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TSCA Environmental Release Application (TERA) for Bradyrhizobium japonicum (B. japonicum)

On May 17, 2000, the Office of Pollution Prevention and Toxics approved a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) under the biotechnology regulations promulgated under TSCA. The strain was given the EPA tracking designation of R-00-01. The TERA pertains to field trials of a mutant strain (Bj 61A273KS) of Bradyrhizobium japonicum (B. japonicum), a common soil bacterium which is used as a commercial seed inoculant (see below). This strain will be tested on soybean plants at a total of eight sites (0.25 to 0.5 acres each). The studies will evaluate the competitive ability of the strain to enhance root-nodulation, improve nitrogen fixing capability and enhance yield. The field tests were set to start in May 2000 and continue three years.

Rhizobia and Nitrogen Fixation)

Rhizobia are a group of bacteria, encompassing genera Rhizobium, Sinorhizobium and Bradyrhizobium, normally found in soil, which establish mutually beneficial (symbiotic) relationships with legumes. Rhizobia form growths called nodules on the roots of the legumes (a process called nodulation) and provide usable nitrogen to the plants. In return, the plants provide a carbon and energy source for the rhizobia.

Nitrogen is an essential nutrient for healthy plant growth. Although abundant in the air and in organic matter in the soil, plants are unable to use nitrogen in these forms. Conventional methods of providing nitrogen to plants in a usable form include adding nitrogen rich fertilizers to the soil, or inoculating seed (i.e., coating the seed) with bacteria able to fix nitrogen. Nitrogen fixation is the process of converting atmospheric into an inorganic form that plants can use. Nitrogen "fixing" bacteria not only provide nitrogen to the plants they nodulate, but also leave behind excess nitrogen in the soil, potentially reducing the need for nitrogen fertilizers in the next growing season.

Strains of rhizobia vary in their ability to fix nitrogen and to nodulate host plants. Furthermore, the more effective nitrogen fixing microorganisms are not always the most successful at nodulation of plant roots. As a result, seed inoculant companies are constantly searching for more effective and competitive strains of rhizobia. This is ordinarily done by screening native bacteria for the desired trait. This practice has yielded limited results, so researchers in industry and academia have recently applied more advanced genetic technologies to try to achieve such goals.

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 reporting 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.

Summary of the Risk Assessment

EPA's review of the TERA identified a concern for the use of antibiotic resistance marker genes. These genes can be of concern if they are transferred to other microorganisms which are human, animal or plant pathogens, against which these antibiotics are used as treatment. EPA determined that for purposes of the small scale field trials, the risk of transfer of the resistance markers is considered low because the markers are integrated into the bacterial chromosome and are, therefore, stable (i.e., they are not likely to transfer). In addition, the naturally occurring parent bacteria are not human inhabiting microorganisms. The genetic modifications did not affect the ability of the modified strains to infect humans. The potential for transfer of the antibiotic resistance genes to humans through direct contact with the microorganisms during their growth or use is, therefore, extremely low.


EPA has determined that the proposed small scale field trials of the intergeneric microorganism will not present an unreasonable risk of injury to health or the environment. The host bacterium, B. japonicum 61A273KS, is well characterized and has a long history of agricultural use with no reports of human, animal, or plant pathogenicity. The approval letter to the submitter identified concerns which may be raised if the microorganisms are used on a commercial scale and lists additional information which may be needed, should the company decide to commercialize one or more of the modified strains. The additional sites and the new strain in the TERA have not changed the Agency's risk assessment. The approval letter to the submitter requests better characterization of Bj 61A273 if a future MCAN is submitted.

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.