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Among bee species, the western honey bee (Apis mellifera) is preferred in monitoring studies performed in the agricultural landscape, while bee matrices, pollen, and honey are mostly a subject of these studies due to their unique composition. A justified question about the relevance of other bee matrices, like larvae, foragers, beebread, and/or wax, has been raised. The ability of different bee matrices (wax, pollen grains, bee bread, foragers, larvae, nectar, and honey) to absorb pesticide residues is subjected in this study. All samples were collected during a crop flowering season (oilseed rape) on intensively managed agricultural land in Slovakia and Germany. The observed high variability in residue levels, profile, and number of detections among studied matrices from Germany, west, and east Slovakia gave us an assumption of the use of different agricultural practices between these two countries. Fungicides clearly dominated across all samples in all sampling regions. The increased pesticide profile positively correlated with the oilseed rape pollen grains in pollen pellets and/or bee bread. Bee wax, pollen, and bee bread showed a high number of detected active substances and total residue concentrations among matrices, indicating their high ability to absorb pesticide residues in the surrounding hive environment.

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The use of coloured pan traps (bee bowls, Moericke traps) for sampling bees (and other pollinators) has continuously increased over the last two decades. Although a number of methodological studies and conceptual frameworks offer guidance on standardised sampling, pan trap setups vary widely in characteristics even when optimised for capturing bees. Moreover, some uncertainty persists as to how local flower abundance and diversity influence sampling. We systematically reviewed peer-reviewed studies that used pan traps for bee collection and that were listed in the Web of Science core collection. To gauge methodological variation, we identified a set of relevant methodological criteria and assessed the studies accordingly. For obtaining evidence that pan trap samples and floral environment around traps are correlated, we screened the relevant studies for such correlations. While some aspects of pan trapping (e.g., trap coloration and elevation) were similar in the majority of studies, other aspects varied considerably (e.g., trap volume/diameter and sampling duration). Few studies used floral abundance and/or diversity as an explanatory variable in their analyses of bee samples. Among these studies, we found a considerable variation in key aspects of floral survey methods, such as time and space between vegetation surveys and pan trap sampling, abundance measures (quantitative, semi-quantitative and presence-absence), and processing of raw data prior to analysis. Often studies did not find any correlation between the floral environment and bee samples. Reported correlations varied markedly across studies, even within groups of studies applying a similar method or analysing a similar group of bees. Our synthesis helps to identify key issues of further standardisation of pan trap methodology and of associated floral surveys. In addition to the few aspects that have been standardised over the past decades, we suggest methodological direction for future research using pan traps as a better standardised method for the collection of wild bees. We encourage further studies to illuminate if and how varying floral resources around traps bias bee samples from pan traps. More generally, our synthesis shows that trapping methodologies should be reviewed regularly when their use increases to ensure standardisation.

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The proposed use of equivalence tests instead of difference tests in the revised guidance on the risk assessment of plant protection products for bees is a reasonable approach given an adverse effect was observed in the lower tier studies, using the hypothesis that there is a risk as the null hypothesis places the burden to prove the opposite on the other side. However, some uncertainties regarding the application of equivalence tests in field studies are discussed in the present study. Here, we compare equivalence and difference testing methods using a control dataset of a honey bee field effect study conducted in northern Germany in 2014. Half of the 48 colonies were assigned to a hypothetical test item group, and the colony strength data were analyzed using t-tests, a generalized linear mixed model (GLMM), and the corresponding equivalence tests. The data reflected the natural variability of honey bee colonies, with initially approximately 12 000 adult bees. Although the t-test and GLMM confirmed that 24 + 24 colonies are sufficient to show "no adverse effect," the equivalence tests of the t-test and GLMM were not able to reject the null hypothesis and classified at least some of the assessments as "high risk," indicating a power that was too low. Based on this, different operating options to reduce the variability are discussed. One possible option, which may provide a more realistic application of equivalence to avoid false high risk, is to consider the lower confidence interval of the control as a baseline and use GLMMs. With this option, we demonstrate a relatively acceptable probability to prove that no high risk for initially similar groups can be achieved. Further studies with different numbers of colonies are still needed to develop and validate the suggested approach. Integr Environ Assess Manag 2024;20:1496-1503. © 2024 SETAC.

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Biopesticides, having as active ingredients viruses, bacteria, or fungi, are developed to substitute or reduce the use of chemical plant protection products in different agrosystems. Though the application of mixtures containing several products is a common practice, interactions between microbial biopesticides and related effects on bees as non-target organisms have not been studied yet. In the current study, we exposed winter bees to five different microbial-based products and their combinations at the maximum recommended application rate to assess their responses. Laboratory oral exposure tests (acute/chronic) to single or binary products were conducted. Survival and food consumption of the tested bees were evaluated over the experimental duration. Our results show that some product combinations have potential additive or synergistic effects on bees, whereas others did not affect the bee's survival compared to the control. Exposure of tested bees to the most critical combination of products containing Bacillus thuringiensis aizawai ABTS-1857 and B. amyloliquefaciens QST 713 strongly resulted in a median lifespan of 4.5 days compared to 8.0 and 8.5 days after exposure to the solo products, respectively. The exposure to inactivated microorganisms by autoclaving them did not differ from their respective uncontaminated negative controls, indicating effects on bee mortality might originate in the treatment with the different microorganisms or their metabolites. Further investigations should be conducted under field conditions to prove the magnitude of observed effects on bee colonies and other bee species.

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In the European registration process, pesticides are currently mainly tested on the honey bee. Since sensitivity data for other bee species are lacking for the majority of xenobiotics, it is unclear if and to which extent this model species can adequately serve as surrogate for all wild bees. Here, we investigated the effects of field-realistic contact exposure to a pyrethroid insecticide, containing lambda-cyhalothrin, on seven bee species (Andrena vaga, Bombus terrestris, Colletes cunicularius, Osmia bicornis, Osmia cornuta, Megachile rotundata, Apis mellifera) with different life history characteristics in a series of laboratory trials over two years. Our results on sensitivity showed significant species-specific responses to the pesticide at a field-realistic application rate (i.e., 7.5 g a.s./ha). Species did not group into distinct classes of high and low mortality. Bumble bee and mason bee survival was the least affected by the insecticide, and M. rotundata survival was the most affected with all individuals dead 48 h after application. Apis mellifera showed medium mortality compared to the other bee species. Most sublethal effects, i.e. behavioral abnormalities, were observed within the first hours after application. In some of the solitary species, for example O. bicornis and A. vaga, a higher percentage of individuals performed some abnormal behavior for longer until the end of the observation period. While individual bee weight explained some of the observed mortality patterns, differences are likely linked to additional ecological, phylogenetic or toxicogenomic parameters as well. Our results support the idea that honey bee data can be substitute for some bee species' sensitivity and may justify the usage of safety factors. To adequately cover more sensitive species, a larger set of bee species should be considered for risk assessment.

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As part of the agricultural landscape, non-target organisms, such as bees, may be exposed to a cocktail of agrochemicals including insecticides and spray adjuvants like organosilicone surfactants (OSS). While the risks of insecticides are evaluated extensively in their approval process, in most parts of the world however, authorization of adjuvants is performed without prior examination of the effects on bees. Nevertheless, recent laboratory studies evidence that adjuvants can have a toxicity increasing effect when mixed with insecticides. Therefore, this semi-field study aims to test whether an OSS mixed with insecticides can influence the insecticidal activity causing increased effects on bees and bee colonies under more realistic exposure conditions. To answer this question a pyrethroid (Karate Zeon) and a carbamate (Pirimor Granulat) were applied in a highly bee attractive crop (oil seed rape) during bee flight either alone or mixed with the OSS Break-Thru S 301 at field realistic application rates. The following parameters were assessed: mortality, flower visitation, population and brood development of full-sized bee colonies. Our results show that none of the above mentioned parameters was significantly affected by the insecticides alone or their combination with the adjuvant, except for a reduced flower visitation rate in both carbamate treatments (Tukey-HSD, p < 0.05). This indicates that the OSS did not increase mortality to a biologically relevant extent or any of the parameters observed on honey bees and colonies in this trial. Hence, social buffering may have played a crucial role in increasing thresholds for such environmental stressors. We confirm that the results of laboratory studies on individual bees cannot necessarily be extrapolated to the colony level and further trials with additional combinations are required for a well-founded evaluation of these substances.

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In 2018 the European Union (EU) banned the three neonicotinoid insecticides imidacloprid, clothianidin (CLO), and thiamethoxam (TMX), but they can still be used if an EU Member State issues an emergency approval. Such an approval went into effect in 2021 for TMX-coated sugar beet seeds in Germany. Usually, this crop is harvested before flowering without exposing non-target organisms to the active ingredient or its metabolites. In addition to the approval, strict mitigation measures were imposed by the EU and the German federal states. One of the measures was to monitor the drilling of sugar beet and its impact on the environment. Hence we took residue samples from different bee and plant matrices and at different dates to fully map beet growth in the German states of Lower Saxony, Bavaria, and Baden-Württemberg. A total of four treated and three untreated plots were surveyed, resulting in 189 samples. Residue data were evaluated using the US Environmental Protection Agency BeeREX model to assess acute and chronic risk to honey bees from the samples, because oral toxicity data are widely available for both TMX and CLO. Within treated plots, we found no residues either in pools of nectar and honey crop samples (n = 24) or dead bee samples (n = 21). Although 13% of beebread and pollen samples and 88% of weed and sugar beet shoot samples were positive, the BeeREX model found no evidence of acute or chronic risk. We also detected neonicotinoid residues in the nesting material of the solitary bee Osmia bicornis, probably from contaminated soil of a treated plot. All control plots were free of residues. Currently, there are insufficient data on wild bee species to allow for an individual risk assessment. In terms of the future use of these highly potent insecticides, therefore, it must be ensured that all regulatory requirements are complied with to mitigate any unintentional exposure. Environ Toxicol Chem 2023;42:1167-1177. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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The western honey bee Apis mellifera is globally distributed due to its beekeeping advantages and plays an important role in the global ecology and economy. In recent decades, several studies have raised concerns about bee decline. Discussed are multiple reasons such as increased pathogen pressure, malnutrition or pesticide use. Insecticides are considered to be one of the major factors. In 2013, the use of three neonicotinoids in the field was prohibited in the EU. Flupyradifurone was introduced as a potential successor; it has a comparable mode of action as the banned neonicotinoids. However, there is a limited number of studies on the effects of sublethal concentrations of flupyradifurone on honey bees. Particularly, the larval physiological response by means of protein expression has not yet been studied. Hence, the larval protein expression was investigated via 2D gel electrophoresis after following a standardised protocol to apply sublethal concentrations of the active substance (flupyradifurone 10 mg/kg diet) to larval food. The treated larvae did not show increased mortality or an aberrant development. Proteome comparisons showed clear differences concerning the larval metabolism, immune response and energy supply. Further field studies are needed to validate the in vitro results at a colony level.

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The response of bee species to various stressors is assumed to depend on the availability of sufficient nutrients in their environment. We compare the response of three bee species (Apis mellifera, Bombus terrestris, Osmia bicornis) under laboratory conditions. Survival, physiology, and sensitivity, after exposure to the fungicide prochloraz, the insecticide chlorantraniliprole, and their mixture with different nutritional resources (sugar only, sugar with amino acids or pollen) were observed. Prochloraz reduced the bee survival of A. mellifera and O. bicornis fed with pollen, but not with other diets. Chlorantraniliprole impaired the survival of A. mellifera fed with sugar or pollen diet, but not with amino acid diet. The mixture impaired survival of A. mellifera and O. bicornis in association with every diet. B. terrestris was only affected by chlorantraniliprole and its mixture with prochloraz fed with sugar diet. The activity of P450 reductase was higher in A. mellifera fed with amino acids in all treatments, whereas no effect emerged in O. bicornis and B. terrestris. Our results indicate that the sensitivity of bee species after exposure to agrochemicals is affected by diet. Thus, balanced and species-dependent nutrition ameliorated the effects. Further field studies are necessary to evaluate the potential effects of such mixtures on bee populations.

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To avoid potential adverse side effects of chemical plant protection products, microbial pest control products (MPCP) are commonly applied as biological alternatives. This study aimed to evaluate the biosafety of a MPCP with the active organism Bacillus thuringiensis ssp. aizawai (strain: ABTS-1857). An in-hive feeding experiment was performed under field-realistic conditions to examine the effect of B. thuringiensis (B. t.) on brood development and the bacterial abundance of the core gut microbiome (Bifidobacterium asteroids, Gilliamella apicola, the group of Lactobacillus and Snodgrasella alvi) in Apis mellifera worker bees. We detected a higher brood termination rate and a non-successful development into worker bees of treated colonies compared to those of the controls. For the gut microbiome, all tested core members showed a significantly lower normalized abundance in bees of the treated colonies than in those of the controls; thus, a general response of the gut microbiome may be assumed. Consequently, colony exposure to B. t. strain ABTS-1857 had a negative effect on brood development under field-realistic conditions and caused dysbiosis of the gut microbiome. Further studies with B. t.-based products, after field-realistic application in bee attractive crops, are needed to evaluate the potential risk of these MPCPs on honey bees.

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Risk assessment of plant protection products (PPPs) will be conducted before authorization for their possible effects on non-target organisms, including honey bees. Tank mixtures are often common practice by farmers, and mostly their effects on honey bees are not routinely assessed. To enable a realistic assessment of laboratory-reported effects of a combination of the insecticide thiacloprid and fungicide prochloraz on honey bees, a large-scale field study with spray application in winter oilseed rape was conducted in four regions in Germany. Several parameters were investigated, including mortality, flight activity, and colony development. Residue analysis of various materials (e.g., dead bees, nectar, and pollen) was conducted to assess exposure level. We observed several intoxication symptoms 2 h after application, including a high number of moribund bees and dead bees on the first day after application (DAA +1) compared to the control. Adverse effects were observed on the number of open brood cells, with a significant reduction of approximately 22% compared to control over the experimental period. High residue concentrations were detected on flowers and dead bees on the day of application, which decreased rapidly within six days. The residue concentrations detected were higher in bee-collected materials than in materials stored in the hive. In conclusion, exposure to a combination containing thiacloprid-prochloraz poses a high risk to honey bees. Thus, the application of such a mixture on flowering crops is restricted in Germany.

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Beeswax is known to have a high capacity to accumulate different contaminants due to its fat-soluble properties. Many surveys in Europe and the USA have shown high levels of contamination in beeswax especially with acaricides used for varroa treatment. In this study, we investigated the transfer pathways of various active substances from beeswax into different matrices under field conditions. Honey, bee bread, larvae, and pupae samples were collected 6-8 weeks after building the experimental colonies on different charges of wax foundations. Identification and quantification of the target substances were performed with an established and validated multi-residue method using LC-MS/MS and GC-MS systems. Nine out of 19 active substances in wax could be detected in the analyzed matrices. Our results confirm the migration of different contaminants from wax into different bee matrices including honey, bee bread, and bee brood. The concentration of detected residues in the different matrices was significantly increased by increasing residue concentration in wax. Therefore, the maximum detected residues in the matrices were almost in wax containing high residual concentrations. Bee bread can be considered as the most important matrix due to relatively high detected concentrations and transfer ratios of the most contaminants. A significant effect of the lipophilicity of active substances on the transfer ratio into bee bread was found, which means that increasing the Log P values has positive effects on the transfer ratio. In conclusion, our results provide the first detailed information regarding the migration of active substances from wax into various matrices under realistic field conditions and are fundamentally important for assessing potential exposure and risks for honey bees.

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The increasing use of Bacillus thuringiensis (Bt)-based plant protection products (PPPs) has recently raised some concerns regarding their environmental accumulation and possible chronic exposure of non-target species, including pollinators, to higher than expected doses. The exposure level of such microbial PPPs in bee's matrices under field conditions has not yet been described. Therefore, the current study aims at evaluating the realistic exposure level and comparing the distributions and persistence of Bt spores under field conditions. A field trial with spray application in oilseed rape (Brassica napus) as a representative bee-attractive crop was conducted. During the experimental period, different matrices, including honeybee-collected and -stored matrices as well as bee larvae and dead bees, were collected and analyzed using newly established methods. The concentration of Bt spores in the various matrices was quantified. The results show high levels of Bt spores in honey sac and pollen pellets with reduction over time but no reduction of Bt spores in the stored matrices within the colony, i.e., nectar and bee bread, over time. Our results show for the first time the exposure level of bees to Bt spores under realistic field conditions and are fundamentally important for assessing potential exposure and risks for pollinators.

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Honey bee health is affected by multiple stressors, such as the exposure to plant protection products (PPPs), dietary limitation, monofloral diets and pressure of diseases and pathogens and their interactions. Here, we analysed the interacting effects of plant protection products and low nutritional pollen source on honey bee health under semi-field conditions. We established a healthy honey bee colony in each of 24 tents, planted either with monofloral maize, maize with a diverse flower strip or with monofloral Phacelia tanacetifolia. To evaluate the interaction between exposure to PPPs and nutritional status, a mixture of the insecticide thiacloprid and the fungicide prochloraz was applied. For each colony, we investigated brood capping rate as well as adult longevity, body and head weight, and enzyme activity of acetylcholinesterase and P450 reductase of newly hatched worker bees. We found a significant reduced capping rate in treated maize compared to flowering strips and Phacelia, but no interaction effect between pesticide treatment and nutritional status on capping rate. The response to treatment on the longevity of adults differed significantly between maize and Phacelia, with flower strips being intermediate, indicating interaction effects of PPP treatment and low pollen quality in maize compared to Phacelia and flowering strip treatments. Head weight of newly hatched worker bees showed significant interaction of nutritional status and treatment of PPPs. PPPs slightly increased body weight in all nutritional statuses, except for Phacelia. Enzyme activity of acetylcholinesterase and P450 reductase showed significant different responses between maize and Phacelia to PPP exposure, but not between maize and flowering strip. Our results support the hypothesis that higher pollen quality promotes development of larvae and pupae, longevity of adults and detoxification of PPPs.

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The side effects from the use of plant protection products and their potential effects on non-target arthropods (NTAs) such as honey bees, other insects within the vegetation layer and epigeic arthropods nowadays receive more attention. However, uncertainties about the factors driving the deposition of active substances (a.s.) into off-crop areas persist, in particular during sowing of treated seeds. Analysing a highly standardised 8-year field experiment, we assessed the importance of various factors potentially affecting dust drift and deposition of a.s., emitted during the sowing process of treated seeds and deposited on fields adjacent to the drilling field, i.e. on the ground, on flowers, and on nonflowering plant parts. Regarding a.s. deposition, the Heubach a.s. value has a predictive capability, which is independent from all other factors taken into account in this study, and can thus be considered as a scenario-independent measure of potential dust deposition. Petri dish samplers, an established standard method for measuring a.s. deposition, were representative of the results from the plant samplers for a given combination of drilling technique and adjacent crop type. Adjacent crop type is likely to impact on a.s. deposition. The present work will enable a more field-realistic exposure assessment for bees and other NTAs.

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This study was aimed at evaluating the effect of a microbial pest-controlling product (MPCP) with the active substance Bacillus thuringiensis ssp. aizawai (strain: ABTS-1857) on adults and larvae of honeybees. To determine the contamination levels of Bt spores in different matrices, a colony-feeding study under semi-field conditions was performed. Furthermore, two chronic adult trials and a chronic larval study were conducted under laboratory conditions to test the effects of different concentrations of the plant protection product (PPP) on the development and mortality. Possible modifications of the chronic oral toxicity test were assessed by additional pollen feeding. Our results showed that Bt spores were detected in all matrices over the entire test duration in different concentrations, decreasing over time. The survival of adult bees and larvae was negatively affected in laboratory conditions after a chronic exposure to the MPCP depending on the tested concentrations. Moreover, the earliest sign of bee mortality, resulting from exposure to ABTS-1857, was recorded only after 96 h at the highest tested concentration. Pollen feeding to adults significantly increased the survival of the treated bees. In conclusion, the PPP with the Bt strain ABTS-1857 showed an effect on the mortality of adults and larvae under laboratory conditions. Further studies with Bt-based PPPs under realistic field conditions are necessary to evaluate the potential risk of those MPCPs on honeybees.

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The ongoing debate about glyphosate-based herbicides (GBH) and their implications for beneficial arthropods gives rise to controversy. This research was carried out to cover possible sublethal GBH effects on the brood and colony development, adult survival, and overwintering success of honey bees (Apis mellifera L.) under field conditions. Residues in bee relevant matrices, such as nectar, pollen, and plants, were additionally measured. To address these questions, we adopted four independent study approaches. For brood effects and survival, we orally exposed mini-hives housed in the "Kieler mating-nuc" system to sublethal concentrations of 4.8 mg glyphosate/kg (T1, low) and 137.6 mg glyphosate/kg (T2, high) over a period of one brood cycle (21 days). Brood development and colony conditions were assessed after a modified OECD method (No. 75). For adult survival, we weighed and labeled freshly emerged workers from control and exposed colonies and introduced them into non-contaminated mini-hives to monitor their life span for 25 consecutive days. The results from these experiments showed a trivial effect of GBH on colony conditions and the survival of individual workers, even though the hatching weight was reduced in T2. The brood termination rate (BTR) in the T2 treatment, however, was more than doubled (49.84%) when compared to the control (22.11%) or T1 (20.69%). This was surprising as T2 colonies gained similar weight and similar numbers of bees per colony compared to the control, indicating an equal performance. Obviously, the brood development in T2 was not "terminated" as expected by the OECD method terminology, but rather "slowed down" for an unknown period of time. In light of these findings, we suggest that chronic high GBH exposure is capable of significantly delaying worker brood development, while no further detrimental effects seem to appear at the colony level. Against this background, we discuss additional results and possible consequences of GBH for honey bee health.

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For the last decade, scientists have reported a loss of honeybee colonies. Multiple factors like parasites, pathogens and pesticides are dealt as possible drivers of honeybee losses. In particular, insecticides are considered as a major factor of pollinator poisoning. We applied sublethal concentrations of four insecticidal substances to honeybee larval food and analyzed the effects on transcriptome. The aim was to identify candidate genes indicating early negative impacts after application of insecticidal substances. Honeybee larvae were kept in-vitro under hive conditions (34-35 °C) and fed with dimethoate, fenoxycarb, chlorantraniliprole and flupyradifurone in sublethal concentrations between day 3-6 after grafting. Larvae at day 4, 6 and 8 were sampled and their transcriptome analyzed. By use of a RT-qPCR array differences in gene expression of selected gene families (immune system, development detoxification) were measured. Targets mainly involved in development, energy metabolism and the immune system were significantly affected by the insecticidal substances tested, selectively inducing genes of the detoxification system, immune response and nutritional stress.

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Recent studies have reported interspecific differences in how bee species respond to various stressors. Evaluating the exposure and responses of different bee species to plant protection products is considered an essential part of their risk assessment. This study was conducted to assess the impacts of thiacloprid-prochloraz mixture on buff-tailed bumblebees (Bombus terrestris) and red mason bees (Osmia bicornis) in a worst-case scenario under semi-field conditions. Bumblebee colonies or solitary bee trap nests were confined in tunnels with flowering oilseed rape. The recommended maximum application rates of 72 g thiacloprid/ha and 675 g prochloraz/ha were applied as a tank mixture during bee flight in full flowering oilseed rape. Several parameters such as flight and foraging activity, population parameters, and exposure level were investigated. Our results show adverse effects of the combination of thiacloprid and prochloraz on the reproductive performance of red mason bees. The number of cocoons produced by O. bicornis was significantly reduced in the treatment compared to the control group. Regarding bumblebees, we found no effects of the thiacloprid-prochloraz mixture on any observed parameters of colony development. The maximum detected concentrations of both active substances three days after application were higher in O. bicornis pollen mass compared to B. terrestris stored pollen. We conclude that this worst-case scenario of thiacloprid-prochloraz exposure poses a high risk to solitary bees and thus the use of such mixture should be restricted.

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