Dennis Schuetzle

The Identification of Polynuclear Aromatic Hydrocarbon (PAH) Derivatives in Mutagenic Fractions of Diesel Particulate Extracts

The soluble organic fraction (SOF) of particulate matter from diesel exhaust (from point sources, ambient air, etc.) contains hundreds of organic constituents. Norman-phase high pressure liquid chromatography (HPLC) has been used to separate the SOF into subfractions suitable for subsequent chemical analysis and bioassays. These fractions consist of non-polar(PAH), moderately polar (transition) and highly polar constituents. The non-polar fractions have been well characterized and consist of PAH and aliphatic hydrocarbons. The specific compounds present in the transition and polar fractions are for the most part unknown. This analytical information has been difficult to obtain since these compounds are highly labile, polar, of low volatility and in very low concentrations when compared to the bulk of material found in the SOF. Mutagenicity tests using the Ames Salmonella typhimurium assay indicate that the transition fraction accounts for most of the mutagenicity when compared to the non-polar (PAH) and polar fractions. A variety of chromatographic and mass spectrometric techniques are described that have been used to determine the composition of the HPLC fractions. More than one hundred species have been identified in the transition fraction of diesel particulate matter using high resolution gas chromatography (HRGC)/high resolution mass spectrometry (HRMS), HPLC and direct-probe high resolution mass spectrometry. It has been found that the transition fraction contains mostly PAH derivatives consisting of hydroxy, ketone, quinone, carboxaldehyde, acid anhydride and dihydroxy derivatives of PAH. Three nitro-PAH species have been tentatively identified and 1-nitropyrene positively identified in the transition fraction. The 1-nitropyrene was found to account for approximately 45% and 30% of the direct-acting mutagenicity observed for the transition fraction and total extract, respectively. The HPLC separation procedure was shown to give better than 95% recovery of the mass and mutagenic activity. The problem of PAH oxidation during the analytical procedures and possible effect on bioassay results are discussed.

Contribution of 1-nitropyrene to direct-acting Ames assay mutagenicities of diesel particulate extracts

Projections of future increases in the number of diesel powered U.S. passenger cars have raised concerns about potential adverse human health effects associated with diesel engine particulate exhaust (Pepelko et al., 1980). One concern has been carcinogenic polycyclic aromatic hydrocarbons (PAH), known for many years to be associated with diesel exhaust particles. A more recent concern, raised by experiments with the Ames Salmonella mutagenesis assay, has been the discovery that PAH may not be the only potential carcinogens in the diesel exhaust particles (Huisingh et al., 1978; Wang et al., 1978; Pepelko et al., 1980). These experiments have shown that organic solvent extracts of diesel exhaust particles are directly mutagenic (i.e., mutagenic without activation by mammalian tissue homogenate), whereas the predominant PAH identified in the extracts are mutagenic in the Ames assay only with activation (Searle, 1976; McCann and Ames, 1976; Gibson et al., 1978). We (Schuetzle et al., 1981) and others (Erickson et al., 1979) have identified many oxygenated derivatives of PAH in these extracts. Many of these derivatives could be direct-acting mutagens, but little is known yet about their mutagenicities or their concentrations in the extracts. One of the PAH derivatives identified was l-nitropyrene (I-NP) (Schuetzle et al., 1982), recently shown to be an extremely potent direct-acting mutagen in the Ames assay (Wang et al., 1980; LOfroth et al., 1980, Rosenkranz et al., 1980). Herein we report a rigorous determination of the percentage contribution of 1-NP to the direct-acting Ames assay mutagenicities of extracts of exhaust particles collected from 3 different diesel powered passenger vehicles. The percentage contribution, P, of 1-NP is given by:

Standard reference materials for chemical and biological studies of complex environmental samples

Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) are often used in methods development and interlaboratory comparison studies since they are homogeneous and readily available to the scientific community. SRM 1649 (urban dust/organics), SRM 1650 (diesel particulate matter), and SRM 1597 (complex mixture of polycyclic aromatic hydrocarbons from coal tar) are three environmental samples which have been used by the scientific community for these purposes. These SRMs were originally developed to assist laboratories in validating analytical procedures for the determination of polycyclic organic compounds in complex mixtures. In addition, these SRMs have been valuable for the comparison of methodologies for bacterial bioassays and the development of bioassay-directed fractionation and bioassay-directed chemical analysis techniques. Most recently these SRMs were chosen for use as test samples in a collaborative study coordinated by the World Health Organization--International Programme on Chemical Safety. This paper provides a summary of much of the work to date (published and unpublished) on the chemical and biological characterization of these three SRMs. Information regarding the availability of other NIST SRMs that might be useful for these types of studies are provided also.

Factors Influencing the Emissions of Nitrated-Polynuclear Aromatic Hydrocarbons(Nitro-PAH) from Diesel Engines

The potential problem of 1-nitropyrene (1-NP) formation during filter sampling of diesel emissions from dilution tubes is assessed. Rates of formation are calculated using data generated from several independent filter exposure studies. A portion of the 1-NP (12% average) found in participates collected from light-duty (LDD) and heavy-duty diesels (HOD) was found to be due to formation on the filter under average sampling conditions of 10-15/1 dilution at 44°C with 3 ppm NO2 for 23 min. On the average, the concentration of 1-NP in participates emitted from HDD is 16 times less than that found in LDD run under similiar transient conditions. The average emission rate of 1-NP generated from the LDD and HDD was 4.7 μg/km and 1.5 μg/km, respectively for the vehicles operating under Federal Test Procedure (FTP)-transient conditions. Under operating conditions which increase exhaust temperature (increased speed and load), the concentration of nitro-PAH is significantly reduced with a concurrent increase in the c...

Gas-phase atmospheric chemistry of 1- and 2-nitronaphthalene and 1,4-naphthoquinone

The gas-phase photolysis of 1- and 2-nitronaphthalene and the reactions of these nitroarenes with OH and NO3 radicals, N2O5 and O3 have been investigated at 298±2K and 1 atm total pressure of air. Since 1,4-naphthoquinone was observed as a photolysis product of 1-nitronaphthalene in air, its photolysis and reactions with OH and NO3 radicals, N2O5 and O3 were also studied. Using relative rate methods for the determination of rate constants for the reactions with OH radicals and NO3 radicals and/or N2O5, and rate constants of k(OH+cyclohexane) = 7.49 × 10−12cm3molecule−1 s−1 and k(NO3 + propene) = 9.4 × 1015 cm3 molecule−1s−1 and the equilibrium constant for the NO3 + NO2ahN2O5 reactions of K = 2.3 × 10−11 cm3molecule−1, the rate constants obtained for these reactions were (in cm3molecule−1s−1 units): for reaction with the OH radical; 1-nitronaphthalene, (5.4±1.8)× 10−12; 2-nitronaphthalene, (5.6±0.9)× 10−12; 1,4-naphthoquinone, (3.1±1.2) × 10−12; for reaction with the NO3 radical; 1-nitronaphthalene, ≤7.2× 10−15; 2-nitronaphthalene, ≤7.2×10−15; 1,4-naphthoquinone, <1 × 10−15; for reaction with N2O5; 1-nitronaphthalene, (1.3±0.7) × 10−18; 2-nitronaphthalene, (1.2±0.5) ×10−18; 1,4-naphthoquinone, <4×10−19; for reaction with O3; 1-nitronaphthalene, include the uncertainties in the rate constants for the reference reactions or in the eq <6×10−19; 2-nitronaphthalene, <6×10−19; 1,4-naphthoquinone, <2×10−19 (where the cited error limits do notuilibrium constant K). The photolysis reactions were investigated under blacklamp irradiation and under natural solar irradiation. With solar irradiation at an NO2 photolysis rate of JNO2 = 4.3 × 10−3s−1, the outdoor photolysis rates were (in s−1 units): 1-nitronaphthalene, (1.37±0.10) × 10−4; 2-nitronaphthalene, (1.06±0.08)× 10−41,4-naphthoquinone, ~ 9 × 10−5. These data show that under atmospheric conditions the dominant loss process of 1- and 2-nitronaphthalene and 1,4-naphthoquinone is photolysis, with calculated photolysis lifetimes of ~2 h for all three compounds. The OH radical reactions are calculated to be an order of magnitude less important than photolysis as an atmospheric loss process, with the N2O5, NO3 radical and O3 reactions being of negligible importance.

Experimental Investigation of the Atmospheric Chemistry of 2-Methyl-1-Nitronaphthalene and a Comparison of Predicted Nitroarene Concentrations with Ambient Air Data

Abstract The atmospheric chemistry of 2-methyl-1-nitronaphthalene and the photolysis of 3- nitrobiphenyl have been investigated. Under atmospheric conditions, photolysis was the dominant loss process for 2-methyl-1-nitronaphthalene, while no photolysis of 3- nitrobiphenyl was observed. These data have been used, together with our previous data concerning the atmospheric reactions of 1-and 2-nitronaphthalene and the formation of nitroarenes from the gas-phase atmospheric reactions of the 2- to 4-ring PAH, to predict ambient nitroarene concentrations. The predicted concentrations of the nitronaphthalenes, 3-nitrobiphenyl, 2-nitrofluoranthene and 2-nitropyrene are compared with ambient air data from Torrance and Glendora, CA, USA.

Design and Implementation of a Collaborative Study of the Mutagenicity of Complex Mixtures in Salmonella Typhimurium

In 1987, the International Programme on Chemical Safety (IPCS) in collaboration with the U.S. Environmental Protection Agency (U.S. EPA) and the U.S. National Institute of Standards and Technology (U.S. NIST) initiated an international collaborative study of the mutagenicity of complex environmental mixtures in the Ames Salmonella typhimurium mutation assay. The objectives of this study were: (1) to estimate the inter- and intra-laboratory variability associated with the extraction of mixtures for bioassay, (2) to estimate the inter- and intra-laboratory variability associated with the Salmonella typhimurium bioassay when applied to complex mixtures, and (3) to determine whether standard reference complex mixtures would be useful in mutagenicity studies and to evaluate whether reference or certified mutagenicity values determined from this collaborative study should be reported. The complex mixtures used in this study were selected from standard reference materials (SRMs) which had previously been issued by the U.S. NIST as SRM 1597 (coal tar), SRM 1649 (diesel particulate matter) and SRM 1650 (urban air particulate matter) with certified values for polycyclic aromatic hydrocarbons. These SRM complex mixtures are available to scientists as reference standards for analytical chemistry research and are under consideration as SRMs for mutagenicity studies of complex environmental mixtures. This paper briefly describes the final study design, protocol, selection of the complex mixtures, and implementation of this international study.

Thermogravimetry of filter-borne diesel particulates

Abstract A direct thermogravimetric (TG) method is described for determining the percentage of volatile components in filter-borne diesel particulates. The procedure gives a quick estimate of total solvent extractable hydrocarbon components. A silica gel fractionation/size exclusion Chromatographic (SGF/SEC) procedure has also been developed to analyze further the solvent-extractable hydrocarbons of the particulates. The solvent extraction SGF/SEC procedure determines the contribution of lubricating oil and unburned fuel to particulates emitted from diesel engines. Results from this study indicate that the TG method yields a comparable measure of adsorbed hydrocarbons with those obtained by solvent extraction.

THE IDENTIFICATION OF MUTAGENIC CHEMICAL SPECIES IN AIR PARTICULATE SAMPLES

Techniques are described for the identification and quantitation of mutagenic chemical species in air particulate samples. The samples were separated into chemical fractions using high performance liquid chromatography (HPLC). Bioassays were used to prioritize these chemical fractions for identification of chemical mutagens. The moderately polar HPLC fractions have been found to represent a major portion of the direct acting mutagenicity (TA 98) for the total particulate extract. Mass spectrometry/mass spectrometry (MS/MS) and high resolution mass spectrometry (HRMS) techniques were used for determining the presence of isomer groups of compounds. Many of these isomer groups potentially contain a large number of specific isomers. Fused silica capillary column GC/MS was found to be the best technique for identification of specific isomers. Quantitative techniques are described for accurately determining the contribution of a chemical species to the total mutagenicity of particulate extracts. Polynuclear aromatic hydrocarbon (PAH) derivatives such as nitrated-PAH were found to account for a major portion of the direct-acting Ames assay mutagenicities in these particulates. Nitro-PAH may be formed during sampling. However, the formation of nitro-PAH is not significant when particulates are collected at low temperatures (<40°C), for short sampling times (<1 hr.), and in effluents containing less than 3 ppm NO2.

Identification of Genotoxic Agents in Complex Mixtures of Air Pollutants

Recent surveys have shown that global environmental effects are ranked as a major issue of public concern (109). Numerous newspaper and magazine articles on the greenhouse effect, ozone depletion, acid rain, hazardous waste, and air toxics are some of the recent issues which have helped to raise this level of concern.