Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-12-2013 to 25-03-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 207 Earthworm, Acute Toxicity Test[M] (2013)

Version / remarks:

The test was conducted in accordance with China HJ/T153 - 2004 Chemical Test Guideline stipulations and 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 Press. 2013: 58-62. The test was also completed under OECD TG 207: Earthworm, Acute Toxicity (1984).

Deviations:

no

Qualifier:

according to guideline

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6200 (Earthworm Subchronic Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): Not applicable.
- Method of mixing into soil (if used): Range finding test: In the range-finding test, 10 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 490 g dry artificial soil for each test container. The medium was thoroughly mixed by an electric mixer avoiding build-up of heat. ; Definitive test or limit test: 50 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 1950 g dry artificial soil for the single test concentration and the Control. Then the total 2000 g test substrate was divided into four replicates with 500 g each. Deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. The complete mixture was moist but not so wet that water appeared when the artificial soil was compressed. The concentrations were: 0 mg/kg bw (with 0 g test item in to 2000 g dry soil) and/or 1000 mg/kg (with 2.002 g test item into 2000 g dry soil).
- Method of application to soil surface (if used): See above.
- Controls: The blank (negative) control group consisted of a test system without application of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evaporation of vehicle before use: Not applicable.

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earth worm.
- Source: Nanjing Earthworm Nursery (China)
- Age at test initiation (mean and range, SD): older than 2 months (with clitellum)
- Weight at test initiation (mean and range, SD): day 1: mean weight 397 mg and range: 383 – 415 mg ; Individual test item concentration groups – mean and individual weights are reported in the full study report.

ACCLIMATION
- Acclimation period: 30 days in soil then precultured in control soil for 24h before start of the test
- Acclimation conditions (same as test or not): Yes (precultured in control soil)
- Health during acclimation (any mortality observed): None reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Remarks:

Sorting the Earthworms from the artificial soil and testing their reaction to a mechanical stimulus at the front end at 7d. Pathological condition were checked and recorded. Any behavioural or pathological symptoms were checked and recorded.

Test temperature:

Definitive study: 20 °C - 22 °C

pH:

pH: pH of the artificial soil was measured at the beginning of the test. Weighed 5 g of air-dried soil sample in 50 mL beaker. 25 mL CO2- free distilled water was added by cylinder. Stirred for 5 min. Standing for 1 h and then measurement of the pH. pH of the artificial soil at the start of the test:
Temperature: 21.8 °C ; First reading pH: 6.46; Second reading pH: 6.46 and Mean pH: 6.46

Moisture:

Definitive study: Moisture: 27% to 35%
Air humidity in chamber was 80% to 85% with continuous lighting of 400 to 800 lux
Soil moisture content is determined by drying a small sample at 105°C and re-weighing.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 Litre; glass
- Amount of soil or substrate: Definitive test 2000 g dry artificial soil (range finding test: 500 g dry soil)
- 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: Not applicable.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Not applicable.
- Sampling depth (cm): Not applicable.
- Composition (if artificial substrate): See below.
- % sand: 70% industrial sand (60% particle size between 50-200 μm)
- % silt: sphagnum peat 10% (no visible plant remains ; pH 6.0, finely ground and dried)
- % clay: 20% kaolin clay (Kaolinite content > 30%)
- Organic carbon (%): not reported
- Maximum water holding capacity (in % dry weight): Deionized water was added to give an overall moisture content of ca. 35% of the dry weight, and the medium was thoroughly mixed
- Pretreatment of soil: Range finding test: In the range-finding test, 10 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 490 g dry artificial soil for each test container. The medium was thoroughly mixed by an electric mixer avoiding build-up of heat. Concentrations of 0, 10, 99.8, 1001 mg/kg dry soil were prepared using 500 g total dry soil ; Definitive test or limit test: 50 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 1950 g dry artificial soil for the single test concentration and the Control. Then the total 2000 g test substrate was divided into four replicates with 500 g each. Deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. The complete mixture was moist but not so wet that water appeared when the artificial soil was compressed. The concentrations were: 0 mg/kg bw (with 0 g test item in to 2000 g dry soil) and/or 1000 mg/kg (with 2.002 g test item into 2000 g dry soil).
- Storage (condition, duration): Not applicable. Artificial soil prepared prior to exposure.
- Stability and homogeneity of test material in the medium: Stable. No instability reported.

OTHER TEST CONDITIONS
- Photoperiod: Continuous light
- Light intensity: The light intensity was 400 to 800 lux.
Other: The humidity was kept constant by the chamber from 80% to 85% during the test period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, behavioural and pathological signs

VEHICLE CONTROL PERFORMED: No.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Definitive study (limit test): 0 (blank control) mg/kg and/or 1000 mg/kg
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Preliminary range-finding study: 0 (blank control), 0, 10, 99.8, 1001 mg/kg dry weight (nominal)
- Results used to determine the conditions for the definitive study: Yes.

Nominal and measured concentrations:

Preliminary range-finding study: 0 (blank control), 0, 10, 99.8, 1001 mg/kg dry weight (nominal)
Definitive study: 0 (blank control) mg/kg and/or 1000 mg/kg dry weight (nominal)

Reference substance (positive control):

yes

Remarks:

chloracetamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% C.I. - mg/kg dry soil

Details on results:

- Mortality at end of exposure period: None in control or in 1000 mg/kg dry weight soil.
- Total mass of adults at beginning of test: Weights are reported in the full study report.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: No differences between worm weight to control was observed.
- No. of offspring produced: Not applicable.
- No. of unhatched cocoons: Not applicable.
- Morphological abnormalities: None reported.
- Behavioural abnormalities: None reported.
- Other biological observations: At the beginning of the test item administration, test worms soon disappeared into the test medium soil. During the test, all earthworms remained in the soil.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Relevant effect levels: 14d LC50 = 25.1 (C.I. 22.3 – 28.27) mg/kg dry weight
- Other: Not applicable.

Reported statistics and error estimates:

The LC50 and 95% confidence limits were calculated by Trimmed Spearman-Karber Method (Version 1.5, US EPA). In this test, no statistical method was utilised due to the no/low toxicity for the test item at the limit concentration.

Test 1. Mortality during the artificial soil test

Group	Number of earthworms	7d			14d
		Mortalities	Mortality (%)	Abnormalities	Mortalities	Mortality (%)	Abnormalitiesl
0 (blank control) mg/kg dry soil	40 (n = 4 replicates ; 10)	0	0	None	0	0	None
1000 mg/kg dry soil	40 (n = 4 replicates ; 10)	0	0	None	0	0	None

Validity criteria fulfilled:

yes

Conclusions:

The test item 14d LC50 was determined to be > 1000 mg/kg dry soil based on nominal loading.

Executive summary:

The acute toxicity of the test item to earth worm (Eisenia foetida) was determined in a 14d acute toxicity test in artificial soil according to OECD TG 207 and the China CRC MEP guidelines, Effects on Biotic Systems, 207 Earthworm, Acute Toxicity Test (2013) referenced in China HJ/T153 - 2004 under GLP. Following a preliminary range finding test conducted at : 0 (blank control), 10, 99.8, 1001 mg/kg dry soil which indicated no mortalities or abnormalities, a definitive limit test of two test groups were conducted with four replicates per group in artificial soil at concentrations of : 0 (blank control) and 1000 mg/kg dry weight (nominal). 50 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 1950 g dry artificial soil for the single test concentration and the Control. Then the total 2000 g test substrate was divided into four replicates with 500 g each. Deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. The complete mixture was moist but not so wet that water appeared when the artificial soil was compressed. The concentrations were: 0 mg/kg bw (with 0 g test item in to 2000 g dry soil) and/or 1000 mg/kg (with 2.002 g test item into 2000 g dry soil).The test temperature was maintained at 20 - 22 °C under continuous light at 400 to 800 lux with a moisture content of soil of 27 to 35%. The test system was validated against a 2-chloracetamide reference item. There was no mortality in the blank control group or at 1000 mg/kg dry weight and no sign of abnormal behaviour. The 14d LC50 was determined to be> 1000 mg/kg dry soil based on nominal loading. All relevant guideline criteria were met.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-10-2009 to 23-12-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met or with acceptable deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

yes

Remarks:

minor deviations in temperature and moisture content that were considered not to impact the integrity of the study.

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

yes

Remarks:

minor deviations in temperature and moisture content that were considered not to impact the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2007 ; signature: March 2009

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): Not applicable.
- Method of mixing into soil (if used): The test item is not soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone. All concentrations to be tested were based on the test item. The test item was dissolved in an amount of acetone sufficient to prepare a stock solution This stock solution was used to produce the various dosage solutions of the test item. An appropriate amount of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil by the solvent solution. After evaporation of the solvent by placing it under a fume hood, the quartz sand was coated by the test item.
Test item Stock solutions:
mixture 1 : 8197.1 mg test item in 100 mL acetone
mixture 2 : 100 mL mixture 1 + 100 mL acetone -> 200 mL
mixture 3 : 100 mL mixture 2 + 100 mL acetone -> 200 mL
mixture 4 : 100 mL mixture 3 + 100 mL acetone -> 200 mL
mixture 5 : 100 mL mixture 4 + 100 mL acetone -> 200 mL
Corresponding concentrations:
Control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw)
Each mixture was stirred for approx. 3 minutes. 50 ml of each mixture was applied onto 200 g quartz sand. After evaporation of the solvent under a fume hood (ea. 1.5 h), the coated quartz sand was incorporated into 1800 g artificial soil to prepare the different test item soil concentrations. The test item coated fraction of the quartz sand was incorporated into the remainder of the artificial soil by thoroughly mixing it in for approx. 10 minutes as described in the guideline. The artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used.
- Controls: The blank (negative) control group consisted of a test system without application of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable. See above.
- Evaporation of vehicle before use: Yes. See above.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earth worm.
- Source: In house laboratory strain, source and strain documented in the full study report.
- Age at test initiation (mean and range, SD): older than 2 months and less than one year (+/- 4 weeks per individual) ; ca. 9 months old with clitellum ; Adult
- Weight at test initiation (mean and range, SD): fresh weight 300 – 600 mg ; day 0: control mean: 434.1 g (SD 61.3) n=10 ; solvent control: 408.0 g (SD 52.6) n= 10 ; test item: 62.5 mg/kg soil (dw) group: 421.5 g (SD 69.1) n=10 ; test item: 1000 mg/kg soil (dw) group: 432.9 g (SD 43.3) n=10

ACCLIMATION
- Acclimation period: worms selected for the test were acclimatised in artificial soil under test conditions at least 24 hours before starting the test
- Acclimation conditions (same as test or not): Yes (and taken from a pre-synchronised culture)
- Health during acclimation (any mortality observed): None reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

28 d

Remarks:

In accordance with the relevant guidelines.

Post exposure observation period:

After 28 days of exposure, the adult worms were removed and the cocoons produced by these individuals were kept for a further 28 days in the treated artificial soil. At the end of the test period (i.e. after 56 days) the juvenile worms hatched from these cocoons were extracted from the artificial soil.

Test temperature:

Definitive study: mean: 19.2 °C and range: 16.7 °C – 20.7 °C – it was noted in the study:
The temperature was for a few short periods of time below 18 °C (guideline recommended) at down to 16.7 °C, however it was considered the test results were not affected. Lighting: 16/8 h light/dark at 521 - 561 lx (recom. 400 to 800 lx)

pH:

pH: pH of the artificial soil was measured at the beginning and end of the definitive test.
Start: Control: 5.7 ; solvent control: 5.7 ; test groups: 5.7
End: Control: 6.4 ; solvent control: 6.3 ; test groups: 5.9 to 6.3
This was within the required pH range : 6.0 +/- 0.5

Moisture:

Soil moisture: was measured at the beginning and end of the definitive test.
Start (% dry mass): Control: 43.2% ; solvent control: 46.4% ; test groups: 44.2% - 45.6%
Start (% of WHCmax): Control: 54.5% ; solvent control: 58.5% ; test groups: 55.7% - 57.5%
End: (% dry mass): Control: 47.7% ; solvent control: 47.1% ; test groups: 44.3% - 49.3%
Start (% of WHCmax): Control: 60.2% ; solvent control: 59.4% ; test groups: 55.3% - 62.2%
Soil moisture at the end of the test was at three treatments (i.e. Water control, 62.5 and 125 mg test item/kg soil (dw)) slightly higher than required by the guideline.
All other requirements concerning test performance have been fulfilled. It was considered the test results were not affected.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 170.5 cm2 and height 6 cm; polystyrol plastic covered by transparent cover
- Amount of soil or substrate: Definitive test: Per test vessel an amount corresponding to 500 g dw (dry weight) was used.
- 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: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Not applicable.
- Sampling depth (cm): Not applicable.
- Composition (if artificial substrate): See below.
- % sand: 69.6% Quartz sand (> 50% particle size between 50-200 μm)
- % silt: sphagnum peat 10% (no visible plant remains ; pH 6.0, finely ground and dried ; adjusted with 0.4% CaCO3)
- % clay: 20% kaolin clay (Kaolinite content > 30%)
- Organic carbon (%): Not reported
- Maximum water holding capacity (in % dry weight): The maximum water-holding capacity (WHCmax) of the artificial soil was determined. Per test vessel an amount corresponding to 500 g dw (dry weight) was used. Soil moisture: was measured at the beginning and end of the definitive test.
Start (% dry mass): Control: 43.2% ; solvent control: 46.4% ; test groups: 44.2% - 45.6%
Start (% of WHCmax): Control: 54.5% ; solvent control: 58.5% ; test groups: 55.7% - 57.5%
End: (% dry mass): Control: 47.7% ; solvent control: 47.1% ; test groups: 44.3% - 49.3%
Start (% of WHCmax): Control: 60.2% ; solvent control: 59.4% ; test groups: 55.3% - 62.2%
Soil moisture at the end of the test was at three treatments (i.e. Water control, 62.5 and 125 mg test item/kg soil (dw)) slightly higher than required by the guideline.
All other requirements concerning test performance have been fulfilled. It was considered the test results were not affected.
- Pretreatment of soil: The test item is not soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone. All concentrations to be tested were based on the test item. The test item was dissolved in an amount of acetone sufficient to prepare a stock solution This stock solution was used to produce the various dosage solutions of the test item. An appropriate amount of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil by the solvent solution. After evaporation of the solvent by placing it under a fume hood, the quartz sand was coated by the test item.
Test item Stock solutions:
mixture 1 : 8197.1 mg test item in 100 mL acetone
mixture 2 : 100 mL mixture 1 + 100 mL acetone -> 200 mL
mixture 3 : 100 mL mixture 2 + 100 mL acetone -> 200 mL
mixture 4 : 100 mL mixture 3 + 100 mL acetone -> 200 mL
mixture 5 : 100 mL mixture 4 + 100 mL acetone -> 200 mL
Corresponding concentrations:
Control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw)
Each mixture was stirred for approx. 3 minutes. 50 ml of each mixture was applied onto 200 g quartz sand. After evaporation of the solvent under a fume hood (ea. 1.5 h), the coated quartz sand was incorporated into 1800 g artificial soil to prepare the different test item soil concentrations. The test item coated fraction of the quartz sand was incorporated into the remainder of the artificial soil by thoroughly mixing it in for approx. 10 minutes as described in the guideline. The artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used.
- Storage (condition, duration): Not applicable. Artificial soil prepared prior to exposure.
- Stability and homogeneity of test material in the medium: Stable. No instability reported.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark at 521 - 561 lx (recom. 400 to 800 lx)
- Light intensity: The light intensity was 400 to 800 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Biomass and Reproduction of Eisenia fetida.

VEHICLE CONTROL PERFORMED: Yes (acetone) with n=8 replicates.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw)
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Preliminary range-finding study: 0 (blank control), 0, 1.0, 10, 100, 1000 mg/kg dry weight (nominal)
- Results used to determine the conditions for the definitive study: Yes. No mortality was observed in the solvent control and at the concentrations of 1.0, 10 and 1000 mg test item/kg soil ( dw). At the concentration of 100 mg test item/kg soil (dw) 5% mortality was observed. Mean values of 77.5 to 91.0 cocoons were found in the solvent control and at the concentrations of 1.0, 10, 100 and 1000 mg test item/kg artificial soil (dw), respectively. The number of juveniles after 28 days reached from 4.0 to 18.5 as a mean value from two replicates in all treatments tested.

Nominal and measured concentrations:

Control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw)

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

28 d

Dose descriptor:

NOEC

Remarks:

mortality

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Remarks:

mortality

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Remarks:

mortality

Effect conc.:

880.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: C.I. 762.5 - 1073.7 mg/kg (dw)

Duration:

28 d

Dose descriptor:

NOEC

Remarks:

Biomass

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

LOEC

Remarks:

Biomass

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

56 d

Dose descriptor:

NOEC

Remarks:

reproduction

Effect conc.:

62.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Remarks:

reproduction

Effect conc.:

125 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Remarks:

reproduction

Effect conc.:

153.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: C.I. 139.6 - 168.8 mg/kg (dw)

Details on results:

- Mortality at end of exposure period: Yes. See tables.
- Total mass of adults at beginning of test: Not applicable. The number and biomass (mg) of the earthworms at the beginning and end of the test at 28 days are documented in the full study report. See above sections.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Control 155.3%; solvent control mean: 160.9% ; 62.5 mg/kg (dw): 166.2% ; 125 mg/kg (dw): 163.6% ; 250 mg/kg (dw): 168.1% ; 500 mg/kg (dw): 145.6% ; 1000 mg/kg (dw): 152.1%
- No. of offspring produced: Control mean: 457.3 +/- 27.7 ; solvent control mean: 471.3 +/- 84.6 ; 62.5 mg/kg (dw): 398.5 +/- 57.0 ; 125 mg/kg (dw): 289.5 +/- 11.1 ; 250 mg/kg (dw): 140.0 +/- 19.4 ; 500 mg/kg (dw): 1.8 +/- 3.5 ; 1000 mg/kg (dw): 0.0 +/- 0.0
- No. of unhatched cocoons: None reported. At 500 and 1000 mg/kg (dw) – unhatched cocoons were observed.
- Morphological abnormalities: None.
- Behavioural abnormalities: Food consumption: Most of the food was consumed by the worms in all concentrations (except the two highest concentration tested) 7, 14, 21 and 28 days after application of the test item. In the second highest concentration half or more than half of the food was consumed and in the highest concentration of the test item tested half or less than half of the food was consumed at 7 14, 21 and 28 days after application of the test item. No other effects on behaviour or morphology of the adult earthworms were observed.
- Other biological observations: None.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (Carbendazim) – full information documented in the full study report
- Relevant effect levels: LOEC(reproduction) = 1.0 mg/kg (dw) ; demonstrating the sensitivity of the test system
- Other: The HCD results of preceding reference item tests at the laboratory are presented in the full study report: LOEC = 1.0 to 3.0 mg/kg soil (dw)

Reported statistics and error estimates:

See: "Overall remarks, attachments"

Table 1.0 - Effects on Mortality, Biomass and Reproduction of Eisenia fetida

Concentration	Adult Mortality (%)	Biomass (% of initial weight)	Number of juveniles (% of solvent control)
Control	0.0	155.3	97.0
Solvent control	0.0	160.9	100.0
62.5	2.5	166.2	84.6
125	0.0	163.6	61.4 #2
250	0.0	168.1	29.7 #2
500	12.5 #1	145.6	0.4 #2
1000	60.0 #1	152.1	0.0 #2

#1 : significantly different to control (Fisher's Exact Binomial Test; 1-sided, p < or = 0.05).

#2 : significantly different to control (Dunnett's test; 1-sided, p < or = 0.05).

Validity criteria fulfilled:

yes

Conclusions:

The test item relevant effect levels were estimated based on nominal loading to be:
28-d NOEC Mortality : 250 mg test item/kg soil (dw)
28-d LOEC Mortality : 500 mg test item/kg soil (dw)
28-d LC50 : 880.7 mg test item/kg soil (dw) (95% C.I.= 762.5 - 1073.7 mg test item/kg soil).
28-d NOEC Biomass : 1000 mg test item/kg soil (dw)
28-d LOEC Biomass : 1000 mg test item/kg soil (dw)
56-d NOEC Reproduction : 62.5 mg test item/kg soil (dw)
56-d LOEC Reproduction : 125 mg test item/kg soil (dw)
56-d EC50 Reproduction : 153.6 mg test item/kg soil (dw) (95% C.I. = 139.6 - 168.8 mg test item/kg soil)

Executive summary:

The toxicity of the test item to the earth worm (Eisenia fetida) was determined in a 28-d toxicity test in artificial soil according to OECD TG 222 and ISO 11268-2 guidelines under GLP. Initially, a preliminary range finding test was conducted at : 0 (solvent control), 0, 1.0, 10, 100, 1000 mg/kg dry weight soil (nominal) which indicated: no mortality in the solvent control and at the concentrations of 1.0, 10 and 1000 mg test item/kg soil (dw). At the concentration of 100 mg test item/kg soil ( dw) 5% mortality was observed. Mean values of 77.5 to 91.0 cocoons were found in the solvent control and at the concentrations of 1.0, 10, 100 and 1000 mg test item/kg artificial soil ( dw), respectively. The number of juveniles after 28 days reached from 4.0 to 18.5 as a mean value from two replicates in all treatments tested. Subsequently, a definitive test was conducted at Blank control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw). Ten Eisenia fetida (clitellate adults) per replicate (4 for the water control, 8 for the solvent control, 4 per test item concentration) were exposed to the test item at the nominal concentrations indicatedat a mean temperature of 19.2°C and 601 to 723 lx. After 28 days of exposure, the adult worms were removed and the cocoons produced by these worms were kept for a further 28 days in the treated artificial soil. At the end of the test period (i.e. after 56 days) the juvenile worms hatched from these cocoons were extracted from the artificial soil. Mortality, biomass and morphological and/or behavioural changes of the adult worms were assessed after 28 days. The number of juvenile earthworms was assessed after 56 days. All validity criteria were considered to have been met. The reference item Carbendazim was tested and the LOEC Reproduction was 1.0 mg/kg artificial soil (dw). The observed effect is within the expected range from the guidelines and within the range of historical data generated at the laboratory. Thereby confirming sensitivity of the test system. The test item relevant effect levels were estimated based on nominal loading to be: 28-d NOEC Mortality = 250 mg test item/kg soil (dw), 28-d LOEC Mortality = 500 mg test item/kg soil (dw), 28-d LC50 = 880.7 mg test item/kg soil (dw) (95% C.I.= 762.5 - 1073.7 mg test item/kg soil), 28-d NOEC Biomass : 1000 mg test item/kg soil (dw), 28-d LOEC Biomass = 1000 mg test item/kg soil (dw) and 56d- NOEC Reproduction = 62.5 mg test item/kg soil (dw), 56-d LOEC Reproduction = 125 mg test item/kg soil (dw), 56-d EC50 Reproduction = 153.6 mg test item/kg soil {dw) (95% C.I. = 139.6 - 168.8 mg test item/kg soil).

Description of key information

1. 14 -d acute toxicity LC50 (earthworm) : > 1000 (C.I. -) mg/kg dry weight nominal, OECD TG 207, 2014

2. 56d- NOEC Reproduction (earth worm) : 62.5 mg test item/kg soil (dw) nominal, OECD TG 222, 2010

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

62.5 mg/kg soil dw

Additional information

Key study : OECD TG 207, 2014 : The acute toxicity of the test item to earth worm (Eisenia foetida) was determined in a 14d acute toxicity test in artificial soil according to OECD TG 207 and the China CRC MEP guidelines, Effects on Biotic Systems, 207 Earthworm, Acute Toxicity Test (2013) referenced in China HJ/T153 - 2004 under GLP. Following a preliminary range finding test conducted at :0 (blank control), 10, 99.8, 1001 mg/kg dry soil which indicated no mortalities or abnormalities , a definitive limit test of two test groups were conducted with four replicates per group in artificial soil at concentrations of : 0 (blank control) and 1000 mg/kg dry weight (nominal). 50 g of a mixture of fine ground quartz sand and quantity of test substance was mixed with 1950 g dry artificial soil for the single test concentration and the Control. Then the total 2000 g test substrate was divided into four replicates with 500 g each. Deionised water was added finally to give an overall moisture content of about 35 per cent of the dry weight. The complete mixture was moist but not so wet that water appeared when the artificial soil was compressed. The concentrations were: 0 mg/kg bw (with 0 g test item in to 2000 g dry soil) and/or 1000 mg/kg (with 2.002 g test item into 2000 g dry soil).The test temperature was maintained at 20 - 22 °C under continuous light at 400 to 800 lux with a moisture content of soil of 27 to 35%. The test system was validated against a 2-chloracetamide reference item. There was no mortality in the blank control group or at 1000 mg/kg dry weight and no sign of abnormal behaviour. The 14d LC50 was determined to be > 1000 mg/kg dry soil based on nominal loading. All relevant guideline criteria were met.

 

Key study : OECD TG 222, 2010 : The toxicity of the test item to the earth worm (Eisenia fetida) was determined in a 28-d toxicity test in artificial soil according to OECD TG 222 and ISO 11268-2 guidelines under GLP. Initially, a preliminary range finding test was conducted at : 0 (solvent control), 0, 1.0, 10, 100, 1000 mg/kg dry weight soil (nominal) which indicated: no mortality in the solvent control and at the concentrations of 1.0, 10 and 1000 mg test item/kg soil (dw). At the concentration of 100 mg test item/kg soil ( dw) 5% mortality was observed. Mean values of 77.5 to 91.0 cocoons were found in the solvent control and at the concentrations of 1.0, 10, 100 and 1000 mg test item/kg artificial soil ( dw), respectively. The number of juveniles after 28 days reached from 4.0 to 18.5 as a mean value from two replicates in all treatments tested. Subsequently, a definitive test was conducted at Blank control, solvent control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil (dw). Ten Eisenia fetida (clitellate adults) per replicate (4 for the water control, 8 for the solvent control, 4 per test item concentration) were exposed to the test item at the nominal concentrations indicated at a mean temperature of 19.2°C and 601 to 723 lx. After 28 days of exposure, the adult worms were removed and the cocoons produced by these worms were kept for a further 28 days in the treated artificial soil. At the end of the test period (i.e. after 56 days) the juvenile worms hatched from these cocoons were extracted from the artificial soil. Mortality, biomass and morphological and/or behavioural changes of the adult worms were assessed after 28 days. The number of juvenile earthworms was assessed after 56 days. All validity criteria were considered to have been met. The reference item Carbendazim was tested and the LOEC Reproduction was 1.0 mg/kg artificial soil (dw). The observed effect is within the expected range from the guidelines and within the range of historical data generated at the laboratory. Thereby confirming sensitivity of the test system. The test item relevant effect levels were estimated based on nominal loading to be: 28-d NOEC Mortality = 250 mg test item/kg soil (dw), 28-d LOEC Mortality = 500 mg test item/kg soil (dw), 28-d LC50 = 880.7 mg test item/kg soil (dw) (95% C.I.= 762.5 - 1073.7 mg test item/kg soil), 28-d NOEC Biomass : 1000 mg test item/kg soil (dw), 28-d LOEC Biomass = 1000 mg test item/kg soil (dw) and 56d- NOEC Reproduction = 62.5 mg test item/kg soil (dw), 56-d LOEC Reproduction = 125 mg test item/kg soil (dw), 56-d EC50 Reproduction = 153.6 mg test item/kg soil {dw) (95% C.I. = 139.6 - 168.8 mg test item/kg soil).