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Ecotoxicological information

Toxicity to terrestrial plants

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Administrative data

Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Toxicity of eight polycyclic aromatic compounds to red clover (Trifolium pratense), ryegrass (Lolium perenne), and mustard (Sinapsis alba)
Author:
Sverdrup LE, Krogh PH, Nielsen T, Kjær C, Stenersen J
Year:
2003
Bibliographic source:
Chemosphere 53, 993-1003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Version / remarks:
Version 1984
Principles of method if other than guideline:
Comparative study including four aromatic and four heteroaromatic compounds using three different plants
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenanthrene
EC Number:
201-581-5
EC Name:
Phenanthrene
Cas Number:
85-01-8
Molecular formula:
C14H10
IUPAC Name:
phenanthrene
Test material form:
solid
Details on test material:
- Substance type: organic
- for additional information see study record
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phenanthrene
- Source of test material: Sigma-Aldrich, Steinheim, Germany
- Analytical purity: > 96 %

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations (initial concentrations in test soil for verification of test substance levels)

Test substrate

Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
- Method of preparation of test solutions: a stock solution in acetone was prepared that corresponded to the highest test dosage. For each test concentration, dilutions from the stock solution with acetone were made such that 250 mL of the resulting acetone solution contained the amount of test material required to produce the final test concentration after mxing with soil.
- Method of mixing into soil: 250 mL acetone solution for each of the treatments (equal volumes) was added to 1.25 kg of soil in an aluminium container using aluminium equipment as required and mixed thoroughly resulting in the desired test concentration. The soil was then transferred to glass plates and left in a fume head for 24 h to evaporate the solvent.
- Addition of water: after test substrate preparation, 200 mL water/kg soil was added to each sample corresponding to approximately 65 % of the water holding capacitiy and mixed thoroughly into the soil.
- Separation into test samples: the bulk humid samples (1.5 kg) were divided into five sub-samples of 300 g each, one for each of the four replicates and one for chemical analysis.
- Controls: controls and solvent solvent (for solvent controls, soil samples were treated the same way as the test samples but using acetone without test substance)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): not specified
- Evaporation of vehicle before use: yes

Test organisms

open allclose all
Species:
Trifolium pratense
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Red clover
- Plant family: Fabaceae
- Variety: no data
- Source of seed: DLF Trifolium (Randers, Denmark)
- Prior seed treatment: before addition of seeds to soil, they were inspected and damaged/discoloured seeds were removed. Seeds of similar size were then selected and used for the experiments.
Species:
Sinapis alba
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: white mustard
- Plant family: Brassicaceae
- Variety: no data
- Source of seed: Ove Rassmussens Planteskole (Silkeborg, Denmark)
- Prior seed treatment: prior to the tests damaged/discoloured seeds were rejected. Seeds of similar size were selected for the experiment.
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: perennial ryegrass
- Plant family: Poaceae/Gramineae
- Variety: no data
- Source of seed: DLF Trifolium (Randers, Denmark)
- Prior seed treatment: prior to the tests damaged/discoloured seeds were rejected. Seeds of similar size were selected for the experiment.

Study design

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:
Exposure period was 19 to 21 days: at least 14 days of exposue after emergence of 50 % of the seeds in the control samples

Test conditions

Test temperature:
Night: 15 °C, day ~25 °C, sometimes higher
pH:
Soil pH: 6.2
Moisture:
Test soils were hydrated to 65 % of the water holding capacity with water (200 mL/kg soil dw).
Details on test conditions:
All three species were tested simultaneously.
TEST SYSTEM
- Testing facility: growth chamber placed in a green house
- Test container: transparent cylinder made of hard plastics (35 cm high, inner diameter 9.5 cm), closed at the top and bottom with lids. The upper lid was perforated with one hole (diameter 0.8 cm).
- Amount of soil: 300 g wet weight per test container
- Method of seeding: planting of seeds into the test soil
- No. of seeds per container: 5
- No. of replicates per treatment group: 4; one additional replicate for test substance analysis
- No. of replicates per control: 5
- No. of replicates per vehicle control: 5
- Plant density: ca. 71 cm²/5 seeds = ca. 14 cm²/seed; 300 g soil wet weight/5 seeds = 60 g soil ww/seed

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil test substrate: Danish agricultural soil
- Geographic location: Askov, Jutland, Denmark
- Soil texture (if natural soil): sandy loam
- Composition: coarse sand (0.2 – 2 mm) 38.4 %,
fine sand (0.063 – 0.2 mm) 23.6 %,
coarse silt (0.02 – 0.063 mm) 10.0 %,
fine silt (0.002 – 0.02 mm) 12.3 %,
clay (<0.002 mm) 13 %,
humus 2.8 %
- Organic carbon content: 1.6 %
- Soil pH: 6.2, no adjustment
- Density: 1.135 g/cm³ dry soil
- Maximum water holding capacity (in % dry weight): no data
- CEC (Cation exchange capacity): 8.14 meq/100 g
- Pretreatment of soil prior to use: drying at 80 °C for 24 h, sieving (2 mm mesh)
- Stability and homogeneity of test material in the medium: no information reported

GROWTH CONDITIONS
- Photoperiod: 16/8 h light/dark
- Light source: daylight from outside of the green house, in addition cultivation lamps Philips SON/TPlus (400 W, 310 - 780 nm), three lamps were employed covering an area of 3 m².
- Light intensity and quality: >= 5 klux, lamps were switched on when natural light dropped below 5 klux and were swiched off when light intensity increased above 25 klux
- Photoflux densitiy of artificial light source: approx. 300 µE/m²/s at the soil surface
- Day/night temperatures: up to and sometimes above 25 °C/15 °C
- Relative humidity (%): 65 % of the water holding capacitiy
- Air exchange rate: no data
- Watering regime and schedules: as needed, test containeres were weighed twice weekly and the lost amount of weight was replaced with water
- Water source/type: no data
- Position of test containers: At each time of watering, test containerws were randomised for their position in the green house.

EFFECT PARAMETERS MEASURED: dry weight, fresh weight, number of germinated seeds by counting the seedlings that protruded above the soil surface

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: no data
Nominal and measured concentrations:
Nominal concentrations: 1 10 100 1000 mg/kg soil dw
Measured concentration: 0.9 9.9 114 1100 mg/kg soil dw
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Species:
Trifolium pratense
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 30 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: original value (24 mg/kg soil dw) recalculated to a standard soil with 2 % organic carbon
Key result
Species:
Trifolium pratense
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 24 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: estimation based on two ECx values and log-logistic dose-response relationship
Key result
Species:
Trifolium pratense
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
37 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 22 - 52 mg/kg soil dw
Key result
Species:
Trifolium pratense
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
79 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 0 - 390 mg/kg soil dw
Key result
Species:
Sinapis alba
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 33 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: original value (26 mg/kg soil dw) recalculated to a standard soil with 2 % organic carbon
Key result
Species:
Sinapis alba
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 26 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: estimation based on two ECx values and log-logistic dose-response relationship
Key result
Species:
Sinapis alba
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
77 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 41 - 110 mg/kg soil dw
Key result
Species:
Sinapis alba
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
480 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 350 - 620 mg/kg soil dw
Key result
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 218 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: original value (174 mg/kg soil dw) recalculated to a standard soil with 2 % organic carbon
Key result
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 174 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: estimation based on two ECx values and log-logistic dose-response relationship
Key result
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
300 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 210 - 400 mg/kg soil dw
Key result
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
760 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
fresh weight
Remarks on result:
other: 95% C.I.: 520 - 990 mg/kg soil dw
Details on results:
SEED GERMINATION
- No effect on seed emergence were observed at any exposure level: LC50 supposed to be >> 1000 mg/kg soil dw.
- Seedling emergence in controls: ≥ 80%
PLANT GROWTH
- The EC10 values are estimates based on EC20 and EC50 values using a log-logistic dose-response relationship (taken from EU RAR Coal-tar pitch, high temperature, Appendix A.4 and A.5
- Difference between vehicle controls and controls without spiking procedure: no significant differences on growth (dry and fresh weight) observed (ANOVA, p > 0.05)
- Normal growth in controls: yes
Reported statistics and error estimates:
Estimates of the concentrations that caused a 20% and 50% reduction of growth ( EC20- and EC50-values, respectively) were done by linear interpolation of the dose-response curve, and the confidence intervals were calculated by the regression procedure of SAS/STAT (SAS, 1999).
Estimates of the concentrations that caused a 50% reduction in seed emergence (LC50 values) was done using probit analysis in SAS/STAT (SAS, 1999).
NOEC values were assessed using ANOVA and Dunnett's procedure (at a 5% significance level) (SAS, 1999) after testing for fulfilment of ANOVA requirements using SAS LAB (SAS, 1992).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
seed emergence ≥ 80 %, normal growth of control seedlings throughout the test