Effects of combined exposure to 17α-ethynylestradiol and dibutyl phthalate on the growth and reproduction of adult male zebrafish (Danio rerio)

• Combined in vivo toxicity of EE2 and DBP was studied in adult male zebrafish.
• Decreased VTG and AOX induction were found with mixed EE2 and DBP.
• Histopathological changes of testis, liver and gill were examined.
• The effects of mixed EE2 and DBP can be either antagonistic or additive.

To evaluate the combined effects of 17α-ethynylestradiol (EE2) and dibutyl phthalate (DBP) on the growth and reproduction of male zebrafish, three-month-old fish were exposed to 0.005 or 0.020 µg/L EE2, 100 or 500 µg/L DBP or their binary mixtures under semi-static conditions. Investigated parameters include the length, weight, condition factor, vitellogenin (VTG) induction, acyl-CoA oxidase (AOX) protein level, histopathological alteration of testis, liver and gill, and reproductive capacity. After 21 d exposure, no statistical difference was found among the weights, lengths and condition factors of different treatment groups. In all binary mixture groups, decreased VTG levels were detected compared to EE2-only groups; and the AOX levels were significantly lower than DBP-only treatments while both chemicals can individually induce AOX synthesis. Therefore, EE2 and DBP may act additively on VTG and antagonistically on AOX induction in males. After 45 d exposure, delayed gametogenesis was observed for the DBP-only groups, indicated by fewer spermatozoa and more spermatocytes, which was further aggravated with the addition of EE2. The developmental delay of testis partially recovered after a 30 d depuration in clean water. Combined exposure also caused liver and gill lesions, which were not alleviated during the 30 d depuration, suggesting a nonreversible harmful effect the same as single exposure. Mixed EE2 and DBP were observed to impair the reproductive capability (the fecundity and fertilization rate) of males, while single exposure did not. Co-exposed to 0.020 µg/L EE2 and 100 µg/L DBP promoted the early hatching of offspring (F1 generation) at 48 h post-fertilization (hpf), but the survival rates of the F1 generation were similar in all treatments. Our findings indicate that the effects of mixed EE2 and DBP at environmentally relevant levels can be either antagonistic or additive relying on the specific toxicological endpoints and the respective doses of each chemical.