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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: short-term (with study design considered suitable for long-term assessment)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-11-26 to 2008-12-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study is reliable with restrictions. High substance concentrations has been tested and physical effects cannot be excluded.
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): Insoluble
- Density: 0.81 g/cm3 (20 °C)


Analytical monitoring:
no
Details on sampling:
No analytical monitoring
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Calculated amounts of the stock solution and additional volumes of acetone were mixed thoroughly with quartz sand in a glass vessel (1 % of the entire soil amount per concentration). After evaporation of the solvent the treated quartz sand was mixed carefully with the soil with a mixer to insure a homogeneous distribution of the test item. Amounts of test item, solvent, quartz sand and tap water added. The treated soil was transferred to the test container and the seeds were sown (see above).
- Controls: A mixture of natural soil and quartz sand
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent, acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 40.0 mL per treatment
- Evaporation of vehicle before use: Yes
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Rape
- Plant family: Brassicaceae
- Variety: Digger
- Source of seed: KWS Lochow GmbH
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature and protected from moisture until use.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oats
- Plant family: Gramineae
- Variety: Flämingsprofi
- Source of seed: KWS Lochow GmbH
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature 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
- Prior seed treatment/sterilization: No
- Seed storage: The seeds were stored dark at the test facility at room temperature and protected from moisture until use.
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
21 d
Test temperature:
12 - 24 °C
pH:
5.4 +/- 0.1 (natural soil)
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: 0.7 kg (dry weight)
- Method of seeding: Test container 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
- No. of replicates per vehicle control: 8


SOURCE AND PROPERTIES OF SUBSTRATE (if soil): LUFA soil No. 2.2 (batch: Sp2.23708)
- Geographic location: Soil collected by LUFA Speyer (67346 Speyer, Germany)
- Pesticide use history at the collection site: No pesticide use for the last five years.
- Collection procedures: acc. to ISO 10381-6, sampling on 2008-09-18
- Sampling depth (cm): 0 - 20 cm
- Soil texture (if natural soil)
- % sand: 81.4 +/- 1.2
- % silt: 12.2 +/- 0.6
- % clay: 6.4 +/- 0.9
- Soil taxonomic classification: loamy sand
- Soil classification system: German DIN
- Organic carbon (%): 2.16 +/- 0.40
- Maximum water holding capacity (in % dry weigth): 48.2 +/- 5.0
- CEC: 10 +/- 1 mval/100 g
- Pretreatment of soil: soil was stored
- Storage (condition, duration): A 2:1 mixture of natural soil (LUFA 2.2) and quartz sand with a C-content of 1.07 %. 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 ‰)
Origin: GRÜNES LANDHAUS,
St. Godehard-Str.23, D-31139 Hildesheim

Nutrient Concentration [%]
Ammonium nitrogen 5
Nitrate nitrogen 10
P2O5 7
Potassium oxide 22
Magnesium oxide 6
Boron 0.03
Iron (Chelate) 0.12
Copper 0.02
Manganese 0.05
Molybdenum 0.005
Zinc 0.01


GROWTH CONDITIONS
- Photoperiod: 16 h
- Light source: Special fluorescent tube Fluora and Daylight (Hoklatherm)
- Light intensity and quality: > 3500 lx
- Day/night temperatures: 22 +/- 10 °C
- Relative humidity (%): 70 +/- 25
- Watering regime and schedules: Bottom watering of the test container 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 untreated control plants.
Observations included all variations, either inhibitory or stimulatory, between the treated replicates and the untreated control. Such variations were phytotoxic symptoms (chlorosis, necrosis, wilting), formative effects and growth and development rates.


- 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 or 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
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/kg soil DW
- Results used to determine the conditions for the definitive study: Yes
Nominal and measured concentrations:
62.5 - 125 - 250 - 500 - 1000 mg/kg soil dry weight (nominal)
Reference substance (positive control):
no
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 height
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 height
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 height
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
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Shoot fresh weight
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
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:
seedling 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:
seedling 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:
seedling emergence
Details on results:
No statistically significant differences were found between the control and the solvent control. Therefore for calculations the replicates of the control and solvent control were summarized in a pooled control group. The EC25 and EC50 values for shoot height, shoot fresh weight and seedling emergence were >1000 mg/L in all three tested species (oats, rape, soybean).
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
The NOEC was determined for inhibition of shoot height, fresh weight and rate of emergence by One Way Analysis of Variance (ANOVA); followed by DUNNETT'S METHOD if statistically significant differences compared to control replicates were found. 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.
EC25 and EC 50 values were calculated via regression analysis
intervals of growth using GraphPadPrism. Calculation of the confidence intervals for EC25 and EC50 values were carried out using standard procedures.

SEEDLING EMERGENCE

Inhibition [%] of Rate of Emergence

Test concentration

[mg/kg DW]

Rate of Emergence [%] 

Oats

Inhib.[%]

Rape

Inhib.[%]

Soybean   

Inhib.[%]

Pooled control

94

-

94

-

93

-

62.5     

83

11

100

-6

98

-5

125

93

1

100

-6

98

-5

250

95

-1

93

1

100

-8

500

98

-5

98

-4

100

-8

1000

98

-5

100

-6

98

-5

 Inhib. = Inhibition                     negative values = promoted growth

Inhibition [%] of Shoot Height

Test concentration

[mg/kg DW]

Shoot Height [cm]

Oats

Inhib.[%]

Rape

Inhib.[%]

Soybean   

Inhib.[%]

Pooled control

37.6

-

15.6

-

24.8

-

62.5

39.9

-6

15.5

1

31.3

-26

125

37.5

0

15.2

3

28.8

-16

250

35.8

5

15.2

3

30.5

-23

500

37.6

0

14.4

8

30.4

-23

1000

36.4

3

15.5

1

30.9

-25

Inhib. = Inhibition                     negative values = promoted growth

Inhibition [%] of Shoot Fresh Weight

Test concentration

[mg/kg DW]

Shoot Fresh Weight [mg]

Oats

Inhib.[%]

Rape

Inhib.[%]

Soybean

Inhib.[%]

Pooled control

1134

-

1739

-

2318

-

62.5                      

1324

-17

1582

9

2431

-5

125

1084

4

1457

16

2345

-1

250

1093

4

1427*

18

2470

-7

500

1124

1

1256*

28

2146

7

1000

1093

8

1458

16

2147

7

Inhib. = Inhibition                     negative values = promoted growth

*) Test item related significantly different, p<0.05, ANOVA and Dunett's Method a=0.05

Validity criteria fulfilled:
yes
Conclusions:
The test item did not cause any test item related effects > 25 % on the shoot height, the shoot fresh weight and the rate of emergence of the plant species oats and soybean at all tested concentrations.
The test item caused test item related effects of 28 % on the shoot fresh weight of the plant species rape at the concentration 500 mg/kg DW. No test item related effects were observed on shoot height and rate of emergence at all tested concentrations.
After 21 days no test item related phytotoxic effects were observed for the tested plant species.
Executive summary:

The phytotoxicity of the test item Tetrabutane 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 nominal concentrations 1000 - 500 - 250 - 125 - 62.5 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. The inhibition of shoot height, shoot fresh weight and rate of emergence was calculated in relation to the pooled control. After 21 days no test item related phytotoxic effects were observed for the tested plant species.

Shoot Height: NOEC, EC25 and EC50Values with (p)

Species

NOEC*

[mg/kg DW]

EC25

[mg/kg DW]

p = 95%

[mg/kg DW]

EC50

[mg/kg DW]

p = 95%

[mg/kg DW]

Oats

1000

> 1000

> 1000

Rape

1000

> 1000

> 1000

Soybean

1000

> 1000

> 1000

Shoot Fresh Weight: NOEC, EC25 and EC50 Values with (p)

Species

NOEC*

[mg/kg DW]

EC25

[mg/kg DW]

p = 95%

[mg/kg DW]

EC50

[mg/kg DW]

p = 95%

[mg/kg DW]

Oats

1000

> 1000

> 1000

Rape

125

> 1000

> 1000

Soybean

1000

> 1000

> 1000

Emerged Seedlings: NOEC, EC25 and EC50 Values with (p)

Species

NOEC*

[mg/kg DW]

EC25

[mg/kg DW]

p = 95%

[mg/kg DW]

EC50

[mg/kg DW]

p = 95%

[mg/kg DW]

Oats

1000

> 1000

> 1000

Rape

1000

> 1000

> 1000

Soybean

1000

> 1000

> 1000

Description of key information

Key value for chemical safety assessment

Additional information

Phytotoxicity of Tetrabutane to three terrestrial plant species has been determined in a seedling emergence test over a period of 21 days, according to OECD 208 (July 2006). The test was conducted with the nominal concentrations1000 - 500 - 250 - 125 - 62.5 mg/kgsoil dry weight (DW). After 21 days no test item related phytotoxic effects were observed for the tested plant species (oats,rape and soybean). The test substance did not cause any test item related effects > 25 % on the shoot height, the shoot fresh weight and the rate of emergence of the plant species oats and soybean at all tested concentrations. The EC50 values for shoot height, shoot fresh weight and seedlings emergence were >1000 mg/kg dw. For the plant species rape, the test substance caused test item related effects of 28 % on the shoot fresh weight at the concentration 500 mg/kg DW. No test item related effects were observed on shoot height and rate of emergence at all tested concentrations.

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