1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Mar - 24 Apr 2018

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:

July 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4100 (Terrestrial Plant Toxicity Tier I (seedling emergence))

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz; Wiesbaden, Germany

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: Samples of the initial test item stock solution (100 g a.s./ha) and of deionized water
- Sampling method: Directly before application
- Sample storage conditions before analysis: Deep frozen

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation: A test item stock solution (equivalent to 100 g a.s./ha and 200 L/ha) was prepared by dissolving a weighed amount of test item in deionized water. The stock solution itself was only used for analytical purposes. The test item solutions were prepared by dilution of the test item stock solution with deionized water (gravimetric determination).
- Method of application to soil surface: The test item solutions and control solution (deionized water) were applied to the soil surface with a linear cabinet track sprayer (SprayLab SLGH 2500, Schachtner, Ludwigsburg, Germany). Type of nozzles: Teejet 8001 EVS (1 nozzle); Spray pressure: 4.0 bar; Application speed: 2 km/h; Distance (nozzle - target area): 30 cm
- Controls: The blank control spray solution was 200 L/ha deionized water.

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sugar beet
- Plant family: Chenopodiaceae
- Variety: 2VK1433
- Source of seed: KWS Saat AG (21 Jun 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 80%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Oilseed rape winter
- Plant family: Brassicaceae
- Variety: Marathon
- Source of seed: Samen Schlereth (18 Mar 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Cucumber
- Plant family: Cucurbitaceae
- Variety: Delikatess
- Source of seed: Enza Zaden (06 Sep 2013)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Soybean
- Plant family: Fabaceae
- Variety: Sultana
- Source of seed: Samen Schlereth (27 Jan 2015)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Helianthus annuus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sunflower
- Plant family: Asteraceae
- Variety: Pacino Cola
- Source of seed: Benary (31 Oct 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 85%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

other: Solanum lycopersicum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Tomato
- Plant family: Solanaceae
- Variety: Sparta F1
- Source of seed: Enza Zaden (06 Feb 2017)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Plant family: Amaryllidaceae
- Variety: Braunschweiger Blutrot
- Source of seed: Enza Zaden (06 Aug 2015)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Oat
- Plant family: Poaceae
- Variety: Flämingsstern
- Source of seed: Samen Schlereth (14 Mar 2011)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 80%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Ryegrass
- Plant family: Poaceae
- Variety: Belevue
- Source of seed: Samen Schlereth (10 Oct 2017)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Corn
- Plant family: Poaceae
- Variety: Saludo
- Source of seed: Bundesamt für Ernährungssicherheit A-1220 Wien (07 Apr 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Remarks:

greenhouse

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Remarks:

21 d after 50% control seedling emergence

Test temperature:

12 - 32 °C

pH:

6.92 CaCl2 (soil)

Moisture:

45 - 95% relative humidity during light and dark cycle

Details on test conditions:

TEST SYSTEM
- Testing facility: Greenhouse conditions Bayer AG
- Test container: Commercial non-porous plastic flower pots (15 cm diameter), filled with approx. 1.2 L soil
- Amount of soil: 1.2 L
- Method of seeding: The seeds were sown 1 d prior to the application of the test item. The commercial plastic flower pots were pre-filled with soil. Only intact and healthy looking seeds of similar size were sowed. The seeds were manually pressed into the soil surface and completely covered with a layer of soil. On the day of the test start, before application of the test item, the soil was top watered to facilitate emergence.
- No. of test groups: 6 (control: 1; test item: 5)
- No. of seeds per container: 2 or 4 seeds, depending on plant species (total: 20 seeds per test group)
- No. of replicates per treatment group: 10 (2 seeds/pot) or 5 (4 seeds/pot), depending on plant species (1 pot = 1 replicate)
- No. of replicates per control: 20 seeds in 5 or 10 replicates (pots), depending on plant species

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil type and geographic location: The soil is a silt loam, a mixture of standard "Riedberg" soil + washed sand (ratio 9/1) from Bayer AG, Horticulture and Services, H 872, Industriepark Hoechst, 65926 Frankfurt am Main, Germany. The soil mixture is named NTP soil.
- Collection procedures: The soil was sieved to 2 mm.
- % sand: 4.9% fine sand (0.063 - 0.20 mm), 20.3% middle sand (0.20 - 0.63 mm), and 6.1% coarse sand (0.63 - 2.0 mm)
- % silt: 4.9% fine silt (0.002 - 0.006 mm), 17.8% middle silt (0.006 - 0.020 mm)) and 28.9% coarse silt (0.02 - 0.063 mm)
- % clay: 17.1% clay (0 - 0.002 mm)
- Organic carbon (%): 0.72%
- Lime content: 0.3% CaCO3
- CEC: 12 meq/100 g
- pH value: 6.92 CaCl2
- Pretreatment of soil: Sterilization by vapour pressure (120 °C, 30 min)
- Fertilization: 2.4 g Blaukorn/L
- Other: The pots of each plant species were placed in the greenhouse in a randomized design with all plants of one species arranged together in a species plot. On Day 7, the pots of each plant species except Allium cepa were rearranged within each species plot. Allium cepa was rearranged on Day 14. Up to 4 d prior to the final assessment, the pots of each plant species were rearranged according to their treatment level to facilitate the final assessment.

GROWTH CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light source: Natural daylight supplemented by artificial lighting
- Light intensity and quality: 173.73 - 537.5 µmol/m²/sec
- Day/night temperatures: 12 - 32 °C during light and dark cycle
- Relative humidity (%): 45 - 95% during light and dark cycle
- Watering regime and schedules: After application of the test item, bottom watering was performed throughout the study period with saucers underneath each pot. Water was supplied and retained within the saucer according to the needs of the plants to maintain an optimal water supply for plant growth. The top soil was kept moist until the controls reached 50% of seedling emergence. Watering was checked daily.
- Fertilization: No additional fertilizer was needed during the study.

EFFECT PARAMETERS MEASURED:
- Emergence: The number of emerged seedlings was counted per replicate for each test group on Day 0 (min 50% emergence of control seedlings) and Days 7, 14 and 21 post emergence of 50% control seedlings. The seedlings were considered as emerged when they reached the BBCH stage 10, except onion. Onion (Allium cepa) was counted as emerged with 1 cm visible shoot.
- Survival: The number of emerged seedlings that survivved was recorded per replicate for each test group on Day 0, 7, 14 and 21 post emergence of 50% control seedlings.
- Phytoxicity: Visual phytotoxicity ratings according to EPPO Standard 135 (e.g. chlorosis, necrosis, bleaching, reddening, deformation, stunting) were recorded per replicate for each test group on Day 0, 7, 14, and 21 post emergence of 50% control plants.
- Phytotoxicity rating system: Phytotoxicity was (subjectively) recorded from the living plants at each assessment time following a 0-E rating system (0: no injury/effect, A: slight symptom(s), B: moderate symptom(s), C: severe symptom(s), D: total-plant symptom(s) and E: moribund). Any plant considered dead was not rated and removed from the pot. Symptoms assessed: a = chlorosis (yellowing of green shoot tissue); b = necrosis (e.g. brown shoot tissue, parts of the plant die); c = bleaching (e.g. shoot tissue without pigmentation); d = deformation (e.g. leaf curl, abnormal leaf shape, abnormal plant habitus); e = stunting (e.g. plant height reduced with shorter internode length, plant growth reduction); f = reddening (reddening of green shoot tissue)
- Growth stages: Growth stages at the final assessment were recorded as minimum and maximum per replicate for each test group according to BBCH-Monograph - Growth stages.
- Shoot dry weight: The shoot dry weight was determined at the final assessment. The dry weight was determined on a replicate basis, i.e. all plants of one pot (representing one replicate) were weighed together. Plants were cut directly at the soil surface and put in pre-weighed bags. The bags containing the plants were dried at 60°C until constant weight and reweighed (considering the tare weight) to obtain the shoot dry weight of each replicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.3
- Range finding study: No

Nominal and measured concentrations:

0.06, 0.14, 0.31, 0.71, 1.64, 3.78, 8.7, and 20.0 g a.s./ha (nominal)
8.0E-05, 1.87E-04, 4.13E-04, 9.47E-04, 2.19E-03, 5.04E-03, 1.16E-02, and 2.67E-02 mg a.s./kg soil dw (nominal, converted values calculated for this entry by technical editor)

Reference substance (positive control):

no

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Survival

Remarks on result:

other: corresponding to 0.06 g a.s./ha (NOER)

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Survival

Remarks on result:

other: 95% CI: 2.80E-04 to 5.87E-04 mg/kg soil dw

Remarks:

result corresponding to 0.30 g a.s./ha (ER50)

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.001 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass (shoot dry weight)

Remarks on result:

other: 95% CI: 2.53E-04 - 1.07E-03 mg/kg soil dw

Remarks:

result corresponding to 0.39 g a.s./ha (ER50)

Details on results:

In the above table the effect concentrations for the most sensitive speicies are listed. For the remaining results please refer to "Any other information on results incl. tables".

Reported statistics and error estimates:

Seedling emergence, survival and shoot dry weight were compared to the untreated control using the ToxRat software for statistical analysis (ToxRatPro version 3.2.1). Statistical analyses were performed to obtain NOER (No Observed Effect Rate), LOER (Lowest Observed Effect Rate), ER25/ER50 effect ratesfor emergence, survival and shoot dry weight.

VALIDITY CRITERIA

The study fulfilled the validity criteria of the guideline and is thus considered valid (Table 1).

 

Table 1. Validity criteria defined by OECD guideline 208.

Criterion	Outcome	Criterion fulfilled
Seedling emergence in the controls is at least 70%.	Control emergence (% of seeds) and survival (% of emerged plants): B. vulgaris: 100 and 100% B. naputs: 95 and 100% C. sativa: 90 and 100% G. max: 100 and 100% H. annus: 90 and 100% S. lycopersicum: 95 and 100% A. cepa: 70 and 100% A. sativa: 95 and 94.7% L. perenne: 85 and 100% Z. mays: 100 and 100%	Yes
No visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, deformatons) in control seedlings and the plants of every particular species exhibit normal variation in growth and morphology.	No visible phytotoxic effects and normal variation in growth, plant development and morphology in control plants.	Yes
The environmental conditions for a particular species are identical and the growing media contain the same amount of soil matrix, support media or substrate from the same source.	The environmental conditions during the test were identical within species. The pots were filled in equal manner with the same soil.	Yes

 

ANALYTICAL RESULTS

The measured test item concentration in the initial test item stock solution was 94% of nominal (HPLC analysis).

 

BIOLOGICAL RESULTS

Typical symptoms observed at the final assessment (21 d after 50% control seedling emergence) in this seedling emergence study include chlorosis, reddening, necrosis, deformation and stunting of the seedlings. The severity and occurrence of the observed phytotoxic symptoms differed among species and test item rates.

 

The NOER, LOER, ER25/ER50 for emergence, survival and shoot dry weight expressed in g a.s./ha are summarized for each plant species in Tables 2, 3 and 4 for the final assessment (21 d after 50% emergence of the control seedlings).

 

Table 2. Emergence

Plant species	ER25	95% confidence limits		ER50 (EC50)	95% confidence limits		LOER	NOER (NOEC)
	g a.s./ha	lower	upper	g a.s./ha (mg/kg soil dw*)	lower	upper	g a.s./ha	g a.s./ha
Beta vulgaris	0.65	0.43	0.85	1.06 (1.41E-03)	0.80	1.39	0.71	0.31
Brassica napus	0.80	0.60	0.97	1.03 (1.37E-03)	0.85	1.26	1.64	0.71
Cucumis sativus	12.34	5.53	7883.73	>8.7^b (>1.16E-02)	n.d.	n.d.	0.71	0.31
Glycine max	2.84	1.76	4.22	6.32 (8.43E-03)	4.24	12.62	3.78	1.64
Helianthus annus	7.11	n.d.	n.d.	12.30 (1.64E-02)	n.d.	n.d.	8.70	3.78
Solanum lycopersicum	0.40	0.28	0.50	0.57 (7.60E-04)	0.45	0.72	0.71	0.31
Allium cepa	1.61	n.d.	n.d.	1.81 (2.41E-03)	n.d.	n.d.	1.64	0.71
Avena sativa	> 8.7^a	n.d.	n.d.	>8.7^a (>1.16E-02)	n.d.	n.d.	>8.7	8.7
Lolium perenne	0.66	0.45	0.87	1.08 (1.44E-03)	0.82	1.59	1.64	0.71
Zea mays	> 20^b	n.d.	n.d.	>20^b (2.67E-02)	n.d.	n.d.	>20	20

* Calculated by technical editor.

n.d.: not determined (outside the tested ranged or mathematical reason)

^a: Not calculated (no negative effect observed up to the highest rates tested)

^b: Not calculated (outside the tested range)

                                              

Table 3. Survival

Plant species	ER25	95% confidence limits		ER50 (EC50)	95% confidence limits		LOER	NOER (NOEC)
	g a.s./ha	lower	upper	g a.s./ha (mg/kg soil dw*)	lower	upper	g a.s./ha (mg/kg soil dw*)	g a.s./ha (mg/kg soil dw*)
Beta vulgaris	1.41	0.71	31.57	2.92 (3.89E-03)	1.36	2754.07	0.71	0.31
Brassica napus	>1.64^a	n.d.	n.d.	>1.64^a (> 2.19E-03)	n.d.	n.d.	>1.64^a	1.64^a
Cucumis sativus	1.68	n.d.	n.d.	2.30 (3.07E-03)	n.d.	n.d.	3.78	1.64
Glycine max	2.70	1.67	4.19	5.09 (6.79E-03)	3.40	11.45	1.64	0.71
Helianthus annus	19.35	7.85	n.d.	>20^b (2.67E-02)	n.d.	n.d.	20	8.70
Solanum lycopersicum	>3.78^b	n.d.	n.d.	>3.78^b (5.04E-03)	n.d.	n.d.	>3.78	3.78
Allium cepa	0.73	0.47	0.99	1.09 (1.45E-03)	0.82	1.82	0.71	0.31
Avena sativa	2.01	1.37	2.63	3.31 (4.41E-03)	2.53	4.44	1.64	0.71
Lolium perenne	0.17	0.11	0.23	0.30 (4.0E-04)	0.21 (2.8E-04)	0.44 (5.87E-04)	0.14	0.06 (8.00E-05)
Zea mays	>20^a	n.d.	n.d.	>20^a (2.67E-02)	n.d.	n.d.	>20	20

* Calculated by technical editor.

n.d.: not determined (outside the tested ranged or mathematical reason)

^a: Not calculated (no negative effect observed up to the highest rates tested)

^b: Not calculated (outside the tested range)

  

Table 4. Shoot dry weight

Plant species	ER25	95% confidence limits		ER50	95% confidence limits		LOER	NOER
	g a.s./ha (mg/kg soil dw*)	lower	upper	g a.s./ha (mg/kg soil dw*)	lower	upper	g a.s./ha (mg/kg soil dw*)	g a.s./ha (mg/kg soil dw*)
Beta vulgaris	1.49	n.d.	n.d.	1.97 (2.63E-03)	n.d.	n.d.	3.78	1.64
Brassica napus	0.82	0.32	2.10	1.26 (1.68E-03)	0.64	2.46	0.31	0.14
Cucumis sativus	1.87^c	n.d.	n.d.	3.78^d (5.04E-03)	n.d.	n.d.	> 3.78	> 1.64^e
Glycine max	1.36^c	0.64	2.87	12.95^c (1.73E-02)	2.37	70.72	0.71	0.31
Helianthus annus	3.87^c	2.52	5.40	15.35^c (2.05E-02)	10.24	31.23	3.78	1.64
Solanum lycopersicum	0.83	0.03	25.61	1.95 (2.60E-03)	0.02	173.05	1.64	0.71
Allium cepa	0.29	0.06	1.51	0.72 (9.6E-04)	0.21	2.50	0.31	0.14 (1.87E-04)
Avena sativa	0.40^c	0.06	0.80	8.47^c (1.13E-02)	3.95	76.32	< 0.31	< 0.31
Lolium perenne	0.20^c	0.11	0.38	0.39^c (5.2E-04)	0.19 (2.53E-04)	0.80 (1.07E-03)	0.31	0.14
Zea mays	5.85^c	3.42	8.92	>20^d (2.67E-02)	19.94	129.80	1.64	0.71

* Calculated by technical editor.

n.d.: not determined (outside the tested ranged or mathematical reason)

^c:Probit analysis, replicates used while fitting

^d: Calculated value was outside the tested range.

^e: Toxrat calculated the NOER as 3.78 g a.s./ha. At this rate only one plant survived. Moreover, this calculation should be considered cautiously since the criteria for normality and homogeneity were not met.

  

CONCLUSIONS

The most sensitive species was Lolium perenne with the lowest ER50 of 0.30 g a.s./ha (EC50 = 4.0 E-04 mg/kg soil dw) based on survival and 0.39 g a.s./ha (EC50 = 5.20 E-04 mg/kg soil dw) based on biomass (shoot dry weight) as well as the lowest NOER of 0.06 g a.s./ha (NOEC = 8.0 E-05 mg/kg soil dw) based on survival.

The second most sensitive species was Solanum lycopersicum with an ER50 of 0.57 g a.s./ha (EC50 = 7.60 E-04 mg/kg soil dw) based on emergence.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Reference 2

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 Jun - 05 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 227 (Terrestrial Plant Test: Vegetative Vigour Test)

Version / remarks:

July 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4150 (Terrestrial Plant Toxicity Tier I (vegetative vigor))

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: Initial test item stock solution (100 g a.s./ha) and of deionized water (control).
- Sampling method: Samples were taken directly before application.
- Sample storage conditions before analysis: Deep frozen

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation: A test item stock solution (equivalent to 100 g a.s./ha and 200 L/ha) was prepared by dissolving the test item in deionized water. The stock solution itself was not applied but used for analytical purposes. The test item concentrations were prepared by dilution of the test item stock solution with deionized water. The amount of deionized required for the preparation of each test item rate water was determined gravimetrically.
- Method of application to soil surface: The serial dilutions of the test item stock solution and the control solution were applied onto the foliage of plants and above-ground portions at a volume rate of 200 L/ha with a calibrated sprayer (SprayLab SLGH 2500, Schachtner, Ludwigsburg, Germany); Nozzel Teejet 8001 EVS (1 nozzle was used); Spray pressure: 3.60 / 4.0 bar; Application speed: 2 km/h; Distance (nozzle - target area): 30 cm. Calibration to 200 L/ha ± 10% was performed by spraying glass plates of known weight and area and weighing these immediately afterwards to determine the actual amount of water applied.
- Controls: 200 L deionized water/ha (spray)
- Other: The plant species were treated at the 2 - 4 leaf stage with 5 test item rates and a water control.

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sugar beet
- Plant family: Chenopodiaceae
- Variety: 2VK1433
- Source of seed: KWS Saat AG (21 Jun 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 80%
- Seed storage:The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Oilseed rape winter
- Plant family: Brassicaceae
- Variety: Marathon
- Source of seed: Samen Schlereth (18 Mar 2016)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Cucumber
- Plant family: Cucurbitaceae
- Variety: Delikatess
- Source of seed: Enza Zaden (06 Sep 2013)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Soybean
- Plant family: Fabaceae
- Variety: Sultana
- Source of seed: Samen Schlereth (27 January 2015)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Helianthus annuus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sunflower
- Plant family: Asteraceae
- Variety: Pacino Cola
- Source of seed: Benary (16 Aug 2017)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

other: Solanum lycopersicum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Tomato
- Plant family: Solanaceae
- Variety: Balkonstar
- Source of seed: Enza Zaden (24 Jul 2015)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Plant family: Amaryllidaceae
- Variety: Braunschweiger Blutrot
- Source of seed: Enza Zaden (06 Aug 2015)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Oat
- Plant family: Poaceae
- Variety: Flämingsstern
- Source of seed: Samen Schlereth (14 Mar 2011)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 80%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Ryegrass
- Plant family: Poaceae
- Variety: Belevue
- Source of seed: Samen Schlereth (10 Oct 2017)
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 95%
- Seed storage: The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Corn
- Plant family: Poaceae
- Variety: Saludo
- Source of seed: Bundesamt für Ernährungssicherheit A-1220 Wien
- Prior seed treatment/sterilization: The seeds had no chemical treatments or pesticide coatings.
- Historical germination of seed (germination of seed lot tested): 100%
- Seed storage:The seeds were stored in closed boxes in a refrigerator (0 - 15 °C).

Test type:

vegetative vigour test

Study type:

laboratory study

Remarks:

greenhouse

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Test temperature:

17 - 32 °C during light and dark cycle

pH:

6.92 CaCl2 (soil)

Moisture:

48 - 94% relative humidity during light and dark cycle

Details on test conditions:

TEST SYSTEM
- Testing facility: Greenhouse conditions Bayer AG
- Test container: Commercial non-porous 15 cm plastic pots filled with approx. 1.2 L soil.
- Amount of soil: 1.2 L
- Method of seeding: To reach the 2-4 leaf stage at the start of testing, the seeds were sown prior to testing. The commercial plastic flower pots were pre-filled with soil and only intact and healthy seeds of similar size were sowed for the test. The seeds were manually pressed into the soil surface and completely covered with a layer of soil to give a seeding depth of approximately 1 - 2 cm. The pots were top watered and only healthy looking plants were used for the test. The plants selected for controls and treatments were comparable.
- No. of test groups: 6 (control: 1 and test item: 5)
- No. of seeds per container: 2 or 4 seeds, depending on plant species (total: 20 plants per test group)
- No. of replicates per treatment group: 10 (2 seeds/pot) or 5 (4 seeds/pot), depending on plant species (1 pot = 1 replicate)
- No. of replicates per control: 20 seeds in 5 or 10 replicates, depending on plant species
- Other: The pots of each plant species were placed in the greenhouse in a randomized design with all plants of one species arranged togheter in a species plot. During the course of the experimental study part, on Day 7 after application, the pots of each plant species were rearranged withing each species plot. Up to 4 d prior to the final assessment, the pots of each plant species were rearranged according to their treatment level to facilitate the final assessment.

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil type and geographic location: The used soil was silt loam, a mixture of standard "Riedberg" soil + washed sand (ratio 9/1) from Bayer AG, Horticulture and Services, H 872, Frankfurt am Main, Germany. The soil mixture is named NTP soil.
- Collection procedures: The soil was sieved to 2 mm.
- % sand: 4.9% fine sand (0.063 - 0.20 mm), 20.3% middle sand (0.20 - 0.63 mm), and 6.1% coarse sand (0.63 - 2.0 mm)
- % silt: 4.9% fine silt (0.002 - 0.006 mm), 17.8% middle silt (0.006 - 0.02 mm), and 28.9% coarse silt (0.02 - 0.063 mm)
- % clay: 17.1% clay
- Organic carbon (%): 0.72% C
- CEC: 12 meq/100 g
- pH value. 6.92 (CaCl2)
- Pretreatment of soil: Sterilization by vapor pressure (120 °C, 30 min)
- Fertilization: 2.4 g Blaukorn/L

GROWTH CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light source: Natural daylight supplemented by artificial lighting
- Light intensity and quality: 200 - 602 µmol/m²/s
- Day/night temperatures: 17 - 32 °C during light and dark cycle
- Relative humidity (%): 48 - 94% during light and dark cycle
- Watering regime and schedules: Bottom watering was performed with saucers beneath each pot throughout the study. Water was supplied and retained within the saucer according to the need of the plants to maintain an optimal water supply for plant growth. Watering was checked daily.
- Any pest control method/fertilization: Liquid fertilizer (Wuxal Universaldünger) was added to the saucers during the watering of all plant species.

EFFECT PARAMETERS MEASURED:
- Survival: Number of plants that survived per replicate. Assessment: Day 7, 14 and 21 after application.
- Phytotoxicity: Visual rating according to EPPO standard 135 (e.g. chlorosis, necrosis, bleaching, deformation, reddening, stunting) of every replicate. Assessment: Day 7, 14 and 21 after application.
- Phytotoxicity rating system: Phytotoxicity was (subjectively) recorded from the living plants at each assessment time following a 0-E rating system (0: no injury/effect, A: slight symptom(s), B: moderate symptom(s), C: severe symptom(s), D: total-plant symptom(s) and E: moribund). Any plant considered dead was not rated and removed from the pot. Symptoms assessed: a = chlorosis (yellowing of green shoot tissue); b = necrosis (e.g. brown shoot tissue, parts of the plant die); c = bleaching (e.g. shoot tissue without pigmentation); d = deformation (e.g. leaf curl, abnormal leaf shape, abnormal plant habitus); e = stunting (e.g. plant height reduced with shorter internode length, plant growth reduction); f = reddening (reddening of green shoot tissue)
- Growth stage: The minimum and maximum BBCH for the test item rates and control. Assessment: At the end of the assessment period.
- Shoot dry weight: The mean shoot dry weight for each replicate compared to the untreated controls. Assessment: At the end of the assessment period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.3
- Range finding study: No

Nominal and measured concentrations:

Control, 0.71, 1.64, 3.78, 8.7 and 20.0 g a.s./ha (nominal test item rates applied with a volume rate equivalent to 200 L/ha)
Control, 9.47E-04, 2.19E-03, 5.04E-03, 1.16E-02, and 2.67E-02 mg a.s./kg soil dw (converted values calculated for this entry by technical editor)

Reference substance (positive control):

no

Species:

other: B. vulgaris, C. sativus, G. max, S. lycopersicum, Z. mays

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 0.001 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Biomass

Remarks:

shoot dry weight

Remarks on result:

other:

Remarks:

corresponding to 0.71 g a.s./ha (NOER)

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.002 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Biomass

Remarks:

shoot dry weight

Remarks on result:

other: 95% CI: 0.01 - 0.032 mg a.s./kg soil dw

Remarks:

corresponding to 11.97 g a.s./ha (ER50)

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.02 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Survival

Remarks on result:

other: 95% CI: 0.015 - 0.034 mg a.s./kg soil dw

Remarks:

corresponding to 15.16 g a.s./ha (ER50)

Reported statistics and error estimates:

Survival and shoot dry weight were compared to the untreated control using the ToxRat software for statistical analysis (ToxRatPro version 3.2.1). Statistical analyses were performed to obtain NOER (No Observed Effect Rate), LOER (Lowest Observed Effect Rate), ER25/ER50 for survival and shoot dry weight.

VALIDITY CRITERIA

The study met the validity criteria laid down by the OECD guideline 227 and is thus considered valid (Table 1).

Table 1. Validity criteria defined by OECD guideline 227 and study performance.

Performance criteria	Outcome	Criterion fulfilled
Seedling emergence is ≥ 70%.	The germination rate of the seeds used in this study was ≥ 70%.	Yes
In the controls, the plants do not exhibit phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and only normal variation in growth and morphology for that particular species.	The control plants of each species showed normal variation in growth, plant development and morphology.	Yes
In the controls, the mean plant survival is ≥ 90% for the duration of the study.	All plant species showed a survival of ≥ 90% in the control.	Yes
The environmental conditions for a particular species are identical and growing media contain the same amount of soil matrix, support media, or substrate from the same source.	The environmental conditions during the test were identical within each species. The pots used for all species were filled in equal manner with the same soil.	Yes

 

ANALYTICAL RESULTS

The measured test item concentration in the initial test item stock solution was 95 to 97% of nominal (HPLC analysis).

BIOLOGICAL RESULTS

Phytotoxic symptoms observed at the final assessment (Day 21 after application) include chlorosis, necrosis, deformation and stunting of the plants. The severity and occurrence of phytotoxic symptoms differed among species and test item rates.

The effect values for survival and shoot dry weight are summarized for each plant species in Table 2 and Table 3 for the final assessment (Day 21 after application).

Table 2. Survival

Plant species	ER25	95% confidence limits		ER50 (EC50)	95% confidence limits		LOER	NOER (NOEC)
	g a.s./ha	lower	upper	g a.s./ha (mg a.s./kg soil dw*)	lower	upper	g a.s./ha	g a.s./ha (mg a.s./kg soil dw*)
Beta vulgaris	15.59	10.41	22.65	23.12^e (0.031)	17.38	63.21	20.0	8.70
Brassica napus	20.0	n.d.	n.d.	22.64^e (0.0030)	n.d.	n.d.	20.0	8.70
Cucumis sativus	> 20^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	> 20	20
Glycine max	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20
Helianthus annus	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20
Solanum lycopersicum	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20
Allium cepa	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20
Avena sativa	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20
Lolium perenne	8.45	5.54	11.66	15.16 (0.020)	11.03 (0.015)	25.64 (0.034)	8.7	3.78 (0.005)
Zea mays	> 20^c	n.d.	n.d.	> 20^c (> 0.027)	n.d.	n.d.	> 20	20

* Calculated by technical editor.

n.d.: Not calculated (outside the tested ranged or mathematical reason)

^b: Not calculated (outside the tested range)

^c: Not calculated (no effect observed up to the highest rate tested)

^e: Extrapolated value (< 10% above the highest rate tested)

 

Table 3. Shoot dry weight

Plant species	ER25	95% confidence limits		ER50 (EC50)	95% confidence limits		LOER	NOER (NOEC)
	g a.s./ha	lower	upper	g a.s./ha (mg a.s./kg soil dw*)	lower	upper	g a.s./ha	g a.s./ha (mg a.s./kg soil dw*)
Beta vulgaris	4.87	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	0.71	< 0.71 (<9.47E-04)
Brassica napus	7.11	n.d.	n.d.	16.29 (0.022)	n.d.	n.d.	3.78	1.64 (2.19E-03)
Cucumis sativus	< 0.71^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	0.71	< 0.71 (<9.47E-04)
Glycine max	> 20^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	0.71	< 0.71 (<9.47E-04)
Helianthus annus	> 20^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	> 20	20
Solanum lycopersicum	> 20^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	0.71	< 0.71 (<9.47E-04)
Allium cepa	> 20^b	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	> 20	20
Avena sativa	> 20^a	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	20	8.7
Lolium perenne	4.01^d	1.79	6.09	11.97^d (0.016)	7.96 (0.011)	24.14 (0.032)	3.78	1.64 (2.19E-03)
Zea mays	18.02	n.d.	n.d.	> 20^b (> 0.027)	n.d.	n.d.	1.64	0.71 (<9.47E-04)

* Calculated by technical editor.

n.d.: Not calculated (outside the tested ranged or mathematical reason)

^a: Calculated values were outside the tested range

^b: Not calculated (outside the tested range)

^d: Probit analysis: replicates used while fitting

 

CONCLUSIONS

Lolium perenne was the most sensitive species with the lowest ER50 of 11.97 g a.s./ha (EC50 = 0.01596 mg a.s./kg soil dw) based on biomass (shoot dry weight) and the second lowest ER50 of 15.16 g a.s./ha (EC50 = 2.02E-02 mg a.s./kg soil dw) based on survival. The lowest NOER of < 0.71 g a.s./ha (NOEC <9.47E-04 mg a.s./kg soil dw) was obtained for Beta vulgaris, Cucumis sativus, Glycine max, Solanum lycopersicum based on biomass (shoot dry weight). 

The second most sensitive species was Brassica napus with an ER50 of 16.29 g a.s./ha (2.17E-02 mg/kg soil dw) based on biomass (shoot dry weight).

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (21 d) = 8.0 E-05 mg a.s./kg soil dw (OECD 208, nominal, L. perenne, survival)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

0 mg/kg soil dw

Additional information

Three studies are available, in which the toxicity of Indaziflam (CAS 950782-86-2) to terrestrial plants was assessed according to OECD guidelines and GLP.

In the first study (M-631211-01-1), a Seedling Emergence and Seedling Growth Test according to OECD guideline 208 was performed. In this test, a total of 10 non-target terrestrial crop plant species (6 dicotylenous and 4 monocotyledonous species from 8 plant families) were sown in silt loam and treated with 5 test item rates ranging from 0.06 – 20.0 g a.s./ha (corresponding to the concentration range of 8.00 E-05 to 2.67E-02 mg a.s./kg soil dw) for 21 d under controlled greenhouse conditions. The test item rates were applied to the soil surface using a calibrated laboratory track sprayer at a volume rate of 200 L/ha (pre-emergence application). Test item concentrations in the stock solution were analytically v erified by HPLC. A water control was run in parallel. On Days 0, 7, 14, and 21 post emergence of 50% of the control seedlings various assessments were made including for seedling emergence, plant survival, visual phytotoxicity, plant growth stage and shoot dry weight.

The measured test item concentration in the stock solution was 94% of nominal. The most sensitive species was Lolium perenne with the lowest ER50 of 0.30 g a.s./ha (EC50 = 4.0 E-04 mg a.s./kg soil dw) based on survival and 0.39 g a.s./ha (EC50 = 5.20 E-04 mg a.s./kg soil dw) based on biomass (shoot dry weight) as well as the lowest NOER of 0.06 g a.s./ha (NOEC = 8.0 E-05 mg a.s./kg soil dw) based on survival.

In the second study (M-633942-01-1), a Vegetative Vigour Test according to OECD guideline 227 was performed. In this test, a total of 10 non-target crop plant species (6 dicotylenous and 4 monocotyledonous species from 8 plant families) were sown in silt loam and treated with 5 test item rates ranging from 0.71 – 20.0 g a.s./ha (corresponding to the concentration range of 9.47E-04 to 2.67E-02 mg a.s./kg soil dw) for 21 d under controlled greenhouse conditions. The plant species were treated at the 2-4 leaf stage by spraying the test item solutions onto the foliage of plants and above-ground plant portions using a calibrated laboratory track sprayer at a volume rate of 200 L/ha. Test item concentrations in the stock solution were analytically verified by HPLC. A water control was run in parallel. On Days 0, 7, 14, and 21 after application plant survival and visual phytotoxicity were assessed.

The measured test item concentration in the stock solution was 95 - 97% of nominal. Lolium perenne was the most sensitive species with the lowest ER50 of 11.97 g a.s./ha (EC50 = 0.01596 mg a.s./kg soil dw) based on biomass (shoot dry weight) and the second lowest ER50 of 15.16 g a.s./ha (EC50 = 2.02E-02 mg a.s./kg soil dw) based on survival. The lowest NOER of < 0.71 g a.s./ha (NOEC < 9.47E-04 mg a.s./kg soil dw) was obtained for Beta vulgaris, Cucumis sativus, Glycine max, Solanum lycopersicum and Zea mays based on biomass. 

In the third study (M-635874-01-1), a seedling emergence and growth test in accordance to OECD 208 was performed with 10 non-target terrestrial crop plant species (6 dicotylenous and 4 monocotyledonous species from 5 plant families). Five seeds of each species were sown in the pots filled with treated silt loam soil and treated with 5 test item rates ranging from 0.72 to 19.48 µg/kg dw (corresponding to 0.13 to 3.79 µg a.s./kg dw). Serial dilutions of the test item were incorporated in soil by using a shaker for pre soil mixtures and a concrete mixer for the final soil mixing. A water control was tested in parallel. On day 0, 7, 15 and 21 post emergence of 50% of control seedlings survival, phototoxicity, growth stages and shoot dry weight were assessed.The most sensitive species was Brassica napus winter with the lowest EC10 (21 d) of 0.39 µg a.s./kg soil dw based on biomass (shoot dry weight). The second most sensitive species was Lolium multiflorum with an EC10 (21 d) of 0.42 µg a.s./kg soil dry weight also based on biomass (shoot dry weight).