Alterations induced by titanium dioxide nanoparticles (nano-TiO2) in fertilization and embryonic and larval development of the tropical sea urchin Lytechinus variegatus.

The release of nanomaterials into the environment is the cause of an emerging concern. Titanium dioxide nanoparticles (nano-TiO2) among the most produced nanomaterials, has been documented in marine coastal areas posing a threat on marine biota. Sea urchin embryos are recognized as suitable bioindicators in ecological risk assessment and recently for nanomaterials. This study investigated the impact of nano-TiO2 on fertilization, embryonic and larval development of the tropical sea urchin Lytechinus variegatus in a range of concentrations (0.005-5 µg/mL) which includes environmentally relevant ones. The behavior of nano-TiO2 in tropical natural seawater was determined by dynamic light scattering (DLS) and toxicity was evaluated through fertilization and embryotoxicity tests, and morphological/morphometric analyses of sea urchin's larvae. Limited toxicity was recorded for nano-TiO2 in tropical sea urchin embryos and larvae, except for effects at the gastrula stage at 0.005 µg/mL. Large agglomerates of nano-TiO2 (5 µg/mL) were observed adhering onto sea urchin larvae thus probably preventing nanoparticles uptake at the highest concentrations (>0.005 µg/mL). Environmental levels of nano-TiO2 are able to cause toxicity on tropical sea urchin L. variegatus embryos with potential consequences on populations and their ecological role in tropical coastal areas.

Textile microfibers in wild Antarctic whelk Neobuccinum eatoni (Smith, 1875) from Terra Nova Bay (Ross Sea, Antarctica).

Antarctica has been affected directly and indirectly by human pressure for more than two centuries and recently plastic pollution has been recognized as a further potential threat for its unique biodiversity. Global long-range transport as well as local input from anthropogenic activities are potential sources of plastic pollution in both terrestrial and marine Antarctic territories. The present study evaluated the presence of microplastics in specimens of the Antarctic whelk Neobuccinum eatoni, a key species in benthic communities of the Ross Sea, one of the largest marine protected areas worldwide. To this aim, a thermo-oxidative extraction method was applied for microplastic isolation and quantification, and polymer identification was performed by manual µ-FTIR spectroscopy. Textile (semi-)synthetic or composite microfibers (length range: 0.8-5.7 mm) were found in 27.3% of whelk specimens, suggesting a low risk of bioaccumulation along Antarctic benthic food webs in the Ross Sea. Their polymer composition (of polyethylene terephthalate and cellulose-polyamide composites) matched those of outdoor technical clothing in use by the personnel of the Italian "Mario Zucchelli" station near Terra Nova Bay in the Ross Sea. Such findings indicate that sewage from base stations may act as potential local sources of textile microplastic fibers in this remote environment. More in-depth monitoring studies aiming at defining the extent of microplastic contamination related to such sources in Antarctica are encouraged.

Behavioural, physiological and molecular responses of the Antarctic fairy shrimp Branchinecta gaini (Daday, 1910) to polystyrene nanoplastics.

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 (-NH2, 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-NH2 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.

Under pressure: Nanoplastics as a further stressor for sub-Antarctic pteropods already tackling ocean acidification.

In the Southern Ocean (SO), plastic debris has already been found in waters and sediments. Nanoplastics (<1 µm) are expected to be as pervasive as their larger counterparts, but more harmful to biological systems, being able to enter cells and provoke toxicity. In the SO, (nano)plastic pollution occurs concomitantly with other environmental threats such as ocean acidification (OA), but the potential cumulative impact of these two challenges on SO marine ecosystems is still overlooked. Here the single and combined effects of nanoplastics and OA on the sub-Antarctic pteropod Limacina retroversa are investigated under laboratory conditions, using two surface charged polystyrene nanoparticles (PS NPs) as a proxy for nanoplastics. Sub-Antarctic pteropods are threatened by OA due to the sensitivity of their shells to changes in seawater carbonate chemistry. Short-term exposure (48 h) to PS NPs compromised the ability of pteropods to counteract OA stress, resulting in a negative effect on their survival. Our results highlights the importance of addressing plastic pollution in the context of climate change to identify realistic critical thresholds of SO pteropods.

Nanoplastics affect moulting and faecal pellet sinking in Antarctic krill (Euphausia superba) juveniles.

Plastic debris has been identified as a potential threat to Antarctic marine ecosystems, however, the impact of nanoplastics (<1 µm) is currently unexplored. Antarctic krill (Euphausia superba) is a keystone species of Southern Ocean pelagic ecosystems, which plays a central role in the Antarctic food webs and carbon (C) cycle. Krill has been shown to rapidly fragment microplastic beads through the digestive system, releasing nanoplastics with unknown toxicological effects. Here we exposed krill juveniles to carboxylic (COOH, anionic) and amino- (NH2, cationic) polystyrene nanoparticles (PS NPs) and we investigated lethal and sub-lethal endpoints after 48 h. The analysis of PS NP suspensions in Antarctic sea water (SW) media showed that PS-COOH formed large agglomerates (1043 ± 121 nm), while PS-NH2 kept their nominal size (56.8 ± 3 nm) during the exposure time. After 48 h, no mortality was found but increase in exuviae production (12.6 ± 1.3%) and reduced swimming activity were observed in juveniles exposed to PS-NH2. The microbial community composition in SW supports the release of krill moults upon PS NP exposure and stimulates further research on the pivotal role of krill in shaping Southern Ocean bacterial assemblages. The presence of fluorescent signal in krill faecal pellets (FPs) confirmed the waterborne ingestion and egestion of PS-COOH at 48 h of exposure. Changes in FP structure and properties were also associated to the incorporation of PS NPs regardless of their surface charge. The effects of PS NPs on krill FP properties were compared to Control 0 h as a reference for full FPs (plastic vs food) and Control 48 h as a reference for more empty-like FPs (plastic vs lack of food). Exposure to PS NPs led to a FP sinking rate comparable to Control 48 h, but significantly lower than Control 0 h (58.40 ± 23.60 m/d and 51.23 ± 28.60 m/d for PS-COOH and PS-NH2; 168.80 ± 74.58 m/d for Control 0 h). Considering the important role played by krill in the food web and C export in the Southern Ocean, the present study provides cues about the potential impact of nanoplastics on Antarctic pelagic ecosystems and their biogeochemical cycles.

Combined effects of nanoplastics and copper on the freshwater alga Raphidocelis subcapitata.

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.

Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype.

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-NH2 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-NH2 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-NH2 (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-NH2-coelomocytes interaction and toxicity. PS-NH2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH2 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.

Episodic records of jellyfish ingestion of plastic items reveal a novel pathway for trophic transference of marine litter.

Invertebrates represent the most plentiful component of marine biodiversity. To date, only few species have been documented for marine litter intake. Here, we report for the first time the presence of macroplastic debris in a jellyfish species. Such novel target to plastic pollution highlights an under studied vector of marine litter along marine trophic web, raising further concern over the impact on marine wildlife.

Co-exposure to titanium dioxide nanoparticles does not affect cadmium toxicity in radish seeds (Raphanus sativus).

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.

Comparative ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer Brachionus plicatilis.

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.

Long-term toxicity of surface-charged polystyrene nanoplastics to marine planktonic species Dunaliella tertiolecta and Artemia franciscana.

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<200nm), caused inhibition of algal growth (EC50=12.97µg/ml) and mortality in brine shrimps at 14 d (LC50=0.83µg/ml). Moreover, 1µg/ml PS-NH2 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-NH2 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.

Evidence for immunomodulation and apoptotic processes induced by cationic polystyrene nanoparticles in the hemocytes of the marine bivalve Mytilus.

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.

Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

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.

Static and dynamic baropodometry to evaluate patients treated by total knee replacement with a mobile meniscus.

It was the purpose of this study to use static and dynamic baropodometry pre- and postsurgery in patients submitted to knee replacement with a mobile bearing. Baropodometric analysis was accompanied by clinical and subjective evaluation. Data obtained from the baropodometer revealed considerable oscillations occurring between pre- and postsurgery. Static examination showed a significant increase in the percentage of loading on the side treated that was not observed in the dynamic evaluation. An analysis of the Knee Society score showed a considerable increase in values between pre- and postsurgery similar to how the interpretation of correlations presurgery revealed statistically significant values between function and length of step, between pre- and postsurgery data for scores for function and length of step and Knee Score. The short-term results obtained are encouraging but only a long-term follow-up will be able to confirm the use of evaluating total knee replacement.

Testosterone replacement, cardiovascular system and risk factors in the aging male.

Investigations concerning the role of testosterone replacement on cardiovascular risk show conflicting results. Treatments with supraphysiological doses seem detrimental in animal models and men. On the other hand, cross-sectional, prospective and angiographic studies frequently find an inverse, favorable relationship between plasma testosterone and cardiovascular events. Testosterone replacement therapy in the hypogonadic elderly has a positive or at least neutral effect on several coronary disease risk factors. Testosterone appears to decrease LDL-cholesterol without adversely affecting HDL cholesterol, and improve insulin sensibility and the thrombotic/fibrinolytic balance; testosterone does not negatively influence the inflammatory response and arterial wall vasoreactivity. These findings provide a measure of reassurance concerning potential adverse heart effects of testosterone substitutional therapy in older men, even if more specific trials than reported are needed to overcome residual suspicions.