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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2017 to February 2019
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:
Please find the detailed deviations observed from Study Plan in background document part. These deviations were not considered to have adversely affected the integrity or validity of the study as the water and solvent control plants were unaffected.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
Chemical analysis was carried out on the treatment solutions to verify the concentrations of the test item in the formulation.
Details on sampling:
A total of 14 pots of three seeds each were sown for cabbage, carrot and lettuce, 20 pots of two seeds for tomato and ten pots of four seeds each for oat and onion. For all species 9.5 cm internal diameter non-porous plastic pots were used and each was filled to a depth of approximately 75 mm, the seeds were spaced out on the soil surface and approximately10 mm of soil was added to cover the seeds. Each pot was labelled with the study number, species name, treatment, rate of application, replicate number, application date and the name of the Study Director. Each application rate was also identified by a colour-coded plastic stick label. After application each pot was placed in an individual water tray (i.e. saucer ) and placed at random within rectangular blocks of evenly spaced pots for each plant species.
Vehicle:
no
Details on preparation and application of test substrate:
The test material was dissolved in acetone. The water and solvent controls were applied for all species before the test material was applied for cabbage, lettuce, tomato and oat at 62.5, 125, 250, 500 and 1000 mg a.i./kg dy soil and at 1000 mg a.i./kg dry soil for carrot and onion.

The solvent solutions were applied to dry soil and when the acetone had evaporated, the dry soil as mixed into the appropriate amount of weighted moist soil. This soil was then moistened with reverse osmosis water and mixed using a handheld mixer to ensure that the soil had a structure suitable for sowing the seeds.

The treated soil was then distributed among labelled replicate pots. For the untreated water and solvent control groups the same amounts of dry soil and moist sil was used as for the treatment groups. For the water control the dry soil was mised into the moist soil and for the solvent control the appropriate volume of acetone was added and allowed to evaporate off. Water was then added as for the treated groups. The pH of the soil was recorded after application.

Chemical analysis was carried out on the treatment solutions to verify the concentrations of the test material. Aliquots of each treatment solution were placed into glass vials. For more details, the method details and results of the analysis are presented in the attached background document part.
Species:
other: Avena sativa, Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Oat (Avena sativa), Family: Poaceae (Gramineae)
Onion (Allium cepa), Family: Liliaceae (Amarylladaceae)

The seeds were purchased from commercial suppliers. They were not treated with any type of fungicides, insecticides or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was = 75% for all species.
Each plant species was sown into treated soil and monitored for 15-18 days following a minimum of 50% germination of the control seedlings.
Species:
other: Brassica oleracea, Daucus carota, Lactuca sariva, Lycopersicon esculentrum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Cabbage (Brassica oleracea), Family: Brassicaceae (Cruciferae)
Carrot (Daucus carota), Family: Apiaceae (Umbelliferae)
Lettuce (Lactuca sativa), Family: Asteraceae (Compositae)
Tomato (Lycopersicon esculentum), Family: Solanaceae

The seeds were purchased from commercial suppliers. They were not treated with any type of fungicides, insecticides or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was = 75% for all species.
Each plant species was sown into treated soil and monitored for 15-18 days following a minimum of 50% germination of the control seedlings.
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Remarks:
The soil used was a sandy loam (batch number GIL 1401). The carbon content (1.89% organic matter), pH and particle size of the soil were analysed according to UK classification and it was sieved prior to use to remove all particles >2 mm.
Limit test:
yes
Total exposure duration:
21 d
Remarks:
Minimum of 21 days after application of the test material.
Test temperature:
19-30.3°C
pH:
7.4-7.6
Moisture:
43.01-94.12%
Details on test conditions:
A total of 14 pots of three seeds each were sown for cabbage, carrot and lettuce, 20 pots of two seeds for tomato and ten pots of four seads each for oat and onion. For all species 9.5 cm internal diameter non-porous plastic pots were used and each was filled to a depth of approximately 75 mm, the seeds were spaced out on the soil surface and approximately -10 mm of soil was added to cover the seeds. Each pot was labelled with the stud number, speies name, treatment, rate of application, replicate number, application date and the name of the Study Director. Each application rate was also identified by a color-coded plastic stick label. After application each pot was placed in an individual water tray and placed at random withn rectangular blocks of evenly spaced pots for each plant species.

Plants were watered as required maintaining seedling health and growht. Plants were maintained in controlled environment rooms providing a continuous photoperiod of 16 hours light per day. Temperatures were maintained at levels suitable for the individual species. Temperature, humidity and light intensity, (initially at soil level and at the top of the canopy after germination) were recorded daily throughout the exposure period. For more details, please see below the summary of the environmental parameters.


SOURCE AND PROPERTIES OF SUBSTRATE (if soil): Soil analysis
pH 7.5
Organic carbon by wet oxidation : 1.1% w/w
Available potassium : 93.8 mg/L
Total potassium : 784 mg/kg
Particle size:
Sand 2.00-0.063 mm : 79% w/w
Silt 0.063-0.002 mm : 10% w/w
Clay <0.002 mm : 11% w/w
Textural class : Sandy loam
- Organic carbon (%): 1,1%
- Maximum water holding capacity (in % dry weigth): the moisture content of the soil was determined to be 5.38% (initial).
- Pretreatment of soil: Sieved prior to use to remove all particles >2 mm. The soil was not pasteurized, or heat treated before use.

- Phytotoxicity rating system : assessment was conducted using a uniform scoring system based on plant size and vigor (for more details, please refers to the document in attached background material).


TEST CONCENTRATIONS
- Range finding study : A preliminary range finding test was carried out at test concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil.
- Results used to determine the conditions for the definitive study: Please find the details on range-finding results in the attached background materials. Based on the results of the range finding test, a definitive dose-response test was conducted at rates of 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil for cabbage, lettuce, tomato and oat and a limit test at 1000 mg a.i./kg dry soil was conducted for carrot and onion.
Nominal and measured concentrations:
62.5, 125, 250, 500 and 1000 mg a.i./kg of dry soil for cabbage, lettuce, tomato and oat and as a limit study at a nominal concentration of 1000 mg a.i./kg dry soil for carrot and onion.
Reference substance (positive control):
yes
Species:
Avena sativa
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
height
Key result
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
dry weight
Species:
other: Cabbage, Carrot, Lettuce, Tomato, Oat, Onion
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and plant survival, growth and health
Remarks:
and survival
Species:
other: Cabbage, Carrot, Lettuce, Tomato, Oat, Onion
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germination and plant survival
Remarks:
and survival
Details on results:

Germination in the water and solvent controls was 100% for all species. There were no treatment related effects on germination in any plant species at any treatment rate.

There were no treatment-related effects on plant survical for any species.

For tomato, a significant reduction in height of 11% was recorded at 1000 mg a.i./kg dry soil in comparison to the water control.

For oat, siginificatn reductions in height of 7%, 8%, 12% and 14% at 125, 250, 500, and 1000 mg a.i./kg, respectively, were recorded in comparison to the water control and 10% and 2% reduction at 500 and 1000mg a.i./kg, respectively, were recorded in comparison to the solvent control.

For lettuce, a significant reduction in dry weight of 32% was recorded at 1000 mg a.i./kg dry soil in comparison to the water control, and 22% and 41% at 500 and 1000 mg a.i./kg dry soil, respectively, in comparison to the solvent control.

For tomato significant reductions in dry weight of 20% and 46% were recorded at 500 and 1000 mg a.i./kg dry soil, respectively, in comparison to the water control and 19%, 21% and 47% reductions at 250, 500 and 1000 mg a.i./kg dry soil, respectively, in comparison to the solvent control were observed.

For oat, significant reductions in dry weight of 15% and 21% at 500 and 1000 mg a.i./kg dry soil, respectively, in comparison to the water control and 20% and 26% at 500 and
1000 mg a.i./kg dry soil, respectively, in comparison to the solvent control.

No significant adverse effects in height or dry weight were observed in cabbage, carrot and onion at any treatment rate compared to the water or solvent control.

Plant growth inhibition of <10% was visually observed in lettuce, tomato and oat. Apart from plant growth inhibition, no visible effects (e.g. phytotoxicity, wilting or leaf deformity) were observed at any treatment rate in any of the species.

INJURY RATING SYSTEM: Plant growth inhibition of <10% was visually observed in lettuce, tomato and oat. Apart from plant growth inhibition, no visible effects (e.g. phytotoxicity, wilting or leaf deformity) were observed at any treatment rate in any of the species (no herbicidal symptoms were recorded). For more details please see the document in attached background material.
Reported statistics and error estimates:
Statistical analysis of mean replicate height and dry weight was carried out using the SAS 9.1.3 programme (SAS Institute Inc. 2002).

Statistical analysis of the height and dry weights of cabbage , carrot, lettuce, oat and onion, in comparison to both the negative (water) and solvent controls, was carried out using the two-sided t-test. Statistical analysis of the height and dry weights of tomato in comparison to the water and solvent controls was conducted using Williams’ test. The results for the water control and solvent control were compared using the t-test.

The dry weight data was log transformed prior to analysis to stabilize the variance and the means are reported as back transformed geometric means.


The IT systems listed below were used on this study: Pristima (SAS 9.1.3: Statistics).

  • Summary of effects on height and dry weight in comparison to the solvent control (see the document in attached background material part of this RSS).

  • Environmental Conditions:

































































 



Environmental parameters



 



Plant species



Air temperature


(°C)



Relative humidity


(%)



Light (16:8) (lux)



 



Nominal; range



Nominal range



 



 



22ºC ± 10 ºC



70% ± 25%



 



Cabbage



19.0 - 30.3



43.01 - 94.12



6390 - 11840



Carrot



19.0 - 30.3



43.01 - 94.12



4420 - 7740



Lettuce



19.0 - 30.3



43.01 - 94.12



4380 - 8980



Tomato



19.0 - 30.3



43.01 - 94.12



4190 - 11440



Oat



19.0 - 30.3



43.01 - 94.12



5310 - 18760



Onion



19.0 - 30.3



43.01 - 94.12



5380 - 11760


Validity criteria fulfilled:
yes
Conclusions:
There were no statistically significant effects on germination or survival in any plant species at any treatment rate. Germination and survival in the water control and solvent control was 100% for all species.

The EC50 values for germination and survival were estimated to be >1000 mg a.i./kg dry soil for all plant species.

The NOEC values for effects on germination and survival were 1000 mg a.i./kg dry soil for all plant species.

Statistically significant reductions in height of 10% and 12%, in comparison to the solvent control, were calculated for oat at 500 and 1000 mg a.i./kg dry soil.

The EC25 and EC50 values for effects on height compared against the solvent control were >1000 mg a.i./kg dry soil for all species.

The NOEC values for effects on height compared against the solvent control were 250 mg a.i./kg dry soil for oat, and 1000 mg a.i./kg dry soil for cabbage, carrot, lettuce, tomato and onion.

Significant reductions in dry weight in comparison to the solvent control were calculated for lettuce and oat at 500 and 1000 mg a.i/kg dry soil and for tomato at 250, 500 and 1000 mg a.i./kg dry soil.

The EC25 values for effects on dry weight compared against the solvent control were 599.34, 534.01 and 837.71 mg a.i./kg dry soil for lettuce, tomato and oat, respectively, and >1000 mg a.i./kg dry soil for cabbage, carrot, and onion.

The EC50 values for effects on dry weight compared against the solvent control were >1000 mg a.i./kg dry soil for all species.

The NOEC values for effects on dry weight compared against the solvent control were 250, 125 and 250 mg a.i./kg dry soil for lettuce, tomato and oat respectivel,y, and >1000 mg a.i./kg dry soil for carrot, cabbage and onion.

The lowest endpoint observed on plant was retained as a key result and is a NOEC = 125 mg a.i/kg dry soil based on a significant reduction of the dry weight on tomato following a soil exposure of 22 days to the test item. 
Executive summary:

A study was conducted according TG OECD 208 and GLP to determine the phytotoxic effects on seedling emergence and growth of six non-target plant species. They consist of four dicotyledons (cabbage, carrot, lettuce and tomato) and two monocotyledons (oat and onion), following exposure to the registered substance in soil during critical early stages in development. Following the initial range finding test conducted at 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil, the definitive study was conducted as a dose-response test at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg a.i./kg of dry soil for cabbage, lettuce, tomato and oat and as a limit study at a nominal concentration of 1000 mg a.i./kg dry soil for carrot and onion. Analysis of the concentrations of the treatment solutions confirmed nominal levels of the test substance were within applied limits ± 20%, confirming accurate formulation. Effect levels are reported based on nominal application rates.


 


Three seeds were sown in each of 14 pots for cabbage, carrot and lettuce, two seeds in each of 20 pots for tomato, and four seeds in each of 10 pots for oat and onion for each application rate and concurrent water and solvent (acetone) controls. The soil used was a sandy loam, sieved to remove all particles > 2mm. After application and sowing, the pots were checked daily, and the numbers of emerged seedlings were recorded. Emergence, survival and sub-lethal effects were recorded daily until at least 50% emergence of control seedlings and thereafter every one to three days. Plants were harvested 15 to 18 days after 50% germination in the water and solvent controls, giving a total exposure period of 22 days for all species. The height of each individual plant was measured, and the plants were then dried before being individually weighed to obtain a measurement of dry weight.


 


Effect on germination and plant survival


Germination in the water and solvent controls was 100% for all species. There were no treatment related effects on germination or survival in any plant species at any treatment rate. The EC50 values for germination and survival were estimated to be >1000 mg a.i./kg dry soil for all plant species. The NOEC values for effects on germination and survival were 1000 mg a.i./kg dry soil for all plant species.


 


Effect on plant growth and health


No visible phytotoxic effects were observed in the water or solvent controls. The results for the water control and solvent control were compared using the t-test and were determined to be not statistically significantly different. Effects of the test item were therefore determined based on statistical comparison with the solvent control group. At harvest, measurement of the height and dry weight for each surviving seedling from each replicate pot was conducted.


No adverse significant effects in height were observed in cabbage, carrot, lettuce, tomato and onion at any treatment rate compared to the solvent control. For oat significant reductions in height of 10% and 12% reductions at 500 and 1000 mg a.i./kg, respectively, were recorded in comparison to the solvent control.


Statistically significant reductions in height of 10% and 12%, in comparison to the solvent control, were calculated for oat at 500 and 1000 mg a.i./kg dry soil.


The EC25 and EC50 values for effects on height compared against the solvent control were >1000 mg a.i./kg dry soil for all species. The NOEC values for effects on height compared against the solvent control were 250 mg a.i./kg dry soil for oat, and 1000 mg a.i./kg dry soil for cabbage, carrot, lettuce, tomato and onion.


 


No adverse significant effects in dry weight were observed in cabbage, carrot and onion at any treatment rate compared to the solvent control.


Significant reductions in dry weight in comparison to the solvent control were calculated for lettuce and oat at 500 and 1000 mg a.i/kg dry soil and for tomato at 250, 500 and 1000 mg a.i./kg dry soil. The EC25 values for effects on dry weight compared against the solvent control were 599.34, 534.01 and 837.71 mg a.i./kg dry soil for lettuce, tomato and oat respectively, and >1000 mg a.i./kg dry soil for cabbage, carrot, and onion. The EC50 values for effects on dry weight compared against the solvent control were >1000 mg a.i./kg dry soil for all species. The NOEC values for effects on dry weight compared against the solvent control were 250, 125 and 250 mg a.i./kg dry soil for lettuce, tomato and oat respectively, and >1000 mg a.i./kg dry soil for carrot, cabbage and onion. 


The lowest endpoint observed on plant was retained as a key result and is a NOEC = 125 mg a.i/kg dry soil based on a significant reduction of the dry weight on tomato following a soil exposure of 22 days to the test item. 


 


 


 

Description of key information

A study according TG OECD 208 and GLP conducted demonstrates that there is no statistically significant effects on germination or survival in any plant species at any treatment rate. Germination and survival in the water control and solvent control was 100% for all species. The EC50 values for germination and survival were estimated to be >1000 mg a.i./kg dry soil for all plant species. The NOEC values for effects on germination and survival is 1000 mg a.i./kg dry soil for all plant species. The NOEC values for effects on height compared against the solvent control were 250 mg a.i./kg dry soil for oat, and 1000 mg a.i./kg dry soil for cabbage, carrot, lettuce, tomato and onion. The NOEC values for effects on dry weight compared against the solvent control were 250, 125 and 250 mg a.i./kg dry soil for lettuce, tomato and oat respectively, and >1000 mg a.i./kg dry soil for carrot, cabbage and onion. The lowest endpoint observed on plant was retained as a key result and is a NOEC = 125 mg a.i/kg dry soil based on a significant reduction of the dry weight on tomato following a soil exposure of 22 days to the test item. 

Key value for chemical safety assessment

Additional information