Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to bees: acute oral

Remarks:

and contact

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jul 2007 to 02 Aug 2007

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)

Version / remarks:

1998

Deviations:

yes

Remarks:

See 'Any other information on results incl. tables'

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Version / remarks:

1998

Deviations:

yes

Remarks:

See 'Any other information on results incl. tables'

GLP compliance:

yes (incl. QA statement)

Application method:

other: oral and contact

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Controls: In the oral toxicity test, the bees of one control group received a 50 % (w/v) aqueous sucrose solution and one additional control group was fed with the aqueous sucrose solution mixed with acetone (ratio 10 : 1 ), which was used as a solvent. In the contact toxicity test, the bees of one control group were treated with tap water, one additional control group was treated with acetone.

- Oral Toxicity Test: For the oral toxicity test, the test substance was dissolved in pure acetone in order to get a stock solution. The highest test item dose (200.0 μg/bee) was prepared by mixing a defined amount of the stock solution with a defined amount of a 62.5 % (w/v) aqueous sucrose solution, such that the dose which was calculated for one bee was found in 20 μL of a 50.0 % (w/v) aqueous sucrose solution. This was done to compensate the dilution of the sugar solution by the test item stock solution.
The lower test doses were prepared by mixing a defined amount of the highest oral solution with a defined amount of a 50 % (w/v) aqueous sucrose solution, such that the dose which is calculated for one bee was found in 20 μL, too. The test item was not completely dissolved in the sugar solution, but it was an emulsion. To avoid a lumped, sticky, brown film on the surface of the test item sugar solutions the feeders for the test item were weighed and offered to the bees immediately after preparation. Before the bees were permitted to feed, they were starved for approximately 2 hours. A quantity of 250 μL of test item and reference item solutions was offered for a maximum of 6 hours to each replicate to ensure sufficient consumption of test or reference item. Bees within a cage shared the test solution and therefore are assumed to have received a similar dose. The amount of test solution consumed by each replicate ( consisting of 10 bees) was determined by weighing the feeders (eppendorf cups) before and after feeding. After the test solutions had been consumed (or latest after 6 hours), the bees were supplied ad libitum with untreated 50 % aqueous sucrose solution.

- Contact Toxicity Test: For the contact toxicity test, the test substance was dissolved in pure acetone. The test item was not completely dissolved, but it was an emulsion. After the bees had been anaesthetised with carbon dioxide they were treated individually by topical application with a micro applicator. Before application the test item solution was agitated well to get a constant emulsion. In each treatment group, i.e. test item, reference item and both controls, 2 μL of the respective solutions were applied dorsally to the thorax of each bee.
Between every application, the outside of the micro applicator needle was cleaned with a mixture of water and a water-wetting agent. This reduced the surface tension of the applied solution and ensured that the drop of the test item solution spread out immediately after application on each bee. After application the bees were returned to the test cages and fed with a 50 % (w/v) aqueous sucrose solution ad libitum.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bee
- Source: Young adult worker bees deriving from a healthy colony, which descended from a breeding line of a beekeeper in Rheinland-Pfalz, Germany
- Bee colonies: The bee colonies were inspected periodically according to good bee keeping practice by an experienced apiarist. Work carried out during inspection was documented in a hive record. Furthermore the colonies were examined for a reportable bee epidemic by an authorised bee specialist.
- Others: One day before the start of the test the bees were collected randomly from the outer combs of the colony for the oral toxicity test and for the contact toxicity test.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Test temperature:

23.0 - 26.5 °C

Humidity:

50 % - 88 %

Photoperiod and lighting:

- Lighting: During the experimental phase the test animals were kept in constant darkness (start of experimental phase in the oral toxicity test: feeding of the bees; start of experimental phase in the contact toxicity test: topical application) except during the assessments. Observations were made under neon light.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Cages made of stainless steel (width: 10 cm; depth: 5.5 cm; height: 8.5 cm). The front side of the cages was equipped with a transparent window so that the bees could be observed. The bottom of the cages consisted of perforated steel, which guaranteed sufficient air supply for the test insects. The test cages were lined with filter paper.

For both oral and contact test:
- No. of organisms per replicate: 10
- No. of replicates per treatment group: 5
- No. of replicates per control: 5
- No. of replicates per positive control: 5

EFFECT PARAMETERS MEASURED
The number of dead bees in the individual test cages was recorded after 4 h, 24 h and 48 h. In case of symptoms of poisoning, the behavioural differences between the bees of the control group and those of the test item treatment were noted at each observation interval.

Nominal and measured concentrations:

- Nominal concentration (Contact exposure): 200 μg/bee
- Nominal concentrations (Oral doses provided): 12.5, 25.0, 50.0, 100, 200 μg/bee
- Nominal concentrations (Oral doses consumed): 14.3, 28.4, 56.1, 111.5 and 192.3 μg/bee

Reference substance (positive control):

yes

Remarks:

Dimethoate 40 EC

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 192 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: oral exposure

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 200 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: contact exposure

Details on results:

An overview of the results is provided in Table - Table in 'Any other information on results incl. tables'.
- Oral toxicity test: The oral toxicity test was carried out with the target dose levels of 12.5, 25.0, 50.0, 100.0 and 200.0 μg/bee. The actual consumption by bees in the oral test was 14.3, 28.43, 56.08, 111.47 and 192.27 μg/bee. After 48 hours exposure, 0% and 6 % of mortality were found in the negative control (sugar solution) and solvent control (acetone). For the test substance treated groups, 2%, 0%, 8%, 6% and 20% mortality were found in the 14.3, 28.43, 58.06, 111.47, 192.27 µg/bee consumption amount, respectively. The corrected mortality for solvent control were -4.3%, -6.4%, 2.1%, 0.0% and 14.9% in the 14.3, 28.43, 58.06, 111.47, 192.27 µg/bee (consumption) dosages.
- Contact Toxicity Test: The contact toxicity test was carried out with the dose level of 200 μg/bee by topical application. After 48 hours exposure, 2% and 0 % of mortality were found in the negative control (tap water) and solvent control (acetone). For the test substance treated groups, 2% mortality was found. The corrected mortality for solvent control was 2% as well.

Results with reference substance (positive control):

- Oral toxicity test: After exposure to the positive control (Dimethoate 40 EC) for 48 hours, 24%, 40%, 78%, and 98% of mortality were found in the 0.09, 0.11, 0.16 and 0.22 µg/bee (consumption) groups. The corrected mortality for negative control were 24%, 68%, 86% and 100%, respectively.
- Contact toxicity test: After exposure to the positive control (Dimethoate 40 EC) for 48 hours, 10%, 54%. 86% and 98% of mortality were found in the 0.10, 0.14, 0.20 and 0.30 µg/bee groups. The corrected mortality for negative control were 8.2%, 53.1%, 85.7% and 98.0%, respectively.

Reported statistics and error estimates:

The average mortality of the five replicates per dose was calculated after correction for control mortality according to the formula of SCHNEIDER-ORELLI (1947). The LD50 values with 95 % confidence intervals of the test item and the reference item treatment were calculated by means of a probit analysis using the statistic program SAS release Version 9.1.3, service pack 4 (2002-2003).

Table 1. Mortality, corrected mortality and total consumption in the oral test with the test item, reference item and control

Treatment (Target dose)	Test item consumed	Mortality [%]		Corrected Mortality[%]
		24 h	48 h	24 h	48 h
Control 1 (sugar solution)	--	0.0	0.0	-	-
Control 2 (acetone sugar solution 1:10)	--	6.0	6.0	-	-
Test item (µg/bee)*
12.5	14.3	0.0	2.0	-6.4	-4.3
25.0	28.43	0.0	0.0	-6.4	-6.4
50.0	56.08	8.0	8.0	2.1	2.1
100.0	111.47	6.0	6.0	0.0	0.0
200.0	192.27	20.0	20.0	14.9	14.9
Reference item: Dimethoate 40 EC
0.08	0.09	14.0	24.0	14.0	24.0
0.10	0.11	40.0	68.0	40.0	68.0
0.14	0.16	78.0	86.0	78.0	86.0
0.21	0.22	98.0	100.0	98.0	100.0

*corrected for control 2 (sugar solution with acetone)

**corrected for control 1 (pure sugar solution)

 

Table 2. LD50 values in the oral test with the test item and the reference item (dimethoate) 

Treatment	24 h		48 h
	LD50	Limits*	LD50	Limits*
	µg/bee
test substance	> 192.27	-	> 192.27	-
	µg/bee
Reference Item	0.12	0.11 to 0.13	0.10	0.10 to 0.11

Table 3. Mortality and corrected mortality in the contact test with the control, the test item and the reference item treatment

Treatment	Mortality [%]		Corrected Mortality [%]
	24 h	48 h	24 h	48 h
Control (tap water) 2 µL application volume	0.0	2.0	-	-
Control (acetone) 2 µL application volume	0.0	0.0	-	-
Test item (µg/bee)
200.0	0.0	2.0	0.0	2.0
Reference item:Dimethoate 40 EC
0.10	2.0	10.0	2.0	8.2
0.14	26.0	54.0	26.0	53.1
0.20	84.0	86.0	84.0	85.7
0.30	96.0	98.0	96.0	98.0

 

Table 4. LD50 values in the contact test with the test item and the reference item (dimethoate) 

Treatment	24 h		48 h
	LD50	Limits*	LD50	Limits*
	µg/bee
Test substance	> 200.0	-	> 200.0	-
	µg/bee
Reference Item	0.16	0.15 to 0.17	0.14	0.13 to 0.15

*upper and lower confidence limits; p = 0.05

Deviation from OECD TG 213 and OECD TG 214

The humidity ranged from 70 - 88%. However, results from the control and toxic standard treatment groups showed that the deviation had no impact on the validity of the study.

Validity of the Test

The study is considered to be valid because:

• the mean mortality of the control in the oral and contact toxicity test was approximately 10 %

• the 24h LD50 of the reference item in the oral toxicity test was within the range of 0.10 to 0.35 μg/bee

• the 24h LD50 of the reference item in the contact toxicity test was within the range of 0.10 to 0.30 μg/bee

Validity criteria fulfilled:

yes

Remarks:

See Validity of the Test in 'Any other information on results incl. tables'

Conclusions:

In an acute oral and contact toxicity test in honeybees, performed in accordance with OECD TG 213 and OECD TG 214, the 48-hour oral LD50 was determined to be 192 µg/bee and the contact LD50 was > 200 µg/bee.

Executive summary:

The toxicity of the test substance to the honeybee (Apis mellifera) was determined in an acute oral (48-h) and contact (48-h) toxicity test. The tests were performed following OECD TG 213 and OECD TG 214, and in compliance with GLP criteria. For the oral toxicity test, the bees (10 bees/replicate and 5 replicates/concentration) were offered an aqueous sucrose solution that contained the test substance at dosages of 12.5, 25, 50, 100 and 200 µg/bee. The actual consumption dosages were determined by weighing the feeders before and after feeding. It was determined that the actual consumption dosages were 14.30, 28.43, 56.08, 111.47 and 192.27 µg/bee. For the contact toxicity test, the bees were exposed to a 2 μL droplet of the test substance solution on the dorsal surface of the thorax. The dose was 200 µg/bee. For both tests, acetone was used as solvent control. In addition, a negative control group was involved in oral (sugar solution) and contact tests (tap water). Dimethoate 40 EC was used as positive control at concentrations of 0.08, 0.10, 0.14, 0.21 µg/bee in oral toxicity test, and 0.10, 0.14, 0.20, 0.30 µg/bee at contact toxicity test.

After 48 hours of exposure, the maximal mortality in control was 6% and 2% in the oral (solvent control) and contact (negative control) toxicity tests, respectively. The test substance caused negligible mortality (≤ 2.1 %) up to a dose of 192 µg/bee. In the contact toxicity test, the mortality was 2%. The 24 -hour contact and oral LD50 values for the positive control were 0.16 and 0.12 μg dimethoate/bee respectively, therefore validity criteria were met.

Based on this information, the 48-hour oral LD50 was determined to be 192 µg/bee, and the contact LD50 was > 200 µg/bee.

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Another study on honeybees is included as supporting study.

48 -hour oral toxicity test, LD50 > 192 µg/bee, and 48 -hour contact toxicity test, LD50 > 200 µg/bee, Apis mellifera, mortality, OECD TG 213 & OECD TG 214, Warmers 2007

Additional information

There are two acute toxicity studies available on honeybees (Apis mellifera), both following standard guidelines and GLP compliant. Both acute exposure experiments were under laboratory conditions and following both oral and contact applications. In the first study, the observed 48-hour LD50 values were > 95.5 µg/bee for oral and > 100 µg/bee for contact application, respectively (Bocksch 2005). In the second study, the observed 48-hour LD50 values were > 192 µg/bee for oral and > 200 µg/bee for contact application, respectively (Warmers 2007). In both studies, the LD50 values were found to be above the highest tested dose. Thus, the higher LD50 value (> 192 µg/L; Warmers 2007) is considered to be closer to the real LD50 and is selected for the CSA.