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TSCA Experimental Release Application Approved for Pseudomonas putida Strains (fact sheet)

Fact Sheet

TSCA Experimental Release Application

Approved for Pseudomonas putida Strains


In 1998, the Office of Pollution Prevention and Toxics approved the TSCA Environmental Release Applications (TERAs) R98-0004/5 under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERAs, submitted by the National Explosives Waste Technology & Evaluation Center (NEWTEC) and the Oak Ridge National Laboratory, involve field trials of two modified strains of Pseudomonas putida (P.putida). P. putida strain RB1500 was given the tracking designation R98-0004, and strain RB1501 was designated R98-0005. Both strains will be tested on a two acre site in South Carolina, in order to determine whether they will produce light in the presence of trinitrotoluene (TNT), and whether the light produced can be used as a practical means for the detection of TNT in soil. The eventual application of such technology would be for the detection of land mines and unexploded ordnance in soils world-wide. The field trials were set for two days in October and December of 1998.

Land Mines and Unexploded Ordnance (UXO)

It has been estimated that there are 70 million land mines presently active in the world, with more added every day. In addition, many areas are endangered by unexploded ordnance (UXO) which include bombs, grenades, shells, raw explosives and other related devices which were fired, but did not detonate. Such devices can remain active for many years after firing. The key to detecting land mines and UXO is detecting the presence of TNT in the soil which has leaked from the devices. If successful, the microorganisms which are the subjects of these TERAs will provide an inexpensive means to detect TNT in the soil.

Regulatory Background

The EPA requirements concerning microbial products subject to TSCA (15 U.S.C. Section 2601, et seq.) are set forth in "Microbial Products of Biotechnology; Final Regulation under the Toxic Substances Control Act" (62 FR 17910 (April 11, 1997)), and are codified at 40 C.F.R. Part 725. Microorganisms resulting from the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera (intergeneric microorganisms) constitute "new" microorganisms subject to TSCA Section 5 notification requirements. Persons who manufacture, import, or process intergeneric microorganisms for commercial purposes subject to EPA jurisdiction under TSCA, are required to submit a Microbial Commercial Activity Notice (MCAN). Persons conducting commercial research and development activities may submit a TERA, instead of an MCAN, before initiation of such testing, instead of an MCAN. EPA conducts a review of these submissions to determine whether the intergeneric microorganisms may present an unreasonable risk to health or the environment. The Agency can impose regulatory controls under section 5 of TSCA.

Genetic Characteristics of Strains RB1500 and RB1501

Both microorganisms were modified from P. putida strain KT2440. The strains were modified so that in presence of TNT, strain RB1500 will bioluminesce (produce light) while strain RB1501 will fluoresce (reflect light which shines on it). The light thus produced can be detected with the proper instruments. In this manner, the location of land mines and UXO can be determined.

Summary of the Risk Assessment

P. putidais a common, wide-spread, soil bacterium that is not pathogenic to plants or animals. Since the genetic modifications involved in creating strains RB1500 and RB1501 are expected to have no effect on pathogenicity (i.e., on the ability of the microorganisms to infect and to cause disease), it is believed that these strains present a low ecological hazard for small-scale field testing.

A major issue in the EPA review of the TERAs was the use of the specific antibiotic resistance marker genes. These genes can be of concern if they can be transferred to other microorganisms which are human, animal, or plant pathogens, for which these antibiotics are used as treatment. Because kanamycin/gentamicin, and penicillin/ampicillin have wide-spread use in such treatment, there is a concern that these resistance genes could be transmitted to other microorganisms in the environment which may be human, animal or plant pathogens. EPA believes, however, that these concerns are mitigated for the field trial because:

  • The field trials will occur on a small site with limited access;
  • The microorganisms will only be applied to the soil on two days, over a two hour timespan. Application will only occur when the winds are below 5 miles per hour, in order to reduce the chance of the microorganisms spreading through the air;
  • All steps from growth through application of the microorganisms will be performed aseptically (no contamination or spread of the microorganisms) and all equipment will be disinfected after use;
  • Because P. putidais not a human or animal pathogen, it is not expected to be found in the same ecological areas as such pathogens. Therefore, the two strains are not expected to come into contact with pathogenic microorganisms, thus mitigating the risk of spreading the antibiotic resistance genes to such microorganisms;
  • Although P. putidais naturally found in the soil, these two strains have been grown in the laboratory, and have adapted to its environment. Therefore, the two strains are not expected to survive long outside of the laboratory, in the soil.


EPA has determined that the proposed small scale field trials of these intergeneric microorganisms will not present an unreasonable risk of injury to health or the environment. However, the following issues will need to be addressed in the event of a future submission of these microorganisms for commercial use in a microbial commercial activity notice (MCAN). These issues have generally been a concern for EPA in its evaluation of environmental releases of other intergeneric microorganisms:

  • Ensure that the added genetic material is stably inserted into the submission microorganisms, so there is less chance of the genetic material transferring to other microorganisms in the environment.
  • Avoid the use of the antibiotic resistance genes associated with antibiotics of clinical value (penicillin/ampicillin and kanamycin/gentamicin).

The following concerns for the land mines and leftover chemicals in the soil were raised by the South Carolina Department of Health and Environmental Control(DHEC):

  • The land mines should be removed when the field tests are completed.
  • Any data on leftover TNT and its breakdown products in the soil should be sent to theDHEC.
  • Any hazardous release of TNT and its breakdown products should be contained and removed from the soil.

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.