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OECD Test Method Development: Mollusc Reproductive Toxicity Tests – Development and Validation of Test Guidelines - CB0467

Since the 1990’s, there has been a significantly growing body of scientific evidence that the reproductive health of mammals, fish and aquatic wildlife may be adversely impacted by certain classes of natural and synthetic chemicals. Many of these chemicals act as disrupters of the normal functioning of the endocrine systems that are essential to development and reproduction, with adverse consequences for populations of both commercially and ecologically important species (especially fish and shellfish). Important examples of the impacts of endocrine disrupting chemicals (‘EDCs’) include feminisation of fish in UK rivers and the collapse of Canadian lakes ecosystems exposed to oestrogens, together with the dramatic impact of the anti-foulant paint ingredient tributyl tin (TBT) on molluscs. Together with human health considerations, concern over the ecosystem impacts of EDCs has become a global issue and has led to important developments to identify and manage the key chemicals of concern in Europe, North America and South East Asia. In 1996, the OECD established a Special Activity on Endocrine Disrupter Testing and Assessment with the objectives of providing information and coordinating activities, developing new and revised existing OECD test guidelines to detect endocrine disrupters; and harmonising hazard and risk characterisation approaches. To date, the OECD has successfully developed or updated a variety of test guidelines for detecting reproductive (including EDC) impacts in mammals, fish, amphibians and arthropods (eg insects). Importantly, an OECD (2010) Detailed Review Paper concluded that there is a need to develop a test guideline to address the impacts of chemicals which can cause reproductive toxicity (including ED impacts) on molluscs. Led by scientists from the UK and Germany, supported by advice from other countries, it was concluded that mollusc-based lifecycle tests are needed, given their major ecological and economic importance and known sensitivity to a number of chemicals (including EDCs). Since 2010, the UK has started a successful, and active, work programme with a number of other OECD countries, especially Denmark, France and Germany, and has sponsored pre-validation studies using mudsnails (P. antipodarum) and pondsnails (L. stagnalis) exposed to reference chemicals. Recent studies have raised important questions which need to be addressed with respect to the basic biology of these species (eg disease and water quality) before it is sensible to proceed into validation work. Therefore, this proposal describes a step-wise approach to resolving these issues, with the aim of leading the UK contribution to the development of a mollusc test guideline and present it to the OECD Working Group of National Co-ordinators (WNT) for its approval in 2015.
Objective 1 – Critical review of on-going work with OECD scientific community and experimental studies to fill critical gaps in the understanding of how confounding factors affect the use of P. antipodarum and L. stagnalis in reproductive toxicity tests.

As highlighted in the OECD (2010)8 DRP on Molluscs Life-Cycle Toxicity Testing, the key challenges arising from studies undertaken thus far include understanding how confounding factors (e.g. temperature, day-length, season, population density, physico-chemical water quality, disease (pathogen) status and food quality) affect the performance of the mollusc reproduction tests. More recent discussions at the OECD VMG-eco meeting in November 2011 on the variability observed in pre-validation studies with P. antipodarum underlined the need to address the impact of latent disease and parasites in snail populations used for the OECD-related work. Research to date (see 8 for review) has highlighted that while the impact of some of these factors on test performance is known, additional data is essential in order to more precisely specify acceptable and optimally sensitive ranges of test conditions. Therefore, under Objective 1, we propose to ensure that there is robust and scientifically sound understanding of the role that critical environmental, biological and experimental factors have on the performance of P. antipodarum and L. stagnalis reproduction test methods.

Firstly, environmental and species-specific biological factors will be addressed in well controlled laboratory experiments using a step-wise approach. Baseline data will be gathered from non-exposed snail populations to build a high quality database of normal reproductive outputs in control animals (an approach widely used for other OECD test guideline applications), supported by histological checks of the pathogen status of both snail species (this will help develop specific pathogen free strains of P. antipodarum and L. stagnalis). In parallel, reference chemical exposure studies will be conducted for both P. antipodarum and L. stagnalis to address the impact of diet, population density and seasonality on the survival, development and reproductive performance of populations of P. antipodarum and L. stagnalis. Previous studies have shown that seasonality and environmental factors (e.g. food abundance) can significantly affect the egg laying capacity of L. stagnalis, with the highest fecundity being observed in mid-May through to mid September9. Diet and population density is also known to influence fecundity in freshwater molluscs in terms of what is often referred to as the “drying pond effect”, whereby increased crowding or reduced water quality may stimulate reproduction in some snail populations10, or reduce it in others according to ecological context11. Predictably, diet has also been shown to have a major impact on the reproductive performance and health of molluscs, while parasitism and disease status may dramatically affect fecundity in freshwater molluscan species. This is exemplified in studies using the North America freshwater pond snail Lymnaea elodes and the castrating trematode, Echinostoma revolutum12. Here researchers demonstrated that diet and infection status had a significant impact on L. elodes growth, with infected snails producing fewer eggs and tending to grow to larger sizes than uninfected snails regardless of diet. In contrast, uninfected snails displayed diet-dependent patterns of growth and reproduction. These findings support the view that molluscs are commonly infected with trematode parasites, some of which are known to disrupt a range of physiological functions, including the host endocrine system, and may be an undetected source variation when conducting reproductive toxicity studies13. Specific to this proposal, Cefas has conducted a successful pilot study investigating parasite burdens and the reproductive histology of P. antipodarum14 as part of the 2011 pre-validation work and is currently doing the same for L. stagnalis strain INRA-2011 supplied by French colleagues. Reflecting best practice in other areas of (eco)toxicology to use pathogen free strains of organisms, a key component of future work will therefore be to understand the impact of disease (pathogen) status on the use of both P. antipodarum and L. stagnalis in reproductive toxicity tests. As an internationally recognized and UK-accredited centre of expertise in invertebrate pathogen diagnostics (UKAS ISO 17025 accredited for the identification of pathogens in molluscs and crustaceans, plus GLP accredited for conducting ecotoxicology studies), Cefas has all the necessary resources and experience to successfully address this important aspect of the work13.

Secondly, experimental factors that have proven to be a challenge in the mollusc work undertaken previously needs also to be tackled in order to provide a robust basis for conducting the P. antipodarum and L. stagnalis reproduction tests. Experimental factors such as temperature and water chemistry (eg hardness) are especially relevant to applying aquatic toxicity test guidelines to a wide range of organic and inorganic chemicals, while there may be a need to use solvent carriers when conducting OECD test guidelines on organic chemicals for environmental risk assessment or water quality criteria setting. Specific priorities for study here include firstly water quality physico-chemical information (e.g. hardness and salinity) and then temperature. Here we propose to first build on our previous review work14 and conduct a mollusc species-specific literature review of the impact of these experimental variables in the context of partial and full lifecycle ecotoxicity studies, followed by targeted experimental work using the (pre-)draft OECD test guidelines for each species. For example, temperature has been shown to influence the impact bisphenol A has on the reproduction output of P. antipodarum, with a temperature dependant decrease of NOECs by a factor of four being observed15. The same study also demonstrated a clear link between temperature and the reproductive success of control groups. As discussed by Sieratowicz15 a number of different photo-periods have been used in previous studies using both P. antipodarum and L. stagnalis. However, the potential influence of this factor on reproductive output is still awaiting a systematic and thorough investigation.

Thirdly, where there are data gaps on the potential effect of important carrier solvents (often used in regulatory ecotoxicology) on the development and reproduction of P. antipodarum and L. stagnalis reproduction tests, we will conduct targeted experiments to fill these gaps. This third phase will encompass currently approved OECD solvents15, plus other solvents where there are no practical alternatives – as has recently occurred in the autumn 2011 pre-validation work with L. stagnalis exposed to tributyltin.

Objective 2 – Collaborating with international partners and coordination of UK laboratories involved in future ring tests.

Since 2009, Cefas has successfully worked with both species of relevance to this work as part of the UK and overseas consortia conducting pre-validation studies for both P. antipodarum and L. stagnalis. Cefas scientists are also active members of the OECD VMG-eco working group and related expert groups, with excellent communication in place with many scientists at the Danish, French, German and other UK laboratories cited in the Defra tender document. Through the OECD and also the UK-Japan cooperation on endocrine disrupters research, Cefas scientists also have relevant cooperation in place that can add significant value to this project in terms of mechanistic invertebrate endocrinology. Under Objective 2, Cefas will identify and approach suitable UK laboratories to participate in validation ring tests with both P. antipodarum and L. stagnalis and managing their involvement via sub-contracts. Once suitable sub-contractors have been identified and agreed with Defra, or its representatives, Cefas understands that the funds required to cover the subcontractors’ experimental work would be additional to the financial figures included in this tender. Importantly, this also reflects the need to consider the financial implications (in terms of analytical chemistry) of reference chemical selection. Specifically, one would expect there to be variable costs per P. antipodarum and L. stagnalis reproduction test in light of which type of chemicals would be used in the future ring- tests (some organic chemicals are relatively expensive to analyse (eg using LC-MS or GC-MS) compared to metals analysed using AAS or ICP-MS). As outlined in the DRP8 Cefas will ensure that work undertaken conforms to test validation principles as outlined in the OECD Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (OECD, 2005). Once initiated Cefas, would lead the technical contribution to the development of the guideline, including evaluating the results generated under objective 1 and 2 and developing a guideline draft which will be presented to the OECD for formal comment.
Time-Scale and Cost
From: 2012

To: 2015

Cost: £441,491
Contractor / Funded Organisations
Fields of Study
Chemicals and Nanotechnology