Population-level Consequences Of Exposure Of Fish To Oestrogenic Wastewater Treatment Works Effluents

Lead Research Organisation: University of Exeter
Department Name: Biosciences


A very significant amount of the water flowing in many UK rivers originates from the effluent of wastewater treatment works (WWTW) and this can affect the native wildlife. We have shown that reproduction in fish living in many UK Rivers, most notably in the roach, a common member of the carp family of fish, has been damaged by exposure to chemicals contained in the WWTW effluents that disrupt the body's sex hormone systems. The chemicals identified responsible for these effects were oestrogens (female sex hormone) and included natural oestrogen hormones, pharmaceutical oestrogens taken by women as part of the contraceptive pill, and some industrial chemicals similar in their structure to natural oestrogens. The presence of feminised fish in the wild is of concern because we have shown that they have a reduced capacity to breed and this may potentially lead to adverse effects for the population and impact the ecosystem more widely. Disorders in human reproductive health occur that are very similar in nature to effects seen in wild fish and some of these effects have been associated with exposure to oestrogens. Adding further concern about WWTW effluents in UK Rivers, long term exposures of fish have found additional adverse health effects including disruption to the immune system and genetic damage. This project seeks to investigate the likely consequences of exposure to oestrogenic WWTW effluents for the sustainability of fish populations living in UK Rivers, using the roach as sentinel species. Although the roach is very common in many polluted water systems, this can sometimes give misleading impressions of the health of the population. Arguably, it is better to count the number of breeding individuals in a population rather than the total number of fish because fish populations with low numbers of breeding fish have low genetic variation and inbreeding and may even be at risk of extinction. Given the results of our experiments performed to date, we think that populations of roach from more polluted sites will have smaller numbers of breeding individuals and will be more inbred than those from clean sites, because a higher proportion of fish will be unable to breed. To investigate this we will first make assessments on the ability of roach to breed in in groups that include fish that have been exposed throughout their whole lives to an oestrogenic WWTW effluent and fish that have been maintained throughout their lives in clean water. We will use a genetic technique (DNA microsatellites) to identify the parents of the offspring produced and in turn determine the reproductive success of individual fish in the competing groups and try to relate this success to their sexual physiology. We will then conduct experiments to investigate how exposure to the oestrogenic WWTW effluent over one and two generations impacts the development of the testes and ovaries and consequent breeding success in the second generation. In the final part of this project we will investigate the underlying genetic health of wild roach populations in UK Rivers using DNA microsatellites for populations collected from rivers sites with different levels of pollution with oestrogenic WWTW effluent. Ultimately, our results will enable us to assess the implications of life long exposure to oestrogenic WWTW effluents on both individuals and populations, advancing significantly knowledge on how the sustainability of roach populations in UK Rivers may be impacted. This work is intended to greatly improve the assessment of the health impacts of exposure to complex mixtures of chemical discharges on fish populations in the natural environment for the better protection of our aquatic resources and biodiversity. The work will have importance in the regulation of discharges and thus is of very wide interest to the government regulatory bodies, environment protection groups, industry and the wider public.


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Brown AR (2014) Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

Description The project set out to determine if there are population-level consequences for fish living in UK rivers contaminated with wastewater treatment works (WwTW) effluent, using roach, Rutilus rutilus, as the study sentinel species. First we set out to determine the effects of life-long exposure to an estrogenic WwTW effluent on the reproductive capabilities of roach. We had already established reproductive failure after chronic exposure of roach to a treated estrogenic wastewater effluent for up to 3.5 years. To understand the reasons for this we then conducted parentage analysis using DNA microsatellites on breeding colonies that included both exposed control fish to determine the reproductive fitness of exposed fish. Histological analysis of the gonads demonstrated that all fish exposed to 100% effluent for 3 years were females.
Presumptive females exposed to sexual maturity to WwTW effluent bred normally, but putative sex-reversed males breeding as females contributed poorly, if at all. The results of this study indicate that exposure to WwTW effluents can have substantial impacts on breeding capabilities, with likely implications at a population level (Environ. Sci. Technol. 2011, 45, 1673-1679).

We then investigated evidence for trans-generational effects by exposing their offspring to either 100% effluent or 100% water. Gonadal histology was examined at 3 years (160 fish) and again at 3 years and 9 months (36 fish). We found no evidence for endocrine disruption in males kept in water, whether the maternal parent was exposed or not. Analysis of the gonads of males exposed to 100% effluent, revealed various degrees of feminisation, confirming estrogenicity of the effluent; no differences between fish with effluent-exposed or water-exposed mothers were found. Surprisingly the majority of feminised male fish had only a few oocytes per gonadal section, in contrast to the all-female population from the previous exposure. Chemical analysis on the effluent demonstrated that ammonia levels were substantially lower during periods of the second exposure and concentrations of steroidal estrogens were low (19ng/L E1, 2.1 ng/LE2 and no EE2 was detected) a likely consequence of improvements to the WwTW works undertaken during the second exposure. In order to evaluate the effects of maternal exposure to a WWTW effluent on reproductive competiveness of the offspring, we conducted a breeding study using 160 fish at the end of the 3rd year, with two breeding scenarios in 8 tanks. Parentage analysis revealed no difference in reproductive success between fish with either effluent exposed or control mothers.
Our final objective was to test for evidence of reduced genetic variation in populations of wild roach inhabiting rivers contaminated with estrogenic WwTW effluents. We genotyped a total of 1769 fish from 32 sites from sites of roach with between 14-19 DNA microsatellites predominantly in the region of the Thames catchment. Analysis of population structure demonstrated that some populations have been confined to some of the most contaminated river stretches over multiple generations, and have not been sustained by immigration. We found no evidence that size of breeding populations (Ne) or genetic diversity was reduced in more contaminated rivers. There was, however, evidence for less variation in (Ne) from the more effluent contaminated rivers, and a 65% reduction at for the most contaminated sites could not be ruled out. These results suggest that roach living in some effluent-contaminated river stretches where feminization is widespread are self-sustaining (BMC Biology 2014 12:1
Exploitation Route The data derived for this work has been used by the UK Environment Agency, UK Water Industry and defra in their deliberations about the impact of wastewater treatment works effluent discharges on fish populations and decisions on investments for improvements in treatment technologies.

This research has formed the ground work for addressing population resilience for the effects of exposure to oestrogen in fish living in UK rivers. Extremely little is known on population level impacts and resilience for any contaminant discharged into the environment.
Sectors Chemicals,Communities and Social Services/Policy,Education,Environment

URL http://www.youtube.com/universityofexeter#p/c/868210A42F3C27E1/6/rbD-8xnbezA
Description This work provided a significant advance in our understanding on the effects of oestrogenic wastewater treatment works (WWTW) effluents on the feminisation of fish (roach, Rutilus rutilus) and consequences for fish populations in UK rivers. It established that long term exposure to a WWTW effluent (full strength) induced complete sex reversal (all males became females). However, the work also established that the intersex condition in wild fish populations did not appear to impact adversely on the genetics (diversity) of wild populations. In this project a range of molecular tools were developed and implemented, spanning targeted qRT-PCR, gene arrays and DNA microsatellites into various laboratories, including an industry laboratory, and these tools are being used to address the mechanisms by which EDCs work and investigate their population level consequences. Information derived from this research on EDCs has been pro-actively supplied to government bodies, industry and international bodies, as evidenced by the many workshops attended/hosted and international meetings at which we have been invited to present our data. A new collaboration was set up with South West Water through this NERC funded project . Our work on EDCs between 2008-2012 has appeared in the Times, Telegraph, Observer, and other national and international newspapers. It has been a feature article in US National geographic and Frontiers in Science. It has also appeared in NERC News, as NERC podcast, as an ACS webinar, as short videos on U-tube (e.g. http://www.youtube.com/universityofexeter#p/c/868210A42F3C27E1/6/rbD-8xnbezA), featured on BBC televisions including Country File and features in various international news and televisions programmes. The main researcher employed on this grant (Dr Patrick Hamilton) secured a further post doctoral research post following on from this grant. The research resulted in further research funding secured from NERC and defra.
First Year Of Impact 2008
Sector Chemicals,Communities and Social Services/Policy,Environment
Impact Types Societal

Description DEFRA - impacts of EE2 and endocrine disruption
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
Description Environment Agency - DNA microsatellite genotypes
Geographic Reach National 
Policy Influence Type Participation in a national consultation
Impact We have worked with the EA to investigate the ability of the microsatellite dataset generated in the project to determine the effectiveness of the EA's restocking practices. We demonstrated that it is a useful tool to distinguish between roach introduced from the Environment Agency's farm fish from native fish in most rivers using their DNA microsatellite genotypes.
Description NERC NE/K004263/1
Amount £400,000 (GBP)
Funding ID NE/K004263/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 04/2013 
End 04/2016
Title Microsatellite genotypes 
Description Microsatellite genotypes for 1,769 roach genotyped in this study are available at http://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-12-1. This has now been expanded in the current NERC-funded project to include rivers in northern England. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact as above 
Description Anglian Water Services NE/G019355/1 
Organisation Anglian Water Services
Country United Kingdom 
Sector Private 
PI Contribution Provision of data on chemical content and biological activity of selected chemicals in wastewater discharges. Reporting
Collaborator Contribution Access to Wastewater treatment works for fish exposure studies, staff support for these exposures.
Impact Research Papers Reports (incl government reports) International presentations
Description AstraZenenca - data, staff training, papers 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Provision of data on chemical effects analysis in fish and other animals Training of AZ staff in molecular methods Development of specific test methods Co-authored research papers Shared research programmes
Collaborator Contribution Finance Access to research facilities Support for grant applications Training of Exeter staff in GLP and industry environment (secondments) Shared research programmes
Impact New test methods for chemicals Strategic partnership Co-authored research papers CASE partnership on PhD projects
Description Brunel NE/G019355/1 
Organisation Brunel University London
Country United Kingdom 
Sector Academic/University 
PI Contribution Shared research projects. Exchange of personnel for training Co-authored research paper outputs
Collaborator Contribution Shared research projects. Exchange of personnel for training Co-authored research paper outpu
Impact Research papers
Description CEH NE/G019355/1 
Organisation UK Centre for Ecology & Hydrology
Country United Kingdom 
Sector Public 
PI Contribution n/a
Collaborator Contribution Provision of essential data on the oestrogenic effluent content in the selected UK rivers using catchment modelling.
Impact as above
Start Year 2009
Description Environment Agency NE/G019355/1 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution DNA analysis of fish
Collaborator Contribution provision of access to databases and background information on the rivers and fish populations.
Impact as above
Start Year 2009
Description NIBB and Ministry of Environment, Japan NE/G019355/1 
Organisation Ministry of the Environment
Department National Institute for Basic Biology (NIBB)
Country Japan 
Sector Public 
PI Contribution Data and scientific analyses
Collaborator Contribution Data and scientific analyses and resources
Impact Significant series of multidisciplinary research papers ( 10) Student exchange Research training
Start Year 2007
Description OECD NE/G019355/1 
Organisation Organisation for Economic Co-operation and Development OECD
Country France 
Sector Public 
PI Contribution Data and scientific advice for supporting chemical risk assessment
Collaborator Contribution Facilitating meetings for policy development in Europe
Impact Development of OECD test guidelines for chemical testing
Start Year 2006
Description UK Government NE/G019355/1 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution defra -data for supporting policy development on chemical risks
Collaborator Contribution funding
Impact Research papers, government reports, changes in policy
Description Axminster and Exeter College visits, Hamilton 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Hamilton (PDRA and Co-I) has given schools talks in 2015 about his research at Axminster College and Exeter College.
Year(s) Of Engagement Activity 2015
Description Population genetic analysis talk, TGAC course Norwich 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Hamilton gave an invited talk on the use of population genetic analysis, in particular RAD-seq to investigate the population impacts of pollution in the aquatic environment. TGAC course on RAD-seq Norwich, June 2015.
Year(s) Of Engagement Activity 2015
Description The Feminisation of Nature - Howard Hughes Lecture USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The Feminisation of Nature. Howard Hughes Lecture - Medical University of South Carolina, USA
Year(s) Of Engagement Activity 2015