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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Version / remarks:
adopted 19 July 2006
Deviations:
yes
Remarks:
No solvent control was performed since no effects of the organic solvent used were expected. The mean temperature and humidity deviated from the range required by the guideline. This did not impact the outcome of the biological results.
Qualifier:
according to
Guideline:
other: EU Method C.31 (Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008, amended by Commission Regulation (EC) No 2016/266
Deviations:
yes
Remarks:
No solvent control was performed since no effects of the organic solvent used were expected. The mean temperature and humidity deviated from the range required by the guideline. This did not impact the outcome of the biological results.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: not applicable (UVCB substance)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 55 g/L (trial 1) and 37.5 g/L (trial 2)
- Sampling method: duplicate samples were taken from two application solutions
- Sample storage conditions before analysis: stored at -20°C before analysis.

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Application solutions for the different test concentrations were prepared by dispersing the test item in the organic solvent dichloromethane and stirring for 10-15 minutes. Adequate volumes of the test item-solvent dispersions were applied on the surface of adequate amounts of quartz sand. After evaporating the solvent overnight, the test item-quartz sand mixtures were thoroughly mixed with soil substrate before the seeds were sown.
- Controls: The control treatment was prepared in the same way as the test item treatments but without solvent and test item. No solvent control was performed since no effects of the organic solvent dichloromethane were expected due to the long time duration of evaporation from quartz sand overnight. Additionally, no effects of dichloromethane on seed germination were reported for lettuce [1] and pine [2] and therefore also not expected for the plant species used in this test.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dichloromethane
- Evaporation of vehicle before use: yes

[1] Anderson J. D., L. L. Triplett, A. H. Haber, H. Meyer, A. M. Meyer (1973): Dichloromethane and Lettuce Seed Germination. DOI: 10.1126/science.179.4068.94. Science 179 (4068): 94-96.
[2] Groot A. (1985): Application of germination inhibitors in organic solvents to conifer seeds. Great Lakes Forestry Centre. Canadian Forestry Service. ISBN 0-662-14434-1. ISSN Q822-210X. Information Report O-X-371: 1-14.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
- Plant family: Amaryllidaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Species:
Triticum aestivum
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: wheat
- Plant family: Poaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Species:
Phaseolus vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: bean
- Plant family: Fabaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Species:
Beta vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: beet
- Plant family: Amaranthaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: oilseed rape
- Plant family: Brassicaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Species:
other: Solanum lycopersicum (synonym: Lycopersicon esculentum)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: tomatoe
- Plant family: Solanaceae
- Source of seed: commercial supplier
- Prior seed treatment/sterilization: none
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Remarks:
21 days after emergence of 50% in the control for all test species, i.e. 25 days after test start for oilseed rape and wheat, 27 days for bean and beet, 28 days for tomato and 32 days for onion.
Test temperature:
mean temperature 17.0 - 19.5°C (see growth conditions)
pH:
4.9
Moisture:
Relative humidity in the greenhouse: 18 - 92 % (for several periods of time, humidity was outside the recommended range of 70 ± 25 % with no impact on the outcome of the study)
Details on test conditions:
TEST SYSTEM
- Testing facility: greenhouse
- Test container (type, material, size): Non-porous plastic pots, inner diameter: 13.0 cm
- Amount of soil: 800 g fresh weight

- No. of seeds per container: 5
- No. of plants (retained after thinning): no thinning performed
- No. of replicates per treatment group: 6
- No. of replicates per control: 6

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: supplied by LUFA Speyer (67346 Speyer, Germany)
- Pesticide use history at the collection site: The soil was not treated with any pesticide or organic fertiliser for four years prior to use

- Sampling depth (cm): 0-20 cm depth
- Soil texture (if natural soil)
- % sand: 86
- % silt: 11.5 (trial 1), 11.1 (trial 2)
- % clay: 2.5 (trial 1), 2.9 (trial 2)
- Soil taxonomic classification: loamy sand
- Soil classification system: USDA
- Organic carbon (%): 0.71%
- Maximum water holding capacity (in % dry weigth): 32.5 and 32.2 g/100 g dry soil (trial 1 and 2, respectively)

- Storage (condition, duration): before use, the soil was stored in plastic bags under ambient room conditions

GROWTH CONDITIONS
- Photoperiod: light:dark regime 16:8 h
- Light intensity and quality: 35 – 99 μE/m2/s (trial 1), 127 – 144 μE/m2/s (trial 2). Although the light intensity was lower than recommended by the guidelines, it was fully sufficient for normal plant growth.
- Day/night temperatures: 10.2 – 34.3 °C (minimum-maximum) with a mean of 19.5 °C (trial 1), 11.4 – 28.6 °C (minimum-maximum) with a mean of 17.0 °C (trial 2), for one short period, temperature was above the upper limit of the recommended range of 18 – 22 °C with no impact on the outcome of the study
- Relative humidity (%): 19 - 92 % (minimum-maximum) with a mean of 54 % (trial 1), 18 - 80 % (minimum-maximum) with a mean of 48 % (trial 2); for several periods of time, humidity was outside the recommended range of 70 ± 25 % with no impact on the outcome of the study
- Watering regime and schedules:
- Water source/type: tap water
- Volume applied: 0.5 - 1 mL per pot, as needed
- Interval of applications: as required
- Method of application: bottom watering
- Any pest control method/fertilization (if used): on one occasion all plants received universal fertilizer (“Wuxal® Universaldünger” , Maag, Switzerland, dilution 1:80 in 0.5 L water; constituents: 8.0 % (w/w) nitrogen with 2.3 % nitrate-N, 3.7 % ammonium-N and 2.0 % urea-N; 8.0 % (w/w) phosphate; 6.0 % potassium oxide; water soluble parts of boron (0.01 % (w/w)), copper (0.004 % (w/w)), iron (0.02 % (w/w)). manganese (0.012 % (w/w)), molybdene (0.001 % (w/w)), zinc (0.004 % (w/w)).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
emergence, visual phytotoxicity (i.e. leaf and stem deformations, chlorosis, necrosis and overall appearance compared with the control) and plant mortality: weekly (after 50% of the control seedlings had emerged);
final fresh weight and the final shoot length: at the end of the test (21 days after 50% of the control seedlings had emerged)

- Phytotoxicity rating system (if used): scoring system for visual effects:
0 (no symptoms): 0% of leaf area damaged,
1 (slight symptoms): ≤ 25% of leaf area damaged,
2 (moderate symptoms): 25-75% of leaf area damaged,
3 (severe symptoms): ≥ 75% of area leaf damaged

VEHICLE CONTROL PERFORMED: no; effects of the organic solvent dichloromethane were not expected due to the long time duration of evaporation from quartz sand overnight. Additionally, no effects of dichloromethane on seed germination were reported for lettuce [1] and pine [2] and therefore also not expected for the plant species used in this test.
[1] Anderson J. D., L. L. Triplett, A. H. Haber, H. Meyer, A. M. Meyer (1973): Dichloromethane and Lettuce Seed Germination. DOI: 10.1126/science.179.4068.94. Science 179 (4068): 94-96.
[2] Groot A. (1985): Application of germination inhibitors in organic solvents to conifer seeds. Great Lakes Forestry Centre. Canadian Forestry Service. ISBN 0-662-14434-1. ISSN Q822-210X. Information Report O-X-371: 1-14.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1, 10, 100 and 1000 mg/kg dry soil
- Results used to determine the conditions for the definitive study: significant effects on fresh weight and shoot length at 1000 mg/kg dry soil; no effects on emergence

Nominal and measured concentrations:
SOIL CONCENTRATIONS
nominal 10, 32, 100, 320, and 1000 mg /kg dry soil

TREATMENT DISPERSIONS
nominal: 55 and 37.5 g test material/L
measured: 50.5 and 37.9 g test material /L (92 and 101% of nominal)
Reference substance (positive control):
no
Key result
Species:
other: all tested species
Dose descriptor:
EC10
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: duration 25-28 days, depending on species
Key result
Species:
other: Solanum lycopersicum (synonym: Lycopersicon esculentum)
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
21 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per replicate
Key result
Species:
other: Solanum lycopersicum
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
34 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per plant
Key result
Species:
Triticum aestivum
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
59 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per replicate
Key result
Species:
Triticum aestivum
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
64 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per plant
Key result
Species:
Brassica napus
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
67 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean shoot lenghth per replicate
Key result
Species:
Brassica napus
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
75.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per replicate
Key result
Species:
Beta vulgaris
Duration:
27 d
Dose descriptor:
EC10
Effect conc.:
138 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per plant
Key result
Species:
Brassica napus
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
190 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean fresh weight per replicate
Key result
Species:
other: Solanum lycopersicum
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
202 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean shoot lenghth per replicate
Key result
Species:
Triticum aestivum
Duration:
25 d
Dose descriptor:
EC10
Effect conc.:
298 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
mean shoot lenghth per replicate
Details on results:
For details see Attachment 1 (Summary table for effect concentrations) and Attachment 2 (Tables 1-37: Detailed results on endpoints).

For evaluation, the EC10 is preferred over NOEC based on the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.11: PBT/vPvB Assessment (version 3.0, June 2017) and the Report of the OECD workshop on statistical analysis of aquatic toxicity data (Series on testing and assessment, N°10. Environmental Health and Safety Publications. Series on testing and Assessment (ENV/MC/CHEM(98)18)).

- SEEDLING EMERGENCE
Emergence rates were 77 – 93% in controls and 57 – 100% in the test item treatments without a concentration-effect relationship and without a statistically significant difference compared to the control for all test species (Tables 1, 7, 13, 19, 26, and 32).

- PHYTOTOXICITY
WHEAT, OILSEED RAPE and TOMATO
No phytotoxicity was observed (Tables 8, 27, and 33).

ONION:
Slight necrosis (i.e. 0-25% of total leaf area per pot) was observed in 3-5 pots per treatment from 32 mg/kg dry soil on (Table 2).

BEAN
In the test item treatments of 32, 100 and 320 mg/Kg dry soil, a few pots showed moderate feeding damages (0-25% of total leaf area per pot) which was not test item treatment-related (Table 14).

BEET
Slight to moderate root rot (i.e. 0-25% and 26-75% of total leaf area per pot) was observed in the control and in all test item treatments (Table 20). These symptoms were due to fungal infection – during the study, beet seedlings emerged successfully from the exposed seeds but afterwards their basal shoot parts were partly infected by a soil fungus. This infection weakened the growth of the basal part of the shoot tissue and induced a bending of the shoot to the soil surface followed partly by the death of the infected plants by wilting (Table 21). The infection of beet root by soil fungi is a well-known phenomenon and difficult to control. In this study, the occurrence of fungal infection was observed in both trials and even the careful control of soil humidity in the second trial could not prevent the infection of the plants. Since the infection was observed across all treatments including the control, the probability of the fungal infection is considered similar for all treatments, which allows the comparison of data on seedling emergence, seedling mortality and seedling growth between the test item treatments and the control.

- PLANT SURVIVAL
Mortality of emerged plants was 0 – 8% in the controls and 0 – 12% in the test item treatments without a concentration-effect relationship and without a statistically significant difference compared to the control for all test species except beet (Tables 3, 9, 15, 28, and 34). For beet, fungal infection was observed in the control and in all test item treatments (see PHYTOTOXICITY above), which resulted in a reduced survival of emerged beet plants, i.e. 78% in the control and 62 – 100% in the test item treatments (Table 21). No concentration-effect relationship was observed and no statistically significant difference between the test item treatments and the control was detected. Since plant survival was directly related to plant mortality due to fungal infection, control and test item treatments did not differ statistically significantly in mortality due to fungal infection (Table 22). Therefore, the occurrence of fungal infection is considered similar across all treatments and does allow a comparison of seedling growth between the test item treatments and the control.

- MEAN FRESH WEIGHT PER PLANT
ONION
The mean fresh weight per plant was 71-105% of the control without a concentration-effect relationship. At 32 mg/kg dry soil, the mean fresh weight was 71% of the control and statistically significantly reduced compared to the control (Table 5). However, this statistically significant result is not considered to be biologically relevant since at the two following test concentrations the mean fresh weight per plant were 94 and 105% of the control, respectively, and did not differ statistically significantly from the control. At 1000 g/kg dry soil, the mean fresh weight was 74% of the control and statistically significantly reduced compared to the control, which is considered a test item treatment-related effect.

WHEAT
The mean fresh weight per plant was 68-93% of the control with a concentration-effect relationship starting from 100 mg/kg dry soil (Table 11). At 320 and 1000 mg/kg dry soil, the mean fresh weight/plant was 87 and 68% of the control, respectively, and statistically significantly reduced compared to the control, which is considered a test item treatment-related effect.

BEAN
The mean fresh weight per plant was 96-136% of the control without a concentration-effect relationship and without a statistically significant difference compared to the control (Table 17).

BEET
The mean fresh weight per plant was 75-97% of the control with a concentration-effect relationship (Table 24). At 1000 mg/kg dry soil, the mean fresh weight was 75% of the control and statistically significantly reduced compared to the control, which is considered a test item treatment-related effect.

OILSEED RAPE

The mean fresh weight per plant was 50-87% of the control without a concentration-effect relationship but with statistically significant differences compared to the control from the lowest test concentration of 10 mg/kg dry soil on (Table 30).

TOMATO
The mean fresh weight per plant was 42-91% of the control with a concentration-effect relationship starting from 100 mg/kg dry soil (Table 36). At 320 and 1000 mg/kg dry soil, mean fresh weight per replicate was 69 and 42% of the control, respectively, and differed statistically significantly from the control, which was considered a test item-related effect.

- MEAN SHOOT LENGHTH
ONION
The mean shoot length was 84-106% of the control without a concentration-effect relationship (Table 6). At 32 mg/kg dry soil, the mean shoot length was 84% of the control and statistically significantly reduced compared to the control. However, this statistically significant result is not considered to be biologically relevant since at the three following test concentrations the mean fresh weight per plant was 96, 106 and 89%, respectively, and did not differ statistically significantly from the control.

WHEAT
The mean shoot length was 79-100% of the control with a concentration-effect relationship (Table 12). At 320 and 1000 mg/kg dry soil, the mean shoot length was 93 and 79% of the control, respectively, and statistically significantly reduced compared to the control, which is considered a test item treatment-related effect.

BEAN
The mean shoot length was 103-143% of the control without a concentration-effect relationship (Table 18). No statistical analysis was performed since the shoot length in all test item treatments up to and including the highest test concentration of 1000 mg/kg dry soil was larger than the control.

BEET
The mean shoot length was 94-100% of the control without a concentration-effect relationship and without a statistically significant difference between the test item treatments and the control (Table 25).

OILSEED RAPE
The mean shoot length was 79-91% of the control without a concentration-effect relationship but with statistically significant differences compared to the control from the lowest test concentration of 10 mg/kg dry soil on (Table 31).

TOMATO
The mean shoot length 78-101% of the control with a concentration-effect relationship (Table 37). At 320 and 1000 mg/kg dry soil, mean shoot length per replicate was 86 and 78% of the control, respectively, and differed statistically significantly from the control, which was considered to be a test item-related effect.

Reported statistics and error estimates:
For emergence and mortality of emerged plants, differences between the test item treatment and the control were tested using the multiple sequentially-rejective Fisher test after Bonferroni-Holm (one-sided greater, α = 0.05). NOEC and LOEC were determined according to the results of the statistics.
For fresh weight per replicate, fresh weight per replicate and plant and shoot height per replicate, differences between the test item treatments and the control were tested using the Williams t-test (one-sided smaller, α = 0.05, concentration-effect relationship established) or the Dunnett t-test (one-sided smaller, α = 0.05, no concentration-effect relationship established). NOEC and LOEC were determined according to the results of the statistics.
Calculations of LC10, LC20 and LC50 as well as EC10, EC20 and EC50 for emergence, fresh weight and shoot height were performed using Probit analysis and maximum likelihood regressions. In case of low values of r2 (quotient of determination which shows the reliability of fit between the calculated and the observed concentration-effect relationship) additional ECx-calculations were performed excluding the results of the two lowest test concentrations in three data sets of oilseed-rape (i.e. fresh weight per replicate, fresh weight per plant and replicate and shoot height per replicate).
Validity criteria fulfilled:
yes
Conclusions:
most sensitive endpoint regarding fresh weight (EC10 21 – 190 mg/kg dw)
most sensitive species regarding: Solanum lycopersicum (tomato; EC10 21-202 mg/kg dw)
not toxic with regard to emergence and survival (LC/EC10 > 1000 mg/kg dw)

Description of key information

Toxicity to terrestrial plants (OECD 208, EU C.31):

most sensitive endpoint: fresh weight (EC10 = 21 – 190 mg/kg dw)

most sensitive species: Solanum lycopersicum (tomato; EC10 = 21-202 mg/kg dw)

not toxic with regard to emergence and survival (LC/EC10 >1000 mg/kg dw)

Key value for chemical safety assessment

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

Additional information

21 mg/kg soil dw was the overall lowest EC10 value observed in this test.