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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:
2016-02-08 to 2018-05-16
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
adopted March 2006/corrected September 2014
Principles of method if other than guideline:
The study has been performed with and without silicone oil as inert carriert in parallel to investigate the bioavailability of the test substance under different conditions.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TOC: 73.5% calculated at test laboratory.
Oxygen conditions:
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
-Test system: inoculum of the aqueous phase of non-adapted activated sludge.
Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal STP in Hildesheim, Germany. The activate sludge is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. Then the settled sludge was resuspended in mineral salts medium, maintainance in aerobic conditions by aeration for 3 days. After homogenization with a blender and sedimentation, the supernatant was decanted and was maintained in an aerobic condition by aeration with C02-free air for 3 days until test start.
- Concentration of sludge: 15 ml/L were used to initiate inoculation
- Initial cell/biomass concentration: CFU 10^5 to 10^8 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
14.2 mg/L
Based on:
test mat.
Initial conc.:
1.14 other: mg test substance/ test vessel (80 mL)
Based on:
test mat.
Test concentration/ test vessel
Initial conc.:
10.4 mg/L
Based on:
Carbon content in the test vessel
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral salts medium according to test guideline
- Solubilising agent (type and concentration if used): The study was run with and without silicone oil in parallel to investigate the bioavailability of the tst item in both conditions.
- Test temperature: 20 +/- 1°C, acutal 19-21 °C, occasionally between 18°C and 21.6 °C for short periods.
- Continuous darkness: ylow light conditions
- Other:

- Culturing apparatus: Headspace flasks (120 mL volume, 80 mL test medium, Headspace 1:2)
- Number of culture flasks/concentration: 3 replicates with and without silicone oil, 5 replicates at test end.
- Method used to create aerobic conditions: aeration of sludge before test start.
- Preparation of test substance replicates: 1.2 µl of the test substance was pipetted at the wall of the test vessel. After addition of 80 mL test medium the test vessel was closed immediately. For preparation of test substance replicates with silicone oil, a stock solution of 14.2 g/L test substance in silicone oil was prepared and 80 µL of the stock solution was tranferred to the test vessel before adding the test medium.
- Measuring equipment: IC analyser
- Test performed in closed vessels due to significant volatility of test substance: Headspace flask
- Details of trap for CO2 and volatile organics if used: Sodium hydroxide was injected and the test vessel were shaken for 1 h before analysis of inorganic carbon concentration.
- Other: Application once at test start.

- Sampling frequency: Analysis at test start and 7 further sampling times (see sampling shedule in details on analytical method)
- Sampling method: addition of NaOH
- Other: TIC of NaOH (blind value) was determined at each sampling time.

- Inoculum blank: mineral medium (80 mL)with inoculum without test substance or reference substance. 80 µL silicone oil was added for the silicone oil inoculum control.
-Functional control: mineral medium (80 mL) with inoculum and reference substance. 80 µL silicone oil was added for the reference item replicates whti silicone oil.
- Toxicity control: mineral medium (80 mL) with inoculum and test substance and reference substance. For toxicity control with silicone oil a stock solution of 14.2 g/L test substance in silicone oil was prepared and 80 µL of the stock solution was transfer into the test vessel before adding the reference substance medium.

STATISTICAL METHODS: Calculation of the ratio theoretical TIC concentration at test start (ThIC=TOC) to net TIC production. Calculation of 95% confidence interval with Sigma Plot.
Reference substance:
benzoic acid, sodium salt
Test concentration 30 mg/L (corresponds to 17.5 mg C/L in the test vessel).
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
95% confidence interval 78-95%
% degradation (CO2 evolution)
Sampling time:
7 d
% degradation (CO2 evolution)
Sampling time:
11 d
% degradation (CO2 evolution)
Sampling time:
14 d
% degradation (CO2 evolution)
Sampling time:
21 d
Details on results:
The toxicity control showed 77% after 14 days and 84% (CI 77-91%) on day 28. In the toxicity control with silicone oil a biodegration rate of 63% after 14 days and 72% after 28 days (CI 69-75%) was observed. Therefore, the biodegradation of the reference substance was not inhibited by the test substance.

The test item replicates without silicone oil reached the 10% level (beginning of biodegradation) within 7 days and the 60% pass level on day 14. The biodegradation reached 86% after 28 days. The 95 % confidence interval on day 28 was 78 - 95%. The test item replicates with silicone oil reached the 10% level (beginning of biodegradation) within 11 days, but did not reach the 60% pass level within the 28-day-period of the study. The biodegradation reached 45% after 28 days (CI 39-51%). Contrary to the expectation, the use of silicone oil did not improve the bioavailability of the test substance, but slowed the biodegradation rate down. The test item is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.
Results with reference substance:
The functional controls containing the reference substance reached the pass level of 60% within 7 days. The functional control without silicone reached a maximum of 91% (CI 88-90% on day 28) on day 14, the functional control with silicone oil reached 89% on day 28 (CI 88-91%).

Biodegradation and confidence interval of the test substance in comparison to the functional control and the toxicity control without silicone oil


Biodegradation (%) on Day 28

Confidence Interval on Day 28







Functional control







Test substance







Toxicity control

(Test substance + reference substance)







Validity criteria fulfilled:
Mean amount of TIC in inoculum controls was 1.48 mg C/L to 1.77 mg C/L (criterion <3 mg C/L). Degradation of functional controls reached 60% within 7 days (criterion >60% after 14 days).
Interpretation of results:
readily biodegradable
The test substance is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.

Description of key information

The ready biodegradability tests according to test guideline OECD 310 and OECD 301 demonstrates that the test substance is readily biodegradable and fulfills the 10 -day window.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information

The biodegradation of the test substance has been examined in two ready biodegradability tests. In the key study according to test guideline OECD310, the biodegradation rate of the test substance reached a biodegradation of 86% after 28 days. The 10 % level was reached within 7 days and the pass level of 60% was reached on day 14. The use of silicone oil to investigate the bioavailability of the test substance, slowed the biodegradation rate down contrary to the expectation. However, based on the replicates without silicone oil, the test substance can be considered as readily biodegradable within the 10 -day-window.

The result of the key study is supported by another study on ready biodegradation according to OECD301. In this study a biodegradation rate of 77% after 28 days has been observed. However, the 10 -day-window has been marginally missed. The 10% level has been reached between day 7 and 8, the pass level of 60% biodegradation has been reached on day 18. Due to the nature of the test substance as an UVCB substance, the 10 day window is not applicable and the test substance has been considered as readily biodegradable.