Mecoprop

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see the read-across justification report in Section 13 of the dossier.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2.636 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 2.092 - 3.322

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.463 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LD50

Effect conc.:

89.4 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 70.9 - 112.7

Duration:

72 h

Dose descriptor:

NOED

Effect conc.:

49.6 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LD10

Effect conc.:

43.7 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 11.4 - 61.5

Duration:

72 h

Dose descriptor:

other: LD20

Effect conc.:

55.7 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 %CL: 21.5 - 72.8

Duration:

72 h

Dose descriptor:

LC10

Effect conc.:

1.29 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 0.336 - 1.814

Duration:

72 h

Dose descriptor:

other: LC20

Effect conc.:

1.641 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CL 0.635 - 2.148

Conclusions:

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Executive summary:

The effect of the test material on the honey bee was assessed in an acute toxicity test according to OECD Test Guideline 237 and in compliance with GLP.

Honeybee (Apis mellifera L.) larvae were exposed to the test material in an acute toxicity test. The toxicity of the test material was determined at doses of 99.2, 49.6, 24.8, 12.4 and 6.2 μg a.s./larva (after correction for purity, corresponding to 107.1, 53.5, 26.8, 13.4 and 6.7 μg test material/larva). The concentrations in the diet were 2.925, 1.463, 0.731, 0.366 and 0.183 g a.s./kg food.

Additionally, honeybee larvae were treated with Dimethoate technial as the toxic reference item at a concentration of 8.8 μg dimethoate/larva. The study design also included a negative control group with an untreated diet and a solvent control group that received the same diet amended with acetone at the same concentration at which it was used in the test material treatments.

After 72 hours of oral exposure, 0.0 % mortality was observed in the solvent-free control (AC), while in BC (untreated diet C with 2 % v/v acetone) 5.6 % mortality occurred. In the test material group, mortalities ranged between 0.0 and 61.1 % after 72 hours. Statistically significant mortality occurred at the highest test material dose (99.2 μg a.s./larva). Mortality in the dimethoate toxic reference treatment (AR) was above 50 % across all replicates (D7/72 h), being 55.6 %.

Other observations such as smaller body size of surviving larvae or/and remaining food on D7 occurred at an increased rate (52.4 % and 16.7 % at the two highest test material groups AT and BT (99.2 and 49.6 μg a.s./larva). In the lower test material groups the rate of remaining food/smaller body size was below or at the level of the solvent control.

The concentration of active substance in the primary test material dosing stock solution A was 101 % of the nominal concentration and therefore within acceptable parameters as defined in the study plan. No test material has been detected in the control specimen.

Because control mortality was ≤ 15 %, corrected mortality in the reference item dose of 8.8 μg a.s./larva was ≥ 50 % and the analytical verification of active substance concentration in the test material stock solution displayed that it was within ± 20 % of the nominal concentration the study can be regarded as valid.

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

 

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Reference 2

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see the read-across justification report in Section 13 of the dossier.

Reason / purpose for cross-reference:

read-across source

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

> 0.714 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

0.357 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

NOED

Effect conc.:

50 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

0.714 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

EC10

Effect conc.:

0.214 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidnce limits 0.061 - 0.367 mg a.i./mL diet.

Duration:

4 d

Dose descriptor:

other: EC20

Effect conc.:

0.429 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 0.194 - 0.663 mg a.i./mL diet.

Duration:

4 d

Dose descriptor:

other: LOED

Effect conc.:

100 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

ED10

Effect conc.:

29.98 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 8.578 - 51.38 μg a.i./bee.

Duration:

4 d

Dose descriptor:

other: ED20

Effect conc.:

59.96 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 27.18 - 92.73 μg a.i./bee.

Conclusions:

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.


Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Executive summary:

The chronic toxicity of the test material when administered to the honey bee (Apis mellifera) during the larval stage I was assessed according to accordance with the OECD Guidance Document 239 under GLP conditions.

Larvae from three colonies were transferred (grafted) to well plates containing untreated artificial diet and held in an incubator. Starting at two days after grafting, artificial diets containing the test material or control substance were provided to larvae. Test diets were provided for a total of four days. Larvae were then held for up to 14 days after the completion of dosing in order to allow emergence of adult bees. A negative control, solvent control and positive control group were maintained concurrently. Each treatment and control group contained 16 larvae from each of the three hives, for a total of 48 larvae per treatment group. Nominal test levels were selected based upon known toxicity data and were 100, 50.0, 25.0, 12.5 and 6.25 μg a.i./bee.

In order to control bias, larvae were impartially distributed to the treatment and control groups. No other potential sources of bias were expected to affect the results of the study. Dosing of larvae with the test material in artificial diet simulates the possible exposure of larval bees in the hive. The no-observed-effect-concentration/dose (NOEC/D) and EC/D10, 20, 50 values were based on adult emergence and weight at test termination.

By day 8, all living larvae had consumed their entire allotted diets. No significant differences between the negative and solvent control means for either mortality or body weight were detected, therefore all statistical comparisons were made to the negative control. Mean mortality in the treatment groups ranged from 14.6 to 43.8 %, and the highest level (100 μg a.i/bee) was significantly different. Reductions in mean body weight relative to the control ranged from 2 to 12 %, and no significant trend of decreasing body weight was detected.

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.

 

 

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.

Reference 3

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2014 to 27 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 237 (Honey bee (Apis mellifera) Larval Toxicity Test, Single Exposure)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Analytical monitoring:

yes

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: The test/reference material was mixed into sterile filtered aqueous sugar solution (Stock A). It was necessary to use acetone in order to dissolve the test material. Several dilutions were prepared by adding further sugar solution (Stock B, C and so on).
Samples of the test material stock A and of the control were taken in duplicate and handed over to the Principal Investigator (PI) for chemical analysis. In order to increase the stability of the active substance, 0.1 % v/v formic acid was added to each specimen before forwarding it to the PI.
Thereafter the royal jelly was added to each stock solution at a ratio of 1:1, based on (w/w), to reach the final test concentrations (AT, BT, CT and so on). Larvae of the negative control treatment received untreated diet C and larvae of the solvent control group were fed with diet C amended with 2 % v/v acetone, corresponding to the solvent concentration present in the diets containing the test material.
Before feeding the final test/reference material sugar solutions were warmed up to 35 °C in a water bath. The larvae were fed with a defined quantity of the respective test material concentration (concentration series).
This test was performed by way of single larva feeding according the scheme described under point “Feeding scheme”. The order of application was the following: Control, test material (from the lowest to the highest concentration) and finally reference material.
After consumption of test/reference material containing diet applied on D4, larvae of all treatment groups were fed with untreated diet C on D5 and D6.
- Chemical name of vehicle: Acetone

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM : Apis mellifera carnica P.
- Common name: Honey bee
- Source: For each test larvae were collected from three different colonies, each representing a replicate in order to avoid genetic influences on the test outcome.
- Age at test initiation: First instar larvae (L1 during grafting) of queen-right colonies maintained at the test facility in good health condition were used for the test.
- Disease free: Yes
- Kept according to standard practices: Yes
- Health condition of the hive: All larvae used in the test derived from healthy (free of clinical symptoms of any disease) and queen-right bee colonies. The larvae were taken from hives that had not received treatments with chemical substances for at least one month. Before application all sick or dead larvae were exchanged for normally developed individuals originating from the respective colony. All plates used in the study were randomised using a scheme, which is added to the raw data. Thereafter the plates were denoted with study number, treatment group and replicate number. Hence it can be assumed that the study was conducted with healthy and unbiased larvae.

ACCLIMATION
- Acclimation period: The bee colonies producing the larvae were held under field conditions in hives including a healthy queen. Brood in egg, larval and pupal stages as well as filled food combs (containing nectar and pollen) were present. A sufficient amount of food was present in the bee hives.
- Acclimation conditions: Climatic chamber (Type: Binder KBF 720), 34.0 – 35.0 °C (according to study plan: 34 – 35 °C), RH 93 – 96 % (according to study plan: around 95 %), Illumination: Constant darkness throughout the test (diffuse artificial light only during handling and assessments); Ventilation: By the air-conditioning equipment of the climatic chamber.
- Feeding: The aqueous sugar solutions as one component of the artificial diets were prepared freshly on the day of their first use and in case of diet C stored in a fridge (below 10 °C) for further use on D5 and D6. The sugar solution was mixed with royal jelly every day before each feeding occasion. Each larva was fed separately using a sterile pipette. The food drop was placed next to the larvae to avoid drowning. Before feeding the final diets were warmed up to about 35°C. During the process the culture plate in operation was placed on a warming plate (at 35 °C).
- Health during acclimation: None specified.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

72 h

Remarks:

Pre-grafting (in vivo): D3 - D0; Grafting: D1; Pre-exposure (in vitro) D1 - D3, Application D4; Post-exposure (in vitro): D5 - D7.

Test temperature:

34.0 – 35.0 °C

Humidity:

93 – 96 %

Photoperiod and lighting:

Constant darkness throughout the test (diffuse artificial light only during handling and assessments).

Details on test conditions:

TEST SYSTEM
- Test container / cage: Crystal polystyrene grafting cells (CNE Nicoplast, internal diameter 9 mm) were placed in 48 well plates. The well plates were filled up to 1/3 with a piece of dental roll. The grafting cells were placed on the wetted and disinfected dental rolls. Well plates were labelled with study number, treatment group and replicate number, placed on an adjustable warming plate (stretching table) and warmed up to 35 °C. Artificial diet A was pipetted into the grafting cells, followed by placing one freshly grafted larva per cell.
- Number of larvae/replicate: 12
- Number of replicates:
3 replicates of each control
3 replicates of each test material dosage
3 replicates of the reference item dose
All replicates of each dosage were placed on one culture plate.
- Number of concentrations (Dose-response test): Control: 2; Test material treatment: 5; Reference material treatment: 1
- Completion of the test: After the last assessment on D7 the culture plates with all organisms participating in the test were placed in a freezer (at -18°C).

EGG CULTURE CONDITIONS
- Method of producing L1 larvae: Each of the three colonies used in the test was treated parallel in the same way: On day -3 (D-3) the respective queen of the colony was confined on an empty brood comb, which was enclosed in an excluder cage and thereafter placed in the hive. The queen laid her eggs solely on this comb. The caging time was approx. 30 h. In the afternoon of day -2 (D-2) the queen was released from the excluder cage. The comb was checked for the presence of freshly laid eggs, was confined in the excluder again in order to prevent any further access and egg laying by the queen, and was placed near to frames containing open brood in the hive. The eggs were incubated within the hive between day -2 (D-2) and day 1 (D1).
- Grafting: On D1 the combs containing larvae were transported from the hives to an acclimatised laboratory room using a polystyrene box. Larvae were transferred from the combs to the cells using a suitable grafting tool (e.g. grafting needle Swiss type). During grafting the C-shaped larvae were placed on the surface of the artificial diet within the grafting cells. Each replicate represents larvae originating from a different colony to exclude colony effects. The grafting was performed on a warming plate maintained at 34 to 35 °C.

INCUBATION CONDITIONS
- Temperature : 34.0 – 35.0 °C
- Relative humidity : 93 – 96 %

EFFECT PARAMETERS MEASURED:
- Mortality: Number of dead larvae (immobile larvae or ones which did not react to a contact stimulus were noted as dead) daily on D5 to D7 (24, 48 and 72 h after application).
- Morphological differences: Compared to the control on D7 (72 h after application).
- Presence of unconsumed food: Qualitatively described on D7.

FOOD CONSUMPTION
- Amount of treated diet consumed per group: After consumption of test/reference material containing diet applied on D4, larvae of all treatment groups were fed with untreated diet C on D5 and D6.

VEHICLE CONTROL PERFORMED: Yes

SANITARY METHODS
- Dental rolls were wetted with 15 % glycerol and a disinfecting agent (Milton sterilising fluid). Grafting cells were disinfected (in a 70 % ethanol bath for approximately 30 minutes) followed by drying of the cells under laminar-flow. Only sterile equipment was used (e.g. culture well plates from Nunclon™ and Eppendorf Biopur one-way pipette tips). Before and during the transfer of larvae grafting tools were disinfected in 70 % ethanol. Sugar solution was sterile filtered at 0.22 μm with a syringe filter before mixing with a proprietary royal jelly obtained from a certified source. The Plexiglass desiccator was cleaned with 70 % ethanol. Laboratory surfaces were disinfected with a ready to use disinfectant for medical devices (Bacillol AF). During grafting personnel used surgical masks. After each application, glass flasks and beakers used were cleaned in the dishwasher.

TEST CONCENTRATIONS
- Range finding study: A preliminary rangefinder test was performed under non-GLP conditions to determine the definitive dosages, whose results are not reported in this Final Report.

Nominal and measured concentrations:

The toxicity of the test material was determined at doses of 99.2, 49.6, 24.8, 12.4 and 6.2 μg a.s./larva (after correction for purity, corresponding to 107.1, 53.5, 26.8, 13.4 and 6.7 μg test material/larva). The concentrations in the diet were 2.925, 1.463, 0.731, 0.366 and 0.183 g a.s./kg food.

Reference substance (positive control):

yes

Remarks:

Dimethoate

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2.636 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 2.092 - 3.322

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.463 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LD50

Effect conc.:

89.4 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 70.9 - 112.7

Duration:

72 h

Dose descriptor:

NOED

Effect conc.:

49.6 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

72 h

Dose descriptor:

LD10

Effect conc.:

43.7 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 11.4 - 61.5

Duration:

72 h

Dose descriptor:

other: LD20

Effect conc.:

55.7 µg per larva

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 %CL: 21.5 - 72.8

Duration:

72 h

Dose descriptor:

LC10

Effect conc.:

1.29 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL: 0.336 - 1.814

Duration:

72 h

Dose descriptor:

other: LC20

Effect conc.:

1.641 other: g a.s./kg food

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CL 0.635 - 2.148

Details on results:

- Mortality: After 72 hours of oral exposure, 0.0 % mortality was observed in the solvent-free control (AC), while in BC (untreated diet C with 2 % v/v acetone) 5.6 % mortality occurred. In the test item group, mortalities ranged between 0.0 and 61.1 % after 72 hours. Statistically significant mortality occurred at the highest test material dose (99.2 μg a.s./larva).
Other observations such as smaller body size of surviving larvae or/and remaining food on D7 occurred at an increased rate (52.4 % and 16.7 % at the two highest test material groups AT and BT (99.2 and 49.6 μg a.s./larva). In the lower test material groups the rate of remaining food/smaller body size was below or at the level of the solvent control.
The concentration of active substance in the primary test material dosing stock solution A was 101 % of the nominal concentration and therefore within acceptable parameters as defined in the study plan. No test material has been detected in the control specimen.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. Mortality in the dimethoate toxic reference treatment (AR) was above 50 % across all replicates (D7/72 h), being 55.6 %.

Reported statistics and error estimates:

Mortality: For each concentration the corrected mortality was calculated according to ABBOTT (1925) modified by SCHNEIDER ORELLI (1947) following the formula in case control mortality had occurred:

Mcorr [%] = ((Mt – Mc) / (100 – Mc)) x 100

Mcorr = corrected mortality [%]
Mc = mortality of the control group [%]
Mt = mortality of the test group [%]

Statistical analysis: For statistical calculation of the mortality results and of the NOEC/NOED the Fisher’s Exact Binomial test (with Bonferroni Correction) was used. The accepted significance level was p ≤ 0.05 (one-sided greater).
The median lethal dose/concentration (LD50/LC50) of the test material along with the 95 % confidence limits were calculated with the Moving Average Computation procedure.
The statistical calculations were performed with the computer program ToxRat Professional 2.10.06 (2010).

Toxicity of the Test Material to Apis mellifera L. in an Acute Larval Toxicity Test

Treatment Group		Dosage [μg a.s./Larva]	Concentration [g a.s./kg Food]	Observations After 72 h (on D7)
				Cumulative Mortality (Mean) [%]		Other Observations (Mean)# [%]
				Absolute	Correct.
Control	AC	-	-	0.0	-	0.0
	BC	-	-	5.6	-	3.0
Test material	AT	99.2	2.925	61.1*	58.8	52.4
	AB	49.6	1.463	5.3	0.0	16.7
	CT	24.8	0.731	0.0	0.0	0.0
	DT	12.4	0.366	2.8	0.0	2.8
	ET	6.2	0.183	5.6	0.0	3.0
Reference material	AR	8.8	0.260	55.6	55.6	22.2

Results are averages based on 3 replicates, containing 12 larvae each.

Correct.: Corrected mortality (according to SCHNEIDER-ORELLI 1947): Test material corrected by BC and reference item corrected by AC, negative values are treated as “0”; Calculation are performed with non-rounded values

* Statistically significant difference in pairwise comparison between treatment and untreated control (Fisher`s Exact Binominal Test with Bonferroni Correction; α=0.05; one sided greater)

# Other observations (large quantities of remaining food, smaller body size of larva)

Validity Criteria

Larvae mortality in the controls:

AC: 0.0 % for larvae across all three replicates (D7/72 h)  

BC: 5.5 % for larvae across all three replicates (D7/72 h)  

Validity criterion met.

Reference material mortality: 55 % for larvae exposed to 8.8 μg a.s./larva at D4/all ref. replicates (D7/72 h). Validity criterion met.

Concentration of a.s. in analysed sample of test material stock solution A: Recovery of 101 % of the nominal concentration of stock solution A. Validity criterion met.

Mortality and Behavioural Abnormalities

Treatment	Replicate	Cumulative Mortality After 24 h (D5)	Cumulative Mortality After 48 h (D6)	Observations after 72 h (D7)
				Cumulative Mortality			Other Observations
		No.	No.	No.	M [%]	CM [%]	Ns.	Ns. [%]	F
AC (Blank)	1	0	0	0	0.0		0	0.0	-
	2	0	0	0	0.0		0	0.0	-
	3	0	0	0	0.0		0	0.0	-
	Average				0.0	-		0.0
BC (Solvent control)	1	0	0	0	0.0		0	0.0	-
	2	0	1	1	8.3		1	9.1	F
	3	0	0	1	8.3		0	0.0	-
	Average				5.6	-		3.0
AT	1	0	6	7	58.3		5	100.0	F
	2	0	7	10	83.3		0	0.0	-
	3	0	4	5	41.7		4	57.1	F
	Average				61.1	58.8		52.4
BT	1	0	0	0	0.0		8	50.0	F
	2	0	1	2	16.7		0	0.0	-
	3	0	0	0	0.0		0	0.0	-
	Average				5.6	0.0		16.7
CT	1	0	0	0	0.0		0	0.0	-
	2	0	0	0	0.0		0	0.0	-
	3	0	0	0	0.0		0	0.0	-
	Average				0.0	0.0		0.0
DT	1	0	1	1	8.3		0	0.0	-
	2	0	0	0	0.0		0	0.0	-
	3	0	0	0	0.0		1	8.3	F
	Average				2.8	0.0		2.8
ET	1	0	0	0	0.0		0	0.0	-
	2	0	1	1	8.3		1	9.1	F
	3	1	1	1	8.3		0	0.0	-
	Average				5.6	0.0		3.0
AR	1	0	3	4	33.3		0	0.0	-
	2	0	4	6	50.0		1	16.7	F
	3	0	7	10	83.3		1	50.0	F
	Average				55.6	55.6		22.2

No.: number of dead larvae; M [%]: mortality; CM [%]: corrected mortality according to SCHNEIDER-ORELLI 1947 (referring to control); Ns.: no of larvae with food left/small size; Ns [%]: relative number of larvae with food left/small size; F: food left, smaller body size Average % of other observations were calculated according to the following formula: Sum of larvae showing effects / Sum of surviving larvae x 100 %; Calculations are performed with non-rounded values; negative values will be set to “0”.

Statistical Analysis – Fisher’s Exact Binomial Test with Bonferroni Correction

Fisher´s Exact Binomial Test with Bonferroni Correction with survival at 72 h: Two-sample comparisons between treatment and control on the multiple significance level (Alpha is 0.05; one-sided greater). Two sample comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm, 1979); Ho (no effect) is accepted, if the probability p > Alpha*; p(exact) is the probability that the increase in category "Dead" observed in the treatments is due to chance.

 

Threshold Dosages for Mortality at 72 h

Treatment [μg Consumed A.S./Larva]	Introduced	Survived	Dead	% Mortality	P	Alpha*	Sign.
Control	36	34	2	5.6
6.2	36	34	2	5.6	0.693	0.050	-
12.4	36	35	1	2.8	0.500	0.017	-
24.8	36	36	0	0.0	0.247	0.013	-
49.6	36	34	2	5.6	0.693	0.025	-
99.2	36	14	22	61.1	< 0.001	0.010	+

+: significant; -: non-significant

A NOED of 49.6 μg a.s./larvae is suggested by the program.

 

Threshold Concentrations for Mortality at 72 h

Treatment [mg A.S./kg Food]	Introduced	Survived	Dead	% Mortality	P	Alpha*	Sign.
Control	36	34	2	5.6
0.183	36	34	2	5.6	0.693	0.050	-
0.366	36	35	1	2.8	0.500	0.017	-
0731	36	36	0	0.0	0.247	0.013	-
1.463	36	34	2	5.6	0.693	0.025	-
2.925	36	14	22	61.1	< 0.001	0.010	+

+: significant; -: non-significant

A NOEC of 1.463 mg a.s./kg food is suggested by the program.

 

Statistical Analysis – Moving Averages Computation

Results of the Moving Averages Computation with survival at 72 h: Parameters and results as obtained from the computations (se: standard error)

 

Lethal Doses After 72 Hours

%Trim chosen: 1

Log LD50: 1.9514

f-Function: 0.8500

Standard Deviation(f): 0.1202

Log distance of doses d: 0.3010

se(Log LD50): 0.0362

 

LD50: 89.404

lower 95%-confidence limit: 70.939

upper c. l.: 112.675

 

Lethal Concentrations After 72 Hours

%Trim chosen: 1

Log LC50: 0.4210

f-Function: 0.8500

Standard Deviation(f): 0.1202

Log distance of doses d: 0.3009

se(Log LC50): 0.0362

LC50: 2.636

lower 95%-confidence limit: 2.092

upper c. l.: 3.322

Statistical Analysis - Probit analysis using linear weighted regression Lethaldosesafter 72 hours

Determination of the dose /response function; data is shown which entered the probit analysis; Log(x): logarithm of the dose; n: number of organisms; Emp. Probit: empirical probit; Reg. Probit: calculated probit for the final function. 

Treatment [μg a.s./larva]	Log(x)	% Mortality	n	Emp. Probit	Weight	Reg. Probit
Control		0.00	36			Excluded
6.2	0.792	0.00	36	-3.7218	0.056	-3.722
12.4	1.093	0.10	36	-3.0926	0.406	-3.093
24.8	1.394	0.10	36	-3.0926	0.406	-3.093
49.6	1.695	0.00	36	-3.7218	0.056	-3.722
99.2	1.997	58.82	36	0.2211	22.514	0.221

Excluded: Value not in line with the chosen function.

 

Parameters of the Probit Analysis: Results of the Regression Analysis

Computation runs: 1

Slope b: 4.20047

Intercept a: -8.17348

Variance of b: 1.83982

Goodness of Fit

Chi^2:0.86261

Degrees of freedom: 3

p(Chi^2): 0.83444

Log LD50: 1.94585

SE Log LD50: 0.04964

g-Criterion: 0.40058

F: 33.352

p(F) (df: 1 ;3): 0.010

 

Chi^2 is a goodness of fit measure. If the probability. p(Chi^2 ). is lower or equal than 0.100, data is much scattering round the computed dose/response function. In this case and with quantal data. Confidence limits are corrected for heterogeneity.

 

Slope function after Litchfield and Wilcoxon 1. 730

(The slope function is derived from the slope. b. of the linearized probit function and computes as S = 10^(1/b); small values refer to a steep dose/response relation and large ones to a flat relation.)

 

Results of the Probit Analysis

Selected effective doses (LDx) of the test material and their 95 % confidence limits(by normal approximation).

Parameter	LD10	LD20	LD50
Value [μg a.s./larva]	43.727	55.653	88.278
Lower 95 % CL	11.394	21.513	63.879
Upper 95 % CL	61.516	72.841	114.356

 

Statistical Analysis-Probit Analysis Using Linear Weighted Regression LethalConcentrationsAfter 72 Hours

Determination of the dose/response function; data is shown which entered the probit analysis; Log(x): logarithm of the dose; n: number of organisms; Emp. Probit: empirical probit; Reg. Probit: calculated probit for the final function. 

Treatment [g a.s./kg Food]	Log(x)	% Mortality	n	Emp. Probit	Weight	Reg. Probit
Control		0.00	36			Excluded
0.183	-0.738	0.00	36	-3.7218	0.056	-3.722
0.366	-0.437	0.10	36	-3.0926	0.406	-3.093
0.731	-0.136	0.10	36	-3.0926	0.406	-3.093
1.463	0.165	0.00	36	-3.7218	0.056	-3.722
2.925	0.466	58.82	36	0.2211	22.514	0.221

Excluded: Value not in line with the chosen function

 

Parameters of the Probit Analysis: Results of the Regression Analysis

Computation runs: 1

Slope b: 4.20186

Intercept a: -1.74578

Variance of b: 1.84085

Goodness of Fit

Chi^2: 0.86161

Degrees of freedom: 3

p(Chi^2): 0.83468

Log LC50:0.41548

SE Log LC50: 0.04962

g-Criterion: 0.40054

F: 33.395

p(F) (df: 1 ;3): 0.010

Chi^2 is a goodness of fit measure. If the probability. p(Chi^2). is lower or equal than 0.100, data is much scattering round the computed dose/response function. In this case and with quantal data. Confidence limits are corrected for heterogeneity.

Slope function after Litchfield and Wilcoxon: 1. 730

(The slope function is derived from the slope. b. of the linearized probit function and computes as S = 10"(1/b); small values refer to a steep dose/response relation and large ones to a flat relation.)

  

Results of the Probit Analysis

Selected effective doses (LCx) of the test material and their 95 % confidence limits (by normal approximation).

Parameter	LC10	LC20	LC50
Value [g a.s./kg food]	1.290	1.641	2.603
Lower 95 % CL	0.336	0.365	1.884
Upper 95 % CL	1.814	2.148	3.372

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

Executive summary:

The effect of the test material on the honey bee was assessed in an acute toxicity test according to OECD Test Guideline 237 and in compliance with GLP.

Honeybee (Apis mellifera L.) larvae were exposed to the test material in an acute toxicity test. The toxicity of the test material was determined at doses of 99.2, 49.6, 24.8, 12.4 and 6.2 μg a.s./larva (after correction for purity, corresponding to 107.1, 53.5, 26.8, 13.4 and 6.7 μg test material/larva). The concentrations in the diet were 2.925, 1.463, 0.731, 0.366 and 0.183 g a.s./kg food.

Additionally, honeybee larvae were treated with Dimethoate technial as the toxic reference item at a concentration of 8.8 μg dimethoate/larva. The study design also included a negative control group with an untreated diet and a solvent control group that received the same diet amended with acetone at the same concentration at which it was used in the test material treatments.

After 72 hours of oral exposure, 0.0 % mortality was observed in the solvent-free control (AC), while in BC (untreated diet C with 2 % v/v acetone) 5.6 % mortality occurred. In the test material group, mortalities ranged between 0.0 and 61.1 % after 72 hours. Statistically significant mortality occurred at the highest test material dose (99.2 μg a.s./larva). Mortality in the dimethoate toxic reference treatment (AR) was above 50 % across all replicates (D7/72 h), being 55.6 %.

Other observations such as smaller body size of surviving larvae or/and remaining food on D7 occurred at an increased rate (52.4 % and 16.7 % at the two highest test material groups AT and BT (99.2 and 49.6 μg a.s./larva). In the lower test material groups the rate of remaining food/smaller body size was below or at the level of the solvent control.

The concentration of active substance in the primary test material dosing stock solution A was 101 % of the nominal concentration and therefore within acceptable parameters as defined in the study plan. No test material has been detected in the control specimen.

Because control mortality was ≤ 15 %, corrected mortality in the reference item dose of 8.8 μg a.s./larva was ≥ 50 % and the analytical verification of active substance concentration in the test material stock solution displayed that it was within ± 20 % of the nominal concentration the study can be regarded as valid.

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

Reference 4

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 August 2017 to 12 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

other: OECD Guideline Guidance Document on Honey Bee Larval Toxicity Test, Repeated Exposure, OECD Series on Testing & Assessment No. 239

Deviations:

yes

Remarks:

with modifications from Schmehl et al. 2016 (Schmehl DR, Tomé HVV, Mortensen AN, Martins GF, Ellis JD 2016. Protocol for the rearing of honey bee (Apis mellifera L.) workers. Journal of Apicultural Research, 2016. DOI: 10.1080/00218839.2016.1203530.

Principles of method if other than guideline:

Modifications include diet composition (more water and less royal jelly in diets A and B), the introduction of a pre-pupal transfer step (transferring of larvae on day 7/8 of development to a new culture plate), and changes to rearing environment (no glycerol/sterilising solution used, lid placed upon plate throughout development, and no emergence box). The changes included in the protocol did not impact the results because survival on negative control was only 10.4 % which confirms that the actual method is appropriate for the proposed study.

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of all the test diets were collected for chemical analysis to measure test concentrations.
- Sampling method: Each sample consisted of 1 mL of the appropriate diet and was placed in a uniquely identified 20 mL scintillation vial. On days 3 and 6, the first and last days of dosing, triplicate samples were collected at the highest and lowest test concentrations, and a single sample was collected from the remaining test material concentrations, the negative control and the solvent control. On days 4 and 5, a single sample was collected from each test material concentration, the negative control and the solvent control. Samples were not collected from the positive control diet. A duplicate set of samples, consisting of the same number and sample volume, was collected in order to provide back-up samples.
- Sample storage conditions before analysis: Samples were stored in a freezer until they were transferred to the analytical laboratory for processing and analysis.

Vehicle:

yes

Details on preparation and application of test substrate:

TEST MATERIAL
On Day 3 of the test, a primary stock solution of test material was prepared by weighing 1.527 g into a tared 10 mL flask. Acetone was then added to the flask. The primary stock solution was clear and yellow with no visible precipitates and served as the primary stock solution to prepare the highest treatment level (100 μg a.i./bee). Subsequent stock solutions were prepared by serial volumetric dilution of the primary stock solution and subsequent test stocks (5.0 mL of the primary brought to 10 mL total volume, 5.0 mL of the secondary brought to 10 mL total volume, etc.) to prepare stocks for the 50, 25, 12.5 and 6.25 μg a.i./bee treatment groups, respectively. The solutions were inverted at least 20 times to mix and appeared homogeneous and clear and yellow with no visible particulates. The test material treatment levels were based on the reported purity.

POSITIVE CONTROL
The positive control stock solution was prepared by dissolving 0.0890 g of dimethoate in 10 mL of acetone. The solution was inverted at least 20 times to mix and was completely dissolved and appeared translucent and homogeneous. The dimethoate treatment level was based on the reported purity. In order to prepare treated test diets, 50.3 and 100.5 μL of the appropriate stock solution were added to 11.1 g of Diet B (density 1.11 g/mL) and 22.8 g of Diet C (density 1.14 g/mL), respectively. The diets were mixed in tared 50 mL glass beakers and stirred for approximately 15 minutes using a stir plate and magnetic stir bar.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honey bee
- Source: Bees used in the test were obtained from hives maintained by the testing facility, located near Alachua, FL.
- Taxonomic confirmation: Identification of the test organisms was made based on Michener (Michener C 2007. Bees of the World. 2nd edition).
- By whom?: Testing facility staff.
- Age at test initiation: Larvae, approximately 72-hours after hatching at initial exposure.
- Cultural background: Larvae for the test were selected from brood frames collected from apparently healthy hives with a known history of apicultural practices and not treated with antibiotics, miticides or other pesticides within the previous four weeks. Larvae from three different hives, one for each replicate, were used.

ACCLIMATION
- Health during acclimation: Larval health was based on a visual assessment of full turgidity and appropriate size. In order to start the test, three replicates of 16 apparently healthy larvae were selected and indiscriminately assigned to each treatment level. Unhealthy larvae were replaced on Day 3 prior the dosing.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

4 d

Post exposure observation period:

14 days

Test temperature:

- Larvae: Average temperature of 33.97 ºC
- Bees: Average temperature of 33.89 ºC

Humidity:

- Larvae: Average relative humidity of 93.13 %
- Bees: Average relative humidity of 68.74 %

Photoperiod and lighting:

Bees were maintained in the dark throughout the test period

Details on test conditions:

TEST SYSTEM
- No. of organisms per container: Each treatment and control group contained 16 larvae
- No. of replicates per treatment group: Three; one from each of the three hives, for a total of 48 larvae per treatment group.
- No. of replicates per control, vehicle control and positive control: Three for each control; one from each of the three hives for a total of 48 larvae for each control.

EGG CULTURE CONDITIONS
The queen from each hive was confined in an excluder for approximately 24 hours on an empty frame of drawn comb in order to isolate the potential area for egg laying and provide more uniform age classes of larvae. After the egg-laying period, the queens were released and the frames were examined for the presence of eggs. The frames with eggs were kept in the hive for approximately 75 hours, near the brood, until the larvae hatched and reached an appropriate size to be transferred to the laboratory.
In the laboratory, larvae were grafted using a Chinese grafting tool under a laminar flow hood into plastic cell cups (internal diameter of 9 mm and a depth of 8 mm) containing artificial diet. Cell cups were placed in 48-well tissue culture plates and covered with lids. The grafting cell cups were positioned at the top of cotton dental roll placed in the well in order to allow air circulation. More larvae than needed were transferred and incubated for approximately 48 hours, at which time the appropriate number of healthy larvae were selected for use in testing. All material used in the larval grafting were pre-sterilised under UV-light. In addition, a space heater was turned on close to the larval frame during the grafting to keep the larvae warmed during the process.

FOOD CONSUMPTION
During incubation, larvae were fed artificial diets prepared with royal jelly purchased from a reputable supplier in the USA, tissue culture grade glucose and fructose, laboratory grade yeast extract powder and well water purified by reverse osmosis. The diets were prepared and supplied to larvae as follows:
Day 1 (grafting): 20 µL of Diet A (This diet is placed in each cell cup prior to grafting larvae)
Day 2: n/a
Day 3: 20 µL of Diet B
Day 4: 30 µL of Diet C
Day 5: 40 µL of Diet C
Day 6: 50 µL of Diet C
Diet A: 44.25 % weight of fresh royal jelly, 44.25 % weight of water, 0.90 % weight of yeast extract, 5.30 % weight of glucose and 5.30 % weight of fructose.
Diet B: 42.95 % weight of fresh royal jelly, 42.95 % weight of water, 1.30 % weight of yeast extract, 6.40 % weight of glucose and 6.40 % weight of fructose.
Diet C: 50 % weight of fresh royal jelly, 30 % weight of water, 2.0 % weight of yeast extract, 9.0 % weight of glucose and 9.0 % weight of fructose.
Diets A and B were prepared on the day the diet was provisioned to the larvae. Diet C was prepared on test Day 4 and provisioned to the larvae on test Day 4, 5, and 6. Unused Diet C was stored refrigerated between feeding intervals and brought to room temperature prior to use on days 5 and 6. The diet was gently mixed prior to feeding to ensure a homogenous diet.

VEHICLE CONTROL PERFORMED: Yes

DOSING OF LARVAL BEES AND TRANSFER TO PUPAL PLATES
Test diets (dosed and un-dosed) were administered with a micropipette directly into the cells containing the larvae. All bees received untreated diets on Day 1. On Days 3, 4, 5 and 6 the treatment group dosed diets were placed next to larva, along the wall of grafting cell with the appropriate concentration of test material. Bees from negative and solvent control (0.5 % acetone) received untreated artificial diet, and positive control bees received artificial diet containing dimethoate. A clean positive displacement pipette tip was used for each treatment and control group. After all diet was consumed, larvae were transferred to clean pupal plates containing paper for absorption of faeces. Typically, this transfer occurred on Day 7, but larvae that had not consumed the diet entirely were transferred on Day 8. Larvae which did not consume diet by Day 8 were considered dead and removed from the study.

HOUSING AND ENVIRONMENTAL CONDITIONS
Larvae in well plates were held in a hermetically sealed Plexiglas desiccator containing a saturated potassium sulfate solution (50 g of K2SO4 into 500 mL H2O) and placed in an incubator. Relative humidity and temperature inside the desiccator was monitored continuously using a traceable datalogger (VWR®). During the test periods, the average relative humidity and temperature were 93.13 % and 33.97 °C, respectively. After pupal transfer on day 7 or 8, the bees were held in a similar hermetically sealed Plexiglas desiccator containing a saturated sodium chloride solution (200 g of NaCl into 500 mL H2O) and placed in an incubator. Relative humidity and temperature inside the desiccator was monitored continuously using a traceable datalogger (VWR®). During the test periods, average relative humidity and temperature were 68.74 % and 33.89 °C, respectively. Significant variations in the relative humidity and temperature were observed only when the desiccator was opened to grafting, feeding procedures and observations. Bees were maintained in the dark throughout the test period, except during the procedures described previously.

EFFECT PARAMETERS MEASURED
Starting on the first day of dosing (day 3), larvae were observed daily until either mortality or adult emergence occurred. Cells containing dead larvae were removed from well plates after mortality was recorded. Observations of sublethal effects, including the presence of uneaten diet and larvae with reduced body size, were recorded on days 7 and 8. Emerged bees from each replicate were collected, and weighed individually in a tared 2 mL microcentrifuge tube in order to evaluate possible sublethal effects on body mass.
In addition, deformities were checked to evaluate sublethal effects on morphology of emerged bees. By day 20, approximately 14 days after final dosing, all bees had either emerged or died. After test termination, bees were transferred to a freezer and held for disposal by incineration.

The data were evaluated to determine effects of the test material on larval survival, pupal survival, adult emergence and body weight of honey bees. These variables were defined as follows:
- Larval survival: The proportion of larvae which consume their diet and are alive for transfer to pupal plates relative to the number of larvae starting the test per replicate or treatment group. Any larvae that failed to consume their entire allotment of diet by day 8 were considered mortalities.
- Pupal survival: The proportion of emerged adult bees relative to the number of larvae surviving at day 8 per replicate or treatment group.
- Adult emergence: The proportion of emerged adult bees relative to the number of larvae starting the test per replicate or treatment group. Any bee that failed to emerge as an adult was considered a mortality.
- Body weight: The average weight of emerged adult bees within each replicate.
Mean adult emergence and body weight of the negative and solvent control groups were compared with a two-sample t-test. Treatment groups were compared to the negative control only. Test data were evaluated to determine the no-observed-effect-concentration/dose (NOEC/D), which was defined as the maximum test material dietary concentration (mg a.i./mL) or cumulative dose (µg a.i./bee) which showed no adverse effects on adult emergence or body weight. Adult emergence data were converted from survival to mortality prior to statistical analysis in order to provide an estimated EC/D50 and NOEC/D. Normality and homogeneity of variance were tested with a Shapiro-Wilk’s and Bartlett’s test, respectively. Treatment group means were compared to the negative control mean using Dunnett and William’s tests in order to determine if a significant trend was detected. A probability of 0.05 was considered the threshold of statistical significance. Results of the Dunnett and William’s test were used to help establish the NOEC/D. All statistical analyses were performed using CETIS version 1.9.1.8. Mean larval survival and pupal survival were calculated but were not analysed statistically. Data collected for sublethal effects other than weight (such as the presence of malformed bees or delays in either diet consumption or pupation) were not statistically analysed due to the low incidence of observed responses.
Effects on adult emergence were described as percent mortality in each treatment group and were calculated with the following equation.

Percent mortality = [number dead / total number starting test] x 100 %

Percent mortality is equivalent to percent reduction in survival. Effects on body weight were described as reductions from the control mean weight and were calculated with the following equation.

Percent inhibition = [(control mean – treatment mean) / control mean] x 100 %

Nominal and measured concentrations:

- Nominal concentrations: 100, 50.0, 25.0, 12.5 and 6.25 μg a.i./bee as 0.715, 0.357, 0.179, 0.089 and 0.045 mg a.i./mL diet.

Reference substance (positive control):

yes

Remarks:

Dimethoate, 6.2 μg a.i./bee

Key result

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

> 0.714 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Key result

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Key result

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

0.357 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Key result

Duration:

4 d

Dose descriptor:

NOED

Effect conc.:

50 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

0.714 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

EC10

Effect conc.:

0.214 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidnce limits 0.061 - 0.367 mg a.i./mL diet.

Duration:

4 d

Dose descriptor:

other: EC20

Effect conc.:

0.429 other: mg a.i./mL diet

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 0.194 - 0.663 mg a.i./mL diet.

Duration:

4 d

Dose descriptor:

other: LOED

Effect conc.:

100 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Duration:

4 d

Dose descriptor:

ED10

Effect conc.:

29.98 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 8.578 - 51.38 μg a.i./bee.

Duration:

4 d

Dose descriptor:

other: ED20

Effect conc.:

59.96 other: μg a.i./bee

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult emergence and weight at test termination.

Remarks on result:

other: 95 % confidence limits 27.18 - 92.73 μg a.i./bee.

Details on results:

MORTALITY AND OBSERVATIONS
By day 8, all living larvae had consumed their entire allotted diets. No significant differences between the negative and solvent control means for either mortality or body weight were detected (two-sample t-test, p>0.05), therefore all statistical comparisons were made to the negative control. Mean mortality in the treatment groups ranged from 14.6 to 43.8 %, and the highest level (100 μg a.i/bee) was significantly different according to Dunnett and Williams Multiple Comparison Test (p < 0.05). Reductions in mean body weight relative to the control ranged from 2 to 12 %, and no significant trend of decreasing body weight was detected (Dunnett and Williams Multiple Comparison Test, p>0.05).
Therefore, the NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively. Both statistical tests were used because Williams and Dunnett’s test are recommended for monotonic and not monotonic data response, respectively. As the data interpretation about monotonicity is very often ambiguous, Williams and Dunnett tests were used in the study.
The EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively.
Since all treatment group mortality was less than 50 %, the EC50 and ED50 were determined to be greater than 0.715 mg a.i./mL and 100 µg a.i./bee, respectively, based on visual evaluation of the data.

VALIDITY CRITERIA
This test was considered valid based on the following criteria:
1. In the negative control plate(s), cumulative larval mortality (prior to pupal transfer) was less than 15 % across replicates.
2. In the negative control group, adult emergence was higher than 70 % on Day 20.
3. In the positive control (dimethoate) group, larval mortality was higher than 50 %.

Results with reference substance (positive control):

In the positive control (dimethoate) group, larval mortality was higher than 50 %.

Reported statistics and error estimates:

The EC/D50 was defined as the theoretical concentration or dose which would result in 50 % mortality prior to adult emergence or a 50 % reduction in body weight. Since there were no effects of 50 % or more, the EC/D50 for adult emergence and weight were determined by visual inspection of the data.

Mean Survival and Adult Emergence

Treatment Group (μg a.i./bee)	Larval Survival¹(%) Day 8	Pupal Survival²(%) Day 20	Adult Emergence³(%) Day 20	Adult Mortality4⁴(%) Day 20
Negative Control	91.7	97.8	89.6	10.4
Solvent Control (0.5 %)	95.8	97.8	93.8	6.3
6.25	83.3	94.4	79.2	20.8
12.5	89.6	95.2	85.4	14.6
25	89.6	93.8	83.3	16.7
50	81..3	91.5	75.0	25.0
100*	70.8	82.0	56.3	43.8
Positive Control	16.7	80.0	10.4	89.6

¹(Number of living larvae on day 8 / Initial number of larvae) x 100 (per treatment group)

²(Number of emerged adults on day 20 / Number of living larvae on day 8) x 100 (per treatment group)

³(Number of emerged adults on day 20 / Initial number of larvae) x 100 (per treatment group)

⁴(Number of dead bees on day 20 / Initial number of larvae) x 100 (per treatment group)

*Treatment group was significantly different from the negative control mean in according to Dunnett and Williams Multiple Comparison Test, p<0.05). Solvent and positive controls were not included in the statistical test.

Honey Bee Adult Mean Weight

Treatment Group (µg a.i./bee)	Replicate	Number of Bees Weighed per Replicate	Adult Weight (g) (average per replicate)	Mean Adult Weight (g)¹	Standard Deviation Weight (g)²	Reductions in Body Weight Relative to Control (%)
Negative Control	1	13	0.108	0.106	0.003	-
	2	16	0.103
	3	14	0.107
Solvent Control (0.5% Acetone)	1	15	0.098	0.103	0.005	3
	2	16	0.107
	3	14	0.106
6.25	1	10	0.106	0.104	0.003	2
	2	16	0.101
	3	12	0.106
12.5	1	14	0.103	0.104	0.003	2
	2	15	0.102
	3	12	0.108
25	1	13	0.098	0.097	0.001	10
	2	14	0.096
	3	13	0.098
50	1	9	0.100	0.100	0.000	6
	2	16	0.100
	3	11	0.100
100	1	7	0.089	0.093	0.005	12
	2	10	0.099
	3	9	0.093
Positive Control (6.2)	1	2	0.092	0.088	0.007	17
	2	2	0.081
	3	1	0.092

¹Mean weight calculated for emerged bees per treatment level.

²Standard deviation calculated based on average per replicate.

All treatment group means were not significantly different from the negative control mean (Dunnett and Williams Multiple Comparison test, p<0.05). Solvent and positive controls were not included in the statistical test.

Measured Concentrations of Test Material in Larval Diet B Samples

Sample ID	Sampling Day	Nominal Test Concentration (μg a.i./bee)	Nominal Test Concentration (μg a.i./mL)	Measured Diet Concentration*† (mg a.i./mL)	Percent of Nominal†	Mean Measured Concentration† (mg a.i./mL)
1	3	Negative control	Negative control	<LOQ	-	-
2	3	Solvent control	Solvent control	<LOQ	-	-
3	3	6.25	0.045	0.0404	89.8	0.0404 ±0.000252 CV = 0.623%
4	3			0.0401	89.1
5	3			0.0406	90.3
6	3	12.5	0.089	0.0829	93.1	-
7	3	25.0	0.179	0.158	88.5	-
8	3	50.0	0.3572	0.332	93.0	-
9	3	100	0.7143	0.647	90.5	0.645 ± 0.00208 CV = 0.323 %
10	3			0.644	90.2
11	3			0.643	90.1

* The limit of quantitation (LOQ) for Larval Diet B was 0.0275 mg a.i./mL, defined as the lowest nominal concentration in

a fortified sample with an acceptable recovery between 70 – 110 %.

†Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

 

Measured Concentrations of Test Material in Larval Diet C Samples

Sample ID	Sampling Day	Nominal Test Concentration (μg a.i./bee)	Nominal Test Concentration (μg a.i./mL)	Measured Diet Concentration*† (mg a.i./mL)	Percent of Nominal†	Mean Measured Concentration† (mg a.i./mL)
12	4	Negative control	Negative control	<LOQ	-	-
13	4	Solvent control	Solvent control	<LOQ	-	-
19	5	Negative control	Negative control	<LOQ	-	-
20	5	Solvent control	Solvent control	<LOQ	-	-
26	6	Negative control	Negative control	<LOQ	-	-
27	6	Solvent control	Solvent control	<LOQ	-	-
14	4	6.25	0.045	0.0436	96.8	0.0438 ± 0.000205 CV = 0.468 %
21	5			0.0440	97.8
28	6			0.0436	96.8
28	6			0.0437	97.1
30	6			0.0440	97.9
15	4	12.5	0.089	0.0890	100	0.0887 ± 0.000379 CV = 0.427
22	5			0.0883	99.2
31	6			0.0889	99.9
16	4	25.0	0.179	0.175	97.7	0.175 ± 0.000577 CV = 0.331 %
23	5			0.174	97.4
32	6			0.175	97.9
17	4	50.0	0.3572	0.346	96.8	0.346 ± 0.000577 CV = 0.167 %
24	5			0.347	97.0
33	6			0.346	96.8
18	4	100	0.7143	0.682	95.4	0.684 ± 0.00377 CV = 0.551 %
25	5			0.680	95.1
34	6			0.685	95.8
35	6			0.690	96.6
36	6			0.684	95.8

* The limit of quantitation (LOQ) for Larval Diet C was 0.0288 mg a.i./mL, defined as the lowest nominal

concentration in a fortified sample with an acceptable recovery between 70 – 110 %.

† Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

 

Total Daily and Cumulative Doses of Test Material Provided to Honey Bee Larvae

Treatment Concentration (μg a.i./ μLdiet)	Daily Dose*†				Cumulative Dose‡ (μg a.i./ bee)
	Day 3	Day 4	Day 5	Day 6
Negative control	0.0	0.0	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0	0.0	0.0
0.045	0.893	1.340	1.787	2.234	6.25
0.089	1.787	2.680	3.574	4.467	12.5
0.179	3.574	5.361	7.148	8.935	25.0
0.357	7.148	10.722	14.296	17.870	50
0.715	14.296	21.444	28.592	35.740	100.1
Positive control Dimethoate 0.04432 μg a.i./ μL)	0.886	1.330	1.773	2.216	6.20

* The daily dose is based on complete consumption of the following:

Day 3 = 20 μL of Diet B; Day 4 = 30 μL of Diet C; Day 5 = 40 μL of Diet C; Day 6 = 50 μL of Diet C.

† The daily dose was calculated as volume of diet provided x concentration.

‡ The cumulative dose is the sum of all daily doses. The numbers were rounded for presentation.

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.

Executive summary:

The chronic toxicity of the test material when administered to the honey bee (Apis mellifera) during the larval stage I was assessed according to accordance with the OECD Guidance Document 239 under GLP conditions.

Larvae from three colonies were transferred (grafted) to well plates containing untreated artificial diet and held in an incubator. Starting at two days after grafting, artificial diets containing the test material or control substance were provided to larvae. Test diets were provided for a total of four days. Larvae were then held for up to 14 days after the completion of dosing in order to allow emergence of adult bees. A negative control, solvent control and positive control group were maintained concurrently. Each treatment and control group contained 16 larvae from each of the three hives, for a total of 48 larvae per treatment group. Nominal test levels were selected based upon known toxicity data and were 100, 50.0, 25.0, 12.5 and 6.25 μg a.i./bee.

In order to control bias, larvae were impartially distributed to the treatment and control groups. No other potential sources of bias were expected to affect the results of the study. Dosing of larvae with the test material in artificial diet simulates the possible exposure of larval bees in the hive. The no-observed-effect-concentration/dose (NOEC/D) and EC/D10, 20, 50 values were based on adult emergence and weight at test termination.

By day 8, all living larvae had consumed their entire allotted diets. No significant differences between the negative and solvent control means for either mortality or body weight were detected, therefore all statistical comparisons were made to the negative control. Mean mortality in the treatment groups ranged from 14.6 to 43.8 %, and the highest level (100 μg a.i/bee) was significantly different. Reductions in mean body weight relative to the control ranged from 2 to 12 %, and no significant trend of decreasing body weight was detected.

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.

Description of key information

Key study: Kleebaum (2014) - Read across (MCPP-P)

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

 

 

Key study: Tomé et al. (2018) - Read across (MCPP-P)

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.

Additional information

Key study: Kleebaum (2014) - Read across (MCPP-P)

The effect of the test material on the honey bee was assessed in an acute toxicity test according to OECD Test Guideline 237 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Honeybee (Apis mellifera L.) larvae were exposed to the test material in an acute toxicity test. The toxicity of the test material was determined at doses of 99.2, 49.6, 24.8, 12.4 and 6.2 μg a.s./larva (after correction for purity, corresponding to 107.1, 53.5, 26.8, 13.4 and 6.7 μg test material/larva). The concentrations in the diet were 2.925, 1.463, 0.731, 0.366 and 0.183 g a.s./kg food.

Additionally, honeybee larvae were treated with Dimethoate technial as the toxic reference item at a concentration of 8.8 μg dimethoate/larva. The study design also included a negative control group with an untreated diet and a solvent control group that received the same diet amended with acetone at the same concentration at which it was used in the test material treatments.

After 72 hours of oral exposure, 0.0 % mortality was observed in the solvent-free control (AC), while in BC (untreated diet C with 2 % v/v acetone) 5.6 % mortality occurred. In the test material group, mortalities ranged between 0.0 and 61.1 % after 72 hours. Statistically significant mortality occurred at the highest test material dose (99.2 μg a.s./larva). Mortality in the dimethoate toxic reference treatment (AR) was above 50 % across all replicates (D7/72 h), being 55.6 %.

Other observations such as smaller body size of surviving larvae or/and remaining food on D7 occurred at an increased rate (52.4 % and 16.7 % at the two highest test material groups AT and BT (99.2 and 49.6 μg a.s./larva). In the lower test material groups the rate of remaining food/smaller body size was below or at the level of the solvent control.

The concentration of active substance in the primary test material dosing stock solution A was 101 % of the nominal concentration and therefore within acceptable parameters as defined in the study plan. No test material has been detected in the control specimen.

Because control mortality was ≤ 15 %, corrected mortality in the reference item dose of 8.8 μg a.s./larva was ≥ 50 % and the analytical verification of active substance concentration in the test material stock solution displayed that it was within ± 20 % of the nominal concentration the study can be regarded as valid.

Under the conditions of the study the LD50 (72 h) was determined to be 89.4 μg a.s./larva, which is equivalent to a LC50 (72 h) of 2.636 g a.s./kg food. The NOED was 49.6 μg a.s./larva and the corresponding NOEC was 1.463 g a.s./kg food.

 

Key study: Tomé et al. (2018) - Read across (MCPP-P)

The chronic toxicity of the test material when administered to the honey bee (Apis mellifera) during the larval stage I was assessed according to accordance with the OECD Guidance Document 239 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Larvae from three colonies were transferred (grafted) to well plates containing untreated artificial diet and held in an incubator. Starting at two days after grafting, artificial diets containing the test material or control substance were provided to larvae. Test diets were provided for a total of four days. Larvae were then held for up to 14 days after the completion of dosing in order to allow emergence of adult bees. A negative control, solvent control and positive control group were maintained concurrently. Each treatment and control group contained 16 larvae from each of the three hives, for a total of 48 larvae per treatment group. Nominal test levels were selected based upon known toxicity data and were 100, 50.0, 25.0, 12.5 and 6.25 μg a.i./bee.

In order to control bias, larvae were impartially distributed to the treatment and control groups. No other potential sources of bias were expected to affect the results of the study. Dosing of larvae with the test material in artificial diet simulates the possible exposure of larval bees in the hive. The no-observed-effect-concentration/dose (NOEC/D) and EC/D10, 20, 50 values were based on adult emergence and weight at test termination.

By day 8, all living larvae had consumed their entire allotted diets. No significant differences between the negative and solvent control means for either mortality or body weight were detected, therefore all statistical comparisons were made to the negative control. Mean mortality in the treatment groups ranged from 14.6 to 43.8 %, and the highest level (100 μg a.i/bee) was significantly different. Reductions in mean body weight relative to the control ranged from 2 to 12 %, and no significant trend of decreasing body weight was detected.

Under the conditions of this study, the EC10 and ED10 were determined to be 0.214 mg a.i./mL (95 % confidence limits 0.061 – 0.367 mg a.i./mL), and 29.98 µg a.i./bee (95 % confidence limits 8.578 – 51.38 µg a.i./bee), respectively. The EC20 and ED20 were determined to be 0.429 mg a.i./mL (95 % confidence limits 0.194 – 0.663 mg a.i./mL), and 59.96 µg a.i./bee (95 % confidence limits 27.18 – 92.73 µg a.i./bee), respectively. The EC/D50 values along with 95 % confidence limits were not determined because the test material did not kill more than 50 % of bees. The NOEC and NOED for mortality were 0.357 mg a.i./mL and 50 µg a.i./bee, respectively. The LOEC and LOED for mortality were 0.715 mg a.i./mL and 100 µg a.i./bee, respectively.

 

 

 

Considering the very close structural similarity between the source and target substances (as justified in the read-across report that is attached to section 13 of the dossier), results from the study performed with mecoprop-p can be extrapolated to mecoprop.