Single exposure of food-derived polyethylene and polystyrene microplastics profoundly affects gut microbiome in an in vitro colon model.

Microplastics (MPs) are widespread contaminants highly persistent in the environment and present in matrices to which humans are extensively exposed, including food and beverages. MP ingestion occurs in adults and children and is becoming an emerging public health issue. The gastrointestinal system is the most exposed to MP contamination, which can alter its physiology starting from changes in the microbiome. This study investigates by an omic approach the impact of a single intake of a mixture of polyethylene (PE) and polystyrene (PS) MPs on the ecology and metabolic activity of the colon microbiota of healthy volunteers, in an in vitro intestinal model. PE and PS MPs were pooled together in a homogeneous mix, digested with the INFOGEST system, and fermented with MICODE (multi-unit in vitro colon model) at loads that by literature correspond to the possible intake of food-derived MPs of a single meal. Results demonstrated that MPs induced an opportunistic bacteria overgrowth (Enterobacteriaceae, Desulfovibrio spp., Clostridium group I and Atopobium - Collinsella group) and a contextual reduction on abundances of all the beneficial taxa analyzed, with the sole exception of Lactobacillales. This microbiota shift was consistent with the changes recorded in the bacterial metabolic activity.

Bioplastic leachates characterization and impacts on early larval stages and adult mussel cellular, biochemical and physiological responses.

Bioplastics are promoted as safer alternatives to tackle the long-term persistence of conventional plastics. However, information on the potential release of additives and non-intentionally added substances (NIAS) in the surrounding environment is limited, and biological effects of the leachates have been little studied. Leachates produced from three bioplastics, i.e. compostable bags (CB), bio-polyethylene terephthalate bottles (bioPET) and polylactic acid cups (PLA), and a control polymeric material, i.e. rubber tire (TR), were examined. The chemical nature of bioplastic polyesters PET, PLA and poly (butylene adipate-co-terephthalate) (PBAT) in CB, was confirmed by analytical pyrolysis. Fragments were incubated in artificial sea water for 14 days at 20 °C in darkness and leachate contents examined by GC-MS and HPLC-MS/MS. Catalysts and stabilizers represented the majority of chemicals in TR, while NIAS (e.g. 1,6-dioxacyclododecane-7,12-dione) were the main components of CB. Bisphenol A occurred in all leachates at a concentration range 0.3-4.8 µg/L. Trace metals at concentrations higher than control water were found in all leachates, albeit more represented in leachates from CB and TR. A dose response to 11 dilutions of leachates (in the range 0.6-100%) was tested for biological effects on early embryo stages of Mytilus galloprovincialis. Embryotoxicity was observed in the whole range of tested concentrations, the magnitude of effect depending on the polymers. The highest concentrations caused reduction of egg fertilization (CB, bioPET, TR) and of larvae motility (CB, PLA, TR). TR leachates also provoked larvae mortality in the range 10-100%. Effects on adult mussel physiology were evaluated after a 7-day in vivo exposure to the different leachates at 0.6% concentration. Nine biomarkers concerning lysosomal functionality, neurotransmission, antioxidant and immune responses were assessed. All lysosomal parameters were affected, and serum lysozyme activity inhibited. Harmonized chemical and biological approaches are recommended to assess bioplastic safety and support production of sustainable bioplastics.

The sub-lethal impact of plastic and tire rubber leachates on the Mediterranean mussel Mytilus galloprovincialis.

Ocean contamination by synthetic polymers can represent a risk for the fitness of marine species due to the leaching of chemical additives. This study evaluated the sub-lethal effects of plastic and rubber leachates on the mussel Mytilus galloprovincialis through a battery of biomarkers encompassing lysosomal endpoints, oxidative stress/detoxification parameters, and specific responses to metals/neurotoxicants. Mussels were exposed for 7 days to leachates from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC), containing organic additives and metals in the ng-µg/L range. The leachate exposure affected general stress parameters, including the neutral lipid content (all leachates), the lysosomal membrane stability (PS, PP, PVC and CTR leachates) and lysosomal volume (PP, PVC and TR leachates). An increased content of the lipid peroxidation products malondialdehyde and lipofuscin was observed in mussels exposed to PET, PS and PP leachates, and PP, PVC and CTR leachates, respectively. PET and PP leachates increased the activity of the phase-II metabolism enzyme glutathione S-transferase, while a decreased acetylcholinesterase activity was induced by PVC leachates. Data were integrated in the mussel expert system (MES), which categorizes the organisms' health status based on biomarker responses. The MES assigned healthy status to mussels exposed to PET leachates, low stress to PS leachates, and moderate stress to PP, CTR and PVC leachates. This study shows that additives leached from selected plastic/rubber polymers cause sub-lethal effects in mussels and that the magnitude of these effects may be higher for CTR, PVC and PP due to a higher content and release of metals and organic compounds.

The hidden threat of plastic leachates: A critical review on their impacts on aquatic organisms.

Plastic products are made from the essential polymer mixed with a complex blend of substances including catalyst remnants, polymerization solvents, and a wide range of other additives deliberately added to enhance the desirable characteristics of the final product. Additives include bisphenols, phthalates, flame retardants, and further emerging and legacy contaminants. With a few exceptions, additives are not chemically bound to the polymer, and potentially migrate within the material reaching its surface, then possibly leach out to the environment. Leachates are mixtures of additives, some of which belong to the list of emerging contaminants, i.e. substances that show the potential to pose risks to the environment and human health, while are not yet regulated. The review discusses the state of the art and gaps concerning the hidden threat of plastic leachates. The focus is on reports addressing the biological impacts of plastic leachates as a whole mixture. Degradation of plastics, including the weathering-driven fragmentation, and the release of additives, are analysed together with the techniques currently employed for chemically screening leachates. Because marine plastic litter is a major concern, the review mainly focuses on the effects of plastic leachates on marine flora and fauna. Moreover, it also addresses impacts on freshwater organisms. Finally, research needs and perspectives are examined, to promote better focused investigations, that may support developing different plastic materials and new regulations.

Chemical composition and ecotoxicity of plastic and car tire rubber leachates to aquatic organisms.

Synthetic polymer-based materials are ubiquitous in aquatic environments, where weathering processes lead to their progressive fragmentation and the leaching of additive chemicals. The current study assessed the chemical content of freshwater and marine leachates produced from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC) microplastics, and their adverse effects on the microalgae Raphidocelis subcapitata (freshwater) and Skeletonema costatum (marine) and the Mediterranean mussel Mytilus galloprovincialis. A combination of non-target and target chemical analysis revealed a number of organic and metal compounds in the leachates, including representing plasticizers, antioxidants, antimicrobials, lubricants, and vulcanizers. CTR and PVC materials and their corresponding leachates had the highest content of tentatively identified organic additives, while PET had the lowest. The metal content varied both between polymer leachates and between freshwater and seawater. Notable additives identified in high concentrations were benzothiazole (CTR), phthalide (PVC), acetophenone (PP), cobalt (CTR, PET), zinc (CTR, PVC), lead (PP) and antimony (PET). All leachates, except PET, inhibited algal growth with EC50 values ranging from 0.5% (CTR) and 64% (PP) of the total leachate concentration. Leachates also affected mussel endpoints, including the lysosomal membrane stability and early stages endpoints as gamete fertilization, embryonic development and larvae motility and survival. Embryonic development was the most sensitive parameter in mussels, with EC50 values ranging from 0.8% (CTR) to 65% (PET) of the total leachate. The lowest impacts were induced on D-shell larvae survival, reflecting their ability to down-regulate motility and filtration in the presence of chemical stressors. This study provides evidence of the relationship between chemical composition and toxicity of plastic/rubber leachates. Consistent with increasing contamination by organic and inorganic additives, the leachates ranged from slightly to highly toxic to mussels and algae, highlighting the need for a better understanding of the overall impact of plastic-associated chemicals on aquatic ecosystems.

The Multixenobiotic resistance system as a possible protective response triggered by microplastic ingestion in Mediterranean mussels (Mytilus galloprovincialis): Larvae and adult stages.

The emerging paradigm on plastic pollution in marine environments is that microsize particles (MPs) have far more subtle effects than bigger fragments, given their size range overlapping with that of particles ingested by filter-feeders. The impacts include gut blockage, altered feeding and energy allocation, with knock-on effects on widespread physiological processes. This study investigated whether ingestion of polystyrene MPs (PS-MPs) triggers protective processes in marine mussels. The Multixenobiotic resistance (MXR) system is a cytoprotective mechanism acting as an active barrier against harmful xenobiotics and a route of metabolite detoxification. Both larvae and adults were employed in laboratory experiments with different concentrations of 3-µm PS-MPs (larvae), and 3-µm and 45-µm PS-MPs (adults) matching size range of planktonic food through the mussel lifecycle. Embryos grown in the presence of 3-µm PS-MPs showed significant reduction of MXR activity and down-regulation of ABCB and ABCC transcripts encoding the two main MXR-related transporters P-glycoprotein and the Multidrug resistance-related protein, respectively. In adults, effects of PS-MPs were assessed in haemocytes and gills, which showed different modulation of MXR activity and ABCB/ABCC expression according to MP size (haemocyte and gills) or particle concentration (haemocyte). These data showed that modulation of MXR activity is part of a generalized response triggered by particle ingestion.

Microplastic exposure and effects in aquatic organisms: A physiological perspective.

The impact of microplastics (MPs) on aquatic life, given their ubiquitous presence in the water compartment, represents a growing concern. Consistently, scientific knowledge is advancing rapidly, although evidence on actual adverse effects is still highly fragmented. This paper summarizes the recent literature on MP impacts on aquatic organisms in an attempt to link routes of uptake, possible alterations of physiological processes, and outcomes at different levels of biological organization. Animal feeding strategies and MP biodistribution is discussed, alongside with relevant effects at molecular, cellular, and systemic level. Pathways from animal exposure to apical physiological responses are examined to define the relevance of MPs for animal health, and to point out open questions and research gaps. Emphasis is given to emerging threats posed by leaching of plastic additives, many of which have endocrine disruption potential. The potential role of MPs as substrates for microorganism growth and vehicle for pathogen spreading is also addressed.

Uptake and transcriptional effects of polystyrene microplastics in larval stages of the Mediterranean mussel Mytilus galloprovincialis.

The widespread occurrence of microplastics (MP) in the marine environment is cause of increasing concerns about the safety of the exposed ecosystems. Although the effects associated to the MP uptake have been studied in most marine taxa, the knowledge about their sub-lethal impacts on early life stages of marine species is still limited. Here, we investigated the uptake/retention of 3-µm polystyrene MP by early stages of the Mediterranean mussel Mytilus galloprovincialis, and the related effects on gut clearance, feeding efficiency, morphological and transcriptional parameters involved in embryo-larval development. Uptake measurements were performed on larvae at 48 h, 3, 6 and 9 days post fertilization (pf) after exposure to a range of 50-10,000 particles mL-1. At all tested pf periods, treatments resulted in a significant and linear increase of MP uptake with increasing concentrations, though levels measured at 48 h pf were significantly lower compared to 3-9 d pf. Ingested MP were retained up to 192 h in larvae's gut, suggesting a physical impact on digestive functions. No change was noted between the consumption of microalgae Nannochloropsis oculata by larvae when administered alone or in the presence of an identical concentration (2000 items mL-1) of MP. The exposure to 50-10,000 MP mL-1 did not alter the morphological development of mussel embryos; however, transcriptional alterations were observed at 50 and 500 MP mL-1, including the up-regulation of genes involved in shell biogenesis (extrapallial protein; carbonic anhydrase; chitin synthase) and immunomodulation (myticin C; mytilin B), and the inhibition of those coding for lysosomal enzymes (hexosaminidase; ß-glucorinidase; catepsin-L). In conclusion, though not highlighting morphological or feeding abnormalities, data from this study revealed the onset of physical and transcriptional impairments induced by MP in mussel larvae, indicating sub-lethal impacts which could increase their vulnerability toward further environmental stressors.

Forensic DNA against wildlife poaching: identification of a serial wolf killing in Italy.

The recent expansion of the Italian wolf population through the Apennine and western Alps, after centuries of contractions, is causing conflicts with human activities leading to a rise in poaching or illegal killings. Here we show how molecular population genetics has been used to identify a suspect serial wolf killer. We analysed DNA extracted from a necklace made of ten presumed wolf canine teeth, confiscated in 2008 to a man living in the northern Italian Apennine (Liguria Region). Individual genotypes were determined using 12 unlinked autosomal microsatellites (STRs), mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and three Y-linked STRs. Results indicate that the teeth belonged to six different individuals (three males and three females), which were assigned to the Italian wolf population with p > 0.90 by Bayesian procedures. One of these genotypes matched with the genetic profile of a male wolf previously found-dead and already non-invasively sampled in the same area. Another genotype matched with that of a female wolf non-invasively sampled twice in the same area 1 year before. These data are being used as forensic genetic evidence in the ongoing criminal trial against the suspect serial wolf killer.

Heavy metals in tissues of loggerhead turtles (Caretta caretta) from the northwestern Adriatic Sea.

Thirty-five specimens of Caretta caretta were collected dead along the Adriatic Sea coast from the Po Delta to the Reno mouth (Italy). Turtles were classified into four size categories ranging from 24.5 to 74 cm, by measuring the minimum straight-line carapace length (MSCL). Cd, Cu, Fe, Mn, Ni, and Zn levels were assessed in liver, lung, muscle and adipose tissue. Cd, Cu and Fe mainly accumulated in the liver (8.9, 23.7 and 1180 mg/kg dry mass [d.w.], respectively), and Mn in the lung (29.5 mg/kg d.w.). Levels of Ni were higher in adipose (22 mg/kg d.w.) than other tissues, while Zn concentrations were higher in muscle (about 140 mg/kg d.w.). Negative correlations with size were established for Zn in liver and Cu in adipose tissue, while positive correlations were observed for Mn and Ni in adipose tissue. Metal concentrations did not differ between males and females, nor between individuals found stranded and those victims of by-catch. On average, Cd, Cu, Mn and Ni concentrations in our specimens were higher than in loggerhead turtles and other species living in other areas. We hypothesize that trace metals could be used as "acquired markers" to help investigate migration routes of C. caretta.