Effects of binary mixtures of xenoestrogens on gonadal development and reproduction in zebrafish

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex ratio, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 microg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE+10 or 100 microg/l NP (EE1+NP10 or EE1+NP100, respectively), 10 ng/l EE+10 or 100 microg/l NP (EE10+NP10 or EE10+NP100, respectively) or solvent control (0.01% acetone, v/v) in a static-renewal system with replacement every 48 h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10+NP10 and EE10+NP100), increasing NP concentration acted antagonistically to the action of EE in terms of VTG induction at 60 dph. Similarly, non-additivity was observed with egg production, where EE1+NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in an additive fashion in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F1 swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after periods of depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects that depend on the particular response being determined and the respective exposure concentrations of each chemical.