1. Roach Picture credit: Tom Pottinger 3-spined stickleback Picture credit: Richard Maunder PROGRESS WITH THE EDCAT PROGRAMME – RISKS OF OESTROGENS FOR FISH POPULATIONS Peter Matthiessen*, Jan Balaam #, Andrew Johnson*, Ioanna Katsiadaki #, Tom Pottinger*, Jim Readman ‡, John Sumpter †, Charles Tyler § and John Zhou ¶ * Centre for Ecology and Hydrology (CEH), UK; # Centre for Environment, Fisheries and Aquaculture Science (Cefas), UK; ‡ Plymouth Marine Laboratory (PML), UK; † Brunel University, UK; § University of Exeter, UK; ¶ University of Sussex For more information, please see the EDCAT website: www.ceh.ac.uk/edcatwww.ceh.ac.uk/edcat or contact Peter Matthiessen, the programme leader: peter@matthiessen.freeserve.co.uk EDCAT sponsors: UK Department for Environment, Fisheries and Rural Affairs (Defra); Environment Agency of England and Wales (EA); United Kingdom Water Industry Research Ltd (UKWIR) ABSTRACT During its first year, EDCAT (Endocrine Disruption in Catchments) has shown that:- 1) Modelled oestrogen concentrations in the River Ray are similar to measured values at some times of year 2) However, in vitro oestrogenic activity and steroid concentrations in water are very variable, with highest activity just downstream of the STW discharge during October 2006 3) To date, stickleback (Gasterosteus aculeatus) populations have only shown minor vitellogenin (VTG) responses to oestrogens, but this may be due to the transience of exposure and to the lack of fish sampling during October. 4) Breeding experiments with roach (Rutilus rutilus) in semi-natural spawning groups have shown that weakly intersex fish are as successful as normal fish in fathering offspring 5) During 2007, we are aiming to improve our exposure modelling in order to correctly reflect seasonal and spatial variation, to continue studies of stickleback populations to establish true oestrogen exposure and populations variables, and to conduct further breeding experiments to assess the effects of intersex in more severely affected roach. INTRODUCTION Fish populations in many UK lowland rivers and estuaries are feminised (vitellogenin, ovotestis, feminised gonoducts, and/or feminised secondary sexual characteristics in males) The EDCAT programme is testing the hypothesis that oestrogen contamination in these rivers is a risk to fish populations. EDCAT (Endocrine Disruption in Catchments) is a multi-funder, multi-institute 6 year (Phases 1 and 2) programme which is studying the River Ray in the United Kingdom. Current work is establishing the baseline situation prior to oestrogen removal from the Swindon (Rodbourne) sewage treatment works (STW) discharge. MODELLING AND EXPOSURE MEASUREMENTS EXAMS modelling of E1, E2 and EE2 in the R. Ray (see near right) predicts highest E2 equivalent concentrations (~11 ng/l) immediately downstream of the STW discharge in summer. Predicted activity is higher than time-averaged values measured in spring with the Yeast Estrogen Screen (YES) (see far right), but similar to those measured in autumn. Most activity is attributable to estrone (E1), oestradiol (E2) and ethinylestradiol (EE2) To improve predictions, the model will be further developed, and re-run using measured values for discharge rates, river flow-rates, and biodegradation rates FISH STUDIES The stickleback populations in the R. Ray are being monitored for their demographic characteristics. To date, plasma and whole-body VTG levels in males are low (see far left), but samples were not taken in October at the time of highest E2-equivalent activity. We have identified 6 variable DNA micro-satellites in roach that have been successfully used to identify the parentage of fry produced in freely-breeding spawning groups in the laboratory In the first breeding experiment, using fish from the R. Bourne, there was no difference in the breeding success of intersex and normal males (see near left). However, the intersex fish were only mildly abnormal (intersex indices up to 3.2), and further experiments are now being performed with more severely affected fish DISCUSSION AND CONCLUSIONS Model predictions of E2-equivalent concentrations in the Ray are in the right range, but do not yet reflect the fact that the level of oestrogenic activity is very variable, both between sites and between seasons. We intend to improve this situation by developing the model further, and by using more realistic input data for sewage and stream flow rates etc. Although oestrogenic activity in October 2006 was high enough to produce a vitellogenin response in stickleback, little response was seen in samples taken earlier in the year. We therefore propose to coordinate the chemical and biological sampling more closely in the coming year. The degree of contamination in October should have been high enough to produce effects on sticklebacks, but it remains to be seen if reproduction, and ultimately population demographics, are indeed affected. We have developed a set of robust DNA microsatellite markers for identifying parentage in roach fry, allowing the reproductive success of freely-breeding male fish to be accurately assessed. An initial experiment, in which mildly intersex fish were allowed to compete with normal fish, showed no differences in reproductive success. We are now conducting further breeding experiments using roach populations containing more strongly intersex individuals. Overall, EDCAT is on target to establish baseline conditions in the River Ray before the Rodbourne STW is upgraded in 2008/09 to remove oestrogens. R. Thames Childrey Brook R. Ock R. Cole R. Ray Diagrammatic map of study area – not to scale Rodbourne STW Site 1 Site 2 Site 3 Site 4/5 Site 6 Site 7 Site 8 Site 9 Site 10 Site 11 Site 13 Site 12 Predicted E2-equivalent concentrations (ng/l) in the River Ray throughout the year, at a range of distances (1-13 km) downstream of the Rodbourne STW discharge E2-equivalent concentrations (ng/l) in the River Ray at 5 sites and 4 sampling dates. Each concentration represents a value time-averaged over a month using POCIS passive samplers and assay of POCIS extracts with the Yeast Estrogen Screen (YES) Male VTG levels in sticklebacks from the R. Ray and the reference site (R. Ock) Reproductive success of freely-breeding male roach from the River Bourne. The degree of intersex was mild (intersex index up to 3.2), and there was no difference between the success of mildly intersex fish and normal males.