Draft Genome Sequence of Zalerion maritima ATCC 34329, a (Micro)Plastic-Degrading Marine Fungus.

Zalerion maritima is a marine fungus that has been studied for the biodegradation of (micro)plastics. Here, we report the draft genome sequence of strain ATCC 34329, which was shown to have a size of 58.4 Mb, a GC content of 44.39%, and 10,802 predicted genes.

Microplastics in Internal Tissues of Companion Animals from Urban Environments.

Companion animals living in urban areas are exposed to environmental contaminants, which may include microplastics. A preliminary study was conducted by collecting postmortem samples from the internal tissue (lungs, ileum, liver, kidney, and blood clots) of 25 dogs (Canis familiaris) and 24 cats (Felis catus) living in an urban environment in Porto metropolitan area, Portugal. Suspected microplastics were found in 80 samples from 35 animals (18 cats and 17 dogs), often occurring in more than one tissue of the same animal (71.4%), primarily under small sizes (50.3% as 1-10 µm). Micro-Raman spectroscopy confirmed a fraction of particles as common polymer types (e.g., polyethylene terephthalate). However, the number of particles was very low. This study highlights the possibilities of the internalization and distribution of microplastics in the internal tissues of terrestrial vertebrates.

Effects of virgin and weathered polystyrene and polypropylene microplastics on Raphidocelis subcapitata and embryos of Danio rerio under environmental concentrations.

Microplastics are ubiquitous contaminants of freshwater ecosystems. However, few ecotoxicity assays have been conducted on freshwater organisms using environmentally relevant concentrations of virgin and weathered microplastics. This work assessed the adverse effects of virgin and artificially weathered fragments of polystyrene and polypropylene on the microalga Raphidocelis subcapitata (72 h growth inhibition assay) and on embryos of the fish Danio rerio (96 h fish embryo assay) under environmentally relevant concentrations (2000-200,000 MP L-1) and high concentrations (12.5-100 mg L-1). Sizes of microplastics were measured as tens (polystyrene) to hundreds (polypropylene) of micrometers, while aging was assessed by measuring the carbonyl index. In the microalga, the tested high concentrations promoted growth, while environmentally relevant concentration induced either growth inhibition or promotion. In zebrafish embryos, environmentally relevant concentrations decreased body length and heart rates. No relevant effects were observed in organisms exposed to high concentrations for mortality, malformations, hatching rates, and swimming bladder inflation. Virgin microplastics presented slightly higher toxicity but direct comparison was hindered by the lack of a linear dose-response curve. Despite the lack of a clear pattern, adverse effects were often observed in the lowest environmentally relevant concentrations, raising concerns over the impacts of microplastics on freshwater ecosystems.

Suspected microplastics in Atlantic horse mackerel fish (Trachurus trachurus) captured in Portugal.

Microplastics have been found in fish, but most studies have focused on the digestive system without considering additional organs. Herein, the objective was to assess the presence of microplastics in internal organs (gills, guts, kidney, heart) of the Atlantic horse mackerel (Trachurus trachurus) captured of the coast of Portugal (Northeast Atlantic Ocean). Suspected microplastics were present in all organs, with particles of larger size (i.e., equivalent diameter) found in the gut and those of lower size in the heart and its luminal blood. Suspected microplastics of 1-10 µm were the most abundant (65.4%), more likely to translocate, owing to their minute size, but more difficult to properly characterize. These results highlight the need to expand the analytical work on organs and tissues for assessing microplastics in organisms, but also emphasize the actual need for developing analytical methods that allow for an accurate isolation, identification, and characterization of microplastics in biota.

Preparation of biological samples for microplastic identification by Nile Red.

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 µm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94-100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97-100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

Considerations when using microplates and Neubauer counting chamber in ecotoxicity tests on microplastics.

The ubiquity of microplastics raises issues regarding contamination control measures and laboratory practices. The objective was to adapt the use of counting chambers and plastic microplates on the ecotoxicity evaluation of microplastics. Counting chambers, originally used to quantify cells, can also be used to count high concentrations of microplastics (<100 µm) used in laboratory assays. By decontaminating the chamber and mixing the test solution with Nile Red (1:1), fluorescent particles can be easily counted under optical microscopy. Microplate wells, due to their composition, can be contaminated or release microplastics to the test medium, which can interfere with the results of ecotoxicity assays or spectroscopy readings. A cleaning method based on ethanol was developed, which effectively removed particles by 91% without interfering with microalgae yield. Besides providing practical applications that can improve ecotoxicity assays, this work intends to raise awareness on the need to adapt laboratory practices when working with microplastics.

Selection of microplastics by Nile Red staining increases environmental sample throughput by micro-Raman spectroscopy.

Nile Red staining enables visual identification and quantification of fluorescent particles as a proxy to microplastics at low cost and high throughput, including those of small sizes (≥2 µm), when preceded by proper natural organic matter removal, but providing no chemical characterization. On the other hand, micro-spectroscopy methods allow chemical characterization of particles based on their spectra, essential for polymer identification, but are costly and time-consuming. This work addresses the combination of both Nile Red staining with micro-Raman spectroscopy for the identification of microplastics. Besides being useful for quantification, Nile Red staining can be advantageously used as an objective criterion for pre-selection of particles for micro-Raman spectroscopy, producing little interference. The use of the 442 nm laser in micro-Raman spectroscopy induces Nile Red luminescence thus allowing to target the specific suspected microplastics when using an orange filter, reducing the number of particles subjected to identification and improving sample throughput. Staining dyes could also be used for mapping suspected microplastics before targeted analysis by micro-Raman spectroscopy. Thus, coupling Nile Red with micro-Raman spectroscopy can be useful to improve time efficiency while using this equipment.

A One Health perspective of the impacts of microplastics on animal, human and environmental health.

Microplastics contamination is widespread in the environment leading to the exposure of both humans and other biota. While most studies overemphasize direct toxicity of microplastics, particle concentrations, characteristics and exposure conditions being used in these assays needs to be taken into consideration. For instance, toxicity assays that use concentrations over 100,000 times higher than those expected in the environment have limited practical relevance. Thus, adverse effects on animal and human health of current environmental concentrations are identified as a knowledge gap. Conversely, this does not suggest the lack of any significant effects of microplastics on a global scale. The One Health approach provides a novel perspective focused on the intersection of different areas, namely animal, human, and environmental health. This review provides a One Health transdisciplinary approach to microplastics, addressing indirect effects beyond simple toxicological effects. Microplastics can, theoretically, change the abiotic properties of matrices (e.g., soil permeability) and interfere with essential ecosystem functions affecting ecosystem services (e.g., biogeochemical processes) that can in turn impact human health. The gathered information suggests that more research is needed to clarify direct and indirect effects of microplastics on One Health under environmentally relevant conditions, presenting detailed knowledge gaps.

Microplastics and fibers from three areas under different anthropogenic pressures in Douro river.

Sources contributing to specific concentration of microplastics and fibers are still not completely understood. This study aimed at assessing the concentrations of microplastics (2-5000 µm) and fibers (18-5667 µm) in three areas of distinct influences in the Douro river, Porto, Portugal: (i) a countryside area; (ii) a wastewater treatment effluent release zone; and (iii) an area in proximity to a boat dock and maintenance station. Nile Red staining coupled with microscopy allowed the identification of small microplastics (≥2 µm) with a median concentration of the three areas of 231 MP L-1. Most were fragments (69%). Sizes <40 µm were the most abundant (84%). Highest concentrations of microplastics were found near the boat dock/maintenance and lowest in the countryside area. Fibers were mostly natural (non-synthetic, 63%). Highest concentrations of fibers were found in the area influenced by the wastewater effluent, especially of synthetic fibers, and lowest in the countryside area. Concentration of all fibers and synthetic fibers was 46 F L-1 and 6 F L-1, respectively. High concentrations of microplastics and fiber contamination suggest that the wastewater treatment plant effluent and boat dock/maintenance are the likely sources originating hotspot areas.

Contamination issues as a challenge in quality control and quality assurance in microplastics analytics.

Microplastics are widely distributed environmental contaminants. To understand their impacts on the environment and health, more high-quality results are needed. Since microplastics are present in every environment, including indoor air, proper precautions must be adopted in order to prevent contamination of samples and overestimation of environmental concentrations. Thus, to guarantee a proper quality of results, researchers must adopt strict contamination control measures. This review was conducted to understand current contamination control practices. A total of 50 studies published in 2019 were reviewed, including sampling of biota, air, soil, sediment, freshwater and saltwater, regarding 10 contamination control parameters. Overall, studies usually only comply with 4 out of 10 of these measures, which include avoiding the use of plastic materials, covering samples with glass lids or aluminum foil, filtering solutions, or running procedural blanks. The importance of these measures is also exemplified with real observation of contamination. Finally, seven measures to control for contamination are suggested in order to improve the quality of results in microplastic sampling in future assessments.

Worldwide contamination of fish with microplastics: A brief global overview.

Widespread contamination of microplastics may lead to internalization in fish. This literature review from March 2019 to March 2020 details that a median of 60% of fish, belonging to 198 species captured in 24 countries, contain microplastics in their organs. Carnivores species ingested more microplastics than omnivores. Only 14% of fish were from aquaculture. Most studies focused on digestive systems, with presence in other organs currently being insufficiently assessed. Based on this assessment, knowledge gaps that should be addressed in future studies were identified.

The importance of contamination control in airborne fibers and microplastic sampling: Experiences from indoor and outdoor air sampling in Aveiro, Portugal.

Airborne microplastics and microfibers are released from daily materials, contaminating both indoor and outdoor air. Sampling in Aveiro, Portugal, revealed concentrations of 6 fibers m-3, with more synthetic fibers found in outdoor than indoor (8.5% vs. 4.1%, n = 6), with variations in fiber characteristics between sampling periods. Suspected microplastics (<10 µm) also followed this trend (12 vs. 5 particles m-3). Synthetic fibers presented peculiar characteristics, with larger median sizes of 513 µm and 90% of lighter colors. Nonetheless, numerous fibers and suspected microplastics were found in field blanks, possibly from sampling contamination, reducing the reliability of results. Few previous works have reported field blanks so far, raising concerns about the quality of their results as well. Thus, quality assurance measures should be more strictly applied when working with airborne fibers and microplastics, while more research should focus on the factors involved in the variation of concentrations and characteristics of airborne fibers.

Identification of microplastics in white wines capped with polyethylene stoppers using micro-Raman spectroscopy.

Beverages, often packaged in plastic, can be a source of microplastics in the human diet. In this study, an improved method for detection of microplastics in white wines capped with synthetic stoppers is explored. Visual quantification in the stereomicroscope or using Nile Red were excluded due to the small size of particles. Quantification in the optical microscope identified up to 5,857 particles.L-1 but lacked chemical characterization. Finally, micro-Raman spectroscopy was used for the first time in complex beverages in the identification of microplastics particles in white wines, allowing identification of at least one synthetic particle for each bottle, except in two cases. Improvements included reduction in volume filtered, selection of aluminum oxide filters, selection of blue laser and lack of H2O2 treatment. Using this method, identification of all particles present in small representative areas of the filter by micro-Raman spectroscopy will allow proper quantification of microplastics in complex beverage matrices.

Environmental status of (micro)plastics contamination in Portugal.

Plastics and microplastics are ubiquitous contaminants in aquatic ecosystems. This critical review is the first attempt at analyzing sources, concentration, impacts and solutions of (micro)plastic litter in Portugal based on all currently available literature. We found that, besides sea-based sources (e.g. shipping, fishing), 5717 t of mismanaged waste and 4.1 trillion microplastics from wastewater, mostly from untreated wastewater, are released to the environment every year. The highest concentrations are found in the North, Center and Lisbon regions, mostly comprised of consumer products, fishing gear and microplastics (<5 mm), especially fragments and pellets. This contamination has resulted in ingestion of plastics by organisms, including mussels, fishes, birds and turtles. Thus, every Portuguese citizen may consume 1440 microplastics a year based on the consumption of mollusks. Awareness campaigns, improvements in waste management and reductions in the release of untreated wastewater are recommended measures to reduce plastic pollution in Portugal.

Major factors influencing the quantification of Nile Red stained microplastics and improved automatic quantification (MP-VAT 2.0).

Automated count of Nile Red fluorescent microplastics allows fast and reliable quantification. However, factors involving staining, digital camera conditions and settings introduce variability to the results. The objective of this paper is to identify and propose solutions to these factors and improve on the previous MP-VAT script. While removal of digital sensor defects had little influence on results and staining can be reduced to 5 min, Nile Red concentrations cannot be reduced <0.01 mg mL-1, the 470 nm LED lantern emission must be >1600 lx, and photographic conditions should be maintained as stable as possible ideally improving the filter membrane area and using the recommended settings of 2 s, ISO100, F5.6. It was also found that Nile Red can be removed from microplastics using acetone or hydrogen peroxide with iron. More importantly, both particles and fluorescent are lost with time and thus quantification should be conducted within a week. Finally, MP-VAT 2.0 was developed to remove unselected areas and to identify only red particles, excluding white reflections from quantification. This updated version of MP-VAT produced improved recovery rates of 98.2 ± 6.9 for spiked samples and 95.9 ± 10.3 on actual environmental samples, presenting a cheap and reliable complementary method for microplastic identification.

An easy method for processing and identification of natural and synthetic microfibers and microplastics in indoor and outdoor air.

Microplastics and microfibers can contaminate every matrix, including in the atmosphere, thus leading to incidental inhalation. However, concentrations of airborne synthetic particle in indoor and outdoor environments are not well understood due to the complexities of sampling, sample processing and identification. This work aims at producing a simple protocol to determine the concentrations of airborne microplastics and fibers. This is accomplished by removing organic matter using hydrogen peroxide (H2O2), followed by removal of mineral matter by density separation with sodium iodide (NaI). Finally, identification of fibers into synthetic or natural under the stereomicroscope can be achieved following a diagram produced by systematically observing the most common textile fibers. This method produces a recovery rate of 94.4 % for spiked samples and has been proven suitable for environmental samples. •Fibers and microplastics in air are easier to identify after carbonaceous matter removal;•No loss of microfiber is expected from the solutions used;•Recovery rates of spiked samples is 94.4 %.

Effects of spatial and seasonal factors on the characteristics and carbonyl index of (micro)plastics in a sandy beach in Aveiro, Portugal.

Coastal environments are highly contaminated with plastics of various sizes. In order to understand the distribution and factors influencing (micro)plastics contamination in the environment, sampling of a sandy beach in Costa Nova, Aveiro, Portugal, was conducted by collecting plastic particles and sediments for density separation in transects from the mean low tide line to the dunes, during wet and dry seasons. For surface collection, microplastics comprised 69.4% of plastics, presenting concentrations of 3-6 items m-2 in the wet season, mostly polyethylene pellets carried ashore by storms, and <1 item m-2 for dry season, lower due to less backwashing, were found. Collection of infrared spectra of these particles allowed characterization by polymer type and carbonyl index of all particles. Variations in carbonyl index were found to be related to season, site and particle color. Density separated microplastics, mostly fibers, presented 23 times higher concentrations than surface collection (22 microplastics kg-1, 280 microplastics m-2), due to the identification of smaller sizes, and with higher concentrations in dry seasons, likely from accumulation in sediment and bathing season. Overall, different sampling methods allowed identification of different particle types and sizes, which may vary according to seasonal and spatial factors.

Environmental exposure to microplastics: An overview on possible human health effects.

Microplastics are ubiquitous environmental contaminants leading to inevitable human exposure. Even so, little is known about the effects of microplastics in human health. Thus, in this work we review the evidence for potential negative effects of microplastics in the human body, focusing on pathways of exposure and toxicity. Exposure may occur by ingestion, inhalation and dermal contact due to the presence of microplastics in products, foodstuff and air. In all biological systems, microplastic exposure may cause particle toxicity, with oxidative stress, inflammatory lesions and increased uptake or translocation. The inability of the immune system to remove synthetic particles may lead to chronic inflammation and increase risk of neoplasia. Furthermore, microplastics may release their constituents, adsorbed contaminants and pathogenic organisms. Nonetheless, knowledge on microplastic toxicity is still limited and largely influenced by exposure concentration, particle properties, adsorbed contaminants, tissues involved and individual susceptibility, requiring further research.