Toxicity assessment of microplastics within the Loire River in the clam Corbicula fluminea exposed to environmentally relevant exposure conditions.

The dispersed pollution caused by microplastics (MPs) represents a current and global concern. While the fragmentation of plastic debris into smaller particles occurs in rivers, little MP research is done on freshwater species and is published compared to the marine environment. The Loire River is the longest river in France and is subject to moderate to high anthropic pressure while it represents major societal and economic issues. However, there are not many studies that have been put forward with regards to the effect of environmental MPs (EMPs) on aquatic organisms and no policies have been enacted to monitor the plastic pollution. In this study, freshwater bivalves, Corbicula fluminea, were exposed for 21 days to environmentally relevant concentrations of a mixture of <200 µm MPs generated from plastic litter collected directly along the banks of the Loire River. This mixture was composed of 40% polyethylene (PE), 40% polypropylene (PP), 10% polyethylene terephthalate (PET) and 10% polyvinylchloride (PVC) (mass percentage). Ecotoxicological effects were assessed from the individual to the molecular levels on several endpoints: condition index, filtration efficiency, enzyme activities, lipid peroxidation, energy reserves and gene expression. The ingestion of EMPs caused damages at the biochemical level. Indeed, we reported an increase in catalase activity in gills and digestive mass, a decrease in TBARs in gills, a decrease in acetylcholinesterase activity in the digestive mass, a decrease of glycogen and lipid contents in the whole organisms and a significant induction of the expression of gst, cat, mp, acp genes. The current results suggest therefore that long-term exposure to realistic doses of EMPs causes toxicity towards freshwater benthic biota. The analysis of biomarker activities and the analysis of gene expression are complementary to prevent the effects of a plastic contamination at higher biological levels in aquatic organisms.

Toxicity assessment of environmental MPs and NPs and polystyrene NPs on the bivalve Corbicula fluminea using a multi-marker approach.

Small plastic particles, microplastics (MPs) and nanoplastics (NPs) represent a major threat in aquatic environments. Freshwater organisms are exposed to MPs and NPs, particularly in industrial and urban areas. The present study aimed to compare the toxicity between polystyrene NPs (PS NPs) and environmental microplastics (ENV MPs) and nanoplastics (ENV NPs) generated from macro-sized debris collected in the Garonne River on the freshwater bivalve C. fluminea. The organisms were exposed to the different plastic particles at three environmentally relevant concentrations: 0.008, 10, and 100 µg L-1 for 21 days. The biological responses of organisms were assessed using a multi-biomarker approach from the sub-individual to the individual level. The results demonstrated that: i) ENV NPs triggered more effects on detoxification processes and immune response, confirming that using manufactured NPs for laboratory exposure can lead to misleading conclusions on the risks posed by plastic particles; ii) effects of ENV MPs were less marked than ENV NPs, emphasizing the importance of testing a size continuum of plastic particles from NPs to MPs; iii) some effects were only observed for the low and/or intermediate concentrations tested, underlining the importance of using environmentally relevant concentrations. In light of these results, laboratory studies should be continued by exposing aquatic species to environmental MPs and NPs. The properties of these particles have to be characterized for a better risk assessment of environmental plastic particles.

Assessment of microplastic contamination in the Loire River (France) throughout analysis of different biotic and abiotic freshwater matrices.

The contamination of microplastics (MP) in freshwater environments represent a major way for the MP transport in the environment. The assessment of MP pollution in freshwater compartments is then important to visualize the pressure and the impacts on medium, and to set up necessary measures. In this context, this study focused on the influence of anthropogenic activities of a medium French city (Angers) on MP levels in samples collected from the Loire River, the longest river in France. Abiotic and biotic matrices were collected upstream and downstream Angers. A first analysis was performed based on microscopy to determine the size, colour and shape of suspected MP and a complementary analysis by µ-FTIR (micro-Fourier Transform InfraRed) was conducted to determine the composition of plastic particles. Three organisms belonging to different trophic levels were studied: when the MP level was expressed per individual, the lowest abundance of MP was found in Tubifex sp. Followed by Corbicula fluminea, while the highest was measured in Anguilla anguilla. To establish the relationship with their habitat, the presence of MP in sediment and water was also analysed. Therefore, this works constitutes a complete overview of the MP levels in freshwater abiotic and biotic matrices. Overall, the presence of MP in analysed samples did not follow a particular pattern, neither in the sites nor matrices: the characteristics depending on a multifactorial outcome (feeding mode, organism size …). However, correlation of MP pattern between clams and sediment was quite evident, while the one between worms and their habitat was not. This demonstrates the relevance of investigating plastic contamination both in biotic and abiotic matrices. Finally, a standardisation of sampling and analytical analysis protocols would be helpful to make comparisons between studies more robust.

Plastic additives: challenges in ecotox hazard assessment.

The risk of plastic debris, and specifically micro(nano)plastic particles, to ecosystems remains to be fully characterized. One particular issue that warrants further characterization is the hazards associated with chemical additives within micro(nano)plastic as they are not chemically bound within the polymers and can be persistent and biologically active. Most plastics contain additives and are therefore potential vectors for the introduction of these chemicals into the environment as they leach from plastic, a process that can be accelerated through degradation and weathering processes. There are knowledge gaps on the ecotoxicological effects of plastic additives and how they are released from parent plastic materials as they progressively fragment from the meso to micro and nano scale. This review summarizes the current state of knowledge of the ecotoxicity of plastic additives and identifies research needs to characterize the hazard they present to exposed biota. The potential ecological risk of chemical additives is of international concern so key differences in governance between the European Union and New Zealand to appropriately characterize their risk are highlighted.

Spermatozoa: A relevant biological target for genotoxicity assessment of contaminants in the estuarine bivalve Scrobicularia plana.

Evaluation of DNA quality of gametes is a relevant method to predict potential consequences of pollutants in the next generations, as it allows to define adverse outcome pathways implicated in pollutant-mediated toxicity for risk assessment. In the present study, a comet assay was developed for the spermatozoa of Scrobicularia plana exposed to 10 and 100µg/L of benzo[a]pyrene (B[a]P) for 24h and 5days. The induction of apoptosis and repair mechanisms was assessed by determining caspase-3 activity and polymerase cell nuclear antigen (PCNA) mRNA expression level. Results showed that B[a]P induced high levels of DNA breaks that were associated with apoptosis for all the conditions tested, indicating that the spermatozoa were sensitive to B[a]P. PCNA gene expression was induced in animals exposed to the highest concentrations of B[a]P, suggesting that defence mechanisms were enhanced in these animals. This preliminary study demonstrated the utility of spermatozoa as a relevant biological target for genotoxicity assessment of contaminants and will enable to predict the effect of contaminants on future generations.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene.

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.