Reaction products of tetrabromophthalic anhydride with 2,2'-oxydiethanol and methyloxirane

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sept 2020 - 16 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants, Growth Test)

Version / remarks:

19 July 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analytical method validation, Sneckenberger et al. 2020

Details on sampling:

- Concentrations: of each dosing stock
- Sampling method: Three 20 mL aliquots were collected using a 10-mL glass graduated pipette. For the lowest and highest test concentration stocks, analytical samples were prepared by transferring each 20.0-mL aliquot to a uniquely identified clear glass vial. For the remaining test concentration stocks, analytical samples were prepared by transferring one 20.0-mL aliquot of each to a uniquely identified clear glass vial. The dosing stocks were continuously stirred as the aliquots were removed.

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Method of mixing into soil: Individual dosing stocks were prepared by mixing the appropriate amount of test substance (9.1407, 18.2813, 36.5627, 73.1254, and 146.2507 grams) with acetone (up to 300 mL) and then mixing 200 mL dosing stock with play sand (500 g) in a 1-L beaker prior to spiking the soil to prepare each test concentration. Mixing occured per hand, with spoon or mixer.
- Controls: yes, vehicle control
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 200 mL
- Evaporation of vehicle before use: yes

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Plant family: Liliacae
- Variety: Yellow Granex Hybrid
- Source of seed: Park Seed Co., Greenwood, SC USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 94%

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Ryegrass
- Plant family: Poaceae
- Variety: Gator 3
- Source of seed: Meyer Seed Co., Baltimore, MD USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 90%

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Soybean
- Plant family: Fabaceae
- Variety: Viking 2265 OG
- Source of seed: Johnny’s Selected Seeds, Winslow, ME USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 88%

Species:

Helianthus annuus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sunflower
- Plant family: Asteraceae
- Variety: Royal Hybrid 1121 F1
- Source of seed: Johnny’s Selected Seeds, Winslow, ME USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 92%

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Tomato
- Actual species name: Solanum lycopersicum
- Plant family: Solanaceae
- Variety: Rutgers
- Source of seed: Meyer Seed Co., Baltimore, MD USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 85%

Species:

Raphanus sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Radish
- Plant family: Brassicaceae
- Variety: Cherry Belle
- Source of seed: Territorial Seed Company, Cottage Grove, OR USA
- Prior seed treatment/sterilization: no
- Historical germination of seed (germination of seed lot tested): 100%

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Test temperature:

17.85 - 35.8 °C (mean 24.28 °C)

pH:

6.8 in water
6.3 in 0.01 M CaCl2

Moisture:

7.67 %

Details on test conditions:

TEST SYSTEM
- Testing facility: Eurofins EAG Agroscience, LLC, Maryland
- Test container: round plastic pots (16 cm in diameter and 12 cm deep)
- Amount of soil: For each test and control soil, a total amount of 105.6 kg fresh weight (97.50048 kg dry weight) of soil was weighed.
- Method of seeding: The seeds for A. cepa, L. perenne, S. lycopersicum, and R. sativus were planted per hand at an approximate depth of 10 mm while the seeds for the remaining species were planted at an approximate depth of 20 mm.
- No. of seeds per container: 5 for A. cepa and L. perenne, 2 for other crops
- No. of replicates per treatment group: 4 for A. cepa and L. perenne; 10 for other crops
- No. of replicates per control: 4 for A. cepa and L. perenne; 10 for other crops
- No. of replicates per vehicle control: 4 for A. cepa and L. perenne; 10 for other crops

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil texture: consisted of kaolinite clay, industrial quartz sand and peat, with limestone added to buffer the pH
- % sand: 88
- % silt: 2
- % clay: 10
- Soil classification system: loamy sand soil
- Organic carbon (%): 1.8
- Organic matter (%): 3.1
- Pretreatment of soil: no

NUTRIENT MEDIUM (if used)
- Description: slow-release fertiliser was added

GROWTH CONDITIONS
- Photoperiod: minimum 16-hour photoperiod
- Light source: Artificial lighting to supplement natural sunlight
- Light intensity and quality: 76.19 - 350.69 µEm²s-1 (mean 163.67 µEm²s-1), second run: 75.59 - 290.39 µEm²s-1 (mean 159.22 µEm²s-1)
- Day/night temperatures: 17.85 - 35.8 °C (mean 24.28 °C), second run: 14.8 - 32.44 °C (mean 21.13 °C)
- Relative humidity (%): 16.4 - 84.5 % (mean 56.2 %), second run: 9.6 - 65.7 % (mean (32.3%)
- Watering regime and schedules: Seedling growth pots for A. cepa and L. perenne were individually placed in 8-inch plastic sub irrigation saucers on the greenhouse bench in a randomised complete block arrangement. Seedling growth pots for all other species were placed in sub irrigation trays on the greenhouse bench in a randomized complete block arrangement.
- Water source/type: well water from the greenhouse facility
- Volume applied: Saucers and trays were filled to a predetermined depth to standardize the amount of water delivered to each test pot.
- Interval of applications: Watering record
Date Description of Watering
21 October 2020 All pots watered by sub-irrigation following application and placement in trays or saucers.
28 October 2020 All trays and saucers filled.
01 November 2020 All trays with sunflower, soybean, tomato, and radish filled.
03 November 2020 All saucers with onion and ryegrass filled.
04 November 2020 All trays with sunflower, soybean, tomato, and radish filled.
06 November 2020 All trays and saucers filled.
07 November 2020 All trays with sunflower, soybean, tomato, and radish filled.
08 November 2020 All trays with sunflower, soybean, tomato, and radish filled.
09 November 2020 All trays with sunflower, soybean, tomato, and radish filled.
10 November 2020 All trays with sunflower, soybean, tomato, and radish filled.

Watering Record
Date Description of Watering
08 January 2021 All pots watered by sub-irrigation following application and placement in saucers.
12 January 2021 All saucers filled.
16 January 2021 All saucers with soybean filled.
17 January 2021 All saucers with onion filled.
20 January 2021 All saucers filled.
23 January 2021 All saucers with soybean filled.
24 January 2021 All saucers filled.
27 January 2021 All saucers with soybean filled.
- Method of application: sub-irrigation
- Any pest control method/fertilisation: no

ACCLIMATION PERIOD: not needed

EFFECT PARAMETERS MEASURED:
Observations documenting seedling emergence for all species were made on Days 7, 14, and 21 after planting. Observations at test termination consisted of assessing effects on seedling growth and condition, and were made on Day 21. Observations for emergence consisted of determining the number of emerged seedlings within each replicate. Emergence was defined as the presence of visible plant tissue at the surface of the soil. Seedling condition was described by noting the presence or absence of possible signs of phytotoxicity such as chlorosis, leaf curl, unshed seed coat, and necrosis. A score of 0 indicated that no effect was observed, and a score of 100 indicated plant mortality. Survival was calculated based on the number of emerged seeds that were living at test termination.
The growth of emerged seedlings was evaluated by assessing the height of seedlings and the dry weight of living seedlings within replicates at test termination

- Phytotoxicity rating system (if used): Seedling condition was described by noting the presence or absence of possible signs of phytotoxicity such as chlorosis, leaf curl, unshed seed coat, and necrosis. A score of 0 indicated that no effect was observed, and a score of 100 indicated plant mortality. Intermediate scores described the severity of conditions that were observed on living seedlings, where an increase in score described a condition of greater detriment. While scores provided numeric data, conditional ratings were derived from subjective and qualitative assessments of phytotoxicity. Survival was calculated based on the number of emerged seeds that were living at test termination. Rating system adopted from Frans, Robert E. and Ronald E. Talbert, 1977.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: 21-Day Non-GLP Range-Finding Test with Saytex RB-79 on Six Species of Plants
- Test concentrations: 1, 10, 00, 1000 mg/kg dry soil and negative control and vehicle control

Nominal and measured concentrations:

nominal: 0 (Negative Control), 0 (Solvent Control), 62.5, 125, 250, 500, and 1000 mg/kg dry soil
Measured Test Soil Concentration (% Nominal): 0 (Solvent control), 63.5 (102), 129 (103), 232 (93), 481 (96), 861 (86) mg/kg dry soil

Reference substance (positive control):

no

Remarks:

historical control data of the lab is available

Species:

Allium cepa

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Helianthus annuus

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growthendpoints

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Allium cepa

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Helianthus annuus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Allium cepa

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Helianthus annuus

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: for all endpoints

Remarks:

seedling emergence, phytotoxicity, growth

Details on results:

The negative and solvent control group means for mean seedling emergence, height, or dry weight were not statistically significantly different (p>0.05) for all species. Since there was 100% survival in the negative and solvent control groups for A. cepa (onion), L. perenne (ryegrass), G. max (soybean), H. annuus (sunflower), and S. lycopersicum (tomato), no statistical comparison analyses were warranted. The negative and solvent control group means for mean survival were not statistically
significantly different (p>0.05) for R. sativus (radish). Therefore, test treatment group means were compared to the solvent control group mean.
Visible phytotoxic effects in the presence (ryegrass, sunflower, tomato, and radish) and the absence of slow-release fertilizer (onion and soybean) in the negative control group included one soybean seedling with severe stem curl, one sunflower seedling with severe necrosis, and one tomato seedling with slight leaf curl. In addition, visible phytotoxic effects in the solvent control group included only one soybean seedling also with severe stem curl. Out of 18 and 17 total emerged seedlings, it is not uncommon for one seedling in a negative or solvent control group, respectively, to occasionally exhibit signs of stem curl in a seedling emergence study conducted in the greenhouse facility at Eurofins-Easton laboratory. In addition, it also is not uncommon for one sunflower seedling out of 20 to exhibit signs of necrosis and one tomato seedling out of 16 to exhibit signs of leaf curl. Because only one seedling in each control group for these species, representing less than 7% of the total emerged, exhibited any visible phytotoxic effects, these effects were considered incidental and to be within the normal variation in plant growth and morphology for that particular species during a seedling emergence study. Therefore, these phytotoxic effects even in the presence of slow-release fertilize did not impact the validity of the study or the interpretation of the biological results.

Seedling Emergence
Statistical analyses weren’t warranted for seedling emergence for G. max (soybean), H. annuus (sunflower), and S. lycopersicum (tomato) since effects were less than or equal to 0% when compared to the solvent control group. For L. perenne (ryegrass), R. sativus (radish), and A. cepa (onion), there was not a statistically significant (p > 0.05) difference when the mean of any treatment group was compared to the solvent control group mean using the Dunnetts multiple comparison test. Therefore, the NOEC, based on seedling emergence for all species, was 1000 mg/kg dry soil and the LOEC was >1000 mg/kg dry soil.

Survival
Statistical analyses weren’t warranted for survival for R. sativus (radish) since effects were less than or equal to 0% when compared to the solvent control group. Since there was 100% survival for A. cepa (onion), G. max (soybean), and S. lycopersicum (tomato) in each control and test treatment group, no statistical analyses were warranted. For L. perenne (ryegrass) and H. annuus (sunflower), there was not a statistically significant (p > 0.05) difference when the mean of any treatment group was compared to the solvent control group mean using the Dunnetts multiple comparison test. Therefore, the NOEC, based on survival for all species, was 1000 mg/kg dry soil and the LOEC was >1000 mg/kg dry soil.

Height
For A. cepa (onion), L. perenne (ryegrass), G. max (soybean), and H. annuus (sunflower) there was not a statistically significant (p > 0.05) difference when the mean of any treatment group was compared to the solvent control group mean using the Dunnetts Multiple Comparison test. For S. lycopersicum (tomato) and R. sativus (radish), there was a statistically significant (p ≤ 0.05) difference when the mean of the 500 mg/kg dry soil treatment group of each species was compared to the solvent control group mean using the Dunnetts multiple comparison test. However, these differences were considered incidental and not treatment-related due to the lack of a dose response effect (no statistically significant difference determined at the 1000 mg/kg dry soil treatment level). Therefore, the NOEC, based on height for all species, was 1000 mg/kg dry soil and the LOEC was >1000 mg/kg dry soil.

Dry Weight
For A. cepa (onion), G. max (soybean), H. annuus (sunflower), and R. sativus (radish) there was not a statistically significant (p > 0.05) difference when the mean of any treatment group was compared to the solvent control group mean using the Dunnetts Multiple Comparison test. For L. perenne (ryegrass), there was a statistically significant (p ≤ 0.05) difference when the mean of the 125 mg/kg dry soil treatment group was compared to the solvent control group mean using the Dunnetts multiple comparison test. For S. lycopersicum (tomato), there was a statistically significant (p ≤ 0.05) difference when the means of the 250 and 500 mg/kg dry soil treatment groups were compared to the solvent control group mean using the Dunnetts multiple comparison test. However, for the ryegrass and tomato, the statistically significant differences were considered incidental and not treatment-related due to the lack of a dose response effect (no statistically significant difference determined at the 1000 mg/kg dry soil treatment level). Therefore, the NOEC, based on dry weight for all species, was 1000 mg/kg dry soil and the LOEC was >1000 mg/kg dry soil.

Results with reference substance (positive control):

not reported

Reported statistics and error estimates:

Statistical analyses were used to evaluate effects of soil-incorporated test substance on seedling emergence, survival, dry weight, and height. Negative and solvent control group means were compared with a t-test at the 0.05 level of significance.
For all species, treatment group means for seedling emergence, survival, dry weight, and height were calculated and compared to the control group mean using the Dunnetts Multiple Comparison Test to determine which group(s), if any, differed significantly from the control at the 0.05 level of significance. Prior to conducting the tests, the seedling emergence, survival, dry weight, and height data were evaluated for normality using the Shapiro-Wilk’s Test for normality test (α = 0.01) and the Levene’s Test for homogeneity of variance (α = 0.01). All statistical analyses were performed using a personal computer and commercially available software.

Measured test concentrations in dosing stocks, October 21, 2020

Nominal Test Soil Concentration (mg/kg dry soil)	Nominal Dosing Stock Concentration (ppm)	Day 0 Measured Monomer (ppm)	Day 0 Measured Dimer (ppm)	Day 0 Measured Trimer (ppm)	Day 0 Measured Total (% Recovery^2) (ppm)
Solvent Control	0	<LOQ	<LOQ	<LOQ	<LOQ
62.5	30469	31300^1	29800^1	30500^1	30700^1(101)
125	60938	64900	62700	63800	64000 (105)
250	121876	108000	104000	104000	106000 (87.0)
500	243751	232000	227000	229000	230000 (94.5)
1000	487502	377000^1	373000^1	376000^1	376000^1(77.1)
The method limit of quantitation (LOQ) for these analyses was set at 5000 ppm, defined as the lowest nominal concentration of a matrix fortification sample for which a mean recovery of 70-110% and relative standard deviation of ≤ 20% has been obtained. ^1Mean, measured concentration of the three samples submitted for analysis. ^2Results were generated using Excel in full precision and rounded to three significant figures based upon accuracy of measurements. Manual calculations may differ slightly.

Measured test concentrations in dosing stocks, January 8, 2021

Nominal Test Soil Concentration (mg/kg dry soil)	Nominal Dosing Stock Concentration (ppm)	Day 0 Measured Monomer (ppm)	Day 0 Measured Dimer (ppm)	Day 0 Measured Trimer (ppm)	Day 0 Measured Total (% Recovery^2) (ppm)
Solvent Control	0	<LOQ	<LOQ	<LOQ	<LOQ
62.5	8791	9220^1	9180^1	9140^1	9190^1(104)
125	17581	18000	18000	17900	18000 (102)
250	35162	35600	35300	35100	35400 (101)
500	70324	70900	70000	69800	70400 (100)
1000	140648	138000^1	136000^1	135000^1	137000^1(97.2)
The method limit of quantitation (LOQ) for these analyses was set at 5000 ppm, defined as the lowest nominal concentration of a matrix fortification sample for which a mean recovery of 70-110% and relative standard deviation of ≤ 20% has been obtained. ^1Mean, measured concentration of the three samples submitted for analysis. ^2Results were generated using Excel in full precision and rounded to three significant figures based upon accuracy of measurements. Manual calculations may differ slightly.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for soil-incorporated Saytex RB-79 was determined to be >1000 mg/kg dry soil in a 21-day seedling emergence test in all species. The NOEC and LOEC were 1000 and >1000 mg/kg dry soil, respectively.

Executive summary:

The objective of this study was to determine the effect of Saytex RB-79 on seedling emergence and growth of six species of terrestrial non-target higher plants according to OECD Guideline 208 and following GLP criteria. The test species were chosen for this study because they are economically important, readily cultivated test organisms widely used in research: Allium cepa (onion), Lolium perenne (ryegrass), Glycine max (soybean), Helianthus annuus (sunflower), Solanum lycopersicum (tomato) and Raphanus sativus (radish).

Seven test groups (five treatment groups, a solvent control, and a negative control) for each test species were exposed to nominal concentrations of 62.5, 125, 250, 500, and 1000 mg/kg dry soil for 21 days. Concentrations were based on a previously-conducted, non-GLP range-finding test. Emergence for onion was <70% and survival for soybean was <90% in the control groups, thus not meeting the validity criteria of this test. Therefore, a second definitive test with these two species was conducted. Data for the onion and soybean from the initial (not valid) definitive test were not reported, but were retained with the study records.

The test substance was mixed with a solvent and then applied to an inert material (sand). After the evaporation of the solvent, the treated sand was incorporated into the soil used for planting test species. The negative control was prepared similarly to the test treatment groups but without the addition of Saytex RB-79 and solvent to the sandand maintained concurrently. The solvent control was prepared similarly to the test treatment groups but only solvent was added to the sand and was also maintained concurrently.

There were twenty planted seeds for each test level. The number of replicates and number of seeds planted per replicate varied by species, and was either four replicates per treatment level, with each replicate containing five planted seeds (A. cepa and L. perenne) or ten replicates per treatment level, with each replicate containing two planted seeds (all other species). Seeds were impartially assigned to pre-labeled growth pots and planted on the day of test initiation. After planting, the replicate pots were placed on benches in the greenhouse in a randomised configuration to minimise bias from microclimates that may exist within the greenhouse. The in-life test duration was a total of 21 days. Observations documenting seedling emergence were made at least weekly during the test, while observations of height and condition were made on test termination. Dry weights were determined from seedling shoots collected at test termination.

 The EC₅₀ value for soil-incorporated Saytex RB-79 was determined to be >1000 mg/kg dry soil in a 21-day seedling emergence test in all species. The NOEC and LOEC were 1000 and >1000 mg/kg dry soil, respectively.

Description of key information

Seedling Emergence and Seedling growth test, OECD 208, GLP, Allium cepa (onion), Lolium perenne (ryegrass), Glycine max (soybean), Helianthus annuus (sunflower), Solanum lycopersicum (tomato) (formerly known as Lycopersicon esculentum) and Raphanus sativus (radish), 21 days in artificial soil, nominal concentrations: 62.5, 125, 250, 500, and 1000 milligrams per kilogram of soil dry weight (mg/kg dry soil);

The EC₅₀ value for soil-incorporated Saytex RB-79 was determined to be >1000 mg/kg dry soil in a 21-day seedling emergence test in all species. The NOEC and LOEC were 1000 mg/kg dry soil and >1000 mg/kg dry soil, respectively.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

1 000 mg/kg soil dw

Additional information

The objective of this study was to determine the effect of Saytex RB-79 on seedling emergence and growth of six species of terrestrial non-target higher plants according to OECD Guideline 208 and following GLP criteria. The test species were chosen for this study because they are economically important, readily cultivated test organisms widely used in research: Allium cepa (onion), Lolium perenne (ryegrass), Glycine max (soybean), Helianthus annuus (sunflower), Solanum lycopersicum (tomato) and Raphanus sativus (radish).

Seven test groups (five treatment groups, a solvent control, and a negative control) for each test species were exposed to nominal concentrations of 62.5, 125, 250, 500, and 1000 mg/kg dry soil for 21 days. Concentrationswere based on a previously-conducted, non-GLP range-finding test. Emergence for onion was <70% and survival for soybean was <90% in the control groups, thus not meeting the validity criteria of this test. Therefore, a second definitive test with these two species was conducted. Data for the onion and soybean from the initial (not valid) definitive test were not reported, but were retained with the study records.

The test substance was mixed with a solvent and then applied to an inert material (sand). After the evaporation of the solvent, the treated sand was incorporated into the soil used for planting test species. The negative control was prepared similarly to the test treatment groups but without the addition of Saytex RB-79 and solvent to the sandand maintained concurrently. The solvent control was prepared similarly to the test treatment groups but only solvent was added to the sand and was also maintained concurrently.

There were twenty planted seeds for each test level. The number of replicates and number of seeds planted per replicate varied by species, and was either four replicates per treatment level, with each replicate containing five planted seeds (A. cepaandL. perenne) or ten replicates per treatment level, with each replicate containing two planted seeds (all other species). Seeds were impartially assigned to pre-labeled growth pots and planted on the day of test initiation. After planting, the replicate pots were placed on benches in the greenhouse in a randomised configuration to minimise bias from microclimates that may exist within the greenhouse. The in-life test duration was a total of 21 days. Observations documenting seedling emergence were made at least weekly during the test, while observations of height and condition were made on test termination. Dry weights were determined from seedling shoots collected at test termination.

 The EC₅₀ value for soil-incorporated Saytex RB-79 was determined to be >1000 mg/kg dry soil in a 21-day seedling emergence test in all species. The NOEC and LOEC were 1000 and >1000 mg/kg dry soil, respectively.