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Ecotoxicological information

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:
20 Oct - 15 Dec 2021
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
GLP compliance:
Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen
Analytical monitoring:
Details on preparation and application of test substrate:
- Method of mixing into soil: The quantity of test item required to obtain the desired concentrations was applied via a portion of quartz sand. For this, the required amount of test item (32.25 – 500 mg per vessel) was applied on 10 g quartz sand. Both ingredients were mixed thoroughly. Subsequently, the mixture was added to 561 g moist test substrate, representing 500 g dry mass and mixed thoroughly. Subsequently, the volume of water needed to adjust the mixture on 55% WHCmax was added and the test matrix was mixed thoroughly.
- Controls: Test substrate adjusted to 40 - 60% of the maximum water holding capacity.
Test organisms (species):
Eisenia andrei
Animal group:
Details on test organisms:
- Common name: Earthworm
- Source: Regenwurmfarm Tacke, Borken, Germany
- Age at test initiation: 2 months – 1 Year
- Weight at test initiation: 250 – 600 mg

- Acclimation period:5 days
- Acclimation conditions: In artificial soil using same food which was also used in the test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
28 d
Post exposure observation period:
28 days, to observe the reproducitve output.
Test temperature:
18 - 21 °C
5.5 - 5.9
50.8 - 55.4 % WHC
Details on test conditions:
- Test container (material, size): Plastic container, 17 x 12 cm; 6 cm height
- Amount of soil or substrate: 500 – 650 g of dry mass artificial soil to ensure a moist substrate depth of about 5-6 cm
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

- Composition:
Sphagnum peat, air-dried, finely ground 10 %
Kaolinite, air-dried 20 %
Industrial quartz sand, air-dried 70 %
- Maximum water holding capacity: 55 %

- Photoperiod: 16 h : 8 h
- Light intensity: 547 – 701 lx

- Physical/Pathological symptoms and changes in behavior
- Survival rate (after 28 d)
- Weight change (after 28 d)
- Reproduction (after 56 d)

- Spacing factor for test concentrations: 2
Nominal and measured concentrations:
Nominal (mg test item/ kg soil dw): 0 (control), 62.5, 125, 250, 500, and 1000
Reference substance (positive control):
Carbendazim (during a separate, sub-acute toxicity tests under non-GLP conditions)
56 d
Dose descriptor:
Effect conc.:
261.2 mg/kg soil dw
95% CI:
>= 58.1 - <= 425.7
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
56 d
Dose descriptor:
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
28 d
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: weight change, survivial rate
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: Weight change, survival rate, reproduction
Details on results:
- Mortality at end of exposure period: The survival of Eisenia andrei was not affected by test item up to and including the highest test concentration. No concentration-effect dependency occurred.
- Total mass of adults at beginning of test: wet mass between 250 mg and 600 mg
- Changes in body weight of live adults (% of initial weight) at end of exposure period: For control, 62.5, 125, 250, 500, and 1000 mg/kg soil dw: 58.2, 62.9, 67.3, 67.4, 68.3 and 63.3 %
- Mean number of offspring produced for control, 62.5, 125, 250, 500, and 1000 mg/kg soil dw: 104.3, 108.8, 106.3, 88.8, 92.3, 80.8 (mean number of juveniles)
- Morphological abnormalities: None.
- Behavioural abnormalities: None.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: In this last reference test, the NOEC for weight change and survival was found to be ≥ 5 mg/kg. No stat. significant concentration/effect dependence was obtained. EC values could not be calculated. Reproduction was stat. significantly affected by carbendazim. The NOEC was suggested to be 1.58 mg/kg. The EC10 and EC50 were 1.2 mg/kg and 2.9 mg/kg, respectively.
Reported statistics and error estimates:
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations are based on the nominal test item concentrations. For each concentration, the percent mortality, the percent loss/increase in biomass of the adults, and the number of offspring produced in the test was determined. The percent deviation was calculated in comparison to the control. Means were compared by appropriate statistical methods (ANOVA followed by Dunnett’s Multiple t-Test or Williams` Multiple Sequential t-Test for metric response after trend analysis of contrasts). Where the test results show inhibition around 50% they were statistically analysed to determine an EC10, EC20 and EC50 value together with 95% confidence intervals using an appropriate statistical tool. If it was not possible to determine EC values because no inhibition was observed, the respective EC value is reported as being > highest nominal (or measured – defined in previous paragraph) concentration tested. All calculations were performed with the computer software ToxRat Professional by ToxRat® Solutions GmbH.

Table 1: Validity criteria for OECD 222 (2016)

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.

104.3 (mean); 83 – 120 (range)


The coefficient of variation of reproduction is ≤ 30%.

14.3 %



Validity criteria fulfilled:
For further details please refer to table 1 within “Any other information on results incl. tables”
After exposure of Eisenia andrei to the test item in a chronic test, the most sensitive NOEC (56 d) was found for the endpoint reproduction and determined to be 500 mg/kg soil dry weight, i.e. the second highest concentration tested. A respective EC10 (56 d) for reproduction was calculated to be 261.2 mg test item/kg soil.

Description of key information

NOEC (56 d): 500 mg test item/kg dw for reproduction (Eisenia andrei, OECD 222)

EC10 (56 d): 261.2 mg test item/kg dw for reproduction (Eisenia andrei, OECD 222)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
261.2 mg/kg soil dw

Additional information

One GLP study according to OECD guideline 222 on the chronic toxicity to earthworms (Eisenia andrei) using artificial soil was performed with the test item. Earthworms were exposed to nominal concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg dry weight at 20 ± 2 °C for 56 days. After the first 28 days adult earthworms were removed and the survival rate and biomass was determined.

The reproduction rate was determined by counting juvenile earthworms 28 days later and thus after 56 days in total. Appropriate concentrations were obtained by mixing the test substance into the test substrate. No analytical verification was performed.

No statistical significant effects on survival and biomass of the adult (changes in body weight) were observed in the control or at the test concentrations. However, a statistical significant difference was observed for the reproduction of the earthworms between the control group and the highest test concentration after 56 d. Thus, the 28-d NOEC for mortality and growth was ≥ 1000 mg test item/kg soil dry weight (nominal) and the 56 d-NOEC for reproduction was 500 mg test item/kg soil dry weight (nominal). The respective 56 d-EC10 for reproduction was determined to be 261.2 mg test item/kg dw. The test was valid according to the OECD guideline 222 validity criteria.