N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

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:

4-17.1.2013

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION (especially for difficult test substances)
Since the test substance is poorly soluble in test medium at concentrations needed for the test, saturated solutions of the test substance in test medium were prepared by adding measured amounts of the test substance to the test medium. The solutions were stirred for period of 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:

TEST ORGANISM
- 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 pre-inoculated from the stock solution and cultivated in flasks with the test medium in indirect daylight at laboratory temperature.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 to 23.5 °C

pH:

7.7 to 8.0

Conductivity:

0.188 mS/m

Nominal and measured concentrations:

- Nominal concentrations: 100, 53, 28, 15, 8, 4 and 2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed with stoppers venting air
- Material, size, headspace, fill volume: 50 mL in each test flask
- Aeration: No
- Initial cell density: 5 000 cells/mL
- Control end cell density: 237 500 in 1 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes
The test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL each of basic stock solutions B, C, D in 1000 mL deionised water. The solutions were kept in the dark at 4 ± 1 °C in fridge. The 4 basic solutions contained the following components and each was made up to 1000 mL with deionised water.
Solution A: NH4Cl 1.5 g, MgC12.6H2O 1.2 g, CaCl2.2H2O 1.8 g, MgSO4.7H2O 1.5 g and KH2PO4 0.16 g
Solution B: FeCl3.6H2O 64 mg and Na2EDTA.2H2O 100 mg
Solution C: H3BO3 185 mg, MnCl2.4H2O 415 mg, ZnCl2 3.0 mg, CoCl2.6H2O 1.5 mg, CuCl2.2H2O 0.01 mg and Na2MoO4.H2O 7.0 mg
Solution D: NaHCO3 50 g

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water with conductivity smaller than 5 μS/cm was used for the preparation of solutions.
- Culture medium different from test medium: No
- Intervals of water quality measurement: At the beginning and the end of the test the pH of the test mixtures was measured. The temperature was measured every 24 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: No data
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: white light, 6800 to 6900 lux (measured every 24 hours)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.9
- Range finding study: Yes
- Test concentrations: 100, 60, 40, 20 and 10 mg/L (nominal concentrations)
- Results used to determine the conditions for the definitive study: Yes. Due to toxicity observed in the preliminary test the full definitive test was performed. The percentage reduction in growth rate observed in the preliminary test was 15.9, 20.1, 22.7, 27.8 and 28.9 % at nominal concentrations of 10, 20, 40, 60 and 100 mg/L.

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.:

< 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limit: 1.0 to 2.8 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limit: 4.1 to 4.9 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limit: 0.0 to 0.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Reduction in the growth rate and inhibition of the yield was seen in every test concentration.
In the main test, the biomass in the control cultures increased more than 45 times during the 72-hour test period.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures is 13.1 % (CV0-24 h = 2.4 %, CV24-48 h = 12.6 % and CV48-72 h = 24.1 %).
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is 2.7 %.

TEST QUALITY CRITERIA
- The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35 %.
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests.
All three criteria were fulfilled.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 72 hour ErC50 = 0.94 mg/L (95 % confidence limit: 0.084 to 1.04 mg/L)
- Other: The inhibition of growth rate by the reference substance obtained from the most recent testing facility reference was in the appropriate range. The sensitivity of test species and proficiency of the laboratory in test performance was therefore verified.

Reported statistics and error estimates:

For the determination EC and NOEC values the nominal concentrations were used.
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 were calculated using the software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
The model fits the regression line to the data (EC x inhibition) and calculates the EC value and 95 % confidence interval for desired % of inhibition.
The determination of NOEC values was done using comparisons in ANOVA (Analysis of Variance) analysis. The ANOVA method used is the part of statistical software QC.Expert 2.5 © 1998-2000.

Table 1: Mean cell Density in Definitive Test

Nominal Concentration (mg/L)	Mean Cell Density in 1 mL
	0 hours	24 hours	48 hours	72 hours
100	5000	18 750	27 083	39 583
53	5000	20 833	29 167	50 000
28	5000	22 917	35 417	54 167
15	5000	27 083	37 500	77 083
8	5000	31 250	43 750	97 917
4	5000	33 333	50 000	133 333
2	5000	43 750	62 500	147 917
Control	5000	60 417	106 250	237 500

 

Table 2: Reduction of Growth and Inhibition of Yield in Definitive Test

Nominal Concentration (mg/L)	Growth rate
	Mean Growth (µ)	Reduction (%)	Mean Yield (Y)	Inhibition (%)
100	0.69	46.4	34 583	85.1
53	0.77	40.4	45 000	80.6
28	0.79	38.3	49 167	78.9
15	0.91	29.1	72 083	69.0
8	0.99	23.0	92 917	60.0
4	1.09	15.0	128 333	44.8
2	1.13	12.3	142 917	38.5
Control	1.29	0.0	232 500	0.0

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC was <2 mg/L.

Executive summary:

The test substance, Solvent Red 19E, was tested for growth inhibition on algae Desmodesmus subspicatus under GLP conditions.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV and OECD Test Guideline No.201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test, Adopted March 23, 2006.

In a preliminary study, algae were exposed the test material at nominal concentrations of 10, 20, 40, 60 and 100 mg/L under static conditions. Following this, a definitive study was carried out at nominal concentrations of 2, 4, 8, 15, 28, 53 and 100 mg/L under static conditions.

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

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

Reduction in the growth rate and inhibition of the yield was seen in every test concentration.

Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L. The 72 h EyC50 was determined to be 4.5 mg/L (95 % condidence limit 4.1 to 4.9 mg/L) and the 72 h NOEC for yield was <2 mg/L.

Description of key information

The 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1.8 mg/L

Additional information

The test substance, Solvent Red 19E, was tested for growth inhibition on algae Desmodesmus subspicatus under GLP conditions.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV and OECD Test Guideline No.201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test, Adopted March 23, 2006.

In a preliminary study, algae were exposed the test material at nominal concentrations of 10, 20, 40, 60 and 100 mg/L under static conditions. Following this, a definitive study was carried out at nominal concentrations of 2, 4, 8, 15, 28, 53 and 100 mg/L under static conditions.

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

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

Reduction in the growth rate and inhibition of the yield was seen in every test concentration.

Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L. The 72 h EyC50 was determined to be 4.5 mg/L (95 % condidence limit 4.1 to 4.9 mg/L) and the 72 h NOEC for yield was <2 mg/L.