picoxystrobin (ISO); methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Application method:

soil

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

A stock solution containing 4 mg/mL test substance was prepared by dissolving 100.0 mg of test substance in 25 mL acetone.

A sequential 1:1 dilution series was prepared, starting with the 4 mg/mL stock solution, and 5 mL of the corresponding solutions were added to 50 g fine quartz sand. The treated sand was left for two hours until the solvent had evaporated. The test item blended sand was added to artificial soil with reduced sand fraction to result in a final net weight of 200 g dry weight (for the control 65 g acetone treated sand was added to artificial soil with reduced sand fraction to result in a final net weight of 260 g dry weight). This composition was moistened with deionised water and mixed with a laboratory mixer to ensure a homogenous distribution. In the course of applying the test item, the soil was moistened with the added water.

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: Predatory soil mites
- Source: ECT Oekotoxikologie GmbH, 65439 Flörsheim a. M., Germany (Cultured by IBACON)
- Age at test initiation: Approximately 12 days after reaching the adult stage (33 days after placing adult females in clean rearing vesselsover a period of 3 days)
- Stage at test initiation: Adults
- Feeding: At experimental start 1 small spatula of cheese mites (Tyrophagus putrescentiae) was placed on the soil surface at test start. After 2, 5, 7, 9 and 12 days, the predatory mites were fed 1-2 small spatulas of cheese mites.

ACCLIMATION
- Acclimation period: 33 days

Study type:

laboratory study

Total exposure duration:

14 d

Test temperature:

18-22°C

pH (if soil or dung study):

At test start: 6.3-6.4
At test end: 6.4

Photoperiod and lighting:

16 h light : 8 h dark (400 to 800 lux)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass containers (volume: 100 mL; diameter: 5 cm)
- Amount of soil: Approximately 20 g ± 1.0 g artificial soil dry weight
- No. of organisms per container: 10 females per unit i.e. 80 individuals per control and 40 individuals per test item group
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Ventilation: All vessels including the additional containers were ventilated on days 2, 5, 7, 9 and 12 by opening the lids for a short period.
- Water content maintenance: Water content was checked on day 7 after the application by re-weighing each test container. It was not necessary to compensate loss of water as it did not exceed 2% of the initial soil wet weight.
- Determination of the number of soil mites: After 14 days of incubation the soil was filled into Millipore pots with attached plastic containers for collecting the escaping mites. These extraction units were placed in a Kempson extractor. The soil including the mites was exposed to temperatures of approximately 25°C and 30°C for approximately 2 days. Escaping mites were collected in a fixation liquid cooled to a temperature of approximately 16°C. The fixation liquid contained glycol and a detergent. Adult animals were counted once, missing adult predatory mites were considered dead due to rapid decomposition under test conditions. Juvenile animals were counted twice under binocular microscopes. None of the replicate counts deviated more than 10% from their mean value.
- Extraction efficiency: The extraction efficiency was checked separately by adding 30 mites (20 juveniles and 10 adults) each to two extraction units containing untreated soil: The number of extracted animals were counted. 59 animals out of 60 were recovered giving an extraction efficiency of 98.3%.

ARTIFICIAL SOIL: Artificial soil was prepared according to OECD 226 before the study started and stored until use in a closed plastic-container.
Ingredients:
- 5% Sphagnum-peat, air-dried and finely ground (≤ 2 mm)
- 20% Kaolin clay, extra pure
- approximately 0.3% chalk (CaCO3) extra pureto adjust pH to 6.0 ± 0.5
- approximately 74.7% fine quartz-sand, F 34, grain size with more than 50% by mass of particle size 0.05-0.2 mm
The artificial soil was moistened to approximately half of the final water content 2 days before the application. The additional water required to achieve the final water content was added when applying the test item.

EFFECT PARAMETERS MEASURED: Mortality and reproduction

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50, and 100 mg test substance/kg

Reference substance (positive control):

yes

Remarks:

dimethoate

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

40.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits 12.7 to 50.7 mg/kg soil dry weight

Key result

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Details on results:

The mean mortality of adult soil mites observed in the test substance groups was 5-15% which was not statistically significantly different compared to the control, where 11% of the soil mites were dead after 14 days of exposure (Fisher's Exact Test, α = 0.05, one-sided greater).

The mean reproduction in the test substance groups was not statistically significantly different compared to the control up to and including the concentration of 25 mg/kg soil dry weight. At the concentration of 50 mg/kg soil dry weight and above reproduction was statistically significantly reduced compared to the control (Williams t-test, α = 0.05, one-sided smaller).

Results with reference substance (positive control):

The reference item containing dimethoate as active substance is tested at least once a year. The most recent test showed statistically significant effects on reproduction at concentrations of 4.3 mg dimethoate/kg artificial soil dry weight; the EC50 for reproduction was calculated to be 4.33 mg dimethoate/kg soil dry weight.

Validity criteria fulfilled:

yes

Conclusions:

EC50: 40.8 mg/kg soil dry weight (95% CI: 12.7 to 50.7 mg/kg dw) (reproduction)
LOEC: 50 mg/kg soil dry weight (mortality and reproduction)
NOEC: 25 mg/kg soil dry weight (mortality and reproduction)

Executive summary:

The effects of the test substance on survival and reproduction of the soil mite, Hypoaspis aculeifer, were measured in a 14-day soil exposure GLP-compliant laboratory study based on OECD 226 (2008). Four replicates (eight in the control) of ten soil mites each were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/kg dry artificial soil and to an untreated control (using the same amount of acetone and fine quartz sand per g substrate as in the test item groups). The test item concentrations were selected based on the results of a non-GLP preliminary range-finding study. After 14 days, the soil mites were assessed for the number of surviving adults (mortality) and the number of juveniles (reproduction).

The reference item containing dimethoate as active substance is tested at least once a year. The most recent test showed statistically significant effects on reproduction at concentrations of 4.3 mg dimethoate/kg artificial soil dry weight; the EC50 for reproduction was calculated to be 4.33 mg dimethoate/kg soil dry weight.

All validity criteria for the study were met. No significant effects on survival were observed up to and including the concentration of 100 mg/kg soil dry weight. Reproduction was not affected up to and including the concentration of 25 mg/kg soil dry weight but was statistically significantly reduced at 50 mg/kg soil dry weight and above.

The 14-day EC50 for test substance, based on reproduction was determined to be 40.8 mg/kg artificial soil dry weight. The overall 14-day Lowest-Observed-Effect Concentration (LOEC) was determined to be 50 mg/kg artificial soil dry weight and the overall 14-day No-Observed-Effect Concentration (NOEC) was determined to be 25 mg/kg artificial soil dry weight.

Reference 2

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

Deviations:

no

GLP compliance:

yes

Application method:

soil

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

A stock solution containing 2.0 mg/mL test substance was prepared by dissolving 50 mg of test substance in 25 mL acetone.

A sequential 1:1 dilution series was prepared, starting with the 2.0 mg/mL stock solution, and 5 mL of the corresponding dilutions were added to 50 g fine quartz sand. The treated sand was left for approximately two hours until the solvent had evaporated. The test item blended sand was added to artificial soil with reduced sand fraction to result in a final net weight of 200 g dry weight (for the control 65 g acetone treated sand was added to artificial soil with reduced sand fraction to result in a final net weight of 260 g dry weight).

The soil was prepared with a reduced sand content so that the final mixture, including the test item, would contain approximately 75% sand.

This composition was moistened with deionised water and mixed with a laboratory mixer to ensure a homogenous distribution. In the course of applying the test item, the soil was moistened with the added water.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: Springtails
- Source: Bred under standardised conditions at IBACON
- Age at test initiation: 10-12 days
- Stage at test initiation: Juveniles, adults
- Feeding: After the introduction of the test organisms, approximately 2 mg (half of a small spatula) of granulated dry yeast were spread over the soil surface at test start. After 14 days, the Collembola were fed granulated dry yeast ad libitum by adding 2 mg (half of a small spatula) to the test vessels.

ACCLIMATION
- Acclimation period: 12 days

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

18-22°C

pH (if soil or dung study):

At test start: 6.1 to 6.4
At test end: 6.1 to 6.4

Photoperiod and lighting:

16 h light : 8 h dark (400 to 800 lux)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass containers (volume: 100 mL; diameter: 5 cm)
- Amount of soil: 30 g ± 1.0 g artificial soil fresh weight
- No. of organisms per container: 10 per unit, i.e. 80 individuals for the control and 40 individuals for the test item treated groups
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Ventilation: All vessels including the additional containers were ventilated two times a week by opening the lids for a short period.
- Water content maintenance: Water content was checked on day 14 after the application by reweighing the additional test containers. It was not necessary to compensate loss of water as it did not deviate by more than 2% from the initial water content.
- Determination of the number of Collembola: The content of the test containers was suspended in water, the suspension was tinted with dark ink and stirred with a fine brush. The Collembola drifted to the surface. Adult animals were counted once visually, juvenile animals were counted two times under binocular microscopes. Missing Collembola were considered to be dead due to rapid decomposition under test conditions. One of the replicates was counted three times because the first two counts deviated more than 10% from their mean value.

ARTIFICIAL SOIL INGREDIENTS: 5% Sphagnum-peat, air-dried and finely ground (≤ 2 mm); 20% Kaolin clay, extra pure, Kaolinite content >30%; approximately 0.3% chalk (CaCO3) extra pure to adjust pH to 6.0 ± 0.5; approximately 74.7% fine quartz-sand, F 34, grain size with more than 50% by mass of particle size 0.05-0.2 mm.

The artificial soil was moistened to approximately half of the targeted final water content 3 days before the application. The additional water required to achieve the final water content was added when applying the test item.

Maximum water holding capacity of the artificial soil: 41% of the dry weight.

EFFECT PARAMETERS MEASURED: Mortality, Behavioural abnormalities and reproduction

Nominal and measured concentrations:

Nominal: 3.125, 6.25, 12.5, 25 and 50 mg/kg dry artificial soil

Reference substance (positive control):

yes

Remarks:

boric acid

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

34.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

25 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Details on results:

The mortality of adult Collembola observed in the test substance groups was 18-20% which was not statistically significantly different compared to the control, where 15% of the Collembola were dead after 28 days of exposure (Fisher's Exact Test, α = 0.05, one-sided greater). No abnormal behaviour was observed with the surviving adult Collembola.

The mean reproduction in the test substance groups was not statistically significantly different compared to the control up to and including the concentration of 12.5 mg/kg artificial soil dry weight. At the concentrations of 25 and 50 mg/kg artificial soil dry weight reproduction was statistically significantly reduced (Williams t-test, α = 0.05, one-sided smaller)

These results show that the test substance does not affect Folsomia candida reproduction when applied up to and including a concentration of 12.5 mg/kg artificial soil dry weight. No abnormal behaviour was observed with the surviving juvenile Collembola.

Results with reference substance (positive control):

The reference item boric acid is tested at least once a year. In the most recent test, the EC50 for reproduction was determined to be 75.7 mg boric acid/kg soil dry weight.

Validity criteria fulfilled:

yes

Conclusions:

EC50: 34.8 mg/kg artificial soil dry weight (reproduction)
LOEC: 25 mg/kg artificial soil dry weight (mortality and reproduction)
NOEC: 12.5 mg/kg artificial soil dry weight (mortality and reproduction)

Executive summary:

The effects of the test substance on survival and reproduction of Collembola, Folsomia candida, was measured in a 28-day soil exposure GLP-compliant laboratory study based on OECD 232 (2009) and ISO 11267 (1999). Eight replicates for the control and four replicates per test item group, containing ten Collembola each (total 80 per control and 40 individuals per test item group) were each exposed for 28 days to the nominal concentrations of 3.125, 6.25, 12.5, 25 and 50 mg/kg dry artificial soil and an untreated control (using the same amount of acetone and fine quartz sand per g substrate as in the test item groups). The test item concentrations were selected based on the results of a non-GLP preliminary range-finding study. After 28 days the Collembola were assessed for the number of surviving adults (mortality) and the number of juveniles (reproduction).

The reference item boric acid is tested at least once a year. In the most recent test performed, the EC50 for reproduction was determined to be 75.7 mg boric acid/kg soil dry weight.

All validity criteria were met. No significant effects were observed up to and including the concentration of 12.5 mg/kg soil dry weight.

The 28-day EC50 for the test substance was determined to be 34.8 mg/kg artificial soil dry weight. The overall 28-day Lowest-Observed-Effect Concentration (LOEC) was determined to be 25 mg/kg artificial soil dry weight and the No-Observed-Effect Concentration (NOEC) based on mortality and reproduction was determined to be 12.5 mg/kg artificial soil dry weight.

Reference 3

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Ministry of Agriculture, Fisheries and Food Health and Safety Executive (1986). Laboratory testing for toxicity to honey bees. Data Requirements for Approval Under the Control of Pesticides Regulations. Working Document 7/3.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OEPP/EPPO (1992). Guideline on test methods for evaluating the side-effects of plant protection products on honeybees. No. 170 Bulletin OEPP/EPPO Bulletin 22:203-215.

Deviations:

no

GLP compliance:

yes

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Method of test material application: single topical dose
- Body part: thorax
- Volume of test solution applied: 1 µL
- Controls: yes
- Chemical name of vehicle: Acetone

- Oral test: Preliminary work showed that the test substance would not form a stable, homogeneous dispersion, at any concentration required to administer doses required in the test, in 50% sucrose solution, even if it was first dissolved in acetone. Thus, it could not be tested by the oral route of administration.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bee
- Source: Jealott's hill colonies
- Cultural background: worker bees
- Kept according to standard practices: yes

RATIONALE FOR SELECTION OF SPECIES: The honey bee was chosen as the test organism, being representative of the pollinating insects, which may be at risk if flowering crops are sprayed with pesticides.

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24.5-25°C

Humidity:

58-89%

Photoperiod and lighting:

Bees were kept in dark except during observation

Details on test conditions:

TEST SYSTEM
- Test container: Cylindrical, stainless steel, wire mesh cages, 170 mm long and 45 mm diameter, were closed at one end by a stainless steel cap and at the other end by a foam rubber bung, slit and fitted with a glass feeding tube. The feeding tube was a 50 mm long, 10 mm diameter flat-bottomed soda glass tube of which the open end had been narrowed, by heating, to a 2 mm diameter hole.
- No. of replicates per treatment group: 3
- No. of replicates per control: 3

EFFECT PARAMETERS MEASURED: Mortality and sublethal effects were assessed at 1, 2, 4, 24 and 48 hours after treatment.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Range finding study: No mortality was observed in this preliminary analysis
- Test concentrations (Nominal): 0.1, 1, 10 and 100 µg/bee
- Results used to determine the conditions for the definitive study: yes

Nominal and measured concentrations:

Definitive study (Nominal): 5, 10, 20, 50, 100 and 200 µg/bee

Reference substance (positive control):

yes

Remarks:

Dimethoate

Key result

Duration:

24 h

Dose descriptor:

LD50

Effect conc.:

> 200 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

>= 200 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Sub-lethal effects

Details on results:

With the test substance there were no dose-related effects, such that the 24 hour and 48 hour LD50 levels were not attained, and the LD50 value at 24 and 48 hours is >200 µg a.i./bee.
Control mortality was 0% at 24 hours. As there was only one bee affected at an intermediate dose rate there was no dose response.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- Relevant effect levels: 24 h LD50: 0.19 µg a.i./bee (contact toxicity)
48 h LD50: 0.17 µg a.i./bee (contact toxicity)
24 h LD50: 0.17 µg a.i./bee (oral toxicity)
48 h LD50: 0.17 µg a.i./bee (oral toxicity)
The LD50 values for the toxic control reference (dimethoate) show that the bees were reacting normally to pesticide doses under the conditions of this test.

Validity criteria fulfilled:

yes

Conclusions:

24 h LD50: >200 µg a.i./bee (mortality)
24 and 48 h NOEL: ≥ 200 µg a.i./bee (sub-lethal effects)

Executive summary:

The acute contact and oral toxicity to honey bees (Apis mellifera) of technical substance was tested in the laboratory. In contact tests a 1 µL drop of acetone was applied to the thorax of each bee, containing a range of concentrations of the test substance. Due to its low solubility in water, the test substance could not be dispersed in 50% w/v sucrose solution and was, therefore, not tested by oral administration.

There were no dose-related effects on the bees during the 48 hours following contact treatment with doses of diluted formulation containing 5 to 200 µg a.i./bee. The 24 hour and 48 hour contact LD50 and No Observed Effect Level (NOEL) for the test substance were, therefore, >200 and ≥200 µg active a.i./bee respectively.

Results obtained for dimethoate, tested concurrently as a toxic reference compound, indicated that the bees were reacting normally under the test conditions.

The results indicate that the test substance is of low toxicity to honey bees under the conditions of this test.

Description of key information

24-hour LD50 (honeybee): >200 μg per animal; OEPP/EPPO (1992). Guideline on test methods for evaluating the side-effects of plant protection products on honeybees. No. 170 Bulletin OEPP/EPPO Bulletin 22:203-215; Reliability = 1

14-day NOEC (predatory mite): 25 mg/kd soil dw; OECD 226; Reliability = 1

28-day NOEL (Collembola): 12.5 mg/kg soil dw; OECD 232; Reliability = 1

Key value for chemical safety assessment

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

12.5 mg/kg soil dw