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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-06-24 to 2015-11-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD standard guideline under GLP with well documented report.
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Remarks:
two minor deviations from the recommended upper temperature range of 32°C and some occasional deviations from the lower limit for relative humidity during the definitive tests, but all the validity criteria for all species tested were met.
GLP compliance:
yes (incl. certificate)
Remarks:
2015-01-12
Analytical monitoring:
yes
Details on sampling:
At Day 0 of the first definitive test, replicated sub samples of the stock solution of NBPT at 26.5 g/L (nominal), used to treat the soil, were analysed at CEMAS using high performance liquid chromatography (HPLC) fitted with a UV detector to confirm the concentrations.
Two additional replicated sub samples were stored frozen.
Vehicle:
yes
Details on preparation and application of test substrate:
A bulk soil (corrected for moisture content) was prepared for each treatment group with a deionised water control and thoroughly mixed in a drum mixer to ensure even distribution of the test item solution throughout the soil. The soil treatments were prepared in ascending order of concentration, starting with the water control. The bulk treated soil was transferred to fill the required number of plant pots in each treatment group (ensuring the same depth of soil is placed into each pot) and the seeds were sown immediately.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
- Plant family: Poaceae (Gramineae)
- Variety: Dalguise
- Source of seed: Frontier Agriculture, Thatcham, UK,
- Historical germination of seed (germination of seed lot tested): 95%
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Ryegrass
- Plant family: Poaceae (Gramineae)
- Source of seed: Herbiseed, Twyford, UK
- Historical germination of seed (germination of seed lot tested): 90%
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Oilseed Rape
- Plant family: Brassicaceae (Cruciferae)
- Source of seed: Harlow Agricultural Merchants Harlow, UK.
- Historical germination of seed (germination of seed lot tested): 85%
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Fabaceae (Leguminosae)
- Variety: Elena
- Source of seed: Soya (UK) Ltd., Southampton, UK
- Historical germination of seed (germination of seed lot tested): 85%
Species:
Helianthus annuus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Sunflower
- Plant family: Asteraceae (Compositae)
- Variety: San Luca
- Source of seed: Syngenta Seeds Ltd, Cambridge,UK
- Historical germination of seed (germination of seed lot tested): 100%
Species:
other: Solanum lycopersicon
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Solanaceae
- Variety: Alicante
- Source of seed: Jungle Seeds Ltd, Watlington,UK:
- Historical germination of seed (germination of seed lot tested): 95%
Test type:
early seedling growth toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
21 d
Remarks:
* after 50% emergence in the controls was attained
Post exposure observation period:
All plants were assessed for emergence until 50% emergence was achieved in the control for each species. Observations on emergence, visual injury/phytotoxicity, survival and growth stage according to the BBCH- scale (see section 18 References) were recorded at 7, 14 and 21 days after 50% emergence in the control.
Visual assessments of phytotoxicity were carried out and were categorised using the scoring table below where appropriate with a general description of the effect, where appropriate.
Following the final assessment at 21 days after 50 % emergence of each species in the control, individual plants in each treatment replicate were harvested at the soil surface and weighed. These shoots were then dried in an oven set at 60oC to determine shoot dry weight.
Test temperature:
Definitive Study – Oat,Ryegrass,Oilseed Rape,Soybean and Sunflower:
range: 13.2 - 35.9°C. On one occasion the temperature rose above 32°C over a 5 hour period on 22 August 2015 with temperatures recorded at over 35°C for 2 hours.*
Definitive Study – Tomato:
Range: 12.4 - 32.2°C. On one occasion the temperature rose above 32oC for less than two hours on 19 September 2015.*
*There were two minor deviations from the recommended upper temperature range of 32oC and some occasional deviations from the lower limit for relative humidity during the definitive tests, but as the validity criteria for all species tested were met, these were considered to have had no impact on the emergence, survival, growth and health of the plants
pH:
Soil chararcteristic:
pH determined in water: Determined on a dry soil basis: 7.8
Moisture:
Definitive Study – Oat,Ryegrass,Oilseed Rape,Soybean and Sunflower:
Humidity Range (%): 27.7 - 91.7: Some excursions from the stated lower range for relative humidity in the Study Plan were recorded on several occasions but these were on average only around a five hour duration

Definitive Study – Tomato
Humidity Range (%): 27.8 – 82.7: Some excursions from the stated lower range for relative humidity in the Study Plan were recorded on several occasions but these were on average only around a five hour duration.

Details on test conditions:
TEST SYSTEM
- Testing facility: Glasshouse
- Test container (type, material, size): non-porous plastic pots (15 cm diameter)
- Method of seeding: seeds were planted approximately 1-2 cm below the soil surface
- No. of seeds per container: 3 or 4
- No. of replicates per treatment group: 5 to 7
- No. of replicates per control: 5 to 7

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: The soil used in the study was a commercially available loamy sand (USDA Classification) supplied by Baileys of Norfolk, Brick Kiln Road, Hevingham, Norfolk NR10 5NL, UK.
- Pesticide use history at the collection site: Artificail soil. No pest control or fertilisation was used during this study.
- % sand: 77%, 0.005- 2.00 mm
- % silt: 13%, 0.002 - 0.05 mm
- % clay: 10%, < 0.002 mm
- Soil taxonomic classification: Sandy Loam
- Soil classification system: USDA Classification

TEST ORGANISMS
The plant seeds were obtained from a commercial source and the seeds and had not been treated with an insecticide or fungicide. They were be stored refrigerated/dark until used.

GROWTH CONDITIONS
- Photoperiod: Glasshouse lighting was set to achieve a 16: 8 hour light: dark cycle for the duration of the study.
- Light source: natural and artificial source

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
Range Finding Bioassay
0.002, 0.02, 0.2, 2.0 and 20 kg NBPT/ha equivalent to 0.00267, 0.0267, 0.267, 2.67 and 26.7 mg NBPT/kg dry soil* (corrected for purity). Deionised water control
Definitive Bioassay
0.02, 0.2, 2.0, 20 and 200 kg NBPT/ha equivalent to 0.0267, 0.267, 2.67, 26.7 and 267 mg NBPT/kg dry soil* (corrected for purity). Deionised water control

* Conversion of the application rate to the soil concentration has been calculated assuming a soil density of 1.5 g/mL and an incorporation depth of 5cm in soil.
Reference substance (positive control):
no
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
26.7 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: survival and dry shoot weight (biomass)
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
184 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: 143.4
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
205 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: biomass
Remarks on result:
other: 175.8
Species:
other: Solanum Lycopersicon
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
26.7 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: dry shoot weight (biomass)
Species:
other: Solanum Lycopersicon
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
and survival
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and biomass
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and biomass
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and biomass
Species:
Helianthus annuus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
267 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and biomass
Details on results:
PHYTOTOXICITY
Visual injury/phytotoxicity was observed in all plant species at the higher rate of 267 mg NBPT/kg dry soil with varying degrees of severity by 21 DAE.The dicotyledonous species were affected more than the monocotyledons, where the leaf edge yellowing was mild. The worst affected species was the soybean where the effect was severe, resulting in death of the plants. The main symptoms of the observed phytotoxicity were stunting and yellowing of the younger leaf tips or cotyledons. No phytotoxic effects were observed in any of the species tested at 26.7 mg NBPT/kg dry soil and below. The plants growth and condition were the same as the untreated control group. 

EMERGENCE
There was no significant difference in the rate of emergence of oat, ryegrass, oilseed rape, soybean, sunflower or tomato at 21 days after 50 % emergence in the control for any tested concentration (Dunnetts’s, two-tail,p>0.05 or Steel’s Many Rank, two-tail, p=0.05).

SURVIVAL
There was no significant difference in the survival of oat, ryegrass, oilseed rape, soybean, sunflower and tomato at 21 days after 50 % emergence in the control (Dunnett’sTest, two-tail,p>0.05 or Steel’s Many Rank, two-tail, p=0.05) for any tested concentration except for the soybean seedlings where there was a significant effect at the highest rate of 267 mg NBPT/kg dry soil (76.2 % of the plants died).

SHOOT DRY WEIGHT
There was no significant difference in the shoot dry weight of oat, ryegrass, oilseed rape and sunflower at 21 days after 50 % emergence in the control at any treatment rate and of soybean and tomato up to 26.7mg NBPT/kg dry soil. However, there was a significant difference between the untreated control and 267 mg NBPT/kg dry soil in the soybean and the tomato (Dunnett’s Test, two-tail,p>0.05).

NBPT CONCENTRATIONS
Confirmatory analysis of the concentrations of NBPT in the highest rate solution prepared, used to treat the soil, was carried out at Day 0. The measured concentration of NBPT in the solution was 99.1 % of nominal concentration and therefore the final results are based on the nominal concentrations/rates of NBPT.
Results with reference substance (positive control):
No applicable
Reported statistics and error estimates:
Emergence, plant survival and shoot dry weight data generated were subject to statistical analysis using ToxCalc (1999) for each of the six plant species using Dunnett’s two-tail hypothesis test or Steels Many-Rank Test (p=0.05, two tail). The EC50 was estimated using Linear Interpolation and the R2 was calculated using linear regression (Microsoft Excel, 2013) for soybean.
See details below in the"Any other information on results incl. tables" below.

Phytotoxicity

Species

Common Name

Treatment Concentration

Control

NBPT

(26.7 mg/kg)

NBPT

(267 mg/kg)

Control

NBPT

(26.7 mg/kg)

NBPT

(267 mg/kg)

Phytotoxicity Summary (severity rating score)a

BBCHb(leaf stage)

Oats

None (0)

None (0)

Slight necrosis leaf tip (2)

13-14

13-14

13-14

Sunflower

None (0)

None (0)

Stunting (3-6)

 

16

16

16-17

Ryegrass

None (0)

None (0)

Slight necrosis leaf tip (1 & 2)

12-13

13-14

13-14

Oilseed Rape

Slight stunting

Slight stunting

Yellowing of the cotyledons (8)

14-15

13-14

13-14

Tomato

None (0)

None (0)

Yellowing of the cotyledons (8)

13-14

13-14

13-14

Soybean

Slight stunting

None (0)

Death (8-10)

12-13

12-13

9-13

 aat 21 days after 50% control emergence, see Section 16.2 Survival and Phytotoxicity

bat 21 days after 50% control emergence, BBCH indicates differences in phenological development stages of the plants using external morphological characteristics (plant leaf stage).

Emergence

Species Common Name

Emergence

% Emergence

Percentage Difference Relative to the Control

NBPT mg/kg dry soil

Control

0.0267

0.267

2.67

26.7

267

Oat

90.0

-

-

-

0.0

-16.7

Ryegrass

95.0

0.0

+5.3

-10.5

0.0

-5.3

Oilseed rape

85.7

+16.7

+5.6

+5.6

-5.6

0.0

Soybean

95.2

+5.0

0.0

+5.0

+5.0

+5.0

Sunflower

100

-

-

-

0.0

0.0

Tomato

81.0

-5.9

+5.9

-5.9

0.0

-5.9

Percentage differences calculated on unrounded values.

* 21 DAE Days after 50% emergence in the control

1significantly different to the control (Dunnett’s test, two tail, p=0.05)

 

 Survival

Species Common Name

Survival

% Survival

Percentage Difference Relative to the control

at 21 DAE*

NBPT mg/kg dry soil

Control

0.0267

0.267

2.67

26.7

267

Oat

100

-

-

-

0.0

0.0

Ryegrass

100

0.0

0.0

0.0

0.0

0.0

Oilseed rape

100

0.0

0.0

0.0

0.0

0.0

Soybean

100

0.0

0.0

0.0

0.0

-76.21

Sunflower

100

-

-

-

0.0

0.0

Tomato

100

0.0

-11.1

0.0

0.0

-12.5

Percentage differences calculated on unrounded values.

* 21 DAE Days after 50% emergence in the control

1significantly different to the control (Dunnett’s test, two tail, p=0.05)

Shoot Dry Weight

 

Common Name

Dry Shoot Weight

Average Shoot Dry Weight g

Percentage Difference Relative to the control

at 21 DAE*

NBPT mg/kg dry soil

Control

0.0267

0.267

2.67

26.7

267

Oat

0.095

-

-

-

-3.0

+2.6

Ryegrass

0.019

+33.0

+10.5

+11.8

+18.7

-9.6

Oilseed rape

0.262

-5.7

-5.9

-9.3

-1.0

-32.2

Soybean

0.394

+5.9

+2.6

+13.1

+12.4

-65.11

Sunflower

0.358

-

-

-

+4.7

+8.0

Tomato

0.145

-12.6

-5.2

+21.5

+15.6

-43.41

Percentage differences calculated on unrounded values.

* 21 DAE Days after 50% emergence in the control

1significantly different to the control (Dunnett’s test, two tail, p=0.05)

Estimated EC50, NOEC and LOEC for the tested effects

Biological

Endpoint

Species Common Name

Treatment Concentration

mg NBPT/kg dry soil

EC50

NOEC

LOEC

Emergence

Oats

-

267a

>267a

Sunflower

-

267b

>267b

Ryegrass

-

267a

>267a

Oilseed Rape

-

267a

>267a

Tomato

-

267a

>267a

Soybean

-

267a

>267a

Survival

Oats

-

267a

>267a

Sunflower

-

267b

>267b

Ryegrass

-

267a

>267a

Oilseed Rape

-

267a

>267a

Tomato

-

267a

>267a

Soybean

184c(143.4)

R20.9784d

26.7a

>267a

Dry Shoot Weight

(Biomass)

Oats

-

267a

>267a

Sunflower

-

267a

>267a

Ryegrass

-

267a

>267a

Oilseed Rape

-

267a

>267a

Tomato

-

26.7a

>267a

Soybean

205c(175.8)

R20.9393d

26.7a

>267a

aDunnett’s Hypothesis Test (p=0.05, two tail)

bSteels Many-Rank Test (p=0.05, two tail)

cLinear Interpolation (Confidence Interval in parenthesis)

Statistical Analysis ToxCalc™ (1999)

dMicrosoft Excel, 2013

Validity criteria fulfilled:
yes
Remarks:
Seedling emergence and survival for all species was at least 70% and 90%, respectively, in the control plants with no visible signs of injury (with the exception of slight stunting to an individual plant).
Conclusions:
The soil incorporation of NBPT up to rates of 20 kg NBPT/ha (26.7 mg NBPT/kg dry soil) to six terrestrial plant species did not produce any statistically significant effects on emergence, survival and shoot dry weight nor phytotoxicity effects of the six species tested compared to the untreated control plants.

The soil incorporation of NBPT at a rate of 200 kg NBPT/ha (267 mg NBPT/kg dry soil) to six terrestrial plant species showed varying degrees of phytotoxicity, however, there was no statistical difference on emergence, survival and biomass in the tested species with the exception of soybean and tomato. There was a statistical difference between the high rate of NBPT and control plants for both survival and plant growth (dry weight) in soybean, whereas only the dry weight in the tomato was statistically different to the control.
Executive summary:

A reliable study was performed to assess the chronic effects of NBPT on six terrestrial plants (monocotyledoneous and dicotyledoneous cultivated plants) according to the OECD guideline 208 under GLP.

A range finder bioassay was carried out using NBPT at rates ranging from 0.002 to 20 kg/ha (equivalent to 0.00267 mg/kg to 26.7 mg NBPT/kg dw) applied by soil incorporation. Oat (Avena sativa), sunflower (Helianthus annuus) and tomato (Solanum lycopersicon) were assessed for effects on emergence, survival, phytotoxicity and final shoot dry weight for a test period of 21 days after 50% emergence in the untreated control (DAE50).

Following the results of the range-finder bioassay, a dose response/limit test bioassay was carried out using 20 and 200 kg NBPT/ha (26.7 and 267 mg NBPT/kg dry soil) for sunflower and oats. A dose response bioassay was conducted at a range of 0.02 to 200 kg NBPT/ha (0.0267 to 267 mg NBPT/kg dry soil) for ryegrass (Lolium perenne), oilseed rape (Brassica napus) soybean (Glycine max (G. soja)) and tomato. Plants were observed for effects on seedling emergence and phytotoxicity up to 21 days (DAE50). After the final observations the dry shoot weight (biomass) was determined. The test was restarted for tomato because it did not achieve 70% emergence in the control treatments. The validity criterion for this species was met on the retest.

The soil incorporation of NBPT at a rate of 200 kg NBPT/ha (267 mg NBPT/kg dry soil) to six terrestrial plant species showed varying degrees of phytotoxicity, however, there was no statistical difference on emergence, survival and biomass in the tested species with the exception of soybean and tomato. There was a statistical difference between the high rate of NBPT and control plants for both survival and plant growth (dry weight) in soybean, whereas only the dry weight in the tomato was statistically different to the control.

The soil incorporation of NBPT up to rates of 20 kg NBPT/ha (26.7 mg NBPT/kg dry soil) to six terrestrial plant species did not produce any statistically significant effects on emergence, survival and shoot dry weight of the six species tested compared to the untreated control plants.

Confirmatory analysis of the concentrations of NBPT in the highest rate solution prepared, used to treat the soil, was carried out at Day 0. The measured concentration of NBPT in the solution was 99.1 % of nominal concentration and therefore the final results are based on the nominal concentrations/rates of NBPT. This study fulfilled the validity criteria cited in the OECD Guideline 208.

The Soybean, a dicotyledoneous species of the Fabaceae (Leguminosae) family has been shown to be the most sensitive species to NBPT and the survival the most sensitive endpoint with an EC50= 184 mg NBPT/kg dry soil and a NOEC= 26.7 mg NBPT/kg dry soil.

Description of key information

The soil incorporation of NBPT up to rates of 20 kg NBPT/ha (26.7 mg NBPT/kg dry soil) to six terrestrial plant species did not produce any statistically significant effects on emergence, survival and shoot dry weight nor phytotoxicity effects of the six species tested compared to the untreated control plants.

The soil incorporation of NBPT at a rate of 200 kg NBPT/ha (267 mg NBPT/kg dry soil) to six terrestrial plant species showed varying degrees of phytotoxicity, however, there was no statistical difference on emergence, survival and biomass in the tested species with the exception of soybean and tomato. There was a statistical difference between the high rate of NBPT and control plants for both survival and plant growth (dry weight) in soybean, whereas only the dry weight in the tomato was statistically different to the control.

Key value for chemical safety assessment

Additional information