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The growing concern on nanoplastics (1 μm) impact on marine life has stimulated a significant amount of studies aiming to address ecotoxicity and disclose their mechanisms of action. Here, we applied an integrative approach to develop an Adverse Outcome Pathway (AOP) upon acute exposure to amino-modified polystyrene nanoparticles (PS-NH

Larval settlement is a critical step for sessile benthic species such as corals, whose ability to thrive on diverse natural and anthropogenic substrates may lead to a competitive advantage in the colonization of new environments with respect to a narrow tolerance for a specific kind of substratum. Plastic debris, widespread in marine waters, provides a large, motile, and solid substratum supporting a highly diverse biological community. Here we present the first observation of a floating plastic bottle colonized by the deep-sea coral Desmophyllum dianthus. The density pattern and co-occurring species composition suggest a pioneer behavior of this coral species, whose peculiar morphologic plasticity response when interacting with the plastic substrate (i.e., low density polyethylene) has not been observed before. The tolerance of D. dianthus for such plastic substrate may affect ecological processes in deep water environments, disrupting interspecific substrate competition in the benthic community.

Nanoplastics are considered contaminants of emerging concern at the global scale. The recent evidence of their occurrence in seawater from the Mediterranean Sea calls for a thorough evaluation of their impact on marine life and in particular on vulnerable life stages such as planktonic embryos. Here, we investigated the impact of increasing nominal concentrations of 50 nm amino-modified (PS-NH

The occurrence and impact of contaminants of emerging concerns (CECs) have been investigated in Antarctica much less than in other parts of the world. Although legacy anthropogenic pollutants can reach Antarctica via long-range transport, CECs mainly originate from local sources. Here, we investigated the ability of a freshwater crustacean, the Antarctic fairy shrimp Branchinecta gaini, to cope with nanoscale titanium dioxide (n-TiO2), a widely used pigment in consumer products (e.g. paintings), including those for personal care (e.g. sunscreens). An in vivo acute short-term exposure study (9 h, n-TiO2 concentration range 50–200 μg ml-1) was performed and the expression levels of several genes involved in stress response were evaluated. No effect on the expression of heat-shock protein chaperone genes was found, with the exception of Hsp70a, which was significantly upregulated at 200 μg ml-1 n-TiO2. Similarly, cytochrome P450 was upregulated at 100 and 200 μg ml-1 n-TiO2, while the expression levels of cathepsin L and of antioxidant genes such as superoxide dismutase and glutathione peroxidase were significantly reduced with increasing concentrations of n-TiO2. This study shows for the first time the responsiveness and sensitivity of an Antarctic freshwater crustacean to n-TiO2 exposure and supports its suitability as a biomonitor of CECs in Antarctica.

Since the first explorers reached Antarctica, their activities have quickly impacted both land and sea and thus, together with the long-range transport, hazardous chemicals began to accumulate. It is commonly recognized that anthropogenic pollution in Antarctica can originate from either global or local sources. Heavy metals, organohalogenated compounds, hydrocarbons, and (more recently) plastic, have been found in Antarctic biota, soil sediments, seawater, air, snow and sea-ice. Studies in such remote areas are challenging and expensive, and the complexity of potential interactions occurring in such extreme climate conditions (i.e., low temperature) makes any accurate prediction on potential impacts difficult. The present review aims to summarize the current state of knowledge on occurrence and distribution of legacy and emerging pollutants in Antarctica, such as plastic, from either global or local sources. Future actions to monitor and mitigate any potential impact on Antarctic biodiversity are discussed.

Marine plastic pollution is a global and pervasive environmental issue. Knowledge on plastic degradation in natural settings is still very limited due to current technological limitations, hampering our understanding of plastic fate (including its breakdown into micro- and nanoplastics) and of its risk for marine ecosystems. Here we present the proof of concept of the Ocean Plastic Incubator Chamber (OPIC), a novel equipment to follow plastic degradation in situ at sea over time. OPIC consists of a frame containing a motorised rotating stage with transparent tubes sub-assemblies where reference plastic materials are incubated and exposed to natural weathering conditions for defined time multi-years period. OPIC has been designed, tested and adapted for deployment with mooring line platforms in the open ocean with potential future application in remote environments at different depths (from shallow waters to deep sea environments). This incubator will allow us to measure different markers of plastic aging in situ in the ocean for the first time, providing new insights into the multiple and locally driven dynamics regulating plastic transformations and fate at sea.

Plastic debris has been recognised as a potential stressor for Antarctic marine organisms. In this study, the effects of surface charged polystyrene nanoparticles (PS NPs) on the immune cells (coelomocytes) of the Antarctic sea urchin Sterechinus neumayeri were assessed through in vitro short-term cultures. The behaviour of anionic carboxylated (PS-COOH) and cationic amino-modified (PS-NH2) NPs in filtered natural sea water (NSW) from King George Island (South Shetland Islands) was characterised by dynamic light scattering. Cellular morphology, NP uptake, phagocytic capacity and gene expression were evaluated after 6 and 24 h of exposure to 1 and 5 µg mL−1 PS NPs. Secondary characterisation showed an initial good dispersion of PS NPs in NSW, followed by nano-scale aggregation after 24 h. Both PS NPs affected cellular phagocytosis and generated an inflammatory response against oxidative stress and apoptosis at the molecular level. Fluorescently labelled PS-COOH aggregates were internalised by phagocytes and associated to the modulation of genes related to external challenges, antioxidant responses and cell protection against stress and apoptosis. Exposure to PS-NH2 caused a strong decrease in phagocytic capacity and the formation of cellular debris at 5 µg mL−1 after 24 h, but low gene modulation, suggesting a threshold in coelomocytes defence ability against PS-NH2. This study represents the first attempt to assess the impact of nanoplastics on Antarctic marine organisms. Our findings demonstrate that PS NPs with different surface charges constitute a challenge for S. neumayeri immune cells.

The number of scientists and tourists visiting Antarctica is on the rise and, despite the management framework for environmental protection, some coastal areas, particularly in the Antarctic Peninsula region, are affected by plastic contamination. The few data available on the occurrence of microplastics (300 µm appears to be scarce and far less abundant than paint chips released from research vessels. Yet, near some coastal scientific stations, the fragmentation and degradation of larger plastic items, as well as microbeads and microfibers released into wastewater from personal care products and laundry, could potentially affect marine organisms. Some studies indicate that, through long-range atmospheric transport, plastic fibers produced on other continents can be deposited in Antarctica. Drifting plastic debris can also cross the Polar Front, with the potential to carry alien fouling organisms into the Southern Ocean. Sea ice dynamics appear to favor the uptake of microplastics by ice algae and Antarctic krill, the key species in the Antarctic marine food web. Euphausia superba apparently has the ability to fragment and expel ingested plastic particles at the nanoscale. However, most Antarctic organisms are endemic species, with unique ecophysiological adaptations to extreme environmental conditions and are likely highly sensitive to cumulative stresses caused by climate change, microplastics and other anthropogenic disturbances. Although there is limited evidence to date that micro- and nanoplastics have direct biological effects, our review aims at raising awareness of the problem and, in order to assess the real potential impact of microplastics in Antarctica, underlines the urgency to fill the methodological gaps for their detection in all environmental matrices, and to equip scientific stations and ships with adequate wastewater treatment plants to reduce the release of microfibers.

Marine plastic pollution is a growing worldwide environmental concern as recent reports indicate that increasing quantities of litter disperse into secluded environments, including Polar Regions. Plastic degrades into smaller fragments under the influence of sunlight, temperature changes, mechanic abrasion and wave action resulting in small particles < 5mm called microplastics (MP). Sea ice cores, collected in the Arctic Ocean have so far revealed extremely high concentrations of very small microplastic particles, which might be transferred in the ecosystem with so far unknown consequences for the ice dependant marine food chain. Sea ice has long been recognised as a transport vehicle for any contaminates entering the Arctic Ocean from various long range and local sources. The Fram Strait is hereby both, a major inflow gateway of warm Atlantic water, with any anthropogenic imprints and the major outflow region of sea ice originating from the Siberian shelves and carried via the Transpolar Drift. The studied sea ice revealed a unique footprint of microplastic pollution, which were related to different water masses and indicating different source regions. Climate change in the Arctic include loss of sea ice, therefore, large fractions of the embedded plastic particles might be released and have an impact on living systems. By combining modeling of sea ice origin and growth, MP particle trajectories in the water column as well as MPs long-range transport via particle tracking and transport models we get first insights about the sources and pathways of MP in the Arctic Ocean and beyond and how this might affect the Arctic ecosystem.

Benzo(a)pyrene (B(a)P) is a well-known genotoxic agent, the removal of which from environmental matrices is mandatory, necessitating the application of cleaning strategies that are harmless to human and environmental health. The potential application of nanoparticles (NPs) in the remediation of polluted environments is of increasing interest. Here, specifically designed NPs were selected as being non-genotoxic and able to interact with B(a)P, in order to address the genetic and chromosomal damage it produces. A newly formulated pure anatase nano-titanium (nano-TiO2), a commercial mixture of rutile and anatase, and carbon black-derived hydrophilic NPs (HNP) were applied. Once it had been ascertained that the NPs selected for the work did not induce genotoxicity, marine mussel gill biopsies were exposed in vitro to B(a)P (2 μg/mL), alone and in combination with the selected NPs (50 µg/mL nano-TiO2, 10 µg/mL HNP). DNA primary reversible damage was evaluated by means of the Comet assay. Chromosomal persistent damage was assessed on the basis of micronuclei frequency and nuclear abnormalities by means of the Micronucleus-Cytome assay. Transmission Electron Microscopy (TEM) was performed to investigate the mechanism of action exerted by NPs. Pure Anatase n-TiO2 was found to be the most suitable for our purpose, as it is cyto- and genotoxicity free and able to reduce the genetic and chromosomal damage associated with exposure to B(a)P.

In Antarctic regions, the composition and metabolic activity of microbial assemblages associated with plastic debris (“plastisphere”) are almost unknown. A macroplastic item from land (MaL, 30 cm) and a mesoplastic from the sea (MeS, 4 mm) were collected in Maxwell Bay (King George Island, South Shetland) and analyzed by Fourier transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR), which confirmed a polystyrene foam and a composite high-density polyethylene composition for MaL and MeS, respectively. The structure and function of the two plastic-associated prokaryotic communities were studied by complementary 16S ribosomal RNA gene clone libraries, total bacterioplankton and culturable heterotrophic bacterial counts, enzymatic activities of the whole community and enzymatic profiles of bacterial isolates. Results showed that Gamma- and Betaproteobacteria (31% and 28%, respectively) dominated in MeS, while Beta- and Alphaproteobacteria (21% and 13%, respectively) in MaL. Sequences related to oil degrading bacteria (Alcanivorax,Marinobacter) confirmed the known anthropogenic pressure in King George Island. This investigation on plastic-associated prokaryotic structure and function represents the first attempt to characterize the ecological role of plastisphere in this Antarctic region and provides the necessary background for future research on the significance of polymer type, surface characteristics and environmental conditions in shaping the plastisphere.

The increasing use and disposal of plastics has become a persistent problem in the marine environment, calling for studies that refer to realistic scenarios to understand their effects on biota. Particularly, the understanding about the effects of co-exposure with nanoplastic particles and metals on aquatic organisms is still limited. The present work aimed to investigate the acute toxicity of amino-functionalized polystyrene nanoparticles (PS-NH2; 50 nm) as proxy for nanoplastics on brine shrimp Artemia franciscana larvae under different culture conditions and at different stages of development, as well as the combined effect with two reference toxicants - potassium dichromate (K2Cr2O7) and copper sulfate (CuSO4). Nauplii (instar II or III larval stages) were exposed to different concentrations of PS-NH2 (0.005 to 5 μg mL-1) for up to 48 h, with or without agitation in order to mimic a more realistic environmental scenario. Larval mobility and PS-NH2 accumulation were monitored under microscopy. PS-NH2 alone showed toxicity only at the highest concentration tested (5 μg mL-1) regardless the incubation method used (61.2 + 3.1% and 65.0 + 4.5% with and without agitation, respectively). Moreover, instar III stage was the most sensitive to PS-NH2 exposure (38.2% immobility in 24 h of exposure; 5 μg mL-1). Evidence of PS-NH2 retention in the gastrointestinal tract in a concentration- and time-dependent manner was also obtained. Mixtures of PS-NH2 (0.005 and 5 μg mL-1) with different concentrations of K2Cr2O7 increased the immobilization rate of the larvae after 48 h of exposure, when compared to the K2Cr2O7 alone. Similar results were observed for CuSO4 in the co-exposure conditions at different concentrations. However, exposing nauplii to a mixture of PS-NH2 (0.005 μg mL-1) and CuSO4 decreased immobilization rate, in comparison to the group exposed to CuSO4 alone. The present work highlights the potential risk posed by nanoplastics to zooplanktonic species through their interaction with other toxicants.

Plastic debris has been identified as a potential threat to Antarctic marine ecosystems, however, the impact of nanoplastics (

The air humidity in Antarctica is very low and this peculiar weather parameter make the use of flame retardants in research facilities highly needed for safety reasons, as fires are a major risk. Legacy and novel flame retardants (nFRs) including polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs), 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE), Dechlorane Plus (DP), and other nFRs were measured in indoor dust samples collected at research Stations in Antarctica: Gabriel de Castilla, Spain (GCS), Julio Escudero, Chile (JES), and onboard the RRS James Clark Ross, United Kingdom (RRS JCR). The GC-HRMS and LC-MS-MS analyses of dust samples revealed ∑PBDEs of 41.5 ± 43.8 ng/g in rooms at GCS, 18.7 ± 11.6 ng/g at JES, and 27.2 ± 37.9 ng/g onboard the RRS JCR. PBDE pattern was different between the sites and most abundant congeners were BDE-183 (40%) at GCS, BDE-99 (50%) at JES, and BDE-153 (37%) onboard the RRS JCR. The ∑HBCDs were 257 ± 407 ng/g, 14.9 ± 14.5 ng/g, and 761 ± 1043 ng/g in indoor dust collected in rooms at GCS, JES, and RRS JCR, respectively. The ∑nFRs were 224 ± 178 ng/g at GCS, 14.1 ± 13.8 ng/g at JES, and 194 ± 392 ng/g on the RRS JCR. Syn- and anti-DP were detected in most of the samples and both isomers showed the highest concentrations at GCS: 163 ± 93.6 and 48.5 ± 61.1 ng/g, respectively. The laboratory and living room showed the highest concentration of HBCDs, DPs, BTBPE. The wide variations in FR levels in dust from the three research facilities and between differently used rooms reflect the different origin of furnishing, building materials and equipment. The potential health risk associated to a daily exposure via dust ingestion was assessed for selected FRs: BDEs 47, 99, and 153, α-, β-, and γ-HBCD, BTBPE, syn- and anti-DP. Although the estimated exposures are below the available reference doses, caution is needed given the expected increasing use of novel chemicals without a comprehensive toxicological profile., This work was supported by Project SENTINEL-CTM2015- 70535-P (JD and BJ), Projects ANID-FONDECYT 1130329 (KP) and 1161673 (KP), 11150548 (CGM), ANID-FONDAP 15110027 (RA-H), ANID-PIA-INACh-ACT192057 (CGM), INACH RT_12_17 (CGM), and INACH RT_08_13 (RA-H), PNRA 2013/C1.04 (SC). We thank the Project PLANET (PNRA 14_0009) in collaboration with the British Antarctic Survey- Ecosystems Team for the collection of samples on board the RRS James Clarke Ross (EB). This research was also supported by the RECETOX Research Infrastructure (Czech Ministry of Education, Youth and Sports: LM2018121), RECETOX RI (CZ.02.1.01/0.0/0.0/16_013/ 0001761), and CETOCOEN EXCELLENCE Teaming 2 project supported by Horizon2020 (857560) and the Czech Ministry of Education, Youth and Sports (02.1.01/0.0/0.0/18_046/0015975).

There is evidence and serious concern that microplastics have reached the most remote regions of the planet, but how far have they travelled in terrestrial ecosystems? This study presents the first field-based evidence of plastic ingestion by a common and central component of Antarctic terrestrial food webs, the collembolan Cryptopygus antarcticus. A large piece of polystyrene (PS) foam (34 × 31 × 5 cm) covered by microalgae, moss, lichens and microfauna was found in a fellfield along the shores of the Fildes Peninsula (King George Island). The application of an improved enzymatic digestion coupled with Fourier transform infrared microscopy (µ-FTIR), unequivocally detected traces of PS (less than 100 µm) in the gut of the collembolans associated with the PS foam and documented their ability to ingest plastic. Plastics are thus entering the short Antarctic terrestrial food webs and represent a new potential stressor to polar ecosystems already facing climate change and increasing human activities. Future research should explore the effects of plastics on the composition, structure and functions of polar terrestrial biota.

Coastal environments can be influenced by water body masses with particular physical, chemical, and biological properties that create favourable conditions for the development of unique planktonic communities. In this study, we investigated a continental shelf front at Ponza Island (Tyrrhenian Sea) and discussed its diversity and complexity in relation to major environmental parameters. Moon phase and current direction were found to play a significant role in shaping species abundance and behaviour. During in situ observations, we also provided the first data on the behaviour of juveniles of a rare mesopelagic species, Trachipterus trachypterus, suggesting the occurrence of Batesian mimicry.

The monitoring of plastic pollution through marine biota is a difficult task, which is receiving increasing attention nowadays. A selection of appropriate bioindicator species for plastic ingestion has been proposed, mostly covering benthic filter-feeding organisms or large marine vertebrates. However, monitoring programs involving a broad range of synergetic bioindicators are still missing. Jellyfish have recently been reported as target organisms for marine litter, being able to internalise a number of anthropogenic fragments, from macro- to microplastics. With this perspective, we consider the trophic role of jellyfish and critically discuss its use as a novel bioindicator for plastic pollution on a global scale. Being a widespread energy source in pelagic and deep-sea food webs worldwide and occurring at high densities, jellyfish may represent the invertebrate bioindicator counterpart to monitor plastic pollution in pelagic waters, along with their common predators, and we recommend its inclusion in future monitoring surveys.

Titanium Dioxide Nanoparticles (TiO2 NPs) could cause alterations in exposed aquatic species, in terms of oxygen consumption, ammonia excretion and tissues functionality therefore, the aim of the present study is to evaluate the effects of acute exposure to different concentrations of TiO2 NPs (0.1, 0.5, 1.0 and 2.5 mgL-1) on routine metabolism (oxygen consumption and ammonia excretion) and histological parameters (branchial and hepatic) in Oreochromis niloticus. After 24 hours, we observed an increase in oxygen consumption of 2.36 and 3.23 times in groups exposed to 1.0 and 2.5 mgL-1 of TiO2 NPs respectively, as well as an increase in ammonia excretion of 3.54, 4.0 and 4.82 times higher in groups exposed to 0.5, 1.0 and 2.5 mgL-1 of TiO2 NPs respectively, compared to the control group. Histological analysis showed, after 72 hours, moderate to severe alterations in both gills and liver of TiO2 exposed fish at concentrations 1.0 and 2.5 mgL-1, the severity and occurrence of the alteration observed was grade 3 (severe and extensive pathological alterations). We concluded that waterborne exposure of Nile tilapia to TiO2 NPs caused alteration in routine metabolism and histological parameters in a dose-dependent manner. Nanoparticulas de Dioxido de Titânio (TiO2NPs) podem causar alteracoes nas especies aquaticas expostas, em termos de consumo de oxigenio, excrecao de amonia e funcionalidade de tecidos, portanto, o objetivo do presente estudo e avaliar os efeitos da exposicao aguda a diferentes concentracoes de TiO2NPs (0,1, 0,5, 1,0 e 2,5 mgL-1) sobre o metabolismo de rotina (consumo de oxigenio e excrecao de amonia) e parâmetros histologicos (branquiais e hepaticos) em Oreochromis niloticus. Apos 24 horas, observamos um aumento no consumo de oxigenio de 2,36 e 3,23 vezes em grupos expostos a 1,0 e 2,5 mgL-1 de TiO2NPs respectivamente, bem como um aumento na excrecao de amonia de 3,54, 4,0 e 4,82 vezes maior nos grupos expostos a 0,5, 1,0 e 2,5 mgL-1 de TiO2NPs respectivamente, em comparacao com o grupo controle. A analise histologica mostrou, apos 72 horas, alteracoes moderadas a severas tanto nas brânquias quanto no figado de peixes expostos a TiO2NPs em concentracoes de 1,0 e 2,5 mgL-1, a gravidade e a ocorrencia da alteracao observada foram de grau 3 (alteracoes patologicas graves e extensas). Concluimos que a exposicao a TiO2NPs da tilapia do Nilo causaram alteracao no metabolismo de rotina e nos parâmetros histologicos de uma maneira dose-dependente.

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-NH2) 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-NH2-formed nano-scale aggregates (Z-Average

The retrieval of a polystyrene macro-plastic piece stranded on the shores in King George Island (South Shetlands, Antarctica) gave the opportunity to explore the associated bacterial flora. A total of 27 bacterial isolates were identified by molecular 16s rRNA gene sequencing and 7 strains were selected and screened for their ability to produce biofilm and antibiotic susceptibility profiles. All the bacterial isolates were able to produce biofilm. The Kirby-Bauer disk diffusion susceptibility test to 34 antibiotics showed multiple antibiotic resistances against the molecules cefuroxime and cefazolin (belonging to cephalosporins), cinoxacin (belonging to quinolones) and ampicillin, amoxicillin + clavulanic acid, carbenicillin and mezlocillin (belonging to beta-lactams). The obtained results suggest that plastics can serve as vectors for the spread of multiple resistances to antibiotics across Antarctic marine environments and underline the relevance of future studies on this topic.

The adverse effects of engineered nanomaterials (ENM) in marine environments have recently attracted great attention although their effects on marine benthic organisms such as foraminifera are still largely overlooked. Here we document the effects of three negatively charged ENM, different in size and composition, titanium dioxide (TiO2), polystyrene (PS) and silicon dioxide (SiO2), on a microbial eukaryote (the benthic foraminifera Ammonia parkinsoniana) using multiple approaches. This research clearly shows the presence, within the foraminiferal cytoplasm, of metallic (Ti) and organic (PS) ENM that promote physiological stress. Specifically, marked increases in the accumulation of neutral lipids and enhanced reactive oxygen species production occurred in ENM-treated specimens regardless of ENM type. This study indicates that ENM represent ecotoxicological risks for this microbial eukaryote and presents a new model for the neglected marine benthos by which to assess natural exposure scenarios.

Micro- (, This work was financed by Generalitat Valenciana (GV-2014/085-Prometeo-II), and talian Antarctic Research Program (PNRA) contract number: PNRA 16_0075 NANOPANTA: Nano-Polymers in the Antarctic marine environment and biota

Polystyrene nanoparticles (PS NPs) are considered an upcoming threat to marine ecosystems. When NPs encounter biological fluids, they are readily covered by a biomolecular coating, named the “protein corona”, which critically drives the biological fate and toxicity of NPs. Here we provide the first attempt to identify key components of protein coronas of charged amino- and carboxyl-functionalized (PS-NH2 and PS-COOH, respectively) PS NPs exposed to coelomic fluid (CF) of the Mediterranean sea urchin Paracentrotus lividus. The formation of protein coronas conferred identical colloidal features to both positively and negatively charged PS NPs, which acquired a negative ζ potential and monodisperse size distribution in sea urchin CF. Proteomic analysis revealed striking functional analogies of proteins identified from NP–coronas, especially entailing the potential to promote cell association and internalization of both NP types, as mediated by adsorbed protein species. Such basal findings strengthen the importance of the biological identity of NPs in the evaluation of the ecotoxicity potential of polymeric NPs.

Nanoplastics are recognized as able to interact with other pollutants including heavy metals, and with natural organic matter, with implications for the potential risks to biota. We investigated the interaction of carboxylated polystyrene nanoparticles (PS–COOH NPs) with copper (Cu) and algal exudates (EPS) and how such interaction could affect Cu toxicity towards the freshwater microalga Raphidocelis subcapitata. PS–COOH NPs behavior in the presence of Cu and EPS was determined by dynamic light scattering (DLS), while PS–COOH NPs surface interaction with Cu ions and EPS was investigated by fluorimetric analysis. ICP-MS was used to test Cu ion adsorption to PS–COOH NPs in the presence and absence of algae. The interaction between PS–COOH NPs and the algal cell wall was assessed by fluorescence microscopy. Short- and long-term toxicity tests were carried out in parallel to assess the impact of PS–COOH NPs on algal growth. Results showed altered nanoparticle surface charge and hydrodynamic diameter following algal EPS exposure, supporting the hypothesis of a protein corona formation. In contrast, no absorption of Cu ions was observed on PS–COOH NPs, either in the presence or absence of algae. No differences on algal growth inhibition were observed between exposure to Cu only, and to Cu in combination with PS–COOH NPs, in short-term as well as long-term tests. However, after 72 h of exposure, the adsorption of PS-COOH NPs to algal cell walls appeared to correspond to morphological alterations, revealing potential disturbances in the mitotic cycle. Our findings confirm the ability of PS–COOH NPs to interact with EPS as shown for other nanomaterials. Environmentally realistic exposure scenarios are thus needed for evaluating nanoplastic toxicity, as nanoparticles will not maintain their pristine nature once released into natural media. Prolonged exposure and use of different end-points such as cell morphological changes and EPS production seem more reliable for the investigation of nanoplastic/algal cell interactions which can drive food chain transfer of nanoplastics and ultimately toxicity.

There is evidence and serious concern that microplastics have reached the most remote regions of the planet, but how far have they travelled in terrestrial ecosystems? This study presents the first field-based evidence of plastic ingestion by a common and central component of Antarctic terrestrial food webs, the collembolan Cryptopygus antarcticus . A large piece of polystyrene (PS) foam (34 × 31 × 5 cm) covered by microalgae, moss, lichens and microfauna was found in a fellfield along the shores of the Fildes Peninsula (King George Island). The application of an improved enzymatic digestion coupled with Fourier transform infrared microscopy (µ-FTIR), unequivocally detected traces of PS (less than 100 µm) in the gut of the collembolans associated with the PS foam and documented their ability to ingest plastic. Plastics are thus entering the short Antarctic terrestrial food webs and represent a new potential stressor to polar ecosystems already facing climate change and increasing human activities. Future research should explore the effects of plastics on the composition, structure and functions of polar terrestrial biota.

The incorporation of silver nanoparticles (AgNPs) in commercial products is increasing rapidly. The consequent release of AgNPs into domestic and industrial wastewater raises environmental concerns due to their anti-microbial properties and toxicity to non-target aquatic organisms. The aim of the present study was to investigate the effects of nanArgen™ (Nanotek S.A.), a AgNP-enabled consumer product, in the marine bivalve Mytilus galloprovincialis. Two environmentally relevant concentrations of nanArgen™ (1 and 10 μg/L) were tested in vivo for 96 h, and Ag was quantified in mussel soft tissue and natural seawater (NSW). nanArgen™ suspensions were characterized via TEM, SEM, EDS, DLS, and UV-vis optical analysis. Several molecular and biochemical responses were investigated in exposed mussels: lysosomal membrane stability by Neutral Red Retention Time (NRRT) assay; micronucleus (MN) frequency in hemocytes; metallothionein (MT) protein content and gene expression (mt10 and mt20); catalase (CAT) and glutathione-S-transferase (GST) activities; malondialdehyde (MDA) accumulation in digestive glands; and efflux activity of ATP-binding cassette transport proteins (ABC) in gill biopsies. SEM, TEM and DLS analyses confirmed the presence of well-defined AgNPs in nanArgen™ which were roughly spherical with an average particle size of approx. 30 ± 10 nm. DLS analysis revealed the formation of AgNP aggregates in nanArgen™ suspension in NSW (Z-average of 547.80 ± 90.23 nm; PDI of 0.044). A significant concentration-dependent accumulation of Ag was found in mussels' whole soft tissue in agreement with a concentration-dependent decrease in NRRT and an increase of MN frequency in hemocytes and GST activities in digestive glands. A significant increase in MDA levels and MT via both molecular and biochemical tests, were also observed but only at the highest nanArgen™ concentration (10 μg/L). No changes were observed in CAT activities. ABC efflux activities in gill biopsies showed a significant decrease (p 0.05) only at the lowest concentration (1 μg/L). On such basis, nanArgen™ is shown to be able to induce toxicity and Ag accumulation in marine mussels similarly to AgNPs and in short-term exposure conditions at environmentally relevant concentrations. AgNP-enabled products, instead of pristine AgNPs, should be the focus of future ecotoxicity studies in order to address any risks associated to their widespread use, disposal and uncontrolled release into the aquatic environment for non target species.

In order to assess the impact of nanoplastics on marine species, polystyrene nanoparticles (PS NPs) have been largely used as model particles. Here we studied the effects of 50 nm amino-modified PS-NH

Recent developments on environmental fate models indicate that as nano waste, engineered nanomaterials (ENMs) could reach terrestrial ecosystems thus potentially affecting environmental and human health. Plants can be therefore exposed to ENMs but controversial data in terms of fate and toxicity are currently available. Furthermore, there is a current lack of information on complex interactions/transformations to which ENMs undergo in the natural environment as for instance interacting with existing toxic compounds. The aim of the present study was to assess the behavior and biological effects of titanium dioxide nanoparticles (n-TiO2) (Aeroxide P25, Degussa Evonik) and its interaction with cadmium (CdCl2) in plants using radish seeds (Raphanus sativus L. Parvus) as model species. Radish seeds were exposed to n-TiO2 (1-1000mg/L) and CdCl2 (1-250mg/L) alone and in combination using a seed germination and seedling growth toxicity test OECD 208. Percentage of seed germination, germination index (GI) and root elongation were calculated. Cell morphology and oxidative stress parameters as glutathione-S-transferase (GST) and catalase activities (CAT) were measured in radish seeds after 5 days of exposure. Z-Average, PdI and Z-potential of n-TiO2 in Milli-Q water as exposure medium were also determined. DLS analysis showed small aggregates of n-TiO2, negative Z-potential and stable PdI in seed's exposure media. Germination percentage, GI and root length resulted affected by n-TiO2 exposure compared to controls. In particular, n-TiO2 at 1mg/L and 100mg/L did not affect radish seeds germination (100%) while at concentration of 10mg/L, 200mg/L, 500mg/L, and 1000mg/L a slight but not significant decrease of germination % was observed. Similarly root length and GI resulted significantly higher in seeds exposed to 10mg/L and 200mg/L compared to 1mg/L, 100mg/L, 500mg/L, 1000mg/L and control (p < 0.05). On the opposite, CdCl2 significantly abolished germination % and GI compared to control seeds and a concentration dependent decrease on root elongation was observed against controls (p < 0.05). As well, significant decrease of germination %, GI and root elongation was observed in seeds co-exposed to n-TiO2 and CdCl2 at the highest concentrations (1000mg/L n-TiO2 and 250mg/L CdCl2) compared to co-exposed seeds at low concentration (1mg/L n-TiO2 and 1mg/L CdCl2) and controls (p < 0.05). Root elongation significantly increase compared to control at the lowest co-exposure concentration (p < 0.05). Similarly at intermediate concentrations of 10 and 100mg/L in co-exposure conditions, n-TiO2 did not affect CdCl2 toxicity. Concerning antioxidant enzymes, a significant increase of CAT activity in seeds exposed to single high n-TiO2 concentration (1000mg/L) was observed while n-TiO2 (1mg/L), CdCl2 (1 and 250mg/L) and co-exposure resulted significantly decreased compared to controls (p < 0.05). Regarding GST activity, a slight increase in seeds exposed to 1000mg/L n-TiO2 but no significantly was observed, however both n-TiO2 and CdCl2 alone (1 and 250mg/L, respectively) or in combinations caused a significant decrease in GST activity (p < 0.05). Therefore, overall data support the hypothesis that the presence of n-TiO2 do not affect the toxicity of CdCl2 at least at the highest concentration (100 and 250mg/L) in radish seeds. Morphological alterations in nuclei, vacuoles and shape of radish root cells were observed upon single Cd exposure and not abolished in the presence of n-TiO2. Nevertheless, although n-TiO2 seems not to reduce Cd toxicity at high concentration (up to 250mg/L), interactions cannot be excluded based on obtained results.

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH2), has been investigated on the marine rotifer Brachionus plicatilis, a major component of marine zooplanktonic species. The role of different surface charges in affecting PS NP behaviour and toxicity has been considered in high ionic strength media. To this aim, the selected media were standardized reconstituted seawater (RSW) and natural sea water (NSW), the latter resembling more natural exposure scenarios. Hatched rotifer larvae were exposed for 24h and 48h to both PS NPs in the range of 0.5-50μg/ml using PS NP suspensions made in RSW and NSW. No effects on lethality upon exposure to anionic NPs were observed despite a clear gut retention was evident in all exposed rotifers. On the contrary, cationic NPs caused lethality to rotifer larvae but LC50 values resulted lower in rotifers exposed in RSW (LC50=2.75±0.67µg/ml) compared to those exposed in NSW (LC50=6.62±0.87µg/ml). PS NPs showed similar pattern of aggregation in both high ionic strength media (RSW and NSW) but while anionic NPs resulted in large microscale aggregates (Z-average 1109 ± 128nm and 998±67nm respectively), cationic NP aggregates were still in nano-size forms (93.99 ± 11.22nm and 108.3 ± 12.79nm). Both PDI and Z-potential of PS NPs slightly differed in the two media suggesting a role of their different surface charges in affecting their behaviour and stability. Our findings confirm the role of surface charges in nanoplastic behaviour in salt water media and provide a first evidence of a different toxicity in rotifers using artificial media (RSW) compared to natural one (NSW). Such evidence poses the question on how to select the best medium in standardized ecotoxicity assays in order to properly assess their hazard to marine life in natural environmental scenarios.

Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 μg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses random...

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Polymeric nanoparticles can reach the marine environment from different sources as weathering of plastic debris and nanowaste. Nevertheless, few data are available on their fate and impact on marine biota. Polystyrene nanoparticles (PS NPs) can be considered as a model for studying the effects of nanoplastics in marine organisms: recent data on amino-modified PS NPs (PS-NH2) toxicity in sea urchin embryos underlined that marine invertebrates can be biological targets of nanoplastics. Cationic PS NPs have been shown to be toxic to mammalian cells, where they can induce apoptotic processes; however, no information is available on their effects and mechanisms of action in the cells of marine organisms. In this work, the effects of 50 nm PS-NH2 were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. Hemocytes were exposed to different concentrations (1, 5, 50 μg/ml) of PS-NH2 suspension in ASW. Clear signs of cytoxicity were evident only at the highest concentrations (50 μg/ml). On the other hand, a dose dependent decrease in phagocytic activity and increase in lysozyme activity were observed. PS-NH2 NPs also stimulated increase in extracellular ROS (reactive oxygen species) and NO (nitric oxide) production, with maximal effects at lower concentrations. Moreover, at the highest concentration tested, PS-NH2 NPs induced apoptotic process, as evaluated by Flow cytometry (Annexin V binding and mitochondrial parameters). The results demonstrate that in marine invertebrates the immune function can represent a significant target for PS-NPs. Moreover, in Mytilus hemocytes, PS-NH2 NPs can act through mechanisms similar to those observed in mammalian cells. Further research is necessary on specific mechanisms of toxicity and cellular uptake of nanoplastics in order to assess their impact on marine biota.

Nano-sized polymers as polystyrene (PS) constitute one of the main challenges for marine ecosystems, since they can distribute along the whole water column affecting planktonic species and consequently disrupting the energy flow of marine ecosystems. Nowadays very little knowledge is available on the impact of nano-sized plastics on marine organisms. Therefore, the present study aims to evaluate the effects of 40 nm anionic carboxylated (PS-COOH) and 50 nm cationic amino (PS-NH2) polystyrene nanoparticles (PS NPs) on brine shrimp Anemia franciscana larvae. No signs of mortality were observed at 48 h of exposure for both PS NPs at naplius stage but several sub-lethal effects were evident. PS-COOH (5-100 mu g/ml) resulted massively sequestered inside the gut lumen of larvae (48 h) probably limiting food intake. Some of them were lately excreted as fecal pellets but not a full release was observed. Likewise, PS-NH2 (5-100 mu g/ml) accumulated in larvae (48 h) but also adsorbed at the surface of sensorial antennules and appendages probably hampering larvae motility. In addition, larvae exposed to PS-NH2 undergo multiple molting events during 48 h of exposure compared to controls. The activation of a defense mechanism based on a physiological process able to release toxic cationic NPs (PS-NH2) from the body can be hypothesized. The general observed accumulation of PS NPs within the gut during the 48 h of exposure indicates a continuous bioavailability of nano-sized PS for planktonic species as well as a potential transfer along the trophic web. Therefore, nano-sized PS might be able to impair food uptake (feeding), behavior (motility) and physiology (multiple molting) of brine shrimp larvae with consequences not only at organism and population level but on the overall ecosystem based on the key role of zooplankton on marine food webs. (C) 2015 Elsevier Inc. All rights reserved.