ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY MEMO Department of Agricultural Chemistry, Oregon State University, Jeff Jenkins, Specialist SUBJECT: B.t. A HISTORY OF B.t. Bacillus thuringiensis, or B.t., was initially isolated in 1902 in Japan from diseased silkworm larvae. The bacterium was isolated again in 1911 from diseased Mediterranean flour moth larvae. B.t was first used as an insecticide in 1938(1) and used commercially in the U.S. for the first time in 1958, when it was granted an exemption from a food tolerance(2). B.t. is now used on food crops, forage crops, and forests for Lepidopteran control. It is also used for mosquito control. B.t. is from the family Bacillaceae, which has two genus divisions: Clostridium, with 205 species, and Bacillus, with 33 species(3). The species Bacillus thuringiensis has over 19 varieties. Of those, five are currently used commercially: kurstaki, used in forestry pest control; israelensis, used for mosquito control; and aiyawai, morrisoni, and san diego, used in crop production(4). The product being used in Oregon forests is Bacillus thuringiensis var. kurstaki, sometimes referred to as B.t.k. In addition, certain crops, such as cotton and potatoes, have been genetically engineered to produce the toxin found in B.t.k. Field trials are currently underway in the U.S. to evaluate this pest control technique. RECENT USE OF B.t. ON FOREST LAND Between 1958 and 1974, a total of 23 thousand acres of U.S. land was sprayed with B.t. to suppress forest insect populations. In recent years the major forestry use of B.t. has been to control spruce budworm in Maine, where 200 thousand acres were treated during the 1980 spray program(2). Between 1984-1991, 450 thousand acres were sprayed with B.t. in Western Oregon. Lane County, Oregon, accounted for 240 thousand acres of sprayed land during the 1985-86 European gypsy moth eradication program(5). Approximately 2,700 pounds of B.t. (active ingredient) were used in Oregon for forestry and agricultural applications, according to a 1987 survey(6). In April 1992, B.t. was sprayed over 10 thousand acres of North Portland for Asian gypsy moth eradication(7). The Asian moth, first trapped in Oregon 1991, is thought to have entered the country from Russia via ship, where it had defoliated vast forests of both conifers and broad-leaf trees in Siberia(8). The Asian gypsy moth is larger than the European variety and feeds on conifers as well as broad-leafed trees. In addition, female Asian moths can fly 20 to 40 kilometers, unlike the European females, which are flightless(7). This broad range of food preferences and the added mobility of the female make the Asian gypsy moth particularly threatening to Northwest forests. B.t. is scheduled to be sprayed on about 200 thousand acres of Northeastern Oregon forests infested by spruce budworm. Spraying is aimed at controlling the budworm in specific areas until long- term solutions can be implemented. Long term solutions include trimming stands and replacing fir trees with pines, which are less susceptible to spruce budworm(9). ALTERNATE CONTROL METHODS Biological insecticides, such as B.t., are often the insecticide of choice for aerial spray programs because they are naturally derived, are host-specific, and they do not persist in the environment. Synthetic insecticides, such as acephate, carbaryl, diflubenzuron, and trichlorfon, are effective in gypsy moth eradication, but they are less acceptable because of public concern over human health risks and effects on nontarget organisms. Another control option is the release of large numbers of sterile males designed to eliminate the next generation of pests. Sterile male release was used against the California medfly but facilities which can rear sterile gypsy moths or sterile spruce budworms are currently not capable of supplying the necessary number of organisms needed(7). COSTS Failure to eradicate the Asian gypsy moth would necessitate a yearly suppression program to deal with the economic and environmental impacts of the moth. Such a suppression program could cost up to $821 million over a 40 year period(10). The loss potential for failure to eradicate the Asian gypsy moth is estimated at $2 billion in recreation and tourism losses and $1.5 billion in commercial timber losses(10). In contrast, Oregon's share of the Asian gypsy moth eradication costs is $500,000(10) with up to $1.3 million in costs for detection(11). MODE OF ACTION OF B.t. B.t. is toxic to organisms in two ways: by activation of the toxin and by germination of the spores, which may lead toinfection. Naturally occurring B.t. is rare and, when found, is predominately in the active or vegetative form. Naturally occurring B.t. does not normally contribute significantly to insect mortality, but for pest control purposes, B.t. is formulated using the dormant or spore form, which contains a crystalline toxin. In leaf-feeding Lepidopteran larvae (the target pest), B.t. spores must first be ingested. As the spores pass through the digestive system, they must dissolve under alkaline conditions in the presence of specific enzymes before the toxin can be activated. The activated toxin must then bind to specific receptors in the midgut lining of the larvae. As a result of this binding, gut function is disrupted, which is the primary cause of death in most susceptible species. The disruption of gut function also allows germinated spores (the vegetative form) to infect vital organs and this second action (infection) is thought to be the primary cause of death in species that are less susceptible to the toxin(12). For B.t. to be toxic at expected environmental concentrations, all of these conditions must be satisfied, thus imparting the specific selectivity for leaf-feeding Lepidopteran larvae. HUMAN HEALTH RISKS The possibility of human exposure to B.t. occurs occupationally, during application procedures, or environmentally, as a result of direct contact with sprayed foliage or other surfaces, or as a result of inhaling spray mist, eating plants or animals contaminated with B.t., or drinking contaminated water (5). B.t. spores can become activated under certain situations, even if the conditions are not present for the crystals to be dissolved. It is this situation that potentially affects non- target organisms, both human and non-human. There are two recorded cases of B.t.-caused infections in humans: in one instance, a technician was accidently stuck with a needle contaminated with B.t.i.; in the other, a farm worker who was handling B.t.k. was splashed in the eye with the formulation(5). Workers exposed to B.t. during formulation production were followed for seven months and remained free of complaints during this period. The literature has no reports of reproductive or neurological effects from B.t. exposure and mutagenicity tests have been negative. No tumor producing effects were seen in a two-year chronic study by Abbott Laboratories, in which rats were given dietary doses of 8,400 mg/kg of B.t. formulation(13). A passive surveillance program was initiated during the 1985-86 Lane County, Oregon, gypsy moth spray program and an unknown number of routine human specimens were cultured for the Bacillus species. There were 55 confirmed B.t. cultures and, of these, 52 were probable contaminants. The remaining three positive cultures were taken from patients with pre-existing medical problems, and B.t. could not be positively ruled out as a pathogen(14). Nonetheless, the medical community has been reluctant to label any bacterium as absolutely non-pathogenic(14). The Oregon Health Division currently recommends that physician- diagnosed patients who are severely immunocompromised consult with their own doctors about whether or not to leave a spray area(15). EFFECTS ON ANIMALS Because of its specificity for leaf-feeding Lepidopteran larvae, the B.t. formulations commonly used in forestry situations have not been implicated in other non-target insect deaths. Honey bees, stiltbugs, flies, and beetles all show few or no effects from spraying, and praying mantis and earthworms were not adversely affected(5). However, one study noted a reduction in the number of some parasitic adult wasps and a Lane County, Oregon study indicated a two year reduction in the numbers of non-target Lepidoptera, but noted recovery by the third year(16). B.t. may be toxic to some shrimp and mussels and, therefore, has not been registered for use in aquatic environments. B.t. appears to be of low toxicity to fish and aquatic insects(2). Canadian Forest Service studies show that song bird populations were not significantly reduced, even at very high B.t. application levels. Mallards and bobwhite quail showed no effects in feeding studies. Chickens evidenced lower weight gains when fed formulations above 2%, but this may have been due to food avoidance(5). A U.S. Forest Service review of the literature indicates that mice, hogs, rats, cattle, dogs, guinea pigs, and rabbits exposed to B.t. by routes similar to those that humans would experience in the environment showed no significant effects(5). FATE IN THE ENVIRONMENT The persistence of formulated B.t. in the environment is strongly dependent on various parameters, including sunlight, humidity, and soil conditions. Reports of the half-life of viable spores on foliage range from less than a day to several weeks, depending on formulation and climactic conditions. B.t. is not toxic to plants. B.t. may remain active in soil for as long as four months. Viable spores, however, may remain dormant for years(2). Few studies have been conducted to determine the fate of B.t. in aquatic environments. One study indicated B.t. disappearance from water four weeks following overspray during a forest application(3). The data on B.t. persistence in air indicates that B.t. thuringiensis var. thuringiensis was present for up to 17 days(3). No information was available for B.t.k. Sunlight is the primary cause of inactivation of B.t. spores in the environment. Susceptibility of B.t. to solar radiation is enhanced under humid conditions(2). AVAILABLE HELP Information on the spraying program and the use of B.t.: Asian Gypsy Moth Spray Project, 1-800-443-MOTH Information on human health effects: Oregon Health Division, (503) 731-4023 Information on follow-up detection: Oregon Department of Agriculture, 1-800-525-0137 Company information on manufacturing or testing of B.t.: Novo- Nordisk Bioindustrials, Inc., (203) 790-2600, 33 Turner Road, PO Box 1907, Danbury, CT, 06813-1907 Fax: (203) 790-2611 For questions about this document or material therein: Jeff Jenkins, Extension Specialist, Oregon State University, (503) 737-5993, Fax: (503) 737-0497, Email address: jenkinsj@oes.orst.edu or, Pat Thomson, Information Specialist, (503) 737-1802, Fax: (503) 737-0497 REFERENCES 1. C. R. Worthington, Ed., The Pesticide Manual, A World Compendium (British Crop Protection Council, Surrey, 1991). 2. M. Ghassemi, et al., Environmental fates and impact of major forest use pesticides, U.S. Environmental Protection Agency, Washington, D.C. (1981). 3. J.P. Friel, Ed., Dorland's Illustrated Medical Dictionary, 26th Edition (W.B. Saunders, Co., Philadelphia, 1981). 4. Richard T. Meister, Ed., Farm Chemicals Handbook '92 (Meister Publishing Co., Willoughby, 1992). 5. Insecticide Background Statement: Bacillus thuringiensis in Pesticide Background Statement, Volume IV. Insecticides Agriculture Handbook Number 685, U.S. Forest Service, (1989). 6. J.W. Rinehold and J.M. Witt, Oregon Pesticide Use Estimates for 1987, Oregon State University Extension Service, Special report 843 (1989). 7. U.S. Department of Agriculture and States of Oregon and Washington Cooperative Asian Gypsy Moth Eradication Project, Environmental Assessment, USDA Animal and Plant Health Inspection Service (1992). 8. Capital Press, March 6, 1992, pp. 1-2. 9. Leroy Kline, Oregon Department of Forestry, Protection Division,Insect and Disease Section, Salem, Or., private communication (May, 1992). 10. U. S. Department of Agriculture and States of Oregon and Washington Cooperative Asian Gypsy Moth Eradication Project, Response to Comments, USDA Animal and Plant Health Inspection Service (1992). 11. Capital Press, March 20, 1992, pp. 1-2. 12. S.S. Gill, E.A. Cowles, and P.V. Pietrantonio, "The Mode of Action of Bacillus thuringiensis endotoxins," Annual Review of Entomology 37,615-36 (1992). 13. Bacillus Thuringiensis (B.t.), Pesticide Information Profile, EXTOXNET (Extension Toxicology Network), Cornell University (1988). 14. M. Green, et al., Public health implications of the microbial pesticide Bacillus thuringiensis: An epidemiological study, 1985-86, American Journal of Public Health, 80(70), 848-52(1990). 15. L.R. Foster, Memo to Tri-County Area Physicians, Gypsy Moth Spraying, Oregon Department of Human Resources, Health Division (April 20, 1992). 16. J.C. Miller, "Field Assessment of the effects of a microbial pest control agent on nontarget Lepidoptera," American Entomologist, Summer, 135-139 (1990). ADDITIONAL REFERENCES 1. F.N. Dost, S.L. Wagner, and J.M. Witt, Toxicological Evaluation of Bacillus thuringiensis, Oregon State University Extension Service Toxicology Information Program, Department of Agricultural Chemistry, Corvallis, Or. (1985). 2. W.J. Hayes, Jr., Pesticides Studied in Man (Williams & Wilkins, Baltimore, 1982). 3. W.J. Hayes, Jr. and E.R. Laws, Jr., Eds., Classes of Pesticides, Handbook of Pesticide Toxicology, Volume 2 (Academic Press, Inc., New York, 1991). 4. Pesticide Fact Sheet, Bacillus thuringiensis (B.t), U.S. Environmental Protection Agency, Office of Pesticides and Toxic Substances, Office of Pesticide Programs, Washington, D.C., Fact Sheet Number 93 (1986). _____________________ Printed January, 1992