Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
1
of
38
BIODEGRADATION
STUDY
REPORT
The
18­
Day
Aerobic
Biodegradation
Study
of
Perfluorooctanesulfonyl­
Based
Chemistries
PROJECT
NUMBER
3M
Project
ID:
Not
Available
Contract
Analytical
Project
ID:
CA097
STUDY
DIRECTOR
Cleston
C.
Lange,
Ph.
D.

SPONSOR
REPRESENTATIVE
James
K.
Lundberg,
Ph.
D.,
3M
Environmental
Laboratories
Bldg
2­
3E­
09,
P.
O.
Box
33331,
St.
Paul,
MN
55133­
3331
CONTRACT
LABORATORY
Pace
Analytical
Services,
Inc.

Science
Solutions
Division
Contract
Analytical
Group
1700
Elm
Street,
Suite
200
Minneapolis,
Minnesota
55414
PROJECT
DATES
Project
Initiation:
September
8,
2000
Project
Completion:
October
6,2000
Final
Report:
Friday,
February
23,
2001
AUTHOR
Cleston
C.
Lange,
Ph.
D.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
2
of
38
Table
of
Contents
Title
Page.........................................................................................................................................
1
Table
of
Contents
.............................................................................................................................
2
List
of
Figures
...................................................................................................................................
3
List
of
Appendices
............................................................................................................................
3
1.0
Project
Personnel........................................................................................................................
4
2.0
Data
Requirements
.....................................................................................................................
4
3.0
Project
Objective
.......................................................................................................................
4­
5
4.0
Test,
Control
and
Reference
Articles
..........................................................................................
5­
7
5.0
Receipt/
Generation
of
Samples....................................................................................................
7
6.0
Methods
6.1
Sample
Preparation
6.1.1
Sludge
Collection
and
Characterization
................................................................................
8
6.1.2
Culture
Setup..................................................................................................................
8­
10
6.1.3
Solid
Phase
Extraction
(SPE).........................................................................................
10­
11
6.2
Instrumental
Analysis................................................................................................................
12
6.3
Data
Transformations
and
Calculations
6.3.1
Molar
Calculations
.............................................................................................................
13
6.3.2
Conversion
of
ng/
mL
to
mM
................................................................................................
13
6.3.3
Mass
Balance
Calculations
................................................................................................
14
6.4
Software
Versions.....................................................................................................................
14
7.0
Results
7.1
Incubator,
Refrigerator,
and
Freezer
Temperature
Log
Data........................................................
15
7.2
Mishaps
recorded
during
the
study
............................................................................................
16
7.3
Sludge
Characterization
Report
7.3.1
Metals
..............................................................................................................................
16
7.3.2
Wet
Chemistry
..................................................................................................................
16
7.4
Quality
Control/
Sample
Matrix
Spike
Results
..............................................................................
17
7.5
LC/
MS
Calibration
and
Calibration
Verification.......................................................................
17­
18
7.6
Blanks
...............................................................................................................................
18
7.7
Sample
Results
7.7.1
Results
of
incubations
with
N­
EtFOSE
alcohol
....................................................................
19
7.7.2
Results
of
incubations
with
N­
EtFOSAA..............................................................................
20
7.7.3
Results
of
incubations
with
N­
EtFOSA
................................................................................
21
7.7.4
Results
of
incubations
with
M556........................................................................................
22
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
3
of
38
7.7.5
Results
of
incubations
with
FOSA.......................................................................................
23
7.7.6
Results
of
incubations
with
PFOSulfinate
............................................................................
24
7.7.7
Results
of
incubations
with
PFOS
.......................................................................................
25
7.7.8
Results
of
incubations
with
PFOA
.......................................................................................
26
9.0
Conclusions
..........................................................................................................................
26­
27
10.0
Sample
and
Data
Retention
......................................................................................................
29
11.0
References
..............................................................................................................................
30
LIST
OF
FIGURES
Figure
1:
Biodegradation
chart
for
N­
EtFOSE
alcohol
.........................................................................
19
Figure
2:
Biodegradation
chart
for
N­
EtFOSAA..................................................................................
20
Figure
3:
Biodegradation
chart
for
N­
EtFOSA
....................................................................................
21
Figure
4:
Biodegradation
chart
for
M556............................................................................................
22
Figure
5:
Biodegradation
chart
for
FOSA
...........................................................................................
23
Figure
6:
Biodegradation
chart
for
PFOSulfinate
................................................................................
24
Figure
7:
Biodegradation
chart
for
PFOS
...........................................................................................
25
Figure
8:
Biodegradation
chart
for
PFOA
...........................................................................................
26
Figure
9:
Proposed
biodegradation
pathway
for
perfluorooctanesulfonyl­
based
chemistries
tested........
28
List
of
Appendices
Appendix
A:
Signatures
of
Project
Personnel
.....................................................................................
31
Appendix
B:
Table
1.
Sample
results
for
SPE
eluate
2
LC/
MS
analysis
.........................................
32­
33
Appendix
B:
Table
2.
Sample
results
for
SPE
eluate
3
LC/
MS
analysis
.........................................
34­
35
Appendix
C:
Table
1.
Sample
results
for
the
sum
of
SPE
eluate
2
and
eluate
3
LC/
MS
analysis
.....
36­
37
Appendix
D:
Table
1.
Final
Results
(mM)
and
mass
balance...............................................................
38
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
4
of
38
1.0
Project
Personnel
1.1
Sponsor
3M
Company
1.2
Sponsor
Representative
Dr.
James
K.
Lundberg
1.3
Contract
Facility
Personnel:

1.3.1
Study
Director
Dr.
Cleston
C.
Lange
1.3.2
Laboratory
Management:
Mr.
Gabe
J.
Lebrun
1.3.3
Sample
Preparation
Analysts
Ms.
Angela
L.
Schuler
1.2.4
HPLC/
MS
Analyst
Mr.
Anthony
E.
Scales
1.2.5
Sludge
Characterization
Report
Mr.
Daryl
K.
Peterson
1.2.6
Sample
Custodian:
Dr.
Cleston
C.
Lange
1.2.7
Quality
Assurance
Manager
Mr.
Dirk
Hoogenboom
1.2.8
Report
Author
Dr.
Cleston
C.
Lange
2.0
Data
Requirements
Conversations
between
the
study
director
and
the
sponsor
representative,
concerning
details
of
the
biodegradation
study
for
N­
EtFOSE
alcohol
(Project
CA058),
were
held
prior
to
September
5,
2000.
Those
conversations
resulted
in
a
decision
that
a
two
sample­
point
biodegradation
study,
approximately
three
weeks
duration,
should
be
conducted
for
each
of
the
perfluorooctanesulfonyl­
based
chemistries
targeted
in
that
study
to
confirm
the
degradation
pathways
proposed
in
the
CA058
final
report
1
.
The
approval
to
proceed
with
the
study
was
granted
via
a
documented
phone
conversation
held
on
September
5,
2000
between
the
study
director
and
sponsor
representative.
The
samples
for
this
study
were
subsequently
prepared,
incubated
for
eighteen
days,
solid
phase
extracted
(SPE),
and
the
SPE
eluates
analyzed
by
quantitative
LC/
MS.
This
study
was
not
conducted
under
the
GLP
regulations.

3.0
Project
Objective
The
objective
of
the
study
was
to
determine
the
products
generated
for
each
of
the
targeted
perfluorooctanesulfonyl­
based
chemistries
when
incubated
as
test
substrates
with
aerobic
municipal
wastewater
treatment
plant
sludge.
The
substrates
tested
were
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
5
of
38
the
perfluorooctanesulfonyl
products
of
N­
EtFOSE
alcohol
biodegradation,
detected
during
the
course
of
project
CA058.
In
this
study,
the
perfluorooctanesulfonyl­
based
substrates
were
individually
incubated
with
aerobic
sludge
for
eighteen
days
at
a
concentration
of
approximately
2.5
mg/
mL.
The
results
from
this
study
helped
to
establish
the
possible
environmental
fate
of
the
following
test
materials:
N­
EtFOSE
alcohol,

NEtFOSAA
N­
EtFOSA,
M556,
FOSA,
PFOSulfinate,
PFOS
and
PFOA.
The
proposed
aerobic
degradation
pathway
from
this
study
supports
the
stepwise
biodegradation
of
NEtFOSE
alcohol
proposed
in
the
Study
CA058
report.
Several
of
the
tested
substrates
presented
special
difficulties
that
may
necessitate
further
testing.

4.0
Test
and
Reference
Articles
All
test
and
reference
articles
for
this
study
were
received
from
the
3M
Environmental
Laboratory
on
June
8,
2000.
Approximately
1
gram
of
each
of
the
neat
materials,
except
M556,
was
received
by
the
study
director
at
Pace
Science
Solutions
(PSS),
Contract
Analytical
Group
(CAG)
Laboratory,
with
an
accompanying
chain
of
custody
(COC
#

14681).
Pertinent
material
and
safety
data
sheets
(MSDS)
and
the
available
purity
information
were
also
included
for
those
materials.
M556
was
provided
without
a
COC,

MSDS
or
purity
information.
Upon
receipt
at
PSS,
each
substance
was
given
a
Pace
test,
control,
&
reference
(TCR)
number.

All
test
and
reference
articles
were
stored
at
–80°
C
±
15.0°
C
during
the
study.
Because
the
purity
of
the
test
and
reference
materials
had
not
been
adequately
established,
all
final
data
in
data
tables
for
this
report
were
calculated
assuming
a
purity
of
100%.
A
lack
of
purity
information
did
not
negatively
impact
the
study.

Test
&
reference
articles
are
described
below
with
their
corresponding
percent
purity
estimation,
3M
identification
number,
and
the
Pace
identification
number.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
6
of
38
NC:
purity
analysis
not
completed.
All
purity
information
regarding
the
control
&

reference
articles
was
maintained
under
lock
and
key
in
the
laboratory
in
a
client
specific
binder.

5.1.
Perfluorooctanoate
Ammonium
Salt
(PFOA)

The
purity
was
determined
at
97­
99%
by
HPLC/
MS
as
part
of
project
CA058.

3M#:
TCR­
99131­
37
Pace
#:
CA­
TCR02­
001
O
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
O
­
NH
4
+

5.2.
Perfluorooctanesulfinate
Potassium
Salt
(PFOSulfinate)

Purity:
NC
3M#:
SD­
007
Pace
#:
CA­
TCR02­
002
K
F
F
F
F
F
F
F
F
F
F
F
F
S
O
O
F
F
F
F
F
5.3.
Perfluorooctanesulfonate
Potassium
Salt
(PFOS)

Purity:
86.4%
3M#:
SD­
009
Pace
#:
CA­
TCR02­
003
F
F
F
F
F
F
F
F
F
F
F
F
S
O
O
F
F
F
F
F
O
­
K
+

5.4.
Perfluorooctanesulfonamide
(FOSA)

Purity:
NC
3M#:
SD­
029
Pace
#:
CA­
TCR02­
004
F
F
F
F
F
F
F
F
F
F
F
F
NH2
S
O
O
F
F
F
F
F
5.5.
N­
Ethyl
perfluorooctanesulfonamide
(N­
EtFOSA)

Purity:
NC
3M#:
SD­
012
Pace
#:
CA­
TCR02­
005
F
F
F
F
F
F
F
F
F
F
F
F
CH3
NH
S
O
O
F
F
F
F
F
BACK
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MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
7
of
38
5.6.
[2­(
Perfluorooctanesulfonamido)
acetic
acid]
(M556)

Purity:
NC
3M#:
00001­
5­
24
Pace#:
CA­
TCR02­
006
F
F
F
F
F
F
F
F
F
F
F
F
NH
S
O
O
F
F
F
F
F
O
H
O
5.7.
[2­(
N­
Ethyl­
perfluorooctanesulfonamido)
acetic
acid]

(N­
EtFOSAA)
and
was
provided
labeled
as
FC­
129
Purity:
NC
3M#:
SE­
038
Pace
#:
CA­
TCR02­
007
F
F
F
F
F
F
F
F
F
F
F
F
N
S
O
O
F
F
F
F
F
O
H
CH3
O
5.8.
[2­(
N­
Ethyl­
perfluorooctanesulfonamido)
ethyl
alcohol]
(N­
EtFOSE
alcohol)

Purity:
99.9%
3M#:
SE­
035
Pace
#:
CA­
TCR02­
008
F
F
F
F
F
F
F
F
F
F
F
F
N
S
O
O
F
F
F
F
F
O
H
CH3
5.0
Receipt/
Generation
of
Samples
All
samples
for
this
study
were
generated
at
Pace.
Sixty­
eight
test
cultures
and
control
cultures
were
prepared
for
this
18­
day,
two­
point
study.
All
sixty­
eight
cultures
were
extracted
by
solid
phase
extraction
(SPE)
to
generate
204
analytical
samples
referred
to
as
SPE
eluates
1,
2
and
3
for
each
culture.
Of
the
three
sample
eluates,
only
eluates
2
and
3
were
analyzed
by
quantitative
LC/
MS.
The
data
obtained
previously
from
project
CA058
1
provided
ample
evidence
to
suggest
that
mass
balance
was
obtained
by
analysis
of
only
eluates
2
and
3.
It
is
assumed
that
no
analytes
were
contained
in
the
aqueous
eluate
1.
BACK
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Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
8
of
38
6.0
Methods
6.1
Sample
Preparation
6.1.1.
Collection
of
Sludge.

To
prepare
test
cultures,
sludge
was
obtained
from
the
primary
municipal
waste
treatment
facility
for
the
Twin
Cities
metropolitan
area.

Arrangements
were
made
for
Pace
personnel
to
retrieve
fresh
mixed
liquor
suspended
solids
(MLSS)
from
the
aeration
units
at
the
Twin
Cites
Metro
Wastewater
Treatment
Facility
(also
referred
to
as
Pig's
Eye
Sewer
Treatment
Plant)
located
in
St.
Paul,
MN.
Six
liters
of
MLSS
was
collected
by
Pace
laboratory
personnel
on
July
31,
2000
and
delivered
as
six
1­
liter
bottles
of
MLSS
with
a
corresponding
chain
of
custody
(Pace
C.
O.
C.
#
465254).
Upon
receipt,
each
bottle
was
labeled
bottles
#1
through
#6,
and
stored
at
4°
C.
Bottle
#1
was
immediately
delivered
to
Mr.
Daryl
Peterson,
of
the
Minnesota
Environmental
Laboratory
at
Pace
Analytical
Services,
for
sludge
characterization.
The
sludge
characterization
report
was
received
on
August
16,
2000
and
is
summarized
in
section
8.3.

The
MLSS
used
for
this
study
was
from
bottle
#3
and
had
been
stored
at
4°
C
for
approximately
5
weeks
prior
to
being
used
for
this
study.
The
settled
sludge
constituted
approximately
20%
of
the
volume,
or
approximately
200
mL
of
settled
sludge
from
a
1­
liter
bottle,
based
on
visual
inspection.

6.1.2.
Culture
Preparation.

All
cultures
were
prepared
on
September
8,
2000
and
the
final
sample
collection
occurred
on
September
26,
2000.
This
study
was
set
up
using
slight
modifications
to
the
original
proposed
methods
included
with
Pace
proposal
000511.1.
Changes
were
made
with
regard
to
culture
volume
BACK
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Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
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Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
9
of
38
(changed
from
20
mL
to
25
mL),
types
of
controls
prepared,
number
of
concentrations
tested,
and
the
number
of
sample
points
employed.

All
substrates
for
this
study
were
tested
for
biodegradability
at
a
final
concentration
of
approximately
2.5
mg/
mL
(part
per
million;
ppm).
The
culture
preparation
procedure
described
below
was
documented
as
standard
operating
procedure
(SOP)
CAG­
SP­
03
3
.

Sample
cultures
were
prepared
using
a
mineral
salts
medium
defined
in
EPA
Guideline
OPPTS
835.3200.
The
medium
pH
was
7.4
and
contained
per
liter,
0.334
g
Na2HPO4­
2H2O,
0.005
g
NH4Cl,
0.2175
g
K2HPO4,
and
0.085
KH2PO4,
0.0275
g
CaCl2­
anhydrous,
0.0225
g
MgSO4­
7H2O,
and
0.00025
g
FeCl3­
6H2O.
Per
one
liter
of
mineral
salts
medium
prepared,
1
mL
of
methanol
was
added.
The
mineral
salts
medium
containing
sludge
was
prepared
fresh
on
September
7,
2000
by
adding
50
mL
of
settled
sludge
to
950
mL
of
mineral
salts
medium
(reagent
ID
CA­
R00­
100).
This
mineral
medium
plus
sludge
solution
was
then
used
to
prepare
test
cultures.
Mineral
salts
medium
without
sludge
was
prepared
on
August
10,
2000
and
was
used
for
the
preparation
of
the
no­
sludge
abiotic
controls
(reagent
ID
CA­
R00­
085).

Cultures
were
prepared
by
transferring
25
mL
of
appropriate
culture
solution
into
labeled,
sterile
125
mL
polycarbonate
Nalgene
culture
flasks.
Appropriate
amounts
of
the
test
substrate
stock
solutions
were
added
to
culture
flasks
containing
culture
solution.
Blank
controls
did
not
receive
test
substrate.

In
total
68
cultures
were
prepared,
and
included
duplicate
sets
of
test
cultures,
control
cultures,
and
appropriate
blanks
for
day
zero
sampling
and
day
18
sampling
for
each
analyte.

The
targeted
final
concentration
of
the
test
substrate
in
each
culture
was
2.5
mg/
mL,
or
approximately
5
mM.
The
actual
tested
concentrations
for
each
were
as
follows:
the
N­
EtFOSE
alcohol
was
tested
at
2.856
mg/
mL
(5.002
mM),
and
prepared
by
adding
6
mL
of
a
11,900
mg/
mL
stock
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solution
CA058­
SS0001
to
a
25
mL
culture
solution;
N­
EtFOSAA
was
tested
at
2.539
mg/
mL
(4.341
mM),
prepared
by
adding
6
mL
of
a
10,580
mg/
mL
stock
solution
CA058­
SS­
009
to
a
25
mL
culture;
the
M556
was
tested
at
2.595
mg/
mL
(4.659
mM),
prepared
by
adding
65
mL
of
a
998
mg/
mL
stock
solution
CA­
TCR02­
006
to
a
25
mL
culture;
N­
EtFOSA
was
tested
at
2.825
mg/
mL
(5.360
mM),
prepared
by
adding
6
mL
of
a
11,770
mg/
mL
stock
solution
CA058­
SS­
008
to
a
25
mL
culture;
FOSA
was
tested
at
2.566
mg/
mL
(5.141
mM),
prepared
by
adding
6
mL
of
a
10,690
mg/
mL
stock
solution
CA058­
SS­
007
to
a
25
mL
culture;
PFOS
was
tested
at
2.455
mg/
mL
(4.564
mM),
prepared
by
adding
6
mL
of
a
10,230
mg/
mL
stock
solution
CA058­
SS­
002
to
a
25
mL
culture;
PFOSulfinate
was
tested
at
2.647
mg/
mL
(5.071
mM),
prepared
by
adding
6
mL
of
a
11,030
mg/
mL
stock
solution
CA058­
SS­
006
to
a
25
mL
culture;
PFOA
was
tested
at
2.645
mg/
mL
(6.136
mM),
prepared
by
adding
6
mL
of
a
11,020
mg/
mL
stock
solution
CA058­
SS­
003
to
a
25
mL
culture.

The
day
zero
samples
were
immediately
placed
in
a
freezer
and
maintained
at
–20°
C
until
final
culture
preparation.
All
of
the
other
test
cultures
and
control
cultures
were
placed
in
a
temperature
controlled
shaking
incubator
maintained
at
25°
C
±
3°
C,
and
shaken
at
200
rpm
for
aeration.
Samples
were
incubated
for
18
days.
After
18
days,
the
cultures
were
removed
from
the
incubator
and
frozen
until
final
sample
preparation
by
SPE.

All
culture
preparation
information,
including
times,
analyte
additions,
etc.

were
recorded
into
sample
preparation
worksheets
and
signed
and
dated
by
the
sample
preparation
analyst.
All
original
data
sheets
and
exact
copies
of
incubator
and
freezer
logs
were
kept
in
a
project
specific
binder
and
labeled
as
project
CA097.
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Friday,
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23,
2001
Page
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38
6.1.3
Solid
Phase
Extraction
of
Cultures
All
Sample
cultures
and
control
cultures
were
prepared
by
solid
phase
extraction
methodology
using
SEP­
VAC
C18
6cc
SPE
cartridges
from
Waters
Corporation
(Part
No.
WAT036905).
A
sample
label
was
applied
to
each
SPE
cartridge
prior
to
use,
and
each
was
packed
with
a
plug
of
quartz
glass
wool
to
deter
plugging
of
the
SPE
cartridge
filter.
Each
SPE
cartridge
was
washed
prior
to
use
by
drawing
5
mL
of
methanol
and
then
5
mL
of
aqueous
1%
acetic
acid
solution
through
the
cartridge.
The
wash
solution
eluates
were
discarded
to
waste.
All
of
the
SPE
eluates
for
samples
were
collected
in
clear
I­
chem
vials
with
labels
that
identified
them
as
eluate
1,
2
or
3,
as
defined
below.

Samples
that
had
been
frozen
were
thawed
at
room
temperature
before
extraction.
Following
the
thawing,
and
prior
to
the
solid
phase
extraction,

0.25
mL
of
glacial
acetic
acid
was
added
to
each
of
the
cultures
yielding
a
final
concentration
of
1%
acetic
acid.
The
content
of
each
acidified
culture
was
swirled
to
mix,
and
then
the
sample
contents
drawn
by
vacuum
through
the
appropriately
labeled
SPE
cartridge.
The
aqueous
eluate
was
collected
in
an
I­
chem
vial
labeled
eluate
1,
removed
from
the
vacuum
manifold,
and
capped.
Then,
25
mL
of
methanol
was
added
to
the
culture
flask,
the
flask
sealed,
and
vigorously
shaken.
The
cap
was
then
removed
from
the
flask,
and
the
methanol
content
(25
mL)
drawn
through
the
SPE
cartridge,
collected
in
an
I­
chem
vial
labeled
eluate
2.

Eluate
2
was
expected
to
contain
a
majority
of
the
analyte
that
was
in
the
original
culture
sample.
As
a
precaution
that
some
analyte
may
be
retained
in
the
SPE
cartridge,
or
in
the
culture
flask,
a
second
25
mL
methanol
step
was
conducted
in
a
similar
fashion
to
that
used
for
eluate
2,
and
collected
in
a
third
I­
chem
vial,
labeled
eluate
3.
Aliquots
of
eluates
2
and
3
were
transferred
to
autovials,
capped,
and
then
quantitatively
analyzed
by
HPLC/
MS.
The
remaining
volume
of
each
eluate
was
stored
at
4°
C
±
3°
C.
All
data
was
recorded
on
appropriated
culture
preparation
worksheets.
The
SPE
procedure
was
documented
as
method
CAG­
SP­
04
2
.
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2001
Page
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38
6.2
Instrumental
Analysis
Quantitative
LC/
MS
analysis
was
conducted
on
an
HP1100
high
performance
liquid
chromatography
with
mass
spectrometer
detector
(HPLC/
MSD)
system.

The
HPLC/
MSD
was
set
up
with
two
columns
(4.6
x
150
mm
Betasil
C8
column
and
a
4
x
35
mm
NG1
guard
column)
and
a
pressure
relief
valve
(Alltech
Part
No.
39025)
that
served
as
a
flow
through
splitter.
This
operating
method
used
was
documented
as
method
CAG­
ORG­
23
4
.
The
MSD
was
operated
in
electrospray
ionization­
negative
mode
with
selected
ion
monitoring
(SIM).
Ions
monitored
included:
m/
z
413
(PFOA);
m/
z
483
(PFOSulfinate);
m/
z
498
(FOSA);

m/
z
499
(PFOS);
m/
z
526
(N­
EtFOSA);
m/
z
556
(M556);
m/
z
584
(N­
EtFOSAA);

and
m/
z
630
(N­
EtFOSE
Alcohol­
acetate
adduct).
All
analysis
was
quantitative,

with
8
calibration
standards
prepared
spanning
approximately
5
to
1000
ng/
mL
(ppb)
and
containing
each
of
the
target
analytes.
Typical
injection
volumes
for
samples
and
calibration
standards
were
50
mL,
except
were
otherwise
noted
for
instrumental
dilutions
in
section
7.5
below.

MSD
Settings:
HPLC
Settings:

Ionization
mode:
API­
ES
negative
Flow:
1
mL/
min,
splitter
used,
near
10:
1
split
Gas
Temp:
300°
C
Time
(min).
%A
%B
Drying
gas:
8.0
L/
minute
0.00
97.0
3.0
Nebulizer
Pressure:
30
psig
0.50
97.0
3.0
Vcap:
3500V
6.00
5.0
95.0
Fragmentor:
70V
8.50
5.0
95.0
EMV
Gain:
2.0
8.51
97.0
3.0
Actual
Dwell
for
each
ion:
146
10.50
97.0
3.0
SIM
resolution:
high
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Friday,
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2001
Page
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38
6.3
Data
Transformations
and
Calculations
6.3.1
Molar
Calculations:

Because
all
data
was
reported
from
the
analysis
as
ng/
mL
(part
per
billion,
ppb),

the
transformation
of
ng/
mL
to
molar
concentrations
had
to
be
conducted
to
obtain
mass
balance
information.
The
mole
conversion
values
used
for
each
of
the
analytes
were
as
follows:

PFOA­
NH4+
salt
=
431
nanogram
(ng)
per
nanomole
(nmole)

PFOSulfinate­
K+
salt
=
522
ng/
nmole
PFOS­
K+
salt
=
538
ng/
nmole
FOSA
=
499
ng/
nmole
N­
EtFOSA
=
527
ng/
nmole
M556
acid
=
557
ng/
nmole
N­
EtFOSE
acid
=
585
ng/
nmole
N­
EtFOSE
alcohol
=
571
ng/
nmole
6.3.2
Conversion
of
ng/
mL
to
micromolar
(m
mM).

(Working
Examples):

500
ng/
mL
PFOSulfinate­
K+
=
(500
ng/
mL)*(
1nmole/
522
ng)
=
0.958
nmole/
mL
=
0.958
mmole/
liter
=
0.958
mM
50
ng/
mL
PFOS­
K+
=
(50
ng/
mL)
*
(0.1
nmole/
522
ng)
=
0.0958
nmole/
mL
=

0.0958
mmole/
L
=
0.0958
mM
=
95.8
nM
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2001
Page
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38
6.3.3
Mass
Balance
Calculations:

All
ng/
mL
values
were
converted
to
their
corresponding
micromolar
concentrations
(mM)
(see
section
7.3.2,
above).
Then,
the
sum
of
the
quantified
molar
values
(mM)
for
all
analytes
in
eluate
2
and
eluate
3
were
calculated
for
each
culture.
The
sum
value
was
then
divided
by
the
known
test
substrate
concentration
added,
and
that
end
value
reported
as
a
percentage
of
the
known
starting
concentration.

(Working
Example):

Starting
EtFOSE­
OH
was
at
1800
mM
After
incubation,
the
following
were
determined:

PFOA
detected
at
50
mM
PFOS
detected
at
100
mM
N­
ETFOSE
ACID
detected
at
500
mM
EtFOSE­
OH
detected
at
1000
mM
Mass
balance
=
[(
50mM
+
100mM
+
500mM
+
1000mM)/
1800mM]
X
100%

Mass
balance
=
(1650mM/
1800mM)
X
100%
=
91.7%

6.4
Software
Versions
Microsoft™
Excel
2000
was
used
for
data
processing
and
producing
tables.

Microsoft™
Word
2000
was
used
for
processing
the
analytical
report
text.

ChemSketch
v.
4.55
was
used
for
preparation
of
chemical
drawings
&
figures
HP
Chemstation
for
HPLC/
MSD
Rev.
A.
07.01was
used
for
analysis.
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2001
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38
7.0
Results
7.1
Incubator,
Refrigerator,
and
Freezer
Temperature
Log
Data.

During
this
study,
one
reciprocal
shaker
incubator
was
used.
The
Pace
ID
number
for
the
incubator­
shaker
was
0270.
The
Shaker
Speed
on
the
incubator
was
set
to
200
rpm,
but
was
not
regularly
verified.

One
–20
°C
freezer,
a
Norlake
freezer,
was
used
during
the
study
for
the
storage
of
cultures
until
they
could
be
prepared
by
solid
phase
extraction.
The
freezer
ID
was
0050
and
was
stored
at
location
SL­
R6
at
Pace.

One
IsoTemp
ultra­
low
freezer
(­
80°
C)
was
used
for
storage
of
test,
control
and
reference
materials
during
the
study.
The
freezer
ID
was
0241
and
was
maintained
at
location
SL­
R4
at
Pace.

Two
refrigeration
units,
a
Carroll
walk­
in
cooler
and
a
True
refrigerator,
were
used
during
the
study
for
storage
of
the
SPE­
prepared
samples
and
storage
of
stock
standards.
The
ID
of
the
coolers
was
ID
0140
(location
SL­
R1)
and
ID
0213
(location
SL­
R8),
respectively.

The
following
average
temperatures
recorded
±
their
standard
deviation
for
each
during
the
timeframe
of
the
study
are
shown
below.
The
average
temperature
and
standard
deviation
are
based
on
the
current
reading,
low
reading,
and
high
reading
recorded
in
the
instrument
logs
each
day
for
the
study,
excluding
weekends
and
holidays.

Incubator­
shaker,
ID
0270,
from
dates
9/
8/
00
to
10/
6/
00:
25.8°
C
±
0.9°
C
Norlake
Freezer,
ID
0050,
from
dates
9/
8/
00
to
10/
6/
00:
­16.2°
C
±
4.5°
C
IsoTemp
Freezer,
ID
0241,
from
dates
9/
8/
00
to
10/
6/
00:
­80.5°
C
±
3.6°
C
Carroll
walk­
in
cooler,
ID
0140,
from
dates
9/
8/
00
to
10/
6/
00:
3.7°
C
±
1.7°
C
True
refrigerator,
ID
0213,
from
dates
9/
8/
00
to
10/
6/
00:
3.9°
C
±
1.1°
C
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Friday,
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23,
2001
Page
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38
7.2
Mishaps
recorded
during
the
Study.

Originally,
the
study
was
intended
for
21
days
of
incubation.
However,
for
convenience
the
samples
were
harvested
after
18
days
of
incubation.
Hence,

samples
and
controls
labeled
for
21­
days
of
incubation
were
actually
18­
day
incubated
samples.

7.3
Mixed
Liquor
Suspended
Solids
Characterization
Report
The
results
of
the
chemical
analysis
of
the
mixed
liquor
suspended
solids
(MLSS)

used
for
this
study
were
as
follows:
The
date
of
collection
of
the
MLSS
was
August
1,
2000
and
the
digestion
date
was
August
3,
2000.
The
Pace
project
number
for
the
MLSS
characterization
report
was
1035161.
The
pH,
5­
Day
BOD,
and
total
sulfur
content
of
the
sludge
were
not
determined,
however,
for
future
studies
it
is
recommended
that
these
parameters
be
measured.

7.3.1
Metals:

Boron:
600
mg/
L
Cadmium:
12.9
mg/
L
Calcium:
124,000
mg/
L
Chromium:
273
mg/
L
Cobalt:
15.6
mg/
L
Copper:
1,450
mg/
L
Iron:
31,000
mg/
L
Lead:
140
mg/
L
Magnesium:
29,700
mg/
L
Manganese:
12,200
mg/
L
Molybdenum:
60
mg/
L
Nickel:
123
mg/
L
Selenium:
28
mg/
L
Potassium:
30,500
mg/
L
Zinc:
1,320
mg/
L
7.3.2.
Wet
Chemistry:

Sulfate:
57.1
mg/
L
Orthophosphate:
6.8
mg/
L
Phosphorous:
56.7
mg/
L
Ammonia:
1.3
mg/
L
Kjeldahl
Nitrogen:
197
mg/
L
Chemical
Oxygen
Demand
(COD):
3,440
mg/
L
Total
Organic
Carbon
(TOC):
120
mg/
L
Total
suspended
solids:
2,280
mg/
L
Total
solids:
3,070
mg/
L
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7.4
Quality
Control/
Sample
Matrix
Spike
Results.

Analyte
recoveries
from
sludge­
containing
sample
matrices
by
the
methods
employed
for
this
study
have
been
determined
twice
previously
for
several
analyte
concentrations,
and
in
both
instances
recoveries
were
near
100%,
with
some
exceptions
for
M556.
Those
recoveries
were
described
in
the
two
reports
submitted
to
3M
concerning
project
CA058.
Analyte
recoveries
from
similar
controls
was
deemed
unnecessary
for
this
study.

7.5
HPLC/
MS
Calibration
and
Calibration
Verification.

The
LC/
MS
calibration
standards
were
multi­
component
and
contained
PFOA,

PFOSulfinate,
PFOS,
FOSA,
N­
EtFOSA,
M556,
N­
EtFOSE
acid
and
N­
EtFOSE
alcohol.
Eight
calibration
standards
were
prepared
in
methanol
at
approximately
5,
10,
20,
40,
100,
200,
500
and
1000
ng/
mL,
and
were
labeled
as
CA058­

MW014
to
CA058­
MW­
021.
The
exact
concentration
values
for
these
standards
were
used
for
quantitation
of
the
analytes
in
the
samples.
Aliquots
of
calibration
standards
(prepared
as
10
mL
solutions)
were
transferred
to
autovials
and
capped
for
use
in
LC/
MS
runs.
Continuing
calibration
verification
(CCV)
was
included
in
sequence
runs.
The
CCVs
used
were
aliquots
of
standards
CA058MW
016
(20
ng/
mL)
and
CA058­
MW­
019
(200
ng/
mL).
The
CCV
results
were
used
for
checking
both
low
and
high
range
curve
data
integrity
and
reliability
of
reported
concentrations
during
a
sequence
run.

Instrument
calibration
was
performed
for
each
analyte
with
8
calibration
standards
injected.
Compounds
were
quantified
by
use
of
either
the
low
5
calibration
points
(5
to
100
ng/
mL)
or
the
high
5
calibration
points
(40
to
1000
ng/
mL).
Values
below
100
ng/
mL
were
reported
based
on
the
values
reported
from
low
calibration
curve
data,
and
all
residuals
in
that
range
were
100%
±
25%.

Values
reported
between
100
and
1000
ng/
mL
were
from
high
calibration
curve
data,
and
again
residuals
on
values
in
that
range
were
100%
±
25%.

Concentrations
reported
above
1000
ng/
mL
were
determined
from
instrument
runs
in
which
10
mL
of
sample
was
injected
and
quantified
off
of
calibration
curves
generated
from
50
mL
of
calibration
standards
being
injected.
Data
from
instrumental
dilutions
were
obtained
using
high
range
calibrations,
with
residuals
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of
100%
±
25%.
The
instrumental
dilutions
resulted
in
a
dilution
factor
of
5
being
used
to
back
calculate
the
true
analytical
concentration
in
the
sample.

Calibration
check
and
verification
standards
(CCVs)
were
used
after
15
sample
injections
and
included
both
calibration
standards
CA058­
MW­
016
(20
ng/
mL)

and
CA058­
MW­
019
(200
ng/
mL)
for
verification
of
low
and
high
range
curves.

CCVs
were
flanked
by
methanol
blanks.
All
quantitation
was
conducted
using
averaged
calibration
curves
flanking
30
samples,
one
set
of
CCVs,
and
the
pertinent
methanol
blanks.
All
calibration
curves
were
slightly
quadratic
and
had
R­
values
of
at
least
0.990.

All
data
was
of
good
to
excellent
quality,
with
residuals
on
curves
and
CCVs
in
the
acceptable
range
of
100%
±
25%.
Data
was
collected
from
external
standard
calibration,
and
not
internal
standard
as
suggested
by
ETS­
8­
136.0.

7.6
Blanks
Methanol
blanks
were
employed
during
the
HPLC/
MS
analysis
and
consisted
of
pure
methanol
injected
onto
the
HPLC/
MS
columns
for
analysis.
Methanol
blanks
were
analyzed
to
determine
the
background
level
of
analytes
in
the
methanol
solvent
used
during
sample
preparation.
The
results
of
methanol
blank
injections
verified
that
there
were
no
detectable
target
analytes
in
the
methanol
used
for
this
study.

Sample
blanks,
consisting
of
mineral
medium
containing
sludge
and
no
analyte,

were
incubated
and
prepared
the
same
as
regular
samples.
Results
obtained
were
used
to
determine
whether
the
sample
matrix
previously
contained
any
of
the
analytes
of
interest.
Analysis
of
the
sample
blanks
verified
that
there
were
no
detectable
fluorochemical
analytes
of
interest
in
sample
blanks
during
this
study.

7.7
Sample
Results
The
term
biodegradation
can
be
used
to
describe
biotransformation
or
biomineralization
(complete
breakdown
to
inorganic
components).
The
biodegradation
described
below
may
be
more
appropriately
termed
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2001
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biotransformation.
Biodegradability
was
arbitrarily
classified
depending
on
how
much
test
substrate
remained
after
18
days
incubation,
and
were
as
follows:
1)

Biodegraded
well
(<
25%
parent
remaining);
2)
Moderately
biodegraded
(25­
75%
of
parent
remaining);
3)
Poorly
biodegraded
(75­
95%
parent
remaining);
and
4)
Not
biodegraded
(95­
110%
of
parent
remaining).

7.7.1
N­
EtFOSE
Alcohol
The
N­
EtFOSE
alcohol
was
biodegraded
well
in
the
18­
day
incubation
experiments
(Figure
1)
with
only
15.9%
of
the
original
N­
EtFOSE
alcohol
remaining
after
18
days
incubation
with
sludge
(Appendix
D,
Table
1).
The
biodegradation
resulted
in
N­
EtFOSAA
as
the
major
biotransformation
product.
Minor
products
were
M556,
N­
EtFOSA,
FOSA,
PFOS,

PFOSulfinate
and
PFOA.
The
results
were
in
good
agreement
with
those
obtained
from
previous
biodegradation
experiments
(see
separate
reports
for
project
CA058).
The
N­
EtFOSE
alcohol
cultures
served
as
a
positive
control
for
this
study
and
supported
the
fact
that
the
biological
activity
of
the
MLSS
used
for
this
study
was
sufficient
for
biodegradation
to
occur.
Mass
balance
for
N­
EtFOSE
alcohol
cultures
ranged
from
88%
to
107%.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
1.
The
biodegradation
of
N­
EtFOSE
alcohol,
showing
N­
EtFOSE
alcohol
biodegraded
well
to
yield
N­
EtFOSAA,
M556
and
PFOS.
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2001
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7.7.2
N­
EtFOSAA
The
N­
EtFOSAA
was
poorly
degraded
in
18­
day
incubation
experiments
(Figure
2).
This
was
an
unexpected
result,
since
degradation
of
N­
EtFOSAA
had
been
observed
in
study
CA058,
and
this
compound
was
expected
to
be
a
good
substrate.
Only
9.3
%
of
the
N­
EtFOSAA
was
degraded,
and
yielded
product
M556,
FOSA,
PFOS
and
PFOA
(see
Appendix
D,
Table
1
for
final
results).
Some
of
the
observed
products
may
be
attributed
to
degradation
of
the
impurities
present
in
the
test
material,
which
included:
N­
EtFOSA,

PFOSulfinate,
and
PFOA.
Both
N­
EtFOSA
and
PFOSulfinate
were
present
at
day
0
but
could
not
be
detected
by
day
18,
and
could
account
for
some,

but
not
all,
of
the
products
observed.
The
low
level
of
biodegradability
detected
could
be
explained
if
N­
EtFOSAA
is
toxic
to
microorganisms
in
sludge.
Alternatively,
the
lack
of
biodegradation
may
have
been
due
to
a
reduced
bioavailability
of
the
substrate
caused
by
a
higher
molecular
polarity
of
N­
EtFOSAA
(an
acid),
which
would
result
in
lowered
membrane
permeability.
Another
possible
explanation
for
the
observed
poor
biodegradability
may
have
been
that
the
substrate
was
unable
to
induce
the
appropriate
genes
and
enzymes
required
for
biodegradation
to
occur.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
2.
The
biodegradation
chart
for
N­
EtFOSAA,
showing
poor
biodegradability
and
low
levels
of
products
formed.
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2001
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38
7.7.3
N­
EtFOSA
After
the
18­
day
incubation,
13.7%
of
the
parent
N­
EtFOSA
substrate
remained,
and
the
biotransformation
products
M556,
FOSA,
PFOSulfinate,

PFOS
and
PFOA
were
formed
(Figure
3).
The
N­
EtFOSA
was
termed
moderately
biodegraded
because
only
31%
of
the
original
N­
EtFOSA
added
to
the
no­
sludge
controls
remained
after
18
days
of
incubation,
and
no
biotransformation
products
were
detected,
see
Appendix
D,
Table
1.
After
18
days,
mass
balance
for
N­
EtFOSA
was
the
poorest
of
all
analytes
tested,

at
approximately
30­
35%
recovery.
These
results
may
indicate
loss
of
the
test
material
by
volatilization
to
the
air,
since
the
vapor
pressure
of
NEtFOSA
while
currently
unknown,
may
be
significant,
and
cultures
were
incubated
with
test
flasks
loosely
capped
to
allow
for
the
exchange
of
air
into
the
flasks.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
3.
The
biodegradation
chart
for
N­
EtFOSA,
showing
the
biotransformation
of
N­
EtFOSA
to
yield
the
products
M556
and
FOSA
as
the
major
products.
Losses
of
N­
EtFOSA
in
both
the
test
cultures
and
the
nosludge
control
cultures
may
be
attributed
to
volatilization,
accounting
for
the
poor
mass
balance
values.
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38
7.7.4
M556
The
test
substrate
M556
was
moderately
biodegraded
after
the
18­
day
incubation
(Figure
4),
with
74.4%
of
the
parent
material
remaining
in
cultures
that
contained
sludge
(see
Appendix
D,
Table
1).
The
M556
did
not
degrade,
and
no
quantifiable
products
were
detected
in
the
no­
sludge
controls
following
18
days
of
incubation.
Mass
balance
for
M556
was
excellent
with
recoveries
between
95
and
112%.
The
results
indicate
that
M556
was
biotransformed
to
FOSA
(major
product),
PFOSulfinate,
PFOS
and
PFOA.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
4.
The
biotransformation
chart
for
M556,
showing
the
moderate
biodegradation
of
M556
with
FOSA
as
the
major
product.
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7.7.5
FOSA
The
test
substrate
FOSA
was
poorly
biodegraded
in
18­
day
incubation
experiments,
with
89.8%
of
the
parent
material
remaining
in
test
cultures
after
incubation
(see
Appendix
D,
Table
1
and
Figure
5).
No
degradation
of
FOSA
was
observed
in
the
no­
sludge
abiotic
controls.
Mass
balance
for
FOSA
was
excellent
with
recoveries
between
94%
and
101%.
The
results
indicate
that
FOSA
was
biotransformed
to
PFOSulfinate,
PFOS
and
PFOA.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
5.
The
biotransformation
chart
for
FOSA,
showing
the
biodegradation
of
FOSA
to
yield
PFOS
as
the
major
product.
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7.7.6
PFOSulfinate
PFOSulfinate
was
observed
to
degrade
in
both
the
test
cultures
and
the
nosludge
abiotic
control
cultures.
However,
the
products
formed
were
dependent
on
whether
sludge
was
present
or
not,
as
demonstrated
in
Figure
6
and
presented
in
the
data
from
Appendix
D,
Table
1.

The
formation
of
PFOA
was
observed
as
the
major
product
from
PFOSulfinate
in
the
abiotic
controls
and
was
likely
caused
by
an
abiotic
mechanism
described
previously
by
Hu
et
al.
(1990)
5
.
This
reaction
occurs
rapidly,
as
approximately
15%
of
the
PFOSulfinate
transformed
in
the
day
zero
abiotic
controls.
After
18
days,
approximately
60%
of
the
PFOSulfinate
was
transformed
to
yield
PFOA
(major
product)
and
PFOS
(minor
product)
at
a
10:
1
mole
ratio.

In
sharp
contrast
to
the
products
observed
in
the
abiotic
controls,
sample
cultures
containing
sludge
resulted
in
84%
of
the
PFOSulfinate
being
biotransformed
to
PFOS
(major
product)
and
PFOA
(minor
product)
at
an
8:
1
mole
ratio,
a
complete
reversal
of
the
behavior
observed
in
abiotic
controls.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
6.
The
transformation
chart
for
PFOSulfinate,
showing
the
biotransformation
of
PFOSulfinate
to
PFOS
as
the
major
product,
and
the
chemical
transformation
of
PFOSulfinate
in
abiotic
controls
to
yield
PFOA
as
the
major
product.
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7.7.7
PFOS
As
shown
in
Figure
7
and
presented
in
the
data
of
Appendix
D,
Table
1,

PFOS
was
not
measurably
biodegraded
in
the
18­
day
incubation
experiments
in
either
the
test
cultures
or
abiotic
controls.
This
could
be
because
it
cannot
be
biodegraded
under
the
test
conditions,
or
because
it
is
toxic
at
the
tested
concentrations.
It
is
also
possible
that
a
lack
of
bioavailability
could
explain
the
lack
of
biodegradation.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
7.
The
transformation
chart
for
PFOS,
showing
no
degradation
with
PFOS
as
a
test
substrate.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
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Lange,
Ph.
D.,
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Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
26
of
38
7.7.8
PFOA
The
PFOA
was
not
measurably
biodegraded
in
the
18­
day
incubation
experiments,
as
shown
in
Figure
8
and
presented
in
the
data
of
Appendix
D,
Table
1.
As
with
PFOS,
the
lack
of
activity
may
be
because
it
cannot
be
biodegraded
under
the
test
conditions,
it
may
be
toxic
at
the
tested
concentrations,
or
it
is
simply
not
bioavailable.
PFOA
was
also
not
degraded
in
controls,
suggesting
it
is
stable
under
all
the
conditions
tested.

PFOA
PFOS
PFOS
sulfinate
FOSA
M556
N­
EtFOSA
N­
EtFOSE
acid
N­
EtFOSE
alcohol
Day
0
biodeg
sample
Day
18
biodeg
sample
Day
0
no­
sludge
control
Day
18
no
sludge
control
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Concentration
(uM)

Figure
8.
The
transformation
chart
for
PFOA,
showing
no
degradation
with
PFOA
as
a
substrate.

9.0
Conclusions
The
18­
day
incubations
of
the
targeted
fluorochemicals
resulted
in
several
interesting
and
unanticipated
discoveries.
First,
the
formation
of
PFOA
from
PFOSulfinate
was
found
to
occur
predominantly
in
the
no­
sludge
abiotic
controls.
This
observed
formation
of
PFOA
from
PFOSulfinate
in
the
abiotic
controls
is
supported
by
a
1990
publication
that
described
a
1­
electron
elimination
process
for
conversion
of
perfluorinated
sulfinic
acids
to
perfluorinated
carboxylic
acids
4
.
In
contrast,
samples
of
PFOSulfinate
that
contained
MLSS
sludge
formed
PFOS
as
the
major
product.
This
was
postulated
to
be
due
to
a
biological
sulfur
oxidation
mechanism.
Further
transformations
of
PFOS
and
PFOA
were
BACK
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Project
CA097
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Day
Aerobic
Biodegradation
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Lange,
Ph.
D.,
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Analytical
Services,
Science
Solutions
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Friday,
February
23,
2001
Page
27
of
38
not
observed.
This
was
supported
by
the
observation
that
no
changes
occurred
in
the
sample
cultures
and
controls
for
the
study
of
PFOS
and
PFOA
after
18
days
of
incubation.
PFOS
and
PFOA
are
probably
endpoints
of
the
biodegradation
of
N­
EtFOSE
alcohol
in
municipal
waste
treatment
facilities.
The
degradation
pathway
deduced
from
this
work
is
represented
in
Figure
9
below.

A
second
significant
discovery
was
the
observation
that
several
of
the
metabolites
studied
which
were
expected
to
be
easily
biotransformed,
were
not
significantly
biotransformed
at
the
concentrations
tested.
This
may
be
explained
by
several
possible
reasons.
First,
the
test
substance
may
be
toxic
to
the
microorganisms
responsible
for
the
biodegradation
activity
at
the
analyte
concentration
tested.
Second,
the
test
substance
may
be
too
polar
to
cross
the
biological
cell
membranes,
or
may
adsorb
to
sludge
or
test
containers,
thus
limiting
the
bioavailability
of
the
analyte
to
the
interior
of
the
cell
where
the
reactions
may
take
place.
Third,
the
test
substrate
may
be
unable
to
successfully
cause
the
induction
of
appropriate
genes
and
enzyme
required
for
biodegradation
activity.
Compounds
that
were
expected
to
be
highly
biodegradable,
but
exhibited
low
biodegradability,
were:
N­
EtFOSAA,
N­
EtFOSA,
and
FOSA.

As
expected,
PFOS
and
PFOA
did
not
biodegrade
during
18
days
of
incubation
with
aerobic
MLSS
sludge,
and
both
are
likely
the
endpoint
products
for
perfluorooctanesulfonyl­
based
chemistries
when
they
enter
into
biological
waste
treatment
facilities.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
28
of
38
F
F
F
F
F
F
F
F
F
F
F
F
S
O
O
F
F
F
F
F
OH
O
OH
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
PFOA
X.
F
F
F
F
F
F
F
F
F
F
F
F
N
S
O
O
F
F
F
F
F
OH
CH
3
F
F
F
F
F
F
F
F
F
F
F
F
N
S
O
O
F
F
F
F
F
O
CH3
N­
EtFOSE
Aldehyde
F
F
F
F
F
F
F
F
F
F
F
F
N
S
O
O
F
F
F
F
F
OH
CH3
O
F
F
F
F
F
F
F
F
F
F
F
F
NH
S
O
O
F
F
F
F
F
OH
O
F
F
F
F
F
F
F
F
F
F
F
F
CH
3
NH
S
O
O
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
NH2
S
O
O
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
O
O
F
F
F
F
F
H
F
F
F
F
F
F
F
F
F
F
F
F
NH
S
O
O
F
F
F
F
F
OH
F
F
F
F
F
F
F
F
F
F
F
F
NH
S
O
O
F
F
F
F
F
O
N­
EtFOSE
Alcohol
N­
EtFOSE
Acid
M556
N­
EtFOSA
PFOS
FOSA
PFOSulfinate
N­
EtFOSA
alcohol
N­
EtFOSA
aldehyde
I.

II.

III.

IV.

V.

VI.

VII.

VIII.

IX.

XI.
Abiotic
mechanism
Figure
9.
The
proposed
biodegradation
pathway
from
project
CA058
for
N­
EtFOSE
alcohol
as
mediated
by
the
microbial
activity
of
aerobic
municipal
sludge.
The
pathway
was
confirmed
by
the
results
of
this
study
by
incubating
each
analyte
with
aerobic
sludge
for
18
days.
PFOS
and
PFOA,
shown
in
rectangles,
did
not
degrade
over
the
course
of
the
study
and
are
probably
deadend
metabolites.
Intermediates
circled
were
not
detected
in
this
study,
nor
project
CA058,
but
were
proposed
intermediates
based
on
the
expected
2­
electron
oxidation
mechanisms
of
several
degradative
enzymes.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
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Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
29
of
38
9.0
Sample
and
Data
Retention
Original
facility
records
and
a
copy
of
all
data
and
the
final
report
will
be
retained
in
the
Pace
Analytical­
Tier
2
data
archives
for
a
period
of
5
years
after
completion
of
the
project.
At
the
end
of
5
years,
the
data
will
be
disposed
of
following
standard
laboratory
procedures
defined
in
the
document
PSS­
ARC­
02.

The
following
will
be
provided
to
3M
personnel:

·
The
original
signed
report
and
any
later
amendments
to
the
report.

·
Copies
of
all
original
raw
data
in
the
form
of
chromatograms,
reduced
data,
and
written
records
concerning
this
project.

·
Copies
of
all
instrumental
raw
data
will
be
archived
onto
CD
and
a
copy
provided
to
3M
Facility
data
will
be
retained
for
a
period
of
10
years.
Facility
data
is
available
for
inspection
and
includes
the
following
records:

§
Training
records
§
Controlled
storage
temperature
logs
§
Standard
preparation
logs
§
Calibration
and
maintenance
logs
§
Chemical
and
solvent
traceability
logs
§
Standard
Operating
Procedures
§
Methods
pertaining
to
the
conduct
of
this
project
The
remaining
sample
extracts
will
be
retained
at
the
Pace
Analytical
facility
for
a
period
of
2
years
after
completion
of
the
project
at
4°
C
in
the
Carroll
walk­
in
cooler
(Pace
ID
0140)
located
in
the
Pace
Analytical­
Tier
2
facility.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
30
of
38
10.0
References
1.
Final
reports
for
Project
CA058:
"2­
week
N­
EtFOSE
Alcohol
Biodegradation
Screen
Study
Report"
and
"Aerobic
Biodegradation
of
N­
EtFOSE
Alcohol
Study
Report",
3M
LIMS
Project
#
E00­
2252.

2.
Pace
Science
Solutions
method
CAG­
SP­
04
"C18
Solid
Phase
Extraction
Procedure
for
Fluorochemicals
Recovery
from
Aqueous/
Sludge
Matrices."

3.
Pace
Science
Solutions
method
CAG­
SP­
03
"Culture
Preparation
for
Assessment
of
Aerobic
Biodegradability
of
Fluorochemicals
Using
Municipal
or
Industrial
Sludge
as
Microbial
Inoculum"

4.
Pace
Science
Solutions
method
CAG­
ORG­
23
"Quantitative
Analysis
of
Fluorochemicals
by
High
Performance
Liquid
Chromatography
with
Mass
Spectrometric
Detection".

5.
Hu,
Chang­
Ming,
Ze­
Qi
Xu,
and
Wei­
Yuan
Huang.
1990.
"Reaction
of
Perhalofluoroalkyl
Sulfinates
with
One­
Electron
Transfer
Oxidants.
A
Facile
Method
for
the
Synthesis
of
Perhalofluorocarboxylic
Acids".
J.
Fluorine
Chemistry.
49:
433­
437.
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
31
of
38
APPENDIX
A
SIGNATURES
OF
PROJECT
PERSONNEL
Project
Title:
18­
Day
Aerobic
Biodegradation
Study
for
Perfluorooctanesulfonate
Chemistries.

Client
Project
ID:
Not
Available
Contract
Analytical
Project
Number:
CA097
The
following
individuals
participated
in
the
conduct
of
this
project:

Study
Director
_____________________________
___________
Cleston
C.
Lange,
Ph.
D.
Date
Laboratory
Management:
_____________________________
___________
Bruce
E.
Warden
Date
Analysts/
Technicians:
_____________________________
___________
Angela
L.
Schuler
Date
_____________________________
___________
Anthony
E.
Scales
Date
Sample
Custodian:
_____________________________
___________
Cleston
C.
Lange,
Ph.
D.
Date
Report
Reviewed
by:
______
_______________________
___________
Dirk
W.
Hoogenboom
Date
Report
Reviewed
by:
_____________________________
___________
Kuruppu
N.
Dharmasiri,
Ph.
D.
Date
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
32
of
38
Appendix
B
TABLE
1.
The
quantitative
LC/
MS
analysis
results
for
eluate
2
(first
SPE
methanol
elution).
A
value
of
zero
implies
the
compound
was
not
detected
or
was
detected
at
a
level
less
than
50%
of
the
MQL
of
5
ng/
mL.

Concentration
(ng/
mL)

Sample
ID
Description
Eluate
PFOA
PFOS
PFOS
Sulfinate
M556
N

EtFOSE
acid
FOSA
N­
EtFOSE

alcohol
N­
EtFOSA
CA097­
0801­
SA­
200
DAY
0
BIODEG
FOR
PFOA
2
2758
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
201
DAY
0
BIODEG
FOR
PFOA
DUPLICATE
2
2673
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
202
DAY
0
BIODEG
FOR
PFOS
2
14.1
2385
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
203
DAY
0
BIODEG
FOR
PFOS
DUPLICATE
2
13.8
2451
0.
0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
204
DAY
0
BIODEG
FOR
PFOSulfinate
2
56.7
78.9
2634
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
205
DAY
0
BIODEG
FOR
PFOSulfinate
DUPLICATE
2
84.7
79.1
2660
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
206
DAY
0
BIODEG
FOR
FOSA
2
3.9
0.0
0.0
0.0
0.0
2518
0.0
0.0
CA097­
0801­
SA­
207
DAY
0
BIODEG
FOR
FOSA
DUPLICATE
2
3.3
0.0
0.0
0.0
0.0
2330
0.0
0.0
CA097­
0801­
SA­
208
DAY
0
BIODEG
FOR
N­
EtFOSA
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2521
CA097­
0801­
SA­
209
DAY
0
BIODEG
FOR
N­
EtFOSA
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2562
CA097­
0801­
SA­
210
DAY
0
BIODEG
FOR
M556
2
0.0
0.0
0.0
2617
0.0
4.8
0.0
0.0
CA097­
0801­
SA­
211
DAY
0
BIODEG
FOR
M556
DUPLICATE
2
0.0
0.0
0.0
2572
0.0
4.5
0.0
0.0
CA097­
0801­
SA­
212
DAY
0
BIODEG
FOR
N­
EtFOSE
acid
2
9.2
0.0
11.2
0.0
2377
0.0
0.0
18.9
CA097­
0801­
SA­
213
DAY
0
BIODEG
FOR
N­
EtFOSE
acid
DUPLICATE
2
9.1
0.0
11.1
0.0
2349
0.0
0.0
21.8
CA097­
0801­
SA­
214
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
2
0.0
0.0
0.0
0.0
0.0
0.0
2810
0.0
CA097­
0801­
SA­
215
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
2945
0.0
CA097­
0801­
SA­
216
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
2
2569
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
217
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
2
2700
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
218
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
2
13.7
2462
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
219
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
2
12.6
2534
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
220
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
2
333.2
72.5
2239
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
221
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
2
384.8
79.4
2158
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
222
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
2
3.3
0.0
0.0
0.0
0.0
2475
0.0
0.0
CA097­
0801­
SA­
223
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
2
3.6
0.0
0.0
0.0
0.0
2416
0.0
0.0
CA097­
0801­
SA­
224
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2142
CA097­
0801­
SA­
225
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2071
CA097­
0801­
SA­
226
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
2
0.0
0.0
0.0
2514
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
227
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
DUPLICATE
2
0.0
0.0
0.0
2638
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
228
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
2
8.5
0.0
11.1
4.6
2207
0.0
0.0
17.0
CA097­
0801­
SA­
229
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
DUPLICATE
2
8.5
0.0
11.2
0.0
1979
0.0
0.0
17.5
CA097­
0801­
SA­
230
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
2
0.0
0.0
0.0
0.0
0.0
0.0
2820
0.0
CA097­
0801­
SA­
231
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
2823
0.0
CA097­
0801­
SA­
232
DAY
0
SLUDGE
BLANK
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
233
DAY
0
SLUDGE
BLANK
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
234
DAY
18
BIODEG
FOR
PFOA
2
2821
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
235
DAY
18
BIODEG
FOR
PFOA
DUPLICATE
2
2618
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
236
DAY
18
BIODEG
FOR
PFOS
2
13.6
2484
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
237
DAY
18
BIODEG
FOR
PFOS
DUPLICATE
2
13.1
2447
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
238
DAY
18
BIODEG
FOR
PFOSulfinate
2
149.9
1444
369.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
239
DAY
18
BIODEG
FOR
PFOSulfinate
DUPLICATE
2
149.6
1404
409.6
0.0
0.0
0.0
0.0
0.0
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
33
of
38
Appendix
B,

TABLE
1
CONTINUED
Concentration
(ng/
mL)

Sample
ID
Sample
Description
Eluate
PFOA
PFOS
PFOS
Sulfinate
M556
N

EtFOSE
acid
FOSA
N­
EtFOSE

alcohol
N­
EtFOSA
CA097­
0801­
SA­
240
DAY
18
BIODEG
FOR
FOSA
2
12.2
73.9
22.1
0.0
0.0
1947
0.0
0.0
CA097­
0801­
SA­
241
DAY
18
BIODEG
FOR
FOSA
DUPLICATE
2
13.1
76.2
19.2
0.0
0.0
2300
0.0
0.0
CA097­
0801­
SA­
242
DAY
18
BIODEG
FOR
N
­EtFOSA
2
0.0
16.9
6.6
72.4
0.0
497.4
0.0
362.5
CA097­
0801­
SA­
243
DAY
18
BIODEG
FOR
N­
EtFOSA
DUPLICATE
2
0.0
16.2
5.7
72.5
0.0
478.3
0.0
332.2
CA097­
0801­
SA­
244
DAY
18
BIODEG
FOR
M556
2
4.7
21.5
6.1
1539
0.0
399.5
0.0
0.0
CA097­
0801­
SA­
245
DAY
18
BIODEG
FOR
M556
DUPLICATE
2
4.9
21.3
7.5
1813
0.0
441.0
0.0
0.0
CA097­
0801­
SA­
246
DAY
18
BIODEG
FOR
N­
EtFOSE
acid
2
13.7
12.0
4.6
31.2
2250
23.4
0.0
7.6
CA097­
0801­
SA­
247
DAY
18
BIODEG
FOR
N­
EtFOSE
acid
DUPLICATE
2
12.2
10.6
4.7
27.7
1929
25.8
0.0
10.3
CA097­
0801­
SA­
248
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
2
20.2
116.9
57.3
73.5
1779
23.4
411.6
9.5
CA097­
0801­
SA­
249
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
DUPLICATE
2
18.4
91.5
25.2
71.8
1727
26.2
430.9
15.0
CA097­
0801­
SA­
250
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
2
2733
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
251
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
2
2755
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
252
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
2
14.1
2366
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
253
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
2
14.2
2529
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
254
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
2
1255
129.9
787.5
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
255
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
2
913.9
128.7
1226
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
256
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
2
3.7
0.0
0.0
0.0
0.0
2482
0.0
0.0
CA097­
0801­
SA­
257
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
2
4.0
0.0
0.0
0.0
0.0
2557
0.0
0.0
CA097­
0801­
SA­
258
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1112
CA097­
0801­
SA­
259
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
397.6
CA097­
0801­
SA­
260
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
2
0.0
0.0
0.0
2645
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
261
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
DUPLICATE
2
0.0
0.0
0.0
2492
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
262
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
2
9.7
0.0
10.8
5.1
2421
0.0
0.0
0.0
CA097­
0801­
SA­
263
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
DUPLICATE
2
9.7
0.0
11.2
0.0
2514
0.0
0.0
0.0
CA097­
0801­
SA­
264
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
2
0.0
0.0
3.7
0.0
7.1
0.0
3398
0.0
CA097­
0801­
SA­
265
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
DUPLICATE
2
0.0
0.0
0.0
0.0
15.0
0.0
2630
0.0
CA097­
0801­
SA­
266
DAY
18
SLUDGE
BLANK
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
267
DAY
18
SLUDGE
BLANK
DUPLICATE
2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
34
of
38
Appendix
B,
Table
1
continued.

TABLE
2.
The
quantitative
LC/
MS
analysis
results
for
eluate
3
(second
SPE
methanol
elution).
A
value
of
zero
implies
the
compound
was
not
detected
or
was
detected
at
a
level
less
than
50%
of
the
MQL
of
5
ng/
mL.

Concentration
(ng/
mL)

Sample
ID
Description
Eluate
PFOA
PFOS
PFOS
Sulfinate
M556
N

EtFOSE
acid
FOSA
N­
EtFOSE

alcohol
N­
EtFOSA
CA097­
0801­
SA­
200
DAY
0
BIODEG
FOR
PFOA
3
59.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
201
DAY
0
BIODEG
FOR
PFOA
DUPLICATE
3
54.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
202
DAY
0
BIODEG
FOR
PFOS
3
0.0
161.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
203
DAY
0
BIODEG
FOR
PFOS
DUPLICATE
3
0.0
127.7
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
204
DAY
0
BIODEG
FOR
PFOSulfinate
3
0.0
6.7
170.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
205
DAY
0
BIODEG
FOR
PFOSulfinate
DUPLICATE
3
0.0
7.1
174.2
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
206
DAY
0
BIODEG
FOR
FOSA
3
0.0
0.0
0.0
0.0
0.0
70.3
0.0
0.0
CA097­
0801­
SA­
207
DAY
0
BIODEG
FOR
FOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
93.8
0.0
0.0
CA097­
0801­
SA­
208
DAY
0
BIODEG
FOR
N­
EtFOSA
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
124.1
CA097­
0801­
SA­
209
DAY
0
BIODEG
FOR
N­
EtFOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
130.0
CA097­
0801­
SA­
210
DAY
0
BIODEG
FOR
M556
3
0.0
0.0
0.0
233.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
211
DAY
0
BIODEG
FOR
M556
DUPLICATE
3
0.0
0.0
0.0
240.4
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
212
DAY
0
BIODEG
FOR
N­
EtFOSE
acid
3
0.0
0.0
0.0
0.0
231.8
0.0
0.0
0.0
CA097­
0801­
SA­
213
DAY
0
BIODEG
FOR
N­
EtFOSE
acid
DUPLICATE
3
0.0
0.0
0.0
0.0
223.9
0.0
0.0
0.0
CA097­
0801­
SA­
214
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
3
0.0
0.0
0.0
0.0
0.0
0.0
196.2
0.0
CA097­
0801­
SA­
215
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
137.3
0.0
CA097­
0801­
SA­
216
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
3
180.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
217
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
3
8.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
218
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
3
0.0
104.7
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
219
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
3
0.0
146.6
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
220
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
3
14.1
4.2
151.7
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
221
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
3
8.6
0.0
71.1
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
222
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
3
0.0
0.0
0.0
0.0
0.0
59.6
0.0
0.0
CA097­
0801­
SA­
223
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
56.1
0.0
0.0
CA097­
0801­
SA­
224
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
172.9
CA097­
0801­
SA­
225
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
157.7
CA097­
0801­
SA­
226
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
3
0.0
0.0
0.0
241.2
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
227
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
DUPLICATE
3
0.0
0.0
0.0
271.4
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
228
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
3
0.0
0.0
0.0
0.0
453.7
0.0
0.0
0.0
CA097­
0801­
SA­
229
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
DUPLICATE
3
0.0
0.0
0.0
0.0
561.8
0.0
0.0
0.0
CA097­
0801­
SA­
230
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
3
0.0
0.0
0.0
0.0
0.0
0.0
169.3
0.0
CA097­
0801­
SA­
231
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
148.6
0.0
CA097­
0801­
SA­
232
DAY
0
SLUDGE
BLANK
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
233
DAY
0
SLUDGE
BLANK
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
234
DAY
18
BIODEG
FOR
PFOA
3
67.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
235
DAY
18
BIODEG
FOR
PFOA
DUPLICATE
3
150.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
236
DAY
18
BIODEG
FOR
PFOS
3
0.0
104.6
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
237
DAY
18
BIODEG
FOR
PFOS
DUPLICATE
3
0.0
143.3
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
238
DAY
18
BIODEG
FOR
PFOSulfinate
3
5.7
130.2
35.3
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
239
DAY
18
BIODEG
FOR
PFOSulfinate
DUPLICATE
3
6.1
121.0
36.4
0.0
0.0
0.0
0.0
0.0
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
35
of
38
Appendix
B
TABLE
2,
CONTINUED
Concentration
(ng/
mL)

Sample
ID
Description
Eluate
PFOA
PFOS
PFOS
Sulfinate
M556
N

EtFOSE
acid
FOSA
N­
EtFOSE

alcohol
N­
EtFOSA
CA097­
0801­
SA­
240
DAY
18
BIODEG
FOR
FOSA
3
0.0
11.5
4.1
0.0
0.0
233.4
0.0
0.0
CA097­
0801­
SA­
241
DAY
18
BIODEG
FOR
FOSA
DUPLICATE
3
0.0
7.4
0.0
0.0
0.0
146.7
0.0
0.0
CA097­
0801­
SA­
242
DAY
18
BIODEG
FOR
N­
EtFOSA
3
0.0
0.0
0.0
13.1
0.0
34.7
0.0
51.6
CA097­
0801­
SA­
243
DAY
18
BIODEG
FOR
N­
EtFOSA
DUPLICATE
3
0.0
0.0
0.0
11.5
0.0
30.7
0.0
31.3
CA097­
0801­
SA­
244
DAY
18
BIODEG
FOR
M556
3
0.0
5.2
0.0
386.8
0.0
75.0
0.0
0.0
CA097­
0801­
SA­
245
DAY
18
BIODEG
FOR
M556
DUPLICATE
3
0.0
0.0
0.0
139.0
0.0
14.7
0.0
0.0
CA097­
0801­
SA­
246
DAY
18
BIODEG
FOR
N­
EtFOSE
acid
3
0.0
0.0
0.0
5.1
191.2
0.0
0.0
0.0
CA097­
0801­
SA­
247
DAY
18
BIODEG
FOR
N­
EtFOSE
acid
DUPLICATE
3
0.0
0.0
0.0
6.8
299.9
0.0
0.0
0.0
CA097­
0801­
SA­
248
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
3
0.0
15.2
7.3
13.8
260.1
0.0
30.1
0.0
CA097­
0801­
SA­
249
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
DUPLICATE
3
0.0
14.1
4.8
15.9
288.2
0.0
33.2
0.0
CA097­
0801­
SA­
250
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
3
36.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
251
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
3
31.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
252
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
3
0.0
100.7
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
253
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
3
0.0
75.7
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
254
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
3
12.9
9.3
43.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
255
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
3
9.7
8.8
66.5
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
256
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
3
0.0
0.0
0.0
0.0
0.0
74.4
0.0
0.0
CA097­
0801­
SA­
257
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
65.5
0.0
0.0
CA097­
0801­
SA­
258
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
94.3
CA097­
0801­
SA­
259
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
33.8
CA097­
0801­
SA­
260
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
3
0.0
0.0
0.0
229.5
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
261
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
DUPLICATE
3
0.0
0.0
0.0
237.5
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
262
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
3
0.0
0.0
0.0
0.0
248.8
0.0
0.0
0.0
CA097­
0801­
SA­
263
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
acid
DUPLICATE
3
0.0
0.0
0.0
0.0
305.9
0.0
0.0
0.0
CA097­
0801­
SA­
264
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
3
0.0
0.0
0.0
0.0
0.0
0.0
173.1
0.0
CA097­
0801­
SA­
265
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
DUPLICATE
3
0.0
0.0
0.0
0.0
4.4
0.0
206.2
0.0
CA097­
0801­
SA­
266
DAY
18
SLUDGE
BLANK
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
CA097­
0801­
SA­
267
DAY
18
SLUDGE
BLANK
DUPLICATE
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
36
of
38
Appendix
C
TABLE
1.
The
summed
results
for
the
SPE
eluate
2
and
eluate
3
collections
from
day
0
and
day
18
samples
for
each
analyte
with
aerobic
sludge.
Note
that
N­
EtFOSE
acid
and
FOSA
were
poorly
degraded
in
18
days
suggesting
either
toxicity
at
the
tested
concentration
or
a
lack
of
bioavailability
to
microorganisms.
Reduced
bioavailability
may
be
a
result
of
increased
polarity
of
the
test
compound
versus
that
of
the
more
neutral
N­
EtFOSE
alcohol,
and
thus
a
decreased
mobility
across
the
hydrophobic
cell
membrane
might
be
expected.
A
value
of
zero
implies
the
compound
was
not
detected
or
was
detected
at
a
level
less
than
50%
of
the
MQL
of
5
ng/
mL.

Concentration
(ng/
mL)

Sample
Description
Sample
ID
ELUATE
PFOA
PFOS
PFOS
sulfinate
M556
N­
EtFOSE
acid
FOSA
N­
EtFOSE
alcohol
N­
EtFOSA
DAY
0
BIODEG
FOR
PFOA
CA097­
0801­
SA­
200
2+
3
2818
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
PFOA
DUPLICATE
CA097­
0801­
SA­
201
2+
3
2728
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOA
CA097­
0801­
SA­
234
2+
3
2889
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOA
DUPLICATE
CA097­
0801­
SA­
235
2+
3
2768
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
CA097­
0801­
SA­
216
2+
3
2750
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
CA097­
0801­
SA­
217
2+
3
2708
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
CA097­
0801­
SA­
250
2+
3
2769
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
DUPLICATE
CA097­
0801­
SA­
251
2+
3
2786
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
PFOS
CA097­
0801­
SA­
202
2+
3
14.1
2546
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
PFOS
DUPLICATE
CA097­
0801­
SA­
203
2+
3
13.8
2579
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOS
CA097­
0801­
SA­
236
2+
3
13.6
2589
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOS
DUPLICATE
CA097­
0801­
SA­
237
2+
3
13.1
2590
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
CA097­
0801­
SA­
218
2+
3
13.7
2567
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
CA097­
0801­
SA­
219
2+
3
12.6
2681
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
CA097­
0801­
SA­
252
2+
3
14.1
2467
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
DUPLICATE
CA097­
0801­
SA­
253
2+
3
14.2
2604
0.0
0.0
0.
0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
PFOSulfinate
CA097­
0801­
SA­
204
2+
3
56.7
85.7
2804
0.0
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
PFOSulfinate
DUPLICATE
CA097­
0801­
SA­
205
2+
3
84.7
86.3
2834
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOSulfinate
CA097­
0801­
SA­
238
2+
3
155.6
1574
404.3
0.0
0.0
0.0
0.0
0.0
DAY
18
BIODEG
FOR
PFOSulfinate
DUPLICATE
CA097­
0801­
SA­
239
2+
3
155.7
1525
446.0
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
CA097­
0801­
SA­
220
2+
3
347.3
76.8
2391
0.0
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
CA097­
0801­
SA­
221
2+
3
393.4
79.4
2229
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
CA097­
0801­
SA­
254
2+
3
1268
139.2
830.5
0.0
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
DUPLICATE
CA097­
0801­
SA­
255
2+
3
923.6
137.5
1292
0.0
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
FOSA
CA097­
0801­
SA­
206
2+
3
3.9
0.0
0.0
0.0
0.0
2589
0.0
0.0
DAY
0
BIODEG
FOR
FOSA
DUPLICATE
CA097­
0801­
SA­
207
2+
3
3.3
0.0
0.0
0.0
0.0
2423
0.0
0.0
DAY
18
BIODEG
FOR
FOSA
CA097­
0801­
SA­
240
2+
3
12.2
85.4
26.2
0.0
0.0
2180
0.0
0.0
DAY
18
BIODEG
FOR
FOSA
DUPLICATE
CA097­
0801­
SA­
241
2+
3
13.1
83.6
19.2
0.0
0.0
2446
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
CA097­
0801­
SA­
222
2+
3
3.3
0.0
0.0
0.0
0.0
2535
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
CA097­
0801­
SA­
223
2+
3
3.6
0.0
0.0
0.0
0.0
2472
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
CA097­
0801­
SA­
256
2+
3
3.7
0.0
0.0
0.0
0.0
2556
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
DUPLICATE
CA097­
0801­
SA­
257
2+
3
4.0
0.0
0.0
0.0
0.0
2623
0.0
0.0
DAY
0
BIODEG
FOR
N
­EtFOSA
CA097­
0801­
SA­
208
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2645
DAY
0
BIODEG
FOR
N
­EtFOSA
DUPLICATE
CA097­
0801­
SA­
209
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2692
DAY
18
BIODEG
FOR
N
­EtFOSA
CA097­
0801­
SA­
242
2+
3
0.0
16.9
6.6
85.5
0.0
532.1
0.0
414.1
DAY
18
BIODEG
FOR
N
­EtFOSA
DUPLICATE
CA097­
0801­
SA­
243
2+
3
0.0
16.2
5.7
84.0
0.0
509.0
0.0
363.6
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSA
CA097­
0801­
SA­
224
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2314
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSA
DUPLICATE
CA097­
0801­
SA­
225
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2228
DAY
18
NO
SLUDGE
CONTROL
FOR
N
­EtFOSA
CA097­
0801­
SA­
258
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1207
DAY
18
NO
SLUDGE
CONTROL
FOR
N
­EtFOSA
DUPLICATE
CA097­
0801­
SA­
259
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
431.4
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
37
of
38
Appendix
C
TABLE
1,
CONTINUED
Concentration
(ng/
mL)

Sample
Description
Sample
ID
ELUATE
PFOA
PFOS
PFOS
sulfinate
M556
N­
EtFOSE
acid
FOSA
N­
EtFOSE
alcohol
N­
EtFOSA
DAY
0
BIODEG
FOR
M556
CA097­
0801­
SA­
210
2+
3
0.0
0.0
0.0
2850
0.0
4.8
0.0
0.0
DAY
0
BIODEG
FOR
M556
DUPLICATE
CA097­
0801­
SA­
211
2+
3
0.0
0.0
0.0
2812
0.0
4.5
0.0
0.0
DAY
18
BIODEG
FOR
M556
CA097­
0801­
SA­
244
2+
3
4.7
26.7
6.1
1926
0.0
474.6
0.0
0.0
DAY
18
BIODEG
FOR
M556
DUPLICATE
CA097­
0801­
SA­
245
2+
3
4.9
21.3
7.5
1952
0.0
455.7
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
CA097­
0801­
SA­
226
2+
3
0.0
0.0
0.0
2755
0.0
0.0
0.0
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
DUPLICATE
CA097­
0801­
SA­
227
2+
3
0.0
0.0
0.0
2909
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
CA097­
0801­
SA­
260
2+
3
0.0
0.0
0.0
2875
0.0
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
M556
CA097­
0801­
SA­
260
2+
3
0.0
0.0
0.0
2729
0.0
0.0
0.0
0.0
DAY
0
BIODEG
FOR
N
­EtFOSE
acid
CA097­
0801­
SA­
212
2+
3
9.2
0.0
11.2
0.0
2609
0.0
0.0
18.9
DAY
0
BIODEG
FOR
N
­EtFOSE
acid
DUPLICATE
CA097­
0801­
SA­
213
2+
3
9.1
0.0
11.1
0.0
2573
0.0
0.0
21.8
DAY
18
BIODEG
FOR
N
­EtFOSE
acid
CA097­
0801­
SA­
246
2+
3
13.7
12.0
4.6
36.3
2441
23.4
0.0
7.6
DAY
18
BIODEG
FOR
N
­EtFOSE
acid
DUPLICATE
CA097­
0801­
SA­
247
2+
3
12.2
10.6
4.7
34.5
2229
25.8
0.0
10.3
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
acid
CA097­
0801­
SA­
228
2+
3
8.5
0.0
11.1
4.6
2661
0.0
0.0
17.0
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
acid
DUPLICATE
CA097­
0801­
SA­
229
2+
3
8.5
0.0
11.2
0.0
2540
0.0
0.0
17.5
DAY
18
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
acid
CA097­
0801­
SA­
262
2+
3
9.7
0.0
10.8
5.1
2670
0.0
0.0
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
acid
DUPLICATE
CA097­
0801­
SA­
263
2+
3
9.7
0.0
11.2
0.0
2820
0.0
0.0
0.0
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
CA097­
0801­
SA­
214
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
3007
0.0
DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
DUPLICATE
CA097­
0801­
SA­
215
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
3082
0.0
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
CA097­
0801­
SA­
248
2+
3
20.2
132.1
64.6
87.4
2038.7
23.4
441.6
9.5
DAY
18
BIODEG
FOR
N
­EtFOSE
alcohol
DUPLICATE
CA097­
0801­
SA­
249
2+
3
18.4
105.6
30.0
87.8
2015.3
26.2
464.1
15.0
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
alcohol
CA097­
0801­
SA­
230
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
2989
0.0
DAY
0
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
alcohol
DUPLICATE
CA097­
0801­
SA­
231
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
2972
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
N
­EtFOSE
alcohol
CA097­
0801­
SA­
264
2+
3
0.0
0.0
3.7
0.0
7.1
0.0
3571
0.0
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
DUPLICATE
CA097­
0801­
SA­
265
2+
3
0.0
0.0
0.0
0.0
19.4
0.0
2836
0.0
DAY
0
SLUDGE
BLANK
CA097­
0801­
SA­
232
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
0
SLUDGE
BLANK
DUPLICATE
CA097­
0801­
SA­
233
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
SLUDGE
BLANK
CA097­
0801­
SA­
266
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DAY
18
SLUDGE
BLANK
DUPLICATE
CA097­
0801­
SA­
267
2+
3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BACK
TO
MAIN
Project
CA097
18­
Day
Aerobic
Biodegradation
of
Perfluorooctanesulfonate­
based
Chemistries
Cleston
Lange,
Ph.
D.,
Pace
Analytical
Services,
Science
Solutions
Division
Friday,
February
23,
2001
Page
38
of
38
Appendix
D
TABLE
1.
The
final
results
table
for
each
perfluorooctanesulfonyl­
based
substrate
tested.
Values
were
converted
to
micromolar
(uM)
concentrations
from
the
data
in
Appendix
C.
The
values
reported
below
are
the
averaged
value
determined
for
duplicate
samples.
The
percent
of
parent
remaining
in
samples
and
the
mass
balance
data
are
calculated
from
the
expected
concentration,
and
was
the
amount
of
test
material
added
to
each
culture
at
the
beginning
of
the
experiment.

Sample
Type
Concentration
(m
mM)

Sample
Description
PFOA
PFOS
PFOS
Sulfinate
FOSA
M556
NEtFOSA
N­
EtFOSAA
N­
EtFOSE
alcohol
Expected
Conc.
(uM)
%
Parent
Remaining
Molar
Mass
Balance
DAY
0
BIODEG
FOR
PFOA
6.434
0.000
0.000
0.000
0.000
0.000
0.000
0.
000
6.136
104.9%
104.9%
DAY
18
BIODEG
FOR
PFOA
6.563
0.000
0.000
0.000
0.000
0.000
0.000
0.000
6.136
107.0%
107.0%

DAY
0
NO
SLUDGE
CONTROL
FOR
PFOA
6.332
0.000
0.000
0.000
0.000
0.000
0.000
0.000
6.136
103.2%
103.2%

DAY
18
NO
SLUDGE
CONTROL
FOR
PFOA
6.445
0.000
0.000
0.000
0.000
0.000
0.000
0.000
6.136
105.0%
105.0%

DAY
0
BIODEG
FOR
PFOS
0.032
4.763
0.000
0.000
0.000
0.000
0.000
0.000
4.564
104.4%
105.1%

DAY
18
BIODEG
FOR
PFOS
0.031
4.813
0.000
0.000
0.000
0.000
0.000
0.000
4.564
105.5%
106.1%
DAY
0
NO
SLUDGE
CONTROL
FOR
PFOS
0.031
4.877
0.000
0.000
0.000
0.000
0.000
0.000
4.564
106.9%
107.5%

DAY
18
NO
SLUDGE
CONTROL
FOR
PFOS
0.033
4.713
0.000
0.000
0.000
0.000
0.000
0.000
4.564
103.3%
104.0%

DAY
0
BIODEG
FOR
PFOSulfinate
0.164
0.160
5.400
0.000
0.000
0.000
0.000
0.000
5.071
106.5%
112.9%

DAY
18
BIODEG
FOR
PFOSulfinate
0.361
2.880
0.814
0.000
0.000
0.000
0.000
0.000
5.071
16.1%
80.0%

DAY
0
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
0.859
0.145
4.425
0.000
0.000
0.000
0.000
0.000
5.071
87.3%
107.1%
DAY
18
NO
SLUDGE
CONTROL
FOR
PFOSulfinate
2.542
0.257
2.033
0.000
0.000
0.000
0.000
0.000
5.071
40.1%
95.3%

DAY
0
BIODEG
FOR
FOSA
0.008
0.000
0.000
5.022
0.000
0.000
0.000
0.000
5.141
97.7%
97.8%
DAY
18
BIODEG
FOR
FOSA
0.029
0.157
0.044
4.636
0.000
0.000
0.000
0.000
5.141
90.2%
94.7%

DAY
0
NO
SLUDGE
CONTROL
FOR
FOSA
0.008
0.000
0.000
5.017
0.000
0.000
0.000
0.000
5.141
97.6%
97.7%

DAY
18
NO
SLUDGE
CONTROL
FOR
FOSA
0.009
0.000
0.000
5.189
0.000
0.000
0.000
0.000
5.141
100.9%
101.1%

DAY
0
BIODEG
FOR
M556
0.000
0.000
0.000
0.009
5.083
0.000
0.000
0.000
4.659
109.1%
109.3%

DAY
18
BIODEG
FOR
M556
0.011
0.045
0.013
0.932
3.481
0.000
0.000
0.000
4.659
74.7%
96.2%
DAY
0
NO
SLUDGE
CONTROL
FOR
M556
0.000
0.
000
0.000
0.000
5.084
0.000
0.000
0.000
4.659
109.1%
109.1%

DAY
18
NO
SLUDGE
CONTROL
FOR
M556
0.000
0.000
0.000
0.000
5.030
0.000
0.000
0.000
4.659
108.0%
108.0%

DAY
0
BIODEG
FOR
N­
EtFOSA
0.000
0.000
0.000
0.000
0.000
5.063
0.000
0.000
5.360
94.5%
94.5%
DAY
18
BIODEG
FOR
N­
EtFOSA
0.000
0.031
0.012
1.043
0.152
0.738
0.000
0.000
5.360
13.8%
36.9%
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
0.000
0.000
0.000
0.000
0.000
4.310
0.000
0.000
5.360
80.4%
80.4%
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSA
0.000
0.000
0.000
0.000
0.000
1.554
0.000
0.000
5.360
29.0%
29.0%

DAY
0
BIODEG
FOR
N­
EtFOSAA
0.021
0.000
0.021
0.000
0.000
0.039
4.429
0.000
4.341
102.0%
103.9%
DAY
18
BIODEG
FOR
N­
EtFOSAA
0.030
0.021
0.009
0.049
0.064
0.017
3.991
0.000
4.341
91.9%
96.3%

DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSAA
0.020
0.000
0.021
0.000
0.004
0.033
4.445
0.000
4.341
102.4%
104.2%

DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSAA
0.023
0.000
0.021
0.000
0.005
0.000
4.693
0.000
4.341
108.1%
109.2%

DAY
0
BIODEG
FOR
N­
EtFOSE
alcohol
0.000
0.000
0.000
0.000
0.000
0.000
0.000
5.332
5.002
106.6%
106.6%
DAY
18
BIODEG
FOR
N­
EtFOSE
alcohol
0.045
0.221
0.091
0.050
0.157
0.023
3.465
0.793
5.002
15.9%
96.9%
DAY
0
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
0.000
0.000
0.000
0.000
0.000
0.000
0.000
5.219
5.002
104.3%
104.3%
DAY
18
NO
SLUDGE
CONTROL
FOR
N­
EtFOSE
alcohol
0.000
0.000
0.004
0.000
0.000
0.000
0.023
5.610
5.002
112.2%
112.7%
BACK
TO
MAIN
BACK
TO
MAIN
