CMEP meeting, Praha the 30th June 2011

1. CMEP Task 3.2 C- Effect Based Monitoring Tools:Effect based tools for hormonally active substancesRobert Kase Petra Kunz Cornelia Kienle Inge Werner contact: Robert.Kase@oekotoxzentrum.ch CMEP meeting, Praha the 30th June 2011

2. Structure • Derivation of EQS and implementation in risk assessment Example of EE2 a very potent estrogen receptor binding substance Overview of current EQS-proposals for waste water relevant micropollutants and EE2, E2, E1 Detection possibilities • How relevant are hormonally active substances and what are their effects? Environmentally relevant questions regarding fish toxicity Results of a Swiss wide risk assessment via biotests Options to reduce risks from estrogenic substances • Which methods can be used to measure hormonally and reproduction toxic effects and what are the costs? Options to measure different mode of actions Some cost and time factors for test procedures • Overall summary and final conclusions Seite 2 I Oekotoxzentrum | Eawag-EPFL

3. Derivation of EQS andimplementation in riskassessment Seite 3 I Oekotoxzentrum | Eawag-EPFL

4. Scope of Environmental Quality Standards (EQS) • AA-EQS (Annual Average-EQS) must be derived as protection against the effects of long-term exposure • MAC-EQS (Maximum acceptable concentration) against the effects of short-term exposure. • Goal: • protection against long-term exposure • Comparison: • with the measured annual average concentration (90th percentile) • Data: • chronic effect data are preferred Because of the exposure scenario and the specific mode of action only an AA-EQS is proposed for EE2 and E2 EQS are based on reliable and relevant effect data. The critical step is to identify and to evaluate them. Seite 4 I Oekotoxzentrum | Eawag-EPFL

5. <1 tolerable risk >1 intolerable risk Source: Knacker 2007 Risk Assessment = Exposure Assessement / Hazard Assessment MEC= Measured environmental concentration (usually the 90th percentile) QC= Quality criteria (usually the AA-EQS) Seite 5 I Oekotoxzentrum | Eawag-EPFL

6. Steps of the development of an EQS-proposal for the FOEN / BAFU Source: Kase et al. 2011 http://www.oekotoxzentrum.ch/qualitaetskriterien Seite 6 I Oekotoxzentrum | Eawag-EPFL

7. Coworking on the priority substances in the (Sub)Working Group E Substances proposed for EQS derivation Leads and associated MS/stakeholders at 28th June 2010 Aim:To develop harmonized Environmental Quality Standards (EQS) and allow a border crossing risk management In the future a regulation of EE2 and E2 is expected.

8. Prioritized wastewater relevant micropollutants from over 250 candidate substances 27% 43% Contact persons: pharmaceuticals, industrial chemicals and hormone active substances / biocides, plant protection productsand chelating agents Robert Kase (robert.kase@oekotoxzentrum.ch)Marion Junghans (marion.junghans@oekotoxzentrum.ch) Seite 8 I Oekotoxzentrum | Eawag-EPFL

9. Hormone active and „endocrine disrupting“ effects estrogenic effect androgenic effect • In both cases, the possible effects are: • changes in the behaviour • changes in the fertility • intersex • population relevant effects Most effects are considered relevant according to the TGD for EQS The steroidal hormone system is highly conserved among different taxa, so there is a widespread effect in animals and humans. Seite 9 I Oekotoxzentrum | Eawag-EPFL

10. Example EE2: SSD approach in the EU dossier The EU EQS guidance notes than an assessment factor in the range of 1-5 should be applied to the HC5 derived. Based on the available dataset it is considered that an assessment factor of 2 is appropriate based on the data for EE2. This AF is warranted because: • The mode of toxic action is well understood. The HC5 has been derived based on data for two of the most sensitive taxonomic groups, i.e. fish and amphibians. • In addition where several studies were available for a species the lowest reliable effect concentration has been used adding a further level of conservatism. The HC5 from this distribution is 0.07 ng/l. Applying an assessment factor of 2 to this HC5 gives an AA-QSfreshwater,eco of 0.035 ng/l. Seite 10 I Oekotoxzentrum | Eawag-EPFL

11. List of existing SwissEQS-proposals and proposed EU EQS 33 EQS derivations were derived according to the Technical Guidance Document for EQS (TGD for EQS). Thedossiers were sent to external reviewers for a plausibilty and quality check. Estrogenic potency: EE2 E2 E1 At least for some of the steroidal hormonactive substances (EE2 and E2) problems with the analytical LOQ are expected in routine methods. Seite 11 I Oekotoxzentrum | Eawag-EPFL

12. Bioanalytical detection limits for hormone active substances Comparison of 5 in vivo and 10 in vitro test procedures. More information on the compared test-procedures can be found in (Kase et al. 2009) Abbreviations: T= testosterone DHT= dihydrotestosterone E2=17-β-estradiol EE2= 17-α-ethinylestradiol Even bioanalytical methods reach detection limits below 1 ng/L. Moreover they can measure estrogenic effects in an integrative way, so they could be suitable as screening methods. With these methods it is possible to measure integrative E2-equivalents (EEQ) with different cell based bioassays Seite 12 I Oekotoxzentrum | Eawag-EPFL

13. How relevant are hormonally active substances and what are their effects? Seite 13 I Oekotoxzentrum | Eawag-EPFL

14. Environmentally relevant questions Environmental Science and Technology 2005 Seite 14 I Oekotoxzentrum | Eawag-EPFL

15. Situation analysis 2005 and theories 2010 2005 John Sumpter 2010, one Sunday morning in May at the Pharmaceutical Advisory Group in Sevilla: • Presentation regarding“Pharmaceuticals in the Environment: What are the key unresolved issues?” • STP effluents entering rivers are the main route of entry of pharmaceuticals into the environment. • So, aquatic organisms, particularly fish, may be at greatest risk.” Fish seem to be exposed at Diclofenac and EE2 concentrations above their effect levels, for many other substances it remains unclear. Seite 15 I Oekotoxzentrum | Eawag-EPFL

16. Estrogenic impacts on fish populations ? • The SSD based EQS for E2 will be at 0.4 ng/L (derived from 11 chronic NOECs of different fish species) • Fish species are the most sensitive taxonomic groups. • The most sensitive study is done with rainbow trout (Lahnsteiner et. al. 2006)  NOEC of 0.5 ng/L (for endpoints: fertilization success, sperm density and volume). • E2,the metabolite estrone (E1) and the pharmaceutical 17-alpha ethinylestradiol (EE2) contribute additionally to the estrogen receptor mediated estrogenicity  there is a cumulative risk. • Additionally the ER receptor binding for several non steroidal substances (Dang 2011) indicate a need of integrative measurement methods to identify estrogenic potentials. Seite 16 I Oekotoxzentrum | Eawag-EPFL

17. Example: Estrogenicity during wastewater treatment steps Results of the pilot study at the STEP de Vidy Lausanne with the YES and ER-Calux assays in 17β-estradiol-equivalents (EEQ) (4th measurement campaign): Legend: Blue: YES-EEQ [ng/L] Red: ER-Calux-EEQ [ng/L] CITEQ = Change Index = (EEQ Treatment Step / EEQ Influent) -1 PAC-UF treatment PAC-UF Secondary clarifier Moving Bed biology Mechanical treatment Primary clarifier PAC-UF Treatment 0.29 ng/L; CIPAC/EN= 126 0.16 ng/L; CIPAC/EN= 61 EN LF Waste-water Biological Treatment 3.03 ng/L; CILF/EN = 12 0.90 ng/L; CILF/EN = 11 Influent 37.15 ng/L 9.8 ng/L Ozonation Sand filtration OZ SF Conclusions: • Compared to the conventional biological treatment a 6-13 times lowered receptor binding estrogenicity results via additional wastewater treatment steps. • Without bioanalytical tests like YES- and ER-Calux a performance control of WWTP regarding the elimination of estrogenicity is hardly or not possible. Ozonation + SF 0.23 ng/L; CIOZ+SF/EN= 160 0.15 ng/L; CIOZ+SF/EN= 65 AA-EQS proposal for E2 = 0.4 ng/L

18. Water quality during wastewater treatment PAC-UF and Ozonation-SF can lead to 6-13 times lowered estrogenicity compared to the conventional biological treatment. Ozonation-SF can lead to more than 73 times lowered risk quotients for Diclofenac. Conclusion: There are options developed and available to reduce ecotoxicological and fishtoxic effects of wastewater insurface waters. • Main conclusions for bioassays applied in the FOEN-Project “Strategy Micropoll” : • A broad range of micropollutants and their effects were eliminated by more than 80% (by PAC-UF and Ozonation AF). • There was no evidence for a toxicity increase due to constant formation of stable toxic ozonation by-products. • An ozonation should be followed by a final filtration step with biological activity. • Quality of treated effluent was significantly improved, leading to improved surface water quality. Report in prep.: Cornelia Kienle, Robert Kase, Inge Werner 2011 Seite 18 I Oekotoxzentrum | Eawag-EPFL

19. Conclusions for steroidal hormone active substances AA-EQS proposal for E2 = 0.4 ng/L Fischnetz: - Downstream SWTP: up to 5 ng/L EEQ NRP50: - Surface Waters: 0.5 - 4 ng/L EEQ Strategie Micropoll: - Effluent Vidy (upgraded): 0.15 - 0.29 ng/L EEQ - Effluent Würi (upgraded): 0.05 - 0.33 ng/L EEQ Source: Kunz et al. 2011 • In conclusion it is very likely that different steroidal estrogens (EE2, E2 and E1) have an impact on fish populations at environmental relevant concentrations(see map before), also at median flow conditions close to WWTPs. • But therearelimitations: • todetectthe different steroidalestrogens due toanalyticalproblemsattheseloweffectlevels. • todetectseveralmodeofactionsforendocrinedisruptors. Seite 19 I Oekotoxzentrum | Eawag-EPFL

20. Which methods can be used to measure hormonally and reproduction toxic effects and what are the costs? Seite 20 I Oekotoxzentrum | Eawag-EPFL

21. Test with invertebrates, e.g. reproduction test with Potamopyrgus antipodarum or Daphnia magna optional biotests Reproduction-toxicity Xenopus laevis metamorphosis assay e.g. fish egg test or a non acute test with invertebrates Thyroidal- modulation Embryo-toxicity in vivo Mode Of Action (MOA) in vitro Receptor-binding if you have strong (cyto)toxic effects Steroidgenesis- and Aromatasemodulation Receptor binding-assays, e.g. ELRA H295R-Steroido-genesis Assay ER/AR receptorbinding YES/YAS Sumpter and modified to Schultis/Metzger or ER/AR-Calux Is it possible to detect different modes of action with a modular test battery? The modular system presented here allows the switching between test modules according to the continuously developing state-of-the-art of science and technology as well as the incorporation of novel developments. Not applicable for all environmental samples! Source: Kase et al. 2009 Or short: In principle yes, but far too expensive for a routine monitoring

22. How much time do you have to invest? • In general: in vivo-tests consume more time than in vitro-tests, scaling days vs. hours • Specific remarks: • in vivo:the reproduction test with Potamorpyrgus antipodarum needs most overall time but minimum working time • in vitro:MVLN and E-Screen have the most overall and working time need Time in days [d] overall time working time Time in hours [h] overall time working time Seite 22 I Oekotoxzentrum | Eawag-EPFL

23. How expensive are the materials for one test run? In vivo and in vitro: # comment: these are commercial tests, so additional costs will occure • In general thein vivo tests are more expensive than in vitro tests regardingmaterials neededand in addition more time consuming. • Since November 2010 aDIN/ISO Standardization for different cell based ER receptor binding assays is ongoing, but there is still a long way to go in order to have standardized methods for regulative purposes available. Seite 23 I Oekotoxzentrum | Eawag-EPFL

24. Overall summaryand final conclusions Seite 24 I Oekotoxzentrum | Eawag-EPFL

25. Summary • Thus, it is safe to say that….. • for environmental sampleswith anunknowncomposition (unknownmixtures) only integrative bioanalyticaltools, usedasscreeningmethods, areabletodetectspecifichormoneactiveeffects • Specifichormoneactiveeffectsareresultingfrom an exposureto a mixtureofknownandunknownsubstanceswithendocrinedisruptingproperties. • These tools are available and have been proposed in the past, for example within: • the international validation efforts by the OECD and the US EPA EDTA for methods to detect endocrine disruptive effects • the Global Water Research Coalition (GWRC) report "Tools to detect estrogenic activity in environmental waters" (Leusch 2008). • an extensive literature search and evaluation in which 15 biological test methods (5 in vivo and 10 in vitro) were evaluated (Kase et al. 2009), comprising 8 OECD methods and 3 out of five in vitromethods mentioned in Leusch (2008 and 2010). Seite 25 I Oekotoxzentrum | Eawag-EPFL

26. Final conclusions • These tools are able to measure the total receptor binding potential of an environmental sample by expressing its potency in EEQ (or EE2Q). These estrogenic potencies are then directly comparable with proposed EQS for E2 or EE2 for a decision for which samples additional analytical measurements are necessary. • Accordingly with effect based tools there is a potential: • To reduce the high costs for specific analytical measurements • To provide reliable information forendocrinedisrupting potentials coveringseveral modes of action • However, forregulatory purposesthe concept of dose addition is a sufficiently accurate model to predict combination effects of groups of endocrine disruptors with similar effects (Kortenkamp, 2007), but in order to do so an extensive data set of analytically measured EDCs has to be available. • In most cases this analytical data set is not available, therefore we recommend cost efficient and sufficiently sensitive effect based tools on a cellular basis to detect hormone active potentials in environmental samples in a first step. Seite 26 I Oekotoxzentrum | Eawag-EPFL

27. But, definitely without risks • Do not hesitate to contact us for further information: • Robert.Kase@oekotoxzentrum.ch • (risk-assessor) • Cornelia.Kienle@oekotoxzentrum.ch • (multiple effect assessment) • Petra.Kunz@oekotoxzentrum.ch • (DIN/ISO standardization) • Inge.Werner@oekotoxzentrum.ch • (Head of Ecotox centre) Remember What’s Good for the Fish is Good for Us Also Thank youfor your attention Source: US-EPA, Lazorchak J 2010 Seite 27 I Oekotoxzentrum | Eawag-EPFL

28. More supporting information and references Abegglen C et al.(2009): Ozonung von gereinigtem Abwasser. Schlussbericht Pilotversuch Regensdorf. Eawag Bericht Dübendorf, 16. Juni 2009 Burkhardt-Holm P, Giger W, Güttinger H, Ochsenbein U, Peter A, Scheurer A, Segner A, Staub E, Suter M J F (2005): Where have all the fish gone? The reasons why fish catches in Swiss rivers are declining. Environmental Science and Technology 39 (21) pp 441-447. Dang Z C, Rub S, Wang W, Rorije E, Hakkert B, Vermeire T (2011): Comparison of chemical-induced transcriptional activation of fish and human estrogen receptors: Regulatory implications. Toxicology Letters 201:152–175. EC (European Commission) (1997): (DG XII – Environment and Climate Research Programme). European Workshop on the Impact of Endocrine Disruptors on human Health and Wildlife. Report of Proceedings of a Workshop 2-4 December 1996, Weybridge, UK. Commission of the European Community, Brussels; Report EUR 17549 FNSNF (2008): Konsensplattform “Hormonaktive Stoffe in Abwassern und Gewässern” Schlussdokument. Nationales Forschungsprogramm “Hormonaktive Stoffe“. http://www.nrp50.ch/final-products/final-reports-consensus-plattforms.html Götz C W, Kase R, Hollender J (2011): Mikroverunreinigungen - Beurteilungskonzept für organische Spurenstoffe aus kommunalem Abwasser. Studie im Auftrag des BAFU. Eawag, Dübendorf. Götz C W, Kase R, Kienle C, Hollender J (2010): Mikroverunreinigungen aus kommunalem Abwasser - Kombination von Expositions- und ökotoxikologischen Effektdaten. Gas Wasser Abwasser, 7/2010. Seite 28 I Oekotoxzentrum | Eawag-EPFL

29. More supporting information and references Götz C W, Stamm C, Fenner K, Singer H, Schärer M, Hollender J (2009): Targeting aquatic microcontaminants for exposure categorization and application to the Swiss situation. Environ Sci Pollut Res. DOI 10.1007/s 1356-009-0167-8 Hutchinson T H, Ankley G T, Segner H, Tyler C R (2006): Screening an Testing for Endocrine Disruption in Fish-Biomarkers As „Signposts“, Not “Traffic Lights” in Risk Assessment. Environmental Health Perspectives 114:106-114 Kase R, Eggen R I L, Junghans M, Götz C, Hollender J (2011): Assessment of Micropollutants from Municipal Wastewater- Combination of Exposure and Ecotoxicological Effect Data for Switzerland, Waste Water - Evaluation and Management, Fernando Sebastián García Einschlag (Ed.), ISBN: 978-953-307-233-3, InTech Kase R, Kunz P, Gerhardt A (2009): Identifikation geeigneter Nachweismöglichkeiten von hormonaktiven und reproduktionstoxischen Wirkungen in aquatischen Ökosystemen. Umweltwiss Schadst Forsch 21(4). DOI :10.1007/s12302­009­0072­2. Kienle C, Kase R, Werner I (2011 in prep.): Evaluation of bioassays and wastewater quality. In vitro and in vivo bioassays for the performance review in the Project "Strategy MicroPoll". Summary. Oekotoxzentrum Eawag/EPFL, Dübendorf. Knacker T (2007): Möglichkeiten der ökotoxikologischen Bewertung für Einzelstoffe und Gemische. Presentation at the IKSR-Workshop Mikroverunreinigung aus der Siedlungswasserwirtschaft 23. Mai 2007, Bonn, Germany. Kortenkamp, A (2007): Ten years of mixing cocktails: a review of combination effects of endocrine disrupting chemicals. Environ. Health Persp 115, Suppl. 1: 98-105. Seite 29 I Oekotoxzentrum | Eawag-EPFL

30. More supporting information and references Kunz P, Kienle C, Homazava N (2011): Endocrine Effects in the Aquatic Environment – The Situation in Switzerland. Presentation at the Endocrine Disruptors Workshop 8. Februar 2011, Zürich, Switzerland.Lahnsteiner F, Berger B, Kletzl M, Weismann T (2006): Effect of 17β-estradiol on gamete quality and maturation in two salmonid species. Aquatic Toxicology 79:124–131. Leusch F (2008): Tools to Detect Estrogenic Activity in Environmental Waters. Global Water Research Coalition 2008 Maack G, Adler N, Bachmann J, Ebert I, Hickmann S, Küster A, Rechenberg B (2010):Environmental Risk Assessment of medicinal products for human use: Does the risk assessment reflect the reality? Presentation May 2010; SETAC Sevilla. TGD for EQS 2009/2010: CHEMICALS AND THE WATER FRAMEWORK DIRECTIVE: TECHNICAL GUIDANCE FOR DERIVING ENVIRONMENTAL QUALITY STANDARDS. Draft 2010. Draft version 6.0, 23 February 2010. US-EPA, Lazorchak J (2010): Results of a 21-Day Fathead Minnow (Pimephales promelas) Fecundity Study Following Exposure to Ethinylestradiol (EE2). Presentation at the SETAC Annual Meeting Portland, OR November 11, 2010. Vermeirssen E L M, Suter M J –F, Burkhardt-Holm P (2006): Estrogenicity Patterns in the Swiss Midland River Lützelmurg in relation to treated domestic sewage effluent discharges and hydrology. Environmental Toxicology and Chemistry 25(9):2413-2422 Seite 30 I Oekotoxzentrum | Eawag-EPFL

31. Annex: Additional information for the proposed test procedures A full summary of available and OECD tests is available in Kase et al. 2009 Seite 31 I Oekotoxzentrum | Eawag-EPFL