Aggregation in experimental studies with microparticles: Bacterial communities in the exposure system affect animal responses to the test particles

The role of microorganisms is frequently overlooked in effect studies with particulate materials, such as microplastics. In addition to the microbes naturally found in the environment, test animals can transfer their microbiome to the surrounding media and establish bacterial communities in the exposure vessels. The interactions between the animals and the bacterial communities during the exposure can influence the animal responses to experimental factors, such as particle abundance, aggregation, and other characteristics. However, the current designs in particle ecotoxicology often overlook these interactions. In our 72-hour experiment, Daphnia magna were exposed to mixed kaolin clay and microplastics (<20-{micro}m polystyrene fragments). We aimed to assess microbial communities derived from Daphnia microbiota, focusing on particle-associated biofilms and non-adherent cells and the effects of the total suspended solids (1-10 mg/l), microplastics contribution (0-10%), dissolved organic matter (agarose; 0 and 20 mg/l), and aggregate size/topology on these communities. Furthermore, we explored the impact of bacterial diversity and community composition on Daphnia mortality and body condition using individual protein content as a proxy. We found a high similarity between bacterial communities and the Daphnia microbiome, indicating the microbiome as the source. Experimental factors had differential effects on the biofilms and non-adherent cells, with total suspended solids and agarose mainly influencing non-adherent cells at the family level (mostly upregulation) and microplastics affecting biofilms (both up- and downregulation). Aggregate size and topology were the key predictors of bacterial alpha diversity and the abundance of the affected families. Finally, the adverse effects on Daphnia were primarily driven by small aggregate size, agarose addition, and high biofilm diversity. These findings underscore the need to consider microbial components and their interactions with particles and species to comprehensively understand microplastic effects and develop ecologically relevant hazard assessment assays.