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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:
19 July, 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
DOSE LEVELS AND REPLICATION:
Six test item concentrations (10.2, 25.6, 64, 160, 400 and 1000) were tested against untreated soil with the species Allium cepa, Avena sativa, Brassica napus, Cucumis sativus, Pisum sativum and Solanum lycopersicum. The test concentrations were based on the results of a previously performed non-GLP range-finding test.
There were four replicates (pots) per treatment with 6 seeds per pot for the monocotyledonous and seven replicates (pots) per treatment with 3 seeds per pot for the dicotyledonous species. Hence, the total number of seeds per species and treatment was at least 24 and 21, respectively.
Species:
other: Solanum lycopersicum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Cultivar: Hellfrucht
- Lot No.: 4292523
- Source of supply: Enza Zaden
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Cultivar: Maxigolt
- Lot No.: 4299518
- Source of supply: Enza Zaden
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Cultivar: Fatum
- Lot No.: 4259008
- Source of supply: Enza Zaden
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Cultivar: Akela
- Lot No.: n.a.
- Source of supply: Freudenberger
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Cultivar: Canyon
- Lot No.: n.a.
- Source of supply: Nordsaat
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Cultivar: Stuttgarter Riesen
- Lot No.: 4301200
- Source of supply: Enza Zaden
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
16 d
Test temperature:
19.8-29-3 °C (23.1 °C, mean value)
pH:
pH of soil (0.01 M CaCl2): 5.8 ± 0.6
Moisture:
Rel. air humidity [%]: 29.9-71.0 (41.9 %, mean value) Although relative air humidity mean values are below the recommendations of the OECD Guideline 208 growth of control plants was normal and all pots were watered sufficiently all the time. Hence, a negative effect of low relative air humidity on plants can be excluded.
Details on test conditions:
TEST SOIL:
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-Palatinate, Germany, and sieved (2 mm). The field had neither received any organic fertilisers nor plant protection substances during the last three years before sampling. The soil was stored at ECT GmbH at ambient temperature until usage.

PROPERTIES OF LUFA SOIL TYPE:
- Soil type (USDA): sandy loam
- Sampling date: 17 November 2017
- Organic carbon content [%]: 0.68 ± 0.04
- Nitrogen [%]: 0.08 ± 0.01
- pH (0.01 M CaCl2): 5.8 ± 0.6
- Cation exchange capacity [meq/100 g]: 7.5 ± 0.8
- Particle size distribution [%] (USDA): < 0.002 mm (7.5 ± 0.4); 0.002 – 0.05 mm (33.6 ± 0.5); 0.05 – 2.0 mm (58.9 ± 0.4)
- Water holding capacity [%]: 35.4 ± 1.0
- Weight per volume [g/1000 mL]: 1307 ± 41

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 495 ± 10 g soil fresh weight and placed in a separate polystyrene beaker (Kastelplast GmbH, Mainz, Germany) serving as water reservoir.

APPLICATION OF THE TEST ITEM:
Defined amounts of the ground test item were weighed out for each treatment separately and ground with fine quartz sand. Afterwards, test item and quartz sand were thoroughly mixed for at least 5 minutes into the 16.2 kg (dw) soil by using a mixer in order to homogeneously distribute the test item in the 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 supplemented with the test item.

TEST START AND MAINTENANCE:
After filling of the pots the seeds were sown into the soil. The seeds were 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. Each pot was labelled with the study number, the treatments code, a consecutive number and the plants species.
Pots containing soil and seeds were distributed randomly in the test room and re-arranged at minimum weekly intervals. After placing the pots in the test room, the water reservoirs were filled with deionised water as necessary to moisten the soil sufficiently. Furthermore each pot was covered with a plastic lid to avoid drying up of the soil during germination of the seeds. As soon as plants had emerged lids were taken off.
During the test, deionised water was added to the reservoirs as needed to maintain appropriate soil moisture. Additionally, a nutrient solution (“Substral®”, 7 ml/L H2O) was added as needed judged by the observation of control plants.
The light intensity [μE m-2 s-1] ranged between 211 and 257.

SEEDLING EMERGENCE AND SEEDLING SURVIVAL:
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. Seedlings were also counted on day 7 to determine the emergence rate, and at the end of the study to determine survival of seedlings.
Survival of seedlings was determined by the difference between the number of seedlings emerged until day 7 and the number of live seedlings at the end of the test.
Seedlings which had emerged after day 7 were excluded from the statistical evaluation and further reporting.

VISUAL APPEARANCE:
On day 7, day 14 and at the end of the study, the number of seedlings was counted and evaluated according to a 6-point scoring system with the categories shown below. The six categories were chosen to facilitate and standardise the description of the visual appearance of the plants.
The allocation to a certain category does not necessarily mean that the observed phenomenon is an effect; especially categories 2 and 3 may be the expression of the normal variation of growth and morphology of the plant.

SHOOT FRESH WEIGHT:
At the end of the test, seedlings were clipped at soil surface and shoot fresh weight of each single plant was determined immediately.

STATISTICAL ANALYSIS:
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. Statistical methods were applied to evaluate whether differences between test item treatments and control were significant.
Arithmetic means for each pot were calculated from the single plants within a pot. Since each pot constituted a replicate, the mean values were used for statistical analysis. Plants which had emerged after day 7 were excluded from the calculations.
Emergence and survival data were checked by the Step-down Cochran-Armitage Test Procedure or in case of a non-monotone dose response with the Fisher's exact binominal test with Bonferroni correction to determine the NOEC and LOEC. Significance level was alpha = 0.05, one sided greater. The 3-Parameter normal cumulative distribution function was used to determine the effective concentrations EC10 and EC50 for the end point shoot fresh weight.
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 either the Williams multiple sequential 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 3.2. (ToxRat Solutions GmbH, 2010).
Nominal and measured concentrations:
- Nominal test concentrations: 10.2, 25.6, 64.0, 160, 400 and 1000 [mg a.s./kg soil dw]
Reference substance (positive control):
no
Key result
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
10.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Key result
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
EC50
Effect conc.:
22.61 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
other: All species
Duration:
7 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:
other: All species
Duration:
7 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
Species:
other: All species
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Species:
other: All species
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
LOEC
Effect conc.:
25.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
25.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
64 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
392.22 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
25.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
64 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Brassica napus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
114.45 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
160 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
400 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Cucumis sativus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
706.55 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
64 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
160 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
Pisum sativum
Duration:
16 d
Dose descriptor:
EC50
Effect conc.:
443.38 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
other: S. lycopersicum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
10.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
other: S. lycopersicum
Duration:
15 d
Dose descriptor:
LOEC
Effect conc.:
25.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Species:
other: S. lycopersicum
Duration:
15 d
Dose descriptor:
EC50
Effect conc.:
55.21 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: Shoot fresh weight
Details on results:
SEEDLING EMERGENCE AND SURVIAL:
- Emergence in the control was at least 70.8 %. Survival of seedlings in the controls was 100 %.
- The requirements defined by the test guideline regarding emergence (≥70%) and survival of seedlings (≥90%) in the control were fulfilled.
- No statistically significant effect on either emergence or survival of seedlings in any of the test species at any test item concentrations up to and including 1000 mg a.s./kg soil dry weight was determined

VISUAL APPEARANCE:
- Typical damages at test end (day 7) were chlorosis, necrosis and deformation of seedlings which was observed at test end at ≥ 10.2 mg a.s./kg soil dry weight for C. sativus, at ≥ 25.6 mg a.s./kg soil dry weight for A. cepa, B. napus, P. sativum and S. lycopersicum and at ≥ 64.0 mg a.s./kg soil dry weight for A. sativa.

SEEDLING SHOOT FRESH WEIGHT:
- Shoot fresh weight was significantly reduced at test item concentrations of ≥ 400 mg a.s./kg soil dry weight in C. sativus, ≥ 160 mg a.s./kg soil dry weight in P. sativum, ≥ 64 mg a.s./kg soil dry weight in A. sativa and B. napus and at ≥ 25.6 mg a.s./kg soil dry weight in A. cepa and S. lycopersicum on day 7.
Validity criteria fulfilled:
yes
Remarks:
For details see "Overall reamrks"
Conclusions:
The effect of the test item Perkalink 900 on the emergence and growth of six species of higher terrestrial plants was tested under laboratory conditions.
The overall lowest NOEC and EC50 were 10.2 mg a.s./kg soil dry weight and 22.61 mg a.s./kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa after 15 days of exposure.
Executive summary:

According to the OECD Guideline for the Testing of Chemicals No. 208; Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test, adopted 19 July 2006 seeds of two monocotyledonous species, Allium cepa (onion) and Avena sativa (oat), 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 either 14 days (A. sativa, B. napus, C. sativus), 15 days (A. cepa, S. lycopersicum) or 16 days (P. sativum) following 50 % emergence of the control plants. Test item concentrations ranged from 10.2 to 1000 mg a.s./kg soil dry weight for all species. Defined amounts of the test item were weighed out separately and ground with fine quartz sand. Afterwards test item and quartz sand were thoroughly mixed for 5 minutes into the test soil by using a mixer in order to homogeneously distribute the test item in the 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 supplemented with the test item. Soils were supplied with water and a nutrient solution as needed by bottom watering. The test was performed in a growth chamber equipped with artificial lighting (211 to 257 μE m-2 s-1, for 16 hours per day) and air conditioning (air temperature ranging from 19.8 to 29.3 °C, mean: 23.1 °C; relative air humidity ranging from 29.9 to 71.0%, mean: 41.9% (A. sativa, B. napus, C. sativus) and 42.4% (A. cepa, P. sativum, S. lycopersicum). 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 the end of the test, (day 14, 15 or 16), seedlings were counted, evaluated visually, and harvested to determine shoot fresh weight. The validity criteria were fulfilled as seedling emergence in the control was ≥ 70.8% and seedling survival in the control was 100% for all six species. No phytotoxicity was observed in the controls. No statistically significant effect on either emergence or survival of seedlings in any of the test species at any test item concentrations up to and including 1000 mg a.s./kg soil dry weight was determined. Typical damages at test end were chlorosis, necrosis and deformation of seedlings which was observed at test end at ≥ 10.2 mg a.s./kg soil dry weight for C. sativus, at ≥ 25.6 mg a.s./kg soil dry weight for A. cepa, B. napus, P. sativum and S. lycopersicum and at ≥ 64.0 mg a.s./kg soil dry weight for A. sativa. Shoot fresh weight was significantly reduced at test item concentrations of ≥ 400 mg a.s./kg soil dry weight in C. sativus, ≥ 160 mg a.s./kg soil dry weight in P. sativum, ≥ 64 mg a.s./kg soil dry weight in A. sativa and B. napus and at ≥ 25.6 mg a.s./kg soil dry weight in A. cepa and S. lycopersicum.

The overall lowest NOEC and EC50 were 10.2 mg a.s./kg soil dry weight and 22.61 mg a.s./kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa.

Description of key information

The effect of the test item Perkalink 900 on the emergence and growth of six species of higher terrestrial plants was tested under laboratory conditions. The overall lowest NOEC and EC50 were 10.2 mg a.s./kg soil dry weight and 22.61 mg a.s./kg soil dry weight, respectively, and were observed with shoot fresh weight of Allium cepa after 15 days of exposure.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:
22.61 mg/kg soil dw
Long-term EC10, LC10 or NOEC for terrestrial plants:
10.2 mg/kg soil dw

Additional information