Development of chronic tests for endocrine active chemicals. Part 2. An extended fish earli-life stage test with an androgenic chemical in the fathead minnow (Pimephales promelas)

The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 ¿g MDHT l-1 for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 ¿g l-1 was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 ¿g l-1. Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 ¿g/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 ¿g l-1 showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 ¿g l-1 up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had mature