1. COMmunity Programme of Research on Environmental Hormones and ENdocrine Disrupters

2. COMPREHEND Objectives Effluent survey across Europe Principal active components Partitioning of components and activity Evidence of impacts on aquatic wildlife Development of new detection systems Application of existing screens to effluents

3. COMPREHEND NETWORK ITM Stockholm FEI Helsinki SLU Uppsala IVL Stockholm RIZA Lelystad RIVO DLO Umuiden NIVA Oslo CEH Windermere CEH Wallingford UBRUN Uxbridge TNO Delft CGE Paris EAWAG Dubendorf

4. Alarm from UK! High incidence of intersex (ovo-testis in roach)

5. Other examples of abnormalities in wild fish UK-intersex in gudgeon and flounder Holland-intersex in bream! Sweden-intersex in roach and skewed sex ratios in eelpout!

6. Key ’end-points’ to detect endocrine disruption Development of the gonads The yolk protein vitellogenin OvaryTestis

7. Exposure protocol design Return to river EffluentRiver water Buffer tanks 500 L tanks rainbow trout 200 L tanks carp 0%25%50% 100% 0%25% 50% 100%

8. Fish bioassay to detect estrogenicity of effluents

9. VTG in zebrafish exposed to the model estrogen ethinylestradiol

10. Sex ratios in zebrafish exposed to ethinylestradiol

11. VTG concentrations in zebrafish exposed to methyltestosterone

12. Percentage F1 generation 0 10 20 30 40 50 60 70 Control2.5%10% Females Males Exposure to a pulp mill effluent

13. Percentage F2 generation 0 10 20 30 40 50 60 70 Control2.5%10% Males Females Exposure to a pulp mill effluent

14. A potential consequence of feminisation Feminised males XY functional females Normal males XY Females XX Males XY Males YY 25% 50% 25% X

15. A potential consequence of masculinisation Masculinised female XX functional males Normal female XX Females XX 100% X

16. Vitellogenin analyses: Conclusions RIA is sensitive but difficult to perform Heterologous ELISAs lack sensitivity Homologous ELISAs are promising Urgent need for calibration of standard VTG

17. Direct in vitro assay of the estrogenicity of unfractionated effluents VitellogeninCarpLiverCARP-HEP Light emissionTroutGonadRTG-2 * ColorimetricHumanYeastYES Light emissionHumanBreast cancerER-CALUX VitellogeninTroutLiverTROUT-HEP ResponseEstrogen Receptor Cell TypeAssay * New assay developed as part of COMPREHEND

18. Bioassay-directed fractionation: Solid phase r All the estrogenic activity in fractions B and C

19. Relative in vitro potency of estrogenic compounds Estradiol (E2)1 Ethinylestradiol (EE2)1 (10-25 in vivo) Estrone (E1)0.5 Estriol (E3)0.01 Nonylphenol0.0001 Nonylphenol ethoxylates0.00005 Bisphenol A0.00005

20. Suppression of E2 activity in the YES assay by NPA and OPA

21. Population impacts Sex ratios of Dutch bream populations 0.7 0.6 0.5 0.4 0.3 0.2 Male Female

22. Vitellogenin levels in wild male bream in the Netherlands

23. 1.1  g l -1 0.6  g l -1 GC-MS/MSBisphenol A 7.0  g l -1 4.2  g l -1 HPLCNP ethoxylates 3.1  g l -1 1.6  g l -1 HPLCNonylphenol (NP) IndustrialDomesticTechniquesDeterminand Effuent analytical chemistry: Industrial chemicals

24. Effuent analytical chemistry: Industrial chemicals 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 00.20.40.60.811.21.4 NP concentration (micrograms per litre) NP 1EO concentration (micrograms per litre)

25. n.d.2 ng l -1 GC-MS/MS GC-High resolution MS Ethinylestradiol (EE2) n.d.17 ng l -1 GC-MS/MS GC-High resolution MS Estriol (E3) <l.o.d.14 ng l -1 GC-MS/MS GC-High resolution MS Estradiol (E2) <l.o.d.51 ng l -1 GC-MS/MS GC-High resolution MS Estrone (E1) IndustrialDomesticTechniquesDeterminand Effuent analytical chemistry: Estrogenic steroids

26. How efficient were the STW studied in COMPREHEND at removing steroid estrogens? In the COMPREHEND main survey only effluent concentrations were measured. To measure effectiveness we also need to know what the influent concentrations were! We can predict the concentrations in untreated sewage based on typical human excretion values and flow.

27. Using the method to predict estrone influent concentrations. Some examples 4045Ternes et al., 1999 Rio de Janeiro in Brasil 8797Johnson et al., 2000 West in Holland 5030Baronti et al., 2000 Roma Est in Italy 5131Baronti et al., 2000 Ostia in Italy 5540Baronti et al., 2000 Cobis in Italy Measured (ng l -1 ) Predicted (ng l -1 ) ReferenceSTW and country

28. Initial observations for COMPREHEND STWs Estradiol (E2) removal was high, median value 95% (range 92-99%) Estrone (E1) removal was more variable, median value 93% (range 36-99%). N.B. One STW in Norway was an exception to the other plants, having only chemical treatment (P precipitation). This failed to remove estrogenic steroids during treatment and had high E2 and E1 levels in the effluent.

29. Estrone removal related to total hydraulic retention time ( COMPREHEND plus other data) y = 0.2277Ln(x) + 0.1086

30. Management of steroid estrogens in the sewage treatment plant The steroid estrogens are naturally biodegraded The greater the degree of biological treatment, as evidenced by hydraulic retention time (HRT), the greater the removal. If HRT cannot be increased, then some other form of tertiary treatment may need to be considered

31. What about the pill – ethinylestradiol (EE2)? EE2 is up to 25 times more potent in vivo than E2 and 75 times more potent than E1 Thus, it could still represent the most active estrogenic compound present in STW effluents, even at concentrations below the detection limit EE2 biodegrades more slowly than the other steroidal estrogens although its potential to sorb is higher

32. Management of steroid estrogens in the river catchment The time and location of greatest endocrine disruption could be modelled It may be that only certain reaches, at certain times of the year are responsible for endocrine disruption of fish Such considerations may assist management of endocrine disruption at the catchment scale

33. Conclusions Only certain STWs, at certain times of the year, may be responsible for mostof the endocrine disruption in fish. Larger biological treatment tanks, to provide more time for biodegradation, will help. If this is not practicable, tertiary biological treatment, PAC, GAC, ozone or UV may be needed to guarantee removal of all endocrine disrupters, including the most potent EE2.