1. Introduction
Renewable diesel fuels can replace petroleum-based diesel in compression ignition (CI) engines (diesel engines) in order to reduce net CO
2 and particulate matter (PM) emissions to the atmosphere [
1,
2,
3,
4]. Diesel exhaust gases such as NO
x and volatile organic species (VOCs) are precursors to smog and secondary aerosol formation [
5]. The PM composition determines the radiative forcing (RF), with Black Carbon (BC) contributing to positive radiative forcing by absorbing incoming solar radiation. BC from on-road diesel exhausts has been estimated to contribute to around 10% of the atmospheric BC burden, with a RF of 44 mW/m
2 [
6]. The exposure to diesel exhaust emissions (PM and gases such as NO
x) has been related to adverse health impacts such as various lung and cardiovascular diseases [
7,
8,
9] where the PM fraction has specifically been attributed to lung cancer in animal models [
10].
Oxidative stress is one established biological response related to the toxic potential of PM [
11] which is initiated when high levels of reactive oxygen species (ROS) overwhelm the antioxidant defence systems within cells. High levels of ROS in cells can trigger a cascade of events associated with inflammation and apoptosis [
12] but also induce oxidative DNA damage, mutations, and pathways towards carcinogenesis. Several studies have shown that a high specific surface area (SSA) of insoluble particles is a key property for toxicological responses, and in particular for the generation of ROS [
13,
14].
Diesel exhaust PM consists primarily of solid, insoluble carbonaceous particles with high SSA (closely related to BC), an organic fraction including polycyclic aromatic hydrocarbons (PAHs) and an ash fraction enriched with metals [
15]. All of these components have been identified to contribute to toxicologically relevant responses in general and to ROS formation specifically [
16,
17,
18]. This has motivated further studies, and in particular, a need to identify what particle properties drive diesel PM toxicity.
Renewable diesel emissions, such as those from hydrotreated vegetable oil (HVO) and rapeseed methyl ester (RME), have shown both higher [
19,
20,
21] and lower [
19,
22] ROS formation potential compared to petroleum diesel emissions. Modifications to the engine combustion conditions can induce large differences in the composition and characteristics of emitted PM. Control of the engine operating conditions is therefore essential for a just comparison of fuel effects on PM properties. It remains unclear if the contradictory results of earlier studies are a result of engine type and operation or a fundamental difference that can be linked to the fuel replacement.
RME is a fatty acid methyl ester (FAME) fuel produced by transesterification of rapeseed oil. It has an oxygen content of about 10%. HVO consists of paraffinic hydrocarbons, chemically similar to petroleum diesel but with a negligible aromatic content and a higher cetane number [
23,
24]. Previous studies show a 50–80% PM mass reduction with FAME fuels [
1,
2] and 20–50% reduction for HVO [
3,
4]. When PM mass emissions were reduced, increased nucleation mode particle number emissions have been observed [
25]. Emission characteristics have also been found to depend on the feedstock from which the fuel was derived [
26,
27,
28]. This includes examples of altered primary particle size and changes to the internal nanostructure depending on FAME substitution level and oxygen content of the FAME fuels [
29,
30].
Exhaust gas recirculation (EGR) is a well-known and commonly used technique that reduces combustion temperatures in order to reduce NO
x emissions from engines [
31]. The effects of EGR and fuel have been widely studied on regulated gas emissions such as NO
x, CO and hydrocarbons (HC), as well as on engine performance and soot emissions. NO
x emissions decrease with increasing EGR (decreasing O
2 concentration) while HC, CO and soot emissions increase [
32,
33]. However, to our knowledge, only a few studies exist on the effects that EGR has on particle composition and characteristics [
34,
35,
36,
37,
38]. The chemical composition, such as PAH and metal emissions, and the physical properties of particle emissions induced by varying the EGR and thus combustion conditions is of particular interest for understanding the drivers of diesel PM toxicity.
Low temperature combustion (LTC) summarizes combustion concepts where flame temperatures are drastically reduced to simultaneously achieve very low NO
x and PM emissions from engines. It is known from basic combustion studies and model flames that soot properties change with decreasing combustion temperature. Non-regulated emissions with health impact such as aromatic compounds [
39], including PAHs, may increase as combustion temperature decreases [
40].
We utilized an experimental heavy-duty diesel engine with precise control of the combustion parameters to investigate how the ROS formation potential and properties of PM depend on the level of EGR and on the fuel formulation. The aim of this work was to quantify the influence of combustion temperature on emission characteristics and particle-induced ROS formation for petroleum diesel and renewable HVO and RME fuels. Based on the results, we present a novel framework of the relation between combustion conditions, particle properties, and the ROS formation potential. In addition, we describe a method to generate and collect engine exhaust particles designed to have varied physicochemical characteristics for future in vivo studies.
2. Materials and Methods
2.1. Emission Generation
The experiments were performed with a modern 6-cylinder 13-L compression ignition engine, modified to operate with a single cylinder. The engine was equipped with a compressed air line, a backpressure valve and an exhaust gas recirculation (EGR) system to re-circulate a well-defined proportion of the exhaust gas back to the cylinder. Detailed engine specifications can be found in [
41]. The engine was operated with three fuels at a fixed low engine load of IMEP
g 6 bar (gross indicated mean effective pressure) and an engine speed of 1200 RPM in all experiments. The fuels tested were Swedish ultra-low sulphur diesel (referred to as petroleum diesel) without addition of biofuels (MK1; B0) and two renewable diesel fuels: hydrotreated vegetable oil (HVO) and rapeseed methyl ester (RME). All fuels were tested without blending.
To allow a direct comparison of emissions between the fuels, the combustion phasing (CA50 position) was held constant at five crank angle degrees (CAD) after top dead center (ATDC), which was achieved by fine tuning the start of injection (SOI) timing. This CA50 position is near the optimum condition where maximum efficiency and reduced emissions can be achieved for the tested engine. The engine was equipped with an XPI common rail injection system with an injector with 10 holes and 148° spray angle. The common rail pressure was set at 1200 bar for all the test conditions throughout the experiments.
In a first stage, EGR sweeps were carried out for each fuel to probe the impact of EGR level (and thereby combustion temperature) on the emission characteristics. In these tests, the averaging time was ~1.5 min and the EGR level was increased in steps (10–12 steps for each fuel), starting with no EGR (ambient O2 concentration of 21%) until the O2 concentration in the engine intake was reduced to ~9%. With no EGR, the global Lambda (λ, excess air ratio) was ~4.6 for all tested fuels. λ was reduced to ~1.2 when the O2 concentration in the engine intake was reduced to 9%.
Six operation points were chosen for repeated longer experiments, with averaging time >30 min (
Table 1). A more extensive analysis of five of these points was carried out allowing quantitative comparisons of the emissions between the fuels. These were for petroleum diesel: ~10% intake O
2 (LTC), ~13% intake O
2 (NO
x reducing EGR) and ~17% intake O
2 (low EGR). All three fuels, i.e., diesel, RME and HVO were sampled at ~13% intake O
2.
2.2. Dilution System
The raw exhaust emissions were sampled from the exhaust pipe before any aftertreatment device with a heated 6 mm stainless steel probe (150 °C), followed by dilution in two stages to roughly simulate atmospherically relevant dilution and relevant partitioning of PAHs. The first dilution stage was performed with two ejector dilutors in parallel diluted with heated air (150 °C) to 1:4. The exhaust was further diluted with filtered room temperature air to a total dilution factor of 1:30–150 (dependent on the experiment) in a stainless steel partial flow dilution tunnel (
Figure A1 in
Appendix A).
2.3. Emission Characterization
The measurement setup is described in
Figure A1 (
Appendix A). The raw gas emissions of NO
x, HC, O
2, CO and CO
2 were measured in samples from the exhaust line with an AMA i60 (AVL, Graz, Austria) emission system. The equivalent Black Carbon (eBC, “soot”) mass concentration in the undiluted exhaust was measured with a photoacoustic technique (Micro Soot Sensor model 483, AVL, Graz, Austria), and used for quantification of eBC. This measure is often used as an online indicative measure of particulate matter (PM) mass emissions from diesel engines [
43]. With the abovementioned raw emission data, fuel flow and in-cylinder pressure measurement the gross indicated specific emissions were calculated by the means of chemical equilibrium, and power output calculations. The full procedure of obtaining the indicated emissions is explained in [
44].
The diluted exhaust emissions were characterized after the dilution tunnel using a soot particle aerosol mass spectrometer (SP-AMS, Aerodyne Inc. Billerica, MA, USA), a fast mobility particle analyzer (model DMS500, Cambustion Ltd., Cambridge, UK) and an aethalometer (model AE33, Magee Scientific, Ljubljana, Slovenia). The absorption wavelength of 880 nm was used to estimate the eBC concentrations measured by the aethalometer, using the standard settings of particle optical properties recommended by the manufacturer. The CO2 in the diluted exhaust was measured with a non-dispersive infrared CO2 analyser (LI-8020, LI-COR, Lincoln, Dearborn, MI, USA). The dilution ratio was monitored with the CO2 measurements in the raw and diluted exhaust. In order to reduce the concentrations to those in the optimal range for the SP-AMS and aethalometer, a third dilution stage where a part of the exhaust gas was first led through a HEPA-filter to remove particulates was used specifically for these two instruments.
2.3.1. Thermal-Optical Carbon Analysis and Transmission Electron Microscopy Imaging
Samples for thermal optical analysis of organic carbon (OC) and elemental carbon (EC) were collected on quartz filters (Pallflex Tissuequartz, 47 mm) and analysed with a thermal optical analyzer (Model 2001, DRI, Chicago, IL, USA) using the EUSAAR_2 protocol [
45]. In addition, extracted samples (described in
Section 2.4) were analysed for the relative fractions of OC and EC. The OC fraction was divided into non-refractory OC (nrOC) and refractory OC (rOC). The nrOC consists of carbon that evolved at temperatures up to 450 °C in inert gas (OC1 to OC3 in the EUSAAR_2 protocol) and refractory OC of carbon that evolved at 650 °C in inert gas and at 500 °C in a 2% O
2 gas mixture (OC4 and Pyrolytic Carbon in the EUSAAR_2 protocol).
To analyse the soot particle aggregate structure and primary particle size, samples were collected with electrostatic precipitation using a nanometer aerosol sampler (model 3089, TSI Inc., Shoreview, MN, USA) on lacey carbon coated Cu-grids and analysed with a transmission electron microscopy (JEOL 3000F). The TEM was operated at 300 kV and equipped with a Schottky FEG and 2 × 2 k CCD.
The TEM images were analysed with the software ImageJ for primary particle size determination. The diameter of clear primary particles without overlap at the edges of the soot agglomerates was measured in TEM images with magnification of minimum 20,000. For two samples, Printex90 (P90) and diesel particles collected at 10% intake O2 (Diesel 10%), partly overlapping primary particles had to be included in order to acquire a large enough number of counted particles. The number of analysed particles varied between 43–167 primary particles, the number of agglomerates varied between 2–17 for the various samples. The sample size of the number of measured primary particles corresponds to a 95% confidence interval. A lognormal distribution was fitted to the primary particle size distribution and the geometric mean was used as the mean primary particle size.
The specific surface area (SSA; m
2/g) for each measured primary particle was estimated by using the diameter (
) and an assumed inherent material density (
) of 1.8 g/cm
3 according to the following equation [
46]:
By assuming point contact between the primary particles in the agglomerates, we estimate that the primary particle SSA is representative as the sample SSA. The primary particle SSA distribution was assumed to be lognormal and the geometric mean was used as an estimate of the sample SSA.
2.3.2. SP-AMS
The SP-AMS is an online instrument that determines that particle chemical composition by sampling the aerosol directly from diluted exhaust. It was operated with a time-resolution of 10 s. The SP-AMS was used to investigate and quantify the chemical composition of the non-refractory organic and refractory carbonaceous PM. To investigate the non-refractory composition, the SP-AMS was run in the standard aerosol mass spectrometer (AMS) mode with vaporization using only thermal desorption on a heated tungsten target (600 °C), followed by electron ionization (70 eV) and detection in a high-resolution time-of-flight mass spectrometer. The particle phase total PAH concentration was calculated from parent ions between 202 Th and 300 Th [
47]. For investigation of refractory components, we used the “dual-vaporizer” configuration [
48], in which a 1064 nm intracavity laser is added for vaporization of rBC. We calibrated the instrument using 300 nm (mobility equivalent) diameter ammonium nitrate (standard AMS mode) and carbon black (Regal Black, Cabot Corp., Alpharetta, GA, USA) particles (dual vaporizer mode). A detailed description of the detection and analysis methods can be found in
Appendix A.
2.4. PM Collection with HVCI and Extraction for ROS, PAH and Metal Analysis
Samples from the five chosen operation points were collected with a High-Volume Cascade Impactor (HVCI 900, BGI by Mesa Labs, Butler, NJ, USA) on Teflon filters followed by methanol extraction. There were three operating points for petroleum diesel (EGR levels corresponding to 10%, 13%, 17% intake O
2) and one operating point each for RME and HVO (13% intake O
2). In order to collect sufficient mass of particulates on the sample filter for analysis and future toxicological studies, each engine operating point could be run for as long as 3 h. PM samples from the dilution tunnel were collected on the final filter of the HVCI with a flow rate of 900 lpm. The cut-off size was 1 µm (PM1) and the final filter (Whatman PTFE, 150 mm, pore size 5 µm) was loaded with 10–20 mg. At least two particle filters were pooled in each case during the extraction. The filters were left to equilibrate in room temperature (RT) over night before weighing the collected masses. The filters were stored in −80 °C before extraction. After sample collection, the PTFE filters were extracted by sonication (3 × 30 min) in analytical grade methanol (<25 °C), followed by low-pressure evaporation (150 mbar, <35 °C) in 10 mL glass vials [
49]. The glass vials were left overnight to equilibrate in RT before weighing, both before and after evaporation. The gravimetric mass extraction efficiency was for all samples ≥85%.
2.4.1. ROS Assay
The ability of the extracted PM to generate ROS was determined using the cells-free version of the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH
2-DA) assay as previously described in greater detail [
50,
51]. However, in the current protocol, all PM was tested for autofluorescence where the ROS probe was replaced by Hank’s balanced saline solution (HBSS). Printex90, a commercially available carbon black, was used as a reference material and was analysed along with the extracted PM samples. Briefly, the DCFH
2-DA (#D399, Invitrogen, Waltham, MA, USA) was chemically hydrolysed in the dark with NaOH to generate 2′,7′-dichlorodihydrofluorescein (DCFH
2), which was further diluted with phosphate buffer (pH 7.4) to 0.04 mM. The PM suspensions were prepared using 16 min sonication (Branson S-450D) in Hank’s balanced saline solution (HBSS, without phenol, #H6648, Sigma Aldrich, St. Louis, MO, USA). The samples were further diluted in HBSS and tested at 0 µg/mL and eight doubling PM concentrations from 1.05 up to 101.25 µg/mL. The final concentration of DCFH
2 at assay start was 0.01 mM. Generated ROS caused formation of 2′,7′-dichlorofluorescein (DCF) from DCFH
2 that was spectrofluorimetrically measured following 3h of incubation in the dark (37 °C and 5% CO
2). Excitation and emission wavelengths were λ
ex = 490 nm and λ
em = 520 nm, respectively (Victor Wallac-2 1420; PerkinElmer, Skovlunde, Denmark).
2.4.2. PAH Analysis
The PM samples in the glass vials and quality controls were extracted and cleaned in accordance with a previously described procedure [
52] with some modifications. Full description of the PAH extraction and analytical purification procedures can be found in
Appendix A. The concentration of 32 native PAHs (including the 16 US EPA priority PAHs) and 16 alkylated species, 17 nitrated and 9 oxygenated PAHs (nitro-PAHs and oxy-PAHs) and 6 dibenzothiophenes (DBTs) were analysed. Full description of compounds investigated, the transitions, limits of detection (LODs), as well as retention times (RT) for all targets are shown in
Table A1,
Table A2 and
Table A3 in
Appendix A. In short, prior to extraction two labelled internal standard mixtures containing 16 deuterated U.S. EPA priority PAHs, and four deuterated nitro-PAHs were spiked to the samples. Particles were extracted with 3 mL dichloromethane for 3 h, using a Sonica Ultrasonic Extractor (Soltec, Milan, Italy). The samples were cleaned using silica columns prior to concentration to a final volume of approx. 30–40 µL under nitrogen flow. Target compounds were separated on an Agilent 5975C mass spectrometer (MS) coupled to a 7890A gas chromatograph (GC, Agilent Technologies, Santa Clara, CA, USA). Electron impact ionization (EI) was performed for PAHs, alkylated PAHs and DBTs. Electron capture negative chemical ionization mode (ECNCI) was used for the nitro- and oxy-PAH species. The MS was operated in selected ion monitoring mode (SIM) for both EI and ECNCI modes.
2.4.3. Metal Analysis
The extracted PM samples were analysed for metal content by ICP-MS as previously described [
53] but with slightly modified extraction times. It was not possible to transfer the amount of ≤1 mg particle matter from the glass vials used in the filter extraction procedure to containers suitable for microwave-assisted acid digestion to completely degrade the PMs. Therefore, acid extractions were performed by adding a volume of 1 mL of 25% (v/v) nitric acid directly to the vials. Samples were first agitated at 600 oscillations per min overnight (Stuart Scientific SF1 shaker), then incubated for approximately 7 h at room temperature without agitation and then shaken for another 72 h and transferred with 6 mL of ultrapure water into polypropylene tubes. Before analysis, the samples were centrifuged for 5 min at 4500 × g (Heraeus Multifuge X3 FR, Thermo Scientific, Waltham, MA, USA), because incomplete digestion of the particles was achieved. A volume of 5 mL of the supernatant was transferred to a new polypropylene tube and diluted 5-fold with 5% nitric acid. A triple quadrupole inductive coupled plasma mass spectrometer (ICP-MS) (Agilent 8900 ICP-QQQ, Santa Clara, CA, USA) equipped with a MicroMist borosilicate glass concentric nebulizer and a Scott type double-pass water-cooled spray chamber was run in no gas (Cd, Hg, Pb, Bi, U) or helium (V, Cr, Mn, Fe, Co, Ni, Cu, Ga, As, Se, Rb, Sr, Ag, In, Cs, Ba, Tl) mode with 0.1–3 s integration time per mass. Quantification was performed based on external calibration. The results should be considered semi-quantitative.
4. Conclusions
The physicochemical properties and emission levels without any aftertreatment devices were investigated as a function of EGR (intake O2 level) and renewable fuel substitution in a highly controlled heavy-duty diesel engine. The study design was developed to test the influence of intake O2 concentration (EGR) and combustion temperatures on particle emission characteristics and toxicity.
Both the emission levels and the physicochemical particle properties were found to depend strongly on the O2 availability (the amount of EGR) and thus flame temperatures in the combustion cylinder. HVO and RME resulted in a general reduction of eBC emissions by 30% and 54% respectively in comparison to petroleum (MK1) diesel at 13% intake O2. The reduction of eBC and PM may be primarily from the reduced particle mobility size at a similar number of emissions for the RME case, while the mass reduction was due to both lower PN emissions (#/kWh) and reduced mobility size for HVO. RME generated the lowest PAH emissions in both, in terms of mass fraction and in absolute emission levels. High levels of EGR (diesel 10% intake O2) resulted in immature soot emissions characterized by refractory organic carbon (rOC) and non-refractory PAHs. Fe and Cu detected in exhaust PM were found to originate from engine wear and were enriched in the lubricating oil. Precautions should be taken to not use alloys or additives in the engine or lubricating oil which potentially could be toxic if they are aerosolized and inhaled.
The soot samples did not show a correlation between the ROS formation potential and metal or PAH content or SSA. Moreover, the ROS formation potential was not significantly increased or reduced by neither renewable HVO nor RME when compared to diesel at the same engine operating condition. However, as the PM mass emission levels were two to three times lower for the renewable fuels compared to diesel, the ROS formation related to fuel consumption (or engine power output) was lower for the renewable fuels compared to diesel.
The ROS formation potential was correlated to the intake O2 concentration (i.e., EGR) and to refractory oxygen-containing fragments in the SP-AMS. This indicates that engine operating conditions, combustion temperatures and the availability of O2 are important engine parameters that can alter the soot emission ROS formation potential, and ultimately the related diesel engine PM toxicity potential. In addition, the SP-AMS results point towards a correlation between the amount of strongly bound surface oxygen functional groups and the soot ROS formation potential. Therefore, based on our results, we propose that the main mechanisms controlling the soot ROS formation potential is (1) a soot maturation process from rOC to rBC that occurs with increasing combustion temperature, and (2) due to late-cycle soot oxidation and the addition of strongly bonded oxygenated functional groups. These observations suggest that further research on the ROS formation potential and carcinogenesis of solid, carbonaceous PM emitted from combustion engines should be directed towards detailed analysis of soot maturity (e.g., the carbon nanostructure) and surface properties. The possibility that surface oxygen functionalization may control part of the ROS formation potential may suggest that aftertreatment, such as diesel oxidation catalysts, could potentially modify the ROS formation potential.
The study was limited to a single engine operation at low load (IMEPg 6 bar) and constant speed (1200 rpm). Therefore, it is not clear to what extent these results can be extrapolated to other engine operating conditions and real-world driving, for example, high load and transient conditions as well as emissions that have passed exhaust aftertreatment systems. The assessment of the ROS formation potential was carried out on particles that were collected on Teflon filters followed by extraction in methanol. To minimize possible alterations in particle properties from the filter collection and extraction procedure, future studies may apply alternative methods that allow particle collection into liquids for direct ROS formation studies. Additionally, the response of different diesel soot components in various types of ROS probes is of interest for future evaluations.