3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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:

September 27, 2001 - January 09, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (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 (AAA 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. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). This diluted activated sludge was used as inoculum to give a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

104 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week
- pH: Prior to test start, the pH was in the range of 7.4 - 7.5 (measured in each test flask after the addition of the activated sludge inoculum). At the end ot the incubation, the pH was measured again in each test flask
- 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) 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 7.7 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 stirring for five minutes and ultrasound dispersion for fifteen minutes was employed. For dosage of the reference item, a stock solution containing 250 mg sodium benzoate per 100 ml test water was prepared. From this 10 ml aliquots were 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 250 ml with test water.

TEST SYSTEM
- Culturing apparatus: The test flasks (500ml Erlenmeyer flasks, labeled with all necessary information to assure unmistakable identification) were incubated under continuous stirring in a SAPROMAT 012 (Veith GmbH, Heidenheim,
Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day.
- 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:
- Sampling method:
- 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 (O2 consumption)

Remarks:

of the test item BLUE GS 5664.80

Value:

-7

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

of reference item sodium benzoate

Value:

96

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

of the toxicity control containing both the test item and the reference item

Value:

48

Sampling time:

28 d

Details on results:

BIODEGRADATION OF THE TEST ITEM
The percent biodegradation of the test item was calculated based on the COD of 1.19 mg O2/mg test item.
The BOD of the test item BLUE GS 5664.80 in the test media was in the range of the inoculum controls.
Consequently, BLUE GS 5664.80 was found to be not biodegradable under the test conditions within 28 days.

Abiotic control:
No significant degradation of the test item occurred in the abiotic control under the test conditions.

BIODEGRADATION IN THE TOXICITY CONTROL
The percent biodegradation in the toxicity control containing both the test item and the reference item was calculated based on the COD of the test item and the ThOD of the reference item.
The biodegradation rate in the toxicity control containing both the test item and the reference item showed a similar course of biodegradation over the 28-day exposure period as the two procedure controls containing the reference item, only. Within 14 days of exposure, a biodegradation rate of 45% was observed. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms because the biodegradation rate in the toxicity control was >25% within 14 days.

Remarks on result:

not measured/tested

Results with reference substance:

BIODEGRADATION OF THE REFERENCE ITEM
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.
In the procedure controls, the reference item was degraded by an average of 88% by exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 96%.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

BLUE GS 5664.80 was found to be not biodegradable within 28 days in OECD 301F and GLP test.

Executive summary:

The test item BLUE GS 5664.80 was investigated for its ready biodegradability in amanometric respirometry test over 28 days based on EU Commission Directive 92/69 EEC, C.4 -D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992).

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 1.19 mg O2/mg test item.

The biochemical oxygen demand (BOD) of the test item BLUE GS 5664.80 in the test media was in the range of the inoculum controls.

Consequently, BLUE GS 5664.80 was found to be not biodegradable under the test conditions within 28 days.

In the toxicity control, containing both BLUE GS 5664.80 and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, BLUE GS 5664.80 had obviously no inhibitory effect on the activity of activated sludge microorganisms.

In the procedure controls, the reference item sodium benzoate was biodegraded by an average of 88% on exposure Day 14, and reached an average biodegradation rate of 96% by the end of the test (Day 28), thus confirming suitability of the activated sludge.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001, December 10 to 2002, March 18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: The COD was determined in RCC Study No. 816120

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Version / remarks:

1981

Deviations:

yes

Remarks:

Activated sludge from one source was used. The test water was slightly changed. Only the biological oxygen demand (BOD) was monitored.

GLP compliance:

yes

Oxygen conditions:

not specified

Inoculum or test system:

activated sludge, domestic (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 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. 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 100 mg dry material per liter

Duration of test (contact time):

28 d

Initial conc.:

31 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

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 7.3.

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 7.6.

Test concentrations
The amounts of test item were directly weighed into the test flasks. No emulsifiers or solvents were used but ultrasound dispersion was employed for fifteen minutes to obtain a clear solution of the test item. For dosage of the reference item, a stock solution containing 75 mg
sodium benzoate per 100 ml test water was prepared. From this 10 ml aliquots were added to the corresponding test flasks. To each flask activated sludge was added .
Finally, the flasks were made up to a volume of 250 ml with test water.

Test Conditions

Apparatus: The test flasks (SOOmL Erlenmeyer flasks, labeled with all necessary information to assure unmistakable identification) were incubated under continuous stirring in a SAPROMAT 012 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day.
Principle: Electro-chemical analysis process: The biodegradation process consumes the dissolved oxygen in the liquid and generates C02. The C02 is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.
Test duration: 28 days
Light regimen: Darkness
Test temperature: 22 °C, maintained with a built-in thermostat and checked once per week.
pH: Prior to test start, the pH was 7.6 (measured in each test flask after the addition of the activated sludge inoculum). At the end of incubation, the pH was measured again in each test flask

Reference substance:

benzoic acid, sodium salt

Remarks on result:

not determinable because of methodological limitations

Details on results:

The percent biodegradation of the test item was calculated based on the COD of 1.19 mg O2/mg test item.
The BOD of the test item BLUE GS 5664.80 in the test media was below the range of the inoculum controls.
Consequently, BLUE GS 5664.80 was found to be not biodegradable under the test conditions within 28 days.

Remarks on result:

not determinable because of methodological limitations

Results with reference substance:

The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg.
In the procedure control, the reference item was degraded by 63% and 74% by exposure Day 7 and 14, respectively; thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by 87%.

Validity criteria fulfilled:

not specified

Interpretation of results:

not inherently biodegradable

Conclusions:

The percent biodegradation of the test item was calculated based on the COD of 1.19 mg O2/mg test item.
The BOD of the test item BLUE GS 5664.80 in the test media was below the range of the inoculum controls.
Consequently, BLUE GS 5664.80 was found to be not biodegradable under the test conditions within 28 days.

Executive summary:

The test item BLUE GS 5664.80 was investigated for its inherent biodegradability in a manometric respirometry test over 28 days based on OECD Guideline for Testing of

Chemicals No. 302 C (1981 ). The following modifications were made: activated sludge from one source was used, the test water was slightly changed, and only the biological oxygen demand (BOD) was monitored.

The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 1.19 mg 02/mg test item.

The biochemical oxygen demand (BOD) of the test item BLUE GS 5664.80 in the test media was below the range of the inoculum controls.

Consequently, BLUE GS 5664.80 was found to be not biodegradable under the test conditions within 28 days.

In the procedure control, the reference item was degraded by 63% and 74% by exposure Day 7 and 14, respectively; thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by 87%.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed