UNITED NATIONS ENVIRONMENT PROGRAMME INTERNATIONAL LABOUR ORGANISATION WORLD HEALTH ORGANIZATION INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 217 BACILLUS THURINGIENSIS This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. Environmental Health Criteria 217 Microbial Pest Control Agent BACILLUS THURINGIENSIS Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. World Health Organization Geneva, 1999 The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organisation (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals. The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. WHO Library Cataloguing-in-Publication Data Bacillus thuringiensis. (Environmental health criteria ; 217) 1.Bacillus thuringinesis - pathogenicity 2.Pest control, Biological - methods 3.Insecticides - chemistry 4.Environmental exposure 5.Occupational exposure I.Series ISBN 92 4 157217 5 (NLM Classification: QW 127.5.B2) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1999 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city, or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR BACILLUS THURINGIENSIS PREAMBLE ABBREVIATIONS 1. SUMMARY 1.1. Identity, biological characteristics, and analytical methods 1.2. Mode of action on target insects 1.3. Habitats 1.4. Commercial products, production and application 1.5. Effects of Bt on non-target organisms 1.6. Exposure and effects of Bt on humans 1.7. Conclusions 2. IDENTITY, BIOLOGICAL PROPERTIES, AND ANALYTICAL METHODS 2.1. Identity 2.1.1. Bacillus thuringiensis (Bt) 2.1.2. Relationship between Bacillus thuringiensis and Bacillus cereus 2.1.3. Crystal composition and morphology 2.1.4. Classification of Bt subspecies 2.1.5. Genetics of ICP 2.1.6. Beta-exotoxin 2.1.7. Other Bt metabolites 2.2. Bioassays 2.2.1. Spore counts 2.2.2. International bioassay for ICPs 3. MODE OF ACTION ON TARGET INSECTS 3.1. Bioactivity of field isolates 3.2. Mechanism of action of Bt formulations 3.3. Resistance of insect populations 4. NATURAL AND TREATED HABITATS 4.1. Natural occurrence of Bt 4.1.1. Bt in insect hosts 4.1.2. Bt in soil 4.1.3. Bt on plant surfaces 4.2. Treated habitats 4.3. Environmental fate, distribution and movement 4.3.1. Distribution and fate of Bt in terrestrial habitats 18.104.22.168 Fate of Bt and ICP on plant surfaces 22.214.171.124 Fate of Bt in soil 4.3.2. Distribution and fate of Bt in aquatic habitats 4.3.3. Transport of Bt by non-target organisms 5. COMMERCIAL PRODUCTION 5.1. History of Bt and its commercial applications 5.1.1. Production levels 5.1.2. Production processes, formulations and quality standards 5.1.3. General patterns of use 126.96.36.199 Applications in agriculture and forestry 188.8.131.52 Applications in vector control 6. EFFECTS ON ANIMALS 6.1. Mammals 6.1.1. Oral exposure 6.1.2. Inhalation exposure 6.1.3. Dermal exposure 6.1.4. Dermal scarification exposure 6.1.5. Subcutaneous inoculation 6.1.6. Ocular exposure 6.1.7. Intraperitoneal exposure 184.108.40.206 Immune-intact animals 220.127.116.11 Immune-suppressed animals 6.1.8. Effects of activated Bt ICP 6.1.9. Studies in wild animals 6.2. Effects on birds 6.3. Effects on aquatic vertebrates 6.4. Effects on invertebrates 6.4.1. Effects on invertebrates other than insects 6.4.2. Effects on non-target insects 18.104.22.168 Aquatic insects 22.214.171.124 Terrestrial insects 126.96.36.199 Honey-bees 188.8.131.52 Parasitoids 7. EXPOSURE AND EFFECTS ON HUMANS 7.1. Bacillus thuringiensis 7.1.1. Experimental exposure of humans 7.1.2. Exposure of workers during manufacture 7.1.3. Exposure of workers in spraying operations 7.1.4. Exposure of human populations by spraying operations over populated areas. 7.1.5. Clinical case reports 7.1.6. Dietary exposure of the general population 7.2. Bacillus cereus 8. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 9. CONCLUSIONS AND RECOMMENDATIONS 10. PREVIOUS EVALUATIONS BY INTERNATIONAL ORGANISATIONS REFERENCES RÉSUMÉ RESUMEN NOTE TO READERS OF THE CRITERIA MONOGRAPHS Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda. * * * A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postage 356, 1219 Châtelaine, Geneva, Switzerland (telephone no. + 41 22 - 9799111, fax no. + 41 22 - 7973460, E-mail firstname.lastname@example.org). * * * This publication was made possible by grant number 5 U01 ES02617-15 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA, and by financial support from the European Commission. Environmental Health Criteria PREAMBLE Objectives In 1973 the WHO Environmental Health Criteria Programme was initiated with the following objectives: (i) to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits; (ii) to identify new or potential pollutants; (iii) to identify gaps in knowledge concerning the health effects of pollutants; (iv) to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results. The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976 and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g., for genetic, neurotoxic, teratogenic and nephrotoxic effects. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR BACILLUS THURINGIENSIS Members Professor D. Calamari, Department of Biology, Structure and Function, University of Milan, Varese, Italy Dr C. Cummins, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom Dr N. Gratz, Commugny, Switzerland (Rapporteur) Dr A. Klier, Unité de Biochimie Microbienne, Département des Biotechnologies, Institut Pasteur, Paris, France Professor P. Lüthy, Microbiological Institute ETH, Zurich, Switzerland Dr C.M. Scanlan, Texas A & M University, Department of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas, USA (Chairman) Observers Dr R.J. Cibulsky, Abbott Laboratories, Chemical and Agricultural Products, North Chicago, Illinois, USA Dr E. Cozzi, Clinical Research and Development, Animal Health Products, Abbott Laboratories, North Chicago, Illinois, USA Dr M. Ronchin, International Centre for Pesticide Safety, Busto Garolfo, Milan, Italy Secretariat Professor M. Maroni, International Centre for Pesticide Safety, Busto Garolfo, Milan, Italy Dr R. Plestina, Zagreb, Croatia ENVIRONMENTAL HEALTH CRITERIA FOR BACILLUS THURINGIENSIS A WHO Task Group on Environmental Health Criteria for the microbial pest control agent Bacillus thuringiensis (Bt) met at the International Centre for Pesticide Safety in Busto Garolfo, Milan, Italy, from 27 to 31 October 1997. Professor M. Maroni, Director of the Centre, welcomed participants on behalf of the Centre, which was responsible for organizing the meeting. Dr R. Plestina, IPCS temporary adviser, opened the meeting and welcomed participants on behalf of Dr M. Mercier, Director of IPCS. The Group reviewed and revised the draft and made an evaluation of the risks for human health and the environment from exposure to Bt products. Drs R. Plestina and A. Aitio of the IPCS Central Unit were responsible for the scientific aspects of the monograph, and Dr P.G. Jenkins for the editing. The assistance of manufacturers, notably Abbott Laboratories, in providing unpublished documentation for the review is greatly appreciated. Microbial Pest Control Agents (MPCAs), notably products of various Bt subspecies, are increasingly used in pest management programmes against the larvae of several insect pests of major agricultural crops and forests, and several insect vectors of human diseases, and some nuisance pests. Bt products have been used worldwide, and their commercial production is about 1% of that of chemical pesticides. A number of reviews have recently been published on various aspects of Bt (Entwistle et al., 1983; McClintock et al., 1995; Cannon, 1996; Dean et al., 1996; Kumar et al., 1996; Schnepf et al., 1998; Nielsen-LeRoux et al., 1998). The activities in preparing this document were recommended by an Informal Consultation on the Safety of Microbial Pest Control Agents held in Geneva in June 1993. The first draft of the monograph was prepared by a drafting group in 1994 and was subsequently amended by Professor C.M. Scanlan (Texas A&M University, Department of Veterinary Pathobiology, The Texas Veterinary Medical Center, Texas, USA). The revised draft was circulated to the IPCS contact points. Based on the comments received, it was amended and updated by Drs B.M. Hansen and N.B. Hendriksen (National Environmental Research Institute, Roskilde, Denmark). The document was finally approved by the Task Group members. ABBREVIATIONS Bc Bacillus cereus Bt Bacillus thuringiensis Bta Bacillus thuringiensis subspecies aizawai Btd Bacillus thuringiensis subspecies darmstadiensis Bte Bacillus thuringiensis subspecies entomocidus Btg Bacillus thuringiensis subspecies galleriae Bti Bacillus thuringiensis subspecies israelensis Btk Bacillus thuringiensis subspecies kurstaki Btko Bacillus thuringiensis subspecies konkukian Btt Bacillus thuringiensis subspecies thuringiensis Btte Bacillus thuringiensis subspecies tenebrionis cfu colony forming unit GILSP good industrial large-scale practice GMO genetically modified organism HPLC high-performance liquid chromatography ICP insecticidal crystal protein ITU international toxic unit IUPAC International Union of Pure and Applied Chemistry MPCA microbial pest control agent NTO non-target organism PCR polymerase chain reaction SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis 1. SUMMARY This monograph deals with microbial pest control agents (MCPAs) based on Bacillus thuringiensis (Bt). This bacterium is also a key source of genes for transgenic expression to provide pest resistance in plants and microorganisms as pest control agents in so-called genetically modified organisms (GMOs). The potential effects on human health and the environment of GMOs involve several aspects that are only remotely or not at all related to Bt products, and they are therefore outside the scope of this monograph. 1.1 Identity, biological characteristics and analytical methods Bt is a facultative anaerobic, gram-positive bacterium that forms characteristic protein inclusions adjacent to the endospore. Bt subspecies can synthesize more than one parasporal inclusion. Bt is genetically indistinguishable from Bc, except for the ability of Bt to produce parasporal crystalline inclusions, which are toxic for certain invertebrates, especially species of insect larvae belonging to the insect orders Coleoptera, Diptera and Lepidoptera. The parasporal inclusions are formed by different insecticidal crystal proteins (ICP). The crystals have various shapes (bipyramidal, cuboidal, flat rhomboid, spherical or composite with two crystal types), depending on their ICP composition. A partial correlation between crystal morphology, ICP composition, and bioactivity against target insects has been established. The basic phenotypic taxon is the subspecies, identified by the flagellar (H) serotype. By 1998, 67 subspecies had been described. The genes that encode the ICPs are mostly on plasmids. Each ICP is the product of a single gene. Most plasmids with ICP genes are readily transferred by conjugation between Bt strains and may be transferred to related species of bacteria. The phenotypic classification has now been complemented by molecular biological characterization, based on the sequence of the crystal (cry and cyt) genes rather than target organism specificity. Different domains of the ICP are responsible for host susceptibility (receptor recognition) and toxicity (pore formation). Techniques commonly used to characterize Bt strains or the ICP itself include cell wall fatty acid analysis, monoclonal antibodies, oligonucleotide DNA probes, plasmid profiles, polymerase chain reaction (PCR) analysis, DNA fingerprinting and SDS-PAGE profiles. Beta-exotoxin, a heat-stable nucleotide, is produced by some Bt subspecies during vegetative growth and may contaminate the products. Beta-exotoxin is toxic for almost all forms of life including humans and the target insect orders. During vegetative growth, various Bt strains produce an assortment of antibiotics, enzymes, metabolites and toxins, including Bc toxins, that may have detrimental effects on both target organisms and non-target organisms (NTOs). Spore counts do not accurately reflect the insecticidal activity of a Bt strain or Bt product. The potency (ITU/mg) of each Bt product is bioassayed using an international standard that uses a specific test insect. 1.2 Mode of action on target insects The sporulated Bt with ICP or spore-ICP complexes must be ingested by a susceptible insect larva. The efficacy of the ICP depends on the solubilization in the midgut, the conversion of the protoxin to the biologically active toxin by proteolytic enzymes, specific membrane receptor binding by the C-terminal domain of the active toxin, and pore formation by the N-terminal domain with subsequent lysis of the epithelial cells. Spore germination and proliferation of the vegetative cells into the haemocoel may result in a septicaemia, contributing to the cause of death. Receptor binding by the ICP is the major determinant of host specificity by the different Bt ICPs. 1.3 Habitats Many different Bt subspecies have been isolated from dead or dying insects mostly from the orders Coleoptera, Diptera and Lepidoptera, but many subspecies have also been isolated from soil, leaf surfaces and other habitats. The carcasses of dead insects often contain large quantities of spores and ICPs that may enter the environment. The coleopteran-active and lepidopteran-active Bt subspecies are primarily associated with the soil and phylloplane (leaf surfaces), whereas the dipteran-active Bt subspecies are commonly found in aquatic environments. In the environment, the spores persist and vegetative growth may occur when conditions are favourable and nutrients are available. 1.4 Commercial products, production and application Conventional Bt products, which utilize naturally-occurring Bt strains, account for approximately 90% of the world MPCA market. Most Bt products contain ICP and viable spores, but in some Bti products the spores are inactivated. Each year some 13 000 tonnes are produced using aerobic fermentation technology. Conventional Bt products have been targeted primarily against lepidopteran pests of agricultural and forestry crops; however in recent years, Bt strains active against coleopteran pests have also been marketed. Strains of Bti active against dipteran vectors of parasitic and viral diseases are being used in public health programmes. Commercial Bt formulations may be applied as an insecticide to foliage, soil, water environments or food storage facilities. After the application of a Bt subspecies to an ecosystem, the vegetative cells and spores may persist at gradually decreasing concentrations for weeks, months or years as a component of the natural microflora. The ICPs, however, are rendered biologically inactive within hours or days. 1.5 Effects of Bt on non-target organisms Studies on mammals, particularly those on laboratory animals, have evaluated possible infectivity and toxicity of various Bt preparations, which include the ICPs, vegetative cells and spores. The ICPs, spores and vegetative cells of the Bt subspecies, which were administered by different routes, were mostly non-pathogenic and non-toxic to the various animal species tested. The vegetative cells and/or spores of Bt were demonstrated to persist for weeks without causing adverse effects. Bt has not been observed to adversely affect birds, fish or many other non-target aquatic vertebrates tested in a large number of laboratory and field studies. Relatively few species of aquatic invertebrates are susceptible to Bt under either laboratory or field conditions. Bt does not adversely affect earthworms. The Bt subspecies have generally been shown to be highly specific in their insecticidal activity for Coleoptera, Diptera and Lepidoptera and have demonstrated little, if any, direct toxicity to non-target arthropods. Most of the existing safety data on non-target arthropods has been generated using the Bt subspecies with activity against Diptera and Lepidoptera. Studies of Bti formulations free of toxic contaminants have not demonstrated deleterious effects on the vast majority of non-target arthropods. Some midges (Diptera: Chironomidae), which are closely related to mosquitos, have been shown to be susceptible to high dosages of Bti, but are not affected by mosquito larvicidal dosages. In field studies, transient decreases or increases in populations of some non-target arthropods have been reported. Many insect orders have been tested in either the laboratory or field, most of which have shown no effect from Btk. Mortality has been observed in honey-bees (Apis mellifera) after exposure to vegetatively growing Btt and Btk, but the effect does not seem to be related to spores or ICPs. In laboratory and field studies Btg demonstrated no adverse effect on honey-bees. Bte strains that produce beta-exotoxin have been shown to have adverse effects on non-target arthropods. 1.6 Exposure and effects of Bt on humans The field application of Bt products can result in considerable aerosol and dermal exposure of workers. Agricultural uses of Bt can result in Bt contamination of potable water and food. With the exception of case reports on ocular and dermal irritation, no adverse health effects have been documented after occupational exposure to Bt products. Human volunteers ingested and inhaled large quantities of a Btk formulation but experienced no adverse health effects. Antibody titres to the vegetative cells, spores and spore-crystal complexes have been demonstrated in workers who spray Bt products; however, no adverse health effects were reported. There have been some case reports on the occurrence of Bt in patients with different infectious diseases. However, none of these studies unequivocally demonstrates an actual risk to human health from the use of Bt. Bt has not been reported to cause adverse effects on human health when present in drinking-water or food. 1.7 Conclusions Owing to their specific mode of action, Bt products are unlikely to pose any hazard to humans or other vertebrates or to the great majority of non-target invertebrates provided that they are free from non-Bt microorganisms and biologically active products other than the ICPs. Bt products may be safely used for the control of insect pests of agricultural and horticultural crops as well as forests. They are also safe for use in aquatic environments including drinking-water reservoirs for the control of mosquito, black fly and nuisance insect larvae. However, it should be noted that vegetative Bt has the potential for the production of Bc-like toxins, the significance of which as a cause of human disease is not known. 2. IDENTITY, BIOLOGICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Commercial Bacillus thuringiensis (Bt) products are microbial pest control agents (MPCAs) containing specific insecticidal crystalline proteins (ICPs) and most often living spores as well as formulating agents. They are processed fermentation products. 2.1.1 Bacillus thuringiensis Bt is a facultative anaerobic, motile, gram-positive, spore-forming bacterium. The formation of parasporal crystals adjacent to the endospore during sporulation stages III to IV distinguishes Bt from other Bacillus species. Bt, like other Bacillus species, has been classified on the basis of its cellular, cultural, biochemical and genetic characteristics (Baumann et al., 1984; Claus & Berkley, 1986; Slepecky & Hemphill, 1992; Carlson & Kolsto, 1993; Hansen et al., 1998). In 1958, Heimpel & Angus (1958) introduced a classification scheme to identify these crystalliferous bacteria based on their morphological and biochemical characteristics. However recent molecular analysis shows that several variations can be found within serotypes, and that specific biochemical characteristics do not always refer to a specific serotype (Helgason et al., 1998; Hansen et al., 1998). 2.1.2 Relationship between Bacillus thuringiensis and Bacillus cereus Bt is a member of the Bc group, which also contains Bacillus cereus (Bc), B. mycoides and B. anthracis. Furthermore, the psychrotolerant B. weihenstephanensis has recently been proposed as a new member of the group (Lechner et al., 1998). Bt can only be distinguished from Bc by the production during the sporulation process of one or more inclusion bodies, which have been found to be toxic for invertebrates, primarily insect species in the orders Coleoptera, Diptera and Lepidoptera (de Barjac, 1981b; Andrews et al., 1987). Several studies have been dedicated to a comparison of Bt and Bc on the basis of characters not related to the production of ICPs (Hendriksen & Hansen, 1998). Phenotypic differentiation of Bt and Bc is not possible on the basis of morphology or utilization of organic compounds (Baumann et al., 1984; Logan & Berkeley, 1984; Priest et al., 1988), characterization of cell content of fatty acids (Väisänen et al., 1991) or sugars (Wunschel et al., 1994), multilocus enzyme electrophoresis (Zahner et al., 1989; Carlson et al., 1994), enterotoxin production (Damgaard et al., 1996a; Hansen & Hendriksen, 1997a), or serological- and phage-typing techniques (Ohba & Aizawa, 1978; 1986; Väisänen et al., 1991; Murakami et al., 1993; Ahmed et al., 1995). Likewise, genotypic differentiation of Bt and Bc is not possible by DNA homology analysis (Kaneko et al., 1978), ribotyping (Priest et al., 1994; Demezas & Bell, 1995), 16S rDNA sequencing (Ash et al., 1991); analysis of the 16S-23S internal transcribed sequence (Wunschel et al., 1994; Bourque et al., 1995), PCR analysis of genes encoding Bc-like toxic products (Damgaard et al., 1996b; Asano et al., 1997; Hansen & Hendriksen, 1997b) or pulsed field gel electrophoresis (Carlson & Kolsto, 1993; Carlson et al., 1994). Giffel et al. (1997) found differences in 16S rDNA sequences between a limited number Bt and Bc. Beattie et al. (1998) were able to discriminate among members of the Bc group by Fourier transform infrared spectroscopy, and Brousseau et al. (1992) were able to distinguish Bt and Bc by random amplified polymorphic DNA fingerprinting. However, the transfer of ICP encoding plasmids from Bt to Bc makes the receptor Bc indistinguishable from Bt, and vice versa (González et al., 1981, 1982). 2.1.3 Crystal composition and morphology The existence of parasporal inclusions in Bt was first noted in 1915 (Berliner, 1915), but their protein composition was not delineated until the 1950s (Angus, 1954). Hannay (1953) detected the crystalline fine structure that is a property of most of the parasporal inclusions. Bt subspecies can synthesize more than one inclusion, which may contain different ICPs. ICPs have also been called delta endotoxins; however, since the term endotoxin usually refers to toxins associated with the outer membranes of gram-negative bacteria, comprising a core lipopoplysaccharide, lipid A and somatic (O) antigens, this term is not used in this monograph. Depending on their ICP composition, the crystals have various forms (bipyramidal, cuboidal, flat rhomboid, or a composite with two or more crystal types). A partial correlation between crystal morphology, ICP composition, and bioactivity against target insects has been established (Bulla et al., 1977; Höfte & Whiteley, 1989; Lynch & Baumann, 1985). 2.1.4 Classification of Bt subspecies The classification of Bt subspecies based on the serological analysis of the flagella (H) antigens was introduced in the early 1960s (de Barjac & Bonnefoi, 1962). This classification by serotype has been supplemented by morphological and biochemical criteria (de Barjac, 1981a). Until 1977, only 13 Bt subspecies had been described, and at that time all subspecies were toxic to Lepidopteran larvae only. The discovery of other subspecies toxic to Diptera (Goldberg & Margalit, 1977), Coleoptera (Krieg et al., 1983) and apparently Nematoda (Narva et al., 1991) enlarged the host range and markedly increased the number of subspecies. Up to the end of 1998, over 67 subspecies based on flagellar H-serovars had been identified (Table 1). Updated lists of the serovarieties can be obtained from the reference centre of the Pasteur Institute in Paris (Unité des Bactéries Entomopathogènes, Institut Pasteur, Paris, France). Table 1. Current classification of 67 Bacillus thuringiensis subspecies based on their flagellar (H) antigensa Flagellar antigens B. thuringiensis Flagellar antigens B. thuringiensis subspecies subspecies 1 thuringiensis 28a, 28c jegathesan 2 finitimus 29 amagiensis 3a, 3c alesti 30 medellin 3a, 3b, 3c kurstaki 31 toguchini 3a, 3d sumiyoshiensis 32 cameroun 3a, 3d, 3e fukuokaensis 33 leesis 4a, 4b sotto 34 konkukian 4a, 4c kenyae 35 seoulensis 5a, 5c galleriae 36 malaysiensis 5a, 5c canadensis 37 anadalousiensis 6 entomocidus 38 oswaldocruzi 7 aizawai 39 brasiliensis 8a, 8b morrisoni 40 huazhongensis 8a, 8c ostriniae 41 sooncheon 8b, 8d nigeriensis 42 jinghongiensis 9 tolworthi 43 guiyanguebsus 10a, 10b darmstadiensis 44 higo 10a, 10c londrina 45 roskildiensis 11a, 11b toumanoffi 46 chanpaisis 11a, 11c kyushuensis 47 wratislaviensis 12 thompsoni 48 balearica 13 pakistani 49 muju 14 israelensis 50 navarrensis 15 dakota 51 xiaguangiensis 16 indiana 52 kim 17 tohokuensis 53 asturiensis 18a, 18b kumamotoensis 54 poloniensis 18a, 18c yosoo 55 palmanyolensis 19 tochigiensis 56 rongseni 20a, 20b yunnanensis 57 pirenaica 20a, 20c pondicheriensis 58 argentinensis 21 colmeri 59 iberica 22 shandongiensis 60 pingluonsis 23 japonensis 61 sylvestriensis 24a, 24b neoleonensis 62 zhaodongensis 24a, 24c novosibirsk 63 bolivia 25 coreanensis 64 azorensis 26 silo 65 pulsiensis 27 mexicanensis 66 gracioensis 28a, 28b monterrey 67 vazensis a Data provided by the Unité des Bactéries Entomopathogènes, Institut Pasteur, Paris, France Crystal serology has shown that a particular crystal type may be produced by more than one H-serovar (Krywienczyk et al., 1978; Smith, 1987). 2.1.5 Genetics of ICP In the early 1980s, it was established that most genes coding for the ICPs reside on large transmissible plasmids, of which most are readily exchanged between strains by conjugation (González & Carlton, 1980; González et al., 1981). Since these initial studies, numerous ICP genes have been cloned, sequenced and used to construct Bt strains with novel insecticidal spectra (Höfte & Whiteley, 1989). The currently known crystal (cry) gene types encode ICPs that are specific to either Lepidoptera (cryI), Diptera and Lepidoptera (cryII), Coleoptera (cryIII), Diptera (cryIV), or Coleoptera and Lepidoptera (cryV) (Höfte & Whiteley, 1989). A separate designation is used for the cytolytic (cyt) genes that encode a nonspecific cytolytic factor, present in Bti ICP and some other Bt subspecies. However, due to the increasing number of characterized ICP genes and inconsistencies in the existing cry gene nomenclature, which is based on insecticidal spectrum, Crickmore et al. (1998) proposed a new nomenclature based on ICP gene sequences. The new cry genes are listed in Tables 2 and 3, and Table 4 is a conversion list from old to new cry gene names. Current identification of Bt employs both the identity of the cry genes, which define the host range, and the H-serovars, which define the subspecies, and recently DNA fingerprinting has been used for further characterization of subspecies (Hansen et al., 1998). The ICP gene sequences provided the basis for the construction of gene-specific probes to screen established Bt strains by hybridization and PCR analysis for the presence of known nucleotide sequences, and for characterizing the ICPs from new Bt isolates (Prefontaine et al., 1987; Juarez-Perez et al., 1997; Bravo et al., 1998; Shevelev et al., 1998). These studies have permitted the distinction of numerous subclasses of ICP genes based on sequence homology and toxicity spectra of the encoded proteins. All ICPs described to date attack the insect gut upon ingestion (see chapter 3). To date, each of the proteolytically activated ICP molecules with insecticidal activity has a variable C-terminal domain, which is responsible for receptor recognition (host susceptibility), and a conserved N-terminal domain, which induces pore formation (toxicity) (Li et al., 1991). Most naturally occurring Bt strains contain ICPs active against a single order of insects. However, conjugative transfer between Bt strains or related species can occur, resulting in new strains with various plasmid contents. Thus the mobility of the cry genes and the exchange of plasmids may explain the diverse and complex activity Table 2. Bacillus thuringiensis crystal protein genes (Crickmore et al., 1998) Name Acc No Reference Journal Coding Cry1aa1 M11250 Schnepf et al., 1985 JBC 260 6264-6272 527-4054 Cry1aa2 M10917 Shibano et al., 1985 Gene 34 243-251 153->2955 Cry1aa3 D00348 Shimizu et al., 1988 ABC 52 1565-1573 73-3603 Cry1aa4 X13535 Masson et al., 1989 NAR 17 446-446 1-3528 Cry1aa5 D17518 Udayasuriyan et al., 1994 BBB 58 830-835 81-3611 Cry1aa6 U43605 Masson et al., 1994 Mol Micro 14 851-860 1->1860 Cry1aa7 AF081790 Osman, 1998 unpublished Cry1aa8 I26149 Liu, 1996 USP 5556784 148-3675 Cry1ab1 M13898 Wabiko et al., 1986 DNA 5 305-314 142-3606 Cry1ab2 M12661 Thorne et al., 1986 J Bact 166 801-811 155-3625 Cry1ab3 M15271 Geiser et al., 1986 Gene 48 109-118 156-3623 Cry1ab4 D00117 Kondo et al., 1987 ABC 51 455-463 163-3630 Cry1ab5 X04698 Hofte et al., 1986 EJB 161 273-280 141-3605 Cry1ab6 M37263 Hefford et al., 1987 J Biotech 6 307-322 73-3540 Cry1ab7 X13233 Haider & Ellar, 1988 NAR 16 10927-10927 1-3465 Cry1ab8 M16463 Oeda et al., 1987 Gene 53 113-119 157-3624 Cry1ab9 X54939 Chak & Jen, 1993 PNSCRC 17 7-14 73-3540 Cry1ab10 A29125 Fischhoff et al., 1987 Bio/technology peptide seq 5 807-813 Cry1ab11 I12419 Ely & Tippett, 1995 USP 5424409 73- Cry1ab12 AF057670 Silva-Werneck et al., 1998 unpublished 41-3505 Cry1ac1 M11068 Adang et al., 1985 Gene 36 289-300 388-3921 Cry1ac2 M35524 Von Tersch et al., 1991 AEM 57 349-358 239-3772 Cry1ac3 X54159 Dardenne et al., 1990 NAR 18 5546-5546 339->2192 Cry1ac4 M73249 Payne et al., 1991 USP 4990332 1-3537 Cry1ac5 M73248 Payne et al., 1992 USP 5135867 1-3534 Cry1ac6 U43606 Masson et al., 1994 Mol Micro 14 851-860 1->1821 Cry1ac7 U87793 Herrera et al., 1994 AEM 60 682-690 976-4512 Cry1ac8 U87397 Omolo et al., 1997 Curr Micro 34 118-121 153-3686 Cry1ac9 U89872 Gleave et al., 1992 NZJCHS 20 27-36 388-3921 Cry1ac10 AJ002514 Sun & Yu, 1997 unpublished 388-3921 Cry1ac11 AJ130970 Makhdoom & Riazuddin, 1998 unpublished 156-3689 Cry1ac12 I12418 Ely & Tippett, 1995 USP 5424409 81->2990 Cry1ad1 M73250 Payne & Sick, 1993 USP 5246852 1-3537 Table 2 (contd). Name Acc No Reference Journal Coding Cry1ad2 A27531 Payne & Sick, 1995 AUP 632335 1-3537 Cry1ae1 M65252 Lee & Aronson, 1991 J Bact 173 6635-6638 81-3623 Cry1af1 U82003 Kang et al., 1997 unpublished 172->2905 Cry1ag1 AF081248 Osman, 1998 unpublished Cry1ba1 X06711 Brizzard & Whiteley, 1988 NAR 16 2723-2724 1-3684 Cry1ba2 X95704 Soetaert, 1996 unpublished 186-3869 Cry1bb1 L32020 Donovan et al., 1994 USP 5322687 67-3753 Cry1bc1 Z46442 Bishop et al., 1994 unpublished 141-3839 Cry1bd1 U70726 Kuo & Chak, 1999 unpublished 842-4534 Cry1be1 Payne et al., 1998 USP 5723758 1-3681 Cry1ca1 X07518 Honee et al., 1988 NAR 16 6240-6240 47-3613 Cry1ca2 X13620 Sanchis et al., 1989 Mol Micro 3 229-238 241->2711 Cry1ca3 M73251 Payne & Sick, 1993 USP 5246852 1-3570 Cry1ca4 A27642 Van Mellaert et al., 1990 EP 0400246 234-3800 Cry1ca5 X96682 Strizhov, 1996 unpublished 1->2286 Cry1cb1 M97880 Kalman et al., 1993 AEM 59 1131-1137 296-3823 Cry1da1 X54160 Hofte et al., 1990 NAR 18 5545-5545 264-3758 Cry1da2 I76415 Payne & Sick, 1997 USP 5691308 1-3495 Cry1db1 Z22511 Lambert, 1993 unpublished 241-3720 Cry1ea1 X53985 Visser et al., 1990 J Bact 172 6783-6788 130-3642 Cry1ea2 X56144 Bosse et al., 1990 NAR 18 7443-7443 1-3516 Cry1ea3 M73252 Payne & Sick, 1991 USP 5039523 1-3516 Cry1ea4 U94323 Barboza-Corona et al., 1998 WJMB 14 437-441 388-3900 Cry1ea5 A15535 Botterman et al., 1994 EP 0358557 54-3566 Cry1eb1 M73253 Payne & Sick, 1993 USP 5206166 1-3522 Cry1fa1 M63897 Chambers et al., 1991 J Bact 173 3966-3976 478-3999 Cry1fa2 M73254 Payne & Sick, 1993 USP 5188960 1-3525 Cry1fb1 Z22512 Lambert, 1993 unpublished 483-4004 Cry1fb2 AB012288 Masuda & Asano, 1998 unpublished 84-3587 Cry1fb3 AF062350 Song & Zhang, 1998 unpublished Cry1fb4 I73895 Payne et al., 1997 USP 5686069 peptide seq Cry1ga1 Z22510 Lambert, 1993 unpublished 67-3564 Cry1ga2 Y09326 Shevelev et al., 1997 Febs Lett 404 148-152 692-4210 Cry1gb1 U70725 Kuo & Chak, 1999 unpublished 532-4038 Cry1ha1 Z22513 Lambert, 1993 unpublished 530-4045 Table 2 (contd). Name Acc No Reference Journal Coding Cry1hb1 U35780 Koo et al., 1995 unpublished 728-4195 Cry1ia1 X62821 Tailor et al., 1992 Mol Micro 6 1211-1217 355-2511 Cry1ia2 M98544 Gleave et al., 1993 AEM 59 1683-1687 1-2160 Cry1ia3 L36338 Shin et al., 1995 AEM 61 2402-2407 279-2438 Cry1ia4 L49391 Kostichka et al., 1996 J Bact 178 2141-2144 61-2217 Cry1ia5 Y08920 Selvapandiyan, 1996 unpublished 524-2680 Cry1ia6 AF076953 Zhong et al., 1998 unpublished 1-2157 Cry1ib1 U07642 Shin et al., 1995 AEM 61 2402-2407 237-2393 Cry1ic1 AF056933 Osman et al., 1998 unpublished 1-2157 Cry1i-like I90732 Payne et al., 1998 See Table 3 peptide seq Cry1ja1 L32019 Donovan et al., 1994 USP 5322687 99-3519 Cry1jb1 U31527 Von Tersch & Gonzalez, 1994 USP 5356623 177-3686 Cry1jc1 I90730 Payne et al., 1998 USP 5723758 peptide seq Cry1ka1 U28801 Koo et al., 1995 FEMS 134 159-164 451-4098 Cry1-like I90729 Payne et al., 1998 See Table 3 peptide seq Cry2aa1 M31738 Donovan et al., 1989 JBC 264 4740-4740 156-2054 Cry2aa2 M23723 Widner & Whiteley, 1989 J Bact 171 965-974 1840-3741 Cry2aa3 D86064 Sasaki et al., 1997 Curr Micro 35 1-8 2007-3911 Cry2aa4 AF047038 Misra et al., 1998 Unpublished 10-1908 Cry2aa5 AJ132464 Yu & Pang, 1999 Unpublished <1-1860 Cry2aa6 AJ132465 Yu & Pang 1999 Unpublished <1-1860 Cry2aa7 AJ132463 Yu & Pang, 1999 Unpublished <1->1611 Cry2ab1 M23724 Widner & Whiteley, 1989 J Bact 171 965-974 1-1899 Cry2ab2 X55416 Dankocsik et al., 1990 Mol Micro 4 2087-2094 874-2775 Cry2ac1 X57252 Wu et al., 1991 FEMS 81 31-36 2125-3990 Cry3aa1 M22472 Herrnstadt et al., 1987 Gene 57 37-46 25-1956 Cry3aa2 J02978 Sekar et al., 1987 PNAS 84 7036-7040 241-2175 Cry3aa3 Y00420 Hofte et al., 1987 NAR 15 7183-7183 566-2497 Cry3aa4 M30503 McPherson et al., 1988 Bio/technology 6 61-66 201-2135 Cry3aa5 M37207 Donovan et al., 1988 MGG 214 365-372 569-2503 Cry3aa6 U10985 Adams et al., 1994 Mol Micro 14 381-389 569-2503 Cry3ba1 X17123 Sick et al., 1990 NAR 18 1305-1305 25-1977 Cry3ba2 A07234 Peferoen et al., 1990 EP 0382990 342-2297 Cry3bb1 M89794 Donovan et al., 1992 AEM 58 3921-3927 202-2157 Cry3bb2 U31633 Donovan et al., 1995 USP 5378625 144-2099 Table 2 (contd). Name Acc No Reference Journal Coding Cry3bb3 I15475 Peferoen et al., 1995 USP 5466597 <1->1291 Cry3ca1 X59797 Lambert et al., 1992 Gene 110 131-132 232-2178 Cry4aa1 Y00423 Ward & Ellar, 1987 NAR 15 7195-7195 1-3540 Cry4aa2 D00248 Sen et al., 1988 ABC 52 873-878 393-3935 Cry4ba1 X07423 Chungjatpornchai et al., 1988 EJB 173 9-16 157-3564 Cry4ba2 X07082 Tungpradubkul et al., 1988 NAR 16 1637-1638 151-3558 Cry4ba3 M20242 Yamamoto et al., 1988 Gene 66 107-120 526-3933 Cry4ba4 D00247 Sen et al., 1988 ABC 52 873-878 461-3868 Cry5aa1 L07025 Sick et al., 1994 USP 5281530 1-4155 Cry5ab1 L07026 Narva et al., 1991 EP 0462721 1-3867 Cry5ac1 I34543 Payne et al., 1997 USP 5596071 1-3660 Cry5ba1 U19725 Payne et al., 1997 USP 5596071 1-3735 Cry6aa1 L07022 Narva et al., 1993 USP 5236843 1-1425 Cry6ba1 L07024 Narva et al., 1991 EP 0462721 1-1185 Cry7aa1 M64478 Lambert et al., 1992 AEM 58 2536-2542 184-3597 Cry7ab1 U04367 Payne & Fu, 1994 USP 5286486 1-3414 Cry7ab2 U04368 Payne & Fu, 1994 USP 5286486 1-3414 Cry8aa1 U04364 Foncerrada et al., 1992 EP 0498537 1-3471 Cry8ba1 U04365 Michaels et al., 1993 WO 93/15206 1-3507 Cry8ca1 U04366 Ogiwara et al., 1995 Curr Micro 30 227-235 1-3447 Cry9aa1 X58120 Smulevitch et al., 1991 FEBS 293 25-28 5807-9274 Cry9aa2 X58534 Gleave et al., 1992 JGM 138 55-62 385->3837 Cry9ba1 X75019 Shevelev et al., 1993 FEBS 336 79-82 26-3488 Cry9ca1 Z37527 Lambert et al., 1996 AEM 62 80-86 2096-5569 Cry9da1 D85560 Asano et al., 1997 AEM 63 1054-1057 47-3553 Cry9Da2 AF042733 Wasano & Ohba, 1998 Unpublished <1->1937 Cry9Ea1 AB011496 Midoh & Oyama, 1998 Unpublished 211-3660 Cry9 like AF093107 Wasano & Ohba, 1998 See Table 3 <1->1917 Cry10Aa1 M12662 Thorne et al., 1986 J Bact 166 801-811 941-2965 Cry10Aa2 E00614 Uorufuiirudo, 1996 JP 1986005098 940-2968 Cry11Aa1 M31737 Donovan et al., 1988 J Bact 170 4732-4738 41-1969 Cry11Aa2 M22860 Adams et al., 1989 J Bact 171 521-530 <1-235 Cry11Ba1 X86902 Delecluse, 1995 AEM 61 4230-4235 64-2238 Cry11Bb1 AF017416 Orduz et al., 1998 BBA 1388 267-272 97-2346 Cry12Aa1 L07027 Narva et al., 1991 EP 0462721 1->3771 Table 2 (contd). Name Acc No Reference Journal Coding Cry13Aa1 L07023 Narva et al., 1992 WO 92/19739 1-2409 Cry14Aa1 U13955 Narva et al., 1994 WO 94/16079 1-3558 Cry15Aa1 M76442 Brown & Whiteley, 1992 J Bact 174 549-557 1036-2055 Cry16Aa1 X94146 Barloy et al., 1996 J Bact 178 3099-3105 158-1996 Cry17Aa1 X99478 Barloy et al., 1998 Gene 211 293-299 12-1865 Cry18Aa1 X99049 Zhang et al., 1997 J Bact 179 4336-4341 1451-3571 Cry19Aa1 Y07603 Rosso & Delecluse, 1996 AEM 63 4449-4455 719-2662 Cry19Ba1 D88381 Hwang et al., 1998 SAB 21 179-184 626-2671 Cry20Aa1 U82518 Lee & Gill, 1997 AEM 63 4664-4670 60-2318 Cry21Aa1 I32932 Payne et al., 1996 USP 5589382 1-3501 Cry21Aa2 I66477 Feitelson, 1997 USP 5670365 1-3501 Cry22Aa1 I34547 Payne et al., 1997 USP 5596071 1-2169 Cry23Aa1 AF03048 Donovan & Slaney, 1998 WO 98/13498 Cry24Aa1 U88188 Kawalek & Gill, 1998 Unpublished 1-2022 Cry25Aa1 U88189 Kawalek & Gill, 1998 Unpublished 1-2028 Cyt1Aa1 X03182 Waalwijk et al., 1985 NAR 13 8207-8217 140-886 Cyt1Aa2 X04338 Ward & Ellar, 1986 JMB 191 1-11 509-1255 Cyt1Aa3 Y00135 Earp & Ellar, 1987 NAR 15 3619-3619 36-782 Cyt1Aa4 M35968 Galjart et al., 1987 Curr Micro 16 171-177 67-816 Cyt1Ab1 X98793 Thiery et al., 1997 AEM 63 468-473 28-777 Cyt1Ba1 U37196 Payne et al., 1995 USP 5436002 1-795 Cyt2Aa1 Z14147 Koni & Ellar, 1993 JMB 229 319-327 270-1046 Cyt2Ba1 U52043 Guerchicoff et al., 1997 AEM 63 2716-2721 287-655 Cyt2Ba2 AF020789 Guerchicoff et al., 1997 AEM 63 2716-2721 <1->469 Cyt2Ba3 AF022884 Guerchicoff et al., 1997 AEM 63 2716-2721 <1->469 Cyt2Ba4 AF022885 Guerchicoff et al., 1997 AEM 63 2716-2721 <1->469 Cyt2Ba5 AF022886 Guerchicoff et al., 1997 AEM 63 2716-2721 <1->471 Cyt2Ba6 AF034926 Guerchicoff et al., 1997 AEM 63 2716-2721 <1->472 Cyt2Bb1 U82519 Cheong & Gill, 1997 AEM 63 3254-3260 416-1204 spectra observed in Bt (González & Carlton, 1980; González et al., 1981; González et al., 1982; Reddy et al., 1987; Jarrett & Stephenson, 1990). New Bt strains have been developed by conjugation that are toxic to two insect orders. 2.1.6 Beta-exotoxin Beta-exotoxin is associated with certain Bt subspecies (Btd, Btg, Btte and Btt), and products made from these Bt subspecies may contain the toxin (Cantwell et al., 1964; Mohd-Salleh et al., 1980). This heat-stable nucleotide, which is composed of adenine, glucose and allaric acid, inhibits RNA polymerase enzymes by acting competitively with ATP (Faust, 1973; Farkas et al., 1977). Since RNA synthesis is a vital process in all life, beta-exotoxin exerts its toxicity for almost all forms of life tested including numerous insect species in the orders Coleoptera, Diptera and Lepidoptera. The presence of beta-exotoxin can be assayed using houseflies (Musca domestica) or high-performance liquid chromatography (HPLC) techniques (Campbell et al., 1987). Bt containing beta-exotoxin is used for the control of houseflies in some countries, but regulatory agencies currently prohibit the use of beta-exotoxin for other purposes. 2.1.7 Other Bt metabolites Commercial Bt products do not contain metabolites that are considered hazardous to humans and the environment. However, Bt, like other bacteria, may produce during the vegetative growth and sporulation stages an assortment of antibiotics, enzymes, metabolites and toxins that are biologically active and may have effects on both target and non-target organisms (NTOs). Using a non-quantitative (Lund & Granum, 1997) commercial Bc enterotoxin immunoassay (Tecra), Damgaard (1995) reported that vegetative cells grown from spores of commercial Bt products excreted a diarrhoeal enterotoxin. Damgaard et al. (1996a) found by Vero cell assay that Bt isolated from food was enterotoxigenic. None of these investigations estimated the quantity or activity of enterotoxins produced by the Bt strains. However, Shinagawa (1990) investigated a number of Bc and Bt isolates with an immunological assay and concluded that 43% of the Bt isolates had the same level of enterotoxins as enterotoxic Bc. Tayabali & Seligy (1997) found that vegetative Bt obtained from commercial products caused extensive damage to cultivated insect cells. At least three enterotoxic activities have been described in Bc (Agata et al., 1995; Lund & Granum, 1997), and some Bc isolates are known to produce an emetic toxin (Andersson et al., 1998). The emetic toxin is primarily associated with the Bc H-1 (Kramer & Gilbert, 1992; Nishikawa et al., 1996) serotype, and has not so far been associated with Bt isolates. Table 3. Bt-associated toxins or toxin-like proteins that have not been assigned a name or entered into the nomenclature for the reasons given Name Accession Journal Coding region Reason Reference Cry1i-like I90732 USP 5723758 Peptide seq Insufficient sequence data Payne et al., 1998 Cry1-like I90729 USP 5723758 Peptide seq Insufficient sequence data Payne et al., 1998 Cry9-like AF093107 unpublished <1->1917 Insufficient sequence data Wasano & Ohba, 1998 40kda M76442 J Bact 174 549-557 45-971 No reported toxicity Brown & Whiteley, 1992 Cryc35 X92691 unpublished 1-981 No reported toxicity Juarez-Perez et al., 1995 Crytdk D86346 unpublished 177-2645 No reported toxicity Hashimoto, 1996 Cryc53 X98616 unpublished 1-1005 No reported toxicity Juarez-Perez et al., 1996 p21med X98794 AEM 63 468-473 1-552 No reported toxicity Thiery et al., 1997 ET34 AF038049 WO 98/13498 No reported toxicity Donovan & Slaney, 1998 Vip3a(a) L48811 PNAS 93 5389-5394 739-3105 Not a crystal protein Estruch et al., 1996 Vip3a(b) L48812 PNAS 93 5389-5394 118-2484 Not a crystal protein Estruch et al., 1996 Table 4. Bacillus thuringiensis holotype toxins Name Old name Name Old name Cry1Aa CryIA(a) Cry5Ac Cry1Ab CryIA(b) Cry5Ba Cry1Ac CryIA(c) Cry6Aa CryVIA Cry1Ad CryIA(d) Cry6Ba CryVIB Cry1Ae CryIA(e) Cry7Aa CryIIIC Cry1Af Cry7Ab CryIIICb Cry1Ag Cry8Aa CryIIIE Cry1Ba CryIB Cry8Ba CryIIIG Cry1Bb ET5 Cry8Ca CryIIIF Cry1Bc PEG5 Cry9Aa CryIG Cry1Bd CryE1 Cry9Ba CryIX Cry1Be Cry9Ca CryIH Cry1Ca CryIC Cry9Da Cry1Cb CryIC(b) Cry9Ea Cry1Da CryID Cry10Aa CryIVC Cry1Db PrtB Cry11Aa CryIVD Cry1Ea CryIE Cry11Ba Jeg80 Cry1Eb CryIE(b) Cry11Bb Cry1Fa CryIF Cry12Aa CryVB Cry1Fb PrtD Cry13Aa CryVC Cry1Ga PrtA Cry14Aa CryVD Cry1Gb CryH2 Cry15Aa 34kDa Cry1Ha PrtC Cry16Aa cbm71 Cry1Hb Cry17Aa cbm72 Cry1Ia CryV Cry18Aa CryBP1 Cry1Ib CryV Cry19Aa Jeg65 Cry1Ic Cry19Ba Cry1Ja ET4 Cry20Aa Cry1Jb ET1 Cry21Aa Cry1Jc Cry22Aa Cry1Ka Cry23Aa Cry2Aa CryIIA Cry24Aa Jeg72 Cry2Ab CryIIB Cry25Aa Jeg74 Cry2Ac CryIIC Cry26Aa Cry3Aa CryIIIA Cry27Aa Cry3Ba CryIIIB Cry28Aa Cry3Bb CryIIIBb Cyt1Aa CytA Cry3Ca CryIIID Cyt1Ab CytM Cry4Aa CryIVA Cyt1Ba Cry4Ba CryIVB Cyt2Aa CytB Cry5Aa CryVA(a) Cyt2Ba "CytB" Cry5Ab CryVA(b) Cyt2Bb Alpha-toxin is a phospholipase C, which primarily affects the cell membrane phospholipids (Heimpel, 1954; Bonnefoi & Béguin, 1959). Gamma-toxin is toxic to sawflies (Tenthredinidae), but the mode of action of this heat-labile toxin has not been determined (Heimpel, 1967). The so called "water-soluble toxin" paralyses Lepidoptera (Fast, 1971), and the "mouse factor exotoxin" is toxic to mice as well as to Lepidoptera (Krieg, 1971). The modes of action of these toxins have not been delineated. A novel Bt vegetative insecticidal protein (Vip3A) has been identified from the culture media of some Bt strains (Estruch et al., 1996). Several Bt and Bc enzymes have been described which may play a role in non-target activity: phospholipase (Damgaard et al., 1996b), sphingomyelinase (Gilmore et al., 1989), protease (Hotha & Banik, 1997), chitinase (Sampson & Gooday, 1998), and haemolysin (Baida & Kuzmin, 1995). 2.2 Bioassays 2.2.1 Spore counts Bacterial spore counts do not necessarily reflect the insecticidal activity of a Bt strain or Bt product because the number and amount of ICPs produced per bacterial cell can vary. 2.2.2 International bioassay for ICPs The final formulation of each Bt product is bioassayed against an accepted international standard using a specific test insect (Dulmage et al., 1981; de Barjac & Larget-Thiery, 1984). Its potency is defined in ITU/mg product. The standardization allows comparison of different formulations in the laboratory. Currently, the larvicidal activity is expressed in terms of lethal doses (LD50) or lethal concentrations (e.g., LC50, LC90) according to the bioassay method used. For example, when susceptible mosquito larvae are exposed to Bti ICP, they have an LC50 of approximately 10 ng/ml water. A Bti whole culture gives an LC50 of approximately 103 cells/ml for susceptible mosquito larvae while a 109 cells/ml culture does not affect any laboratory mammals exposed by various routes. 3. MODE OF ACTION ON TARGET INSECTS 3.1 Bioactivity of field isolates The mode of action of Bt has been reviewed by Schnepf et al. (1998) and can be summarized in the following stages: 1) ingestion of sporulated Bt and ICP by an insect larva; 2) solubilization of the crystalline ICP in the midgut; 3) activation of the ICP by proteases; 4) binding of the activated ICP to specific receptors in the midgut cell membrane; 5) insertion of the toxin in the cell membrane and formation of pores and channels in the gut cell membrane, followed by destruction of the epithelial cells (Cooksey, 1971; Norris 1971; Fast, 1981; Huber & Lüthy, 1981; Lüthy & Ebersold, 1981; Smedley & Ellar, 1996); and 6) subsequent Bt spore germination and septicaemia may enhance mortality (Fig. 1). The specific bioactivity of Bt is dominated by the ICPs that are encoded by the cry genes and are active against susceptible species in the insect orders Coleoptera, Diptera and Lepidoptera. Specific Bt activities against other insect orders (Hymenoptera, Homoptera, Dictyoptera, Mallophaga) and to nematodes (Strongylida, Tylenchida), mites (Acari), flatworms (Digenea) and protozoa (Diplomonadida) have been described (Feitelson, 1993; Zukowski, 1995). The ICP must be ingested to be effective against the target (Visser et al., 1993). 3.2 Mechanism of action of Bt formulations The ICP-spore complexes of Bt are ingested by susceptible insect larvae. In the midgut the parasporal crystalline ICP is dissociated to the protoxin form, and the protoxin is then activated to a holotoxin by gut proteases (Warren et al., 1984; Jaquet et al., 1987; Aronson et al., 1991; Honée & Visser, 1993). Shortly afterwards, the gut becomes paralysed and the larva ceases to feed. The ICP structure and function have been reviewed in detail by Schnepf et al. (1998). Binding of the ICP to putative receptors is a major determinant of ICP specificity and the formation of pores in the midgut epithelial cells is a major mechanism of toxicity (Van Frankenhuyzen, 1993). The active toxin consists of three distinct domains (Höfte & Whiteley, 1989; Li et al., 1991; Grochulski et al., 1995). The three domains interact in a complex manner, but experimental data suggest that the C-terminal and middle domains of the toxin are involved in epithelial cell receptor binding and structural functions, while the N-terminal domain is primarily involved in ion channel and pore formation (Huber et al., 1981; Schnepf et al., 1998; Dean et al., 1996). Binding to specific receptors has been demonstrated to be closely related to the insecticidal spectrum of the ICPs (Denolf et al., 1997). Van Rie et al. (1989) found the affinity of these toxins similar for the tobacco budworm (Heliothis virescens) and the tomato hornworm (Manduca sexta) brush border membrane vesicles, but the number of binding sites differed and reflected varied bioactivity. However, the toxin affinity for binding sites does not appear constant for all insects. Pore or ion channel formation occurs after the binding to the receptor and insertion of the N-terminal domain into the membrane, whereby the regulation of the trans-membrane electric potential is disturbed. This can result in colloid-osmotic lysis of the cells, which is the main cytolytic mechanism that is common to all ICPs (Knowles & Ellar, 1987; Slatin et al., 1990; Schwartz et al., 1991; Schnepf et al., 1998). When the midgut epithelium of the larva is damaged, the haemolymph and gut contents can mix. This results in favourable conditions for the Bt spores to germinate. The resulting vegetative cells of Bt and the pre-existing microorganisms in the gut proliferate in the haemocoel causing septicaemia, and may thus contribute to the mortality of the insect larva. 3.3 Resistance of insect populations A number of insect populations of several different species with different levels of resistance to Bt have been obtained by laboratory selection experiments during the last 15 years (Schnepf et al., 1998). The species include Plodia interpunctella, Cadra cautella, Leptinotarsa decemlineata, Chrysomela scripta, Tricholplusia ni, Spodoptera littoralis, Spodoptera exigua, Heliothis virescens, Ostrinia nubilalis and Culex quinquefasciatus (Schnepf et al., 1998) and resistance is shown to either Btk, Bti, Btte or other Bt subspecies. During the last few years populations of the diamondback moth, Plutella xylostella, resistant to Btk and Bta have been found in heavily treated areas in numerous geographically isolated regions in the world, including Hawaii, Phillippines, Indonesia, Malaysia, Central America and some USA states (Schnepf et al., 1998). It is clear that this widespread appearance of resistance to Bt presents a cautionary tale for the way of using Bt and Bt toxin genes in pest management. Schnepf et al. (1998) have reviewed resistance management of Bt. 4. NATURAL AND TREATED HABITATS The Bt subspecies represents a group of organisms that occur naturally and can be added to an ecosystem to achieve insect control (Andrews et al., 1987; Stahly et al., 1991). In this monograph, a natural habitat is considered to be one where Bt can be isolated when there has been no previous history of application of the organism to that ecosystem, whereas a treated habitat is one where Bt can be isolated after a previous history of application of the organism for insect control. Insecticides formulated with Bt are being manufactured and used worldwide. These commercial Bt products may be applied as an insecticide to foliage, soil, water environments and food storage facilities. After application of Bt to an ecosystem, the organism may persist as a component of the natural microflora. 4.1 Natural occurrence of Bt Members of the Bacillus cereus group can be found in most ecological niches. Hansen et al. (1996) reviewed the occurrence of Bt in the environment. Although the early Bt isolates were pathogenic for insects, it is now apparent that several Bt isolates have no known target (Ohba & Aizawa, 1986; Ohba et al., 1988; Hansen et al., 1996, 1998; Damgaard et al., 1997b). This lack of insecticidal activity may be attributed to the loss of ability to produce ICPs (Gordon, 1977), which may be due to a mutation in the ICP gene that could prevent expression (Klier & Lecadet, 1976; Stahly et al., 1978; Dean, 1984) or to the loss of ICP encoding sequences. Finally, the lack of known activity of a Bt crystalline toxin might simply be explained by a failure to test against the actual target organism. The list of Bt targets is still increasing. Although our knowledge of the activity of Bt populations in the environment is limited, a certain level of turn-over and vegetative growth must occur, as annual and seasonal variations in numbers and subspecies diversity of Bt populations have been observed (Damgaard et al., 1997b; Kim et al., 1998). 4.1.1 Bt in insect hosts Numerous Bt subspecies have been isolated from dead or dying insect larvae and in most cases the isolate has toxic activity to the insect from which it was isolated (Goldberg & Margalit, 1977; de Barjac, 1981b; Hansen et al., 1996). These organisms have a narrow host range in the orders Coleoptera, Diptera and Lepidoptera and can proliferate within the bodies of their host insects. When the infected insect larva dies, the dead insect carcass usually contains relatively large quantities of spores and crystals that may be released into the environment (Prasertphon et al., 1973; Grassi & Deseö, 1984; Aly, 1985; Aly et al., 1985). Growth of Bt in non-target organisms has also been described. Eilenberg et al. (in press) found that Bt multiplication had occurred in non-target insects, which were also infected by insect pathogenic fungi. Akiba (1986) reported recycling of naturally occurring Bt in insect cadavers when competitive microorganisms were at a low density. Outbreaks of Bt in susceptible insect populations occur relatively infrequently; most outbreaks have been limited to situations where the insect density is relatively high, providing better opportunity for establishing the disease within the insect population (Lynch et al., 1976; Burges & Hurst, 1977; Vaòková & Purrini, 1979; Margalit & Dean, 1985). 4.1.2 Bt in soil The spores of Bt persist in soil, and vegetative growth occurs when nutrients are available (DeLucca et al., 1981; Akiba, 1986; Ohba & Aizawa, 1986; Travers et al., 1987; Martin & Travers, 1989). DeLucca et al. (1981) found that Bt represented between 0.5% and 0.005% of all Bacillus species isolated from soil samples in the USA. Martin & Travers (1989) recovered Bt from soils globally. Meadows (1993) isolated Bt from 785 of 1115 soil samples, and the percentage of samples that contained Bt ranged from 56% in New Zealand to 94% in samples from Asia and central and southern Africa. Ohba & Aizawa (1986) isolated Bt from 136 out of 189 soil samples in Japan. 4.1.3 Bt on plant surfaces Bt has been found extensively in the phylloplane. Numerous Bt subspecies have been recovered from coniferous trees, deciduous trees and vegetables, as well as from other herbs (Smith & Couche, 1991; Damgaard et al., 1997b). The Bt isolates have demonstrated a broad diversity both with specific activities to insects from the orders Coleoptera and Lepidoptera and with unknown activities (Smith & Couche, 1991; Damgaard et al., 1997b; Hansen et al., 1998). The bacterium has also been isolated from stored grain products (Meadows et al., 1992). 4.2 Treated habitats Treated habitats are the locations where Bt insecticides (usually a mixture of spores and crystals) have been applied. In Canada, Meadows (1993) estimated that approximately 1015 viable Btk spores per ha were released in a typical spray operation to control spruce budworm (Choristoneura fumiferana). 4.3 Environmental fate, distribution and movement Bt, like other members of the genus Bacillus, has the ability to form endospores that are resistant to inactivation by heat and desiccation and that persist in the environment under adverse conditions (Stahly et al., 1991). When considering the degradation of Bt in the environment, it is important to distinguish between changes in the numbers of viable spores and changes in biocidal activity. The survival and activity in the environment has been reviewed by Hansen et al. (1996). The distribution and environmental transport of applied Bt formulations are influenced by the type of application and various environmental factors (Bulla et al., 1985; Andrews et al., 1987). Bt formulations are used in agriculture and forestry against coleopteran and lepidopteran pests and are usually directed towards the surface of plants, while the Bt formulations for control of dipteran pests (mosquitos and blackflies) are applied to their aquatic, larval habitats. Many Bt insecticides exhibit poor stability under field conditions, and so frequent reapplication is required (Griego & Spence, 1978; Sorenson & Falcon, 1980; Beegle et al., 1981). 4.3.1 Distribution and fate of Bt in terrestrial habitats 184.108.40.206 Fate of Bt and ICP on plant surfaces Solar radiation appears to be the environmental factor most damaging to the stability of Bt ICP (Pinnock et al., 1974; Pinnock et al., 1977; Griego & Spence, 1978; Pusztai et al., 1991). Griego & Spence (1978) demonstrated that Bt spores are inactivated rapidly when exposed to UV radiation, while Pusztai et al. (1991) demonstrated that the tryptophan residues of the Bt protoxin are damaged by solar radiation in the 300-380 nm range. The combined effect of sunlight, leaf temperature and vapour pressure deficit appeared to contribute more to the reduction of bioactivity than any other single factor (Leong et al., 1980). Residue bioactivity may be detected up to 2 weeks after treatment with formulations containing UV protectants (Hostetter et al., 1975). Other studies on the effect of environmental exposure to Bt spores revealed that spore survival can be affected by the surface to which the material is applied. Pinnock et al. (1974) reported that the half-life of Bt spores on leaves of California live oak (Quercus agrifolia) was 3.9 days, as compared to a half-life of 0.63 days when applied to leaves of western redbud (Cercis occidentalis). Ignoffo (1992) summarized data for the reduction of spore viability and ICP activity on leaves of various plants in sunlight: Bt spore viability was reduced 80% in one day on red cedar leaves and 8% on soy bean leaves, while the ICP activity declined by 20% on red cedar leaves but 65% on soy bean leaves. Dent (1993) reported that Bt formulations on foliage frequently have half-lives of up to 10 days. However, unformulated Bt may have a half-life of only a few hours. Pedersen et al. (1995) found that the initial spore half-life was 16 h during the first week after spraying cabbage with unformulated Btk. There is also evidence that plant chemicals can inactivate Bt or influence infectivity. Lüthy (1986) demonstrated that extracts prepared from cotton leaves could inactivate ICPs. Commercially applied Bt may persist at low levels for considerable periods of time. Reardon & Haissig (1983) reported that Btk was still present on balsam fir (Abies balsamea) one year after applications to control spruce budworm. The proliferation of spores through infection of susceptible insects should not be discounted as a source of low levels of Bt in treated areas. Several studies have demonstrated that Bt is able to grow and sporulate in insect cadavers (Meadows, 1993). From dead Egyptian cotton leafworm (Spodoptera littoralis), Jarrett & Stephenson (1990) isolated between 5.0 × 105 and 9.2 × 107 spores per larva. Bt may be lost to the soil by overspray during application or by the action of rain on plant surfaces. Further losses arise from in situ degradation by environmental factors, such as ultraviolet (UV) radiation and microbial activity (Griego & Spence, 1978; Sorenson & Falcon, 1980; Beegle et al., 1981; West et al., 1984a,b). Pedersen et al. (1995) found that Bt was dispersed by rain splash from the soil to the lower leaves of cabbage. 220.127.116.11 Fate of Bt in soil Petras & Casida (1985) reported that Bt spore counts in soil declined by a factor of ten in the first 2 weeks after application and then remained constant for 8 months. The response was similar in spores from commercial and laboratory cultures. In contrast, vegetative cells introduced into the soil persisted for only a short time. Soil pH had little effect on their survival. Spore persistence for more than 2 weeks apparently resulted from the inability of the spores to germinate in the soil. Pedersen et al. (1995) sprayed unformulated Btk (1.2 5 104 cfu per g soil, spontaneous rifampicin-resistant mutant) on soil in 1993, and 2.3 × 103 cfu per g soil remained after 336 days. The field was left undisturbed, and 5´ years later spots with 1.5 × 103 Btk per g soil were found (Hansen & Hendriksen, 1999), but spots with very low Btk numbers were also recorded. These data indicate that the Btk had multiplied. West et al. (1984a,b) reported that vegetative cells in soil disappeared at a rapid, exponential rate, whereas parasporal crystals disappeared at a slower, non-exponential rate, and spore numbers remained unaltered through 91 days of incubation at 25°C, with no detectable germination. The proteinaceous crystalline protoxin of Bt has been shown to be degraded by soil microorganisms at an exponential rate with a half-life of about 3-6 days. Saleh et al. (1970) reported that Bt spores could remain viable for several months in the soil and germinate when soil conditions favoured bacterial growth. Bt spores do not appear to germinate readily in most soils and the crystalline protoxins are metabolized by other microorganisms. West et al. (1984a) reported that Bta in soil showed an exponential loss of insecticidal activity. The rate of loss was greater in non-sterile soil than in autoclaved soil. There was an initial rapid decrease, which stabilized at approximately 10% of the original inoculum level after 250 days incubation, until the cessation of sampling after two years. No loss of insecticidal activity was observed in autoclaved soil, which suggests that soil microorganisms were responsible for the loss of insecticidal activity in the natural, non-sterilized soil. Several studies determined that Bt did not grow under most natural soil conditions (West et al., 1984a,b; Akiba, 1986). The data suggested that this was attributable to a failure of Bt spores to germinate in soil under these conditions. The spore is the only state in which Bt persists in natural soils. An environmental fate study demonstrated no significant spore accumulation in either the organic or the mineral layers of soil over an 11-month period when Bt was applied aerially at 100 times the concentration used for operational programmes (Bernier et al., 1990). Studies have indicated that Bt is relatively immobile in soil. Martin & Reichelderfer (1989) found no vertical movement beyond a 6-cm deep zone in soil and less than 10 m lateral movement, even along drainage courses. Akiba (1991) reported a one-month irrigation study simulating the summer rainy season in Japan. There was no translocation of Bt below a depth of 10 cm. In soils receiving 45 cm simulated rainfall, Bt was detected to a depth of 3-6 cm. In tests using soil columns, Bt did not pass through a column of volcanic ash soil but a few spores were detected in flow-through water from an alluvial sand column. Results suggested that the major factor causing a decrease in the level of Bt was not a physical dilution due to the rainwater, but possibly an affinity of the spores for the soil particles. Venkateswerlu & Stotsky (1992) reported that adsorption and binding of Bt toxin proteins to soil particles were greater on montmorillonite than on kaolinite clays. Maximum adsorption occurred within 30 min, and adsorption was not significantly affected by temperature between 7°C and 50°C. 4.3.2 Distribution and fate of Bt in aquatic habitats Bti is often applied directly to water for the control of mosquitos and blackflies. Rapid sedimentation in all but the fastest flowing streams is regarded as an important limitation on the efficacy of such applications. Sheeran & Fisher (1992) demonstrated that the sedimentation of Bti is facilitated by adsorption onto particulate material. Ohana et al. (1987) found that spores may persist for at least 22 days in sediments, and the spores may be mobilized with such sediments during floods. Btk has been reported to survive in fresh water and in seawater for more than 70 and 40 days, respectively, at 20°C (Menon & De Mestral, 1985). A higher percentage of Btk was found to survive for extended periods in lake water than in tap and distilled water, presumably due to the presence of more nutrients in lake water. Bt has not been isolated from any drinking-water supplies. Spores of Bti remained viable for shorter periods when suspended in moving water than when in static bottles, indicating that static laboratory trials may overestimate the longevity of these spores in the environment (Yousten et al., 1992). Carcases of mosquito larvae killed by Bti have been shown to allow for the complete growth cycle (germination, vegetative growth and sporulation), thus becoming toxic themselves to scavenging yellow fever mosquito (Aedes aegypti) larvae (Khawaled et al., 1990). Contact of Bti with mud can result in an immediate disappearance of larvicidal activity, but it has little influence on spore viability (Ohana et al., 1987). The cessation of toxicity was found to be caused by bacterial adsorption to soil particles, but the inactivation could be reversed by washing the mud away. Special Bti formulations have been developed to prolong residence time of Bt at the surface or in the water column, where target insects feed. Manasherob et al. (1998) found germination, growth and sporulation of Bti in excreted food vacuoles of a protozoan. 4.3.3 Transport of Bt by non-target organisms In a field trial where Btk was sprayed on cabbage and soil, Pedersen et al. (1995) found that the Btk could be transported by non-target insects. Up to 103 cfu per g were found on surface-active insects, and carabid beetles carrying Btk were found up to 135 m from the Btk-treated area. In a study of interactions between Bti and fathead minnows (Pimephales promelas), ingestion of Bt resulted in a high number of recoverable spores in the gastrointestinal tract and faeces (Snarski, 1990). Bti spore counts decreased rapidly after test fish were transferred to clean water, but spores were detected in low numbers in faeces for over 2 weeks. The data indicated that minnows could disperse Bt spores in the freshwater environment. Meadows (1993) reported that, after the application of Bt on land, it may be dispersed by birds and mammals feeding on infected target insects. Some Bt-infected insect larvae may contain 6.6 × 108 to 4.2 × 109 spores per gram dry mass (Burges & Hurst 1977). 5. COMMERCIAL PRODUCTION 5.1 History of Bt and its commercial applications 5.1.1 Production levels Conventional Bt products, which utilize naturally occurring or modified Bt strains, account for approximately 90% of the world MPCA market (Bernhard & Utz, 1993). Current annual production of Bt has been estimated at 3000 or more tonnes in developed countries. In China, up to 10 000 tons are produced annually (personal communication by Guan Xiong, Fujian Agricultural, University, 1997). 5.1.2 Production processes, formulations and quality standards Bt products are produced using fermentation technology (Bernhard & Utz, 1993). Most commercial products contain ICP and viable Bt spores, but the spores are inactivated in some Bti products. During commercial-scale production of Bt products, there is little loss of bioactive components to the environment. The type of loss incurred is a function of the recovery method involved. No significant amount of bioactive component is lost from fermenter harvests if filtration is used to separate the insoluble solids (active ingredients) from the soluble liquid (inert) fraction of the harvest liquor, as shown by complete lack of bioactivity in the resulting liquid fraction. The liquid waste fractions may contain a low concentration of insoluble active component, but this is typically inactivated by processing in on-site waste treatment facilities. Although it is not a physical loss, measurable bioactivity is diminished if the recovered active material is processed through a dryer, due to the exposure of the bioactive components to the high temperatures required for drying. Guidelines for the handling of microorganisms during manufacture have been reviewed by Frommer et al. (1989). Commercial Bt formulations include wettable powders, suspension concentrates, water dispersible granules, oil miscible suspensions, capsule suspensions and granules (Tomlin, 1997). Quality standards for Bt fermentation products have been accepted by IUPAC (Quinlan, 1990). These standards include limits on the concentration of microbial contaminants and metabolites (Table 5). In most cases strains of Bt that produce beta-exotoxins are not approved for commercial application, although some commercial use has been approved for control of certain fly species that are not susceptible to ICPs (Carlberg et al., 1985). Table 5. Maximum allowable levels of microbial contamination in bacterial insecticides (IUPAC Recommendation; Quinlan, 1990) Types of microorganisms Maximum concentrations Viable mesophiles <1 × 105/g Viable yeasts and moulds <100/g Coliforms <10/g Staphylococcus aureus <1/g Salmonella <1/10g Lancefield Group D Streptococci <1 × 104/g 5.1.3 General patterns of use Commercial applications of Bt have been directed mainly against lepidopteran pests of agricultural and forest crops; however, in recent years strains active against coleopteran pests have also been marketed (Table 6). Strains of Bti active against dipteran vectors of parasitic disease organisms have been used in public health programmes. 18.104.22.168 Applications in agriculture and forestry Commercial use of Bt on agricultural and forest crops dates back nearly 30 years, when it became available in France. Use of Bt has increased greatly in recent years and the number of companies with a commercial interest in Bt products has increased from four in 1980 to at least 18 (Van Frankenhuyzen, 1993). Several commercial Bt products with Bta, Btk or Btte have been applied to crops using conventional spraying technology (Table 6). Various formulations have been used on major crops such as cotton, maize, soybeans, potatoes, tomatoes, various crop trees and stored grains. Formulations have ranged from ultralow-volume oil to high-volume, wettable powder and aqueous suspensions. In the main, naturally occurring Bt strains have been used, but transgenic microorganisms expressing Bt toxins have been developed by conjugation and by genetic manipulation, and in some cases, these have reached the commercial market. These modified organisms have been developed in order to increase host range, prolong field activity or improve delivery of toxins to target organisms. For example, the coleopteran-active cryIIIA gene has been transferred to a lepidopteran-active Btk (Carlton et al., 1990). A plasmid bearing an ICP gene has been transferred from Bt to a non-pathogenic leaf-colonizing isolate of Pseudomonas fluorescens; fixation of the transgenic cells produces ICP contained within a membrane which prolongs persistence (Gelernter, 1990). The gene expressing cryIA(c) ICP has been inserted in Clavibacter xyli subspecies cynodontis, a bacterium that colonizes plant vascular systems. This has been shown to deliver the ICP effectively to European corn borer (Ostrinia nubilalis) feeding within plant stems (Beach, 1990). Improvements in performance arising from such modifications are such that transgenic organisms and their products are likely to be used much more widely in the future. 22.214.171.124 Applications in vector control Bti has been used to control both mosquitos and blackflies in large-scale programmes (Lacey et al., 1982; Chilcott et al., 1983; Car, 1984; Car & de Moor, 1984; Cibulsky & Fusco, 1987; Becker & Margalit, 1993; Bernhard & Utz, 1993). For example, in Germany 23 tonnes of Bti wettable powder and 19 000 litres of liquid concentrate were used to control mosquitos (Anopheles and Culex species) between 1981 and 1991 in the Upper Rhine Valley (Becker & Margalit, 1993). In China, approximately 10 tonnes of Bti have been used in recent years to control the malarial vector, Anopheles sinensis. The Onchocerciasis Control Programme of West Africa used more than five million litres of Bti from 1982 to 1997 to control blackflies (Simulium damnosum), the vector of the onchocerciasis filarial worm (Onchocerca volvulus), on the Upper Volta River System. The Programme was initially based solely on the control of the vector, Simulium damnosum sensu lato, by spraying the insecticide at breeding sites on river systems, where larval stages develop. At the peak of larvicidal activities about 50 000 km of rivers were treated in an area of over one million km2. The purpose was to interrupt the transmission of the parasite Onchocerca volvulus. The interruption of the cycle is achieved by destroying larval stages through aerial application of insecticides to breeding sites. Insecticide application was undertaken weekly (Moulinier et al., 1995). In order to assess the environmental impact of such treatments a network of sampling stations throughout the programme area were established. Formulations of Bti range from wettable powder and fluid concentrates applied via conventional spray equipment from ground and air to slow-release briquet and tablet formulations. Examples of commercial Bti products are listed in Table 6. Table 6. Examples of commercial Bt products used against agricultural, forestry and public health pests (Tomlin, 1997; see also the Internet site http://www.sipweb.org/bacteria.htm) Bt sub-species Commercial products Producer Bt (not defined) Rijin Scientific & Technological Development Bt (not defined) Bitayon Jewin-Joffe Industry Ltd Bt (not defined) Delfin, Thuricide SDS Biotech KK Btk Bactospeine, Biobit, Dipel, Foray Abbott (USA) Bollgard Ecogen/Crop Care Bactucide Caffaro Baturad Cequisa Condor, Crymax, Cutlass, Ecogen Foil, Jackpot, Lepinox, Rapax, Raven Jackpot, Lepinox, Rapax Intrachem Cordalene Agrichem Larvo Troy Costar, Delfin, Design, Novartis/Thermo Trilogy Co Javelin, Steward, Thuricide, Vault Ecotech Bio, Ecotech Pro Ecogen/Roussel-Uclaf Halt Wockhardt Bta Xentari, Florbac Abbott Certan Novartis Btte Novodor Abbott Bti Bactimos, Gnatrol, Vectobac Abbott Acrobe Cyanamid JieJueLing, MieJueLing Huazhong Agricultural University Teknar Novartis/ThermoTrilogy Co Bt Ybt-1520 Mianfeng pesticide Huazhong Agricultural University Bt chinesensis Shuangdo preparation Huazhong Agricultural University Btg Spicturin Tuticorin Alkali Chemicals and Fertilisers Ltd 6. EFFECTS ON ANIMALS 6.1 Mammals Microbial pest control agents (MPCA) can, in principle, cause harmful effects via toxicity, inflammation, or a combination of these effects. The presence of bacteria in a specimen derived from tissues does not necessarily mean infection. Colonization refers to the multiplication of MPCA either on the surface or within an animal/human organism without causing any tissue damage. Persistence refers to the ability to recover the inoculum of the MPCA over time. Persistence and transient disturbances of the normal microbial flora are to be expected after exposure of experimental animals to MPCA, since clearance of the inoculum is not instantaneous. Persistence may not be equated with infection (Siegel & Shadduck, 1990). Infection by a MPCA means that there is evidence of the establishment and proliferation of the MPCA in tissues, coupled with tissue damage. Evidence of multiplication includes a measurable increase in the total amount of MPCA, recovery of vegetative stages when spores were administered, and failure of the inoculum to clear. It cannot be determined solely on the basis of lesions since injection of foreign material can elicit an inflammatory process (Siegel et al., 1987). A classification of MPCA toxicity and infectivity has been proposed, in which MPCA is classified as toxic if an oral dose < 106 cfu per mouse causes mortality or clinical or pathological changes (Burges, 1980). However, any classification is very difficult because of the complexicity of the issue when dealing with living organisms (Ignoffo, 1973; Shadduck, 1983). Older reports do not discriminate between different strains of Bt but modern molecular techniques have proven that variability exists within strains with the same serotype (Helgason et al., 1998; Hansen & Hendriksen, 1997a,b). Mammalian toxicity studies on Bt-containing pesticides demonstrate that the tested isolates are not toxic or pathogenic (McClintock et al., 1995), as they occur in the products. Toxicity studies submitted to the US Environmental Protection Agency to support registration of Bt subspecies, and reviewed by McClintock et al. (1995), failed to show any significant adverse effects on body weight gain, clinical observations or upon necropsy. Infectivity/pathogenicity studies have shown that the intact rodent system responds as expected to eliminate Bt gradually from the body after oral, pulmonary or intravenous challenge. However, clearance of Bti and Btk is not instantaneous. An intact immune system is not a prerequisite for clearance of Bti and Btk. 6.1.1 Oral exposure In studies conducted with a single oral dose of laboratory grown Bt and commercial Bt formulations, there was no mortality associated with ingestion of Bti or Btk in mice and rats (Fisher & Rosner, 1959; de Barjac et al., 1980; Shadduck, 1980; Siegel et al., 1987) (Table 7). Additionally, in data summarized by McClintock et al. (1995), no toxicity or infectivity was observed following oral administration of various Bt subspecies at doses of up to 4.7 × 1011 cfu/kg in rats. In a study involving repeated oral exposure of mice and rats for 21 days with laboratory grown Bti, there was no mortality associated with ingestion of Bti and normal weight gain was observed in all treated rodents (de Barjac et al., 1980) (Table 8). Hadley et al. (1987) conducted a study in which sheep were repeatedly treated with two commercial Btk formulations for 60 days. The only clinical sign was loose stools in sheep exposed to one Btk formulation. There was also microscopic evidence of moderate to marked lymphoid hyperplasia of the Peyer's patches in the caecum and colon of two out of six sheep treated with one Btk formulation and in one out of six sheep treated with the other Btk formulation. The authors did not consider these findings clinically significant. Other multiple dose studies with Bt were summarized by McClintock et al. (1995). In rats, no toxicity or infectivity was associated with dietary exposure to Bti (4 g/kg per day ) for 3 months. Administration of 1.3 × 109 Btk spores/kg per day to rats by oral gavage was not toxic or infectious. McClintock et al. (1995) also reported the results of a 2-year study in which a commercial Btk preparation was fed to rats at 8400 mg/kg per day in the diet. Despite this excessive dose, the only effect observed was a decrease in body weight of females during weeks 10-104 of the study. 6.1.2 Inhalation exposure These tests primarily address the potential infectivity of a MPCA. Inhalation is a likely route by which humans and animals may be exposed to Bt during application. De Barjac et al. (1980) exposed 10 female Swiss mice for 12 min to 2 × 108 Bti spores (48-h laboratory grown whole culture). The mice were monitored for clinical signs for 15 days, and then killed. The lungs were removed aseptically and cultured for bacteria, but no Bti was recovered. Siegel et al. (1987) exposed 27 female Sprague-Dawley rats to 2 × 106 spores of a commercial Bti formulation for 30 min. Rats were serially killed over a 27-day period and the lungs were cultured. Table 7. Acute toxicity (single oral exposure) of Bt in experimental animals Subspecies Material tested Test animal Dosea Mortalityb Reference tested Btk Washed cells, 24-h culturec Rat, female, Sprague-Dawley 1.4 × 107cfu 0/6 Shadduck, 1980 Btk Commercial product Ratd 2 × 1011cfu 0/10 Fisher & Rosner, 1959 Bti 48-h culturec Mouse, female, Swiss 1.7 × 108cfu 0/20 de Barjac et al., 1980 Bti 48-h culturec Rat, female, Wistar 3.4 × 107cfu 0/10 de Barjac et al., 1980 Bti Washed cellsc, 24-h culture Rat, female, Sprague-Dawley 6.9 × 107cfu 0/6 Shadduck, 1980 Bti Washed commercial product Rat, female, Sprague-Dawley 4 × 107cfu 0/10 Siegel et al., 1987 a All doses given are per animal b Number dead/number treated c Laboratory grown culture d Breed and sex unknown Table 8. Repeated dose (oral exposure) toxicity of Bt in mice, rats and sheep Subspecies Material tested Test animal Dose Exposure Mortalitya Reference tested time Btk Commercial product Sheep, male, Rambouillet/Merino 1 × 1012cfu 5 months 0/6 Hadley et al., 1987 Btk Commercial product Sheep, male, Rambouillet/Merino 1 × 1012cfu 5 months 0/6 Hadley et al., 1987 Bti 48-h cultureb Mouse, female, Swiss 4.7 × 1010cfu 21 days 0/20 de Barjac et al., 1980 Bti 48-h cultureb Rat, female, Wistar 1.2 × 1011cfu 21 days 0/10 de Barjac et al., 1980 a Number dead/number exposed b Laboratory grown culture Recovery of Bti declined from 5.92 × 103 cfu/g lung tissue at 3 h after exposure to none detected at 7 days after exposure. No gross lung lesions were observed. Fisher & Rosner (1959) exposed 10 mice to 3 × 1010 spores of a commercial Btk product 4 times in a 6-day period. The Btk-treated mice exhibited no clinical signs during the treatment period and no gross pathological changes at necropsy. 6.1.3 Dermal exposure This test is similar to the dermal exposure tests used in chemical toxicology. Bt does not have any external contact toxicity due to its mode of action, as shown in the following studies. De Barjac et al. (1980) applied 5.1 × 107 cfu Bti of a 48-h laboratory grown culture to the skin of 20 female Swiss mice. No mortality was observed and there was no evidence of skin inflammation. Other studies, reviewed by McClintock et al. (1995), indicate that Bt was not toxic or pathogenic to rabbits following dermal exposure to various Bt subspecies at doses of up to 2500 mg/kg. In some cases, mild irritation was observed. 6.1.4 Dermal scarification exposure This test evaluates both the potential toxicity and infectivity of a MPCA. In the case of Bt, toxicity is unlikely due to its mode of action. However, this test also evaluates the importance of intact skin in preventing infection by Bt. Fisher & Rosner (1959) scarified the skin of 4 rabbits, then applied 2.2 × 106 cfu of a commercial Btk formulation. No skin inflammation or sign of infection was observed. De Barjac et al. (1980) applied 3.3 × 1013 cfu of a commercial Bti formulation to the skin of 6 male New Zealand White rabbits. No skin inflammation or sign of infection was observed. 6.1.5 Subcutaneous inoculation This route of exposure is considered a more challenging test of potential infectivity than oral or dermal exposure, because the barrier of the skin is breached. However, subcutaneous exposure may take place only if the skin is damaged by the spraying or is already otherwise damaged. De Barjac et al. (1980) subcutaneously inoculated 20 female Swiss mice and 10 tricolour guinea-pigs, respectively, with 8.5 × 107 cfu and 1.7 × 108 cfu of a 48-h laboratory-grown Bti culture. There was no evidence of infection and no mortality was observed. Siegel et al. (1987) subcutaneously inoculated 15 female BALB/c mice with 1 × 109 cfu of a commercial Bti formulation. Abscesses occurred at the injection site but these were attributed to the introduction of high concentrations of heat-stable foreign material, since they also occurred when autoclaved Bti was injected. There was no evidence of infection and no mortality was observed. 6.1.6 Ocular exposure The primary purpose of this procedure is to test for the irritancy of a MPCA, although this test also evaluates potential infectivity as well. In these tests, Bt may persist for days in rabbit eyes because of the depth of the conjunctival sac coupled with limited tear production by rabbits. De Barjac et al. (1980) inoculated the eyes of six male New Zealand White rabbits with 3.7 × 107 cfu of a 48-h laboratory-grown Bti culture. No conjunctivitis or ocular irritation was observed. Siegel & Shadduck (1990) inoculated 12 female New Zealand White rabbits with 5.4 × 106 cfu of a commercial Bti formulation. No ocular irritation or conjunctivitis was observed and no Bti was recovered by swabbing after one week. In data reviewed by McClintock et al. (1995), only mild irritation was observed following ocular administration of certain Bt subspecies to rabbits. Siegel et al. (1987) inoculated the eyes of 6 male New Zealand White rabbits with 50 mg of a dry powder-commercial Bti formulation for 9 days, and another 6 male New Zealand White rabbits were treated with 50 mg of a laboratory-grown Bti culture for 9 days. No ocular irritation or conjunctivitis was observed in the rabbits that received the commercial powder. The rabbits that received the laboratory culture experienced severe conjunctival congestion and discharge. This was not attributed to Bti but rather to the nature of the inoculum. The laboratory strain was a dry paste with hard clumps while the commercial formulation was a soft powder. 6.1.7 Intraperitoneal exposure The administration of a MPCA by this route is considered a highly challenging route of exposure. Human and animal exposure to Bt by this route is very unlikely to occur during the course of normal application of Bt. This route evaluates the ability of Bt to cause infection or produce toxic metabolites in the peritoneal cavity. Some of the safety studies that utilized this route of exposure also evaluated the clearance of Bt over time (Table 9). Additional studies employing mice have been conducted using this route of exposure, which evaluates the role played by an intact immune system in preventing infection by Bt. These studies were deemed necessary to assess the risk posed by Bt to humans undergoing immunosuppressive chemotherapy and the risk posed by Bt to humans infected with the human immunodeficiency viruses. Immune suppression in mice was accomplished either by use of corticosteroids, which inhibited helper T-cells and selectively damaged B-cell activity, or through the use of athymic mice, which lack the functional T lymphocyte component of their immune system. 126.96.36.199 Immune-intact animals De Barjac et al. (1980) intraperitoneally injected 100 female Swiss mice with 3.4 × 107 cfu of a 48-h laboratory-grown Bti culture and killed groups of 10 mice daily (Table 9). Blood samples were taken by cardiac puncture and Bt was recovered until day 8. No mortality was observed. Fisher & Rosner (1959) intraperitoneally injected 30 mice of unspecified sex and strain with a laboratory-grown Btk culture and withdrew cardiac blood samples 24, 48 and 72 h after injection. There was no mortality and Btk was recovered as late as 48 h after injection from heart blood. Siegel & Shadduck (1990) conducted three clearance studies using female CD-1 mice. In one experiment, 33 females were injected with 2.7 × 107 cfu of a washed commercial Bti formulation and serially killed over 80 days. Bti did not clear and was recovered from the heart blood on days 67 and 80. The investigators noted that the initial inoculum was composed of approximately 95% vegetative cells and that vegetative cells take longer to clear than spores. This was confirmed in a follow-up experiment in which two groups of 16 females each were injected with inocula containing 1.5 × 107 cfu of spores only or a 25% vegetative cell and 75% spore mixture. Both inocula cleared exponentially from the spleens of the mice but the 100% spore inoculum cleared sooner than did the inoculum that contained vegetative cells. These experiments demonstrated that Bti and Btk persist for a variable length of time in mice following injection but that they are cleared over time. These studies also suggest that the nature of the inoculum may play a role in the speed by which it is cleared. Data summarized by McClintock et al. (1995) indicate that toxicity (100% mortality in extreme cases) may be observed following injection of >108 cfu of certain Bt subspecies intraperitoneally in mice. Lower doses (< 107 cfu/mouse) were non-toxic. Death generally occurred shortly after injection, indicating that an infectious process had not occurred. Although the basis for the toxicity observed at doses >108 cfu is not understood, these findings are not considered as evidence of a hazard associated with Bt products, since the route of administration is not relevant to human and animal exposure conditions. Table 9. Acute toxicity of Bt after intraperitoneal injection of guinea-pigs, mice and rats Subspecies Material tested Test animal Dose Mortalitya Reference tested Btk Washed cell, 24-h cultureb Rat, female, Sprague-Dawley 1.4 × 109cfu 0/6 Shadduck, 1980 Btk Commercial product Mouse 3 × 109cfu 0/5 Fisher & Rosner, 1959 Bti 48-h cultureb Mouse, female, Swiss 6.8 × 107cfu 0/20 de Barjac et al., 1980 Bti 48-h cultureb Guinea-pig, female, tricolour 1.7 × 107cfu 0/10 de Barjac et al., 1980 Bti Washed cellb, 24-h culture Rat, female, Sprague-Dawley 6.9 × 107cfu 0/6 Shadduck, 1980 Bti Washed commercial product Rats, male and female Sprague-Dawley 4 × 107cfu 1/20 Siegel et al., 1987 a Number dead/number treated b Laboratory grown culture 188.8.131.52 Immune-suppressed animals Siegel et al. (1987) injected 42 female BALB/c mice with 1.25 mg of a cortisone acetate twice weekly in order to suppress their immune system and subsequently injected them with 3.4 × 107 cfu of a washed commercial Bti formulation. Three cortisone-treated mice but none of the non-cortisone-treated mice died but this mortality was attributed to injury caused by injection. In the remaining 39 mice Bti was still recovered in the spleen 49 days after injection. In a companion experiment, 42 athymic mice were injected with the same dose of a washed commercial Bti formulation. Twenty-six of the 42 died within 5 to 10 h after injection; autopsy did not reveal the cause of death. In the surviving mice, Bti was recovered in the spleen 49 days after the injection. In a follow-up experiment, 30 athymic mice were injected with 2.6 × 107 cfu of another (washed) commercial Bti formulation and serially killed over a 36-day period. No mortality occurred. Bti was still recovered on day 36 after injection. Siegel & Shadduck (1990) injected 24 athymic mice with 2.7 × 107 cfu of a washed commercial Bti formulation and evaluated clearance over a 27-day period. No mortality was observed and clearance was faster in the euthymic than athymic mice. Bti was still recovered 27 days after injection. These experiments demonstrated that an intact immune system is not essential to prevent infection by Bti and Btk, but the kinetics of clearance differ between athymic and euthymic mice as well as between corticosteroid-treated and untreated euthymic mice. Based on these data, immune-suppressed individuals do not face any increased risk of infection by Bt. 6.1.8 Effects of activated Bt ICP It has been demonstrated that the alkali-solubilized ICP from Bti is lethal when injected into mice (Thomas & Ellar, 1983). Alkali-solubilized Bti ICP was also cytolytic to human erythrocytes, mouse fibroblasts, and primary pig lymphocytes in vitro (Thomas & Ellar, 1983; Gill et al., 1987). This activity is attributed to a cytolytic factor encoded by Cyt A gene of Bti. Most other Bt subspecies lack this gene. Human exposure to activated Bti ICP is most unlikely. 6.1.9 Studies in wild animals Numerous studies have been conducted on wild animals as part of the registration process. Most of the data are proprietary and not publicly available. No adverse effects have been reported. In Canada, Innes & Bendell (1989) studied the effect of a commercial Btk formulation on small mammal populations in woodland. Populations of eight species of rodents (Clethrionomys gapperi, Eutamius minimus, Microtus chrotorrhinus, Napaeozapus insignis, Peromyscus maniculatus, Phenacomys intermedius, Tamias striatus and Zapus hudsonius) and four species of shrew (Blarina brevicauda, Sorex cinereus, Sorex fumeus and Sorex hoyii) were studied by trapping over a 3-month period and shown to be unaffected when compared to populations from untreated areas. This suggests that the ingestion of infected insects by shrews had no immediate effects on their populations. 6.2 Effects on birds In a number of studies (Table 10), the acute toxicity and pathogenicity of commercial Bta, Bti, Btk and Btte formulations for young bobwhite quail (Colinus virginianus) and young mallards (Anas platyrhynchus), when administered daily by oral gavage at high dosages, were evaluated (Beavers et al., 1989a,b; Lattin et al., 1990a,b,c,d; Beavers, 1991a,b). The Bt-treated birds showed no apparent toxicity or pathogenicity. In those studies which also evaluated feed consumption and weight gain, the Bt-treated birds showed no effect when compared with the non-treated controls. In Canada, Buckner et al. (1974) assessed the impact of Btk on breeding bird populations (13-14 families, 33-34 species) during a field trial for spruce budworm control. The bird populations in 10-ha control and treated plots were measured before and daily for 3 weeks after application. No differences were detected between the populations in the control and treated plots. In the USA, Gaddis & Corkran (1986) evaluated the effect of a Bt spray programme on the reproductive performance of the chestnut-backed chickadee (Parus rufescens). This study was undertaken to determine if secondary effects on the chickadees would result from the possible reduction of lepidopteran species, which contribute to the diet of this species. The data showed no treatment-related effect on the number of eggs per nest, percentage of eggs hatched, percentage of young fledged, percentage of nests fledging at least one young, or the body weight of the nestlings. 6.3 Effects on aquatic vertebrates The World Health Organization (WHO, 1982) reviewed laboratory and field studies, performed by that time, that examined the impact of Bt on frogs (Hyla regilla, Rana temporaria), goldfish (Carassius auratus), mosquito fish (Gambusia affinis), newts (Taricha torosa, Triturus vulgaris), rainwater killifish (Lucania parva) and toads (Bufo species). No adverse effects were reported. Under static renewal conditions, Boeri (1991) exposed rainbow trout (Oncorhynchus mykiss) to high concentrations (100 mg/litre) of a commercial Bta formulation for 96 h and observed no adverse effects (Table 11). Table 10. Effects of oral 5-day exposure of Bt on birds Materials Species Dose Results Reference testeda Bta Colinus virginianus 1714 mg (3.4 × 1011 cfu)/kg/day no toxicity or Beavers, 1991b pathogenicity observed Anas platyrhynchus 1714 mg (3.4 × 1011 cfu)/kg/day no toxicity or Beavers, 1991a pathogenicity observed Bti Colinus virginianus 3077 mg (3.4 × 1011 cfu)/kg/day no toxicity or Lattin et al., 1990d pathogenicity observed Anas platyrhynchus 3077 mg (6.2 × 1011 cfu)/kg/day no toxicity or Lattin et al., 1990b pathogenicity observed Btk Colinus virginianus 2857 mg (5.7 × 1010 cfu)/kg/day no toxicity or Lattin et al., 1990a pathogenicity observed Anas platyrhynchus 2857 mg (5.7 × 1010 cfu)/kg/day no toxicity or Lattin et al., 1990c pathogenicity observed Btte Colinus virginianus 740 mg (4 × 109 spores)/kg/day no toxicity or Beavers et al., 1989a pathogenicity observed Anas platyrhynchus 740 mg (4 × 109 spores)/kg/day no toxicity or Beavers et al., 1989b pathogenicity observed a commercial preparation Under static renewal conditions, Surprenant (1989) exposed rainbow trout (Oncorhynchus mykiss) to high concentrations (100 mg/litre) of a commercial Btte formulation for 96 h and observed no adverse effects (Table 11). During 30- or 32-day static renewal tests, bluegill sunfish (Lepomis macrochirus), sheepshead minnow (Cyprinodon variegatus) and rainbow trout (Oncorhynchus mykiss) were exposed to commercial Bti, Btk or Btte formulations at aqueous and dietary concentrations from 100 to 500 times the expected environmental concentrations (Table 11) (Christensen, 1990a,b,c,d,e,f,g,h). The results of these studies indicated that exposure to very high concentrations of Bti, Btk and Btte did not adversely affect the survival of these fish, nor did it produce lesions. In the Btk study, the rainbow trout had a 20% mortality during the last 4 days of the study (Christensen,1990b). This effect was attributed to the excessive competition for food that resulted from poor visibility due to the turbidity and the presence of suspended solids encountered in the water. In Canada, Buckner et al. (1974) assessed the impact of Btk on brook trout (Salvelinus fontinalis Mitchell), common white suckers (Catostomus commersoni Lacepede) and smallmouth bass (Micropterus dolomieui Lacepede) during a field trial for spruce budworm control. The fish populations were assessed visually in underwater surveys before and after the spray programme. No effect on their populations was seen. Two analyses of surveys of the impact of the larvicidal campaign in the Onchocerciasis Control Programme of West Africa, which compared fish populations during the programme with the normal yearly fluctuation, observed little or no effects on the non-target populations. However, few details were provided (Yameogo et al., 1988; Levêque et al., 1988; Calamari et al., 1998). 6.4. Effects on invertebrates 6.4.1 Effects on invertebrates other than insects The World Health Organization (WHO, 1982), reviewed laboratory and field studies performed up to that time that examined the impact of Bt on aquatic invertebrates, which included bivalve mollusks (oyster larvae, Crassostrea gigas, Ostrea edulis), copepods, decapods, flatworms, isopods, gastropods and ostracods. Of these organisms, only a few demonstrated any adverse effects. In Canada, Buckner et al. (1974) evaluated the impact of Btk on a number of aquatic invertebrates during a field trial for control of the spruce budworm. Populations of Amphipoda (amphipods), Decapoda (crayfish), Hydracarina (water-mites), Hirudinea (leeches), Table 11. Effects of Bt on fish Material Species Concentration Duration Results Reference testeda Bta Oncorhynchus mykiss 100 mg/litre water 96 h No-observed-effect level Boeri, 1991 Btk Lepomis macrochirus 2.9 × 109cfu/litre waterb 32 days No significant toxicity Christensen, 1990a 1.2 × 1010cfu/g dietc or pathology Oncorhynchus mykiss 2.9 × 109cfu/litre waterb 32 days 20% mortality but Christensen, 1990b 1.1 × 1010cfu/g dietc not infectivity Cyprinodon variegatus 2.6 × 1010cfu/litre waterc 30 days No significant toxicity Christensen, 1990c 3.3 × 109cfu/g dietc or pathology Bti Lepomis macrochirus 1.2 × 1010cfu/litre waterc 30 days No significant toxicity Christensen, 1990f 1.3 × 1010cfu/g dietc or pathology Oncorhynchus mykiss 1.1 × 1010cfu/litre waterc 32 days No significant toxicity Christensen, 1990g 1.7 × 1010cfu/g dietc or pathology Cyprinodon variegatus 1.3 × 1010cfu/litre waterc 30 days No significant toxicity Christensen, 1990h 2.1 × 1010cfu/g dietc or pathology Btte Salmo gairdneri 100 mg/litre water 96 h No-observed-effect level Surprenant, 1989 Oncorhynchus mykiss 1.6 × 1010 cfu/litre waterc 30 days No significant toxicity Christensen, 1990d 1.34 × 1010cfu/g dietc or pathology Cyprinodon variegatus 9.94 × 109cfu/g diet 30 days No significant toxicity Christensen, 1990e or pathology a commercial formulations b nominal concentration c measured average concentration Hydrozoa (freshwater hydra), Nematoda (roundworms), Oligochaeta (segmented worms), Porifera (freshwater sponges), Pulmonata (freshwater snails) and Turbellaria (flatworms) were determined by sampling 14 days prior to and up to 28 days after treatment. The populations of these aquatic invertebrates were not affected by the Btk treatment. Benz & Altwegg (1975) studied the impact of Bt treatment at 100 times the recommended rate on populations of the earthworm Lumbricus terrestris and found no effect. Horsburgh & Cobb (1981) reported that populations of the two-spotted spider mite (Tetranchus urticae) and Panonychus ulmi were not affected by biweekly sprays with a commercial Btk product. Weires & Smith (1977) determined that sprays of a commercial Btk product on apples during a 4-month season had no effect on the two-spotted spider mite (Tetranchus urticae) and Panonychus ulmi, or on two predatory mites (Amblyseius fallacis and Zetzellia mali). 6.4.2 Effects on non-target insects An extensive literature exists on the consequences of exposure of NTOs to Bt, including reports of several long-term field studies. The data have been reviewed periodically (e.g., WHO, 1982; Lacey & Mulla, 1990; Melin & Cozzi, 1990; Molloy, 1992; Otvos & Vanderveen, 1993). The range of non-target species that have been found to be susceptible to direct toxic action of Bt has remained small. A list of non-target species found to be insensitive to Btte was issued by Keller & Langenbruch (1993). In more than 30 years of commercial use, no serious, direct effects on NTOs have been reported as arising from Bt-based MPCAs. Several studies which identified effects of Bt on predators or parasitoids of susceptible insect species are listed by Navon (1993), but the effects have been small. Mortality in bees has been observed after exposure to vegetatively growing Bt but the effect does not seem to be related to spores or ICPs. 184.108.40.206 Aquatic insects Bti is specific in its toxicity to dipterans. Nevertheless, many studies have tested the effect of Bti applications on a wide range of aquatic insects. Lacey & Mulla (1990) summarized a number of studies of the effects of Bti on certain non-target arthropod species and arthropod populations in the laboratory and field (Table 12). The results of representative studies are summarized below. Table 12. Effects of Bti on non-target arthropodsa Arthropod order Type of study Resulta References Coleoptera Laboratory - Schnetter et al., 1981 Field - Mulla et al., 1982; Mulligan & Schaefer, 1982; Mulla, 1988 Diptera Laboratory + Garcia et al., 1980; Ali, 1981; (Chironomidae) Ali & Baggs, 1981; Schnetter et al., 1981 Field + Mulla et al., 1971; Ali, 1981; Mulligan & Schaefer, 1982; Rogatin & Baizhanov, 1984 Field - Miura et al., 1980 Ephemeroptera Laboratory - Ali, 1980; Schnetter et al., 1981; Mulligan & Schaefer, 1982 Field - Schnetter et al., 1981; Mulla et al., 1982, Mulligan & Schaefer, 1982; Mulla, 1988 Heteroptera Field - Schnetter et al., 1981; (Corixidae) Mulligan & Schaefer, 1982 Heteroptera Laboratory - Schnetter et al., 1981; (Notonectidae) Olejnicek & Maryskova, 1986; Aly & Mulla, 1987 Field - Mulla et al., 1982; Mulligan & Schaefer, 1982; Mulla, 1988 Field + Purcell, 1981 Odonata Laboratory - Mulla & Khasawinah, 1969; Mulligan & Schaefer, 1982; Aly, 1985; Aly & Mulla, 1987 Field - Mulla, 1988 a - = no effect reported; + = an effect was reported, but does not necessarily imply that either individual arthropods or populations of arthropods were adversely affected. Four species of chironomid larvae (Chironomus crassicaudatus, Chironomus decorus, Glyptotendipes paripes, Tanytarsus species) were tested with four Bti preparations. The chironomid larvae were less susceptible to Bti, being 13- to 75-fold more tolerant than mosquito larvae to the various Bti preparations (Ali, 1981; Ali & Baggs 1981). Garcia et al. (1980) induced low to high levels of mortality in some nematocerous Diptera, including a variety of taxa in the families Ceratopogonidae, Chironomidae and Dixidae, using dosages of Bti that were 50 to several hundredfold higher than concentrations used for mosquito control. Schnetter et al. (1981) reported complete mortality in chironomid larvae ( Chironomus thummi ) exposed to high levels of Bti for 48 h without food. Field-collected adult aquatic beetles exposed to Bti suffered little or no mortality (Schnetter et al., 1981). Ali (1980) tested a Bti formulation at 20 times the larvicidal dosage for mosquitos and reported no adverse effects against larval mayflies (Baetis species). Schnetter et al. (1981) reported that mayflies (Cloeon species) suffered no mortality when fed Bti at high dosages. Aly & Mulla (1987) fed Bti intoxicated mosquito larvae (Culex quinquefasciatus) to field-collected fourth to fifth instar backswimmers (Notonecta undulata). The predators were fed at the rate of 10 larvae per predator per day for 4 days, then the predators were fed unintoxicated mosquito larvae and observed for 15 to 17 days. The nymph and adult notonectids exhibited no adverse effects. Olejnicek & Maryskova (1986) observed no marked mortality in backswimmers (Notonecta glauca) that were fed Bti intoxicated mosquito larvae. Schnetter et al. (1981) found no mortality in backswimmers (Notonecta glauca) exposed for 48 h to high levels of Bti. Mosquito larvae intoxicated with extremely high dosages of Bti were fed to naiads of the dragonfly Tarnetrum corruptum and damselfly Enallagma civile; the duration of development of the dragonfly and damselfly naiads, from the time of exposure to emergence, was not affected (Aly, 1985; Aly & Mulla, 1987) Merritt et al. (1989) reported no evident of effects on the drift of aquatic invertebrates, or on the numbers of these invertebrates in benthic Surber samples, during a blackfly (Simulium species) control programme. In the USA, Molloy (1992) reviewed ten field trials where Bti was used against blackfly (Simulium species) larvae. He concluded that, although there was a potential for adverse impact of Bti on filter-feeding chironomids, the impact on stream insect communities overall was very small. Over a three-season period, Bti administered at mosquito larvicidal rates had no adverse effects on the larvae of diving beetles (Dytiscidae) or water scavengers (Hydrophylidae) (Mulla et al., 1982; Mulla, 1988). The application of a Bti formulation in a wildlife marsh showed no adverse effects on beetle larvae (Mulligan & Schaefer, 1982). Ali (1981) evaluated the efficacy of various levels of a Bti formulation against chironomids in the families Chironominae and Tanytarsinae and obtained mortality at dosages higher than those employed to control mosquito larvae. Miura et al. (1980), using mosquito larvicidal dosages of a commercial Bti product, showed no reduction in the field populations of chironomids following treatment. Mulla et al. (1971) reported marked reductions in some chironomid populations, using a commercial Bti product at rates of 20 to 40 times the mosquitocidal rates. Mulligan & Schaefer (1982) reported a 40 to 70% reduction in some chironomid species after application of a Bti formulation to a wildlife marsh. Rogatin & Baizhanov (1984) noted a significant reduction of chironomids after Bti exposure. Extensive quantitative observations were made on mayfly nymphs, mostly Callibaetis pacificus, but no notable effects were observed when Bti was applied against mosquito larvae (Mulla et al., 1982; Mulla, 1988). Mulligan & Schaefer (1982) found that Bti did not adversely affect mayfly nymphs (Callibaetis species). Schnetter et al. (1981) reported that mayflies (Cloeon species) were not affected when Bti was used in floodwater mosquito (Aedes vexans) larval habitats. Schnetter et al. (1981) collected water boatmen (Corisella species) from mosquito larval habitats on the upper Rhine river in Germany. The water boatman population was not affected after exposure to Bti for 48 h. Adverse effects were noted on backswimmers (Buenoa species, Notonecta undulata, and Notonecta unifasciata) during field trials with Bti (Mulla et al., 1982; Mulla, 1988). Mulligan & Schaefer (1982) reported that the backswimmer (Notonecta species) populations in a wetland marsh were not adversely affected by the application of a Bti formulation. Purcell (1981) noted reductions in populations of backswimmers (Buenoa elegans, Notonecta indica) after application of Bti, but attributed this to the flying activity of these predators. No adverse effects on naiads of the dragonfly (Tarnetrum corruptum) and damselfly (Enallagma civile) were reported when Bti was used against larval mosquito populations (Mulla & Khasawinah, 1969; Mulla, 1988). No notable reduction in the number of nymphs of several species of dragonfly (Anisoptera) and damselfly (Zygoptera) occurred when Bti was applied in a wetland marsh (Mulligan & Schaefer, 1982). In the follow-up to the Onchocerciasis Control Programme of West Africa (section 220.127.116.11), little or no effect on the non-target populations was observed. However, few details were provided (Yameogo et al., 1988; Levêque et al., 1988; Calamari et al., 1998). 18.104.22.168 Terrestrial insects Melin & Cozzi (1990) summarized a number of studies on the effects of Btk, Btg, Btt and Bte on non-target arthropod species and arthropod populations in the laboratory and field. Representative studies on Btk, Btg, Btt and Bte are listed in Tables 13 and 14. Obadofin & Finlayson (1977) determined that a commercial Btk product had a minimal effect on the ground beetle ( Bembidion lampros ). Wilkinson et al. (1975) evaluated the contact activity of a commercial Btk product for 5 days at levels equivalent to field rates on an adult ladybird beetle (Hippodamia convergens) and found no adverse effects. Workman (1977) exposed earwigs (Labidura riparia) to a commercial Btk product at rates equivalent to 10 times the normal field application rate. No mortality was observed in these predators. Hamed (1978-1979) found that two tachinid species (Bessa fugax and Zenilla dolosa) were not affected after being fed suspensions of a commercial Btk product. Horn (1983) observed a reduction in the number of syrphid larvae on collards sprayed with a commercial Btk product. This effect was attributed to a repellent effect on the syrphid adults. Hamed (1978-1979) found that Picromerus bidens was not adversely affected after feeding upon lepidopteran larvae (Yponomeuta evonymellus) that had fed upon leaves treated with commercial Btk products. Hassan (1983) determined a commercial Btk product to be harmless to adult lacewings (Chrysopa carnea) when they were exposed at normal field rate concentrations. Wilkinson et al. (1975) found negligible mortality in larval or adult lacewings (Chrysopa carnea) when a commercial Btk product was applied as a contact spray at recommended field rates. Yousten (1973) fed lethal quantities of Btk to larval cabbage loopers (Trichoplusia ni) and just prior to death offered these larvae to young Chinese praying mantids (Tenodera aridifolia subspecies sinensis). The mantids were not susceptible to the spore-crystal mixtures in the intact insect host. Asquith (1975) found that black ladybird beetles (Stethorus punctum) on apple trees were not affected by treatment with a commercial Btk product. Buckner et al. (1974) monitored populations of ground beetles following aerial spraying of spruce with two commercial Btk products and found no effect on these predators. Harding et al. (1972) detected no reduction in population levels of ladybird beetles (coccinellids), rove beetles (staphyllinids), or checkered beetles (clerids) in plots treated with a commercial Btk product. Johnson (1974) evaluated several commercial Btk products as both sprays and baits on tobacco. During the 2-year study, the populations of two coccinellids (Hippodamia convergens and Colemegilla maculata) were not affected by the microbial treatments. Wallner & Surgeoner (1974) found no effects on coccinellids (Cycloneda munda, Chilocorus bivulnerus and Adalia bipuncta) following forest sprays with a commercial Btk product. While evaluating a commercial Btk product for the control of gypsy moth (Lymantria dispar) and elm spanworm ( Ennomos subsignacius ), Dunbar et al. (1972) found no adverse effect on Table 13. Effects of Btk on non-target arthropods Arthropod order Type of study Resultsa References Acarina Field - Weires & Smith, 1977; Horsburgh & Cobb, 1981 Coleoptera Laboratory - Wilkinson et al., 1975; Obadofin & Finlayson, 1977 Field - Harding et al., 1972; Buckner et al., 1974; Johnson, 1974; Wallner & Surgeoner, 1974; Asquith, 1975 Dermaptera Laboratory - Workman, 1977 Diptera Laboratory - Hamed, 1978-1979 Laboratory - Horn, 1983 Field - Dunbar et al., 1972; Fusco, 1980 Heteroptera Laboratory - Hamed, 1978-1979 Field - Harding et al., 1972; Elsey, 1973; Jensen, 1974; Wallner & Surgeoner, 1974 Hymenoptera Laboratory - Krieg, 1973 (Honey-bees) Laboratory - Krieg et al., 1980 Field - Buckner et al., 1974 Hymenoptera Laboratory - Wallner & Surgeoner, 1974; (Parasitoids) Laboratory + Hassan & Krieg, 1975; Krieg et al., 1980 Dunbar & Johnson, 1975; Mück et al., 1981; Weseloh & Andreadis, 1982; Wallner et al., 1983; Thomas & Watson, 1986 Field - Dunbar et al., 1972; Buckner et al., 1974; Wanller & Surgeoner, 1974; Hamel, 1977; Morris et al., 1977; Morris et al., 1980; Fusco, 1980 Field + Weseloh et al., 1983 Table 13. (cont'd) Arthropod order Type of study Resultsa References Neuroptera Laboratory - Wilkinson et al., 1975; Hassan, 1983 Dictyoptera Laboratory - Yousten, 1973 (mantis) a - = no effect reported; + an effect was reported, but does not imply that either individual arthropods or populations of arthropods were adversely affected. Table 14. Effects of different Bt strains on non-target arthropods Arthropod order Type of study Resulta References Btg Hymenoptera Laboratory - Cantwell & Shieh, 1981 (Honey-bees) Field - Burges, 1977 Field - Burges & Bailey, 1968 Btt Coleoptera Field + Kazakova & Dzhunusov, 1977 Hymenoptera Laboratory + Krieg & Herfs, 1963; Krieg, 1973 (Honey-bees) Laboratory - Martouret & Euverte, 1964; Cantwell et al., 1966 Hymenoptera Laboratory + Hassan & Krieg, 1975; (Parasitoids) Salama et al., 1982; Hassan, 1983; Salama & Zaki, 1983 Laboratory - Krieg et al., 1980 Table 14. (cont'd) Arthropod order Type of study Resulta References Bte Coleoptera Laboratory - Salama & Zaki, 1983 Laboratory + Salama et al., 1982 Hymenoptera Laboratory + Salama & Zaki, 1983 (Parasitoids) Neuroptera Laboratory + Salama et al., 1982 a - = no effect reported; + = an effect was reported, but does not imply that either individual arthropods or populations of arthropods were adversely affected. two tachinids (Blepharipa scutellata and Parasitigena agilis). Fusco (1980) reported an increased incidence of parasitism by two tachinids (Blepharipa pratensis and Compsilura concinnata) when Btk was applied in a field study. Elsey (1973) reported no detrimental effect on spined stiltbug nymphs or adults (Jalysus spinosus) during a 2-month field study with a commercial Btk product. Harding et al. (1972) conducted a 2 year study to evaluate the effects of Btk on the natural enemies of the bollworm (Helicoverpa zea) on cotton (Gossypium hirsutum). Following applications of Btk against this pest, they reported no detectable effects on Anthocoridae (minute pirate bugs, Orius species), Lygaediae (bigeyed bugs, Geocoris species), Nabidae (damsel bugs, Nabis species), or Reduviidae (assassin bugs). Jensen (1974) used a commercial Btk product on soybeans (Glycine canescens) to control the green cloverworm (Plathypena scabra) and the velvetbean caterpillar (Anticarsis gemmitalis). No adverse effect was observed on Lygaediae (bigeyed bugs, Geocoris species) or Nabidae (damsel bugs, Nabis species). Wallner & Surgeoner (1974) found no effect on the spined soldier bug (Podisus maculiventris), following forest sprays of commercial Btk products to control the oakleaf caterpillar (Heterocampa manteo). Although many data exist, in a review of the effects of the use of Btk in Canada, Addison (1993) concluded that few studies on NTOs had used soil invertebrate species and soil conditions relevant to field conditions in Canadian forests. Salama & Zaki (1983) reared cotton leafworm larvae (Spodoptera littoralis) on a diet containing Bte and then fed these larvae to adult staphylinid beetles (Paederus alferii). Predator longevity was not significantly affected and no difference was seen in acceptability to predators between untreated larvae and those exposed to Bte. Salama et al. (1982) treated aphids with sprays of Bte and provided these treated insects to newly hatched coccinellid larvae (Coccinella undecimpunctata). The survival of larvae of predators was not affected by feeding on the treated prey. However, the duration of predator larval development was increased in the group treated with Bte and there was a definite reduction in prey consumption. Salama et al. (1982) evaluated the effect of Bte on the development of lacewing larvae (Chrysopa carnea) by presenting them with either sprayed aphids or treated cotton leafworm larvae (Spodoptera littoralis). When fed either the sprayed aphids or the treated cotton leafworms, the duration of larval development was significantly extended and prey consumption was significantly reduced. 22.214.171.124 Honey-bees Krieg (1973) observed mortality in adult honey-bees (Apis mellifera) that were fed non-sporulated broth cultures of Btk. The mortalities were attributed to the thermolabile alpha-toxin. Since alpha-toxin is inactivated during sporulation, the toxin would not present a problem in sporulated commercial Btk products. When Krieg et al. (1980) fed fully sporulated cultures of Btk to adult honey-bees at concentrations of 1 × 108 spores and crystal per bee over a 7-day period, no harmful effects were observed. Cantwell & Shieh (1981) fed a 1:20 solution of Btg in a sucrose solution to newly emerged adult honey-bees. After 14 days, there was no difference in mortality between treated and untreated groups. Treatment of hives resulted in no adverse effect on the adult workers or colony life as determined by egg laying, brood production, brood capping, or honey production. Buckner et al. (1974) observed no adverse effects on honey-bees following aerial spraying of spruce (Picea species) with commercial Btk products. Cantwell et al. (1966) fed honey-bees sugar solutions containing Btt spores, Btt culture supernatant with beta-exotoxin, and Btt crystals. The crystals did not harm the bees, but the supernatant caused nearly 100% mortality at day 7. Significant mortality was seen in the spore-treated bees at 8 days and was attributed to bacterial septicaemia. It should be noted that the dosages of each treatment were many times higher than the bees would be exposed to in the course of a lepidopteran control programme. Krieg (1973) reported mortality in honey-bees fed whole nonsporulated cultures of Btt, which was attributed to the presence of beta-exotoxin. Krieg & Herfs (1963) reported that vegetative cells of Btt did not harm honey-bees; however, they reported toxicity in Btt preparations containing the beta-exotoxin. Martouret & Euverte (1964) fed worker honey-bees cultures of Btt incorporated into mixtures of sugar, honey and clay. Complete mortality was seen at 7 days for the spore-crystal-exotoxin preparation and at 14 days for the spore-crystal complex. 126.96.36.199 Parasitoids a) Btk Dunbar & Johnson (1975) collected adult parasitoids (Cardiochiles nigriceps) in the field and fed them suspensions of a commercial Btk product. In the group fed Btk, shorter life spans were reported. Since the investigators could not be sure whether feeding actually took place, starvation may have been the cause of death. Hassan & Krieg (1975) observed no adverse effects on adult chalcid wasps (Trichogramma cacoeciae) that were fed suspensions of a commercial Btk product. Krieg et al. (1980) fed washed spores and crystals of Btk (5 × 107 spores and crystals) for 7 days to adult chalcid wasps (Trichogramma cacoeciae) and observed no mortality or reduced capacity to parasitize. Mück et al. (1981) reported significant mortality in adult braconids (Cotesia glomerata) that were fed a commercial Btk product at rates of 108 and 109 spores per ml, but observed little effect on the adult parasitoids (Pimpla turionellae). They reported midgut epithelial damage in the Pimpla turionellae, which resulted from the ICP. Thomas & Watson (1986) found lower survival in adult ichneumonids (Hyposoter exiguae) fed suspensions of a commercial Btk product. They concluded the mortality was due to the spore-crystal complex. Wallner & Surgeoner (1974) observed no effect on parasitoids following treatments with commercial Btk products for control of the notodontid moth (Heterocampa manteo). Wallner et al. (1983) reported an indirect effect on the braconid Rogas lymantriae when it parasitized gypsy moth (Lymantria dispar) hosts fed Btk. The sex ratio of the parasitoid offspring was skewed towards males in the treated larvae, as the female parasitoids lay more fertilized eggs in larger, untreated host larvae. Weseloh & Andreadis (1982) reported synergism in laboratory tests with gypsy moth larvae (Lymantria dispar) fed a commercial Btk product and exposed to the braconid (Cotesia melanoscelus). The percentage of parasitism was increased in Btk-intoxicated larvae since these grew more slowly and were at the approximate size suitable for parasitism for a longer time. Buckner et al. (1974) reported no detrimental effects on parasitoid populations following field application of a commercial Btk product. Dunbar et al. (1972) reported an increase in the percentage of parasitism of gypsy moth (Lymantria dispar) and elm spanworm (Ennomos subsignarius) larvae in forestry plots treated with a commercial Btk product. Fusco (1980) reported an increase in the percentage of parasitism of gypsy moth (Lymantria dispar) larvae by the braconids Cotesia melanoscelus and Phobocampe unicincta following aerial sprays with a commercial Btk product. Hamel (1977) found that parasitoids attacking early instar western spruce budworm larvae (Choristoneura occidentalis) increased in number following aerial application of a commercial Btk product, while older budworm larvae were reduced in number. In two field studies, commercial Btk products showed no detrimental effects on parasitoid populations (Morris et al., 1977, 1980). Wallner & Surgeoner (1974) demonstrated 6- to 12-fold increases in the percentage of parasitism in gypsy moth larvae (Lymantria dispar) by the braconid Cotesia melanoscelus in forestry plots treated with a commercial Btk product. b) Btt Hassan (1983) observed the chalcid Trichogramma cacoeciae was not affected by exposure to dried surface films of Btt. Hassan & Krieg (1975) fed a suspension of three different commercial Bt products to adult chalcids (Trichogramma cacoeciae) and reported a minor reduction in the capacity to parasitize with Btt, but none with the other Bt products. The effect of the Btt product may have been due to the beta-exotoxin. Krieg et al. (1980) fed washed spores and crystals of Btt to adult chalcids (Trichogramma cacoeciae) for 7 days and observed no mortality or reduced capacity to parasitize. Lowered reproductive potential was observed for both the braconids Microplitis demolitor and Zele chlorophthalma following exposure to Btt (Salama et al., 1982; Salama & Zaki, 1983). Salama & Zaki (1983) reported increased development times for Zele chlorophthalma parasitizing the cotton leafworm Spodoptera littoralis treated with Bte. 7. EXPOSURE AND EFFECTS ON HUMANS There are some case reports on the occurrence of Bt in patients with different infectious diseases. However, none of these studies demonstrate an actual risk to human health by the use of Bt. They emphasize the need for further research on the production of toxins, knowledge of factors causing the genes of the toxins to be expressed, and knowledge on the natural occurrence of Bt and Bc. The medical practice does not discriminate between Bt and Bc as causative agents in infectious diseases. Therefore, the true proportion of Bt in Bc- induced disease is not known. 7.1 Bacillus thuringiensis For aeons, humans have been exposed to Bt in their natural habitats, particularly from soil, water and the phylloplane. However in the recorded scientific literature, only few adverse effects to these environmental Bt levels have been documented. The manufacture and field application of Bt products can result in aerosol and dermal exposure of workers and the human population, especially by spraying programmes in populated areas. Agricultural and horticultural uses of Bt can also result in dietary exposure. 7.1.1 Experimental exposure of humans Eight human volunteers ingested 1 gram of a Btk formulation (3 × 109 spores/g of powder) daily for 5 days. Of the eight volunteers, five also inhaled 100 mg of the Btk powder daily for five days. Comprehensive medical examinations immediately before, after, and 4 to 5 weeks later failed to demonstrate any adverse health effects, and all the blood chemistry and urinalysis tests were negative (Fisher & Rosner, 1959). Pivovarov et al. (1977) reported that ingestion of foods contaminated with Btg at concentrations of 105 to 109 cells/g caused nausea, vomiting, diarrhoea and tenesmus, colic-like pains in the abdomen, and fever in three of the four volunteers studied. The toxicity of the Btg strain may have been due to beta-exotoxin (Ray, 1990). 7.1.2. Exposure of workers during manufacture Many manufacturers of Bt products monitor the exposure and the associated health risks of their workers. Over a period of 30 years of production, there have been no reports of such workers having been adversely affected (RJ Cibulsky, personal communication, 1997). 7.1.3 Exposure of workers in spraying operations Noble et al. (1992) studied aerosol Btk exposure and subsequent nose and throat carriage of Bt by workers during a major spray programme for gypsy moth (Lymantria dispar) control. Spraying down from high lifts, spraying low foliage or spraying with prevailing breezes resulted in lower exposures of spray operators than did spraying upwards into trees. The mean exposure values ranged from 3.0 × 103 to 5.9 × 106 Bt spores/m3 sampled air. Individuals working most shifts during the spray period were exposed to 5.4 × 106 to 7.2 × 107 organisms. Nearly all the workers exposed to higher concentrations for several shifts (5 to 20) were culture-positive for Bt, and the majority of the workers remained culture-positive for 14 to 30 days. Of those who were culture-positive, eight workers reverted to a culture-negative status during the project or within 30 days of project completion. During the spray programme, some workers experienced chapped lips, dry skin, eye irritation, and nasal drip and stuffiness, but no serious health problems resulted. These symptoms were transient and frequently occurred during the beginning of a spray run and when Bt spray concentrations were increased. No significant differences were found with respect to gender or smoking status. In the same study, Noble et al. (1992) evaluated the health records of the general population in the county where the Btk spray programme was conducted. After examining the records of 3500 hospital emergency room admissions, 1140 family practice patients, and over 400 bacterial cultures from 10 hospitals, no evidence for community illness or infections attributed to Btk could be documented. Laferrière et al. (1987) demonstrated antibody titres in 11 of 107 workers exposed to Btk during a 2-year spraying period. By the middle of the spray operation, seven had developed titres to spore-crystal complexes, six to vegetative cells, and one to spores. Their titres tended to be low, but were higher in those exposed for a second year. Two months after the exposure ended, nine workers were retested. Of these workers, five had no detectable antibodies to the spore-crystal complexes, and four who had been among those with the highest titres against vegetative cells had significantly lower titres. Elliott et al. (1988) measured the exposure of individual workers and other individuals within the spraying area on the day of application during an aerial Btk spray programme for gypsy moth (Lymantria dispar) control. Concentrations of spores were measured using personal air sampling devices. The concentration of spores ranged from 0 to 1.1 × 104 cfu/m3 for individual workers, the highest concentration being incurred by a spray card checker who was in brief contact with the material. For non-working individuals, the average Bt exposure was 1.3 × 103 cfu/m3. In the spray area, a general survey showed concentrations of 0 to 4.2 × 103 cfu/m3. 7.1.4 Exposure of human populations by spraying operations over populated areas Btk and Bti have been sprayed over populated areas in several countries, including the USA, Canada and New Zealand. Some of these applications have been followed by public health surveillance programmes. In general, no (or very few) harmful effects have been reported among residents of the sprayed communities. 7.1.5 Clinical case reports Commercial Bt products have been used for over two decades, but Bt has been isolated in only a few cases of human bacterial infection. Samples & Buettner (1983) reported that a farm worker developed a corneal ulcer in one eye. It had been accidentally splashed with a commercial Btk product and Bt was subsequently isolated from the affected eye. The eye was treated with a topical antibiotic and corticosteroid and the corneal ulcer resolved 14 days after treatment. The report attributed the corneal ulcer to Bt infection. However, the possibility that Bt may have been a non-pathogenic contaminant of the ulcer was not considered. There are no other reports of Bt being associated with ocular infections in workers. During the investigation of a gastroenteritis outbreak in a chronic care institution, bacteria were isolated from four individuals and were identified as B. thuringiensis. The Bt isolates showed cytotoxic effects characteristic of B. cereus (Jackson et al., 1995). Damgaard et al. (1997a) isolated Bt in burn wounds in two patients. None of the isolates showed any toxicity to Vero cells. Hernandez et al. (1998) isolated Bt from a war wound; this strain (Bt konkukian) could infect immunosuppressed mice after cutaneous application. Warren et al. (1984) reported that a research worker developed a marked local reaction and lymphadenitis following a needle stick injury when handling Bti. Acinetobacter calcoaceticus and Bt were cultured from the exudate. The condition responded to penicillin. Green et al. (1990) reported that Bt was isolated from body fluids of 55 patients with different infectious diseases. In 52 of them, it was considered a contaminant, while in three cases with pre-existing medical problems, no firm conclusion was established concerning a causal relationship between the infection and Bt. Furthermore, Bt was isolated from the conjunctiva of a worker presenting with conjunctivitis, and with a history of a splash with a Bt product. Despite the widespread use of Bt-based products, only two incidents of possible allergic reactions have been reported to the US EPA (McClintock et al., 1995). After detailed analysis, neither of these was considered to be causally related to Bt. 7.1.6 Dietary exposure of the general population In some Asian countries, Bti has been added to domestic containers of drinking-water for mosquito control. From these high Bt exposures in drinking-water, no adverse effects in humans have been reported. In Africa, some rivers have been dosed with Bti at weekly intervals for blackfly control. No adverse effects in the human populations that drink the river water have been reported. Btk has been reported to survive for 1 to 2 months in fresh water and in seawater. However, viable Bt cultures have not been isolated from drinking-water supplies (Menon & De Mestral, 1985). There is little information on levels of Bt to be found in food, but it is possible that, in view of the widespread prevalence of Bt, its presence in food is common and is not always related to its use on food products. Bt spores have been shown to be unable to germinate in mammalian digestive systems; however, Bt has been isolated from faecal and urinary samples in occupational studies. Noble et al. (1992) reported that 5 out of 10 vegetable samples were positive for Btk. The positive samples were obtained from both supermarkets and from organically grown products. Such results may account for the recovery of Bt from faecal and urinary samples during the occupational studies and may reflect community exposure through food. 7.2 Bacillus cereus The close affiliation between Bt and Bc raises the question of whether strains of Bt can cause human illness during vegetative growth. During vegetative growth Bc can produce different kinds of toxins; these toxins can cause gastrointestinal diseases in humans after ingestion. The emetic toxin is an enzymatically synthesized peptide that causes vomiting (Granum, 1997) a few hours after ingestion. Most Bc strains producing this toxin seem to belong to the same serotype (Mikami et al., 1995; Nishikawa et al., 1996). The enterotoxins are a group of proteins causing abdominal pain and diarrhoea after an incubation period of 8-16 h. The enterotoxins causing gastrointestinal disease are most likely produced in the small intestine. Characteristics of the two types of disease caused by Bc are shown Table 15. Based on analysis of outbreaks the infective dose is believed to vary between 105 and 108 vegetative cells or activated spores per gram, but it may be so low as 104 (Granum, 1997). In addition to the two toxins, Bc can produce different lytic enzymes, e.g., haemolysins, which most likely are involved in the gastrointestinal diseases. In addition to gastrointestinal diseases Bc can cause various diseases, notably in immunosuppressed individuals (Drobniewski, 1993). Analysis of reported foodborne diseases reveals that Bc is frequently diagnosed as the cause of gastrointestinal disorders (Notermans & Batt, 1998) in many countries. Several food-borne disease outbreaks caused by Bc have been reported by Notermans & Batt (1998). However, Bc gastrointestinal diseases are highly under-reported, as both types of illness are relatively mild and usually last less than 24 h (Granum, 1997; Notermans & Batt, 1998). The incidence of Bc in foods varies between 101 and 107, the highest concentrations being found in herbs/spices and boiled rice (Notermans & Batt, 1998). The degree of toxicity of enterotoxins varies from Bc strain to strain, probably due to differences in toxin components (Lund & Granum, 1997). Hassan & Nabbut (1996) found that clinical Bc isolates from human diarrhoeal faeces were strong producers of diarrhoeal enterotoxin, while isolates from blood, wounds, normal faeces, milk and rice were weak producers of diarrhoeal enterotoxin (Hassan & Nabbut, 1996). This variation is reflected in the variable numbers (105-108 viable cells or spores per g) of Bc reported to cause symptoms in humans, and it has been suggested that foods containing more than 104 Bc per g may not be safe for consumption (Granum, 1997). Several European countries have a critical level of 104-105 Bc per g for acceptance of food products (Notermans & Batt, 1998). This critical level will include Bt, as the methods used do not discriminate between Bc and Bt. Table 15. Characteristics of the two types of disease caused by B. cereus (from Granum, 1997) Characteristic Emetic syndrome Diarrhoeal Infective dose (cells/g) 105-108 104-107 Toxin produced Preformed in food In the small intestine Type of toxin Cyclic peptide Protein Incubation period (h) 0.5-5 8-16 (occasionally >24) Duration of illness 6-24 12-24 (occasionally many days) Symptoms Vomiting, nausea, malaise Abdominal pain, diarrhoea 8. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT Owing to their specific mode of action, Bt products are unlikely to pose any hazard to humans or other vertebrates or to the great majority of non-target invertebrates provided that they are free from non-Bt microorganisms and from biologically active products other than the ICPs. Bt products may be safely used for the control of insect pests of agricultural and horticultural crops as well as forests. Bt is also safe for use in aquatic environments including drinking-water reservoirs for the control of mosquito, black fly and nuisance insect larvae. However, it should be noted that vegetative Bt have the potential for the production of Bc-like toxins, the significance of which as a cause of human disease is not known. 9. CONCLUSIONS AND RECOMMENDATIONS * Bt may be safely used for the control of insect pests of agricultural crops and forests. * Bti is safe for use in aquatic environments, including drinking-water reservoirs, for the control of mosquito, blackfly and nuisance insect larvae. * Bt products should contain the ICPs and be free from other microorganisms and biologically active metabolites. * New Bt products based on either new Bt strains and/or new ICPs require appropriate assessment. * FAO and WHO should develop standard specifications for Bt preparations as is done for chemical pesticides. * Good industrial large-scale practice (GILSP) standards should be employed for the production of Bt products. * Standardized valid methods for the assessment of gastrointestinal consequences of vegetatively produced agents should be developed. * The occurrence of resistant insect populations underscores the need for research on the relationships between cry-toxins and the ecology of Bt. * More research on the fate of Bt spores and ICPs in the environment is needed. This should cover the natural occurrence of Bt and Bc in foods and its relationship to exposure to Bt from its pesticide use. * Research into dose-response analysis and the consequent acceptable daily intake levels of Bt in the diet and beverages is a high priority. 10. PREVIOUS EVALUATIONS BY INTERNATIONAL ORGANISATIONS WHO (1985) considered the safe use of MPCA at the Ninth Meeting of the WHO Expert Committee on Vector Control in 1984. The report considered that addition of live microorganisms to drinking-water is undesirable and recommended that the use of Bt H-14 for the control of Aedes aegypti in drinking-water should be restricted to the asporogenic form. At the 1990 meeting, WHO (1991), after reviewing new research data, stated that its previous recommendation was unduly restrictive, provided that properly designed formulations were used. REFERENCES Addison JA (1993) Persistence and nontarget effects of Bacillus thuringiensis in soil: a review. 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Yousten AA (1973) Effect of the Bacillus thuringiensis delta-endotoxin on an insect predator which has consumed intoxicated cabbage looper larvae. J Invertebr Pathol, 21: 312-314. Yousten AA, Genthner FJ, & Benfield EF (1992) Fate of Bacillus sphaericus and Bacillus thuringiensis serovar israelensis in the aquatic environment. J Am Mosq Control Assoc, 8: 143-148. Zahner V, Momen H, Salles CA, & Rabinovitch L (1989) A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis. J Appl Bacteriol, 67: 275-282. Zukowski K (1995) [Laboratory examination of the effectiveness of new biological preparations for reducing populations of cockroaches (Blattella germanica L.)]. Rocz Panstw Zakl Hig, 46: 293-297 (in Polish). RÉSUMÉ La présente monographie traite des agents microbiens de lutte contre les nuisibles utilisant le bacille Bacillus thuringiensis (Bt). Ce bacille, qui est aussi appelé bacille de Thuringe, est également une source très importante de gènes utilisée pour conférer aux plantes et aux microorganismes transgéniques (ou organismes génétiquement modifiés, OGM) la faculté de résister aux ravageurs et autres nuisibles. Les effets que pourraient avoir ces OGM sur la santé humaine et l'environnement présentent divers aspects qui sont sans rapport ou tout au plus en rapport lointain avec les produits à base de Bt et n'entrent pas, par conséquent, dans le cadre de la présente monographie. 1. Identité, caractéristiques biologiques et méthodes d'analyse Bacillus thuringiensis est une bactérie anaérobie facultative, gram-positif, qui forme des inclusions protéiques caractéristiques adjacentes à l'endospore. Certaines sous-espèces de Bt peuvent synthétiser plusieurs inclusions parasporales. Le Bt est génétiquement indiscernable du Bc, exception faite de son aptitude à former des inclusions cristallines parasporales qui sont toxiques pour certains invertébrés, notamment les larves d'insectes appartenant aux ordres suivants: coléoptères, diptères et lépidoptères. Les inclusions parasporales sont constituées de diverses protéines cristallisées insecticides (ICP). Ces cristaux sont de forme variable (bipyramidale, cuboïdale, rhomboïdale plane, sphérique ou composite, c'est-à-dire comportant deux types de cristaux) selon la nature des protéines qui les composent. On a établi une corrélation partielle entre la morphologie des cristaux, la composition en protéines cristallisées et l'activité biologique vis-à-vis des insectes. Le taxon phénotypique fondamental est la sous-espèce, caractérisée par son sérotype flagellaire (H). En 1998, on avait déjà décrit 67 sous-espèces. Les gènes qui codent pour les ICP sont pour la plupart situés sur les plasmides. Chacune de ces protéines n'est le produit que d'un seul gène. La plupart des plasmides porteurs de gènes ICP se transmettent facilement d'une souche bactérienne à l'autre par conjugaison et peuvent aussi passer à une espèce bactérienne voisine. La classification phénotypique est maintenant complétée par une caractérisation basée sur la biologie moléculaire et plus précisément sur la séquence des gènes codant pour les cristaux (cry et cyt) plutôt que sur la spécificité vis-à-vis des insectes cibles. Divers domaines des ICP sont responsables de la sensibilité de l'hôte (reconnaissance des récepteurs) et de la toxicité (formation de pores). Parmi les techniques couramment utilisées pour caractériser les souches de Bt ou les inclusions protéiques elles-mêmes, on peut citer l'analyse des acides gras pariétaux, les anticorps monoclonaux, les sondes d'ADN oligonucléotidiques, les profils plasmidiques, l'analyse par amplification génique (PCR), la technique des empreintes génétiques et les profils électrophorétiques SDS-PAGE (électrophorèse en gel de polyacrylamide en présence de dodécylsulfate de sodium). Certaines sous-espèces de Bt produisent pendant leur croissance une bêta-exotoxine constitué d'un nucléotide thermostable qui peut contaminer les produits. Cette bêta-exotoxine est toxique pour presque toutes les formes de vie, y compris l'Homme et les insectes cibles. Au cours de leur croissance, les diverses souches de Bt produisent toutes sortes d'antibiotiques, d'enzymes, de métabolites et de toxines, y compris des toxines Bc, qui peuvent avoir des effets nocifs sur les organismes visés ou non visés. La numération des spores n'est pas le reflet fidèle de l'activité insecticide d'une souche de Bt ou d'un produit qui en dérive. Pour mesurer l'activité (en unités toxicologiques internationales (ITU) par mg), on procède à un test biologique sur insecte au moyen d'un étalon international. 2. Mode d'action sur les insectes cibles Les Bt sporulés ou les complexes spores-ICP doivent être ingérés par les larves d'insectes appartenant aux espèces sensibles. L'efficacité des ICP dépend de plusieurs facteurs: solubilisation dans l'intestin moyen, conversion de la protoxine en toxine biologiquement active sous l'action des enzymes protéolytiques, fixation de la toxine active aux récepteurs membranaires spécifiques par sa région C-terminale et formation de pores par la région N-terminale entraînant la lyse des cellules épithéliales. La germination des spores et la prolifération de cellules bactériennes végétatives dans l'hémocèle peut entraîner une septicémie qui contribue également à la mort. La spécificité d'hôte des différentes ICP est essentiellement déterminée par leur fixation aux récepteurs. 3. Habitats On a isolé de nombreuses sous-espèces de Bt sur des insectes morts ou mourants appartenant principalement à l'ordre des coléoptères, des diptères et des lépidoptères, mais nombreuses sont également celles qui ont été isolées du sol, de la surface des feuilles ou d'autres habitats. Les cadavres d'insectes contiennent souvent de grandes quantités de spores et d'ICP susceptibles de pénétrer dans l'environnement. Les sous-espèces actives contre les coléoptères et les lépidoptères sont principalement associées au sol et aux surfaces foliaires, alors que les sous-espèces actives contre les diptères se rencontrent communément dans les milieux aquatiques. Dans l'environnement, les spores sont capables de persister et de se développer en présence de conditions favorables et de nutriments appropriés. 4. Produits du commerce. Production et épandage Les produits commerciaux classiques à base de bacille de Thuringe, qui utilisent des souches naturelles, représentent environ 90% du marché mondial des agents microbiologiques de lutte contre les nuisibles. La plupart de ces produits contiennent des spores viables et des ICP, mais dans certains d'entre eux, les spores sont inactivées (Bti). Chaque année, on en produit quelque 13 000 tonnes par la technique de fermentation aérobie. Les produits classiques à base de Bt sont principalement destinés à lutter contre les lépidoptères qui ravagent les cultures et les plantations forestières; toutefois, ces dernières années, on a également commercialisé des souches actives contre les coléoptères. Les programmes de santé publique utilisent également des souches de Bti actives contre les diptères vecteurs de maladies virales ou parasitaires. Les formulations commerciales de bacille de Thuringe peuvent être épandues sur le feuillage, le sol, les étendues d'eaux ou dans les entrepôts de denrées alimentaires pour combattre les insectes. Une fois le produit épandu dans l'écosystème, les cellules bactériennes végétatives et les spores peuvent persister à des concentrations progressivement décroissantes pendant des semaines, des mois ou des années en tant que constituants de la microflore naturelle. Par contre, les ICP perdent leur activité biologique au bout de quelques heures ou de quelques jours. 5. Effets du Bt sur les organismes non visés Les études effectuées sur des mammifères et notamment celles qui ont porté sur des animaux de laboratoire ont consisté a évaluer l'infectiosité et la toxicité éventuelles de diverses préparations à base de Bt contenant notamment des ICP, des spores et des cellules bactériennes en phase végétative. Sous ces trois formes, les différentes sous-espèces de Bt se sont révélées pour la plupart non pathogènes et non toxiques pour les diverses espèces animales utilisées. On a montré que les cellules bactériennes en phase végétative et les spores persistaient pendant plusieurs semaines sans causer d'effets nocifs. En particulier, on a constaté que le Bt n'avait pas d'effets indésirables sur les oiseaux, les poissons et de nombreux autres vertébrés aquatiques non visés, lors d'études en laboratoire ou sur le terrain portant sur un grand nombre de spécimens. Il n'y a que relativement peu d'invertébrés aquatiques qui se révèlent sensibles au Bt en laboratoire ou sur le terrain. Par ailleurs, le bacille de Thuringe n'exerce pas non plus d'effets nocifs sur les lombrics. L'activité insecticide des différentes sous-espèces de Bt présente en général une spécificité d'hôte très marquée vis-à-vis des coléoptères, des diptères et des lépidoptères et on a montré qu'elle n'avait pratiquement aucun effet toxique direct sur les arthropodes non visés. La plupart des données relatives à l'innocuité de ces produits pour les arthropodes non visés concernent les sous-espèces de Bt actives contre les diptères et les lépidoptères. Les études consacrées aux formulations de Bti exemptes de contaminants toxiques ont montré qu'elles étaient sans danger pour la plupart des arthropodes non visés. Certains moucherons (chironomides appartenant à l'ordre des diptères) très proches des moustiques se sont révélés sensibles à de fortes doses de Bti mais ne sont nullement affectés aux doses utilisées pour la destruction des larves de moustiques. Des études sur le terrain ont mis en évidence des cas de réduction ou au contraire d'augmentation de certaines populations d'arthropodes non visés. Les études toxicologiques auxquelles ont été soumis de nombreux ordres d'insectes n'ont, pour la plupart d'entre eux, révélé aucun effet toxique imputable au Btk. On a observé une certaine mortalité chez des abeilles (Apis mellifera) qui avaient été soumises à des bacilles des sous-espèces Btt et Btk en phase végétative, mais il ne semble pas que les spores ou les ICP soient capables de produire un tel effet. En laboratoire et sur le terrain, le Btg n'a aucun effet toxique sur les abeilles. Les souches de Bte productrices de bêta-exotoxine se sont révélées capables d'exercer des effets toxiques sur les arthropodes non visés. 6. Exposition humaine et effets du bacille de Thuringe sur l'Homme Les ouvriers qui épandent des produits à base de Bt peuvent être fortement exposés à ces produits par contact cutané ou par inhalation d'aérosols. L'usage du Bt en agriculture peut entraîner la contamination de l'eau potable et des denrées alimentaires par le bacille. Toutefois, à l'exception de quelques cas d'irritation des yeux ou de la peau, on n'a pas connaissance d'effets nocifs attestés qui résulteraient d'une exposition professionnelle à des produits à base de Bt. Des volontaires qui avaient ingéré ou inhalé de grandes quantités de diverses formulations de Btk, n'ont ressenti aucun effet indésirable. On a mis en évidence des anticorps dirigés contre les cellules bactériennes, les spores et les complexes spores-cristaux chez des ouvriers chargés de l'épandage de produits à base de Bt; aucun effet indésirable n'a cependant été observé. On connaît le cas d'un certain nombre de patients atteints de maladies infectieuses chez lesquels la présence de Bt a été mise en évidence. Toutefois, aucune des études qui leur ont été consacrées n'a permis de conclure de façon certaine que l'utilisation du Bt comporte effectivement un risque pour la santé humaine. Il ne semble pas non plus que la présence de Bt dans l'eau destinée à la consommation ou dans les denrées alimentaires soit à l'origine d'effets indésirables chez l'Homme. 7. Conclusions Compte tenu de la spécificité de leur mode d'action, il est improbable que les produits à base de Bt constituent un danger quelconque pour l'Homme et les vertébrés ni pour la très grande majorité des invertébrés non visés, pour autant qu'ils ne contiennent pas d'autres microorganismes ou de substances biologiquement actives autres que les ICP. On peut utiliser ces produits en toute sécurité pour détruire les insectes qui ravagent les domaines agricoles et horticoles ainsi que les forêts. Ils sont également sans danger pour le milieu aquatique et on peut notamment les épandre dans les réservoirs d'eau potable pour lutter contre les moustiques, les simulies et les larves d'insectes incommodants. Il convient cependant de noter qu'en phase végétative, le Bt est capable de produire des toxines de type Bc dont on ignore si elles sont susceptibles de provoquer des maladies chez l'Homme. 1. RESUMEN Esta monografía trata sobre los plaguicidas microbianos (PM) basados en Bacillus thuringiensis (Bt). Esta bacteria es también una fuente clave de genes cuya expresión transgénica confiere resistencia frente a plagas a plantas y microorganismos, actuando como plaguicida en los denominados organismos modificados genéticamente (OMG). Los posibles efectos de los OMG sobre la salud humana y el medio están poco o nada relacionados con los productos basados en Bt, por lo que quedan fuera del ámbito de esta monografía. 1. Identidad, características biológicas y métodos de laboratorio Bt es una bacteria gram-positiva y anaerobia facultativa que forma inclusiones proteicas características junto a la endospora. Las subespecies de Bt pueden sintetizar más de una inclusión parasporal. Desde el punto de vista genético, Bt es indistinguible de Bc, exceptuando la capacidad de Bt para producir inclusiones parasporales cristalinas que son tóxicas para ciertos invertebrados, en particular para las larvas de insectos pertenecientes a los órdenes Coleóptera, Díptera y Lepidóptera. Dichas inclusiones parasporales están formadas por distintas proteínas cristalinas insecticidas (PCI). Los cristales tienen formas diversas (bipiramidales, cuboides, romboides planos, esféricos o compuestos por dos tipos de cristales), dependiendo de su composición en PCI. Se ha comprobado que existe una correlación parcial entre la morfología del cristal, la composición en PCI y la bioactividad frente a los insectos diana. El taxón fenotípico básico es la subespecie, identificada por el serotipo flagelar (H). Hasta 1998 se habían descrito 67 subespecies. Los genes que codifican las PCI se encuentran fundamentalmente en los plásmidos. Cada PCI es el producto de un solo gen. La mayoría de los plásmidos con genes de PCI se transfieren fácilmente por conjugación entre cepas de Bt y pueden transferirse a especies bacterianas emparentadas. La clasificación fenotípica se ha complementado en la actualidad con la caracterización biomolecular, basada en la secuencia de los genes de las proteínas cristalinas (cry y cyt), no en la especificidad para las especies diana. En las PCI, la susceptibilidad del huésped (reconocimiento de receptores) y la toxicidad (formación de poros) son responsabilidad de dominios distintos de la molécula. Las técnicas utilizadas habitualmente para caracterizar las cepas de Bt o la propia PCI consisten en análisis de los ácidos grasos de la pared celular, anticuerpos monoclonales, sondas de oligonucleótidos de ADN, perfiles de plásmidos, análisis por reacción en cadena de la polimerasa (PCR), estudios del ADN (huella genética) y perfiles de SDS-PAGE (dodecil sulfato sódico -- electroforesis en gel de poliacrilamida). La beta-exotoxina, un nucleótido termoestable, es sintetizada por algunas subespecies de Bt durante el crecimiento vegetativo y puede contaminar los productos. Es tóxica para casi todas las formas de vida, incluidos los seres humanos y los órdenes de insectos diana. Durante el crecimiento vegetativo, varias cepas de Bt producen una gama de antibióticos, enzimas, metabolitos y toxinas, incluidas toxinas de Bc, que pueden tener efectos nocivos tanto en las especies que son objetivo del plaguicida como en las que no lo son. Los recuentos de esporas no reflejan con exactitud la actividad insecticida de una cepa o un preparado de Bt. Se mide la potencia (UTI/mg) de cada producto mediante ensayos biológicos para los que se utiliza un patrón internacional basado en un insecto concreto. 2. Modo de acción en los insectos diana Es preciso que las larvas de los insectos susceptibles ingieran Bt esporulado con PCI o con complejos espora-PCI. La eficacia de la PCI depende de su solubilización en el intestino medio, de la conversión de la protoxina en la toxina biológicamente activa por la acción de enzimas proteolíticas, de la unión específica del dominio C-terminal de la toxina activa al receptor de membrana y de la formación de poros por parte del dominio N-terminal, con la consiguiente lisis de las células epiteliales. La germinación de esporas y la proliferación de células vegetativas en el hemocele puede ocasionar una septicemia y contribuir a la muerte. La unión de la PCI al receptor es el determinante principal de la especificidad de huésped para las distintas PCI de Bt. 3. Hábitats Se han aislado muchas subespecies de Bt a partir de insectos muertos o moribundos, la mayoría pertenecientes a los órdenes Coleóptera, Díptera y Lepidóptera, pero también del suelo, de superficies foliares y de otros hábitats. Los exoesqueletos de insectos muertos contienen a menudo grandes cantidades de esporas y PCI que pueden incorporarse al medio. Las subespecies de Bt activas frente a coleópteros y lepidópteros se asocian fundamentalmente con el suelo y el filoplano (superficies foliares), mientras que las activas frente a dípteros se hallan generalmente en medios acuáticos. En el ambiente, las esporas persisten y pueden entrar en crecimiento vegetativo cuando las condiciones son favorables y hay nutrientes disponibles. 4. Productos comerciales, producción y aplicación Los preparados convencionales de Bt, que utilizan cepas de Bt que aparecen de forma espontánea en la naturaleza, representan aproximadamente el 90% del mercado mundial de los PM. La mayoría de los preparados de Bt contienen PCI y esporas viables, pero en algunos productos de Bti las esporas están inactivadas. Cada año se producen aproximadamente 13.000 toneladas utilizando tecnología de fermentación aerobia. Los preparados convencionales de Bt tienen como objetivos primarios las plagas de lepidópteros que afectan a los cultivos agrícolas y forestales; sin embargo, en los últimos años también se han comercializado cepas de Bt activas frente a plagas de coleópteros. Se están utilizando en programas de salud pública cepas de Bti activas frente a dípteros vectores de enfermedades parasitarias y víricas. Las formulaciones comerciales de Bt pueden aplicarse como insecticidas al follaje, el suelo, el medio acuático o instalaciones de almacenamiento de alimentos. Tras aplicar una subespecie de Bt a un ecosistema, las células vegetativas y las esporas pueden persistir en concentraciones gradualmente decrecientes durante semanas, meses o años como un componente de la microflora natural. Sin embargo, las PCI pierden su actividad biológica en horas o días. 5. Efectos de Bt sobre especies que no son objetivo del plaguicida En estudios con mamíferos, en particular con animales de laboratorio, se ha evaluado la posible infecciosidad y toxicidad de diversos preparados de Bt, que comprenden las PCI, células vegetativas y esporas. Las PCI, las esporas y las células vegetativas de las subespecies de Bt, que se administraron por distintas vías, carecían en su mayoría de patogenicidad y toxicidad para las diversas especies animales estudiadas. Se comprobó que las células vegetativas o las esporas de Bt persistían durante semanas sin causar efectos adversos. No se ha observado que Bt afecte a pájaros, peces o muchas otras especies de vertebrados acuáticos que no son objetivo del plaguicida y se han estudiado en gran número de trabajos de laboratorio y de campo. Son relativamente pocas las especies de invertebrados acuáticos susceptibles a Bt, tanto en condiciones de laboratorio como de campo. Bt no afecta a las lombrices de tierra. En general, las subespecies de Bt muestran gran especificidad en su actividad insecticida frente a Coleóptera, Díptera y Lepidóptera, así como una toxicidad directa escasa, si no nula, frente a los artrópodos que no son su objetivo. La mayor parte de los datos disponibles sobre inocuidad en éstos se han obtenido con las subespecies de Bt activas frente a Díptera y Lepidóptera. Los estudios sobre formulaciones de Bti sin contaminantes tóxicos no han puesto de manifiesto efectos nocivos en la gran mayoría de los artrópodos que no son objetivo del plaguicida. Se ha comprobado que algunas moscas enanas (Díptera: Chironomidae), estrechamente emparentadas con los mosquitos, son sensibles a dosis altas de Bti, pero no se ven afectadas por dosis letales para larvas de mosquito. En estudios de campo se han descrito disminuciones o aumentos transitorios de las poblaciones de algunos artrópodos que no son objetivo del plaguicida. Se han estudiado muchos órdenes de insectos, tanto en el laboratorio como en trabajos de campo, y se ha comprobado que en la mayoría de ellos Btk no tiene efecto. Se ha observado mortalidad en abejas melíferas (Apis mellifera) tras la exposición a Btt y Btk en crecimiento vegetativo, pero el efecto no parece guardar relación con las esporas o las PCI. En los estudios de laboratorio y de campo, Btg no mostró efectos adversos sobre las abejas melíferas. Se ha comprobado que cepas de Bte productoras de beta-exotoxina tienen efectos adversos sobre artrópodos que no son objetivo del plaguicida. 6. Exposición a Bt y efectos sobre los seres humanos La aplicación agrícola de preparados de Bt puede suponer una considerable exposición de los trabajadores, tanto en aerosol como dérmica. Puede, asimismo, causar la contaminación del agua potable y los alimentos por la bacteria. Salvo casos notificados de irritación ocular y dérmica, no se han documentado efectos adversos sobre la salud tras la exposición laboral a preparados de Bt. Individuos voluntarios ingirieron e inhalaron grandes cantidades de una formulación de Btk sin sufrir efectos adversos. Se detectaron títulos de anticuerpos frente a las células vegetativas, las esporas y los complejos espora-cristal en trabajadores que pulverizaban preparados de Bt, pero no se registraron efectos adversos. Se han descrito algunos casos de presencia de Bt en pacientes con diversas enfermedades infecciosas. Sin embargo, ninguno de estos estudios demuestra de forma inequívoca que el uso de Bt entrañe un riesgo real para la salud humana. No se ha demostrado que Bt tenga efectos adversos en seres humanos cuando está presente en el agua potable o los alimentos. 7. Conclusiones Debido a la especificidad de su modo de acción, es improbable que los preparados de Bt entrañen peligro alguno para los seres humanos u otros vertebrados, o para la gran mayoría de los invertebrados que no constituyen su objetivo, siempre y cuando no contengan microorganismos distintos de Bt y productos biológicamente activos distintos de las PCI. Los preparados de Bt pueden utilizarse con seguridad para controlar las plagas de insectos de los cultivos agrícolas y hortícolas, así como las forestales. También es seguro su uso en medios acuáticos, incluidos los depósitos de agua potable, para controlar el mosquito, la mosca negra y las larvas de insectos dañinos. Sin embargo, es preciso señalar que las formas vegetativas de Bt pueden sintetizar toxinas del tipo de las producidas por Bc, cuya importancia como causa de enfermedades humanas se desconoce.