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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Microplastics in the Arctic: A case study with sub-surface water and fish samples off Northeast Greenland.

The Arctic is a unique and fragile ecosystem that needs to be preserved and protected. Despite its remoteness, plastic pollution has been documented in this region. In the coming years, it is likely to worsen since, with climate changes and the opening of new shipping routes, the human presence is going to increase in the whole area. Here, we investigated the presence of microplastics (MPs) in sub-surface water and in two mid-trophic level Arctic fishes collected off Northeast Greenland: the demersal bigeye sculpin, Triglops nybelini, and the pelagic polar cod, Boreogadus saida. Plastics debris were found in the water samples at a concentration of 2.4 items/m3 ±0.8 SD which is higher than in most seas at lower latitudes. Both fish species had eaten MPs with different proportion among the species, 34% for T. nybelini (n = 71) and 18% for B. saida (n = 85). The significant difference in the occurrence of MPs between the two species is likely a consequence of their feeding behavior and habitat. Polyethylene was the main plastic polymer for water samples (41%, n = 17) and polyester (34%, n = 156) for fish samples as analyzed by Fourier Transformed Infrared (FT-IR) spectroscopy. Our data underscore that the Arctic regions are turning into a hotspot for plastic pollution, and this calls urgently for precautionary measures.

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.

Adverse effects of the SSRI antidepressant sertraline on early life stages of marine invertebrates.

Widespread contamination of coastal environments by emerging compounds includes low concentrations of pharmaceuticals. These pollutants are not currently incorporated in monitoring programs despite their effects on non-target organisms are very little documented. Among the selective serotonin reuptake inhibitor (SSRI) antidepressants, sertraline (SRT) is one of the most prescribed globally. In this work, earlier life stages of Amphibalanus amphitrite, Brachionus plicatilis and Mytilus galloprovincialis were exposed to environmental concentrations of SRT in order to study both sub-lethal and lethal responses in 24/48 h-tests. Low concentrations of SRT altered significantly swimming behavior in A. amphitrite and B. plicatilis giving 48 h-EC50 (µg/L) of 113.88 and 282.23, respectively whereas higher values were observed for mortality and immobilization. EC50 embryotoxicity with M. galloprovincialis was 206.80 µg/L. This work add new data about SRT ecotoxicity on marine invertebrates and confirms the applicability of behavioral endpoints to evaluate the environmental impact of antidepressants in marine organisms.

Old model organisms and new behavioral end-points: Swimming alteration as an ecotoxicological response.

Behavioral responses of aquatic organisms have received much less attention than developmental or reproductive ones due to the scarce presence of user-friendly tools for their acquisition. The technological development of data acquisition systems for quantifying behavior in the aquatic environment and the increase of studies on the understanding the relationship between the behavior of aquatic organisms and the physiological/ecological activities have generated renewed interest in using behavioral responses also in marine ecotoxicology. Recent reviews on freshwater environment show that behavioral end-points are comparatively fast and sensitive, and warrant further attention as tools for assessing the toxicological effects of environmental contaminants. In this mini-review, we perform a systematic analysis of the most recent works that have used marine invertebrate swimming alteration as behavioral end-point in ecotoxicological studies by assessing the differences between behavioral and acute responses in a wide range of species, in order to compare their sensitivity.