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 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:

2017-04-06 to 2017-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
The preliminary test (non-GLP)
The preliminary test was performed using the following test item concentrations: 120, 12, 1.2, 0.12 mg/L plus the control. The test item in the amount of 61.2 mg was weighed in a glass crystallizer and quantitatively transferred into flask by multiple washing with the Elendt M7 medium and filled up to 510 mL [SOP/W/7]. The test item concentration of 120 mg/L was visually homogeneous and colored black,
however, sufficiently transparent for an observation of the test organisms. Lower test item concentrations were prepared by sequential dilutions with the Elendt M7 medium in a ratio 1:9 (v/v)
(i.e. 50 mL of higher test item concentration was mixed with 450 mL of the Elendt M7 medium). The color intensity was correlated with the test item concentration.

The definitive test
The definitive test was performed with a single test item concentration of 120 mg/L as a limit test plus the control. The limit test concentration of 120 mg/L was selected to ensure that the mean measured
concentration is above 100 mg/L even in case of slight acceptable deviations from the nominal value. The test item in the amount of 60.1 mg was weighed in a glass crystallizer and quantitatively
transferred into flask by multiple washing with the Elendt M7 medium and filled up to 500 mL [SOP/W/7], (Table 3). The test item concentration of 120 mg/L was visually homogeneous and colored
black, however, sufficiently transparent for an observation of the test organisms. The control was Elendt M7 medium 500 mL). From the test item concentration and the control 50 mL were taken as a
sample for chemical determinations at exposure initiation [SOP/W/83].

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Standard laboratory culture maintained in the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology [SOP/W/67]
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Daphnia magna was cultured in glass beakers of 150 mL volume (one parent per vessel) at room temperature 18 – 22o C with the light : dark ratio of 16 h : 8 h (electronic time programmer, KANLUX, Poland). The culture was maintained in Elendt M7 medium [SOP/W/63, SOP/W/67].
- Feeding during test: none

ACCLIMATION
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Pseudokirchneriella subcapitata algae suspension. Group B vitamins and micronutrients necessary for proper growth were supplied with lyophilized Spirulina sp. suspension.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

18.9-20.7

pH:

7.64-7.75

Dissolved oxygen:

8.9-9.0

Conductivity:

521 µS/cm

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): covered with transparent lids in order to minimize evaporation and to prevent accidental contamination
- Material, size, headspace, fill volume: 150 mL
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16:8 light:dark

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 120, 12, 1.2, 0.12 mg/L plus the control.
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: nominal concentrations verified analytically

Validity criteria fulfilled:

yes

Description of key information

Immobilization of Daphnia magna exposed to the test item, Direct Black 18L NA active dye, was investigated during a 48 -hour static test. The definitive test was performed with a single test item concentration of 120 mg/L as limit test. Four replicates were used for the test item concentration and the control, each with five Daphnia magna. Daphnia magna were observed for immobilization after 24 and 48 h of exposure. During exposure, no immobilization of Daphnia magna was observed in the control and in the test item concentration of 120 mg/L. The test item concentration was stable under test conditions. The mean measured concentration in the limit test concentration was 106.8 mg test item/L and 90.6 mg main ingredient/L. The endpoint values were determined based on the nominal test item concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

120 mg/L