Isabel Oliveira

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.

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.

Biological variables and health status affecting inorganic element concentrations in harbour porpoises (Phocoena phocoena) from Portugal (western Iberian Peninsula).

The coastal preferences of harbour porpoise (Phocoena phocoena) intensify their exposure to human activities. The harbour porpoise Iberian population is presently very small and information about the threats it endures is vital for the conservation efforts that are being implemented to avoid local extinction. The present study explored the possible relation between the accumulation of trace elements by porpoises and their sex, body length, nutritional state, presence of parasites and gross pathologies. The concentrations of arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb), zinc (Zn) and selenium (Se) were evaluated in 42 porpoises stranded in Portugal between 2005 and 2013. Considering European waters, porpoises stranded in Portugal present the highest Hg concentrations and the lowest Cd concentrations, which may reflect dietary preferences and the geographic availability of these pollutants. While no effect of sex on trace element concentrations was detected, there was a positive relationship between porpoise body length and the concentration of Cd, Hg and Pb. Animals in worse nutritional condition showed higher levels of Zn. Harbour porpoises with high parasite burdens showed lower levels of Zn and As in all analysed tissues and also lower levels of renal Ni, while those showing gross pathologies presented higher Zn and Hg levels. This is the first data on the relationship between trace elements and health-related variables in porpoises from southern European Atlantic waters, providing valuable baseline information about the contamination status of this vulnerable population.

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.

Toxicity of emerging antifouling biocides to non-target freshwater organisms from three trophic levels

Antifouling (AF) systems provide the most cost-effective protection against biofouling. Several AF biocides have, however, caused deleterious effects in the environment. Subsequently, new compounds have emerged that claim to be more environment-friendly, but studies on their toxicity and environmental risk are necessary in order to ensure safety. This work aimed to assess the toxicity of three emerging AF biocides, tralopyril, triphenylborane pyridine (TPBP) and capsaicin, towards non-target freshwater organisms representing three trophic levels: algae (Chlamydomonas reinhardtii), crustacean (Daphnia magna) and fish (Danio rerio). From the three tested biocides, tralopyril had the strongest inhibitory effect on C. reinhardtii growth, effective quantum yield and adenosine triphosphate (ATP) content. TPBP caused sub-lethal effects at high concentrations (100 and 250μgL-1), and capsaicin had no significant effects on algae. In the D. magna acute immobilisation test, the most toxic compound was TPBP. However, tralopyril has a short half-life and quickly degrades in water. With exposure solution renewals, tralopyrils toxicity was similar to TPBP. Capsaicin did not cause any effects on daphnids. In the zebrafish embryo toxicity test (zFET) the most toxic compound was tralopyril with a 120h - LC50 of 5μgL-1. TPBPs 120h - LC50 was 447.5μgL-1. Capsaicin did not cause mortality in zebrafish up to 1mgL-1. Sub-lethal effects on the proteome of zebrafish embryos were analysed for tralopyril and TPBP. Both general stress-related and compound-specific protein changes were observed. Five proteins involved in energy metabolism, eye structure and cell differentiation were commonly regulated by both compounds. Tralopyril specifically induced the upregulation of 6 proteins implicated in energy metabolism, cytoskeleton, cell division and mRNA splicing whilst TPBP lead to the upregulation of 3 proteins involved in cytoskeleton, cell growth and protein folding. An ecological risk characterization was performed for a hypothetical freshwater marina. This analysis identified capsaicin as an environment-friendly compound while tralopyril and TPBP seem to pose a risk to freshwater ecosystems. Noneless, more studies on the characterization of the toxicity, behaviour and fate of these AF biocides in the environment are necessary since this information directly affects the outcome of the risk assessment.

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.

Bioaccumulation of trace element concentrations in common dolphins (Delphinus delphis) from Portugal

The common dolphin (Delphinus delphis) is one of the most abundant species in Atlantic Iberia, representing a potentially important tool to assess the bioaccumulation of trace elements in the Iberian marine ecosystem. Nine elements (As, Cd, Cu, Hg, Mn, Ni, Pb, Se and Zn) were evaluated in 36 dolphins stranded in continental Portugal. Dolphins had increasing Hg concentrations (16.72μg·g-1 ww, liver) compared with previous studies in Atlantic Iberia, whereas Cd concentrations (2.26μg·g-1 ww, kidney) fell within reported ranges. The concentrations of some trace elements (including Cd and Hg) presented positive relationships with dolphin length, presence of parasites and gross pathologies. Common dolphins may help biomonitoring more offshore Atlantic Iberian areas in future studies, which would otherwise be difficult to assess.

Nanoparticles-Based Systems for Osteochondral Tissue Engineering

Osteochondral lesions represent one of the major causes of disabilities in the world. These defects are due to degenerative or inflammatory arthritis, but both affect the articular cartilage and the underlying subchondral bone. Defects from trauma or degenerative pathology frequently cause severe pain, joint deformity, and loss of joint motion. Osteochondral defects are a significant challenge in orthopedic surgery, due to the cartilage complexity and unique structure, as well as its exposure to high pressure and motion. Although there are treatments routinely performed in the clinical practice, they present several limitations. Tissue engineering can be a suitable alternative for osteochondral defects since bone and cartilage engineering had experienced a notable advance over the years. Allied with nanotechnology, osteochondral tissue engineering (OCTE) can be leveled up, being possible to create advanced structures similar to the OC tissue. In this chapter, the current strategies using nanoparticles-based systems are overviewed. The results of the studies herein considered confirm that advanced nanomaterials will undoubtedly play a crucial role in the design of strategies for treatment of osteochondral defects in the near future.