pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-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:

2020-11-17 - 2021-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, Method C.2. “Daphnia sp. Acute Immobilisation Test”

Version / remarks:

adopted 30. May 2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13. Apr. 2004: ”Daphnia sp., Acute Immobilisation Test“

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100.7 mg/L nominal concentration of the active substance (limit test).
- Sampling method: Before measurement, 500 μL of the taken samples from the blank control and the concentration 100 mg/L (a. s.) were diluted with 500 μL Acetonitrile (2-fold). 200 μL of the diluted sample of 100 mg/L (a. s.) was filled up to 10 mL with Acetonitrile/ demineralised water (50/50%) (100-fold).
- Sample storage conditions before analysis: the test item was stable in the daphnia immobilization test medium for 2 days (107.6% mean recovery).

Details on test solutions:

A test solution containing 100.7 mg/L nominal concentration of the test item (active substance (a. s.), corresponding to 124.9 mg/L of the test item (based on a purity of 80.6 % given by the sponsor)) in M4-medium was prepared and used for the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Berlin
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Source: Umweltbundesamt Berlin
- Feeding during test: green algae (Desmodesmus subspicatus)

Test type:

static

Water media type:

freshwater

Remarks:

M4-Medium (recipe of ELENDT)

Limit test:

yes

Total exposure duration:

48 h

pH:

The pH values were determined at the beginning and at the end of the test.
Blank control: start: pH 7.9; after 48 h: pH 7.5 //
100 mg/mL test solution: start: pH 7.9; after 48h: pH 7.6

Dissolved oxygen:

The concentration of dissolved oxygen were determined at the beginning and at the end of the test.
Blank control: start: 9.3 mg/L; after 48 h: 8.8 mg/L //
100 mg/mL test solution: start: 9.1 mg/mL; after 48h: 8.8 mg/L

Nominal and measured concentrations:

At the beginning and at the end of the test, the content of the test item in the test solution was determined using HPLC-UV-determination. The concentration determined at the start of the test was 97 % of the nominal concentration. At the end of the test the determined concentration was 108 % of the nominal concentration. Therefore, the determination of the biological results was based on the nominal concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size, headspace, fill volume: glass, nominal volume 50 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 vessels, each containing 20 ± 5 mL test solution
- No. of vessels per control (replicates): 4 vessels, each containing 20 ± 5 mL test solution

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours, using LED’s

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (202005R201) to assure that the test conditions are reliable.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Results with reference substance (positive control):

The 24h-EC50 value was determined as 2.01 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.

Immobility

Nominal Concentration in mg/L (a. s.)	Immobility 48 hours
	absolute				in %
Blank control	1	0	0	0	5
100	0	0	0	0	0

One of the animals was immobilised in the blank control, but this can be stated as not significant. None of the animals were immobilised in the tested concentration.

Validity criteria fulfilled:

yes

Conclusions:

The following results were determined for the test item Bayscript Black TP LXS 51134 (species:
Daphnia magna) and based on the active substance of the test item (purity of 80.6 %).
48h-NOEC ≥ 100 mg/L (a. s.)
48h-LOEC > 100 mg/L (a. s.)
24h-EC50 > 100 mg/L (a. s.)
48h-EC50 > 100 mg/L (a. s.)

Executive summary:

This study was performed as a limit test in order to evaluate the toxicity of the test item Bayscript Black TP LXS 51134 in an acute immobilisation test under static conditions towards Daphnia magna. Daphnia magna STRAUS, was chosen in the guideline as a typical species of zoo-plankton.

Young daphnia, aged less than 24 hours at the start of the test, were exposed to the test item for a period of 48 hours. Immobilisation was recorded at 48 hours and compared with control values. Not more than 10 % of the control daphnia should show immobilisation or other signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water. The results were analysed in order to determine the NOEC, LOEC and EC50 at 24 h and at 48 h and are given in a range.

A positive control was tested in a separate study to assure that the test conditions are reliable.

One valid experiment was performed. The study was performed as a limit test with the concentration of 100 mg/L (active substance).
For the test concentration and the blank control, 20 daphnia were exposed to the
test item for 48 hours in a static test system. After 48 hours, the immobilised daphnia were counted.

At the beginning and at the end of the test, the content of the test item in the test solution was determined using HPLC-UV-determination. The concentration determined at the start of the test was 97 % of the nominal concentration. At the end of the test the determined concentration was 108 % of the nominal concentration. Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations (see OECD 202 (§ 23)).

No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met. The result of the test is considered valid.

The following results were determined for the test item Bayscript Black TP LXS 51134 (species:
Daphnia magna) and based on the active substance of the test item (purity of 80.6 %).
48h-NOEC ≥ 100 mg/L (a. s.)
48h-LOEC > 100 mg/L (a. s.)
24h-EC50 > 100 mg/L (a. s.)
48h-EC50 > 100 mg/L (a. s.)

Description of key information

The following results were determined for the test item Bayscript Black TP LXS 51134 (species:
Daphnia magna) and based on the active substance of the test item (purity of 80.6 %).
48h-NOEC ≥ 100 mg/L (a. s.)
48h-LOEC > 100 mg/L (a. s.)
24h-EC50 > 100 mg/L (a. s.)
48h-EC50 > 100 mg/L (a. s.)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 100 mg/L