Long-Term Effects of Oestrogenic Effluent Exposure on Wild Fish
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LONG-TERM EFFECTS OF OESTROGENIC EFFLUENT EXPOSURE ON WILD FISH POPULATIONS A thesis submitted for the degree of Doctor of Philosophy by Elizabeth Nicol Institute for the Environment Brunel University Uxbridge Middlesex September 2014 ii TABLE OF CONTENTS DECLARATION VIII ABSTRACT IX ACKNOWLEDGEMENTS X LIST OF FIGURES XI LIST OF TABLES XIII ABBREVIATIONS XIV CHAPTER 1 GENERAL INTRODUCTION 1 1.1 CHEMICALS IN THE ENVIRONMENT 3 1.2 SOURCES AND EXPOSURE ROUTES OF EDCS 4 1.3 EDCS IN THE AQUATIC ENVIRONMENT 6 1.4 DELIVERY OF CHEMICALS TO THE AQUATIC ENVIRONMENT 7 1.4.1 DIFFUSE SOURCES 8 1.4.2 POINT SOURCES 9 1.5 HISTORICAL BACKGROUND AND JUSTIFICATION FOR THIS RESEARCH 10 1.6 CHEMICALS AND AQUATIC WILDLIFE POPULATIONS 13 1.7 WATER QUALITY AND FISH POPULATION DYNAMICS 15 1.8 FISH POPULATION QUANTIFICATION 18 1.9 GENETIC VARIATION: IMPORTANCE, USE AND APPLICATION FOR FISH POPULATIONS 19 1.10 GENETIC DIVERSITY AND THE ASSOCIATION WITH FITNESS 21 1.11 MOLECULAR MARKERS IN ECOTOXICOLOGY AND THEIR APPLICATION TO FISH POPULATIONS 22 1.12 USE OF MICROSATELLITES FOR PARENTAGE ANALYSIS 23 1.13 FACTORS AFFECTING GENETICS OF WILD FISH POPULATIONS 25 1.14 LINKS BETWEEN GENETIC DIVERSITY AND POLLUTION 28 1.15 CHALLENGES IN ASSESSING CHEMICAL IMPACTS ON POPULATIONS 29 1.16 OVERALL HYPOTHESIS AND AIMS 33 1.17 KEY OBJECTIVES 33 CHAPTER 2 GENERAL MATERIALS AND METHODS 35 2.1 GEOGRAPHICAL MODELLING 36 2.1.1 INTRODUCTION TO GEOGRAPHICAL INFORMATION SYSTEMS 36 2.1.2 MODELLING WITH GIS 36 2.1.3 EFFLUENT EXPOSURE MODELLING OF UK CATCHMENTS 38 2.1.4 STUDY REGION 39 2.1.5 CHOICE OF STUDY SITES 42 2.1.6 MODEL DESCRIPTION 42 2.1.7 EFFLUENT MODELLING OUTPUT 44 2.1.8 ADDITIONAL PREDICTIONS OF ENVIRONMENTAL CONCENTRATIONS OF OESTROGENS 47 2.1.9 EXTENSION OF GIS WORK: INCORPORATING RESTOCKING AND CHANNEL OBSTRUCTIONS 47 2.2 DNA MICROSATELLITE USE IN POPULATION GENETIC APPLICATIONS 50 2.2.1 HISTORY OF MICROSATELLITE MARKER TECHNOLOGY 50 2.2.2 WHAT ARE THEY? 51 2.2.3 HOW DO THEY WORK? 52 iii 2.2.4 ADVANTAGES OF MICROSATELLITE USE 55 2.2.5 LIMITATIONS OF USING MICROSATELLITES 56 2.2.6 APPLICATION OF MICROSATELLITES TO THE STUDY OF FISH POPULATIONS 57 2.3 DETAILED METHODOLOGY OF MICROSATELLITE USE WITHIN THIS THESIS 58 2.3.1 DEVELOPMENT OF MICROSATELLITES USED IN THIS THESIS (WORK CONDUCTED BY P.HAMILTON) 59 2.3.2 SAMPLE COLLECTION 60 2.3.3 DNA EXTRACTION 60 2.3.4 DNA EXTRACTION USING CHELEX PROTOCOL 61 2.3.5 DNA SAMPLE PREPARATIONS FOR PCR 61 2.3.6 MICROSATELLITE AMPLIFICATION USING PCR 61 2.3.7 RUNNING SAMPLES ON SEQUENCER 62 2.3.8 DATA ANALYSIS 63 CHAPTER 3 POPULATION DIFFERENTIATION AND GENETIC DIVERSITY OF R. RUTILUS ACROSS ENGLAND 67 3.1 STATEMENT OF CONTRIBUTION TO THIS WORK 68 3.2 INTRODUCTION 68 3.2.1 IMPORTANCE OF GENETIC VARIATION 68 3.2.2 APPLICATION OF MOLECULAR MARKERS 68 3.2.3 INTRODUCTION TO PHYLOGEOGRAPHY 71 3.2.4 FAMILY CYPRINIDAE —- THE CARPS AND MINNOWS 73 3.2.5 CYPRINID POPULATION STRUCTURE 73 3.2.6 TEMPORAL STABILITY OF FISH POPULATION DYNAMICS 75 3.2.7 AIMS AND OBJECTIVES 77 3.3 MATERIALS AND METHODS 77 3.3.1 STUDY SPECIES AND SITE SELECTION 77 3.3.2 SAMPLE COLLECTION 80 3.3.3 MICROSATELLITE ANALYSIS 82 3.3.4 POPULATION-GENETIC ANALYSES 82 3.3.5 POPULATION TREES 83 3.3.6 PRINCIPAL COMPONENT AND STRUCTURE ANALYSES 83 3.4 RESULTS 83 3.4.1 GENETIC DIVERSITY 83 3.4.2 GENETIC ANALYSIS OF ROACH IN ENGLISH RIVERS 83 3.4.3 POPULATION-GENETIC STRUCTURE OF ROACH 88 3.5 DISCUSSION 93 3.5.1 ROACH POPULATION SITUATION IN THE THAMES CATCHMENT 94 3.5.2 IMPLICATIONS FOR ROACH POPULATIONS 96 3.6 CONCLUSION 98 CHAPTER 4 EXAMINING THE INFLUENCE OF OESTROGENIC EFFLUENT CONTAMINATION ON WILD R. RUTILUS POPULATIONS 102 4.1 STATEMENT OF CONTRIBUTION TO WORK 103 4.2 INTRODUCTION 103 4.2.1 PRESSURES ON GLOBAL WATER RESOURCES 104 iv 4.2.2 UK RIVERINE ENVIRONMENT 105 4.2.3 OCCURRENCE OF ENDOCRINE DISRUPTING CHEMICALS (EDCS) IN THE AQUATIC ENVIRONMENT 106 4.2.4 INFLUENCE OF CONTAMINANTS ON FISH 108 4.2.5 INFLUENCE OF CONTAMINANTS AT THE GENETIC LEVEL 109 4.2.6 INFLUENCE OF EDCS ON POPULATION-LEVEL ENDPOINTS 112 4.2.7 MEASURING POPULATION IMPACTS USING GENETIC APPROACHES 113 4.2.8 HYPOTHESIS, AIMS AND OBJECTIVES 114 4.3 MATERIALS AND METHODS 115 4.3.1 STUDY LOCATION 115 4.3.2 CHOICE OF SAMPLE SITES 115 4.3.3 EFFLUENT MAPPING FOR SAMPLE SITE PREDICTIONS 115 4.3.4 ROACH AS THE STUDY SPECIES 115 4.3.5 SAMPLE COLLECTION 116 4.3.6 FISH SCALE AGING 118 4.3.7 GENETIC SAMPLE PROCESSING 118 4.3.8 POPULATION-GENETIC ANALYSES 118 4.3.9 DETECTING POPULATION BOTTLENECKS 118 4.3.10 ESTIMATING EFFECTIVE POPULATION SIZE (NE) 120 4.3.11 TEMPORAL NE ESTIMATES (CALCULATED BY P. HAMILTON) 121 4.3.12 STATISTICAL ANALYSIS 122 4.4 RESULTS 122 4.4.1 EVIDENCE FOR POPULATION BOTTLENECKS 122 4.4.2 RELATIONSHIP BETWEEN PREDICTED OESTROGEN CONCENTRATION AND NE 123 4.4.3 TEMPORAL VARIABILITY IN NE 123 4.5 DISCUSSION 127 4.5.1 UNDERSTANDING POPULATION BOTTLENECKS USING DEMOGRAPHIC INFORMATION 127 4.5.2 NE ESTIMATES OF ROACH IN CONTEXT 129 4.5.3 LIMITED EVIDENCE OF EFFLUENTS IMPACTING NE OF ROACH POPULATIONS 130 4.5.4 COMPARING FINDINGS WITH LABORATORY STUDIES AND SIMULATIONS 132 4.5.5 CAVEATS 134 4.5.6 IMPROVEMENTS FOR FURTHER WORK 135 4.6 CONCLUSION 136 CHAPTER 5 INFLUENCE OF MULTI-GENERATIONAL WWTW EFFLUENT EXPOSURE ON THE REPRODUCTIVE SUCCESS OF R. RUTILUS 137 5.1 INTRODUCTION 138 5.1.1 PERSISTENT POLLUTANTS PRESENT IN WWTW EFFLUENTS 138 5.1.2 POTENTIAL CONSEQUENCES OF OESTROGENIC EXPOSURE FOR FISH 140 5.1.3 SENSITIVE WINDOWS OF DEVELOPMENT 142 5.1.4 METHODS FOR INVESTIGATING MULTIGENERATIONAL EFFECTS OF EFFLUENT EXPOSURE 143 5.1.5 TRANSGENERATIONAL AND EPIGENETIC INFLUENCE OF EDCS 144 5.1.6 EXPERIMENTAL AIMS AND HYPOTHESIS 146 5.2 MATERIALS AND METHODS 146 5.2.1 EXPOSURE EFFLUENT 146 5.2.2 FISH ORIGIN AND MAINTENANCE 148 5.2.3 EXPERIMENTAL DESIGN 148 v 5.2.4 BREEDING EXPERIMENT 148 5.2.5 HISTOLOGICAL ANALYSES OF GONADAL DEVELOPMENT 151 5.2.6 FIXATION AND DISSECTION PROCESSING 151 5.2.7 TISSUE PROCESSING 152 5.2.8 EMBEDDING 153 5.2.9 SECTIONING 154 5.2.10 STAINING 155 5.2.11 LIGHT MICROSCOPY AND HISTOPATHOLOGICAL ANALYSIS 156 5.2.12 PARENTAGE ANALYSES 157 5.2.13 STATISTICAL ANALYSIS 158 5.3 RESULTS 160 5.3.1 GONADAL DEVELOPMENT, HISTOLOGICAL EXAMINATION AND SEX RATIOS IN ROACH 160 5.3.2 IMPACTS OF WWTW EFFLUENT EXPOSURE ON BREEDING CAPACITY OF MALE ROACH 163 5.3.3 REPRODUCTIVE SUCCESS OF INDIVIDUALS FISH USING PARENTAGE ANALYSIS 164 5.4 DISCUSSION 165 5.4.1 GROSS MORPHOLOGICAL ENDPOINTS 166 5.4.2 EFFLUENT EXPOSURE EFFECTS ON TESTICULAR DEVELOPMENT 167 5.4.3 SEX RATIOS IN EXPOSED ROACH POPULATIONS 168 5.4.4 BREEDING SUCCESS OF MALE AND FEMALE ROACH 169 5.4.5 IMPLICATIONS FOR ROACH POPULATIONS 170 5.5 CONCLUSION 172 CHAPTER 6 ENVIRONMENTAL INFLUENCES OF STREAM HABITAT AND PROXIMAL LAND USE ON TWO LEVELS OF FRESHWATER FISH DIVERSITY 174 6.1 INTRODUCTION 175 6.1.1 BIODIVERSITY AND THE ENVIRONMENT 175 6.1.2 ECOLOGICAL AND EVOLUTIONARY DRIVERS OF SPECIES AND GENETIC DIVERSITY 175 6.1.3 GENETIC DIVERSITY AND ENVIRONMENTAL CHANGE 177 6.1.4 FACTORS AFFECTING FISH ASSEMBLAGES 179 6.1.5 STUDY AIMS AND HYPOTHESIS 180 6.2 MATERIALS AND METHODS 181 6.2.1 STUDY SYSTEM AND SITE SELECTION 181 6.2.2 SPECIES SELECTION 182 6.2.3 SPECIMEN COLLECTION FOR GENETIC DATA AND SPECIES DIVERSITY CALCULATIONS 183 6.2.4 CHARACTERISING SPECIES DIVERSITY INDICES 183 6.2.5 GENETIC ANALYSES 184 6.2.6 CALCULATING GENETIC DIVERSITY AND INTERNAL RELATEDNESS MEASURES 184 6.2.7 CHARACTERISATION OF ENVIRONMENTAL VARIABLES- INCLUDING LAND USE, WATER QUALITY, HYDROLOGY AND DISTANCE ENDPOINTS 185 6.2.8 STATISTICAL ANALYSIS OF ENVIRONMENTAL VARIATION, GENETIC DIVERSITY AND SPECIES DIVERSITY 190 6.3 RESULTS 191 6.3.1 POPULATION-GENETIC STRUCTURE 191 6.3.2 FISH SPECIES PATTERNS ACROSS THE CATCHMENT 193 6.3.3 RELATIONSHIPS BETWEEN SPECIES DIVERSITY AND GENETIC DIVERSITY 194 6.3.4 GENETIC DIVERGENCE RELATIONSHIPS WITH BIOGEOGRAPHY AND ASSEMBLAGE DIVERGENCE195 vi 6.3.5 LAND USE PATTERNS ACROSS THE CATCHMENT 197 6.3.6 ENVIRONMENTAL PREDICTORS OF GENETIC AND SPECIES DIVERSITY 197 6.4 DISCUSSION 201 6.4.1 RELATIONSHIPS BETWEEN FISH SPECIES DIVERSITY AND GENETIC DIVERSITY OF R. RUTILUS 202 6.4.2 ENVIRONMENTAL PREDICTORS OF SPECIES AND GENETIC DIVERSITY 204 6.4.3 ISSUES AND IMPROVEMENTS 207 6.5 CONCLUSION 209 CHAPTER 7 GENERAL DISCUSSION 211 OVERVIEW 212 7.1 ROACH AS SENTINELS FOR STUDYING POPULATION IMPACTS OF EDCS 212 7.2 GENOTYPING APPLICATION FOR POPULATION GENETICS OF ROACH 214 7.3 INVESTIGATING THE IMPACTS OF EFFLUENT EXPOSURE — BRIDGING THE GAP BETWEEN NDIVIDUALS, POPULATIONS AND MULTIPLE GENERATIONS 216 7.4 EDCS IN CONTEXT — THE ROLE OF ECOLOGY AND GEOGRAPHY INFLUENCING FISH POPULATION PATTERNS 218 7.5 CONTRIBUTION TO SCIENCE 219 7.6 RECOMMENDATIONS AND FUTURE AVENUES OF INVESTIGATION 220 7.7 FINAL CONCLUSION 221 REFERENCES 224 vii DECLARATION The work submitted in this thesis was conducted between 2010 and 2014 at Brunel University (Uxbridge, Middlesex), University of Exeter (Exeter, Devon) and Essex and Suffolk Water Treatment Works (Langford, Essex). This work was carried out independently and has not been submitted for any other degree.