Tetrasodium 2-[[8-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]benzoyl]amino]-1-hydroxy-3,6-disulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From September 20 to October 19, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away 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 ll, Füllinsdorf, Switzerland.
- Pretreatment: the sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material was resuspended in tap water. This procedure was repeated twice. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Preparation of inoculum for exposure: calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g dry material per liter (± 10 %).
- Method of cultivation: during holding, the sludge was aerated at room temperature until use.
- Initial cell/biomass concentration: defined volumes of the diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

113 mg/L

Based on:

test mat.

Initial conc.:

32.1 - 32.5 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: the test water was prepared according to the testing guidelines.
- Dilution water: the flasks were made up to a volume of 1000 ml with test water.
- Vessels: 2000-ml Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with purified water and dried.
- Test temperature: 22-23 °C.
- pH: prior to test start the pH was 7.4 (measured in all test flasks before the addition of activated sludge (inoculum). At the end of incubation the pH ranged from 7.3 to7.4.
- Aeration of dilution water: 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.
- Continuous darkness: the test flasks were incubated in a dark room.

SAMPLING
- Sampling frequency: samples were taken on Day 0 (treatment day), 9,7, 10, 14, 21, 27 and 28 of the incubation period for DOC analysis.
- Sampling amount: one sample of about 10 ml was taken from each test flask per sampling date.
- Sampling treatment: samples were filtered through a 0.45 µm filter and analyzed for DOC on the day of sampling.
- Replicate: triplicate per sample.
- Evaporation: water evaporation losses were determined by weighing the flasks and were compensated by adding purified water prior to sampling.

CONTROL AND BLANK SYSTEM
- Inoculum blank: activated sludge, without test item. 1 replicate.
- Reference item: 51-52 mg/l of reference item (i.e. 30.6 - 32.1 mg/l DOC). 2 replicates.
- Abiotic control: 111 mg/l (i.e. 32.1 mg/l DOC) of test item. Poisoned with mercury dichloride at a concentration of 10 mg/l. 1 replicate.
- Toxicity control: 111 mg/l (i.e. 61.0 mg/l DOC) of test item and 51 mg/l of reference item. 1 replicate.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (DOC removal)

Value:

2 - 8

Sampling time:

28 d

Details on results:

ln the test flasks containing the test item and inoculum the mean concentrations of DOC (dissolved organic carbon) varied between 31.6 and 29.8 mg/l over the exposure period of 28 days and were not significantly different from the initial mean DOC concentration of 32.3 mg/l measured on Day 0. Expressed as percentage DOC-removal, mean values in the range from 2 to 8 % were noted. Therefore, test item was found to be not biodegradable under the test conditions.

ABIOTIC CONTROL
ln the abiotic control containing the test item and poisoned medium, the DOC concentrations varied from 31.9 to 29.4 mg/l over the exposure period of 28 days and were not significantly different from the initial DOC concentration of 32.1 mg/l measured at Day 0. Thus, no significant abiotic degradation occurred under the test conditions.

TOXICITY CONTROL
ln the toxicity control containing the test item (corresponding lo 51.2 % of total DOC), the reference item (corresponding lo 48.8 % of total DOC) and inoculum, the initial DOC concentration of 61.0 mg/l measured on Day 0 decreased by 50 % within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed not to be inhibitory to activated sludge because degradation was higher than 35 % within 14 days.

Results with reference substance:

ln the procedure controls containing the reference item sodium benzoate and inoculum, sodium benzoate was readily biodegraded by 100 % within 7 days of exposure, confirming the suitability of the activated sludge.

DOC-removal	% DOC-removal alter x days
	3	7	10	14	21	27	28
Test item	6	3	7	5	7	9	9
Test item	1	2	5	5	2	7	4
Mean	3	2	6	5	5	8	7
Sodim benzoate	99	100	104	102	100	104	105
Sodim benzoate	99	102	104	94	101	103	106
Mean	99	101	104	98	100	104	105
Toxified solution with test item	2	2	8	1	4	4	3
Test item and sodium benzoate	46	50	53	50	50	54	53

Biodegradation of test item

Vessel	Conc.	mg DOC/l* after x days
		0	3	7	10	14	21	27	28
Test item	113	32.1	30.9	33.1	31.9	32.2	30.8	32.3	31.7
Test item	113	32.5	32.9	34.0	32.9	32.7	33.0	33.2	33.5
Blank	-	0.0	0.7	2.0	1.9	1.7	1.1	3.0	2.4
Average of test item flasks, corrected for the blank	-	32.3	31.2	31.6	30.5	30.8	30.8	29.8	30.2

* Values given represent mean of three determinations

Biodegradation of sodium benzoate

Vessel	Conc.	mg DOC/l* after x days
		0	3	7	10	14	21	27	28
Reference item	51	30.6	1.1	2.0	0.7	1.0	1.1	1.8	**
Reference item	52	28.6	1.0	1.4	0.8	3.3	0.9	2.0	0.8
Blank	-	0.0	0.7	2.0	1.9	1.7	1.1	3.0	2.4
Average of ref. item flasks, corrected for the blank	-	29.6	0.4	-0.3	-1.2	0.5	-0.1	-1.1	-1.5

* Values given represent mean of three determinations

**Value not readable

Biodegradation in the toxicity control

Vessel	Conc.		mg DOC/l* after x days
	Test item	Sodium benzoate	0	3	7	10	14	21	27	28
Toxified solution with test item	111	-	32.1	31.6	31.5	29.4	31.9	30.7	30.7	**
Test item and sodium benzoate	112	51	61.0	33.7	32.6	30.6	32.0	31.8	31.3	31.0
Test item and sodium benzoate correctd for black	-	-	61.0	33.0	30.6	28.7	30.3	30.7	28.3	28.6
Blank	-	-	0.0	0.7	2.0	1.9	1.7	1.1	3.0	2.4

* Values given represent mean of three determinations

**Value not readable

Interpretation of results:

not readily biodegradable

Conclusions:

Not readily biodegradable

Executive summary:

The substance was investigated for the ready biodegradability in a "28-Day DOC Die-Away Test" according to the EU mathod C.4-A and the OECD Guideline 301 A.

In the test flasks containing the test item and inoculum the mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0. Therefore, test item was found to be not biodegradable under the test conditions.

In the abiotic control containing the test item and poisoned inoculum no degradation was noted atter 28 days of exposure (based on DOC-measurements).

The reference item sodium benzoate was biodegraded by 100 % within 7 days of exposure, confirming the suitability of the activated sludge.

In the toxicity control containing the test item, the reference item sodium benzoate and inoculum, the initial DOC decreased by 50 % within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed to be not inhibitory to activated sludge because degradation was higher than 35 % within 14 days.

Conclusion

Not readily biodegradable

Description of key information

Not readily biodegradable

Key value for chemical safety assessment

Type of water:

freshwater

Additional information

The substance is a monoazo dye. Reactive Red 147 is not expected to be ready biodegradable because of its chemical structure and the specific function. Commonly, dyes undergo a primary transformation, i.e. discolourization due to the interrupting the conjugation. Nevertheless, the degradation process involves more steps and take more time.

However, there is no experimental information on Reactive Red 147, therefore the available data on the structural analogous Similar Substance 01 has been taken into consideration. The read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The Similarn Substance 01 was investigated for the ready biodegradability in a "28-Day DOC Die-Away Test" according to the EU mathod C.4-A and the OECD Guideline 301 A. In the test flasks containing the test item and inoculum the mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0. Therefore, test item was found to be not biodegradable under the test conditions.

In the abiotic control containing the test item and poisoned inoculum no degradation was noted atter 28 days of exposure (based on DOC-measurements). In the toxicity control containing the test item, the reference item sodium benzoate and inoculum, the initial DOC decreased by 50 % within 14 days of exposure. Thus, the test item can be assumed to be not inhibitory to activated sludge because degradation was higher than 35 % within 14 days.