Disodium [N-(2-chlorophenyl)-2-[(2-hydroxy-5-nitrophenyl)azo]-3-oxobutyramidato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Remarks:

BOD5

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 25 to December 15, 1993

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:

ISO 5815 (Water quality - Determination of Biochemical Oxygen Demand after 5 Days (BOD5) - Dilution and Seeding Method)

Version / remarks:

corresponding to 92/96/EEC C.5

Deviations:

yes

Remarks:

Aerate over night after addition of the inoculum.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Seeding water of an urban waste water

Details on inoculum:

- Source of inoculum/activated sludge: underrun of a sewage treatment plant
- Preparation of inoculum for exposure: aerated for 2 to 3 days until it is totally ablated and saturated with oxygen
- Initial cell/biomass concentration: 10E5 bacteria/ml
- Water filtered: yes
- Type and size of filter used: Whatman GF/A filter

Duration of test (contact time):

5 d

Initial conc.:

ca. 6.4 - ca. 824.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 20 ± 1 °C

TEST SYSTEM
- Measuring equipment: O2 - Determination measured with an oxygen sensitive electrode system: SYLAND (SAV : 00 107)

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

Reference substance:

other: D(+)-Glucose (Merck Nr. 8337) / L-Glutamic acid (Merck Nr. 291) solution

Test performance:

Precipitation on the fifth day in the test concentrations from 824.6 - 206.2 mg/l

Parameter:

% degradation (O2 consumption)

Remarks on result:

not determinable

Parameter:

BOD5

Value:

ca. 6 mg O2/g test mat.

Parameter:

COD

Value:

ca. 1 113 mg O2/g test mat.

Parameter:

BOD5*100/COD

Value:

ca. 0.5 other: dimensionless value

Results with reference substance:

The BOD5 for reference substance is 210 mg O2/g test material. The results of the reference substance meet the validation criteria.

The reference values out of acceptance criteria are excluded from the BOD5 evaluation.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

BOD5 = 6 mg O2/g.
COD = 113.0 mg O2/g.
BOD5/COD = 0.005

Executive summary:

Method

The test was carried out according to ISO 5815 Second Edition 1989-08-01 (E) corresponding to 92/96/EEC C.5.

This study was conducted in compliance with OECD Good Laboratory Regulations.

The substance tested at different concentrations, from about 6 to 800 mg/l with a spacing factor of 2, was dissolved in a well aerated seeding water of an urban waste water, without suppression of nitrification, and then incubated at a costant 20 °C temperature for 5d. The reference substance D(+)-Glucose / L-Glutamic acid was tested in a concentration of 20 ml/l.

A blank has been determined in a parallel assay containing no test substance.

The BOD5 is determined by the difference in dissolved oxygen content at the beginning and at the end of the test measured with an oxygen sensitive electrode system: SYLAND (SAV : 00 107).

Results

The BOD5 of the test substance corrected by the blank control was 6 mg O2/g. The oxygen consumption after 5 days was approximately between 0 and 1 mg O2/l for all the tested concentrations except for the two highest ones: 412.3 and 824.6 mg test substance/litre dilution water. For these concentrations the oxygen consumption was respectively 1.9 and 4.9 mg O2/l. Moreover a precipitation on the fifth day in the test concentrations from 824.6 - 206.2 mg/l BOD5 was observed. The BOD5 calculated was the mean value of two replicates of the vessels containing 824.6 mg of test substance, the only concentration that fulfilled the acceptance criteria. The BOD5 measured was 6 mg O2/mg. The COD value measured n the same batch results 1113.0 mg O2/g (BOD5/COD = 0.005).

According to the "Guidance on the Application to the CLP Criteria" for biodegradation test performed in less than 28 days, the ready biodegradability of a substance can be assessed calculating the BOD5/COD ratio. If the result is >= than 0.5 the substance can be considered readily biodegradable.

In this case the ratio does not achieve this requirement and the substance cannot be considered as readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 16 December 1993 to 07 March 1994

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:

yes

Remarks:

The sludge concentration in the test was 1040 mg/l instead of 200-1000 mg/l as recommended in the guidelines. For the test drinking water is used instead of deionised water.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Sludge of a biological sewage treatment plant (ARA-Therwil) collected on 04.01.94
- Method of cultivation: according to the specification of the EEC L133 (pp. 99-105) Volume 31 resp. OECD Method 302B
- Storage conditions: 22.0 ± 3°C (room temperature)
- Test concentration: 150mg/l DOC nominal; indirect daylight
- Water filtered: yes

Duration of test (contact time):

28 d

Initial conc.:

ca. 130.9 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: 22.0 ± 3°C (room temperature)
- Continuous darkness: no

TEST SYSTEM
- Number of culture flasks/concentration: 2 flasks for each concentration
- Measuring equipment: Shimadzu TOC Analyzer

SAMPLING
- Sampling frequency:
1 day before addition of sludge
at 0h and 3h after addition of sludge
at 2, 5, 7, 9, 12, 13, 14, 15, 16, 19, 21, 23, 26, 28 days after addition of sludge

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing activated sludge and mineral nutrients but no test substance

Reference substance:

diethylene glycol

Parameter:

% degradation (DOC removal)

Value:

ca. 44.2

Sampling time:

28 d

Remarks on result:

other: Adsorption after h = 25.9 %

Results with reference substance:

The biodegradation curve of the reference substance showed a typical curve with lag and log phases, and a plateau after 12 days with a bioelimination of 98.8 % rached at day 16.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The bioelimination of the substance measured as DOC after 28 days corrected by the blank control is: 44.2 % (average of 2 tests running in parallel)
Adsorption after 3 hours: 25.9 %

Executive summary:

Method

Test was performed according to the OECD Guideline for Testing of Chemicals, No. 302B, Paris 1981 and the EEC-Directive, L133 (pp. 99-105) Volume 31, May 88.

A mixture containing the test substance, mineral nutrients and sludge of a biological sewage treatment plant in aqueous medium was agitated and aerated at 22.0 ± 3 °C in indirect light for up to 28 days. Blank controls, containing the acrtivated sludge and mineral nutrients but no test substance were run in parallel.

The bioelimination at a specific time was calculated from the DOC values of the preliminary test and the main test according.

Results

The concentration in DOC of the test substance in the preliminary test was 145.4 mg/l; at day 0 the DOC of the substance was 130.9 mg/l. Diethylenglycol was used as reference substance. After 3h from the start of the test, the value of adsorption was 25.9 %. The bioelemination of the substance measured as DOC after 28 days was 44.2 %. For the reference substance the bioelimination reached a value of 98.8 % at 16th day. The biolemination curve of the reference substance showed a typical curve with lag and log phases, and a plateau after 12 days. The bioelimination curve of the test substance showed no lag phase; the log phase started almost immediately from the start of the test.

Based on the available information the substance can be considered as not readily biodegradable.

Reference 3

Endpoint:

biodegradation in water: screening test, other

Remarks:

COD

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from January 21 to March 04, 1994

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:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

Version / remarks:

DEV DIN 38'409 - H 41-1 (1980) corresponding to 92/96/EEC C.6

Deviations:

yes

Remarks:

Mean of 2 determinations instead of 3

GLP compliance:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

not specified

Duration of test (contact time):

> 2 h

Initial conc.:

ca. 2.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: Titration (Potentiometric)

Details on study design:

TEST CONDITIONS
- Test temperature: 148 ± 3 °C

Reference substance:

other: potassium hydrogen phthalate

Parameter:

% degradation (O2 consumption)

Remarks on result:

not determinable

Parameter:

COD

Value:

ca. 1 113 mg O2/g test mat.

Validity criteria fulfilled:

yes

Remarks:

The COD of 20 mL of the reference solution shall be 200 mg/l. The result was in an acceptable range of COD 200 ± 8 mg/l.

Conclusions:

COD measured: 1113mg O2/g

Executive summary:

Method

The test was carried out according to DEV DIN 38'409 - H 41-1 (1980) corresponding to 92/96/EEC C.6

This study was conducted in compliance with OECD Good Laboratory Regulations.

2.5 mg of the test substance were dissolved in water and oxidized by potassium dichromate in a strong sulphuric acid medium with silver sulphate as a catalyst under reflux for 2 hours.

The COD of a test substance is calculated from the amount of the unreacted K2Cr2O7, determinated by a titration with (NH4)Fe(SO4)2 • 6H2O.

Endpoint of titration is the color switch from blue-green to red-brown of a Ferroin-indicator solution. Potassium hydrogen phthalate solution was used as reference substance.

Results

The COD measured in this experiment for the substance was 1113 mg O2/g.

Reference 4

Endpoint:

biodegradation in water: screening test, other

Remarks:

BOD5/COD

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 27, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.5 (Degradation: Biochemical Oxygen Demand)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.6 (Degradation: Chemical Oxygen Demand)

GLP compliance:

no

Oxygen conditions:

other: aerobic for BOD and anaerobic for COD

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: underrun of a sewage treatment plant
- Preparation of inoculum for exposure: aerated for 2 to 3 days until it is totally ablated and saturated with oxygen
- Initial cell/biomass concentration: 10E5 bacteria/ml
- Water filtered: yes
- Type and size of filter used: Whatman GF/A filter

Duration of test (contact time):

5 d

Initial conc.:

1 - 500 mg/L

Based on:

test mat.

Initial conc.:

1 g/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

other: potassium hydrogenphthalate

Parameter:

% degradation (O2 consumption)

Remarks on result:

not determinable

Parameter:

BOD5

Value:

0 mg O2/g test mat.

Parameter:

COD

Value:

ca. 1 562.7 mg O2/g test mat.

Parameter:

BOD5*100/COD

Value:

0 other: dimensionless value

Validity criteria fulfilled:

not specified

Interpretation of results:

not readily biodegradable

Conclusions:

BOD5 is 0 mg O2/g.
COD is 1562.7 mg O2/g.

Executive summary:

Method

The study was performed according to internal methods A-15 and A-22, similar to EU method C.5 and EU method C.6 respectively. For the BOD5 determination, the substance tested at different concentrations, from about 1 to 500 mg/l, was dissolved in a well aerated seeding water from the underrun of a sewage treatment plant, and incubated at a costant 20 °C temperature for 5 days

A blank has been determined in a parallel assay containing no test substance.

The BOD is determined by the difference in dissolved oxygen content at the beginning and at the end of the test measured. The BOD5 of the test substance corrected by the blank control after 5 days was 0 mg O2/g.

For the COD determination, 1 g of the test substance was dissolved in one liter of water and oxidized by potassium dichromate in a strong sulphuric acid medium with silver sulphate as a catalyst under reflux for 2 hours.

The COD of a test substance is calculated from the amount of the unreacted K2Cr2O7, determinated by a titration with (NH4)Fe(SO4)2 • 6H2O.

Endpoint of titration is the color switch from blue-green to red-brown of a Ferroin-indicator solution. Potassium hydrogen phthalate solution was used as reference substance.

Results

The COD measured in this experiment for the substance was 1562.7 mg O2/g. The BOD5 value measured in this experiment for the test substance was 0 mg O2/g.

According to the "Guidance on the Application to the CLP Criteria" for biodegradation test performed in less than 28 days, the ready biodegradability of a substance can be assessed calculating the BOD5/COD ratio. If the result is less than 0.5 the substance can be considered as not readily biodegradable. In this case the BOD5/COD ratio is 0 and the substance cannot be considered as readily biodegradable.

Description of key information

Not readily biodegradable

Additional information

The substance under registration has been evaluated for the potential to be biodegradated in aquatic environment.

READY BIODEGRADABILITY

The first ready biodegradability evaluation (Huntsman, 1993; Huntsman, 1994) was made by two studies in which BOD5 and COD were calculated. The studies were conducted following ISO 5815 Second Edition 1989-08-01 (E) corresponding to 92 / 96 / EEC C.5 and DEV DIN 38'409 - H 41-1 (1980) corresponding to 92 / 96 / EEC C.6, respectively.

The tested concentrations for BOD5 assessment ranged from 6.4 and 824.6 mg/l. The concentration of substance for COD assessement was 2.5 mg/l. The BOD5 value was 6 mg O2/g obtained at the maximum concentration of the substance tested; the COD value was 1113 mg O2/g.

In agreement with the "Guidance on the Application to the CLP Criteria", for biodegradation test performed in less than 28 days, the ready biodegradability of a substance can be assessed calculating the BOD5/COD ratio. If the result is >= than 0.5 the substance can be considered readily biodegradable.

In this case the substance cannot be considered as readily biodegradable because the BOD5/COD ratio is below the 0.5 value.

An analogue consideration can be made for the second study (Huntsman, 1984) performed to assess the ready biodegradability of the substance. In this case the BOD5 was tested at concentrations ranging from 1 to 500 mg/l. The COD determination was performed using 1 g/l of substance. The BOD5 value was 0 mg O2/g and the COD value was 1562.7 mg O2/g. As mentioned above the BOD5/COD ratio was below the 0.5, therefore the substance is confirmed to be non readily biodegradable.

INHERENT BIODEGRADABILITY

To assess the potential of the substance under registration to be inherently bodegradable, a study ws performed according to EEC Directive, L133 (pp. 99-105) Volume 31, May 88 (ISSN 0378-6978) according to OECD Guideline for Testing of Chemicals No. 302B, Paris 1981.

The initial concentration of test substance was 150 mg/l approximately. The DOC was determined for 28 days. During the first 3 hours was observed an adsorption of 25.9 %. After 28 days the bioelimination of the substance measured as DOC (mg/l) was 44.2%. Based on the available information the substance can be considered as not readily biodegradable.