Environ. Sci. Technol. 2008, 42, 4229–4235
PAHs, PAH-Induced Carcinogenic Potency, and Particle-ExtractInduced Cytotoxicity of Traffic-Related Nano/Ultrafine Particles
The mean content of the particle-bound total-PAHs/-BaPeqs and the PAH/BaPeq-derived carcinogenic potency followed the order nano > ultrafine > fine > coarse. For a sunny day sample, the cytotoxicity of particle extracts (using 1:1 (v/v) n-hexane/dichloromethane) was significantly higher (p < 0.05) for the nano (particularly the 10-18 nm)/ultrafine particles than for the coarser particles and bleomycin. Therefore, trafficrelated nano and ultrafine particles are possibly cytotoxic.
C H I H - C H U N G L I N , †,‡,§ S H U I - J E N C H E N , * ,†,‡ K U O - L I N H U A N G , †,‡ W E N - J H Y L E E , ‡,§ W E N - Y I N N L I N , | JEN-HSIUNG TSAI,† AND H S O - C H I C H A U N G * ,⊥ Department of Environmental Engineering and Science, National Pingtung University of Science and Technology, Nei Pu, PingTung 91201, Taiwan, Sustainable Environment Research Center, National Cheng Kung University, 70101, Taiwan, Department of Environmental Engineering, National Cheng Kung University, 70101, Taiwan, Institute of Environmental Planning and Management, National Taipei University of Technology, Da An 106, Taipei, Taiwan, and Department of Veterinary Medicine, National Pingtung University of Science and Technology, Nei Pu, PingTung 91201, Taiwan
Introduction
Received December 13, 2007. Revised manuscript received February 26, 2008. Accepted March 17, 2008.
Polycyclic aromatic hydrocarbons (PAHs) bound in nano/ ultrafine particles from vehicle emissions may cause adverse health effects. However, little is known about the characteristics of the nanoparticle-bound PAHs and the PAH-associated carcinogenic potency/cytotoxicity; therefore, traffic-related nano/ ultrafine particles were collected in this study using a microorifice uniform deposition impactor (MOUDI) and a nano-MOUDI. For PM0.056-18, the difference in size-distribution of particulate total-PAHs between non-after-rain and after-rain samples was statistically significant at R ) 0.05; however, this difference was not significant for PM0.01-0.056. The PAH correlation between PM0.01-0.1 and PM0.1-1.8 was lower for the after-rain samples than for the non-after-rain samples. The average particulate totalPAHs in five samplings displayed a trimodal distribution with a major peak in the Aitken mode (0.032-0.056 µm). About half of the particulate total-PAHs were in the ultrafine size range. The BaPeq sums of BaP, IND, and DBA (with toxic equivalence factors g0.1) accounted for ∼90% of the totalBaPeq in the nano/ultrafine particles, although these three compounds contributed little to the mass of the sampled particles. * Corresponding author phone: +886-8-7740263; fax: +886-87740364; e-mail:
[email protected]; hcchaung@ mail.npust.edu.tw. † Department of Environmental Engineering and Science, National Pingtung University of Science and Technology. ‡ Sustainable Environment Research Center, National Cheng Kung University. § Department of Environmental Engineering, National Cheng Kung University. | Institute of Environmental Planning and Management, National Taipei University of Technology. ⊥ Department of Veterinary Medicine, National Pingtung University of Science and Technology. 10.1021/es703107w CCC: $40.75
Published on Web 05/01/2008
2008 American Chemical Society
Alveolar macrophages (AMs) play an important role in the local regulation of inflammatory responses within the alveolar space (1). They may clear infected pathogens or exogenous particle extracts by binding to and ingesting bacteria and environmental particles through specific receptors (2, 3). Atmospheric particles reaching the alveoli are ingested by AMs (4). Lundborg et al. (5) observed that ultrafine carbon particle aggregates were ingested by human or rat AMs. Several epidemiologic studies have demonstrated the association of ambient ultrafine particles (UFPs) with adverse respiratory and cardiovascular effects, resulting in morbidity and mortality in susceptible parts of the population (6–11). Bunn et al. (12) found that AMs obtained from children contained carbonaceous UFPs. UFPs were also found in AMs from healthy nonsmoking adults (13). These observations indicate that AMs are relevant target cells for UFPs. Although nano/ultrafine particles contribute relatively little to the total mass of the particulate matter (PM), they are responsible for most of the numerical concentration of the airborne particles (14, 15). Traffic sources emit numerous fine aerosols (PM2.5) (16), and vehicles may generate exhaust particles with diameters in the 10-300 nm range, with an average of about 60 nm (17). Kittelson et al. (18) indicated that most particles emitted by on-road vehicles had diameters of less than 50 nm. Idealized number- and mass-weighted size distributions of diesel exhaust particles are commonly trimodal and log-normal, respectively (19). The nuclei mode particles may account for 1-20% of the particle mass and for more than 90% (in number) of the particles in typically collected PM (19). Traffic emission is regarded as one of the major contributors to the high concentrations of PAHs in urban air (20, 21). Diesel and gasoline-powered engines emit large amounts of PAHs that partition between gas and particle phases. Zielinska et al. (22) stated that particle-bound semivolatile PAHs and nonvolatile four- to six-ring PAHs dominated the submicrometer particles that were collected on 0.1-0.18, 0.18-0.32, and 0.32-0.56 µm MOUDI stages. Miguel et al. (23) found that significant amounts of some PAH species were present in the ultrafine mode (0.05-0.12 µm) in a tunnel. Recently, heavy-duty diesel vehicles were found to produce quantifiable emissions of 3- to 6-ring PAHs (including coronene) under light driving cycles whereas heavier driving cycles produced only 3- and 4- ring PAHs in quantifiable amounts (24). In 2005 Miguel et al. (25) performed the first measurement of particle size distribution down to 10 nm for 12 USEPA priority PAHs colleted by a Nano-MOUDI sampler behind a MOUDI impactor from ambient air about 70 km downwind from central Los Angeles. Still, little attention has been paid to the carcinogenic potencies and cytotoxicities of traffic-related nanoparticlebound PAHs (10-56 nm, with health significance). Accordingly, this work elucidates the mass concentrations in nano, ultrafine, fine, and coarse particles, and determines the 15 PAHs in sized particles that were collected beside a busy VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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road. The particle size distributions and cumulative mass fractions of particles/PAHs were measured/compared using MOUDI and Nano-MOUDI samplings. The PAHs sampled on sunny days were also compared with those collected on days after rain. The carcinogenic potencies (as equivalent BaP concentrations (BaPeqs)) induced by PAHs in the sizeresolved particles were determined and compared. The particle extracts (containing PAHs) were tested using an AM assay to examine the cytotoxicities derived from the corresponding reduction in cell viability of AMs. The cytotoxicities induced by the particle extracts were statistically compared.
Experimental Section Sampling. All of the atmospheric particulate samples were collected at a heavily trafficked roadside in a city in southern Taiwan. (See A1 in the Supporting Information for details of the sampling site.) A MOUDI and a nano-MOUDI (models 100 and 115, respectively, from MSP Co., Minneapolis, MN), equipped with quartz filters (with diameters of 37 and 47 mm, respectively), were used to collect size-resolved aerosol samples. The height of the sampler inlet above the ground was about 1 m. The impactors in the MOUDIs separated the particulate matter into 13 size ranges (at 50% efficiency) with the following equivalent cutoff diameters: 0.010-0.018, 0.018-0.032, 0.032-0.056, 0.056-0.1, 0.1-0.18, 0.18-0.32, 0.32-0.56, 0.56-1.0, 1.0-1.8, 1.8-3.2, 3.2-5.6, 5.6-10, and 10-18 µm. The particles were divided into four size groups: coarse (PM2.5-10), fine (PM2.5), ultrafine (PM0.01-0.1: 0.01 µm < Dp < 0.1 µm), and nano (PM0.01-0.056: 0.01 µm < Dp < 0.056 µm) according to the literature (18, 26, 27). The sampling flow rates of the MOUDI and Nano-MOUDI were 30 and 10 L min-1, respectively. These devices were operated 15 h (from 7:00 to 22:00) a day. Several consecutive seven-day samplings were performed to collect sufficient atmospheric PAHs and PM to quantify their masses. During each sampling period, a quartz filter was continuously used for a MOUDI (or nano-MOUDI) stage. Collected from each one-week sampling period, each sample on each stage of the MOUDI (and nano-MOUDI) was analyzed separately. Five samplings were conducted and each sampling involved the collection of 13 (MOUDI and nano-MOUDI stages) samples (3, 10, 4, and 3 samples of PM2.5-18, fine (PM0.1-2.5 + ultrafine), ultrafine (PM0.056-0.1 + nano), and nano particles (PM0.01-0.056), respectively) to yield a total of 65 samples. The quartz filters were pretreated before sampling by being heated in a muffle furnace in air for 2.5 h at 900 °C. Prior to being weighed on an electronic six-digit balance ((2 µg) before and after sampling, the filters were conditioned for 24 h at a constant temperature of 25 °C and a relative humidity of 40%. The concentration of suspended particulate matter was determined by dividing the particle mass by the volume of sampled air. PAH Analysis and Quality Control. The identified 15 PAH species included four 4-ring (fluoranthene (FL), pyrene (Pyr), benzo(a)anthracene (BaA), chrysene (CHR)), six 5-ring (cyclopenta(c,d)pyrene (CYC), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(e)pyrene (BeP), benzo(a)pyrene (BaP), perylene (PER)), four 6-ring (indeno(1,2,3,cd)pyrene (IND), dibenzo(a,h)anthrance (DBA), benzo(b)chrycene (BbC), benzo(ghi)perylene (BghiP)), and one 7-ring (Coronene (COR)) PAH compounds. The 4-ring and 5-/6-/ 7-ring PAHs are referred to as middle molecular weight (MMW) and high molecular weight (HMW) PAHs, respectively. In the PAH extraction and analysis, all samples were Soxhlet-extracted with a mixed solvent (1:1 (v/v) n-hexane/ dichloromethane) for 24 h. The extracts were then concentrated, cleaned using a silica column, and reconcentrated by being purged with ultrapure nitrogen (at a flow rate 1.0 L min-1) to 1.0 mL before analysis. 4230
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PAHs were identified and quantified using a (HewlettPackard 5890A) gas chromatograph/mass spectrometer (GC/ MS) equipped with a Hewlett-Packard capillary column (HP Ultra 2- 50 m × 0.32 mm i.d., with a 0.17 µm-thick film) and a mass-selective detector (MSD) (Hewlett-Packard 5972). Controlled by a computer workstation and equipped with a HP-7673A automatic sampler, the GC/MS instrument was operated under the following conditions: injection volume ) 1 µL, splitless injection ) 300 °C, ion source temperature ) 310 °C; oven heating from 50 to 100 at 20 °C min-1, and then from 100 to 290 at 3 °C min-1, which was held for 40 min. A scan mode was employed to determine the masses of molecular and fragment ions of pure PAH standards in the GC/MS analysis. PAHs were quantified by selective ion monitoring (SIM). Analyses of serial dilutions of PAH standards revealed the limits of detection (LODs) of GC/MS to be between 0.023 and 0.106 ng for the 15 PAH compounds. The limit of quantification (LOQ) was defined as the LOD divided by the sampling volume. The LOQ values of the 15 PAH compounds were between 0.122 and 0.561 ng N m3-. (See A2 of the Supporting Information for the quality control procedure of the PAH analysis.) Preparation of Particle Extracts. The particles collected on a sunny day (S4) were extracted by 1:1 (v/v) n-hexane/ dichloromethane. Subsequently, the organic solvent used to extract the particle matters was evaporated by a stream of nitrogen. The residues obtained after evaporating the mixed solvent were then resuspended in dimethyl sulfoxide (DMSO) and stored in a -80 °C freezer until the cytotoxicity assay was performed using porcine alveolar macrophages as an in vitro screening system. (See A3 in the Supporting Information for details of DMSO control tests.) Preparation of Alveolar Macrophage Cell Suspensions. Healthy male piglets weighing 10-12 kg were obtained from the pig farm at the National Pingtung University of Science and Technology. All animals were confirmed to be without infection. Each animal was anesthetized intraperitoneally (ip) with an injection of sodium pentobarbital (10 mg kg-1 body weight) (Abbot, USA). The trachea was cannulated and lungs were then lavaged (to a total lung capacity 8 times that of typical) with Solution A (2 mM CaCl2, 140 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 10 mM HEPES, and 1.3 mM MgSO4 at pH 7.4) for the collection of alveolar macrophages. This lavage was centrifuged (300g, 10 min, 4 °C), and the cell pellets were resuspended in RPMI-1640 (Invitrogen-Gibco, Grand Island, USA) containing 10% heat-inactivated fetal calf serum (FCS), 5 mg mL-1 gentamycine, 1 mg mL-1 kanamycine, 1.2 mM sodium pyruvate, 0.2 mM β-mercaptoethanol, and 2 g L-1 sodium bicarbonate (culturing RPMI-1640). After a 3-h incubation, nonadherent cells were removed and AM cells were retained in a fresh RPMI-1640 medium. These cells were then plated in 96-well tissue culture plates (2 × 105 cells/200 µL/well). Over 95% of these cells were characterized as AM cells, displaying phenotypes of SWC9+ and SWC3highMHC-IhighMHC-IIhigh. A portion of the AM cell suspension was fixed for 2 h at room temperature in a fixing solution composed of 2% glutaraldehyde and 1% osmium tetroxide in a 0.1 M cacodylate buffer (pH 7.4). Then, the cells were centrifuged at 10 000g for 5 min. The cell pellets were postfixed, block stained, and dehydrated in acetone. Thin sections were stained sequentially in aqueous uranyl acetate and alkaline lead citrate, and then examined using an EM-H600 electron microscope (Hitachi). In the cell screening system, bleomycin (Sigma-Aldrich), which can cause alveolar epithelial cell death, was used as a positive control. Bleomycin was dissolved (2 U mL-1) in a phosphate buffer solution (PBS) (containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na2HPO4, and 0.2 g of KH2PO4 in 1 L of H2O) (pH 7.2). These AMs were treated with 8 µL of saline,
TABLE 1. Mean Concentrations of PM (µg m-3), PAH (ng m-3), and BaPeq (ng m-3) with SD Values in Parentheses for Various Particle Sizes (n = 5) nano PAHs
TEFa
PM
-
PAHs FL
0.001
Pyr
0.001
CYC
c
BaA
0.1
CHR
0.01
BbF
0.1
BkF
0.1
BeP
c
BaP
1
PER
c
IND
0.1
DBA
1
BbC
c
BghiP
0.01
COR
c
b
total PAHs a
concn.
ultrafine BaPeq
concn.
fine
BaPeq
concn.
coarse BaPeq
13.1 ((4.48)
-
16.5 ((3.59)
-
93.0 ((30.6)
-
2.02 ((1.40) 2.58 ((2.43) 1.77 ((2.43) 1.16 ((0.75) 1.22 ((0.53) 0.68 ((0.49) 0.70 ((0.53) 1.53 ((1.27) 0.50 ((0.21) 1.63 ((1.08) 1.15 ((0.65) 0.54 ((0.20) 0.85 ((0.37) 1.21 ((0.90) 1.14 ((0.72) 18.7 ((4.95)
0.002 ((0.001) 0.003 ((0.002) 0.116 ((0.075) 0.012 ((0.005) 0.068 ((0.049) 0.070 ((0.053) 0.503 ((0.205) 0.115 ((0.065) 0.539 ((0.201) 0.012 ((0.009) 1.44 ((0.539)
2.27 ((1.56) 2.92 ((2.74) 2.02 ((2.76) 1.27 ((0.73) 1.34 ((0.54) 0.77 ((0.54) 0.79 ((0.55) 1.78 ((1.40) 0.62 ((0.29) 1.85 ((1.14) 1.38 ((0.68) 0.62 ((0.22) 1.04 ((0.36) 1.27 ((0.89) 1.35 ((0.92) 21.3 ((5.51)
0.002 ((0.002) 0.003 ((0.003) 0.127 ((0.073) 0.013 ((0.005) 0.077 ((0.054) 0.079 ((0.055) 0.617 ((0.289) 0.138 ((0.068) 0.616 ((0.222) 0.013 ((0.009) 1.69 ((0.589)
5.53 ((5.22) 6.51 ((7.35) 3.16 ((3.93) 2.12 ((0.92) 2.25 ((0.77) 1.58 ((1.03) 1.47 ((0.82) 3.74 ((2.60) 1.48 ((0.53) 3.77 ((1.36) 3.22 ((1.36) 1.15 ((0.39) 2.54 ((1.27) 2.71 ((0.99) 3.36 ((2.38) 44.6 ((18.6)
0.006 ((0.005) 0.007 ((0.007) 0.212 ((0.092) 0.022 ((0.008) 0.158 ((0.103) 0.147 ((0.082) 1.481 ((0.534) 0.322 ((0.136) 1.150 ((0.391) 0.027 ((0.010) 3.53 ((0.924)
TEF: toxic equivalent factor.
b
concn. 47.1 ((23.5) 0.99 ((0.80) 1.48 ((2.00) 0.53 ((0.59) 0.41 ((0.26) 0.42 ((0.19) 0.35 ((0.27) 0.31 ((0.15) 0.69 ((0.51) 0.29 ((0.15) 0.89 ((0.21) 0.42 ((0.20) 0.21 ((0.05) 0.51 ((0.32) 0.95 ((1.35) 1.03 ((1.25) 9.49 ((6.07)
BaPeq 0.001 ((0.001) 0.001 ((0.002) 0.041 ((0.026) 0.004 ((0.002) 0.035 ((0.027) 0.031 ((0.015) 0.286 ((0.146) 0.042 ((0.020) 0.210 ((0.054) 0.010 ((0.014) 0.662 ((0.181)
s: Not available. c Data from Nisbet and Lagoy (33).
bleomycin, or extracts from the sized particles in a CO2 incubator at 37 °C for 24 h. At the end of incubation, cells were centrifuged at 300g at 4 °C for 10 min, washed using the PBS twice, and then stained with 10 µL of propidium iodide (PI) (Sigma-Aldrich) in 500 µL of PBS on ice for 10 min. After the staining, cells were washed using 1 mL of PBS and centrifuged at 300g at 4 °C for 5min, followed by the addition of 1 mL of PBS to resuspend the cells. The reduction in cell viability (RCV) of AMs was evaluated by counting the PI-positive cells using a flow cytometer (Coulter Epics Altra Flow Cytometry, Beckman Coulter, CA). The RCV of AMs in each treated sample was calculated according to the following formula: RCV (% control) ) (% of PI-positive cells in treatment - % of PI-positive cells in control)/% of PI-positive cells in control.
Results and Discussion Concentrations of Atmospheric Particles Collected beside a Busy Road. The mean concentration (93.0 ( 30.6 µg m-3) of fine particles (PM0.01-2.5) was approximately double that of coarse particles (PM2.5-10), and those of nano, ultrafine, and PM0.01-10 particles were 13.1 ( 4.48, 16.5 ( 3.59, and 140 ( 38.6 µg m-3, respectively (Table 1). Although all five samplings (S1 to S5) were performed on sunny days, S1 and S5 samplings were conducted immediately following heavy rain (which lasted for several days) that scavenged significant amounts of atmospheric particles (particularly coarse particles). Therefore, the PM0.01-10 concentrations of S1 and S5 (103 and 74.1 µg m-3, respectively) were lower than those of
sunny-day samplings (S2-S4) (Figure 1). However, the nano and ultrafine particle concentrations of S1 and S5 were higher than those of S2-S4, so both the nano/PM0.01-10 and the ultrafine/PM0.01-10 ratios of S1 and S5 clearly exceeded those of S2-S4. The nano/PM0.01-10 ratios of S1 and S5 (0.14 and 0.256, respectively) were 2.15 and 3.94 times the average ratio (0.065) of S2-S4, respectively. S1 and S5 exhibited ultrafine/ PM0.01-10 ratios of 0.155 and 0.294, respectively: about 1.74 and 3.30 times that of the S2-S4 average (0.089), respectively. Size Distributions and Accumulation Percentages of Collected Particles and Particle-Bound PAHS (With/ Without Rain Effect). One of our earlier papers indicated that most of the nano and ultrafine particles collected at the same sampling site were mainly contributed by traffic (28). Traffic-related atmospheric particles (PM0.01-18) had a trimodal size distribution, like those of urban area particles (29). The mass concentration of traffic-related particles (PM0.01-18) exhibited a trimodal distribution, with a major, a secondary, and a minor peak in the coarse (3.2-5.6 µm), fine (1-1.8 µm), and nano (0.018-0.032 µm) size ranges, respectively (Figure 2). The particles collected on sunny days (mean of S2-S4) displayed a bimodal distribution with a secondary peak that was larger (1.0-1.8 µm) than that (0.18-0.32 µm) of the particles sampled on sunny days after rain (mean of S1 and S5) (Figure 3). With an additional minor peak in the nano size range (0.018-0.032 µm), the particles collected following rain exhibited a trimodal size distribution. The difference between PM size distributions before and after rain was significant in the size range 0.056-18 µm VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Concentrations and ratios of nano, ultrafine, fine, coarse, and PM0.01-10 particles for the S1 to S5 samples with sampling periods (month/day/year) of 8/3-8/9/2004, 01/17-01/24/2005, 03/02-03/08/2005, 11/28-12/24/2005, and 06/14-06/20/2006, respectively.
FIGURE 2. Size distributions of the mean PM0.01-18 and particle-bound total-PAHs (with one standard deviation in the error bar).
FIGURE 3. Size distributions of the mean PM (with one standard deviation in the error bar) collected on sunny days and sunny days after rain. (p ) 0.0094) but insignificant in the range 0.01-0.056 µm (p ) 0.237) at a significance level of R ) 0.05. The former result follows from the fact that the washout coefficients for PM sizes of 3-10 µm may increase significantly during heavy rain events (30). The latter result may be associated with the fact that the sampling site was close to the traffic emission source and most of the collected nanoparticles were freshly 4232
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FIGURE 4. Size distributions of the particle-bound mean total-PAHs (with one standard deviation in the error bar) collected on sunny days and sunny days after rain. emitted from vehicle tailpipes. Therefore, the effect of rain on the nanoparticle size distribution was not significant. In this study, the distribution of particulate total-PAHs (average of five samplings) was bimodal with a major peak in the Aitken mode (0.032-0.056 µm) and a minor peak in the accumulation mode (0.56-1 µm) (Figure 2). In fact, the particle size distribution of PAHs usually varies with PAH species and sampling conditions/locations. For example, six heavy PAHs from heavy-duty diesel exhaust were all found to be single-modal, peaking in 0.32-0.56 and 0.18-0.32 µm size ranges for COR and the other five PAHs, respectively (24). Miguel et al. (31) observed mainly a single- or bi- modal particle size distribution (peaking at 0-0.18 and/or 2.5-10 µm) for eight heavy PAHs at Claremont, Los Angeles; however, they found bi- or trimodal size distributions (the major peak mostly in the 0.056-0.1 µm range, and two minor ones in the 0.010-0.018 and/or 0.18-2.5 µm ranges) for the same PAH species at Riverside, Los Angeles (25). Both the with/without rain sampling cases in this study displayed trimodal size distributions for particulate totalPAHs (Figure 4); furthermore, the peak in the Aitken size range is wider in this study than that in the work by Miguel et al. (25). On sunny days, the major, secondary, and minor peaks were within the ranges 0.018-0.032, 0.56-1, and 3.2-5.6 µm, respectively (Figure 4), while those for sunny days after rain were within 0.032-0.056, 0.18-0.32, and 5.6-10 µm, respectively. The two cases exhibited a size distribution difference for particle-bound total-PAHs, which
FIGURE 5. Cumulative mass fractions of the PM0.01-18 and particle-bound total-PAHs. was significant in the size range of 0.056-18 µm (p ) 0.013, R ) 0.05) but insignificant in the 0.01-0.056 µm range (p ) 0.192, R ) 0.05). Clearly, rain only weakly influenced the size distributions of the PM and particle-bound total-PAHs for the nano particles; in contrast, rain affected the larger sampled particles although some other environmental conditions (such as wind direction/speed, height of the mixing layer, and source strength) must also be considered. Rain washed out significant fractions of accumulation mode particles (0.1 < Dp < 1 µm) and larger particles (PM1-18) (Figure 3). The rain-scavenging of accumulation mode particles may reduce the surface area (opportunity) for the coagulation of vehicle-emitted nuclei mode particles (Dp < 0.01 µm) or Aitken mode particles (0.01 < Dp < 0.1 µm). Hence, rain reduced the PAH concentrations of accumulation and coarse mode particles. The rain also lowered the PAH correlation between PM0.01-0.1 and PM0.1-1.8. This finding may be partially related to the fact that high molecular PAHs tend
to remain on ultrafine particles with which they are emitted (23, 32) because the hydrophobic PAHs had lower fluxes during volatilization/condensation and required more time to partition onto larger particles and more hygroscopic particles during sunny days after rain, which were more humid than those on sunny days. The cumulative percentages of the particles in nano, ultrafine, fine, and PM0.01-10 size ranges were 8.74, 11.0, 62.2, and 93.7%, respectively, whereas those of particulate totalPAHs in the corresponding size ranges were 33.2, 37.9, 77.9, and 94.8%, respectively (Figure 5). The mass cumulative percentages of particulate total-PAHs in the nano and ultrafine size ranges were 3.80 and 3.45 times that of PM0.01-18, respectively. Figures 2 and 5 show that approximately half of particulate total-PAHs were in the ultrafine size range, associated with heavy traffic. The health impact of these must be considered. Carcinogenic Potencies Induced by PAHs in the SizeResolved Particles. Although 4-ring PAHs were about half of the total-PAHs for particles of each size range (Table 1), they are less carcinogenic than PAH compounds with more rings, such as BaP, DBA, BaA, BbF, BkF, and IND (33). Therefore, the carcinogenic potencies of particle-bound PAHs were evaluated by determining their toxic equivalence factors (TEFs) following the conversion of PAH concentration into equivalent BaP concentration (BaPeq) (33). The concentrations of total-PAHs in nano, ultrafine, fine, and coarse particles were 18.7, 21.3, 44.6, and 9.49 ng m-3, respectively, corresponding to total-BaPeqs of 1.44, 1.69, 3.53, and 0.66 ng m-3, respectively (Table 1). The total-PAHs concentrations for PM2.5 were 22.4-74.0 ng m-3 (average ) 44.6 ng m-3)slower than those (43.7-68.2 ng m-3, average ) 57.0 ng m-3) of the particles collected at a sampling site near three highways at Guangzhou, China (34). The PM10-bound totalPAHs concentrations (27.2-94.0 ng m-3) exceeded the TSPbound values (0.38-11.6 ng m-3) observed at a heavily trafficked suburban site (500 m away from highways) in New Brunswick, NJ (35). In this study, BaP, IND, and DBA (high TEFs) contributed only 9.0, 9.5, 9.4, and 9.2% of the totalPAHs of the nano, ultrafine, fine, and coarse particles, respectively. However, the sums of their BaPeqs accounted
FIGURE 6. Reduction in cell viability (RCV (% control), presented as mean ( SEM for cells incubated in triplicates) of alveolar macrophages and concentrations of total-PAHs and total-BaPeq (multiplied by 10) for the particle extracts of the S4 sample. Cells (2 × 105/200 µL/well) were treated with 8 µL of saline, bleomycin (BLM, 2 U mL-1), or particle extracts and incubated in a CO2 incubator at 37 °C for 24 h. VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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for 88, 89, 90, and 92% of total-BaPeq, respectively, and thus they played an important role in PAH-associated carcinogenic potency with high health concerns. The concentrations of total-PAHs or total-BaPeq in the nano, ultrafine, fine, and coarse particles followed the order fine > ultrafine > nano > coarse (Table 1), but the mean content (µg-PAH or µg-BaPeq per g-particle mass) of particlebound total-PAHs or total-BaPeq (calculated from the PM, total-PAHs, and total-BaPeq data) followed the order of nano > ultrafine > fine > coarse. This carcinogenic potency tendency resulting from PAH-related BaPeq raises health concerns about traffic-related nano and ultrafine particles. Cytotoxicities Induced by Particle Extracts. Figure 6 shows the cytotoxicity responses (expressed as reduction in cell viability (RCV in % control, see the final paragraph of the Experimental Section for details)) induced by particle extracts (containing PAHs) from the S4 sample. The cells treated with extracts from 0.018-0.18 and 5.6-18 µm particles had greater mean RCV values than those from 0.18-5.6 µm particles (p < 0.05). The mean RCV values for the 0.18-0.32, 0.32-0.56, 0.56-1.0, 1.0-1.8, 1.8-3.2, and 3.2-5.6 µm particles were not statistically different. A similar trend was also observed among the mean RCV values for the 0.018-0.032, 0.032-0.056, 0.056-0.1, and 0.1-0.18 µm particles. It is interesting to find that the mean RCV value (128 ( 21%) for bleomycin (BLM) was statistically similar to those for the 0.032-0.056, 0.056-0.1, and 0.1-0.18 µm particles. Among the extracts of the tested size-resolved particles, one of the 0.01-0.018 µm (10-18 nm) nanoparticles exhibited the greatest mean RCV value (381 ( 75%), which was also statistically higher than that of BLM (p < 0.05). Accordingly, it is inferred that among the tested particle extracts containing PAHs, the one from the 10-18 nm nanoparticles displayed the highest AM cytotoxicity (also higher than the BLM-induced one); furthermore, those from the 0.032-0.056, 0.056-0.1, and 0.1-0.18 µm particles were similar to that from BLM. For the particles in each of the 13 size ranges, the variation trends in total concentration (or amount) of extracted PAHs and corresponding total-BaPeq were roughly similar, but were quite different from that in the cytotoxicity response. The total-BaPeq values of PM0.18-0.32, PM0.32-0.56, PM0.56-1.0, and PM1.0-1.8 were higher than those of particles in the five individual size ranges in PM0.01-0.18, but the cytotoxicity seemed to be lower for the former PM than for the latter (particularly the PM0.01-0.018). PAHs (especially BaP) might alter the functions (e.g., endocytosis and phagocytosis) of macrophagic cells (36). On the other hand, it was reported that ultrafine particles are more critical than PM2.5-10 for particle toxicity although PM2.5-10 might also be toxic (37). The phagocytic activity inhibition of macrophagic cells is higher for ultrafine particles than for coarser particles (38). If considering both the toxicant and particle size effects on the cytotoxicity response, the inconsistency between totalBaPeq values (from PAHs) and cytotoxicity responses in this study reveals that some nanoparticles or extractable toxicants (e.g., some organic metals or other organics) were possibly present in the particle extracts. More research (e.g., more sampling and cytotoxicity tests directly on the size-resolved particles) is necessary to further compare the cytotoxicity of traffic-related particles in these size ranges.
Acknowledgments We thank the National Science Council of the Republic of China, Taiwan for financially supporting this research under Contract NSC-94-2211-E-020-001 and NSC-94-2211-E-020008.
Supporting Information Available Sampling site information, quality control of PAH analysis, DMSO control tests, and associated references. This infor4234
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