1 Detailed Review Paper on Mollusc Life-Cycle 2 Toxicity Testing for Endocrine Disrupters and 3 Other Chemicals 4 Final Draft Version 09 – 1 July 2009 FINAL_DRAFT_DRP_V9 1 Content 2 1 Executive Summary ..................................................................................... 6 3 2 Introduction ................................................................................................. 8 4 2.1 Background ............................................................................................. 8 5 2.2 Purpose of the DRP.................................................................................10 6 2.3 Objectives of partial- and full mollusc life cycle tests ...............................12 7 3 Neuroendocrine control of physiological functions in molluscs ....................15 8 3.1 Phylogeny and the divergence of molluscan endocrinology ......................15 9 3.2 (Neuro-)endocrine control in molluscs .....................................................17 10 3.3 Modes of action of endocrine disrupting chemicals (EDCs) in molluscs ....27 11 3.3.1 Possible TBT MOAs: Vertebrate-type steroid hypothesis (aromatase 12 inhibition).......................................................................................................29 13 3.3.2 Possible TBT MOAs: Vertebrate-type steroid hypothesis (inhibited 14 testosterone excretion) ....................................................................................30 15 3.3.3 Possible TBT MOAs: Vertebrate-type steroid hypothesis (modulation of 16 free versus fatty acid-bound testosterone levels) ...............................................30 17 3.3.4 Possible TBT MOAs: Neuropeptide hypothesis ....................................31 18 3.3.5 Possible TBT MOAs: Retinoid X receptor hypothesis ..........................32 19 4 Endocrine disruption in wild molluscs .........................................................34 20 4.1 Effects of tributyltin ................................................................................34 21 4.1.1 Prosobranch molluscs ..........................................................................35 22 4.1.2 Bivalves..............................................................................................36 23 4.2 Effects of estrogens and their mimics ......................................................38 24 4.3 Summary of EDC effects in wild molluscs ...............................................39 25 5 Experimental induction of endocrine disruption in molluscs .........................40 26 5.1 Organotins ..............................................................................................40 27 5.2 Estrogens and their mimics .....................................................................42 28 5.3 Androgens and their mimics ....................................................................44 29 5.4 Others EDCs...........................................................................................45 30 5.5 Summary ................................................................................................45 31 6 Experimental design considerations .............................................................47 32 6.1 Exposure duration ...................................................................................47 2 FINAL_DRAFT_DRP_V9 1 6.1.1 Strength and weaknesses of partial versus full life cycle tests................47 2 6.1.2 Partial life cycle (PLC) tests ................................................................48 3 6.1.3 Full life cycle (FLC) tests ....................................................................50 4 6.2 Possible periods of exposure during development ....................................50 5 6.3 Routes of dosing .....................................................................................52 6 6.3.1 Water .................................................................................................52 7 6.3.2 Sediments ...........................................................................................54 8 6.4 Dose selection ........................................................................................55 9 6.5 Statistical considerations .........................................................................56 10 6.5.1 Controlling variability .........................................................................57 11 6.5.2 Defining the experimental unit.............................................................58 12 6.5.3 Importance of statistical power ............................................................59 13 6.5.4 Data treatment ....................................................................................60 14 6.5.5 NOEC and ECX determination .............................................................61 15 6.5.6 Sample size in FLC tests .....................................................................64 16 7 Candidate test protocols ..............................................................................65 17 7.1 Strengths and weaknesses of potential test species ...................................65 18 7.2 Strengths and weaknesses of different test methods and endpoints ............72 19 7.2.1 Partial life cycle tests ..........................................................................72 20 7.2.2 Full life cycle tests ..............................................................................73 21 7.2.3 Biomarkers .........................................................................................74 22 7.2.4 Conclusions about test methods and endpoints .....................................76 23 7.3 Candidate partial life cycle test methods ..................................................77 24 7.3.1 The test organism Potamopyrgus antipodarum .....................................77 25 7.4 Candidate full life cycle test methods ......................................................84 26 7.4.1 The test organism Lymnaea stagnalis ...................................................84 27 7.4.2 The test organism Crassostrea gigas ....................................................91 28 8 Recommended Protocols – data gaps and validation requirements ................99 29 8.1 Data gaps and research requirements .......................................................99 30 8.1.1 The freshwater mudsnail (Potamopyrgus antipodarum) ........................99 31 8.1.2 The pond snail (Lymnaea stagnalis) ................................................... 100 3 FINAL_DRAFT_DRP_V9 1 8.1.3 The Pacific oyster (Crassostrea gigas) ............................................... 100 2 8.1.4 Other species .................................................................................... 101 3 8.2 Validation requirements ........................................................................ 101 4 9 Culture and handling of recommended species........................................... 104 5 9.1 Methods ............................................................................................... 104 6 9.1.1 The freshwater mudsnail (Potamopyrgus antipodarum) ...................... 104 7 9.1.2 The pond snail (Lymnaea stagnalis) ................................................... 108 8 9.1.3 The Pacific oyster (Crassostrea gigas) ............................................... 111 9 9.2 Data gaps.............................................................................................. 114 10 9.2.1 Adjustment of industrial aquaculture techniques ................................. 114 11 9.2.2 Basic research ................................................................................... 114 12 9.2.3 Sensitivity of genetically different strains ........................................... 115 13 10 Implementation ........................................................................................ 116 14 10.1 Animal welfare ..................................................................................... 116 15 10.2 Facilities and equipment........................................................................ 117 16 10.3 Running costs ....................................................................................... 118 17 10.4 Test reproducibility and confounding factors ......................................... 118 18 10.5 Data interpretation ................................................................................ 119 19 10.5.1 Representativeness and data extrapolation ...................................... 120 20 10.5.2 Environmental relevance ............................................................... 120 21 10.5.3 Hormesis or low dose effects ......................................................... 121 22 10.6 Advantages and disadvantages of the different candidate species ............ 122 23 11 Conclusions .............................................................................................. 125 24 12 References................................................................................................ 129 25 13 List of abbreviations ................................................................................. 166 26 14 Appendix 1............................................................................................... 169 27 28 4 FINAL_DRAFT_DRP_V9 1 Acknowledgements 2 The preparation of this review was solely supported by funding from the German and 3 United Kingdom Governments. Many scientists kindly provided advice on various 4 aspects, but particular assistance was given by Toshihiro Horiguchi, Laurent Lagadic, 5 Gerry LeBlanc,