Are the new atmospheric health risk substances EPFRs oxidatively toxic? : Results from a large urban road dust study in Northwest China

: 10 Atmospheric particulate matter (PM) is an important factor of premature human 11 mortality due to PM inhalation. However, the toxic and hazardous components 12 contained within the atmospheric PM are still not fully identified. Environmental 13 persistent free radicals (EPFRs), a novel environmental health risk substance in the 14 atmosphere, have been hypothesized to be important contributors to human respiratory 15 health risks due to their potential to enhance the oxidation potential of PM, but this 16 hypothesis has not been compelling confirmed in the actual atmospheric environment. 17 Therefore, the fugitive characteristics of EPFRs in dust and their potential oxidative 18 toxicity were investigated by using road dust from a mention city in northwest China. 19 The research results showed that the road dust in Xi'an is rich in C-centered EPFRs 20 ((6.6±5.0) ×10 17 spins/g), and its half-life can reach 4.5 years. Water-insoluble dust was 21 the main contributor (71%) to the oxidative toxicity of road dust, showing a rapid 22 toxicity-producing process, while the rate of oxidative toxicity production was more 23 constant for water-soluble dust. The contribution of the EPFRs dominant factor to the 24 total dust oxidative toxicity was estimated to be 17% based on the positive matrix 25 factorization (PMF) model, and up to 33% for water-insoluble dust oxidative toxicity. 26 Metals and organic carbon were the main contributing components to the oxidative 27 toxicity of the WS fraction. Summarily, this study demonstrated that the EPFRs are an 28 important contributor to the oxidative toxicity of in actual atmospheric PM, and their 29 oxidative toxicity is dependent on the type of free radicals, providing important insights 30 into what other potentially toxic substances contribute to the oxidative toxicity of 31 atmospheric PM.

EPFRs are a kind of radicals with lifetimes ranging from minutes to years and can be 48 existed steadily in the environment 13 , which is proposed in comparison with short-lived 49 radicals such as hydroxyl radicals (•OH). 50 The primary origins of EPFRs in the atmospheric environment are vehicle exhaust 51 emissions, industrial emissions, and emissions from combustion sources such as coal 52 combustion. However, in recent years, several studies have been discovered that EPFRs 53 can also be originated from road dust, and EPFRs produced from road dust contributes 54 up to 27% in urban areas of northwest China 14 . Meanwhile, some research indicated 55 that dust particles can carry EPFRs for long-range transport during dust storms 15 . The 56 metallic minerals in dust particles provide the prerequisite for the formation and 57 existence of EPFRs, such as Fe 2+ , Cu 2+ , Mn 2+ , and Zn 2+ . These metallic minerals 58 possess paramagnetic characteristics and induce the probability to attach with the 59 particles in the EPFRs generation process 16,17,18 . Currently, the EPFRs generation 60 mechanism with the involvement of transition metals described specifically as follows: 61 aromatics adsorbed onto the surface of transition metal oxides, and then the electrons 62 4 of aromatics are transferred to transition metal ions, resulting the metal ions forming 63 stable single-electron structures while being reduced 19,20 . Meanwhile, some research 64 also suggest that EPFRs can still be formed without the involvement of transition metals. 65 For example, the coking process of organics to form graphite-like material could 66 produce EPFRs, and this process is considered to be a main mechanism for the 67 production of EPFRs in atmospheric PM 21 . The health effects of EPFRs, which are 68 generated in large quantities through different mechanisms and attached to atmospheric 69 particulate matter, have been hotly debated. 70 To date, the health risk and toxic mechanism of EPFRs in atmospheric PM is 71 unclear. The paradoxical view is that EPFRs are long-lived free radicals but they are 72 reactive. It is supposed that EPFRs play the role of catalysts to generate ROS. More

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According to the electron paramagnetic resonance (EPR) analysis of the collected 105 samples, it was determined that road dust contained EPFRs with a mass concentration 106 range of (1.4~40) × 10 17 spins/g, and an average mass concentration of (6.6±5.0) × 10 17 107 spins/g, the specific concentration of samples shown in Table S1. As shown in Fig. 1 108 the level of concentration is comparable to the results that have been reported. However, 109 compared with the concentration of EPFRs in PCP-contaminated soil (2.02×10 18 110 spins/g), uncontaminated soil (7×10 16 spins/g), asphaltene (6.7×10 18 spins/g), and Xi'an 111 atmospheric fine PM (2.65×10 18 spins/g) 28 residents, and thus the health risks caused by road dust must be considered need to be 119 taken into account especially in dusty cities in northwest China. As shown in Fig. 1, the 120 city of Xi'an was divided into four regions based on the trajectories of metro lines 1 and 121 2, and found that the mass concentrations of EPFRs in road dust were significantly 122 higher in region II, the eastern part of Xi'an, than in the other three regions. This is due  Based on the signal characteristics of EPFRs, it was found that the EPFRs of road 146 dust is mainly C-centered radical types. As we all know, the g factor is one of the most  185 To investigate the lifetime of EPFRs in road dust, EPR analysis was performed 186 again on samples placed at room temperature and protected from light for 450 days, 187 and it was found that EPFRs are decayable in road dust, and information on the

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The obtained EPR spectra were segmented by using the least squares method to fit 217 10 the spectra of different types of EPFRs, and the signal characteristics (g-factor and △ 218 Hp-p) and intensity of C-centered EPFRs (C-EPFRs) and O-centered EPFRs (O-EPFRs) 219 were obtained, respectively 34 . The results are shown in Fig. 3a  The study used the consumption rate of the reducing substance dithiothreitol (DTT) 236 in simulated lung fluid contains road dust samples to represent its oxidative toxicity 237 11 level, and the results showed that the collected road dust was oxidatively toxic. As 238 shown in Fig. 4, the total oxidative toxicity of road dust (Total-DTT) was (0.34 ± 0.08) 239 μmol/min/g, which is smaller than both the oxidative toxicity of atmospheric PM2.5   The pie charts represent the ratio of DTT consumption to Total-DTT consumption in road dust 290 without fractions. 291

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In this study, it was found that EPFRs contribute to the oxidative toxicity of road 293 dust and that the toxicity of EPFRs is related to their type. The oxidative toxicity of  As shown in Fig. 6, it was found that Total-DTT and WIS-DTT showed a weak 307 correlation with the concentrations of EPFRs after decay, but not with any other 308 components, indicating that EPFRs may contribute to the oxidative toxicity of road dust.

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To concretely investigate the contribution of EPFRs to oxidative toxicity in road dust, contributed 20% of the WS-DTT, but the metals actually contributed much more than 357 that (Fig. S9), as more metal elements were present in the complex-type factor (62% of   and chromium standard, respectively, and mdust represents the road dust mass (g). In this 399 study, the EPR absorption curve area was expressed as the peak-to-peak distance 400 multiplied by the line width squared.

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To determine the decay behavior of EPFRs in road dust, the road dust samples 402 after EPR detection were placed at room temperature (temperature 25°C ± 5°C and 403 relative humidity of 60 ± 5%) and analyzed for EPR again after an interval of 450 days.