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OPEN Differential toxicities of fine

particulate matters from various
sources
Received: 25 June 2018 Minhan Park1, Hung Soo Joo1,2, Kwangyul Lee1, Myoseon Jang3, Sang Don Kim1,
Accepted: 5 November 2018 Injeong Kim1, Lucille Joanna S. Borlaza 1, Heungbin Lim4, Hanjae Shin5, Kyu Hyuck Chung6,
Published: xx xx xxxx Yoon-Hyeong Choi 7, Sun Gu Park7, Min-Suk Bae8, Jiyi Lee9, Hangyul Song1 & Kihong Park1
Fine particulate matters less than 2.5 µm (PM2.5) in the ambient atmosphere are strongly associated
with adverse health effects. However, it is unlikely that all fine particles are equally toxic in view of their
different sizes and chemical components. Toxicity of fine particles produced from various combustion
sources (diesel engine, gasoline engine, biomass burning (rice straw and pine stem burning), and coal
combustion) and non-combustion sources (road dust including sea spray aerosols, ammonium sulfate,
ammonium nitrate, and secondary organic aerosols (SOA)), which are known major sources of PM2.5,
was determined. Multiple biological and chemical endpoints were integrated for various source-specific
aerosols to derive toxicity scores for particles originating from different sources. The highest toxicity
score was obtained for diesel engine exhaust particles, followed by gasoline engine exhaust particles,
biomass burning particles, coal combustion particles, and road dust, suggesting that traffic plays the
most critical role in enhancing the toxic effects of fine particles. The toxicity ranking of fine particles
produced from various sources can be used to better understand the adverse health effects caused by
different fine particle types in the ambient atmosphere, and to provide practical management of fine
particles beyond what can be achieved only using PM mass which is the current regulation standard.

Fine particles in the ambient atmosphere are of significant research interest owing to their impacts on climate
change and detrimental effects on human health. These particles are directly emitted from various sources and
produced by gas-to-particle conversion of secondary products from atmospheric oxidation of SO2, NOx, and
hydrocarbons. Epidemiological and toxicological studies worldwide have suggested a strong link between expo-
sure to fine particles and adverse health effects (e.g., respiratory disease, lung cancer, cardiovascular disease, and
premature mortality)1–3. The World Health Organization (WHO) established guidelines for particulate matter
based on mass concentration of particulate matter less than 2.5 µm (PM2.5)4. However, health risks of PM2.5 are
not fully taken into account with the PM2.5 mass, and all fine particles may not be equally toxic5. Due to multiple
sources and formation pathways, ambient PM2.5 have diverse sizes, shapes, surface charges, surface chemistry and
chemical compositions, leading to differential health effects among particle types. Even different PM2.5 with the
same mass concentrations may exert variable effects on human health. For example, an earlier study by Lelieveld
et al.3 in their assessment of the contribution of outdoor air pollution sources to premature mortality reported
that health impact was dependent on assumptions on toxicity of particles6,7. A recent review on epidemiological
and toxicological literatures related to long-term effects of PM components or source factors suggests that there
has been insufficient information to make clear conclusions about differential health effects among components
or sources8. It was also reported that much more enhanced understanding of exposure and health effects should

1
School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST),
Gwangju, Republic of Korea. 2Department of Environmental Engineering, Anyang University, Anyang, Republic of
Korea. 3Department of Environmental and Global Health, University of Florida, Gainesville, FL, USA. 4Department
of Industrial Plant Science & Technology, Chungbuk National University, Cheongju, Republic of Korea. 5R&D
Headquarter, KT&G, Daejeon, Republic of Korea. 6School of Pharmacy, Sungkyunkwan University, Suwon, Republic
of Korea. 7Department of Preventive Medicine, Gachon University Graduate School of Medicine, Incheon, Republic
of Korea. 8Department of Environmental Engineering, Mokpo National University, Muan, Republic of Korea.
9
Department of Environmental Science and Engineering, Ewha Womans University, Seoul, Republic of Korea.
Correspondence and requests for materials should be addressed to K.P. (email: [email protected])

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be needed before it can be concluded that the control of specific sources or components of PM2.5 should be more
effective to protect human health than the PM2.5 mass as a whole9.
The differential toxicities among particle types according to chemical composition remain to be established
due to the existence of numerous chemical constituents in ambient aerosols. Conduction of complete toxicity
tests for all possible chemical constituents of ambient aerosols and generation of practical chemical groups for
regulatory purposes is a virtually impossible task. Moreover, it is difficult to ascertain the combined roles of indi-
vidual chemical constituents (e.g., synergetic effects between chemical species and transformation of chemicals
through complex cellular mechanisms) in triggering adverse health effects. Thus, determination of source-specific
toxicity may present a more efficient alternative means to elucidate the effects of individual PM2.5 rather than
chemical component-specific toxicity measurements. The number of toxicity tests for hundreds or thousands of
chemical components in PM2.5 (chemical component-specific toxicity test) can be reduced to the smaller particle
groups that are major distinct sources for fine particles (source-specific toxicity test) and a range of biological
responses to source-specific aerosols under comparable conditions (PM generation, collection, exposure and
biological systems) can be assessed. Typically, source apportionment studies are effectively used to determine the
major sources for ambient PM2.5 through measurement of their chemical components10,11. Source apportionment
results can be combined with source-specific toxicity data to evaluate overall toxicity of ambient PM2.5.
The purpose of this study is to assess variability in toxicities of fine particles produced from various com-
bustion sources and non-combustion sources which are known major sources of PM2.5 (Fig. 1). The combus-
tion sources include diesel engine, gasoline engine, biomass burning and coal combustion. The non-combustion
sources include road dust, sea spray aerosols, ammonium sulfate, ammonium nitrate, and secondary organic
aerosols (SOA) produced from the photo-oxidation of toluene, 1,3,5-trimethylbenzene (TMB), isoprene, and
α-pinene in the presence of NOx under natural sunlight. To determine toxicity of various particles, multiple
biological and chemical endpoints (oxidative potential (OP), cell viability, genotoxicity (based on mutagenicity
and DNA damage), oxidative stress and inflammatory response) using human airway cell lines, animal ovary
cell lines and Salmonella strains with preexisting mutations were determined. This method facilitated the direct
comparison of various endpoints linked to health effect on respiratory system with minimization of differences in
exposure and biological systems. It is believed that the developed toxicity score accounts for differential toxicities
of various aerosols linked to adverse health effects on respiratory system. The tested method assigned priority
to PM2.5 sources potentially harmful to human respiratory system. However, the dominant mortality also came
from cardiovascular diseases12. Further incorporation of toxicological data which are related to health effects on
cardiovascular system could improve the toxicity score database. The multiple endpoints were integrated to derive
toxicity scores for particles originating from different sources. The database for toxicity could be used to better
understand health effects caused by different fine particle types of ambient PM2.5. Chemical characterization
of the particles was additionally conducted to relate their major chemical components to the toxicity score for
source-specific aerosols.

Methods
Generation of fine particles from various sources. Exhaust particles were produced from a heavy duty
diesel engine (DL08S, 7640 cc, 1500 rpm, Doosan Infracore, Korea), light duty diesel engine (DL08S, 2740 cc,
1500 rpm, Isuzu motors, Japan), diesel generator engine (192FC, 498 cc, 3000 rpm, Hi-earns, China) and gasoline
generator engine (GXH50, 50 cc, 5500 rpm, Honda, Japan). Table S7 summarizes the specifications of the diesel
and gasoline engines used in this study. The engine exhaust particles were diluted (1:100) by an aerosol diluter
(3302A, TSI Inc., USA) and sampled into PM2.5 filters for determination of chemical composition and toxicity,
and real-time aerosol instruments were used to assess the number size distribution and mass concentration, as
shown in Fig. 1.
A biomass burning chamber mainly consisting of a combustion chamber (0.54 m3), primary dilution chamber
(3.75 m3) and secondary dilution chamber (0.04 m3), was employed for generation of rice straw and pine stem
burning particles (Fig. 1). Clean air was supplied into the combustion chamber using a mass flow controller.
About 25 g of biomass was loaded on the grid of the combustion stove and ignited using a propane torch. Rice
straw and pine stems for biomass were collected from rural and forest areas in Korea. The smoke was drawn into
a primary (3.75 m3) dilution chamber (1:1), followed by a secondary dilution chamber (0.04 m3) (1:10). Several
outlets were used for PM2.5 sampling on filters for chemical and toxicity analyses and for real-time measurements
of number size distribution and mass concentration with further dilution (1:100).
A bench scale high-temperature furnace (HTF55342C, Thermo Electron Corp., USA) with a quartz tube was
employed to produce coal combustion particles (Fig. 1). Bituminous coal used by coal power plants (Korea South
Power Co. Ltd., Hadong, Korea) was pulverized and sieved through a 200-mesh screen (< 75 µm). Pulverized
bituminous coal was fed into a quartz tube (0.00327 m3) using a solid aerosol generator (SAG 410, Topas GmbH,
Germany) at a feeding rate of 3.5 g/h. Several burning temperature conditions (550, 900, and 1100 °C) were used
to generate coal combustion particles. Next, particles from the furnace were diluted with clean air and introduced
into filters for determination of chemical composition and toxicity, and real-time aerosol instruments were used
for determination of number size distribution and mass concentration. Prior to real-time measurements, samples
were further diluted (1:100) using an aerosol diluter.
Road dust was collected from the shoulder of the roadside and walkway in a tunnel near a junction area in
Korea (urban Gwangju). All collected samples were dried in a desiccator for 24 h. A 12-mesh sieve (< 1.7 mm)
was used to remove large particles, and the dust further sieved through a 400-mesh screen (< 38 µm) to col-
lect fine dust samples. Sieved dust was aerosolized using a solid aerosol generator under identical conditions
with an input pressure of 15 psi and feeding rate of 6 g/h, as shown in Fig. 1. Aerosolized dust was subsequently
introduced into the PM2.5 filter sampling system (Fig. 1). Prior to real-time measurements, aerosols were diluted

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Figure 1. Experimental design for generation, physical and chemical characterization, and toxicity tests for
various primary and secondary aerosols from different sources.

(1:100) using an aerosol diluter. Carbon black powder (Cabot Corp., USA), Arizona dust13 and Mongolian dust
collected from roadsides in urban Ulaanbaatar were additionally re-suspended using the above method.
Sea spray aerosols were generated from natural seawater sampled from the South sea of Korea using two
system types, specifically, a marine aerosol reference tank (MART) (Marine Research Systems, USA) and a pro-
totype bubble bursting chamber (Fig. 1). Ammonium sulfate (Sigma-Aldrich, USA) and ammonium nitrate
(Sigma-Aldrich, USA) in deionized water solution were used to produce ammonium sulfate and ammonium
nitrate particles with the aid of an atomizer, as shown in Fig. 1. Particles were dried using a series of diffusion
driers before sampling into filters or introducing into real-time aerosol instruments.

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Secondary organic aerosols (SOA) were produced under natural sunlight using University of Florida
Atmospheric PHotochemical Outdoor Reactor (UF-APHOR) dual chambers (52 m3 for each) located on the roof
of the Black Hall at the University of Florida (Fig. 1). Before sunrise, hydrocarbon was inserted into the chamber
with a U-shaped injector. For photooxidation of toluene, HONO produced from the reaction of 0.1 M NaNO2
and 10% w/w H2SO4 aqueous solution were introduced into the chamber as a source of OH radicals. The initial
mixing ratio of HONO was estimated based on the difference in NO2 concentrations with and without the base
denuder (1% Na2CO3 + 1% glucose)14. Particle size distribution and concentrations of chamber-generated SOA
were monitored with the aid of a scanning mobility particle sizer (SMPS) and calculated to mass concentrations
using SOA density (1.3 g/cm3 for α-pinene SOA and 1.4 g/cm3 for other three types of SOA)15–17. The mass con-
centration of organic carbon (OC) was additionally monitored using a semi-continuous Organic carbon/Element
carbon (OC/EC) aerosol analyzer. Organic matter (OM) concentration was determined by multiplying OC by the
[OM]/[OC] ratio, which was calculated as 1.6 for α-pinene and 2.0 for the other three SOA types18. The chamber
experiment conditions used to produce various organic aerosols and SOA yields are summarized in Table S2.
Generated SOAs were collected using a Particle-Into-Liquid sampler (PILS) (Applikon, Netherlands) and toxici-
ties were examined via chemical and biological assays.

Physical and chemical characterization of fine particles. Number size distribution of fine particles
from various sources was measured with a scanning mobility particle sizer (SMPS) (3081 DMA and 3022A CPC,
TSI Inc., USA) (14 to 660 nm), NanoScan SMPS (3910, TSI Inc., USA) (10 to 420 nm), and optical particle sizer
(OPS) (3330, TSI Inc., USA) (0.3 to 10 μm). PM2.5 mass concentration was assessed with a Dust Trak instrument
(DRX, TSI Inc., USA). For offline chemical analysis, PM2.5 filter samples (Zefluor filter for ions and elements, and
quartz filter for carbonaceous species) were collected from various sources in laboratory and urban (Gwangju),
rural (Jangseoung), roadside (Gwangju), and industrial (Gwangyang) sites in Korea.
Ions, elements, and carbonaceous species (OC and EC) in PM2.5 filter samples were determined via ion chro-
matography (IC) (850 Professional IC, Metrohm, Switzerland), inductively coupled plasma-mass spectrometry
(ICP-MS) (7500ce, Agilent, USA), and an OC-EC carbon analyzer (5L, Sunset laboratory Inc., USA), respectively.
A Zefluor filter was used for ion and element analyses. The filter was equilibrated at a temperature of 21 ± 2 °C and
relative humidity of 35 ± 5% for 24 h before and after sampling. Triplicate pre- and post-weighing was performed
for each filter. After sampling, the Zefluor filter was cut into two equal parts for analyses of ions and elements. One
part of the filter was extracted with 20 mL deionized water under 1 h ultra-sonication and 3 h shaking. The water
extracts were subsequently passed through a polytetrafluoroethylene (PTFE) syringe filter, and 8 water-soluble
ions (SO42−, NO3−, Cl−, NH4+, Na+, K+, Mg2+, and Ca2+) analyzed via IC. Recovery was determined as 87.5–
103.5% using standard samples. The second portion of the sampled filter was digested in PTFE (polytetrafluo-
roethylene) vessels by adding HNO3 and HCl (3:1) in a closed microwave system. The digested solution was
concentrated to 0.5 mL in a heating block and treated with 2% HNO3 up to a volume of 10 mL. The solution was
subsequently filtered using a PTFE syringe filter and 14 elements (Al, As, Ba, Cd, Co, Cu, Fe, Mn, Ni, Pb, Sr, Ti, V,
and Zn) were analyzed via ICP-MS. Recovery was determined as 87.8–118.8% using standard samples. A quartz
filter (Pall corporation., USA) was applied for analysis of carbonaceous species. The filter was prebaked at 450 °C
for 5 h before sampling and stored in a freezer at −20 °C prior to and after sampling. OC and EC were measured
with the OC-EC analyzer employing the thermal-optical transmittance (TOT) method19 based on the National
Institute for Occupational Safety and Health (NIOSH) 5040 temperature protocol20.
For analysis of organic compounds for biomass burning particles, PM2.5 samples were extracted with dichlo-
romethane (DCM) followed by acetone using a Soxhlet extractor. The solvent extract was subsequently reduced
to a volume of 250 μL with a rotary evaporator and nitrogen blowdown. Then, the extract was methylated using
diazomethane (1-methyl-3-nitro-1-nitrosoguanidine, MNNG). It was reacted with silylation reagent to derivat-
ize levoglucosan to trimethylsilyl derivatives (TMS-derivatives). Eight classes of organic compounds (n-alkanes,
hopanes and steranes, PAHs, n-alkanoic acids, benzoic acid, dicarboxylic acids, resin acid and levoglucosan)
and 122 organic species were analyzed using GC-MS (7890A GC, 5975C MSD, Agilent Technologies, USA). For
organic analyses of coal combustion, diesel and gasoline engine exhaust particles, PM2.5 samples were extracted
using the mixture of DCM and methanol (MeOH) (3:1 by volume) with 1 h sonication. The extract was reduced to
a volume of 500 μL with a Turbo Vap concentrator (Turbo Vap II, Caliper Life Sciences, USA) subjected to a gentle
stream of nitrogen (99.9%). Six classes of organic compounds (n-alkanes, hopanes, PAHs, n-alkanoic acids, dicar-
boxylic acids, and levoglucosan (61 organic species)) for coal combustion particles and four classes of organic
compounds (n-alkanes, PAHs, n-alkanoic acids, and levoglucosan (49 organic species)) for diesel and gasoline
engine exhaust particles were analyzed via GC-MS (7890A GC, 5975C MSD, Agilent Technologies, USA).

Biological and chemical responses of fine particles. For assessment of particle toxicity, various chem-
ical and biological assays were conducted. PM2.5 samples on glass fiber filters and PTFE filters (Pall corporation,
USA) were extracted using DI water and dimethylsulfoxide (DMSO) (Sigma Aldrich, USA) at room temperature
and dichloromethane (DCM) (Samchun chemicals, Korea) at 37 °C. For the chemical assay, OP (used to estimate
the capability of PM to generate reactive oxygen species (ROS)) was measured. ROS are reported to cause oxi-
dative stress followed by inflammation and cell death21,22. The OP was measured using two different techniques:
Dithiothreitol (DTT) chemical assay (OP_DTT)23,24 and Electron Spin Resonance spectrometry (OP_ESR)25,26.
OP_DTT evaluates the reaction of ROS via the formation of a DTT-disulfide as a result of electron transfer from
DTT to ROS. DTT is commonly used as a chemical surrogate for cellular reducing agents (NADH and NADPH)
in order to mimic in vivo interactions of PM with biological oxidants. On the other hand, OP_ESR measures the
ability of PM to generate OH radicals via the Fenton oxidation in the presence of H2O2 (hydrogen peroxide) and
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap agent. OP activities measured using both assays were
employed to evaluate the ROS generation capability of PM samples.

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For the biological assay, human airway epithelial (A549, H292, BEAS-2B and SAEC) and Chinese hamster
ovary (CHO-K1) cell lines were used to assess in vitro cell toxicity. A variety of biological responses (cell viability,
genotoxicity (mutagenicity and DNA damage), oxidative stress, and inflammation) that facilitate identification
of the specific PM triggering ROS and inflammatory responses leading to oxidative stress, genotoxicity and cell
death were evaluated for source-specific aerosols27–29. Neutral red uptake (NRU) (Sigma Aldrich, USA)30 and
water-soluble tetrazolium salt (WST-1) assays (Takara, Japan) were utilized to determine cell viability in the pres-
ence of PM2.5. Ames test31 and the Single Cell Gel Electrophoresis (SCGE) Comet assay32 were conducted to
determine mutagenicity and DNA damage of cells induced by PM, respectively. Levels of cellular reactive oxygen
species (ROS) causing oxidative stress in cells were measured via the DCFDA (2′,7′-dichlorofluorescein diacetate)
assay (Abcam, UK)33. Measurements of IL-6 and IL-8 (Abcam, UK) were obtained for determining the expression
of genes related to the inflammatory response34. The initial PM2.5 extracts were diluted 1–150 μg/mL for NRU,
10–500 μg/mL for WST-1, 0.1–1,000 μg/mL for the Ames test, 62.5–500 μg/mL for Comet, 15–150 μg/mL for
DCFDA, and 2–15 μg/mL for the IL-6 and IL-8 assays while undiluted extracts were used for OP assays. Table S8
and Supplementary Information provides details of the chemical and biological assays performed in this study.
The biological effects of PM are not comparable among different studies owing to distinct exposure concentra-
tions, biological models, endpoints, and PM generation methods35. Here, we employed similar exposure and cell
conditions and identical endpoints for various aerosols within the same batch to obtain comparable toxicity data
for PM2.5 from different sources. However, non-linear concentration-response functions for various endpoints
and different exposure concentrations will limit the ability to use toxicological data to predict risks in human
populations. Experimental design for generation, physical and chemical characterization, and toxicity tests for
various primary and secondary aerosols from different sources is illustrated in Fig. 1.

Statistical information. Statistical analysis of all toxicity data performed using SPSS ver. 21.0 (IBM SPSS,
USA). Differences between groups were assessed with Tukey’s post-hoc test following one-way analysis of vari-
ance (ANOVA). Statistical significance was accepted at p < 0.05.

Results
For source-specific aerosols, Fig. 2 illustrates multiple chemical and biological responses (OP, cell viability, geno-
toxicity and DNA damage, oxidative stress, and inflammatory response). The toxicological values employed were
OP activity normalized by PM2.5 mass for OP_DTT using dithiothreitol (DTT) assay and OP-ESR using electron
spin resonance (ESR), half-maximal effective concentration (EC50) for cell viability, specific activity (number of
revertant colonies per PM2.5 mass) for mutagenicity, significant concentration-dependent induction factor (sum-
mation of fold change relative to control divided by dose concentration within the concentration range showing
a significant dose-response relationship) for DNA damage, relative fluorescence intensity (fold change relative to
control for specific dose concentration) for oxidative stress, and relative maximum cytokine production (max-
imum fold change relative to control divided by dose concentration from the dose-response curve) for inflam-
matory response. The dose concentrations among sources were in a similar range for each assay except OP. For
each endpoint, relative toxicological values of source-specific aerosols were plotted as sphere size with standard
deviations. The highest value among the different aerosol types for each response is taken as 100% (maximum size
of the sphere). All measurements of endpoints for source-specific aerosols, except SOAs, were conducted under
similar conditions to facilitate direct comparison of the contributions of the different particles to biological and
chemical responses.
Our results disclosed higher toxicity of combustion than non-combustion aerosols. Among the combustion
aerosols, diesel engine exhaust particles (engine displacement of 2800 cc) were identified as the most toxic based
on chemical and biological responses. Further analyses are required to confirm the statistical significance of this
result as discussed in a toxicity score calculation section. In particular, genotoxicity (mutagenicity) and OP_DTT
of diesel engine exhaust particles were significantly higher than those for other aerosol types. The mutagenic
effects of soot particles are suggested to be associated with the organic components (e.g., PAH) generating reac-
tive oxygen species (ROS) that are able to break DNA strands36. Polar or quinone fractions of PAH in diesel
engine exhaust particles are reported to play an important role in the increased toxic response37. OP_DTT is
sensitive to the amount of quinone, an oxidation product of PAHs and redox recycling agent in particles25,38. The
mutagenicity of particles produced from smaller diesel engines (engine displacement of 498 cc) that emit lower
levels of organic components was relatively low compared to particles from larger engines (2800 cc). A more
in-depth explanation of chemical components is provided in a later section. Gasoline engine exhaust particles
also showed comparable or lower toxicity relative to diesel engine exhaust particles based on various endpoints.
Both rice straw and pine stem burning particles (popular biomasses used in East Asia) showed significant tox-
icity in terms of effects on cell viability (NRU and WST-1) comparable to that of diesel engine exhaust particles.
Pine stem burning particles were associated with higher inflammatory responses than rice straw burning parti-
cles. However, OP_ESR and OP_DTT were higher for rice straw particles. OP_DTT and OP_ESR is reported to
be correlated well with OC and trace elements (Cu and Zn), respectively25,26. Rice straw particles contained higher
fractions of Cu, Zn, and OC than pine stem particles, explaining the higher OP values obtained. Evaluation of the
OP of PM2.5 using multiple assays is essential to provide useful complementary information.
In the case of coal combustion particles (burned at 1100 °C and 550 °C), the biological responses were rela-
tively low compared to other combustion particle types. The bituminous coal was selected because it was the most
abundant type used in the world39. Also, it has been extensively used in power plants in East Asia40,41. Variability
in toxicity among different coal types was found to be small42,43. Two burning temperatures were used for simula-
tion: residential coal combustion (low temperature) and power plant coal combustion (high temperature). Coal
combustion particles generated at a temperature of 550 °C showed relatively higher toxicity in many endpoints
than those at 1100 °C. One potential explanation for this finding is that the higher burning temperature leads to

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Figure 2. Multiple chemical and biological responses (oxidative potential, cell viability, genotoxicity (based on
mutagenicity and DNA damage), oxidative stress and inflammatory response) for source-specific aerosols. The
relative magnitude is plotted as the size of sphere with standard deviation, and the highest value among different
aerosol types for each response is taken as 100% (maximum sphere size). aCoal combustion temperature.
b
Engine displacement.

more complete burning of pulverized coal, leading to lower emission of carbonaceous species. Our data suggest
that residential coal combustion particles typically emitted at lower burning temperatures are more toxic than
power plant coal combustion particles emitted at higher burning temperatures. Levels of carbonaceous species,
including toxic organic compounds (such as PAH), were significantly higher in residential than industrial coal
combustion particles44.
Ammonium sulfate and ammonium nitrate particles which were aerosolized and dried from their solutions
showed little toxicity in almost all endpoints. Limited in vitro toxicity data on these particles are currently availa-
ble. Both neutralized sulfate and nitrate particles exert little or no effects on inflammatory responses and cell via-
bility45,46, and inclusion of strong acids (e.g., sulfuric acid or nitric acid) in particles is proposed to lead to adverse
respiratory effects47,48. The acid aerosols can increase metal solubility yielding the ROS. Earlier epidemiological
studies26,49–51 indicate that sulfate has detrimental effects on human health. It is possible that sulfate in the ambient
atmosphere reacts with other chemical components, leading to modification of the size and chemistry of the par-
ticles, and consequent adverse health effects, in contrast to laboratory-generated sulfate particles52. However, the

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Figure 3. Normalized toxicity scores (0 to 10) for source-specific aerosols with differential weights (CCSD) and
equal weights given to endpoints.

issue of whether sulfate exerts combined effects with other chemical components (e.g., OC and metals) emitted at
the same time remains to be established45,53.
Fine particles (< 2.5 µm) from Arizona dust13 mainly consisting of mineral/soil components showed little
toxicity. Additionally, low toxicity was observed for sea spray aerosols from natural seawater based on OP and cell
viability analyses (data not shown). However, we observed significant toxicity of resuspended fine dust collected
from the roadsides and tunnels at urban sites based on oxidative potential, cell viability, and inflammation end-
points, which may be attributable to carbonaceous species and heavy metals originating from vehicles (engine
exhaust, tire wear, and brake pad). Fine particles from carbon black powder (Cabot Inc., USA) exerted a little
toxicity, suggesting that carbon black itself would not be so harmful but adsorbed components, such as organics
and heavy metals, are the greater contributory factors54.
SOA showed substantial toxicity in OP and inflammatory response (IL-8) assays (Fig. 2). Specifically, OP of
the SOA produced from photochemical reaction of toluene and NOx under natural sunlight (i.e., toluene SOA)
using the UF-APHOR chamber (outdoor smog chamber) was higher than that obtained for other types of SOA
(TMB SOA, isoprene SOA, and α-pinene SOA), and was comparable to biomass (hickory wood) burning parti-
cles. Toluene is the most abundant volatile organic compound (VOC) emitted from motor vehicles (anthropo-
genic source)55,56 while isoprene SOA is globally the highest VOC from natural sources57. The IL-8 response of
toluene SOA was higher than TMB SOA. In our study, no cell toxicity (WST-1) was detected for both aromatic
and biogenic SOA. The range of exposed mass concentrations of SOA are summarized in Table S2.
As discussed in previous sections, biological and chemical responses for source-specific aerosols varied sig-
nificantly among toxicological endpoints, making accurate quantification of the representative toxicity difficult.
For example, chemical components related to inflammatory responses may be distinct from those associated
with bacterial mutagenicity. The relative magnitude of one response would not be feasible to represent toxicity
of particles. Average toxicity data obtained for different types of diesel or gasoline engines, biomasses (rice straw
and pine stem) and coal combustion temperatures were used to represent diesel engine exhaust, gasoline engine
exhaust, biomass burning and coal combustion particles, respectively. A Multiple Attribute Decision Making
(MADM) model was applied to derive toxicity scores of source-specific aerosols from various toxicity data. The
MADM is to make choice of the best alternative among a set of alternatives with multiple and conflicting attrib-
utes58. Determination of the weights of attributes is very important in the process of decision making58. There are
three types of MADM models to determine the weights of attributes (subjective, objective and integrated)58–60.
In this study, Correlation Coefficient and Standard Deviation (CCSD) method, which is the objective decision
making model58, was employed to determine the weights of attributes (endpoints) when the outcome (toxicity
score) was simultaneously influenced by multiple attributes58. In the CCSD method, the endpoint having the
highest standard variation among source types had the highest weight (i.e., differential weights were given to
endpoints) without any subjective judgement. As resulted from the CCSD, the highest weight was found for cell
viability followed by mutagenicity, oxidative potential, inflammatory response, and oxidative stress. The derived
weights were multiplied by normalized values of endpoints to determine the toxicity score of source-specific aer-
osols. The toxicity score was normalized up to 10 (normalized toxicity score of source i = (toxicity score of source
i − minimum toxicity score)/(maximum toxicity score − minimum toxicity score) * 10). Source with the highest
toxicity has the toxicity score value closest to 10. Multiple toxicological data were integrated into the toxicity score
for source-specific aerosols as shown in Fig. 3.
Overall, as shown in Fig. 3, the highest toxicity score was obtained for diesel engine exhaust particles, followed
by gasoline engine exhaust particles, biomass burning particles, coal combustion particles, road dust, desert dust,
ammonium sulfate, and ammonium nitrate, suggesting that traffic plays the most critical role in enhancing the
toxic effects of PM2.5. Toluene SOA toxicity based on OP data was comparable to biomass burning particles. In

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Figure 4. Comparison of daily toxicity score with mass-normalized OP_DTT (pmol/min/µg) for ambient
PM2.5.

general, the concentration of quinones is negligible in SOA. Jiang et al.57 reported that alkylperoxides produced
from photo-oxidation of precursor hydrocarbons were responsible for the OP_DTT response of the resulting
SOA via a non-catalytic reaction mechanism. However, due to the limited number of endpoints for SOA, the
SOA data were not included in the CCSD method. In addition to the CCSD method, a linear combination of nor-
malized values of endpoints (i.e., equal weights were given to endpoints) for each source was used to determine
the toxicity score (0 to 10) of source-specific aerosols. The toxicity ranking with equal weights was similar to that
obtained from the CCSD method with differential weights as shown in Fig. 3. Hereafter, the toxicity score (0 to
10) derived from the CCSD method was used for further calculation.
The aging process of freshly emitted particles in the ambient atmosphere may also modulate toxicity. For instance,
aged combustion particles oxidized by ozone are suggested to exacerbate lung injury and inflammation relative to
non-oxidized particles36,61,62. The effects of atmospheric aging on toxicity of particles require further investigation.
The toxicity score for each source could therefore be continuously improved by adding new data sets from in vitro, in
vivo, and epidemiology studies. The toxicity score results should be useful for decision makers to assess contribution
of PM2.5 sources to human health and to establish PM abatement policies in addition to PM2.5 mass concentration.
Typically, source contribution for ambient PM2.5 is determined from the measured chemical components
using various source apportionment methods10. By combining the toxicity scores for source-specific aerosols with
the mass fractions of sources in ambient PM2.5, toxicity score for ambient PM2.5 can be derived. The toxicity score
for ambient PM2.5 is the summation of values obtained by multiplying toxicity scores for source-specific aerosols
by mass fractions of the corresponding sources (toxicity score for ambient PM2.5 = toxicity score of source 1 *
mass fraction of source 1 + toxicity score of source 2 * mass fraction of source 2 + toxicity score of source 3 *
mass fraction of source 3 + toxicity score of source 4 * mass fraction of source 4+ ….). The summation assumes
that source-specific toxicity scores are additive.
We measured the OP_DTT for ambient PM2.5 samples. The OP is a good indicator of the oxidizing capability
of PM without cell culture and widely used for toxicity testing of ambient aerosols due to its simplicity of applica-
tion29. The oxidizing capability of PM can trigger oxidative stress followed by cell death and can be considered as an
additional health metric29. The daily toxicity score for ambient PM2.5 derived here was compared with the measured
OP_DTT (pmol/min/µg) as shown in Fig. 4. It revealed a moderate correlation between the two values (r = 0.49,
n = 34). The toxicity score or OP_DTT was found to vary significantly even with similar PM2.5 mass concentrations
which was attributable to the significant differences in the contributory sources over the experimental days.
The principal component analysis (PCA) method was applied to relate chemical and biological responses of
source-specific aerosols to their chemical components. Details on chemical data for source-specific aerosols are
included in Methods and Tables S3, S4, S5, and S6. For PCA, 9 samples (diesel engine exhaust particles (heavy and
light duty), gasoline engine exhaust particles, rice straw burning particles, pine stem burning particles, coal com-
bustion particles burned at 550 °C and 1100 °C, dust (tunnel and roadside)) and 30 variables including chemical
components (fractions in PM2.5) and biological and chemical responses (relative magnitude in each endpoint) were
used. The chemical components included 8 ions (ammonium, calcium, magnesium, potassium, sodium, chloride,
nitrate, and sulfate), 14 elements (Al, As, Ba, Cd, Co, Cu, Fe, Mn, Ni, Pb, Sr, Ti, V, and Zn), 2 carbonaceous species
(OC and EC), and 4 OC classes (n-alkanes, PAHs, alkanoic acids, and sugars (levoglucosan only)). The biological
and chemical endpoints were oxidative potential (OP_DTT and OP_ESR), cell viability (NRU (CHO), NRU (H292)
and WST-1 (A549)), genotoxicity (mutagenicity and DNA damage), oxidative stress (DCFDA), and inflammatory
response (IL-6 and IL-8). As shown in Fig. 5 (loading plot), almost all toxicological endpoints were grouped together
with total OC, PAHs, n-alkanes and EC in principal PC1 and PC2. The sum of PC1, PC2 and PC3 explained 78%

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Figure 5. PCA results for relation of chemical and biological responses of source-specific aerosols to their
chemical components.

variance of all data (PC1 = 46%, PC2 = 17% and PC3 = 16%). However, biological responses can be influenced not
only by chemical composition but also interaction among chemical species, bioavailability, and oxidation state.
Compounding effects between chemical species cannot be fully described by correlation analysis.

Discussion
Fine particles were generated from distinct combustion (diesel engine, gasoline engine, biomass burning and
coal combustion) and non-combustion (road dust, sea spray aerosols, ammonium sulfate, ammonium nitrate
and SOA) sources. Measured sizes, chemical components and toxicities of source-specific aerosols varied signif-
icantly among the PM2.5 source types, suggesting that even at the same mass concentration, the effects of PM2.5
on human health are significantly variable. Various biological (cell viability, genotoxicity, oxidative stress and
inflammation in respiratory cells) and chemical (oxidative potential) responses to source-specific aerosols were
measured and statistically integrated to derive toxicity scores using the MADM model which determines the
weights of endpoints when the toxicity score is simultaneously influenced by multiple endpoints. The highest
weight was found for cell viability followed by mutagenicity, oxidative potential, inflammatory response, and
oxidative stress. The weights for endpoints can be further improved by adding new assays with different cells.
Also, the MADM model can be modified by putting more emphasis on specific health endpoints. Diesel engine
exhaust particles had the highest toxicity score followed by biomass burning, gasoline engine exhaust, and coal
combustion particles. Moreover, different size distributions of aerosols from various sources resulted in variable
total and lung deposition efficiencies of particles in the human respiratory system which can be used to estimate
lung dose more accurately. The tested method assigned priority to PM2.5 sources potentially harmful to human
respiratory system. Further incorporation of toxicological data which are related to health effects on cardiovas-
cular system could improve the toxicity score database. In-vivo toxicological data for acute toxicity, sub/chronic
toxicity, carcinogenicity, and other toxicity can also reinforce the toxicity score.
It is not straightforward to use in vitro data (cell viability, genotoxicity, inflammatory response, oxidative
potential and oxidative stress) determined in this study to predict health effects (morbidity and mortality) in
human populations. In general, in vitro data can be used to rank various types of particles in terms of the toxic
potential including possible carcinogenicity. Each of the marker we have studied will help to understand the haz-
ard and differential toxicity of various fine particles.
A priori knowledge of toxicity of particles produced from various sources obtained here can be linked with
source apportionment and exposure level to derive a new health metric for ambient PM2.5 in future work. The
toxicity potential of various types of particles should be integrated with long term or short term health effects at
individual or population level to predict public health impacts with consideration of differential toxicities of fine
particles.

Conclusion
The database for toxicity scores for source-specific aerosols was constructed which can be and used to better
understand the complex detrimental health effects caused by different fine particle types of ambient PM2.5. By
considering differential toxicities of particles although it must be continuously improved, it could be possible to
provide information that is more relevant for decision makers to establish PM2.5 abatement policies rather than
only focusing on PM2.5 mass concentration.

Data Availability
Data from this study are available from the corresponding author upon reasonable request.

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Acknowledgements
This research was supported by the National Leading Research Laborator y program (NRF-
2016R1A2A1A05005532) and the PM 2.5 research consortium (NRF-2014M3C8A5028593 and NRF-
2017M3D8A1092220), both funded by the Ministry of Science and ICT (MSIT) and the National Research
Foundation (NRF) of Korea.

Author Contributions
K.P. initiated and designed the study. K.L., M.B. and J.L. measured the chemical composition of PM2.5. M.P.,
H.S.G., M.J., L.J.S.B., H.L., H.S. and K.H.C. conducted toxicity tests. I.K. and S.D.K. performed toxicity score
derivation. Y.H.C. and S.G.P. conducted epidemiological analysis. K.P., M.P., and H.S.J. analyzed all the data and
wrote the manuscript. All authors discussed and approved the content of the manuscript.

Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-35398-0.
Competing Interests: The authors declare no competing interests.
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