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TSCA Experimental Release Application (TERA) for modified Gordonia terrae, R-13-0001 and modified Rhodococcus jostii, R-13-0002

On April 24, 2013, the Office of Pollution Prevention and Toxics approved the TSCA Experimental Release Applications (TERAs) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERAs, submitted by the US Army Engineer Research and Development Center, Vicksburg, MS and US Army Corps of Engineers, Seattle, WA, involves field trials of modified strains of Gordonia terrae and Rhodococcus jostii. The strains were given the tracking designations of R13-0001 and -0002, respectively. These microorganisms will be used in a field demonstration of bioaugmentation to enhance hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation in contaminated groundwater.

Proposed Use and Field Study

The proposed bioremediation demonstration will be performed in three phases. Phase I, which has been completed, consisted of field site characterization and laboratory testing to select a suitable bioaugmentation culture(s) and optimization of conditions that facilitate growth, RDX-degrading activity, and cell transport under field conditions at the Umatilla Chemical Depot (UMCD) in Hermiston, OR. The UMCD has been identified as an ideal demonstration site. Explosives-contaminated sludge and liquid wastes generated at the UMCD Washout Plant were discharged to unlined lagoons, where infiltration through ~ 50 ft of unsaturated site soil to groundwater resulted in a ~ 300 acre RDX groundwater plume. Maximum RDX and TNT concentrations within the plume have been ~ 200 μg/L and 70 μg/L, respectively, in recent years. A groundwater pump and treat facility with activated carbon adsorption began full-time operations at the site in 1997. Treatment efficiency of the pump and treat facility has significantly declined over the years, which has prompted consideration of bioremediation technology for its potential to optimize the existing remedy.

Phases II and III are the field tests which are the subject of TERAs R-13-0001 and R-13-0002.

A mixed microbial culture of Gordonia terrae KTR9 pGTK2:Km+ (R13-0001) and Rhodococcus jostii RHA1 pGTK2:Km+ (R13-0002), capable of aerobically biodegrading RDX, will be injected into the aerobic RDX plume. For Phase II, in July 2013, a forced-gradient culture transport experiment will be conducted by injecting a tracer/culture (≈ 106 cells/ml) solution in the bioaugmentation well with extraction pumping and sampling from a down-gradient well. This test will confirm that field test parameters previously determined in 2012 are suitable for distributing cells at the field-scale. Phase III, planned for 2014, consists of a bioaugmentation demonstration with subsequent push-pull tests to obtain field-scale performance data on bioaugmentation culture transport, viability, and xplA gene transfer, as well as RDX degradation rates. A field-scale demonstration of the application of the bioaugmentation plot (≈ 100 m3) will be compared with aerobic and anaerobic biostimulation plots. The demonstration will use low concentration (≈ 10 mM fructose) substrate injections in the aerobic biostimulation and aerobic bioaugmentation field plots and culture injection in the bioaugmentation plot only, followed by high concentration (50-100 mM corn syrup or ethanol) substrate injections in the anaerobic biostimulation field plot. In situ RDX degradation rates will be determined by triplicate push-pull tests in injection wells and down-gradient wells within the treatment plots twice over a ≈ 3 month period. Injections will occur at least 190 feet below the surface. The field-scale demonstration will assess bioaugmentation culture transport, viability, xplA gene transfer to other indigenous microorganisms, RDX degradation rates over time, as well as generate data needed to perform a cost-benefit analysis for implementation of the aerobic bioaugmentation approach for RDX treatment at UMCD.

Conclusions of EPA's Review

EPA decided to grant these TERA submissions on June 17, 2013. The potential hazards posed by the submission microorganism in the proposed field tests are low, as are potential exposures. Thus, the small-scale testing of this microorganism for RDX degradation in this contaminated aquifer poses low risk to human health and the environment.

As a condition of EPA approval of these TERAs, quarterly progress reports including descriptions of field methods and preliminary findings for the activities described in the TERA will be provided to the EPA. Microbial data submitted will include polymerase chain reaction (PCR) results and colony forming units (CFUs) per milliliter of groundwater. In addition to the quarterly reports, detailed reports including all field, laboratory, and microbial analysis methods and results will be provided upon completion of Phase II and again upon completion of Phase III, as described in the TERAs.

In addition to the required reporting of Phase II and III data, EPA recommended monitoring of metabolites of concern to assess on-site subsurface concentrations and the potential for aerobically-generated metabolites that migrate beyond the injection well locations: 4-nitro-2,4-diazabutanal (NDAB) and formaldehyde could be monitored. If TNT is present at the test area, then monitoring for 2-amino-4,6-dinitrotoluene, 2-nitro-4,6-diaminotoluene, and methylenedinitramine (MEDINA) may also be of value. The periodicity of metabolite sampling should allow an evaluation of the potential for effects on ecological and human receptors at current and/or future sites. Finally, data-gathering to assess the potential for spread of the introduced bacteria beyond the injection site should be assessed. This data may be beneficial in addressing concerns that may arise in future field tests.

For a copy of the original nonconfidential TERAs and the nonconfidential approval letter, please contact the TSCA Non-Confidential Information Center (NCIC) by phone at (202) 566-0280, or by fax at (202) 566-9744.