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An APS spectrometer (Model 3321, TSI Inc., USA) measured the aerosol size distributions in the ambient air from 0.5 to 20 μm and analyzes the time-of-flight of individual particles in an accelerating flow field (Volckens and Peters, 2005; Peters and Leith, 2003). The experimental system for measuring the separation characteristics of a cyclone using the APS is shown in Fig. 1. The particle size distribution in the air flow before and after the cyclones were measured using the APS. This measurement was conducted six times for 6 min at each flow rate of the cyclones.

Fig. 1. 
Schematic diagram of the APS system to evaluate the particle penetration of the cyclones.

Additional experiments were required to determine the penetration of particles with a size of 0.5 μm or smaller through the cyclones. The system for measuring the cyclone’s separation characteristics using fluorescent PSL particles (Polyscience Inc., Warrington, PA, USA) is shown in Fig. 2. The fluorescent PSL particles were supplied by an atomizer and collected with a filter for 20 min, each with and without a cyclone. The filter sample without a cyclone was designated as a reference filter, and the filter sample after the cyclone was designated as a backup filter (BF). The flow rate for the cyclones and reference filters was measured using a mass flow meter and set to the same value before each test. The flow rate of the introduced clean air was carefully adjusted that the flow of the air contained the PSL particles was 2 L/min, with and without each cyclone. Moreover, we confirmed that the number concentration of the generated PSL particles did not differ significantly with or without cyclones using a condensation particle counter (CPC, Model 3750). Fluorescent particles of 0.1, 0.2, 0.5, and 1.0 μm were supplied. The fluorescent PSL particle solution in the atomizer was diluted 20 times with ultra-pure water from the fluorescent solution of PSL to make it 0.05 mL/mL.

Fig. 2. 
Schematic diagrams of the experimental system to evaluate the performance of the cyclones using the fluorescent PSL monodisperse particles.

The filters collected the PSL particles dissolved in ethyl acetate and shaken at 150 rpm for 120 min. After that, the solution was filtered through a quartz fiber filter to eliminate impurities, and the obtained solution was measured using a spectrofluorophotometer (RF-6000, Shimadzu Corporation, Japan). The fluorescent PSL particle concentration was measured at 441 nm of the excitation wavelength, 470-510 nm of fluorescence wavelength, 600 nm/min scan speed, 1.0 nm of the data interval, auto sensitivity, 5.0 nm of spectral bandwidth excitation side, and 5.0 nm of fluorescence side.

Fig. 3 shows the DMA system (DMA-5180, SHIBATA) for measuring particle penetration through the cyclones. An Am-241 neutralizer between the diffusion dryer and the DMA was also used. Test particles were generated using an atomizer using 4% ammonium sulfate solution. The flow rate of the introduced clean air is the same as in the above method using the fluorescent PSL particles. The particles were classified in order by the DMA in a size range from 100 nm to 500 nm in diameter. The classified monodisperse particles with one diameter were measured the number concentration by CPC at front of and back of the cyclone to calculate the penetration rate. In addition, we used an isokinetic sampling probe to avoid particle separation by the small flow rate of the CPC against that of pump.

Fig. 3. 
Schematic diagrams of the DMA system to evaluate the particle penetration of the cyclones.

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS, iCAP TQ, Thermo Fisher Scientific) was used to analyze the samples in the Atmospheric Environment Innovation Center at Seikan Kensa Center Inc. for ICP-MS analysis. The powder sample was weighed at about 1 mg and decomposed in 5 mL nitric acid, 1 mL hydrogen peroxide, and 2 mL hydrofluoric acid with a microwave decomposition device. The decomposed liquid was transferred to a 50 mL centrifuge tube, and the volume was adjusted to 50 mL. Finally, the sample solutions were analyzed. The quantification of metals (e.g., Al, Ti, V, Cr, Mn, Fe, Cu, Zn, Ni, Pb) was performed. The standard solution was diluted 10, 100, and 1000 times with a mixed solution of 5% nitric acid and 1% hydrofluoric acid to drive for an appropriate concentration for the target element. The LOD for Al, Ti, V, Cr, Mn, Fe, Cu, Zn, Ni, and Pb was 40, 1.9, 0.1, 0.4, 0.4, 5.0, 0.8, 1.4, 0.26, and 0.3 ppmw, respectively. Recovery rate of the target elements ranged from 87% to 97%, measured using standard reference materials (CRM).

Enrichment factors (EFs) were used to assess the contribution of anthropogenic emissions to the atmosphere for each metal in PM_2.5 (Jing et al., 2022; Ny and Lee, 2010). The reference element is often taken to be Al and Fe (for terrestrial sources) and Na (for oceanic sources) (Ny and Lee, 2010; Zoller et al., 1974; Chester and Stoner, 1973). Moreover, Ti and Mn have been used successfully (Jing et al., 2022; Obiajunwa et al., 2002; Loska et al., 1997). In this study, we avoid using of Al or Fe as reference elements because the small and large cyclones are made of aluminium and stainless steel, respectively. We focused on assessing the enrichment levels of V, Cr, Mn, Fe, Cu, Zn, Ni, and Pb in samples because the transition metals mainly come from anthropogenic activities (Chen et al., 2010; Okuda et al., 2007; Song et al., 2006). Therefore, Ti was used as the reference element in the following equation to avoid Al and Fe, which may be contaminated by cyclones, and to focus on heavy metals and transition metals:

where C_i and C_Ti refer to the abundance of each element and Ti in PM_2.5 and the Earth’s crust. The average crustal concentrations were obtained from a study by Taylor (1964).

The reactive oxygen species (ROS) generation ability (or OP) of PM is one of the comprehensive and realistic toxicity indicators which reflects physicochemical properties (e.g., size, surface area, chemical composition) of PM. Redox-active particles deplete antioxidants, damaging biomolecules, and leading to acute and chronic diseases (Gao et al., 2020; Delfino et al., 2011). In particular, the dithiothreitol-measured OP of aerosol particles has been extensively used worldwide as a suitable indicator for evaluating the oxidation properties of ambient particles (Wang et al., 2019). First, to measure the OP of PM_2.5, a phosphate buffer (0.1 M Na₂HPO₄+0.1 M KH₂PO₄) at pH 7.4 was treated with Chelex resin to remove trace metals used as a solvent (Charrier and Anastasio, 2012). Then, the powders of DTT and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB, FUJIFILM Wako Pure Chemical Corporation, Japan) were dissolved in the phosphate buffer to prepare 1.0 mM DTT and 6.25 mM DTNB solutions. The powder samples weighed about 2 mg were sonicated in 19 mL of the phosphate buffer for 15 min at a temperature <15°C. Next, 1.9 mL of each sample solution was transferred to a microtube, and 2 μL of Tween-20 solution (10% in H₂O, BioVision, Inc., USA) was added. The reaction continued at 37°C for 30 min in a Thermo block (ND-M01, Nissin, Japan). A total of 100 μL of the 1.0 mM DTT solution was added to the microtube. After 6 min of the start of the reaction with the DTT solution, 20 μL of the DTNB solution was added quickly to develop color (the reaction stopped there) and put in a refrigerator. More detailed information about the preparation of the standard solution and measurement absorbance is given in the previous study (Kurihara et al., 2022). The precision of the DTT assay was checked using the reference material CRM#28. The sample’s DTT consumption rate (μM/mg/min) was calculated as the regression slope between DTT concentration and time. LOD was 0.09 μM/mg/min; the LOD value was calculated on the standard solution curves set using a linear regression model (Piehler et al., 2020).

Endotoxin (a component of the exterior cell membrane of Gram-negative bacteria) is a group of toxic and proinflammatory compounds of lipopolysaccharide (LPS) responsible for releasing and activating cytokine production (Carty et al., 2003; Heinrich et al., 2003). A certain type of bacteria causes symptoms involving airway inflammation, fever, and lung function decline due to their strong immune-modulating properties (Heinrich et al., 2003; Reed and Milton, 2001). Endotoxins were assayed using the quantitative kinetic chromogenic LAL method. Before experiments, all glassware such as triangular flasks, test tubes with aluminum caps, and reservoirs were sterilized at 250°C for 2 hours. First, a 0.05% Tween solution was prepared using Tween-20 (10% in H₂O, BioVision, Inc., USA) and endotoxin-free distilled water/LAL reagent water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan). Then, 2 mg of each powder sample was dissolved in 4 mL 0.05% Tween solution at room temperature using a horizontal shaker for 1 hour and sonicated at low temperature for 15 min. An endotoxin standard solution was prepared at 7 concentrations (1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.0125 EU mL^-1) using a control standard endotoxin (Associates of Cape Cod. Inc., MA, USA). After 7 min of centrifugation at 5000 rpm, 50 μL of supernatant per sample, blank (LAL reagent water), and standard endotoxin solution was transferred to a 96-well microplate. Then 50 μL of a lysate reagent (Pyro Color-MP: Chromogenic Diazo-Coupling Kit, Associates of Cape Cod. Inc., MA, USA) was added to the microplate using an 8-series pipette and the microplate was stirred manually to mix. The plate was immediately put on an ice pack after warming in 96-well shaking incubators at 37.0°C for 25 min. After the incubation was completed, the PyroColor Diaza Reagent DIA150-MP (Associates of Cape Cod. Inc.) was added to each well. A total of a 50 μL of HCl solution, ammonium sulfamate and N-(1-Naphthyl) polyethylenediamine (NEDA) were added to each well using an 8-unit pipettor and stirred. Absorbance was measured at 540 nm using a microplate reader (AS ONE: MPR-A100). Endotoxin concentrations were expressed as endotoxin units per milligram of sample (EU mg^-1). The correlation coefficient of a calibration curve was taken to 0.980 or more to confirm the experiment’s accuracy. The accuracy of the DTT assay was checked using the reference materials CRM#28 and CRM#30 (Urban Aerosols and Gobi Kosa Dust, NIES, National Institute for Environmental Studies Onogawa, Tsukuba, Japan). LOD was 0.1 EU mg^-1; the LOD value was calculated using a linear regression model fitted to the standard solution curves (Piehler et al., 2020).

The cytotoxicity of the collected particles was evaluated by the viability of human epithelial A549 cells and the IL (interleukin)-6 production of macrophage-like cells differentiated from human leukemia monocytic (THP-1) cells. A549 cells derived from human alveolar cell carcinoma of lung cancer cells are frequently used in toxicity research to evaluate the biological effects of particles on human respiratory systems (Jia et al., 2017). Macrophage cells, which are antigen-presenting cells, are one of the most potent producers of proinflammatory cytokines in the airways and lungs and play roles as the driver of local and systemic inflammatory responses. Moreover, exposure of atmospheric particles to macrophage cells has also confirmed a significant release of IL-6, one of those important cytokines (Jia et al., 2017; Hiraiwa and van Eeden, 2013).

The A549 cells used to measure this viability were purchased from Japanese Cancer Research Resources Bank (JCRB), National Institutes of Biomedical Innovation, Health and Nutrition, Japan. And they were maintained with Minimum Essential Medium (MEM, Thermo Fisher Scientific, Waltham, MA, USA) including 10% deactivated fetal bovine serum (FBS, Origin South America, CCP-FBS-BR-500, Cosmo Bio Co., Ltd., Tokyo, Japan), 1% penicillin-streptomycin solution (PS, FUJIFILM Wako Chemicals Corporation, Osaka, Japan), and 1% MEM Non-Essential Amino Acids (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. The cells were seeded with a concentration of 1,000 cells/well on 96 well plates 24 hours before the particles were exposed. It should be noted that the small seeding concentration and culture time in this study differ from the typical cell density exposed in the subconfluently cell state. However, although the actual effect may be overestimated, the toxicity of the particles can be accurately characterized due to be grasped with higher sensitivity. Incubated cells were exposed for 24 hours to a solution suspended in the above-prepared culture medium at particle concentrations of 7.5, 75, and 150 μg mL^-1. The viability of exposed cells was measured by the Premix Water-soluble tetrazolium salt-1 (WST-1) Cell Proliferation Assay System (MK-400, TAKARA BIO INC., Shiga, Japan). Following a standard protocol in this analysis, we added 10 μL of WST-1 solution to each well, incubated for 4 hours for the reaction, and measured their absorbance at 450 nm and 630 nm. Cell viability was calculated as a ratio and compared with the control cells. Each concentration was repeated in three wells (n=3).

To provide additional data on PM_2.5 cytotoxicity, inflammatory mediator release, cytokines (IL-6) were measured after treating of macrophage-like cells with PM_2.5. IL-6 is promptly and transiently produced during infections and tissue injuries and stimulates acute-phase responses and immune reactions. Moreover, high-level IL-6 exerts pathologically on chronic inflammation and autoimmunity (Tanaka et al., 2014). Before exposure, THP-1 was treated with phorbol 12-myristate 13-acetate (PMA, FUJIFILM Wako Chemicals Corporation, Osaka, Japan) for differentiation into functional macrophages (Starr et al., 2018). Human monocytic leukemia THP-1 cells purchased from the JCRB were maintained in RPMI-1640 (FUJIFILM Wako Chemicals Corporation, Osaka, Japan) containing 1% deactivated FBS, 1% PS and stored in a humidified atmosphere containing 95% air and 5% CO₂ at 37°C. In this measurement, THP-1 cells were seeded at 4.8×10⁴ cells/well on 12 well plates with PMA (200 nM). After incubating for 72 hours, the medium was replaced with a PMA-free medium and additional incubated for 72 hours until the sample is exposed. The exposure time of the sample was set 24 hours, similar to the cell viability described above. The levels of IL-6 in the centrifuged supernatants were measured by Human IL-6 enzyme-linked immunosorbent assay (ELISA) Kit (LBIS Human IL-6 ELISA Kit, AKH-IL6, FUJIFILM Wako Shibayagi, Gunma, Japan) 24 hours after particle exposure, according to the manufacturer’s instructions. The absorbance was observed under the microplate reader at 450 nm and 630 nm. LOD was 0.98 pg/mL; the LOD value was calculated on the standard solution curves set using a linear regression model (Piehler et al., 2020). The amount of cytokine release is determined by the concentration from the absorbance of the measured sample by their calibration curve that was obtained the absorbance measured by seven concentrations of IL-6 standard regent (i.e., positive control). For each experiment, the mean values were obtained from the replicate analysis (e.g., n=3).

T-test was used to compare the differences between cell viability and cytokine levels for a similar dose-manners. *p<0.05 implies statistically significant and **p<0.01 is highly significant.

The results of the redox activity of the PM_2.5 samples collected using the small and large cyclones are summarized in Fig. 8. The DTT loss rates were 0.21 and 0.96 μM/mg/min for the small cyclone samples, and 0.82 and 0.62 μM/mg/min for the large cyclone samples collected from 10 December 2020 to 6 January 2021 and 10 March 2021 to 14 April 2021, respectively.

Fig. 8. 
DTT oxidizing ability of samples collected using the small and large cyclones (n=3).

In this study, the DTT loss rate of the samples collected using small and large cyclones differed in the sampling periods, consistent with other studies (Fang et al., 2017; Fujitani et al., 2017). Fujitani et al. (2017) found that transition metals in the sample were a significant contributor to DTT consumption (more than 80%). In our study, the difference in the DTT oxidization ability is likely associated with the difference between transition metal concentrations (e.g., Cu, Mn, Ni, Fe, Zn, Pb) in the samples.

Fig. 9 shows the endotoxin concentrations in the samples collected using the small and large cyclones. The endotoxin content of the particles was 129.58 EU/mg and 60.49 EU/mg in the large cyclone samples collected from 10 December 2020 to 6 January 2021 and 10 March to 14 April 2021, respectively. Endotoxins were not detected in the small cyclone samples.

Fig. 9. 
Concentration of endotoxin in PM_2.5 collected using the small and large cyclones (n=3).

The endotoxin level in the large cyclone samples was detected significantly high compared to that in the small cyclone samples. This is consistent with other studies (Khan et al., 2018; Schins et al., 2004). Khan et al. (2018) found that concentrations of endotoxin in coarse particles were significantly higher than those in fine particles in Japan. In this study, the abundance of smaller particles in the samples of the small cyclone was higher than in the samples of the large cyclone. This indicates that the distribution of small particles significantly decreased the endotoxin levels in the small cyclone samples.

Fig. 10 shows the cytotoxic effects of PM_2.5 collected using large and small cyclones evaluated with A549 cells in MEM. The results show the ratio of the metabolic activity of exposed cells to the metabolic activity of unseeded cells. The highest toxicity of the large cyclone samples was greater than in the small cyclone samples, except for the samples collected from 10 March to 14 April 2021 at 75 μg/mL and 150 μg/mL of PM_2.5 concentrations. The difference between the small and large cyclone samples was insignificant (p<0.1) at 7.5 μg/mL, significant (p<0.05) at 75 μg/mL and highly significant (p<0.01) at 150 μg/mL.

Fig. 10. 
PM_2.5 concentration-dependent cell-manner viability for A549 cells (*p<0.05, **p<0.01 imply that the difference between the small and large cyclone samples was significant, n=3). The samples were collected (a) from 10 December 2020 to 6 January 2021 and (b) from 10 March to 14 April 2021.

Fig. 11 shows the estimated levels of cytokines using the IL-6 release measurement for macrophage-like cells in the collected PM_2.5. The value of the control shows IL-6 levels in the blank medium used in the experiments. The level of IL-6 released after exposure to the samples collected using the large cyclone was markedly higher than that observed after exposure to the samples collected using the small cyclone.

Fig. 11. 
PM_2.5 concentration-dependent manners in cytokines estimated using IL-6 release measurement for macrophage-like cells (*p<0.05 imply that the difference between the small and large cyclone samples was significant, n=3). The samples were collected (a) from 10 December 2020 to 6 January 2021 and (b) from 10 March to 14 April 2021.

An in vitro toxicity assay of PM_2.5 collected using the small and large cyclones was conducted on lung epithelial A549 cells. The A549 cell viability after exposure to the PM_2.5 collected from both cyclones decreased corresponding with the DTT loss rate, consistent with previous studies (Zou et al., 2016; Cao et al., 2015), which found that smaller particles can enter cells or induce ROS production and damage cells.

To evaluate whether the PM_2.5 collected using small and large cyclones had proinflammatory effects on macrophage-like THP-1 cells, the production of IL-6 was examined. The cytokine level estimated using IL-6 release measurement was higher in the large cyclone samples than in the small ones, which is supported by other studies (Gorbet and Sefton, 2005; Huang et al., 2004; Schins et al., 2004). Schins et al. (2004) found that the cytokine levels were higher in coarse particles than in fine ones due more to their biological components (e.g., endotoxin) than metal content. Gorbet and Sefton (2005) reported that Gram-negative bacteria cause an inflammatory response to local infection by stimulating the synthesis of inflammatory mediators (e.g., IL-6). Zou et al. (2016) showed that the proapoptotic effects of particles are unrelated to their proinflammatory properties, and water-soluble ions and individual soluble metals (V, Ni, Fe) were relatively inactive for apoptosis and inflammation in macrophages.

The chemical composition analyses of PM_2.5 collected using the small and large cyclones showed that the small cyclone samples had higher concentrations of water-soluble ions and transition metals, except for Fe and Ti, than the larger cyclone samples. The comparison of the samples collected using large and small cyclones significantly reflected the significant toxicity of the particles. ROS generation was associated with transition metals and they decreased the viability of the human lung epithelial A549 cells. The endotoxin concentration was detected only in the large cyclone samples and significantly affected the level of cytokines estimated using the IL-6 release measurement.