Schweizerisches Zentrum für angewandte ÖkotoxikologieCentre Suisse d’écotoxigologie appliquée

Eawag-EPFL

Need for monitoring options, exposure of EE2 and E2 and aims of the meeting

presented by:Christiane Heiss (Federal Environment Agency, UBA, DE)& Robert Kase (Swiss Centre for Applied Ecotoxicology, CH)

contact : Christiane.Heiss@uba.deRobert.Kase@oekotoxzentrum.ch

Bioanalytical options for the monitoring of steroidal estrogens in surface water2/th February Federal Institute of Hydrology Koblenz (BfG), DE

Structure of the presentation

Christiane Heiss:Prioritization process in WFD for EE2 and E2 State of present regulatory discussion Monitoring need

Robert Kase:Exposure examples for steroidal estrogens & bioanalytical screening options toreduce the monitoring costs Ecotoxicological relevance of EE2 and E2 and monitoring problems Swiss wide modelling of EE2 and other steroidal estrogens-

which concentrations do we expect and what is the situation in other countries? Bioanalytical measurements of river systems Summary & main conclusions Aims of the meeting

COM-proposal: EE2 and E2 as priority substances

substance use state of play in MS 17 alpha-ethinyl-

estradiol(EE2)

Pharmaceutical; syntheticsteroid hormone used

mainly in oralcontraceptives. No

production data available.Approx 32 million women

in EU use EE2-basedcontraception.

Monitoring databasecontains data from 3countries, 2 showing

exceedance of EQS, 1likely exceedance;literature predicts

exceedances morewidely.

17 betaestradiol(E2)

Steroid hormone: excretednaturally (approx 90%) inhuman and livestock urinebut also (<10%) as a resultof pharmaceutical use (of

which 90% from HRT)

Monitoring database (2countries) andliterature show

exceedance of EQS.

state of political discussion at EP and Council

The pollution of waters and soils with pharmaceutical residues is an emerging environmental concern ( EP, Council, COM agree)

Measures at source are necessary at EU Level ( and MS level) ( EP, Council) Allocation of local measures to limit risks are currently based on measured

concentration in water Current line of argumentation in EP, Council: deletion of EE2 and E2 from Annex I

and request for monitoring in the “watchlist” mechanism Rationale: lack of monitoring methods and high uncertainty on local concentrations of

EE2 and E2 in rivers and lakes

Gap between scientific literature and Suitable tools for investigative monitoring of chemical status for EE2, E2 need

to be standardized as monitoring tools under EQS-D compliance Monitoring with EQS for EE2, E2 not clear High uncertainty on costs and legal practicability for permits

Options to act:

Recital (10a) ( Council prop.) Novel monitoring methods such as passive sampling and […] other tools show promise for future application, and their development should therefore be pursued.

Recital (11) (COM proposal): (...) 2009/90/EC (...) laying down(...) technical specifications for chemical analysis and monitoring of water status , establishes minimum performance criteria for the analytical methods used in monitoring water status. Those criteria ensure meaningful and relevant monitoring information by requiring the use of analytical methods that are sensitive enough to ensure that any exceedance of the EQS can be reliably detected and measured.

(11a) (Council) The implementation of this Directive raises challenges which include the diversity of possible solutions to scientific, technical and practical questions and the incomplete development of monitoring methods, as well as constraints on human and financial resources. To help address some of these challenges, the development of monitoring strategies and analytical methods should be supported by technical work by expert groups under the Common Implementation Strategy for the Water Framework Directive.

Red highlights: CH, underlined phrases: Council

Approach of German ministry for environment

Two position papers, based on expert judgement in two workshops to define

a mandate for CMEP and make a statement on sensitivity and reliability of

existing tools

I. Focal discussion of experts from authorities and academia on bioassays

II. Focal discussion of Laender experts from water laboratories on chemical analyses‘ experience with EE2 and E2

Position papers should document state of the art and recommend a monitoring strategy to support needs of EQS-Directive

Abstracts and presentations are welcomeCoordination: UBA and BfG

Ecotoxicological relevance of EE2 and E2

EE2: n=9, HC5 = 70 pg/L AA-EQS= HC5/2 = 35 pg/l

SSD graph EE2 SSD graph E2

E2: n=11, HC5 = 800 pg/l AA-EQS= HC5/2 = 400 pg/l

EE2 and E2: Specific SSD approaches in the EU dossiersThe EU EQS guidance notes than an assessment factor in the range of 1-5 should be applied to the HC5derived. Based on the available dataset it is considered that an assessment factor of 2 is appropriate based on the data for EE2 and E2.

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, fish and amphibians and is also protective for molluscs. • In addition where several studies were available for a species the lowest reliable effect concentration has been used adding a further level of conservatism.

mainly fishspecies are at risk

Ecotoxicological relevance of EE2 and E2 and monitoring problems

There was significant progress in the hazard assessment of EE2 and E2, but the monitoring of 35 pg/L EE2 and 400 pg/L E2 will require the best available(bio)analytical techniques and cannot be done with routine methods.

We have no high safety factors (only 2), according to sufficient data quality

LOQs for EE2 and E2 which are magnitudes of order away from the AA-EQS will not provide a protection level for all fish populations

Therefore also here: the Limit Of Quantification (LOQ) of methods shouldpreferably lower than the AA-EQS

Despite the ecotoxicological relevance the EQS setting will be postponed.

In the meanwhile we should investigate, discuss, and prepare appropriatemonitoring options for the regulation

Chemical quality assessment in WFD

MEC= Measured environmental concentration (mean, median or 90th percentile)

QC= Quality criteria (usually the AA-EQS)

Question:Is it necessary to measure everywhere EE2, E2 ?

I would say no, because we have sensitive bionanalytical methods which allow a screening and decision to find locations where EQS may be exceeded

The verification of this thesis will follow on the next slides.

?QC

MEC (RQ)nt Riskquotie>1 intolerable risk

<1 tolerable riskSource: Knacker 2007

Which EE2 concentrations do we expect in Swiss water bodies?

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Surface waterEE2 concentration

withoutaccumulation

Surface waterEE2 concentrationwith accumulation

EE2 effluentconcentration

PEC > 350 pg/L

70 pg/L < PEC ≤ 350 pg/L

35 pg/L < PEC ≤ 70 pg/L

3.5 pg/L < PEC ≤ 35 pg/L

0.35 pg/L < PEC < 3.5 pg/L

PEC < 0.35 pg/L

Com

paris

onof

PEC

with

EE2-

AA

-EQ

S at

495

disc

harg

esof

trea

ted

efflu

ent

(wat

erco

urse

s)

Situation analysis of EE2 in Switzerland at low flow conditions (Q347)

AA

-EQ

S m

etA

A-E

QS

exce

edan

ce

Main conclusions for EE2 exposure

EE2 will cause under low flow conditions in around 20-25% of the surface waterbodies an EQS exceedance, the monitoring can be reduced to water bodies whichare significant polluted by WWTPs. Screening methods are an option! (Kienle et al. 2011)

The effluents of WWTPs will have EE2 concentrations between 70-350 pg/L, therefore also the L-YES (LOQ of 2-3 ng/L for E2) should be performed withupconcentrated samples and/or more sensitive bioanalytics should be used asscreening tools, also for surface water (see Hecker and Hollert 2011).

The consumption of EE2 in Switzerland is comparable to neighbouring countries like (Austria, Germany and France) with the exception of Italy where lowerconsumption is reported. In general a comparable EE2 situation is expected.

Country consumption of EE2 / Mio habitantsand year

Austria 0.508

Germany 0.562

France 0.504

(Italy 0.328, lower to around 50 Mio catholics)Adapted form Hanah et al. 2009 and internal UBA data

extrapolated EE2 consumption for CH 0.525 kg/Mio habitants / year

What is with the combined risks of steroidal estrogens at median flow?

• The SSD based EQS for E2 will be at 0.4 ng/l (derived from 11 chronic NOECs of different fishspecies)

• The most sensitive study is done withrainbow trout (Lahnsteiner et. al. 2006) NOEC of 0.5 ng/L (for endpoints: fertilizationsuccess, sperm density and volume).

• E2 the metabolite estrone (E1) and thepharmaceutical 17-alpha ethinylestradiol (EE2) contribute additionally to the estrogenreceptor mediated estrogenicity, so we havea cummulative risk.

• Also industrial chemicals like BPA, Nonylphenol, Octylphenol and somePhthalates could have estrogenic impacts andhave lower receptor binding potentials.

Again, we have in a limited number ofareas a combined and continous risk, the use of screening methods canreduce the measurement costs of high end analytics!

Source: Burkhard-Holm et al. 2005

Examples of bioanalytical detection limits

FSA

XEM

A

Pa-R

epro

1

Pa-R

epro

2

Dm

-Rep

ro

Chi

rote

st

MV

LN

YES

McD

on.

YES

Sum

pter

YAS

Sum

pter

H29

5R

ER

Cal

ux

AR

Cal

ux

E-Sc

reen

ELR

A

AW

AC

CS

Sam

ple

conc

entra

tion

in n

g/L

0.01

0.1

1

10

100

1000

10000

min. LOEC oder ECx max. LOEC oder ECx min. EC50 max. EC50

EE2

kein

e An

gabe

kein

e A

ngab

e

kein

e An

gabe

kein

e An

gabe

kein

e An

gabe

EE2

T

E2

EE2

E2

T

E2

DHT

E2

E2

Even bioanalytical methodsreach detection limits below 1 ng/L. Moreover they can measureestrogenic effects in an integrative way, so they could besuitable as screening methods.

It is possible to measureintegrative E2-equivalents (EEQ)with different cell based bioassays

Comparison of 5 in vivo and10 in vitro test procedures. More information on thecompared test-procedurescould be found in (Kase et al. 2009)

Abbreviations: T= testosteroneDHT= dihydrotestosteroneE2=17-β-estradiolEE2= 17-α-ethinylestradiol

Source: Kase et al. 2009

How does it look with real bioanalytical measurements?

The results show that the estrogenic activity in the Rhine at Lobith was low, with values below 0.2 ng estradiol-equivalents per litre (E2-eq/L). Exceptions were two peak concentrations of 0.4 and 0.8 ng E2-eq/L found in the summer of 2010.

It seems the bioanalytics partially verify our modelling results, the risk is limited to polluted areas and only a few exceptions are above 0.4 ng/L E2-eq.

Rhine at Lobith from RIWA 2012:

AA-EQS for E2 at 0.4 ng/L

How the bioanalytics look in other rhine-stations and Maas?

AA-EQS for E2 at 0.4 ng/L

The results show that the estrogenic activity is also low in other locations in 2010 and 2011 with the exception that Maas has high EQS exceedance potential for E2. In this case the screening enables to distinguish clearly between polluted and unpolluted water bodies.

From RIWA 2012: Meanestrogenicity activity in Rhine at Lobith, Lek atNieuwegein, Amsterdam Rhinechannel and Maas atKeizersveer

Summary & main conclusions

It is highly likely that different steroidal estrogens (EE2, E2 and E1) and some estrogenic industrial chemicals have an impact at environmental relevant concentrations on fish populations, even at median flow conditions close to WWTPs. But this estimated continous risk exists mainly in highly populated areas with low dilution factors for waste water.

At low flow conditions we expect only in a limited number of water bodies (around 20%) AA-EQS exceedances for EE2 in Switzerland, which has a comparable relative EE2 consumption to other Member States (exception Italy). We expect for around 80% of water courses EE2-EQS compliance.

The combined risk at median flow conditions of receptor binding substances is also limited to some polluted areas.

The bioanalytical measurement in the river Rhine indicate only few EEQ above the E2- AA- EQS in 2004-2011 and allows a clear differentiation of estrogenic potentials.

With a validated bioanalytical screening tool we could reduce the costs for high end monitoring measurements

Aims of the meeting

To provide you with relevant information about the usefulness of bioanalyticaltools for surface water screening

To identify and discuss suitable bioanalytical monitoring options which could be candidates for a validation project

To recommend 1-2 bioanalytical tools for a prevalidation project

To identify cooperation partners and to discuss participation (laboratories andCMEP, ISO, CEN)

If we succeed with these steps the CMEP lead could discuss the outcome at thenext WG E meeting in April for a further task discussion

Outlook: The implementation of a validated effect based tool in the WFD? (see presentation of Bernd Gawlik, JRC)

Last but not least

Do you have any suggestions for furtherimprovement ?

Do you have any questions ?

If yes, please do not hesitate to contact us.

contact: Christiane.Heiss @uba.deRobert.Kase@oekotoxzentrum.ch

Source: Gerd Maack, SETAC 2010 Sevilla

Cited and related references

Burkhardt-Holm Patricia, Giger Walter , Guettinger Herbert , Ochsenbein Ueli , Peter Armin, Scheurer Karin , Segner , Helmut, Staub Erich and Suter Marc J.-F. (2005): Where Have All the Fish Gone? Environ. Sci. Technol., 2005, 39 (21), pp 441A–447A. DOI: 10.1021/es053375z

Hannah R., J. D'Aco V., Anderson P.D., Buzby M.E., Caldwell D.J., Cunningham V.L., Ericson J.F.,Johnson A.C., Parke N.J., Samuelian J.H. and Sumpter J.P. (2009).:"Exposure assessment of 17alphaethinylestradiol in surface waters of the United States and Europe.“ Environmental Toxicology and Chemistry 28(12): 2725-2732.

Hecker Markus and Hollert Henner (2011): Endocrine disruptor screening: regulatory perspectives and needs. Environmental Sciences Europe 2011. 23:15. http:www.enveurope.com/content/23/1/5

Kase R., Kunz P., Hollert H., Werner I (2012): Contribution on bioanalytical assays for steroidal estrogens, in EU JRC report by Loos, R. “Analytical methods relevant to the European Commission's 2012 proposal on Priority Substances under the Water Framework Directive”. ISBN 978-92-79-26642-3. Publications Office of the European Union, 2012. Available at CIRCABC or http://publications.jrc.ec.europa.eu/repository/handle/111111111/26936.

Kase Robert, Clayton Helen, Martini Frederique (2012): Science-Policy Interface (SPI) activity on prioritisation of research needs, knowledge availability and dissemination for the Working Group E (Chemical Aspects) 2010-2012. Open available at CIRCABC.

Kase R, Segner H, Maack G (2011): Diclofenac and EE2: Some answers for the key unresolved issues in the aquatic environment. Pharmaceutical Advisory Group, Milan the 18th May 2011

Kase Robert, Eggen Rik I L, Junghans Marion, Götz Christian and Juliane Hollender (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, InTechDownload: http://www.oekotoxzentrum.ch/dokumentation/publikationen/doc/bookchapter

Kase Robert, Kunz Petra, Gerhardt Almut (2009): Identification of reliable test procedures todetect endocrine disruptive and reproduction toxic effects in aquatic ecosystems”. "Identifikation geeigneter Nachweismöglichkeiten von hormonaktiven und reproduktionstoxischen Wirkungen in aquatischen Ökosystemen." Umweltwiss SchadstForsch 21(4): DOI 10.1007/s12302-009-0072-2. Download: http://link.springer.com/article/10.1007/s12302-009-0072-2

Kienle, C., Kase, R., Werner, I. (2011). Evaluation of bioassays and wastewater quality: In vitro and in vivo bioassays for the performance review in the Project "Strategy MicroPoll".Swiss Centrefor Applied Ecotoxicology, Eawag-EPFL, Duebendorf. Download: http://www.oekotoxzentrum.ch/dokumentation/berichte/doc/Bericht_Micropoll.pdf.

Knacker Thomas (2007): Möglichkeiten der ökotoxikologischen Bewertung für Einzelstoffe und Gemische. Vortrag am IKSR-Workshop. Mikroverunreinigung aus der Siedlungswasserwirtschaft, der am 23. Mai 2007 in Bonn gehalten wurde.

Cited and related references

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.

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

RIWA, Authors:Tineke Slootweg and Corin J Houtman (2012): Evaluatie van hormonale activiteit gemeten in de Rijn bij Lobith (2010-2011). http://www.riwa-rijn.org/de/veroeffentlichungen/

Cited and related references

Appendix I: Modelling parameters for EE2 in treated Swiss effluent and river water

4.18 kg/ year EE2 consumed in Switzerland 8 million Swiss habitants 60% metabolised in the human body, meaning 40% EE2 enters Influent 90% degraded during biological treatment 365 days 400 L waste water per person and day

In contrast to this limitations this model is usable for different other waste water driven micropollutants (see Kase et al. 2011) and can be used for risk assessments as a kind of realistic worst case scenario under low flow conditions.

It was also successfully used for situation analysis in Switzerland, NordrhineWestfalia, Baden-Württemberg and lake Konstanz. http://www.oekotoxzentrum.ch/projekte/stofffluss/index

Appendix II: Chemical quality assessment for Switzerland

Appendix II: Riskquotients during waste water treatments

Appendix II: Estrogenicity during waste water treatment

Appendix II: Water quality during waste water treatment