Pollinator monitoring in agroecosystems – general methods for evaluations in field studies

Extensive knowledge of the occurrence, condition and population changes of wild bee communities in agroecosystems is important. The knowledge is needed to understand the complexity of potential exposure routes to plant protection products in specific crops and agricultural scenarios or to evaluate possible impacts of treatments at a landscape scale taking into account other influencing parameters like the cultivation system or management practices.Extensive knowledge of the occurrence, condition and population changes of wild bee communities in agroecosystems is important. The knowledge is needed to understand the complexity of potential exposure routes to plant protection products in specific crops and agricultural scenarios or to evaluate possible impacts of treatments at a landscape scale taking into account other influencing parameters like the cultivation system or management practices

Impact of an Oomen feeding with a neonicotinoid on daily activity and colony development of honeybees assessed with an AI based monitoring device

Feeding experiments are standard tools in the pollinator risk assessment. The design (Oomen et al. 1992) was developed to test insect growth regulators and herbicides. In recent years there was an update (Lückmann & Schmitzer 2015) on the outline in order to also focus on the advantage of different rates making a dose response design possible where exposure levels are known. Additionally, this design gives the possibility to test different rates for honey bee colonies foraging in the same landscape. The main objective of the experiment presented here was to determine the natural variability of foragers losses of hives fed with a sub-lethal neonicotinoid concentration compared to an untreated control. Other objectives were to see if the neurotoxic exposure results in any observable sub-lethal effects and to find out if losses can be correlated to hive development. This was assessed with traditional methods and a novel, visual monitoring device.Feeding experiments are standard tools in the pollinator risk assessment. The design (Oomen et al. 1992) was developed to test insect growth regulators and herbicides. In recent years there was an update (Lückmann & Schmitzer 2015) on the outline in order to also focus on the advantage of different rates making a dose response design possible where exposure levels are known. Additionally, this design gives the possibility to test different rates for honey bee colonies foraging in the same landscape. The main objective of the experiment presented here was to determine the natural variability of foragers losses of hives fed with a sub-lethal neonicotinoid concentration compared to an untreated control. Other objectives were to see if the neurotoxic exposure results in any observable sub-lethal effects and to find out if losses can be correlated to hive development. This was assessed with traditional methods and a novel, visual monitoring device

Available methods for the sampling of nectar, pollen, and flowers of different plant species

Background: The new draft EFSA guidance document introduces additional assessment factors for pollinators other than honey bees. However, there are no standard test protocols available. Therefore, the only way for risk assessment refinements, are a more precise estimate of the potential exposure in nectar and pollen. The aim of the paper is to present available sampling methods of nectar and pollen but also tries to refine methodology for sampling of nectar and pollen mentioned in the guidance document.Results: Nectar can be collected by hand from a wide variety of crop plants. This can be done with the help of capillaries as well as with centrifugation. Pollen can be collected with manual sampling or the help of a suction pump. Bees and bumble bees can be used for both matrices with many plants. Solitary bees are able to collect pollen. More detailed results are presented for oil seed rape and Phacelia.Conclusion: Nectar and pollen can be collected from flowering crop plants visited by pollinators in amounts that are high enough to allow residue analysis. However, the minimum number of bees needed to collect the amount is not 20 but much higher, depending on the species of plant sampled. At least 200 honey bees should be collected for each matrix

Practical experiences with a syrup feeding study design based on a new MRL guideline SANTE11956/2016 rev.9 (2018)

A new study design, according to the guideline SANTE11956/2016 rev:9 (2018), was established to determine the maximum residue level (MRL) of plant protection products in honey. The guideline describes a syrup bee feeding study designed as a worst-case scenario for transferring plant protection products into honey. Previously, field and semi-field studies designs were used. The objectives of this study were to validate the suitability of this feeding semi-field studies according to the new guideline.A new study design, according to the guideline SANTE11956/2016 rev:9 (2018), was established to determine the maximum residue level (MRL) of plant protection products in honey. The guideline describes a syrup bee feeding study designed as a worst-case scenario for transferring plant protection products into honey. Previously, field and semi-field studies designs were used. The objectives of this study were to validate the suitability of this feeding semi-field studies according to the new guideline

Bee colony assessments with the Liebefeld method: How do individual beekeepers influence results and are photo assessments an option to reduce variability?

Colony strength, food storage and brood development are a fundamental part of each honeybee field study. Colony assessments are used to compare and assess those for beehive over time. At present, most colony assessments are made by experienced beekeepers according to Liebefeld method. This method is based on an estimation of areas covered by honeybees, food and brood stages on each side of a comb. Areas are counted from a grid separating the comb side into 8 sections which are protocolled with an accuracy of 0.5 sections. An assessment for a hive takes up to 20 min and even with two field locations, it is necessary to split assessments between beekeepers. So, it is important to make estimates as comparable as possible. For this purpose, beekeepers practice the assessments on pre-determined photographs to “calibrate themselves”. The advantage of the Liebefeld assessment is that the condition of bee hive is estimated with minimum disturbance of the bees. Digital photography is under discussion to gain data with high precision and accuracy with one major disadvantage. To be able to see food and brood stages in photographs, bees have to be removed from combs. This, however, results in a disturbance of the colony – especially if the assessments take place in short time intervals of 7 ± 1 days. An experiment was performed to evaluate the variation between individual beekeepers and to compare the results to data generated with photographs. For the experiment, five colonies were assessed each by four beekeepers independently according to Liebefeld method. Each comb side of the five colonies was photographed with and without honeybees sitting on it for precise analysis at the computer for a number of bees, nectar cells, pollen cells, eggs, open brood and capped brood. The number of bees and cells with the different contents were generated by an area-based assessment in ImageJ as well as a detailed counting with help of HiveAnalyzer® Software. Data from beekeeper estimations were then compared with assessments based on digital photography. With the results of the experiment, we tried to answer several questions. With the study, we wanted to determine the level of variation between the beekeepers for the live stages and food stores estimated.Colony strength, food storage and brood development are a fundamental part of each honeybee field study. Colony assessments are used to compare and assess those for beehive over time. At present, most colony assessments are made by experienced beekeepers according to Liebefeld method. This method is based on an estimation of areas covered by honeybees, food and brood stages on each side of a comb. Areas are counted from a grid separating the comb side into 8 sections which are protocolled with an accuracy of 0.5 sections. An assessment for a hive takes up to 20 min and even with two field locations, it is necessary to split assessments between beekeepers. So, it is important to make estimates as comparable as possible. For this purpose, beekeepers practice the assessments on pre-determined photographs to “calibrate themselves”. The advantage of the Liebefeld assessment is that the condition of bee hive is estimated with minimum disturbance of the bees. Digital photography is under discussion to gain data with high precision and accuracy with one major disadvantage. To be able to see food and brood stages in photographs, bees have to be removed from combs. This, however, results in a disturbance of the colony – especially if the assessments take place in short time intervals of 7 ± 1 days. An experiment was performed to evaluate the variation between individual beekeepers and to compare the results to data generated with photographs. For the experiment, five colonies were assessed each by four beekeepers independently according to Liebefeld method. Each comb side of the five colonies was photographed with and without honeybees sitting on it for precise analysis at the computer for a number of bees, nectar cells, pollen cells, eggs, open brood and capped brood. The number of bees and cells with the different contents were generated by an area-based assessment in ImageJ as well as a detailed counting with help of HiveAnalyzer® Software. Data from beekeeper estimations were then compared with assessments based on digital photography. With the results of the experiment, we tried to answer several questions. With the study, we wanted to determine the level of variation between the beekeepers for the live stages and food stores estimated

A framework for a European network for a systematic environmental impact assessment of genetically modified organisms (GMO)

The assessment of the impacts of growing genetically modified (GM) crops remains a major political and scientific challenge in Europe. Concerns have been raised by the evidence of adverse and unexpected environmental effects and differing opinions on the outcomes of environmental risk assessments (ERA). The current regulatory system is hampered by insufficiently developed methods for GM crop safety testing and introduction studies. Improvement to the regulatory system needs to address the lack of well designed GM crop monitoring frameworks, professional and financial conflicts of interest within the ERA research and testing community, weaknesses in consideration of stakeholder interests and specific regional conditions, and the lack of comprehensive assessments that address the environmental and socio economic risk assessment interface. To address these challenges, we propose a European Network for systematic GMO impact assessment (ENSyGMO) with the aim directly to enhance ERA and post-market environmental monitoring (PMEM) of GM crops, to harmonize and ultimately secure the long-term socio-political impact of the ERA process and the PMEM in the EU. These goals would be achieved with a multi-dimensional and multi-sector approach to GM crop impact assessment, targeting the variability and complexity of the EU agro-environment and the relationship with relevant socio-economic factors. Specifically, we propose to develop and apply methodologies for both indicator and field site selection for GM crop ERA and PMEM, embedded in an EU-wide typology of agro-environments. These methodologies should be applied in a pan-European field testing network using GM crops. The design of the field experiments and the sampling methodology at these field sites should follow specific hypotheses on GM crop effects and use state-of-the art sampling, statistics and modelling approaches. To address public concerns and create confidence in the ENSyGMO results, actors with relevant specialist knowledge from various sectors should be involved

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 an international ring test with the dung fly Musca autumnalis in support of a new OECD test guideline

A standardized bioassay using the face fly, Musca autumnalis L (Diptera: Muscidae), was developed to test the lethal and sublethal toxicity of parasiticide residues in livestock dung. The repeatability of this test was assessed for the parasiticide ivermectin in seven tests performed in four laboratories in Germany and France. Additional results of limit tests were provided by two laboratories from the UK. Test results had an acceptable range of heterogeneity. The calculated effect concentration at which 50% emergence was observed (EC50) averaged 4.65 +/- 2.17 (Standard Deviation (SD) mu g ivermectin/kg fresh dung (range: 1.20-7.7)). Effects on emergence were, with one exception, not observed below the No Observed Effect Concentration (NOEC) ranging between 1.11 and 3.33 mu g ivermectin/kg. No effect on development time was observed. We conclude that the face fly is suitably sensitive, and the methods sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals. Following these considerations, this species was accepted as a possible test organism in a recently published OECD Guideline (No. 228). (c) 2010 Elsevier B.V. All rights reserved

Results of 2-Year Ring Testing of a Semifield Study Design to Investigate Potential Impacts of Plant Protection Products on the Solitary Bees Osmia Bicornis and Osmia Cornuta and a Proposal for a Suitable Test Design

There are various differences in size, behavior, and life history traits of non-Apis bee species compared with honey bees (Apis mellifera; Linnaeus, 1758). Currently, the risk assessment for bees in the international and national process of authorizing plant protection products has been based on honey bee data as a surrogate organism for non-Apis bees. To evaluate the feasibility of a semifield tunnel test for Osmia bicornis (Linnaeus, 1758) and Osmia cornuta (Latreille, 1805), a protocol was developed by the non-Apis working group of the International Commission for Plant-Pollinator Relationships, consisting of experts from authorities, academia, and industry. A total of 25 studies were performed over a 2-yr period testing a replicated control against a replicated positive control using either a dimethoate or diflubenzuron treatment. Studies were regarded to be valid, if ≥30% of released females were found to occupy the nesting units in the night/morning before the application (establishment). Thirteen studies were regarded to be valid and were analyzed further. Parameters analyzed were nest occupation, flight activity, cell production (total and per female), cocoon production (total and per female), emergence success, sex ratio, and mean weight of females and males. Dimethoate was a reliable positive control at the tested rate of 75 g a.i./ha, once >30% females had established, displaying acute effects such as reduction in flight activity, increase in adult mortality (shown by nest occupation), and reproduction ability of the females (total cell and cocoon production). On the other hand, no effects on larval and pupal development were observed. The growth regulator diflubenzuron had statistically significant effects on brood development, causing mortality of eggs and larvae at a rate of approximately 200 g a.i./ha, whereas fenoxycarb did not cause any significant effects at the tested rates of 300 and 600 g a.i./ha. In conclusion, the ring-test protocol proved to be adequate once the study comprised a well-established population of female Osmia bees, and the results improved in the second year as the laboratories increased their experience with the test organism. It is noted that the success of a study strongly depends on the experience of the experimenter, the crop quality, the quality of the cocoons, and the weather conditions. Based on these finding, recommendations for a semifield study design with Osmia spp. are proposed. Environ Toxicol Chem 2020;00:1–15.</p