Nile red staining for rapid screening of plastic-suspect particles in edible seafood tissues.

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02-0.06 h/cm2 filter surface compared to 4.5-14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques.

Microplastics in lakeshore and lakebed sediments - External influences and temporal and spatial variabilities of concentrations.

Microplastics have been predominantly studied in marine environments compared to freshwater systems. However, the number of studies analyzing microplastic concentrations in water and sediment within lakes and rivers are increasing and are of utmost importance as freshwaters are major pathways for plastics to the oceans. To allow for an adequate risk assessment, detailed knowledge concerning plastic concentrations in different environmental compartments of freshwaters are necessary. Therefore, the major aim of this study was the quantification and analysis of temporal and spatial distribution of microplastics (<5 mm) in freshwater shore and bed sediments at Lake Tollense, Mecklenburg-Western Pomerania, Germany. Likewise, it addresses the hypothesis that lakes may serve as long-term storage basins for microplastics. Concentrations were investigated semi-annually over a two-year period at four sandy bank border segments representing different expositions and levels of anthropogenic influence. In addition, lakebed samples were taken along the longitudinal dimension of Lake Tollense. Mean microplastic abundances were 1,410 ± 822 particles/kg DW for lakeshore sediments and 10,476 ± 4,290 particles/kg DW for lakebed sediments. Fragments were more abundant compared to fibers in both sediment compartments. Spatial and temporal variation was especially recognized for lakeshore sediments whereas microplastic abundances in lakebed sediments did not differ significantly between sampling points and sampling campaigns. This can be related to long-term accumulation at the lakebed. Lower microplastic abundances were found within the intertidal zone at lake beaches where constant wave action reduces accumulation. Increased microplastic abundances were recognized at the beach with least anthropogenic influence but in proximity to a tributary, which may serve as microplastic input pathway into Lake Tollense due to its catchment comprising mainly agricultural areas. Furthermore, spatial variations in microplastic concentrations were related to the abundance of macroplastic items at beaches and correlated with pedologic sediment characteristics, namely the content of organic matter.

Anthropogenic litter in freshwater environments - Study on lake beaches evaluating marine guidelines and aerial imaging.

Studies on macroplastic pollution in freshwater systems are rare compared to the marine environment. Nevertheless, freshwater systems are worthy to be equally investigated as they are pathways of plastic to the ocean and lakes may act as (temporary) sinks. The aim of this study was to identify sources for plastics and influences on its distribution in a limnic environment. Anthropogenic litter (>5 mm) was monitored semi-annually over a three-year period at four sandy bank border segments of Lake Tollense in Mecklenburg-Western Pomerania, Germany. The selected beaches represent different expositions and vary in their level of anthropogenic activity. Considering all six samplings, mean abundance of anthropogenic litter is 0.2 ± 0.1 items/m2 or 130.9 ± 91.0 items/100 m beach length. The averaged mass of anthropogenic litter is 0.5 ± 1.0 g/m2 or rather 218.7 ± 284.6 g/100 m. Plastic consistently is the predominate material (72%) and cigarette butts are the most found items. A higher pollution by anthropogenic litter is found at the end of tourist season unveiling the impact of anthropogenic activity on litter abundance. Additionally, litter transport via tributaries into the lake plays a role. Testing the detection of anthropogenic litter via aerial images taken by unmanned aerial vehicles resulted in good recovery rates when minimizing the flight height. Furthermore, the analysis of anthropogenic litter distribution displayed on the images showed litter accumulation areas at the border of sandy beach areas. The deployment of marine guidelines in a freshwater environment did work well, however, small changes in the protocol are suggested for future lake beach studies dealing with anthropogenic litter pollution.

Nile red staining in microplastic analysis-proposal for a reliable and fast identification approach for large microplastics.

A variety of methods concerning the identification of microplastics in environmental samples exist. While visual identification is often used, implemented easily, and cost-efficient but implying biased results, spectroscopic or chromatographic approaches are reliable but time-consuming and need specific equipment. Nile red staining is an available alternative and complement method for identifying microplastics. In this study, Nile red staining and subsequent photographing in a UV light photobox was tested on its reliability and feasibility. The approach was compared with a second identification process using again staining but a fluorescence microscope. Selected identified microplastic particles were analyzed by µ-Raman spectroscopy to prove their polymeric origin. The results show that the presented approach is faster compared with the use of a fluorescence microscope or µ-Raman spectroscopy. Furthermore, it is cost-effective as well as accurate for large microplastics > 0.63 mm and, therefore, may be applied when large sample volumes need to be analyzed. Graphical abstract.

Spatial distribution of atmospheric microplastics in bulk-deposition of urban and rural environments - A one-year follow-up study in northern Germany.

Atmospheric microplastic deposition rates play a crucial role for calculating the input of microplastics in the environment and to further understand pollution patterns. In this study, the spatial and temporal distribution of atmospheric microplastic particles in urban and rural areas of Northern Germany was investigated. Therefore, eleven structurally diverse locations in Hamburg and Mecklenburg-Western Pomerania were equipped with bulk-deposition samplers in triplicates and sampled monthly between August 2019 and July 2020. The resulting 306 samples were treated with hydrogen peroxide (30 %) and sodium hypochlorite (6-14 %) to digest biological organic matter. The filters were subsequently stained with the lipophilic dye Nile Red and underwent visual microplastic identification via fluorescence microscopy. Fragments and fibers were quantified down to a cut-off size of 10 µm. The polymer composition of microplastic particles was investigated along a subset of particles via µ-Raman spectroscopy. The microplastic deposition rate for Northern Germany (89 ± 61 MP/m2/day) is in the same order of magnitude as those reported by previous studies. Significant differences in microplastic deposition rates were found between urban and rural sampling sites. Population density was identified as an important factor for greater amounts of microplastics and higher shares of fibers in urban samples. Special attention was given to the canopy cover at two forested sampling sites and an influence of the comb-out effect on atmospheric microplastic deposition was detected.

Abundance and Distribution of Microplastics in Invertebrate and Fish Species and Sediment Samples along the German Wadden Sea Coastline.

Monitoring strategies are becoming increasingly important as microplastic contamination increases. To find potentially suitable organisms and sites for biota monitoring in the German Wadden Sea, we collected invertebrates (n = 1585), fish (n = 310), and sediment cores (n = 12) at 10 sites along the coast of Lower Saxony between 2018 and 2020. For sample processing of biota, the soft tissue was digested and the sediment samples additionally underwent a subsequent density separation step. Microplastic particles were identified using Nile red and fluorescence microscopy, followed by polymer composition analysis of a subset of particles via µRaman spectroscopy. All investigated species, sediment cores, and sites contained microplastics, predominantly in the morphology class of fragments. Microplastics were found in 92% of Arenicola marina, 94% of Littorina littorea, 85% of Mytilus edulis, and 79% of Platichthys flesus, ranging from 0 to 248.1 items/g. Sediment core samples contained MPs ranging from 0 to 8128 part/kg dry weight of sediment. In total, eight polymers were identified, predominantly consisting of polyethylene, polyvinylchloride, and polyethylene terephthalate. Considering the sampling, processing, and results, the species Mytilus edulis and Platichthys flesus are suitable species for future microplastic monitoring in biota.

Quantification and characterization of microplastics in surface water samples from the Northeast Atlantic Ocean using laser direct infrared imaging.

15 filtration samples were collected at eight locations onboard the RV Sonne (cruise SO279 in 2020) from 6 m water depth using a fractionated stainless-steel filtration unit. The size fraction > 300 µm was visually examined and potential microplastic particles were analyzed by ATR-FTIR spectroscopy. The treatment of size class 20 µm < d < 300 µm was based on enzymatic-oxidative microwave-assisted "one-pot" matrix digestion in conjunction with analysis of the microplastics by time-efficient LDIR imaging. Total number concentrations ranged from 47 to 2154 microplastic particles per m3 (average for all stations: 500 ± 700 microplastic particles m-3 (1 SD; n = 8)). In total, 20 polymer types were identified. The most common polymer types were polyethylene terephthalate (20 %) and acrylates/polyurethane/varnish (15 %). 93 % of the detected microplastics were smaller than 100 µm in length. Analysis of sample replicates indicates high spatio-temporal variations in microplastic pollution within the investigated region.

Microplastic concentrations, characteristics, and fluxes in water bodies of the Tollense catchment, Germany, with regard to different sampling systems.

The widespread presence of microplastics in multiple environmental compartments has largely been demonstrated. Assessing the ecological risk that microplastics pose is, at the present stage, hindered due to methodical differences. Moreover, different methods hamper meaningful comparisons between studies and data on microplastics <300 µm is scarce. Therefore, we focused on microplastics >20 µm in freshwater and sampling-related aspects in this concern. Sampling was conducted between 2018 and 2020 in the Tollense catchment in northeastern Germany and was carried out by in situ pump filtration. Two different sampling systems (cutoff sizes 20 µm and 63 µm) were applied to filter water volumes of 0.075-1.836 m3. Retained particles were analyzed by a combination of Nile red staining and micro-Raman spectroscopy. Thereby, we found microplastic concentrations between 123 and 1728 particles m-3 using the 63-µm cut-off size and between 1357 and 2146 particles m-3 using the 20-µm cut-off size. Local hydrodynamics (discharge and flow velocity) and land cover are likely influencing the observed microplastic concentrations and fluxes. The variability between both sampling systems cannot fully be explained by the different mesh sizes used. We argue that differentiation between a theoretical cut-off size (finest mesh) and a factual cut-off size (reliable quantification) can help to understand sampling related differences between studies.

Microplastics detected in cirrhotic liver tissue.

BACKGROUND: The contamination of ecosystem compartments by microplastics (MPs) is an ubiquitous problem. MPs have been observed in mice tissues, and recently in human blood, stool and placenta. However, two aspects remain unclear: whether MPs accumulate in peripheral organs, specifically in the liver, and if liver cirrhosis favours this process. We aimed to examine human liver tissue samples to determine whether MPs accumulate in the liver. METHODS: This proof-of-concept case series, conducted in Germany, Europe, analyzed tissue samples of 6 patients with liver cirrhosis and 5 individuals without underlying liver disease. A total of 17 samples (11 liver, 3 kidney and 3 spleen samples) were analyzed according to the final protocol. A reliable method for detection of MP particles from 4 to 30 µm in human tissue was developed. Chemical digestion of tissue samples, staining with Nile red, subsequent fluorescent microscopy and Raman spectroscopy were performed. Morphology, size and composition of MP polymers were assessed. FINDINGS: Considering the limit of detection, all liver, kidney and spleen samples from patients without underlying liver disease tested negative for MPs. In contrast, MP concentrations in cirrhotic liver tissues tested positive and showed significantly higher concentrations compared to liver samples of individuals without underlying liver disease. Six different microplastic polymers ranging from 4 to 30 µm in size were detected. INTERPRETATION: This proof-of-concept case series assessed the presence of MPs in human liver tissue and found six different MP polymers in the liver of individuals with liver cirrhosis, but not in those without underlying liver disease. Future studies are needed to evaluate whether hepatic MP accumulation represents a potential cause in the pathogenesis of fibrosis, or a consequence of cirrhosis and portal hypertension. FUNDING: No funding was received for conducting this investigator driven study.

Microplastics in a deep, dimictic lake of the North German Plain with special regard to vertical distribution patterns.

The investigation of microplastics (MPs) in freshwater has received increased attention within the last decade. To date, sampling is mainly conducted at the surface of both rivers and lakes and only a few studies assessed the vertical distribution of MPs in the water column of freshwater bodies. To contribute to the understanding of MP pollution in the water column of freshwater lakes, this study evaluated the vertical profile of MPs in Lake Tollense considering particles between 63 and 5000 µm in size. Sampling was conducted on three occasions at three depths (surface, 7 m and 10 m) along a transect including eight sampling stations. The retrieved samples were digested with hydrogen peroxide and sodium hypochlorite and investigated via Nile Red staining and fluorescence microscopy. Subsequently, a sub-sample of stained particles was verified by µRaman-spectroscopy. The vertical distribution of MPs in Lake Tollense differed considerably between particle shapes (irregular particles (IPs) and fibers). Fibers did not show a noticeable pattern with depth and ranged between 22 fibers m-³ at 0 m to 19 fibers m-³ at 10 m. In contrast, IPs were distinctly less abundant in sub-surface samples with concentrations between 50 IPs m-³ at 0 m to 29 IPs m-³ at 10 m. Concerning IPs, buoyant polymers (mainly PE and PP) and concerning fibers PET and PP dominated the polymeric composition. Besides particle inherent properties, wind-induced mixing is likely affecting the intensity of vertical concentration gradients. This study highlights the need for depth-integrated sampling approaches in order to achieve representative data without over- or underestimating the overall abundances.

Microplastic analysis in the South Funen Archipelago, Baltic Sea, implementing manta trawling and bulk sampling.

Microplastic contamination in surface waters of the South Funen Archipelago in Denmark was assessed. Therefore, ten manta trawls were conducted in June 2015. Moreover, 31 low-volume bulk samples were taken to evaluate, whether consistent results in comparison to the net-based approach can be obtained. Microplastic contamination in the South Funen Archipelago (0.07 ±â€¯0.02 particles/m3) is slightly below values reported before. The sheltered position of the study area, low population pressure on adjacent islands and the absence of any major potential point sources were identified as major factors explaining the low concentration of microplastics. Within the Archipelago, harbors or marinas and the associated vessel traffic are the most probable sources of microplastics. The concentration of microplastics in low-volume bulk samples is not comparable to manta trawl results. This is mainly due to insufficient representativeness of the bulk sample volumes.

Microplastic in beach sediments of the Isle of Rügen (Baltic Sea) - Implementing a novel glass elutriation column.

To extent the understanding on microplastics in the marine environment we performed a case study at four beaches on the Isle of Rügen considering abundance and spatial distribution of microplastics in beach sediments. For the analysis, density separation via a glass elutriation column was implemented. In advance, efficiencies were tested for two polymers, being not buoyant in water. Recovery rates of 80% for PET and 72% for PVC particles in sandy samples were achieved. A median abundance of 88.10 (Q1=55.01/Q3=114.72) microplastic particles per kg dry sediment or 2862.56 (Q1=1787.34/Q3=3727.28) particles per m2 was found at the beaches on Rügen. Fibers were more abundant than fragments at all beaches. In this study, no statistically significant differences but only tendencies were determined between the beaches with different exposition and anthropogenic activity as well as for distribution patterns which showed that microplastic fragments accumulate in topographic depressions, similar to macrolitter items.

Marine litter abundance and distribution on beaches on the Isle of Rügen considering the influence of exposition, morphology and recreational activities.

The abundance, weight and composition of marine debris were determined at the northwest coast of the Isle of Rügen in 2015. A total number of 1115 macrolitter items were registered, resulting in an abundance of 304±88.96 items per 100m of beach length and therefore being greater than the abundances found for other beaches at the Baltic Sea. Macrolitter items were predominantly composed of plastic, on average 83%. The four beaches under investigation have different exposition as well as touristic levels. The differing influence of wind and water currents as well as recreational activities on the macrolitter at these beaches was detectable. The distribution of items within a beach segment was analyzed by implementing D-GPS and drone aerial photography. The results of this analysis suggested that the identity of the substrate as well as the presence of vegetation are both major influencing factors in the macrolitter distribution.

Microplastic pollution in lakes and lake shoreline sediments - A case study on Lake Bolsena and Lake Chiusi (central Italy).

Rivers and effluents have been identified as major pathways for microplastics of terrestrial sources. Moreover, lakes of different dimensions and even in remote locations contain microplastics in striking abundances. This study investigates concentrations of microplastic particles at two lakes in central Italy (Lake Bolsena, Lake Chiusi). A total number of six Manta Trawls have been carried out, two of them one day after heavy winds occurred on Lake Bolsena showing effects on particle distribution of fragments and fibers of varying size categories. Additionally, 36 sediment samples from lakeshores were analyzed for microplastic content. In the surface waters 2.68 to 3.36 particles/m(3) (Lake Chiusi) and 0.82 to 4.42 particles/m(3) (Lake Bolsena) were detected, respectively. Main differences between the lakes are attributed to lake characteristics such as surface and catchment area, depth and the presence of local wind patterns and tide range at Lake Bolsena. An event of heavy winds and moderate rainfall prior to one sampling led to an increase of concentrations at Lake Bolsena which is most probable related to lateral land-based and sewage effluent inputs. The abundances of microplastic particles in sediments vary from mean values of 112 (Lake Bolsena) to 234 particles/kg dry weight (Lake Chiusi). Lake Chiusi results reveal elevated fiber concentrations compared to those of Lake Bolsena what might be a result of higher organic content and a shift in grain size distribution towards the silt and clay fraction at the shallow and highly eutrophic Lake Chiusi. The distribution of particles along different beach levels revealed no significant differences.