2-[[6-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]naphthalene-1,5-disulphonic acid, sodium salt

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

EC Number:

274-700-1

EC Name:

Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Cas Number:

70616-89-6

Molecular formula:

C24H18ClN7O10S3.3Na

IUPAC Name:

trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonato-2-naphthyl}diazenyl)naphthalene-1,5-disulfonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-Finding TestIn 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 TestThe concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7

Test solutions

Vehicle:

yes

Remarks:

Culture medium

Details on test solutions:

Range-finding TestThe 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 TestBased 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 PreparationA 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.

Test organisms

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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test conditions

Test temperature:

24 ± 1 ºC

pH:

7.1 - 9.9

Nominal and measured concentrations:

Range-Finding TestChemical analysis of the 100 mg/L test preparations on Days 5 and 7 (old media) showed measured test concentrations of 101 % and 99 % of nominal respectively indicating that the test item was stable under test conditions. Analysis of the freshly prepared test sample on Day 2 showed a measured test concentration of 48 % nominal. It was noted at the time of analysis that the color of the Day 2 test sample did not match that of the Day 5 and 7 samples suggesting that a possible mix up during sampling had occurred. This was considered to have had no adverse effect on the results obtained as the Day 7 results showed that the test item was stable over the test duration.Definitive TestVerification of Test ConcentrationsChemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 99% to 102% of nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

Experimental Design and Study ConductRange-finding TestThe 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. Data from the control group was shared with similar concurrent studies.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 TestBased 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 PreparationA 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.Exposure ConditionsAs 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

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 8

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 TestThe 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.Definitive TestVerification of Test ConcentrationsChemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to range from 99% to 102% of nominal and so the results are based on nominal test concentrations only.Validation CriteriaThe 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 : 130Growth Data Based on Frond NumberNumbers 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 RateErC10 (frond number) = >100 mg/L ErC20 (frond number) = >100 mg/L ErC50 (frond number) = >100 mg/LWhere: 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.YieldEyC10 (frond number) = >100 mg/L EyC20 (frond number) = >100 mg/L EyC50 (frond number) = >100 mg/LWhere: 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 WeightThe dry weight of Lemna minor in each flask in the definitive test was determined and the 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 RateErC10 (dry weight) = >100 mg/L ErC20 (dry weight) = >100 mg/LErC50 (dry weight) = >100 mg/LWhere: 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.YieldEyC10 (dry weight) = >100 mg/L EyC20 (dry weight) = >100 mg/L EyC50 (dry weight) = >100 mg/LWhere: 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):

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 EffectConcentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)Average Specific Growth Rate Frond Number 3.4 3.1-3.8 0.625 1.25Dry Weight 3.0 2.7-3.2 0.625 1.25Yield Frond Number 1.81.6-2.2 0.625 1.25Dry Weight 1.4 1.2-1.7 0.625 1.25The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Any other information on results incl. tables

The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and 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

Applicant's summary and conclusion

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 gave the following results: Response Variable  Measurement Variable  EC50 (mg/L)  No Observed Effect Concentration (NOEC) (mg/L) Average Specific Growth RateFrond Number >100 100  Dry Weight >100 100 Yield Frond Number >100 100 Dry Weight >100 100

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 99% to 102% 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