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Toxicity to terrestrial plants

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Endpoint:
toxicity to terrestrial plants: short-term (with study design considered suitable for long-term assessment)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 Sep to 2013 to 08 Oct 2013
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.4150 (Terrestrial Plant Toxicity Tier I (vegetative vigor))
Version / remarks:
January 2012
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
A 6.0 mg cation/mL primary spray stock solution was prepared by placing 13.3959 g (3.0007 g as active ingredient) of the test substance in a 500-mL volumetric flask and bringing it to volume with deionized water. The resultant solution was observed to be clear and dark green in color following preparation. Secondary spray stock solutions were prepared from the 6.0 mg cation/mL primary spray stock solution.

FOLIAR APPLICATION
Foliar application was performed after the plants reached the 2- to 4-leaf stage (approximately 2 to 3 weeks from seedling). The replicate pots for each species tested were placed on a stainless steel tray. The appropriate spray stock solution was applied to the pots using an application chamber. The application device incorporated an overhead atomizing spray nozzle (Spray Systems Company, Wheaton, Illinois, 8001-E-SS) calibrated at 20 psi to deliver the stock solution on a revolving belt (229 feet/minute) which transported the pots on a tray (91.4 cm long x 40.6 cm wide = 3.7E-05 hectares) past the spray nozzle. The system was calibrated to deliver 3.47 mL of the spray stock solution per tray. After treatment, pots were randomized by species, within a block (approximately 1.5 m2) on a greenhouse bench once at exposure initiation using computer randomization.
Species:
Zea mays
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: corn
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oats
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: perennial ryegrass
Species:
Phaseolus vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: common bean
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: cucumber
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: oilseed rape
Species:
Raphanus sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: radish
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: soybean
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: tomato
Test type:
vegetative vigour test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
21 d
Test temperature:
The temperature during testing was maintained between 23 and 43 °C by heating and ventilation units which cycled as required to maintain the temperature within the greenhouse.
pH:
6.1
Details on test conditions:
TEST SYSTEM
- Testing facility: The study was conducted in a greenhouse covered with Lexan® (polycarbonate) panels.
- Test container: The exposure vessels consisted of polypropylene pots (Kord Products Ltd.). The pots were 12-cm tall with a top diameter of 14 cm and a bottom diameter of 12 cm. The interior base was fitted with 20 cm filter paper (Rockline, Inc.) to retain the support medium and allow for plant uptake of solution by sub-irrigation.
- Amount of soil: Each pot was filled to a depth of 10 cm with approximately 1.2 kg of untreated support medium (diameter at surface of the support medium = 12 cm; surface area = 104 cm2). Each pot was placed in a polypropylene saucer (Kord Products Ltd.) and received approximately 100 mL of 400 mg/L Peters 20-20-20 fertilizer solution prepared with deionized water and provided to the plants via sub-irrigation
- Method of seeding: Seeds were impartially selected and planted at a depth of approximately 1 to 2 cm below the surface of the support substrate in each pot (40 seeds per application rate and the control).
- No. of seeds per container: 4 (common bean, corn, cucumber, oilseed rape, radish, soybean and tomato) and 8 (oats, onion and perennial ryegrass)
- No. of replicates per treatment group: 5 (oat, onion and perennial ryegrass) and 10 (common bean, corn, cucumber, radish, oilseed rape, soybean and tomato)
- No. of replicates per control: 5 (oat, onion and perennial ryegrass) and 10 (common bean, corn, cucumber, radish, oilseed rape, soybean and tomato)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Preparation: Loam was collected from Rochester, Massachusetts.
- Pesticide use history at the collection site: A representative sample of the loam was analyzed for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected at concentrations that would compromise the results of this study.
- % sand: 91
- % silt: 7
- % clay: 2
- Soil classification system: USDA
- Composition (if artificial substrate): Washed silica sand (Holliston Sand Company, Slatersville, Rhode Island, SMV Lot No. 081513) was combined with loam to produce a mixture containing 70% sand and 30% loam.
- Organic carbon (%): 0.522 (corresponding to 0.9% organic matter)

NUTRIENT MEDIUM
- Description: The plants were sub-irrigated using 400 mg/L Peters 20-20-20 solution (SMV No. 032113, supplied by Griffin Greenhouse Supplies) prepared in deionized water. Approximately 100 mL was provided to all pots by sub-irrigation approximately twice weekly. All additional watering was provided using approximately 100 mL of deionized water daily.

GROWTH CONDITIONS
- Photoperiod: The photoperiod was 16 hours of light and 8 hours of darkness.
- Light intensity and quality: Sodium vapor lights supplemented natural light when necessary to maintain > 800 footcandles (equivalent to > 105 μmol/m2/s) during the light period.
- Air pollution: The potential for air pollution within the greenhouse is believed to be minimal due to the rural location of the laboratory and the lack of other industrial businesses in the area. The greenhouse is located in a relatively isolated section of the laboratory grounds, which reduces the possibility of air contamination from concurrent testing.

TEST CONDITION MEASUREMENTS
Air temperature within the greenhouse was controlled using thermostatically-regulated heating and ventilation systems. Air temperature and relative humidity was constantly monitored using a Hobo data logger to obtain the day/night temperature range. Light intensity was measured daily at each bench using an Apogee radiometer/photometer held at average maximum leaf height for each species. Light intensity (photosynthetically-active radiation, PAR) was measured in μmol/m2/sec.

EFFECT PARAMETERS MEASURED:
True leaf counts (stage of development) of seedlings were made on the day of, but prior to, application of the test substance. Seedlings were observed in a clockwise pattern, starting at the center of the label. Any morphological abnormalities observed and survival were recorded weekly after foliar application. Morphological abnormalities were reported as the percentage of plants exhibiting the abnormality. Shoot length was measured and recorded on days 14 and 21. At test termination (21 days post-treatment), all shoots in a replicate pot were cut at the soil surface and placed in a labeled, pretared container. Shoots were dried in radiant heat ovens at approximately 70 ± 5 ºC for a minimum of three days before determining dry weights. Plants that died during the exposure were not included in statistical calculations.

RANGE-FINDING TEST
A preliminary 21-day range-finding vegetative vigor test was conducted exposing common bean, corn, cucumber, oats, oilseed rape, onion, perennial ryegrass, radish, soybean and tomato to nominal application rates of 1.12, 11.2, 112 and 1120 g cation/ha and a control (three replicates per application rate, four plants per replicate). Following termination of exposure, plant survival, shoot length and dry weight were determined. Results of the preliminary study is presented in 'Any other information on results incl. tables'. Based on these results (inhibition of > 25% in the endpoints), Tier II tests for all ten plant species were conducted. Tier II testing was conducted at nominal application rates of 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha (for common bean, oilseed rape and soybean), 18, 35, 70, 140, 280 and 560 g cation/ha for corn and tomato, 1.1, 4.4, 18, 70 and 280 g cation/ha for cucumber and onion, 18, 35, 70, 140 and 280 g cation/ha for oat, 8.8, 18, 35, 70, 140 and 280 g cation/ha for radish and 2.2, 4.4, 8.8, 18, 35 and 70 g cation/ha for perennial ryegrass.
Nominal and measured concentrations:
Nominal application rates were selected based on preliminary testing:
- Phaseolus vulgaris (Common Bean): 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha
- Zea mays (Corn): 18, 35, 70, 140, 280 and 560 g cation/ha
- Cucumus sativa (Cucumber): 1.1, 4.4, 18, 70 and 280 g cation/ha
- Avena sativa (Oats): 18, 35, 70, 140 and 280 g cation/ha
- Brassica napus (Oilseed Rape): 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha
- Allium cepa (Onion): 1.1, 4.4, 18, 70 and 280 g cation/ha
- Lolium perenne (Perennial Ryegrass): 2.2, 4.4, 8.8, 18, 35 and 70 g cation/ha
- Raphanus sativus (Radish): 8.8, 18, 35, 70, 140 and 280 g cation/ha
- Glycine max (Soybean): 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha
- Lycopersicon esculentum (Tomato): 18, 35, 70, 140, 280 and 560 g cation/ha

The recommended application volume was 10 gallons/A. The test substance stock solutions were applied to the seedlings by calibrating the spray apparatus to deliver the stock solutions at an application volume of 10 gallons/A, which is equivalent to 93.5 L/ha.

- Measured concentrations: The measured concentration ranged from 100 to 120% of nominal concentration during pre-spray application analysis and from 110 to 130% of nominal concentration during post-spray application analysis.
Reference substance (positive control):
no
Species:
other: Lolium perenne, Allium cepa and Glycine max
Duration:
21 d
Dose descriptor:
EC25
Effect conc.:
30.4 g/ha
Nominal / measured:
nominal
Conc. based on:
other: pure test substance
Basis for effect:
germination
Remarks:
shoot length and shoot dry weight
Remarks on result:
other: See 'Any other information on results incl. tables' for a complete overview of the effect values per species
Remarks:
recalculated value, expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation.
Species:
other: Lolium perenne, Allium cepa and Glycine max
Duration:
21 d
Dose descriptor:
EC25
Effect conc.:
22 g/ha
Nominal / measured:
nominal
Conc. based on:
other: test substance cation species
Basis for effect:
growth
Remarks:
shoot length and shoot dry weight
Remarks on result:
other: See 'Any other information on results incl. tables' for a complete overview of the effect values per species
Remarks:
original value presented in study
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6.1 g/ha
Nominal / measured:
nominal
Conc. based on:
other: pure test substance
Basis for effect:
growth
Remarks:
shoot length
Remarks on result:
other: See 'Any other information on results incl. tables' for a complete overview of the effect values per species
Remarks:
recalculated value, expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation.
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.4 g/ha
Nominal / measured:
nominal
Conc. based on:
other: test substance cation species
Basis for effect:
growth
Remarks:
shoot length
Remarks on result:
other: See 'Any other information on results incl. tables' for a complete overview of the effect values per species
Remarks:
original value presented in study
Details on results:
PHASEOLUS VULGARIS
- Appearance: All plants in the 18, 70 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (0.27, 1.1 and 4.4 g cation/ha).
- Survival: Percent survival in the control and all application rates tested averaged 100%. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 43.0 cm. Shoot length for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 40.5, 42.9, 38.8, 41.3, 34.0 and 34.3 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 1.2860 g. Shoot dry weight for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 1.4842, 1.2278, 1.3519, 1.2270, 0.8487 and 1.0315 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to the 70 g cation/ha application rate (34% inhibition) compared to the control. Inhibition of 20% was observed at the highest application rate, 280 g cation/ha and was not significantly reduced from the control.

ZEA MAYS
- Appearance: Several dead plants were observed in the 70, 140, 280 and 560 g cation/ha application rates. One plant in the 35 g cation/ha application rate was observed to be necrotic. Several plants in the 70, 140, 280 and 560 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (18 and 35 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha application rates averaged 100, 100, 83, 63, 70 and 40%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent survival among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 96.1 cm. Shoot length for plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha application rates averaged 89.9, 84.2, 71.0, 52.0, 55.1 and 54.3 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates
≥ 35 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 2.1664 g. Shoot dry weight for plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha application rates averaged 1.9971, 1.3753, 1.0370, 0.5504, 0.6704 and 0.6893 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 35 g cation/ha compared to the control.

CUCUMUS SATIVA
- Appearance: Several dead plants were observed in the 70 and 280 g cation/ha application rates. All surviving plants in the 70 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (1.1, 4.4 and 18 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 100, 100, 100, 68 and 53%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent survival among plants exposed application rates ≥ 70 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 23.3 cm. Shoot length for plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 25.0, 26.8, 25.8, 23.9 and 22.6 cm, respectively. William’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 0.9238 g. Shoot dry weight for plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 0.9717, 0.8650, 0.7965, 0.9346 and 0.7975 g, respectively. Wilcoxon’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

AVENA SATIVA
- Appearance: One dead plant was observed in the 70 g cation/ha application rate. Several dead plants were observed in the 140 and 280 g cation/ha application rates. All surviving plants in the 70, 140 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (18 and 35 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 18, 35, 70, 140 and 280 g cation/ha application rates averaged 100, 100, 98, 65 and 28%, respectively. Cochran-Armitage Trend Step-Down Test determined a significant reduction in percent survival among plants exposed to application rates ≥ 140 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 63.1 cm. Shoot length for plants exposed to the 18, 35, 70, 140 and 280 g cation/ha application rates averaged 61.8, 56.9, 47.0, 32.4 and 22.5 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 0.6670 g. Shoot dry weight for plants exposed to the 18, 35, 70, 140 and 280 g cation/ha application rates averaged 0.6199, 0.5925, 0.3999, 0.2282 and 0.1555 g, respectively. William’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed application rates ≥ 70 g cation/ha compared to the control.

BRASSICA NAPUS
- Appearance: One dead plant each was observed in the 1.1 and 4.4 g cation/ha application rates. All plants in the 70 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (0.27 and 18 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 100, 98, 98, 100, 100 and 100%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 11.8 cm. Shoot length for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 11.0, 11.5, 11.2, 11.1, 11.2 and 10.9 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to the 280 g cation/ha application rate compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 1.0978 g. Shoot dry weight for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 1.0146, 1.0464, 0.9483, 0.9572, 0.6077 and 0.7314 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 70 g cation/ha compared to the control.

ALLIUM CEPA
- Appearance: One dead plant was observed in the 4.4 g cation/ha application rate. Several dead plants were observed in the 70 and 280 g cation/ha application rates and the remaining plants were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (1.1 and 18 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 100, 98, 100, 88 and 73%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent survival among plants exposed to the 280 g cation/ha application rate compared to the control
- Shoot length: Day 21 mean shoot length in the control was 30.4 cm. Shoot length for plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 31.4, 28.8, 28.8, 12.9 and 13.9 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 0.1050 g. Shoot dry weight for plants exposed to the 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 0.1262, 0.0930, 0.1053, 0.0186 and 0.0188 g, respectively. Wilcoxon’s Test with Bonferroni-Holm’s Adjustment determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 70 g cation/ha compared to the control.

LOLIUM PERENNE
- Appearance: Several dead plants each were observed in the 35 and 70 g cation/ha application rates. All surviving plants in the 35 and 70 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (2.2, 4.4, 8.8 and 18 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 2.2, 4.4, 8.8, 18, 35 and 70 g cation/ha application rates averaged 100, 100, 100, 100, 83 and 8%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent survival among plants exposed to application rates ≥ 35 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 34.2 cm. Shoot length for plants exposed to the 2.2, 4.4, 8.8, 18, 35 and 70 g cation/ha application rates averaged 34.7, 34.9, 33.4, 32.1, 23.3 and 13.8 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates ≥ 35 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 0.2130 g. Shoot dry weight for plants exposed to the 2.2, 4.4, 8.8, 18, 35 and 70 g cation/ha application rates averaged 0.2395, 0.1981, 0.2029, 0.2122, 0.0862 and 0.0099 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 35 g cation/ha compared to the control.

RAPHANUS SATIVUS
- Appearance: One dead plant each was observed in the 70 and 140 g cation/ha application rates. Two dead plants were observed in the 280 g cation/ha application rate. All surviving plants exposed to the 70, 140 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (8.8, 18 and 35 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 8.8, 18, 35, 70, 140 and 280 g cation/ha application rates averaged 100, 100, 100, 98, 98 and 93%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 4.9 cm. Shoot length for plants exposed to the 8.8, 18, 35, 70, 140 and 280 g cation/ha application rates averaged 4.7, 4.7, 4.4, 3.9, 3.8 and 3.5 cm, respectively. William’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed application rates ≥ 35 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 0.4432 g. Shoot dry weight for plants exposed to the 8.8, 18, 35, 70, 140 and 280 g cation/ha application rates averaged 0.4600, 0.4739, 0.4584, 0.3688, 0.4243 and 0.3732 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight (e.g.17 and 16% inhibition respectively) among plants exposed to the 70 and 280 g cation/ha application rates tested compared to the control. Inhibition of 4% was observed at the intermediate application rate of 140 g cation/ha and was not significantly reduced relative to the control.

GLYCINE MAX
- Appearance: Two dead plants were observed in the 1.1 g cation/ha application rate. Several dead plants and several necrotic plants were observed in the 18 g cation/ha application rate. Several dead plants were observed in the 70 and 280 g cation/ha application rates. All surviving plants in the 70 and 280 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (0.27 and 4.4 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 100, 95, 100, 90, 80 and 35%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent survival among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 89.9 cm. Shoot length for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 96.1, 79.8, 83.1, 72.1, 54.8 and 48.4 cm, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot length among plants exposed to application rates ≥ 18 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 1.8650 g. Shoot dry weight for plants exposed to the 0.27, 1.1, 4.4, 18, 70 and 280 g cation/ha application rates averaged 2.1057, 1.7411, 2.0044, 1.9068, 1.1644 and 1.1837 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 70 g cation/ha compared to the control.

LYCOPERSICON ESCULENTUM
- Appearance: All plants exposed to the 70 g cation/ha application rate were observed to be necrotic. Several dead plants were observed in the 140, 280 and 560g cation/ha application rates. All surviving plants in the 140, 280 and 560 g cation/ha application rates were observed to be necrotic. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (18 and 35 g cation/ha).
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha application rates averaged 100, 100, 100, 83, 45 and 15%, respectively. Cochran-Armitage Trend Step-Down Test determined a significant reduction in percent survival among plants exposed application rates ≥ 140 g cation/ha compared to the control.
- Shoot length: Day 21 mean shoot length in the control was 35.9 cm. Shoot length for plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha averaged 34.2, 35.1, 28.5, 28.1, 28.1 and 21.0 cm, respectively. Wilcoxon’s Test with Bonferroni-Holm’s Adjustment determined a significant reduction in shoot length among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
- Shoot dry weight: Day 21 mean shoot dry weight in the control was 1.4954 g. Shoot dry weight for plants exposed to the 18, 35, 70, 140, 280 and 560 g cation/ha application rates averaged 1.4187, 1.3504, 0.8539, 0.8287, 0.6150 and 0.5278 g, respectively. William’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to application rates ≥ 70 g cation/ha compared to the control.
Reported statistics and error estimates:
Observations of morphological abnormalities and phytotoxic effects were reported as the percentage of the plants exhibiting the effect. Percent inhibition of the treatment data was calculated relative to the control data.

The NOEC on day 21 percent survival, shoot length and shoot dry weight when compared to the control data was determined. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002) or Cochran-Armitage Trend Step-Down Test (U.S. EPA, 2002) were used to establish treatment effects for percent survival data. Shoot length and shoot dry weight data were first checked for normality using Shapiro-Wilks' Test (U.S. EPA, 2002) and for homogeneity of variance using Bartlett's Test (U.S. EPA, 2002). If the data sets passed the tests for homogeneity and normality, Dunnett's Multiple Comparison Test (U.S. EPA, 2002) or William’s Multiple Comparison Test (U.S. EPA, 2002) was used to determine the NOEC. If the data did not pass the tests for homogeneity or normality, then Wilcoxon’s Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002) was used to determine the NOEC. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' Test and Bartlett's Test, where the 99% level of certainty was applied.

The EC25 value (the concentration of test substance which reduced percent survival, shoot length or shoot dry weight by 25%) and 95% confidence limits were determined compared to the control when ≥ 25% inhibition was observed. The EC values and their 95% confidence limits were determined by linear interpolation of the biological response versus the nominal application rate (Norberg-King, 1993). CETIS™ Version 1.8 (Ives, 2013) was used to assist in these computations. If less than the designated percent inhibition was observed for the noted parameter at the highest application rate tested, the EC value was empirically estimated to be greater than the highest application rate tested.

Table: Results preliminary study

Nominal application rate (g cation/ha)

Endpoint

21-Day percent inhibition(a)

Common bean

Corn

Cucumber

Oats

Oilseed raoe

Onion

Perennial ryegrass

Radish

Soybean

Tomato

1.12

Survival

0

0

0

0

0

0

0

0

0

0

Shoot length

14

-5

-5

-1

5

1

-2

-3

11

-2

Dry weight

18

-8

-1

-2

23

-7

4

9

18

1

11.2

Survival

0

0

0

0

0

0

0

0

0

0

Shoot length

12

-18

-9

-9

3

21

16

11

-5

-20

Dry weight

17

-9

27

0

35

32

33

7

24

6

112

Survival

0

8

83

8

0

8

42

8

25

42

Shoot length

22

3

12

22

1

24

50

24

41

8

Dry weight

65

38

40

33

36

49

88

32

67

38

1120

Survival

75

100

100

100

100

100

100

100

100

100

Shoot length

36

NA(b)

NA

NA

NA

NA

NA

NA

NA

NA

Dry weight

50

NA

NA

NA

NA

NA

NA

NA

NA

NA

a) Percent inhibition relative to the control

b) NA = Not Applicable. No surviving plants were observed in this treatment level.

Table: NOEC and EC25 values of all species tested

Species

Biological parameter

Based on nominal application rates (g cation/ha)

EC25

NOEC

Phaseolus vulgaris

21-Day Percent Survival

> 280

280

21-Day Shoot Length

> 280

18

21-Day Shoot Dry Weight

44

18

Zea mays

21-Day Percent Survival

96

35

21-Day Shoot Length

66

18

21-Day Shoot Dry Weight

27

18

Cucumus sativa

21-Day Percent Survival

51

18

21-Day Shoot Length

> 280

280

21-Day Shoot Dry Weight

> 280

280

Avena sativa

21-Day Percent Survival

110

70

21-Day Shoot Length

68

35

21-Day Shoot Dry Weight

49

35

Brassica napus

21-Day Percent Survival

> 280

280

21-Day Shoot Length

> 280

70

21-Day Shoot Dry Weight

34

18

Allium cepa

21-Day Percent Survival

220

70

21-Day Shoot Length

30

18

21-Day Shoot Dry Weight

22

18

Lolium perenne

21-Day Percent Survival

38

18

21-Day Shoot Length

29

18

21-Day Shoot Dry Weight

22

18

Raphanus sativus

21-Day Percent Survival

> 280

280

21-Day Shoot Length

170

18

21-Day Shoot Dry Weight

> 280

35

Glycine max

21-Day Percent Survival

82

18

21-Day Shoot Length

22

4.4

21-Day Shoot Dry Weight

39

18

Lycopersicon esculentum

21-Day Percent Survival

160

70

21-Day Shoot Length

310

35

21-Day Shoot Dry Weight

48

35

 

Calculation of key result

The doses of the test substance were expressed in the test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

EC25: (100/72.4) x 22 g test substance cation/ha = 30.4 g pure tests substance/ha.

NOEC: (100/72.4) x 4.4 g test substance cation/ha = 6.1 mg pure test substance/ha.

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 21-d EC25 was determined to be 22 g cation/ha in Lolium perenne, Allium cepa (both monocots) and Glycine max (dicot). This value corresponds to a recalculated value of 30.4 g/ha pure substance.
Executive summary:

The toxicity of the test substance to plants was determined in a GLP-compliant study in accordance with OECD TG 227. In this study, ten plant species tested were (40 seedlings/treatment): Phaseolus vulgaris (common bean), Zea mays (corn), Cucumus sativa (cucumber), Avena sativa (oats), Brassica napus (oilseed rape), Allium cepa (onion), Lolium perenne (perennial ryegrass), Raphanus sativus (radish), Glycine max (soybean) and Lycopersicon esculentum (tomato). Plants were exposed to the test substance at nominal application rates ranging from 0.27 to 560 g cation/ha. The maximum recommended application rate of the test substance is 1120 g cation/ha. The concentration of free cation was analytically verified in the stock solutions used to over-spray the seedlings on day 0. Since the measured stock solution concentrations approximated the desired nominal stock concentrations, indicating the stock solutions applied to the plants was properly prepared, the results of this study are based on nominal application rates. Effects on plant health and survival were evaluated 7, 14 and 21 days (test termination) after the application of the test substance to the plant foliage. Shoot length was determined on test days 14 and 21 and shoot dry weight was evaluated on day 21 by harvesting, drying and determining shoot dry weight by replicate. Observations of morphological abnormalities and phytotoxic effects were reported as the percentage of the plants exhibiting the effect. The EC25 as well as the NOEC values were determined.

Based on a comparison of the EC25 values, the most sensitive monocotyledon species tested were perennial ryegrass (Lolium perenne) and onion (Allium cepa), both with an EC25 value of 22 g cation/ha for shoot dry weight. The most sensitive dicotyledon species tested was soybean (Glycine max), with an EC25 of 22 g cation/ha for shoot length. In general, the monocotyledons tested were more sensitive to the test substance than the dicotyledons tested. The responses for bean, cucumber, oilseed rape and radish provided only are EC25 values for the three parameters measured, with the remaining EC25 values being greater than the highest application rate tested for the species. Since the species were not the most sensitive species tested, no further testing at higher application rates was conducted. Based on the results, the 21-d EC25 was determined to be 22 g cation/ha, which corresponds to a recalculated value of 30.4 g/ha pure substance.

Endpoint:
toxicity to terrestrial plants: short-term (with study design considered suitable for long-term assessment)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2013 to 30 Sep 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.4100 (Terrestrial Plant Toxicity Tier I (seedling emergence))
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
A sample of each spray stock solution was collected on the day of test substance application, prior to, and subsequent to application, for analysis of the test substance cation species concentration.
Vehicle:
no
Details on preparation and application of test substrate:
A 12 mg cation/mL primary spray stock solution was prepared by placing 26.7961 g of test substance (6.0023 g as active ingredient) in a 500-mL volumetric flask and bringing it to volume with deionized water. The resultant solution was observed to be clear and dark green in color following preparation. Secondary spray stock solutions were prepared from the 12 mg cation/mL primary spray stock solution.
Species:
Zea mays
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: corn
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oats
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: perennial ryegrass
Species:
Phaseolus vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: common bean
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: cucumber
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: oilseed rape
Species:
Raphanus sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: radish
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: soybean
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: tomato
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
19 - 40 °C
Details on test conditions:
TEST SYSTEM
- Testing facility: The study was conducted in a greenhouse covered with Lexan® (polycarbonate) panels.
- Test container: The exposure vessels consisted of polypropylene pots (Kord Products Ltd.). The pots were 12-cm tall with a top diameter of 14 cm and a bottom diameter of 12 cm. The interior base was fitted with 20 cm filter paper (Rockline, Inc.) to retain the support medium and allow for plant uptake of solution by sub-irrigation.
- Amount of soil: Each pot was filled to a depth of 10 cm with approximately 1.2 kg of untreated support medium (diameter at surface of the support medium = 12 cm; surface area = 104 cm2). Each pot was placed in a polypropylene saucer (Kord Products Ltd.) and received approximately 100 mL of 400 mg/L Peters 20-20-20 fertilizer solution prepared with deionized water and provided to the plants via sub-irrigation
- Method of seeding: Seeds were impartially selected and planted at a depth of approximately 1 to 2 cm below the surface of the support substrate in each pot (40 seeds per application rate and the control).
- No. of seeds per container: 4 (common bean, corn, cucumber, oilseed rape, radish, soybean and tomato) and 8 (oats, onion and perennial ryegrass)
- No. of replicates per treatment group: 5 (oat, onion and perennial ryegrass) and 10 (common bean, corn, cucumber, radish, oilseed rape, soybean and tomato)
- No. of replicates per control: 5 (oat, onion and perennial ryegrass) and 10 (common bean, corn, cucumber, radish, oilseed rape, soybean and tomato)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Preparation: Loam was collected from Rochester, Massachusetts.
- Pesticide use history at the collection site: A representative sample of the loam was analyzed for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected at concentrations that would compromise the results of this study.
- % sand: 91
- % silt: 7
- % clay: 2
- Soil classification system: USDA
- Composition (if artificial substrate): Washed silica sand was combined with loam to produce a mixture containing 70% sand and 30% loam.
- Organic matter (%): 0.9

NUTRIENT MEDIUM
- Description: The plants were sub-irrigated using 400 mg/L Peters 20-20-20 solution prepared in deionized water. Approximately 100 mL was provided to all pots by sub-irrigation approximately twice weekly. All additional watering was provided using deionized water.

GROWTH CONDITIONS
- Photoperiod: The photoperiod was 16 hours of light and 8 hours of darkness.
- Light intensity and quality: Sodium vapor lights supplemented natural light when necessary to maintain > 800 footcandles (equivalent to > 105 μmol/m2/s) during the light period.
- Air pollution: The potential for air pollution within the greenhouse is believed to be minimal due to the rural location of the laboratory and the lack of other industrial businesses in the area. The greenhouse is located in a relatively isolated section of the laboratory grounds, which reduces the possibility of air contamination from concurrent testing.

TEST CONDITION MEASUREMENTS
Air temperature within the greenhouse was controlled using thermostatically-regulated heating and ventilation systems. Air temperature and relative humidity was constantly monitored using a Hobo data logger to obtain the day/night temperature range. Light intensity was measured daily at each bench using an Apogee radiometer/photometer held at average maximum leaf height for each species. Light intensity (photosynthetically-active radiation, PAR) was measured in μmol/m2/sec.

EFFECT PARAMETERS MEASURED:
Each pot was observed on days 0, 7 and 14 days (test termination) after 50% of the control seeds had emerged to determine percent emergence and to record any morphological abnormalities (e.g., chlorosis of leaves) of the emerged shoots. Observations of morphological abnormalities were reported as the percentage of plants exhibiting the abnormality. Shoot length was measured at test termination. Dicotyledons were measured to the tallest new growth and monocotyledons were measured to the longest leaf length using a ruler and measuring to the nearest 0.5 cm. Additionally at test termination (21 days post treatment), the shoots in a replicate pot were cut at the soil surface and placed in a labeled, pretared container. Shoots were dried in an oven at 70 ± 5 ºC for a minimum of three days before determining shoot dry weights. Weights were measured using a four place balance (Sartorius) to the nearest 0.1 mg. Plants that died during the exposure were not included in statistical calculations.

RANGE-FINDING STUDY
A preliminary 14-day range-finding seedling emergence test was conducted exposing Phaseolus vulgaris (common bean), Zea mays (corn), Cucumus sativa (cucumber), Avena sativa (oat), Brassica napus (oilseed rape), Allium cepa (onion), Lolium perenne (perennial ryegrass), Raphanus sativus (radish), Glycine max (soybean) and Lycopersicon esculentum (tomato) to nominal application rates of 1.12, 11.2, 112 and 1120 g cation/ha and a control (three replicates per application rate, four seeds per replicate). Following 14 days of exposure, percent inhibition of emergence, survival, shoot length and dry weight were determined.
- Results: Based on these results (inhibition generally of < 25% in the endpoints at 1120 g cation/ha), Tier II tests for all ten species tested were conducted. Tier II testing was conducted at nominal application rates of 8.8, 18, 70, 280 and 1120 g cation/ha (for corn and oilseed rape); and 70, 140, 280, 560 and 1120 g cation/ha (for common bean, cucumber, oat, onion, radish, perennial ryegrass, soybean and tomato).
Nominal and measured concentrations:
Nominal application rates were selected based on preliminary testing:
- Phaseolus vulgaris (Common Bean): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Zea mays (Corn): 0 (control) 8.8, 18, 70, 280 and 1120 g cation/ha
- Cucumus sativa (Cucumber): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Avena sativa (Oats): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Brassica napus (Oilseed Rape): 0 (control) 8.8, 18, 70, 280 and 1120 g cation/ha
- Allium cepa (Onion): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Lolium perenne (Perennial Ryegrass): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Raphanus sativus (Radish): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Glycine max (Soybean): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha
- Lycopersicon esculentum (Tomato): 0 (control) 70, 140, 280, 560 and 1120 g cation/ha

The recommended application volume was 10 gallons/A. The test substance stock solutions were applied to the seedlings by calibrating the spray apparatus to deliver the stock solutions at an application volume of 10 gallons/A, which is equivalent to 93.5 L/ha.

- Measured concentrations: Measured concentrations were approximately 110% of nominal concentration during pre-spray application analysis and ranged from 110 to 120% of nominal concentration during post-spray application analysis. These results closely approximate the desired nominal concentrations and indicate that the spray stock solutions were properly prepared and that test substance concentrations were stable during the application process.
Reference substance (positive control):
no
Key result
Species:
Lolium perenne
Duration:
14 d
Dose descriptor:
EC25
Effect conc.:
1.3 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: pure test substance
Basis for effect:
other: shoot dry weight
Remarks on result:
other: Recalculated value expressed in mg pure substance/kg soil dw
Species:
Lolium perenne
Duration:
14 d
Dose descriptor:
EC25
Effect conc.:
980 g/ha
Nominal / measured:
nominal
Conc. based on:
other: pure test substance
Basis for effect:
other: shoot dry weight
Remarks on result:
other: Recalculated value, expressed as pure substance. See 'Any other information on results incl. tables' for calculation and a complete overview of the effect values per species
Species:
Lolium perenne
Duration:
14 d
Dose descriptor:
EC25
Effect conc.:
710 g/ha
Nominal / measured:
nominal
Conc. based on:
other: test substance cation species
Basis for effect:
other: shoot dry weight
Remarks on result:
other: original value as presented in the study report
Details on results:
PHASEOLUS VULGARIS
- Abnormalities: One seed each in the control and the 280 and 1120 g cation/ha application rates did not emerge. Two seeds in the 70 g cation/ha application rate did not emerge. One dead plant was observed in the 1120 g cation/ha application rate. No dead plants or plants with morphological abnormalities were observed in the remaining application rates tested (140 and 560 g cation/ha).
- Emergence: Percent emergence in the control averaged 98%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 95, 100, 98, 100 and 98%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 100, 100, 100, 100 and 98%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 16.2 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 18.3, 18.8, 18.6, 18.9 and 18.5 cm, respectively. Wilcoxon’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.3454 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.3569, 0.3560, 0.3583, 0.3330 and 0.3536 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

ZEA MAYS
- Abnormalities: One seed each in the control and the 18 g cation/ha application rate did not emerge. Two seeds in the 280 g cation/ha application rate did not emerge. Several seeds in the 8.8, 70 and 1120 g cation/ha application rates tested did not emerge. Two dead plants were observed in the 18 g cation/ha application rate.
- Emergence: Percent emergence in the control averaged 98%. The day 14 percent emergence among plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 90, 98, 93, 95 and 93%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 100, 94, 100, 100 and 100%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 41.3 cm. Shoot length for plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 41.3, 44.0, 40.7, 41.1 and 45.4 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.2499 g. Shoot dry weight for plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 0.2638, 0.2604, 0.2492, 0.2476 and 0.2899 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

CUCUMUS SATIVA
- Abnormalities: Several seeds in the control and all application rates tested did not emerge. One and two dead plants, respectively, were observed in the 140 and 280 g cation/ha application rates. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested (70, 560 and 1120 g cation/ha).
- Emergence: Percent emergence in the control averaged 85%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 85, 78, 65, 90 and 88%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 100, 95, 94, 100 and 100%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 7.0 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 7.2, 7.0, 5.9, 7.0 and 6.6 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.1783 g. Shoot dry weight for plants exposed to the 7.2, 7.0, 5.9, 7.0 and 6.6 g cation/ha application rates averaged 0.1799, 0.1755, 0.1667, 0.1895 and 0.1740 g, respectively. Wilcoxon’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

AVENA SATIVA
- Abnormalities: Two seeds in the control did not emerge. Several seeds in all application rates tested (70, 140, 280, 560 and 1120 g cation/ha) did not emerge. No dead plants or plants with morphological abnormalities were observed in the control or any of the application rates tested.
- Emergence: Percent emergence in the control averaged 95%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 88, 80, 80, 75 and 73%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined a significant reduction in percent emergence for plants exposed to the 1120 g cation/ha application rate compared to the control.
- Survival: Percent survival in the control and all application rates tested averaged 100%. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 27.2 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 28.4, 28.8, 25.1, 26.6 and
26.8 cm, respectively. Dunn’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.0556 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.0576, 0.0619, 0.0572, 0.0529 and 0.0563 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

BRASSICA NAPUS
- Abnormalities: One seed in the 280 g cation/ha application rate did not emerge. Two seeds each in the 70 and 1120 g cation/ha application rate did not emerge. Several seeds in the control and the remaining application rates tested (8.8 and 18 g cation/ha) did not emerge. One dead plant was observed in the 1120 g cation/ha application rate. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested.
- Emergence: Percent emergence in the control averaged 90%. The day 14 percent emergence among plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 90, 90, 95, 98 and 95%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 100, 100, 100, 100 and 98%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 7.4 cm. Shoot length for plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 7.8, 7.5, 7.5, 7.3 and 7.7 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.1439 g. Shoot dry weight for plants exposed to the 8.8, 18, 70, 280 and 1120 g cation/ha application rates averaged 0.1415, 0.1319, 0.1384, 0.1318 and 0.1447 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

ALLIUM CEPA
- Abnormalities: Several seeds in the control and all application rates tested (70, 140, 280, 560 and 1120 g cation/ha) did not emerge. No dead plants or plants with morphological abnormalities were observed in the control or any of the application rates tested.
- Emergence: Percent emergence in the control averaged 88%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 85, 83, 88, 83 and 80%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control and all application rates tested averaged 100%. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 13.7 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 13.0, 11.8, 13.8, 12.3 and 12.7 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.0108 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.0100, 0.0085, 0.0110, 0.0092 and 0.0095 g, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in shoot dry weight among plants exposed to the 140 g cation/ha application rate compared to the control. However, due to the lack of statistical significance at higher application rates, the effect observed at the 140 g cation/ha application rate was not considered to be toxicant related.

LOLIUM PERENNE
- Abnormalities:Several seeds in the control and all application rates tested (70, 140, 280, 560 and 1120 g cation/ha) did not emerge. No dead plants or plants with morphological abnormalities were observed in the control or any of the application rates tested.
- Emergence: Percent emergence in the control averaged 80%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 80, 88, 75, 78 and 60%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control and all application rates tested averaged 100%. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 16.6 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 17.8, 19.7, 16.6, 18.9 and 14.0 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.0084 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.0122, 0.0147, 0.0083, 0.0121 and 0.0062 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

RAPHANUS SATIVUS
- Abnormalities: Several seeds in the control and all application rates tested (70, 140, 280, 560 and 1120 g cation/ha) did not emerge. One dead plant was observed in the 1120 g cation/ha application rate. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested.
- Emergence: Percent emergence in the control averaged 90%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 93, 88, 90, 93 and 90%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 100, 100, 100, 100 and 97%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 2.7 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 2.7, 2.7, 2.6, 3.0 and 3.1 cm, respectively. Dunn’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.1389 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.1507, 0.1449, 0.1356, 0.1412 and 0.1455 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

GLYCINE MAX
- Abnormalities: One seed each in the 280 and the 1120 g cation/ha application rates did not emerge. Two seeds each in the control and the 70 g cation/ha application rate did not emerge. Several seeds in the 140 and 560 g cation/ha application rates did not emerge. No dead plants or plants with morphological abnormalities were observed in the control or any of the application rates tested.
- Emergence: Percent emergence in the control averaged 95%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 95, 93, 98, 93 and 98%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control and all application rates tested averaged 100%. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 24.8 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 23.1, 23.1, 22.6, 24.2 and 24.7 cm, respectively. Dunn’s Test with Bonferroni-Holm’s Adjustment determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.3660 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.3164, 0.3431, 0.3362, 0.3375 and 0.3403 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.

PERSICON ESCULENTUM
- Abnormalities: Several seeds in the control and all application rates tested (70, 140, 280, 560 and 1120 g cation/ha) did not emerge. One dead plant was observed in the 560 g cation/ha application rate. No dead plants or plants with morphological abnormalities were observed in the control or the remaining application rates tested.
- Emergence: Percent emergence in the control averaged 85%. The day 14 percent emergence among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 80, 80, 90, 90 and 80%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent emergence for plants exposed to any of the application rates tested compared to the control.
- Survival: Percent survival in the control averaged 100%. Percent survival among plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 100, 100, 100, 97, and 100%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in percent survival among plants exposed to any of the application rates tested compared to the control.
- Shoot length: Day 14 mean shoot length in the control was 7.1 cm. Shoot length for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 6.8, 7.2, 6.7, 7.4 and 6.8 cm, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot length among plants exposed to any of the application rates tested compared to the control.
- Shoot dry weight: Day 14 mean shoot dry weight in the control was 0.0589 g. Shoot dry weight for plants exposed to the 70, 140, 280, 560 and 1120 g cation/ha application rates averaged 0.0576, 0.0643, 0.0554, 0.0751 and 0.0680 g, respectively. Dunnett’s Multiple Comparison Test determined no significant reduction in shoot dry weight among plants exposed to any of the application rates tested compared to the control.
Reported statistics and error estimates:
The No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p < 0.05) on day 14 percent emergence, percent survival, shoot length and shoot dry weight when compared to the control data was determined. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002) was used to establish treatment effects for percent emergence and percent survival data. Shoot length and shoot dry weight data were first checked for normality using Shapiro-Wilks' Test (U.S. EPA, 2002) and for homogeneity of variance using Bartlett's Test (U.S. EPA, 2002). If the data sets passed the tests for homogeneity and normality, Dunnett's Multiple Comparison Test (U.S. EPA, 2002) was used to determine the NOEC. If the data did not pass the tests for homogeneity or normality, then Wilcoxon’s Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002) or Dunn’s Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002) was used to determine the NOEC. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' Test and Bartlett's Test, where the 99% level of certainty was applied. The EC25 value (the concentration of test substance which reduced percent emergence, percent survival, shoot length or shoot dry weight by 25%) and 95% confidence limits were determined compared to the control when ≥ 25% inhibition was observed. The EC value and 95% confidence limits were determined by linear interpolation of the biological response versus the nominal application rate (Norberg-King, 1993). CETISTM Version 1.8 (Ives, 2013) was used to assist in these computations. If less than the designated percent inhibition was observed for the noted parameter at the highest application rate tested, the EC value was empirically estimated to be greater than the highest application rate tested.

Calculation of key result

The doses of the test substance were expressed as cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

EC25: (100/72.4) x 710 g test substance cation/ha = 980 g pure test substance/ha.

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 14-d EC25 was determined to be 710 g test substance cation/ha in Lolium perenne based on its shoot dry weight results. This value corresponds to a recalculated value of 980 g pure substance/ha, or 1.3 mg pure substance/kg soil dw.
Executive summary:

The toxicity of the test substance to plants was determined in a GLP-compliant study in accordance with OCSPP 850.4100. In this study, ten plant species were

tested (40 seedlings/treatment): Phaseolus vulgaris (common bean), Zea mays (corn), Cucumus sativa (cucumber), Avena sativa (oats), Brassica napus (oilseed rape), Allium cepa (onion), Lolium perenne (perennial ryegrass), Raphanus sativus (radish), Glycine max (soybean) and Lycopersicon esculentum (tomato). Plants were exposed to the test substance at nominal application rates ranging from 8.8 to 1120 g cation/ha (maximum recommended application rate of the test substance). The concentration of free cation was analytically verified in the stock solutions used to over-spray the seedlings on day 0. Since the measured stock solution concentrations approximated the desired nominal stock concentrations, indicating the stock solutions applied to the plants was properly prepared, the results of this study are based on nominal application rates. Seeds were planted, the soil surface was oversprayed with the test substance, and observations were made 7 and 14 days (termination) after ≥ 50% emergence of the control seedlings occurred. Effects on seedling emergence and survival were evaluated and shoot length measurements were taken at the above intervals. At test termination, after length measurements were taken and the shoots were harvested, plants were dried and shoot dry weight per replicate was determined.

The most sensitive monocotyledon species tested was Lolium perenne (perennial ryegrass), with an EC25 value of 710 g cation/ha for shoot dry weight. All dicotyledon species tested were not affected by the test substance concentrations tested, with an empirically estimated EC25 value of > 1120 g cation/ha, the maximum recommended application rate and the highest application rate tested. Based on the results, the 14-d EC25 was determined to be 710 g cation/ha, which corresponds to a recalculated value of 980 g/ha pure substance. Accordingly, this value is converted to 1.3 mg pure substance/kg soil dw, which is used as effect value.

Description of key information

The 14-d EC25 value is 1.3 mg pure substance/kg soil dw (recalculated), corresponding to 980 g pure substance/ha, determined in a seedling emergence test, OPPTS 850.4100, Martin, 2014.

In the vegetative vigour test, the 21-d EC25 value is 30.4 g pure substance/ha soil dw (recalculated), OECD TG 227, Martin, 2014. This test was not considered relevant for the test substance and therefore not used in the risk assessment.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:
1.3 mg/kg soil dw

Additional information

Table: Overview of available data on the toxicity to plants

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Monocots:

Zea mays (corn)

Avena sativa (oat)

Allium cepa (onion)

Lolium perenne (perennial ryegrass)

Dicots:

Phaseolus vulgaris (common bean)

Cucumis sativa (cucumber)

Brassica napus (oilseed rape)

Raphanus sativus (radish)

Glycine max (soybean)

Lycopersicon esculentum (tomato)

OPPTS 850.4100 (seedling emergence test) / GLP

14-d EC25

1.3 mg/kg soil dw

(corresponding to 980 g pure substance/ha)

(recalculated)

 

Lolium perenne was the most sensitive species

Martin, 2014