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Microplastics are a widespread contaminant found not only in various natural habitats but also in drinking waters. With spectroscopic methods, the polymer type, number, size, and size distribution as well as the shape of microplastic particles in waters can be determined, which is of great relevance to toxicological studies. Methods used in studies so far show a huge diversity regarding experimental setups and often a lack of certain quality assurance aspects. To overcome these problems, this critical review and consensus paper of 12 European analytical laboratories and institutions, dealing with microplastic particle identification and quantification with spectroscopic methods, gives guidance toward harmonized microplastic particle analysis in clean waters. The aims of this paper are to (i) improve the reliability of microplastic analysis, (ii) facilitate and improve the planning of sample preparation and microplastic detection, and (iii) provide a better understanding regarding the evaluation of already existing studies. With these aims, we hope to make an important step toward harmonization of microplastic particle analysis in clean water samples and, thus, allow the comparability of results obtained in different studies by using similar or harmonized methods. Clean water samples, for the purpose of this paper, are considered to comprise all water samples with low matrix content, in particular drinking, tap, and bottled water, but also other water types such as clean freshwater. Graphical abstract

TUM-ParticleTyper is a novel program for the automated detection, quantification and morphological characterization of fragments, including particles and fibers, in images from optical, fluorescence and electron microscopy (SEM). It can be used to automatically select targets for subsequent chemical analysis, e.g., Raman microscopy, or any other single particle identification method. The program was specifically developed and validated for the analysis of microplastic particles on gold coated polycarbonate filters. Our method development was supported by the design of a filter holder that minimizes filter roughness and facilitates enhanced focusing for better images and Raman measurements. The TUM-ParticleTyper software is tunable to the user's specific sample demands and can extract the morphological characteristics of detected objects (coordinates, Feret's diameter min / max, area and shape). Results are saved in csv-format and contours of detected objects are displayed as an overlay on the original image. Additionally, the program can stitch a set of images to create a full image out of several smaller ones. An additional useful feature is the inclusion of a statistical process to calculate the minimum number of particles that must be chemically identified to be representative of all particles localized on the substrate. The program performance was evaluated on genuine microplastic samples. The TUM-ParticleTyper software localizes particles using an adaptive threshold with results comparable to the "gold standard" method (manual localization by an expert) and surpasses the commonly used Otsu thresholding by doubling the rate of true positive localizations. This enables the analysis of a statistically significant number of particles on the filter selected by random sampling, measured via single point approach. This extreme reduction in measurement points was validated by comparison to chemical imaging, applying both procedures to the same area at comparable processing times. The single point approach was both faster and more accurate proving the applicability of the presented program.

Abstract. Eastern boundary upwelling systems (EBUS) are among the most productive marine ecosystems on Earth. The production of organic material is fueled by upwelling of nutrient-rich deep waters and high incident light at the sea surface. However, biotic and abiotic factors can modify surface production and related biogeochemical processes. Determining these factors is important because EBUS are considered hotspots of climate change, and reliable predictions of their future functioning requires understanding of the mechanisms driving the biogeochemical cycles therein. In this field experiment, we used in situ mesocosms as tools to improve our mechanistic understanding of processes controlling organic matter cycling in the coastal Peruvian upwelling system. Eight mesocosms, each with a volume of ∼55 m3, were deployed for 50 d ∼6 km off Callao (12∘ S) during austral summer 2017, coinciding with a coastal El Niño phase. After mesocosm deployment, we collected subsurface waters at two different locations in the regional oxygen minimum zone (OMZ) and injected these into four mesocosms (mixing ratio ≈1.5 : 1 mesocosm: OMZ water). The focus of this paper is on temporal developments of organic matter production, export, and stoichiometry in the individual mesocosms. The mesocosm phytoplankton communities were initially dominated by diatoms but shifted towards a pronounced dominance of the mixotrophic dinoflagellate (Akashiwo sanguinea) when inorganic nitrogen was exhausted in surface layers. The community shift coincided with a short-term increase in production during the A. sanguinea bloom, which left a pronounced imprint on organic matter C : N : P stoichiometry. However, C, N, and P export fluxes did not increase because A. sanguinea persisted in the water column and did not sink out during the experiment. Accordingly, export fluxes during the study were decoupled from surface production and sustained by the remaining plankton community. Overall, biogeochemical pools and fluxes were surprisingly constant for most of the experiment. We explain this constancy by light limitation through self-shading by phytoplankton and by inorganic nitrogen limitation which constrained phytoplankton growth. Thus, gain and loss processes remained balanced and there were few opportunities for blooms, which represents an event where the system becomes unbalanced. Overall, our mesocosm study revealed some key links between ecological and biogeochemical processes for one of the most economically important regions in the oceans.

In the environment the weathering of plastic debris is one of the main sources of secondary microplastic (MP). It is distinct from primary MP, as it is not intentionally engineered, and presents a highly heterogeneous analyte composed of plastic fragments in the size range of 1 µm−1 mm. To detect secondary MP, methods must be developed with appropriate reference materials. These should share the characteristics of environmental MP which are a broad size range, multitude of shapes (fragments, spheres, films, fibers), suspensibility in water, and modified particle surfaces through aging (additional OH, C=O, and COOH). To produce such a material, we bring forward a rapid sonication-based fragmentation method for polystyrene (PS), polyethylene terephthalate (PET), and polylactic acid (PLA), which yields up to 10 5 /15 mL dispersible, high purity MP particles in aqueous media. To satisfy the claim of a reference material, the key properties-composition and size distribution to ensure the homogeneity of the samples, as well as shape, suspensibility, and aging -were analyzed in replicates (N = 3) to ensure a robust production procedure. The procedure yields fragments in the range of 100 nm−1 mm (<20 µm, 54.5 ± 11.3% of all particles). Fragments in the size range 10 µm−1 mm were quantitatively characterized via Raman microspectroscopy (particles = 500-1,000) and reflectance micro Fourier transform infrared analysis (particles = 10). Smaller particles 100 nm−20 µm were qualitatively characterized by scanning electron microcopy (SEM). The optical microscopy and SEM analysis showed that fragments are the predominant shape for all polymers, but fibers are also present. Furthermore, the suspensibility and sedimentation in pure MilliQ water was investigated using ultraviolet-visible spectroscopy and revealed that the produced fragments sediment according to their density and that the attachment to glass is avoided. Finally, a comparison of the infrared spectra from the fragments produced through sonication and naturally aged MP shows the addition of polar groups to the surface of the particles in the OH, C=O, and COOH region, making these particles suitable reference materials for secondary MP.