Investigating the health effects of estrogenic chemicals individually and within wastewater treatment works effluents using an ERE-GFP transgenic zebrafish (Danio rerio) model.

It is now well established globally that oestrogens are a major environmental contaminant in WwTW effluent discharging into receiving waterways. Even in developed countries despite advances in wastewater treatment and a general reduction in the incidence of gross point source pollution, effluent from most WwTWs contain detectable estrogenic activity; around a third of English rivers currently are thought to contain levels of oestrogenic chemicals sufficient to cause some endocrine disruption in fish. These disruptions in fish present themselves in a number of different ways including a sex ratio biased in favour of females, the presence intersex individuals; delayed sexual maturation; and alterations in reproductive behaviour. This illustrate the need for a deeper understanding on how oestrogenic chemicals act within the body to affect wildlife health. Using a transgenic oestrogen response element (ERE), green fluorescent protein (GFP) zebrafish model, this thesis work set out to determine tissue targets and the potential for health impacts of environmental oestrogens and their mixtures within effluents from WwTWs and how these responses may vary both seasonally and for exposures for different developmental life stages. This was measured through a combination of biological endpoints including mortality/hatching rates, length, weight and condition factor (in adults), vitellogenin (vtg) induction (mRNA) in whole body embryos and in the livers of adults, and also through GFP quantification (measured by fluorescence microscopy on target tissues, Western blotting for whole body GFP induction and GFP mRNA induction). The body of thesis work also sought to further establish how fluorescence responses in the transgenic zebrafish model compared in terms of sensitivity for the detection of oestrogens with vitellogenin (VTG) induction, a well-established biomarker for oestrogen exposure in fish. The ontogeny of endogenous GFP expression in individual fish was also followed from early life stages through the period of sexual differentiation using fluorescent microscopy, to determine if endogenous GFP expression could be used as a sex marker in our zebrafish model. In the WwTW effluent studies, differences in GFP tissue patterning (and of other biological endpoints such as vtg induction) were found to be reflective of the varying concentrations of steroidal oestrogens found in the different wastewaters, (both temporally and between treatment works), and they also illustrated the considerable dynamic nature of the oestrogenic potencies of the WwTW effluents. For chronic effluent exposures, biological responses to both the oestrogenic effluent and the synthetic oestrogen EE2 were differed between the sexes particularly in terms of overall growth and sex differentiation parameters. For my investigation into endogenous GFP expression, I found varying levels of fluorescence, throughout the various life stages of our zebrafish to 30dpf. There were four different patterns of GFP expression across the different body tissues recorded, based on the tissues expressing GFP and the timing and duration of that expression. Gonadal sex (measured via histology at 60dph) however was associated only with sex for one of the recorded GFP expression patterns (GFP expression in the liver at 30dph – corresponding to the female phenotype). Overall, the results from the studies presented in this thesis illustrate the potential for differing health effects of both individual exogenous oestrogens and for a single WwTW effluent over time/season and for effluents from different WwTW. The work also highlights the importance in the timing (developmental stage) of exposure on the biological effects seen. The ability of ERE-GFP transgenic zebrafish model to allow for the analysis of individual tissues, such as the heart or brain, through non-destructive fluorescence microscopy, further demonstrates its significant advantages for screening and testing for environmental oestrogens. Whilst these results suggest that GFP induction is less sensitive than VTG induction per se, related (in part) to the greater magnitude of response for VTG induction, there was a good positive correlation between the two variables illustrating further the utility of the GFP induction as a bio-monitor to detect for the presence of exogenous oestrogens contained in real world effluents. These findings further highlight the importance of integrating biomonitoring approaches to complement analytical chemistry techniques in the monitoring of oestrogenic chemicals in wastewater effluents. Differences, in the levels of endogenous GFP expression across the different body tissues during early life in our zebrafish model, and prior to gonadal differentiation, are likely a reflection of the varying functional roles oestrogen receptors perform during development (i.e. they are under spatial-temporal control) and are not thought to provide an accurate marker for the determination of sex in the zebrafish model but could be utilised as a phenotypic marker for identifying females at 30dpf. With a view towards the future, additional steps now need to be taken in order to further corroborate these findings and to maximise the potential of the transgenic ERE-GFP zebrafish model system. These include the need for identifying the molecular mechanisms of oestrogenic chemicals and of oestrogen responsive genes and pathways, particularly in the responsive target tissues identified in this thesis, to better inform on adverse health outcomes. Ultimately, the results of this thesis further highlight how the development and application of transgenic fish models can offer huge potential for more integrative health effects assessments, allowing for an improved understanding of the risks posed by oestrogenic chemicals, and for tailoring future approaches to ERA strategies, specifically in respect of AOP frameworks