The surgical mask used was a 3-ply ear loop model with a melt-blown polypropylene middle layer (SM-3EP-50G, Arista Sensi, Indonesia). The 70% ethanol and 0.5% hydrogen peroxide without stabiliser solutions – semiconductor grade were obtained from Merck (Singapore) and a 3% H2O2 antiseptic solution with stabiliser was obtained from ICM Pharma (Unity Pharmacy, Singapore). Investigation showed no impact on the mask filtration efficiencies between using the 0.5% H2O2 solution prepared from the antiseptic solution versus the semiconductor grade H2O2 solution. Regular household dishwashing detergent based on linear alkylbenzene sulfonates was used for the testing (Mama Lemon, Lion Corporation, exact composition was unknown). A 5% sodium hypochlorite household bleach (FairPrice Anti-Bacterial Bleach – White, Singapore) was used as the stock solution for the bleach treatment study. The UV-C treatment was carried out in a self-built UV cabinet, fitted with two 30 W low pressure germicidal lamp (G30T8, Sankyo Denki, Japan) with wavelength centred at 254 nm. A radiometer with a UV-C probe (Delta OHM H2101.2 and LP471UVC probe, Italy) was used for measuring the UVC power density between 220 to 280 nm within the cabinet. Viral ToxGlo™ Assay (Promega, USA) was used for the anti-viral assays.
The treatment methods investigated include 1) heat treatment by boiling, immersing in hot water and steaming, 2) chemical treatment using hydrogen peroxide, bleach, ethanol and liquid detergent and 3) UV-C treatment. Unless or otherwise stated, the experiments were conducted with the full surgical mask.
The procedure for heat treatment by hot water was as follows. Water was heated using an electric kettle. The water was then transferred to a heat-insulated bowl immediately. The mask was fully submerged into the hot water (80 to 90oC), with weights to hold down the mask. The solution was covered to minimise cooling and left to stand for 10 and 30 min respectively. The temperature of water was found to be 40 to 45oC after 30 min.
The procedure for steaming was as follows. A metal steamer was placed inside a pot and water was filled to just below the bottom of the steamer. The water was brought to a boil. The mask was then placed on the metal steamer with the inner layer facing downwards. The masks were steamed for 10 and 30 min with the lid covered.
Boiling treatment was conducted as follows. Water was brought to a boil in a pot, after which, the mask was submerged inside the boiling water for 10 and 30 min, with the lid of the pot covered.
After each respective heat treatment, the mask was left to dry at room temperature.
Procedures for bleach treatment were as follows. Store-bought bleach was diluted to give 0.05% sodium hypochlorite (bleach: water=1:99) and the mask was submerged fully for 30 min. Similar procedure was carried out for 0.5% sodium hypochlorite solution (bleach: water = 1:9).
Steps for treatment by surfactant-based soap was as follows. Store-bought dishwashing detergent was diluted with water (final volume: 200 mL, final concentration: 5 mg/mL) and the mask was submerged fully for 1 minute.
Steps for hydrogen peroxide treatment was as follows. Mask was submerged fully in 0.3% hydrogen peroxide solution for 30 min. Similar procedure was carried out with 0.5% hydrogen peroxide for 1 hour.
Steps for ethanol treatment was as follows. Mask was submerged fully in 70% ethanol (200 mL) for 10 min before removal.
After each respective chemical treatment, the mask was rinsed with tap water to remove the soaking chemicals and left to dry at room temperature.
Pre-determined locations with receiving power density of 10-15 W/m2 was marked in the UV cabinet. The masks were placed at these locations and irradiated for 15 min. The total mask was subjected to a surface dose of 9 to 13.5 kJ/m2 during each cycle of irradiation. The amount of light that reached the bottom layer was approximately 9.2 ± 0.3 % of the total irradiated light, which is equivalent to a dose of 1.3 kJ/m2.
Off-gassing measurement of chlorine
Measurement of possible off-gassing of chlorine were performed at two stages of the decontamination process: 1) immediately after rinsing of the surgical mask with tap water and 2) after drying of the surgical mask. A chlorine detector with a detection range of 0 to 1.5 ppm with increment units of 0.01 ppm was used (Riken Keiki, SC-8000, Japan). No chlorine was detected when the air sampling probe was placed beside the mask at both stages.
Particle Filtration Efficiency (PFE) and Differential pressure (Delta-P) measurements
The PFE was evaluated based on a modified test method stated in ASTM-F2299 standards. The aerosol filtration test rig system constructed using stainless steel was set up based on Scheme S1. The main deviation from the standard was using a smaller mask coupon of 9.6 cm2 and filtration efficiency was determined by measuring the aerosol concentration before and after insertion of the mask coupon using a single downstream aerosol isokinetic sampling probe.
A 6 port Collison nebuliser (BGI, USA) was used to generate the aerosols. The 0.1 µm diameter polystyrene latex (PSL) beads solution prepared according to manufacturer instructions (Duke Scientific, USA) was used for the PFE testing, while a Di-Ethyl-Hexyl-Sebacat (DEHS) polydispersed aerosols was used to determine the filtration efficiency between 0.05 to 0.6 µm. The polydispersed DEHS has a geometric mean of 0.189 µm and geometric standard deviation of 1.9. The aerosols were passed through a Krython-85 neutralizer (TSI Model 3012A) for the DEHS experiment and not in the PSL experiment. The aerosols were diluted by HEPA filtered laboratory air (22 ± 2 oC with a humidity of 65 ± 10 %) prior to entering into the test rig. All mask coupons were equilibrated in laboratory air environment and tested at a face velocity of 12.3 cm/s controlled via a Mass Flow Controller (Dwyer, United States) in negative suction mode.
Concentration of PSL was measured by a condensation particle counter (TSI Model 3775) with the Differential Mobility Analyser (TSI Model 3081) electromobility diameter set at 0.11 µm. The DEHS concentration was measured in the scanning mobility particle sizer mode from 0.05 to 0.6 µm. Differential pressure across the mask coupons was measured using a micromanometer (TSI Model 5815).
Bacterial Filtration Efficiency (BFE)
BFE was conducted by an external vendor (TUV-SUV-PBS Pte Ltd). ASTM-F2101 test methods for evaluating BFE of medical face mask materials using biological aerosol of S. aureus was followed. The mean particle size of the challenge aerosol was 3 0.3 µm over a flow rate of 28.3 0.3 L/min over a test area of approximately 38.5 cm2. The results of the untreated and treated mask samples were compared against negative control (filtered air) and positive control stream of S. aureus.
Bacterial and Viral culture
S. aureus (ATCC 25923), a Gram-positive bacterium found commonly in the upper respiratory tract, was used. It was cultured in tryptic soy broth (TSB) at 37oC, overnight with shaking. S. aureus was diluted to 1 x 109 colony-forming units CFU/mL using 1:19, TSB in 0.05% Triton X-100 as the inoculating medium.
Influenza A/Puerto Rico/8/1934 (H1N1) was grown in embryonated chicken egg. The virus was harvested 3 days post-infection from the allantoic fluid.
Inoculation of biological agents on test swatch
For anti-bacterial studies, 2 x108 CFU of S. aureus was inoculated onto square swatches (16 cm2) prepared from the surgical masks resulting. For anti-viral studies, 2 x 106 Plague Forming Unit (PFU) H1N1 in virus growth medium was inoculated onto square swatches (1 cm2). The inoculated swatches were allowed to dry at room temperature in a biosafety cabinet for 60 min, before proceeding with the treatment studies.
Treatment of the test swatch for anti-bacterial and anti-viral assays
Briefly, for bleach treatment, the inoculated swatches were submerged fully in sodium hypochlorite solution (0.05% and 0.5%) for 30 min, followed by rinsing with deionised water thrice.
For hydrogen peroxide treatment, the inoculated swatches were submerged fully in H2O2 (0.3% and 0.5%) for 60 min, followed by rinsing with deionised water thrice. The volume to surface area ratio was kept consistent at 0.4 mL solution (hydrogen peroxide or bleach) per centimetre square of test swatch.
For UV-C treatment, the inoculated swatches were placed at the pre-determined locations in the UV cabinet with the outer layer facing the UV lamp described in the earlier section.
After treatment, the swatches were transferred to centrifuge tubes, and 10 mL of extraction buffer (TSB) was added, followed by vortexing for 1 minute. The supernatant was serially diluted, and plated in duplicates on tryptic soy agar (TSA) plates. The TSA plates were incubated for 24 hours before the CFUs on the plates were enumerated to determine the bacterial Log10 reduction for each treatment method, compared to the positive control (bacterial count extracted from inoculated swatches that were not inactivated).
Viral growth medium (VGM) was added to the retrieved swatch and incubated at 37°C, with shaking at 750 rpm, for 1 hour to extract any viable viral particles from the disinfected swatch. Half log serial dilutions of the extracted viral suspension were performed with VGM and added to Mardin-Darby Canine Kidney (MDCK) cells, and then incubated for 72 hours at 37°C, 5% CO2. Viral ToxGlo™ Assay was used to detect viral cytopathic effects as per manufacturer's instructions. The amount of viable virus that caused 50% cell death in the MDCK cells (TCID50, tissue culture infectious dose) was inversely correlated to the measured luminescence, and the TCID50 value at 72 hours was calculated using the Reed and Muench method. The log reduction in viable virus for each inactivation method was determined by calculating the difference between the TCID50 of the respective disinfected swatch and the non-disinfected control swatch. Experimental results were representative of three independent experiments performed in triplicates.
The methods that were proven to retain the masks properties and attained sufficient log reduction for treatment were then selected for the aging test. Untreated masks were subjected to the hydrogen peroxide and bleach treatment for 10 times and UV treatment for 30 times respectively. The PFE and BFE results were then obtained and compared with the original value.
Procedure for hydrogen peroxide and bleach decontamination
The procedure for 0.5% hydrogen peroxide and 0.05% sodium hypochlorite (bleach) is shown in Figure S1. A 200 mL solution was sufficient for 4 masks. The masks should not be wring dry as this would cause some degradations to the mask, which affects their PFEs (Figure S2).