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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2018 to 29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 207 Earthworm Acute Toxicity Test [M].
Version / remarks:
The Editorial Board of “The Guidelines for the Testing of Chemicals” of Chemical Registration Center of the Ministry of Environmental Protection. Second Edition Beijing: China Environmental Press. 2013, Page 58-62.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: China Hazardous Chemicals Management Standardization Technical Committee. GB/T 21809-2008 Chemicals -Test Method of Earthworm Acute Toxicity Test [S].
Version / remarks:
Beijing: China Standard Press, 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Department of Science, Technology and Standards of State Environmental Protection Administration. HJ/T 153 - 2004 The Guidelines of Chemical Testing [S].
Version / remarks:
Beijing: China Environmental Press, 2004.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Redworm
- Source: The test species was purchased from Wuxi Earthworm Breeding Base (Code No.: Earthworm 20171226) and is raised in the test facility of CTI-SET.
- Age at test initiation: Earthworms were adult (at least two months old with clitellum)
- Weight at test initiation: Earthworms with individual wet mass of 307-399 mg
- Justification for selection: Eisenia foetida is recommended as the test species by The Guidelines for the Testing of Chemicals. Characteristics which make this test organism suitable for toxicity testing are its ease of obtaining, raising and sensitivity to a variety of chemical substances.

ACCLIMATION
- Adaptation: Prior to test, the earthworms were placed in the artificial soil for approximately 24 hours under the test conditions.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
18.9 - 20.9 °C
pH:
6.42 - 6.45
Moisture:
33.4 - 35.8 %
Details on test conditions:
TEST ORGANISM
- Common name: Redworm
- Source: The test species was purchased from Wuxi Earthworm Breeding Base (Code No.: Earthworm 20171226) and is raised in the test facility of CTI-SET.
- Age at test initiation: Earthworms were adult (at least two months old with clitellum)
- Weight at test initiation: Earthworms with individual wet mass of 300-600 mg.
- Justification for selection: Eisenia foetida is recommended as the test species by The Guidelines for the Testing of Chemicals. Characteristics which make this test organism suitable for toxicity testing are its ease of obtaining, raising and sensitivity to a variety of chemical substances.

ACCLIMATION
- Adaptation: Prior to test, the earthworms were placed in the artificial soil for approximately 24 hours under the test conditions.

TEST SYSTEM
- Amount of soil or substrate: 750 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per vehicle control: 4
- Before exposure, twenty earthworms will be randomly selected from the population of adapted earthworm and weighed individually. The individual wet weight was in the range of 307-399 mg, and satisfied the required range of 300 mg to 600 mg. A group of ten earthworms for each replicate will be selected, weighed together and randomly assigned to test containers. The test worms were placed on the surface of the soil and allowed to enter into the soil. The containers were covered with perforated plastic film. When the earthworms were placed in the test containers, the exposure was initiated.

SOURCE AND PROPERTIES OF SUBSTRATE
- Preparation of Artificial Soil: The constituents of artificial soil (calculated as dry weight) are listed as follows: 10 % sphagnum peat (no visible plant remains, finely ground, the moisture content was measured to be 150.2 %, the amount of sphagnum peat added was based on dry weight); 20 % kaolin clay; 70 % quartz sand. The artificial soil (calculated as dry weight) was prepared and mixed thoroughly in a mixer. The pH value of the soil was determined to be 6.26. The dry artificial soil was taken and pre-moistened with purified water. Before the test, the wet soil was sampled and dried for about 3 hours at 105 °C to determine the moisture content to be 21.5 %.
- Preparation of the Test Soil:
For the blank control group, 717 g of wet soil (moisture content 21.5 %; dry weight 590 g) was mixed with 10 g quartz sand. 83 mL of purified water was mixed thoroughly with the soil to adjust the moisture content of the test soil to approximately 35 %. Four replicates were prepared.
For solvent control group, 4 mL dichloromethane was mixed with 10 g quartz sand and placed under the fume hood to allow the solvent to evaporate completely. Next, the quartz sand was mixed with 717 g of wet soil (moisture content 21.5 %; dry weight 590 g). 83 mL of purified water was mixed thoroughly with the soil to adjust the moisture content of the test soil to approximately 35 %. Four replicates were prepared.
For test material treatment group, 601.9 mg, 601.2 mg, 605.5 mg and 604.6 mg of test material was weighed, dissolved in 4mL dichloromethane, and mixed with 10 g quartz sand respectively. And then, the mixture was then mixed with 717 g of wet soil (moisture content 21.5 %; dry weight 590 g) to obtain the soil with final test material concentration of 1000 mg/kg. 83 mL the purified water was mixed thoroughly with the soil to adjust the moisture content of the test soil to approximately 35 %. Four replicates were prepared.
About 750 g test soil was weighed and added into each test container. The remaining test soil was used to determine the moisture content and pH value.

OTHER TEST CONDITIONS
- Humidity: 70-88 %
- Light intensity: 508-720 lux at the start of the test

EFFECT PARAMETERS MEASURED:
- The light intensity of the test area was measured at the start of the test. The temperature and humidity was monitored by the temperature and humidity recorder.
- Moisture content of the test soil was measured at the start and end of the test. Four samples (each containing about 10 g of test soil) were taken from each group and dried about 3 hours at 105 °C for moisture content measurement.
- At the start of the test, test soil of each treatment group was sampled, and the pH value was measured after air drying and sieving.
- The survival and toxicity symptoms of earthworm were assessed after 7 days and 14 days of exposure. The earthworm was classified as dead when they did not response to a gentle mechanical stimulus to the front end. The wet mass of the survived worms in each test container was weighed together at 14 days of exposure.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Preliminary Test (Non-GLP): To assist the selection of the test concentrations, a preliminary test (study No.: G18207003P) was performed prior to the definitive test. Due to low water solubility of the test material, dichloromethane was selected as solvent to assist the dispersion of test material on test soil. The preliminary test was conducted using the test concentrations of 100 mg/kg, 500 mg/kg and 1000 mg/kg. Blank control and solvent control groups were set in parallel. The test duration was 14 days. The result of the preliminary test showed that the mortality of earthworm in the control and the test material concentrations of 100 mg/kg, 500 mg/kg and 1000 mg/kg after 7 days and 14 days of exposure were 0 % and no abnormal symptoms were observed. Based on the results of the preliminary test, the limit test was conducted at the concentration of 1000 mg/kg.
Nominal and measured concentrations:
Nominal: 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
2-chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
BIOLOGICAL OBSERVATION AND DEMAND
- The mortality of earthworms in blank control, solvent control and test material treatment group was 0 % after 7 days and 14 days of exposure and there were no abnormal symptoms.
- The mean wet weight of the earthworms in controls and test material treatment group was 310-360 mg at the start of the test. The mean wet weight of the earthworms was 280-307 mg at the end of the test.

LC50
- The 7d-LC50 and 14d-LC50 of test material to earthworm was > 1000 mg/kg.

VALIDITY OF THE TEST
- The mortality in the controls and solvent controls was 0 % at the end of the test (less than 10 %), which satisfied the validity of the test.
Results with reference substance (positive control):
The result of the latest sensitivity test (Study No.:18ST001) conducted from 16 March 2018 to 30 March 2018 indicated that the 14d-LC50 of 2-chloroacetamide to Eisenia foetida was 59.86 mg/kg and its 95 % confidence limits were 55.20-64.92 mg/kg, which was within the recommended range (20-80 mg/kg) based on
GB/T 31270.15-2014.

Validity of the Test

The mortality in the controls and solvent controls was 0 % at the end of the test (less than 10 %), which satisfied the validity of the test.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 14d-LC50 of test material to earthworms (Eisenia foetida) was greater than 1000 mg/kg.
Executive summary:

The toxicity of the test material to earthworms was determined in accordance with the standardised guideline OECD 207 and other Chinese guidelines, under GLP conditions.

Based on preliminary investigation, the study was performed as limit test to evaluate the acute toxicity of test material to earthworm (Eisenia foetida). The test duration was 14 days. The test concentration was set as 1000 mg/kg. Additionally, blank control and solvent control were set in parallel. Four replicates were prepared for each group and each replicate contained 10 earthworms. The survival and toxic symptoms of the test earthworms were assessed after 7 and 14 days of exposure and the LC50 of test material to earthworm was calculated.

The light intensity of the test area was measured at the start of the test, and the temperature and humidity were monitored with the temperature and humidity recorder. Moisture content of the test soil was measured at the start and end of the test. The pH value of the test soil was measured at the start of the test.

The light intensity was in the range of 508-720 lux at the start of the test. During the test, the temperature was in the range of 18.9-20.9 °C and the humidity was in the range of 70-88 %. The moisture content of test soil in the controls and test material concentration groups was in the range of 34.5-35.8 % at the start of the test and 33.4-34.5 % at the end of the test, respectively. The pH value of test soil was in the range of 6.42-6.45 at the start of the test. The mean wet weight of the earthworms in the controls and the test material treatment group was 310-360 mg at the start of the test and 280-307 mg at the end of the test. The mortality of earthworms in both the controls and the test material treatment group of 1000 mg/kg was 0 % after 7 days and 14 days of exposure and there were no abnormal symptoms.

Under the conditions of this study, the 14d-LC50 of test material to earthworms (Eisenia foetida) was greater than 1000 mg/kg.

Description of key information

Under the conditions of this study, the 14d-LC50 of test material to earthworms (Eisenia foetida) was greater than 1000 mg/kg.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw

Additional information

The toxicity of the test material to earthworms was determined in accordance with the standardised guideline OECD 207 and other Chinese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based on preliminary investigation, the study was performed as limit test to evaluate the acute toxicity of test material to earthworm (Eisenia foetida). The test duration was 14 days. The test concentration was set as 1000 mg/kg. Additionally, blank control and solvent control were set in parallel. Four replicates were prepared for each group and each replicate contained 10 earthworms. The survivals and toxic symptoms of the test earthworms were assessed after 7 and 14 days of exposure and the LC50 of test material to earthworm was calculated.

The light intensity of the test area was measured at the start of the test, and the temperature and humidity were monitored with the temperature and humidity recorder. Moisture content of the test soil was measured at the start and end of the test. The pH value of the test soil was measured at the start of the test.

The light intensity was in the range of 508-720 lux at the start of the test. During the test, the temperature was in the range of 18.9-20.9 °C and the humidity was in the range of 70-88 %. The moisture content of test soil in the controls and test material concentration groups was in the range of 34.5-35.8 % at the start of the test and 33.4-34.5 % at the end of the test, respectively. The pH value of test soil was in the range of 6.42-6.45 at the start of the test. The mean wet weight of the earthworms in the controls and the test material treatment group was 310-360 mg at the start of the test and 280-307 mg at the end of the test. The mortality of earthworms in both the controls and the test material treatment group of 1000 mg/kg was 0 % after 7 days and 14 days of exposure and there were no abnormal symptoms.

Under the conditions of this study, the 14d-LC50 of test material to earthworms (Eisenia foetida) was greater than 1000 mg/kg.