Biological responses of the polychaete Hediste diversicolor (O.F.Müller, 1776) to inorganic mercury exposure: A multimarker approach.

Mercury (Hg) is a global priority pollutant given its relevance in terms of environmental damage and threat to human health. Its ecotoxicity was tested using the benthic keystone species Hediste diversicolor as target species. After 10 days of exposure to different levels of inorganic Hg (10 and 50 µg L-1), bioaccumulation and a wide range of biological responses were evaluated at different biological levels, including biomarkers of exposure, neurotoxicity, oxidative stress, genotoxicity and cytochemistry. In controlled laboratory conditions, Hg was taken up by H. diversicolor in a dose-response manner and caused a range of biological responses, including oxidative stress (GSTs, GPx, GSH-2GSSG, and TOSCA), neurotoxicity (AChE), and cellular damages at the membrane level (LFs, NLs, Ca2+-ATPase); however, it did not cause significant DNA damage or mortality. This study confirms the capability of H. diversicolor to tolerate high levels of metals and clarifies the mechanisms underlying the damage caused by waterborne Hg and the defense mechanisms, activated in this species. In particular, detoxification of the inorganic form of Hg in this species was found to be strongly related to glutathione expression and several antioxidant enzymes of the antioxidant system. This process also efficiently minimized negative effects on DNA and prevented death, but was not sufficient to avoid neurotoxicity and some cellular damages, mainly at the intestinal level.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the "mortality" is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Preliminary study to characterize plastic polymers using elemental analyser/isotope ratio mass spectrometry (EA/IRMS).

Plastic waste is a growing global environmental problem, particularly in the marine ecosystems, in consideration of its persistence. The monitoring of the plastic waste has become a global issue, as reported by several surveillance guidelines proposed by Regional Sea Conventions (OSPAR, UNEP) and appointed by the EU Marine Strategy Framework Directive. Policy responses to plastic waste vary at many levels, ranging from beach clean-up to bans on the commercialization of plastic bags and to Regional Plans for waste management and recycling. Moreover, in recent years, the production of plant-derived biodegradable plastic polymers has assumed increasing importance. This study reports the first preliminary characterization of carbon stable isotopes (δ13C) of different plastic polymers (petroleum- and plant-derived) in order to increase the dataset of isotopic values as a tool for further investigation in different fields of polymers research as well as in the marine environment surveillance. The δ13C values determined in different packaging for food uses reflect the plant origin of "BIO" materials, whereas the recycled plastic materials displayed a δ13C signatures between plant- and petroleum-derived polymers source. In a preliminary estimation, the different colours of plastic did not affect the variability of δ13C values, whereas the abiotic and biotic degradation processes that occurred in the plastic materials collected on beaches and in seawater, showed less negative δ13C values. A preliminary experimental field test confirmed these results. The advantages offered by isotope ratio mass spectrometry with respect to other analytical methods used to characterize the composition of plastic polymers are: high sensitivity, small amount of material required, rapidity of analysis, low cost and no limitation in black/dark samples compared with spectroscopic analysis.

Amount and distribution of neustonic micro-plastic off the western Sardinian coast (Central-Western Mediterranean Sea).

A plethora of different sampling methodologies has been used to document the presence of micro-plastic fragments in sea water. European Marine Strategy suggests to improve standard techniques to make future data comparable. We use Manta Trawl sampling technique to quantify abundance and distribution of micro-plastic fragments in Sardinian Sea (Western Mediterranean), and their relation with phthalates and organoclorine in the neustonic habitat. Our results highlight a quite high average plastic abundance value (0.15 items/m(3)), comparable to the levels detected in other areas of the Mediterranean. "Site" is the only factor that significantly explains the differences observed in micro-plastic densities. Contaminant levels show high spatial and temporal variation. In every station, HCB is the contaminant with the lowest concentration while PCBs shows the highest levels. This work, in line with Marine Strategy directives, represents a preliminary study for the analysis of plastic impact on marine environment of Sardinia.

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

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

A methodological approach to understand functional relationships between ecological indices and human-induced pressures: the case of the Posidonia oceanica meadows.

The European Water Framework Directive (WFD) 2000/60/EC requests the achievement of the Good Status for all surface waters, including the coastal waters, by 2015. In order to check compliance with the needs of Directive, Italian national monitoring data on Posidonia oceanica meadows have been explored and the relationships among the Posidonia Rapid and Easy Index (PREI), and human-induced pressures have been analyzed along the Italian coasts. The aim of this work is to establish functional relationships between a response variable (i.e. the PREI) and a set of potential pressure (i.e. land use, potential organic and nutrient loading, pesticides) and status (i.e. transparency, trophic level and stability of the water column) indicators in a quantitative way. The ecological responses of coastal marine environment have been evaluated using appropriate statistical tools, such as the multiple linear regression analyses and "linear programming" techniques. Results show that more than 70% of the variability of the P. oceanica meadows status, expressed as PREI value, is significantly explained only by a few pressure/status indicators (namely: potential organic load, specific nitrogen load, natural areas extent, water column transparency), among all those initially considered in the model. The application of the proposed model could allow decision makers to better address remedial actions and to achieve the environmental targets proposed by the EU Directives.

Long-term lethal toxicity test with the crustacean Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded. . Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Toxicity evaluation of diethylene glycol and its combined effects with produced waters of off-shore gas platforms in the Adriatic Sea (Italy): bioassays with marine/estuarine species.

Diethylene glycol (DEG) is commonly used to dehydrate natural gas in off-shore extraction plants and to prevent formation of gas hydrates. It may be released into the sea accidentally or in discharged produced waters (PWs). PWs samples from off-shore gas platforms in the Adriatic Sea (Italy) have been used in this study. The objectives of the study were: a) to evaluate the toxicity of DEG for marine organisms; b) to evaluate if a high DEG content in PWs may alter their toxicity; c) to verify whether the DEG threshold concentration established by the Italian legislation (3.5 g/l) for PWs discharged at sea is safe for marine environment. Ten different species (Vibrio fischeri, Phaeodactylum tricornutum, Dunaliella tertiolecta, Brachionus plicatilis, Artemia franciscana, Tigropus fulvus, Mytilus galloprovincialis, Crassostrea gigas, Tapes philippinarum and Dicentrarchus labrax) have been exposed to DEG; four of these species were also exposed to PWs in combination with DEG. The results showed that: a) DEG is not toxic at levels normally detected in Adriatic PWs; b) DEG in combination with PW showed mainly additive or synergistic effects; c) short-term bioassays showed that the DEG limit of 3.5 g/l could be acceptable.

Seasonal, spatial and inter-annual variations of trace metals in mussels from the Adriatic sea: a regional gradient for arsenic and implications for monitoring the impact of off-shore activities.

Mussels are widely used as bioindicator organisms for monitoring chemical pollutants including trace metals. These elements are natural constituents in the marine environment and their basal concentrations in the organisms can be influenced by several environmental and biological factors. The aim of this work was to extend our knowledge on the natural variability of trace metals in mussels tissues, focusing on seasonal and inter-annual fluctuations in a coastal reference site of the Adriatic coast (Portonovo); a total of 39 samplings were performed during 5 years, providing an extended data-set for tissue levels of As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn. Concentrations of trace metals in mussels tissues revealed marked seasonal fluctuations with significant differences between various sampling years. Such fluctuations appeared mostly related to phytoplanctonic blooms and especially to reproductive cycle which exhibited a certain inter-annual shift of the gametogenesis period. Lower concentrations were measured in summer months for the majority of elements while a different seasonal cycle was observed for arsenic, not correlated with gonadic development, neither with other elements. Chemical speciation of arsenic was characterized to distinguish compounds of natural origin from those potentially reflecting an anthropogenic impact. Arsenobetaine and arsenocholine were always the predominant forms (up to 85% of total arsenic), while a significant increase of dimethylarsine and trimethylarsine oxide in spring (24% of total arsenic) might reflect the effect of phytoplanctonic bloom on both geochemistry and trophic transfer of this element. A significant inter-annual variability was observed for both the seasonal cycle and the range of values measured for all the elements, with particularly marked differences for arsenic, ranging from less than 10 to more than 40 microg/g in summer periods of different years. Data obtained on reference mussels were used to assess the impact of 41 off-shore platforms distributed along 5 nautical districts in the Northern and Central Adriatic Sea. Organisms sampled on these structures from 2001 to 2005 exhibited a certain enrichment of cadmium and zinc, probably associated to the use of anodic electrodes. Marked annual and geographical variations were measured for concentrations of arsenic in mussels of different platforms. However, the comparison with results of Portonovo, allowed to exclude the anthropogenic impact of exploitation activities and revealed a natural regional gradient of arsenic levels in mussels tissues associated to the changing influence of the Po river runoff on seawater salinity. In particular the higher concentrations measured in organisms sampled from platforms in the Central compared to Northern Adriatic confirmed a significant relationship between salinity and arsenic bioaccumulation, consistent with a role of arsenobetaine as an acquired osmolyte for mussels. The overall results confirmed the importance of natural variability when assessing the potential impact of anthropogenic activities.

Extracellular carbohydrates released by the marine diatoms Cylindrotheca closterium, Thalassiosira pseudonana and Skeletonema costatum: effect of P-depletion and growth status.

A laboratory study was performed on the extracellular production of carbohydrates by the marine diatoms Cylindrotheca closterium, Thalassiosira pseudonana and Skeletonema costatum. The investigation was aimed at elucidating the role of P-starvation and growth status on abundance and chemical characteristics of the released non-attached polysaccharides. Inorganic phosphorus depletion determined an increase of total polysaccharides in all species examined compared to nutrient-replete (complete f/2) conditions. The highest abundance of polysaccharides per unit cell was found in T. pseudonana (28.4 micromol C 10(-6) cells), followed by C. closterium (2.56 micromol C 10(-6) cells) and S. costatum (1.18 micromol C 10(-6) cells). Maximum production rates were found at the transition between exponential and stationary growth phase. Gas-chromatographic analysis of the dissolved fraction showed glucose to be the most abundant monomer in exponentially growing, P-replete cultures (81.6%, 90% and 32% as molar percentage of total aldoses in C. closterium, T. pseudonana and S. costatum, respectively). A strong reduction in glucose was found in C. closterium, but not in T. pseudonana and S. costatum, under P-depleted conditions. Species-specific variations in the amount and aldose signatures of the released polysaccharides according to nutrient status and growth conditions can provide useful insights on the production and persistence of these organic compounds in the water column.