May
7,
2003
MEMORANDUM
Subject:
Consideration
of
non­
target
ecological
effects
for
Bacillus
thuringiensis
var.
tenebrionis
expressing
the
Cry34/
35Ab1
binary
insect
control
protein
as
expressed
in
corn
as
a
part
of
Pioneer
Hi­
Bred
International,
Inc's
application
for
an
Experimental
Use
Permit
(
EUP);
EPA
Reg.
No.
029964­
EUP­
L.

To:
Mike
Mendelsohn,
Regulatory
Action
Leader
Biopesticides
and
Pollution
Prevention
Division,
7511C
From:
Robyn
Rose,
Entomologist
Biopesticides
and
Pollution
Prevention
Division,
7511C
Thru:
Zigfridas
Vaituzis,
Ph.
D.,
Microbiologist
Biopesticides
and
Pollution
Prevention
Division,
7511C
Background
Pioneer
Hi­
Bred
International
Inc.
has
requested
an
experimental
use
permit
(
EUP)
for
the
plant
incorporated
protectant
(
PIP)
Bacillus
thuringiensis
var.
tenebrionis
expressing
the
Cry34/
35Ab1
Construct
PHP17662
in
field
corn
(
hereafter
referred
to
as
Cry34/
35)
to
be
planted
from
March
2003
to
March
2004.
According
to
Pioneer's
EUP
request,
623.907
acres
of
Cry34/
35
corn
will
be
planted
among
19
states
in
a
total
of
97
locations
with
no
more
than
340.25
acres
planted
in
a
state
(
Table
1).

Table
1.
States
and
acreage
of
Cry34/
35
corn
to
be
planted
under
029964­
EUP­
L
State
Total
Acres/
State
Total
Locations/
State
CA
2.875
2
GA
1.700
2
HI
340.250
8
IA
60.386
10
IL
46.776
11
IN
19.880
5
KS
7.336
6
2
Table
1.
Continued
State
Total
Acres/
State
Total
Locations/
State
MI
6.388
3
MN
21.112
7
MO
7.206
3
ND
6.850
3
NE
24.440
5
OH
3.320
6
PA
4.052
3
PR
45.550
6
SD
1.450
3
TN
1.950
2
TX
2.770
5
WI
19.616
7
Total
623.907
97
Several
research
trials
will
be
planted
under
this
EUP
including
a
non­
target
insect
survey.
The
non­
target
insect
trials
will
be
planted
in
five
locations
among
two
states
on
a
total
of
2.576
acres
(
Table
2).
No
more
than
1.25
acres
of
Cry34/
35
corn
will
be
planted
per
location
for
the
nontarget
insect
test.

Table
2.
States
and
acreage
of
Cry34/
35
corn
to
be
planted
for
the
non­
target
insect
trial
State
Number
of
Locations/
State
IA
2
NE
3
Total
5
All
test
plants
will
be
isolated
from
other
parties'
sexually
receptive
corn
in
accordance
with
USDA­
APHIS
Performance
Standards.
Isolation
will
be
achieved
by
planting
Cry34/
35
test
plots
at
least
660
feet
from
other
parties'
sexually
receptive
corn,
bagging
or
removing
tassels
prior
to
pollen­
shed,
or
temporal
isolation.

Studies
Submitted
The
non­
target
organism
studies
submitted
in
support
of
a
Section
3
registration
are
listed
in
Table
3.
These
studies
were
not
reviewed
for
the
EUP,
but
will
be
reviewed
prior
to
granted
a
full
commercial
registration.
3
Table
3.
Non­
target
ecological
effects
studies
submitted
for
Section
3
registration
Title
MRID
PS149B1
Binary
Insecticidal
Crystal
Protein:
An
8­
Day
Dietary
Study
with
the
Rainbow
Trout,
Oncorhynchus
mykiss,
Walbaum
457904­
03
PS149B1
Binary
Insecticidal
Crystal
Protein:
An
Acute
Toxicity
Study
with
the
Daphnid,
Daphnia
magna
Straus
457904­
04
PS149B1
Binary
Insecticidal
Crystal
Protein:
Dietary
Toxicity
to
Parasitic
Hymenoptera
(
Nasonia
vitripennis)
457904­
05
Assessment
of
Chronic
Toxicity
of
Diet
Containing
Bacillus
thuringiensis
PS149B1
Insecticidal
Crystal
Protein
to
Collembola
(
Folsomia
candida)
457904­
06
PS149B1
Insecticidal
Crystal
Protein:
Dietary
Toxicity
of
Green
Lacewing
larvae
(
Chrysoperla
carnea)
457904­
07
Degradation
of
Microbial
Lbinary
PS149B1
Delta­
Endotoxin
in
a
Representative
Soil
from
the
Mid­
Western
USA
Maize­
Growoing
Region
452422­
14
A
lady
beetle
dietary
toxicity
study
(
MRID
45242210),
and
tritrophic
interaction
study
of
PS149B1
corn,
lady
beetles
and
corn
leaf
aphids
(
MRID
45242211)
were
submitted
to
the
Agency
and
reviewed
in
a
memorandum
from
Robyn
Rose
to
Mike
Mendelsohn
May
15,
2001.
The
May
15,
2001
review
also
includes
a
non­
target
species
hazard
assessment.

Conclusions
Based
on
the
limited
total
acreage,
small
individual
plots
and
isolation
requirements,
adverse
effects
to
non­
target
invertebrate
and
vertebrate
organisms
are
not
expected
from
planting
Cry34/
35
corn
under
this
EUP.
However,
additional
studies
to
those
listed
in
Table
1
should
be
conducted
and
submitted
to
the
Agency
for
review
prior
to
a
full
commercial
registration.

Avian
A
six
week
chicken
broiler
study
with
the
chickens
fed
starter
diets
to
day
17,
grower
diets
to
day
31
and
finisher
diets
to
day
42
(
50­
60
%
transgenic
corn)
should
be
conducted
and
submitted
for
EPA
to
review.
A
control
study
with
non­
transgenic
corn
in
the
standard
diets
should
be
run
concurrently.

Mammalian
Wildlife
Acute
oral
toxicity
(
MRID
Nos.
452422­
07,
452422­
08,
452422­
09),
in
vitro
digestibility
(
MRID
No.
452422­
12),
in
vitro
simulated
gastric
fluid
digestibility
(
MRID
No.
455845­
02)
and
a
SDSPAGE
sensitivity
analysis
in
support
of
the
simulated
gastric
fluid
digestion
study
(
MRID
No.
457904­
08)
studies
have
been
submitted
to
the
Agency
in
support
of
registering
Cry34/
35
field
corn.
These
submissions
will
be
considered
as
part
of
the
risk
assessment
for
mammalian
wildlife
toxicity.
4
Non­
target
Plants
Since
the
active
ingredient
in
this
product
is
an
insect
toxin
that
has
never
shown
any
toxicity
to
plants,
these
studies
have
been
waived
for
this
product.
In
addition,
there
is
no
evidence
of
transfer
of
any
gene,
including
introduced
genes,
to
organisms
other
than
those
with
which
corn
is
sexually
compatible.
In
addition,
there
are
no
weedy
relatives
of
corn
in
the
U.
S.

Non­
target
invertebrates
Honey
Bees:
Honey
bee
larval
acute
toxicity
studies
should
be
conducted
and
submitted
to
the
Agency
for
review
prior
to
a
full
commercial
registration.
This
study
should
be
conducted
through
adult
emergence.

Lady
Beetles:
Lady
beetle
studies
were
submitted
(
MRID
Nos.
452422­
10
and
452422­
11)
and
reviewed
in
the
May
15,
2001
memo.
Although
the
adult
lady
beetle
studies
are
acceptable,
the
lady
beetle
larvae
acute
toxicity
test
should
be
repeated
with
purified
protein.
The
test
previously
submitted
to
the
Agency
included
feeding
aphids
that
had
fed
on
Cry34/
35
corn
to
lady
beetles.
However,
it
has
been
shown
that
aphids
do
not
ingest
Bt
protein
from
plants
because
they
are
phloem
feeders
and
the
Bt
protein
is
not
expressed
in
the
phloem.
Therefore,
lady
beetle
larvae
were
not
exposed
to
Bt
protein
in
the
submitted
study
(
MRID
No.
452422­
11).
Since
Cry35
is
not
expressed
in
pollen
of
Cry34/
35
field
corn,
lady
beetle
larvae
tests
should
be
conducted
with
purified
protein.
According
to
the
August
2002
FIFRA
Scientific
Advisory
Panel
(
SAP),
Coleomegilla
maculata
is
the
most
likely
lady
beetle
to
be
affected
by
coleopteran­
active
Bt
proteins.
Therefore,
Tier
1
lady
beetle
dietary
toxicity
studies
should
be
conducted
with
C.
maculata.

Green
Lacewing:
The
Tier
1
green
lacewing
dietary
toxicity
study
was
conducted
by
feeding
lacewing
larvae
moth
egg
diet
coated
with
Bt
Cry34/
35
protein
(
MRID
No.
457904­
07).
However,
the
August
2002
SAP
concluded
that
this
protocol
is
an
inappropriate
and/
or
inadequate
method
to
test
the
potential
impact
of
Cry
proteins
on
the
green
lacewing.
The
SAP
also
recommended,
and
the
Agency
agrees,
that
the
minute
pirate
bug
(
Orius
insidiosus)
is
a
more
appropriate
test
organism
than
the
green
lacewing.
Therefore,
a
Tier
1
dietary
toxicity
test
should
be
conducted
with
the
minute
pirate
bug
prior
to
full
commercial
registration
of
Cry34/
35.

Parasitic
Hymenoptera:
The
August
2002
SAP
stated
that
"[
t]
he
relevancy
of
the
hymenopteran
parasitoid
Nasonia
vitripennis
is
questionable
because
it
is
a
gregarious
pupal
endoparasitoid
of
dipteran
hosts
and
it
rarely,
if
ever,
occurs
in
corn.
A
different
species
of
hymenopteran
might
have
been
appropriate."
Since
the
parasitic
Hymenoptera
Tier
1
dietary
toxicity
study
submitted
to
the
Agency
for
review
utilized
Nasonia
vitripennis
(
MRID
No.
457904­
05),
and
additional
non­
target
insect
study
should
be
conducted
prior
to
full
commercial
registration.
A
more
appropriate
hymenopteran
should
be
considered
such
as
Macrocentrus
grandii
or
Trichogramma
sp.
In
lieu
of,
or
in
addition
to,
conducting
an
additional
Tier
1
dietary
toxicity
study
with
an
additional
parasitic
Hymenoptera,
a
test
could
be
conducted
with
a
carabid
ground
beetle.
A
5
carabid
would
be
an
appropriate
indicator
organism
for
a
coleopteran­
active
Bt
corn
such
as
Cry34/
35.

Field
Studies:
In
addition
to
conducting
additional
Tier
1
laboratory
tests
with
appropriate
indicator
organisms
such
as
Orius
insidiosus,
Macrocentrus
grandii,
Trichogramma
sp.
and/
or
a
Carabid
(
e.
g.,
Pterostichus
sp.),
field
studies
should
be
conducted
that
evaluate
the
potential
adverse
effects
of
growing
Cry34/
35
corn
at
a
commercial
scale.
Field
studies
should
evaluate
potential
effects
at
varying
trophic
levels
(
e.
g.,
plant
feeders,
predators,
parasitoids,
decomposers).
In
addition,
an
appropriate
non­
target
coleopteran
(
e.
g.,
Tetraopes
milkweed
beetle)
that
may
be
unintentionally
exposed
to
Cry34/
35
should
be
evaluated.

Earthworm
and
Collembola
Although
an
earthworm
study
is
not
required
for
full
commercial
registration,
the
Agency
recognizes
their
importance
as
decomposers
living
in
the
soil.
Therefore,
conducting
an
earthworm
dietary
toxicity
study
is
recommended
prior
to
commercialization
of
Cry34/
35.
A
collembola
study
has
been
submitted
to
the
Agency
for
review
(
MRID
No.
457904­
06).

Fish
The
requirement
for
a
static
renewal
toxicity
study
on
freshwater
fish
is
normally
waived
based
on
a
lack
of
exposure
of
fish
to
Bt
proteins
produced
in
corn.
Exposure
from
corn
pollen,
if
it
does
take
place,
will
be
of
a
very
short
duration
and
quantity
and
is
not
expected
to
have
any
detectable
effect
on
freshwater
fish.
An
8­
Day
Dietary
Study
with
the
Rainbow
Trout
(
Oncorhynchus
mykiss)
has
been
submitted
to
the
Agency
for
review
(
MRID
No.
457904­
03)

Aquatic
Invertebrates
Published
and
in­
house
data
for
Bt
crystal
protein
containing
corn
show
that
Bt
crystal
proteins
have
no
measurable
deleterious
effects
on
Daphnia
magna,
a
sensitive
aquatic
invertebrate
species.
An
Acute
Toxicity
Study
with
the
Cledoceran
Daphnia
magna
has
been
submitted
tot
he
Agency
for
review
(
MRID
No.
457904­
04).

Estuarine
and
Marine
Animals
The
estuarine
fish
and
shrimp
studies
are
not
required
for
this
product
because
of
very
low
potential
for
exposure.

Endangered
Species
Based
on
the
previously
submitted
Cry
protein
toxicity
and
exposure
data
there
will
not
be
a
"
may
effect"
situation
for
endangered
mammals,
birds,
plants
and
aquatic
species.
All
endangered/
threatened
insect
species
that
are
susceptible
to
Cry34/
35
(
e.
g.,
coleopterans)
may
be
6
adversely
affected
if
exposed.
In
the
case
of
Cry34/
35
protein,
the
test
plots
for
the
proposed
EUP
program
must
not
be
near
the
habitats
of
endangered
beetle
(
coleopteran)
species
since
these
may
be
adversely
affected
if
exposed
to
the
Bt
protein
in
the
soil
or
through
pollen
consumption.
An
assessment
of
potential
impacts
on
endangered
species
should
be
conducted
and
submitted
to
the
Agency
for
review
prior
to
full
commercial
registration.

Soil
Fate
The
potential
accumulation
and/
or
persistence
of
Cry34/
35
in
soil
should
be
evaluated
prior
to
full
commercial
registration.
This
should
be
done
by
determining
the
DT
50
or
time
to
50%
degradation
of
Cry34/
35
protein
in
varying
soil
types.
In
particular
the
DT
50
in
a
"
worst
case
scenario"
should
be
determined.
Since
Cry
proteins
have
been
shown
to
readily
bind
to
clay
soils
and
persist
longer
at
low
pH,
the
DT
50
of
Cry34/
35
should
be
determined
in
soils
with
a
high
clay
content.
Studies
should
include
insect
bioassays
with
a
sensitive
species
such
as
the
Colorado
potato
beetle
as
well
as
an
ELISA
for
comparative
purposes.

Additional
field
studies
should
be
conducted
after
full
commercial
registration.
Field
studies
should
be
conducted
in
a
variety
of
soil
types
and
should
utilize
fields
that
have
had
Cry34/
35
corn
grown
for
three
consecutive
years.
Persistence
of
Cry34/
35
in
the
soil
should
be
monitored
close
to
the
soil
surface
(
e.
g.,
12
­
18
inches
deep)
as
well
as
deep
in
the
soil
(
e.
g.,
several
feet
below
surface
level)
where
microbial
activity
is
reduced.
Sampling
should
occur
immediately
after
anthesis
as
well
as
post­
harvest
and
should
continue
until
no
Bt
protein
is
detected.
