Cellular effects of microplastics are influenced by their dimension: Mechanistic relationships and integrated criteria for particles definition.

The definition of microplastics (MPs) is nowadays too generic from a biological perspective, since different characteristics of these particles might influence their effects. To provide experimental evidence that size is an important factor to be considered, Mediterranean mussels Mytilus galloprovincialis were exposed to five size classes of polyethylene fragments (PE-MPs, 20-50 µm, 50-100 µm, 100-250 µm, 250-500 µm, 500-1000 µm). After 10 days of exposure, MPs ingestion and mechanistic relationships between particles size and cellular effects were analysed through a wide panel of biological alterations, including immune system responses, cholinergic function, antioxidant system, lipid metabolism and peroxidation. Results were further elaborated through a Weight of Evidence approach, summarizing the overall biological significance of obtained results in a hazard index based on the number and magnitude of variations and their toxicological relevance. PE-MPs 500-1000 µm were identified as the less biologically reactive size class due to the limited ingestion of particles coupled with the lack of biological effects, followed by PE-MPs 250-500 µm, which slightly altered the cholinergic function and lysosomal membranes. Conversely, PE-MPs smaller than 250 µm provoked a more consistent onset of biological alterations in terms of immune system composition and functioning, redox homeostasis, and lipid metabolism. The overall findings of this study highlight the importance of considering the size of particles for monitoring and risk assessment of MPs, introducing a more integrated evaluation of plastic pollution that, beside particles concentration, should adequately weigh those characteristics triggering the onset of biological effects.

Microplastics in urban water cycles: Looking for a more scientific approach for sampling and characterization in wastewater and drinking water treatment plants.

Specific campaigns to detect microplastics (MPs) in the urban water cycle were carried out in three drinking water plants and two wastewater treatment plants. A self-designed sampler for MPs detection in water matrices was in this study preliminary validated and then tested in long term campaigns sampling up to 1000 L. Raw drinking water and wastewater show microplastics (MPs) concentrations of 2-11 and of 480-801 MPs/m3, respectively, and MPs removals of 47-78 % and of 84-98 %, correspondingly. Specific roles of chemical and physical conventional processes in microplastics removals were investigated. Solid-liquid separation, flotation and filtration are the main processes for achieving high microplastics removal. Regarding concentrated matrices, MPs concentrations in sludge samples varied in the range of 5000-500,000 MPs/m3. Finally, shapes, size classes and polymers' typologies were investigated in the extracted MPs. The detected sizes are mainly 0.5-0.1 mm in drinking waters while 5-1 mm in wastewaters. Wastewaters were predominated by synthetic fibers (polyester type), while drinking waters were mainly characterized by fragments and the fibers were mostly of natural origin. Finally, the results of this study supported best practices and guidelines for a representative assessment of MPs in water (sampling methods, extraction procedures, characterization and quantification).

New insights into the impact of leachates from in-field collected plastics on aquatic invertebrates and vertebrates.

The impact of leachates from micronized beached plastics of the Mediterranean Sea and Atlantic Ocean on coastal marine ecosystems was investigated by using a multidisciplinary approach. Chemical analysis and ecotoxicological tests on phylogenetically distant species were performed on leachates from the following plastic categories: bottles, pellets, hard plastic (HP) containers, fishing nets (FN) and rapido trawling rubber (RTR). The bacteria Alivibrio fischeri, the nauplii of the crustaceans Amphibalanus amphitrite and Acartia tonsa, the rotifer Brachionus plicatilis, the embryos of the sea urchin Paracentrotus lividus, the ephyrae of the jellyfish Aurelia sp. and the larvae of the medaka Oryzias latipes were exposed to different concentrations of leachates to evaluate lethal and sub-lethal effects. Thirty-one additives were identified in the plastic leachates; benzophenone, benzyl butyl phthalate and ethylparaben were present in all leachates. Ecotoxicity of leachates varied among plastic categories and areas, being RTR, HP and FN more toxic than plastic bottles and pellets to several marine invertebrates. The ecotoxicological results based on 13 endpoints were elaborated within a quantitative weight of evidence (WOE) model, providing a synthetic hazard index for each data typology, before their integrations in an environmental risk index. The WOE assigned a moderate and slight hazard to organisms exposed to leachates of FN and HP collected in the Mediterranean Sea respectively, and a moderate hazard to leachates of HP from the Atlantic Ocean. No hazard was found for pellet, bottles and RTR. These findings suggest that an integrated approach based on WOE on a large set of bioassays is recommended to get a more reliable assessment of the ecotoxicity of beached-plastic leachates. In addition, the additives leached from FN and HP should be further investigated to reduce high concentrations and additive types that could impact marine ecosystem health.

Comparative microplastic load in two decapod crustaceans Palinurus elephas (Fabricius, 1787) and Nephrops norvegicus (Linnaeus, 1758).

The present work compares microplastics (MPs) contamination in two charismatic crustaceans: European spiny lobster Palinurus elephas and langoustine Nephrops norvegicus. Samples (P. elephas n = 14; N. norvegicus n = 15) were collected between 76 and 592 m depth, from four sites in west Sardinia, Italy. An extraction protocol was applied on stomachs and intestines, separately, and over 500 particles were further characterized through µFT-IR. We document 100 % occurrence in specimens from both species, with P. elephas being significantly more contaminated (9.1 ± 1.75 vs. 3.2 ± 0.45 MPs individual-1), ingesting larger MPs with different polymeric composition. The scavenging-based feeding strategy of both species could explain such exposure to MPs, mostly derived by single-use plastic. The overall results highlight that both species are clearly affected by plastic pollution, being valuable bioindicators and charismatic species that could thus represent excellent flagship species for raising awareness toward the global issue of plastic in the marine environment.

Carassius auratus as a bioindicator of the health status of Lake Trasimeno and risk assessment for consumers.

Fish are good bio-indicators of the health status of the aquatic environment and can be used as biomarkers to assess the aquatic behavior of environmental pollutants, the exposure of aquatic organisms, and the health risk for consumers. Goldfish are a significant bioindicator in the Lake Trasimeno aquatic system (Umbria, Italy). This study aimed to characterize the health status and the chemical and biotic contamination of Lake Trasimeno to define its anthropogenic and natural pressures and the risk associated with consuming its fishery products. 114 determinations were performed on Carassius auratus samples from 2018 to 2020, and the occurrence of brominated flame retardants, non-dioxin-like polychlorinated biphenyls, heavy metals, and microplastics was analytically investigated. Dietary exposure assessment, risk characterization, and benefit-risk evaluation were performed for schoolchildren from 3 to 10 years old. Flame-retardants registered high levels of non-detects (99% for polybrominated diphenyl ether and 76% for hexabromocyclododecanes), while polychlorinated biphenyls were found in all samples with a maximum level of 56.3 ng/g. Traces of at least one heavy metal were found in all samples, though always below the regulatory limit. Microplastics were found with a 75% frequency of fish ingesting at least one particle. Dietary exposure and risk characterization reveal negligible contributions to the reference values of all contaminants, except for mercury, which reached up to 25% of admissible daily intake. The benefit- risk assessment highlighted that the benefits of freshwater fish intake outweigh the associated risks. The examination of goldfish as indicator fish reveals the quality of Lake Trasimeno's aquatic environment and the safety of its products.

Microplastics in seawater and marine organisms: Site-specific variations over two-year study in Giglio Island (North Tyrrhenian Sea).

Geographical and temporal differences of microplastic occurrence were documented in water and fish collected in 2017 and 2019 from the Giglio Island (North Tyrrhenian Sea) close to the area where the Costa Concordia sank in January 2012. Results on water samples showed a site-dependent difference, suggesting the role of surface current dynamics in the microplastic local distribution, while tested Neuston nets (200 µm and 330 µm mesh size) did not influence microplastic retention efficiency. Fish exhibited in 2019 a higher frequency of specimens positive to microplastic ingestion with respect to 2017, with an occurrence higher than those typically observed in other Mediterranean areas. Both in water and fish, fragments were the dominating shape, polypropylene and polyethylene were the prevalent polymers, without particular difference between sites and years. This study highlights the importance of applying microplastic investigation in biotic and abiotic matrices for an effective monitoring of this pollution in the marine environment.

Colonization of plastic debris by the long-lived precious red coral Corallium rubrum: New insights on the "plastic benefits" paradox.

Seafloor macrolitter is ubiquitous in world's oceans; still, huge knowledge gaps exist on its interactions with benthic biota. We report here the colonization of plastic substrates by the Mediterranean red coral Corallium rubrum (L. 1758), occurring both in controlled conditions and in the wild at ca. 85 m depth in the Western Mediterranean Sea. Juveniles settled on seafloor macro-litter, with either arborescent or encrusting morphology, ranging from 0.6 to 3.5 mm in basal diameter and 0.2-7.1 years of age, also including a fraction (20%) of potentially sexually mature individuals. In controlled conditions, larvae settled and survived on plastic substrates for >60 days. Our insights show that marine plastic debris can provide favourable substrate for C. rubrum settlement either in controlled conditions or in the wild, suggesting their possible use in restoration activities. However, we pinpoint here that this potential benefit could result in adverse effects on population dynamics.

Microplastics in real wastewater treatment schemes: Comparative assessment and relevant inhibition effects on anaerobic processes.

The occurrence, fate and removal of microplastics (MPs) in a wastewater treatment plant (WWTP) in Central Italy were investigated together with their potential adverse effects on anaerobic processes. In the influent of the WWTP, 3.6 MPs.L-1 were detected that mostly comprised polyester fibers and particles in the shape of films, ranging 0.1-0.5 mm and made of polyethylene and polypropylene (PP). The full-scale conventional activated sludge scheme removed 86% of MPs, with the main reduction in the primary and secondary settling. MPs particles bigger than 1 mm were not detected in the final effluent and some loss of polymers types were observed. In comparison, the pilot-scale upflow granular anaerobic sludge blanket (UASB) + anaerobic membrane bioreactor (AnMBR) configuration achieved 94% MPs removal with the abatement of 87% of fibers and 100% of particles. The results highlighted an accumulation phenomenon of MPs in the sludge and suggested the need to further investigate the effects of MPs on anaerobic processes. Accordingly, PP-MPs at concentrations from 5 PP-MPs.gTS-1 to 50 PP-MPs.gTS-1 were spiked in the pilot-scale UASB reactor that was fed with real municipal wastewater, where up to 58% decrease in methanogenic activity was observed at the exposure of 50 PP-MPs.gTS-1. To the best of our knowledge, the presented results are the first to report of PP-MPs inhibition on anaerobic processes.

Long-lasting effects of chronic exposure to chemical pollution on the hologenome of the Manila clam.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post-transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas-associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long-lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

Distribution and characterization of microplastic particles and textile microfibers in Adriatic food webs: General insights for biomonitoring strategies.

This study provided a comprehensive characterization on ingestion of different typologies of microplastics in several fish and invertebrate species from the Adriatic Sea, considered as a preferential area of plastic accumulation in the Mediterranean. Almost 500 organisms were sampled in the three sectors of Northern, Central and Southern Adriatic, testing the hypothesis that area of collection, habitat and feeding strategy might influence the occurrence of plastic particles in biota. In this study, the overall characterization considered separately plastic microparticles (MPs) from textile microfibers (MFs) which also included natural and semi-synthetic ones. Ingestion of MPs was a widespread phenomenon, but their number (typically 1 or 2) did not reveal any significant relationship with biometric values, geographical areas or ecological features of the species. Conversely, the frequency of ingestion, ranging from 13 to 35% of organisms containing MPs, appeared a more reliable index to highlight such differences, revealing higher values in species from Central and Southern basins compared to the Northern one, as well as in benthopelagic compared to benthic or pelagic organisms. Geographical differences also occurred in terms of size and typology of ingested particles, suggesting the importance of local river runoffs and surface currents dynamics. Textile microfibers (MFs) were also abundant in Adriatic food webs occurring in all the analyzed species with average numbers (3-10) and frequencies (40-70%) higher than those reported for MPs; further, an elevated percentage of MFs (>80%) was of natural or semi-synthetic origin. Overall, this study provided general insights toward the harmonization of a common biomonitoring strategy, as in the context of MSFD, including the suggestion of a frequency-based index and of a multi-species approach to increase the ecological relevance of assessment, as well as the comparability between different areas and trophic webs.

Precision-Cut Tissue Slices (PCTS) from the digestive gland of the Mediterranean mussel Mytilus galloprovincialis: An ex vivo approach for molecular and cellular responses in marine invertebrates.

The precision-cut tissue slices (PCTS) represent a largely used biological model in mammalian research. This ex vivo approach offers the main advantages of in vitro systems, while maintaining the natural architecture of the tissue. The use of PCTS in toxicological research has been proposed for investigating the cellular effects of xenobiotics or bioactive compounds mostly in mammalian models. Their application is increasing also in marine organisms, but still limited to fish. This work validates the use of PCTS in an invertebrate species, the Mediterranean mussel Mytilus galloprovincialis. Intact tissue slices of different thicknesses (300, 350 and 400 µm) were successfully obtained from the digestive gland. The slices maintained the histological integrity and the viability after 6 h and 24 h incubation in culture medium, with some differences depending on the thickness. The enzymatic activities and mRNA levels of catalase and glutathione S-transferase, chosen as model biological endpoints, were measured until 24 h incubation, revealing the functionality of such systems. This work demonstrates the suitability of mussel PCTS for investigating molecular and cellular responses in ecotoxicological research.

The fate of microplastics in an Italian Wastewater Treatment Plant.

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 ±â€¯0.3 MPs/L in the IN, 0.9 ±â€¯0.3 MPs/L after the SET and 0.4 ±â€¯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 ±â€¯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.