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Ecotoxicological information

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Application of the Test Item:
To prepare a stock solution, 24.7528 g of the test item were made up with acetone to a total volume of 100 mL in a volumetric flask. Aliquots of the stock solution were diluted with acetone to achieve seven different test solutions in a geometric series, ranging from 2.95 mg/mL to 247.5 mg/mL (corresponding to nominal test item concentrations in soil of 7.8 to 500 mg/kg soil dry weight).
The test item was applied via spiked quartz sand as the carrier. Test solutions (test item dissolved in acetone) were mixed into portions fine quartz sand and left to evaporate under mild conditions. After complete evaporation of the solvent, the spiked quartz sand was mixed thoroughly with the test soil of the respective treatment. For the solvent control soil, quartz sand was mixed with acetone without test item and left to evaporate.
Vehicle:
yes
Details on preparation and application of test substrate:
Test Soil:
Standard soil LUFA 2.3, a sandy loam, was purchased from the Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), D-67346 Speyer, Germany. LUFA soil Sp 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 ambient temperature at ECT GmbH until use.

Properties of standard soil LUFA Sp 2.3:
- Soil type (USDA): sandy loam
- Sampling date: 2011-11-24
- Particle size distribution [%]:
< 0.002 mm: 8.7 ± 1.7
0.002 – 0.05 mm: 27.6 ± 3.8
0.05 – 2.0 mm: 63.7 ± 4.4
- Organic carbon content [%]: 0.94 ± 0.10
- Nitrogen [%]: 0.08 ± 0.02
- pH (0.01 M CaCl2): 6.8 ± 0.2
- Cation exchange capacity [meq/100 g]: 10.7 ± 1.4
- Water holding capacity [%]: 35.6 ± 3.0
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Family: Amaryllidaceae
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
Source of supply: Nordsaat Saatzuchtgesellschaft mbH, Granskevitz, Germany
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Brassicaceae
Source of supply: Lufa Speyer, Speyer, Germany.
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Cucurbitaceae
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Solanaceae
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
Source of supply: Enza Zaden Deutschland GmbH & Co. KG, Dannstadt-Schauernheim, Germany.
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
15 d
Test temperature:
Air temperature: 22-27 °C (mean = 23.7 °C)
pH:
6.8 ± 0.2
Moisture:
Rel. humidity: [%]: 31.4-79.7 (mean = 48.2 %)
Details on test conditions:
Methodology:
Seeds of two monocot species, Allium cepa (onion) and Avena sativa (oat), and four dicot species, Brassica napus (rape), Cucumis sativus (cucumber), Lycopersicon esculentum (tomato) and Pisum sativum (pea) were planted in a natural sandy loam soil (standard soil LUFA Sp2.3) immediately after test item application and left to grow under controlled conditions for up to 15 days. Each species was tested at 5 out of 7 test item concentrations (dose response test) ranging from 7.8 to 500 mg/kg soil dry weight. Test item was dissolved in acetone and mixed into portions of fine quarts sand. After complete evaporation of the solvent the spiked sand was mixed into the soil. A control soil and a solvent control soil were prepared by adding either pure sand or acetone spiked sand (without test item) to soil aliquots. Plants were allowed to emerge and grow for up to 15 days following 50 % emergence of the control plants. Soils were supplied with water or nutrient solution by bottom watering. The test was performed in growth chambers equipped with artificial lighting (16 hours per day) and air conditioning (air temperature ranging from 22 to 27 °C The pots were placed randomly at the beginning of the test and were re-arranged weekly. At half-time of the test seedlings were evaluated visually. Final counting, visual evaluation, and harvest to determine shoot length and shoot fresh weight took place at test end on day 14 or 15.
The test with L. esculentum was performed twice since the results of the first test did not allow for determining a NOEC for shoot length. Therefore, the number of test concentrations in the second test was increased from five to seven test item concentrations plus the controls.
Nominal and measured concentrations:
Nominal concentration: 7.8, 15.6, 31.3, 62.5, 125.0, 250.0 and 500.0 [mg/kg soil dw]
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 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:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot lenght
Species:
Allium cepa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Avena sativa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Avena sativa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Species:
Avena sativa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot lenght
Species:
Avena sativa
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Brassica napus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Brassica napus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Species:
Brassica napus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
7.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot length
Species:
Brassica napus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
31.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Cucumis sativus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Cucumis sativus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Species:
Cucumis sativus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot length
Species:
Cucumis sativus
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Lycopersicon esculentum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Lycopersicon esculentum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Species:
Lycopersicon esculentum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
25 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot length
Species:
Lycopersicon esculentum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
55.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Species:
Pisum sativum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Species:
Pisum sativum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
>= 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Species:
Pisum sativum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot length
Species:
Pisum sativum
Duration:
15 d
Dose descriptor:
NOEC
Effect conc.:
125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot fresh weight
Details on results:
The test item had statistically significant adverse effects on plant growth at concentrations of ≥ 15.6 mg/kg soil dw.
The lowest EC50 was 64.7 mg/kg soil dry weight and was observed with the shoot fresh weight of L. esculentum seedlings.
The lowest NOEC was 7.8 mg/kg soil dry weight and was determined for the endpoint shoot length of B. napus seedlings.

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 either day 7 or day 8 for all species, and at the end of the test, i.e. either on day 14 or on day 15.
No significant effect on seedling emergence was observed for any of the six test species.
Results with reference substance (positive control):
Survival of seedlings in the controls was 100 % except for Allium cepa for which survival was 91 %.
Reported statistics and error estimates:
Statistical Analysis:
Probit Analysis was used to determine effective rates EC10, EC20 and EC50. 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 .
Normal distribution was checked by the Kolmogorov-Smirnov-Test, and homogeneity of the variances by Cochran's Test Procedure. NOEC and LOEC were determined by applying the Williams Test for homogeneous variances, and by Welch-t Test for inhomogeneous variances. Significance level was Alpha = 0.05, two-sided.
Statistical evaluations were performed using the ToxRat software Version 2.10 (ToxRat Solutions GmbH, 2010).

Result:

Summary of the ECx LOEC, and NOEC of the endpoints emergence, survival, shoot length, and shoot fresh weight, based on the nominal test item concentrations in soil [mg/kg soil dry weight].

  Allium cepa  Avena sativa Brassica napus Cucumis sativus Lycopersicon esculentum

Lycopersicon esculentum

Pisum sativum

 Emergence *(1)        

A = first Test

 B = second Test  
 EC10  55.4   n.d. 39.3   n.d.   n.d. n.d.   54.3
 EC20  141.0   n.d. 97.4   n.d.   n.d. n.d.   104.3
 EC50   n.d.  n.d.    n.d.  n.d.    n.d. n.d.  364.2
 LOEC  250  >500  >125  >125  >125  >125  500
 NOEC  125  >=500  >=125  >=125  >=125  >=125  250
 Survival *(2)              
  EC10  130.9   n.d.  n.d.    n.d.   n.d.  n.d.   222.2
  EC20  137.2   n.d.  n.d.    n.d.   n.d.  n.d.   325.1
  EC50  150.1   n.d.  n.d.    n.d.   n.d.  n.d.  673.1 *(3)
 LOEC  250  >500  >125  >125  >125  >125  >500
NOEC   125  >=500  >=125  >=125  >=125  >=125  >=500
 Shoot lenght              
  EC10   n.d. 140.8   47.2  67.8  27.0 16.6  77.6
  EC20   n.d. 194.8  62.7  204.5  60.5  55.4  105.0
  EC50   n.d.  362.6  108.0    n.d.   n.d.   n.d.  187.5
 LOEC  125  250  15.6  125  <=7.8 55.9   250
 NOEC  62.5  125  7.8  62.5  <7.8  25.0  125
 Shoot fresh weight              
  EC10  62.5    n.d. 41.5   64.4  4.5 43.0  86.2
  EC10  73.3    n.d. 51.1   81.1  11.3 54.1  104.6
  EC10  99.5    n.d. 76.2   125.9  64.7 84.1   151.6
 LOEC  125  250  62.5  125  15.6 125  250
 NOEC  62.5  125  31.3  62.5  7.8  55.9  125

n. d.: not determined due to mathematical reasons.

(*1) = Emergence until day 7(8); (*2) = survival until the end of the test; (*3) = extrapolated value

Conclusions:

The test item had statistically significant adverse effects on plant growth at concentrations of ≥ 15.6 mg/kg soil dw.

The lowest EC50 was 64.7 mg/kg soil dry weight and was observed with the shoot fresh weight of L. esculentum seedlings.

The lowest NOEC was 7.8 mg/kg soil dry weight and was determined for the endpoint shoot length of B. napus seedlings.

Validity criteria fulfilled:
yes
Remarks:
The validity criteria were fulfilled as seedling emergence in the control was ≥ 70 % and seedling survival in the control was ≥ 90 % for all six test species. No phytotoxicity was observed in the controls.
Conclusions:
The chronic toxicity of the substance to terrestrial plants was tested according to OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test). Five to seven different test item concentrations per species were tested against a solvent control (acetone) with each of the six plant species. No significant effect on seedling emergence or mortality was observed for any of the six test species. Effects were observed for shoot length and fresh weight. Shoot length and shoot fresh weight of B. napus seedlings was significantly reduced at concentrations of 15.6 and 62.5 mg/kg soil dw, respectively. Hence, B. napus was the most sensitive species with regard to shoot length and weight.
Executive summary:

The chronic toxicity of the substance to terrestrial plants was tested according to OECD Guideline 208 (Terrestrial Plants Test. Seedling Emergence and Seedling Growth Test). Five to seven different test item concentrations per species were tested against a solvent control (acetone) with each of the six plant species. Seeds of two monocot species, Allium cepa (onion) and Avena sativa (oat), and four dicot species, Brassica napus (rape), Cucumis sativus (cucumber), Lycopersicon esculentum (tomato) and Pisum sativum (pea) were planted in a natural sandy loam soil (standard soil LUFA Sp2.3) immediately after test item application and left to grow under controlled conditions for up to 15 days. Each species was tested at 5 out of 7 test item concentrations (dose response test) ranging from 7.8 to 500 mg/kg soil dry weight. Test item was dissolved in acetone and mixed into portions of fine quarts sand. After complete evaporation of the solvent the spiked sand was mixed into the soil. A control soil and a solvent control soil were prepared by adding either pure sand or acetone spiked sand (without test item) to soil aliquots. Plants were allowed to emerge and grow for up to 15 days following 50 % emergence of the control plants. Soils were supplied with water or nutrient solution by bottom watering. The test was performed in growth chambers equipped with artificial lighting (16 hours per day) and air conditioning (air temperature ranging from 22 to 27 °C The pots were placed randomly at the beginning of the test and were re-arranged weekly. At half-time of the test seedlings were evaluated visually. Final counting, visual evaluation, and harvest to determine shoot length and shoot fresh weight took place at test end on day 14 or 15.

The test item significantly reduced seedling emergence of A. cepa by 42 % at 250 mg/kg soil dw. At this concentration none of the seedlings survived. Seedling emergence of P. sativum was significantly reduced by 62 % at 500 mg/kg soil dw. For all other tested species adverse effects were observed neither on emergence nor on survival

Visual evaluation revealed discoloured leaves at the highest test concentrations.

Shoot length and shoot fresh weight of B. napus seedlings was significantly reduced at concentrations of 15.6 and 62.5 mg/kg soil dw, respectively. Hence, B. napus was the most sensitive species with regard to shoot length and weight.

Description of key information

The chronic toxicity of the substance to terrestrial plants was tested according to OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test). Five to seven different test item concentrations per species were tested against a solvent control (acetone) with each of the six plant species. No significant effect on seedling emergence or mortality was observed for any of the six test species. Effects were observed for shoot length and fresh weight. Shoot length and shoot fresh weight of B. napus seedlings were significantly reduced at concentrations of 15.6 and 62.5 mg/kg soil dw, respectively. Hence, B. napus was the most sensitive species with regard to shoot length and weight.

Key value for chemical safety assessment

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

Additional information