Micro-by-micro interactions : Aggregation dynamics, biofilms, and ecotoxicological impacts of suspended solids

Abstract: Increasing concerns arise regarding the environmental risk of microplastics (MP) and their interactions with biota, prompting numerous effect studies. However, current ecotoxicology methods fall short when it comes to assessing MP impacts. One intriguing aspect of MP is their propensity to aggregate and interact with various substances in their environment, including bacteria. The unresolved methodological challenges in MP ecotoxicology revolve around aggregation and biofilm formation. Consequently, to establish suitable protocols for MP hazard assessment, it is essential to comprehend how particle behaviour and microbial colonisation affect the responses of test species to MP. This thesis aimed to improve our understanding of MP behaviour in ecotoxicological assays, focusing on the interplay between aggregation, biofilms, and effects on test organisms. First, a metaanalysis of findings from 20 published experimental studies addressing MP effects on algal growth was conducted to identify material and particle characteristics associated with growth inhibition (Paper I). The results revealed poor experimental control over particle aggregation, sedimentation, and algal biofilm formation resulting in conflicting findings across studies, even for the same polymers and particle sizes, and insufficient evidence for growth inhibition due to the MP exposure. These challenges were addressed by applying experimental designs with clay particles typical for natural environments, bacteria and measuring aggregation as a particle size distribution (PSD) in assays with Daphnia magna as a model test organism in incubation systems preventing sedimentation (Papers II-IV). In Paper II, natural biofilms were grown on MP and clay particles that were used in the experiments at high suspended solid concentrations (10-1000 mg/L) and daphnid mortality as the endpoint. We found that MP directly stimulated aggregation in the particle mixture and indirectly daphnid survival because larger aggregates were associated with lower mortality. Moreover, biofilm had positive effects on aggregate size and daphnid survival. In Paper III, environmentally relevant levels of suspended solids (0.1-10 mg/L) and MP (0-10%) were used for aggregate size and diversity analysis by structural equation modeling addressing the effects of dissolved organic matter (DOM) and test animals (D. magna) on the particle aggregation. The main aggregation drivers were DOM and filtration by the daphnids, although the total concentration of suspended solids and MP contribution also promoted it. Moreover, the daphnids introduced microorganisms to the exposure system, resulting in their propagation and biofilm formation on the test particles, which affected the daphnid response to the exposure (Paper IV). The bacterial diversity was mostly affected by the aggregate size and diversity, although the total suspended solids and DOM also contributed, promoting mainly the non-adhering cells, whereas particle-associated biofilms were affected by MP. The adverse effects on Daphnia were induced by small aggregate size, high DOM and biofilm diversity, with no direct MP effects.These findings demonstrate that test organisms, DOM, mineral particles, and MP can affect aggregation and bacterial diversity in the exposure experiments over a few days resulting in non-stable exposure conditions. Moreover, the aggregate diversity and biofilm composition can be the proximate drivers of the test organism responses, with the indirect and often minor role of MP. Assessing PSD and biofilms in ecotoxicity testing of MP and, most likely, any solid waste particles, such as black carbon, will facilitate interpreting the results and developing assays towards ecologically relevant hazard assessment.