Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

see Any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

Analytical monitoring:

yes

Details on sampling:

see Details on analytical methods

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM - Common name: Desmodesmus subspicatus - Strain: Brinkmann - Source (laboratory, culture collection): The Botanic Institute of the Czech Academy of Science, Třeboň (27.5.2016) - Method of cultivation: Inoculum culture was kept 3-4 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test.

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:

preliminary test: 7.4 - 8.0limit test: 7.7 - 8.1

Nominal and measured concentrations:

Preliminary test - nominal concentration: 1 mg/l - determined concentration: 1.1 mg/l (0 h) - determined concentration: 1.0 mg/l (72 h) - nominal concentration: 100 mg/l - determined concentration: 98.3 mg/l (0 h) - determined concentration: 99.2 mg/l (72 h) - nominal concentration: 100 mg/l (without algae) - determined concentration: 100.7 mg/l (0 h) - determined concentration: 100.5 mg/l (72 h)Limit test - nominal concentration: 100 mg/l - determined concentration: 92.5 mg/l (0 h) - determined concentration: 94.5 mg/l (72 h) - nominal concentration: 100 mg/l (without algae) - determined concentration: 94.5 mg/l (0 h) - determined concentration: 95.3 mg/l (72 h)

Details on test conditions:

TEST SYSTEM - Test vessel: Erlenmeyer flasks with stoppers venting air - Aeration: without - Initial cells density: 5000 cells per 1 mL - Control end cells density: Burker´s counting chamber - 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 OTHER TEST CONDITIONS - Sterile test conditions: yes - Adjustment of pH: no - Photoperiod: continuous white light - Light intensity and quality: 4 440 - 8 880 lux EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Burker´s counting chamber TEST CONCENTRATIONS - Test concentrations: 1, 5, 10, 50 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

see Table 1 and 2

Results with reference substance (positive control):

- Reference substance: potassium dichromate - EC50 = 0.87 mg/L (72 h, growth rate) - Interlaboratory data: EC50 = 0.51 - 1.19 mg/L (72 h, growth rate)

Reported statistics and error estimates:

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).

Table 1: Growth rate – preliminary test

Nominal concentration (mg/L)	Replicates	Growth rate for section-by-section			Growth rate 0-72 h
		0-24 h	24-48 h	48-72 h
100	1	1.32	0.69	1.47	1.16
	2	1.32	0.85	1.35	1.17
	3	1.32	0.69	1.47	1.16
50	1	1.83	0.18	1.50	1.17
	2	1.83	0.18	1.58	1.20
	3	1.61	0.41	1.54	1.19
10	1	1.83	1.10	1.44	1.46
	2	1.61	1.32	1.42	1.45
	3	1.83	1.10	1.53	1.49
5	1	2.01	0.92	1.86	1.60
	2	2.01	0.85	1.84	1.57
	3	2.01	0.92	1.85	1.59
1	1	2.17	0.94	1.78	1.63
	2	2.17	1.00	1.65	1.61
	3	2.17	0.89	1.78	1.61
Control	1	2.30	1.25	1.46	1.67
	2	2.17	1.46	1.23	1.62
	3	2.17	1.42	1.44	1.68

Table 2: Growth rate – limit test

Nominal concentration (mg/L)	Replicates	Growth rate for section-by-section			Growth rate 0-72 h
		0-24 h	24-48 h	48-72 h
100	1	2.01	0.15	1.00	1.06
	2	1.83	0.47	0.81	1.04
	3	1.83	0.34	1.05	1.07
	4	1.83	0.34	0.94	1.04
	5	1.83	0.47	0.86	1.06
	6	2.01	0.29	0.86	1.06
Control	1	2.01	1.15	1.51	1.56
	2	2.01	1.15	1.37	1.51
	3	2.17	1.05	1.36	1.53
	4	1.83	1.10	1.76	1.56
	5	2.17	1.10	1.39	1.55
	6	2.01	1.10	1.47	1.53

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 31.7 % in the limit test. Therefore, exact value of EC50 could not be calculated and the value of EC are given in the form of a range.There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values. So the nominal concentrations were used for all evaluations and results.growth rate: EC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)growth rate: NOEC > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

Executive summary:

The test substance, Direct Black 112, 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 (EC) No. 761/2009, Annex IV.

The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg/L. The analytical results showed, that the test substance Direct Black 112 was sufficiently stable in the test medium at conditions of the test in concentration range 1 – 100 mg/L. The highest inhibition of growth rate was 29.7 % and the highest inhibition of yield was 77.6 % in the preliminary test. Based on no toxicity of the test substance found in the preliminary test (measured by growth rate), the limit test was performed subsequently. Only the value of EC50 (72h) based on growth rate is needed for the classification and risk assessment of chemicals, so the value of EC50 (72h) based on yield was not calculated. The concentration of 100 mg/L was tested in the limit test.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study. The nominal concentrations were used for all evaluations and results.

The inhibition of growth rate was 31.7 % in the limit test. Therefore, exact value of EC50 could not be calculated and the value of EC are given in the form of a range.

growth rate: EC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

growth rate: NOEC > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L