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The physical and chemical properties of nanoplastics make them potential carriers for some environmental contaminants, modifying their biological effects. Nevertheless, the change in toxicity caused by pollutant adsorption on nanoplastics is still controversial, depending on the interactions between chemical and physical pollutants, the consequent change in bioavailability, the modification of intake, transport and accumulation in the organisms and also on the characteristics of contaminants. In this context, the aim of the present study was the evaluation of combined effects made by 0.5 μm nanobeads of polystyrene and triclosan adsorbed on their surface in comparison with those caused by single contaminants. The systemic effects of 7 day exposure to nanoplastics, triclosan alone and to the nanoplastic–triclosan complex have been analyzed by employing zebrafish larvae and using a multi-tier approach from the evaluation of cellular and molecular effects to the impact at organism level. Results highlighted by confocal microscopy evidenced nanobead ingestion and translocation in several tissues and organs to guarantee the goodness of the exposure results. Behavioral assays were then conducted to highlight larval swimming defects as a ‘real-time’ readout of the potential effects on the whole organism, while a suite of several biomarkers and functional proteomics was applied to investigate the effects at both cellular and molecular levels. The whole data set pointed out a clear modification in the toxicological effects of the nanoplastic–triclosan complex in comparison with single contaminants, proved by opposite behaviours in the larval swimming activity and modulation of diverse protein classes as well as by different effects on several biochemical endpoints. This means that the interaction between chemical and physical pollutants leads to more complicated responses than additive, synergistic or antagonist models, resulting in a modification of toxicity instead of its increase or decrease.

Despite the pollution induced by plastics become a well-known and documented problem, bringing many countries to adopt restrictions about their production, commercialization and use, the impact of another emerging category of synthetic polymers, represented by the Water-Soluble Polymers (WSPs), also known as "liquid plastics", is overlooked by scientific community. WSPs are produced in large quantities and used in a wide plethora of applications such as food packaging, pharmaceuticals and personal care products, cosmetics and detergents, with a consequent continuous release in the environment. The aim of this study was the investigation of the possible toxicity induced by polyvinyl alcohol (PVA), one of the main produced and used WSPs, on two freshwater model organisms, the crustacean Daphnia magna and the teleost Danio rerio (zebrafish). We evaluated the effects of solubilized standard PVA powder and PVA-based commercial bags for carp-fishing, at 3 different concentrations (1 µg/L, 0.5 mg/L and 1 mg/L), through the exposures for 14 days of D. magna (daphnids; age24 h) and for 5 days of zebrafish embryos (up to 120 h post fertilization - hpf). As acute effects we evaluated the immobilization/mortality of specimens, while for chronic toxicity we selected several endpoints with a high ecological relevance, as the behavioural alteration on swimming performance, in real-time readout, and the activity of monoamine oxidase (MAO), a neuro-enzyme with a potential implication in the organism movement. The results showed the lack of significant effects induced by the selected substances, at all tested concentrations and in both model organisms. However, considering the wide plethora of available WSPs, other investigations are needed to provide the initial knowledge of risk assessment of these compounds contained in some consumer products.

Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics’ toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size = 1 and 10 μm) each one. The mixture at the lowest concentration contained 5 × 105 MP/L of 1 μm and 5 × 105 MP/L of 10 μm, while the higher one was arranged with 2 × 106 MP/L of 1 μm and 2 × 106 MP/L of 10 μm. Proteomics’ analyses of gills showed the complete lack of proteins’ modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress.

Triclosan (TCS, 5‑chloro‑2‑(2,4‑dichlorophenoxy) phenol) is becoming a major surface waters pollutant worldwide at concentrations ranging from ng L−1 to μg L−1. Up to now, the adverse effects on aquatic organisms have been investigated at concentrations higher than the environmental ones, and the pathways underlying the observed toxicity are still not completely understood. Therefore, the aim of this study was to investigate the toxic effects of TCS at environmental concentrations on zebrafish embryos up to 120 hours post fertilization (hpf). The experimental design was planned considering both the quantity and the exposure time for the effects on the embryos, exposing them to two different concentrations (0.1 μg L−1, 1 μg L−1) of TCS, for 24 h (from 96 to 120 hpf) and for 120 h (from 0 to 120 hpf). A suite of biomarkers was applied to measure the induction of embryos defence system, the possible increase of oxidative stress and the DNA damage. We measured the activity of glutathione‑S‑transferase (GST), P‑glycoprotein efflux and ethoxyresorufin‑o‑deethylase (EROD), the level of ROS, the oxidative damage through the Protein Carbonyl Content (PCC) and the activity of antioxidant enzymes. The genetic damage was evaluated through DNA Diffusion Assay, Micronucleus test (MN test), and Comet test. The results showed a clear response of embryos defence mechanism, through the induction of P-gp efflux functionality and the activity of detoxifying/antioxidant enzymes, preventing the onset of oxidative damage. Moreover, the significant increase of cell necrosis highlighted a strong cytotoxic potential for TCS. The overall results obtained with environmental concentrations and both exposure time, underline the critical risk associated to the presence of TCS in the aquatic environment.

This study aimed to assess the combined effects of ocean acidification (OA) and pollution to the polychaete Syllis prolifera inhabiting the CO

The more or less extensive lockdowns, quarantines, smart working and the closure of numerous recreational or personal care activities due to the COVID-19 pandemic have not only heavily changed the habits and behaviors of all of us, but have also had consequences on the release of some types of pollutants. The aim of this study was to evaluate the possible changes due to the indirect effects of the pandemic in the contamination of plastic mixtures sampled in 9 sites of the main watercourses of the metropolitan city of Milan (N. Italy), which is one of the major industrialized and urbanized areas in Italy. To achieve this goal, we carried out two sampling campaigns, the first one carried out in November 2019, before the arrival of the SARS-CoV-2 virus in Italy, the second in November 2020, during a severe regional lockdown that coincided with other restrictions imposed at the national level. The main results showed a difference in contamination of plastics between the two samplings, not so much due to a quantitative variation, but certainly qualitative. We obtained non-homogeneous data with respect to changes in the number of plastics sampled in the different waterbodies, while it was evident that the plastics' contamination has shifted from a primary and industrial origin to one due to a secondary origin of the sampled plastics, linked especially to the fragmentation of common use objects, or deriving from synthetic garments.

The study of the contamination of plastic mixtures sampled in natural environments is currently focused on their qualitative and quantitative assessment, while the evaluation of their effects on organisms is normally performed by experiments carried out at exposure conditions (size, shape, polymers) often far from the environmental ones. To improve the ecological realism, the aim of this study was to collect different plastic mixtures in 9 sampling stations located in 7 watercourses within the metropolitan city of Milan, one of the most anthropized and industrialized European areas, to evaluate both their qualitative and quantitative characteristics and, at the same time, to assess their ecotoxicological effects by exposing for 7 days some specimens of the freshwater bivalve Dreissena polymorpha to the mixtures collected in the sampling sites. The plastic characterization was performed by a Fourier-Transform Infrared spectrometer coupled with an optical microscope (μFT-IR), after several stages aimed to sample cleaning, separation of plastics and visual sorting. The possible effects caused by the plastic mixtures were carried out by the measurements of a biomarker suite to evaluate many cellular and molecular endpoints in mussel tissues. The main results showed a widespread and heterogeneous contamination of plastics in the entire metropolitan area, with contamination peaks found above all in the only two rivers of natural origin (Olona River and Lambro River) where comparable or higher values were reached than plastic concentrations measured in several European rivers. Despite this worrying contamination, the ecotoxicological data obtained after the exposures to the plastic mixtures collected in the selected water bodies showed only a mild effect on oxidative stress and on the variation of some antioxidant enzymes.

The adsorption of biomacromolecules is a fundamental process that can alter the behaviour and adverse effects of nanoparticles (NPs) in natural systems. While the interaction of NPs with natural molecules present in the environment has been described, their biological impacts are largely unknown. Therefore, this study aims to provide a first evidence of the influence of biomolecules sorption on the toxicity of cerium oxide nanoparticles (CeO2NPs) towards the freshwater bivalve Dreissena polymorpha. To this aim, we compared naked CeO2NPs and coated with alginate and chitosan, two polysaccharides abundant in aquatic environments. Mussels were exposed to the three CeO2NPs (naked, chitosan- and alginate-coated) up to 14 days at 100 μg L−1, which is a concentration higher than the environmental one predicted for this type of NP. A suite of biomarkers related to oxidative stress and energy metabolism was applied, and metabolomics was also carried out to identify metabolic pathways potentially targeted by CeO2NPs. Results showed that the coating with chitosan reduced NP aggregation and increased the stability in water. Nonetheless, the Ce accumulation in mussels was similar in all treatments. As for biological effects, all three types of CeO2NPs reduced significantly the level of reactive oxygen species and the activity of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase. The effect was more pronounced in individuals exposed to CeO2NPs coated with alginate, which also significantly induced the activity of the electron transport system. Metabolomics analysis of amino acid metabolism showed modulation only in mussels treated with CeO2NPs coated with alginate. In this group, 25 metabolites belonging to nucleotides, lipids/sterols and organic osmolytes were also modulated, suggesting that the nanoparticles affect energetic metabolism and osmoregulation of mussels. This study highlights the key role of the interaction between nanoparticles and natural molecules as a driver of nanoparticle ecotoxicity.

This study had the dual objective of both the qualitative and quantitative assessment of plastic mixtures sampled in 5 different sites located along the Lambro River (northern Italy), and the contemporarily determination of the ecotoxicological effects of the same mixtures sampled, through 21-day laboratory exposures of the freshwater bivalve Dreissena polymorpha. The monitoring survey was carried out by a Fourier Transform Infrared Microscope System, while the ecotoxicological assessment was performed by the mussel mortality, a biomarker suite and the proteomics. The main results of the monitoring have highlighted some critical points, related to the concentration of plastics detected at Milan and, especially at the southernmost sampling station, where a daily flow of more than 6 million plastic debris has been estimated, ending directly into the Po River, the main Italian river. The ecotoxicological analysis highlighted how the toxicity is not exclusively due to the plastic concentration, but that the different characteristics of the polymers probably become more important. Furthermore, we observed an extensive mortality of bivalves exposed to the sampled mixtures in the two southernmost sampling stations, while the battery of biomarkers and the results of proteomics have highlighted how the sampled plastic mixtures caused an imbalance in the redox state, already indicated as a classic effect due to plastic exposure, but also an impact on energy stock and on some fundamental cellular pathways always linked to energy metabolism.

This study aimed to assess the toxicological consequences related to the interaction of fullerene nanoparticles (C60) and Benzo(α)pyrene (B(α)P) on zebrafish embryos, which were exposed to C60 and B(α)P alone and to C60 doped with B(α)P. The uptake of pollutants into their tissues and intra-cellular localization were investigated by immunofluorescence and electron microscopy. A set of biomarkers of genotoxicity and oxidative stress, as well as functional proteomics analysis were applied to assess the toxic effects due to C60 interaction with B(α)P. The carrier role of C60 for B(α)P was observed, however adsorption on C60 did not affect the accumulation and localization of B(α)P in the embryos. Instead, C60 doped with B(α)P resulted more prone to sedimentation and less bioavailable for the embryos compared to C60 alone. As for toxicity, our results suggested that C60 alone elicited oxidative stress in embryos and a down-regulation of proteins involved in energetic metabolism. The C60 + B(α)P induced cellular response mechanisms similar to B(α)P alone, but generating greater cellular damages in the exposed embryos. Once C60 nanoparticles and B(α)P meet in water, they reciprocally affect their bioavailability and, by consequence, their toxicity to organisms.

Microplastics (MPs), plastic debris smaller than 5mm, are widely found in both marine and freshwater ecosystems. However, few studies regarding their hazardous effects on inland water organisms, have been conducted. For this reason, the aim of our research was the evaluation of uptake and chronic toxicity of two mixtures (MIXs) of virgin polystyrene microbeads (PMs) of 10μm and 1μm in size (MIX 1, with 5×105 of 1μmsizePMs/L and 5×105 of 10μmsizePMs/L, and MIX 2 with 2×106 of 1μmsizePMs/L and 2×106 of 10μmsizePMs/L) on freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia) during 6 exposure days. The PM uptake in the mussel body and hemolymph was assessed using confocal microscopy, while the chronic toxicity of PMs was evaluated on exposed mussels using a comprehensive battery of biomarkers of cellular stress, oxidative damage and neuro- genotoxicity. Confocal microscopy analyses showed that MPs concentrated in the gut lumen of exposed mussels, absorbed and transferred firstly in the tissues and then in the hemolymph. The results revealed that PMs do not produce oxidative stress and genetic damage, with the exception of a significant modulation of catalase and glutathione peroxidase activities in mussels exposed to MIX 1. Regarding neurotoxicity, we observed only a significant increase of dopamine concentration in mussels exposed to both MIXs, suggesting a possible implication of this neurotransmitter in an elimination process of accumulated PMs. This research represents a first study about the evaluation of virgin MP toxicity in zebra mussel and more research is warranted concerning the long term neurological effects of virgin MPs.

Particularly upon their first appearance, Twitter and James Joyce’s unique style had to face similarly infamous charges in that they were both accused of slowly but inexorably corrupting the English language. In fact, the linguistic practices – stylemes in the case of Ulysses – breeding such corruption are often the same: clippings, abbreviations, the dropping out of inflexional endings, omissions, ellipses, the overabundance of acronyms, creative compound words and blends, symbolisations and onomatopoeias, lexicalisations of (often) vernacular pronunciations. This paper sets out to investigate these features, their rhetorical effect and pragmatic function in order to explore the epistemological, perceptive and social context which made it possible a hundred years ago for an Irish modernist to anticipate how English was to be used on today’s social media and on Twitter in particular.

The increasing use of nanomaterials raises several concerns regarding their potential risk for the environment and human health. In particular, the aquatic ecosystems appear highly susceptible. In this context, we investigated the interplay between carbon nanopowder (CNPW) and the common pollutant benzo(α)pyrene (B(α)P) in zebrafish embryos. CNPW was contaminated with B(α)P, and showed significant adsorption towards the hydrocarbon. Embryos were then exposed to CNPW (50 mg L−1) or B(α)P (0.2, 6, 20 μg L−1) alone, or to the CNPW doped with the three B(α)P concentrations. We demonstrated that CNPW helps B(α)P uptake by zebrafish embryos and we also demonstrated that the interaction between CNPW and the hydrocarbon affects the stress response pathways of the organism, so eliciting the toxic effect. In particular, the modulation of genes related to the cellular stress response (cyp1a, hsp70, sod1, sod2) and the measurement of oxidative stress enzyme activities allowed us to identify critical molecular events modulated by the pollutants alone and in co-exposure. Finally, to evaluate the toxic effects due to CNPW interactions with B(α)P, we analyzed biomarkers of cyto-genotoxicity. No significant genotoxicity was induced by B(α)P and CNPW alone, but the co-exposure led to an increase of cytotoxicity, and a higher incidence of necrotic and apoptotic cells. Altogether our data show that nanomaterials, even if they are not toxic per se, could help to enhance the toxicity of common pollutants.

Studies related to the evaluation of plastics in freshwaters have been increasing in recent years because approximately 80% of plastic items found in the sea are from inland waters. Despite the ecological relevance of these surveys, no information has been available until now about the hazard related to plastic mixtures in freshwaters. To fill this knowledge gap, we carried out a study aimed to assess the environmental risk associated with the “cocktail” of plastics and environmental pollutants adsorbed on their surface in one of the larger European freshwater basins. Plastic debris was collected by a manta trawl along one transect each in four of the Italian subalpine great lakes (Lake Maggiore, Como, Iseo and Garda) and administered to zebra mussels (Dreissena polymorpha), a useful freshwater biological model present in all these lakes. We estimated a plastic density from 4908 MPs/km2 (Lake Iseo) to 272,261 MPs/km2 (Lake Maggiore), while the most common polymers found were polyethylene and polypropylene, with percentages varying between 73% and 100%. A biomarkers suite consisting of 10 different endpoints was performed after 7 days of exposure to investigate the molecular and cellular effects of plastics and related adsorbed pollutants. The main results highlighted a diffuse but different toxicity due to plastics for each lake, and there were significant changes in the antioxidant and detoxifying enzyme activities in Lake Maggiore, Iseo and Garda, an increase in protein carbonylation in L. Como, and a cellular viability decrease of approximately 30% for zebra mussels from L. Iseo and Garda. Despite this variability in the endpoints' responses, the application of the biomarker response index showed a similar environmental hazard due to plastics for all the sampled lakes.

Manganese (Mn) might stimulate the valve closure reflex in the freshwater bivalve Anodontites trapesialis, leading to metabolic suppression, whereas zinc (Zn) is not able to modify this behavior. To investigate this particular response, we exposed A. trapesialis specimens to Mn (0.5 mg L-1 ) and Zn (1.0 mg L-1 ) alone, and to their mixture, to measure further endpoints in different clam tissues: glycogen level in gills, and calcium (Ca2+ ), sodium (Na+ ), and chloride (Cl- ) concentrations in the hemolymph. Furthermore, we used cutting-edge technology, proteomics, to evaluate modifications in protein patterns under the 3 exposure tests. The main results highlighted that only Mn caused a clear drop in glycogen levels in gills, an increase in Ca2+ and Na+ , and a simultaneous decrease in Cl- concentration in the hemolymph. The proteomic analysis confirmed that Mn promoted more effects in A. trapesialis than the other tested conditions, because the number of proteins modulated was higher than the results obtained after exposure to Zn and the mixture. Moreover, 11 of the 12 modulated proteins were down-expressed. These results consolidate the hypothesis that Mn might suppress gill metabolic rate in A. trapesialis. Environ Toxicol Chem 2019;38:2480-2485. © 2019 SETAC.

Cannabis is the most used illicit drug worldwide and in some countries a new regulatory policy makes it legal under some restrictions. This situation could lead to a substantial increase in environmental levels of the cannabis active principle (Δ-9-tetrahydrocannabinol [Δ-9-THC]) and its main metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Although previous studies have highlighted the toxicity of Δ-9-THC, the adverse effects of THC-COOH on aquatic organisms is completely unknown, even though such effects could be more significant because the environmental concentrations of THC-COOH are higher than those of the parent compound. The present study aimed to assess oxidative and genetic damage to the zebra mussel (Dreissena polymorpha) because of 14-d exposures to 3 THC-COOH concentrations, mimicking a current environmental situation (100 ng/L), as well as exposure to 2 possible worst-case scenarios (500 ng/L and 1000 ng/L), because of the potential increase in THC-COOH in surface waters. Variations in the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were measured, as well as levels of lipid peroxidation and protein carbonyl content. Genetic injuries were investigated by single-cell gel electrophoresis assay, DNA diffusion assay, and the micronucleus test. A significant imbalance in antioxidant defense enzymes was noted in response to the 3 tested concentrations, whereas oxidative damage was noted only at the higher one. Moreover, an increase in DNA fragmentation in zebra mussel hemocytes, but no fixed genetic damage, was found. Although the results showed that THC-COOH toxicity was lower than that of Δ-9-THC, the increase in cannabis use might increase its levels in freshwaters, enhancing its hazard to bivalves and likely to the whole aquatic community. Environ Toxicol Chem 2017;36:472–479. © 2016 SETAC

Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic ecosystems up to µg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occurrence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Electrophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell-shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation.

Dreissena polymorpha is a widespread filter-feeder species, resistant to a broad range of environmental conditions and different types of pollutants,which has recently colonized Italian freshwaters. Although widely used to monitor pollution in freshwater environments, this species is also an important food source for some fish and water birds. It can also be used to concentrate or remove particulate organic matter to interrupt avian-to-human transmission of pollutants and control health risks for animals and humans. In this study, the accumulation/inactivation in D. polymorpha of human health-related spiked enteric viruses was described. The removal of endogenous Escherichia coli, the classical indicator of fecal contamination,was tested as well.Our preliminary lab-scale results demonstrate that zebra mussels can reduce significantly poliovirus titer after 24 h and rotavirus titer after 8 h. E. coli counts were also reduced in the presence of zebra mussels by about 1.5 log after 4 h and nearly completely after 24 h. The fate of the two enteric viruses after concentration by zebra mussels was also investigated after mechanical disruption of the tissues. To our knowledge, the accumulation from water and inactivation of human health-related enteric viruses by zebra mussels has never been reported.

The improper release of plastic items and wastes is nowadays one of the main environmental and social problems, whose solution or mitigation represents a great challenge worldwide. In this context, the growing use of the so-called biodegradable plastics could represent a possible solution in the short to medium term. The few information known about the ecological impact of these materials on freshwater organisms, especially the ones relative to the micro-debris derived from their aging, prompted us to study the comparison of the sub-lethal effects eventually caused by plastic and biodegradable plastic micro-debris on the mussel Dreissena polymorpha, which represents an excellent biological model for the freshwater ecosystems. We selected two powders of polyvinylchloride (PVC) and Mater-Bi® administered at 1 mg/L to D. polymorpha specimens in semi-static conditions for 14 days. The presence of micro-debris was evaluated on mussel tissues and pseudo-faeces using advanced microscopy techniques. The sub-lethal effects were investigated on exposed mussels at 6 and 14 days using a suite of biomarkers of cellular stress, oxidative damage, and genotoxicity. Lastly, we compared the ecotoxicity of these two materials integrating each endpoint in the Biomarker Response Index. Microscopy observations highlighted the surprising absence of micro-debris in the gut lumen and tissues of exposed mussels, but the presence of both PVC and Mater-Bi® micro-debris in the pseudo-faeces, suggesting a possible efficient elimination mechanism adopted by mussels to avoid the micro-debris gulping. Consequently, we did not observe significant sub-lethal effects, except for the glutathione-S-transferase activity modulation after 6 days of exposure.

The ongoing development of nanotechnology has raised concerns regarding the potential risk of nanoparticles (NPs) to the environment, particularly aquatic ecosystems. A relevant aspect that drives NP toxicity is represented by the abiotic and biotic processes occurring in natural matrices that modify NP properties, ultimately affecting their interactions with biological targets. Therefore, the objective of this study was to perform an ecotoxicological evaluation of CeO2NPs with different surface modifications representative of NP bio-interactions with molecules naturally occurring in the water environment, to identify the role of biomolecule coatings on nanoceria toxicity to aquatic organisms. Ad hoc synthesis of CeO2NPs with different coating agents, such as Alginate and Chitosan, was performed. The ecotoxicity of the coated CeO2NPs was assessed on the marine bacteria Aliivibrio fischeri, through the Microtox® assay, and with the freshwater crustacean Daphnia magna. Daphnids at the age of 8 days were exposed for 48 h, and several toxicity endpoints were evaluated, from the molecular level to the entire organism. Specifically, we applied a suite of biomarkers of oxidative stress and neurotoxicity and assessed the effects on behaviour through the evaluation of swimming performance. The different coatings affected the hydrodynamic behaviour and colloidal stability of the CeO2NPs in exposure media. In tap water, NPs coated with Chitosan derivative were more stable, while the coating with Alginate enhanced the aggregation and sedimentation rate. The coatings also significantly influenced the toxic effects of CeO2NPs. Specifically, in D. magna the CeO2NPs coated with Alginate triggered oxidative stress, while behavioural assays showed that CeO2NPs coated with Chitosan induced hyperactivity. Our findings emphasize the role of environmental modification in determining the NP effects on aquatic organisms. Interactions with environmental molecules influence the fate and ecotoxicity of NPs.

The emerged threat of microplastics (MPs) in aquatic ecosystems is posing a new challenges in environmental management, in particular the civil Wastewater Treatment Plants (WWTPs) which can act both as collectors of MPs from anthropic use and as a source to natural environments. In this study, MP fate was investigated in one of the biggest WWTPs of Northern Italy, built at the beginning of the 2000s and which serves a population equivalent of about 1,200,000, by evaluating their presence at the inlet (IN), the removal efficiency after the settler (SET) and at the outlet (OUT), and their transfer to sludge. Samples were collected in three days of a week and plastic debris was characterized in terms of shape, size and polymer composition using the Fourier Transform Infrared Microscope System (μFT-IR). The number of detected MPs was 2.5 ± 0.3 MPs/L in the IN, 0.9 ± 0.3 MPs/L after the SET and 0.4 ± 0.1 MPs/L in the OUT, indicating a total removal efficiency of 84%. However, considering that this WWTP treats about 400,000,000 L wastewaters/day, the potential release of MPs to the receiving aquatic system would be approximately 160,000,000 MPs/day, mainly polyesters (35%) and polyamide (17%). Furthermore, a great amount of MPs removed from wastewater was detected in the recycled activated sludge, with 113 ± 57 MPs/g sludge dry weight, corresponding to about 3,400,000,000 MPs deposited in the 30 tons of sludge daily produced by this WWTP. Given the possible re-use of WWTP sludge in fertilizers for agriculture, our results highlight that WWTPs could represent a potential source of MPs also to agroecosystems.

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.

This study aimed to assess the toxicological consequences related to the interaction of fullerene nanoparticles (C

DDx, PCBs, PBDEs, Hg, and As contamination in sediments and aquatic organisms of different trophic levels (zooplankton, mussel, fish) were analyzed in Lake Maggiore and Lake Lugano, two large deep perialpine lakes. In the period 2001–2015, we analyzed the spatial and temporal trends of the considered pollutants to detect potential contamination sources and to compare concentrations with Sediment Quality Guidelines (SQGs) or existing Quality Standards (QSs). DDx and Hg contamination deriving from past industrial activities in the Pallanza Basin still exceeded SQGs in sediments and QSs in fish, with potential risks for the ecosystem. Banned in Europe in 1985, PCBs showed low residual values, while recent PBDE peaks resulted in the exceedance of the QSs for biota in both lakes, probably due to current industrial activities. Arsenic mainly derives from geochemical origin. The analysis of the biomagnification of toxicants in a pelagic food chain in Lake Maggiore (zooplankton–fish) according to a stable isotope approach is also presented, according to both the Trophic Magnification Factor and the Trophic Level-adjusted BioMagnification Factor: the importance of seasonality and a Hg > DDx ≈ PBDEs biomagnification capacity were observed. Low PCB bioaccumulation was detected in biota, probably because equilibrium was not reached yet in young fish.

The metallic element pollution is a serious environmental problem but still unsolved since these contaminants are released mainly by human activity, reaching all the environmental compartments. Traditional wastewater treatment plants are very efficient in removing metallic elements only when their concentration is in the order of mg/L, but are not able to remove them until μg/L, as it would be needed to cope with the water quality standards in low flow receptors. Therefore, the aim of our study was to evaluate the potential removal of some recalcitrant metallic elements to the classical treatments, by the natural process of bio-filtration performed by the invasive zebra mussel (Dreissena polymorpha). For this purpose we built a pilot-plant at the Milano-Nosedo wastewater treatment plant, where we placed about 40,000 D. polymorpha specimens appointed to the wastewater bio-filtration. The metallic element removal due to zebra mussel activity was evaluated in the treated wastewater with a plasma optical emission spectrometry (ICP-OES). Data obtained in these experiments showed an encouraging metallic element removal due to D. polymorpha activity; in particular, the total abatement (100%) of Cr after one day of bio-filtration exposure is remarkable. Therefore, this study encourages further research related with the use of bivalves as a new tool for the wastewater depuration process; in this regard, the contaminated mollusks used in the bio-filtration could be incinerated or stored in special landfills, as is also the case of traditional sewage sludge.

Illicit drugs are considered to be emerging aquatic pollutants since they are commonly found in freshwater ecosystems in the high ng L(-1) to low μg L(-1) range concentrations. Although the environmental occurrence of the most common psychoactive compounds is well known, recently some investigations showed their potential toxicity toward non-target aquatic organisms. However, to date, these studies completely neglected that organisms in the real environment are exposed to a complex mixture, which could lead to dissimilar adverse effects. The present study investigated the oxidative alterations of the freshwater bivalve Dreissena polymorpha induced by a 14-d exposure to an environmentally relevant mixture of the most common illicit drugs found in the aquatic environment, namely cocaine (50 ng L(-1)), benzoylecgonine (300 ng L(-1)), amphetamine (300 ng L(-1)), morphine (100 ng L(-1)) and 3,4-methylenedioxymethamphetamine (50 ng L(-1)). The total oxidant status (TOS) was measured to investigate the increase in the reactive oxygen species' levels, while the activity of antioxidant enzymes and glutathione S-transferase were measured to note the eventual imbalances between pro-oxidant and antioxidant molecules. In addition, oxidative damage was assessed by measuring the levels of lipid peroxidation and protein carbonylation. Significant time-dependent increases of all the antioxidant activities were induced by the mixture. Moreover, the illicit drug mixture significantly increased the levels of carbonylated proteins and caused a slight variation in lipid peroxidation. Our results showed that a mixture of illicit drugs at realistic environmental concentrations can impair the oxidative status of the zebra mussel, posing a serious hazard to the health status of this bivalve species.

Synthetic musk compounds (SMCs) are extensively used as fragrances in several personal care products and have been recognized as emerging aquatic pollutants. Among SMCs, galaxolide (HHCB) and tonalide (AHTN) are extensively used and have been measured in aquatic ecosystems worldwide. However, their potential risk to organisms remains largely unknown. The aim of this study was to investigate whether 21-day exposures to HHCB and AHTN concentrations frequently measured in aquatic ecosystems can induce oxidative and genetic damage in Dreissena polymorpha. The lipid peroxidation (LPO) and protein carbonyl content (PCC) were measured as oxidative stress indexes, while the DNA precipitation assay and the micronucleus test (MN test) were applied to investigate genetic injuries. HHCB induced significant increases in LPO and PCC levels, while AHTN enhanced only protein carbonylation. Moreover, significant increases in DNA strand breaks were caused by exposure to the highest concentrations of HHCB and AHTN tested in the present study, but no fixed genetic damage was observed.

One of the fundamentals in the ecotoxicological studies is the need of data comparison, which can be easily reached with the help of a standardized biological model. In this context, any biological model has been still proposed for the biomonitoring and risk evaluation of freshwaters until now. The aim of this review is to illustrate the ecotoxicological studies carried out with the zebra mussel Dreissena polymorpha in order to suggest this bivalve species as possible reference organism for inland waters. In detail,we showed its application in biomonitoring, as well as for the evaluation of adverse effects induced by several pollutants, using both in vitro and in vivo experiments. We discussed the advantages by the use of D. polymorpha for ecotoxicological studies, but also the possible limitations due to its invasive nature.

Introduction Ethiopia has experienced rapid expansion of antiretroviral therapy (ART). However, as long-term retention in ART therapy is key for ART effectiveness, determinants of attrition need to be identified so appropriate interventions can be designed. Methods We used data from the ‘Cohort of African people Starting Antiretroviral therapy’ (CASA) project, a prospective study of a cohort of HIV-infected patients who started ART in seven health facilities (HFs). We analysed the data of patients who had started first-line ART between January 2013 and December 2014. The Kaplan–Meier method was used to estimate the probability of retention at different time points. The Cox proportional hazards model was used to identify factors associated with attrition. Results A total of 1198 patients were included in the study. Kaplan–Meier estimates of retention in care were 83.9%, 82.1% and 79.8% at 12, 18 and 24 months after starting ART, respectively. Attrition was mainly due to loss to follow-up, transferred-out patients and documented mortality. A multivariate Cox proportional hazard model showed that male sex, CD4 count

Illicit drugs have been identified as emerging aquatic pollutants because of their widespread presence in freshwaters and potential toxicity towards aquatic organisms. Among illicit drug residues, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly detected in freshwaters worldwide at concentration that can induce diverse adverse effects to non-target organisms. However, the information of toxicity and mechanisms of action (MoA) of these drugs, mainly of COC metabolites, to aquatic species is still fragmentary and inadequate. Thus, this study was aimed at investigating the toxicity of two concentrations (0.3 and 1.0 μg/L) of COC, BE and EME similar to those found in aquatic ecosystems on zebrafish (Danio rerio) embryos at 96 h post fertilization through a functional proteomics approach. Exposure to COC and both its metabolites significantly altered the protein profile of zebrafish embryos, modulating the expression of diverse proteins belonging to different functional classes, including cytoskeleton, eye constituents, lipid transport, lipid and energy metabolism, and stress response. Expression of vitellogenins and crystallins was modulated by COC and both its main metabolites, while only BE and EME altered proteins related to lipid and energy metabolism, as well as to oxidative stress response. Our data confirmed the potential toxicity of low concentrations of COC, BE and EME, and helped to shed light on their MoA on an aquatic vertebrate during early developmental period.

Pharmaceutical and personal care products (PPCPs) have been recognized as emerging aquatic pollutants. Even if their presence in aquatic ecosystems is well-known, the information regarding the effects towards aquatic non-target organisms is inadequate and is often related to a limited number of conventional model species. The present chapter highlights the suitability of the zebra mussel (Dreissena polymorpha) as a non-conventional model organism for aquatic ecotoxicology to investigate the toxicity of emerging pollutants. We investigated the adverse effects due to 96 hours in vivo exposure to environmental concentrations (1 nM) of diverse PPCPs, namely triclosan (TCS), trimethoprim (TMP), ibuprofen (IBU), diclofenac (DCF) and paracetamol (PCM), on the zebra mussel by using a suite of biomarkers. The analysis of biomarker responses and their integration into a biomarker response index (BRI) showed that TCS was the most toxic for the zebra mussel molecule among the tested PPCPs, followed by TMP, IBU, DCF and PCM. Our findings demonstrate that the sensitivity of the zebra mussel in response to low PPCP concentrations confers to this bivalve the status of a reliable model organism for aquatic ecotoxicology, suggesting that it should be routinely used to evaluate the toxicity of emerging aquatic pollutants.

Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the “particle-specific” effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

a b s t r a c t This study shows the evaluation of the possible use of the freshwater bivalve Dreissena polymorpha for the removal of some recalcitrant contaminants, namely many pharmaceuticals and drugs of abuse that are not sufficiently removed from civil wastewaters. This mollusk has an enormous filtering capability and is highly resistant to natural and anthropogenic stresses and to a significant bioaccumulation of lipophilic contaminants. All these characteristics may be particularly useful for the removal of compounds not easily eliminated by conventional wastewater treatment processes. To verify this hypothesis an experimental study was conducted at the pilot scale using a pilot plant installed in the largest wastewater treatment plant of Milan (Milano-Nosedo, Italy). First, we presented results obtained in several preliminary tests in order to evaluate the capability of zebra mussel specimens to survive in different wastewater mixtures, its filtering capacity and the possible influence of bio- and photo-degradation in the abatement of the molecules of interest. Finally, data obtained in the final tests demonstrated a capacity of this filter-feeder to reduce the concentrations of several pharmaceuticals and drugs of abuse higher than that obtained by the simple natural sedimentation, suggesting a possible implementation of the bio-filtration process in wastewater management. © 2014 Elsevier B.V. All rights reserved.

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.

A fugacity model reported in the literature was applied to a high-altitude pasture in the Italian Alps. The model takes into account three compartments (digestive tract, blood and fat tissues) in unsteady-state conditions using food as the contamination source. Disregarding biotransformation inside cow tissues, the predicted concentrations of 14 polychlorinated biphenyls (PCBs) in milk were in good agreement with the observed data, especially for congeners known for their resistance to biotransformation (e.g., CB-138 and 153). In contrast, the predicted concentrations were clearly overestimated for congeners with high biotransformation susceptibilities. Therefore data measured in milk and faeces were used to calculate the first-order-biotransformation rate constants in dairy cows. The PCB absorption efficiency observed for pasture conditions was lower than that observed in the cowshed. The final version of the model included biotransformation and observed PCB absorption and was able to predict PCB concentrations in cow milk with mean differences between the predicted and measured data below ± 20% for most congeners.

PCBs are a persistent environmental problem due to their high stability and lipophilicity. The non-ortho- and the mono-ortho-substituted PCBs (dioxin-like-PCBs) share a common and well-described toxicity mechanism in vertebrates, initially involving binding to cytosolic AhRs. Invertebrate AhRs, however, show a lack of dioxin binding, and little information is available regarding the mechanism of toxicity of dl-PCBs in invertebrates. In this study, a proteomic approach was applied to analyse the variations in the pattern of the gill proteome of the freshwater mussel Dreissena polymorpha. Mussels were exposed to a mixture of dl-PCBs, and to perform a more in-depth evaluation, we chose to investigate the role of gender in the proteome response by analysing male and female mussels separately. The results revealed significant modulation of the gill tissue proteome: glycolysis and Ca2 + homeostasis appear to be the main pathways targeted by dl-PCBs. In light of the differences between the male and female gill proteome profiles following exposure to dl-PCBs, further in-depth investigations of the role of gender in the protein expression profiles of a selected biological model are required.

The Lake Maggiore (Northern Italy) has been recognized as an aquatic environment heavily contaminated by persistent organic pollutants, mainly organochlorine compounds, but to date limited information is available regarding another class of widespread and hazardous pollutants, such as the polycyclic aromatic hydrocarbons (PAHs). The aim of this study was to investigate seasonal and temporal trends of 18 PAHs accumulated in native Dreissena polymorpha specimens during a 5-year biomonitoring program, as well as to identify the possible PAH emission sources by using isomeric diagnostic ratios. Zebra mussels were sampled both in their pre- (May) and post-reproductive (September) stage over the 2008-2012 period in eight sampling stations covering the whole lake shoreline. PAH concentrations were measured through gas chromatography coupled to mass spectrometry. A notable PAH contamination following an increasing temporal trend was noticed in bivalves from all the sampling stations, with the benzo(α)anthracene as the predominant compound. An overall increase in PAH levels was found in the post-reproductive surveys, indicating a marked seasonality of this contamination probably due to the increase in touristic activity during spring-summer months.

Cannabis is the most used illicit substance worldwide and its main psychoactive compound, the Δ-9-tetrahydrocannabinol (Δ-9-THC), is detected in aquatic environments at measurable concentrations. Even though its occurrence is well documented, no information is available on its hazard to aquatic organisms. The aim of this study was to assess the adverse effects induced to zebra mussel (Dreissena polymorpha) specimens by 14 day exposures to environmentally relevant Δ-9-THC concentrations (0.05 μg/L and 0.5 μg/L) by means of the application of a biomarker suite. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indices. The single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test) were applied to investigate DNA injuries, while the neutral red retention assay (NRRA) was used to assess Δ-9-THC cytotoxicity. The lowest treatment induced negligible adverse effects to bivalves, while 0.5 μg/L Δ-9-THC exposure caused remarkable alterations in D. polymorpha oxidative status, which lead to significant increase of lipid peroxidation, protein carbonylation and DNA damage.

One of the current main challenges faced by the scientific community is concerning the fate and toxicity of plastics, due to both the well-known threats made by larger plastic items spreading in ecosystems and their fragmentation into micro- and nanoparticles. Since the chemical and physical characteristics of these smaller plastic fragments are markedly different with respect to their bulk product, the potential toxicological effects in the environment need to be deeply investigated. To partially fill this gap of knowledge, the aim of this study was to evaluate the polystyrene nanobead intake in the tissues of zebrafish (Danio rerio) embryos and their related toxicity. Embryos at 72 h post fertilization (hpf) were exposed for 48 h to 0.5 μm fluorescent polystyrene nanobeads at a concentration of 1 mg L−1. Confocal microscopy was employed to investigate nanoplastic ingestion and tissue infiltration, while potential sub-lethal effects were evaluated by measuring several endpoints, which covered the adverse effects at the molecular (protein carbonylation), cellular (P-glycoprotein, activity of several antioxidant/detoxifying enzymes) and organism levels by evaluating of possible changes in the embryos' swimming behaviour. Imaging observations clearly highlighted the nanoplastics' uptake, showing nanobeads not only in the digestive tract, but also migrating to other tissues through the gut epithelium. Biomarker analyses revealed a significant decrease in cyclooxygenase activity and an induction of superoxide dismutase. The behavioural test highlighted a significant (p

Illicit drugs have been recently identified as a serious environmental problem because of the growing evidence regarding their occurrence in aquatic environment and potential toxicity towards non-target organisms. Among them, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly measured in freshwaters worldwide at levels that might cause diverse sub-lethal effects to aquatic organisms. Thus, the present study was aimed at investigating the potential adverse effects induced by the exposure to environmental concentrations (0.04, 0.4, 4 and 40 nM) of COC, BE, and EME on zebrafish (Danio rerio) embryos at 96 h post fertilization. Cytotoxicity was assessed by the Trypan Blue exclusion method, while primary and fixed genetic damages were evaluated by the Single Cell Gel Electrophoresis (SCGE) assay, and the DNA diffusion assay together with the Micronucleus test, respectively. The involvement of oxidative stress in the mechanism of action (MoA) of all tested drugs was assessed by measuring the activity of defense enzymes (SOD, CAT, GPx, and GST) and the expression of their encoding genes. Exposure to COC and both metabolites significantly reduced cell viability, increased DNA fragmentation and promoted the onset of apoptotic cells and micronuclei in zebrafish embryos. Results from oxidative stress-related endpoints and gene expression suggested that the observed genotoxicity may be caused by an overproduction of free radicals that imbalanced the oxidative status of embryos. The integration of biomarker responses into a synthetic index showed that at each tested concentration, BE and EME had a similar toxicity and were both more toxic than COC. Our data confirmed the potential toxicity of environmental concentrations of COC, BE, and EME, suggesting the need of further in-depth studies to shed light on their MoA and long-term toxicity towards non-target aquatic species.