Document ID: EPA-HQ-OPPT-2003-0012-0180
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-08-29T04:00Z

CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
2
of
52
STATEMENT
OF
CONFIDENTIALITY
OR
NO
CONFIDENTIALITY
CLAIMS
(
To
be
completed
by
the
Sponsor)
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
5
of
52
PAGE
RESERVED
FOR
40
CFR
158.34
(
c)
(
1)
CERTIFICATION
(
To
be
completed
by
the
Sponsor)
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
6
of
52
CONTENTS
STATEMENT
OF
CONFIDENTIALITY
OR
NO
CONFIDENTIALITY
CLAIMS
2
STATEMENT
OF
THE
STUDY
DIRECTOR
AND
CIT
SCIENTIFIC
MANAGEMENT
3
OTHER
SCIENTIST
INVOLVED
IN
THIS
STUDY
3
STATEMENT
OF
QUALITY
ASSURANCE
UNIT
4
PAGE
RESERVED
FOR
40
CFR
158.34
(
c)
(
1)
CERTIFICATION
5
SUMMARY
8
1.
INTRODUCTION
10
2.
MATERIALS
AND
METHODS
10
2.1
TEST
ITEM
10
2.1.1
Identification
10
2.1.2
Preparation
of
the
test
solutions
10
2.2
TEST
SYSTEM
11
2.2.1
Animals
11
2.2.2
Environmental
conditions
during
culture
11
2.2.3
Environmental
conditions
during
the
test
12
2.2.4
Food
12
2.3
TREATMENT
12
2.3.1
Administration
of
the
test
item
12
2.3.2
Time
schedule
12
2.3.3
Definitions
13
2.3.4
Observations
13
2.4
CHEMICAL
ANALYSIS
13
2.5
DATA
EVALUATION
13
2.5.1
Determination
of
the
EC50
14
2.5.2
Determination
of
the
No
Observed
Effect
Concentration
(
NOEC)
14
2.6
VALIDITY
CRITERIA
14
2.7
ARCHIVING
14
2.8
STUDY
PLAN
ADHERENCE
15
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
7
of
52
3.
RESULTS
16
3.1
VALIDITY
CRITERIA
16
3.2
CHEMICAL
ANALYSIS
16
3.3
WATER
QUALITY
16
3.4
RESULTS
16
4.
CONCLUSION
18
Figure
1:
Total
number
of
living
neonates
per
parent
alive
after
21
days
versus
concentration
19
Table
1:
Physico­
chemical
parameters
over
the
21­
day
period
20
Table
2:
Number
of
immobilized
parents
21
Table
3a:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
­
Control
22
Table
3b:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
4.31
mg/
L
23
Table
3c:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
9.16
mg/
L
24
Table
3d:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
20.0
mg/
L
25
Table
3e:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
44.2
mg/
L
26
Table
3f:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
88.6
mg/
L
27
Table
4:
Living
neonates
per
parent
alive
at
the
end
of
the
test
versus
concentration
of
the
test
item
28
Table
5:
Length
of
parent
alive
at
the
end
of
the
test
29
APPENDICES
30
1.
Analytical
certificate
and
composition
of
the
test
item
31
2.
Preparation
of
M4
reconstituted
water
34
3.
Chemical
analysis
of
test
solutions
37
4.
EC50
estimation
43
5.
21­
day
NOEC
determination
based
on
reproduction
capacity
45
6.
Statistical
analysis
of
Daphnia
length
data
48
to
52
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
8
of
52
SUMMARY
At
the
request
of
APME,
Brussels,
Belgium,
the
toxicity
to
reproduction
of
the
test
item
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
was
evaluated
in
the
Cladoceran,
Daphnia
magna
STRAUS
(
clone
5)
using
a
21­
day
semi­
static
test
according
to
OECD
guideline
No.
211
(
21st
September
1998).
The
criterion
of
toxicity
measured
was
the
effect
of
the
test
item
in
solution
on
the
reproductive
efficacy.
This
is
expressed
as:

 
the
EC50,
the
concentration
required
to
reduce
the
number
of
living
neonates
per
adult
alive
at
the
end
of
the
test
to
50%
of
the
control
value,

 
the
No
Observed
Effect
Concentration
(
NOEC),
obtained
by
analysis
of
variance
from
these
data.

Methods
The
test
item
was
dissolved
in
reconstituted
water
(
M4)
prepared
from
deionized
water
with
a
conductivity
<
10
µ
S/
cm.
The
total
hardness
of
the
reconstituted
water
is
generally
between
238
and
289
mg/
L
(
as
CaCO3)
and
the
pH
is
8.0
±
0.5.

Reproduction
test
Five
concentrations
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
were
used
together
with
a
control
at
0,
6.25,
12.5,
25.0,
50.0
and
100
mg/
L
expressed
as
the
APFO
(
solids)
content
of
the
test
item.

Test
solutions
were
changed
every
2
or
3
days.

Ten
daphnids
(
ten
individual
replicates)
were
exposed
to
each
concentration
for
21
days
and
immobilization
of
adults
and
number
of
dead
and
living
neonates
were
recorded
daily
throughout
the
test.

Environmental
parameters
were:

 
pH:
7.56
to
8.26,

 
temperature:
18.4
°
C
to
20.4
°
C,

 
dissolved
oxygen:
7.8
to
9.2
mg/
L
(>
3
mg/
L
throughout
the
test),

 
water
hardness:
255
to
289
mg/
L
as
CaCO3.

Chemical
analysis
Chemical
analysis
was
undertaken
to
measure
the
concentration
of
the
test
item
in
each
test
solution,
except
for
the
control,
at
the
beginning
of
the
test,
before
and
after
the
medium
renewals
and
at
the
end
of
the
test.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
9
of
52
Results
Chemical
analysis
Some
measured
concentrations
were
not
within
±
20%
of
the
corresponding
nominal
values.
However,
the
maximum
CV
of
the
measured
concentrations
(
at
the
beginning
of
the
test
and
before
medium
renewals)
at
each
of
the
different
nominal
target
values
was
only
8.50%
(
at
6.25
mg/
L
nominal),
indicating
that
the
test
item
was
sufficiently
stable
between
medium
renewals.
All
study
results
were
therefore
based
on
harmonic
means
of
measured
concentrations
in
test
solutions
throughout
the
test,
i.
e.
21
days.
These
harmonic
means
were
calculated
as
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L
expressed
as
the
APFO
(
solids)
content
of
the
test
item,
for
nominal
values
of
6.25,
12.5,
25.0,
50.0
and
100
mg/
L,
respectively.

Mortalities
The
test
item
did
not
induce
significant
mortality
in
parent
animals
at
concentrations
tested.
At
the
end
of
the
test,
the
cumulative
mortality
in
parent
animals
was
20%
(
2/
10)
in
the
control,
10%
(
1/
10)
at
4.31
mg/
L,
0%
(
0/
10)
at
9.16
mg/
L,
0%
(
0/
10)
at
20.0
mg/
L,
10%
(
1/
10)
at
44.2
mg/
L
and
10%
(
1/
10)
at
88.6
mg/
L.

Reproduction
A
significant
inhibition
of
the
reproduction
capacity
(
expressed
as
the
number
of
living
neonates
produced
per
parent
alive
at
the
end
of
the
test)
was
obtained
at
44.2
and
88.6
mg/
L,
compared
to
the
control.

The
reproduction
capacity
values
were
as
follows:
21­
day
EC50:
39.6
mg/
L
Confidence
limits:
36.7
to
42.5
(
p
=
0.05)
No
Observed
Effect
Concentration
(
21­
day
NOEC):
20.0
mg/
L
(
p
=
0.05)

Length
A
significant
inhibition
of
the
length
of
parent
animals
(
alive
at
the
end
of
the
test)
was
obtained
at
88.6
mg/
L,
compared
to
the
control.

The
length
values
were
as
follows:
21­
day
EC50:
>
88.6
mg/
L
No
Observed
Effect
Concentration
(
21­
day
NOEC):
44.2
mg/
L
(
p
=
0.05)

Conclusion
Under
our
experimental
conditions,
the
21­
day
EC50
based
on
reproduction
capacity
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
a
semi­
static
test
system
was
39.6
mg/
L
for
Daphnia
magna.
The
NOEC
after
21
days
based
on
survival
of
the
parent
animals
was
88.6
mg/
L
(
the
maximum
concentration
tested).
The
NOEC
at
21days
based
on
reproduction
capacity
was
20.0
mg/
L.
The
NOEC
at
21
days
based
on
length
of
the
parent
animals
was
44.2
mg/
L.

These
results
are
expressed
as
the
APFO
(
solids)
content
of
the
test
item.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
10
of
52
1.
INTRODUCTION
The
objective
of
this
study
was
to
assess
the
reproductive
toxicity
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
to
Daphnia
magna
(
clone
5)
using
a
21­
day
semi­
static
test
according
to
OECD
guideline
No.
211
(
21st
September
1998).

In
this
test,
the
criterion
of
toxicity
measured
is
the
effect
of
the
test
item
in
solution
on
the
reproductive
efficacy,
expressed
as
the
EC50,
the
concentration
required
to
reduce
the
number
of
living
neonates
per
adult
alive
at
the
end
of
the
test
to
50%
of
the
control
value.
The
No
Observed
Effect
Concentration
(
NOEC)
is
derived
statistically
from
the
data
using
analysis
of
variance.

2.
MATERIALS
AND
METHODS
2.1
TEST
ITEM
2.1.1
Identification
 
supplier:
3M
 
name:
­
Study
plan:
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
­
labeling:
FLUORAD
TM
Both
names
correspond
to
the
same
test
item.

 
batch
number:
10004
 
description
at
receipt:
colorless
liquid
 
description
on
the
analytical
certificate:
3M(
TM)
Fluorad(
TM)
Fluorochemical
Emulsifier
 
containers:
three
drums
 
date
of
receipt:
28
January
2002
 
storage
conditions:
at
room
temperature
 
purity:
19.6%
(
expressed
as
solids)
in
purified
water.

Data
relating
to
the
characterisation
of
the
test
item
are
documented
in
an
analytical
certificate
and
in
the
composition
of
the
test
item
(
presented
in
appendix
1)
provided
by
the
Sponsor.

2.1.2
Preparation
of
the
test
solutions
The
stock
solutions,
for
the
test,
were
prepared
by
dissolving
the
test
item
directly
in
test
water
(
see
§
2.2.2).

The
conditions
of
preparation
of
the
stock
solutions
are
reported
in
the
following
table:

Day
of
addition
Quantity
of
test
item
(
as
received)
(
mg/
L)
Quantity
of
APFO
(
solids)
(
mg)
(
1)
Volume
of
water
(
mL)
Concentration
of
APFO
(
solids)
(
mg/
L)
(
1)
Duration
of
the
agitation
(
minutes)
0,
2,
5,
7,
9,
12,
14,
16
and
19
1020.4
200
2000
100
5
(
1)
expressed
as
APFO
(
solids)
based
on
stated
test
item
composition
of
19.6%
APFO
in
purified
water
The
agitation
was
continued
until
the
stock
solutions
were
used
to
prepare
the
test
solutions.

Test
solutions
were
prepared
by
further
dilution
of
the
stock
solution
with
test
water
to
provide
a
geometric
series
of
concentrations:
0,
6.25,
12.5,
25.0,
50.0
and
100
mg/
L.
CIT/
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22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
11
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52
Test
vessels
containing
daphnids
were
filled
directly
from
the
test
solution
containers
immediately
after
preparation.
Solutions
were
changed
every
2
or
3
days
(
every
Monday,
Wednesday
and
Friday).

The
pH
of
the
test
solutions
remained
within
acceptable
limits
(
between
6.5
and
9
and
in
the
range
±
0.5
unit
of
the
test
water)
after
preparation
and
there
was
no
adjustment
of
pH
before
addition
of
the
animals.

2.2
TEST
SYSTEM
2.2.1
Animals
Species:
Daphnia
magna
STRAUS­
clone
5.
Reason
for
this
choice:
a
species
commonly
used
in
Europe
for
aquatic
toxicity
testing
and
recommended
in
OECD
guideline.
Breeder:
CIT.
Origin:
Centre
Technique
du
Bois
et
de
l'Ameublement,
Paris,
France.
Parentage:
the
parents
of
the
daphnids
to
be
used
in
the
test
were
born
within
the
same
24­
hour
period.
They
had
produced
more
than
three
broods
and
the
first
before
day
12,
prior
to
their
use
as
breeding
stock
to
produce
neonates
for
this
test.
Life
stage:
at
the
beginning
of
the
test,
first
instar
daphnids
between
6
and
24
hours
old
and
produced
from
parthenogenically
reproducing
brood
female
population.
Acclimation:
animals
were
held
in
water
of
the
same
quality
as
the
test
water
from
birth,
until
use
in
the
test.
Allocation
of
the
animals:
animals
were
randomly
placed
first
into
one
beaker
containing
water
of
the
same
quality
and
conditions
as
the
culture
water
and
then
6
hours
later,
the
remaining
daphnids
were
randomly
assigned
to
the
test
item
solutions
using
a
pipette.
Number
of
animals:
Ten
animals
per
concentration
and
in
the
control
with
one
animal
per
vessel.

2.2.2
Environmental
conditions
during
culture
During
the
culture
period,
the
stock
aquarium
conditions
are
as
follows:
Water:
reconstituted
(
M4).
The
pH
of
this
solution
is
8.0
±
0.5;
the
sum
of
Ca
and
Mg
ions
in
the
solution
is
2.5
mmol/
L
(
molar
ratio
Ca:
Mg
is
approximately
4:
1
and
Na:
K
approximately
10:
1);
total
hardness
is
between
238
and
289
mg/
L
as
CaCO3.
Details
of
its
preparation
are
presented
in
appendix
2.
Temperature
in
water:
between
18
°
C
and
22
°
C.
Temperature
is
recorded
continuously
and
checked
generally
twice
a
week.
Light/
dark
cycle:
16/
8
hours.
Forced
aeration:
none.
Oxygen
concentrations
remain
above
80%
of
the
saturation
value
at
the
selected
temperature.
Loading:
the
animals
are
loaded
at
one
animal/
20
mL
of
culture
water.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
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52
The
medium
is
replaced
generally
each
Monday,
Wednesday
and
Friday.
On
each
of
these
occasions,
neonates
are
separated
from
the
adults
and
either
removed
or,
once
monthly,
used
to
start
a
new
culture.
During
holding,
pH,
water
hardness
and
dissolved
oxygen
concentrations
are
checked
in
the
"
new"
and
"
old"
media
when
solutions
are
replaced
generally
twice
a
week.

2.2.3
Environmental
conditions
during
the
test
The
test
conditions
were
as
follows:
Test
water:
see
§
2.2.2.
Water
was
changed
thrice
weekly
during
the
21­
day
test
period.
Temperature:
between
18
°
C
and
22
°
C
throughout
the
test.
The
temperature
was
controlled
using
a
water­
bath
into
which
the
beakers
were
placed
and
the
maximum
and
minimum
temperature
were
recorded
daily
using
a
max­
min
thermometer.
Light/
dark
cycle:
16/
8
hours.
Light
intensity:
less
than
1200
lux.
Duration
of
test:
21
days.
Test
vessels:
glass
beakers.
Loading
rate:
one
daphnid
per
vessel
containing
50
mL
of
solution.
Forced
aeration:
was
not
used
during
the
test.
Dilution
water
was
aerated
prior
to
the
addition
of
the
test
item.
Oxygen
concentrations:
were
maintained
>
3
mg/
L
and
>
60%
of
the
air
saturation
value
throughout
the
test.

Temperature,
pH,
dissolved
oxygen
concentration
and
total
hardness
were
measured
in
the
control
solution
and
the
highest
concentration
(
where
animals
were
still
alive)
at
the
beginning
and
the
end
of
the
test
as
well
as
at
least
once
a
week
before
and
after
the
solution
change.
Temperature
was
also
measured
continuously
in
a
vessel
placed
near
the
test
vessels.

2.2.4
Food
During
culture
and
the
test,
animals
were
fed
daily
(
until
day
20)
with
a
diet
of
approximately
6.106
cells
of
Chlorella
vulgaris
and
6.106
cells
of
Pseudokirchneriella
subcapitata
(
both
strains
are
cultured
at
CIT)
per
daphnid.
The
carbon
content
of
this
diet
corresponded
to
0.16
mg
of
carbon
per
daphnid
per
day
(
until
day
20).

2.3
TREATMENT
2.3.1
Administration
of
the
test
item
Ten
animals
were
used
for
each
concentration
with
five
concentrations
and
one
control.
The
nominal
concentrations
used
were
as
follows:
0,
6.25,
12.5,
25.0,
50.0
and
100
mg/
L
expressed
as
the
APFO
(
solids)
content
of
the
test
item.

2.3.2
Time
schedule
Experimental
starting
date
(
first
day
of
treatment):
6
November
2002,
Experimental
completion
date:
27
November
2002.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
13
of
52
2.3.3
Definitions
Living
progeny
(
or
living
neonates):
offspring
found
alive
in
solution
after
its
birth.
Dead
progeny
(
or
dead
neonates):
offspring
found
dead
in
solution
after
its
birth.
Aborted
egg:
egg
(
or
embryo)
found
in
solution
after
an
abortion.

The
death
is
defined
as
the
inability
of
a
daphnid
to
swim
within
15
seconds
after
gentle
agitation
of
the
test
vessel.

2.3.4
Observations
Observations
were
made
to:
1)
determine
the
number
of
immobilized
or
dead
parent
daphnids
in
each
test
solution
during
the
test
and
the
day
of
death.
Immobilization
is
defined
as
the
inability
of
a
daphnid
to
swim
within
15
seconds
after
gentle
agitation
of
the
test
vessel,
2)
count
the
number
of
neonates
(
alive
and
dead
progeny)
for
each
parent
animal
throughout
the
test
and
per
day
of
test
and
record
the
dates
of
release,
3)
count
the
number
of
aborted
eggs
and
record
the
dates
of
observation,
4)
establish
the
length
of
all
surviving
parent
daphnids
at
the
end
of
the
test
(
to
the
nearest
0.1
mm).

Observations
were
undertaken
everyday.
Neonates
(
alive
and
dead
progeny)
and
aborted
eggs
were
removed
from
the
test
vessels
everyday.

2.4
CHEMICAL
ANALYSIS
Chemical
analysis
was
undertaken
to
measure
the
concentration
of
the
test
item
in
each
test
solution,
except
for
the
control,
throughout
the
test.

Samples
of
5
mL
were
taken
from
each
test
solution
container
(
except
for
the
control)
at
the
beginning
of
the
test.
Samples
totaling
5
mL
were
also
taken
before
and
after
the
medium
renewals
and
at
the
end
of
the
test.
Samples
were
stored
at
­
20
°
C
until
analysis.

The
analytical
procedure
is
presented
in
appendix
3.

2.5
DATA
EVALUATION
The
following
measurements
and
calculations
were
performed
when
possible:

 
the
number
of
parent
animals
which
were
immobilized
or
died
during
the
test
and
day
of
death,

 
the
number
of
neonates
(
live
and
dead
progeny)
for
each
parent
animal
and
dates
of
release,

 
the
number
of
aborted
broods
and
dates
of
observation,

 
the
number
of
neonates
per
parent
throughout
the
test
and
per
day
of
test,

 
the
coefficient
of
variation
for
control
fecundity
(
based
on
the
number
of
neonates
per
parent
animal
alive
at
the
end
of
the
test
per
day
and
based
on
the
total
number
of
living
neonates
per
parent
animal
alive
at
the
end
of
the
test),

 
a
plot
of
total
number
of
living
neonates
per
parent
animal
for
each
replicate
alive
at
the
end
of
the
test
versus
concentration
of
the
test
item,

 
the
results
of
tests
of
homogeneity
of
data,

 
the
results
of
analysis
of
variance
(
Dunnett
test
or
Bonferroni
t­
test)
on
the
number
of
living
neonates
per
surviving
parent
at
the
end
of
the
test
and
the
length
of
these
surviving
parents,
used
to
calculate
the
No
Observed
Effect
Concentration
(
NOEC)
and
also
the
Lowest
Observed
Effect
Concentration
(
LOEC)
after
21
days
for
each
parameter,

 
where
applicable,
an
EC50
based
on
reproduction
capacity
(
or
mortality
or
length)
of
parent
animals
and
confidence
limits,
at
the
end
of
the
test.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
14
of
52
2.5.1
Determination
of
the
EC50
When
for
at
least
two
concentrations,
effect
(
mortality/
inhibition
of
reproduction
capacity
reduction
of
length)
is
>
0%
and
<
100%,
the
EC50
is
calculated
according
to
Probit
analysis
(
i.
e.
Finney's
method,
published
by
E.
Weber,
combined
with
Bliss's
method).
The
confidence
interval
limits
are
calculated
statistically
according
to
Fieller's
method.
When
only
one
partial
effect
is
observed,
the
EC50
is
estimated
(
using
Probit
analysis
or
an
interpolation
procedure,
depending
on
the
data
obtained)
and
the
highest
concentration
causing
no
effect
and
the
lowest
concentration
producing
100%
effect
are
used
as
confidence
limits.
If
at
all
concentrations
effect
is
0%
or
100%,
a
EC50
can
not
be
calculated.
It
is
estimated
using
the
geometric
mean
of
the
highest
concentration
causing
no
effect
and
the
lowest
concentration
producing
100%
effect.

2.5.2
Determination
of
the
No
Observed
Effect
Concentration
(
NOEC)
The
following
method
was
applied
to
determine
the
No
Observed
Effect
Concentration
after
21
days
(
21­
day
NOEC)
based
on
reproduction
capacity.
After
checking
of
the
normality
of
the
data
with
Chi­
square
and
Shapiro­
Wilks
tests
(
normal
at
p
=
0.01)
as
well
as
the
variance
homogeneity
(
Bartlett
test);
(
p
=
0.01),
the
NOEC
was
determined
by
ANOVA
(
Bonferroni
t­
test;
p
=
0.05).

Another
statistical
procedure,
described
in
appendix
5
was
used
to
determine
the
21­
day
NOEC
(
based
on
length
of
parent
animals
at
the
end
of
the
test)
since
length
data,
although
variances
were
homogeneous,
were
not
normally
distributed.

2.6
VALIDITY
CRITERIA
For
the
test
to
be
valid
the
following
criteria
should
be
fulfilled:

 
the
mortality
in
the
controls
(
parent
females)
should
not
exceed
20%
at
the
end
of
the
test,

 
the
average
cumulative
number
of
living
young
produced
per
surviving
parent
female
should
be
 
60
in
the
controls
at
the
end
of
the
test.

2.7
ARCHIVING
The
following
study
materials
are
archived
by
CIT
(
BP
563,
27005
Evreux,
France)
for
5
years
after
the
end
of
the
in
vivo
phase
of
the
study:

 
Study
plan
and
possible
amendments,

 
raw
data,

 
correspondence,

 
final
report
and
possible
amendments.

On
completion
of
this
period,
the
archived
study
materials
will
be
returned
to
the
Sponsor,
or
may
be
archived
at
CIT
for
a
further
period
(
at
additional
cost).
The
total
duration
of
archiving
(
depending
on
regulations)
will
be
the
responsibility
of
the
Sponsor.
In
addition,
raw
data
not
specific
to
the
study
including,
but
not
limited
to,
records
of
environmental
data
and
equipment
calibration,
will
also
be
archived
by
CIT
and
retained
for
at
least
30
years.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
15
of
52
2.8
STUDY
PLAN
ADHERENCE
The
study
was
performed
in
accordance
with
the
Study
plan
No.
22658
ECD
and
subsequent
amendments,
with
the
following
deviation
from
the
agreed
Study
plan:

 
temperature
in
test
solutions
was
between
18
°
C
and
22
°
C
but
varied
slightly
by
more
than
±
1
°
C
during
the
test
(­
1.3
°
C).

This
minor
deviation
was
not
considered
to
have
compromised
the
validity
or
integrity
of
the
study.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
16
of
52
3.
RESULTS
3.1
VALIDITY
CRITERIA
The
two
validity
criteria
were
fulfilled:
 
the
mortality
in
the
controls
was
 
20%
(
2/
10
parents)
at
the
end
of
the
test,
 
the
average
cumulative
number
of
living
young
produced
per
surviving
parent
was
 
60
in
the
controls
at
the
end
of
the
test.

3.2
CHEMICAL
ANALYSIS
Results
are
presented
in
table
A,
appendix
3.

Some
measured
concentrations
were
not
within
±
20%
of
the
corresponding
nominal
values.
However,
the
maximum
CV
of
the
measured
concentrations
(
at
the
beginning
of
the
test
and
before
medium
renewals)
at
each
of
the
different
nominal
target
values
was
only
8.50%
(
at
6.25
mg/
L
nominal),
indicating
that
the
test
item
was
sufficiently
stable
between
medium
renewals.
All
study
results
were
therefore
based
on
harmonic
means
of
measured
concentrations
in
test
solutions
throughout
the
test,
i.
e
21
days.
These
harmonic
means
were
calculated
as
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L
expressed
as
the
APFO
(
solids)
content
of
the
test
item,
for
nominal
values
of
6.25,
12.5,
25.0,
50.0
and
100
mg/
L,
respectively.

3.3
WATER
QUALITY
A
translucent
colorless
solution
was
obtained
at
all
test
item
concentrations.

The
minimum
and
maximum
values
of
parameters
measured
during
the
test
were:

 
pH:
7.56
and
8.26,

 
temperature:
18.4
°
C
and
20.4
°
C,

 
dissolved
oxygen:
7.8
and
9.2
mg/
L
(>
3
mg/
L
throughout
the
test),

 
water
hardness:
255
and
289
mg/
L
as
CaCO3.

Results
of
water
quality
measurements
are
included
in
table
1.

3.4
RESULTS
 
Number
of
parent
animals
which
were
immobilized
or
died
during
the
test
and
day
of
death
(
table
2).

The
test
item
did
not
induce
significant
mortality
in
parent
animals
at
concentrations
tested.
At
the
end
of
the
test,
the
cumulative
mortality
in
parent
animals
was
20%
(
2/
10)
in
the
control,
10%
(
1/
10)
at
4.31
mg/
L,
0%
(
0/
10)
at
9.16
mg/
L,
0%
(
0/
10)
at
20.0
mg/
L,
10%
(
1/
10)
at
44.2
mg/
L
and
10%
(
1/
10)
at
88.6
mg/
L.

The
EC50
value
based
on
mortality
of
parent
animals
after
21
days
was
as
follows:

 
21­
day
EC50:
>
88.6
mg/
L.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
17
of
52
 
Coefficient
of
variation
for
control
fecundity
(
tables
3a
and
4).

The
coefficient
of
variation
for
control
fecundity
was
16.4%
based
on
the
total
number
of
living
neonates
per
parent
animal
alive
at
the
end
of
the
test
(
see
table
4).
The
same
coefficient
was
obtained
using
the
total
number
of
neonates
(
alive
and
dead
progeny)
per
parent
animal
alive
at
the
end
of
the
test
per
day
(
see
table
3a)
since
no
dead
progeny
was
observed
in
the
control
group
during
the
study.

 
Numbers
of
broods,
neonates
(
alive
and
dead
progeny),
aborted
eggs
and
dates
of
observation
(
tables
3a
to
3f,
table
4).

For
parent
animals
of
the
control
and
the
groups
at
4.31
and
9.16
mg/
L,
the
first
brood
occurred
between
day
8
and
day
10.
Four
to
five
broods
were
obtained
in
the
test
solution
replicates
where
the
parent
animal
was
alive
after
21
days.
Between
9.16
and
88.6
mg/
L,
the
test
item
induced
a
dose­
related
effect
on
occurrence
of
the
first
brood
(
delay)
and
also,
on
the
number
of
broods
obtained
in
the
test
solution
replicates
where
the
parent
animal
was
alive
after
21
days
(
decrease).
The
average
number
of
brood
per
parent
animal
alive
at
the
end
of
the
test
and
per
test
solution
was
4.75,
4.67,
4.80,
4.50,
2.25
and
1.22
at
0,
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L,
respectively.
Consequently,
the
total
number
of
neonates
(
alive
and
dead
progeny)
and
aborted
eggs
produced
per
parent
animal
alive
at
the
end
of
the
test
decreased
quickly
between
9.16
and
88.6
mg/
L.

After
21
days,
the
average
number
of
neonates
(
alive
and
dead
progeny)
produced
per
parent
alive
at
the
end
of
the
test
and
per
test
solution
reached
63.3,
70.7,
64.2,
62.9,
27.3
and
0.9
at
0,
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L,
respectively.
Furthermore,
the
number
of
aborted
eggs
produced
per
parent
alive
at
the
end
of
the
test
increased
with
the
test
item
concentration
from
20.0
mg/
L,
corresponding
to
93%
of
total
progeny
at
88.6
mg/
L.
Hence,
the
production
of
neonates
was
highly
affected
by
the
test
item.
Dead
progeny
and
aborted
eggs
in
parent
animals
alive
at
the
end
of
the
test
represented
0%,
0.2%,
0.2%,
1.4%,
12.1%
and
99.2%
of
the
total
progeny
(
including
aborted
eggs)
for
test
solutions
at
0,
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L,
respectively.

 
Number
of
living
neonates
per
parent
alive
at
the
end
of
the
test
versus
concentration
of
the
test
item
(
table
4
and
figure
1).

After
21
days,
the
average
number
of
living
neonates
produced
per
parent
alive
at
the
end
of
the
test
and
per
test
solution
reached
63.3,
70.6,
64.1,
62.3,
24.0
and
0.1
at
0,
4.31,
9.16,
20.0,
44.2
and
88.6
mg/
L,
respectively.
The
level
of
reproduction
in
the
control
(
0
mg/
L)
was
within
the
range
previously
observed
under
these
conditions
in
this
laboratory.
This
average,
as
%
of
control
value,
corresponded
to
112%
at
4.31
mg/
L,
101%
at
9.16
mg/
L,
98%
at
20.0
mg/
L,
38%
at
44.2
mg/
L
and
0%
at
88.6
mg/
L.
Hence,
as
for
the
total
production
of
neonates,
that
of
living
neonates
was
highly
affected
by
the
test
item
and
this
effect
was
statistically
significant
from
44.2
mg/
L,
compared
to
the
control.

It
was
therefore
concluded
that
the
test
item,
when
tested
at
concentrations
of
44.2
and
88.6
mg/
L,
induced
adverse
effect
on
the
reproduction
capacity
of
daphnids.

The
reproduction
capacity
values,
based
on
production
of
living
neonates
per
parent
alive
at
the
end
of
the
test,
were
as
follows:
21­
day
EC50:
39.6
mg/
L
Confidence
limits:
36.7
to
42.5
(
p
=
0.05)
No
Observed
Effect
Concentration
(
21­
day
NOEC):
20.0
mg/
L
(
p
=
0.05)

The
statistical
results
for
the
determination
of
the
21­
day
EC50
and
21­
day
NOEC
based
on
reproductive
capacity
of
parent
animals
are
reported
in
appendices
4
and
5,
respectively.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
18
of
52
 
Length
of
parent
alive
at
the
end
of
the
test
(
table
5)

The
average
length
of
parent
animals
alive
at
the
end
of
the
test,
as
%
of
control
value,
corresponded
to
99%
at
4.31
mg/
L,
94%
at
9.16
mg/
L,
95%
at
20.0
mg/
L,
94%
at
44.2
mg/
L
and
85%
at
88.6
mg/
L.
A
significant
inhibition
of
the
length
of
parent
animals
(
alive
at
the
end
of
the
test)
was
obtained
at
88.6
mg/
L,
compared
to
the
control.

The
length
values
were
as
follows:
21­
day
EC50:
>
88.6
mg/
L
No
Observed
Effect
Concentration
(
21­
day
NOEC):
44.2
mg/
L
(
p
=
0.05)

The
statistical
result
for
the
determination
of
the
21­
day
NOEC
based
on
length
of
parent
animals
is
presented
in
appendix
6.

4.
CONCLUSION
Under
our
experimental
conditions,
the
21­
day
EC50
based
on
reproduction
capacity
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
a
semi­
static
test
system
was
39.6
mg/
L
for
Daphnia
magna.
The
NOEC
after
21
days
based
on
survival
of
the
parent
animals
was
88.6
mg/
L
(
the
maximum
concentration
tested).
The
NOEC
at
21days
based
on
reproduction
capacity
was
20.0
mg/
L.
The
NOEC
at
21
days
based
on
length
of
the
parent
animals
was
44.2
mg/
L.

These
results
are
expressed
as
the
APFO
(
solids)
content
of
the
test
item.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
19
of
52
Figure
1:
Total
number
of
living
neonates
per
parent
alive
after
21
days
versus
concentration

:
replicate;

:
mean
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Concentration
(
mg/
L)
Number
of
neonates
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
20
of
52
Table
1:
Physico­
chemical
parameters
over
the
21­
day
period
Control
Renewal
0
1
3
Day
Solution
new
old
new
old
new
Beaker
1
2
1
2
1
2
3
4
3
4
Oxygen
(
mg/
L)
8.9
8.8
8.1
8.1
8.4
8.3
8.0
7.9
8.1
8.1
Oxygen
(%)
94
94
93
92
96
96
94
92
95
94
pH
8.06
8.12
8.03
8.19
7.56
7.63
7.76
7.96
7.79
7.81
Total
Hardness
(
mg/
L
as
Ca
CO3)
255
255
255
272
255
255
289
272
255
255
Temperature
(
°
C)
19.9
19.9
19.8
19.7
19.7
19.7
18.5
18.6
18.7
18.7
Renewal
8
Day
Solution
old
new
old
new
old
Beaker
5
6
5
6
8
9
8
9
9
10
Oxygen
(
mg/
L)
7.9
7.8
8.3
8.4
7.9
7.8
8.1
8.1
8.0
7.9
Oxygen
(%)
93
93
94
99
91
90
94
93
94
94
pH
8.22
8.26
7.81
7.92
8.22
8.16
8.08
8.07
7.86
7.89
Total
Hardness
(
mg/
L
as
Ca
CO3)
289
272
255
272
272
272
255
255
289
272
Temperature
(
°
C)
19.4
19.2
19.0
19.0
19.3
19.2
19.1
19.3
19.8
19.8
Highest
concentration
(
where
animals
were
still
alive)

Renewal
0
1
3
Day
9
Solution
new
old
new
old
new
Concentration
(
mg/
L)
Beaker
51
52
51
52
51
52
53
54
53
54
Oxygen
(
mg/
L)
9.2
9
8.1
8.2
8.4
8.4
8.1
8.1
8.3
8.3
Oxygen
(%)
96
95
93
94
96
97
93
93
97
97
pH
8.09
8.13
8.24
8.26
7.89
7.86
8.02
8.03
7.73
7.74
Total
Hardness
(
mg/
L
as
Ca
CO3)
255
255
272
255
255
255
272
289
255
255
Temperature
(
°
C)
19.8
19.7
18.9
19.3
19.7
19.5
18.4
18.4
19.0
19.1
Renewal
8
Day
21
Solution
old
new
old
new
old
Concentration
(
mg/
L)
Beaker
55
56
55
56
57
58
57
58
59
60
Oxygen
(
mg/
L)
7.8
7.8
8.8
8.8
8.0
8.0
8.4
8.5
7.9
7.9
Oxygen
(%)
90
90
99
99
91
91
95
96
94
94
pH
8.25
8.22
7.88
7.89
8.22
8.25
8.09
8.11
7.96
8.00
Total
Hardness
(
mg/
L
as
Ca
CO3)
289
272
272
255
289
272
255
255
272
272
Temperature
(
°
C)
19.1
18.9
18.9
19.0
19.1
19.1
19.2
19.3
19.6
19.6
new:
new
test
solution;
old:
old
test
solution
Temperature
in
test
water
throughout
the
test:
minimum
18.4
°
C
/
maximum
20.4
°
C
0
2
9
0
2
16
6
19
88.6
16
6
88.6
19
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
21
of
52
Table
2:
Number
of
immobilized
parents
Concentration
(
mg/
L)
1st
imm.
2nd
imm.
3rd
imm.
4th
imm.
5th
imm.
6th
imm.
7th
imm.
8th
imm.
9th
imm.
10th
imm.
Number
of
daphnids
immobilized
at
the
end
of
the
test
(
d
21)

control
d
09
d
15
2
4.31
d
14
1
9.16
20.0
44.2
d
19
1
88.6
d
21
1
d
:
day
imm.:
immobilization
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
22
of
52
Table
3a:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
­
Control
NEONATES
PER
ADULT
­
Group
1:
0
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
L.
P.+
D.
P.
(
1)
(
1)
per
day
(
1)
1
6
­
­
9
19
­
­
12
18
­
­
15
12
­
­
19
3
­
­
21
58
58
2.76
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
4
­
­
10
14
­
­
13
18
­
­
17
4
­
­
20
­
­
­
­
40
40
1.90
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
­
­
­
+
D9
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
4
8
­
­
9
22
­
­
13
21
­
­
16
11
­
­
19
­
­
­
­
63
63
3.00
1
­
­
10
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
5
10
­
­
9
17
­
­
12
17
­
­
15
17
­
­
19
6
­
­
21
72
72
3.43
­
­
­
­
­
­
­
­
5
­
­
16
­
­
­
­
­
­
­
­
­
­
­
6
8
­
­
8
16
­
­
11
19
­
­
15
17
­
­
18
10
­
­
21
70
70
3.33
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
7
8
­
­
9
15
­
­
12
4
­
­
+
D15
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
7
­
­
9
16
­
­
12
21
­
­
15
15
­
­
18
7
­
­
21
69
69
3.29
­
­
­
­
­
­
­
­
­
­
­
­
3
­
­
19
­
­
­
­
­
­
­
9
7
­
­
8
4
­
­
11
20
­
­
15
14
­
­
18
8
­
­
21
66
66
3.14
1
­
­
9
12
­
­
12
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
7
­
­
8
18
­
­
11
18
­
­
14
16
­
­
18
9
­
­
21
68
68
3.24
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
Total
67
0
0
­
153
0
0
­
161
0
0
­
109
0
0
­
43
0
0
­
506
506
Mean
63.3
63.3
3.01
CV
16.4%

L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CV:
Cefficient
of
Variation
based
on
the
number
of
living
and
dead
progeny
per
parent
alive
at
the
end
of
the
test
and
per
day
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
23
of
52
Table
3b:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
4.31
mg/
L
NEONATES
PER
ADULT
­
Group
2:
4.31
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
(
1)
(
1)
1
4
­
­
9
7
2
3
12
­
­
­+
D14
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
8
­
­
8
25
­
­
11
22
­
­
15
20
­
­
18
7
­
­
21
82
82
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
10
­
­
8
17
­
­
11
7
­
­
14
12
­
­
18
4
­
­
21
50
50
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
4
9
­
­
9
18
1
­
12
19
­
­
15
18
­
­
18
­
­
­
­
64
65
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
5
11
­
­
8
25
­
­
12
23
­
­
15
15
­
­
18
10
­
­
21
84
84
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
6
12
­
­
8
18
­
­
11
19
­
­
14
18
­
­
18
10
­
­
21
77
77
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
7
9
­
­
9
21
­
­
12
13
­
­
15
15
­
­
19
­
­
­
­
58
58
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
12
­
­
8
21
­
­
11
25
­
­
14
18
­
­
18
11
­
­
21
87
87
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
9
10
­
­
9
17
­
­
12
18
­
­
15
17
­
­
18
8
­
­
21
70
70
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
11
­
­
8
12
­
­
12
23
­
­
15
16
­
­
18
­
­
­
­
63
63
­
­
­
­
­
­
­
­
­
­
­
­
1
­
­
19
­
­
­
­
­
­
Total
96
0
0
­
181
3
3
­
169
0
0
­
150
0
0
­
50
0
0
­
635
636
Mean
70.6
70.7
L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
24
of
52
Table
3c:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
9.16
mg/
L
NEONATES
PER
ADULT
­
Group
3:
9.16
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
(
1)
(
1)
1
8
­
­
8
17
­
­
11
11
­
­
15
18
­
­
18
9
­
­
21
63
63
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
10
­
­
9
16
­
­
13
19
­
­
16
6
­
­
19
­
­
­
­
53
53
2
­
­
10
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
5
­
­
8
12
­
­
11
11
­
­
14
18
­
­
18
5
­
­
21
52
52
­
­
­
­
­
­
­
­
­
­
­
­
1
­
­
19
­
­
­
­
­
­
4
9
­
­
9
14
­
­
11
17
­
­
13
21
­
­
16
1
­
­
21
64
65
­
­
­
­
­
1
­
12
­
­
­
­
2
­
­
19
­
­
­
­
­
­
5
11
­
­
8
14
­
­
11
14
­
­
14
13
­
­
17
10
­
­
21
69
69
­
­
­
­
­
­
­
­
­
­
­
­
7
­
­
18
­
­
­
­
­
­
6
8
­
­
9
17
­
­
12
16
­
­
15
12
­
­
18
­
­
­
­
58
58
­
­
­
­
­
­
­
­
1
­
­
16
4
­
­
19
­
­
­
­
­
­
7
9
­
­
8
9
­
­
11
18
­
­
15
16
­
­
18
4
­
­
21
56
56
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
9
­
­
8
12
­
­
11
10
­
­
14
20
­
­
17
12
­
­
20
71
71
1
­
­
9
6
­
­
13
­
­
­
­
1
­
­
18
­
­
­
­
­
­
9
14
­
­
8
15
­
­
11
18
­
­
14
16
­
­
18
9
­
­
21
72
72
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
12
­
­
8
23
­
­
11
19
­
­
14
19
­
­
18
10
­
­
21
83
83
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
Total
98
0
0
­
155
1
0
­
154
0
0
­
174
0
0
­
60
0
0
­
641
642
Mean
64.1
64.2
L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
25
of
52
Table
3d:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
20.0
mg/
L
NEONATES
PER
ADULT
­
Group
4:
20.0
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
(
1)
(
1)
1
5
­
­
8
17
­
­
11
13
­
­
14
25
­
­
18
­
­
­
­
60
60
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
12
­
­
8
19
­
­
11
20
­
­
14
­
1
­
17
7
­
­
21
80
81
­
­
­
­
­
­
­
­
­
­
­
­
22
­
­
18
­
­
­
­
­
­
3
9
­
­
8
11
­
­
11
10
­
­
14
19
­
­
18
2
­
­
21
51
51
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
4
7
­
­
8
15
­
­
11
26
­
­
14
18
­
­
18
­
­
­
­
66
66
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
5
13
­
­
8
18
­
­
11
22
­
­
14
13
­
­
17
9
­
­
21
78
79
­
­
­
­
1
­
­
12
­
­
­
­
2
1
­
18
­
­
­
­
­
­
6
10
­
­
10
4
1
­
13
8
­
3
17
4
­
­
21
­
­
­
­
26
27
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
7
13
­
­
8
16
­
­
11
16
­
­
14
19
­
­
18
9
­
­
21
73
73
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
20
­
­
15
7
2
­
18
­
­
­
­
­
­
­
­
­
­
­
­
27
29
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
9
15
­
­
8
18
­
­
11
23
­
­
14
21
­
­
18
11
­
­
21
88
88
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
12
­
­
8
15
­
­
11
20
­
­
14
19
1
­
18
8
­
­
21
74
75
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
Total
116
0
0
­
141
3
0
­
158
0
3
­
162
3
0
­
46
0
0
­
623
629
Mean
62.3
62.9
L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
26
of
52
Table
3e:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
44.2
mg/
L
NEONATES
PER
ADULT
­
Group
5:
44.2
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
(
1)
(
1)
1
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
8
­
­
8
13
­
­
11
16
2
­
14
18
2
­
18
­
­
­
+
D19
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
3
2
­
14
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
5
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
4
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
5
2
­
­
14
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
2
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
6
5
3
­
9
10
­
­
12
15
­
­
15
18
­
­
19
­
­
­
­
50
53
­
­
­
­
­
­
­
­
2
­
­
16
­
­
­
­
­
­
­
­
­
­
7
1
4
­
9
18
­
­
12
10
5
­
15
15
­
­
19
­
­
­
­
44
53
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
­
2
­
8
15
­
­
11
20
2
­
14
16
­
­
17
4
2
­
21
61
67
­
­
­
­
­
­
­
­
­
­
­
­
6
­
­
18
­
­
­
­
­
­
9
2
6
­
9
15
­
­
12
17
1
­
15
17
­
­
19
­
­
­
­
51
58
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
4
2
­
12
1
1
­
14
­
­
2
21
­
­
­
­
­
­
­
­
5
8
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
Total
21
19
0
­
72
1
0
­
80
10
2
­
90
2
0
­
4
2
0
­
216
246
Mean
24.0
27.3
L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
27
of
52
Table
3f:
Living,
dead
and
aborted
eggs
produced
at
each
brood
release
 
88.6
mg/
L
NEONATES
PER
ADULT
­
Group
6:
88.6
mg/
L
Parent
Brood
1
Brood
2
Brood
3
Brood
4
Brood
5
Total
Total
number
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
D.
P.
A.
E.
Day
L.
P.
L.
P.+
D.
P.
(
1)
(
1)
1
­
5
­
14
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
1
7
1
1
­
16
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
2
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
3
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
4
­
­
3
12
­
­
­
+
D21
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
5
­
­
5
12
­
­
8
21
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
6
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
7
­
­
18
16
­
­
18
19
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
8
­
1
7
12
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
1
­
­
3
16
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
9
­
1
10
12
­
­
15
16
­
­
7
21
­
­
­
­
­
­
­
­
0
1
­
­
9
14
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
10
­
­
9
12
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
0
0
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
­
Total
1
8
64
­
0
0
41
­
0
0
7
­
0
0
0
­
0
0
0
­
1
9
Mean
0.1
0.9
L.
P.:
Living
Progeny
D.
P.:
Dead
Progeny
A.
E.:
Aborted
Eggs
+
=
mortality
of
the
adult
at
the
corresponding
study
day
(
1)
=
for
parent
animals
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
28
of
52
Table
4:
Living
neonates
per
parent
alive
at
the
end
of
the
test
versus
concentration
of
the
test
item
For
parents
alive
at
the
end
of
the
test
Test
item
concentrations
(
mg/
L)
Parent
number
0
4.31
9.16
20.0
44.2
88.6
1
58
­
63
60
0
1
2
40
82
53
80
­
0
3
­
50
52
51
3
0
4
63
64
64
66
0
­
5
72
84
69
78
2
0
6
70
77
58
26
50
0
7
­
58
56
73
44
0
8
69
87
71
27
61
0
9
66
70
72
88
51
0
10
68
63
83
74
5
0
Mean
63.3
70.6
64.1
62.3
24.0
0.1
(
as
%
Control)
­
(
112)
(
101)
(
98)
(
38)
(
0)
CV
16.4%
18.1%
15.3%
34.6%
110%
300%

CV:
Coefficient
of
Variation
based
on
the
number
of
living
progeny
per
parent
alive
at
the
end
of
the
test
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
29
of
52
Table
5:
Length
of
parent
alive
at
the
end
of
the
test
Length
(
mm)
of
parents
alive
at
the
end
of
the
test
Test
item
concentrations
(
mg/
L)
Parent
number
0
4.31
9.16
20.0
44.2
88.6
1
3.3
­
3.1
3.1
3.1
3.0
2
3.4
3.4
3.3
3.1
­
2.8
3
­
3.3
3.1
3.1
3.3
2.8
4
3.3
3.4
3.4
3.4
3.1
­
5
3.4
3.4
3.1
3.1
3.1
2.6
6
3.4
3.3
3.1
3.4
3.0
3.0
7
­
3.3
3.3
3.3
3.3
2.6
8
3.4
3.4
3.1
3.1
3.1
2.8
9
3.3
3.3
3.1
3.1
3.1
3.0
10
3.4
3.1
3.1
3.3
3.3
3.0
Mean
3.4
3.3
3.2
3.2
3.2
2.8
(
as
%
Control)
­
(
99)
(
94)
(
95)
(
94)
(
85)
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
30
of
52
APPENDICES
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
31
of
52
1.
Analytical
certificate
and
composition
of
the
test
item
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
32
of
52
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
33
of
52
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
34
of
52
2.
Preparation
of
M4
reconstituted
water
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
35
of
52
PREPARATION
OF
MEDIUM
M4
RECONSTITUTED
WATER
Trace
elements
Amount
of
trace
elements
for
the
preparation
of
stock
solutions
I,
which
contain
only
one
salt
per
solution.
From
the
different
stock
solutions
I
a
second
stock
solution,
stock
solution
II
is
prepared,
which
contains
all
trace
elements
(
combined
solution
I)
20­
fold
concentrated
(
related
to
M4):

Stock
solution(
s):
I
_____________________________________________________________________________
(
single
substance)
to
prepare
the
combined
20­
fold
amount
concentration
concentrated
stock
added
to
(
in
relation
to
solution
II
add
the
deionized
water
medium
M4)
following
amount
of
(
mg/
L)
stock
solution
I
to
deionized
water
(
mL/
L)
_____________________________________________________________________________
M4
H3BO3
57190
20000­
fold
1.0
MnCl2,
4
H2O
7210
20000­
fold
1.0
LiCl
6120
20000­
fold
1.0
RbCl
1420
20000­
fold
1.0
SrCl2,
6
H2O
3040
20000­
fold
1.0
NaBr
320
20000­
fold
1.0
Na2MoO4,
2
H2O
1260
20000­
fold
1.0
CuCl2,
2
H2O
335
20000­
fold
1.0
ZnCl2
260
20000­
fold
1.0
CoCl2,
6
H2O
200
20000­
fold
1.0
KI
65
20000­
fold
1.0
Na2SeO3
43.8
20000­
fold
1.0
NH4VO3
11.5
20000­
fold
1.0
_____________________________________________________________________________

Na2EDTA,
2
H2O
5000
2000­
fold
­
FeSO4,
7
H2O
1991
2000­
fold
­

Both
solutions
(
1
L)
are
prepared
individually,
poured
together
and
autoclaved
immediately.

This
gives:
Fe­
EDTA
solution
(
2
L)
1000­
fold
20.0
_____________________________________________________________________________

­:
not
applicable
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
36
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52
With
the
20­
fold
concentrated
stock
solution
II
(
which
contains
all
trace
elements),
the
macro­
nutrients
and
vitamins,
M4
is
prepared
as
follows:
_____________________________________________________________________________
Amount
Concentration
Amount
of
stock
added
to
(
related
to
solution
added
deionized
medium
M4)
to
prepare
medium
water
(
mg/
L)
(
mL/
L)
_____________________________________________________________________________
M4
stock
solution
II
(
combined
trace
elements)
20­
fold
50
Macro­
nutrients
stock
solution(
s)
single
substance
CaCl2,
2
H2O
293800
1000­
fold
1.0
MgSO4,
7
H2O
246600
2000­
fold
0.5
KCl
58000
10000­
fold
0.1
NaHCO3
64800
1000­
fold
1.0
Na2SiO3,
9
H2O
50000
5000­
fold
0.2
NaNO3
2740
10000­
fold
0.1
KH2PO4
1430
10000­
fold
0.1
K2HPO4
1840
10000­
fold
0.1
Vitamins
A
vitamin
stock
solution
is
prepared
by
adding
the
three
vitamins
to
one
liter
deionized
water:

mg/
L
M4
thiamine
hydrochloride
750
10000­
fold
­
cyanocobalamine
(
B12)
10
10000­
fold
­
biotine
7.5
10000­
fold
­

combined
vitamin­
stock
­
10000­
fold
0.1
_____________________________________________________________________________
­:
not
applicable
The
vitamin
stock
is
stored
frozen
in
small
aliquots.
Vitamins
are
added
to
the
media
shortly
before
use.

To
avoid
precipitation
of
salts
when
preparing
the
complete
media
add
the
aliquots
of
stock
solutions
to
approximately
500­
800
mL
deionized
water
and
then
complete
to
1
liter.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
37
of
52
3.
Chemical
analysis
of
test
solutions
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
38
of
52
CHEMICAL
ANALYSIS
Principle
An
aliquot
of
each
sample
was
diluted
and
analyzed
by
Ion
Chromatography
with
Electrochemical
Detection
(
Conductimetry).
The
concentrations
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
were
determined
from
a
calibration
curve
of
peak
area
against
concentration
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
standard
solutions.

Sample
preparation
All
samples
were
mixed.
If
necessary,
samples
were
then
diluted
with
Milli­
Q
to
achieve
concentrations
in
the
range
1­
100
mg/
L
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO).

Chromatographic
conditions
Pump
:
GP
50
Gradient
Pump
(
Dionex)

Mobile
phase
:
phase
A:
aqueous
sodium
hydroxide
solution
50
mM*
phase
B:
Milli­
Q
water
phase
C:
acetonitrile
*
Aqueous
sodium
hydroxide
solution
50
mM:
2.6
mL
of
sodium
hydroxide
solution
at
46/
48%
(
Fisher
Chemicals,
Ref.:
S/
4930/
05)
was
added
to
1
L
of
Milli­
Q
Water.

Time
(
min)
phase
A
(%)
phase
B
(%)
phase
C
(%)
curve
0
10
85
5
6
5
40
0
60
6
12
40
0
60
­
13
10
85
5
­
25
10
85
5
­

Flow
rate
:
1
mL/
min
Precolumn
:
Ionpac
ATC­
3,
4
mm
(
Dionex)

Column
:
Ionpac
AS16
(
Dionex)
length
=
250
mm,
inner
diameter
=
4
mm
Temperature
:
30
°
C
Detector
:
ED
50
Electrochemical
Detector
(
Dionex)
Conductimetry
after
eluent
neutralization
CIT/
Study
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22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
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Neutralization
:
ASRS
Ultra
(
Dionex),
electric
current
70
mA
or
AMMS
(
Dionex)

Injector
:
AS50
(
Dionex),
at
10
°
C
Injected
volume
:
25
µ
L
Data
acquisition
software
:
Multichrom
2
(
Fisons
Instruments)

Retention
time
:
APFO,
approx.
9
min
Analysis
time
:
30
min
Calibration
curve
Peak
areas
were
determined
for
standard
solutions
ranging
from
1
to
100
mg/
L
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
(
six
levels).
A
calibration
curve
was
obtained
by
plotting
test
item
peak
areas
against
concentrations.

The
regression
analysis
of
the
calibration
data
gave
an
equation
of
the
following
form:
Y
=
aX
+
b
where
Y
=
test
item
peak
area
(
µ
Vs)
X
=
concentration
of
test
item
(
mg/
L)
a
=
slope
value
b
=
intercept
Assay
Samples
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
were
analyzed
by
Ion
Chromatography
with
Electrochemical
Detection
(
Conductimetry).

One
dilution
was
prepared
for
each
sample
and
one
injection
(
of
25­
µ
L
aliquots)
was
performed
for
each
final
dilution.

The
peak
area
was
determined
for
each
sample.
The
concentration
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
each
sample
was
calculated
using
the
equation
obtained
from
the
calibration
data.

All
the
results
are
expressed
as
mg/
L
of
APFO.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
40
of
52
Table
A:
Concentration
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
the
test
solutions
during
the
study
(
mg/
L)

Nominal
day
0
day
2
day
5
day
7
old
fresh
old
fresh
old
fresh
6.25
3.94
(
63)
4.22
(
68)
4.02
(
64)
3.87
(
62)
4.10
(
66)
3.88
(
62)
4.28
(
68)

12.5
8.97
(
72)
8.50
(
68)
8.57
(
69)
8.68
(
69)
9.81
(
78)

25
19.0
(
76)
19.9
(
80)
19.0
(
76)
19.0
(
76)
18.9
(
76)
18.8
(
75)
21.1
(
84)

50
43.3
(
87)
46.4
(
93)
42.0
(
84)
44.4
(
89)
42.5
(
85)
45.1
(
90)
42.2
(
84)

100
82.6
(
83)
86.1
(
86)
82.0
(
82)
91.5
(
92)
80.0
(
80)
83.5
(
84)
87.9
(
88)

Nominal
day
12
day
19
old
fresh
old
fresh
old
fresh
old
6.25
4.63
(
74)
4.50
(
72)
4.45
(
71)
4.83
(
77)
5.24
(
84)
4.34
(
69)
4.41
(
71)

12.5
9.65
(
77)
9.26
(
74)
9.30
(
74)
9.19
(
74)
9.48
(
76)
9.10
(
73)
9.63
(
77)

25.0
21.2
(
85)
20.9
(
84)
19.8
(
79)
20.0
(
80)
22.4
(
90)
19.5
(
78)
21.7
(
87)

50.0
43.9
(
88)
45.1
(
90)
41.2
(
82)
43.8
(
88)
45.3
(
91)
44.2
(
88)
50.6
(
101)

100
98.0
(
98)
94.8
(
95)
90.8
(
91)
80.7
(
81)
98.8
(
99)
93.6
(
94)
103
(
103)

Nominal
Harmonic
mean
(
days
0­
21)

6.25
12.5
25.0
50.0
100
vd:
value
discarded
(
aberrant
value)
CV:
Coefficient
of
Variation
(
2):
based
on
measured
concentrations
at
the
beginning
of
the
test
and
before
medium
renewals
4.59
4.52
2.20
7.25
day
16
Measured
(
1)

Measured
(
1)
day
21
vd
vd
(
1):
numbers
in
brackets
represent
percentages
of
the
nominal
concentration
Measured
CV
(%)
(
2)

4.31
9.16
20.0
44.2
89.0
8.50
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
41
of
52
VALIDATION
OF
THE
ANALYTICAL
METHOD
The
validation
of
the
analytical
method
was
performed
according
to
CIT
Standard
Operating
Procedures.
The
specificity,
limit
of
quantification,
linearity,
repeatability
of
injections,
accuracy
and
precision
(
Coefficient
of
Variation:
CV
%)
of
the
analytical
method
were
determined.

Specificity
The
specificity
of
the
analytical
method
was
demonstrated
as
follows:

 
analysis
of
a
standard
solution
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
Milli­
Q
water,

 
analysis
of
Milli­
Q
water
or
test
water
(
LC,
M4
or
dechlorinated;
desionized
water)
without
dilution,
No
relevant
interference
between
the
test
item
peak
and
Milli­
Q
water
or
test
water
was
observed
on
chromatograms.

Limit
of
quantification
The
limit
of
quantification
of
the
analytical
method
was
established
as
1
mg/
L
for
a
standard
solution
of
APFO.
This
limit
corresponds
to
a
limit
of
quantification
of
1
mg/
L
for
the
test
item
in
aqueous
phase.

Linearity
Linearity
was
checked
by
analysis
of
three
different
sets
of
six
standard
solutions
containing
1,
5,
10,
20,
50
and
100
mg/
L
of
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)
in
Milli­
Q
water.
Satisfactory
linearity
was
demonstrated
in
the
range
1
to
100
mg/
L
since
the
coefficients
of
determination
obtained
were
higher
than
0.999.

Repeatability
of
injections
Replicate
analysis
(
n
=
10)
of
a
solution
containing
50
mg/
L
of
the
test
item
gave
satisfactory
results
since
the
coefficients
of
variation
values
obtained
were
as
follows:

 
1%
based
on
peak
height,

 
1%
based
on
peak
area.
Based
on
these
results,
the
repeatability
of
injections
of
the
analytical
method
was
validated
taking
into
account
peak
area.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
42
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52
Accuracy
and
precision
Six
analyses
of
solutions
containing
2.03
and
50.7
mg/
L
of
the
test
item
in
Milli­
Q
water
were
carried
out.

The
accuracy
and
the
precision
(
CV%)
obtained
were
as
follows:

Concentration
(
mg/
L)
Dilution
CV
Accuracy
Nominal
Mean
measured*
factor
(%)
(%)
2.03
2.02
1
6
99
50
.7
53.7
1
2
106
*:
mean
values
of
six
determinations
Conclusion
The
analytical
method
was
validated
and
considered
to
be
suitable
for
the
analysis
of
the
samples
of
the
study.
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
43
of
52
4.
EC50
estimation
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
44
of
52
21­
day
EC50
ESTIMATION
BASED
ON
REPRODUCTIVE
CAPACITY
_____________________________________________________________________________
Concentration
Percentage
inhibition
mg/
L
_____________________________________________________________________________
0
4.31
0
9.16
0
20.0
2
44.2
62
88.6
100
_____________________________________________________________________________

Slope:
3.183
Equation:
Y
=
3.183
x
X
 
21.369
21­
day
EC50
LOWER
LIMIT
UPPER
LIMIT
______________________________________________

39.6
(
36.7
­
42.5
)
mg/
L
Significance:
95%
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
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5.
21­
day
NOEC
determination
based
on
reproduction
capacity
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
46
of
52
21­
day
NOEC
determination
based
on
reproduction
capacity
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
47
of
52
21­
day
NOEC
determination
based
on
reproduction
capacity
(
continued)
CIT/
Study
No.
22658
ECD/
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PERFLUOROOCTANOATE
(
APFO)/
APME
48
of
52
6.
Statistical
analysis
of
Daphnia
length
data
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
49
of
52
CIT/
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No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
50
of
52
CIT/
Study
No.
22658
ECD/
AMMONIUM
PERFLUOROOCTANOATE
(
APFO)/
APME
51
of
52
CIT/
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No.
22658
ECD/
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PERFLUOROOCTANOATE
(
APFO)/
APME
52
of
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