EPA Grant Number: R827015-01-0
Title: Development of Relevant Ecological Screening Criteria (RESC) for
Petroleum Hydrocarbon-Contaminated Exploration and Production Sites
Investigators: Roman Lanno, Will Focht, Anne Cross
Institutions: Oklahoma State University
EPA Project Officer: Bala Krishnan
Project Period: July 1, 2000 to June 30, 2001 (N/C Ext. to December 31,
2001)
Project Amount: $117,463
Research Category: Ecorisk assessment
The major accomplishments of this quarter were:
Completion of microbial analysis of hydrocarbon-contaminated site soils
Soil samples were collected for a period of two years from study sites in the Tallgrass Prairie Preserve that had been contaminated by a crude oil spill in 1999, and from control uncontaminated sites. Aerobic heterotrophic bacteria and naphthalene-degrading bacteria were enumerated by dilution and plating on solid media. The number of aerobic heterotrophic bacteria varied little from site to site or seasonally (Fig. 1, top); the number of naphthalene-degrading bacteria was generally considerably greater in the contaminated sites than in the uncontaminated sites (Fig. 1, bottom), suggesting that some hydrocarbons were bioavailable throughout the two years of the study.
Naphthalene-degrading pseudomonad bacteria isolated from three soil samples collected July 1999 were identified to the level of species through DNA sequencing of 16S rRNA. Different species of culturable pseudomonad bacteria were found to dominate a site depending on whether it had been recently contaminated with crude oil, had been contaminated eight years previously, or was uncontaminated.
DNA was also extracted from the samples for PCR amplification of a portion of the 16S rRNA. The amplified PCR products were separated by denaturing gradient gel electrophoresis (DGGE) in order to compare the bacterial species diversity of contaminated versus uncontaminated sites. The DGGE banding pattern was less complex for samples taken from the recently contaminated sites and showed dominance by a few sequences, suggesting that contamination by crude oil decreased bacterial diversity. The most heavily contaminated sites (J6N-NF and J6N-F) still showed this effect two years post contamination, while the less heavily contaminated sites (J6S-F and J6S-NF) had DGGE banding patterns virtually indistinguishable from that of control sites.
Plans for the current quarter include completion of the assessment of nitrifying bacteria in soil samples and the development and application of the derivation process for Relevant Ecological Screening Criteria to the available data to derive a RESC for hydrocarbons in prairie soil.
Supplemental Keywords: hydrocarbon, soil, soil invertebrates, plants, soil screening levels, bioavailability, exposure assessment, toxicity tests
Figure 1. Numbers of culturable bacteria in the spill sites versus uncontaminated sites. Upper figure: Numbers of aerobic heterotrophic bacteria. Lower figure: Numbers of naphthalene-degrading bacteria. Also indicated are the dates when the spill occurred and tilling and nutrient amendment were performed. J6N-NF (North Lobe, not fertilized), J6N-F (North Lobe, fertilized), J6S-NF (South Lobe, not fertilized), J6S-F (South Lobe, fertilized), J6C (tilled, uncontaminated control site), J6P (not tilled, uncontaminated control area, e.g. undisturbed prairie). North Lobe sites had initially much higher TPH levels than did the South Lobe sites.