1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Application method:

contact

Analytical monitoring:

yes

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: Bees shortly anaesthetised with CO2 were treated individually by topical application of the test solution with an Eppendorf Micropipette
- Body part: dorsal thorax of bee
- Volume of test solution applied: 4 µL; According to the practical experience and to guarantee a good penetration of the test item the application
volume of 4 µL or af least 2 µL/bee was more appropriate than 1 µL/bee suggested in the guideline.
- Controls: In the two control treatment groups 4 uL deionised water/bee, 4 uL acetone/bee and 2 pi 0.1 % (v/v) Tween solution/bee (reference
item vehicle), respectively were applied in the same manner.
- Chemical name of vehicle: The test item and the reference item were dissolved in acetone or 0.1 % (v/v) Tween solution.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: honey bee
- Source: purchased from Bienenfarm Kern GmbH Rehbacher Anger 10, D-04249 Leipzig
- Age at test initiation: between 2 and 4 weeks as typical for "summer" bees
- Disease free: yes
- Kept according to standard practices: yes

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24.2 - 26.1 °C

Humidity:

54 - 67 %

Photoperiod and lighting:

Constant darkness throughout the test (diffuse artificial light of about 100 Ix only during handling and assessments)

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): disposable cage of cardboard with holes in the bottom for ventilation and a glass plate in front for observation of the bees (dimensions inside: 30 mm x 45 mm x 65 mm:)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per per control / vehicle control: 3



EFFECT PARAMETERS MEASURED :
Time and frequency of assessments: 4, 24 and 48 hours after application

Reference substance (positive control):

yes

Remarks:

Perfekthion EC 400

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 µg per animal

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 µg per animal

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Reference 2

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Analytical monitoring:

yes

Details on preparation and application of test substrate:

- Details of food source: sucrose solution
- Method of feeding during study: After the test, item-sucrose solution was consumed in the oral toxicity test {after about 1 hour), untreated 50 % (w/v) aqueous sucrose solution was provided as food in both tests. Food was provided ad libitum using a feeding tube. Twice a day the feeding tube was filled up with new sucrose solution before the bees had consumed the whole sucrose solution.
-other: 200 µL sucrose solution/10 bees = 20 µL/bee

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: honey bee
- Source: purchased from Bienenfarm Kern GmbH Rehbacher Anger 10, D-04249 Leipzig
- Age at test initiation: between 2 and 4 weeks as typical for "summer" bees
- Disease free: yes
- Kept according to standard practices: yes

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24,2 - 26,1 °C

Humidity:

54 - 67 %

Photoperiod and lighting:

Constant darkness throughout the test (diffuse artificial light of about 100 Ix only during handling and assessments)

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): disposable cage of cardboard with holes in the bottom for ventilation and a glass plate in front for observation of the bees (dimensions inside: 30 mm x 45 mm x 65 mm:)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per per control / vehicle control: 3



EFFECT PARAMETERS MEASURED :
Time and frequency of assessments: 4, 24 and 48 hours after application

Reference substance (positive control):

yes

Remarks:

Perfekthion EC 400

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 120 µg per animal

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 120 µg per animal

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: TheLC50 of the reference item Perfekthion EC 400 was 0.165 µg a.i. per bee in the oral toxicity test after
24 hours, This value corresponded also to the expected range for the oral 24 h - LD50 (0.10-0,35 µg a.i./bee) cited in the OECD Guideline 213.

Reference 3

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 Oct - 05 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Ringtest, Recommendations of the Hypoaspis Ring-Test group Final Meeting on January 15, 2007 in Utrecht

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of test solutions: The test item was mixed into deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions for application. All application solutions were prepared freshly on the day of the test.
- Method of test material application: The test item was thoroughly mixed into 500 g artificial dry weight artificial soil of each application rate using a laboratory mixer.
- Controls: soil treated with deionised water only deionised water only

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: soil mite
- Source: bred since 2002 at Bayer Crop Science AG, originally obtained from ECT Ecotoxicology GmbH, Flörsheim, Germany
- Age at test initiation: 35 days after start of egg laying
- Date of collection: 10 - 12 Sep 2007
- Breeding conditions: bred on a mixture of plaster of Paris, activated charcoal and demineralised water (11:1:10 w/w) in Bella Plast vessels, feeding: Tyrophagus putrescentiae (cheese mite)

Study type:

laboratory study

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

5.53 - 6.05

Humidity:

17.22% - 18.94%

Photoperiod and lighting:

550 - 665 lux artificial light
16 h light and 8 h darkness

Details on test conditions:

TEST SYSTEM
- Test container: glass vessels (volume 140 mL, diameter 5 cm at the bottom, height 7 cm) covered with glass lids that allow air exchange
- Amount of soil or dung or substrate: 20 g dry weight artificial soil (height: 1.5 cm)
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Feeding: 60 mg Tyrophagus putrescentiae/test vessel, on day 0, 4, 7 and 11 days after test start
- Moisture adaption: After 7 days the loss of water was determined by reweighing the test vessels. The missing amount of water was added.
- Collection of organisms: After 14 days, the surviving adults and the living juveniles were extracted, applying a temperature gradient. The content of each test vessel was transferred to sieve vessels (mesh size approximately 0.8 mm). Each sieve vessel was put onto another vessel containing a fixing liquid. The vessels were positioned in MCFADYEN-Extractor. The temperature was increased from approximately 25 to 40°C within two days. The temperature was increased from approximately 25 to 40°C within two days. Extracted mites were collected in a fixing solution and counted under a binocular.

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: according to OECD 226 ( 74.8% fine quartz sand (partlicle size: 0.2 - 0.05 mm = 73%), 5% sphanum peat (air dried, finely ground), 20% kaolin clay, approx. 0.2% CaCO3)
- Preparation of soil: soil was prepared by mixing the dry components intensely in a laboratory mixer. Three days before test start the dry artificial soil was premoistened with 56 mL deionised water per 500 g dry weight artificial soil. During
the application of the test item the water content was adjusted to a final water content of approximately 50 % of the maximum water holding capacity.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED: adult mortality and number of surviving offspring hatched (day 14)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.77
- Range finding study: no

Nominal and measured concentrations:

0, 100, 178, 312, 562 and 1000 mg test item/kg dry weight (nominal)
0, 45.08, 80.3, 140.7, 253.3 and 450.8 mg a.i./kg dry weight (nominal, converted values calculated for this entry by technical editor)

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 450.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 450.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Details on results:

- Mortality at the end of exposure period: No mortality of Hypoaspis aculeifer was observed after 14 d test duration in the control group and at any application rate, including the highest rate of 1000 mg test item/kg dry weight artificial soil.

Reported statistics and error estimates:

Data of reproduction were tested for normal distribution and homogeneity of variance using Kolmogorroff-Smirnov Test and Cochran-Test (α = 0.05) respectively. Data of reproduction were normally distributed and homogeneity of variances was given, but data were not monotonous. Therefore, Dunnett’s Test (one-sided smaller, α = 0.05) was used to determine NOEC and LOEC values.

Table 1. Validity criteria for OECD 226.

Performance criteria	Outcome	Criterion fulfilled
Mean adult female mortality should not exceed 20% at the end of the test	0%	yes
The coefficient of variation calculated for the number of juvenile mites per replicate should not be higher than 30% at the end of the definitive test	11.9%	yes
The mean number of juveniles per replicate (with 10 adult females introduced) should be at least 50 at the end of the test	309	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”. OECD 226 was not yet available for this test conduction. However, all given validity criteria were fullfilled.

Reference 4

Endpoint:

toxicity to terrestrial arthropods, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Sep - 29 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

With respect to the properties of the test item (Log pow ≥ 2) 5% peat instead of 10 % peat was used considering the influence on bioavailability (EPPO 2002).

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Wetfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of test solutions: The test item (1.2504 g) was mixed into 250 mL deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions (100 mL) for application.
- Volume of test solution applied: 100 mL test item solution was mixed homogeneously into 500 g artificial soil dry weight with a laboratory mixer. In a second step 10 mL of deionised water was introduced in the same way. Approximately 30 g (wet weight) of the test substrate was filled in each test vessel avoiding compression.
- Controls: soil treated with deionised water only deionised water only

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: white rat springtail
- Source: originally obtained from Ibacon GmbH, Rossdorf, Germany and bred at Bayer Crop Science since 2002
- Age at test initiation: 10 - 12 days
Breeding conditions: Bellaplast vessels (9.5 cm diameter) filled with a mixture of Plaster of Paris and activated charcoal and demineralised water (11:1:10 w/w) up to a height of 1 cm and closed with perforated plastic lids, had to be moistened, temerature: 20 - 24 °C, Light cycle: permanent dark, feeding: once a week with dry yeast (once a week).
- Synchronisation of the test organisms: Twelve days before starting the study, egg clusters from the breeding containers were transferred to fresh breeding substrate to obtain Collembola of a uniform age (10 - 12 days old at test start). After 48 h the egg cluster were removed and the remaining Collembola hatched from the eggs were fed with granulated dry yeast.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

5.63 - 6.45

Humidity:

15.70% - 18.19% (water content)

Photoperiod and lighting:

Photoperiod: 16 h light and 8 h dark
Light intensity: 568 - 609 lux

Details on test conditions:

TEST SYSTEM
- Test container: Glass vessels (volume: 140 mL, diameter: 5 cm) covered with glass lids
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 5
- No. of replicates per control: 5
- Feeding regime: 2 mg granulated dry yeast (day 0 and 14)

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: according to OECD 232 guideline, sphagnum peat (5%), kaolin clay (20%), industrial quartz sand (74% - 75%), CaCO3: 0.2% -1%
- Maximum water holding capacity: 45.3 g water/artificial soil dw

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED:
- adult mortality (day 28), number of living young (day 28)
- Method evaluation: soil of each replicate was decanted into a Bellaplast vessel, stirred up with 80 mL of deionised water and the Collembola drifted to the surface. The water was coloured with 10 mL black ink in order to increase the contrast between the water and the white Collembola. From each vessel a digital image was taken. The adult and juvenile Collembola on each digital image were counted automatically. These procedures were carried out with the LemnaTec Scanalizer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2
- Range finding study : no

Nominal and measured concentrations:

0, 31, 63, 125, 250, 500 and 1000 mg test item/kg dw (nominal)
0, 45.08, 80.3, 140.7, 253.3 and 450.8 a.i./kg dry weight (nominal, converted values calculated for this entry by technical editor)

Reference substance (positive control):

yes

Remarks:

Betosip (Phenmedipham 15.4%) is tested once a year as a toxic standard at concentrations of 50, 100 and 200 mg test item/kg artificial soil dry weight.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 450.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 450.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Details on results:

- Mortality at the end of exposure period: No mortality of Folsomia candida was observed after 14 d test duration in the control group and at any application rate, including the highest rate of 1000 mg test item/kg dry weight artificial soil.

Results with reference substance (positive control):

- In the most recent test (non-GLP) the mortality rate of adult Collembola was 8 %, 16 % and 34 % at 50, 100 and 200 mg Betosip/kg artificial soil dry weight. In the treatment groups 100 and 200 mg Betosip/kg artificial soil dry weight the number of juveniles were statistically significant reduced (William’s-T Test, one-sided-smaller, α= 0.05) in comparison to the control.
- NOEC (reproduction): 50 mg Betosip (7.7 mg a.s.)/kg artificial soil dry weight.
- LOEC (reproduction): 100 mg Betosip (15.4 mg a.s.)/kg artificial soil dry weight.
- The study proves the sensitivity of the test system.

Reported statistics and error estimates:

Data of reproduction were tested for normal distribution and homogeneity of variance using Kolmogorroff-Smirnov Test and Cochran-Test (α = 0.05) respectively. Data of reproduction were normally distributed and homogeneity of variances was given, but data were not monotonous. Therefore Dunnett’s Test (one-sided-smaller, α = 0.05) was used to determine NOEC and LOEC values.

Table 1: Validity criteria for ISO 11267.

Criterion from the guideline to be satisfied in the controls	Outcome	Validity criterion fulfilled
Average mortality of the adults in the control after 28 days is < 20%.	0%	yes
The coefficient of variation of reproduction in the control after 28 days is ≤ 30%.	6.1%	yes
Average reproduction rate in the control after 28 days is > 100 juveniles per control vessel.	1228	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Reference 5

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Mar - 19 Apr 2017

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:

yes

Remarks:

Due to technical reasons the duration of the test was 29 days

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of test material application: The test item (2.5023 g) was mixed into 250 mL deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions (250 mL) for application.
- Volume of test solution applied: 50 mL test item solution was mixed into 500 g artificial soil dry weight with a laboratory mixer. Approximately 30 g (wet weight) of the test substrate was filled in each test vessel avoiding compression.
- Controls: soil treated with deionised water only

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: white rat springtail
- Source: bred at Bayer AG, BAG-CS-EnSa-Testing since September 2015, originally obtained from BioChem agrar, Labor für biologische und chemische Analytik GmbH, Gerichshain, Germany
- Age at test initiation: 10 -12 days
- Breeding conditions: Breeding vessels: Plastic vessels (9.5 cm diameter), breeding substrate: Plaster of Paris, activated charcoaland demineralised water (10:1.25:12.5 w/w) filled up to 1 cm, temperature: room temperature, Light cycle: permanent dark, feeding: once a week with bakers dry yeast
Synchronisation of the test organisms: Twelve days before starting the study, egg clusters from the breeding containers. After 2 days the egg clusters were removed and the remaining collembolans hatched from the eggs were fed with granulated dry yeast.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

29 d

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

5.63 - 6.30

Humidity:

Water content: 19.90 - 20.46 %

Photoperiod and lighting:

Photoperiod: 16 h light and 8 h dark
Light intensity: 584 - 692 lux

Details on test conditions:

TEST SYSTEM
- Test container: glass vessels (volume 140 mL, diameter 5 cm) covered with glass lids
- Amount of soil or dung or substrate: 30 ± 1 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Feeding regime: 5 mg granulated dry yeast (day 0 and 14) per test vessel

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: according to OECD guideline 232, sphagnum peat (5%), kaolin clay (20%), industrial quartz sand (74% - 75%), CaCO3 for the adjustment to pH to 6.0 ± 0.5
- Preparation: components were mixed in a laboratory mixer. Four days before test start dry artificial soil was pre-moistened with 72 mL deionized water per 500 g dry weight artificial soil. During the application of the test item the water content was adjusted to a final water content of approximately 50% of the maximum water holding capacity.
- Maximum water holding capacity: 50%

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 584 - 692 lux

EFFECT PARAMETERS MEASURED:
- adult mortality (day 28), number of living young (day 28)
- Method evaluation: After 29 days, the soil of each replicate was transferred into a plastic vessel and stirred up with 80 mL of deionized water in order to drift the collembolans to the surface. The water was coloured with 10 mL black
ink in order to increase the contrast between the water and the white collembolans. From each vessel a digital image was taken and checked. The adult collembolans were visually counted and marked on the digital image, followed by confirmation of the automatically generated juvenile count.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 1.78

Nominal and measured concentrations:

0, 100, 178, 316, 562 and 1000 mg test item/kg dw (nominal)
0, 19.4, 34.53, 61.30, 109.0 and 194 mg a.i./kg dw (nominal, converted values calculated for this entry by technical editor)

Reference substance (positive control):

yes

Remarks:

Boric acid (0, 44, 67, 100, 150 and 225 mg/kg dw)

Duration:

29 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

29 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

29 d

Dose descriptor:

NOEC

Effect conc.:

>= 194 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Duration:

29 d

Dose descriptor:

LOEC

Effect conc.:

> 194 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Details on results:

- Mortality at the end of exposure period: No mortality of Folsomia candida was observed after 29 d test duration in the control group and at any application rate, including the highest rate of 1000 mg test item/kg dry weight artificial soil.

Results with reference substance (positive control):

- EC50 (reproduction) = 82 mg test item/kg dw (95 % CI: 57 - 112 mg/kg dw)
- NOEC (reproduction) < 44 mg/kg dw

Reported statistics and error estimates:

- The software used to perform the statistical analysis was ToxRat Professional 3.2.1.
- Fisher's Exact Binominal Test with Bonferroni Correction, one-sided-greater, α = 0.05, was used to determine NOEC and LOEC values for mortality.
- Data of reproduction were tested for normal distribution and homogeneity of variance using Shapiro-Wilk’s-Test and Levene’s-Test (α = 0.05) respectively. Data of reproduction were normally distributed. The homogeneity of variances was not given even after transformation. Therefore, Welch-t-test after Bonferroni-Holm (one-sidedsmaller, α = 0.05) was used to determine NOEC and LOEC values.

Table 1: Validity criteria for OECD 232

	Outcome	Validity criterion fulfilled
Mean adult mortality should not exceed 20% at the end of the test.	3.8%	yes
The mean number of juveniles per vessel should be at least 100 at the end of the test.	1504.9	yes
The coefficient of variation calculated for the number of juveniles should be less than 30% at the end of the definitive test.	11.0%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Reference 6

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Apr - 02 May 2017

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 (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of test solutions: The test item (2.5019 g) was mixed into deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions for application. All application solutions were prepared freshly on the day of the test.
- Method of test material application: The test item was thoroughly mixed into 500 g artificial dry weight artificial soil of each application rate using a laboratory mixer.
- Controls: soil treated with deionised water only

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: soil mite
- Source: bred since 2002 at Bayer Crop Science AG, originally obtained from ECT Ecotoxicology GmbH, Flörsheim, Germany
- Age at test initiation: 28 days after start of egg laying
- Date of collection: 13 - 16 Mar 2017
- Kept according to standard practices: yes
- Breeding conditions: bred on a mixture of plaster of Paris, activated charcoal and demineralised water (10:1.25:12.5 w/w) in plastic vessels, feeding: Panagrellus redivivus (nematodes), light cycle: permanent dark, temperature: room temperature
- Synchronisation of the test organisms: 2 x 300 adult, female Hypoaspis aculeifer were transferred to fresh breeding vessels. After three days of egg laying, these females were removed. The Hypoaspis aculeifer hatched from the eggs were fed with Panagrellus redivivus.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

5.51 - 5.93

Humidity:

19.53% - 20.25%

Photoperiod and lighting:

478 - 573 lux artificial light
16 h light and 8 h darkness

Details on test conditions:

TEST SYSTEM
- Test container: glass vessels (volume 140 mL, diameter 5 cm at the bottom, height 7 cm) covered with glass lids that allow air exchange
- Amount of soil or dung or substrate: 20 g dry weight artificial soil (height: 1.5 cm)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Feeding: 52 - 124 mg nematodes (Panagrellus redivivus) per test vessel on day 0, 3, 8 and 11 days after test start
- Moisture adaption: After eight days the loss of water was determined by reweighing the test vessels. The missing amount of water was added.
- Collection of organisms: After 14 days, the surviving adults and the living juveniles per test vessel were extracted, applying a temperature gradient. The content of each test vessel was carefully transferred to sieve vessels (mesh size approximately 0.8 mm). The vessels were positioned in MacFadyenExtractor. The temperature was increased from approximately 25 to 40 °C within two days. Extracted mites were collected in a fixing solution (20 % ethylene glycol, 80 % deionised water; 2 g detergent/L). All Hypoaspis aculeifer (adult females and juveniles) were counted under a binocular.

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: according to OECD 226 (75% fine quartz sand (partlicle size: 0.2 - 0.05 mm = 90.8%), 5% sphanum peat (air dried, finely ground), 20% kaolin clay, (30% kaolinite), CaCO3 for pH adjustment to 6.0 ± 0.5
- Preparation of soil: soil was prepared by mixing the dry components intensely in a laboratory mixer. Five days before test start the dry artificial soil was premoistened with 72 mL deionised water per 500 g dry weight artificial soil. During the application of the test item the water content was adjusted to a final water content of approximately 50% of the maximum water holding capacity.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 478 - 573 lux

EFFECT PARAMETERS MEASURED: adult mortality and number of surviving offspring hatched (day 14)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.78
- Range finding study : no

Nominal and measured concentrations:

0, 100, 178, 316, 562 and 1000 mg test item/kg dw (nominal)
0, 19.4, 34.5, 61.3, 109 and 194 active a.i./kg dw (nominal, converted values calculated for this entry by technical editor)

Reference substance (positive control):

yes

Remarks:

Dimethoate

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 194 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 194 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: mortality and reproduction

Remarks on result:

other: converted values calculated for this entry by technical editor

Details on results:

- Mortality at the end of exposure period: No mortality of Hypoaspis aculeifer was observed after 14 d test duration in the control group and at any application rate, including the highest rate of 1000 mg test item/kg dry weight artificial soil.

Results with reference substance (positive control):

LC50: 1.8 mg a.s./kg dry weight artificial soil
NOEC: 3.2 mg a.s./kg dry weight artificial soil
LOEC: 5.6 mg a.s./kg dry weight artificial soil
- Effect concentrations are in the recommended range of the guideline, indicating that an EC50 based on the number of juveniles of 3.0 – 7.0 mg a.s./kg dry weight artificial soil shows that the test organisms are sufficiently sensitive.

Reported statistics and error estimates:

The software used to perform the statistical analysis was ToxRat Professional Version 3.2.1. For the statistical evaluation of mortality Fisher´s Exact Binomial Test with Bonferroni Correction (one-sided-greater, α = 0.05) was used. To test for normal distribution and homogeneity of variance Shapiro-Wilk’s Test and Levene’s Test (α = 0.05), respectively were used. Data of reproduction were normally distributed and homogeneity of variances was given. Williams t-test (one-sided-smaller, α = 0.05) was used to determine NOEC and LOEC values.

Table 1. Validity criteria for OECD 226.

Performance criteria	Outcome	Criterion fulfilled
Mean adult female mortality should not exceed 20% at the end of the test	5%	yes
The coefficient of variation calculated for the number of juvenile mites per replicate should not behigher than 30% at the end of the definitive test	8.4%	yes
The mean number of juveniles per replicate (with 10 adult females introduced) should be at least 50 at the end of the test	323.3	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

Apis mellifera

LD50 (48 h) > 100 µg a.s./bee (contact)

LD50 (48 h) > 120 µg a.s./bee (oral)

Folsomia candida

NOEC (28 d) ≥ 500 mg a.s./kg dry weight

Hypoaspis aculeifer

NOEC (14 d) ≥ 500 mg a.s./kg dry weight

Additional information

Six GLP studies on the toxicity of indaziflam to terrestrial arthropods are available. Two of them were short-term studies testing the toxicity on honeybees according to OECD 214 and 213. The limit tests were conducted with a test concentration of 100 mg a. s. per bee for the contact test and 120 µg a. s. per bee for the oral test. No mortality or effects on the behaviour of the bees (or other sub-lethal effects) were observed in comparison with the control bees in the contact and oral toxicity test. Therefore it is concluded that the LD50 (48 h) is > 100.0 µg a. s. per bee in the contact toxicity test and > 120.0 µg consumed a. s. per bee in the oral toxicity test.

Two long-term studies were conducted with springtail Folsomia candida. One study was performed according to ISO 11267 with nominal test material concentrations in a range of 31 to 1000 mg/kg dry weight (equivalent to 15.5 to 500 mg a.s./kg dry weight). In the second test with F. candida (according to OECD 232) was exposed to test material concentrations ranging from 100 to 1000 which corresponds to 19.4 to 194 mg a.s./kg dry weight. In both studies, no effect of the test item on the mortality and reproduction of F. canida was observed. Therefore, the deduced NOEC (28 d) was ≥ 500 mg a.s./kg dry weight in the first and ≥ 194 mg a.s./kg dry weight in the second test.

Two further terrestrial long-term toxicity tests are available testing the toxicity on the soil mite Hypoaspis aculeifer according to OECD 226. Test item was tested in a range of 100 to 1000 mg/kg dw corresponding to 50 to 500 mg a.s./kg soil and from 19.4 to 194 mg a.s./kg, respectively. In both tests, the test item did not have any lethal or sub-lethal effect on H. acululeifer after 14 days. Therefore, NOEC (14 d) values were ≥ 500 mg a.s./kg and ≥194 mg a.s./kg, respectively.