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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Version / remarks:
2006
Deviations:
no
Principles of method if other than guideline:
Two tests were run in this study, DT1 and DT2. The latter was performed with two test species only, A. sativa and P. sativum, and with different test item concentrations because a NOEC could not be determined in DT1. Seeds of two monocotyledonous species, Avena sativa (oat) and Allium cepa (onion), as well as four dicotyledonous species, Brassica napus (rape), Cucumis sativus (cucumber), Pisum sativum (pea) and Solanum lycopersicum (tomato) were planted in a natural sandy loam soil (standard soil LUFA Sp 2.3) immediately after test item application and left to grow under controlled conditions for 14(16) days following 50 % emergence of the control plants. Five test item concentrations were tested with each species. Rates in DT1 ranged from either 0.4 to 40 mg/kg soil dry weight, or 4.0 to 400 mg/kg soil dry weight or 12.6 to 1000 mg/kg soil dry weight. In test run DT2 the test concentration ranged from 1.0 to 316 mg/kg soil dry weight for both test species.
The appropriate amounts of test item for each treatment was ground together with fine quartz sand and then mixed homogeneously into the test soil. The concentration of sand added to the soil was 1% (i.e. 10 g/kg soil). The control soil received sand which had not been treated with the test item. Soils were supplied with water and a nutrient solution as needed by bottom watering. The test was performed in growth chambers equipped with artificial lighting (208 to 264 μE m-2 s-1, for 16 hours per day) and air conditioning (air temperature ranging from 18.8 to 29.2°C, relative humidity ranging from 35.1 % to 67.3 %). The pots were placed randomly at the beginning of the test and were re-arranged at minimum weekly intervals. On day 7 after 50% emergence of control, seedlings were evaluated visually. At test end (day 14/16), seedlings were counted, evaluated visually, and harvested to determine shoot fresh weight. After termination of the test (DT1) a second test (DT2) was performed with two test species, A. sativa and P. sativum.
GLP compliance:
yes
Remarks:
The previous range-finding test was not performed in compliance with Good Laboratory Practice Principles.
Analytical monitoring:
no
Remarks:
Verification of test concentration will be provided with exact weights of the test item added to the soil. No supporting chemical analysis will be performed.
Vehicle:
no
Details on preparation and application of test substrate:
Pre-treatment of the Test Item
Appropriate quantities of the test item will be weighed out per treatment and ground with fine quartz sand before being mixed into the soil of the respective treatments.
The method of test item application in the definitive test was chosen in order to comply with the method of application used in the previously performed non-GLP range-finding test.

Application of the Test Item
Defined amounts (corresponding to the nominal concentrations given in Table 3) of test item will be weighed out separately and ground with fine quartz sand. Afterwards test item and quartz sand will be thoroughly mixed into the test soil by using a hand mixer (or equivalent) in order to homogeneously distribute the test item in the soil. The concentration of sand added to the soil will be 1% (i.e. 10 g/kg soil). The control soil will receive sand which has not been treated with the test item.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Family: Amaryllidaceae
Cultivar: Stuttgarter Riesen
Lot No.: 4333216
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Family: Poaceae
Cultivar: Canyon
Source of supply: Nordsaat Saatzucht GmbH, Langenstein, Germany
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Brassicaceae
Cultivar: Akela
Source of supply: Feldsaaten Freudenberger, Krefeld, Germany
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Cucurbitaceae
Cultivar: Delikatess
Lot No.: 4291508
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Fabaceae
Cultivar: Rondo
Lot No.: 117024NU
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
Species:
other: Solanum lycopersicum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Solanaceae
Cultivar: Hellfrucht
Lot No.: 4317738
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
16 d
Remarks:
First test run (DT1): Duration of the test was 14 days for A. cepa, A. sativa and S. lycopersicum and 16 days for the remaining species; Second test run (DT2): Duration of the test was 14 days for A. sativa and 16 days P. sativum.
Test temperature:
First Test Run (DT1)
Air temperature [°C]
A. cepa, B. napus, C. sativus, P. sativum (time period: from 29 June to 19 July 2017)
mean: 26.7
min: 21.8
max. 31.6
A. sativa, S. lycopersicum (time period: from 29 June to 17 July 2017)
mean: 26.7
min. 22.6
max. 31.6

Second Test Run (DT2)
Air temperature [°C]
A. sativa (time period: 28 September 2017 – 16 October 2017)
mean: 21.5
min: 18.8
max. 26.1
P. sativum (time period: 28 September 2017 – 20 October 2017)
mean: 21.9
min: 18.8
max. 29.2
pH:
pH of LUFA Soil Type 2.3 used in test run DT1 and test run DT2
pH (0.01 M CaCl2):
Sp2.34316 (DT1): 5.9 ± 0.6
Sp2.32017 (DT2): 5.84 ± 0.6
Moisture:
First Test Run (DT1)
Rel. air humidity [%]
A. cepa, B. napus, C. sativus, P. sativum (time period: from 29 June to 19 July 2017)
Mean: 38.8
Min. 28.2
Max. 64.1
A. sativa, S. lycopersicum (time period: from 29 June to 17 July 2017)
Mean: 39.3
Min. 28.2
Max. 64.1

Second Test Run (DT2)
Rel. air humidity [%]
A. sativa (time period: 28 September 2017 – 16 October 2017)
Mean: 50.8
Min. 39.7
Max. 67.3
Rel. air humidity [%]
P. sativum (time period: 28 September 2017 – 20 October 2017)
Mean: 50.2
Min. 18.8
Max. 29.2
Details on test conditions:
Plant tests will be performed in an air-conditioned room equipped with artificial lighting. Control and treated specimens of a particular plant species will always be tested in parallel and under the same environmental conditions.
Illuminance and relative humidity will deviate from the range suggested by the test guideline such that light intensity will be ≥ 200 μE m-2 s-1 instead of 350 ± 50 μE m-2 s-1. Relative humidity will be in the range of 30 to 70% instead of 70% ± 25% due to air conditioning. These conditions ensure good growing conditions as has been confirmed in earlier studies. Air temperature and air moisture will be recorded. Short-term deviations (< 2 hours) from the recommended temperature and humidity ranges are unavoidable and should not affect the integrity or outcome of the study.

First Test Run (DT1)
Test Soil
Standard soil LUFA Type 2.3, a sandy loam, was purchased from the Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), D-67346 Speyer, Germany. LUFA soil Type 2.3 was collected from an uncultivated field at Offenbach, Rhineland-Palatinate, Germany, and sieved (<=2 mm). The field had neither received any organic fertilisers nor plant protection substances during the last four years before sampling. The soil was stored at ECT GmbH at ambient temperature until usage.
Test Containers
Common plant pots made of polypropylene (diameter 11 cm, height 8.5 cm) were used as test pots. Each pot was filled with 491 ± 10 g soil fresh weight and placed in a separate polystyrene beaker (Kastelplast GmbH, Mainz, Germany) serving as water reservoir.
Light
Light intensity [μE m-2 s-1]: min. 213 and max. 352

Second Test Run (DT2)
Test Soil
Same type and source as in DT1 but different batch
Test Containers
Same as in DT1 but filled with 495 ± 10 g fresh weight soil instead of 491 ± 10 g fresh weight soil.
Plant Species
Test species used in DT2 were Avena sativa and Pisum sativum
Light
Light intensity [μE m-2 s-1]: min. 208 and max. 264



Nominal and measured concentrations:
First Test Run (DT1)
Test item: 0.40 mg/kg soil; 1.26 mg/kg soil; 4.00 mg/kg soil; 12.6 mg/kg soil; 40.0 mg/kg soil; 126 mg/kg soil; 400 mg/kg soil; 1000 mg/kg soil.
Weighed or nominal test item per treatment: 0.0070 g; 0.0230 g; 0.0327 g; 0.2062 g; 0.6545; 1.8325 g; 5.8176 g; 8.1810 g.
Second Test Run (DT2)
Test item: 1.00 mg/kg soil; 3.16 mg/kg soil; 10.0 mg/kg soil; 31.6 mg/kg soil; 100 mg/kg soil; 316 mg/kg soil.
Reference substance (positive control):
no
Remarks:
The test guideline does not recommend the testing of a reference substance. Therefore, the test was performed without a reference item.
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 40 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 40 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Avena sativa
Duration:
14 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
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Brassica napus
Duration:
16 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
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Brassica napus
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Cucumis sativus
Duration:
16 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
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Cucumis sativus
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
> 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: First Test Run (DT1), Seedling emergence and survival
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
12.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
1.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
41.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
< 12.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
<= 12.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
21.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC50
Remarks:
extrapolated value (higher than highest tested concentration)
Effect conc.:
1 911 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Brassica napus
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Brassica napus
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Brassica napus
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
200 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Brassica napus
Duration:
16 d
Dose descriptor:
EC50
Remarks:
Extrapolated value (higher than highest tested concentration)
Effect conc.:
1 020 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Cucumis sativus
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Cucumis sativus
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Cucumis sativus
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
365 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Cucumis sativus
Duration:
16 d
Dose descriptor:
EC50
Remarks:
Extrapolated value (higher than highest tested concentration)
Effect conc.:
1 414 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
< 4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
<= 4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
2.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
EC50
Effect conc.:
174 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
12.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
40 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
19.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
other: S. lycopersicum
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
127 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: First Test Run (DT1)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: Second Test Run (DT2), Seedling emergence and survival
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: Second Test Run (DT2), Seedling emergence and survival
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
>= 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: Second Test Run (DT2), Seedling emergence and survival
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
> 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks on result:
other: Second Test Run (DT2), Seedling emergence and survival
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: Second Test Run (DT2)
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
>= 316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: Second Test Run (DT2)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: Second Test Run (DT2)
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: Second Test Run (DT2)
Key result
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
3.16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Remarks on result:
other: Overall result from conservative evaluation from results of DT1 and DT 2.
Details on results:
First Test Run (DT1)
Statistical Evaluation
Number of emerged and number of survived seedlings and shoot fresh weight at the end of the test were considered for statistical data analysis.
Neither emergence rate nor survival was significantly reduced in any of the test species. NOEC values for the endpoints emergence rate and survival are therefore assumed to be equal to or higher than the highest tested concentration for the respective test species and EC10 and EC50 values are assumed to be higher than the highest tested concentration.
The lowest NOEC and EC10 was observed with P. sativum (NOEC <4.0 mg/kg soil dw) and A. cepa (EC10 = 1.7 mg/kg soil dw).

Second Test Run (DT2)
Statistical Evaluation
Number of emerged and number of survived seedlings and shoot fresh weight at the end of the test were considered for statistical data analysis.
Since neither emergence rate nor survival was significantly reduced for any of the two species NOEC values for emergence rate and survival are assumed to be ≥ 316 mg test item/soil dry weight. EC10 and EC50 values of emergence and survival are assumed to be higher than 316 mg/kg dw.
Results with reference substance (positive control):
The test guideline does not recommend the testing of a reference substance. Therefore, the test was performed without a reference item.
Reported statistics and error estimates:
Number of emerged and number of survived seedlings as well as shoot fresh weight at the end of the test was considered for statistical data analysis.
Arithmetic means for each pot were calculated from the single plants within a pot. Since each pot constituted a replicate, the mean values per pot were used for statistical analysis. Plants which had emerged after day 7 were excluded from calculation of the mean per pot.
Emergence and survival data were checked by the Fisher's exact binominal test with Bonferroni correction to determine the NOEC and LOEC. Significance level was alpha = 0.05, one sided greater.
Probit and Weibull Analysis was used to determine the effective concentrations EC10 and EC50.
Normal distribution and homogeneity of variances were checked by the Kolmogorov-Smirnov Test and Cochran’s test, respectively.
NOEC and LOEC of shoot fresh weight were determined by applying the William’s t-Test (monotonous dose response relationship). In case of inhomogeneous variances Welch t-Test was applied. Significance level was alpha = 0.05, one-sided smaller.
Statistical evaluations were performed using the ToxRat Pro software Version 2.10 (ToxRat Solutions GmbH, 2010).

First Test Run (DT1)

Emergence and Survival

Number of seeds introduced, number of emerged seeds, and number of survived seedlings in the untreated control pots.

 Plant species

 Seeds [No.]1

 Day 0 [d]2

 Emerged [No.]3

 Survived [No.]4

 Emerged [%]

 Survived [%]

 A. cepa

 24

 6

 19

 19

 79.2

 100

 A. sativa

 24

 4

 22

 22

 91.7

 100

 B. napus

 21

 4

 19

 19

 90.5

 100

 C. sativus

 21

 4

 21

 21

 100

 100

 P. sativum

 21

 4

 15

 14

 71.4

 93.3

 S. lycopersicum

 21

 4

 19

 19

 90.5

 100

1) Total number of seeds introduced; 2) day 0 of the test given as days after sowing; 3) emerged until day 7 (plants which emerged after day 7 were not considered); 4) survived until test end.

Shoot Fresh Weight

Shoot fresh weight was statistically significantly reduced at test item concentrations of ≥ 4 mg/kg soil dw (P. sativum), at ≥ 12.6 mg/kg soil dw (A. sativa, A. cepa), at ≥ 40.0 mg/kg soil dw (S. lycopersicum) and at ≥ 1000 mg/kg soil dw (B. napus, C. sativus).

Mean shoot fresh weight [g] at the end of the test.

 Treatment code Test item  C  T1  T2  T3  T4  T5  T6  T7  T8
 [mg/kg soil dw]  0.0  0.40  1.26  4.0  12.6  40  126  400  1000
 Allium cepa                            
 Mean [g]  0.320  0.288  0.316  0.274  0.199*  0.171*   --   --   --
 ± SD  0.037  0.099  0.051  0.045  0.042  0.023   --   --   --
 Reduction [%]  n.a.  10.1  1.6  14.7  37.9  46.5   --   --   --
 Avena sativa                            
 Mean [g]  1.516  --  --  --  1.363*  1.364*  1.199*  0.961*  0.900*
 ± SD  0.085  --  --  --  0.099  0.201  0.093  0.122  0.048
  Reduction [%]  n.a.  --  --  --  10.1  10.0  20.9  36.6  40.6
 Brassica napus                            
 Mean [g]  2.840  --  --  --  2.839  3.518  2.916  2.074  1.485*
 ± SD  0.744  --  --  --  0.462  1.037  0.531  0.251  0.459
 Reduction [%]  n.a.  --  --  --  0.1  -23.8  -2.7  27.0  47.7
 Cucumis sativus                            
 Mean [g]  5.453  --  --  --  5.508  6.330  5.597  4.801  3.432*
 ± SD  0.497  --  --  --  0.632  1.525  1.038  0.706  0.504
 Reduction [%]  n.a.  --  --  --  -1.0  -16.1  -2.6  12.0  37.1
 Pisum sativum                          
 Mean [g]  2.819  --  --  2.318*  2.229*  2.079*  1.623*  0.951*  --
 ± SD  0.435  --  --  0.540  0.356  0.327  0.541  0.102  --
 Reduction [%]  n.a.  --  --  17.8  21.0  26.2  42.4  66.3  --
 S. lycopersicum                            
 Mean [g]  2.268  --  --  2.171  2.022  1.898*  1.096*  0.486*  --
 ± SD  0.422  --  --  0.508  0.273  0.338  0.116  0.165  --
 Reduction [%]  n.a.  --  --  4.2  10.8  16.3  51.7  78.7  --

* Statistically significantly different from control (Williams test / Welch t-test, p≤0.05, one-sided smaller); (--) = concentration not tested; n.a. = not applicable; negative reduction = increase.

Second Test Run (DT2)

Emergence and Survival

Emergence in the control was at least 81.0%. Survival of seedlings in the controls was 100%. Hence, the requirements defined by the test guideline regarding emergence (at least 70%) and survival (at least 90 %) of seedlings were fulfilled.

Number of seeds introduced, number of emerged seeds, and number of survived seedlings in the untreated control pots.

 Plant species

 Seeds [No.] 1

 Day 0 [d] 2

 Emerged [No.] 3

 Survived [No.] 4

 Emerged [%]

 Survived [%]

 A. sativa

 24

 4

 24

 24

 100

 100

 P. sativum

 21

 6

 17

 17

 81

 100

1) Total number of seeds introduced; 2) day 0 of the test given as days after sowing; 3) emerged until day 7 (plants which emerged after day 7 were not considered); 4) survived until test end.

Shoot Fresh Weight

Shoot fresh weight of each live seedling was determined at the end of the test. Shoot weight of dead seedlings as well as seedlings which had emerged after day 7 were not considered. Shoot fresh weight was statistically significantly reduced at 316 mg/kg soil dw (P. sativum). No statistically significant effect on shoot fresh weight was observed in A. sativa at test item concentrations of up to and including 316 mg/kg soil dry weight.

Mean shoot fresh weight [g] at the end of the DT2 test.

 Treatment code, test item  C  T1  T2  T3  T4  T5  T6
 [mg/kg soil dw]  0.0  1.0  3.16  10.0  31.6  100  316
 Avena sativa                     
 Mean [g]  1.197  1.211  1.254  1.245  1.195  1.175  1.138
 ± SD  0.099  0.045  0.145  0.199  0.082  0.225  0.366
 Mean [% of C]  100  101  105  104  100  98.2  95.1
 Pisum sativum                     
 Mean [g]  2.994  3.253  3.342  3.374  2.861  3.042  1.862*
 ± SD  0.482  0.562  0.548  0.275  0.483  0.416  0.256
 Mean [% of C]  100  109  112  113  95.5  102  62.2

* Statistically significantly different from control (Williams test, p≤0.05, one-sided smaller).

Validity criteria fulfilled:
yes
Remarks:
Both tests, DT1 and DT2 fulfilled the validity criteria of the test guideline, as seedling emergence and seedling survival in the control was above the required 70% and 90% minimum, respectively. No phytotoxicity was observed in the controls.
Conclusions:
The potential effects of VULKACIT NZ/TBBS on emergence and growth of higher terrestrial plants were assessed under laboratory conditions.
The test item had no adverse effect on either seedling emergence or seedling survival. It induced, however, adverse effects on shoot fresh weight at concentrations of ≥4.0 mg/kg soil dry weight (= LOEC).
The lowest NOEC of 3.16 mg/kg soil dry weight was estimated by a conservative evaluation of the diverging results of two consecutive test runs with Pisum sativum.
Executive summary:

Two tests were run in this study, DT1 and DT2. The latter was performed with two test species only, A. sativa and P. sativum, and with different test item concentrations because a NOEC could not be determined in DT1. Seeds of two monocotyledonous species, Avena sativa (oat) and Allium cepa (onion), as well as four dicotyledonous species, Brassica napus (rape), Cucumis sativus (cucumber), Pisum sativum (pea) and Solanum lycopersicum (tomato) were planted in a natural sandy loam soil (standard soil LUFA Sp 2.3) immediately after test item application and left to grow under controlled conditions for 14(16) days following 50 % emergence of the control plants. Five test item concentrations were tested with each species. Rates in DT1 ranged from either 0.4 to 40 mg/kg soil dry weight, or 4.0 to 400 mg/kg soil dry weight or 12.6 to 1000 mg/kg soil dry weight. In test run DT2 the test concentration ranged from 1.0 to 316 mg/kg soil dry weight for both test species.

Emergence and Survival

Neither emergence nor survival of the seedlings was statistically significantly affected in any of the species at test item concentrations up to and including 40 mg/kg soil dry weight (A. cepa), 400 mg/kg soil dw (P.

sativum; S. lycopersicum), and 1000 mg/kg soil dw (A. sativa, B. napus, C. sativus), which were the highest tested concentration, respectively.

Visual appearance

Plants growing in the highest applied treatment of each species, respectively, revealed some slight damages such as chlorosis and necrosis which always comprised less than 25% per single plant. A. cepa and C.

sativus did not exhibit any visible test item related damages.

Shoot fresh weight

Shoot fresh weight in test run DT1 was statistically significantly reduced at test item concentrations of ≥ 4 mg/kg soil dw (P. sativum), at ≥ 12.6 mg/kg soil dw (A. sativa, A. cepa), at ≥ 40.0 mg/kg soil dw (S. lycopersicum) and at 1000 mg/kg soil dw (B. napus and C. sativus). As no clear NOEC could be determined in P. sativum and A. sativa, the test was repeated (DT2) for these two species at lower concentrations.

In DT2 shoot fresh weight of P. sativum was statistically significantly reduced at 316 mg/kg soil dw which was the highest tested concentration in DT2, while in A. sativa no adverse effects were seen at up to and including 316 mg/kg soil dw.

Conclusion:

The potential effects of VULKACIT NZ/TBBS on emergence and growth of higher terrestrial plants were assessed under laboratory conditions.

The test item had no adverse effect on either seedling emergence or seedling survival. It induced, however, adverse effects on shoot fresh weight at concentrations of ≥4.0 mg/kg soil dry weight (= LOEC).

The lowest NOEC of 3.16 mg/kg soil dry weight was estimated by a conservative evaluation of the diverging results of two consecutive test runs with Pisum sativum.

Description of key information

The test item had no adverse effect on either seedling emergence or seedling survival. It induced however adverse effects on shoot fresh weight of higher terrestrial plants at concentrations of ≥4.0 mg/kg soil dry weight (= LOEC). The lowest NOEC of 3.16 mg/kg soil dry weight was estimated by a conservative evaluation of the diverging results of two test runs with Pisum sativum.

Key value for chemical safety assessment

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

Additional information

While neither seedling emergence nor seedling survival in test item treated differed significantly from emergence or survival in the control, soil shoot fresh weight was clearly

affected. At test item concentrations of ≥4.0 mg/kg soil dry weight shoot fresh weight was significantly reduced by 17.8% in the test species Pisum sativum (pea) which with regard to the LOEC turned out to be the most sensitive among the six tested species in test run DT1.

Allium cepa (onion) and the monocotyledonous Avena sativa (oats) revealed reduced shoot weight at concentrations of 12.6 mg/kg soil dry weight and above.

The lowest EC10 and EC50 of 1.7 mg/kg soil dw and 41.8 mg/kg soil dw, respectively, was found with A. cepa.

Surprisingly, in the repeated test (DT2) with A. sativa and P. sativum adverse effects on shoot fresh weight occurred only in P. sativum and at the highest tested concentration (316 mg/kg soil dry weight) although seeds, soil and overall growth conditions were the same in both test runs, DT1 and DT2. Due to the diverging results in the DT1 and DT2 test with Pisum sativum there remains some uncertainty regarding the test item concentration that causes an adverse effect on shoot fresh weight. A conservative evaluation would be to consider both, the LOEC from DT1 (4.0 mg/kg soil dw) and the results from DT2. Consequently the overall NOEC would be 3.16 mg/kg soil dw. Visual damages of P sativum seedlings were seen at ≥400 mg/kg soil in DT1 and at ≥100 mg/kg soil in DT2.