Chemical and Bioassay Analyses of Diesel and Biodiesel Particulate Matter:
Pilot Study -- Final Report
By Norman Y. Kado, Robert A. Okamoto and Paul A. Kuzmicky
Department of Environmental Toxicology, University of California
Davis, California, 95616
Howard E. Haines
The Montana Department of Environmental Quality
The U.S. Department of Energy
The Renewable Energy Report Library,
Montana State Library
1515 East Sixth Avenue
Helena, Montana 59620-I 800
Full report (Acrobat file, 3.1Mb)
The exhaust from diesel fuel combustion is known to be a highly complex mixture of toxic compounds. Combustion products from fuel consisting of a mixture of diesel with rapeseed oil ethyl ester (REE) or from 100% REE also is a complex mixture of compounds. Any effort to determine the potential health effects of the emissions from these fuels would require extensive chemical and biological analyses. One approach to help evaluate potential human health effects from the mixture of compounds present in particulate matter is to use a short-term bioassay in conjunction with chemical analyses. Bioassays have been developed to measure a number of different health effects, including effects hypothesized to be at least in part responsible for chronic diseases. For example, some bioassays measure damage to genetic material, or DNA. This damage, referred to as genotoxic activity, is thought to be integral in the process of developing many types of cancer.
In collaboration with the University of Idaho, the Montana Department of Environmental Quality, and the U.S. Department of Energy, we investigated two important health-based components of diesel and biodiesel exhaust: 1) The concentrations of polycyclic aromatic hydrocarbons (PAHs - some suspected animal and human carcinogens and present in these emissions) and 2) The genotoxicity (DNA damaging capability) of the particulate extracts from these emissions.
Four different fuels were tested in a 1995 Dodge 3/4 ton pickup truck Cummins B (5.9 L, Turbo diesel): 1) 100% ethyl ester of rapeseed oil (REE) 2) 100% diesel 2-D low sulfur fuel 3) 20% REE + 80% diesel 4) 50% REE + 50% diesel. Emissions from the truck were collected on filters under the controlled conditions of a chassis dynamometer-dilution tunnel facility at the Los Angeles County Metropolitan Transit Authority (LACMTA) facility. An EPA test cycle was followed throughout. The cycle incorporates two approximately equal sampling times (referred to as Pi and P2 parts of the cycle). Due to the limited amounts of samples, filters were lcut in half to provide samples for chemical analyses and bioassay investigations. For the chemical analyses, filter halves from the PI and P2 filters were pooled and extracted. Deuterated PAH isotopes were added for quantitation of each PAH. The filter extracts were analyzed using a gas chromatograph/mass spectrometer (GC/MS) in the selective ion mode (SIM) which is a specific analyses for selected PAHs. The PAHs can be generally divided into two groups: 1) the semi-volatile PAHs (for example, phenanthrene - three connected benzene rings) and 2) the non-volatile PAHs (for example, benzo(a)pyrene - five connected benzene rings). In diesel emissions, the concentrations of these semi-volatile PAHs have been reported to be higher compared to the heavier non-volatile PAHs. We analyzed for both semi-volatile and non-volatile PAHs.
Use of 100% desel fuel without a catalytic converter and under the condition of a hot start resulted in the highest quantities of PAHs measured per mile. The exception was for benzo(a)pyrene and perylene which had higher total masses per mile with the 100% REE and 50% REE blend than with the 100% diesel fuel. Under the conditions of a cold start without catalyst, emissions of fluoranthene and benzo(ghi)perylene from 100% REE were higher (pg / mile) than that from 100% diesel fuel, but pyrene was lower from the 100% REE fuel.
For the catalyst-equipped engine, PAHs such as phenanthrene, fluoranthene, and pyrene remained at an approximately equivalent emission rate (g/mile) independent of the REE content in the fuel (ranging from 100% diesel to 100% REE). Further, in the catalyst-equipped engine, the more chemically reactive PAHs [for example, benzo(a)pyrene] were emitted at greater levels for the pure REE and some of the blended REE fuels than in emissions from 100% diesel fuel.
For the bioassay analyses, a simple modification of the Salmonella/microsome test (called the microsuspension assay) was used throughout. Each filter half from each part of the EPA cycle (Pl and P2) was tested individually for genotoxicity (the potential to damage DNA). Three doses of each filter extract were tested in duplicate. The slope of the linear portion of the dose-response curve was used to determine the specific activity or potency of each extract. The emissions of mutagenic compounds, expressed as revertant equivalents per mile, were determined from this potency value and the total mass of particulate matter collected. For both the non-catalyst and catalyst-equipped engine, use of the 100% REE fuel produced in the lowest genotoxic (DNA-damaging) activity in the tests. Blended fuels in the non-catalyst-equipped engine produced less emissions than emissions than the 100% diesel fuel.
For the catalyst-equipped engine, the highest emissions were from the cold start 100% diesel fuel when compared to any of the hot start samples. The next highest to the cold start 100% diesel fuel was the 20% REE/diesel blend, followed by either the 50% REE/diesel blend or the hot start 100% diesel. The use of the 100% REE fuel resulted in the lowest emissions compared to the REE blends and 100% diesel fuels.
These pilot studies, differences in the total emission of genotoxic compounds from the catalyst-equipped engine compared to the non-catalyst- equipped engine are apparent. The catalyst-equipped engine in some cases had higher mass emissions (g/mile) of certain PAHs.
These studies would benefit from a replication using larger sample size, and a trapping of the vapor-phase compounds in conjunction with the trapping and analyses of the particulate matter. The vapor-phase mutagenic compounds could then be compared to the particle phase and a more complete profile of emissions could be obtained. Further, the emissions with and without a catalyst need further investigation measuring both particle and vapor-phase. Finally, two procedural approaches are recommended for incorporation into the test plan: 1) tunnel blanks where a sa!mpling of the tunnel without the engine running and conducted for identical times as the test cycle is recommended. 2) tunnel conditioning where filtered ambient air is drawn through the system for specified times prior to testing the next fuel is recommended to be incorporated into the test plan.
The highest relative specific mass mutagenic activity collected during either the hot or cold test cycles was the particulate matter collected from the 100% diesel fuel emissions. The 100% diesel fuel was higher than either the 100% REE or the diesel-REE blends. For the PI phase of the entire cycle, and without catalyst, the next highest in specific mass mutagenic activity is the 20% REE (80% diesel), followed by 50% REE (50% diesel). The lowest relative specific mass mutagenic activity was from the particulate matter collected from emissions of 100% REE fuel.
All particulate matter collected had measureable genotoxic (mutagenic) activity. All extracts of the particulate matter when tested in the Salmonella microsuspension procedure had primarily linear dose-response characteristics which is an indication that mutagenic compounds were present. The relative specific mass mutagenic activity (mutagenic activity per mass of particulate matter) provides a way to analyze relative potency of the particulate matter. This provides a description of the degreee of mutagenicity of a specific compound or complex mixture. Exposure characteristics however, depend on the emissions, or the amount of mutagenic compounds emitted per mile traveled. Since we do not know all the specific mutagenic compounds emitted, we measure mutagelrlic activity as an index of these compounds. The emissions therefore reported as "revertant equivalents per mile" and are dependent on the potency of the particles in combination with the mass of particles emitted. A discussion of the potency, or specific mass mutagenic activity is followed by a discussion of the emissions.
The specific mass mutagenic activity was markedly different depending on the fuel type and if the vehicle was equipped with a catalytic converter for emissions. When there was no catalytic converter, the highest relative specific mass mutagenic activity for particles collected either during the cold or hot test cycle was from the 100% diesel fuel. The specific mass mutagenic activity decreased with the increase of REE, with the 100% REE fuel having the lowest relative activity. The 100% REE activity was approximately 3 to 7 times lower than that of the 100% diesel fuel, depending on whether it was a hot or cold part of the cycle, and whether a catalytic converter was used. The REE produced significantly lower specific mass mutagenic activity than diesel fuel when a catalyst was not used. The 100% REE and REE blends were approximately 3 times less potent per mass of particulate matter than the 100% diesel samples (Table 6).
The specific mass mutagenic activity with a catalyst was higher than the activity without a catalyst (Table 7). For the Pl part of the cycle, mutagenic activity from the 20% REE blend was higher than that of the hot or cold diesel tests. This increase was also seen in the P2 part of the cycle. The P2 part of the cycle overall had higher specific mass mutagenic activity than the Pl part of the cycle, with at least a doubling of activity for all fuels. The 100% REE fuel had approximately 10 times more in activity in the P2 than in the Pl portions of the entire test cycle, but still produced approximately l/4 that produced by less than diesel or the blends. The nature of the P2 portion of the test cycle may have produced this increased activity with a catalyst. The high engine speeds experienced during the P2 portion of the test cycle may produce the greater amounts of mutagenic compounds. However, when no catalyst was in place, the Pl and P2 portions of the cycle appeared to be equivalent (see Table 6). The catalyst used in this study may therefore facilitate the formation of certain mutagenic compounds. The increase in the 20% REE and the similarity of all REE blends compared to the 100% diesel fuel and in the Pl and P2 portions of the test cycle with catalyst, should be further investiaged. A number of mechanisms are possible. For example, an increase in the specific mass mutagenic activity can be the result of enriching for particles that have adsorbed mutagenic compounds and eliminating possibly larger particles.
Although the activity per particle mass is important as an indicator of the potency of the particulate matter, an important component of the analysis of human exposure is to investigate the total emissions of mutagenic compounds, or levels of mutagenic compounds emitted per mile. The total emissions of mutagenic compounds without a catalytic converter followed the rank order of specific mass mutagenic acivity: 100% diesel (cold start) > 100% diesel (hot start) > 50% diesel > 20% REE > 100% REE (cold start) > 100% REE (hot start). The emissions per mile from the hot start 100% REE fuel without a catalytic converter are approximately 4 times lower than the 100% diesel fuel.
The emissions from the catalytst-equipped truck had a rank order pattern similar to those of its specific mass mutagenic activity. The 100% diesel fuel (cold start) had the highest emissions and this was followed by the 20% REE (80% diesel). The hot start 100% diesel and 50% REE were approximately equivalent in emissions, but slightly lower than the 20% REE fuel. The 100% REE had the lowest emissions as was the case when there was no catalyst. The use of catalyst with fuel blended with REE results in a small reduction in the emissions of mutagenic compounds from those found in the 100% diesel fuel. These results suggests that the catalyst is not functioning in the manner intended for diesel particulate matter. The catalyst with the fuels tested here needs further investigation.
These studies would benefit from a replication using larger sample size, and a trapping of the vapor-phase compounds in conjunction with the trapping and analyses of the particulate matter. The vapor-phase mutagenic compounds could then be compared to the particle phase and a more complete profile of emissions could be obtained. Further, the emissions with and without a catalyst need further investigation measuring both particle and vapor-phase. Finally, two procedural approaches are recommended for incorporation into the test plan: 1) tunnel blanks where a sampling of the tunnel without the engine running and conducted for identical times as the test cycle is recommended. 2) tunnel conditioning where filtered ambient air is drawn through the system for specified times prior to testing the next fuel is recommended to be incorporated into the test plan.
Full report (Acrobat file, 3.1Mb)
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