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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1989-02-03 to 1989-03-17
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD 301 B.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Details on test material:
The substance investigated was AF-366, a white powder which was received via
CIVO-TNO Zeist. A 500 ml polyethylene bottie with a red screw cap, labelled:
‘AF-366’, was received from TNO-CIVO on 1988-09-27. The solubility in water
appeared to be poor.
The sample was stored at room temperature. The COD of the sample was found
to be 2.01 mg

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
A sample of activated sludge was taken from an oxidation ditch situated on
the premises of TNO, Delft, The Netherlands. The oxidation ditch is used to
treat domestic sewage.
The original sludge (about 4 g of solid substance.l-1 ) was allowed to settie
for 5 minutes and 10 ml of the supernatant was used to inoculate one litre
of medium (2), yielding a final inoculum concentration of about 0.04 g.l-1.
The inoculated medium was aerated with C02-free air for 24 hours before use.
Duration of test (contact time):
ca. 42 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 10 mg/L
Based on:
Initial conc.:
ca. 20 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The biodegradation test was conducted essentially as detailed in OECD 301 B
(1). Two-litre glass bottles (Schott Duran) closed with a plastic screw cap
were used for the test. A 20 ml glass vial containing the C02-absorbing
fluid was suspended from the screw cap in each bottle.
The bottles were filled with one litre of inoculated medium, and 5 ml of
0.40 M NaOH was placed in the vial.
Two concentrations of test substance (10 and 20 mg.l-1 respectively) were
prepared in triplicate by adding the correct amount of dry test substance to
each bottle containing inoculated medium. A control series without test sub
stance was included to allow correction of the background C02 production by
the inoculum.
In order to determine the inoculuin activity, 50 mg.l-1 of sodium acetate as
a carbon source was added to two additional bottles containing inoculated
medium only.
In order to detect possible toxic effects of the test substance, sodiuin ace
tate (50 mg.l-1) was also acided to further duplicate bottles containing 10
or 20 mg.l-1 of AF-366.
The bottles were incubated on a Braun Pilot Shake orbital shaking machine in
a room kept at 20°C for 42 days. The C02-absorption vials were replaced by
fresh ones after 7, 14, 21, 28 and 42 days. The amount of C02 absorbed was
determined according to OECD 301 B (1) by titration with 0.1 M HCl. The pH
value of the medium was also determined after 7, 14, 21, 28 and 42 days.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradationopen allclose all
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: for 10 mg/L
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: for 20 mg/L
% degradation (CO2 evolution)
Sampling time:
42 d
Remarks on result:
other: for 10 mg/L
% degradation (CO2 evolution)
Sampling time:
42 d
Remarks on result:
other: for 20 mg/L
Details on results:
The biodegradation of acetate in the control series in the absence of test
substance was used as a measure for the microbial activity of the inoculum.
A reduction of the biodegradation of acetate in the presence of the test
substance, would indicate a toxic effect on the inoculum activity.
The results of these tests show that, as expected, acetate was significantly degraded (78%) within
two weeks. No toxic effects of the added AF-366 on the inoculum activity
could be found. In fact, increased C02-production indicated that the test
substance was biodegraded in the presence of acetate. After 28 days, 4 and
12 mg C02 were additionally produced with respectively 10 and 20 mg of AF-
366 per litre added. This is the same C02-production as found in the bio
degradability test proper.
The pH of the medium decreased somewhat during the test (from pH 7.7 (7th
day) to pH 7.4 (42nd day)).
Although some C02-production was found, the biodegradation seemed to be
restricted by the insolubility of the product in water. The chemical struc
ture had not been specified by the sponsor, thus the biodegradability (based
on a theoretical C02-production) could not be calculated. However, an indi
cation of the biodegradability could be derived from the COD-value of the
test substance. In a complete oxydation of a test substance to C02 (other
possible components (H, N, 0 or S) are not considered), 1.38 mg C02 is pro
duced per mg 02 utilized. The COD of 2.01 mg therefore indicates in
that case a maximum theoretical C02-production of 2.8 mg C02 per mg of AF-

Applicant's summary and conclusion

Validity criteria fulfilled:
valid study
Interpretation of results:
inherently biodegradable
The biodegradability of a product coded AF-366 was determined by the method
laid down in the OECO Guideline 301 B; Ready Biodegradability: Modified
Sturm Test (1).
A control test showed that the activity of the inoculum was sufficient and
that no toxic effect of the test substance on its activity could be found.
The test substance was even degraded in the presence of acetate (the amounts
of C02 produced being comparable with those in the biodegradation test
In the biodegradability test itself the biodegradation rate was strongly
dependent on the concentration of AF-366 added, being higher at 20 than at
10 mg AF-366 added per litre. The maximum C02 production was 12 mg.l-1 after
28 days and 19 mg.l-1 after 42 days.
Because the sponsor did not provide any data on the chemical composition of
AF-366, the biodegradability could not be calculated. However, based on the
COD-value of the test substance, indications for a minimum biodegradability
of 21% after 28 days and 34% after 42 days could be derived.
It was therefore conciuded that, although AF-366 is not readily biodegradable,
it could be characterized as inherently biodegradable.