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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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.1 (Acute Toxicity for Fish)

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 the 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, 24-48, 48-72 and 72-96 hours). An analysis of samples was started immediately after delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

DILUTION WATERStandard dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D in 1000 mL of deionized water.A. 117.6 g CaCl2·2H20 in 1L deionized waterB. 49.3 g MgSO4·7H2O in 1L deionized waterC. 25.9 g NaHCO3 in 1L deionized waterD. 2.3 g KCl in 1L deionized waterThe dilution water should have a pH 7.8 ± 0.2, values measured in this study were 7.8 – 8.0 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 2.52 – 2.54 mmol/l in the preliminary semi-static test. Measured values meet the requirements.PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCEThe stock solution of the test substance was prepared in the dilution water. 200 mg of the test substance was weighed into 2000 ml of the dilution water for the preliminary semi-static test. The concentrations of solutions used in the preliminary semi-static test were obtained by dilution of the stock solution with dilution water.

Test organisms (species):

Poecilia reticulata

Details on test organisms:

Species: Poecilia reticulata Supplier: certified supplier EMPLA s.r.o., Hradec Králové (Czech Republic) Date of delivery: 23.3.2016 Length: 2.0 – 3.0 cmAge: 3 - 4 months oldHousing: The fish were kept in all-glass aquaria in aerated drinking water.Diet: “TetraMin” – complete feed for aquarium fish (manufacturer: Tetra GmbH, Germany), once a day.Medical treatment: NoneSensitivity test: Species is periodically monitored by testing with the reference substance - potassium dichromate.Preparation for test: Fish were acclimatized for 7 days in dilution water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

no

Test temperature:

22 ± 2°C, with a variation of ± 1°C at each individual test

pH:

7.6 - 8.4

Dissolved oxygen:

6.4 - 7.8 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

no mortality observed

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 the reference substance, potassium dichromate.The results of verification test with K2Cr2O7 are the following:96 hour: LC50 = 152 mg/l48 hour: LC50 = 178 mg/l INTERLABORATORY TEST DATAFrom the result of the last interlaboratory test arranged by CSlab spol. s r.o. in 2015 we have obtained data for potassium dichromate 96h – EC50 generated at 12 laboratories. The following reference range of values could be calculated from those data:96 hour: LC50 = 78 – 183 mg/l

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 7 fish, there through the conditions of the limit test have been fulfilled. It was demonstrated that the LC50 is higher than this concentration.

Executive summary:

The test substance, Direct Black 112, was tested for acute toxicity on fish Poecilia reticulata. The test was performed according to method C.1 – Acute toxicity for fish, 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. 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, 48, 72 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 the preliminary semi-static test, the study was terminated.The experiment with the concentration of 100 mg/l included in the preliminary semi-static test has been performed using 7 fish, therethrough the conditions of the limit test have been fulfilled. It was demonstrated that the LC₅₀is 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.

Test results:                                     

24 hour – LC₅₀ > 100 mg/l

48 hour – LC₅₀ > 100 mg/l

72 hour – LC₅₀ > 100 mg/l

96 hour – LC₅₀ > 100 mg/l

24 hour – LC₀ = 100 mg/l

48 hour – LC₀ = 100 mg/l

72 hour – LC₀ = 100 mg/l

96 hour – LC₀ = 100 mg/l

 

24 hour – LC₁₀₀ > 100 mg/l

48 hour – LC₁₀₀ > 100 mg/l

72 hour – LC₁₀₀ > 100 mg/l

96 hour – LC₁₀₀ > 100 mg/l

Description of key information

The experiment with the concentration of 100 mg/l included in the preliminary semi-static test has been performed using 7 fish, there through the conditions of the limit test have been fulfilled. It was demonstrated that the LC₅₀ is higher than this concentration.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L