Reaction mass of 5,5'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[3-methylsalicylic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and potassium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxy-7-sulfonaphthalene-2-sulfonate - 2,2',2''-nitrilotriethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol

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:

2018-02-22 and 2018-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 50, 20, 10, 5, 2, 1 and 0.5 μg/mL
- Samples of each test item concentration and the control were collected at exposure initiation and at exposure termination.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The definitive test was performed with the test item concentration 120 mg/L plus the control.
- 480.3 mg of the test item were weighed into a glass crystallizer and quantitatively transferred into a glass aquarium using the test medium. After mixing, the resulting test item concentration 120 mg/L of a total volume 4 L in the aquarium was homogeneous, without visible non-dissolved particles and black-colored.
- The control was the test medium of a total volume 4 L in an aquarium. Seven fish were introduced into every aquarium. Every treatment was tested in one replicate (i.e. a total of 2 test vessels).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: Brachydanio rerio, syn. Danio rerio
- Source: Laboratory of Aquatic Toxicology at the Institute of Industrial Organic Chemistry Branch Pszczyna, initiated from the fish acquired from ‘The Culture of fish in Goczałkowice’
- Age at study initiation: approximately 3 months old
- Length at study initiation (average): 2.51 cm ± 0.13
- Weight at study initiation (average): 0.2 g ± 0.02
- Feeding during test: none

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: yes
- Type and amount of food: flake food and live feed (i.e. Daphnia magna and Artemia salina)
- Health during acclimation (any mortality observed): The mortality in the fish population during the quarantine period was 0.8% (one dead fish out of 125 in the batch). The fish were healthy, free from external parasites and visible deformations.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

233 mg/L CaCO3

Test temperature:

21.7 – 23.5 ºC

pH:

7.35 - 7.61

Dissolved oxygen:

94 – 99%

Salinity:

not applicable

Conductivity:

628 µS/cm

Nominal and measured concentrations:

nominal concentrations: 120 mg/L (101.8 mg/L nom conc of main ingredient)
measured concentrations: 97.2 mg/L (at exposure initiation) and 96.6 mg/L (at exposure termination)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Type: closed (Glass aquaria covered with glass lids in order to minimize evaporation and to prevent accidental loss of fish (due to jumping out) and accidental contamination.)
- Material, size, headspace, fill volume: 4 L
- Aeration: constant aeration
- No. of organisms per vessel: 7

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water according to OECD guideline

EFFECT PARAMETERS MEASURED:
- pH values were measured at exposure initiation, at 24 h of exposure, at 48 h of exposure, at 72 h of exposure and at exposure termination.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 120.0 mg/L
- Results used to determine the conditions for the definitive study: The results of the observations for fish mortality and changes in behaviour, respiration and pigmentation were conducted repeatedly during the exposure after 3, 6, 24, 48, 72 and 96h. In the control and in the test item concentration 120 mg/L neither mortality of fish nor any symptoms of intoxication were observed. Therefore, a limit test was to be performed in the definitive test.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No mortality of fish and no other symptom was observed.

Description of key information

In a 96 hours acute toxicity study, approximately 3 months old zebrafish (Danio rerio) were exposed to the test item, Direct Black 18L NA active dye, in groups of seven animals per treatment in one replicate. The test was of static design. The test item concentration was prepared by mixing the proper amount of the test item with the test medium (i.e. reconstituted water). The resulting test item concentration 120 mg/L was visually homogeneous, colored and without visible non-dissolved particles. The control was test medium.

The content of the main ingredient in the test item concentrations was determined with a validated spectrophotometric method. Samples of the test item concentration 120 mg/L and the control collected at exposure initiation and at exposure termination were chemically analyzed. At exposure initiation the determined content of the main ingredient in the test item concentration was 97.2 mg/L, i.e. 115 mg test item/L and 95.5% of the nominal concentration. At exposure termination the determined content of the main ingredient in the test item concentration was 96.6 mg/L, i.e. 114 mg test item/L and 94.9% of the nominal concentration. Therefore, the test item concentration was stable under test conditions. At exposure termination in the control and in the test item concentration 120 mg/L no mortality of fish nor any symptoms of intoxication were observed.

Results:

The endpoint values:

LC50/96 h is higher than 120 mg/L

LOEC/96 h is higher than 120 mg/L

NOEC/96 h is higher than or equal to 120 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

120 mg/L