CRITERIO OBJETIVO

The work presented in this thesis has contributed to a UK-Australian collaboration on EDCs in the aquatic environment. As a country, Australia has its own unique biota and climatic and environmental conditions which vary regionally. It is currently adapting to water scarcity and this may be exacerbated in the future by changing climate and its increasing population. Consequently, interest in EDCs in Australia has increased in recent years, in the context of climatic threats to the aquatic environment and its unique biota.

Wastewater treatment works effluent (WWTW) effluent contamination of the Australian aquatic environment is widespread across the country from temperate to tropical regions. Like in the UK, this can be characterised by a large contribution of effluent to river flow following discharge to small creeks or during periods of low rainfall, which can reach up to 100% downstream of WWTWs (Kumar et al., 2012). In these effluents, oestrogenic activity is common and the steroid oestrogens have been detected at concentrations up to 54 ng/L E1 (Braga et al., 2005), 18.5 ng/L E2 (Allinson et al., 2010) and 1.3 ng/L EE2 (Ying et al., 2009). Their concentrations show little variance between WWTWs in different regions with different climates, with similar concentrations detected in temperate South Australia, cool temperate Australian Capital Territory and subtropical Queensland (Williams et al., 2007). They also compare well with the range of concentrations observed in the UK and other European countries (Johnson et al., 2007b; Johnson et al., 2005; Ying et al., 2002b). In addition, other oestrogenic contaminants have also been detected, such as bisphenol A (BPA) and the alkylphenols, which are not subject to the same restrictions as in the EU (Ying

et al., 2009). Strong anti-androgenic activity was also detected in effluent from

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exposures (Kumar et al., 2012). However, there is little data available that quantifies this activity in environmental samples in Australia.

As a consequence of effluent discharge, oestrogenic activity has been detected in the aquatic environment and the concentrations of steroid oestrogens have been found to exceed their singular and combined PNECs for endocrine disruption in fish at WWTWs outfalls and downstream river sites (Ying et al., 2009; Young et al., 2004). However, concentrations have also been detected upstream of WWTW discharges, which suggests that multiple inputs should be considered. Indeed, in some localities such as small rural streams, agriculture has been shown to have a significant impact on steroid oestrogen concentrations (Williams et al., 2007). How these discharges impact the aquatic environment is largely unknown, but there is already some evidence of estrogenic endocrine disruption in the pest species, eastern mosquitofish (Gambusia

holbrooki), in effluent contaminated areas (Reitsema et al., 2010; Rawson et al., 2008;

Batty and Lim, 1999). However, there have been no studies to determine the effects on wild native species, in part due to the limited understanding of their baseline biology, endocrinology and the lack of available tools for ecotoxicology testing. This situation is improving and recent research has developed tools for measuring vitellogenin in the barramundi (Lates calcarifer) and black bream (Acanthopagrus butcheri) (Codi King et

al., 2008), which found that they were susceptible to oestrogen exposure. Work has

also been completed on the Murray rainbowfish (Melanotaenia fluviatilis) to assess baseline reproduction and the use of molecular methods and biomarkers has also been investigated (Woods and Kumar, 2011; Pollino et al., 2007; Pollino and Holdway, 2003). This found that these fish could be affected by oestrogens and consequently, the Murray rainbowfish has been proposed for use as an Australian fish model to assess endocrine disruptors. Nonetheless, so far there have been no assessments of intersex in wild native fish in oestrogen contaminated areas. Indeed, the closest example is a study of invasive common carp on the River Yarra, which found no evidence of vitellogenin induction or gonadal abnormalities. However, this was unsurprising due to the dilution of tertiary treated effluent within this catchment, which resulted in no oestrogenic activity being detected at capture sites (Kumar et al., 2012).

In both the UK and Australia, anti-androgenic activity occurring in the environment is an emerging topic. The causes remain largely unknown and so it is important that significant environmental anti-androgens are identified in both countries and their effects assessed in fish models. Additional consideration should also be made for the possibility of mixture effects with steroid oestrogens on common endpoints. Consequently, the overall aim of this thesis project was to identify anti-androgens in UK

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and South Australian catchments and to produce environmentally relevant exposures to assess their impacts on sexual disruption in fish alone and in combination with steroid oestrogens. The hypothesis was that anti-androgens would be detected in the environment and that they could cause sexual disruption in fish under experimental conditions. The null hypothesis was that anti-androgenic compounds would not be detected in the environment and that antiandrogens would not affect sexual disruption in fish in vivo. Work towards the overall project aim was completed under three objectives:

Objective One: Predictive modelling of anti-androgenic pharmaceuticals

This represented a targeted approach to identifying environmental anti-androgens by focussing on a set of pharmaceuticals with an anti-androgenic mode of action. Effluent and river concentrations of these pharmaceuticals were estimated in a UK and a South Australian catchment using predictive modelling techniques. Concentrations of steroid oestrogens were modelled alongside the anti-androgenic pharmaceuticals. The models were then adapted to analyse future trends in their concentrations and associated risks to fish health under climate and population change scenarios up to 2050.

Objective Two: In vivo assessment of pharmaceuticals

The two major anti-androgenic pharmaceuticals, bicalutamide and cyproterone actetate, were applied in two in vivo assessments with two appropriate fish models. The exposure concentrations were informed by the predictive modelling in objective one to produce environmentally relevant exposure scenarios. This aimed to determine the impacts of these pharmaceuticals on sexual disruption alone and in combination with steroid oestrogens, using endpoints including vitellogenin induction, secondary sexual characteristics and intersex.

Objective Three: Effect Directed Analysis of WWTW effluents

As part of a broader approach to identifying anti-androgens, the chemical constituents of UK WWTW effluent and river samples were identified following extraction, fractionation and gas chromatography-mass spectrometry. Potential anti-androgens were identified through literature searches and in vitro bioassays and their contribution to the identified activity was determined.

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