Ethanol, 2,2'-iminobis-, N-(C13-15-branched and linear alkyl) derivs.

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Oct 2012 - 24 Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 22030: Soil quality - Biological methods - Chronic toxicity in higher plants

Deviations:

yes

Remarks:

Temperature and humidity were not in line with recommendations from the guideline

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): One day before application, the application solution of the highest test concentration of the test substance (i.e. 800 mg/kg dry soil for oat) was prepared by dissolving 17.104 mL of test item (corresponding to 15.565 g based on a density of 0.91 g/mL) in 100 mL methanol under continuous stirring for five minutes. This amount corresponded to 3.113 mg, dissolved within 20 mL methanol, and applied to 4.000 kg air dry weight soil (corresponding to 3.891 kg dry weight soil and to 800 mg test item per kg dry soil substrate, see section 3.4). Aliquots of this application solution were subsequently diluted with methanol to obtain application solutions for the lower test concentrations of both species. Then, the volumes of 20 or 40 mL of test item-methanol mixtures (needed for the application of the test item-methanol mixture into soil batches for four replicates of one species or 2 x four replicates of two species) were applied on 80 and 160 g quartz sand, respectively, and the solvent was allowed to evaporate overnight. Afterwards, the test item-sand mixtures were mixed into the respective amounts of soil substrates under continuous stirring for five minutes.
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol
- Evaporation of vehicle before use: yes

Species:

Brassica rapa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: turnip rape
- Plant family: Brassicaceae
- Variety: rapid cycling
- Source of seed: Carolina Biological Supply Company, Burlington, NC, USA

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: oat
- Plant family: Poaceae
- Source of seed: Landi Gelterkirchen, Gelterkirchen, Germany

Test type:

other: Chronic toxicity test based on emergence, vegetative growth and reproductive capacity

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

61 d

Remarks:

32 d for Brassica rapa

Test temperature:

Turnip rape: 14.6 - 39.1 °C (23.0 °C mean)
Oat: 22 Feb - 11 Apr 2013 (09:15 h): 14.6 - 39.3 °C (mean: 23.1 °C); 11 Apr (11:20 h) - 24 Apr 2013: 17.3 - 53.2 °C (mean: 27.1 °C)

pH:

6.8 ± 0.2 (initial)

Moisture:

28.2% of water-holding capacity at start of seeding

Details on test conditions:

TEST SYSTEM
- Testing facility: greenhouse
- Test container (type, material, size): non-porous plastic pots (inner diameter at bottom and top: 9.0 and 12.0 cm, respectively; height: 12.0 cm)
- Amount of soil: 800 g soil (fresh weight)
- No. of seeds per container: 10 for the first 14 d, afterwards 4
- No. of plants (retained after thinning):
- No. of replicates per treatment group: 4 (per treatment and plant species)
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Source: Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), Speyer, Germany (LUFA Speyer 2.3, Batch Sp.2.3.4612)
- Pesticide use history at the collection site: The soil was not treated with any pesticide or organic fertiliser for at least three years prior to use.
- Soil texture (if natural soil)
- % sand: 63.1 ± 5.0
- % silt: 28.4 ± 4.5
- % clay: 8.5 ± 1.7
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- Organic carbon (%): 0.94 ± 0.10
- Maximum water holding capacity (MWC g/100 g dry soil): 37.3 ± 1.8
- Storage (condition, duration): Before use, the soil was stored slightly moist in water-tight containers or plastic bags. During storage, the water content of the air-dried substrate (with a rest of moisture) was 2.8% of the water holding capacity, resulting to 102.8 g air-dried substrate per 100 g dry substrate. For this study, the water content of the fresh substrate was adjusted to 28.2% resulting to 110.5 g water per 100 g dry soil or 110.5 g fresh soil per 100 g dry soil. Therefore, the amount of 7.7 g water had to be added to the 102.5 g air-dried substrate to achieve 110.5 g fresh substrate with 28.2% of water holding capacity.

NUTRIENT MEDIUM
- Description: The fertilizer WUXAL® was used with the following constituents(per L): 100 g N, 100 g P2O5, 75 g K2O, 124 mg B, 50 mg Cu, 248 mg Fe, 149 mg Mn, 12 mg Mo, 50 mg Zn. The application of the fertilizer was conducted as a 0.2% solution on 19-Mar-2013 (DAT 25: 50 ml per pot for both species), on 25-Mar-2013 (DAT 31: 50 mL per pot for turnip rape, 200 mL per pot for oat), on 02-Apr-2013 (DAT 39: 200 mL for oat) and 10-Apr-2013 (DAT 47: 200 mL for oat).


GROWTH CONDITIONS
- Photoperiod: 16 h light, 8 h darkness
- Relative humidity (%): Turnip rape: 13 - 66% (mean: 40%); Oat: 22 Feb - 11 Apr 2013 (09:15 h): 9-66% (mean: 38%); 11 Apr (11:20 h) - 24 Apr 2013: 8-82% (mean: 45%)
- Watering regime and schedules
- Water source/type: local tap water
- Volume applied: 50 - 300 mL per pot
- Interval of applications: 10 occasions for Turnip rape and 20 occasions for oat
- Method of application: bottom irrigation

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of plants emerged were observed at least every second working day during the first week until 50% of the seedlings had emerged. In addition, the plants were observed at least once a week for visual phytotoxicity (e.g., leaf and stem deformations, chlorosis, necrosis and overall appearance compared with the control) and plant mortality until 21 days after application.

At day 14 after 50% of the seedlings in the control had emerged, four out of the eight plants per pot were harvested (i.e. all endpoints at DAT 17 for turnip rape, mortality and symptoms at DAT 17 and biomass at DAT 18 for oat). The following endpoints were determined: Presence/absence of visible flower buds per plant of turnip rape, Number of flowers per plant of turnip rape, Fresh mass per plant of oat and turnip rape directly after cutting, Shoot length per plant of oat and turnip rape directly after cutting, Mortality of both plant species, Symptoms of toxicity. Afterwards, the density of the plants was reduced to four plants per pot.

At day 32 after application (i.e. development of control seed pods of turnip rape), the four turnip rape plants per pot were harvested. The following endpoints were determined: Growth stadium according to BBCH scheme, Number of seed pods carrying fertile seeds (visibly swollen), Shoot length per plant directly after cutting, Fresh mass of shoots (without inflorescences and seed pods) and seed pods directly after cutting, Dry mass of shoots and seed pods, Mortality.

At day 61 after application (i.e. emergence of inflorescences in the control oats) the four oat plants per pot were harvested. The following endpoints were determined: Growth stadium according to BBCH scheme, Total number of flowers per plant for oat, Shoot length per plant directly after cutting, Fresh mass of shoots (without inflorescences) and inflorescences directly after cutting, Dry mass of shoots and inflorescences, Mortality.

Determination of shoot fresh weight was carried out for all individual plants of one pot. The plants were cut directly between root and shoot. For that purpose, each plant was pulled slightly out of the soil to uncover the site of cutting. The length of the cut plants was measured directly after cutting, the fresh weight of the cut plants was determined directly after determination of the plant length. Determination of dry weight of different plant parts was carried out by measuring all shoots, inflorescences and seed buds of one pot (=1 replicate) of the respective treatment.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range-finding study
- Test concentrations: control, 1, 10, 100, 1000 mg/kg soil dw (Oil seed rape (38 d) and Oat (52 d))
- Results used to determine the conditions for the definitive study: No effect values were derived. However, effects were observed at concentrations of > 100 mg/kg soil dw.

Nominal and measured concentrations:

Brassica rapa (nominal): control, 6.25, 12.5, 25, 50, 100, 200 mg/kg soil dw
Avena sativa (nominal): control, 25, 50, 100, 200, 400, 800 mg/kg soil dw

Reference substance (positive control):

no

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of large fruits

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fresh weight (fruits)

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

18.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fresh weight (fruits)

Remarks on result:

other: 95% CL: 5.4 - 61.6 mg/kg soil dw

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight (fruits)

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

23.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight (fruits)

Remarks on result:

other: 95% CL: 8.6 - 65.5 mg/kg soil dw

Species:

Brassica rapa

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 200 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

< 25 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of flowers

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

EC10

Effect conc.:

2.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of flowers

Remarks on result:

other: 95% CL: 0.17 - 37.2 mg/kg soil dw

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

400 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fresh weight (flowers)

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

EC10

Effect conc.:

7.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fresh weight (flowers)

Remarks on result:

other: 95% CL: 0.3 - 210 mg/kg soil dw

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight (flowers)

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

EC10

Effect conc.:

19 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight (flowers)

Remarks on result:

other: 95% CL: 3.0 - 121 mg/kg soil dw

Species:

Avena sativa

Duration:

61 d

Dose descriptor:

NOEC

Effect conc.:

>= 800 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Reported statistics and error estimates:

The effect of the test item on emergence and on survival was expressed as LC10, LC25 and LC50, and the effect on growth (biomass of shoots, inflorescences and seed buds) as EC10, EC25 and EC50, if possible. The calculations were conducted by Probit analysis. The NOEC and LOEC for emergence, mortality and biomass were evaluated by appropriate statistical tests (i.e. data with a concentration-effect relationship: Williams t-test; data without a concentration-effect relationship: Dunnett t-test; data with a non-homogeneous distribution: Welch t-test after Bonferroni correction). Visual phytotoxicity were evaluated directly from the raw data considering a scoring system for visual injury.

The validity criteria were fulfilled.

Table 1: Summary of effects of Atmer 163 on growth and reproduction of rapid-cycling turnip rape after 17 and 32 days of exposure

DAT	Endpoint	NOEC	EC10	95% CL	EC25	95% CL	EC50	95% CL
		mg/kg soil dry weight
17	Number of buds	100	31.9	14.1 - 72.1	87.5	57.3 - 134	269	137 - 527
	Number of flowers	6.25	3.2	0.79 - 13.0	14.1	6.5 - 30.6	73	41.7 - 127
	Fresh weight (plant)	100	35.2	13.1 - 94.6	100	60.0 - 168	320	125 - 821
	Shoot length	50	60.7	42.1 - 87.7	114	93.9 - 138	229	179 - 293
	Mortality	> 200	n.a.	n.a.	n.a.	n.a.	> 200	n.a.
	Symptoms of toxicity	50	34.0	n.a.	50.0	n.a.	65.6	n.a.
32	BBCH stadium	> 200	n.a.	n.a.	n.a.	n.a.	> 200	n.a.
	Number of small fruits	> 200	n.a.	n.a.	n.a.	n.a.	> 200	n.a.
	Number of large fruits	50	n.a.	n.a.	7.7	1.5 - 39.3	73.6	26.7 - 202
	Shoot length	> 200	n.a.	n.a.	n.a.	n.a.	> 200	n.a.
	Fresh weight (shoot)	> 200	149	n.a.	n.a.	n.a.	> 200	n.a.
	Fresh weight ( fruits)	50	18.2	5.4 - 61.6	49.6	25.2 - 97.8	151	76.0 - 302
	Dry weight (shoot)	> 200	69.5	n.a.	n.a.	n.a.	> 200	n.a.
	Dry weight (fruits)	50	23.7	8.6 - 65.5	67.8	39.0 - 118	218	107 - 446
	mortality	> 200	n.a.	n.a.	n.a.	n.a.	> 200	n.a.

DAT: Days after treatment
n.a.: not applicable due to no clear concentration-effect relationship or scattering of data
95% CL: 95% Confidence Limits

Table 2: Summary of effects of Atmer 163 on growth and reproduction of oat after 17/18 and 61 d of exposure

DAT	Endpoint	NOEC	EC10	95% CL	EC25	95% CL	EC50	95% CL
		mg/kg soil dry weight
17	Mortality	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Symptoms of toxicity	200	181	n.a.	221	n.a.	275	n.a.
18	Fresh weight (plant)	100	68	32.9 - 141	192	129 - 287	610	403 - 924
	Shoot length	100	179	111 - 291	722	489 - 1065	> 800	n.a.
61	BBCH stadium	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Number of pre-flowers	100	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Number of flowers	< 25	2.5	0.17 - 37.2	28.9	7.9 - 106	431	174 - 1066
	Shoot length	< 25	110	n.a.	n.a.	n.a.	> 800	n.a.
	Fresh weight (shoot)	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Fresh weight (pre-flowers)	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Fresh weight (flowers)	400	7.6	0.3 - 210	111	33.1 - 375	> 800	179 - >800
	Dry weight (shoot)	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Dry weight (pre-flowers)	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.
	Dry weight (flowers)	50	19	3.0 - 121	106	43.5 - 259	715	247 - 2072
	mortality	> 800	n.a.	n.a.	n.a.	n.a.	> 800	n.a.

DAT: Days after treatment
n.a.: not applicable due to no clear concentration-effect relationship or scattering of data
95% CL: 95% Confidence Limits

Description of key information

NOEC (32 d): 50 mg/kg soil dw based on the fresh/dry weight of fruits of Brassica rapa (ISO 22030)
NOEC (61 d): < 25 mg/kg soil dw based on the number of flowers of Avena sativa (ISO 22030)
EC10 (61 d): 2.5 mg/kg soil dw based on the number of flowers of Avena sativa (ISO 22030)

Key value for chemical safety assessment

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

2.5 mg/kg soil dw

Additional information

One study is available investigating the chronic effects of Atmer 163 (CAS No. 97925-95-6) to the two plant species Brassica rapa and Avena sativa (Schmidt, 2013). The study was performed according to ISO 22030 (GLP). Both plant species were cultivated in artificial soil under controlled conditions in a greenhouse. Five concentrations of the test substance were mixed into soil (up to 200 mg/kg soil dw for B. rapa and up to 800 mg/kg soil dw for A. sativa). Emergence, vegetative growth and reproductive capacity were assessed throughout the exposure period of 32 d for B. rapa and 61 d for Avena sativa. In accordance with the guideline the reproductive endpoints were assessed at the end of exposure (e.g. number and dry-, freshweight of fruits and flowers). Effects of the test substance on all assessed reproductive parameters were observed for B. rapa. Fresh weight and dry weight of fruits was found to be the most sensitive endpoint with a NOEC (32 d) of 50 mg/kg soil dw. The data for A. sativa show a similar pattern. Here the number of flowers was found to be the most sensitive endpoint. A NOEC could not be derived since effects were already observed at the lowest concentration tested. Therefore, the NOEC (61 d) was reported as < 25 mg/kg soil dw. Since no NOEC could be derived, the EC10 was used instead for the Chemical Safety Assessment and the derivation of the PNEC soil. An EC10 (61 d) of 2.5 mg/kg soil dw was calculated. No mortality was recorded for both plant species up to the highest concentration tested.