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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 August - 22 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.4200 (Seed Germination/Root Elongation Toxicity Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen
- Stability under test conditions: Not determined
- Solubility and stability of the test substance in the solvent/vehicle: Assumed stable
- Reactivity of the test substance with the test medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in ethanol which was mixed with quartz sand and homogenised.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item was dissolved in ethanol which was mixed with quartz sand and homogenised.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal 5, 10, 50, 100, 500 and 1000 mg/L
- Sampling method: Subsamples of the 1000 mg/L ethanol stock solution were filtered and submitted for analysis. Filtrate were diluted with ethanol 5 times prior to GC-FID.
- Sample storage conditions before analysis: Not reported
Vehicle:
yes
Remarks:
Ethanol
Details on preparation and application of test substrate:
- Method of application to filter paper (if used): N/A
- Method of mixing into sand (if used): Test item dissolved in ethanol, mixed with quartz sand. Ethanol was evaporated to leave the test item homogenised through the quartz sand. The sand was then adjusted to 60-80 % moisture content.
- Method of application to soil surface (if used): N/A
- Controls: Blank control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Ethanol
- Concentration of vehicle in test medium (stock solution and final test solution): Test item was homogenised in a ethanol stock solution. The ethanol solution was evaporated from the quartz sand after mixing.
- Evaporation of vehicle before use: Yes
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Solanaceae
- Variety: -
- Source of seed: Guandong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Garden cucumber
- Plant family: Cucurbitaceae
- Variety: -
- Source of seed: Guandong, China
- Prior seed treatment/sterilization: No
- 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 (Compositae)
- Variety: -
- Source of seed: Guangdong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
other: Phaseolus radiatus L.
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Mung bean
- Plant family: Fabaceae (Leguminosae)
- Variety: -
- Source of seed: Guandong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Brassica oleracea var. capitata
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Cabbage
- Plant family: Brassicaceae (Cruciferae)
- Variety: -
- Source of seed: Japan
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
other: Citrullus lanatus
Details on test organisms:
- Common name: Watermelon
- Plant family: Not reported
- Variety: -
- Source of seed: Guangdong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
other: Brassicaceae brassica
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Mustard leaf
- Plant family: Brassicaceae (Cruciferae)
- Variety: -
- Source of seed: Guangdong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Oryza sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Rice
- Plant family: Poaceae (Gramineae)
- Variety: -
- Source of seed: Guangdong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Species:
Daucus carota
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Carrot
- Plant family: Apiaceae (Umbelliferae)
- Variety: -
- Source of seed: Australia
- Prior seed treatment/sterilization: No
- 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: Corn/ maize
- Plant family: Poaceae (Gramineae)
- Variety: -
- Source of seed: Guangdong, China
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): Not reported
- Seed storage: Not reported
Test type:
seed germination/root elongation toxicity test
Study type:
laboratory study
Substrate type:
other: quartz sand
Limit test:
no
Remarks:
The test end was defined when at least 65 percent of test seeds in blank and solvent treatment groups had germinated and the mean developed root in each treatment was at least 20 mm long.
Test temperature:
23.9 - 25.6 ºC
pH:
5.93 - 6.04
Moisture:
60-80 % moisture content
Details on test conditions:
TEST SYSTEM
- Testing facility: The Petri dishes were placed in the incubator at a slight angle to facilitate linear root growth.
- Test container (type, material, size): Petri dish
- Amount of sand: Not reported
- Method of seeding: The seeds were positioned on the substrate allowing adequate room for anticipated growth. The radical end of the seed was aligned in the direction of this growth.
- No. of seeds per container: 15
- No. of plants (retained after thinning): N/A
- No. of replicates per treatment group: 15
- No. of replicates per control: 15
- No. of replicates per vehicle control: 15

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Quartz sand

NUTRIENT MEDIUM (if used)
- Description: None used

GROWTH CONDITIONS
- Photoperiod: Darkness
- Light source: N/A
- Light intensity and quality: N/A
- Day/night temperatures: 25° C ± 1 °C
- Relative humidity (%): Not reported
- Wind velocity: N/A
- Air exchange rate: N/A
- Watering regime and schedules: No
- Water source/type: N/A
- Volume applied: N/A
- Interval of applications: N/A
- Method of application: Ethanol stock solution mixed through quartz sand. Solvent evaporated and seeds planted into homogenised quartz sand.
- Any pest control method/fertilization (if used): No

ACCLIMATION PERIOD: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Root length

- Phytotoxicity rating system (if used): N/A

VEHICLE CONTROL PERFORMED: Yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5, 10, 50, 100, 500, 1000 mg/L, demostrating a spacing factor of 2.
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Yes
- Test concentrations: 1, 10, 50, 100, 500, 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Nominal and measured concentrations:
Nominal - 5, 10, 50, 100, 500, 1000 mg/L
Reference substance (positive control):
no
Key result
Species:
Lycopersicon esculentum
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Cucumis sativus
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Lactuca sativa
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
other: Phaseolus radiatus L.
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root growth
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Brassica oleracea var. capitata
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
other: Citrullus lanatus
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
other: Brassicaeae brassica
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root length
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Oryza sativa
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root growth
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Daucus carota
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root growth
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Key result
Species:
Zea mays
Duration:
27 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and root growth
Remarks on result:
other: Note: study duration not reported as the end of the incubation period is determined when the 65 % of control seeds had germinated i.e. no fixed duration. 27 days is based on experimetnal start/end dates.
Details on results:
SEED GERMINATION
- Percent seed germination: >65 % in all control replicates and > 50 % in all test groups.
- Root length: Treatment groups were all comparable to the controls.
- Root discolouration/malformation: Not observed.
- Other effects: None.
- Results with reference substance valid? Not tested.
Reported statistics and error estimates:
The germination, root length and its standard deviation in each treatment were calculated. The germination, root length inhibition rates of the blank and solvent control groups was statistical analyzed (t-test, p < 0.05).
Validity criteria fulfilled:
yes
Conclusions:
Germination and root elongation inhibition rates of the seed treated with 6 test concentrations were all below 50 % and the EC50 of germination and root elongation inhibition rates of 10 seeds were > 1000mg/L (nominal).
Executive summary:

OPPTS 850.4200 (2012) – The inhibitory effect of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on seed germination and root elongation was evaluated in in 10 species of seed (tomato, cucumber, lettuce, mung bean, cabbage, watermelon, mustard leaf, rice, carrot and corn) in accordance with US EPA OPPTS 850.4200 guidance and GLP.

 

As the test substance was insoluble in water, the stock solution was prepared with ethanol. The stock solution was well mixed with the quartz sand. The ethanol was evaporated completely, and test water was added to the sand to adjust the substrate toca. 70-80 % moisture content.

 

At the start of the test, the concentration of the stock solution and the content in the quartz sand of the 1000 mg/L test group was determined. The stock solution was diluted with ethanol 50 times before GC analysis.

 

At the end of the test, the root length of seeds in each group was measured with electronic vernier calipers- the root length above 5 mm was considered as the sign of germination. The developmental state of the seeds was observed and recorded with any abnormal seedling development or appearance such as lesions, enhanced root growth (measured), discoloration, swelling, loss of turgor, reported, where appropriate.

 

At the end of the test, at least 65 percent of test seeds in blank and solvent treatment groups had germinated and the mean developed root in each treatment was at least 20 mm long. And there were no significant differences of germination and root elongation inhibition rates between blank and solvent groups (p≥0.05), which revealed that the solvent treatment had no important effect on the germination and root elongation of the seeds.

 

The test results revealed that the germination and root elongation inhibition rates of the seed treated with 6 test concentrations were all below 50 % and the EC50 of germination and root elongation inhibition rates of 10 seeds were > 1000mg/L (nominal concentration).

Description of key information

Seed germination and root elongation EC50: > 1000 mg/L; US EPA OPPTS 850.4200; Chenfang, M (2012)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:
1 000 mg/kg soil dw

Additional information

OPPTS 850.4200 (2012) – The inhibitory effect ofTetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on seed germination and root elongation was evaluated in in 10 species of seed (tomato, cucumber, lettuce, mung bean, cabbage, watermelon, mustard leaf, rice, carrot and corn) in accordance with US EPA OPPTS 850.4200 guidance and GLP.

 

As the test substance was insoluble in water, the stock solution was prepared with ethanol. The stock solution was well mixed with the quartz sand. The ethanol was evaporated completely, and test water was added to the sand to adjust the substrate toca. 70-80 % moisture content.

 

At the start of the test, the concentration of the stock solution and the content in the quartz sand of the 1000 mg/L test group was determined. The stock solution was diluted with ethanol 50 times before GC analysis.

 

At the end of the test, the root length of seeds in each group was measured with electronic vernier calipers- the root length above 5 mm was considered as the sign of germination. The developmental state of the seeds was observed and recorded with any abnormal seedling development or appearance such as lesions, enhanced root growth (measured), discoloration, swelling, loss of turgor, reported, where appropriate.

 

At the end of the test, at least 65 percent of test seeds in blank and solvent treatment groups had germinated and the mean developed root in each treatment was at least 20 mm long. And there were no significant differences of germination and root elongation inhibition rates between blank and solvent groups (p≥0.05), which revealed that the solvent treatment had no important effect on the germination and root elongation of the seeds.

 

The test results revealed that the germination and root elongation inhibition rates of the seed treated with 6 test concentrations were all below 50 % and the EC50of germination and root elongation inhibition rates of 10 seeds were > 1000mg/L (nominal concentration).

Study conducted in accordance with International guidelines and in accordance with GLP. All guideline validity criteria were satisfied, therefore this study can be regarded as sufficient to conclude on this endpoint.

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