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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.04. – 06.05. 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
no impact on the study (see Any other information ...)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
EC Number:
EC Name:
Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)
Cas Number:
Molecular formula:
tripotassium 18-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-13λ³,15λ³-dioxa-1λ⁴,2-diaza-14-cuprahexacyclo[³,¹².0⁴,⁹.0¹⁶,²⁵.0¹⁷,²²]pentacosa-1,4(9),5,7,10,16,18,20,22,24-decaene-14,14-bis(ylium)-13,15-diide-10,20,24-trisulfonate
impurity 1
Chemical structure
Reference substance name:
Potassium chloride
EC Number:
EC Name:
Potassium chloride
Cas Number:
Molecular formula:
potassium chloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue 234
- Other name: Ostazin Blue H-5R
- Lot/batch No.: 8002
- Expiration date of the lot/batch: unlisted

Study design

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 test substance concentration
Initial conc.:
4.9 mg/L
Based on:
test mat.
(COD 3.40 mg·L-1 )
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: 84 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 0.1008 g·L-1 of deionized water. From this solution 291.6 mL (48.6 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 4.9 mg·L-1 of the test substance, and had COD 3.40 mg·L-1. 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 dihydrate, (Na2HPO4·2H2O) 33.40 g
Ammonium chloride (NH4Cl) 0.50 g
Water up to the volume of 1 000 mL

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 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

The bottles were placed into thermostat.
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

- Method used to create aerobic conditions: The solution was prepared from aerated water
- Measuring equipment: oximeter WTW OXI 730 with membrane probe CellOx 325
- 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 4.9 mg·L-1 of the test substance and 2.10 mg·L-1 of sodium benzoate (COD 6.84 mg·L-1).
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

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.
% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d

BOD5 / COD results

BOD5 / CODopen allclose all
Key result
0.694 other: mg·mg-1
Key result
0.198 other: mg·mg-1
Results with reference substance:
Sodium benzoate COD 1.636 mg·mg-1

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
In this 28-day study of ready biological degradability the degradation of 6.2 % of the test substance, Reactive Blue 234, was attained in the end of study.
Executive summary:

The test substance, Reactive Blue 234, 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 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:             0.694 mg·L-1

COD of the reference substance in medium at the beginning of the main test:  1.636 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 degradability the degradation of 6.2 % of the test substance, Reactive Blue 234, was attained in the end of study.