The in vitro aryl hydrocarbon receptor (AhR) agonist potency of offshore produced water effluents, collected from the United Kingdom Continental Shelf, was determined using the dioxin responsive (DR)-chemically activated luciferase expression (CALUX) assay. Octadecylsilane (C18) solid phase extraction (SPE) extracts of produced water were exposed to DR-CALUX cells for 24h in order to investigate the contribution in potency from compounds that are stable to metabolism by the CALUX cells during exposure. The stable AhR agonist potency determined over 24h was highly variable and ranged from 1 to 430ng TCDD TEQCALUXl−1. These data reflect the highly variable composition of produced water discharges from different production fields. It is recommended that further work be performed to characterise the full range of stable dioxin like AhR agonists present in offshore produced water discharges using techniques such as bioassay-directed analysis.

The DR-CALUX assay has been utilised for the bio-analytical screening of a number of estuarine sediments for dioxin-like activity. Total sediment extracts (samples containing all extracted compounds) and cleaned-up extracts (samples with the most stable compounds isolated from the total extracts) were screened. The concentration of the stable dioxin-like compounds in the cleaned-up sediment extracts was between 1.0 and 106 pgTEQCALUX g(-1) dry weight. The majority of sediments contained levels of dioxin-like compounds that were above concentrations that are considered to be a low risk to aquatic organisms. The CALUX bio-analytical approach showed some disparity with the traditional analytical approach. The reasons for these differences have been identified tentatively: firstly, the DR-CALUX assay responds to all dioxin-like compounds, and secondly, it measures non-additive effects. The dioxin-like activity of compounds in sediment total extracts, which contain both labile and stable compounds, were also assessed and were six orders of magnitude higher than the cleaned-up samples. This suggests the vast majority of the total dioxin-like activity is attributable to labile compounds. Overall, the DR-CALUX assay is shown to be a useful tool in the assessment of dioxin-like activity in estuarine sediments.

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.

The in vitro estrogen receptor (ER) agonist potency and C1 to C9 alkyl substituted phenol content of offshore produced water effluents collected from the UK sector of the North Sea were determined using a combination of bio-analytical and chemical analysis techniques. An in vitro reporter gene assay was used to determine ER agonist potency, whilst gas chromatography coupled to mass spectrometry (GC-MS) was used to quantify the concentration of alkylphenols. The in vitro ER agonist potency was highly variable and ranged from less than the limit of detection (theoretically 0.03 ng 17beta-estradiol (E2) l(-1)) to 91 ng E2 l(-1). C1 to C5 alkylphenol concentrations were also highly variable ranging from 5 to 1600 microg l(-1) with a median concentration of 206 microg l(-1). These data reflect the highly variable composition of produced water discharges from different fields. The observed poor correlation of the alkylphenol isomer content and ER agonist activity suggests that other compounds present in the produced water discharges may be responsible for the ER agonist activity observed. It is recommended that further work be performed to characterise the full range of ER agonists present in offshore produced water discharges.

As a result of the introduction of tighter discharge limits and effluent treatment processes at source, the concentration of alkylphenol ethoxylates and nonylphenol present in the final effluent discharge from a sewage treatment works that treats trade effluent from the textiles industry was reduced. The estrogenic effects of the final effluent discharge to the Aire River were compared over a four‐year period during which various treatment measures were introduced. Male rainbow trout exposed to the effluent on four occasions in consecutive years (1994–1997) showed a reduction in the level of induced vitellogenesis between 1994 and 1997. A marked decrease in gonadosomatic index (GSI) and increase in heptaosomatic index (HSI) was measured in fish exposed to the effluent in 1994. In successive years, these differences diminished, and in the case of the GSI no measurable difference was observed between fish exposed to the final effluent or those in the control group in 1997. However, an increase in HSI was still measurable in 1997 in fish exposed to the final effluent and at sites farther downstream. The reduction in the effects of the effluent paralleled the reduction in the concentration of nonylphenol as well as its mono‐ and diethoxylates, which have been demonstrated to produce estrogenic effects in trout exposed to these compounds in the laboratory. This study demonstrates that the setting of more restricted discharge limits for known estrogenic chemicals of industrial origin can lead to significant reductions in the estrogenic activity of the watercourses into which the effluents are discharged.

This paper summarises results of the EDMAR programme which is investigating oestrogenic and androgenic endocrine disruption in UK coastal waters. Most of the data concern fish. Four species (flounder, viviparous blenny and two sand gobies) are experiencing feminisation in industrialised estuaries. In males this includes vitellogenin (VTG) synthesis, ovotestis induction and/or feminised sexual characteristics. Although reproductive success may be impaired in some cases, implications for fish populations are still unclear. Suspected causative contaminants include natural oestrogenic substances and synthetic oestrogen mimics. The majority of the oestrogenic activity is adsorbed to sediments, and routes of exposure may include benthic food chain transfer. Some natural androgenic substances are also being discharged to estuaries, but their activity appears low.

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 beta-estradiol and estrone. Despite removal of a high proportion of (lie 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.

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 17α‐methyltestosterone and 5α‐dihydrotestosterone over three‐week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 μg/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.

The ICES biological effects monitoring in pelagic ecosystems (BECPELAG) workshop is a multi-national, multi-disciplinary workshop aimed at establishing suitable techniques for monitoring the effects of contaminants on pelagic ecosystems. One of the many activities that have been performed concurrently is the extraction of water samples using semi-permeable membrane devices (SPMDs) and large volume solid phase extraction (SPE) followed by in vitro testing and targeted chemical analysis of the concentrated extracts. The following in vitro assays were used: DR-CALUX, yeast oestrogen and androgen screen (YES & YAS), blue mussel (Mytilus edulis) embryo, Tisbe battagliai, and Skeletonema costatum. Data from the study are presented along with recommendations on procedures for the use of in vitro in the monitoring of the pelagic 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, 5β‐androstane‐3α, 11β‐diol‐17‐one, androsterone, and epi‐androsterone as responsible for 99% of the in vitro activity determined in the effluent.

Twenty-six pesticidal compounds and samples of stormwater from two different agricultural catchments were screened for oestrogenic activity using an in vitro recombinant yeast-based assay. Only six fungicides showed an oestrogenic response with low comparative biological activity of 5000 to 2.5 million times less potent than 17beta-estradiol (E2), a natural steroidal oestrogen. Concentrations of biological activity expressed as E(2) equivalents for the headwater stream stormwater samples ranged from <0.01 to 0.11 ng E2/l. These values are at least one order of magnitude below levels that have been documented to produce oestrogenic effects in fish and are therefore considered to represent a low risk to associated headwater stream communities. The potential sources of the oestrogenic activity measured in the headwater streams are discussed.

This report describes the identification of important estrogenic compounds in surface and sediment pore‐water samples collected from the Tyne and Tees estuaries (United Kingdom) through the application of toxicity identification evaluation (TIE) procedures. The Tyne and Tees estuaries represent estuaries that have been historically impacted by industrial activities and continue to receive treated domestic sewage and industrial effluent. In 1998, Dabholm Gut on the Tees received a mixture of treated and untreated effluent, while Howdon sewage treatment works (STW) discharged primary treated effluents. An estrogenically active water sample collected from Howdon STW on the Tyne was shown to contain 17β‐estradiol, androsterone, and an unknown estrogenic compound(s). Most of the activity contained in a sample collected from the Dabholm Gut combined discharges on the Tees was also due to 17β‐estradiol with additional activity from nonylphenol and (tentatively) bis (2‐ethylhexyl)phthalate. The only sediment pore‐water sample to demonstrate estrogenic activity was collected from Dabholm Gut.

The transient movement of pesticides at biologically active concentrations during storm events is considered to be a cause of biological impoverishment in some headwater streams. The programme of work described sought to identify compounds that are the cause of toxic effects during such events. Along with targeted pesticide analysis, toxicity identification evaluation (TIE) procedures were used to identify compounds with a demonstrated toxic effect. These procedures were specifically directed towards isolating and attributing toxicity to classes of organic contaminants in samples collected from an English headwater stream during a storm event. The organic load was isolated by means of solid-phase extraction (SPE). Bioassay of the SPE extract at ×100 whole water concentrations confirmed that the samples contained substances toxic to Daphnia magna, although the raw samples were not toxic. Targeted pesticide analysis identified simazine and diuron as the major pesticides present and, using a toxicity unit (TU) approach, were shown to be responsible for a significant amount of the observed concentrate toxicity during a runoff event. However, they were not present in sufficient quantities to be totally responsible for a more toxic later event. By simplification of the SPE isolate using reverse-phase HPLC, fractions from which were tested for toxicity, the cause of concentrate toxicity in the later event was isolated to two discrete fractions. GC–MS analysis of these fractions identified nonylphenol, endosulfan sulphate and pendimethalin as present, with the majority of toxicity attributed to nonylphenol (NP). The main advantage of the TIE approach is that it allows biological active compounds with a demonstrated effect to be identified that may not be selected by more traditional techniques.

In order to assess the risk posed by hazardous substances present in the marine environment, investigations are required to characterise the most toxicologically significant substances present. This task can represent finding the proverbial needle in a haystack, since, in many industrially impacted areas, estuarine sediments and surface waters contain a complex mixture of natural and synthetic substances. To help the process, toxicity identification evaluation (TIE) procedures have been developed to isolate and characterise the biologically active compounds present. The TIE procedures have been combined with an acute bioassay using a marine copepod (Tisbe battagliai), mutagenic assay (Mutatox ) and an in vitro yeast-based screen for oestrogenic activity. A wide range of compounds, including pesticides, surfactant metabolites, natural steroids and industrial chemicals, have been identified and used to inform monitoring programmes and risk assessments.