Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]naphthyl]azo]naphthalene-2-sulphonate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.11. – 06.11.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

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.

Details on test solutions:

The stock solution of the test substance was prepared in the dilution water. 100 mg of the test substance was weighed into 1000 mL of the dilution water for the preliminary test. The concentrations of solutions used in the preliminary test were obtained by dilution of the stock solution with dilution water.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus were obtained by aperiodic parthenogenesis from own laboratory breeding. Daphnia were fed by a mixture of algae. The sensitivity of the test species is periodically monitored by testing with the reference substance, potassium dichromate.The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment. At handling a care should be taken of not to penetrate the air under animal’s shell.TEST ORGANISM- Common name: Daphnia Magna- Strain: Straus- Source: own laboratory breeding- Age at study initiation: young daphnia born in 24 hours- Method of breeding: aperiodic parthenogenesis- Feeding during test: noACCLIMATIONThe grow-up gravid females are separated into dilution water.Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment. - Type and amount of food: a mixture of algae- Health during acclimation: healthy

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The content of sum Ca + Mg ions should have the value of 2.50 +/- 0.10 mmol/L, values measured in this study were 2.52 mmol/L in the preliminary test.

Test temperature:

20.0 ± 2.0 °C; with a variation of ± 1°C at each individual test

pH:

7.8 of dilution water

Dissolved oxygen:

higher than 3 mg/L

Nominal and measured concentrations:

1, 5, 10, 50, 100 mg/L + C(control)

Details on test conditions:

TEST SYSTEM- Test vessel: glass beakers 50ml- Type (delete if not applicable): open- Aeration: no- No. of organisms per vessel: 10- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2- Biomass loading rate: at least 2 ml per individualFor holding and culturing of the daphnia the M4 medium was used (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG 202).For test was used dilution water (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG 202). Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1 000 mL of deionized water.A. 117.6 g CaCl2.2H20 in 1 L of deionized waterB. 49.3 g MgSO4.7H2O in 1 L of deionized waterC. 25.9 g NaHCO3 in 1 L of deionized waterD. 2.3 g KCl in 1 L of deionized water

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

Reference testThe sensitivity of the test species and correctness of test performance is periodically verified in six-month period by testing with the reference substance, potassium dichromate.The results of the verification test with K2Cr2O7, carried out in period from 15.4. to 17.4.2015 are the following:24 hour – EC50 = 1.95 mg.L-1(95% confidence limit: 1.75 – 2.15 mg.L-1)48 hour – EC50 = 0.86 mg.L-1(95% confidence limit: 0.70 – 1.02 mg.L-1)

Validity criteria fulfilled:

yes

Conclusions:

The experiment with the concentration of 100 mg·L-1 included in the preliminary test No.2 has been performed using 20 daphnia, therethrough the conditions of the limit test given in guidelines have been fulfilled. It was demonstrated that the EC50 is higher than this concentration.

Executive summary:

The test substance, Acid Black 26, was tested in acute immobilisation test on Daphnia magna.

The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

The preliminary test No.1 was performed in a range of the test substance nominal concentrations 1 – 100 mg·L-1. Due to complicated solubility of the test substance, two stock solutions were prepared for the testing. The stock solutions differed in preparation (solution A – 15 minutes ultrasonicated, solution B – 15 minutes ultrasonicated and 48 hours stirred).

The results of analytical determinations of concentration of the test substance from solution A and B were different, therefore the preliminary test No.2 were prepared. The stock solution (solution C – 15 minutes ultrasonicated and then 7 days stirred) was prepared for preliminary test No.2. The test was performed as static.

The results of preliminary test No. 2 are used for all further evaluations.

Based on no toxicity of the test substance found in preliminary test No.2 the study was terminated. The experiment with the concentration of 100 mg·L-1 included in the preliminary test No.2 has been performed using 20 daphnia, therethrough the conditions of the limit test given in guideline have been fulfilled. It was demonstrated that the EC50 is higher than this concentration.

The analytical results showed that the test substance was not stable in dilution water under given test conditions. The results of individual concentrations were very different. For this reason the nominal concentrations were used for all evaluation and results.

 

Test results:

24 hour – EC50 > 100 mg·L-1          (nominal concentration)

48 hour – EC50 > 100 mg·L-1          (nominal concentration)

24 hour – EC0 = 100 mg·L-1            (nominal concentration)

48 hour –EC0 = 100 mg·L-1             (nominal concentration)

24 hour – EC100 > 100 mg·L-1       (nominal concentration)

48 hour – EC100 > 100 mg·L-1        (nominal concentration)

Description of key information

The test substance was tested in acute immobilisation test on Daphnia magna according to method C.2 – Daphnia sp. Acute Immobilisation Test (GLP study).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The test was performed as static. The nominal concentration were used for all evaluation and results.

24 hour–EC₅₀ >100 mg·L^-1               (nominalconcentration)

48 hour–EC₅₀ >100 mg·L^-1               (nominalconcentration)