Honey bees for pesticide monitoring in the landscape: Which bee matrices should be used?

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.

Honey bee colonies can buffer short-term stressor effects of pollen restriction and fungicide exposure on colony development and the microbiome.

Honey bees (Apis mellifera) have to withstand various environmental stressors alone or in combination in agriculture settings. Plant protection products are applied to achieve high crop yield, but residues of their active substances are frequently detected in bee matrices and could affect honey bee colonies. In addition, intensified agriculture could lead to resource limitation for honey bees. This study aimed to compare the response of full-sized and nucleus colonies to the combined stressors of fungicide exposure and resource limitation. A large-scale field study was conducted simultaneously at five different locations across Germany, starting in spring 2022 and continuing through spring 2023. The fungicide formulation Pictor® Active (active ingredients boscalid and pyraclostrobin) was applied according to label instructions at the maximum recommended rate on oil seed rape crops. Resource limitation was ensured by pollen restriction using a pollen trap and stressor responses were evaluated by assessing colony development, brood development, and core gut microbiome alterations. Furthermore, effects on the plant nectar microbiome were assessed since nectar inhabiting yeast are beneficial for pollination. We showed, that honey bee colonies were able to compensate for the combined stressor effects within six weeks. Nucleus colonies exposed to the combined stressors showed a short-term response with a less favorable brood to bee ratio and reduced colony development in May. No further impacts were observed in either the nucleus colonies or the full-sized colonies from July until the following spring. In addition, no fungicide-dependent differences were found in core gut and nectar microbiomes, and these differences were not distinguishable from local or environmental effects. Therefore, the provision of sufficient resources is important to increase the resilience of honey bees to a combination of stressors.

Exposure of honey bees to mixtures of microbial biopesticides and their effects on bee survival under laboratory conditions.

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.

Nutritional resources modulate the responses of three bee species to pesticide exposure.

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.

Impact of a Microbial Pest Control Product Containing Bacillus thuringiensis on Brood Development and Gut Microbiota of Apis mellifera Worker Honey Bees.

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.

Large-scale study investigating the effects of a tank mixture containing thiacloprid-prochloraz on honey bees (Apis mellifera).

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.

Transfer of xenobiotics from contaminated beeswax into different bee matrices under field conditions and the related exposure probability.

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.

Impact of microorganisms and entomopathogenic nematodes used for plant protection on solitary and social bee pollinators: Host range, specificity, pathogenicity, toxicity, and effects of experimental parameters.

Pollinating bees are stressed by highly variable environmental conditions, malnutrition, parasites and pathogens, but may also by getting in contact with microorganisms or entomopathogenic nematodes that are used to control plant pests and diseases. While foraging for water, food, or nest material social as well as solitary bees have direct contact or even consume the plant protection product with its active substance (e.g., viruses, bacteria, fungi, etc.). Here, we summarize the results of cage, microcolony, observation hive assays, semi-field and field studies using full-size queen-right colonies. By now, some species and subspecies of the Western and Eastern honey bee (Apis mellifera, A. cerana), few species of bumble bees, very few stingless bee species and only a single species of leafcutter bees have been studied as non-target host organisms. Survival and reproduction are the major criteria that have been evaluated. Especially sublethal effects on the bees' physiology, immune response and metabolisms will be targets of future investigations. By studying infectivity and pathogenic mechanisms, individual strains of the microorganism and impact on different bee species are future challenges, especially under field conditions. Overall, it became evident that honey bees, bumble bees and few stingless bee species may not be suitable surrogate species to make general conclusions for biological mechanisms of bee-microorganism interactions of other social bee species. Solitary bees have been studied on leafcutter bees (Megachile rotundata) only, which shows that this huge group of bees (∼20,000 species worldwide) is right at the beginning to get an insight into the interaction of wild pollinators and microbial plant protection organisms.

Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product.

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.

High nutritional status promotes vitality of honey bees and mitigates negative effects of pesticides.

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.

Assessment of the impacts of microbial plant protection products containing Bacillus thuringiensis on the survival of adults and larvae of the honeybee (Apis mellifera).

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.

Overview of the testing and assessment of effects of microbial pesticides on bees: strengths, challenges and perspectives.

Currently, there is a growing interest in developing biopesticides and increasing their share in the plant protection market as sustainable tools in integrated pest management (IPM). Therefore, it is important that regulatory requirements are consistent and thorough in consideration of biopesticides' unique properties. While microbial pesticides generally have a lower risk profile, they present special challenges in non-target organism testing and risk assessment since, in contrast to chemical pesticides, their modes of action include infectivity and pathogenicity rather than toxicity alone. For this reason, non-target organism testing guidelines designed for conventional chemical pesticides are not necessarily directly applicable to microbial pesticides. Many stakeholders have recognised the need for improvements in the guidance available for testing microbial pesticides with honey bees, particularly given the increasing interest in development and registration of microbial pesticides and concerns over risks to pollinators. This paper provides an overview of the challenges with testing and assessment of the effects of microbial pesticides on honey bees (Apis mellifera), which have served as a surrogate for both Apis and non-Apis bees, and provides a foundation toward developing improved testing methods.

Chronic High Glyphosate Exposure Delays Individual Worker Bee (Apis mellifera L.) Development under Field Conditions.

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.

Comparing response of buff-tailed bumblebees and red mason bees to application of a thiacloprid-prochloraz mixture under semi-field conditions.

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.

Assessment of acute sublethal effects of clothianidin on motor function of honeybee workers using video-tracking analysis.

Sublethal impacts of pesticides on the locomotor activity might occur to different degrees and could escape visual observation. Therefore, our objective is the utilization of video-tracking to quantify how the acute oral exposure to different doses (0.1-2ng/bee) of the neonicotinoid "clothianidin" influences the locomotor activity of honeybees in a time course experiment. The total distance moved, resting time as well as the duration and frequency of bouts of laying upside down are measured. Our results show that bees exposed to acute sublethal doses of clothianidin exhibit a significant increase in the total distance moved after 30 and 60min of the treatment at the highest dose (2ng/bee). Nevertheless, a reduction of the total distance is observed at this dose 90min post-treatment compared to the distance of the same group after 30min, where the treated bees show an arched abdomen and start to lose their postural control. The treated bees with 1ng clothianidin show a significant increase in total distance moved over the experimental period. Moreover, a reduction in the resting time and increase of the duration and frequency of bouts of laying upside down at these doses are found. Furthermore, significant effects on the tested parameters are observed at the dose (0.5ng/bee) first at 60min post-treatment compared to untreated bees. The lowest dose (0.1ng/bee) has non-significant effects on the motor activity of honeybees compared to untreated bees over the experimental period.

Impacts of chronic sublethal exposure to clothianidin on winter honeybees.

A wide application of systemic pesticides and detection of their residues in bee-collected pollen and nectar at sublethal concentrations led to the emergence of concerns about bees' chronic exposure and possible sublethal effects on insect pollinators. Therefore, special attention was given to reducing unintentional intoxications under field conditions. The sensitivity of winter bees throughout their long lifespan to residual exposure of pesticides is not well known, since most previous studies only looked at the effects on summer bees. Here, we performed various laboratory bioassays to assess the effects of clothianidin on the survival and behavior of winter bees. Oral lethal and sublethal doses were administered throughout 12-day. The obtained LD50 values at 48, 72, 96 h and 10 days were 26.9, 18.0, 15.1 and 9.5 ng/bee, respectively. Concentrations <20 µg/kg were found to be sublethal. Oral exposure to sublethal doses was carried out for 12-day and, the behavioral functions were tested on the respective 13th day. Although slight reductions in the responses at the concentrations 10 and 15 µg/kg were observed, all tested sublethal concentrations had showed non-significant effects on the sucrose responsiveness, habitation of the proboscis extension reflex and olfactory learning performance. Nevertheless, chronic exposure to 15 µg/kg affected the specificity of the early long-term memory (24 h). Since the tested concentrations were in the range of field-relevant concentrations, our results strongly suggest that related-effects on winter and summer bees' sensitivity should also be studied under realistic conditions.