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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:
2013-03-11 - 2013 - 2013-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name: N-((3(5)-Methyl-1H-pyrazol-1 -yl)methyl)acetamide
Analysed purity: 99.8 %
Water solubility: ca. 8 % = 87 g/L
Appearance: white solid
Analytical monitoring:
no
Vehicle:
yes
Remarks:
deionised water
Details on preparation and application of test substrate:
The test item was incorporated into the soil. Since the test item is soluble in water, the required quantity of
test item was dissolved in a suitable amount of deionised water. The test solutions were then mixed with
the soil by intensive stirring in a mixer.
Preparation of the test solutions
1. 0.2667 g P70/05/S-5 was weighed and filled up to 1000 mL with deionised water yielding stock
solution A.
2. 200.00 mL stock solution A was filled up to 1000 mL with deionised water yielding solution B.
Preparation of the test substrate
1. 2 x 25.0 kg soil dry weight was prepared for each treatment group.
2. 2250 mL water was added to 25.0 kg soil d.w. each. The soil was made with dried ingredients and
was therefore at nominal 0 % moisture before mixing the components.
3. 250 ml test solution was added to 25.0 kg soil d.w. each resulting in test substrate concentrations of
400 and 2000 g test item/ha
Order of application and cleaning procedure
The order of application was as follows:
- test item in ascending order
Application equipment
mixing machine: type: AOX 600 (Rotationsmischer)
mixing time: 2 x 4 minutes for each replicate
rotations per minute: approx. 750
The mixer was cleaned with a brush following application of each test item concentration and with cleaning
agents after the last mixing.
Cleaning agents used were Mucasol (alkaline cleaning agent for laboratory equipment), water,
deionised water and sprit for glass equipment.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
common name: oat
variety: Scorpion
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
common name:onion
variety: Sturon
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
common name: oilseed rape
variety: Visby
Species:
Helianthus annuus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
common name: sunflower
variety:Goldener Neger
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
common name:cucumber
variety: Burpless Tasty Green
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
common name:pea
variety:Gloriosa
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
21 d
Test temperature:
12 – 34 °C (recommended conditions for greenhouse testing: 22 ± 10 °C)
Moisture:
25 – 77 % (recommended conditions for greenhouse
testing: 70 ± 25 %)
Details on test conditions:
Test soil
Origin: agricultural soil from site Gerichshain (batch G 01/2013)
stored for at least 1 year before used in the test
Pesticide and fertiliser use in previous years
(before sampling from the field):
no pesticides or fertilisers were applied on the origin plot
of the test soil for at least 5 years (the plot was fallow
land)
Characteristics
Soil type: loamy sand (classification according to DIN 4220)
pH-value (CaCl2): 6.5
Corg (%): 0.87
Salt content (mg KCl/100 g dry soil): 30.4
Water holding capacity (WHC) (g/100 g dry soil): 30.4
Particle size distribution: < 0.002 mm (clay): 12.6 %
(according to DIN ISO 11277) 0.002 – 0.063 mm (silt): 32.6 %
0.063 – 2.0 mm (sand): 54.8 %
Application rates: 400 and 2000 g test item/ha
corresponding to
0.533 and 2.667 mg test item/kg soil dry weight (not corrected for purity)
400 g test item correspond to a normal mean nitrogen application
of 200 kg nitrogen/ha
2000 g test item/ha are a fivefold increase of mean amount
Test conditions
Test pot: non-porous plastic flower pot ( 15 cm)
amount soil/pot: 1.1 kg fresh soil;
Surface area per pot used in the test: 177 cm2
Number of seeds/100 cm2: Maximal seed density according to guideline: 3-10
depending on the size of the seeds, actual density:
2.2 – 2.8
Number of seeds /pot (= replicate): 5: oat, onion
4: oilseed rape, sunflower, cucumber, pea
Examples of recommended densities are given by
OECD guideline 208 for oilseed rape (3 per 15 cm pot)
for cucumber (1-2 per 15 cm pot). However, based on
the experiences of BioChem agrar with plant testing
adequate growth conditions are provided with densities
of up to 4 seeds per 15 cm pot for these species. With
respect to the statistical evaluation of the results the
increased number of seeds per replicate significantly
decreased the variability between replicates. Thus, this
deviation from the guideline recommendation ensures
an optimal statistical analysis with a high discriminatory
power.
Number of pots (= replicates)/treatment: 6: oat, onion
8: oilseed rape, sunflower, cucumber, pea
Number of seeds/treatment: 30-32
Fertilising: according to good horticultural practice with the liquid fertiliser “Pflanzenvollnahrung“ (origin: Gute Wahl GmbH, 80333 München, Germany)
content: 4% N, 3% P2O5, 3% K2O
cycle: weekly
amount/pot: 100 mL (0.5%)
Irrigation: bottom watering in pot saucers daily with tap water
(pH: 7.3, conductivity: 80 μS/cm)
Environmental test conditions (greenhouse)
Temperature: 12 – 34 °C (recommended conditions for greenhouse testing: 22 ± 10 °C)
Relative humidity: 25 – 77 % (recommended conditions for greenhouse testing: 70 ± 25 %)
Recording: continuously by a thermohygrograph
Illumination: source: daylight/artificial light
light period: 16 h (including 10 h HQL-light)
light intensity: 314 – 382 μE/m2/s (recommended
conditions for greenhouse testing: 350 ± 50 μE/m2/s)
Test duration: 21 days after 50 % seedling emergence

Experimental procedure
The test item was incorporated into the soil. Since the test item is soluble in water, the required quantity of
test item was dissolved in a suitable amount of deionised water. The test solutions were then mixed with
the soil by intensive stirring in a mixer.
Preparation of the test solutions
1. 0.2667 g P70/05/S-5 was weighed and filled up to 1000 mL with deionised water yielding stock
solution A.
2. 200.00 mL stock solution A was filled up to 1000 mL with deionised water yielding solution B.
Preparation of the test substrate
1. 2 x 25.0 kg soil dry weight was prepared for each treatment group.
2. 2250 mL water was added to 25.0 kg soil d.w. each. The soil was made with dried ingredients and
was therefore at nominal 0 % moisture before mixing the components.
3. 250 ml test solution was added to 25.0 kg soil d.w. each resulting in test substrate concentrations of
400 and 2000 g test item/ha

Course of trial
The tests were conducted in a greenhouse. Two monocotyledonous plant species (Avena sativa, Allium
cepa) and four dicotyledonous species (Brassica napus, Helianthus annuus, Cucumis sativa, Pisum
sativum) were used in the test representing 6 families (Poaceae, Liliaceae, Brassicaceae, Asteraceae,
Cucurbitaceae, Fabaceae).
The culturing of the test plants was performed in plastic pots. The number of seeds per pot for each plant
species was four for oilseed rape, sunflower, cucumber and pea plants as well as five for oat and onion
plants.
The soil used was a loamy sand (classification according to DIN 4220) with a content of 0.87 %
organic carbon, pH of 6.5 and a salt content of 30.4 mg/100 g dry soil (corresponding to 0.04 %).
Bottom watering of the pots was performed using pot saucers. The plants were watered daily and the
amount of water volume per pot was adjusted to the respective growth stage.
The test item application rates were tested in comparison with an untreated control group that was
maintained under the same environmental conditions. The treatment of the soil was carried out by
incorporating the test item directly into the soil. The soil was mixed with the quantity of the test item
solution which was necessary to achieve the test concentration in the soil. The soil of the control group
was mixed with the corresponding amount of deionised water only. After thorough mixing and filling the
treated soil into the pots, the seeds were sown on the soil surface and covered with a thin layer of soil. An
initial top watering was stimulate the seed germination. The pots were then set up randomly in a greenhouse
and were repositioned periodically to minimize variability in growth of plants. The duration of the
test was 21 days after 50 % seedling emergence.
During the observation period, i.e. 21 days after 50 % of the control plants had emerged, the plants were
observed weekly for seedling emergence, survival and visual phytotoxicity (e.g. chlorosis). A plant is defined
as dead, when it is photosynthetically inactive, i.e. 100 % necrosis and no chlorophyll.
At the end of the test, emergence, survival (mortality), final shoot fresh weight (biomass of
surviving plants) and visible detrimental effects on different parts of the plants were recorded. The
observations included all variations between treated and untreated plants and also the stage of development.
The recorded effects were used to determine the statistically significant differences for emergence and
shoot fresh weight.

Assessment parameters during the test
Time and parameters for assessments
7 to 21 days after 50 % seedling emergence in the control group, the plants were observed weekly for
emergence, visible phytotoxicity and mortality compared to untreated control plants
Day 7 and 14 after 50 % seedling emergence
 Seedling emergence (number of plants per pot)
 Surviving plants (number of plants per pot)
 Severity of phytotoxic symptoms, e.g. chlorosis, necrosis, growth inhibition (%/plant)
 Any lack of effects (resulting from sum of previous single assessments)
Day 21 after 50 % seedling emergence
 Seedling emergence (number of plants per pot)
 Surviving plants (number of plants per pot)
 Severity of phytotoxic symptoms, e.g. chlorosis, necrosis, growth inhibition (%/plant)
 Any lack of effects (resulting from sum of previous single assessments)
 Shoot fresh weight (only surviving plants per pot)
Nominal and measured concentrations:
400 and 2000 g test item/ha
corresponding to
0.533 and 2.667 mg test item/kg soil dry weight (not corrected for purity)
400 g test item correspond to a normal mean nitrogen application
of 200 kg nitrogen/ha
2000 g test item/ha are a fivefold increase of mean amount
Reference substance (positive control):
no
Key result
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
Helianthus annuus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Helianthus annuus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Cucumis sativus
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Key result
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Key result
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
other: NOER
Effect conc.:
>= 2 000 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Validity criteria fulfilled:
yes
Conclusions:
The soil application of P70/05/S-5 at rates of 400 and 2000 g test item/ha to six terrestrial plant species
caused no adverse effects on seedling emergence and shoot fresh weight.
Executive summary:

The soil application of P70/05/S-5 to oat, onion, oilseed rape, sunflower, cucumber and pea plants caused

no adverse effects on seedling emergence and shoot fresh weight at the tested rates of 400 and 2000 g

test item/ha.

No visible phytotoxic effects were observed on day 21 after 50 % emergence at the tested rates.

No Observed Effect Rates (NOER) for the six tested plant species at test termination 21 days after 50 % emergence application of P70/05/S-5:

Plant species

P70/05/S-5(g test item/ha)

Avena sativa

Allium cepa

Brassicanapus

Helianthus annuus

Cucumissativa

Pisum sativum

Seedling emergence(on day21after50 %emergence)

NOER

> 2000

> 2000

> 2000

> 2000

> 2000

> 2000

      Growth (shoot fresh weight on day 21 after 50 % emergence)              
 NOER  >2000  >2000  >2000  >2000  >2000  >2000

Description of key information

The soil application of P70/05/S-5 at rates of 400 and 2000 g test item/ha to six terrestrial plant species

caused no adverse effects on seedling emergence and shoot fresh weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:
2 000 mg/kg soil dw

Additional information