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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:
22 May to 05 July 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
GLP compliance:
Specific details on test material used for the study:
Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%
Solubility in water: Very low
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant ('Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands)
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment
- Pretreatment: Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 3.5 g/L in the concentrated sludge as used for the test.
- Concentration of sludge: The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 10.5 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
12 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: Tap-water purified by reverse osmosis (Milli- RO), mineral components, and inoculum
- Test temperature: 22 and 23°C
- pH: 7.6 to 7.7
- pH adjusted: Only for the toxicity control at the start of the test (adjusted using 1M HCl)
- Aeration of dilution water: Yes
- Continuous darkness: Yes

- Number of culture flasks/concentration: 2 bottles/test suspension; 2 bottles/inoculum blank control; 1 bottle/procedural control; 1 bottle/toxicity control
- Method used to create aerobic conditions: Synthetic air (CO2 < 1 ppm)
- Measuring equipment: CO2-absorbers

- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the procedure and toxicity control were made over a period of at least 14 days.
Reference substance:
acetic acid, sodium salt
CASRN 127-09-3
% degradation (CO2 evolution)
Sampling time:
28 d
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of the test item (3% and 2%, based on ThCO2).

The Theoretical CO2 production (ThCO2) of the test item was calculated to be 3.16 mg CO2/mg.

Acceptability of the Test:

1.           The reference item was biodegraded by at least 60% (actual result: 81%) within 14 days.

2.           The difference of duplicate values for %-degradation of the test item was always less than 20 (actual result: ≤ 2%).

3.           The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (56.5 mg CO2 per 2 litres of medium, corresponding to 28.3 mg CO2/L).

4.           The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).


Since all criteria for acceptability of the test were met, this study was considered to be valid.

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was not readily biodegradable under the conditions of the modified Sturm test presently performed (3% and 2%, based on ThCO2). In the toxicity control, the test item was found not to inhibit microbial activity.

Description of key information

1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was not readily biodegradable under the conditions of the modified Sturm test presently performed; 3% and 2% in 28 days based on ThCO2. The test item was found not to inhibit microbial activity.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:

Additional information