Document ID: EPA-HQ-OPPT-2002-0051-0015
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2006-03-07T05:00Z

BIODEGRADATION
(ASS/
LAS
Shake
Culture
Test)

TEST
SUBSTANCE
Identity:
Perfiuorooctanoic
acid,
ammonium
salt;
may
also
be
referred
to
as
PFOA
ammonium
salt,
Ammonium
perfluorooctanoate,
PFO,
FC­
116,
FC­
126,
FC­
169,
or
FC­
143.
(Octanoic
acid,
pentadecafluoro­,
ammonium
salt,
CAS
#
3825­
26­
l)
,

.Remarks:
The
test
substance
is
a
white
powder.
The
3M
production
lot
number
was
83.
The
test
sample
is
referred
to
by
the
testing
laboratory
as
FC­
143.
The
purity
of
the
sample
was
not
sufficiently
characterized,
although
current
information
indicates
it
is
a
mi>;
tureof
96.5
­
100%
test
substance
and
0
­
3.5%
C6,
C7,
and
C9
perfluoro
analogue
compounds.

METHOD
Methods:
Shake
Culture
study
modeled
after
the
Soap
and
Detergent
Association's
presumptive
test
for
the
determination
of
ABS/
LAS
biodegradability.
Type:
Aerobic
GLP:
No
Year
completed:
1978
Contact
time
(units):
2.5
months
Innoculum:
Activated
sludge
collected
at
the
3M
Chemolite
Facility,
Cottage
Grove,
MN,
3M
Decatur
Facility,
Decatur
AL,
and
Metro
Wastewater
Treatment
Plant,
St
Paul,
MN
RESULTS
No
biodegradation
was
observed
in
the
2.5
month
shake
culture
biodegradation
study.

DATA
QUALITY
Reliability:
Klimisch
ranking
=
2.
This
study
meets
all
criteria
for
quality
testing,
but
the
analytical
methodology
is
questionable.
The
purity
of
the
sample
was
not
sufficiently
characterized.
REFERENCES
Fate
of
Fluorochemicals
in
the
Environment,
Project
number
9970612613,
E.
A.
Reiner,
July
19,
1978,
3M
Company,
Environmental
Laboratory.

OTHFR
Submitter:
3M
Company,
Environmental
Laboratory,
P.
O.
Box
33336,
St.
Paul,
Minnesota,
55133
Last
changed:
5/
25/
00
­
TECHNICAL
REPORT
SUMMARY
[7/
19/
18
To:
TECHNICAL
COMMUNICATIONS
CENTER
­
201~
XN
hpo*
nt
­
If
rewt
isprintad
M
both
sides
of
m,
snnd
nn,
copies
to
TCC.)

DIVISIOI'

Environmental
Laboratory
(EE
8
PC)
hOjUt
­W­
t
Tw*
Fate
of
Fluorochemicals
in
the
Environment
W­
t.
Numbu
0535
~rolact
Numbar
9970612613
Rmort
Number
T
O
Biodegradation
Studies
of
Fluorocarbons
­
III
:'s
D.
L.
Bacon
AUttW(
Sl
w­
Em~
lovr
NumborW
E.
A.
Reiner
47816
N"­
ma'­*
nc.
44703,
p.
6­
14,
21,
25­
27,
29
35
39­
43
46
.
32­
35;
49400,
p,
llk2.
'
r
,
No.
of
P­
8
including
Covonhaat
45727,
p.
17
ooCURlTY)
o
­
8
cloyd
mgg$
fp
b
NW
Chm#
la
Rapcmad
q
Yu
B
No
KEYWOROS:
ktalact
twnn
fmln
3M
thmaurur.
suw
other
a@
pliabla
arms.
J
(Biodegradation)
EE
&
PC­
Div.
Envir
.
Assess.
Fluorochemical
Degradation
URRENT
OBJECTWE:

To
evaluate
the
susceptibilities
of
FC­
95
and
FC­
143
to
microbial
decomposition.

REPORT
ABSTRACTz
{=
260
wordr)
This
detract
informtkn
ir
dirtributod
by
thr
Technical
Communiutions
(&
I­
try
llart
3wrtr
tu
compurv
R&
O.

A
biodegradatioa
study
is
described
which
allows
the
evaluation
of
the
susceptibility
of
FC­
95
and
FC­
143
to
aerobic
microbial
degradation.
The
culturing
procedures
used
in
this
study
are
modeled
after
the
Soap
and
Detergent
Association's
presumptive
(shake
culture)
test
for
the
determination
of
ABS/
LAS
biodegradability.
Microbial
inocula
were
obtained
from
activated
sludge
collected
at
Chemolite,
Decatur
and
Metro
waste
W;
atrfffnt
plants.
Analytical
procedures
included
GLC,
C­
scintillation
counting
and
analysis
for
released
fl&
ride.
Degradation
of
reference
compounds
demonstrated
the
suitability
of
the
biodegradation
test
conditions.
­2­

SUMMARY
Fluorochemicals
FC­
95
and
Fe­
143
were
shown
to
be
completely
resistant
to
biodegradation
in
a
a&
month
shake
culture
biodegradation
study.
The
mixed
microbial
test
cultures
used
in
this
study
were
derived
from
activated
sludge
inocula
obtained
from
three
waste
treatllent
systems
(Chemolite,
Decatur,
8~
the
Twin
Cities
Metro
plant).
The
cultures
were
maintained
in
dilute
yeast
extract­
basal
salts
media
supplemented
with
the
hydrogen
analog
of
the
respective
fluorochemicals
Test
cultures
also
contained
FC­
95
or
FC­
143.
Phenol
and
1­
dodecene­
derived
linear
alkyl
sulfonate
(LAS)
were
used
as
reference
compounds.
Their
degradation
demonstrated
that
biodegradation
could
occur
under
the
test
conditions.
All
cultures
were
transferred
15
t#
nes
over
the
2&
month
period,
and
temperature
was
controlled
at
25
C..
during
the
latter
half
of
the
experiment.

In
the
final
growth
period,
degradation
products
of
14C
labeled
fluorochemicals
were
assayed
for
by
thin­
layer
chromatography
(TLC)
and
gas
liquid
chromatography
(GLC).
Chemicals
separated
by
TLC
were
visualized
by
TLC­
autoradiograph.
Methylated
and
nonmethylated
cuiture
extracts
separated
by
'GLC
were
detected
by
electron
capture.
No
degradation
products
were
detected.
Scintillation
counting
showed
that
all
radioactivity
associated
with
the
labeled
fluorochemicals
remained
in
the
culture
medium.

In
all
but
the
final
growth
period,
fluorocarbon
biodegradation
was
monitored
simply
by
measuring
the
initial
and
final
fluoride
concentration
in
the
media.
No
increase
in
fluoride
concentration
was
observed
indicating
that
if
biodegradation
did
occur,
it
did
not
result
in
the
release
of
fluoride.
Control
cultures
supplemented
with
fluoride
showed
that
fluoride
is
not
lost
from
the
media
under
the
experimental
conditions
used.

While
this
study
cannot
rule
out
the
possibility
that
conditions
could
be
found
that
would
allow
the
biodegradation
of
these
compounds,
the
results
of
this
study
suggest
that
these
chemicals
are
likely
to
persist
in
the
environment
for
extended
periods
unaltered
by
microbial
catabolism.
­3­
.

INTRODUCTION
The
fluorochemicals
selected
for
this
study,
FC­
143
and
FC­
95,
have
perfluorinated
carbon
chains
and
are
chemically
stable.
The
perfluorinated
portion
of
fluorocarbons
have
not
been
found
to
be
susceptible
to
biological
degradation
(1).
Therefore,
biodegradation
studies
were
conducted
on
these
compounds
primarily
for
the
sake
of
completeness.
Without
such
testing,
it
could
not
be
said
with
certainty
that
these
compounds
would
resist
microbial
modification.

Since
biodegradation
was
unlikely,
the
best
feasible
test
conditions
for
biodegradation
were
selected.
Inocula
were
obtained
from
areas
considered
likely
to
contain
acclimated
microorganisms.
Long
acclimation
periods­
were
used
in
an
attempt
to
select
and
develop
populations
of
microbes
capable
of
degrading
these
compounds,
and
hydrogen
analogs
of
the
fluorocarbons
were
added
to
try
to
select
organisms
that
might
gratuitously
"cometabolize"
the
fluorocarbons.
­4­

METHODS
AND
MATERIALS
Chemicals
FC­
95,
FC­
143,
the
hydrogen
analog
of
FC­
95,
ammonium
octanoate
(the
hydrogen
analog
of
FC­
143),
carbon­
14
labeled
FC­
143,
and
carbon­
14
labeled
FC­
95
were
obtained'from
Commercial
Chemicals
Division.
These
chemicals
were
used
as
received
unless
desi,
gnated
otherwise
(Arthur
Meadel­
Heport
in
Progress).

Standard
linear
alkylate
sulfonate
prepared
for
use
as
a
reference
compound
for
biodegradation
studies
was
obtained
from
the
US/
EPA
Laboratory
in
Cincinnati,
Ohio.
Except
where
noted,
all
other
compounds
were
reagent
grade.

Culture
Media
The
control
medium
used
in
these
studies
had
the
composition
shown
in
TABLE
1.

TABLE1
CONTROL
MEDIUM
COEPOSITION
1)
Basal
salts
solutions:

1.0
g/
l
­
NH4Cl
2.0
g/
l
­
K2HP04
0.25
g/
l
­
IUgS04*
7H20
0.002
g/
l
­
FeS04*
7H20
2)
Well
water
­
25
ml/
l
3)
Yeast
extract
­
0.3
g/
l
4)
Rydrogen
analogs
of
either
FC­
95
or
FC­
143
­
20
mg/
l
Media
were
prepared
from
stock
solutions
which
were
combined
and
brought
to
volume
just
prior
to
each
culture
transfer.
A
fresh
solution
of
FeSO
in
media
preparae­
l
7H2O
was
prepared
and
dry
yeast
extract
was
used
ion
at
each
transfer,
The
pH
of
all
media
was
adjusted
to
7.5
with
1.0
N
HCl
and
if
overshot
adjusted
back
with
1.0
N
NaOH.
The
well
water
was
added
to
insure
an
adequate
supply
of
trace
elements.
Analyses
of
the
well
water
made
during
the
ll­
month
period
prior
to
the
initiation
of
this
study
showed
its
calcium
hardness
to
range
from
92
to
144
mg/
l
expressed
CaCO
itate
resulting
from
the
addition
of
well
water
was
8'
Any
precip­
.
emoved
by
filtration
through
a
#54
Whatman
filter.
­5
The
purified
hydrogen
analogs
of
FC­
95
and
FC­
143
were
used
in
biodegradation
test
media
and
controls.
These
compounds
were
included
in
an
attempt
to
select
a
microbial
population
likely
to
degrade
the
fluorocarbons.
Enzymes
capable
of
catalyzing
defluorination
reactions
are
frequently
identical
to
enzymes
involved
in
carbon­
hydrogen
bond
cleavage
(1).
Additional
components
of
other
specific
media
are
listed
in
TABLE
2.

TABLE
2
GROWTH
MEDIA
FORMULATIONS
Media
FC­
95
FC­
143
Test
Phenol
Controls
LAS
Controls
Fluoride
Controls
14
C­
FC­
95
Test
Components
FC­
95
Control
Medium
+
50
mg/
l
FC­
95
FC­
143
Control
Medium
+
50
mg/
l
FC­
143
FC­
95
or
FC­
143
Control
Medium
+
30
mg/
l
Phenol
FC­
95
or
FC­
143
Control
Medium
+
30
mg/
l
Standard
Linear
Alkylbensenesulfonate
(LAS)
FC­
95
or
FC­
143
Control
Medium
+
33.2
mg/
l
NaF
(15.0
mg/
l
F­)

FC­
95
Control
Medium
+
50
mg/
l
14C­
FC­
95
14C­
FC­
143
Test
.
FC­
143
Control
Medium
+
50
mg/
l
"C­
FC­
143
FC­
95
Control
Medium
+
30
mg/
l
LAS
+
50
mg/
l
FC­
95
FC­
143
Control
Medium
+
30
mg/
l
LAS
+
50
mg/
l
FC­
143
FC­
95
+
LAS
FC­
143
+
LAS
Culturing
Procedures
The
initial
growth
period
was
started
by
inoculating
49
ml
of
each
medium
with
1
ml
of
activated
sludge
supernatant.
The
activated
sludge
used
was
a
mixture
of
two
sludges
collected
on
the
day
of
inoculation.
The
sludge
was
obtained
from
the
Metropolitan
Waste
Control
Commission's
Metro
plant
in
Saint
Paul,
Minnesota,
and
the
Chemolite
Waste
Treatment
Plant
in
Cottage
Grove,
Minnesota.
_­...
^
.­..
"_.
­.
.
.
.
.
.
­
­.
.
..­..
a
­6­

Following
inoculation,
the
cultures
in
polypropylene
Erlenmeyer
flasks
were
shaken
at
200
rpm
on
rotary
shakers
at
room
temperature
(4).

At
the
end
of
each
growth
period,
each
culture
was
transferred
to
identical
fresh
media
using
a
1%
inoculum
from
the
preceding
culture
(i.
e.,
0.5
ml
of
existing
culture
to
49.5
ml
of
identical
new
medium).

The
growth
period
between
transfers
varied
as
is
noted
in
TABLE
3.
A
10
ml
sample
was
taken
from
each
culture
at
10
minutes
after
inoculation
or
culture
transfer
and
at
the
end
of
each
growth
period.
Samples
were
centrifuged
for
10
min.
at
17,000
x
g
prior
to
analysis
of
the
centrifugate.
Deviations
from
this
culturing
procedure
are
noted
in
TABLE
3.

The
final
growth
per­
iod
differed
from
preceding
periods.
Media
were
prepared
with
Carbon­
U­
labeled
FC­
95
and
FC­
143.
One
hundred
ml
cultures
were
grgwn
in
flasks
on
a
rotary
shaker
in
a
growth
chamber
controlled
at
25
C.
+
1.
Twenty
ml
samples
were
taken
at
10
min.,
2
days
and
at
7
days.

Chemical
Analysis
Fluoride
ion
concentrations
were
measured
using
a
fluoride
ion
electrode
(Orion
ion
analyzer
fluoride
electrode
model
96­
OQ),
and
a
standard
curve
drawn
from
the
results
of
measurements
of
accurately
prepared
fluoride
standards.
The
concentrations
of
these
fluoride
standards
bracketed
the
concentrations
present
in
the
experimental
samples.
Fluoride
curves
were
set
up
at
each
sampling
period,
except
for
transfer
1.
For
the
analyses
following
this
transfer,
a
1.0
ppm
fluoride
standard
was
used
to
calibrate
the
instrument
with
the
assumption
that
the
slope
of
the
previous
fluoride
curve
remained
constant.

Phenol
analysis
was
done
according
to
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
14th
Edition,
1975.
Linear.
alkylbenzenesulfonate
(LAS)
was
analyzed
for
by
the
methylene
blue,
chloroform
extraction
method
described
in
the
14th
edition
of
Standard
Methods
(3),
except
in
transfers
8­
14,
LAS
was
analyzed
by
a
modification
of
this
method.
In
this
modified
method,
the
samples
was
diluted
to
100
ml
in
a
separatory
funnel.
Also
added
to
the
separatory
funnel
were
25
ml
of
Standard
Methods
methylene
blue
solution
and
100
ml
of
chloroform.
This
mixture
was
shaken
for
30
seconds,
allowed
to
settle,
swirled,
and
the
chloroform
drawn
off
through
glass
wool
into
a
2.5
cm
diameter,
spec
20
curvette,
Percent
transmittance
was
read
at
652
nm
and
compared
to
a
standard
curve
prepared
with
surfactant
samples
of
known
concentration
treated
in
the
same
manner.
­7­

TABLE
3
SUMMARY
OF
CULTURING
PROCEDURES
USED
IN
THE
SHAKE
FLASK
BIODEGRADATION
STUDY
OF
FC­
95
AND
FC­
143
Culture
Growth
Transfer
#
Period
(days).
Notes
0
3
1
3
Used
activated
sludge
inoculun'from
Metro
and
Chemolite.
.

FC­
143­
hydrogen
analog
added
to
143
cultures
and
controls.

2
4
3
3
4
3
5
6
7
6
8
3
9
10
11
12
13
14
15
3
3
At
the
time
of
culture
transfer,
1
ml
of
Decatur
sludge
supernatant
added
to
cultures.

LAS
replaced
phenol
as
a
reference
compound.
LAS
media
was
inoculated
with
a
mixture
of
control
culture
and
Chemolite
and
Decatur
sludge
supernatant.

The
use
of
fluoride
control
was
discontinued.

Shaker
was
inadvertently
turned
off,
possibly
for
5
days,
during
this
growth
period.

*
ml
of
Metro
sludge
supernatant
was
added
to
all
cultures.

In
this
and
subsequent
growth
periods,
cultures
were
grown
in
a
reciprocating
sbaker­
wateg
bath
at
100
strokes
per
min.
and
25
C.

6
6
8
+6
(2)

7
78
days
=
Total
Enrichment
Period
SCarbon
14
Counting
Techniques
Scintillation
counting
was
pef&
yrmed
on
1
ml
samples
of
culture
centrifugate
added
to
Aquasol
and
counted
with
an
internal
standard
quench
correction.
The'radioac$&;
i;;
;i
i!
e?
g
samples
was
compared
to
known
weight
samples
of
­
­
C­
FC­
143
added
directly
to
Aquasol.

Solid
samples
were
collected
directly
onto
millipore
HA
0.45
pm
filters
composed
of
cellulose
acetate
and
cellulose
nitrate.
The
filters
were
then
washed
with
deioniz
fl
water
and
placed
into
paper
combustion
conffi)
them's
Packard
wet
with
Combustaid?
),
T;~
d~$
ombusted
in
Agri­
combustion
equipment.
CO
resulting
from
combustion
was
trapped
in
a
scintillation
fluid
cgntaining
an
organic
amine
and
count&
d
in
Agrichem's
Packard
scintillation
counter.
Samples
were
recounted
with
an
internal
standard
for
'quench
correction.

Thin­
Layer
Chromatography
(TLC)

Thin­
layer
chro@
ography
was14erformed
to
detect
radioactive
metabolites
of
C­
FC­
95
and
C­
FC­
143.
were
collected
and
immediately
frozen.
Ten
ml
culture
samples
frozen
for
about
f.
month.
These
samples
were
stored
The
samples
were
extracted
immediately
after
thawing
with
10
ml
of
ethyl
acetate.
The
samples
were
then
centrifuged
at
1.7,000
x
g
to
ensure
the
separation
of
the
ethyl
acetate,
water,
and
solids
phases,
The
water
phase
and
portions
of
the
ethyl
acetate
phase
were
evaporated
to
dryness
under
N
The
dried
samples
were
resuspended
in
a
9:
l
hexane:
ethyl
ethes*
mixture.
(Some
samples
which
evaporated
to
dryness
in
air
before
spotting
were
resuspended
in
methanol.)
were
spotted
on
E.
Merck
silica
gel
GF
The
resuspended
samples
residue
were
also
applied
directly
to,@&
e
$!~:~
ss"~~~
l":,~,":~~
were
referenced
against
a
mixture
of
C­
FC­
143
and
C­
FC­
95.
The
plates
were
developed
with
10%
ethanol
in
ethyl
acetate
and
visualized
by
exposing
Kodak
no­
screen
x­
ray
film
on
the
plates
for
one
week.

TLC
was
repeated
on
the
remaining
portion
of
the
Solvent
samples.
The
solvent
was
allowed
to
evaporate
to
dryness
in
air,
and
the
residue
resuspended
in
methanol.
heavily,
developed
as
before,
These
plates
were
spotted
more
2
weeks.
and
visualized
with
x­
ray
film
for
.

a&
3642
­9­

Gas­
Liquid
Chromatography
(GLC)

Ethyl
acetate
extracts
were
prepared
as
described
in
the
thinlayer
chro"
fa;
g;;
pi;
meih@.
Control
solutions
were
made
by
dissolving
­
­
C­
FC­
143
in
ethyl
acetate.
Portions
of
the
ethyl
acetate
extract
samples
and
the
ethyl
acetate
control
solutionswere
also
methylated.
Aliquots
of
the
methylated
and
nonmethylated
ethyl
acetate
extracts
and
controls
were
injected
onto
the
5713
Hewlett
Packard
gas
chromatograph
with
electron
capture
detector.
Methylated
samples
were
injected
within
3
hrs.
of
their
methylation.
The
chromatographic
column
was
12
ft.
x
l/
8"
O.
D.
stainless
steel
packed
with
20%
DC
200
(12,500
CS)
on
10%
Bentone
34
and
;50%
80/
90
mesh
Anakrom
P.
A.
temperature
was
250
C.,
The
injechion
port
and
the
detector
temperature
300
g.
The
column
temperahure
was
grogrammed
to
hold
for
4
min.
at
g5
C.,
to
rise
to
180
C.
at
8
C.
per
min.,
and
to
hold
at
180
C.
The
flow
rate
was
adjusted
to
35
ml/
min.
of
Argon/
methane,
95/
5.

blethylations
were
performed
by
adding
a
20
~1
aliquot
of
a
1
pg/
ml
C
FlgC!
OOH
solution,
as
a
reference
compound
to
each
sample.
Dfazomethane
was
then
added
until
a
yellow
color
persisted.
The
'
samples
were
then
loosely
capped,
swirled
and
allowed
to
stand
for
15
minutes.
Nitrogen
was
blown
over
the
samples
until
the
yellow
color
disappeared,
and
the
sample
was
returned
to
its
original
volume
with
ethyl
acetate.

RESULTS
AND
DISCUSSION
Fluoride
Release
In
all
but
the
final
growth
period,
degradation
of
FC­
95
and
FC­
143
was
monitored
only
by
analysis
of
fluoride
concentration
at
the
beginning
and
end
of
each
culture
period.
It
was
assumed
that
if
the
fluorochemical
portions
o$
these
molecules
were
degraded,
fluoride
ion
would
accumulate
in
the
media.
To
ensure
that
fluoride
was
not
lost
from
the
culture
by
absorption,
precipitation
or
volatilization,
control
cultures
were
grown
with
15
mg/
l
of
fluoride.
This
fluoride
concentration
is
approximately
what
would
result
if
FC­
95
or
FC­
143
underwent
degradation
with
50
percent
fluoride
release.
The
results
of
the
fluoride
analyses
conducted
on
different
days
showed
considerable
variation.
This
was
due
to
the
variable
and
very
sluggish
response
of
the
fluoride
electrode.
TABLE
4a
shows
the
results
obtained
at
each
transfer.
TABLE
4b
shows
the
results
obtained
when
the
same
samples,
which
had
been
stored
in
polyethylene
containers,
were
analyzed
together
after
the
termination
of
the
experiment.,
Despite
the
variability
due
to
the
analytical
technique,
the
results
indicate
that
fluoride,
if
released
to
the
media
through
biodegradation,
would
not
be
lost
from
the
media.

The
results
of
the
fluoride
analysis
on
fluorocarbon­
containing
cultures
and
controls
are
shown
in
TABI&
5.
The
results
show
that
no
biodegradation
with
fluoride
release
occurred.
WI3643
lOTABLE
4a
INITIAL
AND
FINAL
FLUORIDE
CONCENTRATION
(mg/
l)
OF
FLUORIDE
SUPPLEMENTED
CONTROLS
MEASURED
BY
SPECIFIC
ION
ELECTRODE
AT
TRE
TIME
OF
TRANSFER
FC­
95
FC­
143
Fluoride
Control
Transfer
#
Initial
Final
Fluoride
Control
Initial
'Final
0
21
23
20
21
1
23
22
21
20
2
20
22
16
21
3
26
­
17.5
25
16.5
­
4
16.5
19.2
16
17.3
TABLE
4b
INITIAL
AND.
FINAL
FLUORIDE
CONCENTRATION
(mg/
l)
OF
FLUORIDE
SUPPLFWNTED
CONTROLS
MEASURED
BY
SPECIFIC
ION
ELECTRODE
MEASURED
COLLECTIVELY
AT
END
OF
STUDY
I%­
95
FC­
143
Fluoride
Control
Fluoride
Control
Transfer
#
Initial
Final
Initial
Final
0
1614
16.2
15.7
17.0
1
15.6
16.2
15.7
15.0
2
15.6
16.2
14.5
16.4
3
16.2
16.2
16.4
15.6
4
15.6
15.7
15.7
17.0
5
19.3
17.0
15.0
16.4
llTransfer
#
Init.
Final
0
0.46,,
0.51
1
0.50
0.46­

2
0.42
0.66
3
(5)
1.75
1.6
4
0.73
0.71
5
0.72
0.78
6
0.73
0.8
7
0.14
0.17
8
0.90
0.84
9
0.84
0.73
10
0.72
0.81
11
0.81
0.80
12
0.74
0.73
13
0.73
0.84
14
0.81
0.78
TABLE
5
INITIAL
AND
FINAL
FLUORIDE
CONCENTRATION
(q/
l)
OF
FC­
143
AND
FC­
95­
CONTAINING
CULTURES
AND
OF
NONSUF'PLEMENTED
95
AND
143
CONTROL
CULTURES
FC­
95
Test
95
Control
FC­
143
Test
Init.
Final
Init.
Final
0.31
0.33
co.
1
0.36
0.36
x0.1
0.34
0.56.
CO.
1
1.75
1.5
.83
0.68
0.60
40.1
0.61
0.68
<O.
l
0.63
0.70
SO.
1
SO.
1
CO.
1
(0.1
0.66
0.66
40.1
0.72
0.60
co.
1
0.60
0.68
(0.1
0.69
0.62
<O.
l
0.66
0.62
SO.
1
0.64
0.66
40.1
0.66
0.64
Q.
1
<Oh
co.
1
to.
1
(0.1
CO.
1
co.
1
x0.1
co.
1
CO.
1
1
.81
1
qo.
1
KO.
1
x0.1
SO.
1
co.
1
0.56
co.
1
x0.1
<O.
l
(0.1
<O.
l
<O.
l
x0.1
x0.1
co.
1
x0.1
co.
1
<O.
l
SO.
1
so.
1
<O.
l
KO.
1
<O.
l
CO.
1
q0.1
go.
1
co.
1
<o.
l
<o.
l
SO.
1
q0.1
W.
1
<o.
1
:
143
Control
'Init.
Final
­12­

Reference
Compounds
Reference
compounds
were
used
to
demonstrate
that
the
biodegradation
test
conditions
used
were
suitable
to
degrade
compounds
known
to
be
somewhat
resistant
to
degradation.

In
the
first
four
growth
periods,
30
mg/
l
phenol
was
added
to
two
cultures
which
were
identical
to
the
test
cultures,
except
that
they
lacked
fluorocarbons.
Analytical
problems
prevented
the
measurement
of
phenol
concentration
during
the
first
three
growth
periods.
In
the
fourth
growth
period,
phenol
was
found
to
degrade
to
less
than
1.3
mg/
l,
the
limit
of
sensitivity
of
the
method
as
applied.
This
demonstrated
that
the
test
conditions
were
suitable
for
the
biodegradation
of
phenol.

In
the
fifth
through
final
growth
periods,
reference
linear
alkyl
sulfonate
(LAS)
was
used
as
the
reference
compound.
This
compound
is
a
standard
reference
material
used
in
the
Soap
and
Detergent
Association's
biodegradation
test
method
for
anionic
surfactants(
6).
This
material
is
considered
to
be
relatively
easily
degraded.
In
the
Soap
and
Detergent
Association's
shake
flask
biodegradation
test,
the
results
are
considered
invalid
if
the
removal
of
l­
dodecenederived
LAS
is
not
nearly
complete.

The
data
showing
the
extent
of
degradation
of
LAS
in
surfactant
supplemented
controls
are
depicted
in
TABLE
6.
The
data
showing
the
equivalent
amount
of
methylene
blue
active
substances
in
the
controls
not
supplemented
with
LAS
are
depicted
in
TABLE
7.
Little
LAS
degradation
occurred
during
the
first
few
adaptive
transfers.
Three
transfers
were
required
before
the
majority
of
the
LAS
began
to
degrade
in
the
surfactant
supplemented
control
for
FC­
95.
Five
transfers
were
required
for
LAS
degradation
in
the
143
control.
Therefore,
it
appeared
that
organisms
capable
of
degrading
l­
dodecenederived
LAS
were
not
initially
present
in
sufficient
numbers
for
LAS
degradation.
The
test
condition
allowed
for
enrichment
of
these
organisms,
but
enrichment
occurred
at
a
slower
rate
than
had
been
anticipated.
ConsequentlY,
changes
were
made
in
the
procedure
to
increase
the
rate
and
likelihood
of
acclimating
organisms
capable
of
degrading
the
fluorochemicals.
Growth
periods
were
extended
from
3
to,
4­
6
dazs,
and
temperature
was
raised
from
room
temperature
(~
20
to
22
C)
to
a
constant
temperature
of
25OC.
Results
of
LAS
degradation
in
the
final
growth
period
are
shown
in
TABLE
8.

In
the
growth
periods
following
transfers
11
and
12,
an
experiment
was
done
to
determine
if
50
mg/
l
of
FC­
95
or
FC­
143
inhibited
the
degradation
of
LAS.
These
results
are
shown
in
TABLE
9.
FC­
95
appears
to
have
an
inhibiting
effect
on
the
microbial
degradation
of
LAS.
However,
its
presence
was
not
completely
inhibitory.
.
Comparison
with
TABLES
8
and
6
shows
that
the
presence
of
50
mg/
l
of
FC­
95
inhibited
LAS
degradation
by
18%
and
23%
during
these
two
test
periods.
On
the
other
hand,
within
the
limits
of
the
precision
of
our
method,
FC­
143
did
not
appear
to
have
a
significant
effect
on
LAS
degradation.
­13­

In
the
final
growth
period,
50
mg/
l
of
carbon
14­
labeled
FC­
95
and
FC­
143
were
used
as
test
substrates
in
place
of
the
nonlabeled
fluorochemicals.
Both
FC­
95
and
FC­
143
cultures
were
prepared
in
triplicate.
The
concentrations
of
the
radioactive
fluorocarbons
present
in
the
aqueous
phase
as
determined
by
scintillation
counting
are
shown
in
TABLE
10.
The
initial
FC­
95
concentration
is
much
lower
than
expected.
This
low
value
could
have
rer$
ulted
from
a
systematic
error
in
the
collection
of
the
initial
FC­
95
samples.
It
is
also
possible
that
FC­
95
had
not
completely
dissolved
in
the
cultures
when
the
first
sample
was
taken,
but
this
seems
unlikely,
since
the
initial
values
for
FC­
95
concentration
from
all
3
parallel
cultures
were
almost
identical
(30.3,
29,8
and
30.4
mg/
l).
Nevertheless
the
remaining
data
show
that
the
radioactivity
associated
with
FC­
95
and
FC­
143
remained
in
solution
during
the
entire
7­
day
degradation
test­
period..
AntilysG'
of,
the'
biological
solids
showed
some
binding
of
radioactive
material,
but
the
vast
majority
remained
in
the
liquid
phase.

TABLE
6
CQNCXHTRATIti
OF
LAS
(mg/
l)
IN
SUPPLEMENTED
CONTROLS
AND
96
LAS
REXOVED
Transfer
#

4
5
6
7
8
9
10
11
12
13
14
95
­
Surfactant
Control
143
­
Surfactant
Control
LAS
LAS
Init.

31.5
28.3
29.8
25.0
31.2
33.0
32.7
31.0
31.3
31.7
31.3
Final
96
Removal(
7)
Init.

26.8
18.4
35.5
27.5
0.1
32.8
25.5
15.5
27.0
12.0
91.1
25.0
3.75
89.8
37.0
3.17
95.1
38.9
2.33
95.9
42.7
2.0
95.0
39
2.33
96.5
41.3
2.5
93.5
41.3
3.0
92.8
40.3
Final
29.5
25.8
24.0
30.6
35.8
13.7
12.8
13.7
19.7
18.0
13.7
%
Removal
.

19.4
21.2
7.0
­2.0
11.1
94.6
89.7
93.8
77.9
88.3
90.7
­14­

TAi3J.
Z
7
CONCENTRATION
OF
METHYLENE
BLUE
ACTIVE
SUBSTANCE
(mg/
l)
IN
NONSUPPLWENTED
CONTROLS
95
­
Control
143
­
Control
Transfer
#

4
5
6
7
8
9
10
11
12
13
14
Lnit.

1.0
1.15
0.50
5.2s
3.0
5.75
4.17
4.33
3.0
1.33
3.67
0.67
3.67
1.0
Final
1.9
.38
.75
10.2
0.88
1.83
1.17
0.67
Init.

4.5
4.5
7.1
5.0
9.0
10.9
12.3
11.5
12.3
11.3
14.5
Final
4.5
3.5
5.50
10.2
10.9
12.2
9.67
12.0
13.3
14.5
11.3
TABLE
8
CONCENTRATION
OF
MBAS
(mg/
l)
IN
SURFACTANT
SUPPLEMENTED
AND
NONSUPPLEMENTED
CONTROLS
DURING
FINAL
GROWTH
PERIOD
95
Coat
#2
LA:
01s
FC­
143
Controls
Non­
#l
LAS
#2
LAS
NonTime
SUPPl,
SUPPl.
&J&
Suppl.
Suppl.
SUP&

Initial
28.7
29,3
1.0
34.0
36.7
6.5
Day
2
13.0
26.0
1.0
8.0
22.3
5.3
Day
7
1.67
2,0
.7
6.3
6.3
4.0
%
LAS
96.5
95.4
­
91.6
92.3
­
Removal
(7)
­15­

TABLE
9
EFFECT
OF
FC­
95
AND
FC­
143
ON
THE
BIODEGRADATION
OF
LAS
ANALYZED
FOR
AS
MBAS
FC­
95
+
LAS
Culture
FC­
143
+
LAS
Culture
'b
LAS
$8
LAS
Transfer
#
Init.
Final
Removal(
8)
Init.
Final
Removal(
a)

11
58.0
34.7
73.6
53.7
27.3'
97.8
12
64;
3
­4&
3
78.9
53.7
34.0
71.4
TABLE
10
CONCENTRATION
OF
14C­
FC­
95
OR
14C­
FC­
143
IN
THE
CENTRIFUGATE
OF
TEST
CULTURE
DURING
THE
FINAL
GROiVTR
PERIOD
14
C­
FC­
95
Cultures
Standard
14
C­
FC­
143
Cultures
Standard
Concentration
Deviation
Concentration
Deviation
Init.
30.1
mg/
l
0.3
mg/
l
46.2
mg/
l
0.9
mg/
l
Day
2
52.8
0.5
48.0
0.3
Day
7
53.5
3.1
49.7
0.4
­16­

Thin­
layer
chromatography
did
not
reveal
the
presence
of
radioactive
metabolic
products
of
either
FC­
143
or
FC­
95.
Likewise,
gas
liquid
chromatography
of
the
same
culture
extracts,
both
before
and
after
methylation,
showed
no
products
that
were
not
initially
present
or
not
also
present
in
controls.
From
the
combination
of
these
results,
it
can
be
concluded
that
no
biodegradation
of
these
fluorochemicals
occurred.

REFERENCES
AND
FQOTNOTES
(1)
Goldman,
Peter,
Enzymology
of
Carbon­
Halogen
Bonds.
Degradation
of
Synthetic
Organ­
ic
Molecules
in
the
Biosphere,
Nat.
Acad.
of
Sci.,
Washington,
DC
(1972).

(2)
There
was
a
six­
day
period
before
the
onset
of
the
final
growth
period
during
which
the
test
cultures
were
shaken
at
25O
C.
in
the
presence
of
FC­
95
or
FC­
143.

(3)

(4)
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
14th
Edition,
American
Public
Health
Association
(1975)
.

Daytime
temperatures
were
observed
to
range
between
20
and
22
F.
Night
temperatures
were
not
measured
during
that
part
of
the
study
in
which
cultures
were
shaken
at
ambienttemperature
(see
TABLE
3).
However,
measurement
made
near
the
termination
of
this
23­
month
study,
in
Januagy,
showed
that
nighttime
temperature
frequently
drops
to
17
C.

(5)
At
this
transfer,
Decatur
sludge
was
added
which
'contained
a
high
fluoride
concentration.

(6)
Subcommittee
on
Biodegradation
Test
Methods
of
the
Soap
and
Detergent
Association,
A
Procedure
and
Standards
for
the
Determination
of
the
Biodegradability
of
Alkyl
Benzene
Sulfonate
and
Linear
Alkylate
Sulfonate.
J.
of
the
American
Oil
Chemists'
Society,
42:
986
(1966).

(7)
Percent
LAS
removal
was
calculated
as:

%
Removal
­
WB~,,
­
mASCI)
­
(MBASsF
­
mASCF)
x
1oo
mASSI
­
lKBASCI
Where:

blBASSI
­
The
initial
methylene
blue
active
substances
(MBAS)
concentration
of
the
surfactant
supplemented
culture.
MBASCI
=
The
initial
RBAS
concentration
of
the
nonsupplemented
control
(TABLE
7).
MBASSF
=
The
final
MBAS
concentration
of
the
surfactant
supplemented
culture.
MBASCI
=
The
final
MBAS
concentration
of
the
nonsupplemented
control
(TABLE
7).

7)
The
percent
%I
Removal
=

Where:
­17­
.
(&
rOOO~@+)

LAS
Removal
was
calculated
as:
cmt'd
(
mAsSTI
­
­ASCII
­
(mASsTF
­
aASCF)

(mASsI
­
bfBAsCI)
x
100
rnASSTI
­
The
initial
methylene
blue
active
substances
CbfBAS)
concentration
of
the
culture
supplemented
by
both
LAS
surfactant
and
either
FC­
95
or
FC­
143.

MBASCI
=
The­
initial
BBAS
concentration
of
the
noosupplemental
control
(TABLE
7).

BBASSTF
=
The
final
MBAS
concentration
of
cultures
supplemented
with
surfactant
and
fluorocarbon.

BBASCF
=
The
final
MBAS
concentration
of
the
nonsupplemental
control
(TABLE
7).

MBASsI
=
The
initial
MBAS
concentration
of
the
surfactant
supplemental
culture
(TABLE
6).

It
was
assumed
that'MBAS
concentration
due
to
FC­
95
or.
FC­
143
was
not
reduced
by
the
biodegradation
or
other
loss
of
these
compounds.

&A++

EAR/
ten