Challenges inherent to the extraction of micro- and nanoplastics (MNPs) from the environment, combined with the limited range of commercially available MNPs, have prompted an increasing number of researchers to generate in-house reference and test MNPs. The first part of this review provides a comprehensive overview of existing MNP production methods, including top-down and bottom-up fabrication techniques. Strengths and weaknesses of different methods are compared and contrasted, and the potential for optimization and control over MNP properties is discussed. Methods to label and to artificially weather MNPs before, during, or after production, as well as appropriate dispersion protocols for introducing MNPs into different media, are also covered. The second part of this review focuses on how reference and test MNPs have been implemented in different types of studies, categorized as toxicity, uptake, fate, and monitoring. Given the wide range of properties needed to fully define MNPs, we propose a set of essential properties that need to be characterized depending on the study type. Looking forward, we suggest future needs, not only in the creation of reference MNPs, but also in experimental protocols that would help to better understand the behavior and impacts of MNPs. Overall, this review aims to provide the necessary information to guide researchers in decision-making regarding which reference MNPs are most appropriate to answer their specific research questions and to serve as a framework that will contribute to obtaining reliable, benchmarked data urgently needed to develop consensus on the fate and risk posed by MNPs.

Microplastic particles (MPs) are efficiently transferred from wastewater to sludge during wastewater treatment, but it remains unclear to what extent the physical-chemical properties (e.g., polymer type, shape, size) of the MPs affect their incorporation into heteroagglomerates. To assess the latter, different variants of MPs were individually spiked to activated sludge, which was aerated and mixed for durations between 0 and 120 min, followed by 30 min of sedimentation. In experiments with no aeration/mixing, already 70 – 80 % of all MP variants were found in the sedimented sludge. Including 120 min of aeration/mixing resulted in an additional accumulation of 15 – 20 % of the MPs in the sedimented sludge, with steady state conditions being reached after less than 15 min aeration/mixing. Overall, more than 90 % of all MP variants were accumulated in the sedimented sludge and up to 10 % remained in the supernatant. The similar relative affinities of spiked MP variants to sludge flocs derived from time resolved experiments (increasing aeration/mixing time followed by 30 min sedimentation time) agree with the similar behavior of all MPs during the activated sludge process. Images of flocs collected at the end of the experiments showed that MPs were dominantly associated with flocs. The formation of heteroagglomerates was likely favored by the close-to-neutral zeta potential of the MPs in filtered wastewater, suggesting that van der Waals forces control heteroagglomerate formation. Taken together, these results indicate that the removal of MPs during the activated sludge process is driven by the formation of heteroagglomerates, with differential sedimentation being more important than fluid shear.