Registration Dossier

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Currently viewing:

Administrative data

toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-06-25 to 2002-07-10
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
On days 2, 3 and 10 the incubator temperature was outside the temperature range given in the guideline. This minor deviation was not considered to have affected the outcome or integrity of the study.
GLP compliance:

Test material

Test material form:
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: 10 - 30 °C
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Melting point: -29°C (freezing point: -35.5°C)
- Boiling point: test substance showed decomposition above 260°C
- Vapour pressure: 8.5 x 10E-3 p at 25°C
- Water solubility (under test conditions): < 1 mg/L
- Solubility in organic solvents: soluble
- log Pow: > 6
- pKa: not applicable
- Base or acid catalysis of test material: no data
- UV absorption: no data
- Stability of test material at room temperature: stable
- pH dependance on stability: no data

OTHER PROPERTIES (if relevant for this endpoint)
- Adsorption characteristics: no data

Sampling and analysis

Analytical monitoring:

Test substrate

Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Test substance for the total amount of substrate was made up with 10 mL acetone for each exposure concentration. A weighed sub-sample of approx. 100 g wet weight was then taken from each treatment substrate and placed in a weighed container. The test solutions were spread out in vessels and left for approx. 1 hour to allow the solvent to evaporate. The sub-sample was reweighed to determine moisture loss over the evaporation period and any weight loss was replaced with reverse-osmosis (RO) water, where necessary. The sub-sample of test substrate was added to the respective batch of test substrate and thoroughly mixed by hand. RO water was then added to increase the moisture content to the required level (approx. 35% of dry weight).
- Controls: untreated control (not sub-sampled); solvent control (received same quantitiy pf any additive agent and was applied using the same procedures as the treatment doses.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evaporation of vehicle before use: yes

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: earthworm
- Source: Blades Biological, Cowden, Edenbridge, Kent, UK; Batch No. GF010
- Age at test initiation (mean and range, SD): at least two months old, no further details mentioned
- Weight at test initiation (mean and range, SD): 468 mg (mean)

- Acclimation period: approx. 24 hours
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): no mortality mentioned

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
14 d
Post exposure observation period:

Test conditions

Test temperature:
16.8 - 20.8 °C
6.0 +/- 0.5 (adjusted in the basic test substrate before addition of test substance, no measurement of pH furthermore)
35 %
Details on test conditions:
- Test container (material, size): glass crystallising dish of approx. 1L capacity, covered with perforated cling-film
- Amount of soil or substrate: approx. 556 g dry weight of test substrate (nominally equally to 750 g wet weight at the required 35 % moisture content)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4 replicates for 1000 mg/kg, 1 replicate each for 0.1, 1, 10 and 100 mg/kg (combined range-finding and limit test)
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

- Composition (if artificial substrate): basic test substrate containing 10 % sphagnum peat ("Irish Moss Peat" Strikes Garden Centre, Knaresborough, UK), 20 % kaolinite clay (Whitchem Ltd, Staffs, UK) and 70 % industrial sand (Silver grade sand, Hepworth Minerals and Chemicals Ltd, Cheshire, UK)
- Organic carbon (%):
- Maximum water holding capacity (in % dry weigth):
- CEC:
- Pretreatment of soil: The dry weight ratios of constituents were mixed and the pH adjusted to 6.0 +/- 0.5 using calcium carbonate.
- Storage (condition, duration): under ambient room conditions in a suitable covered container
- Stability and homogeneity of test material in the medium: not mentioned

- Photoperiod: continuous lighting
- Light intensity: 493 Lux at the beginning of the test, 662 Lux at the end of the test

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Survival of earthworms was assessed at days 7 and 14, missing earthworms were considered dead, due to rapid decomposition under test conditions. Any observed behavioural (i.e. burrowing time at day 0) or pathological symptoms and morphological changes were also recorded. After assessment at day 7 the test substrate and earthworms were replaced. The vessels were then re-weighed. Prior ot the day 14 earthworm assessment, each vessel was re-weighed to assess comparative moisture loss.


- Spacing factor for test concentrations: not mentioned
- Combined range finding study and limit test
- Test concentrations: 0.1,. 1.0, 10, 100 and 1000 mg/kg
Nominal and measured concentrations:
1000 mg/kg nominal (in the limit test), 0.1, 1.0, 10 and 100 mg/kg nominal (in the range-finding test)
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
14 d
Dose descriptor:
Effect conc.:
ca. 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: weight changes of surviving earthworms
Key result
14 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality at end of exposure period: see table below
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: 19.25 % at control, 12.8 % at solvent control, 29.4 % at 1000 mg/kg (24.5 % at 0.1 mg/kg, 21.9 % at 1 mg/kg, 16.7 % at 10 mg/kg, 26.5 % at 100 mg/kg)
- Morphological abnormalities: no abnormalities detected
- Behavioural abnormalities: no abnormalities detected
- Other biological observations:
Burrowing times at day 0: no earthworm visible on soil surface 1 hour after introduction
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: LC50 (14 d) = 32.84 mg/kg
Reported statistics and error estimates:
The percentage weight change at day 14 was analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among
the dose levels was performed. Where this showed no evidence of heterogenicity (P ¿ 0.01), pair-wise comparisons with control were made using two-sample t-tests.
Treatment with test substance did not result in any statistically significant changes (P ¿ 0.01) in mean weight compared to control.

Any other information on results incl. tables

Mortality of Eisenia foetida over 14 days exposure to Tri (hexyl, octyl, decyl) citrate: range finder / limit test

Treatment[mg/kg] Mortality          
  day 7    day 14    
   Number dead / total number of earthworms  mean [%]  Number dead / total number of earthworms  mean [%]
control   1 / 40  2.5  3 / 40  7.5
 Solvent contreol  0 / 40  0  0 / 40  0
 0.1  0 / 10  0  0 / 10  0
 1.0  0 / 10  0  0 / 10  0
 10  0 / 10  0  1 / 10  10
 100  0 / 10  0  0 / 10  0
 1000   0 / 40  0  0 / 40  0

Applicant's summary and conclusion

Validity criteria fulfilled:
The 14 day LC50 of Tri (hexyl, octyl, decyl) citrate to Eisenia foetida was observed to be > 1000 mg/kg, the highest dose tested.
The no observed effect concentration (NOEC) after 14 days of exposure was calculated to be 1000 mg/kg, based on earthworm weight.
There were no sub-lethal morphological changes observed in the test earthworms. There was not considered to be any evidence of treatment-related repellent effects.
The chloracetamide toxic reference results indicated that the earthworms from the source batch used for the test were responding normally in the test system.
Executive summary:

The acute toxicity of the read-across substance Tri (hexyl, octyl, decyl) citrate to the earthworm Eisenia foetida was determined during a 14 day exposure period according to OECD Guideline of Testing of Chemicals No. 207, adopted April 1984. For the combined range finding and limit test, the test substance was mixed with the OECD recommended artificial soil at nominal concentrations of 0.1, 1.0, 10, 100 and 1000 mg/kg dry substrate. After 7 and 14 days the effects of the test substance on the earthworms` survival (and any observable sub-lethal effects) were examined. The results were used to calculate LC50 values and no observed effect concentations.

The 14 day LC50 of the test substance to Eisenia foetida was observed to be > 1000 mg/kg, the highest dose tested. The NOEC for the test substance after 14 days of exposure was calculated to be 1000 mg/kg, based on a comparison of surviving earhworm weight changes. As there was no observable toxic effects at the 1000 mg/kg treatment rate, no further testing was carried out.

A definitve toxicity test with analytical grade chloroacetamide was carried out under similar conditions used for the definitive test with Tri (hexyl, octyl, decyl) citrate, using the eathworms from the same source and batch number. The 14 day LC50 of chloroacetamide to Eisenia foetida was calculated to be 32.84 mg/kg. Based on published data this is considered to be an acceptable response and indicated that earthworms from the source used in the test were responding normally in the test system.