Tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

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:

02.02. - 16.02.2017

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:

Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 266/2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: at the beginning of the test and at the end of the test in all test concentrations- Sampling method: The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. - Sample storage conditions before analysis: The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)The stock solution of the test substance was prepared in the test medium. 100 mg of the test substance was weighed into 1000 mL of the test medium for the preliminary test and 50 mg of the test substance was weighed into 500 mL for the limit test. The stock solution was ultrasonicated for 5 minutes.- Differential loading: The test was performed with nominal concentrations 100 mg·L-1.- Controls: The test contained six parallel series of the test substance concentrations and six controls without the test substance. - Chemical name of vehicle (organic solvent, emulsifier or dispersant): Test medium - the water with conductivity smaller than 5 μS·cm-1 was used for the preparation of solutions. All chemicals used were of analytical-grade.- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):The volume of the test solution was 20 mL in each testing flask.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM- Common name: freshwater algae Desmodesmus subspicatus- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81 on date 27.5.2016- Source: from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň- Age of inoculum (at test initiation): The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed.- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture.ACCLIMATION- Acclimation period: 3-4 days- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 – 22 °C

pH:

7.6

Conductivity:

1.16 µS·cm-1

Nominal and measured concentrations:

100 mg·L-1 (6 replicates)

Details on test conditions:

Preliminary testRange of nominal concentration: 1-100 mg/lStock solution of the test substance: 100.01 mg/lTest concentrations: 100, 50, 10, 5 and 1 mg/lConductivity of deionized water: 0.88 μS/cmpH of the test medium: 7.6Volume of inoculated algae culture: 0.55 mL in 200 mL of mixtureLighting during the test: 8 345 – 8350 luxTemperature during the test: 22 – 23 °CInicial density: 5000 cells per 1mlLimit testStock solution of test substance: 50.09 mg·L-1 into 500 mL of the test mediumTest concentration: 100 mg·L-1Conductivity of deionized water: 1.16 µS·cm-1pH of test medium: 7.6 Volume of inoculum algae culture: 0.97 mL in 200 mL mixtureLighting during the test: 8 340 – 8350 luxTemperature during the test: 21 – 22°CInicial density: 5000 cells per 1mlThe test contained six parallel series of the test substance concentration and six controls without the test substance. The volume of the test solution was 20 mL in each testing flask. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. The light intensity and temperature were measured every 24 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber.

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

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:

The test quality criteria were fulfilled.

Results with reference substance (positive control):

- Results with reference substance valid? YesThe value EC50 for inhibition of growth rate (72h – ErC50) obtained from our last reference test meets the calculated range from the interlaboratory test.The sensitivity of test species and proficiency of our laboratory in test performance was proved.Reference test: 72 hour – ErC50 = 0.87 mg/L (95% confidence limit: 0.64 – 1.17 mg/L)Interlaboratory test: 72 hour – ErC50 = 0.51 – 1.19 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 21.5 % in the limit test.Therefore exact value of ErC50 could not be calculated and the value of EC is 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). 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 % 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.

Executive summary:

The test substance, Direct Blue 85, 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-1.

The analytical results showed, that the test substance Direct Blue 85 was sufficiently stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L-1 .

The highest inhibition of growth rate was 25.2 % and the highest inhibition of yield was 58.4 % 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 72 hour – ErC50 is needed for the classification and risk assessment of chemicals, so the value of 72 hour – EyC50 was not calculated.

The concentration of 100 mg·L-1 was tested in the limit test. The inhibition of growth rate was 21.5 %.

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.

Test results:

72 hour – ErC50 > 100 mg·L-1        (nominal concentration)

72 hour – NOECr < 100 mg·L-1      (nominal concentration)

Description of key information

The inhibition of growth rate was 21.5 % in the limit test.

Therefore exact value of ErC50 could not be calculated and the value of EC is 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).

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 % 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.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L