Toxicity of gamma aluminium oxide nanoparticles in the Mediterranean mussel (Mytilus galloprovincialis): histopathological alterations and antioxidant responses in the gill and digestive gland.

PURPOSE: Accumulation of Gamma aluminium oxide nanoparticles γ-Al2O3 NPs significant impact on aquatic ecosystems. However, the toxicity of γ-Al2O3 NPs in aquatic organisms has been limited investigated. This study investigated histopathological changes and antioxidant responses induced by different concentrations of γ-Al2O3 NPs in Mytilus galloprovincialis. MATERIAL AND METHODS: In this study, mussels were exposed to different concentrations of 5 nm γ-Al2O3 NPs (0, 5, 20 and 40 mg/L) for 96 h under controlled laboratory conditions. Gill and digestive gland from mussels were assessed to histopathological (light microscopy, histopathological condition indices, digestive gland tubule types), SOD, CAT, GPx activities. RESULTS: Histopathological indices calculated higher, and significantly different in all exposure groups compared to the control group in gill and digestive gland (p < 0.05). Atrophic phase tubules proportion very high in 20 and 40 mg/L γ-Al2O3 NPs exposure groups. No significant changes in CAT activities in the gill and digestive gland (p > 0.05). Superoxide dismutase (SOD) activities significantly different (p ≤ 0.05) in the digestive gland from 20 mg/L γ-Al2O3 NPs exposures, and GPx activities significantly different (p < 0.05) in gill from 40 mg/L γ-Al2O3 NPs exposures. CONCLUSION: These results indicate that contamination of γ-Al2O3 NPs negatively affects the aquatic organism.

Assessing relative sensitivity of marine and freshwater bivalves following exposure to copper: Application of classical and novel genotoxicological biomarkers.

Determination of relative sensitivity of biota following exposures to contaminants including metals is important for environmental protection. Copper (Cu), although biologically essential can be highly toxic to biota if present at higher concentrations in the natural environment. Given its ubiquitous presence within coastal and inland water bodies, we compared Cu-induced genotoxicity in two ecologically important mussel species, the freshwater Dreissena polymorpha (DP) and marine Mytilus galloprovincialis (MG), along with its tissue specific accumulation. Novel biomarker in terms of induction of gamma H2AX (γ-H2AX) foci, along with comet assay and induction of micronuclei (MN) were used to determine DNA damage response (DDR) in these two species following exposure to a range of Cu concentrations (18, 32, 56 µg L-1) for 10 days. Concentration-dependent increases in Cu concentration in gill tissue, as determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), were paralleled by a greater degree of genotoxicity. An induction of γ-H2AX foci was present in all Cu exposure concentrations, proving this technique to be a sensitive and suitable biomarker of genotoxicity in bivalves. The multi-biomarker approach adopted here suggests firstly that in parallel with MG, which is widely used to assess the health of marine and coastal environment, DP is also suitable representative of inland water bodies, and that there is a similar mechanism of action for the induction of genotoxicity between the two species, following exposure to Cu. Secondly, for genotoxicity assessment a battery of responses could simultaneously be assessed in these two bivalve species. Finally, for adequate protection of the environment it is vital to adopt a multi-biomarker, multi-species approach to determine adverse biological effects to gain a holistic understanding of the real threat posed by contaminants to hydrosphere.

In vitro toxicity testing in hemocytes of the marine mussel Mytilus galloprovincialis (L.) to uncover mechanisms of action of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil without and with dispersant.

Dispersants used in oil spills could result toxic to marine organisms and could influence the toxicity of oil compounds. The aim of this work was to uncover the mechanisms of action of the water accommodated fraction (WAF) of a naphthenic North Sea crude oil produced at 10, 15 and 20 °C without and with the dispersant Finasol OSR52 (WAF and WAFD, respectively) using hemocytes of the marine mussel Mytilus galloprovincialis. Primary cultures of hemocytes were exposed in glass-coated microplates to different WAF or WAFD dilutions (0.25, 2.5, 25, 50 and 100%) and to the dispersant alone at the same concentrations present in the WAFD dilutions (1.25, 12.5, 125, 250 and 500 mg/L). Of the two in vitro approaches tested, the second one was selected which involved exposure of hemocytes for 4 h to unfiltered WAF, WAFD and dispersant dilutions without cell culture media. WAF decreased hemocytes viability only at the highest dilution whereas WAFD and the dispersant alone were cytotoxic at the three highest concentrations. Temperature of production of WAF, WAFD and dispersant did not influence their cytotoxicity to hemocytes. WAF increased ROS production and MXR transport activity in hemocytes. Exposure to WAFD and dispersant increased ROS production, provoked plasma membrane and actin cytoskeleton disruption and decreased phagocytic activity. In conclusion, the dispersant tested was toxic to mussel hemocytes and it greatly increased the toxicity of WAFD. The present data could be useful for the environmental risk assessment of oil spills and their remediation strategies in the marine environment.

Biological effects of tris (1-chloro-2-propyl) phosphate (TCPP) on immunity in mussel Mytilus galloprovincialis.

Organophosphate flame retardants (OPFRs) are increasingly produced and used as alternatives of brominated flame-retardants (BFRs) and have become emerging marine environmental contaminants. So far, however, little is known regarding the biological effects of OPFRs in marine organisms. In this study, the biological effects of one of the most abundant OPFRs, tris (1-chloro-2-propyl) phosphate (TCPP), on the immunity in mussel Mytilus galloprovincialis were characterized by testing the reactive oxygen species, apoptosis, antioxidant system and immunity related gene expressions. Results indicated that both TCPP exposures (10 and 100 nmol L-1) significantly (p < 0.01) enhanced reactive oxygen species production and the high dose of TCPP induced more apoptosis and oxidative stress in mussel hemocytes. TCPP also induced an obvious hormesis phenomenon (low dose inhibition and high dose stimulation) in mussel hemocytes, as indicated by the gene expression profiles of caspase 8 and mytimacin. The down-regulated gene expression levels of lysozymes suggested that both TCPP exposures inhibited the innate immunity in mussel M. galloprovincialis. The significantly (p < 0.01) increased gene expression levels of TLR, galectin, PGRP and LITAF demonstrated that TCPP induced dose-dependent immune stress in mussels. Overall, this work suggested that TCPP could influence the immune system in marine mussel M. galloprovincialis.

The physiological effects of oil, dispersant and dispersed oil on the bay mussel, Mytilus trossulus, in Arctic/Subarctic conditions.

Increasing oil development around Alaska and other Arctic regions elevates the risk for another oil spill. Dispersants are used to mitigate the impact of an oil spill by accelerating natural degradation processes, but the reduced hydrophobicity of dispersed oil may increase its bioavailability to marine organisms. There is limited research on the effect of dispersed oil on cold water species and ecosystems. Therefore, spiked exposure tests were conducted with bay mussels (Mytilus trossulus) in seawater with non-dispersed oil, Corexit 9500 and oil dispersed with different concentrations of Corexit 9500. After three weeks of exposure, acute and chronic physiological impacts were determined. The majority of physiological responses occurred during the first seven days of exposure, with mussels exhibiting significant cytochrome P450 activity, superoxide dismutase activity and heat shock protein levels. Mussels exposed to non-dispersed oil also experienced immune suppression, reduced transcription and higher levels of mortality. After 21 days, mussels in all treatments exhibited evidence of genetic damage, tissue loss and a continued stress response. Bay mussels are useful as indicators of ecosystem health and recovery, and this study was an important step in understanding how non-dispersed oil, dispersant and dispersed oil affect the physiology of this sentinel species in Arctic/subarctic conditions.

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.

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.

A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus.

Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420bp, including an ORF of 2169bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and 'EEVD' motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca.

Monitoring follow up of two areas affected by the Prestige oil four years after the spillage.

The sinking of the oil tanker Prestige in November 2002 resulted in the spill of more than 63,000 tonnes of crude oil, and polluted more than 1,000 km of coastline, especially affecting Galicia (northwestern Spain). Four years after the accident, a new biological monitoring study was undertaken of two Galician areas intensely affected by the spill, Lira and Ancoradoiro, previously evaluated in the months following the accident ( Laffon et al. 2006 ). The mussel Mytilus galloprovincialis was employed as bioindicator organism to determine both polycyclic aromatic hydrocarbons (PAH) levels and genotoxic effects. PAH were determined chromatographically in seawater samples and mussel tissues collected from November 2006 to January 2008. The results obtained showed that PAH pollution was still present in these areas, but bioaccumulation of these compounds in mussels was low, compared to reference mussels, and lower than in our previous study. DNA damage assessment was also performed in gills and hemolymph cells by means of the alkaline comet assay. DNA damage levels were higher in mussels from the exposed areas than in reference mussels. DNA damage decreased after a 7-d recovery period in the laboratory, but prolonging the recovery period up to 14 d did not contribute to less DNA damage in gill cells. Hemolymph cells were more sensitive than gill cells to the induction of DNA damage.

Biochemical responses of Mytilus galloprovincialis as biomarkers of acute environmental pollution caused by the Don Pedro oil spill (Eivissa Island, Spain).

In the present work, the potential use of several antioxidant and detoxification biomarkers in the digestive gland of wild mussels (Mytilus galloprovincialis) for biomonitoring the marine pollution induced by the Don Pedro oil spill has been investigated. Two locations from the East to South-East of Eivissa (Ibiza) and Formentera islands were selected, one extensively affected by the oil spill and the other one not affected and considered as the control area. Mussels were sampled one, two and six months after the Don Pedro accident. Polycyclic aromatic hydrocarbon (PAH) levels were significantly increased in the soft tissues of mussels in the affected area one month after the disaster, returning to normal values after six months. Markers of oxidative damage in lipids--malondialdehyde, and in proteins--carbonyl derivates, and antioxidant enzyme--catalase, superoxide dismutase and glutathione peroxidase, activities significantly increased as result of the spill oil after one month, returning to basal values at two month sampling time. Glutathione/glutathione disulfide ratio (GSH/GSSG), as a marker of the redox status, was reduced after one and two months indicating a more oxidized situation. Markers of detoxification--glutathione-S-transferase and cytochrome P4501A activities and metallothionein gene expression--were significantly increased by the oil spill one month after the accident, returning to the basal values at two month sampling time. In conclusion, the Don Pedro accident induced a transient situation of PAHs pollution resulting in enhanced antioxidant and detoxification defense systems in the wild mussel M. galloprovincialis returning to normal levels six months from the spill. The selected biomarkers are a useful tool for biomonitoring the response to acute exposure to pollutants in marine mussels.

Olive oil mill wastewater toxicity in the marine environment: alterations of stress indices in tissues of mussel Mytilus galloprovincialis.

This study investigated the impact of olive mill wastewater (OMW) as a pollutant of the marine environment, via the detection of stress indice alterations in mussels Mytilus galloprovincialis. Due to the absence of data concerning the levels of OMW in the receiving waters, mortality test (96h) was first performed in order to estimate the range of OMW concentration where no mortality occurs. OMW concentrations ranging from 0.01 to 0.1% (v/v) showed no increased mortality and thus were used for the determination of pre-pathological alterations in tissues of mussels. In particular, mussels exposed to either 0.1 or 0.01% (v/v) OMW for 5 days showed significant alterations of stress indices in their tissues. Specifically, decreased neutral red retention (NRR) assay time values, inhibition of acetylcholinesterase (AChE) activity, as well as a significant increase of micronucleus (MN) frequency and DNA damage were detected in haemolymph/haemocytes and gills, compared with values measured in tissues of control mussels. The results of the present study showed that OMW disposal into the marine environment could induce pre-pathological alterations in marine organisms, before severe disturbances, such as disease, mortality, or population changes occur.

Effects of the fuel oil spilled by the Prestige tanker on reproduction parameters of wild mussel populations.

The aim of this work was to assess possible effects of the Prestige oil spill on reproduction parameters of mussels along the Galician and Bay of Biscay coast. Studied endpoints included sex ratio, gonad histology and vitellogenin-like proteins using the alkali-labile phosphate (ALP) method. A high prevalence of haemocytic infiltration of follicles and severe oocyte atresia was found in most localities in April 2003. Spawning gonads were observed in most impacted populations in the same sampling. In April 2004 mature small sized follicles were observed. No histopathological changes were observed in April 2005 and 2006, except a high prevalence of necrotic gametes in 6 out of 22 localities in April 2006. Female ALP levels showed high interindividual variability in April 2004, which was reduced in April 2005 and 2006. No xenoestrogenic effects were observed in male mussels. Overall, gamete alterations were detected during 2003-2004 and a recovery trend was observed afterwards.

The role of selenium-dependent glutathione peroxidase (Se-GPx) against oxidative and genotoxic effects of mercury in haemocytes of mussel Mytilus galloprovincialis (Lmk.).

This study investigated whether mercury (Hg) oxidative and genotoxic effects are related with its ability to inhibit selenium-dependent glutathione peroxidase (Se-GPx) activity in haemocytes of mussel Mytilus galloprovincialis. Se-GPx activity was measured both in Se-free cells' cytosolic fraction and in Se-treated cells, pre-treated with 4 microg/l of Se (as sodium selenite), before the exposure to the metal. Hg at concentrations ranged within 10 or 20 microg/l, thus representing the onset of Hg toxic effects, showed to inhibit Se-GPx activity in Se-free cells, followed by increased levels of superoxide anions (()O(2)(-)) and nitric oxide (NO) generation, lipid peroxidation and DNA damage as well. On the other hand, increased enzymatic activity and a significant attenuation of Hg toxicity were measured in Se-treated cells exposed to Hg in all cases. The results of the present study showed that inhibition of Se-GPx activity by Hg could promote a shift in the balance between oxidants and antioxidants in favor of oxidants, resulted in the enhancement of Hg-induced oxidative and genotoxic effects. In addition, Se bioavailability within phagocytic cells, such as haemocytes, could regulate the antioxidant role of Se-GPx, thus reinforcing haemocytes' immune system against toxic effects induced by pro-oxidants, such as Hg.

Acute toxicity of a shoreline cleaner, CytoSol, mixed with oil and ecological risk assessment of its use on the Galician Coast.

The application of embryo-larval bioassay with the purple sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis at 48 hours, and with neonates of the mysid Siriella armata at 96 hours, was used to evaluate the acute toxicities of the following preparations: (1) the shoreline cleaning agent CytoSol; (2) the water-accommodated fraction of CytoSol plus a light crude oil; and (3) the runoff from a pilot-scale treatment with CytoSol of a rocky coastal substrate impregnated with residues from the Prestige oil spill (which occurred on November 19, 2002). The mussel was the most sensitive organism to CytoSol and runoff effects (EC(50) = 8.0 microL/L and 64.3 mL/L, respectively), and the mysid was the least sensitive to the runoff (EC(50) > 200 mL/L). The predicted no-effect environmental concentration (PNEC) was calculated from the no observed-effect concentration of the species most sensitive to the runoff. The predicted environmental concentration (PEC) was estimated from a simple and reasonable dilution model, and the PEC/PNEC ratio was calculated according to the area treated and the values of the variables considered in the model. Implications for the management of the treatment operations are discussed.

Molecular cloning of the ribosomal P-proteins MgP1, MgP2, MgP0, and superoxide dismutase (SOD) in the mussel Mytilus galloprovincialis and analysis of MgP0 at stress conditions.

The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.

Expression analysis of the molluscan p53 protein family mRNA in mussels (Mytilus spp.) exposed to organic contaminants.

In this study, we report the tissue expression analysis of the p53 protein family mRNA in mussels (Mytilus spp.) by means of quantitative RT-PCR. The tissue specific response was evaluated after 24 h exposure to a sublethal benzo[a]pyrene (B[a]P) concentration (75 nM), showing a 2.6 fold induction in digestive gland cells and a dramatic gene down regulation in circulating hemocytes. The comet assay and DNA gel diffusion tests showed significant effects in hemocytes and negligible differences in the digestive gland nuclei, implicating p53 in DNA damage of molluscan hemocytes. Finally, the kinetics of p53 protein family mRNA expression in the digestive gland of animals exposed to B[a]P and crude oil (0.5 ppm) showed partially overlapping trends, characterised by a common down regulation after 1 week exposure. These data should be carefully considered in view of the biological effects of organic pollutants and particularly following spills.

Comparative petroleum hydrocarbons levels and biochemical responses in mussels from hydrothermal vents (Bathymodiolus azoricus) and coastal environments (Mytilus galloprovincialis).

Aliphatic hydrocarbons and PAHs in the whole soft tissues of Bathymodiolus azoricus from three Mid-Atlantic Ridge hydrothermal vents (Menez-Gwen, Lucky Strike and Rainbow), and Mytilus galloprovincialis from three contaminated coastal sites in South Portugal were analysed, and its effects on the digestive gland microsomes mixed-function oxygenase system (MFO) were assessed. Aliphatic hydrocarbons levels were present in the same magnitude in both coastal and hydrothermal environments, while the UCM (unresolved complex mixture) for coastal mussels were higher than in vent mussels. In general, significantly higher PAHs concentrations were found in coastal mussels, compared to B. azoricus where low molecular weight PAHs (2-3 rings) represented the majority of PAHs contrarily to what was observed in M. galloprovincialis. The MFO components were present in both mussel species, and were detected in vent mussels for the first time. However this system seems to have different roles in species from these contrasting environments. In coastal mussels MFO responded to hydrocarbon contamination while response in hydrothermal organisms appeared to be related mainly to endogenous factors.

Ras gene in marine mussels: a molecular level response to petrochemical exposure.

Mussels are susceptible to numerous toxicants and are often employed as bioindicators. This study investigated the status of the ras proto-oncogene in Mytilus galloprovincialis following petrochemical exposure. A M. galloprovincialis homologue of the vertebrate ras gene was isolated, showing conserved sequence in regions of functional importance and a high incidence of polymorphic variation. Mutational damage was investigated in mussels chronically exposed to the water-accommodated fraction of #4 fuel-oil (WAF), and in mussels collected along the NW coast of Portugal in sites with different levels of petrochemical contamination. A ras gene point mutation was identified in the codon 35 of one individual exposed to 12.5% WAF. No mutations were detected in mussels from the WAF control or environmental samples. This represents the first report of a ras gene mutation, experimentally-induced by petrochemical exposure, in an invertebrate species.

Induction of DNA strand breaks in the mussel (Mytilus trossulus) and clam (Protothaca staminea) following chronic field exposure to polycyclic aromatic hydrocarbons from the Exxon Valdez spill.

In 2002, 13 years after the Exxon Valdez spill, mussels and clams were examined for lingering oil exposure and damage. Known oil patches were sampled at four locations, and compared to nearby reference areas (same bay), and were also compared to "hot reference" sites to verify the methods used (Cordova harbor and fresh diesel spill at Port Chalmers). Passive samplers deployed for a month at the sites, along with tissue samples, confirmed that the oiled sites were oiled (fingerprinting back to Exxon Valdez oil) and that reference sites were clean. The highest PAH loads were detected in sub-surface interstitial waters at oiled sites. Exposure at the surface was generally low level, and probably intermittent. DNA damage was assessed in blood cells using sensitive comet analyses. DNA strand breakage was detected in both mussels and clams, with the highest level of damage detected at "hot reference" sites of Cordova harbor and Port Chalmers. Bioavailability and DNA damage at the oiled sties was low, indicating there has been substantial progress in recovery from the spill 13 years before, yet low level bioavailability and damage were still detectable.

Biochemical responses of the marine mussel Mytilus galloprovincialis to petrochemical environmental contamination along the North-western coast of Portugal.

Following the development of urban and industrial centres petrochemical products have become a widespread class of contaminants. The aim of this study was to investigate the effects of petrochemical contamination in wild populations of mussels (Mytilus galloprovincialis) along the NW Atlantic coast of Portugal by applying antioxidant and energetic metabolism parameters as biomarkers. For that, mussels were collected at five sampling sites presenting different petrochemical contamination levels. To evaluate the mussels' antioxidant status, enzymatic activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione S-transferases, as well as glutathione redox status were evaluated in gills and digestive glands of mussels collected from the selected sites. Lipid peroxidation was determined in the same tissues to quantify cellular oxidative damage. Furthermore, to investigate how energetic processes may respond to these contaminants, the activity of NADP(+)-dependent isocitrate dehydrogenase was determined in mussels' digestive glands, and octopine dehydrogenase was determined in mussels' posterior adductor muscle. Furthermore, the concentrations of aliphatic hydrocarbons, unresolved complex mixture and polycyclic aromatic hydrocarbons (PAHs) were quantified in mussels' tissue, and abiotic parameters were quantified in water samples collected at each site. Several biomarkers showed statistically significant differences among sampling sites. The redundancy analysis (RDA) used to perform the integrated analysis of the data showed a clear separation of the sampling sites in three different assemblages, which are in agreement with the PAHs levels found in mussels tissues. In addition, the RDA indicated that some of the selected biomarkers may be influenced by abiotic parameters (e.g. salinity, pH, nitrates and ammonia). The approach selected for this study seems to be suitable for monitoring petrochemical contamination.