N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

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:

key study

Study period:

30 July 2002 to 2 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPPO 170 (1992)

Deviations:

no

GLP compliance:

yes

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

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: Control and solvent control
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium (stock solution and final test solutions): A 2.5535 g portion (2.4999 g as a.i.) of the test material was placed in a 25 mL volumetric flask and brought to volume with acetone to yield a 100 mg a.i./mL stock solution. The stock solution was observed to be clear and colourless with no visible undissolved test material. Dosing solutions for the exposure were prepared by diluting the stock solution with acetone.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bee
- Strain origin: Carniolan
- Age at test initiation: Young worker honey bees
- Date of collection: The honey bees were collected the day before test initiation at which time they were handpicked from brood frames removed from a single mature hive.
- Cultural background (if honeybees): The honey bees were not previously exposed to pesticide application
- Kept according to standard practices: Yes; the test organisms were supplied with deionised water and 50 % sucrose solution prepared by dissolving an equal portion of sucrose in deionised water (w/w). The sucrose solution was provided in a PVC trough and deionised water from an inverted glass jar with a perforated lid placed on a paper towel.

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24 to 25 °C

Humidity:

58 to 90 % (relative)

Photoperiod and lighting:

A black curtain covered the front of the incubator which maintained the test organisms in near darkness to simulate hive conditions.

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): The test chambers measured 13 x 13 x 13 cm. The chamber frame was constructed from sheet PVC and each chamber was covered with a polyester mesh (approximately 3.5 mm mesh opening). One end of the chamber contained a glass sliding door. Each glass door had a hole with a self-sealing silicone closure large enough to accommodate the glass tube used to add test organisms.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 2 for the 1.0 and 10 µg a.i./bee dose levels; 3 for the 100 µg a.i./bee dose level
- No. of replicates per control / vehicle control: 3

EFFECT PARAMETERS MEASURED:
Observations of the test organisms were made at 4, 24, and 48 hours of exposure. The number of mortalities and any unusual behaviour exhibited (i.e., lethargy, ataxia) were also recorded.

VEHICLE CONTROL PERFORMED: Yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: A preliminary range-finding contact toxicity test was initiated. At test initiation honey bees were exposed to nominal dosages of 1.0, 10 and 100 µg a.i./bee, a solvent control and a control (2 replicates, 10 honey bees per replicate treatment level). When little or no toxicity was observed at 24 hours of exposure, an additional replicate (10 honey bees) was established for the solvent control and 100 µg a.i./bee treatment level so this test would qualify as a limit test should no further mortality be observed. The honey bees used in the additional replicates were from the same source and collected on the same day as the remaining test population. With the additional bees tested, the preliminary test qualified as a limit test.

Nominal and measured concentrations:

- Nominal concentrations: 1.0, 10 and 100 µg a.i./bee

Reference substance (positive control):

yes

Remarks:

dimethoate

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Empirically estimated

Details on results:

Following the 48 hour preliminary contact test, 5 % mortality was observed among honey bees exposed to the 1.0 and 10 µg a.i./bee treatment levels. Mortality of 3 % was observed in the control. No mortality was observed in the solvent control or the 100 µg a.i./bee treatment level. At test termination, no sub-lethal effects (i.e., spasmodic body movements) were observed among honey bees exposed to any of the treatment levels or the control. Since less than 10 % mortality was observed in the 100 µg a.i./bee treatment level, the preliminary test qualified as a limit test and no further testing was performed.

Results with reference substance (positive control):

Following 24 hour dimethoate exposure, mortality of 17, 63 and 93 % was observed among honey bees exposed to the treatment levels of 0.050, 0.10 and 0.20 µg a.i./bee, respectively. All of the surviving honey bees exposed to the 0.10 µg a.i./bee treatment level were lethargic and some had spasmodic body movements.
The test guideline states that the 24 hour contact LD50 for dimethoate is in the range of 0.10 to 0.30 µg a.i./bee (OECD, 1998). The data generated during this test indicated the bees were slightly more sensitive to dimethoate than suggested by the study guideline. These results are typical of previous results generated at the testing laboratory.

Reported statistics and error estimates:

When the mortality in the exposure was less than 50 % in all concentrations tested, the LD50 for these exposures was empirically estimated to be greater than the highest dosage tested (100 µg a.i./bee).

Table 1: Cumulative Percent Mortality

Nominal Dose (µg/bee)	Replicate	Time (Hours)
		4	24	48
Control	A B C Mean	0 0 0 0	0 0 10 3.3	0 0 10 3.3
Solvent Control	A¹ B C² Mean	- - 0 0	0 0 0 0	0 0 0 0
1.0	A B Mean	0 0 0	10 0 5	10 0 5
10	A B Mean	0 0 0	0 0 0	10 0 5
100	A B C² Mean	0 0 0 0	0 0 0 0	0 0 0 0
Positive Control 0.05	A B C Mean	-³ - - -	0 30 20 17	- - - -
Positive Control 0.10	A B C Mean	- - - -	80⁴ 50⁴ 60⁵ 63	- - - -
Positive Control 0.20	A B C Mean	- - - -	90⁴ 90⁵ 100 93	- - - -

¹ Replicates A and B of the solvent control were not dosed until 40 minutes prior to the 4 hour observation period. Therefore, no 4 hour observation was made.

² Additional replicate established at 24 hours of exposure; all observations reported here were made at the requisite interval after the replicate was established.

³ Dimethoate reference test was observed and terminated at 24 hours.

⁴ All surviving bees were observed to have spasmodic body movement.

⁵ All surviving bees were observed to be lethargic.

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.

Executive summary:

The acute toxicity of the test material to the honey bee was investigated in a contact test conducted in accordance with the standardised guidelines OECD 214 and EPPO 170 under GLP conditions.

Apis mellifera (strain origin: Carniolan) were exposed to the test material at nominal dose levels of 1.0, 10 and 100 µg a.i./bee for 48 hours. The test was conducted in near total darkness at a temperature of 24 to 25 °C and relative humidity of 58 to 90 %. Control and solvent control experiments were run concurrently, along with a positive control material, dimethoate, at concentrations of 0.05, 0.10 and 0.20 µg a.i./bee.

Following the 48 hour preliminary contact test, 5 % mortality was observed among honey bees exposed to the 1.0 and 10 µg a.i./bee treatment levels. Mortality of 3 % was observed in the control. No mortality was observed in the solvent control or the 100 µg a.i./bee treatment level. At test termination, no sub-lethal effects (i.e., spasmodic body movements) were observed among honey bees exposed to any of the treatment levels or the control. Since less than 10 % mortality was observed in the 100 µg a.i./bee treatment level, the preliminary test qualified as a limit test and no further testing was performed.

Following 24 hour dimethoate exposure, mortality of 17, 63 and 93 % was observed among honey bees exposed to the treatment levels of 0.050, 0.10 and 0.20 µg a.i./bee, respectively. All of the surviving honey bees exposed to the 0.10 µg a.i./bee treatment level were lethargic and some had spasmodic body movements.

Therefore under the conditions of this study, the 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.

Reference 2

Endpoint:

toxicity to bees: acute oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2002 - 16 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPPO 170 (1992)

Deviations:

no

GLP compliance:

yes

Application method:

oral

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Details of food source: 50 % sucrose solution
- Method of feeding during study: The sucrose solution was provided in a PVC trough
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium (stock solution and final test solutions): A 2.5535 g portion (2.4999 g as a.i.) of the test material was placed in a 25 mL volumetric flask and brought to volume with acetone to yield a 100 mg a.i./mL stock solution. The stock solution was stored in a refrigerator at approximately 4 °C. Dosing solutions for the exposure were prepared by diluting the stock solution with acetone. These acetone stock solutions were then diluted with sucrose solution to produce the concentrations required for the test.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bee
- Strain origin: Carniolan
- Age at test initiation: Young worker honey bees
- Date of collection: The honey bees were collected the day before test initiation at which time they were handpicked from brood frames removed from a single mature hive.
- Cultural background (if honeybees): The honey bees were not previously exposed to pesticide application
- Kept according to standard practices: Yes; the test organisms were supplied with deionised water and 50 % sucrose solution prepared by dissolving an equal portion of sucrose in deionised water (w/w). The sucrose solution was provided in a PVC trough and deionised water from an inverted glass jar with a perforated lid placed on a paper towel.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Test temperature:

24 to 25 °C

Humidity:

61 to 85 % (relative)

Photoperiod and lighting:

A black curtain covered the front of the incubator which maintained the test organisms in near darkness to simulate hive conditions.

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): The test chambers measured 13 x 13 x 13 cm. The chamber frame was constructed from sheet PVC and each chamber was covered with a polyester mesh (approximately 3.5 mm mesh opening). One end of the chamber contained a glass sliding door. Each glass door had a hole with a self-sealing silicone closure large enough to accommodate the glass tube used to add test organisms.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

TEST DESIGN
The toxicity test was initiated upon addition of 200 µL of the appropriate treated sucrose solution contained in a 0.5 mL vessel to each test chamber.
The control and solvent control cages received untreated sucrose solution (1:1 sucrose/deionised water). To assist in verification of food solutions consumption, the vessels containing the solution were weighed on an analytical balance before addition to the test chambers and at the end of the exposure phase. Bees were allowed to feed on the test solutions for four hours before the solutions were removed. The percent of diet consumed was calculated from these measurements. Untreated 50 % sucrose solution was then added to each cage in a clean feeding trough. Fresh sucrose solution was supplied at 24 hours. Untreated, deionised water was also provided from an inverted glass jar with a perforated lid placed on a paper towel throughout the exposure. The study was terminated 48 hours after administration of the treated solutions.

EFFECT PARAMETERS MEASURED:
Observations of the test organisms were made at 4, 24, and 48 hours of exposure. The number of mortalities and any unusual behaviour exhibited (i.e., lethargy, ataxia) were also recorded.

VEHICLE CONTROL PERFORMED: Yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: the dosages for this test were selected on the basis of the contact test performed with the honey bee.

Nominal and measured concentrations:

- Nominal concentrations: 6.3, 13, 25, 50 and 100 µg a.i./bee

Reference substance (positive control):

yes

Remarks:

dimethoate

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Empirically estimated

Details on results:

Based on weights of food vials recorded at the beginning and end of the exposure period (4 hours), the following mean percent of the diet was consumed; 98, 95, 94, 95, 99, 94 and 94 % for the control, solvent control, 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively.
Following the 48 hour exposure, 3.3, 3.3, 10, 3.3 and 6.7 % mortality was observed among honey bees exposed to the 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively.
Following the 48 hour definitive oral test, 10 and 6.7 % mortality was observed among control and solvent control honey bees, respectively.
At test termination, no sub-lethal effects were observed among honey bees exposed to the treatment levels or the controls.

Results with reference substance (positive control):

Following 24 hour dimethoate exposure, mortality of 50, 90 and 100 % was observed among honey bees exposed to 0.090, 0.18, and 0.36 µg a.i./bee treatment levels, respectively. Some of the surviving honey bees exposed to the 0.090 and 0.18 µg a.i./bee treatment levels were observed to be lethargic or had spasmodic movement.
The test guideline states the 24 hour oral LD50 for dimethoate is in the range of 0.10 to 0.35 µg a.i./bee (OECD, 1998). The data generated during this test indicated the bees were slightly more sensitive to dimethoate than suggested by the study guideline.

Reported statistics and error estimates:

When the mortality in the exposure was less than 50 % in all concentrations tested, the LD50 for these exposures was empirically estimated to be greater than the highest dosage tested (100 µg a.i./bee).

Table 1: Cumulative Percent Mortality

Nominal Dose (µg/bee)	Replicate	Time (Hours)
		4	24	48
Control	A B C Mean	0 10 10¹ 6.7	0 10 20 10	0 10 20 10
Solvent Control	A B C Mean	0 10 0 3.3	0 10 0 3.3	0 20 0 6.7
6.3	A B C Mean	0¹ 0 10 3.3	0 0 10 3.3	0 0 10 3.3
13	A B C Mean	0 0 0 0	0 0 0 0	10 0 0 3.3
25	A B C Mean	0 0 10 3.3	0 0 10 3.3	10 10 10 10
50	A B C Mean	0 10 0 3.3	0 10 0 3.3	0 10 0 3.3
100	A B C Mean	10¹ 0 0¹ 3.3	10 0 0 3.3	10 0 10 6.7
Positive Control 0.09	A B C Mean	-² - - -	20³ 40³ 90³ 50	- - - -
Positive Control 0.18	A B C Mean	- - - -	100 100 70⁴ 90	- - - -
Positive Control 0.36	A B C Mean	- - - -	100 100 100 100	- - - -

¹ Some surviving bees were observed to be lethargic.

² Dimethoate reference test was observed and terminated at 24 hours.

³ Some surviving bees were observed to be lethargic.

⁴All surviving bees were observed to have spasmodic body movement.

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.

Executive summary:

The acute toxicity of the test material to the honey bee was investigated in an oral test conducted in accordance with the standardised guidelines OECD 213 and EPPO 170 under GLP conditions.

Apis mellifera (strain origin: Carniolan) were exposed to the test material at nominal dose levels of 6.3, 13, 25, 50 and 100 µg a.i./bee for 4 hours The bees were observed for 48 hours from the time the test material was administered. The test was conducted in near total darkness at a temperature of 24 to 25 °C and relative humidity of 61 to 85 %. Control and solvent control experiments were run concurrently, along with a positive control material, dimethoate, at concentrations of 0.09, 0.18 and 0.36 µg a.i./bee.

Based on weights of food vials recorded at the beginning and end of the exposure period (4 hours), the following mean percent of the diet was consumed; 98, 95, 94, 95, 99, 94 and 94 % for the control, solvent control, 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively.

Following the 48 hour exposure, 3.3, 3.3, 10, 3.3 and 6.7 % mortality was observed among honey bees exposed to the 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively. Following the 48 hour definitive oral test, 10 and 6.7 % mortality was observed among control and solvent control honey bees, respectively. At test termination, no sub-lethal effects were observed among honey bees exposed to the treatment levels or the controls.

Therefore under the conditions of this study, the 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.

Description of key information

The 48 hour LD50 values for acute contact and oral toxicity were empirically estimated to be greater than 100 µg a.i./bee.

Additional information

Contact Toxicity

The acute toxicity of the test material to the honey bee was investigated in a contact test conducted in accordance with the standardised guidelines OECD 214 and EPPO 170 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Apis mellifera (strain origin: Carniolan) were exposed to the test material at nominal dose levels of 1.0, 10 and 100 µg a.i./bee for 48 hours. The test was conducted in near total darkness at a temperature of 24 to 25 °C and relative humidity of 58 to 90 %.

Control and solvent control experiments were run concurrently, along with a positive control material, dimethoate, at concentrations of 0.05, 0.10 and 0.20 µg a.i./bee.

Following the 48 hour preliminary contact test, 5 % mortality was observed among honey bees exposed to the 1.0 and 10 µg a.i./bee treatment levels. Mortality of 3 % was observed in the control. No mortality was observed in the solvent control or the 100 µg a.i./bee treatment level. At test termination, no sub-lethal effects (i.e., spasmodic body movements) were observed among honey bees exposed to any of the treatment levels or the control. Since less than 10 % mortality was observed in the 100 µg a.i./bee treatment level, the preliminary test qualified as a limit test and no further testing was performed.

Following 24 hour dimethoate exposure, mortality of 17, 63 and 93 % was observed among honey bees exposed to the treatment levels of 0.050, 0.10 and 0.20 µg a.i./bee, respectively. All of the surviving honey bees exposed to the 0.10 µg a.i./bee treatment level were lethargic and some had spasmodic body movements.

Therefore under the conditions of this study, the 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.

Acute Oral Toxicity

The acute toxicity of the test material to the honey bee was investigated in an oral test conducted in accordance with the standardised guidelines OECD 213 and EPPO 170 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Apis mellifera (strain origin: Carniolan) were exposed to the test material at nominal dose levels of 6.3, 13, 25, 50 and 100 µg a.i./bee for 4 hours The bees were observed for 48 hours from the time the test material was administered. The test was conducted in near total darkness at a temperature of 24 to 25 °C and relative humidity of 61 to 85 %.

Control and solvent control experiments were run concurrently, along with a positive control material, dimethoate, at concentrations of 0.09, 0.18 and 0.36 µg a.i./bee.

Based on weights of food vials recorded at the beginning and end of the exposure period (4 hours), the following mean percent of the diet was consumed; 98, 95, 94, 95, 99, 94 and 94 % for the control, solvent control, 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively.

Following the 48 hour exposure, 3.3, 3.3, 10, 3.3 and 6.7 % mortality was observed among honey bees exposed to the 6.3, 13, 25, 50 and 100 µg a.i./bee treatment levels, respectively.

Following the 48 hour definitive oral test, 10 and 6.7 % mortality was observed among control and solvent control honey bees, respectively.

At test termination, no sub-lethal effects were observed among honey bees exposed to the treatment levels or the controls.

Therefore under the conditions of this study, the 48 hour LD50 value was empirically estimated to be greater than 100 µg a.i./bee.