disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

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:

October 31,1996 to March 07, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)

Version / remarks:

EEC Publication No. L383 A, December 1992

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

Version / remarks:

adopted July 17, 1992

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Type: Microorganisms from a domestic waste water treatment plant
- Source: ARA Ergolz II, Füllinsdorf I Switzerland
-Conditioning: The activated sludge used for this study was washed three times with tap water and an amount corresponding to 4 g dry material (± 10 %) per litre was mixed with Sorensen buffer solution pH 7 and was then aerated until use. An amount of 0.5 ml sludge (filtered through cotton wool) was added to 1000 ml test medium (with the exception of the abiotic control).

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: 21 - 22 °C. The inoculated flasks were incubated in a shaking water bath. The temperature was checked once per week until week 4 when the temperature was checked on day 27 and day 28.
- pH: 7.4
- pH adjusted: yes
- Continuous darkness: yes
- Test medium: The test medium (30 ml per flask) was prepared according to the OECD Guideline No. 301 E. The stock solutions were:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2H2O, 0.5 g NH4CI dissolved in bidistilled water and filled up to 1000 ml with bidistilled water (pH 7.4).
b) 22.5 g MgSO4 x 7H2O dissolved in bidistilled water and filled up to 1000 ml with bidistilled water.
c) 36.4 g CaCl2 x 2H2O dissolved in bidistilled water and filled up to 1000 ml with bidistilled water.
d) 0.25 g FeCl3 x 6H2O dissolved in bidistilled water and filled up to 1000 ml with bidistilled water. ~ In order to avoid having to prepare this solution immediately before use, 1 (one) drop of concentrated HCI per litre was added.
e) Trace element solution:
30.23 mg MnS04 x H2O, 57.2 mg H3B03, 42.8 mg ZnSO4 x 7H20, 36.85 mg (NH4)6 Mo,024 x 4H2O, 100 mg Fe-chelate (FeCl3, EDTA) dissolved in bidistilled water and filled up to 1000 ml with bidistilled water. The solution was sterilised at 120 °C for 20 minutes.
f) 15 mg yeast extract dissolved in bidistilled water and filled up to 100 ml with bidistilled water. The solution was sterilised by filtration through a 0.2 μm filter.
10 ml of stock solution .a) and one ml each of the stock solutions b) - f) were filled up to 1000 ml with bidistilled water, and the test medium was adjusted to pH 7.4 with a diluted hydrochloric acid solution.

TEST CONCENTRATIONS
Test article: Based on DOC-values which were determined in a pre-test performed within RCC Project No. 639810, the test article was dissolved in the test medium at a concentration of 100 mg/I in duplicate, corresponding to actual amounts of 32.9 and 34.1 mg DOC/I.

TEST SYSTEM
- Culturing apparatus:Shaking water bath
- 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), 7, 14, 21, 27 and 28 of the incubation period.
- Sampling method: Per sampling interval, two flasks of the samples containing the test article or reference compound and one flask of the inoculum blank, the toxicity control and the abiotic control were taken and analyzed for DOC in triplicate. Water evaporation losses were compensated by adding bidistilled water at the respective sampling intervals.
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Untreated test medium was inoculated and used as the control.
- Abiotic sterile control: The concentration of the test article in sterile filtered test medium (0.45 μm filtered) was 100 mg/I, corresponding to an actual amount of 33.8 mg DOC/I.
- Toxicity control: The concentrations of the test article and the reference compound Aniline in the test medium were 50 and 25 mg/I, respectively, corresponding to a total actual amount of 34.8 mg DOC/I. From this, a theoretical amount of 19.4 mg DOC/I originated from Aniline.
- Other:

STATISTICAL METHODS:

Reference substance:

aniline

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the test item Red Rwa 4565

Value:

3

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of reference item Aniline

Value:

102

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Remarks:

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

Value:

52

Sampling time:

28 d

Details on results:

DEGRADATION OF THE TEST ARTICLE
In the test flasks containing the test article and inoculum (test set no. 1 and 2), the mean concentrations of DOC (Dissolved Qrganic Qarbon} varied from 32.8 to 34.7 mg/I over the exposure period of 28 days and were practically equal to the initial mean DOC concentration of 33.5 mg/I measured on day-0. Expressed as percentage DOC-removal, very low values in the range from -4 to 3 % were noted. Therefore, SCARLET RN 1165 was found to be practically nonbiodegradable under the test conditions over the 28-day exposure period to activated sludge from a domestic waste water treatment plant.

Abiotic control:
In the abiotic control (test set no. 6) containing the test article and sterile medium, the DOC concentrations varied from 31.9 to 34.3 mg/I over the exposure period of 28 days and were practically equal to the initial DOC concentration of 33.8 mg/I measured on day 0. Therefore, no abiotic degradation occurred.

DEGRADATION OF THE REFERENCE COMPOUND {ANILINE)
In the procedure controls (test set no. 3 and 4) containing the reference compound Aniline and inoculum, Aniline was readily biodegraded by an average of 99 % within 14 days of exposure, and was fully biodegraded (102 %} by the end of the test (day 28).

DEGRADATION IN THE TOXICITY CONTROL (SCARLET RN 1165 AND ANILINE)
In the toxicity control (test set no. 7) containing the test article (44.3 % as DOC), the reference compound (55.7 % as DOC} and inoculum, the initial DOC concentration of 34.8 mg/I measured on day-0 decreased by 52.0 % to 16.7 mg/I within 28 days of exposure. This indicates nearly complete biodegradation of the reference compound Aniline in the presence of test article, and that the nonbiodegradability of SCARLET RN 1165 is not the result of a toxic effect on the microorganisms. In other words, 50.3 % degradation (based on DOC measured on day-0) occurred within 14 days of exposure; thus, according to the test guidelines the test article can be assumed to not be inhibitory because degradation was >35 % within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

SCARLET RN 1165 was found to be practically nonbiodegradable over the 28-day exposure period to activated sludge from the aeration tank of a domestic waste water treatment plant.
The reference compound Aniline was readily biodegraded by an average of 99 % within 14 days of exposure, and was fully biodegraded (102 %) by the end of the test (day 28); thus confirming suitability of the activated sludge.
In the toxicity control, containing both SCARLET RN 1165 and the reference compound Aniline, no inhibitory effect on the microorganisms was observed.

Executive summary:

SCARLET RN 1165 was investigated for its ready biodegradability in the "28-Day Modified OECD Screening Test".

In the test flasks containing the test article and inoculum (test set no. 1 and 2) the mean concentrations of DOC (Dissolved Organic .Q.arbon) varied from 32.8 to 34.7 mg/I over the exposure period of 28 days and were practically equal to the initial mean DOC concentration of 33.5 mg/I measured on day-0. Expressed as percentage DOC-removal, very low values in the range from -4 to 3 % were noted over the 28-day exposure period. Therefore, SCARLET RN 1165 was found to be practically nonbiodegradable in the "Modified OECD Screening Test".

In the abiotic control (test set no. 6) containing the test article and sterile medium, the DOC concentrations varied from 31.9 to 34.3 mg/I over the exposure period of 28 days and were practically equal to the initial DOC concentration of 33.8 mg/I measured on day-0. Therefore, no abiotic degradation occurred.

In the procedure controls (test set no. 3 and 4) containing the reference compound Aniline and inoculum, Aniline was readily biodegraded by an average of 99 % within 14 days of exposure, and was fully biodegraded (102 %) by the end of the test (day-28); thus confirming suitability of the activated sludge.

In the toxicity control (test set no. 7) containing the test article (44.3 % as DOC), the reference compound (55.7 % as DOC) and inoculum, the initial DOC concentration of 34.8 mg/I measured on day-0 decreased by 52.0 % to 16.7 mg/I within 28 days of exposure. This indicates nearly complete biodegradation of the reference compound Aniline in the presence of test article, and that the nonbiodegradability of SCARLET RN 1165 is not the result of a toxic effect on the microorganisms. In other words, 50.3 % degradation (based on DOC measured on day-0) occurred within 14 days of exposure; thus, according to the test guidelines the test article can be assumed to not be inhibitory 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:

October 29, 1996 - March 11, 1997

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)

Version / remarks:

adopted July 17, 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, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from a domestic waste water treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland)
- Preparation of inoculum for exposure: The activated sludge was centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifuged. This procedure was repeated two times. An aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet sludge to dry sludge calculated. Finally, amounts of wet sludge corresponding to 4 g +/- 10% dry weight basis were added per litre of Soerensen buffer (pH 7) and kept aerated until use six days later.
- Concentration of sludge: An amount of activated sludge from a domestic waste water treatment plant, corresponding to 0.2 g dry material, was added per litre final test medium. The ratio of inoculum and test article (as DOC) was within 2.5:1 and 4:1.

Duration of test (contact time):

28 d

Initial conc.:

200 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS

Test medium
The stock solutions according to the guidelines were:
a) 8.5 g KH2PO4, 21.75 g KrHPOr, 33.4 g NarHPOox ZHrO,0.5 g NH4CI dissolved in bidistilled water and made up to 1000 ml with bidistilled water. The pH was 7.4.
b) 22.5 g MgSOo x TH.O dissolved in bidistilled water and made up to 1000 mlwith bidistilled water.
c) 36.4 g CaCl, x ZH.O dissolved in bidistilled water and made up to 1000 mlwith bidistilled water.
d) 0.25 g FeCl. x 6HrO dissolved in bidistilled water and made up to 1000 ml with bidistilled water. Note: ln order to avoid having to prepare this solution immediately before use, 1 (one) drop of concentrated HCI per litre was added.
10 ml of stock solution a) and 1 ml each of stock solutions b)-d) were combined and made up to 1000 ml with bidistilled water.

Test concentrations

The concentration of the test article was based on its DOC concentration which was determined in a pre-test. In this pre-test the DOC of a solution containing 100 mg SCARLET RN 1165 per 1000 ml test medium was 38.9 mg C/l. Derived from this, two times 400 mg of SCARLET RN 1165 where each weighed into a reactor flask, filled with 500 ml of test medium, and after the addition of 100 ml activated sludge the flasks were filled to the 2-litre mark with test medium. The corresponding mean starting Doc concentration was 71.4 mg C/l (corrected for the blank).
ln the procedure controlthe concentration of the reference compound (Aniline) was 200 mg per 2 litres, corresponding to a theoretical amount of 71.4 mg c/1. ln addition, an untreated lnoculated control (inoculum blank) was run.


Test Conditions

Test duration: 28 days
Light conditions: The study was run under diffuse illumination.The flasks were continously aerated. The oxygen concentration was checked at the start of the test and at the respective sampling intervals, resulting in oxygen concentrations in the range from 8.0 - 8.8 mg O2, per litre.
Test temperature: The test temperature was checked at the respective sampling intervals, and ranged from 22.5 - 23°CC.
pH: The pH was checked at the start of the test and at the respective sampling intervals, and ranged from 7.0 to 7.6.

Reference substance:

aniline

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the test item Scarlet RN 1165

Value:

9

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Remarks:

of the reference item Aniline

Value:

100

Sampling time:

28 d

Details on results:

DEGRADATION OF TEST ARTICLE
During the exposure period of 28 days, the mean DOC concentration slightly decreased from 72.4 mg/l (starting value after 3 hours of exposure to 69.0-62.4 mg/l. No DOC removal was observed during the first three hours of exposure. Expressed as percentage DOC removal, low mean values in the range from 5 to 14% were noted. SCARLET RN 1165 was therefore found to be practically nonbiodegradable by the end of the 28-day exposure period to activated sludge from a domestic waste water treatment plant.

DEGRADATION OF REFERENCE COMPOUND (ANILINE)
The initial DOC concentration (measured after 3 hours of exposure) of the test sotution containing the reference compound Aniline rapidly decreased by 98% within the first seven days of exposure. Consequently, the validity criterion of 70% DOC removal within 14 days of exposure was fulfilled. At the end of the test (day 28), Aniline was completely biodegraded (100%).

Remarks on result:

not determinable because of methodological limitations

Results with reference substance:

The initial DOC concentration (measured after 3 hours of exposure) of the test containing the reference compound Aniline rapidly decreased by 98% within the first seven days of exposure. Consequently, the validity criterion of 70% DOC-removal within 14 days of exposure was fulfilled. At the end of the test (day 28), Aniline was completely biodegraded (100%).

Validity criteria fulfilled:

not specified

Interpretation of results:

not inherently biodegradable

Conclusions:

SCARLET RN 1165 was found to be practically nonbiodegradable in the "Zahn-Wellens / EMPA Test' over the 28-day exposure period to microorganisms from a domestic waste water treatment plant. Additionally, no physico-chemical adsorption occurred on the activated sludge.

The reference compound Aniline ultimately biodegraded by 98 % within seven days of exposure. At the end of the test (day-28), Aniline was completely biodegraded (100 %); thus confirming suitability of the activated sludge.

Executive summary:

SCARLET RN 1 165 was investigated for its biodegradability in the 28-Day Zahn-Wellens/ EMPA Test".

The mean concentration of DOC (Dissolved Organic Carbon) in the test flasks containing the test article slightly decreased from initially 72.4 mg/l to 69.0 -62.4 mg/l over the exposure period of 28 days. Expressed as percentage DOC removal, low mean values in the range from 5 to 14% were noted. Therefore, SCARLET RN 1165 was found to be practically nonbiodegradable over the 28 -day exposure period under the present test conditions. In addition, no DOC-removal was observed during the first three hours of exposure, indicating that no physico-chemical adsorption occured on the activated sludge.

The reference compound Aniline ultimately biodegraded by 98% within seven days of exposure. At the end of the test (day 28), Aniline was completely biodegraded (100%).

Description of key information

SCARLET RN 1165 was found to be non-biodegradable in test performed according to OECD TG 301a and 302b.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed