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Plastic pollution represents an emerging environmental issue in terrestrial Antarctica, especially in the Antarctic Peninsula and Maritime Antarctica, which have been recently recognized as hot spots for plastic litter. In these regions, freshwater (FW) environments such as lakes host isolated ecosystems and species that can be severely affected by increasing environmental and anthropogenic stressors, which include plastics that are still overlooked. In this study, we investigated for the first time the impact of nanoplastics on adults of the fairy shrimp Branchinecta gaini (Order Anostraca) populating Antarctic FW ecosystems, using surface charged polystyrene nanoparticles (PS NPs) as a proxy. Short-term acute toxicity (48 h) was investigated by exposing adults to carboxyl (-COOH, 60 nm) and amino-modified (-NH 2 , 50 nm) PS NPs at 1 and 5 μg mL -1 . Biodisposition of PS NPs and lethal and sub-lethal effects (i.e., swimming, moulting, histology, gene expression) were assessed. Behaviour of PS NPs in Antarctic FW media was monitored through 48 h of exposure showing that both PS NPs kept their nanoscale size in the Antarctic FW media. Survival of fairy shrimp adults over short-term exposure was not affected, on the other hand an increase in moulting rate and alterations in the gut epithelium were observed upon exposure to both PS NPs. Significant alterations at the behavioural (ventilation rate) and molecular (up-regulation of Hsp70mit, Hsp83, Sod, P450) levels were related to PS NP surface charge and associated with PS-NH 2 exposure only. Nanoplastics could represent a threat for Antarctic FW biodiversity and the Antarctic fairy shrimp could be a valuable model for assessing their impact on such remote and pristine aquatic ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 2 ) and 60 nm carboxy-modified (PS-COOH) polystyrene nanoparticles (PS NPs) on the embryonic development of the ascidian Ciona robusta (phylum Chordata), a common benthic invertebrate living in Mediterranean coastal areas with the peculiarity of being an early chordate developmental model. A strong agglomeration of PS-COOH (approx. 1 µm) was observed in natural sea water (NSW) already at time 0, while PS-NH 2 resulted still monodispersed (approx. 130 nm) but largely aggregated after 22 h with a microscale dimension similar to those negatively charged. However, their effect on C. robusta embryos development largely differed at 22 h: PS-COOH did not affect larvae phenotypes nor their development, while PS-NH 2 caused a dose-dependent effect (EC 50 (22 h) of 7.52 μg mL -1 ) with various degrees of phenotype malformations (from mild to severe) and impairment of larval swimming. Embryos (up to 30%) exposed to 15 µg mL -1  PS-NH 2 resulted not developed and the majority was unable to hatch. Calculated PS-NH 2 EC 50 resulted higher than those available for other marine invertebrate species, suggesting a protective role of the egg envelopes surrounding C. robusta embryos toward nanoplastics exposure.

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.

Micro- (<5 mm) and nanoplastics (<1 μm) are emerging threats for marine ecosystems worldwide. Brine shrimp Artemia is recognized as a suitable model among planktonic species for studying the impact of polystyrene nanoparticles (PS NPs) through short and long-term bioassays. Our study aims to evaluate the time-dependent effects of cationic amino-modified PS-NH 2 (50 nm) in A. franciscana after short- (48 h) and long-term exposure (14 days). For this purpose, nauplii were exposed to a concentration range of PS-NH 2 (0.1, 1, 3 and 10 μg/mL) in natural sea water (NSW), and physiological, biochemical and molecular responses were investigated. Short-term exposure to PS-NH 2 caused a decrease in nauplii growth and affected the development in a concentration-dependent manner, long-term exposure impaired the survival, but not the growth and feeding behavior. Oxidative stress was detected after short term exposure as the decrease in the activity of antioxidant enzymes, and was fully evident in the long-term as lipid peroxidation, suggesting an accumulative effect. The decrease in Cholinesterase (ChE) activity observed indicates possible neurotoxic action of PS-NH 2 . Also, Carboxylesterase (CbE) inhibition by PS-NH 2, described for the first time in this study, anticipates potential effects in biotransformation of exogenous and endogenous compounds, being the crustacean juvenile hormone methyl farnesoate (MF) that regulates development and molting, one candidate. Furthermore, short- and long-term exposure to PS-NH 2 affect the expression of genes involved in cell protection, development and molting. Overall, our results reveal that low PS-NH 2 concentrations induce physiological, biochemical and molecular (changes in gene expression) alterations in Artemia, and point at their potential risk for this model organism, supporting the general concern about nanoplastics occurrences in aquatic environments and their ability to represent an ecological threat for aquatic zooplanktonic species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nowadays, the occurrence of a large volume of plastic litter in oceanic and coastal zones has increased concern about its impacts on marine organisms. The degradation of plastic polymers leads to the formation of smaller fragments at both micro and nano scale (<5 mm and <1 μm respectively). Nanoplastics (NPs), due to their smaller size and high specific surface area can establish colloidal interactions with marine microalgae, therefore potential toxicity can be led. . To assess this hypothesis, the aim of the present study is to examine the behaviour of polystyrene nanoparticles (PS NPs) of different sizes (50 and 100 nm) in marine water and their possible effects at different physiological and cellular levels in the marine diatom Phaeodactylum tricornutum. Different biomarkers and stress responses in P. tricornutum were analysed when organisms were exposed to environmentally relevant PS NPs concentrations between 0.1 and 50 mg L -1 . Our results showed significant differences between controls and exposure microalgae, indicating toxicity. After 24 h, an increase in oxidative stress biomarkers, damage to the photosynthetic apparatus, DNA damage and depolarization of mitochondrial and cell membrane from 5 mg L -1 were observed. Further after 72 h the inhibition of population growth and chlorophyll content were observed. Examining effects the effects related to PS NPs size, the smallest (50 nm) induced greater effects at 24 h while bigger PS NPs (100 nm) at72 h. This bigger particles (100 nm) showed more stability (in size distribution and spherical form) in the different culture media assayed, when compared with the rest of particles used. Strong adsorption and/or internalization of PS NPs was confirmed through changes in cell complexity and cell size as well as the fluorescence of 100 nm fluoresbrite PS NPs after washing cell surface., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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 2 on Mediterranean sea urchin Paracentrotus lividus immune system cells (coelomocytes) in the presence of celomic fluid (CF) and at different NP concentrations (1, 5, 10, and 25 μg mL -1 ) and experimental conditions (absence or presence of EDTA). PS-NH 2 acquired a protein corona once incubated with CF, dominated by the toposome precursor protein (TPP). In short-term cultures, a significant concentration- and time-dependent decrease in lysosomal membrane stability and apoptotic-like nuclear alterations were observed in phagocytes upon exposure to PS-NH 2 (10 and 25 µg mL -1 ) in CF but they resulted abolished in the presence of EDTA confirming the role of TPP in triggering PS-NH 2 -coelomocytes interaction and toxicity. PS-NH 2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH 2 to affect pump's efflux activity. Overall results encourage additional studies on positively charged nanoplastics, since the observed effects on sea urchin coelomocytes as well as the TPP corona formation might represent a first step for addressing their impact on sensitive marine species.

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH 2 ), 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 LC 50 values resulted lower in rotifers exposed in RSW (LC 50 =2.75±0.67µg/ml) compared to those exposed in NSW (LC 50 =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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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

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-NH 2 , 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-NH 2 were used to expose sea urchin embryos in natural sea water (PS-NH 2 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 randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH 2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 40nm anionic carboxylated (PS-COOH) and 50nm cationic amino (PS-NH2) polystyrene nanoparticles (PS NPs) on brine shrimp Artemia franciscana larvae. No signs of mortality were observed at 48h of exposure for both PS NPs at naplius stage but several sub-lethal effects were evident. PS-COOH (5-100μg/ml) resulted massively sequestered inside the gut lumen of larvae (48h) probably limiting food intake. Some of them were lately excreted as fecal pellets but not a full release was observed. Likewise, PS-NH2 (5-100µg/ml) accumulated in larvae (48h) 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 48h 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 48h 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.