Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

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:
2.11. - 6.11.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Preliminary semi-static test: The analytical determination of the test substance concentrations were performed at the beginning of test, before renewal of the test solutions, after renewal of the test solutions and at the end of the test. The samples for analysis were taken from the highest (100 mg·L-1) and the lowest test concentration (1 mg·L-1). The test substance precipitated from the solution during the test. The samples for analysis were taken from the clear solution above the precipitate. All samples for analysis were delivered in transport box to analytical laboratory immediately after preparation and after each test period (0 – 24 hours and 24 – 48 hours). 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. 50 mg of the test substance was weighed into 500 mL of the dilution water for the preliminary semi-static test. The stock solution was ultrasonicated for 5 minutes. The concentrations of solutions used in the preliminary semi-static test were obtained by dilution of the stock solution with dilution water. Test solution 1 (prepared 08.02.2017)Stock solution of the test substance:50.05 mg·/500 mLTest solution 2 (prepared 09.02.2017)Stock solution of the test substance:50.04 mg·/500 mL
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:
semi-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 and 2.57 mmol/L in the preliminary semi-static test. Measured values meet the requirements.
Test temperature:
20.0 ± 2.0 °C; with a variation of ± 1°C at each individual test
pH:
pH of dilution water 7.8 ± 0.2; measured was 7.8
Dissolved oxygen:
higher than 3 mg/Lmeasured: 8.5 and 8.3 mg·L-1
Conductivity:
0.88 µS·cm-1
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.Because culturing and test medium are different we included pre-test acclimation period: brood daphnids were maintained in dilution water for 48 hours prior to the start of the test.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.:
> 25.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 25.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
25.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
25.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 25.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 25.6 mg/L
Nominal / measured:
meas. (TWA)
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 19.11. to 11.11.2016 are the following:24 hour – EC50 = 1.95 mg.L-1(95% confidence limit: 1.15 – 1.67 mg.L-1)48 hour – EC50 = 0.74 mg.L-1(95% confidence limit: 0.66 – 0.84 mg.L-1)
Validity criteria fulfilled:
yes
Conclusions:
The experiment with the concentration of 100 mg·L-1 included in the preliminary semi-static test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guidelines have been fulfilled.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 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values.Because concentrations of the test substance have not been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, time-weighted mean concentrations were used for evaluations and results.
Executive summary:

The test substance, Direct Blue 85, 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 was performed in a range of the test substance nominal concentrations 1 – 100 mg·L-1.

The test was performed as semi-static. The test solutions were renewed after 24 hours.

The test substance concentrations was measured at the beginning, after 24 hours (before and after renewal) and at the end of the test at the highest (100 mg·L-1) and the lowest concentration (1 mg·L-1).

The analytical results from the preliminary semi-static test showed, that the test substance was not stable in dilution water at conditions of the test.

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

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 percent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values.

Because concentrations of the test substance have not been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, time-weighted mean concentrations were used for evaluations and results.

 

Test results:

24 hour – EC50 > 25.6 mg·L-1          (time-weighed mean concentration)

48 hour – EC50 > 25.6 mg·L-1          (time-weighed mean concentration)

24 hour – EC0 = 25.6 mg·L-1            (time-weighed mean concentration)

48 hour – EC0 = 25.6 mg·L-1            (time-weighed mean concentration)

24 hour – EC100 > 25.6 mg·L-1        (time-weighed mean concentration)

48 hour – EC100 > 25.6 mg·L-1        (time-weighed mean 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

EC50/LC50 for freshwater invertebrates:
25.6 mg/L

Additional information

The experiment with the concentration of 100 mg·L-1 included in the preliminary semi-static test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guidelines have been fulfilled.

Because concentrations of the test substance have not been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, time-weighted mean concentrations were used for evaluations and results.

24 hour–EC50 > 25.6 mg·L-1               (time-weighted mean concentration)

48 hour–EC50 > 25.6 mg·L-1               (time-weighted mean concentration)