Aggregation in experimental studies with microparticles: Experimental settings change particle size distribution during exposure.

The ubiquitous occurrence of microplastics is raising broad concerns and motivating effect studies. In these studies, however, particle behaviour in the water and aggregation are rarely considered leading to contradictory results reported by different studies. Using an environmentally relevant experimental setup with Daphnia magna as a test organism, we investigated how experimental conditions affect particle aggregation and the aggregate heterogeneity in terms of the particle size distribution. The experimental factors considered were (1) exposure duration (48 h vs 120 h), (2) the total mass of suspended solids (0-10 mg/l) composed of natural mineral particles (kaolin) and microplastics, (3) the proportion of the microplastics in the particle suspension (0-10% by mass), (4) dissolved organic matter (DOM; 0 vs 20 mg agarose/l), and (5) presence of the test organism (0 and 5 daphnids/vial). We found that particle aggregation occurs within the first 48 h of incubation in all treatments, no substantial change in the aggregate heterogeneity is observed afterwards. The median aggregate size was ∼2-fold higher than the nominal average particle size of clay and microplastics in the stock suspensions used to prepare the experimental mixtures. The strongest positive driver of the aggregate size and heterogeneity was DOM, followed by the presence of daphnids and the concentration of the suspended solids in the system. Also, microplastics were found to facilitate aggregation, albeit they were the weakest contributor. Moreover, besides directly increasing the aggregation, DOM relaxed the effects of the total solids and daphnids on the aggregate size. Thus, the particle size distribution was established early during the exposure and shaped by all experimental factors and their interactions. These findings improve our understanding of the processes occurring in the exposure systems when conducting effect studies with microplastics and other particulates and demonstrate the necessity to access the particle size distribution to characterise the exposure. Aslo, relevant experimental designs with microplastics must include relevant natural particulates and DOM to ensure environmentally realistic particle behaviour and adequate particle-biota interactions.