A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex

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:

December 06 1995 to January 04, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

EC Number:

423-940-7

EC Name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

Cas Number:

85585-91-7

IUPAC Name:

4-[2-(7-{2-[4-(ethenesulfonyl)-2-hydroxy-5-methoxyphenyl]diazen-1-yl}-8-hydroxy-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid 4-[2-(8-hydroxy-7-{2-[2-hydroxy-4-(2-hydroxyethanesulfonyl)-5-methoxyphenyl]diazen-1-yl}-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid dicopper tetrasodium hydride

Test material form:

solid

Specific details on test material used for the study:

Identification: Pacified Reactive Black 31
Description: Dark blue crystals
Batch: 9T-55
Purity: 89%
Storage: At room temperature in the dark
Stability under storage conditions: Stable

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

44 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Test vessels
2 litre all-glass brown coloured bottles

CO2-free air
A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 - 1 litre 0.0125 M barium hydroxide solution to trap CO2 which might be present in small amounts. The CO2 free air was sparged through the scrubbing solutions at a constant rate, with the exception that a temporary breakdown of the aeration was noted on day 10 for test ottles A and B. This failure was considered to have no effect on the outcome of the study.

Test concentration
The test substance was tested in duplicate at 88.0 mg per 2 litres, corresponding to ca. 15 mg TOC/l. The organic carbon content was based on the molecular formula.

Type and number of bottles
Test suspension: containing test substance and inoculum (2 bottles)
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance (ca. 40 mg/l sodium acetate) and inoculum (1 bottle)
Toxicity control: containing test substance, reference substance and inoculum (1 bottle)

Measurements
Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2 absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2 abosrber was placed at the far end of the series. Phenolphthalein was used as a pH indicator.

On the 28th day, the pH of the test suspensions were measured and 1 ml of concentrated hydrochloric acid was added to each nottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on Day 29.

Reference substance

Reference substance:

other: sodium acetate

Results and discussion

Test performance:

The ThCO2 production was calculated to be 1.421 mg CO2/mg.

The concentration was 88.0 (A and B) in 2 litres test medium. Hence, the ThCO2 production following complete degradation was 125.0 mg/2 litres for A and B.

The psoitive control contained 80.5 mg sodium acetate resulting in a ThCO2 production followinf complete degradation of 86.4 mg per 2 litres,

The toxicity control contained 80.5 mg sodium acetate and 88.0 mg of Pacified Reactive Black 31 in 2 litres of test medium. Hence, the ThCO2 production following complete degradation of Pacified Reactive Black 31 plus sodium acetate was 211.4 mg/2 litres.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 5 - <= 5.7

Sampling time:

29 d

Details on results:

The relative degradation values calculated from the measurements performed during the test period revealed no significant (>10% deh=gradation) of Pacified Reactive Black 31.

BOD5 / COD results

Results with reference substance:

In the toxicity control, 21% degradation occurred in 14 days. This was below the 25% level. 25% degradation was reached on Day 16.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Executive summary:

Pacified Reactive Black 31 was tested for its ready biodegradability in the CO2 Evolution Test (Modified Sturm Test) at 88.0 mg/2 litres, corresponding to ca. 15 mg TOC/l.

The study procedure was based on EEC directive 92/69, C.4 -C, December 1992, and OECD Guideline No. 301B July 17, 1992.

The theoretical CO2 production of Pacified Reactive Black 31 was calculated to be 1.421 mg/CO2/mg.

The relative degradation values calculated from the measurements performed during the test period revealed no significant (>10%) degradation of Pacified Reactive Black 31.

Pacified Reactive Black 31 was found to have borderline inhibiting properties on microbial activity. However, this was considered to have no effect on the outcome of the study.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, Pacified Reactive Black 31 was not readily biodegradable under the conditions of the modified Sturm test presently performed.