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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:
2011-04-05 to 2011-05-03
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD TG 301F.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. certificate)

Test material


Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, D-31137 Hildesheim

- Pretreatment:
The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with C02-free air for 4 days. 10 mL/L were used to initiate inoculation.

- Concentration of sludge: 10 mL/L
- Initial cell/biomass concentration: 10^7 - 10^8 CFU/L
- Water: demineralized
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
70 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Preparation of Test Flasks:
All test solutions were prepared in measuring flasks in demineralised water with the required amounts of mineral medium stock solutions and 10 mL/L inoculum. The test item was weighed out and demineralised water was added. After ultrasonic treatment it was transferred into the measuring flasks.

The reference item was weighed out and transferred into the measuring flasks with demineralised water.

250 mL measuring flasks were used to place 250 mL of the test solutions in the brown glass bottles.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with Oxi-Top® measuring heads and the measuring system was activated.

The temperature in the incubator was documented continuously throughout the test by a hygrothermograph.
At test start the pH-values of the test solutions in the measuring flasks (before dividing) and at test end the pH-values of the test solutions in the brown glass bottles were determined.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Results of the Functional Control
The pass level of a biodegradation > 60 % was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled

Results of the Toxicity Control
In the toxicity control the biodegradation achieved 49 % after 14 days. After 28 days the biodegradation came to 50 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
% Degradation
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
10 % level (begin of biodegradation): not reached for replicate 1, day 9 for replicate 2
60 % pass level was not reached within 28 days
6 % mean biodegradation after 28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
other: not readily biodegradable
The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.
Executive summary:


The purpose of this assay was to provide information on the biodegradability of the test item in aqueous environment and thus serve as a rational basis for risk assessment for environmental fate in aqueous compartments.

Study Design

For this purpose, the test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganismsduring exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. This study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with activated sludge.


The test item replicate 1 did not reach the 10 % level (beginning of biodegradation), test item replicate 2 reached the 10 % level after 9 days. The 60 % pass level was not reached within 28 days. After 28 days the mean biodegradation was 6 %.

Biodegradation of sodium benzoate
The reference item sodium benzoate was sufficiently degraded to 93 % after 24 days.

Biodegradation in the Toxicity Control
In the toxicity control containing both, the test item and the reference item sodium benzoate, 49 % biodegradation was noted within 14 days and 50 % biodegradation was determined after 28 days of incubation.

The validity criteria of the guideline are fulfilled.


The test item is classified as not readily biodegradable in the 10-d-window and after 28 days.