Reaction mass of Trisodium 4-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate and Trisodium 4-({4-chloro-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonate

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 - 21 September 2016; Experiment completion date - 16 October 2016; Study completion date - 14 December 2016.

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:

March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40000/I TE
Physical state/Appearance: Yellow-orange powder
Batch: MC-13
Purity: 84.1%
Expiry Date: 27 August 2019
Storage Conditions: Stored frozen in the dark

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
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 2 (fresh media) and Day 5 (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
Samples of the control and 100 mg/L test group were taken from the bulk test preparation on Day 0 (fresh media) and from the pooled replicates (R1-R6) on Day 7 (old media) for quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

Test Medium

Details on test solutions:

Range-finding Test
The test concentration to be used in the definitive test was 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). Two replicate flasks each containing 250 mL 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 result of the range-finding test a “limit test” was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD Test Guidelines, no effect on growth was observed

Test organisms (species):

Lemna minor

Details on test organisms:

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:

yes

Total exposure duration:

7 d

Test temperature:

24 ± 1 ºC

pH:

6.5 - 7.6

Nominal and measured concentrations:

Chemical analysis of the 100 mg/L test preparations on Day 2 (fresh media) and Days 5 and 7 (old media) showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Test
The test concentration to be used in the definitive test was 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). Two replicate flasks each containing 250 mL 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) on a black non-reflective surface for 7 days.
On Days 2 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 2, 5 and 7.

Definitive Test
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD Test Guidelines, no effect on growth was observed.

Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give the required test concentration of 100 mg/L.
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 and Day 7.

Exposure Conditions
As in the range-finding test 500 mL glass conical flasks were used. Six flasks each containing 250 mL of solution were prepared for the control and 100 mg/L 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 11 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) on a black non-reflective surface for 7 days.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-Finding Test
The frond counts and percentage inhibition of growth values from the exposure of Lemna minor to the test item during the range-finding test were determined.
The results showed no significant effect on growth at the test concentrations of 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a “limit test” to confirm that at the maximum test concentration given in the OECD Test Guidelines, no effect on growth was observed.
Chemical analysis of the 100 mg/L test preparations on Day 2 (fresh media) and Days 5 and 7 (old media) showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations only.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.73 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: 11
Mean frond number in control cultures at Day 7: 131

Growth Data Based on Frond Number
Numbers of fronds in each flask in the definitive test were determined and the average specific growth rates, yields and percentage inhibition values calculated.
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) =>100 mg/L
ErC20 (frond number) =>100 mg/L
ErC50 (frond number) =>100 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 100 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences between the control and 100 mg/L test concentration (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 100 mg/L.

Yield
EyC10 (frond number) =>100 mg/L
EyC20 (frond number) =>100 mg/L
EyC50 (frond number) =>100 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 100 mg/L test concentration as above. There were no statistically significant differences between the control and 100 mg/L test concentration (P≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 mg/L.

Growth Data Based on Dry Weight
The dry weight of Lemna minor in each flask in the definitive test was determined and average specific growth rates, yield and percentage inhibition values calculated. 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) =>100 mg/L
ErC20 (dry weight) =>100 mg/L ErC50 (dry weight) = >100 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 100 mg/L test concentration as above. There were no statistically significant decreases in dry weight between the control and 100 mg/L test concentration (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 100 mg/L.

Yield
EyC10 (dry weight) =>100 mg/L
EyC20 (dry weight) =>100 mg/L
EyC50 (dry weight) =>100 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 100 mg/L test concentration as above. There were no statistically significant decreases in dry weight between the control and 100 mg/L test concentration (P≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100 mg/L.

Results with reference substance (positive control):

See "Any other information on results incl. tables"

Results:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>100	100
	Dry Weight	>100	100
Yield	Frond Number	>100	100
	Dry Weight	>100	100

Positive control results

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.

Validity criteria fulfilled:

yes

Conclusions:

The average specific growth rate (frond number) EC50 was determined to be > 100 mg/L.

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 a nominal concentration of 100 mg/L (six replicate flasks 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, 3, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations only. Exposure of Lemna minor to the test item based on nominal concentrations gave the following results:

Response Variable	Measurement Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>100	100
	Dry Weight	>100	100
Yield	Frond Number	>100	100
	Dry Weight	>100	100

Description of key information

The 7-day EC50 and NOEC (using growth rate based on frond number) were >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

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 a nominal concentration of 100 mg/L (six replicate flasks 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, 3, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 97% to 105% of nominal and so the results are based on nominal test concentrations only. Exposure of Lemna minor to the test item based on nominal concentrations gave the following results:

Response Variable	Measurement Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>100	100
	Dry Weight	>100	100
Yield	Frond Number	>100	100
	Dry Weight	>100	100