Stable Isotope Insights into Microplastic Contamination within Freshwater Food Webs.

Microplastic pollution and ingestion are ubiquitous phenomena in freshwater ecosystems. However, our understanding of the role of trophic niche in microplastic ingestion is still limited. Here, we quantified the level of microplastic (700 µm to 5 mm) contamination for macroinvertebrates and fish within the Garonne river. We then used stable isotope analyses (δ13C and δ15N) to quantify trophic niches. We first demonstrated that the abundance of ingested microplastics differed between macroinvertebrates and fish and was not significantly related to microplastic pollution. We then found that microplastic characteristics (shape, color, size, and polymer composition) differ between the abiotic (surface waters and sediments) and biotic (ingested by macroinvertebrates and fish) compartments. The abundance of ingested microplastics increased with the size of organisms in both fish and macroinvertebrates and tended to increase with trophic position in macroinvertebrates only. Finally, the origin of the resources consumed by fish significantly affected the abundance of microplastics ingested. Altogether, these results suggest the absence of microplastic bioaccumulation in freshwater food webs and the dominance of direct consumption, most likely accidentally. The use of stable isotope analyses is therefore crucial to improve our understanding of microplastic ingestion by wild organisms.

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.

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.

Microplastic in angling baits as a cryptic source of contamination in European freshwaters.

High environmental microplastic pollution, and its largely unquantified impacts on organisms, are driving studies to assess their potential entry pathways into freshwaters. Recreational angling, where many anglers release manufactured baits into freshwater ecosystems, is a widespread activity with important socio-economic implications in Europe. It also represents a potential microplastic pathway into freshwaters that has yet to be quantified. Correspondingly, we analysed three different categories of industrially-produced baits ('groundbait', 'boilies' and 'pellets') for their microplastic contamination (particles 700 µm to 5 mm). From 160 samples, 28 microplastics were identified in groundbait and boilies, with a mean concentration of 17.4 (± 48.1 SD) MP kg-1 and 6.78 (± 29.8 SD) mg kg-1, yet no microplastics within this size range were recorded in the pellets. Microplastic concentrations significantly differed between bait categories and companies, but microplastic characteristics did not vary. There was no correlation between microplastic contamination and the number of bait ingredients, but it was positively correlated with C:N ratio, indicating a higher contamination in baits with higher proportion of plant-based ingredients. We thus reveal that bait microplastics introduced accidentally during manufacturing and/or those originating from contaminated raw ingredients might be transferred into freshwaters. However, further studies are needed to quantify the relative importance of this cryptic source of contamination and how it influences microplastic levels in wild fish.

Urbanization and hydrological conditions drive the spatial and temporal variability of microplastic pollution in the Garonne River.

Microplastic (MP) pollution represents a novel environmental pressure acting on freshwater ecosystems. Improving our understanding of the dynamics of MP pollution in freshwater ecosystems is therefore a prerequisite for managing and limiting this pollution. In this study, we quantified the spatial and temporal variability of MP (size range 700 µm - 5 mm) pollution in surface water in 14 sites located across the Garonne river catchment (Southwestern France, 6 in the main river and 8 tributaries). MP concentration averaged 0.15 particles.m-3 (± 0.46 SD) and strongly varied both in space and in time. We found that the spatial variation in MP concentration was driven by urbanization and that the temporal variation in MP concentration and MP size was driven by hydrological conditions, with higher concentrations and smaller particles sizes in warm seasons with low discharge. Polyethylene (44.5%), polystyrene (30.1%) and polypropylene (18.2%) were the main polymers and their proportion did not vary significantly across sampled sites. Particle color was associated with polymer type, with a high proportion of white particles in polystyrene. We also found a significant and negative relationship between MP size and the distance to the source in sites located in the main stream. MP pollution across watershed, from headwater tributaries to lowland rivers, is dynamic, and further studies are needed to improve the resolution of our knowledge of spatial and temporal patterns of MP pollution in freshwater ecosystems.

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges.

Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.