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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:
November 19, 2014 to December 11, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD and Chinese test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 207 Earthworm, Acute Toxicity Test[M]. Second edition. Beijing: China Environmental Science Press. 2013: 58-62.
Deviations:
no
Qualifier:
according to
Guideline:
other: China National Technical Committee for Standardization of Dangerous Chemical Management. GB/T 21809-2008 Chemical Earthworm, Acute Toxicity Tests [S]. Beijing: China Standardization Press, 2008.
GLP compliance:
yes
Specific details on test material used for the study:
Physicochemical properties
Melting point: -39 °C at 101.3 kPa
Boiling point 216 °C at 101.3 kPa
Volatility: Non-volatile
Vapour Pressure: 1.22 kPa at 37.8 °C
Solubility in Water: Using the US EPA On-Line EPI Suite™ WSKOW v1.42 model, the water solubility range is predicted to be 2.64E-27 to 0.004421mg/L. This indicates that the substance is insoluble, with water solubilities much lower than 0.1mg/L.
Solubility in Solvent Water: none
Density: 0.82
Odor: None
Date of expiration: July, 2016
Analytical monitoring:
no
Details on sampling:
Not specified in the study report.
Vehicle:
no
Details on preparation and application of test substrate:
Accurately weigh 2.778 g test substance into the beaker and dissolved it with appropriate amount volume of n-hexane. Then it was diluted to 50mL to obtain the 55.56mg/mL of test stock solution for test. Accurate draw 10.0mL of test stock solution, and mixed with and a total of 10g quartz sand respectively. Firstly dry the solvent, and then it was mixed with 740g wet artificial soil in the test container to prepare the test system of 1000 mg/kg dry weight soil. Solvent control was prepared by drawing 10.0mL of solvent and mixed with a total of 10g quartz sand firstly. Then dry the solvent, and it was mixed with 740g wet artificial soil in the test container to prepare the solvent control. Blank control was prepared by mixing about 10g of quartz and 740g wet artificial soil. Four test systems were prepared for each test group.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Species: Eisenia foetida Batch No.: EEf20140704-1 Age: Older than 2 months Weight: 418 mg - 458 mg Amount: 120 individuals Source: Zhuhai Hongming Earthworm Breeding Park Selected reasons: The recommended test species is Eisenia foetida (Michaelsen). They are existed in soil with rich organic matter. Its susceptibility to chemicals resembles that of true soil-inhabiting species. They have a short life cycle and are very prolific. It is available commercially and can be bred readily in a wide range of organic waste materials. Cocoons can be purchased commercially or distributed from a central source to ensure the same strain is used. “The Guidelines for the Testing of Chemicals” recommend the use of this species. Holding: All earthworms were obtained and held in the laboratory for 14 days and more under the same surroundings before used. During the holding period, all the earthworms were healthy. They were breed for 24h in the artificial soil before artificial soil test. Reference substance test of Eisenia foetida: After domestication, the 14d-LC50 of reference substance chloracetamide(C2H4ClNO) was tested for the Eisenia foetida. It was 18.3 mg/kg dry weight, and the 95% confidence limit was between 17.5 mg/kg dry weight and 19.1 mg/kg dry weight, which was within the range of 10 mg/kg dry weight to 100mg/kg dry weight. Report details refer to 2014GST0009R.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Test temperature:
23.9°C
pH:
6.50
Moisture:
Beginning of test: Mean measurement 26.4 - 27.5%End of test: Mean measurement 25.9 - 26.1%
Details on test conditions:
Preparation of artificial soil: The dry constituents were blended in the correct proportions with sphagnum peat 10% (no visible plant remains), kaolin clay 20% (AR, Weight loss on ignition≤15%), industrial sand 70% (particle size between 40-100μm) and mixed thoroughly, either in a large-scale laboratory mixer or small electric cement mixer. Moisture content was then determined by drying a small sample at 105°C ± 5°C and re-weighing. Deionized water was added to give an overall moisture content of 35% of the dry weight, and the medium was thoroughly mixed. The pH was measured as close to pH 5.5 to 6.5 as possible. Test conditions Exposure conditionsTest method: Ten earthworms were placed on the artificial soil surface for each 1L glass beaker. The containers were covered with perforated plastic film to prevent the artificial soil from drying. Test duration: 14d Test concentration: One exposure level of 1000mg/kg dry soil of test was prepared. Control: A blank control group and a solvent group were prepared. Replicates: 4 replicates / blank control, 4 replicates/solvent group and 4 replicates/test substance. Test organism: 120 qualified domesticated earthworms were selected. Each group was assigned 10 earthworms randomly with four replicates. They were washed and dried by filter paper and weighed before use. Test environment conditions Test temperature: The temperature was kept by the thermostatic air conditioning. The temperature range was from 19.1 °C to 21.6 °C during the test period. Test humidity: The humidity was kept by the humidifier. The humidity of surrounding air range was from 66.0% to 97.0% during the test period Light condition: Continuous light, the light intensity was 600 lx at the beginning of the test and 610 lx at the end of the test.Solvent Name: n-hexane Batch: 20130104-2 Source: Guangzhou Chemical ReagentEquipment Beakers, Petri dish, humidifier, blender and so on. Test method Preparation of test substance soil: Accurately weigh 2.778 g test substance into the beaker and dissolved it with appropriate amount volume of n-hexane. Then it was diluted to 50mL to obtain the 55.56mg/mL of test stock solution for test. Accurate draw 10.0mL of test stock solution, and mixed with and a total of 10g quartz sand respectively. Firstly dry the solvent, and then it was mixed with 740g wet artificial soil in the test container to prepare the test system of 1000 mg/kg dry weight soil. Solvent control was prepared by drawing 10.0mL of solvent and mixed with a total of 10g quartz sand firstly. Then dry the solvent, and it was mixed with 740g wet artificial soil in the test container to prepare the solvent control. Blank control was prepared by mixing about 10g of quartz and 740g wet artificial soil. Four test systems were prepared for each test group. Observation and measurement Normal observation: Any behavioural or pathological symptoms were checked and recorded at 7d and 14d. Sorting the worms from the artificial soil and testing their reaction to a mechanical stimulus at the front end. Earthworms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end.Index measurement pH: pH of the artificial soil was measured at the beginning of the test. Weighed 5g of air-dried soil sample in 50mL beaker . Added 25mL CO2-free distilled water by cylinder. Stirred for 5min to allow it fully spread. Laid aside for 1h and then measured the pH.Moisture: Moisture of the artificial soil was measured at the beginning and at the end of the test. 3 Petri dishes with about 10g of soil were placed in the drying oven at 105°C ± 5°C for 4h and cooled in desiccator for 30min. The weight was measured down to 1mg until it reached constant value. When the difference of two consecutive weighing was less than 0.1%, it was considered to have reached a constant quantity. The average from three weighing was calculated. Environmental conditions: The test environment light intensity was determined and recorded above the test container location at the end and the start of the test respectively. The environment temperature and humidity were recorded daily. A temperature hygrometer was added for continuous monitoring of culture temperature and humidity. Weight: Total weight of earthworms was weighted and the average weight of earthworms was obtained by dividing the number of earthworms at the beginning and at the end of the test. Data processing : Calculate the average weight of live earthworm and the moisture content of artificial soil at the beginning and the end of test, and calculate the pH of artificial soil at the beginning. The moisture content of artificial soil (%) = {[(wet soil mass+ dish mass)-(dish mass)]-[(dry soil mass+ dish mass) - (dish mass)]}÷[( dry soil mass+ dishes mass) - (dishes mass)] × 100%. The modified mortality (%) = (Number of dead earthworm in test group- Number of dead earthworm in control group)/(Number of earthworm in test group-Number of dead earthworm in control group)×100%.
Nominal and measured concentrations:
Nominal concentrations: 1000 mg/kg
Reference substance (positive control):
no
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:
The results of artificial soil limit test are shown in Table 1 to Table 5. The mean moisture content of control group and treatment group at start and at the end of test were 27.1% and 25.9%, respectively. The results demonstrated the mortalities in the blank controls and the solvent group were 0%, which did not exceed 10% at the end of the test and fulfilled the criteria for a valid test. The mortality in the test group was 0%. One earthworm was leaking yellow liquid on 14th day for the blank control group, no other obvious physical or pathological symptoms or distinct changes in behaviour observed in the test organisms on 7th day and 14th day. Under the conditions of the present test, the 14d-LC50 of the test substance to earthworm was greater than 1000mg/kg dry soil.
Results with reference substance (positive control):
Reference substance not used in this study.
Reported statistics and error estimates:
The mortalities of earthworm in blank control and the solvent group were 0% respectively, which did not exceed 10% at the end of the test, and fulfilled the criteria for the validity of test.

Mortality of earthworm during the artificial soil limit test (%)

Groups

Number of earthworm

7d

14d

Number of dead earthworm

Mortality (%)

Modified mortality (%)

Number of dead earthworm

Mortality (%)

Modified mortality (%)

Blank control

40

0

0

0

0

0

0

Solvent group

40

0

0

0

0

0

0

1000 mg/kg dry soil

40

0

0

0

0

0

0

 

Pathological symptoms and abnormal behaviour of earthworm during the artificial soil limit test

Groups

7d

14d

Blank control

Earthworms located in the soil below the surface, and all were healthy

One earthworm was leaking yellow liquid, and others were healthy

Solvent group

Earthworms located in the soil below the surface, and all were healthy

Earthworms located in the soil below the surface, and all were healthy

1000 mg/kg dry soil

Earthworms located in the soil below the surface, and all were healthy

Earthworms located in the soil below the surface, and all were healthy

 

The average weight of the earthworm at the start and end of the limit test

Groups

Replicate

Number of earthworm

The beginning

The end

Number of live earthworm

Mean Weight/ Replicate (g)

Mean weight/group (g)

Number of live earthworm

Mean Weight/Replicate (g)

Mean weight/group (g)

Blank control

Replicate 1

10

10

0.424

0.441

10

0.390

0.398

Replicate 2

10

10

0.458

10

0.401

Replicate 3

10

10

0.451

10

0.416

Replicate 4

10

10

0.431

10

0.383

Solvent group

Replicate 1

10

10

0.442

0.437

10

0.405

0.391

Replicate 2

10

10

0.421

10

0.374

Replicate 3

10

10

0.449

10

0.412

Replicate 4

10

10

0.437

10

0.375

1000 mg/kg dry soil

Replicate 1

10

10

0.435

0.437

10

0.386

0.403

Replicate 2

10

10

0.418

10

0.409

Replicate 3

10

10

0.438

10

0.397

Replicate 4

10

10

0.456

10

0.421

The pH of the artificial soil at the start of the limit test

Dry soil (g)

Test temperature (°C)

pH

First reading

Second reading

Mean

5.000

23.9

6.50

6.60

6.50

 

Moisture content of the artificial soil at the start and end of the limit test

Groups

The beginning

The end

Measure (%)

Mean (%)

Measure (%)

Mean (%)

Blank control

Replicate 1

27.76

27.5

26.05

26.01

Replicate 2

27.14

26.41

Replicate 3

27.63

25.98

Solvent group

Replicate 1

27.27

27.4

25.77

25.8

Replicate 2

27.28

25.40

Replicate 3

27.52

26.09

1000 mg/kg dry soil

Replicate 1

26.06

26.4

25.79

25.8

Replicate 2

26.66

25.55

Replicate 3

26.38

26.13

 

Validity criteria fulfilled:
yes
Conclusions:
Under the present test conditions, results indicated that the 14d-LC50 of the test substance to earthworm by artificial soil test was greater than 1000mg/kg dry soil.
Executive summary:

The artificial soil test involves keeping earthworms in samples of a precisely defined artificial soil to which a limit of concentration of the test substance has been applied. Mortality is assessed 7 and 14 days after application. Then the acute toxicity of the test substance is determined by the results of the mortality.

 

Guidelines

[1] Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 207 Earthworm, Acute Toxicity Test[M]. Second edition. Beijing: China Environmental Science Press. 2013: 58-62.

[2] China National Technical Committee for Standardization of Dangerous Chemical Management. GB/T 21809-2008 Chemical Earthworm, Acute Toxicity Tests [S]. Beijing: China Standardization Press, 2008.

[3] OECD.OECD Guidelines for Testing of Chemicals, No. 207 Earthworm, Acute Toxicity Tests. Paris: OECD & OCED, Adopted 4th April, 1984.

 

Test conditions

Test substance: NovaSpec Base Oil

Type of test: Acute toxicity test in artificial soil test

Test organism: Eisenia foetida

Test duration: 14d

Test concentration: One test group of 1000 mg/kg dry soil was designed

Control: A blank control group and a solvent group were prepared.

Replicates: 4 replicates/group

Test organism number: 10 earthworms / replicate

Preparation of test system: Accurately weighed 2.778 g test substance into the beaker and dissolved it with appropriate amount volume of n-hexane. Then it was diluted to 50mL to obtain the 55.56mg/mL of test stock solution for test. Accurately draw 10.0mL of test stock solution and mixed with a total of 10g quartz sand respectively. Firstly dry the solvent, and then it was mixed with 740g wet artificial soil in the test container to prepare the tested system of 1000 mg/kg dry weight soil. Solvent group was prepared by drawing 10mL of solvent and mixed with a total of 10g quartz sand firstly. Then dry the solvent and it was mixed with 740g wet artificial soil in the test container to prepare the solvent control. Blank control was prepared by mixing about 10g of quartz and 740g wet artificial soil. Four replicates were prepared for each test group.

Temperature in incubator: 19.1°C-21.6°C

Light condition: Continuous light, the light intensity was 600 lx at the beginning of the test and 610 lx at the end of the test.

 

Results

14d-LC0 1000mg/kg dry soil

14d-LC50 > 1000mg/kg dry soil

Description of key information

Assessment of toxicity to soil macroorganisms

Key value for chemical safety assessment

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

Additional information

A Klimish one rated GLP study was conducted in accordance with current OECD guidelines is available to assess the effects of toxicity to Eisenia foetida in artificial soil. The toxicity of the test item to the Eisenia foetida has been investigated and have a 14-Day L0 value of 1000 mg/kg dry soil. The 14-Day LC50 gave a value of > 1000 mg/kg dry soil. Earthworms located in the soil below the surface, and all were healthy, therefore indicating that the substance is not toxic to soil macroorganisms.

No classification is applicable.