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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:
16.05.2016 - 17.06.2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
The inoculum contained a mixed population of microorganisms obtained from the secondary effluent of the waste water treatment plant of Pardubice processing predominantly municipal sewage.

The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.

Justification of the test system
The inoculum preparation is in conformity with the recommendations of the test guideline.
Duration of test (contact time):
28 d
Initial conc.:
3.4 mg/L
Based on:
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Test temperature: 20+/-1 °C
- pH of medium: 7.4
- pH adjusted: no
- Illummination: in the dark

- Inoculated medium: The volume of waste water for the inoculation of mineral medium was chosen 1.6 mL per 1 L of medium (COD of waste water: 108 mg/L). 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 48 mL of modified waste water.
- Test substance: The stock solution of the test substance was prepared in concentration 0.1012 g/L
of deionized water.
From this solution 240.0 mL (40.0 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 6 L.
The solution prepared in this way contained 4.05 mg/L of the test substance, and had COD
3.40 mg/L.

- 2 x 9 bottles with the test substance and inoculated medium
- 2 x 9 bottles with the reference substance and inoculated medium for the check of inoculum activity
- 9 bottles with the test substance and inoculated medium for the determination of oxidized nitrogen forms
- 2 x 5 bottles with the test and the reference substance mixture and inoculated medium for the toxicity test
- 2 x 9 bottles with inoculated medium only for the blank determination

- Inoculum blank: yes, the inoculated mineral medium
- Toxicity control: yes, the test and reference substance mixture

Measurement: At the zero-time and at the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of test the
appropriate bottles from each series were taken off and they were analysed for dissolved oxygen by the Winkler method.
Reference substance:
benzoic acid, sodium salt
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Key result
0.148 other: mg/mg
Key result
0.84 other: mg/mg
1.647 other: mg/mg
Remarks on result:
other: with total nitrification
1.098 other: mg/mg
Remarks on result:
other: without total nitrification
Results with reference substance:
COD of reference substance 1.660 and 80.3 %
Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
In this 28-day study of ready biological degradability the degradation of 7.1 % of the test substance, Reactive Red 45:1, was attained in the end of study.
Executive summary:

The test substance, Reactive Red 45:1, was tested forthe ready biological degradability in Closed Bottle Test.


Test performance

The test was performed according to:

Method C.4E - Closed Bottle Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.


The results of biological degradation are related to experimentally determined COD values
of the test and reference substance at the beginning of the test.

The test substance was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out.

Sodium benzoate was used as the reference substance. The dosage was carried out from the stock solution.

COD of the test substance in medium at the beginning of the main test:             3.40 mg·L-1

COD of the reference substance in medium at the beginning of the main test:     3.32 mg·L-1

In parallel to the main test the toxicity test was performed.

Based on the test substance contains nitrogen in the chemical composition, the oxidized nitrogen forms were determined and the correction for nitrification was carried out.

The test was performed at temperature of 20 ± 1 °C with the pH values of solutions 7 - 8
at the beginning of the test.


Validity of the test

The prescribed validity criteria in the test were fulfilled.

The test substance was not inhibiting for the used inoculum.

Since all criteria of acceptability were met, this study is considered to be valid.


Test results

In this 28-day study of ready biological degradabilitythe degradation of 7.1 % of the test substance, Reactive Red 45:1, was attained in the end of study.

Description of key information

Reactive Red 45:1 is considered to be not readily biodegradable under the test condition.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:

Additional information

In the 28-day study of ready biodegradability (Closed bottle test), the degradation of the test substance, Reactive Red 45:1, was 7.1% at the end of the study.