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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:
03 Mar - 02 Apr 1992
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
May 1981
according to
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
according to
other: EEC Directive 84/44
Version / remarks:
Sep 1984
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Secondary effluent from an activated sludge plant collected from the domestic sewage treatment plant Kläranlage Walsrode, Germany.
- Storage conditions: The effluent was kept under aerobic conditions.
- Storage length: The effluent was collected freshly on Day 0 of the test period.
- Preparation of inoculum for exposure: To prepare the inoculum, the sample was filtered through a paper filter. The first 200 mL were discarded and the rest of the filtrate was used for the test.
- Initial cell concentration in the inoculated test vessels: 2.9E+06 colony forming units/mL

Duration of test (contact time):
28 d
Initial conc.:
1.12 mg/L
Based on:
test mat.
Initial conc.:
0.92 mg/L
Based on:
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: Mineral nutrient solution prepared according to OECD 301 D in autoclaved purified water.
- Test temperature: 18.7/19.1 °C
- Aeration of dilution water: Yes, the autoclaved purified water was aerated for 20 h with filtered air.
- Continuous darkness: Yes
- Other: The mean O2-concentration of the dilution water was 8.5 mg/L.

- Culturing apparatus: 100 mL volumetric flask with stopper
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: Aeration of dilution water
- Measuring equipment: O2-detector OXI 96 (WTW). The stirring rate during O2 measurement was about 1000 rpm.

- Sampling frequency: The oxygen content was measured on Days 0, 7, 14, 21 and 28.

- Inoculum blank: Yes, containing dilution water, mineral nutrient solutions and inoculum.
- Abiotic sterile control: Yes, containing dilution water and mineral nutrient solutions without inoculum.
- Toxicity control: Yes, an inhibition test was performed by comparing the BOD value for a particular time interval in the Itr series (test substance + reference substance) with the respective values of the Ir series (reference substance: fatty alcohol with 10 moles ethylene oxide) and T series (test substance).
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d

The calculated values of the theoretical oxygen demand were 2.86 mg O2/mg test substance and 1.67 mg O2/mg reference substance.

Table 1. Ready Biodegradability [%] of the test substance and reference substance (sodium benzoate).


Test substance

Reference substance

7 d



14 d



21 d



28 d




In the inhibition test, all BOD values for the Itr series (test substance + reference substance) were higher than the sum of the respective values for the Ir series (reference substance: fatty alcohol) and the T series (test substance). In conclusion, the test article exerted no inhibitory effect on the microbial biomass under these test conditions.

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The substance is not readily biodegradable according to OECD guideline criteria since the defined 10% threshold for biodegradation was never reached and neither was the 60% pass level within the 10-d thereafter (3.1% in 28 d, OECD 301 D).

Description of key information

Not readily biodegradable (3.1% after 28 d, OECD 301 D)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

There is one GLP study available, in which the ready biodegradability of the test substance was assessed according to the OECD guideline 301 D.

In a closed bottle test microorganisms (2.9E+06 colony forming units) from the secondary effluent of a local domestic sewage treatment plant were inoculated with 1.12 mg/L (measured) of the test substance. A toxicity control was run in parallel to assess the potential inhibitory effect of the test substance on the microbial biomass.

Total organic carbon was analyzed to determine the actual concentration of the substance in the initial stock solution. The biodegradation of the test substance was determined by measuring the dissolved O2 concentration in the test bottles over 28 d. The biodegradability of the test substance was then calculated on the basis of the theoretical oxygen demand.

The results of the toxicity test indicate no inhibitory effect of the substance on microorganisms at the tested concentration of 1.12 mg/L. The biodegradability of the reference substance was 71.9%, confirming the suitability of the test system. The biodegradability of the substance was 3.1% after 28 d. Since the recommended pass level was not attained by the end of the 28 d inoculation period, the substance is regarded as not readily biodegradable according to the OECD 301 guideline criteria.