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

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-10-2010 to 16-11-2010

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 226 (Predatory Mite (Hypoaspis (Geolaelaps) Aculeifer) Reproduction Test in Soil)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

soil

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of mixing into soil or dung: Not applicable. See below.
- Method of application to soil surface: 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 : 819.8 mg test item in 50 mL acetone -> 400 mg test item/kg artificial soil (dw) concentration
mixture 2 : 25 mL mixture 1 + 25 mL acetone -> 50 mL -> 200 mg test item/kg artificial soil (dw)
mixture 3 : 25 mL mixture 2 + 25 mL acetone -> 50 mL -> 100 mg test item/kg artificial soil (dw)
mixture 4 : 25 mL mixture 3 + 25 mL acetone -> 50 mL -> 50 mg test item/kg artificial soil (dw)
mixture 5 : 25 mL mixture 4 + 25 mL acetone -> 50 mL -> 25 mg test item/kg artificial soil (dw)
Corresponding concentrations:
Control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw)
Each mixture was stirred for approx. 1 minute. 5 ml of each mixture was applied onto 20 g quartz sand. After evaporation of the solvent under a fume hood (ea. 1 h), the coated quartz sand was incorporated into 180 g artificial soil to prepare the different test item soil concentrations. The test item concentrations were corrected for purity of the test item (detailed in the full study report). 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. 3 minutes. This was done by using a mixer fitted on a drilling machine. This way and due to the small amount of artificial soil used per concentration (i.e. 200 g dry weight) the artificial soil and the test item coated fraction of the quartz sand were homogenously mixed. The mixed artificial soil was visually and manually checked for homogeneity. Finally, the spiked artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 20 g dw (dry weight) was used which corresponds to approx. 2 cm depth of wet soil in the test vessel.
- 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.
- Volume of test solution applied: See above.

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: Predatory Mite (Hypoaspis (Geolaelaps) aculeifer CANESTRINI (Acari: Laelapidae)
- Source: In house laboratory strain, source and strain documented in the full study report.
- Taxonomic confirmation: Yes.
- By whom? : By laboratory.
- Age at test initiation (mean and range, SD): Adult mated female mites were used to start the test. The mites were of similar age (7 to 14 days after reaching the adult stage) i.e. they were taken from a synchronised culture between the 28th and 35th day after starting the respective culture.
- Stage at test initiation: See above.
- Weight at test initiation (mean and range, SD): Not applicable.
- Date of collection: Documented in the full study report.
- Cultural background (if honeybees): Not applicable.
- Disease free: Yes.
- Kept according to standard practices: Yes.
- Health condition of the hive (if honeybees): Not applicable.

ACCLIMATION
- Acclimation period: Adult mated female mites were used to start the test. The mites were of similar age (7 to 14 days after reaching the adult stage) i.e. they were taken from a synchronised culture between the 28th and 35th day after starting the respective culture.
- Acclimation conditions (same as test or not): See above.
- Feeding: Prey mites (Tyrophagus putrescentiae) at the beginning of the test. Food was checked twice per week and was added ad libitum.
- Health during acclimation (any mortality observed): None reported.

RATIONALE FOR SELECTION OF SPECIES (if other than requested by test guideline): Not applicable.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

14 d

Remarks:

In accordance with the relevant guidelines.

Post exposure observation period:

After an extraction period of 48 hours the number of adults and the number of juveniles were determined

Test temperature:

Definitive study: mean: 19.5 °C and range: 19.1 °C – 19.9 °C
Lighting: 16/8 h light/dark at 539 - 728 lx (recom. 400 to 800 lx)

pH (if soil or dung study):

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

Humidity:

Soil moisture: was measured at the beginning and end of the definitive test.
Start (% dry mass): Control: 28.6% ; solvent control: 29.5% ; test groups: 26.6% - 28.8%
Start (% of WHCmax): Control: 53.5% ; solvent control: 55.2% ; test groups: 52.2 – 55.9%
End: (% dry mass): Control: 27.3% ; solvent control: 28.2% ; test groups: 26.6% - 28.8%
Start (% of WHCmax): Control: 51.1% ; solvent control: 52.8% ; test groups: 49.8% - 53.9%

Photoperiod and lighting:

Lighting: 16/8 h light/dark at 539 - 728 lx (recom. 400 to 800 lx)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass 200 mL volume diameter 5 cm. Covered with perforated parafilm (to allow aeration)
- Amount of soil or dung or substrate: Per test vessel an amount corresponding to 20 g dw (dry weight) was used which corresponds to approx. 2 cm depth of wet soil in the test vessel.
- Depth of dung (cm): Not applicable. Artificial 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: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil or dung)
- Geographic location: Not applicable.
- Pesticide use history or pharmaceutical use history at the collection site: Not applicable.
- Dung source livestock: Not applicable.
- Pesticide use history at the collection site: Not applicable.
- Collection procedures: Not applicable.
- Sampling depth (cm): Not applicable.
- Soil texture (if natural soil) Not applicable.
- Composition (if artificial substrate): See below. A quantity of the quartz sand (10% of the total amount) was separated for the application of the test item. Calcium carbonate was used to adjust the pH-value to 6 ± 0.5. The maximum water-holding capacity (WHCmax) of the artificial soil was determined.
- % sand: 73 - 74% Quartz sand (> 50% particle size between 50-200 μm)
- % silt: sphagnum peat 5% (no visible plant remains ; pH 6.0, finely ground and dried ; adjusted with < 1% CaCO3)
- % clay: 20% kaolin clay (Kaolinite content > 30%)
- Soil taxonomic classification: Not applicable, artificial substrate
- Soil classification system: Not applicable, artificial substrate
- Composition (if artificial substrate): See above
- Organic carbon (%): Not reported
- Nitrogen (%): Not reported.
- C/N ratio (%): Not reported.
- Maximum water holding capacity (in % dry weight): (i) 21.4- 32.0% of the dry weight of the artificial soil ; or (ii) 40-60% of the WHCmax (53.4%) of the artificial soil
A quantity of the quartz sand (10% of the total amount) was separated for the application of the test item. Calcium carbonate was used to adjust the pH-value to 6 ± 0.5. The maximum water-holding capacity (WHCmax) of the artificial soil was determined.
- CEC: Not reported.
- Pretreatment of soil or dung: 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 : 819.8 mg test item in 50 mL acetone -> 400 mg test item/kg artificial soil (dw) concentration
mixture 2 : 25 mL mixture 1 + 25 mL acetone -> 50 mL -> 200 mg test item/kg artificial soil (dw)
mixture 3 : 25 mL mixture 2 + 25 mL acetone -> 50 mL -> 100 mg test item/kg artificial soil (dw)
mixture 4 : 25 mL mixture 3 + 25 mL acetone -> 50 mL -> 50 mg test item/kg artificial soil (dw)
mixture 5 : 25 mL mixture 4 + 25 mL acetone -> 50 mL -> 25 mg test item/kg artificial soil (dw)
Corresponding concentrations:
Control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw)
Each mixture was stirred for approx. 1 minute. 5 ml of each mixture was applied onto 20 g quartz sand. After evaporation of the solvent under a fume hood (ea. 1 h), the coated quartz sand was incorporated into 180 g artificial soil to prepare the different test item soil concentrations. The test item concentrations were corrected for purity of the test item (detailed in the full study report). 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. 3 minutes. This was done by using a mixer fitted on a drilling machine. This way and due to the small amount of artificial soil used per concentration (i.e. 200 g dry weight) the artificial soil and the test item coated fraction of the quartz sand were homogenously mixed. The mixed artificial soil was visually and manually checked for homogeneity. Finally, the spiked artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 20 g dw (dry weight) was used which corresponds to approx. 2 cm depth of wet soil in the test vessel.
- 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 539 - 728 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 ; Reproduction ; Morphology and Behaviour

VEHICLE CONTROL PERFORMED: Yes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw)
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Preliminary range-finding study: 0 (blank control), 0, 0.1, 1.0, 10, 100, 1000 mg/kg dry weight (nominal)
- Test concentrations: Five percent mortality was observed in the control, the solvent control and at the concentrations of 0.1 mg test item/kg soil (dw). At the concentrations of 1.0, 10 and 100 mg test item/kg soil (dw) no mortality was observed. At the concentration of 1000 mg test item/kg soil (dw) 25% mortality was observed. Concerning the number of juveniles mean values of 211.0 and 228.0 were found in the control and in the solvent control, respectively. And at the concentrations of 0.1, 1.0, 10 and 100 mg test item/kg artificial soil (dw) 209.5 to 269.0 juveniles were found, respectively. At the concentration of 1000 mg test item/kg artificial soil (dw) 112.0 juveniles were found. This information was used to justify the definitive test concentrations of: Control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw).

Nominal and measured concentrations:

Control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw)

Reference substance (positive control):

yes

Remarks:

Boric acid

Duration:

14 d

Dose descriptor:

NOEC

Remarks:

Mortality

Effect conc.:

200 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LOEC

Remarks:

Mortality

Effect conc.:

400 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

NOEC

Remarks:

Reproduction

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

14 d

Dose descriptor:

LOEC

Remarks:

Reproduction

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: Yes. See tables.
- Total mass of adults at beginning of test: approx. 180 females and 20 males per unit
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Not applicable.
- No. of offspring produced: See tables.
- No. of unhatched cocoons: Not applicable.
- Morphological abnormalities: No effects on behaviour or morphology of the juvenile and adult predatory mites were observed.
- Behavioural abnormalities: No effects on behaviour or morphology of the juvenile and adult predatory mites were observed.
- Other biological observations: None reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (Boric Acid) – full information documented in the full study report
- Relevant effect levels EC50 = 165.2 (C.I. 131 – 204.0) 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

Reported statistics and error estimates:

See: "Overall remarks, attachments"

Table 1.0 : Effects on Mortality and Reproduction of Hypoaspis aculeifer

Concentration  / mg test item/kg soil (dw)	Number of dead adult female mites	Mortality / %	Number of juveniles / mean	+/- sd	Number of juveniles / % of solvent control	Reproduction / % of solvent control
Control	2	5.0	202.3	9.5	-	-
Solvent Control	8	10.0	211.5	13.1	100.0	100.0
25	5	12.5	228.0	31.7	107.8	107.8
50	5	12.5	230.8	31.7	109.1	109.1
100	3	7.5	166.0	52.4	78.5 #	78.5 #
200	6	15.0	168.8	56.8	79.8 #	79.8 #
400	12	30.0 *	151.5	39.3	71.6 #	71.6 #

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

# = significantly different to solvent control (Williams t-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:
14-d NOEC Mortality : 200 mg test item/kg soil (dw)
14-d LOEC Mortality : 400 mg test item/kg soil (dw)
14-d NOEC Reproduction : 50 mg test item/kg soil (dw)
14-d LOEC Reproduction : 100 mg test item/kg soil (dw)

Executive summary:

The toxicity of the test item to the predaceous mite (Hypoaspis aculeifer) Acari (soil-dwelling predatory mite) was determined in a 14-d toxicity test in artificial soil according to OECD TG 226 guideline under GLP. Initially, a preliminary range finding test was conducted at : 0 (blank control), 0, 0.1, 1.0, 10, 100, 1000 mg/kg dry weight soil (nominal) which indicated: five percent mortality was observed in the control, the solvent control and at the concentrations of 0.1 mg test item/kg soil (dw). At the concentrations of 1.0, 10 and 100 mg test item/kg soil (dw) no mortality was observed. At the concentration of 1000 mg test item/kg soil (dw) 25% mortality was observed. Concerning the number of juveniles mean values of 211.0 and 228.0 were found in the control and in the solvent control, respectively. And at the concentrations of 0.1, 1.0, 10 and 100 mg test item/kg artificial soil (dw) 209.5 to 269.0 juveniles were found, respectively. At the concentration of 1000 mg test item/kg artificial soil (dw) 112.0 juveniles were found as a mean value from two replicates. Subsequently, a definitive test was conducted at blank control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw). Ten adult female Hypoaspis aculeifer per test replicate (4 for the water control, 8 for the solvent control, 4 per test item concentration) were exposed to the test item for 14 days at the nominal concentrations indicated at a temperature of 19.1°C to 19.9°C and 539 to 728 lx. After 14 days of exposure followed by an extraction period of 48 hours the number of adults and the number of juveniles were determined. Mortality, reproduction and morphology and behaviour were determined. All validity criteria were considered to have been met. Boric acid tested as a reference item was EC50 =165.2 (C.I. 131 – 204.0) mg/kg (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. After 14 days of exposure 5.0% mortality was observed in the control and 10.0% in the solvent control. At the concentrations of 25, 50, 100 and 200 mg test item/kg soil (dw) 7.5% to 15.0% mortality was observed. At the concentration of 400 mg test item/kg soil (dw) 30% mortality was observed. Statistical analysis (Fisher's Exact Binomial Test; 1-sided; p< or =0.05) revealed a significant difference between the solvent control and the concentration of 400 mg test item/kg artificial soil ( dw). The fecundity as the number of juveniles per adult female introduced per test vessel (i.e. 10 females per test vessel) was for the water control 19.2 to 21.3 juveniles and for the solvent control 18.9 to 23.0 juveniles. At the concentrations of 25 and 50 mg test item/kg soil (dw) 19.5 to 26.7 and 19.4 to 26.6 juveniles were determined. At the concentrations of 100, 200 and 400 mg test item/kg soil ( dw) 8.8 to 19.9, 9.9 to 23.8 and 11.7 to 20.5 juveniles were found, respectively. Statistical analysis (Williams t-test; 1-sided, p < or =0.05) showed a significant difference concerning the number of juveniles between the solvent control and the concentrations of 100, 200 and 400 mg test item/kg soil (dw). No effects on behaviour or morphology of the juvenile and adult predatory mites were observed. The test item relevant effect levels were estimated based on nominal loading to be:14-d NOEC Mortality was 200 mg test item/kg soil (dw) and 14-d LOEC Mortality was 400 mg test item/kg soil (dw), whereas 14-d NOEC Reproduction was 50 mg test item/kg soil (dw) and 14-d LOEC Reproduction was 100 mg test item/kg soil (dw).

Description of key information

14d- NOEC Reproduction (predatory mite) : 50 mg test item/kg soil (dw) nominal, OECD TG 226, 2010

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

50 mg/kg soil dw

Additional information

Key study : OECD TG 226, 2010 : The toxicity of the test item to the predaceous mite (Hypoaspis aculeifer) Acari (soil-dwelling predatory mite) was determined in a 14-d toxicity test in artificial soil according to OECD TG 226 guideline under GLP. Initially, a preliminary range finding test was conducted at : 0 (blank control), 0, 0.1, 1.0, 10, 100, 1000 mg/kg dry weight soil (nominal) which indicated: five percent mortality was observed in the control, the solvent control and at the concentrations of 0.1 mg test item/kg soil (dw). At the concentrations of 1.0, 10 and 100 mg test item/kg soil (dw) no mortality was observed. At the concentration of 1000 mg test item/kg soil (dw) 25% mortality was observed. Concerning the number of juveniles mean values of 211.0 and 228.0 were found in the control and in the solvent control, respectively. And at the concentrations of 0.1, 1.0, 10 and 100 mg test item/kg artificial soil (dw) 209.5 to 269.0 juveniles were found, respectively. At the concentration of 1000 mg test item/kg artificial soil (dw) 112.0 juveniles were found as a mean value from two replicates. Subsequently, a definitive test was conducted at blank control, solvent control, 25, 50, 100, 200 and 400 mg test item/kg artificial soil (dw). Ten adult female Hypoaspis aculeifer per test replicate (4 for the water control, 8 for the solvent control, 4 per test item concentration) were exposed to the test item for 14 days at the nominal concentrations indicated at a temperature of 19.1°C to 19.9°C and 539 to 728 lx. After 14 days of exposure followed by an extraction period of 48 hours the number of adults and the number of juveniles were determined. Mortality, reproduction and morphology and behaviour were determined. All validity criteria were considered to have been met. Boric acid tested as a reference item was EC50 =165.2 (C.I. 131 – 204.0) mg/kg (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. After 14 days of exposure 5.0% mortality was observed in the control and 10.0% in the solvent control. At the concentrations of 25, 50, 100 and 200 mg test item/kg soil (dw) 7.5% to 15.0% mortality was observed. At the concentration of 400 mg test item/kg soil (dw) 30% mortality was observed. Statistical analysis (Fisher's Exact Binomial Test; 1-sided; p< or =0.05) revealed a significant difference between the solvent control and the concentration of 400 mg test item/kg artificial soil ( dw). The fecundity as the number of juveniles per adult female introduced per test vessel (i.e. 10 females per test vessel) was for the water control 19.2 to 21.3 juveniles and for the solvent control 18.9 to 23.0 juveniles. At the concentrations of 25 and 50 mg test item/kg soil (dw) 19.5 to 26.7 and 19.4 to 26.6 juveniles were determined. At the concentrations of 100, 200 and 400 mg test item/kg soil ( dw) 8.8 to 19.9, 9.9 to 23.8 and 11.7 to 20.5 juveniles were found, respectively. Statistical analysis (Williams t-test; 1-sided, p < or =0.05) showed a significant difference concerning the number of juveniles between the solvent control and the concentrations of 100, 200 and 400 mg test item/kg soil (dw). No effects on behaviour or morphology of the juvenile and adult predatory mites were observed. The test item relevant effect levels were estimated based on nominal loading to be: 14-d NOEC Mortality was 200 mg test item/kg soil (dw) and 14-d LOEC Mortality was 400 mg test item/kg soil (dw), whereas 14-d NOEC Reproduction was 50 mg test item/kg soil (dw) and 14-d LOEC Reproduction was 100 mg test item/kg soil (dw).