Tetrasodium 4,4'-bis[[4-morpholino-6-(p-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

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:

Experimental Starting Date - 26 May 2008; Experimental Completion Date - 27 June 2008; Study Completion Date - 18 Sep 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 66042/A TE
Batch No.: 0582900
Purity: >80 %
Expiration date: 25-Feb-2013
pH: 9.5 at concentration of 10 g/L
Aggregate state / physical form at room temperature: solid
Storage conditions: At room temperature at about 20 °C, away from direct sunlight.

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf / Switzerland) treating predominantly domestic wastewater.
- Storage conditions: The sludge was aerated at room temperature.
- Storage length: Four days prior to use.
- Preparation of inoculum: The sludge was washed twice 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 to dry weight was calculated.
- Concentration of sludge: Based on the ratio "wet to dry ", calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (± 10 %) dry material per liter.
- Ratio inoculum/test item: 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 of 300 mg dry material per liter. The ratio between inoculum and test item (based on the mean DOC measured) was 3.3 : 1.

Duration of test (contact time):

28 d

Initial conc.:

350.4 mg/L

Based on:

DOC

Initial conc.:

351.2 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Water/stock solution: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2P04: 8.50 g/l; K2HP04: 21.75 g/l; Na2HP04 x 2H20: 33.40 g/l; NH4CI: 0.50 g/l. The pH of this solution was 7.4.
2) MgSO4 x 7H2P: 22.50 g/l
3) CaCl x 2H2O: 36.40 g/l
4) FeCl3 x 6H2O 0.25 g/l, stabilized with one drop of concentrated HCI per litre.
- Preparation of mineral medium: To obtain the final test water, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) - 4) were combined and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.
- 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. Each flask was loosely covered with aluminium foil to allow the exchange of air between the flask and the surrounding atmosphere. The test media were continuously stirred by magnetic stirrers.
- Temperature: 20 - 23 °C. The inoculated flasks were incubated in a temperature controlled room. The temperature was checked on each sampling date in one of the inoculum blanks. Additionally, the room temperature was continuously recorded.
- pH: 6.8 - 7.6 (before adjustment). Prior to test start (after the addition of the activated sludge inoculum) and before each sampling, the pH was measured in each test flask. If necessary, the pH was adjusted to 7.5 ± 0.5 with a diluted sulphuric acid solution.
- Light conditions: The test flasks were incubated in the dark.
- Oxygen concentration: 7.2 - 8.7 mg 02/l. The oxygen concentration was measured at the start of the test and before each sampling.

SAMPLING
- Sampling: Per sampling interval, one sample of about 10 mL was taken and analysed for DOC. 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 vessels.
- Sampling dates: Exposure day 0 (0 and 3 hours (± 30 minutes) after the addition of the test chemical), 3, 7, 10, 14,21 and 28, and on at least two occasions during the first fourteen days of exposure.

CONTROL AND BLANK SYSTEM
- Sampling: exposure day 0, 3, 7, 14 and 28, and on at least one occasion during the first seven days of exposure.
- Toxicity control sampling: exposure days 0, 7, 14, and 28.

Reference substance:

diethylene glycol

Remarks:

purity: 100 %

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Replicate 1

Parameter:

% degradation (DOC removal)

Value:

1

Sampling time:

28 d

Remarks on result:

other: Replicate 2

Details on results:

Degradation of the Test Item: In the test flasks containing the test item and activated sludge (inoculum), the mean concentration of DOC varied between 89 and 94 mg/l over the exposure period of 28 days and was not significantly different from the initial mean DOC concentration of 93 mg/l measured on Day 0 (starting value after 3 hours of exposure). Expressed as percentage DOC removal, mean values in the range from -1 to 4 % were noted. Therefore, test item was not biodegradable under the test conditions. No DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.

Degradation of the Reference Item: In the procedure control containing the reference item diethylene glycol and activated sludge
(inoculum), the reference item was completely degraded by 99 % within the first seven days of exposure, thus confirming suitability of the activated sludge.

Degradation in the Toxicity Control: In the toxicity control, containing the test item (corresponding to 46 % of total DOC), the
reference item (corresponding to 54 % of total DOC) and activated sludge (inoculum), the initial DOC concentration of 185 mg/L measured on Day 0 rapidly decreased by 49% within the first 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 351 mg/L because degradation was >35 % within 14 days of incubation.

Measurement of pH, Oxygen and Temperature
During the test the pH was in the range 6.8 -7.6 (before adjustment).
The oxygen concentration was in the range 7.2 - 8.7 mg/L during the test.
The temperature was in the range 20 - 23 °C.

Results with reference substance:

In the procedure control containing the reference item diethylene glycol and activated sludge (inoculum), the reference item was completely degraded by 99 % within the first seven days of exposure, thus confirming suitability of the activated sludge.

Degradation in the Toxicity Control

In the toxicity control, containing the test item (corresponding to 46 % of total DOC), the reference item (corresponding to 54 % of total DOC) and activated sludge (inoculum), the initial DOC concentration of 185 mg/l measured on Day 0 rapidly decreased by 49 % within the first 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 351 mg/l because degradation was >35 % within 14 days of incubation.

Dissolved Organic Carbon Content (DOC) in the Test Flasks

	DOC (mg/l)*
	Test item			Procedure control			Inoculum control			Toxicity control
Time	Replicate N.			Replicate N.			Replicate N.			Replicate No. 1**
	1	2	mean**	1	2	mean**	1	2	mean**
0 h	93.5	96.7	91.7	101.2	100.9	97.7	3.3	3.5	3.4	185.2
3 h	95.4	96.5	92.7	-	-	-	3.6	3	3.3	-
3 d	96.2	98.4	93.8	88.9	89.4	85.6	3.6	3.5	3.6	-
7 d	94.3	95	90.5	5.5	5.5	1.4	4.1	4.2	4.2	100.6
10 d	94.8	95.8	90.8	-	-	-	4.4	4.6	4.5	-
14 d	92.6	93.8	88.6	4.8	4.6	0	4.5	4.8	4.7	93.6
21 d	95.4	96.9	91.6	-	-	-	4.8	4.4	4.6	-
28 d	97.1	97	92.1	4.9	4.9	0	4.3	5.6	5	92.3

*Mean values of at least triplicate measurements per sample.

**Values corrected for the inoculum controls.

- Not determine

Percentage Biodegradation of the Test Item and the Reference Item

	Percentage Biodegradation*
	Test item**			Procedure control			Toxicity control
Time	Replicate N.			Replicate N.			Replicate No. 1
	1	2	mean	1	2	mean
0 h	0	0	0	0	0	0	0
3 h	-1	-2	-1	13	12	12	-
7 d	2	3	2	99	99	99	46
10 d	2	2	2	-	-	-	-
14 d	5	4	4	100	100	100	49
21 d	1	1	1	-	-	-	-
28 d	0	1	1	100	100	100	50

*Corrected for the inoculum controls

**0 days = 3 hours ± 30 minutes after the addition of the test item

- Not determined

Measurement of pH, Oxygen and Temperature

During the test the pH was in the range 6.8 -7.6 (before adjustment).

The oxygen concentration was in the range 7.2 - 8.7 mg/l during the test.

The temperature was in the range 20 - 23 °C.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0 (starting value after 3 hours of exposure). Therefore, test item was not biodegradable under the test conditions.
No DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge.

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 for Testing of Chemicals, No. 302 B (1992) and ED Commission Directive 88/302/EEC, Part C.9. In the test flasks, containing the test item and activated sludge (inoculum), the mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0 (starting value after 3 hours of exposure). Therefore, test item was not biodegradable under the test conditions. No DOC removal was observed during the first three hours of exposure which is an indication that the test item did not adsorb on activated sludge. The reference item diethylene glycol was ultimately and completely degraded by 99 % within the first seven days of exposure, thus confirming suitability of the activated sludge. In the toxicity control, containing the test item, the reference item diethylene glycol and activated sludge (inoculum), the initial DOC decreased by 49 % within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 351mg/L because degradation was >35 % within 14 days.

Description of key information

Under test conditions no biodegradation observed.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Additional information

Several studies on the biodegradability in water are available on the category of Stilbene Fluorescent Whitening Agents and none of the substances is neither readily nor inherently biodegradable in the available studies. The complete overview of the category is reported in the Category Justification Report attached to the point 13 of this dossier. An inherently biodegradation test according to Zahn Wellens method was performed on the substance under registration (CAS 28950-61-0) and indicates that the test substance was presumably not biodegraded (DOC removal, mean values in the range from -1 to 4 %) (Seyfried B., 2008). The result is in agreement with the estimation made using BIOWIN 4.10 on the substance in itself. The most representative studies on ready and inherently biodegradation on analogous substances are also reported in a weight of evidence approach.