Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

Preparation of stock and test solutions: A stock solution has been prepared with a concentration of 1.0 g/L of the material presented (97.54% active matter). Therefore, 0.1000 g of the material has been 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 975.4 mg/L of active matter . The different solutions used in the test have been prepared from this stock solution of 975.4 mg/L of active material of the test product.

Concentrations tested (active matter): 0.98 mg/L, 3.13 mg/L, 9.78 mg/L, 31.3 mg/L, 97.8 mg/L, 312.9 mg/L and 977.7 mg/L
- Actual concentration of the test stock solution (active matter): 977.7 mg/L

Test organisms (species):

Lemna minor

Details on test organisms:

- Source : Federal Enviromental Agency (Berlín, Germany)

Growing conditions:
o Temperature: 24 ± 2ºC
o Intensity and quality of lighting: 6500 Lux

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24±2ºC

pH:

See pH value during the test (Table 1) in section "Any other information on materials and methods incl. tables"

Nominal and measured concentrations:

Concentrations tested: 1, 3,2, 10, 32, 100, 320 y 1000 mg/L
Concentrations tested (active matter): 0.98 mg/L, 3.13 mg/L, 9.78 mg/L, 31.3 mg/L, 97.8 mg/L, 312.9 mg/L and 977.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 ml beaker
- Material, size, headspace, fill volume: glass with 100 ml of test medium

GROWTH MEDIUM
- Standard medium used: yes
Test medium with the following composition:
Macroelements
— Solution 1: KNO3 17,5 g/L H2O; KH2PO4 4,5 g/L H2O; K2HPO4 0,63 g/L H2O
— Solution 2: MgSO4 7H2O 5 g/L H2O
— Solution 3: Ca(NO3)2 4H2O 14,75 g/L H2O

Microelements
— Solution 4: H3BO3 120mg/L H2O
— Solution 5: ZnSO4 7H2O 180 mg/L H2O
— Solution 6: Na2MoO4 2H2O 44,0 mg/L H2O
— Solution 7: MnCl2 4H2O 180,0 mg/L H2O
— Solution 8: FeCl3 6H2O 760,0 mg/L H2O
AEDT Na2 2H2O 1500,0 mg/L H2O

Add to 1000 mL of deionized sterile water:
Solution 1 20 mL
Solution 2 20 mL
Solution 3 20 mL
Solution 4 1 mL
Solution 5 1 mL
Solution 6 1 mL
Solution 7 1 mL
Solution 8 1 mL

- Number of replicates per trial: 3
- Number of test concentrations: 7 + control
- Geometric progression factor used: 3.2

ILLUMINATION:
Culture apparatus: Incubation chamber
Intensity and Quality of light: 6500 Lux
The surface where the test is performed receives uniform fluorescent lighting and continuous (white-cold type).

Reference substance (positive control):

yes

Remarks:

3,5-diclofenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

103 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

575 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The validation criterion considered for this study is:
- The average number of fronds in the control must present a growth rate specific medium of at least 0.275 per day.
- Duplication time of approximately 2.5 days, and multiplication by 7 of the number of fronds, at the end of the study
- The EC50 7d value of 3,5-diclofenol is in the range 2.2 - 3.8 mg/L.

Results with reference substance (positive control):

The reference substance used to verify the response of the test organisms is 3,5-diclofenol. The result of the periodic verification of the response of the test organisms is EC50 7d = 2.9 mg/L.

Growth rate inhibition: The results of the count of fronds at the beginning and at the end of the test are represented in the following table (table 2 of the study report)

Table 2: Number of fronds

Replicas Nº of fronds	control		Concentration (mg/L)
			0,98		3,13		9,78		31,3		97,8		312,9		977,7
	0d	7d	0d	7d	0d	7d	0d	7d	0d	7d	0d	7d	0d	7d	0d	7d
1	12	86	12	80	12	72	12	57	12	52	12	38	12	35	12	31
2	12	87	12	77	12	74	12	54	12	51	12	39	12	36	12	29
3	12	81	12	79	12	70	12	57	12	47	12	43	12	38	12	28
4	12	92	12		12		12		12		12		12		12
5	12	82	12		12		12		12		12		12		12
6	12	88	12		12		12		12		12		12		12
Average	12	86	12	78,7	12	72	12	56	12	50	12	40	12	36,3	12	29,3
Sd	0	4,0	0	1,5	0	2,0	0	1,7	0	2,6	0	2,6	0	1,5	0	1,5
CV	0	4,7	0	1,9	0	2,8	0	3,1	0	5,3	0	6,6	0	4,2	0	5,2

From the difference in the number of fronds at the beginning and at the end of the trial, the growth rates for the control and for each of the concentrations are calculated. The growth rates at 7 days are represented in the following table:

Table 3: Growth rate

Growth rate		Concentration (mg/L)
	control	0,98	3,13	9,78	31,3	97,8	312,9	977,7
	0.281	0.269	0.256	0.220	0.204	0.172	0.158	0.128

In the following table, the percentage of inhibition of the growth rate is represented:

Table 4: % Inhibition of growth rate

Concentration (mg/L)	% inhibition of growth rate
control	0
0,98	4,5
3,13	9,0
9,78	21,8
31,3	27,5
97,7	38,9
312,9	43,7
977,7	54,6

Yield inhibition. The weight of the fronds is determined at the beginning and at the end of the test. The initial weight is calculated from the average of 6 repetitions of weighing 12 fronds. The following table shows the weights obtained for each of the test concentrations.

Table 5: Biomass

Concentration (mg/L)	Initial biomass (0days) (mg)	Final biomass (7days)(mg)	Difference of biomass (Bf-Bi) (mg)
control	2,0	7,9	5,9
0,98	2,0	7,4	5,4
3,13	2,0	6,9	4,9
9,78	2,0	6,4	4,4
31,3	2,0	5,7	3,7
97,7	2,0	4,6	2,6
312,9	2,0	4,3	2,3
977,7	2,0	3,9	1,9

In the following table, the percentage of inhibition of biomass (yield) is represented.

Table 6:% Inhibition of the growth rate

Concentration (mg/L)	% inhibition of biomass growth (yield)
control	0
0,98	8,5
3,13	16,9
9,78	25,4
31,3	37,3
97,7	55,9
312,9	61
977,7	67,8

Validity criteria fulfilled:

yes

Conclusions:

The concentration of the test item that produces an inhibition of the 50% growth rate at 7 days (EC50 7d) in Lemna minor is 575 mg/L.
The concentration of the test item that produces an inhibition of Biomass of 50% at 7 days (EC50 7d) in Lemna minor is 103 mg/L.

Executive summary:

The study of acute toxicity to aquatic plants of Direct Black RBB dye was carried out. For the determination of the inhibition of algal growth, the freshwater aquatic plant Lemna minor has been used and the operative method according to the OECD Guideline 221 (2006) has been followed.

The freshwater aquatic plant Lemna minor has been exposed to the substance at the test concentrations 1; 3,2; 10; 32; 100; 320 and 1000 mg/L based on active material. The total duration of the trial has been 7 days.

The result obtained in this test indicates that the dye Direct Black RBB dye presents an inhibition value of the EC50 growth rate (7d) equal to 575 mg/L and an inhibition value of the biomass EC50 (7d) equal to 103 mg/L after 7 days of the test.

 

 

 

 

Description of key information

There is one study conducted on the substance Direct Black RBB (CAS 68877-33-8) according to the OECD Guideline 221 to determine the toxicity to aquatic plants Lemna minor. Based on the ECHA guidances, this test method is considered more suitable for testing toxicity of coloured test materials to aquatic plants, than the OECD guideline 201 to test the toxicity to aquatic algae.

The EC50 (7d) value 575 mg/L active ingr. obtained in this study, shows that the tested substance is not harmful to this type of aquatic plant.

Key value for chemical safety assessment

EC50 for freshwater plants:

575 mg/L