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

This study was conducted between 17 August 2016 and 23 November 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)

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 were taken for Quantitative analysis from the control and each test group from the freshly prepared bulk test preparation on days 0, 3 and 5, and from pooled old or expired test preparationis on Days 3, 5 and 7. All samples were stored frozen prior to anlaysis. Duplicate samples were taken at each occasion and stored for further analysis if necessary.

Vehicle:

yes

Remarks:

Culture 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 concentra tions 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 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) ere each separately dissolved in culture medium and the volume adjusted to 1liters to give 100 and 32 mg/mL test concentrations 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..

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 i llumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

Not reported

Test temperature:

24 ± 1”C

pH:

6.0-7.7

Dissolved oxygen:

Not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Chemical analysis of the freshly prepared test samples on Days 0, 3 and 5 and of the old or expired test smaples onDays 3, 5 and 7 showed measured concentrations to range from 85% to 106% of nominal and hence it was considered appropriate to calculate the results based on nomknal concentrations only.

Details on test conditions:

Experimental Design and Study Conduct

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 conce ntrations 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 were prepared for each con trol 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 hom ogeneity.
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”C 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 results of the renge-finding test the following test concnetrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/mL


Experimental Preparation
A nominal amount of test item (100 and 32 mg) was dissolved in culture medium and the volume adjusted to 1 li ter to give the 100 and 32 mg/L test concentrations 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 Days 0, 3 and 5 (fresh media) and Days 3, 5 and 7 (old media).

Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of sol ution 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 10 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.
On days 2 and 5 the test solutions were renewed.

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.:

ca. 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.:

ca. 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.:

ca. 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.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-Finding Test
The results showed a flat response in terms of inhibition of growth rate at all test concentrations employed.
Based on this informations test concentrations of 1.0, 3.2, 10, 32 and 100 mg/mL were selected for the definitive test.
Chemical analysis of the freshly prepared test solutions on Day 2 showed measured test concentrations to range from 88% to 102% of nominal. A decline in measured concentrations was observed in the 1.0 mg/L test preparations on Days 3 and 7 indicating that the test item was possibly unstable over the test duration. As such it was considered appropriate to conduct the definitive test using a semi-static testing regime.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared test samples on Days 0, 3 and 5 and of the old or expired test samples on Days 3, 5 and 7 showed measured test concentrations to range from 85% to 106% of nominal and hence it was considered appropriate to calculate the results based on nominal test concentrations only.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.71 days in line with the O ECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at day 0 : 10
Mean frond number in control cultures at day 7 : 127

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 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, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations (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) = 5.1 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 all test concentrations as above. There were no statistically significant differences between the control, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations (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 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 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 all test concentrations as above. There were no statistically significant differences between the control, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations (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) = 16 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 all test concentrations as above There were no statistically significant differences between the control, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations (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 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.

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

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 Rate   Frond 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 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 test solutions were renewed on days 3 and 5.

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 freshly prepared test samples on Days 0, 3 and 5 and of the old or expired test samples on Day 3, 5 and 7 showed measured test concentrations to range from 85% to 106% of nominal and hence it was considered appropriate to calculate the results based on nominal test concentrations only.

Exposure of Lemna minor to the test item based on nominal test 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

EC50 (average specific growth rate based on frond number as well as dry weight) was >100 mg/L. Similarly, EC50 (yield based on frond number and dry weight) was also >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)”. 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 test solutions were renewed on days 3 and 5. 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. Chemical analysis of the freshly prepared test samples on Days 0, 3 and 5 and of the old or expired test samples on Day 3, 5 and 7 showed measured test concentrations to range from 85% to 106% of nominal and hence it was considered appropriate to calculate the results based on nominal test concentrations only. Exposure of Lemna minor to the test item based on nominal test concentrations gave the following results: EC50 and NOEC (growth rate/frond number) was >100 and 100 mg/L, respectively.