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Toxicity to soil macroorganisms except arthropods

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toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:
not specified
Specific details on test material used for the study:
- Source and lot/batch No.of test material: batch E 4201
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated
Analytical monitoring:
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Aqueous imazalil sulphate of 10, 1, 0.1, 0.01 and 0.001 mg/ml were prepared by dilution of the stock solution. An appropriate volume of these solutions was diluted to 75 ml with distilled water and mixed with 675 g of artificial soil (30.5 g moisture/100g) in a Kenwood kitchen machine. The prepared test batches were transferred to 1litre glass beakers.
- Controls: yes
- artificial soi:l: 10% sphagnum peat, 20 % kaolinite clay, 62% industrial quartz sand and 1% calcium carbonate.

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: redworm
- Source: local bait farm (Kalmthout, Belgium)
- Weight at test initiation (mean and range, SD): only worms with body weights exceeding 400 mg were used in the test

- Acclimation period: minimum 14 days, test room of 20°C with a light/dark cycle of 12h/12h
- Acclimation conditions (same as test or not): yes, except for breeding medium
- glass trays containing breeding medium (50% construction sand, 25% peat and 25 % vegetal leavings, brought at pH = 6 with 0.5 N Ca(OH)2
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
14 d
Test temperature:
30.5 g moisture/100g
Details on test conditions:
- Test container (material, size): 1 litre glass beaker covered with parafilm with ventilation holes
- Amount of soil or substrate: 675g soil (with 75 ml test substrate)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 1

- Composition (if artificial substrate): Artificial soil was prepared in accordance with the OECD-guideline No 207 and the EEC-guideline. For each kg of artificial soil substrate (oven-dry equivalent) 0.708 kg of sand, 0.351 kg of peat, 0.201 kg of kaolinite and 0.01 g of CaCO3 were mixed. After mixing, the artificial soil had a moisture content of 30.5 g/100 g (oven-dry soil), a water holding capacity of 52.3 g/100 g, an organic matter content of 11.0% and pH (H2O) and pH of 6.5.
- Storage (condition, duration): 20°C in closed polyethylene bags

- Photoperiod: 12h light / 12h dark
- Light intensity: not indicated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality: worms not responding to a mechanical stimulus to their front were considered as dead. The determination of surviving worms took place on day 7 and day 14.

- Spacing factor for test concentrations: 1.4
- Range finding study: yes
- Test concentrations: 10, 1, 0.1, 0.01, 0.001 mg/ml
- Results used to determine the conditions for the definitive study: yes
Nominal and measured concentrations:
Nominal concentrations: 150, 215, 320, 460, 680, 820 µg/g
Reference substance (positive control):
chloroacetamide and copper sulphate
Key result
14 d
Dose descriptor:
Effect conc.:
271 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% confidence interval: 486 - 577 mg/kg
Details on results:
- Mortality at end of exposure period:
150 µg/g: 0%
215 µg/g: 0%
320 µg/g: 2.5%
460 µg/g: 17.5%
680 µg/g: 60%
820 µg/g: 100%

- Morphological abnormalities: At concentrations near the LC50, surviving worms were extended and limp, while some of the test specimen had lost several posterior segments.

- Behavioural abnormalities: The worms in contact with sublethal concentrations of imazalil sulphate showed signs of hyperactivity and were very sensitive to mechanical stimuli.

- Other biological observations: The dead worm bodies were detected only occasionally because a rapid decomposition semed to occur in the test medium. A typical decay smell, however, was noticed.

- Other: Imazalil was found to disappear from soil with a half-life of 170 days. Consequently, it could be accepted that no significant amounts of test substance were lost from the test containers during the experiments. The worms were actually exposed to the intended test concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
* copper sulphate: LC50 > 1000 µg/g
* chlorocetamide: 100 µg/g (10 µg/g < LC50 < 100 µg/g)

Reported statistics and error estimates:
Probit analysis following the method of Finney was applied: the LC50 and its 95% confidence limits, the LD5 (5% mortality), the LC95 (95% mortality), and the X2-statistic for testing the homogeneity of the data, were calculated by an APL computer program developed by Janssen Information Services.
Validity criteria fulfilled:
The acute toxicity of Imazalil sulphate to Eisenia foetida was determined in a 14 day study according to OECD guideline 207. The LC50 (14 days) of Imazalil sulphate for earthworms of the species Eisenia foetida in an artificial soil was 532 µg/g which corresponds to 541 mg imazalil/kg soild dw. Based on the EC document (2009), the endpoint was corrected to 271 mg imazalil /kg soil dw. The results of the test can be considered reliable without restriction.

Description of key information

The study of Van Leemput (1986), investigating the toxicity of Imazalil sulphate to earthworms (Eisenia foetida) according to the OECD guideline 207, was considered as the key study for endpoint coverage. Based on the EC document (2009), the endpoint was corrected to 271 mg imazalil /kg soil dw which corresponds to 360.43 mg/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
360.43 mg/kg soil dw

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