Development of a short‐term reproductive endocrine bioassay using steroid hormone and vitellogenin end points in the estuarine mummichog ( Fundulus heteroclitus )

We have developed a short‐term gonadal recrudescence test with the estuarine mummichog ( Fundulus heteroclitus ) and determined endocrine end points sensitive to a strong estrogen agonist (ethynylestradiol; EE 2 ) and an antiestrogen (ZM 189,154; ZM) at concentrations of 0 to 1,000 ng/L in three separate experiments. A protocol was developed to ensure a year‐round supply of recrudescing fish. A protocol for determining steroid production (testosterone and 11‐ketotestosterone [11‐KT] in incubated testes tissue and testosterone and 17‐estradiol [E 2 ] in incubated prematurational follicles) was optimized. Recrudescing fish (males, gonadosomatic index = 2%; females = 10%) were exposed to graded doses of EE 2 or ZM for 7 to 15 d using a static daily‐renewal protocol. At high EE 2 (>250 ng/L), the effect on males was depression of androgen steroidogenesis and plasma steroid levels. In females, high EE 2 depressed gonadal production and circulating E 2 levels; however, EE 2 concentrations <100 ng/L caused increased gonadal production and plasma E 2 . Low ZM (<100 ng/L) had little effect on male and female fish, while higher concentrations (>250 ng/L) increased E 2 and 11‐KT production while decreasing plasma 11‐KT and E 2 (1,000 ng/L only). Male and female plasma vitellogenin responded in a concentration‐dependent fashion to EE 2 with no effect by ZM. The low observable effect concentrations for the endocrine parameters were 1 ng/L for EE 2 and 250 ng/L for ZM. The bioassay and results encompassing the environmentally relevant exposure range (1–100 ng/L) will be useful for assessing effects of endocrine‐active contaminants in estuarine environments.