Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No.2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test.

Vehicle:

no

Details on test solutions:

The stock solution of the test substance was prepared in the test medium. 100 mg of the test substance was weighted into 1000 ml of the test medium for the preliminary test and 50 mg of the test substance was weighted into 500 ml of the test medium for the limit test. The stock solutions for the preliminary and limit test were ultrasonicated for 15 minutes.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM - Common name: Desmodesmus subspicatus - Strain: Brinkmann 1953/SAG 86.81 - Source (laboratory, culture collection): The Botanic Institute of the Czech Academy of Science - Method of cultivation: indirect daylight at laboratory temperature

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 - 24°C (controlled at ± 2°C)

pH:

7.6 - 8.0

Nominal and measured concentrations:

see table No. 1

Details on test conditions:

TEST SYSTEM - Test vessel: Erlenmeyer flask - Aeration: stopper venting air - Initial cells density: 500 cells/ml - No. of vessels per concentration (replicates): 3 (preliminary test), 6 (limit test) - No. of vessels per control (replicates): 3 (preliminary test), 6 (limit test) GROWTH MEDIUM - Standard medium used: yes TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water: deionized water - Conductivity: 1.33 – 2.73 µS/cm - Culture medium different from test medium: no OTHER TEST CONDITIONS- Photoperiod: continuous light - Light intensity and quality: 8340 to 8350 lux TEST CONCENTRATIONS - Range finding study: 1, 5, 10, 50, 100 mg/l¨REFERENCE SUBSTANCE- Reference substance: potassium dichromate- Summary formula: K2Cr2O7- CAS No.: 7778-50-9- Batch No.: PP/2014/05543- Content of active component: 99.8 %

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

see table No. 2 and 3

Results with reference substance (positive control):

REFERENCE SUBSTANCE - Reference substance: potassium dichromate - EC(50) = 0.87 mg/l (72 h, growth rate) - Summary formula: K2Cr2O7 - CAS No.: 7778-50-9 - Batch No.: PP/2014/05543 - Content of active component: 99.8 %

Reported statistics and error estimates:

The NOEC values are evaluated by analysis ov variance (pragram ANOVA).

Table No. 2: Mean cell density - preliminary test

nominal concentration	mean cell density in 1 ml
	0 h	24 h	48 h	72 h
100 mg/l	5000	8333	29167	127083
50 mg/l	5000	10417	35417	133333
10 mg/l	5000	16667	37500	147917
5 mg/l	5000	14583	50000	152083
1 mg/l	5000	22917	52083	152083
control	5000	22917	58333	156250

Table No. 3: Mean cell density - limit test

nominal concentration	mean cell density in 1 ml
	0 h	24 h	48 h	72 h
100 mg/l	5000	16667	33333	116667
control	5000	20833	48958	151042

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 7.6 % and inhibition of yield was 23.5 % in the limit test.Therefore exact value of ErC(50) could not be calculated and the value of EC are given in the form of a range.The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic). The analytical results showed that concentrations determined at the beginning of the test decreased to the end of the test more than 20 %. Therefore, the geometric mean of measured concentration was used for evaluations and results presentation.Test results:EC50 > 71.0 mg/l (72 h, growth rate) (geometric mean of measured concentration)EC50 > 71.0 mg/l (72 h, yield inhibition) (geometric mean of measured concentration)NOEC < 71.0 mg/l (72 h, growth rate) (geometric mean of measured concentration)NOEC < 71.0 mg/l (72 h, yield inhibition) (geometric mean of measured concentration)

Executive summary:

The test substance, Direct Black 51, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No. 2016/266.

The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg/l.

The highest inhibition of growth rate was 6.0 % and the highest inhibition of yield was 19.3 % in the preliminary test. Based on no toxicity of the test substance found in the preliminary test, the limit test was performed subsequently.

The concentration of 100 mg/l was tested in the limit test. The inhibition of growth rate was 7.6 % and inhibition of yield was 23.5 %.

The analytical results showed, that the test substance, Direct Black 51, was not stable in the test medium at conditions of the test. With respect to this observation the geometric mean of measured concentration was used for results presentation and evaluation.

Test results:

EC₅₀> 71.0 mg/l (72 h, growth rate) (geometric mean of measured concentration)

EC₅₀> 71.0 mg/l (72 h, yield inhibition) (geometric mean of measured concentration)

NOEC < 71.0 mg/l (72 h, growth rate) (geometric mean of measured concentration)

NOEC < 71.0 mg/l (72 h, yield inhibition) (geometric mean of measured concentration)

Description of key information

The test substance does not meet the classification criteria for short-term aquatic toxicity according to Regulation (EC) No. 1272/2008.

Key value for chemical safety assessment

EC50 for freshwater algae:

71 mg/L

EC10 or NOEC for freshwater algae:

71 mg/L