On December 6, 2003, the Office of Pollution Prevention and Toxics approved the TSCA Environmental Release Applications (TERAs) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERAs, submitted by the University of Tennessee and Micro Systems Technologies, LLC, involve field trials of two intergeneric microorganisms, Pseudomonas fluorescens (P. fluorescens) strains HK44 and 5RL. The HK44 strain was given the tracking designation of R-04-01 (previously reviewed under submissions R02-0001 and P95-1601). The strain 5RL was given the tracking designation of R-04-02, and has not been previously reviewed. The microorganisms will be tested at Fort Carson U.S. Air Force Reserve Station and in a cargo-shipping container aboard a ship bound from Thailand to Vancouver, B.C. Canada to examine the competence of these strains of P. fluorescens in detecting and monitoring naphthalene and methyl salicylate. The eventual application of such technology is the incorporation of these microorganisms into an enclosed sampling device for bioluminescent detection of chemicals of concern in air, water, and soil environments. The field trials were set to begin the first week of March 2004.

Bioluminescence and Biosensors

The field tests will be performed using genetically engineered microorganisms (GEMs) in an encapsulated state on a membrane that is incorporated into an enclosed sampling device. The investigation seeks to continue testing the hypothesis that whole cell bioluminescent microorganisms can be used in bioreporter sensor technology for the positive detection, identification, and quantification of chemicals of concern in the environment. Detection of the chemical of concern will occur via observed bioluminescence of the GEM by electronic sensors embedded within the sampling device. Specifically, P. fluorescens HK44 is a whole-cell bioreporter that produces bioluminescent light when exposed to naphthalene, salicylate, or 4-methyl salicylate. If the field tests are successful, P. fluorescens strains HK44 and 5RL will provide a method to detect naphthalene and methyl salicylate compounds in air, soil, or water.

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 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. EPA conducts a review of these submissions to determine whether the intergeneric microorganisms present an unreasonable risk to health or the environment. The Agency can impose regulatory controls under Section 5 of TSCA.

Genetic Characteristics of Strains Hk44 and Rl5

The two strains of P. fluorescens were modified for use in the proposed field tests. P. fluorescens strain HK44 and plasmid pUTK21 are, respectively, the same intergeneric bacterium and genetic element previously reviewed by EPA for use in similar field tests (submissions R02-0001 and P95-1601). P. fluorescens strain 5RL is an intermediate strain used in the construction of strain HK44. The organism contains an introduced naphthalene degradative plasmid (pKA1) modified by transposon mutagenesis (TN4431) to create a lux gene transcription fusion with the gene for salicylate hydroxylose (pUTK21).

Summary of the Risk Assessment

There is low risk associated with the proposed field tests using the microorganisms P. fluorescens HK44 and 5RL in a metal capsule sampling device for detection of naphthalene or methyl salicylate. Members of the species P. fluorescens are ubiquitous in the environment. These microorganisms and their parent microorganisms are not pathogenic or toxic to humans. There are instances of opportunistic infection in plants and animals but no data for frank pathogenicity.

The genetic construction of the submission microorganisms HK44 and 5RL may be of concern due to the inclusion of a gene that encodes resistance to tetracycline. The use of antibiotic resistance genes of clinically important antibiotics is discouraged for microorganisms to be released to the environment, especially those on mobile genetic elements. However, for the proposed field tests using P. fluorescens HK44 and 5RL as biosensors, there is essentially no possibility that transfer of this antibiotic resistance gene will occur. There is no direct exposure of the microorganism to any other microorganisms out in the environment. Even in the event of an accidental leakage, destruction, or loss of the sampling device, there would still be a low level of exposure of these cells in the environment.

Conclusion

EPA has determined that the proposed field trials of these microorganisms will not present an unreasonable risk of injury to health or the environment. Additionally, EPA previously allowed field trials of P. fluorescens with similar constructs that resulted in no adverse consequences. In the TERA approval letter, EPA reminded the submitter to avoid the use of antibiotic resistance genes of clinically important antibiotics for microorganisms to be released to the environment, especially those on mobile genetic elements. Further information may be needed if the microorganisms are ever proposed for large scale testing or submitted as Microbial Commercial Activity Notice (MCAN).

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.