507 Environmental Toxicology and Chemistry, Vol. 21, No. 3, pp. 507–514, 2002  2002 SETAC Printed in the USA 0730-7268/02 $9.00 + .00 ESTROGENIC ACTIVITY MEASURED IN A SEWAGE TREATMENT WORKS TREATING INDUSTRIAL INPUTS CONTAINING HIGH CONCENTRATIONS OF ALKYLPHENOLIC COMPOUNDS—A CASE STUDY DAVID A. SHEAHAN,*² G EOFF C. BRIGHTY,‡ MIC DANIEL,‡ SONIA J. KIRBY,² M ARK R. HURST,² JOE KENNEDY,‡ STEVEN MORRIS,² E DWIN J. ROUTLEDGE,§ JOHN P. SUMPTER,§ and MICHAEL J. WALDOCK² ²CEFAS, Centre for Fisheries and Aquaculture Science, Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex CMO 8HA, United Kingdom ‡Environment Agency, National Centre for Ecotoxicology and Hazardous Substances, Hawberry Park, Wallingford, Oxon OX10 8BD, United Kingdom §Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom ( Received 13 December 2000; Accepted 17 September 2001) Abstract—Chemical analyses were combined with a biological assay to investigate the main estrogenic chemicals as they passed through a sewage treatment works (STW) and entered a river. The STW studied was unusual in that it received wastewater from the textile trade. This wastewater was shown to contain high concentrations of alkylphenol polyethoxylates and their degradation products, such as nonylphenol. High-performance liquid chromatography fractionation, combined with biological assay, showed that the majority of the estrogenic activity was contributed by the alkylphenolic chemicals and the natural estrogens 17 -estradiol and estrone. Despite removal of a high proportion of the alkylphenolic chemicals by the various treatment processes within the STW, concentrations in the final effluent were still high compared to most other STW effluents in the United Kingdom. The effluent was very estrogenic to caged fish, as was the river water 2 and 5 km downstream of the STW, even though less so. Using various approaches, attempts were made to determine which group of chemicals contributed most to the estrogenic activity of the effluent. The analysis suggested that, in this unusual situation, the alkylphenolic chemicals may contribute the majority of the estrogenic activity of the effluent. However, this conclusion was based on a number of uncertainties that are presently unresolved and hence can be considered only tentative. Keywords—Estrogen Sewage Vitellogenin Alkylphenols Oncorhynchus mykiss INTRODUCTION A growing body of research in the United Kingdom has indicated that treated sewage effluents have the potential to produce estrogen-like effects in fish exposed to the final ef- fluent [1] and also in fish exposed in receiving waters [2,3]. It has been demonstrated that the natural steroids 17 -estradiol and estrone and the synthetic estrogen 17 -ethynyl estradiol are present at sufficient concentrations to account for the es- trogenic activity measured in final effluents from sewage treat- ment works (STWs) processing mainly domestic effluent [4,5]. However, their contribution to the total estrogenic budget in effluents containing significant amounts of industrial waste is less clear. One group of chemicals, nonylphenol ethoxylates (NPEs), is used in a wide variety of industrial processes. For all in- dustrial sectors, the estimated UK usage is 18,000 tpa, and 5% of this use is associated with the textile industry [6]. Long- chain NPEs that enter STWs are degraded primarily by suc- cessive removal of ethoxylate side groups from the hydrophilic side chain to yield nonylphenol (NP), nonylphenol mono- ethoxylate + nonylphenol diethoxylate (NP1EO+NP2EO), and carboxylated compounds [7]. It is the NP and NP1EO and NP2EO that are the most toxic [8], have the highest estrogenic activity [9,10], and generally have the longest environmental persistence [11]. Use of these chemicals in the textile industry in particular * To whom correspondence may be addressed (d.a.sheahan@cefas.co.uk). results in regionalized inputs of NPEs to trade waste received by STWs (82% of the UK textiles and woolen industry is located in the north of the country, 44% in the Yorkshire region alone) [12]. Thus, when suggesting improvements to effluent treatment systems in these regions to mitigate environmental impacts, it is important to assess the contribution of NPEs to the estrogenic activity. This paper describes a case study car- ried out in the Yorkshire region that underpins a project to identify and reduce the concentration of chemicals contributing to the estrogenic activity present in a UK river. The results described in this paper indicate that the breakdown products of alkylphenolic chemicals may make a significant contribu- tion to the estrogenic activity of STW final effluents, partic- ularly at downstream sites in the receiving waters to which they are discharged. MATERIALS AND METHODS The objectives of this study were to determine the concen- tration of NPEs from different trade sources entering a STW receiving textiles trade wastewaters and to evaluate the con- tribution of these compounds to the estrogenic activity of the final effluent. An in vitro technique (a recombinant yeast assay) and an in vivo caged trout bioassay were used to determine the estrogenic activity of the effluent. Study site selection The STW is situated on a site of approximately 0.25 km 2 in the Aire River valley, United Kingdom. It receives an av- erage daily throughput of approximately 10,000 to 40,000 m 3