Tetrasodium [3-[(3-chloro-2-hydroxy-5-sulphophenyl)azo]-5-[[4-chloro-6-(3-sulphoanilino)-1,3,5-triazin-2-yl]amino]-4-hydroxynaphthalene-2,7-disulphonato(6-)]cuprate(4-)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

published in O.J.L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic (adaptation not specified)

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.

Duration of test (contact time):

28 d

Initial conc.:

3.69 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

INSTRUMENTS AND EQUIPMENT- oxygen vessels with glass stopper, volume of approx. 280 mL - large glass bottles of 5 and 10 L volume for the preparation and inoculation of medium- closed thermostat- pH meter WTW Inolab pH 730- oximetr WTW Oxi 730- analytical balance XS 105 DU (Mettler Toledo)- UV/VIS spectrophotometer Helios Alpha- equipment for the determination of nitrite- volumetric flasks, flasks, beakers, pipettes and further common laboratory equipmentPREPARATION OF SOLUTIONS FOR THE TEST - Inoculated mediumThe volume of waste water for the inoculation of mineral medium was chosen 1.9 mL per 1 L of medium (COD of waste water: 79 mg/L). 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 substance The stock solution of the test substance was prepared in concentration 0.1032 g/L of deionized water.From this solution 214.8 mL (35.8 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 3.69 mg/L of the test substance, and had COD 3.40 mg/L. The pH value of solution: 7.5.- Reference substanceThe stock solution of sodium benzoate was prepared in concentration 1.0000 g/L of deionized water. From this solution the 12.6 mL (2.1 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 2.10 mg/L of sodium benzoate, and had COD 3.45 mg/L. The pH value of solution: 7.5. - Test substance for the nitrification determination The stock solution of the test substance for the nitrification determination was prepared in concentration 0.1032 g/L of deionized water.From this solution 143.2 mL (35.8 mL/L) were transferred to a large glass bottle and completed with the inoculated medium to the volume of 4 L.The solution prepared in this way contained 3.69 mg·L-1 of the test substance, and had COD 3.40 mg/L.- Toxicity testThe solution was prepared by dosing 143.2 mL of stock solution of the test substance and 8.4 mL of stock solution of the reference substance into inoculated medium and completed with this medium to the volume of 4 L. The mixture prepared in this way contained 3.69 mg/L of the test substance and 2.10 mg/L of sodium benzoate and had COD 6.85 mg/L.- Control (blank) determinationThe inoculated mineral medium without test substance was used for the blank determination.TEST CONDITIONSTemperature: 20 ± 1.0°C Illumination: no illumination, in the darkExposition time: 28 daysTEST PROCEDURE- Series of bottles for the test2 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 determinationThe bottles were placed into thermostat.- MeasurementAt the zero-time and at the 3rd, 7th, 10th, 14th, 17th, 21st, 24th and 28th day of the test the appropriate bottles from each series were taken off and they were analysed for dissolved oxygen by the electrochemically using oximeter device (oximeter WTW OXI 730).The determination of nitrites was carried out in parallel bottles in these time intervals by means of spectrometric methods.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

3.7

Sampling time:

28 d

Details on results:

see Table No. 1

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In this 28-day study of ready biological degradability the degradation of 3.7 % of the test substance, Reactive Violet 1, was attained in the end of study.

Executive summary:

The test substance, Reactive Violet 1, was tested for the ready biological degradability in Closed Bottle Test.

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.

COD of the reference substance in medium at the beginning of the main test: 3.45 mg/L.

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.

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.

In this 28-day study of ready biological degradability the degradation of 3.7 % of the test substance, Reactive Violet 1, was attained in the end of study.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed