1.22 Weight differences of honey bees after administration of sublethal doses of dimethoate

Background: The aim of this work was to assess honey bee body weight as a possible further parameter to detect effects in a 10 day chronic feeding study according to OECD 2451 following exposure to sublethal concentrations of a plant protection product (i.e. dimethoate). This investigation is based on the assumption that weight differences might be caused by chronic feeding of dimethoate. Two set of tests in two different laboratories (Lab 1 and Lab 2) were conducted in order to investigate possible weight changes of complete adult honey bees and/or parts of their body (honey stomach and intestine) following treatment of dimethoate. Bees were weighed before and after chronic feeding of sub-lethal concentrations of dimethoate. Results: Differences in the number of bees which lost weight following treatment of sublethal concentrations of dimethoate was found in Lab 1, but could not confirmed in Lab 2. The difference in weight between the control group and the dimethoate treatment could only be detected as a statistical significant difference in one lab at the highest concentration (0.4 mg/kg). Assessment of weight changes of parts of the bee body (honey stomach and intestine) shows a very high variation (CV) which makes interpretation of the data of the total body weight questionable. Conclusion: The results of the two laboratories were contradictory and no conclusive assessment can be done following the two sets of experiments. Assessment of bee body weight within a 10-day chronic feeding study is considered questionable for the detection of sublethal effects. Further work with other active ingredients is needed to clarify if body weight change of honey bees can be used as a parameter for sublethal effects.Background: The aim of this work was to assess honey bee body weight as a possible further parameter to detect effects in a 10 day chronic feeding study according to OECD 2451 following exposure to sublethal concentrations of a plant protection product (i.e. dimethoate). This investigation is based on the assumption that weight differences might be caused by chronic feeding of dimethoate. Two set of tests in two different laboratories (Lab 1 and Lab 2) were conducted in order to investigate possible weight changes of complete adult honey bees and/or parts of their body (honey stomach and intestine) following treatment of dimethoate. Bees were weighed before and after chronic feeding of sub-lethal concentrations of dimethoate. Results: Differences in the number of bees which lost weight following treatment of sublethal concentrations of dimethoate was found in Lab 1, but could not confirmed in Lab 2. The difference in weight between the control group and the dimethoate treatment could only be detected as a statistical significant difference in one lab at the highest concentration (0.4 mg/kg). Assessment of weight changes of parts of the bee body (honey stomach and intestine) shows a very high variation (CV) which makes interpretation of the data of the total body weight questionable. Conclusion: The results of the two laboratories were contradictory and no conclusive assessment can be done following the two sets of experiments. Assessment of bee body weight within a 10-day chronic feeding study is considered questionable for the detection of sublethal effects. Further work with other active ingredients is needed to clarify if body weight change of honey bees can be used as a parameter for sublethal effects

Proposal for a new OECD guideline for the testing of chemicals on adult honey bees (Apis mellifera L.) in a 10 day chronic feeding test in the laboratory and results of the recent ring test 2014

Background - Even though the evaluation of potential chronic oral effects on adult honey bees (Apis mellifera L.) is an integral part of the risk assessment according to e.g. the EC Regulation 1107/2009 and the EFSA Guidance Document, (EFSA 2013), there is no validated guideline available for this test system, yet. To address this new requirement and to develop a new test guideline an international ring test group was founded and a ring test was carried out in summer 2014. The ring test was carried out on the basis of a test protocol, which followed the recommendation for the proposed guideline. Results - A validity criterion for the control mortality of ≤ 15 % was met for the untreated control group in all tests and laboratories within the first run. However, for the solvent group this validity criterion could not be met in 7 out of 17 labs. In the reference item treatment group clear doseresponse correlation could be observed with the tested concentration levels and the mean LC50 and LDD50 values could be calculated, as well as the NOEC and NOEDD levels.Conclusion - The results gained in these tests indicate the suitability and reproducibility of the described test method which could serve as a basis for an official test guideline. However, the use of acetone as solvent at the tested concentration level is still questioned. Keywords: chronic toxicity, honey bee, laboratory tes

Laboratory acute contact toxicity test with the leafcutter bee Megachile rotundata

So far little is known about the toxicity of Plant Protection Products (PPPs) to solitary bees other than Osmia spp. as well as the inter- and intra-species sensitivity differences of honey bees and solitary bees.Megachile rotundata is a commercially bred solitary bee which is used worldwide mainly for the pollination of alfalfa. In general, bees can be exposed to PPPs directly by contact spray application (overspray) or indirectly via nectar and pollen. The leafcutter bees additionally can be exposed to (possibly) contaminated leaf pieces which are used for the building of brood cells. Therefore, contact toxicity might be of major importance within leafcutter bee species. Acute contact toxicity tests with M. rotundata based on the existing honey bee testing guideline OECD No. 214 were carried out, to make a first step in the direction of the development of a standard test method and collect data for the comparison of inter- and intra-species contact toxicity sensitivity. The toxic reference substance dimethoate was used as test substance. LD50/24h values of M. rotundata were compared to values of A.mellifera generated in a similar period of time. The low mortality observed in the control also after 96 hours, confirms the feasibility and reliability of the test method. The LD50/24h values of M. rotundata in all four tests were higher compared to those of A. mellifera. Accordingly, M. rotundata appeared to be slightly less sensitive to formulated dimethoate than A. mellifera.So far little is known about the toxicity of Plant Protection Products (PPPs) to solitary bees other than Osmia spp. as well as the inter- and intra-species sensitivity differences of honey bees and solitary bees.Megachile rotundata is a commercially bred solitary bee which is used worldwide mainly for the pollination of alfalfa. In general, bees can be exposed to PPPs directly by contact spray application (overspray) or indirectly via nectar and pollen. The leafcutter bees additionally can be exposed to (possibly) contaminated leaf pieces which are used for the building of brood cells. Therefore, contact toxicity might be of major importance within leafcutter bee species. Acute contact toxicity tests with M. rotundata based on the existing honey bee testing guideline OECD No. 214 were carried out, to make a first step in the direction of the development of a standard test method and collect data for the comparison of inter- and intra-species contact toxicity sensitivity. The toxic reference substance dimethoate was used as test substance. LD50/24h values of M. rotundata were compared to values of A.mellifera generated in a similar period of time. The low mortality observed in the control also after 96 hours, confirms the feasibility and reliability of the test method. The LD50/24h values of M. rotundata in all four tests were higher compared to those of A. mellifera. Accordingly, M. rotundata appeared to be slightly less sensitive to formulated dimethoate than A. mellifera

5.10 How do Regulatory Requirements and Assumptions Correlate to Practical Experience in Residue Studies with Nectar and Pollen?

Residues of pesticides detected in pollen and nectar (bee relevant matrices) represent a realistic research approach to estimate pollinator exposure. Therefore, a robust and reliable method to sample and measure these residues is part of risk assessment schemes in several parts of the world. EFSA guidance for pollinators was the first risk assessment to allow for the refinements of the expected residue values during exposure. EPA as well as IBAMA followed suite and proposed in vivo refinements for residue values. To achieve this goal nectar and pollen from plant species have to be collected in sufficient amounts to allow for residue analysis. Several methods are available for the collection of bee matrices. We list general methods developed to sample pollen and nectar, focus on some common issues encountered during the conduct of these studies and place the measurements derived from these studies into a risk assessment context. With all the information available now it would be a useful task to compare residue levels in matrices collected manually and with the help of pollinators to give advice for guidance document refinements and help to approve the design of studies in the future.Residues of pesticides detected in pollen and nectar (bee relevant matrices) represent a realistic research approach to estimate pollinator exposure. Therefore, a robust and reliable method to sample and measure these residues is part of risk assessment schemes in several parts of the world. EFSA guidance for pollinators was the first risk assessment to allow for the refinements of the expected residue values during exposure. EPA as well as IBAMA followed suite and proposed in vivo refinements for residue values. To achieve this goal nectar and pollen from plant species have to be collected in sufficient amounts to allow for residue analysis. Several methods are available for the collection of bee matrices. We list general methods developed to sample pollen and nectar, focus on some common issues encountered during the conduct of these studies and place the measurements derived from these studies into a risk assessment context. With all the information available now it would be a useful task to compare residue levels in matrices collected manually and with the help of pollinators to give advice for guidance document refinements and help to approve the design of studies in the future

1.17 Comparison of Control and Toxic Reference Data between Honey Bee Laboratory Studies Conducted in Germany and in Spain over the Last Decade

The Draft EFSA Bee Guidance Document (2013) describes various types of bee studies to be part of the risk assessment. Eurofins Agroscience Services (EAS) has been conducting acute toxicity and chronic feeding studies with adult bees over the last decade and larval acute and chronic studies over the last 5 years in Germany and in Spain. The studies are conducted with different subspecies and in different times of the year in the two countries.The aim of the comparison is to find out if season and geographical origin of the bees have any influence on the test outcome, i.e. control/solvent control mortality and reference item 24 h LD50 range with controls and toxic reference data collected over multiple years. The results give an indication how relevant the testing of different subspecies is for the registration of plant protection products in Europe.The Draft EFSA Bee Guidance Document (2013) describes various types of bee studies to be part of the risk assessment. Eurofins Agroscience Services (EAS) has been conducting acute toxicity and chronic feeding studies with adult bees over the last decade and larval acute and chronic studies over the last 5 years in Germany and in Spain. The studies are conducted with different subspecies and in different times of the year in the two countries.The aim of the comparison is to find out if season and geographical origin of the bees have any influence on the test outcome, i.e. control/solvent control mortality and reference item 24 h LD50 range with controls and toxic reference data collected over multiple years. The results give an indication how relevant the testing of different subspecies is for the registration of plant protection products in Europe