disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 06, 2002 - August 12, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater. The
sludge was washed once 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.
- Pretreatment: During holding, the sludge was aerated at room temperature until use.
- Concentration 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. Test water was inoculated with defined volumes of this diluted activated sludge to give a final
concentration of 20 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

101 mg/L

Based on:

test mat.

Initial conc.:

102 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: 22 - 24°C. The inoculated flasks were incubated in a temperaturecontrolled room. The temperature was checked on each sampling date. Additionally, the room temperature was continuously recorded.
- 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 - 7.6 in all test flasks.
- pH adjusted: yes
- Continuous darkness: yes
- Test water
The 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
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.
b) MGSO4 x 7H2O: 22.50 g/L
c) CaCl2 x 2H20: 36.40 g/L
d) FeCl3 x 6H20: 0.25 g/L

In order to avoid having to prepare solution d) immediately before use, one drop of concentrated HCI per liter was added.
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.

TEST CONCENTRATIONS
The amounts of test item were directly weighed into the test flasks. No emulsifiers or solvents were used but the test item flasks were stirred for ten minutes to obtain a clear red solution of the test item. For dosage of the reference item, a stock solution containing 500 mg sodium benzoate per 100 ml test water was prepared. From this a 10 ml aliquot was withdrawn and added to the corresponding test flasks. To each flask (with the exception of the abiotic control) activated sludge was added. Finally, the flasks were made up to a volume of 1000 ml with test water.

TEST SYSTEM
- Culturing apparatus: 2000-ml Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The test vessels were filled with 1000 ml of test medium, or test medium containing test item and/or reference item. 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. The test vessels were labeled with the necessary information to assure unmistakable identif ication.
- Number of culture flasks/concentration:
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency: Samples were taken on Day 0 (treatment day), 3, 7, 14, 21 and 28 of the incubation period for DOC analysis.
- Sampling method: One sample of about 10 ml was taken from each test flask per sampling date. Prior to sampling, water evaporation losses were determined by weighing the flasks and were compensated by adding purified water. Deposits on the test vessels were scraped off and resuspended in the test vessel.
- Sampling preparation: Samples were filtered through a 0.45 µ/m filter, and analyzed Íot DOC on the day of sampling. DOC analyses were performed on a Shimadzu TOC-500 Analyser (in triplicate per sample).
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the test item Red Rwa 4565

Value:

0

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of reference item sodium benzoate

Value:

103

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Remarks:

of abiotic control

Value:

9

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Remarks:

of toxicity control test item and reference item

Value:

59

Sampling time:

28 d

Details on results:

DEGRADATION OF THE TEST ITEM
ln the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) varied between 25.2 and 27.7 mglL over the exposure period of 28 days and was not significantly different from the initial mean DOC concentration of 27.8 mglL measured on Day 0. Expressed as percentage DOC removal, mean values in the range from 0 to 9% were noted. Therefore, Red Rwa 4565 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 trom 26.4 to 32.6 mg/L over the exposure period of 28 days and were not significantly different from the inítial DOC concentration of 31.6 mg/L measured on Day 0. Thus, no significant abiotic degradation occurred under the test conditions.

DEGRADATION OF THE REFERENCE ITEM
ln the procedure controls, containing the reference item sodium benzoate and activated sludge (inoculum), sodium benzoate was readily and completely biodegraded within 3 days of exposure, thus confirming suitability of the activated sludge.

DEGRADATION IN THE TOXICITY CONTROL
ln the toxicity control, containing the test item (corresponding to 52.3% of total DOC), the reference item (corresponding to 47.7% of total DOC) and activated sludge (inoculum), the initial DOC concentration of 61.2 mg/L measured on Day 0 decreased by 59% within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed to not be inhibitory to activated sludge because degradation was clearly >35% within 14 days.

Remarks on result:

not measured/tested

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Red Rwa 4565 was found to be not biodegradable under the test conditions.

Executive summary:

Red Rwa 4565 was investigated for its ready biodegradab¡lity in a"28-Day DOC Die-Away Test" according to the EU Commission Directive 92169 EEC, C.4-4, 1992, and the OECD Guideline for Testing of Chemicals, No. 301 A, 1992.

ln the test flasks containing the test item Red Rwa 4565 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. Therefore, Red Rwa 4565 was found to be not biodegradable under the test conditions.

ln 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).

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

ln the toxicity control, containing the test item, the reference item sodium benzoate and activated sludge (inoculum), the initial DOC decreased by 59% within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed to not be inhibitory to activated sludge because degradation was >35% within 14 days.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 03, 2002 - October 28, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

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)

Version / remarks:

1987

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, FOllinsdorf, Switzerland) treating predominantly domestic wastewater
- Preparation of inoculum for exposure: 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. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g dry material per liter (±10%). During holding, the sludge was aerated at room temperature until use.
- Concentration of sludge: Defined volumes of this activated sludge were added to test water to obtain a final concentration of 200 mg dry material per liter. the ratio between inoculum and test item (added as DOC) was 2.5:1.

Duration of test (contact time):

28 d

Initial conc.:

251 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS

Test water
The test water was prepared according to the testing guidelines. Analytical grade salts were
dissolved in purified water to obtain the following stock solutions:
a) 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.

b) MgSO4 x 7H2O: 22.50 g/L
c) CaCl2 x 2H20: 36.40 g/l
d) FeCl3 x 6H20: 0.25 g/l

In order to avoid having to prepare solution d) immediately before use, one drop of concentrated HCI per liter was added.
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 7.8 to 7.4 with a diluted hydrochloric acid solution.

Test concentrations
The amounts of test item were directly weighed into the test flasks. No emulsifiers or solvents were used. For dosage of the reference item, a stock solution containing 480 mg sodium benzoate per 200 mL test water was prepared. From this a 100 mL aliquot was withdrawn and added to the corresponding test flask. To each flask activated sludge was added. Finally, the flasks were made up to a volume of 2000 mL with test water.

Test Conditions

Test vessels: 2 liter Erlenmeyer flasks equipped with magnetic stirrers and loosely covered with aluminium foil to allow the exchange of air between the flask and the surrounding atmosphere.
Identification: The test vessels were labeled with all necessary information to assure unmistakable identification.
Test duration: 28 days
Light conditions: Darkness
Test temperature: 21 - 24'C. The inoculated flasks were incubated in a temperaturecontrolled room. The temperature was checked on each sampling date.
pH: The pH (measured at the start of the test and at each sampling interval) was in the range of 7.1 - 7.6. No pH adjustments were necessary.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the test item Red Rwa 4565

Value:

7

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the procedure control

Value:

100

Sampling time:

28 d

Details on results:

DEGRADATION OF THE TEST ITEM
ln the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) varied between 78.3 and 86.6 mg/L during the exposure period of 28 days and was not significantly different from the initial mean DOC concentration of 85.4 mg/L measured after three hours of exposure. Consequently, Red Rwa 4565 was found to be not biodegradable under the test conditions.
Physico-chemical adsorption :
No DOC removal was observed during the first three hours of exposure indicating that the test item did not adsorb on activated sludge.

DEGRADATION OF THE REFERENCE ITEM
ln the procedure control containing the reference item sodium benzoate and activated sludge (inoculum), the DOC content rapidly decreased by 98.5% within the first three days of exposure, thus confirming suitability of the activated sludge.

Remarks on result:

not measured/tested

Test concentrations

Identification	Flask no.	Amount of test item (mg/L)	Amount of reference item (mg/L)	Measured amount of DOC (mg/L)
Test item	1	251	-	83.4
Test item	2	251	-	84.1
Inoculum control	4	-	-	2.8
Procedure control	3	-	120	72.2

Validity criteria fulfilled:

not specified

Interpretation of results:

not inherently biodegradable

Conclusions:

ln the test flasks containing the test item Red Rwa 4565 and activated sludge (inoculum) the mean concentrations of DOC (dissolved organic carbon) measured throughout the 28-day exposure period were not significantly different from the initial mean DOC concentration measured after three hours of exposure. Consequently, Red Rwa 4565 was found to be not biodegradable under the test conditions.

Executive summary:

The test item Red Rwa 4565 was investigated for its inherent biodegradability in a Zahn-Wellens / EMPA test over 28 days, based on OECD Guideline for Testing of Chemicals, No. 302 B (1992) and EU Commission Directive 87l302lEEC, Part C (1987).

ln the test flasks containing the test item Red Rwa 4565 and activated sludge (inoculum) the mean concentrations of DOC (dissolved organic carbon) measured throughout the 28-day exposure period were not significantly different from the initial mean DOC concentration measured after three hours of exposure. Consequently, Red Rwa 4565 was found to be not biodegradable under the test conditions. No DOC removal was observed during the first three hours of exposure indicating that the test item did not adsorb on activated sludge. The reference item sodium benzoate was ultimately and completely degraded within the first three days of exposure, thus confirming suitability of the activated sludge.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed