5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to bees: acute contact

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Sep - 25 Sep 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: EPPO guideline 170 (1992)

GLP compliance:

yes (incl. QA statement)

Remarks:

Federal Department of the Interior, 25 Apr 1996

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: topical application
- Body part: thorax
- Volume of test solution applied: 1 µL
- Controls: 20% sucrose solution (blank), acetone (vehicle control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Method of test solution preparation: A superstock with a nominal concentration of 5.38 mg RPA 107382/mL acetone was prepared by diluting 53.7 mg RPA 107382 with acetone to a total volume of 10 mL. Thereafter, 1 mL of the superstock was diluted with acetone to a total volume of 10 mL. This procedure was repeated once more to obtain a primary stock solution with a concentration of 53.8 mg RPA 107382/L (equivalent to 0.05 µg a.i./bee). Thereafter, the primary stock solution was subsequently diluted to prepare six secondary stock solutions with nominal concentrations equivalent to 0.025, 0.0125, 0.00625, 0.00313, 0.00156 and 0.00075 µg RPA 107382/bee. The secondary stock solutions were prepared subsequently by diluting 1 mL of the appropriate stock solution with acetone to a total volume of 2 mL. Acetone was used to dose the test substance with since the test substance showed a low solubility in deionized water.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honeybee
- Source: Bee keeper Mr. O. Keller, Steinach, Switzerland
- Age at test initiation (mean and range, SD): 4 - 14 days
- Disease free: yes

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Feeding: honey

Study type:

laboratory study

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 - 26.5 °C

Humidity:

50 - 59%

Photoperiod and lighting:

darkness

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): Perforated steel panels with one panel consisting of a removable glass plate, cage dimensions were 12.5 cm x 12.5 cm x 12.5 cm
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality and behavioural observations were recorded 24, 48 and 72 h after test start

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study
- Test concentrations: 0.0000558, 0.000558, 0.00558, 0.0558, 0.558, 5.58 and 55.8 ug RPA 107382 a.i./bee
- Results used to determine the conditions for the definitive study: 20, 0 and 20% mortality for the three lowest concentrations, respectively, and 100% mortality for the other concentrations.

Nominal and measured concentrations:

Nominal: 0.000750, 0.00156, 0.00313, 0.00625, 0.0125, 0.0250, 0.0500 µg per bee

Reference substance (positive control):

yes

Remarks:

dimethoate

Duration:

72 h

Dose descriptor:

LD50

Effect conc.:

0.011 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CL = 0.008 - 0.014

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.006 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.013 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- For details on mortality see Table no. 1.
- Mortality of controls was 3.3%.

Results with reference substance (positive control):

Dimethoate was applied at 16 µg/bee and induced 80% mortality after 24, 48 and 72 h contact time.

Table 1: Mean percent mortality and mean percent affected bees recorded after 72 h in the contact test with honeybees (Apis mellifera) exposed to RPA 107382. *, Statistically significant difference between the control and the test substance treatment level (Fisher Exact test; p < 0.05)

Nominal concentrations (µg/bee)	% Mortality	% Affected (sublethal effects)
0 (vehicle)	3.3 ± 5.8	0 ± 0
0 (blank)	3.3 ± 5.8	0 ± 0
0.000750	6.7 ± 5.8	0 ± 0
0.00156	6.7 ± 5.8	0 ± 0
0.00313	6.7 ±11.5	3.3 ± 5.8
0.00625	20.0 ± 10.0	10.0 ± 10.0
0.0125	56.7 ± 5.8*	13.3 ± 11.5
0.0250	86.7 ± 15.3*	10.0 ± 10.0
0.0500	96.7 ± 5.8*	0 ± 0

Table 2: Validity criteria for OECD 214

Criterion from the guideline	Outcome	Validity criterion fulfilled
The average mortality for the total number of controls must not exceed 10% at the end of the test (0.10 – 0.30 µg a.i./bee, 24 h).	3.3%	yes
The LD50 of dimethoate standard meets the specified range (LD50 (24 h): 0.10 - 0.30 µg a.i./bee).	16 µg/bee and induced 80% mortality after 24 hours	Not applicable

Validity criteria fulfilled:

not specified

Remarks:

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

Reference 2

Endpoint:

toxicity to bees: acute oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 Sep - 25 Sep 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: EPPO guideline 170 (1992)

GLP compliance:

yes (incl. QA statement)

Remarks:

Federal Department of the Interior, 25 Apr 1996

Application method:

oral

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of feeding during study: The food was placed in a weighing boat positioned beneath the test cages and the bees had access to the food through the perforations in the cages. The food containing the test substance was placed in the cages in a polypropylene vial.
- Method of test solution preparation: A superstock with a nominal concentration of 3.58 mg/g honey was prepared by blending 35.8 mg RPA 107382 with 10 g of honey. Thereafter, 1 g of the superstock was diluted with honey to a total volume of 10 g. This procedure was repeated twice to obtain a primary stock solution concentration of 0.00358 mg RPA 107382/g honey (equivalent to 0.1 µg a.i./bee). The primary stock solution was subsequently diluted to prepare six secondary stock solutions with nominal concentrations equivalent to 0.05, 0.025, 0.0125, 0.00625, 0.00313 and 0.00156 µg a.i./bee. The secondary stock solutions were prepared subsequently by diluting 2 g of the appropriate stock solution with honey to a total weight of 4 g. Honey was used since the test substance showed low solubility in deionized water.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honeybee
- Source: Bee keeper Mr. O. Keller, Steinach, Switzerland
- Age at test initiation (mean and range, SD): 4 - 14 days
- Disease free: yes

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Feeding: honey

Study type:

laboratory study

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 - 26.5 °C

Humidity:

50 - 59%

Photoperiod and lighting:

darkness

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): Perforated steel panels with one panel consisting of a removable glass plate, cage dimensions were 12.5 cm x 12.5 cm x 12.5 cm
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3
- No. of replicates per control: 3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality and behavioural observations were recorded 24, 48 and 72 h after test start

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study
- Test concentrations nominal: 0, 0.0002, 0.002, 0.02, 0.2, 2, 20 and 200 µg RPA 107382 a.i./bee
- Test concentrations calculated: 0, 0.00014, 0.002, 0.02, 0.113, 1.07, 10.0 and 66.7 µg a.i./bee
- Results used to determine the conditions for the definitive study: 0, 20 and 50% mortality for the three lowest concentrations, respectively, and 100% mortality for the four highest concentrations.

Nominal and measured concentrations:

Nominal: 0, 0.00156, 0.00313, 0.00625, 0.0125, 0.0250, 0.0500 and 0.1000 µg per bee
Calculated: 0, 0.00156, 0.00313, 0.00590, 0.0114, 0.0192, 0.0317 and 0.0644 µg per bee

Reference substance (positive control):

yes

Remarks:

dimethoate

Duration:

72 h

Dose descriptor:

LD50

Effect conc.:

0.033 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CL = 0.019 - 0.064 µg/bee

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.011 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.019 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- For details on mortality see Table no. 1.
- Mortality of the control was 6.7%.

Results with reference substance (positive control):

Dimethoate was applied at 0.18 µg/bee and induced 73.3% mortality after 72 h.

Reported statistics and error estimates:

- Calculation of LC50: The nominal doses for RPA 107382 tested and the corresponding biological-response data (mortality) derived from the oral toxicity test were used to estimate median lethal-dose concentrations (LD50) and 95% confidence intervals using linear and non-linear regression models. The computer Program used for this was modified from C. Stephan (Peltier and Weber, 1985). It estimates the LD50 values using three statistical methods: probit analysis, moving average angle method and binomial probability. The method which provides the strongest calculation (the smallest confidence interval span and highest goodness of fit probability) is used.
- Calculation of NOEC/LOEC: The effect of treatment with RPA 107382 was tested against the control using the Fisher Exact test (Zar, 1984). The computer programme used to run the statistical analyses was “Statistics for Windows”, by StatSoft Inc.

Table 1: Mean percent mortality and mean percent affected bees recorded after 72 h in the oral test with honeybees (Apis mellifera) exposed to RPA 107382. *, Statistically significant difference between the control and the test substance treatment level (Fisher Exact test; p < 0.05)

Nominal concentrations (µg/bee)	% Mortality	% Affected (sublethal effects)
0	6.7 ± 5.8	0 ± 0
0.00156	10.0 ± 10.0	0 ± 0
0.00313	6.7 ± 11.5	6.7 ±11.5
0.00590	0 ± 0	3.3 ± 5.8
0.0114	6.7 ±11.5	10.0 ± 10.0
0.0192	26.7 ± 20.8*	16.7 ± 11.5*
0.0317	46.7 ± 5.8*	20.0 ± 0.0*
0.0644	86.7 ± 5.8*	10.0 ± 0.0

Table 2: Validity criteria for OECD 213

Criterion from the guideline	Outcome	Validity criterion fulfilled
The average mortality for the total number of controls must not exceed 10% at the end of the test.	6.7%	yes
The LD50 of the toxic standard meets the specified range (0.10 – 0.35 µg a.i./bee, 24 h).	LD50 not reported	Not applicable

Validity criteria fulfilled:

not specified

Remarks:

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

Reference 3

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Oct - 15 Oct 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Contact toxicity in direct application to 2 to 3 day-old spiderlings (second instar hatchlings) in covered petri dishes containing humidified quartz sand. Mortality was recorded after 24 and 48 hours.

GLP compliance:

no

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

AVI-210 (bulk) 0.1 g was weighed into a conical flask and carefully dissolved with acetone 10 mL; the test substance solution was then prepared by adding 1 mL of this solution to a 100 mL graduated flask and making up to volume with distilled water to which 0.1% of polyoxyethylene (20) sorbitan monolaurate had been added. The control substance solution was similarly prepared by dissolving dimethoate (standard material) 43 mg in 1 mL acetone and making up to 100 mL with distilled water containing 0.1% of polyoxyethylene (20) sorbitan monolaurate.

Test organisms (species):

other: Pardosa pseudoannulata

Animal group:

Araneae (soil-dwelling lycosid spider)

Details on test organisms:

TEST ORGANISM
- Common name: Pond wolf spider
- Source: Adults had been collected in fields around the rice paddy at the Experimental Station. Spiderlings were obtained by keeping adult females bearing an egg sac in the laboratory. On the day before exposure, spiderlings were separated from the adult spider by stimulation with a brush.
- Age at test initiation (mean and range, SD): 2 to 3 day old spiderlings (second instar hatchlings)
- Date of collection: October 2001

ACCLIMATION
On the day before treatment, the second instar hatchlings on top of the adult female abdomen were stimulated with a brush to effect separation from the adult spider. The spiderlings were released 5 per test container and acclimated in a thermostatic room (22 °C, 16 h light/8 h dark, relative humidity ≥ 80%).

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

22 °C

Humidity:

≥ 80%

Photoperiod and lighting:

16 h light and 8 h dark

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): high-sided glass petri dishes, 9 cm diameter, 7.5 cm depth, closed with a plastic lid containing a 5.7 cm diameter hole covered with gauze; the dishes contained 100 g of quartz sand and 24 mL of distilled water (70% water content)
- No. of organisms per container (treatment): 5
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 5

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The numbers of viable, distressed and dead individuals were examined 24 and 48 h after start of exposure.

FEEDING
- Several neanurid springtails

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

Rate of application: 6 mg of 100 ppm solution/cm2 = 600 L of 100 ppm solution/ha

Reference substance (positive control):

yes

Remarks:

dimethoate

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

100 ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: The application rate of the test substance at 100 ppm was 600 L/ha.

Details on results:

- Mortality at end of exposure period: 93.3% mortality after 48 h (and already after 24 h)
- No mortality was observed in the untreated group.

Results with reference substance (positive control):

100% mortality after 48 h (and already after 24 h).

Validity criteria fulfilled:

not applicable

Remarks:

No guideline followed

Reference 4

Endpoint:

toxicity to bees: acute contact

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 Jul - 06 Jul 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Version / remarks:

1998

Deviations:

yes

Remarks:

-Range of test room humidity was 49.8% - 63.0%, instead of 50 - 70%. Period of deviation was not significant, validation criterion reached.

GLP compliance:

yes (incl. QA statement)

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: bees were anesthetized with notrogen gas (approx. 30 sec) and doses with 1 µL of the test solution on the dorsal side of the thorax. Control group was exposed with 1 µL of deionized water.
- Body part: thorax, dorsal
- Volume of test solution applied: 1 µL
- Controls: deionized water (negative control), solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 1% in highest test concentration and solvent control, 0.5, 0.25, 0.12, 0.06% acetone in 0.05, 0.025, 0.013, and 0.006 mg/mL nominal test concentration, respectively
- Method of test solution preparartion: a stock solution with the concentration of 10 mg/mL was prepared by weighing in 0.5 g of the test substance into 50 mL of acetone. For obtaining the highest test concentration, 0.5 mL of the stock solution was diluted in 49.5 mL deionized water. The following test concentrations were obtained by a serial dilution of 25 mL of the previuos test concentration in 25 mL of deionized water.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: worker bees
- Source: honeybees were collected from hives located in the Apiario Cacilda Alves Moreira (Santana de Parnaiba, SP) in an area away from agricultural crops. Young adult worker bees were collected from the same frame on the test day with fumigation help.
- Age at test initiation (mean and range, SD): approximately 1 to 4 weeks old
- Stage at test initiation: Young adult worker bees
- Date of collection: 22 Aug 2016
- Disease free: yes
- Kept according to standard practices: yes
- Health condition of the hive (if honeybees): The animals were collected only from healthy colonies. The colonies did not receive any treatment (acaricides or antibiotics) at any season of the year.
- Transport: during transport and before test start, bees were kept in a gauze-covered plastic bucket and fed ad libitum candy paste.

ACCLIMATION
- Acclimation period: approximately 2 hours
- Acclimation conditions (same as test or not): Same as in test
- Feeding: ad libitum supply of sugar syrup (candy paste) and water

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.5 to 25.5 °C

Humidity:

49.8 to 63.0%

Photoperiod and lighting:

Darkness (except on evaluation periods)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass beakers (250 mL) covered with a cloth net on top
- No. of organisms per container (treatment): 10 bees per replicate, impartially chosen
- No. of replicates per treatment group: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3
- No. of replicates per positive control: 3


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and abnormal behaviour were recorded after4, 24, 48, and 72-hours of exposure. Assessment for mortality and behaviour was recorded at 4 and 24 hours for the reference substance

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study : yes
- Test concentrations: control, 0.001, 0.010, 0.10, and 1.0 µg/bee
- Results used to determine the conditions for the definitive study: observed mortalities were 0, 23, 30, 90, and 100%

Nominal and measured concentrations:

- Nominal: control, solvent control, 0.006, 0.013, 0.025, 0.050, 0.100 µg/bee

Reference substance (positive control):

yes

Remarks:

Dimethoate technical grade was used as reference substance

Toxic reference:

- The test with the toxic reference dimethoate was conducted with the following concentrations: control, 0.075, 0.150, 0.300, and 0.600 µg a.i./bee

Duration:

72 h

Dose descriptor:

LD50

Effect conc.:

0.03 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 0.020 - 0.050 µg/bee

Details on results:

- Behavioural abnormalities: Abnormal behaviour as slow movements were observed in the survivors at all tested doses, control group, solvent group, and moribund bees at 0.025 µg/bee.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 24-h LD50 with the reference substance dimethoate was 0.21 µg/bee (95% CI: 0.17 - 0.30 µg/bee)

Reported statistics and error estimates:

- The median lethal dose (LD50) and the 95% confidence limits were estimated by using the trimmed Spearman-Karber method. Fisher's Exact Test was used to compare the mortality observed in the control to the test substance treatments.

Table 1: Results of the contact toxicity test with honey bees (Apis mellifera) for the test substance Ethiprole at 72 hours

Nominal dose (µg/bee)	Number of dead bees	Total number of bees	Mortality (%)	Behaviour
Control	3	30	10	25A; 2C
Solvent control	3	30	10	26A; 1C
0.006	8	30	27	16A; 6C
0.013	11	30	37	12A; 7C
0.025	12	30	40	10A; 7C; 1D
0.050	18	30	60	12C
0.100	27	30	90	3C

A = normal; B = agitated; C = lethargy; D = moribund

Table 2: Validity criteria for OECD 214

Validity criterion	Outcome	Validity criterion fulfilled
The average mortality for the total number of controls must not exceed 10% at the end of the test (0.10 – 0.30 µg a.i./bee, 24 h).	10%	Yes
The LD50 of dimethoate standard meets the specified range (LD50 (24 h): 0.10 - 0.30 µg a.i./bee).	LD50 = 0.21 µg a.i./bee	Yes

Validity criteria fulfilled:

yes

Remarks:

see Table 2 in 'any other information on results incl. tables'

Reference 5

Endpoint:

toxicity to bees: acute oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Aug - 01 Sep 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: Bees were starved for approximately 2 hours before test start and then were anesthetized with nitrogen gas (N2) for approximately 30 seconds. After anesthetization period a bee feeder with 200 µL of 50% sucrose sugar syrup solution mixed with test substance at the chosen doses was offered during 4 hours. The control was only exposed to 50% sucrose sugar syrup solution without test substance. Solvent control was exposed to 50% sucrose solution with 1% of acetone. The amount of test substance consumed was determines by weighing the test feeder at the start and end of the 4-hour exposure time.
- Controls: negative control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 1% in highest test concentration and solvent control, 0.5, 0.25, 0.12, 0.06% acetone in 6.105, 3.053, 1.526, and 0.763 µg/mL nominal test concentration, respectively
- Method of test solution preparartion: a stock solution with the concentration of 1221 µg/mL was prepared by weighing in 0.1221 g of the test substance into 100 mL of acetone. For obtaining the highest test concentration, 0.5 mL of the stock solution was diluted in 25 mL deionized water. The following test concentrations were obtained by a serial dilution of 25 mL of the previuos test concentration in 25 mL of deionized water.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: worker bees
- Source: honeybees were collected from hives located in the Apiario Cacilda Alves Moreira (Santana de Parnaiba, SP) in an area away from agricultural crops. Young adult worker bees were collected from the same frame one day before the test with fumigation help.
- Age at test initiation (mean and range, SD): approximately 1 to 4 weeks old
- Stage at test initiation: Young adult worker bees
- Date of collection: 29 Aug 2016
- Disease free: yes
- Kept according to standard practices: yes
- Health condition of the hive (if honeybees): The animals were collected only from healthy colonies. The colonies did not receive any treatment (acaricides or antibiotics) at any season of the year.
- Transport: during transport and before test start, bees were kept in a gauze-covered plastic bucket and fed ad libitum candy paste.

ACCLIMATION
- Acclimation period: approximately 1 day
- Acclimation conditions (same as test or not): Same as in test, bees were kept in the dark under test temperature
- Feeding: ad libitum supply of sugar syrup (candy paste) and water until 2 hours before test

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Test temperature:

22.7 - 26.1 °C

Humidity:

53.8 - 63.3%

Photoperiod and lighting:

Darkness

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass beakers (250 mL) covered on top with a cloth net
- No. of organisms per container (treatment): 10 impartially chosen young adult bees per repllicate
- No. of replicates per treatment group: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3
- No. of replcates per positive control: 3
- Feeding during test: after the 4 h exposure period, bee feeders were replaced with ones that contained candy paste ad libitum

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and behaviour were assessed at 4, 24, and 48 hours after the exposure to the solutions (controls and test substance), and at 4 and 24 hours after the exposure to the reference substance.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study : yes
- Test concentrations: solvent control, 0.001, 0.010, 0.100, and 1.00 µg/bee
- Results used to determine the conditions for the definitive study: Observed mortalities were 10, 10, 60, 100, and 100%, respectively

Nominal and measured concentrations:

- Nominal concentrations: control, solvent control, 0.002, 0.004, 0.008, 0.015, and 0.030 µg/bee

Reference substance (positive control):

yes

Remarks:

Dimethoate technical was used as reference substance with the same batch of bees.

Toxic reference:

The positive control substance dimethoate was tested in the following nominal concentrations: 0.075, 0.150, 0.300, and 0.600 µg a.i./bee

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

0.01 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 0.008 - 0.012 µg/bee

Details on results:

- Behavioural abnormalities: Symptom as slow movements was observed in the survivors of all tested doses, control group, solvent group and moribund bees were observed at the dose 0.015 µg/bee.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The nominal LD50 for the reference substance dimethoate was 0.13 µg a.i./bee (95% CI: 0.11 - 0.17 µg a.i./bee)

Reported statistics and error estimates:

- The median lethal oral dose (LD50) and the 95% confidence limits were estimated by using the trimmed Spearman-Karber method. Fisher's Exact Test was used to compare the mortality observed in the control to the test substance treatments. T-test was used for comparison of food uptake of sucrose solution with test substance versus control.

Table 1: Results of the oral toxicity test with honey bees (Apis mellifera) for the test substance Ethiprole at 48 hours

Nominal dose (µg/bee)	Number of dead bees	Total number of bees	Mortality (%)	Food uptake (%)	Behaviour
Control	0	30	0	98.61	28A; 2C
Solvent control	3	30	10	98.78^1	26A; 1C
0.002	5	30	17	98.05^1	22A; 3C
0.004	8	30	27	98.44^1	16A; 6C
0.008	12	30	40	97.40^1	11A; 7C
0.015	21	30	70	97.97^1	2A; 6C; 1D
0.030	29	30	97	95.30	1C

A = normal; B = agitated; C = lethargy; D = moribund;¹= t-test, p>0.05

Table 2: Validity criteria for OECD 213

Validity criterion	Outcome	Validity criterion fulfilled
The average mortality for the total number of controls must not exceed 10% at the end of the test.	0% in control, 10% in solvent control	Yes
The LD50 of dimethoate standard meets the specified range (LD50 (24 h): 0.10 - 0.35 µg a.i./bee).	LD50 = 0.13 µg a.i./bee	Yes

Validity criteria fulfilled:

yes

Remarks:

see Table 2 in 'any other information on results incl. tables'

Additional information

One acute study with honeybees (Apis mellifera) is available according to EPPO Guideline No. 170 (adopted in 1992) and GLP. The study covers both contact and oral toxicity testing during a period of 72 hours. For contact toxicity, an LD50 (72h) of 0.011 µg/bee and a NOEC (72h) of 0.00625 µg/bee was measured. For oral toxicity, the LD50 (72h) was 0.0334 µg/bee and the NOEC (72h) 0.0114 µg/bee, both values are based on calculated concentrations according to the amount of test substance containing honey that was consumed. Two additional studies are available on the acute toxicity to bees, both studies were conducted under GLP conditions. The first study was conducted according to OECD guideline 214 (bee acute contact toxicity) and the second study was conducted according to OECD guidleine 213 (bee acute oral toxicity). The obtained LD50 values were 0.03 µg/bee (72 h) for contact toxicity and 0.010 µg/bee (48 h) for oral toxicity based on nominal concentrations.

Additionally, one study with 2 to 3-day old second instar hatchlings of the pond wolf spider (Pardosta pseudoannulata) reports 100% mortality when the substance was applied at 600 L/ha. The study report does not mention GLP or any guideline.