Temporal evolution of imposex and butyltin contamination in Gemophos viverratus from São Vicente (Cabo Verde) - a countercurrent trend on the world scenario.

Organotin (OT) based Antifouling Systems (AFS) were globally banned by the AFS Convention since 2008, but the Republic of Cabo Verde did not ratify this Convention, nor did it develop a national legislation to control OT-AFS. Gemophos viverratus imposex and butyltin tissue contamination were assessed around the São Vicente Island (Cabo Verde) in 2019 and compared with the data available from 2012. The vas deferens sequence index (VDSI), the relative penis length (RPL), the percentage of females with imposex (%I) and the percentage of sterilized females (%S) in 2019 ranged from 0 to 4.0, 0-84.4%, 0-100% and 0-5.1%, respectively, whilst TBT reached maximum values of ≈30 ng TBT-Sn g-1 dw in the whelk tissues. These values are very similar to those registered in 2012, which indicates that TBT pollution has not decreased over the years, in contrast to the declining trend observed worldwide.

Long-term monitoring of Nucella lapillus imposex in Ria de Aveiro (Portugal): When will a full recovery happen?

Nucella lapillus imposex has been monitored in Ria de Aveiro (Portugal) in order to understand the evolution of organotin (OT) pollution after the EU ban on OT-based antifouling systems (OT-AFS). A rapid decrease of imposex occurred, but a countercurrent tendency upsurged recently (2018-2019) with a slight increase of imposex at some sites, instead of a continuous decline towards a complete recovery. In 2018 the vas deferens sequence index ranged between 0.6 and 1.0 across stations and the percentage of females with imposex varied from 63 to 100%. In 2019 these parameters varied between 0.4 and 1.0 and 37-100%, while the concentration of butyltins (TBT, DBT, MBT) in the dogwhelk tissues were < 1 ng Sn g-1 dw. Although N. lapillus imposex levels are low they are indicative of OT pollution. The full recovery of N. lapillus imposex in the future is uncertain and a cause for concern.

Vulnerability of Tritia reticulata (L.) early life stages to ocean acidification and warming.

Ocean acidification and warming (OA-W) result mainly from the absorption of carbon dioxide and heat by the oceans, altering its physical and chemical properties and affecting carbonate secretion by marine calcifiers such as gastropods. These processes are ongoing, and the projections of their aggravation are not encouraging. This work assesses the concomitant effect of the predicted pH decrease and temperature rise on early life stages of the neogastropod Tritia reticulata (L.), a common scavenger of high ecological importance on coastal ecosystems of the NE Atlantic. Veligers were exposed for 14 days to 12 OA-W experimental scenarios generated by a factorial design of three pH levels (targeting 8.1, 7.8 and 7.5) at four temperatures (16, 18, 20 and 22 °C). Results reveal effects of both pH and temperature (T °C) on larval development, growth, shell integrity and survival, individually or interactively at different exposure times. All endpoints were initially driven by pH, with impaired development and high mortalities being recorded in the first week, constrained by the most acidic scenarios (pHtarget 7.5). Development was also significantly driven by T °C, and its acceleration with warming was observed for the remaining exposure time. Still, by the end of this 2-weeks trial, larval performance and survival were highly affected by the interaction between pH and T °C: growth under warming was evident but only for T °C ≤ 20 °C and carbonate saturation (pHtarget ≥ 7.8). In fact, carbonate undersaturation rendered critical larval mortality (100%) at 22 °C, and the occurrence of extremely vulnerable, unshelled specimens in all other tested temperatures. As recruitment cohorts are the foundation for future populations, our results point towards the extreme vulnerability of this species in case tested scenarios become effective that, according to the IPCC, are projected for the northern hemisphere, where this species is ubiquitous, by the end of the century. Increased veliger mortality associated with reduced growth rates, shell dissolution and loss under OA-W projected scenarios will reduce larval performance, jeopardizing T. reticulata subsistence.

Characterization and comparison of transcriptional activities of the retinoid X receptors by various organotin compounds in three prosobranch gastropods; Thais clavigera, Nucella lapillus and Babylonia japonica.

Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway.

Tralopyril bioconcentration and effects on the gill proteome of the Mediterranean mussel Mytilus galloprovincialis.

Antifouling (AF) systems are used worldwide as one of the most cost-effective ways of protecting submerged structures against heavy biofouling. The emergence of environmentally friendly AF biocides requires knowledge on their environmental fate and toxicity. In this study we measured the bioconcentration of the emerging AF biocide tralopyril (TP) in the Mediterranean mussel Mytilus galloprovincialis and investigated the effects of TP on the mussel gill proteome following acute (2days) and chronic (30days) exposure, as well as after a 10-day depuration period. The experiments were carried out with 1µg/L TP; blank and solvent (5×10(-5)% DMSO) controls were also included. Proteomics analysis was performed by mass spectrometry-based multidimensional protein identification technology (MudPIT). Differentially expressed proteins were identified using a label-free approach based on spectral counts and G-test. Our results show that TP is rapidly accumulated by mussels at concentrations up to 362ng/g dw (whole tissues), reaching steady-state condition within 13days. Ten days of depuration resulted in 80% elimination of accumulated TP from the organism, suggesting that a complete elimination could be reached with longer depuration times. In total, 46 proteins were found to be regulated in the different exposure scenarios. Interestingly, not only TP but also DMSO alone significantly modulated the protein expression in mussel gills following acute and chronic exposure. Both compounds regulated proteins involved in bioenergetics, immune system, active efflux and oxidative stress, often in the opposite way. Alterations of several proteins, notably several cytoskeletal ones, were still observed after the depuration period. These may reflect either the continuing chemical effect due to incomplete elimination or an onset of recovery processes in the mussel gills. Our study shows that exposure of adult mussels to sublethal TP concentration results in the bioconcentration of this biocide in the tissues and modulates the expression of several proteins that may intervene in important metabolic pathways.

LC-MS/MS determination of tralopyril in water samples.

A targeted analytical method was established to determine tralopyril (4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile) in water. This compound has been recently introduced as a biocide in ship antifouling paints, becoming a potential new environmental contaminant. The method presented here allows for the first time the direct determination of tralopyril in environmental samples without the need of a pre-concentration step. The injected sample is separated by a 30 min HPLC-gradient on a reversed phase column and the compound identified and quantified by negative ion LC-MS/MS. Tralopyril solutions in DMSO, seawater, river Glatt water and E3 medium (used for zebrafish experiments) were analysed to demonstrate the applicability of the method. The method provides good retention time reproducibility and a quantitation limit (LOQ) of 0.025 µg L(-1) for DMSO, seawater and E3 exposure medium and 0.05 µg L(-1) for river Glatt water. Calculated tralopyril half-lives were 6.1 h for seawater, 8.1 h for river Glatt water and 7.4 h for E3 medium at 18 °C.

Acute toxicity of tralopyril, capsaicin and triphenylborane pyridine to marine invertebrates.

A need for environmentally acceptable alternative antifouling (AF) biocides has arisen through restrictions in the use of many common biocides in the European Union through the Biocidal Product Regulation (Regulation EU No. 528/2012). Three such alternatives are triphenylborane pyridine (TPBP), tralopyril and capsaicin. This study aims at extending the available information on the toxicity of these three emerging AF biocides to key marine invertebrates. Here we investigate the toxicity of tralopyril and capsaicin to the early life stages of the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus and also of tralopyril, capsaicin and TPBP to the early life stages of the copepod Tisbe battagliai. The EC50 that causes abnormal development of mussel's D-veliger larvae and impairs the growth of sea urchin pluteus larvae are respectively 3.1 and 3.0 µg/L for tralopyril and 3,868 and 5,248 µg/L for capsaicin. Regarding the copepod T. battagliai, the LC50 was 0.9 µg/L for tralopyril, 1,252 µg/L for capsaicin and 14 µg/L for TPBP. The results obtained for the three substances are compared to a reference AF biocide, tributyltin (TBT), and their ecological risk evaluated. These compounds pose a lower environmental risk than TBT but still, our results suggest that tralopyril and TPBP may represent a considerable threat to the ecosystems.

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.

Combined use of Nassarius reticulatus imposex and statolith age determination for tracking temporal evolution of TBT pollution in the NW Portuguese continental shelf.

Spatial and temporal trends of tributyltin (TBT) pollution on the northern continental shelf of Portugal (5-34 m depth) were assessed using Nassarius reticulatus as a bioindicator. Imposex levels were determined in 2006 and 2010, and integrated with age readings from statoliths for a better chronological analysis of the data. Females affected with imposex were detected in 84% of the sites surveyed in 2006, with levels higher than the Ecological Quality Objective (EcoQO) set by the OSPAR Commission for N. reticulatus (VDSI < 0.3) in approximately half of those sites. In 2010, imposex was only detected in 20% of the surveyed sites and, when detected, the VDSI were lower than the EcoQO, except for one location. The comparison between the current results and the levels previously reported for 2004 and 2005 in the same offshore area evidences a clear decline of imposex in 2010 that confirms the efficacy of Regulation (EC) No. 782/2003 and the IMO AFS-Convention (entered into force in 2003 and 2008, respectively). Moreover, the age of the whelks used in the monitoring is estimated in 3-5 years, indicating that actual TBT water contamination in 2010 may in fact be lower than that extrapolated from the imposex levels. We conclude that the legislation was indeed very effective in reducing TBT pollution in the NW Portuguese continental shelf, not only in terms of the magnitude but also in terms of fast field repercussion.

Hydrobia ulvae imposex levels at Ria de Aveiro (NW Portugal) between 1998 and 2007: a counter-current bioindicator?

Imposex expression in prosobranch gastropods has been widely used as a biomarker of tributyltin (TBT) pollution. Estuaries have been described as the most affected areas by this problem since they usually enclose the main TBT sources--ports, dockyards and marinas--resulting from the compound's application as a biocide in antifouling paints on ships. Using Hydrobia ulvae as a bioindicator, the current work addresses the most reliable methods to reduce the influence of critical variables, such as the animals' size, on imposex levels assessment for TBT pollution monitoring and presents its temporal trends from 1998 to 2007 in Ria de Aveiro (NW Portugal) to evaluate the effectiveness of recent legislation applied to reduce TBT environmental levels. H. ulvae imposex levels did not decrease in this estuarine system during the last decade despite the implementation of the EU Regulation No. 782/2003. Instead, there was a global significant increase in the percentage of females affected by imposex and a slight increase of the vas deferens sequence index (VDSI), contrasting with what has been described for other bioindicators in the same study area. These results show that different biology/ecology traits determine distinct routes of TBT uptake and/or bioaccumulation, pointing the importance of choosing the bioindicator depending on the compartment that is being monitored (sediment vs. water). Sediment ingestion as feeding habit is discussed and pointed as a reason to choose H. ulvae as a bioindicator of TBT pollution persistence in sediment. It is therefore predicted that the response of different prosobranch species around the world may diverge according to the compartment that is being monitored and that female masculinisation may not be completely eradicated in the near future due to TBT persistence in sediments.

Factors affecting RPSI in imposex monitoring studies using Nucella lapillus (L.) as bioindicator.

Nucella lapillus (L.) is a marine gastropod mollusc widely used as a bioindicator of TBT pollution in the North Atlantic coastlines. The species reproductive cycle and the male penis length seasonal/spatial variation were studied at a single site at Aveiro seashore (NW Portugal) between December 2005 and June 2007. The main objective of this work is to assess if the "Relative Penis Size Index" (RPSI)--an important imposex assessment index--varies seasonally and spatially in the same sampling site and how this can affect results obtained in imposex monitoring programmes. Animals able to reproduce were found every month but a seasonal pattern in N. lapillus reproductive cycle was evident. Female gametogenic maturation varied seasonally and a decrease in capsule gland volume and condition factor occurred in late summer/early autumn. The gametogenesis in males did not show a significant seasonal variation as in females but the condition factor, penis length, amount of sperm and prostate volume also diminished in late summer/early autumn. On the other hand, males that were close to egg capsules clusters had larger penises than those far away from clusters. The temporal and spatial male penis length variation introduces a bias on imposex assessment results when using RPSI and the magnitude of the error involved is evaluated for different TBT pollution levels scenarios. We consider that RPSI provides interesting and complementary information that should not be excluded from monitoring programmes, but temporal or spatial comparisons of imposex should be based on other more reliable imposex indices like the VDSI.

Occurrence and sources of selected phenolic endocrine disruptors in Ria de Aveiro, Portugal.

BACKGROUND, AIM AND SCOPE: Ria de Aveiro (Portugal) is a shallow coastal lagoon of high economic and ecological importance. Hardly any data on its chemical pollution by polar organic pollutants are available in literature. This study focused on the presence and sources of a series of phenolic endocrine-disrupting compounds (EDCs) in this area, including parabens, alkylphenolic compounds and bisphenol-A (BPA). A number of possible sources of pollution are present in the area, including the large harbours present in the lagoon, the city of Aveiro and the rivers discharging into the area. A recently constructed submarine wastewater outfall, located a few kilometres from the lagoon inlet has also been suggested as a possible source of pollution to Ria de Aveiro in several publications. The aim of the current field study was to investigate the occurrence and main sources of phenolic endocrine disruptors in Ria de Aveiro. MATERIALS AND METHODS: An extensive sampling campaign was performed, with surface water and wastewater grab samples taken at over 50 locations, in duplicate on different days. Samples were treated using solid phase extraction and analysed by liquid chromatography tandem mass spectrometry. RESULTS AND DISCUSSION: Concentrations in lagoon water were generally low: not exceeding 20 ng/L for most analytes. Levels in river water exceeded those in the lagoon by a factor 3 to 500 (o-phenylphenol (PhP) and nonylphenoxy ethoxy acetic acids (A9PEC), respectively), with concentrations up to 700 ng/L for BPA and 7,300 ng/L for A9PEC. Samples from the harbours showed EDC levels similar to those in the rest of the lagoon, but in the city of Aveiro, elevated concentrations were observed for alkylphenol ethoxylates (A9PEO), A9PEC, PhP and BPA. Wastewater effluents showed low levels for parabens, whilst alkylphenolic compounds reached several micrograms per litre. The effluents are discharged into the ocean via a submarine outfall, but as marine water near the outfall showed slightly elevated concentrations only for A9PEO, it does not seem to be a significant source of these EDCs for the area. CONCLUSIONS: All the studied phenolic EDCs were detected in the study area, with high levels found in some of the rivers discharging into the lagoon, and generally low concentrations in the lagoon itself. The main sources for all investigated EDCs were the rivers Caster and Antuã which discharge into the lagoon. The city of Aveiro was identified as a secondary source. As the tidal water exchange volume is much larger than the freshwater input from the rivers, concentrations of phenolic EDCs remained low in the lagoon.

Chemical and biological characterization of estrogenicity in effluents from WWTPs in Ria de Aveiro (NW Portugal).

Effluents from wastewater treatment plants (WWTPs) are responsible for the input of estrogenic contaminants into aquatic ecosystems, leading to widespread effects in wildlife. In the present work, levels of estrone (E1), 17alpha- and 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2), bisphenol A (BPA), and nonylphenol (NP) were quantified in effluents from WWTPs located in Ria de Aveiro (NW Portugal), as well as in the final effluent discharged into the Atlantic Ocean through the S. Jacinto submarine outfall. Reference sites, located at the entrance of the estuarine system and at the seaside, were also included. Samples were collected under summer (June 2005) and winter (February 2006) conditions. For the summer survey samples, estrogenicity and androgenicity were evaluated using the yeast estrogen screen (YES) and the yeast androgen screen (YAS) assay. Estrone levels varied from 0.5 to 85 ng/L in the summer survey and between

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.

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.