Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 24 to August 25, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of activated sludge: ARA Ergolz II, Füllinsdorf, Switzerland.
- Pretreatment: the sludge was washed three timed with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet dry weight was calculated.
- Amount of sludge: calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (± 10 %) dry material per liter.
- Storage conditions: during holding, the sludge was aerated at room temperature until use.
- Preparation of inoculum for exposure: before use, the dry weight of this diluted activated sludge was determined again and defined amounts were added to test water to obtain a final concentration.
- Concentration of sludge: 300 mg dry material per liter.
- Loading rate: the ratio between inoculum and test item (based on the mean DOC measured) was 3: 1.

Duration of test (contact time):

28 d

Initial conc.:

240.2 - 241 mg/L

Based on:

test mat.

Initial conc.:

87.3 - 87.6 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test vessels: 2000-ml Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The final volume was 1000 ml per flask.
- Coverage: each flask was loosely covered with aluminum foil to allow the exchange of air between the flask and the surrounding atmosphere.
- Stirring: the test media were continuously stirred by magnetic stirrers.
- Test temperature: 21 - 24 °C
- pH: 6.8 - 7 .4
- Light conditions: the test flasks were incubated in the dark.
- Oxygen concentration: 7.5 - 8.9 mg O2/l.

TEST MEDIUM
Test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
a) KH2PO4 8.50 g/l, K2HPO4 21.75 g/l, Na2HPO4 x 2H2O 33.40 g/l, NH4CI 0.50 g/l. The pH of this solution was 7.4.
b) MgSO4 x 7H2O 22.50 gll
c) CaCl2 x 2H2O 36.40 g/l
d) FeCl3 x 6H2O 0.25 g/l, stabilized with one drop of concentrated HCI per liter.
To obtain the final test water, 10 ml of stock solution a) and 1 ml each of stock solutions b) - d) were combined and made up to 1000 ml with purified water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution.

SAMPLING
- Sampling frequency: samples were taken on Exposure Day 0 (0 hours and 3 hours ± 30 minutes after the addition of the test and/or reference item) and on Exposure Day 3,7, 11, 14,21 and 28 of the incubation period.
- Sample storage before analysis: prior to sampling, water evaporation losses were compensated by adding purified water up to the mark. Deposits on the test vessels were resuspended in the test vessel.
- Sampling amount: per sampling interval, one sample of about 10 ml was taken and analyzed for DOC.
- Sample Preparation: samples were filtered through a 0.45 µm filter. The first 3-4 ml of the filtrate was discarded. Thereafter, the samples were analyzed for DOC on the day of sampling.

CONTROL AND BLANK SYSTEM
- Procedure control: duplicate. 220 mg/l of reference item, corresponding to 97.8-98.5 mg/l DOC.
- Inoculum blank: duplicate. 3.1 - 3.5 mg/l of DOC
- Abiotic sterile control: one replicate. 240.1 mg/l of test item (91.7 mg/l DOC). Poisoned with mercury dichloride at a concentration of 10 mg/l.
- Toxicity control: one replicate. 240.1 mg/l of test item (91.7 mg/l DOC), 220 mg/l of reference item (180.2 mg/l DOC).

Reference substance:

diethylene glycol

Parameter:

% degradation (DOC removal)

Value:

5

Sampling time:

28 d

Details on results:

ln the test flasks, containing the test item and activated sludge (inoculum), the mean concentration of DOC (dissolved organic carbon) varied between 78 and 88 mg/l over the 21-day exposure period, end was not significantly different from the initial mean DOC concentration of 86 mg/l, measured on Day 0 (starting value after 3 hours of exposure). Therefore, test item was not biodegradable under the test conditions within 28 days.
No DOC removal was observed during the first three hours of exposure, which is an indication that the test item did not adsorb on the activated sludge (inoculum).

ABIOTIC CONTROL
No significant elimination of the test item occurred in the abiotic control under the test conditions within 28 days.

TOXICITY CONTROL
In the toxicity control, containing the test item (corresponding to 45 % of total DOC), the reference item (corresponding to 55 % of total DOC) and activated sludge (inoculum), the initial DOC concentration of 178 mg/l measured on Day 0 (after three hours) rapidly decreased by 50 % within the first 14 days of exposure. Thus, the test item was not inhibitory to activated sludge at the tested concentration of 240 mg/l, because degradation was > 35 % within 14 days of incubation (based on the test guidelines).

Results with reference substance:

ln the procedure controls, containing the reference item diethylene glycol and activated sludge (inoculum), the reference item completely degraded by 100 % within the first seven days of exposure, thus confirming suitability of the activated sludge.

Dissolved organic carbon content (DOC, mg/l) in the test flasks

Time (d)	Test item			Procedure control			lnoculum control			Abiotic control	Toxicity control*
	Repl. 1	Repl. 2	Mean*	Repl. 1	Repl. 2	Mean*	Repl. 1	Repl. 2	Mean
0 hours	90.9	90.6	87.5	101.8	101.1	98.2	3.5	3.1	3.3	91.7	180.2
3 hours	89.3	88.9	86.0	100.9	99.9	97.3	3.3	2.9	3.1	91.1	178.0
3	90.0	90.5	87.3	91.8	89.0	87.4	3.2	2.8	3.0	91.5	169.6
7	76.4*	88.0	77.7	4.4	4.6	0.0	4.1	4.9	4.5	89.5	83.9
11	86.0	83.8	81.2	4.4	5.1	1.1	3.7	3.7	3.7	91.5	88.9
14	90.9	90.4	87.0	4.4	4.1	0.6	3.7	3.7	3.7	90.6	89.4
21	86.3	85.9	83.1	3.5	3.1	0.3	3.0	3.0	3.0	88.0	84.5
28	85.3	84.7	81.4	4.0	3.8	0.3	3.5	3.8	3.7	90.7	83.3

*: values corrected for the mean of the inoculum controls

**: the value should be regarded with care. For unknown reasons, it is obviously too low with respect to the other values.

Percentage biodegradation of the test item and the reference item

Time (d)	Test item			Procedure control			Abiotic control	Toxicity control*
	Repl. 1	Repl. 2	Mean*	Repl. 1	Repl. 2	Mean*
0*	0	0	0	0	0	0	0	0
3	-1	-2	-1	9	11	10	0	5
7	17**	3	10	100	100	100	2	53
11	4	7	6	99	98	99	0	50
14	-1	-1	-1	99	100	99	1	50
21	3	3	3	99	100	100	3	53
28	5	6	5	100	100	100	0	53

*3 hours ± 30 minutes after the addition of the test and/or reference item

**: the value should be regarded with care. For unknown reasons, it is obviously too low with respect to the other values.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The substance was not biodegradable under the test conditions within 28 days.

Executive summary:

The test item was investigated for its potential (inherent) ultimate biodegradability in a Zahn-Wellens / EMPA test over 28 days, based on the OECD Guideline No. 302 B (1992) and EU Commission Directive 88/302/EEC, Part C.9 (1988).

In the test flasks containing the test item and inoculum, the mean concentration of DOC (dissolved organic carbon) remained constant during the exposure period of 28 days. Consequently, the substance was not biodegradable under the test conditions within 28 days.

No DOC removal was observed during the first three hours of exposure, which is an indication that the test item did not adsorb on the activated sludge (inoculum).

The reference item diethylene glycol ultimately and completely degraded within the first seven days of exposure, thus confirming suitability of the activated sludge. In the abiotic control, containing the test item and poisoned mineral medium, no significant degradation was noted after 28 days of exposure (based on DOC measurements). In the toxicity controt, containing the test item, the reference item diethylene glycol and activated sludge (inoculum), the initial DOC decreased by 50 % within 14 days of exposure. The substance was not inhibitory to activated sludge at the tested concentration of 240 mg/l, because degradation was higher than 35 % within 14 days (based on the test guidelines).

Conclusion

The substance was not biodegradable under the test conditions within 28 days.

Description of key information

Neither readily nor inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The test item was investigated for its potential (inherent) ultimate biodegradability in a Zahn-Wellens / EMPA test over 28 days, based on the OECD 302 B. In the test flasks containing the test item and inoculum, the mean concentration of DOC (dissolved organic carbon) remained constant during the exposure period of 28 days; consequently, the substance was not biodegradable under the test conditions within 28 days. No DOC removal was observed during the first three hours of exposure, which is an indication that the test item did not adsorb on the activated sludge (inoculum).