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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:
25 March 2015 to 17 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study According to GLP
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Remarks:
See "details on results" for a list of the deviations. None were considered to have affected the integrity of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Two samples of stock solution (equivalent to the highest treatment solution) were taken immediately prior to application to soil, and stored frozen until chemical analysis. One of the duplicate samples was shipped under frozen conditions to the Principal Investigator for chemical analysis.
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The test item was dissolved in ethanol, spiked onto quartz sand and, after evaporation of the solvent, mixed thoroughly into the soil.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Concentration of vehicle in test medium (stock solution and final solutions): Stock solution = 204 mg/mL. Aliquots were diluted with ethanol to achieve the test concentrations of 2.6 (treatment T1), 9.51 (T2), 28.5 (T3) , 85.5 (T4) and 128 (T5) mg/mL. For treatment T6, the stock solution was used directly.
- Evaporation of vehicle before use: Yes
- Application details: A test solution volume of 20, 60 and 80 mL was homogeneously spiked on 31.5, 122.5 and 154 g quartz sand for the treatments T1, T6 and T2 to T5, respectively. After complete evaporation of the solvent, the test-item coated quartz sand was thoroughly mixed with 3.15 (T1) and 15.4 kg soil dry weight (T2 to T6), respectively.
- Controls: The control was untreated soil, while the solvent control was soil (15.4 kg dry weight) mixed analogously to the treatments with 154 g of ethanol spiked quartz sand without test item.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion
- Plant family: Amaryllidaceae
- Variety: Stuttgarter Riesen
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim Germany
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oats
- Plant family: Poaceae
- Variety: Champion
- Source of seed: Saaten Union GmbH, Hannover, Germany
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: oilseed rape
- Plant family: Brassicaceae
- Variety: Liforum
- Source of seed: Landwirtschaftliche, Untersuchungs- und Forschungsanstalt (LUFA), Speyer, Germany
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: cucumber
- Plant family: Cucurbitaceae
- Variety: Delicatess
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim Germany
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: peas
- Plant family: Fabaceae
- Variety: Rondo
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim Germany
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Species: Solanum lycopersicum (formerly specified as Lycopersicon esculentum)
- Common name: tomato
- Plant family: Solanaceae
- Variety: Hellfrucht
- Source of seed: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim Germany
Test type:
other: seedling emergence and early seedling growth
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Remarks:
The test item was mixed into the soil via ethanol spiked on quartz sand immediately before the seeds of the test species were planted.
Post exposure observation period:
The number of seedlings in the controls was checked daily until the day on which at least 50% of the plants had emerged. This day was defined as the day 0 of the study for each species. Plants were allowed to emerge and grow for 14 days following 50% emergence of the control plants.
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH
- Test container (type, material, size): Common plant pots made of polypropylene (diameter 10 cm, height 8 cm, and diameter 11 cm, height 8 cm, respectively)
- Amount of soil: Each pot was filled with 377 ± 10 g soil fresh weight and 485 ± 10 g soil fresh weight, respectively.
- Method of seeding: The seeds were randomly picked with a pair of tweezers and pressed into the soil at a uniform distance between seeds. The seeds had neither been dressed with fungicides nor imbibed with water.
Allium Cepa and Avena sativa
- No. of seeds per container: 6
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4
Brassica napus, Cucumis sativus, Solanum lycopersicum, Pisum sativum
- No. of seeds per container: 3
- No. of replicates per treatment group: 7
- No. of replicates per control: 7
- No. of replicates per vehicle control: 7

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Standard soil LUFA Type 2.3, a sandy loam, was purchased from the Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), D-67346 Speyer, Germany. Information on the soil properties was provided by the supplier of the soil. LUFA soil Type 2.3 was collected from an uncultivated field at Offenbach, Rhineland-Palatine, Germany, and sieved (<=2 mm).
- Pesticide use history at the collection site: The field had neither received any organic fertilisers nor plant protection substances during the last four years before sampling.
- Soil texture (if natural soil): sandy loam
- Particle size distribution [%]: < 0.002 mm [6.3% ± 1.9]; 0.002 – 0.05 mm [33.8% ± 0.2]; 0.05 – 2.0 mm [59.9% ± 1.9]
- Organic carbon [%]: 0.67% ± 0.04
- Nitrogen [%]: 0.08% ± 0.01
- pH (0.01 M CaCl2): 5.8 ± 0.7
- Cation exchange capacity [mval/100 g]: 7.3 ± 1.0
- Water holding capacity [%]: 35.6 ± 1.7
- Storage (condition, duration): at ambient temperature until usage.

NUTRIENT MEDIUM (if used)
- Description: The nutrient solution was prepared by diluting a commercially available liquid fertilizer, Substral® (Scotts Celaflor GmbH, Mainz, Germany) with deionised water (6.0 mL/L water).

GROWTH CONDITIONS
- Photoperiod:16 hours per day
- Light source: artificial lighting
- Light intensity and quality: 206 - 360 μE m-2 s-1
- Day/night temperatures: air temperature ranging from 16.0 – 27.5°C
- Relative humidity (%): 22.0 - 54.5%
- Watering regime and schedules: Soils were supplied with water or nutrient solution by bottom watering. Each test container (common plant pot) was placed in a separate polystyrene beaker serving as water reservoir.
- Water source/type: deionised water
- Volume applied: Deionised water was added to the reservoirs as necessary to maintain the soil sufficiently moist.
- Any pest control method/fertilization (if used): Seedlings were fertilised twice during the test by adding volumes of 50 mL, each, of a nutrient solution to the reservoirs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Measurement endpoints were emergence, survival, shoot length, shoot fresh weight, and visual detrimental effects (mortality, chlorosis, 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.
- Emergence was defined as the number of seedling emerged on day 7 (A. cepa, P. sativum and S. lycopersicum) or day 8 (A. sativa, B. napus and C. sativus).
- Visual detrimental effects were assessed weekly.
- Shoot length and fresh weight were evaluated at the end of the test (day 14).

VEHICLE CONTROL PERFORMED: yes
- the solvent control was mixed with the same amount of ethanol spiked quartz sand without test item.

TEST CONCENTRATIONS
- 16.5, 49.4, 148, 444, 667 and 796 mg test item/kg soil dry weight
- Range finding study
- Test concentrations: 0.10, 1.00, 10.0, 100 and 1000 mg/kg soil dry weight.
- Results used to determine the conditions for the definitive study - see attachment
Nominal and measured concentrations:
Nominal concentrations:
16.5, 49.4, 148, 444, and 667 mg test item/kg soil dry weight for Solanum lycopersicum
49.4, 148, 444, 667 and 796 mg test item/kg soil dry weight for all other species
control and solvent control
Reference substance (positive control):
no
Remarks:
not required
Species:
Allium cepa
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Allium cepa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Avena sativa
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Brassica napus
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
148 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
444 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
254 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% CL: not determined
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
other: EC20
Effect conc.:
749 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% CL: not determined
Species:
Cucumis sativus
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Pisum sativum
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil ww
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Pisum sativum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Pisum sativum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Pisum sativum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 796 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Lycopersicon esculentum
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 667 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
seedling emergence
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 667 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
other: survival
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
94.8 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Remarks on result:
other: 95% CL: 11.8-176
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
other: EC20
Effect conc.:
191 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Remarks on result:
other: 95% CL: 57.7-290
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
148 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
49.4 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
shoot length
Key result
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
70.5 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% CL: 2.4-144
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
other: LC20
Effect conc.:
112 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% CL: 10.3-199
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
272 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% CL: 122-482
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
148 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Species:
Lycopersicon esculentum
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
49.4 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
growth
Remarks:
fresh weight
Details on results:
ANALYTICAL VERIFICATION OF THE TEST SOLUTION
The contents of Benzyl Salicylate in the test solution used to spike the quartz sand for the highest test concentration (nominal 204 mg/mL) was verified by chemical analysis (analysed in threefold, results = 205.8, 203.2 and 202.2 mg/L). The mean measured concentration was 99.8% of nominal.

SEEDLING EMERGENCE AND SURVIVAL
- The day on which at least 50% of the seedlings had emerged in the untreated control of the respective plant species was defined as day 0 (starting point) of the test.
- The number of live seedlings was determined on day 7 (A. cepa, P. sativum and S.lycopersicum) or day 8 (A. sativa, B. napus and C. sativus) and at the end of the test.
- Emergence of seedlings in the controls was ≥ 86% for all six test species.
- Survival of seedlings in the controls was always 100% in all species.
- No effect on seedling emergence on day 7 or 8 was observed at ≤667 mg/kg in S. lycopersicum and at ≤796 mg/kg soil in B. napus, C. sativus, A. cepa, A. sativa and P.sativum.
- Survival of the seedlings was not significantly affected in any of the species by any of the treatments.

VISUAL APPEARANCE
Seedlings were evaluated visually on day 7 or 8 and test end. Typical damages were reduced growth and death of seedlings which was observed at higher test item concentrations in S. lycopersicum. Plants exhibited stunted growth and curly leaves at test item concentrations of ≥444 mg/kg soil dry weight, while these effects seemed to become smaller from day 7 to test end.

SEEDLING SHOOT LENGTH AND SHOOT FRESH WEIGHT
- Shoot length and shoot fresh weight of each live seedling was determined at the end of the test.
- Shoot length was significantly reduced at test item concentrations of ≥148 mg/kg soil in S.lycopersicum. No effects on shoot length were observed in A. cepa, A. sativa, B. napus, C.sativus and P. sativum at concentrations up to and including 796 mg/kg soil dry weight.
- Shoot fresh weight was significantly reduced at ≥49.4 mg/kg soil dry weight in S.lycopersicum and at ≥444 mg/kg soil in B. napus. No effects on shoot fresh weight were observed in A. cepa, A. sativa, C. sativus and P. sativum at concentrations up to and including 796 mg/kg soil dry weight.

DEVIATIONS
- The highest test concentration (T6) was 796 mg/kg soil instead of 1000 mg/kg soil due to an error in the amount of soil used for this treatment.
- In T6 of C. sativus, 18 instead of 21 seeds had been sown.
- Visual inspection of A. sativa, P. sativum and S. lycopersicum was done on day 8 instead of day 7.
- During the study, the relative air humidity sometimes was below the lower limit of 45% (measured minimum 22%) given by the guideline. Although the values for relative air humidity in the growth chamber were partly below the range recommended by the guideline for greenhouses, the environmental conditions in the growth chamber were appropriate as confirmed by normal growth of the plants in the controls.

It is considered that the deviations had no impact on the conduct or integrity of the study.
Validity criteria fulfilled:
yes
Remarks:
More than 70% of the controls emerged, less than 10% of the control plants died and no phytotoxic effects were observed in the controls.
Conclusions:
No effect on seedling emergence and survival was observed at ≤667 mg/kg in S. lycopersicum and at ≤796 mg/kg soil in B. napus, C. sativus, A. cepa, A. sativa and P. sativum.

Shoot length was significantly reduced at test item concentrations of ≥148 mg/kg soil in S.lycopersicum while no effects were observed up to and including 796 mg/kg soil in B. napus,C. sativus A. cepa, A. sativa and P. sativum.

Shoot fresh weight was significantly reduced at ≥148 mg/kg soil in S. lycopersicum and at ≥444 mg/kg soil in B. napus.

The lowest LOEC (148 mg/kg soil dry weight) and NOEC (49.4 mg/kg soil dry weight) were observed for shoot length and shoot fresh weight in S. lycopersicum.

The lowest EC10 (70.5 mg/kg soil dry weight) and EC50 (272 mg/kg soil dry weight) were obtained for shoot fresh weight in S. lycopersicum.

Description of key information

The effects of Benzyl Salicylate on the seedling emergence and early growth of higher terrestrial plants has been determined. The lowest EC10 was 70.5 mg/kg soil dry weight.

Key value for chemical safety assessment

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

Additional information

The seedling emergence and seedling growth test (OECD 208) covers a sensitive stage in the life-cycle of a plant and therefore data obtained from this study can be used as estimates of long-term toxicity (ECHA Chapter R.7c, section R.7.11.3.1). For long-term toxicity testing, ECHA considers six species as the minimum with two monocotyledonous species and four dicotyledonous species.


 


The effect of Benzyl Salicylate was tested with six test species representing six plant families. Test species comprised two monocotyledonous, Avena sativa (oats) and Allium cepa (onion), and four dicotyledonous species, Brassica napus (oilseed rape), Cucumis sativus (cucumber), Pisum sativum (peas) and Solanum lycopersicum (formerly specified as Lycopersicon esculentum, tomato).


The testing was performed in a Speyer 2.3 soil with an Organic Carbon content of 0.67%.


No effect on seedling emergence and survival was observed at ≤667 mg/kg in S. lycopersicum and at ≤796 mg/kg soil in B. napus, C. sativus, A. cepa, A. sativa and P. sativum.


 


Shoot length was significantly reduced at test item concentrations of ≥148 mg/kg soil in S.lycopersicum while no effects were observed up to and including 796 mg/kg soil in B. napus, C. sativus, A. cepa, A. sativa and P. sativum.


 


Shoot fresh weight was significantly reduced at ≥148 mg/kg soil in S. lycopersicum and at ≥444 mg/kg soil in B. napus.


 


The lowest LOEC (148 mg/kg soil dry weight) and NOEC (49.4 mg/kg soil dry weight) were observed for shoot length and shoot fresh weight in S. lycopersicum. The lowest EC10 (70.5 mg/kg soil dry weight) and EC50 (272 mg/kg soil dry weight) were obtained for shoot fresh weight in S. lycopersicum.


 


The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC depends on the experiment design (e.g. the concentrations used in the test). Therefore the lowest EC10 of 70.5 mg/kg soil dry weight has been chosen as the key value for chemical safety assessment.