Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

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:

23.6.2016 - 21.7.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

There was deviation from the study plan and guideline. The value of EyC50(72h) was not calculated.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory.
An analysis of samples was started immediately after delivery.
The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing.
An analysis of samples was started immediately after delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

The stock solution of the test substance was prepared in the test medium.
100 mg of the test substance was weighed into 1000 mL of the test medium for the preliminary and the limit test

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 on date 27.5.2016
- Age of inoculum (at test initiation): 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

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21-24°C

pH:

7,6-7,7

Nominal and measured concentrations:

1-100 mg/L

Details on test conditions:

Preliminary test
Range of nominal concentration: 1-100 mg/l
Stock solution of the test substance: 100.09 mg/l
Test concentrations: 100, 50, 10, 5 and 1 mg/l
Conductivity of deionized water: 1.86 µS/cm
pH of the test medium: 7.6
Volume of inoculated algae culture: 0.33 mL in 200 mL of mixture
Lighting during the test: 8 315 – 8 345 lux
Temperature during the test: 21.5 – 23.0 °C
Inicial density: 5000 cells per 1ml
Testing 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.
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.

Limit test
Stock solution of the test substance: 25.02 mg/l
The test concentration: 100 mg/l
Conductivity of deionized water: 1.47 µS/cm
pH of the test medium: 7.6
Volume of inoculum algae culture: 2.22 mL in 200 mL mixture
Lighting during the test: 8 315 – 8 340 lux
Temperature during the test: 21.5 – 23.0 °C
Testing 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 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.

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.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 18.04. to 21.04. 2016, are the following:

72 hour – ErC50 = 0.87 mg·L-1 (95% confidence limit: 0.64 – 1.17 mg·L-1)

Interlaboratory test data
From the results of the last interlaboratory test arranged by CSlab spol. s r.o. in 2015 we have obtained data for potassium dichromate 72h – ErC50 generated at 14 laboratories. The following reference range of values could be calculated from those data:

72 hour – ErC50 = 0.51 – 1.19 mg·L-1

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 20.4 % in the limit test.
Therefore exact value of ErC50 could not be calculated and the value 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 per cent
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 (nominal concentration)

72 hour – NOECr < 100 mg/L (nominal concentration)

Executive summary:

The test substance, Reactive Red 45:1, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according tomethod 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 the test substance nominal concentrations 1 – 100 mg·L^-1.

The analytical results showed, that the test substanceReactive Red 45:1was sufficiently stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L^-1.

The highest inhibition of growth rate was 32.1 % and the highest inhibition of yield was
70.2 % in the preliminary test.Based on no toxicity of the test substance found in the preliminary test (measured by growth rate), the limit test was performed subsequently.Only the value of E_rC₅₀(72h) is neededforthe classification and risk assessment of chemicals, sothe value of E_yC₅₀(72h) was not calculated.

The concentration of 100 mg·L^-1was tested in the limit test.The inhibition of growth rate was 20.4 %.

 

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in 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.

Description of key information

The 72h-ErC50 of Reactive Red 45:1 in Desmodesmus subspicatus was >100 mg/L.

The 72h-NOErC50 of Reactive Red 45:1 in Desmodesmus subspicatus was <100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The test substance, Reactive Red 45:1, 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 the test substance nominal concentrations 1 – 100 mg/L.

The analytical results showed that the test substance Reactive Red 45:1was sufficiently stable in the test medium at conditions of the test in concentration range 1 – 100 mg·L-1.

The highest inhibition of growth rate was 32.1 % and the highest inhibition of yield was 70.2 % in the preliminary test. Based on no toxicity of the test substance found in the preliminary test (measured by growth rate), the limit test was performed subsequently. Only the value of ErC50(72h) is needed for the classification and risk assessment of chemicals, so the value of EyC50(72h) was not calculated.

The concentration of 100 mg/L was tested in the limit test. The inhibition of growth rate was 20.4 %.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in 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.