Increasing demand for size-resolved identification and quantification of microplastic particles in drinking water and environmental samples requires the adequate validation of methods and techniques that can be used for this purpose. In turn, the feasibility of such validation depends on the existence of suitable certified reference materials (CRM). A new candidate reference material (RM), consisting of polyethylene terephthalate (PET) particles and a water matrix, has been developed. Here, we examine its suitability with respect to a homogeneous and stable microplastic particle number concentration across its individual units. A measurement series employing tailor-made software for automated counting and analysis of particles (TUM-ParticleTyper 2) coupled with Raman microspectroscopy showed evidence of the candidate RM homogeneity with a relative standard deviation of 12% of PET particle counts involving particle sizes >30 µm. Both the total particle count and the respective sums within distinct size classes were comparable in all selected candidate RM units. We demonstrate the feasibility of production of a reference material that is sufficiently homogeneous and stable with respect to the particle number concentration. Graphical Abstract

Accurate quantification of small microplastics in environmental and food samples is a prerequisite for studying their potential hazard. Knowledge of numbers, size distributions and polymer type for particles and fibers is particularly relevant in this respect. Raman microspectroscopy can identify particles down to 1 $${\upmu }$$μ m in diameter. Here, a fully automated procedure for quantifying microplastics across the entire defined size range is presented as the core of the new software TUM-ParticleTyper 2. This software implements the theoretical approaches of random window sampling and on-the-fly confidence interval estimation during ongoing measurements. It also includes improvements to image processing and fiber recognition (when compared to the previous software TUM-ParticleTyper for analysis of particles/fibers $$> 10$$> 10 $${\upmu }$$μ m), and a new approach to adaptive de-agglomeration. Repeated measurements of internally produced secondary reference microplastics were evaluated to assess the precision of the whole procedure.

Detecting microplastics (MPs) in marine organisms is vital for understanding the ecological impact of MP pollution. Free-living marine nematodes, key players in benthic ecosystems, are often employed as bioindicators because of their sensitivity to environmental changes and thus hold promise as bioindicators for MP pollution too. This study investigated the detection of MPs in nematodes using µ-Raman spectroscopy combined with a tailored digestion protocol, targeting MPs in size ranges between 1 and 15 µm. While this is the first documented attempt to detect MPs in field-collected nematodes, significant challenges were identified. Contamination, particularly from airborne MPs and plastic-based laboratory materials, posed a major obstacle. We found higher numbers of <5 µm particles of polypropylene (PP), polyethylene terephthalate (PET), polylactic acid (PLA), polymethyl methacrylate (PMMA), and polystyrene (PS) in a natural community of nematodes compared to blank controls, suggesting the potential ingestion of small-sized MPs by nematodes in the real world. However, small MPs exhibited greater contamination challenges, underscoring the need for improved contamination control measures, such as open-air filters and plastic-free workflows. Despite these challenges, this study highlights the potential of µ-Raman spectroscopy as a valuable tool for detecting small-sized MPs in field-collected marine invertebrates, provided contamination risks are minimized. The likelihood of nematodes encountering MPs in marine sediments is high, but whether this translates to significant ingestion remains uncertain pending on the analysis of more field samples and the application of efficient measures of contamination reduction.