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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:
2018-01-11 to 2018-07-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no analytical validation of concentrations
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
April 4, 1984
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
n.a.
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of application to filter paper (if used): n. a.
- Method of mixing into soil (if used): For test item treatment group, 602.3 mg, 605.1 mg, 602.2 mg and 604.9 mg of test item was weighed, dissolved in 5 mL acetone, and mixed with 10 g quartz sand respectively. And then, the mixture of test item and quartz sand was placed under the fume hood to allow the solvent to evaporate completely. The mixture was then mixed with 711.0 g of wet soil (the result of moisture content was 20.5%, the dry weight was 590 g.) to obtain the soil with final test item concentration of 1000 mg/kg. 89 mL the purified water was mixed thoroughly with the soil to adjust the moisture content of the test soil to approximately 35%. 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.
- Method of application to soil surface (if used): n. a.
- Controls: For blank control group, 711.0 g of wet soil (the result of moisture content was 20.5%, the weight was 590 g based on dry weight) was mixed with 10 g quartz sand. 89 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.

- - Chemical name of vehicle (organic solvent, emulsifier or dispersant), concentration of vehicle in test medium (stock solution and final test solution) and evaporation of vehicle before use:
For solvent control group, 5 mL acetone was mixed with 10 g quartz sand and placed under the fume hood to allow the solvent to evaporate completely. And then, the quartz sand was mixed with 711.0 g of wet soil (the result of moisture content was 20.5%, the dry weight was 590 g). 89 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.

The constituents of artificial soil (calculated as dry weight) are listed as follows:
- 10% sphagnum peat (no visible plant remains, finely ground, dried and the moisture content was measured to be 10.4%);
- 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 5.83. The dry artificial soil was taken and pre-moistened with purified water. Before the test, the wet soil was sampled, four replicates, and dried for about 3 hours at 105 °C to determine the moisture content to be 20.5%.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Source: The test species was purchased from Wuxi Earthworm Breeding Base (Code No.: Earthworm 20171016) and is raised in the test facility of CTI-SET.

Result of sensitivity test: The result of the latest sensitivity test (Study No.:17ST007) conducted from 25 October 2017 to 08 November 2017 indicated that the 14d-LC50 of 2-chloroacetamide (CAS No.: 79-07-2) to Eisenia fetida was 58.7 mg/kg and its 95% confidence limits was 50.5-68.2 mg/kg, which was within the recommended range (20-80 mg/kg) based on GB/T 31270.15-2014.

Adaptation: Prior to use, the earthworms were placed in the artificial soil about 25 hours under test condition.

Characteristic requirement: Earthworms were adult (at least two months old with clitellum) with an individual wet mass of 300 mg to 600 mg.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
Test temperature: 20±2 °C
pH:
6.29-6.38 at the start of the test.
Moisture:
72-88%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): not reported
- Amount of soil or substrate: About 750 g test soil was weighed and added into each test container.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

Exposure
Groups of ten earthworms were selected, weighed individually and randomly assigned to test containers. The test earthworms were placed on the surface of the test soil and allowed to enter into the soil. Thereafter, the containers were covered with perforated plastic film. When the earthworms were placed into the test containers, the exposure began.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- 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, dried and the moisture content was measured to be 10.4%);
- 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 5.83. The dry artificial soil was taken and pre-moistened with purified water. Before the test, the wet soil was sampled, four replicates, and dried for about 3 hours at 105°C to determine the moisture content to be 20.5%.
- PREPARATION OF TEST SOIL
For blank control group, 711.0 g of wet soil (the result of moisture content was 20.5%, the weight was 590 g based on dry weight) was mixed with 10g quartz sand. 89mL 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, 5mL acetone was mixed with 10g quartz sand and placed under the fume hood to allow the solvent to evaporate completely. And then, the quartz sand was mixed with 711.0g of wet soil (the result of moisture content was 20.5%, the dry weight was 590g). 89mL 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 item treatment group, 602.3mg, 605.1mg, 602.2mg and 604.9mg of test item was weighed, dissolved in 5mL acetone, and mixed with 10g quartz sand respectively. And then, the mixture of test item and quartz sand was placed under the fume hood to allow the solvent to evaporate completely. The mixture was then mixed with 711.0g of wet soil (the result of moisture content was 20.5%, the dry weight was 590g.) to obtain the soil with final test item concentration of 1000mg/kg. 89mL the purified water was mixed thoroughly with the soil to adjust the moisture content of the test soil to approximately 35%.
About 750g 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
- Photoperiod and light intensity: Continuous fluorescent light with light intensity of 400-800 lux
- Test humidity: 70-90%
- Feeding: There was no feeding during the test
The light intensity was in the range of 534-746 lux at the start of the test.
During the test, the temperature was in the range of 19.2-20.5°C and the humidity was in the range of 72-88%.
The moisture content of test soil in the controls and test item treatment group was in the range of 33.7-34.4% at the start of the test and 33.7-34.3% at the end of the test, respectively.
All the pH value of test soil in the controls and test item treatment group was in the range of 6.29-6.38 at the start of the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality. The survivals 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 individual wet mass of the survived earthworms of each replicate was weighed on day 14 day of the exposure. The report does not present a statistical analysis of the weight.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: n.a.
- Justification for using less concentrations than requested by guideline: Based on results from the range finding study a Limit test was conducted. The test concentration was set to be 1000 mg/kg, blank control and solvent control was set in parallel.

RANGEFINDING STUDY: yes
- Test concentrations: 100 mg/kg, 500 mg/kg and 1000 mg/kg
- Results used to determine the conditions for the definitive study: Yes
Nominal and measured concentrations:
1000 mg/kg
Reference substance (positive control):
no
Remarks:
Sensitivity test regularly conducted. Most recent results see below.
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
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 mortality of earthworms in blank control, solvent control and test item treatment group was 0 % after 7 days and 14 days of exposure and there were no abnormal symptoms. The result of mortality is listed in Table 1.The 7d-LC50 and 14d-LC50 of test item to earthworm was greater than 1000 mg/kg.
Results with reference substance (positive control):
No positive control was performed. Sensitivity is tested in regularly intervals. Result of sensitivity test
The result of the latest sensitivity test (Study No.:17ST007) conducted from 25 October 2017 to 08 November 2017 indicated that the 14d-LC50 of 2-chloroacetamide (CAS No.: 79-07-2) to Eisenia foetida was 58.7 mg/kg and its 95% confidence limits was 50.5-68.2 mg/kg, which was within the recommended range (20-80 mg/kg) based on GB/T 31270.15-2014.
Reported statistics and error estimates:
n.a.

The moisture content of test soil in the controls and test item treatment group was in the range of 33.7-34.4 % at the start of the test and 33.7-34.3 % at the end of the test, respectively.

All the pH value of test soil in the controls and test item treatment group was in the range of 6.29-6.38 at the start of the test.

Table 1: Acute toxicity of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate

Concentration [mg/kg]

Replicate

Number of exposed earthworm

7 days of exposure

14 days of exposure

 

 

Dead number

Mortality (%)

Cumulative dead number

Cumulative mortality (%)

Blank control

1

10

0

0

0

0

2

10

0

0

3

10

0

0

4

10

0

0

Solvent control

1

10

0

0

0

0

2

10

0

0

3

10

0

0

4

10

0

0

1000

1

10

0

0

0

0

2

10

0

0

3

10

0

0

4

10

0

0

The mean weights or earthworms are shown in Table 2.

Table 2: Mean weight of earthworm

Concentration [mg/kg]

Replicate

Earthworm total weight (mg)

Earthworm mean weight (mg)

0 d

14 d

0 d

14 d

Blank control

1

5027

4073

503

407

2

4808

3845

481

385

3

5054

4223

505

422

4

4913

4240

491

424

Solvent control

1

5123

4516

512

452

2

5026

4083

503

408

3

4990

4265

499

427

4

4920

4419

492

442

1000

1

4521

4215

452

422

2

4309

4007

431

401

3

4277

3841

428

384

4

4801

3925

480

393

Validity criteria fulfilled:
yes
Remarks:
The mortality in the controls was 0 % at the end of the test (less than 10%).
Conclusions:
In this study, the 14d-LC50 of test item 1,3,5-Tris-(3-mercaptopropyl)isocyanurate to earthworm (Eisenia foetida) was greater than 1000 mg/kg.
Executive summary:

In a 14-day acute toxicity study, earthworms (Eisenia fetida) were exposed to 1,3,5-Tris-(3-mercaptopropyl)isocyanurate at 1000 mg/kg dry weight of soil (nominal value). No reference chemical was used. Sensitivity is checked regularly with 2-chloroacetamide. The result of the latest sensitivity test (Study No.:17ST007) conducted from 25 October 2017 to 08 November 2017 indicated that the 14d-LC50 of 2-chloroacetamide (CAS No.: 79-07-2) to Eisenia fetida was 58.7 mg/kg and its 95% confidence limits was 50.5-68.2 mg/kg, which was within the recommended range (20-80 mg/kg) based on GB/T 31270.15-2014. The experiment was carried out in accordance with OECD guideline 207. No mortality was observed during the time of exposure. The 14-day NOEC and LOEC based on mortality was > 1000 mg/kg dw of soil. The concentrations of the test item were not verified analytically.

 

Worms were assessed for mortality after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14. The mean weight of the earthworms in the control and the test item treatment groups did not vary noticeable. It was of 428 - 512 mg at the start of the test and 384 - 452 mg at the end of the test. No abnormal symptoms were observed.

It can be concluded that the acute earthworm 14d-LC50 of test item1,3,5-Tris-(3-mercaptopropyl)isocyanurate was greater than 1000 mg/kg.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with earthworms according to OECD guideline 207.

 

Results Synopsis

 

Test organism size/age (mean weight or length): Earthworms were adult (at least two months old with clitellum) with an individual wet mass of 300 mg to 600 mg.

 

Test Type: Limit test, static

 

14-day LC50: > 1000 mg/kg dw of soil

14-day NOEC: > 1000 mg/kg dw of soil

 

Endpoint(s) Effected: Mortality; Weight.

Description of key information

An acute toxicity study with earthworm (Eisenia fetida) was carried out in accordance with OECD guideline 207. No mortality was observed during the time of exposure (14d). The 14-day NOEC and LOEC based on mortality was > 1000 mg/kg dw of soil. The concentrations of the test item were not verified analytically.

Key value for chemical safety assessment

Additional information