Environ. Sci. Technol. 1084, 18, 428-434
GC/MS and MS/MS Studies of Diesel Exhaust Mutagenicity and Emissions from Chemically Defined Fuelst Thomas R. Henderson,* James D. Sun, Albert P. LI, Ray L. Hanson, and Willlam E. Bechtold Inhalation Toxicology Research Instltute, Albuquerque, New Mexico 87 185
1. Michael Harvey, Jeffry Shabanowitz, and Donald F. Hunt Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904
rn Selected polycyclic aromatic hydrocarbons (PAHs) dissolved in an aliphatic solvent (hexadecane) were utilized as fuels in a single cylinder diesel engine to examine relationships between diesel fuel aromaticity, PAH content, and the mutagenic activities associated with diesel soot. The direct-acting mutagenic response to Salmonella and the percent extractable organics associated with soot particles was 3-4-fold lower when pure hexadecane was combusted compared to reference diesel fuel. Certain PAHs (pyrene and phenanthrene) when added to aliphatic fuel caused increased emissions of the same PAH and increased nitro-PAH emissions corresponding to the parent PAHs but no increase in soot production. Other PAHs (1-methylnaphthalene,acenaphthene, and benzo[a]pyrene) increased the overall emissions of several PAHs and soot and altered patterns of nitro-PAH emissions. It was concluded that the incomplete combustion of PAHs through soot-forming mechanisms may significantly influence exhaust emissions of PAHs and nitro-PAHs from diesel engines, and the concentration on soot particles reflects both the boiling point and the relative stability of individual compounds. Introduction Use of diesel engines in light-duty vehicles has been increasing because of increased fuel efficiency and low taxes on diesel fuels in some countries. Diesel engines are also capable of operation on a wider variety of fuels than other engines. Shale oils, vegetable oils, alcohols, and coal microparticles are being considered as fuels or fuel extenders. However, the greater particulate emissions by diesel engines compared to gasoline engines and the association of direct-acting mutagens with diesel soot particles have raised concern about the potential health effects that may occur from increasing use of diesel engines in light-duty vehicles (1-5). The effect of variations in operating conditions on the emission characteristics of diesel engines has been investigated to some degree. Engine operation on both nitrogen-free air and pure aliphatic fuels was reported to reduce mutagenic activity in the emissions (6). Operation of an Oldsmobile V8 diesel engine on fuels with varying aromatic content showed that the mutagenicity of the soot particles was increased when fuels with increased concentrations of higher boiling polycyclic aromatic hydrocarbons (PAHs) were combusted (5). With a Peugeot engine, the differences following combustion of the same fuels were much smaller. Unburned fuel PAHs may contribute to exhaust mutagenicity by reacting with NO, to form nitro-PAHs. Correlations were noted between the PAHs present in reference diesel fuels and the nitro-PAHs present in exhaust soot extracts (7). Comparison of the soot extracts from a single-cylinder diesel and a V8 diesel engine folPresented in preliminary form at the 1982 American Society for Mass Spectroscopy Meeting, Honolulu, HI, June 6-11, 1982. 428
Environ. Sci. Technol., Vol. 18, No. 6, 1984
lowing analysis by tandem triple-quadrupole mass spectrometry (MS/MS) showed that the nitro-PAH profiles of exhaust extracts were very similar when the engines were operated on the same reference fuel, except that the single-cylinder engine exhaust contained detectible amounts of dinitropyrenes and was more mutagenic ( 4 ) . In this work, a single-cylinder diesel engine was used to investigate the emissions from chemically defined fuels as a way of distinguishing combustion products from unburned fuel components. This approach allowed direct comparison between fuel composition and the composition of the organic mixtures associated with exhaust soot particles. The studies also allowed discerning which PAHs were formed in the combustion processes and the influences of variations in fuel composition. Materials and Methods A standard no. 2 diesel reference fuel (Phillips Petroleum) was utilized in these experiments for comparison with chemically defined fuels. The aromatic content of this no. 2 diesel fuel was 19.2%, and the PAHs recovered from this fuel by dimethyl sulfoxide (Me,SO) extraction were predominately methylated naphthalenes (7). The hexadecane (cetane) utilized for chemically defined fuel studies was reagent grade (Fisher Chemical Co.). When analyzed gas chromatography/mass spectrometry (GC/MS), only a single aliphatic peak was obtained with no aromatic hydrocarbons detected by direct GC/MS analysis or after extraction with Me,SO to concentrate aromatic hydrocarbons as described previously (3). Spikes of 0.2 mg each of pyrene-dlo and benzo[a]pyrene-d,, were added as internal standards to 100 mL of hexadecane before MezSO extraction. The pyrene or benzo[a]pyrene content of the hexadecane calculated from the isotopic internal standards was less than 0.1 ppm. For mixed aliphatic/aromatic fuels, 0.01, 0.10, and 1.0 w t % of selected PAHs (Table I) were dissolved in hexadecane, and 300 mL of each of these fuels was burned for each sample collection. The PAHs used were obtained from Aldrich Chemical Co. and recrystallized, and the purity was confirmed, with only a single peak being detectable by GC/MS. The single-cylinder engine used was a direct-injection Swan engine (Jintang Diesel Engine Works, The Peoples Republic of China) coupled to an electrical generator. The engine was operated at 2000 rpm (75% of maximum) with a 900-W electrical load (75% of the maximum). Total soot produced from the combustion of 300 mL of each fuel mixture was collected on Pallflex T60A20 filters after a 10-fold dilution and cooling with air in a dilution tunnel. At the point of filter sample collections, the temperature of the exhaust stream was approximately 33 "C, while the average NO, concentration was approximately 900 ppm in the undiluted exhaust stream or 90 ppm at the filter. The flow rate of the exhaust stream through sampling filters ranged from 66 to 70 cfm. Following sample collection, the soot was determined by weighing the filters
0013-936X/84/0918-0428$01.50/0
0 1984 American Chemical Society
before and after collection, and the filters were stored a t -80 "C. The filters were then ultrasonically extracted with dichloromethane as described previously (5). After filtration through Millipore filters (0.45 pm), the extracts were concentrated by rotary evaporation, and the last few milliliters of solvent was removed under a stream of nitrogen. Residues were stored a t -20 "C with GC/MS analyses conducted within 1 week after extraction. Each PAH additive tested was run with the low, medium, and high concentrations in that order to minimize carry-over. The engine was run 15 min on pure hexadecane between PAH spiked fuels to further minimize PAH cross-contamination. The weight of soot collected per liter of reference fuel combusted showed less than 10% variation from day-to-day. The PAH spiked fuels were not run in duplicate (300 mL/run) because only limited quantities of hexadecane were available. In order to ensure that the extraction procedures quantitatively recovered benzo[a]pyrene,duplicate exhaust filter segments were cut in half. One was spiked with 0.5 mg of benzo[a]pyrene-dlzin 0.05 mL of toluene spread over the filter before extraction. The total extract was spiked in the second. The benzo[a]pyrene content was 33.1 f 0.1 pg/g of extract whether the benzo[a]pyrene-d12spike was added before or after extraction. The dichloromethane extracts of diesel soot particles were fractionated with MezSO (3). A 10-100-mg sample of dried extract was dissolved in 10 mL of pentane and extracted 3 times with 10 mL of Me2S0. The 30-mL layer of Me2S0 was diluted with 2 volumes of water, cooled, and extracted 3 times with pentane (20 mL total). The pentane layers were pooled, washed with water to remove residual Me2S0, and evaporated to dryness. This "aromatic" fraction was dissolved in 0.25-0.5 mL of toluene for GCIMS analysis. Toluene was used as it was found to improve separations of closely related PAHs such as benzo[e]pyrene and benzo[a]pyrene. Aliquots of aromatic fractions were prepared and dissolved in MezSO for bioassay or dissolved in CHZCl2for MS/MS analysis. For quantitation of PAHs in diesel extracts, pyrene-dlo (98 atom % D) and benzo[a]pyrene-dlz (97 atom % D) were obtained from Merck & Co., Los Angeles, CA, for use as internal standards. A solution containing 1 mg/mL of each was prepared in toluene. A 0.2-mL spike was added to each soot extract before fractionation with Me2S0. Integration of the single ion chromatogram peaks was accomplished by manual quantitation programs on the INCOS data system. Response fractors vs. the internal standards and retention times were determined for PAHs for which deuterated standards were not available (phenanthrene and fluoranthene) to allow quantitation of these PAHs. The average response factors for pyrene/pyrene-dlo was 0.93 over a concentration ratio of 1/10 to l O / l , and the response factor was reproducible from day-to-day within &lo%. A standard solution of 0.10% (w/v) benzo[a]pyrene standard dissolved in hexadecane was used to verify the linearity and recovery of the benzo[a]pyrene following spiking with the dI2 standard and fractionation with Me2S0. The benzo[a]pyrene measured was 0.098 f 0.014% over a range of sample sizes from 10 to 500 mg of hexadecane solution. The GC/MS analyses were performed with a Finnigan Model 4023 GC/MS operated in the electron impact mode. A 20-m SP-2100 fused silica column was used for GC separation. The end of the column was inserted directly into the ion source. The transfer and separator ovens and the injector were maintained a t 250 "C. The GC was
temperature programmed from 50 to 290 "C at 10 "C/min. A 0.5-pL sample was injected in the splitless mode with a 2-min hold a t 50 "C before the initiation of the temperature program. The MS was interfaced to a Finnigan InCOS data system with standard 3.1 C software. The scan parameters were 1.9 s up with a 0.1 s bottom hold over a mass range of 50-400 amu using 70 eV electron ionization conditions. The MS/MS analyses were carried out on a Finnigan Model 4500 tandem triple quadrupole. Samples a t 0 "C in an ice chest were transported between Albuquerque, NM, and Charlottesville, VA, via airplane, and the MS/ MS analyses were done the day after departure. The analysis for nitro-PAHs was as described previously (4, 7) by using (M - 17) neutral loss scans to analyze nitro-PAHs by the reaction PAH-NO2
isobutane
7 PAH-N02-H+
argon
PAH-N+O
+ OH.
The program was a l-s scan, 100-400 amu, with the third quadrupole scanning 17 amu behind quadrupole 1. The second quadrupole was operated with RF voltage only with argon as the collision gas. The second part of the program was a 0.1-s MID (multiple ion detection) scan for nitropyrenes/nitrofluoranthenes with quadrupole 1set at 248 amu and quadrupole 3 a t 231 amu. This was followed sequentially by a 0.1-s MID scan for nitropyrene-d, with quadrupole 1 holding a t 257 amu and quadrupole 3 held a t 240 amu. MID scans for dinitropyrene/dinitrofluoranthene were also attempted with 0.1-s scans, quadrupole 3 set a t 276 and quadrupole 1 set a t 293. No detectable m/z 276 ions above background noise levels were detected. Dinitropyrene standards were detected only with low sensitivity (10-100 ng needed for detection). Salmonella mutagenicity assays were conducted as previously reported (5, 8). Strain TA98 was used throughout. Cultures were incubated in the dark for 48 h and colonies counted with an Artek Colony Counter. Sodium azide, l-aminoanthracene, and 2-nitrofluorene were used as positive controls. Assays were run in triplicate and averaged, and the specific activity in revertants per microgram of extract was calculated by linear regression analyses of the data at regions of linear doseresponse. Relative standard deviation of triplicate plates consistently averaged less than lo%, and correlation coefficients were greater than 0.99 for the slope of dose-response curves. Results The GC/MS analyses of filter extracts prepared from soot collected from the single-cylinder diesel engine running on pure hexadecane fuel showed the predominant component to be unburned hexadecane (Figure 1). The smaller, secondary peaks were identified as phenanthrene, fluoranthene, pyrene, and traces of other PAHs from the mass spectra and retention times. Comparison of the mass spectrum of the hexadecane used as fuel with the GC/MS peak from soot extracts showed that they were identical (Figure 2). This hexadecane peak appeared to be about 60% of the mass of all soot extracts by peak height analysis compared to a hexadecane standard. When hexadecane fuel was spiked with 1% pyrene and combusted in the single-cylinder diesel engine, GC/MS analysis of the soot extracts showed a pronounced pyrene peak in addition to the hexadecane peak (Figure 3). The mass spectrum of the pyrene peak was identical with a pyrene standard spectrum as were the retention times. The pyrene content of this extract was calculated to be 11.4% . Similar increased concentrations of phenanthrene Environ. Sei. Technol., Vol. 18, No. 6, 1984
429
Table I. Diesel Soot Mutagenicity vs. Fuel Composition fuel combusted base fuel reference fuel hexadecane hexadecane hexadecane hexadecane
additives none
revertants/ revertants/ Mg of Pg of extract extract ( direct)a (indirect)b 20
9.3
total sootC
total extractable direct fraction, % r e v e r t a n d 33
11
total indirect revertantsd
74
35
none 8.1 14 20 12 18 11 1 0 g/L pyrene 20 8.7 8.3 8.0 13 5.8 1 0 g/L phenanthrene 6.4 4.0 7.5 8.0 3.9 2.4 18 31 29 5.8 31 52 1 g/L 1-methylnaphthalene hexadecane 1 g/L acenaphthene 11 11 20 7.8 18 17 hexadecane 1 0 g/L benzo [a Ipyrene 2.5 25 29 8.4 6.0 61 a Slope of the linear portion of the dose-response curve in Salmonella TA-98 without the addition of S-9 liver enzyme preparations. Correlation coefficients > 0.99. Triplicate plate counts agreed within 10%. All samples were run on same day with the same TA-98 inocula. Standards of 2.5 pg/plate benzo[a]pyrene with S-9 resulted in 201 t 3 revertantslplate. A 1 . 5 pg/plate 2-nitrofluorene standard showed 163 t: 11revertants/plate in the absence of S-9. Slope of the linear portion of the dose-response curve in Salmonella TA-98 with the addition of S-9. Total grams of soot per liter of fuel combusted. Total revertants = revertants per liter of fuel combusted x l o 6 .
lmnl
’00,01
-
RlCi
RIC-
Scan 200
Flgure 1. Reconstructed ion chromatogram (RIC) of a soot extract from a single-cylinder diesel engine running on hexadecane. The temperature program was 50-290 OC at 10 OC/min. The MS scan conditions were 1.9 s up, 0.1 s bottom hold, and a mass range of 50-450 amu. The instrument was a Flnnigan 4023 GUMS. The peak at scan 859 is hexadecane.
I
F I
Figure 2. Comparison of mass spectrum of aliphatic peak 859 on Figure 1 with the mass spectrum of a hexadecane standard.
and benzo[a]pyrene were noted in soot extracts after addition of these PAHs to fuels, but no methylnaphthalenes or acenaphthene were detected in soot extracts after their addition to fuel. The effects of different fuel combinations on the mass of soot collected, the percent extractable by dichloro430
Environ. Scl. Technol., Vol. 18, No. 6, 1984
M7
UB
~can200
’
600
Im
1000
1115 1l.W
‘3
IWl
400
l7Bl
1800
Flgure 3. Reconstructed ion chromatogram (RIC) of a soot extract from a single-cylinder engine runnlng on hexadecane plus 1% pyrene. Conditions were as in Figure 1. The peak at scan 868 corresponds to hexadecane, and the peak at scan 1331 corresponds to pyrene.
methane and the mutagenicity of the extracts were determined, and results are summarized in Table I. There were obvious differences between the emissions from commercial reference fuel and hexadecane fuel. The specific mutagenic activity of the two extracts was similar, but the differences in the extractable fraction of the particles resulted in a %fold higher total mutagenicity with the reference fuel than with the hexadecane fuel. The data in Table I represent PAH concentrations in hexadecane which resulted in detectable alterations in particle emissions and/or mutagenicity of extracts. The chemical nature of the PAH added appeared to be more important in influencing emissions than the concentration added, once a minimum threshold concentration was reached. The minimum concentrationsof individual PAHs required to elicit a detectable alteration in emissions ranged from 0.1 to 1.0%, and 0.01% concentrations resulted in minor changes in emissions. Changes in direct mutants per microgram of extract also showed no trends with concentration of added PAHs except a minimum concentration was required. Two types of responses were noted when PAHs were added to hexadecane fuel (Table I). Some PAHs (pyrene and phenanthrene) did not result in increased soot emissions (“nonsoot promoting”), Another group of “sootpromoting” PAHs (1-methylnaphthalene, acenaphthene,
Table 11. PAH Emissions vs. Fuel Composition mg of PAH emissions/L of fuel combustedb fuel combusted base fuel
additives
phenanthrene
fluoranthrene
pyrene
benzo[ a 1pyrene
reference fuel hexadecane hexadecane hexadecane hexadecane hexadecane hexadecane standard deviation,a %
none none 1 0 g/L pyrene 1 0 g/L phenanthrene 1 g/L 1-methylnaphthalene 1 g/L acenaphthene 1 0 g/L benzo[a]pyrene
22 1.4 1.7 8.3 30 13 73
23 3.1 3.1 4.5 30 18 68
41 6.2 75 7.0 50 47 98
2.6 2.5 0.40 0.17 8.4 3.0 92
8.3
6.8
7.9
8.3
Total PAH content recovered from soot partia Average of standard deviations for duplicate analyses on each sample. cles collected on filters and converted to a per liter basis. The quantitation was carried out by GUMS, after addition of pyrene-d,, and benzo[a]pyrene-d,, internal standards and fractionation with Me,SO.
-
and benzo[a]pyrene) increased particulate emissions significantly compared to hexadecane alone. Pyrene and 1-methylnaphthalene were the only PAHs which did not decrease the specific activity of direct mutagens in soot extracts when added to fuels (Table I). Other PAH supplements resulted in extracts with somewhat lower direct-acting mutagenicity (specific activity), but similar total yields of mutagenic activity. The lowest total yields of direct mutagenic activity were noted with phenanthrene (80% decrease compared to hexadecane control) and benz[a]pyrene (70% decrease). The extracts shown in Table I were spiked with deuterated PAH internal standards and fractionated with MezSO to concentrate aromatic compounds for quantitative PAH analyses. Phenanthrene, fluoranthene, pyrene, and benzo[a]pyrene were selected for quantitation as they were well separated on fused silica capillary columns, were the major components in soot extracts, and were close in retention times to the deuterated internal standards. No naphthalenes were detected in these extracts, even from combustion of fuels spiked with 1-methyllnaphthalene and acenaphthene. The PAH analyses of soot extracts are presented in Table 11. The emissions per liter of fuel combusted normalize for variations in mass of soot particles collected and the percent extractable organics, enabling direct comparisons of the effects of added PAHs to the fuel. The total yields of each PAH were considerably higher from combustion of reference fuel than from hexadecane (Table 11). Pyrene addition to the fuel increased the pyrene concentrations in soot extracts over the whole concentration range, as phenanthrene addition to fuel increased phenanthrene concentrations in soot extracts. In the case of the soot-promoting PAHs, (l-methylnaphthalene, acenaphthene, and benzo[a]pyrene), fuel additions resulted in increased overall emissions of all PAH measured (Table 11). Most of the emissions were intermediate between the emissions from pure hexadecane and reference fuel, but the PAH emissions from 1% benzo[alpyrene surpassed even the reference fuel. The increased mutagenicity after the addition of S-9 was correlated with increased benzo[a]pyrene emissions (Table I). In view of the variation in direct mutagenicity of soot extracts from different PAH supplemented fuels, MS/MS analyses were carried out to determine if the patterns of nitro-PAHs present in the extracts were distinctive. In particular, the possible association of nitropyrenes with direct mutagenicity was investigated. Table I11 shows that there were no quantitative correlations between calculated nitropyrenes in selected aromatic fractions and the direct mutagenicity of these extracts. The concentrations of nitropyrenes were increased by pyrene supplementation
Table 111. Nitropyrene and/or Nitrofluoranthene Concentrations and Mutagenicitya
fuel combusted base fuel additive reference fuel hexadecane hexadecane hexadecane hexadecane
none
none 1 0 g/L pyrene 1 0 g/L phenanthrene 10 g/L 1-methylnaphthalene hexadecane 10 g/Lacenaphthene hexadecane 10 g/L benzo[a]pyrene
Pg of nitrodirect mrenes/ muta-g of genicity,b aromatic revertfraction ants/mg 342
53
590 1000 400 130
55 47 31 15
46 15
24 2
a MS/MS MID analyses using nitropyrene-d, internal standard, 100 ng/sample, added t o 1-10 mg of extract dissolved in 100 pL of CH,Cl,. An aliquot of 2-5 pL was applied t o the solid probe capillary and volatilized into the MS source by a 5-min temperature program from 25 to 350 "C. All analyses were performed on weighed aliquots of aromatic fractions recovered from crude extracts by Me,SO fractionation. The reproducibility of MS/MS quantitation was better than 25% for samples frun in Mutagenicity assays and controls are deduplicate. scribed in Table I.
of the fuel, but the mutagenicity of the aromatic fractions was unchanged. Methylnaphthalene and acenaphthene fuel additives also produced decreased mutagenicity, but the nitropyrene levels were not affected proportionally. MS/MS does not differentiate between isomers of the same molecular weight such as nitropyrenes and nitrofluoranthenes, and from the PAH analyses in Table I1 it was apparent that both nitropyrenes and nitrofluoranthenes might be present. The MS/MS response factor for 1-nitropyrene relative to nitropyrene-d9 was verified to be linear with a range of standards added to a typical sample, and the reproducibility was &lo%. No nitrofluoranthene standards were available. The use of aromatic fractions rather than whole extracts removes dinitropyrenes/dinitrofluoranthenes which might contribute to total mutagenicity ( 4 ) . The Finnigan 4500 MS/MS was not sensitive enough to detect dinitropyrenes in unfractionated extracts under the conditions used in this experiment. Table IV shows that the ratio of nitropyrenes/pyrene in soot extracts decreased as the pyrene content of soot extracts increased and the ratio was not linear with fuel concentration. A 100-foldincrease in pyrene concentration in fuel resulted in a 10-fold increase in soot pyrene concentration but only a 5-fold increase in nitropyrene concentration. Environ. Scl. Technol., Vol. 18,No. 6, 1984
431
Table IV. Nitropyrene vs. Pyrene Concentration in Unfractionated Soot Extracts nitropyrenes,b ni tr opyrenesl fuela PPm PPm pyrene 0.01% pyrene 0.10% pyrene 1.0% pyrene
47
1 0 000
70
20 000 t 2 100
0.0034
23 0
110 000 t 1 5 000
0.0020
t
260
0.0045
50
i
Hexadecane Fuel 55 revlpg
'f
'i
I Yj
iTT
a In hexadecane. Nitropyrene analysis by MS/MS as described in Table 111. Pyrene analysis by GC/MS as described in Table 11. Figures represent mean of duplicate analyses r standard deviation.
loo]
'i:
'I'
1% Pyrene Fuel
TT 0
50
0.01% Pyrene Fuel
1
I
I
/
50
MI€
100
140
180
220
240
Flgure 4. Comparison of MS/MS spectra of unfractionated soot extracts. The top spectrum is the summed spectra from 250 scans on a Finnigan 4500 MS/MS. The soot extract was from the combustion of 1% (w/v) pyrene dissolved in hexadecane. The bottom spectrum was of a soot extract from the combustion of 0.01% w/v pyrene dissolved in hexadecane. The scans represent (M - 17)-linked scans with quadrupole 3 scanning 17 amu behind quadrupole 1.
In view of the wide range of mutagenicities observed in Table 111,MS/MS scans for total nitro-PAH ions were also compared to see if other compounds besides nitropyrenes were detectable in these samples. The results of the summed MS/MS spectra obtained on selected samples is shown in Figures 4 and 5. Figure 4 shows the MS/MS spectra of unfractionated extracts from 0.01 % pyrene and 1.0% pyrene fuels. The differences between the two summed spectra were small, and the major differences noted were in the m / e 231 peak, (corresponds to nitropyrene/ nitrofluoranthene daughter ions) which was increased in relative intensity in extracts from 1%pyrene fuel compared to 0.0170 pyrene fuels. The 0.1% pyrene fuel extracts (not shown) were intermediate. Quantitative values for nitropyrenes in unfractionated extracts are shown in Table IV. The (M - 17)' spectra from the other nonsoot-promoting PAH added to hexadecane fuel appeared similar. That is, phenanthrene did not change the overall patterns of nitro-PAH emissions, but 1% phenanthrene addition to fuel showed slightly increased m / e 207 and m / z 252 ion intensities, corresponding to nitrophenanthrenes and dinitrophenanthrenes. The MS/MS spectra from soot-promoting PAHs reflect the changes in overall PAH emissions elicited by the addition of these compounds to fuels (Table 11). Figure 5 shows a comparison of the nitro-PAH emissions between hexadecane fuel and hexadecane supplemented with benzo[a]pyrene. Benzo[a]pyrene supplementation almost completely suppressed the m / z 231 peak (nitropyrenes) 432
Environ. Sci. Technol., Vol. 18, No. 6, 1984
Flgure 5. Comparison of MS/MS spectra of aromatic fractions from soot extracts. Conditions were as in Figure 4. The top spectrum was an aromatic fraction from soot collected following the combustion of pure hexadecane fuel. The bottom scan was an aromatic fraction collected following the combustion of hexadecane plus 1% w/v benzo[a]pyrene.
as was seen in Table 111. The major peak seen at m/z 202 corresponded to dinitronaphthalenes, compounds that are known to have low activity in the Ames bioassay (9). The other peaks corresponded to single-ring compounds, mononitro (odd mass numbers) and dinitro (even mass numbers) compounds such as nitrostyrenes, alkylated benzenes, or alkylated nitrocyclohexadienes. None of the single ring compounds are known to have appreciable activity in the Ames bioassay and thus are not likely to contribute to the total mutagenicity observed. A comparison of Figure 4 and 5 shows that the concentration of nitro-PAHs by Me,SO fractionation facilitates MS/MS comparisons ( 4 ) .
Discussion In this study, a single-cylinder diesel engine and chemically defined fuels were used to determine which fuel components appeared to influence the chemical composition and mutagenicity of diesel soot particles. Hexadecane was selected for use as the experimental fuel since extracts of exhaust particles following its combustion consisted largely of unaltered hexadecane. Hexadecane chromatographed well on capillary GC columns was well separated from PAHs and was readily identified by its mass spectrum. It was also commerically available free from contamination with aromatic compounds. Analysis of soot extracts from hexadecane combustion showed that a major portion (-60%) consisted of unburned fuel. How much of the extractable fraction of soot is unburned fuel is not discernible from the combustion of complex standard diesel reference fuels because of the spectrum of high-boiling aliphatic hydrocarbons present. The nature of the other -30% nonvolatile material in soot extracts from combustion of pure hexadecane was not determined. The addition of PAHs to hexadecane diesel fuel demonstrated PAHs associated with soot particles were increased by the spiking of fuels with these compounds as observed previously for pyrene (4). The concentration of some PAHs in soot extracts over their concentrations in starting fuels may be due to the known resistance of aromatic compounds to combustion in diesel engines (IO). The concentration of pyrene in soot extracts was not di-
rectly related to concentration in fuels as shown in Table IV. Volatile PAHs may be lost during soot particle collection on filters, since fuel spikes of 1-methylnaphthalene and acenaphthene (bp 240 and 278 “C) (11) resulted in increased particulate emissions, but they were not detected in soot extracts. Schuetzle et al. (12, 13) reported that PAHs as large as phenanthrene (bp 340 “C) (11) were associated with both the particulate and vapor-phase diesel emissions. Thus, lower molecular weight PAHs may be concentrated in the vapor phase of emissions, while the high boiling nitro-PAHs may condense on the soot particles. The addition of each nitro group to naphthalene increases the boiling point -90 OC (11). Thus, it is not surprising that nitro derivatives of some single- or double-ring PAHs were detected in soot extracts by MS/MS while the parent PAHs were not detected. Increased emissions of many PAHs from combustion of soot-promoting PAHs cannot be explained simply by condensation of unburned fuel residues on soot particles. Soot formation has been proposed to involve the formation of multiring PAHs from smaller aromatic hydrocarbons and final dehydrogenation to yield soot (10, 14-17). The soot-promoting PAHs also increase the emissions of high molecular weight PAHs ( m / z >270), which was interpreted as evidence that there PAHs are related to the processes of soot formation (18). Addition of PAHs to fuel would be expected to alter the ratio of PAHs present in exhaust streams and thus the type of nitro-PAHs associated with soot particles. Tables I11 and IV show that this appears to be largely the case with pyrene addition to fuel. Table I1 shows pyrene emissions were increased 10-fold when 1%pyrene was combusted compared to hexadecane alone. The ratio of pyrene/ fluoranthene increased from 2/1 to 25/1. The nitropyrenes increased 5-fold. Since the mutagenicity was not changed, it appears that the increased nitropyrene emissions were associated with decreased emissions of other nitro-PAHs which were not discerned by MS/MS. The more likely candidates are nitrofluoranthenes, which appear to have higher mutagenic potency than nitropyrenes (9) and which are not differentiated from nitropyrenes by MS/MS. The addition of fluoranthenes to hexadecane fuel has been found to result in increased fluoranthene and nitrofluoranthene emissions, along with decreased nitropyrene emissions (18). Also, polynitro-PAHs may have been reduced. This interpretation suggests that the concentration of active NO, species in exhaust streams may be limiting, since the ratio of nitropyrenes/total pyrene decreases with increasing pyrene emissions (Table IV). Addition of PAHs which form less active nitro-PAHs (9) would be expected to decrease the mutagenicity of exhaust extracts. Table I shows this appears to be the case for addition to the fuel of phenanthrene, acenaphthene, and benzo[a]pyrene, where both the specific activity of soot extracts and total mutagen emissions were reduced but the corresponding nitro-PAHs were not detected by MS/MS except in traces. Addition of 4-methylbiphenyl to hexadecane fuel resulted in 90% reduction of direct mutagenicity in extracts (19). Nitromethylbiphenyl isomers have only low direct mutagenic specific activity in the Ames bioassay. The best explanation for differences in mutagenic emissions with PAH additions to fuel is that the reactions of PAHs with NO, may differ. That is, certain PAHs may be more readily oxidized than nitrated by NO, in exhaust streams, or the nitro derivatives formed may be unstable a t the exhaust temperatures. This appears to be the ex-
planation for the very low direct mutagenicity of soot extracts from combustion of 1% benzo[a]pyrene (Tables I and 111). MS/MS showed very low nitropyrene concentrations, but only traces of nitrobenzopyrenes. Studies of benzo[a]pyrene reaction with NOz on MS solid probe tips showed that it is prone to oxidation (19). The reaction of NOz has been studied with over 100 PAHs, and the (M + H+ - 17) fragmentation has been observed to be the major mode of collision-induced fragmentation under the conditions used here. The reactions of NOz with PAHs in the (M - 17) MS/MS analysis are sufficiently specific and quantitative that it has been used to quantitatively analyze environmental extracts for PAHs (19). The data reported here were obtained with a singlecylinder, direct-injection engine run under optimal, steady-state conditions. While the patterns of nitro aromatic emissions measured by MS/MS were quite similar (9), the proportion of unburned fuel in particulate emissions may be higher than is found with automobile engines. Quantitative differences have been noted in different engine designs and operating cycles (@,as well as differences in particulate emissions when fuels of increased aromaticity were combusted (20). Conclusions Studies with an experimental diesel engine have shown that it is possible to study the relative stability of PAHs under combustion conditions by addition to aliphatic diesel fuels. The more stable PAHs such as pyrene appear more likely to contribute to the formation of mutagenic nitroPAHs on exhaust particles. Certain PAHs appear to be prone to oxidation and/or soot formation, rather than formation of nitro-PAHs. These include PAHs such as benzo[a]pyrene. The formation of nitro-PAHs on exhaust particulates also appears to be limited by the availability of active NO, species. Acknowledgments We are grateful to R. F. Henderson, J. S. Dutcher, Y. S. Cheng, A. L. Brooks, C. E. Mitchell, W. E. Hadley, and R. 0. McClellan for discussions and review of the manuscript and Amy Federman and Nicole Dumont for technical assistance. Registry No. Pyrene, 129-00-0; phenanthrene, 85-01-8; 1methylnaphthalene, 90-12-0; acenaphthene, 83-32-9; benzo[a]pyrene, 50-32-8.
Literature Cited Huisingh, J. S.; Bradow, R.; Jungers, R.; Claxton, L.; Zweidinger, R.; Tejada, S.; Bumgarner, J. E.; Duffield, F.; Water, M.; Simmon, V. F.; Hare, C.; Rodriguez, S.; Snow, L. “Application of Biossays to the Characterization of Diesel Particle Emissions”; U.S.Environmental Protection Agency: 1978; EPA-60019-78-027. Pederson, T. C.; Siak, J. S. JAT, J. Appl. Toxicol. 1981, I, 54-60. Henderson, T. R.; Li, A. P.; Royer, R. E.;Clark, C. R. Enuiron. Mutat. 1981, 3, 211-220. Henderson, T. R.; Royer, R. E.; Clark, C. R.; Harvey, T. M.; Hunt, D. F. Enuiron. Sci. Technol. 1983, 17, 443. Clark, C. R.; Henderson, T. R.; Royer, R. E.; Brooks, A. L.; McClellan, R. 0.;Marshall, W. F.; Naman, T. M. Fundam. Appl. Toxicol. 1982, 2, 38-43. Yergey, J. A.; Risby, T. H; Lestz, S. S. “The Chemical Characterization of Diesel Particulate Matter”, paper presented at the 1981 EPA Diesel Emissions Symposium, Raleigh, NC, Oct 5-8, 1981. Henderson, T. R.; Royer, R. E.; Clark, C. R.; Harvey, T. M.; Hunt, D. F. JAT J . App. Toxicol. 1982, 2, 231-237. Envlron. Sci. Technol., Vol. 18,
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Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31. 347-364.
Tokiwa, H.; Nakagawa, R.; Ohnishi, Y. Mutat. Res. 1981, 91, 321-325. Graham, S. C.; Homer, J. B.; Rosenfeld, J. L. J. Proc. R. SOC.London, Ser. A 1975, 344,259-285. "Handbook of Chemistry and Physics", 47th ed.; Chemical Rubber Co.: Cleveland, OH, 1967. Schuetzle, D.; Riley, T. C.; Prater, T. J.; Harvey, T. M.; Hunt, D. F. Anal. Chem. 1981,54, 265-271. Schuetzle, D. paper presented at the EPA Symposium on the Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures, Chapel Hill, NC, Jan 25-27, 1982. Wagner, H. G. G. Symp. (Znt.) Combust., [Proc.] 1979,16. Longwell, J. P. Symp. (Znt.) Combust., [Proc.] 1977, 16. Pasternak, M.; Zinn, B. T.; Browner, R. F. Symp. (Znt.) Combust., [Proc.] 1981, 18.
(17) Bittner,J. D.; Howard, J. B. Symp. (Int.)Combust., [Proc.]
1981, 18. (18) Henderson, T. R.; Sun, J. D.; Brooks, A. L. and Bechtold, W. E. paper presented at the 31st Annual Conference on Mass Spectrometry and Applied Topics, Boston, MA, May 5-13, 1983; p 404. (19) Hunt, D. F.; Shabonowitz, J.; Harvey, T. M.; Coates, M. paper presented at the 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI, June 1982; pp 800-801. (20) Seizinger, D. E.; Naman, T. M.; Marshall, W. F.; Clark, C. R.; McClellan, R. 0. paper presented at the Society for Automotive Engineers Meeting, Troy, MI, June 1982;paper no. 820813.
Received for review May 24,1983. Accepted November 10,1983. Research performed under US.Department of Energy Contract DE -AC04- 76EV01013.
Fate and Metabolism of Isopropylphenyl Diphenyl Phosphate in Freshwater Sediments Michael A. Heltkamp," James N. Hucklns, Jimmie D. Petty, and James L. Johnson U.S. Department of the Interior, Fish and Wildlife Service, Columbia National Fisheries Research Laboratory, Columbia, Missouri 65201
w The aerobic and anaerobic biodegradation of isopropylphenyl diphenyl phosphate (IPDP) was determined in freshwater sediments with both di[14C]phenyl- and is~propyl[~~C]phenyl-labeled IPDP. Mineralization of IPDP was slow in these sediments, as only about 8% was degraded to I4CO2after 4 weeks. The degradation rates were not affected by oxygen tension, chemical concentration, or seasonal differences in sediment. Chemical analyses of aerobic samples by gas chromatography and mass spectrometry resulted in the tentative identification of nine minor degradation products which suggested pathways involving stepwise demethylation of the isopropyl moiety and enzymatic hydrolysis of both substituted and unsubstituted phenyl moieties, as well as methylation of some intermediates. The diverse microflora occurring in these freshwater sediments appeared to be responsible for the rate and pathway of IPDP degradation. This study indicates that the half-life of IPDP in some freshwater sediments may be greater than previously expected.
Introduction The use of triaryl phosphates (TAPS)in fire-resistant hydraulic fluids and as fire retardant plasticizers has increased steadily over the last 30 years, and the 1977 annual production is believed to have approached 104 million pounds ( I ) . Until the mid-l960s, tricresyl phosphate and cresyl diphenyl phosphate were the predominantly used TAPs. However, these TAPs containing o-cresyl have been associated with neurotoxic effects in mammals (2-4) and poisoning in humans (5-7); furthermore, they are dependent for production on the availability of 0-cresol, and production costs are relatively high. Consequently, isopropylphenyl diphenyl phosphate (IPDP) was introduced as a substitute for the cresyl TAPs. Its production has steadily increased since 1970 ( I ) while that of the cresyl TAPShas decreased. If this trend continues, the annual *Address correspondence to this author at the National Center for Toxicological Research, Jefferson, AR 71602. 434
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production of IPDP is expected to total 60-65 million pounds by 1986 ( I ) . It has been estimated that 70% of the annual production of TAPS is eventually discharged into the environment ( I ) , and most of this industrial effluent goes directly into freshwater ecosystems. Environmental residues of TAPShave been reported in fish tissues (8),water and sediment (9, IO), and drinking water (11). In view of the increased industrial use of IPDP, there is some concern about its environmental fate. The toxicity of IPDP has been determined for freshwater invertebrates (12) and fishes ( I 3 ) ,and although the compound is not acutely toxic a t reported environmental concentrations (9, IO), it is relatively lipophilic (14) and could bioaccumulate through the food chain. The purpose of this study was to determine the rate and probable pathway of IPDP biodegradation in freshwater sediments.
Experimental Section Radiolabeled Compounds. We purchased di[14C]phenyl- and isopropyl[14C]phenyl-labeled2-IPDP from Pathfinder Laboratories Inc., St. Louis, MO. Both lots of IPDP were uniformly ring labeled with a specific activity of 28.97 pCi/mg, and thin-layer and gas chromatography indicated that purity exceeded 99%. The I4C-ring-labeleddiphenyl phosphate (DP) standard was the generous gift of Dr. Derek Muir, Freshwater Institute, Winnipeg, Manitoba, Canada. It had a specific activity of 5.1 pCi/mg, and it was purified by reverse-phase thin-layer chromatography. The purity was verified by gas chromatography after methylation with diazomethane. Low-Exposure Microcosms. Microcosms consisted of 250-mL Erlenmeyer flasks containing 10 g (moist weight) of sediment and 90 mL of lake water (pH 7.1). Sediment was collected with an Ekman dredge from the littoral zone of Little Dixie Reservior, Callaway County, MO. This reservior is slightly eutrophic, well characterized (Table I), and representative of reservoirs in the tilled-plain areas of the Midwest (15,16). The upper 3 cm of sediment was removed, homogenized at low speed in a Waring blender, overlaid with lake water, and stored aerobically in the dark for several days at 22 "C. Each low-exposure microcosm
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1984 by the
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