We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17a-methyltestosterone and 5alpha-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 micorg/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment.

Environmental androgens are a group of compounds that to date have received very little attention. In this study, a yeast-based androgen screen (YAS) was used to determine the level of in vitro androgenic activity in seven United Kingdom estuaries. Surface water, sediment pore water, and sediment particulate material solvent extracts collected from Southampton Water, the Thames, Mersey, Tees, Tyne, Clyde, and Forth were tested for in vitro androgenic activity. Eleven of the 41 surface water samples collected displayed androgenic activity >2 ng dihydrotestosterone (DHT) equivalents/L (3-9 ng DHT/L), while eight of the 39 sediment pore waters collected showed activity >45 ng DHT/L (51-187 ng DHT/L). High levels of androgenic activity were determined in the solvent extracts of sediments, with 10 of 39 samples exhibiting a level of androgenic activity >454 ng DHT/kg (1,020-15,300 ng DHT/kg). In vitro YAS testing of five selected sewage treatment works (STW) effluents entering these estuaries showed that measurable levels (34-635 ng DHT/L) of androgenic activity were observed in those receiving only primary treatment (Howdon STW and Irvine Valley Sewer) at the time of the survey. A toxicity identification evaluation (TIE) study of Irvine Valley Sewer effluent using the YAS assay was used to identify the natural steroids/steroid metabolites dehydrotestosterone, androstenedione, androstanedione, 5beta-androstane-3alpha,11beta-diol-17-one, androsterone, and epi-androsterone as responsible for 99% of the in vitro activity determined in the effluent.

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We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17α-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 μg/L), linuron (at 150 μg/L), and fenitrothion (at 15 and 150 μg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption.

The estrogen receptor (ER) agonist potency of offshore produced water discharges was examined via bioassay-directed chemical analysis. The in vitro estrogen receptor (ER) and androgen receptor (AR) agonist potency of five produced water samples collected from oil-production platforms in the British and Norwegian sectors of the North Sea was determined by using the yeast estrogen and androgen screens. Produced water samples were extracted in situ on the production platforms by using large-volume solid-phase extraction. All five extracts tested positive for the presence of ER agonists, whereas no AR agonist activity could be detected. By using the yeast estrogen screen assay in association with bioassay-directed fractionation, attempts were made to identify the ER agonist compounds present in the produced water extracts. The fractionation procedure used cyano-amino-bonded silica normal-phase high-performance liquid chromatography to isolate estrogenic compounds from produced water extract followed by full-scan gas chromatography-electron-impact mass spectrometry (GC-(EI)MS) to identify them. Isomeric mixtures of C1 to C5 and C9 alkylphenols contributed to the majority of the ER agonist potency measured in the samples.