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

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:

2017-03-29 to 2017-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Working solutions of Direct Black 18L NA active dye at the concentrations of 50, 20, 10, 5, 2, 1, and 0.5 μg of the main ingredient/mL were analyzed spectrophotometrically and the absorbance was recorded. The standard curve (peak area versus quantity of the standard) is linear with coefficient of 0.99975. The range of linearity of the analytical graph is from 0.5 μg/mL to 50 μg/mL.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item in the amount of 2250.1 mg was weighed in a glass beaker and quantitatively transferred into a flask by multiple washing with the 20X AAP medium and filled up to 2250 mL [SOP/W/7]. The
test item concentration of 1000 mg/L was visually homogeneous and colored black. Lower test item concentrations were prepared by sequential dilutions with the 20X AAP medium in a ratio 1:2 (v/v), (Table 4). The control was the 20X AAP medium (2500 mL).

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba CPCC 310
- Source: Cultivation in a standard laboratory culture at the Institute of Industrial Organic Chemistry Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology. The culture of Lemna gibba on agar bevels was received from Canadian Physiological Culture Centre, Department of Biology, University of Waterloo, Ontario, Canada.
- Age of inoculum (at test initiation): The dry weight of the representative sample of the duckweed culture used as the inoculum.
- Method of cultivation: Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated in 22 - 26°C with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week.

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.2 24.5 °C

pH:

at exposure initiation: 77.27 – 7.58
at exposure termination: 8.32 – 8.95

Conductivity:

1559 μS/cm

Nominal and measured concentrations:

nominal concentrations: 1000, 333, 111, 37, 12.3, 4.12, 1.37, 0.46 mg/L plus the control
measured concentrations:
- According to guideline the separating factor should not exceeded 3.2, a separation factor of 3.0 was used.

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: beakers
- Material, fill volume: glass, 400 mL
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (20X AAP medium)

TEST MEDIUM / WATER PARAMETERS
- according to guideline (20X AAP medium)

OTHER TEST CONDITIONS
- Light intensity and quality: 7885 – 8108 lux

EFFECT PARAMETERS MEASURED:
- Determination of frond number: on days 2, 4 and at exposure termination
- Determination of biomass: dry weight at exposure termination

RANGE-FINDING STUDY
- Test concentrations: 0, 1.2, 12 and 120 mg/L
- Results used to determine the conditions for the definitive study: The range of the test item concentrations to be used in the definitive test was determined on the basis of the results of the preliminary test. In addition, in the preliminary test in the modified 20X AAP medium (control) changes in morphology were observed when compared to the normal development of colonies in the 20X AAP medium. Therefore, in the definitive test the 20X AAP medium was used as the test medium.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

101.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Remarks on result:

other: 95% CL: 75.04 – 135.54

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

7.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate (based on frond number)

Remarks on result:

other: 95% CL: 4.11 – 11.73

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

197.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Remarks on result:

other: 95% CL: 141.57 - 289.84

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

5.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Remarks on result:

other: 95% CL: 2.87 - 7.80

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield (based on frond number)

Remarks on result:

other: 95% CL: 0.31 - 1.41

Details on results:

The morphology of plants was observed in each test item concentration and the control on days 2, 4 and at exposure termination. The morphological effects were compared with appearance of colonies in the control. After 2 day of exposure, in the test item concentrations of 37, 12.3, 4.12, 1.37, 0.46 mg/L, no distinctive changes from the development of plants in the control were observed. In the test item concentration of 111 mg/L slightly bending down of fronds were observed. In the test item concentrations of 1000, 333 mg/L slightly bending down of fronds and colored fronds were observed.
After 4 day of exposure, in the test item concentrations of 12.3, 4.12, 1.37, 0.46 mg/L, no distinctive changes from the development of plants in the control were observed. In the test item concentration of 37 mg/L shorter roots were observed. In the test item concentration of 111 mg/L slightly bending down of fronds, colored fronds and shorter roots were observed. In the test item concentrations of 1000, 333 mg/L slightly bending down of fronds, shorter roots, colored fronds and roots were observed.
At exposure termination, in the test item concentrations of 12.3, 4.12, 1.37, 0.46 mg/L, no distinctive changes from the development of plants in the control were observed. In the test item concentration of 37 mg/L shorter roots were observed. In the test item concentrations of 1000, 333, 111 mg/L slightly bending down of fronds, shorter roots, colored fronds and roots were observed.

Results with reference substance (positive control):

- Relevant effect levels: ErC50 = 16.117 mg/L, ErC20 = 11.294 mg/L, ErC10 = 9.378 mg/L, EyC50 = 12.250 mg/L, EyC20 = 8.552 mg/L, EyC10 = 7.087 mg/L based on frond number (at exposure termination)

Results of observations on day 7, definitive test

 

Nominal test item concentration [mg/L]	Observations
Control	normal size, color, shape of fronds, normal appearance of roots
0.46	no changes
1.37	no changes
4.12	no changes
12.3	no changes
37	shorter roots
111	slightly bending down of fronds, shorter roots, colored fronds and roots
333	slightly bending down of fronds, shorter roots, colored fronds and roots
1000	slightly bending down of fronds, shorter roots, colored fronds and roots

Validity criteria

In the definitive test, the following validity criteria specified in the OECD Guideline No. 221 (2006) were met:

-  The doubling time of frond number in the control was 2.3 days, criterion: less than 2.5 days (the factor of frond number in the control between 0 and 7 day was8.5).

-  The average specific growth rate in the control between day 0 and day 7 was 0.305 d^-1(minimum requirement: higher than 0.275 d^-1).

Validity criteria fulfilled:

yes

Description of key information

The growth of Lemna gibba exposed to the test item, Direct Black 18L NA active dye, was investigated in a 7 day static test. The definitive test was performed using eight test item concentrations: 1000,

333, 111, 37, 12.3, 4.12, 1.37, 0.46 mg/L plus the control. The test item concentrations were split up into three replicates, the control was split up into six replicates. At exposure termination, in the test item concentrations of 12.3, 4.12, 1.37, 0.46 mg/L, no distinctive changes from the development of plants in the control were observed. The endpoint values were determined based on the nominal test item concentrations.

Results

Endpoint values of growth rate based on nominal test item concentrations, definitive test

 

Endpoint value [mg/L]	Frond number			Dry weight
	0-2 d	0-4 d	0-7 d	0-7 d
ErC10	5.55 (0.84 – 15.09)	27.55 (10.95 – 49.45)	47.83 (26.16 – 73.41)	7.40 (4.11 – 11.73)
ErC20	40.22 (14.67 – 78.19)	152.19 (95.04 – 225.77)	301.69 (218.90 – 419.88)	101.42 (75.04 – 135.54)
ErC50	> 1000	> 1000	> 1000	> 1000
LOEC	111	37	37	12.3
NOEC	37	12.3	12.3	4.12

Calculations according to [4], [SOP/W/68] ( - ) - 95% confidence limits

 

Endpoint values of yield based on nominal test item concentrations, definitive test

 

Endpoint value [mg/L]	Frond number			Dry weight
	0-2 d	0-4 d	0-7 d	0-7 d
EyC10	1.93 (0.26 – 5.82)	8.11 (1.86 – 18.78)	7.73 (2.85 – 14.99)	0.74 (0.31 – 1.41)
EyC20	13.86 (4.20 – 29.15)	37.12 (15.21 – 66.02)	37.96 (20.78 – 59.09)	5.02 (2.87 – 7.80)
EyC50	604.70 (292.19 – 1912.28)	680.94 (388.42 – 1600.34)	797.19 (506.36 – 1490.18)	197.41 (141.57 – 289.84)
LOEC	4.12	37	37	4.12
NOEC	1.37	12.3	12.3	1.37

Calculations according to [4], [SOP/W/68] ( - ) - 95% confidence limits

Key value for chemical safety assessment

EC50 for freshwater plants:

1 000 mg/L