[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)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:

2003

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:

1984

Qualifier:

according to guideline

Guideline:

other: Real Decreto 363/1995 (BOE núm. 133 de Junio de 1995)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

Test concentrations: 1, 2.2, 4.8, 10, 22, 48, 100, 220, 480 and 1000 mg / L, plus one control (culture medium only)
Preparation of the stock and test solutions: A stock solution with a concentration of 1.022 g/L of the test material presented was prepared. Therefore, 0.1022 g of the test material was dissolved in 100 mL of culture medium in a volumetric flask, Class A of 100 mL by shaking, so we have a stock solution of 1022 mg/L. Due to the fact that the study material contains an average of 97.87% of active material, the stock solution has a concentration of 1000 mg/L of active ingredient. The different solutions used in the test have been prepared from this stock solution of 1000 mg L.
Actual concentration of the test stock solution: 1001.2 mg/L of active material.

Test organisms (species):

Daphnia magna

Details on test organisms:

Strain: Daphnia magna, Strauss (Cladocera, Crustacea)
Age: ≤ 24-hours life, in good physiological conditions
Origin: Stirling University, United Kingdom
Culture conditions to obtain the test organisms:
- Temperature: between 18 and 22 ºC
- Photoperiod of 16 hours of light / 8 hours of darkness
- No aeration
- Feeding: Type of food: Chlorella (freshwater microalgae) and marine algae extract
-Frequency of feeding: 3 times / week

The reference substance used to verify the response of the test organisms is potassium dichromate (K2Cr2O7). The result of the periodic verification of the response of the test organisms is EC50 24h = 0.7 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 ± 2 ºC

pH:

average value pH at the beginning of the test: 7.5 (interval 7.62-8.31)
average value pH at the end of the test: 7.54 (interval 7.64-7.72)

Dissolved oxygen:

average value O2 at the beginning of the test: 8.52 mg/L (interval 8.55-8.54 mg/L)
average value O2 at the end of the test: 8.34 mg/L (interval 8.31-8.32 mg/L)

Nominal and measured concentrations:

Nominal: 1 mg/L, 2,2 mg/L, 4,8 mg/L, 10 mg/L, 22 mg/L, 48 mg/L, 100 mg/L, 220 mg/L, 480 mg/L and 1000 mg/L
Measured: 1,01 mg/L, 2,22 mg/L, 4,83 mg/L, 10,07 mg/L, 22,15 mg/L, 48,35 mg/L, 100,7 mg/L, 221,5 mg/L, 483,4 mg/L and 1007 mg/L.

Details on test conditions:

Photoperiod: darkness
Test vessels: Test tubes (40 mL) capped with parafilm.
Test volume used: 10 mL
Number of test vessels per concentration: 4
Number of individuals per concentration: 20, divided into 5 individuals per test vessel
Age of individuals: less than 24 hours of life

Composition of culture medium OECD 202:
- Solution 1: CaCl 2*2H2O 11.76 g/L H2O
- Solution 2: MgSO4*7H2O 4.93 g/L H2O
- Solution 3: NaHCO3 2.59 g/L H2O
- Solution 4: KCl 0.23 g/L H2O
Add to 1000 mL of deionized water:
Solution 1: 25 mL
Solution 2: 25 mL
Solution 3: 25 mL
Solution 4: 25 mL

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

The number of individuals that show mobility are counted after 24 h and after 48 h and used to calculate the % of inhibition of mobility after 24h and after 48h.
At the maximum concentration tested, the test substance does not produce inhibition of mobility in the Daphnia magna species.
The validation criterion considered for this study is: - the percentage of immobilization observed in the control vessels is lower or equal to 10% - EC50-24h of potassium dichromate is between 0.6 mg/L and 1.7 mg/L.

Results with reference substance (positive control):

The result of the periodic verification of the response of the test organisms is EC50 24h = 0.7 mg/L.

Table 1: Number of Individuals showing mobility after 24 h

Concentration (mg/L)	Number of individuals showing mobility
	replicates				Total
	1	2	3	4
Control	5	5	5	5	20
1	5	5	5	5	20
2.2	5	5	5	5	20
4.8	5	5	5	5	20
10	5	5	5	5	20
22	5	5	5	5	20
48	5	5	5	5	20
100	5	5	5	5	20
220	5	5	5	5	20
480	5	5	5	4	19
1000	5	5	4	4	18

 

Table 2: Number of Individuals showing mobility after 48 h

Concentration (mg/L)	Number of individuals showing mobility
	replicates				Total
	1	2	3	4
Control	5	5	5	5	20
1	5	5	5	5	20
2.2	5	5	5	5	20
4.8	5	5	5	5	20
10	5	5	5	5	20
22	5	5	5	5	20
48	5	5	5	5	20
100	5	5	5	5	20
220	5	5	5	5	20
480	5	5	5	4	19
1000	5	5	4	4	18

 

Table 3: % inhibition of mobility

conc. (mg/L)	% inhibition of mobility (24h)	% inhibition of mobility (48h)
control	0	0
1	0	0
2.2	0	0
4.8	0	0
10	0	0
22	0	0
48	0	0
100	0	0
220	0	0
480	5	5
1000	10	10

Validity criteria fulfilled:

yes

Conclusions:

The result obtained in this study indicates that the substance has an EC50 (24h) higher than or equal to 1000 mg/L and an EC50 (48h) higher or equal to 1000 mg/L in Daphnia magna.

Executive summary:

The study of acute toxicity in Daphnia of the substance has been carried out. For the determination of the inhibition of mobility, the species of crustaceans Daphnia magna have been used and the OECD 202 (1984) and Royal Decree 363/1995 (BOE No. 133 of June 1995) operating methods have been followed.
The freshwater crustacean Daphnia magna has been exposed to the substance at test concentrations: 1, 2.2, 4.8, 10, 22, 48, 100, 220, 480 and 1000 mg / L of active ingredient. The total duration of the trial was 48 hours.
The result obtained in this study indicates that the substance has a value of EC50 (24h) greater than or equal to 1000 mg/L and an EC50 value (48h) greater than or equal to 1000 mg/L of active ingredient.

Description of key information

There is one study available to determine the short-term toxicity of the substance Direct Black RBK (CAS 2196165 -14 -5) to Daphnia according to the OECD Guideline 202. At the maximum concentration tested (EC50 (48h) >1000 mg/L act.ingr.) the substance is not harmfulf to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 1 000 mg/L