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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June 2013 to 23 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Remarks:
At the request of the sponsor, the application rate was separated by a factor of 4 to cover the necessary range of concentrations. This differs from the recommended spacing of 3, specified in the OECD guideline.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The highest test concentration was prepared by grinding the test item to a uniform fine powder in a pestle and mortar. An aliquot of 40.8991 g of the ground test item was weighed and mixed homogenously with sand to give 1000 g of treated sand. Using a separation factor of 4, the lower concentrations were prepared by serial dilution of treated sand from a higher rate and mixing homogenously with untreated sand. A second series of test substrates, using an initial highest concentration of 6135g of ground test item made up to a total of 1000 g of treated sand, was prepared for Glycine max (Soya bean) due to the original application failing the validity criteria as the untreated controls exhibited visible phytotoxic effects for the species. The final treated soil was prepared by mixing the 500g of treated sand with 6.15 kg of untreated sand and 0.8 litre of water using a cement mixer. 13.35 kg of loam was then added and mixed thoroughly to give a final soil mix of 20 kg. For the second series of test substrates, 500 g of treated sand was mixed with 0.5 kg of untreated sand, 0.15 litre of water and 2.0 kg of loam to give a final soil mix of 3.0 kg.
- Controls: The untreated control which was prepared first as above except that 6.65 kg (first application) or 1 kg (second application) of untreated sand was added to the soil mix.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable, no vehicle used
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Onion
- Plant family: Liliaceae
- Variety: White Lisbon
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
- Plant family: Poaceae
- Variety: Dalguise
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Ryegrass
- Plant family: Poaceae
- Variety: Not applicable
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: not reported
Species:
Zea mays
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Maize
- Plant family: Poaceae
- Variety: Minipop
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Beta vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: sugar beet
- Plant family: Chenopodiaceae
- Variety: Boogie
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Oilseed rape
- Plant family: Brassicaceae
- Variety: Astrid
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Daucus carota
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Carrot
- Plant family: Apiaceae
- Variety: Early Nantes
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: not reported
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soya bean
- Plant family: Fabaceae
- Variety: Elena
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Lettuce
- Plant family: Asteraceae
- Variety: Little Gem
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Solanaceae
- Variety: Moneymaker
- Source of seed: Commercial supplier
- Prior seed treatment/sterilization: None
- Historical germination of seed (germination of seed lot tested): Not reorted
- Seed storage: Not reported
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
21 d
Remarks:
The test duration was 21 days after 50% emergence in the controls for all species tested except onion, which continued until 24 days after 50% emergence in the controls.
Post exposure observation period:
Not applicable
Test temperature:
20/06/13 - 22/07/13 Max: 31.5°C Min: 10.9 °C, Mean: 20.5 °C
27/07/13 - 19/08/13 Max: 33.4°C Min: 12.6 °C, Mean: 20.9 °C
pH:
7.8
Moisture:
20/06/13 - 22/07/13 Max: 91.0 % Min: 27.9 %, Mean: 66.5 %
27/07/13 - 19/08/13 Max: 94.1 % Min: 27.7 %, Mean: 68.6 %
Details on test conditions:
TEST SYSTEM
- Testing facility: Greenhouse at test facility
- Test container (type, material, size): Non-porous plastic pots , 8x8x8 cm
- Amount of soil: 370 g of soil mix per pot
- Method of seeding: Seeds sown directly into pots at a depth of 1 - 2 cm
- No. of seeds per container: Four seeds ( 6 for onion)
- No. of plants (retained after thinning) : No thinning reported
- No. of replicates per treatment group: Five
- No. of replicates per control: Five
- No. of replicates per vehicle control: Not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Not applicable, artificial soil
- Pesticide use history at the collection site: Not applicable, artificial soil
- Collection procedures: Not applicable, artificial soil
- Sampling depth (cm): Not applicable, artificial soil
- Composition (if artificial substrate): sand: 62 % w/w (2.00 - 0.063 mm),silt: 21 % w/w (0.063 - 0.002 mm), clay: 17 % w/w (< 0.002 mm)
- Organic carbon (%): 1.1 % w/w (by wet oxidation)
- Maximum water holding capacity (in % dry weight): Not reported
- CEC: 17.9 meq/100g
- Pretreatment of soil: Not reported
- Storage (condition, duration): Not reported

GROWTH CONDITIONS
- Photoperiod: Ambient light for at least 16 hours
- Light source: Natural sunlight
- Light intensity and quality: Max: 59.6 kilo lux (Kl) (66.5 Kl) , mean ambient light intensity for the period was 18.6 Kl. (16.6 Kl) The light level for the duration of the study was well in excess of the 2000 lux, giving good growing conditions for the plants.
- Day/night temperatures: 20/06/13 - 22/07/13 Max: 31.5°C Min: 10.9 °C, Mean: 20.5 °C. 27/07/13 - 19/08/13 Max: 33.4°C Min: 12.6 °C, Mean: 20.9 °C
- Relative humidity (%): 20/06/13 - 22/07/13 Max: 91.0 % Min: 27.9 %, Mean: 66.5 % 27/07/13 - 19/08/13 Max: 94.1 % Min: 27.7 %, Mean: 68.6 %
- Wind velocity: Not applicable
- Air exchange rate: Not applicable
- Watering regime and schedules:
- Water source/type: Not reported
- Method of application: All pots were placed in saucers filled with enough water to ensure that the pots were kept moist at all times.
- Any pest control method/fertilization (if used): None

ACCLIMATION PERIOD: Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The plants were assessed at 7, 14 and 21 days after 50% emergence in controls (10, 17 and 24 days for onion) for emergence, mortality and visual phytotoxicity expressed as a percentage of healthy untreated control plants.

The height of all live plants above soil level to the top of the tallest leaf was recorded in cm at the final assessment. The height and dry weight of dead plants were not measured or weighed but were recorded as “0”. All plants in one treatment pot were cut at soil level and placed in paper bags for drying. This procedure was repeated for all the treatment pots in the five replicates of a species. Dead plants were not harvested. The species were dried in an oven for following number of days: Allium cepa (Onion), Glycine max (Soya bean) and Lycopersicon esculentum (Tomato) were dried in an oven for 2 days, Daucus carota (carrot) was dried in an oven for 3 days and the remaining tested species were dried for 5 days in an oven. All plant material from all the pots was dry. Once the plant material was dry the contents of each bag were weighed.

- Phytotoxicity rating system (if used): 0 %: no phytotoxicity; 1 - 39 %: slight phytotoxicity; 40 -69 %: moderate phytotoxicity: 70 -99% severe phytotoxicity; 100 %: all plants dead

VEHICLE CONTROL PERFORMED: Not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 4
- Justification for using less concentrations than requested by guideline: Not applicable
- Test concentrations: Nominal concentration; 0 ( untreated control), 0.244, 0.977, 3.91, 15.6, 62.5, 250 and 1000 mg test substance /kg soil.
Nominal and measured concentrations:
Nominal concentration; 0 ( untreated control), 0.244, 0.977, 3.91, 15.6, 62.5, 250 and 1000 mg test substance /kg soil dw.
Reference substance (positive control):
no
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Zea mays
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Zea mays
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(height)
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(height)
Species:
Allium cepa
Duration:
24 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Allium cepa
Duration:
24 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(height)
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(height)
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight, height and emergence
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight, height)
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
719 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight)
Remarks on result:
other: 95% CL 566 - 935 mg/kg soil
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight, height)
Species:
Daucus carota
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
550 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight)
Remarks on result:
other: 95% CL 472 - 651 mg/kg soil
Species:
Daucus carota
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight, height)
Species:
Beta vulgaris
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
276 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight)
Remarks on result:
other: 95% Cl: 206 - 384 mg/kg soil
Species:
Beta vulgaris
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight, height)
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
275 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight)
Remarks on result:
other: 95% CL 182 - 446 mg/kg soil
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
(dry weight, height)
Details on results:
SEED GERMINATION
No details reported

SEEDLING EMERGENCE
The EC50 values based on final emergence for all the tested species are greater than the highest concentration of test item tested. In these cases the EC50 values are given as > 1000 mg test substance /kg soil.

VEGETATIVE VIGOUR
Darcus carota (carrot) shows a 62 % reduction as compared to the control in the final dry weight at 1000 mg test substance/kg soil, the highest concentration tested. Using the four top concentrations, which shows a consistent decline in dry weight (15.6, 62.5, 250 and 1000 mg test substance/ kg soil), the EC50 value was calculated as 550 mg test substance/kg soil for final dry weight. However, the most sensitive species tested was Lycopersicon esculentum (tomato) a 82 % reduction at 1000 mg test substance/ kg soil. The calculated EC50 was 275 mg test substance/ kg soil.
Beta vulgaris (sugar beet), Brassica napus (oilseed rape) and Daucus carota (carrot) shows 54 %, 60 % and 55 % reduction in the final height respectively at the highest concentration tested. Using the four top concentrations, which show a consistent decline in final height (15.6, 62.5, 250 and 1000 mg test substance/kg soil); the EC50 value were calculated as 863, 843 and 976 mg test substance/kg soil respectively for final height.
The high tolerance to the test substance meant that the EC50 values based on final dry weight and final height for all the tested species apart from Beta vulgaris (sugar beet), Brassica napus (oilseed rape) and Daucus carota (carrot) and final height for Lycopersicon esculentum (tomato) are greater than the highest concentration of tested.
The final visual phytotoxicity symptoms oberved for all species are shown in the table below.

INJURY RATING SYSTEM:
See above
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The descriptive statistics for calculating Analysis of Variance AOV Means using ARM 8.0 software were Least Significant Difference (LSD) with 5% significance level.
The 50% Effect Concentration (EC50) values were calculated using audited mean values of final height, final emergence and dry weight per treatment and ARM 8.0 software using simple probit – maximum likelihood estimation method with 95% confidence level. The estimation algorithms were provided courtesy of an education establishment..
The No Observed Effect Concentration (NOEC) was the highest concentration of the test substance at which no adverse effect was observed. In this test, the concentration corresponding to the NOER, had no statistically significant adverse effect (p ≤0.05) when compared with the control.
Validity criteria fulfilled:
yes
Remarks:
At least 70% emergence in the controls. Control seedlings did not exhibit any phytotoxic effects. Mean survival of the emerged control seedlings was at least 90% . The environmental conditions were identical for all the tested species.
Conclusions:
Lycopersicon esculentum (tomato) was the most sensitive species, with a 21-day EC50 of 275 mg test substance/kg soil dw based on foliar dry weight with 95% confidence limits of 182 to 446 mg test substance/kg soil dw.
Executive summary:

The effect of the test substance to four monocotyledon and six dicotyledon plant species was measured in a GLP-compliant test following OECD guideline 208. The test substance was pre-mixed in artificial soil at seven nominal test concentrations ranging from 0.244 to 1000 mg test substance/kg soil dw plus a control. The test duration was 21 days (24 days for onion) after 50% emergence in the controls.


The21-day EC50 values range from 275 to >1000 mg test substance/kg soil based on foliar dry weight and from 843 to >1000 mg test substance/kg soil dw based on final plant height. Tomato was the most sensitive species, with a 21-day EC50 of 275 mg test substance/kg soil dw based on foliar dry weight with 95% confidence limits of 182 to 446 mg test substance/kg soil dw.

Description of key information

Lowest 21-day EC50: 275 mg/kg soil dw (OECD TG 208, Bramby-Gunary 2013)

Lowest 21 -day NOEC: 15.6 mg/kg soil dw (OECD TG 208, Bramby-Gunary 2013)

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:
275 mg/kg soil dw
Long-term EC10, LC10 or NOEC for terrestrial plants:
15.6 mg/kg soil dw

Additional information

A single, fully reliable GLP compliant terrestrial plant toxicity study conducted to OECD TG 208 is available. The most sensitive species was tomato with an EC50 of 275 mg/kg soil dw. The NOEC was 15.6 mg/kg soil dw, based on reduced plant growth indicated by reduced dry weight and plant height. The results are considered reliable and adequate for risk assessment. Ten plant species have been tested, and the results of the study thus can be interpreted as information on the long-term toxicity of the substance to terrestrial plants. 

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