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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:
09-05-2016 to 30-06-2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate
EC Number:
Cas Number:
Molecular formula:
2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate
Test material form:
other: liquid
Details on test material:
2-({[3-(isocyanatomethyl)-3, 5, 5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate of Evonik Degussa GmbH Batch SB 4195 of 28 March 2016

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany. The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
189.6 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The study was performed in a manometric Test System with test flasks containing a volume of approximately 500 mL. The test flasks were incubated at 22°C * 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used. The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
In the toxicity control containing both the test item and the reference item sodium benzoate, 37% and 34% biodegradation was noted within 14 days and 41% and 37% biodegradation after 28 days of incubation based on ThODNH4 and ThODNO3, respectively. According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
pH-Value of Test Solutions: 7.6 (measured at the start of the test), 7.5 to 7.8 (measured at the end of the test)

BOD5 / COD results

Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 89% after 14 days and to 90% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.
The mean biodegradation after 28 days was 0% (THODNH4, ThODNO3). Therefore, the test item is considered to be not readily biodegradable.
Executive summary:

The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 17.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.


The pH-value of the test item flasks at the end of the test was 7.7 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline (Table4).

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded by more than 60% after 4 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 0% (ThODNH4, ThODNO3). The validity criterion was fulfilled.

Toxicity Control:

If in a toxicity test, containing both the test substance and a reference compound less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 34% (ThODNO3) at day 14; the test item was not inhibitory.