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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data available
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Details on test material:
no data available
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data available

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Fresh sludge samples were collected from ten sites in Japan, such as municipal sewage-treatment plants, rivers, lakes and seas.
The filtered supernatant of an activated sludge (cultivated sludge for 3 months) were mixed with an equal volume of the filtered supernatant of freshly collected ten-source mixture when used.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Thirty mg of the test substance or aniline (reference substance) and 9 mg as MLSS (mixed liquor suspended solid) of activated sludge were added to 300 ml of test medium. A concentration of inoculum was 30 mg/l as MLSS. A concentration of the test substance was 100 mg/l. A volume of mixture was 300 ml. The test and reference solutions were cultivated in BOD meter together with the inoculum blank and abiotic control ones at 25 °C for 28 days, during which the oxygen consumption was continuously measured.
Reference substance
Reference substance:

Results and discussion

Preliminary study:
no data available
% Degradation
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
Results of test substance and specific chemical analysis by HPLC at end of test (after 28 days) are as follows:

Residual amount Breakdown products
of test substance
o-Chlorobenzyl chloride o-Chlorobenzylalcohol o-Chlorobenzaldehyde o-Chlorobenzoic acid

Water + test substance solution
[mg] 0 26.3 0 0
[%] 0 99 0 0
Sludge + test substance solution (Values are expressed as mean of three times)
[mg] 0 24.5 0.7 0.9
[%] 0 92 2 3
Theoretical Value
[mg] 30.0 26.6 26.2 29.2

Results of carbon analysis by TOC and of BOD at end of test (after 28 days) are as follows:

Water + test substance solutions Sludge + test substance solutions
Residual amount of DOC [mg] 15.8 16
(Theoretical Value: 15.7 mg C)
Residual amount of DOC [%] 100 102
BOD [mg] 0 0

o-Chlorobenzyl chloride (OCBC) was not detected at both water plus test substance and sludge plus test substance solutions after 28 days. In water plus test substance solutions, o-chlorobenzyl alcohol was detected by LC-MS after 28 days. Production rate of o-chlorobenzyl alcohol was 99 % from the measurement by HPLC. Therefore, it is considered that OCBC hydrolyzed into o-chlorobenzyl alcohol. In sludge plus test substance solutions, o-chlorobenzyl alcohol, o-chlorobenzaldehyde and o-chlorobenzoic acid were detected respectively after 28 days. Mean production rates of o-chlorobenzyl alcohol, o-chlorobenzaldehyde and o-chlorobenzoic acid were 92, 2 and 3 % respectively. Consequently it is considered that OCBC is hydrolyzed and produces o-chlorobenzyl alcohol, and then the o-chlorobenzyl alcohol is slowly biodegradable to o-chlorobenzaldehyde and o-chlorobenzoic acid. Degradation pathway of o-chlorobenzyl chloride is as follows: o-chlorobenzyl alcohol, o-chlorobenzaldehyde, o-chlorobenzoic acid

BOD5 / COD results

0 g O2/g test mat.
Results with reference substance:
68 % degradation after 7 days
74 % degradation after 14 days

Applicant's summary and conclusion

Interpretation of results:
under test conditions no biodegradation observed
In this test, the determination by the BOD method showed 0% degradation of OCBC through 28 days
Executive summary:

OCBC was assessed in a modified MITI test over a period of 28 days, according the OECD guideline 301 C. Under the test conditions biodegradation of OCBC was not detected after 28 days, based on the oxygen consumption. Therefore, the test item was considered not ready biodegradable.