N-{[Benzyl(ethyl)amino]-[(chloro-cyano-nitrophenyl)substituted]phenyl}acetamide

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:

17 Jan 2020 to 8 May 2020

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)

Analytical monitoring:

yes

Details on sampling:

The test concentration along with the control and vehicle control was analyzed for the test item concentration at the beginning and at the end of the test. For analysis, single composite sample was drawn from prepared test concentration and from the control and vehicle control.

Vehicle:

yes

Details on test solutions:

Test item concentration of 100 mg/L (G3) was prepared by mixing 100 mg of test item and volume was made up to 1000 mL using test medium and acetone at 0.01 % v/v in a volumetric flask.

Test item was ground using 0.1 mL acetone and then little volume of test medium before making up to final volume. The prepared test item concentration was sonicated for 15 minutes.
For vehicle control (G2), 0.1 mL acetone was added and made up to 1 L using test medium in a volumetric flask.

The test was performed on a black, non-reflecting surface due to coloured nature of test medium as per the OECD Guidance Document.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna minor
- Source: Eurofins Agroscience Services Ecotox GmbH, Aquatic Toxicology, Eutinger Strasse 24, D-75223 Niefern Öschelbronn, Germany.

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: Swedish Standard (SIS) medium. Monocultures (culture with one plant species) of Lemna plants were maintained aseptically between 22 and 26 °C under a continuous illumination (of light intensity 6500 to 10000 Lux). Periodically (7 - 10 days), a number of young, light-green plants were sub-cultured using glass beakers containing fresh medium at the test facility.
- Any deformed or abnormal cells/plants observed: none

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

Range finding test: 23.2 to 24.7 degC
Definitive test: 23.2 to 24.6 degC

pH:

Range finding test: 6.69 to 8.05
Definitive test: 7.07 to 8.09

Nominal and measured concentrations:

Range finding test: 0.1, 1, 10, 50 and 100 mg/L.
Definitive test: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: Glass beaker.
- Type: closed
- Material, size, headspace, fill volume: Glass beaker of 250 mL capacity.
- Type of cover: Petri plate
- Aeration: yes
- Agitation: no
- Renewal rate of test solution: Not renewed.
- No. of colonies per vessel: Three
- No. of fronds per colony: Four
- No. of vessels per concentration (replicates): Two for range finding test, Six for definitive test.
- No. of vessels per control (replicates): Two for range finding test, Six for definitive test.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: Range finding test - 7090 to 7320, Definitive test - 7260 to 7400.

EFFECT PARAMETERS MEASURED:
- Determination of frond number: Manual counting
- Determination of biomass: Dry weight

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: No adverse effects observed in range finding tests at any concentration.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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:

NOEC

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:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Fronds were found to be normal in appearance in control, vehicle control and test item treated group at the end of the test. Test medium was clear in control and vehicle control and blackish grey colour with little settleable solids in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.

Results with reference substance (positive control):

The inhibition of growth rate for frond numbers was 0,0.8, 25.3, 36.7 and 100% and yield was 0, 2.1, 52.0, 66.1 and 100% for dry weight was 0, 0.5, 29.5. 43.7 and 100% and yield was 0, 1.1, 58.0, 73.5 and 100% at the tested coocentralions of 0.3, 1, 3, 9 and 27 rng/L, respectively as compared with the control.

Reported statistics and error estimates:

The statistical analysis of the growth rate and yield data was evaluated using licensed copies of SYSTAT Statistical package version 12.0. The growth rate and yield for frond number and dry weight was tested for normality (Shapiro-Wilk test) and homogeneity of variance before performing further analysis. The growth rate and yield for frond number and dry weight data were found to be normal and homogeneous and performed two sample t-test (for NOEC evaluation).

Solubility: Test item was tested for its solubility in acetone and test medium along with sonication and formed homogenous semi-transparent greyish black colour suspension with settable solids at 0.1 mg/mL (performed as a non-GLP test).

Analysis of Test Concentrations:

The test item was recoverable using the analytical method at nominal concentrations of 1.140 and 246.0 mg/L in the test medium. The stability test results concluded that the test item was found to be stable in the test medium at the test conditions up to 7 days at nominal concentrations of 1.140 and 246.0 mg/L.

Validity of the Test

The Lemna growth inhibition test fulfills the validity criteria of the OECD guideline 221: The factor of frond number increase, measured in the control between day 0 and day 7, was 13.4 corresponding to an average specific growth rate for frond number in the control of 0.371 per day and to a doubling time of 1.871 days (validity criterion: growth rate ≥ 0.275 per day, doubling time < 2.5 days).

The factor of frond number increase, measured in the vehicle control between day 0 and day 7, was 13.4 corresponding to an average specific growth rate for frond number in the control of 0.371 per day and to a doubling time of 1.868 days (validity criterion: growth rate ≥ 0.275 per day, doubling time < 2.5 days).

The results of active ingredient analysis of 100 mg/L showed that overall percent agreement was 112.86% at the start of test (day 0) and 116.99% at the end of test (day 7) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%). No measurable concentration of test item was recorded in the control and vehicle control samples.

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for growth rate and yield (for frond number and dry weight) on the basis of the nominal concentration of the test item was higher than 100 mg test item/L.

Executive summary:

The effect of FAT 41047/A TE was tested on the growth of freshwater vascular aquatic plant, Lemna minor. This test was conducted in accordance with OECD test guideline 221, in a GLP certified laboratory.

 

A range finding test was conducted at concentration of 0 (control), 1, 10, 50 and 100 mg/L. No toxicity was observed at maximum concentration.

Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with a control (each group had six replicates).

Lemna was exposed in static mode with no renewal of test medium. Frond observations were made on days 3, 5 and 7. Dry weight was determined at the start (day 0) and at the end of the test (day 7).

3,5-Dicholorphenol was used as a positive control. The test medium served as vehicle control. Analysis of test item concentration in the test samples was carried out using HPLC.

Fronds were found to be normal in appearance in control and test item treated group at the end of the test. Test medium was clear in control and vehicle control and blackish grey colour with little settleable solids in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.

The test item was recoverable using the analytical method at nominal concentrations of 1.140 and 246.0 mg/L in the test medium. The stability test results concluded that the test item was found to be stable in the test medium at the test conditions up to 7 days at nominal concentrations of 1.140 and 246.0 mg/L. The results of active ingredient analysis of 100 mg/L showed that overall percent agreement was 112.86% at the start of test (day 0) and 116.99% at the end of test (day 7) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of <20%). No measurable concentration of test item was recorded in the control and vehicle control samples.

Based on the findings of the study, the NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 100 mg/L.

 

The NOECr (growth rate of frond numbers) was found to be 1 mg/L in Positive control (3,5- Dicholorophenol).

Description of key information

NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 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

The effect of FAT 41047/A TE was tested on the growth of freshwater vascular aquatic plant,Lemna minor. This test was conducted in accordance with OECD test guideline 221, in a GLP certified laboratory.

 

A range finding test was conducted at concentration of 0 (control), 1, 10, 50 and 100 mg/L. No toxicity was observed at maximum concentration.

Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with a control (each group had six replicates).

Lemnawas exposed in static mode with no renewal of test medium. Frond observations were made on days 3, 5 and 7. Dry weight was determined at the start (day 0) and at the end of the test (day 7).

3,5-Dicholorphenol was used as a positive control. The test medium served as vehicle control. Analysis of test item concentration in the test samples was carried out using HPLC.

Fronds were found to be normal in appearance in control and test item treated group at the end of the test.Test medium was clear in control and vehicle control and blackish grey colour with little settleable solids in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.

Based on the findings of the study, the NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 100 mg/L.

 

The NOECr (growth rate of frond numbers) was found to be 1 mg/L in Positive control (3,5- Dicholorophenol).