N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

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:

26.11.-20.12.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
Since the test substance is poorly soluble in test medium at concentrations needed for the test, the saturated solutions of the test substance in test medium were prepared for preliminary and full test. The saturated solutions were prepared by adding measured amounts of the test substance to the test medium. The solutions were stirred for period 72 hours on a shaft stirrer and consequently filtered through 0.45 μm filter. These solutions were used for testing.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): The collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3 days
- 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.

ACCLIMATION
- Acclimation period: 3 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:

23.0-23.5°C

pH:

7.8

Nominal and measured concentrations:

100, 56, 31, 17, 9, 5, and 3 mg.L-1 (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyers flasks with stoppers venting air
- Aeration: no
- Initial cells density: 5000 cells/1 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water with conductivity smaller than 5 μS.cm-1 was used for the preparation of solutions.
- All chemicals used were of analytical grade.

OTHER TEST CONDITIONS
- Lighting: continuous white light, 6900 lux to 6950 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The density of algae culture was evaluated microscopically at 24, 48 and 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.

TEST CONCENTRATIONS
- Preliminary test concentrations: 100, 60, 40, 20 and 10 mg.L-1 (nominal concentration)
- On the basis of preliminary test results the test was performed.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

58.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 53.0 – 64.1 mg•L-1

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limit: 3.0 – 5.0 mg•L-1

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 1.4 – 5.0 mg•L-1

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limit: 0 – 1.2 mg•L-1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

- Results with reference substance valid? yes The value EC50 for inhibition of growth rate (72h – ErC50) obtained from 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 = 0.94 mg.L-1 (95% confidence limit: 0.84 – 1.04 mg.L-1)

The values of ErC50 and EyC50 were calculated using the software “Toxicita” (Water Research Institute, Ostrava, Czech Republic).

The values and confidence intervals of ErC10 and EyC10 for the test substance were calculated by the software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd. Czech Republic).

The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).

For the evaluation EC and NOEC values the nominal concentrations were used.

Table: Calculation of EC50 values

	EC50 mg·L^-1	Confidence limit	Approach function
ErC50(72 h)	58.5	53.0 – 64.1	Log-linear
EyC50(72 h)	4.0	3.0 – 5.0	Log-linear
ErC10(72 h)	3.1	1.4 – 5.0	Linear regression
EyC10(72 h)	0.2	0 - 1.2	Linear regression

Validity criteria fulfilled:

yes

Executive summary:

The test substance, Solvent Yellow 124, 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.

Since the test substance is poorly soluble in test medium at concentrations needed for the test, the saturated solutions of the test substance in test medium were prepared for preliminary and full test. The saturated solutions were prepared by adding measured amounts of the test substance to the test medium. The saturated solutions were prepared by stirring the initital solution for period 72 hours on a shaft stirrer. Finally they were filtered through 0.45 μm filter.

The preliminary test was performed in range of test substance nominal concentrations 10 – 100 mg•L-1. Based on the toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

Full test was performed in the range of test substance nominal concentrations 3 – 100 mg•L-1.

The nominal concentrations were used for all evaluations and results because study was performed without analytical determination of test substance concentrations. No suitable analytical method was found.

 

Test results:

72 hour – ErC50 = 58.5 mg•L-1      (nominal concentration)

(95% confidence limit: 53.0 – 64.1 mg•L-1)

 

72 hour – EyC50 = 4.0 mg•L-1       (nominal concentration)

(95% confidence limit: 3.0 – 5.0 mg•L-1)

 

72 hour – ErC10 = 3.1 mg•L-1        (nominal concentration)

(95% confidence limit: 1.4 – 5.0 mg•L-1)

 

72 hour – EyC10 = 0.2 mg•L-1       (nominal concentration)

(95% confidence limit: 0 – 1.2 mg•L-1)

 

72 hour – NOECr < 3.0 mg•L-1       (nominal concentration)

72 hour – NOECy < 3.0 mg•L-1       (nominal concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

58.5 mg/L

EC10 or NOEC for freshwater algae:

3 mg/L