We recommend microcosm studies generally only when one must provide site specific chemical degradation rates or when there is considerable skepticism about the biodegradation of contaminant materials. Skeptics should first be directed to the unequivocal evidence found in the literature that biodegradation of fuel hydrocarbons does occur under natural conditions.  Several of the many available references may be provided upon request. If more evidence of bioremediation of fuel hydrocarbons is required than the tests described herein (endpoint, kinetic assay, comparative population counts, etc.) then a microcosm study using site-specific aquifer materials and contaminants may be undertaken by the MiL, inc. for its clients.  Column studies, Shelby tube tests and “kettle” studies can be provided upon request.  However, the slurry assay is often requested by agencies and is generally the most cost effective particularly when preceded by our biofeasibility package which can determine setup conditions before embarking on these more involved tests.

Slurry Testing Assay

In general accordance with procedures presented in “Guide for Conducting Treatability Studies Under CERCLA, Biodegradation Remedy Selection - Interim Guidance” (EPA/540/R-93/519a), slurry testing is conducted to evaluate biodegradation under four conditions as follows:  1) Abiotic Control whereby biologic processes are minimal or halted; 2) Unamended conditions utilizing unmodified Site soil and groundwater to simulate natural (unenhanced) biodegradation; 3) Oxygen Enriched conditions whereby Site groundwater is oxygenated to stimulate biological activity; and 4) Oxygen and Nutrient Enriched conditions whereby Site groundwater is enriched with oxygen, nitrogen, and phosphorus to stimulate biological activity.  Results of this test are frequently used to confirm that in situ bioremediation is (or is not), occurring at the Site.
 

General Test Procedure:

1.  Bubble oxygen [Puritan-Bennett oxygen concentrator is employed] through the Site groundwater at 3.0 l/min for 30 minutes to oxygenate.

2. Spike Site groundwater with contaminant hydrocarbons or chlorohydrocarbons to a final concentration determined by site contaminant conditions as previously defined.

3. 64 serum vials with Teflon-lined seals, or equivalent containers, are divided into four groups of 16.  Container capacities must be determined by our laboratory, based in part, on the nature and types of chemical analyses to be performed at each time step (see below)

               -Abiotic Control #1 [sequential vial number]

               -Unamended #2 [sequential vial number]

               -Oxygen Enriched #3 [sequential vial number]

               -Oxygen and Nutrient Enriched #4 [sequential vial number]

 
4. To each of the four groups of containers (16 containers per group), site soil is added and site groundwater prepared in Steps 1 and 2 is added in a ratio of (1:4 w/v).  Soil and groundwater volumes are maintained the same for all 64 containers.  An addition of hydrogen peroxide or other form of oxide [determined in consultation with client] as well as nitrogen/phosphate is added to two groups of containers as summarized below:

             -Abiotic Control: site soil and site groundwater prepared in steps 1&2

             -Unamended: site soil and site groundwater prepared in steps 1&2

             -Oxygen Enriched: site soil, site groundwater prepared in steps 1&2 and hydrogen peroxide in the site groundwater

             -Oxygen and Nutrient Enriched: site soil, site groundwater as prepared in steps 1&2, 1% w/ hydrogen peroxide or appropriate equivalent and nitrogen/phosphate in stoichiometric ratios and balanced as determined in the MiL, inc.'s Biofeasibility assay

5. All containers are agitated at moderate speed for the remainder of the test.  The 16 Abiotic Control containers are placed at 5^oC and the 48 containers in the other 3 groups at 15 ^oC to 25 ^oC [depending upon site conditions] for the duration of the assay.

6. Within 30 minutes following completion of Step 5 (time t_o), four containers from each group are sacrificed [harvested] and centrifuged to separate particles from water.  The supernatant from three containers in each group are analyzed typically for TCL volatile organic compounds.  The soil from the three containers in each group is also analyzed for the same components.  The water fraction from the fourth vial of each group is analyzed for dissolved oxygen content.  Supernatant and soil from the fourth container of each group is analyzed by Total heterotrophic plate counts modified method 9215C under aerobic and/or anaerobic conditions [depending upon degradation scenario].  A more informative approach is to employ the MiL inc.’s comparative population enumeration assay at this point.

7. After one week of incubation (time t₁), four more containers from each group are harvested and step 6 is repeated.

8. After two weeks of incubation (time t₂), four additional containers from each group are harvested and step 6 is repeated.

9.  After four weeks of incubation (time t₃), four containers from each group and harvested and step 6 is repeated. This protocol calls for:

                -48 analyses of soil to appropriate chemistry

                -48 analyses of water for appropriate chemistry

                -16 analyses of dissolved oxygen content in waters

                -32 or 64 analyses of Total aerobic and/or anaerobic counts 

An Option may be the following when approved by the regulatory oversight:

 
Composite Sampling during test results in a reduction in analyses                                                 

            -16 analyses of composite group soil for chemistry

            -16 analyses of composite water for chemistry

            -16 analyses of dissolved oxygen content in waters

            -32 or 64 analyses of Total aerobic and/or anaerobic counts

Data Analysis and Interpretation

Microbial population data and TCL volatile [and semivolatile if desired] results for soil and supernatant samples at t_o through t₃ are presented on tables and graphs to enable comparison of the results.   Reductions in the concentrations of spiked compounds [or client’s free phase material] which may be present in the Site soil will be calculated in absolute terms and relative to the abiotic control.  The mean concentrations for each group at each time step are used for these calculations.