Jan Balaam

Identification of in vitro estrogen and androgen receptor agonists in North Sea offshore produced water discharges

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.

Characterisation of potentially genotoxic compounds in sediments collected from United Kingdom estuaries.

The mutagenic activity bioassay Mutatox was used to assess the mutagenic activity associated with sediments collected from five UK estuaries. Assays were performed on extracts of sediment pore water and residual particulate material isolated from sediment samples collected from the rivers Tyne, Tees, Mersey, and Thames as well as Southampton Water. No mutagenic activity was associated with the pore water extracts, however, 7 of the 28 organic solvent extracts of sediment particulate material contained potential genotoxins. By using Mutatox in association with bioassay-directed fractionation, attempts were made to identify the mutagenic compounds present in the extracts. The fractionation procedure used normal phase solid phase extraction, C18 reverse phase HPLC and cyano/amino bonded silica normal phase HPLC. GC-MS (EI and NICI) analysis was used to identify polycyclic aromatic hydrocarbons (PAH), alkyl substituted PAH, nitro-polycyclic aromatic compounds (nitro-PACs), polycyclic aromatic ketones, oxygenated-PACs, and other known mutagens contributing to the genotoxicity measured in the samples. Some potentially genotoxic compounds remain unidentified.

Potency and characterization of estrogen‐receptor agonists in United Kingdom estuarine sediments

The activity of estrogen-receptor (ER) agonists in sediments collected from the United Kingdom (UK) estuaries was assessed using the in vitro recombinant yeast estrogen screen (YES assay). The YES assay was successfully used to determine the in vitro ER agonist potency of pore waters and solvent extracts of sediments collected from UK estuaries. Estrogen-receptor agonists were detected in 66% of the pore water samples and in 91% of the sediment solvent extracts tested. The pore waters tested had ER agonist potencies from less than 2 to 68 ng 17beta-estradiol (E2) L(-1), whereas sediment extracts had potencies from less than 0.2 to 13 microg E2 kg(-1). A toxicity identification evaluation approach using bioassay-directed fractionation was used in an attempt to identify the ER agonists in extracts of sediments collected from the Tyne and Tees estuaries (UK). Gas chromatography-mass spectrometry was used to provide lists of compounds in the fractions obtained that were evaluated for known ER agonist activity using published data and an ER quantitative structure-activity relationship model. Toxicity identification evaluation characterization failed to identify any ER agonists in pore water extracts; however, three compounds in sediment solvent extracts were identified as ER agonists. Nonylphenol, cinnarizine, and cholesta-4,6-dien-3-one were identified in the sample collected from the Tyne estuary. Important ER agonist substances that contaminate marine sediments remain unidentified. The present study as well as previous work on effluents point toward the involvement of natural products in the estrogenic burdens of marine sediments. Further work is required to establish the relative contribution of natural products and anthropogenic chemicals to current environmental impacts in the context of the Oslo and Paris Commission strategy to eliminate hazardous substances by 2020.

Endocrine disrupting activities in sewage effluent and river water determined by chemical analysis and in vitro assay in the context of granular activated carbon upgrade

As part of endocrine disruption in catchments (EDCAT) programme, this work aims to assess the temporal and spatial variations of endocrine disrupting chemicals (EDCs) in River Ray, before and after the commissioning of a full-scale granular activated carbon (GAC) plant at a sewage treatment works (STW). Through spot and passive sampling from effluent and river sites, estrogenic and anti-androgenic activities were determined by chemical analysis and in vitro bio-assay. A correlation was found between chemical analyses of the most potent estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2)) and yeast estrogen screen (YES) measurement, both showing clearly a reduction in estrogenic activity after the commissioning of the GAC plant at the STW. During the study period, the annual average concentrations of E1, E2 and EE2 had decreased from 3.5 ng L(-1), 3.1 ng L(-1) and 0.5 ng L(-1) to below their limit of detection (LOD), respectively, with a concentration reduction of at least 91%, 81% and 60%. Annual mean estrogenic activity measured by YES of spot samples varied from 1.9 ng L(-1) to 0.4 ng L(-1) E2 equivalent between 2006 and 2008 representing a 79% reduction. Similarly, anti-androgenic activity measured by yeast anti-androgen screen (anti-YAS) of spot samples was reduced from 148.8 to 22.4 μg flutamide L(-1), or by 85%. YES and anti-YAS values were related to each other, suggesting co-existence of both types of activities from chemical mixtures in environmental samples. The findings confirm the effectiveness of a full-scale GAC in removing both estrogenic and anti-androgenic activities from sewage effluent.

Effects of sewage effluent remediation on body size, somatic RNA: DNA ratio, and markers of chemical exposure in three-spined sticklebacks.

Body mass, fork length, RNA:DNA ratio, specific growth rate, and hepatic EROD activity and CYP1A expression, were measured in three-spined sticklebacks in the River Ray (south west England) at sites downstream of an urban waste water treatment works (WWTW) prior to, and following, remediation of the effluent with granular activated carbon (GAC) tertiary treatment. During the same two-year period fish were also sampled from a neighbouring reference river (R. Ock). The WWTW effluent elevated water temperatures and nutrient content in the R. Ray and rendered a direct comparison of fish populations in the two rivers untenable. Instead, the stability of population parameters within each river during matched pre- and post-remediation periods was compared. Stickleback populations in both rivers were annual but fish in the R. Ray spawned earlier and were larger than those in the R. Ock. In the R. Ray fish gained mass throughout the winter months whereas in the R. Ock growth was much reduced during this period. In fish from the R. Ray the somatic RNA:DNA ratio remained elevated during May-November after remediation, rather than declining as in the same period pre-remediation and as was the case for fish in the R. Ock during both periods. The specific growth rate of the first post-remediation generation of sticklebacks in the R. Ray was higher than that of the previous pre-remediation generation. Following remediation there was no decline in hepatic EROD activity or in the abundance of hepatic CYP1A transcripts in fish in the R. Ray suggesting that the primary route of exposure to contaminants for these fish was not via the water column, and that the change in performance of the fish post-remediation was not impeded by continued exposure to contaminants. Both EROD activity and CYP1A expression increased in fish in the R. Ock during the later stages of the study suggesting that the fish in this river were exposed to an unidentified contaminant episode. This may have been linked with the poorer performance of fish in the R. Ock during the post-remediation period. The improved performance of fish in the R. Ray suggest that there may be factors in good quality secondary treated sewage effluent which can adversely influence the performance of fish populations, directly or indirectly, and which can be removed by tertiary treatment.

The use of modelling to predict levels of estrogens in a river catchment: How does modelled data compare with chemical analysis and in vitro yeast assay results?

Effluent discharges at Rodbourne sewage treatment works (STWs) were assessed using chemical and in vitro biological analysis as well as modelling predictions. Results showed that Rodbourne STW discharged less estrone (E1) than expected, but similar 17beta-estradiol (E2) and 17alpha-ethinyl estradiol (EE2) to those predicted by a widely cited effluent prediction model. The Exposure Analysis Modelling System (EXAMS) model was set up using measured effluent concentrations as its starting point to predict estrogen concentrations along a 10 km length of the receiving water of the River Ray. The model adequately simulated estrogen concentrations along the river when compared to July 2007 measured data. The model predicted combined estrogen equivalents in reasonable agreement with estrogenicity as measured by passive sampler (POCIS) extracts using the yeast estrogen screen. Using gauged mean flow values for 2007 the model indicated that the most important determinand for estrogen exposure in the Ray was not season, but proximity to the Rodbourne effluent. Thus, fish in the first 3 km downstream of Rodbourne were typically exposed to two or even three times more estrogens than those living 7-10 km further downstream. The modelling indicated that, assuming the effluent estrogen concentrations measured in February 2008 were typical, throughout the year the whole length of the Ray downstream of Rodbourne would be estrogenic, i.e. exceeding the 1 ng/L E2 equivalent threshold for endocrine disruption.

Bio-analytical and chemical characterisation of offshore produced water effluents for estrogen receptor (ER) agonists

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.

Identification of nonregulated pollutants in North Sea-produced water discharges.

Produced water collected from oil and gas platforms on the United Kingdom Continental Shelf was characterized for nonregulated pollutants through an effects-directed analysis procedure. Produced water samples were characterized for the presence of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) agonists using the dioxin-responsive, chemical-activated luciferase gene expression assay (DR-CALUX) and yeast estrogen screen (YES) bioassays. The AhR and ER agonists were then isolated by normal-phase, high-performance liquid chromatography and identified using gas chromatography coupled to mass spectrometry in a number of formats. The identified compounds were cross-referenced with those compounds routinely analyzed and regarded by the Oslo and Paris (OSPAR) Commission for the North East Atlantic as priority hazardous substances. The occurrence in produced water of a number of nonregulated compounds with demonstrable potential environmental effects is presented, to our knowledge for the first time. These include persistent organic contaminants, such as hexachlorobenzene, decachlorobiphenyl, and octachlorodibenzofuran.

Indices of stress in three-spined sticklebacks Gasterosteus aculeatus in relation to extreme weather events and exposure to wastewater effluent

Whole-body concentrations of cortisol and glucose were measured in three-spined sticklebacks Gasterosteus aculeatus from two rivers (Rivers Ray and Ock) in southern England during a 30 month period in order to assess effects on the stress axis of (1) remediation of a wastewater treatment works (WWTW) effluent (River Ray) and (2) episodic changes in flow rate arising from periods of high rainfall (Rivers Ray and Ock). The postcapture concentrations of cortisol and glucose in fish from both rivers did not exhibit a seasonal periodicity but did show significant between-sample, between-site and between-river variation, superimposed upon a consistent downward trend for each analyte during the monitoring period. Corticosteroid and glucose concentrations following capture were inversely linked with a progressive increase in condition of the fish during this period. Site-dependent trends possibly related to exposure to the WWTW effluent were detected for both analytes in fish from the River Ray. For fish in the River Ray, a significant proportion of variation in both corticosteroid and glucose concentrations, additional to the downward trend with time, was accounted for by temporal proximity of the sample to exceptional flow events arising from episodes of high rainfall and high turbidity. This relationship was not statistically significant for fish from the River Ock. These data suggest that the responsiveness of the stress axis in free-living G. aculeatus may be altered by exposure to WWTW effluent and by exposure to physical changes in the aquatic environment such as those arising from extreme weather events. The magnitude of these effects may be increased by exposure to both stressors concurrently.

Bioanalytical characterisation of estrogen and arylhydrocarbon receptor agonists in transplanted blue mussels (Mytilus edulis): proof of concept

The characterisation of estrogen (ER) and arylhydrocarbon (AhR) receptor agonists was performed in extracts of the tissues of transplanted blue mussels (Mytilus edulis). The yeast estrogen screen (YES) was used to detect the presence of ER agonists, whilst the DR-CALUX assay was used to detect AhR agonists. The concentration of ER agonists in mussel tissue from the Tees estuary, Brancaster and River Crouch (UK) were below the limits of detection for the YES assay (0.87 pg E2 g(-1)). AhR agonists were measured at concentrations of between 1 and 950 pg TCDD g(-1) in mussel tissue. A bioassay-directed fractionation of the sample extracts, followed by advanced broad spectrum gas chromatography-mass spectrometry analysis was then used in an attempt to identify the AhR agonists present. This showed that a complex mixture of AhR agonists occurs in the samples and that further work will be required in order to isolate and identify the individual compounds responsible.