REPORT TO DEFRA

ON

ENDOCRINE DISRUPTERS IN THE

ENVIRONMENT - LINKING RESEARCH AND

POLICY

A JOINT UK GOVERNMENT

SETAC EUROPE (UK BRANCH)

AND SETAC EUROPE MEETING

31 March - 1 April 2003

AND

UK-JAPAN GOVERNMENT RESEARCH

CO-OPERATION ON ENDOCRINE DISRUPTION

IN THE AQUATIC ENVIRONMENT WORKSHOP

2-3 April 2003

CENTRAL SCIENCE LABORATORYYORK

Report prepared by Dr Helen Thompson, Central Science

Laboratory, Sand Hutton, York, YO41 1LZ

10 May 2003

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This report is divided into 2 sections the Section 1 gives an overview of the 2 day SETAC Europe/ SETAC Europe UK Branch/UK government meeting (31 March-1 April 2003), section 2 details the proceedings of the 1.5 day UK/Government workshop on research co-operation in endocrine disrupter research (2-3 April 2003)

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SECTION 1

ENDOCRINE DISRUPTERS IN THE

ENVIRONMENT - LINKING RESEARCH AND

POLICY

A JOINT UK GOVERNMENT

SETAC EUROPE (UK BRANCH)

AND SETAC EUROPE MEETING

31 March - 1 April 2003

CENTRAL SCIENCE LABORATORYYORK

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Introduction....................................................................................................................5

Session 1 - Endocrine Disruption in the Freshwater Environment.........................5

Endocrine Disruption in freshwater fish: the past, the present and the future..............5

Mixtures of estrogenic contaminants in fish exposed to sewage treatment effluents....6

Reproductive effects of oestrone in a fathead minnow pair-breeding test.....................6

Development of an excretion and transformation model to predict concentrations of steroid oestrogens in sewage effluents and in vulnerable catchments...........................7

Estimating the effects of 17a-ethinylestradiol on populations of the fathead minnow Pimephales promelas: are conventional toxicological endpoints adequate?..................8

Session 2 - Endocrine Disruption in the Marine Environment:..............................9

Overview of UK Endocrine Disruption Research in the Aquatic Environment............9

Oestrogen and androgen receptor agonists: Identification and measurement of in vitro activity in the aquatic environment................................................................................9

Biomarkers of oestrogenic endocrine disruption in fish: The story in UK estuaries.. .10

The three-spined stickleback as the European sentinel species for (anti)-androgenic and oestrogenic xenobiotics.........................................................................................11

The use of oestrogenic exposure markers as predictors of population level reproductive success in an estuarine fish.....................................................................11

Biological Effects Techniques For Monitoring Endocrine Disrupting Chemicals In The Offshore Oil Industry............................................................................................12

Session 3 Current European programmes and future perspectives:....................13

Community Action in Drafting Policies and Supporting Research in the Endocrine Disrupter Field.............................................................................................................13

Overview of Danish field investigations on endocrine disruption in fish (brown trout, roach and flounder)......................................................................................................14

Results of the Dutch national investigation on estrogenic compounds in the aquatic environment..................................................................................................................14

The ENDIS-RISKS project: Endocrine disruption in the Scheldt estuary; distribution, exposure and effects.....................................................................................................15

Session 4 Effects in other species..............................................................................16

Screening of potential endocrine disruptors for (ant)agonist activity in the Drosophila melanogaster BII ecdysteroid bioassay.........................................................................16

Endocrine disruption in freshwater arthropods, molluscs and nematodes...................17

The Elucidation of Annetocin in Earthworms; Mechanistic Link Between Gene and Life-cycle Parameters Following Exposure to EDCs..................................................18

Session 5 Science issues and the way forward?.......................................................19

Gender bent but not in the mind of fish.......................................................................19

Molecular Approaches to Unravelling Sexual Disruption in Fish...............................19

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Something from nothing? - combination effects of multi-component mixtures of estrogenic chemicals....................................................................................................20

Methods for identifying endocrine disruptors in complex mixtures............................21

OECD endocrine disrupter assessment & ecotoxicology – progress so far.................22

Concluding session.....................................................................................................23

Appendix 1 Programme.............................................................................................24

Appendix 2 List of participants................................................................................26

Appendix 3 Poster Abstracts.....................................................................................38

A Sensitive Ribonuclease Protection Assay to Detect Multiple Gene Targets in Fish Endocrine Disrupter Research......................................................................................38

Differential display PCR analysis for detecting genes regulated by endocrine disrupters in fish...........................................................................................................38

Development of an hybridisation protection assay for detecting aromatase P450 gene expression.....................................................................................................................39

Rapid bioconcentration of 17b-oestradiol in the common mussel (Mytilus edulis): implications for biomonitoring....................................................................................40

Effects of endocrine disrupters on the egg production of Acartia tonsa......................40

Determination of endocrine effects in sediments with prosobranch snails..................41

A bioassay for assessing toxicant effects on growth and development of individual Lymnaea stagnalis embryos.........................................................................................42

Xenopus tropicalis larvae as a model for the determination of the effects of endocrine disruptors on amphibian development.........................................................................42

Vitellogenin and plasma zinc as a markers of avian exposure to oestrogenics...........43

The effects of fenoxycarb and diflubenzuron on honeybee (Apis mellifera) colonies 44

Cadmium and zinc effects associated with the Aznalcóllar mining spill on Procambarus clarkii : vitellogenin/vitellin, ovarian and hepatopancreatic indexes and histopathology as biomarkers of physiological disruption...........................................45

Ubiquitous imposex in the marine gastropod Nucella lapillus (L.) in Galicia (NW Spain)...........................................................................................................................45

First monitoring of the occurrence of endocrine disruption in inland populations of eel (Anguilla anguilla), roach (Rutilus rutilus), rudd (Scardinius erythrophtalmus) and tench (Tinca tinca) in Flanders (Belgium)...................................................................46

A search for evidence of endocrine disruption of reproduction in top predator fish.. .47

Fish microsomal cyp450 activities and endocrine disruption screening......................48

The fate of alkylphenolpolyethoxylates and alkylphenols in roach (Rutilus rutilus). .48

Accounting for differences in and responsiveness and sensitivity of fish to oestrogenic effluents........................................................................................................................49

Successful detection of environmental (anti-)androgens using a fathead minnow (Pimephales promelas) non-spawning assay................................................................49

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Uncertainties in evaluating results of a yeast estrogenic assay....................................50

How predictive are in vitro tests of in vivo responses to endocrine disrupting chemicals......................................................................................................................51

Comparison of several tests for endocrine/estrogenic activity....................................51

In vitro and in vivo estrogenicity of substituted benzenes...........................................52

Inclusion of Endocrine Disruption in the Environmental Risk Assessment of Chemicals.....................................................................................................................53

Factors to consider when examining uncertainty, exposure and impact in risk assessments for potential EDCs...................................................................................53

ACE -Analysing combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms.............................................................................................54

Determination and fate of conjugated steroid estrogens in the sewage treatment process..........................................................................................................................55

Behaviour of Endocrine Disrupting Chemicals Bisphenol A, Nonylphenol and Short-Chain Nonylphenolethoxylates during Anaerobical Mesophile Treatment of Municipal Sewage Sludge............................................................................................56

Mobility and Fate of Endocrine Disrupting Compounds (EDC) in Soil after Application of Sewage Sludge to Agricultural Land...................................................56

Effective Removal of Natural and Synthetic Steroids from Sewage Sludge during Aerobic and Anaerobic Sludge Stabilisation...............................................................57

Xenoestrogen Removal from Sewage Sludge..............................................................58

Recycled Paper Distinctly Contributes to the Bisphenol A, Nonylphenol Ethoxylate, and Nonylphenol Load of Municipal Wastewater.......................................................58

Degradation of the radioactive and non-labelled branched 3',5'-dimethyl 3'-heptyl-phenol nonylphenol isomer by Sphingomonas TTNP3...............................................59

Assessment of estrogenic activity in WWTP effluents using chemical analysis and biological endpoints.....................................................................................................60

Release of Nonylphenol, Octylphenol, and Bisphenol A with Leachate from Municipal Landfill Simulation Reactors......................................................................60

The Determination of Alkylphenols in Food...............................................................61

Uptake of nonylphenols by plants in water solution....................................................62

Analysis of endocrine-disrupting phenolic compounds and steroids with GC-MS/MS......................................................................................................................................63

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Introduction

The SETAC meeting “Endocrine Disruptors in the Environment: Linking Research and Policy” held on 31 March and 1 April at the Central Scisnce Laboratory, York was attended by 112 participants from across Europe from academia, government and industry. The organising committee comprised Helen Thompson (CSL), Mike Roberts (Defra), Kathleen Cameron (Defra), Geoff Brighty (EA), John Sumpter (Brunel University) and Tom Hutchinson (Astrazeneca, BEL). The aim was to present the significant amount of work funded by government (DEFRA, BBSRC and Environment Agency) in the UK on endocrine disrupters in the environment and to put it in the context of other work funded within Europe. Thus the meeting provided a forum for wider discussion of government, industry and EU funded endocrine disrupter research.

Dr Helen Thompson (CSL) welcomed the participants to Central Science Laboratory and gave a brief overview of its work. Dr Thompson then introduced the meeting and thanked the sponsors (Defra and the Environment Agency). The meeting was structured to provide an overview of the work funded by UK government of day 1 with sessions on “Endocrine Disruption in the Freshwater Environment” and “Endocrine Disruption in the Marine Environment”. This was introduced by Kathy Cameron with a brief review of the co-ordination of UK Research (including the forthcoming Defra call for tenders on endocrine disruption) and the Government Interdepartmental Group on Endocrine Disrupters. Other European research was the focus of day 2 with sessions on “Current European programmes and future perspectives”, “Effects in other species” and “Science issues and the way forward?” with a final discussion session on analysis of the key future issues.

Session 1 - Endocrine Disruption in the Freshwater Environment Chair: Helen Thompson

Endocrine Disruption in freshwater fish: the past, the present and the future.

John P Sumpter

Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K.

Research on endocrine disruption has exploded since Theo Colborn and co-workers first suggested it, back in 1992, as a common mechanism to explain a number of examples (in birds, fish, and molluscs) of developmental and/or reproductive problems in wildlife. However, in reality the study of endocrine disruption has been in progress for well over 50 years. Probably nobody would have predicted the explosion in research activity in this field which has occurred in the last 10 years, precipitated primarily, I think, by a few case studies, in particular developmental problems in some alligator populations in the U.S. and reproductive problems indicative of exposure to “oestrogens” in fish in British rivers and estuaries. This could be considered a case of the field getting “fatter”, but is it progressing? My answer is yes, although possibly not at the rate I would like. Possibly too much time

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and effort has gone into learning more and more about what we already know something about, and not enough into trying to move the field forward. Now that many people, from different disciplines, are working in the area of endocrine disruption, it is probably appropriate that larger, multi-disciplinary projects attempt to tackle some of the key unanswered questions. Many of these questions, such as “are there population-level effects caused by endocrine disruption?”, “can the effects of complex mixtures be reliably predicted?” and “what are wildlife exposed to?” will require broadly-based approaches if they are to be addressed successfully. There is still plenty for us all to do!

Mixtures of estrogenic contaminants in fish exposed to sewage treatment effluents.

Gibson, R.(1) Spary, C. (2) Tyler, C.R.(2) Sumpter, J.P.(3) and Hill, E.M.(1)

(1) University of Sussex, Brighton, (2) University of Exeter, (3) Brunel University, Uxbridge

Effluent from sewage treatment works (STWs) contains estrogenic substances that may contribute towards the high incidence of intersex fish in many U.K rivers. A range of estrogenic chemicals have been identified in these effluents but there is little information on which chemicals accumulate in fish. To address this, we exposed rainbow trout to either tap water or STW effluent for 10 days and investigated the estrogenic activity of the bile and induction of vitellogenin biomarker. The estrogenic activity of hydrolysed bile was measured using a yeast estrogen receptor transcription screen (YES assay). Glucuronidase treatment of bile from effluent exposed fish increased estrogenic activity by 11-56 fold. There was no induction in plasma vitellogenin levels in tapwater exposed fish and the estrogenicity (estradiol equivalents μg/mL) of the hydrolysed bile was 0.05 ±0.01 (mean ± s.d.). Levels of vitellogenin were markedly induced in effluent exposed male and female fish and the estrogenic activity in bile of both sexes was 1.3 ±0.3 μg/mL which was 13,000-50,000 fold higher than the activity of the effluent. Bile samples were fractionated by RP-HPLC, screened in the YES assay and analysed by GC-MS. The bile of reference fish contained only estradiol whereas bile of effluent-exposed fish contained high concentrations of estradiol, estrone, ethynylestradiol and alkylphenolics as well as an unknown estrogenic component. There was no difference in estrogenic profiles between bile of male and female fish in the two treatments. The results indicate that estrogenic contaminants bioconcentrate to a high degree in fish bile and that TIE analysis can be used to determine the nature of these estrogenic mixtures in fish tissues.

Reproductive effects of oestrone in a fathead minnow pair-breeding test.

Thorpe K.L.1,2, Benstead, R.3, Hutchinson, T.H.2 Cummings R.I.2 and Tyler, C.R1.

1University of Exeter, Devon, UK. 2AstraZeneca, Devon, UK. 3Environment Agency, Waterlooville, Hants, UK.

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The natural steroid oestrogens, oestradiol and oestrone, and the synthetic steroid ethinyloestradiol, are known to contaminate the aquatic environment throughout Europe. The oestrogenic effects of the oestradiol-17b and the ethinyloestradiol, have been well characterised in fish. These steroids are present in effluents at concentrations that induce vitellogenin synthesis and in some cases at concentrations that cause reproductive effects. Oestrone is known to be a weaker oestrogen compared with oestradiol-17b, but is often found in effluents from STWs at far higher concentrations than oestradiol-17b (up to 10-fold higher). Little is known about the effects of exposure to exogenous oestrone on reproduction in fish. In this study pair-breeding fathead minnow were exposed to oestrone at concentrations ranging between 32 and 1000ng/L. Measured exposure oestrone concentrations were greater than 70% of nominal throughout. Oestrone exposure induced a concentration-dependent induction of VTG. Exposure to the highest dose of oestrone for 3 weeks resulted in a lower gonadosomatic index in both the males and females and caused a suppression of male secondary sex characteristics (tubercle number and fatpad index). Egg production was reduced at exposure concentrations of 320ng/L and 1000ng/L. There were no trans-generational effects on hatching success, sex ratio, growth or development in the F1 generation.

Development of an excretion and transformation model to predict concentrations of steroid oestrogens in sewage effluents and in vulnerable catchments

Andrew C. Johnson and Richard J. Williams

Centre for Ecology and Hydrology, Wallingford, Oxon, OX10 8BB, United Kingdom

Given the significance of steroid oestrogens as endocrine disrupters, it is clearly important for water quality regulators and managers to be able to quantify the steroid oestrogens entering a catchment. Unfortunately, measuring steroid oestrogen concentrations in sewage influent and effluent remains an extremely difficult and expensive undertaking. Thus, a predictive model for assessing steroid oestrogen (17ß-oestradiol, oestrone and 17a-ethinyloestradiol) load reaching an STW has been developed. As the population served by a STW is usually known, it should be possible to calculate the quantity of steroid estrogens that a population would generate on a daily basis. The influent concentrations could then be predicted using the best available flow data for the STW. To achieve a good prediction a wide range of complex factors need to be assessed including conjugation and metabolism within the body together with quantities excreted in the urine and faeces by different members of the population. These data are then combined with fate and behaviour information for the conjugates and hormones themselves to predict concentrations at sewage works inlet following sewer transit. When a degradation factor for the sewage works and hydrological information is incorporated then effluent and river concentrations can be predicted. The method has proved to be reasonably accurate when tested against a recent comprehensive data set. Thus, the model can be used to deliver predicted steroid oestrogen concentrations for effluents of selected STW and combined with dilution factors as a first tier of a risk assessment exercise. The model can be connected to sophisticated GIS hydrological catchment models (e.g. the GREAT-ER model) to predict river concentrations for catchments combining the impact of

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multiple discharges. Comparing predicted levels with appropriate known biological effect levels allows the identification of high-risk sites.

The basic principles of this model suggest it could be adapted to predict concentrations of selected pharmaceuticals and personal health care products using the same approach.

Estimating the effects of 17a-ethinylestradiol on populations of the fathead minnow Pimephales promelas: are conventional toxicological endpoints adequate?

Eric Grist, N. Claire Wells, Paul Whitehouse, Geoff Brighty and Mark Crane

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK.WRc-NSF, Henley Road, Medmenham, Marlow, Buckinghamshire, SL7 2HD, UKEnvironment Agency, National Centre for Ecotoxicology and Hazardous Substances, Howbery Park, Wallingford, Oxon.

Environmental benchmarks have recently been proposed for several steroids including the synthetic steroid, 17a-ethinyloestradiol (EE2). These benchmarks are based on extrapolation from studies involving long-term exposure of various fish species to EE2 (1). One of the critical studies was a complete life-cycle experiment performed with the fathead minnow Pimephales promelas over a 289 day exposure period (2). The lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) for gonad histology were 4 and 1 ngl-1, respectively. This was because no testicular tissue could be found in any fish exposed to 4 ngl-1. In the present paper, the survival and reproduction data from that study are re-analysed to determine the effects of EE2 on the intrinsic rate of population growth (r = ln (l)), a parameter of demographic importance. We estimate critical threshold concentrations with respect to r and compare these with those previously derived from conventional toxicity test summaries. Further, we assess the influence of individual variability on threshold estimates using a combination of bootstrap and regression approaches, together with a suite of perturbation analyses. These yield ErC100 values (the concentration estimated to reduce intrinsic growth rate to zero) of 3.11 ngl-1 (linear model) and 3.41 ngl-1 (quadratic model), comparable with a maxiumum acceptable toxicant concentration of 2 ngl-1 for feminisation of exposed fish calculated by Laenge et al (2). Our results indicate that reduction in population growth rate with increasing concentration occurred more through EE2 acting to reduce fertility than survival rates. The significance of these summary statistics when deriving environmental benchmarks for steroid oestrogens are discussed in the context of affording protection to populations following long-term exposure.

References1. Young W.F., Whitehouse P, Johnson I, and Sorokin N. Proposed Predicted No Effect Concentrations (PNECs) for Natural and Synthetic Steroid Oestrogens in Surface Waters. Environment Agency Technical Report P2-T04/1, Environment Agency, Bristol, UK, 2002.

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2. Laenge R.; Hutchinson T.H.; Croudace C.; Siegmund F.; Schweinfurth H..; Hampe P.; Panter G.H.; Sumpter J.P.; Environ. Toxicol .Chem. 2001, 20, 1216.

Session 2 - Endocrine Disruption in the Marine Environment: Chair Mike Waldock

Overview of UK Endocrine Disruption Research in the Aquatic Environment

Yvonne Allen

Centre for Environment, Fisheries and Aquaculture Science, Burnham Laboratory, Remembrance Avenue, Burnham on Crouch, Essex, CM0 8HA, UK.

The issue of endocrine disrupters and their effects in the UK aquatic environment first emerged when the presence of hermaphrodite fish in the lagoons of sewage treatment works (STWs) was reported. Subsequent freshwater-based research aiming to assess the full extent of the effects on fish and identify the causative substances, found that some STWs contain oestrogenic compounds that were having a feminising effect on male fish, as manifested by induction of vitellogenin and appearance of oocytes in the testes. In the light of the findings of these studies, further work was funded in the UK to determine if oestrogenic effects were also occurring in the marine environment. It was discovered that several estuaries were heavily contaminated with oestrogens, manifested as feminisation of male flounder. This work catalysed the initiation of the Endocrine Disruption in the Marine Environment (EDMAR) programme, funded by a consortium of UK Government Departments and Agencies and the European Chemical Industry Association. This programme sought to investigate evidence for changes in the reproductive health of marine fish and invertebrates due to endocrine disruption, and the possible causes. To achieve this objective, the work broadly comprised development of new methods for detecting endocrine disruption, measurement of biological responses in the field and identification of causal substances and sources. The results of these research programmes highlighted areas which require further work and identified gaps in knowledge. These were discussed at a Defra (Department for Environment, Food and Rural Affairs) sponsored workshop in 2001, the outcome of which was a list of recommendations for future research. Thispresentation will provide an overview of UK aquatic ED research and present the workshop's recommendations for further research.

Oestrogen and androgen receptor agonists: Identification and measurement of in vitro activity in the aquatic environment.

K.V. Thomas, M.R. Hurst, J. Balaam and J.E. Thain

CEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UK.

Effects consistent with exposure to oestrogenic and androgenic substances have been observed in wild fish populations. In the UK, this has been predominantly observed as

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the occurrence of elevated plasma vitellogenin (VTG) concentrations in certain freshwater and estuarine species (e.g. flounder). In order to assess the risk that oestrogenic and androgenic substances pose to the aquatic environment, and to regulate activities that may be releasing such compounds, there is a need to identify which compounds are responsible for these effects. It is possible to identify the compounds responsible for in vitro effects by using a combination of in vitro receptor based assays and chemical analysis. For example, steroid oestrogens have been identified as responsible for the majority of the activity seen in marine sewage treatment works (STW) effluents. Targeted chemical analysis and bioanalytical monitoring can then be applied to assess the effectiveness of additional treatment processes in reducing the oestrogenic activity of STW effluents. This and other applied examples are used to show how oestrogenic and androgenic substances can be identified in the environment, how bio- and chemical analysis can be used to assess their effects in the environment and how the information obtained can be used in both regulatory and policy frameworks.

Biomarkers of oestrogenic endocrine disruption in fish: The story in UK estuaries.

M.F. Kirby 1 , Y.T. Allen1, S.W. Feist2, I. Katsiadaki2, P. Matthiessen3, A.P. Scott2 and J.E.Thain1.

1. CEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UK.2. CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, UK.3. CEH, Far Sawrey, Ambleside, Cumbria, UK.

CEFAS has utilised biomarkers of endocrine disruption for monitoring effects in UK estuarine fish populations since 1996. Plasma vitellogenin (VTG) concentrations and the presence of the ovotestis condition in male flounder (Platichthys flesus) have been assessed with respect to the temporal trends of these biomarkers in a number of estuaries. These data confirm that plasma VTG concentrations in male flounder remain significantly elevated in several UK estuaries. However, analysis of the time series data suggests that oestrogenic contamination, as measured by plasma VTG, have reduced in the Tyne and Mersey. The data is also assessed with respect to specific point sources. The occurrence of the ovotestis intersex condition has continued to be detected at a low but consistent levels - with the majority of cases being noted in specimens from the Tyne and Mersey estuaries.This presentation also describes the development of other estuarine fish species, during the Endocrine Disruption in the Marine Environment (EDMAR) programme, as sentinels for the detection of oestrogenic effects. In particular, the sand goby (Pomatoschistus sp.) and the viviparous blenny (Zoarces viviparus) were chosen as representatives that have an entire life-cycle that is estuarine-based. Data is presented for the presence of ovotestes and the induction of VTG mRNA in both species but of most note was the discovery of a morphological marker in the sand goby denoted morphologically intermediate papilla syndrome (MIPS). MIPS represents feminisation of the male uro-genital papilla and has been associated with areas of known oestrogenic contamination and has been induced by exposure to oestrogens in the laboratory.

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Finally, a brief overview of the current research on endocrine disruption is given which is focussed on understanding the confounding factors and influences on VTG induction to enable us to interpret monitoring data with greater knowledge and so more confidently inform appropriate policy.

The three-spined stickleback as the European sentinel species for (anti)-androgenic and oestrogenic xenobiotics

I. Katsiadaki, A. P. Scott and M. R. Hurst.

CEFAS, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK

The male three-spined stickleback (Gasterosteus aculeatus) produces a glue protein, spiggin, for the construction of a nest during the breeding period. Spiggin production is under the control of androgens and is naturally found only in the kidney and urinary bladder of breeding males. A specific ELISA for spiggin has been developed and has shown that female sticklebacks exposed to waterborne androgens and the androgenic pulp mill effluent also produce spiggin in a dose response manner. Here, we describe a system for the detection of anti-androgens, based on inhibition or reduction of spiggin production by simultaneous treatment of female sticklebacks with model androgens (5dihydrotestosterone, 17-methyltestosterone) and the pure anti-androgen flutamide as well as a series of pesticides with suspected anti-androgenic activity. The pesticides examined for expressing anti-androgenicity in vivo included 2, 4 DDE, 4,4 DDE, Linuron, Diazinon, Procymidon and Vinclozolin. The development of an ELISA for stickleback vitellogenin and its application to whole body homogenates and tissue extracts is also described and the advantages of the stickleback as a model for endocrine disruption research in Europe are highlighted.

The use of oestrogenic exposure markers as predictors of population level reproductive success in an estuarine fish.

C. D. Robinson 1 , J. A. Craft2, C. F. Moffat1, I. M. Davies1, E. S. Brown2, M. F. Kirby3

and C. Megginson1

1Fisheries Research Services, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen. AB11 9DB, UK.2School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow. G4 0BA, UK3Centre for Environment, Fisheries and Aquaculture Science, Remembrance Avenue, Burnham-on-Crouch, Essex. CM0 8HA, UK

Many freshwater fish populations, and some estuarine ones, are subject to exposure to oestrogenic substances. The consequent effects on individual fish include altered gene expression, male production of egg proteins, intersex, and impairment of sperm quality. There is continuing debate as to the wider population and ecological significance of these exposures. A population-level effect occurs when a certain proportion of all the individuals in a population are showing an effect, or, more

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classically, when there are changes to population structure and/or size such as may arise from altered reproductive success. In order to investigate potential population-level effects, we conducted a series of prolonged exposures of a common UK estuarine fish (the sand goby, Pomatoschistus minutus Pallas) to oestrogenic substances (sewage effluent, octylphenol, oestradiol [E2] and ethynyl oestradiol) and measured a range of exposure indicators. Effects were seen on male maturation indices, vitellogenin (VTG) mRNA expression, and the morphology of uro-genital papillae (UGP). In an E2 dose-response breeding experiment, several of these indicators were related to subsequent individual and population-level reproductive success. E2 exposed populations showing reduced gonad size, sperm duct gland size, and male colouration, together with feminised male UGPs and increased VTG mRNA expression, subsequently had significantly reduced fertile egg production, due to fewer pairs breeding, smaller broods being produced and reduced fertility. Furthermore, there were clear relationships between nuptial colouration, sperm duct gland size and VTG mRNA expression in pre-breeding males and subsequent population-level fertile egg production. Neither ovary nor testes size were predictive of reproductive success. In UK estuaries, sand gobies show few effects of oestrogenic exposure, even where other species have elevated plasma VTG titres. However, some populations show increased incidence of abnormal UGP morphology and these may be at risk of having reduced reproductive success.

Biological Effects Techniques For Monitoring Endocrine Disrupting Chemicals In The Offshore Oil Industry

J.E.Thain1, K.V. Thomas1, S.W. Feist2, K. Hylland3, I. Katsiadaki2, A.P. Scott2, W. Reynolds

1. CEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UK.2. CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, UK.3. NIVA P.O.Box 173, Kjelsas, N-0411 Oslo, Norway.

OSPAR has included endocrine disruption as a criterion for defining hazardous substances 'of concern' whose discharges to the marine environment are to be reduced to near-zero by 2020. But, there is currently no reliable information regarding the occurrence of endocrine disruption in offshore waters and it is impossible to assess the likelihood that offshore chemicals pose an environmental endocrine disrupting compound problem. The deployment of a number of biological effects techniques in an offshore international workshop, ICES BECPELAG (International Council for the Exploration of the Seas, Biological Effects of Contaminants in Pelagic Ecosystems), has partly contributed to filling the information gap. These included the use of in situ caging of cod and mussels around an oil platform in the Northern North Sea and reference sites and bioassay and TIE studies on oil platform produced water. The results of these investigations will be presented pertinent to the potential use of the techniques for monitoring endocrine disruption eg VTG in cod, histology of tissues, yeast screening assays (YES), TIE and chemical analysis. Recommendations for a future monitoring strategy for endocrine disruption in relation to the offshore chemical industry will be discussed. The use of in situ techniques and technology in offshore environments will be presented also.

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Session 3 Current European programmes and future perspectives: Chair Peter Matthiessen

Community Action in Drafting Policies and Supporting Research in the Endocrine Disrupter Field

Dr Tuomo Karjalainen,

Scientific Officer, European Commission, Research Directorate-General, Directorate E: Biotechnology, Agriculture and Food Unit E2: Food Quality, Square de Meeus 8, Office SDME 8/29, 1050 Brussels

My talk will cover four topics :1. Community startegy on endocrine disrupters : This strategy, resulting from

public concern over potential hazards of endocrine disrupting chemicals, is specifically detailed in two Communications published by the European Commission [COM(1999)706 and COM(2001)262]. These Communications outline the need for: (i) further research; (ii) international cooperation; (iii) communication to the public; (iv) policy action; and (v) short-, medium-, and long-term strategy

2. ED projects funded by the 4th Framework Programme (1994-1998). The 18 projects funded by this programme covered four main areas:

(i) development of test methods; (ii) monitoring of ED chemicals in the environment; (iii) ecological effects; and (iv) human exposure and health effects

3. ED projects funded by the 5th Framework Programme (1998-2002). ED related projects were funded by the Quality of Life Programme (Key action 4 «Environment and Health ») as well as the Energy, Environment and Sustainable Development Programme. The former, focusing on human health effects of EDCs, has financed 14 projects with a total budget of 34 million euros, whereas the latter, focusing on environmental aspects of endocrine disruption, has sponsored 7 projects with a budget of 16 million euros.

4. Place of ED research in the 6th Framework Programme (2002-2006). ED related projects will be sponsored mainly by the Thematic Priority 5 (Food Quality and safety), although other priorities may also touch upon certain aspects of endocrine disruption.

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Overview of Danish field investigations on endocrine disruption in fish (brown trout, roach and flounder)

Lisette B. Christiansen, Bodil Korsgaard, Thomas Aagaard, Louise L. Madsen,Poul Bjerregaard

Institute of Biology, University of Southern Denmark, 55 Campusvej, DK-5230Odense M, Denmark

One study has examined the gonadal histology and vitellogenin induction of a salmonid species, brown trout Salmo trutta, from Danish streams with low to moderate load of primarily domestic sewage effluent. A high occurrence of males with vacuolated testes and only presence of early spermatogenetic stages were found at one particular stream. The cell type containing the vacuoli appeared to be the sertoli cells. Measurements of estrogens and a number of estrogenic compounds in the sewage effluent were performed but did not reveal a clear connection between the observed effects and the concentration of the hormones and chemicals in the water. Both male and female brown trout from the stream, in which testicular effects among males were observed, did, however, have a significantly higher level of plasma vitellogenin - measured by ELISA - compared to fish from control sites. This implies that the fish had been exposed to compounds with estrogenic action. Whether the morphological changes in the testes were a hormonal effect or a delay in the testicular development ascribed to population differences remain to be elucidated. Intersex roach (Rutilis rutilis) were found in streams receiving effluents, but always at a frequency less than 30%. Juvenile rainbow trout (Oncorhynchus mykiss) caged in the streams where brown trout and roach were affected, did not show increases in plasma vitellogenin concentrations. Plasma vitellogenin concentrations in male flounders from different locations in Danish coastal areas and fiords showed fairly large differences, and also at some sites, very high – largely unexplainable – differences within the population were found.

Results of the Dutch national investigation on estrogenic compounds in the aquatic environment

A.D.Vethaak1, S.M. Schrap2, P. de Voogt 3

1 RIKZ, Ministry of Transport, Public Works and Water Management, The Hague, The Netherlands2 RIZA, Ministry of Transport, Public Works and Water Management, Lelystad, The Netherlands3 Environmental and Toxicological Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.

The ‘National investigation into the occurrence and effects of estrogenic compounds in the aquatic environment’ (Dutch acronym, LOES), is a base-line study on the occurrence of a number of natural and synthetic estrogen active substances in the aquatic environment and the associated estrogenic effects in fish in surface waters. The LOES study shows that almost all the selected compounds were present at low

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concentrations in the Dutch aquatic environment. At some locations, they were found at higher levels.

Levels of natural hormones in surface water were below the LOD except for estrone, which was found above the LOD in half of the samples. Phthalates and bisphenol-A were found in almost all samples taken, but levels are unlikely to have caused any estrogenic effects. Nonylphenol and the nonylphenol ethoxylates were found in sufficiently high concentrations to cause estrogenic effects in fish. PBDEs were found in suspended matter in the Western Scheldt, with extremely high concentrations of BDE 209.

Field studies indicate that estrogenic effects were not observed in male fish (flounder) in open sea and in Dutch estuaries. Minor to moderate estrogenic effects were observed in fish (bream and flounder) in the major inland surface waters. The prevalence of feminizing effects in male fish is largest in small regional surface waters that are strongly influenced by sources of emission of potential hormone-disrupting compounds, such as specific industrial wastewater streams, biologically-treated and untreated municipal wastewater and run-off and leaching from agricultural manure.

Very high concentrations of vitellogenin were found in the blood plasma of male bream in waters receiving wastewater treatment plant effluents. Also a high prevalence of intersexuality was observed. Through additional research with in vitro and in vivo bioassays, both in the laboratory and on location (in the wwtp effluent and the receiving surface water), the hormones and nonylphenol(ethoxylate)s appear to be responsible for the estrogenic effects found.

The ENDIS-RISKS project: Endocrine disruption in the Scheldt estuary; distribution, exposure and effects

Verslycke Tim* 1 , Ghekiere A.1, Fockedey N.2, Roose P.3, De Wash K.4, Vethaak D.5, Mees J.6, Monteyne E.3, Noppe H.4, Deneudt K.6, Vanden Berghe W.6, Vincx M. 2, De Brabander H.4 and Janssen C.R. 1

1Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Belgium 2Marine Biology Section, Ghent University, Belgium3Management Unit of the North Sea Mathematical Models (BMM), Belgium 4Laboratory of Chemical Analysis, Ghent University, Belgium5National Institute for Coastal and Marine Management (RIKZ), The Nederlands6Flanders Marine Institute (VLIZ), Belgium

The first indications of possible effects of endocrine disrupting substances and the presence of these substances in the Scheldt estuary have recently been published. The industrial areas of Ghent and Antwerp are to a large extent responsible for this pollution. Although these preliminary studies indicate an extensive pollution with a wide variety of known endocrine disrupters, no detailed exposure and effect data is available for the Scheldt estuary. Furthermore, a detailed knowledge of the distribution and long-term effects of these substances is needed in the framework of

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future-oriented policy measures at the national and European level. The ENDIS-RISKS project, a multidisciplinary consortium between five different research institutes, will evaluate the distribution, exposure and effects of endocrine disrupters in the Scheldt estuary. An extended list of endocrine disrupters and the in vitro estrogen and androgen potency will be analysed in water, sediment and suspended solids. In addition, body burdens of these endocrine disrupters will be analysed in biota (mysid shrimp and gobies) and linked with in situ biomarker responses and population effects in resident mysid populations. Field samples and in situ studies will run over four years (3 campaigns per year, 2002-2006). The field study will allow an identification of potential problem chemicals with endocrine disruptive activity in the Scheldt estuary. These chemicals will be further evaluated through laboratory exposures with mysid shrimp to evaluate the acute and chronic effects on endocrine regulated processes in these animals (energy and (ecdy)steroid metabolism, vitellogenesis, specific protein expression,…). The results of laboratory and field studies will be linked to come to an integrated risk estimation for endocrine disrupters in the Scheldt estuary. The ENDIS-RISKS project, its website, project partners, goals and planning as well as the results of the first sampling campaign will be presented.

Session 4 Effects in other speciesChair Peter Matthiessen

Screening of potential endocrine disruptors for (ant)agonist activity in the Drosophila melanogaster BII ecdysteroid bioassay.

Ms. Frances H. Cary 1, Dr. Laurence N. Dinan 1, Dr. Rod W. Wilson 1, Dr. Thomas H. Hutchinson 2 and Dr. Lisa Tattersfield 3

1 University of Exeter Exeter Devon ; 2 AstraZeneca Global Safety, Brixham Environmental Laboratory Brixham Devon ; 3 Syngenta, Jealott's Hill Research Station, Bracknell Berkshire

There is mounting evidence that a wide variety of compounds can have endocrine disrupting effects on humans and wildlife. However, investigations so far have focused primarily on exposure to human and other higher vertebrates, with invertebrate findings largely restricted to marine molluscs. Ecdysteroids are insect steroid hormones involved in the control of moulting and development. The BII cell bioassay is based on an ecdysteroid-responsive cell line from Drosophila melanogaster and is capable of detecting potential invertebrate endocrine disrupting compounds acting as ecdysteroid agonists or antagonists. There have been reports of endocrine disruption resulting from exposure to bisphenol A, a compound widely used in plastics. These reports have included weak antagonism (EC50 = 1 x 10-4 M vs. 5 x 10-8 M 20-hydroxyecdysone) and ligand binding to the ecdysteroid receptor. We therefore tested 3 other related compounds in the BII bioassay. Thirteen of the tested compounds were found to be weak ecdysteroid antagonists, including bisphenol A tetramethyl (EC50 = 6.9x10-4 M), 2-hydroxydiphenylmethane (EC50 = 1.8x10-4 M) and 2,4-dihydroxybenzophenone (EC50 = 1.4x10-4 M). However, many compounds of a closely related structure had no effect. None of the compounds was found to act as ecdysteroid agonists. Statistical analysis revealed a significantly lower molecular

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mass amongst the active compounds. The results of investigations into any quantitative structure activity relationships (QSAR) among the compounds tested will also be presented. Comparisons of these data with in vitro work using vertebrate hormone assays indicate that some bisphenols have been previously identified as estrogenic, i.e. agonists, illustrating the stark differences between vertebrate and invertebrate responses where activities may be different or absent. The EC50 values presented are much higher than those likely to be found in the environment. However, some in vivo exposures of aquatic insects suggest developmental effects at low levels of exposure.

Endocrine disruption in freshwater arthropods, molluscs and nematodes

Lennart Weltje 1, Conny Scholz 1, Christian Vogt 1, Sebastian Höss 2, Jacco van Doornmalen 1, Bernd Markert 1 and Joerg Oehlmann 3

1 International Graduate School (IHI), Ecotoxicology Group, Markt 23, D-02763 Zittau, Germany2 Ecossa, Thierschstrasse 43, D-80538 Munich, Germany3 J.W. Goethe University, Faculty of Biology and Informatics, Siesmayerstrasse 70, D-60054 Frankfurt am Main, Germany

Invertebrates are underrepresented in endocrine-disruptor research. Although they make up the dominant part of the animal diversity in freshwater ecosystems, effects of endocrine disruptors have mostly been studied in vertebrates or with in vitro systems derived from them. Our research is aimed at elucidating effects of some well-known (vertebrate) endocrine disruptors on freshwater invertebrates. Therefore,we assessed the effects of several pesticides ( e.g. fenarimol,vinclozolin, methoprene) and pharmaceuticals (e.g. ethynylestradiol, cyproterone acetate, tamoxifen) on non-biting midges Chironomus riparius, isopods Asellus aquaticus, pondsnails Lymnaea stagnalis, mudsnails Potamopyrgus antipodarum and nematodes Caenorhabditis elegans. This selection of organisms not only offers sexually reproducing species (C.riparius and A. aquaticus), but also hermaphrodites (L. stagnalis and C.elegans) and parthenogens (P. antipodarum). Since our studies were focused on endocrinically-mediated effects and not on non-specific toxicity, we worked at aqueous concentrations in the pico- to nanomolar range and chose to analyse developmental and reproductive endpoints. For the arthropods these were moulting (isopods), sex-ratio, egg-rope production and metamorphosis (midges) and for the snails, egg development and egg or embryo production. For nematodes, growth and number of juveniles was determined. Furthermore, we studied the sex-dependency of moulting frequency, body weight and adult emergence in arthropods. Most of the substances evoked effects at one or more concentrations, often in more than one species. Considering that the endocrine systems of invertebrates are different from those of vertebrates, it may be questioned if these effects are really caused by endocrine mediation. However, sex-dependency of effects in arthropods and stimulation of reproduction in snails and nematodes are indicative of endocrine disruption. It is concluded that invertebrates offer many targets for endocrine disruptors, which may substantially differ from those in vertebrates. As a consequence, the list of (potential) endocrine disrupting substances will become longer when invertebrates are taken into account.

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The Elucidation of Annetocin in Earthworms; Mechanistic Link Between Gene and Life-cycle Parameters Following Exposure to EDCs

Huw J. Ricketts1, Jason M. Weeks2, Ian. Johnson2, David J. Spurgeon3, A. John Morgan1, Peter Kille1

1School of Biosciences, Cardiff University, PO Box 911, Cardiff, CF10 911Email RickettsHJ@Cardiff.ac.uk2WRc-NSF ltd., Henley Road, Medmenham, Marlow, Buckinghamshire, SL7 2HD3CEH, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, PE28 2LS

In recent years there has been growing concern of the possible harmful consequences of exposure to xenobiotic compounds that are capable of modulating or disrupting the endocrine system. This concern with Endocrine Disrupting Chemicals (EDCs) is directed at both wildlife and humans with the alteration of endocrine function being caused by interference with the synthesis, secretion, transport, binding, action or elimination of natural steroid hormones in the body. These natural substances are responsible for the maintenance of homeostasis and reproductive drive and influence development and/ or behaviour. To date little attention has been focussed on endocrine disruption effects in terrestrial invertebrates. Advancement within the field of biomarker research has been rapid and not surprisingly the application of the molecular biology tool kit has enabled the development of new and exciting endpoints; which have allowed a sensitive measurement of gene expression within model organisms such as the earthworm. Subsequently, this has in turn facilitated the mechanistic link between exposure or dose and biological effect. To date, no reproductive biomarker has been forthcoming. One such candidate gene is annetocin, which has previously been characterised as a member of the oxytocin/vasopressin superfamily of neuropeptides. It is expressed within those tissues of the earthworm containing reproductive organs and is involved in osmoregulation and the induction of egg-laying behaviour. The human homologue of annetocin, oxytocin, is under the expressional control of an oestrogen-receptor. This gives further evidence that annetocin is explicitly involved in reproductive events within the earthworm. This presentation reports on the elucidation of the annetocin gene in the earthworm Eisenia andrei and attempts to link gene changes to observed toxicity and lifecycle parameters following earthworm exposures using the test substances 17 ethinyloestradiol, 17 oestradiol, testosterone, bisphenol A and nonylphenol.

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Session 5 Science issues and the way forward? Chair John Sumpter

Gender bent but not in the mind of fish

Jon Nash

Laboratory of Aquatic Ecology, University of Leuven, Charles de Bériotstraat 32B-3000 Leuven, Belgium

There is strong evidence that environmental exposure to endocrine disrupting chemicals (EDCs) is resulting in significant alterations to the reproductive system of many wild fish populations. Most of these studies measure chemically induced changes to the endocrine system (i.e. vitellogenin) or reproductive morphology (i.e. intersex) and mostly provide a simple marker of exposure. The heightened concern over the effects of EDCs is however primarily driven by the hypothesis that disruption to the reproductive system may have serious deleterious consequences on reproductive success. There is therefore an urgent need to understand the impact of endocrine disruption at the population level. To address this need we have conducted extensive multigenerational studies on breeding populations of zebrafish in our laboratory. These experiments have shown that exposure to environmentally relevant levels of EDCs, cause very significant reductions in reproductive success. Lifetime exposure to 5ng/l of ethynylestradiol, for example, caused complete reproductive failure. Moreover, these reproductive failures were not generally caused by exposure proximate to the timing of spawning but by developmental disruption during embryonic and larval development. Histology revealed that the adult male gonads had not completely differentiated into functional testes. Significantly, these sterile males still initiated spawning in females and resulted in unfertilised eggs. This differential in the sensitivity of behaviour when compared to gonadal disruption raises important issues in understanding the implications of endocrine disruption in wild populations. This and how these laboratory experiments may be translated so as to understand the impact of endocrine disruption on reproductive success in wild populations will form the major theme of the talk I am presenting.

Molecular Approaches to Unravelling Sexual Disruption in Fish.

Tyler,C.R., E.M.Santos, G.Paull, G Riley, J.Ball, J.P.Nash, M. Fenske, R. van Aerle.

Environmental and Molecular Fish Biology, University of Exeter, Prince of Wales Road, Exeter, EX4 4PS. UK.

Exposure to chemicals that mimic hormones or interfere with the process of sex hormone synthesis/metabolism (endocrine disrupting chemicals – EDCs) can lead to alterations of sexual function in fish. A wide range of genes that play central roles in reproduction have been cloned with a view to unravelling the mechanisms of sexual disruption in fish including, vitellogenin, vitelline envelope proteins, steroid receptors, various enzymes involved with sex hormone biosynthesis and gonadotrophins. Expression of some of these genes are being used as biomarkers for

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EDC exposure in both the field and laboratory. The methodologies employed for quantifying these mRNAs have included various PCR techniques and hybridisation protection assays. EDCs (and other chemicals) can have combination (e.g. additive) effects and they can also interact with the nervous system to alter development, reproduction and growth. Furthermore, some chemicals can act at multiple targets to disrupt physiological function (e.g. nonylphenol). Development of more comprehensive molecular approaches are therefore needed if we are to more fully appreciate the interactions of chemicals with the endocrine system and identify pathways and mechanisms of endocrine disruption. Projects are ongoing in the UK, more widely in Europe, in Japan and the USA, to develop and apply fish arrays with this challenge in mind. Other molecular techniques that will be discussed in the context of developing our understanding on population level consequences of endocrine disruption in fish are the use and application of DNA microsatellite markers.

Something from nothing? - combination effects of multi-component mixtures of estrogenic chemicals

Andreas Kortenkamp, Nissanka Rajapakse and Elisabete Silva

Centre for Toxicology, School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UK

An argument frequently used to dismiss concerns of health effects associated with estrogenic chemicals is that endogenous steroidal estrogens are too potent for xenoestrogens to contribute significantly to estrogenic effects. We have tested this idea experimentally by assessing the ability of combinations of xenoestrogens to modulate the actions of 17-estradiol. Great care was taken to ensure that each xenoestrogen was present at a level well below its no-observed effect concentration (NOEC). Next, concentration-response relationships for each xenoestrogen and 17-estradiol were recorded. These data were used to predict entire concentration-response curves of mixtures of xenoestrogens with 17-estradiol, assuming additive combination effects. The experimentally observed responses were in excellent agreement with the model predictions for additivity. The joint additive effect of the xenoestrogens led to a dramatic enhancement of the hormone’s action, even when each single agent was present below its NOEC. Our results show that not even sub-NOEC levels of xenoestrogens can be considered to be without effect on potent steroidal estrogens, when they act in concert with a large number of similarly acting chemicals.

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Methods for identifying endocrine disruptors in complex mixtures

Mona Weideborg *, Eilen Arctander Vik*, Liv Bruås*, Keith Roebuck**

*Aquateam-Norwegian water technology centre AS, P. O.Box 6875,0504 OSLO, Norway** Norske Shell, P.O. Box 40, 4090 Tananger, Norway.

Aquateam investigated direct and indirect methods for monitoring endocrine disruptors in complex mixtures and this presentation includes results from these studies.

The direct method employed was analysing for nonylphenol in marine fish tissue. Results were generated from parallel analysis by three accredited laboratories and compared. Both muscle and liver tissue from fish caught near offshore platforms and coastal waters were analysed. The results were compared with analytical results of produced water discharged from the platforms and recipient water near the platforms. No significant differences in the nonylphenol content were found between fish caught at different locations. The study showed, however, differences in nonylphenol concentrations between the three laboratories (1-2 log units) depending on analytical method, isomers, sample preparation, contamination sources, and standards. It was concluded that future monitoring of nonylphenols should focus on agreed target isomers and methodology.

Presently, the Norwegian offshore industry has agreed on monitoring four nonylphenol and four octylphenol isomers in produced water. These isomers are only a small fraction of the total concentration. This approach may overlook the more potent endocrine disrupting isomers.

The indirect method was used to study produced water discharges and removal efficacy of water treatment technology on hormone disrupting compounds in produced water. The influent and effluent was first fractionated (a TIE approach) by solid phase in different n-octanol/water partitioning ranges, and then analysed for both two isomers of nonylphenol and octylphenol as well as for the estrogenic mimics (in vitro induction of vitellogenin) of chemicals in different log POW-fractions. The result showed that chemicals in the n-octanol/water partitioning range of 3-6 were mainly responsible for the estrogenic activity. The method appears promising but needs further development in combination with direct methods to identify the biologically active endocrine disrupting chemicals in the produced water.

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OECD endocrine disrupter assessment & ecotoxicology – progress so far.

Tom Hutchinson1 & Anne Gourmelon2

1AstraZeneca Brixham Environmental Laboratory, UK & Chairman OECD Validation Management Group for Ecotoxicology 2OECD Secretariat, rue André Pascal, Paris.

As part of the Endocrine Disrupter Testing & Assessment (EDTA) activity, the OECD has established a Validation Management Group for mammalian test guidelines (‘VMG-mamm’) and a similar group for ecotoxicology test guidelines (‘VMG-eco’). The VMG-eco has been charged to examine recommendations for new protocols on amphibians, birds, fish and invertebrates (crustaceans). Based on recommendations from OECD technical expert meetings held since 1998, the VMG-eco is currently working on the pre-validation of a non-spawning fish assay for screening chemicals for potential endocrine modulating activity in oviparous species. Other proposals now being considered by the VMG-eco include the US EPA’s short-term fish reproduction (partial life-cycle) test and a fish full life-cycle test. The technical and logistical challenges involved in validating these three fish assays will be reviewed from an individual perspective. A brief update will also be provided on the other species of interest to the OECD EDTA activity (amphibians, birds and invertebrates).

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Concluding session Chairs John Sumpter and Peter Matthiessen

The discussion session on day 2 aimed to provide an analysis of the key future issues seen as areas urgently requiring further research. All members of the audience were allocated one vote and the results were:

1. Population level effects (23 votes)2. Effluent cleanup technologies (16 votes)3. EDC in catchments (fate/behaviour/effects) and comparison with non-EDCs (15

votes)4. Other modes of action, e.g. thyroid (11 votes)5. Invertebrate endocrinology (9 votes)6. Mixtures (different modes of action and in vivo effects) (8 votes)7. Other issues, e.g. test methods (5 votes)8. Low dose effects/ hormesis (0 votes)9. Is risk assessment possible/regulation by hazard (0 votes)

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Appendix 1 Programme

Monday, 31 March 2003: UK Government –Funded Research09:15 Registration and Coffee

09:50 Welcoming Remarks and Introduction, Helen Thompson

10:00 Co-ordination of UK Research – the Government Interdepartmental Group on Endocrine Disrupters, Kathy Cameron

Session 1 - Endocrine Disruption in the Freshwater Environment Chair: Helen Thompson10:10 Endocrine disruption in freshwater fish: the past, the present and the

future. John Sumpter10:50 Mixtures of estrogenic contaminants in fish exposed to sewage treatment

effluents, Richard Gibson, et al11:10 Reproductive effects of oestrone in a fathead minnow pair-breeding test.

Karen Thorpe et al.

11:30 Coffee

11:50 Development of an excretion and transformation model to predict concentrations of steroid oestrogens in sewage effluents and in vulnerable catchments, Andrew Johnson and Richard Williams

12:10 Estimating the effects of 17a-ethinylestradiol on populations of the fathead minnow Pimephales promelas: are conventional toxicological endpoints adequate?Eric Grist et al

12:30 Lunch

Session 2 - Endocrine Disruption in the Marine Environment: Chair Mike Waldock13:40 Overview of UK Endocrine Disruption Research in the Aquatic

Environment Yvonne Allen

14:00 Oestrogen and androgen receptor agonists: Identification and measurement of in vitro activity in the aquatic environment. Jan Balaam et al

14:20 Biomarkers of oestrogenic endocrine disruption in fish: The story in UK estuaries.Mark Kirby et al

14:40 The three-spined stickleback as the European sentinel species for (anti)-androgenic and oestrogenic xenobiotics Ioanna Katsiadaki et al.

15:00 Tea and poster session

15:50 The use of oestrogenic exposure markers as predictors of population level reproductive success in an estuarine fish. Craig Robinson et al

16:10 Biological Effects Techniques For Monitoring Endocrine Disrupting Chemicals In The Offshore Oil Industry William Reynolds et al

16:30 General Discussion/ summing up: Mike Waldock17:00 - 1800 Poster social

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Tuesday 1 April 2003: Current European programmes and future perspectives: Chair Peter Matthiessen

0915 Opening remarks: Peter Matthiessen

0920 Community Action in Drafting Policies and Supporting Research in the Endocrine Disrupter Field, Tuomo Karjalainen

Other National Programmes 0950 Overview of Danish field investigations on endocrine disruption in fish

(brown trout, roach and flounder) Poul Bjerregaard et al

1010 Results of the Dutch national investigation on estrogenic compounds in the aquatic environment Pim de Voogt et al

1040 The ENDIS-RISKS project: Endocrine disruption in the Scheldt estuary; distribution, exposure and effects Tim Verslycke et al

1100 Coffee

Effects in other species1130 Screening of potential endocrine disruptors for (ant)agonist activity in the

Drosophila melanogaster BII ecdysteroid bioassay. Frances Cary et al

1150 Endocrine disruption in freshwater arthropods, molluscs and nematodes Lennart Weltje et al

1210 The Elucidation of Annetocin in Earthworms; Mechanistic Link Between Gene and Life-cycle Parameters Following Exposure to EDCs. Huw Ricketts et al

1230 Lunch

Science issues and the way forward? Chair John Sumpter1330 Gender bent but not in the mind of fish, Jon Nash

1350 Molecular Approaches to Unravelling Sexual Disruption in Fish. Charles Tyler et al

1410 Something from nothing? - combination effects of multi-component mixtures of estrogenic chemicals Andreas Kortenkamp et. al

1430 Tea1500 Methods for identifying endocrine disruptors in complex mixtures

Mona Weideborg et al

1520 Structure-Based Prediction of Oestrogenic Activity: Use of Molecular Quantum Similarity Analysis Ana Gallegos Saliner et al

1540 OECD endocrine disrupter assessment & ecotoxicology – progress so far. Tom Hutchinson & Anne Gourmelon

1600 Gaps in our science knowledge: A regulators perspective and Panel Discussion

1630 Close

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Appendix 2 List of participantsYvonne AllenCEFAS Burnham Laboratory, Remembrance Avenue, Burnham on Crouch, Essex, CM0 8HA, UK.

y.t.allen@cefas.co.uk

Angela ArchCentral Science LaboratorySand HuttonYork YO41 1LZ, UKa.arch@csl.gov.uk

apr. Katrien ArijsGhent University Laboratory of Environmental Toxicology Jozef Plateaustraat 22 9000 GENT Belgium Tel : 32 9 264 37 07 Fax : 32 9 264 37 66 Email : katrien.arijs@rug.ac.be

Prof Koji ArizonoPrefectural University Kumamoto 3-1-100 Tsukide 862-8502 KUMAMOTO Japan Tel : 81 (81)-96-383-6062 Fax : 81 (81)-96-383-6062 Email : arizono@pu-kumamoto.ac.jp

Jan BalaamCEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UK.J.L.Balaam@cefas.co.uk

Rachel BensteadEnvironment AgencyWaterlooville, Hants, UK

Rachel.benstead@environment-agency.gov.uk

Professor Poul BjerregaardUniversity of Southern Denmark Institute of Biology 55, Campusvej 5230 ODENSE Denmark Tel : 45 6550 2456 Fax : 45 6593 0457 Email : poul@biology.sdu.dk

Dr Naomi BlakeCambridge Environmental Assessments Battlegate Road Boxworth CAMBRIDGE CB3 8NN United Kingdom Tel : 44 (0)1954 268287 Fax : 44 (0)1954 267659 Email : naomi.blake@cea-res.co.uk

Ms Catherine BothamMRC Institute for Environment & Health Environmental Toxicology Group 94 Regent Road LEICESTER LE1 7DD United Kingdom Tel : 44 0116 223 1632 Fax : 44 0116 223 1601 Email : cab15@le.ac.uk

Jayne BrianDepartment of Biological SciencesBrunel University,Uxbridge, Middlesex UB8 3PH, UKJayne.brian@brunel.ac.uk

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Geoff BrightyEnvironment AgencyNational Centre for Ecotoxicology and Hazardous Substances, Howbery Park, Wallingford, Oxon.Geoff.Brighty@environment-agency.gov.uk

Dr Fabrice BroeckaertMonsanto Europe SA Toxicology Avenue de Tervuren 270-272 1150 BRUSSELS Belgium Tel : 32 2 7764354 Fax : 32 2 7764444 Email : fabrice.broeckaert@ea.monsanto.com

dr Kate BuchananCardiff University Cardiff School of Biosciences Cardiff University, Main Building Park Place CARDIFF CF10 3TL United Kingdom Tel : 44 02920 875200 Fax : 44 02920 874305 Email : BuchananKL1@cf.ac.uk

Mr Bruce CallowJSC International Ltd Osborne House 20 Victoria Avenue HARROGATE HG1 5QY United Kingdom Tel : 44 01423 520245 Fax : 44 01423 520297 Email : bruce.callow@jsci.co.uk

Dr Kathleen CameronCGMPDefraAshdown House123 Victoria StreetLondonKathleen.Cameron@defra.gov.gsi.uk

Miss Frances CaryUniversity of Exeter School of Biological Sciences Prince of Wales Road EXETER EX4 4PS United Kingdom Tel : 44 01392 263795 Fax : 44 01392 263700 Email : F.H.Cary@ex.ac.uk

Mr. Daire CaseyUniversity of Wales, Cardiff School of Biosciences PO Box 915 CARDIFF CF10 3TL United Kingdom Tel : 44 (029) 20875061 Fax : 44 (029) 20874305 Email : caseyd@cardiff.ac.uk

Professor John CraftGlasgow Caledonian University Biological Sciences Charles Oakley Laboratories Cowcaddens Road GLASGOW G4 0BA United Kingdom Tel : 44 0141 331 3220 Fax : 44 0141 331 3208 Email : j.a.craft@gcal.ac.uk

Mark CroninSchool of Pharmacy and Chemistry, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UKm.t.cronin@livjm.ac.uk

Ph.D Gert-Jan De MaagdMinistry of Transport, Public Works and directorate-general water P.O. Box 20906 Johan de Wittlaan 3-7 2500 EX DEN HAAG Nederland Tel : 31 0031703519033 Fax : 31 0031703519078 Email : g.j.dmaagd@dgw.minvenw.nl

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Dr Pim De VoogtUniversity of Amsterdam Environmental & Toxicological Chemistry Nieuwe Achtergracht 166 1018 WV AMSTERDAM Nederland Tel : 31 +31 20 525 6565 Fax : 31 +31 20 525 6522 Email : pdevoogt@science.uva.nl

Kate DempseyGlasgow Caledonian University Biological Sciences Charles Oakley Laboratories Cowcaddens Road GLASGOW G4 0BA United Kingdom Tel : 44 0141 331 3222 Fax : 44 0141 331 3208 Email : k.dempsey@gcal.ac.uk

Miss Kathryn EllisUniversity of Liverpool Human Anatomy and Cell Biology New Medical School Ashton Street LIVERPOOL L69 3GE United Kingdom Tel : 44 0151 794 7425 Fax : 44 0151 794 5517 Email : k.ellis@liverpool.ac.uk

Mr Eamonn FarrellyBraddan Scientific Ltd 8 Wheatlands Road HARROGATE HG2 8AZ United Kingdom Tel : 44 01423 705 168 Fax : 44 01423 705 169 Email : Eamonn.Farrelly@bradden-scientific.com

Alwyn FernandesCentral Science LaboratorySand HuttonYork YO41 1LZ, UKa.fernandes@csl.gov.uk

Miss Anel FloresSussex University CPES Sussex University Falmer BRIGHTON BN1 9QJ United Kingdom Tel : 44 01273872507 Email : A.M.Flores@sussex.ac.uk

Mr. Andrew FogartyAthlone Institute of Technology Toxicology Dublin Road Athlone 00000 CO. WESTMEATH Ireland Tel : 353 902-24434 Fax : 353 902-24492 Email : afogarty@ait.ie

Dr. Hector GaliciaSpringborn Smithers Labs (Europe) AG Seestrasse 21 9326 HORN Switzerland Tel : 41 0041718446970 Fax : 41 0041718418630 Email : hgalicia@springbornsmithers.ch

Mr. John GaultInstitute of Technology, Sligo Environmental Science Ballinode SLIGO Ireland Tel : 353 071-55254 Email : gault.john@itsligo.ie

Martin GehringDresden University of Technology Waste Management Pratzschwitzer Str. 15 01796 PIRNA Germany Tel : 49 3501-530027 Fax : 49 3501-530022 Email : martin.gehring@mailbox.tu-dresden.de

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Richard GibsonUniversity of Sussex, Brightonrwg20@sussex.ac.uk

Miss Rachel GomesImperial College Environmental Science & Technology RSM Building Prince Consort Road LONDON SW7 2BP United Kingdom Tel : 44 +44 (0)20 759 47398 Fax : 44 +44 (0)20 759 46016 Email : rachel.gomes@ic.ac.uk

Dr. Raimund GrauBayer CropScience Nobel-Str. 50 D-40789 MONHEIM Germany Tel : 49 +49 2173 38 4777 Email : raimund.grau@bayercropscience.com

Eric GristSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK.

e.grist@vla.defra.gsi.uk

Mr John HandleySafepharm Laboratories Ltd Ecotoxicology PO Box 45 DERBY DE1 2BT United Kingdom Tel : 44 01332 792896 Fax : 44 01332 799018 Email : jhandley@safepharm.co.uk

Prof Tony HardyCentral Science LaboratorySand HuttonYork YO41 1LZ, UKa.hardy@csl.gov.uk

Dr Elizabeth HillUniversity of Sussex CPES Falmer East Sussex BRIGHTON BN1 9QJ United Kingdom Tel : 44 01273 678382 Fax : 44 01273 677196 Email : e.m.hill@sussex.ac.uk

Miss Amelia HinchcliffeBraddan ScientificLtd 8 Wheatlands Road HARROGATE HG2 8AZ United Kingdom Tel : 44 01423 705 168 Fax : 44 01423 705 169 Email : Eamonn.Farrelly@braddan-scientific.com

Mr Philip HolmesMRC Institute for Environment and Health Environmental Toxicology University of Leicester 94 Regent Road LEICESTER LE1 7DD United Kingdom Tel : 44 0116 223 1627 Fax : 44 0116 223 1601 Email : ph14@le.ac.uk

Dr Karen HowardSenior Environmental ChemistExponent International Ltd2D Hornbeam Park OvalHarrogateNorth YorksHG2 8RB Tel: 01423 853200Fax: 01423 810431 email: khoward@exponent.uk.com

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Mr. Mark HurstCEFAS Remembrance Avenue BURNHAM-ON-CROUCH CM0 8HA United Kingdom Tel : 44 01621 787200 Email : m.r.hurst@cefas.co.uk

Tom HutchinsonBrixham Environmental LaboratoryAstraZenecaBrixhamDevonTom.Hutchinson@astrazeneca.com

Taisen IguchiOkazaki National Research InstitutesJapan

Hiroshi IshibashiPrefectural University of KumamotoJapan

M.Tech.Sc. Pavel IvashechkinRWTH Aachen (Technical University) ISA (Environmental Engineering) Mies-van-der-Rohe Str. 1 52074 AACHEN Germany Tel : 49 241 80-91514 Fax : 49 241 80-92499 Email : ivashechkin@isa.rwth-aachen.de

Andrew JohnsonCentre for Ecology and Hydrology, Wallingford, Oxon, OX10 8BB, UKajo@ceh.ac.uk

Dr Tuomo Karjalainen, Scientific Officer, European Commission, Research Directorate-General, Directorate E: Biotechnology, Agriculture and Food Unit E2: Food Quality, Square de Meeus 8, Office SDME 8/29, 1050 Brussels, Belgiumtuomo.karjalainen@cec.eu.int

Ioanna KatsiadakiCEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset,I.Katsiadaki@cefas.co.uk

Carole KellyCGMPDefraAshdown House123 Victoria StreetLondonCarole.Kelly@defra.gov.gsi.uk

Mark KirbyCEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UK.m.f.kirby@cefas.co.uk

Andreas KortenkampCentre for Toxicology, School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UKandreas.kortenkamp@ulsop.ac.uk

Dr. ir. Joost LahrAquaSense Consultants Ecological Risk Assessment P.O. Box 95125 1090 HC AMSTERDAM Nederland Tel : 31 + 31.20.5922244 Fax : 31 + 31.20.5922249 Email : joost.lahr@aquasense.nl

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Mrs Gwynne LyonsWWF-UK Toxics Programme Panda House Weyside Park GODALMING GU7 1XR United Kingdom Tel : 44 1483 412 518 Email : lyonswwf@aol.com

Predoc Student Laura Martín DíazUniversidad de Cádiz Departemento de Química Física Polígono Río San Pedro s/n 11510 PUERTO REAL (CáDIZ) Spain Tel : 34 956016449 Fax : 34 956016040 Email : laura.martin@uca.es

Peter MatthiessenCEH, Far Sawrey, Ambleside, Cumbria, UKpmatt@ceh.ac.uk

Mr Richard MaunderCEH Windermere Fish Biology The Ferry House, Far Sawrey Ambleside CUMBRIA LA22 0LP United Kingdom Tel : 44 015394 42468 Fax : 44 015394 46914 Email : rjmau@ceh.ac.uk

Dr Andrew McEwenBioDynamics Research Limited Metabolic Chemistry Pegasus Way, Crown Business Park Rushden, Northants RUSHDEN NN10 6ER United Kingdom Tel : 44 01933 319900 Fax : 44 01933 319990 Email : andrew.mcewen@biodynamics.co.uk

Toxicologist Odile MercierSumitomo Chemical Agro Europe Regulatory Affairs 2 rue Claude Chappe 69370 SAINT DIDIER AU MONT D'OR France Tel : 33 (0) 478 64 32 61 Fax : 33 (0) 478 47 70 05 Email : odile@lyon.sumitomo-chem.de

Dr. Eline MeulenbergELTI Support Drieskensacker 12-10 6546 MH NIJMEGEN Nederland Tel : 31 +31 24 3778261 Fax : 31 +31 24 3774290 Email : E.Meulenberg@eltisupport.nl

Frank MikkelsenBiosense Laboratories HIB-Thormohlensgt. 55 N-5008 BERGEN Norway Tel : 47 0047 55543966 Fax : 47 0047 55543771 Email : customer@biosense.no

Prof Ian MorrisUniversity of Manchester PPT, Biological Sciences G38 Stopford Oxford Road MANCHESTER M13 9PT United Kingdom Tel : 44 01612755492 Fax : 44 0161 2755600 Email : ian.morris@man.ac.uk

Dr Jon NashUniversity of Leuven Laboratory of Aquatic Sciences Charles de Bériotstraat 32 B3000 LEUVEN Belgium Tel : 32 16232416 Email : jon.nash@bio.leuven.ac.be

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Yuji OkayasuPublic Works Research InstituteJapan

Dr Kirsty ParkUniversity of Stirling Biological Sciences University of Stirling Stirling STIRLING FK9 4LA United Kingdom Tel : 44 01786 467799 Fax : 44 01786 464994 Email : k.j.park@stir.ac.uk

Mr Gregory PaullUniversity of Exeter Biological Sciences Hatherly laboratories Prince of Wales Road EXETER EX4 4PS United Kingdom Tel : 44 01392 263701 Fax : 44 01392 263700 Email : g.c.paull@exeter.ec.uk

Dr Mika PeckSussex University CER CPES Falmer BRIGHTON BN1 9QJ United Kingdom Tel : 44 01273 273852 Email : m.r.peck@sussex.ac.uk

Dr Jean-Marc PorcherINERIS Ecotoxicology Unit Parc Technologique ALATA BP - 2 60550 VERNEUIL EN HALATTE France Tel : 33 344 55 65 84 Fax : 33 344 55 67 67 Email : jean-marc.porcher@ineris.fr

Dr Nissanka RajapakseCentre for Toxicology, School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UKNissanka.rajapakse@ulsop.ac.uk

Dr Christine ReteunaRHODIA SERVICES RESPONSIBLE CARE PRODUCT STEWARDSHIP ETOILE PART-DIEU - 190 AVENUE THIERS 69457 LYON CEDEX 6 France Tel : 33 04 37 24 88 78 Fax : 33 04 37 24 88 51 Email : CHRISTINE.RETEUNA@EU.RHODIA.COM

William ReynoldsCEFAS Burnham Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex, UKw.j.reynolds@cefas

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Mr Huw RickettsCardiff University School of Biosciences Main Building (BIOSI 1) Museum Avenue, P.O. Box 915 CARDIFF CF10 3TL United Kingdom Tel : 44 02920876680 Email : rickettshj@cardiff.ac.uk

Mr Gavin RileyUniversity of Exeter Biological sciences Hatherly Laboratories Prince of Wales Road EXETER EX4 4PS United Kingdom Tel : 44 01392 264389 Fax : 44 01392 263700 Email : G.Riley@exeter.ac.uk

Prof Mike RobertsCentral Science LaboratorySand HuttonYork YO41 1LZ, UKM.Roberts@csl.gov.uk

Mike RobertsCGMPDefraAshdown House123 Victoria StreetLondonMike.Roberts@defra.gov.gsi.uk

Craig RobinsonFisheries Research Services, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen. AB11 9DB, UK.c.robinson@marlab.ac.uk

Martin RoseCentral Science LaboratorySand HuttonYork YO41 1LZ, UKm.rose@csl.gov.uk

Dr Andrew RutterMLT Research Ltd R&D 5 Chiltern Close Cardiff Industrial Park CARDIFF CF14 5DL United Kingdom Tel : 44 029 2074 7033 Fax : 44 029 2074 118 Email : andy.rutter@mltresearch.com

Dr. María Quintela SánchezUniversidade da Coruña Ecology Campus da Zapateira s/n 15071 A CORUñA Spain Tel : 34 981-167000-2028 Fax : 34 981-167065 Email : marqui@mail2.udc.es

Dr Eduarda SantosUniversity of Exeter Biological Sciences Hatherly Laboratories Prince of Wales Road EXETER EX4 4PS United Kingdom Tel : 44 01392263796 Fax : 44 01392263700 Email : e.santos@exeter.ac.uk

Anja SchreerUFZ Environmental Research Center Molecular Animal Cell Toxicology Permoserstrasse 15 04318 LEIPZIG Germany Tel : 49 341-2352318 Fax : 49 341-2352401 Email : anja.schreer@uoe.ufz.de

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Miss Asa SjostromImperial College Environmental Science and Technology Imperial College at Silwood Park ASCOT SL5 7PY United Kingdom Tel : 44 020 75942289 Email : asa.sjostrom@imperial.ac.uk

Mr Michael SmithUniversity of Sussex Chemistry, physics and environmental sci Post grad pigeon holes, CPES, University of Sussex BRIGHTON BN1 9QJ United Kingdom Tel : 44 07967 316 747 Email : m.d.smith@sussex.ac.uk

Mr Colin SparyExeter University School of Biological Sciences Hatherley Laboratories Prince of Wales Road EXETER EX4 4PS United Kingdom Tel : 44 01392 264 389 Fax : 44 01392 263700 Email : cjspary@exeter.ac.uk

Manabu SumiMinistry of EnvironmentJapan

Prof John SumpterDepartment of Biological SciencesBrunel University,Uxbridge, Middlesex UB8 3PH, UKJPS@brunel.ac.uk

Dr. Marc J.-F. SuterEAWAG AQU Ueberlandstr. 133 PO Box 611 CH-8600 DUEBENDORF Switzerland Tel : 41 +41 1 823 54 79 Fax : 41 +41 1 823 53 11 Email : marc.suter@eawag.ch

Dr Mark TaylorEnglish Nature Somerset & Gloucestershire Team Roughmoor Bishops Hull TAUNTON TA1 5AA United Kingdom Tel : 44 01823-283211 Email : mark.taylor@english-nature.org.uk

Hiroaki TanakaPublic Works Research InstituteJapan

Mr. Lars TennhardtDresden University of Technology Department of Waste Management Pratzschwitzer Str. 15 01796 PIRNA Germany Tel : 49 +49 3501 530027 Fax : 49 +49 3501 530022 Email : lars.tennhardt@web.de

Karen ThorpeBrixham Environmental LaboratoryAstraZenecaBrixhamDevonkaren.thorpe@brixham.astrazeneca.com

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Helen ThompsonCentral Science LaboratorySand HuttonYork YO41 1LZ, UKH.Thompson@csl.gov.uk

Mr Andy TindallThe University of Manchester PPT, Biological Sciences G38, PPT, Biological Sciences, Oxford Rd., The Univeristy of Manchester MANCHESTER M13 9PT United Kingdom Tel : 44 0161 275 5727 Email : Andrew_Tindall@hotmail.com

Mr Terry ToobyJSC International Limited Director Osborne House 20 Victoria Avenue HARROGATE HG1 5QY United Kingdom Tel : 44 +44 (0) 1423 520245 Fax : 44 +44 (0) 1423 520297 Email : Terry.Tooby@jsci.co.uk

Prof Charles TylerEnvironmental and Molecular Fish Biology, University of Exeter, Prince of Wales Road, Exeter, EX4 4PS. UK. c.r.tyler@exeter.ac.uk

ir. Bram VersonnenRUG Laboratory of Environmental Toxicology J. Plateaustraat 22 9000 GENT Belgium Tel : 32 9 264 37 07 Fax : 32 9 264 37 66 Email : bram.versonnen@rug.ac.be

drs. ir. Tim VerslyckeGhent University Laboratory of Environmental Toxicology J. Plateaustraat 22 9000 GHENT Belgium Tel : 32 +32(0)92643707 Fax : 32 +32(0)92643766 Email : tim.verslycke@rug.ac.be

Emma VineDepartment of Biological SciencesBrunel University,Uxbridge, Middlesex UB8 3PH, UKemma.vine@brunel.ac.uk

Mr Dirk VogelDresden University of Technology Department of Waste Management Pratzschwitzer Strasse 15 01796 PIRNA Germany Tel : 49 +49 3501 53 00 35 Fax : 49 +49 3501 53 00 22 Email : Dirk.Vogel@mailbox.tu-dresden.de

Mike WaldockCEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset,m.j.waldock@cefas.co.uk

Dr Jacqui WarintonSyngenta Ecological Sciences Jealott's Hill International Research Centre BRACKNELL RG42 6EY United Kingdom Tel : 44 01344 414944 Fax : 44 01344 414124 Email : jacqui.warinton@syngenta.com

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Dr Jason WeeksWRc-NSF Henley Road, Medmenham Marlow, Buckinghamshire MARLOW SL7 2HD United Kingdom Tel : 44 +44 (0)1491 636525 Fax : 44 +44 (0)1491 636501 Email : weeks_j@wrcplc.co.uk

M.sc. Mona WeideborgAquateam-Norwegian water techn. centre Hasleveien 10 P.O. Box 6875 Rodeløkka N-0504 OSLO Norway Tel : 47 4722358118 Fax : 47 4722358110 Email : mona.weideborg@aquateam.no

Dr. Lennart WeltjeInternational Graduate School (IHI) Ecotoxicology Markt 23 D-02763 ZITTAU Germany Tel : 49 +49-3583-771526 Fax : 49 +49-3583-771534 Email : weltje@ihi-zittau.de

Jim WharfeEnvironment Agency, National Centre for Ecotoxicology and Hazardous Substances, Howbery Park, Wallingford, Oxon.Jim.wharfe@environment-agency.gov.uk

Mr James WheelerRoyal Holloway, University of London School of Biological Sciences Egham Hill EGHAM TW20 OEX United Kingdom Tel : 44 01784 41 41 96 Fax : 44 01784 47 07 56 Email : j.wheeler@rhul.ac.uk

Mr Matthew WilkinsonWWF-UK Toxics Programme Panda House Weyside Park GODALMING GU7 1XR United Kingdom Tel : 44 01483 426 444 Email : mwilkinson@wwf.org.uk

M.Sc. Leah WollenbergerTechnical University of Denmark Environment and Resources DTU Bygningstorvet DTU - Building 115 2800 KGS. LYNGBY Denmark Tel : 45 45251593 Fax : 45 45252859 Email : lew@er.dtu.dk

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Alex FordSchool of Life Sciences, Napier University, 10 Colinton Road, Edinburgh,UK. EH10 5DTTel:(+44) 0131 455 2372e-mail: a.ford@napier.ac.uk

Melanie Gross-SorokinNational Centre for Ecotoxicologyand Hazardous SubstancesEnvironment AgencyEvenlode HouseHowbery ParkWallingfordOX10 8BD

tel: 01491 828540fax: 01491 828427

Melanie.gross-sorokin@environment-agency.gov.uk

Grace PanterBrixham Environmental LaboratoryAstraZenecaBrixhamDevonUK

Grace.panter@brixham.astrazeneca.com

Philip LightowlersENDS Report, Environmental Data ServicesFinsbury Business Centre40 Bowling Green LaneLondon EC1R 0NE, UKTel: + (0) 20 7814 5313Fax: + (0) 20 7415 0106plightowlers@ends.co.uk

John GarrodCGMPDefraAshdown House123 Victoria StreetLondonJohn.garrod@defra.gsi.gov.uk

Paul LeonardFisheries and Aquatic Science UnitScience DirectorateDefraRm. 405, Cromwell HouseDean Stanley StreetLondon SW1P 3JHTel: 020 7238 1583 E-mail: paul.leonard@defra.gsi.gov.uk

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Appendix 3 Poster Abstracts

A Sensitive Ribonuclease Protection Assay to Detect Multiple Gene Targets in Fish Endocrine Disrupter Research

Wheeler J.R.1, Lopez-Juez E.1, Morritt D.1, Gimeno S.2, and Crane M.3

1School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK.2Procter & Gamble Eurocor, Temselaan 100, B-1853 Strombeek-Bever, Belgium.3Crane Consultants, Chancel Cottage, 23 London Street, Faringdon, Oxon, SN77AG, UK.

In recent years there has been a drive towards understanding of the molecular basis of the endocrine systems of fishes. Much of this work has been from a toxicological perspective, funded by research interest in endocrine disruption. Many fish genes have been characterised and subsequently used as endpoints in toxicity tests. These have centred on oestrogen responsive genes such as the fish egg yolk protein, vitellogenin and the egg shell proteins, zona radiata. Advances in molecular biology have resulted in many more genes being sequenced and so the potential for (eco-) toxicogenomics has never been greater. We have developed a sensitive Ribonuclease Protection Assay (RPA) to measure mRNA expression of oestrogen receptor subtypes alpha and beta, vitellogenin and a control gene simultaneously. Gene expression is quantified by its ability to protect a complementary labelled probe from nuclease digestion. This complex is then separated on a polyacrylamide gel and visualised by a chemiluminescent reaction. Multiple RNAs can be quantified in one reaction as long as they differ in size resolvable by an acrylamide gel. As all the transcripts are measured in one assay, costs are minimised and the number of animals reduced for in vivo studies. Receptor levels have been mapped in the major fish tissues providing baseline data. Here we report results using this assay and Zebrafish (Danio rerio) exposed to 17b-oestradiol. We also discuss the application of this approach to other standard toxicity test species e.g. Fathead minnow (Pimephales promelas) and Carp (Cyprinus carpio) and measurement of other gene targets (e.g. the androgen receptor) to encompass a range of endocrine modes of action.

Differential display PCR analysis for detecting genes regulated by endocrine disrupters in fish

Anja Schreer, Stefan Scholz, Kristin Schirmer

UFZ Centre for Environmental Research, Junior Research Group of Molecular Animal Cell Toxicology, Leipzig, Germany

It is well known that numerous man-made chemicals in the aquatic environment are mimicking the effects of the natural hormone 17b-estradiol. Examples of such chemicals are 17a-ethinylestradiol and 4-nonylphenol. These compounds can demonstrably affect ontogenesis and hormone homoestasis. Likely, these alterations can be detected as changes in gene expression prior to the onset of effects at the

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cellular or organism level. Based on this assumption we use fluorescent differential display reverse transcriptase (dd-rt) PCR as a method to observe disruption of the hormonal system at the level of gene expression. This approach is based on the amplification of cDNA with fluorescently labelled random primers. Changes in gene expression patterns cause a change in band patterns of the amplified cDNA if subjected to polyacrylamid gel electrophoresis. Subsequent re-amplification and sequencing of bands with altered density allows to identify responsive genes. One application of this approach is the derivation of typical gene expression fingerprints for several potential endocrine disrupting chemicals. The second application is the use of the dd-rtPCR as a method to obtain sensitive genes as targets on a cDNA-Array. We currently apply the fluorescent dd-rtPCR technique to 3 test systems. Firstly, we use primary hepatocyte cultures from rainbow trout to investigate the effects of single substances to obtain gene expression fingerprints. Secondly, we examine the patterns of gene expression in livers of fish previously exposed to industrial and sewage treatment plant effluents. Finally, the third application comprises the characterization of endocrine disrupting chemicals in vitro on a whole organism level in medaka embryos.

Development of an hybridisation protection assay for detecting aromatase P450 gene expression.

Riley, G.*, Thomas-Jones, E. & Tyler, C.R.*

*School of Biological Sciences, University of Exeter, Exeter, EX4 4PS+Molecular Light Technology Research LTD, Cardiff, UK, CF14 5DL

Measurements of specific mRNAs for genes that play central roles in reproductive function are being used with greater prevalence as biomarkers of endocrine disruption. This has been pioneered in fish for detecting oestrogenic chemicals using vitellogenin and vitelline envelope proteins and these have proved to be both sensitive and reliable indicators of exposure to exogenous oestrogens and their mimics. Disruptions in the oestrogen pathway, leading altered sexual function, may also come about by alterations in steroid synthesis and metabolism, as well as via interaction with the oestrogen receptor. Aromatse P450's (gonad and brain) play important roles in oestrogen biosynthesis and this is potentially a site for endocrine disruption. Here we report the development and validation of a specific, rapid and highly sensitive method for the quantitative detection of aromatase P450 transcripts in the fathead minnow (Pimephales promelas). The method is based on the hybridisation protection assay. A chemiluminescent-labelled probe is reacted with samples of either total or mRNA where it hybridises to any complementary target present. Following a selection step to remove any unreacted probe, the chemiluminescent reaction is activated and the light emitted is proportional to the concentration of specific mRNA transcript in the sample.

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Rapid bioconcentration of 17b-oestradiol in the common mussel (Mytilus edulis): implications for biomonitoring

Dr Mika R Peck & Dr Elizabeth M Hill,

Centre for Environmental Research, Sussex University, Falmer, Brighton & Hove, East Sussex. BN1 9QG

The reported endocrine disrupting effects of contaminants in certain estuarine/marine species highlight the urgent need to assess the potential exposure of organisms to these chemicals. Common mussels (Mytilus edulis) were exposed to artificial seawater spiked with radiolabelled (14C) 17b-oestradiol at a concentration of 9.4 ng/L (95% confidence interval(CI) ± 2.9 ng/L) for two weeks. They were then allowed to depurate in clean water for a further two weeks. Mussels were regularly sampled, sexed and their body burden of radioactive oestrogen analysed. A significant uptake was observed over 13 days of exposure, resulting in a final body burden of 22.8 (95% CI ± 2.4) ng 14C oestrogen/g wet weight, equivalent to a bioconcentration factor of 2,428 (CI± 257). During the time period of the experiment, body burdens did not reach steady state conditions and there was no significant difference in uptake between males and females. After 10 days of exposure, most of the radiolabel was detected in the gills (55% of the total), and of the remainder, 37% was in the digestive gland and 8% in the gonads. Following four days of depuration, the total radioactivity in whole tissue only decreased by 20%. The RP-HPLC profiles of radiolabelled oestrogens within the different tissues are presented.

This study shows that M. edulis can bioconcentrate oestrogens such as 17b-oestradiol to a high degree and as such, this organism may be a useful sentinel species in monitoring the levels of environmental oestrogens in the estuarine/marine food chain.

Effects of endocrine disrupters on the egg production of Acartia tonsa

Leah Wollenberger, Jane Bergstrøm, K. Ole Kusk

Environment & Resources DTU Technical University of Denmark Bygningstorvet 115 DK-2800 Kgs. Lyngby Denmark

The calanoid copepod Acartia tonsa is widely distributed and dominant in coastal ecosystems and is currently used as a test species to assess environmental hazards. We have previously shown that larval development of A. tonsa was a highly sensitive towards sublethal concentrations of endocrine disrupters and other specifically acting compounds. In the present study, we have investigated a further sublethal endpoint, the egg production of A. tonsa, and compared the sensitivity of this test parameter to results from larval development tests. Fertile A. tonsa have no egg pouch, eggs are released directly and continuously into the water (up to 50 eggs per female and day). Test organisms were exposed for 6 days and the number of eggs produced per female within 24 hours was recorded on day 4 and on day 6. Test compounds comprised synthetic and naturally occurring vertebrate and invertebrate hormones as well as industrial chemicals with known endocrine effect in vertebrates. Additionally,

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reference compounds and secondary effluents from Danish municipal sewage treatment plants were assessed. The following LC10(mg/l) for the inhibition of egg production were found: 0.27 (17b-estradiol), 0.01-0.1 (progesterone), >0.1 (estrone), >0.1 ethinylestradiol, >0.1 (diethylstilbestrol), 0.03 (testosterone), 0.46 (methyltestosterone), >1.0 (flutamide), 0.1 (ecdysone), 0.002 (juvenile hormone III), 0.02 (bisphenol A), 0.11 (tetrabromobisphenol A), 0.74 (3,4-dichloroaniline), 0.15 (3,5-dichlorophenol) and 0.8 (potassium dichromate). Generally, larval development was more sensitive than egg production.

Determination of endocrine effects in sediments with prosobranch snails

Michaela Tillmann*,Martina Duft*, Ulrike Schulte-Oehlmann** & Jörg Oehlmann**

*International Graduate School Zittau, Department of Ecotoxicology, Markt 23, D-02763 Zittau **J.W. Goethe University Frankfurt, Department of Ecology and Evolution –Ecotoxicology, Siesmayerstraße 70, D-60054 Frankfurt

Sediments, which are often a sink for lipophilic compounds like most endocrine disruptors, exhibit a high ecotoxicological relevance. However, organismic test systems for the determination of endocrine disrupting chemicals in whole-sediment samples are rare. For an endocrine evaluation of sediments special emphasis should be laid on typically benthic organisms which are in direct contact with the contaminants. In the last years, our working group developed new test systems with two benthic prosobranch snails, which proofed to be especially sensitive for the identification of xenohormons in artificial sediments as well as in field samples. The first one is the parthenogenetic and ovoviviparous freshwater mudsnail Potamopyrgus antipodarum, which lives in the upper layers of freshwater and brackish sediments. The number of newly produced embryos in its broodpouch turned out to be the most sensitive endpoint to identify sediment components, such as xeno-androgens, xeno-estrogens and substances with generally adverse effects on reproduction. While exposure to xeno-estrogens resulted in a marked increase of the embryo number, xeno-androgens led to a lower embryo production. Another response to androgenic pollution is the expression of the virilisation phenomenon “imposex” in prosobranch gastropods. Imposex is characterised as the additional formation of male reproductive organs like penis and/or vas deferens in females. The netted whelk Nassarius reticulatusis one of the marine prosobranch species expressing imposex. The phenotypic imposex expression in N. reticulates will b e demonstrated by means of studies with various androgens as well as real environmental samples. The so-called “uterotrophic snail assay” will be presented as a possible endpoint to give first information on the estrogenic potential of spiked artificial sediments and field samples. Additionally, the responses of the two species exposed to field sediments from large German rivers will be compared.

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A bioassay for assessing toxicant effects on growth and development of individual Lymnaea stagnalis embryos

Casey D. 1, Pascoe D. 1, Tattersfield L.J. 2, Hutchinson T.H. 3. 1. School of Biosciences, Cardiff University, PO Box 915, Cardiff, CF10 3TL, UK; 2. Ecological Sciences, Syngenta, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK; 3.AstraZeneca Global Safety, Health and Environment, Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon TQ5 8BA, UK.

There is growing evidence that invertebrates are at risk from endocrine disrupting chemicals present in the environment. Molluscs have been shown to be particularly sensitive to endocrine disruption in the marine environment, although studies on freshwater species have been inconclusive. The present study introduces a novel bioassay focusing on the effects of toxicants upon growth and development of individual pond snail (Lymnaea stagnalis) embryos. In previous investigations embryos have been exposed whilst within the egg mass, thereby relying upon group information with its inherent response variability to gauge the impact upon the individual. This method involves dissecting individual eggs from the egg mass and placing them in the individual wells of a 96-well microtitre plate (1 egg/well) where they are grown under clean or exposed conditions until hatching. Mortality, developmental rate and percentage deformity are used as endocrine-mediated endpoints to assess the impact of toxicants upon development. The effect of separation (from the egg mass) upon egg survival was studied and comparison of survival made following separation when embryos were freshly laid, half-hour old or one-day old. Other factors such as water evaporation and oxygen consumption rates were examined, as was the influence of water hardness upon development. A standard toxicant (cadmium) and a known endocrine disruptor (17 a-ethinylestradiol) were assessed for developmental toxicity with reference to the embryo age. The bioassay provides information about the normal and toxicant-induced development of this key invertebrate species. This methodology allows the observation of individual animals throughout their development with emphasis upon identification of particular stages of embryonic development.

Xenopus tropicalis larvae as a model for the determination of the effects of endocrine disruptors on amphibian development

Andrew Tindall1, Dan Pickford2, Lisa Tattersfield3 & Ian Morris4

1School of Biological Sciences, University of Manchester, Manchester,2AstraZeneca, Global Safety Health and Environment, Brixham Environmental Laboratory, Brixham, 3Ecological Sciences, Syngenta, Jealott’s Hill, Bracknell, 4Hull York Medical School, University of York, York, UK

A decline in global amphibian populations over the past 50 years has led to a search for causative factors. Several hypotheses have been put forward which include exposure to UV radiation, loss of habitat and environmental contamination by toxins and endocrine disruptors.

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Our current study aims to characterise the potential for interactions between environmental oestrogens and the thyroid axis. Impaired development could severely affect reproduction in amphibia. The model species we have chosen is Xenopus tropicalis which offers a number of advantages over the more commonly used Xenopus laevis including shorter generation time and diploid rather than tetraploid chromosomes.

X. tropicalis larvae (120 per treatment) were continuously exposed in flow-through equipment to: dilution water control; solvent control (methanol 100ìl/l); thyroxine (T4) 1.287nM; methimazole (MMI) 0.876ìM and ethinyl estradiol (EE2) 0.337nM. Exposure began at Nieuwkoop and Faber stage 45- 48 and larvae were sampled at stage 53-56.

Following T4 treatment statistically significant decreases were seen in body weight (P<0.01), snout-vent length (P<0.001), and total length (P<0.001) at stage 53-55. The EE2 treated larvae showed a statistically significant increase in snout-vent length at stage 55 when compared to the solvent control group (P<0.05).

An increased developmental rate was observed for T4 (log rank significance <0.0001) and EE2 (<0.0001). The MMI and solvent control treatments caused a decrease in developmental rate (<0.05 and <0.0001 respectively).

The well-established effect of T4 in amphibians was recapitulated in our system, evident as a decrease in the size and weight of the larvae, therefore, thyroid activation by pollutants in the natural environment could lead to increased mortality from starvation or predation. EE2 was shown to cause an increase in developmental rate in X. tropicalis larvae, indicating interaction between oestrogen and thyroid axes. Future work will determine whether persistent disruption of the endocrine system occurs which affects later development.

Vitellogenin and plasma zinc as a markers of avian exposure to oestrogenics

Angela Arch1, Ioanna Katsiadaki2, Helen Thompson1 and Sandy Scott2

1 Central Science Laboratory, Sand Hutton, York2 CEFAS, Weymouth Laboratory, Barrack Road, Weymouth

The objective of this part of a larger study on the effects of oestrogenics on birds was to develop an ELISA for vitellogenin (VTG) in plasma of Japanese quail (a model species) and to assess the use of plasma zinc as an indirect measure of VTG in quail.

Young male Japanese quail were injected with 17β-oestradiol and blood samples taken for the vitellogenin (VTG) preparation. The vitellogenin was purified from plasma by FPLC and SDS-PAGE of E2-treated plasma samples revealed the presence of at least three VTG sub-units (, , ). A highly specific and sensitive ELISA was developed for the purified quail VTG with a detection limit of 0.02g/ml of plasma VTG. The vitellogenin levels in 6 week old male control birds (0.3-2.85 μg/ml VTG) is lower than control female birds (0.2-17500 μg/ml VTG). The wide variability in

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females may be due to the variations in the timing of sexual maturity of female birds at this stage of development.

Plasma zinc levels were determined to assess whether it could be used as an indirect measure of vitellogenin to allow monitoring of levels across species. VTG has been suggested to be a carrier of zinc in blood and therefore zinc may be a useful indirect measure where species specific antibodies for VTG are unavailable. Comparison of levels of vitellogenin and zinc in male and female birds shows that significantly higher levels of zinc are detected in plasma when vitellogenin levels are above 100 μg/ml in female birds (p<0.001). This suggests that zinc can be used as an indicator of plasma vitellogenin levels when they are above a threshold. This threshold is of 100 μg/ml in female quail but may be lower in males. Plasma zinc may therefore be a useful indicator of high levels of VTG in birds when species-specific antibodies are unavailable.

The effects of fenoxycarb and diflubenzuron on honeybee (Apis mellifera) colonies

Helen Thompson, Selwyn Wilkins, Alastair Battersby, Ruth Waite and David Wilkinson

Central Science Laboratory, Sand Hutton, York

The objective of this study was to assess the effects of exposure to two insect growth regulating (IGR) insecticides: a juvenile hormone analogue, fenoxycarb and a chitin synthesis inhibitor, diflubenzuron, on the development of honeybee colonies, workers and queens.

Test colonies headed by queens of similar age and were housed in a single chamber wooden Smith hive. The IGRs were diluted with 50% w/v sucrose to a rate equivalent to their maximum application rate. 100 eggs were marked on a single frame from the centre of each colony on the day of dosing, 1, 2, 3 and 5 weeks later. On day 17 after each marking the contents of each cell was identified to determine their fate. All colonies were also assessed for levels of brood and numbers of bees prior to the day of test item application up to a year after treatment.

Significantly greater replacement/removal of marked eggs in the fenoxycarb and diflubenzuron treated colonies was recorded in the first 2 weeks after treatment. There were short term reductions in the numbers of adult bees and brood after treatment with diflubenzuron when compared with controls. Colonies treated with fenoxycarb declined earlier during the season than the control colonies and started the season slower.

Sister queen pupae were raised and transferred to individual Apidea (a small nucleus colony) containing treated or control fondant (icing sugar). The numbers of mated queens were determined by recording egg production. All mated queens were then introduced into larger colonies and the number of eggs laid in a 24 hour period recorded. Numbers of mated queens produced were 17/23 (74%) in the control, 17/24 (71%) in the diflubenzuron treated and 0/23 (0%)in the fenoxycarb treated. In the

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fenoxycarb treated 17/23 queens were present but showed virgin queen characteristics, e.g. small abdomen, suggesting they had not been mated. There was a significant reduction in the numbers of eggs laid by queens treated with diflubenzuron and no eggs laid by any queens treated with fenoxycarb.

Cadmium and zinc effects associated with the Aznalcóllar mining spill on Procambarus clarkii : vitellogenin/vitellin, ovarian and hepatopancreatic indexes and histopathology as biomarkers of physiological disruption

Martín-Díaz, M.L.1, Tuberty S. R. 2, McKenney C. L., Jr. 3, Sales, D.4 and DelValls, T.A.1

1Facultad de Ciencias del Mar, Departamento de Química Física, Campus Río San Pedro s/n, 11510 Puerto Real (Cádiz) Spain 2University of West Florida, Center for Environmental Diagnostics and Bioremediation, One Sabine Island Dr., Gulf Breeze, FL, USA 325613US Environmental Protection Agency NHEERL, Golf Ecology Division, One Sabine Island Dr., Golf Breeze, FL, USA 325614Facultad de Ciencias del Mar, Departamento de Ingeniería Química, Tecnología de los Alimentos y Tecnología del Medio Ambiente, Campus Río San Pedro s/n, 11510 Puerto Real, Spain

Females red swamp crayfish, Procambarus clarkii, were exposed in the laboratory during 21 days to zinc and cadmium at concentrations determined in the Guadiamar River after the Aznalcóllar mining spill (SW, Spain): 1000 μg·L-1 and 3000 μg·L-1 of zinc chloride, 10 μg·L-1 and 30 μg·L-1 cadmium chloride. Hemolymph samples were obtained every seven days for vitellogenin/vitellin determination through an Enzyme Linked Inmunosorbent Assay. At the end of the environmental simulation animals were dissected and females Ovarian Index (OI) and Hepatopancreatic Index (HsI) for the different treatments were analysed. Ovary samples were taken for an histopathological study. The presence of these metals at the concentration found in the field led to disruption of vitellogenesis and ovarian maturation specially for the highest concentration of zinc exposed to crabs.

Ubiquitous imposex in the marine gastropod Nucella lapillus (L.) in Galicia (NW Spain)

Quintela, M.; Ruiz, J.M.; Barreiro, R.

Área de Ecoloxía, Facultade de Ciencias, Universidade da Coruña. Campus da Zapateira, E-15071 A Coruña, Spain

Tributyltin (TBT) is a biocide used in antifouling paints. It causes a type of endocrine disruption named “imposex” consisting on the superimposition of male sexual characters, notably penis and sperm duct (i.e. vas deferens), in female gastropods. These characters constitute biomarkers of the level of TBT contamination and are quantified by two indexes: RPSI (based on penis size) and VDSI (based on vas deferens development).

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The most appropriate species for biomonitoring TBT pollution, although restricted to rocky substrates, is Nucella lapillus. Severe development of sperm duct can occlude genital papilla causing female sterilisation. Since this is an extremely sensitive species, moderate contamination levels can lead to population extinction.

Surveys were conducted between 1996 and 2002 to assess the extent and impact of imposex over Galician coastline. For those sites visited several times the evolution of imposex is shown. All populations sampled were found to be affected by imposex, even those in open shores and far from shipping facilities. Values for RPSI, VDSI and % sterilisation ranged from 6 to 57, from 3.2 to 4.6 and from 0 to 54 respectively. None of the populations is considered to be at risk of extinction. The correlation between % of sterilisation and RPSI (r=0.73***) was the weakest of those the could be established between any pair of these data.

The bioaccumulation of several organotin species was also determined in 20 of those samples. While TBT (36-974 ppb Sn) and DBT (169-909 ppb Sn) were detected in every sample, MBT (65-387 ppb Sn) and TPhT (39-250 ppb Sn) were quantifiable in only 15 and 11 of them, respectively.

First monitoring of the occurrence of endocrine disruption in inland populations of eel (Anguilla anguilla), roach (Rutilus rutilus), rudd (Scardinius erythrophtalmus) and tench (Tinca tinca) in Flanders (Belgium).

Versonnen B.J.1, Goemans G.2, Verslycke T. 1, Arijs K. 1, Belpaire C.2 & JanssenC.R.1

1Ghent University, Laboratory of Environmental Toxicology and Aquatic Ecology, J. Plateaustraat 22, B-9000 Gent, Belgium2Institute for Forestry and Game Management, Duboislaan 14, B-1560 Groenendaal,Belgium To evaluate the potential effects of endocrine disrupters on fish in Belgian surface waters, 165 eels (Anguilla anguilla), 472 roach (Rutilus rutilus), 177 rudd (Scardinius erythrophtalmus) and 155 tench (Tinca tinca) were sampled between September 1998 and October 2001. Length, weight and gonadal weight of all fish were determined. In addition, 65 % of the fish were sampled for blood. Histological sections of a number of roach were made. Gonadosomatic indices (GSI) (not for eel), condition factors (Cf) and whole blood, plasma or liver vitellogenin (VTG) concentrations were determined and correlated with pollutant levels in the water and sediment (for roach, rudd and tench) or to the chemical body burdens (for eel). VTG concentrations were analysed using protein electrophoresis and Western blotting. As positive controls, a number roach and eel were exposed to 0.1 to 1 µg ethinylestradiol/L in a series of laboratory experiments. Exposure to ethinylestradiol lead to significant increases in VTG content in roach, as well as in eel. However, results of the field study with eel indicated that this species is rather insensitive to contamination. Despite high internal concentrations of PCBs, metals and pesticides, no increased mortality, pathological deformities or enhanced VTG concentrations were noted. Hence, no correlations between the biological parameters and the internal concentration of pollutants were detected. Only

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minor changes in Cf, GSI and VTG content were observed in the different populations of roach, rudd and tench, except for the fish collected at one of the 14 sampling sites. Resident male as well as female fish at this site – the highly polluted canal of Beverlo – exhibited high VTG concentrations. Intersex was only observed in a limited number of roach.

A search for evidence of endocrine disruption of reproduction in top predator fish

Emma Vine, Professor John Sumpter and Professor Charles Tyler Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH

Aquatic organisms may receive greater exposures of endocrine disrupting chemicals than terrestrial organisms, as the aquatic environment is one of the ultimate sinks for most chemicals. Jobling et al. (1998) showed that in some of the worst affected rivers in the UK, 100% of the male roach examined downstream of sewage treatment works were intersex. However, no work to date in the British Isles (and extremely little elsewhere) has been carried out on top predatory fish, and as yet we do not know if top predatory fish can be affected by endocrine disrupting chemicals in any way. Given the very serious effects to predatory birds caused by bioaccumulation of some pesticides in the 1950's to 1970's, it is important to know whether endocrine disrupting chemicals are causing adverse effects to top predators due to them accumulating high concentrations of these chemicals. The three species of top predator fish examined in this study are gonochoristic, namely, perch (Perca fluviatilis), pike (Esox lucius) and zander (Stizostedion lucioperca). In other studies and in aquaculture the effects caused by endocrine disrupting chemicals in other species of fish have been found to be, for example, elevated plasma vitellogenin levels and intersexuality, found especially in male fish as a result of feminisation (1,2,3). This study will investigate these effects, as well as observing the total oestrogenic content in bile samples.These endpoints are being examined from fish upstream and downstream of sewage treatment works. In the 'control' fish we have studied so far, no endocrine disrupting effects have been found.

REFERENCES:

(1) Jobling, S., M. Nolan, et al. (1998). Widespread sexual disruption in wild fish. Environmental Science and Technology 32(17): 2498-2506(2) Allen Y., A.P. Scott, P. Matthiessen, S. Haworth, J.E. Thain, S.Feist, (1999). Survey of estrogenic activity in United Kingdom estuarine and coastal waters and its effects on gonadal development of the flounder Platichthys flesus. Environmental Toxicology and Chemistry 18 (8): 1791-1800 (3) Folmar, L. C., N.D. Denslow, K. Kroll, E.F. Orlando,J. Enblom, J. Marcino, C. Metcalfe, L.J. Guillette, (2001). Altered serum sex steroids and vitellogenin induction in walleye (Stizostedion vitreum) collected near a metropolitan sewage treatment plant. Archives of Environmental Contamination and Toxicology 40 (3): 392-398

This worked was financed by DEFRA

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Fish microsomal cyp450 activities and endocrine disruption screening

Antony Strong, Andrew Deacon, Andrew McEwen, Guy Webber and Stuart Wood

BioDynamics Research Ltd, Rushden, Northants, NN10 6ER, UK.

There is increasing concern in the media, among politicians and within environmental organisations, about the effects of chemicals in our environment that affect the endocrine systems of fish, other wildlife and humans. The chemicals in question are diverse (pesticides, PCBs, plasticisers, petrochemicals, and a variety of industrial chemicals) and have been implicated in some serious human endocrine-mediated changes. A great deal of pressure has been placed on environmental protection agencies to devise regulatory tests for the effects of these chemicals and to require limitations on their manufacture and release. Fish are increasingly recognised as an excellent model for such tests, in that the aquatic environment is readily controlled and may provide early warnings of the effects that these chemicals will have on human health. In addition, the large number of eggs which fish produce provides an excellent model to examine the effects on female fertility. Although many of these environmental chemicals and endocrine steroids are metabolised by the CYP450 enzymes, there is still only limited information available on the activities of "classical" drug-metabolising enzymes in fish. This poster presents information on the metabolic activity of the major CYP450 enzymes in fish liver microsomes, conducted as part of a validation of the use of fish in endocrine disruption screening studies. These data are compared to those in rat liver microsomes.

The fate of alkylphenolpolyethoxylates and alkylphenols in roach (Rutilus rutilus)

Michael D. Smith and Elizabeth M. Hill

Centre for Environmental Research, School of Chemistry, Physics and Environmental Science, University of Sussex, Brighton, BN1 9QJ, UK.

Nonylphenols (NPs) and their ethoxylates (NPEOs) enter the aquatic environment through the degradation of alkylphenolpolyethoxylate surfactants in sewage treatment works, and have been shown to retard testicular growth and stimulate vitellogenin synthesis in fish. In order to understand the fate of these contaminants in freshwater fish, roach were exposed to either 14C NP (mixture of branched chain nonyl isomers, at 5 micrograms/L), or 14C NPEOs (either NP4avEO or NP9avEO as mixtures of alkyl isomers and EO oligomers, at 12 micrograms/L) for 4 days in a flow-through system. In each treatment, the radiolabel was distributed throughout all the major tissues and was highly concentrated in bile. Mean bioconcentration factors (BCFs) of radiolabelled residues in bile were 34,000, 5,500, and 3,800, for NP, NP4avEO, NP9avEO respectively, however these residues in bile were identified as mainly Phase I metabolites. In contrast, residues in muscle and gonads consisted only of the parent compounds, and BCFs for NP, NP4avEO, and NP9avEO were 40, 10, and 4 in muscle, and 81, 20 and 8 in ovaries, respectively. Analysis of these latter tissues showed that only the lower ethoxylate oligomers (<5EO) were present, suggesting that there was less uptake of higher NPEO oligomers. This study shows that in roach,

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mixtures of alkylphenolics can bioconcentrate in tissues including gonads and that bioconcentration is lower for NPEOs than for NPs primarily due to differences in rates of uptake of the individual NPEO oligomers.

Accounting for differences in and responsiveness and sensitivity of fish to oestrogenic effluents.

Spary, C.J.(1), Gibson, R.(2), Hill, E.M.(2), Sumpter, J.P (3) and Tyler, C.R. (1)

(1) School of Biological Sciences, Hatherley Laboratories, University of Exeter, Prince of Wales Road, Exeter, EX4 4PS,

(2) The School of Chemistry, Physics and Environmental Sciences, University of Sussex, Brighton, BN1 9QJ,

(3) Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH.

It is now well established that effluents from sewage treatment works (STWs) are oestrogenic to fish, inducing a range of feminising effects in male fish. Little is known, however, to account for the reported inter- or intra-species differences in responsiveness and sensitivity for these oestrogenic effects. In this study roach (Rutilus rutilus) and rainbow trout (Oncorhynchus mykiss) were exposed to sewage treatment effluent discharges containing different concentrations of oestrogenic chemicals (for 10 days) and the oestrogenic activity in bile and induction of plasma VTG (as a biomarker for oestrogenic response) measured in individual fish. Trout were found to be more responsive (and sensitive)species and for the most potent effluent tested there was a 10-fold higher induction of VTG compared with roach (in males). Vitellogenic responses in male fish exposed to the same effluent differed by more than 2 (roach) and 3 (trout) orders of magnitude. The oestrogen activity of hydrolysed bile in trout exposed to more potent effluent (measured using a recombinant oestrogen receptor-transcription screen) was 26-times higher than in the control (tap-water exposed) fish. Elevated concentrations of plasma VTG correlated with the amount of estrogenic activity in the bile, indicating that differences in the vitellogenic responses of trout are related to differences in the amount of oestrogenic chemicals taken up from the water.

Successful detection of environmental (anti-)androgens using a fathead minnow (Pimephales promelas) non-spawning assay.

Panter G.H.1, T.H. Hutchinson1, K.S. Hurd1, R. Lànge2, A.J. Sherren3, R.D. Stanley1 J.P.Sumpter4 and C.R. Tyler5.

1AstraZeneca Global Safety, Health & Environment, Brixham, UK; 2Schering AG, Berlin, Germany;3Shell Global Solutions, Chester, UK; 4Brunel University, Uxbridge, UK;5Exeter University, Exeter, UK.

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Screening assays have been successfully developed for the detection of (anti)oestogenic substances in several fish species, including the fathead minnow. However, a validated screen for detecting environmental (anti-)androgens and other endocrine active substances (EASs) in the fathead minnow is lacking. Current work is aimed at developing a short-term in vivo non-spawning assay for the screening of (anti-)androgenic substances in this fish species. Sub-adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to reference substances for up to 21 days, at 25*C. Male and female fish were held in separate tanks. The reference substances used to evaluate (anti-) androgenic activity were the androgen dihydrotestosterone (DHT) and the anti-androgen flutamide. After 14 and 21 (for flutamide only) days exposure, fish from each treatment were sampled and evaluated for secondary sexual characteristics (SSC), gonadosomatic index (GSI), whole-body vitellogenin (VTG) concentrations determined by enzyme-linked immunosorbent assay (ELISA) and gonad histology. Evaluation of SSC appeared to be a rapid and sensitive endpoint for measuring (anti-)androgen exposure. Results obtained indicate that in the presence of DHT there was an induction of nuptial tubercles (male characteristic) in both the males (more abundant) and females. In male fish exposed to flutamide, a decline in male nuptial tubercles was observed. The assay developed employing fathead minnow sub-adults can thus be used to test for both androgens and anti-androgens.

Uncertainties in evaluating results of a yeast estrogenic assay.

Arijs K. 1 , Dhooge W.2, Versonnen B.J.1, D’Haese I.3, Comhaire F.2, Verstraete W.3, Janssen C.R.1

1 Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J. Plateaustraat 22, 9000 Gent, Belgium

2 Laboratory of Hormonology and Endocrinology, Ghent University, De Pintelaan 185, 9000 Gent, Belgium

3 Laboratory of Microbial Ecology and Technology, Ghent University, Coupure links 653, 9000 Gent, Belgium

Over the past decade, a number of yeast-cell based assays have been developed to screen for endocrine disruptive effects. In this study, a previously described yeast estrogenic screen (YES) with Saccharomyces cerevisiae was used to perform a ringtest with three laboratories. The chemicals studied were: 4,4’-DDE, benzyl butyl phthalate, bisphenol-A, methoxychlor, α,β-endosulfan, lindane, permethrin, genistein and 17β-estradiol. Two different solvents were tested: ethanol and dimethylsulfoxide. All test solutions were run at least twice in each laboratory. The intra-laboratory variability, expressed as the coefficient of variation, was below 10 % in all laboratories. The inter-laboratory variability was below 20 %, except for three test solutions (4,4’-DDE and lindane dissolved in ethanol and α,β-endosulfan dissolved in DMSO) which did not produce an estrogenic response in one laboratory. A number of issues that could lead to the mislabeling of chemicals as endocrine disruptive substances in the YES were subsequently investigated. The use of different solvents (ethanol or dimethylsulfoxide), the initial yeast cell number, the initial yeast cell stage and background concentrations of estrogenic compounds all affected the data interpretation and led to uncertainties in the evaluation of the test results. These findings illustrate that great care must be taken to the test conditions when performing

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the YES. Suggestions are made for future use of this assay in the study of the endocrine disruptive potency of chemicals.

How predictive are in vitro tests of in vivo responses to endocrine disrupting chemicals

Holmes P, Harrison PTC, Harris R

The growing concerns in Europe that environmental exposure to chemicals may be affecting the health of wildlife and humans through interference with the endocrine system have many important implications for both regulators and scientists. Extensive programmes of research are now in progress in Europe, the USA and other regions, aimed at providing a better understanding of the scientific basis for endocrine disruption, and international efforts, involving the OECD, are underway to develop suitable testing strategies and methodologies. Together, these actions should facilitate the regulatory assessment of the extent of any risks posed by chemicals suspected of being endocrine disrupters. However, progress in developing agreed, validated test protocols capable of clearly defining a chemical's hormonal activity profile has been slow and bedevilled by problems. In particular, the debate as to the value of currently available in vitro bioassay methods to predict the actual hazard posed by a chemical, compared with the results of bioassays using intact animals or the range of endpoints considered in traditional regulatory toxicity test protocols, remains an important area of uncertainty.

In this presentation, examples will be given of comparisons of the consistency of response shown by a number of anthropogenic and naturally occurring chemicals when tested using different in vitro and in vivo test methods. The data used in these analyses were extracted from published peer reviewed papers using standardised recording criteria, and comparisons were facilitated by use of a Microsoft Access based application, the Relational Database of Information on Potential EndocrineDisrupters (REDIPED) developed at the MRC Institute for Environment and Health. Attention will be focused on results relating to agonistic and antagonistic activities. Implications of the design of test batteries will also be discussed.

Comparison of several tests for endocrine/estrogenic activity.

Eline P. Meulenberg,

ELTI Support, Drieskensacker 12-10, 6546 MH Nijmegen, The Netherlands

Hormone disruption due to contaminating chemicals in the environment and in particular water is a source of concern. Given the enormous number of different compounds present in surface water, not only wild life, but also man may suffer from effects on hormonal systems, especially in the case of surface water intended for the production of drinking water. To assess an estrogenic activity of water samples several tests have been developed. Of these we selected two different yeast assays, one from München (University of München Weihenstephan) and the other from Wageningen (Rikilt). In addition, an estrogenreceptor (ER) assay was performed in

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two modifications, in an enzyme format as well as in a luminescent format. As a reference 17ß-estradiol was used. The samples consisted of natural river water, half purified water intended for the production of drinking water, and spiked samples of various matrices that were passed through a battery of immunoaffinity columns. It appeared that the sensitivity of each test was high, but results were very different. Half purified water showed no estrogenic activity in any test. The values found for the other samples are discussed in relation to the compounds present and the cross-reactivity of each test as determined by the respective institute and university. Further, the performance and applicability of the tests are discussed.

In vitro and in vivo estrogenicity of substituted benzenes

Arijs K., Versonnen B.J., Janssen C.R.

Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J.Plateaustraat 22, 9000 Gent, Belgium

The yeast estrogenic screen (YES) is a rapid and sensitive in vitro assay, allowing high numbers of chemicals to be tested at a reasonable cost. To evaluate the structural features of a chemical which could influence estrogenic activity, 67 substituted benzenes were tested with the YES. Structural features important for estrogen receptor binding were identified. A phenolic ring structure and ring structures with functional groups able to form hydrogen bonds with the estrogen receptor promoted estrogenicity. Furthermore, para-substituted benzenes are more likely to exert xenoestrogenic effects than ortho- or meta-substituted benzenes. Since in vitro tests are preferably used in combination with in vivo test systems, two groups of isomers were selected for subsequent in vivo testing: o-, m- and p-dichlorobenzene and o-, m- and p-nitrophenol. To examine the in vivo estrogenic activity of these six chemicals, plasma vitellogenin production was measured in zebrafish after 14 days exposure to these compounds. Following exposure, blood samples were taken and plasma was analyzed with protein electrophoresis to determine the relative vitellogenin content. The results of the in vivo assays indicate that the structural elements found to be responsible for estrogenic potency in the YES, did not affect vitellogenesis in zebrafish to the same extent. The full data set will be presented and discussed in the context of the relevance and use of a tiered approach for the testing of endocrine disrupters.

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Inclusion of Endocrine Disruption in the Environmental Risk Assessment of Chemicals

José María Navas Antón, Mª Victoria Pablos, Mª del Mar Babín, José Vicente Tarazona

INIA. Dpt. of Environment, Laboratory of Ecotoxicology. Ctra de la Coruña, Km 7. 28040 Madrid.

Environmental/Ecological risk assessment (ERA) protocols are considered the essential element for introducing a scientifically sound evaluation in the management, authorization and register of new and used chemicals, so that their use permits an adequate protection of the environment. In Europe, these protocols are essential aspects in the regulation of chemicals, including industrial chemicals, pesticides, biocides, pharmaceuticals, etc., as well as the activities which produce, handle and/or use these substances. Risk assessments are conducted as tiered protocols, where lower tier estimations focus on a direct comparison of the levels/concentrations of the chemical expected in environmental compartments vs. the toxicity observed in a set of species under laboratory conditions. One of the major problems of the systems used for the ERA of chemicals is that they do not take into account the possible effects of endocrine disruption on animal reproductive performance. At present, various projects are running in our laboratory with the main objective of establish the methodology that would permit to include in the ERA protocols the deleterious effects caused by endocrine disruption on organisms and populations. The first step in these projects is to determine differences between concentrations inducing toxic effects and those inducing endocrine disruption. If toxic effects are detected in some species at chemical concentrations below those inducing endocrine disruption, then the classical ERA protocol is also covering the endocrine disruption effects. At the contrary, if endocrine disruption occurs at concentrations below those resulting toxic, then the ERA protocol should be modified to take into account endocrine disruption. In the present work a comparison between concentrations inducing endocrine disruption and those detected as toxic for different species and in different experimental systems is presented. Implications for the development of ERA protocols are commented.

Acknowledgement: this work is financially supported by project REN2002-00639/GLO, Spanish Ministry of Science and Technology.

Factors to consider when examining uncertainty, exposure and impact in risk assessments for potential EDCs.

E. Farrelly, A. Hinchcliffe,

Braddan Scientific Ltd, 8 Wheatlands Road, Harrogate, North Yorkshire

Field observations of sexual disruption within wildlife populations, associated with anthropogenic inputs into the environment, have become a significant concern over the last decade. The potential impactsby synthetic chemicals, in addition to naturally occurring ones, are undergoing regulatory evaluation. Recent laboratory and field studies of wildlife, including mammals, amphibia, reptiles, birds, fish and aquatic

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invertebrates, have investigated the relationship between chemical exposure and reproductive problems. However, in most cases the causal link between environmental exposure and population impact is limited. Furthermore it is recognised that field observations have a critical spatial component resulting in localised impacts. Significant data gaps exist for the integration of environmentally relevant chemical information such as biotic and abiotic degradation, reactivation, metabolism, sediment absorption, bioavailability and ecosystem transport fluxes.

The correlation of these factors with mode of action, biochemical response, mechanistic data and interspecies sensitivity is required in understanding and demonstrating causality. In addition, it is important to recognise the current uncertainties that exist regarding the environmental and biochemical triggers for both sexual determination and changes in reproductive strategies (e.g. altered fecundity) for stability of populations within ecosystems. These should be part of the selection of representative indicator species for testing within a hazard identification strategy.

The basis of confidence in both assessing and managing any adverse ecological risks of wildlife exposure to Endocrine Disrupting Compounds (EDC’s) and their consequences will be a better understanding of the environmental and pharmacokinetic mechanisms by which the chemical(s) are bioavailable. This poster highlights key factors that should be considered when assessing and managing risks posed by potential endocrine disrupting chemicals, including: bioavailabilty, bioconcentration potential, exposure pathways, environmental biotransformation, ecosystem transport fluxes, biomass considerations, exposure duration..........amongst others.

ACE -Analysing combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms.

Brian, J.V. and the ACE Consortium

Dept. of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PHU.K.

Increasing numbers of chemicals are being found to possess endocrine disrupting properties. Concern is growing that such chemicals may be capable of disturbing reproductive function in wildlife. Although significant numbers of studies have attempted to address this issue, the majority of data published thus far have emanated from studies conducted using single chemicals. In reality, wildlife are exposed to ill-defined mixtures of large numbers of chemicals throughout their lifetime. The objective of ACE is to contribute to the hazard assessment of endocrine disrupting chemicals in the aquatic environment by assessing the effects of mixtures of estrogenic chemicals on fish, as well as in a number of in vitro assays. The observed effects will be compared to the effects predicted by the model of. Concentration Addition, in order to establish whether such a model might be used in future risk assessment practices. In addition, data from the in vitro assays will be compared to those of the in vivo assays, as an indication of whether effects in fish can be predicted by studies conducted in rapid, inexpensive cell-based assays (thus reducing the need for in vivo trials in such situations). Analytical chemistry support will be provided

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throughout the project. For this, existing methods will be optimized to allow analyses of extremely low concentrations of environmental estrogens (representative of the concentrations found in surface waters). In addition, methods will be developed for the simultaneous analysis of mixtures of estrogenic compounds in water samples. On completion of the project, we hope to have an improved understanding of the relevance of current environmental guidelines (based on single chemical effects), and the importance of accounting for joint effects of endocrine disrupters.

Determination and fate of conjugated steroid estrogens in the sewage treatment process

Rachel L. Gomes, Mark D. Scrimshaw and John. N. Lester

Environmental Processes and Water Technology Research Group, Department of Environmental Science and Technology, Imperial College of Science, Technology and Medicine, London SW7 2BP, UK

Natural and synthetic steroid estrogens have been implicated as the major contributors to estrogenic activity in sewage effluent and receiving surface waters. The majority of estrogenic material is excreted from the human body within urine in its biologically inactive, conjugated form. However, estrogens in the free, deconjugated state have been observed in sewage treatment effluent, implying that deconjugation occurs prior to, or during the sewage treatment process.

A liquid chromatography/mass spectrometry (LC/MS) method using negative electrospray ionisation (ESI) has been developed for the direct determination of nine selected free and conjugated steroid estrogens. Solid phase extraction (SPE) was employed for isolation and concentration, with recoveries between 85 to 95 ± 2.4 %. Both methods were sufficiently robust and selective for use in analysing 2.5 L samples containing conjugated and free steroid estrogens spiked to 0.5 and 1 ng/L levels within several wastewater matrices, including sewage influent and effluent from a sewage treatment works, and mixed liquor (biological sludge) developed in the laboratory from a synthetic sewage. Investigations into the deconjugation of sulphated and glucuronide steroid estrogens in mixed liquor and sewage influent determined the process to be biotic and moiety dependent. The main site for glucuronide deconjugation is in the sewerage system with concentrations of 5 mg/mL being completely deconjugated in sewage influent within 24 hours. For sulphated conjugates, minimal deconjugation occurred in both sewage influent and mixed liquor matrices. Long periods in the sewerage system may allow for further deconjugation, though it is likely that sulphate conjugates will be present during the sewage treatment process and may persist in effluent.

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Behaviour of Endocrine Disrupting Chemicals Bisphenol A, Nonylphenol and Short-Chain Nonylphenolethoxylates during Anaerobical Mesophile Treatment of Municipal Sewage Sludge.

Pavel Ivashechkin,

Aachen Technical University, Eckertweg 20/3 Zi. 79 52074 Aachen Germany

Bisphenol A (BPA), nonylphenol (NP) and short-chain nonylphenolethoxylates (NPEO) are contained in municipal and industrial waste water. During the treatment the chemicals are only partly mineralised. They are mostly absorbed by the sludge and the rest is discharged into the river. In case of the agricultural use of sewage sludge the chemicals can reach the surface water, contaminate the groundwater or enter food chains of terrestrial organisms.

At many waste water treatment plants (WWTP) sewage sludge is digested anaerobically. In our experiment two laboratory digesters (60 L each) were continuously fed with fresh sewage sludge from a municipal WWTP during five months and spiked with methanol solution of BPA and NPEO (0,5 g/kg dry matter). The temperature was held at 37-38°C, the average retention time was 24 days.

After the first month the methane production, the pH and the concentration of volatile organic acids had stabilised, which indicates a good quality of digestion. After 3-4 months the concentrations of NP and BPA in the output did not change anymore. Around 70-80% of BPA was mineralised and about 15% of NPEO was transformed into NP in both digesters. Tests are now being carried out to see if NP itself has been partly mineralised.

The digested sludge was centrifuged in order to determine how much of the chemicals stay in the sludge and are brought to the field, and how much is directed with the liquid back to the input of WWTP. About 15% of BPA and less than 5% of NP was found in the liquid phase.

The results of the experiment show that anaerobical digestion can reduce BPA concentration in sludge but initiates the formation of NP with higher endocrine potential than that of NPEO, and that NP and BPA are mostly associated with the solid phase.

Mobility and Fate of Endocrine Disrupting Compounds (EDC) in Soil after Application of Sewage Sludge to Agricultural Land.

Dirk Vogel, Martin Gehring, Lars Tennhardt, Diethelm Weltin, Bernd Bilitewski

Dresden University of Technology, Department of Waste Management, Germany

Lysimeter experiments were conducted over a period of 2 years, run-off experiments were carried out in summer 2000, in order to investigate the behaviour of EDCs in agricultural soils after application of sewage sludge. The soils were either loaded with digested sludge, EDC spiked digested sludge, or solely an EDC mixture

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encompassing 4-nonylphenol, 4-tert-octylphenol, bisphenol A, 17b-estradiol, and 17a-ethynylestradiol. In lysimeter experiments mobilisation of the alkylphenols and towards deeper soil horizons was observed. For example, 14 to 316 µg/kg of OP and 30 to 93 µg/kg of EE2 were detected within the upper 30 cm of soil. In general, EDC concentrations decreased with soil depths. No considerable concentrations of EDCs were detected in the leachate. Adsorption to the soil matrix and/or biodegradationprevents a direct transport to groundwater.

In run-off experiments one sandy soil and one loamy soil were examined. The results show an extensive transport of EDCs with the run-off. Mass portions of EDCs from sandy soil were higher than those emitted from loamy soil, except in the case of 17a-ethynylestradiol which showed an inverse behaviour. Adsorption of EDCs to soil was more extensive in loamy soil than in the sandy soil.

Effective Removal of Natural and Synthetic Steroids from Sewage Sludge during Aerobic and Anaerobic Sludge Stabilisation

Lars Tennhardt , Martin Gehring, Dirk Vogel, Diethelm Weltin, Bernd Bilitewski

Dresden University of Technology, Department of Waste Management, Germany

Endocrine disruption has become one of the most important environmental issues causing extensive scientific, political, legislative, and administrative action. Adverse ecological effects downstream of wastewater treatment plant outlets are mainly attributed to very potent natural and synthetic steroids. Corresponding to their respective hydrophobicity, some of these substances accumulate during wastewater treatment in the sewage sludge. Therefore, the removal of 17b?estradiol (E2), estrone (E1), estriol (E3), 17a?ethinylestradiol (EE2) and mestranol (ME) during sewage sludge treatment was studied under varying conditions.

Under anaerobic–mesophilic conditions, the elimination rates of E2 amounted to 94.9 to 97.3 %. A high simultaneous increase of the E1 concentration was observed. EE2 was moderately eliminated by 57.5 to 69.2 %.

Under aerobic–thermophilic conditions, about 92 % of the spiked E2 was eliminated within the first 16 hours whilst EE2 proved to be stable.

Under aerobic–psychrophilic conditions, E2 and E1 were eliminated by approximately 91 % after 6 days and by 99 % after 34 days. An extensive removal of EE2 and ME was observed amounting to 98 % elimination after 20 days and 99 % after 34 days.

In experiments simulating simultaneous aerobic sludge treatment, the E2 elimination rates amounted to 98.6 to 99.6 %. About 68.4 to 90.3 % of the E2 was already removed in the first, anoxic denitrification vessel. At the same time, formation of E1 was observed. On molar basis, the overall elimination rate of the sum of E2+E1 amounted to 96.1 to 98.0 %.

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We can summarise that the natural steroids were rapidly eliminated by more than 90 % under all oxygen and temperature conditions studied. E2 degradation lead to the well–known metabolite E1. A moderate to very good elimination of EE2 and ME was observed under anaerobic and aerobic–psychrophilic conditions. Surprisingly, EE2 was stable under aerobic–thermophilic conditions.

Xenoestrogen Removal from Sewage Sludge

Martin Gehring, Lars Tennhardt, Dirk Vogel, Diethelm Weltin, Bernd Bilitewski

Dresden University of Technology, Department of Waste Management, Germany

Bisphenol A (BPA), alkylphenol polyethoxylates (APEOs), and alkylphenols (AP) are widespread environmental contaminants and exert, in part, estrogenic and antiandrogenic activity. They are frequently classified as endocrine disrupting compounds (EDCs), endocrine active compounds (EACs), or (suspected) environmental estrogens (EEs). Transport with industrial and municipal wastewater to aquatic ecosystems is the major emission pathway of these compounds. Because of their lipophilic character, AP and BPA predominantly adsorb to particles like suspended solids, sediments, and sewage sludge. AP mainly derive from APEO degradation. AP as well as BPA are not biodegradable under anaerobic conditions.

The removal of BPA, 4-nonylphenol (NP), and 4-tert-octylphenol (OP) during sewage sludge treatment was studied in a series of laboratory experiments simulating different treatment technologies frequently applied at German wastewater treatment plants (WWTPs). Corresponding to the literature and results of a field study at full–scale WWTPs in Germany, these compounds were not degraded under anaerobic conditions. But, in the most cases, aerobic and anoxic conditions lead to a more or less extensive elimination of all target compounds.

Simultaneous aerobic, aerobic–thermophilic, aerobic–psychrophilic, and anaerobic–mesophilic sludge treatment technologies have been studied. The data derived are discussed in relation to costs, applicability, and the actual frequency of application of these technologies at German WWTPs. Recommendations are made how to optimise the performance of municipal wastewater and sewage sludge treatment facilities with regard to BPA, NP, and OP.

Recycled Paper Distinctly Contributes to the Bisphenol A, Nonylphenol Ethoxylate, and Nonylphenol Load of Municipal Wastewater

Martin Gehring , Lars Tennhardt, Dirk Vogel, Diethelm Weltin, Bernd Bilitewski

Dresden University of Technology, Department of Waste Management, Germany

2,2-Bis(4-hydroxyphenyl)propane (bisphenol A, BPA) and 4-nonylphenol (NP) are known to cause estrogenic effects in humans and wildlife and are regarded as (suspected) environmental endocrine disrupters (EDs). NP detected in environmental

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samples is mostly the degradation product of anionic 4-nonylphenol polyethoxylate tensides (NPEOs).

BPA and NP are frequently detected in municipal wastewater and sewage sludge as well as in landfill leachates, organic wastes, surface waters, and even the atmosphere. One of the main mass fluxes into wastewater treatment plants (WWTPs) is hygiene paper with approximately 0.5 million tons/a released into wastewater in Germany. Because of the use of BPA e. g. in the colour–developing layers of temperature–sensitive papers and of NPEOs for pulping and de-inking wastepaper during the recycling process, BPA, NPEOs and NP are expected to be widespread contaminants in recycled paper products. There-fore, hygiene papers, graphic wastepaper fractions, and cellulose have been analysed for BPA and, in part, for NP, NPEOs, and 4-tert-octylphenol (OP), respectively.

The results show that the three sorts of toilet paper studied and made from 100 % recy-cled paper are highly contaminated with BPA, NP, NPEOs, and OP, respectively. The ED concentrations determined in seven wastepaper fractions collected in the city of Dresden, Germany, were 2 to 3 orders of magnitude lower than in toilet paper. The BPA concentrations obviously correspond to the percentage of wastepaper used for production. In all but one case, the concentrations of all target compounds in the cellulose samples studied were not detectable or below the limit of quantification.

Degradation of the radioactive and non-labelled branched 3',5'-dimethyl 3'-heptyl-phenol nonylphenol isomer by Sphingomonas TTNP3.

Philippe François-Xavier CORVINI & Max Dohmann

Institut für Siedlungswasserwirtschaft Aachen, RWTH Aachen, Mies van der Rohe Strasse 1, 52074 AACHEN GERMANY.

In the environment, the endocrine disrupting chemical nonylphenol (NP) is a very persistent xenobiotic, which can only be eliminated through microbial degradation processes. NP is essentially found as technical NP product, which consists of a complex mixture of alkyl chain isomers. Since each NP isomer may lead to its own range of metabolite, the study of the respective metabolites concentration and isolation are strongly impaired. By synthesising and studying the degradation of a defined isomer of the NP mixture, this difficulty can be partly overcome. The presence and the possible degradation of the 3',5'-dimethyl 3'-heptyl-phenol (p353NP) nonylphenol isomer has been assessed during cultures of Sphingomonas TTNP3 which were fed with the technical mixture of nonylphenol. Then, the radioactive and non-labelled form of this p353NP isomer have been both synthesised. For the radioactive isomer the synthese was achieved with [ring-U-14C]-labelled phenol and 3,5-dimethyl-3-heptanol according to the alkylation of Friedel and Crafts. The degradation kinetics have been performed with and without radioactive p353NP. A radioactive mass balance has been calculated from the measured radioactivity: in fractions of various polarity, in the biomass and in the form of CO2 as mineralisation product. The partition of the 14C bound radioactivity shows that the p353NP isomer is degraded and serves in a quite inefficient way as carbon source for the biomass

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production of the Sphingomonas strain. Beside, the presence of the nonanol corresponding to the nonyl chain has been confirmed and the concentration of this metabolite has been determined during the fermentation course. Beside no further radioactive catabolites other than the parent compounds could be detected in the extracellular medium.

Assessment of estrogenic activity in WWTP effluents using chemical analysis and biological endpoints.

MJ-F Suter, HR Aerni, B Kobler, BV Ruthishauser, FE Wettstein, R Fischer,W Giger, A HungerbÃhler, A Peter, R SchÃnenberger, AC VÃgeli, RIL Eggen

Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 DÃbendorf, Switzerland

Natural and anthropogenic chemicals that interact with the endocrine system have become an increasingly important issue for environmental scientists and regulatory bodies. Among these so-called endocrine disrupting chemicals (EDCs), the natural and synthetic steroid hormones are the most potent found in the aquatic environment. This is illustrated by the fact that adult male rainbow trout start producing vitellogenin (VTG), an egg yolk precursor protein normally found in female fish, at 17b-estradiol (E2) concentrations of 1 – 10 ng/L. Expected environmental concentrations for E2 in effluent receiving waters are in the same range. Concentrations as low as 0.1 ng/L of 17a-ethinylestradiol (EE2), a synthetic steroid hormone used as contraceptive, already induce vitellogenesis. Based on an estimated consumption of 4 kg/year and elimination in the wastewater treatment plant (WWTP) of more than 70%, predicted environmental concentrations for EE2 in Swiss effluent receiving surface waters are around 0.3 ng/L, clearly indicating a potential problem. Adult male rainbow trout were exposed in triplicates to WWTP effluent, river water and lake water. Both the laboratory control and river water fish showed a general trend towards reduced VTG concentration after the exposure. The fish exposed to the WWTP effluent on the other hand showed a significantly increased VTG concentration in two out of five exposures, indicating the presence of estrogenic compounds in the effluent. The estrogenicity measured with the yeast screen correlated with the concentrations determined for the natural and synthetic steroid hormones. All five effluents tested positive with the yeast estrogen screen.

Release of Nonylphenol, Octylphenol, and Bisphenol A with Leachate from Municipal Landfill Simulation Reactors

Dirk Vogel, Martin Gehring, Lars Tennhardt, Diethelm Weltin, Bernd Bilitewski

Dresden University of Technology, Department of Waste Management, Pratzschwitzer Str. 15, D–01796 Pirna, Germany Laboratory scale lysimeters have been operated using municipal waste obtained from a local landfill in order to simulate long term real life conditions. The objective is to determine the influence of aeration on the quality of the leachate. A large scale

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application at German landfills offers reduction of costs and timeframe of maintenance after closing a landfill. Further lysimeter trials have been carried out using mechanically and biologically pre–treated municipal waste.

Waste of several horizons of depth and age was excavated from the landfill and placed in four duplicate sets of lysimeters, each with a volume of 100 l. Leachate is recirculated and samples are collected weekly. After three months of operation under anaerobic conditions, one of each duplicate reactors of the four sets was switched to aerobic mode. The concentrations of the xenoestrogenes Bisphenol A (BPA), 4–Nonylphenol (NP) and 4–tert–Octylphenol (OP) in the leachate are investigated.

At the beginning of the experiment NP was only detectable in the drainage of waste disposed of after 1992. Over the period of anaerobic operation an increase of the NP concentrations in the leachate was observed. At the end of the anaerobic phase concentrations of NP were in the range of 300 to 1500 ng/l. After changing over to aerobic mode a slight decrease of the NP concentrations in the drainage occurred. In order to confirm this trend the analysis of the subsequent samples needs to be awaited.

In contrast to NP BPA was detected in concentrations ranging from 65 to 1600 ng/l at the start of the experiment. A decrease of BPA release was observed at the end of the three months anaerobic phase. Moreover, BPA could not be detected in the drainage from waste disposed of before 1990. Generally, more BPA as well as NP was released from waste disposed of after 1992 compared to the leachate from older waste. The drop of BPA concentrations after the change–over to aerobic mode seems to be not as steep as for NP. Again, for confirmation of the trend the subsequent samples still need to be analysed.

The Determination of Alkylphenols in Food

Alwyn Fernandes*, Claire Costley and Martin Rose Central Science Laboratory, Sand Hutton, York, North Yorkshire, YO41 1LZ.A method for the determination of alkylphenols in food using cold solvent extraction, followed by a two-stage chromatographic purification and GC-MS analysis was developed. The method was validated and used to measure concentrations of octylphenol (OP) and nonylphenol (NP) congeners in UK duplicate diet samples.

Alkylphenols (APs) are a group of degradation products derived mainly from the hydrolytic breakdown of alkylphenol ethoxylates. The open-ended usage of these compounds results in their entry to waste-water and sewage treatment systems where biodegradative treatment inadvertently provides the transformation to persistent, lipophilic and more toxic products, the APs.

A procedure using internal standardisation was used for the analysis. The food sample was fortified with 13C labelled NP. The full range of OP and NP isomers were isolated from the matrix by maceration and methanol extraction. The extract was concentrated, solvent exchanged to cyclohexane and purified by open column chromatography using deactivated neutral alumina followed by Florisil. The purified extracts were concentrated and analysed by gas chromatography-mass spectrometry (GC-MS). The method was validated and used to measure

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concentrations of the range of OP and NP congeners in UK duplicate diet samples. In addition to the specificity intrinsic to MS, other quality control measures included the use of blanks, internal standards, analyte recovery monitoring and replicate analyses.

Individual p-n-octylphenol and p-n-nonylphenol isomers were also measured although these were not detected in any of the samples. Only one sample showed OP at 8.7 µg/kg, but levels of NP ranged from < 3.8 µg/kg to 25 µg/kg. This range of concentrations is lower than that reported earlier for items of food (Sasaki et al. 1999). Tests carried out on the stability of the alkylphenols in a duplicate diet matrix over a 6 month period suggested that some analyte depletion may have occurred during extended frozen storage which may have in part accounted for the relatively lower concentrations detected, although the extent of usage of these compounds also needs to be taken into consideration.

ReferencesSasaki, K., Takatsuki, S., Nemoto, S., Imanaka, M., Eto, S., Murakami, E., and Toyoda, M., 1999, Determination of Alkylphenols and 2,4-Dichlorophenol in Foods. Journal of the Food Hygienic Society of Japan, 40(6), 460 – 472.

Uptake of nonylphenols by plants in water solution

Å. Sjöström, C. Collins, G. Shaw

Department of Environmental Science and Technology, Imperial College LondonSilwood Park Campus, Ascot, Berkshire SL5 7PY

Nonylphenol polyethoxylates (NPEOs) are complex mixtures of isomers produced in large volumes and used widely as surfactants. Numerous studies have shown that persistent breakdown products of NPEOs, such as nonylphenol (NP), display oestrogenic activity contributing to endocrine disturbance in fish populations. During sewage treatment, NP and NPEOs adhere strongly to sewage sludge, and the subsequent application of sludge to agricultural land may create a pathway into the foodchain via the uptake of nonylphenols by crop plants. This study measures the potential for uptake of NP and nonylphenol12ethoxylate (NP12EO) by three major food crops. Mature carrot, wheat and bean plants were exposed to high concentrations of NP or NP12EO in a hydroponic system for three days. The plant material was extracted with methanol, cleaned up on alumina and analysed by high performance liquid chromatography (HPLC) with fluorescence detection. The results show that considerable accumulation of both compounds into roots is taking place in all three species, as well as translocation into other plant parts.

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Analysis of endocrine-disrupting phenolic compounds and steroids with GC-MS/MS Jacco van Doornmalen (1), Anke Laufer (1), Lennart Weltje (1), Bernd Markert (1), Joerg Oehlmann (2)

(1) International Graduate School (IHI) Zittau, Ecotoxicology Group, Markt 23, D-02763 Zittau, Germany(2) J.W. Goethe University, Faculty of Biology and Informatics, Siesmayerstrasse 70, D-60054 Frankfurt am Main, Germany

For risk-assessment purposes, endocrine-disrupting compounds (EDCs) need to be analysed in a variety of samples, like surface waters and biota. EDCs comprise a group of very diverse substances, of which phenolic compounds (e.g. alkylphenols, bisphenol-A) and steroids (e.g. ethynylestradiol, methyltestosterone) are well-known examples. Due to their relatively high polarity, the latter substances are hard to analyse directly with gas chromatography (GC). A common practise to overcome this problem is to derivatise the substances, prior to analysis. Silylating agents are becoming quite popular for this purpose, although rest products that accumulate in some parts of the system may interfere with the analysis on the long term. We chose to use another derivatising agent, viz. trimethylsulfonium hydroxide (TMSH), which methylates hydroxyl groups as a result of a pyrolytic reaction in the hot injector of the GC. A SilcoSteel injector was installed to reduce contamination of the system as much as possible, which allowed us to analyse relatively dirty samples (e.g. extracts of sewage sludge and biota). Tandem mass spectrometry (MS/MS) was used to detect the compounds of interest with high sensitivity. Generally, TMSH proved to be a good alternative for the derivatisation of phenolic compounds and steroids, and is presently used for the analysis of these substances in freshwater and freshwater invertebrates.

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