Page
1
of
5
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
WASHINGTON,
DC
20460
OFFICE
OF
PREVENTION,
PESTICIDES
AND
TOXIC
SUBSTANCES
MEMORANDUM
PC
Code
No.
129106
DP
Barcode:
D283014
SUBJECT:
EFED
Review
of
Relyea
Paper
Entitled
"Predator­
induced
stress
makes
the
pesticide
carbaryl
more
deadly
to
gray
treefrog
tadpoles
(Hyla
versicolor)
"

TO:
Anthony
Britten,
Chemical
Review
Manager
Betty
Shackleford,
Product
Manager
Special
Review
and
Reregistration
Division
FROM:
Thomas
M.
Steeger,
Ph.
D.,
Senior
Biologist
Environmental
Risk
Branch
IV/
EFED
(7507C)

Through:
Betsy
Behl,
Branch
Chief
Environmental
Risk
Branch
IV/
EFED
(7507C)

The
Environmental
Fate
and
Effects
Division
(EFED)
has
completed
its
review
of
the
research
article
entitled
"Predator­
induced
stress
makes
the
pesticide
carbaryl
more
deadly
to
gray
treefrog
tadpoles
(Hyla
versicolor)"
published
in
the
February
2001
issue
of
the
Proceedings
of
the
National
Academy
of
Science.
The
paper,
authored
by
Rick
Relyea
and
Nathan
Mills
(Department
of
Biology,
University
of
Pittsburg)
provides
data
demonstrating
that
prolonged
sub­
acute
exposure
of
gray
treefrog
tadpoles
to
carbaryl
at
3
to
4%
of
the
reported
LC50
(2.5
­
20.6
mg/
L)
killed
10
to
60%
of
the
tadpoles.
Furthermore,
the
paper
claims
that
in
the
presence
of
"predatory
cues"
carbaryl
was
2
to
4
times
more
lethal
to
tadpoles.
The
authors
conclude
that
"under
more
realistic
conditions
of
increased
exposure
times
and
predatory
stress
[simulated
in
their
study],
current
application
rates
for
carbaryl
can
potentially
devastate
gray
treefrog
populations"
and
that
given
the
common
mechanism
of
action,
i.
e.,
acetylcholinesterase
inhibition,
of
carbaryl
with
other
widely
used
pesticides
(carbamates
and
organophosphates),
the
"negative
impacts
may
be
widespread
in
nature."
While
EFED
concurs
that
biotic
and
abiotic
effects
do
impact
the
toxicity
of
chemicals,
we
do
not
concur
with
the
author's
contention
that
their
protocol
is
indicative
of
"more
realistic
ecological
conditions"
than
EFED's
current
battery
of
acute
and
chronic
toxicity
tests;
all
of
these
studies
are
conducted
under
rigidly
controlled
laboratory
conditions
and
are
not
intended
to
be
representative
of
all
of
the
variables
that
may
affect
the
toxicity
of
a
compound
in
the
field.
Furthermore,
the
EFED
environmental
fate
and
ecological
risk
assessment
chapter
on
carbaryl
submitted
in
support
of
the
re­
registration
eligibility
decision
does
attempt
to
account
for
carbaryl's
risk
to
amphibians
and
is
to
some
extent
protective
to
amphibians
at
the
concentrations
discussed
Page
2
of
5
in
the
Relyea
and
Mills
paper.
However,
while
the
authors
are
correct
that
a
cumulative
assessment
of
the
effects
of
all
chemicals
acting
through
a
similar
mode
of
action
may
be
more
realistic,
the
logistics
of
conducting
such
an
evaluation
would
require
additional
resources
than
are
currently
available
in
EFED.

The
EFED
environmental
fate
and
ecological
risk
assessment
chapter
on
carbaryl
contains
both
acute
and
chronic
amphibian
toxicity
data
(see
Attachment
1
for
excerpt
on
amphibians
from
chapter).
Although
bullfrogs
(Rana
catesbeiana)
are
relatively
inured
(LD50
>
4,000
mg/
Kg)
to
carbaryl
on
an
acute
oral
exposure
basis,
leopard
frog
tadpoles
(Rana
blairi)
exhibited
a
90%
reduction
in
swimming
activity
at
carbaryl
concentrations
in
the
3.5
­
7.2
mg/
L
range.
The
chapter
notes
that
such
an
impairment
would
likely
render
the
tadpoles
[prey]
vulnerable
to
predation
provided
the
predators
were
not
similarly
impaired.
Furthermore,
the
chapter
notes
that
chronic
exposure
of
southern
leopard
frogs
(Rana
sphenocephala)
to
carbaryl
led
to
developmental
and
growth
effects
and
that
the
long­
term
effects
of
short­
term
carbaryl
exposures
to
amphibians
during
critical
life
stages
was
uncertain
and
could
potentially
lead
to
population­
level
effects.
Therefore,
the
EFED
risk
assessment
does
discuss
qualitatively
the
potential
susceptibility
of
amphibians
following
both
acute
and
chronic
exposure
to
carbaryl.

EFED
does
not
typically
evaluate
risk
to
aquatic
animals
on
a
species­
by­
species
or
class­
byclass
basis
but
rather
relies
on
surrogate
species
as
representatives
of
broad
ranges
of
aquatic
organisms.
As
with
most
screening­
level
risk
assessments
conducted
by
EFED,
the
carbaryl
chapter
used
fish
toxicity
data
as
a
surrogate
for
amphibians.
Toxicity
values
for
freshwater
fish
ranged
from
0.25
to
20
mg/
L;
the
most
sensitive
species,
i.
e.,
Atlantic
salmon
(Salmo
salar)
with
a
96­
hour
LC50
value
of
0.25
mg/
L,
was
selected
for
calculating
risk
quotient
(RQ)
values
used
in
EFED's
assessment
of
ecological
risk
to
freshwater
vertebrates.
The
salmon
LC50
value
represents
roughly
10%
of
the
lower
LC50
range
(2.5
to
20.6
mg/
L)
for
amphibians
reported
in
Relyea
and
Mills
paper.
Given
that
EFED's
levels
of
concern
(LOC),
i.
e.,
the
ratio
of
expected
environmental
concentrations
(EEC)
to
the
LC50
value,
for
endangered
is
0.05,
if
the
EEC
was
greater
than
0.01
mg/
L,
it
would
exceed
EFED's
LOC.
Therefore,
the
ecological
risk
assessment
for
aquatic
vertebrates
is
protective
for
species
with
96­
hour
LC50
values
greater
than
0.01
mg/
L.
(0.04%
of
the
range
reported
by
Relyea
and
Mills).

EFED
concurs
with
the
study
authors
that
biotic
and
abiotic
effects
can
impact
the
toxicity
of
pesticides
and
that
it
is
difficult
to
account
for
these
effects
on
the
basis
of
the
limited
laboratory
tests
that
are
typically
available
for
evaluating
the
effects
of
pesticides.
EFED
also
concurs
with
the
authors
that
chemicals
with
similar
modes
of
action
may
have
additive
toxicities
and
that
cummulative
assessments
may
better
account
for
toxicity;
however,
the
practicality
of
implementing
such
evaluations
is
limited
for
screening­
level
assessments.

EFED
is
uncertain
regarding
how
representative
the
Relyea
and
Mills
article
is
of
field
effects
though
or
of
the
direct
effects
of
carbaryl
and
predatory
cues.
The
experimental
design
included
10
tadpoles
in
10­
liter
polyethylene
tubs
containing
filtered
tapwater.
In
a
10­
day
static
renewal
study,
they
changed
water
on
days
3
and
7.
In
16­
day
static­
renewal
exposures,
they
changed
water
every
4
days.
Water
quality
parameters
(dissolved
oxygen,
temperature,
pH
and
Page
3
of
5
ammonia)
were
measured
midway
through
the
16­
day
exposure
studies.
Predator
treatments
consisted
of
a
larval
salamander
(Ambystoma
maculatum)
housed
within
a
250­
ml
plastic
cup,
covered
with
a
fiberglass
window
screening,
in
each
of
the
exposure
tanks;
controls
consisted
of
the
plastic
cup
alone.
Nominal
carbaryl
concentrations
ranged
from
0.045
to
0.54
mg/
L;
both
negative
and
solvent
(acetone)
controls
were
run
concurrently.
The
results
demonstrate
that
increased
ammonia
concentrations
were
associated
(P<
0.0001,
range
of
means
=
0.21
­
0.99
mg/
L)
with
carbaryl
concentration,
an
effect
attributed
to
the
presence
of
dead
tadpoles
and
excess
unconsumed
food.
A
regression
analysis
of
survival
against
ammonia
was
significant
(P
<
0.001,
but
not
particularly
predictive
(R
2
=
0.395).
Predators
had
no
effect
on
ammonia
(P
>
0.1)
and
only
had
small
effects
on
oxygen
and
pH
(9%
decrease
in
oxygen,
P
<
0.0001;
5%
decrease
in
pH,
P
=
0.019).
Given
that
water
quality
parameters
were
only
measured
midway
through
the
study
and
that
both
tadpoles
and
thus
feeding
rates
were
likely
increasing
throughout
the
study,
ammonia
levels
may
have
been
considerably
higher
toward
the
end
of
the
studies.
Thus
it
is
unclear
whether
ammonia,
pH
and
dissolved
oxygen
had
an
effect
on
the
toxicity
of
carbaryl
to
tadpoles.
It
is
noteworthy
that
the
Relyea
and
Mills
data
showed
precipitous
declines
in
tadpole
survival
after
5
days
of
exposure.

Although
it
is
difficult
to
design
a
study
that
can
accurately
reflect
field
conditions
and
particularly
predator­
prey
relationships,
EFED
is
not
convinced
that
the
Relyea
and
Mills
study
could
be
interpreted
as
more
representative
of
field
conditions.
Typically,
prey
demonstrate
predator
avoidance
behavior
in
the
presence
of
a
perceived
threat.
In
this
study,
tadpoles
were
unable
to
escape
their
perceived
threat;
predatory
cues,
i.
e.,
seeing
a
predator
(visual
cues)
may
have
protracted
their
response
well
beyond
the
chemical
cues
released
following
the
salamander's
consumption
of
tadpoles.
It
is
questionable
whether
tadpoles
would
have
remained
in
view
of
a
potential
predator
under
more
realistic
conditions.

In
refined
ecological
assessments,
EFED
oftentimes
has
mesocosm
study
data
available
to
assess
the
risk
of
pesticides
under
"field
conditions".
These
studies,
while
considerably
more
expensive
that
the
Relyea
and
Mills
protocol,
may
represent
the
most
accurate
reflection
of
controlled
field
studies.
It
is
interesting
to
note
though
that
while
mesocosm
studies
may
yield
LC50
values
similar
to
laboratory
studies,
they
rarely
provide
LC50
values
showing
enhanced
toxicity.
Test
species
within
these
studies
are
better
able
to
rely
on
compensatory
mechanism
to
shield
themselves
from
the
toxic
effects
of
chemicals.

In
addition,
the
environmental
fate
of
pesticides
is
often
different
under
field
conditions.
Under
alkaline
conditions,
i.
e.,
pH
>
7,
carbaryl
undergoes
hydrolysis
with
half­
lives
ranging
from
0.15
to
12
days.
While
Relyea
and
Mills
accurately
note
carbaryl's
susceptibility
to
hydrolysis,
they
fail
to
mention
that
under
aearobic
conditions,
carbaryl
is
also
microbially
degraded
in
the
aquatic
environment
with
a
half
life
of
approximately
5
days.
It
is
likely
that
gray
treefrogs
in
the
Relyea
and
Mills
study
were
exposed
to
carbaryl
concentrations
considerably
lower
than
nominal
after
3
to
4
days.
Thus
the
actual
exposure
regime
may
have
been
more
representative
of
pulsed
exposures
to
declining
concentrations
of
carbaryl
and
increasing
concentrations
of
ammonia.
While
it
is
clear
that
predators
had
an
effect
on
the
response
of
tadpoles
to
the
exposure
regime,
EFED
does
not
concur
that
the
test
results
are
representative
of
the
effects
of
predation
on
carbaryl
toxicity
alone.
Page
4
of
5
EFED
concurs
with
Relyea
and
Mills
that
both
biotic
and
abiotic
factors
impact
the
toxicity
of
pesticides
and
that
current
screening
methods
do
not
account
for
the
full
range
of
these
effects
nor
do
screening
level
assessments
take
into
account
aggregate
effects
from
exposure
to
chemicals
with
similar
modes
of
action.
Screening­
level
assessments
attempt
to
identify
where
EFED's
LOCs
are
exceeded
and
where
EFED
has
uncertainties
regarding
risk.
With
respect
to
amphibians,
the
chapter
discusses
the
likelihood
of
acute
and
chronic
effects
from
current
uses
of
carbaryl.
Page
5
of
5
Attachment
1.
Excerpt
on
Amphibians
from
the
Environmental
Fate
and
Ecological
Risk
Assessment
for
the
Reregistration
of
Carbaryl
Chapter
According
to
an
available
supplemental
study
with
a
50%
carbaryl
formulation,
the
LD50
for
the
bullfrog
(Rana
catesbeiana)
is
greater
than
4,000
mg/
kg,
or
practically
nontoxic
(MRID
00160000).
A
single
acute
exposure
of
plains
leopard
frog
tadpoles
(Rana
blairi)
to
carbaryl
concentrations
in
the
3.5
­
7.2
mg/
L
range
led
to
a
90%
reduction
in
swimming
activity,
including
sprint
speed
and
sprint
distance,
activity
ceasing
completely
at
7.2
mg/
L
(Bridges
1997).
This
reduction
in
activity
and
swimming
performance
may
result
in
increased
predation
rates
and,
because
activity
is
closely
associated
with
feeding,
may
result
in
slowed
growth
that
could
lead
to
failure
to
complete
metamorphosis.
Acute
exposure
to
low
carbaryl
levels
may
not
only
affect
immediate
survival
of
tadpoles
but
also
impact
critical
life
history
functions.

On
a
chronic
basis,
carbaryl
has
been
shown
to
have
the
potential
to
adversely
affect
amphibians.
In
a
recent
study,
nearly
18%
of
southern
leopard
frog
(Rana
sphenocephala)
tadpoles
exposed
to
carbaryl
during
development
exhibited
some
type
of
developmental
deformity,
including
both
visceral
and
limb
malformations,
compared
to
a
single
deformed
(<
1%)
control
tadpole
demonstrating
that
carbaryl
exposure
can
result
in
amphibian
deformities
(Bridges,
2000).
Although
the
length
of
the
larval
period
was
the
same
for
all
experimental
groups,
tadpoles
exposed
throughout
the
egg
stage
were
smaller
than
their
corresponding
controls.
Because
exposure
to
nonpersistent
chemicals
may
last
for
only
a
short
period
of
time,
it
is
important
to
examine
the
long­
term
effects
that
short­
term
exposure
has
on
larval
amphibians
and
the
existence
of
any
sensitive
life
stage.
Any
delay
in
metamorphosis
or
decrease
in
size
at
metamorphosis
can
impact
demographic
processes
of
the
population,
potentially
leading
to
declines
or
local
extinction.
