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After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC 50 ) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6-44.9) and 92.5 mg/L (87.7-97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity.

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 (LC 50 ≥ 10 times reference toxicant), while only one dispersant met the Italian approval criterion (EC 50 > 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., (Copyright © 2017. Published by Elsevier Inc.)

Plastic pollution has been globally recognized as a critical issue for marine ecosystems and nanoplastics constitute one of the last unexplored areas to understand the magnitude of this threat. However, current difficulties in sampling and identifying nano-sized debris make hard to assess their occurrence in marine environment. Polystyrene nanoparticles (PS NPs) are largely used as nanoplastics in ecotoxicological studies and although acute exposures have been already investigated, long-term toxicity on marine organisms is unknown. Our study aims at evaluating the effects of 40nm PS anionic carboxylated (PS-COOH) and 50nm cationic amino-modified (PS-NH 2 ) NPs in two planktonic species, the green microalga Dunaliella tertiolecta and the brine shrimp Artemia franciscana, respectively prey and predator. PS NP behaviour in exposure media was determined through DLS, while their toxicity to microalgae and brine shrimps evaluated through 72h growth inhibition test and 14 d long-term toxicity test respectively. Moreover, the expression of target genes (i.e. clap and cstb), having a role in brine shrimp larval growth and molting, was measured in 48h brine shrimp larvae. A different behaviour of the two PS NPs in exposure media as well as diverse toxicity to the two planktonic species was observed. PS-COOH formed micro-scale aggregates (Z-Average>1μm) and did not affect the growth of microalgae up to 50μg/ml or that of brine shrimps up to 10μg/ml. However, these negatively charged NPs were adsorbed on microalgae and accumulated (and excreted) in brine shrimps, suggesting a potential trophic transfer from prey to predator. On the opposite, PS-NH 2 -formed nano-scale aggregates (Z-Average<200nm), caused inhibition of algal growth (EC 50 =12.97μg/ml) and mortality in brine shrimps at 14 d (LC 50 =0.83μg/ml). Moreover, 1μg/ml PS-NH 2 significantly induced clap and cstb genes, explaining the physiological alterations (e.g. increase in molting) previously observed in 48h larvae, but also suggesting an apoptotic pathway triggered by cathepsin L-like protease in brine shrimps upon PS-NH 2 exposure. These findings provide a first insight into long-term toxicity of nanoplastics to marine plankton, underlining the role of the surface chemistry in determining the behaviour and effects of PS NPs, in terms of adsorption, growth inhibition, accumulation, gene modulation and mortality. The use of long-term end-point has been identified as valuable tool for assessing the impact of nanoplastics on marine planktonic species, being more predictable of real exposure scenarios for risk assessment purposes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, long-term effects of Ni, a widespread heavy metal in the aquatic ecosystems, have been determined on growth and lethality of the clam Ruditapes philippinarum, a known bioindicator of the marine environment. Three/four-month-old bivalves have been exposed to different concentrations of Ni dissolved in synthetic seawater. Growth and lethality as endpoints after 28 days of treatment have been observed. Obtained results are the following: EC 25  = 3.97 ± 0.94 and 9.45 ± 1.59 mg/L and NOEC = 1.56 and 6.25 mg/L for growth and mortality, respectively. Moreover, this study can be considered a new tool for the evaluation of fitness of bivalve clam, together with other biological responses following to the biological impacts of metal pollution.

Diethylene glycol (DEG) is a chemical compound used during offshore oil activities to prevent hydrate formation, and it may be released into the sea. A full ecotoxicological characterization is required according to European and Italian regulations for chemical substances. We have evaluated long-term toxic effects of DEG on indicator species of the marine environment as algae (Phaeodactylum tricornutum), crustaceans (Artemia franciscana), molluscs (Tapes philippinarum) and fish (Dicentrarchus labrax). A range of no observed effect concentrations (365-25,000 mg/L) has been identified. Based on the toxicity results and the ratio between predicted environmental concentration and predicted no-effect concentration, we have estimated the maximum allowable value of DEG in the marine environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Acute heavy metal toxicity was compared in nauplii of an autochthonous Artemia parthenogenetica (Branchiopoda, Anostraca) from Saline di Cervia (Ravenna, Italy) and of a non native commercially available A. franciscana. No significant difference in sensitivity was detected between two species.

The availability of standardized protocols for both organism culture and bioassay with ecologically relevant species is of great concern in ecotoxicology. Acartia tonsa represents an important, often dominant, member of zooplankton communities and meets all the practical criteria suggested for model species. New standardized procedures for laboratory culturing of the copepod A. tonsa and standardized methods for acute (24- and 48-h) and semichronic (7-d, static-renewal) toxicity tests with the nauplius stage are described. In both cases, eggs are the starting stage, and nauplius immobilization is the endpoint. The methods were the object of an intercomparison test involving nine laboratories, and nickel was the reference toxicant. Relative reproducibility was 24, 25, and 34% for 24-h, 48-h, and 7-d tests, respectively., (Copyright © 2012 SETAC.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.