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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 December 2016 - 20 February 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
according to guideline
other: Ministry of environmental protection of the people's Repulic of China, The Guidelines for Testing of Chemicals - Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test
Version / remarks:
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
C21 H30 N6 O6
Constituent 2
Chemical structure
Reference substance name:
2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate
Molecular formula:
C18 H30 N4 O5
2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate
Test material form:
liquid: viscous
Details on test material:
Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.

Sampling and analysis

Analytical monitoring:

Test substrate

Details on preparation and application of test substrate:
- Method of preperation of test substrate: per treatment group, approx. 2,5 kg of substrate was prepared and evenly distributed into four containers (approx 750 g wet weight/container).
2521.1 mg of the test substance was dissolved in 10 mL acetone to give the solvent stock solution. This solution was inverted several time to ensure adequate mixing and homogeneity. An aliquot solvent solution was seperately added to a certain amount of silica sand and the solvent allowed to evaporate off prior to incorporation into formulated artificial soil with demonized reverse osmosis water. These were mixed by a mixer to give the blank control, solvent control, and test substrate, with a nominal moisture content of 40-60% by mass of the total water holding capacity with the demonized water.
- Method of mixing into soil (if used): mixer
- Controls: yes, blank control and solvent control
- Chemical name of solvent: 2-propanone (acetone)
- Evaporation of solvent before use: yes
- Concentration of vehicle in test medium: approx. 4 mL/kg dry soil weight in the initial mixture before the solvent was evaporated off.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Common name: earthworm
- Source: Jiang Su province Jurong Stock Farm
- Age at test initiation: adult earthworms, with clitellum from the same population, were used
- Weight at test initiation: 300 - 600 mg (wet weight)

- Quarantaine period: all newly aquired earthworms were quarantined and observed for 14 days prior to use in the test
- Breeding: earthworms were bred on a substrate consisting of 50% by volume peat and 50% by volume cow manure and kept under 20 ±2°C for 7 days prior to testing.
- Acclimation period: 24 hours
- Acclimation conditions: yes; earthworms were not exposed to test substance
- Health during acclimation: no mortality or other health defects were observed during the acclimation period

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
Total exposure duration:
14 d

Test conditions

Test temperature:
20.1 - 20.3°C
Start: 6.25 - 6.28
End: 6.24 - 6.27
Start: 25.07 - 26.38%
End: 24.30 - 26.10%
Details on test conditions:
- Test container: 1L breathable glass bottles with cover
- Amount of soil or substrate: 750 g (wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 1
- No. of replicates per solvent control: 1

- Composition (artificial substrate): Spagnum peat (10% w/w), Kaolinite clay (20% w/w) and Silica sand (screened with 0.2mm sieve; 70% w/w)
- Maximum water holding capacity (in % dry weigth): 49%
- Pretreatment of soil: the pH was adjusted to 6.0 ± 0.5 and moisture conent was adjusted to 40-60% by mass of the total water holding capacity with deionized water
- Storage (condition, duration): not indicated
- Stability and homogeneity of test material in the medium: not indicated

- Photoperiod: the vessels were covered and kept under continuous illumination
- Light intensity: 595-622 lux
- Feeding during exposure: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality at day 7 and at day 14.
- Other measurements: temperature and light intensity (daily); soil moisture contents and pH value of the blank control group, solvent conrol group and the treatment group, in one container randomly (at the beginning and at the end of the test).


- Spacing factor for test concentrations: not applicable since the test was performed as a limit test.
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/kg
- Results used to determine the conditions for the definitive study: yes, based on the results of the range findng study, a limit test was carried out using a nominal concentration of 1000 mg/kg
Nominal and measured concentrations:
Nominal concentration: 1000 mg/kg dry soil
Reference substance (positive control):
chloroacetamide (test date: 27 Sept. 2016 - 11 Oct. 2016)

Results and discussion

Effect concentrations
Key result
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 mortality occured at the blank control group and at the solvent control group. In the treatment group, mortality was 2.5% at the end of the exposure period (day 14).
- Total mass of adults at the beginning of the test:
* blank controls: 2095
* solvent controls:2134
* treatment group: 2065
- Total mass of adults at the end of the test:
* blank controls: 1839
* solvent controls: 1886
* treatment group: 1699
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
* blank controls: 12.25 ± 1.45%
* solvent controls: 11.64 ± 0.85%
* treatment group: 17.7 ± 6.39%
- No abnormalities were observed in any of the treatment groups.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: LC50: 21.4 mg/kg dry soil weight (95% CI: 18.3-24.8 mg/kg dw
- Other: the LC50 range for tests performed during 2009 and 2015 was 3.1 - 30.3 mg/kg. The obtained result in the reference study was within the historical range.
Reported statistics and error estimates:
Differences of the average body weight of the surviving earthworms in each group during the test were determined by using Excel.

Applicant's summary and conclusion

Validity criteria fulfilled:
No mortality occured in the control groups.
The LC50 for acute toxicity to earthworms (14-day exposure) was > 1000 mg/kg dry soil weight.
Executive summary:

In an acute toxicity test, performed according to OECD guideline 207 and GLP principles, Eisenia fetidae (earthworms) were exposed to a limit concentration (1000 mg/kg dry weight) of STABiO D-376N for 14 days. A blank control and a solvent control group were included. The treatment group and the control groups consisted of in 4 replicates of 10 worms each. Acetone was used as a solvent to apply the test substance. The test was performed in artificial soil consisting of peat (10%), clay (20%) and silica sand (70%). Based on the results of a range finding study in which no mortality occured in all tested concentrations (10, 100 and 1000 mg/kg dry soil), a nominal concentration of 1000 mg/kg dry soil was used in the final test. Mortality was recorded at day 7 and at day 14. No mortality occured in the blank control group and in the solvent control group. In the treatment group, no mortality occured after 7 days and 2.5% mortality occured after 14 days. The LC50 was determined to be >1000 mg/kg dry weight, based on the nominal test concentration.