The spectra from the Fourier-transform infrared spectroscopy of the inner frontal, middle filtering and outer layers of the mask materials had absorption bands at the same positions and with the same relative intensities as spectra obtained for polypropylene, from the internal database of the Slovenian National Building and Civil Engineering Institute (Supplementary Information Fig. S1). This confirmed that the source mask indeed contained polypropylene.
Two different shapes of microplastics were obtained from the three layers: fibres resulted from milling of the inner and outer layers, while milling of middle layer resulted in irregularly-shaped fragments (FE-SEM Zeiss Ultra Plus; Fig. 1 D-F). This is not surprising given the fact that the composition of the intact source medical mask material was different between the layers: the inner layer and outer layer were very similar in shape being composed of a mesh of fibres with very uniform diameters (21.2 ± 1.5 μm and 22.1 ± 1.6 μm for inner and outer layer, respectively), while the fibres in the middle layer were significantly thinner (4.3 ± 2.2 μm). The fibres in middle layer had various diameters and the mesh was more compact and interwoven (Fig. 1 A-C). This difference is in line with the fact that these layers are produced using different technological approaches (see introduction and ref. (9)). Also, Ellison et al. [10] reported that thinner fibres are formed by melt-blown process used for the middle layer than by the spun-bond processes used for the inner and outer layers.
Fig. 1Representative scanning electron microscopy images of the intact mask layers (A-C) and milled microplastics (D-F) derived from the medical mask inner frontal layer, middle filtering layer and outer layer. White bars on the images represent 100 μm
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We observed that cryo-milling deforms the shape of the original fibres in medical mask. It is unclear how relevant these particles are for ecotoxicity testing in comparison to those released in the environment. Wang et al. [14] reported that different shapes of microplastics, mostly fragments of fibres, were released from the three layers of medical masks after UV weathering. Interestingly, the middle layer was more susceptible to UV than the inner and outer layers. Extraction of fibre fragments from the water after aging could be an option to obtain relevant testing materials, but the recovery in this case is low and would not be sufficient for large scale experiments or for soil toxicity testing where large quantities are needed. Therefore cryo-milling remains the most common approach to produce microplastics for research as it enables sufficient amount of testing material to be produced. Other approaches that had been used previously to produce microplastics from larger plastic items all include some mechanical fragmentation, these are: cutting with scissors, grinding with mortar and liquid nitrogen, and cutting with cryogenic microtome (Table 1).
Table 1 Results of the GC-MS analysis for the three layers of the disposable medical mask, with total numbers of peaks detected, combined mass fraction of extracted compounds detected and a list of compounds with ≥90% quality and at levels of > 10 μg/g microplastics. The possible functions of chemicals were extracted from Zimmermann et al. [37] and Groh et al. [38] through the database “Chemicals associated with plastic packaging”. For the chemicals which were not listed in any of these two publications, the function was summarised from the PubChem database. Where available, detection in other plastic samples was described (after Zimmermann et al. [37])Full size table
The particle size distributions obtained by laser diffraction analysis (Microtrac S3500 Bluewave) were very similar for the inner and outer layers, with mean sizes (±standard deviation; expressed as the equivalent diameters of spherical particles) of the fibres of 45.1 ± 21.5 μm and 42.0 ± 17.8 μm, respectively. The fragments of the middle filtering layer were slightly larger, at 55.6 ± 28.5 μm. As can be seen from the size distributions shown in Fig. 2, for the inner, middle and outer layers, 99.1%, 97.6% and 99.4% of the particles, respectively, were < 176 μm, which is not too surprising given that they were sieved through a 250-μm fine-mesh sieve.
Fig. 2Numerical particle size distributions of the milled microplastics derived from the medical mask inner frontal layer (A), middle filtering layer (B) and outer layer (C), as determined by laser diffraction analysis
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Wang et al. [14] reported that the particle size distributions differed between the layers of medical mask weathered in water. The particles from the outer layer were mainly distributed in the range of 20–100 μm and 100–500 μm, particles from the inner layer were mainly distributed in 30–100 μm and 100–500 μm, and for the middle layer, the particle size of the microplastics was 50–200 μm. Most of the particles released were less than 200 μm in size for all three mask layers, with this trend being particularly pronounced for the middle layer, where this size distribution accounted for 91.2% of the total concentration. This means that the size range of particles obtained by cryo-milling in our case study is within the environmentally relevant values, although the particle sizes will largely depend on the choice of the parameters used for the milling method as well as for sieving.
We analysed extracts from the milled microplastics from the inner frontal, middle filtering and outer layers of the medical mask. GC-MS chromatograms solvent (methanol), procedural blank and of the extracts are presented in Figs. S2-S6 (Supplementary information). The data for the different compounds identified from the three layers of the medical mask are given in Table 1. This revealed several long-chain hydrocarbons; however, many of these are not listed in Table 1 because the identification of long-chain hydrocarbons is not reliable.
Among most common groups of chemicals were: antioxidants, such as 2,4-di-(tert-butyl) phenol; 2,6-di-(tert-butyl)-4-(methoxymethyl)phenol and methyl-3,5-bis (1,1-dimethylethyl)-4-hydroxy-benzene-propanoate (also known as Metilox); and lubricants: e.g. methyl palmitate; methyl-3,5-bis (1,1-dimethylethyl)-4-hydroxy-benzene-propanoat;, eicosane; methyl stearate and (E)-9-octadecenamide (i.e., oleamide). Some oleamide was detected in the procedural blank as well (Supplementary Fig. S3). Of particular interest, some of the compounds detected from the extraction of the inner layer are commonly used as food flavourings and antimicrobial agents (e.g. 2,4-dimethylanisole; 2,4-dimethylphenol; benzothiazole; heptadecane). The total amounts of the extracted compounds detected by GC-MS were similar for each of the layers of the medical masks (Table 1), but there were indications of many more compounds in the GC-MS chromatogram from the extraction of the inner frontal layer (Supplementary Fig. S2).
It has been reported that a number of different compounds can leach from polypropylene products [39]. For example, a total of 107 analytes were identified in leachates from polypropylene food containers [39]. Among these, the most abundant groups were antioxidants and their degradation products (tris (2,4-di-tert-buthylphenyl)phosphite; 2,6-Di-tert-butyl-4-ethyl-phenol), plasticizers (e.g. bis-(2-ethylhexyl) phthalate; dibutyl phthalate), cross-linking agents (e.g. 2-mercaptobenzothiazole; benzothiazole) and other additives (e.g., antistatic agents; lubricants; non-ionic surfactants) [39]. Similarly, Zimmermann et al. [37] reported a number of chemicals in PP products, for example 18, 5 and 22 different chemicals in gummy candy packaging, handkerchief packaging, and shampoo bottle, respectively. Some of these chemicals were also detected in the medical masks characterised in this study (Table 1). We could not find data specific to medical masks, but there are some records that medical masks might contain formaldehyde and bromo-2-nitropropane-1,3-diol (bronopol), which can cause acute dermatitis in healthcare workers [40], but these were not identified in the present samples.
We observed no effects on the mobility and survival of D. magna exposed to the three types of microplastics that were milled from the three layers of the medical mask at 1 mg L− 1-100 mg L− 1 for 48 h. However, there was attachment of these microplastics to the body surface and ingestion of the microplastics by D. magna (Fig. 3). This is in line with our previous work where no acute effects of polyethylene cosmetic beads and polyester textile fibres on D. magna were recorded, but these microplastics were as well found in the gut [41, 42]. Acute effects were however observed in the case of polyester textile fibres when the exposure was prolonged for additional 24 h as the daphnids could not recover from the exposure [41]. We would thus suggest the need for further studies on chronic effects of microplastics from medical masks. Of note, chronic studies have already shown numerous effects of other types of microplastics on D. magna [43, 44]. Furthermore, multigenerational studies with daphnids have shown that some effects, such as decreased reproduction, can persist over at least two generations without further exposure to the microplastics [43]. The choice of exposure scenarios and endpoint selection in future polypropylene microplastics ecotoxicity studies with D. magna should also consider the physicochemical properties that appear particular for this type of test material [45], as well as testing for the expected chemically and physically induced interactions of microplastics with the test organisms, for example adsorption onto the body surface and interference with moult.
Fig. 3Representative light microscopy images of Daphnia magna after 48 h exposure to the medical mask microplastics. Left: Microplastics from the middle filtering layer of the medical mask attached to the body surface of a D. magna. Right: Microplastics from the inner frontal layer of the medical mask in the gut of a D. magna (white arrow)
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Although several plastics-associated chemicals were identified in methanol extracts from medical mask microplastics (Table 1), obviously the concentrations in test medium during the acute exposure of D. magna were not high enough to cause acute lethal effects. Similarly, when leachates from 26 different plastic products were tested (analysed at 100–250 g plastics L− 1 water), none of the leachates from polypropylene were toxic to the water flea D. magna [46]. Also, in another study, leachates from polypropylene showed the lowest inhibition of the survival and settlement of the barnacle Amphibalanus amphitrite when compared to high-density and low-density polyethylene, polyvinylchloride, polycarbonate, polyethylene terephthalate, polystyrene (all analysed at 1000–5000 cm2 L− 1 with water; equivalent to 100–500 g plastics L− 1 water) [47].
Currently, there is only one very recent ecotoxicity study available on the polypropylene microplastics from medical masks [48]. The authors report the effect of microplastics obtained from FFP2 medical mask on springtails Folsomia candida and earthworms Eisenia andrei. The reproduction and growth of juvenile springtails and spermatogenesis of earthworm were decreased already at environmentally relevant concentration (Table 2). No induction of oxidative stress and effects on survival were found for both species. To our knowledge, no data on the effects of medical mask microplastics for aquatic organisms currently exists.
Table 2 Overview of the ecotoxicity studies on microplastics derived from polypropyleneFull size table
Many literature reviews have indicated that polypropylene microplastics are among the least studied microplastics in laboratory ecotoxicity studies [77,78,79]. This is surprising given the fact that polypropylene is the second largest European and global plastic resin in terms of production volume [80, 81] and polypropylene microplastics are among the most common found in the environment [77]. For example, of the total of 157 peer-reviewed ecotoxicity articles published by 2018 with 612 different microplastics on aquatic organisms, only 12.1% included polypropylene [33]. Our literature search using the keywords “polypropylene” and “microplastics” (September 2021) resulted in 688 hits within the category Environmental Sciences of the Web of Science knowledge base and 2003 hits within ScienceDirect. For the keyword combination “polypropylene” and “microplastics” and “toxic” the number of hits was 59 and 1175 for Web of Science (WoS) and ScienceDirect, respectively (Table S1 Supplementary information). After abstract inspection, in total 27 studies were identified as ecotoxicity studies including species being relevant for this review. An additional 3 were found in the review by De Sá et al. [33] dealing with microplastics from fishing ropes which were not identified during our search in WoS or ScienceDirect. Interestingly, 44% of studies included in our review were recently published (2021) which indicates that the number of studies on polypropylene microplastics has increased significantly (Supplementary information Table S1, Table 2).
Three types of polypropylene microplastics have been studied in terms of their shapes and sources: fibres from the cutting of fishing rope; fragments obtained from cryo-milling of different products; and purchased fragments (pellets) from polymer producing companies. A comparative analysis of the reported adverse effects across the test species for exposure concentrations and with other microplastic polymer types is very difficult, because the various studies have used a range of test materials of different dimensions (fibres: length 20–1000 μm, width 15–200 μm; fragments: diameter ~ 10–3000 μm), and according to different concentration metrics (particle mass/volume, particle number/volume, particle mass/mass sediment or soil, particle mass/surface area of agar plate) and toxicity endpoints. Also, it was not possible to find a difference between the effects of polypropylene fibres and fragments, although it was suggested previously that the shape of the microplastics has a predominant role in some adverse effects, with fibres showing greater toxicity [49, 50].
Nevertheless, it can be concluded that both polypropylene fibres and fragments have the potential to induce adverse effects on organisms at concentrations that can already be found in the environment [49, 52, 53, 55], although some studies also tested unrealistically high microplastics levels [50] (Table 2). The environmental relevance of some of the test concentrations is difficult to assess, as the measurement metrics are different from those most commonly reported in monitoring studies (i.e., particles volume− 1 or km− 2) [82]. The potentially adverse effects induced by polypropylene microplastics are similar to those induced by other types of microplastics [33], and include: their retention in the gut; decreased feeding and growth rates; changed metabolic rates and metabolic processes; changes in moult process; decreased reproduction; stress induction, oxidative stress and antioxidant responses; induction of immune responses; alteration of the gut microbiome; and (very rarely) mortality (Table 2). However, as Rochman et al. [83] emphasised, microplastics represent a diverse suite of contaminants that show a range of different molecular structures, monomer compositions, chemical additives, sizes, shapes and colours, with many of these potentially involved in their toxicity potential. It is therefore imprecise to generalise toxicity data across microplastics types, even within the same polymer group. This implies the need for new experimental data for polypropylene microplastics from medical masks as currently only such study exists [48]. In particular, such studies should be directed towards the investigation of microplastics from weathered medical masks. Weathering affects not only the surface properties of the particles, but also the release of the additives and the plastic-derived intermediates, as well as the sorption of other environmental pollutants [18, 84, 85]. This can lead to alterations to the behaviour of the microplastics and to their bioavailability to organisms (i.e., the form in which they are available for organisms to ingest), and ultimately to their hazard potential [18, 86, 87].
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