Size dependent uptake and trophic transfer of polystyrene microplastics in unicellular freshwater eukaryotes.

Microplastics (MP) have become a well-known and widely investigated environmental pollutant. Despite the huge amount of new studies investigating the potential threat posed by MP, the possible uptake and trophic transfer in lower trophic levels of freshwater ecosystems remains understudied. This study aims to investigate the internalization and potential trophic transfer of fluorescent polystyrene (PS) beads (0.5 µm, 3.6 × 108 particles/mL; 6 µm, 2.1 × 105 particles/mL) and fragments (<30 µm, 5 × 103 particles/mL) in three unicellular eukaryotes. This study focuses on the size-dependent uptake of MP by two freshwater Ciliophora, Tetrahymena pyriformis, Paramecium caudatum and one Amoebozoa, Amoeba proteus, serving also as predator for experiments on potential trophic transfer. Size-dependent uptake of MP in all three unicellular eukaryotes was shown. P. caudatum is able to take up MP fragments up to 27.7 µm, while T. pyriformis ingests particles up to 10 µm. In A. proteus, small MP (PS0.5µm and PS6µm) were taken up via pinocytosis and were detected in the cytoplasm for up to 14 days after exposure. Large PS-MP (PS<30µm) were detected in A. proteus only after predation on MP-fed Ciliophora. These results indicate that A. proteus ingests larger MP via predation on Ciliophora (PS<30µm), which would not be taken up otherwise. This study shows trophic transfer of MP at the base of the aquatic food web and serves as basis to study the impact of MP in freshwater ecosystems.

Influence of the polymer type of a microplastic challenge on the reaction of murine cells.

Due to global pollution derived from plastic waste, the research on microplastics is of increasing public interest. Until now, most studies addressing the effect of microplastic particles on vertebrate cells have primarily utilized polystyrene particles (PS). Other studies on polymer microparticles made, e.g., of polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), or poly (ethylene terephthalate) (PET), cannot easily be directly compared to these PS studies, since the used microparticles differ widely in size and surface features. Here, effects caused by pristine microparticles of a narrow size range between 1 - 4 µm from selected conventional polymers including PS, PE, and PVC, were compared to those of particles made of polymers derived from biological sources like polylactic acid (PLA), and cellulose acetate (CA). The microparticles were used to investigate cellular uptake and assess cytotoxic effects on murine macrophages and epithelial cells. Despite differences in the particles' properties (e.g. ζ-potential and surface morphology), macrophages were able to ingest all tested particles, whereas epithelial cells ingested only the PS-based particles, which had a strong negative ζ-potential. Most importantly, none of the used model polymer particles exhibited significant short-time cytotoxicity, although the general effect of environmentally relevant microplastic particles on organisms requires further investigation.

Polystyrene microparticle distribution after ingestion by murine macrophages.

The impact of microplastic particles on organisms is currently intensely researched. Although it is well established that macrophages ingest polystyrene (PS) microparticles, little is known about the subsequent fate of the particles, such as entrapment in organelles, distribution during cell division, as well as possible mechanisms of excretion. Here, submicrometer (0.2 and 0.5 µm) and micron-sized (3 µm) particles were used to analyze particle fate upon ingestion of murine macrophages (J774A.1 and ImKC). Distribution and excretion of PS particles was investigated over cycles of cellular division. The distribution during cell division seems cell-specific upon comparing two different macrophage cell lines, and no apparent active excretion of microplastic particles could be observed. Using polarized cells, M1 polarized macrophages show higher phagocytic activity and particle uptake than M2 polarized ones or M0 cells. While particles with all tested diameters were found in the cytoplasm, submicron particles were additionally co-localized with the endoplasmic reticulum. Further, 0.5 µm particles were occasionally found in endosomes. Our results indicate that a possible reason for the previously described low cytotoxicity upon uptake of pristine PS microparticles by macrophages may be due to the preferential localization in the cytoplasm.

Tailor-Made Protein Corona Formation on Polystyrene Microparticles and its Effect on Epithelial Cell Uptake.

Microplastic particles are pollutants in the environment with a potential impact on ecology and human health. As soon as microplastic particles get in contact with complex (biological) environments, they will be covered by an eco- and/or protein corona. In this contribution, protein corona formation was conducted under defined laboratory conditions on polystyrene (PS) microparticles to investigate the influence on surface properties, protein corona evolution, particle-cell interactions, and uptake in two murine epithelial cells. To direct protein corona formation, PS particles were preincubated with five model proteins, namely, bovine serum albumin (BSA), myoglobin, ß-lactoglobulin, lysozyme, and fibrinogen. Subsequently, the single-protein-coated particles were incubated in a cell culture medium containing a cocktail of serum proteins to analyze changes in the protein corona profile as well as in the binding kinetics of the model proteins. Therein, we could show that the precoating step has a critical impact on the final composition of the protein corona. Yet, since proteins building the primary corona were still detectable after additional incubations in a protein-containing medium, backtracking of the particle's history is possible. Interestingly, whereas the precoating history significantly disturbs particle-cell interactions (PCIs), the cellular response (i.e., metabolic activity, MTT assay) stays unaffected. Of note, lysozyme precoating revealed one of the highest rates in PCI for both epithelial cell lines. Taken together, we could show that particle history has a significant impact on protein corona formation and subsequently on the interaction of particles with murine intestinal epithelial-like cells. However, as this study was limited to one cell type, further work is needed to assess if these observations can be generalized to other cell types.

Pristine and artificially-aged polystyrene microplastic particles differ in regard to cellular response.

Microplastic particles (MP), arising from the gradual decomposition of plastics in the environment, have been identified as a global problem. Most investigations of MP cytotoxicity use pristine spherical particles available from commercial sources when evaluating their impact on mammalian cells, while only limited data is available for the more relevant "weathered microplastic". In this study, we exposed murine macrophages to polystyrene MP either after up to 130 days of accelerated ageing or in pristine condition. Weathered and pristine MP were physicochemically characterized, and their cytotoxicity was investigated using biological assays, transcriptome analysis, and metabolic pathways prediction. Whereas the response to pristine MP is mainly dominated by a TNF-α release, sharp-edged weathered MP induce broader adverse cellular reactions. This study stresses the importance of including more realistic test particles (e.g., weathered particles) in combination with a broad range of biological assays when evaluating the potential risk of microplastic exposure.

Shape, size, and polymer dependent effects of microplastics on Daphnia magna.

The effects of microplastic (MP) pollution on organisms are gaining increasing attention. To date, a variety of polymers of different shapes and sizes are used in ecotoxicology. Although polystyrene (PS) is the predominant polymer type used in effect studies, it is still unclear whether the observed effects derive from the polymer itself or from a certain particle shape and size. To elucidate whether the effects are polymer specific, we conducted a systematic study on Daphnia magna by comparing various PS-MPs to nonplastic control particles with similar properties. In chronic exposure experiments, we used PS beads (6 µm; 20 µm), fibers (Ø 3 µm, length: 75.5 µm), and fragments (5.7 µm; 17.7 µm) in two different size classes and two different concentrations (500 and 5000 particles ml-1) and in-house-produced control particles of comparable size, shape, concentration and, if possible, density. Although most PS properties did not elicit effects on the tested endpoints, we observed sublethal effects on D. magna life history and morphology for small PS beads and fragments. Interestingly, no adverse effects were detected for any of the control particles. Hence, the observed effects are polymer-specific, related to the size and shape of the polymer, and do not result from particle exposure per se.

In vitro cultivation of primary intestinal cells from Eisenia fetida as basis for ecotoxicological studies.

The earthworm Eisenia fetida is a commonly used model organism for unspecific soil feeders in ecotoxicological studies. Its intestinal cells are the first to encounter possible pollutants co-ingested by the earthworm, which makes them prime candidates for studies of toxic effects of environmental pollutants on the cellular as compared to the organismic level. In this context, the aim of this study was to demonstrate the suitability of preparations of primary intestinal E. fetida cells for in vitro ecotoxicological studies. For this purpose, a suitable isolation and cultivation protocol was established. Cells were isolated directly from the intestine, maintaining >85% viability during subsequent cultivations (up to 144 h). Exposure to established pollutants and soil elutriates comprising silver nanoparticles and metal ions (Cu2+, Cd2+) induced a significant decrease in the metabolic activity of the cells. In case of microplastic particles (MP particles), namely 0.2, 0.5, 2.0, and 3.0 µm diameter polystyrene (PS) beads as well as 0.5 and 2.0 µm diameter polylactic acid (PLA) beads, no active uptake was observed. Slight positive as well as negative dose and size dependent effects on the metabolism were seen, which to some extent might correlate with effects on the organismic level.