Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]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:

17.03. – 28.04.2016

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:

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: at the beginning of the test, after 24, 48 hours and at the end of the test in all test concentrations- Sampling method: The samples for analysis were taken at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory. The samples for analysis at 24, 48, 72 hours were delivered to analytical laboratory immediately after the end of appropriate testing period. - 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 1 000 mL of the test medium for the preliminary test and the full test and for the full test No.2. The stock solution for the full test was ultrasonicated for 15 minutes and then stirred for period 30 minutes on a shaft stirrer. The stock solution for the full test No.2 was ultrasonicated for 15 minutes and then stirred for period 10 days on a shaft stirrer. - Differential loading: The test was performed in range nominal concentrations from 9 mg·L-1 to 100 mg·L-1. For the test was used geometric concentration series of the test substance with factor 1.5. - Controls: The test contained three parallel series of the test substance concentrations and three 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: 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:

no

Total exposure duration:

72 h

Test temperature:

21.5 – 22.5 °C

pH:

7.7

Conductivity:

0.99 µS·cm-1

Nominal and measured concentrations:

100, 67, 45, 30, 20, 13 and 9 mg·L-1

Details on test conditions:

Preliminary testRange of nominal concentration: 1-100 mg/lStock solution of the test substance: 100.04 mg/lTest concentrations: 100, 50, 10, 5 and 1 mg/lConductivity of deionized water: 1.23 μS/cmpH of the test medium: 7.7Volume of inoculated algae culture: 0.85 mL in 200 mL of mixtureLighting during the test: 8 360 – 8 380 luxTemperature during the test: 22.5 – 23 °CInicial density: 5000 cells per 1mlFull test No.2The full test was performed in a range of the test substance nominal concentrations 9 – 100 mg·L-1. It was observed that the test substance was not completely dissolved and quality criterion 2 was not fulfilled. Therefore the full test No.2 was performed. The test was performed in range nominal concentrations from 9 mg·L-1 to 100 mg·L-1. For the test was used geometric concentration series of the test substance with factor 1.5. The volume of the test solution was 20 mL in each testing flask. The full test No.2 conditions: Stock solution of test substance: 100.09 mg·L-1Test concentration: 100, 67, 45, 30, 20, 13 and 9 mg·L-1Conductivity of deionized water: 0.99 µS·cm-1pH of test medium: 7.7 Volume of inoculum algae culture: 0.60 mL in 200 mL mixtureLighting during the test: 8 310 – 8330 luxTemperature during the test: 21.5 – 22.5 °CInicial density: 5000 cells per 1mlTesting mixtures were incubated in Erlenmeyers’ flasks. The test contained three parallel series of the test substance concentrations and three controls without the test substance.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.:

>= 37.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 7.8 mg/L

Nominal / measured:

meas. (geom. mean)

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 = 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 analytical results showed that concentrations determined at the beginning of the test decreased to the end of the test more than 20 % in the two lowest concentrations. Therefore the geometric mean of measured concentrations should be used for evaluations and results presentation. The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat professional Version 3.2.1. (see Table " Calculation of EC50 values" in box Any other information ...)

Executive summary:

The test substance, Acid Black 26, 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 test substance nominal concentrations 1 – 100 mg·L-1.

The analytical results showed, that the test substance Acid Black 26 was not stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L-1 . Based on the toxicity of the test substance found in the preliminary test, the full test was performed in appropriate concentration range.

 

The full test was performed in a range of the test substance nominal concentrations 9 – 100 mg·L-1.

The full test had not fulfilled the quality criterion 2 and there was problem with solubility of the test substance, the test substance was not completely dissolved. For these reasons the full test No.2 was performed arranged as follows.

The full test No.2 was performed in a range of the test substance nominal concentrations 9 – 100 mg·L-1. Due to complicated solubility of the test substance, the stock solution for the testing was prepared as follows: 15 minutes ultrasonication and then 10 days stirring.

The samples for analytical determination were taken at the beginning, after 24 hours, 48 hours and at the end of the test.

The results of full test No. 2 was used for all further evaluations.

The analytical results showed that the test substance Acid Black 26 was not stable in the test medium at conditions of the test in the two lowest concentrations. With respect to this observation the geometric mean of measured concentrations were used for results presentation and evaluation.

 

Test results:

72 hour – ErC50 = 37.1 mg·L-1       (95% confidence limit: 19.7 – 68.4 mg·L-1)

(geometric mean of measured concentration)

72 hour – EyC50 = 7.2 mg·L-1         (95% confidence limit: 7.0 – 7.4 mg·L-1)

(geometric mean of measured concentration)

72 hour – ErC10 = 3.0 mg·L-1         (95% confidence limit: 1.8 – 5.0 mg·L-1)

(geometric mean of measured concentration)

72 hour – EyC10 = 1.5 mg·L-1         (95% confidence limit: 1.4 – 1.5 mg·L-1)

(geometric mean of measured concentration)

 

72 hour – NOECr ˂ 7.8 mg·L-1       (geometric mean of measured concentration)

72 hour – NOECy ˂ 7.8 mg·L-1       (geometric mean of measured concentration)

Description of key information

The analytical results showed that concentrations determined at the beginning of the test decreased to the end of the test more than 20 % in the two lowest concentrations. Therefore the geometric mean of measured concentrations should be used for evaluations and results presentation.

The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat professional Version 3.2.1.

Table “Calculation of EC50 values”

	EC50 mg·L^-1	Confidence limit	Approach function
ErC50(72 h)	37.146	19.707 – 68.424	Nonlinear regression
EyC50(72 h)	7.208	7.005 – 7.424	Nonlinear regression
ErC10(72 h)	3.014	1.819 – 4.994	Nonlinear regression
EyC10(72 h)	1.468	1.430 – 1.507	Nonlinear regression

72 hour – NOECr ˂ 7.8 mg·L-1       (geometric mean of measured concentration)

72 hour – NOECy ˂ 7.8 mg·L-1       (geometric mean of measured concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

37.15 mg/L

EC10 or NOEC for freshwater algae:

7.8 mg/L