Registration Dossier

Administrative data

Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-01 to 2011-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed according to relevant guidelines and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No analytical monitoring

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The test item amounts for each treatment were weighed out on calculated amounts of quartz sand (1 % of the entire soil amount per concentration), acetone was added and everything was mixed thoroughly. After evaporation of the acetone the treated quartz sand was mixed thoroughly with the soil to ensure a homogeneous distribution of the test item. The treated soil was transferred to the test container and the seeds were sown
- Controls: Control: Untreated quartz sand, Solvent Control: Acetone (evaporated) and quartz sand
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Evaporation of vehicle before use: yes

Test organisms

open allclose all
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Rape
- Plant family: Brassicaceae
- Variety: Kadore
- Source of seed: KWS SAAT AG, D-37555 Einbeck, Germany
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature (20 ± 10 °C) and protected from moisture until use.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oats
- Plant family: Gramineae
- Variety: Ivory
- Source of seed: Saaten Union GmbH, D-30916 Isernhager HB, Germany
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature (20 ± 10 °C) and protected from moisture until use.
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Leguminosae
- Variety: Merlin
- Source of seed: Saatbau Linz, A-4021 Linz, Austria
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature (20 ± 10 °C) and protected from moisture until use.

Study design

Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
21 d

Test conditions

Test temperature:
nominal: 22 +/- 10 °C
actual: 13 - 23 °C
pH:
5.5 +/- 0.1 (natural soil)
Moisture:
- humidity: nominal: 70 +/- 25 %, actual: 45 - 91 %
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): Plastic container (standard flower pots) with a diameter of ca. 12 cm
- Amount of soil:
Control, solvent control, 1000 mg/kg DW: 15.0 kg DW
- Method of seeding: Test containers were filled each with the same amount of treated soil and seeding holes were made with a seedling pistil. One seed was given in each hole and covered with soil.
- No. of seeds per container: 5
- No. of replicates per treatment group: 8
- No. of replicates per control: 8



SOURCE AND PROPERTIES OF SUBSTRATE (if soil): A 2:1 mixture of natural soil (LUFA 2.2, batch: Sp2.23010, Sp2.24110) and quartz sand.
- Geographic location: Soil collected by LUFA Speyer (67346 Speyer, Germany), quartz sand collected by Dörentrup Quarz GmbH Co. KG (31089 Duingen, Germany)
- Pesticide use history at the collection site: No pesticide use for the last five years.
- Soil texture (if natural soil)
- grain size: - Soil taxonomic classification: loamy sand (IS)
- Soil classification system: German DIN
- Organic carbon (%): 1.87 +/- 0.2
- CEC: 9.9 +/- 0.7 mval/100 g
- Storage (condition, duration): The soil has been stored at the test facility at green house conditions until use.


NUTRIENT MEDIUM (if used)
- Description:
Flory 9 hydro (0.5 ‰) and Hakapos® Soft Spezial 16+8+22(+3) (0.5 ‰)
Origin: Grünes Landhaus, St. Godehard-Str.23, D-31139 Hildesheim

Nutrient
Concentration [%]
(Hakaphos®)
Ammonium nitrogen 9.7
Nitrate nitrogen 6.3
P2O5 8
Potassium oxide 22
Magnesium oxide 3
Boron 0.03
Iron (Chelate) 0.15
Copper 0.02
Manganese 0.05
Molybdenum 0.005
Zinc 0.015



GROWTH CONDITIONS
- Photoperiod: 16 h
- Light source: Special-fluorescent tube Fluora and Daylight (OSRAM)
- Light intensity and quality: nominal: > 3500 lx, actual: 5752 ± 284
- Day/night temperatures: 22 +/- 10 °C, actual: 13 - 23°C
- Relative humidity (%): 70 +/- 25, actual: 45 - 91
- Watering regime and schedules: Bottom watering of the test containers with nutrient solution as necessary. The watering interval based on visual observations of soil surface and plants and was checked daily.




EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
During the 21 day observation period the plants were observed every 7 days for number of emerged seedlings, number of dead plants and visual phytotoxic effects. The rating of the treated plants was done in relation to the pooled control / solvent control plants.
Observations included all variations, either inhibitory or stimulatory, between the treated test replicates and the untreated control replicates. Such variations were phytotoxic symptoms (chlorosis, necrosis, wilting), formative effects and growth and development rates.
At the end of the study shoot height and shoot fresh weight were measured.
Day 0: Application as specified above.
Every working day: The test containers were bottom watered and fertilised as needed.
Day 7, 14, 21: The replicates were observed for visual phytotoxic effects number of emerged seedlings and number of dead plants.
Test end (day 21): Final shoot heights and fresh weight measurements were done.



- Phytotoxicity rating system (if used):

(1) Alteration of form and appearance (deformation)
(1.1) Curling / twisting / sticking or other deformation of the leafs
(1.2) Change of appearance
(1.3) Shoot deformations
(1.4) Wilting
(1.5) Leaf shedding
(1.6) Shedding of the leaf apex
(1.7) Stunted growth
(1.8) Lacking of cotyledons

(2) Discolouration of the leafs
(2.1) Lighter green (2.2) Darker green
(2.3) Chlorosis (2.3.1) Spots
(2.3.2) Whole leaf
(2.3.3) Veins
(2.3.4) Between veins
(2.3.5) Marginal
(2.4) Loss of colour
(2.5) Discolouration

(3) Alteration of the leaf cells
(3.1) Necrosis (3.1.1) Spots
(3.1.2) Marginal
(3.1.3) Apical
(3.1.4) Between veins
(3.1.5) Whole leaf

(E) Dead plant (Number of dead plants)


Assessment of the Effects

- = no effects observed / normal growth
+/- = slight effects
+ = medium effects
++ = strong effects


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Range finding study
Results of the Preliminary Test: Inhibition of Shoot Fresh Weight and Emergence on day 21

Test
concentration Inhibition of shoot fresh weight [%] Inhibition of emergence
[%]
[mg/kg DW] Oats Rape Soybean Oats Rape Soybean
10 -3 10 6 0 -8 -27
100 -22 14 2 -7 -8 -27
1000 -18 2 -14 0 -8 -19

Nominal and measured concentrations:
nominal: 1000 mg/kg soil dry weight (DW)

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
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:
growth
Remarks:
(Shoot fresh weight, shoot height) and emergence
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:
growth
Remarks:
(Shoot fresh weight, shoot height) and emergence
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:
growth
Remarks:
(Shoot fresh weight, shoot height) and emergence
Details on results:

SEEDLING EMERGENCE
- Percent seedling emergence:
Inhibition [%] of Rate of Emergence
Test concentration
[mg/kg DW] Rate of Emergence [%]
Oats Inhibition[%] Rape Inhibition[%] Soybean Inhibition[%]
Pooled control 94 - 93 - 96 -
1000 93 1 85 9 100 -4

Inhib. = Inhibition negative values = promoted growth

- Plant height:
Inhibition [%] of Shoot Height
Test concentration
[mg/kg DW] Shoot Height [cm]
Oats Inhibition[%] Rape Inhibition[%] Soybean Inhibition[%]
Pooled control 43.2 - 14.5 - 25.0 -
1000 42.8 1 13.9 4 25.4 -2

- Fresh weight:
Inhibition [%] of Shoot Fresh Weight
Test concentration
[mg/kg DW] Shoot Fresh Weight [mg]
Oats Inhibition[%] Rape Inhibition[%] Soybean Inhibition[%]
Pooled control 1375 - 1586 - 2174 -
1000 1340 3 1536 3 2200 -1



Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
The percent inhibition of biomass growth (shoot height, shoot fresh weight) and rate of emergence were calculated in relation to the control. No statistically significant differences were determined between the control and solvent control data. Therefore, all calculations are based on the pooled control data.

Statistical significant differences were determined for inhibition of shoot height, shoot fresh weight and rate of emergence by One Way Analysis of Variance (ANOVA), between control and solvent control. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Any other information on results incl. tables

Oats: Visual Observations and Number of Dead Plants on Day 21

Test
concentration

Phytotoxic effects

Phytotoxic

Replicate

­[mg/kg DW]

effects

1

2

3

4

5

6

7

8

pooled
control

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1000

-

-

-

-

-

-

-

-

Rape: Visual Observations and Number of Dead Plants on Day 21

Test
concentration

Phytotoxic effects

Phytotoxic

Replicate

[mg/kg DW]

effects

1

2

3

4

5

6

7

8

pooled
control

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1000

-

-

-

-

-

-

-

-

Soybean: Visual Observations and Number of Dead Plants on Day 21

Test
concentration

Phytotoxic effects

Phytotoxic

Replicate

[mg/kg DW]

effects

1

2

3

4

5

6

7

8

pooled
control

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1000

-

-

-

-

-

-

-

-

Oats: Shoot Height on Day 21

Pooled control

Limit concentration [mg/kg DW]

Control

Solvent control

1000

Repl.

Height

[cm]

Mean ± SD

Height

[cm]

Mean ± SD

Height

[cm]

Mean ± SD

35.1

39.3 ± 7.2

48.9

39.5 ± 8.7

33.1

38.4 ± 3.3

44.7

44.3

39.1

1

39.6

26.9

42.1

47.5

42.6

39.1

29.6

35.0

38.6

47.0

46.2 ± 1.9

5.8

40.3 ± 19.3

46.8

44.8 ± 5.3

48.9

50.2

40.5

2

44.0

47.3

40.4

45.6

51.0

51.4

45.3

47.0

 -

44.6

46.4 ± 5.2

44.2

41.6 ± 6.6

47.8

43.6 ± 2.7

51.0

38.1

41.2

3

49.6

45.0

42.3

38.0

32.0

44.7

48.6

48.8

42.0

20.0

44.7 ± 15.6

44.1

40.2 ± 8.0

19.2

43.6 ± 14.1

55.3

33.2

45.7

4

54.2

33.9

53.7

55.8

49.5

46.1

38.1

 -

53.2

53.4

46.7 ± 7.4

40.0

44.3 ± 5.3

42.2

44.4 ± 2.9

54.8

45.0

42.0

5

38.0

38.6

45.2

46.4

51.9

49.0

41.0

45.8

43.4

48.6

45.9 ± 4.1

48.1

49.5 ± 1.3

40.0

44.5 ± 5.7

44.3

51.1

41.2

6

40.7

48.2

44.0

51.2

50.0

52.7

44.8

50.3

 -

40.2

43.0 ± 4.7

43.8

45.8 ± 4.4

43.0

42.8 ± 1.3

44.3

51.8

42.4

7

38.5

41.6

44.7

49.1

45.9

41.2

 -

 -

42.9

40.6

44.6 ± 5.2

38.0

33.1 ± 14.0

39.2

40.6 ± 3.9

44.5

41.4

36.7

8

51.9

40.9

46.0

41.3

12.2

40.6

 -

 -

 -

Mean   ± SD

44.6 ± 2.5

41.8 ± 4.9

42.8 ± 2.2

43.2 ± 4.0

Sig. Dif. P 0.05

No1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No biological significant effects were observed on the shoot height, the shoot fresh weight and the rate of emergence of all tested plant species. The NOEC is 1000 mg/kg DW for shoot height, shoot fresh weight and rate of emergence for all tested plant species.

After 21 days no test item related phytotoxic effects were observed for all tested plant species at the limit concentration 1000 mg/kg DW.
Executive summary:

The phytotoxicity of the test item to three terrestrial plant species was determined in a seedling emergence test over a period of 21 days according to OECD 208 (July 2006). The test was conducted with the limit concentration 1000 mg/kg soil dry weight (DW). Test systems were one monocotyledon, oats (Gramineae), and two dicotyledons, rape (Brassicaceae) and soybean (Leguminosae). The test item was incorporated into the soil in which the seeds were sown afterwards. The test containers were bottom watered with nutrient solution as needed. The toxic effects of the test item were determined on day 7, 14 and 21 by visual observations (number of emerged seedlings and phytotoxic effects) and on day 21 by shoot height and shoot fresh weight determination. As there were no statistically significant differences between the control and the solvent control, they were pooled for further calculations.

No biological significant effects were observed on the shoot height, the shoot fresh weight and the rate of emergence of all tested plant species (Table 1).The NOEC is 1000 mg/kg DW for shoot height, shoot fresh weight and rate of emergence for all tested plant species.

After 21 days no test item related phytotoxic effects were observed for all tested plant species at the limit concentration 1000 mg/kg DW.

Inhibition and NOEC of Shoot Height, Shoot Fresh Weight and
Rate of Emergence in Comparison to the Pooled Control

Shoot height

Shoot fresh weight

Rate of Emergence

Species

Inhibition [%]

NOEC [mg/kg DW]

Inhibition [%]

NOEC [mg/kg DW]

Inhibition [%]

NOEC [mg/kg DW]

Oats

1

1000

3

1000

1

1000

Rape

4

1000

3

1000

9

1000

Soybean

-2

1000

-1

1000

-4

1000