1-[(2-chloro-4-nitrophenyl)azo]-2-naphthol

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:

02.08. to 15.08.2019

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. No.2016/266

Deviations:

yes

Remarks:

without impact (see Any other information on ... methods )

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solutions of the test item were prepared in the test medium. 100 mg of the test item was weighed into 1000 mL of the test medium for the preliminary and limit test. The stock solutions were ultrasonicated for 30 minutes and stirred for period 1 hours. The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with test medium.

Preliminary test: 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 item nominal concentrations 1 – 100 mg·L-1. Based on low toxicity of the test item found in the preliminary test, the limit test with the test item nominal concentration 100 mg·L-1 was performed subsequently.

- Controls: The test contained six parallel series of the test item concentration and six controls without the test item.

- 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: The green freshwater algae 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:

22.0 – 22.5 °C

pH:

8.1

Conductivity:

1.66 µS·cm-1

Nominal and measured concentrations:

100 mg·L-1
The study will be performed without analytical determination of the test item concentrations.

Details on test conditions:

Preliminary test
Range of nominal concentration: 1-100 mg/l
Stock solution of the test substance: 100.06 mg / 1000 mL-1
Test concentrations: 100, 50, 10, 5 and 1 mg/l
Conductivity of deionized water: 1.58 μS/cm
pH of the test medium: 8.1
Volume of inoculated algae culture: 1.07 mL in 200 mL of mixture
Lighting during the test: 7442 – 7450 lux
Temperature during the test: 22.0°C
Inicial density: 5000 cells per mL

Limit test
Stock solution of the test substance: 100.05 mg·L-1 / 1000 mL-1
The test concentration: 100 mg·L-1 nominal
Conductivity of deionized water: 1.66 µS·cm-1
pH of the test medium: 8.1
Volume of inoculum algae culture: 4.0 mL in 500 mL mixture
Lighting during the test: 7438 – 7450 lux
Temperature during the test: 22.0 – 22.5 °C
Inicial density: 5000 cells per 1ml

Testing mixtures were incubated in the Erlenmeyers’ flasks. The test contained six parallel series of the test item concentration and six controls without the test item.
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? Yes

The 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 = 1.02 mg·L-1 (95% confidence limit: 0.84 – 1.23 mg·L-1)
Interlaboratory test: 72 hour – ErC50 = 0.51 – 1.19 mg·L-1

Mean cell density - limit test

Nominal concentration mg·L^-1	Mean cell density in 1 mL
	0 hours	24 hours	48 hours	72 hours
100	5000	17708	75000	154167
Control	5000	23957	86458	187500

Percentual reduction of mean growth rate and percentual inhibition of mean yield – limit test

Nominal concentration mg·L^-1	Growth rate		Yield
	Mean growth µ	Reduction %	Mean area Y	Inhibition %
100	1.14	5.4	149167	18.3
Control	1.21	-	182500	-

Growth curves and other details on results are in attached file PigRed4_157-19-23_Algae_result tables.pdf

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 5.4 % and the inhibition of yield was 18.3 % 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 statistical software ToxRat Professional Version 3.2.1

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 item, Pigment Red 4, 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 item nominal concentrations 1 – 100 mg·L-1.

The highest inhibition of growth rate was 4.3 % and the highest inhibition of yield was 18.8 % in the preliminary test. Based on low toxicity of the test item found in the preliminary test (measured by growth rate), the limit test was performed subsequently. Only the value of 72 hour – ErC50 is needed for the classification and risk assessment of chemicals.

The concentration of 100 mg·L-1 was tested in the limit test. The inhibition of growth rate was 5.4 % and inhibition of yield was 18.3 %.

The study was performed without the analytical determination of the test item concentrations. The suitable analytical method for the determination of the test item concentration is not available.

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)

The determination of NOEC value was done by evaluation of the results from preliminary test using statistical software ToxRat Professional Version 3.2.1.

Description of key information

The inhibition of growth rate was 5.4 % and the inhibition of yield was 18.3 % 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 statistical software ToxRat Professional Version 3.2.1

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