Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial plants: long-term

Remarks:

Seedling emergence

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Mar 2008 to 13 Jun 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants, Growth Test)

Version / remarks:

July 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4100 (Terrestrial Plant Toxicity Tier I (seedling emergence))

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Spray mixtures were sampled after their preparation to provide material for analytical confirmation of the test concentrations. Triplicate samples were collected from the test concentration, and a single sample was collected from the negative control on each day of preparation.

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of Spray Mixtures: The first spray mixture for the 150 g a.s./ha application rate was prepared by diluting 3.07 mL of the test substance to 500 mL with well water purified by reverse osmosis, and mixing. After two months, the second spray mixture for the 150 g a.s./ha application rate was prepared by diluting 1.23 mL of the test substance to 200 mL with well water purified by reverse osmosis, and mixing. Negative control spray mixtures consisted of well water purified by reverse osmosis.
- Application of Spray Mixtures: Application of the test substance was made with a Sprayer (spray booth). The booth was used to simulate inadvertent direct application or off-target drift resulting from actual field use. The test substance spray mixture was placed into a 500 mL polyethylene spray bottle that was pressurized to 1.38 bars. The spray bottle was connected to a moveable spray nozzle that was used to spray the plants in the test pots. Pots containing planted seeds were placed in the spray booth, which served as a protected area for spraying. The spray nozzle was suspended approximately 41 cm above the pots during delivery of the spray mixture. Applications first were made to the control group, followed by the treatment group. This procedure was used to avoid cross-contamination. Applications were made at a nominal spray volume of 200 L/ha. The spray coverage appeared uniform over the spray area.
- Sprayer calibration: Sprayer calibration was performed in the following manner. A clean, tared piece of absorbent paper (0.078 m2) was sprayed with water under application conditions. The amount of water collected on the paper was determined by weighing. This procedure was repeated three times on each day of application. The mean weight of water collected in the three trials was used to calculate the applied spray volume.

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: onion
- Family: Liliaceae

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: ryegrass
- Family: Poaceae

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: wheat
- Family: Poaceae

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: corn
- Family: Poaceae

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: sugarbeet
- Family: Chenopodiaceae

Species:

Brassica oleracea var. capitata

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: cabbage
- Family: Brassicaceae

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: soybean
- Family: Fabaceae

Species:

Lactuca sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: lettuce
- Family: Asteraceae

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: tomato
- Family: Solanaceae

Species:

Raphanus sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: radish
- Family: Brassicaceae

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Test temperature:

16.01 – 38.39 ˚C

pH:

7.2

Moisture:

10.23% – 90.50%

Details on test conditions:

TEST SYSTEM
- Test container: Plastic pots (approximately 16 cm in diameter and 12 cm deep)
- Amount of soil: 1940 cm3 soil volume
- Method of seeding: Soil was added to the pots and a template was used to gently compact the soil and leave ten uniform holes for planting. After planting the seeds, holes were closed by depressing the soil surface. T. aestivum, Z. mays, and G. max seeds were planted at an approximate depth of 20 mm, while seeds of all other species were planted at an approximate depth of 6 mm.
- No. of seeds per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control group: 4

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil taxonomic classification: Sandy loam
- Composition: was composed of kaolinite clay (16%), industrial quartz sand (71%), and silt (13%). A slow-release fertilizer was added to provide nutrients essential for plant growth, and limestone was added to buffer the pH.
- Organic carbon: 1.2%
- pH: 7.2

GROWTH CONDITIONS
- Photoperiod: Minimum 16-hour photoperiod.
- Light source: Artificial lighting (high pressure sodium)
- Water source and procedure: Water for seedling growth was supplied by subirrigation with well water from the glasshouse facility. Seedlings were subirrigated to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were filled to a predetermined depth to help standardize the amount of water delivered to each tray.
- Fertilization: A slow-release fertilizer was added to the soil to provide nutrients essential for plant growth.
- Pesticide and Metal Screening of Well Water and Soil: The well water and soil used for plant studies are periodically screened for pesticides and metals. No analytes were measured at levels that were expected to have an impact on the study.

EFFECT PARAMETERS MEASURED
Observations of the test were made 7 and 14 DAA to document seedling emergence. Observations were made 21 DAA to document seedling emergence and growth, and to determine the general condition of seedlings. Observations consisted of noting whether emergence had or had not occurred and assessing the condition of each seedling. Emergence was defined as the presence of visible plant tissue at the surface of the soil. Seedling condition was described by noting the presence or absence of possible signs of phytotoxicity such as necrosis, leaf curling, or chlorosis. Each emerged seedling was then assigned a numerical score that described the plant condition. Condition score is a subjective or qualitative assessment that determines whether damage is slight, moderate, or severe. A score of 10 does not mean that 10% of the plant is showing the effect (e.g. chlorosis), merely that the severity of the effect (e.g. chlorosis) is very slight.
The growth of emerged seedlings was evaluated 21 DAA by assessing the height and biomass of seedlings. Plant biomass was estimated by measuring the total dry weight of the shoots within each replicate. Seedling height was measured with a ruler to the nearest whole centimetre from the surface of the soil to the apical meristem (G. max and L. esculentum), or to the tip of the tallest leaf (all other species). Dead or non-emerged seedlings were assigned a height of 0 cm, and seedlings less than 1 cm tall were assigned a height of 0.25 cm for calculation of means. Seedlings were then clipped at soil level, the shoots of all living seedlings within a replicate were placed in a labelled paper container, dried in an oven, and weighed as a group. Mean seedling height and replicate biomass were determined for each treatment group containing living seedlings at test termination.

Nominal and measured concentrations:

Nominal application rate: 150 mg a.s./ha (corresponding to 1172 g formulation/ha)

Reference substance (positive control):

no

Key result

Species:

other: Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleracea, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

150 g/ha

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

survival, height and biomass

Remarks on result:

other: equivalent to 0.2 mg a.s./kg soil dw

Key result

Species:

other: Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleracea, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus

Duration:

21 d

Dose descriptor:

ER50

Effect conc.:

> 150 g/ha

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

seedling emergence

Remarks:

survival, height and biomass

Remarks on result:

other: equivalent to > 0.2 mg a.s./kg soil dw

Details on results:

An overview of the results is provided in Table 1 – Table 4 in ‘Any other information on results incl. tables’
- Analytical Verification of Spray Mixtures: Samples collected from the first and second spray mixture to demonstrate homogeneity and verify test substance concentrations for the 750 ppm a.s. spray mixtures had means and standard deviations of 868 ± 5.29 and 835 ± 6.51 ppm a.s. with coefficients of variation of 0.609% and 0.780%, respectively. These values represent 116% and 111% of nominal concentrations, respectively.

- Seedling Emergence: No significant effects on the seedling emergence was observed in any of the plant species at 150 g a.s./ha treatment. The mean reduction levels of seedling emergence compare to the control were -16%, -3%, 5%, -10%, -9%, 15%, 3%, -21%, 15%, and -3% for Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleracea, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus, respectively.

- Survival: 100% survive of Lolium perenne, Triticum aestivum, Zea mays, Brassica oleracea, Glycine max, Lactuca sativa, and Raphanus sativus was found at the end of the test. The reduction of survival level for Allium cepa, Beta vulgaris and Lycopersicon esculentum was 0%, 4% and 1%, respectively, compare to the control

- Biomass: The mean reduction levels of biomass compare to the control were -55%, 5%, 10%, -16%, -15%,1%, 4%, 15% 0% and -3% for Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleracea, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus, respectively.

- Height: The mean reduction levels of height compare to the control were -22%, -3%, 2%, -14%, -1%, 18%, 11%, 0%, 18% and -8% for Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleracea, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus, respectively.

- Phytoxicity: Visual signs of phytotoxicity (necrosis, leaf curl or chlorosis) were assessed, scored on a scale of 0-100, with 0 indicating no phytoxicity and 100 indicating maximum effect (dead seedling). Very slight toxicity was recorded in tomatoes, soybean, cabbage and onion but similar results were noted for the corresponding controls, therefore, it is considered that there were no phytotoxic effects attributed to exposure to the test substance during this study.

Overall, There were no observed effects on the emergence , survival, biomass, or height of any test species.

Table 1. Analytical Results

Date of application (analyte)	Sample number	Spray mixture concentration (ppm a.s.)
		Nominal	measured	mean	% of nominal
March 14 2008	1	0	< LOQ	-	-
	2	750	872	868	116
	3	750	862
	4	750	870
May 21 2008	5	0	< LOQ	-	-
	6	750	835	835	111
	7	750	829
	8	750	842

 

Table 2. Effects of test substance on Seedling Emergence, Survival, Biomass and Height of Ten Plant Species in a 21-Day Seedling Emergence Test

Species: Treatment Group (g a.s./ha)	Number Emerged Mean ± SD (% Reduction)	% Survival Mean ± SD (% Reduction)	Biomass (g) Mean ± SD (% Reduction)	Height (cm) Mean ± SD (% Reduction)
Allium cepa(Onion):
Negative Control	7.75 ± 1.50	90.9 ± 6.27	0.099 ± 0.0134	10.6 ± 1.81
150	9.00 ± 1.15 (-16%)	91.3 ± 5.95 (0%)	0.153 ± 0.0494 (-55%)	13.0 ± 4.02 (-22%)
Triticum aestivum(Wheat):
Negative Control	10.00 ± 0.00	100.00 ± 0.00	1.91 ± 0.289	36.1 ± 2.70
150	9.50 ± 0.58 (5%)	100.0 ± 0.00 (0%)	1.72 ± 0.155 (10%)	35.5 ± 1.88 (2%)
Zea mays(Corn):
Negative Control	7.75 ± 0.96	100.00 ± 0.00	3.28 ± 0.368	34.7 ± 3.89
150	8.50 ± 1.29 (-10%)	100.0 ± 0.00 (0%)	3.80 ± 0.355 (-16%)	39.7 ± 5.52 (-14%)
Beta vulgaris(Sugarbeet):
Negative Control	8.00 ± 1.15	97.2 ± 5.56	1.92 ± 0.263	15.8 ± 2.19
150	8.75 ± 0.50 (-9%)	93.8 ± 12.50 (4%)	2.20 ± 0.481 (-15%)	16.0 ± 3.07 (-1%)
Brassica oleracea(Cabbage):
Negative Control	8.50 ± 0.58	100.00 ± 0.00	1.34 ± 0.249	11.8 ± 1.40
150	7.25 ± 0.96 (15%)	100.0 ± 0.00 (0%)	1.32 ± 0.141 (1%)	9.7 ± 1.15 (18%)
Glycine max(Soybean):
Negative Control	7.75 ± 1.26	100.0 ± 0.00	3.12 ± 0.357	13.4 ± 2.01
150	7.50 ± 0.58 (3%)	100.0 ± 0.00 (0%)	2.99 ± 0.354 (4%)	11.9 ± 2.39 (11%)
Lactuca sativa(Lettuce):
Negative Control	7.25 ± 0.96	100.0 ± 0.00	0.78 ± 0.128	7.8 ± 0.95
150	8.75 ± 1.50 (-21%)	100.0 ± 0.00 (0%)	0.66 ± 0.204 (15%)	7.8 ± 1.48 (0%)
Lycopersicon esculentum(Tomato):
Negative Control	8.25 ± 1.50	86.7 ± 16.33	1.37 ± 0.457	12.1 ± 1.69
150	7.00 ± 0.82 (15%)	85.6 ± 1.71 (1%)	1.37 ± 0.458 (0%)	9.9 ± 2.92 (18%)
Raphanus sativus (Radish):
Negative Control	9.00 ± 0.00	100.0 ± 0.00	2.02 ± 0.226	12.2 ± 0.73
150	9.25 ± 0.50 (-3%)	100.0 ± 0.00 (0%)	2.08 ± 0.267 (-3%)	13.2 ± 1.52 (-8%)

 

Table 3. Effect values of the test substance on 21-Day Emergence and Survival 

Species	21 -Day Emergence (mg a.s./ha)				21 -Day Survival (mg a.s./ha)
	LOER	NOER	ER25	ER50	LOER	NOER	ER25	ER50
Monocots
Allium cepa(onion)	>150	150	>150	>150	>150	150	>150	>150
Lolium perenne(ryegrass)	>150	150	>150	>150	>150	150	>150	>150
Triticum aestivum(wheat)	>150	150	>150	>150	>150	150	>150	>150
Zea mays(corn)	>150	150	>150	>150	>150	150	>150	>150
Dicots
Beta vulgaris(sugarbeet)	>150	150	>150	>150	>150	150	>150	>150
Brassica oleracea(cabbage)	>150	150	>150	>150	>150	150	>150	>150
Glycine max(soybean)	>150	150	>150	>150	>150	150	>150	>150
Lactuca sativa(lettuce)	>150	150	>150	>150	>150	150	>150	>150
Lycopersicon esculentum(tomato)	>150	150	>150	>150	>150	150	>150	>150
Raphanus sativus(radish)	>150	150	>150	>150	>150	150	>150	>150

Table 4. Effect values of the test substance on 21-Day Height and Biomass

Species	Height (mL/ha)				Biomass (mL/ha)
	LOER	NOER	ER25	ER50	LOER	NOER	ER25	ER50
Monocots
Allium cepa(onion)	>150	150	>150	>150	>150	150	>150	>150
Lolium perenne(ryegrass)	>150	150	>150	>150	>150	150	>150	>150
Triticum aestivum(wheat)	>150	150	>150	>150	>150	150	>150	>150
Zea mays(corn)	>150	150	>150	>150	>150	150	>150	>150
Dicots
Beta vulgaris(sugarbeet)	>150	150	>150	>150	>150	150	>150	>150
Brassica oleracea(cabbage)	>150	150	>150	>150	>150	150	>150	>150
Glycine max(soybean)	>150	150	>150	>150	>150	150	>150	>150
Lactuca sativa(lettuce)	>150	150	>150	>150	>150	150	>150	>150
Lycopersicon esculentum(tomato)	>150	150	>150	>150	>150	150	>150	>150
Raphanus sativus(radish)	>150	150	>150	>150	>150	150	>150	>150

 

Validity of the Test

The test is considered valid based on the following criteria:

1) Mean emergence in the control group was greater than 70% for all species;

2) There were no phytotoxic effects observed in the control groups;

3) Mean survival in the control group was greater than 90% for all species;

4) The environmental conditions and growing medium for each species was the same for all test groups.

Survival of L. esculentum in the control group was 86.7% of the emerged seedlings. Although slightly below the established criterion described above, the survival was considered adequate for the purpose of the study. Incidental signs of toxicity were observed in the control group for T. aestivum, B. oleracea, G. max, and L. esculentum. In spite of these incidental signs, the controls were considered adequate for the test.

Validity criteria fulfilled:

yes

Remarks:

See validity of the test in 'Any other information on results incl. tables'

Conclusions:

In a seedling emergence test performed according to OECD TG 208 in ten plant species, no toxic effects of the test substance were observed in any of the test species at the 150 g a.s./ha application rate (equivalent to 0.2 mg a.s./kg dw soil) for seeding emergence, survival or growth (height and biomass).

Executive summary:

In this study performed according to OECD TG 208 and in compliance with GLP, ten species of higher plants, four monocot (Allium cepa (onion), Lolium perenne (ryegrass), Triticum aestivum (wheat) and Zea mays (corn)) and six dicot (Beta vulgaris (sugarbeet), Brassica oleracea (cabbage), Glycine max (soybean), Lactuca sativa (lettuce), Lycopersicon esculentum (tomato) and Raphanus sativus (radish)). The test plants were treated with 150 g a.s./ha of the test substance formulation and a negative (water) control using a sprayer. The spray mixture was applied at a nominal spray volume of approximately 200 L/ha. The treatment and control groups each consisted of four replicate test pots, with each pot containing ten planted seeds of one species. Seeds were sown in plastic pots in sandy loam soil. The test was performed in a glasshouse with 16.01 – 38.39 ˚C temperature and 37.53% relative humidity. Artificial lighting was used to provide a minimum 16-hour photoperiod. The test duration was 21 days after the application of the test substance. Observations of emergence were made on Days 7, 14, and 21, while observations of height and condition were made only on Day 21 at test termination. Dry weights were determined from seedling shoots collected on Day 21 and dried to a constant weight.

All validity criteria of the OECD guideline were considered met. Mean emergence in the control group for all species was > 70% and mean survival for all control groups was > 90%, except for L. esculentum, where 86.7% of the emerged seedlings survived. Since 10 different species were tested, this rate of survival was considered acceptable for the purpose of the study. Exposure to the test substance at a rate of 150 g a.s./ha did not result in adverse effects on seedling emergence, survival or biomass compared to the control for any of the species tested. The most sensitive species tested for seedling emergence were tomato and cabbage. The seedling emergence of these two species decreased by 15% compared to the controls. Based on the findings, the NOEC value was determined to be 150 g a.s./ha (equivalent to 0.2 mg a.s./kg soil dw) for seedling emergence, growth (biomass and height) and survival, based on the nominal application rate.

Reference 2

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Mar 2008 to 18 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 227 (Terrestrial Plant Test: Vegetative Vigour Test)

Version / remarks:

July 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4150 (Terrestrial Plant Toxicity Tier I (vegetative vigor))

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The spray mixture was sampled after its preparation to provide material for analytical confirmation of the test concentration. Triplicate samples were collected from the test concentration, and a single sample was collected from the negative control.

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of Spray Mixtures: The spray mixture for the 150 g a.s./ha application rate was prepared by diluting 3.07 mL of the test substance to 500 mL with well water purified by reverse osmosis, and mixing.
- Application of Spray Mixtures: Application of the test substance was made with a Sprayer (spray booth). The booth was used to simulate inadvertent direct application, or off-target drift resulting from actual field use. The test substance spray mixture was placed into a 500 mL polyethylene spray bottle that was pressurized to 1.38 bars. The spray bottle was connected to a moveable spray nozzle that was used to spray the plants in the test pots. Pots containing test plants were placed in the spray booth, which served as a protected area for spraying. The spray nozzle was suspended approximately 41 cm above the target (plant canopy) during delivery of the spray mixture. Applications first were made to plants in the control group, followed by plants in the treatment group. This procedure was used to avoid cross contamination. Applications were made using a nominal spray volume of 200 L/ha. The spray coverage appeared uniform over the spray area.

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Family: Liliaceae

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: ryegrass
- Family: Poaceae

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: wheat
- Family: Poaceae

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: corn
- Family: Poaceae

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: sugarbeet
- Family: Chenopodiaceae

Species:

Brassica oleracea var. capitata

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: cabbage
- Family: Brassicaceae

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: soybean
- Family: Fabaceae

Species:

Lactuca sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: lettuce
- Family: Asteraceae

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: tomato
- Family: Solanaceae

Species:

Raphanus sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: radish
- Family: Brassicaceae

Test type:

vegetative vigour test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Test temperature:

16.93 - 33.60 ˚C

pH:

7.2

Moisture:

12.38 % - 81.60%

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): Plastic pots (depth of 10 cm and top diameter of 11 cm)
- Amount of soil: Approximately 710 cm3
- No. of seeds per container: 5
- No. of replicates per treatment group: 6
- No. of replicates per control group: 6

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil taxonomic classification: sandy loam
- Composition: kaolinite clay, industrial quartz sand and peat, with slow release fertiliser (71% sand, 13% silt, and 14% clay)
- Organic carbon: 1.2%
- pH: 7.2

GROWTH CONDITIONS
- Photoperiod: a minimum 16-hour photoperiod
- Light source: Artificial lighting
- Pesticide and Metal Screening of Well Water and Soil: The well water and soil used for plant studies are periodically screened for pesticides and metals. No analytes were measured at levels that were expected to have an impact on the study.
- Watering of seedlings: Water lost through transpiration and evaporation was replaced by subirrigation with well water from the glasshouse facility. Seedlings were subirrigated to minimize the potential for the leaching of the test substance through the soil. Subirrigation trays were filled to a predetermined depth to help standardize the amount of water delivered to each tray.

EFFECT PARAMETERS MEASURED:
- Plant Condition: Observations of plant condition were made prior to application and at 7, 14, and 21 DAA. After application, the condition of each seedling was assessed using a numerical score to indicate the severity of apparent signs of phytotoxicity.
- Phytotoxicity rating system: The condition score is a subjective and qualitative assessment that describes the severity of observed damage. A score of 0 indicated that there was no evidence of phytotoxicity. Scores of 10 to 30 indicated that the effects were slight and not apparently detrimental. Moderate effects were given scores ranging from 40 to 60, whereas severe effects, such as death of leaves, were rated from 70 to 90. Plant deaths were recorded as 100. The mean condition score for each replicate was calculated on each day of observation.
- Plant Height: Observations of plant height were made prior to application and at 21 DAA. Prior to application, the approximate height or range of heights in the test groups was recorded. At test termination, individual plant heights were measured to the nearest whole centimeter from the surface of the soil to the apical meristem (L. esculentum, G. max and C. sativa) or to the tip of the tallest leaf (all other test species). Dead plants were assigned a height of zero centimeters. The mean height for each replicate and each treatment group was calculated.
- Plant Weight: Measurements of plant weight were made only after test termination, since the procedures used were destructive. Living plants were clipped at soil level, combined by replicate, dried in an oven for at least 24 hours, and weighed. Dead plants were not collected. The total shoot dry weight of the living plants, or biomass, for each replicate was used to determine the mean biomass for each treatment group.

VEHICLE CONTROL PERFORMED: No

Nominal and measured concentrations:

Nominal application rate: 150 mg a.s./ha (corresponding to 1172 g formulation/ha)

Reference substance (positive control):

no

Key result

Species:

other: Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleraceae, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

150 g/ha

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

height and biomass

Remarks on result:

other: equivalent to 0.2 mg a.s./kg soil dw

Key result

Species:

other: Allium cepa, Lolium perenne, Triticum aestivum, Zea mays, Beta vulgaris, Brassica oleraceae, Glycine max, Lactuca sativa, Lycopersicon esculentum and Raphanus sativus

Duration:

21 d

Dose descriptor:

ER50

Effect conc.:

> 150 g/ha

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

height and biomass

Remarks on result:

other: equivalent to > 0.2 mg a.s./kg soil dw

Details on results:

An overview of the results is provided in Table 1– Table 3 in ‘Any other information on results incl. tables’
- Analytical Verification of Spray Mixtures: The control samples showed no indication of the presence of the test substance or of the presence of a co-eluting substance at the characteristic retention time of the test substance. Samples collected at the start of the experiment to demonstrate homogeneity and verify test substance concentrations for the 750 ppm a.s. spray mixtures had a mean and standard deviation of 881 ± 4.00 ppm a.s., with a coefficient of variation of 0.454%. This value represents 117% of nominal concentration.

Table 1.Analytical Results

Date of application	Sample number	Spray mixture concentration (ppm a.s.)
		Nominal	measured	Mean1	% of nominal
March 24 2008	1	0	< LOQ	-	-
	2	750	885	881	117
	3	750	881
	4	750	877

1. The limit of quantitation (LOQ) was 100 μg a.s./mL, calculated as the product of the concentration of the lowest calibration standard (1.00 μg a.s./mL) and the dilution factor of the negative control samples (100).

Table 2. Effects of the test substance on Mean Height and Biomass of Ten Plant Species in a 21-Day Vegetative Vigour Test

Species: treatment Group (g a.s./ha)	Height (cm) mean ± SD (% reductipon)			biomass (g) mean ± SD (% reductipon)
	Day 7	Day 14	Day 21	Day 21
Allium cepa(Onion):
Negative Control	16.7 ± 1.67	21.9 ± 2.59	27.6 ± 2.72	0.49 ± 0.112
150	16.6 ± 2.28 (1%)	21.0 ± 2.03 (4%)	26.9 ± 1.56 (2%)	0.46 ± 0.117 (5%)
Lolium perenne(Ryegrass):
Negative Control	22.1 ± 1.31	26.0 ± 1.54	29.3 ± 1.22	6.95 ± 0.639
150	21.7 ± 1.45 (2%)	26.0 ± 2.37 (0%)	30.0 ± 1.68 (-3%)	6.31 ± 1.007 (9%)
Triticum aestivum(Wheat):
Negative Control	40.1 ± 1.18	44.9 ± 1.78	55.4 ± 2.09	7.40 ± 0.913
150	40.6 ± 0.73 (-1%)	44.2 ± 1.14 (2%)	55.5 ± 3.41 (0%)	6.90 ± 1.053 (7%)
Zea mays(Corn):
Negative Control	55.5 ± 3.68	70.1 ± 4.66	73.0 ± 7.20	11.3 ± 2.17
150	59.3 ± 4.44 (-7%)	74.1 ± 4.19 (-6%)	78.4 ± 7.84 (-7%)	12.7 ± 1.32 (-13%)
Beta vulgaris(Sugarbeet):
Negative Control	21.0 ± 0.94	26.9 ± 1.78	31.2 ± 2.09	13.5 ± 1.48
150	20.7 ± 1.29 (2%)	26.2 ± 2.37 (2%)	30.6 ± 2.88 (2%)	12.6 ± 1.30 (6%)
Brassica oleracea(Cabbage):
Negative Control	16.7 ± 1.23	18.9 ± 1.79	20.6 ± 2.14	17.2 ± 3.28
150	17.3 ± 1.29 (-4%)	19.9 ± 1.47 (-5%)	21.0 ± 1.61 (-2%)	18.4 ± 2.18 (-7%)
Glycine max(Soybean):
Negative Control	18.8 ± 0.67	26.8 ± 0.55	43.4 ± 3.12	24.7 ± 2.00
150	19.1 ± 0.47 (-2%)	26.2 ± 1.32 (2%)	40.0 ± 4.19 (8%)	23.6 ± 1.30 (5%)
Lactuca sativa(Lettuce):
Negative Control	11.6 ± 0.28	13.9 ± 0.97	14.9 ± 1.20	16.8 ± 1.23
150	11.5 ± 0.88 (1%)	13.9 ± 1.43 (0%)	14.7 ± 1.38 (1%)	14.7 ± 1.93 (12%)
Lycopersicon esculentum(Tomato):
Negative Control	18.2 ± 1.85	27.8 ± 3.06	36.5 ± 1.90	24.4 ± 2.39
150	18.1 ± 1.59 (0%)	28.0 ± 1.74 (-1%)	36.0 ± 2.90 (1%)	27.5 ± 2.69 (-13%)
Raphanus sativus(Radish):
Negative Control	16.5 ± 0.65	18.2 ± 1.25	22.1 ± 2.04	6.59 ± 0.677
150	17.0 ± 0.53 (-3%)	19.6 ± 0.81 (-8%)	23.3 ± 2.54 (-5%)	6.35 ± 0.489 (4%)

 

Table 3. Effect values of the test substance on 21 days seedling height and biomass

Species	21-day height (mg a.s./ha)				21-day dry weight (mg a.s./ha)
	LOER	NOER	ER25	ER50	LOER	NOER	ER25	ER50
Monocots
Allium cepa (onion)	>150	150	>150	>150	>150	150	>150	>150
Lolium perenne (ryegrass)	>150	150	>150	>150	>150	150	>150	>150
Triticum aestivum (wheat)	>150	150	>150	>150	>150	150	>150	>150
Zea mays (corn)	>150	150	>150	>150	>150	150	>150	>150
Dicots
Beta vulgaris (sugarbeet)	>150	150	>150	>150	>150	150	>150	>150
Brassica oleracea (cabbage)	>150	150	>150	>150	>150	150	>150	>150
Glycine max (soybean)	>150	150	>150	>150	>150	150	>150	>150
Lactuca sativa (lettuce)	>150	150	>150	>150	>150	150	>150	>150
Lycopersicon esculentum (tomato)	>150	150	>150	>150	>150	150	>150	>150
Raphanus sativus (radish)	>150	150	>150	>150	>150	150	>150	>150

Validity of the Test

The test was considered valid based on the following criteria:

1) Germinability of seeds used in the test was greater than 70% for all species;

2) There were no phytotoxic effects observed in the control groups;

3) Survival of control plants was greater than 90% for all species;

4) The environmental conditions and growing medium for each species was the same

for all test groups.

Slight necrosis was observed on one A. cepa seedling. In spite of this incidental sign, the

controls were considered adequate for the test.

Validity criteria fulfilled:

yes

Remarks:

See validity of the study in 'Any other information on results incl. tables'

Conclusions:

In a vegetative vigour test performed according to OECD TG 227 in ten plant species, no toxic effects of the test substance were observed in any of the test species at the 150 g a.s./ha application rate (equivalent to 0.2 mg a.s./kg dw soil) for growth (height and biomass).

Executive summary:

The impact of the test substance on the vegetative vigour of ten species was studied according to OECD TG 227, in compliance with GLP criteria. The planted seedlings of four monocot species: Allium cepa (onion), Lolium perenne (ryegrass),Triticum aestivum (wheat) and Zea mays (corn), and six dicot species: Beta vulgaris (sugarbeet), Brassica oleracea (cabbage), Glycine max (soybean), Lactuca sativa (lettuce), Lycopersicon esculentum (tomato) and Raphanus sativus (radish) were sprayed with a single test concentration of the test substance formulation, and a negative (water) control. The nominal concentration for each species was 150 g a.s./ha, corresponding to 1172 g of formulated product per hectare. The spray mixture was applied at a nominal spray volume of approximately 200 L/ha. The treatment and control groups each consisted of six replicated experimental units. Each replicate comprised of five individually planted seedlings of one species, hence 30 plants of each species were exposed at each treatment. Seedlings were sown in plastic pots in a sandy loam soil. After the spray mixture had been applied to the seedlings the experimental units were impartially assigned to sub-irrigation trays which were arranged randomly on glass house benching on the day of test initiation. The test was performed in a glasshouse with 16.93 – 33.60 ˚C temperature and 42.57% mean relative humidity. Artificial lighting was used to provide a minimum 16-hour photoperiod. The test duration was 21 days after application of the test substance. Observations of seedling condition and growth were made on Days 0, 7, 14, and 21, while observations of height were made only on Day 21. Seedling dry weights were determined from shoots collected on Day 21 and dried to a constant weight.

Mean emergence and seedling survival in the control group for all species was > 70% and > 90%, respectively. Slight necrosis was recorded in one seedling in the onion control group, which was considered incidental and did not impact on the outcome of the study. After 21 days of exposure, soybean showed the largest reduction (8%) in height compare to the control group. The most sensitive species tested for biomass change was lettuce, in which the plant fresh weight of the treated seedlings decreased by 12% compared to the control group. Visual signs of phytotoxicity (necrosis, leaf curl or chlorosis) were assessed, scored on a scale of 0-100, with 0 indicating no phytoxicity and 100 indicating maximum effect (dead seedling). Very slight toxicity was recorded in radish and 2 seedlings died in the lettuce treatment group. Given the numbers of seedlings and range of species tested, it is considered that there were no phytotoxic effects attributed to exposure to the test substance during this study. Based on these findings, it is concluded that the post-emergence application of the test substance formulation at 150 g a.s./ha (corresponding to 1172 g formulation/ha) resulted in no adverse effects on any of the species tested. Therefore, the ER50 was determined to be > 150 g a.s./ha and NOEC was determined to be 150 g a.s/ha (equivalent to 0.2 mg a.s./kg soil dw) for growth (height and biomass), based on nominal application rate.

Description of key information

21-d, NOEC = 150 g a.s./ha (equivalent to 0.2 mg a.s./kg dw soil), seeding emergence, survival and biomass, OECD TG 208, Porch 2008

21-d, ER50 > 150 g a.s./ha (equivalent to > 0.2 mg a.s./kg dw soil), seeding emergence, survival and biomass, OECD TG 208, Porch 2008

21-d, NOEC = 150 g a.s./ha (equivalent to 0.2 mg a.s./kg dw soil), growth (biomass and height), OECD TG 227, Porch 2008

21-d, ER50 > 150 g a.s./ha (equivalent to > 0.2 mg a.s./kg dw soil), growth (bimass and height), OECD TG 227, Porch 2008

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

0.2 mg/kg soil dw

Additional information

Two studies are available for this endpoint. One study focuses on seedling emergence (OECD TG 208; GLP compliant) and the other one focuses on vegetative vigour (OECD TG 227; GLP compliant). Both were selected as key studies. Ten species were used in these studies: four monocots (Allium cepa (onion), Lolium perenne (ryegrass), Triticum aestivum (wheat) and Zea mays (corn)) and six dicots (Beta vulgaris (sugarbeet), Brassica oleracea (cabbage), Glycine max (soybean), Lactuca sativa (lettuce), Lycopersicon esculentum (tomato) and Raphanus sativus (radish)). In both studies, these plants were exposed to 150 g a.s./ha of the test substance formulation (nominal application rate; corresponding to 1172 g formulation/ha) and a negative (water) control using a sprayer for 21 days. No significant effects were observed up to the highest test concentration in both studies.

In the seedling emergence study, the seeds were sown in plastic pots in sandy loam soil. The treatment (spray application to the soil surface) and control groups each consisted of four replicated experimental units. Each replicate is comprised of ten individually planted seedlings of one species. The test was performed in a glasshouse with 16.01 – 38.39 ˚C temperature and 37.53% mean relative humidity. Artificial lighting was used to provide a minimum 16-hour photoperiod. Observations of emergence were made on Days 7, 14, and 21, while observations of height and condition were made only on Day 21 at test termination. Dry weights were determined from seedling shoots collected on Day 21 and dried to a constant weight. The results show that exposure to 150 g a.s./ha of the test substance did not result in adverse effects on seedling emergence, survival, or growth (biomass and height) of the plant compared to the control for any of the species tested. Therefore, the NOEC value was determined to be 150 g a.s./ha (equivalent to 0.2 mg a.s./kg soil dw) for seedling emergence, growth (biomass and height) and survival based on the nominal application rate.

In the vegetative vigour study, the seeds were sown in plastic pots in sandy loam soil. The treatment (spray application to emerged seedlings) and control groups each consisted of six replicated experimental units. Each replicate is comprised of five individually planted seedlings of one species. The test was performed in a glasshouse with 16.93 – 33.60 ˚C temperature and 42.57% mean relative humidity. Artificial lighting was used to provide a minimum 16-hour photoperiod. The test duration was 21 days after the application of the test substance. Observations of seedling condition and growth were made on Days 0, 7, 14, and 21, while observations of height were made only on Day 21. Seedling dry weights were determined from shoots collected on Day 21 and dried to a constant weight. After 21 days of exposure, no significant adverse effects on seedling emergence compared to the control for any of the species and parameters tested. As well as survival, plant fresh weight and plant height were not affected by the application of the test substance. Therefore, the ER50 was determined to be > 150 g a.s./ha and NOEC was determined to be 150 g a.s/ha (equivalent to 0.2 mg a.s./kg soil dw) for growth (height and biomass), based on nominal application rate.