Size-dependent effects of polystyrene microplastics on anaerobic digestion performance of food waste: Focusing on oxidative stress, microbial community, key metabolic functions

Nanomaterials (NM) exhibit novel physicochemical properties that determine their interaction with biological substrates and processes. Three metal oxide nanoparticles that are currently being produced in high tonnage, TiO(2), ZnO, and CeO(2), were synthesized by flame spray pyrolysis process and compared in a mechanistic study to elucidate the physicochemical characteristics that determine cellular uptake, subcellular localization, and toxic effects based on a test paradigm that was originally developed for oxidative stress and cytotoxicity in RAW 264.7 and BEAS-2B cell lines. ZnO induced toxicity in both cells, leading to the generation of reactive oxygen species (ROS), oxidant injury, excitation of inflammation, and cell death. Using ICP-MS and fluorescent-labeled ZnO, it is found that ZnO dissolution could happen in culture medium and endosomes. Nondissolved ZnO nanoparticles enter caveolae in BEAS-2B but enter lysosomes in RAW 264.7 cells in which smaller particle remnants dissolve. In contrast, fluorescent-labeled CeO(2) nanoparticles were taken up intact into caveolin-1 and LAMP-1 positive endosomal compartments, respectively, in BEAS-2B and RAW 264.7 cells, without inflammation or cytotoxicity. Instead, CeO(2) suppressed ROS production and induced cellular resistance to an exogenous source of oxidative stress. Fluorescent-labeled TiO(2) was processed by the same uptake pathways as CeO(2) but did not elicit any adverse or protective effects. These results demonstrate that metal oxide nanoparticles induce a range of biological responses that vary from cytotoxic to cytoprotective and can only be properly understood by using a tiered test strategy such as we developed for oxidative stress and adapted to study other aspects of nanoparticle toxicity.

Microplastics are anthropogenic contaminants which have been found in oceans, lakes and rivers. Investigations focusing on drinking water are rare and studies have mainly been using micro-Fourier Transform Infrared Spectroscopy (μ-FT-IR). A major limitation of this technique is its inability to detect particles smaller than 20 μm. However, micro-Raman spectroscopy is capable of detecting even smaller particle sizes. Therefore, we show that this technique, which was used in this study, is particularly useful in detecting microplastics in drinking water where particle sizes are in the low micrometer range. In our study, we compared the results from drinking water distributed in plastic bottles, glass bottles and beverage cartons. We tested the microplastic content of water from 22 different returnable and single-use plastic bottles, 3 beverage cartons and 9 glass bottles obtained from grocery stores in Germany. Small (-50-500 μm) and very small (1-50 μm) microplastic fragments were found in every type of water. Interestingly, almost 80% of all microplastic particles found had a particle size between 5 and 20 μm and were therefore not detectable by the analytical techniques used in previous studies. The average microplastics content was 118 ± 88 particles/l in returnable, but only 14 ± 14 particles/l in single-use plastic bottles. The microplastics content in the beverage cartons was only 11 ± 8 particles/l. Contrary to our assumptions we found high amounts of plastic particles in some of the glass bottled waters (range 0-253 particles/l, mean 50 ± 52 particles/l). A statistically significant difference from the blank value (14 ± 13) to the investigated packaging types could only be shown comparing to the returnable bottles (p < 0.05). Most of the particles in water from returnable plastic bottles were identified as consisting of polyester (primary polyethylene terephthalate PET, 84%) and polypropylene (PP; 7%). This is not surprising since the bottles are made of PET and the caps are made of PP. In water from single-use plastic bottles only a few micro-PET-particles have been found. In the water from beverage cartons and also from glass bottles, microplastic particles other than PET were found, for example polyethylene or polyolefins. This can be explained by the fact that beverage cartons are coated with polyethylene foils and caps are treated with lubricants. Therefore, these findings indicate that the packaging itself may release microparticles. The main fraction of the microplastic particles identified are of very small size with dimensions less than 20 μm, which is not detectable with the μ-FT-IR technique used in previous studies.

In this study, we evaluated accumulation and adverse effects of ingestion of microplastics in the monogonont rotifer (Brachionus koreanus). The dependence of microplastic toxicity on particle size was investigated by measuring several in vivo end points and studying the ingestion and egestion using 0.05-, 0.5-, and 6-μm nonfunctionalized polystyrene microbeads. To identify the defense mechanisms activated in response to microplastic exposure, the activities of several antioxidant-related enzymes and the phosphorylation status of mitogen-activated protein kinases (MAPKs) were determined. Exposure to polystyrene microbeads of all sizes led to significant size-dependent effects, including reduced growth rate, reduced fecundity, decreased lifespan and longer reproduction time. Rotifers exposed to 6-μm fluorescently labeled microbeads exhibited almost no fluorescence after 24 h, while rotifers exposed to 0.05- and 0.5-μm fluorescently labeled microbeads displayed fluorescence until 48 h, suggesting that 6-μm microbeads are more effectively egested from B. koreanus than 0.05- or 0.5-μm microbeads. This observation provides a potential explanation for our findings that microbead toxicity was size-dependent and smaller microbeads were more toxic. In vitro tests revealed that antioxidant-related enzymes and MAPK signaling pathways were significantly activated in response to microplastic exposure in a size-dependent manner.