male tilapia exposed to 0.2 μg·L-1 methomyl compared to controls. Thus it would appear the 0.2 μg·L-1 methomyl might be considered as the no observed adverse antioxidant defense system impairment effect level. However, the significant changes in the activities and levels of all the antioxidant parameters in male tilapia exposed to 2 μg·L-1,20 μg·L-1 and 200 μg·L-1 were observed. With respect to the response of antioxidants in different tissues and organs to methomyl, the change trends of time-dependent and dose-dependent in serum, kidney and liver were similar to one another to some extent. For it is easier to sample serum and determine serum antioxidants compared to liver and kidney, so the contents and activities of antioxidants in serum can be used to reflect the contents and activities of antioxidants in liver and kidney of fish exposed to methomyl. However, the times at which the activities and levels of antioxidants increase and decrease significantly in tissues and organs were different from one another, suggesting the impairments degree in serum, kidney and liver influenced by methomyl were different from one another. The impairments degree followed the order of liver> kidney> blood according to the changes of SOD, GST, GR, GPx, GSH and GSSG in serum, kidney and liver, and the impairments degree followed the order of kidney> blood> liver according to the changes of CAT in serum, kidney and liver. With respect to the change trends of antioxidants in different tissues and organs, GPx activities and GSSG levels in serum, kidney and liver of male tilapia exposed to 2 μg·L-1,20 μg·L-1 and 200 μg·L-1 were all induced to increase significantly (p<0.05), furthermore, GPx activities and GSSG levels increased with increasing of methomyl concentrations and delaying of exposure time. On the contrary, GSH levels in serum, kidney and liver of male tilapia exposed to 2 μg·L-1,20 μg·L-1 and 200 μg·L-1 were all inhibited to decrease significantly (p<0.05), furthermore, GSH levels decreased with increasing of methomyl concentrations and delaying of exposure time. However, the change trends of CAT, SOD, GST, GR activities in serum, kidney and liver of male tilapia exposed to 2 μg·L-1,20 μg·L-1 and 200 μg·L-1 were different according to the methomyl concentrations and exposure time, that is CAT, SOD, GST, GR activities in serum, kidney and liver of male tilapia exposed to 2 μg·L-1 were induced to increase significantly (p<0.05), furthermore, they increased with delaying of exposure time; and CAT, SOD, GST, GR activities in serum, kidney and liver of male tilapia exposed to 20 μg·L-1 and 200 μg·L-1 were induced to increase significantly (p<0.05) then were inhibited to decrease significantly (p<0.05) with delaying of exposure time. As reported, the levels or activities of antioxidants are potential biomarkers revealing a contaminant-mediated biological effect on the organism, Therefore, the significant changes of the activities of CAT, SOD, GST, GR, GPx and the contents of GSH, GSSG in the present study reflected the oxidative stress by balancing ROS in tilapia, suggesting methomyl with concentrations no less than 2 μg·L-1 could result in antioxidant defense system impairment to male tilapia.The results of the recovery tests showed that the toxicity produced by lower concentration of methomyl (no more than 20 μg·L-1) was reversible and the toxicity produced by higher concentration of methomyl (200 μg·L-1) was irreversible within 18 days after stimulus withdraw. So it could be concluded 200 μg·L-1 might besuggested as the threshold dose for methomyl-induced irreversible oxidative damage and endocrine disruption to male tilapia.The research results showed methomyl is of very high toxicity to fish according to the standard on the grade of acute toxicity of pesticide. Therefore, it is very easy for methomyl to make fish die. And the results of the antioxidant defense system impairment effects of methomyl to male tilapia showed that the 0.2μg·-1 methomyl might be considered as the no observed adverse antioxidant defense system impairment effect level and methomyl with concentrations no less than 2 μg·L-1 could result in antioxidant defense system impairment to male tilapia. The results of the endocrine disruption mechanism of methomyl to male tilapia showed that 2 μg·L-1 methomyl might be considered as the no observed adverse endocrine disruption effect level and methomyl with concentrations no less than 20 μg·L-1 could disrupt multiple sites in HPG axis, leading to the changes of GSI and E2, T,11-KT, VTG levels in serum of male tilapia and resulting in reproduction dysfunction to fish. With respect to the sensitivity of GSI, E2, T,11-KT and VTG to methomyl, it could be concluded from our results that they followed the order:VTG> E2> 11-KT> T> GSI, suggesting VTG is the most sensitive biomarker to indicate methomyl pollution. The results of the recovery tests showed that the toxicity produced by lower concentration of methomyl (no more than 20 μg·L-1) was reversible and the toxicity produced by higher concentration of methomyl (200 μg·L-1) was irreversible within 18 days after stimulus withdraw. So it could be concluded 200 μg·L-1 might besuggested as the threshold dose for methomyl-induced irreversible oxidative damage and endocrine disruption to male tilapia. |
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