Reproductive effects in two species of native freshwater gastropod mollusc exposed to 17 β- oestradiol or an environmentally relevant mixture of oestrogenic chemicals in outdoor mesocosms A Thesis submitted for the degree of Doctor of Philosophy By Alice Louise Baynes Institute for the Environment Brunel University September 2009 ii Declaration The work submitted in this thesis was carried out between 2004 and 2008 at Brunel University (Uxbridge, Middlesex) and Essex and Suffolk Water Treatment Works (Langford, Essex). This work was carried out independently and has not been submitted for any other degree. iii Abstract Institute for the Environment Brunel University Recent evidence suggests that molluscs may be sensitive to the effects of endocrine disrupting chemicals (EDCs) in a similar manner to vertebrates, such as fish. Despite this (with the exception of TBT-induced imposex in marine gastropods), molluscs have been largely overlooked in the field of endocrine disruption. Life-cycle studies were conducted in which two species of native UK freshwater gastropod molluscs (the hermaphrodite Planorbarius corneus and the gonochorist Viviparus viviparus ) were exposed to either 17 β-oestradiol or environmentally relevant mixtures of chemicals known to be oestrogenic to vertebrates and to be present in UK treated sewage effluents (TSE) and rivers. Adult snails were exposed for four months in outdoor mesocosms, fed by river water, over the spring and summer (breeding season) in order to examine effects on reproductive output, growth and mortality. Furthermore, offspring (F1s) were also developmentally exposed over the same period. F1 juvenile snails were then depurated in river water for nine months (over winter) after which time their growth, survival, and reproductive success were measured in further un-dosed river water mesocosm studies in the following spring/summer. Histopathology was used to determine immediate effects of chemical exposure on adult and F1 snails’ reproductive health. Histopathology was also used to determine long lasting effects of chemical exposure on depurated F1s. Exposure to oestrogenic chemicals resulted in a range of effects, including modulated fecundity and growth in F0 adults, to retardation of growth, sexual development and fecundity in developmentally exposed F1s. Exposure to mixtures of oestrogenic chemicals also resulted in possible modulation of the immune system, resulting in increased parasitism and over winter mortality of exposed F1s compared to snails exposed to river water alone. Differences in sensitivity and response to exposure between the two species and the generations were also observed. iv Acknowledgements I would like to take this opportunity to acknowledge my supervisors Dr Edwin Routledge and Dr Susan Jobling for all their advice, guidance and inspiration throughout my PhD research. I would like to thank the Department for Environment, Food and Rural affairs (DEFRA) for their funding and giving me the opportunity to conduct this research. I would also like to acknowledge all the staff at the Institute for the Environment at Brunel University, and Essex and Suffolk Water Treatment works at Langford, Essex, for their day to day help during my practical research, and I would like to thank Dr Burkard Watermann from Limnomar, Germany, for his invaluable histopathological advice and expertise. I would like to say a special thank you to my fellow snail researcher Rachel Benstead for all her advice, hard work and insane amount of egg counting during the mesocosm studies and beyond. I would also like to take this opportunity to thank my friends and family for all their support, especially Sonia Chapman and Dr Jon Bolland, for all the good times and for always being their when I needed you most. Lastly I would like to thank Graham Harris. Not least for his snail wrangling skills, but, also for all his support, words of encouragement, and generally keeping me as sane as possible throughout the last push of research and the thesis writing. Thank you. v Contents DECLARATION ................................................................................................................................................ II ABSTRACT ..................................................................................................................................................... III ACKNOWLEDGEMENTS ................................................................................................................................. IV CONTENTS ..................................................................................................................................................... V LIST OF FIGURES .......................................................................................................................................... IX LIST OF TABLES ........................................................................................................................................... XI 1 INTRODUCTION ..................................................................................................................................1 1.1 INTRODUCTION ................................................................................................................................2 1.2 THE (VERTEBRATE ) ENDOCRINE SYSTEM ........................................................................................3 1.3 ENDOCRINE DISRUPTION (ED) .........................................................................................................8 1.4 POSSIBLE ENDOCRINE DISRUPTING CHEMICALS (EDC S).................................................................9 1.5 EVIDENCE OF ENDOCRINE DISRUPTION IN THE ENVIRONMENT ......................................................16 1.6 EFFECTS OF KNOWN VERTEBRATE EDC S ON MOLLUSCS : FIELD AND LABORATORY STUDIES .........22 1.7 THE MOLLUSC ENDOCRINE SYSTEM ..............................................................................................30 1.8 AIMS ..............................................................................................................................................36 2 INTRODUCTION TO THE TEST SPECIES AND PRELIMINARY WORK ..............................37 2.1 INTRODUCTION TO GASTROPODS ...................................................................................................38 2.1.1 Pulmonates...............................................................................................................................38 2.1.2 Prosobranchs ...........................................................................................................................44 2.2 INTRODUCTION TO THE TEST SPECIES .............................................................................................47 2.2.1 Test species 1 – Planorbarius corneus (Pulmonate) ................................................................47 2.2.1.1 Ecology, habitat and distribution ...................................................................................................47 2.2.1.2 Life cycle and reproduction ...........................................................................................................48 2.2.1.3 Reproductive Morphology and Physiology....................................................................................49 The Hermaphrodite system.................................................................................................................................49 The ovotestis.......................................................................................................................................................50 2.2.2 Test species 2 – Viviparus viviparus (Prosobranch) ................................................................50 2.2.2.1 Ecology, habitat and distribution ...................................................................................................50 2.2.2.2 Life cycle and reproduction ...........................................................................................................52 Females...................................................................................................................................................55 Males ......................................................................................................................................................57 Embryos..................................................................................................................................................58 2.2.2.3 Reproductive Morphology and Physiology....................................................................................59 Male........................................................................................................................................................59 Female ....................................................................................................................................................59 2.3 PRELIMINARY WORK AND OPTIMISATION .......................................................................................60 2.3.1 Planorbarius corneus...............................................................................................................60 2.3.1.1 Planorbarius corneus acquisition ..................................................................................................61 2.3.1.2 General laboratory culture conditions............................................................................................61 2.3.1.3