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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:

Test material

Constituent 1
Reference substance name:
Constituent 2
Test material form:
solid: particulate/powder
migrated information: powder

Sampling and analysis

Analytical monitoring:

Test substrate

Details on preparation and application of test substrate:
An amount of test substances were dissolved in the dichloromethane to obtain solvent stock solution. Each solvent stock solution was inverted several times to ensure adequate mixing and homogeneity
An aliquot solvent stock solution were separately added to certain amount of silica sand and the solvent were allowed to evaporate off prior to incorporation into formulated artificial soil with deionised reverse osmosis water. These were mixed by hand to obtain the test concentrations of 1000 mg/kg respectively.
The solvent controls were maintained under identical condition but not exposed to the test substance. The blank controls were maintained under identical condition but not exposed to solvent or the test substance.
For the controls and each test concentration 10 earthworms were added to each 1 liter closed glass vessel containing approximately 750 wet weight of substrate. The vessels were covered and maintained under continuous illumination (508 - 525 lux) in a climate chamber at 19.8 to 21 .0°C for a period of 14 days. The earthworms received no food during exposure. The total gross weights of the worm added to each beaker of the controls and test groups were recorded on Day 0 before addition to the test or control vessels. The surviving earthworms of each beaker were weighed on Day 14.
Any mortalities were recorded at 7 and 14 days after the start of treatment.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
Details on test organisms:
- Source: Shanghai Yinquan Agriculture Development Co., Ltd.
- Age at test initiation (mean and range, SD): > 2 month
- Weight at test initiation (mean and range, SD): 300 - 600 mg wet weight

- All newly acquired earthworms were quarantined and observed for 14 days prior to use in the test.
- Earthworms are 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.
- The test organisms were with clitellum and were transferred into artificial soil (without test substance) approximately 24hours prior to the start of the test.

Study design

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

Test conditions

Test temperature:
19.8 to 21.0 °C
test start: 6.09 - 6.21
test end: 5.93 - 6.09
test start: 23.94 - 25.63%
test end: 22.32- 24.10%
Details on test conditions:
Sphagnum peat: 10% w/w
Kaolinite clay: 20% w/w
Silica sand (screened with 0.2 mm sieve): 70% w/w

The pH of the artificial soil was adjusted to 6.0 + 0.5 by the addition of calcium carbonate. These ingredients were weighed and mixed in the above proportions. Moisture content of the test substrate was adjusted to 40-60% by mass of the total water-holding capacity with the deionized water.
The measured value was 44%, so the moisture content of the artificial soil should be in the range of 18%-26%.

Mortality was assessed at 7 and 14 days after application.
Nominal and measured concentrations:
The nominal concentration of the test substance was 1000 mg/kg
Reference substance (positive control):

Results and discussion

Effect concentrations
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:
No mortalities occurred in the control group at the 7th and 14th day after application, the mortalities of S-control group was 0% at the 7th and 2.5% at the 14th day after application and the mortalities of treatment group was 2.5% at the 7th and 7.5% at the 14th day after application. According to the result of Homoscedastic t-test, compared with the blank control group, the blank control group and the 1000 mg/kg treatment group had no significant difference)(p=0.1625, p>0.05).
Results with reference substance (positive control):
The most recent test with chloroacetamide was performed from 16 Oct. 2013 to 30 Oct. 2013. The 14-days LCso for chloroacetamide
was 15 mg/kg dry soil weight.
Reported statistics and error estimates:
The differences of the average body weight of the surviving earthworms between the control group and the trealment groups were determined by using the Homoscedastic t-Test at the end of the study. The data analyses were carried out with Toxcalc software v5.0.32.

Applicant's summary and conclusion

Validity criteria fulfilled:
Mortality rate in the blank control group and solvent control group were 0% and 2.5% which was not more than 10 per cent, so the test was valid.
The 14-day LC50 value was more than 1000 mg/kg dry soil weight.

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