REPORT to Defra 2007 UK‐wide survey of TBT‐induced imposex in dogwhelks (Nucella lapillus). Contributors: John Thain, Centre Environment Fisheries and Aquaculture Science (Cefas), Burnham Laboratory, Remembrance Avenue, Burnham‐on‐Crouch, Essex, UK. CM0 7AH. Mathew Gubbins, Fisheries Research Services (FRS), Marine Laboratory, 375 Victoria Road, Aberdeen, UK. AB11 9DB. Lynn McIlroy, Environment and Heritage Service (EHS), 17, Antrim Road, Lisburn, Northern Ireland, UK. BT28 3BT Brian Miller, Scottish Environment Protection Agency (SEPA), Marine Science, 5 Redwood Crescent, Peel Park, East Kilbride, UK. G74 5PP. 1. Introduction Imposex in gastropods is the imposition of male sexual organs in female whelks and periwinkles and has been found to be a very sensitive indicator of TBT exposure, the effect is dose related and severe imposex can lead to sterility in females and detrimental reproductive effects on individuals and populations. TBT‐specific biological effects monitoring was first established in the mid 1980s when thickening of shells of the Pacific Oysters (Crassostrea gigas) and the development of imposex in dogwhelks (Nucella lapillus) and periwinkles (Littorina littorea) was attributed to this compound (Waldock and Thain, 1983, Gibbs and Bryan, 1986, Thain and Waldock,1986, and Gibbs et al 1987). The data collected by 1987 using these two species provided the evidence for environmental damage and subsequently, led to a UK ban on the use of TBT on small boats and in aquaculture. In 1989, the European Union imposed a similar ban (EU, Council Directive (76/769/EEC). Over the past fifteen years extensive surveys have been conducted to measure the prevalence of imposex in the UK. In 1992, in preparation for the 1993 North Sea Quality Status Report the Fisheries Research Services (FRS), Aberdeen, conducted a survey around the North Sea. To compliment this study a further survey was conducted using a similar sampling strategy, by laboratories in the countries around the Celtic Sea with the added emphasis on including some ʹhot spotʹ monitoring. In 1998, Defra funded FRS to conduct a further North Sea study that included some TBT chemical analysis of the flesh of animals. Under the OSPAR JAMP (Oslo Paris Commission Joint Assessment Monitoring Programme), the UK has an obligation to report data on the incidence of imposex in UK waters. It is included in the OSPAR CEMP (Coordinated Environmental Monitoring Programme) and is category 1 rated i.e. is mandatory since it is fully supported by Analytical Quality Control (AQC) procedures and assessment criteria. AQC procedures have been developed through QUASIMEME and BEQUALM for the measurement of imposex with regular intercalibration studies being conducted Europe wide. OSPAR guidelines for TBT‐specific biological effects monitoring are now firmly in place, including assessment criteria. An International Maritime Organisation (IMO) International Convention on the Control of Harmful Anti‐fouling Systems agreed at a Diplomatic Conference, in October 2001, to prohibit the application or re‐application to ships of organostanic compounds as biocides in antifouling systems from 1st January 2003. This has now been implemented in the EU by Council Directive 2002/62/EC and, in the UK, approval for use of organostanic compounds acting as biocides in antifouling systems, granted under the Food and Environment Protection Act (FEPA) or the Control of Pesticides Regulations (COPR), have now been revoked. More recently the IMO ban has included the prohibition of use (or presence) on any ship’s hull and this will take effect from 1st September 2008. In the light of the EU ban and the revocation of approvals in the UK for the application of TBT, a benchmark survey of the effects and residual concentrations of TBT in UK waters was conducted UK‐wide in 2004. This survey was conducted by the Contracting Monitoring Authorities (CMAs) within the UK CSEMP (Clean and Safe Seas Environmental Monitoring Programme) funded by Defra. This data was to provide a baseline for further monitoring(trends), and provide the basis for a UK‐wide strategy to be developed following the total prohibition of use on the hull of any ship. The OPSAR strategy for the monitoring of imposex states that the measurements should be conducted at least three yearly, and in this respect a repeat survey is reported here for 2007. The aims of the 2007 survey were to provide; • an indication of the effectiveness of the IMO ban following the implementation of EU Council Directive 2002/62/EC. • comply with the requirements of OSPAR • provide important data for WFD purposes • and allow the data to be ready and assessed for reporting at the next OSPAR QSR(Quality Status Report) and CP2 (Charting Progress 2). 2. Consortium Approach The 2004 UK‐wide survey of imposex in whelks was conducted by CMAs within the CSEMP (previously UK National Marine Monitoring Programme [NMMP]): Centre for Environment Fisheries and Aquaculture Science (Cefas); Fisheries Research Services (FRS, Aberdeen); Scottish Environmental Protection Agency (SEPA, East Kilbride); and Environment and Heritage Service DOE NI (EHS, Lisburn). This was appropriate because the skill base existed within the NMMP CMAs, all were experienced and compliant with Analytical Quality Control (AQC) procedures via QUASIMEME, some CMAs were conducting regular localised surveys and could contribute to the sampling and design of the programme and all were a part of the CSEMP which facilitated the submission of data to the International Council for the Exploration of the Seas (ICES) data base and assessment by the Oslo Paris Commission ( OSPAR). The 2004 survey was successfully completed using this consortium of CMAs and it was therefore felt appropriate to approach this survey in 2007 in the same manner. The programme was conducted by the consortium with each CMA responsible for conducting the survey in each of their regions. Regular meetings of the consortium partners were held, prior to, during and post sampling in order to ensure that the programme was meeting its objectives. 3. Objectives The objectives of the programme were; Objective 1. To conduct UK‐wide imposex/intersex surveys in accordance with the OSPAR guidelines at shoreline sites and major ports previously surveyed in the UK in 2004. Objective 2. Compare 2007 data with previous surveys in order to establish the extent of improvement in levels of imposex achieved by the controls on TBT and to establish a baseline for future monitoring following the implementation of IMO ban implemented by the EU by council Directive 2002/62/EC. Objective 3. To assess the data collected, and to report on the findings (and make these available for OSPAR, WFD purposes and QSR) and to advise on the development of the UK strategy for TBT monitoring in future. 4. Method 4.1 Survey design One of the major aims of the survey was to measure imposex in dogwhelks and to assess if there had been changes in imposex since the previous survey in 2004. Therefore it was imperative that stations sampled in 2004 were again sampled in 2007 and this to include the site of special interest at Sullom Voe in Scotland. In addition, sites were also identified which provided information on important hot spots not covered in the 2004 survey and for sites in England and Wales where there were Water Framework Directive (WFD) TBT issues. Each CMA took responsibility for sampling in its region, and the OSPAR sampling strategy and analysis was adhered to. 4.2 Sampling The protocol followed that described by OSPAR (2002). Dogwhelks were sampled during low tide, by hand, from the shoreline between spring low water and mid‐tide levels with sample sizes of 40 specimens per station. Specimens were collected that were sexually mature and yet not too old, in order to minimize former and possibly higher contamination levels. This involved collecting specimens of around 12 to 25 mm in shell height and that dogwhelks were toothed. At the time of collection supporting information was collected, this included, the time taken to collect the 40 individuals in order to give an indication of species abundance and the presence of juveniles or egg capsules was also noted as this can be a valuable indication of continued reproductive activity in populations. After collection the 40 individuals were transported live to the laboratory in cool damp conditions and assessed for imposex within seven days of collection. 4.3 Determination of imposex At the laboratory Nucella, were examined live, without narcotisation. Before breaking the shell with a vice, individual measurements for shell heights were measured to the nearest 0.1 mm with a vernier calliper and recorded. Specimens parasitised by trematodes or other endoparasites were excluded from further analyses. Under the microscope in imposex‐affected species the following observations and measurements (with an accuracy for organ extensions of 0.1 mm) were undertaken, according to the OSPAR guideline described below (see also Oehlmann et al., 1991): a. the imposex stage for each female, and b. the length of penis in Nucella males and females (if present). Measurement of penis length in Nucella should be done immediately after removing it from the shell, as the penis enlarges after its death. On the basis of these measurements the following calculations of imposex intensities and indices of possible interference with the reproductive capability in prosobranch populations was calculated: a. Vas Deferens Sequence Index (VDSI) in Nucella, as the mean value of the imposex stages in a population: b. the proportion of sterile female Nucella; After all the individuals were measured the Vas deferens sequence index (VDSI) was calculated to assess the OSPAR class for each site, (Table 4.3.1.), OSPAR (2004)). Vas Deferens Sequence Index (VDSI): = Sum of VDS values of all females sampled / Number of females sampled. VDSI values > 4,0 indicate that at least some of the females in the sample are sterilised due to imposex development; Table 4.3.1.