Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

New insights on the impacts of e-waste towards marine bivalves: The case of the rare earth element Dysprosium.

With the technological advances and economic development, the multiplicity and wide variety of applications of electrical and electronic equipment have increased, as well as the amount of end-of-life products (waste of electrical and electronic equipment, WEEE). Accompanying their increasing application, there is an increasing risk to aquatic ecosystems and inhabiting organisms. Among the most common elements present in WEEE are rare earth elements (REE) such as Dysprosium (Dy). The present study evaluated the metabolic and oxidative stress responses of mussels Mytilus galloprovincialis exposed to an increasing range of Dy concentrations, after a 28 days experimental period. The results obtained highlighted that Dy was responsible for mussel's metabolic increase associated with glycogen expenditure, activation of antioxidant and biotransformation defences and cellular damage, with a clear loss of redox balance. Such effects may greatly impact mussel's physiological functions, including reproduction capacity and growth, with implications for population conservation. Overall the present study pointed out the need for more research on the toxic impacts resulting from these emerging pollutants, especially towards marine and estuarine invertebrate species.

Contaminants bioaccumulation and pathological assessment in Mytilus galloprovincialis in coastal waters facing the brownfield site of Bagnoli, Italy.

We studied the bioaccumulation of metals and PAHs, the pathological conditions, regressive phenomena and pathogens in wild Mytilus galloprovincialis taken along the North Pier facing the former second Italian largest steelworks of Bagnoli. There was no Cd and Pb bioaccumulation with respect to the EU role 221/2002. Metal shell index decreased as follows: Pb > Ni > Zn > Cu and correlates with the pollution state. The level of BaP was up to thirtysixfold higher the EU rule 835/2011. The sum of 4 hydrocarbons, PAH4, were up to seventeen-fold the rule. PAH levels increased toward the coast. Prevalence values of tissue necrosis and inflammatory lesions were between 50 and 100%. In May animals showed lesion like granulocytomas and inflammatory capsules. Signs of atresia, necrotic oocytes and diffused cases of hermaphroditism were detected. An appropriate localization of farming to avoid contamination from sediment turbulence and risks for consumer health is needed.

The impact of propranolol, 17α-ethinylestradiol, and gemfibrozil on early life stages of marine organisms: effects and risk assessment.

Pharmaceuticals are ubiquitously detected in the marine environment at the ng-µg/L range. Given their biological activity, these compounds are known to induce detrimental effects on biota at relatively low exposure levels; however, whether they affect early life stages of marine species is still unclear. In this study, a set of bioassays was performed to assess the effects of propranolol (PROP), 17-α ethinylestradiol (EE2), and gemfibrozil (GEM) on gamete fertilization and embryonic development of mussels (Mytilus galloprovincialis) and sea urchins (Paracentrotus lividus), and on the survival of seabream (Sparus aurata) larvae. Treatments of PROP (500, 5000, 50,000 ng/L), EE2 (5, 50, 500 ng/L), and GEM (50, 500, 5000 ng/L) were selected to encompass levels comparable or superior to environmental concentrations. Obtained data were tested for dose-response curve fitting and the lowest EC10/LC10 used to calculate risk quotients (RQs) based on the MEC/PNEC. No alteration was induced by PROP on the mussel gamete fertilization, while inhibitory effects were observed at environmental levels of EE2 (500 ng/L) and GEM (5000 ng/L). Fertilization was significantly reduced in sea urchin at all PROP and EE2 dosages. The 48-h exposure to all pharmaceuticals induced the onset of morphological abnormalities in either mussel or sea urchin embryos. Alterations were generally observed at environmentally relevant dosages, except for PROP in mussels, in which alterations occurred only at 50,000 ng/L. A decreased survival of seabream larvae was recorded after 96-h exposure to PROP (all treatments), EE2 (50-500 ng/L), and GEM (500 ng/L). A median RQ > 1 was obtained for all pharmaceuticals, assigning a high risk to their occurrence in marine environments. Overall, results showed that current levels of contamination by pharmaceuticals can impact early stages of marine species, which represent critical junctures in the resilience of coastal ecosystems.

Marine environmental risk assessment and acute water quality criterion for pentachlorophenol in coastal waters.

Pentachlorophenol (PCP) is a organochlorine biocide that, unlike most other organochlorines, is still in use as timber preservative. Its water solubility, high toxicity, bioaccumulation potential, and the concentrations reported in estuarine waters (up to 0.1 µg L-1) indicate it may pose a risk in coastal environments. Aquatic environrmental regulations are commonly based on standard freshwater organisms that may not represent the sensitivity of marine species. The present study consists of a water quality criteira reevalutation of PCP in coastal waters based on toxicity tests conducted recording sensitive endpoints of marine species representative of coastal ecosystems, following QA/QC standard procedures. The toxicity thresholds (EC10) found were 4.69 µg L-1 for Paracentrotus lividus sea-urchin embryos, 6.47 µg L-1 for Mytilus galloprovincialis mussel larvae, and 78.4 µg L-1 for Isochrysis galbana cells. Therefore, there is only one order of magnitude between the predicted no-effect concentration (PNEC) for early life stages of bivalves and echinoderms and the maximum concentrations actually recorded in coastal water, which yields a remarkable risk quotient for PCP in these highly productive marine habitats. In addition, we have reviewed the ecotoxicological data on PCP toxicity on marine species representative of the main systematic groups, from algae to chordates, and derived a probabilistic acute saltwater quality criterion of 2.66 µg L-1, intended to protect 95% of the marine species. Lack of adequate protection for marine ecosystems in some current PCP national guidelines has been identified.

Risk assessment of some heavy metals in mussels (Mytilus galloprovincialis) and veined rapa whelks (Rapana venosa) for human health.

The purpose of the present study was to analyze the concentrations of lead, cadmium and mercury in mussels (Mytilus galloprovincialis) and veined rapa whelks (Rapana venosa) caught in the Varna Bay of Black Sea and to evaluate the risk for human health from the presence of the three heavy metals. The highest average concentrations in mussels were those of cadmium (0.280 mg/kg), followed by lead (0.251 mg/kg) and mercury (0.017 mg/kg). Veined rapa whelks also showed highest levels of cadmium (1.113 mg/kg), followed by lead (0.045 mg/kg) and mercury (0.034 mg/kg). EDI values for adults consuming mussels and veined rapa whelks were below the published RfDo and PTWI values. All THQ and HI values were below 1. The consumption of M. galloprovincialis and R. venosa caught in the Varna Bay, Black Sea, did not pose any risk for the health of adult people as lead, cadmium and mercury were concerned.

Assessment of the effects of sulfated polysaccharides extracted from the red seaweed Irish moss Chondrus crispus on the immune-stimulant activity in mussels Mytilus spp.

Seaweeds contain a number of health enhancing and antimicrobial bioactive compounds including sulfated polysaccharides (SP). In the present study, SP extracted from a European red seaweed Irish moss Chondrus crispus was chemically analyzed, SP content extracted and the immune-response effect on wild Irish mussels Mytilus spp. investigated for the first time. A high percent yield of SP was extracted from C. crispus and the immune-stimulant activity of SP was assessed in a laboratory trial with mussels exposed to three different treatments of low (10 µg mL-1), medium (20 µg mL-1) and high (50 µg mL-1) SP dose concentrations and a control mussel group with no exposure to SP. An initial mussel sample was processed prior to the trial commencing and mussels were subsequently sampled on Days 1, 2, 3, 4, 7, and 10 post SP exposure. Both cell, humoral and immune related gene responses including haemocyte cell viability, haemocyte counts, lysozyme activity and expression of immune related genes (defensin, mytimycin and lysozyme mRNA) were assessed. No mussel mortalities were observed in either the treated or non-treated groups. Mussels exposed with SP showed an increase in haemocyte cell viability and the total number of haemocytes compared to control mussels. Lysozyme activity was also higher in treated mussels. Additionally, up-regulated expression of defensin, mytimycin and lysozyme mRNA was observed in SP treated mussels shortly after exposure (on Days 1, 2, and 3) to SP. These results indicate that a high quality yield of SP can be readily extracted from C. crispus and more importantly based on the animal model used in this study, SP extracted from C. crispus can rapidly induce health enhancing activities in Mytilus spp. at a cellular, humoral and molecular level and with a prolonged effect up to ten days post treatment.

Dioxin-like compounds bioavailability and genotoxicity assessment in the Gulf of Follonica, Tuscany (Northern Tyrrhenian Sea).

The Gulf of Follonica (Italy) is impacted by the chemical pollution from ancient mining activity and present industrial processes. This study was aimed to determine the bioavailability of dioxin-like compounds (DLCs) in coastal marine environment and to assess the genotoxic potential of waste waters entering the sea from an industrial canal. Moderately high levels of DCLs compounds (∑ PCDDs + PCDFs 2.18­29.00 pg/g dry wt) were detected in Mytilus galloprovincialis transplanted near the waste waters canal and their corresponding Toxic Equivalents (TEQs) calculated. In situ exposed mussels did not show any genotoxic effect (by Comet and Micronucleus assay). Otherwise, laboratory exposure to canal waters exhibited a reduced genomic template stability (by RAPD-PCR assay) but not DNA or chromosomal damage. Our data reveal the need to focus on the levels and distribution of DLCs in edible species from the study area considering their potential transfer to humans through the consumption of sea food.

Metabolomics assessment of the effects of diclofenac exposure on Mytilus galloprovincialis: Potential effects on osmoregulation and reproduction.

The presence of pharmaceutically active compounds in aquatic environments has become a major concern over the past 20years. Elucidation of their mode of action and effects in non-target organisms is thus now a major ecotoxicological challenge. Diclofenac (DCF) is among the pharmaceutical compounds of interest based on its inclusion in the European Union Water Framework Directive Watch List. In this study, our goal was to investigate the potential of a metabolomic approach to acquire information without any a priori hypothesis about diclofenac effects on marine mussels. For this purpose, mussel's profiles were generated by liquid chromatography combined with high resolution mass spectrometry. Two main metabolic pathways were found to be impacted by diclofenac exposure. The tyrosine metabolism was mostly down-modulated and the tryptophan metabolism was mostly up-modulated following exposure. To our knowledge, such DCF effects on mussels have never been described despite being of concern for these organisms: catecholamines and serotonin may be involved in osmoregulation, and in gamete release in mollusks. Our results suggest potential impairment of mussel osmoregulation and reproduction following a DCF exposure.

Assessment of the fitness of the mussel Mytilus galloprovincialis two years after the Hebei Spirit oil spill.

In December 2007, >150km of the West coast of Korea were heavily polluted by crude oil leaked from the oil tanker Hebei Spirit, leading to mass mortality of bivalve mollusks on the intertidal areas. Two years after, mussels Mytilus galloprovincialis were collected from two impacted sites to investigate sub-lethal effects of the oil spill. Tissue content in polycyclic aromatic hydrocarbons (PAHs), hemocyte parameters, reproductive status and energetic reserves were analyzed. PAHs in tissues of mussels as well as hemocyte parameters were not different between impacted and control sites. Energetic reserves were altered in mussels from the impacted sites. Glycogen content remained low at polluted sites, whatever the season. Two years after the Hebei Spirit oil spill, mussels then presented altered energetic metabolism. Further investigations are thus warranted to monitor the sustainability of mussel populations on the oil spilled West coast of Korea.

Comparative assessment of cardiac activity and DNA damage in haemocytes of the Mediterranean mussel Mytilus galloprovincialis in exposure to tributyltin chloride.

This study gives an insight in sensitivity of heart rate (Hr) of Mytilus galloprovincialis as a physiological biomarker. Impact of tributyltin chloride (TBT-Cl) on Hr was studied in parallel with evaluation of mutagenic, genotoxic and cytotoxic potential of TBT-Cl (10, 100 and 1000µg/L) within 96h treatment in static conditions. Mutagenic potential was assessed by SOS/umuC assay while genotoxicity was assessed in haemocytes of M. galloprovincialis by using the comet assay and the micronucleus test. Benzo(a)pyrene (B(a)P) was used as a positive control. Hr variations detected in TBT-Cl treatments can be linked to data obtained in the genotoxicological assays indicating that Hr can be considered and used as a reliable physiological biomarker for detecting the presence of organotin compounds. However despite the observed genotoxic potential of B(a)P, a noteworthy Hr response was not observed which further questions the potential of Hr in the detection of different types of pollutants.

Free fatty acid profiling of marine sentinels by nanoLC-EI-MS for the assessment of environmental pollution effects.

The present work aims to elucidate the free fatty acid (FFA) profile of the mussel Mytilus galloprovincialis caged in an anthropogenically impacted area and in a reference site through an innovative and validated analytical approach for the assessment of biological alterations induced by marine pollution. The FFA pattern is involved in the regulation of different cellular pathways and differs with respect to metabolic stimuli. To this purpose, the lipid fraction of mussels coming from both sampling areas was extracted and the FFA fractions were isolated and purified by a solid phase extraction; then, nano-scale liquid chromatography coupled to electron ionization mass spectrometry (nanoLC-EI-MS) was employed for the characterization of the two samples. A total of 19 and 17 FFAs were reliably identified in the mussels coming from the reference and polluted site, respectively. Significant qualitative and quantitative differences found in saturated, monounsaturated and polyunsaturated species may be exploited as typical pollution biomarkers (e.g. alteration of the fatty acid biosynthetic system and lipotoxicity) and explain adverse and compromising effects (e.g. oxidative stress and inflammatory processes) related to environmental pollution.

Histopathological assessment and inflammatory response in the digestive gland of marine mussel Mytilus galloprovincialis exposed to cadmium-based quantum dots.

Although tissue-level biomarkers have been widely applied in environmental toxicology studies, the knowledge using this approach in marine invertebrates exposed to engineered nanomaterials (ENMs) remains limited. This study investigated histopathological alterations and inflammatory responses induced by Cd-based quantum dots (QDs), in comparison with their dissolved counterparts, in the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and dissolved Cd at the same concentration (10µg Cd L(-1)) for 14days and a total of 15 histopathological alterations and 17 histomorphometric parameters were analysed in the digestive gland along with the determination of histopathological condition indices (Ih). A multivariate analysis showed that the mussel response to QDs was more related to exposure time, inflammatory conditions (frequency of haemocytic infiltration and granulocytomas) and changes of cell-type composition (especially the rate between basophilic and digestive cells) when compared to dissolved Cd, while the response to dissolved Cd was associated with histomorphometric parameters of the epithelium and lumen of digestive tubules and increase of the atrophic tubule frequency. Both Cd forms induced higher Ih compared to unexposed mussels indicating a significant decrease in the health status of digestive gland in a Cd form and time-dependent pattern. Results indicate that the multiparametric tissue-level biomarkers in the digestive gland provide a suitable approach to assess the ecotoxicity and mode of action of metal-based ENMs in marine bivalves.

Combined effects of short-term exposure to elevated CO2 and decreased O2 on the physiology and energy budget of the thick shell mussel Mytilus coruscus.

Hypoxia and ocean acidification are two consequences of anthropogenic activities. These global trends occur on top of natural variability. In environments such as estuarine areas, short-term acute pH and O2 fluctuations are occurring simultaneously. The present study tested the combined effects of short-term seawater acidification and hypoxia on the physiology and energy budget of the thick shell mussel Mytilus coruscus. Mussels were exposed for 72 h to six combined treatments with three pH levels (8.1, 7.7 and 7.3) and two dissolved oxygen (DO) levels (2 mg L(-1), 6 mg L(-1)). Clearance rate (CR), food absorption efficiency (AE), respiration rate (RR), ammonium excretion rate (ER), O:N ratio and scope for growth (SFG) were significantly reduced, and faecal organic dry weight ratio (E) was significantly increased at low DO. Low pH did not lead to a reduced SFG. Interactive effects of pH and DO were observed for CR, E and RR. Principal component analysis (PCA) revealed positive relationships among most physiological indicators, especially between SFG and CR under normal DO conditions. These results demonstrate that Mytilus coruscus was sensitive to short-term (72 h) exposure to decreased O2 especially if combined with decreased pH levels. In conclusion, the short-term oxygen and pH variation significantly induced physiological changes of mussels with some interactive effects.

Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis.

Nanoparticles (NPs), due to their increased application and production, are being released into the environment with unpredictable impact on the physiology of marine organisms, as well as on entire ecosystems and upcoming effects on human health. The aim of the present study was to evaluate and compare the oxidative responses of the mussel Mytilus galloprovincialis after exposure to iron oxide NPs and to iron oxide NPs incorporated into zeolite for 1, 3 and 7 days. Our results showed that both effectors induced changes on animal physiology by causing oxidative stress in hemocytes of exposed mussels compared to control animals. This was shown by the significant increase in reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, ubiquitin conjugates and DNA damage. In addition an increase in prooxidant levels as measured by the prooxidant-antioxidant balance (PAB) assay was observed in exposed mussels' hemolymph. The results show that ROS, DNA damage, protein and lipid oxidation, ubiquitin conjugates and PAB could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors. In addition, more studies are needed in order to ensure the safety of these NPs on various biomedical applications, since it is critical to design NPs that they meet the demands of application without causing cellular toxicity.

Combined use of chemical, biochemical and physiological variables in mussels for the assessment of marine pollution along the N-NW Spanish coast.

This study undertakes an overall assessment of pollution in a large region (over 2500 km of coastline) of the N-NW Spanish coast, by combining the use of biochemical (AChE, GST, GPx) and physiological (SFG) responses to pollution, with chemical analyses in wild mussel populations (Mytilus galloprovincialis). The application of chemical analysis and biological techniques identified polluted sites and quantified the level of toxicity. High levels of pollutants were found in mussel populations located close to major cities and industrialized areas and, in general, average concentrations were higher in the Cantabrian than in the Iberian Atlantic coast. AChE activities ranged between 5.8 and 27.1 nmol/min/mg prot, showing inhibition in 12 sampling sites, according to available ecotoxicological criteria. GST activities ranged between 29.5 and 112.7 nmol/min/mg prot, and extreme variability was observed in GPx, showing activities between 2.6 and 64.5 nmol/min/mg prot. Regarding SFG, only 5 sites showed 'moderate stress' (SFG value below 20 J/g/h), and most sites presented a 'high potential growth' (>35 J/g/h) corresponding to a 'healthy state'. Multivariate statistical techniques applied to the chemical and biological data identified PCBs, organochlorine pesticides and BDEs as the main responsible of the observed toxicity. However, the alteration of biological responses caused by pollutants seems to be, in general, masked by biological variables, namely age and mussel condition, which have an effect on the mussels' response to pollutant exposure.

A multidisciplinary weight of evidence approach for environmental risk assessment at the Costa Concordia wreck: Integrative indices from Mussel Watch.

A complex framework of chemical, biological and oceanographic activities was immediately activated after the Costa Concordia shipwreck, to assess possible contamination events and the environmental impact during both emergency and wreck removal operations. In the present paper, we describe the results obtained with caged mussels, Mytilus galloprovincialis, chosen as bioindicator organisms to detect variations of bioavailability and the early onset of molecular and cellular effects (biomarkers). Seven translocation experiments were carried out during the first year from the incident, with organisms deployed at 2 depths in 3 different sites. After 4-6 weeks, tissue concentrations were measured for the main classes of potentially released chemicals (trace metals, polycyclic aromatic hydrocarbons, volatile and aliphatic hydrocarbons, polychlorinated biphenyls, halogenated pesticides, organotin compounds, brominated flame retardants, anionic surfactants); a wide battery of biomarkers covered responses indicative of exposure, detoxification, oxidative stress, cell damage and genotoxic effects. Results excluded serious contamination events or a consistent increase of environmental pollution although some episodic spills with reversible effects were detected. Data were elaborated within a quantitative weight of evidence (WOE) model which provided synthetic hazard indices for each typology of data, before their overall integration in an environmental risk index, which generally ranged from slight to moderate. The proposed WOE model was confirmed a useful tool to summarize large datasets of complex data in integrative indices, and to simplify the interpretation for stakeholders and decision makers, thus supporting a more comprehensive process of "site-oriented" management decisions.

Mutagenicity test using Vibrio harveyi in the assessment of water quality from mussel farms.

This work analyses the mutagenicity of seawater from mussel farms using the Vibrio harveyi mutagenicity test and its relationship with the accumulated pollutants and the development of gonadal neoplasia in mussels. Histological disorders identified as germinoma were observed in the gonad of Mytilus galloprovincialis during the period of study. The prevalence of this pathology is significantly correlated with certain levels of pollutants accumulated in mussels, mainly of PAHs and PCBs, whose toxic equivalents were calculated as EROD induction equivalency. The mutagenicity and toxicity of the water surrounding mussel's farms is clearly correlated with the pollutants accumulated and with the neoplasia prevalence in mussels. Such correlations are corroborated by a multivariate analysis. Our results conclude with the utility of V. harveyi test as an optimal and rapid method in the monitoring of the quality of the water from mussel farms and as a tool to control the risks of pollution on mussel production and its safety for human food.

Marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the Prestige oil spill "Mussel Watch".

Five integrative biomarker indices are compared: Bioeffects Assessment Index (BAI), Health Status Index (HSI), integrated biological response (IBR), ecosystem health condition chart (EHCC) and Integrative Biomarker Index (IBI). They were calculated on the basis of selected biomarker data collected in the framework of the Prestige oil spill (POS) Mussel Watch monitoring (2003-2006) carried out in Galicia and the Bay of Biscay. According to the BAI, the health status of mussels was severely affected by POS and signals of recovery were evidenced in Galicia after April-04 and in Biscay Bay after April-05. The HSI (computed by an expert system) revealed high levels of environmental stress in 2003 and a recovery trend from April-04 to April-05. In July-05, the health status of mussels worsened but in October-05 and April-06 healthy condition was again recorded in almost all localities. IBR/n and IBI indicated that mussel health was severely affected in 2003 and improved from 2004 onwards. EHCC reflected a deleterious environmental condition in 2003 and a recovery trend after April-04, although a healthy ecosystem condition was not achieved in April-06 yet. Whereas BAI and HSI provide a basic indication of the ecosystem health status, star plots accompanying IBR/n and IBI provide complementary information concerning the mechanisms of biological response to environmental insult. Overall, although the integrative indices based on biomarkers show different sensitivity, resolution and informative output, all of them provide coherent information, useful to simplify the interpretation of biological effects of pollution in marine pollution monitoring. Each others' advantages, disadvantages and applicability for ecosystem health assessment are discussed.

Assessment of the effects of a marine urban outfall discharge on caged mussels using chemical and biomarker analysis.

To assess the presence of endocrine disruptors in treated marine outfall discharges and their possible effects, mussels (Mytilus galloprovincialis) were caged in the environmental mixing zone of the outfall of the Santander sanitation system and in one control area. After 30, 60 and 90 days, samples were collected to perform chemical analyses (metals, anionic surfactants, alkylphenols, bisphenol A, phthalates and estrogenic hormones), biomarkers of general stress (lysosomal membrane stability-LMS, histopathology) and biomarkers of endocrine disruption (vitellogenin-like proteins and gonad index). There were no significant differences between outfall and control sites on contaminant levels, except for 4-tert-octylphenol which was higher in the outfall site. Bacteriological counts were higher in the outfall area. No relevant differences in biomarkers were detected between treated and control mussels. A significant reduction in LMS occurred in both groups after 90 days caging, indicating a stress situation possibly related to caging or to post-spawning reproductive state.