BIODEGRADATION
STUDY
REPORT
`
14­
c'

­~

Revision
I
Wednesday,
November
06,
2002
Biodegradation
Screen
Study
for
Telomer­
Type
Alcohols
PROJECT
NUMBER
3M
Projects
ID:
E0I­
0684
Pace
Contract
Analytical
Projects
ID:
CA085
STUDY
DIRECTOR
Cleston
C.
Lange,
Ph.
D.

CLIENT
James
K.
Lundberg,
Ph.
D.,
3M
Environmental
Laboratory
Bldg
2­
3E­
09,
P.
O.
Box
33331,
St.
PauI,
MN
551
33­
3331
CONTRACT
LABORATORY
Pace
Analytical
Services,
Inc.
Bio­
Analytical
Services
Group
1700
Elm
Street,
Suite
200
Minneapolis,
Minnesota
55414
PROJECT
DATES
Project
Initiation:
August
9,
2000
Project
Completion:
September
18,2001
AUTHOR
Cleston
C.
Lange,
Ph.
D.

Page
1
of
38
Pace
Project
CAO8S:
Biodegradation
Screen
Study
for
Telomer
AIcohols~
 
Revision
1
Study
Director:
Cleston
C.
Lange.
Ph.
D.
Pace
Analytical
Services,
Sb­
Analytical
Services
Group
Wednesday.
November
06,
2002
CONTAIN
NO
CBI
°
Ø00003
Table
of
Contents
Title
Page
­
1
Table
of
Contents
2
List
of
Tables
&
Figures
3
List
of
Appendices
4
Executive
Summary
5
1.0
Project
Personnel
6
2.0
Data
Requirements
and
Revision
Justification
6
3.0
Project
Objective
6
4.0
Test
Article
7­
8
5.0
Reference
Articles
9
6.0
Receipt/
Generation
of
Samples
9
7.0
Methods
9
7.1
Sample
Preparation
9
7.1.1
Collection
of
Sludge
9­
10
7.1.2
Culture
Preparation
11­
12
7.1.3
Solid
Phase
Extraction
(
SPE)
12­
13
7.2
Instrumental
Analysis
(
HPLC/
MS)
13
7.2.1
Instrument
Parameters
13­
14
7.2.2
Qualitative
Analysis
of
Parents
and
Products
14­
15
7.2.3
Quantitative
Analysis
16
7,3
Data
Transformations
and
Calculations
16
7.3.1
MolarCalculations
16
7.3.2
Conversion
of
ng/
mL
to
micromolar
and
nanomolar
16
7.3.3
Molar
Mass
Balance
Calculations
17
7.4
Software
Versions
17
8.0
Results
18
8.1
Sludge
Characterization
18
8.2
Quality
Control/
Sample
Matrix
Spike
Results
18
8.3
Analytical
Blanks
Results
18
8.4
HPLC/
MS
Analysis
19
8.4.1
Qualitative
I­
IPLC/
MS
and
HPLC/
MS/
MS
Results
19­
24
8.4.2
Quantitative
Analysis
Results
for
PFOA
24­
25
9.0
Conclusions
27
10.0
Literature
Cited
28
11.0
Sample
and
Data
Retention
29
Page
2
of
38
Pace
Project
CAO8S:
Biodegradation
Screen
Study
for
Telomer
Alcohol&
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,2002
000004
List
of
Tables
&
Figures
TABLES
Table
1.
Parent
telomer
alcohol
expected
ions
14
Table
2.
Expected
product
fluorinated
fatty
acid
products
ions
15
Table
3.
HPLC/
MS
Data
Table
for
Parent
and
Product
Ions
observed
23
Table
4.
HPLC/
MS/
MS
Data
for
n­
Oxidation
Product
Ions
observed
24
FIGURES
Chemical
Representation
of
Telomer
Alcohols
8
Chemical
Structure
of
perfluorooctanoate
(
PFOA)
9
Plotted
HPLC/
MS
Integrated
Peak
Areas
for
C8,
C10
and
C12
Telomer
Alcohols
20
Plotted
HPLC/
MS
Integrated
Peak
Areas
for
C5,
C14
and
C15
Telomer
Alcohols
20
Plotted
HPLC/
MS
Integrated
Peak
Areas
for
Perfluorinated
Fatty
Acids
Detected
22
Plotted
Peak
Areas
for
Transient
Polyfluorinated
Fatty
Acids
Detected
22
Plotted
PFOA
Concentrations
Measured
in
Biodegradation
Samples
(
SPE
eluate
2)
25
Proposed
Biodegradation
Pathway
for
Telomer
Alcohols
26
Figure
1.
Figure
2.
FIgure
3.
FIgure
4.
Figure
5.
Figure
6.
Figure
7.
Figure
8.

Page
3
of
38
Pace
Project
CA085:
8iodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06.
2002
000005
List
of
Appendices
Appendix
A:
Approval
Signatures
30
Appendix
B:
Final
Quantitative
Results
for
PFOA
(
ng/
mL)
Table
1.
Test
Cultures
Final
Results
31
Table
2.
Abiotic
Cultures
Final
Results
31
Table
3.
Blank
Cultures
Final
Results
31
Appendix
C:
Integrated
Chromatogram
Peak
Area
Data
for
the
Telomer
Alcohols
Table
1.
Telorner
Alcohols
Peak
Areas
32
Table
2.
Percentage
of
Peak
Areas
For
Telomer
Alcohols
Remaining
at
Time
Points
32
Appendix
D:
Table
1.
Integrated
LC/
MS
(
SIR)
Peak
Area
Data
for
Perfluorinated
Fatty
Acids
33
Appendix
E:
Table
I.
Peak
Area
Data
for
Transiently
Formed
Polyfluorinated
Fatty
Acids
34
Appendix
F:
Table
I.
Total
Ion
Chromatogram
(
TIC)
for
SIR
Data
for
Biodegradation
Samples
35
Appendix
G:
Table
I.
TICs
for
SIR
Data
for
Day
0
and
Day
16
Biodegradation
Samples
36
Appendix
H:
Table
1.
TICs
for
SIR
Data
for
Day
0
and
Day
16
Abiotic
Control
Samples
37
Appendix
I:
Table
I.
TICs
for
Extracted
Ion
Data
for
Transient
Polyfluorinated
Fatty
Acids
38
Page
4
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000006
Executive
Summary
A
screening
studywas
undertaken
to
determine
whether
the
fluorochemical
telonier
intermediate
(
telomer
alcohol)
biodegrades
when
exposed
to
municipal
wastewater
treatment
sludge.
The
study
included
the
preparation
of
cultures
for
a
six
sample­
point
comparative
study,
and
included
test
cultures,
blank
cultures,
and
control
cultures.
The
microbial
inoculum
for
cultures
was
sludge
obtained
from
the
Twin
Cities
Metro
Wastewater
Treatment
Facility.
Telomeralcohol
test
substance
was
added
to
cultures
at
approximately
5MM
concentrations.
Cultures
were
incubated
with
shaking
at
25
°
C.
Solid
phase
extraction
of
cultures
for
recovery
of
biodegradation
products
and
parent
analytes
was
employed.
An
HPLCIMS
analytical
method
was
developed
for
analysis
of
the
telomer
alcohols
and
expected
products.
The
I­
IPLC/
MS
analysis
of
culture
extracts
provided
strong
evidence
that
the
telamer
alcohols
were
biodegraded.
The
observed
loss
of
telomer
alcohols
occurred
concomitant
with
the
appearance
of
several
expected
perfluorinated
carboxylic
acids.
Unexpectedly,
transient}
y
formed
polyfluorinated
p­
oxidation
intermediates
were
observed.
The
3­
oxidation
pathway
was
suspected
to
be
the
major
route
of
biodegradation
resulting
in
the
observed
even­
numbered
carbon
chain
length
perfluorinated
fatty
acids.
Minor
end
products,
as
odd­
numbered
carbon
chain
length
perfluorinated
fatty
acids,
were
also
observed
and
were
likely
the
products
of
fatty
acid
a­
oxidation,
a
minor
pathway
of
fatty
acid
metabolism
usually
observed
in
branched­
chain
fatty
acids.
After
the
l6daytest
period,
perfiourinated
carboxylic
acids
ranging
from
C5
to
C,
2
were
observed
in
the
test
cultures.
These
compounds
were
not
in
controls
or
blank
cultures,

Perfluorooctanoate
(
PFOA),
was
the
only
compound
quantitatively
analyzed
and,
based
on
mass
balance
data
accounted
for
approximately
6­
7%
of
the
total
telomer
alcohols
initially
present
in
the
test
cultures.

Page
5
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000007
1.0
Proiect
Personnel
1.1
Sponsor
Company
1.2
Sponsor
Representative
1.3
Contract
Facility
Personnel:

1.3.1
Study
Director
1.3.2
Laboratory
Management:

1.3.3
Sample
Preparation
3M
Dr.
James
K.
Lundberg
Or,
Cleston
C.
Lange
Mr.
Bruce
E.
Warden
Ms.
Angela
L.
Schuler
Dr.
Cleston
C.
Lange
Ms.
Angela
L.
Schuler
Dr.
Cleston
C.
Lange
Dr.
Cleston
C.
Lange
2.0
Data
Requirements
and
Revision
Justification
The
sponsor
representative
desired
an
aerobic
biodegradability
screening
study
to
be
conducted
using
the
telomer
alcohol
mixture
as
test
substrate,
Thestudy
was
initiated
on
August
9,
2000
as
a
non­
GLP
study.

The
report
issued
Friday,
November
16,
2001
is
revised
to
substitute
"
Dupont
Zonyl
BA
type
Telomer,
or
Zonyl
type
Telomer"
with
Telomer.
The
reason
for
the
revision
is
to
more
accurately
reflect
the
impact
of
results
to
the
general
class
of
telomer
alcohols.

3.0
Project
Objective
This
study
was
conducted
in
order
to
elucidate
whether
the
fluorotelomer
intermediate
(
telomer
alcohol)
is
biodegradable
under
aerobic
conditions
using
a
microbial
rich
inoculurn
of
municipal
wastewater
treatment
sludge.
This
study
was
stnilar
to
earlier
studies
conducted
for
3M
by
Pace
as
Pace
projects
CA058
and
CAO9T
2
and
CA1O4
~,

CAl05
`,
and
CAl
32
~.
The
development
of
an
analytical
method
and
the
analysis
of
parent
analytes
and
possible
products
were
critical
parameters
for
the
determinatiofto­
f
biodegradation.

Page
6
of
38
Pace
Project
CAO85:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Ciroup
Wednesday,
November
06,
2002
1.3.4
1,3.5
1,3.6
HPLC/
MS
Analyst
Sample
Custodian:

Report
Author
000008
4.0
Test
Article
The
test
article
used
for
this
study
was
the
Zonyl
BA­
type
telomer
alcohol
fluorochernic­&

surfactant
intermediate.
Two
sources
of
test
material
were
used
for
this
study,
The
first
source
was
provided
by
the
sponsor
company,
3M,
as
approximately
2.5
g
of
crystalline
solid
test
material
labeled
Zonyl
BA­
N
telomer
alcohol,
3M
tracability
number
TN­
A­
2186,

on
July
7,
2000.
The
sponsor
did
not
provide
an
MSDS,
a
chain
of
custody,
the
purity
information
for
the
test
article,
noran
expiration
date
for
the
material.
This
material
was
given
a
test,
control,
and
reference
(
TCR)
number
at
Pace
as
CA­
TCRO2­
009
and
was
stored
at
4
°
C.
Material
CA­
TCRO2­
009
was
used
primarily
for
method
development
purposes
of
the
study,
including
the
first
set
of
cultures
prepared
and
original
LC/
MS
method
development.

A
commercial
vendor,
Aldrich
Chemical
Company,
provided
the
second
source
of
telomer
alcohol
used.
Material
was
purchased
as
Zonyl
BA­
L
fluorotelomer
intermediate
(
1999
Aldrich
Catalog
number
42,151­
0),
also
called
perfluoroalkylethanol.
The
material
characteristics
were
boiling
point
145­
245
°
C,
F(
CF2)~
CH2CH2OH
where
n
equals
approximately
7
to
8,
and
molecular
weight
was
reported
as
M~
ca.
443.70
wt.
An
expiration
date
and
the
purity,
or
percent
composition,
of
the
material
were
not
provided.

The
purchased
material
was
received
at
Pace
on
September
22,
2000.
Upon
receipt
at
Pace,
the
material
was
given
tracability
number
CA­
TNCOO­
254
and
was
stored
at
4
°
C,

or
less.
A
representative
chemical
compilation
of
the
Zonyl
BA­
type
telomer
alcohol
is
shown
in
Figure
1.

Page
7
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telonier
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday.
November
06,
2002
00(
2009
F
F
F
F
F
F
F
I
I
h__
OH
F
F
F
F
F
F
F
F
F
F
F
F
J
~~
OH
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
I
I
I
I
Molecular
formula
=
C5
H5
F130
8
Molecular
Weight
=
364.104
EM
+
CH3COO
1'
=
423
Molecular
formula
=
C10
H5
F17
0
10
Molecular
Weight
=
464.119
(
M
+
CH3COO
]`
=
523
a"
Molecularformula
=
C24
H5
F250
`
14
Molecular
Weight
=
664.149
[
M
+
CH3COO
~
`
=
723
Figure
1.
The
telomer
alcohols.
Based
on
the
HPLC/
MS
average
peak
area
response
observed
in
the
six
abiotic
control
cultures,
the
composition
of
the
test
material
was
6.4%
±
0.3%
as
C5
telomer
alcohol,
39.3%
±
1.5%
as
C8,
27.7%
±
2.1%

as
C10,
17.5%
±
1.2%
as
C12,
7.2%
±
1.0%
as
C54,
and
2.0%
±
0.2%
as
C16.

Page
8
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services.
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
ooooi~
O
F
F
F
F
F
Ce
F
F
F
F
Molecular
formula
=
C5
H5
F9
0
Molecular
Weight
=
264.069
P
(
M
*
Cl­
13C00
7
`=
323
I
I
I
I
I
I
F
F
F
F
F
F
F
F
F
F
F
F
F
F
1FF
F
F
F
ThJ~
l
C
Molecular
formula
=
C12
H5
F21
0
12
Molecular
Weight
=
564.134
[
M
+
CH3COO
1
`=
623
`
IIIflIIIIIII\

F
F
F
F
F
F
F
F
F
F
F
F
OH
I
£
11
111111111
~
ft"~
 
OH
Molecular
formula
=
016
H5
F29
0
16
Molecular
Weight
=
764.164
[
M
CH3COO]
`=
823
5.0
Reference
Materials
The
sponsor
provided
reference
material.
The
neat
material
was
stored
at
­
80
°
C.

NS:
Expiration
date
not
specified
5.1.
Perfluorooctanoate
Ammonium
Salt
(
PFOA)

Purity:
95.2%;
3M#:
TCR­
99131­
3?;
Pace
#:
CA­
TCRO2­
001;
Expires:
NS
F
F
F
F
F
F
F
6
NH
FLLL~
I~
IX~

Figure
2­
Perfluorooctanoate
ammoniurn
salt
6S
Receipt/
Generation
of
Samples
Samples
were
not
received,
but
were
generated
as
an
inherent
part
of
this
study.
All
of
the
experimental
cultures
prepared
for
each
culture
set
were
extracted
by
solid
phase
extraction
(
SPE)
methodology
to
generate
three
analytical
samples
per
culture.
The
analytical
samples,
as
SPE
eluates
collected
for
each
culture,
were
labeled
as
SPE
eluates
1,
2
or
3.
Of
the
three
eluates
generated
for
each
sample,
eluate
1
was
the
25
mL
aqueous
sample
eluate
and
eluates
2
and
3
were
25
mL
methanol
eluates
from
the
SPE
cartridge.
Qualitative
and
semi­
quantitative
HPLC/
MS
analysis
was
conducted
for
eluate
2
only,
and
the
resulting
data
was
evaluated
to
determine
whether
biodegradation
occurred.

7.0
Methods
7.1
Sample
Preparation
7,1.1.
Collection
of
Sludge.

The
sludge
for
this
study
was
obtained
from
the
Twin
Cities
Municipal
waste
treatment
facility
in
a
manner
consistent
with
the
sludge
collected
for
other
studies
listed
in
section
30.
The
sludge
used
for
the
preparation
of
the
first
set
of
cultures
was
collected
on
July31,
2000
and
Page
9
of
38
Pace
Project
cAOa5:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analyticat
Services
Group
000011
Wednesday,
November
06,
2002
delivered
with
Pace
chain
of
custody
(
CCC)
form
465254.
The
sludge
for
the
second
set
of
cultures
was
collected
on
September
18,
2000
and
was
accompanied
with
COC
#
528791.
Sludge
used
for
the
second
set
of
cultures
in
this
study
was
also
used
in
preparation
of
cultures
for
other
fluorochemical
biodegradation
studies
(
CA1O5
and
CA132
5),
and
was
shown
to
be
active
for
biodegradation
of
other
fluorochemical
compounds
in
those
studies.

To
prepare
cultures,
sludge
was
obtained
from
the
primary
municipal
waste
treatment
facility
in
the
Twin
Cities
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
located
in
St.
Paul,
MN.
Typically,
Four
liters
of
MLSS
was
collected
by
Pace
laboratory
personnel
and
delivered
as
four
1­
liter
Nalgene
polypropylene
bottles
containing
MLSS,
and
were
accompanied
with
a
corresponding
chain
of
custody
with
date
collected.
Upon
receipt
at
Pace
Science
Solutions,
the
individual
bottles
were
labeled
#
1
through
#
4,
The
suspended
solids
in
the
bottles
were
allowed
to
settle
at
least
24
hours
at
4
°
C
±
3
°
C.
The
settled
solids
"
sludg&
were
then
used
to
prepare
MLSS
plus
sludge
for
use
in
preparing
test
cultures.
A
sludge
characterization
analysis
was
not
conducted
as
pad
of
this
screening
study.

The
settled
sludge
in
each
bottle
constituted
approximately
20%
of
the
volume,
or
approximately
200
mL
volume
in
a
I­
liter
bottle,
based
on
visual
observations­
and
was
consistent
with
observation
of
earlier
sludge
collections.

Page
10
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000012
7.1.2.
Culture
Preparation.

The
first
set
of
cultures
were
prepared
August
9,
2000
and
incubation
of
those
cultures
continued
with
intermittent
culture
harvests
until
the
final
harvest
on
September
13,
2000,
for
a
total
incubation
time
of
35
days.

The
second
set
of
cultures
were
prepared
on
September
27,
2000
and
incubation
of
those
cultures
continued
with
intermittent
culture
harvests
until
the
final
harvest
on
October
12,
2000,
for
a
total
of
16
days
incubation.
The
culture
preparation
procedure
described
below
was
used,
and
was
documented
as
Pace
standard
operating
procedure
(
SOP)
CAG­
SP­
03
~

Cultures
were
prepared
using
a
mineral
salts
medium
defined
by
EPA
Guideline
OPPTS
835.3200.
The
mineral
salts
medium
pH
was
7.4
and
contained
per
liter,
0,334
g
Na2HPO4­
2H20,
0.005
g
NH4CI,
0.2175
g
K2HPO4,
and
0.085
KH2PO4,
0.0275
g
CaCI2­
anhydrous,
0.0225
g
MgSO4­
7H2O,
and
0.00025
g
FeCl3­
6H20.

Two
liters
of
a
mineral
salts
medium
containing
100
mL
of
settled
sludge
was
prepared
and
contained
1
mL
of
methanol.
To
each
test
culture
was
added
25
mL
of
this
mineral
medium
containing
sludge.

A
mineral
salts
medium,
un­
sterilized
and
without
sludge,
was
prepared.

This
mineral
salts
medium
without
sludge
was
used
to
prepare
the
25
mL
no­
sludge
(
abiotic)
control
cultures.

All
cultures
were
prepared
by
dispensing
25
mL
of
appr3priate
mineral
salts
medium
solution
into
clear
sterile
125
mL
Nalgene
polycarbonate
culture
flasks
containing
labels
with
appropriate
identification
information.

Note:
The
mineral
salts
medium
that
contained
sludge
had
to
be
swirled
regularly
during
dispensing
in
order
to
keep
the
mixture
homogenous
and
prevent
the
sludge
from
settling
out
of
the
solution.

The
test
substance,
either
Zonyl
BA­
N
telomer
alcohol
for
culture
set
one
(
Stock
ID
CA058­
SS­
004
at
10,280
~.
tg/
mLin
methanol)
or
Zonyl
BA­
L
Page
11
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
ooool3
telomer
alcohol
for
culture
set
two
(
Stock
ID
CAO8S­
SW­
001
at
7,288
~
Lg/
mLin
methanol),
was
added
to
the
test
cultures
by
transferring
5
.
iL
and
8J.
LL,
respectively,
to
the
appropriate
test
cultures.
The
final
test
concentration
in
cultures
was
2.056
pg/
mL
of
Zonyl
BA­
N
telomer
alcohol
for
set
one,
and
2.332
pglmL
of
Zonyl
BA­
L
telomer
alcohol
for
set
two.

Blank
control
cultures
received
25
mL
of
mineral
medium
solution,
as
did
the
test
culture,
but
without
addition
of
the
test
substrate.

All
of
the
day
zero
cultures
were
prepared
and
immediately
frozen
at
­
20
°
Cfor
storage
until
SPE
preparation
could
be
conducted.
All
other
cultures
were
placed
in
temperature
controlled
shaking
incubators
that
were
maintained
at
25
°
C
±
3
°
C.
Cultures
were
removed
from
the
incubators
at
designated
time
points.
Upon
removal
from
the
incubator,

cultures
were
either
immediately
frozen,
or
immediately
prepared
for
analysis
by
solid
phase
extraction.

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

were
recorded
in
sample
preparation
worksheets
and
signed
and
dated
by
the
preparation
analyst.
All
original
data
sheets
were
maintained
in
project
specific
binder
labeled
as
Project
CA085.

7.1.3.
Solid
Phase
Extraction
of
Cultures
The
solid
phase
extraction
procedure
described
below
was
documented
as
Pace
standard
operating
procedure
(
SOP)
CAG­
SP­
04
~.

All
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
cartridge
was
packed
with
plug
of
quartz
glass
wool
to
deter
plugging.
Each
SPE
cartridge
was
washed
prior
to
use
by
drawing
5
niL
of
methanol
and
then
5
mL
of
aqueous
1%

acetic
acid
solution
through
the
cartridge.
These
wash
solution
eluates
were
discarded
to
waste.
All
of
the
SPE
eluates
for
this
study
were
collected
in
clear
1­
Chem
vials
with
labels
that
identified
them
as
eluate
1,
2
or
3,
as
defined
below.

Page
12of38
Pace
Project
CAOB5:
`
Biodegradation
Screen
Study
for
Telonier
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services.
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000014
Frozen
cultures
were
thawed
at
ambient
room
temperature
before
extraction.
Following
thawing,
and
prior
to
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
drawn
by
vacuum
through
the
appropriately
labeled
SPE
cartridge
by
carefully
pouring
the
contents
of
the
culture
flask
into
the
SPE
cartridge.
The
aqueous
eluate
was
collected
in
an
1­
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
1­
Chem
vial
labeled
eluate
2.

Eluate
2
was
expected
to
contain
a
majority
of
the
analyte
that
was
in
the
original
culture.

As
a
precaution
that
some
analyte
may
be
retained
in
the
SPE
cartridge
or
in
the
culture
flask,
a
second
25
mL
methanol
eluate
was
collected
in
a
similar
fashion
to
that
collected
for
eluate
2,
and
was
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
±
2
°
C.

The
final
SPE
extractions
of
the
day
7,
14
and
16
day
cultures
from
set
two
occurred
on
September
24,
2001.

7.2
Instrumental
Analysis
(
LC/
MS)

7.2.1
Instrument
Parameters
The
HPLC/
MS
method
used
was
a
modified
version
of
Pace
method
CAGORG
23
as
described
below.

Analysis
of
culture
extracts
from
culture
set
one,
prepared
with
Zonyl
BA­
N
telomer
alcohol,
was
conducted
using
instrument
"
STING"
which
included
an
HPI100
HPLC
pump
with
Gilson
215
liquid
handling
system
in
line
with
a
Micromass
Quattro
II
triple­
quadrapole
mass
spectrometer
detector.

Page
13
of
38
Pace
Project
CAOS5:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06.
2002
000015
Analysis
of
culture
extracts
from
culture
set
two,
prepared
with
Zonyl
BA­
L
telomer
alcohol,
were
analyzed
both
on
instrument
"
STING'
and
instrument
"
1
OLCMSO3"
which
consisted
of
a
Waters
2690
HPLC
system
in
line
with
a
Micromass
Quattro
II
triple­
quadrapole
mass
spectrometer
detector,
Typical
conditions
for
analysis
of
telomer
alcohols
and
fluorochemical
acid
products
were
as
follows:

Typical
injection
volumes
for
samples
and
calibration
standards
were
50
ML.
The
4.6
x
150
mm
Betasil
CS
column
used
for
the
quantitation
of
extracts
from
culture
set
two
had
serial
number
1101567H
and
the
4
x
35
mm
NG1
column
used
had
serial
number
15104.
A
pressure­
regulated
splitter
was
used
with
an
approximate
split
ration
of
2:
1
(
Waste:
MS)

7.2.2
Qualitative
Parent
Analyte
and
Products
Analysis.

Parent
Analyte
Expected
tbns
(
mix)
I
C6
telomer
alcohol
acetate
adduct
423
C10
telomer
alcohol
acetate
adduct
523
C12
telomer
alcohol
acetate
adduct
623
C~
4telomer
alcohol
acetate
adduct
723
C16
telomer
alcohol
acetate
adduct
823
Table
1.
Parent
telomer
alcohols
analyzed.

Page
14
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
Instrument
conditions
for
HPLC/
MS
analysis.

Mass
Spectrometer
HPLC
Ionization
mode:
API­
ES
negative
Desolvation
Temperature:
200
°
C
Source
Block
Temperature:
150'
C
RF
lens:
0.2
Extractor:
3
Cone:
B
Capillary:
3.50
LM
Resolution:
14
HM
Resolution:
14
Ion
Energy:
2.5
Lens
6:
2
MS1
Multiplier:
650
MS/
MS:
Collision
Energy­
varied
from
MS/
MS
Collision
Gas:
Argon
MS2
Multiplier:
750
Time
(
mm).
~
A
0.00
97.0
3.0
0.50
97.0
3.0
5.00
5.0
95.0
11.00
5.0
95.0
11.50
97.0
3.0
15.00
97.0
3.0
Flow:
1
mLlmin,
splitter
approx.
3:
1
Solvent
A=
2
mM
ammonium
acetate
Solvent6=
Methanol
column
Temperature:
ambient
10
to
40
V
for
individual
Experiments,

000016
To
conduct
the
analysis
of
parenl
compounds
and
expected
biodegradation
products,
the
HPLC/
MS
system
was
set
up
with
the
same
chromatographic
configuration
as
that
described
in
method
CAG­
ORG­

23
~.
The
method
utilized
two
columns
in
tandem
with
flow
to
a
pressure
relief
valve
that
serves
as
a
flow
through
splitter
to
the
mass
spectrometer
Z­
spray
source.

Specific
MS
conditions
for
mass
analysis
of
the
telomer
alcohols
and
acid
products
were
developed
during
this
study.
The
mass
spectrometer,

Micromass
triple­
quadrapole
mass
spectrometer
with
Z­
spray
ion
source,

was
operated
with
electrospray
ionization
in
either
selected
ion­
recording
(
SIR)
mode
or
with
mass­
range
(
150­
1000
m/
z)
scanning
on
MS1.

Discrete
chromatographic
peaks
with
singly
charged
negative
ions,
(
M
+

Acetate~
for
parent
telomer
alcohols
and
(
M
­
H'~'
for
expected
acid
biodegradation
products,
were
observed
and
monitored:

Expected
Product
Expected
anion
structure
CF3(
CF2)
2COO~
Expected
anions
(
mix)

Perfluorobutyrate
(
C4)
213,169
Perfiuoropentanoate
(
C5)
CF3(
CF2)
2C00'
263,
219
Perfluorohexanoate
(
C6)
CF3(
CF2J~
COO'
313,
269
Perfluoroheptanoate
(
C7)
CF3(
CF2)
5C00'
363,
319
Perfluorooctanoate
(
C8,
PFOA)
CF3(
CF2)
sCOO~
413,
369
Perfluorononanoate
(
C9)
CFa(
CF2)
7C00'
463,
419
Perfluorodecanoate
(
C10)
CF3(
CF2)
5C00'
513,
469
Perfluoroundecanoate
(
C11)
CF3(
CF2)
9COO~
563,
519
Perfluorododecanoate
(
C12)
CF3(
CF2)
10C00'
613,
569
2K,
2H­
perfiuorooctanoate
(
C3)
CF3(
CF2)
5CH2C00'
377
2K,
2H­
perfluorodecanoate
(
C10)
CF3(
CF2)
7CH2COO'
477
2H.
2H­
perfluorododecanoate
(
C12)
CF3(
CF2)
9CH2COO'
577
Table
2.
Expected
products,
and
expected
anions
for
each.

Page
15
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Sludy
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000017
7.2.3
Quantitative
Analysis.

QuanUtative
analysis
was
conducted
only
for
perfluorooctanoate
(
PFOA)

and
only
on
extracts
of
culture
set
two.
Quantitative
analysis
was
performed
by
the
external
standard
method
using
SPE
extracted
calibration
standards
(
eluate
2
only)
and
quadratic
calibration
curves,

The
HPLCIMS
analysis
of
culture
set
one
extracts
was
used
primarily
for
method
development
purposes.

Typical
injection
volumes
for
samples
and
calibration
standards
were
50
ML.
The
4.6
x
150
mm
Betasil
C8
column
used
for
the
quantitation
of
extracts
from
culture
set
two
had
serial
number
1101
567H
and
the
4
x
35
mm
NG1
column
used
had
serial
number
15104.
The
pressureregulated
splitter
had
no
identifying
number
to
distinguish
it.
The
part
number
for
ordering
the
pressure
relief
valve
(
splitter)
was
Alitech
catalog
part
number
39025.

7.3
Data
Transformations
and
Calculations
7.3.1
Molar
Calculations:

Because
all
data
was
collected
on
an
ng/
mL
basis
(
part
per
billion,
ppb),

a
b'ansformation
from
ng/
mL
to
molar
concentrations
had
to
be
conducted
to
obtain
mass
balance
information
when
applicable.
The
mole
conversion
values
for
each
analyte
are
as
follows:

PFOA
molecular
weight,
as
ammonium
salt,
is
431.10
Zoriyl
BA­
L
telomer
alcohol
average
molecular
weight
is
434.70.

7.3.2
Conversion
of
ng/
mL
to
micromolar
(
MM)
and
nanomolar
(
nM).

(
Working
Examples):

2,232
ng/
mL
telomer
alcohol
=
(
2,232
ng/
mL)
t(
lnmole
/
434.7
ng)
=

5.135
nmole/
mL
=
5.135
gmole/
liter
=
5.135
MM
 
assuming
100%
purity.

150
ng/
mL
PFOA
(
Nl­
14+
salt)
=
(
150
ng/
mL)
*
(
1
nmole
/
431,1
ng)

=
0.3479
nmolelmL
=
0.3479
Mmole/
L
=
0.3479
MM
=
347.9
nM
Page
16of38
Pace
Project
CAOBS:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Gb­
Analytical
Services
Group
Wednesday,
November
06,
2002
000018
7.3.3
Molar
Mass
Balance
Calculations
(
Theoretical
Yield):

Convert
all
ng/
mL
values
to
their
corresponding
molar
concentrations
as
aM
or
nM
(
see
section
7.3.2,
above).
Divide
the
sum
of
the
analyte
concentrations
by
the
known
concentration
of
starting
compound
and
represent
the
final
result
as
a
percentage
of
the
known
starting
concentration.

(
Working
Example):

If,
the
starting
concentration
of
BA­
L
telomer
alcohol
was
at
5.135MM
And,
after
incubation,
the
following
was
determined:

If,
PFOA
was
detected
at
0.3479gM
Then,
the
mass
balance
is
as
follows:

Mass
balance
=
[(
0.3479
MM)
/
5.135
MMIX
100%
Mass
balance
=
[
0.0678]
X
100%
=
6.78%

Or,
6.78%
of
the
telomer
BA­
L
alcohol
was
oxidized
to
form
PFOA.

7.4
Software
Versions
Microsoft~
Excel
2000
was
used
for
data
processing
and
producing
tables.

MicrosoftlM
Word
2000
was
used
for
processing
the
analytical
report
text.

Adobe
Acrobat
4.0
was
used
for
generation
of
the
final
electronic
report.

Masslynx
version
3.2
was
used
for
data
collection
and
peak
integration.

ACD
Chemsketch
version
4.0
was
used
for
chemical
drawings.

Page
17
of
38
Pace
Prcject
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Sludy
Director
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Gb­
Analytical
Services
Group
Wednesday,
November
06,
2002
000019
8.0
Results
8.1
Sludge
Characterization
A
chemical
analysis
of
the
mixed
liquor
suspended
solids
used
for
the
samples
prepared
on
August
9,
2000
was
conducted,
and
characterization
information
can
be
found
in
the
final
report
for
project
CA058'.
The
sludge
for
samples
prepared
on
September
27,
2000
was
not
characterized,
but
was
obtained
from
the
Twin
Cities
Municipal
waste
treatment
facility
in
a
manner
consistent
with
the
sludge
collected
and
used
for
project
CA058.

8.2
Quality
Control/
Sample
Matrix
Spike
Results,

The
determination
of
the
analyte
recoveries
from
sample
matrix
spikes
was
not
included
as
part
of
this
screening
study.
Recoveries
are
only
reflected
in
the
ability
to
achieve
molar
mass
balance
based
on
expected
parent
and
product
yields
from
samples
and
by
the
use
of
a
sludge­
extracted
curve
for
semiquantitative
analysis
of
PFOA.

8.3
Analytical
Blanks
Methanol
solvent
blanks
were
injected
onto
the
HPLC/
MS
column
and
quantitatively
analyzed
to
determine
the
instrument
background
analyte
concentration
and
carry
over
during
the
analysis.

The
no­
sludge
(
abiotic)
controls
containing
mineral
salts
medium
with
test
analyte,
and
culture
blanks,
containing
mineral
salts
medium
with
test
analyte
and
no
sludge,
were
prepared
and
analyzed.
Abiotic
controls
and
blank
cultures
were
prepared
and
incubated
in
an
identical
manner
to
biodegradation
test
cultures.
The
results
were
used
to
determine
whether
the
sample
matrix
contained
any
of
the
analytes
of
interest
and
whether
biodegradation
could
be
attributed
to
the
bioactivity
of
the
sludge.
Fluorochemical
analytes
were
not
detected
in
blanks
and
biodegradation
did
not
occur
in
abiotic
controls.

Page
18of38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday.
November
06,
2002
000020
8.4
HPLC/
MS
Analysis
Results
Semi­
quantitative
analysis
was
conducted
only
for
the
SPE
eluate
2
extracts
from
the
second
set
of
cultures,
prepared
September
27,
2000,
and
were
performed
on
September
of
2001
as
sequence
CA085fi92401b.
spl
(
with
full
scan
MS
data),
and
CAOB5_
092701a.
spl
(
SIR
data)
on
instrument
1OLCMSO3
in
the
Bio­

Analytical
services
group
at
Pace.
The
qualitative
HPLC/
MS
analyses
and
HPLC/
MS
method
development
for
this
study
were
conducted
in
September
and
October
of
2000
as
analytical
sequence
runs
S091500.
spl,
S09I800.
spl,

SO92000.
spl,
SO92100.
spl,
S092600.
spl,
S1000200.
spl,
and
S100400.
spl
on
Pace
instrument
"
STING".

8.4.2
Qualitative
HPLCIMS
and
HPLC/
MS/
MS
Results
The
first
set
of
test
cultures
containing
sludge
and
BA­
N
telomer
alcohol
substrate
provided
evidence
that
biodegradation
occurred.
The
HPLC/
MS
data
showed
near
total
loss
of
all
of
the
telomer
alcohol
peaks
in
the
HPLC/
MS
chromatogram,
with
formation
of
chromatographic
peaks
that
had
mass
spectra
consistent
with
perfluorinated
acids.
However,
at
that
time,
the
analytical
test
method
was
not
complete.
Concerns
about
the
integrity
of
the
test
material
resulted
in
a
second
set
of
test
cultures
being
prepared
using
commercially
purchased
Zonyl
BA­
L
telomer
intermediate.

Although
each
sample,
control,
and
blank
for
the
second
set
of
cultures
was
prepared
and
incubated
in
duplicate,
analysis
was
conducted
for
SPE
eluate
2
extracts
from
one
sample,
control
and
blank
pertime
point.

The
data
showed
that
the
sample
test
cultures
underwent
rapid
loss
of
the
C5,
C9,
C10,
and
C12­
telomer
alcohols,
and
moderate
loss
of
the
C14­
telomer
alcohol
over
the
16
days
of
incubation,
as
determined
by
decreasing
peak
area
response
for
each
telomer
alcohol
ion
at
the
sequential
sampling
time
points
(
Figures
3
and
4).
The
C16­
telorner
alcohol
was
degraded
very
slowly
and
showed
little
loss.

Page
19
of
38
Pace
Project
CAO85:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday.
November
06,
2002
000021.
35000
30000
25000
20000
15000
10000
5000
0
0
5
10
15
20
°
C8
TA
peak
area
AdO
TA
peak
area
°°
C12
TA
peak
area
Figure
3.
HPLCIMS
integrated
peak
areas
for
the
most
abundant
telomer
alcohols
(
TA).
Near
complete
degradation
of
all
three
was
observed.
Data
plotted
with
the
best
manual
fit
of
the
data,

6000
5000
4000
~
C6TA
peak
area
3000
°
uC14
TApeak
area
°
C16
TA
peak
area
~
2000
0
a­

1000
0
Incubaiton
time
(
days)

Figure
4.
HPLC/
MS
integrated
peak
areas
for
the
less
abundant
telomer
alcohols
(
TA).
Near
complete
degradation
was
observed
for
the
C6­
TA.
Moderate
degradation
of
the
C1~­
TAwas
observed
and
little
degradation
of
the
C19­
TA
was
observed.
Data
plotted
with
the
best
manual
fit
of
the
data.

Page
20
of
38
Pace
Project
CAO8S:
~
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000022
0
5
10
15
20
Concomitant
with
the
loss
in
telomer
alcohols
was
the
formation
of
compounds
with
HPLC/
MS
ions
and
retention
times
consistent
with
perfluorinated
adds
(
PFAs),
inc!
uding
perfluorooctanoate
(
PFO~
A,
CrPF.
A),

which
was
confirmed
as
an
end
product.
The
transformation
of
telomer
alcohols
to
PFAs
was
rapid,
with
a
large
increase
in
all
of
the
PFA
peak
area
responses
at
day
1,
and
continuing
increase
through
day­
i6
(
Figure
5).
No
degradation
to
form
PFAs
was
observed
in
the
abiotic
no­
sludge
controls.

Although
PFOA
was
accurately
quantified,
many
other
perfluorinated
acids
were
qualitatively
observed
and
their
relative
concentrations
determined
based
on
the
observed
increasing
peak
area
response.
The
observed
perfluorinated
fatty
acid
end
products
were:
perfluoropentanoic
acid
(
C5
PFA),
per­
fluorohexanoic
acid
(
C6­
PFA),
perfiuoroheptanoic
acid
(
CrPFA),

perfluorooctanoic
acid
(
C8­
PFA,
PFOA),
perfluorononanoic
acid
(
CrPFA),

perfluorodecanoic
acid
(
C,
0­
PFA),
perfluoroundecanoic
acid
(
C11­
PFA)
and
perfiuorododecanoic
acid
(
C1rPFA).

Transientlyformed
intermediate
compounds
were
also
observed
at
early
time
points
following
the
initial
exposure
of
the
sludge
to
the
telomer
alcohol
substrate
(
Figure
6).
The
transient
compounds
were
suspected
to
be:
2H,
2H­
perfluorooctanoate;
2H,
2H
 
perfluorodecanoate;
2H,
2H
Perfluorododecanoate
and
the
possible
f3­
oxidation
pathway
intermediates:

21­
l­
perfluoro­
2­­
octenoate,
2H­
perfluoro­
2­
decenoate,
and
2H­
perfluoro­
2­

dodecenoate.
Table
3
shows
the
MS
ions
and
retention
times
observed
for
the
polyfluorinated
and
perfluorinated
acid
products
and
the
telomer
alcohol
parent
substrates.

HPLC/
MS/
MS
Data
(
Table
4)
support
the
identification
of
the
suspected
metabolites..

Page
21
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000023
FIgure
5.
Integrated
peak
area
responses
for
the
pertluorinated
fatty
acids
(
PFAs)

observed
in
test
culture
extracts.
Even
numbered
carbon
chain
length
carboxylic
acids
were
the
most
abundant
carboxylic
acid
peaks
and
were:
perfluorohexaooate
(
C5­
PFA),

perfluorooctanoate
(
C3­
PFA)
and
perfluorodecanoate
(
Cio­
PFA),

Figure
6.
Transient
polyfluorinated
fatty
acid
intermediates
observed.
The
2Hperfluorinated
olefinic
fatty
acids
are
suspected
~.
oxidationintermediates
that
formed
rapidly
upon
initial
exposure
to
telomer
alcohols,
and
then
were
transformed
to
their
corresponding
pertluorinated
carboxylic
acids
end
products
Page
22
of
38
Pace
Project
CA085:
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000024
250000
200000
C,

k9
a
a.
150000
100000
50000
01
0
 
 
2
4
6
8
10
12
Incubation
time
(
days)
IA
11
14
16
18
rAnal~
esObserved
Chemical
Formula
Retention
Time
LCIMS
anions
F
observed
ft~
ieiorneralcohol*
CF3(
CF2)
3CH2CH2OH..
.

CH3COO"
7.63
323
1
C6­
telomer
alcohol
CF3(
CF2)
6CH2CH2OH..
CH3COGY
8.13
423
010­
telorner
alcohol*
CF3(
CF2)
7CH2CH2OH.
..
CH3COO~
8.50
523
C,
2­
telomer
alcohol'
CF3(
CF2)
9CH2CI­
120H...
CH3COQ
­
8.96
623
C,
4­
telomer
alcohol'
CF3(
CF2)
jiCl­
42CH2OH...
CH3COO~
9.53
723
C,
6­
telomer
alcohol'
CF3(
CF2)
13CH2CH2OH..
.
CH3COO
10.25
923
2H,
21­
f­
perfluorooctanoate'
CF3(
CF2)
5CH2COO~
7.17
377
2H,
2H­
pertluorodecanoate'
CF3(
CF2)
7CR2C00'
7.38
477
2H,
2H
Perfluorododecanoate'
CF3(
CF2)
gCH2COO~
749
577
2H­
perfluoro­
2­
octenoate'
CF3(
CF2)
4CF=
CHCOO~
7.14
357293
2H­
perfluoro­
2­
decenoate'
CF3(
CF2)
6CF=
CHCOO~
735
457393
2H­
Perfluoro­
2­
dodecenoate'
CF3(
CF2)
8CF=
CHCOO~
752
557,493
Perfluoropentanoate
CF3(
CF2)
3COO~
6.62
263,
219
Pertluorohexanoate
CF3(
CF2)
4C00"
6.88
313,
269
Pertluoroheptanoate
CF3(
CF2)
5C00
7.07
363,
319
Pertluorooctanoate
CF3(
CF2)
5C00
­
7.22
413,
369
Perfluorononanoate
Pertluorodecanoate
CF3(
CF2)
7C00
CF3~
CF2)
6COO"
7.30
7.41
463,
419
513,
469
1
Perfluorouridecanoate
CF3(
CF2)
9C00
7.48
563.
519
Perfluorododecanoate
QF3(
CF2),
oCOO~
7.52
613,
569
Only
anions
corresponding
to
even
chain
length
molecules
were
observed.

Table
3.
Parent
and
products
anions
observed
in
by
HPLC/
MS
analysis
of
SPE
eluate2
extracts
from
test
cultures
containing
sludge
and
telomer
alcohol
substrate.
The
parent
anion
signals
decreased
in
test
cultures
over
time
concomitant
with
increases
in
product
signals.
Many
of
the
telomer
alcohol
signals
decreased
below
detection
limits
in
test
cultures.

Page
23
of
38
Pace
Project
CAOB5:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Anafyticat
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000025
Product
Analytes
Observed
Chemical
Formula
Retention
Time
LC/
MS/
MS
LC/
MS/
MS
daughter
Parent
anion
anions
observed
2H
,2H~
perfiuorooctanoate*
CF3(
CF2)
5CH2COcY
7.17
377
333,31
3,293
21­
l,
2H­
perfluorodecanoate
CF3(
CF2)
7CH2COO
­
7.38
477
413,393
2H,
2H
Perfluorododecanoate*
CF3(
CF2)~
CH2COO
7,49
577
.­

2H.
perf1uoro~
2~
octenoate*
CF3(
CF2)
4CF=
CHCOO
7.14
357
313,293,243,143,119
2H.
perfluoro~
2~
decenoate*
CF3(
CF2)
6CFCHCOO
7.35
457
413,393.343
2H­
Perfluoro­
2­
dodecenoate
CF3(
CF2)
5CF=
CHCOO
­
I
`
I~

Table
4.
HPLC/
MS!
MS
data
for
suspected
­
oxidation
products.
Parent
and
daughter
anions
from
theHPLC/
MS/
MS
analysis
of
the
SPE
eluate
2
extract
from
the
1­
day
biodegradation
sample
(
CA085­

0927­
SA­
033
E2).
The
parent
anions
were
observed
as
transiently
formed
anions
in
early
sample
point
biodegradation
samples
only,
with
the
most
intense
signals
for
the
ions
observed
at
day
one.
The
MS/
MS
data
collected
for
each
product
was
consistent
with
the
predicted
chemical
formula.
Common
mass
loss
from
the
parent
anion,
attributed
to
loss
of
CO2
(
mass
44)
and
HF
(
mass
20),
were
observed.

8.4.2
Quantitative
Analysis
of
PFOA
The
HPLC/
MS
multi­
component
calibration
standards
used
in
this
study
for
product
quantitation
contained
the
target
analytes:
PFOA,
PFOSulfinate,
PFOS,

FOSA,
N­
MeFOSA,
M556,
M570,
and
N­
MeFOSE
alcohol.
Five
calibration
standards
were
prepared
by
addition
of
a
known
amount
o(,
perfluorinated
analytes
to
a
culture
medium
with
sludge,
and
then
extraction
by
solid
phase
extraction
in
manner
identical
to
the
treatment
of
samples.
The
PFOA
was
the
only
target
analyte
of
that
mixture
that
was
used
for
this
study.
The
exact
concentrations
of
the
standards
were
used
in
calibration
curves
for
quantitative
analysis.

Page
24
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000026
All
calibration
standards
and
sanip~
eeluates
were
stored
at
4
°
Cin
refrigerator
ID
0213
until
analysis.
Aliquots
were
transferred
to
autovials
and
capped
for
use
in
HPLC/
MS
runs.
The
instrument
calibrations
were
performed
for
PFOA
by
use
of
a
five
point
calibration
with
quadratic
fit
of
the
data.
The
LLOQ
was
11.0
ng/
mL
PFOA,
and
the
coefficient
of
determination
(
r)
for
PFOA
was
0.965.
Quantitative
sequence
runs
contained
calibration
standards
at
the
beginning
and
end
of
the
run.

The
biodegradation
of
the
telomer
alcohols
resulted
in
all
increase
in
the
measured
levels
of
PFOA
during
the
16­
day
study
(
Figure
7).

Figure
7.
The
measured
perfluorooctarioate
(
PFOA)
in
cultures
containing
sludge
and
fluorotelomer
alcohol.
The
PFOA
was
not
measurable
ri
cultures
that
did
not
contain
sludge.
Data
plotted
with
the
best
manual
fit
of
the
data
and
concentrations
shown
were
not
adjusted
for
reference
material
purity
of
95.2%.

Page
25
of
38
Pace
Project
CA085:
`
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
B(
o­
Analytical
Services
Group
Wednesday,
November
06,
2002
180.0
160.0
140.0
j'
120.0
100.0
80.0
a.
60.0
40.0
20.0
0.0
0
2
4
6
8
10
12
14
16
15
Incubafion
time
(
days)

000027
F
 
`
j'
dsMra9wlase
F
F
F
F
HH
F
F
F
F
0
Oxidatlve
decarboxylatlon
h~
tcøan
(
a­
oxIdation)

F
F
F
F
F
F
F
F
OH
Dial
F)
I
I!
K
F
F
F
F
F
F
F
F
0
&
lase
n­
OxidatIon
0~

F
F
F
F
Ilibi
~

F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
111a2
F
4
F
F
F
F
F
P
F
F
0
Perftuorortonanoate
÷
Co2
F
111111!
IH
F
F
F
F
F
F
F
F
,~,
4efl~
0coq~
a~
e
4,
P.
ket~
c~
l
FE
P
F
F
F
F
0
IHb3
F________________________

F
F
F
F
F
F
0
Perfi
uorooctanoate
+

M
3
C~~
Q
Acetate
Figure
8.
Proposed
biodegradation
pathway
for
telomer
alcohols,
shown
for
C10­
telomer
alcohol
as
example.
The
p­
oxidation
pathway
branch
(
Ilib)
involves
primary
formation
of
a
fatty
acyl­
CoA
thioester
that
is
not
shown,
to
clearly
depict
the
products
observed
by
HPLC/
MS.
The
fatty
acyl­
CoA
thiol
bond
is
readily
hydrolyzed
to
yield
the
corresponding
fatty
acids
observed.

Page
26
of
38
Pace
Project
CA085:
~
BiodegradationScreen
Study
for
Telorner
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Sb­
Analytical
Services
Group
Wednesday,
November
06,
2002
000028
F
I
I
.
L
I
I
I
I
I
~`~
0K
C,
Tetomer
Aicohol
diydrog&'
ase
~
r
r
i
ii
ii,
9.0
Conclusions
This
study
has
demonstrated
that
fluorochernical
telomer
alcohots
are
biodegradable
and
are
transformed
to
perfluorinated
acid
end
products.
Although
the
telomer
alcohols
are
supplied
only
as
even
numbered
carbon
chain
compounds,
the
end
product
perfluorinated
acids
consist
of
both
even
number
and
odd
number
carbon
chains,
with
even
numbered
carbon
chain
length
acids
predominating
as
the
major
end
products.

This
observation
suggests
that,
following
the
initial
oxidation
of
the
hydroxyl­
carbon
to
torn,
the
primary
carboxylic
acid
(
telomer
carboxylata),
two
oxidation
mechanisms
exist
as
shown
in
Figure
8,
III.
The
first,
less
utilized
route
(
Figure
8,
lila),
involves
oxidation
of
the
a­
carbon
concomitant
with
decarboxylation
(
oxidative
decarboxylation)
to
form
an
odd
chain
length
perfluorinated
carboxylic
acid,
which
does
not
undergo
further
biotransformation.
The
second
observed
route
of
oxidation
(
Figure
8,
IlIb)
is
the
more
common
route
of
fatty
acid
~­
oxidation,
as
evidenced
by
the
formation
of
detectable
transient
p­
oxidation
polyfluorinated
fatty
acid
intermediates
and
the
more
abundant
even
number
carbon
chain
length
carboxyiic
acids
Although
toxicity
of
the
telomer
alcohols
was
not
addressed
as
part
of
this
study,
the
microbial
degradation
observed
here
over
the
entirety
of
the
16­
day
study
suggests­
that
microorganisms
present
in
the
sludge
were
not
significantly
inhibited
by
the
tested
concentration
of
the
test
material.

Page
27
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
forTelomer
Alcohols'
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
PaceAnalytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000029
8.0
Literature
Cited
1.
Final
Reports
for
Pace
Project
CA058
(
3M
ft
E00­
2252),
"
2­
Week
N­
EtFOSE
Alcohol
Biodegradation
Screen
Study
Report"
and
"
Aerobic
Biodegradation
of
N­
EtFOSE
alcohol
Study
Report'.
Author:
Cleston
C.
Lange,
Ph.
D.

2.
Final
report
for
Pace
Project
CA097
(
3M
#
E01
­
0415),
"
The
18­
Day
Aerobic
Biodegradation
Study
of
Perfluorooctanesulfonyl­
based
Chemistries."
Author:

Cleston
C.
Lange,
Ph.
D.

3.
Final
report
for
Pace
Project
CAIO4
(
3M
#
E01­
0444),
"
The
35­
Day
Aerobic
Biodegradation
Study
of
Perfluorooctanesulfonate
(
PFOS)."
Author:
Cleston
C.

Lange.
Ph.
D.

4.
Final
Report
for
Pace
Project
CAl
05
(
3M
ft
E01­
0683),
"
Fluorochemical
Adipate
(
LI
5468)
Biodegradation
Screen
Study.'
Final
report
issued
July
10,
2001.

Study
Director:
Cleston
C.
Lange,
Ph.
D.

5.
Final
Report
for
Pace
Project
CAl
32
(
3M
ft
E01­
0682).
"
The
Aerobic
Biodegradation
Study
ot
the
Fluorochemical
FC807­
Diester".
Final
report
issued
May
29,
2001.
Study
Director:
Cleston
C.
Lange,
Ph.
D.

6.
Pace
method
CAG­
SP­
03.
"
Culture
Preparation
for
Assessment
of
Aerobic
Biodegradability
of
Fluorochemicals
Using
Municipal
or
Industrial
Sludge
as
Microbial
Inoculum."

7.
Pace
method
CAG­
SP­
04.
"
C18
Solid
Phase
Extraction
Procedure
for
Fluorochemicals
Recovery
from
Aqueous/
Sludge
Matrices."

8.
Pace
method
CAG­
ORG­
23
"
Quantitative
Analysis
of
Fluorochernicals
by
High
Performance
Liquid
Chromatography
with
Mass
Spectrometric
Detection."

Page
28
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
I
Study
Director:
Cleston
C.
Lange.
Ph.
D.
Pace
Analytical
Services,
Blo­
Analytical
Services
Group
Wednesday,
November
06,
2002
ooooao
9.0
Sample
and
Data
Retention
At
a
minimum,
one
copy
of
all
pertinent
raw
data
and
one
copy
of
the
signed
final
report
will
be
retained
in
the
Pace
Analytical­
Tier
2
data
archives
for
a
minimum
period
of
2
years
after
completion
of
the
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
°
Cin
the
Carroll
walk­
in
cooler
(
Pace
ID
0140)
located
in
the
Pace
Analytical­
Tier
2
facility.

The
following
will
be
provided
to
the
sponsor
(
3M):

°
The
original
signed
analytical
report
and
one
copy
of
the
signed
original.

°
The
final
scanned
report
(
read
only)
and
pertinent
electronic
data
on
a
CD.

°
All
original
Data,
correspondence,
chromatograms,
sample
&
standards
prep
sheets,
etc.

*
Upon
request
before
2
years,
the
stored
samples
may
be
sent
to
the
sponsor.

All
electronic
copies
of
the
instrumental
raw
data
will
be
archived
onto
CD
disks
and
one
copy
provided
to
3M
and
one
copy
retained
at
Pace.

Facility
data
will
be
retained
for
a
period
of
10
years.
Facility
data
is
available
for
inspection
and
includes
thefollowing
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
Page
29
of
38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Anatytical
Services,
Sb­
Analytical
Services
Group
Wednesday,
November
06,
2002
000031
APPENDIX
A
APPIOVAL
S~
eNATuREs
Project
Title:
Biodegradation
Screen
Study
for
Telomer
Alcohols
Client
Project
ID:
EUl
­
0684
Contract
Analytical
Project
Numben
0A085
Report
Rev~
sedby:

MouractRahi_
1k7­
0z
Date
Laboratory
Management:

Bruce~
j~
den
 
Signature
Page
30
of
38
Pace
Project
CAO8S:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
PaceAnalytical
Services,
BJo­
AnaJyticaJ
Services
Group
Wednesday,
November
06,
2002
000032
Appendix
B
Final
Quantitative
Results
for
PFOA
in
samples,
controls
and
blanks
Table
1.
The
measured
PFOA
concentrations
in
test
cultures
during
the
16­
day
screening
study
for
telorner
alcohol
aerobic
biodegradability.

JConc.
ntnffco,
n~
4,
t~
Eluat.
21
Snh
PF0A
DaySb~
d~
n4a5on
sacn,
CA085'
0927.
SA'DDt.
02
0.0
Day
0~
deVa4asonsriiyla.
C4085­
0027­
SA.
033
02
252
Day
2
bndeo,
adat,
rn,
aampl°,
C4085­
002?­
SA'
035,
02
650
Cay
5
bcdOnd~'
onsample,
CAOaSO92l'.
SA.~
1.02
I
1.4
Day
7
b~
ecradrionsample.
cA085­
0027.
SA.
039
02
`
39.8
Day
I.
scdearadaelon
sample,
CAOSS­
0027.
SA­
04I
02
`
Ste
Day
IS
S
da'adaelon
sample.
CAOO5­
0921­
SA­
043,
02
1600
Table
2.
The
measured
PFOA
concentrations
in
the
abiotic
control
cultures
during
the
16­
day
screening
study
fortelomer
alcohol
aerobic
biodegradability.

Coticentnfton
OnGkaLj
[
Elssat~
2
SampM
PPOA
Days
r~
sludge
~
mroI,
CAOSS­
0927.
SA.
053,
22
°
0
lay
I
~
slid
0
,
co,
st,
sl.
0A08S0921.
SA.
065,
02
0.0
lay
2
Im
sludge
wlslrOI,
CA085.0927.
SA.
056
22
0.0
layS
Im
didse
~
mlol,
CAOSSOO27.
SAOSQ
22
0.0
lay?
to
sltdQe
~
mml,
CA085'
0027.5A­
060.
22
0.0
lay
14
rIO
slidIm
toMiol,
CAOOS­
0927.
SA­
062
22
0.0
lay
IS
to
sludge~
r4roI.
CAOOS­
0027.
SA­
®
4,
22
0.0
Table
3.
The
final
data
results
for
sludge
blanks
during
the
16­
day
screening
study
for
telomer
alcohol
aerobic
biodegradability.
PFOA
was
not
detected
in
any
of
the
sludge
blanks.

Concentrallon
(
ngknL)
IEluaO.
2J
Sam~*
PFOA
Dayssludge
Slayic.
n_
A0a5.0021.
SA.
045
02
0.0
Day
I
sludge
04*.
Aoe5~
092T­
SA­
o4a.
02
00
Day2
sludge
slaM.
0A01S0027­
SA­
047.
02
0.0
Day
S
sludge
5124*.
CAO85­
0927­
S404e.
02
00
Day?
sludge
slr*.
CAOBS.
0927'
SA­
DiQ,
22
0.0
Day
4
sludge
bla',
k,
CAO5S.
0927­
SA.
050.
22
00
Day
II
sludge
blaik,
CA085'
0927­
SA.
051.
E2
00
Page
31
of38
Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bia­
Analytical
Services
Group
Wednesday,
November
06,
2002
000033
Appendix
C
Integrated
Chromatogram
Peak
Area
Data
for
telomer
alcohols
Table
1.
Integrated
LC/
MS
peak
area
raw
data
for
the
parent
telomer
alcohols
substrates.
Data
acquired
from
sequence
run
CA085_
092701a.
spl
as
SIR
data.

Telomer
Alcohol
Integraled
Peslc
Mess
SampI.
PIle
lb
CS
CS
do
c12
c14
Cli
SUM
lay_
0_
a,
0de~
ada,
FDnsampe,
CA08S0927.$
A.
031,
22
CAOSS
092701a
008
3503
21704
26040
9446
5550
320
326
lay
I
b'od'gadau.
op
sampo,
C4055'
0927'$
A.
033,
E2
Day
2
5,03eoradat!
0l
sample.
CA065~
092?~
SA.
03S,
62
cAO8S
0927Db
000
ND
SI
laos
0833
4766
0310
1340
CAGeS
092701a
010
ND
298
262
88fl
4403
1213
1213
Days
s,
sdsqradaoou
ssmme
Ca0e5~
092?­
Sa.
037
22
CAOO5
092701
a
011
12
090
03
3070
3701
1140
1140
0807
Olsde5radalIonsampl.,
CA005'
012?.
SA'
039,
62
~
A005
092701a
012
ND
S31
NO
1263
3240
5343
`
363
Day
14
b'odeorsdatOl
sample.
CA065­
092?.$
A.
D41,
62
~
A085D92701a
013
ND
594
NO
670
1124
1093
0093
Day
10b,
odeoradlioy
sample
CAOS5­
®
2?.
SA­
043.
62
~
slL,~
C0,
Il0l.
cAoasoO27'
SA'
063.
22
CA085_
09270l8
014
cAoes
ognoia
016
ND
5072
043
30841
II?

22594
085
63734
1276
5014
lIST
1192
113?

1412
Day
I
to
sk,
Oto0074101,
dAOS5'
092?­
SA'OSS,
22
CAO0S
092701
a
017
4890
31305
23373
1273
4712
147?
1477
Day?
oo
slu~
ecor4lol,
cAOSS.
0927.
SA.
050
22
CA005
092701a
010
5108
31002
26464
`
3848
5379
1462
162
Days
so
sluOgecorIml,
CA085.0927.
SA.
059,
22
CAO8S
092701a
019
4303
27486
22470
12856
4941
`
393
393
Day?
no
sluadgecor4ml.
CA0oS.~
7'
SA'O60,22
CA065
092701a
020
3515
21400
13621
10429
4252
$
095
1095
Day
4cm
sludge
6001704.
CAoa5.092?­
SA.
062
22
CAOOS
092701a
021
3310
21284
14368
10228
~
992
$
265
0205
Day
1Gm
sludge
consul,
C4081­
092Z24.084,
62
DayS
sludge
blank.
CA08S0027.
SA'OdS.
22
CA085
D92?
Ola
022
CAOOS
D92701a
044
4602
ND
27329
17
27946
ND
12265
ND
5230
ND
1470
ND
5470
NO
Day
I
slados
stalk
C5085.0927'
SA­
046
62
CAOO5
092701a
045
ND
ND
lID
NO
NO
ND
ND
Day
2
slsoçe
blank.
CA08S.
0927.
SA­
047.
22
CAOSS
D9270Ia
040
ND
ND
lID
ND
NO
ND
ND
DayS
sIsoge
5193k,
cA085.0027­
SA­
048.
02
CA085
092701a
047
ND
ND
140
ND
ND
ND
ND
Day?
sliaage
blank
CAOSS.
003?­
SA­
049.
02
cADSS
092701a
040
3?
102
ND
ND
NO
ND
ND
Day
4
lludge
blank.
CA095.0927.
SA.
050,
22
CAO8S
0927018
049
ND
211
ND
ND
ND
ND
ND
Day
lb
sludge
blank,
CA015.0937­
SA­
0Sl.
62
CAO6S
0927D1a
05D
I?
08
NO
269
ND
ND
ND
Table
2.
Percentage
of
integrated
peak
area
remaining
at
different
time
points
with
respect
to
the
peak
area
at
time
zero
for
their
respective
culture
types.
telomer
alcohols
(
TA)
was
observed
in
test
cultures
only.
for
all
except
C10
TA,
which
was
79.4%.
Near
complete
degradation
of
several
Near
90%
remained
in
control
cultures
Sample
Description
.
fl~
L
%
TA
Remaining
versus
Day
0
c.
TA
C,
TA
C10TA
c13TA
C14TA
c,
0TA
Day
0
biodegradation
sample,
CAO8S­
0927­
SA­
031,
E2
0
100.0%
100.0%
100.0%
100,0%
100,0%
100.0%
)
ay
1
biodegradation
sanipJe.
CAO8S­
0927­
SA­
033,
E2
0.0%
0.4%
4.5%
46.8%
85.8%
101.1%
)
ay
2
biode~
adationsample,
CA085­
0927­
SA­
035,
E2
2
0.0%
1.2%
0.9%
36.2%
80,7%
91.5%
Day
5
biodegradation
sample,
CA055­
0927­
sA­
037.
E2
5
0.3%
3.2%
0.4%
16.6%
66.6%
86.0%
Day
7
biodegradation
sample,
cAo8s­
0927­
SA­
039,
E2
7
0.0%
2,9%
0.0%
6.8%
58.4%
101.3%
Day
14
biodegradation
sample,
cAO85­
0927­
SA­
041.
E2
14
0.0%
2.7%
0.0%
3.6%
`
23.8%
82.4%
Day
16
biodegradation
sample,
CA085­
0927­
SA­
043,
E2
Day
0
no
sludge
control,
CA085­
0927­
SA­
053,
E2
16
 
0
0.0%

100.0%
3.0%

100.0%
0,4%

100.0%
3,7%

100.0%
23.0%

100.0%
85.7%

100.0%
Day
I
no
sludge
control,
CA085­
0927­
SA­
055,
E2
1
96.4%
101.5%
90.2%
82.1%
94.0%
99.0%
Day
2
no
sludge
control,
CA065­
0927­
SA­
056.
E2
2
102.5%
103.3%
108.1%
101.0%
107.3%
98.0%
DayS
no
sludge
control,
CA085­
0927­
SA­
05g,
E2
5
86.4%
89.1%
99.5%
93.6%
98.5%
93.4%
Day
7
no
sludge
control,
cA085­
0927­
SA­
060,
E2
7
69.3%
69.6%
60.3%
75.9%
84.8%
73,4%
Day
14
no
slud~
control.
cAoss­
o927­
SA­
o62
E2
14
65,4%
69.0%
63.6%
74.5%
99.6%
84.8%
)
ay
16
no
slud~
control.
cA085­
0927­
SA­
064,
E2
16
90.7%
86.6%
79.4%
88.9%
104.3%
99.1%

Page
32
of
38
Pace
Project
CA085:
1Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000034
Appendix
D
Integrated
Peak
Area
Data
for
Periluorinated
Acid
End
Products
Table
1.
Integrated
LC/
MS
peak
area
raw
data
for
the
end
products
formed.
Data
acquired
from
sequence
run
CA085_
092701a.
spl
as
SIR
data.

Inteorats
Peak
Arose
Sample
­,
,

Fll'lD~'.
Perflu&
op
ntanoata
Pethooroh
xanoate
Pertluoro.
hepunoat.
Pertluorooctanost
P.
rfluorononasnoata
Pertluorod
caoo.
te
P.
dluoro.
undetafloale
Pertluoro.
lodocanoate
SayG
b,
nulflbdakon
oample.
0A08S0827'
5A'
031.
02
C4085
0927008
008
276
3576
12127
2715
8818
253
0589
375
Say
I
b.
od.
gradaiioo
samp..
04185.0027'
5A'
033.
02
CASeS
092701a
009
13796
46683
15484
4582$
8388
6120
0290
505
Say?
g.
oaegnaoatios
lamp.,
CA085­
D921'
SA'
035,
22
DAllaS
0027053
010
483$
4
127201
25641
88508
6267
17586
1378
1192
Say
S
slCu900adaliOn
san'p°
CAO8S.
0927'
SA'ODT,
22
04085
0921008
Oil
63648
204221
3574?
40809
9375
44419
1841
2605
Day?
bodegraoalso
nampe
c4005.0921.5A.
03e.
ES
CAO8S
0921018
012
72810
210569
43227
170923
13751
75377
2944
*
047
Day
14
S'odadslioii
lamp.,
C406s0927­
SAo41.
£
2
05056
092701a
013
70978
221543
47751
$
90923
15382
92188
3071
11376
ay
lb
Skodegnadel101
9801*
cAoos.
092T'SA'oIa.
62
DAOOS
0037018
014
79828
208870
47305
692075
05069
96834
3279
10603
ay
0cm
gas~
e600004.
CMOSOOV.
SA'
053,
92
04000
0927018
048
193
060
9244
1007
4382
134
0253
00
al
Ito
Sll.~
00011110,
C436S092?.
SA­
065,
92
ay2
cm
sl,~
emood,
CA085.0927.
SA.
050.
62
04088
0927818
017
288
0722
16885
1695
5518
233
1692
51
04085
092701a
058
351
0437
17394
1649
5953
064
1178
20
aySm
stsd9e0000,00,
CA08S0927'
SA'
059.
62
04085
092701a
019
478
1744
17948
1883
7120
270
1926
81
al
7cm
slIme00000100,
05085'
0927'
84'
060.
62
DAllaS
092701a
020
457
0643
05212
1583
8328
173
lOIS
25
ay
08
cm
sludge
01,00050.
0408S0927.
SA.
06282
04085
0037018
020
580
173$
10978
1880
7129
204
0675
98
Day
08cm
lsudge6010051.
cA08s0927­
SA'
084.
£
2
04085
0027018
022
­
811
185$
17010
1037
7203
233
1342
34
Day
0
sludge
Sank.
cAO8s.
1927.54445,
62
CAO85_
092701a
044
818
525
31
II?
`
8
3
0
3
Day
I
nludge
blank.
ca00s.
oo27.
SA.
a4~
62
CAO8S_
0927013_
045
883
152
75
`
60
262
0
0
0
Day
2
01110469
blInk.
04085.0927.54.047.
62
04085
0927018
046
686
510
51
229
722
0
0
48
Days
5111009
bllnb.
C4085.0827.
SA.
D48.
02
04005
0927018
047
972
320
66
223
392
0
0
0
Day'
sludge
bla,
kn,
C4065,0027.
SA'D40,
02
CAGeS
0027318
048
737
248
82
500
247
0
24
42
Day
14
sludge
menu.
04085.0927.54.050.
62
04085
092701a
049
676
237
86
731
314
0
24
29
Day
10
sludge
blank.
CAOS5.0927.
SA'
061.
62
04085
0927008
050
634
285
81
645
397
0
20
0
Page
33
of
36
Pace
Project
CAOBS:
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
000035
Wednesday,
November
06,
2002
Appendix
E
Integrated
Peak
Area
Data
for
Transiently
formed
Polyfluorinated
Acid
Products
Table
1.
Integrated
LC/
MS
peak
area
data
for
transient
products.
Data
from
sequence
CA085092401b.
spl
as
full
scan
MS
data
from
rn/
i
100
to
rn/
i
1000.

­
lnteQrated
Peak
Areas
­­
­
`:.­:
­
­­
~
­
­
,
­
~
~­­

­
Sample
­:
FllelQ::~.­:
2W
porfluoro­
2­
octenoale
2H'
p.
rfluoro.
2­
decenoale
2W
1H,
IW
IH,
IHperfluoro
2'
pnfluoro­
perfluorododecenoate
octanoate
decanoat,
1H,
1H.

perfluorododocanoate
ayO
b,
od6~
adaIionsaes~
e.
04685.0927.84.031
­
62
~
A085_
09240Ib
008
1040
ND
ND
3085
200
ND
Day
I
sodooradaeonsamNe,
04685.0927­
54.033
62
05085
09240W
008
3470
780
249
ISO2S
7005
958
Day
Sblod,
craiaoionsamøe,
0A695'
0027'
SA'
025,62
Day
S
biodognadaoion
68,1144..
0A085'
0837.
SA'
037.
22
04005
092401b
010
223
ND
ND
0020
1802
203
04086
0024006
001
ND
ND
ND
738
ND
ND
Day?
biodeorldali,
n
68.80.
0*
085­
0927­
54.039
62
0*
385
0924006
002
ND
ND
ND
057
ND
ND
Day
04
buld.
901da1000saa,
pOe,
C*
OeSO9Rl'SA'
041
22
05086
602400b
003
ND
ND
ND
88
ND
NP
Day
1650d.
gcadMloaealnpoe,
C4085.0027.
SA'
043.
62
04086
0824016
014
ND
ND
NO
128
ND
ND
Day
acm
altdge01MM.
04086­
0927'
SA.
063.
22
04085
0924016
008
ND
ND
NO
,...
J~
Q,.,._..
ND
ND
Day
Inc
906mGconlosl.
C*
085.0027.
SA'
065.
62
C*
085_
092401b
017
ND
ND
NO
ND
ND
ND
Day
2cm
slLmg0
mnl,
d,
0A085'
0927.
SA.
066,
62
0*
085
0924016
018
ND
ND
ND
ND
ND
NO
DayS
nolIudgeconI,
dI
04085.0037.
SA.
069,
22
Day?
nooludgeconInol.
C*
D8S0027.
S*.
060.
22
0*
006
0924016
018
ND
NO
ND
ND
ND
ND
0*
085
0924066
020
ND
ND
ND
ND
ND
ND
lay
04cm
Oludge
0001001,
0*
W.
0927.
SA'
002
62
Say
00cm
akudgemovol.
0A065'
0927'
M'
064,
62
04305
0924016
020
0*
085
0924000
022
ND
ND
040
20
ND
NO
ND
ND
ND
NO
ND
ND
Say0
sludge
biaS
04065.0937,54045
62
0*
085
092400b
030
ND
S~
NO
NO
ND
ND
Say
I
sludge
bin
cAo6a.
o927.
sA.
ooe,
62
0*
085
0924016
031
ND
ND
NO
,,..
J~
9.,.,,,,,,,
ND
ND
Say2
sludge
blank.
04085.0927­
84,047,62
0*
085
0924016
032
ND
ND
ND
ND
ND
ND
SayS
sludge
blailk,
C4085'
0927'
SA.
04e.
62
CAO8S
092401b
033
ND
ND
ND
,_,,
J4Q,,,,,,,,,
ND
ND
Daylooudgeblagok.
QADOS'
0927.
SA­
040
62
,...,
0&
065_
092401b
034
ND
ND
ND
ND
ND
ND
Say
74
lIMe
blanA,
0A085.0027.
SA­
050.
62
Say
~
6
ojudgeblank
CAO8S­
0027'
SA­
051.
ES
05085
0920016
035
0*
065
0024016
026
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Page
34
of
36
Pace
Project
CAOB5:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
000036
Wednesday,
November
06.
2002
Appendix
F
Typical
Total
Ion
Chromatograms
for
SIR
Data
Showing
Telomer
Alcohols
and
Polyfluorinated
Fatty
Acid
Products
from
Day­
O
to
Day­
16
for
HPLC/
MS
Analysis
of
Biodegradation
Culture
SPE
eluate
2.

SIR
of
17
anions:
rn/
i
263,
313,
363,
413,
463,
513,
563,
and
613
for
pm­
fluorinated
acids;
mlz
377,
477
and
577
for
polyfluorinated
acids;
and
rn/
i
323,
423,
523,
623,
723,
and
823
for
telomer
alcohols,

Pertluorinated
Fatty
Acid
Peaks
cA385.81274*
a42
22.
dey
II
bIOde9
simple
04245
092
lOla
011
100­
r
~

L
~~"`
I.!,
.:,
lllas_
os7'/
ooa,
713
100,
91,720
742
Day­
14',
~
­
~

04065_
202701a_
617
SIR
oflichann.
l.
ES.
l.
a
Sloe
Day­
7
"
1
100­
6~
7733
1.1
9:
00
:/
LL,.,__,,__
3___~_.
08080_
092701a_
211
SIR
oil?
Cliannoli
ESNC
1.5346
0'
I
722
01k
~
)
U
~

SIR
oil?
Cyanrslesort
I
02,6
Pace
Project
CA085:
asiodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bia­
Analytical
Services
Group
Wednesday,
November
06,
2002
000037
Day­
li"
`.
8!
8,000110hannsls
ES'
lIT.
1.08,6
SIR
00
1105an
11
InIS
55.
TIC
1.81MG
722
02
~
J,
Day~
5~

CAo8S_
0927011_
000
100.

Day­
2
L
_.~..

lbl7oIa
I­
lu
SIR
of';
Channels
Es.
050
9'
.
rc
90
`
747
Day­
i
:
.....
P2
~
79"~~
Q~,,:
­

III
o'NIyil
`
1,8,01,
ES
100
[`
 
I
TIC
Day­
a
:
J0~'~_?~~
`~
t~_'­
L
:,...,
ttr2,4
,.
,~_._.­

100
2.00
300
400
5.00
5.02
700
0.00
900
1200
11.02
12100
0300
7420
Telomer
Alcohol
Peaks
Page
35
of
38
Appendix
G
SIR
data
Showing
Ions
for
Telomer
Alcohols
at
Day­
O
and
Day­
i
6
from
Analysis
of
the
Biodegradation
Culture
SPE
eluate
2.

CA085­
0927­
84421
22.
day
a
biodeg
sample
CAE
105
015.'
YE
1,4
lOS
ISO
0S
SIR
of
olcnaon,
4o
ES­
431000001,
S
OdeS
SIR
of
17C$
oannels
6$'
S23.
ce
2.000a
50065
Pace
Project
CAOB5:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols'
 
Revision
1
Study
Director:
Cleston
C.
Lange,
PhD.
Pace
Analytical
Services,
Blo­
Analytical
Services
Group
Wednesday,
November
06,
2002
SIR
or
Il011annOIs
53'
TIC
50065
000038
7.50
913
C4500
270
a
`
108
1
SIR
or
IT
004,0201,05­
ice
~
23N1
I
0000
50065
44
`
53
o
­
...­.
 
­
,­
­
 
­
"~
 
­
.­
C4005092701a,.
006
SIR
of?
Channels
ES.
000
523.00
1
CODa
5.0005
tan
,~
~,

06215,.
C92?
Ola..
I08
SIR
oil?
ChofInelo
ES­
523.00
0,
ceCa
5,0045
14
`­.­­

05065
09270ia~
SIRoil?
ChaRlEs
65,
00
4230010009
50065
S
e.,
2
­­.`
 
,
.­
 
­
 
­
.
 
 
­
 
­
­
 
_
.­­­
,.
~
­­

CROSS
(
52702a
008
SIR
Oil?
Channels
ES­
000
32300
I
CODS
5.0005
S
0
­­­­­­­­­
­­­­­
 
­
 
­
­
r
,.,
 
­
~~­,
r
­­
­­­
CAEOS_
0927OIafi08
SIR
0117
ChanloelS
ES'

cm.
000
I
.~
1~~
nnzr~=
tr
 
tme
.00
2.00
3.00
4,00
5.00
6.00
7.00
6.00
9.00
50.00
11.00
52.00
300
14.00
cAOas­
0927­
sA­
043
22,
day
it
blodeg
sampia
CASES
0h275I~
014
SIR
0127
0114(
100.1%
ES.

100
6230015000
Scales
9.

00585
51112701a
0t4
SIR
oIl?
CTIOSEOOIS
ES.
ICE
723.00
SODa
5
00e6
S
cAoos_
oo2?
o'a_
o'.
.
SIR
oil?
C6,
o,
vlolo
ES'
ice
023.00
i.
000a
SOOeS
S
CREOS_
092701
9,014
Ice
S
CAII05_
41927010_
014
SIR
all
y
CNan,
eIs
ES'
00
41220
I
0034
50045
94
SIR
or?
O'annos
ES'

100
`
U
lb
0
~
­­
 
`
201145
1412'
OIal­
I
ice
 
~

S
;­,;
942
4.20
`
""­`
J
4
'_._
.,___..
._,~
,,.
 
0
­­
 
­
 
 
 
11150
000
2.00
3.00
ON
5.00
6.00
7,00
8.00
9.00
1000
11.00
1200
2200
1400
Page
36
of
38
Appendix
H
SIR
data
Showing
Ions
for
Perfluorinated
Fatty
Acids
at
Day­
O
and
Day­
I
6
from
Analysis
of
the
Biodegradation
CuLture
SPE
eluate
2.

C408S'
092?­
SA.
O31
E2,
day
0
biude~,
ampie
Cs065_
091701a_
008
51R
of
17
CEanwIcOs
ESco
813001.000S
45
2.
OOeô
c.,
ootaor7oIaj~
.
­
.
­
Sl~
0(
I?
CnannOla
ES'
003
500.00
CODa
2.
CEleO
0'­~­­'.`
 
 
.
`"­
 
 
­
`
_
 
`
.`
 
 
`
 
­
`­..
 
`
 
,
 
`
­..
 
 
_
r__..
 
'
.
 
­
 
'
­
 
.
 
 
 
t.
 
­
.,.',
 
 
,
.­
 
 
_
­'_.'_.
 
­
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Pace
Project
CA085:
"
Biodegradation
Screen
Study
for
Telomer
Alcohols"
 
Revision
I
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services.
Bio­
Analytical
Services
Group
Wednesday,
November
06,
2002
000039
Appendix
I
Chroniatograms
of
Extracted
ions
m/
z
377,
477,
and
577
and
Mass
Spectra
for
Each
Peak
for
Polyfluorinated
Acid
Intermediates
Formed
Transiently
in
Biodegradation
Cultures
at
Day­
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Day­
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Day­
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and
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Suspected
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Page
38
of
38
­
­.
rer,

Pace
Project
CA065:
"
Biode9radation
Screen
Study
for
Telomer
Alcohols
 
Revision
1
Study
Director:
Cleston
C.
Lange,
Ph.
D.
Pace
Analytical
Services,
Bio­
Analytical
Services
Group
Wednesday1
November
06,
2002
000040
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