Honeybee brood testing under semi-field and field conditions according to Oomen and OECD GD 75: is there a difference of the brood termination rate?

According to current European regulations on the risk assessment of plant protection products, the risk on honey bee larvae or honey bee brood has to be addressed. If the assessment indicates, that a potential risk cannot be excluded based on data derived from laboratory studies, two higher-tier options are given by the EFSA bee Guidance Document to refine this under more realistic conditions: the Oomen bee brood feeding test and brood studies performed according to the OECD Guidance Document 75. Both study types focus on the brood termination rate (BTR) as the key endpoint. While the Oomen brood test investigates the brood development after the acute or chronic administration of a test item spiked sugar solution to unconfined colonies, brood studies according to OECD GD 75 are performed under semi-field confined exposure conditions and examine potential effects on the bee brood after the overspray of a bee attractive flowering crop. However, the evaluation of historical data from semi-field studies according to OECD GD 75 showed a strong variability of the BTR of pre-imaginal stages developing from marked eggs (BTReggs) in the control. As an alternative, field studies according to EPPO 170 which comprise bee brood evaluations according to OECD GD 75 were considered to produce more reliable termination data. The statistical analysis of available control data shows that Oomen feeding studies and bee brood studies performed under field conditions lead to significantly lower BTReggs of = 20% compared to semi-field bee brood studies for which a mean BTR of about 30% is observed. Moreover, studies with unconfined colonies show a high proportion of control replicates with BTReggs =30% and =40% indicating a higher reliability compared to semifield studies. A comparison of the possibilities and limitations of the three methods shows the strength of each method. In Oomen studies, the exposure of the brood and of the hive bees only can be regarded as artificial. However, the test concentrations can be adjusted to specific needs and to different feeding durations of at least one (acute) or 9 days (chronic). Furthermore, the absence of ‘caging effects’, the low dependency on climatic or crop conditions, the potential to test also herbicides which control dicotyledonous plants (since no crop plant is adversely affected by its mode of action) and an exposure period of at least nine days in chronic Oomen studies are crucial advantages. In contrast, the exposure scenarios of the two other methods are much more realistic and especially for semi-field studies a worst-case situation. Moreover, they also include exposure via pollen and exposure levels and durations, which strongly depend on the application rate and the flowering period of the treated crop. Whereas a dilution of plant protection product residues cannot be excluded during the exposure period in studies with unconfined colonies due to the shift to untreated flowering plants in the surrounding, this is not given for semi-field studies.According to current European regulations on the risk assessment of plant protection products, the risk on honey bee larvae or honey bee brood has to be addressed. If the assessment indicates, that a potential risk cannot be excluded based on data derived from laboratory studies, two higher-tier options are given by the EFSA bee Guidance Document to refine this under more realistic conditions: the Oomen bee brood feeding test and brood studies performed according to the OECD Guidance Document 75. Both study types focus on the brood termination rate (BTR) as the key endpoint. While the Oomen brood test investigates the brood development after the acute or chronic administration of a test item spiked sugar solution to unconfined colonies, brood studies according to OECD GD 75 are performed under semi-field confined exposure conditions and examine potential effects on the bee brood after the overspray of a bee attractive flowering crop. However, the evaluation of historical data from semi-field studies according to OECD GD 75 showed a strong variability of the BTR of pre-imaginal stages developing from marked eggs (BTReggs) in the control. As an alternative, field studies according to EPPO 170 which comprise bee brood evaluations according to OECD GD 75 were considered to produce more reliable termination data. The statistical analysis of available control data shows that Oomen feeding studies and bee brood studies performed under field conditions lead to significantly lower BTReggs of = 20% compared to semi-field bee brood studies for which a mean BTR of about 30% is observed. Moreover, studies with unconfined colonies show a high proportion of control replicates with BTReggs =30% and =40% indicating a higher reliability compared to semifield studies. A comparison of the possibilities and limitations of the three methods shows the strength of each method. In Oomen studies, the exposure of the brood and of the hive bees only can be regarded as artificial. However, the test concentrations can be adjusted to specific needs and to different feeding durations of at least one (acute) or 9 days (chronic). Furthermore, the absence of ‘caging effects’, the low dependency on climatic or crop conditions, the potential to test also herbicides which control dicotyledonous plants (since no crop plant is adversely affected by its mode of action) and an exposure period of at least nine days in chronic Oomen studies are crucial advantages. In contrast, the exposure scenarios of the two other methods are much more realistic and especially for semi-field studies a worst-case situation. Moreover, they also include exposure via pollen and exposure levels and durations, which strongly depend on the application rate and the flowering period of the treated crop. Whereas a dilution of plant protection product residues cannot be excluded during the exposure period in studies with unconfined colonies due to the shift to untreated flowering plants in the surrounding, this is not given for semi-field studies

ICPPR WG Semi-field and field Report and Discussion

The ICPPR Semi-Field/Field Testing (SF/FT) workgroup consists of several ‘writing groups’ that are focused developing technical guidance that is focused on 4 separate but related topics: 1) designing and conducting pollen and nectar residue studies, 2) conducting large scale colony feeding studies, 3) updating guidance for conducting semi-field tunnel studies, and 4) design and interpretation of full field studies with bees. What follows is the current status of each of these activities.The ICPPR Semi-Field/Field Testing (SF/FT) workgroup consists of several ‘writing groups’ that are focused developing technical guidance that is focused on 4 separate but related topics: 1) designing and conducting pollen and nectar residue studies, 2) conducting large scale colony feeding studies, 3) updating guidance for conducting semi-field tunnel studies, and 4) design and interpretation of full field studies with bees. What follows is the current status of each of these activities

Higher TIER bumble bees and solitary bees recommendations for a semi-field experimental design

The publication of the proposed EFSA risk assessment guidance document of plant protection products for pollinators highlighted that there are no study designs for non-Apis pollinators available. Since no official guidelines exist for semi-field testing at present, protocols were proposed by the ICPPR non-Apis working group and two years of ring-testing were conducted in 2016 and 2017 to develop a general test set-up. The ringtest design was based on the draft EFSA guidance document, OEPP/EPPO Guideline No. 170 and results of discussions regarding testing solitary bees and bumble bees during the meetings of the ICPPR non-Apis workgroup. Ring-tests were conducted with two different test organisms, one representative of a social bumble bee species (Bombus terrestris L; Hymenoptera, Apidae) and one representative of a solitary bee species (Osmia bicornis L; Hymenoptera, Megachilidae). The species are common species in Europe, commercially available and widely used for pollination services. Several laboratories participated in the higher-tier ring tests. 15 semi-field tests were conducted with bumble bees and 16 semi-field tests were done with solitary bees in 2016 and 2017. Two treatment groups were always included in the ringtests: an untreated control (water treated) and the treatment with dimethoate as a toxic reference item (optional other i.e. brood-affecting substances fenoxycarb or diflubenzuron). The toxic reference items were chosen based on their mode of action and long term experience in honey bee testing. A summary of the ringtest results will be given and the recommendations for the two semi-field test designs will be presented.The publication of the proposed EFSA risk assessment guidance document of plant protection products for pollinators highlighted that there are no study designs for non-Apis pollinators available. Since no official guidelines exist for semi-field testing at present, protocols were proposed by the ICPPR non-Apis working group and two years of ring-testing were conducted in 2016 and 2017 to develop a general test set-up. The ringtest design was based on the draft EFSA guidance document, OEPP/EPPO Guideline No. 170 and results of discussions regarding testing solitary bees and bumble bees during the meetings of the ICPPR non-Apis workgroup. Ring-tests were conducted with two different test organisms, one representative of a social bumble bee species (Bombus terrestris L; Hymenoptera, Apidae) and one representative of a solitary bee species (Osmia bicornis L; Hymenoptera, Megachilidae). The species are common species in Europe, commercially available and widely used for pollination services. Several laboratories participated in the higher-tier ring tests. 15 semi-field tests were conducted with bumble bees and 16 semi-field tests were done with solitary bees in 2016 and 2017. Two treatment groups were always included in the ringtests: an untreated control (water treated) and the treatment with dimethoate as a toxic reference item (optional other i.e. brood-affecting substances fenoxycarb or diflubenzuron). The toxic reference items were chosen based on their mode of action and long term experience in honey bee testing. A summary of the ringtest results will be given and the recommendations for the two semi-field test designs will be presented

Results of Ring-Testing of a Semifield Study Design to Investigate Potential Impacts of Crop Protection Products on Bumblebees (Hymenoptera, Apidae) and a Proposal of a Potential Test Design

In Europe, the risk assessment for bees at the European Union or national level has always focussed on potential impacts on honeybees. During the revision of the European Food Safety Authority bee guidance it was explicitly stated that bumblebees and solitary bees should be considered as well and consequently concerns were raised regarding the representativeness of honeybees for these other bee species. These concerns originate from differences in size as well as differences in behavioral and life history traits of other bee species. In response to this concern, the non-Apis working group of the International Commission for Plant-Pollinator Relationships initiated a ring-test of a semifield tunnel study design using the bumblebee Bombus terrestris. Nine laboratories participated, validating and improving the proposed design over a 2-year period. The intention of the ring-test experiments was to develop and if possible, establish a test protocol to conduct more standardized semifield tests with bumblebees. In the present study, the results of the ring-tests are summarized and discussed to give recommendations for a promising experimental design. Environ Toxicol Chem 2022;00:1–17