Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

adopted July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Test organisms
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2017-07-24
- Concentration of inoculum: 30 mg/L suspended solids

Pre-treatment of inoculum
− Before use, the inoculum was stored for three days at room temperature under continuous stirring with aeration.
− The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
− An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
− The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Exposure conditions
- Test volume: 250 mL
- Test apparatus: OxiTopControl System (WTW)
- Mixing: 1 magnetic stirrer per test vessel
- Incubation time: 28 days
- Incubation temperature: 22 ± 1 °C
- Test design: test item (3 replicates), reference compound (2 replicates), toxicity control (1 replicate), inoculum blank (3 relicates)
- pH was measured in the flasks at end of incubation

Pre-treatment of the test item
- 29 mg = 25 mg (active ingredient) of the test item were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg active ingredient of the test item/L

Pre-treatment of the reference compound sodium benzoate
- 25 mg of the reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg reference compound/L

Pre-treatment of the toxicity control
- 25 mg of the active ingredient (= 29 mg of the test item) and 25 mg of the reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium
- volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg active ingredient of the test item and 100 mg of the reference compound/L

Reference substance:

benzoic acid, sodium salt

Remarks:

Acros Organics, purity: 99.9 %, batch no. A0357641

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Details on results:

The oxygen consumed by nitrification was not determined at the end of the study, because poor degradation of the test item was observed and the correction for oxygen uptake by nitrification would not have an influence on the results.
pH values after 28 days: 7.4 - 8.1.

Parameter:

BOD5

Value:

>= 2.9 - <= 4.3 other:

Remarks on result:

other: blank corrected BOD value after 28 days; The oxygen consumed by nitrification was not determined at the end of the study, because the correction for oxygen uptake by nitrification would not have an influence on the results.

Remarks:

mg O2/L

Parameter:

ThOD

Value:

1.185 other: mg O2/mg active ingredient

Remarks on result:

other: Test concentration: 100 mg/L (active ingredient)

Results with reference substance:

The reference compound sodium benzoate showed 91 % degradation after 14 days and 95 % degradation after 28 days (ThOD: 1.665 mg O2/ mg; Test concentration: 100 mg/L).

Experimental Procedure

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate.

The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.

The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start of the test. The oxygen consumed by nitrification was not determined after 28 days, because poor degradation of the test item was observed.

Degradation of toxicity control

Test concentration test item	100 mg/L (active ingredient)
Theoretical oxygen demand	1.185 mg O2/mg
Test concentration reference compound	100 mg/L
Theoretical oxygen demand	1.665 mg O2/mg

time	blank-corrected BOD	% degradation
[d]	[mg O2/L]
28	152.8	54

The used concentration of the test item is not toxic to bacteria.

Validity criteria fulfilled:

yes

Remarks:

biodegradation of reference compound ≥ 60 % within 14 d, degradation of toxicity control > 25 % within 14 d, difference in replicates with test item <= 20 %, oxygen uptake of inoculum blank ≤ 60 mg/L, pH independent

Interpretation of results:

not readily biodegradable

Conclusions:

After 28 d a biodegradation of 3 % was found for Direct Yellow 86. Thus, it is considered to be "not readily beiodegradable". A biodegradation of 91 % was found for the reference compound sodium benzoate after 14 d.

Executive summary:

A study was performed to assess the ready biodegradability of Direct Yellow 86 in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008). This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992).

A suspension of 100 mg/L Direct Yellow 86 (active ingredient) in a mineral medium was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days under aerobic conditions in the dark. During this period, degradation was followed by continuous automated BOD determinations.

Direct Yellow 86 (active ingredient) showed:

1 % degradation after 7 days

2 % degradation after 14 days

2 % degradation after 21 days

3 % degradation after 28 days

Therefore, Bayscript Gelb BR (active ingredient) is considered to be “Not Readily Biodegradable“.

The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. A toxicity control (test item and reference compound mixed, one replicate) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms. Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate.

At the end of incubation, the pH was measured in the flasks.

The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start of the test. The oxygen consumed by nitrification was not determined after 28 days, because poor degradation of the test item was observed.

Direct Yellow 86 (active ingredient) showed 3 % degradation after 28 days. Therefore, Direct Yellow 86 (active ingredient) is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 91 % degradation after 14 days.

Description of key information

A study was performed to assess the ready biodegradability of Direct Yellow 86 in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008). This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992).

A suspension of 100 mg/L Direct Yellow 86 (active ingredient) in a mineral medium was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days under aerobic conditions in the dark. During this period, degradation was followed by continuous automated BOD determinations.

Direct Yellow 86 (active ingredient) showed:

1 % degradation after 7 days

2 % degradation after 14 days

2 % degradation after 21 days

3 % degradation after 28 days

Therefore, Bayscript Gelb BR (active ingredient) is considered to be “Not Readily Biodegradable“.

The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. A toxicity control (test item and reference compound mixed, one replicate) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms. Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate.

At the end of incubation, the pH was measured in the flasks.

The test item is an N-containing substance. Therefore, the concentration of nitrite and nitrate was determined at the start of the test. The oxygen consumed by nitrification was not determined after 28 days, because poor degradation of the test item was observed.

Direct Yellow 86 (active ingredient) showed 3 % degradation after 28 days. Therefore, Direct Yellow 86 (active ingredient) is considered to be “Not Readily Biodegradable“.

The reference compound sodium benzoate showed 91 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed