Background: Ecological impacts of micro- and nanoplastics particles (MNP) are among the most discussed environmental concerns. In algae, MNP are commonly hypothesized to reduce growth, which is a standard ecotoxicological endpoint. However, the reported test outcomes vary, with both growth inhibition and stimulation being observed. Due to this conflict of information, a data synthesis for MNP potential to cause growth inhibition in toxicity testing is needed.

Methods: We performed a meta-analysis study to assess the effect of MNP exposure on algal growth. Twenty studies published between 2010 and 2020 and representing 16 algal species and five polymer materials administered as particles in size range 0.04–3,000 μm were included in this meta-analysis. A random-effect model was used to estimate the effect size in three datasets: (1) Low concentration range (<100 mg/L), (2) High concentration range (≥100 mg/L), and (3) Full range model (0.004–1,100 mg/L), which encompassed all studies using the combination of experimental settings (test species, MNP concentration, polymer material, and particle size) yielding the highest effect size within a study.

Results: The exposure to MNP was not significantly associated with growth inhibition in any of the models tested. However, a high heterogeneity between the studies was found in all three models. Neither MNP concentration nor polymer material contributed significantly to the heterogeneity, whereas polymer density had a significant moderating effect, with a higher risk of growth inhibition at lower densities. We also identified a publication bias, with small studies that reported significant inhibition being overrepresented in our dataset.

Conclusions: The meta-analysis found limited evidence for MNP effect on microalgal growth in the standard algal growth inhibition test. The heterogeneity and varying methodological quality of studies limited the interpretation and the confidence in the findings. For hazard assessment, standardization and controlled exposure are needed as well as more sensitive endpoints that can inform us about the effect mechanisms. Finally, using particle-free controls in such tests cannot account for the presence of inert particulates in the test system, and, hence, does not allow to attribute observed effects to the test polymers.

Assessment of microplastic impacts in biota is challenging due to the complex behavior of the test particles and their interactions with other particulates, including microorganisms, in the environment. To disentangle responses to microplastic exposure from those to other suspended solids, both microplastic and natural particles must be present in the test system. We evaluated how microplastic, non-plastic particles, and biofilms interacted in their effects on survivorship using acute toxicity assay with Daphnia magna. The animals were exposed to microplastic and kaolin at different concentrations of suspended solids (SS; 10, 100, and 1000 mg/L) with a varying microplastic contribution (%MP; 0 to 80%) and biofilm (presence/absence) associated with the solids. Also, we examined how these exposure parameters (SS, %MP, and Biofilm) affected aggregate formation that was analyzed using particle size distribution data. Under the exposure conditions, Daphnia mortality was primarily driven by SS concentration but ameliorated by both microplastic and biofilm. The ameliorating effects were related to increased particle aggregation in the presence of biofilm and high %MP. In addition, a weak yet significant positive effect of the biofilm on the survivorship was observed, presumably, due to microbial food supply to the daphniids in the exposure system; the bacteria were utilized at the absence of other food. Therefore, the effects of both natural and anthropogenic particulates depend on the particle behavior and aggregation in the water governed by microbial communities and physicochemical properties of the particles, which must be taken into account in the hazard assessment of plastic litter.

The ubiquitous occurrence of microplastics is raising broad concerns and motivating effect studies. In these studies, however, particle behavior in the water and aggregation are rarely considered leading to contradictory evidence of the effects 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 regarding their size and diversity. 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 size of clay and microplastics in the stock suspensions used to prepare the experimental mixtures. The strongest positive driver of the aggregate size and variability 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 to the overall process. 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. Overall, this study strengthens our understanding of the processes occurring in the exposure systems when conducting effect studies with microplastics and other particulates and demonstrates the necessity to access the particle size distribution to characterize the exposure. These findings also suggest that relevant experimental designs with microplastics must include relevant natural particulates and DOM to ensure environmentally plausible particle behaviour and adequate particle-biota interactions.

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-µm 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.