New Markers For Monitoring The Biodegradation of Petroleum Fuels in Soil

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Peter Child, Louise Vinnai (Investigative Science Inc., Burlington, Ontario, Canada), Wendy Mortimer (Bell Canada, Toronto, Ontario, Canada),
James Ford (Investigative Science Incorporated).

ABSTRACT: We have investigated the use of per-deuterated and phenyl-labeled alkanes as indicators of the biological degradation of petroleum fuels. In laboratory scale reactors containing soil, peat moss and the target compounds, both compounds were completely degraded at a similar rate to diesel fuel within 16 days at room temperature. Degradation slowed markedly in a nitrogen purged reactor and was virtually stopped by incubation at 4 ºC and by the biocide Kathon®.

Corresponding with the disappearance of the starting materials from the reactors was the appearance of fatty acids possessing the labels present in the original compounds. 2H-tetradecanoic-d27 acid arising from 2H-tetradecane-d30 and 12-Phenyldodecanoic acid formed from phenyldodecane were identified. The fatty acid metabolites were found to be incorporated into complex polar lipids and were not found as free acids.

The results of this study demonstrate that 2H-tetradecane and phenyldodecane in soil are both biologically degraded, converted into the corresponding fatty acids and incorporated into higher lipids. The labels in both target molecules survived the metabolic process allowing the metabolites to be traced directly to the parent molecules. The findings show that these labeled compounds can be used as sensitive monitors for the biodegradation of petroleum fuels.

INTRODUCTION
It is generally recognized that bioremediation, the biological degradation of organic pollutants, is a powerful tool for the cleanup of petroleum contaminated water and soil. Without proper verification, however, losses attributed to biodegradation may actually be due to failure to account for sampling variability, analytical variability or abiotic losses such as sorption, leaching or volatilization (Madison, 1991).

One of the most reliable ways of verifying that biodegradation has occurred is to demonstrate the appearance of metabolites that can be unequivocally traced to the pollutant (Madison 1991, Shannon and Unterman 1993, Beller et al 1995) . For most petroleum fuels, however, this has proven to be difficult. This is in part because the main expected products; carbon dioxide, water, acetate and fatty acids (Atlas 1981) are common in the environment and are difficult to trace to the fuel.

Aggarwal and Hinchee (1991) have explored the use of stable isotopes to link the production of carbon dioxide to the biodegradation of petroleum fuel in contaminated soil. The approach was based on the premise that the ratio of the stable isotopes 13C and 12C in fuels is different than that of carbon dioxide present in the air today. Others (Beller et al 1995) have identified products of the anaerobic biodegradation of alkyl benzenes in groundwater contaminated with gasoline.

A different way to link metabolic products to specific fuel components is to spike the fuel with parent molecules possessing recognizable labels that can be traced to the daughter products. Several reports, reviewed by Atlas (1981), describe the addition of radioactively-labeled precursors, such as 14C-labelled hexadecane, to spilled fuels followed by monitoring of the 14C-labelled carbon dioxide produced. This approach has also been widely used to investigate the degradation of herbicides and pesticides in soil (Fuhr 1985). These experiments provide firm evidence of biological degradation of the substrate but possess obvious containment and licensing problems when used in the field.

In the present work, we have investigated the biodegradation of phenyldodecane and fully-deuterated tetradecane, analogues of n-dodecane and n-tetradecane, typical of the straight chain alkanes present in diesel fuel and home heating oil. These analogues were chosen because they are chemically stable, biodegradable and possess readily identifiable mass spectral characteristics. As well they are expected to survive the metabolic process with labels intact and are closely similar to normal components of diesel fuel. These features make the analogues ideal for extending the technology to the field.

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