6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2017 to 03 May 2017

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:

OECD Guideline for the testing of chemicals. Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test. OECD Test Guideline 208. Adopted 19 July 2006).

Deviations:

yes

Remarks:

see "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

not specified

Details on sampling:

Difficulties were encountered in the range finder test during the evaporation of the acetone off of the find sand which did not leave a well distributed residue and therefore, had to be grind further at the treatment level of 1000 mg/kg LOWINOX® 44B25 dry soil. For this reason the test item dose was prepared by the direct addition of weighed quantities of LOWINOX® 44B25 to sand and subsequent mixing of this into the soil. As such, treatment concentrations were calculated from the weight of LOWINOX® 44B25 added to the soil.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

Soil Treatment for Range Finding and Definitive Tests
For the range finding test treatment rates of 0.1, 1.0, 10.0, 100 and 1000 mg/kg dry soil, LOWINOX® 44B25 solutions were prepared in acetone, applied onto fine sand, and mixed thoroughly and the acetone allowed to evaporate. The control treatment soils were treated with the equivalent amount sand (acetone treated and untreated) and deionised water. Treated sand for each treatment group was incorporated into the soil at a rate of 100 g sand/kg dry soil.
For Run 1 and 2* the definitive test the soil treatment was prepared by direct addition of LOWINOX® 44B25 to dry fine quartz sand at a rate of 100 g sand/kg dry soil and the weights used are presented in the table below. Direct addition was used as the preferred method of soil soil incorporation of the test item. Although the test item was soluble in acetone at the test concentration of 1000 mg/kg dry soil used in the range finder the test item did not coat the fine sand effectively once evaporated, drying to a cake that had to be ground further to produce a homogenous mix. A more even distribution was achieved by direct addition and mixing. LOWINOX® 44B25 was added carefully to the sand and mixed thoroughly before incorporation in to the soil.
*the definitive test was restarted for all species with the exception of maize six days after the first run on 06 April 2017 as all the seeds failed to germinate due to unexpectedly high temperatures in the glasshouse.
A bulk sample of soil was weighed for each treatment group and the treated sand added the soil in a drum mixer. The treated sand and soil were mixed continuously for at least 10 minutes to ensure an even distribution of the test item in the soil. Treatments were prepared in the order of the control (untreated sand) followed by the LOWINOX® 44B25 treatment.

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Species:

Helianthus annuus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Species:

other: Solanum lycopersicum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

The plant species were selected using Annex 2 of the OECD Guideline 208 and following consultation with the Sponsor. Seeds were stored refrigerated prior to use and full details of the seed batches, including emergence data are documented in the raw data.
Further details regarding the test species can be found under “Any other information”

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

21 d

Remarks:

Seeds were sown in soil which had been amended with LOWINOX® 44B25 and then assessed for effects up to 21 days after 50% emergence of the seedlings in the control group.

Post exposure observation period:

As above.

Test temperature:

20.8 - 22.8 °C

pH:

6.8

Moisture:

47.7 - 54.5 %

Details on test conditions:

Seed Sowing
The labelled plant pots were filled with the appropriate soil and the seeds planted following the regime described in Section 4. Once filled and sown, the plant pots were placed on saucers and ordered in a random block pattern in the glasshouse. The blocks were re-randomised weekly thereafter in the glasshouse. The plant pots were watered overhead with sufficient water to moisten the soil and to aid seed germination and care was taken to avoid leaching of the test item. Once emerged, watering was continued from the bottom of the plant pot only, at regular intervals over the test period. No pest control or soil nutrients were used during either phase of the study.

Glasshouse/Growth Chamber Environmental Conditions
The temperature, humidity and light levels throughout the study were monitored using IceSpy System 5 monitoring equipment (Skye Instruments Ltd, UK) in the glasshouse and growth chamber at hourly intervals for both phases of the study.
Plants were grown using a 16 h light: 8 h dark photoperiod of direct light or supplemented light.

Observations and Final Assessments
The number of seeds emerged were counted on a daily basis until emergence in the control treatment reached 50 % for each species. Additionally, the plants were checked at least weekly and the treatments were assessed for emergence, visual injury (phytotoxicity) and mortality at 7, 14 and 21 days after 50 % emergence in the control treatment. Visual assessments of phytotoxicity were carried out and were categorised using the scoring table below:
0 = excellent, good colour, no defects
1 = very slight / marginal leaf distortion and/or discolouration
2-3 = slight, but identifiable leaf distortion and/or discolouration
4-5 = moderate foliage discolouration and/or slight stunting
6-7 = stunted and/or very pale foliage
8 = very sickly, yellow or very pale leaves and/or stunted in size/growth
9 = almost dead
10 = all dead
At 21 days after emergence of 50% of the seeds in the control group, after the final assessments, the plants in each treatment replicate were harvested at the soil surface, dried in an oven set at 60oC until dry and weighed to determine shoot dry weight of that treatment replicate.

Nominal and measured concentrations:

Difficulties were encountered in the range finder test during the evaporation of the acetone off of the find sand which did not leave a well distributed residue and therefore, had to be grind further at the treatment level of 1000 mg/kg LOWINOX® 44B25 dry soil. For this reason the test item dose was prepared by the direct addition of weighed quantities of LOWINOX® 44B25 to sand and subsequent mixing of this into the soil. As such, treatment concentrations were calculated from the weight of LOWINOX® 44B25 added to the soil.

Reference substance (positive control):

no

Key result

Species:

Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival & shhot dry weight

Key result

Species:

Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Helianthus annuus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Helianthus annuus

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

other: Solanum lycopersicum

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

other: Solanum lycopersicum

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Brassica napus

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Zea mays

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Key result

Species:

Zea mays

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: emergence, survival and shoot dry weight

Details on results:

Range Finding Test
The range finding test was conducted using soybean, oilseed rape, tomato, sunflower, oat and maize. Five test concentrations of LOWINOX® 44B25 (0.1, 1.0, 10, 100 and 1000 mg/kg dry soil) along with an untreated control and solvent (acetone) control group were assessed. The results from the range finding test were used to identify suitable test concentrations for use in the definitive test.
There was no statistical difference between the untreated control and the acetone control for the emergence, survival and shoot dry weight data. Therefore, the results from the LOWINOX® 44B25 treatments were compared with combined control values. All species reached over 77% emergence relative to the control treatments across the treatment range with most species and treatments achieving higher rates. The survival data showed 100 % survival of the plants in all treatments at the final assessments giving a clear indication of no treatment effect. Dry shoot weight data was compared to the combined control results and showed no apparent treatment effect from 0.1 to 1000 mg LOWINOX® 44B25/kg dry soil. No phytotoxicity or plant injury due to treatment was evident in any of the species.

Definitive Test
Soybean, oilseed rape, tomato, sunflower, oat and maize were exposed to a single concentration of LOWINOX® 44B25 (1000 mg/kg dry soil), along with an untreated control group in the definitive test.
Although there were several excursions from the specified growing conditions for the study, in terms of temperature and relative humidity, this did not impact on the growth and health of the plants.

Emergence
The NOEC for emergence for all species was 1000 mg LOWINOX® 44B25/kg dry soil and the LOEC was > 1000 mg LOWINOX® 44B25/kg dry soil.
There was greater than 70% emergence for all species in the control treatment.

Survival
The NOEC for survival for all species was 1000 mg LOWINOX® 44B25/kg dry soil and the LOEC was > 1000 mg LOWINOX® 44B25/kg dry soil.
There was 100% survival of all emerged plants for all species in the control treatment.

Shoot Dry Weight
The NOEC for shoot dry weight for oilseed rape, soybean, sunflower,tomato, maize and oat was 1000 mg LOWINOX® 44B25 /kg dry soil and the LOEC was >1000 mg LOWINOX® 44B25/kg dry soil, respectively.

Phytotoxicity
No phytotoxic effects or visual injury which could be attributed to a treatment effect were observed in any of the species tested up to 1000 mg LOWINOX® 44B25/kg dry soil. All plants appeared healthy with good vigour.
The seedling emergence for all species in the control treatment was at least 70% and the control plants showed no visible signs of injury or phytotoxicity and overall the control plants maintained growth throughout. The mean plant survival was 100% for the control plants for all species.

Results with reference substance (positive control):

None

Reported statistics and error estimates:

Statistical analysis of the data showed there was no significant difference in the emergence endpoint between the control treatment and the LOWINOX® 44B25 treatment of 1000 mg/kg dry soil for any species. (Fisher’s Exact test (two sided, p=0.05)).
Statistical analysis of the data showed there was no significant difference in the survival endpoint between the control treatment and the LOWINOX® 44B25 treatment for any species. (Fisher’s Exact test (two sided, p=0.05)).
Statistical analysis of the data showed there was no significant difference in the shoot dry weights between the control treatment and the LOWINOX® 44B25 treatment of 1000 mg/kg dry soil for all species. (Dunnett’s hypothesis (two sided, p=0.05)).

Summary of Results in the Range Finding Test

Species	Treatment (mg LOWINOX® 44B25/kg dry soil) % Emergence Relative to the Combined Controls
	Water Control	Acetone Control	0.1	1.0	10.0	100	1000
Oilseed Rape	NA	NA	87	99	87	104	96
Soybean	NA	NA	145	91	109	145	164
Tomato	NA	NA	88	100	88	88	113
Sunflower	NA	NA	75	113	113	100	113
Maize	NA	NA	113	100	100	100	88
Oat	NA	NA	87	100	100	100	100

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Species	Treatment (mg LOWINOX® 44B25/kg dry soil) % Survival Relative to the Combined Controls
	Water Control	Acetone Control	0.1	1.0	10.0	100	1000
Oilseed Rape	NA	NA	104	104	94	104	104
Soybean	NA	NA	109	87	109	109	109
Tomato	NA	NA	100	100	100	100	100
Sunflower	NA	NA	100	100	100	100	100
Maize	NA	NA	100	100	100	88	88
Oat	NA	NA	100	100	100	100	100

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Species	Treatment (mg LOWINOX® 44B25/kg dry soil) % Dry Shoot Weight Relative to the Combined Controls
	Water Control	Acetone Control	0.1	1.0	10.0	100	1000
Oilseed Rape	NA	NA	118	92	144	116	100
Soybean	NA	NA	83	104	87	87	85
Tomato	NA	NA	98	106	106	97	90
Sunflower	NA	NA	102	109	109	100	93
Maize	NA	NA	116	127	113	164	137
Oat	NA	NA	102	110	107	108	114

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Species	Treatment (mg LOWINOX® 44B25/kg dry soil) Visual Injury (Phytotoxicity Score)
	Water Control	Acetone Control	0.1	1.0	10.0	100	1000
Oilseed Rape	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)
Soybean	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)
Tomato	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)
Sunflower	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)
Maize	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)
Oat	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)	None (0)

Data taken at 21 days after 50% emergence in the control groups

 

Summary of Environmental Conditions from Range Finder and Definitive Test

 

Glasshouse Conditions Range Finder

Time Period	Mean and Range Temperature Range (°C)	Mean and Range Humidity (%)	Mean and Range Light Intensity (K Lux)^a
23 February 2017 to 25 March 2017	20.8 14.5 – 35.8	47.7 13.5 – 70.5	7.88 0.46 – 76.2

^aK Lux x 0.001 = Lux, mean includes hours of darkness

NB: Temperature, humidity and light intensity values are based on hourly averages.

 

Glasshouse/Growth Room Conditions Definitive Test Run 1 & 2

Time Period	Mean and Range Temperature Range (°C)	Mean and Range Humidity (%)	Mean and Range Light Intensity (K Lux)^a
31 March 2017 to 02 May 2017	22.8 11.5 – 38.2	54.5 15.2 – 78.0	9.81 0.46 – 81.8

^aK Lux x 0.001 = Lux, mean includes hours of darkness

NB: Temperature, humidity and light intensity values are based on hourly averages.

 

Oilseed Rape – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	4	4	4	4	0.143
	2	2	2	2	2	0.138
	3	3	3	3	3	0.131
	4	3	3	3	3	0.092
	5	4	4	4	4	0.136
	6	4	4	4	4	0.137
	7	3	3	3	3	0.137
	8	3	3	3	3	0.119
1000	1	4	4	4	4	0.130
	2	3	3	3	3	0.144
	3	3	3	3	3	0.134
	4	4	4	4	4	0.088
	5	3	3	3	3	0.116
	6	3	3	3	3	0.132
	7	4	4	4	4	0.111
	8	4	4	4	4	0.096

¹Seeds sown per replicate = 4

²DAE, days after 50% emergence in the control group

 

Oilseed Rape – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	3.25	81%	3.25	100%	0.13
1000	3.43	88%	3.43	100%	0.12

¹Seeds sown per replicate = 4

Data taken 21 days after 50% emergence in the control groups.

 

Oilseed Rape – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	105%	100%	92%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Soybean – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	2	2	2	2	0.501
	2	3	3	3	3	0.524
	3	3	3	3	3	0.502
	4	1	1	1	1	0.410
	5	2	2	2	2	0.440
	6	1	1	1	1	0.417
	7	3	3	3	3	0.507
	8	3	3	3	3	0.451
	9	3	3	3	3	0.483
	10	2	2	2	2	0.407
1000	1	3	3	3	3	0.384
	2	2	2	2	2	0.397
	3	2	2	2	2	0.363
	4	3	3	3	3	0.440
	5	3	3	3	3	0.490
	6	1	1	1	1	0.457
	7	3	3	3	3	0.403
	8	2	2	2	2	0.387
	9	2	2	2	2	0.433
	10	2	2	2	2	0.520

¹Seeds sown per replicate = 3

²DAE, days after 50% emergence in the control group

 

Soybean – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	2.30	77%	2.30	100%	0.46
1000	2.30	77%	2.30	100%	0.43

¹Seeds sown per replicate = 3

Data taken 21 days after 50% emergence in the control groups.

 

Soybean – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	100%	100%	92%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Sunflower – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	3	3	3	3	0.190
	2	3	3	3	3	0.202
	3	3	3	3	3	0.219
	4	3	3	3	3	0.205
	5	2	2	2	2	0.358
	6	2	2	2	2	0.235
	7	3	3	3	3	0.250
	8	3	3	3	3	0.243
	9	3	3	3	3	0.287
	10	3	3	3	3	0.247
1000	1	3	3	3	3	0.280
	2	3	3	3	3	0.262
	3	3	3	3	3	0.332
	4	3	3	3	3	0.276
	5	3	3	3	3	0.250
	6	3	3	3	3	0.299
	7	3	3	3	3	0.281
	8	3	3	3	3	0.267
	9	3	3	3	3	0.276
	10	2	2	2	2	0.326

¹Seeds sown per replicate = 3

²DAE, days after 50% emergence in the control group

 

Sunflower – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	2.80	93%	2.80	100%	0.24
1000	2.90	97%	2.90	100%	0.28

¹Seeds sown per replicate = 3

Data taken 21 days after 50% emergence in the control groups.

 

Sunflower – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	104%	100%	117%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Tomato – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	3	3	3	3	0.144
	2	3	3	3	3	0.147
	3	3	3	3	3	0.133
	4	3	3	3	3	0.117
	5	2	2	2	2	0.157
	6	3	3	3	3	0.156
	7	3	3	3	3	0.144
	8	3	3	3	3	0.153
	9	3	3	3	3	0.143
	10	3	3	3	3	0.130
1000	1	3	3	3	3	0.194
	2	3	3	3	3	0.126
	3	3	3	3	3	0.149
	4	2	2	2	2	0.183
	5	3	3	3	3	0.163
	6	3	3	3	3	0.154
	7	3	3	3	3	0.140
	8	3	3	3	3	0.147
	9	3	3	3	3	0.146
	10	3	3	3	3	0.169

¹Seeds sown per replicate = 3

²DAE, days after 50% emergence in the control group

 

Tomato – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	2.90	97%	2.90	100%	0.14
1000	2.90	97%	2.90	100%	0.16

¹Seeds sown per replicate = 3

Data taken 21 days after 50% emergence in the control groups.

 

Tomato – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	100%	100%	110%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Maize – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	3	3	3	3	0.463
	2	3	3	3	3	0.215
	3	3	3	3	3	0.249
	4	2	2	2	2	0.553
	5	3	3	3	3	0.271
	6	3	3	3	3	0.148
	7	3	3	3	3	0.349
	8	3	3	3	3	0.327
	9	3	3	3	3	0.331
	10	3	3	3	3	0.316
1000	1	3	3	3	3	0.334
	2	3	3	3	3	0.327
	3	3	3	3	3	0.284
	4	3	3	3	3	0.269
	5	2	2	2	2	0.354
	6	3	3	3	3	0.371
	7	3	3	3	3	0.391
	8	3	3	3	3	0.268
	9	3	3	3	3	0.236
	10	3	3	3	3	0.302

¹Seeds sown per replicate = 3

²DAE, days after 50% emergence in the control group

 

Maize – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	2.90	97%	2.90	100%	0.32
1000	2.80	93%	2.80	100%	0.31

¹Seeds sown per replicate = 3

Data taken 21 days after 50% emergence in the control groups.

 

Maize – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	97%	100%	97%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

 

Oat – Emergence, Survival and Mean Dry Shoot Weight

Treatment (mg/kg soil)	Replicate Number	Seeds Emerged by 21 DAE^1,2	Plants Alive at			Mean Shoot Dry Weight (g)^2
			7 DAE^2	14 DAE^2	21 DAE^2
Untreated Control	1	3	3	3	3	0.097
	2	5	5	5	5	0.072
	3	5	5	5	5	0.078
	4	4	4	4	4	0.072
	5	5	5	5	5	0.088
	6	5	5	5	5	0.082
	7	5	5	5	5	0.077
1000	1	5	5	5	5	0.077
	2	5	5	5	5	0.087
	3	5	5	5	5	0.083
	4	5	5	5	5	0.068
	5	4	4	4	4	0.081
	6	5	5	5	5	0.075
	7	5	5	5	5	0.082

¹Seeds sown per replicate = 5

²DAE, days after 50% emergence in the control group

 

Oat – Mean Emergence, Survival and Dry Weight

Treatment (mg/kg soil)	Mean Emergence^1	% Emergence	Mean Survival^1	% Survival	Mean Shoot Dry Weight (g)
Untreated Control	4.57	91%	4.57	100%	0.081
1000	4.86	97%	4.86	100%	0.079

¹Seeds sown per replicate = 5

Data taken 21 days after 50% emergence in the control groups.

 

Oat – Visual Injury and Percentage Emergence, Survival, Dry Weight relative to Control

Treatment (mg/kg soil)	% Emergence Relative to Control	% Survival Relative to Control	% Shoot Weight Relative to Control	Overall Visual Injury (Phytotoxicity Score)
Untreated Control	NA	NA	NA	0 (None)
1000	106%	100%	98%	0 (None)

Data taken at 21 days after 50% emergence in the control groups

NA = not applicable

Validity criteria fulfilled:

yes

Conclusions:

The soil incorporation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol (LOWINOX® 44B25) at 1000 mg product/kg dry soil on the six terrestrial non-target plant species did not produce statistically significant differences on the emergence, survival and dry shoot weight of the species selected.
The NOEC and LOEC for emergence, survival and shoot dry weight for all species was 1000 mg LOWINOX® 44B25/kg dry soil and >1000 mg LOWINOX® 44B25/kg dry soil, respectively.

Executive summary:

The aim of the study was to determine the effects on seedling emergence and early growth on selected higher plants, following exposure to 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, hereafter referred to as LOWINOX® 44B25, in soil. Seeds were sown in soil which had been amended with LOWINOX® 44B25 and then assessed for effects up to 21 days after 50% emergence of the seedlings in the control group. This study has been conducted to meet the requirements specified in the OECD Guideline 208 (2006).

 

Maize (Zea mays), oat (Avena sativa), oilseed rape (Brassica napus), soybean (Glycine max (G. soja)), sunflower (Helianthus annuus) and tomato (Solanum lycopersicon) were assessed for effects on emergence, survival and phytotoxicity for a test period of 21 days after 50% emergence in the control group. After the final observation, at 21 days after the emergence of 50% in the control group for each species (21 DAE), the shoot dry weights were determined.

 

A range finding test was conducted with each species at test concentrations of 0.1, 1, 10, 100 and 1000 mg LOWINOX® 44B25/kg dry soil and as a result a limit test was carried at 1000 mg LOWINOX® 44B25/kg dry soil, along with an untreated control in the definitive phase. The number of replicates in the definitive test was increased to give at least 30 seeds per treatment to provide a more powerful test. LOWINOX® 44B25 was directly incorporated into the soil.

The endpoint data were analysed in order determine if the concentration of 1000 mg/kg LOWINOX® 44B25 had a significant effect on the emergence, survival and dry shoot weight in addition to the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for LOWINOX® 44B25 in soil was determined.

 

Statistical analysis of the data showed there was no significant difference in the emergence and survival for all species tested compared to the control treatment. There was no significant difference in the shoot dry weight data between the control treatment and the LOWINOX® 44B25 treatments for all species.

 

The NOEC and LOEC results for seedling emergence, survival and shoot dry weight are summarised in the following tables:

 

Endpoint	Species	Concentration (mg LOWINOX® 44B25/kg dry soil)
		NOEC	LOEC
Emergence	Oilseed Rape	1000	>1000
	Soybean	1000	>1000
	Sunflower	1000	>1000
	Tomato	1000	>1000
	Maize	1000	>1000
	Oat	1000	>1000

NOEC and LOEC (Fisher’s Exact Test, two sided, p=0.05)

 

Endpoint	Species	Concentration (mg LOWINOX® 44B25/kg dry soil)
		NOEC	LOEC
Survival	Oilseed Rape	1000	>1000
	Soybean	1000	>1000
	Sunflower	1000	>1000
	Tomato	1000	>1000
	Maize	1000	>1000
	Oat	1000	>1000

NOEC and LOEC (Fisher’s Exact Test, two sided, p=0.05)

 

Endpoint	Species	Concentration (mg LOWINOX® 44B25/kg dry soil)
		NOEC	LOEC
Dry Weight	Oilseed Rape	1000	>1000
	Soybean	1000	>1000
	Sunflower	1000	>1000
	Tomato	1000	>1000
	Maize	1000	>1000
	Oat	1000	>1000

NOEC and LOEC (Dunnett’s Hypothesis Test, two sided, p=0.05)

 

No phytotoxicity or plant injury due to treatment was evident in any of the species

 

This study fulfilled the validity criteria cited in the OECD Guideline 208; seedling emergence for all species was at least 70% in the control plants. Control plants showed no visible signs of injury or phytotoxicity and overall the control plants maintained growth throughout.

The mean plant survival in the control treatment was 100% for the study duration, with identical environmental conditions for any particular species and with growing medium containing the same amount of soil matrix. These results meet the validity criteria of > 90% survival in the controls.

 

CONCLUSION

The soil incorporation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol (LOWINOX® 44B25) at 1000 mg product/kg dry soil on the six terrestrial non-target plant species did not produce statistically significant differences on the emergence, survival and dry shoot weight of the species selected.

 

The NOEC and LOEC for emergence, survival and shoot dry weight for all species was 1000 mg LOWINOX® 44B25/kg dry soil and >1000 mg LOWINOX® 44B25/kg dry soil, respectively.

Description of key information

The NOEC and LOEC for emergence, survival and shoot dry weight for all species was 1000 mg LOWINOX® 44B25/kg dry soil and >1000 mg LOWINOX® 44B25/kg dry soil, respectively.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

The aim of the study was to determine the effects on seedling emergence and early growth on selected higher plants, following exposure to 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol, hereafter referred to as LOWINOX® 44B25, in soil. Seeds were sown in soil which had been amended with LOWINOX® 44B25 and then assessed for effects up to 21 days after 50% emergence of the seedlings in the control group. This study has been conducted to meet the requirements specified in the OECD Guideline 208 (2006).

 

Maize (Zea mays), oat (Avena sativa), oilseed rape (Brassica napus), soybean (Glycine max (G. soja)), sunflower (Helianthus annuus) and tomato (Solanum lycopersicon) were assessed for effects on emergence, survival and phytotoxicity for a test period of 21 days after 50% emergence in the control group. After the final observation, at 21 days after the emergence of 50% in the control group for each species (21 DAE), the shoot dry weights were determined.

 

A range finding test was conducted with each species at test concentrations of 0.1, 1, 10, 100 and 1000 mg LOWINOX® 44B25/kg dry soil and as a result a limit test was carried at 1000 mg LOWINOX® 44B25/kg dry soil, along with an untreated control in the definitive phase. The number of replicates in the definitive test was increased to give at least 30 seeds per treatment to provide a more powerful test. LOWINOX® 44B25 was directly incorporated into the soil.

The endpoint data were analysed in order determine if the concentration of 1000 mg/kg LOWINOX® 44B25 had a significant effect on the emergence, survival and dry shoot weight in addition to the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for LOWINOX® 44B25 in soil was determined.

 

No phytotoxicity or plant injury due to treatment was evident in any of the species

 

CONCLUSION

The soil incorporation of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol (LOWINOX® 44B25) at 1000 mg product/kg dry soil on the six terrestrial non-target plant species did not produce statistically significant differences on the emergence, survival and dry shoot weight of the species selected.

 

The NOEC and LOEC for emergence, survival and shoot dry weight for all species was 1000 mg LOWINOX® 44B25/kg dry soil and >1000 mg LOWINOX® 44B25/kg dry soil, respectively.