Behind conventional (micro)plastics: An ecotoxicological characterization of aqueous suspensions from End-of-Life Tire particles.

Million tons of tires become waste every year, and the so-called End-of-Life Tires (ELTs) are ground into powder (ELT-dp; size < 0.8 mm) and granules (ELT-dg; 0.8 < size < 2.5 mm) for recycling. The aim of this study was to evaluate the sub-lethal effects of three different concentrations (0.1, 1, and 10 mg/L) of aqueous suspensions from ELT-dp and ELT-dg on Danio rerio (zebrafish) larvae exposed from 0 to 120 h post-fertilization (hpf). Chronic effects were assessed through biomarkers, real-time PCR, and proteomics. We observed a significant increase in swimming behavior and heart rate only in specimens exposed to ELT-dp suspensions at 1 and 10 mg/L, respectively. Conversely, the activities of detoxifying enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) showed significant modulation only in specimens exposed to ELT-dg groups. Although no effects were observed through real-time PCR, proteomics highlighted alterations induced by the three ELT-dp concentrations in over 100 proteins involved in metabolic pathways of aromatic and nitrogen compounds. The results obtained suggest that the toxic mechanism of action (MoA) of ELT suspensions is mainly associated with the induction of effects by released chemicals in water, with a higher toxicity of ELT-dp compared to ELT-dg.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of uptake and chronic toxicity of virgin polystyrene microbeads in freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia).

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.

Inhibition of vaginal transmission of HIV-1 in hu-SCID mice by the non-nucleoside reverse transcriptase inhibitor TMC120 in a gel formulation.

OBJECTIVE: The development of drugs that can be used as topical microbicides is currently recognized as a priority area of research. DESIGN: A preclinical evaluation of the potential effectiveness of TMC120, a non-nucleoside reverse transcriptase inhibitor (NNRTI), as a topical microbicide to prevent vaginal HIV-1 transmission in a humanized severe combined immunodeficient (hu-SCID) mouse model. METHODS: Reconstituted mice received an intravaginal application of a TMC120-containing gel 20 min prior to a non-invasive vaginal challenge with cell-associated HIV. The possible cytotoxic effect of TMC120-containing-gel on lymphocytes was assessed and their in vivo migration was followed using fluorescently labelled human lymphocytes. Systemic infection was monitored by p24 antigen detection in culture supernatant from cocultured intraperitoneal cells using antigen capture enzyme-linked immunosorbent assay test and by the presence of integrated proviral HIV-1 DNA in DNA extracted from spleen cells. In vivo migration of labelled lymphocytes was examined by analysis of cells isolated from regional lymph nodes. RESULTS: In this model, systemic infection was successfully inhibited by the presence of TMC120-containing gel at vaginal level. The in vivo migration of human lymphocytes from the vagina to regional lymph nodes, following the deposition of TMC120-containing gel, excluded the possibility that inhibition of systemic infection was a result of NNRTI toxicity. CONCLUSIONS: Vaginal transmission of HIV was successfully prevented by the application of a gel formulation containing TMC120. This is the first evidence of the in vivo effectiveness of a microbicide preparation containing an NNRTI against cell-associated HIV.