Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

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:

18.04.-29.04.2016

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)

Deviations:

yes

Remarks:

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 preliminary semi-static test.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

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 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 organisms (species):

Poecilia reticulata

Details on test organisms:

TEST ORGANISM- Common name: Poecilia reticulata- Source: certified supplier EMPLA s.r.o., Hradec Králové (Czech Republic)- Age at study initiation: 3-4 months- Date of delivery:23.3.2016- Length at study initiation: 2.0-3.0 cm- Method of breeding: in all-glass aquaria in aerated drinking water.- Feeding during test: without feedingACCLIMATION- Acclimation period: 7 days in dilution water- Acclimation conditions: same as test- Food type: “TetraMin” - feed for aquarium fish (manufacturer: Tetra GmbH, Germany)- Frequency: once a day- Health during acclimation (any mortality observed): mortality control, The sensitivity of test species isperiodically monitored by testing with the reference substance - potassium dichromate.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Ca+Mg ions 2.5-2.53 mmol.L-1

Test temperature:

22±2°C

pH:

7.8±0.2

Dissolved oxygen:

air saturation value at 22°C is 8.74

Nominal and measured concentrations:

100, 50, 10, 5, 1 mg/l

Details on test conditions:

TEST SYSTEM - Test vessel: - Type (delete if not applicable): open - Material, size, headspace, fill volume: - Aeration: no - Renewal rate of test solution (frequency/flow rate): 24h - No. of organisms per vessel: 7 fishes for each concetration - No. of vessels per concentration (replicates): 1 - No. of vessels per control (replicates): 1 - No. of vessels per vehicle control (replicates): 1 - Biomass loading rate: 1g/l Standard 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 7.7 – 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-1, values measured in this study were 2.50 – 2.53 mmol.L-1 in the preliminary semi-static test. OTHER TEST CONDITIONS - Light intensity: natural variation of daily light and dark EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish mortality and ambient temperature was observed and recorded each 24 hours

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 79.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 79.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 78.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

The 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, carried out in period from 11.04. to 15.04. 2016, are the following:48 hour – LC50 = 178 mg•L-1 (95% confidence limit: 163 – 240 mg.L-1)96 hour – LC50 = 152 mg•L-1 (95% confidence limit: 139 – 168 mg.L-1)

Sublethal observations / clinical signs:

No mortality was observed in the preliminary semi-static test. The exact values of LC₅₀could not be calculated and the values of LC are given in the form of a range. The LC₁₀₀and LC₀values were 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.

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.

Table No.: 5-Calculation of the time-weighted mean concentration

Nominal conc. mg·L^-1	Time hours	Conc 0	Conc 1	ln (conc 0)	ln (conc 1)	Area	Total area	TW mean mg·L^-1
100	0-24	98.9	92.2	4.5941	4.5240	95.5108	319.0729	79.77
	24-48	97.0	55.4	4.5747	4.0146	74.2683
	48-72	97.7	55.3	4.5819	4.0128	74.4999
	72-96	98.7	55.1	4.5921	4.0091	74.7939
1	0-24	0.92	0.90	-0.0834	-0.1054	0.9100	3.5706	0.89
	24-48	1.00	0.87	0.0000	-0.1393	0.9335
	48-72	0.88	0.86	-0.1278	-0.1508	0.8700
	72-96	0.85	0.85	-0.1625	-0.1625	0.8571

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 7 fish, therethrough 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 Blue 78, 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 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 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^-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 the preliminary semi-static test, the study was terminated.The experiment with the concentration of 100 mg·L^-1included 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.

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 – LC₅₀>79.8 mg·L^-1        (time-weighted mean concentration)

48 hour – LC₅₀>79.8 mg·L^-1        (time-weighted mean concentration)

72 hour – LC₅₀>79.8 mg·L^-1        (time-weighted mean concentration)

96 hour – LC₅₀>79.8 mg·L^-1        (time-weighted mean concentration)

 

24 hour – LC₀=79.8 mg·L^-1         (time-weighted mean concentration)

48 hour – LC₀=79.8 mg·L^-1         (time-weighted mean concentration)

72 hour – LC₀=79.8 mg·L^-1         (time-weighted mean concentration)

96 hour – LC₀=79.8 mg·L^-1         (time-weighted mean concentration)

 

24 hour – LC₁₀₀>79.8 mg·L^-1       (time-weighted mean concentration)

48 hour – LC₁₀₀>79.8 mg·L^-1       (time-weighted mean concentration)

72 hour – LC₁₀₀>79.8 mg·L^-1       (time-weighted mean concentration)

96 hour – LL₁₀₀>79.8 mg·L^-1       (time-weighted mean concentration)

Description of key information

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.

Only one study is available. GLP study. Klimish score 1.

96 hour – LC₅₀>79.8 mg·L^-1        (time-weighted mean concentration)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

78.9 mg/L