key: cord-0811508-8cj6q5zd
authors: Liu, Ze; Wang, Jianqun; Yang, Xuetong; Huang, Qian'en; Zhu, Kecheng; Sun, Yajiao; Van Hulle, Stijn; Jia, Hanzhong
title: Generation of environmental persistent free radicals (EPFRs) enhances ecotoxicological effects of the disposable face mask waste with the COVID-19 pandemic()
date: 2022-02-18
journal: Environ Pollut
DOI: 10.1016/j.envpol.2022.119019
sha: 63cfcbc37482af864afade558db019e29c41a304
doc_id: 811508
cord_uid: 8cj6q5zd

A large amount of disposable plastic face masks (DPFs) is produced and used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which results in an inevitable consequence of the dramatic increase of DPFs waste. However, the impact of DPFs exposure to the environment on their toxicity is rarely considered. In this study, a range of 76–276 items/L microplastics (MPs) was detected in the DPFs leachates, and fibrous ([Formula: see text] 80.3%) and polypropylene (PP, [Formula: see text] 89.2%) MPs were dominant. Co, Cu, Ni, Sr, Ti and Zn, were commonly detected in all leachates of the tested DPFs. Organics, such as acetophenone, 2,4-Di-tert-butylphenol, benzothiazole, bisphenol-A and phthalide, were found in the DPFs leachate, which were including organic solvents and plasticizer. Besides, we first found an emerging environmental risk substance, namely environmentally persistent free radicals (EPFRs), was generated in the DPFs leachates. The characteristic g-factors of the EPFRs was in a range of 2.003–2.004, identified as mixture of carbon- and oxygen-centered radicals. By means of in vitro toxicity assay, the DPFs leachate were confirmed to cause cytotoxicity and oxidative stress. Significantly, it is found that the formed EPFRs could contribute more toxic effects. Furthermore, when compared to N95 respirators, the tested surgical masks tend to release more MPs, leach more metals and organics, and generate more EPFRs. Surgical masks were thus showed higher risk than N95 respirators after exposure to water. This work highlights the importance of understanding the chemical complexity and possible toxicity of DPFs for their risk assessment.

color, morphology and types (SI, section S2). In this study, only MPs with a diameter 140 of less than 5 mm were measured by visible observation. The identification of 141 polymers was analysis with using Fourier Transform Infrared Spectroscopy (FTIR) 142 with the Attenuated Total Reflectance (ATR) accessory (Thermo Nicolet FTIR, 143

Nexus) (Wang et al., 2020b) . Throughout the study, several laboratory quality control 144 measures were implemented to circumvent the contamination of samples from plastic 145 material and the environment and more information has been provided in the Section 146 S3 in the supplement. 147 148

Organics in the leachates was analyzed by gas chromatography-mass spectrometry 150 (GC-MS, an Agilent 6890 GC Series coupled to a Hewlett Packard 5973 mass 151 selective detector) using non-target and target screening method (SI, section S4 and 152 Table S3 -S4) after leachates extraction. More details on leachate extraction for GC-153 MS analysis has been shown in the Supporting Information (SI, section S5). NIST 154 library was used for organics qualitative analysis by extracting best hits. 155

For the analysis of trace metals in the DPFs leachate, a Thermo Scientific ICAP 7200 156

Inductive Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analyzer was 157 used. External calibration was performed for the quantification of trace metals. More 158 details on methods and quality control for ICP-OES analysis can be found in the 159 appendix (SI, section S6 and Table S5 ). 160

The control samples were set without adding DPFs for comparing with the leachate 161 samples, which were also prepared with deionized water. The control samples were 162 subjected to a non-target screening by ICP-OES and GC-MS for identifying the 163 chemical components. All compounds detected in the control samples were removed 164 from data set when results discussing. 165 166

The leachates were detected by EPR analysis for quantifying the possible generated 168

EPFRs. Specifically, the obtained leachate was first transferred and placed in an I.D. 169 quartz tube. Afterwards, the quartz tube with the leachate was inserted into the cavity 170 of the EPR instrument for EPFRs analysis. EPR measurements were conducted with 171 using a Bruker EMXmicro-6/1/P/L spectrometer (Karlsruhe, Germany) at room leachate sample was taken and mixed with 500 μL DMPO solution (100 mM), 500 μL 175 DMPO/DMSO solution (100 mM) and 500 μL TEMP solution (of 100 mM) for 176 detecting  OH, O2 − and 1 O2, respectively. Then, the obtained mixture was used for 177 the measurement of EPR. The parameters of EPR measurement were based on our 178 previous study , and also provided in the supplement (SI, To identify the cytotoxicity induced by EPFRs, N-acetyl-l-cysteine (NAC, 5 mM, 197

Sigma-Aldrich, St. Louis, MO) as a free-radical scavenger was used (Aldini et al., 198 2018), which has non-impact on the cell viability (Jia et al., 2020 results of microscopy analysis showed that only fibrous MPs (80.3~97.4%) and 253 particle MPs (< 10%) were found in the DPF leachates and their main colors were 254 blue and transparent (SI- Figure S2 ). It appeared that no obvious differences 255 characteristics of MPs were found in the leachates for the DPFs which were same 256 type but from different brand. presented in the leachates. The abundance of MPs of < 40 μm in leachates tended to be 279 increased with time, but the increase was still not significant (p=0.14). According to the 280 microscope observation, only particles and fibrous MPs were found in the leachates and 281 more than 90% MPs were fibrous in the leachates, which was also consistent with the 282 SEM analysis. Interestingly, fibrous and particles MPs were all reported to be 283 Various heavy metals were identified in the DPFs leachates. As shown in Table 1 leachates for Mask 1, 2, 3, 6 and 7 and Mask 1, 2, 3, 4 and 5, respectively. 300

Comparatively, for N95 respirators, only less than 1 μg/L Cr was found in the 301 leachates of Mask 7. Meanwhile, the concentrations of all detected heavy metals in 302 the surgical masks leachates were commonly higher than those in N95 respirators 303

leachates. It indicates that the amount of DPFs used and discarded could lead to a 304 potential environmental risk that more heavy metals possibly enter the environment 305 via leaching from DPFs waste. It is reasonable to speculate that surgical masks waste 306 more easily leach heavy metals into the aquatic environmental than N95 respirators 307 waste. Furthermore, some of these heavy metals are potentially bio-accumulated by 308 organisms and could cause acute and chronic adverse effects on human health by 309 ingestion, dermal contact and inhalation exposure. As such, contact allergy to Cr, Ni Therefore, the huge amount of DPFs waste generated during the COVID-19 pandemic 316 may not only cause that more heavy metals are potentially released into the 317 environment and accumulated by organisms, but also give rise to a risk of exposure of 318 heavy metals to human by regularly wearing DPFs. 319 (Table  362 2) may have an adverse impact on the environment and human health when leaching 363 from DPFs. 364

N95 respirators leachates from different brands, as a consequence, surgical masks 366 leachates tend to contain more MPs, heavy metals and organics than N95 respirators 367 leachate. Moreover, for DPFs of the same type but from different brands, no 368 significant differences on the characteristics of the released MPs and leached 369 components were observed in their leachate. 370

As hypothesized, EPFRs is suspect to be generated during DPFs exposing in water. 373

To test this hypothesis, as there were no significant differences on the characteristics 374 of the leachates for the DPFs which were the same type but from different brands 375 based on the above results of the DPFs leachates, one kind of surgical masks (Mask 1) 376 and one kind of N95 respirators (Mask 7) was used to analyze the possible generation 377 of EPFRs. The DPF leachates were detected by EPR after 5 d-, 10 d-and 15 d-378

soaking (at room temperature ~25℃) and the obtained EPR spectra are shown in 379 Therefore, it could be inferred that chemical bond cleavage is not the dominant 409 process for EPFRs formation on the DPFs. 410

It was observed that g-factor and spin density in DPFs were slightly decreased as a 411 function of soaking time (from 2.00317 to 2.00338, Figure 3A ). This could be due to 412 a higher contribution of carbon-centered radicals which was suspected to lead to a 413 decreased g-factor due to the electrons transfer of organic additives. The slightly 414 decreased spin density seems to indicate that the generated EPFRs decayed slowly. Therefore, it can be concluded that EPFRs are constantly produced during the surgical 427 mask soaking process and retained in the leachates. Meanwhile, the reaction of 428

EPFRs occurred during surgical mask exposing to water is able to generate reactive 429 radicals (e.g. O2 •− ), which may cause more potential environmental risks. determined. According to the results shown in Figure S6 , cytotoxicity was suppressed 483 by using NAC (85.3% for 15 d-leachates with adding NAC). As discussed above, the 484 generated EPFRs induced the generation of reactive radicals (e.g. O2 •− and • CH3 in 485 

In view of the ecotoxicity of the DPF waste exposure to the aquatic environment, our 504 investigation and assessment with taking surgical masks and N95 respirators exposing 505 in water for 15 days is certainly not representative. Nevertheless, as far as we know, it 506 represents the most comprehensive study of evaluating on the potential risk of DPFs 507 waste exposing to the environment. In previous studies, DPFs leachate is mainly 508 restricted to the analysis chemical composition leaching and MP releasing. In this 509 study, in addition to tentatively identify MPs, metals and organics, we also focus on 510 the generation of EPFRs, with providing data on the DPFs leachate triggering an 511 oxidative stress response. The results of this study indicate that the leachates 512 containing chemicals and EPFRs inducing unspecific toxicity are prevalent in DPFs 513 waste, especially during the COVID-19 pandemic in which the widely use of DPFs is 514 unprecedented. It is worth to highlight that the aim is not to draw a conclusion 515 regarding that the wide use of DPFs has a potential impact on health, but to assess the 516 concentrations of the leaching of the DPFs to the overall pollutions and to evaluate 517 the risks of the excessive exposure of DPFs waste to the aquatic environment during 518 and after the COVID-19 pandemic from a new perspective: the basis of reducing 519

DPFs' environmental impact is to acknowledge their chemical complexity and 520 possible toxicity. However, more accurate methods of MPs counting and 521 identification of plastic-associated chemicals may be limited and the current detection 522 identify origin and assessing toxicity of EPFRs was challenging and hampered by the 524 lack of related valid research data. Correspondingly, the first step to address the 525 limitations is developing a standard of MPs sampling and detecting, as well as 526 scientific and comprehensive approaches for compound identification. More research 527 on the generation, transformation and occurrence of EPFRs and more bioassays 528 assessment for various of EPFRs to explore the toxic effect is helpful to address the 529 limitations. To a large extent, we need to perform effect-directed analysis to identify 530 the compounds causing the toxicity which are not only leached but also generated 531 during the DPF waste exposure to water. 532 533

This study provides new insight into the potential health risk of the disposable face 535 mask waste with the COVID-19 pandemic by not only leaching metals and organics 536 and releasing MPs but also the formed EPFRs. More MPs were released to the 537 leachates from the DPFs with the increasing of the exposure time, for example, the 538 abundance of MPs of surgical mask was enhanced from 28 items/L in 5 d-leachate to 539 231 items/L in 15 d-leachates. It is found that MPs were fragmented with soaking 540 time as the abundance of smaller MPs (e.g. < 40 μm) in leachates was increased. 541

Metals (including Co, Cu, Ni, Sr, Ti and Zn) and organics (including acetophenone, 542 DTBP and bis(2-ethylhexyl) phthalate) were commonly presented in the leachates. 543

Furthermore, more various metals (like Cd, Cr, Mn and Pb) and organics (like tributyl 544 acetylcitrate and benzaldehyde) were found in surgical masks leachates than in N95 545 respirators leachates. From the perspective of plastic-related chemicals, the DPFs was 546 confirmed as a source of MPs, metals and organics, and hence could cause an 547 ecosystem risk after exposing to the aquatic environment. Significantly, the carbon-548 centered EPFRs were detected to be generated on the DPFs, which could be attributed 549 to the electron-transfer reaction between metals and organics contained in the DPFs. 550

In order to estimate the potential adverse effects of the DPF waste, we further 551 conducted in vitro toxicity assessment and demonstrated that the DPFs leachate could 552 cause cytotoxicity and oxidative stress. Noteworthy, the generated EPFRs enhanced 553 the toxic effects of DPFs leachates. Overall, this work provides detailed data and 554 systemic estimation to the generated EPFRs after DPFs exposure to water. These 555 findings contributed to bridging the knowledge gap in better understanding the 556 environmental effects and toxic risk of DPFs waste exposure to the aquatic 557 environment. 558 559

Methodology on MPs characterization, organics and metals analysis in the DPFs 561

leachates. Information of the tested DPFs, quality assurance and quality control for 562 sampling, pH of the leachates and EPR analysis. Further data on the characteristics of 563

MPs in the DPFs leachates, FTIR-ATR results, SEM images of DPFs, GC-MS 564 analysis, EPR spectra and in vitro toxicity. 565

Sections S1-S6, Tables S1-S7, Figures S1-S7. 566

Ze Liu, Jianqun Wang, and Xuetong Yang contributed to the work equally and were 568 regarded as co-first author. This study was funded by the National Natural Science 569 

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