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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:
29.05. – 30.06.2017
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:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): 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. - Preparation of inoculum for exposure: The fresh collected waste water was filtered through paper filter. COD was determined and waste water was aerated till using.The inoculum preparation is in conformity with the recommendations of the test guideline.
Duration of test (contact time):
28 d
Initial conc.:
2.5 mg/L
Based on:
test mat.
(ThOD 1.625 mg·L-1 )
Details on study design:
The volume of waste water for the inoculation of mineral medium was chosen 1.9 mL per 1 L of medium (COD of waste water: 73 mg·L-1). 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 57 mL of modified waste water. The pH value of medium: 7.4.TEST CONDITIONSThe stock solution of the test substance was prepared in concentration 0.1020 g·L-1 of deionized water. From this solution 147 mL (24.5 mL·L-1) 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 2.5 mg·L-1 of the test substance. The pH value of solution: 7.6.- Composition of medium:Preparation of 1 litre of mineral medium: 1 mL of solutions a), b), c) and d) were mixed with approx. 500 mL of water, the mixture was replenished with water up to 1 000 mL. The solution was prepared from aerated water. Solution a) Monopotassium dihydrogen orthophosphate, (KH2PO4) 8.50 gDipotassium monohydrogen orthophosphate, (K2HPO4) 21.75 gDisodium monohydrogen orthophosphate dihydrate, (Na2HPO4·2H2O) 33.40 gAmmonium chloride (NH4Cl) 0.50 g Water up to the volume of 1 000 mLSolution b) 22.50 g Magnesium sulphate heptahydrate, (MgSO4·7H2O) in 1 000 mL of waterSolution c) 27.50 g Calcium chloride, anhydrous, (CaCl2) in 1 000 mL of waterSolution d) 0.25 g Iron (III) chloride hexahydrate, (FeCl3·6H2O) in 1 000 mL of water - Solubilising agent (type and concentration if used): The deionized water with conductivity less than 5 uS·cm-1 was used for the preparation of all solutions. - Test temperature: 20±1.0 °C - pH: 7.4 - pH adjusted: The right composition of medium was checked by the determination of pH value, which must be 7.4. - Aeration of dilution water: yes - Continuous darkness: yes TEST SYSTEMThe bottles were placed into thermostat.2 x 9 bottles with the test substance and inoculated medium2 x 9 bottles with the reference substance and inoculated medium for the check of inoculum activity9 bottles with the test substance and inoculated medium for the determination of oxidized nitrogen forms2 x 5 bottles with the test and the reference substance mixture and inoculated medium for the toxicity test2 x 9 bottles with inoculated medium only for the blank determination - Method used to create aerobic conditions: the solution was prepared from aerated water - Measuring equipment: oximeter WTW OXI 730 - Test performed in open system: no SAMPLING - Sampling frequency: at the zero-time and at the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test - Sampling method: the appropriate two bottles from each series were taken off CONTROL AND BLANK SYSTEM - Inoculum blank: yes, the inoculated mineral medium - Toxicity control: yes, with the mixture contained 2.5 mg·L-1 of the test substance and 2.10 mg·L-1 of sodium benzoate.
Reference substance:
benzoic acid, sodium salt
Test performance:
Degradation is determined by analysis of dissolved oxygen over a 28-day period.The following determinations were done simultaneously under the same conditions:-check of inoculum activity with the reference substance-toxicity test of the test substance for used inoculum in mixture of the test and reference substance-determination of oxygen consumption for nitrification-determination of blank (inoculated mineral medium)The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
0.701 other: mg·mg-1
Remarks on result:
(without nitrification)
Key result
1.625 other: mg·mg-1
Remarks on result:
(with total nitrification)
0.294 other: mg·mg-1
0.033 other: mg·mg-1
Results with reference substance:
Sodium benzoate ThOD 1.665 mg·mg-1COD 1.643 mg·mg-1
Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Reactive Yellow 85, was attained in the end of study.
Executive summary:

The test substance, Reactive Yellow 85, was tested for the 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 determine ThOD values of the test and reference substance.

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.

Specific ThOD of the test substance in medium:                       1.625 mg·mg-1

Specific ThOD of the reference substance in medium:             1.665 mg·mg-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 degradability the degradation of 0% of the test substance, Reactive Yellow 85, was attained in the end of study.

Description of key information

In this 28-day study of ready biological degradability the degradation of 0 % of the test substance, Reactive Yellow 85, was attained in the end of study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information