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Petroleum Hydrocarbon Efficacy & Aquatic Toxicity Testing of HC-2000 in Freshwater and Soil Environments

Powdered Clay Slurry Bioremediation
Efficacy Tests

Efficacy Biodegradation Bench-Scale Tests


This paper presents range finding aquatic toxicities and biostimulation efficacy testing of HC-2000 to provide guidance on the appropriate selection and application of HC-2000 in soil and freshwater environments.

Parallel efficacy and toxicity tests were also conducted on select NCP listed agents including Micro-blaze, Biosolve, and F-500 (Fuel Buster) for comparison purposes.

Current NCP List testing protocols address bioremediation agents efficacy of crude oil degradation in saltwater ocean environments. Over 60% of fuel spills occur in inland freshwater environments. Microbial populations in inland environments differ from marine environments. In addition, refined products have different ratios of alkanes and aromatic compounds than crude oil. EPA is currently proposing revisions to include freshwater and freshwater organisms (10), albeit do not specifically address biostimulation of indigenous soil microorganisms that vary at each site.

Remtech's range finding bioremediation efficacy testing was modified from a Test Protocol (soil slurry reactors) for Evaluating the Capability of Indigenous Microorganisms to Degrade Target Compounds cited in John T. Cookson, Bioremediation Engineering - Design & Application, McGraw-Hill, Inc., 1995 (1). Range finding aquatic toxicity screens were performed using fathead minnows and an abbreviated ASTM E729-96 method.

Biostimulation versus Enhanced Bioremediation

Numerous publications conclude that very little scientific data demonstrates that enhanced degradation (microbial addition) works on full-scale cleanups. According to peer-reviewed literature, bioaugmentation appears to have little benefit for the treatment of spilled petroleum hydrocarbons in the environment. Microbial addition has not been shown to work better than nutrient addition alone in many filed trials (2). Biostimulation has been proven to be a promising tool to accelerate and enhance the natural attenuation process.

Range Finding Bioassays

Remtech conducted range finding aquatic toxicity tests (abbreviated ASTM E729-96 method) using freshwater Fathead minnows and an untreated control for HC-2000 and Micro-blaze. Test concentrations were prepared from 6% solutions of both products. Organism fatalities were recorded during a 96-hour exposure period. LC50s were calculated using Trimmed Spearman-Karger Method (4).

The LC50 for HC-2000 was determined to be 5,764 ppm (11,528 ppm at 3%) and Micro-blaze 1,414 ppm. (8) Published aquatic 48-hour toxicity tests for Micro-blaze by Aqualabs (LC50 - 1,390 ppm for Silverside, (11)) compared favorably to Remtech's range finding test results considering variation in species toxicity and the additional exposure time.

Biostimulation Efficacy Tests

Indigenous bacteria bioremediation tests were conducted to provide relative comparisons for select listed compounds on the NCP List (Micro-blaze, F-500, and BioSolve) and HC-2000. Total Petroleum Hydrocarbons (TPH) concentrations were measured with EPA Method 9071B at independent testing labs. The test protocol was a modified version of the Test Protocol for Evaluating the Capability of Indigenous Microorganisms (1) and using EPA inorganic nutrient preparation (, 40 CRF, Part 300 Appendix C, (5)) and untreated control. This CSTR (Continuously Stirred Reactor) method facilitates non-sacrificial reactor sampling at increasing time intervals (especially in view of the fact that the biosurfactants present in HC-2000 prevents adsorption of petroleum hydrocarbons to the glass reactor sidewalls). Each reactor was charged with distilled water and either diesel fuel or crude oil. Crude oil was obtained from EPA (ANS521 Crude, recd. 20 BERC 4/27/93, 9 April 2010 ELH, outside canister 485380, ATCC 1081 Univ. Blvd., Manassas, VA 20110 (6)). EPAs inorganic nutrient control and an untreated control were run in parallel with each agent.

Percent biostimulation efficacy tests were conducted in glass erlenmeyer flasks with magnetic stirrers covered with foil tops or stoppers. Native soil heterotrophs were obtained from sandy CLAY or powdered clay. Nutrient concentrations were not checked in source soils. Note that crude oil degradation tests with the EPA nutrient and non-treated controls had a tendency to adsorb on reactor sidewalls due to the lack of surfactants. Dawn detergent was injected to these controls in a effort to prevent adsorption, albeit was unsuccessful. The degradation efficacy percentages of the controls are therefore elevated (to high) and would infer that HC-2000 degradation performance compared to controls are higher than reported.

HC-2000 bioremediation efficiency outperformed Micro-blaze, F-500, BioSolve, and EPAs synthetic inorganic fertilizer control for the petroleum hydrocarbons tested. In addition, HC-2000 proved to be less toxic than that the other products tested by having the largest increase in total heterotrophic bacteria counts following inoculation. Note that plate counts actually decreased following inoculation with Biosolve and F-500. HC-2000 has a freshwater 96-Hr aquatic toxicity that is 4.1 times less than Micro-blaze.


     1.     Cookson, John T., Bioremediation Engineering - Design & Application, McGraw-Hill, Inc., 1995.

     2.     Venosa, Albert d., et. al, Literature Review on the Use of Commercial Bioremediation Agents for Cleanup of Oil-Contaminated Estuarine Environments, USEPA, National Risk Management Research Laboratory, Cincinnati, OH, July, 2004.

     3.     Nikolopoullou, M., Kalogerakis, N., Enhanced Bioremediation of Crude Oil Utilizing Lipophilic Fertilizers Combined with Biosurfactants and Molasses, Marine Pollution Bulletin 56 (2008) 1855-1861., Elsevier Ltd, 2008.

     4.     Trimmed Spearman-Karger Estimation of LC50 Values in Toxicity Bioassays, Montana State University, Environmental Science Technology, 1978.

     5.     40 CFR, Part 300, Appendix C, 4.0 Bioremediation Agent Effectiveness Test, 7-1-003 Edition.

     6.     ANS521 (Alaska North Slope) crude oil, Crude Oil - ANS521 Crude, recd. 20 BERC 4/27/93, 9 April 2010 ELH, outside canister 485380, ATCC 1081 Univ. Blvd., Manassas, VA 20110.

     7.     Ryckman, M.D., Remtech Engineers, An Evaluation of Fire Suppression & Bioremediation Products for Spill Control in Non-Fire Situations, November, 2000.

     8.     Ryckman, M.D., Remtech Engineers, Range Finding Freshwater Aquatic Toxicities of HC-2000 & Micro-blaze, June, 2010.

     9.     Venosa, Albert D., Proposed Changes in the Protocol for Listing Bioremediation Agents on the NCP Product Schedule, USEPA, National Risk Management Research Laboratory, Cincinnati, OH, PowerPoint Presentation, 2010.

     10.     Nichols, W.J., Venosa, Albert D., Summary of the Literature on the Use of Commercial Bioremediation Agents for Cleanup of Oil-Contaminated Environments, USEPQ National Risk Management Research Laboratory, Cincinnati, OH, PowerPoint Presentation

     11.     Coastal Bioanalysts, Inc, Micro-Blaze 6% solution NCP Toxicity Bioassay Aquatic Toxicity Test Results, M. Beryllina, 1390, January 7, 1998.

     12.     Federal Aviation Administration, Screening of Fire Fighting Agents, Applied Research Associates, Inc., Tyndall AFB, FL, 2003., MicroblazeOut+, 949 ppm.