Research

Krista A. McCoy,1'2 Lauriel J. Bortnick,2

and Colette M. St Mary12
Resources

of Natural

1School

BACKGROUND: Many
evidence of endocrine

and

sial. Typically,
studies
sites.
unpolluted

and department

Heather

of Zoology,

of Florida,

University

gonadal

EDC-induced

to address

the rela
this question
by explicidy
quantifying
and habitats characterized
by differing
degrees of agricul

ductive
areas

to

exposed

secondary
11536

sexual

to
which

(EDCs),

endocrine-disrupting
alter normal
hormonal

morphology

Zal?

chemicals
signal
can per

so EDCs

conserved,

highly

can
across

functions

physiological

(Crews and McLachlan

affect

that

taxa

2006; McLachlan

studies have shown

that
Indeed, many
to EDCs
is responsible
for changes
exposure
in anatomy,
and reproductive
behavior,
sys

2001).

tem

in a wide

function

array

of vertebrate

taxa, including fishes (Jobling et al. 2006),

et al.

(Hayes

amphibians

2003),

reptiles

(Guillette and Iguchi 2003), and mammals

et

(Sonne

al.

2006),

(Guillette et al. 2006; Swan 2006).

Despite this growing body of literature
much
endocrine
disruption,
remains about whether
controversy
reproduc
in wild
tive abnormalities
observed
popula
tions are caused by EDCs
and not some other

documenting

natural

process.

This

controversy

is fueled,

in

large part, by the difficulty of directly linking

endocrine-disrupting
laboratories
with

effects
field

identified

observations.

Wild

in

and

generate
controlled

novel

patterns

of

toxic

particular
not

pathologies
studies.

laboratory

pathologies
because many

agent
identified

or
in

Investigating
in nature
is further

studies are designed

sites against
reference
polluted
that
sites, yet there are likely no sites on Earth
are not affected
For
by pollution.
example,
are known
that
agricultural
pesticides
are
hun
endocrine
disruptors
transported
challenged
to compare

dreds of miles

through air before being

into once pristine

by precipitation
deposited
areas such as Isle
Park (Lake
Royale National

Superior)
Thurman

humans

including

can

abnormalities

normal

diverse

to
of
complex mixtures
exposed
et al. 2006)
and other envi
(Hayes
et al. 2006)
conditions
(Edwards
a causal
link between
obfuscate

ronmental

2004). Moreover,
many
are
the endocrine
system

of

are

organisms
chemicals

and Penn

components

natural

gonadal

malformations

contaminants

(including

processes.
found no

atrazine)

and
argue
result
For
con

agricultural
and the inci

or testicular
hermaphroditism
In
with
testicular
their
oocytes.
study,
frogs
at both
found
oocytes were
(pol
agricultural
sites
and nonagricultural
luted)
(reference)
of

dence

are
et al. (2006)
by Murphy
reported
or to
to be
naturally
hermaphroditic
as adults
have
testicular
oocytes
commonly

tive behavior (Guillette et al. 1995;McLachlan

2001;

observed

such

species
known

embryonic
development,
adult
system
reproductive
change
as well as
and function,
reproduc

ing during
manently

of
and

(Murphy et al. 2006). None of the amphibian

via

http://dx.doi.org/ [Online 3 July 2008]

Exposure

that
from

found

agriculture
and they

developmental
et al. (2006)
instance, Murphy
sistent
between
relationship

traits.

available

not

have
between

relationships
abnormalities,
reproductive

traits correlate with

repro
success. These
repro
reproductive
in
declines
occurring
population

intersex,
pesticides,
disruption,
116:1526-1532
(2008).
doi:10.1289/ehp.

amphibians,
Health
Perspect

Environ

decline
global
Indeed, Davidson

researchers

strong

sexual

endocrine

that

abnormalities

reproductive
to the

populations.

Other

contaminants.

agricultural

KEY WORDS:

from
relative

are one causal factor

pesticides
driving amphib
in some
ian
declines
locations
by
population
that the degree
of protection
demonstrating
was a
from windborne
pesticides
significant
of Rana muscosa distributions.
predictor

increased with agriculture

RESULTS: The number of abnormalities
and frequency
of intersex gonads
were associated with
in a dose-dependent
altered gonadal
fashion. These
abnormalities
gonadal
were altered and secondary
sexual
concentrations
function. Testosterone,
but not 17?-estradiol,
or demasculinized
traits were either feminized
skin motding)
(reduced forearm width
(increased
in intersex toads. Based on the end points we examined,
female morphol
and nuptial pad number)
areas had hormone
males from agricultural
did not differ across sites. However,
ogy and physiology
intersex toads and non
concentrations
intermediate
between
and secondary
sexual traits that were
at the most
site was not sexually dimorphic;
toads. Skin coloration
agricultural
agricultural male
males had female coloration.

ductive

USA

Knapp (2007) recendy showed thatwindborne

func
and measures
of gonadal
of gonadal abnormalities
the occurrence
quantified
tion in at least 20 giant toads (Bufo marinus) from each of five sites that occur along a gradient of
land use from 0 to 97%.
increasing agricultural

concentrations
and secondary
hormone
activity and success, so affected toads likely have reduced
to amphibian
contribute
abnormalities
could certainly

contribute

amphibian

METHODS: We

Steroid

Florida,

Gainesville,

could
approach
abnormalities

Jr.,1'2

and from
those
collected
laboratory
areas
atrazine-contaminated
agricultural
to reference
and
sites,
they suggested

However,
systems of wildlife.
areas has been controver
in
living
agricultural
on wildlife
to compare polluted with
attempt

tural activity.

CONCLUSIONS:

Louis J. Guillette

J. Hamlin,2

endocrine

disrupt

in wild amphibians
disruption
on the effects of
pollutants

took a novel

OBJECTIVES: We
tionship between

M. Campbell,2

Chelsey

Environment,

contaminants

agricultural

in the Toad Bufo marinus

Form and Function

Gonadal

Alters

Agriculture

(Nations

and Hallberg

1992;

and Cromwell

2000).
and
laboratory

recent
Many
have
that agricultural
suggested
are associated
with
amphibian
and
abnormalities
population

field

studies

contaminants
reproductive
declines

(Davidson and Knapp 2007; Ouellet et al.

1997; Sparling et al. 2001). Hayes et al. (2003)

of gonadal
abnormali
incidences
higher
male
and
ties and
(intersex:
hermaphroditism
in
tissue in the same individual)
female gonadal
to the herbicide
in the
atrazine
frogs exposed
found

1526

volume

(Hayes 1998). However, Murphy et al. (2006)

that 25-50%
of adults from three dif
reported
ferent species had testicular oocytes.
Such pro
in naturally
of testicular
oocytes
portions
gonochoristic
result of a rare
the

sites

Murphy

adults

are not

genetic

anomaly.
as
nonagricultural
were
nevertheless

categorized
et al. (2006)

to be the
likely
It is likely that

lutedwith some type(s) of EDC.

by
pol

to K.A. McCoy,
Smithsonian
correspondence
Research
Institute, Unit 0948, APO AA
Tropical
E-mail:
34002 USA. Telephone:
507-6-461-0821.
kristam@bu.edu
L. Babonis, and R. Harris
We
thank M. McCoy,
and
for constructive
feedback on the manuscript
A. Amick, P. Barnes Jr., M. Fromowitz, M. McCoy,
M. Rehrer, V. Bender, K. Hopkins,
and H. Wineman
Address

for help with field collections.

This work was supported in part by grants from the
Institute of Environmental
Health Sciences
National
to LJ.G. and the Rewald, Olowo,
(R21 ES014053)
Society for Integrative and Comparative
Biology, and
Sigma Xi grants in aid of research and the University
of Florida Institute of Food and Agricultural Sciences
Women's
Club Fellowship to K.A.M.
The funding sources had no influence in the study
or
design; collection, analysis, interpretation of data;
writing of the paper.
The authors declare
they have no competing
financial interests.
Received 3 April 2008;

1161 number 111 November

2008

accepted

Environmental

3 July 2008.

Health

Perspectives

Gonadal

we
took a novel
present
study,
to
gross
asking whether
gonadal
of an anuran amphibian
species
are associated with
{Bufo marinus)
agricultural
most
Unlike
studies, we
exposure.
previous
across a range of sites
this
question
explored
that vary in degree
of agricultural
intensity.
In

the

approach
abnormalities

at our
the proportion
of agriculture
was
to the
sites
related
inversely
study
degree
Stressors.
of urbanization
and its associated
In fact,

we
explicitly
are
abnormalities

Therefore,

gonadal
uted across
are

tested

with

distrib

randomly

human-modified

associated

whether

to

across

sites

five

that differed in degree of agricultural activity

and determined

the total number

We

found

and

types of
incidence

as well

as the

across

this continuum.
in

tural

to the
gonads

relative

of
nonagricul
sex
compared
circulating
and as more
concentrations,
integrated

toads. We

steroid

endocrine
system function,
signals of seasonal
we
three sex-hormone-dependent
compare
sexual characteristics.
secondary

Materials

and Methods

of

intensity

we

exposure,

agricultural

imported Google Earth (Google Inc.,

Mountain View, CA) digital satellite images
a scale

and

into

the

program

analysis

Pro

Image

Plus

House

(Image

image
A/S,

(Crosbie

Twenty

body
each
effect

in more

streetlights.
an overdose
with

B. marinus

et al.
is approximately
2 km2
(Zug
so this measure
reflects the area experi
1975),
enced by collected
toads.

sites an
assigned
"agricultural
on the
rank
of
based
1-5
intensity"
percentage
area around
in the 5.6-km2
of agriculture
the
then

site or

its proximity

to

agriculture

when

no

agricultural land (0%) fellwithin the 5.6-km2

area. We
tests

used

these

ranks for all Kendall's

tau

of association.

Lake Worth
(LW) and
no
contained
(WT)
Wellington
agriculture
(0%). LW was 22 km away from an agricul
tural area and was ranked
was
1, whereas WT
5.18
ranked

km

and was
agriculture,
34%
(HS) contained

from
away
2. Homestead

agricultural

land, which

we

ranked

3. Canal

Point (CP; 51%, bordered on one side by

Lake Okeechobee)
and Belle Glade (BG;

Environmental

Health

Perspectives

volume

of

at

at

collected

tri

0.3%
7

pH

(Sigma
and
photographed,

St. Louis, MO),

for snout-vent

Aldrich,
measured

from

mercury
vapor
was
euthanized

the anesthetic

methanesulfonate

length

(nearest

1mm). Approximately 3 mL of whole blood

was

via

collected

trifuged.

cardiac

Plasma

samples
back to the

transported
and stored

at -80?C

until

puncture
were frozen
laboratory
hormone

and

cen

at -20?C,
on
dry ice,
assays were

One

from each individual

gonad
was fixed in neutral buffered
formalin
for gross
were
All
analysis.
morphologic
procedures
of Florida's
institu
by the University
approved
care and use committee
tional animal
(permit
not
they did
Analyses
evaluated

We

were

animals

D699);

treated

(ANOVA)

humanely,

suffer any pain.

sexual
of primary

and

characteristics.

all primary
sexual characteristics
individuals
First, we evaluated

in two ways.
each abnormality

Kruskal-Wallis

We

under
feeding,
Each
individual

type

across

sites with

of variance
analysis
if the occurrence
of

one-way
to determine

individuals
based on
categorized
We
type of gonadal
abnormality.
scores and evaluated
them based on

also

quantity

and

assigned
the total

number

of abnormalities

across sites. The

differed
specific abnormalities
test does not
test
Kruskal-Wallis
explicitly
whether
effects increase with
increasing agricul
ture, so we used a one-tailed
Kendall's
tau-b

a measure

is considered
intersexed

ran all Kruskal-Wallis

tau tests

in R

free

statistics

IL),
soft

ware (Wessa 2007).

We

in females,

gonadal
morphology
males
with
Bidder's

gross
morphologic
so all
on
analyses
include
males,
only

abnormalities
organ
and intersexes.
These
toads
(Bidder's males),
=
were
a sex
1,
(males
group category
assigned
=
=
Bidder's males
and
differ
2, intersexes
3),
ences

in the mean

evaluated

as described

1161 number 111 November

the

to increased
corresponded
agricul
an
As
of our ranking
intensity.
example
an individual
testes
with misshaped
system,
a
in the testes
(one abnormality
category),
Bidder's
(one
organ
abnormality
vitellogenic
and

sites

in the Bidder's
the presence
(two

genic

organ
of

received

category)

and ovaries
(in
category),
that were
also vitello

testes)
abnormalities

in the ovary/oviduct

a score of 4.

next
Our
of gonadal
Analyses
function.
was
to determine
if
from
the
toads
objective
areas had altered
most
agricultural
gonadal
relative to those from
function
nonagricultural
areas
to assess whether
intersex
and
and
Bidder's

males

from

males
the

from

nonagricultural
groups occurred
at

occurred
not

agricultural
demasculinized

and

feminized

compare

same
areas.

site

and

sites were
to

relative
those

from

In fact, not all

gonadal
site (no intersex toads

at every

sites), so we could
across all sites.
groups

nonagricultural

gonadal
for each measure

of
func
gonadal
sex
toads into six different
grouped
on
based
gross gonadal morphology
categories
in
and their occurrence
(LW,
nonagricultural

Therefore,
tion, we

WT) or agricultural (BG, CP) sites: a) non

agricultural females (NAgFs), b) agricultural
intersex
toads
c) agricultural
(AgFs),
males
Bidder's
(intersexes),
a) agricultural
(Bidder's males),
e) agricultural males
(AgMs),

females

and/)
did

not

nonagricultural males (NAgMs). We

include

from

toads

the

intermediate

site

in these
(HS, 34%)
could not be considered

or as
AgMs.
NAgM
Sex hormone
concentrations.

We
analyzed
testosterone
and
(E2)
a validated
radio

17?-estradiol
circulating
concentrations
with
immunoassay

using

the 96-well

FlashPlate

PLUS system (PerkinElmer,Waltham, MA).

no

observed

abnormalities

of

organs

tural

sponded with agricultural intensity (Siegel and

and Kendall's

severity
c) Bidder's

ber of gonadal abnormalities differed among

as

to
test whether
site
explicitly
corre
in sex group
statistically

tests in SPSS 15.0 (SPSS Inc.,

Chicago,

the

In addition,
the total number
of abnor
were
summed
evaluated
and similarly
to determine
num
if the mean
and maximum

test of association

We

of
and

condition),

differences

1988).

of

three
tissue
a) testes,
types:
following
in the presence
of testes
b) ovaries/oviducts
and developmental
(number
stage of female
in the presence
tissues found
of testes
gonadal

agricultural
intensity
analyses because
they

Castellan

in each

the

malities

1993;

collected

than 1 year.
all locations were

from

while

night,

caine

were

We

agriculture

(Figure 1).

Year had no
period.
or
types of abnormal
at sites where
collections

ities documented
occurred

and

total percentage
of agricul
tural land
and sugarcane
(vegetable
farming,
no livestock)
a 5.6-km2
area around
within
each
collection
site. The
home
of
range

(Cohen and Alford

et al. 2006)
site over a 2-year
on the
percentage

with

the

of

with
pesti
typically
polluted
and Chow-Fraser
1999).
or more
adult
toads
(> 91 mm

length)

Copenhagen, Denmark) on 20 August 2007

calculated

suites

(Standley
areas are more

conducted.

sur
sites and collection
We
Study
techniques.
that varied
in
veyed five sites in south Florida
to
habitat
suburban
type from
completely
To quantify
the
completely
agricultural.

different

induce

con
Polyaromatic
hydrocarbon
are
areas
in urbanized
ubiquitous
et al. 2000),
whereas
agricultural

pathologies.
centrations

Toads

abnor
gonadal
as
so
malities
increased,
activity
agricultural
we went
to determine
if the
further
of
gonads
areas had altered
in
toads
agricultural
living
function

increase

strong

4 and

to

expected

Meshaka

specifically.
agriculture
We
B. marinus
surveyed

abnormalities
gonadal
of intersex
individuals

are

cides

exposure

increasing

land;
agricultural
5, respectively.
the presence
of specific contami
Although
nants at each type of site
certainly
overlapped,
are
and agricultural
urbanized
habitats
pol
of chemicals
luted with
different
milieus
that

or

landscapes

the most

contained
97%)
these were
ranked

are associated with

abnormalities

sex group
above.

2008

1527

across

sites were

were

to meet
the
transformed
log10
of normality
and
of
assumptions
homogeneity
a linear mixed
variances and then analyzed
using
where
and assay by sex
model,
assay
(plate)
Data

interaction
We

used

were
SPSS

identified
default

as the random
post

hoc

effects.

comparisons
(least significant
difference).
E2 to testosterone
were
ratios (E2/testosterone)
log10 transformed
to meet
the assumptions
of normality
and

^^H

H?

McCoy et al.
of variances
homogeneity
We
ANOVA.
one-way
comparisons
Secondary

and

evaluated

conducted

test.
using Tukey's
sexual characteristics.

to compare
photographs
number
of nuptial
pads,

using
hoc

post

relation
We
were

We

used

body color pattern,

size in
and forelimb

to the six sex groups

all photographs,

identified

renamed
blind

toads were

to the sex and

We

collected.

and

above.

observers

site from which

analyzed

the

all statistics

using SPSS 15 and conducted post hoc com

parisons

using Tukey's

tests.

the number
pattern. We
quantified
across
that occurred
changes
(mottling)
a transect
line drawn
from the left eye to the
vent of each
toad using
Image
photographed
Color

of color

sex
across
Plus and compared
mottling
In addition,
groups using a one-way ANOVA.
we
scores
and female mottling
analyzed male
across sites
a two-way ANOVA
separately using
to test for
across
in color
dimorphism
changes
sites (sex X site).
Pro

size. We

Forelimb

across

the forelimb
hum?rus
using

the width

perpendicular
Pro Plus and tested
an

using

arm

to the

of

to the

distal

radioulna

and

Image
sex groups

among

measured
the

axis

for differences
of covari

analysis

ance (ANCOVA), with sex group as the fixed

effect, log^forelimb width) as the dependent
as the covariate
and log^body
factor,
length)
to account
for the influence
of body size. We
to lin
conducted
natural
log transformations
earize

were
slopes
homogeneous;
the interaction
from
the

the data. All

thus, we

removed

analysis and used the simplified ANCO VA to

estimate

means

marginal
group. We

of each
snout-vent

of

length

the elbow

compared
120.2 mm.

pads. No

Nuptial

for

sex groups

width
at a

females had nuptial

were not included

in this
pads and thus
analy
to determine
sis. We
used one-way
ANOVA
if the mean
number
of nuptial
pads per limb
was different
sex groups.
among

Results
sexual
Primary
rank increased

characteristics.
as a function

sex group

Mean
of

agriculture.

More intersex individuals (Figure 1C) occurred

at the

two most

sites than at the

= 21.
6, p< 0.001;

agricultural

sites

other

(Kruskal-Wallis4
=
=
1, p
0.01;
2A).
tau-b3
Figure
Testicular
abnormalities
IB), such as
(Figure
common
and
testes, were
relatively
misshaped
did not vary across sites or increase with
agri
=
=
cultural
5.1, p
0.272;
(KW4
intensity
=
=
Kendall's
the
0.4, p
0.23). However,
tau-b3
Kendall's

number

abnormalities

of female-tissue
or oviducts

ovaries

in the presence
different
among

were

(e.g.,
of

testes)
sites and

significantly
increased with
intensity
increasing
agricultural
=
=
=
Kendall's
17.3, p
0.002;
(KW4
tau-b3
=
0.9, p
0.02;
IC, 2B).
Figures
Importantly,

Bidder's
abnormalties

the number

and developmental
stage of female
in the presence
found
of testes
gonadal
are considered
a measure
of the severity of the
tissues

intersexed
of gonadal phenotypes
observed. Abbreviations:
B, Bidder's organ; C, con
Figure 1. Four broad categories
nective tissue; K, kidney; OV, ovary; OD, oviduct; T, testis. (A) Toads that had no obvious gross gonadal
as having normal gonadal morphology.
abnormalities were categorized
(Left) "Males" had testes, a non
Bidder's organ, and no ovaries or oviducts.
vitellogenic
(Right) "Females" had ovaries, oviducts, nonvitel
logenic Bidder's organs, and no testes; ovaries in various stages of oog?nesis were collected
(oog?nesis
as "male" in
and accumulation
of vitellogenin
increases from left to right). {B) Toads that were categorized
some cases had testicular abnormalities.
(Left) Multiple testes.
(Center) Abnormally shaped testes.
(Right)
Abnormally small testes. (0 Intersex toads had both testes and oviducts or ovaries. (Left)Well-developed
oviduct and testis, but no ovary. (Center) Nonvitellogenic
ovary that is distinct from the Bidder's organ and
testis. (Right) Intersex with a highly vitellogenic
ovary. {D) "Bidder's males" had multiple (left), early vitel
= 0.5 cm.
logenic (center), or late vitellogenic
(right) Bidder's organs but no ovaries or oviducts. Bars

1528

volume

toads

condition.

became

more

culture

increased.

tended

to vary

the

agricultural

that measure,
Applying
as
intersexed
severely
agri
Bidder's
organ abnormalities

across
sites

sites
(KW4

and was
=
9.0,

highest
=
0.06;

at

Figures ID, 2C) but did not correspond signif

to
(Kendall's
icantly
increasing
agriculture
=
=
0.1; Figures
ID, 2C). The
0.6, p
tau-b3
total
number
of gonadal
abnormalities
increased
21.2,

1161 number 111 November

with

p<

(KW4
increasing
agriculture
=
Kendall's
0.001; mean
0.8,
tau-b3

2008

Environmental

Health

Perspectives

Gonadal

maximum

0.04;

Kendall's

tau-b3

0.9,

We

found

E2 concentra
=
<
sex
23.74,
groups
p
(^5,157
were
these differences
driven
in plasma

differences

significant
tions among

but

in females
by high E2 concentrations
sex
to all other
3A).
groups
(Figure
were not
differ
and NAgFs
AgFs
significantly
ent from one another.
entirely
relative

testosterone

Plasma
also

significantly

were

concentrations

different

among

sex

groups

=
<
(^5,162 1 !-26>P ?-001; Figure 3B). NAgFs
testos
in
did not differ
and AgFs
circulating
terone concentrations,
but had
significantly
testosterone
levels than did NAgMs.
lower
concen
testosterone
Intersex toads had plasma
trations
similar to NAgFs
and were
that were
not

from

different

Bidder's

but were

males

and AgMs,

lower

significantly
males
Bidder's

Both

/>= 0.02; Figure 2D).

concentrations.
Sex hormone

0.001),

and AgMs

not

than NAgMs.
were
signifi

cantly different from females, but only AgMs

had

significantly

tive toNAgMs
The

ratio

testosterone

lower

levels

rela

concen

to testosterone

was
(E2/testosterone)
significantly
=
sex
different
48.46,
groups
among
(^5,150
< 0.001;
were
not
Females
3C).
p
sig
Figure
tration

nificantly

from

different

E2/testosterone
The
groups.
intersex
toads was

one

another

ratios
E2/testosterone

that

significantly
of females

Bidder's

groups
males
and

AgMs,
than NAgMs.
On
average,
more
E2 than testosterone

but had
to all

relative

higher
other

in both

different

significantly

from

Color

pattern.
of sex

function

ratio
lower
and

but was
intersex

of

than

similar

to

higher
toads had

(mean E2/testos
terone
males
ratio > 1; Figure
3C). Bidder's
were
between
and
and AgMs
intermediate

mottled
highly
Bidder's
males
females

and

NAgMs.
females

intersexes

score

Mottling
category

(/^

and

similar.
similar

<

and AgFs were

and
to

in

mottling
mottled

more

significantly
were

as a

varied
=
15.6,

128

Intersexes

and
were

AgMs
but more

than
than

less mottled

mottled

than

NAgMs.
were
the only group
that was signifi
than all other sex groups.
less mottled

NAgMs
cantly
We

found
sex and

between

SHI

agriculture

(Figure 3C).

NAgMs

0.001; Figure 4B). NAgFs

(Figure 3B).
of E2

are associated with

abnormalities

a
significant
site on mottling

interaction
=
2.7,
Cr\io3

- 0.03;
Figure 4C). Although female mot
across
sites,
change
in
became
mottling

not

did

tling

the

sexual

less pro
increased

dimorphism
as
nounced
intensity
agricultural
were
in coloration
males
feminized
because

(Figure4C).
Forelimb size. Mean forelimb width was
different

significantly

sex groups

among

(F^jo

= 33.8,
p < 0.001; Figure 4D). Females had
smaller

forelimbs

Intersex

individuals

any other
group.
arm widths
that were

between

intermediate
ent from

than
had

females

and
significantly
and NAgMs.
Bidder's

and AgMs had larger forelimbs

but were

females
not

significantly

from

NAgMs.
different

significantly

and
or

intersexes

pad number was

pads. Nuptial

Nuptial

males

than did

between

intermediate
different

differ

among

sex groups

(we

did not consider females in this analysis) (^3,76

=

6.0,

0.001;

4E).

Figure

Intersex

toads

had significantly fewer nuptial pads than did

whereas

numbers

of nuptial
pads
were
inter
and AgMs
and not
between
differ
significantly
ent from those of either intersex or
NAgMs.

NAgMs,

Bidder's

among
mediate

LW WT
HS
Increasing agriculture

CP

LW

BG

Increasing

WT

HS

CP

Discussion

BG

agriculture

Here

manner.
in a dose-dependent
abnormalities
increase with
increasing
agriculture
as males, males with Bidder's organ abnormalities
individuals classified
(Bidder's), or
of individuals with testes and a specific number of female tissues
site. {B) Percentage
of oog?nesis;
the number of female tissues found in toads with testes (0-3) is a meas
of toads with (1) or without
of the intersex condition.
(0) Bidder's organ
(0 Percentage
at each site.
number of gonadal
abnormalities
each collection
site. {D) Maximum
intensity increases along the x-axis from left to right for each graph.

Figure 2. Gonadal
of
{A) Percentage
intersexes at each
or advanced
stage
ure of the seventy
at
abnormalities
Agricultural

2,500

we

time, that repro

in primary
sexual traits
are
asso
of wild
(feral) B. marinus
statistically
increase
and
ciated with
activity
agricultural
in a
of agricultural
with
exposure
percentage
manner.
of
The
severity
dose-dependent
maximum
number
of
and
the
intersexuality
ductive

show,

for

the first

abnormalities

2,000

males

1,000
800

1,500
600
1,000

400

500
0

t5

200

r^

.?

r^

NAg
Ag Intersex Bidder's Ag
Female Female
male Male

NAg
Male

jfL

Vo

NAg
Ag Intersex Bidder's Ag
male Male
Female Female

NAg
Male

A
Intersex Bidder's Ag
Ag
NAg
male Male
Female Female

and agricultural sites separated

of toads from nonagricultural
by sex group. {A) E2 concentrations.
Figure 3. Sex hormone concentrations
from nonagricultural
ratio across sex groups. NAg, toads collected
centrations.
areas; all other toads were collected
(Q E2/testosterone
on transformed
data. Statistical analyses were conducted
intervals around the mean of the untransformed
Error bars are 95% confidence

Environmental

Health

Perspectives

volume

1161 number 111 November

2008

(B) Testosterone
from agricultural
values.

NAg
Male
con
areas.

1529

HB

McCoy et al.
increase

abnormalities
gonadal
tural intensity.
Gonadal

with

has

agricul
such

abnormalities,
as those
are
to reduce the
likely
reported here,
success
of affected
individuals
reproductive

and could help explain why

studies

have

populations
or have
ing

documented

vitellogenin(s)

we

phologic
sentinel

documented

advertising
endocrine
distributed

that an

the

We

disruption.
and therefore

to
provide
large scale
about
the distribution

associated

with

various

can

widely
at
studied

of

land

endocrine
lated via

use

genin(s)
only known

the Bidder's
to occur

after

organ

in males

and

pad

number

are

areas,

again
is compro

are maintained

characteristics

via

are

and

typically modu
in the
produced
gonads
Female
colora
1999).

signaling
sex steroids

color pattern
for estrogenic

is estro
{Hyperolius argus)
of
induction
female
early

has been

as a biomarker

proposed

activity

typical

B. marinus

of female

is due

to dense

that are modulated

concentrations

melanin

at

mammals,

in part,

least

in

estrogens

by

(McLeod et al. 1994; Shah andMaibach 2001;

Slominski et al. 2004; Thorton
2002).
we

that color pattern

hypothesized
as a biomarker
function
of endoge
nous
concentration.
However,
E2
estrogen
were not
concentrations
different
significantly
between
intersex toads and less
highly mottled

Therefore,

differences

mottled NAgMs. Although the loss of sexually

with
seem

not

does

in B. marinus
is
patterns
increased
there
agriculture,

coloration

dimorphic
associated

to be a clear association

plasma E2 concentrations
testosterone
Plasma
tling.
in all
reduced
agricultural
was

male-typical

B. marinus.

and

between
mot

increased

were

concentrations

testes) as
sexually dimor
testos
be associated with
toads

could
than

(with

so

coloration,

phic coloration
terone
rather

in

concentrations

E2

of P450
Alternatively,
expression
in the skin and local aromatization

aromatase

to estrogens
of androgens
for body color variation.

and Hayes

(Noriega

2000). The dark and mottled skin coloration

is

which

castration,

nuptial

from

(Hayes and Menendez

tion in the reed
frog
and
gen dependent,

uses.
of agricultural
diversity
from
sites in this study
agricultural
have
deformities
that clearly
alter
gonadal
function.
Accumulation
of vitello
gonadal
within

of

that naturally
sexually dimor
sexual traits such as skin color,
and

in toads

Sexual

including
Toads

are

the Bidder's organ

Pancak-Roessler

2002;

agricultural
that testicular
function
suggesting
in toads from these sites.
mised

informa
important
of abnormalities
forms

found

altered

are
be

also

phic secondary
forearm width,

the effects
of
studying
as visual
serve
"tags"
individual
has experienced
B. marinus

testes

sites have
the agricultural
oocytes
vitellogenic
within
their Bidder's
that
organs,
suggesting
the testes of these toads are not
functioning
to suppress Bidder's
organ oog?nesis.
normally

for
species
traits
These

EDCs.

very
tion

have

of sexually dimorphic
external mor
an ideal
traits that makes
B. marinus

within

the

accumulation

Norris 1991; Zaccanti et al. 1994).We found

that approximately 20% of the individuals at

independent

that

amphibian
are declin
to
exposed
pesticides
extinct
Davidson
and
gone
(e.g.,

Furthermore,

et al.

(Brown

that

to suppress

in males

necessary

Knapp 2007; Sparling et al. 2001).

alteration

to the conclusion

led

forearm

Furthermore,
are

number

pad
function

in

could

be

width

responsible
and

androgen

dependent
et al.
(Emerson

mating

nuptial
and
1999;

Epstein and Blackburn 1997; Lynch and

Blackburn 1995; Rastogi and Chieffi 1971;
Thomas and Licht 1993; Thomas et al. 1993;
van Wyk et al. 2003; Wetzel
and Kelley

Qt?

1983). We
in intersex
sex toads

show

these

individuals,
do not build

or

lature

that

associated

are reduced

traits

that

suggesting

inter

muscu

hypertrophied

develop
nuptial
pads
with
normal
levels of

typically

androgens
testes
(Lee and

and properly
functioning
Correales
intersex
toads have
Indeed,
2002).
more
testosterone
reduced
concentrations,
to females

similar
width

and

ers of

NAg
Female

Intersex Bidder's Ag
Ag
Female
male
Male

LW

NAg
Male

Increasing
a

WT
agriculture

HS
?

arm
Thus,
NAgMs.
are biomark
number

pad
status.

androgen

development
toads do not

to

than

nuptial

Because

nuptial
pad
and nonreproductive

is seasonal,
possess

pads, decreased
intersex animals

well-developed

nuptial

nuptial
in

pad development
that they are not

suggests
as
active

as

Moreover,
NAgMs.
are under
width
pads and forearm
selection
(Lee and Correales
2002).
in this study
the abnormalities
found
Thus,
also likely influence
the nature
and action
of
reproductively

3.0

nuptial
sexual

1.5

sexual

selection.

The

occurrence
arise

could

through
of
masculinization
Intersex Bidder's Ag
Ag
NAg
Female Female
male
Male

Intersex

Bidder's
male

Ag
Male

sexual traits are altered at agricultural sites. {A) Photographs

of
Figure 4. Sexually dimorphic secondary
female
the number of color
(F), intersex (I), and male (M) toads. We measured
mottling
by counting
that occurred along the yellow transect
line, and the portion of the forelimb that was measured
changes
can have nuptial pads on the first three digits, whereas
and compared
is shown as a black line. Males
females have none. {B) Mean mottling score across each individual's body separated
by sex group. (0
Mean mottling score for females and males across sites; agricultural
intensity increases along the x-axis
from left to right. (/?) Mean body-size-corrected
forelimb width for each sex group. (?) Mean number of
from nonagricultural
col
areas; all other toads were
nuptial pads by sex groups. NAg, toads collected
lected from agricultural

sites.

Error bars

indicate 95% confidence

intervals.

1530

volume

masculinized,

intersex

of

individuals

feminization

If females

females.

we would

have

found

traits
wider
this.

female
relatively
complete
tracts and some
combination
(e.g., nuptial
small
arms,
Instead,

normal-sized

intersex
testes,

pads,
testes).

individuals
but

reproduc
of male

decreased
We

varied

were

individu

als with
tive

or

of males

did

mottling,
not find

typically
in ovary

had
size

no
In addition,
of oog?nesis.
stage
females
and oviducts,
but no testes)
(ovaries
or solid coloration.
had nuptial
Females
pads
and

1161 number 111 November

2008

Environmental

Health

Perspectives

Gonadal

not

did

across sites, but

pattern
col
mottled
(feminized
areas, and we did not
agricultural
in mot
typical sexual dimorphism
most
site?most
toads
agriculture
male
4C). Therefore,
(Figure
are feminized
and demasculinized

differ
were

males
oration)

in

observe

the

at the

tling
were mottled
B. marinus
at

in color

more

sites.

agricultural
of the
Many

associ
abnormalities
gonadal
in this study are organi
agriculture
the toads'
and remain
throughout

ated with
zational
adult

lives.

Intersex

tinct

ovarian

and

not

reproductive
altered. This
sue
or

after

of

suggesting

that

is fundamentally
to other
that
studies

development
is similar

both ovarian
reported
from
in frogs obtained

have

of dis
that were

tissues

similar,

bilaterally

always

consisted

gonads
testicular

tis

testicular

and

areas
agricultural
to atrazine
tadpoles

exposure
et al. 2003).
These
(Hayes
to
not exclusive
agricultural

are

observations
areas

in other

studies (Murphy et al. 2006), but differences

in study
sites are defined)
how
(e.g.,
design
in the responsiveness
and potential
differences
of subpopulations
for some of
could account
these

An

differences.
concerning
abnormalities

such

environmental
not

question
at which

important
the frequencies
occur
under

remains

conditions.
address

explicitly
examine
populations
we
However,
impact.

Our

this because
with

"normal"
study does
we did not

little

found

the

and fluctua

are
impor
effects
phenotypic
et al. 2006; Guillette
et al. 1995;
(Edwards
a
et al. 2006). Therefore,
Milnes
single mecha
nism of toxicity,
in controlled
identified
labo
tant factors

that determine

to be
is unlikely
alone
ratory settings,
acting
under natural conditions.
Thus, we should not
to
mecha
expect
identify
chemical-specific
nisms of toxicity
in wild
animals. A
occurring
used at our
agricultural
and have well
disruption
studied mechanisms
of toxicity.
Glyphosate
are used at CP and
atrazine
and
(Roundup)

few

the pesticides
endocrine

of

sites cause

BG

and

are known

to

disrupt
Soso

steroidogenesis
et al. 2007).
acute
Glyphosate
disrupts
steroidogenic
regula
across diverse
tory (StAR) protein
expression
taxonomic
the initial
groups, which modulates
et al.

(Oliveira

step

in the
and

2000)
mone

2007;

(Walsh
steroidogenic
pathway
leads to reductions
in steroid
both

et al.
hor

testosterone

production,
including
across several ver
and E2. Research
conducted
tebrate classes has shown that atrazine exposure

can

inhibit

estrogens
(Crain

androgen

production
aromatase

by increasing
et al. 1997; Fan

Environmental

Health

and

increase

transcription
et al. 2007;
Sanderson

Perspectives

have
we

not measured

volume

As
in

expect,
toads are not

might
feral

our

vary across sex groups.

concentrations
hormone

the patterns

consistent
with
entirely
in the
of toxicity
documented
of these well-studied
for either

laboratory
Altered
phenotypes,
including
compounds.
in this study are a
hormone
concentrations,
to several chemi
result of multiple
exposures
over the lifetime
concentrations
cals at various
of the toads.
We

found

in the

anuran

altered

asking
with

and function

B.

amphibian
land

by

dependent
the focus

form

that gonadal

use

in a dose

agricultural
our
fashion. Therefore,
study
literature debate
of the current

if

abnormalities

are

marinus

shifts
from

are associated

gonadal
to a new
line of questions
agriculture
focused on identifying
affected
species, chemi
cal causes,
and developmental,
physiological,
and

ecological

implications

of

exposures.

Pesticides are distributed globally and have

been explicitly linked to amphibian population
declines (Davidson and Knapp 2007; Sparling
et al. 2001).
It is likely
that contaminant
induced
abnormalities
contribute
reproductive
to such declines.

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with
absent
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activity
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zero
that
agriculture,
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intersexes
do not occur at
in
high frequencies
areas unaffected
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The
timing, amount, mixture,
tion of contaminant
concentrations

et al. 2002). We
is
that testosterone
found
sex
not.
is
It
but
modulated
E2
groups
among
is still possible
that other
that we
estrogens

human

that

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