Physicochemical characterization and cytotoxicity of ambient coarse, fine, and ultrafine particulate matters in Shanghai atmosphere

Atmospheric Environment 45 (2011) 736e744

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Physicochemical characterization and cytotoxicity of ambient coarse, fine, and ultrafine particulate matters in Shanghai atmosphere Senlin Lu a, b, *, Man Feng a, b, Zhenkun Yao a, b, An Jing b, Zhong Yufang b, Minghong Wu a, b, *, Guoying Sheng b, Jiamo Fu b, Shinich Yonemochi c, Jinping Zhang d, Qingyue Wang e, Ken Donaldson f a

Shanghai Applied Radiation Institute, Shanghai University, Shanghai 200444, China Institute for Environmental pollution and health, Shanghai University, Shanghai 200444, China Center for Environmental Science in Saitama, Saitama 374-0115, Japan d Shanghai Environmental Monitoring Center, Shanghai 200030, China e School of Science and Engineering, Saitama University, Saitama 338-8570, Japan f ELEGI Colt Laboratory, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2010 Received in revised form 7 September 2010 Accepted 8 September 2010

Epidemiological studies have demonstrated positive relationships between increases in air pollution and adverse health effects. Physicochemical characterization and toxicity of ambient coarse particles (1.8e10 mm diameter), fine particles (1.8e10 mm diameter) and ultrafine particles (<0.1 mm diameter) collected in Shanghai as major air pollutants were investigated. It was found that mass concentrations of different size ambient particles in Shanghai urban atmosphere were higher than those in suburban atmosphere. In addition, the mass concentrations among the different size particles were different. The coarse particles consisted of minerals, while the fine particles were mainly composed of soot aggregates and sulfates; ultrafine particles contained only small amounts of particulates. Crustal elements were mainly distributed in coarse particles, and the anthropogenic elements were mainly found in fine particles. Significant amounts of calcium and magnesium were found in ultrafine particles. Fine particles were found to generate more free radical than coarse and ultrafine particles. Moreover, the results of the cell proliferation assay indicated that ultrafine particles were more cytotoxic than fine and coarse particles. Further investigations are needed to study the mechanism of cytotoxic induced by the ambient particles. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Shanghai Ambient size-resolved particles Physicochemical characterization Free radicals Toxicity

1. Introduction Numerous epidemiological and toxicological studies have demonstrated that increased air pollutants could contribute to adverse health effects (Donaldson et al., 1997, 2005; Mills et al., 2009). Physicochemical characterization and toxicity of ambient inhalable particles such as PM2.5 and PM10, which are major pollutants in the atmosphere with aerodynamic diameters less than 2.5 mm and 10 mm, respectively, have been reported (Lu et al., 2006a, b, 2008; Shao et al., 2007; Li et al., 2009). However, there have been relatively few studies about ambient ultrafine particles (UPF); these particles have aerodynamic diameter <0.1 mm (Geiser et al., 2005; Lin et al., 2005). More recently, it was found that UFPs can efficiently penetrate the respiratory system and even transfer to the extrapulmonary organs,

* Corresponding authors. Shanghai Applied Radiation Institute, Shanghai University, Shanghai 200444, China. Tel.: þ86 21 66137502; fax: þ86 21 66137787. E-mail addresses: [email protected] (S. Lu), [email protected] (M. Wu). 1352-2310/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2010.09.020

including the central nervous system (Oberdörster et al., 2004). Other studies also indicated that UFP exposure was linked to adverse respiratory and cardiovascular health, where stronger associations were found for PM2.5. Furthermore, ultrafine particles have 102e103 times more surface area than fine particles and approximately 105 times more surface area than coarse particles at a given mass (Oberdorster, 2001); the higher surface area-to-mass ratio and the deposition efficiency of UPF are also higher in the alveolar (Duffin et al., 2007). Since size and chemical composition of ambient particles strongly influence their effects on human health, it is crucial to investigate the physicochemical characterization of ultrafine particles in the atmosphere and to evaluate their potential toxicity (Murra and Garza, 2009). We previously reported the physicochemical characterization and potential toxicity of Shanghai PM2.5 and found that the toxicity of urban PM2.5 was greater than suburban PM2.5, while the toxicity of the airborne particles was found to be associated with heavy metals in the particles (Lu et al., 2008). In the current study, the focus was on the comparisons of physicochemical characterization and toxicity of coarse, fine and ultrafine particles in Shanghai’s atmosphere. To our

S. Lu et al. / Atmospheric Environment 45 (2011) 736e744

knowledge, other than the few reports on ambient inhalable particles in Shanghai (Ye et al., 2003; Wang et al., 2005; Fu et al., 2008; Zhang et al., 2009), this is the first study on the physicochemical characterization and bioreactivity of size-resolved particles in Shanghai, which is the biggest commercial city in China. 2. Method and experiment 2.1. Sampling sites Sampling was carried out in Xujiahui (XJH, 311104100 ; 121250 4800 ), the commercial center of Shanghai, and in the satellite city of Jiading (JD, 31220 49.3700 ; 121140 28.6900 ) as shown in Fig. 1. The XJH sampling site was near to the main road (Zhaojiabang Rd.) with almost 30,000 vehicles passing by every day. MOUDI 110 sampler and Nano-MOUDI 125B sampler (MSP Co., Minneapolis, MN, USA) were installed on the 11th floor (about 30 m above ground) of the Shanghai Environmental Monitoring Center building. The JD sampling site was located in Shanghai University (Jiading campus). The distance between this sampling site and the main road (Chengzhong Rd.) was about 200 m. There are nearly 10,000 vehicles travelling on the road every day. The two MOUDI samplers were mounted on the 6th floor (about 15 m above ground) of the No.1 teaching building. The distance between the two sampling sites was about 40 km and there were no visible pollution sources around the two locations.

737

18e10, 10e5.6, 5.6e3.2, 3.2e1.8, 1.8e1.0, 1.0e0.56, 0.56e0.32, 0.32e0.18, 0.18e0.1, 0.1e0.056. The MOUDI 125B impactor could effectively separate these particles into 13 ranges, which included all the size ranges of the MOUDI 110 and also smaller size ranges of 0.056e0.032, 0.032e0.018, 0.018e0.010 mm. Accordingly, the particles were divided into three size groups: coarse particles (1.8e10 mm), fine particles (0.1e1.8 mm), and ultrafine particles (0.1e0.056 mm). Since the concentration of PM0.056e0.032, PM0.032e0.018 and PM0.018e0.010 were low, these particles were not included in the ultrafine particles group. The flow rate of MOUDI 110 and MOUDI 125B sampler was 30 L min1 and 10 L min1, respectively. The particulate matters were collected onto polycarbonate filters (Millipore, UK) with pore size of 0.6 mm. The MOUDI 110 impactor was used continuously (24 h per day) to collect size-resolved particles for 14 days at the JD site and for 21 days at the XJH site. Sampling of the particulate samples was conducted five times at each site and at each time 10 samples were collected from the MOUDI 110, for a total of 100 samples. The NanoMOUDI 125B was concurrently used to collect particles for 48-h sample for Scanning Electron Microscopy (SEM) analysis and the total number of samples collected were 52 (by sampling twice at each site). During sample collection, temperature, humidity, wind speed and wind direction were also recorded (Table 1). After sample collection, the polycarbonate filters were preconditioning for 48 h at constant humidity (40e42%) and temperature (20e22  C), then weighed. The concentration by weight of different size particulate matter was determined by dividing the corresponding weight by the sampled air volume.

2.2. Sampling protocol 2.3. Experimental design The two MOUDI samplers equipped with polycarbonate filters (47 mm diameter) were employed to collect size-resolved ambient particles from December, 2007 to January, 2008. The MOUDI 110 impactor effectively separated the particulate matters into 10 ranges (at 50% efficiency) with the following equivalent cutoff diameter (mm):

The scheme of the experiment is shown in Fig. 2. In the experiment, half of the 24-h samples were analyzed by ICP-MS/AES and the other half were used for Electron Paramagnetic Resonance (EPR) analysis and MTT (3-(4,5-Dimethylthiazol-2-y1)-2,5diphenyl tetrazolium bromide) assay. Coarse, fine and ultrafine particle samples (48-h samples) collected by MOUDI 125B at the two sites were randomly selected for SEM analysis. 2.4. Experimental methods 2.4.1. Particle solution preparation In order to move the particles from the filter, polycarbonate filters were immersed in 5 mL deionized water in eppendorf tubes for 1 h and then sonicated (300 W, 30 kHz) for 30 min. The filters were allowed to air dry at room temperature. After the dried filters were weighed, weight of particles in the solution was calculated,

Table 1 Meteorological data by site during sampling campaign. Sampling site

Fig. 1. Map of sampling sites in urban (Xujiahui) and suburban (Jiading) region of Shanghai. The distance between the two sites is about 40 km.

Sampling date

Temp.( C)

Hum.(%)

max.

min.

max.

min.

Wind speed (km h1)

Jiading (JD)

07/11/22e07/11/23 07/11/27e07/11/28 07/11/28e07/11/29 07/11/29e07/11/30 07/12/1e07/12/2 07/12/3e07/12/4 07/12/5e07/12/6

18 12 12 14 17 11 12

7 10 4 4 9 6 3

81 71 70 76 88 76 87

42 33 35 47 55 44 44

7 15 10 9 10 13 8

Xujiahui(XJH)

08/1/4e08/1/5 08/1/5e08/1/6 08/1/6e08/1/7 08/1/8e08/1/9 08/1/16e08/1/17 08/1/18e08/1/19 08/1/21e08/1/22

12 13 16 11 4 6 5

1 1 5 5 2 1 3

87 93 87 93 81 93 87

28 35 51 66 56 61 70

3 4 10 7 17 9 15

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Selected samples

Divide the sampled filters into 3 groups, i.e. coarse /fine /ultrafine particles and conduct SEM observation

Chemical elements analysis by using of ICP-MS/AES

Free radical investigation by EPR

Toxicological assessment by MTT assay

Conclusions Fig. 2. Flow diagram of the study.

and then stock solution of the particles was calculated as well. This was mass dosage of whole sample. The sample was centrifuged at 3000 rpm for 30 min. The supernatant (2 mL) was carefully removed and used for ICP-AES/MS, EPR and MTT assay. 2.4.2. Cell culture The type II human alveolar epithelial cell line A549 was maintained in continuous culture in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FCS), 2 mM glutamate, 100 IU mL1 streptomycin and 100 mg mL1 penicillin. Cells grew to confluency at 37  C in a humidified atmosphere containing 5% CO2, and washed with phosphate-buffered saline (PBS), then harvested with Trypsin-EDTA. Cells were seeded at 0.7  106 in 96-well culture plates in triplicates for assessment of cell viability. 2.4.3. Scanning electron microscopy The method was described by Lu et al. (2006a) in detail. Briefly, SEM images were obtained on a HITACHI SEM (Hitachi-4700) equipped with an energy dispersive X-ray system (EDX). The EDX spectrometer was a HITACHI SEM/EDX integration system with a Si (Li) detector which allows X-ray detection from elements higher than carbonate (Z > 6). Operation conditions were 20 keV accelerating voltage and 600 pA beam current with spectral acquisition times of 30e100 s. 2.4.4. ICP-MS/AES analysis The supernatant (2 mL) extracted from the filter was diluted to 8 mL with 2% HNO3 for ICP-MS and ICP-AES analyses. The remaining solution was transferred to a vessel and enriched in an ETHOS1600 microwave lab station (Milestone, USA). After enrichment, the ambient particles in the vessel were digested by concentrated HNO3 (61%) for 1 h and then the digestion was repeated again, followed by washing with 2% HNO3 for 3 times. This solution was filtered through a membrane filter (0.45 mm) and added with 200 ppb indium to make a 25 mL solution for analysis. A total of 17 elements in the particle sample solutions were analyzed by ICP-AES (K, Na, Ca, Mg, Al, Fe), and ICP-MS analysis (As, Cu, Zn, Pb, Cd, Cr, Mn, Ba, Sr, Ni, Se). Considering that sensitivity of ICP-AES is lower than that of ICP-MS, we selected ICP/AES for crustal elements analysis and ICP/MS for anthropogenic elements analysis. 2.4.5. EPR detection Samples were prepared in double-distilled water for EPR investigation. The generation of free radicals from the particles suspensions was studied in the presence of spin trap Tempone-H (Alexis Co., Bingham, UK). A solution of 5 mM Tempone-H (10 mL) in

EDTA was mixed with an aqueous particle suspension (495 mL) in an eppendorf tube at 37  C, then transferred immediately to a 50 mL glass capillary and analyzed on the Bruker EMX-E EPR spectrometer (Bruker, Germany). Hydrogen peroxide (100 mM) was selected as a positive control. The EPR-spectra were recorded at room temperature using the following conditions: microwave frequency was 9.39 GHz, magnetic field was 3501.8 G, sweep width at 100 G, scan time of 30 s, number of scans was 3, modulation amplitude was 1.0 G, receiver gain was 7.96  104, and the g value was 2.0059. 2.4.6. MTT assay Viability of the epithelia cell (A549) was determined using the MTT assay. MTT-solution (25 mL) was added to cell culture medium (100 mL) in the wells of a 96-well plate. The plate was incubated for 4 h at 37  C. Thereafter, DMSO-extraction buffer (100 mL) was added and incubated overnight at 37  C. MTT-test measures the ability of the cells to transform MTT to formazan which can be spectrophotometrically detected at 570 nm on a microplate reader. Viability of the cells was calculated as a percentage by comparing the absorbance from cell suspensions exposed to particulate samples with those from corresponding control cell suspensions. 2.4.7. Statistical analysis The data for mass concentration of ambient particles and ICPMS/AES were analyzed by Excel and expressed as means  STD (n ¼ 5). The MTT assay data was analyzed using SPSS software version 13.0 (SPSS Inc. Chicago, IL, USA). 3. Results 3.1. Mass concentration of ambient coarse, fine and ultrafine particles collected in Shanghai The monitoring data revealed that the average concentrations of different size range air particles collected from the JD and XJH sites were different. Average mass concentrations of XJH size-resolved particles were higher than those of JD particles except PM0.1e0.18 and PM0.56e1.0 (Fig. 3). The highest average mass concentration was found in the 0.32e0.56 mm particle size range, while the lowest average mass level of ambient particles was found in the range of 0.18e0.32 mm and 0.56e1.0 mm for the JD and XJH samples, respectively. When these different size particles were divided into coarse, fine and ultrafine particles size groups, the corresponding average mass concentration in the JD and XJH air samples were

Fig. 3. Average mass concentration of size-resolved particles collected by MOUDI 110 sampler in Shanghai urban and suburban site.

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Fig. 4. Microscopic characterization of size-resolved particles collected in Jiading air. a e particles with diameter 3.2e5.6 mm, scale bar, 5 mm; b e particles with diameter 1.0e1.8 mm, scale bar, 2 mm; c e particles with diameter 0.32e0.56 mm, scale bar, 4 mm; d e particles with diameter <0.1 mm scale bar, 10 mm; and in Xujiahui air, e e particles with diameter 3.2e5.6 mm, scale bar, 2 mm; f e particles with diameter 1.0e1.8 mm, scale bar, 4 mm; g e particles with diameter 0.32e0.56 mm, scale bar, 5 mm; h e particles with diameter <0.1 mm scale bar, 1 mm.

6.0  3.3, 7.8  4.6, 4.6  3.1 mg m3 and 16.2  3.8, 12.9  8.4, 2.7  1.4 mg m3, respectively. 3.2. Comparison of microscopic characterization of different size particles in Shanghai urban and suburban atmosphere The SEM results revealed that microscopic characterization of Shanghai ambient size-resolved particles showed differently (Fig. 4). As shown in Fig. 4-a,e, minerals were the main particles found in the coarse particles (3.2e5.6 mm), while minerals with regular microshape, fly ashes and soot aggregates were the main components in fine particles with size ranges of 1.0e1.8 mm and 0.32e0.56 mm (Fig. 4-b,c,f,g). Soot aggregates were the main components in

ultrafine particles of <0.1 mm (Fig. 4-d,h). The SEM results also revealed that the number and phases of particles from the suburban (JD) samples (Fig. 4-aed) were less than those of urban (XJH) samples (Fig. 3-eeh), suggesting that pollution in suburban site is lower compared to urban atmosphere.

3.3. Concentrations of chemical elements The concentration of chemical elements in ultrafine particles collected on one filter was below the detection limits of the ICP-MS/ AES. Therefore, the ultrafine particle filters were combined into one group for analysis.

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3.3.1. Mass concentration of elements found in whole sample solution The ICP-AES/MS results (Fig. 5) showed that total mass concentrations of crustal elements and anthropogenic elements distributed in different particle size groups were different among samples collected from the urban site and suburban site. The highest concentration of crustal elements (9082.06 ng m3) was found in JD coarse particles, while the highest concentration of anthropogenic elements (676.9 ng m3) was found in XJH fine particles. The lowest concentration of crustal elements (3254.2 ng m3) was in XJH coarse particles, and the lowest concentration of anthropogenic elements (4.17 ng m3) was in JD coarse particles. The total mass level of crustal elements in the sample solutions was ranked in the following decreasing order: coarse > fine > ultrafine particles; the order of the anthropogenic elements was ranked as follows: fine particles > coarse particles > ultrafine particles. Calcium was the most abundant crustal elements, where its concentration in the coarse, fine and ultrafine particles of JD atmosphere was 3387.49  3155.19, 3513.89  2333.16 and 2616.23 ng m3, respectively. Correspondingly, the calcium concentrations in XJH coarse, fine and ultrafine particles were 1413.54  1059.6, 2241.35  1214.41 and 1999.90 ng m3, respectively. The mass level of the other crustal elements was ranked in the order: Mg > Na > Fe > K > Al. Zinc has the highest mass concentration among the anthropogenic elements in XJH fine particles at 211.28  104.26 ng m3, while its concentration in XJH coarse and ultrafine particles was 138.63  78.68 and 110.78 ng m3, respectively. The Zn concentration in JD coarse, fine and ultrafine particles was 4.96  4.23, 113.21  108.6 and 16.67 ng m3, respectively. The major metallic elements found in XJH fine particles were Cr, Se, Cu, Mn, Pb, Ni, Ag and Co at 133.39  16.09,

109.02  12.13, 95.37  10.97, 35.62  22.67, 28.94  17.47, 14.55  4.46, 12.06  1.89 and 11.26  1.55 ng m3, respectively. The major metallic elements in JD fine particles were Cr, Mn, Cu, Se, Pb, Ni and As at 103.47  42.76, 76.62  70.13, 66.69  21.3, 58.84  27.09, 52.64  42.96, 30  24.23 and 13.96  12.02 ng m3, respectively. 3.3.2. Mass concentration of elements in soluble fraction The ICP-MS/AES results (Fig. 5) showed that the mass concentration of total crustal elements and total anthropogenic elements in the soluble fraction of JD air were 12,333.4 and 771.31 ng m3, and in XJH air were 11,549.64 and 909.59 ng m3, respectively. Mass levels of crustal elements in JD coarse, fine and ultrafine particles were 4420.95, 3646.51 and 4254.94 ng m3, respectively (Fig. 5-c); their mass levels in XJH coarse, fine and ultrafine particles were 3254.2, 4078.92 and 4216.52 ng m3, respectively (Fig. 5-g). Mass levels of anthropogenic elements in JD coarse, fine and ultrafine particles were 4.17, 553.04, and 214.1 ng m3, respectively; their levels in XJH coarse, fine and ultrafine particles were 369.12, 359.26 and 181.21 ng m3, respectively. The highest mass concentration of crustal elements in the soluble fraction was found in JD coarse particles (4420.95 ng m3), and the lowest mass concentration was in XJH coarse particles (3254.2 ng m3). The mass concentration of anthropogenic elements was highest in JD fine particles (553.04 ng m3) and lowest in coarse particles (4.17 ng m3). Mass level of crustal elements in the soluble fraction of the JD particles can be ordered as coarse particles (4420.95 ng m3) > ultrafine particles (4254.94 ng m3) > fine particles (3646.51 ng m3); their corresponding concentration in XJH particles was ranked as ultrafine particles (4216.52 ng m3) > fine particles (4078.92 ng m3) > coarse particles (3254.2 ng m3). In general, the mass concentrations of anthropogenic elements in the soluble fraction

Fig. 5. Mass concentration of chemical elements in Shanghai size-resolved particles a,b e crustal elements and anthropogenic elements in JD particles (whole sample solution); c,d e crustal elements and anthropogenic elements in JD particles (soluble fraction); e,f e crustal elements and anthropogenic elements in XJH particles (whole sample solution); g,h e crustal elements and anthropogenic elements in XJH particles (soluble fraction).

S. Lu et al. / Atmospheric Environment 45 (2011) 736e744

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were found in the soluble fraction and whole sample of XJH coarse and fine particles. At 50 mg mL1, MTT values for the whole sample of JD coarse and fine particles were 87  6% and 84  4%, while the MTT values for their soluble fraction were 83  16% and 78  5%, respectively. Similarly, MTT levels for the whole sample of XJH coarse, fine and ultrafine particles were 77.3  11%, 80  10% and 72  8%, respectively, and the levels for their soluble fraction were 88.3  14%, 90.5  11% and 78  13%, respectively (Fig. 9). 6. Discussion 6.1. Mass concentration of coarse/fine/ultrafine particles

Fig. 6. Free radical intensity induced by whole sample and soluble fraction of XJH coarse, fine, ultrafine particles after incubation with the generic spin trap Tempone-H for 40 min at 37  C. H2O2 (100 mM) as a control.

Mass concentration of ambient particles in Shanghai atmosphere showed seasonal variations (Lu et al., 2008), but meteorological condition is relatively stable in winter season and thus more

of JD particles and XJH particles have the same order: fine particles > coarse particles > ultrafine particles.

120%

4. EPR activity of PM

100%

soluble fraction

5. MTT assay

80%

40% 120%

b 100%

The MTT assay results showed that coarse/fine/ultrafine particles (XJH whole sample solution) produced decreases in MTT levels at 11.25, 25, 50 and 100 mg mL1 (because the maximum mass stock dose of ultrafine particle solution were 50 mg mL1, decrease in MTT level caused by ultrafine particles could not be compared with others) respectively (Fig. 8). No significant decreases in MTT values

80%

60%

40% 120%

c

8000

100% cell viability%

Free radical int.(a.u.)

a

60%

cell viability%

Ambient particles from XJH atmosphere were selected for the EPR study. After the ambient particles solution was incubated with spin trap Tempone-H, free radicals generated by the particles can be detected and the intensity of free radicals would be increased with time (data not shown). As shown in Fig. 6, the EPR results indicate that fine particles could generate stronger free radical signals than those of coarse and ultrafine particles. Moreover, free radical signals generated by the soluble fraction of XJH particles were stronger than the corresponding whole sample fraction. It is noteworthy that XJH ultrafine particles can produce much stronger free radical signals compared with its whole sample fraction. The results also demonstrated that more radicals were generated by XJH particles than by JD particles (Fig. 7). It was interesting that free radicals signal generated by JD particles (whole sample solution) was in the order: coarse z fine > ultrafine, which were different with that of XJH particles.

cell viability%

whole sample

6000

4000

80%

60% 2000

40% 0 H2O2

JD-C

JD-F

JD-UF

XJH-C

XJH-F

XJH-UF

Treatments Fig. 7. Comparison of intrinsic ability of free radicals induced by JD and XJH ambient size-resolved particles (whole sample solution) after incubation with the generic spin trap Tempone-H for 60 min at 37  C. W e whole sample solution, and S e soluble fraction, H2O2 (100 mM) as a control.

0

12.5

25

50

100

mass dosage ( µg/ml) Fig. 8. Size-resolved ambient particles (Xujiahui whole sample) cause dose-related cytotoxicity. a e ultrafine particles, b e fine particles, c e coarse particles. Each bar shows the mean percentage of viable cell SEM of three independent 4-h exposures in duplicate compared to unexposed control cell cultures.

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cell viability %

120%

solbule fraction

whole sample

80%

40%

0% JD-Coarse

JD-Fine

XJH-Coarse

XJH-Fine

XJHUltrafine

Treatments Fig. 9. Cell viability as assessed by the MTT-test after a 4-h incubation of A549 epithelial cell with JXH and JD, the whole sample and the soluble fraction at 50 mg mL1. Each bar shows the mean percentage of viable cell SEM of three independent 4-h exposures in duplicate compared to unexposed control cell cultures.

particles could be easily collected. Therefore, the air particle sampling was performed during winter (from November, 2007 to January, 2008). Temperature, relative humidity (RH) and wind speed at the two sampling sites are summarized in Table 1. The meteorological data were representative of prevailing winter conditions in Shanghai. The sampling data showed that average mass concentrations of the particles in urban atmosphere were higher than those in suburban atmosphere. In particular, particles with sizes ranging from 0.32e0.56 to 1.0e1.8 mm had higher mass concentrations. This agrees with Gnauk’s report (Gnauk et al., 2008), which showed that the higher daytime mass concentration in the atmosphere of Pearl River Delta, China, was found in samples with particle size of 0.32e0.56 mm. The higher mass could be caused by emissions from vehicles and domestic fires. Considering the large volume of traffic on Zhaojiabang Road (30,000 day1) and Chengzhong Road (10,000 vehicles day1), a higher mass level of fine particles in the atmosphere is reasonable. Compared with the mass level of coarse (6.3  0.88 mg m3), fine (5.8  0.95 mg m3) and ultrafine (1.7  0.67 mg m3) particles near the I-710 freeway in Los Angeles (Ntziachristos et al., 2007), the airborne particle pollution in Shanghai urban atmosphere is serious. It is worth noting that mass concentration of ultrafine particles in suburban region (JD, 4.56  3.13 mg m3) was slightly higher than in urban area (XJH, 2.71  1.44 mg m3). This phenomenon was probably caused by a higher number of ultrafine particles and more secondary particles being formed in suburban atmosphere as demonstrated by the SEM images shown in Fig. 4. 6.2. Physicochemical characterization of size-resolved particles in Shanghai atmosphere Microscopic characterization of urban ambient particles using SEM/EDX analysis has been reported (Shi et al., 2003a; Lu et al., 2007, 2008), but few studies have reported microscopy of ambient ultrafine particles. Our microscopic characterization results indicated that different size particles in Shanghai atmosphere have different chemical components. The coarse particles mainly consisted of minerals (Fig. 4-a,e), which originated from crustal sources. The fine particles were composed of minerals and soot aggregates (Fig. 4-d,h), which were mainly from vehicle exhaust emission, fly ashes emitted by coal combustion (Fig. 4-b,c,f,g), while ultrafine particles consisted of soot aggregates (Fig. 4-d,h). Furthermore, the mineral particles found in Shanghai air can be divided into irregular shaped mineral particles (Fig. 4-a) and regular shaped mineral particles (Fig. 4-e). The regular particles with crystal shape consisted of Ca, S, N and O (data not shown), suggesting that these crystals might be sulfates formed by atmospheric reactions, similar

to particles seen in a case study in Phoenix Arizona reported by Katrinak et al. (1995). The irregular shaped minerals consisted of Al, Si, O and Ca, which were most likely from geological sources (Shi et al., 2003b; Lu et al., 2007), and therefore, microscopic characterization of different size particles can be used to identify particle sources. Chemical elements in Shanghai size-resolved particles were divided into crustal elements (K, Na, Ca, Mg, Al, Fe) and anthropogenic elements (As, Cu, Zn, Pb, Cd, Cr, Mn, Ba, Sr, Ni, Se). Our results showed that crustal elements were mainly distributed in coarse particles, while the anthropogenic elements were richer in fine particles. Chemical elements in the ultrafine particles were relatively lower than those in coarse and fine particles. The highest concentrations of elements were found in the PM0.18e2.5 fraction as previously reported (Ntziachristos et al., 2007). When mass level of individual elements was compared, most of the chemical elements fell into the following concentration profile with particle size. First, mass concentration of several elements such as Na, Al, K, Fe, Cr and Se (in XJH sample) was found to increase with increasing particle size (Fig. 5-a,e,h). In contrast, the mass levels of Ca and Mg in JD soluble fraction decreased with increasing particle size (Fig. 5-c). Similar trends were also reported (Ntziachristos et al., 2007) for Na and Fe in Los Angeles atmosphere, and for Na, Al, Fe near a busy road in Taiwan (Lin et al., 2005). These elements mainly originated from mechanical processes or crustal materials (Ntziachristos et al., 2007). Calcium was reported as the most abundant elements in Shanghai PM2.5 (Lu et al., 2008). In this study, the mass concentration of calcium was found to be the highest among the measured elements and had no significant difference in different size particles and in different fractions. This suggests that (1) the calcium originated from crustal sources and was mainly distributed in minerals in the coarse particles; (2) Ca could participate in atmospheric reactions and lead to the formation of secondary mineral particles in fine particles (Fig. 3-g) and ultrafine particles (SEM images not shown). The abundant Ca distribution in Shanghai ultrafine particles was similar to the ultrafine particles in Bakersfield, California, which was found to contain significant amount of alkaline elements, such as calcium (Chung et al., 2001). The second characterization of the chemical element mass level does not uniformly increase with particle size. They were much richer in fine particles than in coarse particle or in ultrafine particles. This is the case for trace elements, such as Zn, Pb, Cu, Mn, Ni. Our results were in agreement with the findings from Lin et al. (2005), where they reported a high concentration of sub-major trace metals (Zn, Pb, Mn, Cu, Ni) from a busy road was distributed in the 1.0e1.8 mm particle size fraction. Mass concentration of Zn in the different fractions varied widely. The highest zinc content was found in XJH fine particles (whole sample, 211.28  104.3 ng m3) and the lowest zinc level was in JD coarse particles (soluble fraction, 2.92  2.09 ng m3), suggesting that Zn was easily absorbed on fine particles. Higher mass concentration of Zn in ambient particles has also been reported in the atmosphere of Cardiff (Moreno et al., 2004), Beijing (Lu et al., 2006a, b) and Shanghai (Lu et al., 2008; Fu et al., 2008). As shown in Fig. 5, Pb was mainly distributed in Shanghai fine particles, which was in agreement with a previous report (Li et al., 2009). However, compared with the results from 2002 to 2006 where the average mass concentration of Pb in JD PM2.5 was 109 ng m3, the concentration of Pb in JD whole sample has significantly decreased to 52.64  42.96 ng m3. This could be a result of the ban on leaded gasoline since 1997 (Fu et al., 2008). The concentration of Ni in JD fine particles (30  24.3 ng m3) was higher than in XJH fine particles (14.6  4.5 ng m3), which was above the range (2.6e4.0 ng m3) for PM10-Ni set by the WHO for unit carcinogenic risk (WHO, 2000). The highest Mn

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concentration was found in JD fine particles (76.62  70.13 ng m3), but was below the WHO’s guideline value of 150 ng m3. The concentrations of V and Cr were lower than the WHO guideline value of 1000 ng m3 for PM10-V, Cr. The third characterization of mass concentration of Cr, Cu and Se in XJH soluble fraction of the coarse and fine particles showed no significant difference. The source of these anthropogenic elements needs further investigation. Mass levels of the other elements, such as As, Cd, Mn, Ba and Sr were higher in urban ambient particles than in suburban airborne particles. Furthermore, comparison of the mass concentration of trace elements found no significance difference between the whole sample and the soluble fraction, suggesting that the trace elements in the samples were soluble. 6.3. Free radicals generation The particle’s ability to generate free radicals could be used to assess their potential toxicity (Shi et al., 2003b; Donaldson, 2005). In this study, Tempone-H, a highly sensitive but non-specific spin trap, was selected to explore the intrinsic ability of the size-resolved ambient particles to generate free radicals. The results shown in Figs. 6 and 7 indicate that fine particles (whole sample and soluble fraction) could generate stronger free radical signals than coarse and ultrafine particles. Many studies have reported that free radicalegenerating potential of ambient particles is influenced by the chemical compositions in the particles, such as the presence of transition metals, PAH or Quinones (Li et al., 2008). For example, hydroxyl radicals can be generated by available iron via the Fenton reaction ðFe2þ þ H2 O2 þ Hþ /Fe3þ þ H2 O þ OHÞ and several other “Fenton active” transition metals that usually occur in ambient particles, such as chromium, vanadium, and copper are also known to induce OH formation (Donaldson et al., 1997; Shi et al., 2003a). Therefore, we hypothesized that higher content of transition metals would lead to higher free radical signals. In general, the free radical signals generated by XJH whole sample fraction can be ranked in the following order: fine > coarse > ultrafine. This trend was in agreement with the trace metals distribution, where higher concentrations of trace elements were found in fine particles. However, free radical signals produced by the XJH soluble fraction showed no significant difference. This was partly due to the mass concentration of trace metals (i.e. Cr, Cu and Se) was similar in coarse and fine particles. Meanwhile, the relatively high free radical signals produced by the soluble fraction of XJH ultrafine particles with lower mass concentration could be explained by the “ultrafine hypothesis” (Seaton et al., 2009), which proposed that ultrafine particles have larger surface areas and thus higher activity than coarse and fine particles. However, the free radical signals generated by the whole sample fraction of XJH ultrafine particles were lower than those of the soluble fraction. The results were unexpected and require further investigation to explain. We also noticed that free radicals signals generated by JD coarse particles and fine particles were stronger than those of JD ultrafine particles (Fig. 7), which could be attributed to a lower concentration of transition metals in the JD ultrafine particles. 6.4. Cytotoxitiy of ambient coarse/fine/ultrafine particles The MTT assay was used to determine the cytotoxicity of chemicals such TiO2 and carbon back from the particles (Renwick et al., 2001). We proposed that cytotoxicity of the ambient particles was related to their chemical compositions and the generation of free radicals was the main toxicological mechanism, in which ultrafine particles have a greater surface area and thus greater ability to generate free radicals and induce oxidative stress compared to larger particles (Donaldson et al., 1998). The MTT results (Fig. 8) showed that a low concentration (11.25 mg mL1) of ambient size-resolved particles could decrease

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the viability of lung epithelium cells (A549). Moreover, the MTT value was found to decrease with increasing particles concentration; but no significant decreases in MTT values were observed from the soluble fraction and whole sample of XJH coarse and fine particles (Fig. 9). This is consistent with the distribution of anthropogenic elements (transition heavy metals) in XJH particles, where there is no significant difference in mass concentration of anthropogenic elements in the whole sample solution and the soluble fraction (Fig. 5). It is worth noting that XJH ultrafine particles decreased the cell viability significantly compared to that of XJH coarse particles and fine particles at the same concentration. This could be attributed to the higher surface area of ultrafine particles. However, mass level of trace elements and free radical generation capability were comparably lower in XJH ultrafine particles. Therefore, more studies are needed to explain the surface area effects caused by ultrafine particles. 7. Conclusions Our results indicated that (1) mass concentrations of ambient size-resolved particles collected in Shanghai urban atmosphere are higher than those in suburban atmosphere, and the mass level of different size particles are different. The highest mass concentration of ambient particles was found in PM0.56e0.32, while the lowest was found in PM1e0.56 and in particles with size diameter less than 0.056 mm (2) Coarse particles in Shanghai atmosphere were composed minerals, fine particles were mainly composed of soot particles and sulfates, and only few particles were found in the ultrafine fraction. (3) Crustal elements in Shanghai atmosphere were mainly distributed in coarse particles, while the anthropogenic elements were concentrated in fine particles. Significant amounts of calcium and magnesium were found in Shanghai ultrafine particles. (4) The free radical signals generated by ambient fine particles were higher than those of coarse and ultrafine particles. In general, free radicals signal of urban ambient particles was stronger than that of suburban ambient particles. (5) Ultrafine particles were found to be more cytotoxic to A549 cells than fine and coarse particles. However, further investigations are needed to elucidate the mechanism of toxicity due to the different size particles fractions. Acknowledgements The research was granted by the NSFC (Grant No.40675080, 10775094, 40973072) and by Shanghai Pujiang Talent Program, the Shanghai Committee of Science and Technology (Grant No. 10JC1405500), Innovation Program of Shanghai Municipal Education Commission (Grant No. 11ZZ80). Shanghai Leading Academic Discipline Project (No. S30109). We thank the two anonymous reviewers for their suggestions. References Chung, A., Herner, J.D., Kleeman, M.J., 2001. Detection of alkaline ultrafine atmospheric particles at Bakersfield, California. Environmental Science and Technology 35, 2184e2190. Donaldson, K., Brown, D.M., Mitchell, C., Dineva, M., Beswick, P.H., Gilmour, P., MacNee, W., 1997. Free radical activity of PM10: iron-mediated generation of hydroxyl radicals. Environmental Health Perspectives 105, 1285e1289. Donaldson, K., Li, X., MacNee, W., 1998. Ultrafine (nanometer) particle mediated lung injury. Journal of Aerosol Science 29, 553e560. Donaldson, K., Tran, L., Jimenez, L.A., Duffin, R., Newby, D.E., Mills, N., MacNee, W., Stone, V., 2005. Combustion-derived nanoparticles: a review of their toxicology following inhalation exposure. Particle and Fibre Toxicology 2, 10. doi:10.1186/ 1743-89977-2-10. Duffin, Rodger, Tran, Lang, Brown, David, Stone, Vicki, Donaldson, Ken, 2007. Proinflammogenic effects of low-toxicity and metal nanoparticles in vivo and in vitro: highlighting the role of particle surface area and surface reactivity. Inhalation Toxicology 19 (10), 849e856. Fu, Qingyan, Zhuang, Guoshun, Wang, Jing, Xu, Chang, Huang, Kan, Li, Juan, Hou, Bing, Lu, Tao, Streets, D.G., 2008. Mechanism of formation of the heaviest

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