Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

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:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

119 mg test item/L were freshly prepared with dilution water preparation. The dispersion was agitated until the solution was visually clear.
Out of the stock solution 5 concentrations were tested in a test item solutions geometric series with a dilution factor of 2:
119 – 59.5 – 29.8 – 14.9 – 7.44 mg test item/L, corresponding to 100 – 50.0 – 25.0 – 12.5 – 6.25 mg a.s./L.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS, obtained from continuous laboratory cultures.
Reason for the selection: Daphnia magna is the preferred species in accordance with the of the test system test guideline and is bred at the test facility.
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, 16 hours illumination; light intensity of max. 1500 lx
Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), is used.
Feeding of the culture stocks: The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

261 mg [CaCO3/L]

Test temperature:

20.9°C

pH:

7.88

Dissolved oxygen:

8.72 [mg/L]

Conductivity:

718 [μS/cm]

Nominal and measured concentrations:

119 – 59.5 – 29.8 – 14.9 – 7.44 mg test item/L, corresponding to 100 – 50.0 – 25.0 – 12.5 – 6.25 mg a.s./L.

Details on test conditions:

Experimental Procedure
Test item: Acid Black 234
Stock solution, media preparation: 119 mg test item/L were freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear.
Preparation of the test item solutions: Out of the stock solution 5 concentrations were tested in a geometric series with a dilution factor of 2:
119 – 59.5 – 29.8 – 14.9 – 7.44 mg test item/L, corresponding to 100 – 50.0 – 25.0 – 12.5 – 6.25 mg a.s./L.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).
Control: Dilution water without test item incubated under the same conditions as the test groups
Reference Test: A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.
Test method: The study was performed under static conditions.
Test duration: 48 hours
Test vessels: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
Test volume: 20 mL

Dilution water: Same composition as the culture medium
Number of daphnids and replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration and the control.
Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
Acclimatization: Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.
Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.
Test temperature (target): 18 - 22 °C, constant within ± 1 °C
Illumination (target): Diffuse light, light intensity of max. 1500 lx
Photoperiod (target): 16/8 hours light/dark cycle
Feeding: The daphnids were not fed during the study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 58.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The concentrations of the test item were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure
(48 hours) in all concentration levels and in the control.
The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 95 to 106% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were in the range of 56 to 95% of the nominal values.
The measured test item concentrations were not within ± 20% of the nominal concentrations of the test item during the test period. Therefore, the effect concentrations are based on the geometric mean measured concentrations of the test item.

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item Acid Black 234, the 48-hour EC50 for Daphnia magna was 58.9 mg/L (95% confidence limits: 28.9 – 114 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Acid Black 234 were determined at the test facility according to OECD 202.

The study was conducted under static conditions over a period of 48 hours with five nominal concentrations of the test item Acid Black 234 prepared in a geometric series with a separation factor of 2 (nominal concentrations of 7.44 to 119 mg test item/L).

All tested concentration levels were visually clear and blue-black to light blue depending on the concentration level throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item Acid Black 234 were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in all concentration levels and in the control.

The measured concentrations of the test item at the start of the exposure (0 hours) were in the range of 95 to 106% of the nominal values. At the end of the exposure period (48 hours), the measured concentrations were within the range of 56 to 95% of the nominal values. The geometric mean measured concentrations are: 6.22 – 11.5 – 27.6 – 55.2 – 115 mg/L.

The measured test item concentrations were not within ± 20% of the nominal concentrations of the test item during the test period. Therefore, the effect concentrations are based on the geometric mean measured concentrations of the test item Acid Black 234.

Based on the geometric mean measured concentrations of the test item Acid Black 234, the 48-hour EC50 for Daphnia magna was 58.9 mg/L.

Description of key information

EC50 (48h): 58.9 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

58.9 mg/L

Additional information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Acid Black 234 were determined according to OECD 202.

Based on the geometric mean measured concentrations of the test item Acid Black 234, the 48-hour EC50 for Daphnia magna was 58.9 mg/L.

Moreover, a supporting test on similar substance 1 is available. The test shows no mortality at the tested concentration of 180 mg/L. Since the sample concentration is 83% of the nominal concentration, the EC50 has been set at 150 mg/L.