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Toxicity to terrestrial plants

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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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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 DAE1,2

Plants Alive at

Mean Shoot Dry Weight

(g)2

7 DAE2

14 DAE2

21 DAE2

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 Emergence1

% Emergence

Mean Survival1

% 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.