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Fungicides may interact synergistically with insecticides. However, our understanding of the impacts of sublethal insecticide-fungicide combinations on solitary bees is mostly restricted to laboratory studies, providing no information about potential consequences on behavior and reproductive success. We analyzed the effects of a fungicide application, alone and in combination with sublethal levels of an insecticide, on the nesting behavior and reproductive output of the solitary bee Osmia cornuta. We released individually-marked females into oilseed rape field cages, and subsequently sprayed the plants with four treatments: control (water), fungicide (tebuconazole), insecticide (acetamiprid at a sublethal concentration), and mixture (fungicide + insecticide). We recorded nesting activity before and after the sprays and assessed post-spray individual reproductive success. Bees of the single pesticide treatments were unaffected by the sprays and did not differ from control bees in any of the parameters measured. The longevity of bees of the mixture treatment was unaffected. However, these bees showed reduced foraging activity, shorter in-nest pollen-nectar deposition times, and increased difficulty recognizing their nesting cavity, leading to a decrease in provisioning rate, parental investment, and offspring production. Our study demonstrates that co-exposure to a fungicide with otherwise harmless levels of an insecticide caused behavioral effects with consequences on reproductive success. Because longevity was unaffected, these effects would not have been easily detected in a chronic laboratory test. Our results have important implications for bee risk assessment, which should account for exposure to multiple compounds and address behavioral effects and reproductive output under semi-field and/or field conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pollinators are essential for crop productivity. Yet, in agricultural areas, they may be threatened by pesticide exposure. Current pesticide risk assessments predominantly focus on honey bees, with a lack of standardized protocols for solitary bees. This study addresses this gap by developing a long-term oral exposure protocol tailored for O. bicornis. We conducted initial trials to determine optimal container sizes and feeding methods, ensuring high survival rates and accurate syrup consumption measurements. A validation test involving five laboratories was then conducted with the insecticide Flupyradifurone (FPF). Control mortality thresholds were set at ≤ 15% at 10 days. Three laboratories achieved ≤10%, demonstrating the protocol's effectiveness in maintaining healthy test populations. The seasonal timing of experiments influenced control mortality, underscoring the importance of aligning tests with the natural flight period of the population used. Our findings revealed dose-dependent effects of FPF on syrup consumption, showing stimulatory effects at lower concentrations and inhibitory effects at higher ones. The 10-day median lethal daily dose (LDD50) of FPF for O. bicornis (531.92 ng/bee/day) was 3.4-fold lower than that reported for Apis mellifera (1830 ng/bee/day), indicating Osmia's higher susceptibility. Unlike other insecticides, FPF did not exhibit time-reinforced toxicity. This study introduces a robust protocol for chronic pesticide exposure in solitary bees, addressing a critical gap in current risk assessment. Based on its low risk to honey bees and bumblebees, FPF is approved for application during flowering. However, our results suggest that it may threaten Osmia populations under realistic field conditions. Our findings underscore the need for comparative toxicity studies to ensure comprehensive protection of all pollinators and the importance of accounting for long term exposure scenarios in risk assessment. By enhancing our understanding of chronic pesticide effects in solitary bees, our study should contribute to the development of more effective conservation strategies and sustainable agricultural practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In agricultural environments, bees are routinely exposed to combinations of pesticides. For the most part, exposure to these pesticide mixtures does not result in acute lethal effects, but we know very little about potential sublethal effects and their consequences on reproductive success and population dynamics. In this study, we orally exposed newly emerged females of the solitary bee Osmia cornuta to environmentally-relevant levels of acetamiprid (a cyano-substituted neonicotinoid insecticide) singly and in combination with tebuconazole (a sterol-biosynthesis inhibitor (SBI) fungicide). The amount of feeding solution consumed during the exposure phase was lowest in bees exposed to the pesticide mixture. Following exposure, females were individually marked and released into oilseed rape field cages to monitor their nesting performance and assess their reproductive success. The nesting performance and reproductive success of bees exposed to the fungicide or the insecticide alone were similar to those of control bees and resulted in a 1.3-1.7 net population increases. By contrast, bees exposed to the pesticide mixture showed lower establishment, shortened nesting period, and reduced fecundity. Together, these effects led to a 0.5-0.6 population decrease. Female establishment and shortened nesting period were the main population bottlenecks. We found no effects of the pesticide mixture on nest provisioning rate, offspring body weight or sex ratio. Our study shows how sublethal pesticide exposure may affect several components of bee reproductive success and, ultimately, population growth. Our results calls for a rethinking of pollinator risk assessment schemes, which should target not only single compounds but also combinations of compounds likely to co-occur in agricultural environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Pesticide exposure is an important driver of bee declines. Laboratory toxicity tests provide baseline information on the potential effects of pesticides on bees, but current risk assessment schemes rely on one species, the highly social honey bee, Apis mellifera, and there is uncertainty regarding the extent to which this species is a suitable surrogate for other pollinators. For this reason, Osmia cornuta and Osmia bicornis have been proposed as model solitary bee species in the EU risk assessment scheme. The use of solitary bees in risk assessment requires the development of new methodologies adjusted to the biology of these species. For example, oral dosing methods used with honey bees cannot be readily applied to solitary bees due to differences in feeding behaviour and social interactions. In this study, we describe the "petal method", a laboratory feeding method, and validate its use in acute and chronic exposure oral tests with Osmia spp. We conducted five experiments in which we compared the performance of several artificial flowers combining visual and olfactory cues against the petal method, or in which variations of the petal method were confronted. We then use the results of these experiments to optimize the feeding arenas and propose standardized methods for both acute and chronic exposure tests. The petal method provides high levels of feeding success, thus reducing the number of bees needed. It works with a wide variety of petal species and with both female and male Osmia spp., thus ensuring reproducibility across studies. To validate the use of the petal method in ecotoxicology tests, we assess the toxicity of a standard reference insecticide, dimethoate, in O. cornuta adults and determine LD50 values for this species. The petal method should facilitate the inclusion of solitary bees in risk assessment schemes therefore increasing the protection coverage of pesticide regulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Residues detected in pollen collected by honey bees are often used to estimate pesticide exposure in ecotoxicological studies. However, for a more accurate assessment of pesticides effect on foraging pollinators, residues found directly on flowers are a more realistic exposure approximation. We conducted a multi-residue analysis of pesticides on pollen and nectar of melon flowers collected from five fields. The cumulative chronic oral exposure Risk Index (RI) was calculated for Apis mellifera, Bombus terrestris and Osmia bicornis to multiple pesticides. However, this index could underestimate the risk since sublethal or synergistic effects are not considered. Therefore, a mixture containing three of the most frequently detected pesticides in our study was tested for synergistic impact on B. terrestris micro-colonies through a chronic oral toxicity test. According to the result, pollen and nectar samples contained numerous pesticide residues, including nine insecticides, nine fungicides, and one herbicide. Eleven of those were not applied by farmers during the crop season, revealing that melon agroecosystems may be pesticide contaminated environments. The primary contributor to the chronic RI was imidacloprid and O. bircornis is at greatest risk for lethality resulting from chronic oral exposure at these sites. In the bumblebee micro-colony bioassay, dietary exposure to acetamiprid, chlorpyrifos and oxamyl at residue level concentration, showed no effects on worker mortality, drone production or drone size and no synergies were detected when pesticide mixtures were evaluated. In conclusion, our findings have significant implications for improving pesticide risk assessment schemes to guarantee pollinator conservation. In particular, bee pesticide risk assessment should not be limited to acute exposure effects to isolated active ingredients in honey bees. Instead, risk assessments should consider the long-term pesticide exposure effects in both pollen and nectar on a range of bees that reflect the diversity of natural ecosystems and the synergistic potential among pesticide formulations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In agricultural ecosystems, bees are exposed to combinations of pesticides that may have been applied at different times. For example, bees visiting a flowering crop may be chronically exposed to low concentrations of systemic insecticides applied before bloom and then to a pulse of fungicide, considered safe for bees, applied during bloom. In this study, we simulate this scenario under laboratory conditions with females of the solitary bee, Osmia bicornis L. We studied the effects of chronic exposure to the neonicotinoid insecticide, Confidor® (imidacloprid) at a realistic concentration, and of a pulse (1 day) exposure of the fungicide Folicur® SE (tebuconazole) at field application rate. Syrup consumption, survival, and four biomarkers: acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), and alkaline phosphatase (ALP) were evaluated at two different time points. An integrated biological response (IBRv2) index was elaborated with the biomarker results. The fungicide pulse had no impact on survival but temporarily reduced syrup consumption and increased the IBRv2 index, indicating potential molecular alterations. The neonicotinoid significantly reduced syrup consumption, survival, and the neurological activity of the enzymes. The co-exposure neonicotinoid-fungicide did not increase toxicity at the tested concentrations. AChE proved to be an efficient biomarker for the detection of early effects for both the insecticide and the fungicide. Our results highlight the importance of assessing individual and sub-individual endpoints to better understand pesticide effects on bees., (© 2022. The Author(s).)

The recent EU ban of the three most widely used neonicotinoids (imidacloprid, thiamethoxam and clothianidin) to all outdoors applications has stimulated the introduction of new insecticides into the market. Sulfoxaflor is a new systemic insecticide that, like neonicotinoids, acts as a modulator of nicotinic acetylcholine receptors. In agro-environments, bees can be exposed to this compound via contaminated pollen and nectar for long periods of time. Therefore, it is important to assess the potential effects of chronic exposure to sulfoxaflor, alone and in combination with fungicides, on pollinators. In this study, we tested the effects of chronic exposure to two field concentrations of sulfoxaflor (20 and 100 ppb) alone and in combination with four concentrations of the fungicide fluxapyroxad (7500, 15,000, 30,000 and 60,000 ppb) on syrup consumption and longevity in females of the solitary bee Osmia bicornis L. Exposure to 20 ppb of sulfoxaflor, alone and in combination with the fungicide, stimulated syrup consumption, but did not affect longevity. In contrast, syrup consumption decreased in bees exposed to 100 ppb, all of which died after 2-6 days of exposure. We found no evidence of synergism between the two compounds at any of the two sulfoxaflor concentrations tested. Comparison of our findings with the literature, confirms that O. bicornis is more sensitive to sulfoxaflor than honey bees. Our results highlight the need to include different bee species in risk assessment schemes., Competing Interests: Declaration of competing interest The authors declare they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The sulfoximine insecticide sulfoxaflor is regarded as a potential substitute for neonicotinoids that were recently banned in the EU due to their side effects on bees. Like neonicotinoids, sulfoxaflor acts as a competitive modulator of nicotinic acetylcholine receptors. In agricultural environments, bees are commonly exposed to combinations of pesticides, and neonicotinoids are known to interact synergistically with fungicides. The objective of our study is to assess the acute oral toxicity of sulfoxaflor alone and in combination with a single dose of fluxapyroxad, a succinate dehydrogenase inhibitor (SDHI) fungicide, in three bee species: Apis mellifera, Bombus terrestris and Osmia bicornis. Because synergism may be dose-dependent, we tested a range of sulfoxaflor doses. Synergistic effects were assessed using three different approaches: Bliss criterion of drugs independence, ratio test comparing LD 50 s and model deviation ratio. Osmia bicornis was the most sensitive species to sulfoxaflor and both O. bicornis and A. mellifera showed significant synergism between the insecticide and the fungicide. For the most part, these synergistic effects were weak and only occurred at early assessment times and intermediate sulfoxaflor doses. The potential ecological relevance of these effects should be confirmed in field and/or cage studies. Overall, our laboratory results demonstrate that sulfoxaflor is somewhat less toxic than the recently banned neonicotinoids imidacloprid, thiamethoxam and clothianidin, but much more toxic than other neonicotinoids (acetamiprid, thiacloprid) still in use in the EU at the time this study was conducted.

Solitary bees are frequently exposed to pesticides, which are considered as one of the main stress factors that may lead to population declines. A strong immune defence is vital for the fitness of bees. However, the immune system can be weakened by environmental factors that may render bees more vulnerable to parasites and pathogens. Here we demonstrate for the first time that field-realistic concentrations of the commonly used neonicotinoid insecticide thiacloprid can severely affect the immunocompetence of Osmia bicornis. In detail, males exposed to thiacloprid solutions of 200 and 555 µg/kg showed a reduction in hemocyte density. Moreover, functional aspects of the immune defence - the antimicrobial activity of the hemolymph - were impaired in males. In females, however, only a concentration of 555 µg/kg elicited similar immunosuppressive effects. Although males are smaller than females, they consumed more food solution. This leads to a 2.77 times higher exposure in males, probably explaining the different concentration thresholds observed between the sexes. In contrast to honeybees, dietary exposure to thiacloprid did not affect melanisation or wound healing in O. bicornis. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of O. bicornis, possibly leading to an impaired disease resistance capacity.

Pollinators in agroecosystems are often exposed to pesticide mixtures. Even at low concentrations, the effects of these mixtures on bee populations are difficult to predict due to potential synergistic interactions. In this paper, we orally exposed newly emerged females of the solitary bee Osmia bicornis to environmentally realistic levels of clothianidin (neonicotinoid insecticide) and propiconazole (fungicide), singly and in combination. The amount of feeding solution consumed was highest in bees exposed to the neonicotinoid, and lowest in bees exposed to the pesticide mixture. Ovary maturation and longevity of bees of the neonicotinoid and the fungicide treatments did not differ from those of control bees. By contrast, bees exposed to the pesticide mixture showed slow ovary maturation and decreased longevity. We found a synergistic interaction between the neonicotinoid and the fungicide on survival probability. We also found an interaction between treatment and emergence time (an indicator of physiological condition) on longevity. Longevity was negatively correlated to physiological condition only in the fungicide and the mixture treatments. Delayed ovary maturation and premature death imply a shortened nesting period (highly correlated to fecundity in Osmia ). Our findings provide a mechanism to explain the observed dynamics of solitary bee populations exposed to multiple chemical residues in agricultural environments., (© 2018 The Author(s).)

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD 50 ) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (-50%). Nutritional and pesticide stressors reduced also food consumption (-48%) and haemolymph levels of glucose (-60%) and trehalose (-27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection., (© 2017 The Author(s).)

Background: Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory., Results: We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD 10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin., Conclusion: Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)