John E. Thain

The extent of oestrogenic contamination in the UK estuarine and marine environments — further surveys of flounder

In 1996, The Centre for Environment, Fisheries and Aquaculture Science (CEFAS) initiated a project to establish whether oestrogenic materials are present in UK estuarine and marine waters at biologically significant concentrations, and to investigate some of the possible effects which these may have in flounder (Platichthys flesus). Early results are published elsewhere; this paper describes the results of a second wider survey of vitellogenin and reproductive abnormalities in UK flounder. Vitellogenin levels in male blood plasma in the period from spring to winter 1997 were found to be significantly elevated (in comparison with a clean reference site on the Alde estuary) in at least one sample from most of the 11 estuaries investigated. The exceptions were the Tamar and the Dee where all fish appeared entirely normal. In broad terms, the degree of oestrogenic contamination as measured by male vitellogenesis in the various estuaries was ranked in the following descending order: Tees > Mersey > Tyne > Wear = Humber = Clyde = Southampton Water = Thames > Dee = Tamar. VTG concentrations in Tees, Mersey and Tyne male fish were extremely high (> 100,000 ng/ml), and often exceeded those normally found in sexually mature females. At most locations, ovotestis conditions in male flounder were entirely absent but 9% of male Mersey fish and 7% of male Tyne fish contained ovotestis. In a few cases, eggs were fully developed with yolk granules. Most testes did not show gross morphological abnormalities related to oestrogenic exposure, although one testis from a Mersey fish appeared to be almost entirely composed of eggs. Abnormal sex ratios were not seen in any estuary. The paper concludes by outlining a new research programme which will be addressing the biological significance of these observations.

SURVEYS OF PLASMA VITELLOGENIN AND INTERSEX IN MALE FLOUNDER (PLATICHTHYS FLESUS ) AS MEASURES OF ENDOCRINE DISRUPTION BY ESTROGENIC CONTAMINATION IN UNITED KINGDOM ESTUARIES: TEMPORAL TRENDS, 1996 TO 2001

Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recorded in male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrine disruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrations in male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered by this study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, has decreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from the Forth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific point sources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured near the Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma of male fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustained decline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, with the majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to be drawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influences are discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these.

The impact of oestrogenic and androgenic contamination on marine organisms in the United Kingdom: summary of the EDMAR programme

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.

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.

Hepatic EROD Activity in Flounder (Platichthys flesus) as an Indicator of Contaminant Exposure in English Estuaries

Abstract To investigate the presence of sub-lethal contaminant effects in flounder ( Platichthys flesus ) populations in English estuaries, 16 sites from 7 estuaries were sampled in September–December 1997. Determination of hepatic ethoxyresorufin O-deethylase (EROD) activity revealed that significant ( p r =−0.84 and −0.80, respectively. It is suggested that the ‘out of season’ correlation of reproductive parameters with EROD could be linked to contaminant interference, perhaps by xeno-oestrogens. It is clear that flounder populations in English industrialised estuaries are exhibiting widespread sub-lethal contaminant effects and more research is essential to understand the potential implications of this for reproduction and disease that could be affecting the future viability of these populations.

Biomarker Responses in Mussels, an Integrated Approach to Biological Effects Measurements

Biological effects techniques have been used with the aim to further integrate biological effects measurements with chemical analysis and apply these methods to provide an assessment of mussel health status. Live native mussels were collected from selected coastal and estuarine sites around the British Isles, including the rivers Test, Thames, Tees, and Clyde, and Lunderston Bay. A suite of biological effects techniques was undertaken on these mussels, including whole organism responses (scope for growth), tissue responses (histopathology), and subcellular responses (lysosomal stability, multi-xenobiotic resistance [MXR], and Comet assay). In addition, whole mussel homogenates were used to measure organic (polycyclic aromatic hydrocarbons [PAH], polychlorinated biphenyls [PCB]) and metal concentrations. Overall the mussels collected from the Thames were in relatively poor health, based on histopathological markers, significantly higher DNA damage, and elevated expression of MXR detoxifying proteins. In contrast, the mussels collected from the River Test were in the best health, based on histopathological markers, respiration rate (SFG), and low frequency of DNA damage. In conclusion, the biological effects techniques were able to distinguish between relatively contaminated and clean environments, with the Thames mussels in worst health. Mussel tissue chemistry data were not able to explain the variations in biological response. Evidence indicates that the difference in the health of the mussels between the different sites was due to either effects of contaminants that were not measured, or the combined effects of mixture toxicity resulting in a threshold effect.

Intra- and interlaboratory calibration of the DR CALUX® bioassay for the analysis of dioxins and dioxin-like chemicals in sediments

In the Fourth National Policy Document on Water Management in The Netherlands, it is defined that in 2003, in addition to the assessment of chemical substances, special guidelines for the assessment of dredged material should be recorded. The assessment of dredged material is based on integrated chemical and biological effect measurements. Among others, the DR CALUX (dioxin responsive-chemically activated luciferase expression) bioassay has tentatively been recommended for inclusion in the dredged material assessment. To ensure the reliability of this bioassay, an intra- and interlaboratory validation study, or ring test, was performed, organized by the Dutch National Institute for Coastal and Marine Management (RIKZ) in cooperation with BioDetection Systems BV (BDS). The intralaboratory repeatability and reproducibility and the limit of detection (LOD) and quantification (LOQ) of the DR CALUX bioassay were determined by analyzing sediment extracts and dimethyl sulfoxide (DMSO) blanks. The highest observed repeatability was found to be 24.1%, whereas the highest observed reproducibility was calculated to be 19.9%. Based on the obtained results, the LOD and LOQ to be applied for the bioassay are 0.3 and 1.0 pM, respectively. The interlaboratory calibration study was divided into three phases, starting with analyzing pure chemicals. During the second phase, sediment extracts were analyzed, whereas in the third phase, whole sediments had to be extracted, cleaned, and analyzed. The average interlaboratory repeatability increased from 14.6% for the analysis of pure compound to 26.1% for the analysis of whole matrix. A similar increase in reproducibility with increasing complexity of handlings was observed with the interlaboratory reproducibility of 6.5% for pure compound and 27.9% for whole matrix. The results of this study are intended as a starting point for implementing the integrated chemical-biological assessment strategy and for systematic monitoring of dredged materials and related materials in the coming years.

Relationship between sex steroid and vitellogenin concentrations in flounder (Platichthys flesus) sampled from an estuary contaminated with estrogenic endocrine-disrupting compounds.

High concentrations of vitellogenin (VTG; egg yolk protein) have previously been found in male flounder (Platichthys flesus) from several UK estuaries; these levels have been ascribed to the presence of estrogenic endocrine-disrupting compounds (EDCs). Gonadal abnormalities, including intersex, have also been recorded in these estuaries. However, there is no firm evidence to date that these two findings are causally linked or that the presence of estrogenic EDCs has any adverse population effects. In the present study, we examined the relationship between concentrations of VTG and sex steroids (11-oxotestosterone in males and 17β-estradiol in females) in specimens of flounder captured from the estuary of the River Mersey. We first questioned whether the high concentrations of VTG in male and immature female flounder were indeed caused by a direct effect of exogenous EDCs and not indirectly via the endogenous secretion of 17β-estradiol. The data favored the direct involvement of estrogenic EDCs. We then questioned whether the presence of estrogenic EDCs not only stimulated inappropriate VTG synthesis but whether it might also have had a negative effect on endogenous steroid secretion. It should be noted that the predicted consequences of a drop in steroid secretion include smaller gonads, smaller oocytes, fewer numbers of sperm, and depressed spawning behavior. This question was more difficult to answer because of the strong effect of the seasonal reproductive cycle and stage of maturation on steroid concentrations. However, matched by month of capture and stage of maturation, both 17β-estradiol in females and 11-keto-testosterone in males were in most cases significantly lower in those years when VTG concentrations were higher.

Integrated indicator framework and methodology for monitoring and assessment of hazardous substances and their effects in the marine environment.

Many maritime countries in Europe have implemented marine environmental monitoring programmes which include the measurement of chemical contaminants and related biological effects. How best to integrate data obtained in these two types of monitoring into meaningful assessments has been the subject of recent efforts by the International Council for Exploration of the Sea (ICES) Expert Groups. Work within these groups has concentrated on defining a core set of chemical and biological endpoints that can be used across maritime areas, defining confounding factors, supporting parameters and protocols for measurement. The framework comprised markers for concentrations of, exposure to and effects from, contaminants. Most importantly, assessment criteria for biological effect measurements have been set and the framework suggests how these measurements can be used in an integrated manner alongside contaminant measurements in biota, sediments and potentially water. Output from this process resulted in OSPAR Commission (www.ospar.org) guidelines that were adopted in 2012 on a trial basis for a period of 3 years. The developed assessment framework can furthermore provide a suitable approach for the assessment of Good Environmental Status (GES) for Descriptor 8 of the European Union (EU) Marine Strategy Framework Directive (MSFD).

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K.

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-β-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.