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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:
11. Jan. - 9. Feb. 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:
yes (incl. QA statement)
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge obtained from sewage treatment plant Abwasserzweckverband Staufener Bucht treating predominantly domestic sewage
- Preparation of inoculum for exposure: washed twice with tap water.
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium according to guideline
- Test temperature: 20.0 – 23.7°C.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no

- Culturing apparatus: 2 L gas-washing bottles with a septum-closed opening sideways
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: aeration with CO2 free air.
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: 2 absorber flasks with 0.2 M NaOH solution
- Measuring equipment: TOC analyser (TOC-L Shimadzu)

- Sampling frequency: sampling of NaOH at days 0, 4, 7, 11, 14, 21, 28 and 29
- Sampling method: 6 mL NaOH solution was collected from the absorber flasks and the inorganic carbon (IC) was determined. Samples were closed using parafilm to avoid CO2 absorption from the air. 2 mL of a 4 M HCl solution were added to the test vessels on day 28 to outgas CO2 from the water phase and to stop the degradation process.

- Inoculum blank: 3
- Other: reference substance: 3
Reference substance:
benzoic acid, sodium salt
20 mg TOC/L
% degradation (CO2 evolution)
Sampling time:
29 d
Remarks on result:
other: mean of three replicates
Results with reference substance:
Reference substance: 91.7% degradation after 28 days

Table 1: Mean degradation rates

 Day   [% ThCO2]
 0  0
 4 48.67
 7 69.77
 11 77.23
 14 80.87
 21 82.43
28 83.4
 29 82.2

Table 2: Validity criteria for OECD 301.

Criterion from the guideline


Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.



Percentage degradation of the reference compound has reached the pass levels by day 14.



The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

 not performed


The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.



The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

<40 mg/L



Validity criteria fulfilled:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
readily biodegradable

Description of key information

Readily Biodegradable: 82.2% in 28 d (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information

The ready biodegradability of Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) was tested in a guideline study following OECD 301B (GLP). Activated sludge (domestic, non-adapted) was used as inoculum. The degradation of the substance was followed by measuring the CO2 evolution of inoculum. After an inoculation period of 28 days the substance was degraded by 82.2% and the ten-day window criterion was met. Thus, the substance is readily biodegradable according to OECD criteria.