1,3-Benzenedisulfonic acid, 4-[[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]azo]-6-[[4-[[3-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-sulfophenyl]amino]-6-chloro-1,3,5-triazin-2-yl]amino]-, trisodium salt

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:

05.05. – 18.05.2017

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. 761/2009, Annex IV

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006 (Annex 5 corrected: 28 July 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The analytical determination of the test substance concentration was performed at the beginning and at the end of the test. The samples for analysis were taken from the stock solution (100 mg·L-1) with and without algae.- 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 were analyzed on the day of delivery. 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.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTIONThe stock solution of the test substance was prepared in the test medium. 50 mg of the test substance was weighed into 500 mL of the test medium for the preliminary test and 100 mg of the test substance was weighed into 1000 mL of the test medium for the limit test. The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with test medium. The limit test was performed with nominal concentration 100 mg·L-1. Testing mixtures were prepared by dosing of stock solution of the test substance and inoculum into volumetric flasks. The volume of the test solution was 50 mL in each testing flask. The initial density of 5 000 cells per mL was used in the test. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. 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. The growth rates (u) and the yield (Y) and subsequently percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.- Differential loading: The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg·L-1.Based on low toxicity of the test substance found in the preliminary test, the limit test with the test substance nominal concentration 100 mg·L-1 was performed subsequently.- Controls: The test contained six parallel series of the test substance concentration 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 50 mL in each testing flask.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM- Common name: Desmodesmus subspicatus- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81- 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:

21.5 – 23.0 °C

pH:

7.6

Conductivity:

1.26 µS·cm-1

Nominal and measured concentrations:

100 mg·L-195.4 (0 h); 96.6 (72 h)96.4 (0 h); 97.7 (72 h) without algae

Details on test conditions:

Preliminary testStock solution of the test substance: 50.09 mg / 500 mL-1Test concentrations: 100, 50, 10, 5 and 1 mg/lConductivity of deionized water: 1.12 μS/cmpH of the test medium: 7.6Volume of inoculated algae culture: 1.02 mL in 200 mL of mixtureLighting during the test: 7620 – 7650 luxTemperature during the test: 21.5 – 22 °CInicial density: 5000 cells per 1mlLimit testStock solution of the test substance: 50.07 mg / 500 mL-1The test concentration: 100 mg·L-1 nominalConductivity of deionized water: 1.26 µS·cm-1pH of the test medium: 7.6Volume of inoculum algae culture: 4.76 mL in 500 mL mixtureLighting during the test: 7 630 – 7 665 luxTemperature during the test: 21.5 – 23.0 °CInicial density: 5000 cells per 1mlTesting mixtures were incubated in Erlenmeyers’ flasks. The test contained six parallel series of the test substance concentration and six controls without the test substance. The volume of the test solution was 50 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. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. 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.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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 = 1.06 mg·L-1 (95% confidence limit: 0.74 – 1.49 mg·L-1)Interlaboratory test: 72 hour – ErC50 = 0.54 – 1.26 mg·L-1

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 11.4 % and the inhibition of yield was 31.8 % in the limit test. Therefore exact value of ErC50 and EyC50 could not be calculated and the values 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). 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.72 hour – ErC50 > 100 mg·L-1(nominal concentration)72 hour – EyC50 > 100 mg·L-1(nominal concentration)72 hour – NOECr < 1 mg· L-1(nominal concentration)72 hour – NOECy < 1 mg· L-1(nominal concentration)

Executive summary:

The test substance, Reactive Yellow 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 Reactive Yellow 85 was sufficiently stable in the test medium at conditions of the test in the concentration range 1 – 100 mg·L-1 .

The highest inhibition of growth rate was 13.9 % and the highest inhibition of yield was 38.0 % 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-1 was tested in the limit test. The inhibition of growth rate was 11.4 % and the inhibition of yield was 31.8 %.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in the 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 – EyC50 > 100 mg·L-1 (nominal concentration)

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

72 hour – NOECy ˂ 1 mg·L-1       (nominal concentration)

Description of key information

The inhibition of growth rate was 11.4 % and the inhibition of yield was 31.8 % in the limit test. Therefore exact value of ErC50 and EyC50 could not be calculated and the values 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).

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.

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

72 hour – EyC50 > 100 mg·L-1       (nominal concentration)

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

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

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L