1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to bees: chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul 2018 to 03 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 245 (Honey Bee (Apis mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding)

Version / remarks:

2017

Deviations:

yes

Remarks:

See Deviations from the guidelines in 'Any other information in results incl. tables'

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Analytical monitoring:

yes

Details on sampling:

Analytical samples and retain samples of the solvent control, all test item treatment groups, pure solvent (acetone) and the stock solution were taken every day directly after preparation. The sample size was 2 mL for the stock solution and acetone and 5 mL for the feeding solutions (analytical and retain sample, respectively). No samples of the reference item feeding solutions were taken.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

The amount of test item needed for the daily preparation of the stock solution(s) was weighed for several days in advance and stored tightly closed under dark and dry conditions (ambient; 5 °C - 30 °C) until use. Test item stock solutions (ST1 to ST5) were prepared and diluted once every day for each test item concentration. Acetone was used as the solvent. The definitive feeding solutions for the test item treatments were freshly prepared every day diluting the respective stock solution by adding 50 % (w/v) aqueous sucrose solution.
The feeding solutions were offered to the test organisms of each test unit in feeders (plastic syringes, approx. 5 mL). The tip of each feeder was removed so that the bees had access to the feeding solution. A volume of about 3-4 mL of feeding solution was offered to the bees and guaranteed an ad libitum feeding during each 24 h feeding interval. The bees in one cage shared the feeding solution and thus received similar doses (trophallaxis). Freshly prepared feeding solution replaced the feeding solution of the previous day by changed feeders. The amount of feeding solution(s) consumed was determined by weighing the feeders before and after feeding using calibrated equipment. The syringes of 8 additional cages were filled with ~ 3-4 mL of pure 50 % (w/v) aqueous sucrose solution or 50 % (w/v) aqueous sucrose solution containing 5 % acetone and weighed daily for the determination of the evaporation. The untreated sucrose solution and the sucrose solution containing 5 % acetone were stored under cool and dark conditions in the refrigerator (target 6 ± 2° C) for a maximum period of up to 4 days.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Worker bees
- Source: Obtained from a healthy colony of the test facilities’ own stock in Niefern-Öschelbronn, Germany.
- Age at test initiation: Newly hatched; 1 to 2 days old
- Disease free: Yes
- Kept according to standard practices: Yes
- Feeding: 50 % (w/v) aqueous sucrose solution was used as food ad libitum during acclimatisation.

ACCLIMATION
Up to two days prior to exposure to the test item, brood combs containing capped cells which were expected to hatch on the same day were taken out of a honey bee colony and transferred into the climatic chamber. The combs were kept under test conditions. One day prior to exposure to test item the 0 - 1 day old bees were picked off the combs, transferred to the test cages and kept under test conditions until the start of the test. Replacement of moribund or dead bees before start of exposure was not necessary.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

10 d

Test temperature:

31.6 – 33.4°C

Humidity:

35.7 – 65.6 %

Details on test conditions:

TEST SYSTEM
- Test cage: Made of stainless steel (base: 8 cm x 4 cm; height: 6 cm).
- Details of the test cage: The front side of the cages was equipped with a transparent pane to enable observation. The bottom of the cages consisted of perforated steel, which guaranteed sufficient air supply. The cages were lined with filter paper.
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
- Others: Eight test units without bees but with full food syringes (containing 50 % (w/v) aqueous sucrose solution or 50 % (w/v) aqueous sucrose solution containing 5 % acetone) were placed in the climatic chamber for evaluation of evaporation of feeding solution.

EFFECT PARAMETERS MEASURED
Mortality and behavioural abnormalities were recorded every 24 hours (± 2 hours) after application (start of feeding).
The consumption of feeding solution per bee per day was calculated by dividing the total daily consumption per replicate by the number of living bees at the beginning of the respective feeding interval. For each treatment group, the mean consumption of feeding solution per bee per day was calculated by averaging the replicate values.

VEHICLE CONTROL PERFORMED: Yes

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 31.25, 62.5, 125., 250 and 500 mg test substance/kg food

Reference substance (positive control):

yes

Remarks:

dimethoate, 0.9 mg test substance/kg

Key result

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

10 d

Dose descriptor:

NOED

Effect conc.:

8.2 µg per animal per day

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

LD50

Effect conc.:

> 8.2 µg per animal per day

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

> 500 mg/kg diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

An overview of the results are provided in Table - Table in 'Any other information on results incl. tables'.
- Analytical Results: The actual mean concentrations of the test substance in all the feeding solution were in the range from 89 to 104 % of the nominal concentrations; therefore results are based on nominal values. No residues of the test substance above the LOD (0.90 mg/kg) were found in any of the control samples.
- Cumulative Mortality and Behavioural Abnormalities: In the control group fed with pure 50 % (w/v) aqueous sucrose solution no mortality was observed. In the solvent control group fed with 50 % (w/v) aqueous sucrose solution containing 5 % acetone, 7.5 % mortality was observed at the final assessment at 10 days. At the concentrations of 31.25, 62.5, 125, 250 and 500 mg test substance/kg, 0.0, 25.0, 10.0, 5.0 and 27.5 % mortality (corrected mortality:-8.1,18.9, 2.7, -2.7, 21.6 %) was observed, respectively. During the 10-day test period, single moribund and affected bees were observed at the concentration levels of 62.5, 125 and 500 mg test substance/kg feeding solution but without relation to concentration/dose or time of assessment.
- Food Consumption and Uptake of Test Item: The overall mean daily consumption of feeding solution (i.e. the average consumption/bee over 10 days) at the test item concentrations of 31.25, 62.5, 125, 250 and 500 mg a.i./kg was 24.1, 22.9, 20.1, 17.4 and 16.4 mg/bee/day, respectively. Compared to the control and solvent control group (36.6 and 24.8 mg/bee/day, respectively) there was reduced consumption of the test items that was concentration related. The values of food consumption were corrected for evaporation.
After 10 days of continuous exposure, the mean accumulated uptake of the test substance at the treatment levels of 31.25, 62.5, 125, 250 and 500 mg test substance/kg feeding solution was 7.53, 14.3, 25.2, 43.6 and 82.0 μg test substance/bee, respectively. The corresponding average daily dose was therefore 0.75, 1.43, 2.52, 4.36 and 8.20 μg test substance/bee/day.

Results with reference substance (positive control):

After 10 days of exposure, the mortality was 100% in the reference substance treatment group.

Reported statistics and error estimates:

Fisher's exact test with Bonferroni-Holm adjustment (one-sided greater, α = 0.05) was used to evaluate whether there were significant differences between the mortality data of the control and the test item treatment group and to determine the NOEC and NOEDD based on mortality, respectively. To justify the use of the Fisher's exact test with Bonferroni-Holm adjustment a trend analysis by contrasts using proportions was performed as well as a Tarone’s test to test for extra-binomial variance.

Table 1. Cumulative Mortality

Treatment [mg a.i./kg]	Cumulative mortality [%]
	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10
Control(s)
C (0)	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
CS(0)	0.0	0.0	0.0	0.0	0.0	0.0	0.0	2.5	5.5	7.5
Test item
31.25	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
62.5	0.0	0.0	2.5	10.0	10.0	10.0	10.0	10.0	15.0	25.0
125	0.0	0.0	0.0	0.0	0.0	0.0	2.5	5.0	7.5	10.0
250	0.0	0.0	0.0	0.0	5.0	5.0	5.0	5.0	5.0	5.0
500	0.0	0.0	0.0	2.5	2.5	5.0	15.0	20.0	22.5	27.5
Reference item
0.9	0.0	0.0	5.0	10.0	30.0	57.5	85.0	92.5	97.5	100.0

Table 2. Corrected Cumulative Mortality

Treatment [mg a.i./kg]	Cumulative corrected mortality [%]
	E1	E2	E3	E4	E5	E6	E7	E8	E9	E10
Test item
31.25	0.0	0.0	0.0	0.0	0.0	0.0	0.0	-2.6	-5.3	-8.1
62.5	0.0	0.0	2.5	10.0	10.0	10.0	10.0	7.7	10.5	18.9
125	0.0	0.0	0.0	0.0	0.0	0.0	2.5	2.6	2.6	2.7
250	0.0	0.0	0.0	0.0	5.0	5.0	5.0	2.6	0.0	-2.7
500	0.0	0.0	0.0	2.5	2.5	5.0	15.0	17.9	18.4	21.6
Reference item
0.9	0.0	0.0	5.0	10.0	30.0	57.5	85.0	92.5	97.5	100.0

Table 3. Mean Consumption of Feeding Solution

Treatment [mg a.i./kg]	Mean consumption of feeding solution [mg/bee/day]
	A1	A2	A3	A4	A5	A6	A7	A8	A9	A10	mean
Control(s)
C (0)	26.6	29.1	35.1	41.3	49.2	41.6	37.3	35.2	32.9	37.8	36.6
CS(0)	19.2	26.3	30.4	33.9	26.5	21.5	18.4	26.7	19.2	25.5	24.8
Test item
31.25	14.6	22.7	23.1	30.3	26.2	24.6	17.0	27.6	19.4	35.2	24.1
62.5	15.3	19.5	26.3	25.3	21.1	23.3	22.4	29.2	21.1	25.3	22.9
125	22.7	21.2	23.7	21.5	19.2	18.7	12.4	25.8	17.9	18.3	20.1
250	11.3	21.4	20.1	14.8	20.5	16.8	12.7	15.8	17.8	23.2	17.4
500	3.6	23.0	19.1	18.2	19.4	16.0	14.7	17.3	12.5	20.4	16.4
Reference item
0.9	26.8	14.3	18.0	18.6	17.9	11.6	20.5	8.0	5.3	19.7	17.1

X = Overall mean consumption of feeding solution (calculation based on the replicate values)

Table 4. Mean Uptake of Test and Reference Item

Treatment [mg a.i./kg]	Mean uptake per day [µg a.i./bee/day]
	A1	A2	A3	A4	A5
Test item
31.25	0.46	0.71	0.72	0.95	0.82
62.5	0.96	1.22	1.64	1.58	1.32
125	2.84	2.65	2.96	2.68	2.40
250	2.83	5.35	5.03	3.69	5.14
500	1.78	11.50	9.56	9.08	9.68
Reference item
0.9	0.03	0.01	0.02	0.02	0.02

 

Treatment [mg a.i./kg]	Mean uptake per day [µg a.i./bee/day]					DD
	A6	A7	A8	A9	A10
Test item
31.25	0.77	0.53	0.86	0.61	1.10	0.75
62.5	1.46	1.40	1.83	1.32	1.59	1.43
125	2.34	1.55	3.22	2.24	2.29	2.52
250	4.20	3.17	3.94	4.45	5.81	4.36
500	7.98	7.35	8.66	6.23	10.21	8.20
Reference item
0.9	0.01	0.02	0.01	0.00	0.02	0.02

DD = dietary dose (calculation based on the replicate values)

VALIDITY CRITERIA

The study is considered valid because:
- The mean mortality in the controls was ≤ 15 % at the end of the test.

- The mean mortality in the reference item group was ≤ 50 % at the end of the test.

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In a chronic toxicity study in honeybees, performed in accordance with OECD TG 245, the 10-d oral NOEC was determined to be 500 mg test substance/kg food (corresponding to 8.20 μg test substance/bee/day).

Executive summary:

The toxicity of the test substance to the honeybee (Apis mellifera) was determined in a chronic oral (10-d) toxicity test. The test was performed following OECD TG 245 and in compliance with GLP criteria. For the oral toxicity test, the bees (10 bees/ replication and 4 replicates/concentration or controls) were fed ad libitum with a 50 % (w/v) aqueous sucrose solution containing 5 % acetone and the test substance at the concentration levels of 31.25, 62.5, 125, 250 and 500 mg test substance/kg feeding solution. The control groups were exposed for the same period under identical exposure conditions to untreated 50 % (w/v) aqueous sucrose feeding solution and 50 % (w/v) aqueous sucrose feeding solution containing 5 % acetone. The test conditions were: temperature 31.6 - 33.4 ˚C and humidity 35.7 - 65.6%. Assessments of mortality and behavioural abnormalities were carried out daily during the 10-day test period in the control and test item treatment groups. Furthermore, the daily food uptake was determined.

The actual mean concentrations of the test substance in all test item feeding solutions ranged from 89 to 104 % of the nominal concentrations. In the control group fed with pure 50 % (w/v) aqueous sucrose solution, no mortality was observed. In the solvent control group fed with 50 % (w/v) aqueous sucrose solution containing 5 % acetone, 7.5 % mortality was observed at the final assessment at 10 days. In 31.25, 62.5, 125, 250 and 500 mg test substance/kg treatment groups,  0.0, 25.0, 10.0, 5.0 and 27.5 % mortality (corrected mortality: -8.1,18.9, 2.7, -2.7, 21.6 %) was observed, respectively. During the 10-day test period, single moribund and affected bees were observed at the concentration levels of 62.5, 125 and 500 mg test substance/kg feeding solution but without relation to concentration/dose or time of assessment. Compared to the control and solvent control groups (36.6 and 24.8 mg/bee/day, respectively), there was reduced consumption of the test items that were concentration related. After 10 days of continuous exposure, the mean accumulated uptake of the test substance at the treatment levels of 31.25, 62.5, 125, 250 and 500 mg test substance/kg feeding solution was 7.53, 14.3, 25.2, 43.6 and 82.0 μg test substance/bee, respectively. The average daily dose was 0.75, 1.43, 2.52, 4.36 and 8.20 μg test substance/bee/day. Based on the findings, the 10-d NOEC for mortality was determined to be 500 mg test substance/kg. The corresponding NOEDD, based on the actual consumption of the respective feeding solutions, was determined to be 8.20 μg test substance/bee/day.

Reference 2

Endpoint:

toxicity to bees: acute oral

Remarks:

and acute contact

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Working Document D3 of the United Kingdom Pesticides Safety Precautions Scheme "Laboratory testing for toxicity to honey bees"

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:

- Contact toxicity test: Applying 1.0 µL droplets of acetone solutions of the test substance to each bee using a micrometer syringe. One cage of bees at a time were lightly anaesthetised with carbon dioxide and laid ventral side up on a filter paper in a petri dish. A 1.0 µL droplet of the appropriate solution was placed on the ventral surface of the thorax of each bee. The bees were then replaced in the cage and supplied with 20% sucrose in water.
- Oral toxicity: The appropriate concentration was administered as a single dose of 0.2 mL to a group of 10 bees in a cage. The dose was introduced by syringe into a glass tube 50 x 8 mm with a 1.5 mm opening. The tube was inserted open end down through the top of the cage. It has been established experimentally that bees will share the 0.2 mL among themselves and so receive similar doses of 20 µL per bee. The 0.2 mL consisted of 1 part of the appropriate dilution of test substance in acetone mixed with 19 parts of sucrose in water. Two cages of bees were treated with 0.2 mL of 20% sucrose in water containing 5% acetone as controls. When the bees had taken all the test solutions (about 3 hours) the dosage tubes were replaced with tubes containing 20% sucrose in water.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

The worker honeybees were obtained from an experienced apiarist in Cambridgeshire and used for test on the day of removal from the hive.

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24 ± 1°C

Photoperiod and lighting:

In darkness

Details on test conditions:

TEST SYSTEM
- Test cage: 2 mm aperture wire mesh, cylinders 11.5 cm long and 4.0 cm in diameter
- No. of organisms per cage: 10
- No. of replicates per treatment group: 10
- No. of replicates per vehicle control: 2

Nominal and measured concentrations:

- Nominal concentrations: 0 (solvent control), 100 µg/bee

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LD50

Remarks:

Oral

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

LD50

Remarks:

Contact

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.
- Mortality: In the oral toxicity test, the mortality at 24 and 48 hours was 21% and 28%, respectively. In the contact toxicity test, the mortality at 24 and 48 hours was 26% and 31%, respectively.
Because only one dose was tested, no dose-related response was obtained and no statistical analyses were required. The 48-h LD50 was concluded to be > 100 µg/bee.

Table 1. Mortality of bees after oral and contact treatment

	Group	Oral		contact
		24 hour	48 hour	24 hour	48 hour
Treated	1	3	3	1	1
	2	5	5	3	3
	3	4	4	5	6
	4	1	2	6	7
	5	2	2	1	2
	6	0	1	2	2
	7	1	6	0	2
	8	1	1	2	2
	9	1	1	2	2
	10	3	3	4	4
mortality		21%	28%	26%	31%
Control	1	0	0	1	1
	2	1	1	0	0

Validity criteria fulfilled:

not specified

Conclusions:

In an oral and contact acute toxicity study to honeybees, performed in accordance with United Kingdom national standard methods with acceptable restrictions, the 48-hour oral and contact LD50 values were determined to be > 100 µg/bee.

Executive summary:

The acute toxicity of the test substance to the honeybee (Apis mellifera) was determined in acute oral (48-h) and contact (48-h) toxicity tests. The tests were performed following Working Document 3 of the United Kingdom Pesticides Safety Precaution Scheme “Laboratory testing for toxicity to honey bees” and in compliance with GLP criteria. Based on the results from preliminary dose-finding tests, 100 µg/bee was selected as the only testing dose for both oral and contact toxicity tests. In the oral toxicity test, a single dose of 0.2 mL was applied to a group of 10 bees in a cage (10 cages/treatment). It has been established experimentally that bees will share the 0.2 mL among themselves and receive similar doses of 20 µL per bee. The 0.2 mL consisted of 1 part of the appropriate dilution of the test substance in acetone and 19 parts of sucrose in water. Two cages of bees were treated with 0.2 mL of 20% sucrose in water containing 5% acetone as controls. When the bees had taken all the test solution, the dosage tubes were replaced with tubes containing 20% sucrose in water. In the contact toxicity test, the bees (10 bees/ replication, 10 replicates/treatment, 2 replicate/solvent control) were lightly anaesthetised with CO2 and laid ventral side up on a filter paper in a petri dish. A 1.0 µL droplet of the acetone solution with the test substance was placed on each bee's ventral surface of the thorax. The bees were then replaced in the cage and supplied with 20% sucrose in water.

After 48 hours of exposure, one bee died in the control groups for both the oral and contact toxicity test. The 48-h mortality was 28% and 31% for oral and contact toxicity tests, respectively. Based on this information, the 48-hour oral and contact LD50 values were determined to be > 100 µg/bee.

Description of key information

All available data was assessed. One chronic study and one acute study on honeybees are included here as the key studies. Another acute study on honeybees in included as a supporting study.

48-h, LC50 > 100 µg test substance/bee, Apis mellifera, mortality, UK national methods, Cole 1986

10-d, NOEC = 500 mg test substance/kg diet (corresponding to 8.2 µg test substance/bee/day), Apis mellifera, mortality, OECD TG 245, Kling 2018

Additional information

Two acute and one chronic toxicity GLP studies on honeybees (Apis Mellifera) are available for this endpoint. The chronic oral toxicity study followed OECD TG 245 guideline (Reliability 1) and was selected as a key study. The bees (10 bees/ replication and 4 replicates/concentration or controls) were fed ad libitum with a 50 % (w/v) aqueous sucrose solution containing 5 % acetone and the test substance at the concentration levels of 31.25, 62.5, 125, 250 and 500 mg test substance/kg feeding solution for 10 days. The test conditions were: temperature 31.6 - 33.4 ˚C and humidity 35.7 - 65.6%  The 10-d NOEC for mortality was determined to be 500 mg test substance/kg (corresponding to 8.20 μg test substance/bee/day) (Kling 2018).

The LD50 values in both acute toxicity studies were determined to be above the highest tested concentration. The acute toxicity study performed following United Kingdom national standard methods with acceptable restrictions is selected as a key study since a higher testing concentration (100 µg/bee) is used in this study, and the study was performed including both oral and contact exposure. In the oral toxicity test of the key study, a single dose of 0.2 mL was applied to a group of 10 bees in a cage (10 cages/treatment). In the contact toxicity test, the bees (10 bees/replication, 10 replicates/treatment, 2 replicate/solvent control) were lightly anaesthetised and then applied with a 1.0 µL droplet of the acetone solution with the test substance. The 48-hour oral and contact LD50 values were determined to be > 100 µg test substance/bee (Cole 1986, Reliability 2). Only contact toxicity was included in the supporting study. The 48-h contact LD50 was determined to be > 25 µg test substance/bee (Palmer et al., 1994, Reliability 1).