Reaction mass of 5-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide and 3-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide

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:

Experiment start date - 28 July 2017; Experiment end date - 03 August 2017; Study completion date - 09 February 2018.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 31016/T
Physical state/Appearance: Dark green iridescent liquid
Batch: AT-0042774700
Purity: 80 %
Expiry Date: 07 September 2021
Storage Conditions: Room temperature in the dark
Test material description: Test material used in this study is the methyl sulfate salt of Basic Red 046.

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days.

Definitive Test
Verification of Test Concentrations
Samples were taken from the control and each test group from the freshly prepared bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days.
The test was conducted in glass conical flasks (500 mL) each containing 250 mL of test preparation. Two replicate flasks were prepared for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
Nominal amounts of test item (100 and 32 mg) were each separately dissolved in 1 liter of culture medium to give 100 and 32 mg/L test concentrations respectively from which a series of dilutions was made to give further test concentrations of 10, 3.2 and 1.0 mg/L. Each of the prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 (fresh media) and Day 7 (old media)

Test organisms (species):

Lemna minor

Details on test organisms:

The test was carried out using Lemna minor. A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 °C

pH:

6.5 - 7.2 °C

Nominal and measured concentrations:

Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 0.68 to 78 mg/L. A concentration dependent decline in measured test concentrations was observed on Day 7 (old media) in the range of 0.14 to 78 mg/L (21 % to 99 % of the Day 0 measured test concentrations).
Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:
Nominal Test Concentration (mg/L) - Geometric Mean - Measured Test Concentration (mg/L) Expressed as a Percentage of the Day 0 Measured Test Concentration
1.0 0.31 46
3.2 1.7 73
10 6.3 87
32 25 99
100 78 99

Details on test conditions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days. The test was conducted in glass conical flasks (500 mL) each containing 250 mL of test preparation. Two replicate flasks were prepared for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity. The control group was maintained under identical conditions but not exposed to the test item. At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for 7 days on a black non-reflective surface.
On Days 3 and 5 the test solutions were renewed, and observations on the test organisms were recorded on Days 0, 3, 5 and 7. In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 0 (fresh media) and Day 3 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
Nominal amounts of test item (100 and 32 mg) were each separately dissolved in 1 liter of culture medium to give 100 and 32 mg/L test concentrations respectively from which a series of dilutions was made to give further test concentrations of 10, 3.2 and 1.0 mg/L. Each of the prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 (fresh media) and Day 7 (old media).

Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Each control and test flask was inoculated with 3 colonies of Lemna minor (total 12 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) for 7 days on a black non-reflective surface. A static testing regime was employed.

Assessments
Test Organism Observations
The number of fronds present in each test and control culture was recorded on days 0, 2, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.
In addition the dry weight of the fronds in each control and treatment group was determined on day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C to constant weight.

Water Quality Criteria
The pH of each control and test flask was recorded on Day 0 and Day 7. The temperature and light intensity in the incubator were recorded daily.

Verification of Test Concentrations
Samples were taken from the control and each test group from the freshly prepared bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 5.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding Test
The results showed no significant effect on growth at the test concentration of 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test. Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 3.2 to 336 mg/L. A slight decline in measured test concentrations was observed in the aged test preparations on Day 3 in the range of 1.9 to 313 mg/L and on Day 7 in the range of 2.4 to 309 mg/L. Examination of the data could find no cause for the higher than expected test concentrations as the correct quantities of test item had been shown to have been weighed out.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 0.68 to 78 mg/L. A concentration dependent decline in measured test concentrations was observed on Day 7 (old media) in the range of 0.14 to 78 mg/L (21 % to 99 % of the Day 0 measured test concentrations). Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (mg/L) Geometric Mean - Measured Test Concentration (mg/L) Expressed as a Percentage of the Day 0 - Measured Test Concentration
1.0 0.31 46
3.2 1.7 73
10 6.3 87
32 25 99
100 78 99

Validation Criteria
The following data show that the doubling time of the control cultures was 2.00 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at day 0: 12
Mean frond number in control cultures at day 7: 80

Growth Data Based on Frond Number

Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the frond number data:

Average Specific Growth Rate
ErC10 (frond number) = 4.7 mg/L
ErC20 (frond number) = 8.9 mg/L
ErC50 (frond number) = 27 mg/L; 95 % confidence limits 18 to 38 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x %.
Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 0.31 and 1.7 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 1.7 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 6.3 mg/L.

Yield
EyC10 (frond number) = 1.2 mg/L
EyC20 (frond number) = 2.8 mg/L
EyC50 (frond number) = 11 mg/L; 95 % confidence limits 8.0 to 15 mg/L
Where:
EyCx = the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out for the control and all test concentrations as above. There were no statistically significant differences between the control, 0.31 and 1.7 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 1.7 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 6.3 mg/L.

Growth Data Based on Dry Weight
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:
Average Specific Growth Rate
ErC10 (dry weight) = 1.7 mg/L
ErC20 (dry weight) = 3.9 mg/L
ErC50 (dry weight) = 16 mg/L; 95 % confidence limits 12 to 21 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x %.
Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations as above. There were no statistically significant differences between the control, 0.31 and 1.7 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 1.7 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 6.3 mg/L.

Yield
EyC10 (dry weight) = 0.64 mg/L
EyC20 (dry weight) = 1.4L
EyC50 (dry weight) =5.2 mg/L; 95 % confidence limits 4.0 to 6.7 mg/L
Where:
EyCx = the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out for the control and all test concentrations as above. There were no statistically significant differences between the control, 0.31 and 1.7 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 1.7 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 6.3 mg/L

Observations
All test and control cultures were inspected on days 0, 2, 5 and 7. At the start of the test, and on Days 2 and 5 all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from light pink solutions at 0.31 mg/L through to very dark red/pink solutions at 78 mg/L. On Day 7 all control cultures were observed to be clear colorless solutions with some algal growth present. The test cultures were observed to range from light pink solutions with some algal growth evident at 0.31 mg/L through to very dark red/pink solutions at 78 mg/L.

Water Quality Criteria
The pH values of each test and control flask were in the range 6.5 - 7.2. Temperature was maintained at 24 ± 1 ºC throughout the test.

Results with reference substance (positive control):

A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Lemna minor to the reference item gave the following results:
Response Variable Measurement Variable EC50 (mg/L) 95% Confidence Limits No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate Frond Number 3.4 3.1-3.8 0.625 1.25
Dry Weight 3.0 2.7-3.2 0.625 1.25
Yield Frond Number 1.8 1.6-2.2 0.625 1.25
Dry Weight 1.4 1.2-1.7 0.625 1.25
The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Table 1            Frond Numbers and Percentage Inhibition of Growth from the Range-finding Test

 

Nominal Concentration (mg/L)		Number of Fronds				Average Specific Growth Rate		Yield
		Day 0	Day 3	Day 5	Day 7	(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	R1	9	16	23	46	0.233	-	37	-
	R2	9	19	24	44	0.227		35
	Mean	9	18	24	45	0.230		36
1.0	R1	9	21	25	60	0.271	[12]	51	[28]
	R2	9	20	24	49	0.242		40
	Mean	9	21	25	55	0.257		46
10	R1	9	14	18	26	0.152	38	17	56
	R2	9	12	15	23	0.134		14
	Mean	9	13	17	25	0.143		16
100	R1	9	9	10	10	0.015	90	1	94
	R2	9	10	11	11	0.029		2
	Mean	9	10	11	11	0.022		2

R₁– R₂= Replicates 1 and 2

[Increase in growth compared to controls]

- = Not applicable

 

Table 2            Frond Numbers from the Definitive Test

 

Geometric Mean Measured Test Concentration (mg/L)		Number of Fronds
		Day 0	Day 2	Day 5	Day 7
Control	R1	12	23	51	89
	R2	12	21	49	84
	R3	12	21	40	67
	Mean	12	22	47	80
0.31	R1	12	21	49	78
	R2	12	22	40	88
	R3	12	18	53	62
	Mean	12	20	47	76
1.7	R1	12	21	44	66
	R2	12	18	46	74
	R3	12	19	40	67
	Mean	12	19	43	69
6.3	R1	12	21	39	59
	R2	12	21	43	59
	R3	12	18	34	51
	Mean	12	20	39	56
25	R1	12	18	21	38
	R2	12	19	24	34
	R3	12	21	29	35
	Mean	12	19	25	36
78	R1	12	16	18	18
	R2	12	14	15	15
	R3	12	15	15	15
	Mean	12	15	16	16

R₁– R₃= Replicates 1 to 3

 

Table 3            Inhibition of Average Specific Growth Rate and Yield Based on Frond Numbers

Geometric Mean Measured Test Concentration (mg/L)		Average Specific Growth Rate		Yield
		(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	Mean	0.270	-	68	-
	SD	0.021		12
0.31	Mean	0.262	3	64	6
	SD	0.025		13
1.7	Mean	0.250	7	57	16
	SD	0.009		4
6.3	Mean	0.221	18	44	35
	SD	0.012		5
25	Mean	0.156	42	24	65
	SD	0.008		2
78	Mean	0.041	85	4	94
	SD	0.015		2

SD= Standard Deviation

- = Not applicable

 

Table 4            Dry Weights from the Definitive Test

 

Geometric Mean Measured Test Concentration (mg/L)		Dry Weight (mg)
		Day 0*	Day 7
Control	R1		9.0
	R2		6.7
	R3		5.7
	Mean	0.78	7.1
0.31	R1		6.1
	R2		7.6
	R3		9.4
	Mean	0.78	7.7
1.7	R1		5.1
	R2		6.5
	R3		4.9
	Mean	0.78	5.5
6.3	R1		3.2
	R2		4.2
	R3		3.0
	Mean	0.78	3.5
25	R1		1.6
	R2		2.4
	R3		2.4
	Mean	0.78	2.1
78	R1		1.5
	R2		0.9
	R3		0.9
	Mean	0.78	1.1

*Mean value determined from 6 replicate weighings

R₁– R₃= Replicates 1 to 3

 

Table 5            Inhibition of Average Specific Growth Rate and Yield Based on Dry Weight

Geometric Mean Measured Test Concentration (mg /L)		Average Specific Growth Rate		Yield
		(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control	Mean	0.313	-	6.4	-
	SD	0.033		1.7
0.31	Mean	0.325	[4]	6.9	[8]
	SD	0.031		1.7
1.7	Mean	0.278	11	4.7	27
	SD	0.022		0.9
6.3	Mean	0.212	32	2.7	58
	SD	0.026		0.6
25	Mean	0.142	55	1.4	78
	SD	0.033		0.5
78	Mean	0.044	86	0.3	95
	SD	0.042		0.3

SD– Standard Deviation

[Increase in growth compared to controls

* = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and based on the geometric mean measured test concentration gave the following results.
Response Variable Measurement Variable EC50 (mg/L) 95% Confidence Limits No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate
Frond Number 27 18 - 38 1.7 6.3
Dry Weight 16 12 - 21 1.7 6.3

Yield
Frond Number 11 8.0 - 15 1.7 6.3
Dry Weight 5.2 4.0 - 6.7 1.7 6.3

Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp.Growth Inhibition Test (March 2006)”.

Methods

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. 

The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 0.68 to 78 mg/L. A concentration dependent decline in measured test concentrations was observed on Day 7 (old media) in the range of 0.14 to 78 mg/L (21 % to 99 % of the Day 0 measured test concentrations). Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.31, 1.7, 6.3, 25 and 78 mg/L.

Exposure of Lemna minor to the test item based on the geometric mean measured test concentrations gave the following results:

Response Variable	Measurement Variable	EC50 (mg/L)	95% Confidence Limits			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	27	18	-	38	1.7	6.3
	Dry Weight	16	12	-	21	1.7	6.3
Yield	Frond Number	11	8.0	-	15	1.7	6.3
	Dry Weight	5.2	4.0	-	6.7	1.7	6.3

 

Description of key information

EC₅₀ (growth rate based on frond number) for toxicity to Lemna was determined to be 27 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

27 mg/L

Additional information

A study was performed to assess the effect of FAT 31016/T on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”. Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (geometric mean measured test concentrations were determined to be 0.31, 1.7, 6.3, 25 and 78 mg/L, respectively) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. Based on the findings of the study, EC₅₀ growth rate based on frond number observations was determined to be 27 mg/L. The test material used for this test was a different salt form of the dye, a methyl sulphate instead of a bromide was used. The two salt forms differ in water solubility, with the methyl sulfate variant showing higher solubility (cs=734 g/L) compared to the bromide variant (cs=7.9 g/L). Therefore the substance concentration of the free dye is higher for the methyl sulfate variant compared with the bromide variant representing a worst case approach. Regarding the counter ions it has been demonstrated for various dyes and different classes of dyes that the counter ion does not contribute to the aquatoxic properties of the dye. In any case the intrinsic properties of the dye molecule will dominate toxic effects to the aquatic system. Therefore, this approach is justified.