Work this quarter consisted of characterizing soil microbial community assessment and beginning the analysis of the dialysates from SPMDs deployed on site during the previous quarter. TPH analysis of the soils was also completed.

Soil samples were taken for microbial enumeration in March, June, and July. Recently contaminated areas in the north lobe of the spill site (S1, S2, S3, S4) exhibited somewhat higher densities of culturable aerobic heterotrophic bacteria and much higher numbers of bacteria able to use a representative aromatic hydrocarbon (naphthalene) as a sole carbon source, than an uncontaminated, tilled site nearby, or sites which had been contaminated with crude oil eight years previously. Characterization of a subset of the culturable bacteria is ongoing. Soil gas measurements were attempted for the July sample but a complete set could not be obtained because compaction and dryness of the soil prevented accurate measurements. Another sample is scheduled to be taken this fall, in coordination with the rest of the research team.

Initial analysis of dialysates from SPMDs deployed in the north lobe of the spill site accumulated aromatic hydrocarbons (Fig. 1). Analysis by GC-FID showed the presence of hydrocarbons in the C18-24 range. Further analysis by HPLC showed the presence of a number of PAHs. Levels of a number of PAHs present in the SPMDs are currently being quantified by comparison to known standards.

As seen in previous studies of crude-oil contaminated sites in the Tallgrass Prairie (Duncan et al., 1999), the numbers of hydrocarbon-degrading bacteria (here represented by those aerobic heterotrophs capable of growth on naphthalene) were elevated in comparison to samples taken from the tilled, uncontaminated prairie and a nearby aged spill site (SO, Figure 2).

In general, TPH levels decreased over the course of the season, as expected (Figure 3), with the exception of the Oct. 9 Petroflag^TM samples. Soil samples collected shortly before tilling and fertilizer application took place confirm that there was no contamination with brine (Table 1).

SPMDs deployed in the north lobe of the spill site accumulated PAHs from the soil. These results are significant in demonstrating that PSDs, such as SPMDs, can accumulate lipophilic contaminants from soil under field conditions. Further chemical analysis is necessary to determine if other hydrocarbons (e.g. aliphatic) were also absorbed by the SPMDs. Under laboratory conditions, SPMDs have been observed to take up greater levels of hydrocarbons, so the conditions that are required for enhanced uptake of hydrophobic contaminants for soil (e.g., moisture content) need to be determined. Work in progress also involves the determination of bioavailable hydrocarbons from contaminated soils using solid-phase microextraction (SPME) techniques and also the tests examining the bioaccumulation and toxicity of hydrocarbons from these soils by earthworms.

The microbial data confirm the SPMD results that hydrocarbons are bioavailable at this time. Interestingly, a sample taken in October suggests that the number of naphthalene-degraders in the tilled site and the aged spill site may be elevated in comparison to the number in undisturbed prairie. Work to be completed includes evaluating differences in bacterial species composition at the various sites, using traditional methods of characterization as well as molecular genetic techniques, in order to assess with greater sensitivity and precision the microbial response to hydrocarbons, and to distinguish such a response from that resulting from disturbance (e.g., tilling).

Heterogeneity of the distribution of oil prevents strict comparisons between soils sampled at different times using alternative methods of measuring TPH levels, nevertheless, the discrepancy between the Oct. 9 levels measured by Petroflag^TM, and those expected from continuing the downward trend seen with GC and IR, make us hesitant to recommend the exclusive use of Petroflag_ for TPH measurements at this time in spite of its low cost and rapidity.

Plans for the current quarter include:

Table 1: Soil chemistry

* values in ppm

Note: samples were collected before the area was tilled and fertilizer applied. The site designations refer to manipulations that were performed after the soil samples were collected for chemical analysis.

N-t: North Lobe, tilled

N-t, f: North Lobe, tilled and fertilized

S-t: South Lobe, tilled

S-t, f: South Lobe, tilled and fertilized

Prairie 1, Prairie 2: adjacent, uncontaminated control areas