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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: short-term (with study design considered suitable for long-term assessment)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jul 2018 to 24 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Version / remarks:
Jul 2006
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
SAMPLING DESCRIPTION
Four soil samples (each of 100 g) were taken before application from all test item groups of the respective trial and the solvent and tap water control. One for the analytical dose verification (A) and three retain samples (R). Specimen were put in plastic shells, sealed tightly and labelled correspondingly. The samples were stored deep frozen ≤ - 18 °C at the greenhouse immediately after sampling until transport to the analytical laboratory. The transport of the A-samples of the solvent control and the respective highest test item group was performed in a cool box with ice packs, respectively. In the analytical laboratory, the samples were stored again deep frozen until analysis.
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
- Method of mixing into soil: after evaporation of the solvent the treated quartz sand was thoroughly mixed for 5 to 10 minutes into the substrate using an electrical drilling machine. Immediately after thorough mixing the test soil was distributed to the pots.
- Method of application to soil surface: the test item application solution was added drop-wise to an appropriate amount of quartz sand
- Controls: untreated quartz
- Chemical name of vehicle: acetone

Species:
Beta vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: sugar beet
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: rape
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: tomato
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: pea
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: perennial ryegrass
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
artificial soil
Remarks:
Sandy loam: mixture of natural soil with sand
Limit test:
no
Total exposure duration:
21 d
Test temperature:
- Air temperature (min/max/mean) [°C]: 20.27 / 35.02 / 26.11
- Air temperature for the plant species Brassica napus (min/max/mean) [°C]: 19.69 / 31.39 / 26.42
pH:
Soil type with a pH of 7.75
Moisture:
- Relative humidity (min/max/mean) [%]: 46.06 / 85.05 / 70.40
- Relative humidity for the plant species Brassica napus(min/max/mean) [%]: 56.21 / 84.59 / 71.03
Details on test conditions:
TEST SYSTEM
- Test container: Plant pots (diameter: 15 cm)
- Amount of soil: 1.11 kg approx.
- Method of seeding: Untreated seeds from the test species were planted at an appropriate depth and approximately equally spaced in each pot.
- No. of seeds per container: 2 for all dicotyledonous species and 4 for monocotyledonous species
- No. of seeds: In each treatment group a total of 20 seeds were sown.
- No. of replicates per treatment group: 10 for all dicotyledonous species and 5 for monocotyledonous species
- No. of replicates per control: 10 for all dicotyledonous species and 5 for monocotyledonous species
- No. of replicates per vehicle control: 10 for all dicotyledonous species and 5 for monocotyledonous species

SOURCE AND PROPERTIES OF SUBSTRATE:
- Soil texture (if natural soil)
- % sand: 77.94 %
- % silt: 13.13 %
- % clay: 8.93 %
- Composition (if artificial substrate): Sandy loam
- Organic carbon (%): 0.73 % (1.25 % organic matter).
- Maximum water holding capacity (in % dry weigth): 40%
- Storage (condition, duration): deep frozen, up to 30 days between sampling and analysis

TEST CONDITIONS:
For all species apart from the plant species Brassica napus
- Photoperiod (light /dark) [h]: 16 / 8
- Light intensity [μmol/m²/s]: 820 – 1250

For the plant species Brassica napus
- Photoperiod (light /dark ) [h]: 16 / 8
- Light intensity [μmol/m²/s]: 395 - 1140
Nominal and measured concentrations:
- Test rates for all species except Pisum sativum: 0 (control), 0 (solvent control), 0.198, 0.593, 1.78, 5.33, 16.0 mg a.i./kg sdw
- Test rates for Pisum sativum: 0 (control), 0 (solvent control), 16.0, 48.0, 96.0, 288, 864 mg a.i./kg sdw
Analytical Rate Verification in Soil: The analysed concentration of TONALID in the highest test item concentration corresponded to 96 % of the target concentration for trial of all species apart from Brassica napus and 103 % for trial of Brassica napus.
Reference substance (positive control):
no
Key result
Species:
Beta vulgaris
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
< 0.198 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Species:
other: all species apart from Pisum Sativum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
other: all species apart from Pisum sativum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
>= 16 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 864 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
other: Beta vulgaris, Brassica napus, Lycopersicon esculentum, Lolium perenne
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: post-emergence mortality
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 864 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: post-emergence mortality
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: post-emergence mortality
Species:
other: Beta vulgaris, Brassica napus, Lycopersicon esculentum, Lolium perenne
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: post-emergence mortality
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 864 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: post-emergence mortality
Species:
other: Brassica napus, Lycopersicon esculentum, Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.78 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Species:
Phaseolus aureus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
< 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Species:
Beta vulgaris
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.29 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL (0.97-1.55)
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
8.32 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL (7.55 - 9.12)
Species:
Lycopersicon esculentum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
3.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL ( 2.81-4.16)
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
77.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL (54.3-105)
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
4.89 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Remarks on result:
other: 95% CL (4.21-5.97)
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: shoot dry weight
Details on results:
Seedling Emergence:
Seedling emergence was statistically significantly reduced compared to the control for Pisum sativum (Cochran-Armitage test with Rao-Scott adjustment; one-sided greater, α = 0.05). The highest inhibition of seedling emergence was shown in Pisum sativum with 45.0 % at 864 mg a.i./kg sdw.

Post-Emergence Mortality:
Post-emergence mortality was statistically significantly increased compared to the control for Allium cepa (Cochran-Armitage test; one-sided greater, α = 0.05). The highest inhibition compared to the control occurred for Allium cepa with 50.0 % at 16 mg a.i./kg sdw.

Phytotoxicity:
Symptoms of phytotoxicity were observed in all species tested. The observed symptoms were stunted growth, chlorosis, leaf deformation and necrosis. The highest mean phytotoxicity occurred for the species Beta vulgaris and Allium cepa each with 90.0 % at 16.0 mg a.i./kg sdw.

Growth Stage:
Differences in the growth stage of the plants (=BBCH stage) compared to the solvent control were observed for all species tested at least at the highest test item group. The biggest differences in the growth stage between the solvent control and the test item treated plants occurred for Pisum sativum with a BBCH growth stage of 60 in the solvent control group, in comparison to 10 to 15 at 864 mg a.i./kg sdw.

Shoot Dry Weight:
Statistically significant effects on shoot dry weight were detected for all species tested. Inhibition > 80 % occurred at least at the highest test item group for all plant species except Lolium perenne.
Reported statistics and error estimates:
The data of seedling emergence and post emergence mortality were analyzed with the multiple Fisher`s exact test with Bonferroni-Holm adjustment in case of no monotone dose-response relationship. In case of a monotone dose-response a test on variance of the data was conducted. If the variety of the data was non-homogenous, the Cochran-Armitage test with Rao-Scott adjustment was conducted. Otherwise, the Cochran-Armitage test was conducted. The data of shoot dry weight were tested for normality and homoscedasticity using Shapiro-Wilk`s Test and Levene-Test followed by a William`s test in case that both requirements were fulfilled and the trend analysis by contrast was significant. The multiple Welch`s t-test with Bonferroni-Holm adjustment was conducted in case that the data were non-homogenous. In case the data were not normal distributed but homoscedasticity and trend analysis by contrast were significant the Jonckheere-Terpstra test was used. Statistical analyses of seedling emergence, post-emergence mortality and shoot dry weight also included the determination of effect rates (EC50) and their 95 % confidence limits by Probit analysis using linear max. likelihood regression, where possible.

Table 1: Shoot dry weight per living plant [g] of dicotyledonous species

21 DA50E

Application rate [mg a.i./kg sdw]

Mean value [g]

± SD [g]

Inhibition (% compared to control) #

Beta vulgaris

Control (0)

0.237

0.084

-

Solvent control (0)

0.284

0.036

-

0.198

0.226

0.036

20.4

0.593

0.239

0.07

15.8

1.78

0.114

0.013

59.9

5.33

0.016

0.005

94.4

16

0.011

0.004

96.1

Brassica napus

Control (0)

0.989

0.176

-

Solvent control (0)

0.984

0.128

-

0.198

1.014

0.118

-3

0.593

1.048

0.198

-6.5

1.78

0.968

0.167

1.6

5.33

0.785

0.103

20.2

16

0.11

0.067

88.8

Lycopersicon esculentum

Control (0)

0.532

0.072

-

Solvent control (0)

0.526

0.083

-

16

0.439

0.043

16.5

48

0.342

0.101

35

96

0.234

0.138

55.5

288

0.07

0.08

86.7

864

0.073

0.088

86.1

Allium cepa

Control (0)

0.038

0.004

-

Solvent control (0)

0.033

0.005

-

0.198

0.033

0.006

0

0.593

0.029

0.006

12.1

1.78

0.031

0.002

6.1

5.33

0.015

0.003

54.5

16

0.001

0

97

Lolium perenne

Control (0)

0.059

0.017

-

Solvent control (0)

0.054

0.012

-

0.198

0.043

0.004

20.4

0.593

0.058

0.014

-7.4

1.78

0.045

0.01

16.7

5.33

0.045

0.004

16.7

16

0.039

0.006

27.8

Table 2: LOEC, NOEC and EC50 of TONALID for shoot dry weight 21 days after at least 50 % of the seedlings

LOEC

NOEC

EC50 (95% CL)

Dicotyledonous species

Beta vulgaris

0.198

< 0.198

1.29 (0.970 - 1.55)

Brassica napus

5.33

1.78

8.32 (7.55 - 9.12)

Lycopersicon esculentum

5.33

1.78

3.5 (2.81 - 4.16)

Pisum sativum

16

< 16

77.5 (54.3 - 105)

Monocotyledonous species

Allium cepa

5.33

1.78

4.89 (4.21 – 5.97)

Lolium perene

16

5.33

> 16

Validity criteria fulfilled:
yes
Conclusions:
The target concentration of the test item in the highest test item concentration was confirmed for both trials by the analytical dose verification in soil. The study was conducted to determine the effects of the test item on the seedling emergence and early growth of four dicotyledonous and two monocotyledonous species. Since the validity criteria were met for all species tested on day 21, the study can be regarded as valid. Statistically significant differences on seedling emergence could be observed for Pisum sativum. The LOEC was 48.0 mg a.i./kg sdw, the NOEC 16.0 mg a.i./kg sdw. An EC50 could not be calculated for any of the species tested. Statistically significant differences on post-emergence mortality could be observed for Allium cepa. The LOEC was 16.0 mg a.i./kg sdw, the NOEC 5.33 mg a.i./kg sdw. A reliable EC50 could not be calculated for any of the species tested. Statistically significant differences on shoot dry weight could be calculated for all species tested. The most sensitive species with regard to the EC50 was Beta vulgaris. The EC50 with 95 % confidence intervals was 1.29 (0.970 – 1.55) mg a.i./kg sdw. The NOEC based on shoot dry weight for the most sensitive species (Beta vulgaris) could not be determined and was considered < 0.198 mg a.i./kg sdw.
Executive summary:

The toxicity to terrestrial plants was determined in a Seedling Emergence and Seedling Growth Test according OECD guideline No 208 and in compliance with GLP criteria. The test item was tested on non-target terrestrial plant species. Four dicotyledonous and two monocotyledonous species were sown in soil treated with the test item at defined concentrations of 0 (control), 0 (solvent control), 0.198, 0.593, 1.78, 5.33, 16.0 mg a.i./kg sdw (applied for all species except Pisum sativum, 16.0, 48.0, 96.0, 288, 864 mg a.i.//kg sdw (applied for Pisum sativum). In each treatment group a total of 20 seeds were sown. The test duration was from application until 21 days after at least 50 % of the seeds in the solvent control had emerged in each species. During this period, plants were assessed for seedling emergence, post emergence mortality and phytotoxicity symptoms on day 7, 14 and 21. The effects on plant growth stage and shoot dry weight were determined for day 21. Results were compared to the solvent control.

The analysed concentration of the test item in the highest test item concentration corresponded to 96 % of the target concentration and 103 % for trial applied specifically for Pisum sativum. Seedling emergence was statistically significantly reduced compared to the control for Pisum sativum. The highest inhibition of seedling emergence was shown in Pisum sativum with 45.0 % at 864 mg a.i./kg sdw. Post-emergence mortality was statistically significantly increased compared to the control for Allium cepa. The highest inhibition compared to the control occurred for Allium cepa with 50.0 % at 16 mg a.i./kg sdw. Symptoms of phytotoxicity were observed in all species tested. Differences in the growth stage of the plants (=BBCH stage) compared to the solvent control were observed for all species tested at least at the highest test item group. The biggest differences in the growth stage between the solvent control and the test item treated plants occurred for Pisum sativum with a BBCH growth stage of 60 in the solvent control group, in comparison to 10 to 15 at 864 mg a.i./kg sdw. Statistically significant effects on shoot dry weight were detected for all species tested. Inhibition > 80 % occurred at least at the highest test item group for all plant species except Lolium perenne.

The target concentration of the test item in the highest test item concentration was confirmed for both trials by the analytical dose verification in soil. The study was conducted to determine the effects of the test item on the seedling emergence and early growth of four dicotyledonous and two monocotyledonous species. Since the validity criteria were met for all species tested on day 21, the study can be regarded as valid. Statistically significant differences on seedling emergence could be observed for Pisum sativum. The LOEC was 48.0 mg a.i./kg sdw, the NOEC 16.0 mg a.i./kg sdw. An EC50 could not be calculated for any of the species tested. Statistically significant differences on post-emergence mortality could be observed for Allium cepa. The LOEC was 16.0 mg a.i./kg sdw, the NOEC 5.33 mg a.i./kg sdw. A reliable EC50 could not be calculated for any of the species tested. Statistically significant differences on shoot dry weight could be calculated for all species tested. The most sensitive species with regard to the EC50 was Beta vulgaris. The EC50 with 95 % confidence intervals was 1.29 (0.970 – 1.55) mg a.i./kg sdw. The NOEC based on shoot dry weight for the most sensitive species (Beta vulgaris) could not be determined and was considered < 0.198 mg a.i./kg sdw.

Description of key information

The 21-d NOEC was determined by exposing terrestrial plants to AHTN in artificial soil. The effects of the test item on seedling emergence and seedling growth were determined. The 21-d NOEC for the most sensitive species (Beta vulgaris) was determined to be < 0.198 mg/kg dws. This value was recalculated using the LOEC to 0.099 mg/kg dws (see 'Additional information').

Key value for chemical safety assessment

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

Additional information

Chronic effect value can be derived from a 21-day OECD 208 study as it is assumed to cover a sensitive stage in the life-cycle of a plant and therefore data obtained form this. However, the most critical NOEC (based on shoot dry weight for the most sensitive species (Beta vulgaris)) could not be determined and was considered < 0.198 mg a.i./kg sdw. The LOEC for this endpoint was therefore used to calculate a NOEC value by dividing it by 2, as recommended in ECHA'sGuidance on information requirements and chemical safety assessment Chapter R.10: Characterisation of dose [concentration]-response for environment (May 2008). The 21-d NOEC was therefore calculated to be 0.099 mg a.i./kg dws.