Registration Dossier

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January 2015 to 25 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004. The guidelines for the testing of chemicals [S]. Beijing: State Environmental Protection Administration, 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals-Effects on Biotic Systems. [M]. The second edition. Beijing: China Environment Press. 2013: 58-62
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21809-2008, Chemicals-Test method of earthworm acute toxicity test [S]. Beijing: Standardization Administration of China, 2008
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
TEST SOIL
- Artificial soil test substrate was prepared by mixing:
(a) 10 % sphagnum peat (Pindstrup Mosebrug A/S; pH 6.0, no visible plant remains, finely ground, dried)
(b) 20 % kaolin clay (CAS: 1332-58-7; Lot number: 20140708; Sinopharm Chemical Reagent Co. Ltd.; kaolinite content preferably above 30 %).
(c) 70 % quartz sand (CAS: 14808-60-7; Lot number: 20140514; Sinopharm Chemical Reagent Co. Ltd.; fine sand should be dominant with 80 % of the particles between 50 and 200 microns).
- The dry constituents were blended in the correct proportions and mixed thoroughly with a large-scale laboratory mixer.

PREPARATION OF THE TEST SUBSTRATE
- A stock solution of 112 g/L was prepared by dissolving 2.8060 g test substance in 25.0 mL
acetone.
- Test substrates of different concentrations were prepared by mixing stock solution into 10 g quartz sand.
- After evaporating the acetone at the room temperature, 10 g quartz sand was then mixed with 550 g dry weight of soil for each test container.
- The medium was thoroughly mixed and deionised water was added to give an overall moisture content of about 35 % of the dry weight.
- Moisture content was determined by drying a small sample at 105 °C and re-weighing.
- Concurrent blank control and solvent control groups were included. The solvent control group was prepared with the maximum amount of acetone used in test sample preparations (1000 mg/kg artificial soil dw) and the blank control group did not contain added acetone.
- Details on the preparation of the test substrates at chosen concentrations are given in the table below.


Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
- Worms were obtained from Nanjing Earthworm Nursery, Pudong community, Changlu Street, Dachang District, Nanjing, China.
- The earthworms newly obtained in the lab were released from quarantine after 14 days and then the batch was turned into pre-culture (Batch No.: E20150204E).
- Before the test, the worms were acclimated in test soil for 34 days.
- Average weight of worms for this study was in the range 307 to 452 mg.
- The worms were pre-cultured in test soils for another 24 hours, washed quickly with distilled water and surplus water absorbed on filter paper before use.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
Not applicable
Test temperature:
20.6 to 22.3 °C
pH:
5.65 to 6.47 (see Table 1, attached)
Moisture:
26 to 35 % (see Table 1, attached)
Details on test conditions:
RANGE-FINDING TEST
- A pre-test was performed with test substance concentrations of 100, 500 and 1000 mg/kg artificial soil (dry weight) to determine the range of concentrations to be tested in the study.
- Simultaneously one blank control and one solvent control were established under the same conditions.
- One test batch for each concentration and control with 10 individuals each was used.
- The test worms (10 worms each) were firstly placed on the surface of test substrate (dry weight: 560 g each; Moisture content: 35% dry soil weight).
- After having been sealed with plastic films, containers were put into the chambers.
- The test duration was 14 days, and the test temperature was maintained at 20.1°C to 22.5°C with humidity of 80% to 85% and continuous light (intensity of 400 lux to 800 lux).

DEFINITIVE TEST
- Based on the results of the range-finding test, a Definitive test was undertaken with test concentration of 10, 25, 62.5, 156, 390 mg/kg (dry weight of soil), blank control and solvent control.
- Four replications for each concentration and control, each with 10 individuals were used.
- The test worms (10 worms each) were firstly placed on the surface of test substrate (dry weight: 560 g each; Moisture content: 35% dry soil weight).
- After having been sealed with plastic films, containers were put into the chambers.
- The test duration was 14 days, and the test temperature was 20.6°C to 22.3°C with the humidity of 80% to 85% and continuous light (intensity of 400 lux to 800 lux).

OBSERVATIONS
- On the 7th day of exposure, mortality was assessed by emptying test medium onto a stainless plate, sorting worms from the medium and testing their reaction to a mechanical stimulus at the front end.
- Worms were classified as dead when they do not respond to a gentle mechanical stimulus to the front end. Due to rapid decomposition under test conditions, missing earthworms were also considered dead.
- At the completion of this assessment, soils were replaced in the test container and the surviving earthworms were replaced on the same test substrate surface for the 14th day assessment.
- In addition, the number and type of behavioural and other pathological abnormalities (such as. lethargic or rigid worms) were determined during each mortality assessment on day 7 and day 14.
- On day 0 and 14, the weight of the worms, the pH and the moisture were measured.
- The pH value was determined according to "Soil and waste pH (Method 9045D)" (US EPA).
- The moisture content was determined by weighing and then drying a small sample at 105°C and re-weighing.
- The average weight of the worms were weighed on Day 0 prior to assigning to the test containers of both control and treatment groups.The worms were washed prior to weighing (with deionised water) and the excess water removed by placing the worms briefly on filter paper. Surviving worms were weighted with the same methods on day 14.

VALIDITY OF TEST – CONTROL GROUP
- A blank control group, comprising the same number of test worms as each treatment, was also treated with the same methods while without using the test substance.
- Simultaneously, a solvent control group was established. The solvent control group was administrated with the maximum amount of acetone used in the test sample preparations (1000 mg/kg artificial soil (dry weight)).

VALIDITY OF TEST – REFERENCE CONTROL
- The 7-day and 14-day LC50 values of a reference substance (2-chloracetamide) were determined three times a year to assure that the laboratory test conditions were adequate and had not changed significantly. The results of this study are shown in Table 3 and Figure 1 (attached). The 7-days LC50 and 14-days LC50 for reference substance were 33.6 mg/kg and 25.1 mg/kg, respectively.

VALIDITY OF TEST RESULT
- Mortality in the untreated control group at the end of the study must not exceed 10%. Otherwise the test should be considered invalid.
Nominal and measured concentrations:
- Nominal concentrations of 0, 10, 25, 62.5 156 and 390 mg/kg soil dw
Reference substance (positive control):
yes
Remarks:
2-chloroacetamide (CAS 79-07-2; purity ≥ 98.5 %; Lot number 20130905; Sinopharm Chemical Reagent Co Ltd)
Duration:
7 d
Dose descriptor:
LC0
Effect conc.:
10 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
57 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits 47.5 to 68.5 mg/kg soil dw
Duration:
14 d
Dose descriptor:
LC0
Effect conc.:
10 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
30 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits 24.9 to 36.1 mg/kg soil dw
Details on results:
TEST CONDITIONS
- During this test, artificial soils was used with the moisture content of the test medium controlled in range of 26% to 35% with pH form 5.65 to 6.47 (see Table 1, attached).
- Test conditions were maintained as follows: temperature of 20.6°C to 22.3°C, humidity of 80% to 85% and continuous light with intensity of 400 lux to 800 lux.
- No unusual circumstances that might have affected the integrity and quality of the study were observed.
- Mortality was not observed in the control at the end of the study.
- The test system was considered to be valid.

EARTHWORM MORTALITY
- The average weights of the worms at start and end of exposure are given in Table 2 (attached).
- The number of dead earthworms after 7 and 14 days is shown in Table 4 and Table 5 (attached).
- No dead worms were observed in the blank control and solvent control groups.
- No dead worms were observed in treatments of 10.0 mg/kg soil dry weight, while all worms were dead in the test treatments of 156 and 390 mg/kg soil dry weight after 14 days.
- The results showed that the 7 d-LC50 was 57.0 mg/kg dry weight soil, and the 95% confidence interval was 47.5 to 68.5 mg/kg dry weight soil; the 7 d-LC0 was 10 mg/kg dry weight soil. The 14 d-LC50 was 30.0 mg/kg dry weight soil, and the 95% confidence interval was 24.9 to 36.l mg/kg dry weight soil, 14 d-LC0 was 10 mg/kg dry weight soil.

BEHAVIOURAL EFFECTS
- At the beginning of administration, the worms dug into the soil as soon as contacting with the test material, and the survived worms remained in the soil during the course of the test.
Results with reference substance (positive control):
- The test with the reference substance is performed three times a year as a means of ensuring that the laboratory test conditions are adequate and have not changed significantly.
- The results of the most recent test are shown in Table 3 and Figure 1 (attached).
Reported statistics and error estimates:
- The LC50 and 95% confidence intervals were calculated by the Trimmed Spearman-Karber Method (Version 1.5, USEPA).
Validity criteria fulfilled:
yes
Conclusions:
The highest concentration causing no mortality 7 d-LC0 was 10.0 mg/kg dry weight soil. 7 d-LC50 was 57.0 mg/kg dry weight soil, and the 95% confidence interval was 47.5 to 68.5 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 10.0 mg/kg dry weight soil.14-d LC50 was 30.0 mg/kg dry weight soil, and the 95% confidence interval was 24.9 to 36.1 mg/kg dry weight soil.
Executive summary:

GUIDELINE

The test was conducted to determine the acute toxicity of test substances to earthworm according to "The guidelines for the testing of chemicals" (HJff 153-2004) and "The Guidelines for the Testing of Chemicals-Effects on Biotic Systems" (the 2nd edition. Beijing: China Environment Press. 2013); with reference to Procedure 207 of the ' Guidelines for Testing of Chemicals' of the OECD "Earthworm, Acute Toxicity Tests" (1984) etc.

 

METHODS

During this test, the artificial soils was used with the moisture content of the test medium controlled in range of 26% to 35% with pH from 5.65 to 6.47. The test conditions were maintained as follows: temperature of 20.6 °C to 22.3 °C, humidity of 80 % to 85 % and continuous light with intensity of 400 lux to 800 lux. Based on the result of range-finding test and definitive test, the 7 day and 14 day LC50 values were both determined for the test substance in the artificial soil

 

RESULTS

The highest concentration causing no mortality 7 d-LC0 was 10.0 mg/kg dry weight soil. 7 d-LC50 was 57.0 mg/kg dry weight soil, and the 95% confidence interval was 47.5 to 68.5 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 10.0 mg/kg dry weight soil.14-d LC50 was 30.0 mg/kg dry weight soil, and the 95% confidence interval was 24.9 to 36.1 mg/kg dry weight soil.

Description of key information

The highest concentration causing no mortality 7 d-LC0 was 10.0 mg/kg dry weight soil. 7 d-LC50 was 57.0 mg/kg dry weight soil, and the 95% confidence interval was 47.5 to 68.5 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 10.0 mg/kg dry weight soil.14-d LC50 was 30.0 mg/kg dry weight soil, and the 95% confidence interval was 24.9 to 36.1 mg/kg dry weight soil (The guidelines for the testing of chemicals" (HJ/T 153-2004) and "The Guidelines for the Testing of Chemicals-Effects on Biotic Systems" (the 2nd edition. Beijing: China Environment Press. 2013 plus OECD 207 and EU Method C.8 ).

Key value for chemical safety assessment

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

Additional information

GUIDELINE

The test was conducted to determine the acute toxicity of test substances to earthworm according to "The guidelines for the testing of chemicals" (HJ/T 153-2004) and "The Guidelines for the Testing of Chemicals-Effects on Biotic Systems" (the 2nd edition. Beijing: China Environment Press. 2013); with reference to Procedure 207 of the ' Guidelines for Testing of Chemicals' of the OECD "Earthworm, Acute Toxicity Tests" (1984) etc.

 

METHODS

During this test, the artificial soils was used with the moisture content of the test medium controlled in range of 26% to 35% with pH from 5.65 to 6.47. The test conditions were maintained as follows: temperature of 20.6 °C to 22.3 °C, humidity of 80 % to 85 % and continuous light with intensity of 400 lux to 800 lux. Based on the result of range-finding test and definitive test, the 7 day and 14 day LC50 values were both determined for the test substance in the artificial soil

 

RESULTS

The highest concentration causing no mortality 7 d-LC0 was 10.0 mg/kg dry weight soil. 7 d-LC50 was 57.0 mg/kg dry weight soil, and the 95% confidence interval was 47.5 to 68.5 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 10.0 mg/kg dry weight soil.14-d LC50 was 30.0 mg/kg dry weight soil, and the 95% confidence interval was 24.9 to 36.1 mg/kg dry weight soil.