High concentrations of phthalates affect the early development of the sea urchin Paracentrotus lividus.

The toxicity of three phthalates (PAEs) - butylbenzyl phthalate (BBP), diethyl phthalate (DEP), and di-(2-ethylhexyl) phthalate (DEHP) - was tested on the Mediterranean sea urchin Paracentrotus lividus. Fertilized eggs were exposed to environmental and high PAE concentrations for 72 h. The potential toxic effects on larval development and any morphological anomalies were then assessed to estimate PAEs impact. Environmental concentrations never affected development, while high concentrations induced toxic effects in larvae exposed to BBP (EC50: 2.9 ×103 µg/L) and DEHP (EC50: 3.72 ×103 µg/L). High concentrations caused skeletal anomalies, with a slight to moderate impact for DEP/DEHP and BBP, respectively. PAE toxicity was: BBP>DEHP>DEP. In conclusion, the three PAEs at environmental concentrations do not pose a risk to sea urchins. However, PAE concentrations should be further monitored in order not to constitute a concern to marine species, especially at their early developmental stages.

Nanoplastic uptake temporarily affects the pulsing behavior in ephyrae of the moon jellyfish Aurelia sp.

The aim of this study is to investigate for the first time the uptake and ecotoxicological effects of nanoplastics (NPs) in a marine cnidarian. Ephyrae of the moon jellyfish Aurelia sp. of different ages (0 and 7 days old) were exposed to negatively charged polystyrene NPs for 24 h; then, the uptake was assessed through traditional and novel techniques, namely microscopy and three-dimensional (3D) holotomography. Immobility and behavioral responses (frequency of pulsations) of ephyrae were also investigated to clarify if NP toxicity differed along the first life stages. NP uptake was observed in ephyrae thanks to the 3D technique. Such internalization did not affect survival, but it temporarily impaired the pulsation mode only in 0 day old ephyrae. This may be ascribed to the negative charged NPs, contributing to jellyfish behavioral alteration. These findings promote 3D holotomography as a suitable tool to detect NPs in marine organisms. Moreover, this study recommends the use of cnidarians of different ages to better assess NP ecotoxicological effects in these organisms, key components of the marine food web.

Cold storage effects on lethal and sublethal responses of Amphibalanus amphitrite Nauplii.

Bioassays are extensively used in ecotoxicology and there is a constant need for even more sensitive, reliable and easy to rear and obtain model organisms. Larvae of the crustacean Amphibalanus amphitrite are a good ecotoxicological model, for their high sensitivity to a wide range of toxicants and emerging contaminants. A standardized protocol for this toxicity bioassay has been recently proposed. Nevertheless, a limit of this model organism is the lack of resting stages and the need to use larvae immediately after their release from adults, thus increasing laboratory efforts related to the maintenance of adults. The aim of this work is to verify if short-term cold storage of A. amphitrite larvae prior to use in ecotoxicological tests may affect the ecotoxicological responses of these organisms. Three end-points (mortality, immobilization and swimming speed alteration) were measured on nauplii after storing them at 4 ± 1 °C for different times (24, 72 and 120 h) before bioassay set-up. Bioassays were set up using: (i) clean filtered natural sea water (0.22 µm FNSW), (ii) a reference toxicant (Cadmium Nitrate) and (iii) an environmental matrix (sediment elutriate). Results show that mortality, differently from the other two endpoints, was not affected by cold-storage. Even after 5 days of larvae storage at 4 ± 1 °C before bioassay set up, mortality data were comparable to those obtained for non-cold-stored organisms. Moreover, larval sensitivity to the reference toxicant and sediment elutriate did not change. Regarding the other two end points, low cadmium concentrations significantly changed immobility and swimming activity in cold-stored nauplii compared to larvae used immediately after larval release. In conclusion, short-term cold storage of A. amphitrite nauplii before bioassay set up is an appropriate procedure in ecotoxicological testing if mortality is the endpoint to be considered for final evaluation.

Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms.

Microplastics are ubiquitous in aquatic ecosystems, but little information is currently available on the dangers and risks to living organisms. In order to assess the ecotoxicity of environmental microplastics (MPs), samples were collected from the beaches of two islands in the Guadeloupe archipelago, Petit-Bourg (PB) located on the main island of Guadeloupe and Marie-Galante (MG) on the second island of the archipelago. These samples have a similar polymer composition with mainly polyethylene (PE) and polypropylene (PP). However, these two samples are very dissimilar with regard to their contamination profile and their toxicity. MPs from MG contain more lead, cadmium and organochlorine compounds while those from PB have higher levels of copper, zinc and hydrocarbons. The leachates of these two samples of MPs induced sublethal effects on the growth of sea urchins and on the pulsation frequency of jellyfish ephyrae but not on the development of zebrafish embryos. The toxic effects are much more marked for samples from the PB site than those from the MG site. This work demonstrates that MPs can contain high levels of potentially bioavailable toxic substances that may represent a significant ecotoxicological risk, particularly for the early life stages of aquatic animals.

Microplastics ingestion in the ephyra stage of Aurelia sp. triggers acute and behavioral responses.

For the first time, we report a correspondence between microplastics (MP) ingestion and ecotoxicological effects in gelatinous zooplankton (Cnidarian jellyfish). The ephyra stage of the jellyfish Aurelia sp. was exposed to both environmental and high concentrations of fluorescent 1-4 µm polyethylene MP (0.01-10 mg/L). After 24 and 48 h, MP accumulation, acute (Immobility) and behavioral (Frequency pulsation) endpoints were investigated. MP were detected by confocal and tomographic investigations on gelatinous body and mouth, either attached on the surface or ingested. This interaction was responsible for impairing ephyrae survival and behavior at all tested concentrations after 24 h. Acute and behavioral effects were also related to mechanical disturbance, caused by MP, triggering a loss of radial symmetry. Contaminated ephyrae exposed to clean seawater showed full recovery after 72 h highlighting the organisms without the microspheres, attached on body jellyfish surface around the mouth and lappets. In conclusion, short-term exposure to MP affects ephyrae jellyfish health, impairing both their survival and behavior. Polyethylene MP temporarily affect both Immobility and Frequency of pulsation of Aurelia sp. jellyfish. This study provides a first step towards understanding and clarifying the potential impacts of MP contamination in gelatinous zooplankton.

A short-term swimming speed alteration test with nauplii of Artemia franciscana.

The presence of toxicant needs to be assessed within short time in order to effectively protect the aquatic environment from serious threat. Based on the observation that at high temperatures aquatic organisms become more vulnerable to stressors than those maintained at room temperature, a new test was developed. The proposed bioassay consisted in the evaluation of the swimming speed alteration (SSA) of nauplii of Artemia franciscana incubated at 39°C (± 1) for 6h, using a Swimming Behavior Recorder system (SBR). A comparative ecotoxicological study between the 6h SSA test and the 24h mortality test was carried out in order to validate the new method in terms of sensitivity by means of EC50 values. The bioassay was applied to screen different toxicants: K2Cr2O7, Cu(SO4)2, NaClO, SDS and Sertraline hydrochloride. The EC50s calculated for the short-term SSA test and those of the mortality test showed comparable values. For all toxicants, the 6h SSA test was proved to be as sensitive as the 24h mortality test. The method developed in this study is the first temperature-based toxicity test with nauplii of Artemia franciscana and it represents an attractive assay in ecotoxicology because of its convenience in terms of time and costs, feasibility and sensitivity.

Effects of polystyrene microbeads in marine planktonic crustaceans.

Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10mgL-1) for 24 and 48h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1mgL-1) after 48h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans.

Temperature and salinity effects on cadmium toxicity on lethal and sublethal responses of Amphibalanus amphitrite nauplii.

The official protocol of an ecotoxicological assay employing larvae of the crustacean Amphibalanus amphitrite as a model organism has recently been published by the Italian regulatory authority UNICHIM. Such assay is now one of the applicable tests for water quality assessment under Italian law. While specific temperature and salinity values are recommended by ecotoxicology bioassay protocols for test set up, little information is available on response changes in case of parameter variations. In particular, information is totally lacking for this innovative model organism. Under the standard test protocol, 20°C and 37‰ temperature and salinity, respectively, are required to be set in A. amphitrite bioassay. In order to evaluate the environmental relevance of the test, laboratory experiments simulating the effect on larval responses due to variations of temperature and salinity expected in field collected samples were carried out. The effect of temperature and salinity changes on different end-points, involving increasing sensitivity levels, has been investigated, with and without the presence of cadmium nitrate, Cd(NO3)2, as a reference toxicant, to determine the possible interactions between pollutants and environmental parameters fluctuations. Three end-points - mortality, immobilization, and swimming speed alteration - were measured in order to evaluate the impact of a wide range of temperature (5, 10, 15, 20, 25, 30, 35, 40°C) and salinity values (10, 20, 30, 37, 40, 50, 60, 70‰) on response variation after 24 and 48h of exposure. For each parameter, a Non-Effect Range (NER) - namely the limit values within which no effect related to environmental parameter changes is observed - has been defined. For both parameters, NER resulted to be wider for the less sensitive end-points - such as mortality and immobilization - and for shorter exposure time (24h). Later, the same end-points have been evaluated by exposing the same organisms to a reference toxic compound, Cd(NO3)2 (0, 0.2, 0.4, 0.8, 1.6, 3.2mg/L), within the detected NER both for temperature and salinity. LC50 and EC50 values have been calculated for each end-point after 24 and 48h. Cadmium toxicity was shown to decrease at higher salinity values and increase at higher temperatures. Obtained results offer a better bioassay characterization, and the possibility of a more realistic estimation of ecotoxicological assessments performed on field collected samples. Further studies are needed, especially to investigate the effects of simultaneous salinity and temperature changes on end-points.

Antifouling activity of synthetic alkylpyridinium polymers using the barnacle model.

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

The power of Posidonia oceanica meadows to retain microplastics and the consequences on associated macrofaunal benthic communities.

In the coastal environment, a large amount of microplastics (MPs) can accumulate in the sediments of seagrass beds. However, the potential impact these pollutants have on seagrasses and associated organisms is currently unknown. In this study, we investigated the differences in MPs abundance and composition (i.e., shape, colour and polymer type) in marine sediments collected at different depths (-5 m, -15 m, -20 m) at two sites characterized by the presence of Posidonia oceanica meadows and at one unvegetated site. In the vegetated sites, sediment samples were collected respectively above and below the upper and lower limits of the meadow (-5 m and -20 m), out of the P. oceanica meadow, and in the central portion of the meadow (-15 m). By focusing on the central part of the meadow, we investigated if the structural features (i.e. shoots density and leaf surface) can affect the amount of MPs retained within the underlying sediment and if these, in turn, can affect the associated benthic communities. Results showed that the number of MPs retained by P. oceanica meadows was higher than that found at the unvegetated site, showing also a different composition. In particular, at vegetated sites, we observed that MPs particles were more abundant within the meadow (at - 15 m), compared to the other depths, on unvegetated sediment, with a dominance of transparent fragments of polypropylene (PP). We observed that MPs entrapment by P. oceanica was accentuated by the higher shoots density, while the seagrass leaf surface did not appear to have any effect. Both the abundance and richness of macrofauna associated with P. oceanica rhizomes appear to be negatively influenced by the MPs abundance in the sediment. Overall, this study increases knowledge of the potential risks of MPs accumulation in important coastal habitats such as the Posidonia oceanica meadows.

New insights into the impact of leachates from in-field collected plastics on aquatic invertebrates and vertebrates.

The impact of leachates from micronized beached plastics of the Mediterranean Sea and Atlantic Ocean on coastal marine ecosystems was investigated by using a multidisciplinary approach. Chemical analysis and ecotoxicological tests on phylogenetically distant species were performed on leachates from the following plastic categories: bottles, pellets, hard plastic (HP) containers, fishing nets (FN) and rapido trawling rubber (RTR). The bacteria Alivibrio fischeri, the nauplii of the crustaceans Amphibalanus amphitrite and Acartia tonsa, the rotifer Brachionus plicatilis, the embryos of the sea urchin Paracentrotus lividus, the ephyrae of the jellyfish Aurelia sp. and the larvae of the medaka Oryzias latipes were exposed to different concentrations of leachates to evaluate lethal and sub-lethal effects. Thirty-one additives were identified in the plastic leachates; benzophenone, benzyl butyl phthalate and ethylparaben were present in all leachates. Ecotoxicity of leachates varied among plastic categories and areas, being RTR, HP and FN more toxic than plastic bottles and pellets to several marine invertebrates. The ecotoxicological results based on 13 endpoints were elaborated within a quantitative weight of evidence (WOE) model, providing a synthetic hazard index for each data typology, before their integrations in an environmental risk index. The WOE assigned a moderate and slight hazard to organisms exposed to leachates of FN and HP collected in the Mediterranean Sea respectively, and a moderate hazard to leachates of HP from the Atlantic Ocean. No hazard was found for pellet, bottles and RTR. These findings suggest that an integrated approach based on WOE on a large set of bioassays is recommended to get a more reliable assessment of the ecotoxicity of beached-plastic leachates. In addition, the additives leached from FN and HP should be further investigated to reduce high concentrations and additive types that could impact marine ecosystem health.

Assessing the toxicity of aegerolysin-based bioinsecticidal complexes using the sea urchin Paracentrotus lividus as model organism.

The use of alternative solutions for pest management to replace pesticides in agriculture is of great interest. Proteinaceous complexes deriving from edible oyster mushrooms were recently proposed as environmentally friendly bioinsecticides. Such complexes, composed of ostreolysin A6 (OlyA6) and pleurotolysin B (PlyB), target invertebrate-specific membrane sphingolipids in insect's midgut, causing death through the formation of transmembrane pores. In this work, the potential impact of OlyA6/PlyB complexes was tested in the Mediterranean sea urchin Paracentrotus lividus, as an indicator of environmental quality. The ability of the fluorescently tagged OlyA6 to bind sea urchin gametes (sperm, eggs), the lipidome of sea urchin gametes, and the potential toxic effects and developmental anomalies caused by OlyA6/PlyB complexes on P. lividus early development (embryo, larvae) were investigated. The binding of the fluorescently tagged OlyA6 could be observed only in sea urchin eggs, which harbor OlyA6 sphingolipid membrane receptors, conversely to sperm. High protein concentrations affected sea urchin fertilization (>750 µg/L) and early development (> 375 µg/L in embryos; >100 µg/L in larvae), by causing toxicity and morphological anomalies in embryos and larvae. The main anomalies consisted in delayed embryos and incorrect migration of the primary mesenchyme cells that caused larval skeletal anomalies. The classification of these anomalies indicated a slight environmental impact of OlyA6/PlyB complexes at concentrations higher than 750 µg/L. Such impact should not persist in the marine environment, due to the reversible anomalies observed in sea urchin embryos and larvae that may promote defense strategies. However, before promoting the use of OlyA6/PlyB complexes as bio-pesticides at low concentrations, further studies on other marine coastal species are needed.

An integrated approach to characterize deep sediment toxicity in Genoa submarine canyons (NW Mediterranean).

The aim of this study was to evaluate deep sediment toxicity in Genoa submarine canyons (Northwestern Mediterranean), for the first time, by using an integrated approach that combined chemistry and ecotoxicology. Sediments were collected from the main submarine canyons in the Gulf of Genoa (Polcevera and Bisagno) and along the adjacent Western Open Slope. A multi-endpoint ecotoxicological approach was taken by exposing two crustacean larvae (Amphibalanus amphitrite and Artemia sp.). Lethal and sub-lethal responses (mortality, swimming behavior) were investigated. Chemical analysis showed that this area is characterized by metal enrichment, including lead, cadmium, chromium, and nickel. Ecotoxicological tests highlighted that elutriates from the different submarine canyons were toxic only for A. amphitrite nauplii: Polcevera Canyon and Western Open Slope sediments induced stronger lethal and sub-lethal ecotoxicological effects than those from Bisagno Canyon. No direct correlation was found between the outcome of chemical and ecotoxicological characterization. However, barnacle was the most prone species to metal contamination: lethal and sub-lethal responses found in this species may be linked to an increase in the concentration of some metals (i.e., Cr, Ni) from offshore to coastal waters, probably due to anthropogenic activity. These findings suggest that the proposed approach can be a suitable tool for deep-sea sediment contamination monitoring; however, the use of a battery of bioassays involving multiple species and endpoints is recommended to better clarify the dynamics of contaminants in marine sediments at very high depths.

Ecotoxicity of Polyvinylidene Difluoride (PVDF) and Polylactic Acid (PLA) Microplastics in Marine Zooplankton.

The aim of this study was to investigate the ecotoxicity of polyvinylidene difluoride (PVDF) and polylactic acid (PLA) microplastics (MPs) in two marine zooplankton: the crustacean Artemia franciscana and the cnidarian Aurelia sp. (common jellyfish). To achieve this goal, (i) MP uptake, (ii) immobility, and (iii) behavior (swimming speed, pulsation mode) of crustacean larval stages and jellyfish ephyrae exposed to MPs concentrations (1, 10, 100 mg/L) were assessed for 24 h. Using traditional and novel techniques, i.e., epifluorescence microscopy and 3D holotomography (HT), PVDF and PLA MPs were found in the digestive systems of the crustaceans and in the gelatinous tissue of jellyfish. Immobility was not affected in either organism, while a significant behavioral alteration in terms of pulsation mode was found in jellyfish after exposure to both PVDF and PLA MPs. Moreover, PLA MPs exposure in jellyfish induced a toxic effect (EC50: 77.43 mg/L) on the behavioral response. This study provides new insights into PLA and PVDF toxicity with the potential for a large impact on the marine ecosystem, since jellyfish play a key role in the marine food chain. However, further investigations incorporating additional species belonging to other trophic levels are paramount to better understand and clarify the impact of such polymers at micro scale in the marine environment. These findings suggest that although PVDF and PLA have been recently proposed as innovative and, in the case of PLA, biodegradable polymers, their effects on marine biota should not be underestimated.

Microplastics in seawater and marine organisms: Site-specific variations over two-year study in Giglio Island (North Tyrrhenian Sea).

Geographical and temporal differences of microplastic occurrence were documented in water and fish collected in 2017 and 2019 from the Giglio Island (North Tyrrhenian Sea) close to the area where the Costa Concordia sank in January 2012. Results on water samples showed a site-dependent difference, suggesting the role of surface current dynamics in the microplastic local distribution, while tested Neuston nets (200 µm and 330 µm mesh size) did not influence microplastic retention efficiency. Fish exhibited in 2019 a higher frequency of specimens positive to microplastic ingestion with respect to 2017, with an occurrence higher than those typically observed in other Mediterranean areas. Both in water and fish, fragments were the dominating shape, polypropylene and polyethylene were the prevalent polymers, without particular difference between sites and years. This study highlights the importance of applying microplastic investigation in biotic and abiotic matrices for an effective monitoring of this pollution in the marine environment.

Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys.

In this study, we investigated the possibility to improve a new behavioural bioassay (Swimming Speed Alteration test-SSA test) using larvae of marine cyst-forming organisms: e.g. the brine shrimp Artemia sp. and the rotifer Brachionus plicatilis. Swimming speed was investigated as a behavioural end-point for application in ecotoxicology studies. A first experiment to analyse the linear swimming speed of the two organisms was performed to verify the applicability of the video-camera tracking system, here referred to as Swimming Behavioural Recorder (SBR). A second experiment was performed, exposing organisms to different toxic compounds (zinc pyrithione, Macrotrol MT-200, and Eserine). Swimming speed alteration was analyzed together with mortality. The results of the first experiment indicate that SBR is a suitable tool to detect linear swimming speed of the two organisms, since the values have been obtained in accordance with other studies using the same organisms (3.05 mm s(-1) for Artemia sp. and 0.62 mm s(-1) for B. plicatilis). Toxicity test results clearly indicate that swimming speed of Artemia sp. and B. plicatilis is a valid behavioural end-point to detect stress at sub-lethal toxic substance concentrations. Indeed, alterations in swimming speed have been detected at toxic compound concentrations as low as less then 0.1-5% of their LC(50) values. In conclusion, the SSA test with B. plicatilis and Artemia sp. can be a good behavioural integrated output for application in marine ecotoxicology and environmental monitoring programs.

Microplastics do not affect standard ecotoxicological endpoints in marine unicellular organisms.

In this study, the acute toxicity of microplastics (MPs) on unicellular organisms as marine decomposers and microalgae was assessed, by evaluating standards endpoints included in International Standard Organization (ISO) protocols. The bacteria Vibrio fischeri and the diatom Phaeodactylum tricornutum were exposed to different sizes (1-500 µm) of polyethylene MPs in order to evaluate bioluminescence inhibition and microalgal growth. No acute toxicity was found on bacteria or microalgae in an order of magnitude above environmentally relevant concentrations, suggesting that tested MPs did not affect the investigated biological processes. In conclusion, standard ecotoxicological endpoints are not sufficiently sensitive to assess the potential effects of MPs on decomposers and primary producers, conversely to nanoplastics. These findings highlight that the current approach for MP risk assessment in unicellular species should be revised, by providing alternative endpoints to be included in standardized protocols, able to monitor the fate and biological effects of MPs.

New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in Hexaplex trunculus (Gastropoda, Muricidae) with different stressors.

Imposex, i.e. the development of additional male sex organs (penis and/or vas deferens), in females of gonochorist marine and freshwater gastropods, is known to be caused by tributyltin (TBT), and it has been widely used as a biomonitoring tool in environmental surveys for TBT pollution assessment. In this study, we experimentally tested the potential to induce imposex by another endocrine disruptor (polychlorinated biphenyls [PCBs] mixture--Aroclor 1260). Adults of Hexaplex trunculus with low imposex level, coming from an Italian Marine Protected Area, were injected separately with different doses of tributyltin chloride (TBTCl) and Aroclor 1260. The compounds were dissolved in ethanol and the organisms were narcotised by immersion in MgCl(2) solution before injection. Before and after the experiment, butyltin compounds (BuTs) and PCB tissue concentrations were determined. A significant increase in imposex with respect to non-treated organisms was observed in all treatments, including artefact controls. No clear correlation was observed between BuTs and PCB tissue concentrations and indices of imposex incidence. Based on these results, no assumption can be formulated about PCB effect on imposex development. Nevertheless, they suggest that the imposex level increase, at least in H. trunculus, in laboratory conditions might not be caused by TBT only, but it would rather be a non-specific response to different stress stimuli.

A new approach to testing potential leaching toxicity of fouling release coatings (FRCs).

Fouling release coatings (FRCs) are today the main environment-friendly alternative to traditional self-polishing coatings, that continuously release biocides and/or heavy metals into water. FRCs are available on the market as environmentally friendly AF paints and most of them do not contain bioactive agents, however no complete and reliable assessment of their environmental impact has yet been carried out. Only few literature data proving their AF efficacy combined with a demonstrated lack of toxicological effects are available. Ecotoxicological bioassays are commonly used to predict the potential environmental impact of traditional AF paints. Standardized methodologies to obtain leaching products from biocide-based paints are available, while few studies propose experimental methods to assess the potential effects of biocide-free FRCs leachates on non-target organisms. The aim of this work is to propose an experimental protocol to obtain leaching products from biocide-free FRCs in order to evaluate the potential release of substances having toxic effects, by means of an ecotoxicological bioassay. Two ecotoxicological end-points with different sensitivity levels were considered (multi-end-point approach). Five silicone-based commercial coatings were used: their leaching products were collected after different immersion times following the developed experimental method and then two ecotoxicological end-points were evaluated on II stage nauplii of the crustacean Amphibalanus amphitrite as model organism. Moreover, chemical analyses were performed on leachates collected after each immersion time, focusing on the presence of metals in leaching products. From the results obtained from the bioassay, even if not indicative of the real environmental impact of FRCs, a release of toxic substances was observed from tested coatings during early immersion stages, likely to affect the exposed model organism. The potential leaching toxicity of the five tested products was compared. No clear correspondence could be identified between the concentrations of metals detected in leachates and the obtained ecotoxicological data, thus suggesting that other active components might be released by FRCs responsible for the toxic effects pointed out on A. amphitrite larvae.

Ecotoxicological effects of polystyrene microbeads in a battery of marine organisms belonging to different trophic levels.

The aim of this study was to detect ecotoxicological effects of 0.1 µm polystyrene microbeads in marine organisms belonging to different trophic levels. MP build up, lethal and sub-lethal responses were investigated in the bacterium Vibrio anguillarum (culturability), in the green microalga Dunaliella tertiolecta (growth inhibition), in the rotifer Brachionus plicatilis (mortality and swimming speed alteration) and in the sea urchin Paracentrotus lividus (immobility and swimming speed alteration) exposed to a wide range of microplastic (MP) concentrations (from 0.001 to 10 mg L-1). Survival was not affected in all organisms up to 10 mg L-1, while algal growth inhibition, rotifer and sea urchin larvae swimming behaviour alterations were observed after exposure to MPs. Ingestion was only observed in rotifers and it was directly correlated with sub-lethal effects. These results account for the ecotoxicological risk associated to the polystyrene microbeads, which are able to affect different endpoints in primary producers and consumers (rotifers and sea urchins) since no effects were observed in decomposers. This study points out the importance of using a battery of marine organisms belonging to different trophic levels by studying acute toxicity of MPs at low and high contamination levels, and investigating sub-lethal responses. Further investigations aimed at studying the transfer of these materials through the web are particularly recommended.