Development of Eco-Friendly Hydrophobic and Fouling-Release Coatings for Blue-Growth Environmental Applications: Synthesis, Mechanical Characterization and Biological Activity.

The need to ensure adequate antifouling protection of the hull in the naval sector led to the development of real painting cycles, which involve the spreading of three layers of polymeric material on the hull surface exposed to the marine environment, specifically defined as primer, tie coat and final topcoat. It is already well known that coatings based on suitable silanes provide an efficient and non-toxic approach for the hydrophobic and antifouling/fouling release treatment of surfaces. In the present work, functional hydrophobic hybrid silica-based coatings (topcoats) were developed by using sol-gel technology and deposited on surfaces with the "doctor blade" method. In particular, those organic silanes, featuring opportune functional groups such as long (either fluorinated) alkyl chains, have a notable influence on surface wettability as showed in this study. Furthermore, the hydrophobic behavior of this functionalized coating was improved by introducing an intermediate commercial tie-coat layer between the primer and the topcoat, in order to decrease the wettability (i.e., decreasing the surface energy with a matching increase in the contact angle, CA) and to therefore make such coatings ideal for the design and development of fouling release paints. The hereby synthesized coatings were characterized by optical microscopy, contact angle analysis and a mechanical pull-off test to measure the adhesive power of the coating against a metal substrate typically used in the nautical sector. Analysis to evaluate the bacterial adhesion and the formation of microbial biofilm were related in laboratory and simulation (microcosm) scales, and assessed by SEM analysis.

Design and Development of Fluorinated and Biocide-Free Sol-Gel Based Hybrid Functional Coatings for Anti-Biofouling/Foul-Release Activity.

Biofouling has destructive effects on shipping and leisure vessels, thus producing severe problems for marine and naval sectors due to corrosion with consequent elevated fuel consumption and higher maintenance costs. The development of anti-fouling or fouling release coatings creates deterrent surfaces that prevent the initial settlement of microorganisms. In this regard, new silica-based materials were prepared using two alkoxysilane cross-linkers containing epoxy and amine groups (i.e., 3-Glycidyloxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane, respectively), in combination with two functional fluoro-silane (i.e., 3,3,3-trifluoropropyl-trimethoxysilane and glycidyl-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorononylether) featuring well-known hydro repellent and anti-corrosion properties. As a matter of fact, the co-condensation of alkoxysilane featuring epoxide and amine ends, also mixed with two opportune long chain and short chain perfluorosilane precursors, allows getting stable amphiphilic, non-toxic, fouling release coatings. The sol-gel mixtures on coated glass slides were fully characterized by FT-IR spectroscopy, while the morphology was studied by scanning electron microscopy (SEM), and atomic force microscopy (AFM). The fouling release properties were evaluated through tests on treated glass slides in different microbial suspensions in seawater-based mediums and in seawater natural microcosms. The developed fluorinated coatings show suitable antimicrobial activities and low adhesive properties; no biocidal effects were observed for the microorganisms (bacteria).

Functional Nanohybrids and Nanocomposites Development for the Removal of Environmental Pollutants and Bioremediation.

World population growth, with the consequent consumption of primary resources and production of waste, is progressively and seriously increasing the impact of anthropic activities on the environment and ecosystems. Environmental pollution deriving from anthropogenic activities is nowadays a serious problem that afflicts our planet and that cannot be neglected. In this regard, one of the most challenging tasks of the 21st century is to develop new eco-friendly, sustainable and economically-sound technologies to remediate the environment from pollutants. Nanotechnologies and new performing nanomaterials, thanks to their unique features, such as high surface area (surface/volume ratio), catalytic capacity, reactivity and easy functionalization to chemically modulate their properties, represent potential for the development of sustainable, advanced and innovative products/techniques for environmental (bio)remediation. This review discusses the most recent innovations of environmental recovery strategies of polluted areas based on different nanocomposites and nanohybrids with some examples of their use in combination with bioremediation techniques. In particular, attention is focused on eco-friendly and regenerable nano-solutions and their safe-by-design properties to support the latest research and innovation on sustainable strategies in the field of environmental (bio)remediation.

Antifouling Systems Based on Copper and Silver Nanoparticles Supported on Silica, Titania, and Silica/Titania Mixed Oxides.

Silica, titania, and mixed silica-titania powders have been used as supports for loading 5 wt% Cu, 5 wt% Ag, and 2.5 wt% Cu-2.5 wt% Ag with the aim of providing a series of nanomaterials with antifouling properties. All the solids were easily prepared by the wetness-impregnation method from commercially available chemical precursors. The resulting materials were characterized by several techniques such as X-ray diffraction analysis, X-ray photoelectron spectroscopy, N2 physisorption, and temperature-programmed reduction measurements. Four selected Cu and Ag SiO2- and TiO2-supported powders were tested as fillers for the preparation of marine antifouling coatings and complex viscosity measurements. Titania-based coatings showed better adhesion than silica-based coatings and the commercial topcoat. The addition of fillers enhances the resin viscosity, suggesting better workability of titania-based coatings than silica-based ones. The ecotoxicological performance of the powders was evaluated by Microtox luminescence tests, using the marine luminescent bacterium Vibrio fisheri. Further investigations of the microbiological activity of such materials were carried out focusing on the bacterial growth of Pseudoalteromonas sp., Alteromonas sp., and Pseudomonas sp. through measurements of optical density at 600 nm (OD600nm).

Assessment of the effectiveness of a novel BioFilm-Membrane BioReactor oil-polluted wastewater treatment technology by applying biomarkers in the mussel Mytilus galloprovincialis.

Petrochemical industries and oil refineries are sources of hazardous chemicals into the aquatic environments, and often a leading cause of reduced oxygen availability, thus resulting in adverse effects in biota. This study is an expansion of our previous work on the assessment of the BioFilm-Membrane Bioreactor (BF-MBR) to mitigate the impact of oil-polluted wastewater on marine environments. Specifically, this study evaluated the reduction of selected chemical constituents (hydrocarbons and trace metals) and toxicity related to hypoxia and DNA damage to mussels Mytilus galloprovincialis, before and after treatment of oil-polluted wastewater with the BF-MBR. The application of a multidisciplinary approach provided evidence of the efficiency of BF-MBR to significantly reducing the pollutants load from oily contaminated seawaters. As result, the health status of mussels was preserved by a hypoxic condition due to oily pollutants, as evidenced by the modulation in the gene expression of HIF-1α and PHD and changes in the level of hypotaurine and taurine. Moreover, ameliorative effects in the energy metabolism were also found in mussel gills showing increased levels of glycogen, glucose and ATP, as well as a mitigated genotoxicity was revealed by the Micronucleus and Comet assays. Overall, findings from this study support the use of the BF-MBR as a promising treatment biotechnology to avoid or limiting the compromise of marine environments from oil pollution.

Characterization of Five Psychrotolerant Alcanivorax spp. Strains Isolated from Antarctica.

Five psychrotolerant Alcanivorax spp. strains were isolated from Antarctic coastal waters. Strains were screened for molecular and physiological properties and analyzed regarding their growth capacity. Partial 16S rDNA, alk-B1, and P450 gene sequencing was performed. Biolog EcoPlates and the API 20E test were used to evaluate metabolic and biochemical profiles. Bacterial growth in sodium acetate was determined at 4, 15, 20, and 25 °C to evaluate the optimal temperature. Furthermore, the ability of each strain to grow in a hydrocarbon mixture at 4 and 25 °C was assayed. Biosurfactant production tests (drop-collapse and oil spreading) and emulsification activity tests (E24) were also performed. Concerning results of partial gene sequencing (16S rDNA, alk-B1, and P450), a high similarity of the isolates with the same genes isolated from other Alcanivorax spp. strains was observed. The metabolic profiles obtained by Biolog assays showed no significant differences in the isolates compared to the Alcanivorax borkumensis wild type. The results of biodegradative tests showed their capability to grow at different temperatures. All strains showed biosurfactant production and emulsification activity. Our findings underline the importance to proceed in the isolation and characterization of Antarctic hydrocarbon-degrading bacterial strains since their biotechnological and environmental applications could be useful even for pollution remediation in polar areas.

New insights into the structure and function of the prokaryotic communities colonizing plastic debris collected in King George Island (Antarctica): Preliminary observations from two plastic fragments.

In Antarctic regions, the composition and metabolic activity of microbial assemblages associated with plastic debris ("plastisphere") are almost unknown. A macroplastic item from land (MaL, 30 cm) and a mesoplastic from the sea (MeS, 4 mm) were collected in Maxwell Bay (King George Island, South Shetland) and analyzed by Fourier transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR), which confirmed a polystyrene foam and a composite high-density polyethylene composition for MaL and MeS, respectively. The structure and function of the two plastic-associated prokaryotic communities were studied by complementary 16S ribosomal RNA gene clone libraries, total bacterioplankton and culturable heterotrophic bacterial counts, enzymatic activities of the whole community and enzymatic profiles of bacterial isolates. Results showed that Gamma- and Betaproteobacteria (31% and 28%, respectively) dominated in MeS, while Beta- and Alphaproteobacteria (21% and 13%, respectively) in MaL. Sequences related to oil degrading bacteria (Alcanivorax,Marinobacter) confirmed the known anthropogenic pressure in King George Island. This investigation on plastic-associated prokaryotic structure and function represents the first attempt to characterize the ecological role of plastisphere in this Antarctic region and provides the necessary background for future research on the significance of polymer type, surface characteristics and environmental conditions in shaping the plastisphere.

Genomic characterization of a polyvalent hydrocarbonoclastic bacterium Pseudomonas sp. strain BUN14.

Bioremediation offers a viable alternative for the reduction of contaminants from the environment, particularly petroleum and its recalcitrant derivatives. In this study, the ability of a strain of Pseudomonas BUN14 to degrade crude oil, pristane and dioxin compounds, and to produce biosurfactants, was investigated. BUN14 is a halotolerant strain isolated from polluted sediment recovered from the refinery harbor on the Bizerte coast, north Tunisia and capable of producing surfactants. The strain BUN14 was assembled into 22 contigs of 4,898,053 bp with a mean GC content of 62.4%. Whole genome phylogeny and comparative genome analyses showed that strain BUN14 could be affiliated with two validly described Pseudomonas Type Strains, P. kunmingensis DSM 25974T and P. chloritidismutans AW-1T. The current study, however, revealed that the two Type Strains are probably conspecific and, given the priority of the latter, we proposed that P. kunmingensis DSM 25974 is a heteronym of P. chloritidismutans AW-1T. Using GC-FID analysis, we determined that BUN14 was able to use a range of hydrocarbons (crude oil, pristane, dibenzofuran, dibenzothiophene, naphthalene) as a sole carbon source. Genome analysis of BUN14 revealed the presence of a large repertoire of proteins (154) related to xenobiotic biodegradation and metabolism. Thus, 44 proteins were linked to the pathways for complete degradation of benzoate and naphthalene. The annotation of conserved functional domains led to the detection of putative genes encoding enzymes of the rhamnolipid biosynthesis pathway. Overall, the polyvalent hydrocarbon degradation capacity of BUN14 makes it a promising candidate for application in the bioremediation of polluted saline environments.

Phenotypic Variations of Oleispira antarctica RB-8(T) in Different Growth Conditions.

The peculiar biotechnological applications of Oleispira spp. in the natural cleansing of oil-polluted marine systems stimulated the study of the phenotypic characteristics of the Oleispira antarctica RB-8(T) strain and modifications of these characteristics in relation to different growth conditions. Bacterial abundance, cell size and morphology variations (by image analysis) and hydrocarbon degradation (by gas chromatography with flame ionization detection, GC-FID) were analysed in different cultures of O. antarctica RB-8(T). The effects of six different hydrocarbon mixtures (diesel, engine oil, naval oil waste, bilge water, jet fuel and oil) used as a single carbon source combined with two different growth temperatures (4° and 15 °C) were analysed (for 22 days). The data obtained showed that the mean cell volume decreased with increasing experimental temperature. Three morphological bacterial shapes were identified: spirals, rods and cocci. Morphological transition from spiral to rod and coccoid shapes in relation to the different substrates (oil mixtures) and/or growth temperatures was observed, except for one experimental condition (naval oil waste) in which spiral bacteria were mostly dominant. Phenotypic traits and physiological status of hydrocarbon-degrading bacteria showed important modifications in relation to culture conditions. These findings suggest interesting potential for strain RB-8(T) for ecological and applicative purposes.

Innovative, ecofriendly biosorbent-biodegrading biofilms for bioremediation of oil- contaminated water.

Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes. In this study, innovative and ecofriendly biosorbent-biodegrading biofilms have been developed in order to remediate oil-contaminated water. This was achieved by immobilizing hydrocarbon-degrading gammaproteobacteria and actinobacteria on biodegradable oil-adsorbing carriers, based on polylactic acid and polycaprolactone electrospun membranes. High capacities for adhesion and proliferation of bacterial cells were observed by scanning electron microscopy. The bioremediation efficiency of the systems, tested on crude oil and quantified by gas chromatography, showed that immobilization increased hydrocarbon biodegradation by up to 23 % compared with free living bacteria. The resulting biosorbent biodegrading biofilms simultaneously adsorbed 100 % of spilled oil and biodegraded more than 66 % over 10 days, with limited environmental dispersion of cells. Biofilm-mediated bioremediation, using eco-friendly supports, is a low-cost, low-impact, versatile tool for bioremediation of aquatic systems.

Assessing the Effect of Contaminated and Restored Marine Sediments in Different Experimental Mesocosms Using an Integrated Approach and Mytilus galloprovincialis as a Model.

The research presented here was conducted to ascertain the effectiveness of recovery technologies in remediating a compromised marine environment. The multidisciplinary approach aims to integrate traditional chemical-physical analysis and to assess the biological parameters of Mytilus galloprovincialis within different experimental mesocosms (W, G, and B). In particular, this system was designed to reproduce sediment resuspension in a marine environment, which is thought to be one cause of contaminant release. The study combined morphological and ultrastructural observations with DNA damage assessment and mRNA expression of those genes involved in cellular stress responses. The tissues of mussels maintained in the polluted mesocosm showed a higher accumulation of Pb and Hg than in those maintained in restored mesocosm. This observation correlates well with mRNA expression of MT10 and data on DNA damage. The outcome of the biological evaluation consolidates the chemical characterization and supports the concept that the remediation method should be evaluated at an early stage, both to analytically determine the reduction of toxic components and to assess its ultimate impact on the biological system.

Mesocosm System to Evaluate BF-MBR Efficacy in Mitigating Oily Wastewater Discharges: an Integrated Study on Mytilus galloprovincialis.

This work presents the results of recovery efficacy of the system "BioFilm Membrane BioReactor" (BF-MBR), in the treatment of oily contaminated seawaters. To this aim, we proposed a multidisciplinary approach that integrates traditional chemical-physical measures together with the assessment on biological sentinel Mytilus galloprovincialis, maintained in a medium-scale artificial system named mesocosm. The setup included: (1) a mesocosm consisting of uncontaminated seawater; (2) a mesocosm composed of an untreated oily wastewater discharge; and (3) a mesocosm receiving the same oily wastewater previously treated by a BF-MBR pilot scale plant. The multidisciplinary approach that included traditional chemical measures on mesocosms together with the evaluation of morphological organization, mRNA expression of those genes involved in cellular stress response, immunohistochemistry and metabolomic analysis on mussel tissues, was able to provide a robust and holistic evidence of how the proposed treatment is able to reduce the overall impact of oily wastewater discharges on the marine ecosystem.

The genome of Alcaligenes aquatilis strain BU33N: Insights into hydrocarbon degradation capacity.

Environmental contamination with hydrocarbons though natural and anthropogenic activities is a serious threat to biodiversity and human health. Microbial bioremediation is considered as the effective means of treating such contamination. This study describes a biosurfactant producing bacterium capable of utilizing crude oil and various hydrocarbons as the sole carbon source. Strain BU33N was isolated from hydrocarbon polluted sediments from the Bizerte coast (northern Tunisia) and was identified as Alcaligenes aquatilis on the basis of 16S rRNA gene sequence analysis. When grown on crude oil and phenanthrene as sole carbon and energy sources, isolate BU33N was able to degrade ~86%, ~56% and 70% of TERHc, n-alkanes and phenanthrene, respectively. The draft genome sequence of the A. aquatilis strain BU33N was assembled into one scaffold of 3,838,299 bp (G+C content of 56.1%). Annotation of the BU33N genome resulted in 3,506 protein-coding genes and 56 rRNA genes. A large repertoire of genes related to the metabolism of aromatic compounds including genes encoding enzymes involved in the complete degradation of benzoate were identified. Also genes associated with resistance to heavy metals such as copper tolerance and cobalt-zinc-cadmium resistance were identified in BU33N. This work provides insight into the genomic basis of biodegradation capabilities and bioremediation/detoxification potential of A. aquatilis BU33N.

Do plastics serve as a possible vector for the spread of antibiotic resistance? First insights from bacteria associated to a polystyrene piece from King George Island (Antarctica).

The retrieval of a polystyrene macro-plastic piece stranded on the shores in King George Island (South Shetlands, Antarctica) gave the opportunity to explore the associated bacterial flora. A total of 27 bacterial isolates were identified by molecular 16s rRNA gene sequencing and 7 strains were selected and screened for their ability to produce biofilm and antibiotic susceptibility profiles. All the bacterial isolates were able to produce biofilm. The Kirby-Bauer disk diffusion susceptibility test to 34 antibiotics showed multiple antibiotic resistances against the molecules cefuroxime and cefazolin (belonging to cephalosporins), cinoxacin (belonging to quinolones) and ampicillin, amoxicillin + clavulanic acid, carbenicillin and mezlocillin (belonging to beta-lactams). The obtained results suggest that plastics can serve as vectors for the spread of multiple resistances to antibiotics across Antarctic marine environments and underline the relevance of future studies on this topic.

Effects of microplastics on trophic parameters, abundance and metabolic activities of seawater and fish gut bacteria in mesocosm conditions.

Plastic pollution is an emerging threat with severe implications on animals' and environmental health. Nevertheless, interactions of plastic particles with both microbial structure and metabolism are a new research challenge that needs to be elucidated yet. To improve knowledge on the effects played by microplastics on free-living and fish gut-associated microbial community in aquatic environments, a 90-day study was performed in three replicated mesocosms (control-CTRL, native polyvinyl chloride-MPV and weathered polyvinyl chloride-MPI), where sea bass specimens were hosted. In CTRL mesocosm, fish was fed with no-plastic-added food, whilst in MPV and MPI food was supplemented with native or exposed to polluted waters polyvinylchloride pellets, respectively. Particulate organic carbon (POC) and nitrogen, total and culturable bacteria, extracellular enzymatic activities, and microbial community substrate utilization profiles were analyzed. POC values were lower in MPI than MPV and CRTL mesocosms. Microplastics did not affect severely bacterial metabolism, although enzymatic activities decreased and microbes utilized a lower number of carbon substrates in MPI than MPV and CTRL. No shifts in the bacterial community composition of fish gut microflora were observed by denaturing gradient gel electrophoresis fingerprinting analysis.

Microbial communities of polluted sub-surface marine sediments.

Microbial communities of coastal marine sediment play a key role in degradation of petroleum contaminants. Here the bacterial and archaeal communities of sub-surface sediments (5-10 cm) of the chronically polluted Priolo Bay (eastern coast of Sicily, Italy), contaminated mainly by n-alkanes and biodegraded/weathered oils, were characterized by cultural and molecular approaches. 16S-PCR-DGGE analysis at six stations, revealed that bacterial communities are highly divergent and display lower phylogenetic diversity than the surface sediment; sub-surface communities respond to oil supplementation in microcosms with a significant reduction in biodiversity and a shift in composition; they retain high biodegradation capacities and host hydrocarbon (HC) degraders that were isolated and identified. HC-degrading Alfa, Gamma and Epsilon proteobacteria together with Clostridia and Archaea are a common feature of sub-surface communities. These assemblages show similarities with that of subsurface petroleum reservoirs also characterized by the presence of biodegraded and weathered oils where anaerobic or microaerophilic syntrophic HC metabolism has been proposed.

Evaluation of biomarkers in Mytilus galloprovincialis as an integrated measure of biofilm-membrane bioreactor (BF-MBR) system efficiency in mitigating the impact of oily wastewater discharge to marine environment: a microcosm approach.

The large volumes of oily wastewater discharged to marine environment cause heavy impacts on the coastal marine ecosystem. The selection of an appropriate technology to reduce these impacts should be based on the respect of the discharge limits and on the effective assessment and monitoring of its effects on biological organism preservation. To this aim, we set up a controlled microcosm-scale system to compare the effects of a treated and untreated oily wastewater discharge in which the restore process is performed through a Membrane Bio-Reactor. The system is completed by other three microcosms to control and isolate any possible concurrent effect on the Mytilus galloprovincialis, used as sentinel organism. Mytilus galloprovincialis have been kept in all these microcosms, and then mRNA expression and morphology were evaluated on gills and digestive gland. The genes considered in this work are Heat Shock Protein 70 and Metallothionein 10, involved in response to physicochemical sublethal stressors, Superoxide dismutase 1, Catalase, and Cytochrome P450 involved in oxidative stress response. Our results evidenced a significant overexpression, both in gills and digestive gland, of HSP70 in samples maintained in the microcosm receiving the untreated effluent, and of MT10 in those animals kept in microcosm where the effluent was treated. Even though the mRNA modifications are considered "primary" and transient responses which do not always correspond to protein content, the study of these modifications can help to gain insights into the mechanisms of action of xenobiotic exposure. Morphological analysis suggested that, although different, depending on the microcosm, the most serious damages were found in the gill epithelium accompanied with severe haemocyte infiltration, whilst in digestive gland the tissue architecture alterations and the haemocyte infiltration were less pronounced. These observations suggest that the immune system was activated as a general response to stressful stimuli such as the presence of toxic compounds. Moreover, the results indicate that the treatment process is useful. In fact, samples derived from the microcosm receiving the treated effluent, even though presenting some signs of stress, seemed to partially recover the normal structure, although their mRNA expression indicated some cellular suffering.

Multilevel characterization of marine microbial biodegradation potentiality by means of flow-modulated comprehensive two-dimensional gas chromatography combined with a triple quadrupole mass spectrometer.

The present research is focused on the use of a triple quadrupole mass spectrometer (QqQ MS) coupled with flow modulated comprehensive two-dimensional gas chromatography (FM GC × GC) for a multilevel elucidation of biodegradation potentiality of natural marine microbial populations during a bioremediation (biostimulation) treatment. The crude oil used for the evaluation of the bioremediation process, namely Dansk Blend Pier E1, represents a very complex sample. Hence, in order to understand the metabolic activity of microbial populations during the bioremediation process, a GC × GC system was used. The high separation power has allowed a detailed characterization of the different chemical families; moreover, thanks to the high acquisition frequency of the QqQ MS spectrometer, both full scan and multiple reaction monitoring (MRM) data were acquired in the same run. By using this system, both qualitative analysis of untargeted hydrocarbons mixture (crude oil) and qualitative analysis of biomarker compounds, present in low amount and often hindered under the bulk of the sample (i.e. adamantanes, diamantanes, steranes and hopanes), were performed simultaneously. The bioremediation capability of biostimulated bacteria was evaluated at four (T4), eight (T8) and fourteen (T14) days. Progressive degradation of linear, branched, and aromatic hydrocarbons, adamantanes, and diamantanes has been showed, whereas, results underline the lack of any kind of activity against steranes, and hopanes.

Shell fluctuating asymmetry in the sea-dwelling benthic bivalve Mytilus galloprovincialis (Lamarck, 1819) as morphological markers to detect environmental chemical contamination.

Investigations on asymmetries showed that deviations from perfect bilateral symmetry are interpreted as environmental changes inducing developmental instability. Since morphological abnormalities increase with pollution, deformations may be considered indicators of the organism exposition to pollution. Therefore, the onset of asymmetry in otherwise normally symmetrical traits has been used as a measure of some stresses as well. In this context, we studied how marine pollution affects the valve morphological alterations in the mussel Mytilus galloprovincialis. We used 180 specimens (30 per site) from the aquaculture area of Goro (River Po delta, northern Adriatic Sea), translocated, and released within 50 × 50 × 50 cm cages in five sites: two disturbed and one undisturbed near Naples (eastern Tyrrhenian Sea), and one disturbed and one undisturbed near Siracusa (western Ionian Sea). Disturbed sites were stressed by heavy industrialization and heavy tankers traffic of crude and refined oil, and were defined basing on sediment contamination. In particular, by the cone-beam computed tomography we obtained 3D virtual valve surfaces to be analyzed by the geometric morphometric techniques. Specifically, we focused the levels of the shell shape fluctuating asymmetry in relation to the degrees of marine pollution in different sites of the Tyrrhenian Sea. The Mahalanobis distances (interpreted as proxy of the individual shape asymmetry deviation from the mean asymmetry) significantly regressed with the sediment contamination gradient. Indeed, although the left-right differences were normally distributed in each studied site, the individual asymmetry scores (IAS) significantly varied amongst the investigated sites. IAS showed higher values in disturbed areas than those of undisturbed ones in both Tyrrhenian and Ionian Sea. Our results are consistent with past studies on molluscans and other taxa, demonstrating some detrimental effects of chemicals on organisms, although the investigated morphological marker did not discriminate the real disturbance source. Our findings indicate that the mussels act as a prognostic tool for sea pollution levels driving detrimental effects on benthic community.

Effects of petrochemical contamination on caged marine mussels using a multi-biomarker approach: Histological changes, neurotoxicity and hypoxic stress.

This work was designed to evaluate the biological effects of petrochemical contamination on marine mussels. Mytilus galloprovincialis, widely used as sentinel organisms in biomonitoring studies, were caged at the "Augusta-Melilli-Priolo" industrial site (eastern Sicily, Italy), chosen as one of the largest petrochemical areas in Europe, and Brucoli, chosen as reference site. Chemical analyses of sediments at the polluted site revealed high levels of PAHs and mercury, exceeding the national and international guideline limits. In mussels from the polluted site, severe morphological alterations were observed in gills, mainly involved in nutrient uptake and gas exchange. Changes in serotonergic and cholinergic systems, investigated through immunohistochemical, metabolomics and enzymatic approaches, were highlighted in gills, as well as onset of hypoxic adaptive responses with up-regulation of hypoxia-inducible factor transcript. Overall, the application of a multi-biomarker panel results effective in assessing the biological effects of petrochemical contamination on the health of aquatic organisms.