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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:
other: OECD guideline 208 conform study, conducted under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Lot number: 1437
Purity: 99.94%
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0 (vehicle control), 0.00565, 0.0169, 0.0508, 0.152, 0.457, 1.37, 4.12, 12.3, 37.0, 111, 333, and 1000 mg/kg dry soil
- Sampling method: A 5-mL sample volume was collected in duplicate from the vehicle control, and all dosing solutions and transferred to an appropriately labeled culture tube. Samples and fortifications were diluted with 50:50 ACN:HPLC water to provide final sample concentrations within the analytical standard concentration range ( 0.101 to 101 mg/L for the definitive test). Quality control fortifications were prepared at concentrations of 0.000505 and 117 mg/mL for the definitive test.

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of application (carrier material): Sand as carrier (9:1 dry weight mix of base soil and sand carrier)
- Method of mixing into soil: Mixing with rotary mixer (15 min)
- Vehicle Control: 778 mL of acetone added to 8640g sand as carrier.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Evaporation of vehicle before use: Yes
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Cucumber
- Plant family: Cucurbitaceae
- Variety: Straight Eight
- Source of seed: NE Seed
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 85%
- Seed storage: Under refrigeration prior to use.
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Lettuce
- Plant family: Asteraceae
- Variety: Grand Rapids
- Source of seed: Sustainable Seed Company
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 98%
- Seed storage: Under refrigeration prior to use.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
- Plant family: Poaceae
- Variety: Cayuse
- Source of seed: Hearne Seed, L.A. Hearne Co.
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 97 %
- Seed storage: Under refrigeration prior to use.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
- Plant family: Amaryllidaceae
- Variety: Yellow Granex Hybrid 33
- Source of seed: Park Seed Co.
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 94 %
- Seed storage: Under refrigeration prior to use.
Species:
Raphanus sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Radish
- Plant family: Brassicaceae
- Variety: Crimson Giant
- Source of seed: Sustainable Seed Company
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 99%
- Seed storage: Under refrigeration prior to use.
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Fabaceae
- Variety: Williams 82
- Source of seed: Missouri Foundation Seeds
- Prior seed treatment/sterilization: No
- Historical germination of seed (germination of seed lot tested): 85/89 %
- Seed storage: Under refrigeration prior to use.
Test type:
seedling emergence toxicity test
Study type:
other: greenhouse study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No
Test temperature:
- Average: 24.9°C (2.6°C STD)
- Max.: 33.9°C
- Min.: 16.9°C
- Relative humidity: 89%
pH:
6.2
Moisture:
- Moisture at 1/3 Bar: 13.4%
- Moisture at 15 Bar: 6.3%
Details on test conditions:
TEST SYSTEM
- Testing facility: Greenhouse 4, abc Laboratories, Columbia, Missouri, US
- Test container (type, material, size): Round plastic pots, 16.5 cm (diameter), 11.5 cm depth
- Method of seeding: Soil in pots was allowed to settle slightly overnight, to avoid the potential for soil compaction to affect seedling emergence. Seeds were then planted in study pots in ascending treatment order.
- No. of seeds per container: 7
- No. of replicates per treatment group:6
- No. of replicates per control: 5 (One negative control replicate pot within the lettuce test was not planted at initiation. This resulted in lettuce evaluations using a 5 replicate negative control (35 seeds planted) versus 6 replicate (42 seeds planted) vehicle control and all test substance treatments.)
- No. of replicates per vehicle control: 6

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Lime Springs, Iowa
- Pesticide use history at the collection site: Soil source has been in the Conservation Reserve Program (CRP) of the USDA for more than 10 years, with no fertilizer or chemical applications during this period.
- Soil texture (if natural soil)
- % sand: 77
- % silt: 13
- % clay: 10
- Soil taxonomic classification: Sandy loam
- Soil classification system: USDA textural class
- Composition (if artificial substrate): Base soil : grade 2040 silica sand
- Organic carbon (%): 1.2
- Bulk density: 1.39 g/cc disturbed
- CEC: 4.4 meq/100g
- Pretreatment of soil: Sieving through various coarse sieves, ending with a #10ASTM (2mm) sieve, and each tub was sampled for moisture analysis.

GROWTH CONDITIONS
- Light: Natural sunlight with ambient photoperiod provided lighting for study days 0 – 4 of definitive testing. Subsequent lighting was natural sunlight supplemented with 1000 watt high-pressure sodium grow lights set on a 16-hr light to 8-hr dark photoperiod.
- Watering regime and schedules: Top-watered post-planting, bottom watered daily.
- Interval of applications: At initiation.
- Method of application: Incorporation into soil.
- Any pest control method/fertilization (if used): Commercial water-soluble fertilizer (i.e., Peter’s 20 20 20 ½ tablespoon per gallon) was applied via sub-irrigation twice during the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Emergence, Survival, Phytotoxicity (Phytotoxicity effects observed included chlorosis, damping off, leaf cupping, and stunting. Phytotoxic effects were considered severe, if phytotoxicity ratings >70.), Shoot length, Shoot dry weight
-Observations of numbers of seedlings present, cumulative emergence and mortality, and phytotoxicity ratings (i.e., visual injury assessments) were performed for all emergent seedlings on a weekly basis for all species. A seedling was considered emerged upon discernible emergence of plant material from the soil .

VEHICLE CONTROL PERFORMED: Yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Non-symmetric treatment scheme for different species, based on range-finder.
- Range finding study
- Test concentrations: 0 (Control), 0 (Vehicle control), 0.1, 1, 10, 100, 1000 mg/kg dry soil
- Results used to determine the conditions for the definitive study:
#Emergence and Phytotoxicity
Ctrl V. Ctrl 0.1 1 10 100 1000
Cucumber
% Emergence 86 88 93 93 79 100 36
Std. Dev. 18.1 14.1 10.1 10.1 10.1 0.0 23.5
Visual Rating b 0 3 15 10 10 20 92
Lettuce
% Emergence 90 90 86 93 100 50 10
Std. Dev. 7.4 7.4 20.2 10.1 0.0 10.0 11.7
Visual Rating b 0 5 10 10 10 80 95
Oat
% Emergence 98 93 93 100 100 100 50
Std. Dev. 5.8 7.8 10.1 0.0 0.0 0.0 19.7
Visual Rating b 0 5 10 10 10 20 87
Onion
% Emergence 88 83 93 93 79 21 0
Std. Dev. 14.1 21.0 10.1 10.1 10.1 10.1 0.0
Visual Rating b 0 3 0 0 0 90 100
Radish
% Emergence 93 100 100 100 93 100 43
Std. Dev. 7.8 0.0 0.0 0.0 10.1 0.0 12.7
Visual Rating b 0 3 15 15 15 30 87
Soybean
% Emergence 100 98 100 100 100 86 71
Std. Dev. 0.0 5.8 0.0 0.0 0.0 0.0 9.0
Visual Rating b 0 0 0 0 0 20 77

#Mean Percent Post-Emergence Survival
Ctrl V. Ctrl 0.1 1 10 100 1000
Cucumber
% Survival 98 95 100 100 100 100 45
Std. Dev. 5.8 8.0 0.0 0.0 0.0 0.0 29.8
Lettuce
% Survival 100 95 100 100 100 100 100
Std. Dev. 0.0 8.0 0.0 0.0 0.0 0.0 0.0
Oat
% Survival 100 100 100 100 100 100 97
Std. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 8.2
Onion
% Survival 100 100 100 100 100 100 N/A
Std. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 N/A
Radish
% Survival 90 95 86 100 100 100 82
Std. Dev. 8.1 7.4 0.0 0.0 0.0 0.0 21.3
Soybean
% Survival 100 100 100 100 100 100 87
Std. Dev. 0.0 0.0 0.0 0.0 0.0 0.0 24.1
Nominal and measured concentrations:
- Nominal: of 0 (control), 0 (vehicle control), 0.00565, 0.0169, 0.0508, 0.152, 0.457, 1.37, 4.12, 12.3, 37.0, 111, 333, and 1000 mg/kg dry soil
Reference substance (positive control):
not specified
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
114 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicity
Remarks on result:
other: 95%CL: 38.4-188 mg/kg
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: NOEC > 1000 mg/kg; EC5 > 1000 mg/kg
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Remarks on result:
other: 95%CL 70.9-142; NOEC=111 mg/kg
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
57.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weigth
Remarks on result:
other: 95%CL 26.7-88.9 mg/kg
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
701 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicity
Remarks on result:
other: 95%CL not calculated
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: NOEC > 1000 mg/kg; EC5 > 1000 mg/kg
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
34.1 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Remarks on result:
other: 95%CL 17.5-50.7 mg/kg
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
37 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weight
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicity
Remarks on result:
other: 95%CL -14.2 - 204)
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: NOEC > 1000 mg/kg; EC5 > 1000 mg/kg
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
29 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Key result
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
13.1 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weight
Remarks on result:
other: 95% CL 6.83-24.9 mg/kg. Based upon observed negative control variation producing a 14% C.V., the lowest reliable estimator of toxicity upon oat dry shoot weight is deemed to be the next larger incremental EC statistically calculated, which is the EC20.
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
37 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicty
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
37 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: EC10=139 mg/kg 95%CL 30.8-10735 mg/kg
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
35.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Remarks on result:
other: 95%CL 34.6-36.6 mg/kg
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
EC25
Effect conc.:
24.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weight
Remarks on result:
other: 95% CL 14.3-35.2 mg/kg
Species:
Raphanus sativus
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
389 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicity
Remarks on result:
other: 95%Cl 254-490 mg/kg
Species:
Raphanus sativus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
111 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: EC10=567 mg/kg 95% CL 257-97286
Species:
Raphanus sativus
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
33.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Remarks on result:
other: 95%CL 23.4-47.2 mg/kg
Species:
Raphanus sativus
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
23.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weight
Remarks on result:
other: 95% CL 15.7-34.8 mg/kg
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
981 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and phytotoxicity
Remarks on result:
other: 95%CL not calculated
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Remarks on result:
other: EC10=691 mg/kg 95%CL 186-89743 mg/kg
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
205 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot length
Remarks on result:
other: 95% CL 156-269 mg/kg
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
119 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot weight
Remarks on result:
other: 95% CL 74.6-163 mg/kg
Details on results:
Greater than 50% emergence was observed in all control groups at Day 7. Final percent emergence for the control groups (negative and vehicle) ranged from 74 to 100% for all species. There was a significant (p = 0.05) reduction in emergence as compared to the negative control for all species tested: cucumber, lettuce, oat, onion, radish, and soybean. The NOEC values for these species were 111, 333, 333, 37.0, 333, and 333 mg/kg dry soil, respectively.
There were no phytotoxic effects observed in the controls for any of the species tested. Phytotoxic effects were severe (phytotoxicity ratings >70) in at least the highest treated level for all species tested: cucumber, lettuce, oat, onion, radish, and soybean. Phytotoxicity effects observed included chlorosis, damping off, leaf cupping, and stunting. Visual effects are presented in Table 10, but are not considered appropriate for determination of study results, since these values are based on subjective evaluations.
Post-emergence survival ranged from 94 to 100% in the controls for all species. There was a significant (p = 0.05) reduction in survival as compared to the negative control in onion, radish, and soybean. The NOEC values for these species were 37.0, 111, and 333 mg/kg dry soil, respectively.
There was a significant (p = 0.05) reduction in shoot length as compared to the negative control for all species tested: cucumber, lettuce, oat, onion, radish, and soybean. The NOEC values for these species were 111, 37.0, 1.37, 37.0, 4.12, and 111 mg/kg dry soil, respectively.
There was a significant (p = 0.05) reduction in shoot dry weight as compared to the negative control for all species tested: cucumber, lettuce, oat, onion, radish, and soybean. The NOEC values for these species were 4.12, 37.0, 0.0508, 12.3, 1.37, and 111 mg/kg dry soil, respectively.

Study protocol deviation:
One negative control replicate pot within the lettuce test was not planted at initiation. This resulted in lettuce evaluations using a 5 replicate negative control (35 seeds planted) versus 6 replicate (42 seeds planted) vehicle control and all test substance treatments. The reason for this deviation was oversight at planting. While less than protocol specification, a five replicate (35 seeds planted) negative control exhibiting 70% emergence or greater meets OECD 208 requirements and is sufficient to allow statistical evaluation of the four biological endpoints of interest, including calculation and reporting of study results. Therefore it can be concluded that this deviation has no effect on the validity and outcome of the study.


Results with reference substance (positive control):
Not specified.
Reported statistics and error estimates:
For data sets for species/measured parameters for which NOEC analysis indicated significant effects, further analysis was performed to determine effective/inhibition rates if possible. Emergence and post-emergent survival data was analyzed using Probit methods, and plant shoot length and dry weight data was analyzed using non-linear regression dose-response models (Bruce, Versteeg Weighted Probit, Schabenberger Weighted Hormetic, OECD Model 1, and OECD Model 2, all models fitted to the data using the Marquardt method), in effort to elicit corroboration of potential threshold effects, and to determine the EC5, EC10, EC20, EC25, and EC50 values, and their 95% confidence levels, if possible. Regression analysis was not performed upon data sets for species/measured parameters that did not exhibit significance per NOEC analysis. In the report tables, percent emergence, percent survival, shoot length, and visual ratings are rounded to the nearest whole number. Shoot dry weight is reported to the precision as collected.
Results in control (Ctrl) and in vehicle controls (V. Ctrl) with the respective standard deviations
Emergence/Phytotoxicity Effects Mean Percent Post-Emergence Survival Mean Shoot Length Mean Shoot Dry Weight
  Ctrl V. Ctrl   Ctrl V. Ctrl   Ctrl V. Ctrl   Ctrl V. Ctrl
Cucumber
% Emergence 100 90 % Survival 98 100 Length (mm) 312 340 Weight (g) 6.2943 6.4602
Std. Dev. 0 11.7 Std. Dev. 5.8 0 Std. Dev. 43 32 Std. Dev. 1.0838 0.7644
            % Inhibition --- -9 % Inhibition --- -3
NOEC (mg/kg dry soil):  111 NOEC (mg/kg dry soil):  1000 NOEC (mg/kg dry soil):  111 NOEC (mg/kg dry soil):  4.12
EC05 (mg/kg dry soil): 9.48(95% CI -85.7 – 82.2)c EC05 (mg/kg dry soil): >1000(95% CI not calculated) EC05 (mg/kg dry soil): 41.0(95% CI 23.2 – 72.3)b EC05 (mg/kg dry soil): 13.3(95% CI 6.13 – 20.4)b
EC10 (mg/kg dry soil): 114(95% CI 38.4 - 188)c EC10 (mg/kg dry soil): >1000(95% CI not calculated) EC10 (mg/kg dry soil): 61.5(95% CI 38.6 – 97.9)b EC10 (mg/kg dry soil): 27.3(95% CI 12.6 – 42.0)b
EC20 (mg/kg dry soil): 242(95% CI 169 - 337)c EC20 (mg/kg dry soil): >1000(95% CI not calculated) EC20 (mg/kg dry soil): 100(95% CI 70.9 - 142)b EC20 (mg/kg dry soil): 57.8(95% CI 26.7 – 88.9)b
EC25 (mg/kg dry soil): 290(95% CI 214 - 398)c EC25 (mg/kg dry soil): >1000(95% CI not calculated) EC25 (mg/kg dry soil): 121(95% CI 89.1 - 164)b EC25 (mg/kg dry soil): 74.5(95% CI 34.4 - 115)b
EC50 (mg/kg dry soil): 485(95% CI 380 - 659)c EC50 (mg/kg dry soil): >1000(95% CI not calculated) EC50 (mg/kg dry soil): 256(95% CI 213 - 309)b EC50 (mg/kg dry soil): 256(95% CI 213 - 309)b
Lettuce
% Emergence 74 98 % Survival 100 100 Length (mm) 83 90 Weight (g) 0.2742 0.4254
Std. Dev. 18.6 5.8 Std. Dev. 0 0 Std. Dev. 12 21 Std. Dev. 0.0939 0.0782
            % Inhibition --- -9 % Inhibition --- -55
NOEC (mg/kg dry soil):  333 NOEC (mg/kg dry soil):  1000   NOEC (mg/kg dry soil):  37 NOEC (mg/kg dry soil):  37
EC05 (mg/kg dry soil): 664(95% CI not calculated)c EC05 (mg/kg dry soil): >1000(95% CI not calculated) EC05 (mg/kg dry soil): < 0.00565(95% CI not calculated)c EC05 (mg/kg dry soil): 33.0(95% CI 31.3 – 34.8)c
EC10 (mg/kg dry soil): 701(95% CI not calculated)c EC10 (mg/kg dry soil): >1000(95% CI not calculated) EC10 (mg/kg dry soil): 16.1(95% CI 8.24 – 23.9)c EC10 (mg/kg dry soil): 34.2(95% CI 32.4 – 36.1)c
EC20 (mg/kg dry soil): 748(95% CI not calculated)c EC20 (mg/kg dry soil): >1000(95% CI not calculated) EC20 (mg/kg dry soil): 34.1(95% CI 17.5 – 50.7)c EC20 (mg/kg dry soil): 35.7(95% CI 33.9 – 37.7)c
EC25 (mg/kg dry soil): 767(95% CI not calculated)c EC25 (mg/kg dry soil): >1000(95% CI not calculated) EC25 (mg/kg dry soil): 43.9(95% CI 22.5 – 65.4)c EC25 (mg/kg dry soil): 36.4(95% CI 34.5 – 38.4)c
EC50 (mg/kg dry soil): 847(95% CI not calculated)c EC50 (mg/kg dry soil): >1000(95% CI not calculated) EC50 (mg/kg dry soil): 106(95% CI 54.2 - 157)c EC50 (mg/kg dry soil): 38.9(95% CI 36.9 – 41.0)c
Oat
% Emergence 100 98 % Survival 100 100 Length (mm) 394 417 Weight (g) 0.9355 0.9679
Std. Dev. 0 5.8 Std. Dev. 0 0 Std. Dev. 37 21 Std. Dev. 0.1264 0.0521
            % Inhibition --- -6 % Inhibition --- -3
NOEC (mg/kg dry soil):  333 NOEC (mg/kg dry soil):  1000 NOEC (mg/kg dry soil):  1.37 NOEC (mg/kg dry soil):  0.0508
EC05 (mg/kg dry soil): <0.00565 (95% CI not reported)c  EC05 (mg/kg dry soil): 588(95% CI not calculated)b EC05 (mg/kg dry soil): 14.8(95% CI 8.46 – 25.7)b EC05 (mg/kg dry soil): 2.87(95% CI 1.09 – 7.61)c
EC10 (mg/kg dry soil): 101(95% CI -14.2 - 204)c EC10 (mg/kg dry soil): >1000(95% CI not calculated)b EC10 (mg/kg dry soil): 29.0(95% CI 18.5 – 45.3)b EC10 (mg/kg dry soil): 5.70(95% CI 2.50 – 13.0)c
EC20 (mg/kg dry soil): 251(95% CI 150 - 385)c EC20 (mg/kg dry soil): >1000(95% CI not calculated)b EC20 (mg/kg dry soil): 65.6(95% CI 47.6 – 90.4)b EC20 (mg/kg dry soil): 13.1(95% CI 6.83 – 24.9)c
EC25 (mg/kg dry soil): 308(95% CI: 204 - 462)c EC25 (mg/kg dry soil): >1000(95% CI not calculated)b EC25 (mg/kg dry soil): 89.4(95% CI: 67.8 - 118)b EC25 (mg/kg dry soil): 17.9(95% CI: 9.98 – 32.0)c
EC50 (mg/kg dry soil): 537(95% CI:  399 - 796)c EC50 (mg/kg dry soil): >1000(95% CI not calculated)b EC50 (mg/kg dry soil): 313 (95% CI: 262 - 373)b EC50 (mg/kg dry soil): 63.7 (95% CI: 44.3 – 91.6)c
Onion
% Emergence 88 83 % Survival 100 94 Length (mm) 149 142 Weight (g) 0.0683 0.0472
Std. Dev. 10.8 16.7 Std. Dev. 0 13.6 Std. Dev. 8 35 Std. Dev. 0.0153 0.0255
            % Inhibition --- 5 % Inhibition --- 31
NOEC (mg/kg dry soil):  37 NOEC (mg/kg dry soil):  37   NOEC (mg/kg dry soil):  37 NOEC (mg/kg dry soil):  12.3
EC05 (mg/kg dry soil): 32.4(95% CI 0.0226 – 96.7)c EC05 (mg/kg dry soil): 19.6(95% CI 2.33 - 129)b EC05 (mg/kg dry soil): 34.3(95% CI 33.4 – 35.3)b EC05 (mg/kg dry soil): < 0.152(95% CI not calculated)d
EC10 (mg/kg dry soil): 51.0(95% CI 0.144 - 129)c EC10 (mg/kg dry soil): 139(95% CI 30.8 - 10735)b EC10 (mg/kg dry soil): 35.6(95% CI 34.6 – 36.6)b EC10 (mg/kg dry soil): 9.08(95% CI 5.25 – 12.9)d
EC20 (mg/kg dry soil): 88.1(95% CI 1.32 - 186)c EC20 (mg/kg dry soil): >1000(95% CI not reported)b EC20 (mg/kg dry soil): 37.2(95% CI 36.2 – 38.3)b EC20 (mg/kg dry soil): 19.2(95% CI 11.1 – 27.3)d
EC25 (mg/kg dry soil): 108(95% 3.03 - 217)c EC25 (mg/kg dry soil): >1000(95% CI not reported)b EC25 (mg/kg dry soil): 37.8(95% CI 36.8 – 38.9)b EC25 (mg/kg dry soil): 24.8(95% CI 14.3 – 35.2)d
EC50 (mg/kg dry soil): 251(95% CI 65.1 - 532)c EC50 (mg/kg dry soil): >1000(95% CI not reported)b EC50 (mg/kg dry soil): 40.5(95% CI 39.3 – 41.6)b EC50 (mg/kg dry soil): 59.7(95% CI 34.5 – 84.9)d
Radish
% Emergence 95 98 % Survival 100 100 Length (mm) 235 253 Weight (g) 2.647 2.8
Std. Dev. 7.4 5.8 Std. Dev. 0 0 Std. Dev. 15 13 Std. Dev. 0.255 0.46
            % Inhibition --- -8 % Inhibition --- -6
NOEC (mg/kg dry soil):  333 NOEC (mg/kg dry soil):  111 NOEC (mg/kg dry soil):  4.12 NOEC (mg/kg dry soil):  1.37
EC05 (mg/kg dry soil): 332(95% CI 200 - 432)c EC05 (mg/kg dry soil): 316(95% CI 63.4 - 1775)b EC05 (mg/kg dry soil): 18.4(95% CI 12.0 – 28.1)b EC05 (mg/kg dry soil): 7.87(95% CI 4.46 – 13.9)c
EC10 (mg/kg dry soil): 389(95% CI 254 - 490)c EC10 (mg/kg dry soil): 567(95% CI 257 - 97286)b EC10 (mg/kg dry soil): 33.2(95% CI 23.4 – 47.2)b EC10 (mg/kg dry soil): 12.9(95% CI 7.88 – 21.0)c
EC20 (mg/kg dry soil): 473(95% CI 338 - 575)c EC20 (mg/kg dry soil): >1000(95% CI not reported)b EC20 (mg/kg dry soil): 68.0(95% CI 52.0 – 88.8)b EC20 (mg/kg dry soil): 23.4(95% CI 15.7 – 34.8)c
EC25 (mg/kg dry soil): 509(95% CI 376 - 613)c EC25 (mg/kg dry soil): >1000(95% CI not reported)b EC25 (mg/kg dry soil): 89.2(95% CI 70.4 - 113)b EC25 (mg/kg dry soil): 29.3(95% CI 20.3 – 42.2)c
EC50 (mg/kg dry soil): 685(95% CI 559 - 811)c EC50 (mg/kg dry soil): >1000(95% CI not reported)b EC50 (mg/kg dry soil): 268(95% CI 232 - 308)b EC50 (mg/kg dry soil): 73.0(95% CI 57.1 – 93.3)c
Soybean
% Emergence 93 93 % Survival 100 98 Length (mm) 483 512 Weight (g) 5.877 6.131
Std. Dev. 12 12 Std. Dev. 0 5.8 Std. Dev. 71 61 Std. Dev. 0.536 0.897
            % Inhibition --- -6 % Inhibition --- -4
NOEC (mg/kg dry soil):  333 NOEC (mg/kg dry soil):  333 NOEC (mg/kg dry soil):  111 NOEC (mg/kg dry soil):  111
EC05 (mg/kg dry soil): 833(95% CI not calculated)c EC05 (mg/kg dry soil): 144(95% CI 21.4 - 952)b EC05 (mg/kg dry soil): 108(95% CI 72.5 - 160)b EC05 (mg/kg dry soil): < 1.37(95% CI not calculated)d
EC10 (mg/kg dry soil): 897(95% CI not calculated)c EC10 (mg/kg dry soil): 691(95% CI 186 - 89743)b EC10 (mg/kg dry soil): 144(95% CI 103 - 202)b EC10 (mg/kg dry soil): 56.1(95% CI 35.2 – 77.0)d
EC20 (mg/kg dry soil): 981(95% CI not calculated)c EC20 (mg/kg dry soil): >1000(95% CI not reported)b EC20 (mg/kg dry soil): 205(95% CI 156 - 269)b EC20 (mg/kg dry soil): 119(95% CI 74.6 - 163)d
EC25 (mg/kg dry soil): >1000(95% CI: not calculated)c EC25 (mg/kg dry soil): >1000(95% CI not reported)b EC25 (mg/kg dry soil): 319(95% CI: 312 - 326)b EC25 (mg/kg dry soil): 153(95% CI: 96.2 - 210)d
EC50 (mg/kg dry soil): >1000(95% CI:  not calculated)c EC50 (mg/kg dry soil): >1000(95% CI not reported)b EC50 (mg/kg dry soil): 402 (95% CI: 343 - 472)b EC50 (mg/kg dry soil): 369 (95% CI: 232 - 507)d
Validity criteria fulfilled:
yes
Conclusions:
Shoot weight in Oat is the most sensitive endpoint with a derived EC20 of 13.1 mg/kg dry soil. The lowest obtained effect concentration for shoot length is found in oat as well with an EC10 of 29 mg/kg dry weight. For both, emergence and survival, onion is the most sensitive species with a NOEC of 37.0 mg/kg dry weight.
The most relevant endpoints were selected for each species and endpoint based on the standard deviation of the controls. The shoot weight endpoint is by far the most variable and uncertain effect parameter due to a very high standard deviation already in the controls for certain species.
Executive summary:

A Tier I/II seedling emergence non-target study was conducted with the test substance 2,4 di tert-butylphenol. The test protocol was based on the OECD Guideline 208. Six species of plants were selected based on taxonomic criteria and at the suggestion of the Study Sponsor. The species tested were cucumber – Cucumis sativus, lettuce – Lactuca sativa, oat – Avena sativa, onion – Allium cepa, radish – Raphanus sativus, and soybean – Glycine max. The dosing solutions were applied to sand which was then mixed into the test soil. The nominal concentrations for the tier II (definitive test) were 0 (control), 0 (vehicle control), 0.00565, 0.0169, 0.0508, 0.152, 0.457, 1.37, 4.12, 12.3, 37.0, 111, 333, and 1000 mg/kg dry soil. A vehicle of acetone was used to dissolve and apply the test item. Vehicle control pots were dosed with equivalent amounts acetone. Negative control consisted of dry test soil only. The data collected included percent emergence, post-emergent plant survival, visual injury (i.e., phytotoxicity), shoot length, and replicate shoot dry weight at termination. The data were analyzed using Fisher’s Exact and Cochran-Armitage for emergence and survival, and Dunnett’s and Jonckheere-Terpstra (shoot length and shoot dry weight) to compare treatments to the control to determine the no effect concentration (NOEC). Where possible, non-linear regression analysis was used to estimate the effect concentration to elicite a 25 and 50% detrimental effect (EC25 and EC50) for survival, and the inhibitory concentration to elicit a 25 and 50% detrimental effect (EC25 and EC50) for length and weight data. Per Sponsor request, additional effect concentrations (EC5, EC10, and EC20) were also estimated.

The NOEC, EC5, EC10, EC20, EC25, and EC50 concentrations (mg/kg dry soil) for the effects of 2,4-di-tert-butylphenol on seedling emergence, plant survival, shoot height, and shoot dry weight for the seedling emergence test are summarized in the tables above.

The lowest relevant effect values for all endpoints were derived as follows,

Emergence:             NOEC (onion) = 37.0 mg/kg dry soil

Survival:                  NOEC (onion) = 37.0 mg/kg dry soil

Shoot length:           EC10 (oat) = 29.0 mg/kg dry soil

Shoot weight:           EC20(oat) = 13.1 mg/kg dry soil

The most relevant endpoints were selected for each species and endpoint based on the standard deviation of the controls. The shoot weight endpoint is by far the most variable and uncertain effect parameter due to a very high standard deviation already in the controls for certain species.

Description of key information

Shoot weight in Oat is the most sensitive endpoint with a derived EC20 of 13.1 mg/kg dry soil. The lowest obtained effect concentration for shoot length is found in oat as well with an EC10 of 29 mg/kg dry weight. For both, emergence and survival, onion is the most sensitive species with a NOEC of 37.0 mg/kg dry weight.

Key value for chemical safety assessment

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

Additional information

The lowest effect concentration determined in the Seedling Emergence study following OECD test guideline 208, assessing the toxicity of 2,4 -DTBP towards mono- and dicotyledonous plant species is the EC20 of 13.1 mg/kg dry soil for shoot weight reduction in oat. The lowest obtained effect concentration for shoot length is found in oat as well with an EC10 of 29 mg/kg dry weight. For both, emergence and survival, onion is the most sensitive species with a NOEC of 37.0 mg/kg dry weight.