Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

06 April, 2016 ro 19 September, 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 21036/G TE
Physical state/Appearance: Blue solid
Batch: AT-0033945200
Purity: 64.5%
Expiry Date: 13 April 2020
Storage Conditions: Room temperature in the dark from 14 March 2016 to 15 March 2016, thereafter approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the 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:

yes

Remarks:

Culture medium

Details on test solutions:

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.

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:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 °C

pH:

6.5 ± 0.2

Nominal and measured concentrations:

Definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Material, size, headspace, fill volume: 500mL Glass flask.
- No. of colonies per vessel: 3 colonies per vessel
- No. of fronds per colony: 4 fronds per colony
- No of replicates (control and test): Triplicate

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: 1M HCl or NaOH.
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting
- Determination of biomass: dry weight

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no significant effect on growth at the test concentrations of 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.

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:

EC50

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:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Results with reference substance (positive control):

3,5-dichlorophenol concentration: 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
The EC50 was reported as follows;
Average specific growth rate (frond number): 3.4 mg/L
Average specific growth rate (dry weight): 3.0 mg/L
Yeild (frond number): 1.8 mg/L
Yeild (yeild): 1.4 mg/L

Reported statistics and error estimates:

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), were carried out on the average specific growth rate and yield data at 7 days for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

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 showed no significant effect on growth at the test concentrations of 1 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L. Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test. Chemical analysis of the 10 and 100 mg/L test preparations on Days 2, 5 and 7 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 be near nominal with the exception of the 6.25 mg/L test sample on Day 7 where a measured concentration of 66 % of nominal was obtained. Given that this concentration was below the No Observed Effect Concentration (NOEC) this was considered to have had no adverse effect on the outcome of the test and as such 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 1.61 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: 199

Growth Data Based on Frond Number

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

Average Specific Growth Rate

ErC10 (frond number) = 36 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%.

Yield

EyC10 (frond number) = 8.7 mg/L

EyC20 (frond number) = 17 mg/L

EyC50 (frond number) = >100mg/L

Where:

EyCx = the test concentration that reduced yield by x%.

Growth Data Based on Dry Weight

The dry weight of Lemna minor in each flask in the definitive test was determined.  Average specific growth rates, yield and percentage inhibition values were 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) = 30 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%.

Yield

EyC10 (dry weight) = 7.9 mg/L

EyC20 (dry weight) = 28 mg/L

EyC50 (dry weight) = >100 mg/L

Where:

EyCx = the test concentration that reduced yield by x%.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for Lemna minor test was found to be greater than 100 mg/L based on Average Specific Growth Rate (frond number). The NOEC was reported to be 50 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 was the one 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 concentrations of 6.25, 12.5, 25, 50 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. Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to be near nominal with the exception of the 6.25 mg/L test sample on Day 7 where a measured concentration of 66 % of nominal was obtained. Given that this concentration was below the No Observed Effect Concentration (NOEC) this was considered to have had no adverse effect on the outcome of the test and as such it was considered appropriate to calculate the results based on nominal test concentrations only. Exposure of Lemna minor to the test item gave the following results based on the nominal test concentrations:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	>100	50	100
	Dry Weight	>100	50	100
Yield	Frond Number	>100	50	100
	Dry Weight	>100	100	NA

Description of key 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 concentrations of 6.25, 12.5, 25, 50 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. Based on the findings of the study, following results were obtained:

EC50: >100 mg/L (Average specific growth rate - frond number and dry weight)

EC50: >100 mg/L (Yield: Frond Number, dry weight)

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

50 mg/L