Microplastics in Limnic Ecosystems - Investigation of Biological Fate and Effects of Microplastic Particles and Associated Contaminants in Zebrafish (Danio rerio)-

Abstract

Given the continually increasing global polymer production, environmental pollution from plastic debris has been widely perceived as an ecological threat with potentially adverse (eco-)toxicological impacts on various species and ecosystems. Especially small fragments of plastic debris measuring 1 ≤ 1000 µm in size − so-called microplastics (MPs) − can pose a risk to biota by physical or chemical hazards due to ingestion, sorption and transfer of anthropogenic environmental pollutants (e.g., pesticides, polycyclic aromatic hydrocarbons, pharmaceuticals). In recent years, there has been extensive research on the impact of streams and rivers as main entry routes for MPs into the marine environment. However, the evidence of adverse effects caused by the intake of MPs and the transfer of contaminants sorbed to MPs into freshwater ecosystems is still not conclusively documented. Therefore, this thesis addressed the impact of microplastic particles in limnic ecosystems by investigating the biological fate and effects of MPs and associated anthropogenic pollutants on different life stages of zebrafish (Danio rerio). To this end, effects of two common environmental pollutants (benzo(k)fluoranthene, chlorpyrifos) and two synthetic polymers (polystyrene, polymethyl methacrylate) were investigated with regard to alterations of complementary biomarkers during acute and chronic exposure, as well as the trophic transfer of MPs and MP-sorbed contaminants from invertebrate organisms to zebrafish and acute toxic effects of MPs pre-exposed in a natural aquatic ecosystem. Overall, the sorption of both anthropogenic pollutants to MPs could be confirmed under various exposure scenarios. In contrast to recent study results, no adverse effects were observed when zebrafish were exposed to clean, pristine polystyrene and polymethyl methacrylate. The ingestion of MPs was documented in the gastrointestinal tract of zebrafish and did not induce any physiological impairment. The uptake of both pollutants was verified for all investigated species by GC-MS and fluorescence measurements. In the acute exposure scenario using the fish embryo toxicity test (FET), zebrafish embryos displayed sublethal effects and morphological deformities related to the underlying mode of action of benzo(k)fluoranthene and chlorpyrifos. Exposure to MP-associated contaminants significantly reduced malformations and attenuated biomarker responses of acetylcholine esterase activity and CYP450 induction and hence indicated a reduced bioavailability of both pollutants for zebrafish embryos. Chronic exposure of adult zebrafish to both types of MPs and pollutants was designed to assess the potential impact of long-term exposure and possible bioaccumulation of these contaminants of emerging concern. However, exposure to MP-sorbed pollutants failed to alter biomarker responses over the prolonged experimental period. In addition, a novel approach was applied to monitor the uptake of benzo(k)fluoranthene in the intestinal epithelium using confocal laser scanning microscopy. However, no significant bioaccumulation or biotransformation of benzo(k)fluoranthene and chlorpyrifos in brain and liver tissue could be demonstrated using ultra performance liquid chromatography coupled with fluorescence detection and with a high-resolution mass spectrometer (UPLC-FLD/HRMS). In order to address the possible vector effects of MPs, the transfer of benzo(k)fluoranthene and polymethyl methacrylate particles in a simplified limnic food web were investigated, consisting of zooplankton (Daphnia magna), sediment-dwelling invertebrates (Chironomus riparius larvae) and zebrafish as highlevel predator. However, the trophic transfer of benzo(k)fluoranthene via MPs could not be confirmed by a combination of highly sensitive fluorescence tracking using CLSM, hepatic CYP450 induction, and advanced chemical-analytical methods. To account for the demand for environmentally relevant exposure scenarios and to improve risk assessment of MPs, a mixture of the most commonly used synthetic polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride) was exposed in a well-monitored surface water body. To evaluate the potential transfer of hazardous pollutants under natural conditions, the toxicity of the MP mixture was assessed in terms of acute toxic effects (FET), potential neurotoxic effects (AChE activity, larval visual motor response test) and effects of dioxin-like substances (EROD assay) using zebrafish embryos. In conclusion, the exposed MP mixture did not elicit significantly different effects than the natural particles from sediment and suspended matter samples. Eventually, it could only be confirmed that MPs could act as carriers for environmental contaminants following ingestion by various organisms. However, increased deleterious effects of MP-sorbed contaminants on various sensitive life stages of zebrafish could not be corroborated by acute or prolonged exposure, trophic transfer, or natural exposure. The findings suggest that the sorption of pollutants to MPs is more likely to reduce the bioavailability as a result of slow desorption within the organism. Although, MPs offer an alternative exposure route for aquatic organisms via ingestion. Even chronic exposure at environmentally relevant concentrations did not induce bioaccumulation of both highly lipophilic substances in zebrafish tissues. Lastly, the sorption behavior of MPs under realistic environmental exposure conditions was more likely to approximate the behavior of natural particles. Consequently, MPs might pose only a limited risk to limnic communities, especially with regard to the comparatively small fraction of plastic particles in freshwater ecosystems compared to the multitude of naturally occurring (a)biotic particles.