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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th February 1996 to 15 February 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:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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 in vehicle: Stable in water for at least 96 hours

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling for analysis of test concentrations
During the final test, duplicate samples were taken from 0, 10 and 220 mg/l for analysis. Singlular samples were taken from the other test solutions.

Sampling frquency
at t = 0 h and t = 48 h

volume
10 ml from the approximate centre of the test vessel

Storage
The samples were stored in a deep-freeze until analysis.

sample pre-treatment
The frozen samples wre deforsted at room temperature. The entire volume of the samples was transferred quantitatively into suitable volumetric flasks. The flasks were filledup to the mark with 0.5% tetrabutyl ammonium bromide in Milli-Q water.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions
The test solutions were prepared using stock solutions of 250 mg/l by adding quantitatively 128.3 mg of test substance to 500 ml ISO-medium. Exat aliquots were taken from this stock and dilited up to 250 ml of test medium to provide for the required test concentrations. The final test solutions ranged from clear to dark purple solutions without visible precipitation.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Characteristics
For the test, selection of young Daphnia with an age of <24 hours.

Breeding
Start of each batch
With newborn animals, i.e. < 3-days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.

Maxium age of the cultures
4 weeks

Renewal of the cultures
After 7 days of cultiavtion half of the medium twice a week.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Not stated in study report

Test temperature:

18-22°C, consant within +/- 1°C

pH:

At he beginning and end of the test, for all concentrations and the control(s).

Dissolved oxygen:

At he beginning and end of the test, for all concentrations and the control(s).

Salinity:

Not applicable as freshwater study.

Nominal and measured concentrations:

10, 22, 46, 100 and 220 mg/l

Details on test conditions:

Test vessels
100 ml, all-glass

Medium
ISO, prepared in Milli-RO water

Number of Daphnia
20 per concentration

Loading
10 per vessel containing 80 ml medium

Light
16 hours photoperiod daily

Feeding
No feeding

Aeration
No aeration of the test solution

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

85 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Analysis of the samples taken at the start (t=0) of the final test showed measured conenrations of 19 mg/l (86%), 40 mg/l (87%), 90 mg/l (90%) and 270 mg/l (123%) at the nominal concentrations of 22, 46, 100 and 220 mg/l respectively. analysis of samples taken after 48 hours from 22 and 220 mg/l showed that the measured concentrations remained >80 of both initial and nominal concentrations.

Any other information on results incl. tables

Range-finding test

In the range finding 30% immobility of Daphnia was recorded at 100 mg/l. No immobility was seen at or below 10 mg/l.

Acceptability of the test

In the control, no daphnia became immobilized or trapped at the surface of the water.

The analytical program provided clear evidence that the actual test concentration had been maintained at 80& of the initial concentration.

Further, all test conditions remained within the ranges prescribed by the protocol.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Pacified Reactive black 31 did not onduce acute immobilisation of Daphnia magna at or below 22 mg/l corresponding to a measured concentration of 19 mg/l, after 48 hours of exposure (NOEC).

The 24-hour EC50 was >100 mg/l (no observation of daphnids possible at 220 mg/l).

The 28-hour EC50 was 85 mg/l with 95% fidcial limits of 71-108 mg/l.

Executive summary:

The study procedures described in this report were based on the EEC directive 92/69/EEC, Part C.2. "Acute Toxicity for Daphnia" and the OECD guideline No. 202: "Daphnia sp., Acute immobilisation Test", adopted april 4, 1984.

Daphnia were expopsed for a maximum of 48 hours to a range of nominal concentrations from 10 to 220 mg/l. The test was performed in duplicate with 10 daphnia per vessel.

Analysis of the samples taken during the final test showed that the measured test concentrations ranged from 85% to 123% relative to nominal. Measured concentrations remained stable suring the48 -hour test period.

Pacified Reactive Black 31 did not induce acute immobilisation of Daphnia magna at or below 22 mg/l corresponding to a measured concentration of 19 mg/l, after 48 hours of exposure (NOEC).

The 24h-EC50 was >100 mg/l (no obsevrations of Daphnids possible at 220 mg/l).

The 48 -hr EC50 was 85 mg/l with 95% fiducuial imites of 71 -108 mg/l.