Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper (2+) pentasodium 2-[2-{2-amino-5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxyphenyl}diazen-1-yl]-5-({3-carboxy-4-[2-{5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl]phenyl}methyl)benzoate and sodium chloride

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

EC Number:

948-009-7

Molecular formula:

Not applicable for a multi-constituent substance

IUPAC Name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

Test material form:

solid: particulate/powder

Details on test material:

Acid brown 161 batch no. ID151860 CAS no. 85338-16-5
Composition of the muti-constituent substance is included in the report as an extract of the analytical report.

Specific details on test material used for the study:

Designation in Test Facility: 18011801G
Date of Receipt: 18. Jan. 2018
Condition at Receipt: Room temperature, in proper conditions

Specification
The following information concerning identity and composition of the test item was provided by the sponsor.
Name Acid Brown 161
Batch no. ID151860
Appearance dark brown powder
Composition see extract of analytical report (page 26)
CAS No. 85338-16-5
EINECS-No. 286-689-0
Molecular formula not stated
Molecular weight not stated
Purity see extract of analytical report (page 26)
Homogeneity homogeneous
Vapour pressure not stated
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Production date Jan. 2017
Expiry date Jan. 2019
Storage Room Temperature (20 ± 5°C)

Storage
The test item was stored in a tightly closed vessel at room temperature (20 ± 5°C).

Pre-Treatment
The carbon content of 18.18 % was determined by elemental analysis under non-GLP con-ditions, performed at the accredited laboratory Mikroanalytisches Labor Pascher, An der Pulvermühle 1, 53424 Remagen, Germany. The test item was added directly, the amounts were calculated from the carbon content of the test item.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Specification
Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage.

Source and Pre-Treatment of inoculum
Source
The sludge was taken from the activation basin of the sewage treatment plant, In den Seewiesen, 67480 Edenkoben.
Date of collection: 18. May 2018, batch no: 20180518.
Pre-Treatment
The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 4520 mg of sus-pended solids/L.

Duration of test (contact time):

ca. 29 d

Initial test substance concentration

Initial conc.:

ca. 110.6 mg/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its DOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 96 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental Parameters
Flask volume 1500 mL
Apparatus blanks 2, containing mineral medium only
Blank Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing test item, mineral medium and inoculum
Abiotic control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, positive control, mineral medium and in-oculum
Inoculum concentration: 25.0 mg/L
Temperature 20.4 - 23.0 °C without direct lighting
Duration 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L of the test item.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 0

Sampling time:

29 d

Details on results:

The test item Acid Brown 161 is considered as “not readily biodegradable“.
 The degree of biodegradation reached 0 % after 28 days.
 The 10-day-window was not detected.
 Because the test item is a mixture, the 10-day window has not to be taken into account.
 For the test item Acid Brown 161 no biodegradation was observed after 28 days. The 10-day window could not be determined. The criterion of reaching 60% of degradation after 28 days was not met. Acid Brown 161 is therefore considered as “not readily biodegradable” following OECD 301B and EU C.4-C respectively.
 Abiotic degradation was not observed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degrada-tion was not observed. Both replicates of the test item showed very good correspondence.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be consid-ered as toxic towards the inoculum. As degradation in the toxicity flask was 34.6 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 111.2 mg/L”.
For pure substances ready biodegradability is defined in the guidelines as degradation sur-passing 60 % within 10 days after reaching a level of 10 %.
Because the test item is a mixture, the 10-day window has not to be taken into account.
For the test item Acid Brown 161 no biodegradation was observed after 28 days. The 10-day window could not be determined. The criterion of reaching 60% of degradation after 28 days was not met. Acid Brown 161 is therefore considered as “not readily biodegradable” following OECD 301B and EU C.4-C respectively.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.