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Toxicity to soil macroorganisms except arthropods

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toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Mar - 11 May 1999
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
according to guideline
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Version / remarks:
lower peat content (5%)
GLP compliance:
yes (incl. QA statement)
Delivered by : Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, on 04 March 1999 for categories: 4, 5 and 7
Analytical monitoring:
Details on preparation and application of test substrate:
For the study, 50 mg of the test substance were added to 1000 mL of deionised water and stirred on a magnetic stirrer for 10 minutes (stock solution 1). Then, 100 mL of stock-solution 1 were added to 1000 mL deionised water and also stirred on a magnetic stirrer for 10 minutes (stock solution 2). The dosages for this study were prepared by mixing equivalent parts of stock-solution 2 with water. The test concentrations were prepared by mixing the different amounts of these solutions (each dissolved in 100 mL deionised water) into the test substrate with a domestic mixer.
Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
To obtain earthworms for the study, adult earthworms of breeding boxes were transferred to freshly prepared culture substrate for reproduction. The earthworms were removed after 4 weeks. After further 12 months, adult earthworms, which developed from cocoons, were selected for the study.
One day prior to the start of the study, earthworms were removed from the breeding substrate and kept in the test soil (without test substance) under the test conditions for acclimatization. They were removed from this artificial soil and transferred to test boxes after mixing the test concentrations into the test substrate. 10 earthworms were placed in each test box in a randomized test procedure. At the start of the study, they had a mean weight of 0.40 g.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
56 d
Test temperature:
20 ± 2 °C
6.2 - 6.7
53.4 to 54.5 % of the WHC at the start
Soil moisture was maintained in acceptable ranges throughout the test
Details on test conditions:
Artificial soil:
The test soil consisted of 74 % fine quartz sand (84% of the sand has a particle size of 0.06 - 0.2 mm), 5 % dried, finely ground peat (sphagnum peat; pH 2 - 4), 20 % kaolin (kaolinite content of about 36%, pH value ca 7), 1% dried, finely ground cattle manure and approximately 0.25 % calcium carbonate (pure) to adjust the pH value to 6 ± 0.5.

Test system:
The artificial soil was first mixed dry from these components with the added food in a plough-share mixer. The mixture was then moistened with the volume of water required for the final artificial soil. 500 g dry weight soil (equivalent to 675 g wet weight) were prepared for each test container. The test containers were 1.2 L plastic boxes of about 16.5 cm length, 12 cm width and 6 cm height (inner size). Each box was covered with a fine mesh gauze (mesh size 0.5 mm) and labelled. The artificial soil was acclimatized for 1 day under test conditions before the test substance was mixed in.
Four replicates were prepared for each test concentration.
After 4 weeks the number of surviving animals and their weight alteration was determined. They were then removed from the artificial soil. After further 4 weeks, the number of offspring was determined.
Nominal and measured concentrations:
Nominal: 56, 75, 100, 133 and 178 µg test substance/kg dry weight soil.
Reference substance (positive control):
Key result
56 d
Dose descriptor:
Effect conc.:
>= 178 µg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
No mortality nor a body weight reduction of adult earthworms was observed at any concentration. The number of offspring was not reduced at any concentration: the test material did not affect the body weight and reproduction of earthworms.
Results with reference substance (positive control):
Mortality or body weight reduction of adult earthworms as compared to control organisms was not observerd at any dosage. Only the highest dosage of 0.5 kg formulation/ha reduced the numbers of juvenile earthworms by 60 %. The lowest observed-effect-level (LOEL) 0.5 kg/ha (= 0.079 kg active ingredient/ha), equivalent to 0,105 mg a.s./kg dry soil.

Please refer to "overall remark/ attached background material" field for result tables.

Table 1: Validity criteria as reported from the report

Criterion from the guideline to be satisfied in the controls


Validity criterion fulfilled

Adult mortality over the initial 4 weeks of the test is ≤ 10%.

0 %


Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.

114 juveniles


Soil moisture

Maintained in acceptable range



Validity criteria fulfilled:
According to the ones mentioned in the report. For further details please refer to “Any other information on results incl. tables”.
The present guideline study was conducted in compliance with GLP. Under the test conditions used, the reproduction NOEC for Eisenia fetida was ≥178 µg a.s./kg dry soil.

Description of key information

The overall long-term NOEC for the soil macroorganisms except arthropods (Eisenia fetida) is 178 µg a.s./kg dry weight soil.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
10.7 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
178 µg/kg soil dw

Additional information

In the key study (1999), the chronic toxicity of test material technical to earthworms (Eisenia fetida) was investigated after 56-day exposure according to ISO 11268-2 guideline. Ten earthworms, in each of the four replicate, were exposed to concentrations of 56, 75, 100, 133, 178 µg test substance/kg dry weight soil, alongside with a control. The reproduction NOEC was determined to be 178 µg test item/kg soil d.w. the highest concentration tested.


A supportive acute toxicity study according to OECD 207 was performed with Eisenia andrei as test organism. In this test, the LC50 (14-d) was 10.7 mg a.s./kg dws.