Baseline data from the EDCAT research programme on oestrogens and fish populations

The EDCAT programme now has over two year’s baseline data on the chemistry and fish biology of an English river, the Ray, which receives oestrogenic sewage effluent from the city of Swindon. It has developed models which predict oestrogen concentrations in the river throughout the year, and has successfully corroborated these with data on oestrogenic substances, in vitro oestrogenic activity, and non-oestrogenic contaminants. It has also gathered data on the dynamics and oestrogenic responses of stickleback populations in the river during a period when the sewage discharge received both normal dilution, and high dilution from unusually large river flows. Finally, breeding experiments with wild intersex roach have been conducted in which the breeding success of each individual has been tracked by microsatellite analysis of offspring. The Swindon (Rodbourne) sewage treatment plant has now been upgraded with granular activated carbon filtration technology to remove oestrogens, and future work of the EDCAT programme will monitor the rate of recovery of fish populations in the River Ray

Use of the Three-Spined Stickleback (Gasterosteus aculeatus) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17α-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 μg/L), linuron (at 150 μg/L), and fenitrothion (at 15 and 150 μg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption

The impact of oestrogenic sewage discharges on fish populations

For the last 3 years, this multi-institute programme has been addressing the question of whether oestrogenic discharges from sewage treatment plants are able to damage fish populations. It has been known for some time that such discharges are able to feminise individual male fish of several species in rivers and estuaries, causing induction of various oestrogen biomarkers and altering the development of testicular tissue. It has been hypothesised that feminised (intersex) fish may be reproductively compromised, and this has been demonstrated in life cycle experiments in the laboratory. Furthermore, in vitro experiments have shown that sperm from strongly intersex male roach Rutilus rutilus has reduced ability to fertilise eggs. However, impacts of oestrogens on wild fish populations caused by treated sewage have not been demonstrated to date, although an experiment in a Canadian lake treated with ethinylestradiol at environmentally-relevant concentrations caused the collapse of a fathead minnow Pimephales promelas population. The programme described in this presentation (Endocrine Disruption in Catchments – EDCAT) has tackled this issue by studying wild 3-spined stickleback Gasterosteus aculeatus populations in a sewage-contaminated river, and has conducted semi-natural group-breeding experiments with R. rutilus, in which normal males have been allowed to compete with intersex fish. These studies have been supported by detailed chemical analytical and in vitro bioassay measurements, and by the development of improved oestrogen exposure models. The project has focused on the River Ray in southern England whose lower reaches are largely composed of treated sewage effluent from the city of Swindon, and a reference river (the Ock) which is similar to the Ray but receives far less effluent. The Ray was chosen because the Swindon sewage discharge (Rodbourne) was due to be upgraded with granular activated carbon (GAC) treatment plant in early 2008 which was expected to lead to a big reduction in oestrogenic (and other) inputs to the river. This change in inputs provided a good opportunity to study any recovery of fish populations. However, the intersex roach for the breeding experiments have been obtained from several other UK rivers including the Rivers Arun, Bourne, Aire and Nene

Hepatic transcriptional responses to copper in the three-spined stickleback are affected by their pollution exposure history

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Some fish populations inhabiting contaminated environments show evidence of increased chemical tolerance, however the mechanisms contributing to this tolerance, and whether this is heritable, are poorly understood. We investigated the responses of two populations of wild three-spined stickleback (Gasterosteus aculeatus) with different histories of contaminant exposure to an oestrogen and copper, two widespread aquatic pollutants. Male stickleback originating from two sites, the River Aire, with a history of complex pollution discharges, and Siblyback Lake, with a history of metal contamination, were depurated and then exposed to copper (46μg/L) and the synthetic oestrogen ethinyloestradiol (22ng/L). The hepatic transcriptomic response was compared between the two populations and to a reference population with no known history of exposure (Houghton Springs, Dorset). Gene responses included those typical for both copper and oestrogen, with no discernable difference in response to oestrogen between populations. There was, however, some difference in the magnitude of response to copper between populations. Siblyback fish showed an elevated baseline transcription of genes encoding metallothioneins and a lower level of metallothionein induction following copper exposure, compared to those from the River Aire. Similarly, a further experiment with an F1 generation of Siblyback fish bred in the laboratory found evidence for elevated transcription of genes encoding metallothioneins in unexposed fish, together with an altered transcriptional response to 125μg/L copper, compared with F1 fish originating from the clean reference population exposed to the same copper concentration. These data suggest that the stickleback from Siblyback Lake have a differential response to copper, which is inherited by the F1 generation in laboratory conditions, and for which the underlying mechanism may include an elevation of baseline transcription of genes encoding metallothioneins. The genetic and/or epigenetic mechanisms contributing to this inherited alteration of metallothionein transcription have yet to be established.This work was funded by the UK NERC postgenomic and proteomic programme grant NE/C507661/1 and by a Fisheries Society of the British Isles research grant to EMS. Birmingham functional genomics facilities were funded by BBSRC grant 6/JIF13209. We thank R.E. Godfrey, S. Jondhale, A. Jones, and L. Klovrza for technical assistance, J.K. Chipman for help and support, and the Environment Agency for provision of water chemistry data

Knowledge Hub on the Integrated Assessment of Chemical Contaminants and their Effects on the Marine Environment

In a time of environmental awareness, spurred on by the possibility that our world is threatened by climate change, it is important to remember that there are other anthropogenic pressures, which are also essential for addressing the protection of the marine and coastal environment. Pollution is a global, complex issue that contributes to biodiversity loss and poor environmental health and comes from the production and release of many of the synthetic chemicals that we use in our daily lives. Chemical contaminants are often underrepresented as a major contributor of environmental deterioration. The Joint Programming Initiative Healthy and Productive Seas and Oceans (JPI Oceans) established in 2018 the JPI Oceans Knowledge Hub on the integrated assessment of chemical contaminants and their effects on the marine environment. The purpose of the Knowledge Hub was to provide recommendations on how to improve the methodological basis for marine chemical status assessment. The work has resulted in the following policy paper which focuses on improving the efficiency and implementation of integrated assessment methodology of effects of chemicals of emerging concern. Substantial additional knowledge of biological effects is needed to achieve Good Environmental Status (GES) of our oceans and coastal areas. The Knowledge Hub is represented by highly skilled scientists and policy makers, appointed by the JPI Oceans Management Board, to ensure that the recommendations provided are useful for policy making

Towards a System Level Understanding of Non-Model Organisms Sampled from the Environment: A Network Biology Approach

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations