Endocrine Disruption Of Tebuconazole On The HPG Axis Of Adult Zebrafish

In recent years,triazole fungicides have been widely used because of their high activity.At present,triazole fungicide is one of the largest market share pesticides,in which tebuconazole occupies the topest and top second usage amount of triazole fungicides and all fungicides,respectively.However,the environmental and health hazards due to the extensive use of tebuconazole have not been given enough attention.Nowaday,the environmental toxicology research on tebuconazole is rarely reported.At present study,zebrafish(Danio rerio)is used as a model organism,the bio-accumulation,retention and the distribution in different tissue of tebuconazole in zebrafish are evaluated by UHPLC-MS/MS.Based on it,in order to comprehensively reveal the reproductive endocrine disrupting effect of tebuconazole on the HPG axis of adult zebrafish and preliminarily explore it possible disrupting mechanism,the changes of the activities of oxidases,hormone content,steroidogenic metabolism related gene expression,gonadal pathological examination and offspring toxicity effect are investigated after tebuconazole exposure and post-exposure(cleaning)using different concentrations.The main results are displayed as follows.1.In order to reveal the bio-accumulation,retention and the distribution in different tissue of tebuconazole in zebrafish,adult zebrafish are exposed to tebuconazole with the concentration of 0.18 mg/L last for 28 d followed by a cleaning process for 30 d.Subsequently,the residues of tebuconazole in zebrafish or tissues are detected by UHPLC-MS/MS.Results show that tebuconazole tends to equilibrium after 28d exposure with the equilibrium concentration of 3.69 mg/kg.The BCF is 21.7,which means that tebuconazole is a moderate bioconcentration pesticide.The bioconcentration kinetic fits the pseudo first-order kinetic model.The order(from high concentration to low concentration)of tebuconazole distributing in different tissues is liver(5.71 mg/kg)>gonad(3.46 mg/kg)>whole body(3.03 mg/kg)>pars trunci(2.50 mg/kg)>brain(0.37 mg/kg).The highest concentration(31-fold of the exposure level)of tebuconazole is found in liver,while the concentration in gonad is 19-fold of the exposure level.It means that tebuconazole can be easily accumulated in liver and gonad of zebrafish.Compared to control,the body weight,body length,LSI and GSI are decreased during the exposure.It is also found that tebuconazole could be metabolized slowly and retained in zebrafish for a long time.The concentration of tebuconazole is 0.49 mg/kg(2.7-fold of the initial exposure level)after 30 d post-exposure.Compared to control,the body weight of female fish is still decreased(p<0.01),the LSI and GSI decline but without significant difference.2.The changes of the activity of the oxidases in zebrafish are investigated after exposure for 28 d and post-exposure for 30 d of tebuconazole at 0.18,0.92 and 1.84 mg/L levels.Results showed that the activities of SOD,CAT,POD and GST are up-regulated(1.11 to 3.43-fold(p<0.05),compared to control)with positive dose-effect relationship both in female and male adult zebrafish after exposure,which shows that tebuconazole has obvious activation induced effect on oxidase.The activities of SOD remain up-regulation(1.26 to 1.87-fold(p<0.05),compared to control)both in male and female fish.The activities of CAT remain up-regulation at low exposure level,while there is no significant difference between other treatment groups and control.The up-regulation of the activity of POD is only found in high exposure level.For GST,the activities are up-regulated in medium and high exposure levels in female fish,but in male fish it is recovered to control level.Result indicates that the activity of the oxidase is gradually recovered accompany with the concentration of tebuconazole in zebrafish goes down,but the effect is not completely eliminated during the post-exposure period.3.The influences of tebuconazole on hormones content,steroidogenic metabolism related genes expression are evaluated after exposure and post-exposure at 0.18,0.92 and 1.84 mg/L levels.It is found that hormone E2 tends to decline with dose-effect relationship at the three concentrations after exposure and it can recover to normal level at the end of post-exposure,but no influence is found for hormone T.Moreover,the expressions of vtgl,er?,star,cyp17,cyp19a,cyp19b,hsdl7b1,hsdl7b3,lhr and fshr mRNAs are down-regulated(p<0.05)in female zebrafish.After 30 d post-exposure,there is no significant difference of vtgl expression between each treatment group and control in the liver of female zebrafish.The expressions of era and star in female gonad are recovered to normal levels in all treatments except down-regulation at 1.84 mg/L group,but er? expressions in all treatments remain down-regulation(p<0.01).The expressions of cyp11a are up-regulated(p<0.05)both in 0.92 and 1.84 mg/L groups,and cyp17,cyp19b,hsd1763,lhr and fshr are up-regulated(p<0.05)in all treatments(all compared to control).4.The influence of tebuconazole on parental fecundity and offspring development are investigated after parental exposure and post-exposure.It is found that the mean fecundity of F0 generation significantly decreased and the malformation rate of juvenile zebrafish increases both after tebuconazole exposure and post-exposure.Compared to control,hatching time of embryo is prolonged after exposure,but this prolong effect is recovered in post-exposure group.Result indicates that the disrupting effect might be delivered from parental exposure to non-exposure offspring.5.No significant pathological change is found in female adult zebrafish ovary after tebuconazole exposure and post-exposure.However,tebuconazole exposure could influent the development of germ cells.Compared to control,the percentage of primary oocyte(PO)significantly increases by 4.99%,10.05%and 14.48%(p<0.05)for 0.18,0.92 and 1.84 mg/L exposure groups,respectively.But the percentage of late/mature oocyte(LPO)significantly decreasesby 21.62%,25.38%and 28.13%(p<0.01)for 0.18,0.92 and 1.84 mg/L exposure groups,respectively.At post-exposure groups,PO significantly increases by 9.44%,7.41%and 10.18%(compared to control,p<0.05)for 0.18,0.92 and 1.84 mg/L groups,respectively,whereas LMO recovers to normal level.Pathological observation provides physiological basis for fecundity toxicity after tebuconazole exposure.Summarily,tebuconazole is a moderate bioconcentration pesticide,it is not easy to be metabolized in zebrafish.Long-term low concentration exposure to tebuconazole could lead to endocrine disrupting effect on the HPG axis of zebrafish,and hence to bring adverse effect on parental reproduction and offspring development.This adverse effect can go on even if the exposure is escaped and cannot be restored and eliminate in a short term.Result preliminarily suggests tebuconazole may play as an anti-estrogenic chemical on zebrafish by inhibiting the mRNA expression of cyp19,and further lead to the down-regulation of hormone E2.