-i-^^

RE\/\E\N OF

BACILLUS THURINGIENSIS

VAR. KURSTAKI (BTK)

FOR USE IN

FOREST PEST MANAGEMENT

PROGRAMS IN ONTARIO -

WITH SPECIAL EMPHASIS

ON THE

AQUATIC ENVIRONMENT

APRIL 1990

Environment
Environnement

Ontario Jlm Bradley, Minister/ministre

ISBN 0-7729-6453-X

REVIEW OF BACILLUS THURINGIENSIS VAR . KURSTAKI (BTK)
FOR USE IN FOREST PEST MANAGEMENT
PROGRAMS IN ONTARIO - WITH SPECIAL EMPHASIS
ON THE AQUATIC ENVIRONMENT

Report prepared for:

Aquatic Criteria Development Conunittee

Water Resources Branch

Report prepared by:

Dr. G. A. Surgeoner

M. J. Farkas

Department of Environmental Biology

University of Guelph

Guelph, Ontario

NIG 2W1

APRIL 1990

Q

Copyright: Queen's Printer for Ontario, 1990
This publication may be reproduced for non-commercial purposes
with appropriate attribution.

PREAMBLE

In response to the Ministry of Natural Resources' preparation of
Class Environmental Assessment for timber management on crown lands,
the Ontario Ministry of the Environment (MOE) commissioned the
Environmental Review Of Bacillus thurinqiensis var. kurstaki (Btk)
for Use in Forest Pest Management Programs of Ontario With Special
Empasis On The Aquatic Environment.

This review focuses on the aquatic ecosystem effects of Btk as they
relate to the control of insect infestations on crown lands. The
review also considers the effects of Btk on non-target terrestrial
biota (wildlife and humans) as they relate to the exposure of these
lifeforms through the contact and ingestion of contaminated water
and/or biota.

The intent was to develop a Provincial Water Quality Objective
(PWQO) from the information provided in the review document.
However, the difficulty in enumerating Btk in water has prohibited
the development of a numerical PWQO. Instead, a narrative
ENVIRONMENTAL IMPACT STATEMENT has been prepared and is provided on
the following pages.

ENVIRONMENTAL IMPACT STATEMENT

The accxomulated information suggests that no significant impacts on
aquatic or on other beneficial uses of Provincial surface waters
would result from the properly planned, controlled and supervised
use of Bacillus thuringiensis var. kurstaki (Btk) for the control
of forest pests.

RATIONALE

INTRODUCTION

Bacillus thuringiensis (Bt) is a gram positive, mobile, aerobic
bacteria closely related to the ubiquitous soil bacteria Bacillus
cereus . There are at least 20 recognized sub-species (serotypes,
varieties) and 800 strain isolates of Bt (de Barjac 1981, pp. 40-
41) . Bacillus thuringiensis var. kurstaki (Btk) is a strain selected
for high potency against Lepidoptera larvae.

In Canada, 17 formulations of Btk have been registered for the
control of Lepidopterous pests. Formulations exist in Canada for
control of Lepidopterous pests on a variety of commercial vegetables
as well as for home and garden use. In Ontario, the Ministry of
Natural Resources uses Btk (when required) for the control of the
spruce budworm, (Choristoneura fumif erana) , the jack pine
budworm, (Choristoneura pinus) , and the gypsy moth, (Lymantria
dispar) . Most forestry applications in Ontario are applied under
contract to the Ministry of Natural Resources.

Btk is grown in a media which contains carbon, nitrogen and trace
minerals. In response to reduced available nutrients the bacteria
sporulates forming at one end of the cell a dormant endospore and
at the other end the protein crystal, which contains the toxin,
delta-endotoxin, causing Lepidoptera mortality. Once sporulation has
been completed the media is treated to destroy vegetative cells.
Endospores and crystals in approximately equal numbers are separated
from the fermentation broth (growth media) and spray dried to form
a fine technical powder. Each fermentation batch of Btk undergoes
quality assurance tests for microbiological contamination and a
mouse test to ensure no effects against mammalian organisms.lt is
the concentrations of certain proteins making up the crystal and
endospore coat that determine the potency of Btk, not the number of
protein crystals and endospores . The rate of application for a
typical treatment v/ould be approximately 30 BIU per hectare.

11

ENVIRONMENTAL FATE

After application, Btk looses 50% of its insecticidal activity in
1-3 days. However, some viable endospores have been recovered from
foliage one year after ground application. Persistence of Btk on
foliage is dependent on many environmental factors including length
of exposure to sunlight (ultra-violet radiation) , leaf temperature
and vapour pressure deficit.

Bacillus thuringiensis (Bt) can survive as endospores in most types
of soils, although Bt will not grow at pH below 4.8 . Bt may persist
in soils for up to several months. The fate of Bt in soils is
dependent upon microbial competition. Studies have shown that the
relative number of Bt compared to other soil bacilli was reduced
from 20-40% to about 10% over a 12 month period indicating that Bt
is not well adapted to the soil environment. Bt has also been shown
to be immobile in soils.

Btk may directly enter the aquatic environment during spray
operations since no spray buffer zones between application areas and
surface waters are required. Btk may be transported to surface water
bodies in the wash-off from treated trees and the subsequent surface
run-off. Btk may persist as viable spores in lake waters for a
considerable length of time (>70 days) . However, in streams and
rivers Btk is far less persistent.

EFFECTS ON TERRESTRIAL LIFE

Of the beneficial arthropods which were exposed to Dipel" (Btk) , only
one species, Syrphus showed slight effects. At the minimum
recommended field dose, Btk (as Thuricide HPC") had little or no
effect on parasites and predators of the spruce budworm. Post
treatment longevity of Cardiochiles niqriceps (a tobacco budworm
parasitoid) was decreased after ingestion of Btk (Dipel" and Biotrol'
XK) dissolved in sugar water. The researchers contend that C.
niqriceps would not feed on Btk in the field, and only fed on it in
the laboratory since the sugar acted as an attractant . Bees and wasps
exposed to Btk showed no effects. Btk also did not reduce emergence
of parasites or percentage parasitism of the European corn borer.

To determine the effects of Btk on natural enemies of the western
spruce budworm, parasite studies were conducted by Niva et al . ,
(1987) during field tests of two Btk formulations (San 41S and
Thuricide 32 LV) . The results indicated that infected parasites

matured sufficiently to exit from their hosts, but were unable to
complete their development. However, premature parasite mortality
may have been due to lack of nutrition caused by the early deaths
of their hosts, rather than microbial infection.

Significant mortality (<13.4%) was noted in adult Chrysopa carnea
and Hippodamia converqens 3 to 7 days after exposure to Btk, at
dosages approximately 4 to 8 times greater than that which would
reach insects under normal field applications.

No significant reductions in bird populations (74 species
representing 21 families) were apparent in areas treated with Btk
(with and without chitinase) , in Canadian spruce-fir forest
treatment plots, located in Spruce Woods, Manitoba and Algonquin
Park, Ontario. Laboratory tests with pheasants, partridges, cornish
hens, and quail, exposed orally to Bt , yielded no effects. Sparrows
and juncos administered 0.25 ml of 3 x 10' spores/ml daily by syringe
for 2 weeks exhibited no mortality, weight loss or change in the
appearance .

The possibility of indirect effects to birds, specifically spruce
grouse, due to reduced food availability has been raised. However,
it was shown that the year after spraying, the number of mating
pairs observed were not significantly different than the previous
year.

Tests exposing rats, mice, guinea pigs, young swine and hogs orally
to Bt by placing it directly into the stomachs or administering it
in the feed resulted in no symptoms of toxicity.

Sensitizing doses of Thuricide' were given to guinea pigs by
intracutaneous injection, the abrasion patch technique and topical
application on intact skin. Local irritation was found in injected
and abraded animals. No effects were observed from challenging
doses given two weeks after the last sensitizing treatment. Topical
applications also had no effect.

In addition to the oral, intraperitoneal, respiratory, dermal and
hypersensitivity tests. Bacillus Thuringiensis has passed the eye
exposure (in rabbits) and mutagenicity (in vitro) safety tests.

Field studies on plots treated with Bt in Algonquin Park, Ontario
and Spruce Woods, Manitoba indicated that small mammals continued
breeding through treatment periods and trapping data indicated that
the application of Bt treatments (with and without chitinase) did
not harm the small mammal complex inhabiting treatment areas.

EFFECTS ON AQUATIC LIFE

In the review of Btk toxicity to fish, few published scientific
papers pertaining to modern Bt formulations were found. The older

formulations presumably contained beta-exotoxins which are more
toxic to vertebrates. Therefore, studies conducted with the older
formulations would indicate greater toxicity to fish than would be
found with Bt formulations currently on the market today.

The toxic concentrations of Bt to fish in laboratory tests were
typically 1,000 fold higher than those derived from field
applications. Little or no effects were observed when rainbow trout,
yellow perch, mosquito fish or black bullhead fingerlings were
exposed for 4 days to concentrations of 4.5 to 6.5x10' spores/ml.
Effects probably due to the petroleum carrier occurred at a dose of
> 6x10^ spores/ml.

A field evaluation of Thuricide 16B applied in Algonquin Park,
Ontario at an estimated rate of 17.6 BIUs per hectare, showed no
adverse effects to fish populations (brook trout, white suckers,
smallmouth bass) and bottom fauna populations, in rivers for up to
four weeks post treatment. The lack of any documented fish kills
resulting from any of the forestry or agricultural spray programs
involving Btk across Canada and the United States is additional,
and probably the most valid evidence, of the safety of Btk to fish.

In a laboratory study a variety of aquatic invertebrates were
exposed to Btk (Thuricide 32LV) at 2 , 20 and 200 times the worst-
case transitory concentration expected in water after aerial
spraying. Only the black fly (Simulium vittatum) was clearly
affected at the highest exposure (430 lU/ml) with 75% mortality to
the test population. Some impact was suggested for Prosimul ium
Fuscum/mixtum (Simuliidae) and the midge Tanytarus (Chironomidae)
in the same study. Monitoring of water bodies during spray programs
have shown that Btk had no measurable effects on a large variety of
aquatic invertebrates. The most susceptible aquatic invertebrates
are black fly and chironomid larvae. However, no effects have been
reported at concentrations occurring from aerial applications.

HUMAN HEALTH EFFECTS

Despite wide spread use of Bt throughout the world only one
incidence of a corneal ulcer in a farm worker, apparently caused
when a Btk formulation was splashed into the eye, has been reported.
This is the only documented infection in humans and other
vertebrates. Antibiotic treatment resulted in complete recovery of
the farm worker. No effects were observed in a study, in which
eighteen volunteers ingested Ig of Thuricide'/day for 5 days at a
concentration of 3x10" viable spores/g ; and 5 of the test subjects
also inhaled an additional lOOmg of the powder daily over the 5 day
period and showed no effects. All people tested remained healthy and
all the extensive laboratory tests were negative.

Table of Contents

PREAMBLE i

ENVIRONMENTAL IMPACT STATEMENT ii

TABLE OF CONTENTS vi

ACKNOWLEDGEMENTS 1

CONCLUSIONS 2

1 , 0 INTRODUCTION 4

2.0 GENERAL DESCRIPTION

2 . 1 Taxonomy 5

2 . 2 Discovery 5

2 . 3 Presence in Nature 6

2.4 Epizootics in Nature 7

3 . 0 PRODUCTION PROCESS

3 . 1 Production 8

3.2 Commercial Products

Containing Btk 11

3 . 3 Additives to Formulations 13

3.4 Quality Assurance 15

3 . 5 Formulation Potency 15

3.6 Impurities in Commercial

Formulations 17

4.0 HOST SPECIFICITY 18

5 . 0 MODE OF ACTION 2 0

6 . 0 MUTAGENICITY 24

7 . 0 SPRAYING/APPLICATION PRACTICES 2 5

8.0 METHODS FOR ENUMERATING BTK FROM

ENVIRONMENTAL SAMPLES 3 0

9 . 0 ENVIRONMENTAL FATE

9. 1 Foliage 3 3

9.2 Soil 3 5

9.3 Water 3 6

VI

10.0 AQUATIC TOXICITY

10.1 Expected Exposure Levels 38

10.2 Fish Toxicity 38

10.3 Aquatic Invertebrates 41

11.0 HEALTH EFFECTS (TO HUMANS) 45

12.0 EFFECTS ON NON-TARGET TERRESTRIAL ANIMALS

12 . 1 Insects 4 6

12 . 2 Earthworms 4 9

12.3 Birds 5 0

12 . 4 Mammals 51

13.0 LITERATURE CITED 55

14.0 APPENDICES

Appendix 1 63

Appendix 2 71

Appendix 3 75

Appendix 4 86

ACKNOWLEDGEMENTS

The authors wish to thank the numerous personnel from the
Federal Pesticides Directorate, Forest Pest Management Institute,
Ministry of Natural Resources, and industry, who assisted in
answering questions. Particular thanks to Joe Churcher (Ministry
of Natural Resources) , Peter Kingsbury (Forest Pest Management
Institute) and Barry Tyler (Abbott Laboratories) ,

We would also like to thank the staff of the Ontario Ministry
of the Environment: C. Cherwinsky, C. de Barros, E. Leggatt, D.
Poirier, J. Ralston, and D. Rokosh who assisted in the review of the
draft manuscript and provided many useful comments.

CONCLUSIONS

1) The scientific literature indicates that Bacillus thuringiensis
var. kurstaki directly affects only Lepidoptera larvae.

2) In Ontario, the use of this bacteria has been extensive, v.'ith
over 1.2 million hectares of forest treated in the past 7 years for
control of the spruce budworm (Choristoneura fumif erana) , the jack
pine budworm (Choristoneura pinus) , and the gypsy moth (Lynantria
dispar) .

3) The use of Bacillus thuringiensis var. kurstaki, for forestry
purposes in Ontario, is restricted to licensed applicator personnel.

4) Based on the scientific literature, the use of Bacillus
thuringiensis var. kurstaki does not represent a detectable impact
to the quality of surface waters in the Province of Ontario.

5) The use of Bacillus thuringiensis var. kurstaki may reduce non-
target Lepidoptera, some of which may be rare. Studies should be
initiated to determine the effects of aerial applications of Btk to

Lepidoptera larvae, particularly those feeding on understory
vegetation.

6) The reduction of Lepidoptera larvae caused by Bacillus
thurinqiensis var. kurstaki may result in reduced food availability
to certain birds (e.g. spruce grouse) thus affecting nesting
success. Studies should be continued to determine if indirect
effects to birds are significant mortality factors.

1 . 0 INTRODUCTION

The Ontario Ministry of Natural Resources has recently
completed a draft, Class Environmental Assessment, for timber
management on crown lands. Under this Class Environmental
Assessment, the Ministry of Natural Resources proposes (when
required) to use Bacillus thuringiensis var. kurstaki (Btk) for
control of the spruce budworm, Choristoneura fumiferana , the jack
pine budworm, Choristoneura pinus , and the gypsy moth, Lymantria
dispar .

The potential for this "biological" insecticide to enter
surface water during and after application to timber lots is of
potential concern to the Ontario Ministry of the Environment. The
Ontario Ministry of the Environment has been mandated to develop,
and where appropriate, revise the Provincial Water Quality
Objectives and Policies to protect the provinces 's water resources.
These water quality objectives and policies are designed to assure
that "...surface v;aters in the province are of a quality which is
satisfactory for aquatic life and recreation".

In order to formulate a sound environmental policy for Btk,
the available scientific data must be procured and analyzed. The
purpose of this document is to provide the current information known
about this compound with particular emphasis on protecting surface
waters .

2.0 GENERAL DESCRIPTION

2.1 Taxonomy

Bacillus thurinqiensis (Bt) is a gram positive, mobile, aerobic
bacteria closely related to the ubiquitous soil bacteria Bacillus
cereus . Vegetative cells of Bt are approximately 1 um wide by 5 urn
long. The species is separated from B. cereus by its greater
pathogenicity to insects and its ability to produce a bipyrmidal
inclusion body (protein crystal) during sporulation (Heimpel 1967,
pp. 288-291) .

The term Bacillus thurinqiensis in many ways is misleading
since there are at least 20 recognized subspecies (serotypes,
varieties) and 800 strain isolates (de Barjac 1981, pp. 40-41). Any
documentation associated with Bt must include both the serotype and
strain being used and a potency comparison to an accepted
international reference standard. Bacillus thurinqiensis var.
kurstaki is a strain selected for high potency against Lepidoptera
larvae. It does not contain "beta-exotoxins" which exhibit some
mammalian toxicity and potential mutagenicity. This toxin is found
in certain varieties of Bt, i.e. Bacillus thurinqiensis
thurinqiensis . The various toxins associated with Bacillus
thurinqiensis are described in Section 5.0 Mode of Action.

2.2 Discovery

Bacillus thurinqiensis was first isolated from diseased
silkworm larvae in Japan in 1901. It was subsequently given

taxonomic validity by Berliner who isolated bacteria from diseased
Mediterranean flour moths from the Province of Thuringen in East
Germany. It was thus given the scientifically valid name of
Bacillus thurinaiensis Berliner. This original strain is now
designated Bacillus thurinqiensis var. thuringiensis . The isolate
Bacillus thurinqiensis var. kurstaki was first isolated in France
by Kurstak in 1962 but the strain used for commercial North American
production was isolated by Dulmage in 1970 from a laboratory colony
of diseased pink bollworm larvae in the U.S. The particular isolate
of Btk used in commercial formulations is known as Btk-HDl strain.
It is approximately 15X more potent to Lepidoptera larvae as
previous Bt isolates. The HD refers to Howard Dulmage who made the
original isolate.
2.3 Presence in Nature

Bacillus thurinqiensis can grow in moist soils, deriving
nutrients from decaying plants and can grow in tissues of infected
insects (Ghassemi et al. 1981) . In natural soils, Btk only exist
in the spore state (Akiba 1986) . Strains of Bt have been discovered
in many regions of the world, ie. Japan (silkworms); Germany (flour
moths) ; France (original Btk) ; Israel (Bti) ; Kenya (Bt kenyae) ; the
United States (Btk HD-1) and Canada (Bt canadensis) . This indicates
that the bacteria has a natural cosmopolitan distribution wherever
insects occur. Krieg and Langenbruch (1981, p. 841) list the
origins for 24 varieties of Btk.

In Japan, Bacillus thurinqiensis is readily isolated from the
litter beneath sericultural farms (11.4% of bacterial isolates).

It is, however, relatively rare in the forest soils of Japan with
a frequency of 2.7% of bacterial isolates (Ohba and Aizawa 1986).
In the United States, DeLucca et al. (1981) in a survey of soils
never previously treated with Bt, found that it occurred with a
frequency of 0.75% in the approximately 32,000 bacteria isolates
obtained, indicating that it is relatively rare in "natural soils".
It was, however, found in 17% of soils tested, in a wide variety of
soil types, ie. grass, rocky wooded area, and in soil pH ' s ranging
from 4.9 to 8.0. Various strains of Bt have been commonly found in
grain elevators and grain dusts. DeLucca et aJ. (1982) reported Bt
isolates from 55% of settled dust samples and 16.9% of respirable
dust samples in four large grain elevators near New Orleans. There
was no evidence of insects in any of the elevators at the time of
sampling.
2.4 Epizootics in Nature

The original isolation of Bt was made during investigation of
"sotto disease" affecting commercial silkworm production in Japan
(DeLucca et aj^. 1981). It was next isolated in Germany by Berliner
in association with an infestation of Mediterranean flour moths in
stored grains. These natural epizootics and others reported, ie.
sericulture in Japan, stored grains in Kenya and insect cultures,
all indicate that natural epizootics of Bt occur only when insects
are maintained in high densities within a confined area (DeLucca et
al. 1981) . Dulmage and Aizawa (1982) cite a study by Van der Laan
and Wassink (1969) where they attempted to initiate infections from
Bt-killed larvae into susceptible hosts. It was found that natural

infections from sick or dead larvae to susceptible hosts did not
occur. Epizootics caused by Bt rarely occur in nature although
background levels exist in soil samples and grain dusts (Dulmage and
Aizawa 1982). Like chemical insecticides, Btk should not be
expected to have any carry-over effects the following year.
Unexplained benefits to plots treated with Btk for spruce budworm
have been observed in the year following treatment (Dimond and Spies
1981) .

3.0 PRODUCTION PROCESS

3 . 1 Production

The Bacillus thurincfiensis var. kurstaki used for insecticide
formulations are produced in large fermentation vats (up to 130,000
litres) similar to those used for the commercial production of
antibiotics. For North America, there are three primary suppliers
of Btk: 1) Abbott Laboratories with production facilities in
Chicago, Illinois, 2) Duphar with primary production in Belgium,
and 3) Sandoz with primary production near San Francisco,
California. The active ingredient Btk is not produced in Canada but
some formulating does occur in association with Chemagro in Ontario.

The media for bacterial growth and actual media environment
(ie. pH, temperature, aeration level) are proprietary information
unavailable for this review. Each company would have slightly
different methodologies of production. The basic production concept
is similar for all companies (Fig.l).

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The media for bacterial growth must contain carbon, nitrogen
and trace minerals. In discussions with companies these are
obtained from normal commodity raw products. Typical nitrogen
sources may include fish meals, cotton seed flour, soybeans,
autolyzed yeasts and casein. Carbohydrate sources include
hydrolyzed corn products, starch and dextrose. Trace minerals would
be available in these carbon and nitrogen sources. The fermentation
medium ingredients for the U.S. reference standard include: generic
tryptone (10 g/L) , powdered corn (5 g/L) , generic yeast extract (2
g/L, K,H PO, and KH, PO, each at 1 g/L) (Beegle et al. 1986) .

Once the media has been formulated it is steam sterilized to
prevent microbial contamination. Isolates of Btk HD-1 strain are
inoculated into the media to initiate cell growth. Inoculum amounts
range from 2 to 5% of the fermenter volume. The media is then
slowly agitated and provided with an abundant supply of sterilized
air. The pH of the fermenter tanks is typically between 7.2 and
7.6. The temperature of the tanks is maintained at approximately
SCC.

After approximately five days of vigorous vegetative grov;th
the bacteria in response to reduced available nutrients begin to
sporulate forming at one end of the cell a dormant endospore and at
the other end the protein crystal, which contains the toxin (delta-
endotoxin) , causing Lepidoptera mortality.

Once sporulation has been completed the media is treated to
destroy vegetative cells. The spore-crystal complex can be exposed
to temperatures of lOO'C for short periods of time without

10

degradation. Endospores and crystals in approximately equal numbers
are concentrated from the fermentation broth by either
centrifugation or filtration. The resultant spores and crystals are
spray dried to form a fine technical powder. This powder is then
bioassayed against the cabbage looper Trichoplusia ni (Hubner) using
accepted standard bioassay techniques. Individual batches from
fermentaters will vary somewhat in their potency. Potency is not
directly related to the number of endospores or the number of
protein crystals. It is the concentrations of certain proteins
making up the crystal and endospore coat that determine the potency
of the Btk. The technical powders are allowed by international
agreement + 20% variance from the stated potency. Each bioassay
will give slightly different results with the same technical powder.
3.2 Commercial Products Containing Btk

In Canada, 17 formulations of Btk have been registered for
control of Lepidopterous pests in forestry (Table 1). In addition,
other formulations exist in Canada for control of Lepidopterous
pests on a variety of commercial vegetables. Formulations also
exist which have a domestic registration for availability to home
gardeners. There is no post-harvest interval or re-entry time
associated with the use of Btk, i.e. control of cabbage worm can be
made up to the day of harvest (OMAF 1988) .

In the United States, the major use for Btk is against
agricultural pests of cole crops, alfalfa and cotton, and

11

Table 1. Commercial Products of Btk With Potential Forestry Uses

Name

Manufacturer

Under PCPA'

Guarantee'

Dipel F

Abbott

13299

16.0 BlU/mg

Dipel SC

Abbott

14082

9.9 BlU/g

Dipel 88

Abbott

16873

8.5 BIU/L

Dipel 132

Abbott

17954

12.7 BIU/L

Dipel 176

Abbott

20599

12.7 BIU/L

Novabac - 3

Duphar

B

V.

17133

8.6 BIU/L

Futura XLV

Duphar

B

V.

20861

12.7 BIU/L

Futura

Duphar

B

V.

17778

14.4 BIU/L

Bactospeine F

Duphar

B

V.

17782

9.7 BIU/L

Novabac - 3

Cyanamid

15084

8.6 BIU/L

Pfizer

Pfizer

18334

9.7 BIU/L

Evirobac ES

Thuricide HPC

Sandoz

11302

4.2 BIU/L

Thuricide48LV

Sandoz

17980

12.7 BIU/L

1) PCPA: Pest Control Act

2) Guarantee is evaluated in terms of potency which is measured in
Billion International Units (BIU) per unit volume of weight,
reflecting the biological activity of the formulation against larvae
of the cabbage looper, Trichoplusia ni in a standardized bio-assay.

12

approximately 3% is used against forestry pests (Dubois and Lewis
1981). Over one million pounds of formulated Btk are applied
annually in the United States (DeLucca et aJ. 1981).

3.3 Additives to Formulations

Initial products with Btk were crude powders of spores, crystals,
growth media and inert ingredients. Formulations had suspendabil ity
problems, clogged spray systems and provided inconsistent control.
Today, Btk formulations are primarily spore and crystal
concentrates, prepared for use primarily as water suspensions or oil
emulsions. Additives such as thickening agents have been
incorporated into formulations to provide uniform suspensions,
wetting agents to obtain better leaf coverage, antievaporants ,
stickers to increase retention of spray deposits and sun screens to
reduce the degradation of crystals by UV radiation. A product such
as "Thuricide 16B" contains 0.34% Btk, 48.15% culture media, 1.5%
xylene, 0.01% Chevron sticker and 49.99% water (Menon and Mestral
1985) . (It should be noted that "Thuricide 16B is no longer in use
in Canada and no forestry Btk forestry products presently contain
xylene) . Concern has been expressed about potential toxicity of
these inert ingredients (Orton 1987) . Although individual
components of carriers are not listed, it has been shown that
carrier fluid of non-aqueous oil formulations caused less than 2.1%
mortality to non-target insects in laboratory trials (Haverty 1982) .
The Dipel oil vehicle also did not affect target spruce budworm
larvae (Morris 1983). Since target organisms and non-target insects

13

are unaffected by "oil carriers", it is reasonable to assume that
they will not affect larger organisms. Product labels indicate the
potency guarantee of the Btk but do not include concentrations of
other "inert" ingredients. These "inert" ingredients are critical
to the success of new formulations and are proprietary rights of
companies .

As shown by Fortin et al. (1986) (see aquatic toxicity section) and
the concern of micro contaminants (Agriculture Canada letter,
Appendix 1) the "inert ingredients" are perhaps the most toxic
components of Btk formulations. Their toxicities will be minor v/hen
one considers the amounts applied in operational control programs,
and published studies done on "vehicle carriers" in the literature
(Haverty 1982, Morris 1983). Proprietary toxicology data does exist
on many of these standard "inerts" and on the formulated product.
Federal agencies such as the Environmental Protection Service,
Health and Welfare, and Fisheries and Ocean have reviewed this
documentation. With permission of companies this information could
likely be obtained from the companies or Pesticides Directorate in
Ottawa .

Most formulations are now being applied in mixtures with water
(often at a 50/50 ratio) but the increasing tendency to reduce
application costs has been to spray the products "neat", ie.
undiluted. In 1988, all aerial applications in Ontario were made
using undiluted product at the rate of 2.4 L/ha (pers. comm. C.
Howard) . These products were applied primarily as oil-based
emulsions .

14

3.4 Quality Assurance

Each fermentation batch of Btk undergoes two quality assurance
tests, the first of which is mammalian (mouse). Five laboratory
white mice weighing 17-23 gm are injected subcutaneously with one
million spores of Btk from each batch lot. The mice are observed
for seven days. If there is any evidence of infection, skin
reaction or mortality the tests would be repeated. If reactions
continue the batch would be destroyed. Primary product released for
formulation must have no effect on mice injected subcutaneously with
one million spores. The other quality assurance check involves
examination for microbial contaminants and is discussed under
"Impurities in commercial formulations."

3.5 Formulation Potency

Standardization of Btk products is accomplished by bioassays rather
than analytical procedures since the active ingredient (s) is unknown
(proteins) and may actually consist of several different proteins.
Numbers of bacterial spores or parasporal crystals do not provide
reliable standards of potency. The accepted potency standard is the
International Unit (lU) which is derived from bioassays.
The reference standard (designated E-61) maintained by the Pasteur
Institute was derived from Bacillus thurinqiensis var. thurinqiensis
and was given an arbitrary potency of 1,000 lU/mg (Luthy et al .
1982) .

15

In 1970, Btk became the subspecies for production of commercial
formulations of Bt in North America. This strain derived from pink
bollworms was called the HD-1 strain after its discoverer Hov^ard
Dulmage. The lack of beta-exotoxins and high potency to Lepidoptera
larvae (15X higher than previous isolates) made this strain
commercially feasible and desirable.

In 1972, a preparation of HD-1, named HD-l-S-1971 was adopted as the
primary United States and Canadian reference standard. It was
assigned a potency of 18,000 lU/mg (Dulmage 1973). This standard
was subsequently changed to HD-l-S-1980 due to depletion of the HD-
l-S-1971 stock and differential activity of this strain between the
cabbage looper Trichoplusia ni and tobacco budworm Hel iothis
virescens . This reference standard to which current Btk production
is compared has a potency of 16,000 lU/mg (Beegle et a^. 1986) . The
standard is stored at -16'C at USDA, Brownsville in 25 g aliquots and
is available free of charge upon request.

All batches of Btk from commercial fermenters are compared to this
HD-l-S-1980 standard. Thus, if a particular batch in bioassays was
found to be twice as toxic to Trichoplusia ni as the reference
standard, it would be assigned a value of 32,000 lU/mg.
Many products use this potency in the name of the product. Thus,
Dipel 176 would have a potency of 17,600 lU/mg or Dipel 132 a
potency of 13,200 lU/mg. The primary powder is then diluted with
vehicle carriers and a potency on the basis of Billion International
Units per litre of product is provided, ie.

16

Base Powder Final Product

Futura Suspension 12,000 lU/mg 14.4 BIU/L

Dipel 132 13,200 lU/mg 12.7 BIU/L

Thuricide 48 LV 12,000 lU/mg 12.7 BIU/L

It is the potency of the final product in litres, ie. number of

Billion International Units (BIU) per litre which best reflects the

toxicity of the specific product to lepidopterous larvae. The lU/mg

is an explanation of the potency for the technical material,

whereas, the BIU ' s per litre is a measure of toxicity for the

product used in the spray programs.

3.6 Impurities in Commercial Formulations

In 1987, concerns were expressed regarding the presence of
microcontaminants, specifically fecal Streptococcus (S. f aecium) in
formulations of Btk (Cabana and Pelletier 1986) . The relevance of
these contaminants in regard to human health were determined by an
advisory committee of the Pesticides Directorate, Ottawa to be
minor. Streptococcus bacteria are common in many non-sterile foods,
ie. yogurt, and in the environment. S. faecium is commonly found
in the intestines of man and animal. The contaminants "represented
either non- or low-order pathogenicity and exposure under typical
forestry use applications would not likely produce adverse health
effects" (Agriculture Canada letter. Appendix 1) . Nevertheless, an
informal (not regulated) monitoring system for microcontaminants has
been established by companies and monitoring is being done as well

17

by Agriculture Canada's Laboratory Services.

The following potential contaminants and acceptable limits are being

analyzed for (pers. comm. , Glen Dalke, Abbott Laboratories).

Salmonella sp. -zero organisms in 2 5 gm primary powder

3
Coliform bacteria - less than 10 organisms/gm powder

5
Staphylococcus aureus - less than 10 organisms/gm powder

5
Clostridium perfrinqens - less than 10 organisms/gm powder

5
Pseudomonas aeruginosa - less than 10 organisms/gm powder

3
Streptococci/Enterococci - less than 10 organisms/gm powder

4.0 HOST SPECIFICITY

In this section, the review will discuss why Btk is host-specific
and the susceptibility of various insect groups. The toxicity of
Btk to birds, fish, mammals and humans will be discussed elsewhere.
Burger j on and Martouret (1971) provide an early review of the host
spectrum of Bacillus thurinqiensis . Krieg and Langenbruch (1981)
provide detailed information (see pp. 841-885) on the relative
susceptibility of various invertebrates to different strains of Bt.
Btk is shown to be non-toxic to 8 species of Coleoptera, slightly
toxic to a few species of Diptera (not at operational treatment
rates) , non-toxic to the ephemeropteran Picromerus bedens, non-toxic
to any Homoptera or Hemiptera, non-toxic to any Hymenoptera
including honeybees, but with significant toxicity to a wide variety
of Lepidoptera. Operational field trials indicate that use of Btk
enhances natural parasitism (Ticehurst et al. 1982) .
The various strains of Bt are also listed as being of zero to low

18

The various strains of Bt are also listed as being of zero to low
toxicity to Isoptera, Mallophaga, Neuroptera, Orthoptera,
Thysanoptera and Trichoptera. Krieg and Langenbruch also cite
personal unpublished data in this review indicating that Btk is non-
toxic to Daphnia sp. and Cyclops sp.

For all practical purposes, at field dosage rates, Btk and its
delta-endotoxin affect only Lepidoptera. The specificity of Btk is
determined by a variety of factors. The first is that Btk must be
ingested before it can kill a susceptible insect. Topical
applications of Btk to susceptible species would not result in
mortality (Wilkinson et al. 1975). Secondly, in order to release
the toxic delta-endotoxin from the protein crystal, the crystal must
be exposed to an alkaline pH of 9.0 to 10.5 and appropriate
digestive enzymes (Falcon 1971). This combination of requirements,
ingestion, alkaline gut pH, proteolytic enzymes, plus innate
susceptibility of the host, restrict the toxic effects of Btk to
Lepidoptera. Other strains of Bt do cause mortality in other orders
of insects (Heimpel 1967), e.g. Bti to mosquitoes and blackflies and
B.t. var thuringiensis to Diptera including house flies.
Within the Lepidoptera, there are varying degrees of susceptibility
(Heimpel 1967). Highly susceptible Lepidoptera, e.g. Bombyx mori ,
are classified as Type 1 insects. In these insects, delta-
endotoxins cause a rapid paralysis and change in gut pH . Most
Lepidoptera belong to the Type II group. These insects suffer rapid
gut paralysis, but there is no gut leakage, so these insects starve
and die due to septicemia. Type III insects require both delta-

19

endotoxin and endospores to cause death (see Heimpel 1967, p. 298).
In addition, under operational field conditions, mortality of target
insects, e.g. jackpine budworm, varies between trials but rarely
exceeds 90% mortality (e.g. jackpine budworm - 71% mortality)
(Cadogan et aJ. 1986) . Detailed data on the effects due to Btk on
non-target Lepidoptera were not found. It is expected that
approximately 10% of the Btk droplets would reach understory
vegetation (pers. comm. Nick Payne and Terry Innis, Forest Pest
Management Institute, Sault Ste. Marie). This reduced amount
reaching lower foliage would likely result in reduced mortality.
Research at the Forest Pest Management Institute (K. Barber) has
been initiated to answer the question of non-target Lepidoptera
mortality, but data is still pending.

5.0 MODE OF ACTION

The primary mechanism by which Bacillus thurinqiensis kills insects
is a combination of the delta-endotoxin associated with crystals and
potentially vegetative growth and septicemia in larvae. Mortality
is normally caused by toxins associated with the parasporal crystal
and to a limited extent, the endospores. Fast (1977) showed that
in spruce budworm (Choristoneura fumif erana) , spores enhanced the
toxicity of crystals two-fold. However, Fast (1977) concluded that
"spores play little or no role in mortality of spruce budworm
induced by Bacillus thurinqiensis insecticide", i.e. Btk. With
other strains of Bt, both spores and crystals are required to cause
mortality (Burges et al. 1976) . Fast (1976) found the LD,,, dose for

20

newly moulted 6th instar spruce budworm to be 0.6 lU or 1280
crystals and the LD<,o dose to be 3.5 lU or 7000 crystals.
Application of Btk does not result in natural epizootics of target
insects, nor does it control larvae in years subsequent to
application although reduced defoliation in following years has been
noted (Dimond and Spies 1981) . Increased development time,
debility, decreased pupal weights, reduced fecundity, fertility and
adult life span of survivors, has been suggested as possible
mechanisms to explain the few carry-over effects noted in nature
(Morris and Hildebrand 1974).

The chemical structure of the toxin (s) which are derived from Btk
have not been defined. This prevents analysis of formulation on an
analytical chemistry basis and requires that potency be determined
on a bioassay basis and residues on a microbiological basis.
The various toxins found in strains of Bt are proteins. Commercial
formulation of Btk contain the protein crystal and endospores v;ith
the primary toxin being delta-endotoxin, associated with the protein
crystal .

Other toxins include alpha-exotoxin . This toxin is found in the
supernatants of some Bt fermentations. It is water-soluble and
heat-labile. It is produced by bacteria other than B.t. This
material is toxic if fed to mice and has been called the mouse
factor or thermosensitive toxin (Kreig 1971) . Since commercial
formulations are spun dry and heat-treated, alpha-exotoxin is not
considered of significance in commercial formulations.
Beta-exotoxin is a water-soluble, heat-stable protein that is highly

21

toxic to Diptera. Many of the original insecticide formulations of
Bacillus thurinqiensis var. thurinqiensis contained beta-exotoxin .
Synonyms for this toxin include "fly knock-down factor" or
McConnell-Richards factor, and thuringiensin (Dubois and Lewis
1981) . Beta-exotoxin is toxic to vertebrates and can cause
teratogenic effects in insects (Sebesta et ai. 1981). Regulatory
authorities in Canada and the United States have banned the use of
formulations containing beta-exotoxins since 1971. Products
containing beta-exotoxins are still used in Europe and the Soviet
Union. Toxicity data to non-target organisms published in
scientific literature prior to 1971 generally refer to Bt strains
containing beta-exotoxin. These data, although not directly related
to Btk, would represent toxicities more potent than the modern Btk
strain which does not contain beta-exotoxin.

The louse-factor is an exotoxin reported by Gingric et al. (1974)
as being pathogenic to biting lice, and was derived from Btk HD-1
strain. Its significance in modern formulations of Bt is considered
minor and little work has been published on this toxin.
The principal insecticidal component of modern Bacillus
thurinqiensis formulations is the delta-endotoxin associated with
the protein crystal. The crystal itself is not toxic until it is
dissolved in vitro under specific conditions or in the midgut of the
larvae. The toxic protein is relatively small, between 50-100,000
daltons. In spray programs, Btk is not toxic unless ingested by
larvae . Three factors influence the potency of delta-endotoxin: 1)
the strain-related origin of the toxin. 2) the degree of solubility

22

in the gut juice, and 3) the intrinsic susceptibility of the insect

to the toxin (Jaquet et al. 1987) .

The toxic protein is insoluble at acidic or neutral pH , but is

soluble at alkaline pH. The most susceptible insects are those

having a gut pH of 9.0 to 10.5 and appropriate enzymes to dissolve

the crystals (Falcon 1971) . It is essentially only Lepidoptera

larvae which have this combination of digestive enzymes and alkaline

pH.

The toxin once released acts at the surface of the gut epithelial

cells to cause a rapid loss (within 5 minutes of feeding) of ATP

from cells stimulating respiration and glucose uptake. Within

minutes, microvilli of the gut swell and cell apices swell into the

gut lumen. Feeding is inhibited within as little as 2 minutes after

feeding.

Metabolic breakdown of epithelial cells is complete within 10-15

minutes and ions leak from gut lumen to haemolymph. Paralysis

and/or death results from ionic imbalance in the haemolymph. Often

changes in gut and haemolymph permit vegetative propagation of gut

microbial flora and Bacillus thurinqiensis resulting in septicemia

which can cause death of larvae in 24-48 hours.

Dependent on susceptibility of larvae and amount of Btk ingested,

death may be due to ionic imbalances, caused by the toxin due to

disruption of the gut, or by septicemia due to vegetative growth of

Btk and or other gut microflora. The susceptibility to Btk is

related to the age and biomass of the insect. Younger larvae are

generally more susceptible (See Fast 1981 for detailed description) .

23

At sub-lethal doses, anorexia does occur but recovery from feeding
inhibition can occur in 8 hours or less (Retnakaran et ai. 1983) .
There is no evidence of resistance developing to Btk by forest
insects although laboratory selection of stored product Lepidoptera
have resulted in increased tolerance.

6.0 MUTAGENICITY

Being a living organism, the potential exists for mutagenic changes
in Btk. The fact that there are over 20 recognized subtypes and 800
strains (Dulmage and Cooperators 1981) indicates that genetic
variability exists. With the advances of modern molecular biology,
there is, and will continue to be efforts made to isolate and create
more potent strains of Btk (Faust and Bulla 1982). All modern
registered formulations contain "natural" isolates of Btk and there
are no "genetically-engineered" products available. Salama et al .
(1984) selected for UV resistant, high temperature resistant, and
antibiotic resistant strains of Btk. The selection of UV resistant
individuals after 4 minutes exposure to UV light was 0.001-0.2%
dependent on strain. Heat selection at Ib^C for 120 minutes yielded
0.02% and 0.16% mutant survival. Similar low yields of penicillin,
streptomycin and chloramphenicol resistant mutants was obtained.
What is the possibility that strains pathogenic to vertebrates or
other non-target organisms would develop? Bacillus thuringiensis
is closely related to Bacillus cereus which has chemical affinities
to Bacillus anthrax. The probability of mutations affecting non-
insect organisms is exceedingly small. In all laboratory toxicity

24

studies done to date, there was no evidence of vegetative growth in
non-insect lines (Burges 1981) . Each batch of Btk produced by
commercial companies is quality checked by injection of a million
spores into mice (see Production). Fisher and Rosner (1959)
attempted to induce harmful mutations by serial passage of Bt in
mammals. There was no increase in virulence to mammals. Steinhaus
(1959) considered that the chances of mutation of Bacillus
thuringiensis to an anthrax-like organism "are exceedingly small".
Forsberg (1976) appears to agree with this conclusion.
Pathogenic mutations of Bacillus thuringiensis are rare, as natural
epizootics do not occur in nature or in years following spray
programs. Btk appears to be specially adapted to insects, and
poorly competitive against other microbes. The lack of widespread
natural epizootics would indicate that it is genetically a
relatively stable organism with little chance of non-insect
infection.

7.0 SPRAYING/APPLICATION PRACTICES

Bacillus thuringiensis var. kurstaki is registered in Canada for
control of a variety of lepidopterous larvae. Uses include not only
protection of forest trees, but protection of a variety of
vegetables, e.g. cabbages, brussel sprouts. Domestic class
registrations for use by home owners also exist, as do use patterns
for municipal parks and urban areas.
For forestry pests, typical target insects, application timing, and

25

application rates are outlined in Appendix 2. In forestry
management in Ontario, the three major pests currently being
controlled by Btk are 1) Spruce budworm (Choristoneura f umif erana)
2) Jackpine budworm (Choristoneura pinus pinus) and 3) Gypsy moth
(Lymantria dispar) (Appendix 2). Since 1980, a total of 1.23
million hectares of forest lands have been protected in Ontario
using Btk products (2.1 million litres) (Appendix 2). Control of
spruce budworm and jackpine budworm normally occurs in commercial
forestry tracts not associated with human habitation. Control of
gypsy moth may include recreational areas and farm woodlots
primarily in eastern Ontario, but with expanding range into other
areas of southern Ontario, i.e. Simcoe.

Control of lepidopterous larvae is intended to protect trees from
defoliation. Larvae must ingest Btk crystals. Consequently, all
programs are timed to treat trees while larvae are mid-instars and
openly feeding on foliage (Table 2). For spruce budworm, spraying
is done soon after bud flush before larvae reach fifth instar.
Similarly, jackpine budworm is treated after needle pairs begin
escaping from sheaths. Gypsy moth larvae are treated when 40-50%
leaf expansion (typically oaks) occurs and before larvae reach third
instar. In Ontario, application times will vary somewhat with the
phenological development of trees and target insect, but essentially
all spray programs for control of major pests will take place
between mid-May and the first week of July.

Most forestry applications of Btk in Ontario will be applied
aerially under contract to the Ministry of Natural Resources.

26

Table 2. Forestry uses for Btk in Canada (from Thuricide 48 LV Label)

Pest

Crop

Timing

BIU

Litres/ha

Bagworm

Ornamentals
Shade trees

when larvae
feeding

13-19

1-1.5

Elm/Fall

Hardwoods

after leaf

9-19

0.7-1,

.5

spanworm

flush

Gypsy moth

Forest trees

40-50% leaf
expansion

20-30

1.6-2

.4

Spring/Fall

Hardwoods

after leaf

6-9

0.5-0

.7

cankerworm

flush in
spring

Spruce

Conifers

soon after

15-30

1.2-2

.4

budworm

bud flush

Jackpine

Jackpine

at needle

30

2.4

budworm

flush

Tent

Deciduous

at leaf

6-9

0.5-0

.7

caterpillar

trees

flush

27

The Ministry of Natural Resources will have on site a variety of
personnel including application supervisors, safety and
communication personnel. The current applications supervisor is
Craig Howard, Natural Resources, Sault Ste. Marie (705-759-5727) .
There are detailed management plans in place for all aerial spray
programs (Carrow et al. 1981) including public meetings to outline
proposed spray operations. The Ministry of Natural Resources is to
be commended for the detailed planning and execution of their spray
programs. All aerial applications are restricted to licensed aerial
applicators .

The majority of published papers on Btk spray operations deal with
control of spruce budworm. Application normally involves the use
of fixed wing aircraft or large helicopters (i.e. Cessna Ag-truck,
Piper Pawnees, Grumman Ag-Cat and Bell 206 helicopters) (see Morris
1982, Table 3) .

The objective of spray application is to thoroughly cover foliage
with deposits of Btk. Typically, application is made in the early
morning or late evening when winds average less than 10 km/hr.
Aircraft and helicopters make applications from ca . 10 m above the
canopy using conventional boom and nozzle spray systems or Micronair'
rotary atomizers (Morris 1982, Cadogan et aJ. 1986). Ideally,
material should be applied at droplet sizes of 50-200 microns.
Spray deposits of 10-20 droplets/cm" are desirable. For the gypsy
moth, LD,5 levels are 10.8, 2.2 and 0.9 drops per cm*' for 100, 200 and
300 micron droplets respectively (Bryant and Yendol 1988) . Ideally,
conditions should be humid and warm with lepidopterous larvae

28

actively feeding. There should be no rain for at least 24 hours
after spraying to prevent washing off of spray deposits and ensuring
ingestion by larvae.

Typically, treatments are made to provide a coverage of 30 BIU per
hectare involving 1-3 litres of product per hectare if undiluted and
2-6 litres per hectare if diluted in water. There is increasing
tendency to spray formulations undiluted to reduce application
costs. There is a no buffer zone associated with water when
spraying with Btk in Ontario or other provinces. However, in the
public hearing process, efforts will be made to accommodate those
with concerns and, for control of gypsy moth, small island cottage
sites will not be treated.

Dependent on densities of larvae and success of initial spray, areas
may be retreated 5-14 days after initial application. With spruce
budworm and jack pine budworm, a second treatment is required in
about 5% of the cases. With the gypsy moth, three treatments spaced
approximately one week apart may be required in a small percentage
of cases. Most gypsy moth treatments require two treatments due to
asynchronous development of larvae. Thus, certain areas i.e. high
density infestations of gypsy moth, may have 90 BIU's per hectare
applied over a three week period. Success of treatments with modern
formulations has generally been satisfactory, providing significant
larval mortality of approximately 75%, and good foliage protection
of >50%. Success of programs has varied and meteorological
conditions after spraying probably influence results as well as
synchrony in target insect development. Fast and Regniere (1984)

29

showed that extending the exposure period from 1 day to an exposure
of 6 days resulted in a 500 fold increase in the LC=,„ of Btk on
spruce budworm larvae. With better formulations, better application
technology (i.e. droplet size) and repeat sprays, control has been
more consistent than in the past.

8.0 METHODS FOR ENUMERATING BTK FROM ENVIRONMENTAL SAMPLES

Since the chemical structure of the delta-endotoxin of Btk has not
been determined, monitoring of Btk residues can not be made using
analytical chemistry techniques. Enumeration of Btk deposits is
done by several methods including 1) deposit sampling at the time
of application 2) bioassay procedures of foliage which has been
treated 3) microbiological plating techniques and more recently 4)
ELISA and immunofluorescence techniques.

Morris (1982, pp. 247-51) describes the various methods for
assessing Bt deposits at the time of spray application. One method
is placement of a petri plate with nutrient agar amended for Bt
growth at various locations in the forest canopy and floor. These
collect bacterial droplets as they descend and plates are then
incubated for 18 hours at 29"C and colonies counted. This method
underestimates deposits since any colony may represent 150 viable
spores. Deposits can be collected on glass plates, rinsed v;ith
distilled water, diluted with 0.1 % peptose water, and serially
diluted onto agar media (tryptic soy). This technique was 2,000
times more sensitive than the petri plate method.

30

Kromekote cards can be used to analyze spray deposits when Btk
formulations contain marker dyes. Droplets can be analyzed
microscopically or using image analyzers. Similarly, dyed deposits
can be rinsed from glass plates (1.5 ml of 0.1% NaOH) and eluates
measured colormetrically (Cadogan et ai. 1986) .

The detection and quantification of Btk endospores and vegetative
cells from various substrates can be made using various methods
involving plating substrates onto appropriate growth media. Reardon
and Haissig (1984) simply collected foliage (needles) and made
impressions of the foliage into tryptic soy agar media (GBCO,
Madison, Wisconsin) . Plates were stored at room temperature for 48
hours. Bacillus-like colonies were identified and spore stains
(Benz and Boursiewiez 1963) made of random sub-samples. Faust and
Bulla (1982 p. 94) describe a variety of staining techniques for
Bacillus thurinqiensis .

Petras and Casida (1985) recovered Btk from soil by placing lOg
samples in a blender with 30 ml of sterile tap water. The material
was then serially diluted and 0.1 ml aliquots of various dilutions
were spread onto Heart infusion broth-Gelute plates (Kelco, San
Diego, California) . These were incubated for 1 week at 27'C. Young
Bt colonies were white and thin with a cottony appearance and
irregular edge. Bt colonies after 6 days incubation could be
checked for spore and crystal formation.

Menon and Mestral (1985) isolated and enumerated Btk from water and
shellfish by spread plate method using a nutrient agar containing
4 ppm penicillin and 5 ppm polymixin B sulfate. Plates were

31

incubated for 48 hours at sVC and Btk identified by colony
morphology (description not provided) . Colonies were confirmed by
taking a thin film aqueous suspension of colonies onto a clean
slide. This was air dried and heat-fixed. A thin layer of reagent
A (Admidoschawartz- 1.5g, 98% methanol 50 ml, acetic acid 10 ml and
distilled water 40 ml) was pipetted onto the smear while still warm.
This was rinsed off after 70 seconds. The smear was then covered
with Zihl's carbol fuchsin for 20 seconds, rinsed in tap water and
dried. Stained crystals were lilac blue and endospores pink under
dark field, phase contrast microscopy.

In the literature, there are numerous methods of isolating and
enumerating Btk from media and the above mentioned simply
demonstrate some of the techniques.

The newest method of Btk detection and enumeration is by the ELISA
(enzyme-linked immunosorbent assay) technique (Reardon and Hassig
1984) using B.t. kurstaki toxin as the assay. This technique is
described as extremely sensitive, accurate and highly specific (Wie
et al . 1981) . This has a sensitivity of 3 ug/g (3 ppm) of needle
tissue due to interfering cross-reactivity in needle tissue. It is
probable that in water, the sensitivity would be greater due to
reduced interference. Smith and Ulrich (1983) describe in detail
the ELISA technique for detection of the crystal toxin of Btk. West
et al. 1984 describe a detection method for Btk in soil using
immunofluorescence which compared favourably with bioassay and
plating techniques.

32

9.0 ENVIRONMENTAL FATE

9.1 Foliage

Forsberg (1976, Table 3 pp. 32-39) summarizes various laboratory
studies on the field and laboratory persistence of Bacillus
thurinqiensis. Similarly, Morris (1982) has summarized the
persistence of Btk on foliage when used in forest applications
(Table 3). In general, Btk loses 50% of its insecticidal activity
in 1-3 days, often necessitating a second spray application for
insects such as the gypsy moth, spruce budworm and jackpine budv/orm.
In some studies (e.g. McLeod et al. 1983, Beckwith and Stelzer 1987)
longer residual activity (i.e. 10 days) has been reported.
Persistence of Btk on foliage is dependent on many environmental
factors. Leong et al. 1980 concluded that sunlight exposure, leaf
temperature and vapour pressure deficit contribute most to endospore
decay. Sunlight, particularly ultra-violet radiation, inactivates
50% of Btk cells within 30 minutes and 80% within 60 minutes (Krieg
1975) .

The inactivation of both spores and crystals is believed due to
production of peroxide or peroxide radicals produced by UV
irradiation of amino acids (Ignoffo and Garcia 1978) . In situations
where prolonged activity of Btk has been reported (i.e. McLeod et
al. 1983) persistence is believed due to deposits in cracks and
crevices protected from direct sunlight. Some viable endospores of
Btk have been recovered from foliage after ground application of Btk
(1 BlU/tree) one year after treatment (Reardon and Haissig 1984).

33

•<t z. —

c 1. -w

- S TT F

3 t — , 1;

s I e

r - S

R3 -r- — ir
TD C S< :^

C S C C

£ S

5 -^

I 2

iS:o a:o 3^: =a: =a:

=: 5 ^

34

9,2 Soil

An excellent review of the fate of Bacillus thuringiensis in soil
is provided by Dulmage and Aizawa (1982). They concluded that Bt
as applied to ecosystems will persist for several months in the
soil. Bacillus thuringiensis can survive as endospores in most
types of soil (Saleh et al. 1970) although at pH below 4.8, Bt will
not grow. Repeated applications of Bt results in no increased
accumulation of the organism (Dulmage and Aizawa 1982, p. 223).
There appears to be a maximum carrying capacity in most soils.
The fate of Bt in soil is dependent on microbial competition. In
unsterilized soils, numbers of Btk rapidly diminish but may increase
in sterilized soils (Akiba et a],. 1977). When soil was treated at
10^ cells per gram, Bt persisted at lO' cells/g for 12-16 months.
However, the relative number of Bt compared to other soil bacilli
was reduced from 20-40% to about 10% indicating that Bt is not well-
adapted to soil environments. DeLucca et al. (1981) showed that Bt
does not move in the soil, as two serotypes sprayed in close
proximity did not become cross-contaminated. Pruett et aX- (1980)
have shown that in soil, although 38% of the endospores remained
viable after 63 days, only 3% insecticidal activity remained; after
135 days, there were 23% of the original spores and no insecticidal
activity. This strongly suggests that delta-endotoxin decays far
more rapidly than endospores. Finally, West et aA. (1984) have
shown that Bt will not grow (i.e. vegetative growth) under most
natural soil conditions, emphasizing that it is not a well adapted

35

member of the soil microbial community, but requires specialized
habitat of insect bodies.

9.3 Water

Menon and Mestral (1985) reported that after Btk operational spray
programs in Nova Scotia (4.7-9.4 L/ha of Thuricide 16B) , Btk could
be found in stream and reservoir water for 8-12 days after spraying.
Laboratory studies with high inoculation (10' cells/ml) indicated
approximately 30% survival of Btk after 70 days in distilled and tap
water (Fig. 2). In lake water, approximately 50% of cells remained
viable after 70 days. Better survival in lake water is believed due
to the presence of nutrients. In sea water, less than 10% of Btk
cells survived after 40 days. Although viable spores were found in
the study by Menon and Mestral (1985) and colony counts were
confirmed by identification of parasporal bodies, parasporal
endotoxin activity against Lepidoptera was not evaluated. Since
water was sterilized and kept in the dark, two major decomposition
factors, UV radiation and microbial degradation, did not occur.
These survival levels thus represent levels unlikely to occur in the
field, especially when considering steams and shallow lakes. Menon
and Mestral (1985) also reported that Btk was resistant to
chlorination at low concentrations of 0.1 to 0.5 mg/L.
Buckner et aJ. (1974) found that samples of river water 30 minutes
after spraying contained 1730 spores per ml, one month later, no
spores were found.

36

DISTILLED WATER
TAP WATER
LAKE WATER
SEA WATER

TIME (days)

Fig. 2, Survival of B.t. var. kurstaki in distilled water, tap

water, lake water and sea water at 20°C (Menon and Mestral 1985)

37

10.0 AQUATIC TOXICITY

10.1 Expected Exposure Levels

In forest spray operations, it is expected that approximately 30 BIU
(Billion International Units) of toxicity will be applied per
hectare. The proposed worst case scenario for Btk concentrations
in water is believed to be equivalent to approximately 2.14 lU/ml
of water or 2,140 lU per litre (Eidt 1985). This figure was derived
by comparing amounts of active ingredient, i.e. permethrin,
f enitrothion , entering aquatic systems during operational and
experimental spray programs. Similarly, if one assumes direct
application to a hectare of water a meter deep, there would be a
concentration of 3 lU per ml of water. Depending on the formulation
of Btk used, this would represent extremely low volumes of material,
for example, if 2.4L of Dipel"l32 were applied to a hectare of v/ater
with a mean depth of 1 metre, this would represent 0.24 ul of
product per litre. If spraying is repeated 7-10 days after initial
application, it is unlikely that there would be accumulation of Btk
due to stream flow and rapid inactivation of Btk by microorganisms.

10.2 Fish Toxicity

In reviewing the toxicity of Bt to fish, Forsberg (1976 - pg.52)
cites several studies with older formulations of Bt and gives
toxicity values. All of these trials report results of non-Btk
strains which presumably contained beta-exotoxins . Tests did not
specify toxicity levels (i.e. lU) of Bt formulations. They have

38

little value in the current review of Btk other than to provide
bench marks of toxicity which are probably greater than those of
Btk which does not contain beta-exotoxin.

Few published scientific papers on Btk toxicity to fish v/hich
pertain to modern formulations were found. According to Ignoffo
(1973), little or no effects were observed when rainbow trout,
yellow perch, mosquito fish, or black bullhead fingerlings v/ere
exposed for 4 days to 4.5 - 65 X 10' spores/ml. Effects that were
probably due to the petroleum carrier occurred at doses greater than
6 X lO' spores/ml. Some juvenile coho salmon died at the high dose
when they were exposed for 7 days to doses ranging from 5.2 X 10 to
26.4 X 10" spores/ml. Buckner et al.. (1974) reports concentrations
of spores in river water 30 minutes after treatment as 1,730 per ml,
indicating that laboratory toxicities were typically 1,000 fold
higher than field concentrations. Studies cited by Forsberg (1976,
pg. 63-64) refer to Bt formulations which probably contain beta-
exotoxins. Typically, the studies reported no mortalities or LC
values of formulated product Thuricide at 600 ppm or greater. Since
these formulations would contain the beta-exotoxins which are more
toxic to vertebrates, they represent toxicities significantly
greater than those of modern Btk formulations which contain no beta-
exotoxin. It appears that solvents and emulsifiers would be more
toxic than the active ingredient. Potency of formulations was not
reported.

Todd and Jackson (1961) found no adverse effects on Coho salmon fry
or on aquatic insects using formulations of Bt. Buckner et al. 1974

39

evaluated the aquatic impact of Thuricide 16B at the rate of ca.
17.6 BIU per hectare in Algonquin Park, Ontario. They reported that
fish, i.e. brook trout (Salvelinus fontinalis) , white suckers
(Catostomus commersoni) and small mouth bass (Micropterus dolomieui )
and bottom fauna populations (wide variety of organisms) in rivers
suffered no adverse effects up to four weeks after treatment.
Viable Bt spores were present in river water (1,730 spores/ml) and
clams immediately after treatment, but disappeared in four weeks.
Spores were not detected in crayfish collected from the river
(Buckner et al. 1974) .

Proprietary studies on the various formulations exist in the
Pesticides Directorate, Ottawa. These studies have been reviev;ed
by Fisheries and Ocean personnel, and Environment personnel. The
studies met with approval of these ministries. The lack of any
documented fish kills in any of the forestry or agricultural spray
programs involving Btk across Canada and the United States may be
the most valid argument for fish safety. Since the mid 1970 's,
several million hectares of coniferous forest have been sprayed for
spruce budworm and jack pine budworm and several hundred thousand
hectares for the gypsy moth (L. dispar) . There is no evidence of
fish kill in any trials. In Ontario, there has been approximately
1.3 million hectares of forest lands sprayed with Bt between 1980-
1987 (Churcher, pers . comm.), and to our knowledge, no fish effects
have been reported.

In examining fish toxicity, an additional indication of lack of
toxicity with Btk and toxicity of emulsifiers is reflected in

40

published toxicity studies of the closely related Bacillus
thurinqiensis var. israeliensis (Bti) . In Quebec, Fortin et al .
(1986) showed that at extremely high concentrations (4,500 to 6,000
mg/L) of formulated Bti (Tecknar') , mortality of brook trout
(Salvelinus f ontinalis) was 20 and 86.4% respectively. However,
fish exposed to 6,000 mg of freeze-dried Tecknar" were not affected.
Fish fed for 48 h with chow contaminated with 30% W/W Tecknar' did
not show any response. The authors concluded that mortality at high
concentrations with liquid formulation was due to 2% xylene which
was used as a preservative in the formulation. (It should be noted
that no Btk forestry product currently contain xylene) .
Forsberg (1976, pg. 52) suggested that the primary route of exposure
of Btk to fish may be through ingestion of spruce budworm larvae
falling into the stream. His concern was the sensitivity of fish
to Btk vegetative cells. The study by Fortin et al. 198 6 v.'here
fish were exposed to 30% W/W formulated Bti may alleviate some of
this concern. If vegetative cells adversely affect fish to any
significant degree, this would have become apparent in widespread
forestry spray programs already conducted.

Impact studies of Bt on six species of fish carried out by Buckner
et al. (1974) in Canadian spruce-fir forests, revealed no abnormal
effects 3 days and 1 month after spray application.

10.3 Aquatic Invertebrates

Although there is no suggestion of direct mortality to fish, an
expressed concern has been the potential effects of Btk on fish

41

through effects on their food organisms, most important of which are
aguatic insects. Eidt (1985) evaluated the toxicity of Btk to
aguatic insects using the commercial formulation of Thuricide'32 LV
containing 8.45 BIU/L.

Insect immatures were exposed to concentrations of 4 . 3 , 43, and 4 30
lU/ml. This represented 2, 20 and 200 times the expected maximum
field exposure. Immatures were maintained in 300 ml of water, and
air was bubbled into those jars containing stream insects to provide
aeration and circulation. The appropriate concentrations of Btk
were added to water after 24 h and immatures were then treated v.'ith
the appropriate concentrations of Btk. It should be emphasized that
immatures were maintained with Btk for ca. 15 days, whereas in field
situations, maximum pesticide exposure would be for less than 6
hours and rare after 24 hours (Poirier and Surgeoner 1988) . The
results of Eidt's (1985) toxicity evaluations are summarized in
Table 4.

The conclusions of the study by Eidt 1985 were as follows: "There
is no hazard of Btk. aerial sprays at rates adequate for spruce-
budworm control to fish-food organisms in streams. Deposits in
shallow ponds are of little concern in New Brunswick, and measurable
hazard to non-target organisms is unlikely, with the possible
exception of mosquito larvae and related insects with similar
feeding strategies. Subsequent to these experiments, in May 1984,
the New Brunswick regulations were changed so that buffer zones were
not reguired around water bodies for aerial spraying with Btk."

42

Table 4. Effects of Btk (Thuricide 32LV) on Representative
Aquatic Insect Fauna (Eidt 1985) .

Taxa

Effect

Prosimulium

fus cum /mix turn
(Simuliidae)

-some impact suggested,
-high mortality after 5 days

for all concentrations and

control .

Simulium vittatum
(Simuliidae)

■effects at 430 lU/ml (75%
mortality) .
-no effect at lower dosages.

Hydropsyche

(Trichoptera: Hydropsychidae)

water.

-no effect.

-better survival in treated

Chimarra

(Trichoptera: Philopotamidae)

Tanytarsus
(Chironomidae)

-any effect unlikely.

-possible impact at 430 lU/ml .

Procladius ruris
(Chironomidae)

■no effect.

-best survival at highest
concentration.

Stoneflies
(Plecoptera)

-no effect.

Leptophlebia

(Ephemeroptera: Leptophlebiidae)

-no effect.

Nijgronia serricornis
(Megaloptera: Corydalidae)

-no effect

43

Buckner et al. (1974) in monitoring programs of a stream in
Algonquin Park where the forest was treated with 17.6 BIU of
Thuricide also found that Btk had no measurable effect on
Trichoptera (caddisf lies) , Ephemeroptera (mayflies) , Plecoptera
(stoneflies) , Odonata (dragonf lies) , Coleoptera (water beetles) and
Diptera (flies). These authors also found no effect to Turbellaria
(planaria, flatworms) , Nematoda (nematodes, roundv;orms) ,
Olighochaeta (earthworms), Hirudinea (leeches), Amphipoda
(crustaceans) , Decapoda (crayfish) , Hydracarina (water mites) ,
Gastropoda (snails), and Pelecypoda (clams, mussels). Survivorship
of crayfish and clams kept in baskets in spray and non-spray plots
was not significantly different.

In a study by Menon and Mestral (1985), shellfish were collected
from two experimental spray sites in Nova Scotia treated in June.
The Btk formulation used was Thuricide 16 B (0.34% Btk, 48.15%
culture medium, 1.5% xylene, 0.01 % Chevron sticker and 49.99%
water) at rates of 4.7-9.4 L/ha Btk enumeration of shellfish samples
was based on colony morphology (using nutrient-polymyxin-penicil 1 in
agar plates) and microscopic examination of stained smears of all
suspected colonies for parasporal crystals. No Btk was recovered
from any of the shellfish examined. Morris (1982) cites a paper by
Feng (1966, J. Invert. Pathol. 23:280-4) indicating no effect to
American oysters despite high exposure to Bt . He also cites Alzieu
et al. (1975) who found no effect to shrimp, mussels, oysters and
periwinkles (Sci. Peche Bull. Inst. Peche Maut. #250 pp. 11-18).

44

Krieg and Langenbruch (1981) cite unpublished data indicating that
Btk is not toxic to Cyclops sp. or Daphnia sp.

11,0 HEALTH EFFECTS (TO HUMANS)

Samples and Buettner (1983) report on a corneal ulcer apparently
caused by Btk when formulated suspension was accidentally splashed
into the eye of a farm worker. Despite widespread usage throughout
the world (e.g. 1 million pounds annually in the U.S.A.), this is
the only reported infection in humans or other vertebrates. Warren
et al. (1984) report on a laboratory worker who developed infection
after injection of Bacillus thuringiensis var. israeliensis and
Actinobacter calcoaceticus into his finger. In both cases,
antibiotic treatment resulted in complete recovery.

Heimpel (1971) cites a study by Fisher and Rosner (1959) where
eighteen volunteers ingested 1 g of Thuricide'/day for 5 days, at a
concentration of 3 X 10" viable spores/g. In addition, 5 of the
test subjects inhaled 100 mg of the powder daily for 5 days. All
subjects were then thoroughly examined and again 4-5 weeks later.
All people tested remained healthy and all the extensive laboratory
tests were negative. In addition, there are records of eight
employees who were exposed to the bacterial preparation during the
course of product manufacturing. The exposure periods totaled 25
hours in some cases with the result that all exposed personnel were
in good health and showed no harm from the exposure.

45

12.0 EFFECTS ON NON-TARGET TERRESTRIAL ANIMALS

12 . 1 Insects

A valid concern when dealing with an insect pathogen such as Btk is
whether or not it will harm beneficial insects. From the literature
available, it is evident that Bt is safe for beneficial insects.
A number of studies have dealt with this topic, and several of them
are summarized here.

Work done by Hassan et al. (1983) concluded that Dipel' (Btk) was
harmless to most of the beneficial arthropods tested, i.e.
Trichogramma, Pales, Phygadeuon, Leptomastix, Coccygminus, Chrysopa ,
Encarsia. Amblyseius, and was only slightly harmful to Syrphus .
Evaluation was carried out by measuring the reduction in beneficial
capacity (i.e. parasitism) or performance (i.e. egglaying) compared
to the controls. It was concluded that Dipel' can be recommended for
use in integrated control programs. Another study by Wilkinson et
al. (1975) demonstrated that Btk (Thuricide HPC') applied at the
minimum recommended field dose has little or no effect on
parasitoids such as chalcids, ichneumonids, braconids and tachinids,
or on predators such as chrysopids and coccinelids. Buckner et al .
(1974) reported no effect on parasites of the spruce budworm.
Other studies by Dunbar and Johnson (1975) showed that Btk (Dipel'WP
16,000 lUP/mg and Biotrol'XK, WP 7,500 lUP/mg) decreased post-
treatment longevity of Cardiochiles nigriceps (a tobacco budv/orm
parasitoid) when ingested as dissolved active ingredient in sugar
water. The authors are quick to point out, however, that these

46

parasites probably would not feed on Btk in the field, and only fed
on it in the laboratory since the sugar acted as an attractant.
When C. niqriceps adults were topically treated with Btk, no
difference occurred in post-treatment longevity between untreated
and Dipel'-treated wasps. Also, wasps exposed to tobacco leaves
treated with Dipel were not affected.

Tipping and Burbutis (1983) studied the effect of Btk (Thuricide,
4,000 lU/mg) residues on adult emergence and parasitism of the egg
parasitoid Trichoqramma nubilale. Tests were conducted on 1-, 7-,
14- and 21-day greenhouse spray residues from pepper plants at 2
dosage rates, i.e. 9.8 X 10' lU of Al/ha (recommended dose) and 1.96
X 10"* lU of Al/ha. Btk was found not to reduce emergence from, or
parasitism of, European corn borer.

To determine the effects of Bt on natural enemies of western spruce
budworm, parasite studies were conducted by Niwa et ai. (1987)
during field tests of two Btk formulations (San-415 and Thuricide
32 LV) applied at 2 dosages (20 and 30 BlU/ha) against western
spruce budworm in Oregon. Microscopic examination showed that three
parasitic species contained Btk, namely Glypta fumif eranae ,
Apanteles fumif eranae , and Phytodietus fumif eranae. Occurrence of
parasites infected or contaminated with Bt was extremely low
(37/3000) and did not seem to be related to any of the treatment
applications. Infected parasites matured sufficiently to exit from
their hosts, but were not able to complete development. It is
possible that premature parasite mortality was due to insufficient
nutrition caused by the early deaths of their hosts, rather than by

47

microbial infection. In addition, there was no significant
difference between total parasitism, nor the species distribution
of the parasite complex, between the control and the sprayed plots
during any of the sample periods. Despite the fact that Btk may
affect a small number of parasites developing within infected hosts,
it was concluded that field application of Btk for control of
western spruce budworm would not be detrimental to the associated
parasite complex.

Studies by Haverty (1982) have concentrated on evaluating the effect
of the carrier of Dipel 4L'' against non-target insects i.e. the
common green lacewing (Chrysopa carnea) , the convergent lady beetle
(Hippodamia converqens) and an aphelinid (Aphytis melinus) . Three
aerosol spray treatments were evaluated: 1) water only, 2) Dipel'
carrier and water (1:3) at 9.4 L/ha and 3) Dipel'' carrier and water
(1:3) at 18.7 L/ha. The concentrations of the 2 treatments selected
were representative of the optimal maximum rates of Dipel 4L'
aerially applied in forests for the control of spruce budworm and
western spruce budworm. The 9.4L/ha rate did not result in a
significant increase in mortality (never exceeded 2.1%) for any
species tested up to 7 days post-spray. The 18.7 L/ha rate resulted
in a significant increase in mortality for C. carnea and H.
converqens adults 3-7 days after treatment (although mortality never
exceeded 13.4% for any species). The amount of formulation that
actually reached test insects in the experimental spray chamber used
was estimated at 4-8 times greater (depending on dose) than that
which would be expected to reach insects when applied in a forest.

48

These experiments suggest that the carrier in Dipel 4L'' could cause
slight increases in mortality of non-target insects, but only v;hen
applied in excessive amounts. It was concluded that any potential
disadvantages for increased mortality are outweighed by the benefits
of having a microbial insecticide such as Bt for control of forest
defoliators.

Buckner et al. (1974) reported no dramatic reduction of non-target
insects on any of the Algonquin Park areas treated with Btk (see
aquatic toxicity section) .

Many studies on the effect of Bt on honey bees (Apis mellif era) have
been undertaken and are summarized in Bailey (1971). The overall
conclusion of these studies is that there is no evidence that bees
will be harmed by Btk in practice. According to Atkins et al .
(1975), Btk was non-toxic to honeybees at levels as high as 726,000
spores/bee. Buckner et al. (1974), working in Algonquin Park v.'ith
7 honeybee colonies in an operational experimental program involving
Dipel concluded "There was no evidence from the data collected that
any of the Bt formulations affected domestic honeybee colonies, even
when the entire foraging area was treated."

12.2 Earthworms

To test the effect of Btk on earthworms, Benz and Altwegg (1975)
applied Dipel" (16,000 lU of potency (lUP/m.g) in 3 concentrations:
60, 600 and 6,000 mg/m' on field plots measuring 9 m" (recommended
dose for field application = 60 mg/m') . Before treatment, a 50 cm
square (30 cm deep) was dug out and all earthworms counted. After

49

3, 6 and 9 weeks, other 50 cm squares were dug out on the plots and
the earthworms counted and examined for signs of disease. There v;as
no significant difference in worm density between untreated and Bt-
treated plots demonstrating that field treatments using normal
concentrations of Dipel' would have no adverse effect on earthv/orm
populations .

12.3 Birds

There were no significant reductions in bird populations (74 species
representing 21 families) apparent in areas treated with Btk with
and without chitinase in Canadian spruce-fir forest treatment plots,
located in Spruce Woods, Manitoba and Algonquin Park, Ontario
(Buckner et al. 1974). Ignoffo (1973, p. 152) cites several studies
(primarily with old Bt formulations) indicating that Bt had no
effect on pheasants, partridge, Cornish hens or chickens at high
levels in the diet, e.g. 0.2-570 X lO' spores/bird/day for 28-70
days .

Forsberg (1976) cites unpublished reports provided by Abbott
Laboratories to the NRC committee indicating that 250 X 10' spores
of Btk/kg body weight, orally administered for 21 days, caused no
mortality or weight reductions in quail (Stephens 1970, see Forsberg
1976, p. 69) . Twelve pullets fed 38 X lO' spores/kg for 14 days
showed reduced weight gains but no mortality (Couch 1973, cited by
Forsberg 1976). Smirnoff and MacLeod (1961) administered 0.25 ml
of 3 X 10" spores/ml daily to juncos, sparrows and starlings by
syringe for 2 weeks. There was no mortality, weight loss or change

50

in appearance to birds. Bacteria could be detected in faeces for
up to 4 days after feeding had been discontinued.

The possibility of indirect effects to birds due to reduced food
availability has been raised. Szuba et al. (1988, unpublished data)
from the University of Toronto indicate increased mortality
(approximately 30%) of spruce grouse chicks in Jackpine plots
treated with Btk. This is believed due to reduced Lepidoptera
numbers. It was shown, however, that the year after spray, mating
pairs of spruce grouse were not significantly different from the
year before.

12.4 Mammals

According to Heimpel (1971), most of the initial experimental work
carried out on the safety of Bt has been done by Fisher and Rosner
(1959) using the product Thuricide", at a concentration of 9 X 10
viable spores/g, a product which would have contained beta-exotoxin .
Results are summarized as follows:

1) Virulence of Thuricide following serial passage through mice.
Mice injected intraperitoneally with Thuricide had blood taken 6
hours later by cardiac puncture. This was injected
intraperitoneally into a second group of mice and serial passage
repeated with 5 more groups of mice and none of the terminal group
showed abnormal symptoms.

b) Persistence of B.t. in blood of mice. White mice were injected
intraperitoneally with Bt. Although Bt was found in the blood at

51

48 hours, it had disappeared by 72 hours. There was no vegetative
growth.

c) Pathogenicity of B.t. by parenteral administration into guinea
pigs. Guinea pigs injected intraperitoneally with vegetative cells
of B.t. from broth culture could withstand large doses.

d) Inhalation toxicity of B.t. in mice. Mice exposed 4 X to gram
quantities of Thuricide'' while confined in a 30 cm square chamber
showed no symptoms or change in feeding or weight. Gross
pathological examinations were negative.

e) Allergenicity of Thuricide in guinea pigs. Sensitizing doses of
Thuricide" were given to guinea pigs by intracutaneous injection, the
abrasion-patch technique, and by topical application on intact skin.
Local irritation was found in injected and abraded animals as
demonstrated by a slight erythema. No effect was observed from
challenging doses given two weeks after the last sensitizing
treatment. Topical applications had no effect.

f) Acute oral toxicity of Thuricide. Suspensions (33%) of Thuricide'
were placed directly into the stomachs of rats (up to 24 g of
product (2 X 10'"' spores/kg body wt) ) . After a week, there were no
deaths or symptoms of toxicity. Gross pathological examinations
were also negative.

Other studies involving acute and chronic feeding tests show that
no toxicity was apparent in young swine, hogs, chicks, laying hens
and fish are cited by Heimpel (1971). Forsberg (1976) provides a
table on pp. 56-66 outlining toxicology tests to mammals, birds and
fish (Appendix 3), as does Ignoffo (1973, p. 152). Ghassemi et ai.

52

(1981) provide a tabular summary on laboratory studies of toxicity
of Btk to fish, birds and mammals (Appendix 4). Nishiitsutsuj i-Uwo
et aX. (1980) reported that mammalian cells showed no morphological
changes even at the ultra structure level when exposed to Btk. Som
et al. (1986) report that Btk can not flourish in the
gastrointestinal tract of guinea pigs.

According to the WHO (1981), in addition to the aforementioned
tests, i.e. oral, intraperitoneal, respiratory, dermal and
allergenicity and hypersensitivity, the recommended safety tests
that have to be passed by bacterial pest control agents also include
eye exposure (with rabbits) and mutagenicity tests (in vitro).
Bacillus thurinqiensis has passed these as well, in addition to a
subcutaneous and 3-week feeding study. Rogoff (1982) provides a
history of regulatory safety data required for Bt to satisfy the
needs of the EPA. Siegel et ai. (1987) provide a summary of
mammalian toxicity data for the closely related variety, Bti. They
conclude that Bti is not a virulent invasive mammalian pathogen, and
that it can be used safely in environments where human exposure is
likely to occur.

Studies by Buckner et aJ. (1974) on plots treated with Bt in
Algonquin Park, Ontario and Spruce Woods, Manitoba indicated that
small mammals continued breeding through the treatment periods, and
trapping data indicated that the application of Bt treatments v/ith
or without chitinase did not harm the small mammal complex
inhabiting treatment areas. Mammals included in the study were
woodland jumping mice (Napaeozapus insiqnis) , deer mice (Peromyscus

53

maniculatus) , short-tailed shrews (Blarina brevicauda) , common
shrews (Sorex cinereus) , red-backed voles (Clethrionomys gapperi ) ,
and eastern chipmunks (Tamias striatus) .

In addition, proprietary data exists with Pesticides Directorate,
Ottawa, on acute toxicities of various formulations.

54

13.0 LITERATURE CITED

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., Y. Sekijima, K. Aizawa and N. Fujiyoshi. 1977. Microbial
ecological studies on Bacillus thuringiensis II. Dynamics of
Bacillus thuringiensis in sterilized soil. Jap. J. App . Entomol.
Zool. 21: 41-46.

Atkins, E.L. et al. 1975. Toxicity of pesticides and other
agricultural chemicals to honeybees- Laboratory Studies. University
of California. Dev. Agric. Sci. Leaflet 2287.

Bailey, L. 1971. The safety of pest-insect pathogens for beneficial
insects. In: Microbial control of insects and mites. H.D. Burges
and N.W. Hussey, eds. Academic Press, New York. pp. 491-505.

Beckwith, R.C. and M.J. Stelzer. 1987. Persistence of Bacillus
thuringiensis in two formulations applied by helicopter against the
western spruce budworm (Lepidoptera : Tortricidae) in north central
Oregon. J. Econ. Entomol. 80: 204-207.

Beegle, C.C., T.L. Couch, R.T. Alls, P.L. Versoi and B.L. Lee. 1986.
Standardization of HD-l-S-1980: U.S. standard for assay of
lepidopterous-active Bacillus thuringiensis . Bull. Entomol. See.
Amer, 32: 44-45.

Beegle, C.C, H.T. Dulmage, D.A. Wolfenbarger and E. Martinez. 1981.
Persistence of Bacillus thuringiensis Berliner insecticidal activity
on cotton foliage. J. Environ. Entomol. 10: 400-401.

Benz, G. and A. Altwegg. 1975. Safety of Bacillus thuringiensis for
earthworms. J. Invert. Pathol. 26: 125-126.

Benz, G. and K. Boursiewiez. 1963. A method for the differential
staining of spores and parasporal bodies of Bacillus thuringiensis
Berliner and Bacillus f ribocergensis . J. Insect Pathol. 5: 393-94.

Bryant, J.E. and W.G. Yendol . 1988. Evaluation of the influence of
droplet size and density of Bacillus thuringiensis against Gypsy
moth larvae (Lepidoptera: Lymantriidae) . J. Econ. Entomol. 81: 130-
134.

Buckner, C.H., P.D. Kingsbury, B.B. McLeod, K.L. Mortensen and
D.G.H. Ray. 1974. Evaluation of commercial preparations of Bacillus
thuringiensis with and without chitinase against spruce budworm.
F. Impact of aerial treatment on non-target organisms. Chem. Cent.
Res. Inst. Info. Rep. CC-X-59.

Burgerjon, A. and D. Martouret. 1971. Determination and

55

significance of the host spectrum of Bacillus thuringiensis . In:
Microbial Control of Insects and Mites. H.D. Burges and N.W.
Hussey, eds . Academic Press, New York. pp. 305-322.

Burges, H.D. 1981. Safety, safety testing and quality control of
microbial pesticides. In: Microbial Control of Pests and Plant
Diseases 1970-1980. H.D. Burges, ed. Academic Press, New York. pp.
737-767.

Burges et al. 1966. The standardization of Bacillus thuringiensis
tests on three candidate reference materials. In: Insect pathology
and microbial control. P. A. van der Laan, ed. North-Holland,
Amsterdam, pp. 314-338.

Burges, H.D., E.M. Thompson and R.A. Latchford. 1976. Importance
of spores and endotoxin protein crystals of Bacillus thuringiensis
in Galleria mellonella. J. Invert. Pathol. 27: 87-94.

Cabana, J. and M. Pelletier. 1986. Surveillance des pulverisations
aeriennes d' insecticides centre la tordeuse des bourgeons de
I'epinette au Quebec, en 1985. Controle de la qualite effectue, en
1985, sur les preparations a base de Bacillus thuringiensis . I.
Recherche de pathogenes et identification de I'espece insecticide.
Ministere de I'Energie et des Resources, Quebec.

Cadogan, B.L., B.F. Zylstra, C. Nystrom, P.M. Ebling and L.B.
Pollock. 1986. Evaluation of a new Futura formulation of Bacillus
thuringiensis on populations of jackpine budworm, Choristoneura
pinus pinus (Lepidoptera : Tortricidae) . Proc. Entomol . Soc. Ont .
117: 59-64.

Carrow, J.R., S.A. Nicholson and R.A. Campbell. 1981. Aerial
spraying for forest management- an operational manual. Ont. Min.
Nat. Res. 44 pp.

de Barjac, H. 1981. Identification of H-serotypes of Bacillus
thuringiensis. In: Microbial control of pests and plant diseases.
H.D. Burges, ed. Academic Press, New York. pp. 36-43.

DeLucca. A.J. II, M.S. Palmgren and A. Ciegler. 1982. Bacillus
thuringiensis in grain elevator dusts. Can. J. Microbiol. 28: 452-
56.

DeLucca, A.J. II, J.G. Simonson and A.D. Larson. 1981. Bacillus
thuringiensis distribution in soils of the United States. Can. J.
Microbiol. 27: 865-70.

Dimond, J.B. and C.J. Spies III. 1981. Two-year effects of a
Bacillus thuringiensis treatment on spruce budworm, Choristoneura
fumiferana (Lepidoptera: Tortricidae). Can. Entomol. 113: 661-663.

56

Dubois, N.R. and F.B. Lewis. 1981. What is Bacillus thuringiensis?
J. Arboriculture 7: 233-240.

Dulmage, H.T. 1973. Bacillus thuringiensis U.S. assay standard.
Report on the adoption of a primary U.S. reference standard for
assay of formulations containing the delta-endotoxin of Bacillus
thuringiensis. Bull. Entomol. Soc. Amer. 19:200-202.

Dulmage, H.T. and K. Aizawa. 1982. Distribution of Bacillus
thuringiensis in nature. In: Microbial and viral pesticides. E.
Kurstak, ed. Marcel Dekker Inc. New York. pp. 209-237.

Dulmage, H.T. and Cooperators. 1981. Insecticidal activity of
isolates of Bacillus thuringiensis and their potential for pest
control. In: Microbial control of pests and plant diseases 1970-
1980. H.D. Burges, ed. Academic Press, New York. pp. 193-222.

Dunbar, J. P. and A.W. Johnson. 1975. Bacillus thuringiensis :
effects on the survival of a tobacco budworm parasitoid and predator
in the laboratory. Environ. Entomol. 4: 352-354.

Eidt, D.C. 1985. Toxicity of Bacillus thuringiensis var. kurstaki
to aquatic insects. Can. Entomol. 117: 829-837.

Falcon, L.A. 1971. Use of bacteria for microbial control of
insects. In: Microbial control of insects and mites. H.D. Burges
and N.W. Hussey, eds. Academic Press, New York. pp. 67-95.

Fast, P.G. 1981. The crystal toxin of Bacillus thuringiensis. In:
Microbial control of pests and plant diseases 1970-80. H.D. Burges,
ed. Academic Press, New York. pp. 223-248.

Fast, P.G. 1977. Bacillus thuringiensis delta endotoxin, on the
relative roles of spores and crystals in toxicity to spruce budworm
(Lepidoptera: Tortricidae) . Can. Entomol. 109: 1515-18.

Fast, P.G. 1976. Some calculations relevant to field applications
of Bacillus thuringiensis. Bi-monthly Research Notes 32:21. Insect
Pathology Research Institute. Sault Ste. Marie, Ontario, Canada.

Fast, P.G. and J. Regniere. 1984. Effect of exposure time to
Bacillus thuringiensis on mortality and recovery of the spruce
budworm (Lepidoptera: Tortricidae). Can. Entomol. 116: 123-160.

Faust, R.M. and L.A. Bulla Jr. 1982. Bacteria and their toxins as
insecticides. In: Microbial and viral pesticides. E. Kurstak, ed.
Marcel Dekker Inc. New York. pp. 75-209.

Fisher, R. and L. Rosner. 1959. Toxicology of the microbial
insecticide thuricide. J. Agric. Food Chem. 7: 686-688.

57

Forsberg, C.W. 1976. Bacillus thuringiensis : its effects on
environmental quality. Natl. Res. Coun. Can. Public No. NRCC 15385.
134pp.

Fortin, C. , D. Lapointe and G. Charpentier. 1986. Susceptibility
of brook trout (Salvelinus f ontinalis) fry to a liquid formulation
of Bacillus thuringiensis serovar. israelensis (Teknar") used for
blackfly control. Can. J. Fish. Aquat. Sci. 43: 1667-1670.

Ghassemi, M. , L. Fargo, P. Painter, P. Painter, S. Quinlivan, R.
Scofield, A. Takata. 1981. Environmental fates and impacts of major
forest use pesticides. EPA Contract No. 68-02-3174.

Gingric, R.E., N. Allen and D.E. Hopkins. 1974. Bacillus
thuringiensis: Laboratory tests against four species of biting lice
(Mallophaga: Trichodectidae) . J. Invert. Pathol. 23: 232-36.

Hassan, S.A., F. Bigler, H. Bogenschutz, J.U. Brown, S.I. Firth, P.
Huang, M.S. Ledieu, E. Naton, P. A. Oomen, W.P.J. Overmeer, V\' .
Rieckmann, L. Samsoe-Petersen, G. Viggiani, A.Q. van Zon. 1983.
Results of the second joint pesticide testing programme by the
lOBC/WPRS-working group "pesticides and beneficial arthropods."
Zeitschrift fur Angewandte Entomologie 95:151-158.

Haverty, M.I. 1982. Sensitivity of selected non-target insects to
the carrier of Dipel 4L in the laboratory. Environ. Entomol . 11:
337-338.

Heimpel, A.M. 1971. Safety of insect pathogens for man and
vertebrates. In: Microbial control of insects and mites. H.D.
Burges and N.W. Hussey, eds. Academic Press, New York. pp. 469-489.

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

Ignoffo, CM. 1973. Effects of entomopathogens on vertebrates. In:
Regulation of insect populations by microorganisms. L.A. Bulla Jr.
Ed. Ann. N.Y. Acad. Sci. 217. pp. 141-172.

Ignoffo, CM. and C Garcia. 1978. UV-photoinactivation of cells
and spores of Bacillus thuringiensis and effects of peroxidase on
inactivation. Environ. Entomol. 7: 270-2.

Jaquet, F. , R. Hutter and P. Luthy. 1987. Specificity of Bacillus
thuringiensis delta endotoxin. Appl . Env. Microbiol. 53: 500-4.

Krieg, A. 1975. Photoprotection against inactivation of Bacillus
thuringiensis spores by ultraviolet rays. J. Invert. Pathol. 25:
267-268.

Krieg, A. 1971. Concerning exotoxin produced by vegetative cells

58

of Bacillus thurinqiensis and Bacillus cereus . J. Invert. Pathol.
17: 134-135.

Krieg, A. and G.A. Langenbruch. 1981. Susceptibility of arthropod
species to Bacillus thurinqiensis In: Microbial control of pests
and plant diseases 1970-1980. H.D. Burges, ed. Academic Press, New
York. pp. 837-896.

Leong, K.L.H., R.J. Cano and M. Kubinski. 1980. Factors affecting
Bacillus thurinqiensis total field persistence. Environ. Entomol .
9: 593-99.

Luthy, P., J.L. Cordier and H.M. Fischer. 1982. Bacillus
thurinqiensis as a bacterial insecticide: Basic considerations and
application. In: Microbial and viral pesticides. E. Kurstak, ed.
Marcel Dekker Inc., New York. pp. 35-74.

McLeod, P.J., W.C. Yearian and S.Y. Young. 1983. Persistence of
Bacillus thurinqiensis on second year lobolly pine cones. Environ.
Entomol. 12: 1190-92.

Menon, A.S. and J. de Mestral. 1985. Survival of Bacillus
thurinqiensis var. kurstaki in waters. Water, Air and Soil Poll.
25: 265-74.

Morris, O.N. 1983. Comparative efficacy of Thuricide 16B and Dipel
88 against the spruce budworm, Choristoneura fumiferana
(Lepidoptera: Tortricidae) in balsam fir stands. Can. Entomol. 115:
1001-1006.

Morris, O.N. 1982. Bacteria as pesticides: Forest application. In:
Microbial and viral pesticides. E. Kurstak, ed. Marcel Dekker Inc.
N. Y. pp. 239-287.

Morris, O.N. 1977. Long term study of the effectiveness of Bacillus
thurinqiensis-acephate combinations against spruce budworm
Choristoneura fumiferana (Lepidoptera: Tortricidae). Can. Entomol.
109: 1239-48.

Morris, O.N. and M.J. Hildebrand. 1974. Evaluation of commercial
preparations of Bacillus thurinqiensis with and without chitinase
against spruce budworm. , E. Assessment of effectiveness of aerial
application, Algonquin Park, Ontario. Can. For. Ser. Inf. Rpt . CC-
X-59. p. E14.

Nishiitsutsuji-Uwo, J., Y. Endo and M. Himeno. 1980. Effects of
Bacillus thurinqiensis delta endotoxin on insect and mammalian cells
in vitro. Appl . Entomol. Zool. 15: 133-139.

Niwa, C.G., M.J. Stelzer, R.C. Beckwith. 1987. Effects of Bacillus
thurinqiensis on parasites of western spruce budworm (Lepidoptera:
Tortricidae). J. Econ. Entomol. 80: 750-753.

59

Ohba, M. and K. Aizawa. 1986. Distribution of Bacillus
thuringiensis in soils of Japan. J. Invert. Pathol. 47: 277-282.

OMAF (Ontario Ministry of Agriculture and Food). 1988. Vegetable
production recommendations. Public. 363. 78 pp.

Orton, D. 1987. The case against forest spraying with the bacterial
insecticide Bt. Alternatives 15: 28-35.

Petras, S.F. and L.E. Casida Jr. 1985. Survival of Bacillus
thuringiensis spores in soil. Appl . Environ. Microbiol. 50: 1496-
1501.

Poirier, D.G. and G.A. Surgeoner. 1988. Evaluation of a field
bioassay technique to predict the impact of aerial applications of
forestry insecticides on stream invertebrates. Can. Entomol . 120:
627-37.

Pruett, C.J.H., H.D. Burges and C.H. Wyborn. 1980. Effect of
exposure to soil on potency and spore viability of Bacillus
thuringiensis. J. Invert. Pathol. 35: 168-174.

Reardon, R.C. and K. Haissig. 1984. Efficacy and persistence of
Bacillus thuringiensis after ground application to balsam fir and
white spruce in Wisconsin. Can. Entomol. 116: 153-158.

Retnakaran, A., H. Lauzon and P. Fast. 1983. Bacillus thuringiensis
induced anorexia in the spruce budworm, Choristoneura fumiferana .
Entomol. Expt. Appl. 34: 233-239.

Rogoff, M.H. 1982. Regulatory safety data requirements for
registration of microbial pesticides. In: Microbial and viral
pesticides. E. Kurstak, ed. Marcel Dekker Inc., New York. pp. 645-
679.

Salama, H.S., M. Foda and M. Selim. 1984. Isolation of B.
thuringiensis mutants resistant to physical and chemical factors.
Z. Ang. Entomol. 97: 139-145.

Saleh, S.M., R.F. Harris and O.N. Allen. 1970. Fate of Bacillus
thuringiensis in soil. Effect of soil pH and organic amendment.
Can. J. Microbiol. 16: 677-80.

Samples, J.R. and H. Buettner. 1983. Corneal ulcer caused by a
biologic insecticide (Bacillus thuringiensis) . Am. J. Opthalmol . 95:
258-60.

Sebesta, K. , J. Farkas and K. Horska. 1981. Thuringiensin, the beta
exotoxin of Bacillus thuringiensis. In: Microbial control of pests
and plant diseases 1970-1980. H.D. Burges, ed. Academic Press, New
York. pp. 249-281.

60

Siegel, J. P., J. A. Shadduck and J. Szabo. 1987. Safety of the
entomopathogen Bacillus thuringiensis var. israelensis for mammals.
J. Econ. Entomol. 80: 717-723.

Sitting, M. 1980. Pesticide manufacturing and toxic materials
control encyclopedia. Park Ridge, New Jersey. 810 pp.

Smirnoff, W.A. and C.F. McLeod. 1961. Study of the survival of
Bacillus thurinqiensis var. thurinqiensis Berliner in the digestive
tracts and in feces of a small mammal and birds. J. Insect Pathol.
3: 266-270.

Smith, R.A. and J.T. Ulrich. 1983. Enzyme-linked immunosorbent
assay for quantitative detection of Bacillus thurinqiensis crystal
protein. Appl . Environ. Microbiol. 45: 586-90.

Som, N.C., B.B.G. Hosh and M.K. Majumdar. 1986. Effects of Bacillus
thurinqiensis and insect pathogen, Pseudomonas aeruqinosa, on
mammalian gastrointestinal tract. Ind. J. Exp. Biol. 24: 102-107.

Steinhaus, E.A. 1959. On the improbability of Bacillus
thurinqiensis Berliner mutating to forms pathogenic to vertebrates.
J. Econ. Entomol. 52: 506-8.

Szuba, K.J., B.J. Naylor and J.F. Bendell. 1988. Indirect effects
of a bacterial insecticide (B.t.) on wild spruce grouse chicks.
Unpublished manuscript in preparation.

Ticehurst, M. , R.A. Fusco and E.M. Blumenthal. 1982. Effects of
reduced rates of Dipel 4L, Dylox 1.5 Oil and Dimilin W-25 on
Lymantria dispar (L.) (Lepidoptera : Lymantriidae) parasitism, and
defoliation. Environ. Entomol. 11: 1058-62.

Tipping, P.W. and P.P. Burbutis. 1983. Some effects of pesticide
residues on Trichoqramma nubilae (Hymenoptera : Trichogrammatidae) .
J. Econ. Entomol. 76: 892-896.

Todd, I.S. and K.J. Jackson. 1961. The effects on salmon of a
program of forest insect control with DDT on Northern Moresby
Island. Can. Fish. Cult. 30: 15-38.

Warren, R.E., D. Rubenstein, D.J. Ellar, J.M. Kramer and R.J.
Gilbert. 1984. Bacillus thurinqiensis var. israelensis protoxin
activation and safety. The Lancet, March 1984: 678-679.

West, A.A. , N.E. Crook and H.D. Burges. 1984. Detection of Bacillus
thurinqiensis in soil by immunofluorescence. J. Invert Pathol. 43:
150-155.

WHO (World Health Organization). 1981. Mammalian safety of
microbial agents for vector control: A WHO memorandum. Bull. WHO
59: 857-863.

61

Wie, S.I., R.E. Andrews, B.D. Hammock, R.M. Faust and L.A. Bulla.
1981. Enzyme linked immunosorbent assays for detection and
quantification of the entomocidal parasporal crystalline protein of
Bacillus thurinqiensis subspp, kurstaki and israelensis. Appl .
Environ. Microbiol. 43: 891-4.

Wilkinson, J.D., K.D. Biever and CM. Iqnoffo. 1975. Contact
toxicity of some chemical and bioloqical pesticides to several
insect parasitoids and predators. Entomophaqa 20: 113-120.

62

Appendix 1 ,

^f)^l{•lllIl

■ ^^ Ar)ri('ii!tiH(

FqciJ ProfliJf lirj'i and Direction gotuMnle

inspcclioM B('i'iclt Prodijclion el nspeclion de'j al.menii

Pesticides Directorate

Ottawa, Ontario
KIA 0C6

February 22 , 198?

f'le Voire letfit'nce

Our tile Noire 'e'e'C'Jce

MEMORANDUM TO; Bt Manufacturers

Federal and Provincial Forestry Authorities

Federal and Provincial Health Authorities

Federal and Provincial Environment Authorities

Re; Bacillus thur ingiensis (B.t.)

Over the last number of years, there has been a continual
increase in the use and acceptance of B.t, for various control
programs. Against this background, we have been encouraged to
develop quality assurance guidelines for the presence of
extraneous microorganisms. We are endeavouring to develop in
cooperation with Health and Welfare Canada colleagues, industry
and provincial governments, a product profile which will
characterize B.t. formulations.

This program represents a new regulatory initiative not
previously undertaken for B.t., or any other microbial
product. At the present time, standards have not been
established in the Pest Control Products Regulations. Thus,
our current efforts should be viewed as an administrative
guideline which will serve as an interim reference and,
hopefully, lead to the establishment of more definitive, formal
standards in the future.

It is recognized that there is a degree of variability inherent
in large scale manufacturing operations, even within the same
plant. Therefore, some baseline or hands on experience in
typical product profiles is essential to establish a practical
regulatory standard. Based on experiences in 1987 and
subsequent wide ranging discussions, interim "target values" as
outlined in the attached document have been developed. These
initial parameters reflect background information on quality
standards in other areas and an assessment of currently
available manufacturing technology taking into consideration
the projected 1988 provincial forestry program demands.

/2

Canada

63

Methodology :

a) Sampling: 125 ml screw top, plastic containers as well as
detailed instructions re: sampling, packing and shipping
procedures will be provided by Ag Canada's Laboratory Services
to Regional staff and to provincial authorities.

Samples: Bulk Shipment: 1 (x2)* sample/tanker compartment

Barrels: NO. OF SAMPLES / LOT SIZE

4 (x2)*
6

10

/ 1-30 barrels

/ 31-60

/ 61-90

/ 91-120 "

In order maintain a reference sample and for the sake of
precaution re: handling, duplicate samples will be taken for
each compartment/barrel as indicated above.

Laboratory Methodology:

Salmonella :

Streptococci / Enter ococci

Coli forms :

Clostridium perfringens:

Staphylococcus aureus:
Pseudomonas aeruginosa:

- MF-HPB-20

- m-Enterococcus Agar

- Pour Plate or HGMF

- Violet Red Bile Glucose (VRBG) Agar

- Pour Plate or Hydrophobic Grid Membrane
Filtration (HGMF) Method

- MF-HPB-23: Methods Food - Health
Protection Branch - 23

- Pour Plate of HGMF Method

- MF-HPB — 21

- m-PA-C Agar

- HGMF Method

In order to ensure compatabi lity of results. Agriculture Canada will
provide the BT companies and provincial agencies with the detailed
laboratory methodology for each of the selected microorganisms.

W.E. Stewart, Ph.D.
Product Management
Pesticides Directorate

2087E

64

BACKGKOUNUKU uN UTU M IL'KOCONTAM I NAT 1 ON

In Che early summer of 1987, concerns were raised regarding the
presence of micr ocontaminants (specifically fecal
streptococcus), found in formulations of BT . In response to
the concerns. Agriculture Canada carried out an extensive
sampling program of representative batches of BT that were
being used in the major forestry operations across Canada.
These samples were analysed for fecal streptococci by our
Laboratory Services and results compared to those carried out
by several provinces and by one of the Health & Welfare Canada
laboratories. Discussions regarding the significance of the
levels of streptococcus indicated that these organisms axe
common in many non-sterile foods and in the natural
environment, represented either non- or low-order pathogenicity
and that exposure under typical forestry use applications would
not likely produce adverse health effects.

Additional questions have since been raised by some of the
forestry community regarding the possibility of additional
raicrocontaminants being present and whether Canadian standards
(for BT) should be established.

While no specific standards have ever been set for BT
manufacturing, there has always been the Internationally
accepted understanding/guarantee by companies that BT products
would contain no exotoxin and would be free of the human/animal
pathogens Sa Imonela/Shigel la and Vibrio. At present however,
the 7 day mouse test (measuring presence/absence of exotoxin)
appears to be the only test carried out regulary by most
companies .

In order to address the questions raised by last summer's
experiences, it was necessary to review the manufacturing
process for BT with the various companies and to try to
establish where in the process microcontamination could occur
and how contamination might be controlled and prevented.
Therefore, an informal, multi-department committee was
established consisting of members from: Ag Can: Laboratory
Services, Pesticides Directorate; Canadian
Forestry Service; H&W: Bureau of Microbial Hazzards,
Environmental Health Directorate. Foods Directorate, Laboratory
Centre for Disease Control).

./2

65

Early discussions centered on the historical aspects of the use
oC BT (especially in Forestry), the formulations presently
available, and on the events of last summer. The following
meetings were organized and held with the major BT
companies: -October 27, 1987 - Abbott Laboratories.

-November 26, 1987 - Duphar /ChemAgr o Ltd.

-December 7, 1987 - Sandoz Inc.

Companies were asked to present their BT manufacturing and
formulating processes, their quality control process, and
information on storage stability. The topic of ^

microcontaraina t ion was discussed in terms of what extraneous
( bio ) contaminants companies tested for: -routinely, -in 1987;
what stages of the manufacturing process the microcontaminants
could occur; and what steps the companies were taking to
control microcontarainat ion . The possibility and practicality
of testing for miccocontaminat ion using indicator organisms
similar to those used in food manufacturing was discussed.

Summary:

All BT is manufactured using the classical fermentation
process .

Neither the fermentation equipment nor the raw materials
are considered to be potential sources of contamination
since the fermentation equipment is mainly stainless steel
and is thoroughly cleaned between batches and the raw
material components and additives of the manufacturing
process are typically of high quality.

Manufacturing process may be divided into two stages:

a) production of the technical or most concentrated form
- either a thick pasty concentrate or a dry primary
powder .

b) production of the formulated/end use products which
are derived from the concentrate or primary powder.

Much of the process for producing the technical material is
carried out under aseptic conditions, however, at certain
stages of the process, e.g.. centr if ugat ion. it is not
possible to prevent exposure of the material to air and
hence possible contamination.

./3

66

Q

The production of the final end use (formulated) product(s)
occurs under non sterile conditions and as potential for
exposure and therefore presents opportunity for
contamination to occur.

When BT is packaged and shipped in barrels only new
containers are used, therefore, it is unlikely that
significant contamination would result from packaging.

For bulk shipments of BT (up to 20,000 or more litres),
bulk tankers are normally steam cleaned but may in fact
present an opportunity for contamination.

Cone lus ions :

On the part of the manufacturer, the most likely potential
for contamination would be:

a) during concentration of the technical material

b) during production of final end use (formulated)
products

c) from bulk shippraent equipment.

Once the formulated product(s) is delivered, the
opportunity for the introduction of microcontaminants may
result from:

a) the user's storage tanKs/f acilities

b) mixing procedures/equipment

c) handling of formulated product.

W.E. Stewart. Ph.D.
Product Management
Pesticides Directorate

iJES/sn
•OOIE

67

(.)

Based on the limited experience available to date, and the
advice emerging from government /i ndustry consultation, we would
expect all supplies to fall within the proposed target values.
Indeed, typical properties of any particular production lot are
expected to be below these figures.

It is reasonable to assume process and quality assurance
techniques can markedly influence contaminant levels. These
features may vary among manufacturers. Agencies interested in
establishing more restrictive quality assurance parameters may
wish to discuss this element with potential suppliers and
define their specific requirements in tender proposals.

In responding to representations for quality assurance
parameters on B.t., we have attempted to reflect a reasonable
and practical regulatory approach. We have endeavoured to take
into consideration safeguards regarding human health as well as
practical realities that exist. It is hoped that this approach
will accommodate the needs of all parties involved and
encourage industry to upgrade product quality.

W.E. Stewart

Insecticide Evaluation Officer

Product Management Division

SWO/sn
(1.78)0098e

68

II. PROPOSED MONITORING BY MANUFACTURER

It is proposed that B.t. producers carry out a monitoring
program of their own and provide results re: selected
organism testing (as per methodology provided) on each lot
of formulated product sampled at the point of delivery/
transfer .

III. ADDITIOr^AL MONITORING AND RECOMMENDATIONS

a) To further define quality assurance procedures after

delivery to users, Agriculture Canada would like to gather
additional monitoring information on the storage and
composition of formulated B.t., e.g.:

Report on provincial B.t. storage tanks re: their
structure, condition and cleaning procedures/facilities.

- Sample and test B.t. after it has been transferred to
holding tanks and/or mixed and held prior to intended
use .

b) B.t. producers may wish to consider additional monitoring
for the presence of the selected microorganisms, e.g.:

- The general manufacturing environment
( fermenting /formula ting facilities)

- Each lot of technical powder or concentrate.

Each lot of formulated product at the point of packaging
i.e., when barrels and/or bulk containers are being
filled.

- Bulk tankers, to assure that cleaning procedures e.g.,
steam, are carried out and are effective.

- Additional Organism(s): Companies are asked to provide
Agriculture Canada with specific details of any
organism(s) which may have been used as part of their ^t,
manufacturing or development processes.

EVALUATION OF RESULTS:

- Laboratory results from industry, the provinces and Ag

Canada will be reviewed and compared by Ag Canada and our
advisors in H&W.

69

AVAILABILITY OF RESULTS:

Consistent with our operating practices, specific test
results obtained by Agriculture Canada from the 1988
monitoring program will be made available.

William E. Stewart, Ph.D.
Product Management
Pesticides Directorate

70

Appendix 2. Aerial Application of Bacillus thuringiensis in Ontario by
MNR, 1980-1987 (1,228,727 ha treated with 2,156,830+ Litres
Btk) . (Data provided by J. Churcher, OMNR) .

1980-Spruce Budworm

Total Ha
Sprayed

Ha Bt Product
Sprayed Used

Application
Rate

Bt (L)
Used

10,310

4,640

Thuricide 16B
Dipel 88
Novabac 3-e

1980-Oak Leaf Shredder

Total Ha
Sprayed

Ha Bt
Sprayed

Product
Used

Application
Rate

Bt (L)
Used

848

N/A

N/A

N/A

1981 -Spruce Budworm

Total Ha
Sprayed

9,972

Ha Bt

Product

Application

Bt (L)

Sprayed

Used

Rate

Used

296

Dipel 88

24 BIU/7L/ha

835

324

Thuricide

32B

20 BIU/4.7L/ha

760

1,099

Dipel 88

20 BIU/5.9LAia

2,585

862

Dipel 88

20 BIU/2.4LAia

2,070

670

Thuricide

16B

20 BIU/7.2LA>a

3,190

810

Thuricide

16B

20 BIU/7L/ha

3,855

846

Dipel 88

20 BIU/5.1L/ha

1,990

1,731

Thuricide

16B

16 BIU/6.2LAia

6,595

262

Thuricide

16B

16 BIU/6.2L/ha

1,995

6,900

(2 X)

1982-Spruce Budworm

Total Ha
Sprayed

3,454

Ha Bt

Product

Application

Bt (L)

Sprayed

Used

Rate

Used

75

Novabac 3-

■e

20 BIU/7L/ha

175

172

Novabac 3-

-e

20 BIU/5.9LAia

400

2,439

Dipel 88

20 BIU/5.9L/ha

5,740

77

Thuricide

32B

20 BIU/5.9L/ha

180

305

Dipel 88

13 BIU/5.9L/ha

465

25

Thuricide

48B

30 BIU/2.4L/ha

60

3,093

71

1983 -Spruce Budworm

Total Ha

Ha Bt

Product

Application

Bt

(L)

Soraved

Sprayed

Used

Rate

Used

3,502

30

Dipel 88

30 BIU/3.5L/ha

105

40

Dipel 6L

30 BIU/2.3L/ha

95

60

Dipel 6L

30 BIU/4.7L/ha

140

50

Dipel 8L

30 BIU/1.8L/ha

90

60

Dipel 8L

30 BIU/3.5L/ha

105

40

Dipel 88

30 BIU/7L/ha

140

1,513

Dipel 88

20 BIU/5.9L/ha

3,560

1,250

Novabac 3-e

20 BIU/5.9L/ha

2,910

60

Bactospiene

30 BIU/4.7L/ha

210

3,103

1983-Oak Leaf

Shredder

Total Ha

Ha Bt

Product

Application

Bt

(L)

Sprayed

Sprayed

Used

Rate

Used

579

64

1984 -Spruce Budworm

Total Ha
Sprayed

3,697

Dipel

40 BIU/4.7L/ha

300

Ha Bt

Product

Application

Bt (L)

Sprayed

Used

Rate

Used

49

Dipel 176

40 BIU/3.7L/ha

115

64

Dipel 176

30 BIU/1.8L/ha

115

53

Dipel 8L

30 BIU/1.8L/ha

95

47

Dipel 64AF

30 BIU/2.3L/ha

85

58

Dipel 64AF

30 BIU/3.5LAia

100

61

Dipel 6L

30 BIU/2.3L/ha

145

47

Dipel 132

30 BIU/2.3L/ha

110

30

Thuricide 415

30 BIU/2.3L/ha

70

2.688

Dipel 88

20 BIU/5.9L/ha

6,325

3,097

198 5 -Spruce Budworm

Total Ha
Sprayed

29,370

Ha Bt Product
Sprayed Used

Application
Rate

20-30 BIU/
1.6-2.4L/ha

24,370 Dipel 132

5.000 Thuricide 48LV 20 BIU/1.6L/ha
29,370

Bt (L)
Used

42,215
7,875

72

1985 -Jack Pine Budworm

Total Ha

Ha Bt

Product

Application

Bt (L)

Sprayed

Sprayed

Used

Rate

Used

220,000

137,000

Dlpel 132

20 BIU/1.6L/ha

215,750

5,000

Dipel 132

20 BIU/3.2L/ha

7,875

78.000

Thuricide

48LV

20 BIU/1.6L/ha

122,835

220,000

1985-GvDsv

Moth

Total Ha

Ha Bt

Product

Application

Bt (L)

Sprayed

Sprayed

Used

Rate

Used

170

170 Dipel 88

40 BIU/12LAia

800

1986-Spruce Budworm

Total Ha
Sprayed

150,633

Ha Bt
Sprayed

150,633

Product
Used

Application
Rate

Bt (L)
Used

Dipel 132 + 20 BIU/1.6L/ha 237,220
Thuricide 48LV

1986 -Jack Pine Budworm

Total Ha
Sprayed

482,032

Ha Bt Product
Sprayed Used

Application
Rate

482,032 Dipel 132 + 20 BIU/1.6L/ha
Thuricide 48LV

Bt (L)
Used

482,035

1986-Gvpsv Moth

Total Ha
Sprayed

Ha Bt Product
Sprayed Used

103,094 103,094
1987-Spruce Budworm

Application
Rate

Dipel 132 + 30 BIU/6L/ha
Thuricide 48LV (2 X)

Bt (L)
Used

487,060

Total Ha

Ha Bt

Product

Application

Bt (L)

Sprayed

Sprayed

Used

Rate

Used

76,819

76,819

Dipel 132

20-30 BIU/
1.6-2.4L/ha

151,2

73

1987 -Jack Pine Budworm

Total Ha Ha Bt Product
Sprayed Sprayed Used

105,463 105,463 Dipel 132

Application
Rate

20 BIU/1.6L/ha

Bt (L)
Used

166,085

1987-GvpSY Moth

Total Ha
Sprayed

Ha Bt
Sprayed

40,249

Product
Used

Application
Rate

40,249 Dipel 132 + 30 BIU/6L/ha
Thuricide 48LV (2 X)
+ Futura XLV

Bt (L)
Used

190,150

74

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Appendix 4. (from Ghassemi et^ al- 1981).

SUMMAF.Y OF IN VIVO TESTS COirjUCTED TO EVALUATE SAFETY' OF BACILLUS
THIT.INGIEN'SIS PREPARATIONS TO FISHES, BIRDS. ANT) MAM>L^LS

Numbers
of

Inocul'jr

Species Animals (b' 1 1 ion/l-g) Route** Results

Fishes

Rainbow trout, black
bullhead, yellow

perch, mosquito fish

40

<0.01-4.5

TE

negative

Coho sa1nx)n juvenile

20

300-1800

TE

toxicity at

higher doses

Birds

Wild pheasant

9

3600

diet

negative

Partridge

2

5800

diet

negative

Cornish chick

190

0.2

diet

negative

Chick

48

300-1900

diet

negative

New Hampshire

laying

hen

16

' 1000-3000

diet

negative

CocKeral , Van*

.ress

cross

60

480-15,500

diet

negative

Matmals

Mouse

48

0.1-0.3 cells

ip

lethal dose,

50^

48

0.04-2 cells

sc

lethal dose.

5C-

48

0.8-7.8 spores

ip

lethal dose.

5C%

48

20-160 spores

sc

lethal dose,

50°.

10

77

po

negative

10

15

ip

abdominal irri-
tation, some

deatn

10

20,000

ih

neg* ti v(?

Rat

10

200C

po

necati ve

lo:

770C

diet

n<rJa t : ve

20

0.01-1.0

ip

negative

Guinea pig

10

77

diet

negative

10

40

ip

nega five

10

4000

ip

negative

10

0.3

sc

local izec red
at injectic

CtlO"
ir S'tc

ic

16

DA

si iqht cry tncTic on

abradfcc sm

n

Swine, duroc

3

185

diet

negative

HuT^n

18

0.2

PC

neq.i live

5

0.0?

1 *"

nenjtive

'Viable spore count of whole culture preraration trat inrljdta si>0'-es, veae-
tative cells, crystals, and wro'.r culture L-ol'i, un!'-: c.'^e-v. iSc- ':o: '• ■■•-•-
Estimated body w^ign* n^: uSP'2 v.nji. ...;ti*" body v.ei'j'!". w,:s nc -. p'-o^ '.cieJ.

'PC per OS; id, intr.i; ■•'■ . lontM" ; ir, m^ia ' ; t ion ; s:, s-b:'jtare,)us, TE, lou'
e^iposure; DA, j-ttoI c;:. 1 ;ca t iop .

86

Appendix 4. (from Ghassemi e_t al. 1981).

SUMMARY OF IN VIVO TESTS CONTlUCTED TO EVALUATE SAFETY OF BACILLUS
THmiNGIENSIS PREPARATIONS TO FISHES, BIRDS, AND MAMMALS

Species

Numbers

of
Animals

Inoculum*

(billion/kg) Route'

Results

Fishes

Rainbow trout, black
bullhead, yellow
perch, mosquito fish

Coho salmon juvenile

Birds

Wild pheasant

Partridge

Cornish chick

Chick

New Hampshire laying
hen

Cockeral , Vantress
cross

Maninals
Mouse

Rat

Guinea pig

Swine, duroc
Human

40

<0.01-4.5

IE

negative

20

300-1800

TE

toxicity
higher do

at
ses

9

3600

diet

negative

2

5800

diet

negative

190

0.2

diet

negative

48

300-1900

diet

negative

16

■ 1000-3000

diet

negative

60

480-15,800

diet

negative

48
48
48
48
10
10

0.1-0.3 cells
0.04-2 cells
0.8-7.8 spores
20-160 spores
77
15

sc

ip
sc
po
ip

lethal dose, 50'
lethal dose, 50"
lethal dose, 50*»
lethal dose, 501
negative
abdominal irri-

10

20.000

ih

tation,
death
negative

some

10

10C

20

2000
7700
0.01-1.0

po

diet

ip

negative
negative
negative

10
10
10

77

40
4000

diet

ip

ip

negative
nega ti ve
neaati ve

10

0.3

sc

local i/ea

reactio"

10

16

DA

at injection s'te
si ight rryincma on
abraded skin

3

185

diet

negative

18

0.2

po

negative

5

0.02

ih

negative

•Viable spore count of wr.ole cult'jre preparation that included swes, vege-
tative cells, crystals, and wnolc cultu-e broth, unl'?,s otnerv. iso :.:'ec 1 1 'l-J.
Estimated body wignt «^: uspd '.-.ncn .-Otivo body ^-eiiiit was no: nrpvided.
**PG, per OS; io, intr.M-?-- 1 tonea' ; in, inhalation; :c, ^..bcjtjr-cou'. . T[, loc^ical
e;iposure; DA, oermai aci.'l ication.

87

I