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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:
20.11. – 19.12. 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)
Version / remarks:
published in O. J. L 142, 2008
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.:
2.51 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The volume of waste water for the inoculation of mineral medium was chosen 1.8 mL per 1 L of medium (COD of waste water: 76 mg·L-1). 30 L aerated mineral medium was prepared for the test and before the beginning of the test it was inoculated with 54 mL of modified waste water. The pH value of medium: 7.4.

The stock solution of the test substance was prepared in concentration 0.1002 g·L-1 of deionized water. From this stock solution 150 mL (25 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 2.51 mg·L-1 of the test item. The pH value of solution: 7.5.
- 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 g
Dipotassium monohydrogen orthophosphate, (K2HPO4) 21.75 g
Disodium monohydrogen orthophosphate dodecahydrate, (Na2HPO4·12H2O) 67.21 g
Ammonium chloride (NH4Cl) 0.50 g
Water up to the volume of 1 000 mL
The right composition of medium was checked by the determination of pH value, which must be 7.4.

Solution b) 22.50 g Magnesium sulphate heptahydrate, (MgSO4·7H2O) in 1 000 mL of water
Solution c) 27.50 g Calcium chloride, anhydrous, (CaCl2) in 1 000 mL of water
Solution 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: of medium 7.4
- Aeration of dilution water: yes
- Continuous darkness: yes

Test substance series: 2 x 9 bottles with test item and inoculum
Reference substance series: 2 x 9 bottles with reference item and inoculum
Blank series: 2 x 9 bottles with inoculated medium only
Toxicity test series: 2 x 5 bottles with test item, reference item and inoculum for toxicity test
Series for nitrification determination: 1 x 9 bottles with test item and inoculum
The prepared dosing solutions for each series were filled in two parallel bottle replicates (except series for nitrification determination).
The bottles were placed into thermostat.

- 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 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 bottles from each series were taken off

- Inoculum blank: yes, the inoculated mineral medium
- Toxicity control: yes, with the mixture contained 2.51 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 COD.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Key result
0.349 other: mg·mg-1
0.124 other: mg·mg-1
Results with reference substance:
Sodium benzoate COD 1.638 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 item, 5-aminotetrazole, was attained in the end of study.
Executive summary:

The test item, 5-aminotetrazole, 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 test item was sufficiently soluble in used mineral medium so the dosage from the stock solution was carried out.

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

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

Because the test item 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.

The calculations of biological degradation are based on ThOD values of the test and reference item.


Validity of the test

The prescribed validity criteria in the test were fulfilled.

The test item 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 item, 5-aminotetrazole, 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 item, 5 -aminotetrazole, was attained in the end of study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information