Physicochemical properties
The characterization of the five different combustion particles in terms of particle mobility size in air, morphology by transmission electron microscopy (TEM), elemental carbon (EC)/organic carbon (OC) to total carbon (TC) ratio, PAH and metal contents are shown in Table 1 as previously reported [31]. Overall, combustion conditions were the most important determinant for all particle characteristics. The combustion conditions heavily affected the mobility size, OC, EC, metal and PAH contents. The engine emissions measured as total PM1 were reduced by 65% for the renewable diesel fuels compared to the fossil diesel fuel (Table 1).
Analysis of OC, EC, metal- and PAH content was carried out on extracted particles, whereas TEM and mobility size distribution analyses was done on diluted exhaust particles.
Electron microscopy
The five different particle samples from experimental combustion emissions and the CB reference sample were visualized by transmission electron microscopy (Figure 1). The morphology of the particles generated with 13% intake O2 concentration, namely DEP13, HVO13 and RME13 (Figure 1 b, d, e) and DEP17 with 17% intake O2 concentration (Figure 1 c) were all similar in appearance and showed typical soot agglomerates (diameter ~50-300 nm) of smaller primary particles (diameter ~10-30 nm). In contrast, the soot agglomerates from low temperature combustion (DEP9.7) had less defined primary particles and appeared more fused (bridging between primary particles) compared to the other samples (Figure 1 a).
Organic and elemental carbon, primary particle size and specific surface area
Primary particle size and specific surface area (SSA) were estimated and calculated by measuring the diameters of well-defined spherical primary particles from the TEM images (assuming no voids inside the primary particles). The primary particle size decreased with the intake O2 concentration for the MK1 diesel samples. The largest primary particle size was found for HVO13, 23 nm, followed by 17 nm for DEP13 and 16 nm for RME13 (Table 1). Estimated SSA was overall rather similar, with the largest SSA for RME13 with 222 m2/g and the lowest for DEP9.7 with 152 m2/g. The EC fraction was highest for the particles generated at 13% intake O2, and the lowest for 9.7% intake O2. The EC fraction was higher for DEP13 compared to HVO13 and RME13.
Table 1. Mass, size, carbon composition and surface area of particles
|
Particles
|
DEP
|
HVO
|
RME
|
Ref. Carbon black Printex90
|
Fuel
|
MK1 low sulfur diesel
|
Hydrotreated vegetable oil
|
Rapeseed methyl ester
|
-
|
Intake O2 (%)
|
9.7
|
13
|
17
|
13
|
13
|
-
|
Average emitted exhaust PM1 mass concentration (mg/m3)a
|
23
|
96
|
3
|
34
|
34
|
-
|
Particle mobility diameter (GMD) (nm)#
|
55 ± 9
|
104 ±7
|
62 ± 4
|
90 ± 5
|
70 ± 3
|
-
|
Primary particle diameter (GMDp) (nm) *
|
22 [21,23]
|
17 [16,19]
|
16 [15,17]
|
21 [19, 23]
|
15 [14, 16]
|
15 [14, 15]
|
Estimated specific surface area (SSA) (m2 /g)*
|
152 [143, 161]
|
191 [177, 206]
|
207 [191, 224]
|
160 [146, 174]
|
222 [203, 243]
|
230 [217, 243]
|
Elemental to total carbon (EC/TC)
|
0.35
|
0.88
|
0.60
|
0.72
|
0.68
|
|
Organic to total carbon (OC/TC)
|
0.65
|
0.12
|
0.40
|
0.28
|
0.32
|
|
aThe average exhaust PM mass concentration (mg/m3), before dilution and particle mobility diameter (nm) with ± 1 std. dev. in the time series. The GMD of the soot primary particle size was estimated from the TEM images and the specific surface area (SSA) was estimated from the primary particle size distributions. For the primary particle diameter and SSA, intervals in brackets represent the 95% confidence interval of the distribution parameters in the lognormal fitting procedure. The elemental carbon (EC) and organic carbon (OC) to total carbon (TC) fractions are measured in the extracted PM.
#Mobility particle (agglomerate) size based on the number concentration. Measured with the DMS in the dilution tunnel when the particles were airborne. *Geometric mean diameter. Measured on collected samples without the extraction process.
The specific surface area (SSA) was estimated by using the primary particle size (d_pp) distribution and diesel soot density (ρ_pp) of 1.77 µg/m3 [32] with the formula SSA =6/(ρ_pp · d_pp ). Data from Gren et al. [31].
|
Metal contents
Semi-quantitative analysis of elemental contents by inductive coupled plasma mass spectrometry (ICP-MS) showed the highest mass fractions for Cu and Fe (Table 2). For Cu, Fe and several other trace elements, DEP17 showed 5-17 fold higher metal mass fractions compared to the other four samples. The emitted exhaust metal mass concentrations (µg/m3) were within a factor 2 for all operation points, however the PM1 mass emissions varied strongly (Table 1) and hence the metal mass fraction will be higher for the low mass emitting operation point (DEP17). DEP13 had the lowest mass fraction of Cu. RME13 and DEP13 had the lowest mass fraction of Fe.
Table 2. Extracted elemental mass fractions (µg/g)
|
Particles
|
DEP
|
HVO
|
RME
|
Reference values
|
Fuel
|
MK1 low sulfur diesel
|
Hydrotreated vegetable oil
|
Rapeseed methyl ester
|
NIST2975a
|
CBa
|
Ref. b NIST2975
|
Ref. c CB
|
Intake O2 %
|
9.7
|
13
|
17
|
13
|
13
|
V
|
14
|
6
|
ND
|
3
|
2
|
ND
|
ND
|
-
|
-
|
Cr
|
8
|
7
|
52
|
11
|
7
|
7/4
|
ND
|
-
|
-
|
Mn
|
92
|
53
|
ND
|
39
|
43
|
6/3
|
1/0
|
-
|
-
|
Fe
|
220
|
137
|
2,115
|
247
|
116
|
663/516
|
9/12
|
0.0±0.0
|
<1
|
Co
|
2
|
1
|
88
|
1
|
1
|
0/0
|
ND
|
-
|
<1
|
Ni
|
15
|
6
|
118
|
9
|
25
|
4/4
|
ND/1
|
-
|
-
|
Cu
|
2.349
|
629
|
13,160
|
1,632
|
2,291
|
23/13
|
10/1
|
0.0±13
|
11
|
Ga
|
1
|
1
|
1
|
1
|
1
|
ND
|
ND/0
|
0.1±0.1
|
<1
|
As
|
ND
|
ND
|
ND
|
ND
|
ND
|
ND
|
1/2
|
0.5±0.7
|
<2
|
Se
|
2
|
0
|
ND
|
ND
|
0
|
ND
|
ND
|
0.9±0.6
|
<1
|
Rb
|
2
|
1
|
1
|
1
|
1
|
13,926/17,003
|
ND
|
16±4
|
<2
|
Sr
|
99
|
54
|
ND
|
41
|
37
|
2/1
|
1/1
|
-
|
-
|
Ag
|
0
|
0
|
1
|
0
|
0
|
0/0
|
0/0
|
-
|
<2
|
Cd
|
ND
|
ND
|
ND
|
ND
|
ND
|
ND
|
ND/0
|
-
|
<10
|
In
|
0
|
0
|
0
|
0
|
0
|
0/0
|
0/0
|
-
|
-
|
Cs
|
0
|
0
|
ND
|
0
|
0
|
ND
|
ND
|
-
|
-
|
Ba
|
15
|
10
|
ND
|
9
|
6
|
26/ND
|
ND
|
-
|
-
|
Hg
|
0
|
0
|
ND
|
0
|
ND
|
0/0
|
0/0
|
-
|
<0.4
|
Tl
|
0
|
0
|
0
|
0
|
0
|
ND
|
ND
|
-
|
-
|
Pb
|
ND
|
ND
|
ND
|
ND
|
ND
|
21/4
|
3/8
|
-
|
-
|
Bi
|
0
|
0
|
0
|
0
|
0
|
0/0
|
0/0
|
-
|
-
|
U
|
ND
|
0
|
ND
|
ND
|
ND
|
ND
|
ND
|
-
|
<0.2
|
Elemental mass fractions determined by semi-quantitative analysis by ICP-MS (µg/g particle) (ND = not detectable). Blank concentrations were subtracted. NIST2975 and CB were analyzed in duplicates (separated by slash).aResults previously published in Bendtsen et al. (2019) [12]. bReference values from Ball et al. (2000) [33] (the study only analyzed Co, Cu, Fe, Ni, V, and Zn). Note that we extracted for significantly longer time (several days vs. overnight) and with 25% nitric acid instead of 0.1 M phosphate buffer. cReference values from the MAK‐Collection for Occupational Health and Safety (written communication of unpublished data of Degussa) [34].
|
Content of polycyclic aromatic hydrocarbons (PAHs) in the collected particles
The samples were analyzed for native PAHs and PAH derivatives by gas chromatography–mass spectrometry (GC-MS). In total, particle extracts were analyzed for 20 native PAHs, 13 alkylated PAHs (alkyl-PAHs), 14 nitrated PAHs (nitro-PAHs), 10 oxygenated (oxy-PAHs) and 6 dibenzothiophenes (DBTs). Table 3 shows the total amount of different groups of PAH derivatives in µg per g collected particle mass (PM). Values for the individual compounds are given in Additional File A. DEP9.7 showed the highest mass fractions of native PAHs, which was expected due to the low temperature combustion mode caused by the lower intake O2 concentration. DEP9.7 also contained the highest levels of nitro-PAHs.
The highest level of the sum of all PAHs were found in DEP9.7, followed by HVO13, DEP13, RME13 and DEP17. The PAH content decreased with increased intake O2 for DEP9.7, DEP13, and DEP17, which agrees well with a more complete combustion at higher intake O2 concentration.
Compared to DEP13, HVO13 particles contained higher amounts of all PAHs, especially native PAHs and oxy-PAHs, while RME particles contained lower mass fractions of total PAHs.
The levels of DBTs followed a different trend than the other PAH derivatives, by increasing with increasing intake O2 concentrations. DBT levels were highest for DEP17, followed by DEP13 and DEP9.7, while DBT levels were similar for DEP13, HVO13 and RME13.
Table 3. Summary of PAH content (µg/g) in the PM samples. A full list of all PAH derivatives can be found in Additional File A.
|
Particles
|
DEP
|
HVO
|
RME
|
NIST2975
|
Fuel
|
MK1 low sulfur diesel
|
Hydrotreated vegetable oil
|
Rapeseed methyl ester
|
|
Intake O2 %
|
9.7
|
13
|
17
|
13
|
13
|
-
|
Native PAHs
|
23700
|
2470
|
858
|
9960
|
1180
|
52
|
Alkyl-PAHs
|
400
|
483
|
77
|
644
|
150
|
7
|
DBTs
|
47
|
78
|
128
|
86
|
94
|
10
|
Nitro-PAHs
|
131
|
21
|
8
|
65
|
40
|
34
|
Oxy-PAHs
|
2490
|
1450
|
314
|
2630
|
596
|
265
|
Total PAHs
|
26800
|
4500
|
1390
|
13400
|
2060
|
369
|
BaPeq* (µg/g)
|
4685
|
165
|
59
|
1067
|
60
|
3
|
*Sum of BaPeq for 12 PAH (see Additional File L) out of in total 63 different measured PAHs and PAH derivatives.
Levels of native PAHs and PAH derivatives analyzed by GC-MS analysis (µg/g particles). Particle samples, blank control filters and standard reference material NIST2975 were analyzed for 20 native PAHs, 13 alkylated PAHs (alkyl-PAHs), 14 nitrated PAHs (nitro-PAHs), 10 oxygenated PAHs (oxy-PAHs) and 6 dibenzothiophenes (DBTs). PAH concentrations of blank control filters were substracted. A full list of all PAH derivatives can be found in Additional File A.
|
Reactive oxygen species (ROS) generation
Reactive oxygen species generation by the five different particle samples and by CB was measured acellularly, where generated ROS causes formation of 2′,7′ dichlorofluorescein (DCF) from DCFH2 which can be spectrofluorimetrically measured. The initial slope of the curve (alfa values) of measured fluorescence of the five particles are given in Table 4. In comparison, the alfa value of CB was 41554. ROS data were reported previously [31]. The ROS formation potential increased with the intake O2 concentration independently of fuel type. This indicates that engine operating conditions, combustion temperatures and the availability of O2 are important engine parameters that can alter the ROS formation potential of the soot [31].
Table 4. ROS generation (fluorescence per µg)
|
Particles
|
DEP
|
HVO
|
RME
|
Fuel
|
MK1 low sulfur diesel
|
Hydrotreated vegetable oil
|
Rapeseed methyl ester
|
Intake O2 %
|
9.7
|
13
|
17
|
13
|
13
|
ROS (alfa)
|
2685
|
14682
|
24039
|
19998
|
14457
|
The alfa values represent the initial slope of the dose-response curve of measured fluorescence in relation to particle mass..
|
Particle size distribution in dispersion
For the in vivo study, the diesel exhaust particles were collected on Teflon filters with a PM1 pre-separator, extracted using methanol, and dispersed in vehicle and diluted. The particles were dispersed in 0.1% Tween in Nanopure water and sonicated to achieve stable dispersions [35, 36]. The hydrodynamic number and intensity size distributions were measured by Dynamic Light Scattering (DLS). Similar distributions of particles sizes corresponding to agglomerates/aggregates were observed for the five particles, in the same size range as seen for CB and NIST2975 (Additional File, B).
Pulmonary exposure of C57BL/6 mice
Mice were exposed by intratracheal instillation to 6, 18, and 54 µg of dispersed particles and euthanized on day 1, 28 and 90. Exposure to the vehicle (Nanopure water with 0.1% Tween) was included as exposure control (vehicle). In addition, exposure to blank filter extraction dispersed in 0.1%Tween was included as blank filter extraction control (extract). Carbon black Printex90 particles dispersed in the same vehicle (0.1% Tween) were included at a single dose level as reference particle (CB) to enable comparison with previous studies [12, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46].
Pulmonary histopathology
Pulmonary histopathology was evaluated on day 28 (Figure 2) and day 90 (not shown). Generally, only minor histopathological changes were observed. Most particle retention on day 28 was observed in DEP13 and HVO13 exposed mice (Figure 2b, d). Histopathological changes observed for these particles were related to macrophage and lymphocyte infiltration. For DEP9.7, DEP17 and RME13, particles were scarce and no apparent histological changes were observed (Figure 2a, c, e). All five particle types appeared as black micron-sized agglomerates mainly phagocytized in macrophages (Figure 2a1-e1). In addition, some larger dense aggregates were observed for RME13 (Figure 2e2).
Cell composition in bronchoalveolar lavage (BAL) fluid
Pulmonary inflammation was evaluated 1, 28 and 90 days post-exposure by differential cell count of BAL fluid cell composition (Figures 3 and 4, and Additional File C and D).
Day 1 post-exposure
Neutrophil influx was significantly increased in mice exposed to 54 µg of DEP9.7, DEP13, DEP 17, and HVO13 compared to vehicle control with similar, significant dose-response relationships. The 54 µg doses exceeded the level of response to the CB reference at the same dose level. DEP9.7 exposure induced high response (Figure 3 a). In contrast, RME13 did not cause significantly increased neutrophil influx as compared to vehicle. No consistent differences were found for lymphocytes and macrophages compared to vehicle (Figure 3 b and c). For eosinophils, only DEP13 at 54 µg significantly increased the influx compared to vehicle (Figure 3 d).
Day 28 post-exposure
On day 28, some particle exposures seemingly resulted in reverse dose response relationships for neutrophil influx, with significant increase for 6 µg of RME13 and DEP13 compared to vehicle (Additional File, D 1). For 18 µg DEP13, there was a very low response, even significantly decreased compared to vehicle mice. CB exposure was not statistically different from vehicle for neutrophils. However, lymphocytes were significantly increased for CB exposed mice compared to vehicle (Additional File, D 2). No statistical differences were found for macrophages and eosinophils (Additional File, D 3 and 4).
Day 90 post-exposure
On day 90 following exposure, DEP13 had a significantly increased numbers of neutrophils and lymphocytes compared to vehicle (Figure 4 a and b). Presence of macrophages was also observed for all exposed mice at levels around 40’000 cells, including vehicle mice, where HVO13 had a noteworthy lower cell number (Figure 4 c). No statistically significant differences were seen for eosinophils (Figure 4 d).
Serum Amyloid A in lung
Day 1 post-exposure
Saa3 mRNA levels were used as biomarker of acute phase response [19, 46, 47] in lung tissue. On day 1, significant, dose-dependent increase in Saa3 mRNA levels compared to vehicle was observed in lung tissue for all exposures, except for RME13 (Figure 5 a). DEP9.7 (p<0.0003), DEP13 (p<0.0003), DEP17 (p<0.0003), and HVO13 (p<0.0001) of 54 µg all exceeded the level of CB. DEP13 (p=0.0110) and HVO13 (p=0.0074) of 18 µg were also significantly increased compared to vehicle. Saa3 mRNA levels in lung correlated well with neutrophil influx (R2=0.5902, p=0.0002) (Additional File E).
Test for linear dose-response was significant for all exposures, except for RME13 (Figure 5 a).
Day 28 and 90 post-exposure
On day 28, DEP13 of 54 µg (p=0.0034) and CB (p=0.0007) were still increased compared to vehicle (Figure 5 b). No significant differences were seen on day 90 (Figure 5 c).
DNA damage
Genotoxicity was evaluated as DNA strand break levels in the comet assay, using comet tail length and % tail DNA in BAL derived cells, lung cells and liver cells on day 1, day 28 and day 90 post-exposure (Figure 6 and 7, and Additional File F). Generally, variations were observed between exposures and within the vehicle control groups across doses and time points. No increases in DNA stand break levels were observed as compared to vehicle. There were no differences on day 1 (data not shown). There was a significant difference between vehicle and blank filter extraction control, especially for liver on day 28 (p=0.0023), and for all tissues on day 90 (p-values: 0.0005-0.0305), with blank filter extraction control samples having significantly lower DNA strand break levels compared to vehicle (Figure 6 and 7).
When compared to the blank filter extraction control, DEP13 at 6 µg was increased on day 28 for tail length in BAL cells (p=0.078). For tail length in liver cells on day 28, RME13 (p=0.0017), HVO (p=0.004), DEP13 (6 and 18 µg: p=0.0480), DEP17 (18 µg: p=0.0072; 54 µg: p<0.0001), and CB were increased (p=0.0405) (Figure 6). On day 90, DEP17 and HVO13 were increased in both lung (DEP17: p=0.0008; HVO13: p=0.0187) and liver cells (DEP17: p=0.0026; HVO13: p=0.0062) compared to blank filter extraction control (Figure 7).
Correlations
Linear regression analyses were carried out in order to assess physicochemical properties as predictors of inflammation and acute phase response. For this, the SSA of CB was estimated to 230 m2/g, although other values have been reported [5, 11, 48].
Neutrophil influx correlated well with estimated deposited SSA on day 1 (Figure 8 a), where 50-60% of the variation in neutrophil influx could be explained by estimated deposited SSA. To compare with known reference particles, the plot was made with either inclusion (R2 = 0.6388, p<0.0001) or exclusion (R2=0.5523, p=0.0010) of previously published data on surface area and neutrophil influx on standard reference material (SRM) NIST2975 and NIST1650, which are diesel exhaust particles derived from a diesel-powered industrial forklift and a heavy duty truck, respectively. The vehicle used for these historical data was Nanopure water, without the addition of Tween80 [12]. In a previous study comparing the effect of vehicle on carbon black-induced neutrophil influx, there was no differences between 0.1%Tween80 and Nanopure water [35]. Similar significant correlations with neutrophil influx, with 40-50 % of the variation explained, were seen when deposited elemental carbon (EC), organic carbon (OC) and total PAH dose were used as dose metrics (EC: R2=0.522, p=0.0016; OC: R2 =0.4688, p=0.0049), Total PAHs: R2 = 0.4944, p=0.0035) (Figures 8 b-d). Deposited metals (sum of most abundant metals measured) were poorly correlated with neutrophil influx on day 1 (Additional File G a).
Saa3 mRNA levels in lung tissue on day 1 correlated well with deposited EC (R2=0.5041, p=0.0021), but less with deposited surface area, OC, and PAHs (surface area: R2=0.3847, p=0.0104; OC: R2=0.1771, p=0.1182, PAHs: R2=0.3429, p=0.0218) (Figure 9 a-d).
As no significant differences were seen on day 28 for neutrophil influx and for genotoxicity on day 1, these data were not subjected to correlational analysis.
On day 28, ROS formation correlated with genotoxicity in lung (R2=0.3476, p=0.0162) (Additional File H), whereas Total PAHs (Additional File I) and BaPeq (not shown) did not correlate well with genotoxicity, measured as Tail length in BAL, lung and liver in the Comet assay. On day 90, Tail length did not correlate with Total PAHs (Additional File J), or BaPeq (not shown) (Figure 10 a-b). However, ROS correlated well with Tail length in the liver (Figure 10 c) (R2=0.8348, p=0.0301). On day 90, neutrophil influx did not correlate with surface area or deposited elemental carbon (Additional File G b-c).