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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
Study period:
2006-04-20 - 2006-06-28
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP
according to
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
adopted April 04, 1984
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on preparation and application of test substrate:
- Method of mixing into soil: For each test vessel, 552 g of the prepared substrate (air dry, corresponding to 546 g dry weight) was thoroughly mixed with 10 g sand/test item mixture (see Section 2.4.3), 2.8 g calcium carbonate, and 189 mL purified water in a laboratory mixer. By addition of 189 mL purified water to the test substrate of each test vessel, the moisture content was brought to approx. 35 %.

- Controls:
A control was tested in parallel. The control substrate was prepared analogous to the test substrates. A 50 g sand portion was treated with 10 mL acetone (but without test item) identical to the application of the test item in the treatment groups.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 278 mg test item/ mL acetone
- Evaporation of vehicle before use: yes
Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: Earthworm
- Source: from a breeding stock maintained at RCC
- Age at test initiation: The worms used were adults with a clitellum and were approximately five months old.
- Mean body wet weights at test initiation: 341 - 434 mg

- Acclimation period: one day prior to the test start
- Acclimation conditions: same as test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
14 d
Test temperature:
ranged from 19-20 °C over the 14-day exposure period
between 6.1 and 6.2 during test.
between 33 % and 34 % during test.
Details on test conditions:
- Test container (material, size): Cylindrical glass vessels (diameter 10 cm, height 14 cm), volume: ca. 1 L
- Amount of soil or substrate: 556 g artificial soil (dry weight, corresponding to about 750 g wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

- Composition: The artificial soil (air dry components, without calcium carbonate) was prepared in a batch of 20 kg by intensely mixing 2 kg sphagnum peat, 4 kg kaolinite clay, and 14 kg sand (dry weights) in a cement mixer.
- Pretreatment of soil: no
- Storage (condition, duration): not specified

- Photoperiod: continous illumination
- Light intensity: 510-700 Lux

Mortality and Symptoms of Toxicity
After 7 and 14 days of exposure, the content of each single test vessel was emptied onto a plate. The surviving worms were counted and inspected for abnormal behavior or other symptoms of toxicity (worms not moving after gentle mechanical stimulus to their front end are considered to be dead). After the 7-day assessment, the soil of each test vessel was refilled into the test vessel and the worms were replaced into the same test substrate.

Body Weight
The mean body wet weights of the test organisms were determined at the start and at the end of the test. For this purpose, the surviving worms of each test vessel were weighed all together and the weight was divided by the number of surviving worms. Before weighing, the worms were quickly washed with water and surplus water was adsorbed on filter paper. For each test vessel, the difference of the mean body wet weight of the surviving test organisms behween the start and the end of the test was calculated.

Soil Moisture
Prior to the start of the test, the artificial soil moisture was adjusted to approximately 35 % (based on dry weight). After addition of the worms, the vessels were covered with glass lids to reduce evaporation. At the end of the test, the soil moisture was checked at all test concentrations and the control. Aliquots from the substrates of the four replicates per test concentration and control were mixed. The soil moisture in the mixed samples (of approximately 40 g wet weight) was calculated from the determination of the dry weight of the samples.

pH and Temperature
At the start of the test, pH was measured in the untreated artificial soil. At the end of the test, the pH of the soil was checked again for all test concentrations and the control. Aliquots from the substrates of the four replicates per test concentration and control were mixed. The pH was determined in these mixed samples: an aliquot of about 10 g was shaken with 25 mL KCl solution (1 M) for 30 minutes and the pH was measured in this suspension by a pH-electrode. The test temperature (room temperature) was continuously monitored by a temperature recorder.


- Range finding study
- Results used to determine the conditions for the definitive study: The test concentrations were based on the results of a range-finding test (without GLP) and selected in consultation with the Sponsor. However, nominal concentrations in excess of 1000 mg/kg of dry soil were not tested.
Nominal and measured concentrations:
Nominal: 0, 62.5, 125, 250, 500, 1000 mg/kg dry soil
Reference substance (positive control):
14 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
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: no
- Total mass of adults at beginning of test:
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: The average change of the mean wet weight of the surviving test
organisms in the treated soil was in the range from - 1 % to 3%. Compared to the control, the changes in mean body wet weight of the worms were not statistically significantly different up to and including the highest test concentration of 1000 mg test item.
- Morphological abnormalities: not observed
- Behavioural abnormalities: not observed
- Other biological observations: not observed
Results with reference substance (positive control):
2-chloroacetamide was tested as positive control but no details on results are provided.
Reported statistics and error estimates:
The changes in mean body weight of the surviving worms were compared to the control, and were statistically evaluated by means of a multiple Dunnett-test.

Description of key information

No toxic effects have been observed in studies with soil living organisms.

Key value for chemical safety assessment

Additional information

Acute toxicity of the test item to the earthworm Eisenia fetida was determined in a 14-day test according to OECD guideline 207. The EC50 was estimated to be higher than 1000 mg test item/kg soil dw.