TUM-ParticleTyper: A detection and quantification tool for automated analysis of (Microplastic) particles and fibers.

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.

Raman microspectroscopic identification of microplastic particles in freshwater bivalves (Unio pictorum) exposed to sewage treatment plant effluents under different exposure scenarios.

We investigated the uptake of microplastic (MP, <5 mm) particles by using freshwater bivalves (Unio pictorum) as biological samplers in the environment. They were exposed either directly to the biologically purified sewage of a North Bavarian sewage treatment plant (STP) or placed in a small river up- and downstream of the wastewater discharge for 28 days and 6 months, respectively. A control group was maintained in a pond. After acid digestion, the soft tissue was analyzed for MP particles by means of Raman microspectroscopy (RM, over 3000 particles individually measured), which allows for identification and quantification of particles down to 1 µm. Only in the bivalve collective exposed to STP effluents MP was found, however a very small amount (maximum of nine MP particles in the bivalve sample exposed for 6 months). In the bivalves up- and downstream of the wastewater discharge and in control organisms from a pond, no microplastic was identified. The amount of microplastic particles was small in absolute terms and small in relative terms (ca. 1:100 (6 months) and below 1:1000 (28 days)) as hundreds of particles per sample were analyzed which turned out to be non-plastic. Including the results for the river, this indicates a rather low MP contamination level for organisms in close vicinity to a sewage treatment plant.

Variation in plastic abundance at different lake beach zones - A case study.

Plastic particles in marine and freshwater environments span from macroscopic to microscopic size classes. Each may have a different impact on individuals, populations and ecosystems, but still the wide variety of methods used in beach sediment sampling inhibit comparisons among studies and therefore hampers a risk assessment. A large portion of the uncertainties is due to differing sampling strategies. By quantifying the alongshore distribution of macro- and microplastic particles within five beaches of Lake Garda, we aim to shed light on the accumulation behavior of microplastic particles at an exemplary lake which might give indications for potential sampling zones. The identification of plastic at the single particle level with a spatial resolution down to 1µm was performed by Raman microspectroscopy. Given the time consuming approach we reduced the number of samples in the field but increased the spatial area where a single sample was taken, by utilizing a transect approach in combination with sediment cores (5cm depth). The study revealed that, in comparison to the water line and the high-water line, the drift line of all five beaches always contained plastic particles. Since the drift line accumulate particulate matter on a relatively distinct zone, it will enable a comparable sampling of microplastic particles. The applied sampling approach provided a representative method for quantifying microplastic down to 1µm on a shore consisting of pebbles and sand. Hence, as first step towards a harmonization of beach sediment sampling we suggest to perform sampling at the drift line, although further methodological improvements are still necessary.

Pigments and plastic in limnetic ecosystems: A qualitative and quantitative study on microparticles of different size classes.

Recently, macroplastic (>5 mm) and especially microplastic (<5 mm) particles have been reported as emerging contaminants in marine and limnetic ecosystems. Their coloration is gained by the addition of pigments to the polymer blend which is the major component of the respective product. However, color is also a feature of paint and coatings whereby the pigment is the major component. Once abraded from a surface, paint particles may enter the environment via similar pathways as microplastic particles. So far no detailed studies of microplastic particles (pigmented and non-pigmented) as well as paint particles have been performed focusing on very small microparticles (1-50 µm), in either marine or limnetic ecosystems. Using Raman microspectroscopy with a spatial resolution down to 1 µm, we report a remarkable increase in the occurrence of (pigmented) microplastic particles below 500 µm. Among those, most particles were found at a size of ∼130 µm in a freshwater ecosystem (subalpine Lake Garda, Italy). Moreover, our qualitative and quantitative analyses revealed that the number of paint microparticles significantly increased below the size range of 50 µm due to their brittleness (the smallest detected paint particle had a size of 4 µm). Inductively coupled plasma mass spectrometry measurements showed that both colored particles found in nature as well as virgin particles contain a high variety of metals such as cadmium, lead and copper. These additives may elicit adverse effects in biota ingesting these microparticles, thus paints and associated compounds may act as formerly overlooked contaminants in freshwater ecosystems.