Evaluating the uptake and effects of polystyrene nano plastic pollution on stress gene response in the zebrafish (Danio rerio) embryo




Marbach, Sandra

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Plastic pollution in aquatic environments has become a global problem over the past 50 years. Large plastic debris can be degraded into micro-and nano-sized plastics via chemical, biological, and mechanical processes. Nanoplastic pollution is a current threat to human health and the environment that affects nearly every ecosystem on the planet, from Alpine snow to deep-sea sediments. Nanoplastics have been found in the gastrointestinal tract and viscera of aquatic organisms, where polystyrene nanoplastics (PNPs) have been found to embed in tissue, cause oxidative damage to cells, and negatively affect the organism’s metabolism and feeding behavior. Currently, there are no methods to detect nanoplastics in environmental samples due to their small size and ability to accumulate in different areas of the water column, depending on their chemical make-up and density. Therefore, it has been difficult to accurately determine environmentally relevant concentrations of nanoplastics for laboratory studies. For this study, we used three concentrations (0.1 ppm, 1ppm, 10 ppm) and two sizes (0.03 μm, 0.1 μm) of polystyrene nanoplastics, to gain a better understanding of the effects of PNP pollution on the development and xenobiotic stress response of the freshwater teleost fish Danio rerio (zebrafish). The D. rerio embryos were collected immediately after spawning and were exposed to fluorescent PNPs of sizes 0.03 μm and 0.1 μm at concentrations 10 ppm, 1 ppm, 0.1 ppm, and 0ppm. Embryos were collected at 24-, 48-, 72-, and 96-hours post-treatment (hpt) start. Nanoplastic absorbance from treatment and uptake by the embryo, from 0 to 48 hpt, was visualized and estimated using a fluorescence plate reader and fluorescence microscopy. To quantify the health threat posed by PNPs on developing D. rerio embryos from 0 to 96 hpt, we assessed the relative gene expression changes of seven key genes related to xenobiotic stress, and embryonic development, using the quantitative polymerase chain reaction (qPCR). Inert alginate nanoparticles (ANPs) of size 0.1 μm were used as non-toxic controls for gene expression analysis. Fluorescent nanoparticle uptake of the embryo was shown by using a fluorescence plate reader, showing that fluorescent PNPs were being absorbed from the treatment throughout the 96-hour exposure time. By using fluorescent microscopy, we were able to show PNP adsorption to the fish chorion after as few as 24 hours of exposure and embedding of PNPs in the chorion walls after treatments ended. Scanning electron microscopy evidence shows, that PNPs are likely to cross the protective chorion, bypassing through the chorion’s pores and adsorbing to the embryo’s tissue. Gene expression of xenobiotic stress genes was affected by the PNP exposure, leading to changes in nearly all the tested xenobiotic stress response genes. Mortality rates and physical development were not significantly affected by the treatments. Our results indicated that nanoplastic exposure, especially chronic exposure at high concentrations during critical stages of development, can lead to tissue inflammation and xenobiotic stress and can lead to PNP embedding in embryonic tissue. The growing problem of plastic pollution affects aquatic environments at an exceptionally high rate and can be transported through the food chain where they may pose a significant threat to aquatic organisms from plants to macrofauna. Therefore, it is of utmost importance that plastic production, recycling, disposal, and use are well regulated.



Environmental Health, Freshwater Pollution, Polystyrene Nanoplastic Pollution, Stress Response, Zebrafish



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