Mutagenicity and Cytotoxicity of Particulate Matter Emitted from

Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Mutagenicity and Cytotoxicity of Particulate Matter Emitted from Biodiesel-Fueled Engines Avinash Kumar Agarwal,*,† Akhilendra P. Singh,† Tarun Gupta,‡ Rashmi A. Agarwal,‡ Nikhil Sharma,† Prashant Rajput,‡ Swaroop K. Pandey,§ and Bushra Ateeq§ †

Engine Research Laboratory, Departments of Mechanical Engineering, ‡Civil Engineering and §Biological Sciences & Bioengineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India

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S Supporting Information *

ABSTRACT: Biodiesel engines produce several intermediate species, which can potentially harm the human health. The concentration of these species and their health risk potential varies depending on engine technology, fuel, and engine operating condition. In this study, experiments were performed on a large number of engines having different configurations (emissions norms/fuel used), which were operated at part load/full load using B20 (20% v/v biodiesel blended with mineral diesel) and mineral diesel. Experiments included measurement of gaseous emissions, and physical, chemical, and biological characterization of exhaust particulate matter (PM). Chemical characterization of PM was carried out for detecting polycyclic aromatic hydrocarbons (PAH’s) and PM bound trace metals. The biological toxicity associated with PM was assessed using human embryonic kidney 293T cells (HEK 293T). The mutagenic potential of the PM was tested at three different concentrations (500, 100, and 50 μg/mL) using two different Salmonella strains, TA98 and TA100, with and without liver S9 metabolic enzyme fraction. PM samples exhibited cytotoxic effect on HEK 293T cells (IC50 < 100 μg/mL) and there was significant potential for reactive oxygen species (ROS) generation. Comparison of different engines showed that modern engines (Euro-III and Euro-IV compliant) produced relatively cleaner exhaust compared to older engines (Euro-II compliant). Biodiesel-fueled engines emitted lower number of particles compared to diesel-fueled engines. However, chemical characterization revealed that biodiesel-fueled engines exhaust PM contained several harmful PAHs and trace metals, which affected the biological activity of these PM, as reflected in the biological investigations. Mutagenicity and cytotoxicity of PM from biodiesel-fueled engines were relatively higher compared to their diesel counterparts, indicating the need for exhaust gas after-treatment.

1. INTRODUCTION Extensive utilization of nonrenewable energy resources has led to a serious threat to sustainability of global energy resources. Diesel exhaust particles are declared as “potential occupational carcinogen” and “likely human carcinogen via inhalation pathways”.1−3 In modern vehicles, application of aftertreatment devices such as diesel particulate filters (DPFs) has gained significance in order to meet regulatory emission standards. However, regeneration of DPFs to remove deposited carbonaceous particles can potentially generate a large number of newer and smaller nanoparticles (NP, Dp < 10 nm), which exists as a major concern.4 DPF can reduce the PM mass emissions, which is beneficial in reducing the short-term (acute) exposure to diesel exhaust via inhalation; however, its long-term (chronic) effects such as exposure to higher NP needs to be further investigated comprehensively. Besides health hazards, the global fuel crisis (by the late 1970s) motivated researchers to explore alternative fuels to meet increasing energy demand. Toward this, biofuels seem to be an attractive option for achieving clean combustion. In © XXXX American Chemical Society

earlier studies, it has been reported that the engines operated with blends of mineral diesel and biodiesel show nearly similar combustion and performance characteristics along with reduced PM emissions compared to mineral diesel-fueled engines.5,6 Biodiesel-fueled engines also produce a reduced amount of unburnt hydrocarbons (HCs), carbon monoxide (CO), and polycyclic aromatic hydrocarbons (PAHs).6−8 It is worthwhile mentioning here that biodiesel-fueled engines emit relatively higher oxides of nitrogen (NOx) that act as a precursor for ozone formation, and thus, have potential to cause harmful health effects.9−11 Brito et al.12 conducted experiments and reported that the exhaust from biodiesel was more toxic as compared to mineral diesel. Higher number of NP from biodiesel-fueled engines offer higher surface area-tomass ratio, enhanced atmospheric chemical reactivity (via Received: June 19, 2018 Revised: November 9, 2018 Accepted: November 20, 2018

A

DOI: 10.1021/acs.est.8b03345 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

different configurations fueled with mineral diesel and biodiesel were investigated for mutagenicity and cytotoxicity, in addition to the trace metals and morphological investigations of PM. An integrated approach including physical and chemical characterization of PM emitted from different engine operating conditions, engine configurations, fuels and their human health effects makes this study quite versatile.

uptake of various chemical species), undergo long-range transport (owing to higher residence time) and deeper penetration in to the human alveoli. Previous studies have shown that NP have a higher potential to affect human health (in comparison to accumulation mode particles (AMP, 50 nm < Dp< 1000 nm) and they act as a carrier of toxic species such as PAHs, dioxin derivatives, quinones, aldehydes, and trace metals.8−11 The differences in combustion conditions and agglomeration of gaseous species result in different PAHs emission profile. Moreover, the atmospheric chemical reactivity of certain PAHs with OH radical, O3, and NOx results into derivatives such as oxy- and nitro-PAHs with higher molecular weight and mutagenic and carcinogenic behavior.13 Gaseous emissions of aldehydes and various moieties of organic compounds emitted from biodiesel-fueled engine exhibit potential toward oxidative stress.13 Recent studies showed increasingly higher risk of engine-out emission such as isocyanic acid (HNCO), which have detrimental effects on the human health. Protein carbamylation and associated inflammatory responses caused by isocyanic acid and cyanate (NCO−) are shown to be common biochemical pathways for cardiovascular impairment and chronic diseases.14 Roberts et al.15 reported that even a very low concentration (1 ppbv) is sufficient for carbamylation reactions in the human body. The use of biodiesel in production grade engines is quite favorable owing to relatively cleaner emissions (of gaseous pollutants) in the exhaust. However, there is a lack of understanding on adverse human health effects in terms of mutagenicity and cytotoxicity caused by the biodiesel combustion. Lung is the first organ to be affected by PM exposure and most toxicological studies have been done on the lungs to assess the effect of PM exposure. Cardio-respiratory effect of PM is well-studied and is found to be associated with ROS generation and inflammation.16−19 Several independent studies have compared the toxic effect of diesel and biodiesel engine exhaust. Mutlu et al.20 studied the effect of biodiesel utilization in a modern diesel engine and reported that blending of biodiesel with mineral diesel resulted in lower mutagenic potential of exhaust emissions compared to that of baseline mineral diesel. They explained that this reduction was mainly because of reduction in concentrations of highly polar mutagens, including PAHs, aromatic amines, nitroarenes, and oxy-PAHs. However, their conclusion was applicable to small engines only, and these trends could possibly vary depending on the engine technology, size, and operating conditions. Steiner et al.21 showed that biodiesel engine exhaust PM were safer for human lung cells than their mineral diesel counterparts, while Yanamala et al.22 reported higher toxicity of biodiesel engine exhaust PM than mineral diesel exhaust PM when mice lungs were exposed. According to recent studies, kidney is another vital organ, which is particularly vulnerable to toxic effects from environmental pollutants such as PM and heavy metals since it filters entire bloodstream and all the toxins eventually reach it. Long-term exposure of PM causes severe kidney damage, acute renal failure and related deaths, especially in developing countries.23−29 To the best of our knowledge, no such comparative study of exposure of biodiesel-fueled engine exhaust PM is done on kidney or kidney cells. Moreover, there is a dearth of information that can relate the cytotoxicity and mutagenicity potential of PM emitted from biodiesel-fueled engines of different emission norms operated at different engine loads. In this study, three diesel engines of

2. EXPERIMENTAL SETUP In this study, PM sampling was carried out from three engines with different emission norm compliances, namely Euro-II, Euro-III, and Euro-IV (Table 1). These represent typical mix Table 1. Technical Specifications of the Test Engines test engine emission norm fuel injection system number of cylinders bore/stroke compression ratio cubic capacity (cc) maximum engine output maximum torque application type

engine 2

engine 3

Euro-II diesel indirect injection (IDI) 4

engine 1

Euro-III diesel common rail direct injection (CRDI)

Euro-IV diesel common rail direct injection (CRDI)

2

4

75/79.5 mm 22

83/84 mm 18.5

85/96 mm 17.5

1405

909

2200

39 kW @ 5000 rpm

20 kW @ 3600 rpm 103 kW @ 4000 rpm

85 N m @ 2500 rpm medium-duty

55 N m @ 1800−2200 rpm medium-duty

320 N m @ 1700−2700 rpm heavy-duty

of diesel engines with different emission compliances, having mixed traffic of both older and modern technology vehicles on the roads of any mega-city (such as Delhi).30 Higher emission compliance engines and vehicles have been ignored since their population density would be quite low and so will be their environmental impact. All engine experiments were conducted at a constant representative engine speed of 2000 rpm at 50% and 100% engine loads for the sake of scientific comparison. The experiments were performed using two test fuels namely B20 (20% (v/v) biodiesel blended with mineral diesel) and baseline mineral diesel. Important properties of these test fuels are given in Supporting Information (SI) Table S1. The schematic of collection and characterization scheme of PM from different engines is shown in Figure 1. A raw exhaust gas emission analyzer (Horiba: EXSA-1500) was used for the measurement of the regulated emission species and a Fourier transform infrared (FTIR) analyzer (Horiba; FTIR6000-FT(E)) was used for the measurement of unregulated emission species.7 Physical characterization of particles emitted from the engines was carried out using engine exhaust particle sizer (EEPS) spectrometer (TSI Inc.; EEPS3090), which measured particles size-number distribution in the size ranging from 5.6−560 nm, up to a maximum number concentration of 108 #/cc at 10 Hz frequency.31 A partial flow dilution tunnel was used for collecting the PM samples from the engine exhaust on a preconditioned quartz filter substrate.32 For each test engine, test fuel, and engine load combination, PM sampling was done for 30 min. The B


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Figure 1. Experimental scheme (B20:20% v/v blend of biodiesel with mineral diesel, IDI: indirect injection, CRDI: common rail direct injection).

identification of even high molecular weight compounds. The samples were prepared by dissolving PM-laden quartz filter segments in an organic solvent such as acetonitrile (CH3CN), for 48 h under stirring condition at room temperature. This ensured extraction of nearly all species from the PM samples. The filtrates from PM laden filter papers contained dissolved hydrocarbons, which were used as test samples for further analysis. 3.3. Morphological Investigations. The carbonaceous structure of PM emitted from different test engines and test fuels were determined using transmission electron microscope (TEM) (FEI, Tecnai G2 12 Twin TEM 120 kV). The PM laden quartz filters were cut into pieces and then treated with solvent (benzene).34−37 This detached the soot particles from the filter paper and they formed a suspension in benzene. The benzene drops including suspended PM were then deposited on a TEM copper grids (300 mesh size) using a microsyringe and the solvent (benzene) was allowed to evaporate (under inert condition), leaving the PM deposited on the TEM grid for morphological investigations in the TEM. 3.4. Biological Investigations. For biological assay, organic extracts of samples were prepared using the method described in previous article.30 PM laden filter paper was cut into small pieces, dissolved overnight in dichloro-methane (DCM). Then DCM was allowed to evaporate under inert conditions (using N2 purge) at room temperature. The residue was redissolved in dimethyl-sulfoxide (DMSO) and kept at −20 °C for further analysis. 3.4.1. Toxicity Assay and Oxidative Stress Measurement. Human embryonic kidney (HEK293T) cells were cultured in a 75 cm3 cell culture flask, supplemented with minimum essential media (MEM) and 10% fetal bovine serum, provided with 5% CO2 at 37 °C. For cytotoxicity assay, cells were trypsinized, counted using a Coulter counter (Beckman, Indianapolis, IN) and then resuspended in the culture medium. The cells were then seeded in 96-well cell culture plate (∼4000 cells/well) and were allowed to attach overnight. Primary screening was done by exposing the cells at three concentrations of the test samples (50, 100, and 250 μg/mL)

particulate mass collected from each test fuel and test engine combination is given in SI Table S2.

3. EXPERIMENTAL METHODOLOGY PM laden filters were subjected to various chemical characterizations including trace metals, PAH analysis, morphological and biological investigations using different analytical techniques, as described in following subsections.32,33 3.1. PM Bound Trace Metals. High purity chemicals (Seastar, Fischer) viz. HF (47−51%, Suprapure), HNO3 (67− 69%, Suprapure), H2O2 (>30%, Trace analysis), and Milli-Q water (Resistivity: 18.2 MΩ.cm) were used for digestion of trace metals and sample preparation. Aerosol samples (n = 28; full filter of 47 mm diameter; collected onto quartz substrate) digestions were achieved in closed Teflon vials (Savillex) on a hot plate (Graphite coated with PFA; per-fluoro-alkoxy, Analab Scientific Instruments). Briefly, each aerosol sample was treated with HF: HNO3: H2O2: H2O in a stoichiometric proportion of 1:8:3:6 (v/v).33 Subsequently, the capped vials were kept on the hot plate and the temperature of 150 °C was maintained for ∼24 h to ensure complete digestion. Thereafter, the temperature of hot plate was reduced to 100 °C and digested samples were allowed to undergo evaporation until near dryness. Finally, a 250 μL of 8N HNO3 was added to the residue and the total solution volume was made up to 10.25 mL using Milli-Q water. Subsequently, each sample was filtered through a 0.22 μm Teflon filter and trace metals analysis was performed using inductively coupled plasma-optical emission spectrometer (ICP-OES; iCAP 6300 Thermo Scientific). Quality control of the trace metals data was done by analyzing procedural blanks (n = 5) digested in the same batch along with the aerosol samples. The concentration of each trace metal reported herein represents the blank corrected values for full filter. 3.2. Mass Spectrometry. PAHs in the test samples were analyzed by quadrupole time of flight (Q-TOF) mass spectrometer (Waters; HAB213) equipped with electro-spray ionization (ESI) technique. The mass spectrometer separates ions according to their mass-to-charge ratio (m/z), facilitating C


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Figure 2. (a) Regulated (carbon monoxide (CO), hydrocarbon (HC), oxides of nitrogen (NOx)), and (b) unregulated (sulfur dioxide (SO2), formaldehyde (HCHO) and isocyanic acid (HNCO)) gaseous emissions from mineral diesel and biodiesel-fueled test engines.

linear portion of the dose−response curves, as described earlier.39 Data presented as mean ± SEM. P-values calculated using two-sided Student’s t test. Two-way analysis of variance (ANOVA) with Tukey post hoc test using GraphPad Prism was employed for determining the statistical significance of the PM showing mutagenic potential.

for 48 h. The cell viability was examined by adding Resazurinbased fluorescent dye to each well and incubated for 4 h. The plates were then read under a florescence reader at 530/590 nm excitation/emission wavelengths. IC50 values of the samples showing toxicity at 50, 100, or 250 μg/mL were calculated by exposing the HEK 293T cells to 2-fold serial dilution for 48 h, followed by Resazurin assay as described above. The IC50 values were determined by analyzing the dose−response curve. The cellular levels of ROS were determined using 5-(and-6)chloro-methyl-2′,7′-dichloro-dihydro-fluorescein diacetate, acetyl ester (CM-H2DCFDA). The HEK 293T cells were treated with the test samples, DMSO (solvent control), and 50 μM H2O2 (positive control) for 24 h, followed by CMH2DCFDA (Invitrogen, Waltham, MA) treatment for 4 h. Subsequently, cells were processed using lysis buffer (SigmaAldrich, C-2978). The fluorescence readings were taken at an excitation/emission wavelength of 492/517 nm. Experiment was performed in triplicate with three biological replicates. 3.4.2. Ames Test for Mutagenicity. The mutagenicity assay was performed using Salmonella strains TA98 and TA100, with and without metabolic activation by the microsomal fraction of Aroclor-induced rat liver homogenates (S9) using Ames MPF kit (Xenometrix, Switzerland) according to manufacturer’s instructions. Experiments were performed in 384 well plates in duplicate (48 wells for each sample). The known mutagenic agents (4-nitroquinoline-N-oxide for TA98 and 2-nitrofluorene for TA100 in absence of S9 fraction; 2-aminoanthracene for both the strains in the presence of S9 fraction) were used as positive controls at the recommended dosage. The concentration of a sample/positive control producing two times higher number of revertants above the baseline was considered as mutagenic.38 The baseline was taken as a sum of a number of revertants obtained in DMSO (solvent) control and the standard deviation. The experiment was performed in duplicate and repeated thrice. The mutagenic potencies were determined in terms of rev/μg by linear regression over the

4. RESULTS AND DISCUSSION For all investigations, comparisons between different parameters namely engine technologies (Euro-II, Euro-III, and EuroIV); engine load (50% and 100%) and test fuels (B20 and mineral diesel) were made. For statistical significance of the results, two-tailed Student’s t-test was performed. Results with p-values 10; p < 0.0001). In contrast, CRDI engines emitted slightly lower CO at 100% load than at 50% load. Biodieselfueled engines emitted relatively lower CO compared to mineral diesel-fueled engine (except Euro-IV engine) due to better oxidation of CO into CO2.41 Diesel-fueled Euro-IV engine emitted the lowest CO among all test engines and test fuels. In sharp contrast, HC emissions from Euro-II engine at 50% load were higher than that at 100% load (t = 5; p < 0.001), however Euro-III and Euro-IV engines did not show significant difference in HC emissions at different loads. Significantly lower NOx emissions from Euro-III and EuroIV engines compared to Euro-II engine was another important observation.42 In Euro-II engine, NOx emissions at 50% load were significantly higher than that of 100% load (t > 8; p < 0.0001); however, differences in NOx emissions from CRDI D


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Figure 3. (a) Number-size distribution, and (b) count mean diameter (CMD), total particle number (TPN), and total particle mass (TPM) emitted from mineral diesel and biodiesel-fueled test engines.

Brady et al.48 suggested that higher CO emission promotes formation of isocyanic acid. Lower CO emission along with lower isocyanic acid emission from Euro-IV engine is in line with the trend suggested by Brady et al.48 In Euro-II engine, higher engine load resulted in lower isocyanic acid emission, which is in contrast to Euro-III and Euro-IV engines, where increasing engine load resulted in higher isocyanic acid emission. Slightly higher isocyanic acid emission from biodiesel-fueled engines (notably in Euro-II engine) compared to mineral diesel-fueled engine is another important observation from this study. Overall, older engines emitted higher concentration of unregulated gaseous emission compared to modern engines, when biodiesel was used as an alternative fuel. 4.2. Physical Characterization of Particles. Physical characterization of particles included assessment of particle number-size distribution, in different size ranges namely NP, nucleation mode particles (NMP, 10 nm < Dp< 50 nm) and AMP. Figure 3(a) shows that Euro-III and Euro-IV engines emitted higher number of particles (especially larger size particles) compared to Euro-II engine. At 50% load, all engines emitted relatively lower number of particles compared to that at 100% load, which were mainly distributed in NMP range. Increasing the engine load resulted in formation of higher number of particles; however entire particle number-size distribution curve shifted toward larger size particles. Biodieselfueled engines emitted relatively lower number of particles compared to that from mineral diesel. Presence of oxygen in biodiesel may be an important factor responsible for this trend, which could have improved soot oxidation after their formation. Higher number of NP emitted by biodiesel-fueled test-engine compared to mineral diesel is another important observation of this study (SI Figure S1).

engine at different engine loads were not significant. At 50% load, NOx emissions from biodiesel-fueled engines were slightly higher than mineral diesel-fueled engines (t > 1; p < 0.05) (Figure 2a), however at 100% load, these differences were not statistically significant (p > 0.05). According to Kisin,43 biodiesel blend showed higher NOx emissions, having higher mutagenicity compared to baseline mineral diesel. Figure 2(b) shows the concentrations of unregulated gaseous species emitted from different test engines and test fuels. SO2 emissions were the lowest in case of Euro-IV engine fueled with diesel and biodiesel at both the loads. Euro-II engine showed significantly higher SO2 emission at 100% load compared to 50% load (t > 25; p < 0.0001). At 50% load, all biodiesel-fueled test engines emitted slightly higher SO2 compared to their mineral diesel counterparts however at 100% load; this difference was not statistically significant. Bunger et al.44 reported that diesel-fueled engines emitted lower SO2, leading to lower mutagenicity. Formaldehyde emission was the highest from Euro-II engine fueled with biodiesel at both the loads, which is in line with trend reported by Bunger et al. 44 Formaldehyde is an intermediate combustion product, which is formed due to partial combustion/pyrolysis of the fuel and lubricating oil. Euro-IV engine fueled with mineral diesel and biodiesel showed relatively lower formaldehyde emissions compared to Euro-II and Euro-III test engines. Compared to 50% engine load, test engines emitted relatively lower traces of formaldehyde at 100% load. Previous studies showed that use of biodiesel increased aldehyde emissions, which in turn increased the mutagenicity and toxicity of the engine-out exhaust.45−47 Isocyanic acid emission was the lowest from Euro-IV test engine. Isocyanic acid is formed in higher quantities when NOx, CO, and H2/ NH3 react over precious metal catalysts.48 E


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Figure 4. Particulate matter bound trace metals emitted by mineral diesel and biodiesel-fueled test engines. (Al: aluminum, Cu: cupper, Fe: iron, Zn: zinc, Ni: nickle, Cr: chromium, Pb: lead, As: arsenic).

Figure 3(b) shows count mean diameter (CMD) of particles, which represents the average size of particles emitted from the engine.31 CMD trends showed that Euro-II engine emitted relatively larger particles compared to Euro-III and Euro-IV engines (Figure 3a). CMD of particles at 100% load was relatively larger than that at 50% load for all test engines and test fuels (t > 20; p < 0.0001). At 50% load, effect of biodiesel was clearly visible in CMD trends, and it significantly increased (t > 30; p < 0.0001). However, at 100% load, the increase in CMD of particles emitted from biodiesel-fueled engines was not so significant. Euro-II engine emitted relatively lesser total particle number (TPN) compared to Euro-III and Euro-IV test engines. Among all test engines, Euro-III engine at 100% load showed the maximum TPN. Biodiesel-fueled Euro-II engine emitted higher TPN compared to diesel-fueled Euro-II engine; however, Euro-III and Euro-IV engines emitted lesser TPN from biodiesel compared to mineral diesel. Total particle mass (TPM) was calculated from the data of mass-size distribution of particles.31,49 Results showed that Euro-III engine emitted the highest TPM followed by Euro-II and Euro-IV engines. All engines emitted relatively higher TPM at 100% load, compared to 50% load (except biodieselfueled Euro-IV engine). At 50% load, all biodiesel-fueled engines emitted relatively higher TPM compared to dieselfueled engines however at 100% load, only Euro-II engine followed this trend. This exhibited less favorable emission characteristics of biodiesel-fueled older engines (such as Euro-

II) compared to relatively more modern engines (such as Euro-III and Euro-IV). 4.3. Chemical Characterization of PM. Figure 4 shows the PM bound trace metals emitted by different mineral diesel and biodiesel-fueled test engines. These trace metals are represented in miligram/gram of the PM collected from the engine exhaust. PM samples contain few trace metals, for example, Cr, As, and Ni, which are considered to be “probable carcinogens”.30 There are three main sources of trace metals in the engine exhaust PM namely: the fuel, friction and wear generated debris from the engine components, and lubricting oil additives. First category of harmful trace metals inculdes Al, Cu, Fe, and Zn (Figure 4a). These trace metals in the PM originate from the pyrolysis of lubricting oil and wear of moving engine components. Higher concentration of these trace metals have potential to increase the ROS activity level in the cells, which results in an elevated oxidative stress level.50,51 In the PM from all test engines, Al and Fe concentrations were relatively higher compared to other trace metals. Zn and Fe were relatively higher in particultes from Euro-II engine, however, Al and Cu were present in higher concentrations in PM from Euro-III and Euro-IV engines. Due to higher in-cylinder temperatures and higher wear in the engine components, biodiesel-fueled engines emitted relatively higher trace metals in the PM compared to mineral diesel-fueled engines (t > 10; p < 0.005). F


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Figure 5. (a) Transmission electronic microscopy (TEM) images, and (b) primary particle diameter (Dp) of PM emitted by mineral diesel and biodiesel-fueled test engines at 50% load.

species were below the detection limits. However, diesel-fueled Euro-III engine at 100% load exhibited presence of fluoranthene in abundance. The mass spectrum of biodieselfueled Euro-III engine at 100% load revealed that asphaltenes of highest molecular weight were present in the test sample, whereas low molecular weight asphaltenes were detected in diesel-fueled Euro-III engine at 100% load. The diesel and biodiesel-fueled Euro-IV engine at 50% load emitted lower number of PAHs (aliphatic nitro compounds, anthrax-quinone, trinitro-naphthalene, benzo-fluorene,benzo-fluoranthene, benzanthracene, dibenzo-perylene, and alpha-hydroxyl ethers) with low molecular weights. It has been demonstrated previously that anthraquinone exhibits not only antibacterial activity but also shows anticancer properties.54,55 Similarly, glucuronides are generally inactive species derived from pyrene. Diesel or biodiesel-fueled Euro-IV engine at 100% load showed the presence of PAHs with higher molecular weights. The mass spectra of each sample is also presented in SI Table S3. 4.4. PM Morphological Analysis. In this study, only bulk phenomenon of PM has been focused because nanostructures of PM aggregates dispersed in benzene cannot be exactly identical to those of engine-out PM aggregates. In all images (Figure 5a), fused primary particles are surrounded by condensed hydrocarbons, which are soluble in benzene therefore can be quantified by benzene soluble organic fraction (BSOF). Primary particles in the PM clusters from both Euro-II and Euro-III engines were of comparable size and shape (SI Figure S2). Larger PM clusters comprised of short, branched structures of only a few primary particles,

The second category of trace metals includes toxic metals, such as Ni, Cr, Pb, and As (Figure 4b). Presence of Ni containing compounds as additives in the lubricating oil is the main source of Ni in the PM. Cr, As, and Pb originate from all three sources of trace metals. Ni and Cr trace concentrations were the maximum in PM from Euro-II engine; however, Euro-III and Euro-IV engines emitted slightly higher traces of Pb and As. Presence of Ni in the engine exhaust contributes to its toxicity because Ni can react with SO2 and form nickel sulfide (NiS), which has been identified as a “carcinogen”.52 A relatively lower concentration of all trace metals at 100% load was another important observation. Biodiesel-fueled Euro-II engine emitted significantly higher (except As at 50% load) trace metal concentrations in the PM compared to mineral diesel-fueled engine (t > 5; p < 0.005). However, the difference between trace metal concentrations emitted from mineral diesel and biodiesel-fueled Euro-III and Euro-IV engines were not significant. Several PAHs in the samples (∼12) corresponding to different test engines/fuels/loads were measured using Q-TOF mass spectrometry (SI Table S2). The results showed that PM from diesel-fueled Euro-II engine has Asphaltenes, however, biodiesel-fueled Euro-II engine has Asphaltenes of higher molecular weight. Asphaltenes are made up of poly aromatic hydrocarbon rings with O, N, and S heteroatoms combined with heavy trace metals, particularly V and Ni.53 Relative intensity of naphthalene (2-ring structured) and fluoranthene (4-ring structured) was highest for all test engines, except biodiesel-fueled Euro-IV engine at 50% load, where both G


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Figure 6. ROS generation potential of particulate from different engines fueled with mineral diesel and biodiesel. Bars represents mean ± SEM (n = 3). p-values derived from two-sided Student’s t test, **p < 0.001, *p < 0.05 versus dimethyl-sulfoxide (DMSO) control.

content and absence of aromatic structures may be other possible reasons for relatively smaller Dp of biodiesel-fueled engines.57 4.5. Biological Characterization of PM. 4.5.1. Cytotoxicity and ROS Generation by PM. Cytotoxicity and ROS generation potential of PM was performed in HEK 293T cells. The PM samples from mineral diesel-fueled Euro-II and EuroIV engines at 50% load were found to be toxic to HEK 293T cells at concentration

Mutagenicity and Cytotoxicity of Particulate Matter Emitted from

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