S. Pawlowski ^a,b, A. Sauer^b, J. A. Shears^c, C. R. Tyler^c and T. Braunbeck^b

^aInstitut fur Biologische Analytik und Consulting IBACON GmbH, Arheilger Weg 17, D-64380, Rossdorf, Germany. ^bDepartment of Zoology, Section V: Morphology/Ecology, Aquatic Toxicology and Ecotoxicology Group, University of Heidelberg, Im Neuenheimer Feld 230, D-69120, Heidelberg, Germany. ^cEnvironmental and Molecular Fish Biology, The Hatherly Laboratory, School of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS, UK.

Corresponding author. Tel.: +49-6154-697-348; fax: +49-6154-697-371.;

Abstract

The effects of the androgen, 17a-methyltestosterone were assessed on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) using a gonadal recrudescence assay. In this assay, mature male and female fathead minnow, previously kept under simulated winter conditions (15 °C; 8:16 h light:dark regime) were transferred to simulated summer conditions (25 °C water temperature; 16:8 h light:dark regime) to induce gonadal recrudescence. To assess sexual development fish were exposed to nominal concentrations of 0, 0.1, 1, 5 and 50 μg/L 17α-methyltestosterone. After 3 weeks of chemical exposure, effects on condition (condition factor, CF), plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI) and gonad histology were investigated. Reproductive performance, including reproductive output (egg production), spawning behaviour, and fertilisation rate were measured over a subsequent 3-week-period in breeding adults maintained in clean water. 17α-Methyltestosterone had no effects on the condition of fish at any of the doses tested. 17a-Methyltestosterone induced both androgenic and estrogenic effects with females generally more affected by 17α-methyltestosterone than males: atretic follicles and male-specific sex characteristics (androgenic effect) were induced in females at ≥0.1 and ≥1 pg/L 17≥- methyltestosterone, respectively. An inhibitory effect on ovary growth occurred at an exposure concentration of 50 μg/L 17≥-methyltestosterone. In males 1 μg/L 17α-methyltestosterone induced a concentration-response induction of plasma vitellogenin (estrogenic effect) likely due to its conversion into 17α-methylestradiol, rather to the competition with endogenous steroids and their cross reactivity with the estrogen receptor. In the fish breeding studies, concentration-dependent reductions in egg number, fertilisation rate and increases in abnormal sexual behaviour in females were observed. All of these effects occurred at exposure concentrations of ≥5 pg/L 17α- methyltestosterone. Thus, it could be assumed that the observed estrogenic effects in male fathead minnow were likely to the conversion of 17α-methyltestosterone into the estrogen 17α- methylestradiol, rather to the acting of 17α-methyltestosterone itself. In conclusion to this, showing hormonally activity of 17α-methyltestosterone in fish down to 100 ng/L, indicates that its potency was close to the range of several naturally occurring estrogens.

Author Keywords: Fathead minnow (Pimephales promelas); Gonadal recrudescence assay; 17α - Methyltestosterone; Endocrine disruption; Biomarker.