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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:
21.02. - 04.07.2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of the application solution were taken and stored deep-frozen and protected from light until analysis.
Vehicle:
yes
Details on preparation and application of test substrate:
- Preparation of the application mixtures:
Due to its low solubility in water, the test item was added to acetone, applied to quartz sand and mixed with the soil. The test item was homogeneously dissolved in the organic solvent. The application solution for the test item concentration was prepared by dissolving 40 mL of the test item completely in acetone to a volume of 80 mL (nominal concentration of the application solution: 500 mg/mL). The density of the test item is 1.003 g/cm. Therefore, the volume of 40 mL corresponded to 40 g test item. For the second test with carrot plants, a volume of 15 mL (15 g) was dissolved in acetone and filled up to a volume of 30 mL (nominal concentration of the application solution: 500 mg/mL).
The application solutions were prepared as follows:
- For the test with onion, wheat, rape, lettuce and soybean: 40 mL test item filled up to a volume of 80 mL. Of the application solution and the control a volume of 40 mL was poured onto 200 g sand to obtain the sand mixtures for the application.
- For the test with carrot: 15 mL test item filled up to a volume of 30 mL. A volume of 7 mL of the application solution or the control was poured onto 35 g sand to obtain the sand mixtures for the application.
The application mixtures were at both application dates clear solutions with a slight yellow coloration.

- Method of mixing into soil:
The solvent was completely evaporated at room temperature under a hood for approximately 3 to 4 hours. Three aliquots of 66.7 g of the sand mixture were incorporated uniformly into 7293 g of soil (corresponding to 6600 g on a dry weight basis) for 30 minutes using a stainless steel planetary mixer. The soil was continuously mixed during application. Then, the three portions were mixed. In this way, the whole lot of soil needed for all plants was treated as one batch. The ratio of sand to soil was 1% (w/w, dry weight) for all treatments including the control.
- Controls:
- Chemical name of vehicle: acetone
- Evaporation of vehicle before use: yes
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Onion
- Plant family: Alliaceae
- Source of seed: Sativa Rheinau GmbH, CH-8462 Rheinau, Switzerland
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Species:
Triticum aestivum
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Wheat
- Plant family: Poaceae
- Source of seed: Sativa Rheinau GmbH, CH-8462 Rheinau, Switzerland
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Oilseed rape
- Plant family: Brassicaceae
- Source of seed: Landi Oberbaselbiet AG, CH-4460 Gelterkinden, Switzerland
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Species:
Daucus carota
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Carot
- Plant family: Umbelliferae
- Source of seed: Sativa Rheinau GmbH, CH-8462 Rheinau, Switzerland
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Lettuce
- Plant family: Compositae
- Source of seed: Sativa Rheinau GmbH, CH-8462 Rheinau, Switzerland
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Leguminosae
- Source of seed: Saatzuchtgenossenschaft Düdingen, CH-3186 Düdingen, Switzerland
- Prior seed treatment/sterilization:
- Historical germination of seed (germination of seed lot tested): The germination rates of the seeds were determined in non-GLP experiments.
- Seed storage: at 2 to 8 °C prior to use.
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
21 d
Test temperature:
between 16 °C and 32 °C during the test
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): Non-porous plastic pots were used (inner diameter 13 cm).
- Amount of soil: 7293 g of soil (corresponding to 6600 g on a dry weight basis)
- Method of seeding:
- No. of seeds per container: Per replicate (pot) 10 seeds were sown for onion and carrot, 8 seeds for wheat, 7 seeds for lettuce and rape and 5 seeds for soybean. In total 30 to 50 individual plants were used per treatment and plant species.
- No. of replicates per treatment group: 6 for soy bean, 5 for other species
- No. of replicates per control: 6 for soy bean, 5 for other species

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Source: Standard soil, supplied by Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), Speyer/Germany.
- Pesticide use history at the collection site: The soil has not been treated with any pesticides or organic fertilizers for at least five years.
- Organic carbon (%): 1.02
- Maximum water holding capacity (in % dry weigth): 35 +/- 3
- Pretreatment of soil: The soil was sieved through a 2-mm sieve by the supplier
- Storage (condition, duration): kept slightly moist in closed plastic boxes or plastic bags at room temperature.

NUTRIENT MEDIUM
- Description: The plants were fertilized by addition of 20 mL nutrient solution per pot at a concentration of 0.8 g nutrient powder/L.

GROWTH CONDITIONS
- Photoperiod: light-dark cycle of 16 hours light and 8 hours darkness.
- Light source: 400 W sodium grow lights (Osram Plantastar E40 4Y) and 400 W metal halogen lamps (Philips MH Britelux)
- Light intensity and quality: The mean values for the photosynthetically active radiation (PAR) ranged from 6 to 210 µmol/m²/s
- Day/night temperatures:
- Relative humidity (%): varying between 15 and 70 %
- Water source/type:
- Volume applied: The application solution for the test item concentration was prepared by dissolving 40 mL of the test item completely in acetone to a volume of 80 mL. The density of the test item is 1.003 g/cm³. Therefore, the volume of 40 mL corresponded to 40 g test item. For the second test with carrot plants, a volume of 15 mL (15 g) was dissolved in acetone and filled up to a volume of 30 mL.
- Interval of applications: approximately 4 weeks
- Method of application: Due to its low solubility in water, the test item was added to acetone, applied to quartz sand and mixed with the soil.

ACCLIMATION PERIOD: no data

EFFECT PARAMETERS MEASURED:
The number of plants emerged per replicate and phytotoxic symptoms (e.g. leaf and stem deformations, chlorosis, necrosis and overall appearance compared with the control) were assessed at Day 7, 14 and 21 after 50 % of the control plants had emerged. At the end of the test (Day 21), the plant height of all individual plants was measured. Determination of shoot dry weight was carried out at the end of the test for all plants of one pot as one replicate. The plants were cut directly between root and shoot with a pair of scissors. For that purpose, each plant was pulled slightly out of the soil to uncover the site of cutting. Immediately after cutting, the plants were dried at 60°C for minimum four days to a constant weight and weighed immediately with an analytical balance.

- Phytotoxicity rating system: according to EPPO standard PP 1/135 (2) Phytoxicity assessment

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Range finding study: not performed
- Justification for using less concentrations than requested by guideline: A limit test was performed in compliance with the test guideline to demonstrate that the test item has no toxic effect on the test organisms up to at least this concentration.
Nominal and measured concentrations:
Nominal: 1000 mg test item/kg soil (dry weight)
Reference substance (positive control):
no
Species:
Allium cepa
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:
seedling emergence
Species:
Brassica napus
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:
seedling emergence
Species:
Triticum aestivum
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:
seedling emergence
Species:
Daucus carota
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:
seedling emergence
Species:
Lactuca 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:
seedling emergence
Species:
Glycine max (G. soja)
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:
seedling emergence
Details on results:
SEED GERMINATION
- Root discolouration/malformation: No
- Results with reference substance valid?

SEEDLING EMERGENCE
- Percent seedling emergence: between 100 % and 106 % (compared to control)
- Abnormal seed development or appearance: No
- Lesions: not observed
- Swelling: not observed
- Loss of turgour: not observed
- Discoloration: not observed
- Unusual leaf/plant shape or size: not observed

VEGETATIVE VIGOUR
- Abnormal seed development or appearance: not observed
- Lesions: not observed
- Swelling: not observed
- Loss of turgour: not observed
- Discoloration: not observed
- Unusual leaf/plant shape or size: not observed
- Dead plants: not observed

INJURY RATING SYSTEM: not specified
Results with reference substance (positive control):
No reference substance tested.
Reported statistics and error estimates:
For data evaluation, the dry weight per plant for each treatment was calculated as percentage of the dry weight of untreated plants. Additionally, the mean value (x), the standard deviation (s) and the coefficient of variation (CV%) were calculated.
The LC50, EC50 and their 95% confidence limits could not be calculated in this limit test with one test concentration and were determined directly from the raw data. For the determinafion of the LOEC and NOEC, the rate of emergence, the shoot height and the mean shoot dry weight per plant at the tested treatments were tested on significant differences to the control values by a student's t-test (a = 0.05, one-sided smaller).

The analytically determined test item concentrations in the application solutions of the two application time points were 97% or 107% of the nominal values, based on the mean test item concentrations. Consequently, the reported biological results are based on the nominal concentration of the test item in the soil.

Description of key information

No toxic effects have been observed in studies with soil living organisms.

Key value for chemical safety assessment

Additional information

The test item was tested for its influence on the seedling emergence and seedling growth of two monocotyledonous plant species and four dicotyledonous plant species in accordance to OECD guideline 208. A limit test was performed in accordance with the test guideline to demonstrate that the test item has no toxic effect on the test organisms up to and including a nominal concentration of 1000 mg of test item per kg soil (dry weight) after 21 days of exposure. The analytically determined test item concentrations in the application solutions were 97% or 107% of the nominal values, based on the mean test item concentrations.