* Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, New Mexico 87108; SKS Consulting Services, 3942 Rives Chapel Rd., Siler City, North Carolina;
Southwest Research Institute, 6220 Culebra Rd., San Antonio, Texas 78238-5166; and
Desert Research Institute, Atmospheric Sciences Division, 2251 Ragio Parkway, Reno, Nevada 98512-1095
1 To whom correspondence should be addressed. Fax: (505) 348-8567. E-mail: jseagrav{at}LRRI.org.
Received April 1, 2005; accepted June 9, 2005
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ABSTRACT |
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Key Words: engine emissions; compressed natural gas; comparative toxicology; intratracheal instillation; bacterial mutagenicity.
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INTRODUCTION |
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Previously, we compared the toxicity of collected PM and vapor-phase semivolatile organic compounds (SVOC) from a series of gasoline and diesel vehicles using bacterial mutagenicity testing and intratracheal instillation of these emission samples into rats (Seagrave et al., 2002). The current study was conducted to extend this comparison to emissions from heavy-duty CNG vehicles. The study tests the hypotheses that CNG emissions are less toxic than gasoline or diesel emissions, and that high emitter CNG vehicle emissions are more toxic than those from low emitters.
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MATERIALS AND METHODS |
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Emissions Collections and Sample Processing
Vehicles were operated at an ambient temperature of 25°C on a chassis dynamometer. Samples were collected over several days, with each day beginning with a cold-start U.S. Environmental Protection Agency heavy-duty urban dynamometer driving schedule (HD-UDDS) followed by three central business district cycles (Federal Transit Administration of the U.S. Department of Transportation). This series was repeated seven times each day with hot-start HD-UDDS (after the first). Emissions passed directly into a constant volume dilution tunnel to a final dilution of approximately 1:35. The dilution ratios were within 3% of each other for the three buses. Approximately 25% (23.9 to 24.8%) of the total emission stream was directed through a 20- x 20-in. Teflon-impregnated glass fiber filter (Pallflex Product Corp., Putnam, CT) that was followed by a canister containing a 4-in. plug of polyurethane foam (PUF), 20 g cross-linked divinyl benzene (XAD-4), and a second PUF plug. The temperature at the filter collection point was
35 to 40°C. The filters were extracted into acetone by sonication and gentle brushing, and the PUF/XAD-4/PUF fractions were extracted into acetone by Soxhlet extraction. Both fractions were concentrated by evaporation under nitrogen, and the two fractions were recombined. An equivalent volume of air from the tunnel in the absence of an emission source was collected on to the media and extracted similarly as the Tunnel Background (TB) sample.
Chemical Characterization of Emission Samples
PM mass collected on the 20- x 20-in. filters was measured on an analytical balance following subtraction of the precollection filter weight. PM emission rates were calculated based on the PM mass, volumetric flow rate through the filters, and calculated vehicle miles traveled over the course of the collections. Masses of the recovered PM and SVOC samples were determined by weighing the residual mass following evaporation of the solvent at room temperature. Emission rates for recovered SVOC were determined identically to the PM.
The chemical composition of the PM and SVOC fractions of each of the samples was analyzed at the Desert Research Institute (Reno, NV), as described in detail previously for diesel- and gasoline-powered motor vehicle samples (Zielinska et al., 2004). Separate samples for chemical analysis were collected on media optimized for the different chemical analyses in parallel to samples collected and processed for the toxicity testing. Specifically, PM collected on 47-mm-quartz fiber filters (Pall Corp., East Hills, NY) was analyzed for organic and elemental carbon by thermal/optical reflectance (Chow et al., 2001
). The filters were then extracted into purified water for determination of inorganic ions (sulfate, nitrate) by ion chromatography. Teflon membrane filters were used to collect PM for analysis of elements (metals and associated analytes) using x-ray fluorescence (XRF). To analyze organic compounds, we collected PM on Teflon-coated glass fiber filters (Pall) and SVOC on PUF/XAD-4/PUF cartridges downstream from the filters. The filters were sequentially extracted into dichloromethane and acetone, and the cartridges were extracted into acetone. The samples were concentrated under nitrogen, transferred to acetonitrile, and analyzed by gas chromatography/mass spectrometry. The organic analysis focused on chemicals used in previous studies to illustrate differences among motor vehicle and other types of emissions. These included polycyclic aromatic hydrocarbons ([PAH], 75 species), nitrogenated PAH (25 species), and hopanes and steranes (37 species). Speciated organics included vapor-phase SVOC, compounds that distribute between both vapor/particle phase, and compounds that are exclusively in the particle phase. To conserve space, individual data for all of the organic and inorganic species are not presented. Instead, chemical classes are summarized as fractions of either particle mass or fractions of specific chemical classes. The speciation data can be obtained by request from the authors.
Measurement of Bacterial Mutagenicity
Suspensions of the recovered PM were combined with the recovered SVOC extracts and transferred to dimethylsulfoxide and shipped overnight at 0°C to BioReliance Corp. (Rockville, MD) for mutagenicity testing using the Ames Salmonella plate incorporation assay (Ames et al., 1975). Mutagenicity was assessed in Salmonella strains TA98 and TA100, each with and without metabolic activation by the microsomal fraction of Aroclor-induced rat liver homogenates (S9) (Maron and Ames, 1983
). Each sample was tested in duplicate over a 200-fold range of concentrations in parallel with negative (solvent) and positive controls. The positive controls were 2-nitrofluorene for TA98-S9, sodium azide for TA100-S9, and 2-aminoanthracene for TA98+S9 and TA100+S9. Colonies on plates with sufficient precipitate to interfere with electronic counting were counted by hand; other plates were counted using an automated colony counter. All samples met the response criteria of dose-related increases in mean revertants per plate, rising to at least twice the negative control value. However, high concentrations were toxic to the bacteria and suppressed the observed revertant values. Responses were, therefore, quantified as the slope of the initial linear portion of the dose-response curve fit to the linear model as described under statistical analysis of data below.
Measurement of In Vivo Toxicity
Animals.
Male F344/Crl BR rats were purchased from Charles River Laboratories (Wilmington, MA). The rats, 8 ± 1 weeks old at receipt, were quarantined for three weeks before dosing and were 11 ± 1 weeks old at the time of instillation. Serological testing confirmed the absence of common rodent pathogens. The rats were housed two per shoebox cage with hardwood chip bedding and filter caps. The light/dark cycle was 12 h light/12 h dark with lights on at 0600 h. Food (Harlan Teklad Lab Blox, Madison, WI) and water were provided ad libitum. The room temperature was maintained at 2022°C, with a relative humidity of 2050%. All procedures involving animals were approved by the Institutional Animal Care and Use Committee.
Reagents and supplies.
General chemicals and reagents were purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise specified.
Sample preparation.
The PM/SVOC mixtures for toxicological testing were prepared by dilution (at least 100-fold from the stock in acetone) and sonication in sterile 0.9% NaCl containing 0.01% Tween-80 as previously described (Seagrave et al., 2002). The final acetone content of these solutions was adjusted to 1%. Dilutions were made in 0.9% NaCl with 1% acetone and 0.01% Tween-80. Three dilutions of the TB control were prepared similarly from the concentrated acetone extract of filters and PUF/XAD-4/PUF canisters exposed to equivalent volumes of the tunnel dilution air. As a positive control, National Institutes of Standards (NIST) Standard Research Material 2975 (fork lift diesel soot: [DS]) was prepared in the same manner.
Intratracheal instillation.
Rats were anesthetized using 5% halothane in oxygen with nitrous oxide. Once anesthetized, each rat was intubated orally with an intratracheal cannula and instilled with an emission sample or control material suspended in a total volume of 0.5 ml saline. The rats were allowed to recover and returned to their cages.
Groups of five rats for each dose were instilled with the combined sample, with the highest dose determined by a preliminary range-finding experiment to cause responses without lethality. The first experiment tested the responses to material collected from 0.3, 3, or 30 m3 of the TB and 3, 6, or 12 m3 of NE emissions. The second experiment tested responses to material collected from 6, 12, or 24 m3 of NT; 0.75, 1.5, 3 m3 of HE; and 24 m3 of NE. Each experiment included a positive control of a 1 mg/rat of DS and a blank instillation consisting of 0.01% Tween-80 and 1% acetone in 0.9% saline.
Euthanasia and processing.
Previous studies indicated that the strongest inflammatory and cytotoxic effects were observed 24 h after the instillations (Seagrave et al., 2002). Rats were therefore killed by pentobarbital overdose 24 h after instillation, and body weights were recorded. The lungs were removed and weighed. The right cranial lobe was tied off and the left bronchus was temporarily clamped. The right middle, caudal, and accessory lobes were then lavaged with two, 3-ml aliquots of Dulbecco's phosphate buffered saline. The recovered bronchoalveolar lavage fluid (BALF) was combined, the volume was recorded, and the BALF was kept on ice until processed. After lavage, the right cranial lobe was removed and snap frozen in liquid nitrogen. The left bronchus was then unclamped and the lungs were gently instilled with 10% neutral buffered formalin to approximate normal volume, the trachea was ligated, and the lungs were immersed in the same fixative for at least 48 h.
Lung lavage cells and fluid were collected for analyses. An aliquot of BALF was diluted in red blood cell lysing buffer (Roo's solution: 9 g/l NH4Cl2 + 1 g/l K2CO3 + 0.0372 g/l EDTA) and 0.02% (final) Trypan blue to determine total cell numbers and cell differentials. The total number of leukocytes was determined using a hemocytometer. Cytocentrifuge preparations were used for evaluation of differential cell counts. The slides were stained with Wright-Giemsa using a Hema-Tek Slide Stainer, and the percentages of macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes in 300 cells per sample were counted using a 20X objective.
The remaining BALF was centrifuged (10 min at 1000 rpm). Lactate dehydrogenase (LDH) (Gay et al., 1968), total BALF protein (Watanabe et al., 1986
), and alkaline phosphatase were measured in the supernatant using a Hitachi 911 (Roche Diagnostics, Basel Switzerland) clinical chemistry analyzer.
Histopathology was assessed in the fixed left lung by light microscopy. Three transverse slices about 3-mm thick (one through the lung cranial to the hilus, one through the main airway just caudal to the hilus, and one near the end of the main axial airway) were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin. Histologic grading was performed by a board certified veterinary pathologist (A.P.G.) based on the responses of the lung to the instilled material. Briefly, the character, severity, and distribution of pathologic responses such as inflammatory infiltrates (e.g., suppurative bronchiolitis, mononuclear perivascular cuffing), edema, Type II pneumocyte hypertrophy or hyperplasia, fibrosis, and others were graded using a scale from 0 (no pathology; absence of that particular response/diagnosis) to 5 (extreme pathology; severe and widespread presence of that particular response/diagnosis). For analysis, the specific histopathologic diagnoses were then pooled into categories of responses indicating cytotoxicity (e.g., diagnosis of necrosis), inflammation (e.g., bronchiolitis), or parenchymal changes (e.g., fibrosis) as previously described (Seagrave et al., 2002). For each animal, the sum of the individual scores for each pathologic response/diagnosis within a category was calculated. The sum of all scores was also analyzed as an overall pathological response.
Statistical Analysis of Data
We plotted the responses of each emission sample as a function of the doses (in m3 of exhaust) of each emission sample. None of the TB sample doses gave responses greater than the control in any of the measured parameters, so the data from all three doses were pooled with the solvent control as the zero dose values. Response functions were fit to the data at three different dose levels per emission sample (plus the baseline control values), and the slope coefficients from these fitted dose-response curves were used to make comparisons among the toxicological potencies of the different emissions. This technique makes optimal use of the entire dose-response curve, providing substantially more statistical power to discriminate among the potencies of the different emissions samples than would be possible from individual dose-to-dose comparisons.
For each endpoint of the toxicity parameters evaluated in rats, a single regression model with emission-specific slope coefficients was utilized:
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For variables that could exhibit values of zero, log (Yki + 1) was analyzed. Although linear models (i.e.,) fit most of the lavage data equally well, log-linear models were used to obtain potency estimates that were comparable to those of a previous study (Seagrave et al., 2002
). For the mutagenicity data, which showed strongly linear initial dose-responses up to the level of toxicity to the bacteria, Yki was substituted for log (Yki) in this model. We used generalized least squares regression to estimate the dose-response coefficients for endpoints that exhibited a relationship between sample mean and standard deviation, using squared reciprocals of fitted values from estimated standard deviation functions as weights (Neter et al., 1996
); ordinary least squares regression was employed for all other endpoints. Statistical significance of individual emission-specific toxicological potency estimates (bk
0) was assessed using t-tests associated with the regression model. Comparisons between pairs of emission-specific toxicological potencies (bk) were evaluated with F-test contrasts (Searle, 1971
), calculated by the SAS software system (Cary, NC). To evaluate differences among emission samples for each endpoint, p-values from the pair-wise F-tests were adjusted for multiple comparisons (three paired comparisons among the three samples) using the modified Bonferroni procedure devised by Hochberg (1988)
. Statistical significance was assessed at p = 0.05.
Exposure Metrics and Interpretation of Potency Factors
In a previous study comparing mutagenicity and in vivo toxicity of gasoline- and diesel-powered vehicle emissions (Seagrave et al., 2002), we characterized exposure-related effects relative to the mass of administered emission material (either per plate or per animal). A significant disadvantage to this metric is that it does not incorporate the relative emission rates into the potency factors. Thus, a high emitter and low emitter vehicle with similar mass-based potency factors might not be perceived as different, even though the hazard is greater from the high emitter simply because it produces more mass. In the current study, we have chosen to use equal volumes of the equivalently diluted emissions as the metric, thus incorporating emission rates directly into the potency factors. However, to facilitate comparisons with results from the previous paper, we also present results as mass-based potency factors. These two metrics provide different types of useful information: biological responses as a function of mass dose provide a means to investigate differences in the potencies of the emissions materials themselves, while the volume-based metric provides insight into effects associated with differences in the amounts of materials generated by different emissions sources at equal units of operation and exposure.
It should be noted that conversion between these different metrics is straightforward for the linear dose response curves (mutagenicity): for instance, a potency factor in revertants per microgram can be converted to revertants per m3 by multiplying the mass-based potency factor by the µg/m3. However, because the dose is contained in the exponent of the potency factors derived from exponential fits of the data, the conversion is more complex. More importantly, the response is not a simple linear function of the conversion factors. For more detail, please see the on-line supplementary information.
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RESULTS |
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The lavage fluid from one animal in the highest group of NE (24 m3) contained noticeable red blood cells. This animal also had exceptionally high levels of LDH, protein, and total nucleated cells (more than 10 times the levels in any of the other animals in this group). However, the histopathology from this rat did not reflect these effects. We speculate that despite efforts to prevent influx of blood during the removal of the lungs, the lavage fluid was contaminated with blood from the thoracic cavity. In addition, three other animals (one in the diluent control, one in the high-dose TB, and one in the mid-high NE group) had red blood cells in the lavage fluid and levels of at least three lavage parameters that met statistical criteria as outliers (most of these values were also more than 10 times the values of the other animals in the respective group), and these data have been omitted from the analysis (along with the animal in the highest concentration of NT that died before the scheduled sacrifice).
The two primary acute cytotoxicity indicators, lavage total protein (shown in Fig. 4) and LDH, showed very similar dose-response curves, with the effects of the HE sample being pronounced at low exposures when plotted on the basis of the equivalent volumes (Fig. 4A). The NE and NT samples showed much lower potencies. However, on a per unit mass basis (Fig. 4B), the HE sample was similar in potency to the other two CNG samples for these parameters, indicating that the HE toxicity (volume-based) related to a higher emissions rate, rather than increased toxicological potency of emissions substances. The potency factors for alkaline phosphatase (AP) and the histopathological indications of cytotoxicity followed similar response patterns for these three emission samples (Fig. 5).
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The mutagenicity of these samples can be compared with the data previously reported using identical methods for the PM + SVOC fractions of diesel and gasoline vehicles by the conversion process described in Materials and Methods. It is important to note that the previous set of emission samples were from light-duty gasoline and diesel vehicles, while only CNG buses (heavy-duty vehicles) were used in this study. However, all samples from both studies were obtained from vehicles operated under their typical operating conditions, and these comparisons provide a starting point for comparing the toxicity of the emissions produced from vehicles utilizing these three fuel types.
The mutagenicity in revertants per µg, shown in Table 3, indicates that the much greater potency of the HE sample compared with the NE sample on a volumetric basis (Fig. 3) was primarily due to the greater mass of the emissions, since less than two-fold differences in mutagenicity per unit mass were observed between the HE and NE samples in both strains with and without metabolic activation. However, the NT sample contained substantially less mutagenic activity per unit of mass (as well as per emission volume). Table 3 also shows data for selected samples from the previous study. Like the gasoline and diesel emissions samples, the range of mutagenicity caused by the CNG emissions samples was fairly limited (less than a 10-fold difference between the least and most potent conditions). Comparing the normal emitters of the three classes, effects in TA98 with or without metabolic activation were greater for the NE sample than for the gasoline or diesel normal emitter samples. The HE sample had comparable mutagenicity to the high emitter diesel sample and white smoker gasoline sample, while the NE sample was the least mutagenic sample in this strain. Metabolic activation in TA98 had little effect on the CNG or diesel samples, but increased the potency for the gasoline samples. In TA100 without metabolic activation, the HE sample was the most potent of the CNG samples, comparable to previously reported potencies for the white smoke gasoline and high emitter diesel samples. The NE sample resembled the normal emitter diesel and black smoker gasoline samples, and the NT sample was similar in mutagenic potency to the normal emitter gasoline sample. Metabolic activation in this strain decreased mutagenicity by 5070% for the three CNG samples. Reduced mutagenicity with metabolic activation was also observed in TA100 for the high emitter diesel and white smoker gasoline, but not for the other gasoline and diesel emission samples.
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DISCUSSION |
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One important result of this study was that under all assay conditions (either strain of Salmonella and with or without metabolic activation), the mutagenic potencies, as revertants per m3 (thus taking into account the differences in emission rates) of the combined PM + SVOC fractions of the HE vehicle were much greater than for the NT vehicle, with the NE sample having intermediate activity. These observations confirm and extend the previous data for an organic extract of the PM fraction of a CNG-powered heavy duty truck (Lapin et al., 2002), showing that emissions from CNG-fueled vehicles contain substantial amounts of direct-acting mutagens. This was confirmed by the presence of nitro-PAH in both the HE and NE samples that have been shown in several previous studies (McDonald et al., 2004
) to be associated with bacterial mutagenicity. An important consideration for both these samples and the previously reported data (McDonald et al., 2004
) is that many of these nitro-PAH compounds can be formed by PAH reacting with NOx that pass through the filter and sorbent during sample collection (Arey et al., 1988
). This artifact was also suggested for diesel and gasoline emission samples reported previously (McDonald et al., 2004
), but likely is even more important for CNG samples (especially low emitters) because low vehicle mass emissions required the samples to be collected for longer time periods (up to 50 h for the NE sample) to obtain enough sample for the toxicity and mutagenicity studies. An even longer time was required to collect sufficient sample of the NT emissions (63 h), which had a lower proportion of nitro PAHs than the NE vehicle. However, the NOx emission rate of the NT vehicle was only 0.01 g/mi, compared with 0.253 for the NE and 2.05 for the HE.
Intratracheal instillation of collected materials, as used in this study, is widely accepted as a useful method for screening relative toxicity (Henderson et al., 1995), thus justifying further investigations. Although some variation of deposition into different lung lobes may occur with the instillation method, in our hands reasonably reproducible responses are achieved, comparable in value to those achieved following inhalation studies (Seagrave et al., in press).
The HE sample produced greater lung toxicity than the NE and NT samples for nearly all parameters when data were adjusted for the difference in emission rates. There was little difference between the NE and NT emission sample potencies on the basis of mass. Interestingly, the HE sample did not have the highest PAH concentrations (relative to total mass), but had somewhat higher proportions of hopanes and steranes, two classes of organic compounds that are unique to oil and were shown in our previous work to be statistically associated with lung toxicity of emission samples (McDonald et al., 2004).
Despite this study advancing our knowledge of the lung toxicity and mutagenicity of natural gas emissions, it has several limitations. The use of collected, acetone-extracted, and processed samples (as opposed to a study conducted by inhalation) was a limitation of this study. Although attempts were made to quantitatively remove 100% of the PM from the filters, only 1520% of the PM could be removed. This was much less than the expected efficiency, based on results obtained using the same procedures with gasoline and diesel emissions, perhaps suggesting very different composition of the original exhaust. There may be several explanations for the sub-optimal extraction. First, the organic solvent may not have efficiently removed the large amounts of metallic/ionic elements that were associated with the PM from the CNG vehicles. Second, the organic PM appeared to be stickier than previous emission samples, making removal of the material from the filters by mechanical agitation less effective. However, more vigorous physical manipulations were avoided to prevent fragmentation of the filters, that would have resulted in release of fibers into the suspensions. Additional losses of PM during evaporation/concentration may explain some of the differences as well.
Toxicity potency per unit of total mass (including that which could not be extracted) could be either greater or less than the values reported, depending on whether extraction was selective for more or less toxic agents. If the extracted material was representative of the whole mixture, the mass-based potency factors of the extracts would accurately represent the potency of the whole. If most of the toxic materials were extracted, and only relatively inert materials were left on the filter, the mass-based potency of the whole would be less than the potency of the extracts since the denominator (mass) would increase without increasing the numerator (toxicity). In contrast, if the most toxic constituents were not extracted, the potency of the whole would be greater than the potency of the extracts. However, the toxicity and mutagenicity, reported as potency factors per unit volume of emissions, are unlikely to be less than those indicated, since more effective extraction would only add constituents to the mixture without changing the denominator.
In summary, these data demonstrate that collected PM + SVOC emission samples from CNG-fueled vehicles contain both mutagenic and toxic constituents, and serve to place the potential hazard from these emissions in the context of potential health effects from diesel and gasoline-fueled vehicles' emissions.
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SUPPLEMENTARY DATA |
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ACKNOWLEDGMENTS |
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