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VERIFICATION OF BIOREMEDIATION OF PAH CONTAMINATED SOILS THROUGH MONITORING BIOLOGICAL METABOLITES OF PYRENE

Typically, disappearance of parent compound has been used as a measure of site remediation. Loss of parent compound, however, may be due to physical/chemical processes of phase transfer including: volatilization, leaching, or sorption, rather than destruction processes including biodegradation, photodegradation, and/or hydrolysis.

Because biodegradation is considered to be the major pathway for PAH destruction in soil and water environments, a proposed method for the tracking, monitoring and verification that parent compounds have degraded is to monitor the production of known metabolites of the PAH parent compound(s). Limited research has identified metabolites of different PAHs such as pyrene, naphthalene, and phenanthrene.

Through monitoring of metabolites and the use of a mass balance, one can verify that biodegradation is indeed taking place within the soil. However, there is very little information regarding PAH metabolite production and monitoring in complex soil systems. The majority of available information has been generated in the laboratory using pure culture methods.

Soil from the land farming area of the Champion International Superfund wood preserving site at Libby, Montana, was added to biometer flasks and spiked with a known amount of 14C Pyrene. KOH was used within the biometer flasks to trap 14CO2, and a polyurethane plug was used to trap volatile metabolites and other volatile organics. Samples of KOH were analyzed periodically and used to monitor mineralization of the 14C Pyrene. Soil samples were sacrificed at 30 and 60 days.

The soil samples, as well as the polyurethane plugs, were processed through a sequential extraction procedure which involved (1) a water extract to determine water soluble metabolites, bio-available compounds, and also to act an indicator of leaching potential; (2) a solvent extraction performed for evaluation of soil-associated PAHs as well as metabolites; and (3) an MIBK (methyl iso-butyl ketone) extraction procedure employed to determine the fraction of parent compound bound to the soil humus/humin fraction (not used on polyurethane plugs). Items (1) and (2) are the focus of this report.

Samples were then analyzed by reverse phase HPLC. The presence and/or increase in metabolites of pyrene, as well as reduction in parent compound concentrations were determined. Fractions from HFLC analysis were collected and analyzed for 14C, which were then used in the identification of metabolites and also as input for the mass balance . Verification of metabolites collected was made by comparison to: (1). previous research, (2) 14C fractions, (3) GC/MS, (4) IR Spectroscopy, and (5) NMR.

Results were used to generate a 14C mass balance in order to account for all 14C pyrene added to the soil. Also, the production of pyrene metabolites showed that microbial degradation of pyrene had indeed taken place.

Key words: pyrene, polycyclic aromatic hydrocarbons (PAH), bioremediation, biometer, metabolite, mass balance, creosote