Assessment of the eutrophication status at Mediterranean sub-basin scale, within the European Marine Strategy Framework Directive.

The aim of this work is to define harmonized reference conditions and assessment thresholds for selected criteria elements of the Marine Strategy Framework Directive (MSFD) Descriptor 5 (Eutrophication) in the Eastern Mediterranean Sea and to test if a tool for integrated assessment of the status of marine systems can be used as a common methodological approach. In this frame, we tested two statistical approaches in order to set threshold values for four criteria of Descriptor 5: nutrients, chlorophyll a, transparency and dissolved oxygen in the bottom waters. It is noteworthy that this work revealed the need to apply common procedures in data treatment and assessment evaluation. This is the first attempt to set common methods for the assessment of eutrophication in the Eastern Mediterranean, which is essential in marine environments, especially those shared by several countries. To this end, we have applied common criteria and metrics and established thresholds "Good" and "Moderate" for nutrients, chlorophyll a, transparency and dissolved oxygen in the bottom waters for the different Water Types of the Adriatic and Aegean Seas (I, II, IIIW, IIIE), based on datasets provided by Italy, Slovenia, Croatia and Greece. The selected criteria elements were common for all countries, providing a unified approach to GES assessment of two case study areas: the Adriatic Sea and the Saronikos Gulf. Dissolved Inorganic Nitrogen (DIN) threshold values of 15.6, 6.85, 1.61 and 2.11 µmol L-1 were set for the Water Types I, II, IIIW and IIIE, respectively. We also tested if an aggregation tool for GES assessment, such as Nested Environmental status Assessment Tool (NEAT), could be used as a common methodological approach. The comparison of NEAT with TRIX showed good comparability. In this end, NEAT can be used as a useful and much needed assessment tool for assessing eutrophication status of the marine.

Vulnerability to bioinvasions: Current status, risk assessment and management of ballast water through a regional approach - the Adriatic Sea.

The United Nations recognised the transfer of invasive species across natural barriers as one of the greatest pressures to the world's oceans and seas. The BWM Convention sets the global standards on ballast water management (BWM) requirements, while recognising that regional and local specifics have to be considered for its effective implementation. In the Adriatic Sea cross-border activities were conducted to provide for essential information/data and tools to support a regionally coordinated implementation of the BWM Convention. This special issue contains 18 publications that include results and recommendations from studying the ballast water and management issues through sampling of ballast water on vessels, risk assessment for exemptions and BWM, biological and chemical port baseline surveys and monitoring conducted in ports along the Adriatic Sea coast, oceanographic conditions, ballast water sediment issues and their management in ports, and the implementation options of the BWM Convention through the Adriatic States' environmental law and institutions cooperation. Essential data and tools to support a regional approach in the implementation of the BWM Convention were provided, and are therefore available to the administrations of the Adriatic countries to enable protection of the Adriatic Sea environment, human health property and resources from negative impacts of ballast water being discharged in the area. Data, approaches and tools provided here may be helpful in any other region to support an effective BWM Convention implementation.

Strategy of port baseline surveys (PBS) in the Adriatic Sea.

Port baseline surveys (PBS) provide species inventories in and around ports, with a focus on non-indigenous species that may have been introduced by vessels, primarily via ballast water. PBS are an essential tool to support effective management strategies for non-indigenous as well as native harmful aquatic organisms and pathogens (HAOP). This paper describes the methodology of PBS that were conducted in 12 Adriatic ports. The PBS employed existing protocols that were adapted to meet the characteristics of the Adriatic sites. Their results are reported in several papers included in this special issue, each of which is devoted to a specific community. An overview of existing surveys protocols - which provide valuable support to decision-making and to design effective monitoring of non-indigenous species - is also supplied.

Dispersant approval procedures in France and Italy: A comparative ecotoxicity study.

A research project has been performed to the request of the RAMOGE Executive Secretariat to identify differences between dispersant approval procedures in France and Italy and propose ways to harmonize them. A collaborative study has been conducted by CEDRE (Centre of Documentation, Research and Experimentation on Accidental Water Pollution) and ISPRA (Italian Institute for Environmental Protection and Research) to: a) compare current approval procedures in Italy and France with identification of differences and commonalities; b) carry out toxicity tests using both procedures on two selected dispersants; c) propose a common approach between Italy and France. The results showed that, because of the differences in ecotoxicological tests and in the evaluation criteria used, the outcomes on the same products could be different in Italy and in France. Both tested dispersants met the French requirements for approval (LC50 ≥ 10 times reference toxicant), while only one dispersant met the Italian approval criterion (EC50 > 10mg/L). A possible way of harmonizing the approval procedures could be to increase the number of test organisms in the French procedure, which currently only uses one crustacean species. Furthermore, a common criterion for toxicity assessment should be discussed and agreed.

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.

Modulation of CYP1A and genotoxic effects in European seabass (Dicentrarchus labrax) exposed to weathered oil: a mesocosm study.

The aim of this study was to assess medium-term toxicity of weathered oil on European seabass. A mesocosm system reproducing an oil spill at sea was applied. Fish were collected after 48 h, 7, 30 and 60 days. Cyp1a gene transcription, EROD and UDPGT activities, bile PAHs metabolites and micronuclei frequency were investigated. A progressive disappearance of low molecular weight n-alkanes and PAHs in the water of the mesocosm occurred during the experimentation. Fishes exposed to oil displayed a significant increase of cyp1a expression and EROD activity during the entire experiment as well as higher concentrations of PAHs metabolites in bile. Micronulei frequency resulted significantly higher during all experiment in oil exposed sea bass compared to controls. The results highlight the environmental risk associated with the release of oil products at sea and confirm the adopted parameters as useful tools for studying the impact of accidental oil spills on fish.

The use of a test battery in marine ecotoxicology: the acute toxicity of sodium dodecyl sulfate.

For a toxicity assessment of substances entering the marine environment, it is preferable to carry out ecotoxicological tests on a base-set of taxa utilizing target species belonging to different trophic levels. In this study a battery composed of Vibrio fischeri (bacteria), Dunaliella tertiolecta (algae), Tigriopus fulvus (crustacea), Paracentrotus lividus (echinodermata), and Dicentrarchus labrax (pisces) was used for acute toxicity testing of sodium dodecyl sulfate (SDS). SDS is an anionic surfactant widely employed in industry, agriculture, and domestic usage and therefore is found in abundance in the environment, particularly in the sea. The mean values of EC50 obtained were 2.6, 4.8, 7.4, 3.2, 7.3 mg L(-1), respectively, for V. fischeri, D. tertiolecta, T. fulvus, P. lividus, and D. labrax. The results indicate the high acute toxicity of SDS with respect to all the trophic levels represented by the target species. In addition, they highlight the usefulness of employing a base-set of taxa rather than a single species in toxicological tests, in order to obtain more reliable information for the evaluation of toxicity and potential hazards to the marine environment of selected substances.

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.

Bacterial uptake of DOM released from P-limited phytoplankton.

The growth and the structure of a coastal bacterioplankton community were monitored in short-term bottle experiments in order to investigate the bacterial uptake of extracellular organic carbon released by the diatom Cylindrotheca closterium grown under P-balanced and P-depleted conditions. Bacterial specific growth rates and carbon demand were significantly lower in the exudates from P-depleted algae (24% and 30% reduction, respectively). The origin of the extracellular carbon appeared also to affect the taxonomic composition of the bacterioplankton assemblage, mainly reducing the development of gamma-Proteobacteria. This pattern of bacterial carbon uptake could contribute to a longer persistence of the exudates released in P-depleted conditions affecting the dynamics of the carbon cycle in marine environments.