Plastic debris exposure and effects in rivers: Boundaries for efficient ecological risk assessment.

Until recently, plastic pollution research was focused on the marine environments, and attention was given to terrestrial and freshwater environments latter. This discussion paper aims to put forward crucial questions on issues that limit our ability to conduct reliable plastic ecological risk assessments in rivers. Previous studies highlighted the widespread presence of plastics in rivers, but the sources and levels of exposure remained matters of debate. Field measurements have been carried out on the concentration and composition of plastics in rivers, but greater homogeneity in the choice of plastic sizes, particularly for microplastics by following the recent ISO international standard nomenclature, is needed for better comparison between studies. The development of additional relevant sampling strategies that are suited to the specific characteristics of riverine environments is also needed. Similarly, we encourage the systematic real-time monitoring of environmental conditions (e.g., topology of the sampling section of the river, hydrology, volumetric flux and velocity, suspended matters concentration) to better understand the origin of variability in plastic concentrations in rivers. Furthermore, ingestion of microplastics by freshwater organisms has been demonstrated under laboratory conditions, but the long-term effects of continuous microplastic exposure in organisms are less well understood. This discussion paper encourages an integrative view of the issues involved in assessing plastic exposure and its effects on biota, in order to improve our ability to carry out relevant ecological risk assessments in river environments.

Barau's petrel, Pterodroma baraui, as a bioindicator of plastic pollution in the South-West Indian Ocean: A multifaceted approach.

Marine plastic pollution is well described by bioindicator species in temperate and polar regions but remains understudied in tropical oceans. We addressed this gap by evaluating the seabird Barau's petrel as bioindicator of plastic pollution in the South-West Indian Ocean. We conducted a multifaceted approach including necropsies of birds to quantify plastic ingestion; GPS tracking of breeding adults to identify their foraging areas; manta trawling of plastic debris to measure plastic pollution at sea and modelling of plastic dispersal. We developed a spatial risk index of seabird exposure to plastic ingestion. Seventy-one percent of the analysed birds had ingested plastic. GPS tracking coupled with manta trawling and dispersal modelling show that adults consistently foraged at places with high level of plastic concentration. The highest ingestion risk occurred in the northwest of Reunion Island and at latitude 30°S. Our findings confirm that Barau's petrel is a reliable bioindicator of plastic pollution in the region.

Small microplastics have much higher mass concentrations than large microplastics at the surface of nine major European rivers.

Understanding the fates and impacts of microplastics requires information on their sizes, polymer types, concentrations, and spatial and temporal distributions. Here, we focused on large (LMPs, 500 µm to 5 mm) and small (SMPs, 25 to 500 µm) microplastics sampled with the exact same protocol in nine of the major European rivers during the seven months of the Tara Microplastic Expedition. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and pyrolysis coupled with gas chromatography and mass spectrometry (Py-GC-MS) analyses were used to determine the microplastics contents by number and mass. The median LMP concentration was 6.7 particles m-3, which was lower than those in other regions of the world (America and Asia). The SMP mass concentration was much higher to the LMP concentrations, with SMP/LMP ratios up to 1000 in some rivers. We did not observe a systematic positive effect of urban areas for the two size classes or polymers; this could be explained by the fact that the transport of microplastic is highly heterogeneous in rivers. We believe that this study has important implications for predictive models of plastics distribution and fate in aquatic environments.

Untargeted metabolomic insights into plastisphere communities in European rivers.

Every year, rivers introduce a staggering amount of hundred kilotons of plastic into the Oceans. This plastic is inhabited by microorganisms known as the plastisphere, which can be transferred between different ecosystems through the transport of microplastics. Here, we simulated the microbial colonization of polyethylene-based plastic pellets that are classically used to manufacture large-scale plastic products. The pellets were immersed for 1 month in four to five sampling stations along the river-to-sea continuum of nine of the major European rivers. This study presents the first untargeted metabolomics analysis of the plastisphere, by using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The plastisphere metabolomes were similar in the Rhine and Rhone rivers, while being different from the Tiber and Loire rivers, which showed greater similarity to the Thames and Seine rivers. Interestingly, we found a clear distinction between plastisphere metabolomes from freshwater and marine water in most of the river-to-sea continuum, thus suggesting a complete segregation in plastisphere metabolites that is not consistent with a major transfer of microorganisms between the two contrasted ecosystems. Putative annotations of 189 discriminating metabolites suggested that lipid metabolism was significantly modulated. These results enlightened the relevance of using environmental metabolomic as complementary analysis to the current OMICs analysis.

Niche partitioning and plastisphere core microbiomes in the two most plastic polluted zones of the world ocean.

Plastics are offering a new niche for microorganisms colonizing their surface, the so-called "plastisphere," in which diversity and community structure remain to be characterized and compared across ocean pelagic regions. Here, we compared the bacterial diversity of microorganisms living on plastic marine debris (PMD) and the surrounding free-living (FL) and organic particle-attached (PA) lifestyles sampled during the Tara expeditions in two of the most plastic polluted zones in the world ocean, i.e., the North Pacific gyre and the Mediterranean Sea. The 16S rRNA gene sequencing analysis confirmed that PMD are a new anthropogenic ocean habitat for marine microbes at the ocean-basin-scale, with clear niche partitioning compared to FL and PA lifestyles. At an ocean-basin-scale, the composition of the plastisphere communities was mainly driven by environmental selection, rather than polymer types or dispersal effect. A plastisphere "core microbiome" could be identified, mainly dominated by Rhodobacteraceae and Cyanobacteria. Predicted functions indicated the dominance of carbon, nitrogen and sulfur metabolisms on PMD that open new questions on the role of the plastisphere in a large number of important ecological processes in the marine ecosystem.

Among-colony variation in plastic ingestion by Yellow-legged gulls (Larus michahellis) across the western Mediterranean basin.

The Mediterranean region is both a hotspot for biodiversity and for the accumulation of plastic pollution. Many species are exposed to this pollution while feeding, including a wide diversity of seabirds. Our objective was to investigate spatial variation in the quantity and types of plastic ingested by Yellow-legged gulls using information obtained from regurgitated pellets collected in 11 colonies. Anthropogenic debris, and particularly plastic, was found in pellets from all colonies, but the amount varied considerably. This among-colony difference was stable over the two years of study. The presence of marine prey and the proportion of agricultural area around the colonies significantly influenced the number of ingested plastics. As landfills close and garbage management improves, the availability of anthropogenic waste should decline. Following the response of gulls to these changes will be particularly useful for monitoring plastic pollution and for understanding the response of opportunistic wildlife to environmental modifications.

Inferring microplastics origins in the Mediterranean Sea by coupling modelling and in-situ measurements.

This work focuses on the dynamics and retention of microplastics (MP) in the Mediterranean. MP manta-net trawls were performed in autumn 2019 north of the Balearic Islands and along the Balearic Front (BF). Lagrangian modelling was used to find the MP collected origin during the campaign. These combined results show that North of Mallorca is a temporary retention zone of 3 months variability, with MP origin being the Northern Current (NC) and the Gulf of Lion (GOL). Retention processes were less clear along the BF, due to frontal dynamics together with the strong northerly winds. However, it appears that the origin can differ between the North (i.e. the large North-Westerly basin, including the GOL and the NC path) and the South (short distances around the zone) of this front. In both areas, the wind and the current variability are strongly conditioning the existence and position of the MP concentration zones.

Do loggerhead sea turtle (Caretta caretta) gut contents reflect the types, colors and sources of plastic pollution in the Southwest Indian Ocean?

We analyzed plastic debris ingested by loggerheads from bycatch between 2007 and 2021 in the Southwest Indian Ocean (SWIO). We also analyzed plastic debris accumulated on beaches of the east coast of Madagascar as a proxy for ocean plastics to compare the characteristics of beached plastics and plastic ingested by turtles. We conducted a "brand audit" of the plastics to determine their country of origin. An oceanic circulation model was used to identify the most likely sources of plastics in the SWIO. In total, 202 of the 266 loggerheads analyzed had ingested plastics. Plastics categorized as "hard" and "white" were equally dominant in loggerheads and on beaches, suggesting no diet selectivity. Both the brand audit and circulation modeling demonstrated that Southeast Asia is the main source of plastic pollution in the region. This study demonstrates that loggerheads can be used as bioindicators of plastic pollution in the SWIO.

A critical review on the evaluation of toxicity and ecological risk assessment of plastics in the marine environment.

The increasing production of plastics together with the insufficient waste management has led to massive pollution by plastic debris in the marine environment. Contrary to other known pollutants, plastic has the potential to induce three types of toxic effects: physical (e.g intestinal injuries), chemical (e.g leaching of toxic additives) and biological (e.g transfer of pathogenic microorganisms). This critical review questions our capability to give an effective ecological risk assessment, based on an ever-growing number of scientific articles in the last two decades acknowledging toxic effects at all levels of biological integration, from the molecular to the population level. Numerous biases in terms of concentration, size, shape, composition and microbial colonization revealed how toxicity and ecotoxicity tests are still not adapted to this peculiar pollutant. Suggestions to improve the relevance of plastic toxicity studies and standards are disclosed with a view to support future appropriate legislation.

Mission Tara Microplastics: a holistic set of protocols and data resources for the field investigation of plastic pollution along the land-sea continuum in Europe.

The Tara Microplastics mission was conducted for 7 months to investigate plastic pollution along nine major rivers in Europe-Thames, Elbe, Rhine, Seine, Loire, Garonne, Ebro, Rhone, and Tiber. An extensive suite of sampling protocols was applied at four to five sites on each river along a salinity gradient from the sea and the outer estuary to downstream and upstream of the first heavily populated city. Biophysicochemical parameters including salinity, temperature, irradiance, particulate matter, large and small microplastics (MPs) concentration and composition, prokaryote and microeukaryote richness, and diversity on MPs and in the surrounding waters were routinely measured onboard the French research vessel Tara or from a semi-rigid boat in shallow waters. In addition, macroplastic and microplastic concentrations and composition were determined on river banks and beaches. Finally, cages containing either pristine pieces of plastics in the form of films or granules, and others containing mussels were immersed at each sampling site, 1 month prior to sampling in order to study the metabolic activity of the plastisphere by meta-OMICS and to run toxicity tests and pollutants analyses. Here, we fully described the holistic set of protocols designed for the Mission Tara Microplastics and promoted standard procedures to achieve its ambitious goals: (1) compare traits of plastic pollution among European rivers, (2) provide a baseline of the state of plastic pollution in the Anthropocene, (3) predict their evolution in the frame of the current European initiatives, (4) shed light on the toxicological effects of plastic on aquatic life, (5) model the transport of microplastics from land towards the sea, and (6) investigate the potential impact of pathogen or invasive species rafting on drifting plastics from the land to the sea through riverine systems.

Impacts of microplastics and the associated plastisphere on physiological, biochemical, genetic expression and gut microbiota of the filter-feeder amphioxus.

Oceanic plastic pollution is of major concern to marine organisms, especially filter feeders. However, limited is known about the toxic effects of the weathered microplastics instead of the pristine ones. This study evaluates the effects of weathered polystyrene microplastic on a filter-feeder amphioxus under starvation conditions via its exposure to the microplastics previously deployed in the natural seawater allowing for the development of a mature biofilm (so-called plastisphere). The study focused on the integration of physiological, histological, biochemical, molecular, and microbiota impacts on amphioxus. Overall, specific alterations in gene expression of marker genes were observed to be associated with oxidative stresses and immune systems. Negligible impacts were observed on antioxidant biochemical activities and gut microbiota of amphioxus, while we highlighted the potential transfer of 12 bacterial taxa from the plastisphere to the amphioxus gut microbiota. Moreover, the classical perturbation of body shape detected in control animals under starvation conditions (a slim and curved body) but not for amphioxus exposed to microplastic, indicates that the microorganisms colonizing plastics could serve as a nutrient source for this filter-feeder, commitment with the elevated proportions of goblet cell-like structures after the microplastic exposure. The multidisciplinary approach developed in this study underlined the trait of microplastics that acted as vectors for transporting microorganisms from the plastisphere to amphioxus.

Changes in plastic ingestion by yellow-legged gulls (Larus michahellis) over the breeding season.

Gulls can be particularly vulnerable to ingesting plastics when using anthropogenic food sources, with potential consequences for survival and reproductive success. Although birds are known to switch foraging habitats over the breeding season to provide higher quality food for chick provisioning, it is unclear what this means regarding the ingestion of plastics. Here, we tested whether breeding gulls decrease the amount of plastic ingested during reproduction by collecting pellets from a series of monitored nests at a large yellow-legged gull (Larus michahellis) colony in southern France. We found at least one plastic item in 83.9 % of the analyzed pellets, with the most abundant plastic type being polyethylene-based sheet plastic. As predicted, we found a slight decrease in the number of plastic items in pellets at chick hatching. These results suggest that gulls, like other birds, may adjust foraging habits to provide more digestible, less risky, food to chicks.

Discussion about suitable applications for biodegradable plastics regarding their sources, uses and end of life.

This opinion paper offers a scientific view on the current debate of the place of biodegradable plastics as part of the solution to deal with the growing plastic pollution in the world's soil, aquatic, and marine compartments. Based on the current scientific literature, we focus on the current limits to prove plastic biodegradability and to assess the toxicity of commercially used biobased and biodegradable plastics in natural environments. We also discuss the relevance of biodegradable plastics for selected applications with respect to their use and end of life. In particular, we underlined that there is no universal biodegradability of plastics in any ecosystem, that considering the environment as a waste treatment system is not acceptable, and that the use of compostable plastics requires adaptation of existing organic waste collection and treatment channels.

Ecotoxicity of Heteroaggregates of Polystyrene Nanospheres in Chironomidae and Amphibian.

Due to their various properties as polymeric materials, plastics have been produced, used and ultimately discharged into the environment. Although some studies have shown their negative impacts on the marine environment, the effects of plastics on freshwater organisms are still poorly studied, while they could be widely in contact with this pollution. The current work aimed to better elucidate the impact and the toxicity mechanisms of two kinds of commercial functionalized nanoplastics, i.e., carboxylated polystyrene microspheres of, respectively, 350 and 50 nm (PS350 and PS50), and heteroaggregated PS50 with humic acid with an apparent size of 350 nm (PSHA), all used at environmental concentrations (0.1 to 100 µg L-1). For this purpose, two relevant biological and aquatic models-amphibian larvae, Xenopus laevis, and dipters, Chironomus riparius-were used under normalized exposure conditions. The acute, chronic, and genetic toxicity parameters were examined and discussed with regard to the fundamental characterization in media exposures and, especially, the aggregation state of the nanoplastics. The size of PS350 and PSHA remained similar in the Xenopus and Chironomus exposure media. Inversely, PS50 aggregated in both exposition media and finally appeared to be micrometric during the exposition tests. Interestingly, this work highlighted that PS350 has no significant effect on the tested species, while PS50 is the most prone to alter the growth of Xenopus but not of Chironomus. Finally, PSHA induced a significant genotoxicity in Xenopus.

Interactive effect of urbanization and flood in modulating microplastic pollution in rivers.

Freshwater ecosystems play an important role in transporting and accumulating microplastics. Spatial and temporal variability in microplastic pollution can create critical spots and moments of elevated pollution, however, the consequences of their interaction are still poorly understood. This study aimed to assess the interaction between urbanization and flood episodes on river microplastic pollution. The water surface was sampled in two sites of the Garonne River, upstream and downstream a large urban area, during two flood episodes. Samples were chemically digested to facilitate particles isolation, and microplastics (700 µm-5 mm) were characterized through infrared spectroscopy (ATR-FTIR). Microplastic concentration increased by 5-8 fold during flood episodes, driven by river discharge. This increase was more significant in the downstream site. During the flood, there was an overall increase of larger particles on water surface, but only in the downstream site microplastic colours and polymeric compositions significantly varied. Principal component analysis of infrared spectra from polyethylene microplastics revealed that the main variance in the spectral region corresponded to hydroxyl and carbonyl groups. The carbonyl content in microplastics was significantly higher for particles collected during the flood, likely indicating a higher level of degradation. Urbanization modulates freshwater microplastic pollution during floods, and changes in microplastic physicochemical profile should be further integrated within toxicity studies to evaluate risks potentially elevated to animal and human health.

Determination of the microplastic content in Mediterranean benthic macrofauna by pyrolysis-gas chromatography-tandem mass spectrometry.

The Mediterranean Sea water bodies are ones of the most polluted, especially with microplastics. As the seafloor is the ultimate sink for litter, it is considered a hotspot for microplastic pollution. We provide an original analytical development based on the coupling of tandem mass spectrometry to pyrolysis-gas chromatography to improve the detection of plastic contamination in marine organisms. Due to the high selectivity of the mass spectrometer, a straightforward sample preparation consists uniquely of potassium hydroxide digestion. The quantification of six common polymers is possible in one run. The method was applied to analyze the plastic content from 500 µm down to 0.7 µm in the whole body of seven benthic species with variable feeding modes. Plastic was detected in all samples, with an almost systematic detection of polypropylene and polyethylene. Our method presents a major development in determining the levels of plastic contaminations in samples with rich organic matter content.

The role of microplastics in microalgae cells aggregation: A study at the molecular scale using atomic force microscopy.

Plastic pollution has become a significant concern in aquatic ecosystems, where photosynthetic microorganisms such as microalgae represent a major point of entry in the food chain. For this reason an important challenge is to better understand the consequences of plastic pollution on microalgae and the mechanisms underlying the interaction between plastic particles and cell's interfaces. In this study, to answer such questions, we developed an interdisciplinary approach to investigate the role of plastic microparticles in the aggregation of a freshwater microalgae species, Chlorella vulgaris. First, the biophysical characterization, using atomic force microscopy, of the synthetic plastic microparticles used showed that they have in fact similar properties than the ones found in the environment, with a rough, irregular and hydrophobic surface, thereby making them a relevant model. Then a combination of optical imaging and separation experiments showed that the presence of plastic particles in microalgae cultures induced the production of exopolysaccharides (EPS) by the cells, responsible for their aggregation. However, cells that were not cultured with plastic particles could also form aggregates when exposed to the particles after culture. To understand this, advanced single-cell force spectroscopy experiments were performed to probe the interactions between cells and plastic microparticles; the results showed that cells could directly interact with plastic particles through hydrophobic interactions. In conclusion, our experimental approach allowed highlighting the two mechanisms by which plastic microparticles trigger cell aggregation; by direct contact or by inducing the production of EPS by the cells. Because these microalgae aggregates containing plastic are then consumed by bigger animals, these results are important to understand the consequences of plastic pollution on a large scale.

Bacterial Abundance, Diversity and Activity During Long-Term Colonization of Non-biodegradable and Biodegradable Plastics in Seawater.

The microorganisms living on plastics called "plastisphere" have been classically described as very abundant, highly diverse, and very specific when compared to the surrounding environments, but their potential ability to biodegrade various plastic types in natural conditions have been poorly investigated. Here, we follow the successive phases of biofilm development and maturation after long-term immersion in seawater (7 months) on conventional [fossil-based polyethylene (PE) and polystyrene (PS)] and biodegradable plastics [biobased polylactic acid (PLA) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV), or fossil-based polycaprolactone (PCL)], as well as on artificially aged or non-aged PE without or with prooxidant additives [oxobiodegradable (OXO)]. First, we confirmed that the classical primo-colonization and growth phases of the biofilms that occurred during the first 10 days of immersion in seawater were more or less independent of the plastic type. After only 1 month, we found congruent signs of biodegradation for some bio-based and also fossil-based materials. A continuous growth of the biofilm during the 7 months of observation (measured by epifluorescence microscopy and flow cytometry) was found on PHBV, PCL, and artificially aged OXO, together with a continuous increase in intracellular (3H-leucine incorporation) and extracellular activities (lipase, aminopeptidase, and ß-glucosidase) as well as subsequent changes in biofilm diversity that became specific to each polymer type (16S rRNA metabarcoding). No sign of biodegradation was visible for PE, PS, and PLA under our experimental conditions. We also provide a list of operational taxonomic units (OTUs) potentially involved in the biodegradation of these polymers under natural seawater conditions, such as Pseudohongiella sp. and Marinobacter sp. on PCL, Marinicella litoralis and Celeribacter sp. on PHBV, or Myxococcales on artificially aged OXO. This study opens new routes for a deeper understanding of the polymers' biodegradability in seawaters, especially when considering an alternative to conventional fossil-based plastics.

Protocol for microplastic pollution monitoring in freshwater ecosystems: Towards a high-throughput sample processing - MICROPLASTREAM.

Robust and reproducible quantification of microplastic pollution in freshwater ecosystems requires the processing of a large amount of samples collected in varying environmental conditions. Such samples are characterized by a high amount of organic matter compared to microplastics and are highly variable in terms of the quantity and the composition of matrices, requiring a standardized analytical protocol for sample treatment and analysis. However, two important and time-consuming steps for microplastic recovery are the elimination of organic matter and microscopic inspection of samples. Here, we developed and validated a protocol, targeting particles with length ranging from 700 µm to 5 mm, that includes a double-step digestion of organic matter, consisting of incubation with potassium hydroxide followed by hydrogen peroxide solutions, and two stereomicroscopic analyses. In addition, we developed several technical improvements allowing reducing the time needed to process samples, such as the design of an adapted filter-cap to improve the content transfer. The absence of physical and chemical alterations in the investigated microplastic pellets and the average reduction of 65.8% (± 9.59 SD) of organic matter in real samples demonstrated that our protocol is fit for purpose. We recommend a second stereomicroscopic analysis to avoid underestimating microplastic concentration and particle size distribution biased towards larger particles. When used for a large-scale monitoring of microplastic pollution, this protocol resulted in an estimated time of 38 h for one person for the treatment of a batch of 24 samples, allowing a higher throughput sample processing and reproducible quantification. • Protocol customization towards high-throughput sample processing • Double step digestion to improve organic matter elimination • Importance of stereomicroscopic analysis for microplastic recovery.