INTOX Home Page



    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
                        4.3.1.1   Fate of Bt and ICP on plant surfaces
                        4.3.1.2   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
                        5.1.3.1   Applications in agriculture and forestry
                        5.1.3.2   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
                        6.1.7.1   Immune-intact animals
                        6.1.7.2   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
                        6.4.2.1   Aquatic insects
                        6.4.2.2   Terrestrial insects
                        6.4.2.3   Honey-bees
                        6.4.2.4   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 irptc@unep.ch).

                             * * *

         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. Other publications have been
    concerned with epidemiological guidelines, evaluation of short-term
    tests for carcinogens, biomarkers, effects on the elderly and so
    forth.

         Since its inauguration the EHC Programme has widened its scope,
    and the importance of environmental effects, in addition to health
    effects, has been increasingly emphasized in the total evaluation of
    chemicals.

         The original impetus for the Programme came from World Health
    Assembly resolutions and the recommendations of the 1972 UN Conference
    on the Human Environment. Subsequently the work became an integral
    part of the International Programme on Chemical Safety (IPCS), a
    cooperative programme of UNEP, ILO and WHO. In this manner, with the
    strong support of the new partners, the importance of occupational
    health and environmental effects was fully recognized. The EHC
    monographs have become widely established, used and recognized
    throughout the world.

         The recommendations of the 1992 UN Conference on Environment and
    Development and the subsequent establishment of the Intergovernmental
    Forum on Chemical Safety with the priorities for action in the six
    programme areas of Chapter 19, Agenda 21, all lend further weight to
    the need for EHC assessments of the risks of chemicals.

    Scope

         The criteria monographs are intended to provide critical reviews
    on the effect on human health and the environment of chemicals and of
    combinations of chemicals and physical and biological agents. As such,
    they include and review studies that are of direct relevance for the
    evaluation. However, they do not describe  every study carried out.
    Worldwide data are used and are quoted from original studies, not from
    abstracts or reviews. Both published and unpublished reports are
    considered and it is incumbent on the authors to assess all the
    articles cited in the references. Preference is always given to
    published data. Unpublished data are only used when relevant published
    data are absent or when they are pivotal to the risk assessment. A
    detailed policy statement is available that describes the procedures
    used for unpublished proprietary data so that this information can be
    used in the evaluation without compromising its confidential nature
    (WHO (1990) Revised Guidelines for the Preparation of Environmental
    Health Criteria Monographs. PCS/90.69, Geneva, World Health
    Organization).

         In the evaluation of human health risks, sound human data,
    whenever available, are preferred to animal data. Animal and
     in vitro studies provide support and are used mainly to supply
    evidence missing from human studies. It is mandatory that research on
    human subjects is conducted in full accord with ethical principles,
    including the provisions of the Helsinki Declaration.

         The EHC monographs are intended to assist national and
    international authorities in making risk assessments and subsequent
    risk management decisions. They represent a thorough evaluation of
    risks and are not, in any sense, recommendations for regulation or
    standard setting. These latter are the exclusive purview of national
    and regional governments.

    Content

         The layout of EHC monographs for chemicals is outlined below.

    *    Summary -- a review of the salient facts and the risk evaluation
         of the chemical
    *    Identity -- physical and chemical properties, analytical methods
    *    Sources of exposure
    *    Environmental transport, distribution and transformation
    *    Environmental levels and human exposure
    *    Kinetics and metabolism in laboratory animals and humans
    *    Effects on laboratory mammals and  in vitro test systems
    *    Effects on humans
    *    Effects on other organisms in the laboratory and field
    *    Evaluation of human health risks and effects on the environment
    *    Conclusions and recommendations for protection of human health
         and the environment

    *    Further research
    *    Previous evaluations by international bodies, e.g., IARC, JECFA,
         JMPR

    Selection of chemicals

         Since the inception of the EHC Programme, the IPCS has organized
    meetings of scientists to establish lists of priority chemicals for
    subsequent evaluation. Such meetings have been held in: Ispra, Italy,
    1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North
    Carolina, USA, 1995. The selection of chemicals has been based on the
    following criteria: the existence of scientific evidence that the
    substance presents a hazard to human health and/or the environment;
    the possible use, persistence, accumulation or degradation of the
    substance shows that there may be significant human or environmental
    exposure; the size and nature of populations at risk (both human and
    other species) and risks for environment; international concern, i.e.
    the substance is of major interest to several countries; adequate data
    on the hazards are available.

         If an EHC monograph is proposed for a chemical not on the
    priority list, the IPCS Secretariat consults with the Cooperating
    Organizations and all the Participating Institutions before embarking
    on the preparation of the monograph.

    Procedures

         The order of procedures that result in the publication of an EHC
    monograph is shown in the flow chart. A designated staff member of
    IPCS, responsible for the scientific quality of the document, serves
    as Responsible Officer (RO). The IPCS Editor is responsible for layout
    and language. The first draft, prepared by consultants or, more
    usually, staff from an IPCS Participating Institution, is based
    initially on data provided from the International Register of
    Potentially Toxic Chemicals, and reference data bases such as Medline
    and Toxline.

         The draft document, when received by the RO, may require an
    initial review by a small panel of experts to determine its scientific
    quality and objectivity. Once the RO finds the document acceptable as
    a first draft, it is distributed, in its unedited form, to well over
    150 EHC contact points throughout the world who are asked to comment
    on its completeness and accuracy and, where necessary, provide
    additional material. The contact points, usually designated by
    governments, may be Participating Institutions, IPCS Focal Points, or
    individual scientists known for their particular expertise. Generally
    some four months are allowed before the comments are considered by the
    RO and author(s). A second draft incorporating comments received and
    approved by the Director, IPCS, is then distributed to Task Group
    members, who carry out the peer review, at least six weeks before
    their meeting.

         The Task Group members serve as individual scientists, not as
    representatives of any organization, government or industry. Their
    function is to evaluate the accuracy, significance and relevance of
    the information in the document and to assess the health and
    environmental risks from exposure to the chemical. A summary and
    recommendations for further research and improved safety aspects are
    also required. The composition of the Task Group is dictated by the
    range of expertise required for the subject of the meeting and by the
    need for a balanced geographical distribution.

         The three cooperating organizations of the IPCS recognize the
    important role played by nongovernmental organizations.
    Representatives from relevant national and international associations
    may be invited to join the Task Group as observers. While observers
    may provide a valuable contribution to the process, they can only
    speak at the invitation of the Chairperson. Observers do not
    participate in the final evaluation of the chemical; this is the sole
    responsibility of the Task Group members. When the Task Group
    considers it to be appropriate, it may meet  in camera.

         All individuals who as authors, consultants or advisers
    participate in the preparation of the EHC monograph must, in addition
    to serving in their personal capacity as scientists, inform the RO if
    at any time a conflict of interest, whether actual or potential, could
    be perceived in their work. They are required to sign a conflict of
    interest statement. Such a procedure ensures the transparency and
    probity of the process.

         When the Task Group has completed its review and the RO is
    satisfied as to the scientific correctness and completeness of the
    document, it then goes for language editing, reference checking, and
    preparation of camera-ready copy. After approval by the Director,
    IPCS, the monograph is submitted to the WHO Office of Publications for
    printing. At this time a copy of the final draft is sent to the
    Chairperson and Rapporteur of the Task Group to check for any errors.

         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.

    FIGURE 1

    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).

    FIGURE 1

         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

    4.3.1.1  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.

    4.3.1.2  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.

    5.1.3.1  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.

    5.1.3.2  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.

    6.1.7.1  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
    

    6.1.7.2  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.

    6.4.2.1  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 5.1.3.2), 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).

    6.4.2.2  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.

    6.4.2.3  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.

    6.4.2.4  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. Can J Forensic Res, 23: 2329-2342.

    Agata N, Ohta M, Arakawa Y, & Mori M (1995) The  bceT gene of
     Bacillus  cereus encodes an enterotoxic protein. Microbiology, 141:
    983-988.

    Ahmed R, Sankar-Mistry P, Jackson S, Ackermann H-W, & Kasatiya SS
    (1995)  Bacillus cereus phage typing as an epidemiological tool in
    outbreaks of food poisoning. J Clin Microbiol, 33: 636-640.

    Akiba Y (1986) Microbial ecology of  Bacillus thuringiensis: VI.
    Germination of  Bacillus thuringiensis spores in the soil. Appl
    Entomol Zool, 21: 76-80.

    Akiba Y (1991) Assessment of rainwater-mediated dispersion of
    field-sprayed  Bacillus thuringiensis in the soil. Appl Entomol Zool,
    26: 477-483.

    Ali A (1980) Nuisance chironomids and their control: a review. Bull
    Entomol Soc Am, 26: 3-16.

    Ali A (1981)  Bacillus thuringiensis serovar.  israelensis 
    (ABG-6108) against chironomids and some nontarget aquatic 
    invertebrates. J Invertebr Pathol, 38: 264-272.

    Ali A & Baggs RD (1981) Susceptibility of some Florida chironomids and
    mosquitoes to various formulations of  Bacillus thuringiensis serovar
     israelensis de Barjac. J Econ Entomol, 74: 672-677.

    Aly C (1985) Germination of  Bacillus thuringiensis var.  israelensis
    spores in the gut of  Aedes larvae (Diptera: Culicidae). J Invertebr
    Pathol, 45: 1-8.

    Aly C & Mulla MS (1987) Effect of two microbial insecticides on
    aquatic predators of mosquitoes. J Appl Entomol, 103: 113-118.

    Aly C, Mulla MS, & Federici BA (1985) Sporulation and toxin production
    by  Bacillus thuringiensis var.  israelensis in cadavers of mosquito
    larvae (Diptera: Culicidae). J Invertebr Pathol, 46: 251-258.

    Andersson MA, Mikkola R, Helin J, Andersson MC, & Salkinoja-Salonen M
    (1998) A novel sensitive bioassay for detection of  Bacillus cereus
    emetic toxin and related depsipeptide ionophores. Appl Environ
    Microbiol, 64: 1338-1343.

    Andrews RE Jr, Faust RM, Wabiko H, Raymond KC, & Bulla LA Jr (1987)
    The biotechnology of  Bacillus thuringiensis. CRC Crit Rev
    Biotechnol, 6: 163-232.

    Angus TA (1954) A bacterial toxin paralysing silkworm larvae. Nature
    (Lond), 173: 545-546.

    Aronson AI, Han ES, McGaughey W, & Johnson D (1991) The solubility of
    inclusion proteins from  Bacillus thuringiensis is dependent upon
    protoxin composition and is a factor in toxicity to insects. Appl
    Environ Microbiol, 57: 981-986.

    Asano S-I, Nukumizu Y, Bando H, Hzuka T, & Yamamoto T (1997) Cloning
    of novel enterotoxin genes from  Bacillus cereus and  Bacillus
    thuringiensis. Appl Environ Microbiol, 63: 1054-1057.

    Ash C, Farrow JA, Dorsch M, Stackebrandt E, & Collins MD (1991)
    Comparative analysis of  Bacillus anthracis,  Bacillus cereus, and
    related species on the basis of reverse transcriptase sequencing of
    16S rRNA. Int J System Bacteriol, 41: 343-346. 

    Asquith D (1975) Response of the predaceous black lady beetle
     Stethorus punctum (DeConte) to apple orchard insecticide treatments
    (Experiment No. D986-4007). Chicago, Illinois, Abbott Laboratories.

    Baida GE & Kuzmin NP (1995) Cloning and primary structure of a new
    hemolysin gene from  Bacillus cereus. Biochim Biophys Acta, 1264:
    151-154.

    Baumann L, Okamoto K, Unterman BM, Lynch MJ, & Bauman P (1984)
    Phenotypic characterization of  Bacillus thuringiensis and  Bacillus
     cereus. J Invertebr Pathol, 44: 329-341.

    Beach RM (1990) Application for an experimental use permit to ship and
    use a pesticide for experimental purposes only -- Permit No.
    58788-EUP-4 for InCideM 586. Hanover, Maryland, Crop Genetics
    International.

    Beattie SH, Holt C, Hirst D, & Williams AG (1998) Discrimination among
     Bacillus cereus, B. mycoides and  B. thuringiensis and some other
    species of the genus  Bacillus by Fourier transform infrared
    spectroscopy. FEMS Microbiol Lett, 164: 201-206.

    Beavers JB (1991a) ABG-6305: An avian oral pathogenicity and toxicity
    study in the mallard (Project No. 161-118). Easton, Maryland, Wildlife
    International Ltd, pp 1-20 (Unpublished Abbott document).

    Beavers JB (1991b) ABG-6305: An avian oral pathogenicity and toxicity
    study in the bobwhite (Project No. 161-117). Easton, Maryland,
    Wildlife International Ltd, pp 1-21 (Unpublished Abbott document).

    Beavers JB, Larsen AC, & Jaber M (1989a)  Bacillus thuringiensis var.
     tenebrionis: An avian single dose oral toxicity and pathogenicity
    study in the bobwhite (Project No. 161-109). Eston, Maryland, Wildlife
    International Ltd, pp 1-19 (Unpublished Abbott document).

    Beavers JB, Clauss BS, & Jaber M (1989b)  Bacillus thuringiensis var.
     tenebrionis: An avian single dose oral toxicity and pathogenicity
    study in the mallard (Project No. 161-108). Easton, Maryland, Wildlife
    International Ltd, pp 1-19 (Unpublished Abbott document).

    Becker N & Margalit J (1993) Use of  Bacillus thuringiensis
    israelensis against mosquitoes and blackflies. In: Entwistle PF, Cory
    JS, Bailey MJ, & Higgs S ed.  Bacillus thuringiensis, an
    environmental biopesticide: Theory and practice. Chichester, New York,
    Toronto, Wiley & Sons, pp 147-170.

    Beegle CC, Dulmage HT, Wolfenbarger DA, & Martinez E (1981)
    Persistence of  Bacillus thuringiensis Berliner insecticidal activity
    on cotton foliage. Environ Entomol, 10: 400-401.

    Benz G & Altwegg A (1975) Safety of  Bacillus thuringiensis for
    earthworms. J Invertebr Pathol, 26: 125-126. 

    Berliner E (1915) [About the sleep sickness of the  Ephestia kühniella
     Zell. and its vector  Bacillus thuringiensis.] Z Angew Entomol, 2:
    29-56 (in German).

    Bernhard K & Utz R (1993) Production of  Bacillus thuringiensis
    insecticides for experimental and commercial uses. In: Entwistle PF,
    Cory JS, Bailey MJ, & Higgs S ed.  Bacillus thuringiensis, an
    environmental biopesticide: Theory and practice. Chichester, New York,
    Toronto, Wiley & Sons, pp 255-267.

    Bernier RL Jr, Gannon DJ, Moser GP, Mazzarello M, Griffiths MM, &
    Guest PJ (1990) Development of a novel Bt strain for the control of
    forestry pests. In: Brighton Crop Protection Conference -- Pests and
    Diseases -- 1990: Proceedings. Farnham, Surrey, British Crop
    Protection Council, pp 245-252.

    Boeri RL (1991) Acute toxicity of ABG-6305 to the rainbow trout
     (Oncorhynchus mykiss) (Project No. 9107-A). Hampton, New Hampshire,
    Resource Analysts Inc, Enviro Systems Division, pp 1-26 (Unpublished
    Abbott document).

    Bonnefoi A & Béguin S (1959) Recherches sur l'action des cristaux de
     Bacillus thuringiensis souche "anduze". Entomophaga, 4: 193-199.

    Bourque SN, Valero JR, Lavoie MC, & Levesque RC (1995) Comparative
    analysis of the 16S-23S ribosomal intergenic spacer sequences of
     Bacillus thuringiensis strains and subspecies and of closely related
    species. Appl Environ Microbiol, 61: 1623-1626.

    Bravo A, Sarabia S, Lopez L, Ontiveros H, Abarca C, Ortiz M, Lina L,
    Villalobos FJ, Pena G, Nunez-Valdez M-E, Soberón M, & Quintero R
    (1998) Characterization of  cry genes in a Mexican  Bacillus
     thuringienis strain collection. Appl Environ Microbiol, 64:
    4965-4972.

    Brousseau R, Saint-Onge A, Prefontaine G, Masson L, & Cabana J (1992)
    Arbitrary primer polymerase chain reaction, a powerful method to
    identify  Bacillus thuringiensis serovars and strains. Appl Environ
    Microbiol, 59: 114-119.

    Buckner CH, Kingsbury PD, McLeod BB, Mortensen KL, & Ray DGH (1974)
    Impact of aerial treatment on non-target organisms, Algonquin Park,
    Ontario, and Spruce Woods, Manitoba, Section F. In: Evaluation of
    commercial preparations of  Bacillus thuringiensis with and without
    chitinase against spruce budworm. Ottawa, Ontario, Canadian Forestry
    Service, Chemical Control Research Institute, pp 1-72 (Information
    report CC-X-59).

    Bulla LA Jr, Kramer KJ, & Davidson LI (1977) Characterization of the
    entomocidal parasporal crystal of  Bacillus thuringiensis. J
    Bacteriol, 130: 375-383.

    Bulla LA Jr, Faust RM, Andrews R, & Goodman N (1985) Insecticidal
    bacilli. In: Dubnau DA ed. The molecular biology of the bacilli. New
    York, London, Academic Press Inc., vol 2, pp 185-209.

    Burges HD (1977) Control of the wax moth  Galleria mellonella on
    beecomb by H-serotype V  Bacillus thuringiensis and the effect of
    chemical additives. Apidologie, 8: 155-168.

    Burges HD (1980) Safety, safety testing and quality control of
    microbial pesticides. In: Burges HD ed. Microbial control of pests and
    plant diseases 1970-1980. New York, London, Academic Press Inc., pp
    738-767.

    Burges HD & Bailey L (1968) Control of the greater and lesser wax
    moths  (Galleria mellonella and  Achroia griesella) with  Bacillus
     thuringiensis. J Invertebr Pathol, 11: 184-195.

    Burges HD & Hurst JA (1977) Ecology of  Bacillus thuringiensis in
    storage moths. J Invertebr Pathol, 30: 131-139.

    Calamari D, Yameogo L, Hougard J-M, & Levêque C (1998) Environmental
    assessment of larvicide use in the onchocerciasis control programme.
    Parasitol Today, 14: 485-489.

    Campbell DP, Dieball DE, & Brackett JM (1987) Rapid HPLC assay for the
     ß-exotoxin of  Bacillus thuringiensis. J Agric Food Chem, 35: 
    156-158.

    Cannon RJC (1996)  Bacillus thuringiensis use in agriculture: a
    molecular perspective. Biol Rev Cambridge Philos Soc, 71: 561-636.

    Cantwell GE & Shieh TR (1981) CertanTM: A new bacterial insecticide
    against the greater wax moth. Am Bee J, 121: 424-431.

    Cantwell GE, Heimpel AM, & Thompson MJ (1964) The production of an
    exotoxin by various crystal-forming bacteria related to  Bacillus
     thuringiensis var.  thuringiensis Berliner. J Insect Pathol, 6:
    466-480.

    Cantwell GE, Knox DA, Lehnert T, & Michael AS (1966) Mortality of the
    honey bee,  Apis mellifera, in colonies treated with certain
    biological insecticides. J Invertebr Pathol, 8: 228-233.

    Car M (1984) Laboratory and field trials with two  Bacillus
     thuringiensis var.  israelensis products for  Simulium (Diptera:
    Nematocera) control in a small polluted river in South Africa.
    Onderstepoort J Vet Res, 51: 141-144.

    Car M & de Moor FC (1984) The response of Vaal River drift and benthos
    to  Simulium (Diptera: Nematocera) control using  Bacillus
     thuringiensis var.  israelensis (H-14). Onderstepoort J Vet Res, 51:
    155-160.

    Carlberg G, Kihamia CM, & Minhar J (1985) Microbial control of flies
    in latrines in Dar es Salaam with a  Bacillus thuringiensis (serotype
    1) preparation, Muscabac. J Appl Microbiol Biotechnol, 1: 33-44.

    Carlson CR & Kolsto A-B (1993) A complete physical map of a  Bacillus
     thuringiensis chromosome. J Bacteriol, 175: 1053-1060.

    Carlson CR, Caugant DA, & Kolstr A-B (1994) Genotypic diversity among
     Bacillus cereus and  Bacillus thuringiensis strains. Appl Environ
    Microbiol, 60: 1719-1725.

    Carlton BC, Gawron-Burke C, & Johnson TB (1990) Exploiting the genetic
    diversity of  Bacillus thuringiensis for the creation of new
    bioinsecticides. In: Proceedings of the 5th International Colloquium
    on Invertebrate Pathology and Microbial Control. Adelaide, Australia,
    Society for Invertebrate Pathology, pp 18-22.

    Chilcott CN, Pillai JS, & Kalmakoff J (1983) Efficacy of  Bacillus
     thuringiensis var.  israelensis as a biocontrol agent against
    larvae of Simuliidae (Diptera) in New Zealand. NZ J Zool, 10: 319-326.

    Christensen KP (1990a) Dipel technical material  (Bacillus
     thuringiensis var.  kurstaki): Infectivity and pathogenicity to
    bluegill sunfish  (Lepomis macrochirus) during a 32-day static
    renewal test. Wareham, Massachusetts, Springborn Laboratories Inc., pp
    1-53 (Unpublished Abbott document No. 90-1-3211).

    Christensen KP (1990b) Dipel technical material  (Bacillus
     thuringiensis var.  kurstaki): Infectivity and pathogenicity to
    rainbow trout  (Oncorhynchus mykiss) during a 32-day static renewal
    test. Wareham, Massachusetts, Springborn Laboratories Inc., pp 1-57
    (Unpublished Abbott document No. 90-2-3219).

    Christensen KP (1990c) Dipel technical material  (Bacillus
     thuringiensis var.  kurstaki): Infectivity and pathogenicity to
    sheepshead minnow  (Cyprinodon variegatus) during a 30-day static
    renewal test. Wareham, Massachusetts, Springborn Laboratories Inc.,
    vol 2, pp 253-308 (Unpublished Abbott document No. 90-5-3317).

    Christensen KP (1990d)  Bacillus thuringiensis var.  tenebrionis:
    Infectivity and pathogenicity to rainbow trout  (Oncorhynchus mykiss)
    during a 30-day static renewal test. Wareham, Massachusetts,
    Springborn Laboratories Inc., pp 1-54 (Unpublished Abbott document No.
    90-3-3263).

    Christensen KP (1990e)  Bacillus thuringiensis var.  tenebrionis:
    Infectivity and pathogenicity to sheepshead minnow  (Cyprinodon
     variegatus) during a 30-day static renewal test. Wareham,
    Massachusetts, Springborn Laboratories Inc., pp 1-50 (Unpublished
    Abbott document No. 90-6-3348).

    Christensen KP (1990f) Vectobac technical material  (Bacillus
     thuringiensis var.  israelensis): Infectivity and pathogenicity to
    bluegill sunfish  (Lepomis macrochirus) during a 30-day static
    renewal test. Wareham, Massachusetts, Springborn Laboratories Inc., pp
    1-55 (Unpublished Abbott document No. 90-2-3228).

    Christensen KP (1990g) Vectobac technical material  (Bacillus
     thuringiensis var.  israelensis): Infectivity and pathogenicity to
    rainbow trout  (Oncorhynchus mykiss) during a 32-day static renewal
    test. Wareham, Massachusetts, Springborn Laboratories Inc., pp 1-55
    (Unpublished Abbott document No. 90-2-3242).

    Christensen KP (1990h) Vectobac technical material  (Bacillus
     thuringiensis var.  israelensis): Infectivity and pathogenicity to
    sheepshead minnow  (Cyprinodon variegatus) during a 30-day static
    renewal test. Wareham, Massachusetts, Springborn Laboratories Inc., pp
    1-57 (Unpublished Abbott document No. 90-4-3288).

    Cibulsky RJ & Fusco RA (1987) Recent experiences with Vectobac for
    black fly control: An industrial perspective on future developments.
    In: Kim KC & Merritt RW ed. Black flies: Ecology, population
    management, and annotated world list. University Park, Pennsylvania,
    Pennsylvania State University Press, pp 419-424.

    Claus D & Berkeley RCW (1986) Genus  Bacillus Cohn 1872. In: Sneath
    PHA, Mair NS, Sharp ME, & Holt JG ed. Bergey's manual of systematic
    bacteriology. Baltimore, Maryland, Williams & Wilkins, vol 2, pp
    1105-1139.

    Cooksey KE (1971) The protein crystal toxin of  Bacillus
     thuringiensis: Biochemistry and mode of action. In: Burges HD &
    Hussey NW ed. Microbial control of insects and mites. New York,
    London, Academic Press Inc., pp 247-274.

    Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J, Lereclus
    D, Baum J, & Dean DH (1998) Revision of the nomenclature for the
     Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol
    Biol Rev, 62: 807-813.

    Damgaard PH (1995) Diarrhoeal enterotoxin production by strains of
     Bacillus thuringiensis isolated from commercial  Bacillus
     thuringiensis -- based insecticides. FEMS Immunol Med Microbiol, 12:
    245-250.

    Damgaard PH, Granum PE, Bresciani J, Torregrossa MV, Eilenberg J, &
    Valentino L (1997a) Characterization of  Bacillus thuringiensis
    isolated from infections in burn wounds. FEMS Immunol Med Microbiol,
    18: 47-53.

    Damgaard PH, Hansen BM, Pedersen JC, & Eilenberg J (1997b) Natural
    occurrence of  B. thuringiensis on cabbage foliage and in insects
    associated with cabbage crops. J Appl Bacteriol, 82: 253-258.

    Damgaard PH, Larsen HD, Hansen BM, Bresciani J, & Jorgensen K (1996a)
    Enterotoxin-producing strains of  Bacillus thuringiensis isolated
    from food. Lett Appl Microbiol, 23: 146-150.

    Damgaard PH, Jacobsen CS, & Sorensen J (1996b) Development and
    application of a primer set for specific detection of  Bacillus
     thuringiensis and  Bacillus cereus in soil using magnetic
    capture-hybridization and PCR amplification. System Appl Microbiol,
    19: 436-441.

    de Barjac H (1981a) Identification of H-serotypes of  Bacillus
     thuringiensis. In: Burges HD ed. Microbial control of pests and
    plant diseases 1970-1980. New York, London, Academic Press Inc., pp
    35-43.

    de Barjac H (1981b) Insect pathogens in the genus  Bacillus. In:
    Berkley RCW & Goodfellow M ed. The aerobic endospore-forming bacteria:
    Classification and identification. New York, London, Academic Press
    Inc., pp 241-250.

    de Barjac H & Bonnefoi A (1962) Essai de classification biochimique et
    sérologique de 24 souches de  Bacillus de type  thuringiensis.
    Entomophaga, 7: 5-31.

    de Barjac H & Larget-Thiery I (1984) Characteristics of IPS 82 as
    standard for biological assays of  Bacillus thuringiensis H-14
    preparations. Geneva, World Health Organization (WHO/VBC/84.892).

    de Barjac H, Larget I, Bénichou L, Cosmao V, Viviani G, Ripouteau H, &
    Papion S (1980) Test d'innocuité sur mammifères avec du sérotype H 14
    de  Bacillus thuringiensis. Geneva, World Health Organization
    (WHO/VBC/80.761).

    Dean DH (1984) Biochemical genetics of the bacterial insect-control
    agent  Bacillus thuringiensis: Basic principles and prospects for
    genetic engineering. Biotechnol Genet Eng Rev, 2: 341-363.

    Dean DH, Rajamohan F, Lee MK, Wu SJ, Chen XJ, Alcantara E, & Hussain
    SR (1996) Probing the mechanism of action of  Bacillus thuringiensis
    insecticidal proteins by site-directed mutagenesis - a minireview.
    Gene, 179: 111-117.

    DeLucca AJ II, Simonson JG, & Larson AD (1981)  Bacillus thuringiensis
    distribution in soils of the United States. Can J Microbiol, 27:
    865-870.

    Demezas DH & Bell J (1995) Evaluation of low molecular weight RNA
    profiles and ribotyping to differentiate some  Bacillus species.
    System Appl Microbiol, 18: 582-589.

    Denolf P, Hendrickx K, Vandamme J, Jansens S, Peferoen M, Degheele D,
    & Vanrie J (1997) Cloning and characterization of  Manduca sexta and
     Plutella xylostella midgut aminopeptidase N enzymes related to
     Bacillus thuringiensis toxin-binding proteins. Eur J Biochem, 248:
    748-761.

    Dent DR (1993) The use of  Bacillus thuringiensis as an insecticide.
    In: Jones DG ed. Exploitation of microorganisms. London, Chapman &
    Hall, pp 19-44.

    Drobniewski FA (1993)  Bacillus cereus and related species. Clin
    Microbiol Rev, 6: 324-338.

    Dulmage HT & cooperators (1981) Insecticidal activity of isolates of
     Bacillus thuringiensis and their potential for pest control. In:
    Burges HD ed. Microbial control of pests and plant diseases 1970-1980.
    New York, London, Academic Press Inc., pp 193-223. 

    Dunbar JP & Johnson AW (1975)  Bacillus thuringiensis: Effects on the
    survival of a tobacco budworm parasitoid and predator in the
    laboratory. Environ Entomol, 4: 352-354.

    Dunbar DM, Kaya HK, Doane CC, Anderson JF, & Weseloh RM (1972) Aerial
    application of  Bacillus thuringiensis against larvae of the elm
    spanworm and gypsy moth and effects on parasitoids of the gypsy moth.
    Connecticut Experiment Station (Bulletin No. 735).

    Eilenberg J, Damgaard PH, Hansen BM, Pedersen JC, Bresciani J, &
    Larsson R (in press) Natural interactions between  Strongwellsea
     castrans,  Cystosporogenes deliaradicae and  Bacillus thuringiensis
    in the host,  Delia radicum. J Invertebr Pathol.

    Elliott LJ, Sokolow R, Heumann M, & Elefant SL (1988), An exposure
    characterization of a large scale application of a biological
    insecticide,  Bacillus thuringiensis. Appl Ind Hyg, 3: 119-122.

    Elsey KD (1973)  Jalysus spinosus effect of insecticide treatments on
    this predator of tobacco pests. Environ Entomol, 2: 240-243.

    Entwistle PF, Cory JS, Bailey MJ, & Higgs S (1983)  Bacillus
     thuringiensis, an environmental biopesticide: Theory and practice.
    Chichester, New York, Toronto, John Wiley & Sons, 311 pp.

    Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, & Koziel MG
    (1996) Vip3A, a novel  Bacillus thuringiensis vegetative insecticidal
    protein with a wide spectrum of activities against lepidopteran
    insects. Proc Natl Acad Sci (USA), 93: 5389-5394.

    Farkas J, Sebesta K, Horská K, Samek Z, Dolejs L, & Sorm F (1977)
    Structure of thuringiensin, the thermostable exotoxin from  Bacillus
     thuringiensis. Coll Czech Chem Commun, 42: 909-929.

    Fast PG (1971) Isolation of a water-soluble toxin from a commercial
    microbial insecticide based on  Bacillus thuringiensis. J Invertebr
    Pathol, 17: 301.

    Fast PG (1981) The crystal toxin of  Bacillus thuringiensis. In:
    Burges HD ed. Microbial control of pests and plant diseases 1970-1980.
    New York, London, Academic Press Inc., pp 223-248.

    Faust RM (1973) The  Bacillus thuringiensis ß-exotoxin: Current
    status. Bull Entomol Soc Am, 19: 153-156.

    Feitelson JS (1993) The  Bacillus thuringiensis family tree. In: Kim
    L ed. Advanced engineered pesticides. New York, Basel, Marcel Dekker
    Inc., pp 63-71.

    Fisher R & Rosner L (1959) Toxicology of the microbial insecticide,
    Thuricide. J Agric Food Chem, 7: 686-688.

    Frommer W, Ager B, Archer L, Brunius G, Collins CH, Donikian R,
    Frontali C, Hamp S, Houwink EH, Kuenzi MT, Krämer P, Lagast H, Lund S,
    Mahler JL, Normand-Plessier F, Sargeant K, Tuijnenburg-Muijs G, Vranch
    SP, & Werner RG (1989) Safe biotechnology: III. Safety precautions for
    handling microorganisms of different risk classes. Appl Microbiol
    Biotechnol, 30: 541-552.

    Fusco RA (1980) Field evaluation of a commercial preparation of
     Bacillus thuringiensis, DIPEL 4L: Progress report. Pennsylvania
    Bureau of Forestry, Gypsy Moth Pest Management Methods Development
    Project.

    Gaddis PK & Corkran CC (1986) Secondary effects of Bt spray on avian
    predators: the reproductive success of chestnut-backed chickadees.
    Northwest Ecological Research Institute (Unpublished Research Report
    No. 86-03).

    Garcia R, Des Rochers B, & Tozer W (1980) Studies on the toxicity of
     Bacillus thuringiensis var.  israelensis against organisms found in
    association with mosquito larvae. Berkeley, California, University of
    California, Division of Biological Control.

    Gelernter WD (1990) Targeting insecticide-resistant markets: New
    developments in microbial-based products. In: Green MB, Moberg WK, &
    LeBaron H ed. Managing resistance to agrochemicals: From fundamental
    research to practical strategies. Washington, DC, American Chemical
    Society, pp 105-117 (ACS Series 421).

    Giffel MC, Beumer RR, Klijn N, Wagendorp A, & Rombouts FM (1997)
    Discrimination between  Bacillus cereus and  Bacillus thuringiensis
    using specific DNA probes based on variable regions of 16S rRNA. FEMS
    Microbiol Lett, 146: 47-51.

    Gill SS, Hornung JM, Ibarra JE, Singh GJP, & Federici BA (1987)
    Cytolytic activity and immunological similarity of  Bacillus
     thuringiensis subsp.  israelensis and  Bacillus thuringiensis
    subsp.  morrisoni isolate PG-14 toxins. Appl Environ Microbiol, 53:
    1251-1256.

    Gilmore MS, Cruz-Rodz AL, Leimeister-Wächter M, Kreft J, & Goebel W
    (1989) A  Bacillus cereus cytolytic determinant, cereolysin AB, which
    comprises the phospholipase C and sphingomyelinase genes: Nucleotide
    sequence and genetic linkage. J Bacteriol, 171: 744-753.

    Goldberg LJ & Margalit J (1977) A bacterial spore demonstrating rapid
    larvicidal activity against  Anopheles sergentii,  Uranotaenia
     unguiculata,  Culex univitattus,  Aedes aegypti and  Culex
     pipiens. Mosq News, 37: 355-358.

    González JM Jr & Carlton BC (1980) Patterns of plasmid DNA in
    crystalliferous and acrystalliferous strains of  Bacillus
     thuringiensis. Plasmid, 3: 92-98.

    González JM Jr, Dulmage HT, & Carlton BC (1981) Correlation between
    specific plasmids and delta-endotoxin production in  Bacillus
     thuringiensis. Plasmid, 5: 351-365. 

    González JM, Jr Brown BJ, & Carlton BC (1982) Transfer of  Bacillus
     thuringiensis plasmids coding for delta-endotoxin among strains of
     B.  thuringiensis and  B. cereus. Proc Natl Acad Sci (USA), 79:
    6951-6955.

    Gordon RE (1977) Some taxonomic observations on the genus  Bacillus.
    In: Briggs JB ed. Biological regulations of vectors: The saprophytic
    and aerobic bacteria and fungi. Washington, DC, US Department of
    Health, Education and Welfare, pp 67-82 (Publication NIH 77-1180).

    Granum PE (1997)  Bacillus cereus. In: Doyle MP, Beuchat LR, &
    Montville TJ ed. Food microbiology fundamentals and frontiers.
    Washington, DC, ASM Press, pp 327-336.

    Grassi S & Deseö KV (1984) [The natural occurrence of  Bacillus
     thuringiensis Berl. And its importance in the plant protection.] In:
    [Proceedings of Seminar on Phytopathology, Sorrento, Italy, 26-29
    March 1984.] Bologna, Italy, Clueb Publishing Co., vol 2, pp 424-433
    (in Italian).

    Green M, Heumann M, Sokolow R, Foster LR, Bryant R, & Skeels M (1990)
    Public health implications of the microbial pesticide  Bacillus
     thuringiensis: An epidemiological study, Oregon, 1985-1986. Am J
    Publ Health, 80: 848-852.

    Griego VM & Spence KD (1978) Inactivation of  Bacillus thuringiensis
    spores by ultraviolet and visible light. Appl Environ Microbiol, 35:
    906-910.

    Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz JL,
    Brousseau R, & Cygler M (1995)  Bacillus thuringiensis CryIA(a)
    insecticidal toxin: crystal structure and channel formation. J Mol
    Biol, 254: 447-464.

    Hadley WM, Burchiel SW, McDowell TD, Thilsted JP, Hibbs CM, Whorton
    JA, Day PW, Friedman MB, & Stoll RE (1987) Five-month oral (diet)
    toxicity/ infectivity study of  Bacillus thuringiensis insecticides
    in sheep. Fundam Appl Toxicol, 8: 236-242.

    Hamed AR (1978-79) [Effects of  Bacillus  thuringiensis on parasites
    and predators of  Yponomeuta evenymellus (Lep., Yponomeutidae).] Z
    Angew Entomol, 87: 294-311 (in German).

    Hamel DR (1977) The effects of  Bacillus thuringiensis on parasitoids
    of the western spruce budworm,  Choristoneura occidentalis
    (Lepidoptera: Tortricidae), and the spruce coneworm,  Dioryctria
     reniculelloides (Lepidoptera: Pyralidae), in Montana. Can Entomol,
    109: 1409-1415.

    Hannay CL (1953) Crystalline inclusions in aerobic spore-forming
    bacteria. Nature (Lond), 172: 1004.

    Hansen BM & Hendriksen NB (1997a)  Bacillus thuringiensis and  B.
    cereus enterotoxins: Proceedings of the 6th European Meeting of the
    International Organization for Biological and Integrated Control of
    Noxious Animals and Plants/West Paleaearctic Regional Section,
    Copenhagen, 10-15 August 1997. Copenhagen, Denmark, Royal Veterinary
    and Agricultural University, Department of Ecology and Molecular
    Biology.

    Hansen BM & Hendriksen NB (1997b) Comparative PCR analysis of  B.
     thuringiensis and  B. cereus: Abstract for the International
    Workshop on Molecular Biology of  B. cereus,  B. anthracis and 
     B. thuringiensis, Soria Moria, Oslo, 23-25 May 1997.

    Hansen BM & Hendriksen NB (1999) Ecological aspects of the survival in
    soil of spray released  Bacillus thuringiensis subsp.  kurstaki. In:
    Abstract for the IOBC Symposium on Indirect Ecological Effects of
    Biological Control, Montpelier, 17-20 October 1999. Montpelier,
    France, Agropolis International.

    Hansen BM, Damgaard PH, Eilenberg J, & Pedersen JC (1996)  Bacillus
     thuringiensis, ecology and environmental effects of its use for
    microbial pest control (Environmental Project No. 316). Copenhagen,
    Denmark, Ministry of Environment and Energy, Danish Environmental
    Protection Agency.

    Hansen BM, Damgaard PH, Eilenberg J, & Pedersen JC (1998) Molecular
    and phenotypic characterization of  Bacillus thuringiensis isolated
    from leaves and insects. J Invertebr Pathol, 71: 106-114.

    Harding J, Wolfenbarger D, Dupnik T, & Fuchs T (1972) Large scale
    tests comparing  Bacillus thuringiensis with methyl-parathion for
    cotton insect control, field test report. Chicago, Illinois, Abbott
    Laboratories (Unpublished document).

    Hassan SA (1983) Results of laboratory testing of a series of
    pesticides on egg parasites of the genus  Trichogramma (Hymenoptera,
    Trichogrammatidae). Nachr.bl Dtsch Pflanzenschutzdienstes
    (Braunschweig), 35: 21.

    Hassan S & Krieg A (1975) [ Bacillus thuringiensis preparations
    harmless to the parasite  Trichogramma  cacoeciae (Hym.:
    Trichogrammatidae).] Z Pflanzenkr Pflanzenchutz, 82: 515-521 (in
    German).

    Hassan G & Nabbut N (1996) Prevalence and characterization of
     Bacillus  cereus isolates from clinical and natural sources. J Food
    Prot, 59: 193-196.

    Heimpel AM (1954) Investigations of the mode of action of strains of
     Bacillus cereus Frankland and Frankland pathogenic for the larch
    sawfly,  Pristiphora chrichsonii (Htg.). Kingston, Canada, Queen's
    University, 155 pp (Doctoral dissertation).

    Heimpel AM (1967) A critical review of  Bacillus thuringiensis var.
     thuringiensis Berliner and other crystalliferous bacteria. Annu Rev
    Entomol, 12: 287-322.

    Heimpel AM & Angus TA (1958) The taxonomy of insect pathogens related
    to  Bacillus cereus Frankland and Frankland. Can J Microbiol, 4:
    531-541.

    Helgason E, Caugant DA, Lecadet M-M, Chen Y, Mahillon J, Lövgren A,
    Hegna I, Kvaloy K, & Kolsto A-B (1998) Genetic diversity of  Bacillus
     cereus/ B. thuringiensis isolates from natural sources. Curr
    Microbiol, 37: 80-87.

    Hendriksen NB & Hansen BM (1998) Phylogenetic relations of  Bacillus
     thuringiensis: Implications for risks associated to its use as a
    microbiological pest control agent. IOBC Bull, 21: 5-8.

    Hernandez E, Ramisse F, Ducoureau J-P, Cruel T, & Cavallo J-D (1998)
     Bacillus thuringiensis subsp.  konkukian (serotype H34)
    superinfection: Case report and experimental evidence of pathogenicity
    in immunosuppressed mice. J Clin Microbiol, 36: 2138-2139.

    Höfte H & Whiteley HR (1989) Insecticidal crystal proteins of
     Bacillus  thuringiensis. Microbiol Rev, 53: 242-255.

    Honée G & Visser B (1993) The mode of action of  Bacillus
     thuringiensis crystal proteins. Entomol Exp Appl, 69: 145-155.

    Horn DJ (1983) Selective mortality of parasitoids and predators of
     Myzus persicae on collards treated with malathion, carbaryl, or
     Bacillus thuringiensis. Entomol Exp Appl, 34: 208-211.

    Horsburgh R & Cobb L (1981) Effect of Dipel WP and Dipel + Guthion
    tank mix combination for control of variegated leafroller, turfed
    apple bud moth, and red-banded leafroller larvae on apples in a full
    season and late season program in Virginia (Experiment No.
    D-986-4240). Chicago, Illinois, Abbott Laboratories Inc.

    Hostetter DL, Ignoffo CM, & Kearby WH (1975) Persistence of
    formulations of  Bacillus thuringiensis spores and crystals on
    eastern red cedar foliage in Missouri. J Kansas Entomol Soc, 48:
    189-193.

    Hotha S & Banik RM (1997) Production of alkaline protease by  Bacillus
     thuringiensis H 14 in aqueous two-phase systems. J Chem Technol
    Biotechnol, 69: 5-10.

    Huber HE & Lüthy P (1981)  Bacillus thuringiensis delta-endotoxin:
    Composition and activation. In: Davidson EW ed. Pathogenesis of
    invertebrate microbial diseases. Totowa, New Jersey, Allanheld-Osmun
    Publishers, pp 209-234.

    Huber HE, Lüthy P, Ebersold HR, & Cordier JL (1981) The subunits of
    the parasporal crystal of  Bacillus thuringiensis: size, linkage and
    toxicity. Arch Microbiol, 129: 14-18.

    Ignoffo CM (1973) Effects of entomopathogens on vertebrates. Ann NY
    Acad Sci, 217: 141-164.

    Ignoffo CM (1992) Environmental factors affecting persistence of
    entomopathogens. Fla Entomol, 75: 516-525.

    Innes DGL & Bendell JF (1989) The effects on small-mammal populations
    of aerial applications of  Bacillus thuringiensis, fenitrothion, and
    Matacil(R) used against jack pine budworm in Ontario. Can J Zool,
    67: 1318-1323.

    Jackson SG, Goodbrand RB, Ahmed R, & Kasatiya S (1995)  Bacillus
     cereus and  Bacillus thuringiensis isolated in a gastroenteritis
    outbreak investigation. Lett Appl Microbiol, 21: 103-105.

    Jaquet F, Hütter R, & Lüthy P (1987) Specificity of  Bacillus
     thuringiensis delta-endotoxin. Appl Environ Microbiol, 53: 500-504.

    Jarrett P & Stephenson M (1990) Plasmid transfer between strains of
     Bacillus thuringiensis infecting  Galleria mellonella and
     Spodoptera  littoralis. Appl Environ Microbiol, 56: 1608-1614.

    Jensen R (1974) Comparison of various insecticides including DIPEL WP
    for control of the velvetbean caterpillar  (Anticarsia gemmatalis)
    and the green cloverworm  (Plathypena scabra) on soybeans and the
    effect on non-target species (Experiment No D911-1582). Chicago,
    Illinois, Abbott Laboratories Inc. 

    Johnson AW (1974)  Bacillus thuringiensis and tobacco budworm control
    on flue-cured tobacco. J Econ Entomol, 67: 755-759.

    Juarez-Perez VM, Ferrandis MD, & Frutos R (1997) PCR-based approach
    for detection of novel  Bacillus thuringiensis cry genes. Appl
    Environ Microbiol, 63: 2997-3002.

    Kaneko T, Nozaki R, & Aizawa K (1978) Deoxyribonucleic acid
    relatedness between  Bacillus anthracis,  Bacillus cereus and
     Bacillus  thuringiensis. Microbiol Immunol, 22: 639-641.

    Kazakova SB & Dzhunusov KK (1977) The effect of Bitoxibacillin-202 on
    certain orchard insects in the Issyk-Kul' depression. Rev Appl
    Entomol, A65: 5987.

    Keller B & Langenbruch G-A (1993) Control of coleopteran pests by
     Bacillus thuringiensis. In: Entwistle PF, Cory JS, Bailey MJ, &
    Higgs S ed.  Bacillus thuringiensis, an environmental biopesticide:
    Theory and practice. Chichester, New York, Toronto, Wiley & Sons, pp
    171-191.

    Khawaled K, Ben-Dov E, Zaritsky A, & Barak Z (1990) The fate of
     Bacillus thuringiensis var.  israelensis in  B.  thuringiensis
    var.  israelensis-killed pupae of  Aedes aegypti. J Invertebr
    Pathol, 56: 312-316.

    Kim HS, Lee DW, Woo SD, Yu YM, & Kang SK (1998) Seasonal distribution
    and characterization of  Bacillus thuringiensis isolated from
    sericultural environments in Korea. J Gen Appl Microbiol, 44: 133-138.

    Klier AF & Lecadet MM (1976) Arguments based on
    hybridization-competition experiments in favor of the  in vitro
    synthesis of sporulation-specific mRNAs by the RNA polymerase of
     Bacillus thuringiensis. Biochem Biophys Res Commun, 73: 263-270.

    Knowles BH & Ellar DJ (1987) Colloid-osmotic lysis is a general
    feature of the mechanisms of action of  Bacillus thuringiensis
    delta-endotoxins with different insect specificity. Biochim Biophys
    Acta, 924: 509-518.

    Krieg A (1971) Concerning alpha-exotoxin produced by vegetative cells
    of  Bacillus thuringiensis and  Bacillus cereus. J Invertebr Pathol,
    17: 134-135.

    Krieg A (1973) About toxic effects of cultures of  Bacillus cereus
    and  Bacillus thuringiensis on honey bees  (Apis mellifera). 
    Z Pflanzenkr Pflanzenschutz, 80: 483-486.

    Krieg A & Herfs W (1963) The effects of  Bacillus thuringiensis on
    honey bees. Entomol Exp Appl, 6: 1-9.

    Krieg A, Hassan S, & Pinsdorf W (1980) Comparison of the effects of
    the variety  israelensis with other varieties of  B. thuringiensis
    on non-target organisms of the order Hymenoptera:  Trichogramma
     cacoeciae and  Apis mellifera. Anz Schädlingsk Pflanz Umweltschutz,
    53: 81-83.

    Krieg A, Huger AM, Langenbruch GA, & Schnetter W (1983)  Bacillus
     thuringiensis var.  tenebrionis, a new pathotype effective against
    larvae of Coleoptera .. Z Angew Entomol, 96(5): 500-508.

    Krywienczyk J, Dulmage HT, & Fast PG (1978) Occurrence of two
    serologically distinct groups within  Bacillus thuringiensis serotype
    3ab variety  kurstaki. J Invertebr Pathol, 37: 372-375.

    Kumar PA, Sharma RP, & Malik VS (1996) The insecticidal protein of
     Bacillus thuringiensis. Adv Appl Microbiol, 42: 1-43.

    Lacey LA & Mulla MS (1990) Safety of  Bacillus thuringiensis ssp.
     israelensis and  Bacillus sphaericus to nontarget organisms in the
    aquatic environment. In: Laird M, Lacey LA, & Davidson EW ed. Safety
    of microbial pesticides. Boca Raton, Florida, CRC Press, pp 169-188.

    Lacey LA, Escaffre H, Philippon B, Sékétéli A, & Guillet P (1982)
    Large river treatment with  Bacillus thuringiensis (H-14) for the
    control of  Simulium damnosum s.l. in the Onchocerciasis Control
    Programme. Tropenmed Parasitol, 33: 97-101.

    Laferrière M, Bastille A, & Nadeau A (1987) Etude immunologique
    impliquant les composantes de l'insecticide biologique  Bacillus
     thuringiensis var.  kurstaki. St. Henri Rivière-du-Loup, Québec,
    Department of Public Health, Grand-Portage Regional Hospital Center,
    pp 1-19 (Unpublished report).

    Lattin A, Grimes J, Hoxter KA, & Smith GJ (1990a) Dipel technical
    material  (Bacillus thuringiensis var.  kurstaki): An avian oral
    toxicity and pathogenicity study in the bobwhite (Project No.
    161-112). Easton, Maryland, Wildlife International Ltd, pp 1-25
    (Unpublished Abbott document).

    Lattin A, Grimes J, Hoxter K, & Smith GJ (1990b) VectoBac technical
    material  (Bacillus thuringiensis var.  israelensis): An avian oral
    toxicity and pathogenicity study in the mallard (Project No. 161-115).
    Easton, Maryland, Wildlife International Ltd, pp 1-24 (Unpublished
    Abbott document).

    Lattin A, Hoxter K, Driscoll C, Grimes J, & Jaber M (1990c) Dipel
    technical material  (Bacillus thuringiensis var.  kurstaki): An
    avian oral toxicity and pathogenicity study in the mallard (Project
    No. 161-113). Easton, Maryland, Wildlife International Ltd, pp 1-28
    (Unpublished Abbott document).

    Lattin A, Hoxter K, & Smith GJ (1990d) VectoBac technical material
     (Bacillus thuringiensis var.  israelensis): An avian oral toxicity
    and pathogenicity study in the bobwhite (Project No. 161-114). Easton,
    Maryland, Wildlife International Ltd, pp 1-27 (Unpublished Abbott
    document).

    Lechner S, Mayr R, Francis KP, Prüß BM, Kaplan T, Wießner-Gunkel E,
    Stewart GSAB, & Scherer S (1998)  Bacillus weihenstephanensis sp.
    nov. is a new psychrotolerant species of the  Bacillus cereus group.
    Int J System Bacteriol, 48: 1373-1378.

    Leong KLH, Cano RJ, & Kubinski AM (1980) Factors affecting  Bacillus
     thuringiensis total field persistence. Environ Entomol, 9: 593-599.

    Levêque C, Fairhurst CP, Abbau K, Pangy D, Curtis MS, & Traoré K
    (1988) Onchocerciasis control programme in West Africa: ten years of
    monitoring fish populations. Chemosphere, 17: 421-440.

    Li J, Carroll J, & Ellar DJ (1991) Crystal structure of insecticidal
    (delta)-endotoxin from  Bacillus thuringiensis at 2.5 Å resolution.
    Nature (Lond), 353: 815-821.

    Logan NA & Berkeley CW (1984) Identification of  Bacillus strains
    using the API system. J Gen Microbiol, 130: 1871-1882.

    Lund T & Granum PE (1997) Comparison of biological effect of the two
    different enterotoxin complexes isolated from three different strains
    of  Bacillus cereus. Microbiology, 143: 3329-3336.

    Lüthy P (1986) Insect pathogenic bacteria as pest control agents.
    Fortschr Zool, 32: 201-216.

    Lüthy P & Ebersold HR (1981) The entomocidal toxins of  Bacillus
     thuringiensis. Pharmacol Ther, 13: 257-283.

    Lynch MJ & Baumann P (1985) Immunological comparisons of the crystal
    protein from strains of  Bacillus thuringiensis. J Invertebr Pathol,
    46: 47-57.

    Lynch RE, Lewis LC, & Brindley TA (1976) Bacteria associated with eggs
    and first-instar larvae of the European corn borer: Isolation
    techniques and pathogenicity. J Invertebr Pathol, 27: 325-331.

    McClintock JT, Schaffer CR, & Sjoblad RD (1995) A comparative review
    of the mammalian toxicity of  Bacillus thuringiensis-based
    pesticides. Pestic Sci, 45: 95-105.

    Manasherob R, Ben-Dov E, Zaritsky A, & Barak Z (1998) Germination,
    growth, and sporulation of  Bacillus thuringiensis subsp.
     israelensis in excreted food vacuoles of the protozoan  Tetrahymena
     pyriformis. Appl Environ Microbiol, 64: 1750-1758.

    Margalit J & Dean D (1985) The story of  Bacillus thuringiensis var.
     israelensis. J Am Mosq Control Assoc, 1: 1-7.

    Martin PAW & Travers RS (1989) Worldwide abundance and distribution of
     Bacillus thuringiensis isolates. Appl Environ Microbiol, 55(10):
    2437-2442.

    Martin WF & Reichelderfer CF (1989)  Bacillus thuringiensis:
    Persistence and movement in field crops. In: Abstracts of the SIP
    XXIInd Annual Meeting, Centre for Adult Education, University of
    Maryland, College Park, Maryland, 20-24 August 1989. Society for
    Invertebrate Pathology, p 25.

    Martouret D & Euverte G (1964) The effect of  Bacillus thuringiensis
    Berliner preparations on the honey bee under conditions of forced
    feeding. J Insect Pathol, 6: 198-203.

    Meadows MP (1993)  Bacillus thuringiensis in the environment: Ecology
    and risk assessment. In: Entwistle PF, Cory JS, Bailey MJ, & Higgs S
    ed.  Bacillus thuringiensis, an environmental biopesticide: Theory
    and practice. Chichester, New York, Toronto, Wiley & Sons, pp 193-220.

    Meadows MP, Ellis DJ, Butt J, Jarrett P, & Burges HD (1992)
    Distribution, frequency and diversity of  Bacillus thuringiensis in
    an animal feed mill. Appl Environ Microbiol, 58(4): 1344-1350.

    Melin BE & Cozzi EM (1990) Safety to nontarget invertebrates of
    Lepidopteran strains of  Bacillus thuringiensis and their
    (ß)-exotoxins. In: Laird M, Lacey LA, & Davidson EW ed. Safety of
    microbial insecticides. Boca Raton, Florida, CRC Press, pp 149-167.

    Menon AS & De Mestral J (1985) Survival of  Bacillus thuringiensis
    var.  kurstaki in waters. Water Air Soil Pollut, 25: 265-274.

    Merritt RW, Walker ED, Wilzbach MA, Cummins KW, & Morgan WT (1989) A
    broad evaluation of Bti for black fly (Diptera:Simuliidae) control in
    a Michigan river: efficacy, carry and nontarget effects on
    invertebrates and fish. J Am Mosq Control Assoc, 5: 397-415.

    Mikami T, Horikawa T, Murakami T, Sato N, Ono Y, Matsumoto T, Yamakawa
    A, Murayama S, Katagiri S, & Suzuki M (1995) Examination of toxin
    production from environmental  Bacillus cereus and  Bacillus
     thuringiensis. J Pharm Soc Jpn, 115: 742-748.

    Miura T, Takahashi RM, & Mulligan FS III (1980) Effects of the
    bacterial mosquito larvicide,  Bacillus thuringiensis serotype H-14
    on selected aquatic organisms. Mosq News, 40: 619-622.

    Mohd-Salleh MB, Beegle CC, & Lewis LC (1980) Fermentation media and
    production of exotoxin by three varieties of  Bacillus thuringiensis.
    J Invertebr Pathol, 35: 75-83.

    Molloy DP (1992) Impact of the black fly (Diptera: Simuliidae) control
    agent  Bacillus thuringiensis var.  israelensis on Chironomids
    (Diptera: Chronomidae) and other non-target insects: results of ten
    field trials. J Am Mosq Control Assoc, 8: 24-31.

    Morris ON, Armstrong JA, & Hildebrand MJ (1977) Aerial field trials
    with a new formulation of  Bacillus thuringiensis against the spruce
    budworm,  Choristoneura fumiferana (Clem.). Ottawa, Canada, Chemical
    Control Research Institute (Report CC-X-144).

    Morris ON, Hildebrand MJ, & Armstrong JA (1980) Preliminary field
    studies on the use of additives to improve deposition rate and
    efficacy of commercial formulations of  Bacillus thuringiensis
    applied against the spruce budworm,  Choristoneura fumiferana
    (Lepidoptera: Tortricidae). Sault Ste. Marie, Ontario, Canada, Forest
    Pest Management Institute, pp 1-23 (Report FPM-X-32).

    Moulinier CL, Mas JP, Moulinier Y, De Barjac H, Giap G, & Couprie B
    (1995) Étude de l'innocuité de  Bacillus thuringiensis var.
     israelensis pour les larves d'huitre. Bull Soc Pathol Exot, 74(4):
    381-391.

    Mück O, Hassan S, Huger AM, & Krieg A (1981) Effects of  Bacillus
     thuringiensis Berliner on the parasitic hymenoptera  Apanteles
     glomeratus  L. (Braconidae) and  Pimpla turionellae  (L.)
    (Ichneumonidiae). Z Angew Entomol, 92: 303-314.

    Mulla MS (1988) Activity, field efficacy and use of  Bacillus
     thuringiensis (H-14) against mosquitoes. In: de Barjac H &
    Sutherland DJ ed. Bacterial control of mosquitoes and blackflies. New
    Brunswick, New Jersey, Rutgers University Press.

    Mulla MS & Khasawinah AM (1969) Laboratory and field evaluation of
    larvicides against chironomid midges. J Econ Entomol, 62: 37-41.

    Mulla MS, Norland RL, Fanara DM, Darwazeh HA, & McKean DW (1971)
    Control of chironomid midges in recreational lakes. J Econ Entomol
    64(1): 300-307.

    Mulla MS, Federici BA, & Darwazeh HA (1982) Larvicidal efficacy of
     Bacillus thuringiensis serotype H-14 against stagnant-water
    mosquitoes and its effects on nontarget organisms. Environ Entomol,
    11: 788-795.

    Mulligan FS III & Schaefer CH (1982) Integration of a selective
    mosquito control agent  Bacillus thuringiensis serotype H-14, with
    natural predator populations in pesticide-sensitive habitats. Proc
    Calif Mosq Vector Control Assoc, 49: 19-22.

    Murakami T, Hiraoka T, Matsumoto T, Katagiri S, Shinagawa K, & Suzuki
    M (1993) Analysis of common antigen of flagella in  Bacillus cereus
    and  Bacillus thuringiensis. FEMS Microbiol Lett, 107: 179-184.

    Narva KE, Payne JM, Schwab GE, Hickle LA, Galasan T, & Sick AJ (1991)
    Novel  Bacillus thuringiensis microbes active against nematodes, and
    genes encoding novel nematode-active toxins cloned from  Bacillus
     thuringiensis isolates: European patent application EP0462 721A2.
    Munich, Germany, European Patent Office.

    Navon A (1993) Control of Lepidopteran pests with  Bacillus
     thuringiensis. In:  Bacillus thuringiensis, An Environmental
    Biopesticide: Theory and Practice. Eds, Entwistle PF, Cory JS, Bailey
    MJ & Higgs S. Chichester, New York, Toronto, Wiley & Sons, pp 126-146.

    Nielsen-LeRoux C, Hansen BM, Henriksen NB (1998) Safety of  Bacillus
     thuringiensis. IOBC Bull, 21: 269-272.

    Nishikawa Y, Kramer JM, Hanaoka M, & Yasukawa A (1996) Evaluation of
    serotyping, biotyping, plasmid banding pattern analysis, and HEp-2
    vacuolation factor assay in the epidemiological investigation of
     Bacillus  cereus emetic-syndrome food poisoning. Int J Food
    Microbiol, 31: 149-159.

    Noble MA, Riben PD, & Cook GJ (1992) Microbiological and
    epidemiological surveillance programme to monitor the health effects
    of Foray 48B BTK spray. Vancouver, Canada, Ministry of Forests of the
    Province of British Columbia, pp 1-63.

    Norris JR (1971) The protein crystal toxin of  Bacillus
     thuringiensis: biosynthesis and physical structure. In: Burges HD &
    Mussey NW ed. Microbial control of insects and mites. New York,
    London, Academic Press Inc, pp 229-246.

    Notermans S & Batt CA (1998) A risk assessment approach for food-borne
     Bacillus cereus and its toxins. J Appl Environ Microbiol Symp,
    84(suppl): 51S-61S.

    Obadofin AA & Finlayson DG (1977) Interactions of several insecticides
    and a carabid predator  (Bembidion lampros (Hrbst)) and their effects
    on  Hylemya brassicae (Bouché). Can J Plant Sci, 57: 1121-1126.

    Ohana B, Margalit J, & Barak Z (1987) Fate of  Bacillus thuringiensis
    subsp.  israelensis under simulated field conditions. Appl Environ
    Microbiol, 53: 828-831.

    Ohba M & Aizawa K (1978) Serological identification of  Bacillus
     thuringiensis and related bacteria isolated in Japan. J Invertebr
    Pathol, 32: 303-309.

    Ohba M & Aizawa K (1986) Distribution of  Bacillus thuringiensis in
    soils of Japan. J Invertebr Pathol, 47: 277-282. 

    Ohba M, Yu YM, & Aizawa K (1988) Occurrence of noninsecticidal
     Bacillus thuringiensis flagellar serotype 14 in the soil of Japan.
    Appl Microbiol, 11: 85-89.

    Olejnicek J & Maryskova B (1986), The influence of  Bacillus
     thuringiensis on the mosquito predator  Notonecta glauca. Folia
    Parasitol (Prague), 37: 279.

    Otvos IS & Vanderveen S (1993) Environmental report and current status
    of  Bacillus thuringiensis var.  kurstaki use for control of forest
    and agricultural insect pests. Victoria, British Columbia, Ministry of
    Forests, Forestry Canada, pp 1-81.

    Pedersen JC, Damgaard PH, Eilenberg E, & Hansen BM (1995) Dispersal of
     Bacillus thuringiensis var.  kurstaki in an experimental cabbage
    field. Can J Microbiol, 41: 118-125.

    Petras SF & Casida LE Jr (1985) Survival of  Bacillus thuringiensis
    spores in soil. Appl Environ Microbiol, 50: 1496-1501.

    Pinnock DE, Brand RJ, Jackson KL, & Milstead JE (1974) The field
    persistence of  Bacillus thuringiensis spores on  Cercis occidentalis
    leaves. J Invertebr Pathol, 23: 341-346.

    Pinnock DE, Milstead JE, Kirby ME, & Nelson BJ (1977) Stability of
    entomopathogenic bacteria. In: Environmental stability of microbial
    insecticides: Miscellaneous publications. Lanham, Maryland,
    Entomological Society of America, vol 10, pp 77-97.

    Pivovarov Yu P, Ivashina SA, & Padalkin VP (1977) [Hygienic assessment
    of investigation of insecticide preparations.] Gig i Sanit, 8: 45-48
    (in Russian).

    Prasertphon S, Areekul P, & Tanada Y (1973) Sporulation of  Bacillus
     thuringiensis in host cadavers. J Invertebr Pathol, 21: 205-207.

    Prefontaine G, Fast P, Lau PCK, Hefford MA, Hanna Z, & Brousseau R
    (1987) Use of oligonucleotide probes to study the relatedness of
    delta-endotoxin genes among  Bacillus thuringiensis subspecies and
    strains. Appl Environ Microbiol, 53: 2808-2814.

    Priest FG, Goodfellow M, & Todd C (1988) A numerical classification of
    the genus  Bacillus. J Gen Microbiol, 134: 1847-1882.

    Priest FG, Kaji DA, Rosato YB, & Canhos VP (1994) Characterization of
     Bacillus thuringiensis and related bacteria by ribosomal RNA gene
    restriction fragment length polymorphisms. Microbiology, 140:
    1015-1022.

    Purcell BH (1981) Effects of  Bacillus thuringiensis var.
     israelensis on  Aedes taeniorhynchus and some non-target organisms
    in the salt marsh. Mosq News, 41(3): 476-484.

    Pusztai M, Fast P, Gringorten L, Kaplan H, Lessard T, & Carey PR
    (1991) The mechanism of sunlight-mediated inactivation of  Bacillus
     thuringiensis crystals. Biochem J, 273: 43-47.

    Quinlan RJ (1990) Registration requirements and safety considerations
    for microbial pest control agents in the European Economic Community.
    In: Laird M, Lacey LA, & Davidson EW ed. Safety of microbial
    pesticides. Boca Raton, Florida, CRC Press, pp 11-18.

    Ray DE (1990) Pesticides derived from plants and other organisms. In:
    Hayes WJ & Laws ER ed. Handbook of pesticide toxicology. New York,
    London, Academic Press, pp 585-636.

    Reardon RC & Haissig K (1983) Spruce budworm (Lepidoptera:
    tortricidae) larval populations and field persistence of  Bacillus
     thuringiensis after treatment in Wisconsin. J Econ Entomol, 76:
    1139-1143.

    Reddy A, Battisti L, & Thorne CB (1987) Identification of
    self-transmissible plasmids in four  Bacillus thuringiensis
    subspecies. J Bacteriol 169(11): 5263-5270.

    Rogatin AB & Baizhanov M (1984) [Laboratory study of  Bacillus
     thuringiensis israelensis serotype H-14 on various groups of
    hydrobionts.] Izv Akad Nauk Kaz SSR Ser Biol, D6: 22-25 (in Russian).

    Salama HS & Zaki FN (1983) Interaction between  Bacillus thuringiensis
    Berliner and the parasites and predators of  Spodoptera littoralis
    in Egypt. Z Angew Entomol, 95: 425-429.

    Salama HS, Zaki FN, & Sharaby AF (1982) Effect of  Bacillus
     thuringiensis Berl. on parasites and predators of the cotton
    leafworm  Spodoptera littoralis (Boisd). Z Angew Entomol, 94:
    498-504.

    Saleh SM, Harris RF, & Allen ON (1970) Fate of  Bacillus thuringiensis
    in soil: Effect of soil pH and organic amendment. Can J Microbiol,
    16: 677-680.

    Samples JR & Buettner H (1983) Corneal ulcer caused by a biologic
    insecticide  (Bacillus thuringiensis). Am J Ophthalmol, 95(2):
    258-260.

    Sampson MN & Gooday GW (1998) Involvement of chitinases of  Bacillus
     thuringiensis during pathogenesis in insects. Microbiology, 144:
    2189-2194.

    Schnepf E, Crickmore N, van Rie J, Lereclus D, Baum J, Feitelson J,
    Zeigler DR, & Dean DH (1998)  Bacillus thuringiensis and its
    pesticidal crystal proteins. Microbiol Mol Biol Rev, 62: 775-806.

    Schnetter W, Engler S, Morawcsik J, & Becker N (1981) [Efficacy of
     Bacillus  thurigiensis  var. israelensis against mosquito larvae
    and non-target organisms.] Mitt Dtsch Ges Allg Angew Entomol, 2:
    195-202 (in German).

    Schwartz JL, Garneau L, Masson L, & Brousseau R (1991) Early response
    of cultured lepidopteran cells to exposure to  delta-endotoxin from
     Bacillus thuringiensis: involvement of calcium and anionic channels.
    Biochem Biophys Acta, 1065: 250-260.

    Shadduck JA (1980)  Bacillus thuringiensis serotype H-14 maximum
    challenge and eye irritation safety tests in mammals. Geneva, World
    Health Organization, pp 1-21 (WHO/VBC/80.763).

    Shadduck JA (1983) Some considerations on the safety evaluation of
    nonviral microbial insecticides. WHO Bull, 6(1): 117-128.

    Sheeran W & Fisher SW (1992) The effects of agitation, sediment and
    competition on the persistence and efficacy of  Bacillus thuringiensis
    var.  israelensis (Bti). Ecotoxicol Environ Saf, 24: 338-346.

    Shevelev AB, Lewitin E, Novikova SI, Wojciechowska YaA, Usacheva EA,
    Chestukhina GG, & Stepanov (1998) A new PCR-based approach to a fast
    search of a wide spectrum of  cry genes from  Bacillus thuringiensis
    strains. Biochem Mol Biol Int, 45: 1265-1271.

    Shinagawa K (1990) Purification and characterization of  Bacillus
     cereus enterotoxin and its application to diagnosis. In: Pohland AE,
    Dowell VR Jr, & Richard JL ed. Microbial toxins in foods and feeds:
    Cellular and molecular modes of action. New York, Plenum Press, pp
    181-193.

    Siegel JP & Shadduck JA (1990) Clearance of  Bacillus sphaericus and
     Bacillus thuringiensis ssp.  israelensis from mammals. J Econ
    Entomol, 83: 347-355.

    Siegel JP, Shadduck JA, & Szabo J (1987) Safety of the entomopathogen
     Bacillus thuringiensis var.  israelensis for mammals. J Econ
    Entomol, 80: 717-723.

    Slatin SL, Abrams CK, & English L (1990) Delta-endotoxins form
    cation-selective channels in planar lipid bilayers. Biochem Biophys
    Res Commun, 169: 765-772.

    Slepecky RA & Hemphill HE (1992) The genus  Bacillus-nonmedical. In:
    Balows A, Truper HG, Dworkin M, Harder W, & Schleifer K-H ed. The
    prokaryotes, 2nd ed. New York, Basel, Springer-Verlag, vol II, pp
    1663-1696.

    Smedley DP & Ellar DJ (1996) Mutagenesis of 3 surface-exposed loops of
    a  Bacillus thuringiensis insecticidal toxin reveals residues
    important for toxicity, recognition and possibly membrane insertion.
    Microbiology, 142: 1617-1624.

    Smith RA (1987) Use of crystal serology to differentiate among
    varieties of  Bacillus thuringiensis. J Invertebr Pathol, 50: 1-8.

    Smith RA & Couche GA (1991) The phylloplane as a source of  Bacillus
     thuringiensis variants. Appl Environ Microbiol, 57: 311-315.

    Snarski VM (1990) Interactions between  Bacillus thuringiensis subsp.
     israelensis and fathead minnows,  Pimephales promelas Rafinesque,
    under laboratory conditions. Appl Environ Microbiol, 56: 2618-2622.

    Sorenson AA & Falcon LA (1980) Comparison of microdroplet and high
    volume application of  Bacillus thuringiensis on pear suppression of
    fruit tree leaf roller  Acrhips argyrospilus and coverage on foliage
    and fruit. Environ Entomol, 9: 350.

    Stahly DP, Dingman DW, Bulla LA Jr, & Aronson AI (1978) Possible
    origin and function of the parasporal crystals in  Bacillus
     thuringiensis. Biochem Biophys Res Commun, 84(3): 581-588.

    Stahly DP, Andrews RE, & Yousten AA (1991) The genus  Bacillus-insect
    pathogens. In: Balows A, Truper HG, Dworkin M, Harder W, & Schleifer
    K-H ed. The prokaryotes, 2nd ed. New York, Basel, Springer-Verlag, vol
    II, pp 1697-1745.

    Surprenant DC (1989) Acute toxicity of  Bacillus thuringiensis var.
     tenebrionis technical material to rainbow trout  (Salmo gairdneri)
    under static renewal conditions. Wareham, Massachusetts, Springborn
    Life Sciences Inc., pp 1-19 (Unpublished Abbott document).

    Tayabali AF & Seligy VL (1997) Cell integrity markers for  in vitro
    evaluation of cytotoxic responses to bacteria-containing commercial
    insecticides. Ecotoxicol Environ Saf, 37: 152-162.

    Thomas WE & Ellar DJ (1983)  Bacillus thuringiensis var.  israelensis
    crystal delta-endotoxin: effects on insect and mammalian cells  in
     vitro and  in vivo. J Cell Sci, 60: 181-197.

    Thomas EM & Watson TF (1986) Effect of Dipel  (Bacillus thuringiensis)
    on the survival of immature and adult  Hyposoter exiguae
    (Hymenoptera: Ichneumonidae). J Invertebr Pathol, 47: 178-183.

    Tomlin CDS ed. (1997) The pesticide manual, 11th ed. Farnham, Surrey,
    British Crop Protection Council, 1606 pp.

    Travers RS, Martin PAW, & Reichelderfer CF (1987) Selective process
    for efficient isolation of soil  Bacillus spp. Appl Environ
    Microbiol, 53: 1263-1266.

    Väisänen OM, Mwaisumo NJ, & Salkinoja-Salonen MS (1991)
    Differentiation of dairy strains of the  Bacillus cereus group by
    phage typing, minimum growth temperature, and fatty acid analysis. J
    Appl Bacteriol, 70: 315-324.

    Van Frankenhuyzen K (1993) The challenge of  Bacillus thuringiensis.
    In: Entwistle PF, Cory JS, Bailey MJ, & Higgs S ed.  Bacillus
     thuringiensis, an environmental biopesticide: Theory and practice.
    Chichester, New York, Toronto, Wiley & Sons, pp 1-35.

    Vaòková J & Purrini K (1979) Natural epizooties caused by bacilli of
    the species  Bacillus thuringiensis and  Bacillus cereus. Z Angew
    Entomol, 88: 216-221.

    Van Rie J, Jansens S, Höfte H, Degheele D, & Van Mellaert H (1989),
    Specificity of  Bacillus thuringiensis delta-endotoxins: Importance
    of specific receptors on the brush border membrane of the mid-gut of
    target insects. Eur J Biochem, 186: 239-247.

    Venkateswerlu G & Stotzky G (1992) Binding of the protoxin and toxin
    proteins of  Bacillus thuringiensis subsp.  kurstaki on clay
    minerals. Curr Microbiol, 25: 225-233.

    Visser B, Bosch D, & Honée G (1993) Domain-function studies of
     Bacillus thuringiensis crystal proteins: a genetic approach. In:
    Entwistle PF, Cory JS, Bailey MJ, & Higgs S ed.  Bacillus
     thuringiensis, an environmental piopesticide: Theory and practice.
    Chichester, New York, Toronto, Wiley & Sons, pp 71-88.

    Wallner W & Surgeoner G (1974) Control of oakleaf caterpillar,
     Heterocampa manteo, and the impact of controls on nontarget
    organisms. Chicago, Illinois, Abbott Laboratories (Unpublished
    document).

    Wallner WE, Dubois NR, & Grinberg PS (1983) Alteration of parasitism
    by  Rogas lymantriae (Hymenoptera: Braconidae) in  Bacillus
     thuringiensis-stressed gypsy moth (Lepidoptera: Lymantriidae) hosts.
    J Econ Entomol, 76: 275-277.

    Warren RE, Rubenstein D, Ellar DJ, Kramer JM, & Gilbert RJ (1984)
     Bacillus thuringiensis var. israelensis: Protoxin activation and
    safety. Lancet: March 24: 678-679.

    Weires RW & Smith GL (1977) Apple mite control. Hudson Valley, New
    York, New York State Agricultural Experimental Station.

    Weseloh RM & Andreadis TG (1982) Possible mechanism for synergism
    between  Bacillus thuringiensis and the gypsy moth (Lepidoptera:
    Lymantriidae) parasitoid,  Apanteles melanoscelus (Hymenoptera:
    Braconidae). Ann Entomol Soc Am, 75: 435-438.

    Weseloh RM, Andreadis TG, Moore REB, & Anderson JF (1983) Field
    confirmation of a mechanism causing synergism between  Bacillus
     thuringiensis and the gypsy moth parasitoid,  Apanteles
    melanoscelus. J Invertebr Pathol, 41: 99-103.

    West AW, Burges HD, & Wyborn CH (1984a) Effect of incubation in
    natural and autoclaved soil upon potency and viability of  Bacillus
     thuringiensis. J Invertebr Pathol, 44: 121-127.

    West AW, Burges HD, White JR, & Wyborn CH (1984b) Persistence of
     Bacillus thuringiensis parasporal crystal insecticidal activity in
    soil. J Invertebr Pathol, 44: 128-133.

    WHO (1982) Data sheet on the biological control agent  Bacillus
     thuringiensis serotype H-14 (de Barjac 1978). Geneva, World Health
    Organization (WHO/VBC/79.750 Rev. 1).

    WHO (1985) Ninth report of the WHO Expert Committee on Vector Biology
    and Control -- Safe Use of Pesticides. Geneva, World Health
    Organization (WHO Technical Report Series No. 720).

    WHO (1991) Fourteenth report of the WHO Expert Committee on Vector
    Biology and Control -- Safe Use of Pesticides. Geneva, World Health
    Organization (WHO Technical Report Series No. 813).

    Wilkinson JD, Biever KD, & Ignoffo CM (1975) Contact toxicity of some
    chemical and biological pesticides to several insect parasitoids and
    predators. Entomophaga, 20: 113-120.

    Workman RB (1977) Pesticides toxic to striped earwig, an important
    insect predator. Proc Fla State Hortic Soc, 90: 401-402.

    Wunschel D, Fox KF, Black GE, & Fox A (1994) Discrimination among the
     B. cereus group, in comparison to  B. subtilis, by structural
    carbohydrate profiles and ribosomal RNA spacer region PCR. System Appl
    Microbiol, 17: 625-635.

    Yameogo L, Levêque C, Traoré K, & Fairhurst CP (1988) Dix ans de
    surveillance de la faune aquatique des rivières d'Afrique de l'Ouest
    traitées contre les Simulides (Diptera: Simuliidae), agents vecteurs
    de l'onchocercose humaine. Nat Can, 115: 287-298.

    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.