Products released from surgical face masks can provoke cytotoxicity in the marine diatom Phaeodactylum tricornutum

Products released from surgical face masks can provoke cytotoxicity in the marine diatom Phaeodactylum tricornutum Marta Sendra a, ⁎, Araceli Rodriguez-Romero b ,MaríaPilarYeste c , Julián Blasco d , Antonio Tovar-Sánchez d a Department of Biotechnology and Food Science, Faculty of Sciences, University of Burgos, Plaza Misael Bañuelos, 09001 Burgos, Spain b Department of Analytical Chemistry, Faculty of Marine and Environmental Sciences, Marine Research Institute (INMAR), University of Cadiz, Cadiz, Spain c Department of Material Science, Metallurgical Engineering and Inorganic Chemistry, University of Cádiz, Spain d Department of Ecology and Coastal Management, Institute of Marine Sciences of Andalusia (CSIC), Campus Río S. Pedro, 11510, Puerto Real, Cádiz, Spain HIGHLIGHTS •Surgical face masks (FMs) can be a source of Mn, Zn and Ni in marine environments. •The release of chemicals and microfibres from FMs to water depends on the stage of disintegration. •Water from face masks provoke cellular changes in the diatom P. tricornutum. •Water from fragments showed a higher toxicity than water from a whole face mask. •Photosynthetic apparatus was the most sensitive response tested in the microalgae. GRAPHICAL ABSTRACT ABSTRACTARTICLE INFO Editor: Thomas Kevin V Surgical face masks are more present than ever as personal protective equipment due to the COVID-19 pandemic. In this work, we show that the contents of regular surgical masks: i) polypropylene microfibres and ii) some added metals such as: Al, Fe, Cu, Mn, Zn and Ba, may be toxic to some marine life. This work has got two objectives: i) to study the release rate of the products from face masks in marine water and ii) to assess the toxicity in Phaeodactylum tricornutum of these by-products. To achieve these two objectives, we performed release kinetic experiments by adding masks in different stages of fragmentation to marine water (i.e. whole face masks and fragments of them 1.52 ± 0.86 mm). Released microfibres were found after one month in shaking marine water; 0.33 ± 0.24 and 21.13 ± 13.19 fibres·mL −1 were collected from the whole and fragmented face masks, respectively. Significant amounts of dissolved metals such as Mn, Zn and Ni, as well as functional groups only in the water containing the face mask fragments were detected. Water from both treatments was employed to study its toxicity on the marine diatom. Only the water from the face mask fragments showed a significant, dose-dependent, decrease in cell density in P. tricornutum; 53.09 % lower than in the controls. Although the water from the face mask fragments showed greater effects on the microalgae population than the water from the whole face mask, the latter treatment did show significant changes in the photosynthetic apparatus and intrinsic properties of the cells. These results indicate that during fragmentation and degradation face masks a significant chemical print can be observed in the marine environment. Keywords: Ytotoxicity Pandemic secondary effects Phaeodactylum tricornutum Phytoplankton pollutants released PPE degradation 1. Introduction During the COVID-19 pandemic, the use of personal protective equipment (PPE), such as face masks, has contributed to significant advances in controlling the spread of the SARS-CoV-2 virus (Howard, 2020;Worby Science of the Total Environment 841 (2022) 156611 ⁎Corresponding author. E-mail address: [email protected] (M. Sendra). http://dx.doi.org/10.1016/j.scitotenv.2022.156611 Received 4 May 2022; Received in revised form 6 June 2022; Accepted 6 June 2022 Available online 9 June 2022 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/). Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv and Chang, 2020). Estimates of the annual demand for PPE has increased 207 times between 2019 and 2020 with a value of about US $166 billion on the global market (Roberts et al., 2021). Currently, face masks are considered one of the main measures to avoid the progression of the virus and their use is a legal requirement both indoors and outdoors in public spaces in many countries. Data indicate a consumption of approximately 129 billion face masks for 7.8 billion people across the globe per month during the year 2020 (Kalina et al., 2020). To deal with the unexpected and enormous amount of waste generated, an appropriate strategy to manage this threat is mandatory to avoid potential environmental impacts. The situation has led to illegal plastic waste disposals (increasing by280 % worldwide), these inherently alter the adjacent natural habitats (Reportlinker, 2021). From the beginning of the pandemic, a high number of face masks were already found in terrestrial and aquatic systems (Ammendolia et al., 2021). Chowdhury et al. (2021) predicted that an amount of between 0.15 and 0.39 million tonnes of PPE waste entered the ocean during the first year of the pandemic. This was observed in different places; for example, >105,000 t of face masks per month are dumped into the environment by Africans (Benson et al., 2021a, 2021b), compiled monitoring studies on COVID-19 PPE plastic waste in urban and natural areas, and found a density of PPE from 0.001 items·m −2 up to 0.1 items·m −2 in beach transects. Face masks have already been found in natural compartments, reaching >70 surgical face masks along 100 m of shoreline, as reported by the Oceans Asia Organization in Hong Kong. For example, 54 items·m −2 were found in 40 days on sandy and rocky beaches in Bushehr (Iran) (Akhbarizadeh et al., 2021). Plastic polymers are the main composition of surgical face masks. The daily use of face masks and an improper management of their disposal make the policies promoted before the COVID-19 pandemic concerning plastics more complicated to carry out (such as the Basel Convention, United Nations Convention on the Law of the Sea, International Convention for the Prevention of Pollution from Ships, Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection and the United Nations Global Partnership on Marine Litter). Due to their composition, surgical face masks are a large source of plastic pollution and the additives associated to their manufacturing processes such as: stabilisers, antioxidants and UV stabilisers, plasticisers, curing agents, brominated flame retardants, biocides, blowing agents, fillers, inorganic pigments, organic pigments, heat stabilisers, soluble colorants, reinforcements and slip agents (Sendra et al., 2021a, 2021b) must be taken into consideration too. It has been demonstrated in a recent work by Liu and Mabury (2021) that the three layers of a surgical face mask are composed of chemical additives, used to maintain their high quality, that are plastic-containing products such as synthetic antioxidants, synthetic phenolic antioxidants and organophosphate antioxidants. In a recent work by Sendra et al. (2022) it was revealed that surgical face mask fragments in freshwater and under conditions that included UV-C for 7 days are able to leach: i) inorganic (Fe, Cu and Zn) and ii) organic bycompounds (pyridine 2,4,6-trimethyl, 1-dodecanamine N,N-dimethyl and alanine N-methyl-N-methoxycarbonyl-undecyl ester) into water. These soluble compounds leave a chemical footprint in the water that could trigger unwanted effects within the aquatic ecosystem. Moreover, when face masks reach the natural environment, environmental agents initiate their degradation and fragmentation processes that reduce the mask into smaller pieces called microplastics (MPs < 5mm;(Thompson et al., 2004)) and nanoplastics (NPs <100 nm; (Jambeck et al., 2015)) in the form of fibre fragments. In a recent work by Ma et al. (2021) it was reported that more than one billion NPs and MPs were released from each surgical or N95 face mask in 1 L of ultrapure water over 3 min of shaking. Irregularly-shaped particles were found in the water collected, ranging from 5 nm to 600 μm, but most of them were nano scale sized <1μm. These authors also found differences in the fibres released among the layers. The middle layers of the masks released more particles than the outer and inner layers. Furthermore, De Felice et al. (2022) have also shown that a during just one wash, face masks released an average of 284.94 ± 73.66 microfibres, independently of the fabrics. These by-products (inorganic, organic and fibres) released and/or formed from face mask degradation in water could affect aquatic organisms. Therefore, a study concerning the effects of face-mask products should be performed to protect the environment and aquatic ecosystems. However, very few data are available about the toxicity and effects of face masks on aquatic organisms. The work by Sendra et al. (2022) has revealed that face mask products can upand down-modulate several biological processes in zebrafish larvae by modulating differentiated expressed genes found in the results of an RNA-seq analysis. Among the most modulated biological functions, reproductive processes were the most affected. The study of the potential and real effects of face masks is relevant for the most representative groups of organisms. Nevertheless, ecotoxicological studies concerning primary producers are also very important due to their ecological relevance in the food web. Some changes in microalgae communities could provoke unwanted effects in higher organisms. It was demonstrated in the work by Ma et al. (2021), that the microfibres released from face masks may be adsorbed by diatoms. This could indicate that the interactive force between the cells and micro and nanofibres could be similar to nanoscale interaction (Nel et al., 2009). The literature available about primary producers has revealed that they can adand absorb nanoplastics thus provoking unwanted effects at cellular and molecular levels, especially in the photosynthetic apparatus (Déniel et al., 2020; Hazeem et al., 2020;Natarajan et al., 2020;Sendra et al., 2019;Venâncio et al., 2019;Yang et al., 2021). Given everything that has been stated above, the main aims of thiswork are i) to identify and quantify the inorganic compounds released from a whole surgical face mask as well as fragments of a face mask that have been agitated in marine water for one month to mimic natural processes, and ii) to assess the cytotoxicity of the water from whole or fragmented face mask experiments for the marine diatom Phaeodactylum tricornutum. 2. Materials and methods 2.1. Fragmentation and degradation of surgical face masks The experimental fragmentation and degradation of face masks is shown in Fig. 1.Commercialsurgicalfacemasks(LyncMed,Beijing, China) sold in a local pharmacy in Spain were used for this study. Two conditions simulated two different stages of face-mask disintegration; i) a whole surgical face mask submerged in marine water, and ii) fragments of a face mask floating on and/or submerged in marine water. Both conditions did not consider the fasteners of the face masks. The surface marine water used for the experiments was collected by a boat in a clean offshore area of Cadiz Bay (Spain; 36.499109 N, −6.253689 W) and filtered using two consecutive filters of 0.47 and 0.22 μmfilter prior to the experiments. The physico-chemical characterisation of the marine water collected was pH: 8.1, the salinity recorded with a seawater refractometer HI 96822 (HANNA) was 36 psu and organic matter measured as TOC with a TOC analyser Shimadzu TOC-V CHS was 1.5 mg L −1 . The fragmentation of the face mask was performed with a scissor previously cleaned with ethanol. Each face mask was cut into pieces of approximately 2 mm. Three replicates were established in each condition: controls (marine water), a whole single face mask and face mask fragments. A single face mask (whole or fragmented) was placed to an acid-cleaned Erlenmeyer with 2 L of filtered marine water. The whole face masks were dunked into the marine water with a glass rod. The Erlenmeyer flasks were connected to a tube of PVC (D.I. × D.E. = 3/ 16 in. × 5/16 in.) to make the sampling over time easier and covered with parafilm. The Erlenmeyer flasks were in agitation (50 rpm) and in a photoperiod of 12 h of light/12 h of dark at room temperature (20 ± 2 °C). A total of 10 samples of water, according to each treatment, were collected over 1 month, seven of them over the first week, and the other three once a week. 30 mL were collected for inorganic analysis and 15 mL for analysis of the fibres released. M. Sendra et al. Science of the Total Environment 841 (2022) 156611 2 2.2. Analysis of surgical face masks and water collected during the experiments The analysis and behaviour of the surgical face masks are explained in this section and the process is shown in Fig. 2. The different layers of the surgical face masks were analysed prior to the experiment. The materials of the different layers were analysed using Fourier transformed infrared spectroscopy (FTIR) through the Bruker Alpha System. The inorganic compounds of the blue and white layers of the face masks (n:5) were analysed by ICP-MS (ICAP-Q, Thermo-Fisher) after acid microwave digestion (CEM model Mars 5) according to the SW-846 EPA Method 3051A (USEPA, 1987). Furthermore, the nominal size of the face-mask fragments was measured by an optical microscope with an n= 200. After the whole face mask or fragments had been submerged in marine water for one month the number of fibres, the inorganic elements and functional group released from the whole face mask and face mask fragments into the water were measured. 15 mL of water was collected in a flask for the analysis of the fibres and the fragments. The samples were filtered using a black polycarbonate filter of 0.47 μm. The black filter was measured in an optical microscope (5×) and all fragments and fibres were counted as well as the area and length of the fragments and fibres. Samples of the inorganic compounds were collected in a 30 mL metal free flask with a syringe and a 0.22 μmfilter (both previously washed with HNO 3 ). The samples were kept at 4 °C until analysis. The concentrations of metals (i.e. V, Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb) were analysed using ICP-MS (ICAP-Q, Thermo-Fisher) after a pre-concentration using the seawater pre-concentration system (seaFAST). The accuracy of the analysis was established using Seawater Reference Material for trace metals (CASS-6, NRC-CNRC) (recoveries of 109 %, 88 %, 100 %, 90 %, 90 %, 96 %, 100 %, 99 %, and 121 % for V, Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb, respectively were obtained). These last samples filtered by 0.22 μm were also analysed by Fourier-transformed infrared spectroscopy (FTIR) to study the functional groups released into the water from the whole masks and fragments of face masks after one month. 2.3. Microalgae toxicological assay After the analysis of the behaviour of the face masks (with blue and white layer) in marine water, 1350 mL was put in an Erlenmeyer flask to perform the toxicological analysis. This water was filtered using a 0.22 μm cellulose filter and 4 dilutions (with a dilution factor of two) of the marine water that had been in contact with the face masks was performed. Therefore, a total of 5 treatments and the controls were considered. The marine diatom Phaeodactylum tricornutum (Bohlin, 1987) was obtained from the ICMAN Marine Microalgae Culture Collection (IMMCC). The microalgae selected was chosen because it is a standard species used in toxicology tests (ISO 10253:2006). Cells were grown in a filtered (0.2 μm) marine culture medium (pH: 8.2) and F/2 marine medium without EDTA for two weeks prior to the experiment (Fábregas et al., 2000). The experimentswere performed in 100 mL Erlenmeyer flasks (total volume of the experiment 30 mL) under continuous visible light (300 μE −2 s −1 ). Growth inhibition bioassays were developed following the OECD procedure (OECD, 1994), in order to determine the microalgae density after 72 h. The initial cell density of microalgae was 10 4 cells mL −1 . All treatments and controls were carried out in triplicate. 2.4. Flow cytometry analysis The flow cytometer used in this work was a BD Accuri C6 equipped with an emission laser at 488 nm and different detectors: side scatter (SSC), forward scatter (FSC), detector FL1 [band pass (BP) 533/30nm], FL2 [band pass (BP) 585/40nm] and FL3 FL3 <670 nm. Fig. 1. Face mask characterisation and experimental design. Chemical and ecotoxicological experiments. M. Sendra et al. Science of the Total Environment 841 (2022) 156611 3 Fig. 2. Chemical characterisation and behaviour of face mask products after 1 month in natural marine water. Panel A shows FTIR of the three layers of surgical face masks. Panel B shows thenominal size of the facemask fragments used in this work. Panel C and D show the inorganic elements of the face masks and released into themarinewater after 1 month (asterisks represent significant differences in respectto the controls; ANOVA post hoc Dunnett´stest p<0.05). Panel E shows the functional groups found in the marine water exposed to the face masks over 1 month by FTIR. Panel F shows the total items (fragments or fibres) released into the marine water in a mL and Panel G shows the length of microfibres released into the marine water after one month of exposure. M. Sendra et al. Science of the Total Environment 841 (2022) 156611 4 All the responses measured were collected after 24, 48 and 72 h. The cell density of the microalgae population was determined using SSC related to cell complexity and the long pass filter of 670 nm (FL3) related to autofluorescence of chlorophyll a(Shapiro, 2005). Furthermore, the volume of microalgae was determined using a FSC detector related to microalgae volume. The data for cell density was provided by the flow cytometer as cells·μL −1 . The data for cell complexity, cell volume and Chl a is given as the median of each response in accordance with the population of microalgae selected. Production of intracellular ROS (reactive oxygen species: superoxide, hydroxyl and hydrogen peroxide) was quantified using the 2′-7′- dichlorofluorescein diacetate (DCFH-DA), following the procedures of Stachowski-Haberkorn et al. (2013) and Sendra et al. (2017a, 2017b). In brief, the samples were incubated with the fluorophore at a final concentrationof2mMandindarknessatroomtemperaturefor over 20 min. The ROS present in the cells was measured using an FL1 detector[bandpass(BP)533/30nm]asthepositivepercentageof microalgae with ROS in relation to the total microalgae population. 2.5. Photosystem II analysis The effective quantum yield ofphotosynthetic energy conversion in PSII (EQY), total chlorophyll aconcentration (μg·L −1 ) and apparent electron transport rate (ETR) were measuredfluorometrically using a Phyto-PAM instrument (Heinz Walz GmbH) equipped with an ED-101 US/MP Optical Unit (Schreiber et al., 1995). The samples were acclimated in darkness for 15 min before the analysis. 24 h Control 16 8 4 2 1 Cell density (Cells mL -1 ) 0 2e+4 4e+4 6e+4 8e+4 1e+5 48 h Control 16 8 4 2 1 Cell density (Cells mL -1 ) 0 1e+5 2e+5 3e+5 4e+5 5e+5 72 h Marine water dilutions Control 16 8 4 2 1 Cell density (Cells mL -1 ) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 1e+6 2e+6 Water from whole face mask Water from fragments of face mask AB * * 24 h Control 168421 % of ROS 0 20 40 60 80 100 48 h Control 16 8 4 2 1 % of ROS 0 20 40 60 80 100 72 h Control 16 8 4 2 1 % of ROS 0 20 40 60 80 100 * * * ** * * * Marine water dilutions Fig. 3. Cell density and percentage of intracellular ROS of Phaeodactylum tricornutum after 24, 48 and 72 h of exposure to the water from a whole face mask and water from fragments of a face mask; Panels A and B respectively. Asterisks (*) show significant differences with respect to the controls (ANOVA test with Dunnett’sposthoc;p<0.05). M. Sendra et al. Science of the Total Environment 841 (2022) 156611 5 3. Results 3.1. Face mask characterisation and behaviour in marine water Fig. 2 shows the chemical composition of the surgical face masks employed in this work, as well as their behaviour in natural marine water over time. Thethreelayersofthesurgicalfacemaskareformedbypolypropyleneas a plastic polymer (Fig. 2.A). The face masks were studied either whole or in fragments in this work, the fragments had a nominal size of 1.52 ± 0.86 mm (Fig. 2.B). Further chemical characterisation was performed to identify the inorganic elements in the white and blue layers of the face masks. Some elements such as Fe, Cu and Ba showed a higher concentration in the blue layer in respect to the white layer with values of 29.22 ± 7.90 ppm for Fe, 41.61 ± 18.16 ppm for Cu and 166.00 ± 70.85 ppm for Ba in the blue layer (Fig. 2.C). On the other hand, Zn (7.37 ± 5.36 ppm) was only detected in the white layer. Focusing on these elements present in the surgical face masks,thewaterfromwholeandthefragmentsoffacemaskswasanalysed after 1 month in natural seawater and constant shaking to study the leaching processes from the facemasks to the water (Fig. 2.D and Table S1). No significant differences were found between the controls and both treatments with face masks in the marine water. However, there were significant differences with regard to three elements (Mn, Zn and Ni) between the water from the controls (2.7 ± 0.15, 3.07 ± 0.35 and 0.38 ± 0.01 ppm for Mn, Zn and Ni respectively) with respect to the water that had contained the fragments of the face masks (3.55 ± 0.15, 4.03 ± 0.21 and 0.45 ± 0.02 ppm for Mn, Zn and Ni respectively) after 28 days (p<0.05). The released kinetic of the three metals (Mn Zn and Ni) was studied over time (four weeks; 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days). The metals released were measured in water from the whole and the fragments of the face masks and compared to the control concentrations, which are considered as the background level (Fig. S1). The three elements were only released from the face mask fragments. Mn showed higher concentrations in the water from day 3, whereas an increase of Zn was reported in the third and fourth weeks. Finally, Ni showed an increase (1.18 fold change in relation tocontrols; with a concentration of0.45 ± 0.02 ppm) in relation to the background levels at 21 and 28 days. Fig. 2.E shows the FTIR spectra of the marine water controls, and water from the whole and the fragments of face masks. The marine water controls did not show any peaks in the spectrum when the samples were measured. However, both treatments (water from the whole face mask and water from the fragments of the face masks) indicated the presence of alcohols. The characteristic bands are at 3400 cm −1 (O\\H stretching), 1120 cm −1 (C-OH stretching) and 610 cm −1 (O\\H bending). In addition, amine compounds may also be contained in the masks. The characteristic bands are at 1635 cm −1 (N\\H bending) and 1120 cm −1 (C\\N bending). Fig. 2.F shows the total fragments and fibres released into the water for both face mask treatments. Total fragments showed a similar number of fragments released by mL; 0.53 ± 0.23 and 0.56 ± 0.04 fragments·mL −1 respectively. However, the number of fibres was significantly higher in the water from the fragments of the face masks (21.13 ± 13.19 fibres·mL −1 ) than the water from the whole face masks (0.33 ± 0.24 fibres·mL −1 ). With respect to the length of the fibres found in both treatments (Fig. 2. G), the water from the whole face mask had fibres of 0.22 ± 0.3 mm, while the length of fibres from the water from the fragments of the face masks was 0.93 ± 0.28 mm. 3.2. Cytotoxic responses of the microalgae exposed to face mask products Fig. 3.A shows the cell density of a Phaeodactylum tricornutum population exposed to water from a whole face mask and water from fragments of a face mask over 24, 48 and 72 h. The microalgae population exposed to water from the whole face mask did not show any significant decrease in cell density with respect to the controls over the time of the experiments (p>0.05). However, the microalgae population exposed to the water from the fragments of a face mask showed a dose-dependent response when the water was less diluted. A significant decrease in cell density was observed after 24 and 48 h of exposure to this water with the face mask fragments (p<0.05). After 48 h of exposure, the microalgae population showed a cell density 7.11 times lower than the controls. However, this trend was not observed after 72 h under face mask fragments treatment. This indicates a recovery in the cell population after a chronic exposure (72 h) to fragments of a face mask; (Fig. 3.A). In some bars of the graphs, we can find high standard deviation. This is due to the biological variability among biological samples. These differences are higher when the effects of the treatments are not quite severe. However, when a high toxicological response is found in microalgae this variability is lower due to the biological response in microalgae population is similar. Fig. 3.B shows the percentage of intracellular ROS in Phaeodactylum tricornutum populations exposed to water from the whole face mask and water from the fragments of a facemask over 24, 48 and 72 h. An increasing trend in the percentage of ROS was observed after 24 and 48 h after exposure to 16 to 4 dilutions of water from a whole face mask and 48 and 72 h after exposure of water from the fragmentsof a face mask. However, this increasing trend was significant only after 72 h of exposure to water from the fragments of a face mask, it was 6.98 times higher than the controls. The percentage of ROS decreased significantly when the microalgae were exposed to treatments without dilutions, such as after 24 h of the microalgae being exposed to the water from the fragments of a face mask (21.41 times lower than controls) and 48 and 72 h of exposure to water from a whole face mask (Fig. 3.B). Fig. 4 shows the intrinsic properties of the microalgae such as cell complexity (SSC, Fig. 4.A) and cell size (FSC, Fig. 4.B). Concerning cell complexity, the microalgae exposed to the water from a whole face mask showed a trend to increase at higher dilutions of this treatment (16 and 8 factor dilutions increased significantly respect to the controls; (p<0.05). After 48 and 72 h the trend under this treatment was different showing a decrease in SSC at all factor dilutions after 48 h (p<0.05) and significant differences under 2 and 1 factor dilutions after 72 h. In relation to the changes in cell complexity of the microalgae exposed to water from the fragments of a face mask, a significant increase was found after 48 h at 8 dilution factors (p<0.05). However, a significant decrease in cell complexity was found in the microalgae exposed to no dilution of water from the fragments of a face mask (p<0.05) and 2 and 1 dilution factors after 72 h of exposure to this treatment (p<0.05). In relation to the cell size of the microalgae (Fig. 4.B), it was observed that after 24 h the microalgae decreased their cell size when they were exposed to all dilution tested of water from a whole face mask (p<0.05) and from 4 to 1 dilutions tested from the fragments of a face mask (p<0.05). A trend to increase cell complexity at higher dilutions after 48 and 72 h of exposure to both treatments was observed; however, the cell complexity decreased when the dilution factors were lower (p<0.05). Fig. 5 shows the autofluorescence of chlorophyll a(Fig. 5.A) and the total chlorophyll of the microalgae population (Fig. 5.B) exposed to both treatments of water from the face masks over time. We can observe that, in general, FL3 was a more sensitive response than total chlorophyll. After 24 h of exposure, the autofluorescence of the microalgae decreased when the microalgae were exposed to all dilutions of the water from a whole face mask (p<0.05) and from 4 to 1 dilutions of the water from the fragments of a face mask (p<0.05). This trend was observed when measuring total chlorophyll but it was only significant in the microalgae exposed to non-diluted water from the fragments of a face mask (p<0.05). After 48 and 72 h an increasing trend in both responses was observed in the microalgae exposed to higher dilutions (16, 8 and 4) in the case of the microalgae exposed to water from a whole face mask after 48 and 72 h of exposure (p<0.05), and the microalgae exposed to 16, 8, 4 and 2 dilution factors of water from the fragments of a face mask after 48 and 72 h of exposure (p<0.05). However, a different trend was observed for these two responses when the microalgae were exposed to both treatments without any dilution. A significant decrease in autofluorescence and total chlorophyll was observed after 24, 48 and 72 h of exposure (Fig. 5,p<0.05). M. Sendra et al. Science of the Total Environment 841 (2022) 156611 6 Fig. 6 shows the effects on the photosynthetic apparatus in microalgae exposed to water from face mask products through of effective quantum yield of photosystem II (EQY, Fig. 6.A) and apparent electron transport of photosystem II (ETR, Fig. 6.B). The EQY showed a significant decrease in the microalgae, which had been exposed to water from the fragments of the face mask after 24, 48, 72 h (p<0.05). For the microalgae exposed to water from a whole face mask, significant differences for this treatment without any dilution were found after 24 and 72 h (Fig. 6.A;p<0.05). With respect to the ETR responses, there were significant changes in comparison with the controls for both treatments and all dilutions after 48 h (Fig. 6.B; p<0.05). The microalgae populations recovered their ETR at all dilutionsof the waterfrom a whole face maskexcept for the treatment without dilution (p<0.05) after 72 h of exposure. The microalgae exposed to the water from the fragments of a face mask showed a significant decrease at dilutions 16, 8, 4 and 1 dilutions of this treatment (p<0.05). 4. Discussion Surgical face masks have become the personal protective equipment for medical staff and the general population during the COVID-19 pandemic situation and, like most other “rubbish”, they can end up in natural aquatic compartments, which affects their natural populations (Sangkham, 2020). The surgical face masks used in this study are fabricated of mainly plastic polymer such as polypropylene and inorganic compounds. It is not easy for most of the inorganic compounds to be released into water from the treatments involving whole face masks. However, significant amounts of 24h Control 16 8 4 2 1 Median of SSC (a.u.) 0 1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 Water from whole face mask Water from fragments of face mask 48 h Control 16 8 4 2 1 Median of SSC (a.u.) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 1e+6 2e+6 72 h Marine water dilutions Control 16 8 4 2 1 Median of SSC (a.u.) 0 1e+5 2e+5 3e+5 4e+5 5e+5 * * ***** * ** ** * 24h Control 16 8 4 2 1 Median of FSC (a.u.) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 48 h Control 168421 Median of FSC (a.u.) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 72 h Control 168421 Median of FSC (a.u.) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 Marine water dilutions ***** *** **** * * * *** * **** AB Fig. 4. Intrinsic properties of Phaeodactylum tricornutum after 24, 48 and 72 h of exposure to water from a whole face mask and water from fragments of a face mask. Cell complexity (Panel A) and cell size (Panel B). Asterisks (*) show significant differences with respect to the controls (ANOVA test with Dunnett’sposthoc;p<0.05). M. Sendra et al. Science of the Total Environment 841 (2022) 156611 7 Mn, Zn and Ni were released into the water from fragments of the face masks. These elements showed significant differences (p<0.05) in their concentrations between the water from the controls and from the water that had included fragments of face masks; indicating that these elements are released from the beginning of the experiments in the case of Mn, and from 21 to 28 days of the experiments in the case of Zn and Ni (p<0.05). In a recent study by Sendra et al. (2022) it was shown that a highly degraded face mask placed in freshwater released inorganic compounds such as B, Al, Ti, Fe, Cu and Sr over a 7-days period of degradation which included UVC. Although some of these compounds are also present in the face masks used in the present study, these compounds were not released into natural marine water under our specific conditions. Although Sullivan et al. (2021) have reported that the leaching of chemical compounds is a characteristic of this type of face mask, the period of time, the fragments of the face mask, the matrix and the degradation process are essential factors to study the leaching of chemical compounds from face masks into aquatic systems. In addition to the inorganic compounds, functional groups such as alcohols (O\\H stretching, C-OH stretching, O\\H bending) and amines (N\\HbendingandC\\N bending) were identified as the functional groups in a soluble form in our study. In the study by Sendra et al. (2022) it was shown that organic compounds did not leach from face masks into freshwater; however, three organic byproducts were recognised (pyridine 2,4,6-trimethyl, 1-dodecanamine N, N-dimethyl and alanine N-methyl-N-methoxycarbonyl-undecyl ester). The infrared spectrum of these compounds was downloaded from the NIST chemistry webbook site. Although, organic compounds were measured by 24h Control 168421 Median of FL3 (a.u.) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 Water from whole face mask Water from fragments of face mask 48 h Control 168421 Median of FL3 (a.u.) 0 2e+5 4e+5 6e+5 8e+5 1e+6 1e+6 1e+6 72 h Marine water dilutions Control 168421 Median of FL3 (a.u.) 0.0 2.0e+5 4.0e+5 6.0e+5 8.0e+5 1.0e+6 1.2e+6 1.4e+6 * * ** * * * 24h Control 168421 Chla ( gL -1 ) 0 1e+1 2e+1 3e+1 4e+1 5e+1 48 h Control 16 8 4 2 1 Chla ( gL -1 ) 0 50 100 150 200 250 300 72 h Control 16 8 4 2 1 Chla ( gL -1 ) 0 200 400 600 800 1000 Marine water dilutions *** *** * ** * *** * ** * ** * ** * * * AB Fig. 5. Autofluorescence of chlorophyll a(Panel A) and total chlorophyll content (Panel B) in Phaeodactylum tricornutum after 24, 48 and 72 h of exposure to the water from a whole face mask and water from the fragments of a face mask. Asterisks (*) show significant differences with respect to the controls (ANOVA test with Dunnett’sposthoc; p<0.05). M. Sendra et al. Science of the Total Environment 841 (2022) 156611 8 IR but they not measured by chromatography the IR spectrums with amine groups identified in the present work could coincide with the organic compounds measured by chromatography identified in freshwater by Sendra et al. (2022). Independently of the inorganic and organic compounds released into the water from the face masks, these PPEs are a source of microplastic fibres. A recent study by Saliu et al. (2021) estimated the release of microfibres from a surgical mask dumped into a marine environment. An experimental analysis was carried out under weathering experiments with artificial seawater. Data indicated that under 180 h UV-light irradiation and vigorous stirring a whole surgical face mask released about 173,000 fibres·day −1 . Furthermore, microscope and FTIR analyses carried out using face masks found on beaches have indicated similar morphological and chemical degradation as that observed in relation to the face masks used in the laboratory experiments (Saliu et al., 2021). In this study by Saliu et al. (2021) the degradation of a whole face mask was analysed; however, we studied both whole, and fragments of face masks over a long-term experiment (four weeks). In the marine environment, degradation can be enhanced by abrasion due to sediments and waves, UV radiation and microorganisms (Chubarenko et al., 2018). Therefore, the quantity of microfibres released from face masks could be underestimated under laboratory conditions in relation to natural conditions. In the present study, the microfibres were measured and observed with an optical microscope (5×); however, smaller fragments and fibres at nanoscale level may be present in the marine water employed for the experiments, and they were not identified. Once the microfibres are released into natural environments, they could suffer 24 h Control 16 8 4 2 1 EQY 0.0 0.2 0.4 0.6 0.8 1.0 Water from whole face mask Water from fragments of face mask A 48 h Control 16 8 4 2 1 EQY 0.0 0.2 0.4 0.6 0.8 1.0 72 h Marine water dilutions Control 16 8 4 2 1 EQY 0.0 0.2 0.4 0.6 0.8 1.0 ** ** ** ** * ** 24 h Control 16 8 4 2 1 ETR 0 2 4 6 8 10 B 48 h Control 16 8 4 2 1 ETR 0 2 4 6 8 10 72 h Marine water dilutions Control 168421 ETR 0 2 4 6 8 10 Fig. 6. Changes in the photosynthetic apparatus of Phaeodactylum tricornutum after 24, 48 and 72 h of exposure to the water from a whole face mask and water from the fragments of a face mask. Effective quantum yield of photosystem II (Panel A) and apparent electron transport of photosystem II (Panel B). Asterisks (*) show significant differences with respect to the controls (ANOVA test with Dunnett’sposthoc;p<0.05). M. Sendra et al. Science of the Total Environment 841 (2022) 156611 9