Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 15 Apr 2010

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:

Adopted 21st Sept 1998

Deviations:

yes

Remarks:

Due to the low solubility of the test item in sugar solution the max. nominal dose level which was tested in the oral test was 50 µg a.i./bee (nominal).

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Version / remarks:

Adopted 21 Sept 1998

Deviations:

yes

Remarks:

Minor deviations only

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz (Wiesbaden, Germany, 14 Dec 2009)

Application method:

other: Acute Contact & Oral

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

CONTACT TEST
- Preparation of the test item solutions: A stock solution was prepared by dissolving 215 mg test item in 10 mL acetone. 5 µL stock solution contained 100 µg of the active ingredient.
- Volume of test solution applied: A single 5 µL droplet of test item in Acetone was applied to each bee (corresponding to 100 µg a.i./bee). A 5 µL droplet was chosen in deviation to the guideline recommendation of a 1 µL droplet. The test facility experience has proven that higher volumes are suitable since a more reliable dispersion of the test item is ensured without adversely affecting the study outcome [poster 2002, ICPBR Bee Protection Group meeting, Bologna, Italy].
- Body part: Dorsal bee thorax (Burkard applicator)
- Anaesthetization: In the contact test, the bees were anaesthetized for ca. 20 s with CO2 until they were completely immobilized immediately before application.
- Controls: One 5 µL droplet of a) tap water containing 0.5% Adhäsit and b) pure acetone. The reference item was also applied in a 5 µL droplet (dimethoate made up in acetone).
- Chemical name of vehicle: Acetone
- Evaporation of vehicle before use: No
- Wetting agent: Adhäsit. Adhäsit improves spreading and adhesion of the test droplet on the water-repellent hairs on the thorax of bees. Adhäsit is not toxic to honey bees.

ORAL TEST
- Preparation of the test item solutions: A stock solution 1 was prepared by dissolving 536 mg test item in 5 g Acetone. After the 1st dilution step (0.5 g stock solution 1 + 4.5 g water + 5.0 g ready-to-use syrup) the stock solution precipitated in the aqueous sugar solution. In a next step, the stock solution 1 was diluted with acetone (1:1) in order to obtain stock solution 2. The 2nd dilution step (0.5 g stock solution 2 + 4.5 g water + 5.0 g ready-to-use syrup) produced a completely solved solution. 20 mg of dilution no. 2 contained 50 µg a.i.
- Application (oral test): Appropriate amounts of the test item or reference item dilutions in acetone were mixed with syrup (ready-to-use syrup (30% Saccharose, 31% Glucose, 39% Fructose), Apiinvert, Südzucker, Ochsenfurt, Germany). The treated food was offered in syringes, which were weighed before and after introduction into the cages (duration of uptake: 3.05 h for treatments).
- Starvation time: In the oral test, all treatment groups underwent a 20 minute starvation time prior to application.
- Solvent control: The same proportion of syrup, water and acetone was used (50% w/w sugar solution, 45% water, 5% acetone)
- Negative control: 50% w/w aqueous syrup solution (with tap water)
- Treatment time: After a max. of 3.05 h, the syringes containing the treated food were removed, weighed and replaced by ones containing fresh, untreated food.
- Dosing: The target dose level (e.g. 50 µg a.i./bee, nominal) would have been obtained if 20 mg/bee of the treated food was ingested. In practice, higher dose levels (55.8 µg a.i./bee) were obtained as the bees had a higher or lower uptake of the test solutions than the nominal 20 mg/bee (i.e. 22 and 23 mg treated food per bee).

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bees
- Species: Apis mellifera carnica
- Source: Queen-right honey bee colonies bred by the testing facility
- Stage and sex: Adult, female worker bees
- Collection: With glass tubes, from the outer honeycombs (away from the brood), without the use of smoke and without anaesthetics.
- Date of collection: The morning of use
- Disease free: Yes
- Food: Commercial, ready-to-use syrup (Apiinvert: 30% saccharose, 31% glucose, 39% fructose) ad libitum. Was given directly after treatment using syringes. No replacement of the food was necessary during the course of the test (48 h).

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

25 °C

Humidity:

35 - 61% relative humidity

Photoperiod and lighting:

Darkness (except during observation)

Details on test conditions:

TEST SYSTEM
- Test container: Stainless steel cages (L: 10 cm x W: 8.5 cm x H: 5.5 cm ), with a removable glass sheet on the front side, inner walls lined with filter paper (Co. Schleicher & Schuelle, Dassel, Germany) and a perforated bottom with 98 ventilation holes (1 mm in diameter).
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 5
- No. of replicates per control: 5
- No. of replicates per vehicle control: 5
- Food: Commercial ready-to-use syrup ad libitum (30% saccharose, 31% glucose, 39% fructose, Apiinvert). No replacement of the food was necessary during the experimental phase.

OTHER TEST CONDITIONS
- Test design: Solvent and ngegative control, test item, reference item
- Photoperiod: Continuous darkness (except during observation)
- Ventilation: Ventilation was assured to avoid accumulation of pesticide vapour
- Surrounding: Incubator
- Other: The test conditions were continuously recorded with an electronic data logger and documented in the raw data.

EFFECT PARAMETERS MEASURED:
- Mortality: After 4, 24 and 48 h
- Behavioural abnormalities: After 4, 24 and 48 h (i.e. vomiting, apathy, intense cleaning)

VEHICLE CONTROL PERFORMED:
- Yes, vehicle controls for Adhäsit and acetone were included.

TEST CONCENTRATIONS
- Range finding study : No

Nominal and measured concentrations:

Contact test: 100 µg a.i./bee (nominal)
Oral test: 50 µg a.i./bee (nominal)

Reference substance (positive control):

yes

Remarks:

Dimethoate

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:

Remarks:

Contact toxity test

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 55.8 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr. (total fraction)

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

Oral toxicity test

Details on results:

- Mortality at end of exposure period: No mortality occured in either the contact toxicity test or the oral toxicity test.
- Morphological abnormalities: No
- Behavioural abnormalities: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels contact test: LD50 (24 h) = 0.15 µg a.i./bee (95% confidence limit: 0.04 - 0.28 µg a.i./bee) and LD50 (48 h) = 0.13 µg a.i./bee (95% confidence limit: 0.12 - 0.14 µg a.i./bee)
- Relevant effect levels oral test: LD50 (24 h) = 0.15 µg a.i./bee (95% confidence limit: 0.12 - 0.14 µg a.i./bee) and LD50 (48 h) = 0.13 µg a.i./bee (95% confidence limit: 0.12 - 0.14 µg a.i./bee)

Reported statistics and error estimates:

Results obtained with the bees treated with test item and the reference item were compared to the results obtained with the control in both the contact and oral tests. The contact and oral LD59 values of the reference item were estimated by Probit Analysis according to Finney 1971. The software used to perform the statistical analysis was ToxRat Professional v. 2.10 (ToxRat Solutions GmbH, 2009).

VALIDITY CRITERIA

The contact and oral toxicity tests fulfilled the criteria defined by OECD guidelines 213 and 214, respectively (Table 1). Therefore, the study is considered valid and reliable.

Table 1: Validity criteria for OECD 213 and 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.	The control mortality in the contact and oral test was < 10%.   Contact test: CO2/water control: 0% CO2/acetone control: 0%   Oral test: Water/sugar control: 2.0% Acetone/sugar control: 0%	Yes
The LD50 of the toxic standard meets the specified range.   Contact test: 0.10 – 0.30 µg a.i./bee (24 h)   Oral test: 0.10 – 0.35 µg a.i./bee (24 h)	The LD50 values obtained with dimethoate were within the required ranges:   Contact test: 0.15 µg a.i./bee   Oral test: 0.13 µg a.i./bee	Yes

 

DEVIATIONS TO THE STUDY PLAN

Two deviations to the study plan occurred, namely concerning the storage of the test item (1) and the dose level of the test item (2).

(1): The test item was cooler than the desired 25 ± 5 °C.

(2): It was technically not feasible to obtain a maximum dose level of 100 µg a.i./bee in the oral test. The maximum dose level which could be obtained without precipitation was a nominal concentration of 50 µg a.i./bee, which resulted in an actual intake of 55.8 µg a.i./bee.

 

However, neither deviation is presumed to have negatively affected the study outcome because the test item is considered stable at the stated storage conditions of the present study according to the sponsor (1) and because there was no mortality at the achieved dose level of 55.8 µg a.i./bee (2).

 

BIOLOGICAL RESULTS

CONTACT TEST

After 48 h, there was no mortality at 100 µg a.i./bee (Table 2). No mortality occurred in the water control (water + 0.5% Adhäsit) and in the solvent control (acetone).

Table 2. Mortality and behavioural abnormalities of the bees in the contact toxicity test.

Dosage	After 4 h		After 24 h		After 48 h
	mortality	behave. abnorm.	mortality	behave. abnorm.	mortality	behave. abnorm.
	mean [%]	mean [%]	mean [%]	mean [%]	mean [%]	mean [%]
test item: 100 µg a.i./bee	0	0	0	0	0	0
water	0	0	0	0	0	0
solvent	0	0	0	0	0	0
reference item:
0.30 µg a.i./bee	42	50	98	2	100	0
0.20 µg a.i./bee	18	8	86	10	96	0
0.15 µg a.i./bee	4	0	60	0	68	0
0.10 µg a.i./bee	0	0	2	0	12	0

Results are averages of 5 replicates (ten bees each) per dosage/control.

Behave. Abnorm.             = behavioural abnormalities

Water                                 = CO2/water-treated control

Solvent                               = CO2/solvent control

 

ORAL TEST

The maximum dose level of 100 µg a.i./bee could not be achieved because even though the test item was perfectly solved in acetone, the test item/acetone solution precipitated after transfer into the aqueous sugar solution. The maximum dose level which could be obtained without precipitation was 50 µg a.i./bee (nominal), which resulted in an actual intake of 55.8 µg a.i./bee. This dose level led to no mortality after 48 h (Table 3). In the solvent control (acetone) no mortality occurred and there was 2.0% mortality in the water control (50% sugar solution). No behavioural effects were observed at any time.

Table 3. Mortality and behavioural abnormalities of the bees in the oral toxicity test.

Dosage	After 4 h		After 24 h		After 48 h
	mortality	behave. abnorm.	mortality	behave. abnorm.	mortality	behave. abnorm.
	mean [%]	mean [%]	mean [%]	mean [%]	mean [%]	mean [%]
test item: 100 µg a.i./bee	0	0	0	0	0	0
water	0	0	0	0	0	0
solvent	0	0	0	0	0	0
reference item:
0.30 µg a.i./bee	34	34	100	0	100	0
0.20 µg a.i./bee	2	8	70	10	72	2
0.15 µg a.i./bee	0	0	2	0	2	0
0.10 µg a.i./bee	0	0	0	0	0	0

Results are averages of 5 replicates (ten bees each) per dosage/control.

Behave. Abnorm.             = behavioural abnormalities

Water                                 = water control

Solvent                               = solvent control

Validity criteria fulfilled:

yes

Description of key information

LD50 (48 h) > 100 µg a.i./bee (nominal, OECD 214, acute contact toxicity test, Apis mellifera)

LD50 (48 h) > 55.8 µg a.i./bee (nominal, OECD 213, acute oral toxicity test, Apis mellifera)

Additional information

One study is available on the short-term contact and oral toxicity of triafamone (CAS 874195-61-6) to the honey bee Apis mellifera. In this study, an acute contact toxicity test and an acute oral toxicity test were performed according to OECD guidelines 213 and 214 as well as GLP.

In the contact test, no mortality was observed after 48 h at the limit concentration of 100.0 µg a.i./bee. In the oral test, the maximum dose level of 100 µg a.i./bee could not be achieved due to precipitation of the test item in sugar water. The highest dose level which could be achieved was 50 µg a.i./bee (nominal), which resulted in an actual intake of 55.8 µg a.i./bee. After 48 h, no mortality was observed at this dose level. In both tests, no significant mortality occurred in the water and solvent controls and no behavioural or other biological effects were observed.

Therefore, the LD50 (48 h) for the contact toxicity test was > 100 µg a.i./bee and the LD50 (48 h) for the oral toxicity test was > 55.8 µg a.i./bee.