1
Environmental
Effects
Assessment
for
WideStrike
 
,

MXB­
13
Cotton
Zig
Vaituzis,
Ph.
D.,
Senior
Scientist
Biopesticides
and
Pollution
Prevention
Division
2
Outline
 
Introduction
 
Background
of
Risk
Assessment
Process
 
Summary
of
WideStrike
 
Environmental
Assessment
 
SAP
Questions
on
Environmental
Effects
Assessment
3
INTRODUCTION
 
Pesticide:

 
WideStrike
 
cotton
expressing
Cry1F
and
Cry1Ac
to
control
several
lepidopteran
pests
4
INTRODUCTION
 
Assessment
includes:

 
Effects
on
wildlife
 
Gene
flow
to
related
wild
plants
 
Development
of
weediness
 
Fate
of
Cry1F/
Cry1Ac
in
environment
 
Effects
on
endangered
species
 
Assessment
based
on:

 
Data
submitted
to
the
Agency
 
FIFRA
SAP
recommendations,

 
Consultations
with
scientific
experts,
and
 
Public
comments
5
HAZARD
ASSESSMENT
PROCESS
 
Assess
toxicity
to
representative
non­
target
organisms
by
a
tiered
testing
system
 
Start
with
Tier
I
single
species,
high
dose
laboratory
data
 
Mortality
as
end
point
 
Maximum
Hazard
Dose
(
MHD)
approach
developed
by
AIBS
°
1996­
approved
by
SAP
for
naturally
occurring
and
altered
MPCAs
and
toxins,

°
1999­
approved
by
SAP
for
protein
Plant
Incorporated
Protectants
(
PIP).

°
[
Cry
proteins
are
bacterial
toxins]
6
HAZARD
ASSESSMENT
PROCESS
 
Negative
results
by
Tier
I
limit
testing
provide
a
high
degree
of
confidence
that
no
unreasonable
adverse
effects
are
likely
to
occur
 
(
PIP)
Bt
Cry
proteins
are
tested
by
Tier
I
for
short
term
hazard
assessment
 
Long
term
adverse
effects
are
ascertained
by
higher
Tier
long
term
field
testing
 
2000
SAP
and
the
NAS
(
2000)
recommended
direct
field
testing
 
2002
SAP
recommended
Tier
I
and
field
testing
with
emphasis
on
Tier
I
testing
of
agricultural
field
invertebrates
7
RISK
ASSESSMENT
PROCESS
 
Risk
assessment
based
on:

Safety
Factor
X
Expected
Environmental
Concentration
(
EEC)

 
Data
that
establishes
an
LC50,
ED50,
or
LD50
that
is
>

MHD
(
e.
g.
LD50
>
10
X
EEC)
is
sufficient
to
evaluate
adverse
effects
 
Levels
of
concern
(
LOC):

 
Terrestrial:
LD
50
<
5X
EEC
(
EEC/
LD
50
=
RQ
=
0.2)

 
Aquatic:
LC
50
<
10X
EEC
(
EEC/
LC
50
=
RQ
=
0.1)

 
When
LD50
is
below
the
safety
factor
(
a
LOC),
lower
dose
testing
is
necessary
to
establish
a
definitive
LD50
and
a
Lowest
Observable
Effect
Concentration
(
LOEC)
8
RISK
ASSESSMENT
PROCESS
 
Risk
assessment
is
made
by
comparing
the
LOEC
to
the
EEC
 
When
EEC
is
<
LOEC,
a
"
no
adverse
effect"
assessment
is
made
 
2000
SAP
thought
that
Tier
I
laboratory
tests
showing
no
detrimental
effects
are
sufficient
to
make
a
short
term
hazard
assessment
and
field
studies
(
as
higher
Tier
testing)
be
conducted
when
Tier
I
tests
show
toxicity
 
Bt
Cry
endotoxins
are
proteins
and
do
not
have
the
potential
to
bioaccumulate
resulting
in
delayed
adverse
effects
 
Accumulation
through
the
food
chain
is
not
expected
to
take
place
and
therefore,
chronic
effects
testing
of
protein
substances
is
not
routinely
performed.
9
NON­
TARGET
WILDLIFE
TOXICITY
TESTING
 
SAP
reports
(
2000
&
2002
)
recommended:

 
testing
of
species
related
to
the
susceptible
pest
 
focus
on
invertebrates
exposed
to
the
crop
 
However,
testing
was
performed
to
comply
with:

 
all
published
Tier
I
microbial
toxin
requirements
 
Direct
field
census
data
on
the
abundance
of
invertebrates
in
the
field
were
also
requested,

received
and
evaluated
to
supplement
the
findings
of
the
MHD
Tier
I
testing
10
NON­
TARGET
WILDLIFE
TOXICITY
TESTING
 
Mammalian
&
avian
wildlife
 
Aquatic
organisms
 
Plants
 
Adult
&
larval
honey
bees
 
Parasitic
wasp
 
Green
lacewings
 
Lady
beetles
 
Collembola
(
springtail)

 
Monarch
butterfly
 
Earthworms
Toxicity
has
been
evaluated
for:

Reproductive
and
developmental
observations
made
on:

 
Collembola,
honey
bee
and
green
lacewing
larval
maturation
studies
11
TEST
SUBSTANCE
 
Form
of
test
substances
used
in
the
studies:

 
plant
material
(
leaves,
pollen)

 
purified
bacterially­
produced
Cry1F/
Cry1Ac
(
separately
and
in
combination)

 
2000
SAP
guidance:

 
plant
material
preferred
 
bacterially­
derived
protein
also
valid
where:

°
the
test
animals
do
not
consume
plant
tissue
°
large
amounts
of
Cry
protein
are
needed
for
MHD
testing
°
plant
material
toxic,
e.
g
gossypol
in
cotton
 
Adult
insect
studies
are
generally
of
30
days
duration
or
until
negative
control
mortality
reaches
20%.

 
Larval
studies
are
through
pupation
and
adult
emergence
12
MAMMALIAN
WILDLIFE
 
No
significant
toxicity
to
rodents
from
acute
oral
testing
at
the
MHD
(
Cry1F
>
600
mg
a.
i./
kg
and
Cry1Ac
>
700
mg
a.
i./
kg.)

 
No
hazard
to
mammalian
wildlife
is
anticipated
13
AVIAN
HAZARD
 
Acute
single
MHD
oral
LD50
for
Cry1F/
Cry1Ac
(
7.9:
1
ratio)
is
>
128
mg
a.
i./
kg
 
Avian
8
day
dietary
with
10
%
cotton
seed
showed
clinical
symptoms
attributed
to
gossypol,
(
8X­
10X
the
maximum
allowable
in
animal
feeds)
LC50
>
100,000
ppm
 
8
day
study
insufficient
to
assess
hazards
from
chronic
exposure
 
Requested
a
6­
week
broiler
study
with
Cry1F/
Cry1Ac­
corn
(
no
gossypol)
14
AQUATIC
SPECIES
 
No
evidence
for
sensitivity
of
aquatic
(
including
endangered)
species
to
antilepidopteran
Cry
proteins
 
Aquatic
exposure
from
Bt
cotton
is
extremely
small

Freshwater
and
Estuarine
and
Marine
Animal
Hazard
 
Eight
day
dietary
toxicity
to
the
rainbow
trout
with
a
basal
fish
diet
with
100­
mg
a.
i./
kg
diet
of
Cry1F/
Cry1Ac
in
a
ratio
of
7.9:
1
 
No
mortality
or
sublethal
effects
observed
 
No
fish
hazard
is
expected
from
the
proposed
uses
of
WideStrike
 
cotton
15
AQUATIC
SPECIES

Aquatic
Invertebrate
Hazard
 
Acute
toxicity
of
microbial­
produced
510

g
Cry1F/
L
+
2500

g
Cry1Ac/
L
of
test
solution
to
Daphnia
magna
was
>
298X
and
>
23,000X
the
EEC
for
Cry1F
and
Cry1Ac
protein
 
No
immobility
or
other
adverse
effects
were
seen
during
the
study
 
No
hazard
to
aquatic
invertebrates
is
expected
from
incidental
exposure
to
WideStrike
 
cotton
pollen
16
AQUATIC
SPECIES

Aquatic
and
Terrestrial
Plant
Hazard
 
The
active
ingredient
is
an
insect
toxin
(
Bt
endotoxin)
that
has
never
shown
any
toxicity
to
plants
 
Studies
have
been
waived
 
Outcrossing
issues
will
be
addressed
separately
17
TERRESTRIAL
INVERTEBRATE
HAZARD
 
Soil
Invertebrates
 
General
issues
 
October
2000
SAP
concluded
that
earthworms
and
springtails
(
Collembola)
are
appropriate
indicator
species
for
Cry
protein
testing
despite
the
specific
nature
of
the
Cry
protein
toxicity
to
select
target
species.

 
Such
soil
organisms
may
be
subject
to
long­
term
exposure.

However,
data
received
by
EPA
indicate
that
such
proteins
degrade
rapidly
in
field
soils.

 
Therefore
the
potential
for
significant
soil
buildup
and
adverse
effects
to
non­
target
soil
organisms
are
not
anticipated.
Post­
registration
soil
persistence
confirmatory
data,
however,
are
required.
18
TERRESTRIAL
INVERTEBRATE
HAZARD
 
Single
Species
Laboratory
Testing
 
General
Comments
 
The
test
material
in
several
of
the
studies
is
purified
microbial
Cry1F
and
Cry1Ac
protein
in
a
ratio
equivalent
to
the
Cry1F
and
Cry1Ac
proteins
in
whole
WideStrike
 
cotton
plants.

 
In
Summary:
Toxicity
tests
conducted
with
non­
target
arthropods
and
soil
invertebrates
indicate
that
no
adverse
effects
are
expected
when
exposed
to
Cry1F
and
Cry1Ac
protein
concentrations
exceeding
the
EECs.
19
TERRESTRIAL
INVERTEBRATE
HAZARD

Effects
on
Honey
Bee
Larvae
 
Methods:

°
Exposed
to
sucrose
solutions
containing:

 
genetically
modified
pollen
(
Cry1F
only)

 
genetically
modified
pollen
(
Cry1Ac
only)

 
Bt
with
both
Cry1F
and
Cry1Ac
expression
°
Potassium
arsenate
served
as
a
positive
control
°
Honey
bee
larva
were
scored
for
survival
to
capping
and
adult
emergence
 
Results:

°
Treatment
groups
were
not
statistically
different
from
the
negative
control
(
sucrose
only)
which
had
a
survival
of
95%
to
capping
and
93.75%
to
adult
emergence
20
TERRESTRIAL
INVERTEBRATE
HAZARD

Effects
on
Honey
Bee
Larvae
 
Results
(
cont.):

°
At
1.98

g
Cry1F
+
11.94

g
Cry1Ac
per
mL
sugar
water
no
effect
on
survival
of
larvae
to
adult
emergence
was
seen
°
LC50
is
>
4X
expression
in
pollen.

°
No
hazard
to
honey
bee
larvae
and
adult
bee
emergence
is
anticipated.
21
TERRESTRIAL
INVERTEBRATE
HAZARD

Effects
on
Parasitic
Hymenoptera
(
wasp)*

 
A
40%
mortality
was
observed
in
the
Cry1F
+

Cry1Ac
group
at
32X
the
EEC.

 
The
EPA
LOC
for
terrestrial
wildlife
is
an
LC50
less
than
5X
the
field
exposure
(
EEC/
LC50
=
RQ
>
0.2)

 
The
LC50
in
this
study
is
greater
than
32X
the
EEC
 
No
hazard
to
parasitic
Hymenoptera
is
expected
at
field
exposures
(
which
are
minimal
to
nonexistent
*
The
August
27,
2002
SAP
concluded
that
parasitic
Hymenoptera
testing
was
not
appropriate.
Testing
another
beneficial
organism
rather
than
a
parasitoid
was
recommended
as
more
suitable.
22
TERRESTRIAL
INVERTEBRATE
HAZARD

Effects
on
Green
Lacewing
Larvae
 
Mortality
was
increased
and
pupation
was
affected
in
the
Cry1F/
Cry1Ac
at
32X
the
concentration
found
in
pollen
(
LC50
>
14X
pollen
expression)

 
No
effect
is
noted
at
Cry
protein
levels
expressed
in
pollen
that
would
be
encountered
by
green
lacewings
in
the
field
 
August
2002
SAP
questioned
the
appropriateness
of
testing
green
lacewing
and
recommended
testing
an
alternate
natural
enemy
such
as
the
minute
pirate
bug
(
Orius
insidiosus)
because:

°
typically
occurs
in
fields
as
egg
predators
°
typically
feed
on
pollen.
23
INVERTEBRATE
HAZARD
ASSESSMENT

Effects
on
Lady
Beetles
 
No
adverse
effect
was
seen
at
limit
dose
testing
(
LC
50
>
780X
Cry1F
pollen
expression
and
>
8X
Cry1Ac
pollen
expression)

 
No
hazard
to
H.
convergens
is
expected
when
feeding
on
WideStrike
 
cotton
pollen
in
the
field

Effects
on
Collembola
(
spring
tail)

 
Lyophilized
Cry1Ac
cotton
leaf
at
5%
or
50%
of
the
diet
did
not
cause
mortality
or
have
adverse
effects
on
reproduction
 
Combination
of
Cry1F
and
Cry1Ac
showed
no
effect
at
up
to
10X
the
anticipated
field
level
of
expression.

 
No
hazard
to
decomposers
represented
by
Collembola
is
expected
from
exposure
to
WideStrike
 
cotton
in
the
field
24
INVERTEBRATE
HAZARD
ASSESSMENT

Effects
on
Earthworms
 
14­
day
limit
dose
study
was
conducted
on
earthworms
exposed
to
soils
treated
with
microbial­
produced
Cry1Ac
and
Cry
1F,
individually
and
in
combination
 
There
were
no
overt
signs
of
toxicity
to
earthworms
exposed
to
soils
containing
nominal
concentrations
of
Cry1F
and
Cry1Ac
at
50x
the
expected
worst
case
EEC

Monarch
Butterfly
Risk
Assessment
 
Studies
published
in
the
Proc.
Nat.
Acad,
Sci.
(
98
[
21])
were
used
to
show
that
the
density
of
cotton
pollen
on
milkweed
leaves
(
11
grains
of
MXB­
13
pollen
per
cm2)
is
10X
less
than
the
minimum
pollen
density
required
to
elicit
subchronic
or
developmental
effects
on
monarch
butterfly
larvae
 
Therefore
monarch
larvae
are
not
expected
to
be
adversely
affected
by
exposure
to
WideStrike
 
cotton
pollen
in
the
field
25
INVERTEBRATE
HAZARD
ASSESSMENT

Activity
Spectrum
Study
 
The
insecticidal
activity
spectrum
of
Cry1F
and
Cry1Ac
was
determined
for
nine
insect
species
 
Insects
represent
taxonomically
diverse
cotton
pests
 
Both
Cry1F
and
Cry1Ac
activity
was
restricted
to
lepidopteran
insects,
lending
support
to
the
contention
that
the
combination
of
two
Cry
proteins
did
not
expand
the
insect
host
range
Gelechiidae
Curculionidae
Miridae
Noctuidae
Coleoptera
Heteroptera
Lepidoptera
26
INVERTEBRATE
HAZARD
ASSESSMENT

Field
Evaluation
­
Effects
on
Invertebrates
 
Beneficial
arthropods
in
field
plots
of
WideStrike
 
cotton
were
compared
with
plots
of
non­
transgenic
cotton
and
those
with
and
without
insecticide
application
in
LA
and
AZ
 
Preliminary
results
show
no
adverse
effect
on
the
numbers
of
insects
from
over
50
taxa.
(
Chemical
insecticide
treatment
reduced
the
population
of
some
taxa
at
certain
times
of
sampling).

 
Field
monitoring
supports
the
the
Tier
I
single
species
data
showing
a
lack
of
adverse
effects
to
non­
target
invertebrates.
27
ENVIRONMENTAL
FATE
 
Soil
Degradation
 
The
soil
DT50
of
the
plant
expressed
Cry1F
and
Cry1Ac
was
1.3
days
in
a
laboratory
insect
bioassay
with
soil
from
a
cotton
growing
region
(
Wayside,
MS)

 
Soil
incorporation
rates
with
cotton
leaf
tissue
were
0.072
mg
a.
i.
Cry1Ac
and
0.853
mg
a.
i.
Cry1F
per
kg
of
oven
dry
soil
(
approximately
3.2
X
the
EEC
for
incorporation
of
defoliated
whole
plants
of
MXB­
13
into
the
top
15­
cm
of
soil).

 
EPA
is
requesting
additional
studies
on
multiseason
persistence
of
Cry
proteins
in
soils
under
actual
field
conditions
28
EFFECTS
ON
SOIL
MICROORGANISMS
 
Published
studies
performed
by
the
EPA
Office
of
Research
and
Development
on
the
impact
of
transgenic
Cry
cotton
and
other
plants
indicates
that
adverse
effects
on
soil
microorganisms
are
unlikely
 
No
effects
were
seen
due
to
the
protein
itself,

and
only
a
minimal,
transient
increase
in
soil
microbes
attributed
to
the
transgenic
cotton
plant
tissue,
rather
than
the
Cry
protein
expressed
in
that
tissue,
was
observed
29
GENE
FLOW

Horizontal
gene
transfer
(
hgt)
from
Bt
Crops
to
Soil
Organisms
 
Several
experiments
published
in
the
scientific
literature
have
been
conducted
to
assess
the
likelihood
of
hgt
and
have
been
unable
to
detect
gene
transfer
under
typical
field
conditions.

 
Hgt
has
only
been
detected
under
laboratory
conditions
designed
to
favor
DNA
transfer.

 
The
genes
that
have
been
engineered
into
the
Bt
crops
have
their
origin
in
soil
inhabiting
bacteria.

Transfer
of
these
genes
or
toxins
to
other
microorganisms
or
plants
is
not
known
to
occur.

 
In
considering
these
data,
the
Agency
concludes
that
there
is
no
significant
risk
of
hgt
from
the
transgenes
found
in
Cry1F/
Cry1Ac
cotton.
30
GENE
FLOW

Gene
Flow
and
Weediness
Potential
 
In
2001
the
Agency
reviewed
the
potential
for
gene
capture
and
expression
of
the
Bt
endotoxins
by
wild
or
weedy
relatives
of
cotton
in
the
U.
S.,

its
possessions
or
territories.

°
Results
of
review:
1.
Southern
Arizona
2.
Hawaiian
islands
3.
Southern
Florida
4.
Puerto
Rico
 
There
is
presently
either:

1.
No
production
of
commercial
cotton
in
these
locations,

or
2.
A
restriction
on
commercial
cultivation
has
been
placed
where
outcrossing
may
occur
Cultivated
cotton
has
the
opportunity
to
outcross
to
wild
or
feral
species
31
ENDANGERED
SPECIES
CONSIDERATIONS
 
Reviewed
non­
target
data
confirm
that
WideStrike
 
cotton
will
have
no
adverse
effect
on
endangered
and/
or
threatened
species
listed
by
the
US
Fish
and
Wildlife
Service,
including
mammals,
birds
or
terrestrial
and
aquatic
plants
and
invertebrate
species.

 
Comparison
of
the
county­
level
distribution
of
endangered
lepidopteran
species
relative
to
cotton
producing
counties
in
the
US
indicate
that
there
will
be
no
overlap
with
cotton
cultivation
and
endangered
lepidopteran
breeding
habitats.

 
No
consultation
with
the
USFWS
is
required
under
the
Endangered
Species
Act.
32
CONCLUSIONS
 
This
assessment
finds
no
hazard
to
the
environment
from
cultivation
of
Cry1F
and
Cry1Ac
protein
expressing
cotton
for
a
timelimited
registration
period.

 
However,
there
are
issues
that
could
be
looked
at
in
greater
depth.
The
Agency
believes
these
issues
were
not
reasonably
foreseeable
at
the
time
of
registration
application
and
initial
data
development.

 
These
supplemental,
primarily
long
term
effects
studies
would
provide
added
weight
to
support
the
Agency's
conclusions.
33
Additional
Data
Requirements:

 
A
six
week
broiler
dietary
study
(
Cry1F/
Cry1Ac
Corn
may
be
used)

 
A
Tier
I
laboratory
toxicity
test
with
Orius
insidiosus
(
minute
pirate
bug)
­­
a
non­
target
insect
more
appropriate
for
cotton
fields
 
Additional
invertebrate
field
surveys
on
large
plots
that
have
been
planted
with
Cry1F/
Cry1Ac
cotton
for
at
least
three
consecutive
years
 
Additional
long
range
soil
persistence
field
studies
34
Questions
on
the
Environmental
Effects
Assessment
1)
WideStrike
cotton
is
a
product
expressing
pyramided
Cry1F
and
Cry1Ac
Bt
proteins.
The
submitted
non­
target
effects
studies
examined
the
effects
of
the
Cry1F
and
Cry1Ac
proteins
separately
and
in
combination
to
detect
any
synergistic
effects
on
non­
target
wildlife.
No
synergistic
effects
or
increase
in
non­
target
host
range
were
seen
as
a
result
of
combining
these
two
proteins
in
the
same
product.


The
Panel
is
requested
to
comment
on
the
need
for
nontarget
hazard
data
development
on
the
combinations
of
Cry
proteins
being
considered
for
registration
when
data
on
the
effects
of
the
individual
Cry
proteins
are
readily
available
and
show
no
adverse
effects.
35
Questions
on
the
Environmental
Effects
Assessment
2)
The
weight
of
evidence
from
the
reviewed
data
indicates
that
there
will
not
be
a
hazard
to
wildlife
from
the
commercialization
of
WideStrike
cotton.
Although
the
Bt
proteins
expressed
by
WideStrike
are
known
to
affect
only
lepidopteran
insect
species,
the
Agency
evaluated
studies
of
potential
effects
on
a
wide
variety
of
non­
target
organisms
that
might
be
exposed
to
the
Cry1F
and
Cry1Ac
protein,
i.
e.,
wild
mammals,
birds,
invertebrates,
and
aquatic
species.
EPA
concluded
that
aquatic
and
terrestrial
wildlife
was
not
likely
to
be
harmed
and
that
WideStrike
cotton
was
not
likely
to
threaten
the
long­
term
survival
of
any
non­
target
wildlife
populations.


The
Panel
is
requested
to
comment
on
the
Agency's
analysis
of
the
currently
available
data
on
the
potential
impacts
of
WideStrike
cotton
on
non­
target
species.
36
Questions
on
the
Environmental
Effects
Assessment
3)
The
Agency
has
sufficient
information
to
conclude
that
there
is
no
hazard
from
the
proposed
uses
of
WideStrike
 
cotton
to
non­
target
wildlife,
aquatic
and
soil
organisms.
However,
the
Agency
is
requesting
additional,
primarily
long
term
effects
data
that
were
recommended
by
previous
Panels
for
PIP
corn.

The
supplementary
studies
would
provide
additional
weight
to
support
the
Agency's
conclusions.


The
Panel
is
asked
to
comment
on:

(
a)
the
scientific
value
of
the
proposed
additional
studies
that
are
identified
at
the
end
of
the
Environmental
Assessment
section,
including
avian
chronic
exposure
testing
and
multi­
year
field
and
soil
persistence/
terrestrial
expression
studies,
and
(
b)
the
applicability
of
these
data
to
PIP
cotton.
