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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:
Experiment starting date: Mar. 30, 2016 Experiment completion date: May 20, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: "The guidelines for the testing of chemicals" (HJ/T 153-2004) and "The Guidelines for the Testing of Chemicals -Effects on Biotic Systems" (the 2°d edition. Beijing: China Environment Press. 2013);
Version / remarks:
The test was conducted to determine the acute toxicity of test substances (The test substance) 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 2°d edition. Beijing: China Environment Press. 2013); while with reference to Procedure 207 of the 'Guidelines for Testing of Chemicals' of the OECD: "Earthworm, Acute Toxicity Tests" (1984) etc.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
The test was conducted to determine the acute toxicity of test substances (The test substance) 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 2°d edition. Beijing: China Environment Press. 2013); while with reference to Procedure 207 of the 'Guidelines for Testing of Chemicals' of the OECD: "Earthworm, Acute Toxicity Tests" (1984) etc.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone.
Details on preparation and application of test substrate:
Preparation of the Test Substrate
Range-finding Test
The stock solution of 100 g/L was prepared by dissolving 5.0009 g test substance into 50.0 mL acetone.
Test substrates at different concentrations were prepared by adding the stock solution to 10g quartz sand and mixed. After evaporating the acetone at the room temperature, 10g quartz sand then mixed with 550g dry weight of soil for each test container. The medium was thoroughly mixed and deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. Moisture content was determined by drying a small sample at 105°C and re-weighing.
Simultaneously blank control group and solvent control group were established. The solvent control group was administrated with the maximum amount of acetone in the treatment (1000 mg/kg artificial soil (dry weight)), and the blank control group added none acetone. Details about the preparation of the test substrates at chosen concentrations were given as follows.
preparation of the test substrates at chosen concentrations were given as follows.

Test Type Concentration of the test The amount of liquid Quantity of the dry soil (g)
substance (mg/kg (dry soil)) reserves_(mL)

Range-finding test 0 0 560
finding test 0 (acetone) 0 (5.6 mL acetone) 560
100 0.56 560
500 2.80 560
1000 5.60 560
Definitive test
The stock solution of 100 g/L was prepared by dissolving 25.0013 g test substance into 250 mL acetone.
Test substrates at different concentrations were prepared by adding the stock solution to 10g quartz sand and mixed. After evaporating the acetone at the room temperature, 10g quartz sand then mixed with 550g dry weight of soil for each test container. The medium was thoroughly mixed and deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. Moisture content was determined by drying a small sample at I05°C and re-weighing.

Simultaneously blank control group and solvent control group were established. The solvent control group was administrated with the maximum amount of acetone in the treatment (2000 mg/kg artificial soil (dry weight)), and the blank control group added none acetone. Details about the preparation of the test substrates at chosen concentrations were given as follows.

Test Type Concentration of the test The amount of liquid Quantity of the dry soil (g)
substance (mg/kg (dry soil)) reserves_(mL)

Definitive test 0 0 560
0 (acetone) 0 (11.2 mL acetone) 560
800 4.48 560
1000 5.60 560
1200 6.72 560
1500 8.40 560
2000 11.2 560

Test organisms (species):
other: Eisenia foetida
Animal group:
annelids
Details on test organisms:
Test Organism
Eisenia foetida was adopted as the test species. The worms were obtained from an earthworm nursery of Nanjing city in China.
Name: Nanjing Earthworm Nursery
Adress: Pudong community, Changlu Street, Dachang District, Nanjing

Acclimation of the Test Worms
The earthworms newly obtained in the lab will be released from quarantine for 14 days and then this batch was turned into pre-culture (Batch No.: E20160318). Before the test, the worms were acclimated in test soil for 48 days. Range of worms average weight for this study were 362 - 426 mg.
The worms were then pre-cultured in test soils for another 24 h. The worms were 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
Test temperature:
Temperature of 19.8°C to 21.3°
pH:
pH from 6.13 - 6.37
Moisture:
moisture content of the test medium controlled in range of 27% to 35%
Details on test conditions:
Artificial soil test substrate was prepared as follows:
a) 10 per cent sphagnum peat (Pindstrup Mosebrug A/S; pH 6.0, no visible plant remains, finely ground, dried).
b) 20 per cent kaolin clay (CAS: 1332-58-7; Lot number: 20150805; Sinopharm Chemical Reagent Co. Ltd.; kaolinite content preferably above 30 per cent).
c) 70 per cent quartz sand (CAS: 14808-60-7; Lot number: 20160309; Sinopharm Chemical Reagent Co. Ltd.; fine sand should be dominant with 80 per cent of the particles between 50 and 200 microns).
The dry constituents were blended m the correct proportions and mixed thoroughly with a large-scale laboratory mixer.

Facilities
(1) Electronic balance (MS105DU, METTLER TOLEDO, Switzerland);
(2) Glass spoutless beakers (1 L, <1>12 cm, Nanjing Jinzheng Teching Instrument Factory);
(3) Plastic films with small ventilation holes (25 cmx38 cm, Suzhou Surong plastic product company);
(4) Climatic chambers (RXZ-600B, Ningbo Jiangnan Instrument Factory, controllable to ± 2°C with a light intensity of 400 to 800 lux);
(5) Stainless plates (40 cmx25 cm, Nanjing Jinzheng Teching Instrument Factory);
(6) Filter paper: (<1>9 cm, approximately 0.2 mm thick, medium grade, Hangzhou Whatman-Xinhua Filter Paper Co., Ltd.);
(7) deionised water (resistivity 18.2 Mn.cm).
(8) pH meter (HACH HQ 30d);
(9) Oven (DHG-9146A, Shanghai Jinghong laboratory devices CO., LTD.)
3.5 Reference Substance
Reference substance: 2-Chloracetamide, CAS: 79-07-2; purity: 99.96%; Lot number : LT90075; J&K Scientific Ltd.
The test with the reference substance is performed three times a year as a means of assuring that the laboratory test conditions are adequate and have not changed significantly. The results of the most recent test are shown in the attached Table 3 and Figure 1.

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, blank control and solvent 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 19.6°C to 21 .2°C with the humidity of 78% to 81% 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 800, 1000, 1200, 1500 , 2000 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 19.8°C to 21.3°C with the humidity of 78 % to 81% and continuous light (intensity of 400 lux to 800 lux).

Observations
On the 7th day of exposure, the 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 14 th day assessment.

In addition, the number and type of behavioural and other pathological abnormalities ( e.g. lethargic or rigid worms) were determined during each mortality assessment on 7 d and 14 d.
On day O 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 O prior to assigning to the each 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. The surviving worms were weighted with the same methods on day 14.

Validity of Test
(I) 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.

Reference control
The 7-day and 14-day LC50 values of a reference substance (2-chlo racetamide) were determined 3 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 the attached Table 3 and Figure I. The 7-days LC50 and 14-days LC50 for reference substance were 35.1 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.


Data Processing
The LC 50 and 95% confidence intervals were used to calculate by Trimmed Spearman- Karber Method (Version 1.5, USEPA).



Nominal and measured concentrations:
Based on the results of the range-finding test, a Definitive test was undertaken with test concentration of 800, 1000, 1200, 1500 , 2000 mg/kg (dry weight of soil), blank control and solvent control. Four replications for each concentration and control, each with 10 individuals were used.
Reference substance (positive control):
yes
Remarks:
2-Chloracetamide, CAS: 79-07-2; purity: 99.96%; Lot number : LT90075; J&K Scientific Ltd.
Key result
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil ww
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Behavioural Effects were also checked
Remarks on result:
other: The 7 d-LC50 was >1000 mg/kg dry weight soil
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
1 544 mg/kg soil ww
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Behavioural Effects were also checked
Remarks on result:
other: The 14 d-LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight
Details on results:
Test Results
Test Conditions
During this test, artificial soils was used with the moisture content of the test medium controlled in range of 27% to 35% with pH form 6.13 to 6.37 (see the attached1) . The test conditions were maintained as follows: temperature of 19.8°C to 21.3°C, humidity of 78% to 81% 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. Thus, the test system was considered to be valid .

Earthworm Mortality
The average weights of the worms at start and end of exposure see the attached table 2. Number of the dead earthworms after 7 and 14 days is shown in the attached Table 4 and Table 5. No dead worms were observed in treatments of control. No dead worms were observed in treatments of 800 mg/kg soil dry weight, and worms were dead in the test treatments of 1000, 1200, 1500 and 2000 mg/kg soil dry weight after 14 d. The results showed that the 7 d-LC50 was>1000 mg/kg dry weight soil. The 14 d-LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight soil.

Behavioural Effects
At the beginning of the administration, test worms soon disappeared into the test soil medium . During the test, all survival earthworms remained in the soil.
Results with reference substance (positive control):
The test with the reference substance is performed three times a year as a means of assuring that the laboratory test conditions are adequate and have not changed significantly. The results of the most recent test are shown in the attached Table 3 and Figure 1.
Validity criteria fulfilled:
yes
Conclusions:
During this test, artificial soils were used with the moisture content of the test medium controlled in range of 27% to 35% with pH form 6.13 - 6.37. The test conditions were maintained as follows: temperature of 19.8°C to 21.3°C humidity of 78% to 81% and continuous light with intensity of 400 lux to 800 lux.
Based on the result of range-finding test and definitive test, the 7 d and 14 d-LC50 of the test substance in the .artificial soil were both determined. The highest concentration causing no mortality 7 d LC0 were 1000 mg/kg dry weight soil, and 14 d LC0 were 800 mg/kg dry weight soil. The 7 d-LC50 was >1000 mg/kg dry weight soil. The 14 d-LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight soil.
Executive summary:

The test was conducted to determine the acute toxicity of test substances (The test substance) 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 2°d edition. Beijing: China Environment Press. 2013); while with reference to Procedure 207 of the 'Guidelines for Testing of Chemicals' of the OECD: "Earthworm, Acute Toxicity Tests" (1984) etc.

During this test, the artificial soils was used with the moisture content of the test medium controlled in range of 27% to 35% with pH from 6.13 to 6.37. The test conditions were maintained as follows: temperature of l 9.8°C to 21.3°C, humidity of 78% to 81% and continuous light with intensity of 400 lux to 800 lux.

Based on the result of range-finding test and definitive test, the 7d and 14 d-LC50 of the test substance (The test substance) in the artificial soil were both determined. The highest concentration causing no mortality 7 d-LC0 was 1000.0 mg/kg dry weight soil. 7 d-LC50 was > 1000 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 800 mg/kg dry weight soil.14-d LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight soil.

Description of key information

Based on the result of range-finding test and definitive test, the 7 d and 14 d-LC50 of the test substance  in the .artificial soil were both determined. The highest concentration causing no mortality 7 d LC0 were 1000 mg/kg dry weight soil, and 14 d LC0 were 800 mg/kg dry weight soil. The 7 d-LC50 was >1000 mg/kg dry weight soil. The 14 d-LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight soil.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 544 mg/kg soil dw

Additional information

The test was conducted to determine the acute toxicity of test substances (The test substance) 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 2°d edition. Beijing: China Environment Press. 2013); while with reference to Procedure 207 of the 'Guidelines for Testing of Chemicals' of the OECD: "Earthworm, Acute Toxicity Tests" (1984) etc.

During this test, the artificial soils was used with the moisture content of the test medium controlled in range of 27% to 35% with pH from 6.13 to 6.37. The test conditions were maintained as follows: temperature of l 9.8°C to 21.3°C, humidity of 78% to 81% and continuous light with intensity of 400 lux to 800 lux.

Based on the result of range-finding test and definitive test, the 7d and 14 d-LC50 of the test substance (The test substance) in the artificial soil were both determined. The highest concentration causing no mortality 7 d-LC0 was 1000.0 mg/kg dry weight soil. 7 d-LC50 was > 1000 mg/kg dry weight soil. The highest concentration causing no mortality 14 d-LC0 was 800 mg/kg dry weight soil.14-d LC50 was 1544 mg/kg dry weight soil, and the 95% confidence interval was 1411 to 1690 mg/kg dry weight soil.