Carlos M. Barroso

Imposex and organotin prevalence in a European post-legislative scenario: temporal trends from 2003 to 2008.

Imposex levels and organotin tissue concentrations were assessed in Nassarius reticulatus females collected between June and August 2008 at 23 sites along the Portuguese coast. Females with imposex were present at all sampling sites with highest levels inside main harbors. Imposex parameters across sampling stations varied between 6.3% and 100% for %I, 0.2-4.4 for VDSI, 0.1-7.8mm for FPL, 0.3-88.9% for RPLI and 0.0-1.1 for AOS. TBT levels varied between 3.5 and 380 ngSng(-1) dw, representing an average proportion of 50.4% of total butyltins (summation operator BTs=MBT+DBT+TBT). Sterile females were detected at two locations. Highly significant correlations between imposex and TBT levels were found. The efficacy of the EU legislation banning the use of TBT-based antifouling paints since 2003 (EC Regulation 782/2003) was evaluated by comparing the levels of imposex observed in 2008 with those reported for 2003. OTs tissue concentrations were also determined in preserved samples collected in 2003. There was a decrease in imposex and TBT tissue contamination between 2003 and 2008 indicating that a decline in TBT pollution has occurred in the Portuguese coast since the implementation of the legislation. Considering that the EC Regulation 782/2003 is an anticipation of the IMO AFS Convention, a global scale decrease in TBT pollution can be expected in the near future. Despite the rapid amelioration in the Portuguese coast, TBT pollution is still a problem as the Ecological Quality Objective (EcoQO) proposed by OSPAR Commission was not achieved in 91% of the surveyed sites.

Spatial and temporal changes of TBT pollution along the Portuguese coast: inefficacy of the EEC directive 89/677.

Nucella lapillus imposex (superimposition of male characters onto prosobranch females) and organotin body burden were surveyed in the Portuguese coast in 1997 and 2000. Time comparisons of the available data on Nucella lapillus imposex in the Portuguese coast indicate a global increase of TBT pollution in recent years, revealing the inefficacy of the 1993 legislation that bans the use of TBT paints on small boats (<25 m). The absence or scarcity of the species inside harbours, which are the most polluted sites in Portugal, is probably a consequence of extinction due to female sterilisation by TBT.

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel, Nucella lapillus.

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this ‘nonmodel’ species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator–activated receptor (PPAR) pathways. Application of rosiglitazone, a well‐known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT‐induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR‐responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.

Reproductive Cycle of Nassarius Reticulatus in the Ria De Aveiro, Portugal: Implications for Imposex Studies

The reproductive cycle of Nassarius (Hinia) reticulatus was investigated in the Ria de Aveiro on the north-west Portuguese coast during 1992 and 1993. A clear seasonal pattern was observed with the beginning of gametogenesis in August followed by ripening of the gonads in January and February and spawning from February to July. The increase of water temperature above 11°C in February 1993 was associated presumably with the release of gametes. Recruitment inside the Ria of juveniles with 6·5 mm mean shell height was detected in February 1993 and these individuals were spawned in the previous year. Males of N. reticulatus exhibited a considerable variation in length of the penis throughout the reproductive cycle. The accuracy of the use of relative penis length (RPL) index for intersite comparisons of imposex is discussed and the magnitude of the error that might be involved when comparisons are made using populations in different phases of the reproductive cycle is evaluated.

Distribution of synthetic organotins and total tin levels in Mytilus galloprovincialis along the Portuguese coast

Despite the huge amount of literature available on butyltins (BuTs), few studies addressed the environmental levels of phenyltins (PhTs), octyltins (OcTs) and total tin (Sn(T)) in environmental samples. In 2006 a mussel watch survey was developed for the Portuguese coast (total of 29 sampling sites) in order to describe the concentrations of BuTs , PhTs, OcTs and Sn(T) in the whole tissues of Mytilus galloprovincialis (Lamarck, 1819). BuTs were detected in all analyzed samples accounting, in average, for 98.6% of total organotins (Sigma OTs=BuTs+PhTs+OcTs), and presented highest values in the vicinity of harbors. Tributyltin (TBT) was the dominant butyltin, representing, in average, 62% of summation operatorBuTs (Sigma BuTs=TBT+DBT+MBT) suggesting that fresh inputs of TBT are still occurring in the Portuguese coast, particularly near harbors. The contribution of organotin compounds derived from antifouling paints to the total tin levels in M. galloprovincialis is discussed.

Imposex levels and tributyltin pollution in Ria de Aveiro (NW Portugal) between 1997 and 2007: evaluation of legislation effectiveness

Nucella lapillus imposex levels and organotin (OT) concentrations in water and female tissues were measured in samples collected from the Ria de Aveiro (NW Portugal) between 1997 and 2007. Vas deferens sequence index (VDSI), relative penis size index (RPSI), mean female penis length (FPL) and percentage of imposex affected females (%I) were used to determine imposex levels at each site. A significant temporal decline in imposex intensity was observed during the assessed period. Imposex decrease was evident after 2003 although improvements were most notable from 2005 to 2007, probably due to the implementation of the EU Council Regulation no.782/2003 forbidding further application of tributyltin (TBT) antifouling on vessels carrying EU flags. Despite these improvements, OT analysis in N. lapillus female tissues and water indicate there are still recent TBT inputs into the study area.

Spatial and temporal evolution of imposex in dogwhelk Nucella lapillus (L.) populations from North Wales, UK

Nucella lapillus imposex and organotin tissue contamination were assessed, during 2006, at twenty sites in North Wales, between Anglesey and Shell Island on the Lleyn Peninsula. Vas Deferens Sequence Index (VDSI), Relative Penis Size Index (RPSI) and the percentage of affected females (%I) were used to assess imposex levels which varied between 0.5 and 3.8 for VDSI, 0.0 and 11.5% for RPSI and 49 and 100% for %I. Tributyltin (TBT) and triphenyltin (TPT) concentrations in whole tissues varied between 0.8 and 39 and 0.4 and 2.1 ng Sn/g dry weight, respectively. TBT represented the higher fraction of butyltin compounds in the tissues, suggesting that TBT inputs continue to occur. Comparisons with nineteen years of data collected during previous studies demonstrated that there had been a significant reduction in imposex levels over the last two decades following the introduction of legislative restrictions in the U.K. regarding the use of organotin based antifouling paints.

Assessment of inshore/offshore tributyltin pollution gradients in the northwest portugal continental shelf using Nassarius reticulatus as a bioindicator

Imposex and organotin tissue contamination were assessed in Nassarius reticulatus (L.) populations in the northwest Portuguese continental shelf between 2002 and 2005 over an area of 735 km2, involving 366 sampling sites. The objective was to evaluate the dispersion of tributyltin (TBT) from inshore sources inside the Ria de Aveiro estuary into the adjacent deeper sea and to assess endocrine disruption in the netted whelk N. reticulatus, using imposex as a biomarker. The highest levels of TBT tissue contamination and imposex were found in the whelks inside the Ria de Aveiro, and these declined logarithmically with distance from the mouth of this estuary. Remarkably, we found that offshore populations were also extensively affected: TBT (the dominant organotin) tissue concentration was above the detection limits at all sites where whelks were analyzed (12-356 ng TBT-Sn/g dry wt), and imposex occurred at 80% of the sampling stations, with relatively high values at some sites from the deepest area surveyed. This work shows clearly that TBT pollution is not restricted to the Ria de Aveiro but affects a significant part of the adjacent continental shelf as well. The ecological impacts of TBT pollution on offshore ecosystems are discussed.

Inshore/offshore gradients of imposex and organotin contamination in Nassarius reticulatus (L.) along the Portuguese coast.

Imposex and organotin (OT) tissue contamination of the netted whelk Nassarius reticulatus (L.) were assessed in the continental shelves around the main estuaries of the central coast of Portugal (Lisbon: Tagus estuary; Setúbal: Sado estuary) and the main coastal lagoon in the Southern of Portugal (Faro: Ria Formosa). Pollution levels were higher in areas of more intense boat traffic and shipyard activities and imposex showed a clear decreasing gradient from the estuaries to the offshore, in relation to a similar gradient of tissue contamination by tributyltin. Remarkably, imposex was extensively spread over the adjacent continental shelves of Tagus and Sado estuaries. The current work shows that TBT pollution is undoubtedly a matter of concern not only for the above estuaries where harbours are implanted but also for the adjacent continental shelves, regardless the massive dilution of contaminants that may occur in these deeper areas.

Organotin levels in Nazaré canyon (west Iberian Margin, NE Atlantic) and adjacent coastal area

Organotin compounds (OTs) are ubiquitous in the marine environment and high concentrations (μg g(-1) range) in sediments from different coastal areas around the world have been reported. However, few reports have described the OTs contamination status in the offshore and deep sea environment. This work investigated organotin levels in Nazaré canyon for the first time. Levels of monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), diphenyltin (DPT), triphenyltin (TPT), dioctyltin (DOT) and trioctyltin (TOT) were quantified in sediment samples from the upper flanks of the canyon and from the adjacent coastal area. TBT levels detected in the canyon flanks are about two to three orders of magnitude lower than those found in the coastal area. Nevertheless, when quantifiable, TBT levels in the canyon samples were higher than the Environmental Assessment Criteria set for TBT in sediments by the OSPAR Commission indicating that at those locations negative ecological impacts are likely to occur.