Bruce R. Clark

Polycyclic aromatic primary amines as determinant chemical mutagens in petroleum substitutes

Petroleum crude oils and coal- and shale-derived petroleum substitutes have been separated by chemical class and the class fractions have been subjected to bacterial mutagenicity testing. Alkaline constituents of the petroleum substitutes are found to make major contributions to their mutagenicities. High-resolution chromatographic and spectroscopic analysis of alkaline subfractions enriched in mutagenic activity show the causative agents to be polycyclic aromatic primary amines. The amines may be responsible for the increased biological activity of coal- and shale-derived petroleum substitutes relative to petroleum.

Analytical and biological analyses of test materials from the synthetic fuel technologies: IV. Studies of chemical structure-mutagenic activity relationships of aromatic nitrogen compounds relevant to synfuels

Abstract Nitrogen-containing organic compounds from environmental sources are receiving increasing attention because of uniquely active mutagens which have been found in this class (Chrisp et al., 1978; Nagao and Sugimura, 1978; Guerin et al., 1980). Differences in mutagenic activities among the various organo-nitrogen compounds, i.e., pyrrole types, pyridine types and aniline types, have been noted consistently. Furthermore, differences among homologs of a particular compound type are often striking. Information in this paper engages the question of chemical structure/biological activity relationships. Activity data for several N -heterocyclic, nitro-, amino- (primary, secondary and tertiary), and amino- N -heterocyclic aromatic compounds are presented. The number of fused rings and the substituent type affect the mutagenic activities greatly. The trends observed are discussed generally with reference to molecular structural features.

Distribution of mutagenic activity in petroleum and petroleum substitutes

Abstract Several petroleum crude oils and shale- and coal-derived petroleum substitutes have been fractionated by chemical class and the fractions have been tested for mutagenic activity. Crude petroleum substitutes tend to exhibit greater mutagenicities than do petroleum crude oils but the mutagenicity is reduced to comparable levels in low-boiling distillates and samples which have been hydrotreated. The mutagenicity is caused by alkaline and neutral constituents of the petroleum substitutes, but only neutral constituents for the petroleum crudes. The mutagenic components constitute less than ten per cent of the mass of the samples.

Separation of neutral nitrogen compounds from synthetic crude oils for biological testing

Isolation of neutral N-PAHs (PAH homologues containing one or more ring nitrogens) is achieved in three steps using acid-base extraction, gel filtration on Sephadex LH-20, and adsorption chromatography on silicic acid. Gas chromatographic/mass spectrometric analysis of the neutral N-PAH fractions indicated the following as major components: C1-C3 phenylpyrroles, indole, C1-C6 indoles, C1-C3 phenylindoles, carbazole, C1-C5 carbazoles, benzocarbazoles, and C1-C3 benzocarbazoles. The neutral N-PAH fractions were subjected to mutagenicity tests using Salmonella typhimurium/microsomal activation systems devised by Ames. The neutral N-PAH fraction of the coal-derived oil had a specific activity more than twice that of the PAH fraction of the same oil, whereas the shale oil neutral N-PAH fraction showed no activity. These results are discussed in the context of previous work with these oils and with some pure neutral N-PAH compounds.

Separation and Identification of Mutagenic Constituents of Petroleum Substitutes

Abstract A study combining chemical separations, mutagenicity testing, and spectroscopic identifications is underway to isolate and identify mutagens in coal-and shale-derived oils. Ether-aqueous partition combined with Sephadex LH-20 chromatography of the resulting neutral fraction is introduced as a preferred class fractionation procedure. The uniquely important role of polycyclic aromatic primary amines in the mutagenicity of petroleum substitutes is reviewed. Questions are raised concerning the role of polycyclic aromatic hydrocarbons in the mutagenicity of the neutral fraction of petroleum substitutes.

Direct analysis of organic compounds in aqueous by-products from fossil fuel conversion processes: oil shale retorting, Synthane coal gasification, and COED coal liquefaction

Abstract Whole water samples are injected directly into a gas Chromatograph equipped with a packed Tenax‐GC column. Polar compounds are separated with good resolution under the temperature programming conditions employed. The by‐product water from oil shale retorting contains carboxylic acids in the homologous series ranging from acetic to decanoic acid. Various amides, cresols and phenol are present in trace amounts. Coal conversion by‐product waters also contain carboxylic acids, but in trace amounts (except acetic). Major components among the dissolved organics in coal conversion samples are phenol, o‐cresol, m‐cresol and p‐cresol. Present at lower levels are several other alkyl substituted phenols and napthols.

Embryotoxic and teratogenic effects of aqueous extracts of tar from a coal gasification electrostatic precipitator

Aqueous extracts of tar from a coal gasification electrostatic precipitator were tested for its toxic and teratogenic potential in vitro on embryos of the amphibian Xenopus laevis. The 96-h LC50 and EC50 were determined to be 0.83% and 0.48%, respectively. The developmental stage of normal-appearing exposed embryos is not affected by increasing concentrations of the extract. Embryo growth, however, is significantly reduced at concentrations as low as 0.25%. Motility and pigmentation were effectively reduced relative to controls by extract concentrations of 0.5% and greater. Exposed embryos are shorter and stockier than controls. Malformations of head, eyes, viscera, and spine are common, and cartilage formation is abnormal. The epidermis is often hyperplastic, and large blisters occur over the somatic surface. The severity of abnormal development is directly related to the concentration of the toxicant to which the embryos are exposed. Chemical analysis shows that the aqueous extracts contain phenols, furans, monoaromatic and diaromatic hydrocarbons, and mono- and diazaarenes and/or monoaromatic amines.

Analytical and biological analyses of test materials from the synthetic fuel technologies III. Use of sephadex LH-20 gel chromatography technique for the bioassay of crude synthetic fuels

To determine the health effects associated with newly emerging energy technologies, we have subjected a group of synthetic fuels to mutagenicity evaluation, using the Ames Salmonella assay. Coupling of chemical fractionation to the mutagenicity assays was necessary. Fractions obtained by use of Sephadex LH-20 gel chromatography on crude-coal-derived oils and shale oil were tested for mutagenicity with strain TA98 (with Aroclor S9 mix). Mutagenicity results obtained with synthetic fuels were compared with those from a mixture of natural petroleum crude oils. Merits of the Sephadex LH-20 separation technique and precautions in interpreting experimental results are discussed.

Short-Term Bioassay of Complex Organic Mixtures: Part II, Mutagenicity Testing

The feasibility of using short-term mutagenicity assays to predict the potential biohazard of various crude and complex test materials has been examined in a coupled chemical and biological approach. The principal focus-of the research has involved the preliminary chemical characterization and preparation for bioassay, followed by testing in the Salmonella histidine reversion assay described by Ames (1). The mutagenicity tests are intended to (a) act as predictors of profound long-range health effects such as mutagenesis and/ or carcinogenesis, (b) act as a mechanism to rapidly isolate and identify a hazardous biological agent in a complex mixture, and (c) function as a measure of biological activity correlating baseline data with changes in process conditions. Since complex mixtures can be fractionated and approached in these short-term assays, information reflecting on the actual compounds responsible for the biological effect may be accumulated. Thus, mutagenicity tests will (d) aid in identifying the specific hazardous compounds involved and in establishing priorities for further valid testing, testing in whole animals, and more definitive chemical analysis and monitoring.

Mutagenicity of Nitrogen Compounds from Synthetic Crude Oils: Collection, Separation, and Biological Testing

Short-term mutagenesis assays have been used to test complex environmental mixtures, in order to 1) serve as predictors of long-range health effects, 2) guide chemical separation procedures for the isolation and concentration of biologically active materials, 3) identify chemical agents responsible for biological activity, and 4) determine priorities for further, extensive testing. Organic extraction coupled with chemical-class fractionation is a prerequisite for most of these assays.