Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

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 in O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

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). 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. The analytical results showed that the test substance was stable in dilution water under current test conditions.

Vehicle:

no

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 concentrations of solutions used in the preliminary semis-static test were obtained by dilution of the stock solution with dilution water. The exact concentrations were determined using HPLC.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM - Common name: Daphnia magna - Strain/clone: Straus - Source: own laboratory breeding - Age: max. 24 hours

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

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

pH:

7.7 - 8.3

Dissolved oxygen:

7.0 - 7.7 mg/L

Nominal and measured concentrations:

Nominal concentration: 100 mg/L 0 hours: 94.8 mg/L 24 hours (old): 93.8 mg/L 24 hours (new): 95.4 mg/L 48 hours: 96.5 mg/LNominal concentration: 1 mg/L 0 hours: 1.0 mg/L 24 hours (old): 1.0 mg/L 24 hours (new): 1.0 mg/L 48 hours: 0.8 mg/L

Details on test conditions:

TEST SYSTEM - Test vessel: laboratory glass beaker - Renewal rate of test solution (frequency/flow rate): 24 hours - No. of organisms per vessel: 10 - No. of vessels per concentration (replicates): 2 - No. of vessels per control (replicates): 2 TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water: M4 medium OTHER TEST CONDITIONS - Adjustment of pH: no - Photoperiod: daylight

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

Details on results:

see Table 1

Results with reference substance (positive control):

Reference substance: K2Cr2O7EC50 = 1.17 mg/L (24 h)EC50 = 0.62 mg/L (48 h)Interlaboratory test: EC50 = 0.46 – 1.06 mg/L (48 h, K2Cr2O7)

Table 1: 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	10	10	10	10	10
Number of mobile animals	24 hours/2^ndseries	10	10	10	10	10	10
Number of mobile animals	24 hours/total	20	20	20	20	20	20
Immobilisation %	24 hours	0	0	0	0	0	0
Number of mobile animals	48 hours/1^stseries	10	10	10	10	10	10
Number of mobile animals	48 hours/2^ndseries	10	10	10	10	10	10
Number of mobile animals	48 hours/total	20	20	20	20	20	20
Immobilisation %	48 hours	0	0	0	0	0	0

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 EC50 is higher than this concentration.No immobilisation was observed in the preliminary semi-static test. Therefore exact values of EC50 could not be calculated and the values of EC are given in the form of a range. The EC0 and EC100 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.EC50 > 100 mg/L (24 h, nominal concentration)EC50 > 100 mg/L (48 h, nominal concentration)EC0 = 100 mg/L (24 h, nominal concentration)EC0 = 100 mg/L (48 h, nominal concentration)EC100 > 100 mg/L (24 h, nominal concentration)EC100 > 100 mg/L (48 h, nominal concentration)

Executive summary:

The test substance, Direct Black 112, 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 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 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) and the lowest concentration (1 mg/L). The analytical results from the preliminary semi-static test showed, that the test substance was 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 included in the preliminary semi-static test has been performed using 20 daphnia, therethrough the conditions of the limit test given in guideline have been fulfilled. It was demonstrated that the EC₅₀is 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.

EC₅₀> 100 mg/L (24 h, nominal concentration)

EC₅₀> 100 mg/L (48 h, nominal concentration)

EC₀= 100 mg/L (24 h, nominal concentration)

EC₀= 100 mg/L (48 h, nominal concentration)

EC₁₀₀> 100 mg/L (24 h, nominal concentration)

EC₁₀₀> 100 mg/L (48 h, nominal concentration)

Description of key information

The experiment with the nominal 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 guideline have been fulfilled. It was demonstrated that the EC₅₀is higher than this concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L