5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

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:

04 - 11 Apr 2014

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:

adopted 2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0 (solvent control), 0.0476, 0.152, 0.488, 1.56 and 5.00 mg a.i./L (day 0, day 3 (old and newe solutios), day 7)
- Sampling method: 10 mL samples were taken

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution of 50.0 g a.i./L prepared by diluting 0.5183 g test item into 10 mL dimethylformamide (DMF). 100 µL of the stock solution were transferred into 1 L distilled water to obtain the desired highest test concentration solution. Lower test concentration solutions were made via serial dilution (3.2x dilution factor) from the next higher test concentration. An additional 69 µL of DMF was added to these solutions. All vessels were brought to a volume of 1 L with filter sterilized 1 X AAP Media after adding the appropriate volume of the next higher test solution.
- Differential loading: no
- Controls: control: dilution water only (control) and dilution water containing 100µL DMF/L (solvent control)
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: stock solution: 100%, final test solutions: 100µL/L, control: 0%, solvent control: 100µL/L
- Evidence of undissolved material: Test and stock solution were clear and colorless with no precipitates

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: gibbous duckweed
- Strain: G3
- Source: Received from United States Department of Agriculture Fruit Laboratory, Beltsville, Maryland , USA on 03 Jun 2014
- Method of cultivation: Maintained under test conditions in an environmental chamber for at least 14 days immediately prior to experimental initiation. Culture renewed at least once each week. Light intensity: 5800 ± 15% lux, temperature: 25.0 ± 2.0 °C

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.7 - 25.7°C

pH:

7.5 - 8.6

Conductivity:

604 - 1756 µmos/cm

Nominal and measured concentrations:

Nominal: 0 (control), 0 (solvent control), 0.0476, 0.152, 0.488, 1.56 and 5.00 mg a.i./L
Mean measured: < LOQ (control), < LOQ (solvent control), 0.0390, 0.123, 0.408, 1.36, and 4.07 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: crystallization dishes
- Type (delete if not applicable): closed
- Material: borosilicate glass, size: 250 mL, headspace, fill volume: 100 mL
- Type of cover: sterile glass Petri dish lids
- Renewal rate of test solution: one test solution renewal on day 3
- No. of fronds per vessel: 12
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filter sterilized (0.20 µm filter) 20xAAP medium according to OECD 221
- Culture medium different from test medium: no
- Intervals of water quality measurement: ph and conductivity: day 0, 3 (old and new solutions) and 7 from all test levels, temperature: continuously measured

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 6540 - 6710 lux (mean: 6619 lux)

EFFECT PARAMETERS MEASURED:
- Determination of frond number: visual enumeration (day 0, 3, 5 and 7)
- Determination of biomass: dry weight (day 0 and 7, plants were dried at approximately 60°C for approximately 3 days, transferred to a desiccator to cool to ambient temperature, and then weighed)
- Other: phytotoxic effects (daily)

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0 (solvent control), 0.040, 0.20, 1.0 and 5.0 mg a.i./L
- Results used to determine the conditions for the definitive study: inhibition based on frond counts compared to controls: -2.6, 1.6, 0.0 and 31.2%

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

> 4.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: not determinable

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 1.92 - 2.59 mg a.i./L

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 0.991 - 2.76 mg a.i./L

Duration:

7 d

Dose descriptor:

IC50

Effect conc.:

> 4.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: not determinable

Duration:

7 d

Dose descriptor:

other: IC20

Effect conc.:

3.94 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 2.58 - n.a.

Duration:

7 d

Dose descriptor:

other: IC20

Effect conc.:

> 4.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: not determinable

Details on results:

- Any visual signs of phytotoxicity (abnormalities): Fronds appeared normal and healthy in the controls and in the four lowest test levels during the entire study.
- Other: On study days 3 through termination, fronds in the highest treatment level were curled.

Reported statistics and error estimates:

Raw or transformed data from treatment groups were compared to controls for normality and homogeneity of variance using the Shapiro-Wilks test and Bartletts's equality of variance test, respectively. If normality and homogeneity of variance were demonstrated for the raw or transformed values, then parametric analyses were conducted using analysis of variance (ANOVA) followed by Dunnett's test. If normality and/or homogeneity of variance were not demonstrated on raw or transformed values, nonparametric procedures were used. The IC10, IC20, and IC50 values, and the respective 95% confidence intervals, were calculated using linear interpolation.

Table 1: Mean Values for Frond Number Yield and Frond Growth Rates During the Exposure of Lemna gibba to the test item

Mean Measured Concentration (mg a.i./L)	Frond Number Yield	% Inhibition		Frond Number Growth Rate (fronds/hour)	% Inhibition
Control	179.3	NA		0.0165	NA
Solvent Control	171.0	NA		0.0162	NA
Pooled Controls	175.1	NA		0.0163	NA
0.0390	173.3	1.1		0.0163	0.37
0.123	159.8	8.8	*	0.0158	3.1	*
0.408	157.8	9.9	*	0.0158	3.5	*
1.36	145.8	16.8	*	0.0153	6.2	*
4.07	103.3	41.0	*	0.0135	17.7	*

*Statistically significant difference from control (Dunnett's one-tailed test; p ≤ 0.05).

% Inhibition=100-((Treatment group parameter mean / pooled control parameter mean)*100). Calculations were performed in Microsoft Excel on unrounded values. Manual calculations may vary slightly.

Table 2: Mean Values for Dry Weight Yield and Dry Weight Growth Rates During the Exposure of Lemna gibba to the test item 

Mean Measured Concentration (mg a.i./L)	Biomass Yield	% Inhibition		Biomass Growth Rate (grams/hour)	% Inhibition
Control	0.0227	NA		0.0168	NA
Solvent Control	0.0251	NA		0.0173	NA
Pooled Controls	0.0239	NA		0.0170	NA
0.0390	0.0224	6.2		0.0167	2.1
0.123	0.0243	-1.7		0.0171	-0.50
0.408	0.0277	-16.0		0.0179	-5.3
1.36	0.0203	14.9		0.0162	4.9
4.07	0.0128	46.5	*	0.0137	19.8	*

*Statistically significant difference from control (Dunnett's one-tailed test; p ≤ 0.05).

% Inhibition=100-((Treatment group parameter mean / pooled control parameter mean)*100). Calculations were performed in Microsoft Excel on unrounded values. Manual calculations may vary slightly.

Table 3: Measured Test Concentrations During the Exposure of Lemna gibba to the test item 

Nominal Concentration (mg a.i./L)	Day 0 Measured Concentration (mg a.i./L)	Day 0 % Nominal	Day 3 (old) Measured Concentration (mg a.i./L)	Day 3 (old) % Nominal	Day 3 (new) Measured Concentration (mg a.i./L)	Day 3 (new) % Nominal	Day 7 Measured Concentration (mg a.i./L)	Day 7 % Nominal	Mean Measured Concentration (mg a.i./L)	Percent Mean Measured Concentration
Control	< 0.005	---	< 0.005	---	< 0.005	---	< 0.005	---	< 0.005	---
Solvent Control	< 0.005	---	< 0.005	---	< 0.005	---	< 0.005	---	< 0.005	---
0.0476	0.0475	100%	0.0412	86%	0.0365	77%	0.0307	64%	0.0390	82%
0.152	0.152	100%	0.130	85%	0.115	76%	0.0965	64%	0.123	81%
0.488	0.499	102%	0.425	87%	0.386	79%	0.323	66%	0.408	84%
1.56	1.56	100%	1.35	86%	1.34	86%	1.17	75%	1.36	87%
5.00	4.98	100%	4.34	87%	3.81	76%	3.16	63%	4.07	81%

LOQ = 0.005 mg a.i./L

Mean measured values are based on unrounded numbers using Microsoft Excel. Manual calculations using rounded data may vary slightly.

Table 4: Validity criteria for OECD 221.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.	doubling time: 1.77 days or 42 hours	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

IC50 (7 d) = > 4.07 mg/L (mean measured, Lemna gibba, OECD 221)

NOEC (7 d) = 0.039 mg/L (mean measured, Lemna gibba, OECD 221)

Key value for chemical safety assessment

EC50 for freshwater plants:

4.07 mg/L

EC10 or NOEC for freshwater plants:

0.039 mg/L

Additional information

Toxicity to aquatic plants was investigated in a study performed according to OECD 221 with duckweed Lemna gibba G3 under static-renewal conditions.

L. gibba was exposed to the test item to nominal concentrations between 0.0476 and 5.0 mg a.i./L, a control and solvent control containing 100 µLN, N-dimethylformamide/L. Testing solutions were renewed once (after 3 days) during the test. Testing solutions were prepared up to the limit of solubility in the test system. Mean measured test item concentrations were between 81% and 87% of nominal concentrations.

Derived IC50 values based on growth rate inhibition (frond number and dry weight) was > 4.07 mg/L (mean measured). Growth rate NOEC values based on frond number and dry weight were 0.0390 mg/L (mean measured) and 1.36 mg/L (mean measured), respectively.