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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:
18 February to 18 March 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
July 1992
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
Official Journal of the European Communities L383A, 211 - 215 (1992)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic wastewater. The activated sludge was transported in a plastic bottle and aerated until use.
Relevant data are:
Date of activated sludge collection: 11 February 2003
Date of use: 17 February 2003
Settling time of the sludge: 50 min
Used supernatant for inoculation: 5ml/l
Total volume inoculated mineral medium: 50l
Duration of test (contact time):
28 d
Initial conc.:
4.27 mg/L
Based on:
test mat.
Initial conc.:
8.28 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Theoretical Oxygen Demand (ThOD) calculation: Based on the empirical formula, the Theoretical Oxygen Demand (ThODNID) value of T-1063FM was calculated to be 0.61 mg O2/mg.

Test substance concentrations: The determination was performed with two concentrations of T-1063FM: 4.27 and 8.28 mg/l corresponding to a ThOD of 2.6 and 5.1 mg O2/l respectively.
Preparation of the biodegradation test bottles: Two stock solutions were made by dissolving 0.2134 g and 0.4139 g of test substance T-1063FM in 100 millilitres of ultrapure water resulting in two solutions respectively named Stock I and Stock II. Suitable volumes of this stock solutions were added to inoculated medium to obtain test substance concentrations of 4.27 (Stock I) and 8.28 (Stock II) mg/l. These series were completed with an inoculum blank, containing only inoculated mineral medium.
Preparation of the activity control: The final test concentration of sodium acetate was prepared by dissolving 0.3999 g of the reference substance in 100 ml of ultrapure water. From this stock solution a dilution was made in inoculated mineral medium to give a final nominal test concentration of 4.00 mg/l sodium acetate.
Preparation of the toxicity control: Dilutions of the test substance stock II solution and the reference substance stock solution (described above) were prepared in inoculated mineral medium. The toxicity control (contained 8.28 mg/l T-1063FM and 4.00 mg/l sodium acetate.

Test substance
Concentrations: 0 (Inoculum blank), 4.27 and 8.28 mg/l
Number of replicate bottles per sample point: 4
Oxygen concentration determined after 0, 7, 14, 21, 28 days.

Inoculum activity control
Concentration of the reference substance: 4.00 mg/l
Number of replicate bottles per sample point: 4
Oxygen concentration determined after 0, 7 and 14 days.

Toxicity control
Concentration of reference substance: 4.00mg/l
Concentration of test substance: 8.28 mg/l
Number of replicate bottles per sample point: 4
Oxygen concentration determined after 0, 7 and 14 days.

The filled BOD bottles were closed and incubated at about 20 °C in the dark after measurement of the oxygen concentration in one bottle of each series. The oxygen concentrations were measured using an oxygen electrode. Thereafter a separate set of four bottles was sacrificed for oxygen measurements at each time-point. The temperature in one bottle of each treatment was measured at each time-point indicated above. The pH in one bottle of each treatment was measured at the start of the test and after 28 days of incubation, except for the control bottles with sodium acetate and the toxicity controls with sodium acetate and test substance that were measured after 14 days of incubation.

Calculations were performed as given in the test Guidelines. The oxygen depletion (in mg O2/l) in each test bottle after 7, 14, 21 and 28 days was calculated by subtracting each measured oxygen concentration from the mean oxygen concentration measured at the start of the test. The oxygen depletion due to the test or control substance at each time was calculated by subtracting the mean oxygen consumption in the blanks from that in the bottle under consideration.
These crude values were then converted to values per mg substance (biological oxygen demand, BOD). The percentage biodegradation of the test substance was calculated as BOD/COD x 100.
Reference substance:
acetic acid, sodium salt
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: conc. 4.27 mg/l
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: conc. 8.28 mg/l
Details on results:
The pH of the medium remained fairly constant during the test (6.5-7.0). The temperature measured in one of the bottles of each treatment (including the activity control and the blank control test) varied between 19.4 and 20.2 °C during the test.

The measured data of the determinations of the oxygen concentrations in the various bottles of the control test on the inoculum activity and toxicity, as well as the biodegradation test are given in Annex B. The calculated results are given in Annex C (see attachment “T-1063FM - measured data and results biodegredation test.pdf).

The mean oxygen depletion values of the inoculum blanks, the inoculum activity control (4.00 mg/l sodium acetate) and the toxicity control (4.00 mg/l sodium acetate and 8.28 mg/l T- 1063FM) are given in Table 1 (attachment “T-1063FM - ThOD and BOD biodegradation test.pfd”).
The oxygen depletion in the inoculum blank was 1.99 mg O2/l after 28 days of incubation. This is slightly higher than the limit given in the guideline of 1.5 mg O2/l. However, this is not considered to have influenced the degradation of the test substance.
The oxygen depletion in the toxicity control (sodium acetate and test substance) was 4.23 mg O2/l after 14 days, which was higher than that of the inoculum activity control with sodium acetate (4.04 mg O2/l). This indicates that the test substance did not inhibit the biodegradation of sodium acetate. After 14 days of incubation the degradation found in the toxicity control was 33 %, indicating that T-1063FM was not toxic to the inoculum according to the definition given in the Guideline.

Tables 2 and 3 (attachment “T-1063FM - ThOD and BOD biodegradation test.pfd”) show the relevant mean Biochemical Oxygen Demand (BOD) values and the percentage biodegradation of the T-1063FM concentrations of 4.27 and 8.28 mg/l, calculated from the ThOD (0.61 mg 0 of the test substance.
For the concentrations of 4.28 mg/l of T-1063FM a maximum biodegradability of 15 % was reached after 7 days of incubation. For the concentrations of 8.28 mg/l of T-1063FM a maximum biodegradability of 12 % was reached after 28 days of incubation. This means that T-1063FM is not ready biodegradable.
Results with reference substance:
The results show the expected degradation of sodium acetate (> 60 % within 14 days). The calculated Biochemical Oxygen Demand (BOD) of sodium acetate after 7 and 14 days was 0.55 and 0.60 mg O2/mg respectively, which corresponds with its Theoretical Oxygen Demand (ThOD) of 0.68 mg O2/mg
Validity criteria fulfilled:
see overall remarks
Interpretation of results:
under test conditions no biodegradation observed
T-1063FM did not pass the criteria given in the guideline (i.e. > 60% degradation reached within a 10 day period counting from the day that the level of degradation exceeded 10%), and is therefore assessed to be not readily biodegradable in this test.

Description of key information

no biodegredation after 28 days - O2 consumption (EU Method C4-E and OECD guideline 301D)

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:

Additional information