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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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Remarks:
Minor deviations, which were judged to have no impact on the integrity or outcome of the study
Qualifier:
according to
Guideline:
other: Terrestrial (Non-target) Plant Test: 208A: Seedling Emergence and Seedling Growth Test, OECD Proposal for Updating Guideline 208 (July 2000)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 200 mg/mL as stock solution in acetone and as fortification sample
- Sampling method: Acetone stock solutions containing test substance were evaporated by nitrogen convection to remove all traces of acetone. The residue was reconstituted in hexane containing the internal standard dioctyl phthalate. They were then interspersed with standard solutions containing the internal standard of dioctyl phthalate and submitted for GC analysis.
- Sample storage conditions before analysis: no data

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): known volumes of the test substance dissolved in vehicle were mixed with 200 g (± 0.1 g) of laboratory sand each using a glass stirrer, the dried sand mixture for each dose treatment was then mixed with a known weight of soil. Then the moisture content of the prepared soils for treatment was raised to 40% by gradually addition of distilled water whilst mixing through in order to achieve even water distribution.
- Controls: yes, blank and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: yes

Test organisms

open allclose all
Species:
Triticum aestivum
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: wheat
- Plant family: Gramineae (Poaceae)
- Variety: ZBB 065
- Source of seed:
For rangefinder test: Blades Biological (Cowden, UK)
For definitive test: Mr Fothergill's (Harrogate, UK)
- Prior seed treatment/sterilization: none
- Historical germination of seed (germination of seed lot tested): yes, germination rate > 93 % was achieved within 8 days
- Seed storage: in a dark dry environment at 20 ± 10 °C
Species:
Lepidum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: cress
- Plant family: Cruciferae (Brassicaceae)
- Variety: ZBB 015 (rangefinder test), Fine Curled (definitive test)
- Source of seed:
For rangefinder test: Blades Biological (Cowden, UK)
For definitive test: Mr Fothergill's (Harrogate, UK)
- Prior seed treatment/sterilization: none
- Historical germination of seed (germination of seed lot tested): yes, germination rate > 93 % was achieved within 8 days
- Seed storage: in a dark dry environment at 20 ± 10 °C
Species:
Phaseolus aureus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: mung bean
- Plant family: Leguminosae (Fabaceae)
- Variety: ZBB 035
- Source of seed:
For rangefinder test: Blades Biological (Cowden, UK)
For definitive test: Mr Fothergill's (Harrogate, UK)
- Prior seed treatment/sterilization: none
- Historical germination of seed (germination of seed lot tested): yes, germination rate > 93 % was achieved within 8 days
- Seed storage: in a dark dry environment at 20 ± 10 °C

Study design

Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Remarks:
17 - 22 d
Post exposure observation period:
none

Test conditions

Test temperature:
no data
pH:
no data
Moisture:
40 %
Details on test conditions:
TEST SYSTEM
- Testing facility: Covance Laboratories Ltd, England
- Test container (type, material, size): planting pots with nominally 15 cm diameter, no further details mentioned
- Amount of soil: soil was added to lower brim of the empty plant pot and firmed by hand, no further details mentioned
- Method of seeding: each seed was placed in a planlting hole and covered with soil already in the pot, the depth of sowing being equivalent to the relative size of the seed (approx. the same depth of soil covering as the width of each seed)
- No. of seeds per container: 5
- No. of plants (retained after thinning): no thinning performed
- No. of replicates per treatment group:
Rangefinder test: 4 for highest dose group (1000 mg/kg), 2 for intermediate treatment groups
Definitive test: 8 for each treatment group
- No. of replicates per control:
Rangefinder test: 4
Definitve test: 8
- No. of replicates per vehicle control:
Rangefinder test: 4
Definitve test: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: from a site of general agricultural quality ("Im Bildgarten", Nr. 587) in Offenbach, Rheinland-Pfalz, Germany; collected by LUFA Speyer, Germany
- Pesticide use history at the collection site: no pesticides in last 5 years
- Collection procedures: in accordance with ISO 10381-6
- Sampling depth (cm): no data
- Soil texture (if natural soil)
- % sand: 55 (Rangefinder test), resp. 59 (Definitive test)
- % silt: 35 (Rangefinder test), resp. 32 (Definitive test)
- % clay: 10 (Rangefinder test), resp. 9 (Definitive test)
- Soil taxonomic classification: sandy loam
- Soil classification system: UK & BBA
- Organic carbon (%): 1.2 (Rangefinder test), resp. 1.0 (Definitive test)
- Maximum water holding capacity (in % dry weigth): 36 (Rangefinder test), resp. 35.2 (Definitve test) at 0.001 bar
- CEC: 6.2 mEq/100 g (Rangefinder test), resp. 5.9 mEq/100 g (Definitive test)
- Pretreatment of soil: Prior to use the soil was thoroughly mixed and passed through a 2 mm-mesh sieve.
- Storage (condition, duration): The soil was allocated a unique number and stored between 1 and 10°C in a loosely tied plastic bag in accordance with ISO 13081-6. Storage duration was approx. 11 weeks for the Rangefinder test, resp. approx. 3 weeks for the Definitive test.


NUTRIENT MEDIUM (if used)
- Description: not used


GROWTH CONDITIONS
glasshouse under natural lighting, additional lighting and heating were employed to provide optimun conditions for germination and growth of the species under test, the position of individual replicates was randomised to eliminate bias (no further details mentioned)
- Watering regime and schedules:
Each pot stood on a disc of capillary matting inside a dedicated plastic dish, thereby eliminating the possibility of cross-contamination from leachates generated after watering. Distilled water was added to each disc of capillary matting to wet. Following the initial set up of each pot, all watering was performed from beneath by drenching the capillary matting at period throughout the incubation period to maintain the soil moisture. (no further details mentioned)
- Any pest control method/fertilization (if used): not used


ACCLIMATION PERIOD: Seeds were sown in the pots within 24 hours of the incorporation of the test substance into the soil, the observation period started beyond the day on which 50% of the control seedlings had emerged.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The number of emerged seedlings in each replicate was recorded at daily intervals throughout the exposure period up until the time of harvest. Emergence counts were expressed as percentages of the number of seeds sown.
- At the end of the observation period, all the seedlings were cropped at the soil surface and immediately weighed. All the plants harvested from a given pot were weighed together to obtain mean seedling wet biomass data for each pot.
- Qualitative assessments of chlorosis (yellowing/discoloration), necrosis (death of tissue) and vigour (health and standing) were made at daily intervals, whereby each species was assessed relevant to the effect to the control.


- Phytotoxicity rating system (if used): Chlorosis and necrosis were marked on a scale of 0 to 10, where 0 was no effect and 10 was total effect. Vigour was marked on a decreasing scale of 10 to 0, where 10 was full vigour.


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: according to guideline
- Range finding study
- Test concentrations: 0.01 / 0.1 / 1 / 10 / 100 / 1000 mg/kg
- Results used to determine the conditions for the definitive study: The plant species identified being most sensitive to the test substance was chosen for the conduct of the definitive test. Significant effects were noted from wet biomass results for cress. Following estimation of the EC20 and EC80, five dose rates were prepared.
Nominal and measured concentrations:
Rangefinder test: 0.001, 0.1, 1, 10, 100 and 1000 mg/kg
Definitive test: 2.5, 75, 150, 600 and 1000 mg/kg
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Species:
Lepidum sativum
Duration:
17 d
Dose descriptor:
EC50
Effect conc.:
81.88 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Result from Definitive study
Remarks on result:
other: 18.50 - 237.12 mg/kg (95% fiducial limits)
Species:
Lepidum sativum
Duration:
17 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
Result from Definitive study
Species:
Triticum aestivum
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Result from Rangefinder study
Species:
Triticum aestivum
Duration:
22 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
Result from Rangefinder study
Species:
Phaseolus aureus
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Result from Rangefinder study
Species:
Phaseolus aureus
Duration:
22 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
seedling emergence
Remarks:
Result from Rangefinder study
Details on results:
SEED GERMINATION
- Percent seed germination:
- Root length:
- Root discolouration/malformation:
- Other effects:
- Results with reference substance valid?


SEEDLING EMERGENCE
- Percent seedling emergence:
- Percent survival:
- Plant height:
- Dry weight:
- Abnormal seed development or appearance:
- Lesions:
- Swelling:
- Loss of turgour:
- Discoloration:
- Unusual leaf/plant shape or size:
- Other effects:


VEGETATIVE VIGOUR
- Percent seed germination:
- Plant height:
- Dry weight:
- Abnormal seed development or appearance:
- Lesions:
- Swelling:
- Loss of turgour:
- Discoloration:
- Unusual leaf/plant shape or size:
- Dead plants:
- Other effects:


INJURY RATING SYSTEM:
Results with reference substance (positive control):
no reference substance tested
Reported statistics and error estimates:
Rangefinder study: For each species the decrease in biomass for each dose level was calculated as a percentage of that for the control group. Probit analysis was used on the percentage decrease to obtain an estimate of the EC50

Applicant's summary and conclusion

Executive summary:

These results showed that cress is the most sensitive of the three tested species to Tri (hexyl, octyl, decyl) citrate. Cress was thus chosen for the conduct of the Definitive test. Following estimation of the EC20 and EC80, five dose rates were prepared for the Definitive test. These were 2.5, 75, 150, 600 and 1000 mg/kg. The EC50 for cress was 81.88 mg/kg (18.50 to 237.12 limits). The emergence results indicated the LC50 was greater than the highest dose tested, >1000 mg/kg. Analysis of both the qualitative and quantitative results indicate a NOEC (No Observed Effect Concentration) at Treatment 3: a concentration of 2.5 mg/kg dry soil. These results suggest that Tri (hexyl, octyl, decyl) citrate has no effect on emergence, but can affect development and growth of seedlings of sensitive species from a concentration of 81.88 mg/kg dry soil.