Joint Toxicity Of EE2and DBP On The Growth And Development Of Zebrafish

With the occurrence of endocrine disruption in the environment increasinglyserious, the effects of endocrine disrupting chemicals (EDCs) on the growth anddevelopment of organisms have gradually become the focus of research. Ethinylestradiol (EE2) and dibutyl phthalate (DBP), as two important EDCs, have been theconcern of many scientists. Studying the toxicity of phthalates and estrogens cancontribute to a good understanding of the basic mode of action of the interference ofmain EDCs on fish, and provide the basis for the assessment of their potential risks tothe environment.The single and joint toxicity of EE2and DBP on the embryonic development ofzebrafish were studied by observing the changes of the embryonic development andcalculating mortality and hatchability. The type of joint toxic effect was discussed.The results illustrate that zebrafish embryos are sensitive to the toxic effects of EE2and DBP, thus demonstrating a significant sensitivity both to the exposure dose and tothe exposure duration. The embryos begin to develop defects as well (e.g., tail defects,edema, spine curvature, delayed hatching). The toxicity of DBP is higher than that ofEE2; the96-hour median lethal concentration (LC50) of DBP and EE2are2.219mg/Land3.975mg/L, respectively. The joint-dose of DBP and EE2produce an antagonisticeffect on mortality of zebrafish at80hours but a synergistic effect at96hours.To understand the mixed endocrine disrupting effects of these two compounds onzebrafish, vitellogenin (VTG) induction was used as the toxicological endpoint. Theeffect of various exposure on VTG levels in the whole body homogenate of both juvenile and adult male zebrafish was investigated．A factorial design was employedto determine the type of their interactions on VTG induction in juvenile zebrafish. Theresults show that long term exposure to EE2can lead to VTG synthesis which showsa dose-effect relationship. No impacts of DBP on VTG levels was observed injuvenile zebrafish compared with the control. VTG levels in zebrafish exposed to thebinary mixture were significantly elevated compared with those exposed to DBPalone, but no significant interactions were observed, suggesting low dose of EE2andDBP can produce independent estrogenic effect in terms of VTG induction. After a21d exposure period using three-month-old male zebrafish, it was found that DBP mayact additively on VTG induced by EE2.For a more comprehensive understanding of long-term effects of binary mixturesof EE2and DBP on the growth, development and sexual differentiation of zebrafish,partial life-cycle tests (20-115dpf) were conducted to study the effects of single andcombined exposure to EE2and DBP on growth index (body length, body weight,condition factor), gonadal development, histopathological lesions of liver and gill andsex ratio. The results show that fish exposed to EE2alone and binary mixturesdisplayed a reduction in length and increase in condition factor and got fatter. Fishco-exposed to DBP and EE2had a significant increase in the percentage of femaleswhen compared with DBP alone (p<0.05). No statistically different growth index andsex ratio was observed among binary mixtures and EE2-only treatments, suggestingthat they were mainly affected by EE2. Exposure to0.5mg/L DBP alone causedfewer proportions of spermatozoa and retardation of gonadal development. Delayedtesticular development was found to be more severe with the addition of EE2thanDBP alone. An increased number of perinucleolar stage oocytes (Poc) and corticalvesicular stage oocytes (Coc) was found in fish exposed to EE2alone, indicating thatovary development may be slightly inhibited. Co-exposure to DBP and EE2alsoinduced certain damage to ovary, showing that DBP cannot alleviate the developmentretardance of ovary induced by EE2. There were no significant alterations in the liverof fish exposed to single chemicals. The hepatocytes began to swell, and nuclear atrophy and deformation were found in the liver co-exposed to5ng/L EE2and0.5mg/L DBP. Vacuolization of the liver was the main histological change in fishexposed to20ng/L EE2both alone and in combination with DBP (0.1or0.5mg/L),which tended to be more prevalent with elevating concentration of DBP. Fusion ofsecondary lamellae and severe clubbings at the tips of the secondary lamellae werenoticed in gills exposed to20ng/L EE2in combination with0.5mg/L DBP, indicatingmore severe impairment to the gill compared to the two chemicals alone.Histopathological results showed that EE2and DBP displayed synergistic effects ongonad development as well as liver and gill pathology.The combined effects between EE2and DBP were also evaluated in adult malezebrafish (Danio rerio). The results show that DBP may act antagonistically to theaction of EE2in terms of acyl-CoA oxidase (AOX) induction. After transferring toclean water for30days, fecundity and fertilization rate showed no significantdifference between the control and single exposure groups, while males co-exposed toEE2and DBP showed a significant decrease (P<0.05). The hatchability of F1generation at48hpf increased significantly in fish co-exposed to20ng/l EE2and0.1mg/l DBP, indicating that it might promote early hatching. There was no obviousdifference in the survival of the offspring (F1generation) in any treatment, ifcompared to the solvent control. EE2could aggravate the developmental delay oftestis induced by DBP, the extent of which displayed an EE2-concentration manner.The development of testis was partially recovered after30d depuration in clean water,but there was still a reduced number of spermatozoa. Combined exposure could alsocause liver and gill lesions which were as serious as those with the single exposure.After transferring to clean water for30days, the damage was not alleviated,suggesting the harmful effect of EE2and DBP on liver and gill seemed nonreversible.The effects of mixed EE2and DBP can be either antagonistic or additive relying onthe specific toxicological endpoints. In summary, this study evaluted joint toxic effects and potential toxicmechanisms of EE2and DBP on zebrafish in different stages. Embryonicdevelopmental toxicity experiment and chronic exposure experiments on juvenile andadult zebrafish were conducted in combination with various biomarkers, whichprovided seientific evidence for evaluation of the ecological safety of long-termexposure to various pollutants on living organisms.