Document ID: EPA-HQ-OPP-2004-0182-0005
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-10-11T04:00Z

Section
3.0
3
­
1
3
­
Environmental
Assessment
3
­
Comment
Excerpts
Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509B­
084000
Excerpt
Number:
2
Excerpt
Text:
In
many
respects,
the
potential
for
biodiversity
damage
that
may
ensue
from
a
corn
varietal
that
secretes
Bt
toxins
in
its
roots­
seems
even
greater
to
me
.......
affecting
the
biological
integrity
of
the
soil
ecosystem
in
some
of
the
richest
and
most
biodiverse
soils
on
this
planet.
Gene
stacking
seems
particularly
troublesome
and
fraught
with
long
term
toxicological
uncertainty.

EPA
Response:
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
been
evaluated.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
As
expected,
longer
degradation
times
occur
in
corn
tissues
due
to
the
time
required
for
the
tissue
to
decay
and
for
the
Cry3Bb1
protein
to
move
from
tissue
to
soil.
Limited
data
do
not
indicate
that
Cry
proteins
have
any
measurable
effect
on
microbial
populations
in
the
soil
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5).
Horizontal
transfer
from
transgenic
plants
to
soil
bacteria
has
not
been
demonstrated.
Published
studies
of
Bt
Cry
protein
in
soil
show
no
effect
on
bacteria,
actinomyces,
fungi,
protozoa,
algae,
nematodes,
springtails
or
earthworms
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
In
addition,
new
plants
planted
in
Bt
Cry
protein
containing
soil
do
not
take
up
the
Bt
protein
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6).
However,
because
the
Agency
believes
that
additional
studies
would
be
useful
in
completing
the
database
for
long
term
effects
assessment,
it
is
requiring
additional
supplementary
multi­
year
studies
regarding
Cry3Bb1
protein
degradation
and
persistence
in
various
agricultural
soils
after
tree
year
commercial
planting
.

Commenter
Name:
Berg,
Mark
Commenter
Organization
Name:
Foundation
E.
A.
R.
T.
H.
Comment
Number:
30509­
078000
Excerpt
Number:
6
Excerpt
Text:

­
It
is
apparent
that
these
producers
have
concerns
with
usage
of
insecticides.
While
12%
have
personally
become
ill
due
to
exposure
to
insecticides,
20%
claim
that
a
family
member
or
other
associate
has
such
an
experience.

­
By
indicating
either
a
8,
9,
or
10,
exposure
to
the
insecticide
when
filling
insecticide
boxes
(
76%)
and
exposure
of
other
family
members
to
insecticides
(
72%)
surfaced
as
the
primary
concerns
of
growers
with
current
soil
applied
insecticides
­
as
they
are
well
aware
of
the
potential
health
hazards
associated
with
Section
3.0
3
­
2
these
chemicals.
About
half
are
extremely
concerned
about
the
impact
of
insecticide
on
the
environment
(
53%)
and
exposure
to
livestock
(
47%).

EPA
Response:
The
MON
863
corn
will
alleviate
the
concerns
expressed
by
this
comment.
EPA
has
considered
the
potential
for
reductions
in
use
of
conventional
pesticides
in
its
Benefits
Assessment
of
the
Cry
3Bb1
registration
application.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
9
Excerpt
Text:
Introduction
of
Cry
3Bb
corn
hybrids
may
lead
to
impacts
on
soil
health
and
productivity.
If
approved,
Monsanto's
Cry3Bb
corn
could
be
adopted
widely
within
a
few
years
on
millions
of
acres
(
Gray,
2000).
It
will
deliver
into
the
root
zone
concentrations
of
Cry3Bb
toxins
that
are
orders
of
magnitude
above
what
occurs
naturally,
possibly
triggering
profound
effects
on
soils,
soil
food
webs,
and
soil
borne
pest
dynamics.

EPA
Response:
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
also
been
evaluated.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
Limited
data
do
not
indicate
that
Cry
proteins
have
any
measurable
effect
on
microbial
populations
in
the
soil
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5).
Horizontal
transfer
from
transgenic
plants
to
soil
bacteria
has
not
been
demonstrated.
Published
studies
of
Bt
Cry
protein
in
soil
show
no
effect
on
bacteria,
actinomyces,
fungi,
protozoa,
algae,
nematodes,
springtails
or
earthworms
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
In
addition,
new
plants
planted
in
Bt
Cry
protein
containing
soil
do
not
take
up
the
Bt
protein
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6).

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
16
Excerpt
Text:
Environmental
Fate
and
Ecosystem
Impacts­
Cry3Bbl
protein
dissipates
rapidly
in
soil
with
90%
degraded
in
7.9
to
9.2
days.

­
Cry3Bb1
protein
is
not
likely
to
be
secreted
into
the
corn
root
zone
and
does
not
accumulate
in
the
soil
environment.
­
No
adverse
effects
were
observed
with
Cry3Bbl
protein
under
maximum
expected
environmental
exposures
for
nontarget
organisms.
Section
3.0
3
­
3
­
The
levels
of
Cry3Bbl
protein
in
various
plant
tissues
of
MON
863
were
not
found
to
cause
any
adverse
effects
in
animal
and
nontarget
organism
feeding
studies.
­
The
maximum
worst­
case
exposure
to
earthworms
is
at
least
four­
fold
lower
than
a
level
that
is
known
not
to
cause
adverse
effects.

­
Potential
tritrophic
impacts
on
predatory
species
have
been
assessed
and
there
is
minimal
risk
to
insect
predators,
parasitoids
or
other
animals.

­
Beneficial
insect
populations
have
been
shown
to
be
higher
in
Bt
crop
fields
(
i.
e.
corn
and
cotton)
compared
to
fields
treated
with
conventional
broad
spectrum
insecticides.

EPA
Response:
The
data
received
by
EPA
to
date
show
that
the
above
comments
appear
to
be
accurate.
Additional
and
long
range
confirmatory
studies
are
in
progress
and
are
expected
to
be
completed
prior
to
expiration
of
the
current
registration.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
12
Excerpt
Text:
Environmental
Fate
and
Ecosystem
Impacts
­
Laboratory
and
field
studies
consistently
show
no
adverse
effects
on
ecologically
and
economically
important
nontarget
species
at
or
above
the
maximum
expected
environmental
concentration
to
which
the
organisms
would
be
exposed.

­
Bt
products
have
a
40­
year
history
of
safe
use
in
traditional
and
organic
agricultural
systems
and
the
Cry3
class
of
Bt
proteins
has
been
used
during
the
past
decade.

­
The
Cry3Bbl
protein
is
less
toxic
to
nontarget
organisms
than
all
organophosphate
and
pyrethroid
currently
insecticides
registered
and
commonly
used
to
control
corn
pests,
including
CRW.

­
Studies
submitted
by
Monsanto
followed
EPA
protocols
and
provided
sufficient
information
to
establish
the
safety
of
MON
863
for
nontarget
organisms,
including
the
adult
honey
bee,
wasp,
earthworm
and
C
maculata.

­
Field
data
validate
the
results
of
the
maximum
hazard
dose
laboratory
studies
and
demonstrate
that
MON
863
poses
a
minimal
risk
to
nontarget
organisms,
including
beetle
families
commonly
found
in
agricultural
settings.

­
Extensive
laboratory
and
field
tests
demonstrate
that
only
beetles
in
the
family
Chrysomelidae
are
susceptible
to
the
Cry3Bb1
protein.

­
A
preliminary
observation
that
MON
863
may
reduce
pest
nematode
populations,
if
confirmed,
would
Section
3.0
3
­
4
represent
an
unexpected
benefit
rather
than
an
unintended
ecological
risk.

­
Cry3Bb1
protein
dissipates
rapidly
in
soil
with
90%
degraded
in
7.9
to
9.2
days.

­
The
maximum
worst­
case
exposure
to
earthworms
is
at
least
four­
times
lower
than
a
level
that
is
known
not
to
cause
adverse
effects.

­
Some
variation
among
individual
plants
in
a
composite
sample
would
be
expected,
and
in
extreme
cases
where
actual
exposure
exceeds
the
maximum
expected
environmental
exposure
concentration,
then
the
uncertainty
factor
used
in
the
risk
assessment
(
i.
e..
10­
100X)
will
accommodate
these
excursions.

EPA
Response:
The
data
received
by
EPA
to
date
show
that
the
above
comments
appear
to
be
accurate.
EPA
has
considered
the
potential
for
reductions
in
use
of
conventional
pesticides
in
its
Benefits
Assessment
of
the
Cry
3Bb1
registration
application.

Commenter
Name:
Lance
Meinke
Commenter
Organization
Name:
NCR­
46
Memebers
Comment
Number:
OPP02­
0016
Excerpt
Number:
10
Excerpt
Text:
­
The
ECB
has
a
broader
host
range
than
the
predominant
corn
rootworm
pest
species.
Other
than
corn,
only
a
few
weedy
grasses
serve
as
hosts
for
the
western,
Mexican,
and
northern
corn
rootworms.
The
contribution
of
rootworm
adults
from
these
alternate
hosts
that
provide
a
refuge
from
toxins
is
unknown.

EPA
Response:
This
comment
on
the
comparative
plant
host
range
of
the
ECB
and
the
CRW
is
duly
noted
and
addressed
by
the
EPA
in
its
Insect
Resistance
Management
section
of
the
Cry
3Bb1
registration
application.
Section
3.1
3
­
5
3.1
­
Non­
target
Organism
Effects
3.1
­
Comment
Excerpts
Commenter
Name:
Gerald
Wilde
Commenter
Organization
Name:
Kansas
State
University
­
Department
of
Entomology
Comment
Number:
30509­
055000
Excerpt
Number:
1
Excerpt
Text:
Having
been
involved
in
the
testing
of
their
product.
I
have
seen
first
hand
the
remarkable
level
of
western
corn
rootworm
control
this
product
can
deliver.
At
the
same
time
I
am
aware
of
numerous
studies
which
have
been
conducted
to
deal
with
the
insect
resistance
management
aspect
of
this
product
and
the
studies
to
determine
any
possible
adverse
effects
on
non­
target
organisms.
These
studies
and
others
lead
me
to
believe
that
this
transgenic
rootworm
protected
corn
product
should
be
registered
as
it
will
be
a
valuable
pest
management
tool
for
the
corn
producers
of
the
U.
S.
and
other
parts
of
the
world.

EPA
Response:
This
comment
is
consistent
with
EPA's
conclusions.

Commenter
Name:
Caroline
Cox
Commenter
Organization
Name:
The
Northwest
Coalition
for
Alternatives
to
Pesticides
Comment
Number:
30509­
103000
Excerpt
Number:
2
Excerpt
Text:
NCAP
is
particularly
concerned
about
the
levels
of
the
Bt
Cry3Bb
proteins
that
will
be
expressed
in
Cry3Bb
corn.
The
application
refers
to
the
expression
levels
of
the
Cry
3Bb.
Protein
as
moderate
to
low."
(
MRID
#
451845­
01,
page
5).
This
characterization
of
"
moderate
to
low"
is
made
relative
to
the
ability
of
Cry3Bb
endotoxins
at
the
levels
expressed
in
plant
roots,
to
control
corn
rootworm
larvae,
the
principal
target
pest
at
its
most
vulnerable
stage.
But
from
the
perspective
of
likely
environmental
impacts,
the
expression
levels
are
remarkably
high.
Relatively
high
levels
exist
in
plant
leaf
tissue,
pollen
and
grain,
where
they
are
not
needed
and
can
do
much
harm.
Potential
impacts
on
nontarget
organisms,
already
identified
when
monarch
butterflies
and
green
lacewings
are
exposed
to
Bt
proteins,
thus
are
serious
concerns
that
need
to
be
evaluated
carefully.

EPA
Response:
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
also
been
evaluated.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
Limited
data
do
not
indicate
that
Cry
proteins
have
any
measurable
effect
on
microbial
populations
in
the
soil
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5).
Horizontal
transfer
from
transgenic
plants
to
soil
bacteria
has
not
been
demonstrated.
Published
studies
of
Bt
Cry
protein
in
soil
show
no
effect
on
bacteria,
actinomyces,
fungi,
protozoa,
algae,
nematodes,
springtails
or
earthworms
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
In
addition,
Section
3.1
3
­
6
new
plants
planted
in
Bt
Cry
protein
containing
soil
do
not
take
up
the
Bt
protein
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6).
Two
season
field
studies
provide
further
evidence
that
the
Cry3Bb1
protein
in
corn
event
MON
863
produces
no
short
term
risk
of
unreasonable
adverse
effects
for
the
environment
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b).
Because
the
Agency
believes
that
additional
studies
would
be
useful
in
completing
the
database
for
long
term
effects
assessment,
it
is
requiring
additional
supplementary
studies
regarding
Cry3Bb1
protein
degradation
and
persistence
in
various
agricultural
soils
after
three
year
commercial
planting.
The
EPA
green
lacewing
and
monarch
butterfly
hazard
assessment
shows
that
the
effect
on
these
species
in
the
field
is
negligible
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B.
3.
a.
viii).

Commenter
Name:
Thomas
O'Connor
Commenter
Organization
Name:
National
Grain
and
Feed
Association
Comment
Number:
30509­
112000
Excerpt
Number:
2
Excerpt
Text:
It
is
our
understanding
that
Monsanto
intends
to
plant
the
new
product
on
a
limited
number
of
acres
for
pre­
commercial
inbred
seed
propagation
and
hybrid
seed
production
registration.
While
the
amount
of
total
acreage
planted
to
the
corn
under
the
requested
registration
is
relatively
minor,
the
acreage
is
being
planted
in
a
wide
range
of
states.
For
this
reason,
we
would
raise
the
following
issues
with
which
we
believe
regulators
should
have
some
concern:

­
Is
the
planned
buffer
strip
between
these
crops
and
commercial
corn
crops
adequate
to
prevent
pollen
drift
from
the
fields
planted
with
the
new
event
from
inadvertently
mixing
with
commercial
crops?
The
SAP
for
StarLink
corn
raised
concerns
that
the
660­
foot
buffer
foot
zone
may
not
be
adequate.

­
Is
there
any
plan
to
notify
neighboring
farms
adjacent
to
these
fields,
so
that,
if
necessary,
proper
precautions
can
be
taken?
Is
there
any
plan
to
notify
first
buyers
in
the
marketing
chain
that
crops,
no
yet
intended
for
commercial
use
in
food
and
feed,
are
being
planted
in
the
area?
Should
there
be?
Notification
may
be
of
particular
interest
in
areas
that
export
bulk
corn
to
foreign
markets
that
may
not
have
approved
the
event
for
commercial
use.

EPA
Response:
EPA's
response
to
gene
flow
to
conventional
crops
is
addressed
in
the
gene
flow
issues
section
of
this
document.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
4
Excerpt
Text:
The
expression
levels
in
the
current
commercial
Bt
crop
varieties
are
considered
'
high
dose',
i.
e.
a
multifold
concentration
necessary
to
kill
99%
of
the
susceptible
target
pest
species
is
expressed.
Thus,
'
high
dose'
is
very
narrowly
defined
in
terms
of
lethal
effects
exerted
on
the
target
pest
species.
The
target
pests
of
the
transgenic
Cry3Bb
corn
are
CRW
larvae
feeding
on
the
roots
of
corn.
Although
mean
concentration
levels
of
41
mg/
g
fresh
weight
of
root
tissue
translate
into
a
large
amount
of
toxin
compared
to
other
Bt­
Section
3.1
3
­
7
producing
plants,
it
is
below
the
reported
LC50
of
75
ppm
according
to
the
Monsanto
documentation
i.
e.
it
is
not
expected
to
kill
50%
of
the
CRW.
Therefore,
Monsanto
Company
describes
it
as
a
low
to
moderate
expressing
plant.
Cry3Bb
concentration
levels
in
leaves
and
pollen
are
substantially
higher,
2
and
1.5
times
of
the
concentration
achieved
in
roots,
respectively.
In
an
environmental
risk
assessment
context,
such
large
amounts
of
toxin
call
for
rigorous
testing
of
nontarget
effects,
including
also
pollen­
feeding
nontarget
insects,
such
as
honey
bees,
certain
predator
species,
in
particular
coleopteran
species
like
Coleomegilla
maculata,
known
to
feed
extensively
on
corn
pollen,
but
also
non­
coleopteran
species
like
syrphids
and
butterflies,
e.
g.
monarchs
and
swallowtails.
It
is
not
advisable
to
simply
rely
upon
the
described
specificity
of
Bt
proteins
published
in
the
literature.
The
employed
protocols
and
narrow
definitions
of
'
susceptibility'
are
typically
designed
to
screen
for
efficacy
of
Bt
toxins
in
herbivorous
pest
species,
i.
e.
short
term
bioassays
aimed
at
detecting
and
quantifying
lethal
effects
often
with
an
underlying
interest
to
identify
their
potential
economic
utility
for
pest
management
(
MacIntosh
et
al.
1990,
Moar
et
al.
1995).
After
all,
whether
or
not
Bt­
induced
effects
on
nontarget
organisms
will
occur
depends
aside
of
presence
and
density
of
receptors
and
gut
enzyme
composition
also
on
dose,
i.
e.
the
plain
amount
of
toxin
an
organism
ingests.

EPA
Response:
The
above
concerns
were
addressed
by
the
maximum
hazard
dose
(
limit
dose)
toxicity
testing
performed
on
representative
beneficials
from
several
taxa
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B).
Testing
at
the
maximum
hazard
dose
level
takes
into
account
the
variability
in
Cry
Bb1
protein
levels
in
Bt
corn
fields.
The
toxicity
of
the
Cry3Bb1
protein
has
been
evaluated
following
challenge
of
several
species
of
invertebrates
including:
adult
and
larval
honey
bees,
a
parasitic
hymenopteran
(
Nasonia),
green
lacewings,
lady
beetles,
collembola,
monarch
butterfly,
and
earthworms
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B).
Reproductive
and
developmental
observations
were
also
made
on
collembola,
honeybee
and
lady
beetle
larva
maturation
studies
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
3.
a.
iv
and
C.
I.
B).
Insect
host
range
bioassay
and
field
survey
data
were
used
to
assess
the
hazard
to
additional
species
not
directly
tested
in
the
laboratory.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
9
Excerpt
Text:
Nontarget
safety
assessment
Much
of
the
ecotoxicology
data
with
nontarget
organsims
followed,
like
previous
such
regulatory
trials,
an
approach
that
has
been
developed
for
the
side­
effect
testing
of
synthetic
pesticides.
In
our
previous
EcoStrat
Report
on
'
Nontarget
Organisms
and
Bt
Plants'
(
2000)
we
discuss
the
short
comings
of
this
approach
in
detail
and
would
like
to
refer
to
that
part
in
that
report
and
present
only
a
brief
summary
here:

"
These
testing
procedures
are
designed
for
pesticides
and
their
mode
of
release
­
e.
g.
foliar
application
potentially
causing
drift
into
neighboring
habitats,
presence
for
a
limited
time
period
due
to
degradation,
etc.
­
but
these
tests
alone
are
insufficient
for
assessing
ecological
effects
of
transgenic,
insecticidal
plants
on
nontarget
organisms.
By
making
a
plant
produce
an
extra
and
novel
protein
(
e.
g.
Bt)
important
characteristics
of
Bt's
mode
of
release
are
changed:
the
temporal
and
spatial
presence
of
the
insecticidal
toxin
is
extended
due
to
constitutive
expression
and,
thereby,
the
mode
of
exposure
to
herbivorous
insects
and,
consequently,
to
predators
and
parasitoids
is
altered.
Furthermore,
the
altered
plant's
metabolism
(
by
Section
3.1
3
­
8
virtue
of
producing
a
novel
protein)
may
in
itself
affect
interactions
between
the
plants,
their
herbivores
or
pathogens
and
organisms
of
higher
trophic
levels.
Therefore,
we
consider
it
necessary
to
adapt
and
modify
the
testing
procedures
for
transgenic
plants
by
including
more
parameters
than
only
for
acute
toxicity
testing,
such
as
testing
protocols
for
chronic
lethal
and
sublethal
toxicity."
(
EcoStrat
Report
2000,
'
Summary'
and
'
2.1.
General
comments
on
ecotoxicological
studies')

EPA
Response:
EPA
has
taken
the
approach
outlined
in
this
comment.
The
non­
target
insect
testing
has
been
expanded
to
include
species
more
likely
to
be
affected
by
the
particular
active
ingredient
and
testing
of
species
more
likely
to
be
encountered
in
the
particular
agroecosystem
in
question.
Protocols
for
laboratory
testing
of
several
coleopteran
species
are
being
developed.
In
addition,
extensive
field
testing
is
being
performed
to
assess
the
abundance
of
the
fauna
and
flora.
Also,
monitoring
of
general
ecosystem
effects
is
being
required
during
the
duration
of
the
registration.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
14
Excerpt
Text:
Resistance
management
The
target
pest
CRW
has
an
outstanding
track
record
of
adaptation
to
various
management
practises
applied
for
its
control
including
one
that
was
or
still
is
considered
to
be
difficult
to
break,
i.
e.
crop
rotation.
By
current
definition
with
respect
to
the
target
pest
(
see
above
for
discussion
of
that
issue),
the
Cry3Bb
corn
expresses
a
low
to
moderate
dose
of
Bt
toxin
and
therefore
does
not
comply
with
the
requirements
for
current
resistance
management
strategies
designed
for
the
use
of
'
high
dose'
plants.
To
our
knowledge,
no
effective
low
dose
expression
resistance
management
plans
have
been
developed
or
tested
with
transgenic
Bt
plants
yet.
However,
it
might
be
conceivable
that
the
lower
impact
on
the
target
pest
CRW
may
translate
into
lower
selection
pressure.
Therefore,
these
plants
may
in
fact
present
a
good
chance
to
test
whether
a
low
dose
approach
for
pest
management
via
transgenic
plant
technology
may
not
in
fact
be
superior
approach
compared
to
high
dose.
The
'
high
dose'
strategy
accepts
an
'
overkill'
from
a
pest
management
standpoint
and
resembles
more
a
prophylactic
blanket
coverage
of
a
pesticide.
However,
the
question
arising
with
these
plants
is
to
what
degree
the
'
low
dose'
for
CRW
also
is
a
low
dose
other
nontarget
organisms
since
in
terms
of
quantity
much
toxin
is
produced
than
in
any
previous
Bt
crop
plants.
In
any
regard,
these
approaches
need
first
to
be
explored
in
carefully
designed,
smaller
scale
field
trials
and
laboratory
trials
before
large
scale
commercialization
is
advisable.

EPA
Response:
The
above
mentioned
low
dose
issue
is
addressed
in
detail
in
EPA's
Insect
Resistance
Management
section
of
this
document.
The
low­
dose
effects
on
non­
target
organisms
is
also
indirectly
assessed
during
the
field
surveys
being
performed
to
observe
the
abundance
of
invertebrate
fauna.
Also,
monitoring
of
any
effects
on
the
ecosystem
in
general
is
being
required
during
the
duration
of
the
registration.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Section
3.1
3
­
9
Excerpt
Number:
20
Excerpt
Text:
The
massive
input
of
highly
bioactive
toxic
proteins
like
those
of
Bacillus
thuringiensis
(
Bt)
into
the
soil
ecosystem
due
to
commercial
production
of
fransgenic
Bt
crop
plants
can
potentially
exert
effects
on
all
levels
of
the
soil
ecosystem
via
direct
or
indirect
exposure
of
soil
macro­
and
micro­
organisms
to
the
Bt
toxins.
The
fact
that
Bt
is
a
soil
bacteria
does
not
preclude
effects
on
ecosystem
components
and
food
web
organisms
when
the
Bt
proteins
are
released
in
such
enormous
quantities
and
in
a
more
activated
form
that
were/
was
not
experienced
before.
Specificity
of
Bt
proteins
has
largely
been
determined
for
plant
herbivores
and
in
terms
of
lethal
effects.
With
the
production
of
vast
acreages
of
Bt
crop
plants
expressing
the
Bt
toxins
constitutively
(
i.
e.,
throughout
the
season)
and
in
high
concentration,
sublethal
longterm
effects
on
soil
inhabiting
macro­
and
micro
organisms
become
important,
in
particular,
with
respect
to
their
function
in
recycling
of
nutrients
and
soil
ecosystem
health,
i.
e.,
the
intricate
coupling
of
these
processes
and
the
balance
between
beneficial
and
detrimental
organisms.
From
the
overuse
of
insecticides,
the
detrimental
consequences
of
uncoupling
these
processes
on
soil
health
and
soil
fertility
are
well­
known
and
include
a
decline
in
soil
fertility,
disease­
suppression
capacity
and
the
subsequent
buildup
of
soil­
borne
plant
diseases
and
plant­
feeding
pest
organisms.

EPA
Response:
Unlike
the
effects
on
soil
fertility
by
conventional
pesticides,
Bt
Cry
proteins
which
are
biodegradable
act
as
additional
nutrients
for
soil
microbes
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
4).
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
also
been
evaluated.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
4).
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
Limited
data
do
not
indicate
that
Cry
proteins
have
any
measurable
effect
on
microbial
populations
in
the
soil
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
5).
Horizontal
transfer
from
transgenic
plants
to
soil
bacteria
has
not
been
demonstrated.
Published
studies
of
Bt
Cry
protein
in
soil
show
no
effect
on
bacteria,
actinomyces,
fungi,
protozoa,
algae,
nematodes,
springtails
or
earthworms
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
In
addition,
new
plants
planted
in
Bt
Cry
protein
containing
soil
do
not
take
up
the
Bt
protein
(
USEPA.
2002.
MON
863
BRAD
Sections
C.
I.
B.
6).
However,
additional
long
term
soil
degradation
and
persistence
studies
in
various
agricultural
soils
after
a
3
year
commercial
planting
are
being
required
by
EPA.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
25
Excerpt
Text:
3)
Effects
on
soil
organisms
The
three
studies
analyzed
(
studies
12,
14,
and
15)
investigated
the
effect
of
different
plant­
expressed
Bt
toxins
on
microorganisms,
protozoa
and
arthropods
(
Appendix
2,
Table
2).
In
study
12,
the
impact
of
three
Bt
cotton
lines
expressing
either
of
two
different
Bt
toxins
(
Cry
lAb
or
CrylAc)
on
soil
microorganisms,
and
protozoa
was
in
by
mixing
leaf
tissues
of
the
different
plant
lines
or
Section
3.1
3
­
10
purified
toxins
in
various
combinations
in
two
soil
types.
Over
a
period
of
28
and
56
days
­
depending
on
the
experiment
­
persistence
and
activity
of
the
toxin
and
effects
on
populations
of
protozoa
and
microorganisms
was
measured.
No
differences
between
the
numbers
of
protozoa
were
found.
However,
changes
in
bacterial
diversity,
and
bacterial
and
fungal
population
levels
were
observed
(
measured
by
species
identification,
substrate
utilization,
and
DNA
fingerprints).
In
a
number
of
experiments,
incorporation
of
transgenic
Bt
cotton
plant
material
into
soil
led
to
significantly
increased
bacterial
and
fungal
population
levels
compared
with
the
parental
lines.
These
changes
were
described
as
'
transient'
because
they
were
observed
only
during
a
limited
period
of
the
entire
experimental
time.
They
disappeared
until
the
end
of
the
experimental
period,
except
in
one
experiment
(
examining
the
transgenic
Bt­
cotton
line
249)
where
fungal
population
levels
were
always
higher
in
the
transgenic
treatment.
But,
except
for
one
treatment
in
one
experiment,
this
phenomena
(
temporary
increase
of
bacterial
and
fungal
activity
in
the
transgenic
treatment)
occurred
consistently
in
all
experiments.
Since
none
of
the
purified
toxins
or
control
plants
showed
similar
effects
on
microbial
populations,
it
is
suggested
that
other,
unintended
characteristics
of
the
transgenic
plants
may
be
involved
in
this.
These
'
transient'
effects
were
observed
after
one
soilincorporation
event
of
Bt
proteins
only.
Further
investigations
need
to
reveal
whether
after
repeated
soilincorporation
of
Bt
proteins,
constant
Bt
protein
exposure
or
accumulating
Bt
protein
concentrations
these
'
transient',
significant
effects
may
turn
into
permanent,
significant
effects.

EPA
Response:
The
issues
raised
by
this
comment
will
be
addressed
by
studies
of
MON
863
Cry
protein
degradation
rates
over
a
three
year
period
in
a
variety
of
MON
863
corn
field
soils
with
and
without
decaying
Bt
corn
vegetation.
These
studies
and
are
in
developmental
stages.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
26
Excerpt
Text:
In
a
field
study
using
Cry3A­
potatoes
(
study
14),
the
leaf
microflora
and
soil­
borne
pathogens
were
investigated.
Three
treatments
were
applied:
transgenic
Bt
potatoes,
a
non
Bt
potato
variety
(
Russet
Burbank;
probably
not
isogenic)
treated
with
microbial
Btt
and
a
non
Bt
potato
variety
(
Russet
Burbank;
probably
not
isogenic)
treated
with
a
systemic
insecticide.
No
untreated
control
for
comparison
was
included,
in
the
experiment
but
only
'
treatments'
were
compared.
For
a
number
of
reasons,
we
considered
the
omission
of
an
untreated
control
in
favor
of
another
treatment
in
an
attempt
to
simulate
'
common
agronomic
practice'
(
i.
e.
insecticide
treatment)
as
scientifically
problematic
because
it
introduces
a
'
normative
control'.
The
particular
choice
of
treatments,
in
this
case
the
choice
of
a
systemic
insecticide
instead
of
an
untreated
control,
will
bias
the
outcome
and
none
of
the
observed
effects
can
be
clearly
attributed
to
an
individual
treatment.
The
authors
concluded
that
Cry3A­
producing
potato
plants
did
not
consistently
affect
the
species
composition
and
density
levels
of
microorganisms
associated
with
plant
leaves
or
the
incidence
of
plant
diseases.
However,
the
data
provided
do
not
fully
support
this
conclusion.
The
authors
did
report
significantly
higher
disease
infections
of
Verticillium
dahliae
and
viruses
(
PLRV
and
PVY)
in
the
transgenic
treatments.
The
reasons
why
the
authors
believed
this
should
be
interpreted
cautiously
(
i.
e.
effects
possibly
confounded
with
the
surprisingly
early
die­
back
of
the
insecticide­
treated
plants
and
the
insecticide
treatment
itself)
may
well
be
valid
but
do
not
allow
for
a
blanket
dismissal
.
Further,
this
provided
a
case
in
point
why
the
omission
of
an
untreated
control
is
scientifically
problematic;
Section
3.1
3
­
11
the
interactions
between
insecticide
treatment,
insect
virus
vector
control
and
the
surprising
die­
back
of
the
insecticide­
treated
plants
could
not
be
analyzed
property
because
there
was
no
untreated
(
isogenic?)
control
for
comparison.
Important
information
was
lost
and
the
reasons
for
this
phenomenon
remained
speculative.
Therefore,
the
repetition
of
the
whole
experiment
in
a
second
and
third
growing
season
is
recommended,
including
an
untreated
control.
It
was
suggested
that
surveying
leaf
microflora
should
be
considered
as
indicator
for
ecological
effects
in
monitoring
programs.

EPA
Response:
The
inclusion
of
Cry3A­
producing
potato
plant
data
on
comments
regarding
Cry3Bb
corn
is
unusual.
However,
the
recommendation
that
field
survey
data
in
general
should
include
an
untreated
isogenic
control
has
already
been
implemented
by
the
EPA.
The
field
studies
submitted
for
MON863
have
isogenic
controls.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
27
Excerpt
Text:
The
last
study
(
study
15)
investigated
the
effect
of
Bt
cotton
expressing
CrylAb
and
CrylAc
proteins
and
potatoes
expressing
Cry3A
on
two
arthropod
species,
the
springtail
Folsomia
candida
and
the
mite
Oppia
nitens.
The
life
history
parameters
oviposition,
number
of
eggs
produced,
body
length,
etc.,
were
measured
over
a
period
of
7
to
8
weeks.
No
effects
on
any
of
these
parameters
were
found.
However,
it
could
not
be
demonstrated
whether
this
was
due
to
insensitivity
of
the
test
organisms
to
the
Bt
or
whether
the
Bt
toxins
were
not
ingested
or
only
in
low
concentrations.
No
gut
analyses
of
the
two
test
species
were
conducted
to
confirm
the
uptake
of
the
Bt­
toxin.
It
was
assumed
that
the
Bt­
toxin
was
ingested
because,
at
least
for
0.
nitens,
'
green
color
was
seen
in
the
whitish
insect
gut...'.
Both
test
species
primarily
feed
on
the
fungi
growing
on
the
decaying
plant
material.
However,
it
has
not
yet
been
demonstrated
whether
or
not
fungi
growing
on
decaying
Bt
plant
material
also
contain
Bt­
toxins
or
not.
Further,
through
the
addition
of
brewers
yeast
as
food,
the
organisms
probably
consumed
a
mixture
of
the
different
food
types.
Based
on
this
study,
no
evidence
was
found
that
the
transgenic
cotton
lines
and
potato
lines
investigated
had
adverse
effects
on
F.
candida
and
0.
nitens
after
the
2­
month
testing
period.
Since
slightly
lower
(
but
not
statistically
significant)
reproduction
rates
for
0.
nitens
were
observed
in
the
transgenic
treatment
than
in
the
control
after
2
months,
long­
term
effects
under
persistent
exposure
to
Bt
proteins
cannot
be
excluded
and
still
need
to
be
investigated.

EPA
Response:
The
inclusion
of
comments
investigating
the
effect
of
Bt
cotton
expressing
CrylAb
and
CrylAc
proteins
and
potatoes
expressing
Cry3A
in
comments
on
Cry3Bb
corn
is
not
strictly
appropriate.
However,
the
recommendation
that
two
arthropod
species,
the
springtail
Folsomia
candida
and
the
mite
Oppia
nitens
be
looked
at
for
long
term
effects
is
included
in
the
USDA
and
EPA
sponsored
research
,
as
well
as
the
requirement
that
Monsanto
conduct
long
term
non­
target
effects
in
the
field
during
the
MON
863
registration
period.

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Section
3.1
3
­
12
Comment
Number:
30509­
114000
Excerpt
Number:
1
Excerpt
Text:
In
my
article,
I
criticize
EPA
and
the
GMO
Companies
for
totally
inadequate
toxicology
testing
on
nontarget
organisms.
This
is
an
even
greater
problem
here.
EPA
has
no
business
in
approving
these
company
applications
without
having
bonafide
and
comprehensive
toxicology
testing.

EPA
Response:
The
toxicology
studies
for
protein
Plant
Incorporated
Protectants
(
PIP)
done
by
the
EPA
on
non­
target
organisms
have
been
submitted
to
a
Scientific
Advisory
Panel
(
SAP)
review
in
by
the
December
9,
1999
SAP
and
found
to
be
acceptable
(
SAP
Report
No.
99­
06,
February
4,
2000)..

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509­
114000
Excerpt
Number:
5
Excerpt
Text:
In
their
article,
"
Transgenic
Pollen
Harms
Monarch
Larvae,"
the
Cornell
authors
asked:
Could
windblown
corn
pollen
accumulate
on
plants
that
grow
extensively
in
and
adjacent
to
cornfields
and,
like
conventional
insecticides,
inadvertently
kill
native
insects
that
are
not
pests?
To
test
this
question,
they
chose
the
monarch
as
their
nontarget
species.
Female
monarchs
lay
eggs
on
wild
milkweed
plants,
the
only
plants
that
their
caterpillars
can
eat.
In
their
experiment,
conducted
in
the
laboratory,
the
authors
dusted
pollen
gathered
from
one
of
the
Bt
corn
strains
onto
the
leaves
of
the
common
milkweed.
They
established
that
caterpillars
that
fed
on
the
dusted
leaves
ate
less,
grew
more
slowly,
and
suffered
higher
mortality
than
caterpillars
reared
on
milkweed
leaves
dusted
with
pollen
from
a
non­
Bt
corn
strain.
The
scientists
were
circumspect
about
their
results
and
stated
clearly
that
more
research
was
needed
to
determine
the
impact
of
the
toxic
pollen
on
monarchs
in
the
natural
environment.

EPA
Response:
Mon
863
Cry3Bb1
is
a
coleopteran
active
protein
that
is
not
expected
to
affect
lepidopterans
such
as
the
monarch
butterfly.
In
addition,
extensive
research
conducted
on
the
potential
affects
of
monarch
feeding
on
lepidopteran­
active
Bt
corn
pollen
has
shown
a
lack
of
concern
for
subchronic
toxicity.
Nevertheless,
Monsanto
sponsored
a
monarch
butterfly
feeding
study
which
demonstrated
that
corn
pollen
expressing
the
Cry3Bb1
protein
will
not
result
in
toxic
or
developmental
effects
to
monarch
larvae.
In
addition,
the
August
27,
2002
SAP
concluded
that
the
monarch
butterfly
was
not
an
appropriate
indicator
organism
to
be
tested
with
MON
863
pollen
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002)
.

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509­
114000
Excerpt
Number:
9
Excerpt
Text:
Other
studies
warned
of
new
threats.
One
determined
Bt
pollen
to
be
toxic
to
later­
stage
monarch
larvaesignificant
because
older
caterpillars
had
been
assumed
to
be
less
sensitive
than
the
young
ones.
Clarifying,
a
contentious
point
of
the
1999
symposium,
new
data
fed
into
revised
computer
models
now
led
to
Section
3.1
3
­
13
predictions
that
pollen
shedding
and
monarch
breeding
happen
simultaneously
over
wide
geographic
areas.
This
finding
was
made
all
the
more
important
by
new
data
showing
that
extensive
monarch
breeding
occurs
on
milkweed
growing
inside
cornfields.
This,
in
turn,
underscored
the
devastating
effects
that
the
long­
term
use
of
herbicides,
and
genetically
manipulated
organisms
such
as
Round­
up
Ready
crops,
will
have
as
their
use
totally
eliminates
milkweeds
from
the
fields.

EPA
Response:
Extensive
studies
performed
under
the
auspices
of
the
USDA
in
the
US
and
Canada,
and
published
in
the
proceedings
of
the
NAS
show
that
the
amount
of
pollen
deposited
from
the
currently
approved
corn
producing
anti­
lepidopteran
Cry
protein
has
no
detrimental
effect
on
the
development
of
monarch
larvae
(
Proc.
Natl.
Acad.
Sci.
USA,
10.1073)
MON
863
Cry3Bb1
it
is
a
coleopteran
active
protein
that
is
not
expected
to
affect
lepidopterans
such
as
the
monarch
butterfly.
Nevertheless,
Monsanto
sponsored
a
monarch
butterfly
feeding
study
which
has
demonstrated
that
corn
pollen
expressing
the
Cry3Bb1
protein
will
not
result
in
toxic
or
developmental
effects
to
monarch
larvae.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
5
Excerpt
Text:
These
data
show
clearly
that
Cry
3Bb
expression
levels
are
extraordinarily
high
relative
to
previously
approved
Bt­
corn
events.
In
addition,
relatively
high
levels
exist
in
plant
leaf
tissue,
pollen
and
grain,
where
they
are
not
needed
and
can
do
harm
to
nontarget
organisms.
High
levels
in
these
tissues
are
useless
because
the
corn
rootworm
is
only
susceptible
to
the
Cry
3Bb
endotoxin
in
its
larval
stage,
as
it
feeds
in
the
soil
on
plant
roots.
Adults
are
essentially
not
susceptible
and
hence
any
feeding
later
in
the
season
will
have
no
appreciable
impact
on
pest
levels.

EPA
Response:
The
above
mentioned
high­
dose
issue
is
addressed
in
detail
in
EPA's
Insect
Resistance
Management
section
of
this
document.
However,
the
August
2002
SAP
commented
that
the
Cry3Bb
expression
levels
in
MON
863
are
"
not
high
dose".
In
addition,
monitoring
of
the
ecosystem
non­
target
effects
in
general
is
being
required
during
the
duration
of
the
registration
which
will
indirectly
address
any
detrimental
effects
of
the
Cry3Bb
expression
levels
in
plants.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
11
Excerpt
Text:
5.
High
Toxin
Levels
May
Jeopardize
Margins
of
Safety
for
Soil
Organisms
Monsanto
reports
that
the
maximum
environmental
concentration
expected
in
the
soil
is
13.3
mg/
kg,
based
on
the
assumption
that
corn
plants
are
tilled
into
the
top
six
inches
of
soil
when
leaf
tissue
expression
levels
are
peaking.
Yet
exposure
levels
are
likely
to
be
much
higher
for
organisms
that
move
through
the
soil
and
seek
out
the
nutrient
enriched
zone
within
a
few
centimeters
of
roots.
Moreover,
plant
residues
and
stalks
Section
3.1
3
­
14
worked
into
the
soil
will
create
an
uneven
distribution
of
Cry
3Bb
in
the
soil
profile;
organisms
that
happen
to
be
feeding
in
the
vicinity
of
decomposing
plant
tissues
will
be
exposed
to
relatively
higher
levels.

Cry
3Bb
endotoxins
are
lethal
to
earthworms
at
57
mg/
kg
in
the
soil,
resulting
in
just
a
4­
fold
difference
between
the
known
high­
level
concentrations
in
soil.
Such
a
narrow
margin
of
safety
is
not
acceptable
in
any
other
regulatory
context.
While
under
"
normal"
conditions,
a
4­
fold
margin
of
safety
may
spare
most
earthworms
in
fields
planted
to
corn
varieties
expressing
the
Cry
3Bb
endotoxin,
many
factors
can
enhance
the
vulnerability
of
earthworms
­
or
other
nontarget
organisms
­
to
the
effects
of
any
toxin.

Earthworms
moving
through
"
hot
spots"
could
be
exposed
to
lethal
levels.
Exposure
to
other
insecticides
or
herbicides
or
drought
stress
also
can
make
earthworms
more
vulnerable
than
normal.
Tillage
systems,
irrigation,
or
soil
conditions
might
alter
their
movement
and
feeding
habits,
increasing
or
decreasing
their
exposures
levels.

EPA
Response:
Contrary
to
the
submitted
comment,
a
maximum
hazard
dose
LC50
for
earthworms
exposed
to
Cry3Bb1
protein
in
soil
was
determined
to
be
greater
than
570
mg
Cry3Bb1
protein/
kg
dry
soil,
the
only
concentration
tested.
((
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
a.
vii,
MRID
No.
449043­
16
).
This
dose
is
greater
than
10
times
the
maximum
soil
concentration
of
the
cry
protein.
The
data
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein
producing
corn
plants.
The
comments
on
the
theoretically
envisioned
adverse
effects
of
Cry3Bb
protein
on
earthworms
are
also
not
supported
by
field
survey
data.
The
number
of
earthworms
collected
did
not
differ
between
Bt
and
non­
Bt
plots,
but
there
were
significantly
less
earthworms
in
the
plots
treated
with
foliar
insecticides
than
the
other
insecticide
regimes.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b),
Nevertheless,
the
EPA
has
required
Monsanto
to
conduct
additional
long
term
field
surveys
during
the
MON
863
registration
period.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
28
Excerpt
Text:
1.
Data
on
Impacts
on
Nontarget
Organisms
Are
Inadequate
The
need
for
a
much
more
thorough
assessment
of
nontarget
impacts
is
among
the
lessons
learned
from
the
EPA's
review
and
approval
of
Bt­
corn
varieties
engineered
to
control
the
European
corn
borer.

Most
pesticides,
including
traditional
Bt
insecticides,
have
been
tested
in
standard
laboratory
assays
involving
a
half­
dozen
to
as
many
as
ten
indicator
species.
Such
assays
are
reasonably
useful
as
screening
tools
to
determine
whether
there
is
much
risk
of
adverse
impacts
on
populations
of
beneficial
or
nontarget
organisms.
But
there
are
profound
differences
in
Cry3Bb
exposure
pathways
and
levels
and
qualitatively
different
studies
will
be
needed
to
more
fully
assess
impacts
on
nontargets.
A
British
research
team
led
by
T.
H.
Schuler
has
published
an
excellent
review
of
the
impacts
on
Bttransgenic
crops
on
arthropod
natural
enemies,
which
points
out
that
­
Section
3.1
3
­
15
"
Feeding
by
herbivores
induces
changes
in
the
emission
of
plant
volatiles
(
termed
herbivore­
induced
synomones)
making
the
plant
more
attractive
to
parasitoids.
Most
current
transgenes
target
the
digestive
system
of
insects
and
could
therefore
affect
this
induction
process"
(
Schuler
et
al.,
1999).

Monsanto's
registration
application
states
that
the
Cry3Bb
proteins
are
active
against
species
in
"
several
families
of
Coleoptera,"
some
of
which
are
likely
to
play
beneficial
roles
as
predators
or
part
of
food
webs.
Yet,
Monsanto
has
submitted
a
bioassay
on
only
one
Coleoptera
species
(
ladybird
beetles)
in
a
family
that
is
generally
known
not
to
be
susceptible
to
Cry3Bb
toxins.
Much
more
comprehensive
nontarget
organisms
testing
is
needed
and
must
include
both
acute
and
sub­
acute
effects,
as
well
tritrophic
level
studies.

EPA
Response:
Contrary
to
the
claim
in
this
comment,
EPA
has
16
studies
on
a
wide
range
of
non­
target
species.
In
addition
insecticidal
activity
spectrum
bioassays
of
six
Families
of
the
Order
Coleoptera
and
two
Lepidoptera
species
detected
activity
only
against
beetle
species
of
the
family
Chrysomelidae
(
corn
root
worm
and
Colorado
potato
beetle)
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
The
October
2000
and
August
2002
SAP
reports
commented
that
wide
spectrum
non­
target
testing
is
not
necessary
for
the
Cry
proteins
and
should
be
focused
on
groups
of
non­
targets
exposed
and
susceptible
to
the
crop
being
registered
(
SAP
Report
No.
2000­
07.
March
12,
2001);
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
The
Agency
has
determined
that
the
non­
target
organisms
most
likely
to
be
exposed
to
the
protein
in
transgenic
corn
fields
were
beneficial
insects
feeding
on
corn
pollen
and
nectar,
and
soil
invertebrates,
particularly
Coleoptera
spp.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
The
field
studies
performed
to
date
do
not
show
an
effect
on
non­
target
coleopteran
species
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b).
Protocols
for
laboratory
testing
of
carabid
beetles
and
other
significant
coleopteran
soil
species
are
not
available
and
are
under
development
by
the
Agricultural
Biotechnology
Stewardship
Technical
Committee
(
ABSTC).
Additional
multi­
year
field
surveys
to
address
the
long
range
effects
on
significant
soil
beetles
are
in
progress.
A
decreased
abundance
of
parasitoids
in
fields
where
the
pest
larvae
have
been
eliminated
is
a
well
known
phenomenon
and
not
unique
to
the
cultivation
of
Cry
protein
containing
crops.
Comments
from
the
August
2002
SAP
recommended
against
tritrophic
studies
on
green
lacewings
in
corn
because
the
lacewing
prey
in
corn
fields
does
not
have
Bt
cry
toxin
because
they
feed
on
corn
phloem
which
does
not
contain
Cry
proteins
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002)
The
EPA
will,
however,
require
tritrophic
studies
on
a
case­
by­
case
basis
where
they
would
appear
to
be
warranted
by
the
ecology
of
the
crop
in
question.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
29
Excerpt
Text:
The
adverse
impacts
of
Cry3Bb
on
nontarget
organism
will
be
largely
indirect,
the
result
of
changes
in
species
composition
and
food
webs.
Work
by
Angelika
Hilbeck
and
colleagues
at
the
Swiss
Research
Station
for
Agroecology
and
Agriculture
has
shown
that
Bt­
transgenic
corn
can
have
direct
and
indirect
impacts
on
both
nontarget
organisms
and
beneficial
insects.

Two
studies
published
in
1998
showed
that
the
Cry1
toxins
expressed
in
Bt
corn
plant
tissue
can
have
an
adverse
impact
on
the
development
and
populations
of
Chrysoperla
carnea
(
green
lacewings),
a
common
Section
3.1
3
­
16
generalist
predator
(
Hilbeck
et
al.,
1998a;
Hilbeck
et
al.,
1998b).
Mortality
as
high
as
two­
thirds
was
observed
among
green
lacewing
larvae
reared
on
insects
that
had
fed
on
corn
plants
expressing
Bt
toxins.
Similar
morality
was
found
when
activated
Bt
toxins
(
the
truncated
form
of
Bt
expressed
in
corn
plant
tissues)
were
fed
to
green
lacewing
larvae
in
an
artificial
diet
(
Hilbeck
et
al.,
1998a).

In
both
studies,
Hilbeck
and
colleagues
used
Egyptian
cotton
leafworms.
These
insects
fed
on
the
Bt
corn,
or
diets
containing
the
activated
Bt
toxins,
and
suffered
only
minor,
non­
lethal
effects.
But
when
the
lacewings
fed
on
the
cotton
leafworms,
they
suffered
both
impaired
development
and
mortality.
In
addition,
green
lacewings
feeding
on
Bt­
fed
prey
progressed
through
developmental
stages
more
slowly
than
the
controls
fed
on
prey
not
exposed
to
Bt
toxins.

One
of
the
study's
intriguing
findings
is
that
passage
of
Bt
toxins
and
protoxins
through
the
gut
of
certain
insects,
in
this
case
Egyptian
cotton
leafworms,
appears
to
potentiate
the
effects
of
the
Bt
toxins
when
another
insect
(
i.
e.,
green
lacewings)
preys
upon
the
cotton
leafworms
(
Hilbeck
et
al.,
1999).
This
and
other
findings
led
the
team
to
conclude
"
tritrophic
level
studies
are
necessary
to
assess
the
long­
term
compatibility
of
insecticidal
plants
with
common
natural
enemies"
(
Hilbeck
et
al,
1999)
and
to
make
a
critical
point:

"
The
ubiquitous
and
temporally
extended
availability
of
B.
thuringiensis
proteins
in
the
field
in
addition
to
its
modified
form
of
release,
makes
it
necessary
to
verify
and
monitor
the
compatibility
of
this
new
pest
management
strategy
with
natural
enemies.
The
long­
term
agroecological
safety
of
the
combined
use
of
transgenic
crop
plants
and
B.
thuringiensis
insecticides
cannot
simply
be
deduced
from
the
past
record
of
safe
B.
thuringiensis
insecticide
use
when
B.
thuringiensis
compounds
were
available
in
the
field
only
during
short
periods."
(
Hilbeck
et
al.,
1999).

EPA
Response:
Comments
from
the
the
August
2002
SAP
which
addressed
the
tritrophic
feeding
issue
indicate
that
the
tritrophic
scenario
described
above
does
not
exist
in
corn
fields
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
Comments
from
the
the
August
2002
SAP
recommended
against
tritrophic
studies
on
green
lacewings
in
corn
because
the
lacewing
prey
in
corn
fields
does
not
have
any
Bt
cry
toxin
because
they
feed
on
corn
phloem
which
does
not
contain
Cry
proteins
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
The
EPA
will,
however,
require
tritrophic
studies
on
a
case­
by­
case
basis
where
they
are
warranted
by
the
ecology
of
the
crop
in
question.
The
USDA
and
EPA
are
also
funding
long
term
effects
studies
to
address
the
effects
of
season­
long
exposures
to
Bt
Cry
proteins
on
invertebrate
communities.
In
addition
EPA
has
required
Monsanto
to
conduct
long
term
non­
target
effects
studies
in
the
field
during
the
MON
863
registration
period
Commenter
Name:
Douglas
Tallamy
Commenter
Organization
Name:
University
of
Delaware
­
Deparment
of
Entomology
and
Applied
Ecology
Comment
Number:
30509­
117000
Excerpt
Number:
2
Excerpt
Text:
Effects
on
Nontarget
Insects
Section
3.1
3
­
17
In
1999
John
Losey,
Linda
Rayor
and
Maureen
Carter
of
Cornell
University
painted
milkweed
leaves
with
pollen
from
BT
corn
and
fed
those
leaves
to
the
larvae
of
monarch
butterflies.
Some
of
these
larvae
died.
Some
grew
more
slowly,
and
some
developed
with
no
ill
effects.
This
study
sparked
a
national
concern
for
the
monarch's
well­
being
that
a
century
of
mowing
or
paving
over
milkweed
habitat,
the
annual
slaughter
of
millions
of
migrating
monarchs
on
our
nation's
highways,
the
continued
logging
of
the
monarch's
over
wintering
habitats
in
Mexico,
and
monarch
death
from
insecticide
drift
was
never
able
to
muster.
Although
the
Losey
study
has
been
criticized
as
not
depicting
actual
field
conditions
to
which
monarch
larvae
near
BT
corn
are
exposed,
we
need
not
discredit
the
study
to
find
a
flaw
in
the
argument
that
BT
corn
will
threaten
the
monarch.
The
question
is
not
whether
some
monarchs
might
die
if
millions
of
acres
of
BT
are
planted.
Instead
we
should
ask
whether
BT
plants
will
kill
more
monarchs
than
conventional
corn
plantings
that
rely
on
repeated
insecticide
applications
for
their
success.
How
many
monarchs
and
other
nontarget
organisms
that
are
not
quite
as
flashy
die
every
time
insecticides
are
sprayed
on
corn
from
airplanes?
Most
opponents
to
BT
corn
do
not
realize
that
more
insecticide
is
deployed
against
the
western
corn
rootworm
than
any
other
single
pest
in
the
world.
Ironically,
the
people
opposed
to
BT
corn
are
often
the
same
people
concerned
about
the
use
of
pesticides.

EPA
Response:
EPA
recognizes
that
conventional
pesticides
potentially
pose
a
greater
hazard
than
PIPs
to
monarch
butterflies,
other
invertebrates,
and
their
interdependent
interactions
in
the
agroecosystem,
as
well
as
to
animal
and
plant
terrestrial
and
aquatic
wildlife.

Commenter
Name:
Douglas
Tallamy
Commenter
Organization
Name:
University
of
Delaware
­
Department
of
Entomology
and
Applied
Ecology
Comment
Number:
30509­
117000
Excerpt
Number:
3
Excerpt
Text:
Wise
use
of
BT
corn
against
corn
rootworms
would
considerably
reduce
the
pesticides
applied
to
US
corn
overnight,
and,
as
a
direct
consequence,
reduce
monarch
mortality
associated
with
conventional
corn
plantings.
The
reduction
of
pesticide
has
been
a
national
goal
for
decades.
Now
that
we
have
the
technology
to
attain
this
goal,
registration
of
this
technology
is
threatened
because
of
misguided
national
hysteria.
I
urge
that
we
return
to
logic
and
reason
in
this
matter.

EPA
Response:
EPA
recognizes
the
fact
that
PIPs
have
greatly
reduced
the
use
of
conventional
chemical
pesticides
with
resultant
reduction
in
exposure
of
wildlife
to
the
more
toxic
chemicals.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
6
Excerpt
Text:
Cry
3Bb
endotoxins
are
lethal
to.
earthworms
at
57
mg/
kg
in
the
soil,
resulting
in
just
a
4­
fold..
difference
between
the
known
high­
level
concentrations
in
soil.
While
under
"
normal"
conditions,
a
4­
fold
margin
of
safety
may
spare
most
earthworms
in
fields
planted
to
corn
varieties
expressing
the
Cry
3Bb
endotoxin,
Section
3.1
3
­
18
many
factors
can
enhance
the
vulnerability
of
earthworms
­
or
other
non­
target
organisms
­
to
the
effects
of
any
toxin.

For
example,
if
the
average
level
in
the
top
six
inches
of
soil
is
13.3
mg/
kg,
there
surely
will
be
places
within
the
rhizosphere
where
levels
are
as
much
as
two
orders
of
magnitude
higher.
Earthworms
moving
through
such
"
hot
spots"
could
be
exposed
to
lethal
levels.
Exposure
to
other
insecticides
or
herbicides
or
drought
stress
also
can
make
earthworms
more
vulnerable
than
normal.
Tillage
systems,
irrigation,
or
soil
conditions
might
alter
their
movement
and
feeding
habits,
increasing
or
decreasing
their
exposures
levels.

EPA
Response:
Contrary
to
the
submitted
comment,
a
maximum
hazard
dose
LC50
for
earthworms
exposed
to
Cry3Bb1
protein
in
soil
was
determined
to
be
greater
than
570
mg
Cry3Bb1
protein/
kg
dry
soil,
the
only
concentration
tested.
((
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
a.
vii,
MRID
No.
449043­
16
).
This
dose
is
greater
than
10
times
the
maximum
soil
concentration
of
the
cry
protein.
The
data
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein
producing
corn
plants.
The
comments
on
the
theoretically
envisioned
adverse
effects
of
Cry3Bb
protein
on
earthworms
are
also
not
supported
by
field
survey
data.
The
number
of
earthworms
collected
did
not
differ
between
Bt
and
non­
Bt
plots,
but
there
were
significantly
less
earthworms
in
the
plots
treated
with
foliar
insecticides
than
the
other
insecticide
regimes.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b),
Nevertheless,
the
EPA
has
required
Monsanto
to
conduct
additional
long
term
field
surveys
during
the
MON
863
registration
period.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
7
Excerpt
Text:
Indirect
Impacts
Must
Be
Assessed
The
adverse
impacts
of
Cry3Bb
on
non­
target
organism
will
be
largely
indirect,
the
result
of
changes
in
species
composition
and
food
webs.
Work
by
Angelika
Hilbeck
and
colleagues
at
the
Swiss
Research
Station
for
Agroecology
and
Agriculture
has
shown
that
Bt­
transgenic
corn
can
have
direct
and
indirect
impacts
on
both
non­
target
organisms
and
beneficial
insects.

Two
studies
published
in
1998
showed
that
the
Cry1
toxins
expressed
in
Bt
corn
plant
tissue
can
have
an
adverse
impact
on
the
development
and
populations
of
Chrysoperla
carnea
(
green
lacewings),
a
common
generalist
predator
(
Hilbeck
et
al.,
1998a;
Hilbeck
et
al.,
1998b).
Mortality
as
high
as
two­
thirds
was
observed
among
green
lacewing
larvae
reared
on
corn
plants
expressing
B.
thuringiensis.
Similar
morality
was
found
when
activated
Bt
toxins
(
the
truncated
form
of
Bt
expressed
in
corn
plant
tissues)
were
fed
to
green
lacewing
larvae
in
an
artificial
diet
(
Hilbeck
et
al.,
1998a).

More
recent
research
by
the
Hilbeck
team
explores
prey­
mediated
impacts.
Such
impacts
can
arise
when
predatory
insects
like
green
lacewings
feed
on
a
herbivore
that
has,
in
turn,
been
feeding
on
B1
corn
plants.
In
both
the
earlier
and
new
work,
Hilbeck
and
colleagues
used
Egyptian
cotton
leafworms.
These
insects
fed
on
the
Bt
corn,
or
diets
containing
the
activated
B1
toxins,
and
suffered
only
minor,
non­
lethal
effects.
Section
3.1
3
­
19
But
when
the
lacewings
fed
on
the
cotton
leafworms,
they
suffered
both
impaired
development
and
mortality.
In
addition,
green
lacewings
feeding
on
Bt­
fed
prey
progressed
through
developmental
stages
more
slowly
than
the
controls
fed
on
prey
not
exposed
to
Bt
toxins.

These
and
other
findings
led
the
team
to
conclude
"
tritrophic
level
studies
are
necessary
to
assess
the
long
term
compatibility
of
insecticidal
plants
with
common
natural
enemies"
(
Hilbeck
et
al,
1999)
and
to
make
a
critical
point:

"
The
ubiquitous
and
temporally
extended
availability
of
B.
thuringiensis
proteins
in
the
field
in
addition
to
its
modified
form
of
release,
makes
it
necessary
to
verify
and
monitor
the
compatibility
of
this
new
pest
management
strategy
with
natural
enemies,
The
long­
term
agroecological
safety
of
the
combined
use
of
transgenic
crop
plants
and
B.
thuringiensis
insecticides
cannot
simply
be
deduced
from
the
past
record
of
safe
B.
thuringiensis
insecticide
use
when
B.
thuringiensis
compounds
were
available
in
the
field
only
during
short
periods."
(
Hilbeck
et
al.,
1999).

EPA
Response:
Comments
from
the
the
August
2002
SAP
which
addressed
the
tritrophic
feeding
issue
indicate
that
the
tritrophic
scenario
described
above
does
not
exist
in
corn
fields
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
Comments
from
the
the
August
2002
SAP
recommended
against
tritrophic
studies
on
green
lacewings
in
corn
because
the
lacewing
prey
in
corn
fields
does
not
have
any
Bt
cry
toxin
because
they
feed
on
corn
phloem
which
does
not
contain
Cry
proteins
(
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
The
EPA
will,
however,
require
tritrophic
studies
on
a
case­
by­
case
basis
where
they
are
warranted
by
the
ecology
of
the
crop
in
question.
The
USDA
and
EPA
are
also
funding
long
term
effects
studies
to
address
the
effects
of
season­
long
exposures
to
Bt
Cry
proteins
on
invertebrate
communities.
In
addition
EPA
has
required
Monsanto
to
conduct
long
term
non­
target
effects
studies
in
the
field
during
the
MON
863
registration
period
Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
8
Excerpt
Text:
Consulting
with
the
United
States
Fish
and
Wildlife
Service
There
are
many
Coleoptera
that
are
listed
as
threatened
or
endangered
under
the
Endangered
Species
Act,
The
may
be
impacted
in
­
some
way
by
the
EPA's
new
regulation
due
to
the
non­
target
impacts
of
Cry
3Bb
corn.
Any
federal
agency
that
initiates
new
policies
that
may
impact
endangered
or
threatened
species
must
consult
with
the
United
States
Fish
and
Wildlife
Service.
The
consultation
is
required
under
the
Act.
Some
of
these
species
may
need
special
consideration
because
of
their
small
population
size
or
because
their
populations
are
declining.
A
thorough
review
of
the
endangered
species
that
may
be
impacted
by
Cry
3Bb
corn
should
be
instituted
to
determine
the
impact
on
them.
Impacts
on
Soil
Health
and
Productivity
Must
Be
Assessed
EPA
Response:
EPA
conducted
a
hazard
assessment,
exposure
assessment
and
risk
characterization
to
demonstrate
that
Section
3.1
3
­
20
Cry3Bb1
does
not
pose
a
risk
to
endangered
Coleoptera
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
C).
It
was
determined
that
no
adverse
affects
from
Cry3Bb1event
MON
863
are
expected
to
endangered/
threatened
Coleoptera
species
listed
by
the
USFWS
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
C).
Non­
coleopteran
endangered
species
are
not
expected
to
be
susceptible
to
MON
863.
Therefore
since
no
"
may
effect"
finding
was
made,
according
to
the
Endangered
Species
Act,
a
consultation
with
the
USFWS
is
not
required.

Additional
studies
on
soil
health
will
be
conducted
for
a
three
year
period.

Commenter
Name:
Janet
Cutrona
Commenter
Organization
Name:
Janet
Cutrona
Comment
Number:
30509B­
030004
Excerpt
Number:
1
Excerpt
Text:
The
studies
in
this
project
showed
that
monarch
caterpillars
have
to
be
exposed
to
pollen
leaves
greater
than
1,000
grains/
cm.
2
to
show
toxic
effects.

Caterpillars
were
found
to
be
present
on
milkweed
during
the
one
to
two
weeks
that
pollen
is
shed
by
corn,
but
corn
pollen
levels
on
milkweed
leaves
were
found
to
average
only
about
170
pollen
grains/
cm2
in
corn
fields.

Reports
from
several
field
studies
show
concentrations
much
lower
than
that
even
within
the
cornfield.
In
Maryland,
the
highest
level
of
pollen
deposition
was
inside
and
at
the
edge
of
the
corn
field,
where
pollen
was
found
at
about
50
grains/
cm2.
In
the
Nebraska
study,
pollen
deposition
ranged
from
6
grains/
cm2
at
the
field
edge
to
less
than
1
grain/
cm.
2
beyond
10
meters.
Samples
collected
from
fields
in
Ontario
immediately
following
the
period
of
peak
pollen
shed
showed
pollen
concentrations
averaged
78
grains
at
the
field
edge.

EPA
Response:
Extensive
studies
performed
under
the
auspices
of
the
USDA
in
the
US
and
Canada,
and
published
in
the
proceedings
of
the
NAS
show
that
the
amount
of
pollen
deposited
from
the
currently
approved
corn
producing
anti­
lepidopteran
Cry
protein
has
no
detrimental
effect
on
the
development
of
monarch
larvae
(
Proc.
Natl.
Acad.
Sci.
USA,
10.1073).
MON
863
Cry3Bb1
is
a
coleopteran
active
protein
that
is
not
expected
to
affect
lepidopterans
such
as
the
monarch
butterfly.
Nevertheless,
Monsanto
sponsored
a
monarch
butterfly
feeding
study
which
has
shown
that
exposure
to
corn
pollen
expressing
the
Cry3Bb1
protein
will
not
result
in
toxic
or
developmental
effects
in
monarch
larvae..

Commenter
Name:
Richard
Hellmich
Commenter
Organization
Name:
USDA­
ARS
Comment
Number:
30509B­
034000
Excerpt
Number:
3
Excerpt
Text:
As
with
the
lepidopteran
Bt
corn,
there
will
be
questions
about
non­
target
effects
and
insect
resistance
management.
Science
cannot
prove
a
negative
­
that
there
will
never
be
any
non­
target
effects.
But
I
believe
most
of
the
significant
questions
have
already
been
addressed
through
the
registration
process.
Section
3.1
3
­
21
Most
important,
positive
and
negative
impacts
of
new
technologies
must
be
compared
with
those
of
existing
technologies,
not
simply
judged
in
a
vacuum.
AR
possible
impacts
of
any
technology
or
farming
practice
are
impossible
to
foresee,
but
we
can
focus
on
known
and
probable
risks.
When
risks
of
a
technology
are
characterized
as
low,
based
on
actual
data,
then
the
potential
impact
should
be
evaluated
proportional
to
that
level
of
concern.
This
reasonable
approach
should
reduce
the
chances
of
rejecting
safe
technologies
simply
because
they
are
new
and
unfamiliar.

EPA
Response:
The
comment
does
not
require
a
response.

Commenter
Name:
Paul
Mitchell
Commenter
Organization
Name:
Texas
A&
M
University
­
Department
of
Agriculture
Economics
Comment
Number:
30509B­
082000
Excerpt
Number:
6
Excerpt
Text:
The
risk
of
significant
monarch
and
other
non­
target
insect
problems
has
proven
minor
(
see
a
nice
summary
of
the
issue
at
http:
www.
nbiap.
vt.
edu/
news/
200j/
newsO2.
apr.
html#
aprO2O4).
The
StarLink
food
contamination
is
not
an
issue,
since
the
new
event
has
or
will
be
approved
for
human
consumption,
as
have
most
of
the
other
Bt
events.
The
recent
report
of
the
spread
of
other
Bt
event
genes
into
wild
populations
in
Mexico
seems
to
have
serious
scientific
flaws
as
did
the
original
claim
that
Bt
corn
was
killing
a
substantial
number
of
monarch
larvae
(
see
http://
www.
isb.
vt.
edu/
news/
2002/
news02.
mar.
html#
mar0202).
I
am
glad
that
people
are
looking
for
possible
problems
with
these
transgenic
crops.
However,
I
believe
that
proper
science
should
be
used
to
make
policy
relevant
claims.

EPA
Response:
The
comment
does
not
require
a
response.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
3
Excerpt
Text:
Comment
or
Question:
Cry3Bbl
endotoxins
are
lethal
to
earthworms
at
57
mg/
kg
soil,
resulting
in
just
a
four­
fold
difference
between
the
known
high­
level
concentration
in
soil
Such
a
narrow
margin
of
safety
is
not
acceptable
in
any
other
regulatory
context.

Scientific
Background:
Contrary
to
what
has
been
alleged,
no
mortality
of
earthworms
(
or
any
other
effects)
was
observed
at
the
57
mg/
kg
soil
treatment
level.
Mortality
was
only
observed
at
a
ten­
fold
higher
treatment
level.
The
57
mg/
kg
level
was
established
as
a
no
observable
effect
concentration.
Therefore,
the
maximum
worst­
case
exposure
to
earthworms
is
at
least
four­
fold
lower
than
a
level
that
is
known
not
to
have
an
effect
on
earthworms.
Under
actual
use
conditions,
the
crop
will
be
harvested
and
not
tilled
into
the
top
6
inches
of
soil.
Moreover,
the
protein
is
known
to
degrade
rapidly
in
soil.
Therefore,
MON
863
corn
is
expected
to
pose
no
more
than
minimal
risk
to
earthworms.

EPA
Response:
Section
3.1
3
­
22
An
independent
review
of
the
available
data
by
the
EPA
has
reached
the
same
conclusion.
The
laboratory
data
and
field
surveys
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein­
producing
corn
plants.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
5
Excerpt
Text:
Comment
or
Question:
There
is
a
lack
of
data
on
nontarget
species
that
may
play
beneficial
roles
as
predators
or
part
of
food
webs.
The
data
submitted
were
limited
to
a
bioassay
on
only
one
Coleopteran
species
(
ladybird
beetles)
in
a
family
generally
known
not
to
be
susceptible
to
Cry3Bb1
toxins.

Scientific
Background:
A
standard
battery
of
nontarget
organism
tests
was
conducted
for
the
Cry3Bb1
protein
as
required
by
EPA.
This
battery
of
tests
included
the
ladybird
beetle,
which
is
generally
recognized
as
an
important
biological
control
agent.
Therefore,
the
test
was
performed
to
document
the
absence
of
effects
on
this
important
beetle.
Monsanto
has
tested
other
coleopteran
families
in
addition
to
the
Chrysomelidae
(
target
species)
and
Coccinellidae
(
nontarget)
families.
These
families
include
Bruchidae,
Tenebrionidae,
and
Curculionidae.
[
Footnote
6:
Monsanto
report
MSL­
17614;
MRID
455770­
03]
No
effects
are
predicted
in
these
non­
target
coleopteran
families.
Moreover,
no
apparent
affects
on
beetles
or
other
species
were
observed
in
the
first
year
of
a
two­
year
field
study
with
MON
863
corn,
[
Footnote
7:
Monsanto
report
MSL­
17179;
MRID
455382­
06]
nor
in
university
studies
conducted
at
various
locations.
[
Footnote
8:
Monsanto
reports
99­
894E
and
00­
CR­
032E­
7;
MRIDs
453485­
02
and
456530­
03]

Comment
or
Question:
More
comprehensive
testing
of
non­
target
organisms
is
needed
and
must
include
both
acute
and
subacute
effects,
as
well
as
tritrophic
studies.
Tritrophic
studies
are
necessary
to
assess
the
long­
term
compatibility
of
insecticidal
plants
with
common
natural
enemies.

Scientific
Background:
A
comprehensive
ecological
risk
assessment
was
conducted
for
the
Cry3Bb1
protein
as
outlined
in
Subdivision
M
of
the
U.
S.
EPA
FIFRA
regulations.
Crops
with
pesticidal
traits
are
required
to
undergo
acute
and
subacute
evaluation
for
a
series
of
surrogate
species.
Potential
adverse
effects
to
nontarget
organisms
from
exposure
to
Cry3Bb1
were
evaluated
in
a
series
of
studies
with
representative
avian,
aquatic
and
terrestrial
beneficial
invertebrate
species.
These
studies
establish
that
the
potential
risk
to
nontarget
organisms
is
negligible.

Tritrophic
studies
are
mainly
designed
to
examine
the
effects
of
food
chain
transfer
of
the
toxin
and
to
examine
possible
secondary
effects
on
predator
populations
due
to
changes
in
the
prey
(
pest)
populations.
Since
the
­
toxicity
of
Bt
proteins
has
been
assessed
by
directly
feeding
predators
such
as
lacewings
and
ladybird
beetles
(
as
per
EPA's
Subdivision
M),
this
information
is
sufficient
to
assess
risk
from
food
chain
transfer.
Comparison
of
no
observed
effect
levels
to
maximum
exposure
concentrations
indicates
minimal
risk
to
insect
predators,
parasitoids
or
other
animals.
Recent
FIFRA
Scientific
Advisory
Panels
concluded
that
tritrophic
studies
were
not
needed
as
part
of
the
ecological
risk
assessment.

Laboratory
studies
conducted
by
Hillbeck
et
al
[
Footnote
9:
Hillbeck
et
al.
(
1998).
Effects
of
transgenic
Section
3.1
3
­
23
Bacillus
thuringiensis
corn­
fed
prey
on
mortality
and
development
time
of
immature
Chrysoperla
carnea
(
Neuroptera:
Chrysopidea).
Environ.
Entomol.
27:
480­
487]
showed
that
green
lacewing
larvae
feeding
on
Bt
toxin­"
intoxicated"
preys
(
Spodoptera
littoralis
larvae)
had
about
25%
more
mortality
than
those
feeding
on
healthy
("
nonintoxicated")
preys.
However,
recent
studies
by
Dutton
et
al.
[
Footnote
10:
Dutton
et
al.
(
2002).
Update
of
Bt­
toxin
by
herbivores
feeding
on
transgenic
maize
and
consequences
for
the
predator
Chrysoperla
carnea.
Ecological
Entolmol.
(
in
press)]
indicated
that
this
adverse
effect
on
the
lacewing
larvae
is
largely
due
to
the
"
sickness"
of
the
Bt
toxin­
intoxicated
preys,
rather
than
ingested
Bt
toxins.
They
showed
that
an
alternative
prey
(
Tetranychus
uticae
Koch)
feeding
on
Bt
corn
had
a
2
to
3
fold
higher
body
burden
of
Bt
proteins
than
S.
littoralis,
yet
produced
no
adverse
effect
on
the
development
and
survival
of
the
green
lacewing
larvae
tested.

EPA
Response:
An
independent
review
of
the
available
data
by
the
EPA
has
reached
the
same
conclusions.
In
addition,
EPA
intends
to
ask
for
tritrophic
studies
where
they
would
appear
to
be
warranted
by
the
ecology
of
the
crop
in
question.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
6
Excerpt
Text:
An
ideal,
precision­
engineered
transgenic
corn
for
rootworm
control
would
express
in
and
through
roots
a
"
high­
dose"
for
control
of
corn
rootworms
for
the
first
two
months
of
the
corn
growth
cycle,
with
little
or
no
expression
in
other
tissues
or
later
in
the
season.
The
degree
to
which
Cry
3Bb
proteins
are
expressed
in
the
second
half
of
the
growth
cycle,
in
any
tissue,
adds
nothing
to
efficacy
technology
and
increases
the
potential
for
risks,
including
impacts
on
nontarget
organisms
and
the
emergence
of
resistance.
It
will
also
expose
adult
rootworm
beetles
to
sublethal
levels
of
Cry
3Bb
toxins
via
leaf
tissues
during
a
second
stage
of
life,
adding
new
complexity
to
the
assessment
and
management
of
resistance.

EPA
Response:
The
above
mentioned
high­
dose
issue
is
addressed
in
detail
in
EPA's
Insect
Resistance
Management
section
of
this
document.
Monitoring
of
the
agro­
ecosystem
effects
in
general
is
being
required
during
the
duration
of
the
registration
which
will
indirectly
show
any
detrimental
effects
of
the
Cry3Bb
expression
levels.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
9
Excerpt
Text:
Limited
Field
Study
Data
Indicate
Possible
Adverse
Impacts.

A
report
of
the
one
field
study
on
ecological
impacts
of
MON
863
corn
on
nontarget
organisms
(
Bhatti
et
al.,
2001)
states
that
"
Preliminary
field
data
were
collected
from
large­
scale
corn
plots
in
1999"
whereas
the
Dudin
et
al.
report
on
those
1999
field
trials
explains
that
in
each
location,
a
limited
number
of
corn
plants
could
be
sampled
for
Cry
3Bb1
expression
levels
because
of
the
scarcity
of
seeds
and
viable
plants.
Section
3.1
3
­
24
The
1999
trials
apparently
were
not
"
large­
scale"
as
the
term
is
usually
used
to
describe
corn
experiments.

EPA
Response:
The
reported
discrepancy
between
reported
test
plot
sizes
in
is
duly
noted
by
the
EPA.
Research
to
develop
protocols
for
statistically
valid
large
scale
field
studies
is
currently
in
progress.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
11
Excerpt
Text:
A
Flawed
Analysis
of
Bt
Toxin
Concentration
in
Soil
Underestimates
Risks
to
Earthworms
and
Other
Soil
Organisms.

In
the
ecological
impacts
section
of
the
January
8,
2002,
summary
document
(
Ward,
2002;
Monsanto
00­
CR­
032E­
6),
the
author
states
that
no
adverse
impacts
on
nontarget
organisms
should
be
expected
at
"
Maximum
Expected
Environmental
Concentrations"
(
MEECs).
In
the
case
of
pollen,
the
MEEC
was
determined
to
be
93
ug/
g,
a
level
probably
no
more
than
one­
half
the
true
MEEC,
since
the
93
ug/
g
value
was
based
on
a
composite
sample
containing
pollen
from
many
plants
collected
over
seven
days
(
see
discussion
above).

The
MEEC
for
soil
dwelling
organisms
is
estimated
to
be
13.3
mg/
kg
(
or
13.3
ug/
g)
"
based
on
the
assumption
that
corn
plants
are
tilled
into
the
top
six
inches
of
soil
at
the
time
of
maximum
Cry
3Bb1
concentration
(
i.
e.,
93
ug/
g)."

This
method
for
calculating
the
soil
MEEC
is
flawed
and
results
in
a
far
lower
soil
MEEC
than
the
actual
level
of
Cry
3Bb1
protein
that
many
soil­
dwelling
organisms
will
be
exposed
to,
at
least
during
the
first
60
to
90
days
of
the
corn
plant
growth
cycle.
Impacts
on
organisms
that
spend
part
of
their
life
cycle
near
or
in
the
rhizosphere
(
zone
of
soil
immediately
surrounding
roots
and
modified
by
root
exudates)
will,
in
particular,
be
exposed
to
much
higher
concentrations
than
other
soil
organisms.
According
to
Monsanto
scientists,
Cry
3Bb1
protein
levels
are
highest
in
the
parts
of
roots
that
are
growing
rapidly
(
Vaughn
et
al.,
2001).
The
areas
around
root
tips
­
clearly
a
fastgrowing
area
­­
also
likely
emit
the
highest
soil
concentrations
of
Cry
3Bb1
protein
exudates,
again
especially
when
corn
plants
are
growing
rapidly.
For
these
reasons,
and
drawing
on
the
work
of
Saxena
et
al.
(
1999),
the
levels
of
Cry
3Bb1
proteins
in
the
few
centimeters
of
soil
around
the
end
of
rapidly
growing
roots
will
be
far
higher
than
at
any
other
place
or
at
any
other
time.
Mixing
the
Cry
3Bb1
protein
in
a
corn
plant
in
the
top
six
inches
of
soil
obviously
results
in
significant
dilution
of
the
protein,
and
hence
an
MEEC
low
by
perhaps
orders
of
magnitude
compared
to
levels
in
the
rhizosphere
for
the
first
two
months
of
plant
growth.

It
is
worth
emphasizing,
as
well,
that
data
submitted
to
date
by
Monsanto
report
the
concentration
of
Cry
3Bb1
protein
in
fresh
root
tissue,
but
not
in
the
soil
immediately
surrounding
roots.
Root
exudates,
not
levels
in
the
roots,
will
drive
adverse
impacts
on
a
wide
range
of
soil
organisms.
But
even
with
the
far­
toolow
soil
MEEC,
Monsanto's
ecological
effects
studies
have
documented
potentially
serious
impacts
on
earthworms,
an
organism
with
a
MOE
of
only
4.3
(
the
"
No
Observable
Effect
Concentration"
is
4.3
times
higher
than
the
MEEC
in
soil).
In
most
ecological
impact
studies,
the
EPA
strives
to
assure
a
minimum
Section
3.1
3
­
25
MOE
of
10.

Two
other
nontarget
organisms
have
MOEs
under
10,
based
on
relevant
MEECs.
The
adult
honey
bee,
a
key
pollinator,
has
an
MOE
of
just
3.8;
the
common
parasitic
wasp
Nasonia
vitripennis
has
an
MOE
of
only
4.3,
again
based
on
the
pollen
MEEC
of
93
ug/
g.
While
not
an
important
beneficial
insect
in
corn
production
systems,
Nasonia
vitripennis
is
a
common
test
organism
that
is
easily
reared
in
the
laboratory.
Impacts
on
this
organism
may
be
indicative
of
impacts
on
other
parasitic
wasps
that
are
important
in
corn
production
fields.

EPA
Response:
Contrary
to
the
claims
by
this
commenter
of
possible
adverse
effects
because
of
localized
high
soil
Cry3Bb
protein
concentration,
submitted
laboratory
toxicity
data
performed
at
a
maximum
hazard
dose
(
which
takes
into
account
localized
high
concentrations),
and
field
survey
results,
as
well
as
published
reports
of
effects
on
soil
microbial
and
invertebrate
flora
do
not
show
significant
adverse
effects.
However,
additional
multi­
year
field
surveys
are
being
conducted
to
confirm
this
conclusion.
Examination
of
the
submitted
nontarget
studies
shows
that
these
were
performed
with
20x
or
higher
safety
margins,
except
where
pure
pollen
or
plant
tissue
were
used
where
the
concentration
of
the
Cry
protein
cannot
be
increased.
The
parasitic
hymenoptera
were
in
fact
tested
at
only
1X
field
concentration
in
plants
rather
than
10X.
However,
parasitic
hymenoptera
are
not
expected
to
feed
directly
on
corn
plant
tissue.
For
this
reason
the
SAP
commented
that
parasitic
hymenoptera
are
not
an
appropriate
test
species
for
MON863
corn.
The
LC50
for
adults
honey
bees
was
determined
to
be
greater
than
the
highest
concentration
tested,
360
ug
Cry3Bbl
protein/
g
diet,
or
4.3­
times
the
MEEC
(
i.
e.,
93
ug
Cry3Bbl
protein/
g
pollen).
Since
no
adverse
effects
were
noted
at
360
ug/
g
(
and
this
exposure
will
not
be
realized
under
field
conditions),
minimal
risk
to
adult
honey
bees
is
expected.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
42
Excerpt
Text:
B.
An
Inappropriate
Testing
Paradigm,
Poorly
Designed
Experiments,
and
Insufficient
Data
Render
Monsanto's
Assessment
of
the
Environmental
Risks
of
MON
863
Inadequate.
[
Footnote
13:
See
Appendix
A
for
additional
comments
on
environmental
risk
assessment.]

A
proper
risk­
benefit
assessment
of
MON
863
corn
must
analyze
this
technology's
risks
and
benefits
both
in
isolation
and
relative
to
other
accessible,
proven
control
measures.
Data
submitted
by
Monsanto
on
the
nontarget
organism
impacts
of
MON
863
corn,
while
limited,
raise
serious
concerns
about
this
product's
risks
to
the
environment.

Many
organizations
and
experts,
including
scientific
advisory
panels
convened
by
EPA,
have
stressed
that
a
conceptual
framework
different
from
the
one
traditionally
used
to
evaluate
impacts
of
chemical
pesticides,
and
a
different
set
of
questions,
should
serve
as
the
foundation
for
the
ecological
risk
assessment
of
crops
engineered
to
express
toxins
like
Cry
3Bb.
[
Footnote
14:
For
an
overview
of
why
a
new
conceptual
framework
is
needed,
see
comments
by
Dr.
Angelika
Hilbeck
and
Matthias
Meier
on
the
ecological
risks
of
Bt­
transgenic
plants
at
www.
ucsusa.
org/
food/
by_
ren_
app1.
pdf.]
Section
3.1
3
­
26
The
ecological
risk
assessment
of
chemical
pesticides
is
generally
limited
to
acute
lethality
in
test
animals.
Simple
toxicological
experiments
are
designed
to
establish
the
dose
at
which
50
percent
or
90
percent
of
a
test
population
is
killed.
Little
or
no
effort
is
made
to
explore
other
mechanisms
of
toxicity
or
adverse
impacts
other
than
death.
There
are
virtually
no
studies
carried
out
on:

­
Reproduction
and
development,
impacts
that
are
likely
to
occur
at
much
lower
doses
that
acute
poisoning;
[
Footnote
15:
Recent
evidence
of
the
adverse
impact
of
atrazine
on
frog
development
documents
developmental
abnormalities
at
levels
two
to
three
orders
of
magnitude
lower
than
required
to
kill
50
percent
of
adult
frogs
(
Hayes
et
al.,
2002).]

­
Impacts
on
classes
of
nontarget
organisms
that
play
key
roles
in
food
chains
and/
or
plant­
pest­
soil
microbial
community
interactions;

­
Unique
impacts
during
a
portion
of
the
year
or
under
conditions
of
environmental
stress
like
drought,
excessive
heat,
or
in
the
presence
of
soil
mineral
or
chemical
imbalances;
and
­
The
synergistic
impacts
of
exposures
to
multiple
toxins,
chemicals,
and
stresses.

EPA
Response:
Contrary
to
the
commenter's
claims,
the
data
reviewed
on
the
non­
target
organism
impacts
of
MON
863
corn,
do
not
raise
"...
serious
concerns
about
this
product's
risks
to
the
environment".
The`
new
concept'
proposed
by
the
commenter
in
risk
assessment
for
genetically
engineered
plants
is
in
fact
being
implemented
to
supplement
single
species
laboratory
testing.
New
protocols
for
more
appropriate
nontarget
species
are
being
developed
and
a
heavy
reliance
of
ecosystem
effects
as
determined
by
field
surveys
have
been
implemented,
and
additional
protocols
for
large­
scale
multi­
year
field
studies
are
being
developed
concurrently
with
on­
going
field
surveys
on
MON863
corn.
These
field
studies
would
also
detect
reproductive
and
interactive
non­
target
effects.

The
comment
that
"...
a
conceptual
framework
different
from
the
one
traditionally
used
to
evaluate
impacts
of
chemical
pesticides,
and
a
different
set
of
questions,
should
serve
as
the
foundation
for
the
ecological
risk
assessment
of
crops
engineered
to
express
toxins
like
Cry
3Bb"
is
inconsistent
with
the
1999,
2000
and
2002
SAP
comments
which
agreed
with
he
EPA
approach
of
single
species
tier
I
testing
followed
by
field
testing
when
adverse
effects
were
seen.
While
recognizing
that
different
specific
studies
are
appropriate
for
different
B.
t.
crop
risk
assessment,
the
SAPs
do
not
conclude
or
imply
that
EPA's
ecological
risk
assessment
methods
for
these
plants
are
"
fatally
flawed"
(
SAP
Report
No.
99­
06,
February
4,
2000;
SAP
Report
No.
2000­
07.
March
12,
2001;
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
43
Excerpt
Text:
Genetically
engineered
crops
expressing
toxins
in
all
or
most
plant
tissues
expose
nontarget
organisms
within
a
given
agroecosystem
to
qualitatively
and
quantitatively
different
risks
over
a
more
extended
timeframe,
in
contrast
to
chemicals
(
Hilbeck
and
Meier,
2001;
Hilbeck
et
al.,
1999).
An
applied
chemical
Section
3.1
3
­
27
typically
has
a
predictable
environmental
fate
and
the
pest
manager
has
control
over
when
it
is
applied;
it
is
generally
known
where
the
chemical
is
likely
to
wind
up,
whether
and
how
it
breaks
down,
how
long
it
lasts,
and
therefore,
which
organisms
are
exposed
to
what
levels
and
for
how
long.
With
transgenic
organisms
however,
the
environmental
fate
of
toxins
is
much
more
complicated,
since
toxin
levels
are
determined
by
the
development
and
growth
patterns
of
living
organisms.
A
myriad
of
feedback
mechanisms
within
the
environment
govern
plant
development
and
growth,
as
well
as
reproductive
success.
The
farmer
has
little
control
over
these
interactions
once
a
transgenic
crop
is
planted.
For
these
reasons,
the
simple,
one­
dimensional
toxicological
models
EPA
has
adapted
from
its
chemical
testing
and
applied
to
GMO
crops
are
likely
to
fail
to
detect
most
of
the
possibly
important
ecological
impacts
of
transgenic
crops.

Monsanto
studies
on
the
impacts
of
Cry
3Bb
endotoxins
on
nontarget
organisms
suggest
potential
impacts
but
are
far
too
limited
in
sample
size
and
statistical
design
to
support
any
firm
conclusions.

For
example,
the
November
5,
2001
submission
included
a
study
by
Head
et
al.
entitled
"
Insecticidal
Spectrum
of
Activity
for
Cry
3Bb
Protein
in
vitro"
(
Monsanto
Number
455382­
07).
This
study
assesses
impacts
on
a
range
of
nontarget
organisms
including
Coleoptera
from
six
families;
two
species
each
of
Lepidoptera,
Hymenoptera,
and
Neuroptera;
and
one
species
of
Collembola.
Six
to
eight
concentrations
of
Cry
3Bb
toxins
from
1
ppm
to
200
ppm
were
used
in
assays
performed
in
Monsanto's
laboratory
covering
the
Colorado
potato
beetle,
corn
rootworm,
boll
weevil,
corn
earworm,
ECB
and
monarch
butterfly.

It
is
not
possible
to
determine
sample
sizes
and
statistical
reliability
with
the
limited
details
offered
on
the
Standard
Operating
Procedure
(
SOP,
i.
e.,
experimental
protocols),
which
is
covered
in
Appendix
1
of
the
Head
et
al.
document.
The
document
references
the
SOP
to
Stone
et
al.,
1989,
which
is
incorrect,
since
there
is
no
Stone
et
al.,
1989
in
the
references.
There
is
a
similar
reference­
Stone
et
al.,
1991.
In
any
event,
the
SOP
describes
a
method
for
mixing
purified
Cry
3Bb
toxins
into
artificial
media,
which
are
then
fed
to
insects
at
varying
Cry
3Bb
concentrations.
Exposing
insect
larvae
in
a
laboratory
bioassay
to
toxins
via
an
artificial
media
is
very
different
from
larval
exposure
in
crop
fields
as
they
feed
on
plant
tissues
(
Hilbeck
and
Meier,
2001).
It
is
not
possible
to
predict,
at
this
time,
how
relevant
or
accurate
such
bioassays
are
in
determining
the
toxicity
of
MON
863
corn
under
field
conditions.

Monsanto
contracted
with
various
laboratories
to
do
all
other
nontarget
insect
testing
on
key
species
including
honey
bees,
parasitoid
wasps,
ladybird
beetles
and
Collembola.
According
to
the
Head
et
al.
report
(
page
5):

These
assays
were
carried
out
according
to
GLP
at
contract
laboratories
as
part
of
the
nontarget
organism
bioassays
required
for
an
EPA
regulatory
package .
Briefly,
one
or
two
test
concentrations
of
Cry
3Bb
were
used
in
these
assays.
The
maximum
concentration
used
in
each
case
was
based
upon
estimates
of
the
highest
level
of
Cry
3Bb
to
which
that
species
might
be
exposed
if
Cry
3Bbexpressing
crops
were
deployed.

EPA
Response:
The
concept
of
evaluating
multi
year
effects
of
non­
target
invertebrates
in
the
field
and
their
interactions
is
in
fact
being
implemented
to
supplement
single
species
laboratory
testing.
New
protocols
for
more
appropriate
non­
target
species
are
being
developed
and
a
heavy
reliance
of
ecosystem
effects
as
determined
by
field
surveys
have
been
implemented,
and
additional
protocols
for
large­
scale
multi­
year
field
studies
are
Section
3.1
3
­
28
being
developed
concurrently
with
on­
going
field
surveys
on
MON863
corn.
These
field
studies
could
also
detect
reproductive
and
interactive
non­
target
effects.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
44
Excerpt
Text:
The
upper­
end
feeding
rate
­
200
ppm
­
is
just
over
twice
the
maximum
concentration
reported
in
various
corn
tissues.
Ecological
impact
studies
are
typically
carried
out
at
2X,
5X,
and
even
10X
or
more
of
documented
field
concentrations
to
compensate
for
the
generally
low
sample
sizes
used
in
such
experiments
and
in
order
to
establish
a
margin
of
exposure
(
MOE)
at
a
known
effect
level.
The
dose
levels
in
these
contract
studies
are
inadequate
to
establish
MOEs.

Head
et
al.
goes
on
to
explain
that
"
concentration­
mortality
regressions
were
estimated
using
a
probit
model
and
weight
values"
to
estimate
LC50
values.
The
appropriateness
of
such
a
methodology
in
a
laboratory
bioassay
including
just
two
concentration
levels
is
subject
to
question.
Indeed,
in
the
Head
et
al.
study,
the
single
table
reporting
maximum
concentrations
tested
and
estimated
LC50
values
contains
only
two
values
­
one
for
the
Colorado
potato
beetle
and
the
other
for
western
corn
rootworm.

A
March
1,
2001,
study
carried
out
by
Monsanto
scientists
(
Duan
et
al.,
2001)
assessed
the
impact
of
the
variant
Cry
3Bb1
protein
on
larvae
of
the
ladybird
beetle.
The
summary
states:

Test
pollen,
containing
a
variant
of
the
Cry
3Bb1
protein
at
a
level
of
93
ug/
g
fresh
weight
pollen,
was
collected
from
corn
rootworm
protected
corn
event
MON
863.

In
the
Dudin
et
al.
study
on
MON
863
expression
levels,
the
highest
level
reported
of
Cry
3Bb1
protein
in
pollen
was
93
ug/
g.
This
level
was
found
in
the
single
sample
collected
from
U.
S.
grown
corn
(
collected
at
the
Monmouth,
Illinois
test
site).
This
sample
is
a
composite
of
samples
from
many
plants
collected
over
seven
days.
The
Dudin
et
al.
report
does
not
make
clear
how
many
plants
were
sampled
each
day
and
hence
there
is
no
way
of
knowing
the
number
of
plants
in
the
composite
nor
the
variation
in
levels
above
and
below
the
average
composite
level,
93
ug/
g.
But
since
the
93
ug/
g
is
an
average,
it
is
clearly
not
the
maximum
concentration
in
samples
collected.
The
Argentina
pollen
testing
suggests
that
the
range
of
Cry
3Bb1
levels
in
pollen
could
easily
be
twice
the
mean
level,
i.
e.,
some
values
in
a
composite
will
be
one­
half
or
less
the
composite
mean
and
some
will
be
twice
or
more
higher.
Accordingly,
the
actual
maximum
environmental
concentration
documented
in
the
one
U.
S.
test
probably
is
above
150
ug/
g
and
could
approach
200
ug/
g.

In
addition
to
inadequate
dose
levels,
the
sample
sizes
(
10
larvae
per
replicate)
in
the
Duan
et
al.
study
were
also
too
small
to
detect
subtle
impacts.
Appropriate
dose
levels
would
range
up
to
at
least
3­
times
the
maximum
expected
level
in
a
single
plant.
In
addition,
the
study
protocol
was
too
narrow
in
its
exclusive
focus
on
mortality.
Data
are
also
needed,
for
example,
on
the
developmental
and
reproductive
success
of
treated
insects
through
at
least
one
full
generation.

EPA
Response:
Section
3.1
3
­
29
Examination
of
the
submitted
non­
target
studies
shows
that
these
were
performed
with
20x
or
higher
safety
margins,
except
where
pure
pollen
or
plant
tissue
were
used
where
the
concentration
of
the
Cry
protein
cannot
be
increased.
This
high
safety
margin
showing
no
adverse
effects
is
indicative
of
the
absence
of
subtle
effects
at
lower
concentrations
encountered
in
the
field.
However,
additional
multi­
year
field
surveys
are
being
conducted
to
confirm
the
no­
effect
conclusions
drawn
from
high
dose
laboratory
studies.
The
lady
beetle
study
cited
by
the
commenter
was
followed
up
by
three
other
lady
bird
studies
which
showed
no
detrimental
effects.

Commenter
Name:
Jeff
Boeger
Commenter
Organization
Name:
Jeff
Boeger
Comment
Number:
30509B­
092031
Excerpt
Number:
2
Excerpt
Text:
As
for
environmental
effects,
since
corn
in
the
USA
is
grown
exclusively
as
monocultures;
in
environments
with
dissimilar
plants
around
them,
the
chance
of
any
interbreeding
with
native
plant
species
is
very
low.
This
can't
be
said
of
all
crops,
but
it
is
true
of
corn.
The
risk
is
extremely
minimal
compared
to
the
great
good
this
modification
can
do
for
farmers.

EPA
Response:
The
information
available
to
date
indicates
that
the
above
comments
appear
to
be
accurate.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
1
Excerpt
Text:
Many
of
the
public
comments
fail
to
fully
consider
the
collective
evidence
of
laboratory
and
field
studies
submitted
by
Monsanto
that
consistently
demonstrate
no
adverse
effects
of
Cry3Bb1
on
ecologically
and
economically
important
nontarget
species
at
or
above
the
maximum
expected
environmental
concentration
(
MEEC)
to
which
such
organisms
would
be
exposed.
Another
important
consideration,
sometimes
overlooked,
is
the
40­
year
history
of
safe
agricultural
use
of
Bt
products
that
collectively
express
a
diverse
range
of
insecticidal
crystal
proteins.
For
example,
Cry3
Bt
products
(
e.
g.,
Raven
Oil
Flowable
Bioinsecticide)
have
been
used
during
the
past
decade
in
traditional
and
organic
agricultural
systems.
Finally,
any
analysis
of
the
potential
risks
and
benefits
for
a
new
technology
must
be
considered
in
the
context
of
existing
pest
management
practices
and
systems.
The
Cry3Bb1
protein
produced
in
MON
863
corn
is
less
toxic
to
nontarget
organisms
than
all
organophosphate
and
pyrethrold
insecticides
currently
registered
and
commonly
used
to
control
corn
pests,
including
CRW.
In
addition
to
the
low
toxicity
of
the
Cry3Bbl
protein,
the
in­
planta
delivery
system
reduces
exposure
to
nontarget
organisms
since
they
must
ingest
the
plant
tissue
to
be
exposed.

EPA
Response:
The
data
received
by
EPA
to
date
show
that
the
above
comments
appear
to
be
accurate.
Additional
and
long­
range
confirmatory
studies
are
in
progress.

Commenter
Name:
Dennis
Ward
Section
3.1
3
­
30
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
3
Excerpt
Text:
Comment
or
Question:
Nontarget
organism
studies
were
not
conducted
for
important
beetle
species
closely
related
to
the
target
pest
(
also
a
beetle).

Scientific
Background:
Laboratory
studies
with
nontarget
beetle
species
closely
related
to
CRW
(
family
Chrysomelidae),
an
analysis
of
potential
impacts
to
endangered
beetles
from
three
additional
families,
as
well
as
multi­
location
field
investigations
by
Monsanto
and
university
researchers,
demonstrate
that
beetles
in
only
the
family
Chrysornelidae
are
susceptible
to
the
Cry3Bb1
protein.
Monsanto's
initial
data
package
included
the
nontarget
test
species
as
required
in
the
EPA
Subdivision
M
(
1989)
and
OPPTS
(
1996)
guidelines.
Pursuant
to
EPA's
Science
Advisory
Panel
recommendation
[
Footnote
3:
SAP
report
No.
2000­
7,
March
12,
2001],
Monsanto
generated
additional
toxicity
information
for
species
closely
related
to
the
target
species
(
corn
rootworm
beetles
in
this
case).
Initially,
one
species
of
beetle
was
tested
(
adult
Hippodamia
convergens)
and
no
effects
were
observed
at
8,000
ppin
Cry3Bb1
protein
in
the
diet.
Subsequent
studies
were
conducted
with
adult
and
larvae
ladybird
beetles
of
another
species,
Coleomegilla
maculata.
No
adverse
effects
were
observed
in
this
specie
when
fed
MON
863
pollen
at
up
to
50%
of
the
diet
(
the
maximum
exposure
level
in
the
field).

In
addition
to
data
on
Coccinellidae
(
nontarget)
and
Chrysomelidae
(
target
species),
Monsanto
has
conducted
studies
on
representative
species
within
the
Bruchidae,
Tenebrionidae,
and
Curculionidae
families.
[
Footnote
4:
Monsanto
report
C3NTO;
MRID
455382­
07]
These
data
demonstrate
minimal
potential
risk
for
endangered
beetle
species.

Finally,
field
investigations
were
initiated
in
year
2000
by
Monsanto.
as
well
as
by
several
university
researchers.
across
multiple
locations
to
validate
the
prediction
of
minimal
risk­
to
nontarget
beetles
in
the
field
based
on
the
laboratory
data.
An
interim
report
summarizing
the
data
collected
for
key
nontarget
and
target
species
was
submitted
to
EPA
in
2001.
[
Footnote
5:
Monsanto
report
99­
894E;
MRID
453484­
02]
These
data
indicate
no
significant
adverse
effects
of
MON
863
on
carabid
beetles
or
other
nontarget
beetles,
while
adverse
effects
of
currently
used
conventional
insecticides
were
identified.
Thus,
the
field
data
validate
the
results
of
the
maximum
hazard
dose
laboratory
studies
and
demonstrate
that
MON
863
poses
minimal
risk
to
nontarget
organisms,
including
beetle
families
common1y
found
in
agricultural
settings.

EPA
Response:
The
data
received
by
EPA
to
date
show
that
the
above
comments
appear
to
be
accurate.
Additional
and
long­
range
confirmatory
studies
are
in
progress.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
4
Excerpt
Text:
Comment
or
Question:
Additional
nematode
studies
are
needed
since
preliminary
delta
showed
significant
Section
3.1
3
­
31
nematode
population
reductions
in
a
field
study.

Scientific
Background:
Any
reduction
in
the
pest
nematode
population
related
to
use
of
MON
863
would
represent
an
unexpected
benefit
rather
than
an
unintended
ecological
risk.
Consequently,
additional
maximum
hazard
dose
studies
with
pest
and
other
nontarget
nematodes
would
not
be
necessary.
A
preliminary
report
indicated
the
potential
for
corn
expressing
Cry3Bb1
to
impact
certain
nematode
species.
[
Footnote
6:
Monsanto
report
00­
CR­
032E­
7,
Appendix
F,
MRID
456530­
03]
However,
to
put
the
observation
in
proper
context,
the
author
of
the
report
stated
that
the
results
of
this
preliminary
study
"...
make
it
seem
very
unlikely
that
Bt
corn
would
have
an
impact
on
nematode
fauna
not
associated
with
the
corn
plant
itself,
in
other
words
a
nematode
that
feeds
on
the
plant
in
some
way."
­
This
qualification
makes
it
clear
that
the
potential
for
impacts
on
nematodes
is
focused
on
nematode
corn
pest
species;
consequently,
any
effects
would
represent
a
potential
benefit
rather
than
an
unintended
risk.
Based
on
this
preliminary
research
and
the
researcher's
statement,
there
is
no
basis
for
conducting
additional
nontarget
organism
studies
with
nematodes.

EPA
Response:
EPA
does
not
believe
that
impacts
on
nematodes
are
sufficient
to
constitute
an
unreasonable
adverse
effect.
Nonetheless,
to
address
this
issue
conclusively,
EPA
has
requested
additional
data
in
order
to
make
a
risk
assessment.

Commenter
Name:
Lance
Meinke
Commenter
Organization
Name:
NCR­
46
Memebers
Comment
Number:
OPP02­
0016
Excerpt
Number:
9
Excerpt
Text:
­
Both
corn
rootworm
adults
and
larvae
feed
on
the
corn
plant,
which
potentially
exposes
both
life
stages
to
any
toxin
that
is
expressed
throughout
the
plant.
This
could
lead
to
extended
selection
for
resistance.
In
contrast,
only
ECB
larvae
are
exposed
to
CrylAb
as
ECB
moths
do
not
feed
on
corn
tissue.

EPA
Response:
The
above­
mentioned
resistance
issue
is
addressed
in
detail
in
EPA's
Insect
Resistance
Management
section
of
this
document.

Commenter
Name:
Clifford
Habig
Commenter
Organization
Name:
Exponent,
Inc.
Comment
Number:
OPP02­
0017
Excerpt
Number:
3
Excerpt
Text:
EPA
currently
requires
a
suite
of
laboratory
studies
to
support
the
registration
of
PIPs.
Several
of
these
studies,
such
as
those
conducted
on
avian
and
aquatic
invertebrate
species,
flow
directly
from
standard
chemical
and
microbial
testing
on
non­
target
organisms.
Interestingly,
EPA's
evaluation
of
conventional
chemical
pesticides
includes
very
few
data
requirements
concerning
non­
target
insects
and
soil
invertebrates,
whereas
the
microbial
testing
guidelines
require
more
extensive
testing
of
beneficial
insects
and
soil
­
invertebrates.
Similar
to
microbial
pesticides,
most
non­
target
testing
with
PIPs
has
focused
on
non­
target
insects
and
soil
invertebrate
species.
These
data
requirements
represent
a
refinement
of
the
Section
3.1
3
­
32
Series
885
Microbial
Testing
Guidelines,
which
require
testing
on
several
non­
target
insect
species,
including
honeybees,
and
a
representative
series
of
beneficial
insects,
such
as
lady
bird
beetles
and
lacewings.

EPA
guidance
and
representative
testing
protocols
for
non­
target
insects
are
not
very
detailed.
Guidance
on
duration
of
the
tests,
type
of
testing
material,
appropriate
test
endpoints,
and
standard
acceptability
criteria
are
limited
and
registrants
must
interpret
the
available
guidance
when
designing
studies.
The
current
studies
are
different
from
many
of
the
traditional
chemical
beneficial
insect
tests,
such
as
those
used
in
Europe,
in
that
the
PIP
tests
focus
on
dietary
exposure
rather
than
contact
and
spray
exposure,
and
many
of
the
PIP
non­
target
insect
tests
tend
to
be
conducted
for
longer
exposure.
times.
For
example,
beneficial
insects
in
PIP
testing
are
typically
exposed
to
high
concentrations
of
dietary
Bt
proteins
as
the
sole
dietary
source,
for
10
to
30
days,
depending
on
the
test
species.
There
are
also
questions
concerning
the
appropriate
selection
of
laboratory
test
organisms.
Given
the
small
number
of
validated
protocols
for
nontarget
insect
testing,
one
possible
approach
involves
having
a
core
set
of
test
species
for
all
PIPs
that
is
supplemented
by
additional
species
representative
of
those
groups
most
likely
to
be
impacted
by
the
specific
plant
pesticide
product.

EPA
Response:
The
registrants
do
not
need
to
be
in
the
dark
about
interpretation
of
the
available
guidance
for
protocol
development.
They
are
not
left
without
guidance
in
that
the
case­
by­
case
protocol
developed
includes
consultation
and
guidance
from
EPA
scientists.
The
selection
of
a
core
of
appropriate
non­
target
species
to
be
tested
has
been
addressed
in
several
workshops
and
research
proposals
by
the
EPA
and
USDA.
In
addition,
in
2003
EPA
has
commenced
the
development
of
testing
guidelines
specifically
for
PIPs.

Commenter
Name:
Clifford
Habig
Commenter
Organization
Name:
Exponent,
Inc.
Comment
Number:
OPP02­
0017
Excerpt
Number:
4
Excerpt
Text:
EPA
has
traditionally
employed
a
risk
quotient
approach
to
risk
assessment,
in
which
toxicity
endpoints
are
compared
to
estimated
exposure
to
determine
whether
an
adequate
margin
of
safety
exists
for
labeled
or
proposed
uses
of
a
product.
Toxicity
end
points
used
for
acute
risk
assessments
are
typically
LD50,
LC50,
or
EC50
values,
while
toxicity
endpoints
used
for
subchronic
or
chronic
(
longer­
term)
risk
assessments
are
typically
no­
observed
effect
concentrations
(
NOECs)
or
no­
observed
adverse
effect
concentrations
(
NOAECs).
In
rare
cases,
sufficient
data
are
available
to
calculate
ECX
values
(
e.
g.,
EC05,
EC
10,
EC20)
to
use
as
toxicity
endpoints
for
risk
assessment.
Exposures
of
non­
target
organisms
are
typically
estimated
by
environmental
modeling
techniques
or
calculations
based
on
generic
databases.
Risk
assessments
are
conducted
based
on
the
results
of
laboratory
testing
and
the
estimated
exposure;
field
testing
data,
when
available
is
primarily
used
to
collaborate
or
confirm
risk
conclusions
based
on
laboratory­
derived
data.

Non­
target
organism
risk
assessments
for
conventional
chemical
pesticides
are
conducted
for
birds,
mammals,
and
aquatic
organisms
(
fish
and
aquatic
invertebrates).
For
conventional
chemical
pesticides,
risk
assessments
for
terrestrial
arthropods
and
soil
invertebrates
are
limited
to
a
hazard
assessment
for
honeybees.
Toxicity
endpoints
are
typically
selected
based
on
data
for
the
most
sensitive
species
tested
Section
3.1
3
­
33
among
representative
species
for
each
taxonomic
group
evaluated.
Assessments
of
multiple
application
scenarios
are
generally
based
on
a
longer­
term
exposure
estimate
that
incorporates
a
dissipation
component
following
each
application.

Risk
assessments
for
conventional
microbial
pesticides
are
conducted
for
birds,
mammals,
aquatic
organisms,
and
non­
target
terrestrial
arthropods
and
soil
invertebrates.
However,
these
risk
assessments
are
generally
simplified
by
using
the
maximum
hazard
dose
approach
to
testing,
so
that
if
no
adverse
effects
occur
at
the
maximum
hazard
dose
(
concentration),
the
product
is
determined
to
present
low
risk
to
non­
target
organisms.
Thus,
for
microbial
products,
the
exposure
component
of
the
risk
assessment
is
built
into
the
dosing
regime
used
for
testing.

The
nature
of
PlPs
results
in
unique
exposure
scenarios
compared
to
conventional
chemical
or
microbial
pesticides.
Exposures
to
conventional
products
focus
on
off­
site
movement
of
pesticide
residues
to
water
bodies
and
estimated
residues
in/
on
feed
items
and
in
soil.
For
PIPs,
the
specificity
of
the
plantincorporated
proteins
and
incorporation
into
specific
plant
tissues
focuses
exposure
of
non­
target
organisms
on
dietary
ingestion
of
plant
products
and
exposure
in
soil.
Patterns
of
expression
of
PIPs
in
plant
tissues
over
the
course
of
the
growing
season
represent
a
key
component
of
the
exposure
assessment;
depending
on
the
pattern
of
expression
some
off­
site
exposure
(
eg.,
pollen
expression)
is
also
possible.
However,
off­
site
movement
through
processes
such
as
drift
or
runoff
is
greatly
reduced
compared
to
conventional
products,
resulting
in
reduced
risk
to
several
key
groups
of
non­
target
organisms.

EPA
Response:
This
comment
does
not
require
a
response.

Commenter
Name:
Clifford
Habig
Commenter
Organization
Name:
Exponent,
Inc.
Comment
Number:
OPP02­
0017
Excerpt
Number:
9
Excerpt
Text:
Conclusions
­
Non­
target
organism
testing
requirements
for
PIPs
should
include
both
a
core
set
of
studies
required
for
all
PIPs
and
a
supplemental
set
of
studies
that
reflect
the
specific
properties
of
any
given
PIP.
The
testing
strategy
for
MON
863
followed
this
type
of
approach.
­
Current
testing
and
risk
assessment
guidelines
for
PIPs,
with
the
exception
of
the
field
data
requirements,
are
similar
to
well
tested
processes
for
conventional
chemical
and
microbial
pesticides.
Risk
assessment
procedures
used
for
MON
863
follow
this
guidance.
­
Risk
assessment
decisions
for
PIPs
should
be
consider
not
only
the
overall
safety
of
the
individual
PIP
product,
but
should
also
be
compared
to
risks
associated
with
existing
conventional
pest
control
technologies.

EPA
Response:
This
comment
does
not
require
a
response.

Commenter
Name:
Monsanto
Section
3.1
3
­
34
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
2
Excerpt
Text:
In
summary,
Monsanto
supports
a
scientific
approach
to
risk
assessment
where
well
designed
laboratory
studies
with
surrogate
species
that
represent
the
spectrum
of
potentially
exposed
species
are
used
in
an
early
tier
assessment
of
ecological
risk
for
Cry
proteins.
If
suitable
laboratory
surrogate
species
tests
are
not
available
then
semi­
field
or
field
studies
focused
on
particular
indicator
organisms
may
be
appropriate.
If
field
studies
are
undertaken,
the
specific
questions
to
be
answered
should
be
clearly
stated
prior
to
initiation.

EPA
Response:
This
commenter
mirrors
the
approach
that
EPA
is
taking
in
non­
target
risk
assessment
for
PIPs.
The
comments
from
the
October
2000
and
August
2002
SAP
also
endorse
this
approach.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
4
Excerpt
Text:
Question
2:
Duration
of
Field
Abundance
Studies
Please
comment
on
the
adequacy
of
the
2­
year
field
abundance
study
for
making
determination
of
the
potential
risks
from
commercial
use
of
event
MON
863.

Response:
Monsanto
believes
that
a
two­
year
field
abundance
study
is
adequate
for
assessing
the
risk
of
corn
event
MON
863
to
nontarget
organisms.
Results
from
the
first
and
second
year
field
study
conducted
by
Monsanto
indicated
no
adverse
effects
in
key
beneficial
insects
including
families
of
nontarget
beetles.
Collaborative
nontarget
organism
studies
have
been
conducted
with
university
researchers
in
Illinois,
Iowa,
Kansas,
Nebraska,
New
York,
and
South
Dakota.
The
results
of
these
studies
were
consistent
across
sites
and
across
years,
and
confirmed
the
safety
of
the
MON
863
event
to
nontarget
organisms.
[
Footnote
5:
Head,
G.
(
2002).
Research
on
the
Effects
of
Corn
Rootworm
Protected
Transgenic
Corn
Events
on
Nontarget
Organisms:
Preliminary
Results.
An
unpublished
report
prepared
by
Monsanto
Company.
MRID
456530­
03]

A
thorough
laboratory­
based
risk
assessment
using
quail,
earthworms,
Collembola,
ladybird
beetles,
parasitoid
wasps,
lacewing,
honeybees,
monarch
butterfly,
daphnia
and
catfish,
demonstrated
minimal
risk
of
MON
863
to
nontarget
organisms.
The
field
study
undertaken
by
Monsanto
to
provided
additional
information
to
support
the
laboratory
based
risk
assessment,
especially
for
nontarget
beetles.
The
field
study
was
conducted
in
Monmouth,
Illinois
and
utilized
60x60
ft
plots
replicated
four
times
per
treatment.
Conventional
corn
rootworm
control
programs
using
seed
insecticide,
soil
incorporated
insecticide
and
foliar
insecticide
treatments
were
included.
The
first
year
of
the
Monsanto
data
for
key
beneficial
insects
was
collated
and
summarized
in
an
interim
report
that
was
submitted
to
EPA.
These
data
showed
no
adverse
effects
on
important
beneficial
insects
and
closely
related
nontarget
insects.
[
Footnote
8:
Bhatti,
M.
A.,
C.
L.
Pilcher,
M.
J.
McKee,
T.
E.
Nickson,
G.
P.
Head
and
C.
D.
Pilcher
(
2001).
Field
Evaluation
for
Section
3.1
3
­
35
the
Ecological
Impact
of
Corn
Rootworm
Insect­
Protected
Corn
on
Nontarget
Organisms.
Report
MSL­
17179,
an
unpublished
study
conducted
by
Monsanto
Company.
MRID
455382­
06.]
The
second
year
of
field
data
have
been
compiled
and
it
is
our
conclusion
that
the
findings
confirm
the
first
year
data
in
that
there
were
no
adverse
effects
observed
in
the
second
year.
[
Footnote
7:
Preliminary
results
for
two­
year
field
study,
Monsanto
unpublished
data
(
2002)]

EPA
Response:
EPA
has
reviewed
the
two­
year
field
data
summarized
by
this
commenter
for
the
registration
of
MON
863.
However,
with
the
advice
of
the
SAP
and
other
public
commenters,
EPA
is
requiring
studies
that
extend
beyond
a
two
year
period.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
31
Excerpt
Text:
A)
Please
comment
on
the
relative
strengths
and
weaknesses
of
such
field
data
vs.
laboratory
feeding
studies
performed
on
a
limited
number
of
indicator
organisms,
for
purposes
of
hazard
assessment.

Response:
Monsanto
supports
a
scientific
approach
to
risk
assessment
where
a
well­
designed
laboratory
assessment
with
surrogate
species
that
represent
the
spectrum
of
potentially
exposed
species
is
used
in
a
tiered
assessment
of
ecological
risk
for
Cry
proteins.
If
suitable
laboratory
surrogate
species
tests
are
not
available,
or
if
adverse
effects
are
observed
in
these
laboratory
studies,
then
semi­
field
or
field
studies
focused
on
particular
indicator
organisms
may
be
appropriate.
In
the
case
of
the
Cry3Bb1
protein
analysis,
a
field
assessment
was
conducted
to
provide
supplemental
information
and
reinforce
the
minimal
risk
conclusion
reached
based
on
the
laboratory
studies.
Since
this
is
a
beetle­
active
Cry
protein,
additional
information
was
desired
for
nontarget
Coleoptera
species
closely
associated
with
the
soil
environment.
Field
studies
were
conducted
to
address
potential
effects
on
nontarget
beetles,
such
as
staphylinid
or
carabid
beetles.
Results
of
the
field
study
indicated
no
adverse
effects
on
nontarget
beetle
or
other
nontarget
insect
populations.
[
Footnote
2:
Bhatti,
M.
A.,
C.
L.
Pilcher,
M.
J.
McKee,
T.
E.
Nickson,
G.
P.
Head
and
C.
D.
Pilcher
(
2001).
Field
Evaluation
for
the
Ecological
Impact
of
Corn
Rootworm
Insect­
Protectant
Corn
on
Nontarget
Organisms.
Report
MSL­
17179,
an
unpublished
study
conducted
by
Monsanto
Company.
MRID
455382­
06.]

Laboratory
and
field
approaches
to
nontarget
organism
risk
assessment
are
valid
and
complementary.
Traditionally,
data
collection
and
risk
assessment
are
approached
in
a
tiered
analysis,
with
higher
tier
studies
being
conducted
where
risk
is
indicated
at
the
lower
tiers.
Monsanto
believes
that
well
designed
laboratory
assessments
with
surrogate
species,
representing
the
spectrum
of
potentially
exposed
species,
are
a
valid
method
to
assess
ecological
risk
of
Cry
proteins.
If
laboratory
testing
guidelines/
study
protocols
are
not
available
for
certain
surrogate
species,
then
small­
scale
semi­
field
or
field
studies
could
be
used
to
supplement
the
laboratory
data.
Though
not
necessary
or
required
for
the
Cry3Bb1
protein
analysis,
a
field
assessment
was
conducted
to
provide
supplemental
information
and
reinforce
the
minimal
risk
conclusion
from
the
laboratory
assessment.

EPA
Response:
Section
3.1
3
­
36
EPA
supports
the
proposal
to
perform
laboratory
non­
target
effects
testing
supplemented
by
field
survey
data
on
those
invertebrates
that
cannot
be
propagated
in
a
laboratory
setting.
The
August
2002
SAP
has
also
recommended
this
approach.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
32
Excerpt
Text:
Laboratory
studies,
compared
to
field
studies,
have
the
advantage
of
rigorous
control
of
the
amount
of
protein
to
which
the
organisms
are
exposed.
In
the
nontarget
organism
laboratory
studies,
the
diet
containing
the
Bt
protein
is
fed
directly
to
the
nontarget
organism.
The
bioactivity
of
the
protein
is
confirmed
in
the
diet
using
a
known
susceptible
species.
A
laboratory
test
can
more
adequately
characterize
the
hazard
potential
(
intrinsic
toxicity)
of
the
protein
than
in
a
field
study
since
the
ingestion
of
the
material
in
the
diet
can
be
readily
observed/
monitored.
Using
results
from
these
studies
and
'
comparing
them
to
conservative
estimates
of
field
exposure
provides
a
highly
conservative
approach
to
assessing
ecological
risk.
[
Footnote
3:
Suter,
G.
W.
(
1993).
Ecological
Risk
Assessment.
Lewis
Publishers,
Chelsea,
Michigan]
[
Footnote
4:
EPA(
1998).
Guidelines
for
Ecological
Risk
Assessment.
EPA/
630/
R­
95/
002F,
April,
1998.
Risk
Assessment
Forum,
U.
S.
Environmental
Protection
Agency,
Washington,
DC]
A
potential
disadvantage
of
laboratory
testing
is
that
validated
testing
protocols
may
not
be
available
for
all
taxonomic
groups
that
need
to
be
included
to
meet
the
objectives
of
the
ecological
risk
assessment.

For
the
Cry3Bb1
protein,
the
focus
of
the
laboratory
analysis
was
on
beneficial
arthropods,
particularly
pollen
feeders,
and
soil
invertebrates.
For
pollen
feeders,
standard
laboratory
tests
available
to
assess
potential
effects
of
Bt
proteins,
including
honeybee
larva
and
adults,
are
ladybird
beetle
larvae
and
adults,
and
adult
parasitoid
wasp.
These
nontarget
organisms
were
not
affected
at
the
maximum
estimated
exposure
level
Cry3Bb1
protein
in
the
field.
Where
no
effects
are
observed
in
organisms
ingesting
proteins
at
levels
above
what
they
will
see
in
the
field,
minimal
risk
is
indicated.

EPA
Response:
The
above
commenter
is
summarizing
the
EPA
approach
to
non­
target
risk
assessment;
no
response
is
necessary.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
33
Excerpt
Text:
For
soil
invertebrates,
standard
tests
are
available
for
Collembola
(
soil
detritivore)
and
earthworms,
as
these
species
are
routinely
used
to
assess
the
toxicity
of
chemical
insecticides.
Results
of
the
studies
where
Collembola
were
fed
tissues
containing
the
Cry3Bbl
protein
and
earthworm
were
exposed
to
the
protein
in
the
soil,
clearly
indicated
that
no
toxicity
would
be
expected
at
maximum
estimated
exposure
levels
in
the
field.

Typically,
field
studies
are
hi­
her
tier
and
would
only
be
conducted
where
risk
is
not
excluded
at
lower
Section
3.1
3
­
37
tiers.
Minimal
risk
of
Cry3Bbl
protein
to
nontarget
organisms
is
concluded
from
the
standard
laboratory
assessment.
However,
since
this
is
a
beetle­
active
Cry
protein,
Monsanto
desired
additional
information
for
nontarget
Coleoptera
species
closely
associated
with
the
soil
environment.
Since
reliable
and
validated
laboratory
feeding
studies
for
nontarget
Coleoptera
such
as
beetles
in
the
families
Carabidae
and
Staphyllinidae
were
not
available,
field
studies
were
conducted
to
address
potential
effects
on
these
nontarget
beetles,
such
as
staphylinid
or
carabid
beetles.
Results
of
the
field
study
indicated
no
unexpected
effects
on
nontarget
beetles
or
any
other
nontarget
organism.
[
Footnote
2:
Bhatti,
M.
A.,
C.
L.
Pilcher,
M.
J.
McKee,
T.
E.
Nickson,
G.
P.
Head
and
C.
D.
Pilcher
(
2001).
Field
Evaluation
for
the
Ecological
Impact
of
Corn
Rootworm
Insect­
Protectant
Corn
on
Nontarget
Organisms.
Report
MSL­
17179,
an
unpublished
study
conducted
by
Monsanto
Company.
MRID
455382­
06.]

In
addition
to
Monsanto
conducted
studies,
a
number
of
collaborative
nontarget
studies
also
have
been
conducted
with
university
researchers
in
Illinois,
Iowa,
Kansas,
Nebraska,
New
York,
and
South
Dakota.
All
studies
compared
the
impacts
of
MON
863
corn
expressing
the
Cry3Bbl
protein,
with
a
conventional
corn
isoline
and
the
same
isoline
treated
with
the
soil
insecticide
used
locally
to
control
corn
rootworm.
These
studies
included
broad
surveys
of
the
arthropods
present,
as
well
as
studies
that
concentrated
on
particular
taxonomic
groups
like
carabids.
Plot
sizes
were
greater
than
an
acre
in
several
cases.
In
all
of
these
studies,
no
consistent
significant
differences
have
been
seen
between
the
MON
863
plots
and
those
with
the
untreated
isoline
with
respect
to
any
nontarget
species.
[
Footnote
5:
Head,
G.
(
2002).
Research
on
the
Effects
of
Corn
Rootworm
Protected
Transgenic
Corn
Events
on
Nontarget
Organisms:
Preliminary
Results.
An
unpublished
report
prepared
by
Monsanto
Company.
MRID
456530­
03]
However,
the
soil
insecticides
adversely
impacted
a
number
of
nontarget
species.
Thus,
the
results
of
these
studies
were
as
predicted
from
the
Tier
1
tests.

EPA
Response:
EPA
has
reviewed
the
studies
described
in
this
comment
and
incorporated
the
findings
into
the
MON
863
environmental
risk
assessment.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
34
Excerpt
Text:
B)
The
panel
is
requested
to
comment
on
the
logistics,
validity,
cost
and
expected
scientific
gain,
if
any,
of
conducting
a
census
of
the
invertebrate
community
vs.
concentrating
the
studies
on
specific
indicator
organisms.
In
addition,
please
comment
on
the
suggested
indicator
groups
such
as
Carabids
and
Staphyllinids
in
the
case
of
Cry3Bb1,
that
would
be
most
likely
to
provide
the
Agency
with
meaningful
data
for
assessing
potential
hazards
to
nontarget
invertebrates
from
corn
rootworm
PIPS.

Response:
Monsanto
believes
the
value
of
conducting
an
in­
field
census
analysis
of
nontarget
arthropods
is
limited
because
much
of
the
sample
collection,
handling
and
identification
resources
are
expended
on
species
already
well
documented
to
be
at
minimal
risk
and
the
variability
in
field
populations
is
high,
thereby
making
it
difficult
to
draw
conclusions
from
field
insect
population
studies.
Therefore,
there
would
be
limited
scientific
benefit
for
studies
that
are
very
time
and
resource
intensive
to
conduct.
Monsanto
believes
that
of
appropriate
surrogates
for
potentially
exposed
species
is
an
acceptable
way
to
approach
the
Section
3.1
3
­
38
ecological
risk
assessment.
If
potentially
exposed
and
susceptible
species
are
identified
and
cannot
be
tested
in
laboratory
assays,
then
focused
semi­
field
or
field
studies
would
be
a
reasonable
alternative.
The
interpretation
of
any
semi­
field
or
field
studies
should
be
made
relative
to
the
conventional
pest
management
program.
As
more
knowledge
is
gained
from
current
laboratory
and
field
assessment
programs,
the
need
for
field
studies
to
assess
the
ecological
risk
of
plant­
incorporated
protectants
should
decrease.

Census
of
the
invertebrate
community
requires
collection
of
invertebrate
samples,
analysis
of
specimens
and
interpretation
of
data.
Arthropod
communities
are
assemblages
resulting
from
complex
interactions
with
the
physical
environment
as
well
as
with
other
species.
For
a
community
analysis,
interpretation
of
census
data
must
consider
these
interactions
as
well
as
biases
created
by
the
type
of
collection
method
used
(
i.
e.,
some
species
are
more
likely
to
be
trapped
by
some
methods
than
other
species)
and
by
taxonomic
resolution.
Ideally
all
specimens
should
be
identified
to
the
species
level,
however,
the
taxonomy
is
sometimes
complex
or
expertise
may
be
limited.
Exposure
of
organisms
to­
the
protein
in
the
field
is
also
complex
which
makes
it
difficult
to
relate
changes
in
the
trapping
success
for
individual
species
to
exposure.
Interpretation
of
in­
field
census
data
is
often
complicated
by
a
lack
of
understanding
for
the
impacts
of
agricultural
processes
such
as
present
and
past
tillage
practices,
fertilizer
inputs,
and
crop
varieties
on
arthropod
populations.
Many
of
these
factors
have
caused
difficulties
in
the
interpretation
of
field
data
in
EPA's
Office
of
Pesticide
Program
initiative
to
use
field
tests
to
assess
impacts
of
chemical
pesticides
(
e.
g.,
mesocosm
assessments).
In
balance,
minimal
scientific
gain
relative
to
risk
assessment
will
be
obtained
by
conducting
a
census
of
in­
field
arthropod
communities
for
Cry
proteins.

EPA
Response:
The
above
comments
are
reasonably
consistent
with
the
conclusions
of
the
August
2002
SAP.
As
a
result
efforts
are
under
way
to
identify
indicator
invertebrate
species
or
physiological
groups
that
can
be
identified
in
semi­
field
studies.
Protocols
for
these
and
for
full
field
studies
with
appropriate
statistical
power
are
in
various
stages
of
development.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
35
Excerpt
Text:
Where
additional
information
is
considered
necessary
to
supplement
the
standard
laboratory
assessment,
the
information
can
be
obtained
through
additional
laboratory
testing
or
through
focused
semi­
field
or
field
studies.
For
the
Cry3Bb1
protein,
prior
laboratory
testing
indicated
that
only
beetles
are
highly
susceptible
to
the
Cry3Bb1
protein,
specifically
beetles
in
the
family
Chrysomellidae.
[
Footnote
6:
Head,
G.,
M.
Pleau,
S.
Sivausupramanian
and
T.
Vaughn
(
2001).
Insecticidal
Spectrum
of
Activity
for
CryBb1
Protein
in
vitro.
Report
C3NTO,
an
unpublished
study
conducted
for
Monsanto
Company.
MRID
455382­
07]
Therefore,
potential
risk
to
nontarget
species
closely
related
to
the
target
organism
was
included
by
Monsanto
as
part
of
the
assessment.

One
taxonomic
group
specifically
focused
on
in
the
Cry3Bb1
protein
assessment
was
ladybird
beetles,
since
these
species
are
important
biological
control
agents
in
agricultural
ecosystems.
To
evaluate
potential
effects
of
Cry3Bb1
protein
on
ladybird
beetles
Monsanto
conducted
four
laboratory
studies
with
both
Section
3.1
3
­
39
purified
Cry3Bb1
protein
and
MON
863
pollen
using
two
species,
Hippodamia
convergens
and
Coleomegilla
maculata.
Additionally,
Monsanto
conducted
a
two­
year
field
study
that
estimated
the
abundance
of
nontarget
organisms
in
the
soil,
on
the
soil
surface
and
on
foliage.
Five
species
of
ladybird
beetle,
including
Harmonia
axyridis,
Coccinella
septenipunctata,
H.
convergens,
Cycloneda
munda
and
C.
maculata
were
assessed
in
this
field
study.
These
data
clearly
demonstrate
that
larval
and
adult
ladybird
beetles
will
not
be
at
risk
from
the
corn
rootworm
product.
The
adoption
of
MON
863
will
be
a
clear
benefit
to
in­
field
ladybird
beetle
populations
where
adult
corn
rootworm
foliar
insecticide
control
programs
significantly
can
reduce
ladybird
beetle
populations.
[
Footnote
7:
Preliminary
results
for
twoyear
field
study,
Monsanto
unpublished
data
(
2002)]

Other
nontarget
Coleoptera
taxa
that
could
be
potentially
exposed
include
carabid
and
staphyllinid
beetles.
Since
dietary
feeding
test
guidelines
for
representatives
of
these
families
were
not
available,
Monsanto
collected
information
on
their
general
abundance
in
cornfields.
Results
of
these
studies
indicate
that
abundance
of
nontarget
Coleoptera
families
was
no
different
on
MON
863
compared
with
the
untreated
nontransgenic
corn
isoline.
Conventional
insecticide
chemical
the
control
programs
did
tend
to
decrease
the
abundance
of
some
of
these
nontarget
Coleoptera
families.

EPA
Response:
EPA
has
reviewed
the
studies
described
in
this
comment
and
incorporated
the
findings
into
the
MON
863
environmental
risk
assessment.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
36
Excerpt
Text:
Question
3:
Green
Lacewing
Larva
Test
The
Agency
solicits
the
Panel's
comments
on
an
appropriate
design
for
evaluating
the
toxicity
of
Cry3Bb1
proteins
to
lacewing
larvae.

Response:
Monsanto
believes
that
the
green
lacewing
study
conducted
with
Cry3Bb1
protein
treated
moth
egg
diet
is
both
practically
and
scientifically
justified.
The
no
observable
effect
concentration
(
NOEC)
from
the
lacewing
study
greatly
exceeds
the
maximum
expected
environmental
concentration
(
MEEC),
indicating
minimal
risk
from
exposure
to
MON
863.
Results
of
field­
scale
assessment
of
nontarget
organism
abundance
(
see
question
2)
confirm
minimal
risk
to
green
lacewing
larvae
under
field
conditions.

The
green
lacewing
study
conducted
by
Monsanto
involved
exposing
newly
hatched
larvae
to
moth
egg
diet
treated
with
purified
Cry3Bbl
protein
at
the
maximum
diet
concentration
of
8,000
ppm
for
a
period
of
ten
days.
To
determine
if
this
exposure
regime
was
capable
of
detecting
an
adverse
effect
of
the
test
substance,
moth
ego,
diet
treated
with
arsenate
(
a
known
stomach
poison)
was
concurrently
tested
as
a
positive
control.
No
significant
differences
in
mortality
were
detected
between
Cry3Bb1
protein
and
water
control
diet
treatments
(
23%
vs
27%),
while
the
arsenate
diet
treatments
resulted
in
significant
mortality
of
the
test
larvae
(
43%
at
1,000
ppm
and
100%
at
10,000
ppm).
The
significant
mortality
of
the
lacewing
larvae
exposed
to
the
arsenate­
treated
diets
indicated
that
the
test
system
used
in
the
study
was
capable
of
Section
3.1
3
­
40
detecting
adverse
effects
of
a
toxicant
applied
to
the
moth
egg
diet.

Arsenical
compounds
have
historically
been
designated
as
insect
stomach
poisons,
as
opposed
to
contact
poisons,
because
contact
with
arsenicals
does
not
provided
control
of
insect
pests;
the
insect
pests
must
ingest
these
toxins
to
achieve
adequate
control.
[
Footnote
9:
Metcalf,
C.,
W.
Flint
and
R.
Metcalf
(
1962).
Pp.
1087.
In
Destructive
and
Useful
Insects:
Their
Habits
and
control.
McGraw­
Hill
Book
Company,
New
York,
New
York.]
Although
this
observation
does
not
preclude
the
possibility
of
dermal
penetration,
it
does
indicate
that
dermal
penetration
does
not
take
place
at
a
rate
capable
of
producing
substantial
lethality.
Furthermore,
the
arsenate
treated
moth
egg
diet
used
in
the
study
was
supplied
to
the
lacewing
larvae
on
a
very
limited
area
of
the
test
arena
(
only
on
the
bottom
of
the
test
arena),
and
direct
tarsal
contact
with
arsenate­
treated
moth
egg
diet
by
test
larvae,
if
any,
would
have
been
limited.
Thus,
the
significant
lethal
effect
of
the
arsenate
treatment
detected
in
this
study
was
likely
to
have
resulted
from
ingestion
of
lethal
doses
of
the
toxicant
by
test
larvae
via
feeding
the
treated
moth
egg
diet,
rather
than
from
tarsal­
contact
to
the
treated
diet.

EPA
Response:
Comments
at
the
August
2002
SAP
cast
doubt
on
the
validity
of
the
use
of
moth
eggs
for
lacewing
larval
studies.
It
was
noted
that
the
Cry
protein
coats
the
outside
of
the
eggs,
while
the
lacewing
larvae
pierce
the
eggs
and
feed
on
the
egg
contents,
thus
it
is
not
clear
whether
the
lacewing
larvae
actually
ingest
Cry
protein
coating
the
outside
of
the
egg.
Likewise,
arsenate
was
said
to
very
likely
diffuse
into
the
egg
contents
and
give
a
false
sense
of
a
positive
control,
whereas
the
Cry
protein
would
appear
to
give
negative
results
simply
because
of
the
inability
to
get
into
the
eggs.
As
a
result
efforts
are
in
progress
to
develop
an
artificial
lacewing
larva
diet
which
can
be
mixed
with
Cry
protein.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
37
Excerpt
Text:
Some
published
studies
have
evaluated
the
toxicity
of
other
Bt
Cry
proteins
by
using
either
encapsulated
Bt­
Cry
protein­
treated
liquid
artificial
diet
[
Footnote
10:
Hilbeck,
A.,
W.
J.
Moar,
M.
Pusztai­
Carey,
A.
Filippini
and
F.
Bigler
(
1998).
Toxicity
of
Bacillus
thuringiensis
Cry1Ab
Toxin
to
the
Predator
Chrysoperla
carnea
(
Neuroptera:
Chrysopidae).
Environ.
Entomol.
27:
1255­
1263.]
or
natural
prey
pre­
fed
with
Bt
Cry
protein
treated
diet.
[
Footnote
11:
Hilbeck,
A.,
M.
Baumgartner,
P.
M.
Fried
and
F.
Bigler
(
1998).
Effects
of
Transgenic
Bacillus
thuringiensis
Corn­
fed
Prey
on
Mortality
and
Development
Time
of
Immature
Chrysoperla
carnea
(
Neuroptera:
Chrysopidae).
Envrion.
Entomol.
27:
480­
487.]
[
Footnote
12:
Hilbeck,
A.,
W.
J.
Moar,
M.
Pusztai­
Carey,
A.
Filippini
and
F.
Bigler
(
1999).
Prey­
mediated
Effects
of
Cry1Ab
Toxin
and
Protoxin
and
Cry2A
Protoxin
on
the
Predator
Chrysoperla
carnea.
Entomologia
Experimentalis
et
Applcata
91:
305­
316.]
[
Footnote
13:
Meier,
S.
M.
and
A.
Hilback
(
2001).
Influence
of
Transgenic
Bacillus
thuringiensis
Corn­
fed
Prey
on
Performance
of
Immature
Chrysoperla
carnea
(
Neuroptera:
Chyrsopidae).
Basic
Appl.
Ecol.
2:
35­
44.]
Hilbeck
et
al.
fed
the
green
lacewing
larvae
with
natural
prey
(
larvae
of
Spodoptera
littoralis)
reared
on
the
diet
containing
a
lepidopteran­
active
Cry
protein,
and
observed
adverse
effect
of
the
"
Bt­
Cry
protein
intoxicated"­
prey
on
the
mortality
of
test
lacewing
larvae.
However,
recent
studies
indicate
that
this
adverse
effect
from
secondary
exposure
to
Bt
toxins
on
the
lacewing
larvae
is
largely
due
to
the
"
sickness"
of
the
"
Bt
toxin"­
Intoxicated
prey,
rather
than
Section
3.1
3
­
41
ingested
Bt
toxins
per
se.
[
Footnote
14:
Dutton,
A.,
H.
Klein,
J.
Romeis
and
F.
Bigler
(
2002).
Uptake
of
Bt­
toxin
by
Herbivores
on
Transgenic
Maize
and
Consequences
for
the
Predator
Chrysoperla
carnea.
Ecological
Entomol.
(
in
press)]
Therefore,
tri­
trophic
studies
with
natural
prey
exposed
to
Cry
proteins
may
not
be
appropriate
in
determining
the
hazard
of
the
Bt
Cry
protein
toxin
to
green
lacewings
in
the
firsttier
toxicity
studies.
In
addition,
recent
studies
indicated
that
some
of
the
natural
prey
preferred
by
the
green
lacewing,
such
as
aphids,
feed
from
the
phloem
sap
of
transgenic
Bt
plants,
and
do
not
have
detectable
levels
of
the
Cry
protein
in
their
body.
[
Footnote
15:
Raps,
A.,
J.
Kehr,
P.
Gugerli,
E.
J.
Moar,
F.
Bigler,
and
A.
Hilbeck.
2001.
Immunological
analysis
of
phloem
sap
of
Bacillus
thiringiensis
corn
and
of
the
nontarget
herbivore
Rhopalosiphum
padi
(
Homoptera:
Aphididae)
for
the
presence
of
Cry1Ab.
Molecular
Ecology
10:
525­
533.]
[
Footnote
16:
Head,
G.
P.,
C.
R.
Brown,
M.
E.
Groth
and
J.
J.
Duan.
(
2001).
Cry1Ac
Protein
Levels
in
Phytophagous
Insects
Feeding
on
Transgenic
Corn:
Implications
for
Secondary
Exposure
Risk
Assessment.
Entomologia
Experimentalis
et
Applicata:
99:
37­
45.]
Thus
in
nature,
exposure
to
plant­
produced
Cry
proteins
for
lacewing
via
tri­
trophic
interactions
(
i.
e.,
secondary
exposure)
would
be
extremely
limited.

In
summary,
we
believe
that
the
green
lacewing
study
conducted
by
Monsanto
with
Cry3Bbl
proteintreated
moth
egg
diet
is
both
practically
and
scientifically
Justifiable
and
was
deemed
acceptable
by
EPA.
Based
on
our
laboratory
study
as
well
as
field
studies,
minimal
risk
of
event
MON
863
is
expected
for
the
green
lacewing.

EPA
Response:
An
independent
review
of
the
cited
published
studies
by
the
EPA
has
reached
the
same
conclusions
which
is
that
the
tritrophic
studies
on
green
lacewing
do
not
apply
to
the
corn
field
environment
where
the
prey,
such
as
aphids,
do
not
ingest
BT
toxin
while
feeding
on
the
phloem
of
the
corn
plant.
In
addition,
EPA
has
determined
that
in
the
reported
tritrophic
studies
the
prey
were
fed
excessive
doses
of
Cry
protein
in
an
artificial
diet,
which
far
exceeded
the
amounts
present
in
corn
plants.
Nevertheless,
EPA
intends
to
ask
for
tritrophic
studies
where
they
would
appear
to
be
warranted
by
the
ecology
of
the
crop
in
question.
EPA
disagrees
with
the
commenter
on
the
usefulness
of
the
lacewing
testing
as
performed
by
Monsanto.
Comments
at
the
August
2002
SAP
cast
doubt
on
the
validity
of
the
use
of
moth
eggs
for
lacewing
larval
studies.
It
was
noted
that
the
Cry
protein
coats
the
outside
of
the
eggs,
while
the
lacewing
larvae
pierce
the
eggs
and
feed
on
the
egg
contents,
thus
it
is
not
clear
whether
the
lacewing
larvae
actually
ingest
Cry
protein
which
cannot
get
into
the
eggs.
Likewise,
arsenate
was
said
to
very
likely
diffuse
into
the
egg
contents
and
give
a
false
sense
of
a
positive
control,
whereas
the
Cry
protein
would
appear
to
give
negative
results
simply
because
of
the
inability
to
penetrate
the
outside
of
the
eggs.
As
a
result
efforts
are
in
progress
to
develop
an
artificial
lacewing
larva
diet
which
can
be
mixed
with
Cry
protein.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
38
Excerpt
Text:
Question
5:
Preliminary
Risk
Assessment
for
Nontarget
Invertebrates
and
Soil
Fate
Please
comment
on
the
Agency's
nontarget
invertebrate
and
soilfate
assessment.
Section
3.1
3
­
42
Response:
The
collective
evidence
of
laboratory
and
field
studies
submitted
by
Monsanto
consistently
showed
no
adverse
effects
on
nontarget
species,
including
nontarget
beetle
families,
at
maximum
estimated
environmental
concentrations
of
the
CRW
protein.
The
process
by
which
Monsanto,
in
communication
with
EPA,
approached
the
ecological
risk
assessment
for
MON
863
was
logical,
utilized
state
of
the
art
methods
for
assessing
ecological
risk,
and
incorporated
input
from
recent
EPA
Science
Advisory
Panel
reports.
The
initial
laboratory­
based
risk
assessment
was
conducted
similar
to
that
for
past
plant­
incorporated
pesticide
products.
Since
Cry3Bb1
is
a
known
beetle
active
protein,
the
need
was
identified
to
supplement
the
standard
laboratory
assessment
with
more
data
on
nontarget
beetles.
Since
standard
laboratory
dietary
tests
did
not
exist
for
nontarget
beetles,
Monsanto
conducted
a
field
study
to
provide
the
additional
information,
as
well
as
encouraged
independent
academic
institutions
to
engage
in
field
research.
The
overwhelming
conclusion
from
all
of
these
data
is
that
use
of
event
MON
863
will
result
in
minimal
risk
to
nontarget
organisms.

The
laboratory
assessment
for
MON
863
was
based
on
EPA
guidance
and
previous
testing
with
pesticide
incorporated
pesticides.
Risk
characterization
was
based
on
Office
of
Pollution
Prevention
and
Toxic
Substances
(
1996)
885.4000
Guidelines
which
state
(
p.
3),
"...
data
that
establishes
an
LC50,
ED50,
or
LD50
that
is
greater
than
the
maximum
hazard
dosage
level
(
e.
g.,
LD50>
1,000
mg/
kg)
would
often
be
adequate
for
the
purposes
of
hazard
assessment".
For
all
of
the
nontarget
organism
studies
conducted
with
the
Cry3Bbl
protein,
the
LC50
was
found
to
be
greater
than
the
highest
concentration
tested
(
i.
e.,
maximum
hazard
dose),
indicating
minimal
risk.
The
ratio
of
the
LC50:
MEEC
was
greater
than
10
for
all
species
tested
(
including
wasp,
earthworm
and
ladybird
beetle.),
except
the
adult
honey
bee.
The
LC50
for
adults
honey
bees
was
determined
to
be
greater
than
the
highest
concentration
tested,
360
ug
Cry3Bbl
protein/
g
diet,
was
4.3­
times
the
MEEC
(
i.
e.,
93
ug
Cry3Bbl
protein/
g
pollen).
Since
the
NOEC
is
360
ug/
g
and
this
exposure
will
not
be
realized
under
field
conditions,
minimal
risk
is
indicated
for
adult
honey
bees
as
well.
In
addition
to
standard
GLP
data
on
Coccinellidae
(
nontarget),
Monsanto
has
conducted
laboratory
bioassays
with
representative
species
of
the
Bruchidae,
Tenebrionidae,
and
Curculionidae
families
and
found
no
evidence
of
toxicity
in
these
nontarget
Coleoptera
families.
[
Footnote
19:
Head,
G.,
M.
Pleau,
S.
Sivausupramanian
and
T.
Vaughn
(
2001).
Insecticidal
Spectrum
of
Activity
for
Cry3Bb1
Protein
in
vitro.
Report
C3NTO,
an
unpublished
study
conducted
for
Monsanto
Company.
MRID
455382­
07.]
Consequently,
the
laboratory
studies
conducted
by
Monsanto
indicate
minimal
risk
of
MON
863
to
nontarget
organisms.

EPA
Response:
The
above
commenter
is
summarizing
the
EPA
approach
to
non­
target
risk
assessment.
EPA
is
in
agreement
with
these
comments.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
39
Excerpt
Text:
Monsanto
has
submitted
a
soil
degradation
study
for
Cry3Bbl
protein
using
field­
collected
soil
that
indicates
the
protein
will
dissipate
rapidly
under
field
conditions.
[
Footnote
17:
Martin,
J.
W.,
M.
J.
McKee,
S.
Dubelman
and
Y.
A.
Dudin
(
2000).
Aerobic
Soil
Degradation
of
the
B.
t.
Protein
11098
as
a
Component
of
Insect
Protected
Corn.
Report
MSL­
16440,
an
unpublished
study
conducted
by
Monsanto
Company.
Section
3.1
3
­
43
MRID
451568­
04.]
The
design
of
this
study
employed
exaggerated
doses
to
simulate
worst­
case
soil
deposition
from
the
variety
of
potential
mechanisms,
including
potential
secretion,
shedding
of
root
hairs,
degradation
of
biomass
and
pollen
deposition.
The
calculated
DT50
(
time
to
50%
degradation)
was
2.4
to
2.8
days,
and
the
calculated
DT90
(
time
to
90%
degradation)
was
7.9
to
9.2
days.
There
was
no
detection
of
Cry3Bbl,
by
either
ELISA
or
insect
bioassay,
in
any
sample
incubated
for
over
21
days.

Field
investigations
were
initiated
in
2000
by
Monsanto
and
separately
by
a
number
of
university
researchers
across
multiple­
locations
to
validate
the
prediction
of
minimal
risk
to
nontarget
arthropods
in
the
field.
Interim
reports
summarizing
the
data
collected
for
key
nontarget
and
target
species
were
submitted
to
EPA
in
2001
and
2002.
These
data
indicate
no
significant
effects
of
MON
863
on
carabid
beetles
or
other
nontarget
beetles,
while
adverse
effects
of
currently
used
conventional
insecticides
were
identified.
Thus,
the
field
data
validate
the
results
of
the
maximum
hazard
dose
laboratory
studies
and
demonstrate
that
MON
863
poses
a
minimal
risk
to
nontarget
organisms,
including
beetle
families
commonly
found
in
agricultural
settings.

The
collective
evidence
of
laboratory
and
field
studies
submitted
by
Monsanto
consistently
showed
no
adverse
effects
on
nontarget
species
at
maximum
estimated
environmental
concentrations
of
the
Cry3Bb1
protein.
Another
important
consideration,
sometimes
overlooked,
is
the
long
history
of
safe
agricultural
use
of
Bt
products
in
general,
and
specifically,
the
Cry3
class
of
Bt
products
presently
used
in
traditional
and
organic
agricultural
systems
(
e.
g.,
Raven
Oil
Flowable
Bioinsecticide).
Finally,
any
analysis
of
the
potential
risks
and
benefits
of
a
new
technology
must
be
considered
in
the
context
of
existing
pest
management
practices
and
systems.
The
Cry3Bbl
protein
is
less
toxic
to
nontarget
organisms
than
existing
organophosphate
and
pyrethroid
insecticides
currently
approved
and
commonly
used
to
control
corn
pests,
including
CRW.
Furthermore,
in
addition
to
the
lower
toxicity
of
the
Cry3Bb1
protein,
the
in­
plant
delivery
system
further
reduces
exposure
to
nontarget
organisms
since
target
organisms
must
be
closely
associated
with
the
plant
tissue
to
be
exposed.

EPA
Response:
The
data
reviewed
by
EPA
to
date
show
that
the
above
comments
appear
to
be
accurate.
Additional
and
long
range
confirmatory
studies
are
in
progress.

Commenter
Name:
John
Foster
Commenter
Organization
Name:
John
Foster
Comment
Number:
OPP02­
0027
Excerpt
Number:
1
Excerpt
Text:
Having
been
involved
with
the
Bt
corn
and
issues
focused
on
the
monarch
butterfly
also
made
several
things
clear
to
me.
First,
it
is
easy
to
focus
on
potential
hazards
of
new
technologies
and
lose
sight
of
its
potential
benefits
(
and
the
risks
associated
with
existing
technologies).
Secondly,
with
a
new
technology,
appropriate
field
studies
are
the
only
way
to
really
understand
the
performance
of
a
product.
Over
the.
past
3
years,
we
have
been
conducting
field
research
on
the
impact
of
MON
863
on
non­
target
arthropods
at
multiple
sites
in
Nebraska.
We
have
compared
MON
863
with
standard
used
soil
insecticides,
we
have
used
increasing
plot
sizes
as
seed
became
available,
and
variety
of
standard
sampling
techniques,
and
we
have
focused
on
a
number
of
groups
of
arthropods
that
are
of
ecological
and
economic
importance,
and
that
might
be
impacted
by
a
transgenic
coleopteran­
active
protein
that
is
expressed
in
the
roots
and
above­
Section
3.1
3
­
44
ground
tissues
of
a
corn
plant.

To
date,
when
comparing
the
arthropods
found
in
plots
of
MON
863
with
those
in
plots
containing
untreated
isolines,
we
have
seen
no
significant
adverse
effects
of
MON
863
on
any
non­
target
group.
We
have
seen
some
differences
in
the
communities
among
sites
but
nosignificant
impacts
of
the
variety
(
MON
863
compared
to
its
isoline).
Our
analyses
have
included
ground­
dwelling
beetles,
Collembola,
and
spiders.
Furthermore,
I
also
have
communicated
with
other
academics
performing
similar
studies,
with
comparable
results,
over
the
past
3
years.
With
locations
ranging
from
New
York
to
South
Dakota
to
Kansas,
and
a
variety
of
techniques
and
points
of
focus,
and
with
very
large
plot
sizes
in
some
cases,
these
studies
represent
a
comprehensive
investigation
of
the
potential
impact
of
MON
863
on
agro­
ecosystems.
In
all
of
the
studies
that
I
am
aware
of,
no
significant
adverse
impacts
have
been
observed.
Together
with
the
results
generated
by
my
students
in
my
laboratory,
these
studies
give
me
confidence
that
MON
863
is
a
safe
product
with
respect
to
non­
targets,
and
hence,
the
EPA's
ecological
assessment
is
an
accurate
one.
Finally,
I
know
what
the
currently
used
soil
insecticides
can
do
to
agro­
ecosystems.
In
Nebraska,
current
technologies
used
to
control
corn
rootworm
have
obvious
adverse
effects
on
many
non­
target
species.

EPA
Response:
The
above
commenter
confirms
EPA's
ecological
risk
assessment
on
MON
863
as
accurate
from
his
personal
experience
and
asserts
that
MON
863
is
much
friendlier
to
the
ecosystem
than
conventional
pesticide
treatments.
EPA
concurs
with
these
comments.

Commenter
Name:
John
Foster
Commenter
Organization
Name:
John
Foster
Comment
Number:
OPP02­
0027
Excerpt
Number:
2
Excerpt
Text:
I
believe
that
the
introduction
of
MON
863
has
the
potential
to
bring
clear
and
measurable
ecological
benefits
to
corn
production
systems
in
terms
of
reduced
insecticide
usage
and
hence
reduced
worker
exposure
EPA
Response:
EPA
concurs
with
this
comment.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
5
Excerpt
Text:
3.
Monsanto
did
not
conduct
non­
target
organism
toxicity
testing
according
to
EPA
guidelines.
Several
of
the
non­
target
organism
tests
used
to
predict
environmental
risks
were
not
conducted
with
sufficiently
high
doses
of
Cry3Bb1.
Monsanto
should
repeat
non­
target
testing
at
maximum
hazard
doses
(
at
least
10
fold
higher
than
the
maximum
expected
environmental
exposure)
where
those
doses
were
not
used.

EPA
Response:
Section
3.1
3
­
45
Contrary
to
the
claims
by
this
commenter,
examination
of
the
submitted
non­
target
studies
shows
that
these
were
performed
with
20x
or
higher
safety
margins,
except
where
pure
pollen
of
plant
tissue
were
used
and
where
concentration
of
the
Cry
protein
cannot
be
increased.
The
parasitic
hymenoptera
were
in
fact
tested
at
only
1X
field
concentration
in
plants
rather
than
at
10X.
However,
parasitic
hymenoptera
are
not
expected
to
feed
directly
on
corn
plant
tissue.
For
this
reason
the
SAP
commented
that
parasitic
hymenoptera
are
not
an
appropriate
test
species
for
MON863
corn.
The
LC50
for
adults
honey
bees
was
determined
to
be
greater
than
the
highest
concentration
tested,
360
ug
Cry3Bbl
protein/
g
diet,
or
4.3­
times
the
MEEC
(
i.
e.,
93
ug
Cry3Bbl
protein/
g
pollen).
Since
no
adverse
effects
were
noted
at
360
ug/
g
(
and
this
exposure
level
will
not
be
realized
under
field
conditions),
minimal
risk
to
adult
honey
bees
is
expected.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
6
Excerpt
Text:
4.
Monsanto
did
not
conduct
maximum
hazard
dose
tests
of
important
non­
target
beetle
and
nematode
species.
Additional
beetles
species,
such
as
ground
beetles,
should
be
tested
because
beetles
are
most
closely
related
to
CRW
and
therefore
most
likely
to
be
harmed
by
Cry3Bb1.
Monsanto
only
tested
two
ladybird
beetle
species,
and
they
were
not
tested
at
a
maximum
hazard
dose.
In
addition,
further
testing
of
free­
living
nematodes
should
be
conducted
since
preliminary
tests
showed
significant
population
reduction
of
two
nematode
species
when
exposed
to
MON
863
corn.

EPA
Response:
Contrary
to
the
claims
by
this
commenter,
one
lady
beetle
study
was
conducted
at
the
maximum
hazard
dose
(
20xEEC),
and
three
other
lady
beetle
studies
were
conducted
with
pollen
where
the
Cry
protein
content
cannot
be
manipulated
in
the
laboratory.
Protocols
for
testing
of
additional
ground
beetles
are
being
developed
.
In
the
meantime
EPA
made
a
risk
assessment
on
these
species
from
field
survey
results.
EPA
has
requested
additional
data
in
order
to
make
a
risk
assessment.
An
initial
observation
is
that
an
adverse
effect
on
parasitic
nematodes
in
the
corn
root
zone
may
have
beneficial
effects.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
18
Excerpt
Text:
According
to
EPA's
Microbial
Test
Guidelines
(
8),
EPA
can
require
the
testing
of
different
non­
target
organisms
than
are
specified
if
the
agency
believes
that
those
other
organisms
would
be
more
appropriate.
EPA
should
require
Monsanto
to
perform
maximum
hazard
dose
tests
with
several
additional
Coleoptera,
such
as
ground
beetles
and
other
species,
to
gain
confidence
that
MON
863
will
not
harm
beneficial
insects
found
in
corn
fields.

Only
two
species
of
Coleoptera,
both
ladybird
beetles,
have
been
tested
for
sensitivity
to
MON
863,
and
in
at
least
one
case,
the
concentration
of
Cry3Bb1
was
too
low.
Since
Coleoptera
are
more
closely
related
to
the
target
organisms
than
most
of
the
indicator
species
currently
used,
and
therefore
have
a
higher
Section
3.1
3
­
46
likelihood
of
being
susceptible
to
Cry3Bbl,
EPA
should
require
appropriate
maximum
hazard
dose
testing
of
additional
beetle
species.

In
addition,
preliminary
results
strongly
suggest
that
free­
living
nematodes
may
be
harmed
by
CryBb1
(
10).
The
highest
concentration
used
in
those
tests
was
about
10
times
less
than
EEC
(
based
on
the
root
concentration
of
Cry3Bb1
in
MON
863),
and
was
therefore
100­
1000
fold
below
the
appropriate
maximum
hazard
dose.
Several
species
of
free­
living
nematodes
should
be
included
in
additional
maximum
hazard
dose
tests
(
see
section
III.
C.).

EPA
Response:
EPA
has
required
additional
non­
target
beetle
testing.
Protocols
for
these
are
being
developed
under
the
auspices
of
the
ABSTC.
In
the
meantime
EPA
made
a
risk
assessment
on
these
species
from
observations
of
their
abundance
in
corn
fields.
Multi­
year
field
surveys
are
also
addressing
this
issue.
Regarding
the
lady
beetle
comment,
one
lady
beetle
study
was
conducted
at
the
maximum
hazard
dose
(
20xEEC),
and
three
other
lady
beetle
studies
were
conducted
with
pollen
where
Cry
protein
content
cannot
be
manipulated
in
the
laboratory.
EPA
requires
maximum
hazard
dose
testing
whenever
the
testing
conditions
permit
it.
However,
the
commenter
should
note
that
in
the
final
risk
assessment
when
the
LD50
and
the
EEC
are
factored
together,
an
LD50
greater
than
5xEEC
is
considered
as
acceptable.
EPA
has
requested
additional
data
in
order
to
make
a
risk
assessment.
The
initial
observations
also
indicate
that
an
adverse
effect
on
parasitic
nematodes
in
the
corn
root
zone
may
have
beneficial
effects.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
30
Excerpt
Text:
A.
High
Dose
Non­
Target
Organism
Acute
Toxicity
Tests
These
tests
were
not
performed
in
accordance
with
EPA's
microbial
test
guidelines
that
are
also
used
to
evaluate
genetically
engineered
plant­
incorporated
protectants
(
8),
or
in
accordance
with
the
recommendations
of
an
EPA
convened
SAP
that
evaluated
thhose
guidelines
(
9).
Monsanto
did
not
use
maximum
hazard
doses
in
several
of
its
non­
target
organism
tests.

Acute
maximum
hazard
dose
toxicity
tests
should
be
performed
at
a
high
multiplicity
of
expected
exposure
to
increase
the
possibility
that
any
adverse
effects
would
be
detected
in
those
short
term
tests.
Also,
use
of
a
high
dose
increases
the
chance
that
adverse
effects
on
a
less
sensitive
test
species
may
be
detected.
Different
levels
of
sensitivity
between
species
have
commonly
been
recognized
with
Cry
proteins,
such
as
between
Colorado
potato
beetle
and
CRW
in
response
to
Cry3Bb1.

Instead
of
using
the
accepted
definition
of
maximum
hazard
dose,
which
is
typically
at
least
10­
100
times
the
estimated
environmental
concentration
(
EEC),
Monsanto's
tests
are
based
on
the
absolute
concentration
of
Cry3BbI
in
the
plant
for
non­
soil
organisms
(
11).
Monsanto
characterizes
this
as
"...
very
high
dietary
concentrations
(
Table
7)"
(
12).
While
Table
7
of
Monsanto's
safety
assessment
document
(
12)
confirms
that
high
concentrations
of
Cry3Bb1
were
used
compared
to
some
tests
for
previous
Bt
toxins,
the
table
fails
to
disclose
that
those
other
Bt
toxins
were
expressed
at
many
times
lower
concentrations
in
crop
plants
Section
3.1
3
­
47
than
Cry3Bb1
in
MON
863.
The
absence
of
comparative
expression
data
in
Table
7
gives
a
false
impression
of
greater
margins
of
exposure
for­
MON
863
in
toxicity
tests
than
actually
occur.

EPA's
SAP
stated
the
importance
of
maximum
hazard
dose
testing,
saying
"...
the
higher
the
maximum
dose
the
better."
The
Panel
recognized
"...
that
it
is
not
always
practical
to
use
the
100X
[
EEC]
level"
but
that
the
maximum
hazard
dose
should
remain
at
least
10
times
the
EEC
(
9).

EPA
Response:
The
EPA
testing
guidelines
have
been
reviewed
by
two
previous
SAPs
(
1996,
1999).
The
hazard
assessment
process
is
explained
in
Section
C.
I.
A
of
the
MON
863
BRAD
Document.
Briefly,
the
process
is
as
follows:
the
testing
is
performed
at
the
prescribed
maximum
hazard
dose
(
MHD),
mortality
is
the
end
point,
and
if
greater
tan
50
%
mortality
is
seen
at
the
MHD,
then
a
LD50
has
to
be
determined.
If
the
LD50
is
at
least
5x
the
EEC
(
RQ
0.2)
then
a
no­
risk
finding
is
made.
It
is
notable
that
the
guidelines
state
that
the
reason
for
requiring
MHD
testing
is
to
reduce
the
cost
of
LD50
determinations,
not
to
detect
low
level
effects
as
claimed
by
the
commenter.
This
process
also
makes
it
apparent
that
testing
at
only
5x
without
apparent
mortality
can
also
be
used
for
risk
assessment
under
extenuating
circumstances.
Examination
of
the
submitted
non­
target
studies
for
MON863
shows
that
these
were
performed
with
20x
or
higher
safety
margins,
except
where
pure
pollen
of
plant
tissue
were
used
where
concentration
of
the
Cry
protein
cannot
be
increased.
The
parasitic
hymenoptera
were
in
fact
tested
at
only
1X
field
concentration
in
plants
rather
than
at
10X.
However,
parasitic
hymenoptera
are
not
expected
to
feed
directly
on
corn
plant
tissue.
For
this
reason
the
SAP
commented
that
parasitic
hymenoptera
are
not
an
appropriate
test
species
for
MON863
corn.
The
LC50
for
adults
honey
bees
was
determined
to
be
greater
than
the
highest
concentration
tested,
360
ug
Cry3Bbl
protein/
g
diet,
which
was
4.3­
times
the
MEEC
(
i.
e.,
93
ug
Cry3Bbl
protein/
g
pollen).
Since
no
adverse
effects
were
noted
at
360
ug/
g,
and
this
exposure
will
not
be
realized
under
field
conditions,
minimal
risk
to
adult
honey
bees
is
expected.
The
goal
of
risk
assessment
is
to
determine
what
the
effects
are
likely
to
be
at
the
field
concentration.
Toxicity
at
levels
higher
than
the
field
concentration
are
not
indicative
of
hazard
in
the
field.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
31
Excerpt
Text:
While
Monsanto's
use
of
MON
863
corn
tissue
in
several
non­
target
tests
is
useful
for
detecting
possible
unintended
deleterious
changes
in
the
plant,
the
dose
of
Cry3Bb1
delivered
in
those
tests
is
not
high
enough
for
acute
maximum
hazard
testing.
In
tests
with
some
organisms,
Monsanto
used
purified
Cry3BbI
protein
at
higher
concentrations
than
occur
in
MON
863,
but
those
concentrations
remain
below
the
recommendations
of
both
EPA
testing
guidelines
and
the
SAP.
EPA
should
require
Monsanto
to
repeat
maximum
hazard
toxicity
tests
at
100
times
higher
than
the
EEC
for
the
following
invertebrates
where
the
test
dose
was
less
that
10
times
higher
than
the
EEC:
adult
honeybee,
parasitic
hyrnenoptera,
earthworm,
and
the
ladybird
beetle
(
Coleoineggilla
maculam).
The
SAP
recognized
that
a
100X
dose
may
sometimes
not
be
achievable.
If
that
is
the
case
with
some
of
the
above
organisms,
a
dose
between
10X
and
I00X
should
be
used
and
justified.

In
addition,
Monsanto
should
be
required
to
repeat
those
tests
for
other
non­
target
insects
that
were
Section
3.1
3
­
48
conducted
at
less
than
I
00
times
EEC,
unless
the
dose
used
by
Monsanto
can
be
justified.
Those
organisms
include:.
collembola,
larval
honeybee,
ladybird
beetle
(
Hippodainia
covergens),
and
green
lacewing.

The
method
used
by
Monsanto
to
determine
the
soil
EEC
of
13.3
ug
Cry3Bbl/
g
soil
underestimates
the
exposure
of
soil
organisms
(
see
section
II.
D.
"
Persistence
of
MON
863
in
the
Soil").
The
EEC
should
be
based,
as
a
minimum,
on
the
concentration
of
Cry3Bb1
in
the
roots
of
MON
863
corn,
or
58
ug
Cry3Bbl/
g
(
13),
since
root
herbivores,
detritus
feeders,
or
their
predators
and
parasites
could
be
exposed
to
those
levels
of
Cry3Bbl.
On
this
basis,
the
tests
performed
by
Monsanto
on
collembola
were
only
about
15
times
the
more
realistic
EEC,
while
the
earthworm
test
was
done
at
EEC
levels.

In
summary,
the
only
tested
non­
target
invertebrate
where
the
dose
met
­
or
exceeded
the
desired
100
times
EEC
was
daphnia.
Even
if
doses
between
10
times
and
100
times
EEC
are
accepted,
only
four
terrestrial
insect
species
were
adequately
tested,
including
only
a
single
beetle
species.
While
EPA
microbial
pesticide
test
guidelines
only
recommend
testing
of
three
non­
target
insect
species,
those
guidelines
are
not
always
appropriate
for
plant
pesticides.
The
SAP
(
9)
recognized
that
plant
pesticides
differ
significantly
from
microbial
pesticides
and
may
require
testing
of
more
than
three
non­
target
insect
species.
It
is
important
to
test
additional
Coleoptera
species
since
important
non­
target
species
related
to
the
target
corn
rootworm.
(
Coleoptera)
are
often
common
in
corn
fields
(
see
section
III.
B.).

EPA
Response:
Testing
of
aquatic
invertebrates
at
100x
the
terrestrial
EEC
is
not
a
requirement
when
aquatic
exposure
is
low
to
nonexistent.
The
EPA
testing
guidelines
have
been
reviewed
by
two
previous
SAP's
(
1996,
1999).
The
hazard
assessment
process
is
explained
in
Section
C.
I.
A
of
the
MON
863
BRAD
Document.
Briefly,
the
process
is
as
follows:
the
testing
is
performed
at
the
prescribed
maximum
hazard
dose
(
MHD),
mortality
is
the
end
point,
and
if
greater
tan
50
%
mortality
is
seen
at
the
MHD,
then
a
LD50
has
to
be
determined.
If
the
LD50
is
at
least
5x
(
10x
for
aquatics)
the
EEC
(
RQ
of
0.2
and
0.1
respectively)
then
a
no­
risk
finding
is
made.
It
is
notable
that
the
guidelines
state
that
the
reason
for
requiring
MHD
testing
is
to
reduce
the
cost
of
LD50
determinations,
not
to
detect
low
level
effects
at
unrealistically
high
doses
as
claimed
by
the
commenter.
This
process
also
makes
it
apparent
that
testing
of
terrestrial
animals
at
only
5x
without
apparent
mortality
can
also
be
used
for
risk
assessment
under
extenuating
circumstances.
Examination
of
the
submitted
non­
target
studies
for
MON863
shows
that
these
were
performed
with
20x
or
higher
safety
margins,
except
where
pure
pollen
of
plant
tissue
were
used
(
where
the
concentration
of
the
Cry
protein
cannot
be
increased).

The
parasitic
hymenoptera
were
in
fact
tested
at
only
1X
field
concentration
in
plants
rather
than
at
10X.
However,
parasitic
hymenoptera
are
not
expected
to
feed
directly
on
corn
plant
tissue.
For
this
reason
the
SAP
commented
that
parasitic
hymenoptera
are
not
an
appropriate
test
species
for
MON863
corn.
The
LC50
for
adults
honey
bees
was
determined
to
be
greater
than
the
highest
concentration
tested,
360
ug
Cry3Bbl
protein/
g
diet,
which
was
4.3­
times
the
MEEC
(
i.
e.,
93
ug
Cry3Bbl
protein/
g
pollen).
Since
no
adverse
effects
were
noted
at
360
ug/
g,
and
this
exposure
will
not
be
realized
under
field
conditions,
minimal
risk
to
adult
honey
bees
is
expected.
The
goal
of
risk
assessment
is
to
determine
what
the
effects
are
likely
to
be
at
the
field
concentration.
Toxicity
at
levels
higher
than
the
field
concentration
are
not
indicative
of
hazard
in
the
field.
The
EPA
has
the
option
to
agree
to
testing
conditions
that
deviate
from
the
testing
guideline
requirements,
and
EPA
agreed
that
Monsanto
could
test
the
collembolla
and
some
lady
beetles
in
the
presence
of
50%
MON863
plant
tissue.
Monsanto
did
perform
an
earthworm
study
at
10x
Section
3.1
3
­
49
EEC
concentration
(
570
mg
Cry3Bb1
protein/
kg
dry
soil)
(
MRID#
449043­
16).
Protocols
for
additional
soil
insects
are
being
developed,
and
multi­
year
field
surveys
are
being
commenced
to
evaluate
the
effects
on
the
soil
invertebrates
and
their
interactions.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
32
Excerpt
Text:
Greater
reliance
on
non­
target
field
data
was
recommended
by
a
recent
SAP,
and
is
a
welcome
part
of
the
environmental
assessment
of
MON
863.
However,
only
preliminary
results
of
those
field
tests
are
currently
available,
and
those
results
are
insufficient
to
draw
conclusions
about
environmental
impacts
of
MON
863.
Field
testing
typically
needs
to
be
conducted
for
several
years
before
enough
data
can
be
altered,
because
it
involves
more
uncontrollable
variables
than
laboratory
tests.
In
addition,
environmental
conditions
that
may
influence
results
are
variable
and
often
sporadic,
and
may
only
be
encountered
after
several
years.
Therefore
it
is
not
possible
to
predict
whether
Monsanto's
proposed
two
years
of
field
testing
will
be
sufficient.
EPA
should
require
Monsanto
to
continue
its
field
tests
for
as
long
as
necessary
to
acquire
acceptable
data
rather
than
for
a
period
fixed
two
years
as
proposed
by
Monsanto.

In
addition
to
concern
about
the
duration
of
the
field
tests,
Monsanto's
experimental
design
does
not
adequately
address
certain
limitations
of
small
field
tests.
For
example,
edge
effects,
such
as
the
immigration
of
certain
beneficial
organisms,
can
mask
adverse
effects
occurring
in
small
field
tests.
Several
of
the
current
field
tests
do
not
address
the
issue
of
possible
edge
effects.
Monsanto
should
explicitly
address
such
effects
to
the
extent
feasible
in
all
of
the
current
field
tests,
or
at
least
attempt
to
address
how
uncontrollable
edge
effects
may
influence
those
tests.

In
preliminary
experiments,
two
of
three
species
of
nematodes,
including
a
plant
pathogen
and
a
soil
inhabiting
(
free­
living)
species,
suffered
significantly
reduced
populations
when
exposed
to
MON
863.
The
free­
living
species
was
exposed
to
a
root
homogenate
(
root
extract)
that
was
diluted
10
fold
compared
to
the
Cry3Bbl
concentration
in
MON
863
roots
(
10).
The
investigator
concluded
that
because
the
free­
living
nematode
species
were
not
harmed
by
a
MON
863
soil
leachate
used
in
another
test,
free­
living
species
probably
were
not
at
risk.
This
conclusion
was
based
on
the
investigators'
unsupported
assessment
that
the
leachate
more
accurately
represents
the
likely
means
of
exposure
of
free­
living
nematodes
in
MON
863
fields.

That
conclusion
does
not
account
for
the
many
untested
species
of
nematodes
that
occur
in
soil
and
occupy
many
different
important
ecological
niches.
For
example,
species
that
feed
on
detritus
containing
MON
863
roots
may
be
exposed
to
higher
levels
of
Cry3Bb
I
than
occur
in
the
leachate
or
root
extracts.
Some
untested
species
of
nematodes
may
be
more
sensitive
than
the
tested
species,
and
therefore
susceptible
to
the
concentrations
lower
than
found
in
root
extracts.
It
should
also
be
noted
that
some
Cry5
and
CryI4
proteins
are
toxic
to
nematodes.
Cryl4AI
is
toxic
to
nematodes
and
CRW,
so
it
may
not
be
surprising
that
Cry3Bbl
is
toxic
to
both
CRW
and
nematodes
(
14).

EPA
Response:
EPA
did
require
Monsanto
to
continue
its
field
surveys
to
evaluate
the
effects
on
the
soil
invertebrates
and
Section
3.1
3
­
50
their
interactions.
USDA
and
also
EPA
are
sponsoring
research
to
develop
statistically
appropriate
large
scale
field
testing
protocols
to
overcome
effects
like
the
"
edge
effects
"
mentioned
by
this
commenter.
EPA
has
requested
additional
data
in
order
to
make
a
nematode
risk
assessment.
However,
the
initial
observations
also
indicate
that
an
adverse
effect
on
parasitic
nematodes
in
the
corn
root
zone
may
have
beneficial
effects.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
33
Excerpt
Text:
Furthermore,
the
leachate
method
may
greatly
underestimate
the
concentration
of
Cry3Bb1
in
MON
863
corn
field
soils.
That
is
because
the
method
used
to
make
the
leachate
­
suspension
of
soil
that
contained
MON
863
corn
plants
in
an
aqueous
so
solution
followed
by
centrifugation,
where
the
supernatant
is
the
leachate
­
would
probably
leave
most
of
the
Cry3Bb1
in
the
soil
based
on
the
tight
binding
of
active
Cry
protein
to
soil
clay
particles
(
15,16).
Previous
studies
have
shown
that
aqueous
solutions
similar
to
that
used
in
the
Monsanto
submission
are
leave
much
of
the
active
Cry
protein
in
the
soil
(
15,16).
Notably,
the
researchers
did
not
indicate
that
they
tested
the
leachate
for
the
presence
or
concentration
of
Cry3Bbl.

In
addition,
the
a
second
free­
living
nematode
species
unaffected
by
the
MON
863
root
extract
was
reported
not
to
be
feeding.
Since
ingestion
is
required
for
Cry
protein
toxicity,
that
test
was
not
valid.
Therefore,
both
of
the
species
that
were
actually
exposed
to
Cry3Bb1
in
a
realistic
manner
were
harmed.
Those
results
suggest
that
many
other
nematode
species
might
also
be
harmed
by
Cry3Bb1.

Since
nematodes
are
important
and
abundant
soil
organisms,
EPA
should
require
Monsanto
to
perform
additional
testing
of
nematodes
to
determine
the
impact
of
Cry3Bb1
prior
to
registration.
Such
testing
must
include
the
nematode
species
already
examined,
field
studies
on
nematode
populations,
as
well
as
maximum
hazard
tests
and
field
tests
on
additional
free­
living
nematode
species.

Finally,
even
after
several
years
of
testing,
the
data
from
field
experiments
often
result
in
a
substantial
amount
of
statistical
variation
that
can
mask
adverse
effects.
In
addition,
some
environmental
impacts
may
take
many
years
to
develop.
A
recently
published
report
by
the
National
Research
Council
(
17)
recognized
those
limitations
of
small
scale
field
trails
and
recommended
follow­
up
monitoring
programs.
EPA
should
work
with
USDA/
APHIS
to
develop
such
programs.

EPA
Response:
USDA
and
also
EPA
are
sponsoring
research
to
develop
statistically
appropriate
large
scale
field
testing
protocols
to
overcome
new
insect
incursion
effects.
EPA
has
requested
additional
data
in
order
to
make
a
nematode
risk
assessment.
However,
the
initial
observations
also
indicate
that
an
adverse
effect
on
parasitic
nematodes
in
the
corn
root
zone
may
have
beneficial
effects.
Complete
nematode
effects
assessment
was
not
required
prior
to
registration.
Current
agricultural
practices
such
as
use
of
fertilizers,
soil
pH
manipulation,
chemical
pesticide
and
herbicide
uses,
as
well
as
soil
fumigation
and
sterilization
practices
all
have
a
detrimental
impact
on
nematode
populations.
Therefore
immediate
resolution
of
the
possible
effects
on
nematodes
by
MON863
corn
does
not
appear
critical.
Section
3.1
3
­
51
Commenter
Name:
Teresa
Gruber
Commenter
Organization
Name:
Council
for
Agricultural
Science
and
Technology
Comment
Number:
OPP02­
0037
Excerpt
Number:
4
Excerpt
Text:
In
general,
the
EPA
has
identified
and
considered
a
reasonable/
rational
set
of
taxa
and
species
for
pestincorporated
protectants.
Some
additional
attention
to
specific
species
related
to
the
target
pest
maybe
appropriate.
In
the
case
of
Cry3Bb1,
the
protein
is
an
effective
toxin
to
a
few
species
of
Coleoptera,
therefore
some
emphasis
should
be
placed
on
other
Coleoptera
typically
present
in
cornfields
or
in
the
soil
with
some
history
of
corn
production.
Thus,
the
Coccinelids
(
ladybird
beetles)
were
logical
choices
for
study
because
of
their
relatedness
to
the
target
pest
and
their
utility
as
natural
enemies
in
cornfields.
Tests
and
resulting,
decisions
should
emphasize
concentrations
of
the
toxin
likely
to
be
encountered
by
natural
enemies
and
other
nontarget
organisms
under
natural/
field
conditions.

EPA
Response:
EPA
has
required
additional
non­
target
beetle
testing.
Protocols
for
these
are
being
developed
under
the
auspices
of
the
ABSTC.
In
the
meantime
EPA
made
a
risk
assessment
on
these
species
from
observations
of
their
abundance
in
corn
fields.
Additional
multi
year
field
surveys
are
also
addressing
this
issue.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
18
Excerpt
Text:
Current
knowledge
of
Bacillus
thuringiensis
spores
and
crystal
proteins,
and
their
inactivation
in
soil
Bacillus
thuringiensis
seems
to
be
indigenous
to
many
different
environments
since
it
has
been
isolated
worldwide
from
many
habitats
such
as
soils,
insects,
stored­
product
dusts.
and
deciduous
and
coniferous
leaves
(
Schriepf
et
al.
1998).
It
produces
large
amounts
of
insecticidal
delta­
endotoxins
during
sporulation.
In
these
bacteria,
the
endotoxins
are
present
in
a
crystalline,
biologically
inactive
form.
Microbial
Btinsecticides
targeting
lepidopteran
pests
typically
contain
Bt
proteins
of
the
Cryl
class.
In
addition
to
Cryl
6­
endotoxin
crystals,
spores
produced
by
Bacillus
during
the
fermentation
process
also
are
present
in
these
insecticides
(
Feitelson
et
al.
1992).
Other
potential
bioactive
substances
occur
in
many
Btinsecticides,
such
as
VIPs
(
vegetative
insecticidal
proteins),
phospholipases,
zwittermycin,
carrier
and
formulation
ingredients,
all
of
which
exert
an
insecticidal
effect
in
concert.
This
potpourri
of
biochemicals
is
ingested
by
the
insects.
The
Bt
crystals
must
undergo
a
complex
cascade
of
biochemical
reactions
before
they
become
active
against
susceptible
insects.
Firstly,
the
crystals
must
be
dissolved
in
an
alkaline
midgut
milieu
and
the
presence
of
certain
enzymes
to
yield
a
biologically
inactive
protoxin
of
130
kDa
in
molecular
weight
for
the
type
of
Bt
toxin
produced
in
currently
commercially
available
lepidopteran­
toxic
Bt­
com
(
except
Cry9C
corn)
and
Bt­
cotton.
Secondly,
again
in
a
stepwise
chain
of
reactions,
a
65
kDa
toxic
fragment
is
proteolytically
cleaved
from
the
protoxin.
This
fragment,
commonly
referred
to
as
the
activated
toxin,
binds
to
receptors
located
on
the
epithelium
of
the
insect
midgut.
The
receptor­
bound
toxin
then
induces
pore
formation
leading
to
lysis
of
the
midgut
which
results
in
the
death
of
the
insect.
For
induction
of
death
of
a
susceptible
insect,
binding
and
pore
formation
are
essential.
However,
all
steps
proceeding
pore
formation
are
equally
crucial
for
determining
the
specificity
of
the
Bt
proteins
in
killing
insects
Section
3.1
3
­
52
(
Goldburg
and
Tjaden
1990)
(
Figure
2).
Lu
transgenic
plants,
the
truncated
semi­
activated
protein
is
expressed
and
will
be
ingested
by
the
herbivorous
insects
feeding
on
it.
Consequently,
no
crystal
solubilization
and
relatively
little
protoxin­
toxin
conversion
is
necessary
within
the
insect
gut.
It
is
conceivable
that
insects
lacking
the
appropriate
midgut
enzyme
composition
or
pH
milieu
may
well
have
appropriate
receptors.
[
see
PDF
file
for
"
Figure
2.
Differences
Bt­
insecticides
and
Bt
plants"]

EPA
Response:
This
comment
does
not
require
a
response.
The
above
commenter
is
summarizing
the
mode
of
action
of
Bt
Cry
proteins
and
soluble
toxins.

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509­
114000
Excerpt
Number:
2
Excerpt
Text:
Another
biological
approach
was
to
manipulate
the
soil
bacterium
Bacillus
thuringlensis.
The
Bt
bacterium
secretes
a
protein
that,
when
ingested
by
a
sensitive
insect,
cases
the
larval
gut
to
break
down
and
a
gooey,
black
death
ensues.
Industrial
and
academic
scientists
have
selected
numerous
Bt
strains
that
are
toxic
to
the
larvae
of
different
groups
of
insects.
The
Bt
kurstaki
strain
is
lethal
to
the
caterpillars
of
virtually
all
moths
and
butterflies
and
is
produced
in
mass
cultures
that
are
harvested
and
sold
as
Dipel.
Used
in
home
gardens
as
a
"
natural"
toxic
powder
to
kill
tomato
hornworm
is
and
cabbage
caterpillars.
Dipel
is
also
sprayed
to
kill
gypsy
moth
caterpillars
in
the
eastern
deciduous
forests,
spruce
budworms
HI
the
northern
boreal
forests.
and
tussock
moth
caterpillars
in
the
western
Douglas
fir
forests.
Extensive
sprayings
of
Dipel
and
its
derivatives,
along
with
repeated
releases
of
exotic
parasitic
insects.
Have
severely
reduced
the
populations
of
many
benign
and
beneficial
native
insects,
including
several
of
the
New
England
silk
moths
renowned
for
their
elegance
and
bizarre
caterpillars
The
danger
to
nonpest
species
was
raised
to
a
far
more
sophisticated
level
by
the
new
science
of
genetic
engineering,
which
makes
it
possible
to
transfer
genes
between
any
species
on
earth.
When
successful,
the
transferred
genes
give
the
recipient
species
the
ability
to
synthesize
proteins
that
were
specific
to
the
donor
species.
An
obvious
strategy
would
be
to
insert
various
Bt
genes
into
crop
plants.
Then
as
the
seeds
of
the
genetically
modified
strain
sprout
and
grow,
the
inserted
DNA
would
express
itself
in
every
single
cell
of
the
growing
seedling.
Wonder
of
wonders,
the
roots,
stems,
leaves,
and
seeds
of
the
plant
contain
the
Bt
toxin
and
are
toxic
to
virtually
all
caterpillars.
Agricultural
companies
introduced
the
Bt
genes
into
several
crops,
including
potatoes,
soybeans,
cotton,
and
corn.
One
major
target
was
the
European
corn
borer
moth,
an
economically
damaging
species
that
is
found
throughout
the
eastern
United
States
and
southern
Canada.

EPA
Response:
This
comment
does
not
require
a
response.
The
above
commenter
is
summarizing
the
theoretical
consequences
of
use
of
spray
and
plant­
incorporated
Bt
toxins
on
insect
wildlife.

Commenter
Name:
Lincoln
Brower
Section
3.1
3
­
53
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509­
114000
Excerpt
Number:
4
Excerpt
Text:
Many
biologists
heralded
the
new
Bt
corn
technology
because
they
believed
it
would
mitigate
the
need
to
spray
insecticidal
chemicals.
The
corporations
involved
in
marketing
the
seeds
(
for
the
most
part
the
same
ones
that
had
developed
the
synthetic
insecticides
several
decades
earlier)
sponsored
a
multimillion­
dollar
campaign
touting
them
as
an
environmental
panacea.
The
response
was
stunning:
by
the
1998
season,
twenty­
five
percent
of
the
total
U.
S.
corn
crop
(
of
eighty
million
acres)
was
planted
with
Bt
corn.

EPA
Response:
This
comment
does
not
require
a
response.

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509­
114000
Excerpt
Number:
8
Excerpt
Text:
Aware
that
new
data
and
more
sophisticated
analyses
would
be
forthcoming,
the
Union
of
Concerned
Scientists
and
eleven
other
public­
interest
organizations
made
a
request
to
the
EPA:
to
postpone
the
next
Scientific
Advisory
Panel
(
SAP)
meeting
until
more
data
were
collected
and
made
available
to
the
public,
including,
the
scientists'
findings
gathered
over
the
summer
of
2000.
The
EPA,
however,
held
the
SAP
meeting
on
October
19,
a
month
before
the
scientific
symposium
was
scheduled
to
take
place.
Prior
to
the
SAP
meeting,
the
EPA
had
allowed
several
corporations
to
review
the
agency's
preliminary
assessment
and
suggest
modifications.
In
addition,
the
EPA
allowed
the
companies
to
withhold
important
data
as
confidential
business
information.
One
of
the
principal
documents,
contained
approximately
forty
deletions
of
so­
called
"
proprietary"
data.
It
was
therefore
impossible
for
the
EPA
panel
or
independent
scientists
to
evaluate
the
data.
Both
industry
and
the
EPA
documents
also
ignored
relevant
data
readily
available
in
the
scientific
literature.
Thus,
without
considering
the
new
information
that
would
be
presented
the
following
month,
and
drawing
passages
almost
verbatim
from
documents
prepared
by
industry,
the
EPA's
interim
assessment
of
the
risks
and
benefits
presented
to
the
SAP
stated
that
"
the
published
preliminary
monarch
toxicity
information
is
not
sufficient
to
cause
undue
concern
of
harmful
widespread
effects
to
monarch
butterflies
at
this
time."

EPA
Response:
Extensive
studies
performed
under
the
auspices
of
the
USDA
in
the
US
and
Canada,
and
published
in
the
proceedings
of
the
NAS
show
that
the
amount
of
pollen
deposited
from
the
currently
approved
corn
producing
anti­
lepidopteran
Cry
protein
has
no
detrimental
effect
on
the
development
of
monarch
larvae
(
Proc.
Natl.
Acad.
Sci.
USA,
10.1073).
MON
863
Cry3Bb1
is
a
coleopteran
active
protein
that
is
not
expected
to
affect
lepidopterans
such
as
the
monarch
butterfly.
Nevertheless,
Monsanto
sponsored
a
monarch
butterfly
feeding
study
which
has
shown
that
exposure
to
corn
pollen
expressing
the
Cry3Bb1
protein
will
not
result
in
toxic
or
developmental
effects
in
monarch
larvae.
Section
3.2
3
­
54
3.2
­
Environmental
Fate/
Persistence/
Indirect
Effects
on
Soil
Biota
3.2
­
Comment
Excerpts
Commenter
Name:
Dan
Bolling
Commenter
Organization
Name:
Dan
Bolling
Comment
Number:
30509­
045000
Excerpt
Number:
1
Excerpt
Text:
Although
we
all
agree
on
the
goal
of
reducing
the
use
of
pesticides
­
for
environmental
and
economic
reasons
­
I
believe
we
should
not
assume
that
the
proposed
adoption
of
Bt
protected
corn
is
necessarily
the
best
approach.
Undoubtedly
it
is
the
approach
with
the
greatest
potential
economic
return
to
its
developers.
And
it
may
yet
prove
to
be
the
environmentally
preferred
approach,
as
well.

But
as
yet
the
assessment
of
risk
from
Bt
resistant
pest
development,
Bt
soil
residuals,
and
unintended
impacts
on
beneficial
species
have
not
been
adequately
communicated
to
the
public.
Many
thoughtful
voices
have
suggested
that
this
reflects
the
fact
that
inadequate
risk
assessment
has
been
performed.

EPA
Response:
The
toxicology
studies
for
protein
Plant
Incorporated
Protectants
(
PIP)
done
by
the
EPA
on
non­
target
organisms
have
been
submitted
to
a
Scientific
Advisory
Panel
(
SAP)
review
in
December,
1999
and
August
2002.
The
SAPs
found
to
studies
adequate
with
recommendations
that
EPA
test
more
species
appropriate
to
the
agroecosystems
in
question.
(
SAP
Report
No.
99­
06,
February
4,
2000;
SAP
Meeting
Minutes
No.
2002­
05,
November
6,
2002).
These
recommendations,
as
well
as
the
recommendation
to
do
long
range
field
monitoring,
are
being
implemented
by
the
EPA.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
8
Excerpt
Text:
III.
Degradation/
dissipation
of
material
From
the
little
information
on
the
employed
protocols
provided
in
the
Monsanto
Document,
the
presented
degradation
studies
appear
to
have
been
conducted
according
to
the
protocols
described
in
studies
1
and
2
of
the
EcoStrat
report
on
'
Bt
proteins
in
soils'
(
2001).
Also
here,
unrealistic
short
half­
life
dissipation
times
(
2.37
and
2.76
days)
were
reported
based
on
fitted
data
calculated
from
insect
bioassay
results
with
the
Colorado
potato
beetle.
Additionally,
ELISA
analyses
were
conducted.
From
that
we
speculate
that
the
trials
were
not
carried
out
with
unprocessed
plant
material
but
probably
with
microbial
derived
Cry3Bb
protein
or
processed
plant
powder
similar
to
the
reviewed
studies
1
and
2
mentioned
above.
However,
because
also
no
information
is
provided
in
the
Monsanto
Document
on
the
experimental
conditions
like
temperature,
soil
moisture
and
ambient
air
humidity
levels,
no
independent
scientific
evaluation
is
possible.
If
similar
settings,
materials
and
methods
were
employed
as
described
in
the
studies
1
and
2
mentioned
above,
the
same
short
comings
apply
of
course
that
were
outlined
there
(
EcoStrat
Report
2001).
In
an
agricultural
ecosystem,
in
particular
in
temperate
regions,
unprocessed
plant
material
will
enter
the
soil
Section
3.2
3
­
55
upon
harvest
at
the
end
of
the
vegetation
period
during
fall
when
temperatures
drop
and
the
metabolism
of
all
organisms
slows
down
to
an
absolute
minimum
in
order
to
survive
the
cold
winter
season.
Therefore,
it
must
be
expected
that
also
dissipation
and
degradation
of
plant
material
and
the
respective
Bt
proteins
are
reduced
and
will
remain
much
longer
in
the
soil
ecosystem
than
observed
under
optimal
laboratory
conditions.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
29
Excerpt
Text:
1.
Require
Monsanto
to
submit
data
on
Cry
3Bb
expression
levels
in
MON
863
tissues
under
a
range
of
environmental
conditions
and
on
the
quantity
and
fate
of
Cry
3Bb
proteins
exuded
into
the
soil.

Commenter
Name:
Rissler
Commenter
Organization
Name:
UCS
Comment
Number:
30509B­
089000
Excerpt
Number:
31
Excerpt
Text:
4.
Direct
its
Scientific
Advisory
Panel
(
SAP)
to
determine
whether
the
technology
currently
exists,
or
is
likely
to
exist
within
five
years,
to
direct
the
expression
of
Bt
endotoxins
toward
plant
roots
such
that
levels
in
roots
exceed
levels
in
grain,
pollen
and
leaf
tissue
by
10­
fold
or
more.

EPA
Response:
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
been
evaluated.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
As
expected,
longer
degradation
times
occur
in
corn
tissues
due
to
the
time
required
for
the
tissue
to
decay
and
for
the
Cry3Bb1
protein
to
move
from
tissue
to
soil.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
However,
because
the
Agency
believes
that
additional
studies
would
be
useful
in
completing
the
database
for
long
term
effects
assessment,
it
is
requesting
supplementary
multi­
year
studies
regarding
Cry3Bb1
protein
degradation
and
persistence
in
various
agricultural
soils.

The
Agency
has
also
required
the
submission
of
additional
expression
data
in
terms
of
dry
weight,
as
the
amount
of
protein
present
in
the
given
tissue.
Tissues
for
which
expression
data
must
be
provided
include:
leaf,
root,
pollen,
seed,
and
whole
plant.
In
addition,
data
for
each
of
these
tissues
should
be
provided
for
young
plants
in
rapid
growth,
during
flowering,
and
mature
plants
before
harvest
when
that
part
of
the
plant
is
present.
Data
obtained
for
roots
should
also
include
Section
3.2
3
­
56
typical
times
when
corn
rootworm
would
be
feeding.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
17
Excerpt
Text:
II.
Routes
of
input
and
amount
of
transgenic
plant
material
and
plant­
expressed
novel
protein
into
the
soil
ecosystem
In
our
Ecostrat
report
on
'
Bt
in
soils'
(
2001),
we
describe
the
various
routes
transgenic
Bt
plant
material
enters
the
soil
ecosystem.
In
order
to
identify
possibly
exposed
nontarget
organisms
and,
subsequently
study
whether
they
will
be
affected
or
not
by
the
released
Bt
toxins,
tissue­
specific
information
on
Bt
concentration
in
plant
residues
and
plant
excretions
as
well
as
their
dissipation/
degradation
in
soils
is
important.
Tissue­
specific
Bt
concentration
levels
have
been
discussed
above
already
and
were
provided
in
the
Monsanto
Document.
However,
no
data
was
provided
regarded
Bt
concentrations
in
plant
excretions
like
root
exudates
or
mucilage
produced
by
the
roots
(
see
Introduction
in
EcoStrat
report
2001
on
'
Bt
in
soils').
These
excretions
are
produced
throughout
the
life
time
of
the
plant
in
addition
to
small
amounts
of
broken­
off
plant
debris
and
continuously
enter
the
soil
ecosystem.
Many
micro­
organisms
but
also
organisms
like
nematodes
or
macrofauna
like
earthworms
etc.
are
thereby
continuously
exposed
to
these
toxins.
In
particular
when
considering
the
large
amounts
of
Cry3Bb
toxin,
it
must
be
of
primary
interest
to
assure
that
such
organisms
providing
very
important
services
for
soil
health
and
soil
ecosystem
function
will
not
be
adversely
affected
by
the
high
input
of
Bt
toxin.
However,
no
data
on
this
is
provided
in
the
Monsanto
Document.
Further,
also
no
data
is
provided
on
binding
of
Cry3Bb
proteins
to
surface­
active
particles.
In
particular
with
regard
to
the
on­
going
production
of
transgenic
Cry1Ab
corn
on
large
areas
lasting
several
years
already,
this
becomes
an
interesting
and
probably
scientifically
challenging
task
to
study,
since
both
types
of
toxins
may
interact,
for
example
by
competing
for
binding
sites
on
surface­
active
particles.
From
the
results
of
previous
such
studies
with
Cry1Ab
corn,
it
cannot
be
excluded
that
Bt
Cry1Ab
levels
in
soils
are
already
elevated.
Therefore,
and
additionally
because
Monsanto
Company
recommends
to
grow
both
types
of
transgenic
Bt
corn
varieties,
Cry3Bb
and
Cry1Ab
corn,
simultaneously
for
resistance
management
purposes,
soil
nontarget
testing
must
account
for
interaction
effects
of
Cry3Bb
and
Cry1Ab.
To
date,
this
has
not
at
all
been
addressed.

EPA
Response:
The
fate
of
Cry3Bb1
protein
in
soils
and
indirect
effects
on
soil
biota
have
been
evaluated
by
the
EPA.
Test
data
show
that
most
of
the
Cry
protein
deposited
into
soil
is
quickly
degraded,
although
a
residual
amount
may
persist
in
biologically
active
form
for
a
much
longer
period
of
time.(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
It
is
also
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeat
Bt
spray
applications
when
compared
to
soil
exposed
to
growing
Bt
crop.(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Soil
heath
evaluation
from
limited
data
do
not
indicate
that
Cry
proteins
have
any
detrimental
effect
on
microbial
populations
in
the
soil.(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5).
Horizontal
transfer
from
transgenic
plants
to
soil
bacteria
has
not
been
demonstrated
except
under
artificial
laboratory
conditions
created
to
favor
DNA
transfer.
Published
studies
of
Bt
Cry
protein
in
soil
show
no
effect
on
bacteria,
actinomyces,
fungi,
protozoa,
algae,
Section
3.2
3
­
57
nematodes,
springtails
or
earthworms.(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5).
In
addition,
new
plants
planted
in
Bt
Cry
protein
containing
soil
do
not
take
up
the
Bt
protein.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6).
Two
season
field
studies
provide
further
evidence
that
the
Cry3Bb1
protein
in
corn
event
MON
863
produces
no
short
term
risk
of
unreasonable
adverse
effects
for
the
environment.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b.
iii).
Because
the
Agency
believes
that
additional
studies
would
be
useful
in
completing
the
database
for
long
term
effects
assessment,
it
is
requesting
additional
supplementary
studies
regarding
Cry3Bb1
protein
degradation
and
persistence
in
various
agricultural
soils.
Additive
effects
of
several
Cry
proteins
have
been
looked
at
by
the
EPA
in
laboratory
studies
on
non­
target
organism
effects.
No
additional
effects
by
the
combination
were
seen
on
susceptible
insect
species.
The
root
exudate
issue
has
been
indirectly
addressed
by
testing
the
springtails
and
earthworms
at
Cry
protein
doses
exceeding
any
possible
soil
concentrations
from
plant
decay
and
root
exudation
combined.

Some
evidence
suggests
that
Cry
proteins
while
bound
to
some
soil
components,
e.
g.
clays
and
humic
acids,
are
recalcitrant
to
degradation
by
soil
microorganisms,
but
without
eliminating
their
insect
toxicity.
Several
factors
influence
either
the
affinity
of
binding
or
the
rate
of
degradation.
Low
pH
favors
mineral
binding,
while
pH
near
neutrality
generally
substantially
increases
degradation.
Corn
does
not
grow
well
below
~
pH
5.6
and
therefore
most
corn
growing
soils
are
expected
to
be
at
a
higher
pH.
Therefore,
under
most
production
conditions,
corn
would
not
be
grown
on
soils
that
would
favor
Cry
protein
binding
to
clay
particles
and
accumulation
in
soil.
Therefore,
corn
production
would
not
inhibit
the
rate
of
degradation
compared
to
what
is
seen
in
soil
of
lower
pH.
Nevertheless,
these
issues
are
being
evaluated
on
a
case­
bycase
basis
by
environmental
fate
studies
designed
to
determine
the
rate
of
Cry
protein
degradation
over
sufficiently
long
periods
to
assure
an
accurate
assessment
of
degradation
in
agricultural
soils
of
various
compositions
and
pH.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
18
Excerpt
Text:
Soil
nontarget
studies
presented
in
Monsanto
Document
Only
two
soil
inhabiting
organisms
were
studied,
the
collembola
Folsomia
candida
and
the
earthworm
(
probalby
Eisenia
foetida).
No
details
on
the
protocols
applied
were
presented
but
from
the
little
information
given,
we
speculate
that
largely
the
same
protocols
were
applied
as
in
other
previous
regulatory
studies.
These
have
been
reviewed
by
us
and
severe
short
comings
were
identified.
If
our
assumption
of
similar
protocols
is
correct,
the
same
short
comings
will
apply
for
the
studies
presented
here
(
EcoStrat
Report
2000).

"
In
the
experiments,
no
plant
foliage
was
tested
but
...
lyophilized
corn­
leaf
protein
(
a
powder)
was
used
as
dietary
test
material.
E.
foetida
was
added
to
a
mixture
of
soil
and
lyophilized
leaf­
protein
powder,
although,
this
species
hardly
eats
soil
and
prefers
organic
material.
F.
candida
feeds
on
saphrophytic
fungi
growing
on
decaying
plant
material
but
was
also
offered
lyophilized
leaf­
protein
powder
mixed
into
soil.
In
acknowledgment
of
the
feeding
preference
of
this
species
yeast
was
also
added.
But
it
is
unclear
whether
F.
candida
actually
ingests
any
of
the
leaf­
protein
powder
when
ingesting
the
yeast
cells."
Section
3.2
3
­
58
"
None
of
the
ecotoxicological
tests
considered
multitrophic
interactions
between
plants,
herbivores
or
pathogens
and
natural
enemies."

The
regulatory
trials
with
E.
foetida
reviewed
in
the
EcoStrat
Report
(
2000)
lasted
only
14
days
although
these
organisms
can
live
for
more
than
a
year
and
the
Bt
plants
grow
in
the
field
for
several
months
and
there
is
strong
evidence
that
the
Bt
proteins
and
the
transgenic
plant
material
remain
even
longer
in
the
soil.

EPA
Response:
There
are
reports
in
the
open
literature
that
earthworms
tested
for
ingestion
of
Cry
protein
from
soils
do
ingest
it,
and
Cry
protein
is
also
detected
in
earthworm
casts.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
a.
vii).
Lyophilized
leaf
tissue
is
actually
dehydrated
plant
tissue,
therefore,
tissue
was
tested.
The
collembola
in
the
tests
performed
exhibited
a
greenish
color
indicative
of
leaf
tissue
ingestion.
However,
even
if
collembola
do
not
ingest
plant
tissue,
no
hazard
to
collembola
would
exist.
Testing
of
collembola
for
more
than
14
days
(
chronic
testing)
is
not
deemed
appropriate
at
this
time
because
the
collembola
populations
are
evaluated
during
field
testing.
In
addition,
the
collembola
studies
did
go
through
a
reproductive
cycle.
This
amounts
to
a
chronic
and
a
reproductive
test.

The
August,
2002
SAP
cast
doubt
on
the
usefulness
of
tritrophic
studies,
nevertheless,
EPA
intends
to
request
tritrophic
studies
where
they
appear
to
be
warranted
by
the
ecology
of
the
crop
in
question.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000
Excerpt
Number:
19
Excerpt
Text:
It
appears
that
in
the
E.
foetida
study
presented
in
the
Monsanto
Document
microbial­
derived
Cry3Bb
protein
was
added
to
the
soil
while
F.
candida
was
actually
provided
transgenic
Cry3Bb
plant
material.
But
a
10­
fold
safety
margin
is
not
outstanding
for
a
laboratory
ecotoxicity
trial.
In
such
studies
larger
safety
margins
are
commonly
applied,
certainly
for
human
exposure.
Additionally,
the
calculations
relating
to
expected
exposure
of
soil
inhabiting
organisms
presented
in
the
Monsanto
Document
are
unconvincing
and
speculative.
On
page
25
of
56
it
is
stated:

'
The
maximum
environmental
concentration
for
soil­
dwelling
organisms
is
predicted
to
be
13.3
mg/
kg
based
on
the
assumption
that
corn
plants
are
tilled
into
the
tip
6"
of
soil
at
the
time
of
maximum
leaf
expression
for
Cry3Bb1.11098
(
i.
e.,
93
?
g/
g).
The
measured
NOECs
form
these
tests
exceed
the
maximum
predicted
environmental
concentration
by
3­
to
140­
fold,
demonstrating
an
adequate
margin
of
safety
for
these
organisms.'

However,
in
particular
E.
foetida,
commonly
called
the
compost
worm
and
sold
commercially
for
improvement
of
garden
composts
consisting
almost
exclusively
of
organic
material,
and
other
earthworms
in
the
field
prefer
to
feed
on
organic,
i.
e.
plant,
material
and
ingest
little
soil.
So,
the
expected
exposure
is
higher
and
closer
to
the
actual
expression
levels
in
the
transgenic
plant
residues.
At
the
time
of
harvest
expression
levels
recorded
in
the
Monsanto
Document
on
page
24
of
29
(
Table
2)
ranged
between
13
­
35
?
g/
g
(
mean
25
?
g/
g)
for
whole
plants
and
3.2
­
36
?
g/
g
(
mean
24
?
g/
g)
for
roots
(
both
at
day
126
after
planting),
so
in
fact
up
to
3­
times
higher
than
suspected
in
the
Monsanto
Document.
These
concentrations
Section
3.2
3
­
59
add
to
the
Cry3Bb
and
Cry1Ab
toxin
concentrations
potentially
present
already
in
the
soil
due
to
root
exudation
and
previous
Bt
corn
production.
In
the
Monsanto
Document
a
NOEC
of
57
mg/
g
which
is
the
equivalent
of
57
?
g/
g
is
reported
above
which
effects
must
be
expected.
Therefore,
the
safety
margin
has
decreased
to
0
­
1.5
fold
and
adverse
effects
on
this
organism
seem
likely
and
merit
further
laboratory
and
field
experimentation
with
relevant
organisms,
more
realistic
exposure
levels
and
preferred
diet,
i.
e.
unprocessed
plant
material.

However,
no
details
on
rearing
and
experimental
conditions
nor
experimental
design
were
given.
Therefore,
no
final
evaluation
of
the
data
or
conclusions
can
be
drawn
from
these
studies.

We
would
also
like
to
point
out
that
soil
organisms
like
earthworms
may
accumulate
fairly
high
concentrations
of
Bt
and
serve
as
mediators
of
high
Bt
doses
to
their
natural
enemies
like
birds,
rodents
and
other
small
mammals
even
if
the
toxin
does
not
affect
the
earthworms
themselves.
To
our
knowledge,
this
issue
has
not
been
addressed
yet
neither
in
private
nor
public
sector
research.

EPA
Response:
The
available
data
do
not
support
these
comments.
There
are
reports
in
the
open
literature
that
earthworms
tested
for
ingestion
of
Cry
protein
in
the
system
used
by
Monsanto
do
ingest
Cry
protein,
and
Cry
protein
is
also
detected
in
earthworm
casts.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
3.
a.
vii)
The
earthworms
were
not
affected
by
the
Cry
proteins.
Monsanto
testing
was
done
with
purified
cry
protein
in
order
to
expose
the
earthworms
to
large
doses
exceeding
the
possible
soil
concentration.
Birds
and
mammals
fed
Cry
proteins
do
not
show
any
adverse
effects,
therefore
ingestion
of
earthworms
with
cry
protein
would
not
pose
a
hazard.
The
calculations
presented
by
this
commenter
are
not
based
on
the
same
figures
supplied
with
the
earthworm
studies.

The
study
referred
to
by
the
above
commenter
(
MRID
No.
449043­
16)
shows
that
the
570mg/
kg
soil
was
the
highest
concetration
tested,
so
that
the
LC50
is
above
that
concentration,
not
at
that
concentration.
This
study
has
a
10
x
safety
factor.
The
data
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein
producing
corn
plants.
The
calculations
for
soil
concentration
in
the
top
6"
were
done
by
standard
EPA
procedures
that
have
been
approved
by
Scientific
Advisory
Panels.
As
for
the
suggestion
that
a
test
concentration
greater
than
10x
is
required
to
assess
toxicity,
the
EPA
hazard
evaluation
procedure
considers
an
LD50
(
not
a
NOEC)
at
5x
the
environmental
concentration
(
EEC)
as
indicative
of
no
hazard
to
terrestrial
organism
populations,
while
in
the
submitted
studies
there
was
no
LD50,
or
mortality
seen
at
10
x,
the
highest
concentration
tested.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
1
Excerpt
Text:
1)
Only
a
limited
number
of
studies
is
currently
available
that
explored
input.
persistence/
degradation
of
Bt
proteins
from
transgenic
plants
and/
or
their
effects
on
soil
organisms.
These
studies
were
conducted
in
four
different
laboratories
and
do
not
yet
allow
a
meaningful
assessment
of
the
ecological
impact
of
transgenic
Bt
plants
in
soil
ecosystems
for
the
following
reasons:
Section
3.2
3
­
60
a)
The
studies
analyzed
are
difficult
to
compare,
because
of
the
different
experimental
conditions,
methods
and
materials
used.
For
example,
in
some
studies,
Bt
toxins
and
protoxins
derived
from
commercial
formulations
were
used,
in
others
intact
and/
or
frozen
plant
material
was
used,
and
in
others,
processed,
sieved
plant
powder
was
used
to
explore
persistence/
degradation
and
impact
on
soil
organisms.
Furthermore,
different
and
often
rather
unspecified
soil
types
were
used.

b)
Temperatures
for
incubation
were
in
some
studies
high,
which
provides
for
maximal
microbial
activity
and
are
not
representative
for
soils
in
temperate
regions.
Therefore,
it
must
be
assumed
that
microbial
degradation
under
field
conditions
is
reduced
compared
with
what
was
observed
under
high
humidity
and
high
temperature
conditions
in
the
laboratory.
Consequently,
toxin
persistence
can
be
expected
to
be
greater
at
least
in
temperate
regions
with
distinct
winter
seasons
and
Bt
crops
harvested
at
the
end
of
the
growing
season
when
temperatures
begin
to
drop.

For
these
reasons,
it
is
difficult
to
combine
the
information
provided
in
these
studies
for
a
final
synthesis
and
conclusions.

EPA
Response:
EPA
agrees
that
the
long
range
effects
data
base
needs
to
be
enlarged
over
a
longer
time
period
to
supplement
the
existing
information.
To
this
end,
the
EPA
is
granting
time­
limited
registrations
and
is
requiring
multi­
year
Cry
protein
soil
accumulation
studies
in
a
variety
of
soil
and
temperature
conditions.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
F)

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
2
Excerpt
Text:
2)
From
the
few
studies,
it
cannot
be
excluded
that
Bt
proteins
may
accumulate
in
the
soil
under
continuous
large­
scale
production
of
transgenic
Bt
plants.
After
analyzing
the
relevant
studies,
there
is
reason
to
believe
that
Bt
toxins
can
persist
in
soil
for
a
long
time;
according
to
the
data
collected
in
the
laboratory
trials
possibly
for
more
­
than
eight
months.
This
will
depend
on
the
soil
type,
predominantly
on
the
content
of
surface­
active
particles
like
clay
minerals
or
humic
acids.
There
is
further
evidence
that
coupled
with
persistence,
the
insecticidal
activity
of
the
Bt
toxins
is
retained
at
ecologically
relevant
levels.
In
temperate
regions,
the
toxin
may
well
persist
over
the
winter
months.
In
this
context,
root
exudation
of
Bt
from
transgenic
corn
becomes
an
important
aspect
to
consider
because
Bt
toxins
are
excreted
already
in
the
seedling
stage,
i.
e.,
early
in
the
season.
In
practice,
this
may
mean
that
before
the
incorporated
Bt
toxin
from
the
previous
growing
season
is
fully
degraded,
more
Bt
toxin
from
the
following
vegetation
period
is
introduced
again.
Over
many
years,
this
could
potentially
lead
to
an
accumulation
of
Bt
toxins
in
some
soils,
i.
e.
with
high
content
of
surface­
active
particles.
No
coordinated
efforts
are
under
way
to
monitor
the
Bt­
contents
in
the
soils
of
those
areas
where
Bt
crops
are
produced
since
several
years,
nor
has
any
research
been
conducted
and
published
to
date
studying
fate
and
movement,
of
free
and
bound
Bt
proteins
in
such
soils
with
high
content
of
surface­
active
particles.

3)
The
evidence
for
persistence
of
Bt
proteins
in
soils
and
their
retained
insecticidal
activity
poses
potential
hazards
for
soil
micro­
and
macro­
organisms.
Firstly,
nontarget
effects
could
arise
that
may
impact
soil
Section
3.2
3
­
61
food
web
structure
and
biological
regulation
mechanisms.
Further,
it
could
also
select
for
reduced
susceptibility
of
exposed
nontarget
soil
anthropods,
e.
g.
soil
inhabiting
lepidopteran
and/
or
coleopteran
larvae.
This
may
have
repercussions
for
future
transgenic
Bt
plants
designed
to
control
such
soil­
inhabiting
lepidopteran
or
coleopteran
larvae
that
are
serious
pests
of
crop
plants,
e.
g.
Diabrotica
species.
However,
enhanced
unintended
control
of
soil­
inhabiting
pest
species
may
occur
as
well,
as
a
'
positive'
side
effect,
at
least
until
resistance
develops,
in
some
of
the
reviewed
studies,
temporary,
statistically
significant
or
slight,
statistically
insignificant
effects
on
soil
micro­
or
macroorganisms
were
observed.
However,
the
research
conducted
to
date
is
far
too
limited
to
allow
for
'
safe'
conclusions
yet,
in
particular,
with
respect
to
long­
term
effects
under
constant
and/
or
repeated
exposure
to
Bt
proteins.

4)
Soil
ecology
is
so
poorly
understood
that
interpretation
of
the
limited
number
of
studies
is
almost
impossible
due
to
the
fundamental
lack
of
knowledge
about
functioning
and
ecological
interactions
in
soils.

EPA
Response:
The
information
available
to
date
indicates
that
soil
accumulation
of
significant
amounts
of
Bt
Cry
protein
and
ecosystem
perturbation
does
not
take
place.
However,
EPA
recognizes
the
value
of
additional
multiyear
studies
to
confirm
these
findings.
To
this
end
the
EPA
is
requiring
multi­
year
non­
target
effects
and
Cry
protein
soil
accumulation
studies
in
a
variety
of
soil
and
temperature
conditions.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
F).
These
issues
are
also
one
of
the
reasons
why
the
Agency
is
granting
time
limited
registrations
of
Cry
protein
containing
crops.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
3
Excerpt
Text:
Pre­
commercial
release
research
Research
activities
in
basic
soil
ecology
and
soil
(
bio­)
chemistry
and,
specifically,
on
all
aspects
necessary
for
hazard
identification,
including
input,
fate
and
impact
of
Bt
proteins
should
be
increased
significantly.
It
would
further
be
desirable
if
standard
protocols
could
be
developed
and
agreed
upon
to
facilitate
the
comparison
of
the
various
studies
in
the
future.
Pre­
commercial
release
research
should
also
investigate
the
movement
of
the
Bt
proteins
released
from
transgenic
plants
in
soils,
including
soils
that
are
already
in
Bt
plant
production
since
many
years.
Further,
the
impact
of
Bt
proteins
from
transgenic
plants
on
the
soil
macro­
and
microbiota
diversity
and
population
dynamics
should
be
assessed,
and,
vice
versa,
also
the
degree
to
which
varying
communities
of
microorganisms
affect
persistence/
degradation
of
Bt
proteins
in
different
soil
environments.
Because
rhizosphere
microorganisms
are
very
important
for
plant
health
and
development,
it
is
essential
that
proper
testing
of
these
microorganisms
with
Bt,
is
undertaken
prior
to
the
large­
scale
release
of
the
transgenic
Bt
plants.
In
particular
with
regard
to
modem,
sustainable
agricultural
production
methods
aiming
at
reducing
external
input
like
fertilizers,
rhizosphere
microorganisms
will
become
increasingly
important
again.

Post­
commercial
release
continued
oversight
The
most
obvious
and
straightforward
approach
to
study
the
fate
of
Bt
toxins
in
soil
ecosystems
is
to
monitor
the
content
of
Bt
proteins
in
soils
that
have
been
under
continuous
Bt
plant
production
for
several
Section
3.2
3
­
62
years.
Mathematical
modeling
and
computer
simulations
might
be
useful
in
the
design
of
monitoring
schemes.
Many
of
the
questions
posed
in
this
document
could
be
answered
by
now
if
monitoring
had
been
in
place
from
the
beginning
of
commercial
field
production
of
transgenic
Bt
plants.
Today,
in
the
6~
h
year
of
Bt
crop
production,
it
may
be
difficult
to
find
appropriate
control
fields
for
comparison.
However,
this
is
only
true
for
the
United
States
and,
perhaps,
Canada,
where
most
of
the
transgenic
Bt
crops
are
grown.
Therefore,
determination
of
natural
background
levels
of
Bt
protein
in
soils
not
yet
in
production
of
transgenic
Bt
plants
in
Europe
and
most
other
countries
of
the
world
should
be
initiated
before
their
largescale
commercialization.

EPA
Response:
The
information
available
to
date
indicates
that
soil
accumulation
of
significant
amounts
of
Bt
Cry
protein
and
ecosystem
perturbation
does
not
take
place.
However,
EPA
recognizes
the
value
of
additional
multiyear
studies
to
confirm
these
findings.
To
this
end
the
EPA
is
requiring
multi­
year
non­
target
effects
and
Cry
protein
soil
accumulation
studies
in
a
variety
of
soil
and
temperature
conditions.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
F).
These
issues
are
also
one
of
the
reasons
why
the
Agency
is
granting
time
limited
registrations
of
Cry
protein
containing
crops.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
5
Excerpt
Text:
1)
Input
of
Bt­
proteins
from
transgenic
plants.
Bt­
proteins
from
transgenic
plants
entering
the
soil
ecosystem
stem
from
both
living
plants
and
dead
plant
debris/
residues.
The
temporal
distribution
of
the
amounts
of
Bt­
toxins
entering
the
soil
ecosystem
varies
considerably:
­
input
through
root
exudation,
root
leakage
and/
or
root
debris
left
behind
during
growth
is
a
permanent
process
of
probably
small
amounts
(
not
quantified
yet).
­
input
via
plant
parts
(
pollen,
leaves,
etc.)
falling
to
the
ground
throughout
the
season
prior
to
harvest
is
also
a
permanent
process
of
smaller
amounts.
­
input
via
plant
residues
(
dead
or
alive,
e.
g.
as
volunteer
seeds)
remaining
in
the
field
after
harvest,
and,
possibly,
incorporated
into
soil
is
a
cyclic
process
of
large
amounts.

2)
Fate
of
Bt­
proteins
from
transgenic
plants.
Depending
on
the
environmental
conditions
and
soil
type,
Bt­
proteins
can
either
persist
or
degrade
more
or
less
quickly.
Points
to
consider
here
are:
­
Toxin
inactivation.
­
Mechanisms
of
persistence.
­
Chemical
degradation.
­
Microbiological
degradation.
­
Removal
from
soil
(
e.
g.
through
leaching).
­
Uptake
by
higher
soil
organisms
or
plants.

3)
Effects
on
soil
organisms.
Bt
proteins
released
from
transgenic
plants
may
have
various
impacts
on
the
soil
biota.
Section
3.2
3
­
63
­
Impact
on
biodiversity
or
biomass
of
soil
organisms
(
e.
g.
abundance,
population
dynamics,
community
composition).
­
Impact
on
soil
and
plant
health
if
check­
balance
systems
of
pathogenic
and
beneficial
microorganisms
are
disrupted.

EPA
Response:
The
October
2000
SAP
concluded
that
invertebrates
such
as
earthworms
and
springtails
(
collembola)
are
appropriate
indicator
species
for
Cry
protein
testing
because
of
the
specific
nature
of
the
Cry
protein
toxicity
to
select
target
species.
(
SAP
Report
No.
2000­
07.
March
12,2001)
When
it
initially
reviewed
the
applications
for
PIP
products
that
were
registered
in
1995,
EPA
considered
requiring
studies
evaluating
effects
upon
the
representative
beneficial
soil
invertebrates
Collembola
and
earthworms.
The
Agency
was
concerned
(
1)
that
such
soil
organisms
may
be
subject
to
long­
term
exposure
as
a
result
of
soil
incorporation
of
crop
residues
or
when
crop
residues
are
left
on
the
soil
surface
and
(
2)
that
adverse
effects
on
such
soil
organisms
could
result
in
an
accumulation
of
plant
detritus
in
fields.
Recent
reports
of
exudation
of
Cry
proteins
by
corn
roots
throughout
the
growing
season
add
to
this
concern.
However,
the
Agency
understands
that
routine
agronomic
practices
have
included
the
long
term
use
of
chemical
insecticides,
which
have
adverse
effects
on
soil
organisms,
but
there
has
not
been
an
accumulation
of
significant
amounts
of
plant
detritus
in
soils.
Thus,
Cry3Bb1
corn,
which
is
expected
to
have
less
impact
on
these
species
than
chemical
pesticides,
should
not
result
in
any
increased
build­
up
of
plant
detritus
or
Cry
proteins
at
toxic
levels.
Supporting
this
conclusion
are
data
received
by
EPA
that
indicate
that
such
proteins
are
known
to
degrade
rapidly
in
field
soils.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
Cry
proteins
that
are
bound
to
soil
particles
have
been
shown
to
be
rapidly
degraded
by
soil
microbes
upon
elution
from
the
soil
particles.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
Therefore,
the
potential
for
significant
soil
buildup
and
adverse
effects
to
non­
target
soil
organisms
are
not
anticipated.
It
has
been
confirmed
in
published
literature
that
Bt
Cry
protein
released
from
root
exudates
and
biomass
of
Bt
corn
plants
has
no
apparent
effect
on
earthworms,
nematodes,
protozoa,
algae,
bacteria,
actinomyces
and
fungi
in
soil
in
spite
of
the
fact
that
enough
detectable
Cry
protein
is
bound
to
soil
particles
to
show
toxicity
to
the
target
pest.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5)
These
results
suggest
that
despite
its
presence
in
soil,
the
Cry
protein
released
in
root
exudates
of
Bt
corn,
or
from
the
degradation
of
the
biomass
of
Bt
corn,
is
not
toxic
to
a
variety
of
organisms
in
the
soil
environment.
It
has
also
been
reported
that
the
same
degree
of
Bt
Cry
protein
persistence
takes
place
in
soils
that
have
been
exposed
to
repeated
Bt
microbial
spray
applications.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
In
addition,
new
plants
grown
in
Bt
containing
soil
do
not
take
up
the
Bt
protein.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6)
Nevertheless,
data
on
insects
closely
related
to
the
target
pest,
as
well
as
other
studies
to
address
the
published
data
requirements
for
registration
of
microbial
toxins
(
40
CFR
§
158)
have
been
received
and
reviewed.
However,
EPA
is
a
party
to
ongoing
research
to
address
the
long­
range
concerns
expressed.
In
addition,
the
resolution
of
these
questions
are
also
one
of
the
reasons
why
the
Agency
is
granting
time
limited
registrations
of
Cry
protein
containing
crops.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
9
Excerpt
Text:
­
Degradation
Section
3.2
3
­
64
Purified
microbial
and
plant
produced
Bt
toxin:
Seven
studies
provided
information
about
the
retained
insecticidal
activity
of
purified
toxin,
protoxin
derived
from
microbial
Bt­
preparations
and
of
plantproduced
toxin
in
soil
(
studies
6­
9,
11
and
12).
The
experimental
period
in
these
studies
varied
from
50
hours
to
234
days,
and
in
most
of
the
studies,
the
toxin
could
be
detected
at
every
time
point.

Studies
6­
8
investigated
binding
and
insecticidal
activity
of
purified
Bt
toxins
obtained
from
microbial
Bt
preparations
when
bound
on
surface­
active
particles
such
as
clay
minerals
or
humic
acids
in
the
presence
of
microorganisms.
In
all
studies,
it
was
demonstrated
that
Bt
toxins
adsorbed
rapidly
on
the
clay
minerals,
thus,
confirming
the
results
of
earlier
studies
(
see
above,
studies
3
to
5
and
7).
In
study
6,
it
was
shown
that
Bt
toxins
were
resistant
to
microbial
degradation
when
bound
on
the
clay
minerals
montmorillonite
and
kaolinite
or
on
the
clay­
size
fraction
of
different
soils.
Insecticidal
activity
was
retained
after
40
days
(
longest
time
period
measured)
when
toxin­
containing
soils
were
incubated
with
microbial
cultures.
Similar
results
were
obtained
with
humic
acids,
i.
e.,
protection
from
biodegradation
and
retained
insecticidal
activity
(
study
7).
In
study
8,
soils
containing
kaolinite
and/
or
montmorillonite
were
incorporated
with
Btktoxins
and
supematants
from
fresh
garden
soil
to
enhance
microbial
activity.
Again,
insecticidal
activity
was
retained
after
234
days
of
incubation
(
240C).
During
this
time,
for
example,
the
LC50
of
Manduca
sexta
to
Btk
bound
on
kaoliite
increased
fourfold
over
the
LC50
measured
before
incubation.
Hence,
activity
had
declined
to
25%
of
the
initial
activity
of
the
toxin.
The
amount
of
retention
of
insecticidal
activity
varied
with
soil
type,
clay
mineral
content,
clay
mineral
composition
and
pH.

Study
11
describes
a
method
to
optimize
extraction
and
immunological
detection
of
Bt
toxin
from
soils.
Highest
recovery
of
Bt
was
found
in
soils
with
Low
clay
content
and
low
organic
matter.
Buffer
characteristics,
such
as
ionic
interactions
and
pH,
were
important,
e.
g.
neutral
or
alkaline
conditions
promoted
recovery,
and
there
was
also
an
interaction
between
buffer
characteristics
and
soil
type.

In
study
13,
Bt
toxins
rapidly
degraded
to
a
low
concentration
at
the
beginning
of
the
experiment
and
then
remained
unchanged
for
several
weeks.
The
authors
speculated
that
this
may
have
been
the
result
of
binding
of
the
proteins
on
soil
particles,
which
protected
them
from
biodegradation
(
see
above
'
mechanisms').
A
similar
observation
was
made
in
study
7
when
free
toxin
was
mixed
with
free
humic
acids.
After
4
to
6
hours,
microbial
growth
was
lower
in
a
medium
(
DCMM)
containing
both
humic
acids
and
free
toxin
than
in
a
medium
containing
free
toxin
only;
i.
e.,
without
humic
acids.
The
authors
concluded
that
this
effect
was
probably
the
result
of
adsorption
of
the
toxin
to
the
humic
acids
which
resulted
in
their
protection
from
biodegradation.

EPA
Response:
EPA
recognizes
the
fact
that
Cry
proteins
do
adsorb
to
clay
particles,
but
are
quickly
inactivated
by
soil
microbes
upon
elution.
EPA
also
recognizes
that
Cry
proteins
within
plant
tissues
may
remain
active
for
the
same
long
period
of
time
that
it
takes
for
the
plant
tissue
to
decay.
The
long
term
implications
of
the
fate
of
Cry
proteins
is
soil
after
several
seasons
of
Cry
protein
containing
crop
cultivation
are
being
addressed
by
environmental
fate
studies
designed
to
determine
the
rate
of
Cry
protein
degradation
over
sufficiently
long
periods
to
assure
an
accurate
assessment
of
degradation
in
agricultural
soils
of
various
compositions
and
pH.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Section
3.2
3
­
65
Comment
Number:
30509­
113000Part2
Excerpt
Number:
10
Excerpt
Text:
Differences
between
transgenic
plant
toxins
and
purified
microbial
toxins.
In
three
studies
(
11,
12,
and
13)
purified
and
transgenic
plant
toxin
were
compared.
In
studies
11
and
13,
transgenic
plant
toxin
degraded
faster
than
purified
toxin
suggesting
that
the
purified
toxin
was
more
stable.
The
authors
speculated
that
this
could
have
been
due
to
degrading
enzymes
and
compounds
released
when
plants
decay,
or
due
to
the
toxin
being
more
accessible
to
degrading
soil
microorganisms
than
the
purified
toxin,
and/
or
due
to
the
increase
in
microbial
populations
observed
when
plant
material
was
added
(
typically,
a
100­
1000
fold
increase).
However,
when
purified
toxin
was
added
to
non­
Bt
plant
material,
the
purified
toxin
had
the
slowest
degradation
rate
of
all
treatments
measured.
This
is
an
indication
that
other
factors
may
have
a
role
and
that
it
is
important
whether
the
toxin
is
within
or
outside
of
the
plant
material.

In
contrast,
in
study
12,
purified
CrylAb
and
CrylAc
degraded
a
little
faster
than
plant­
expressed
toxin:
purified
toxin
was
detected
after
28
days~
but
not
after
56
days,
whereas
plant­
expressed
toxins
and
purified
toxins
together
with
plant
material
from
parental
plant
lines
were
detected
after
56
days.

In
summary,
a
general
pattern
of
Bt
toxin
degradation
emerged:
initially,
a
rapid
decline
of
the
extractable
toxin
concentration
is
observed,
followed
by
a
more
gradual
decline
to
low
concentrations
of
the
toxin,
which
then
remained
unchanged
for
several
weeks.
This
is
somewhat
similar
to
the
observations
made
in
early
studies
on
crystalline
Bt
protein
degradation
(
see
'
Introduction').
However,
the
results
are
inconsistent,
and
it
is
not
clear
what
influences
the
somewhat
different
degradation
of
purified
microbial
toxins
versus
transgenic
plant­
expressed
toxins.
According
to
Stotzky
(
2000),
adsorption
of
the
toxins
increased
with
their
concentration
and
then
leveled
off,
suggesting
that
the
clays
became
saturated
with
the
toxins,
as
observed
with
other
proteins
and
organic
compounds.
However,
it
still
remains
to
be
investigated
to
what
extent
this
occurs
in
the
field,
whether
or
not
satiation
of
surface­
active
particles
in
the
field
can
be
reached
and
how
free
Bt
toxin
in
soils
move;
especially,
in
soils
that
are
under
continuous
Bt
plant
production
since
several
years
where
surface­
active
particles
are
perhaps
exhibiting
increasing
Bt
satiation
levels?

EPA
Response:
This
comment
is
a
summary
of
soil
degradation
findings
which
were
considered
by
the
EPA
during
the
risk
assessment
process.
Additional
research
on
the
long
term
accumulation
and
effects
of
Bt
proteins
in
soils
are
in
progress.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
11
Excerpt
Text:
Toxin
in
plant
powder.
The
reported
half­
time
for
degradation
of
1.6
days
in
study
1
is
short.
Most
likely,
the
optimal
experimental
conditions
led
to
such
a
fast
degradation.
Leaf
tissue
of
transgenic
Bt­
corn
and
isogenic
non
transgenic
corn
was
lyophilized,
ground
and
sieved.
Thereby,
also
'
hard'
leaf
material
was
well­
prepared
for
microbial
degradation,
i.
e.,
offering
a
large
surface
area
for
attack
by
microorganisms.
The
plant
powder
used
and
the
favorable
laboratory
settings
(
high
temperature)
make
it
difficult,
if
not
impossible,
to
draw
conclusions
about
degradation
under
field
conditions.
Section
3.2
3
­
66
However,
despite
the
optimal
conditions,
20%
of
the
initial
toxin
activity
was
still
detected
after
15
days
in
study
1
using
transgenic
Bt­
corn,
and
after
120
days
with
transgenic
Pt­
cotton
in
study
2.
In
study
2,
the
authors
gave
no
information
whether
the
3ctivity
could
be
measured
after
43
days,
which
represented
the
end
of
the
experimental
period.
Again,
this
supports
the
observations
made
by
others
that
the
toxin
may
persist
in
low
concentrations
for
a
long
time
in
the
soil.

Further,
the
degradation
data
provided
were
not
experimentally
obtained
but
estimated
indirectly
based
on
a
number
of
assumptions
whose
validity
are
unproven.
Only
larval
growth
of
H
virescens
larvae
was
experimentally
measured,
i.
e.,
bioactivity.
H
virescens
larvae
were
sublethally
affected
when
feeding
on
an
artificial
diet
that
contained
a
soil
solution
that
had
been
incubated
with
1.46
j.
ig
Bt
plant
powder/
g
soil
for
up
to
43
days
in
a
time
series
experiment.
The
authors
used
these
data
to
carry
out
a
nonlinear
regression
analysis
by
fitting
a
3­
parameter
function
and,
thereby,
obtained
an
EC50
value,
i.
e.,,
'
effective
concentration
of
test
suspension'
at
which
larval
growth
of
H
virescens
larvae
was
only
half
of
that
in
the
control.
The
authors
assumed
that
the
EC50
value
is
inversely
proportional
(
i.
e.,,
linear
relationship)
to
the
concentration
of
biologically
active
Bt
protein
in
the
soil
and
that
dissipation
follows
first
order
kinetics.
No
data
or
references
are
given
in
support
of
these
assumptions.
First
order
kinetics
in
dissipation
processes
is
commonly
assumed
for
pesticides
and
other
chemicals
yielding
half­
life
values.
These
half­
life
values
are
then
often
used
for
extrapolation
of
long­
term
dissipation
processes.
Even
for
pesticides
and
chemicals,
this
assumption
often
does
not
hold.
For
proteins,
this
certainly
needs
to
be
verified
experimentally,
because
they
are
far
more
complex
in
their
molecular
structure
and
behavior,
and
protein
degradation
in
soil,
in
general,
is
far
less
explored.
A
first
order
kinetics
rate
law
would
imply
a
linear
relationship
between
ln(
1/
EC5o)
and
incubation
time.
However,
the
figures
shown
in
both
papers
suggest
a
non­
linear
relationship
indicating
that
a
simple
first
order
kinetics
rate
law
may
be
inadequate
to
describe
fully
the
dissipation
of
Bt
toxin.
Additionally,
the
data
were
associated
with
large
variances,
which
also
urge
for
cautious
interpretation.
The
authors
used
the
relationship
between
In(
1/
EC5o)
and
incubation
time
to
estimate
the
DT50/
90
values.
However,
because
the
basic
assumptions
are
questionable,
the
DT50/
90
values
are
also
questionable
and
should
be
interpreted
cautiously.

The
conclusions
drawn
by
the
authors:
'
CrylAb
protein
is
likely
to
degrade
rapidly
under
the
reduced
cultivation
practices
associated
with
no­
till
corn
production
as
well
as
under
conventional
cultivation
in
which
corn
plant
residue
is
plowed
into
the
soil.'
are
not
supported
by
these
experiments
that
did
not
test
degradation
under
such
field
conditions
nor
measured
it
directly
under
the
optimal
conditions
in
the
laboratory.

EPA
Response:
EPA
is
aware
of
the
non­
linear
degradation
of
Cry
proteins
in
soils
with
the
resultant
`
tailing'
effect.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
However,
studies
to
confirm
the
long
term
ecological
effects
experimentally,
and
the
time
for
complete
inactivation
of
Cry
proteins
in
agricultural
fields
over
several
planting
seasons
are
in
progress.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
12
Section
3.2
3
­
67
Excerpt
Text:
­
Uptake
by
plants
One
study
(
study
10)
investigated
the
uptake
of
Bt
toxins
from
soil
by
different
plants.
Soils
were
used
in
which
previously
either
Bt­
or
isogenic
corn
had
been
grown,
or
to
which
Bt­
or
isogenic
biomass
or
purified
Bt
toxin
had
been
added.
The
test
plants
were
corn,
carrot,
radish
and
turnip
plants
grown
for
120
to
180
days
in
the
differently
treated
soils.
Additionally,
isogenic
corn
was
grown
in
a
soil­
free,
hydroponic
medium
(
Hoagland's
solution)
in
which
previously
Bt­
corn
had
been
grown
for
15
days.

All
experiments
indicated
that
plants
do
not
take
up
Bt
toxin
released
into
the
soil
or
hydroponic
solution
from
root
exudation,
degradation
of
Bt­
corn
biomass
or
purified
toxin.
However,
the
study
reconfirmed
results
of
previous
studies
conducted
by
the
same
group
investigating
the
persistence
of
Bt
toxins
in
soils
(
studies
3­
7)
when
bound
to
surface­
active
particles
and
corn
root
exudation
of
Bt
toxin
into
soir
(
study
Q).
In
the
experiments
Bt
toxin
was
still
detectable
after
180
days
after
its
release
(
i.
e.
the
longest
time
evaluated).

To
our
knowledge,
this
is
the
first
report
about
experiments
conducted
to
test
for
uptake
of
Bt
toxins
by
plants.
Because
of
the
large
acreages
grown
to
Bt
plants
in
the
United
States,
this
topic
becomes
increasingly
relevant.
Such
studies
could
serve
as
a
model
testing
system
for
future
transgenic
plants
releasing
novel
pesticidal
compounds
into
the
soils
either
via
exudation
or
biomass
incorporation.

EPA
Response:
The
above
comments
confirm
the
EPA
determination
that
plants
do
not
take
up
Bt
protein
released
into
the
soil.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
6).
The
testing
approach
recommended
by
the
commenter
extends
beyond
a
finding
of
an
`
unreasonable
adverse
environmental
effects
determination'
and
into
the
areas
of
academic
research
and
protocol
development.
Currently,
EPA
is
limiting
the
testing
process
to
that
recommended
by
the
FIFRA
Scientific
Advisory
Panels.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
14
Excerpt
Text:
Soil
ecosystems
are
complex
and
little
explored
to
date.
Changes
taking
place
in
soil
ecosystems
can
often
not
be
observed
directly,
and
it
may
take
years
before
they
become
evident.
Early
indications
are
subtle,
and
a
good
farmer
for
example
must
be
an
alert
observer
to
detect
such
subtle
changes
which,
later,
may
result
in
drastic
effects
when
they
go
unobserved.

Soil
organisms
contribute
to
the
maintenance
of
soil
quality
in
that
they
promote
plant
growth,
influence
decomposition
of
plant
and
animal
materials,
influence
the
biogeochemical
cychng,
control
the
formation
of
soil
structure
and
the
fate
of
organic
material
(
Turco
et
al.
1994;
Linden
et
al.
1994).
Soil
contains
unknown
numbers
of
different
species
of
microorganisms
(
Angle
1994).
Of
all
soil
bacteria,
it
is
claimed
that
approximately
90%
cannot
be
isolated
or
cultured
on
laboratory
media
(
Turco
et
al.
1994).
Therefore,
determination
of
microbial
biomass
is
often
restricted
to
indirect
measures
like
metabolic
activity
(
respiration
by
measuring
CO2
emissions,
etc.).
Recently,
good
and
improving
methods
to
account
for
unculturable
microbes
are
being
developed
also
caused
by
an
increasing
demand
of
such
methods
for
the
Section
3.2
3
­
68
ecological
risk
assessment
of
transgenic
organisms
released
into
the
environment
(
see
below
"
Known
impacts
of
other
transgenic
organisms
in
soil
ecosystems").
However,
depending
on
the
methods
used,
'
microbial
communities'
or
microbial
biomass
are
investigated
which
must
be
cautiously
interpreted
under
full
consideration
of
the
constraints
of
each
method.

EPA
Response:
The
USDA
and
EPA
are
funding
long­
term
effects
studies
to
address
these
and
similar
issues.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
19
Excerpt
Text:
Past
knowledge
on
Bt
protein
degradation
and
inactivation
stems
from
research
with
microbial
Btinsecticides
Twenty
years
ago,
using
liquid
formulations
of
the
commercial
Btinsecticide
'
Thuricide',
Pruett
et
al.
(
1980)
showed
that
the
activity
of
crystals
and
spores
declined
relatively
fast,
with
crystals
loosing
their
activity
faster
than
spores.
Although
the
majority
of
spores
could
still
be
recovered
after
one
month,
50%
of
activity
was
lost
within
the
first
week
arid
95%
within
one
month
(
31
days).
The
authors
speculated
that
microorganisms
may
not
alone
be
responsible
for
degradation
and
hypothesized
that
other
soil
bacteria
could
also
impact
the
activity
of
Bt.
For
instance,
Kiselek
(
1974)
found
that
of
50
species
of
soil
bacteria
tested,
24%
were
found
to
be
potentially
antagonistic
to
Bt.
In
another
study,
West
(
1984)
found
a
reduction
of
Bt
insecticidal
activity
of
93
.4
to
99.7%
after
23
days
and
a
half­
life
of
2.7­
5.8
days
depending
on
the
supplements
added
to
the
test
soils
(
CaCO3
and/
or
organic
material).
The
author
concluded
from
his
experiments
that
protein
crystals
were
decomposed
primarily
by
soil
microorganisms.
West
et
al.
(
1984)
reported
that
loss
of
crystal
insecticidal
activity
declined
in
a
natural
soil
at
a
complex,
non­
exponential
rate.
Initial
rapid
decrease
gradually
slowed,
and
the
level
of
activity
stabilized
at
10%
of
the
initial
inocculum
after
250
days
(
sampling
was
terminated
then).
In
autoclaved
soil,
they
did
not
find
a
loss
of
crystal
activity,
suggesting
again
that
microorganisms
were
primarily
responsible
for
degradation.

The
underlying
pattern
in
the
decline
of
insecticidal
activity
seems
to
be
an
initial,
rapid
reduction
followed
by
a
gradually
slower
inactivation
to
a
low
level,
which
remains
constant
for
a
long
time.
However,
there
is
no
clear
picture
as
to
what
factors
were
involved
in
inactivation
of
Bt
crystals.
Clearly,
microorganisms
were
of
primary
importance.
However,
there
also
seem
to
be
other
factors
that
influence
the
fate
of
Bt
spores
and
crystals
in
soil.
Competition
by
other
microorganisms
was
suggested.
Different
soil
types
may
also
be
responsible
for
variability
of
inactivation.

EPA
Response:
The
USDA
and
EPA
are
funding
long
term
effects
studies
to
address
various
aspects
of
the
degradation
of
Cry
proteins
in
agricultural
soils.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
20
Excerpt
Text:
Section
3.2
3
­
69
The
massive
input
of
highly
bioactive
toxic
proteins
like
those
of
Bacillus
thuringiensis
(
Bt)
into
the
soil
ecosystem
due
to
commercial
production
of
fransgenic
Bt
crop
plants
can
potentially
exert
effects
on
all
levels
of
the
soil
ecosystem
via
direct
or
indirect
exposure
of
soil
macro­
and
micro­
organisms
to
the
Bt
toxins.
The
fact
that
Bt
is
a
soil
bacteria
does
not
preclude
effects
on
ecosystem
components
and
food
web
organisms
when
the
Bt
proteins
are
released
in
such
enormous
quantities
and
in
a
more
activated
form
that
were/
was
not
experienced
before.
Specificity
of
Bt
proteins
has
largely
been
determined
for
plant
herbivores
and
in
terms
of
lethal
effects.
With
the
production
of
vast
acreages
of
Bt
crop
plants
expressing
the
Bt
toxins
constitutively
(
i.
e.,
throughout
the
season)
and
in
high
concentration,
sublethal
longterm
effects
on
soil
inhabiting
macro­
and
micro
organisms
become
important,
in
particular,
with
respect
to
their
function
in
recycling
of
nutrients
and
soil
ecosystem
health,
i.
e.,
the
intricate
coupling
of
these
processes
and
the
balance
between
beneficial
and
detrimental
organisms.
From
the
overuse
of
insecticides,
the
detrimental
consequences
of
uncoupling
these
processes
on
soil
health
and
soil
fertility
are
well­
known
and
include
a
decline
in
soil
fertility,
disease­
suppression
capacity
and
the
subsequent
buildup
of
soil­
borne
plant
diseases
and
plant­
feeding
pest
organisms.

EPA
Response:
There
is
no
evidence
that
Bt
toxins
are
released
into
the
soil
in
"
enormous
quantities".
Data
reviewed
by
the
Agency
show
that
the
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Data
available
to
date
also
do
not
show
any
adverse
effects
on
the
soil
biota.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
These
aspects
will,
however,
be
investigated
over
several
years
to
provide
confirmatory
long
term
effects
information.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
23
Excerpt
Text:
1)
Input
of
Bt
proteins
from
transgenic
plants
­
Root
exudation
Only
one
study
investigated
an
input
route
(
Table
1)
by
studying
Bt
proteins
exuded
from
roots
into
the
rhizosphere.
It
is
well
known
that
roots
exudate
carbohydrates
and
amino
acids
(
see
below
'
Introduction').
However,
only
few
plants
are
known
that
excrete
large
molecules
like
proteins
via
their
roots.
This
paper
was
the
first
demonstrating
exudation
of
Bt
proteins
from
roots
of
transgenic
Bt­
com
seedlings.
It
was
shown
in
a
soil­
free
nutrient
solution
that
Bt
toxin
is
released
as
exudate
from
roots.
In
the
soil,
it
was
not
clear
from
the
experimental
description
how
discrimination
between
leakage
from
injured
roots
and
exudated
toxin
was
achieved.
However,
the
ecologically
meaningful
new
outcome
of
this
study
was
that
Bt
proteins
are
continuously
released
into
the
rhizosphere.
Therefore,
the
microorganisms
living
in
or
nearby
the
root
rhizosphere
are
exposed
to
Bt­
toxins
throughout
the
plants'
lifespan.
Because
rhizosphere
microorganisms
are
very
important
for
plant
health
and
development,
it
is
essential
that
proper
testing
of
these
microorganisms
with
the
respective
released
insecticidal
compound
is
undertaken
prior
to
the
largescale
production
of
such
insecticidal
transgenic
plants.

2)
Fate
of
Bt
proteins
from
transgenic
plants:
Mechanisms
of
persistence
and
degradation
Section
3.2
3
­
70
­
Mechanisms
of
persistence
Adsorption.
Laboratory
studies
with
protoxins
and
toxins
of
Bacillus
thuringiensis
isolated
from
microbial
cultures
or
commercial
formulations
showed
that
the
toxins
bind
with
varying
affinity
to
different
soil
components.
The
clay­
size
fraction
of
soil
(
studies
3­
5)
and
humic
acids
(
study
7)
are
especially
effective
in
binding
the
toxins,
whereas
the
silt­
and
sand­
size
fraction
of
soil
have
a
minor
role
(
study
5).

Clay
minerals.
In
study
3,
more
Btk­
toxin
(
Bacillus
thuringiensis
subsp.
kurstaki)
adsorbed
on
the
clay
mineral
montmorillonite
than
on
the
clay
mineral
kaolinite
(
with
a
mixed
cation
complement).
Maximum
adsorption
on
the
clay
minerals
occurred
within
30
minutes
(
studies
3
and
4).
The
pH
had
a
significant
role
in
binding:
In
one
study
(
study
3),
adsorption
of
the
toxin
(
Btk)
on
montmorillonite
decreased
as
the
pH
increased
from
4.4
to
10.0.
In
another
study
(
study
4)
adsorption
of
Btk­
and
Btt­
proteins
(
Bacillus
thuringiensis
subsp.
Tenebrionis)
on
montmorillonite
and
kaolinite
decreased
as
pH
increased
from
6.0
to
11.0.
Below
pH
60.
adsorption
decreased
also
(
study
4).
On
clays
coated
with
hydrous
ferric
oxides
(
HFO­
coated;.
in
the
study
called
'
dirty'
clays)
maximal
adsorption
occurred
at
pH
5.0
(
Btk)
(
study
4).

EPA
Response:
EPA
receives
data
on
soil
degradation
of
Cry
proteins
performed
at
high
Cry
protein
doses
that
would
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
The
authors
of
the
studies
quoted
above
have
also
shown
that
Cry
proteins
adsorbed
to
soil
minerals
are
quickly
metabolized
by
soil
microbes
upon
elution.
The
data
reviewed
by
the
Agency
show
that
the
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels.
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
the
soil
biota
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3).
These
aspects
will,
however,
be
investigated
in
greater
detail
over
several
years
to
provide
confirmatory
long
term
effects
information.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
24
Excerpt
Text:
The
Bt
proteins
did
not
significantly
intercalate
the
clay
minerals
montmorillonite
and
kaulinite
when
bound.
Therefore,
it
can
be
assumed
that
the
proteins
bind
primarily
on
the
external
surface
of
these
clay
minerals.

Humic
acids.
As
for
the
clay
minerals,
pH
and,
additionally,
the
content
of
phenolic
groups
were
primarily
responsible
for
differences
in
the
amount
of
Bt­
proteins
bound
(
study
7).
Lower
pH
and
higher
content
of
phenolic
groups
resulted
in
higher
amounts
of
proteins
bound.
Maximum
adsorption
occurred
within
I
to
2
hours
on
humic
acids
from
forest
soils
and
within
4
to
8
hours
on
humic
acids
from
cultivated
soils.

Desorption
of
clay
minerals.
In
study
3,
most
of
the
adsorbed
toxin
(
Btk)
was
desorbed
from
kaolinite
by
two
washes
with
double
distilled
water.
In
the
same
study,
toxins
bound
on
montmorillonite
were
not
desorbed
with
double
distilled
water.
In
study
4,
larger
amounts
of
Btt
were
desorbed
by
one
or
two
washes
with
double
distilled
water
than
of
Btk,
indicating
that
the
Btk
proteins
had
a
higher
affinity
for
the
clays
Section
3.2
3
­
71
tested.

Desorption
of
humic
acids.
Larger
amounts
of
Bt
proteins
could
be
desorbed
with
double
distilled
water
from
huniic
acids
at
higher
pH
and
lower
content
of
phenolic
groups
(
study
7).

All
toxins,
whether
bound
to
clay
minerals
or
humic
acids,
retained
their
insecticidal
activity
(
studies
3,
5­
8).

EPA
Response:
This
comment
is
a
summary
of
soil
binding
findings
which
were
considered
during
the
EPA
risk
assessment
process.
Additional
research
on
the
long
term
accumulation
and
effects
of
Bt
proteins
in
soils
are
in
progress.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
25
Excerpt
Text:
3)
Effects
on
soil
organisms
The
three
studies
analyzed
(
studies
12,
14,
and
15)
investigated
the
effect
of
different
plant­
expressed
Bt
toxins
on
microorganisms,
protozoa
and
arthropods
(
Appendix
2,
Table
2).
In
study
12,
the
impact
of
three
Bt
cotton
lines
expressing
either
of
two
different
Bt
toxins
(
Cry
lAb
or
CrylAc)
on
soil
microorganisms,
and
protozoa
was
in
by
mixing
leaf
tissues
of
the
different
plant
lines
or
purified
toxins
in
various
combinations
in
two
soil
types.
Over
a
period
of
28
and
56
days
­
depending
on
the
experiment
­
persistence
and
activity
of
the
toxin
and
effects
on
populations
of
protozoa
and
microorganisms
was
measured.
No
differences
between
the
numbers
of
protozoa
were
found.
However,
changes
in
bacterial
diversity,
and
bacterial
and
fungal
population
levels
were
observed
(
measured
by
species
identification,
substrate
utilization,
and
DNA
fingerprints).
In
a
number
of
experiments,
incorporation
of
transgenic
Bt
cotton
plant
material
into
soil
led
to
significantly
increased
bacterial
and
fungal
population
levels
compared
with
the
parental
lines.
These
changes
were
described
as
'
transient'
because
they
were
observed
only
during
a
limited
period
of
the
entire
experimental
time.
They
disappeared
until
the
end
of
the
experimental
period,
except
in
one
experiment
(
examining
the
transgenic
Bt­
cotton
line
249)
where
fungal
population
levels
were
always
higher
in
the
transgenic
treatment.
But,
except
for
one
treatment
in
one
experiment,
this
phenomena
(
temporary
increase
of
bacterial
and
fungal
activity
in
the
transgenic
treatment)
occurred
consistently
in
all
experiments.
Since
none
of
the
purified
toxins
or
control
plants
showed
similar
effects
on
microbial
populations,
it
is
suggested
that
other,
unintended
characteristics
of
the
transgenic
plants
may
be
involved
in
this.
These
'
transient'
effects
were
observed
after
one
soilincorporation
event
of
Bt
proteins
only.
Further
investigations
need
to
reveal
whether
after
repeated
soilincorporation
of
Bt
proteins,
constant
Bt
protein
exposure
or
accumulating
Bt
protein
concentrations
these
'
transient',
significant
effects
may
turn
into
permanent,
significant
effects.

EPA
Response:
This
comment
is
a
summary
of
studies
of
the
effects
of
Cry
protein
containing
plant
tissue
on
soil
microbial
composition.
These
findings
were
considered
during
the
EPA
risk
assessment
process.
Additional
research
Section
3.2
3
­
72
is
ongoing
on
the
long­
term
accumulation
and
effects
of
Bt
proteins
in
soils.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
26
Excerpt
Text:
In
a
field
study
using
Cry3A­
potatoes
(
study
14),
the
leaf
microflora
and
soil­
borne
pathogens
were
investigated.
Three
treatments
were
applied:
transgenic
Bt
potatoes,
a
non
Bt
potato
variety
(
Russet
Burbank;
probably
not
isogenic)
treated
with
microbial
Btt
and
a
non
Bt
potato
variety
(
Russet
Burbank;
probably
not
isogenic)
treated
with
a
systemic
insecticide.
No
untreated
control
for
comparison
was
included,
in
the
experiment
but
only
'
treatments'
were
compared.
For
a
number
of
reasons,
we
considered
the
omission
of
an
untreated
control
in
favor
of
another
treatment
in
an
attempt
to
simulate
'
common
agronomic
practice'
(
i.
e.
insecticide
treatment)
as
scientifically
problematic
because
it
introduces
a
'
normative
control'.
The
particular
choice
of
treatments,
in
this
case
the
choice
of
a
systemic
insecticide
instead
of
an
untreated
control,
will
bias
the
outcome
and
none
of
the
observed
effects
can
be
clearly
attributed
to
an
individual
treatment.
The
authors
concluded
that
Cry3A­
producing
potato
plants
did
not
consistently
affect
the
species
composition
and
density
levels
of
microorganisms
associated
with
plant
leaves
or
the
incidence
of
plant
diseases.
However,
the
data
provided
do
not
fully
support
this
conclusion.
The
authors
did
report
significantly
higher
disease
infections
of
Verticillium
dahliae
and
viruses
(
PLRV
and
PVY)
in
the
transgenic
treatments.
The
reasons
why
the
authors
believed
this
should
be
interpreted
cautiously
(
i.
e.
effects
possibly
confounded
with
the
surprisingly
early
die­
back
of
the
insecticide­
treated
plants
and
the
insecticide
treatment
itself)
may
well
be
valid
but
do
not
allow
for
a
blanket
dismissal
.
Further,
this
provided
a
case
in
point
why
the
omission
of
an
untreated
control
is
scientifically
problematic;
the
interactions
between
insecticide
treatment,
insect
virus
vector
control
and
the
surprising
die­
back
of
the
insecticide­
treated
plants
could
not
be
analyzed
property
because
there
was
no
untreated
(
isogenic?)
control
for
comparison.
Important
information
was
lost
and
the
reasons
for
this
phenomenon
remained
speculative.
Therefore,
the
repetition
of
the
whole
experiment
in
a
second
and
third
growing
season
is
recommended,
including
an
untreated
control.
It
was
suggested
that
surveying
leaf
microflora
should
be
considered
as
indicator
for
ecological
effects
in
monitoring
programs.

EPA
Response:
The
above
comment
is
a
discussion
of
a
transgenic
potato
experiment
recommending
use
of
untreated
control
plots
in
assessing
effects
of
Cry
proteins.
This
comment
is
not
relevant
to
the
MON
863
registration.
The
field
studies
submitted
for
Cry3Bb
MON863
corn
did
include
data
from
untreated
control
plots.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
27
Excerpt
Text:
The
last
study
(
study
15)
investigated
the
effect
of
Bt
cotton
expressing
CrylAb
and
CrylAc
proteins
and
potatoes
expressing
Cry3A
on
two
arthropod
species,
the
springtail
Folsomia
candida
and
the
mite
Oppia
Section
3.2
3
­
73
nitens.
The
life
history
parameters
oviposition,
number
of
eggs
produced,
body
length,
etc.,
were
measured
over
a
period
of
7
to
8
weeks.
No
effects
on
any
of
these
parameters
were
found.
However,
it
could
not
be
demonstrated
whether
this
was
due
to
insensitivity
of
the
test
organisms
to
the
Bt
or
whether
the
Bt
toxins
were
not
ingested
or
only
in
low
concentrations.
No
gut
analyses
of
the
two
test
species
were
conducted
to
confirm
the
uptake
of
the
Bt­
toxin.
It
was
assumed
that
the
Bt­
toxin
was
ingested
because,
at
least
for
0.
nitens,
'
green
color
was
seen
in
the
whitish
insect
gut...'.
Both
test
species
primarily
feed
on
the
fungi
growing
on
the
decaying
plant
material.
However,
it
has
not
yet
been
demonstrated
whether
or
not
fungi
growing
on
decaying
Bt
plant
material
also
contain
Bt­
toxins
or
not.
Further,
through
the
addition
of
brewers
yeast
as
food,
the
organisms
probably
consumed
a
mixture
of
the
different
food
types.
Based
on
this
study,
no
evidence
was
found
that
the
transgenic
cotton
lines
and
potato
lines
investigated
had
adverse
effects
on
F.
candida
and
0.
nitens
after
the
2­
month
testing
period.
Since
slightly
lower
(
but
not
statistically
significant)
reproduction
rates
for
0.
nitens
were
observed
in
the
transgenic
treatment
than
in
the
control
after
2
months,
long­
term
effects
under
persistent
exposure
to
Bt
proteins
cannot
be
excluded
and
still
need
to
be
investigated.

EPA
Response:
The
issue
of
actual
ingestion
of
the
test
material
by
test
species
has
been
presented
by
the
EPA
to
the
August
2000
SAP.
As
a
result,
new
testing
protocols
are
being
developed
to
directly
address
this
issue.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
28
Excerpt
Text:
Where
plant
meets
soil
In
agroecosystems,
living,
plants
and
dead
plant
material
enter
the
soil
ecosystem
in
numerous
ways,
as
roots,
tubers,
etc.,
and
plant
residues/
debris
entering
the
soil
ecosystem
constantly
throughout
the
season,
and
with
distinct
cyclic
peaks
upon
harvest
when
great
amounts
of
plant
residues
are
left
behind.
Another
route
that
plant
compounds
can
enter
the
soil
are
root
exudates.

Root
exudation
The
rhizosphere
comprises
the
outer
tissues
of
the
root
(
cortex
and
epidermis),
the
root
surface,
and
the
soil
around
the
root.
It
can
be
thought
of
as
the
microbial
continuum
that
stretches
out
from
the
endoderniils
of
the
root
(
a
layer
of
cells
within
the
root)
into
the
soil.
The
plants
stimulate
microbial
colonization
by
secreting
carbohydrates
and
other
compounds
that
can
be
used
by
the
microorganisms
as
a
source
of
carbon
and
energy.
In
return,
the
plants
benefit
from
some
of
these
associations
through
a
greater
availability
of
mineral
nutrients.
Beneficial,
neutral,
and
harmful
bacteria,
fungi,
algae,
protozoa,
nematodes,
and
microarthropods
live
in
the
rhizosphere
in
an
intricate
food
web
(
see
below).
This
entire
underground
ecosystem
depends
for
its
food
primarily
on
carbohydrates
and
other
molecules
secreted
by
the
roots
into
the
soil,
collectively
called
'
exudates'.
The
health
of
this
underground
ecosystem
can
be
profoundly
affected
by
agricultural
practices,
tipping
the
balance
in
favor
of
beneficial
or
harmful
organisms.
The
interactions
are
enormously
complex,
and
we
know
only
little
about
them
(
Chrispeels
and
Sadava
1994).
Section
3.2
3
­
74
A
number
of
microorganisms
in
the
rhizosphere
have
specific
functions
that
benefit
the
plant,
such
as
the
nitrogen
fixation
in
low
input
agricultural
systems
or
phosphate
uptake
(
see
below).
Others
simply
aid
in
decomposition
of
organic
matter
and,
consequently,
the
release
and
recycling
of
important
nutrients.

EPA
Response:
The
above
comment
is
a
statement
on
the
role
of
microbes
in
the
root
zone
of
plants
and
on
the
symbiotic
relationship
between
the
plant
and
the
soil
biota.
The
effects
on
soil
biota
were
considered
by
the
EPA
during
the
MON
863
corn
risk
assessment
process.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
29
Excerpt
Text:
Every
root
tip
is
covered
by
a
root­
cap,
a
specialized
tissue
that
forms
a
cap­
like
structure
at
the
end
of
the
root
tip.
Cell
divisions
in
this
meristem
assure
that
root
cap
cells
are
continuously
produced.
As
the
cells
mature,
they
are
shed
or
abraded
from
the
cap
surface
between
the
soil
particles
as
the
root
grows.
For
instance,
the
cap
of
corn
roots
produces
up
to
20'
000
cells
during
the
plants
life.
These
root
cap
cells
are
differentiated
cells
whose
main
function
is
to
produce
mucilage
or
slime
that
serves
as
protection
or
a
lubricant
for
the
delicate
young
root
tissue.
Corn
roots,
for
example,
produce
so
much
of
this
slime
that
it
forms
small
droplets
at
the
root
tips.

In
addition
to
these
slimy
secretions,
the
root
itself
secretes
soluble
molecules,
mainly
low
molecular
organic
acids,
amino
acids,
and
sugars.
This
is
an
active
process
and
not
a
passive
leakage.
As
roots
grow,
they
leave
behind
debris
consisting
of
broken­
off
root
hairs,
mucilage,
root­
cap
cells,
and
other
molecules,
all
of
which
provide
food
for
microorganisms
that,
in
turn,
make
certain
nutrients
available
to
plants.

Symbiotic
relations
between
plant
roots
and
microorganisms
Mycorrhiza.
Most
higher
plants
have
fungal
hyphae
closely
associated
with
their
actively
growing
roots.
Such
an
association
is
called
'
mycorrhiza'.
It
is
a
true
symbiotic
relationship.
The
flow
of
photosynthetic
products
from
the
shoot
to
the
roots
benefits
the
fungus,
which
uses
these
products
as
a
source
carbon.
The
plant,
in
turn,
benefits
from
the
capability
of
the
fungus
to
take
up,
for
example,
phosphate
or
other
nutrients
in
otherwise
nutrient­
poor
soils
and
make
them
available
to
the
plant.
Without
mycorrhizae,
these
plants
grow
very
poorly.
One
reason
is
that
fungi
are
able
to
ramify
in
the
soil
with
their
delicate
hyphae
much
farther
than
plant
roots
can.
Therefore,
they
can
draw
nutrients
from
a
much
larger
volume
of
soil.
Another
reason
is
that
mycorrhizae
produce
acids
that
solubilize
inorganic
soil
compounds
making
them
available
to
the
plants.

EPA
Response:
The
above
comment
is
a
statement
on
the
role
of
mycorrhizal
fungi
in
the
root
zone
of
plants
and
on
the
symbiotic
relationship
between
the
plant
and
the
soil
biota.
The
effects
on
soil
biota
were
considered
by
the
EPA
during
the
MON
863
corn
risk
assessment
process.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).
Section
3.2
3
­
75
Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
30
Excerpt
Text:
Nitrogen
fixation.
Besides
photosynthesis,
biological
nitrogen
fixation
is
the
single
most
important
biological
process
that
supports
life
on
Earth
(
Chrispeels
and
Sadava
1994).
Crucial
in
nitrogen
fixation
are
Rhizobium,
bacteria
(
belonging
to
several
species)
that
live
symbiotically
with
leguminous
plants.
They
live
in
nodules
of
plant
roots
where
they
are
able
to
fix,
i.
e.,,
reduce,
molecular
nitrogen
to
ammonia,
which
they
transfer
to
plants.
Rhizobium
spp.
are
free­
living
bacteria
that
attach
themselves
to
the
surface
of
the
root
hairs
of
legume
plants
and
cause
the
root
hairs
to
engulf
them.
The
interaction
between
Rhizobium
and
its
legume
host
is
very
specific.
Usually
one
species
of
Rhizobium
can
infect
only
one
or
a
few
closely
related
plant
species.

Soil
Ecosystems
and
Soil
Food
Webs
As
soil
ecosystems
are
poorly
understood,
it
is
difficult
to
assess
and
interpret
potential
impacts
of
a
steady
input
of
an
insecticidal
protein
like
Bt
from
transgenic
plants
on
populations
and
communities
of
soil
organisms.
Via
direct
and
indirect
exposure
of
soil
micro­
and
macro­
organisms
to
the
Bt
toxins,
vital
processes
like
decomposition
of
organic
matter
and
nutrient
cycling
can
potentially
be
affected
in
the
long
run.
In
undisturbed
ecosystems,
these
processes
are
tightly
coupled
with
plant
growth,
and
in
disturbed
ecosystems,
this
coupling
is
lost
or
reduced
and,
hence,
human
intervention
becomes
increasingly
necessary
for
their
compensation.
Lal
and
Stewart
(
1992)
suggest
that
soil
organism
losses
correlate
with
detrimental
ecosystem
changes.
Therefore,
monitoring
the
development
of
structure
and
function
of
soil
food
webs
and
assessment
of
potential
toxic
impact
should
be
mandatory
for
assessing
ecosystem
health
after
introduction
of
great
amounts
of
bioactive
compounds
like
Bt
toxins.

Soil
food
web
research
is
a
fairly
new
field,
and
we
are
just
at
the
beginning
to
understand
how
it
influences
plant
communities
and
how
in
turn
plant
communities
influence
soil
food
web
structure
(
Hendrix
et
al.
1986).
However,
it
can
be
safely
stated
that
their
importance
for
plant
health
and
ecosystem
functioning
can
hardly
be
overestimated.

EPA
Response:
This
comment
is
a
summary
of
the
beneficial
effects
of
nitrogen
fixing
bacteria
and
the
need
to
ensure
that
introduction
of
Cry
protein
containing
plant
tissue
does
not
disrupt
these
processes.
These
issues
were
considered
by
the
EPA
during
the
risk
assessment
process.
To
date
there
is
no
evidence
that
such
disruption
is
taking
place
by
Cry
proteins,
especially
since
corn
is
not
a
legume.
Nevertheless,
additional
research
on
the
long
term
accumulation
and
effects
of
Bt
proteins
on
soil
processes
are
in
progress.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
31
Excerpt
Text:
Organic
matter
in
soil
is
derived
from
the
remains
of
plants,
animals,
and
microorganisms
that
live
in
or
on
the
soil.
Leaf
litter
accumulates
on
the
soil
surface,
and
as
it
decays,
it
is
gradually
mixed
in
the
top
layer
Section
3.2
3
­
76
of
soil.
Soil
surface
inhabiting
animals
such
as
earthworms,
centipedes,
millipedes,
etc.,
ingest
large
parts
of
plant
material
or
chew
1arge~
and
tough
plant
parts
tQ
smaller
pieces,
thereby
preparing
the
plant
material
for
further
degradation
by
soil
microorganisms.
Some,
e.
g.
earthworms,
also
actively
incorporate
plant
material
from
the
soil
surface
into
the
soil
by
pulling
the
plant
material
downwards
through
small
soil
channels.
Earthworms
are
major
agents
in
the
process
of
soil
creation
through
the
formation
of
clay­
humus
complexes.
The
eaten
portions
of
the
plant
material
are
excreted
by
the
soil
organisms
as
a
black
organic
residue.
This
process
of
transforming
organic
residues
into
humus
is
completed
by
soil
microorganisms
that
secrete
cell­
wall­
degrading
enzymes
that
convert
insoluble
complex
carbohydrates
and
other
compounds
into
soluble
products
that
they
can
take­
up
and
further
transform
(
Chrispeels
and
Sadava
1994).
Primary
and
secondary
minerals
derived
from
weathered
rocks
and
organic
particles
are
intimately
mixed
in
soil,
forming
various
types
of
soil
aggregates,
which
are
found
in
association
with
air
(
i.
e.,
the
soil
atmosphere)
and
soil
water
(
i.
e.,
the
soil
solution)
in
the
pores
that
exist
between
particles
and/
or
aggregates.
Other
soil
arthropods
living
below
the
soil
surface
include,
for
example,
the
larvae
of
several
lepidopteran,
coleopteran,
and
dipteran
species,
collembolans,
and
mites.
Among
these
are
also
a
number
of
pest
species
such
as
Diabrotica
spp.
(
corn
root
worms),
Melolontha
spp.
(
chafer
larvae),
Agrotis
spp.
(
cutworms),
or
Agriotes
spp.
(
wireworms).

Viruses
and
microorganisms,
including
bacteria,
fungi,
algae,
and
protozoa,
are
found
in
large
numbers
in
soil
­
usually
between
one
and
ten
million
microorganisms
are
present
per
gram
of
soil
­
with
bacteria
and
fungi
being
the
most
prevalent
in
agricultural
soils.
Microorganisms
are
important
as
almost
every
chemical
transformation
taking
place
in
soil
involves
active
contributions
from
soil
microorganisms.
In
particular,
they
have
an
active
role
in
soil
fertility,
as
a
result
of
their
involvement
in
the
cycle
of
nutrients,
such
as
carbon
and
nitrogen,
which
are
required
for
plant
growth.
Saprophytic
fungi
and
bacteria
are
the
base
of
the
detrital
food
web
(
Figure
1).
Other
soil
microorganisms
increase
the
availability
of
mineral
nutrients
(
e.
g.
phosphorus)
t6
plants
(
e.
g.
mycorrhizal
fungi,
see
above)
or
can
transform
mtrogen
gas
present
in
the
atmosphere
into
soluble
nitrogenous
compounds
that
plants
can
utilize
for
growth
(
e.
g.
nitrogen­
fixing
bacteria,
see
above).

EPA
Response:
This
comment
is
a
summary
of
the
beneficial
effects
of
soil
biota
on
the
formation
of
fertile
agricultural
soil.
These
issues
were
considered
by
the
EPA
during
the
MON863
corn
risk
assessment
process.
Additional
research
on
the
long
term
accumulation
and
effects
of
Bt
proteins
in
soils
is
in
progress.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
32
Excerpt
Text:
Protozoa
are
important
in
maintaining
plant­
available
N
and
mineralization
processes,
and
as
bacteria
feeders
they
are
important
in
controlling
bacteria
populations
(
Coleman
1985,
Foissner
1986).
Protozoa
can
act
as
indicators
of
the
presence
of
certain
hazardous
wastes
and,
therefore,
may
be
highly
useful
indicators
of
certain
types
of
environmental
impacts
(
Foissner
1986).
Collembolans
feeding
on
saprophytic
fungi
are
therefore
also
important
regulators
of
these
processes
(
Figure
1).

[
see
PDF
for
"
Figure
1.
Schematic
soil
food
web.
Arrows
indicate
the
direction
of
flows
of
nutrients"]
Section
3.2
3
­
77
Nematodes
are
one
of
the
most
ecologically
diverse
groups
of
animals
and
grouped
in
various
trophic
categories,
most
importantly
plant­
feeding
and
predatory
nematodes,
but
there
are
also
bacteria­
feeders
or
those
that
eat
protozoa
and
fungi.

Nematodes
and
protozoa
function
as
main
regulators
of
mineralization
processes
in
soil
(
Coleman
1985).
They
release
a
large
portion
of
N
when
feeding
on
their
various
prey
groups.
On
the
other
hand,
plantfeeding
nematodes
are
among
the
worst
pest
species
in
agriculture
and
with
many
of
them
becoming
increasingly
resistant
against
the
synthetic
nematicides,
they
may
increase
in
meaning
in
the
future.
However,
the
activities
of
these
groups
and
predator­
prey
interactions,
(
which
determine
the
rate
at
which
mineralization
occurs)
are
in
turn
affected,
and
perhaps
controlled
by,
higher
level
predators
such
as
centipedes,
ground
beetles,
spiders,
and
small
mammals.

Known
impacts
of
other
transgenic
organisms
in
soil
ecosystems
EPA
Response:
This
comment
is
a
discussion
of
the
role
played
by
nematodes
in
the
soil
and
the
agroecosystem.
This
issue
was
considered
by
the
EPA
during
the
MON863
corn
risk
assessment
process.
Preliminary
data
were
presented
that
show
possible
beneficial
effects
of
MON
863
by
controlling
some
pathogenic
nematodes.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
b).
Conclusive
data
on
the
effects
of
Cry3Bb
on
nematodes
was
requested
by
EPA.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
F).

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
33
Excerpt
Text:
In
a
field
study
conducted
by
Donegan
et
al.
(
1999),
three
genotypes
of
alfalfa
(
2
transgenic
and
one
parental)
were
planted
in
an
agricultural
field.
Immediately
prior
to
planting,
the
roots
of
these
plants
were
either
left
uninoculated
or
were
inoculated
with
a
wild­
type
strain,
a
recombinant
strain
with
antibiotic
resistance,
or
a
recombinant
strain
with
antibiotic
resistance
and
enhanced
nitrogen­
fixation
capability
(
Sinorhizobium
meliloti).
Effects
on
the
soil
micro­
organisms
were
measured
(
metabolic
and
DNA
fingerprinting,
soil
microbial
respiration;
population
counts,
nematode
and
micro­
arthropod
diversity)
as
well
as
plant
and
shoot
growth,
enzyme
activities
and
soil
chemistry.
The
researchers
found
significant
differences
in
the
soil
bacterial
communities
associated
with
these
alfalfa
genotypes.
Most
distinct
differences
were
observed
for
one
transgenic,
lignin
peroxidase
producing
alfalfa
genotype.
Significant
differences
in
the
microbial
community
composition
were
observed
that
were
associated
with
differences
in
the
respective
microbial
activities,
e.
g.
soil
enzyme
activities
and
pH
levels.
This
resulted
also
in
altered
plant
growth.
To
evaluate
the
effects
regarding
their
overall
effect
on
soil
fertility
and
soil
productivity
requires
further
experimentation.

In
an
earlier
study,
researchers
from
the
same
laboratory
studied
the
decomposition
of
genetically
engineered
proteinase­
inhibitor
producing
tobacco
plants
under
field
conditions
(
Donegan
et
al.
1997).
They
buried
litterbags
containing
transgenic
and
parental
tobacco
plants
m
field
plots
over
a
5
month
period.
Proteinase­
inhibitor
I
protein
was
detectable
and
immunologically
active
for
at
least
57
days.
Further,
decomposing
parental
and
transgenic
plant
material
differed
in
quality
(
carbon
content)
and
in
the
Section
3.2
3
­
78
response
of
exposed
soil
organisms
(
collembola
and
nematodes).
Collembola
populations
in
the
soil
surrounding
the
transgenic
plant
litterbags
were
significantly
lower
than
in
the
non­
transgenic
control.
Nematode
populations
in
the
soil
surrounding
the
litterbags
of
transgenic
plants
were
greater
than
m
those
of
the
non­
transgenic
control
and
had
a
different
trophic
composition
(
higher
ratio
of
ftingal
feeding
vs.
bacterial
feeding
nematodes).
Again,
an
evaluation
of
these
effects
regarding
their
significance
for
soil
health
and
fertility
requires
further
experimentation.

In
a
recent
study
Lukow
et
al.
(
2000)
tested
whether
T­
RFLP
analysis
(
terminal
restriction
fragment
length
polymorphism)
represents
an
appropriate
technique
for
monitoring
highly
diverse
soil
bacterial
communities,
i.
e.
spatial
and
temporal
variation
in
bacterial
commumty
structure.
To
do
this,
the
authors
used
two
transgenic
potato
and
one
commercial
non­
transgenic
potato
variety
('
Bintje').
It
was
unclear
whether
or
not
the
commercial
potato
variety
'
Bintje'
was
the
isogenic
parent
or
control
variety
for
the
two
transgenic
potato
lines
used.
The
study
revealed
spatial
and
temporal
effects,
as
well
as
space
x
time
interaction
effects
on
the
structural
composition
of
the
bacterial
communities
where
both
transgenic
varieties
differed
significantly
from
the
non­
transgenic
commercial
variety.
The
authors
concluded
that
the
T­
RFLP
technique
proved
to
be
a
suitable
method
for
monitoring
highly
diverse
soil
microbial
communities
for
changes
over
space
and/
or
time.

EPA
Response:
This
comment
is
a
discussion
of
soil
biota
population
differences
in
soils
growing
transgenic
and
nontransgenic
alfalfa,
potato
and
tobacco
plants.
There
were
reported
differences
in
numbers
and
types
of
soil
microbes
and
invertebrates.
The
significance
of
these
differences
is
unclear
in
light
of
the
lack
of
baseline
"
normal"
soil
population
definitions,
the
great
natural
variability
in
soil
biota
populations,
and
the
long
established
fact
that
that
different
crops
(
i.
e
different
organic
nutrients)
have
differing
soil
biota
associations.
The
USDA
and
EPA
have
allocated
research
funds
to
determine
the
significance
of
such
shifts
in
soil
biota
populations.

Commenter
Name:
Angelika
Hilbeck
Commenter
Organization
Name:
Greenpeace
Comment
Number:
30509­
113000Part2
Excerpt
Number:
34
Excerpt
Text:
In
a
microcosm
study,
lnghain
et
al.
(
1999)
wanted
to
assess
potential
effects
of
a
transgenic
ethanolproducing
micro­
organism
on
soil
biota
and
plant
growth.
In
their
experiments,
the
authors
added
a
genetically
engineered
bacteria
(
Klebsiella
planticola
(
SDF2O))
and
the
untransformed
parental
line
(
SDF
15)
to
a
sandy
soil.
In
one
half
of
the
treatments
wheat
plants
were
grown
in
the
soil
and
in
the
other
no
plants
were
grown.
When
SDF2O
was
added
to
the
soil
with
plants,
the
numbers
of
bacteria
and
fungal
feeding
nematodes
increased
significantly
which
coincided
with
the
death
of
the
plants.
In
contrast,
when
SDF15
was
added
to
the
soils
with
plants,
only
the
number
of
bacterial
feeding
nematodes
increased
but
the
plants
did
not
die.
When
no
plants
were
grown
in
the
soil,
the
nematode
community
remained
unchanged.
The
authors
suspected
that
substrates
from
the
plant
roots
may
have
been
essential
for
the
observed
effects..
They
also
stressed
that
their
results
demonstrate
the
importance
of
using
experimental
test
systems
that
incorporate
biological
interactions
and
include
direct
measurements
of
soil
biota
to
assess
the
effects
of
genetically
engineered
micro­
organisms
released
into
soils.
Section
3.2
3
­
79
The
few
studies
conducted
to
date,
revealed
that
the
observed
impacts
differed
from
case
to
case,
were
unexpected,
complex
and
largely
difficult
to
understand
and
evaluate
with
our
little
knowledge
of
the
soil
ecosystem.
Or
in
other
words,
the
studies
support
a
case­
by­
case
safety
assessment
approach
and,
equally
important,
highlight
again
the
urgent
need
for
developing
meaningful
and
proven
scientific­
technical
biosafety
testing
guidelines
involving
whole
organisms,
multiple
species
and
trophic
interactions.
The
studies
also
allow
for
an
optimistic
view
in
that
promising
methodologies
seem
to
emerge
so
that
with
adequate
financial
and
scientific
committment,
the
development
of
such
new
biosafety
testing
guidelines
should
be
a
feasible
task
in
the
future.

Meanwhile
also
some
reports
regarding
environmental
impact
from
the
commercial
production
of
transgenic
plants
have
appeared.
Increased
and
frequent
use
of
glyphosate
associated
with
transgenic
glyphosate­
resistant
crop
production
leads
aside
from
an
observed
weed
shift
to
more
glyphosate­
tolerant
species
(
Progressive
Farmer
June
12,
2000
or
http://
www.
biotechjpjo
net/
weed_
shift.
html),
apparently
also
to
an
increased
frequency
of
Fusartuin
on
soybean
roots
(
up
to
5­
fold)
4
weeks
after
application
of
glyphosate
herbicides
(
Kremer
et
al.
2001).
Yield
levels
were
so
far
unaffected.
However,
a
built­
up
of
the
Fusarium
level
in
the
soil
should
be
monitored.
Fusarium
species
are
responsible
for
a
disease
in
soybean
called
'
sudden
death
syndrome'
to
which
many
transgenic
soybean
varieties
are
susceptible.

EPA
Response:
This
comment
is
a
discussion
of
changes
in
soil
biota
and
plant
health
in
the
presence
of
ethanol
producing
microbes;
a
need
for
development
of
study
guidelines
which
would
include
studies
to
address
the
observed
effects;
and
glyphosate­
resistant
plant
development
with
an
observed
increase
in
Fusarium
fungus.
The
USDA
and
EPA
have
allocated
research
funds
to
determine
the
significance
of
such
effects
on
soil
biota
populations
and
plants.
EPA
also
has
commenced
development
of
testing
guidelines
for
protein
plantincorporated
protectants.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
7
Excerpt
Text:
2.
No
Data
Are
Presented
on
Root
Exudates
Nowhere
in
the
submission
does
Monsanto
present
information
on
the
levels
of
Cry
3Bb
endotoxin
exuded
through
root
systems
and
into
the
soil.
The
lack
of
such
information
is
curious,
given
that
root
exudates
would
presumably
enhance
efficacy
and
surely
have
been
measured
by
Monsanto.

EPA
should
insist
that
data
on
root
exudates
is
submitted,
along
with
properly
conducted
soil
assays
to
determine
the
persistence
of
Cry
3
Bb
proteins
in
the
soil.
Information
on
the
total
flow
of
Cry
3Bb
and
NPTII
proteins
into
the
rhizosphere
via
roots
and
root
exudates,
and
the
environmental
fate
of
these
proteins,
are
essential
to
determine
the
possible
impact
on
soil
microbial
communities.

EPA
Response:
EPA
requires
data
on
soil
degradation
of
Cry
proteins
performed
at
high
Cry
protein
doses
that
would
Section
3.2
3
­
80
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
FIFRA
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
The
data
reviewed
by
the
Agency
show
that
the
Cry
protein
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels
regardless
of
the
Cry
protein
source
source.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
soil
biota.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).
These
aspects
will,
however,
be
investigated
over
several
years
to
provide
confirmatory
long
term
effects
information.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
11
Excerpt
Text:
5.
High
Toxin
Levels
May
Jeopardize
Margins
of
Safety
for
Soil
Organisms
Monsanto
reports
that
the
maximum
environmental
concentration
expected
in
the
soil
is
13.3
mg/
kg,
based
on
the
assumption
that
corn
plants
are
tilled
into
the
top
six
inches
of
soil
when
leaf
tissue
expression
levels
are
peaking.
Yet
exposure
levels
are
likely
to
be
much
higher
for
organisms
that
move
through
the
soil
and
seek
out
the
nutrient
enriched
zone
within
a
few
centimeters
of
roots.
Moreover,
plant
residues
and
stalks
worked
into
the
soil
will
create
an
uneven
distribution
of
Cry
3Bb
in
the
soil
profile;
organisms
that
happen
to
be
feeding
in
the
vicinity
of
decomposing
plant
tissues
will
be
exposed
to
relatively
higher
levels.

Cry
3Bb
endotoxins
are
lethal
to
earthworms
at
57
mg/
kg
in
the
soil,
resulting
in
just
a
4­
fold
difference
between
the
known
high­
level
concentrations
in
soil.
Such
a
narrow
margin
of
safety
is
not
acceptable
in
any
other
regulatory
context.
While
under
"
normal"
conditions,
a
4­
fold
margin
of
safety
may
spare
most
earthworms
in
fields
planted
to
corn
varieties
expressing
the
Cry
3Bb
endotoxin,
many
factors
can
enhance
the
vulnerability
of
earthworms
­
or
other
nontarget
organisms
­
to
the
effects
of
any
toxin.

Earthworms
moving
through
"
hot
spots"
could
be
exposed
to
lethal
levels.
Exposure
to
other
insecticides
or
herbicides
or
drought
stress
also
can
make
earthworms
more
vulnerable
than
normal.
Tillage
systems,
irrigation,
or
soil
conditions
might
alter
their
movement
and
feeding
habits,
increasing
or
decreasing
their
exposures
levels.

EPA
Response:
The
available
data
do
not
support
these
speculative
comments.
There
are
reports
in
the
open
literature
that
earthworms
tested
for
ingestion
of
Cry
protein
do
ingest
Cry
protein,
and
that
Cry
protein
is
also
detected
in
earthworm
casts.
The
earthworms
are
not
affected
by
the
Cry
proteins
going
through
their
system.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
3.
vii).
Monsanto
testing
was
done
with
purified
Cry
protein
in
order
to
expose
the
earthworms
to
large
doses
exceeding
the
possible
soil
concentration.
The
study
referred
to
by
the
above
commenter
(
MRID
No.
449043­
16)
shows
that
the
570mg/
kg
soil
was
the
highest
concentration
tested,
so
that
the
LC50
is
above
that
concentration,
not
at
that
concentration
as
stated
by
the
commenter.
This
study
has
a
10
x
safety
factor.
The
data
show
that
no
adverse
effects
to
earthworms
are
expected
from
exposure
to
Cry3Bb1
protein
producing
corn
plants.
The
EPA
hazard
evaluation
procedure
considers
an
LD50
(
not
a
NOEC)
at
5x
the
environmental
concentration
(
EEC)
as
Section
3.2
3
­
81
indicative
of
no
hazard
to
terrestrial
organism
populations,
while
in
the
submitted
studies
there
was
no
LD50
or
mortality
seen
at
10
x,
the
highest
concentration
tested.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
30
Excerpt
Text:
2.
Impacts
on
Soil
Health
and
Productivity
Must
Be
Assessed
Healthy
soil
is
a
prerequisite
for
profitable
farming
on
a
sustained
basis.
Soil
quality
is
generally
defined
as
the
capacity
of
a
soil
to
take
in,
store
and
purify
water,
to
hold
and
recycle
nutrients,
to
support
a
diverse
and
robust
biotic
community,
and
to
suppress
pathogens
and
other
pests.

Soil
quality
can
and
has
been
degraded
through
erosion,
excessive
tillage,
compaction,
use
of
broadspectrum
insecticides
and
soil
fumigants,
depletion
of
nutrients,
and
the
build
up
of
salt
and
other
minerals.
Research
over
50
years
has
documented
the
significant
and
largely
irreversible
impacts
of
soil
loss
and
degradation
on
average
attainable
corn
yields
(
NRC,
1993).
Plus,
recent
work
by
soil
ecologists
is
beginning
to
explain
the
many
ways
that
soil
microbial
communities
can
impact
plant
growth
and
development,
and
perhaps
even
more
important,
contribute
to
the
microbial
biocontrol
of
soil
borne
pathogens
and
microarthropods.

Kerry
has
highlighted
the
impact
of
plant
root
exudates
on
nematode
populations
"
by
influencing
both
the
dynamics
of
the
nematode
host
and
structure
and
dynamics
of
the
community
of
antagonists
and
parasites
in
the
rhizosphere"
(
Kerry,
2000).
He
stresses
that
little
is
known
about
how
root
exudates
impact
soil
microbial
communities,
as
well
as
microbial
biocontrol
processes.
Introduction
of
Cry
3Bb
corn
hybrids
will
add
another
layer
of
complexity
for
soil
ecologists.

A
key
1998
study
in
Nature
by
van
der
Heijden
and
colleagues
showed
that
the
diversity
of
mycorrhizal
fungi
plays
a
key
role
in
determining
the
productivity
of
soil
ecosystems
(
van
der
Heijden
et
al.,
1999).
An
overview
by
Read
in
the
same
issue
speculates
that
greater
fungal
biodiversity
expands
the
range
of
mechanisms
through
which
microbial
interactions
can
help
plants
deal
with
various
sources
of
stress
and
competition
(
Read,
1999).

EPA
Response:
The
Bt
crops
are
a
very
likely
beneficial
solution
to
the
soil
quality
problems
caused
by
long
term
use
of
chemical
additives
in
agriculture
as
quoted
by
this
commenter.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
31
Excerpt
Text:
Persistence
of
Cry3Bb
Toxins
in
the
Soil
Must
Be
Measured
Section
3.2
3
­
82
A
disadvantage
of
foliar
Bt
products
has
always
been
a
constraint
on
their
commercial
development
­
foliar
Bt
sprays
break
down
quickly
when
exposed
to
sunlight,
and
become
inactive
within
48
hours
to
a
few
days.
Engineering
Bt
into
plant
tissues
helps
solve
this
problem.
Varieties
like
Cry
3Bb
corn
that
express
Bt
in
roots,
and
presumably
as
well
through
root
exudates,
will
assure
even
slower
breakdown
of
Bt
proteins.

Important
work
by
Stotzky
and
colleagues
reported
in
Nature
shows
that
Bt
proteins
are
exuded
from
the
roots
of
Bt
corn
and
can
bind
with
clay
soil
particles
or
humic
acid
and
remain
active
for
over
120
days
in
the
soil
(
Saxena
et
al.,
1999).
This
work
needs
to
be
repeated
with
Cry3Bb
corn
varieties
to
document
the
extent
to
which
they
exude
Cry3Bb
protein
toxin
and
whether
it
too
binds
to
soil
particles.

Stotzky's
team
has
shown
that
Bt
proteins
can
bind
to
clay
particles
and
humic
acids
and
become
very
stable
in
the
soil
(
Saxena
et
al.,
1999).
In
some
farming
systems
and
on
certain
soils,
bound
Bt
may
move
off
fields
with
eroding
soil
and
enter
streams,
ponds
and
lakes,
and
aquatic
ecosystems.
Understanding
the
environmental
fate
and
movement
of
such
bound
Cry
3Bb
endotoxins
will
require
careful
field
research.

If
approved,
Monsanto's
Cry3Bb
corn
could
be
adopted
widely
within
a
few
years
on
millions
of
acres
(
Gray,
2000).
It
will
deliver
into
the
root
zone
concentrations
of
Cry3Bb
toxins
that
are
orders
of
magnitude
above
what
occurs
naturally,
possibly
triggering
serious
effects
on
soils,
soil
food
webs,
and
soil­
borne­
pest
dynamics.

EPA
Response:
EPA
receives
data
on
soil
degradation
of
Cry
proteins
performed
at
high
Cry
protein
doses
that
would
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
FIFRA
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
The
authors
of
the
studies
quoted
above
have
also
shown
that
Cry
proteins
adsorbed
to
soil
minerals
are
quickly
metabolized
by
soil
microbes
upon
elution.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4)
The
data
reviewed
by
the
Agency
show
that
the
Cry
protein
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
level.
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
the
soil
biota.
These
aspects
will,
however,
be
investigated
over
the
life
of
the
limited
MON863
corn
registration
to
provide
confirmatory
long
term
soil
biota
effects
information.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
36
Excerpt
Text:
When
the
first
Bt­
transgenic
plant
applications
were
reviewed,
there
was
little
evidence
that
antibiotic
marker
genes
might
trigger
resistance
problems.
Now
the
transfer
of
antibiotic
marker
genes
in
plant
cells
to
soil
bacteria
has
been
demonstrated
(
Gerbhard
and
Smalla,
1998).
In
a
key
article
in
the
journal
Applied
and
Environmental
Microbiology,
Gebhard
and
Smalla
hypothesize
that:

" 
the
introduction
of
bacterial
genes
into
the
plant
genome
leads
to
a
higher
probability
of
gene
transfer
from
plants
to
bacteria
due
to
the
presence
of
homologous
sequences.
However,
until
now,
there
has
been
a
Section
3.2
3
­
83
lack
of
clear
experimental
evidence
that
successful
gene
transfer
from
plants
to
bacteria
can
occur
at
all."
(
Gebhard
and
Smalla,
1998).

EPA
Response:
The
above
quoted
transfer
of
genes
appears
to
be
a
laboratory
phenomenon
under
conditions
deliberately
created
to
favor
such
gene
transfer.
There
is
no
evidence
of
this
occurring
in
agricultural
fields.
This
aspect
is,
however,
among
the
research
items
to
be
investigated
by
USDA
and
EPA
funded
research
projects.

Commenter
Name:
Charles
Benbrook
Commenter
Organization
Name:
Union
of
Concerned
Scientists
Comment
Number:
30509­
115000
Excerpt
Number:
38
Excerpt
Text:
Also
contrary
to
conventional
wisdom,
genes
that
confer
antibiotic
resistance
have
been
shown
to
move
readily
between
people
and
from
bacteria
in
the
gut
of
farm
animals
to
people
(
Tschape,
1994).
A
team
in
Denmark
has
shown
that
indigenous
soil
bacteria
can
serve
as
a
sink
for
plasmid­
borne
antibiotic
resistance
traits
from
E.
coli
entering
agricultural
soils
from
animal
manure
or
other
wastes
(
Sorensen
et
al.,
1999).

Until
recently
it
was
also
thought
that
foreign
DNA,
such
as
the
gene
constructs
in
Cry
3Bb
corn,
would
pass
through
the
mammalian
digestive
without
being
activated
and
without
consequence.
The
transfer
of
foreign
DNA
in
food
into
the
blood
and
organ
systems
in
mice
has
now
been
demonstrated
(
Schubbert
et
al.,
1997).
In
addition
some
of
the
transgenic
DNA
was
found
to
covalently
bind
to
mouse
DNA,
in
effect
becoming
a
part
of
the
mouse
genome.
The
authors
modestly
summed
up
this
article
by
stating
­­
"
The
medical
and
evolutionary
implications
of
these
observations
may
be
considerable"
(
Schubbert
et
al.,
1997).

EPA
Response:
The
above
quoted
research
is
confined
to
bacteria­
host
interactions.
The
reported
incorporation
of
antibiotic
resistance
genes
into
mammalian
DNA
has
not
been
verified.
This
issue
is
among
the
research
items
to
be
clarified
by
USDA
and
EPA­
funded
research
projects.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
5
Excerpt
Text:
Monsanto
reports
that
the
maximum
environmental
concentration
expected
in
the
soil
is
13.3
mg/
kg,
based
on
the
assumption
that
corn
plants
are
tilled
into
the
top
six
inches
of
soil
when
leaf
tissue
expression
levels
are
peaking.
Yet
exposure
levels
are
likely
to
be
much
higher
for
organisms
that
move
through
the
soil
and
seek
out
the
nutrient
enriched
zone
within
a
few
centimeters
of
roots.

EPA
Response:
The
available
data
do
not
support
these
comments.
The
calculations
for
soil
concentration
in
the
top
6"
were
done
by
standard,
published
EPA
procedures.
Monsanto
testing
was
done
with
purified
Cry
protein
in
order
to
expose
the
earthworms
to
large
doses
far
exceeding
the
possible
soil
concentration.
The
study
has
a
10
x
safety
factor.
Also,
no
adverse
or
reproductive
effects
were
noted
in
springtail
studies
Section
3.2
3
­
84
conducted
with
50%
leaf
tissue.
The
EPA
hazard
evaluation
procedure
considers
an
LD50
(
not
a
NOEC)
at
5x
the
environmental
concentration
(
EEC)
as
indicative
of
no
hazard
to
terrestrial
organism
populations,
while
in
the
submitted
earthworm
studies
there
was
no
LD50
or
mortality
seen
at
10x,
the
highest
concentration
tested.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
10
Excerpt
Text:
Research
over
50
years
has
documented
the
significant
and
largely
irreversible
impacts
of
soil
loss
and
degradation
on
average
attainable
corn
yields
(
NRC,
1993).
Plus,
recent
work
by
soil
ecologists
is
beginning
to
explain
the
many
ways
that
soil
microbial
communities
can
impact
plant
growth
and
development,
and
perhaps
even
more
important,
contribute
to
the
microbial
biocontrol
of
soil
borne
pathogens
and
microarthropods.
Kerry
has
highlighted
the
impact
of
plant
root
exudates
an
nematode
populations
"
by
influencing
both
the
dynamics
of
the
nematode
host
and
structure
and
dynamics
of
the
community
of
antagonists
and
parasites
in
the
rhizosphere"
(
Kerry,
2000).
He
stresses
that
little
is
known
about
how
root
exudates
impact
soil
microbial
communities,
as
well
as
microbial
biocontrol
processes.

EPA
Response:
The
Bt
crops
are
a
very
likely
beneficial
solution
to
the
soil
quality
problems
caused
by
long
term
use
of
chemical
additives
in
agricultural
soils
quoted
by
this
commenter.

Commenter
Name:
Scott
Black
Commenter
Organization
Name:
The
Xerces
Society
Comment
Number:
30509­
L03
Excerpt
Number:
11
Excerpt
Text:
Persistence
of
Cry3Bb
Toxins
in
the
Soil
Must
Be
Measured
The
EPA
needs
to
study
Cry3Bb
corn
varieties
to
document
the
extent
to
which
they
exude
Cry3Bb
protein
toxin
and
whether
it
binds
to
soil
particles.
Work
by
Stotzky
and
colleagues
reported
in
Nature
shows
that
Bt
proteins
are
exuded
from
the
roots
of
Bt
corn
and
can
bind
with
clay
soil
particles
or
humic
acid
and
remain
active
for
over
120
days
in
the
soil
(
Saxena
et
al.,
1999).
In
some
fanning
systems
and
on
certain
soils,
bound
Bt
may
move
off
fields
with
eroding
soil
and
enter
streams,
ponds
and
lakes,
and
aquatic
ecosystems.
Understanding
the
environmental
fate
and
movement
of
such
bound
Cry
3Bb
endotoxins
will
require
careful
field
research.

EPA
Response:
EPA
requires
data
on
soil
degradation
of
Cry
proteins
performed
at
high
Cry
protein
doses
that
would
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
FIFRA
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
The
authors
of
the
studies
quoted
above
have
also
shown
that
Cry
proteins
adsorbed
to
soil
minerals
are
quickly
metabolized
by
soil
Section
3.2
3
­
85
microbes
upon
elution.
The
data
reviewed
by
the
Agency
show
that
the
Cry
protein
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels
regardless
of
the
Cry
protein
source
source.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
soil
biota.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).
These
aspects
will,
however,
be
investigated
over
several
years
to
provide
confirmatory
long
term
effects
information.

Commenter
Name:
Lincoln
Brower
Commenter
Organization
Name:
Sweet
Briar
College
Comment
Number:
30509B­
084000
Excerpt
Number:
2
Excerpt
Text:
In
many
respects,
the
potential
for
biodiversity
damage
that
may
ensue
from
a
corn
varietal
that
secretes
Bt
toxins
in
its
roots­
seems
even
greater
to
me
.......
affecting
the
biological
integrity
of
the
soil
ecosystem
in
some
of
the
richest
and
most
biodiverse
soils
on
this
planet.
Gene
stacking
seems
particularly
troublesome
and
fraught
with
long
term
toxicological
uncertainty.

EPA
Response:
EPA
requires
data
on
soil
degradation
of
Cry
proteins
performed
at
high
Cry
protein
doses
that
would
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
FIFRA
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
Several
studies
have
also
shown
that
Cry
proteins
adsorbed
to
soil
minerals
are
quickly
metabolized
by
soil
microbes
upon
elution.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
The
data
reviewed
by
the
Agency
show
that
the
Cry
protein
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels
regardless
of
the
Cry
protein
source
source.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
soil
biota.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).
These
aspects
will,
however,
be
investigated
over
several
years
to
provide
confirmatory
long
term
effects
information.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
25
Excerpt
Text:
1.
ENVIRONMENTAL
FATE
AND
ECOSYSTEM
IMPACTS
Comment
or
Question:
There
is
a
lack
of
empirical
data
showing
the
concentrations
of
Cry3Bbl
within
the
soil
profile
due
to
root
exudation
and
plant
decomposition,
and
a
lack
of
data
on
soil
fate,
persistence
and
the
potential
for
off­
field
movement
to
streams,
ponds,
lakes
and
aquatic
ecosystems.

Scientific
Background:
A
recent
EPA
Scientific
Advisory
Panel
(
SAP)
report
[
Footnote
2:
SAP
Report
#
2000­
07b,
March
12,
2001]
discussed
the
issue
of
root
exudation
from
Bt
corn
and
provided
the­
following
(
verbatim)
comments:
"
The
panel
agreed
that
for
the
purpose
of
risk
assessment,
the
principal
issues
are
the
amount
of
Cry
protein
that
enters
the
soil
over
time
and
the
length
of
time
that
the
protein
persists
in
a
biologically­
active
form.
The
mechanism
by
which
Cry
proteins
enter
the
soil,
for
example
by
secretion,
shedding
of
root
hairs,
degradation
of
biomass,
pollen,
etc.,
were
to
be
considered
of
secondary
importance,
Section
3.2
3
­
86
and
although
relevant,
knowledge
of
these
might
not
be
necessary
for
completing
a
risk
assessment."

Monsanto
has
submitted
a
soil
degradation
study
for
Cry3Bbl
protein
in
support
of
corn
event
MON
863
registration.
[
Footnote
3:
Monsanto
report
MSL­
16440;
MRID
451568­
04]
The
design
of
this
study
employed.
exaggerated
doses
to
simulate
worst­
case
soil
deposition
from
the
variety
of
potential
mechanisms
identified
by
the
SAP.
Cry3Bb1
protein
was
added
to
the
soil
as
plant
material
at
levels
exceeding
the
upper
estimates
of
the
amount
of
plant
tissue
and
Cry3Bb
I
protein
that
might
possibly
enter
the
soil
through
tillage
of
Bt
crops
or
through
decay
of
corn
roots.
In
fact,
the
plant
tissue
used
contained
the
highest
level
of
Cry3Bbl
protein
found
in
any
tissue
at
the
peak
time
of
Cry
protein
production.
The
study
included
two
exaggerated
dose
levels
of
Cry3Bbl
protein
in
soil
(
52.4
ug/
g
soil
and
174.5
ug/
g),
representing
3%
and
10%
tissue
by
weight
of
soil,
respectively.
These
doses
greatly
exceed
the
amount
of
protein
that
can
reach
soil
(
at
any
one
time)
via
any
conceivable
mechanism,
including
exudation.
Since
the
average
level
of
Cry3Bb1
in
root
tissue
is
41
ug/
g
tissue,
[
Footnote
4:
Monsanto
report
MSL­
17181;
MRID
454240­
01]
a
total
of
4.26
gram
of
fresh
corn
root
tissue
would
have
to
be
mixed
with
(
or
decay
into)
each
gram
of
soil
in
the
root
zone,
an
obvious
impossibility
in
an
agricultural
field.
Thus,
the
dose
of
CrY3]
3b1
protein
used
in
this
study
exceeds
all
possible
estimates
of
Cry3Bb1
protein
in.
the
root
zone,
even
if
exudation
were
to
provide
an
additional
source
of
Cry3Bb1
protein
to
the
soil.

EPA
Response:
At
the
present
time
EPA
is
following
the
EPA
Scientific
Advisory
Panel
(
SAP
Report
#
2000­
07b,
March
12,
2001)
approach
evaluating
the
persistence
of
Cry
proteins
in
soil.
The
amounts
of
Cry
protein
in
soil,
however,
will
be
evaluated
over
the
life
of
the
limited
MON863
registration
to
provide
confirmatory
long
term
soil
biota
effects
information.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
086000
Excerpt
Number:
26
Excerpt
Text:
The
results
of
this
study
showed
that
Cry3Bb1
protein
degrades
rapidly
in
soil.
The
calculated
DT50
(
time
to
50%
degradation)
was
2.4
to
2.8
days,
and
the
calculated
DT90
(
time
to
90%
degradation)
was
7.9
to
9.2
days.
There
was
no
detection
of
Cry3Bbl,
by
either
ELISA
or
insect
bioassay,
in
any
soil
sample
incubated
for
over
21
days.

Comment
or
Question:
The
concentration
of
MON
863
in
the
area
immediately
adjacent
to
the
root
or
other
plant
material
in
soil
will
be
higher
than
the
worst
case
prediction
of
13.3
mg
Cry3Bb1
protein/
kg
soil.

Scientific
Background:
The
Cry3Bb1
protein,
as
engineered
into
MON
863,
contains
no
secretory
peptide
sequences
and
has
no
known
chemical
mechanisms
for
exudation.
As
such,
any
protein
in
the
root
zone
is
expected
to
be
present
by
adventitious
mechanisms
such
as
damage
to
roots.
In
addition,
soil
degradation
experiments
demonstrate
that
Cry3Bb1
has
a
short
half­
life
(
DT50,
2.4­
2.8
days)
in
soil.
Monsanto
calculated
a
conservative
exposure
level
of
13.3
mg
Cry3Bb1
protein/
kg
soil
based
on
a
worst­
case
assumption
that
the
entire
aboveground
mass
of
a
corn
plant
could
be
incorporated
into
the
top
six
inches
of
soil
at
the
time
of
maximum
Cry3Bb1
expression.
Given
that
Cry3Bbl
protein
is
not
likely
to
be
secreted
into
the
root
zone
and
that
this
protein
has
a
short
half­
life,
there
is
no
reason
to
believe
that
Section
3.2
3
­
87
exposure
will
approach,
far
less
exceed,
the
conservative
estimate
of
13.3
Cry3Bb1
mg
protein/
kg
soil.
Furthermore,
NOEC
levels
for
specific
soil
organisms
had
significant
safety
margins
beyond
this
conservative
exposure.

EPA
Response:
The
current
EPA
view
is
that
the
soil
persistence
and
degradation
data,
together
with
the
soil
invertebrate
testing
at
artificially
high
doses
of
Cry
protein,
show
that
no
hazard
to
soil
biota
is
expected.
The
accumulation
of
residual
amounts
of
Cry
protein
in
agricultural
field
soil,
however,
will
be
evaluated
over
the
life
of
the
limited
MON863
corn
registration
to
provide
confirmatory
long
term
soil
biota
effects
information.

Commenter
Name:
Dennis
Ward
Commenter
Organization
Name:
Monsanto
Comment
Number:
30509B­
106000
Excerpt
Number:
6
Excerpt
Text:
Comment
or
Question:
Studies
have
not
been
conducted
to
determine
the
persistence
of
Cry3Bb1
protein
in
soil
under
field
conditions.

Scientific
Background:
Monsanto
has
submitted
a
soil
degradation
study
for
Cry3Bb1
protein
using
fieldcollected
soil
that
indicates
the
protein
will
dissipate
rapidly
under
field
conditions.
{
Footnote
8:
Monsanto
report
MSL­
16440;
MRID
451568­
04]
The
design
of
this
study
employed
exaggerated
doses
to
simulate
worst­
case
soil
deposition
from
the
variety
of
potential
mechanisms,
including
potential
secretion,
shedding
of
root
hairs,
degradation
of
biomass
and
pollen
deposition.
The
study
showed
that
Cry3Bb1
protein
degrades
rapidly
in
soil.
The
calculated
DT50
(
time
to
50%
degradation)
was
2.4
to
2.8
days,
and
the
calculated
DT90
(
time
to
90%
degradation)
was
7.9
to
9.2
days.
There
was
no
detection
of
Cry3Bbl,
by
either
ELISA
or
insect
bioassay,
in
any
sample
incubated
for
over
21
days.

In
the
soil
fate
study,
Cry3BbI
protein
was
added
to
the
soil
as
plant
material
at
levels
exceeding
the
upper
estimates
of
the
amount
of
plant
tissue
and
Cry3BbI
protein
that
might
possibly
enter
the
soil
through
tillage
of
Bt
crops
or
through
decay
of
corn
roots.
In
fact,
the
plant
tissue
used
contained
the
highest
level
of
Cry3Bb1
protein
found
in
any
tissue
at
the
peak
time
of
crystal
protein
production.
The
study
included
two
exaggerated
dose
levels
of
Cry3BbI
protein
in
soil
(
52.4
pg/
g
and
174.5
pg/
c,
soil),
representing
3%
and
10%
tissue
by
weight
of
soil,
respectively.
These
doses
greatly
exceed
the
amount
of
protein
that
can
reach
soil
at
any
one
time
via
any
conceivable
mechanism,
including
exudation.
Since
the­
average
level
of
Cry3Bb
I
in
root
tissue
is
41
ug/
g
tissue,
[
Footnote
9:
Monsanto
report
MSL­
17181;
MRID
454240­
01]
a
total
of
4.26
gram
of
fresh
corn
root
tissue
would
have
to
be
mixed
with
(
or
decay
into)
each
gram
of
soil
in
the
root
zone,
an
obvious
impossibility
in
an
agricultural
field.
Thus,
the
dose
of
Cry3Bb1
protein
used
in
this
study
exceeds
all
possible
estimates,
of
Cry3Bb1
protein
in
the
root
zone
and
provides
a
conservative
estimate
of
Cry3Bb1
degradation
in
soil.

EPA
Response:
The
accumulation
of
residual
amounts
of
Cry
protein
in
agricultural
field
soil
will
be
evaluated
over
the
life
of
the
limited
MON863
corn
registration
to
provide
confirmatory
long
term
soil
biota
effects
information.
Section
3.2
3
­
88
Commenter
Name:
Lance
Meinke
Commenter
Organization
Name:
NCR­
46
Memebers
Comment
Number:
OPP02­
0016
Excerpt
Number:
15
Excerpt
Text:
­
Unlike
ECB,
corn
rootworm
populations
can
be
scouted
the
previous
year
to
determine
whether
or
not
management
efforts,
including
transgenics,
are
needed
in
a
specific
field.
Scouting
techniques
and
thresholds
are
available
for
continuous
corn,
extended
diapause
of
northern
corn
rootworms,
and
western
corn
rootworm
oviposition
in
soybean.
These
methodologies
would
enable
the
use
of
transgenic
varieties
in
a
prescriptive,
pest
management
approach.
­
The
majority
of
corn
acreage
"
at­
risk"
from
corn
rootworm,
is
treated
with
insecticides
in
contrast
to
prevailing
grower
tolerance
of
ECB
infestations
throughout
much
of
the
Corn
Belt.
Farmers
will
expect
the
option
of
protecting
corn
roots
from
corn
rootworms
if
refuge
areas
are
a
required
part
of
an
IRM
plan.
Currently,
either
the
larval
or
adult
stage
is
targeted
when
insecticides
are
applied
to
control
corn
rootworms
and
manage
root
injury.
Soil
insecticides
or
seed
treatments
targeted
at
larvae
are
typically
used
at
planting
to
reduce
larval
injury
and
the
probability
that
plant
lodging
will
occur.
However,
because
placement
of
these
products
is
restricted
to
the
seed
furrow
or
a
narrow
band
over
the
row,
roots
growing
outside
of
the
insecticide
treated
zone
will
often
produce
many
adults
(
goal:
protection
of
nodal
roots
near
the
plant
stem,
not
rootworm
population
management).
Unlike
the
larval
control
strategy,
the
goal
of
adult
control
programs
is
to
greatly
suppress
beetle
densities
(
population
management)
with
broadcast
insecticide
treatments
to
corn
foliage
and
reduce
oviposition
so
that
larval
densities
the
following
year
will
not
cause
economic
loss.
Use
of
the
adult
management
strategy
would
significantly
reduce
beetle
production
from
a
refuge.

EPA
Response:
The
above
commenter
discusses
the
current
control
strategies
for
corn
rootworm
larvae
and
adults.
No
request
for
a
response
from
EPA
is
apparent.
Insect
resistance
management
issues
are
addressed
in
a
separate
section
Commenter
Name:
Clifford
Habig
Commenter
Organization
Name:
Exponent,
Inc.
Comment
Number:
OPP02­
0017
Excerpt
Number:
6
Excerpt
Text:
Environmental
fate
and
expression
can
be
incorporated
into
a
field
program,
including
sampling
of
soil
and
plants,
and
specific
plant
parts
at
various
time
points
during
the
growing
season,
and
can
therefore
compliment
laboratory
studies
on
the
degradation
of
PIP
products.
However,
incorporating
environmental
fate
and
expression
analyses
into
a
field
study
program
(
or
laboratory
testing
on
environmental
fate
and
expression)
needs
to
include
considerations
of
a
practical
nature,
such
as
currently
available
analytical
methods,
the
limits
of
detection
and
quantification
of
those
methods,
and
the
utility
of
continued
sampling
and
analyses
once
soil
concentrations
fall
below
levels
of
detection/
quantification
for
two
or
three
consecutive
sampling
time
points.

EPA
Response:
Section
3.2
3
­
89
The
commenter's
suggestions
on
the
protocol
parameters
for
long
term
field
sampling
for
residual
soil
Cry
protein
determination
have
been
considered
and
generally
accepted
as
valid
by
the
EPA.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
6
Excerpt
Text:
Question
4:
Soil
Degradation/
Accumulation
of
Cry3Bb1
A)
The
Panel
is
requested
to
comment
on
the
advisability
of
testing
additional
soil
types
and
for
having
soil
persistence
studies
for
up
to
3
years.

Response:
Monsanto
believes
that
this
laboratory
soil
degradation
study
demonstrates
that
Cry3Bb1
protein
has
the
propensity
to
degrade
rapidly
in
soils,
and
is
sufficient
for
preliminary
fate
assessment.
Monsanto
will
provide
supplemental
post­
registration
studies
with
soils
of
soils
of
higher
clay
and
silt
content,
as
well
as
field
persistence
studies.

EPA
has
reviewed
a
soil
degradation
study
on
a
sandy
loam
soil,
which
contained
61%
sand,
28%
silt,
10%
clay
and
2.3%
organic
matter.
The
study
showed
a
DT50
of
2.4
days
by
insect
bioassay,
and
2.8
days
by
enzyme­
linked
immunosorbent
assay
(
ELISA).
The
DT90
was
7.9
days
by
insect
bioassay
and
9.2
days
by
ELISA.
[
Footnote
17:
Martin,
J.
W.,
M.
J.
McKee,
S.
Dubelman
and
Y.
A.
Dudin
(
2000).
Aerobic
Soil
Degradation
of
the
B.
t.
Protein
11098
as
a
Component
of
Insect
Protected
Corn.
Report
MSL­
16440,
an
unpublished
study
conducted
by
Monsanto
Company.
MRID
451568­
04.]

EPA
Response:
The
accumulation
of
residual
amounts
of
Cry
protein
in
agricultural
field
soil
will
be
evaluated
over
the
life
of
the
limited
MON863
registration
to
provide
confirmatory
long
term
soil
biota
effects
information.
Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
7
Excerpt
Text:
B)
What
soil
types
would
need
to
be
tested
and
what
duration
is
needed
for
soil
persistence
studies?

Response:
Monsanto
will
provide
supplemental
post­
registration
studies
with
soils
of
higher
clay
and
silt
content.
Given
the
rapid
dissipation
of
the
Cry3Bbl
protein
in
the
laboratory
soil
degradation
study,
Monsanto
suggests
that
the
duration
of
post­
registration
field
persistence
studies
follow
a
tiered
design
(
i.
e.,
a
one­
year
study
would
be
adequate
unless
Cry3Bb1
protein
persistence
were
observed
in
soil).

EPA
Response:
Additional
field
studies
will
be
conducted
that
include
the
incorporation
of
all
non­
harvested
plant
tissue
in
a
variety
of
soil
types
particularly
areas
high
in
clay
(
26%
already
tested)
and
humic
acids.
These
studies
will
be
conducted
for
at
least
one
growing
season
after
harvest
and
continue
until
no
Cry3Bb1
protein
is
detected.
In
addition
the
persistence
of
the
Cry3Bb1
protein
under
less
than
optimum
conditions
(
e.
g.,
high
Section
3.2
3
­
90
or
low
temperatures;
high
or
low
soil
moisture
content)
will
be
examined.
Soil
in
fallow
fields
after
at
least
three
seasons
of
MON
863
corn
cultivation
will
also
be
tested.
These
studies,
in
addition
to
ELISA,
will
include
an
insect
bioassay
utilizing
a
known
sensitive
species
such
as
the
Colorado
potato
beetle.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
8
Excerpt
Text:
C)
Are
these
studies
truly
expressing
the
time
to
50%
or
90%
degradation
of
Bt
protein
in
soil
or
whether
they
are
only
determining
the
level
of
detection
of
Cry3Bbl
protein
in
soil?
Discuss
the
acceptability
of
these
studies
for
a
preliminary
risk
assessment
to
evaluate
the
fate
of
Cry3Bbl
in
soil.

Response:
The
two
detection
methods
used
in
Monsanto's
soil
degradation
study,
an
insect
bioassay
and
an
ELISA
method,
yielded
nearly
identical
DT50
and
DT90
values,
suggesting
that
these
values
are
robust,
and
that
either
method
can
be
used
in
such
studies.

The
soil
degradation
study
was
performed
by
dosing
soils
with
exaggerated
quantities
of
tissues
containing
Cry3Bbl
protein,
and
using
both
insect
bioassay
and
ELISA
methods.
Soil
samples
collected
at
the
various
sampling
intervals
showed
Cry3Bbl
protein
residues
declining
exponentially
as
a
function
of
sampling
time,
but
well
above
the
detection
limit
of
both
methods
used
to
quantify
Cry3Bbl
protein
residues
in
soil.
The
DT50
and
DT90
values
were
calculated
primarily
based
on
detectable
responses
at
the
various
sampling
intervals,
and
method
detection
limits
were
not
an
obstacle
to
proper
determination
of
DT50
and
DT90
values.
Insect
bioassay
methods
have
been
frequently
used
to
measure
soil­
bound
Bt­
protein
residues.
[
Footnote
18:
Saxena,
D.,
S.
Flores
and
G.
Stotsky
(
2002).
Bt
Toxin
is
Released
in
Root
Exudates
from
12
Transgenic
Corn
Hybrids
Representing
Three
Transformation
Events.
Soil
Biol.
Biochem.
34:
133­
137.]

EPA
Response:
An
insect
bioassay
and
an
ELISA
method,
the
two
methods
for
detection
of
Cry
protein
in
soils
used
in
Monsanto's
soil
degradation
studies,
are
currently
acceptable
to
EPA.
Bt
Cry
proteins
adsorbed
to
clay
and
humic
acid
are
detectable
by
the
insect
bioassay
method.

Commenter
Name:
Monsanto
Commenter
Organization
Name:
Monsanto
Comment
Number:
OPP02­
0021
Excerpt
Number:
9
Excerpt
Text:
D)
What
if
any
difference
would
it
make
in
the
values
of
these
ELISA
based
studies
if
clay
particles
to
which
Cry3Bbl
protein
might
bind
are
present
in
the
soil
being
tested
that
measures
should
be
taken
to
ensure
that
the
test
is
not
measuring
inactive
protein
fragments?

Response:
The
ELISA
method
quantifies
the
total
amount
of
Cry3Bbl
protein,
active
and
inactive,
free
or
soil
bound,
present
in
soil
samples.
Inactive
Cry3Bbl
protein
may
consist
of
small
protein
fragments
that
are
not
biologically
active
against
insects
and
consequently
ELISA
quantification
may
potentially
exaggerate
the
amount
of
Cry3Bbl
protein
residue
present
in
soil.
Monsanto
believes
that
the
insect
Section
3.2
3
­
91
bioassay
and
ELISA­
based
DT50
and
DT90
values
obtained
in
the
soil
degradation
studies
provide
a
conservative
estimate
of
the
soil
fate
of
the
Cry3Bbl
protein
derived
from
corn
containing
event
MON
863.

Clay
and
silt
particles
were
present
in
the
soil
used
to
determine
DT50
and
DT90
values
for
Cry3Bb1
protein
(
10%
clay,
28%
silt).
Monsanto
acknowledges
that
soils
with
higher
silt
and
clay
content
should
be
tested
to
verify
that
the
rapid
degradation
pattern
is
not
adversely
affected
by
the
presence
of
additional
silt
and
clay.
The
ELISA
method
quantifies
the
total
amount
of
Cry3Bb1
protein,
active
and
inactive,
present
in
soil
samples.
The
insect
bioassay
method
quantifies
only
toxicologically
active
Cry3BbI
protein.
Toxicologically
active
protein
can
be
free
or
soil­
bound.
Inactive
protein
may
consist
of
small
protein
fragments
that
are
not
biologically
active
against
insects,
but
may
theoretically
still
contain
the
immunophoric
sequence
detected
by
the
ELISA
method.
The
ELISA
method
was
used
in
the
soil
degradation
studies
because
it
is
more
sensitive
than
the
most
sensitive
insect
bioassay
method
(
Colorado
potato
beetle)
currently
available
for
determination
of
Cry3Bb1
protein
residues
in
soil.
The
ELISA
method
could
therefore,
provide
data
for
longer
sampling
intervals
(
i.
e.,
when
most
of
the
Cry3Bb1
protein
had
already
degraded
to
levels
below
insect
toxicity
thresholds).
Indeed,
in
the
cited
study,
the
insect
bioassay
provided
quantitative
data
for
the
first
four
sampling
points,
and
the
ELISA
method
provided
quantitative
data
for
six
sampling
intervals,
extending
the
limits
of
qualification
for
two
additional
weeks
of
sample
incubation.
As
mentioned
above,
the
DT50
and
DT90
values
were
almost
identical
by
both
methods
(
DT50
of
2.4
days
by
insect
bioassay,
and
2.8
days
by
ELISA;
DT90
of
7.9
days
by
insect
bioassay
and
9.2
days
by
ELISA).
This
suggests
that
the
ELISA
method
also
quantifies
mostly
active
Cry3Bb1
protein.
However,
if
the
ELISA
method
were
assumed
to
quantify
both
active
and
inactive
protein,
then
it
would
potentially
exaggerate
the
amount
of
Cry3Bb1
protein
residue
present
in
soil.
In
such
case,
Cry3Bbl
protein
degradation
may
be
even
faster
(
less
risk)
than
indicated
by
the
ELISA­
derived
DT50
and
DT90
values.
Monsanto
believes
that
the
insect
bioassay
and
ELISA­
based
DT50
and
DT90
values
obtained
in
the
soil
degradation
studies
provide
a
conservative
estimate
of
the
soil
fate
of
the
Cry3Bb1
protein
derived
from
corn
containing
event
MON
863.

EPA
Response:
An
insect
bioassay
and
an
ELISA
method,
the
two
methods
for
detection
of
Cry
protein
in
soils
used
in
Monsanto's
soil
degradation
studies,
are
currently
acceptable
to
EPA.
Bt
Cry
proteins
adsorbed
to
clay
and
humic
acid
are
detectable
by
the
insect
bioassay
method.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
8
Excerpt
Text:
6.
Monsanto
has
not
conducted
studies
of
tile
persistence
of
Cry3Bb1
in
the
soil
under
field
conditions.
The
recent
Bt
reassessment
Scientific
Advisory
Panel
(
SAP)
stated
that
it
is
important
to
determine
the
persistence
of
Bt
protein
in
the
soil
under
field
conditions.
Monsanto
should
conduct
soil
persistence
experiments
for
Cry3Bb1
under
field
conditions
and
measure
Cry3Bb1
concentrations
both
close
to
and
distant
from
MON
863
corn
roots.

EPA
Response:
Additional
field
studies
will
be
conducted
that
include
the
incorporation
of
all
non­
harvested
plant
tissue
in
Section
3.2
3
­
92
a
variety
of
soil
types
particularly
areas
high
in
clay
(
26%
already
tested)
and
humic
acids.
These
studies
will
be
conducted
for
at
least
one
growing
season
after
harvest
and
continue
until
no
Cry3Bb1
protein
is
detected.
In
addition
the
persistence
of
the
Cry3Bb1
protein
under
less
than
optimum
conditions
(
e.
g.,
high
or
low
temperatures;
high
or
low
soil
moisture
content)
will
be
examined.
Deep
and
shallow
soils,
as
well
as
soil
close
to,
and
away
from
roots
will
be
examined.
It
is
understood,
however,
that
soil
taken
from
the
root
zone
will
likely
be
contaminated
by
exudates
from
fine
roots
damaged
during
the
soil
collection
process.
Soil
in
fallow
fields
after
at
least
three
seasons
of
MON
863
corn
cultivation
will
also
be
tested.
These
studies,
in
addition
to
ELISA,
will
include
an
insect
bioassay
utilizing
a
known
sensitive
species
such
as
the
Colorado
potato
beetle.

Commenter
Name:
N/
A
Commenter
Organization
Name:
Center
for
Science
in
the
Public
Interest
Comment
Number:
OPP02­
0028
Excerpt
Number:
20
Excerpt
Text:
Monsanto
has
determined
that
Cry3Bb1
degrades
at
a
rate
of
about
50%
in
less
than
three
days
in
soil
under
laboratory
conditions
after
a
single
incorporation
of
finely
ground
MON
863
leaves.
However,
the
Bt
reassessment
SAP
recommended
that
the
rate
of
degradation
of
Cry
proteins
should
be
determined
under
field
conditions
(
18).
EPA
should
require
Monsanto
to
determine
the
rate
of
degradation
of
MON
863
in
the
field
for
at
least
one
growing
season
including
sufficient
time
after
harvest
to
determine
how
quickly
Cry3
Bb1
degrades.

Furthermore,
single
incorporation
studies
to
determine
the
fate
of
Cry
toxins
were
considered
insufficient
by
the
Bt
reassessment
SAP
because
actual
deposition
likely
occurs
on
a
continuous
basis
both
from
root
biomass,
as
well
as
through
possible
exudation
of
Cry3Bb1
through
the
roots.
If
exudation
occurs
at
a
sufficient
rate,
it
is
possible
that
the
concentration
of
Cry3Bb1
in
the
soil
could
increase,
at
least
through
the
growing
season,
because
breakdown
of
Cry
proteins
may
be
slowed
by
binding
onto
clay
particles.

Conducting
field
degradation
studies
is
important
because
Monsanto
reported
that
expression
of
MON
863
is
high
in
the
root
tip
(
20).
Corn
in
particular
is
noted
for
its
ability
to
exude
proteins
through
the
root
cap
(
18),
so
there
is
particular
concern
that
MON
863
corn
may
exude
Cry3Bb1.
In
addition,
Monsanto
calculated
the
average
concentration
of
Cry3Bb
I
in
the
soil
based
on
even
distribution
of
a
single
incorporation
of
corn
plant
residue
to
a
depth
of
six
inches.
That
concentration
was
used
as
a
soil
EEC
to
determine
the
Cry3Bb1
concentration
used
in
non­
target
soil
organism
toxicity
tests.
Due
to
root
growth
and
possible
exudation,
Monsanto
likely
underestimates
the
actual
concentration
of
Cry3Bbl
that
soil
organisms
will
encounter.
While
a
single
incorporation
of
Bt
plant
material
would
expose
soil
organisms
to
steadily
decreasing
amounts
of
Cry3Bbl,
actual
exposure
during
the
growing
season
will
be
continuous.
When
the
primary
exposure
to
Cry3Bb1
is
ingestion
of
root
material
or
root
exudates
by
herbivores
or
detritovors,
or
indirectly
by
their
predators
or
parasites,
exposure
to
higher
concentrations
of
Cry3Bb1
than
estimated
by
Monsanto
can
occur.

Finally,
while
not
a
current
requirement,
EPA
should
require
testing
of
the
impact
of
Cry3Bb1
on
soil
microbes.
Soil
microbes
are
vital
for
maintaining
the
properties
of
soil
responsible
for
crop
productivity
and
such
environmentally
critical
issues
as
water
conservation.
Data
was
generated
on
microbial
impact
for
currently
registered
Bt
crops,
and
the
Bt
reassessment
SAP
recognized
the
importance
of
evaluating
Section
3.2
3
­
93
possible
impact
of
transgenic
pesticidal
crops
on
soil
microorganisms
and
encouraged
additional
effort
in
evaluating
such
impacts.
Unfortunately,
EPA
has
not
developed
guidelines
to
assess
impact
on
soil
microorganisms
so
it
is
not
clear
what
testing
should
be
conducted
to
assess
impact
on
soil
microbes.

EPA
Response:
Additional
field
studies
will
be
conducted
that
include
the
incorporation
of
all
non­
harvested
plant
tissue
in
a
variety
of
soil
types
particularly
areas
high
in
clay
(
26%
already
tested)
and
humic
acids.
These
studies
will
be
conducted
for
at
least
one
growing
season
after
harvest
and
continue
until
no
Cry3Bb1
protein
is
detected.
In
addition
the
persistence
of
the
Cry3Bb1
protein
under
less
than
optimum
conditions
(
e.
g.,
high
or
low
temperatures;
high
or
low
soil
moisture
content)
will
be
examined.
During
the
growing
season
deep
and
shallow
soils,
as
well
as
soil
close
to,
and
away
from
roots
will
be
examined.
It
is
understood,
however,
that
soil
taken
from
the
root
zone
will
likely
be
contaminated
by
exudates
from
fine
roots
damaged
during
the
soil
collection
process.
Soil
in
fallow
fields
after
at
least
three
seasons
of
MON
863
corn
cultivation
will
also
be
examined.
These
studies,
in
addition
to
ELISA,
will
include
an
insect
bioassay
utilizing
a
known
sensitive
species
such
as
the
Colorado
potato
beetle.

Data
on
soil
degradation
of
CryBb
proteins
currently
at
EPA
was
obtained
at
high
Cry
protein
doses
that
would
account
for
the
amount
of
Cry
protein
released
by
continuous
exudation
by
plant
roots.
In
addition,
it
is
general
knowledge
that
microbial
degradation
of
proteins
in
soil
is
ubiquitous.
This
observation
was
reaffirmed
by
the
FIFRA
SAP
(
SAP
Report
No.
99­
06,
February
4,
2000).
Several
studies
have
also
shown
that
Cry
proteins
adsorbed
to
soil
minerals
are
quickly
metabolized
by
soil
microbes
upon
elution.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
The
data
reviewed
by
the
Agency
show
that
the
Cry
protein
degradation
in
soil
occurs
within
days
to
a
barely
detectable
background
levels
regardless
of
the
Cry
protein
source
source.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
4).
Data
available
to
date
also
do
not
show
any
detrimental
effects
on
soil
biota.
(
USEPA.
2002.
MON
863
BRAD
Section
C.
I.
B.
5,6).
These
aspects
of
soil
accumulation
and
runoff
of
Cry
proteins
will,
however,
be
investigated
over
several
years
to
provide
confirmatory
long
term
effects
information.