Fact Sheet

TSCA Experimental Release Application

Approved for Bradyrhizobium japonicum Strains

On May 6, 1998, the Office of Pollution Prevention and Toxics approved the first three TSCA Environmental Release Applications (TERAs) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). These three strains were given the EPA tracking designations R98-0001, R98-0002 and R98-0003. The TERAs pertain to field trials of three modified strains (Bj 5019, JH 359, and TN 119(12)) of Bradyrhizobium japonicum (B. japonicum), a common soil bacterium which is often used as a commercial seed inoculant (see below). The new strains will be tested on soybean plants at a total of eight sites in Ohio (0.5 acre each) and Wisconsin (0.23 acre each). The studies will evaluate the ability of the modified strains to enhance nitrogen fixation and to compete for nodulation relative to the parent strains (USDA 110 and USDA 110-I) as well as commercial strains. The field trials were set to start in the Spring of 1998, and continue for three years.

Rhizobia and Nitrogen Fixation

Rhizobia are a group of bacteria, encompassing the 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 (this process is 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 perform a process called nitrogen fixation. Nitrogen fixation is the process of converting atmospheric nitrogen 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 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, sore searchers in industry and academia have recently applied more advanced genetic technologies to try to achieve these 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 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 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 TSCA Experimental Release Application (TERA), instead of an MCAN, before initiation of such testing. 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 Bj 5019, JH 359, and TN 119(12)

Each of the three strains was altered to provide different abilities. Base preliminary studies in the laboratory, strain Bj 5019 has the ability to out compete indigenous Bradyrhizobia for nodulation of soybean plants; strain JH 359 has the ability to fix higher levels of nitrate than its parent strain; and finally, strain Tn 119(12) has the ability to both out compete for nodulation,and to fix higher amounts of nitrogen. In addition, all three strains have introduced antibiotic resistance genes, which are used as markers to identify the strains in laboratory and environmental studies.

Summary of the Risk Assessment

The principle issue examined by EPA during its review of these TERAs concerned the presence 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, for 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 out of the three modified recipient strains). In addition, the naturally occurring parent bacteria are not human inhabiting microorganisms. The genetic modifications which have been made 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.

Additionally, the Agency reviewed the effects of these microorganisms on "non-target" plants. Historically, different species of rhizobia have been grouped into categories known as "cross-inoculation groups" which consist of the particular rhizobial species and those leguminous plants which it typically nodulates. The interaction between a rhizobial strain and a plant is very specific, being controlled by both various genes within the microorganism and by chemicals produced by the plant. For this reason, the number of legumes a particular rhizobial species is capable of nodulating is limited. When testing a strain of rhizobia as a seed inoculant,there is usually a specific, agriculturally important crop plant (the "target" plant) with which it is tested. However, there are some cases when the particular rhizobial species can nodulate other leguminous plants ("nontarget") within or outside its normal cross-inoculation group. In the case of B. japonicum, nodulation can occur not only on soybeans, but also on sirato, a pasture legume of great importance in other countries, but with only limited use in the U.S. as reserve winter pasture. Because sirato is not an important crop in this country, it was decided that there was minimal concern for any potential effects on nontarget plants.

Conclusion

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. The host bacteria, B. japonicum USDA 110/I-110, are well characterized and have a long history of agricultural use with no reports of human, animal, or plant pathogenicity. In addition, EPA has previously allowed field trials of several other intergeneric strains of B. japonicum with similar constructs, with no adverse consequences. The approval letter to the submitter identifies 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 information which may be required for a Microbial Commercial Activity Notice (MCAN) would include: the potential effects of the bacteria on non-target species (plants other than soybean which may host the bacteria); greater detail in the genetic characterization of the strains; detection methods for the microorganisms in the soil; and the benefits of the microorganisms.

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.