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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 17 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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition 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 in vehicle: Stable in water for at least 96 hours

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Storage
All samples not analysed on the same day of sampling were frozen until analysis.

Sample pre-treatment
The frozen samples were defrosted at room temperature.

50 mg trabutylammonium bromide was added to the samples containing 0 and 1.0 mg/l test substance.

The entire volume of the samples containing more than 1.0 mg/l test substance, was transferred quantitatively into suitable volumetric flasks. The flasks were filled up to the mark with 0.5% tetrabutyl ammonium bromide with Milli-Q water.

Samplin for analysis of test concentrations
during the final test samples were taken from three concentrations, i.e. 1.0, 4.6 and 46 mg/l and the blank control for analysis.

Sampling frequency
at t = 0 h and t = 72 hours

volume
10 ml

Storage
Samples were transferred to analytical chemistry directly after sampling

Test solutions

Vehicle:

no

Details on test solutions:

The test solutions were prepared using a stock solution in ISO-medium. For this purpose the test substance was added quantitatively to obtain a nominal concentration of 100 mg/l. Exact aliquots of this stock solution were diluted upto 200 ml of ISO-medium providing test substance concentrations of a factor 2 greater than the required concentrations. Subseqently, these solutions wer mixed with ISO-medium containing 2E=04 algal cells/ml at a ratio of 1:1. The final volume of the test solutions was 50 ml per vessel. At the start of the tst all test solutions were clear and ranged from colourless to purple. One extra replica without algae was included to correct for background absorbence.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species
Selenastrum capricornutum, strain CCAP 278/4.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/l (24 mh CaCO3/l)

Test temperature:

Temperature of test medium measured every day in a temperature-control vessel.

21.5 to 23°C.

pH:

Measurements at the beginning and end of the tst. The pH of the solutins should prefereably not deviate by more than 1.5 units during the test.

Dissolved oxygen:

Not stated

Salinity:

Not applicable as freshwater study

Conductivity:

Not stated

Nominal and measured concentrations:

Nominal test concentrations
1.0, 2.2, 4.6, 10, 22 and 46 mg/l

Details on test conditions:

Cell density
An initial cell density of 1E+04 cells/ml

Illumination
Continuously using TLD lamps of 18 watts yiedliing 7000 - 7500 lux

Inculation
during incubation the algal cells were kept in suspnesion bvy continuous shaking

Controls
Test medium without test substance or other additives (blank)

Replicas
3 replicas of each test cocentration
6 replicas of the blank control
1 replica of the highest concentration without algae
1 replica of each test cocentration without algae

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Measured test substance contrations
At the start of the test, the actual test concentrations were in agreement with nominal at 4.6 and 46. mg/l, whereas the recovery was 87% at 1.0 mg/l.

At the end of the 72-hour period the measured concentration had not decreased by more than 20% in any of the samples taken including those with algae present.

Inhibition of cell growth and reduction of growth rate
Inhibition of cell growth incraesed with increasing concentration of Pacified Reactive Black 31 from 1.0 mg/l upwards resulting in 86% inhibition at 46 mg/l. Statistically significant inhibition of cell growth was found at test concentrations of 1.0 mg/l and higher.

Growth rate reduction increased with increasing concentration of Pacified Reactive Black 31 from 2.0 mg/l upwards resulting in almost 100% reduction at 46 mg/l during the last 48 hours of the test period (24 - 72 hours). Statistically significant reduction of growth rate was found at test concentrations of 1.0 mg/l and higher.

Any other information on results incl. tables

In the range-fonding test, the extinction values indicated no inhibition of cell growth at concentrations up to and including 1 mg/l with significant inhibition at 10 mg/l and total inhibition at 100 mg/l.

Indirect exposre to the coloured test substance solutions showed that substantial inhibition of cell growth occurred at 100 mg/l, whereas the solutions containing 1.0 or 10 mg/l induced no or only slight inhibition of algal growth by adsoprtion of wave lengths ncessary for normal cell growth.

Acceptability of the test

In the control, cell density increased by an average factor of 36 within 3 days. Analysis of samples showed that the actual exposure concentration remained above 80% relative to the initia concentration.

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, Pacified Reactive Black 31 inhibited cell growth and reduced growth rate of this fresh water alage specied significantly at 1.0 mg/l and higher.

Although the coloyr of the test solution inhibited algal growth owing to adsorption of wave lengths necessary for normal algal growth, this was only significant at the higher concentrations. Hence, the inhibition of algal growth oberved at lower concentrations was considered to be a toxic effect.

The EC50 for cell growth inhibition was 1.3 mg/l and the EC10 for cell growth inhibition could only be estimated as the lowest response was 42%. The estimated value for EbC10 was 0.04 mg/l.

The EC50 for growth rate reduction was 13.5 mg/l for the total test period. However, based on the last 48 hours of exposure the EC50 for growth rate reduction was 8.2 mg/l. The EC10 for gorwth rate reductions was calculated to be 0.84 mg/l for the total tets period and 1.2 mg/l for the last 48 hours.

Comparing the EC-values for growth rate reduction with those for growth inhibition shows that especially the EbC10:0-72 hours reflects an over-estimation of the toxiicty of Pacified Reactive Black 31 to fresh water algae, because it is a factor of 20 to 30 below the EC10 values for growth rate reduction. Instead, the values for groowth rate appear to eb more relaible as they reflect the ability of algae to grown at the lower concentrations in spite of the fact that these concentrations remained stable during the test period.

Executive summary:

The study procedures dwere based on the EEC Directive 92/69/EEC, Publication No. L383 Part C-3, adopted December, 1992; OECD guideline No. 201, Adopted june 7, 1984; and ISO Standard B692, First edition, 15 November, 1989.

In a range-finding test, the extinction values indicated that no inhibitin of cell growth at concentrations up to an including 1 mg/l with significant inhibition at 10 mg/l and total inhibition at 100 mg/l. Indirect exposure to the coloured test substance solutins was tested in a pilot test subsequently performed. The results showed that substantial inhibition of cell growth occurred at 100 mg/l, whereas the solutions containing 1.0 or 10 mg/l induced no or only slight inhibition of algal growth by adsorptionof wave lengths necessary for normal cell growth.

A final EC50 test was performed exposing exponentially growing akgal cultures to Pacified Reactive Black 31 concentrations ranging from 1.0 to 46 m/l, increasing with a factor of 2.2. The initial cell density was 1E+04 cells/ml. The total test period was 72 hours. Samples for analysis of test concentrations were taken at the start and the end of the test period.

Pacified Reactive Black 31 inhibited cell growth and reduced growth rate of the freshwater algae Selenastrum capricornutum signficiantly at 1.0 mg/l and higher.

Comparing the EC-values for growth rate reduction with those for griwth inhibition showed that especially the EbC10; 0 -75 h reflected an overestimation of the toxicity of Pacified Reactive Black 31 to fresh water algae, because it was a factor of 20 to 30 below the EC10 values for growth rate reduction. Instead, the values for growth rate appear to be more relaible as they reflect the ability of algae to grow at the lower concentrations in spite of the fact that those concentrations remained stable during the test period.

The EC50 for gorwth rate reduction was 13.5 mg/l for the total test period (ErC50:0 -75h) with a 95% confidence interval ranging from 10 to 20.5 mg/l. Based on the last 48 housr of exposure the EC50 for gorwth rate reduction was 8.2 mg/l (ErC50: 24 -72h) with a 95% confidence interbal ranging from 3.2 to 25 mg/l.

The EC10 for growth rate reduction was calculated to be 0.84 mg/l for the total test period (ErC10: 0 -72h) and 1.2 mg/l for the last 48 hours (ErC10: 24 -72h).