Characteristics and Potential Inhalation Exposure Risks of Environmentally Persistent Free Radicals in Atmospheric Particulate Matter and Solid Fuel Combustion Particles in High Lung Cancer Incidence Area, China

Environmentally Abstract: Environmentally persistent free radicals (EPFRs) were previously considered an unrec-ognized composition of air pollutants and might help explain the long-standing medical mystery of why non-smokers develop tobacco-related diseases such as lung cancer. However, there is no investigated on EPFRs in Xuanwei rural areas, especially in high prevalence of lung cancer areas. In this study, we selected six types of coal and three types of biomass in Xuanwei, then conducted simulated combustion, and six group of atmospheric particulate matters (APMs) to explore the content and particle size distribution pattern of EPFRs and a new health risk assessment method to evaluate the risk of EPFRs in PM for adults and children. Our results show that the contribution of EPFRs for biomass combustion, coal combustion and APMs were mainly distributed in the size range of <1.1 µ m, which accounted for 76.15 ± 4.14%, 74.85 ± 10.76%, and 75.23 ± 8.18% of PM3.3. The mean g factors and ∆ Hp-p indicated that the EPFRs were mainly oxygen-centered radicals in PM in Xuanwei. The results suggest that the health risk of EPFRs is signiﬁcantly increased when the particle size distribution of EPFRs is taken into account, and coal combustion particulate matter (174.70 ± 37.86 cigarettes for an adult, 66.39 ± 14.39 cigarettes per person per year for a child) is more hazardous to humans than biomass combustion particulate matter (69.41 ± 4.83 cigarettes for an adult, 26.37 ± 1.84 cigarettes per person per year for), followed by APMs (102.88 ± 39.99 cigarettes for an adult, 39.10 ± 15.20 cigarettes per person per year for) in PM 3.3 . Our results provides a new perspective and evidence for revealing the reason for the high incidence of lung cancer in Xuanwei, China.


Introduction
Lung cancer remains the most prevalent cancer (11.6% of all cases) and the leading cause of cancer deaths worldwide (18.4% of all cancer deaths) [1]. Smoking is the largest preventable cause of lung cancer and contributes to more than 80% of cases of this disease on a global scale [2]. However, women have the highest incidence of lung cancer among non-smokers in Xuanwei, China [3]. The mortality of lung cancer in Xuanwei's rural areas are 27.7/100,000 for men and 25.3/100,000 for women, almost five times that of China's national average (4.97/100,000 for both sexes) [4], it ranks among the top in the world for female lung cancer mortality [5]. However, the etiology of lung cancer in the region

Materials and Methods
Xuanwei country located in Yunnan Province, southwest China, has the highest incidence and mortality rate of lung cancer [34]. After field investigation and data search, we found that wild pine and poplar trees are widely distributed, and 1.78 million hectares of corn was planted in Yunnan, accounting for 25 34.57% of total energy consumption in Yunnan Province, China (http://stats.yn.gov.cn/; accessed on 22 September 2021).
Then, simulated combustion on raw coal and biomass was conducted via a burning system, which was simulated according to local residents' combustion mode in our laboratory (in the Supplementary Materials Figure S2). In addition to the above, six sample groups of APMs (A-H) were conducted by a high-volume air sampler (Shibata Science Co., Ltd., Saitama, Japan) at a flow rate of 566 L/min in Xuanwei local rural residences in February and March in 2017 (Table S1), and the aerodynamic diameters were <1.1 µm, 1.1-2.0 µm, 2.0-3.3 µm, 3.3-7.0 µm, and >7.0 µm. The detailed information of raw coal, biomass, and APMs is shown in Table 1. More information on the burning system and pre-treatment and post-treatment of samples can be found in our previous study [28].

Detection of EPFRs
Electron spin resonance (ESR) spectroscopy was used to determine EPFRs. To determine EPFRs, quartz fiber filters were cut into strips of 3-4 mm, then added to a quartz EPR tube and measured on the corresponding instrument. The relevant parameters were as follows: sweep time, 120 s; center field 324.74 mT; sweep width, 25 mT; modulation frequency, 100 kHz; modulation Width, 0.05 mT; microwave frequency 9105.26 MHz; and microwave power, 0.998 mW.

Data Processing and Calculation of the Absolute Number of Spins
ESR tests were performed on standards containing Mn (II) to calibrate the absolute number of spins and then characteristic parameters such as the g factor, and ∆Hp-p of the EPFRs were obtained. The formula used to calculate the spin numbers and g factors are shown in Equations (1) and (2) [35][36][37]: Spins sample (spins/g) = 3.02 × 10 14 (spins/g) × Integral sample /Integral standard (2) where g is the electron spin g-factor of the particle, ň (MHz) represents the microwave frequency, H (mT) is the resonance magnetic field strength during the measurements; 3.02 × 10 14 is the total spins of the standard Mn, Spins sample is the spin concentration of the unknown sample, Integral sample and Integral standard represent the signal integration areas of the sample and Mn (II) standard, respectively [37]. The sample atmospheric spin concentrations of EPFRs (spins/m 3 ) were calculated as the total spin divided by the total sample volume. The EPFR spin concentrations in PM masses (spins/g) were determined as the total spin divided by the collected PM mass.

EPFRs Exposure Evaluation
To date, there is no internationally accepted method to assess the health risk of EPFRs in PM. Several methods are used to assess the health risks of EPFRs [33,[38][39][40]; however, all of them evaluate EPFRs inhalation risk for adults, not children. In our study, we use the equivalent number of cigarettes to evaluate the potential health risks of EPFRs in PM for Xunawei residents [31].The exposure level is provided in Equations (3) and (4): where Inh PM is the daily EPFRs exposure from inhaled PM (spins/g/day); F is the conversion from g to micrograms (1 × 10 −6 ) [31], Fr is the alveolar fraction retained in the lung (0.75). PC PM is the concentration of PM (µg/m 3 ) and R inhalation represents the daily amount of air inhaled (20 m 3 /day for an adult [41], 7.6 m 3 /day for a child) (https://www.epa.gov/risk/risk-assessment-guidance-superfund-rags-part; accessed on 24 September 2021). N cig represents the number of cigarettes (person/year), 30 represents 30 days per month. RCcig (4.75 × 10 16 spins/g) [38] indicates the concentration of free radicals in cigarette tar, and Ctar (0.013 g/cig) indicates the amount of tar per cigarette [31].

Results and Discussion
The size of particulate matter is directly related to their potential to cause health problems. Fine particles (PM 2.5 ) pose the greatest health risk. These fine particles can penetrate deep into the lungs and some may even enter the bloodstream, and can affect a person's lungs and heart. Coarse particles (PM 2.5-10 ) are of less concern, although they can irritate a person's eyes, nose, and throat. Therefore, in this study we focus on particle sizes <1.1 µm, 1.1-2.0 µm and 2.0-3.3 µm. Numerous studies have been conducted on EPFRs in atmospheric particulate matter [22,26,32,42], but there is a lack of studies on EPFRs in particulate matter emitted directly from raw coal combustion and biomass combustion.

EPFRs and PM Concentrations in Biomass Combustion Particles
The concentration distributions of EPFRs and PM in simulated particulate matter emitted from biomass combustion (corncobs, pine, poplar) were provided in Figure 1 and Table S2. The concentrations of EPFEs and PM emitted from the three-biomass combustion were significantly different in the particle size ranges <1.1, 1.1-2.0 and 2.0-3.3 µm. Both the EPFRs and the PM concentration reach their maximum at <1.1 µm, while the lowest concentration is found at the particle size 2.0-3.3 µm. The atmospheric concentrations of EPFRs percentage mean value of PM 1.1 , PM 1.1-2.0 , and PM 2.0-3.3 were 76.25 ± 4.14%, 13.69 ± 3.95%, and 10.06 ± 0.23%, which corresponded to PM mass concentrations were 2948.77 ± 1438.66 µg/m 3 , 1415.44 ± 712.85 µg/m 3 and 1087.57 ± 504.44 µg/m 3 , respectively ( Table 2). The atmospheric concentrations of EPFRs in the PM 1.1 were 4.51 × 10 17 , 4.27 × 10 17 and 3.26 × 10 17 spins/m 3 for corncob, pine, and poplar, respectively, while the mean atmospheric concentrations in PM 1.1 were found to be several times PM 1.1-2.0 (6.34 ± 2.67) and PM 2.0-3.3 (7.60 ± 0.57). The EPFR concentrations in the PM 1.1 at the three sites were 3.11 × 10 15 , 3.37 × 10 15 and 1.08 × 10 15 spins/g for corncob, pine and poplar, respectively (shown in Table S2). It has been reported that the EPFR concentrations in PM 2.5 from the corn straw, rice straw, jujube wood, and pine wood (four biomass, purchased from Jiangsu province) were in the range of 0.9 × 10 19 spins/g to 6.1 × 10 19 spins/g [33], the average radical intensities in PM emissions from fatwood and pine wood were 1.2 × 10 18 , and 9.1 × 10 17 spins/gram, respectively [43]. Compared with previous studies, the EPFR concentrations in PM 3.3 (the range of 1.99 × 10 15 spins/g to 5.50 × 10 15 spins/g) were 2-4 orders of magnitude lower than those reported in a previous study. EPFRs were mainly concentrated in the size range of <1.1 µm, which accounted for 76.25 ± 4.15% of PM 3.3 , indicating that the PM 1.1 emitted biomass combustion is more harmful to the human body than PM 2.0-3.3 and PM 2.0-3.3 ; therefore, deserves more in-depth study. therefore, deserves more in-depth study.   Table 3 lists the size distribution of EPFRs and PM in simulated coal combustion pa ticles (%). Figure 2 and Table S3 shows the EPFRs and PM concentrations in coal combu tion particles. The atmospheric EPFRs concentrations in PM1.1, PM2.0-3.3 and PM2.0-3.3 ma the contribution to PM3.3 74.85 ± 10.76%, 13.10 ± 7.66%, and 12.05 ± 7.25%, respectively. Th average atmospheric concentrations in PM1.1 were found to be several times the PM1.1-(8.81 ± 6.70) and PM2.0-3.3 (8.07 ± 3.68). The concentration distribution of EPFRs and PM coal combustion emission particulate matter is similar to that of biomass combustion pa ticulate matter, both mainly concentrated in the <1.1 um particle size. The mean atmo pheric concentrations of EPFRs in the PM1.1, PM2.0-3.3 and PM2.0-3.3 were 2.16 × 10 17 ± 5.82   Table 3 lists the size distribution of EPFRs and PM in simulated coal combustion particles (%). Figure 2 and Table S3 shows the EPFRs and PM concentrations in coal combustion particles. The atmospheric EPFRs concentrations in PM 1.1 , PM 2.0-3.3 and PM 2.0-3.3 make the contribution to PM 3.3 74.85 ± 10.76%, 13.10 ± 7.66%, and 12.05 ± 7.25%, respectively. The average atmospheric concentrations in PM 1.1 were found to be several times the PM 1.1-2.0 (8.81 ± 6.70) and PM 2.0-3.3 (8.07 ± 3.68). The concentration distribution of EPFRs and PM in coal combustion emission particulate matter is similar to that of biomass combustion particulate matter, both mainly concentrated in the <1.1 um particle size. The mean atmospheric concentrations of EPFRs in the PM 1 Table S3). Compared with previous studies, the EPFR concentrations in PM 3.3 (the range of 4.13 × 10 15 spins/g to 1.78 × 10 16 spins/g) which were 1-4 orders of magnitude lower than those reported in a previous study for the total particular matter from bituminous coal (4.4 × 10 17 spins/g), anthracite (2.3 × 10 17 spins/g) [43], and bituminous (10 19 spins/g), which were purchased from Henan Province.  Table S3). Compared with previous studies, the EPFR concentrations in PM3.3 (the range of 4.13 × 10 15 spins/g to 1.78 × 10 1 spins/g) which were 1-4 orders of magnitude lower than those reported in a previous study for the total particular matter from bituminous coal (4.4 × 10 17 spins/g), anthracite (2.3 × 10 17 spins/g) [43], and bituminous (10 19 spins/g), which were purchased from Henan Province.   Figure 3 and Table S4). As shown in Table 4, the PM1.1 fraction contributed 46.56% ± 9.67% of the PM3.3 mass, which indicated that much more PM in the atmosphere was present in smaller size fractions.

EPFRs and PM Concentrations in Coal Combustion Particles
The

EPFRs and PM Concentrations in Atmospheric Particulate Matters
The mass concentrations of three PM fractions classified as PM <1.1 , PM 1.1-2.0 , and PM 2.0-3.3 , were 42.92 ± 16.50 µg/m 3 , 26.33 ± 5.66 µg/m 3 , and 20.42 ± 2.68 µg/m 3 , respectively (shown in Figure 3 and Table S4). As shown in Table 4, the PM 1.1 fraction contributed 46.56% ± 9.67% of the PM 3.3 mass, which indicated that much more PM in the atmosphere was present in smaller size fractions.
The mean atmospheric concentrations of EPFRs in PM 1.1 , PM 1.1-2.0 , and PM 2.0-3.3 were 7.03 × 10 15 ± 5.29 × 10 15 spins/m 3 , 9.05 × 10 14 ± 2.50 × 10 14 spins/m 3 , and 8.35 × 10 15 ± 3.06 × 10 15 spins/m 3 , respectively, while the mean concentrations in PM were in the range of 2.16 × 10 17 ± 9.73× 10 16 spins/g, 5.38 × 10 16 ± 1.72 × 10 16 spins/m 3 , and 8.35 × 10 14 ± to 3.06 × 10 14 spins/m 3 , respectively. Several other studies have reported EPFR concentrations in atmospheric particulate matter, in PM 2.5 in Taif, Saudi Arabia. Saudi Arabia ranged from 1.6 × 10 16 to 5.8 × 10 16 spins/m 3 [44], and in PM 2.5 in Xuanwei, China ranged from 3.20 × 10 17 to 3.10 × 10 19 spins/g [45]. In PM 2.5 in Xi'an ranged from 9.8 × 10 11 to 6.9 × 10 14 spins/m 3 [38]. In PM 2.5 samples from Baton Rouge, ranged from 2.46 × 10 16 to 2.79 × 10 17 spins/g [31]. The concentrations of EPFRs were lower. The level of EPFR concentration in PM in Xuanwei was several times smaller than that in previous studies. However, in our study, the levels of EPFRs in the PM were dozens of times lower than previous reported EPFR concentrations. EPFRs were mostly present in the PM 1.1 fraction, in which the EPFR concentration was 3.43-19.85 times higher than that in PM 1.1-2.0 and 4.09-15.47 times higher than that in PM 2.0-3.3 , which were in agreement with previous research [46]. In addition, it is worth noting that PM 2.0 can enter the lungs and even the bloodstream more deeply than coarse particles, which may induce harmful reactive oxygen species (ROS) and DNA damage [47].   In our study, the simulated combustion experiments with raw coal and biomass were carried out in a relatively closed room, so the mass concentrations of collected particulate matter were much higher than those collected in open areas. Our results indicate that EP FRs attach more readily to fine particles, which may be due to the fact that fine particles have larger surface areas and more porous surfaces, leading to higher adsorption and re tention of EPFRs [17,46,48]. The distribution pattern in Figures 2-4 showed that EPFR con centrations in each PM fraction increased as the particle size decreased. The main reason for the low concentration of EPFRs in our samples may be that our samples have been  In our study, the simulated combustion experiments with raw coal and biomass were carried out in a relatively closed room, so the mass concentrations of collected particulate matter were much higher than those collected in open areas. Our results indicate that EPFRs attach more readily to fine particles, which may be due to the fact that fine particles have larger surface areas and more porous surfaces, leading to higher adsorption and retention of EPFRs [17,46,48]. The distribution pattern in Figures 2-4 showed that EPFR concentrations in each PM fraction increased as the particle size decreased. The main reason for the low concentration of EPFRs in our samples may be that our samples have been stored for too long, resulting in partial degradation. The above results suggest that the concentration of EPFRs in atmospheric particulate matter varies across regions and different combustion sources. However, to the best of our knowledge, the current studies on

Potential Health Risk of EPFRs
In our study, the main types of EPFRs in PM were phenoxyl and semiquinone radicals. The spectral characteristics of EPFRs were compared with that of cigarette tar, both of them were similar to semiquinone radicals, and identified as semiquinone radicals [56,57], which associated with a quinone/hydroquinone redox cycle capable of producing reactive oxygen species (ROS), to be involved in the carcinogenicity [40].
In this study, to assess the potential health risk of EPFRs in biomass combustion

EPFRs Species Characteristics
The g-factor and peak width (referred to as ∆Hp-p, Gauss) were important parameters for identifying the type of free radicals [17,44], the average ∆Hp-p was calculated to find the distance between the maximum and minimum y-axis values on the x-axis [37]. According to previous reports, carbon-centered radicals are generally less than 2.003, oxygen-centered Atmosphere 2021, 12, 1467 9 of 13 radicals are generally greater than 2.0040, and g factors in the range of 2.0030-2.0040 are believed to correspond to a mixture of carbon-and oxygen-centered radicals [23,49,50].
A comparison of the EPR spectra of EPFRs in different PMs (as shown in Figure 4) indicates that the g-factors in PMs were different and the signal intensity of EPFRs is also different (The g-value for the other samples can be seen in Figures S3-S5). The mean g factor and ∆Hp-p of the EPFRs (Table S5) were ranged from 2.0036 to 2.0040 and 5.8519 to 5.8885 G for PM from coal combustion, ranged from 2.0040 to 2.0041 and 3.7846 to 6.9807 G for PM from biomass combustion, and ranged from 2.0042 to 2.0043 and 5.6444 to 8.7616 G for APMs, indicating that the samples were mainly oxygen-centered radicals (phenoxyl and semiquinone radicals) in Xuanwei. In addition, the small ∆Hp-p variability of EPFRs in biomass combustion particulate matter, raw coal combustion particulate matter, and APMs also indicates that EPFRs are of the same type, but contains various organic species or organometallic combinations [27,51].
Research found that coal has a high g-value of 2.0046 in Xuanwei [45], and the g-factors of EPFRs in atmospheric particles vary from 2.0030 to 2.0047 and ∆H p-p of 4.7-7.9 G [27], which are typical of oxygen-centered or oxygen-containing EPFRs, for example, phenoxyl and semiquinone radicals [45,46,52]. In general, both oxygen-centered and carbon-centered radicals are present in atmospheric particulate matter, as oxygen-centered radicals tend to adhere to fine particles, while carbon-centered radicals mostly adhere to coarse particles. For fine particles, more of the porous structure is exposed, thus providing more available active and adsorption sites for EPFRs [53].
Moreover, the presence of semiquinone and phenoxy radicals may lead to activated species in the fine particulate matter in the environment [54,55]. Thus, oxygen-centered radicals appear to be more toxic with fine particles because of their direct effects on the human body; carbon-centered radicals on coarse particles should also be emphasized because of their environmental impact [46,53].

Potential Health Risk of EPFRs
In our study, the main types of EPFRs in PM were phenoxyl and semiquinone radicals. The spectral characteristics of EPFRs were compared with that of cigarette tar, both of them were similar to semiquinone radicals, and identified as semiquinone radicals [56,57], which associated with a quinone/hydroquinone redox cycle capable of producing reactive oxygen species (ROS), to be involved in the carcinogenicity [40].
In this study, to assess the potential health risk of EPFRs in biomass combustion particulate matter, coal combustion particulate matter and APMs for Xuanwei residents, we used the equivalent of cigarettes to represent the potential health risk of EPFRs for adults and children per person per year (  [22]. The above results indicate that the potential health risks of EPFRs in PM varies from region to region and from one combustion source to another.
The results of this study suggest that the health risk of EPFRs is significantly increased when the particle size distribution of EPFRs is taken into account, and coal combustion particulate matter is more hazardous to humans than combustion particulate matter, followed by APMs.

Conclusions and Limitations of the Study
Coal combustion, biomass burning and APMs are considered to be important sources of EPFRs, and in addition, there is little information on individual exposure levels of inhaled EPFRs in the high lung cancer prevalence area of Xuanwei, China. However, the most important thing is that the mechanism of the high lung incidence is not clear. In this study, we conducted simulated combustion experiments (six types of coal, three types of biomass), and six groups of atmospheric particulate matter were collected to explore the content and particle size distribution pattern of EPFRs and potential health risk of EPFRs for adults and children, providing new perspectives and evidence to reveal the high incidence of lung cancer in Xuanwei.

Conclusions
(1) The contribution of EPFRs for biomass combustion, coal combustion and APMs were mainly distributed in the size range of <1. The results suggest that the health risk of EPFRs is significantly increased when the particle size distribution of EPFRs is taken into account, and coal combustion particulate matter is more hazardous to humans than biomass combustion particulate matter, followed by APMs.

Limitations of the Study
There are significant differences in the concentrations and potential health risks in particles of different sizes, and these differences are due to the influence of the source and generation process. In view of the complexity and diversity of the formation process of EPFRs in the actual atmospheric particulates, and EPFR is a formation mechanism that should be more comprehensively studied in the future. There is a lack of information on the ROS generated by the EPFRs transition metal oxide combination through the cellular matrices and tissue. Some attempts should be performed in cell-free and cell-based experiments to obtain well-characterized information about the ROS generated by the EPFRs transition metal oxide combination and to better address the health effects of EPFRs.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/atmos12111467/s1, Figure S1: Sampling sites, Figure S2: Sketch of sampling system, Figure S3: The EPR spectra of EPFRs in APMs particulate matters, Figure S4: The EPR spectra of EPFRs in biomass burning particulate matters, Figure S5: The EPR spectra of EPFRs in coal burning particulate matters, Table S1: Records of atmospheric particulate matter collected in Yunnan residential areas in 2017, Table S2: EPFRs and PM concentrations in biomass combustion particles, Table S3: EPFRs and PM concentrations in coal combustion particles, Table S4: EPFRs and PM concentrations in atmospheric particulate matters, Table S5: g-values and ∆H p-p of the EPFRs produced by different PM from Xuanwei, Table S6; The potential health risk of EPFRs for adults and child per year.