Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

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:

21.04.-06.05.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

See Any other information...

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples of test solutions were prepared and delivered by Ecotoxicity group. The samples were analyzed on the day of delivery. The samples were analysed non-diluted.

Details on test solutions:

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 25 mg of the test substance was weighted into 250 mL of the test medium for the limit test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM- Common name: Desmodesmus subspicatus- Source (laboratory, culture collection): Brinkmann 1953/SAG 86.81 obtained from the collection ofautotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 3.6.2014.- Age of inoculum (at test initiation): Inoculum culture was kept 3-4 days under conditions at which thetest was performed.- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated inflasks with the test medium on indirect daylight at laboratory temperature.ACCLIMATION- Acclimation period: 3-4 days- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21-24°C±2°C

pH:

7.6-7.7

Conductivity:

5 µS.cm-1

Nominal and measured concentrations:

100, 50, 10, 5, 1 mg/l

Details on test conditions:

TEST SYSTEM- Test vessel: Erlenmayer flask with stoppers venting air- Material, size, headspace, fill volume: glass, 50 ml- Aeration: without- Initial cells density: 5000 cells in 1 ml- Control end cells density:-No. of vessels per concentration (replicates): 3OTHER TEST CONDITIONSTemperature: 21 - 24 °C controlled at ± 2 °CLighting: under continuous white light, 4 440 lux to 8 880 luxExposition time: 72 hoursVolume of tested mixture: 50 mLInitial concentration of algae culture: 5 000 cells in a 1 mLWithout aeration.Agitation of algae culture by shaking.The light intensity and temperature were measured every 24 hours. The density of algae culture wasevaluated microscopically at 24, 48 and 72 hour.The cell density was measured by direct counting ofliving 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

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

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference substance, potassium dichromate.The results of the verification test with K2Cr2O7, carried out in period from 18.04. to 21.04. 2016, are the following: 72 hour – ErC50 = 0.87 mg.L-1 (95% confidence limit: 0.64 – 1.17 mg.L-1)

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 38.9 % in the limit test.Therefore exact value of ErC50 could not be calculated and the value of EC are givenin 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 per centof 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.72 hour – ErC50 > 100 mg.L-1 (nominal concentration)72 hour – NOECr < 100 mg.L-1 (nominal concentration)

Executive summary:

The test substance, Direct Blue 78,was tested for growth inhibition on algaeDesmodesmus subspicatus.The test was performed according tomethod 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^-1.

The analytical results showed, that the test substance Direct Blue 78 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 39.5 % and the highest inhibition of yield was
81.3 % in the preliminary test.Based on no toxicity of the test substance found in preliminary test (measured by growth rate), the limit test was performed subsequently.Only the value of E_rC₅₀(72h) is neededforthe classification and risk assessment of chemicals, sothe value of E_yC₅₀(72h) was not calculated.

The concentration of 100 mg·L^-1was tested in the limit test. The inhibition of growth rate was 38.9 %.

 

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 of this study.

The nominal concentrations were used for all evaluations and results.

72 hour-ErC50>100 mg/l

72 hour -NOECr<100 mg/l

Description of key information

The test was performed according tomethod C.3. -Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV.

Only one study is available.

GLP study.

Klimish score 1.

72 hour-ErC50>100 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L