1
EPA/600/A-QR/noR
XX.X Effects of Cotton Expressing the
Bacillus thuringiensis var. kurstaki Endotoxin
on Soil Microorganisms.
Katherine K. Donegan1 and Ramon J. Seidler2.
1 Introduction
The genetic engineering of plants has facilitated the production of agronomically-desirable crops that
exhibit increased resistance to pests, herbicides, pathogens and environmental stress and
enhancement of qualitative and quantitative crop traits (Gasser and Fraley 1992). Along with these
many benefits, however, comes the potential for adverse ecological effects because of the often
sustained expression in the genetically engineered (transgenic) plant of the engineered trait(s) and the
persistence of the transgenic plant or plant residue in the environment. Consequently, we have
undertaken research to evaluate the potential ecological effects of transgenic plants and their
products.
Some of our research has included microcosm studies with cotton plants that are genetically
engineered to produce the Bacillus thuringiensis var. kurstaki (B.t.k.) endotoxin (Perlak et al. 1990).
Many agriculturally important plants have been engineered to produce endotoxins from different
subspecies of the bacterium Bacillus thuringiensis (B.t.) (Vaeck et al. 1987; Delanney et al. 1989;
Perlak et al. 1990; Lundstrum 1992; Koziel et al. 1993). The endotoxin of Bacillus thuringiensis var.
kurstaki (Btk) has demonstrated insecticidal activity against lepidopterans (Hofte and Whiteley
1989). Although high specificity has been assumed for most B.t. endotoxins, their effects on non-
target organisms have not been fully evaluated. Studies have been performed exposing non-target
invertebrates to various ^/-producing bacterial strains and have demonstrated such detrimental
effects as mortality and reduced fecundity (Ali et al. 1973; Tolstova et al. 1976; Molloy and
Jamnback 1981; Mulla et al. 1982; James et al. 1993; Flexner et al. 1986; Miller 1990). In
preliminary experiments where transgenic Btk cotton plants were placed in natural soils and
decomposed (Pratt et al. 1993; Palm et al 1994), we discovered that tht B.t.k. endotoxin persisted
and retained its immunological and biological activity at levels similar to what has been observed
with microbially-produced Bt endotoxins. Therefore, we considered it important to determine the
impact of the B.t.k endotoxin in decomposing transgenic plants on soil microorganisms because of
the ubiquity of microorganisms in soil and the crucial role they play in soil processes.
Most concern about the environmental release of plants containing Bt endotoxins has been for the
development of resistance in the target pests (Fox 1991; Johnson and Gould, 1992; USDA 1992) or
for gene flow and plant invasiveness (Umbeck et al. 1991, Manasse 1992; Crawley et al. 1993;
1 Dynamac Corporation and2 U.S. Environmental Protection Agency, WED, NHERL, 200 S.W.
35th Street, Corvallis, OR, 97333 USA

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Kareiva et al. 1994; Klinger and Elstrand 1994). Some studies have considered non-target effects of
the Bt endotoxin but have used microbial Bt strains rather than plants that produce Bt toxins (Molloy
and Jamnback 1981; Flexner et al. 1986; Miller 1990). Only a few studies have used transgenic
plants to assess the potential direct or indirect effects of Bt endotoxins on plant and soil ecosystems
(Donegan et al. 1995, Donegan et al. 1996a; Donegan et al. 1996b).
In addition to the potential effects of Bt endotoxins produced by transgenic plants, there is the
possibility that other plant characteristics may be unintentionally altered during the insertion of the
transgene (Lange 1990, MacKenzie 1990; The Economist 1990; Jenkins et al. 1991; Gene Exchange
1992; Yamada 1992). These types of alterations in plant characteristics caused by genetic
manipulation (e.g., changes in plant enzyme production and biomass), that are independent of
expression of the inserted gene, may also produce ecological effects.
In this chapter we describe four experiments that investigated the biological and molecular changes
in microbial populations following the incorporation of purified B.t.k. endotoxin or B. t.k. -producing
cotton into natural soils. Microbial populations were monitored for changes in the total numbers and
species composition of culturable bacteria and fungi, in the substrate utilization of the bacterial
community and in the total DNA content and DNA fingerprints of the eubacteria.
2 Experimental Studies
2.1 Plant Propagation, Sample Preparation and Experimental Design
Seeds of parental and transgenic cotton were provided by the Monsanto Company (St. Louis, MO).
The parental cotton line was Coker 312. The transgenic cotton lines producing B.t.k. endotoxin were
line 81, HD-1, CrylA(b); line 247, HD-73, CrylA(c); and line 249, HD-73, CrylA(c). Cotton seeds
were propagated in a microcosm and harvested when flowers began forming. Only cotton leaves
were used in these experiments because studies showed they have the highest expression level of the
B.t.k. endotoxin (Ream et al. 1992). For treatments containing plant material, parental or transgenic
cotton leaves were incorporated at 1:3 by weight (leaves:soil) into a fine sandy loam soil (Wasco)
that was obtained from the USD A Cotton Research Station in Shafter, CA. In Experiment 4 only, an
additional soil, a clay loam soil (Panoche) that was received from the USDA Agricultural Research
Station in Fresno, CA, was used. For the purified endotoxin treatments, the HD-1 or HD-73
endotoxin provided by the Monsanto Company was added at a concentration of 0.05 ng toxin/g soil,
calculated as the equivalent of the endotoxin concentration in the transgenic plant treatments. Sterile
water was added to all treatments to bring the soil to 45% water holding capacity. Three replicates
were prepared for each treatment and sampled on days 0, 7, 14, 21 and 28 in experiments 1, 2, and 3
and on days 0, 7, 14, 28 and 56 in experiment 4.
Four experiments with the following combinations of treatments and bioassays were conducted:

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EXPERIMENT TREATMENTS
ASSAYS
1 and 2	Soil only (Wasco sandy loam)
+Purified HD-73 toxin
+Parental cotton
+Transgenic 249 cotton
3	Soil only (Wasco sandy loam)
+Purified HD-1 toxin
+Parental cotton
+Parental cotton+HD-1 toxin
+Transgenic 81 cotton
4	Soil only (Wasco sandy loam)
+Purified HD-73 toxin
+Parental cotton
+Parental cotton+HD-73 toxin
+Transgenic 247 cotton
+Transgenic 249 cotton
Soil Only (Panoche clay loam)
+Purified HD-73 toxin
+Parental cotton
+Parental cotton+HD-73 toxin
+Transgenic cotton 247
+Transgenic cotton 249
Bacterial populations
Fungal populations
ELISA
Bacterial populations
Fungal populations
ELISA
Bacterial populations
Fungal populations
Amoebae populations
Ciliate populations
Flagellate populations
Target insect bioassay
Biolog/Bacterial community
Biolog/Species ID
ELISA
Total DNA content
DNA fingerprints
2.2	Determination of endotoxin concentration
Endotoxin concentrations in the samples were determined immunologically by enzyme-linked
immunoabsorbent assay (ELISA) according to Palm et al. (1994) and by determining bioactivity
using bioassays with Heliothis virescens according to Pratt et al. (1993).
2.3	Determination of microbial populations, bacterial species composition and DNA
fingerprints.
Population levels of total culturable bacteria and fungi were determined by plating samples on
selective media. Numbers of protozoa (amoebae, ciliates and flagellates), performed only in
experiment 4, were determined at the Microbial Biomass Service at Oregon State University,
Corvallis, OR, USA according to the method of Darbyshire et al. (1974).
In experiment 4, for the parental, transgenic 247 and transgenic 249 treatments in Wasco and
Panoche soil on sample days 0, 14 and 56, bacterial species identification was performed. Bacterial
colonies were subcultured, Gram stained and identified biochemically based on substrate utilization

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of 95 carbon sources in Biolog Gram negative or Gram positive microtiter plates (Biolog 1992) In
all of the experiments, fungal colonies were examined visually for qualitative morphological
differences
In experiment 4, the parental, transgenic 247 and transgenic 249 cotton treatments in Wasco soil
were evaluated for differences in bacterial community composition. Diluted soil samples that were
used for the total bacteria and fungi plating were also placed in Biolog Gram negative microtiter
plates to determine bacterial community utilization of 95 carbon sources (Garland and Mills 1991).
In experiment 4, 3 replicate samples of Wasco and Panoche soil treatments were analyzed for total
DNA content. The method of Porteous et al. (1994) was used for DNA extraction, polymerase
chain reaction (PCR) amplification and DNA fingerprinting.
2.4 Statistical analyses
Total bacterial, fungal and protozoan population levels were analyzed in SAS with ANOVA and
repeated measures analysis to determine significant (p < 0.05) differences among treatments.
The Log CFU/g means of the treatments were compared within each sample day with Tukey's
Studentized Range Test (SAS Institute 1989).
Principal components analysis (PCA) was used to analyze the bacterial community substrate
utilization assays for each sample day in order to distinguish differences in patterns of carbon source
utilization among the parental cotton treatment and the transgenic 247 and 249 cotton treatments
(Garland and Mills 1991).
3 Results
3.1 Enzyme-linked, insect, and protozoan assays
Both immunological and biological activity of the B.t.k. toxin persisted in the purified toxin
treatments and in the transgenic plant treatments. Purified B.t.k. HD-1 and HD-73 toxins and B.t.k.
toxin in the transgenic lines 81 and 279 cotton plants were determined by ELISA to persist at
detectable levels (the ELISA detection limit is 0.5 ng toxin/g soil) for up to 28 days in experiments 1,
2 and 3. In experiment 4, the purified B.t.k. toxin was detectable by ELISA up to 28 days and the
purified toxin + parental plant, and the B.t.k. toxin in the transgenic 247 and 249 cotton plants, were
detectable by ELISA up to 56 days in the Wasco soil. In experiment 4, the purified B.t.k. toxin and
the B.t.k. toxin in the transgenic 247 and 249 cotton plants were detectable by ELISA up to 28 days
and the purified toxin + parental cotton plant was detectable by ELISA up to 56 days in the Panoche
soil. The target insect bioassays (performed only in experiment 4) indicated biological activity of the
B.t.k. toxin remained up to 28 days in the purified B.t.k. toxin treatment and in the transgenic 247
and 249 plant treatments (day 56 samples were not assayed) in the Wasco soil and in the Panoche
soil. Further discussion of the persistence of the immunological and biological activity of the B.t.k.
toxins are provided in publications on ELISA studies (Palm et al. 1994) and target insect bioassay
studies (Pratt et al 1993).
No significant differences occurred on any sample day in the number of amoebae, ciliates or
flagellates per g dry weight of soil among the transgenic 247, transgenic 249 and parental cotton

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treatments for the protozoan assays that were performed in experiment 4
3.2	Culturable bacteria and fungi counts
Results were compared among treatments without plant material (with the soil only treatment as the
control) and among treatments with plant material (with the soil+parental cotton treatment as the
control). This was done because the addition of the cotton leaves, whether they were parental or
transgenic, nearly always caused a significant increase in bacterial and fungal numbers. This effect
was due to the nutrients from the added cotton leaves promoting microbial growth; plant material
added to soil often results in high microbial counts (Broder and Wagner 1988, Parr and Papendick
1978).
The addition of purified B.t.k. toxin never caused a detectable effect on microbial populations. No
significant differences in bacterial and fungal population levels were observed between the soil only
treatment and the purified HD-1 toxin treatment or purified HD-73 toxin treatment on any of the
sample days in all four experiments.
In contrast, the addition of two of the three lines of transgenic cotton caused detectable, and often
significant, changes in microbial population levels. In experiment 1, bacterial population levels on
sample days 7, 14 and 21 and fungal population levels on sample days 7 and 14 were significantly
higher in the transgenic 249 cotton treatment than in the parental cotton treatment (Figure 1). In
experiment 2, bacterial population levels on sample days 7 and 14 and fungal population levels on
sample days 7, 14, 21 and 28 were significantly higher in the transgenic 249 cotton treatment than in
the parental cotton treatment (Figure 2). In experiment 3, there were no significant differences in
bacterial or fungal population levels on any sample days between the transgenic 81 cotton treatment
and the parental cotton treatment, or the parental cotton treatment + purified HD-1 toxin treatment.
In the experiment 4 treatments with Wasco soil, bacterial populations in the transgenic 247 cotton
treatment were significantly higher than in the parental cotton+purified HD-73 toxin treatment on
sample days 7, 14 and 28. The transgenic 249 cotton treatment was significantly higher than the
parental cotton treatment in bacterial population levels on sample day 56. The transgenic 249 cotton
treatment was also significantly higher than the parental cotton+purified HD-73 toxin treatment in
bacterial population levels on sample days 7 and 56 and in fungal population levels on sample days 7
and 14. For the treatments in experiment 4 with Panoche soil, bacterial populations in the transgenic
247 cotton treatment were significantly higher than those in the parental cotton treatment on sample
day 7 .
3.3	Bacterial species identification
Identification of subcultures from the plates used for the bacterial counts in experiment 4 indicated
that differences in bacterial species composition developed among the transgenic 247 cotton
treatment, transgenic 249 cotton treatment and the parental cotton treatment over the course of the
experiment in the Wasco soil and to a lesser extent in the Panoche soil. Differences among the
treatments in bacterial species composition were not observed at the start of the experiment; on
sample day 0, all colonies subcultured from the parental, transgenic 247 and transgenic 249 cotton
treatments in Wasco or Panoche soil were Gram positive and there were few differences in genus or

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species composition (colonies were mainly Actinomycete or Bacillus spp. in the Wasco soil and
mainly Streptococcus or Bacillus spp. in the Panoche soil).
The greatest differences in Gram identification and species composition in the Wasco soil were
observed on sample day 14. The parental cotton treatment had a larger number of Gram positive
bacteria (34%) than the 247 transgenic treatment (0%) and the 249 transgenic treatment (4%), and
had mainly Bacillus spp (25% of the identified Gram positive colonies), Enterobacter cloacae B
(25% of the identified Gram negative colonies) and Enterobacter spp (25% of the identified Gram
negative colonies) In the transgenic 247 cotton treatment, the predominant species were E. cloacae
B (23% of the identified Gram negative colonies) and Pseudomonas corrugata (35% of the identified
Gram negative colonies) and in the transgenic 249 cotton treatment Enterobacter asburiae (86% of
the identified Gram negative colonies) was the dominant species. On sample day 56 in the Wasco
soil, bacterial composition was most similar between the parental and transgenic 249 cotton
treatments; both had a predominance of Gram positive bacteria (64% for the parental treatment and
75% for the transgenic 249 treatment) that were mainly Bacillus spp. (36% for the parental
treatment and 39% for the transgenic 249 treatment of the identified Gram positive colonies) and
also relatively high numbers of Pseudomonas diminuta (23% for the parental treatment and 8% for
the transgenic 249 treatment of the identified Gram negative colonies) In contrast, in the transgenic
247 cotton treatment on day 56, the majority of colonies were Gram negative and were identified as
Alcaligenes denitrificans (20% of the identified Gram negative colonies) and P. corrugata (25% of
the identified Gram negative colonies).
In the Panoche soil on sample day 14, the parental, transgenic 247 and transgenic 249 cotton
treatments were similar and had a fairly equal distribution of Gram positive and Gram negative
bacteria and mainly Bacillus spp. and E. asburiae colonies. On sample day 56, there was a
predominance of Gram positive bacteria in the Panoche soil, particularly for the parental (69%) and
transgenic 249 (78%) cotton treatments. The most prevalent species in Panoche soil on sample day
56 in the parental treatment were Bacillus spp. (19% of the identified Gram positive colonies)
whereas the transgenic 247 and 249 cotton treatments did not generally have predominant species, in
part because a high number (62%) of the Gram positive colonies in the transgenic 249 cotton
treatment could not be identified.
Based on fungal colony morphology, differences were also observed in fungal species composition
between the parental and transgenic cotton treatments on a few of the sample days in experiments 1
and 2. For example, in experiment 2 on sample days 21 and 28, all the fungal colonies isolated from
the transgenic 249 cotton treatment wertMucor sp. whereas only a few Mucor sp. were isolated
from the parental cotton treatment.
3.4 Bacterial community metabolic substrate utilization
Differences in bacterial species composition were also observed in the Biolog assays for substrate
utilization measured for the diluted soil samples. The Principal Components Analysis of these assay
results indicated differences in microbial usage of metabolic substrates among the parental,
transgenic 247 and transgenic 249 cotton treatments on sample days 7 and 14. Differences in
utilization of the amino acids L-asparagine, L-aspartic acid and L-glutamic acid on sample days 7 and
14 were important in the separation of the parental and transgenic 247 and 249 treatments as
evidenced by their large contribution to the PCA score. No other single metabolic substrates were as

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consistently used to separate the treatments. The utilization of these 3 amino acids was significantly
higher in the transgenic 247 and 249 treatments than in the parental treatment on sample days 7 and
14.
3.5 Total DNA content and eubacteriai DNA fingerprints
Differences among the treatments in bacterial population levels and species composition were also
indicated by the DNA extractions and fingerprints that were performed in experiment 4. As observed
with the total bacterial and fungal counts, the addition of plant material caused an increase in
microbial levels; two to five times more total DNA was extracted from the treatments containing
parental or transgenic plant material than from the treatments containing soil only or soil+purified
HD-73 toxin in both the Panoche and the Wasco soils on sample days 7, 14, 28 and 56. For the
Wasco soil on sample days 7, 14 and 28, the highest DNA content was measured in the transgenic
247 cotton treatment. For example, on day 28 the A260 readings indicated the following DNA
recoveries: 20 to 35 ug DNA per g of the soil or soil+purified HD-73 toxin treatments, 50 to 65 jug
DNA per g of the soil+parental cotton or soil+parental cotton+purified HD-73 toxin treatments, 50
to 75 Mg DNA per g of the soil+transgenic 249 treatment, and 80 to 100 ng DNA per g of the
soil+transgenic 247 cotton treatment.
Differences among treatments in the composition of microbial populations were indicated by
eubacteriai DNA fingerprint patterns (restriction endonuclease digested fragment patterns) of
amplified rDNAs from Wasco soil containing parental, transgenic 247 and transgenic 249 cotton on
sample days 14 and 56. Fingerprint patterns for the treatments varied in the size or length of
fragments, the number, intensity, or absence of bands and the reoccurring presence of a series of
fragments in the fingerprint patterns. For example, on sample day 14 the fragment patterns for the
parental treatment showed intense bands at 600 - 800 bp which were not observed in the fingerprints
of either transgenic treatment. On sample days 14 and 56, the transgenic 247 treatment fingerprints
contained a unique series of 4 intense bands between 150 - 300 bp. In addition, the fingerprint for the
transgenic 249 treatment on sample day 56 had bright 650 bp fragment bands present that were not
observed in the fingerprints for the other treatments.
4. Discussion
The addition of purified B.t.k. endotoxin to natural soil did not have any measurable effects on the
indigenous soil microorganisms. In all of the experiments, no changes were observed in the levels of
culturable, aerobic soil bacteria, fungi or protozoa (effects on protozoa were evaluated only for HD-
73) when purified B.t.k. HD-1 or HD-73 toxin was added to natural soils. In contrast, the increase in
culturable, aerobic bacterial and fungal populations that occurred when leaves of cotton were added
to soil was significantly higher in the transgenic cotton treatments relative to the parental cotton
treatment. This significant stimulation in microbial populations from the transgenic cotton, however,
was only observed with the addition of the 247 and 249 lines, but not the 81 line, of the transgenic
B. t.k.-producing cotton plants. The increase in microbial populations observed with the plate counts
from the addition of cotton leaves was also confirmed with the bulk DNA extractions performed in
experiment 4; the amounts of DNA extracted in the treatments with parental or transgenic plant

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leaves increased as total microbial plate counts increased. For example, the greatest amount of DNA
extracted and the highest bacterial counts coincided, they both occurred in the transgenic 247
treatment on sample day 28 in the Wasco soil in experiment 4.
Qualitative changes in the soil microbial community were also observed with the addition of the
247 and 249 lines of transgenic B.t.k. cotton to Wasco or Panoche soil. The metabolic substrate
utilization assays and species identifications of subcultures performed in experiment 4 indicated
differences in microbial species composition among the treatments. For example, the community
substrate utilization assays indicated major differences on sample days 7 and 14 among the parental,
transgenic 247 and transgenic 249 cotton treatments in the type and level of substrates utilized; there
was significantly greater utilization of asparagine, aspartic acid and glutamic acid in the transgenic
247 and 249 cotton treatments than in the parental cotton treatment even though the population
levels of the treatments were not significantly different. Interestingly, these substrates are important
intermediates, along with glutamine (which is not included as a substrate in the Biolog plates), in
nitrogen assimilation reactions. Species identifications of subcultures from plates used for the total
bacterial populations in experiment 4 similarly showed the greatest differences between the parental
treatment and the transgenic 247 and 249 treatments on sample day 14 (species identifications were
not done for sample day 7). DNA fingerprints of amplified rDNAs also reflected qualitative
differences among the treatments in eubacterial population. Distinct differences in fragment
molecular weight and number and location of bands were observed among the Wasco soil parental,
transgenic 247 and transgenic 249 treatments in experiment 4. These differences between the
parental treatment and the transgenic treatments were pronounced on sample day 14, as occurred
with the substrate utilization measurements and species identifications.
It is somewhat surprising that the line 81 transgenic cotton plant did not cause the same impact on
microbial populations that was observed with the line 247 and line 249 transgenic cotton plants. The
transgenic cotton lines produce endotoxins (HD-1 for line 81 and HD-73 for lines 247 and 249) that
are coded for by genes from closely related, bacterial strains and the HD-1 and HD-73 endotoxins
share 86% amino acid identity. It seems unlikely that the small difference in endotoxins between the
transgenic plant line 81 and the lines 247 and 249 would account for the difference in effects from
the transgenic plants because the purified endotoxins did not differ in their effects and neither had
any detectable impacts on microbial populations.
One possible explanation for the lack of effects on the total microbial population levels from the
addition of the purified HD-1 and HD-73 B.t.k. toxins and the transgenic cotton line 81 is that there
were characteristics of the transgenic 247 and 249 cotton plants, other than production of the B.t.k.
toxin, that impacted soil microorganisms. In most of the experiments, the stimulatory effects of the
transgenic 247 and 249 cotton plants on bacterial and fungal populations were short term, suggesting
that the transgenic plants may have decomposed faster than the parental plants and thus more rapidly
provided nutrients for microbial growth. This potential increase in decomposition rate of the
transgenic 247 and 249 cotton plants may have resulted from genetic manipulation of the plants.
Unanticipated changes in plant quality occurring from insertion of genes have been documented. In
several laboratory and field studies there have been unintentional changes in plant characteristics due
to genetic manipulation and position effects from site(s) of insertion (Gene Exchange 1992). In
addition, there may be genes used to confer a specific trait but which are actually pleiotropic and
change several plant traits (Fitzpatrick 1993). Finally, the practice of tissue culturing, used for both
traditional and genetically engineered plant breeding, frequently produces plant aberrations, some of

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which may not be detected during screening for efficacy of the added trait(s).
These sorts of risk assessment studies for transgenic plants need to be performed under a variety of
environmental conditions. The greater quantitative and qualitative impacts of the transgenic plant
lines in the Wasco soil as compared to the Panoche soil and the higher recovery of DNA from the
Wasco soil than the Panoche soil demonstrate the importance of one environmental variable, soil
type. The clay and organic matter content of the Panoche soil is higher than that of the Wasco soil
and probably resulted in greater binding of the transgenic plant material, making it less available to
exert an impact on the soil microorganisms (Stotzky 1986). In past ELISA studies, we have obtained
much higher recovery of the purified toxin and the transgenic plant toxin from the Wasco soil than
from the Panoche soil (Palm et al. 1996). Similarly, target insect bioassays with Heliothis virescens
have shown higher bioactivity of the same quantity of purified toxin or transgenic plant toxin when
measured in the Wasco soil relative to the Panoche soil (Pratt et al. 1993).
In these studies with B.t.k. producing transgenic cotton plants, we have demonstrated both
quantitative and qualitative changes in exposed soil microorganisms The quantitative effects were
generally transient and not what are typically considered detrimental (i.e, population levels were
stimulated rather than depressed). The qualitative effects of apparent changes in the microbial species
composition, however, has a potential to impact soil processes and may be of ecological significance.
These results are valid only for the methods and test conditions used and additional research is
necessary to determine their scope, extent and significance.
In the continuing debate about the degree and types of risks posed by the environmental release of
transgenic plants, the argument has been made that transgenic plants pose no more risk than the
transgenic compounds they produce and that plants should not be evaluated based on their having
originated from genetic engineering. We believe our results challenge this argument; two of the
transgenic B. t. k. -producing cotton lines impacted soil microorganisms yet no effects were observed
from the purified B.t.k. endotoxins. We suggest that the risk assessment of transgenic plants should
address and monitor for potential ecological effects that may result from changes in plant
characteristics other than expression of the inserted gene(s).

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Acknowledgments
The authors thank Dr. Roy Fuchs, Sharon Berberich, Joel Ream and Bill Schuler of the Monsanto
Company, St. Louis, MO, USA, for providing the cotton seeds, purified toxin and technical
information. The authors would like to acknowledge the excellent technical and scientific support
provided for this study by Valerie Fieland, Lisa Ganio, Curt Palm, Arlene Porteous, Grahame Pratt,
Lynn Rogers and Debbie Schaller.

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Fig. 1. Numbers of total bacteria (A) and fungi (B) in experiment 1, averaged
from duplicate plates of 3 treatment replicates per sample day, of soil alone,
soil+purified HD-73 toxin, soil+parental cotton and soil+transgenic 249 cotton.
Significant (P< 0.05) differences among treatments were determined with
Tukey's Studentized Range Test. The minimum significant differences in log
values calculated for bacterial populations were 0.42 (day 0), 0.29 (day 7), 0.95
(day 14), 0.42 (day 21). 0.66 (day 28) and for fungal populations were 0.44 (day
0), 0.14 (day 7), 0.19 (day 14), 0.28 (day 21), 0.34 (day 28).

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Sample day
¦
| Soil


+HD-1 toxin

+Parental cotton


+Transgenic 249 cotton

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Fig. 2. Numbers of total bacteria (A) and fungi (B) in experiment 2, averaged
from duplicate plates of 3 treatment replicates per sample day, of soil alone,
soil+purified HD-73 toxin, soil+parental cotton and soil+transgenic 249
cotton. Significant (P<0.05) differences among treatments were determined
with Tukey's Studentized Range Test. The minimum significant differences in
log values calculated for bacterial populations were 0.23 (day 0), 1.40 (day 7),
0.87 (day 14), 1.07 (day 21), 0.40 (day 28) and for fungal populations were
0.51 (day 0), 0.91 (day 7), 0.79 (day 14), 0.36 (day 21), 0.32 (day 28).

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0	7	14
Sample day

¦
Soil
| [ +HD-1 toxin


+Parental cotton


+Transgenic 249 cotton

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NHEERL-C0R-2028A
TECHNICAL REPORT DATA
(Please read instructions on the reverse before completingj
1. REPORT NO.
EPA/600/A-96/095
2.
3. RECIPI
4. TITLE AND SUBTITLE
Effects of cotton expressing the Bacillus thuringiensis var. kurstaki endotoxin
on soil microorganisms
5. REPORT DATE
8. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
K. K. Donegan', R. J. Seidler2
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
'Dynamac Corporation, Corvallis, OR
JU.S. Environmental Protection Agency, NHEERL, Corvallis, OR
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
US EPA ENVIRONMENTAL RESEARCH LABORATORY
200 SW 35th Street
Corvallis, OR 97333
13. TYPE OF REPORT AND PERIOD COVERED
Book chapter
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
1996. To be published as a book chapter in "Biotechnology in Agriculture and Forestry"
/
A
ABSTRACT
Many agriculturally important plants have been engineered to produce endotoxins from different subspecies of the
bacterium Bacillus thuringiensis {B. t.). The endotoxin Bacillus thuringiensis var. kurstaki [B.t.k.) has demonstrated
insecticidal activity against lepidopterans. Although high specificity has been assumed for most B.t. endotoxins, their
effects on non-target organisms have not been fully evaluated. This chapter describes experiments that investigated
the biological and molecular changes in microbial populations following the incorporation of purified B.t.k. endotoxin or
B.t.k.-producing cotton into natural soils. Microbial populations were monitored for changes in the total numbers and
species composition of culturable bacteria and fungi, in the substrate utilization of the bacterial community and in the
total DNA content and DNA fingerprints of the eubacteria. -
»
1 7. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
sediments, ecological risk
assessment


18. DISTRIBUTION STATEMENT
19. SECURITY CLASS (This Report)
21. NO. OF PAGES
18
20. SECURITY CLASS [This page)
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION IS OBSOLETE

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