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

Lead Research Organisation: Brunel University London
Department Name: Life Sciences

Abstract

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 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.
 
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 a native species - 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 that reproductive failure occurs after chronic exposure of roach to an estrogenic WwTW effluent for up to 3.5 years. To understand the reasons for this reproductive failure we assessed the gonad development of the fish in the breeding colonies that included both WwTW exposed and unexposed "control" fish. Histological analysis of the gonads demonstrated that all fish exposed to 100% effluent for 3 years were females. Using this analysis two types of female were found; those that looked like normal females (i.e. full sexually mature) and those that seemed to not have developed normally and were considered to possibly be sex-reversed males. Using a technique called DNA microsatellite analysis we conducted parentage analysis of the offspring of fish from all our breeding colonies to determine their reproductive capacity, i.e. how many of the off-spring came from each adult fish. We found that 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 after 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 the period of the second exposure and concentrations of steroidal estrogens were also lower, 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 competitiveness of the offspring, we conducted a breeding study using the 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 river sites 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, mainly due to barriers to migration such as weirs, and have therefore not been sustained by immigration (movement of fish from other stretches of river). We found no evidence that size of breeding populations or genetic diversity was reduced in more contaminated rivers. There was, however, evidence for less variation in breeding populations 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 still self-sustaining (BMC Biology 2014 12:1).
Exploitation Route During this and other Exeter/Brunel Collaborations with roach we have developed an extensive panel of SNPs (6000) for use in genotyping. These can be used for investigating adaptation, population genetic structure and for use in the practical management of the species (e.g. restocking). Some of these SNPs may be of use in related species such as rudd, ide and bream.
Sectors Environment

 
Description The issues relating to the feminising effects on fish of wastewater treatment works (WwTW) effluents enter our rivers has been studied by our group at Brunel University and our collaborators at Exeter University for a number of years. We now know estrogenic wastewaters entering our rivers have implications to fish reproduction. individual male fish can be feminised, which can compromise reproductive output. At high levels this can result in complete feminisation and significantly affect their contribution to the next generation. Because of the risk to wild fish the UK Environment Agency and the Water industry have run a demonstration programme to evaluate technologies to improve effluents and remove estrogens in order to protect wildlife. Some of the technologies tested were effective at removing the estrogens and their effects on fish. However, the technologies tested were also found to be extremely costly both financially and in terms of carbon use. Therefore there is a need to fully understand the risk posed, to make sure any investments in improving effluents are cost effective. due to our research finding over the last few decades and of other researcher in the field, 17-beta-estrdiol (natural steroid estrogen) and ethinylestradiol (contraceptive pill hormone) are on an EU watch list within the Water Framework Directive. This means countries across Europe will need to monitor the levels of these compounds in receiving waters, with the intention of fully characterising the concentration(s) and therefore the risk these compounds might pose to wild fish.
First Year Of Impact 2003
Sector Environment
Impact Types Societal,Economic

 
Description Evidence - Tender
Amount £94,997 (GBP)
Funding ID project_21373 
Organisation Environment Agency 
Sector Public
Country United Kingdom
Start 09/2017 
End 03/2018
 
Description Bob Burn, Exeter and Brunel - modelling population level effects of oestrogens on roach 
Organisation University of Exeter
Department Centre for Ecology and Conservation (CEC)
Country United Kingdom 
Sector Academic/University 
PI Contribution Jobling collaborated extensively with statistician Bob Burn ( ex Reading ) and with Exeter university on modelling population level effects of oestrogens on roach
Collaborator Contribution Bob Burn - modeller and statistician has been working with Jobling (Brunel) and Exeter team to develop a baysian model of population level effects of oestrogens on roach
Impact model still under development.
Start Year 2011
 
Description Brunel and CEH 
Organisation UK Centre for Ecology & Hydrology
Country United Kingdom 
Sector Public 
PI Contribution Hydrological modelling of Thames catchment rivers (in collaboration with CEH) to provide sites with high or low WwTW effluent (estrogenic) pollution levels for population analysis. Field work to collect fish and/or fish tissue samples from rivers with high and low pollution (WwTW) levels to compare population genetics.
Collaborator Contribution Training of research assistant Elizabeth Nicol in GIS and use of CEH's hydrological model. Using of the model to give predicted river concentrations of steroid estrogens present if WwTW effluents to inform field work locations to samples wild fish for NERC funded projects.
Impact In November 2013 Brunel University, Exeter University and CEH, with the support of the Institute of Fisheries Managers (IFM), organised a one day mini-conference for Environment Agency fisheries staff, IFM members and local angling trusts to inform them of the work both universities had been conducting into the issue of endocrine disrupting chemicals and fish. Publications so far from this NERC funded collaboration, DOI: 10.1186/1741-7007-12-1 Disciplines involved in this project are: environmental toxicology, endocrine disruption, wildlife biology, population genetics, hydrological modelling.
Start Year 2006
 
Description Exeter and Brunel (2010 Project) 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution Hydrological modelling of Thames catchment rivers (in collaboration with CEH) to provide sites with high or low WwTW effluent (estrogenic) pollution levels for population analysis. Interrogation and compilation of Environment Agency fish record data to provide historic and current population densities at chosen river sites. Conducted the field work (in collaboration with Environment Agency fisheries teams) to obtain biological samples (fin clips/scales) from wild roach across Thames catchment for genetic population analysis to assess the impacts of inhabiting rivers contaminated with estrogenic WwTW effluents compare to 'clean' sites. Aging of these fish using scales from wild roach inhabiting WwTW effluent impacted or non-impacted sites. In collaboration with Exeter staff sampled roach from the two WwTW exposure breeding studies. Histopathology processing and analysis of fish tissues arising from the WwTW effluent exposure and breeding studies. Worked with Exeter University collaborators to prepare and analyse the tissues for DNA microsatellite analysis.
Collaborator Contribution Running of the two WwTW exposure breeding studies at the Water treatment work site. In collaboration with Brunel University staff sampled roach from the two WwTW exposure breeding studies. Prepare and analyse the tissues arising from field survey sites and breeding studies for DNA microsatellite analysis. Hosted Engagement event at Exeter University.
Impact In October 2011 Exeter University (in collaboration with Brunel University) hosted a meeting entitled 'Are Chemicals Impacting on Fish Populations?' which was attended by UK, European and American researcher in fish biology as well as member of staff from DEFRA, the Environment Agency, Water Companies and the pharmaceutical Industry. A summary of the outcomes of this meeting has been written as review paper and has been submitted to review in Fish and Fisheries. So far two publications have arisen from this project: DOI: 10.1021/es103232q and DOI: 10.1186/1741-7007-12-1 An additional NERC funded project in collaboration between Exeter and Brunel University has also been awarded (NE/K004581/1) to follow on this research. Disciplines involved in this project are: environmental toxicology, endocrine disruption, wildlife biology, population genetics, hydrological modelling.
Start Year 2006
 
Description EDCs and Fish Day (Leeds) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact In November 2013 Brunel University and Exeter University, with the support of the Institute of Fisheries Managers (IFM), organised a one day mini-conference for Environment Agency fisheries staff, IFM members and angling trusts to inform them of the work both universities had been conducting into the issue of endocrine disrupting chemicals and fish.

There was interest from IFM members and EA staff for more information relating to the environmental impacts endocrine disruption in wild fish. A report entitles 'Troubled Waters- The impact of water-polluting chemicals on fish populations' was written and circulated to delegates and other interested parties.
Year(s) Of Engagement Activity 2013
 
Description NERC workshop: Are Chemicals Impacting on Fish Populations? - Exeter 2011 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Transfer of knowledge between researchers from a range of fish research fields e.g. population genetics, evolution, environmental stressors and pollution.
Stimulated discussions between the researcher from differing background on the topics of:
1. How accurately can we model fish populations?
2. What is the real evidence for chemical impacts on wild fish at the level of the population?
3. How can genetic methods enhance our understanding of wild fish populations and chemical impacts?
4. What are the critical factors needed to advance our understanding on chemical impacts in wild fish populations?

A summary review paper resulting from this workshop is currently in review in the journal Fish and Fisheries.
Year(s) Of Engagement Activity 2011