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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:
30 Apr - 25 Jun 2013
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:
yes (incl. QA statement)
Staatsministerium für Umwelt und Landwirtschaft, Germany
Analytical monitoring:
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The test solutions were made by dissolving weighed amounts of the test item in deionised water, immediately prior to application. The test item was dissolved in sufficient deionised water such that the addition of the test solutions to the test substrate resulted in a final water content of 40-60% of WHC. The treated substrate was thoroughly mixed using a laboratory mixer immediately after application.
- Controls: yes, artificial soil without application of test item
Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: earthworm
- Source: reared under ambient laboratory conditions in the test facility (original breeding of animals was purchased from “W. Neudorff GmbH KG”, Emmerthal, Germany)
- Age at test initiation (mean and range, SD): adult worms (approximately 3 months old with clitellum)
- Weight at test initiation (mean and range, SD): 304 - 468 mg/worm

- Acclimation period: approximately 24 h
- Acclimation conditions (same as test or not): same as test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
56 d
Test temperature:
18.5 - 21.8 °C
test start: 6.13 - 6.21
test end: 5.80 - 5.85
Water content (%, pooled replicates per treatment group)
test start: 34.9 – 35.1 % (equivalent to 55.8 – 56.1 % of WHC)
test end: 34.5 – 34.8 % (equivalent to 55.1 – 55.6 % of WHC)
Details on test conditions:
- Test container (material, size): plastic vessel of Bellaplast (inside dimensions: about 16.5 cm x 12 cm x 6 cm) with a lid pervious to air and light
- Amount of soil or substrate: 810 g wet weight corresponding to 600 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 10

Composition of artificial soil:
- 10 % sphagnum peat; origin: Torfwerk Moorkultur Ramsloh, 26683 Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant parts remained, finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content > 30 %); type: Kaolin W, origin: ERBSLÖH Lohrheim GmbH, 65558 Lohrheim, Germany - 0.5 % calcium carbonate; origin: MERCK KGaA, 64271 Darmstadt, Germany
- 69.5 % industrial quartz sand; type: Millisil W3, origin: Quarzwerke GmbH, 50207 Frechen, Germany (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- deionised water

- Photoperiod: light : dark = 16 h : 8 h
- Light intensity: 610 lux artificial light (Lumilux L58W)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- individual fresh weight (mg/worm) at test start and after 4 weeks
- behaviour of earthworms and pathological symptoms weekly
- number of surviving adult earthworms per replicate after 4 weeks
- number of surviving juveniles per replicate after 8 weeks

Nominal and measured concentrations:
Nominal: control, 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight
Reference substance (positive control):
Nutdazim 50 FLOW (Carbendazim, SC 500)
56 d
Dose descriptor:
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
56 d
Dose descriptor:
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
56 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 statistically significant mortality compared to the control was observed at any test item concentration (<= 5%)
- Initial fresh weight / worm:
control: 364.0 mg
62.5 mg/kg soil dw: 364.7 mg
125 mg/kg soil dw: 366.8 mg
250 mg/kg soil dw: 367.4 mg
500 mg/kg soil dw: 365.3 mg
1000 mg/kg soil dw: 363.1 mg
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
control: 45.8
62.5 mg/kg soil dw: 43.7%
125 mg/kg soil dw: 40.9%
250 mg/kg soil dw: 46.8%
500 mg/kg soil dw: 46.5%
1000 mg/kg soil dw: 45.7%
The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control treatment at any concentration tested.
- No. of offspring produced (mean):
control: 98.9
62.5 mg/kg soil dw: 104.3
125 mg/kg soil dw: 106.5
250 mg/kg soil dw: 92.8
500 mg/kg soil dw: 81.8
1000 mg/kg soil dw: 60.3
Statistically significant effects on number of juveniles compared to the control group were recorded at a concentration of 1000 mg test item/kg soil dry weight.
- Behavioural abnormalities: No effects on behaviour (including feeding activity) of the worms were observed during the test.
Results with reference substance (positive control):
In the most recent study with Nutdazim 50 FLOW (BioChem project No. R 12 10 48 004 S, dated October 29, 2012), the number of juveniles was reduced by 72.7 and 98.8 % at concentrations of 5 and 10 mg product/kg soil dry weight (mean number of juveniles = 23 and 1) after 8 weeks of test duration when compared to control (mean number of juveniles = 84).
Reported statistics and error estimates:
The statistical analysis was performed with the software ToxRat Professional 2.10.06 (Ratte 2010). The EC10, EC20 and EC50 values (number of juveniles) were estimated by Probit analysis using the maximum likelihood method (Finney 1971). Fisher`s Exact Binomial Test with Bonferroni Correction and the Williams-t-test were used to compare the control with the independent test item groups. For statistical evaluation of the biomass change, the changed mean fresh weight of surviving worms per replicate was used.
Validity criteria fulfilled:

Description of key information

NOEC (56 d) = 500 mg/kg soil dw (Eisenia fetida; OECD 222)

Key value for chemical safety assessment

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

Additional information

One study is available on the toxicity of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid to soil macroorganisms. The study was conducted according to OECD Guideline 222 and GLP (Friedrich, 2013). The test organism Eisenia fetida was exposed to the test substance at nominal concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight for 56 days. No statistically significant effect on mortality was observed at any test item concentration after 28 d. However, significant effects on number of juveniles compared to the control group were recorded at a concentration of 1000 mg test item/kg soil dry weight. The NOEC (56 d) was thus determined to be 500 mg/kg soil d.w..