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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.

Micro- and nanoplastic contamination is becoming a growing concern for environmental protection and food safety. Therefore, analytical techniques need to produce reliable quantification to ensure proper risk assessment. Raman microspectroscopy (RM) offers identification of single particles, but to ensure that the results are reliable, a certain number of particles has to be analyzed. For larger MP, all particles on the Raman filter can be detected, errors can be quantified, and the minimal sample size can be calculated easily by random sampling. In contrast, very small particles might not all be detected, demanding a window-based analysis of the filter. A bootstrap method is presented to provide an error quantification with confidence intervals from the available window data. In this context, different window selection schemes are evaluated and there is a clear recommendation to employ random (rather than systematically placed) window locations with many small rather than few larger windows. Ultimately, these results are united in a proposed RM measurement algorithm that computes confidence intervals on-the-fly during the analysis and, by checking whether given precision requirements are already met, automatically stops if an appropriate number of particles are identified, thus improving efficiency.

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Abstract. Oxygen minimum zones (OMZs) are characterized by enhanced carbon dioxide (CO2) levels and low pH and are being further acidified by uptake of anthropogenic atmospheric CO2. With ongoing intensification and expansion of OMZs due to global warming, carbonate chemistry conditions may become more variable and extreme, particularly in the Eastern Boundary Upwelling Systems. In austral summer (Feb–Apr) 2017, a large-scale mesocosm experiment was conducted in the coastal upwelling area off Callao (Peru) to investigate the impacts of on-going ocean deoxygenation on biogeochemical processes, coinciding with a rare coastal El Niño event. Here we report on the temporal dynamics of carbonate chemistry in the mesocosms and surrounding Pacific waters over a continuous period of 50 days with high temporal resolution observations (every 2nd day). The mesocosm experiment simulated an upwelling event in the mesocosms by addition of nitrogen (N)-deficient and CO2-enriched OMZ water. Surface water in the mesocosms was acidified by the OMZ water addition, with pHT lowered by 0.1–0.2 and pCO2 elevated to above 900 μatm. Thereafter, surface pCO2 quickly dropped to near or below the atmospheric level (405.22 μatm in 2017, NOAA/GML) mainly due to enhanced phytoplankton production with rapid CO2 consumption. Further observations revealed that the dominance of dinoflagellate Akashiwo sanguinea and contamination of bird excrements played important roles in the dynamics of carbonate chemistry in the mesocosms. Compared to the simulated upwelling, natural upwelling events in the surrounding Pacific waters occurred more frequently with sea-to-air CO2 fluxes of 4.2–14.0 mmol C m−2 d−1. The positive CO2 fluxes indicated our site was a local CO2 source during our study, which may have been impacted by the coastal El Niño. However, our observations of DIC drawdown in the mesocosms suggests that CO2 fluxes to the atmosphere can be largely dampened by biological processes. Overall, our study characterized carbonate chemistry in near-shore Pacific waters that are rarely sampled in such temporal resolution and hence provided unique insights into the CO2 dynamics during a rare coastal El Niño event.