Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations

Feminization of fish caused by certain estrogenic compounds e.g. 17 alpha-ethinylestradiol (EE2) has been shown to be partly reversible. So far it has not been studied if this applies for androgenic compounds too. The androgenic steroid trenbolone acetate (TbA) is used as growth promoter in beef cattle in the United States, South America, and Australia. TbA metabolites are stable in animal waste and have been detected in surface waters associated with feedlot areas and studies on both fish and mammals have demonstrated a strong androgenic effect of those metabolites. Zebrafish (Danio rerio) were exposed to environmentally relevant concentrations of the TbA metabolite 17beta-trenbolone from 0 to 60 days post-hatch (dph) and either sacrificed at 60 dph, transferred to clean water for 170 days or kept in exposure for 170 days. At 60 dph gonadal histology and vitellogenin analyses revealed all-male populations in groups exposed to 15.5 and 26.2 ng/L, and at 9.2 ng/L a skewed sex ratio towards males was observed. After the depuration period no sign of reversibility was observed. Environmentally relevant concentrations of 17beta-trenbolone cause a strong and irreversible masculinization of zebrafish and that raises concern about the effects of androgenic discharges in the aquatic environment. In addition this study also aids in understanding of the so far unknown sex determination process in zebrafish.