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Despite the increasing interest for pharmaceuticals in the marine environment, their accumulation in wild organisms and consequent environmental hazards are still poorly known. The Mediterranean Sea is highly challenged by the density of coastal populations, large consumption of pharmaceuticals and their often limited removal by Wastewater Treatment Plants (WWTPs). In this respect, the present study aims to provide the first large-scale survey on the distribution of such contaminants of emerging concern in native mussels, Mytilus galloprovincialis from Italian coasts. Organisms were collected from 14 sites representative of relatively unpolluted marine waters along the Adriatic and Tyrrhenian Sea and analysed for 9 common pharmaceuticals including Non-Steroidal Anti-Inflammatory Drugs (NSAIDs: Diclofenac DIC, Ibuprofen IBU, Ketoprofen KET and Nimesulide NIM), the analgesic Acetaminophen AMP, the antiepileptic Carbamazepine CBZ, the antihypertensive Valsartan VAL, the anxiolytic Lormetazepam LOR and the antidepressant Paroxetine PAR. Results indicated the widespread occurrence of the majority of pharmaceuticals in mussel tissues: CBZ was measured in >90% of analysed samples, followed by VAL (>50%), PAR (>40%), and DIC (>30%), while only AMP and KET were never detected. Heterogeneous tissue concentrations ranged from a few units up to hundreds of ng/g (d.w.), while seasonal and interannual variability, investigated over 4 years, did not highlight any clear temporal trend. Limited differences obtained between the Adriatic and Tyrrhenian Sea, as well as coastal versus off-shore sampling sites, suggest that analysed levels of pharmaceuticals in mussels tissues should be considered as baseline concentrations for organisms collected in unpolluted areas of the Mediterranean. This study provided the first unambiguous evidence of the widespread occurrence of pharmaceuticals in marine mussels from Italian coasts, giving novel insights on the potential ecotoxicological hazard from such compounds in marine species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Increasing microplastics pollution of marine and terrestrial water is a concerning issue for ecosystems and human health. Nevertheless, the interaction of microplastics with freshwater biota is still a poorly explored field. In order to achieve information concerning the uptake, distribution and effect of microplastics in planarians, Dugesia japonica specimens have been fed with mixtures of food and differently shaped and sized plastic particles. Feeding activity and food intake were non-altered by the presence of high concentrations of different types of plastic particles. However, the persistence of microplastic within the planarian body was a function of size/shape, being small spheres (<10 μm in diameter) and short fibers (14 μm large and 5/6 μm length) more persisting than larger spheres and longer fibers which were eliminated almost entirely by ejection in a few hours. Transmission electron microscopy analysis demonstrated that at least part of microplastics was phagocytized by the enterocytes. Chronic exposure to small plastic did not alter the regenerative ability but caused a significant reduction of the gut epithelium thickness and lipid content of enterocytes, together with the induction of apoptotic cell death, modulation of Djgata 4/5/6 expression and reduced growth rate. The ability of microplastic to perturb planarian homeostasis is concerning being them extremely resilient against mechanical and chemical insults and suggests possible harmful effects upon other more susceptible species in freshwater ecosystems., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 2.18–29.00 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.

High-latitude marine ecosystems are ranked to be among the most sensitive regions to climate change since highly stenothermal and specially adapted organisms might be seriously affected by global warming and ocean acidification. The present investigation was aimed to provide new insights on the sensitivity to such environmental stressors in the key Antarctic species, Adamussium colbecki, focussing also on their synergistic effects with cadmium exposure, naturally abundant in this area for upwelling phenomena. Scallops were exposed for 2 weeks to various combinations of Cd (0 and 40 μgL-1), pH (8.05 and 7.60) and temperature (-1 and +1 °C). Beside Cd bioaccumulation, a wide panel of early warning biomarkers were analysed in digestive glands and gills including levels of metallothioneins, individual antioxidants and total oxyradical scavenging capacity, onset of oxidative cell damage like lipid peroxidation, lysosomal stability, DNA integrity and peroxisomal proliferation. Results indicated reciprocal interactions between multiple stressors and their elaboration by a quantitative hazard model based on the relevance and magnitude of effects, highlighted a different sensitivity of analysed tissues. Due to cellular adaptations to high basal Cd content, digestive gland appeared more tolerant toward other prooxidant stressors, but sensitive to variations of the metal. On the other hand, gills were more affected by various combinations of stressors occurring at higher temperature., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study investigated the influence of nano-TiO(2) (1 mg L(-1)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) (46 pg L(-1)) bioconcentration and toxicity in the European sea bass (Dicentrarchus labrax) during 7 days in vivo exposure. A multimarkers approach was applied in different organs: detoxification in liver; innate immunity and pro-inflammatory response and adaptive immunity in gills and spleen; genotoxicity in peripheral erythrocytes and muscle. Bioconcentration of 2,3,7,8-TCDD in presence of nano-TiO2 was investigated in liver, skin and muscle as well as interaction between nano-TiO2 and organic pollutants in artificial sea water (ASW). Nano-TiO2 negatively influenced immune response induced by 2,3,7,8-TCDD in spleen but not in gills and reduced the DNA damage induced by 2,3,7,8-TCDD in erythrocytes. nano-TiO2 did not interfere with 2,3,7,8-TCDD detoxification and bioconcentration according to the observed no interaction of the nano-TiO2 with organic pollutants in ASW.

In the present study, the genotoxic potential of nanosized TiO2 anatase and micro-sized rutile on bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes was investigated. Human and mouse cells were also studied in order to compare susceptibility to TiO2 in different mammalian species. Cell lines were exposed for 4, 24, and 48 h to different concentrations of TiO2 (20, 50, 100, 150 μg/ml) and DNA damage was investigated by single cell gel electrophoresis (Comet assay). Both anatase and rutile induced increased DNA damage, even though statistically significant effects were scattered according to species and cell lines. Bottlenose dolphin leukocytes and murine fibroblasts exhibited increased DNA damage after rutile exposure at some doses/times, while human fibroblasts showed a significant dose-response effect after a 4 h exposure to anatase. Human leukocytes were tolerant to both anatase and rutile. Ultrastructural investigation showed that TiO2 particles entered the cell and were compartmentalized within membrane-bound vesicles., (Copyright © 2014. Published by Elsevier Ltd.)

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The freshwater painter's mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painter's mussels for environmental quality assessment in freshwater., (2010 Elsevier B.V. All rights reserved.)

Background, Aim, and Scope: Exceptionally high levels of trace metals have been reported in specific tissues of certain polychaetes. In the present study, the Antarctic fan worm Perkinsiana littoralis was shown to hyperaccumulate vanadium in the branchial tissues, and the hypothesis of an antipredatory strategy has been investigated., Materials and Methods: Trace metals (Ag, Al, As, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V, Zn) were measured by atomic absorption spectrophotometry in tissues of P. littoralis and, only for V, in two Antarctic bivalves and in various Mediterranean polychaetes. Subcellular distribution of vanadium was investigated in P. littoralis after differential centrifugations; feeding trials with the Antarctic rock cod Trematomus berancchii were performed to test the palatability of P. littoralis., Results and Discussion: Analyses of trace metals in tissues of P. littoralis confirmed the naturally high bioavailability of cadmium due to upwelling phenomena in the investigated area and revealed extremely high concentrations of vanadium up to 10,000 microg/g, in the branchial crowns; much lower concentrations were measured in the body portions and even less in the Antarctic bivalves and in Mediterranean polychaetes. The subcellular distribution indicated that this metal was associated in branchial crowns with both heavy components and vanadium binding proteins; the latter predominated in body tissues, although with a different pattern of molecular weight. Feeding trials suggested that the elevated levels of vanadium in branchial crown of P. littoralis act as chemical deterrents against predation in more exposed tissues., Recommendations and Perspectives: The hyperaccumulation of toxic metals might represent a common antipredatory strategy for unpalatable branchial crowns of sabellid polychaetes, as recently hypothesized also for the high concentrations of arsenic in the Mediterranean Sabella spallanzanii. The evolution of such adaptation and the reasons behind the possibility for different species to accumulate different metals represent a stimulating field of investigation for future studies.

The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.

Biological interactions between various classes of pollutants are of great relevance for the Antarctic marine environment, where the naturally elevated bioavailability of metals like cadmium might indirectly influence sensitivity of endemic organisms toward other environmental pollutants, e.g. polycyclic aromatic hydrocarbons (PAHs). To further investigate reciprocal effects of different chemicals, the fish Trematomus bernacchii was exposed to trace metals (Cd, Cu, Hg, Ni, Pb) and benzo[a]pyrene (BaP, as a model PAH), dosed alone and in combinations. Co-exposures revealed that BaP did not influence the accumulation of metals, while these elements caused significant changes on tissue levels of the PAH. The marked EROD induction caused by BaP was completely suppressed by co-exposure with Cd and Cu, but no effects were observed with Ni, Hg and Pb. Similar results were confirmed at the protein level by Western blot analyses while CYP1A1 mRNA levels were reduced only during Cd co-exposures. Clear evidence of oxidative perturbations was observed in fish co-treated with Cd and BaP and the reduced capability to absorb peroxyl and hydroxyl radicals suggested some oxidative pathways by which this element might indirectly modulate the biotransformation efficiency of Cytochrome P450. Partly different and post-transcriptional mechanisms of action could be hypothesized for Cu, while moderate oxidative effects of Hg, Ni and Pb during co-exposures would confirm their limited influence on metabolism of PAHs. In general, the overall results revealed a complex pathway of interactions between different chemicals during co-exposures and the importance of oxidative status in modulating induction and expression of CYP1A1.

Synthesis of vitellogenin (VTG) in male fish is a widely recognized effect for estrogenic pollutants in temperate environments, while similar investigations are still lacking for Antarctic organisms. In this study, a preliminary characterization of vitellogenin gene expression was performed by RT-PCR in the key species Trematomus bernacchii sampled in different phases of reproductive cycle and food availability. Females exhibited the highest gene expression during the spawning period, but VTG mRNA was always detected also in males; a significant increase of gene expression was observed both in males and females at the end of the feeding season. These results were not fully supported by a differential exposure to phyto- or anthropogenic estrogens during the planctonic cycle; on the other side, the endocrine properties of cadmium, naturally elevated in Terra Nova Bay and increasing during algal bloom, would explain both the presence of VTG mRNA in males and the seasonal changes of gene induction. Laboratory exposures did not reveal short-term estrogenic effects of cadmium while an elevated responsiveness of T. bernacchii was observed toward a classical estrogenic receptor agonist (17beta-estradiol). Different hypotheses were considered to suggest delayed endocrine effects of cadmium, including the early interaction with other cellular detoxification systems or alterations at multiple levels of the hypothalamus-pituitary-gonad-liver axis. Although molecular mechanisms of VTG gene expression in males of T. bernacchii remain unclear, obtained results provide interesting insights on this species which should stimulate future research activities.

In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as "pilot basin" within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1+/-7.6) than in transplanted (9.3+/-4.7) and reference ones (5.8+/-5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.

The Antarctic silverfish Pleuragramma antarcticum is a key organism in the ecology of Southern Ocean. Eggs with fully developed yolk-sac embryos and newly hatched larvae have been recently observed to occur in the platelet ice accumulating below the sea-ice layer. This environment has strong pro-oxidant characteristics at the beginning of austral spring, when the rapid growth of algal ice communities, the massive release of nutrients and the photoactivation of dissolved organic carbon and nitrates, all represent important sources for oxyradical formation. Such processes are concentrated in a short period of a few weeks which overlaps with the final development of P. antarcticum in platelet ice. The aim of this work was to characterize the antioxidant system in embryos of P. antarcticum and the responsiveness toward the natural increase of pro-oxidant conditions in early spring. Considering the lack of ecotoxicological data on this species and its pivotal importance in the ecosystem of Southern Ocean, the sensitivity of its early life stages was also evaluated after laboratory exposures to environmentally relevant doses of benzo(a)pyrene, as a model chemical potentially released from anthropogenic activities. Obtained results revealed a marked temporal increase of antioxidants in embryos of P. antarcticum as adaptive counteracting responses to oxidative conditions of platelet ice. Particularly prompt responses were observed for glutathione metabolism which, however, did not prevent formation of increasing levels of lipid peroxidation products; from the analysis of total oxyradical scavenging capacity (TOSC), the overall efficiency to neutralize peroxyl radicals remained almost constant while slightly lower TOSC values were obtained toward hydroxyl radicals at the end of sampling period. Laboratory exposures to 0.5-5 microg/l BaP caused a significant accumulation of this PAH but no significant effects on the activity of cytochrome P450. Antioxidants of exposed embryos showed less marked variations than embryos in field conditions suggesting that the elevated pro-oxidant challenge, to which these organisms are naturally adapted, might be responsible for the moderate responsiveness to pro-oxidant chemicals.

The relationship between DNA damage and the exposure of marine organisms to environmental contaminants was examined in the Göteborg harbour area. This research is part of a wider ecotoxicological study planned to evaluate the biological impact of chemical contamination in the River Göta estuary, following a bunker oil (10-100 tonnes) spill occurred in June 2003. Here we present data on the DNA strand breaks derived using the comet assay and the presence of apoptotic cells using the diffusion assay in nucleated erythrocytes of the eelpout (Zoarces viviparus) from the study area and at a clean reference site. Polycyclic aromatic hydrocarbon metabolites were also analyzed in the bile of exposed fish. The results showed a high level of damaged DNA, paralleled by a peak in bile PAH metabolites, in fish from the most impacted site, 3 weeks after the oil spill. A significant recovery was observed in specimens from the spill site, 5 months later, but not in fish caught in the middle part of Göteborg harbour, which is chronically subjected to heavy chemical pollution. The levels of apoptic cells did not show any marked variations, but a significant recovery was observed in fish from the oil impacted site 5 months after the spill.

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study several biomarkers were investigated in marine mussels caged for 4 weeks (June-July 2001) into an industrialized harbour of NW Italy. Organisms were collected at different time-intervals to better characterize the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralize specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidized bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with an increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment.

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study, several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of north-west Italy. Organisms were collected at different time intervals to better characterise the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralise specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidised bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with no variation or increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of reactive oxygen species (ROS) mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.