2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

Toxicity to soil macroorganisms except arthropods

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:

07 September 2009 to 11 November 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 207 Earthworm, Acute Toxicity Test. China Environmental Science Press.

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

SENSITIVITY TEST

- One blank control group and five reference substance test groups were designed and 60 qualified domesticated earthworms were selected. Each group was assigned 10 earthworms randomly.
- Worms was kept on moist filter paper for three hours before being placed in test vials so they could void their gut contents. They were then washed and dried before use.
- The total and the average weight of earthworms was obtained by dividing by the number of earthworms at the beginning of test.
-1 mL of reference substance solution was pipetted into each vial and evaporated to dryness under a slow stream of filtered compressed air, the vial being rotated horizontally as it dries in order to access to subjects on the deposition of material in the filter paper evenly.
- The control vial should be treated with 1 mL of deionised water.
- After drying, 1 mL of deionised water was added to each vial to moisten the filter paper. Each test-tube contains one earthworm. Each vial was sealed with a cap or plastic film with a small ventilation hole.
- Any behavioural or pathological symptoms and mortality were reported at 24h, at the end of the test, any behavioural or pathological symptoms and mortality of the test material should be reported. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The total weight and the average weight of earthworms was obtained by dividing the number of earthworms.
- The 48h-LC50 of chioracetamide with filter paper test method in order to verify the sensitivity of the Eisenia foerida within the range of 1.5 x 10^-3 mg/cm^2 to 4.5 x 10^-3 mg/cm^2.

FILTER PAPER TEST

Preparation of test material:
-Weigh 1.75 g of test material in small beaker and add acetone to dissolve it. Transfer the solution to a 25 mL volumetric flask and dilute to volume with acetone to a concentration of: 7.00 x 10^-2 g/mL (A).
-Transfer 7.9 mL of solution A to a 25 mL volumetric flask, dilute to volume with acetone to a concentration of 2.21 x 10^-2 g/mL (B).
-Transfer 2.5 mL of solution A to a 25 mL volumetric flask, dilute to volume with acetone to a concentration of 7.00 x 10^-3 g/mL (C).
-Transfer 7.9 mL of solution C to a 25mL volumetric flask, dilute to volume with acetone to a concentration of 2.21 x 10^-3 g/mL (D).
-Transfer 2.5 mL of solution C to a 25 mL volumetric flask, dilute to volume with acetone to a concentration of 7.00 x 10^-4 g/mL (E).

Method:
- One blank control group, one solvent group and five test material groups were designed and 70 qualified domesticated earthworms were selected. Each group was assigned 10 earthworms randomly.
- Worms were kept on moist filter paper for three hours before being placed in test vials so they could void their gut contents. They were then washed and dried before use.
- 1 mL of test material solution was pipetted into each vial and evaporated to dryness under a slow stream of filtered compressed air, the vial being rotated horizontally as it dried in order to access to subjects on the deposition of material in the filter paper evenly. The control vial should be treated with 1 mL of deionised water or 1 mL acetone.
- After drying, 1 mL of deionised water was added to each vial to moisten the filter paper. Each test-tube contained one earthworm. Each vial was sealed with a cap or plastic film with a small ventilation hole.
-Any behavioural or pathological symptoms noted and mortality was reported at 24 h. At the end of the test, any behavioural or pathological symptoms and mortality of the test material should be reported. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. Weighing the total weight of earthworms and the average weight of earthworms was obtained by dividing by the number of earthworms.

ARTIFICAL SOIL TEST

Preparation of test material:
-Weigh 2.000 g of test material in a small beaker and add 2.000 g acetone to dissolve it, a 1:1 (mass ratio ) test stock solution of test material and acetone was obtained.

Method:
-One blank control one solvent group and five test material groups were designed and 120 qualified domesticated earthworms were selected. Each group was assigned 10 earthworms randomly with four parallels.
- Earthworms had been domesticated for 24 hours in artificial soil and then washed quickly before use. Worms should be kept on moist filter paper for three hours before being placed in test vials so they could void their gut contents. They were then washed and dried before use.
- Quantity of test material corresponding to 750 g wet weight of artificial soil were mixed with 740 g wet artificial soil and 10 g of a mixture of fine ground quartz sand. The acetone must be ventilated before use. The amount of water evaporated should be replaced. 100 mg/kg test material was prepared.
-Four solvent control and four test groups were designed. Ten gram of a mixture of fine ground quartz sand and a quantity of test material corresponding to 750 g wet weight of artificial soil are mixed with 740 g wet artificial soil. Ten earthworms were placed on the test medium surface for each test container. The test medium must be ventilated before use. The amount of water evaporated should be replaced.
- The containers were covered with perforated plastic film to prevent the test medium from drying and kept under the test conditions for 14 days.
- pH of the artificial soil was measured at the beginning of the test, humidity of the artificial soil was measured at the beginning and at the end of the test, total weight of earthworms was weighted and the average weight of earthworms obtained by dividing the number of earthworms at the beginning and at the end of the test. Any behavioural or pathological symptoms should be checked and recorded at days 7 and 14. Sorting the worms from the medium 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.

- Chemical name of vehicle: Acetone

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Source: Leshui Fishing Firm of Guangzhou
- Age at test initiation: 2 to 3 months
- Weight at test initiation: 300 to 600 mg
- Justification for selection: The recommended test species was Eisenia foetida (Michaelsen). Although this was not a typical soil species, it occurred in soil rich in organic matter. Its susceptibility to chemicals resembles that of true soil-inhabiting species, it had a short life cycle. It was very prolific. It was available commercially and could be bred readily in a wide range of organic waste materials. Cocoons could be purchased commercially or distributed from a central source to ensure the same strain was used.

ACCLIMATION
- Acclimation period: All earthworms were obtained and held in the laboratory for 7 days under the same surroundings before being used. During the holding period, all the earthworms were healthy. They were breeding for 24 h in the artificial soil before the test.

Study type:

laboratory study

Substrate type:

other: Filter paper and artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 ± 2°C

pH:

6.86 to 6.92

Details on test conditions:

TEST SYSTEM (FILTER PAPER TEST)
- Test containers were selected and cleaned appropriately for the test.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 1
- No. of replicates per control: 1
- No. of replicates per vehicle control: 1

TEST SYSTEM (ARTIFICIAL SOIL TEST)
- Amount of soil or substrate: A quantity of test material corresponding to 750 g wet weight of artificial soil were mixed with 740 g wet artificial soil and 10 g of a mixture of fine ground quartz sand.
- 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

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Preparation of artificial soil:
The dry constituents were blended in the correct proportions with sphagnum peat 10%, kaolin clay 20%, industrial sand 70% 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 and re-weighing. Deionised water was added to give an overall moisture content of about 35% of the dry weight, and the medium was thoroughly mixed.
- Determining the pH of artificial soil:
5 g air-dried soil samples were placed in a 50 mL beaker, 25 mL distilled water was added and then stirred for 1 min to fully spread. The samples were laid aside for 1 hour and then the pH measured.
- Determining the moisture content of artificial soil
Two Petri dishes with 1 g soil were placed at 104 ± 1°C for 4 h and cooled in a desiccator for 30 min. When the two consecutive weighing differences was less than 0.1%, it was considered to have reached a constant quality. The average was calculated from three results. The deviation was less than 1% for each dish.
Calculation: water content % = {[(wet soil quality + dish quality)-(dish quality)] - [(dry soil + dish quality) - (dish quality )]} - [(dry soil + dishes quality) - (dishes quality)] x 100%.

OTHER TEST CONDITIONS
- Filter paper test: Dark conditions.
- Artificial soil test: 80% humidity with continuous light.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

Nominal
- Test concentrations for filter paper test: 0.07, 0.0221, 0.007, 0.00221 and 0.0007 g/mL.
- Test concentrations for artificial soil test: 100 mg/kg

Reference substance (positive control):

yes

Remarks:

Chloracetamide

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.076 mg/cm²

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

FILTER PAPER TEST
-The results demonstrated the mortality in the controls did not exceed 10% at the end of either test which fulfilled the criteria for a valid test.
- Under the conditions of the test, the results indicated that the 48 h LC50 of test material to earthworm by skin contamination was 0.076 mg/cm^2.

ARTIFICIAL SOIL TEST
- The results demonstrated the mortality in the controls was not exceed 10% at the end of either test which fulfilled the criteria for a valid test.
- The mean moisture content of artificial soil at start and at the end of test was 29.5 and 27.2%, respectively.
- Under the conditions of the present test, the 14 d LC50 of the test substance to earthworm was higher than 100 mg/kg.

Results with reference substance (positive control):

SENSITIVITY TEST
-The 48 h-LC50 of reference substance chloracetamide was 0.00384 mg/cm^2 which is within the range of 0.001.5 mg/cm^2 to 0.004.5 mg/cm^2.

Table 1: Mortality of the test material during the filter paper test

Concentration (g/mL)	Number of earthworms	24 hours		48 hours
		No. of mortalities	Mortality (%)	No. of mortalities	Mortality (%)
Blank control	10	0	0	1	10
Solvent group	10	0	0	1	10
0.07	10	7	70	10	100
0.0221	10	4	40	9	90
0.007	10	0	0	8	80
0.00221	10	0	0	1	10
0.0007	10	0	0	1	10

 

Table 2: Mortality of the test material during the artificial soil test

Concentration (mg/kg)	Number of earthworms	7 days			14 days
		No. of mortalities	Mortality	Modified mortality	No. of mortalities	Mortality	Modified mortality
Blank control	40	0	0	0	0	0	0
Solvent group	40	0	0	0	0	0	0
100	40	0	0	0	1	2.5	2.5

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the results indicated that the 48 h LC50 of the test material to earthworm by skin contamination was 0.076 mg/cm^2, and the 14 d LC50 of the test material to earthworms by oral exposure was >100 mg/kg.

Executive summary:

The acute toxicity of the test material to earthworms (Eisena foetida) was investigated in a test compromising both a 48 hour filter paper test and a 14 day artificial soil test, in accordance to standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 207 Earthworm Acute toxicity test" and OECD 207 under GLP conditions. Five concentrations (0.0007, 0.00221, 0.007, 0.0221 and 0.07 g/mL) were examined in a 48-hour filter paper test along with a 14 day artificial soil limit test performed at 100 mg/kg dry soil. The mortality of the earthworms to the test material and controls as well as behavioural and pathological symptoms were recorded. The validity of the test was confirmed with (i) the results from the reference substance, chloracetamide, lying within the expected range, (ii) the mortality of earthworms in the control group not exceeding 10% at the end of the test and (iii) the average weight of earthworms in the control group falling no more than 30%. Under the conditions of the study, the results indicated that the 48 h LC50 of the test material to earthworms by skin contamination was 0.076 mg/cm², and the 14 d LC50 of the test material to earthworm by oral exposure was > 100 mg/kg.

Description of key information

Under the conditions of the study, the results indicated that the 48 h LC50 of the test material to earthworms by skin contamination was 0.076 mg/cm², and the 14 d LC50 of the test material to earthworm by oral exposure was > 100 mg/kg.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

100 mg/kg soil dw

Additional information

The acute toxicity of the test material to earthworms (Eisena foetida) was investigated in a test compromising both a 48 hour filter paper test and a 14 day artificial soil test, in accordance to standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 207 Earthworm Acute toxicity test" and OECD 207 under GLP conditions. Five concentrations (0.0007, 0.00221, 0.007, 0.0221 and 0.07 g/mL) were examined in a 48 hour filter paper test along with a 14 day artificial soil limit test performed at 100 mg/kg dry soil. The mortality of the earthworms to the test material and controls, as well as behavioural and pathological symptoms, were recorded. The validity of the test was confirmed with (i) the results from the reference substance, chloracteamide, lying within the expected range, (ii) the mortality of earthworms in the control group not exceeding 10% at the end of the test and (iii) the average weight of earthworms in the control group falling no more than 30%. Under the conditions of the study, the results indicated that the 48 h LC50 of the test material to earthworms by skin contamination was 0.076 mg/cm², and the 14 d LC50 of the test material to earthworm by oral exposure was > 100 mg/kg.