ENDOCRINE DISRUPTORS IN THE IRISH AQUATIC ENVIRONMENT - EPA

191 Pages·2005·4.02 MB·English

Endocrine Disruptors in the Irish Aquatic Environment

ENDOCRINE DISRUPTORS IN THE
IRISH AQUATIC ENVIRONMENT

FINAL REPORT

(Project: Estimation of Estrogenic Compounds in Irish Surface and Waste Waters)

Prepared for the Environmental Protection Agency
by
Department of Biological Sciences, Cork Institute of Technology
Aquaculture Development Centre, University College Cork
Centre for Ecology and Hydrology, Wallingford, UK

Authors:

Heloise Tarrant, Neville Llewellyn, Anne Lyons,
Nicholas Tattersall, Suzanne Wylde, Geriasmos Mouzakitis,
Michelle Maloney and Craig McKenzie.

ENVIRONMENTAL PROTECTION AGENCY
An Ghníomhaireacht um Chaomhnú Comhshaoil
PO Box 3000, Johnstown Castle, Co.Wexford, Ireland

Telephone: [phone] Fax: [phone]
Email: [email protected] Website: www.epa.ie

© Environmental Protection Agency 2005

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Endocrine Disruptors in the Irish Aquatic Environment

ACKNOWLEDGEMENTS

This report has been prepared as part of the Environmental Research Technological
Development and Innovation Programme under the Productive Sector Operational
Programme 2000-2006. The programme is financed by the Irish Government under
the National Development Plan 2000-2006. It is administered on behalf of the
Department of the Environment and Local Government by the Environmental
Protection Agency, which has the statutory function of co-ordinating and promoting
environmental research.

DISCLAIMER

Although every effort has been made to ensure the accuracy of the material contained
in this publication, complete accuracy cannot be guaranteed. Neither the
Environmental Protection Agency nor the author(s) accept any responsibility
whatsoever for loss or damage occasioned or claimed to have been occasioned, in part
or in full, as a consequence of any person acting, or refraining from acting, as a result
of a matter contained in this publication. All or part of this publication may be
reproduced without further permission, provided the source is acknowledged.

ENDOCRINE DISRUPTORS IN THE IRISH AQUATIC ENVIRONMENT

FINAL REPORT

Published by the Environmental Protection Agency, Ireland

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Endocrine Disruptors in the Irish Aquatic Environment

Table of Contents

Executive Summary ix
List of Abbreviations x
Special Acknowledgements xiii
1. Literature Review 1
1.1 General Introduction 2
1.2 General Mechanisms of Endocrine Disruption 3
1.2.1 Overview of the Endocrine System 3
(i) The Hypothalmic-Pituitary-Gonadal Axis
(ii) The Hypothalmic-Pituitary-Adrenal Axis
(iii) The Hypothalmic-Pituitary-Thyroid Axis
1.2.2 Main Classes of Endocrine Disruptors 7
(i) Estrogens
(ii) Androgens
(iii) Progestins
(iv) Aryl Hydrocarbon Receptor Agonists
(v) Disruptors of Thyroid Hormone
1.2.3 Disruption of the Hormone Dynamic Pathway 13
1.2.4 Dose Sensitivity and the Effect of Chemical Mixtures 14
1.3 Mechanisms of Endocrine Disruption in Fish 16
1.3.1 The Teleost Endocrine System 16
1.3.2 Disruption of Hypothalamic and Pituitary Function 17
1.3.3 Disruption of the Reproductive System 17
1.3.4 Disruption in Eggs, Embryos, Larvae and Juvenile Fish 18
1.3.5 Disruption of Liver Function 18
1.4 In vivo Bioassays for the Evaluation of Endocrine Disruption
in Fish 19
1.4.1 Intersex 19
1.4.2 Gonad Weight 20
1.4.3 Liver Weight 20
1.4.4 Vitellogenesis 20

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Endocrine Disruptors in the Irish Aquatic Environment

(i) Radioimmunoassay for Plasma Vitellogenin
(ii) Enzyme Linked Immunosorbent Assay
(iii) SDS Polyacrylamide Gel Electrophoresis
1.4.5 In vivo Studies to Determine the Effects of EDCs in Fish 22
(i) Wild Fish Studies
(ii) Tank Studies
(iii) Cage Studies
1.5 In vitro Bioassays for the Evaluation of EDCs in Environmental
Samples 24
1.5.1 Human Cell Line Assays 25
(i) MCF-7 Cell Lines
(ii) Vitellogenin Assays
1.5.2 Competitive Binding Assays 27
1.5.3 Yeast Screen Assays 28
(i) The Yeast Estrogen Screen (YES) 29
1.6 The Environmental Chemistry of Suspected EDCs 33
1.6.1 Introduction 33
1.6.2 Alkylphenol Ethoxylates 34
1.6.2.1 Useage and Legislative Status 34
1.6.2.2 Chemistry of Alkylphenol Ethoxylates and
Metabolites 36
1.6.2.3 Environmental Behaviour 37
(i) Behaviour in WWTP
(ii) Behaviour Following Release in the
Aquatic Environment
(ii) Concentrations in WWTP and Environmental
Samples
1.6.3 Steroid Sex Hormones and Related Synthetic Compounds 50
1.6.3.1 Useage 50
1.6.3.2 Chemistry of Steroid Sex Hormones and Related
Synthetic Compounds 50
1.6.3.3 Environmental Behaviour 52

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Endocrine Disruptors in the Irish Aquatic Environment

(i) Behaviour in WWTP
(ii) Behaviour in the Aquatic Environment
1.6.4 Bisphenol A 56
1.6.4.1 Chemistry, Useage and Legislative Status of
Bisphenol A 56
1.6.4.2 Environmental Behaviour 56
(i) Behaviour in WWTP
(ii) Behaviour in the Aquatic Environment
(iii) Concentrations in WWTP and Environmental
Samples
1.6.5 Phthalates 59
1.6.5.1 Useage and Legislative Status 59
1.6.5.2 Environmental Behaviour 61
(i) Behaviour in WWTP
(ii) Behaviour in the Aquatic Environment
(iii) Environmental Concentrations
1.6.6 Other Potential Endocrine Disrupting Compounds 65

2. Development of Methodology and Validation 67
2.1 Introduction 68
2.2 In Vitro Bioassay and Toxicity Identification and Evaluation
(TIE) Methodologies 68
2.2.1 Semi-quantitative Detection of Salmonid Vitellogenin in
Wild Brown Trout Plasma by Enzyme Immunoassay 68
2.2.2 Quantitative Detection of Estrogenic Chemicals
using a Recombinant Yeast Bioassay 69
2.2.3 Quantitative Detection of (Anti)Androgenic Chemicals
using a Recombinant Yeast Bioassay 71
2.2.4 Toxicity Identification and Evaluation Methods 74
2.3 In vivo Studies Methodology 78
2.3.1 Site Selection 78
2.3.1.1 Caged Fish Study 78
2.3.1.2 Wild Brown Trout Survey 79
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Endocrine Disruptors in the Irish Aquatic Environment

2.3.2 Sampling Methodology 82
2.3.2.1 Fishing Methods 82
2.3.2.2 Tissue Sampling 84
2.3.3 Tissue Analysis 87
2.3.3.1 Histology 87
2.3.3.2 Vitellogenin Analysis 89
2.3.3.3 Condition Index 89
2.3.3.4 Gonado Somatic Index 90
2.3.3.5 Liver Analysis and Hepatosomatic Index 90
2.3.3.6 Statistical Analysis 90
2.3.4 Experimental Design for Tank Studies 90
2.3.5 Experimental Design for Caged Fish Studies 92

3. Estrogenic Activity of Ballincollig Wastewater Treatment Plant 94
3.0 Introduction 95
3.1 Characteristics of the Site for the In Vivo Caged Fish Study 96
3.2 Results and Discussion 98
3.2.1 Impact of Ballincollig WWTP on Estrogen Levels in the
River Lee 98
3.2.2 Induction of Vitellogenesis in Male Rainbow Trout;
Flow-Through Tank Study 100
3.3 Conclusions 101

4. Survey of Irish Wild Brown Trout for Evidence of Endocrine
Disruption 102
4.1 Introduction 103
4.2 Selection of Sites for the Wild Brown Trout Survey 104
4.3 Results of the Survey of the Wild Brown Trout Population in the
Rivers Lee and Bandon, and the Killarney Lakes. 105
4.3.1 River Bandon 105
(i) Sex Ratio
(ii) Condition
(iii) Hepatosomatic Index
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Endocrine Disruptors in the Irish Aquatic Environment

(iv) Intersex
4.3.2 River Lee 110
(i) Sex Ratio
(ii) Condition
(iii) Hepatosomatic Index
(iv) Intersex
4.3.3 Killarney Lakes 112
(i) Intersex
4.3.4 Other Indices of Reproductive Health 113
(i) Gonadosomatic Index
(ii) Semi-Quantitative Determination of Plasma Vitellogenin
4.4 Survey of the Wild Brown Trout Population in the River Liffey,
Upstream and Downstream of the Osberstown STW. 115
(i) Intersex
(ii) Semi-Quantitative Determination of Plasma Vitellogenin
(iii) Hepatosomatic Index
4.5 Discussion 118
4.6 Conclusions 122

5. In Vitro Bioassay Analysis of WWTP Effluents and Receiving Waters
for Estrogenic Chemicals 124
5.0 Introduction 125
5.1 Characteristics of Sample Sites 125
5.2 Survey of Estrogenicity of WWTP Effluents and Receiving Waters 126
5.2.1 Dose-Response Curves of Extracts of WWTP Effluents
in the YES 126
5.2.2 Non-Parallel Dilution of Sample Extracts in the YES 129
5.2.3 Estrogenicity of WWTP Treated Final Effluents and
Receiving Waters. 132
5.3 Modelling of Effluent and Receiving Water Concentrations of
Estrone, Estradiol and Ethinylestradiol at Selected WWTP 135
5.3.1 Sources of Data Used for Modelling 136
5.3.2 WWTP Modelled 136
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Endocrine Disruptors in the Irish Aquatic Environment

5.3.3 Results for Sewage Effluent Modelling 138
5.3.4 Results for Receiving Waters 142
5.4 Conclusions 146

6. Concluding Remarks 147

7. Bibliography 152

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Endocrine Disruptors in the Irish Aquatic Environment

EXECUTIVE SUMMARY

The objective of this study was to provide an assessment of the potential risk from
endocrine disrupting compounds (EDCs) to Irish freshwaters, their ecosystems and
associated drinking water resources. The different elements of the study are integrated
in a manner designed to answer the following questions:
1. Do Irish rivers contain EDCs?
2. If so, at what concentrations?
3. Are these levels likely to pose a threat to aquatic ecosystems, particularly
wildfish?
4. Is there a risk to drinking water supplies?

Chapter 1 thoroughly reviews the current literature in the area and demonstrates that no
research, prior to this study, had been carried out to address these questions in an Irish
context.

Chapter 2: Details of the methodologies applied in this study are described.

Chapter 3: An in vivo caged fish study was performed to determine the effects of
wastewater treatment plant (WWTP) effluent on exposed fish populations in Irish rivers,
and any associated risk to drinking water resources. To this end, cages of male rainbow
trout were deployed at test and control sites on the river Lee, and levels of plasma
vitellogenin were used as a sensitive indicator of exposure to environmental estrogens.
No evidence of estrogen exposure was found at any of the test or control sites, including
the site at the intake to the Lee Road water treatment works, which supplies drinking
water to Cork City.

Chapter 4 presents the results of a survey of feral Brown trout populations in the rivers
Liffey, Lee and Bandon, and the Killarney Lakes. There was no evidence of exposure to
environmental estrogens in any of the sites surveyed, with the exception of the Liffey
site, located immediately downstream of Osberstown WWTP. Raised plasma
vitellogenin levels in male fish taken from this site indicated estrogenic activity in this
region of the river.

Chapter 5: An in vitro bioassay analysis of point sources of estrogens; namely WWTP
effluents, and their impact on receiving waters, was performed. Estrogen levels in
effluents ranged from 1-17 ng/l while receiving waters were less than 3 ng/l in all cases.
These levels compared favourably with similar size plants investigated in other
countries, including the UK. A model recently developed (2004) at CEH Wallingford
predicts estrogenicity of effluents and receiving waters impacted by domestic waste.
This model was applied for the first time in an Irish context. Generally, for effluents,
agreement was good between measured and predicted values for estrogenicity.
However, the model consistently under-predicted the measured concentration of
estrogens in the receiving waters. Given the predominantly rural nature of the
catchments involved, it is suggested that the additional estrogenic signal is derived from
intensive livestock agriculture.

Chapter 6 presents a summary of the general conclusions from this study.
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Endocrine Disruptors in the Irish Aquatic Environment

LIST OF ABBREVIATIONS

4t-OP 4-tert-octyl phenol
ACTH Adrenocorticotropin
ADC Aquaculture Development Centre
AFP Alpha-fetoprotein
Ah Aryl hydrocarbon receptor
AP Alkylphenol
AP EC Alkyl phenol ethoxy carboxylate
2
APEO Alkylphenol polyethoxylate
AR Androgen receptor
ARE Androgen response element
ARNT Ah receptor nuclear translocator
BBP Butylbenzyl phthalate
BCF Bioconcentration factor
BCHP Butyl cyclohexyl phthalate
BOD-5 5-Day biological oxygen demand
BPA Bisphenol-A
CBG Corticosteroid binding globulin
CNS Central nervous system
CPRG β-D-Galactopyranoside
CRH Corticotropin-releasing hormone
DBP Dibutyl phthalate
DDE Dichlorodiphenyldichloroethylene
DDT Dichlorodiphenyltrichloroethane
DEHP Di(ethylhexyl)phthalate
DEP Diethyl phthalate
DES Diethylstilbestrol
DHEA Dehydroepiandrosterone
DHT Dihydro-testosterone
DIBP Diisobutyl phthalate
DINP Diisononyl phthalate
DnBP Di-n-butyl phthalate
DPhP Diphenyl phthalate
DTDP Ditridecyl phthalate
E2 17β-Estradiol

E2 Estradiol Equivalent
eq
E3 Estrone
EA Environment Agency
EFB Eastern Fisheries Board
EDC Endocrine disrupting chemical
EE 17α-Ethinylestradiol
2
EE 17α-Ethinylestradiol
2
EIA Enzyme immunoassay
ELISA Enzyme linked immunosorbent assay
EPA Environmental Protection Agency

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