Survey of estrogenic and androgenic disruption in Swedish coastal waters by the analysis of bile fluid from perch and biomarkers in the three-spined stickleback.

The potential for endocrine disruption close to sewage treatment plant and pulp mill effluent discharge points along the Swedish Baltic Sea coast was explored using a dual survey strategy employing two stationary fish species. The levels of vitellogenin and spiggin as biomarkers of endocrine disruption were determined in juvenile three-spined sticklebacks (Gasterosteus aculeatus L.) together with the sex ratios and the presence of intersex. As an indication of exposure, estrogenic and androgenic substances were analysed by GC-MS in bile from perch (Perca fluviatilis L.). Spiggin and vitellogenin levels in juvenile three-spined sticklebacks were generally low, and, for most sampling sites no deviation in gonad type ratios were observed. No remarkable levels of natural or synthetic estrogens or androgens were observed in bile fluid from perch, while bisphenol A and 4-nonylphenol were detected in perch from both reference sites and exposed sites. Taken together, the results did not indicate estrogenic or androgenic disruption in the investigated waters.

The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption. I. Sexual differentiation.

Juvenile three-spined stickleback (Gasterosteus aculeatus L.) is introduced as a unique model organism for both androgenic and oestrogenic endocrine action. Intersex is often used as an indicator for disruption of sexual differentiation in fish exposed to different kinds of effluents from human activities. In wild fish it has exclusively been reported in terms of feminisation due to xenoestrogens in the environment. The assumption that the intersex individuals are feminised genetic males can only be proven by genetic sex identification of the intersexual individuals. Intersex and gonadal sex reversal were induced in three-spined sticklebacks by treatment with natural and synthetic steroid hormones. Juvenile sticklebacks were exposed to three nominal concentrations of 17 beta-oestradiol (E2); i.e. 0.01, 1.0 and 10.0 microg/L; which were administered to the water either continuously from hatching to the end of the experiment (39-58 days post hatch), during the first 2 weeks after hatching only, from 14 days after hatching onwards, or during the chorionated embryo stage until hatching. Other groups were exposed to 17 alpha-ethinylestradiol (EE2) at 0.05 microg/L and 17 alpha-methyltestosterone (MT) at 1.0 microg/L (nominal concentrations). MT was applied continuously, during the first 2 weeks post hatch only, or from 14 days after hatching onwards. Gonad histology was examined and the genetic sex was identified with male sex-linked PCR markers. Treatment with oestrogens caused feminisation at the two highest E2 concentrations and with EE2. Exposure to E2 before hatching had no effect. Intersexual individuals from oestrogen treatments were genetic males. The genetic sex marker identified apparent total reversal of the gonad type of genetic males. Treatment with MT did not reveal a clear picture, since intersex was observed in both genetic females and males. MT also caused severe testis abnormalities, mainly the development of large branched cavities with unidentified origin. The process of sex differentiation is most sensitive to the influence of external steroids during the first 2 weeks after hatching. A lower incidence of intersex could also be induced in sticklebacks exposed from 14 days after hatching by E2 treatment, but not with MT. The combination of gonad histopathology with genetic sex identification in juvenile sticklebacks is suggested as a tool for detecting endocrine disruption in laboratory studies, and might become very useful in field surveys.

The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruption II--kidney hypertrophy, vitellogenin and spiggin induction.

This study investigated the suitability of juvenile three-spined sticklebacks, Gasterosteus aculeatus L., for detecting both androgen- and oestrogen-induced endocrine disruption. The investigated endpoints were kidney hypertrophy and the induction of the protein markers spiggin and vitellogenin. Juveniles were exposed to steroid hormones 17 beta-oestradiol (E2: nominal 0.01, 1.0 and 10 microg/L), 17 alpha-ethinylestradiol (EE2: nominal 0.05 microg/L) and 17 alpha-methyltestosterone (MT: nominal 1.0 microg/L) from the day of hatching until the termination of the experiments between 39 and 58 days after hatching. E2 (10 microg/L) and MT were applied during different time windows: (a) 14 days after hatching only and (b) continuously with start 14 days after hatching. Kidney hypertrophy is an androgen-dependent secondary sexual character in adult male sticklebacks and corresponds to the production of the glue protein spiggin during the breeding season. The kidneys were hypertrophied and spiggin levels were elevated in juvenile sticklebacks after treatment with MT. Paradoxically, slightly elevated spiggin levels and kidney hypertrophy were observed also in fish treated with high dose E2. Levels of vitellogenin, the oestrogen-inducible yolk precursor protein, were elevated in juvenile sticklebacks after E2 medium and high dose and EE2 treatment. The tested endpoints are suitable for the study of endocrine disruption in juvenile sticklebacks, a fish species that is easy to handle in laboratory and relevant for temperate geographical regions.