1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 31 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 226 (Predatory Mite (Hypoaspis (Geolaelaps) Aculeifer) Reproduction Test in Soil)

Version / remarks:

October 03, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil or dung: The artificial soil was prepared by mixing the dry components intensely in a laboratory mixer. Three days before test start the dry artificial soil was pre-moistened with 80 mL deionised water per 500 g dry weight artificial soil. During the application of the test item the water content was adjusted to a final water content of approximately 50% of the maximum water holding capacity by mixing 50 mL deionised water into 500 g dry weight artificial soil for the control group and 50 mL test item solution for the treatment groups.

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: soil predatory mite
- Source: The culture of the soil mite Hypoaspis aculeifer (Acari: Laelapidae) used in this test has been bred at Bayer CropScience AG since 2002. The strain was originally obtained from ECT Oekotoxikologie GmbH, Flörsheim am Main, Germany. Mites from a synchronised culture at an age of 28 days after start of egg laying were used in this study.
- Breeding: The mites were bred on a mixture of Plaster of Paris and activated charcoal and demineralised water (10:1.25:12.5 w/w). Plastic vessels (9.5 cm diameter) were filled up to a height of approximately 1 cm with this mixture and are closed with lids. The Hypoaspis aculeifer were fed with Tyrophagus putrescentiae (cheese mites) which were bred on brewer’s yeast. The breeding culture was kept at room temperature and permanent darkness
- Synchronisation of the test organisms: In order to obtain adult, female Hypoaspis aculeifer of a uniform age on 2014-02-10 2x300 adult, female Hypoaspis aculeifer were transferred to fresh breeding vessels. On 2014-02-13, after three days of egg laying, these females were removed. The Hypoaspis aculeifer hatched from the eggs were fed with Tyrophagus putrescentiae.
- Age at test initiation (mean and range, SD): 28 days after start of egg laying for three days
- Stage at test initiation: adult, fertilized, female
- Date of collection: after 14 days exposure

Study type:

laboratory study

Limit test:

no

Total exposure duration:

14 d

Remarks:

The duration of the study was 14 days for exposition to the test item at 20 ± 2 °C plus 2 days of extraction of the mites.

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

start:5.82 - 5.94
end: 5.59 - 6.69

Humidity:

start: 19.90 - 20.76% water content (46.35 - 48.87% of WHCmax)
end:20.20 - 22.14% water content (47.22 - 53.07% of WHCmax)

Photoperiod and lighting:

at start 780 lux
after 3 days 653 lux
after 7 days 654 lux
after 10 days 652 lux
after 14 days 661 lux

Details on test conditions:

TEST SYSTEM
- Test container (material, size): reusable glass vessels (Weck Mini-Sturzglas, volume 140 mL, diameter 5 cm at the bottom, height 7 cm); covered with glass lids to prevent Hypoaspis aculeifer from escaping but allowing aeration during the test period
- Amount of soil or dung or substrate: 20 g dry weight artificial soil
- Depth of dung (cm): approximately 1.5 cm
- No. of organisms per container (treatment): 10 (all adult, fertilzed, female)
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- one additional vessel for each application rate for measurement of pH value and moisture of the artificial soil at the end of the test not loaded with Hypoaspis aculeifer.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil or dung)
- Composition (if artificial substrate) (percentage distribution on dry weight basis):
75% fine quartz sand (sort F 36, particle size 0.2 – 0.05 mm = 91.35%)
5% Sphagnum peat, air dried and finely ground
20% Kaolin clay (content of Kaolinite: = 30.2%)
Calcium carbonate (CaCO3) for the adjustment to pH to 6.0 ± 0.5
- Maximum water holding capacity (in % dry weight): 53.60 g water/100 g dry weight artificial soil

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness photo period
- Light intensity: 400 - 800 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): For the determination of mortality and reproduction surviving adults and living juveniles were extracted and counted after 14 days exposure


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Nominal and measured concentrations:

control, 100, 178, 316, 562, and 1000 mg/kg dw (nominal)
control, 18.5 32.9, 58.5, 104 and 185 mg a.i. mg/kg dw (calculated)

Reference substance (positive control):

yes

Remarks:

Dimethoate

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 185 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: In the control group 2.9% of the adult Hypoaspis aculeifer died which is below the allowed maximum of ≤ 20 % mortality. Mortality between 2.5 and 10.0 % occurred in the test item groups.
- No. of offspring produced: see Table 2 under "Any other information on material and methods incl. tables"

Results with reference substance (positive control):

The toxic standard reference test, with the reference test item mixed into the artificial soil, was performed in 2014 (Report No.LAR/HR-14/14, 2014-03-11; NON-GLP)
- Results with reference substance valid? yes
- Relevant effect levels: LC50 of 3.51 mg a.s./kg (95 % confidence limits from 3.46 mg a.s./kg to 3.57 mg a.s./kg) for mortality of the adult mites. NOEC, LOEC, and EC50 for reproduction were calculated to be 3.2, 5.6, and 5.28 mg a.s./kg dry weight artificial soil (95% confidence limits from 4.02 mg a. s./kg to 6.47 mg a. s./kg), respectively. The results of the reference test indicated that the test system was sensitive to the reference test item.

Reported statistics and error estimates:

The calculation of mean, SD and % mortality of the control and treatment groups with Excel sheets (Microsoft Excel 2010). The software used to perform the statistical analysis was ToxRat Professional 2.10 released February 20, 2010, (Ratte, 2010; ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf).
For reproduction: For the determination of normal distribution and homogeneity of variance Shapiro-Wilk’s Test and Levene’s Test (alpha = 0.05), respectively were used. After transformation to y`=a.ln(y+1) were data of reproduction normally distributed and homogeneity of variances was given. Therefore William's-t test (one-sided-smaller, alpha = 0.05) was used to determine NOEC and LOEC values. The EC10, 20 values could not be determined due to mathematical reasons or inappropriate data.

Table 1: Survival of adult, female Hypoaspis aculeifer after 14 days

Adults/vessel	Control	mg test item/kg dry weight artificial soil
		100	178	316	562	1000
Replicate 1	10	10	11*	9	10	10
Replicate 2	10	10	10	10	10	8
Replicate 3	9	10	10	10	9	10
Replicate 4	10	9	9	10	10	8
Replicate 5	10	--	--	--	--	--
Replicate 6	12*	--	--	--	--	--
Replicate 7	10	--	--	--	--	--
Replicate 8	9	--	--	--	--	--
Mean	9.7	9.8	9.7	9.8	9.8	9.0
Standard Deviation	0.5	0.5	0.6	0.5	0.5	1.2
Coefficient of Variation	5.0	5.1	6.0	5.1	5.1	12.8
% Mortality	2.9	2.5	3.3	2.5	2.5	10.0

* replicates were excluded from all calculations since more than 10 adults were found

 

Table 2. Reproduction of Hypoaspis aculeifer 14 days after test start (juveniles/replicate)

Adults/vessel	Control	mg test item/kg dry weight artificial soil
		100	178	316	562	1000
Replicate 1	298	285	319*	222	287	294
Replicate 2	307	319	273	313	304	222
Replicate 3	234	289	272	291	325	299
Replicate 4	294	287	296	362	331	245
Replicate 5	295	--	--	--	--	--
Replicate 6	371*	--	--	--	--	--
Replicate 7	288	--	--	--	--	--
Replicate 8	282	--	--	--	--	--
Mean	285.4	295.0	280.3	297.0	311.8	265.0
Standard Deviation	24.0	16.1	13.6	58.1	20.2	37.6
Coefficient of Variation	8.4	5.5	4.8	19.6	6.5	14.2
% of control	100	103.4	98.2	104.1	109.2	92.8
Statistics (*)		-	-	-	-	-

(*)=William's t-test one sided smaller; α=0.05; “-“: non-significant; “+”: significant.

* replicates were excluded from all calculations since more than 10 adults were found

Table 3: Overall findings

mg test item/Kg dry weight artificial soil	% mortality (Adults)	Mean number of juveniles per test vessel ± standard dev.	Reproduction (% of control)	Significance (*)
Control	2.9	285.4 ± 24.0	--
100	2.5	295.0 ± 16.1	103.4	-
178	3.3	280.3 ± 13.6	98.2	-
316	2.5	297.0 ± 58.1	104.1	-
562	2.5	311.8 ± 20.2	109.2	-
1000	10.0	265.0 ± 37.6	92.8	-

Table 4: Validity criteria for OECD 226

Criterion from the guideline	Outcome	Validity criterion fulfilled
Mean adult female mortality should not exceed 20% at the end of the test	2.9%	yes
The mean number of juveniles per replicate (with 10 adult females introduced) should be at least 50 at the end of the test;	285.4	yes
The coefficient of variation calculated for the number of juvenile mites per replicate should not be higher than 30% at the end of the definitive test	8.4%	yes

 

Validity criteria fulfilled:

yes

Remarks:

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

Reference 2

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Mar - 14 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of test material application: The test item was mixed into 250 mL deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions (250 mL) for application.
- Volume of test solution applied: 50 mL test item solution was mixed into 500 g artificial soil dry weight with a laboratory mixer. Approximately 30 g (wet weight) of the test substrate was filled in each test vessel avoiding compression.
- Controls: deionized water only

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: white rat springtail
- Source: bred at Bayer AG , BAG-CS-EnSa-Testing since January 2012, originally obtained from Ibacon, Institute for Analytic and Consulting GmbH, Rossdorf, Germany.
- Age at test initiation: 10 -12 days
- Breeding conditions: breeding vessels: plastic vessels (9.5 cm diameter), breeding substrate: Plaster of Paris, activated charcoaland demineralised water (10:1.25:12.5 w/w) filled up to 1 cm, temperature: room temperature, Light cycle: permanent dark, feeding: once a week with bakers dry yeast
Synchronisation of the test organisms: Twelve days before starting the study, egg clusters from the breeding containers were transferred to fresh breeding substrate. After 2 days the egg clusters were removed and the remaining collembolans hatched from the eggs were fed with granulated dry yeast.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

pH (if soil or dung study):

1st test run: 5.82 - 5.94
2nd test run: 5.77 - 6.14

Humidity:

1st test run: 18.92% - 20.98% (water content)
2nd test run: 20.50% - 22.09% (water content)

Photoperiod and lighting:

Photoperiod: 16 h light and 8 h dark (in both test runs)
Light intensity: 1st test run: 589 - 673 lux, 2nd test run: 603 - 638 lux

Details on test conditions:

Test conditions were identical in both test runs

TEST SYSTEM
- Test container: glass vessels (volume 140 mL, diameter 5 cm) covered with glass lids
- Amount of soil or dung or substrate: 30 ± 1 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Feeding regime: 2 - 10 mg granulated dry yeast (day 0 and 14) per test vessel

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: according to OECD guideline 232, sphagnum peat (5%), kaolin clay (20%), fine quartz sand (75%), CaCO3 for the adjustment to pH to 6.0 ± 0.5
- Preparation: components were mixed in a laboratory mixer. Three days before test start dry artificial soil was pre-moistened with 80 mL deionized water per 500 g dry weight artificial soil. During the application of the test item the water content was adjusted to a final water content of approximately 50 % of the maximum water holding capacity.
- Maximum water holding capacity: 50%

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED:
- adult mortality (day 28), number of living young (day 28)
- Moisture content (calculated from the weight difference before and after drying of soil)
- Method evaluation: After 28 days, the soil of each replicate was transferred into a plastic vessel and stirred up with 80 mL of deionized water in order to drift the collembolans to the surface. The water was coloured with 10 mL black
ink in order to increase the contrast between the water and the white collembolans. From each vessel a digital image was taken and checked. The adult collembolans were visually counted and marked on the digital image, followed by confirmation of the automatically generated juvenile count.

Nominal and measured concentrations:

1st test run: control, 100, 178, 316, 562, 1000 mg/kg dw (nominal)
1st test run control, 18.5 32.9, 58.5, 104 and 185 mg a.i. mg/kg dw (calculated)
2nd test run: control, 18, 32, 56, 100, 178, 316, 562, 1000 mg/kg dw (nominal)
2nd test run: 3.33, 5.92, 10.4, 18.5 32.9, 58.5, 104 and 185 mg a.i. mg/kg dw (calculated)

Reference substance (positive control):

yes

Remarks:

Boric acid: 44, 67, 100, 150, 225 mg/kg dw

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

316 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

58.46 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

562 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

In the control group 7.5 % (1st run) and 5.0 % (2nd run) of the adult Folsomia candida died. Mortality in the treatment groups was between 5% and 10% in the 1st test run and between 7.5% and 17.5% in the 2nd test run.

Results with reference substance (positive control):

EC50 (reproduction) = 90 mg test item/kg artificial soil dw (95 %-CI: 68 mg - 119 mg /kg artificial soil dw)
NOEC (reproduction) = < 44 mg/kg artificial soil dw
LOEC (reproduction) = 44 mg/kg artificial soil dw

Table 1. Survival of adult collembolans after 4 weeks treatment (n=10/replicate)

1st run:

mg test item/kg artificial soil dry weight Replicates1) Control 100 178 316 562 1000 1 10 9 10 9 10 10 2 9 9 10 10 10 8 3 9 9 9 7 8 10 4 8 9 9 9 9 9 5 9           6 10           7 10           8 9                         Mean2) 9.3 9.0 9.5 8.8 9.3 9.3 SD2) 0.7 0.0 0.6 1.3 1.0 1.0 % Mortality3) 7.5 10.0 5.0 12.5 7.5 7.5

2nd run:

	mg test item/kg artificial soil dry weight
Replicates1)	Control	18	32	56	100	178	316	562	1000
1	10	10	10	8	9	8	8	9	8
2	10	7	9	10	9	10	6	10	11*
3	10	10	8	10	7	9	10	7	8
4	9	9	7	9	9	9	9	9	9
5	10
6	9
7	10
8	8
Mean2)	9.5	9.0	8.5	9.3	8.5	9.0	8.3	8.8	8.3
SD2)	0.8	1.4	1.3	1.0	1.0	0.8	1.7	1.3	0.6
% Mortality3)	5.0	10.0	15.0	7.5	15.0	10.0	17.5	12.5	16.7

Calculations were done with un-rounded values.

1surviving adult/replicate, each vessel contained a total of 10 collembolans at the test start

2mean and standard deviation (SD) of 8 replicates of the control group and 4 replicates for the treatment groups

3formula: ((initial placed organisms per vessel – mean of surviving adults per vessel) / 10) * 100

*11 instead of 10 collembolans inserted by start of the study, replicate excluded from further calculations

Table 2. Reproduction of the collembolans after 4 weeks treatment (juveniles/replicate)

1strun:

	mg test item/kg artificial soil dry weight
Replicates1)	Control	100	178	316	562	1000
1	1536	1201	1512	1449	1329	1367
2	1453	1362	1252	1298	1209	1246
3	1480	1541	1408	1430	1155	1174
4	1802	1511	1574	1565	1255	1106
5	1777
6	1484
7	1528
8	1660
Mean2)	1590.0	1403.8	1436.5	1435.5	1237.0	1223.3
SD2)	138.2	156.2	140.8	109.4	73.7	111.6
CV3)	8.7
% of Control 4)		88.3	90.3	90.3	77.8	76.9

2ndrun:

	mg test item/kg artificial soil dry weight
Replicates1)	Control	18	32	56	100	178	316	562	1000
1	1518	1272	1459	1202	1206	1155	1195	1362	1263
2	1406	1088	1269	1287	1118	1462	1147	1245	1619*
3	1463	1675	1229	1211	1034	1326	1569	1085	1353
4	1140	1398	1445	1244	1175	1384	1298	1552	1369
5	1602
6	1542
7	1300
8	1166
Mean2)	1392.1	1358.3	1350.5	1236.0	1133.3	1331.8	1302.3	1311.0	1328.3
SD2)	173.5	246.6	118.5	38.5	75.5	130.3	188.7	196.7	57.1
CV3)	12.5
% of Control 4)		97.6	97.0	88.8	81.4	95.7	93.5	94.2	95.4

Calculations were done with un-rounded values

1juveniles produced per test vessel, each test vessel contained a total of 10 collembolans at the test start

2mean and standard deviation (SD) of 8 replicates of the control group and 4 replicates for the treatment groups

3Coefficient of Variation

4formula: mean number of juveniles per treatment group * 100 / mean number of juveniles per control group

Table 3: Validity criteria for OECD 232

	Outcome 1st run	Validity criterion fulfilled	Outcome 2nd run	Validity criterion fulfilled
Mean adult mortality should not exceed 20% at the end of the test.	7.5%	yes	5.0%	yes
The mean number of juveniles per vessel should be at least 100 at the end of the test.	1590	yes	1392	yes
The coefficient of variation calculated for the number of juveniles should be less than 30% at the end of the definitive test.	8.7%	yes	12.5%	yes

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

NOEC (28 d, reproduction) = 58.46 mg test a.i./kg soil dw nominal (Folsomia candida, OECD 232)

NOEC (14 d, reproduction) ≥ 185 mg a.i./kg soil dw nominal (Hypoaspis aculeifer, OECD 226)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

58.46 mg/kg soil dw

Additional information

Two reproduction tests on terrestrial arthropods according to GLP are available. One test was performed with Folsomia candida (M-498367-01-1) and the second study tested the effects of the substance to Hypoaspis aculeifer (M489573-01-1).

The first study was conducted according to OECD 226 with adult soil mite Hypoaspis aculeifer exposed to nominal concentrations from 100 to 1000 mg test item/kg soil dw (corresponding to 18.5 to 185 mg a.i. mg/kg dw). After a period of 14 days, the surviving adults and the living juveniles were collected and counted. Mortality rates were between 2.5 and 10.0 % in all exposure groups. Concerning the number of juveniles, no significant difference between control and the treatment groups was observed . Therefore, the NOEC for reproduction is ≥ 185 mg a.i./kg soil dw.

In the second study conducted according to OECD 232, 10-12 days old springtails Folsomia candida were exposed to test item concentrations between 100 and 1000 mg/kg soil dw (corresponding to 18.5 to 185 mg a.i. mg/kg dw) for 28 days. Statistically significant differences were observed between the control and all treatment groups. The significant effects on reproduction at 104 and 185 mg/kg soil dw (22.2% and 23.1%) can be considered as biologically relevant while the observed statistically significant differences at 58.5, 104 and 185 mg a.i. mg/kg dw were very low and should not be regarded as biologically relevant. Furthermore, no dose-response relationship was observed. Therefore, a second test run was started, testing the same test concentrations and at the same time 3 lower test concentrations ranging from 18 and 1000 mg/kg soil dw (corresponding to 3.33 to 185 mg a.i. mg/kg dw). No significant difference between control and any test item treatment group in the second test run. Therefore, the overall NOEC for reproduction is reported as 58.5 mg a.i./kg soil dw.

 

Furthermore, five laboratory studies are available on the toxicity of the test item towards bees and bumblebees according to GLP (M-441758-01-1, M-548937-01-2, M-483352-01-3, M-491456-01-1, M-571449-01-1). Since additional studies are not entered as robust study summaries, a more detailed summary is supplied in this section. Three studies were performed with honey bee (Apis mellifera) and two studies with bumblebee (Bombus terrestris). The studies were performed according to the OECD guidelines OECD 213, OECD 214 and US EPA OCSPP 850.

In the first study (M-441758-01-1) effects on mortality of honeybees were determined after oral or contact exposure. 30 worker bees (Apis mellifera) per treatment level were exposed for 96 hours to doses of 2.0, 1.0, 0.50, 0.25, 0.13 and 0.063 µg a.s./bee by topical application (contact dose response test) and 30 worker bees per treatment level were exposed for 72 hours to doses of 0.14, 0.069, 0.031, 0.020, 0.012 and 0.0067 µg a.s./bee by feeding (oral dose response test, value based on the actual intake of the test item). The contact test was prolonged for further 48 hours up to 96 hours and the oral test was prolonged for further 24 hours up to 72 hours due to increasing mortality between 24/48 (contact and oral) and 48/72 (contact) hours. In addition, negative controls [oral: a) 50% aqueous sugar syrup solution and b) 50% sugar syrup solution with acetone; contact test: a) tap water + 0.5% Adhaesit and b) acetone] and a toxic reference (dimethoate; 400 g/L nominal) at nominal dose rates of 0.30, 0.20, 0.15 and 0.10 µg dimethoate/bee (contact test) and 0.30, 0.15, 0.08 and 0.05 µg dimethoate/bee (oral test) were tested. The contact LD50 values (48, 72 + 96 h) of the test item were 1.25, 0.46 and 0.41 µg a.s./bee, respectively. Over the entire time of the test (96 hours) behavioural abnormalities (e.g. movement coordination problems and/or apathy) were observed in the 2.0, 1.0, 0.50 and 0.25 µg a.s./bee dose level groups (except after 96 hours in the 2.0 µg a.s./bee dose level). There was 3.3% mortality in the control group (water + 0.5% Adhaesit) and the solvent control group, respectively. The oral LD50 values (24 h, 48 h + 72 h) of the test item were 0.012, 0.010 and 0.010 µg a.s./bee, respectively. During the 4 hours and 24 hours assessments apathy was observed in all treatment groups. Thereafter no more test item related behavioural abnormalities were found until the end of the test. No mortality occurred in the water and solvent control groups, respectively.

 

For the second study (M-548937-01-2) the acute oral toxicity of the test item to the bumblebee (Bombus terrestris) was tested. Under laboratory conditions 30 worker bumblebees (Bombus terrestris) per treatment level were exposed for 48 hours to doses of 0.19, 0.10, 0.05, 0.03 and 0.013 µg a.s./bumblebee by feeding (oral dose-response test, value based on the actual intake of the test item). In addition, negative controls (water control: 50% (w/v) aqueous sucrose solution, 500 g sucrose/L tap water; solvent control: 50% w/v sucrose solution with 5% acetone and 1% Tween80) and a toxic reference item (dimethoate) at an actual dose rate of 2.5 µg dimethoate/bumblebee were tested. The 72 h oral LD50 value was 0.04 µg a.s./bumblebee. The 72 h oral NOED value was 0.013 µg a.s./bumblebee.

 

For the third study (M-483352-01-3) the effects of the test item on the bumble bee (Bombus terrestris L.) after contact exposure was determined. 30 young adult worker bumble bees per treatment group were exposed for 96 hours to doses of 6.25, 12.5, 25, 50 and 100 µg a.s./bumble bee, respectively, by topical application (the unit µg a.s./bumble bee refers to the analysed content of test item, 89.6% w/w). In addition, two negative controls, a water control and a solvent control (acetone), and a toxic reference (Perfekthion EC containing active substance dimethoate: 411.7 g/L analysed) were tested. The toxic reference test dose was 11 µg dimethoate/bumble bee. Mortality and sub-lethal effects were assessed 24, 48, 72 and 96 hours after treatment. In both control groups no mortality was observed during the 96 h test period. In the reference item group, mortality was ≥ 50% at the end of the test. During the 96 hour observation period, dead, affected, apathetic or moribund bumble bees were observed at the dose levels from 12.5 µg to 100 µg a.s./bumble bee. No remarkable effects were observed at the lowest tested dose level. A mortality rate of 96.7% was observed at the highest dose level of 100 µg a.s./bumble bee at the final assessment after 96 hours. After the same period of time, no mortality was observed at the tested dose level of 6.25 µg a.s./bumble bee. The 96 h contact LD50 value for the test item was determined to be 21.9 µg a.s./bumble bee. The 96 h NOED for mortality was determined to be 6.25 µg a.s./bumble bee.

 

For the fourth study (M-491456-01-1) the effects of the test item on honey bee larvae, Apis mellifera carnica, after a single exposure (feeding) event, using spiked diet on day +4 in an in vitro laboratory testing design were determined. The test item was incorporated into the artificial diet in order to determine, the No Observed Effect Dose (NOED) as well as the LD50 value. As reference item, dimethoate (tech.) was used. As assessment endpoint mortality of the honey bee larvae was recorded during the test period from day +4 until day +7, and additionally on day +8. The purpose of the analytical part of this study was to quantify the concentration of the test item in spiked exposure diets, which were used to feed the larvae. This oral toxicity test was performed as a limit test with a single exposure in an in vitro laboratory testing design. Synchronised first instar larvae of Apis mellifera carnica, from three different honey bee colonies, each representing a replicate, were tested in an in vitro laboratory testing design, according to the OECD Draft Test Guideline on Honey Bee (Apis mellifera) Larval Toxicity Test and the current draft version of the Post–WNT25. At day +1 (day 0 was the anticipated day of larval hatching), first instar bee larvae (Apis mellifera carnica) were transferred from their bee hive into an artificial in vitro testing system. The bee larvae were fed with standardised amounts of artificial diet at day +1, +3, +4, +5 and +6. On day +4, the artificial diet was treated according to the respective test groups. In the test item treatment groups, the test item was incorporated into the artificial diet at the nominal test doses of 1.1, 3.3, 10, 30 and 90 ng a.s./larva, corresponding to the nominal test concentrations of 34, 101, 303, 909 and 2727 µg a.s./kg diet, respectively. In the reference item treatment group, dimethoate was incorporated into the artificial diet at a nominal dose of 8.8 µg a.s./larva, corresponding to 266.7 mg a.s./kg diet. In the control groups, water and acetone was incorporated into the artificial diet. The honey bee larvae were incubated at about 35°C during their development. The relative humidity inside the incubator was on average 95 ± 5% from day +1 to +8. Mortality was determined on day + 5, +6, +7 (according to the study plan), and additionally on day +8 (by amendment). Dead test animals were discarded for sanitary reasons. The determination of residues of the test item in the stock solution was performed according to analytical method 01402, using reversed phase High Performance Liquid Chromatography (HPLC) coupled with electrospray and mass spectrometry (MS/MS). The amount of the test item in the stock solution was determined for the test item groups. The actual concentration of the test item in the stock solution was 103% of the nominal concentration. The NOED for the larval mortality was determined to be 3.3 ng a.s./larva (based on nominal). The LOED for the larval mortality was determined to be 10 ng a.s./larva (based on nominal). The LD50 for the larval mortality was determined to be 17.2 ng a.s./larva until day +7 and 12.8 ng a.s./larva until day +8. All validity criteria were met.

 

For the fifth study (M-571449-01-1) potential adverse effects of the test item on the development of honey bee larvae (Apis mellifera) to adulthood in an in vitro oral toxicity test over 22 days were assessed. The study was performed as dose-response test according to the OECD Draft Guidance Document for Testing Chemicals – Honey Bee (Apis mellifera) Larval Toxicity Test, Repeated Exposure (Revision following the April 2015 meeting, 20 July 2015), with modifications. At day +1 (day 0 was the anticipated day of larval hatching), first instar bee larvae (Apis mellifera) were transferred from their bee hive into an artificial in vitro testing system. The bee larvae were fed with standardized amounts of untreated artificial diet at day +1. The test was performed as a dose response test with a repeated exposure (day +3 to day +6) to artificial diets with nominal test concentrations of 2.4, 7.2, 21.6, 64.9 and 194.8 ng a.s. test item/g diet, resulting in total nominal feeding doses of 0.37, 1.1, 3.3, 10 and 30 ng a.s. test item/larva (cumulative, based on the nominal sum of four feeding days (day +3, day +4, day +5 and day +6)). In the reference item group bee larvae were fed from day +3 to day +6 to artificial diets with a nominal test concentration of 48 µg a.s. dimethoate/g diet, resulting in a total feeding dose of 7.4 µg a.s. dimethoate/larva (cumulative, based on the nominal sum of four feeding days (day +3, day +4, day +5 and day +6)). In the control group and solvent control group, water and acetone respectively were incorporated into the artificial diets. Mortalities and other abnormal effects were determined on day + 4, +5, +6, +7, +8, +15 and +22. Dead test animals were discarded for sanitary reason. Assessment endpoint was the emergence of adult honey bees until day 22. During the development of the honey bees from day +1 to +22, they were incubated on average about 34 to 35 °C. From day +1 to +8, the relative humidity inside the incubator was on average about 95 ± 5% and from day +8 to +22 the mean relative humidity was about 80 ± 5%. Overall, it can be concluded that the No Observed Effect Dose (NOED) on day +22, determined in this in vitro honey bee larvae study is 10 ng a.s. test item/larva and the Lowest Observed Effect Dose (LOED) is 30 ng a.s. test item/larva. Furthermore, 12.09 ng a.s. test item/larva was determined as LD50 value.