Registration Dossier

Administrative data

Endpoint:
toxicity to terrestrial plants: long-term
Remarks:
Seedling emergence and seedling growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 19 December 2016 Experimental completion date 21 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear, colourless liquid
Details on test material:
Identification: Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8)
Description: Clear colorless liquid
Batch: 0905137109
Label: Trigonox 29 0905137109 2kg
Purity: 93.4%
Date Received: 18 December 2012
Expiry Date: 01 June 2019
Storage Conditions: Stored frozen at approximately -20 °C; used/formulated at ambient temperature <30 °C
Specific details on test material used for the study:
Name: TRIGONOX 29
Chemical name: 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane
CAS Number: 6731-36-8
Source: Sponsor
Batch Number: 0905137109
Purity: 1,1-di(tert-butylperoxy)-3,3,5-trimethylcyclohexane 93.4%
(3,3,5-trimethylcyclohexane 2.56%)
Storage: Refrigerated (2 - 8°C)
Expiry Date: 01 June 2019

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Two x 5 mL aliquots from the first sample were analysed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed if ince satisfactory results were achieved.

Mean concentrationss were within +/- 4% of the nominal concentration, confirming the accuracy of formulation.

Test substrate

Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
Please see the section below "Any other information on materials and methods"

Test organisms

open allclose all
Species:
Brassica oleracea var. capitata
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.
Species:
Daucus carota
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
The seeds were purchased from commercial suppliers. The seeds were not treated with any type of fungicides, insecticides, or any other type of pesticidal compound. They were stored dry in glass bottles and kept in darkness prior to the start of the study. Germination trials indicated that seed viability was ≥ 75% for all species. Mean weight/seed was also determined.

Study design

Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Remarks:
Loamy sand. The carbon content (1.89% organic matter), pH and particle size of the soil were analysed according to UK classification and it was sieved prior to use to remove all particles >2 mm. The soil was not pasteurized or heat treated before use.
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Each plant species was sown into treated soil and monitored for 16 - 21 days following a minimum of 50% germination of the control seedlings.

Test conditions

Test temperature:
Cabbage 18.1 – 29.2°C
Carrot 18.1 – 29.2°C
Lettuce 16.7 – 28.0°C
Tomato 14.1 – 28.9°C
Oat 16.7 – 28.0°C
Onion 19.1 – 26.2°C
pH:
7.5
Moisture:
Relative humidity (%)
Range Mean range
Cabbage: 36.34 – 79.63 63.37 –69.51
Carrot: 26.75 – 79.63 62.39 – 69.51
Lettuce: 29.81 – 74.24 49.87 – 61.31
Tomato: 39.02 – 90.77 62.97 – 70.72
Oat: 31.96 – 74.24 52.14 – 61.31
Onion: 38.07 – 93.52 48.74 – 87.37
Details on test conditions:
Experimental Design
A total of 14 pots of three seeds each were sown for cabbage, carrot and lettuce, 20 pots of two seeds for tomato and ten pots of four seeds each for oat and onion. For all species 9.5 cm internal diameter plastic pots were used and each was filled to a depth of approximately 75 mm, the seeds were spaced out on the soil surface and approximately 5 mm of soil was added to cover the seeds. Each pot was labelled with the study number, species name, treatment, replicate number, application date and the name of the Study Director. Each application rate was also identified by a colour-coded plastic stick label. After application each pot was placed in an individual water tray and placed at random within rectangular blocks of evenly spaced pots for each plant species.
Nominal and measured concentrations:
Range-finding: 0.1, 1.30 10, 100 and 1000 mg a.i./kg of dry soil
(Cabbage, carrot and lettuce)

Based on the results of the range finding, a dose response test was conducted at rates of 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil for onion with a limit study at 1000 mg a.i./kg dry soil for all other species.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Species:
other: Cabbage, carrot, lettuce, tomato and oat
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Key result
Species:
other: All species tested
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Survival
Key result
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333.33 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Key result
Species:
other: All species tested
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
Height and dry weight
Key result
Species:
other: All species tested
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
phytotoxicity
Details on results:
Please see below "Any other information on results incl. tables"
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

Range finding

There were no treatment related effects on the germination or survival of any species.

No treatment related visual effects, phytotoxicity or growth inhibition, were observed.

 

The only statistically significant adverse effect was a reduction in dry weight of onion at 1000 mg a.i./kg dry soil.

 

Pot trial

There was no visible evidence of any test substance residue on the interior surface of the pots or of any damage to the pot surface at any treatment rate 21 days after application, in comparison to pots containing untreated soil.

It was concluded that it would be acceptable to run the definitive study in the same type of impermeable plastic pot as used in the range finding study.

Formulation analysis

Analysis of the concentrations of Trigonox 29 in the application treatment solutions confirmed nominal levels of the test substance were within applied limits ± 20% for all treatment groups, confirming accurate formulation.

Definitive

Effect on Germination

Germination was recorded daily until there was >50% in both water and solvent controls. There were no effects on germination of cabbage, carrot, lettuce, tomato or oat. The NOEC for germination of these species was therefore 1000 mg a.i./kg dry soil.

For onion there were reductions in germination of 5.4, 10.8 and 29.7% at 12.35, 111.11 and 1000 mg a.i./kg dry soil respectively in comparison to the solvent control. The effect was statistically significant (p= 0.003**) at 1000 mg a.i./kg dry soil. The NOEC was 333.33 mg a.i./kg dry soil.

 Effect on Plant Survival

There were no treatment-related effects on plant survival for any species. 

Effect on Plant Growth and Health

A visual assessment based on the overall condition of surviving seedlings for each replicate was conducted at the end of the study and at regular intervals throughout. This assessment was conducted using a uniform scoring system based on plant size and vigor where 0 indicated no abnormal effects and 10 for all plants within a replicate being dead.

No signs of phytotoxicity were observed in the treatment groups compared to either of the control groups. The NOEC for phytotoxicity was therefore 1000 mg a.i./kg dry soil.

For onionslight plant growth inhibition (10 – 20%) was recorded in two replicates at 111.11 mg a.i./kg dry soil with 3% plant mortality. At 1000 mg a.i./kg dry soil 30 – 40% plant growth inhibition was recorded in three replicates with slight phytotoxicity in two replicates and 7.7% plant mortality.

At harvest, measurement of the height and dry weight for each seedling from each replicate pot was conducted. There were no statistically significant adverse treatment related effects on the height or dry weight of any species in comparison to the solvent control. 

Validation Criteria

The protocol requirement for control seedling emergence of a minimum of 70% for all species was achieved as there was 100% germination in both water and solvent controls for cabbage, carrot, cucumber, lettuce, tomato and oat, with 85 and 92.5% germination of onion in water and solvent controls respectively.

No visible phytotoxic effects were observed in the water or solvent controls.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
There was no statistically significant effect on the germination or survival of cabbage, carrot, lettuce, tomato or oat following application of Trigonox 29 at 1000 mg a.i./kg dry soil. The NOEC for germination for these species was therefore 1000 mg a.i./kg dry soil. The NOEC for survival for all species was 1000 mg a.i./kg dry soil.

For onion there was a statistically significant reduction in germination at 1000 mg a.i./kg dry soil (p = 0.003**). The NOEC for germination for onion was therefore 333.33 mg a.i./kg dry soil.

For onion 3.0 and 7.7% plant mortality was recorded at 111.11 and 1000 mg a.i./kg dry soil respectively.

There were no statistically significant adverse treatment related effects in the height or dry weight of cabbage, carrot, lettuce, tomato and oat in comparison to the solvent control following application of Trigonox 29 at 1000 mg a.i./kg dry soil. The NOEC for height and dry weight for these species was therefore 1000 mg a.i./kg dry soil.

There were no statistically significant adverse treatment related effects in the height or dry weight of onion at 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil. The NOEC for height and dry weight for onion was therefore 1000 mg a.i./kg dry soil.

No signs of phytotoxicity were observed in the treatment groups compared to either of the control groups. The NOEC for phytotoxicity was therefore 1000 mg a.i./kg dry soil for all species.

The study was considered valid as the validation criteria were met and no visible phytotoxic effects were observed in the controls.
Executive summary:

 Summary

This study was carried out with four dicotyledons, (cabbage, carrot, lettuce and tomato) and two monocotyledons, (oat and onion), to determine the toxicity of Trigonox 29 to seedling emergence and seedling growth of non-target plant species during early critical stages in their development, in a dose response test designed to evaluate the phytotoxic effects of the test substance. 

Following initial range finding at 0.1, 1, 10, 100 and 1000 mg a.i./kg dry soil, the definitive study was conducted as a dose response test at 12.34, 37.04, 111.11, 333.33 and 1000 mg a.i./kg of dry soil for onion and as a limit study at 1000 mg a.i./kg dry soil for all other species. 

Each plant species was sown into treated soil and monitored for 16 - 21 days following a minimum of 50% germination in the controls.

A total of 14 pots of three seeds each were sown for cabbage, carrot and lettuce, 20 pots of two seeds each for tomato and 10 pots of four seeds each for oat and onion for each application rate ofTrigonox 29and the water and acetone controls. The soil used was a sandy loam, sieved to remove all particles > 2mm.

After application and sowing, the pots were checked daily and the numbers of emerged seedlings were recorded. Emergence, survival and sub-lethal effects were recorded daily up to a minimum of 50% emergence of control seedlings and thereafter every one to three days. Plants were harvested 16 - 21 days after 50% control germination, giving a total exposure period of 21 days for cabbage, tomato and oat and 26 days for carrot, lettuce and onion. The height of each individual plant was then measured before the plants were dried and weighed to obtain a measurement of dry weight.

Effect on germination and plant survival

There was no effect on the germination or survival of cabbage, carrot, lettuce, tomato or oat.

For onion there were reductions in germination of 5.4, 10.8 and 29.7% at 12.35, 111.11 and 1000 mg a.i./kg dry soil respectively in comparison to the solvent control. The effect was statistically significant (p= 0.003**) at 1000 mg a.i./kg dry soil. The NOEC for germination for onion was therefore 333.33 mg a.i./kg dry soil.

 

There was no effect on the survival of cabbage, carrot, lettuce, tomato or oat. For onion there were reductions in survival of 3.0 and 7.7% at 111.11 and 1000 mg a.i./kg dry soil respectively in comparison to the solvent control. The NOEC for survival for all species was 1000 mg a.i./kg dry soil.

Effect on plant growth and health

At harvest, measurement of the height and dry weight for each seedling from each replicate pot was conducted. There were no statistically significant adverse treatment related effects on the height or dry weight of any species in comparison to the solvent control. 

The effects on height and dry weight are summarised in the tableb below.

Percent effect onheight and dry weight inc omparisonto thes olventcontrol

Species

Treatment

Application rate

(mg a.i./kg dry soil)

Geometric mean height (cm)

% increase (+) or reduction

(-) in mean height

p

Geometric mean dry weight (mg)

% increase (+) or reduction (-) in mean dry weight

p

Cabbage

Water control

0

3.625

-5

0.451T

32.913

+5

0.677T

Solvent control

0

3.807

-

-

31.337

-

-

Trigonox 29

1000

4.474

+18

0.016*T

35.803

+14

0.262T

Carrot

Water control

0

1.961

-2

0.710T

9.518

+4

0.632T

Solvent control

0

2.001

-

-

9.160

-

-

Trigonox 29

1000

2.279

+14

0.021*T

10.694

+17

0.059T

Lettuce

Water control

0

1.480

-4

0.463T

19.698

+6

0.779T

Solvent control

0

1.544

-

-

19.146

-

-

Trigonox 29

1000

1.878

+22

0.001**T

21.758

+14

0.211T

Tomato

Water control

0

4.583

-5

0.322T

34.949

+4

0.647T

Solvent control

0

4.827

-

-

33.675

-

-

Trigonox 29

1000

4.917

+2

0.723T

33.269

-1

0.881T

Oat

Water control

0

35.391

-1

0.608T

53.058

-4

0.509T

Solvent control

0

35.809

-

-

55.507

-

-

Trigonox 29

1000

35.406

-1

0.621T

55.952

+1

0.907T

Onion

Water control

0

 10.267

 -5

0.517T

 4.564

 -15

0.137T

Solvent control

0

 10.808

-

-

 5.379

-

-

Trigonox 29

12.35

 11.885

+10

0.688W

 5.741

+7

0.964D

37.04

 10.739

 -1

0.688W

 5.100

 -5

0.984D

111.11

 11.805

+9

0.683W

 6.728

+25

0.162D

333.33

 11.431

+6

0.683W

 6.225

+16

0.537D

1000

 10.968

+1

0.683W

 5.559

+3

0.998D

pvalues are for the comparison with solvent control using Williams' test (W), Dunnett's test (D) and thet-test (T)

*p<0.05, **p<0.01,

- not applicable

The visual assessment, based on the overall condition of surviving seedlings for each replicate, was conducted at the end of the study and at regular intervals throughout. This assessment was conducted using a uniform scoring system based on plant size and vigour where 0 indicated no abnormal effects and 10 for all plants within a replicate being dead.

There were no visual herbicidal effects (e.g. chlorosis, necrosis, abnormal plant growth) on any species in water and solvent control groups or on cabbage, carrot, lettuce, tomato and oat treated at 1000 mg a.i./kg dry soil. 

For onionslight plant growth inhibition (10 – 20%) was recorded in two replicates at 111.11 mg a.i./kg dry soil with 3% plant mortality. At 1000 mg a.i./kg dry soil 30 – 40% plant growth inhibition was recorded in three replicates with slight phytotoxicity in two replicates and 7.7% plant mortality. 

Conclusion

There was no statistically significant effect on the germination or survival of cabbage, carrot, lettuce, tomato or oat following application of Trigonox 29 at 1000 mg a.i./kg dry soil. The NOEC for germination for these species was therefore 1000 mg a.i./kg dry soil. The NOEC for survival for all species was 1000 mg a.i./kg dry soil.

For onion there was a statistically significant reduction in germination at 1000 mg a.i./kg dry soil (p= 0.003**). The NOEC for germination for onion was therefore 333.33 mg a.i./kg dry soil.

For onion 3.0 and 7.7% plant mortality was recorded at 111.11 and 1000 mg a.i./kg dry soil respectively.

There were no statistically significant adverse treatment related effects in the height or dry weight of cabbage, carrot, lettuce, tomato and oat in comparison to the solvent control following application of Trigonox 29 at 1000 mg a.i./kg dry soil. The NOEC for height and dry weight for these species was therefore 1000 mg a.i./kg dry soil.

There were no statistically significant adverse treatment related effects in the height or dry weight of onion at 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil. The NOEC for height and dry weight for onion was therefore 1000 mg a.i./kg dry soil.

No signs of phytotoxicity were observed in the treatment groups compared to either of the control groups. The NOEC for phytotoxicity was therefore 1000 mg a.i./kg dry soil for all species.

The study was considered valid as the validation criteria were met and no visible phytotoxic effects were observed in the controls.