Abstract-It was recently demonstrated that most, if not all, effluents of sewage-treatment works (STWs) in the United Kingdom are estrogenic to fish. As many STWs discharge into rivers, it is possible that some stretches of rivers downstream of where the effluent enters might also be estrogenic. To assess this possibility, the induction of vitellogenin synthesis in caged male trout placed at various distances downstream of the effluent entry point was used as a biomarker of estrogen exposure. Individual discharges into five rivers in England were studied. In four cases, fish placed in the neat effluent, or close to where it entered a river, showed very marked and rapid increases in their plasma vitellogenin concentrations, demonstrating that the effluent was estrogenic. In two of these four cases, none of the downstream sites were estrogenic, whereas in one of the four, fish placed at a site 1.5 km downstream did respond by synthesizing appreciable amounts of vitellogenin, although sites further downstream were not estrogenic. The situation in the fourth river was quite different; not only was the effluent extremely estrogenic (a maximum vitellogenin response in the mg/ml range was attained), but so were all the other study sites on the river, the last of which was 5 km downstream of where the effluent entered. This particular river receives trade effluent from wool-scouring mills, which contains much higher concentrations of alkylphenolic chemicals than any of the other discharges studied. It is suggested that these chemicals probably account for the estrogenic activity of this river. The final (fifth) river showed no estrogenic activity, not even in the neat effluent. This discharge comes from a very small STW, which receives no trade waste, and one or both of these factors may account for why the effluent (and hence the river) was not estrogenic.

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Abstract-Vitellogenin, a yolk-precursor protein normally found only in the blood plasma of sexually mature female teleosts and other egg-laying vertebrates, was used as an indicator of exposure of male rainbow trout (Oncorhynchus mykiss) to exogenous estrogens. Vitellogenin concentrations were measured using a specific radioimmunoassay for trout. Cages containing adult male trout were placed at the points of discharge and at varying distances downstream of treated sewage effluent outfalls into the River Lea (UK) during the summer and winter months of 1992. After 3 weeks exposure at the majority of sites, fish held up to 15 km downstream of inputs showed an increase in plasma vitellogenin concentration, with statistically significant elevations up to 4.5 km downstream. A repeat survey in November 1992 below Harpenden sewage treatment works showed that the only two stations to give a significant response were 3 m and 1.6 km downstream of the outfall. This reduced effect compared to the first survey is thought to be due to dilution of both the influent sewage to the treatment works and of the river water itself by increased rainfall, the overall increase in dilution being approx. 36%. Trout were also placed in 15 raw water storage reservoirs in southeast England during the summer of 1993 for an exposure period of 6 weeks. No biologically significant increases in plasma vitellogenin concentration were observed in any of the reservoirs.

Existing in vivo tests (with the exception of the full lifecycle test) are not adequate for assessing the reproductive effects of endocrine disrupting chemicals (EDCs) on fish, and hence the need for partial life-cycle tests has been recognized internationally. In this paper we describe the development of a short-term (6 week) reproductive performance test for EDCs using pair-breeding fathead minnows (Pimephales promelas). In the test, reproductive performance in paired fish is assessed over two 3 week periods, one with exposure to the test chemical and one without. The test is highly integrative and measures effects of exposure to chemicals on fecundity, gonadosomatic index (GSI), vitellogenin (VTG) induction, and secondary sexual characteristics (fat pad and tubercles in males). In this test, exposure to butyl benzyl phthalate (BBP) at a nominal concentration of 100 µg/L (measured concentration between 69 µg/L and 82 µg/L) had no discernible effects on reproductive performance. In contrast, all reproductive parameters measured were affected by exposure to 4-NP, albeit some (e.g. VTG induction and reduction in the prominence of secondary sexual characteristicslowest effective dose between 0.65 µg/L and 8.1 ( 1 µg/L [measured]) were more sensitive than others (e.g. number of eggs and spawnings, where the lowest effective dose was between 8.1( 1 µg/L and 57.7 ( 3 µg/L [measured]). Concentrations of 4-NP at or above 48 µg/L [measured] inhibited reproduction completely.

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Sewage treatment works (STW) effluents throughout the U.K. have been shown to be estrogenic, inducing vitellogenin (VTG) synthesis in caged and wild fish for considerable distances (up to several kilometers) downstream of the effluent discharge. Thresholds for vitellogenin induction in caged fish in those studies have been derived from shortterm exposures, and may not necessarily be representative of thresholds for estrogenic responses in wild fish living in rivers that contain STW effluent. In addition, very little is known about the long-term fluctuations in the concentrations of the estrogenic components of STW effluents. In this paper, it was established that the concentrations of natural steroid estrogens and xenoestrogens (alkylphenolic chemicals) in a treated sewage effluent fluctuated temporally (over 8 months), from between 36 and 308 ng/L and between <0.2 (detection limit) and 10.7 µg/L, respectively. Long-term exposure of maturing adult roach to a graded concentration of this effluent (0, 9.4, 17.5, 37.9, and 100% v/v) demonstrated that the vitellogenic response was both dose and time dependent. After 1 month exposure, the response threshold was 37.9 ( 2.3% treated sewage effluent, whereas after 4 months exposure, a significant induction of VTG occurred at an effluent concentration of 9.4 ( 0.9%. The data presented suggests that estrogenic responses in wild fish living in U.K. rivers cannot necessarily be predicted from short-term exposures using caged fish. The functional significance and/or ecological consequences of induction of vitellogenin resulting from exposure to STW effluent has yet to be determined in wild fish.

Rainbow trout were exposed for 3 weeks (in a flow‐through system) to various dilutions of treated effluent (25, 50, 75, and 100%) from Harpenden and Chelmsford sewage treatment works (STW) during November 1994 and August 1996, respectively, and the induction of plasma vitellogenin was measured. Significant (p < 0.05) increases in plasma vitellogenin concentrations occurred in fish held in effluent from Harpenden STW at concentrations of 50% and above. At Chelmsford STW, the effluent appeared to be more potent, causing induction of vitellogenin in fish at concentrations as low as 25% (compared with 50% for the Harpenden effluent). Exposure to lower concentrations of effluent emanating from Chelmsford in April of the following year indicated that estrogenic activity was diluted to no‐effect levels at this STW at concentrations between 12 and 25%.

Abstract-Rainbow trout were exposed for 3 weeks (in a flow-through system) to various dilutions of treated effluent (25, 50, 75, and 100%) from Harpenden and Chelmsford sewage treatment works (STW) during November 1994 and August 1996, respectively, and the induction of plasma vitellogenin was measured. Significant ( p Ͻ 0.05) increases in plasma vitellogenin concentrations occurred in fish held in effluent from Harpenden STW at concentrations of 50% and above. At Chelmsford STW, the effluent appeared to be more potent, causing induction of vitellogenin in fish at concentrations as low as 25% (compared with 50% for the Harpenden effluent). Exposure to lower concentrations of effluent emanating from Chelmsford in April of the following year indicated that estrogenic activity was diluted to no-effect levels at this STW at concentrations between 12 and 25%.