Environmental safety of second and third generation bioplastics in the context of the circular economy.

Bioplastics derived from organic materials other than crude oil are often suggested as sustainable solutions for tackling end-of-life plastic waste, but little is known of their ecotoxicity to aquatic species. Here, we investigated the ecotoxicity of second and third generation bioplastics toward the freshwater zooplankton Daphnia magna. In acute toxicity tests (48 h), survival was impacted at high concentrations (g.L-1 range), within the range of salinity-induced toxicity. Macroalgae-derived bioplastic induced hormetic responses under chronic exposure (21 d). Most biological traits were enhanced from 0.06 to 0.25 g.L-1 (reproduction rate, body length, width, apical spine, protein concentration), while most of these traits returned to controls level at 0.5 g.L-1. Phenol-oxidase activity, indicative of immune function, was enhanced only at the lowest concentration (0.06 g.L-1). We hypothesise these suggested health benefits were due to assimilation of carbon derived from the macroalgae-based bioplastic as food. Polymer identity was confirmed by infra-red spectroscopy. Chemical analysis of each bioplastic revealed low metal abundance whilst non target exploration of organic compounds revealed trace amounts of phthalates and flame retardants. The macroalgae-bioplastic disintegrated completely in compost and biodegraded up to 86 % in aqueous medium. All bioplastics acidified the test medium. In conclusion, the tested bioplastics were classified as environmentally safe. Nonetheless, a reasonable end-of-life management of these safer-by-design materials is advised to ensure the absence of harmful effects at high concentrations, depending on the receiving environment.

Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.

The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly - regardless the time-intervals - at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight "day" effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the "day" effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails' immunocompetence.

Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.

Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (ß-naphthoflavone (ß-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on ß-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard.

Deep dive into the chronic toxicity of tyre particle mixtures and their leachates.

Particles from the tread of vehicle tyres are a global pollutant, which are emitted into the environment at an approximate rate of 1.4 kg.year-1 for an average passenger-car. In this study, popular tyre brands were used to generate a tyre tread microparticle mixture. The chronic toxicity of both particles and chemical leachates were compared on a planktonic test species (Daphnia magna). Over 21 days of exposure, pristine tyre tread microparticles were more toxic (LC50 60 mg.L-1) than chemical lechates alone (LC50 542 mg.L-1). Microparticles and leachates showed distinct effects on reproduction and morphological development at environmentally relevant concentrations, with dose-dependent uptake of particles visible in the digestive tract. Chemical characterization of leachates revealed a metal predominance of zinc, titanium, and strontium. Of the numerous organic chemicals present, at least 54 were shared across all 5 tyre brands, with many classified to be very toxic. Our results provide a critically needed information on the toxicity of tyre tread particles and the associated chemicals that leach from them to inform future mitigation measures. We conclude that tyre particles are hazardous pollutants of particular concern that are close to or possibly above chronic environmental safety limits in some locations.

Internalization and cytotoxicity of graphene oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2.

BACKGROUND: Graphene and graphene derivative nanoplatelets represent a new generation of nanomaterials with unique physico-chemical properties and high potential for use in composite materials and biomedical devices. To date little is known about the impact graphene nanomaterials may have on human health in the case of accidental or intentional exposure. The objective of this study was to assess the cytotoxic potential of graphene nanoplatelets with different surface chemistry towards a human hepatoma cell line, Hep G2, and identify the underlying toxicity targets. METHODS: Graphene oxide (GO) and carboxyl graphene (CXYG) nanoplatelet suspensions were obtained in water and culture medium. Size frequency distribution of the suspensions was determined by means of dynamic light scattering. Height, lateral dimension and shape of the nanoplatelets were determined using atomic force and electron microscopy. Cytotoxicity of GO and CXYG nanoplatelets was assessed in Hep G2 cells using a battery of assays covering different modes of action including alterations of metabolic activity, plasma membrane integrity and lysosomal function. Induction of oxidative stress was assessed by measuring intracellular reactive oxygen species levels. Interaction with the plasma membrane, internalization and intracellular fate of GO and CXYG nanoplatelets was studied by scanning and transmission electron microscopy. RESULTS: Supplementing culture medium with serum was essential to obtain stable GO and CXYG suspensions. Both graphene derivatives had high affinity for the plasma membrane and caused structural damage of the latter at concentrations as low as 4 µg/ml. The nanoplatelets penetrated through the membrane into the cytosol, where they were concentrated and enclosed in vesicles. GO and CXYG accumulation in the cytosol was accompanied by an increase in intracellular reactive oxygen species (ROS) levels, alterations in cellular ultrastructure and changes in metabolic activity. CONCLUSIONS: GO and CXYG nanoplatelets caused dose- and time-dependent cytotoxicity in Hep G2 cells with plasma membrane damage and induction of oxidative stress being important modes of toxicity. Both graphene derivatives were internalized by Hep G2, a non-phagocytotic cell line. Moreover, they exerted no toxicity when applied at very low concentrations (< 4 µg/ml). GO and CXYG nanoplatelets may therefore represent an attractive material for biomedical applications.

Immunocompetence analysis of the aquatic snail Lymnaea stagnalis exposed to urban wastewaters.

Wastewater treatment plant effluents from urban area are a well-known source of chronic multiple micropollution to the downstream living organisms. In this study, ecologically relevant laboratory-bred freshwater gastropods, Lymnaea stagnalis, were exposed for 29 days to raw effluents of a wastewater treatment plant in Lyon area (France). A time-course analysis of individual markers of immunocompetence (hemocyte density and viability, hemocyte NADPH activity, phenol oxidase activity, and capacity of phagocytosis) has shown slight trends of inflammatory-like responses induced by the 100% effluents. So far, no short-term hazard for L. stagnalis can be revealed. However, over the long term, such environmental stress-stimulating immune responses could provoke deleterious life history trade-offs because the immune system is known to be highly energy-consuming.