May
5,
2003
Page
1
of
10
Draft
Protocol
STUDY
PROTOCOL
INHERENT
BIODEGRADABILITY
OF
<
TEST
SUBSTANCE>
USING
THE
ZAHN­
WELLENS/
EMPA
TEST
PROCEDURE
TESTING
FACILITIES:
To
be
determined
SPONSOR
STUDY
DIRECTOR:

STUDY
NUMBER:
XXXX
Draft
Protocol
1.0
SUMMARY
The
Zahn­
Wellens/
EMPA*
Test
evaluates
the
inherent
biodegradability
of
organic
substances
in
water
under
aerobic
conditions.
Activated
sludge,
mineral
nutrients,
and
the
test
material
(
concentrations
corresponding
to
Dissolved
Organic
Carbon
(
DOC}
values
in
the
range
of
50
to
400
mg
DOC/
L
as
the
carbon
source
in
an
aqueous
solution
are
placed
together
in
a
4­
L
glass
vessel
equipped
with
an
agitator
and
an
aerator.
The
mixture
is
agitated
and
aerated
at
20
to
25o
C
under
diffuse
illumination
or
in
a
dark
room
for
up
to
28
days.
Blank
controls,
containing
activated
sludge
and
mineral
nutrients
but
no
test
substance
are
run
in
parallel.
The
degradation
process
is
monitored
by
determination
of
DOC
(
or
COD;
Chemical
Oxygen
Demand)
in
filtered
samples
taken
at
daily
or
other
time
intervals.
Alternatively,
the
biodegradation
process
can
be
monitored
by
determination
of
the
test
substance
and
potential
degradation
products
in
the
test
solution
at
regular
time
intervals.
The
ratio
of
eliminated
DOC,
COD,
and/
or
test
substance
after
each
interval
to
the
value
at
the
start
is
expressed
as
percentage
biodegradation
and
serves
as
the
measure
for
the
rate
of
degradation.
The
result
is
plotted
versus
time
to
give
the
biodegradation
curve.
Test
chemicals
giving
a
result
of
greater
than
20%
loss
in
this
test
may
be
regarded
as
inherent,
primary
biodegradability,
whereas,
a
result
of
greater
than
70%
loss
is
evidence
of
ultimate
biodegradability.

2.0
TEST
SUBSTANCE
Name:
To
be
provided
by
sponsor
Synonym:
Active
substance(
s)
CAS
Name:
CAS
Number(
s):
Molecular
formula:
Solubility
at
25
°
C.:
Vapor
pressure:
Stability:
Appearance/
Color:
Storage
Conditions:
Safety
Precautions:

3.0
TEST
SYSTEM
3.1
Test
System
Justification
The
test
system
is
outlined
by
OECD
guideline
302B
and
was
requested
by
the
sponsor.

*
EMPA:
Swiss
Federal
Laboratories
for
Materials
Testing
and
Research
Page
2
of
10
Draft
Protocol
3.2
Description
of
Test
System
The
test
system
consists
of
uniquely
labeled
glass
test
vessels
with
a
volume
of
from
1
to
4
L.
The
test
vessel
contains
2.5
mL/
L
mineral
nutrient
solution,
activated
sludge
in
an
amount
corresponding
to
0.2­
1.0
g/
L
(
normally
0.2
or
1.0
g/
L)
dry
matter
in
the
final
mixture,
sufficient
stock
solution
of
the
Test
Substance
to
be
tested
so
that
a
DOC
concentration
of
50
to
250
mg/
L
results
in
the
final
mixture.

The
vessels
contain
a
mineral
nutrient
solution
made
according
to
the
test
guidelines
found
in
OECD
302B
adopted
July
17,
1992.

The
test
system
is
mixed
using
a
magnetic
stirrer
with
an
appropriate
stirring
rod.
The
system
is
aerated
by
using
a
glass
tube
of
2
to
4
mm
inner
diameter
to
introduce
air.
The
opening
of
the
tube
should
be
about
1
cm
above
the
bottom
of
the
vessel.

3.3
Route
of
Administration
and
Justification
As
specified
in
the
test
guidelines,
the
test
substance
will
be
added
to
the
test
system
from
a
stock
solution.
The
stock
solution
will
contain
an
appropriate
amount
of
the
test
substance
dissolved
in
either
water
or
in
an
appropriate
solvent
that
is
miscible
in
water,
such
as
methanol.
If
a
solvent
other
than
water
is
used,
the
final
concentration
of
the
solvent
should
not
exceed
0.1%
of
the
total
volume
of
the
test
solution.

4.0
TEST
PROCEDURES
4.1
Microbial
Inoculum
Activated
sludge
from
a
biological
treatment
plant
is
washed
by
(
repeatedly)
centrifuging
or
testing
with
test
water
(
Test
water
is
deionized
water
with
an
organic
carbon
content
of
less
than
5
mg/
L).
The
activated
sludge
must
be
in
an
appropriate
condition.
Such
sludge
is
available
from
a
properly
working
sewage
treatment
plant.
In
order
to
check
the
activity
of
the
inoculum,
the
use
of
a
functional
control
substance
is
required.

4.2
Functional
Control
Substance
A
vessel
with
a
known
substance
will
be
run
parallel
with
each
test
series
in
order
to
check
the
functional
capacity
of
the
activated
sludge.
For
this
purpose,
compounds
such
as
diethyleneglycol,
sodium
benzoate,
and
aniline
are
recommended.
The
functional
control
substance
will
be
documented
in
the
study
records
and
included
in
the
final
report.

Page
3
of
10
Draft
Protocol
The
Functional
Control
Substance
Solution
(
in
duplicate)
is
made
up
as
follows:

a.
To
the
Control
vessel,
add
500
mL
of
test
water,
2.5
mL/
L
mineral
nutrient
solution,
and
activated
sludge
in
an
amount
corresponding
to
0.2
to1.0
g/
L
(
normally
0.2
or
1.0
g/
L)
dry
matter
in
the
final
mixture.
b.
Add
sufficient
stock
solution
of
the
Control
material
to
be
tested
that
a
dissolved
organic
carbon
(
DOC)
concentration
of
50
to
400
mg/
L
results
in
the
final
mixture.
c.
Make
up
with
test
water
to
a
total
volume
of
from
2
to
4
L.
The
total
volume
to
be
chosen
is
dependent
on
the
number
of
samples
to
be
taken
for
DOC
determination
and
the
volumes
necessary
for
the
analytical
procedure.
Normally
a
volume
of
2
L
can
be
regarded
as
satisfactory.
Exact
volumes
used
will
be
recorded
in
the
study
records.

For
the
control
chemical,
the
loss
of
DOC
should
be
greater
than
70%
within
28
days;
otherwise
the
test
is
regarded
as
invalid
and
should
be
repeated
using
an
inoculum
from
a
different
source.

4.3
Test
Solutions
and
Blank
Controls
The
Test
Solution
(
in
duplicate)
is
made
up
as
follows:

a.
To
the
Test
vessel,
add
500
mL
of
test
water,
2.5
mL/
L
mineral
nutrient
solution,
and
activated
sludge
in
an
amount
corresponding
to
0.2­
1.0
g/
L
(
normally
0.2
or
1.0
g/
L)
dry
matter
in
the
final
mixture.
b.
Add
sufficient
stock
solution
of
the
Test
Substance
to
be
tested
so
that
a
DOC
concentration
of
50
to
250
mg/
L
results
in
the
final
mixture.
c.
Make
up
with
test
water
to
a
total
volume
of
2
to
4
L.
The
total
volume
to
be
chosen
is
dependent
on
the
number
of
samples
to
be
taken
for
DOC
determination
and
the
volumes
necessary
for
the
analytical
procedure.
Normally
a
volume
of
2
L
can
be
regarded
as
satisfactory.
.
Exact
volumes
used
will
be
recorded
in
the
study
records.

A
Chemical
Blank
Control
(
in
duplicate)
will
be
set
up
to
run
in
parallel
with
each
test
series.
This
control
contains
only
activated
sludge
and
mineral
nutrient
solution
made
up
with
test
water
to
the
same
total
volume
as
in
the
test
vessels.

Determination
of
Time
0
DOC
value
for
the
Test
Chemical
and
Functional
Control
Chemical:
Inoculation
Blank
Controls
(
in
duplicate)
of
the
Test
Substance
and
Functional
Control
Substance
will
be
tested
at
Time
0
only.
These
controls
will
contain
only
Test
Chemical
(
or
Functional
Control
Chemical)
and
mineral
nutrient
solution
made
up
with
test
water
to
the
same
total
volume
as
in
the
Test
(
or
Functional
Control)
vessels.
At
Time
0,
the
DOC
value
will
be
determined
and
compared
to
the
DOC
concentration
in
the
Test
(
or
Functional
Control)
Vessels
at
3
h.
If
differences
in
the
DOC
values
are
observed,
then
it
can
be
attributed
to
adsorption
Page
4
of
10
Draft
Protocol
of
the
Test
(
or
Functional
Control)
material
by
the
activated
sludge.
For
test
substances
that
are
not
water­
soluble,
the
test
substance
concentration
in
the
test
vessel
will
be
determined
using
appropriate
analytical
methods,
which
will
be
documented
in
the
study
records.

Inhibition
Control
Test
(
10
day
Test):
It
is
recommended
that
this
test
should
be
performed
prior
to
the
start
of
the
Zahn­
Wellens
Test.
The
Inhibition
Control
Test
allows
for
the
establishment
of
inhibition
threshold
levels.

a.
Series
1:
Test
material
with
a
DOC
concentration
of
400
mg/
L
in
the
final
mixture.
b.
Series
2:
Functional
Control
Chemical
with
a
DOC
concentration
of
400
mg/
L
in
the
final
mixture.
c.
Series
3:
Series
1
plus
Series
2.

If
the
DOC
value
of
Series
3
correspond
to
the
sum
of
those
of
Series
1
and
2,
then
the
test
substance
does
not
display
inhibitory
effects.

4.4
Procedure
The
test
vessels
are
agitated
with
magnetic
stirrers
under
diffuse
illumination
or
in
a
dark
room
at
22o
C
(+
3o
C).
Aeration
is
accomplished
by
using
compressed
air
(
organic­
free
filtered).
The
sludge
must
not
settle
during
the
test
and
the
oxygen
concentration
must
not
fall
below
2
mg/
L.
The
pH
value
must
be
checked
at
regular
intervals
(
e.
g.,
Monday
thru
Friday)
and
adjusted
to
pH
7
to
8
with
NaOH
or
H2
SO4,
if
necessary.
Losses
from
evaporation
are
made
up
just
before
each
sampling
with
deionized
water
in
the
required
amounts.
The
best
procedure
is
to
mark
the
liquid
level
on
the
vessel
before
starting
the
test
and
after
each
sampling
(
without
aeration
and
stirring).
The
first
samples
are
always
taken
3
h
after
the
start
of
the
test
in
order
to
detect
adsorption
of
the
test
material
by
the
activated
sludge.
The
elimination
of
the
Test
(
and
Functional
Control)
materials
is
followed
by
DOC
determinations.

The
vessel
is
sampled
by
using
a
glass
pipet
to
remove
50
mL
of
sample
from
the
test
(
or
blank)
vessels
into
a
100
mL
glass
beaker.
The
test
and
blank
samples
are
removed
from
the
beaker
using
a
glass
syringe
(
Luer­
Lok
tip)
and
then
filtered
through
a
syringe
pre­
filter
(
glass
fiber)
with
an
attached
0.2
µ
m
syringe
filters
(
PTFE
or
PVDF).
The
first
5
mL
of
test
(
or
blank)
solution­
filtrate
is
returned
to
the
test
(
or
blank)
vessel.
Sludge
difficult
to
filter
may
be
separated
before
filtering
by
centrifugation.
DOC
determination
in
the
sample
filtrates
is
determined
at
least
in
duplicate
by
means
of
the
Total
Organic
Carbon
(
TOC)
Analyzer.
Test
substance
and
potential
transformation
products
are
determined
using
appropriate
analytical
methods.
The
test
will
run
for
up
to
28,
42,
or
60
days.
If
complete
degradation
(>
70%
loss
of
DOC
or
test
substance
concentration)
is
attained
before
the
test
time
is
over
and
this
result
is
confirmed
by
a
second
analysis
on
the
next
day,
then
the
test
can
be
concluded.
The
length
of
time
that
the
test
is
conducted
will
be
recorded
in
the
study
records
Page
5
of
10
Draft
Protocol
5.0
ANALYTICAL
METHODS,
REFERENCE
STANDARDS,
REAGENTS,
AND
SOLVENTS
All
samples
will
be
analyzed
by
the
appropriate
analytical
method.
The
analytical
method
used
will
be
documented
in
the
study
records
and
the
final
report.
Analytical
reference
standards
that
are
used
to
aid
the
identification
of
the
test
substance
and
its
potential
degradation
products
will
be
documented
in
the
study
records.
All
chemicals
and
solvents
will
be
reagent
grade
or
purer.
Sources
will
be
documented
in
the
study
records.
Deionized
water
produced
at
the
testing
facility
will
be
used
throughout
in
the
study.
All
samples
will
be
stored
frozen
unless
analyzed
with
1
to
2
days.

6.0
DATA
ANALYSIS
The
amount
of
degradation
attained
at
the
end
of
the
test
is
reported
as
the
"
Inherent
Biodegradability
in
the
Static
Test
(
after
x
days)":

DT
(%)
=
1
­
[(
CT
­
CB
)
/
CA
]
x
100
where:
DT
=
Biodegradation
(%)
at
time
T
(
days)

CA
=
Initial
value
(
DOC
in
the
test
mixture
calculated
from
the
DOC
value
in
the
stock
solution,
(
mg/
L)

CB
=
DOC
value
of
the
blank,
(
mg/
L)

The
degradation
rates
are
rounded
to
the
nearest
full
percent.

If
the
result
of
analysis
of
the
first
sample
(
3
h
after
starting
the
test)
is
significantly
different
from
the
theoretical
or
measured
time
0
value,
the
amount
of
deficient
test
substance
(
measured
by
determining
DOC
or
directly)
will
be
reported
as
"
adsorbed
by
the
activated
sludge".

The
significant
points
of
the
degradation
curve
are
to
be
reported
as:
a.
adaptation­
phase
(
days)
b.
degradation­
phase
(
days)
c.
endpoint
of
degradation
reached
after
...
days
7.0
STATISTICAL
METHODS
AND
CONTROL
OF
BIAS
Statistical
methods
including
means,
standard
deviations,
and
regression
lines
will
be
used
as
appropriate.
Bias
will
be
effectively
controlled
through
duplicate
sampling,
replicate
analysis,
sample
spiking,
and
maintenance
of
material
balance.

Page
6
of
10
Draft
Protocol
8.0
REPORTING
OF
DATA
Interim
reports
may
be
written
and
submitted
to
the
study
sponsor
during
the
various
phases
of
the
preliminary
and
definitive
studies.
A
draft
of
the
final
report
will
be
submitted
to
the
sponsor
approximately
one
month
after
the
conclusion
of
all
of
the
definitive
studies.
The
final
report
will
include,
but
not
be
limited
to:
a.
Items
cited
in
EPA
FIFRA
GLPs
40
CFR
160.185.
b.
Study
information
containing
the
identification
of
testing
facility,
location
of
the
raw
data,
study
dates,
and
name
of
the
study
director.
c.
Names
of
principal
study
personnel
and
the
management.
d.
Information
on
the
test
substance
and
information
on
analytical
standards.
Method
of
preparation
of
dosing
solution,
the
amount
and
identity
of
any
cosolvents
or
extractants
used,
and
the
amount
of
test
substance
used.
e.
Description
of
experimental
design
and
experimental
procedures
and
any
deviations
from
the
procedures
stated
in
the
protocol.
f.
The
inoculum
(
sampling
of
the
inoculum,
concentration,
status
of
adaptation)
g.
Description
of
analytical
methods
and
recoveries.
h.
Results
and
discussion
to
include,
but
not
be
limited
to,
material
balance,
adaptation
phase,
degradation
phase,
and
the
degree
of
biodegradation
attained
at
the
end
of
the
test
(
28,
42,
or
60
days)
or,
if
complete
degradation
is
attained
in
less
than
28,
42,
or
60
days,
then
the
biodegradation
is
reported
as
the
"
Inherent
Biodegradability
in
the
Static
Test
(
after
x
days)"

i.
Complete
set
of
calculations
for
one
sampling
point
and
representative
raw
data
9.0
STUDY
RECORD
MAINTENANCE
AND
RETENTION
All
original
paper
data
generated
during
the
study,
the
original
signed
final
report
and
all
original
facility­
specific
raw
data
will
be
retained
by
in
the
DuPont
CR&
D
Environmental
&
Microbiological
Sciences
and
Engineering
Laboratory
(
EMSE)
archive
according
to
40
CFR
792
and
40
CFR
Part
160.
At
least
on
copy
of
the
signed
final
report
will
be
provided
to
the
sponsor.

10.0
GLP
COMPLIANCE
This
study
will
be
conducted
and
reported
in
accordance
with
U.
S.
EPA
Good
Laboratory
Standards
(
40
CFR
792
and
40
CFR
Part
160)
which
are
consistent
with
the
OECD
Principles
of
Good
Laboratory
Practice
published
in
ENV/
MC/
CHEM(
98)
17
(
References
4,
5).
A
statement
of
compliance
will
be
included
in
the
final
report.

Page
7
of
10
Draft
Protocol
11.0
REFERENCES
11.1
OECD
Guidelines
for
Testing
of
Chemicals;
Section
3:
Degradation
and
Accumulation;
Inherent
Biodegradability:
302
B
Zahn­
Wellens/
EMPA
Test:
OECD.
Adopted:
July
17,
1992.

11.2
Chemical
Fate
Testing
Guidelines:
Subpart
D
­
Transformation
Processes,
Part
796.3360,
Inherent
Biodegradability:
Modified
Zahn­
Wellens
Test.
1990.
U.
S.
Environmental
Protection
Agency,
40
CFR
Part
796.
Organization
for
Economic
and
Cooperative
Development
(
OECD)
Guideline
for
the
Testing
of
Chemicals
106,
Adsorption/
Desorption
(
January
21,
2000).
11.3
TSCA
GLP
reference
40
CFR
792
11.4
U.
S.
Environmental
Protection
Agency.
1989.
Good
Laboratory
Practice
Standards,
40
CFR,
Part
160,
Final
Rule.
EPA,
Washington,
D.
C.

Page
8
of
10
Draft
Protocol
PROPOSED
DATES
Experimental
phase
Date
Start:
Finish:

Report
Draft:

Page
9
of
10
Draft
Protocol
Page
10
of
10
STUDY
PROTOCOL
APPROVAL
Study
Director:
Date
Analytical
Chemist:
Date
Research
Manager:
Date
Sponsor:
Date
