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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:
April- May 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage.
Source: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 30. Mar 2012, batch no: 20120330.
Pre-Treatment: The sludge was filtrated, washed with tap water twice, then washed with and resuspended in test medium. It was then aerated for  12 hours. The dry matter was determined with 3740 mg suspended solids/litre.
Duration of test (contact time):
28 d
Initial conc.:
27 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
for CO2 evolution
Details on study design:
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its TOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

Flask volume 1500 mL
Inoculum concentration: 24.9 mg/L
Temperature 20.0 – 21.6 °C
Duration 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L

From each front scrubber flask, ten samples were taken in order to determine the emitted CO2. (on days 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
On day 28, 5 mL HCl 2-m. were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken
Reference substance:
% degradation (inorg. C analysis)
Sampling time:
28 d
Remarks on result:
other: 10 day-windows not fulfilled
Details on results:
As degradation in the toxicity flask was 68% after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 27.3 mg/L".
Biodegradation of the test item could be observed. One test replicate reached 59.8% degradation at the end of the test.
However, ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test item can not be considered as “readily biodegradable”. In the toxicity control, in presence of aniline the degradation of the test item was faster than in the test replicates.
Results with reference substance:
Degradation of positive control 76% at 14 days

Validity criteria:






IC content of test item solution in medium

<= 5% of TC

< 1 %


CO2emitted by the controls

< 70 mg/L



Difference within replicates

<= 20%

17 %


Degradation of positive control > 60%

< 14 days

9 days


Degradation in the toxicity flask on day 14

> 25%

68 %


Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
under test conditions, biodegradation could be observed but not up to 60% within 28 days
Under test conditions, biodegradation could be oberved reaching 51 % at the end of the test. However, as the level of 60% within 28 days was not reached, then the substance cannot be strictly considered as readily biodegradable.
Executive summary:

The test item was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 27.3 mg test item /L) in test medium following OECD 301B and EU-Method C.4-C.

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 24.9 mg dry matter/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 61 % after nine days.

The following data were determined for the test item :

- 10-day-window: day 12 – 22
- degradation at the end of 10-day-window:
 36 %
- degradation at the end of the test:
51 %
- pass level following guideline:
 60% at the end of 10-day-window

Therefore, regardless of the 10-day-window, the substance is not readily biodegradable following OECD 301B/EU C.4-C.

Description of key information

The ultimate aerobic biodegradability of the phosphorous acid, triC12-C14 alkylesters was investigated in a GLP-compliant ready biodegradability screening study performed in accordance with OECD Guideline No. 301B. The test item achieved 36% of degradation at the end of 10-day-window and 51 % at the end of the test The phosphorous acid, triC12-C14 alkylesters is thus considered not 'readily biodegradable'.

Key value for chemical safety assessment

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

Additional information

The ultimate aerobic biodegradability of the phosphorous acid, triC12-C14 alkylesters was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint