Plastics in biogenic matrices intended for reuse in agriculture and the potential contribution to soil accumulation.

The spread of biogenic matrices for agricultural purposes can lead to plastic input into soils, raising a question on possible consequences for the environment. Nonetheless, the current knowledge concerning the presence of plastics in biogenic matrices is very poor. Therefore, the objective of the present study was a quali-quantitative characterization of plastics in different matrices reused in agriculture as manures, digestate, compost and sewage sludges. Plastics were quantified and characterized using a Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in Attenuated Total Reflectance mode. Our study showed the presence of plastics in all the investigated samples, albeit with differences in the content among the matrices. We measured a lower presence in animal matrices (0.06-0.08 plastics/g wet weight w.w.), while 3.14-5.07 plastics/g w.w. were measured in sewage sludges. Fibres were the prevalent shape and plastic debris were mostly in the micrometric size. The most abundant polymers were polyester (PEST), polypropylene (PP) and polyethylene (PE). The worst case was observed in the compost sample, where 986 plastics/g w.w. were detected. The majority of these plastics were compostable and biodegradable, with only 8% consisting of fragments of PEST and PE. Our results highlighted the need to thoroughly evaluate the contribution of reused matrices in agriculture to the plastic accumulation in the soil system.

Unveiling the multilevel impact of four water-soluble polymers on Daphnia magna: From proteome to behaviour (a case study).

The ubiquitous presence of water-soluble polymers (WSPs) in freshwater environments raises concerns regarding potential threats to aquatic organisms. This study investigated, for the first time, the effects of widely used WSPs -polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), and polyethylene glycol (PEG)- using a multi-level approach in the freshwater biological model Daphnia magna. This integrated assessment employed a suite of biomarkers, evaluation of swimming behaviour, and proteomic analysis to investigate the effects of three environmentally relevant concentrations (0.001, 0.5, and 1 mg/L) of the tested WSPs from molecular to organismal levels, assessing both acute and chronic effects. Our findings reveal that exposure to different WSPs induces specific responses at each biological level, with PEG being the only WSP inducing lethal effects at 0.5 mg/L. At the physiological level, although all WSPs impacted both swimming performance and heart rate of D. magna specimens, PAA exhibited the greatest effects on the measured behavioural parameters. Furthermore, proteomic analyses demonstrated altered protein profiles following exposure to all WSPs, with PVA emerging as the most effective.

Monthly variability of floating plastic contamination in Lake Maggiore (Northern Italy).

The monitoring of plastics in freshwater ecosystems has witnessed a significant increase in recent years, driven by the awareness that approximately 80 % of marine plastic litter originates from terrestrial sources transported to the seas through lakes and rivers. Consequently, it is imperative to develop monitoring plans that offer a comprehensive understanding of plastic contamination in these aquatic environments, given their seasonal variations in hydrochemical characteristics and anthropogenic sources. Historically, most global lake monitoring campaigns have been limited to one-time or, at most, seasonal sampling. In this context, the primary objective of the present study was to assess the quantitative and qualitative monthly variations of floating plastics in Lake Maggiore, a large European lake with high ecological and economic significance. Twelve transverse transects were conducted from January to December 2022 using a Manta-net with a 100 µm mesh. Characterization of each plastic particle was performed using a µ-Fourier Transform Infrared Spectroscope (µFT-IR). The results revealed relatively low levels of contamination in Lake Maggiore when compared with other lakes worldwide exclusively from a secondary origin. However, a considerable heterogeneity was observed, both quantitatively and qualitatively. Notably, we identified a 13-fold difference between the minimum (0.02 plastics/m3 in September) and maximum (0.29 plastics/m3 in December) concentrations of plastics, accompanied by significant variations in polymer composition. Our monitoring underscored the necessity of also considering the temporal variation as a potential factor influencing plastic contamination in a lake. Moreover, frequent sampling emerged as a crucial requirement to accurately gauge the extent of plastic pollution, yielding robust and valuable data essential for effective environmental management.

To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

Comparison of plastic pollution between waters and sediments in four Po River tributaries (Northern Italy).

The monitoring of plastic contamination in freshwaters is still pioneering in comparison with marine environments, and few studies analyzed the distribution of these pollutants in both aqueous and bottom compartments of continental waters. Therefore, the aim of this study was the comparison of plastic pollution in both waters and sediments of four Po River tributaries (Ticino, Adda, Oglio and Mincio Rivers), which outflow from the main Italian sub-alpine Lakes, in order to establish the strengths and weaknesses of both matrices. The main results pointed out a heterogeneous plastic contamination, with the lowest values in Ticino (0.9 ± 0.5 plastics/m3 in waters and 6.8 ± 4.5 plastics/kg dry weight - d.w. - in sediments) and the highest in Mincio (62.9 ± 53.9 plastics/m3 in waters and 26.5 ± 13.3 plastics/kg d.w in sediments), highlighting a plastic amount in sediments four times higher than waters. Plastic pollution, mainly due to microplastics, was associated principally to a domestic input in both waters and sediments of Ticino and Adda Rivers, as well as in sediments of Oglio, while an industrial pollution was found in waters and sediments of Mincio and Oglio waters. Our data clearly highlighted as the monitoring of both matrices provide complementary information for a holistic risk assessment of these emerging contaminants in freshwaters: the aqueous matrix provides an instantaneous picture of contamination, while sediments the history of pollution.

Assessment of behavioural effects of three water-soluble polymers in zebrafish embryos.

The water-soluble polymers (WSPs) are widely used in many industrial applications and are present in several commonly used products due to their physical-chemical characteristics: as their name indicates, despite being synthetic polymers, they are able to solubilize in water. Because of this peculiar property, both the qualitative-quantitative evaluation in aquatic ecosystems and their potential (eco)toxicological effects have been neglected until now. The aim of this study was to evaluate the possible effects of three of the most widely used WSPs as polyacrylic acid (PAA), polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) on the swimming behaviour of zebrafish (Danio rerio) embryos after the exposure to different concentrations (0.001, 0.5, 1 mg/L). The exposure lasted from the eggs' collection up to 120 h post fertilization (hpf) also using three different light intensity (300 lx, 2200 lx, 4400 lx) to better evaluate any effects related to different gradients of light/dark transitions. In order to analyze individual behavioural changes in embryos, their swimming movements were tracked and a number of parameters for locomotion and directionality were quantified. The main results showed that all three WSPs resulted in significant (p ≤ 0.05) variations in different movement parameters, suggesting a possible toxicity scale: PVP > PEG â‰« PAA.

First comparative assessment of contamination by plastics and non-synthetic particles in three bivalve species from an Italian sub-alpine lake.

This study aimed to compare the contamination from plastics and non-synthetic particles in the three freshwater bivalve mollusks Unio elongatulus, (native) and Corbicula fluminea and Dreissena polymorpha (invasive), collected in Lake Maggiore, the second greatest Italian lake. Organisms were collected from eight sites located throughout the lake, during three years (2019-2021). The quali-quantitative characterization of particles has been carried out using a Fourier Transform Infrared Microscope System (µFT-IR). Results showed that both plastics and non-synthetic particles released in the water are taken up by bivalves, even though low intake-up to 6 particles/individuals-were measured for all the three species. Microfibers of both synthetic (polyester, polyamide) and natural (cellulose) origin represented the particles mostly ingested by bivalves. A significant decrease of particle loads was observed in 2020 with respect to 2019 and 2021, significantly different for D. polymorpha and U. elongatulus, suggesting a transient reduction of the particle release in the lake in this year. Our findings highlight the need to improve the understanding of the mechanisms of uptake and clearance of these contaminants by filter feeding organisms, and their adverse consequences in realistic environmental conditions.

Packaging contaminants in former food products: Using Fourier Transform Infrared Spectroscopy to identify the remnants and the associated risks.

Food waste and feed-food competition can be reduced by replacing traditional feed ingredients such as cereals, with former food products (FFPs) in livestock diets. These foodstuffs, initially intended for human consumption, are recovered, mechanically unpacked, and then ground. Despite this simple and inexpensive treatment, packaging contaminants (remnants) are often unavoidable in the final product. To maximize the exploitation of FFPs and to minimize the associated risks, packaging remnants need to be quantified and characterized. This study tested the efficacy of the Fourier Transform Infrared Spectroscopy coupled with an optical microscope (µFT-IR) in identifying packaging remnants in 17 FFP samples collected in different geographical areas. After a visual sorting procedure, presumed packaging remnants were analyzed by µFT-IR. The results showed significant differences (p < 0.05) between the FFPs in terms of the total number of foreign particles found (plastics, cellulose and aluminum remnants, ranging from 4 to 19 particles per 20 g fresh matter), and also regarding the number of cellulose and aluminum particles. These data clearly demonstrate the need for sensitive instruments that can characterize the potential contaminants in the FFPs. This would then help to reduce the overestimation of undesirable contaminants typical of simple visual sorting, which is currently the most common method.

Surviving in a changing ocean. Tolerance to acidification might affect the susceptibility of polychaetes to chemical contamination.

This study aimed to assess the combined effects of ocean acidification (OA) and pollution to the polychaete Syllis prolifera inhabiting the CO2 vent system of the Castello Aragonese (Ischia Island, Italy). We investigated the basal activities of antioxidant enzymes in organisms from the acidified site and from an ambient-pH control site in two different periods of the year. Results showed a limited influence of acidified conditions on the functionality of the antioxidant system. We then investigated the responsiveness of individuals living inside the CO2 vent compared to those from the control to face exposure to acetone and copper. Results highlighted a higher susceptibility of organisms from the vent to acetone and a different response of antioxidant enzymes in individuals from the two sites. Conversely, a higher tolerance to copper was observed in polychaetes from the acidified-site with respect to controls, but any significant oxidative stress was induced at sublethal concentrations.

Are "liquid plastics" a new environmental threat? The case of polyvinyl alcohol.

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; age < 24 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.

Can COVID-19 pandemic change plastic contamination? The Case study of seven watercourses in the metropolitan city of Milan (N. Italy).

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.

A realistic approach for the assessment of plastic contamination and its ecotoxicological consequences: A case study in the metropolitan city of Milan (N. Italy).

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.

Alginate coating modifies the biological effects of cerium oxide nanoparticles to the freshwater bivalve Dreissena polymorpha.

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.

Characterization of plastics and their ecotoxicological effects in the Lambro River (N. Italy).

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.

Plastics and biodegradable plastics: ecotoxicity comparison between polyvinylchloride and Mater-Bi® micro-debris in a freshwater biological model.

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.

Natural molecule coatings modify the fate of cerium dioxide nanoparticles in water and their ecotoxicity to Daphnia magna.

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.

Hazard evaluation of plastic mixtures from four Italian subalpine great lakes on the basis of laboratory exposures of zebra mussels.

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.

Evaluation of the infiltration of polystyrene nanobeads in zebrafish embryo tissues after short-term exposure and the related biochemical and behavioural effects.

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 < 0.05) variation in the turn angle between the control and exposed embryos. This study points out the capability of nanoplastics to infiltrate zebrafish embryo tissues, even after a short exposure, thus suggesting the need for deeper investigations following longer exposure times, and highlighting the potential of nanoplastics to cause toxicological effects on freshwater organisms, at the organism level.

Single and Combined Effects of Zinc and Manganese on the Bivalve Anodontites trapesialis: Complementary Endpoints to Support the Hypothesis of Manganese Promoting Metabolic Suppression in Gills.

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.

Environmental concentrations of triclosan activate cellular defence mechanism and generate cytotoxicity on zebrafish (Danio rerio) embryos.

Triclosan (TCS, 5­chloro­2­(2,4­dichlorophenoxy) 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 glutathione­S­transferase (GST), P­glycoprotein efflux and ethoxyresorufin­o­deethylase (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.