Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows ( Pimephales promelas) using easily measured endpoints of sexual development

Abstract

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow ( Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25 °C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6) μg l −1, for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6) μg l −1 and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5) μg l −1. After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6 μg l −1, day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5 μg l −1), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6 μg l −1), but inhibited in female fish (6.0 μg l −1), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens.