COMPARATIVE
EVALUATION
OF
FATHEAD
MINNOW
ASSAYS
FOR
DETECTING
ENDOCRINE­
DISRUPTING
CHEMICALS
COMPARATIVE
EVALUATION
OF
FATHEAD
COMPARATIVE
EVALUATION
OF
FATHEAD
MINNOW
ASSAYS
FOR
DETECTING
MINNOW
ASSAYS
FOR
DETECTING
ENDOCRINE
ENDOCRINE­
DISRUPTING
CHEMICALS
DISRUPTING
CHEMICALS
Endocrine
Disruptor
Methods
Validation
Subcommittee
August
2003
Presented
by:

Dr.
Irv
Schultz
and
Michael
L.
Blanton
2
COMPARATIVE
EVALUATION
OF
FATHEAD
MINNOW
ASSAYS
FOR
DETECTING
ENDOCRINE­
DISRUPTING
CHEMICALS
COMPARATIVE
EVALUATION
OF
FATHEAD
COMPARATIVE
EVALUATION
OF
FATHEAD
MINNOW
ASSAYS
FOR
DETECTING
MINNOW
ASSAYS
FOR
DETECTING
ENDOCRINE
ENDOCRINE­
DISRUPTING
CHEMICALS
DISRUPTING
CHEMICALS
WORK
PERFORMED
BY
On
behalf
of
the
United
States
Environmental
Protection
Agency
EPA
CONTRACT
NUMBER
68­
W­
01­
023
3
Background
Background
Background
EDSTAC
(
1998)
recommends
inclusion
of
fish
(
fathead
minnow)
recrudescence/
reproduction
assay
as
one
of
five
Tier
1
screening
tests
for
EDCs
that
could
affect
the
HPG
axis
Evaluations
conducted
by
EPA
scientists
at
Duluth
Mid­

Continent
Ecology
Division
(
MED;
1998­
1999)
suggest
that
recrudescence
assay
would
be
problematic
for
routine
testing
due
to
logistic
constraints
Alternative
short­
term
reproduction
assay
with
fathead
minnow
described
by
Ankley
et
al.
(
2001;
Environ.
Toxicol.

Chem.
20:
1276)

Detailed
standard
guaidance
published
by
USEPA
(
2002;
A
Short­
Term
Test
Method
for
Assessing
the
Reproductive
Toxicity
of
Endocrine­
Disrupting
Chemicals
using
the
Fathead
Minnow
(
Pimephales
promelas),
EPA/
600/
R­

01/
067)
4
Background
cont.

Background
cont.

Background
cont.

Assay
system
evaluated/
validated
using
materials
reflective
of
MOA
of
concern:


Estrogens
(
methoxychlor,
Ankley
et
al.
2001;
E2,
Harries
et
al.
20001)


Androgens
(
methyltestosterone,
Ankley
et
al.
2001;

20032)


Anti­
androgens
(
vinclozolin,
Makyenen
et
al.
20003;

flutamide,
Jensen
et
al.
4)


Aromatase
inhibitor
(
fadrozole,
Ankley
et
al.
20025)


1Environ.
Sci.
Technol.
34:
3003
(
conducted
with
pairedspawners


2Environ.
Toxicol.
Chem.
22:
1350

3
Aquat.
Toxicol.
48:
461

4Aquat.
Toxicol.
Submitted

5Toxicol.
Sci.
67:
121
5
Background
cont.

Background
cont.

Background
cont.

Organization
for
Economic
Cooperation
and
Development
(
OECD)
forms
EDTA
(
Endocrine
Disruptor
Testing
Advisory)
oversight
group
with
goal
of
international
harmonization
of
EDC
testing
(
1998)
Validation
Management­
Ecology
(
VMG­
eco)

assembled
as
working
group
under
EDTA
(
2001)

to
focus
on
EDC
testing
with
non­
mammalian
vertebrates
(
and
invertebrates)

Fish
Drafting
Group
(
FDG)
one
of
several
formed
to
provide
technical
input
to
VMG­
eco
(
2001)

International
ring
testing
of
draft
fish
protocols
initiated
(
2003)
6
Background
cont.

Background
cont.

Background
cont.

Draft
protocols
provided
for
consideration
to
FDG
for
fish
EDC
testing:


­
Short­
term
(
14
d)
juvenile
vitellogenin
induction
assay
(
fathead
minnow)*


­
Short­
term
(
21­
d)
reproduction
assays
with
fathead
minnow
and
medaka
as
described
by
Ankley
et
al.

(
2001)


­
Zebrafish
(
40­
60
d)
early
developmental
assay

­
Non­
spawning
(
21­
d**)
assay
with
fathead
minnow
and
medaka
conducted
under
conditions
similar
to
spawning
assay

*
Emphasis
decreased
due
to
limitations
in
MOA
detected
(
estrogens)


*
*
Initially
proposed
for
14­
d
as
opposed
to
21­
d
7
Purpose
of
this
Study
Purpose
of
this
Study
Purpose
of
this
Study
Evaluate
short­
term
screening
assays
designed
to
detect
substances
that
interfere
with
the
estrogen
and
androgen
systems
of
fish

evaluate
the
transferability
and
sensitivity
of
short­
term
reproduction
assays
with
the
fathead
minnow
to
identify
specific
modes
of
action
of
endocrine
disruptors
using
four
model
compounds

conduct
a
side­
by­
side
comparison
of
the
21­
day
fathead
minnow
short­
term
reproduction
assay
(
EPA
2001)
with
two
separate
14­
day
assays:
one
a
shortened
version
of
the
21­

day
assay
with
less
intensive
monitoring
of
reproductive
performance
and
the
other
an
assay
using
non­
spawning
fathead
minnows
(
OECD
Draft
31
December
2001)
8
Model
Chemicals
used
and
Modes
of
Action
Model
Chemicals
used
and
Model
Chemicals
used
and
Modes
of
Action
Modes
of
Action
Methoxychlor
is
considered
a
weak
estrogen
that
is
biotransformed
into
an
estrogen
like
metabolite.

Trenbolone
is
an
anabolic
steroid
that
mimics
11­
KT
and
testosterone.
This
is
expected
to
cause
masculinization
of
females
and
perhaps
enhance
growth.

Flutamide
is
an
established
mammalian
anti­
androgen.
If
this
effect
occurs
in
fish,
then
the
normal
effectiveness
of
testosterone
and
11­
KT
would
be
reduced.

Fadrozole
inhibits
aromatase,
which
is
the
key
step
in
E2
synthesis.
Methods
Methods
Methods
10
Experimental
Test
Concentrations
and
Chemical
Analyses
Experimental
Test
Concentrations
and
Experimental
Test
Concentrations
and
Chemical
Analyses
Chemical
Analyses
Methoxychlor:
analyzed
by
gas
chromatograph
with
an
electron
capture
detector
(
GC­
ECD)

Trenbolone
:
control
and
low
samples
analyzed
by
GC
with
mass
selective
detection
(
MSD);
mid­
and
high
samples
analyzed
by
highperformance
liquid
chromatograph
(
HPLC)
analysis
with
a
fluorescence
detector
Flutamide
:
analyzed
by
high
performance
liquid
chromatography
(
HPLC)
with
a
UV/
VIS
detector
at
the
220­
nm
wavelength
Fadrozole
:
analyzed
by
HPLC
with
a
UV/
VIS
detector
Methoxychlor
&
flutamide
prepared
using
a
saturator
column
(
Kahl
et
al.

1999)
5.0
2.5
1.0
Methoxychlor
50
25
5
Fadrozole
650
350
6.0
Flutamide
1.0
0.5
0.1
Trenbolone
High
Mid*

Low
Chemical
(
ug/
L)
11
Proportional
Diluter
Proportional
Diluter
Proportional
Diluter
Continuous
flow
proportional
diluter
used
for
chemical
delivery
Adjusted
to
deliver
three
concentrations
(
including
control)
with
four
replicates
per
concentration
for
the
EPA
assays.

Second
diluter
modified
for
the
nonspawning
assay
to
deliver
four
concentrations
(
including
control)
with
two
replicates
per
concentration.

Chemical
stock
solution
metered
into
the
mixing
cell
of
the
diluter
using
a
fluid
metering
pump.
Diluter
set
to
add
chemical­
laden
water
to
the
test
chamber
every
12
min,
equal
to
six
volume
exchanges
of
water
per
day.
12
Animal
Husbandry
Animal
Husbandry
Animal
Husbandry
4­
month­
old
P.
promelas
were
obtained
from
EC&
T
for
use
in
the
methoxychlor
experiments
Based
on
methoxychlor
results
it
was
decided
that
younger
P.

promelas
should
be
purchased
and
a
in
house
culture
established
at
Battelle
30­
to
60­
day
old
P.
promelas
were
obtained
from
ABC
Laboratories
for
use
in
the
trenbolone,
flutamide,

and
fadrozole
experiments
 
Water
conditions
were
maintained
at
24
°
C
to
26
°
C.
A
flow­
through
system
provided
adequate
volume
replacement
while
maintaining
required
constant
temperature.
Gentle
aeration
was
provided
to
the
tanks.
P.
promelas
housed
in
55
 
gal
tanks
13
Summary
of
the
14­
day
Nonspawning
Assay
Summary
of
the
14
Summary
of
the
14­
day
Nonspawning
Assay
day
Nonspawning
Assay
Protocol
:
OECD
draft
proposal
 
31
December
2001
(
OECD
2001)


Healthy,
sexually
dimorphic
nonspawning
adults
(
males
and
females
contained
in
separate
chambers

Two
replicate
tanks
(
one
for
each
gender)

per
treatment
with
four
treatments:
a
dilution
water
control,
and
low,
medium,

and
high
concentrations

Gross
morphological
conditions,
GSI
determinations
and
histological
analyses
were
performed

Plasma
samples
analyzed
for
sex
steroids
and
VTG.

Excising
gonads
14
Summary
of
the
14­
day
EPA
Assay
Summary
of
the
14
Summary
of
the
14­
day
EPA
Assay
day
EPA
Assay
Pre­
exposure
(
7days)
four
females/
two
males
No
quantitative
measures
of
fecundity
Larval
hatching
conducted
once
during
preexposure
and
once
during
chemical
exposure
Successful
breeding
pairs
transferred
to
the
chemical
exposure
Four
replicate
containers,
a
dilution­
water
control,
and
a
low
and
high
concentration
Behavior,
fecundity
and
routine
water
chemistry
measurements
assessed
Gross
morphological
conditions,
GSI
and
histological
analyses
were
performed
Plasma
samples
analyzed
for
sex
steroids
and
VTG.
Terracotta
tiles
and
screen
used
for
egg
collection
Eggs
on
tile
15
Summary
of
21­
day
EPA
Assay
Summary
of
21
Summary
of
21­
day
EPA
Assay
day
EPA
Assay
21­
day
assay
paralleled
14­
day
assay
with
some
exceptions:

Larval
hatching
once
during
preexposure
phase
and
three
times
during
chemical
exposure
(
Days
7,
14
and
21)

Pre­
exposure
conducted
for
14­
days;

quantitative
counts
of
fecundity
Successful
breeding
pairs
used
for
chemical
exposure
Test
conducted
using
same
stock
solution,
proportional
diluter,
and
water
table
as
the
14­
day
EPA
assay
a)
View
of
pre­
exposure
tanks
b)
fecundity
counts
 
Appearance,
behavior,
and
fecundity
assessed
daily
 
Gross
morphological
conditions,
GSI
and
histological
analyses
performed
 
Plasma
analyzed
for
sex
steroids
and
VTG.
Histology
Histology
Histology
17
Female
Histology
Methods
Female
Histology
Methods
Female
Histology
Methods
General
Ovarian
Staging

General
development
scored
from
1
to
5
(
18
sections)

Quantitative
Staging

Oogonia
and
oocytes
were
typed;
100
cells
from
each
of
three
sections
were
rated
according
to
developmental
stage
Atretic
Follicles

Proportion
of
atretic
follicles/
100
cells
was
determined
Corpora
Lutea

Proportion
of
corpora
lutea/
100
cells
was
determined
18
Male
Histology
Methods
Male
Histology
Methods
Male
Histology
Methods
General
Testes
Staging

General
development
scored
from
1
to
5
(
12
sections)

Quantitative
Testes
Staging

spermatic
cells
were
typed;
100
cells
were
from
each
of
the
three
slides
were
rated
according
to
developmental
stage.

Diameter
of
the
seminiferous
tubule
was
measured
Other
changes
were
noted,
including
changes
to
the
interstitial
tissues

proliferation
of
Sertoli
or
Leydig
cells

premature
shedding
of
spermatocytes

presence
of
any
ovatestes
or
patterns
of
testicular
atrophy

foci
of
necrotic
spermatocytes
Methoxychor
Methoxychor
20
Methoxychlor
Cumulative
Fecundity
Methoxychlor
Cumulative
Fecundity
Methoxychlor
Cumulative
Fecundity
EPA
21­
Day
Assay
0
5,000
10,000
15,000
20,000
25,000
­
15
­
10
­
5
0
5
10
15
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
21
Methoxychlor
Cumulative
Fecundity
Methoxychlor
Cumulative
Fecundity
Methoxychlor
Cumulative
Fecundity
EPA14
Day
Assay
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
22
Methoxychlor
Male
Vitellogenin
(
mg/
mL)

Methoxychlor
Male
Vitellogenin
Methoxychlor
Male
Vitellogenin
(
mg/
mL)

(
mg/
mL)
NS
NS
NS
Significant
?
0.22
0.16
9
 
10.89
1
0.65
0.56
4
High
0.001
0.001
10
 
 
 
 
 
 
Medium
0.003
0.002
10
0.001
0.001
7
<
0.001
<
0.001
7
Low
0.001
<
0.001
10
<
0.001
<
0.001
7
<
0.001
<
0.001
7
Control
SD
Mean
n
SD
Mean
n
SD
Mean
n
Non­
Spawning
Adult
21­
Day
14­
Day
23
Methoxychlor
Steroid
Results
Methoxychlor
Steroid
Results
Methoxychlor
Steroid
Results
MED
Data:
5
µ
g/
L
 
E2
in
Females;
T/
11KT
in
males.

EPA
21­
Day
assay:
no
dose­
related
effects.

EPA
14­
Day
assay:
no
dose­
related
effects;
low
dose
reduced
E2
in
females
and
T
in
males.

Non­
spawning
Adult
assay:
no
dose­
related
effects;

mid
(
2.0
µ
g/
L)
dose
increased
T
in
females.

High
variability
within
treatments.
24
Methoxychlor
Histology
Results
Methoxychlor
Histology
Results
Methoxychlor
Histology
Results
EPA
21­
Day
assay:
no
noticeable
effects
on
gonad
histology.

EPA
14­
Day
assay:
no
noticeable
effects
on
gonad
histology.

Non­
spawning
Adult:
Low
(
0.8
µ
g/
L)
and
mid
(
2.0
µ
g/
L)
concentrations
had
noticeable
effects
on
male
gonad
histology.
25
Effects
of
Methoxychlor
on
Fathead
Effects
of
Methoxychlor
on
Fathead
Minnow
Reproduction
EPA
Minnow
Reproduction
EPA
 
MED
MED
­
15
­
10
­
5
0
5
10
15
20
25
Time
(
d)

0
1000
2000
3000
4000
5000
6000
7000
8000
Eggs
Spawned
Cumulative
Number
of
Methoxychlor
(
µ
g/
L)

Control
0.5
5
*
26
Methoxychlor
Effects
on
Male
VTG
EPA­
MED
Methoxychlor
(
µ
g/
L)

Control
0.5
5
.0000
.0025
.0050
4
5
6
Vit
ellogenin
(

mg/

ml)
*
Trenbolone
Trenbolone
28
Trenbolone
Cumulative
Fecundity
Trenbolone
Cumulative
Fecundity
Trenbolone
Cumulative
Fecundity
EPA21­
Day
Assay
0
5,000
10,000
15,000
20,000
25,000
30,000
­
14
­
12
­
10
­
8
­
6
­
4
­
2
1
3
5
7
9
11
13
15
17
19
21
Day
Cumulative
Number
of
Eggs
Control
Low
High
29
Trenbolone
Cumulative
Fecundity
Trenbolone
Cumulative
Fecundity
Trenbolone
Cumulative
Fecundity
EPA
14­
Day
Assay
0
2,000
4,000
6,000
8,000
10,000
12,000
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
30
Trenbolone
Female
Vitellogenin
(
mg/
mL)

Trenbolone
Female
Vitellogenin
Trenbolone
Female
Vitellogenin
(
mg/
mL)

(
mg/
mL)
Control
>
High;
Medium
Low
>
Medium
*

Control
>
High
Low
>
High
Control
>
High
Low
>
High
Significant
?
0.15
0.11
9
0.27
0.11
15
0.08
0.04
16
High
0.24
0.13
9
 
 
 
 
 
 
Medium
1.12
0.91
10
1.09
1.22
14
1.80
2.24
14
Low
1.02
2.81
8
1.16
1.29
13
0.59
2.61
16
Control
SD
Mean
n
SD
Mean
n
SD
Mean
n
Non­
Spawning
Adult
21­
Day
14­
Day
31
Trenbolone
Steroid
Results
Trenbolone
Steroid
Results
Trenbolone
Steroid
Results
MED
Data:
0.5
µ
g/
L
 
E2,
T
in
females
EPA
21­
Day
assay:
low
dose
(
0.041
µ
g/
L)
reduced
T
in
females;
high
dose
(
0.60
µ
g/
L)
reduced
E2
in
females
versus
low
dose.

EPA
14­
Day
assay:
high
dose
(
0.78
µ
g/
L)
reduced
E2
and
T
in
females.

Non­
spawning
Adult
assay:
low
dose
(
0.071
µ
g/
L)

reduced
E2,
T,
and
KT
in
males,
perhaps
reduced
T
in
females;
mid
dose
(
0.45
µ
g/
L)
reduced
E2
in
females
and
T
in
males
and
perhaps
females;
high
dose
(
0.86
µ
g/
L)
reduced
E2
and
T
in
females.

High
variability
within
treatments.
32
Trenbolone
Histology
Results
Trenbolone
Histology
Results
Trenbolone
Histology
Results
EPA
21­
Day
assay:
High
dose
(
0.60
µ
g/
L)
reduced
the
proportion
of
corpora
lutea
and
increased
the
proportion
of
atretic
follicles
in
the
ovaries.

EPA
14­
Day
assay:
High
dose
(
0.78
µ
g/
L)
reduced
the
proportion
of
atretic
follicles
in
the
ovaries
compared
to
the
control
and
low
dose;
progression
to
late
vitellogenic
stage
inhibited.
Testes
in
males
from
the
high
dose
showed
a
later
general
developmental
stage
than
those
from
the
control
and
low
dose.

Non­
spawning
Adult:
Low
(
0.07
µ
g/
L),
mid
(
0.45
µ
g/
L),
high
(
0.86
µ
g/
L)
doses
less
advanced
ovarian
staging;
low
reduced
atretic
follicles.
33
Effects
of
Trenbolone
on
Fathead
Minnow
Effects
of
Trenbolone
on
Fathead
Minnow
Reproduction
EPA
Reproduction
EPA­
MED
MED
0
2
4
6
8
10
12
14
16
18
20
Exposure
(
d)

0
800
1600
2400
3200
4000
Cumulative
Number
of
Eggs
Spawned
Control
0.005
0.05
0.5
5.0
50
Trenbolone
(
µ
g/
L)
*

***
34
Control
0.005
0.05
0.5
5.0
50
0
5
10
15
20
25
Tubercle
Score
*
*
*
*

Trenbolone
(
µ
g/
l)

Effects
of
Trenbolone
on
Female
Tubercles
EPA­
MED
35
Control
0.005
0.05
0.5
5.0
50
0
6
12
18
24
30
Vitellogenin
(

mg/

ml)
a
a
b
c
c
b,
c
Trenbolone
(
µ
g/
l)

*
*
*
*

Trenbolone
Effects
on
Females
EPA­
MED
36
Masculinization
by
Trenbolone
EPA­
MED
Control
male
and
female
Female
0.05
µ
g/
L
Flutamide
Flutamide
Flutamide
38
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
EPA
21­
Day
Assay
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
­
14
­
12
­
10
­
8
­
6
­
4
­
2
1
3
5
7
9
11
13
15
17
19
21
Day
Cumulative
Number
of
Eggs
Control
Low
High
39
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
EPA
14­
Day
Assay
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
18,000
20,000
0
2
4
6
8
10
12
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
40
Flutamide
Steroid
Results
Flutamide
Steroid
Results
Flutamide
Steroid
Results
MED
Data:
T
 
in
Females.

EPA
21­
Day
assay:
high
dose
(
510
µ
g/
L)
reduced
E2
and
increased
T
and
KT
in
males;
increased
T
in
females.

EPA
14­
Day
assay:
high
dose
(
519
µ
g/
L)
reduced
E2
in
males
versus
low
dose
(
46
µ
g/
L)
.

Non­
spawning
Adult
assay:
no
dose­
related
effects.

High
variability
within
treatments.
41
Flutamide
Histology
Results
Flutamide
Histology
Results
Flutamide
Histology
Results
EPA
21­
Day
assay:
high
dose
(
510
µ
g/
L)
increased
the
proportion
of
atretic
follicles
in
the
ovaries
compared
to
the
control
and
low
dose.

EPA
14­
Day
assay:
high
dose
(
519
µ
g/
L)
increased
the
proportion
of
atretic
follicles
and
decreased
the
proportion
of
corpora
lutea
in
the
ovaries.
Males
from
high
dose
showed
more
advanced
testicular
staging.

Non­
spawning
Adult:
no
noticeable
dose­
related
effects
on
histology;
several
males
in
all
three
doses
had
abnormal
testes
histology.
42
Summary
of
EPA
Flutamide
Results
Summary
of
EPA
Flutamide
Results
Summary
of
EPA
Flutamide
Results
Dose
of
650
µ
g/
L
slightly
increased
VTG
in
males
and
females,
and
slightly
increased
plasma
T
in
females.

Dose
of
650
µ
g/
L
increased
early­
stage
follicles
and
atretic
oocytes
relative
to
controls.

Dose
of
650
µ
g/
L
caused
abnormal
gonadal
histology
in
males,
with
increased
incidence
of
pychnotic
and
degenerating
spermatocytes
among
healthy
cysts.
43
Summary
of
Battelle
Flutamide
Results
Summary
of
Battelle
Flutamide
Results
Summary
of
Battelle
Flutamide
Results
There
was
no
apparent
effect
on
plasma
VTG
in
males
or
females
from
any
of
the
three
designs.

The
high
dose
(
510­
519
µ
g/
L)
decreased
plasma
E2
in
males,
increased
T
in
females
(
21­
Day
assay),
and
KT
in
males
(
21­
Day
assay).

The
high
dose
(
510­
519
µ
g/
L)
increased
the
proportion
of
atretic
follicles
in
the
ovaries
compared
to
the
control
and
low
dose.

Non­
spawning
assay
showed
abnormal
male
histology
that
was
not
related
to
flutamide
dose.
44
0
2
4
6
8
10
12
14
16
18
20
22
Exposure
(
d)

0
1000
2000
3000
4000
5000
6000
Cumulative
Number
of
Eggs
Spawned
Control
62.8
649
Flutamide
(
µ
g/
L)

Effects
of
Flutamide
on
Fathead
Minnow
Reproduction
EPA­
MED
*
45
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
Flutamide
Cumulative
Fecundity
EPA
14­
Day
Assay
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
18,000
20,000
0
2
4
6
8
10
12
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
Fadrozole
Fadrozole
Fadrozole
47
Fadrozole
Cumulative
Fecundity
Fadrozole
Cumulative
Fecundity
Fadrozole
Cumulative
Fecundity
EPA
21­
Day
Assay
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
­
14
­
12
­
10
­
8
­
6
­
4
­
2
0
2
4
6
8
10
12
14
16
18
20
Day
Cumulative
Number
of
Eggs
Control
Low
High
48
Fadrozole
Cumulative
Fecundity
Fadrozole
Fadrozole
Cumulative
Fecundity
Cumulative
Fecundity
EPA
14­
Day
Assay
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
18,000
20,000
0
2
4
6
8
10
12
14
Day
Cumulative
Number
of
Eggs
Control
Low
High
49
Fadrozole
Female
Estradiol
(
pg/
mL)

Fadrozole
Female
Estradiol
Fadrozole
Female
Estradiol
(
pg/
mL)

(
pg/
mL)
Control
>
High
*

Control
>
Low
Control
>
High
Low
>
High
Significant
?
129
142
10
 
 
0
86
39
16
High
457
320
9
 
 
 
 
 
 
Medium
524
727
10
756
1,343
7
468
1,088
16
Low
1,132
1,519
10
1,492
2,861
16
1,176
2,064
15
Control
SD
Mean
n
SD
Mean
n
SD
Mean
n
Non­
Spawning
Adult
21­
Day
14­
Day
50
Fadrozole
Female
Vitellogenin
(
mg/
mL)

Fadrozole
Female
Vitellogenin
Fadrozole
Female
Vitellogenin
(
mg/
mL)

(
mg/
mL)
Control
>
High;
Medium
Low
>
High;
Medium
Control
>
High;
Low
Low
>
High
Control
>
High;
Low
Low
>
High
Significant
?
0.02
0.01
9
0.011
0.005
16
0.002
0.002
16
High
0.03
0.02
10
 
 
 
 
 
 
Medium
0.45
0.79
10
0.75
1.20
16
0.70
1.15
16
Low
2.62
4.93
10
2.25
5.95
16
1.06
5.36
16
Control
SD
Mean
n
SD
Mean
n
SD
Mean
n
Non­
Spawning
Adult
21­
Day
14­
Day
51
Fadrozole
Steroid
Results
Fadrozole
Steroid
Results
Fadrozole
Steroid
Results
MED
Data:
 
E2
in
Females;
T,
11KT
 
in
Males
at
50
µ
g/
L.

EPA
21­
Day
assay:
low
(
5.2
µ
g/
L)
and
high
(
55.7
µ
g/
L)

doses
increased
T
in
females
and
males
and
increased
KT
in
males.
High
dose
increased
KT
in
females.
Low
dose
decreased
E2
in
females.

EPA
14­
Day
assay:
low
(
5.5
µ
g/
L)
and
high
(
59.9
µ
g/
L)

doses
increased
T
in
females
and
perhaps
males
and
increased
KT
in
males
and
perhaps
females.
High
dose
decreased
E2
in
females.

Non­
spawning
Adult
assay:
High
dose
(
57.0
µ
g/
L)
perhaps
increased
KT
and
T
in
males,
increased
KT
and
reduced
E2
in
females.

High
variability
within
treatments.
52
Fadrozole
Histology
Results
Fadrozole
Histology
Results
Fadrozole
Histology
Results
EPA
21­
Day
assay:
high
(
55.7
µ
g/
L)
dose
females
showed
less
advanced
staging;
low
(
5.2
µ
g/
L)
and
high
doses
had
increased
proportions
of
atretic
follicles
in
the
ovaries;
high
decreased
proportion
of
corpora
lutea.
Males
from
high
dose
had
increased
tubule
diameter.
Some
males
had
abnormal
testes
histology.

EPA
14­
Day
assay:
low
(
5.5
µ
g/
L)
and
high
(
59.9
µ
g/
L)

doses
showed
less
advanced
staging
and
decreased
corpora
lutea;
high
increased
proportion
of
atretic
follicles
in
the
ovaries.

Non­
spawning
Adult:
mid
(
31.7
µ
g/
L)
and
high
(
57.0
µ
g/
L)

doses
showed
less
advanced
quantitative
staging;
high
increased
proportion
of
atretic
follicles.
Several
males
in
all
three
doses
had
abnormal
testes
histology.
53
Fadrozole
Effects
on
Fathead
Minnow
Reproduction
EPA­
MED
­
20
­
18
­
16
­
14
­
12
­
10
­
8
­
6
­
4
­
2
0
2
4
6
8
10
12
14
16
18
20
Exposure
(
d)

0
2
4
6
8
10
(

Thousands)

Cumulative
Number
of
Eggs
Spawned
Control
2
10
50
Fadrozole
(
ug/
L)
*

**
54
8
0
2
4
6
E2
(

ng/

ml)
*
*

0
10
20
Vtg
(

mg/

ml)
*
*
*

Control
2
10
50
Fadrozole
effects
on
female
Fathead
Minnow
EPA­
MED
Fadrozole
(
ug/
l)
Spawning
Tile
and
Egg
Dish
Comparison
Spawning
Tile
and
Spawning
Tile
and
Egg
Dish
Comparison
Egg
Dish
Comparison
56
Proportional
Difference
of
Eggs
on
Tiles
Versus
Eggs
on
Dishes
Proportional
Difference
of
Eggs
on
Tiles
Versus
Proportional
Difference
of
Eggs
on
Tiles
Versus
Eggs
on
Dishes
Eggs
on
Dishes
High
<
Control
High
<
Low
High
<
Control
NS
NS
Significant
?
9%
 
41%

14%
 
60%

21%
 
83%

6%
 
342%

CVs
0.72
0.69
0.61
0.72
High
0.87
0.91
0.88
0.87
Low
0.87
0.94
0.92
0.90
Control
EPA
21­
Day
NS
NS
NS
NS
Significant
?
20%
 
95%

9%
 
54%

15%
 
68%

23%
 
115%

CVs
0.65
0.86
0.86
0.75
High
0.86
0.91
0.93
0.92
Low
0.89
0.90
0.91
0.86
Control
EPA
14­
Day
Fadrozole
Flutamide
Trenbolone
Methoxychlor
57
Major
Conclusions
Major
Conclusions
Major
Conclusions
Good
agreement
between
EPA
and
Battelle
for
key
apical
and
diagnostic
data
generated
for
four
different
chemicals
using
21­
d
reproduction
protocol
for
fathead
minnow

Methoxychlor

Fecundity
reductions
comparable

Effects
on
male
VTG
induction
qualitatively
similar

Trenbolone

Fecundity
reductions
comparable

Changes
in
secondary
sex
characteristics
comparable

Decreases
in
female
VTG
similar

Flutamide

Fecundity
reductions
comparable

Subtle
VTG
effects
and
histological
responses
not
similar

Fadrozole

Fecundity
reductions
comparable

VTG
and
E2
reductions
in
females
similar
58
Suggested
Protocol
Suggested
Protocol
Suggested
Protocol
14­
day
spawning
protocol
shows
promise
for
use
as
a
fish
endocrine­
disruptor
screening
protocol
based
on:


Consistency
in
overall
response
to
the
21­
day
protocol

Insufficient
need
for
quantitative
pre­
exposure
fecundity
measurements

Reduced
cost
compared
with
the
21­
day
protocol

Lack
of
response
at
the
low­
exposure
level
and
frequent
negative
responses
at
the
high­
exposure
level
in
the
non­
spawning
protocol.
This
observation
suggests
a
higher
risk
of
false­
negative
results,
particularly
with
weak­
acting
endocrine
disruptors.
59
It
is
recommended
that
additional
development
of
the
abbreviated
14­
day
spawning
protocol
be
performed
to
evaluate
how
well
the
assay
performs
in
comparison
to
the
full
21­
day
protocol
with
weaker
acting
and/
or
high
log­
P
compounds
(
agonists/
antagonists
spanning
range
of
MOA
(
including
mixed
MOA),
as
well
as
"
negative"
chemicals)


A
Mutli­
Chemical
Evaluation
is
currently
underway
with
the
following
chemicals
to
help
provide
additional
information.

°
Atrazine
°
DDE
°
Bisphenol
A
°
Perchlorate
°
Dibutyl
Phthalate
°
Cadmium
Additional
Research,
Testing
and
Questions
Additional
Research,
Testing
and
Additional
Research,
Testing
and
Questions
Questions
60
Work
on
endpoint
measurement
techniques
to
standardize/
reduce
variability

Fecundity
(
capture
of
detached
eggs?)


Histopathology
(
quantification?)


Steroids
(
RIA
vs.
ELISA?)


Others?
Additional
Research,
Testing
and
Questions
cont.

Additional
Research,
Testing
and
Additional
Research,
Testing
and
Questions
cont.

Questions
cont.
