Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

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:

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)

Version / remarks:

published i O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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) and the lowest test concentration (1 mg/l).

Vehicle:

no

Details on test solutions:

For 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 deionized waterB.49.3 g MgSO4·7H2O in 1 L deionized waterC.25.9 g NaHCO3 in 1 L deionized waterD.2.3 g KCl in 1 L deionized waterThe dilution water should have a pH 7.8 ± 0.2, values measured in this study were 7.9 in the preliminary semi-static test. The content of sum Ca + Mg ions should have the value of 2.50 ± 0.10 mmol/l, values measured in this study were 2.47 and 2.52 mmol/l in the preliminary semi-static test. Measured values meet the requirements.

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 type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

approx. 2.50 mmol/l (Ca + Mg ions)

Test temperature:

20±2°C, with a variation of ± 1°C at each individual test

pH:

7.7 - 8.4

Dissolved oxygen:

7.5 - 8.2 mg/l

Nominal and measured concentrations:

see table No. 1

Details on test conditions:

REFERENCE SUBSTANCE- Reference substance: potassium dichromate- Summary formula: K2Cr2O7- CAS No.: 7778-50-9- Batch No.: PP/2014/05543- Content of active component: 99.8 %INSTRUMENTS AND EQUIPMENT- oximeter WTW Oxi 730 with membrane probe CellOx 325- pH meter WTW pH 539 - conductivity meter FE30 (Mettler Toledo)- analytical balance AG 135 (Mettler Toledo)- ultrasound Bandelin Sonorex- equipment for keeping and checking of temperature in test area- beakers, volumetric flasks, pipettesSTUDY CONDITIONS- Temperature: 20±2°C, with a variation of ± 1°C at each individual test- Exposure time: 48 hours- Lighting: daylight- Number of test animals:20 for each concentration and control - Volume of test substance solution: 50 ml (least 2 ml per individual)- Concentration of dissolved oxygen: higher than 3 mg/l- Design of test: semi-static- Without feeding, without aeration.PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCEThe 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 concentrations of solutions used in the preliminary semis-static test were obtained by dilution of the stock solution with dilution water.PRELIMINARY SEMI-STATIC TESTThe stock solution of the test substance in dilution water was prepared for the preliminary semi-static test. The test was performed in a range nominal concentrations from 1 mg/l to 100 mg/l. The test solutions were prepared by dosing of appropriate volumes of the stock solution into volumetric flasks and replenishment up to mark with dilution water. Control test was performed concurrently with dilution water only. The test was performed in two replicates. The volume of the test solution was 50 ml in each test beaker. 10 animals were put into each beaker. Total number of daphnia used for each test concentration and control was 20. The daphnia immobilisation and ambient temperature were observed and recorded after 24 and 48 hours. Ambient temperature was measured at the beginning of the test and at the end of the test. At the beginning, before renewal of the test solutions, after renewal of the test solutions and at the end of the test the oxygen content and pH value were measured. The test was performed as semi-static.Test solution 1Stock solution of test substance: 50.09 mg/0.5 lTest concentration: 100, 50, 10, 5, 1 mg/lConductivity of deionized water: 2.34 µS/cmpH of dilution water: 7.9Oxygen content of dilution water: 8.0 mg/lContent of (Ca+Mg) in dilution water: 2.47 mmol/lTest solution 2Stock solution of test substance: 50.07 mg/lTest concentration: 100, 50, 10, 5, 1 mg/lConductivity of deionized water: 2.34 µS/cmpH of dilution water: 7.9Oxygen content of dilution water: 7.8 mg/lContent of (Ca+Mg) in dilution water: 2.51 mmol/lOn the basic of the preliminary semi-static test results the study was terminated.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 39.6 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.:

> 39.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

39.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

39.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 39.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No significant immobilisation (10 %) was observed in the preliminary semi-static test. Therefore, exact values of EC50 could not be calculated. The EC(0) and EC(100) values are identified by direct observation.As the 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 all evaluations and results.

Results with reference substance (positive control):

24 h: EC(50) = 1.17 mg/l48 h: EC(50) = 0.62 mg/l

Table No. 2: Immobilisation of daphnia - preliminary semi-static test

Nominal concentration	mg/l	100	50	10	5	1	C
Number of mobile animals	24 hours/1^stseries	10	9	9	10	9	9
Number of mobile animals	24 hours/2^ndseries	10	10	9	10	10	9
Number of mobile animals	24 hours/total	20	19	18	20	19	18
Immobilisation %	24 hours	0	5	10	0	5	10
Number of mobile animals	48 hours/1^stseries	10	9	9	10	9	9
Number of mobile animals	48 hours/2^ndseries	10	10	9	10	9	9
Number of mobile animals	48 hours/total	20	19	18	20	18	18
Immobilisation %	48 hours	0	5	10	0	10	10

Validity criteria fulfilled:

yes

Conclusions:

The experiment with the concentration of 100 mg/l 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. It was demonstrated that the EC(50) is higher than this concentration.No significant immobilisation (10 %) was observed in the preliminary semi-static test. Therefore, exact values of EC(50) could not be calculated. The EC(0) and EC(100) values are identified by direct observation.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. As the 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 all evaluations and results.24 h: EC(50) > 39.6 mg/l(time-weighted mean concentration)48 h: EC(50) > 39.6 mg/l(time-weighted mean concentration)24 h: EC(0) = 39.6 mg/l(time-weighted mean concentration)48 h: EC(0) = 39.6 mg/l(time-weighted mean concentration)24 h: EC(100) > 39.6 mg/l(time-weighted mean concentration)48 h: EC(100) > 39.6 mg/l(time-weighted mean concentration)

Executive summary:

The test substance, Direct Black 51, 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 semi-static test was performed in range of the test substance nominal concentrations 1 – 100 mg/l. The test was performed as semi-static. The test solutions were renewed after 24 hours. The test substance concentrations were measured at the beginning, after 24 hours (before and after renewal) and at the end of the experiment at the highest (100 mg/l) and the lowest (1 mg/l) concentration.

The analytical results of the preliminary test showed that the test substance was not stable in dilution water at the conditions of the test. It was observed that the test substance precipitated from the solution during the test.

The experiment with the concentration of 100 mg/l 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. It was demonstrated that the EC(50) is higher than this concentration.

No significant immobilisation (10 %) was observed in the preliminary semi-static test. Therefore, exact values of EC(50) could not be calculated. The EC(0) and EC(100) values are identified by direct observation.

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. As the 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 all evaluations and results.

24 h: EC(50) > 39.6 mg/l (time-weighted mean concentration)

48 h: EC(50) > 39.6 mg/l (time-weighted mean concentration)

24 h: EC(0) = 39.6 mg/l (time-weighted mean concentration)

48 h: EC(0) = 39.6 mg/l (time-weighted mean concentration)

24 h: EC(100) > 39.6 mg/l (time-weighted mean concentration)

48 h: EC(100) > 39.6 mg/l (time-weighted mean concentration)

Description of key information

The test substance does not meet the classification criteria for short-term aquatic toxicity according to Regulation (EC) No. 1272/2008.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

39.6 mg/L