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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:
08 Dec 2016 - 17 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Version / remarks:
July 2006
Deviations:
yes
Remarks:
See detailes in section "Any other information on material and methods incl tables"
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: Test item were weighed out for each treatment separately and ground with fine quartz sand and afterwards mixed into the test soil; concentration of sand added to the soil was 1% (i.e. 10 g/kg soil dw).
- Controls: Control soil received sand which has not been treated with the test item.
- Others: Application rate in detail is shown in Tables 3-5 in section "Any other information on material and methods incl tables".
Species:
other: Solanum lycopersicum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Solanaceae
- Lot number: 4317738
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
- Cultivar: Hellfrucht
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Pea
- Plant family: Fabaceae
- Lot number: 117024NU
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
- Cultivar: Rondo
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Cucumber
- Plant family: Cucurbitaceae
- Lot number: 4291508
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany
- Cultivar: Delikatess
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: rape
- Plant family: Brassicaceae
- Lot number: n.a.
- Source of seed: Feldsaaten Freudenberger, Krefeld, Germany
- Cultivar: Akela
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Onion
- Plant family: Amaryllidaceae
- Lot number: 4333216
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany;
- Cultivar: Stuttgarter Riesen
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oat
- Plant family: Poaceae
- Lot number: n.a.
- Source of seed: Nordsaat Saatzucht GmbH, Langenstein, Germany
- Cultivar: Canyon
- Seed storage: under cool and dry conditions in the dark
- Pretreatment of seeds: seeds had neither been dressed with fungicides nor imbibed with water
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
21 d
Remarks:
Plants were left to grow under controlled conditions for a period of 14 to 21 days following seedling emergence of at least 50% in the control.
Test temperature:
mean air temperature ranging from 17.1 to 26.4 °C

pH:
5.9 ± 0.6 (soil, 0.01 M CaCl2)
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT GmbH
- Test container (type, material, size): polypropylene pots (diameter 11 cm, height 8.5 cm), placed in a separate polystyrene beaker (Kastelplast GmbH, Mainz, Germany) serving as water reservoir
- Amount of soil: 468 ± 10 g soil fresh weight
- Method of seeding: seeds were randomly picked with a pair of tweezers and pressed into the soil at a uniform distance between seeds
- No. of seeds per container: 6 (for monocotyledonous) and 3 (for dicotyledonous)
- No. of replicates per treatment group: 4 (for monocotyledonous) and 7 (for dicotyledonous)
- No. of replicates per control: at least 4 (for monocotyledonous) and 7 (for dicotyledonous)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: LUFA Soil Type 2.3. (Batch F 2.3 4316) was collected from an uncultivated field at Offenbach, Rhineland-Palatinate, Germany on 24 Oct 2016 (information on soil properties was provided by the Landwirtschaftliche Untersuchungs-und Forschungsanstalt (LUFA) Speyer, Germany)
- Pesticide use history at the collection site: field had neither received any organic fertilisers nor plant protection substances during the last four years before sampling
Soil texture (if natural soil)
- % sand (0.05 – 2.0 mm): 59.6 ± 1.4
- % silt (0.002 – 0.05 mm): 33.6 ± 0.5
- % clay (< 0.002 mm): 6.8 ± 1.6
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- Nitrogen (%): 0.08 ± 0.01
- Organic carbon (%): 0.67 ± 0.03
- Maximum water holding capacity (in % dry weigth): 35.6 ± 1.4
- CEC: 7.6 ± 0.8 meq/100 g
- Pretreatment of soil: soil was sieved (≤2 mm)
- Storage (condition, duration): Soil was stored at ECT GmbH at ambient temperature until usage.

NUTRIENT MEDIUM (if used)
- Description: nutrient solution (“Substral®”, 6 ml/L H2O) was added several times during the trial

GROWTH CONDITIONS
- Photoperiod: 16 h
- Light source: artificial lighting (SON-T-Agro high pressure metal halide lamps, Philips GmbH, Hamburg, Germany)
- Light intensity [μE m-2 s-1]: See Table 2 in section "Any other information on material and methods incl tables".
- Day/night temperatures: See Table 2 in section "Any other information on material and methods incl tables".
- Relative humidity (%): See Table 2 in section "Any other information on material and methods incl tables".
- Watering regime and schedules: See Table 1 in section "Any other information on material and methods incl tables".
- Volume applied: Deionised water was added to the reservoirs as necessary to maintain the soil sufficiently moist.
- Any pest control method/fertilization (if used): See Table 1 in section "Any other information on material and methods incl tables".

EFFECT PARAMETERS MEASURED: emergence rate, survival of seedlings, shoot fresh weight, and visual detrimental effects (chlorosis, necrosis, development abnormalities). Day 0 of the test was defined as the day by which at least 50% of the seedlings in the untreated control pots of a particular species had emerged. Visual detrimental effects were assessed on day 7, day 14, and at the end of the test, i.e. earliest on day 19 (day 15 for A. sativa), latest on day 21. Shoot fresh weight was evaluated only at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
Range finding study (non-GLP)
- Test concentrations: between 1.0 and 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP range-finding test the main test was designed (results are summarized in table 1 in section "Any other information on results incl tables".
Nominal and measured concentrations:
Control, 10, 31.6, 100, 316 and 1000 mg test item /kg soil dw
Reference substance (positive control):
no
Species:
other: all six species
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:
other: emergence, survival
Species:
other: all species except S.lycopersicum
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:
other: shoot fresh weight
Species:
other: S.lycopersicum
Duration:
21 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
Species:
other: S.lycopersicum
Duration:
21 d
Dose descriptor:
EC25
Effect conc.:
745 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
other: S.lycopersicum
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
78.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
other: all six species
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival, shoot fresh weight
Remarks on result:
other: ECX assumed to be higher than 1000 mg/kg dw
Species:
other: all six species
Duration:
21 d
Dose descriptor:
EC25
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and shoot fresh weight (except S.lycopersicum)
Remarks on result:
other: ECX assumed to be higher than 1000 mg/kg dw
Species:
other: all six species
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: emergence, survival and shoot fresh weight (except S.lycopersicum)
Remarks on result:
other: ECX assumed to be higher than 1000 mg/kg dw
Details on results:
SEEDLING EMERGENCE
- Percent seedling emergence (untreated control pot):
75.0 % (A.cepa)
91.7 % (A. sativa)
95.2 % (B. napus)
100 % (C.sativus)
81.0 % (P. sativum)
85.7 % (S.lycopersicum)

- Percent survival (untreated control pot):
100 % (A.cepa)
100 % (A. sativa)
90.0 % (B. napus)
100 % (C.sativus)
94.1 % (P. sativum)
100 % (S.lycopersicum)

-Visual appearance: Plants growing in treated soil did not differ in their visual appearance from plants growing in non-treated control soil and did not exhibit any visible test item related damages.

- Others: See section" Any other information on results incl tables for further biological results"



Results of non-GLP range-finding Test

Table 1: Shoot fresh weight of seedlings grown in test item treated soil as determined on day 14 of the range-finding test; data given as percentage of the control.

Species

Test concentration [mg/kg soil]

1

10

100

1000

Shoot fresh weight [% of control]

A. cepa

87.6

168.5

146.1

98.9

A. sativa

100

101.0

85.0

108.3

B. napus

113.1

76.9

99.5

97.9

C. sativus

114.9

119.7

125.4

107.4

P. sativum

59.5

80.1

71.1

56.9

S. lycopersicum

82.7

85.5

79.9

97.1

 

Results of main test:

The test with A. sativa was repeated, since the validity criteria had not been met in first run. Only the results of the repeated test with A. sativa are reported. The tests with the species A. cepa, P. sativum and S. lycopersicum (only results of 2nd run are shown here) were both valid considering the validity criteria given by the test guideline. Consequently the results of both test runs were evaluated separately and are reported.

 

Seedling Emergence and Survival

Emergence in the control was at least 75%. Survival of seedlings in the controls was at least 90%. Hence, the requirements defined by the test guideline regarding emergence and survival of seedlings were fulfilled. Neither emergence nor survival of the seedlings was statistically significantly affected in any of the species at test item concentrations up to and including 1000 mg/kg soil dry weight which was the highest tested concentration.

Seedling Shoot Fresh Weight

Shoot fresh weight of plants growing in test item treated soil was not statistically significantly different from shoot weight of the plants growing in untreated control soil for any of the six species at concentrations up to and including 1000 mg/kg soil dry weight. In the repeated test, however, shoot fresh weight of S. lycopersicum was statistically significantly reduced by 20% and 27% at test item concentrations of 316 and 1000 mg/kg soil dw, respectively.

Table 1: Seedling emergence (emergence after day 7 not considered)

Treatment code

C

T1

T2

T3

T4

T5

Test item [mg/kg soil dw]

0.0

10

31.6

100

316

1000

 

Number of seeds planted

A. cepa (2nd run)

24

24

24

24

24

24

A. sativa

24

24

24

24

24

24

B. napus

21

21

21

21

21

21

C. sativus

21

21

21

21

21

21

P. sativum (2nd run)

21

21

21

21

21

21

S. lycopersicum (2nd run)

21

21

21

21

21

21

 

Number of emerged seeds per treatment on day 7

A. cepa (2nd run)

21

20

19

17

21

18

A. sativa

22

22

23

22

23

23

B. napus

20

21

20

19

19

20

C. sativus

21

20

20

20

20

20

P. sativum (2nd run)

20

19

19

18

20

17

S. lycopersicum (2nd run)

19

21

20

21

20

21

 

Relative number of emerged seeds [% of seeds sown]

A. cepa (2nd run)

87.5

83.3

79.2

70.8

87.5

75.0

A. sativa

91.7

91.7

95.8

91.7

95.8

95.8

B. napus

95.2

100

95.2

90.5

90.5

95.2

C. sativus

100

95.2

95.2

95.2

95.2

95.2

P. sativum (2nd run)

95.2

90.5

90.5

85.7

95.2

81.0

S. lycopersicum (2nd run)

90.5

100

95.2

100

95.2

100

 

Mean % emergence as % of mean emergence of the control

A. cepa (2nd run)

100

95.2

90.5

80.9

100

85.7

A. sativa

100

100

104

100

104

104

B. napus

100

105

100

95.1

95.1

100

C. sativus

100

95.2

95.2

95.2

95.2

95

P. sativum (2nd run)

100

95.1

95.1

90.0

100

85.1

S. lycopersicum (2nd run)

100

111

105

111

105

111

*Accidentally no seeds sowed in one of the pots of this treatment.

Table 2: Seedling survival (emergence after day 7 not considered)

Treatment code

C

T1

T2

T3

T4

T5

Test item [mg/kg soil dw]

0.0

10

31.6

100

316

1000

 

Number of emerged seeds per treatment on day 7

A. cepa (2ndrun)

21

20

20

17

21

18

A. sativa

22

22

23

22

23

23

B. napus

20

21

20

19

19

20

C. sativus

21

20

20

20

20

20

P. sativum (2ndrun)

20

19

19

18

20

17

S. lycopersicum (2ndrun)

19

21

20

21

20

21

 

Number of live seedlings at test end

A. cepa (2ndrun)

21

19

18

17

20

18

A. sativa

22

22

23

22

23

23

B. napus

18

20

20

18

18

20

C. sativus

21

20

20

19

20

20

P. sativum (2ndrun)

20

19

19

18

20

17

S. lycopersicum (2ndrun)

19

21

20

21

20

20

 

Relative number of live seedlings at test end [% of emerged]

A. cepa (2ndrun)

100

95.0

90.0

100

95.2

100

A. sativa

100

100

100

100

100

100

B. napus

90.0

95.2

100

94.7

94.7

100

C. sativus

100

100

100

95.0

100

100

P. sativum (2ndrun)

100

100

100

100

100

100

S. lycopersicum (2ndrun)

100

100

100

100

100

95.2

 

Mean % survival as % of mean survival of the control

A. cepa (2ndrun)

100

95.0

90.0

100

95.2

100

A. sativa

100

100

100

100

100

100

B. napus

100

106

111

105

105

111

C. sativus

100

100

100

95

100

100

P. sativum (2ndrun)

100

100

100

100

100

100

S. lycopersicum (2ndrun)

100

100

100

100

100

95.2

Validity criteria fulfilled:
yes

Description of key information

EC10 (21 d): 78.5 mg/kg soil (Solanum lycopersicum, shoot fresh weight, OECD 208, nominal)

Key value for chemical safety assessment

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

Additional information

One experimental study is available investigating the potential effects of the test item (CAS 16260-09-6) on seedling emergence and growth (ECT, 2017) of plants. The studies were performed according to OECD guideline 208 under GLP conditions. For this study six species of non-target terrestrial plants (2 monocots and 4 dicots) were planted in a natural sandy loam soil (standard soil LUFA Sp 2.3) immediately after test item application. Five test item concentrations were tested with each species: 10, 31.6, 100, 316 and 1000 mg test item /kg soil dw. Plants were left to grow under controlled conditions for a period of 14 to 21 days following seedling emergence of at least 50% in the control. Test item concentrations were applied within artificial 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 (201 to 352 μE m-2 s-1, for 16 hours per day) and air conditioning (mean air temperature ranging from 17.1 to 26.4 °C, relative humidity ranging from 17.0% to 58.1%). The pots were randomly placed at the beginning of the test and were re-arranged at minimum weekly intervals. On day 7 and day 14 (or day 17 for A. cepa) after 50% emergence of control, seedlings were evaluated visually. At the end of the test, seedlings were counted, evaluated visually, and harvested to determine shoot fresh weight. At concentrations up to and including 1000 mg/kg soil dry weight the test item had no statistically significant adverse effect on either seedling emergence, seedling survival or shoot fresh weight of any of the six test species. Shoot fresh weight of the test species Solanum lycopersicum in the second test run was reduced at concentrations of ≥ 100 mg/kg soil dry weight (P<0.05). Therefore the lowest LOEC (lowest observed effect concentration) and NOEC (no observed effect concentration) were determined to be 316 mg/kg soil dw and 100 mg/kg soil dw, respectively. Due to the lack of either adverse effects or a concentration response relationship effective concentrations could not be calculated and are assumed to be higher than 1000 mg/kg dw except for shoot fresh weight of S. lycopersicum (second test run). For S. lycopersicum the EC10 and EC25 were calculated to be 78.5 mg/kg soil dry weight and 745 mg/kg soil dry weight, respectively, while the EC50 was >1000 mg/kg soil dry weight under the conditions of this test.