EXPERIMENTAL SECTION
Reactor Design.
Laboratory scale reactors were comprised of 0.5 litre Mason jars containing 100-200g Guelph sandy loam soil, 1 g commercially available peat moss and the target compounds. The target compounds 2H-tetradecane and phenyldodecane were added to the soil dissolved in 1-2 ml of acetone giving final concentrations of 200-1300 µg/g. Where diesel fuel was employed, it was added directly to the soil and mixed well. Except in the case of nitrogen-purged controls, the Mason jar lid inserts were replaced with circles of filter paper to allow the passage of air into the jar. Nitrogen- control reactors were purged with nitrogen and sealed with a standard Mason jar lid. Kathon® controlled reactors were treated with 46 µg/g biocide. The reactors were kept hydrated and incubated at either room temperature (25 ºC) or refrigerated (4 ºC) depending on the regimen required. Soil samples (2 grams) removed from the reactors were flushed with nitrogen and stored at -20 ºC prior to extraction and analysis.

Extraction and Analysis of Soil.
Extraction of the non-polar target compounds and diesel fuel from soil was done by sonication using dichloromethane as a solvent (USEPA SW-846). Polar lipids were extracted from the soil using a Bligh & Dyer (chloroform/methanol/water) extraction (Kates 1972).

The target compounds in dichloromethane extracts of soil were determined by combined gas chromatography-mass spectrometry (GCMS). Quantification was done using 2H-dodecane-d26 as an internal standard. Under these conditions fatty alcohols and aldehydes are also extracted and are detectable if present. Fatty acids present in chloroform/methanol/water extracts of soil were analyzed as the methyl esters by GCMS employing the same chromatographic conditions as above.

Identification of Neutral and Polar Lipids.
Complex lipids present in chloroform/methanol/water extracts of soil were fractionated by thin layer chromatography using silica gel plates eluted with polar lipid and neutral lipid solvent systems as described (Kates 1972). The compounds were visualized by ultraviolet light or by charring with sulphuric acid. For analysis, the thin layer bands were scraped and the lipid material converted into fatty acid methyl esters.

RESULTS AND DISCUSSION
Evidence of Target Compound Depletion.
In initial experiments, reactors were spiked with 400 µg/g each 2H-tetradecane and phenyldodecane. The soil had a moisture content of about 20%. The reactors were incubated under aerobic conditions at 4 ºC and 25 ºC. One set of reactors was flushed with nitrogen, sealed and incubated at 25 ºC. The soil was periodically sampled over a three week period, extracted and analyzed by GCMS.

The results, summarized in Figure 1, show that under aerobic conditions at room temperature, both target compounds are completely degraded within about 16 days. The initial rate of degradation of deuterated tetradecane displayed apparent first order kinetics with a half life of about 87 hours. There was a reproducible time lag of about 48 hours in the degradation of phenyldodecane during which time the rate was negligible. The period of maximum degradation for this compound occurred during 92 and 192 hours.

FIGURE 1: Degradation of 2H-Tetradecane and Phenyldodecane in Laboratory Scale Reactors


Evidence for Biological Degradation.
Evidence that the loss of target molecules from soil containing reactors was the result of biological action was collected in two ways. First, experiments were conducted to examine the effect of metabolic inhibitors on the reactor. Second, the reactors were examined for metabolic products.

As illustrated in Figure 1, the degradation of 2H-tetradecane and phenyldodecane was markedly slower in soil reactors under a nitrogen atmosphere. About 50% of the material was degraded during the three week test period. No loss of either target compound was detected in the 4 ºC reactors before 200 hours of incubation. During the subsequent 300 hour period, the concentrations of both compounds decreased to 60-70% of their starting values.

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