Field-Level Evaluation of Honey Bee Exposure and Risk from Pesticides Used in Maize Production

Field-Level Evaluation of Honey Bee Exposure and Risk from Pesticides Used in Maize Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Field-Level Evaluation of Honey Bee Exposure and Risk from Pesticides Used in Maize Production Zeliha Şimşek, Mustafa Kösoğlu, Melis Yalçın, Nalan Turgut, Cafer Turgut This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8384368/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Maize seeds are coated with systemic fungicides and insecticides to control pests in maize production. The risk assessments are not well covered for their effects on non-target arthropods, e.g., honey bees, in real farm conditions. The effects of seed-applied thiamethoxam, cyantraniliprole, and a fludioxonil + metalaxyl-M mixture were investigated on honey bees using complementary field, semi-field, and laboratory experiments. Pesticide Residues were analysed in guttation fluid, honey, pollen, and dead bees, and acute toxicity bioassays were conducted with freshly collected guttation droplets from treated maize plants. The mortality rate was higher than in the control across all pesticide treatments in semi-field, and it was highest with thiamethoxam, followed by cyantraniliprole and the fungicide mixture. Field experiments showed lower overall effects, but cyantraniliprole and the fungicide mixture still caused significantly increased mortality. Thiamethoxam reached very high concentrations in guttation fluid (2364–2565 µg L⁻¹) soon after plant emergence, resulting in rapid mortality of over 80% within 4 hours. Cyantraniliprole-contaminated guttation droplets also caused acute toxicity, with mortality often exceeding 60–90% within 24 to 72 hours. Guttation droplets are a critical, yet underappreciated, exposure route for seed-coated pesticides and can deliver lethal doses to honeybees in maize fields. Incorporating guttation exposure into pesticide risk assessment schemes and strengthening integrated pest management strategies are essential to reduce non-target impacts while maintaining effective pest control. Pesticides Maize Honey Bee Residue integrated pest management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Maize ( Zea mays L.) is a significant cereal crop, ranking third after wheat and rice, with an annual production of approximately 1 billion tons (FAO 2023 ). It is used for food and livestock, and is also widely used to produce biofuel, starch, sweeteners, and additives. Maize is produced in many countries due to its adaptability to different climates (Unay et al. 2021 ). Pesticides are used to control pests, diseases, and weeds in maize production. Especially, seed coating is a key protection for seeds and emerging seedlings against soil-borne diseases and pests (Girolami et al. 2009 ; Yalçin et al. 2021 ). Thiamethoxam (THX), a neonicotinoid, and cyantraniliprole (CYN), a diamide, are among the most used systemic insecticides in maize seed treatments worldwide (Sparks et al. 2019 ) Seed coating provides clear agronomic benefits by ensuring seed protection and early-stage pest control without the need for foliar spraying. These same systemic properties of seed-coated pesticides enable active compounds to persist within the plant and translocate to multiple tissues, including leaves, stems, and corn (Özşirvan et al. 2024 ). Thiamethoxam and cyantraniliprole can be uptaken and translocated to the roots, stem, and leaves, and the bioconcentration factor for thiamethoxam reached up to 4500 during peak periods (Özşirvan et al. 2024 ). Such levels raise questions about unintended exposure pathways for non-target organisms. The translocated seed-coated pesticides occur in guttation, which is characterized by guttation droplets formed under conditions of high soil moisture and low transpiration. These droplets exude from hydathodes located at the leaf margins (Chen and Chen 2007 ; Yalçin et al. 2021 ). These droplets can contain substantial concentrations of systemic pesticides because of the upward flow of xylem sap (Girolami et al. 2009 ; Reetz et al. 2011 ). Recent studies confirmed that maize guttation fluid can contain residues of thiamethoxam and cyantraniliprole at levels sufficient to pose both acute and chronic risks to honey bees and other beneficial insects (Analysis et al. 2012 ; Reetz et al. 2016 ). Honey bees, which are key pollinators of both wild flora and many agricultural crops, are known to forage for water sources near their hives to maintain colony thermoregulation and dilute honey stores (Visscher et al. 1996 ; Joachimsmeier et al. 2012 ). When alternative water sources are limited, bees can collect guttation droplets directly from maize or other plants (Shawki et al. 2006). Single corn cultivation is practiced as monoculture, especially over large areas. Unlike nectar or pollen, this water is not stored but used immediately within the hive, meaning bees may be repeatedly exposed to any pesticides dissolved in guttation fluid (Whitehorn et al. 2012 ). Although the environmental fate of neonicotinoids has been studied extensively in parts of Europe and North America, a notable lack of data remains for regions such as Türkiye, where local bee races and dense beekeeping activity may create unique exposure profiles (YALÇIN and TURGUT 2017 ). The local agricultural conditions e.g. climate, soil type, and cropping practices, may influence the uptake and translocation of systemic pesticides in plant tissues (Özşirvan et al., 2024 ; Reetz et al., 2016 ). In addition to guttation, pesticide residues can accumulate in pollen and nectar, further elevating the risk to foraging bees. Multiple surveys have detected neonicotinoid residues in bee-collected pollen and wildflower samples adjacent to treated fields (Pohorecka et al. 2013 ; David et al. 2016 ). The pesticide residues in pollen and nectar pose a risk to queen production and can cause colony collapse, even at sublethal concentrations (Whitehorn et al. 2012 ; Sgolastra et al. 2017 ). Moreover, the simultaneous exposure to insecticides and fungicides can produce synergistic effects, amplifying toxicity (Thompson et al. 2014 ). Despite this growing body of evidence, studies examining the combined pathways of exposure, including guttation, pollen, nectar, and direct contact, remain limited in the Eastern Mediterranean and neighboring temperate regions. The lack of region-specific monitoring is concerning, given Turkiye’s status as one of the world’s largest honey producers (FAO 2023 ) and the widespread reliance on local subspecies of Apis mellifera anatoliaca (Yalçin et al. 2021 ). Cyantraniliprole is a diamide insecticide from the anthranilic diamide class, known for its systemic action and strong efficacy against a wide range of chewing and sucking pests (Özşirvan et al., 2024 ). Its mode of action targets ryanodine receptors, leading to uncontrolled release of calcium in insect muscle cells, which causes paralysis and death. While cyantraniliprole has been marketed as a selective compound with relatively low non-target toxicity, recent studies have highlighted that its systemic behavior allows it to move into guttation droplets, pollen and nectar, posing risks for pollinators that forage or collect water from treated crops (Girolami et al. 2009 ; Shepherd et al. 2024 ). Thiamethoxam, a neonicotinoid insecticide, exhibits systemic activity and is widely used for controlling a broad range of sucking and chewing insect pests. Its mode of action involves binding to nicotinic acetylcholine receptors in the insect central nervous system, leading to overstimulation, paralysis, and eventual death (Sandrock et al., 2013; Shareefdeen & Elkamel, 2024). Although thiamethoxam is valued for its high efficacy and relatively selective toxicity, its systemic properties facilitate translocation into guttation droplets, pollen, and nectar. This translocation raises concerns about potential adverse effects on pollinators that forage on treated plants or collect water from these sources (Stoner et al. 2019 ). Metalaxyl-M, a systemic phenylamide fungicide, is widely used to control oomycete pathogens that cause diseases such as downy mildew and late blight. Its mode of action involves inhibiting RNA synthesis in target fungi, thereby disrupting their growth and reproduction. Metalaxyl-M exhibits high efficacy and selectivity, with systemic properties that allow translocation within plant tissues to protect new growth. However, its systemic nature also raises concerns about the presence of residues in plant fluids, which may affect non-target organisms and environmental exposure. Although primarily targeting fungi, the potential for metalaxyl-M residues in pollen and nectar suggests a need to assess possible risks to pollinators foraging on treated plants. Fludioxonil, a non-systemic fungicide belonging to the phenylpyrrole class, is widely used to control a broad spectrum of fungal pathogens, particularly those causing post-harvest diseases and seed-borne infections. Its mode of action involves disrupting fungal signal transduction pathways, thereby inhibiting spore germination and mycelial growth. Unlike systemic fungicides, fludioxonil remains primarily on the plant surface, minimizing translocation within plant tissues. This characteristic reduces concerns about residues in internal plant fluids; however, its widespread use necessitates evaluation of potential environmental impacts, including effects on non-target organisms, such as pollinators. Field, semi-field, and laboratory studies generate valuable data to prevent colony losses and improve pest management strategies that protect pollinator populations and comparable agroecosystems. Therefore, the primary objective of this research is to identify the risks posed to honeybees by the translocation of seed treatment pesticides through guttation fluid, honey, and pollen, and to categorize these pesticides according to their associated risks. 2. Materials and Methods 2.1. Field and Semi-Field Experiments The field studies were established in designated maize plots using a randomized complete block design with a single factor with three replicates per treatment. Maize seeds were planted using a 4-row precision pneumatic seeder. Prior to sowing, maize seeds were coated with the pesticides specified in ESM, Table S1 . Untreated seeds served as the control group. The field and semi-field experiments were conducted at Aydın Adnan Menderes University, Faculty of Agriculture, Research and Field Trial Farm (37°45'32" N 27°45'35" E). Following emergence, semi-field tunnels were installed over selected plots using galvanized steel frames at a height of 2.75 meters to ensure bee flight and control environmental exposure. Each tunnel was covered with fine-mesh netting (mesh size ≤ 3 mm) that permitted air, light, and rainfall to pass through while preventing bees from escaping. Twelve tunnels were covered for the semi-field maize trial: three for the untreated control and nine for the different seed treatment combinations. For the open-field plots, colonies were directly exposed without any enclosure to mimic realistic foraging conditions. One day prior to guttation sampling, one beehive was positioned at the midpoint of the upper left edge of each semi-field tunnel and at the center of each open-field plot. Colonies contained approximately 10,000 worker bees, headed by sister queens to maintain genetic consistency. Each hive comprised 5–7 frames, including frames with stored honey and pollen for nutrition, as well as brood frames to ensure robust colony development. Brood and food resources were balanced across colonies at the start to maintain uniform strength. 2.2. Guttation Fluid Collection Guttation samples were collected daily from maize leaves during early morning hours, starting at sunrise (approximately 6:30 a.m.) and continuing until guttation droplets had fully evaporated under daylight. Fluid samples were drawn directly from leaf tips using sterile Pasteur pipettes and transferred into labeled Falcon tubes for laboratory analysis. 2.3. Dead Bee Monitoring and Sampling Dead bees were collected daily from designated collection boards placed at hive entrances and throughout the surrounding plot area. The dead bees were recorded in clean sample containers and stored in a deep freezer at − 20°C until extraction and analysis of pesticide residues. 2.4. Toxicity Bioassays with Guttation Fluid To assess the direct toxicity of maize guttation droplets, laboratory bioassays were performed in accordance with OECD Test Guideline 213 (OECD 1998 ). Anatolian honey bees ( Apis mellifera anatoliaca ) were used from healthy, disease-free colonies and carefully selected to ensure uniform worker age. Ten bees were housed in custom stainless-steel cages with glass panels for ventilation and observation. Prior to exposure, the bees were acclimated for 24 hours at 25°C and 50–70% relative humidity. After acclimation, 1–2 mL of freshly collected guttation fluid was introduced into each cage through a sterile syringe, serving as the bees’ sole water source. Mortality was monitored at 4-, 24-, 48-, and 72-hours post-exposure. All tests were conducted in triplicate, including untreated controls to account for natural mortality and environmental factors. 2.5. Pesticide Extraction and Residue Analysis 2.5.1. Extraction from Guttation Fluid, bees, honey and pollen For the determination of pesticide residues in guttation fluid, the modified QuEChERS method was used. A 5 mL aliquot of guttation fluid was mixed with 10 mL of acetonitrile containing 1% acetic acid, vigorously vortexed, and combined with 4 g MgSO₄ and 1 g sodium acetate. After shaking and centrifugation, the supernatant was cleaned with MgSO₄ and PSA, filtered through a Teflon membrane filter, and stored at − 20°C prior to analysis. Dead bees (5 g), pollen (2 g), and honey (5 g) samples were weighed and processed with a modified QuEChERS method. After adding 10 mL of distilled water, the samples were vortexed and shaken. Then, 10 mL of acetonitrile was added, followed by MgSO₄, NaCl, and citrate buffers. After centrifugation and PSA cleanup, extracts were reduced, filtered, and analyzed by LC–MS/MS. 2.5.3. Extraction of pesticides in soil Soil samples were taken three weeks prior to planting at five random locations per plot (at a 30 cm depth) to ensure that no pesticide residues were present before planting. Five grams of soil samples were placed in 50 mL Falcon tubes and mixed with a 1:1 acetone: dicloromethane solution. The tubes were then stored for one night with the lids closed. The next day, the mixture was extracted ultrasonically for 60 minutes and then centrifuged for 5 minutes at 4000 rpm. The supernatant was then transferred and filtered for pesticide analysis. 2.5.4. Analytical Conditions All residue analyses were conducted under Good Laboratory Practice (GLP) conditions using validated instruments (Shimadzu LC–MS/MS). Pesticide identification was based on molecular ion masses, and quantification was performed by comparing sample peak areas to certified standards (Turgut et al. 2010 ; Soydan et al. 2021 ). 2.6. Quality Assurance/Quality control The analysis method used for thiamethoxam and cyantraniliprole, metalaxyl-M, fludioxonil has been validated and quality-controlled according to SANTE (SANTE 2019 ). The LOD and LOQ values were established to ensure the method's reliability (ESM, Tab. S2). Calculating LOD and LOQ typically involves statistical methods based on the response standard deviation and the slope of the calibration curve. This method assumes a linear relationship between analyte concentration and the analytical instrument's response. The signal-to-noise (S/N) ratio for the lowest analyte concentration was determined using a ratio of 1:3. Repeated analyses were performed to achieve consistent results (n = 5). 2.7. Colony Development monitoring in bee colonies Colony strength and brood area were assessed at three-week intervals during the trial. Bee-covered frames and brood areas were measured to monitor population dynamics and production efficiency, calculated according to the standard ellipse formula (Kösoğlu 2021 ). 2.8. Statistical Analysis Statistical evaluation of daily bee mortality was performed using the Wilcoxon signed-rank test to identify significant differences between treatments and controls. Other measured parameters were compared using one-way ANOVA followed by Tukey’s post hoc test. All tests were conducted using IBM SPSS Statistics software at a significance level of p < 0.05. 3. Results 3.1. Bee mortality in the semi-field and field experiment In this semi-field experiment, daily and cumulative honey bee ( Apis mellifera anatolica ) mortalities were monitored to assess the effects of three pesticide treatments cyantraniliprole, fludioxonil + metalaxyl-M, and thiamethoxam compared with an untreated control (Fig. 1 ). According to the Wilcoxon test, all pesticide treatments caused significantly higher cumulative bee mortality than the control (p < 0.05). The total number of deaths clearly showed that the control group had the lowest bee losses (622), while the treated groups had notably higher totals: fludioxonil + metalaxyl-M (1231), cyantraniliprole (1523), and thiamethoxam, with the highest (1696). Bee mortality showed significant treatment- and time-dependent variation, with the guttation-active first 28 days representing the primary risk window in field studies (Fig. 2 ). During this early phase, all pesticide treatments produced markedly higher mortality than the control, with Thiamethoxam and Cyantraniliprole exhibiting the highest peaks (up to 125 and 173 bees per hive, respectively) and consistently elevated daily means relative to the control, which remained below 20 bees on most days. Fludioxonil + Metalaxyl-M also resulted in significantly greater mortality than the control, though at a lower magnitude than the other treatments. After Day 28, mortality declined across all groups; however, pesticide treatments continued to exceed control levels, indicating residual or chronic exposure effects. Cumulative mortality over the full observation period confirmed strong treatment effects, with totals of 1523 bees for Cyantraniliprole, 1231 for Fludioxonil + Metalaxyl-M, and 1696 for Thiamethoxam compared with 622 in the control, highlighting that guttation-related exposure substantially amplified early-phase mortality while longer-term impacts persisted at reduced intensity. All three pesticide treatments posed a significant risk compared to the control, but with different impact profiles. Thiamethoxam clearly caused the highest and most continuous daily mortality, while cyantraniliprole produced sharp local peaks that contributed to its high total mortality. The effect of fungicide coated seeds influence slowly but steady increase indicating that repeated low-level exposure can also accumulate to dangerous levels. 3.2. Pesticide residues in dead bees, honey, pollen, and guttation fluid The residue of Metalaxyl-M was found only in dead bees collected from hives located in maize open-fields. Neither cyantraniliprole nor thiamethoxam was detected in any samples. The average residue level of Metalaxyl-M was significantly higher in hives in semi-field plots at 40 ± 23.27 µg/kg, compared to 0.16 ± 16 µg/kg in open-field plots. Statistical analysis confirmed this difference was significant (F = 4.2, p < 0.05 for semi-field; F = 18.7, p < 0.01 for open field) (Table 1 ). Table 1 Pesticide residues in dead bees samples (µg/kg ) Metalaxyl-M Cyantraniliprole Thiamethoxam Fludioxonil F P Semi-field 40 ± 23.27a nd nd nd 4.2 < 0.05 Field 0.16 ± 16.00b nd nd nd 18.7 < 0.01 nd: not detected Analysis of honey samples from hives located in maize fields showed detectable residues of thiamethoxam, cyantraniliprole, and Metalaxyl-M. Fludioxonil was not detected in any of the samples. The mean concentration of thiamethoxam in semi-field plots was 13.213 µg/kg, while in open-field samples it was 10.458 µg/kg. The concentration of Cyantraniliprole was 8.461 µg/kg in semi-field plots and 7.423 µg/kg in the field. Metalaxyl-M concentration in honey samples was higher (14.023 µg/kg) in the samples from the field located hives compared to semi-field samples (0.210 µg/kg) (Table 2 ). Table 2 Pesticide residues in honey bee samples (µg/kg) Pesticides Semi-field Field Thiamethoxam 13.21 ± 1.305 10.45 ± 1.094 Cyantraniliprole 8.46 ± 3.030 7.42 ± 1.819 Metalaxyl-M 0.21 ± 0.067 14.02 ± 4.858 Fludioxonil nd nd Analysis of pollen samples showed that seed treatment pesticides can contaminate pollen collected by bees. Thiamethoxam was detected at 1.000 µg/kg in pollen from semi-field plots and 3.827 µg/kg in field pollen, indicating approximately four times higher exposure in open fields. Metalaxyl-M was also present in pollen, albeit at lower levels: 0.077 µg/kg in semi-field and 0.062 µg/kg in field (Table 3 ). Table 3 Pesticide residues in maize pollen (µg/kg) Pesticides semi-field Field Thiamethoxam 1.00 ± 0.00 3.827 ± 0.391 Cyantraniliprole nd nd Metalaxyl-M 0.077 ± 0.006 0.062 ± 0.005 Fludioxonil nd nd Guttation fluid in maize showed that thiamethoxam initially reached very high levels (2364 µg/L on day 1) and then gradually declined to 59–73 µg/L by days 25–27 after treatment. This decreasing trend reflects the systemic uptake and subsequent dilution or degradation of the compound as the plants developed. Cyantraniliprole was detected at 1–30 µg/L during the first few days and was undetectable after day 10. The concentration of Metalaxyl-M was very low, starting at 5 µg/L and remaining at approximately 1 µg/L for most of the sampling period, whereas fludioxonil was not detected at any point, indicating negligible systemic transfer. Thiamethoxam residue was found in early guttation samples at levels as high as 2364 µg/L (Fig. 3 ). 3.3. Toxicity of guttation fluid to honey bees Toxicity bioassays demonstrated that guttation liquid collected from maize plants grown from thiamethoxam-treated seeds poses a severe acute hazard to honey bees. During the first five days of the experiment, even 4 hours of exposure resulted in more than 80% worker bee mortality (Fig. 4 ). Mortality decreased slightly in samples collected after day 5, it remained consistently above 50% until day 10. Even in the later stages of plant growth, guttation liquid collected up to day 27 still caused high bee mortality. This shows that guttation remains a risk for an extended period. Bee mortality remained very high after 24 hours of exposure. When bees were exposed to guttation liquid collected during the first nine days, all of them died within 24 hours. In samples collected later, more than half of the bees still died within 24 hours. Longer exposures of 48 and 72 hours indicated that the risk continued to be severe. Guttation liquid from any day in the trial resulted in over 80% mortality after 72 hours in almost all cases, demonstrating that the toxic effect persisted throughout the study. The mortality levels showed clear temporal and day-to-day variability, but a consistent pattern of rapid and severe acute toxicity was observed throughout the experimental period (Fig. 5 ). The mortality ranged between approximately 10% and 80%, with the highest peaks recorded on Days 1, 6–10, and 24–26 in 4 hours of exposure. On days 6–9 and at 25 days, the mortality rate exceeds 70%, demonstrating strong acute toxicity even with early exposure durations. After 24 hours, mortality values generally increased by 10–20%, maintaining high lethality above 60% on most days. At 48 and 72 hours, bee mortality continued to rise slightly or stabilized, suggesting that the compound exerts both acute and residual effects. In some periods (such as Days 17–19 and 23–27), cumulative mortality reached or surpassed 90%, confirming the persistence of toxic residues. Mortality in honey bees exposed to the fungicide mixture of fludioxonil and metalaxyl-M varied considerably throughout the 27-day experimental period but consistently showed acute toxic responses during the initial days following exposure (Fig. 6 ). The highest mortality levels occurred during the first four days, with 4-hour mortality reaching up to 90–100%, indicating strong initial toxicity immediately after contact or ingestion. Although mortality decreased sharply after Day 5, periodic peaks were observed on Days 11, 15, 19, and 24–25, suggesting that exposure intensity fluctuated with environmental or application conditions. The cumulative increase from 4 to 72 hours in most sampling days reflects both rapid and residual toxic effects of the fungicide mixture. 4. Discussion Honey bee mortality was significantly increased in open-field studies by all three pesticide treatments compared with the untreated control, confirming the risks posed by systemic insecticides and fungicide seed coatings. Thiamethoxam-treated colonies exhibited the highest and most continuous daily mortality, with early peaks followed by sustained losses over multiple weeks, consistent with its neurotoxic effects documented under laboratory and field conditions (Laurino et al. 2011 ; Krupke et al. 2012 ). Cyantraniliprole also caused repeated mortality spikes, notably on day 26, indicating that newer generation insecticides can reach biologically relevant concentrations in foraging environments through guttation droplets or treated foliage (Cutler et al. 2014 ; David et al. 2016 ). Fludioxonil + metalaxyl-M produced lower but continuous daily deaths, highlighting that fungicide residues, often considered harmless, can accumulate and interact with other stressors to impair colony health (David et al. 2016 ). These results demonstrate that both acute and chronic exposure under realistic agricultural conditions contribute to substantial colony-level mortality, emphasizing the limitations of laboratory-only toxicity assessments. The variation between the two experimental datasets further illustrates the influence of environmental factors on exposure outcomes. In semi-field studies, thiamethoxam mortality was similar to that of the control, whereas cyantraniliprole and the fungicide mixture continued to elevate losses, suggesting that local foraging behavior, flowering phenology, and weather conditions modulate exposure intensity. Sporadic mortality peaks in thiamethoxam and unusually high daily counts for fludioxonil + metalaxyl-M (e.g., day 56) imply that repeated or combined exposure exacerbates risks. Overall, these findings show that systemic insecticides and fungicide seed treatments can produce cumulative and potentially sublethal effects on colony structure, including reduced brood development and queen vitality. This underscores the need for integrated pest management, stricter residue monitoring, and practical mitigation strategies to protect honey bees and ensure sustainable crop production (Williams et al. 2015 ). Therefore, continuous field-based risk assessments are essential to complement laboratory toxicity tests and inform pollinator conservation policies. The markedly higher Metalaxyl-M residues detected in dead bees from the semi-field (covered greenhouse) compared to the open-field plots suggest that restricted foraging conditions increased exposure and subsequent accumulation. Similar semi-field tunnel studies have shown that limited flight space can lead to higher pesticide and fungicide residues in bees due to prolonged contact within a confined environment (Shepherd et al. 2024 )(Shepherd et al., 2024 ). In contrast, the absence of cyantraniliprole, thiamethoxam, and fludioxonil in all samples is consistent with the possibility that these compounds were either not present at appreciable levels in the environment or were rapidly degraded or metabolized before they could accumulate in bee tissues (Cutler et al. 2014 ; Fu et al. 2021 ). The elevated levels of Metalaxyl-M in dead bees highlight a potential risk to honey bee health, underscoring the importance of considering pesticide application practices, environmental conditions, and bee foraging behavior when assessing exposure pathways and implementing measures to reduce risk to pollinators (Mörtl et al. 2020 ). The detection of thiamethoxam, cyantraniliprole, and Metalaxyl-M in honey from both semi-field and open-field maize plots indicates that foraging bees were exposed to these seed-applied pesticides either directly from treated maize plants or indirectly through contaminated environmental sources. The absence of fludioxonil across all samples may reflect low environmental presence or rapid degradation prior to incorporation into honey. Overall, the elevated pesticide residues observed here highlight the importance of considering crop management and exposure pathways when evaluating risks to honey bees and the quality of their products. The presence of thiamethoxam in maize pollen from both semi-field and open-field plots, with concentrations nearly four times higher in the open field, indicates that bees are more likely to encounter contaminated pollen when they can forage freely across larger areas. This aligns with previous findings showing that planter dust and contaminated pollen are primary exposure pathways for neonicotinoids originating from treated seeds (Cutler et al. 2014 ; Fu et al. 2021 ). The detection of Metalaxyl-M at low levels in both systems further demonstrates that fungicide residues also enter pollen, although to a lesser extent. The absence of cyantraniliprole and fludioxonil suggests limited systemic translocation into maize pollen or rapid degradation prior to bee collection. Recent studies have similarly shown that seed-applied insecticides can move into pollen and nectar at biologically relevant concentrations, potentially contributing to sublethal effects on bee behavior and colony performance (Thompson et al. 2022 ). Overall, the pollen residue patterns observed here reinforce that maize grown from treated seed can serve as a meaningful exposure source for honey bees, highlighting the need to consider pollen contamination when evaluating risks associated with seed-treatment pesticides. The high concentrations of thiamethoxam detected in maize guttation droplets during the first days after emergence demonstrate that guttation represents an acute and significant short-term exposure route for honey bees, particularly when residues reach levels far exceeding lethal doses (EFSA, 2015). The sharp initial peak followed by a steady decline is consistent with previous observations showing that neonicotinoid-treated maize can produce guttation droplets containing extremely high pesticide concentrations shortly after germination (Girolami et al., 2009 ; Tapparo et al., 2012). The rapid disappearance of cyantraniliprole after day 10 and consistently low levels of Metalaxyl-M, together with the absence of fludioxonil, indicate lower systemic mobility or faster dissipation of these compounds. Comparisons with earlier studies reveal that although the residue levels measured here fall within reported ranges, they remain sufficiently high to pose risks to bees collecting water from treated crops, reinforcing conclusions that guttation can be a critical exposure pathway in the early growth stages of seed-treated maize (Tapparo et al., 2012; Lin et al., 2021). The findings of this study show that guttation liquid from maize grown from thiamethoxam-treated seeds constitutes a sustained and severe hazard to honey bees, with mortality exceeding 80% during the first five days after emergence and remaining high throughout the 27-day sampling period. The rapid lethality observed within just a few hours matches earlier studies showing that neonicotinoid-contaminated guttation droplets can kill bees within minutes (Girolami et al., 2009 ; Tapparo et al., 2012), while the ongoing presence of toxicity over time supports field observations of extended neonicotinoid presence in maize guttation (Reetz et al., 2011 ). Environmental factors such as temperature and humidity may have contributed to fluctuations in mortality levels, consistent with reports that guttation production and solute concentration vary with external conditions (Nikolakis; Wirtz et al. 2018 ). Overall, the results reinforce the conclusion that guttation is an important but often overlooked exposure route for systemic insecticides, capable of delivering neonicotinoid concentrations that exceed lethal thresholds for honey bees under real agricultural conditions (Bonmatin et al. 2005 ; Simon-Delso et al. 2018 ). The results of this study show that guttation liquid from maize grown from cyantraniliprole-treated seeds poses a substantial and persistent hazard to honey bees, with mortality ranging from moderate to extremely high across all exposure intervals and throughout the 27-day sampling period. The rapid onset of toxicity observed within the first 4 hours—often exceeding 60–70% mortality on several days—indicates that cyantraniliprole can exert strong acute effects shortly after ingestion, a pattern consistent with its systemic behavior that allows rapid translocation into guttation droplets, nectar, and pollen (Özşirvan et al., 2024 ; Girolami et al., 2009 ; Shepherd et al., 2024 ). Mortality remained high after 24, 48, and 72 hours, with cumulative values frequently approaching or surpassing 90%, demonstrating that bees continued to experience lethal exposure over time, even when residue levels in plant tissues may have declined. The observation that residues were sometimes undetectable in dead bees while mortality remained high aligns with recent findings that conventional residue analyses may underestimate real exposure because cyantraniliprole and its metabolites can act rapidly and degrade before detection. This supports the growing evidence that guttation represents a realistic and often overlooked exposure pathway for systemic insecticides, particularly during early-morning water foraging. 5. Conclusion This study clearly shows that seed-coated insecticides and fungicide seed treatments used in maize farming pose serious short- and long-term risks to honey bee colonies under real agricultural conditions. Treatments with thiamethoxam and cyantraniliprole resulted in high and ongoing bee mortality, with guttation droplets identified as a key exposure route that delivered lethal doses soon after the plants emerged. Fungicide residues, especially Metalaxyl-M + fludioxonil, also contributed to steady colony losses and built up more in environments with limited foraging space, highlighting their often-overlooked role in the decline of bee health. Pesticide residues in pollen and honey confirm that bees are exposed through multiple routes, meaning both direct contact and indirect contamination harm colony structure and vitality. This exposure involves a complex mix of environmental and behavioral factors that affect both the extent and duration of bee exposure. The presence of these pesticides in hive products raises broader concerns about pollination services and honey quality. These findings emphasize that laboratory toxicity tests alone are insufficient, as they fail to capture the complexity of real-world conditions. Differences in mortality and residue levels between open-field and semi-field studies demonstrate how factors such as foraging behavior, weather, and space constraints influence exposure. Guttation droplets, especially from thiamethoxam-treated maize, pose an acute hazard, with residue concentrations far higher shortly after plant emergence, leading to rapid and severe mortality. Cyantraniliprole, often seen as low risk, also showed strong acute and chronic toxicity through guttation exposure, challenging assumptions about its safety. Given these diverse risks, the study highlights the need for integrated pest management strategies that reduce reliance on systemic insecticides and fungicide seed treatments. Improved residue monitoring that accounts for environmental variation and multiple exposure paths is crucial. Targeted mitigation measures such as adjusting application timing, developing bee-friendly formulations to protect pollinators and promote sustainable farming. Overall, this thorough field-based research provides strong evidence that systemic pesticides in maize seed treatments threaten honey bee colonies through multiple exposure routes. It calls for ongoing, field-relevant risk assessments to complement lab studies and guide effective pollinator protection policies. Safeguarding honey bees from these chemical threats is critical not only for colony health but also for the resilience of agricultural ecosystems and food security. Declarations Acknowledgment This research was funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK), project no 118O522. The authors sincerely thank TÜBİTAK for its valuable support during the study and the development of this manuscript. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Cafer Turgut], [Zeliha Şimşek], [Mustafa Kösoğlu], [Melis Yalçın] and [Nalan Turgut]. The first draft of the manuscript was written by [Cafer Turgut] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.” Funding This study was funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK), project no 118O522. Data Availability Data is provided in the supplementary information files Conflict of interest The authors certify that they have no affiliations or involvement in any organization or entity with any financial or non financial interest in the subject matter or materials discusses in this manuscript. Ethical approval All applicable international, national and/or instutional guidelines for the care and use of animals were followed. References Analysis P, Protection SP, Joachimsmeier I, et al (2012) Assessment of risks to honey bees posed by guttation. 11th International Symposium of the ICP-BR Bee Protection Group, Wageningen (The Netherlands) 437:87–90. https://doi.org/10.5073/jka.2012.437.020 Bonmatin JM, Marchand PA, Charvet R, et al (2005) Quantification of imidacloprid uptake in maize crops. J Agric Food Chem 53:. https://doi.org/10.1021/jf0479362 Chen CC, Chen YR (2007) Study on laminar hydathodes of Ficus formosana (Moraceae) III. Salt injury of guttation on hydathodes. Bot Stud 48:215–226 Cutler GC, Scott-Dupree CD, Sultan M, et al (2014) A large-scale field study examining effects of exposure to clothianidin seed-treated canola on honey bee colony health, development, and overwintering success. PeerJ 2:e652. https://doi.org/10.7717/peerj.652 David A, Botías C, Abdul-Sada A, et al (2016) Widespread contamination of wildflower and bee-collected pollen with complex mixtures of neonicotinoids and fungicides commonly applied to crops. Environ Int 88:169–178. https://doi.org/10.1016/j.envint.2015.12.011 FAO (2023) FAO Statistical Yearbook 2023. In: Crop production statistics. Food and Agriculture Organization: Rome. Fu Y, Wang Q, Zhang L, et al (2021) Ecotoxicology and Environmental Safety Dissipation, occurrence, and risk assessment of 12 pesticides in Dendrobium officinale Kimura et Migo. Ecotoxicol Environ Saf 222:112487. https://doi.org/10.1016/j.ecoenv.2021.112487 Girolami V, Mazzon L, Squartini A, et al (2009) Translocation of Neonicotinoid Insecticides From Coated Seeds to Seedling Guttation Drops: A Novel Way of Intoxication for Bees. J Econ Entomol 102:1808–1815. https://doi.org/10.1603/029.102.0511 Joachimsmeier I, Pistorius J, Heimbach U, et al (2012) Water collection by honey bees – How far will foragers fly to use water sources like guttation drops? A first distance trial using cereals and oilseed rape. Julius-Kühn-Archiv 437:82–86. https://doi.org/10.5073/jka.2012.437.019 Kösoğlu M (2021) Honey Bee Breeding and Some Basic Approaches. MAS Journal of Applied Sciences 8:. https://doi.org/10.52520/masjaps.107 Krupke CH, Hunt GJ, Eitzer BD, et al (2012) Multiple routes of pesticide exposure for honey bees living near agricultural fields. PLoS One 7:. https://doi.org/10.1371/journal.pone.0029268 Laurino D, Porporato M, Patetta A, Manino A (2011) Toxicity of neonicotinoid insecticides to honey bees: Laboratory tests. Bull Insectology 64:107–113 Mörtl M, Takács E, Klátyik S, Székács A (2020) Appearance of thiacloprid in the guttation liquid of coated maize seeds. Int J Environ Res Public Health 17:. https://doi.org/10.3390/ijerph17093290 Nikolakis A, JKMM& RS 2014. Neonicotinoid seed treatment products – Occurrence and relevance of guttation for honeybee colonies, 160–167ʺ.. 12th International Symposium of the ICP-PR Bee Protection Group (15–17 September 2014, Ghent, Belgium), 450 pp. OECD (1998) OECD GUIDELINES FOR THE TESTING OF CHEMICALS Honeybees, Acute Oral Toxicity Test Özşirvan G, Yalçın M, Turgut N, et al (2024) Insights into the uptake, translocation, and accumulation dynamics of cyantraniliprole and thiamethoxam seed coating pesticides in maize plants. Environmental Science and Pollution Research 31:44900–44907. https://doi.org/10.1007/s11356-024-34135-7 Pohorecka K, Skubida P, Semkiw P, et al (2013) Effects of exposure of honey bee colonies to neonicotinoid seed-treated maize crops. J Apic Sci 57:199–208. https://doi.org/10.2478/jas-2013-0029 Reetz JE, Schulz W, Seitz W, et al (2016) Uptake of neonicotinoid insecticides by water-foraging honey bees (Hymenoptera: Apidae) through guttation fluid of winter oilseed rape. J Econ Entomol 109:31–40. https://doi.org/10.1093/jee/tov287 Reetz JE, Zühlke S, Spiteller M, Wallner K (2011) Neonicotinoid insecticides translocated in guttated droplets of seed-treated maize and wheat: A threat to honeybees? Apidologie 42:596–606. https://doi.org/10.1007/s13592-011-0049-1 SANTE (2019) Guidance document on analytical quality control and method validation for pesticide residues analysis in food and feed. Safety of the Food Chain Pesticides and Biocides European Commission 1–48 Sgolastra F, Medrzycki P, Bortolotti L, et al (2017) Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species. Pest Manag Sci 73:1236–1243. https://doi.org/10.1002/PS.4449 Shepherd S, Park Y gyun, Krupke CH (2024) Effects of common co-occurring pesticides (a neonicotinoid and fungicide) on honey bee colony health in a semi-field study. Heliyon 10:. https://doi.org/10.1016/j.heliyon.2024.e29886 Simon-Delso N, San Martin G, Bruneau E, Hautier L (2018) Time-to-death approach to reveal chronic and cumulative toxicity of a fungicide for honeybees not revealed with the standard ten-day test. Sci Rep 8:. https://doi.org/10.1038/s41598-018-24746-9 Soydan DK, Turgut N, Yalçın M, et al (2021) Evaluation of pesticide residues in fruits and vegetables from the Aegean region of Turkey and assessment of risk to consumers. Environmental Science and Pollution Research 28:27511–27519 Sparks TC, Wessels FJ, Lorsbach BA, Nugent BM (2019) The New Age of Insecticide Discovery - The Crop Protection Industry and the Impact of Natural Products. Pestic Biochem Physiol 161:12–22. https://doi.org/10.1016/j.pestbp.2019.09.002 Stoner KA, Cowles RS, Nurse A, Eitzer BD (2019) Tracking Pesticide Residues to a Plant Genus Using Palynology in Pollen Trapped from Honey Bees (Hymenoptera: Apidae) at Ornamental Plant Nurseries. Environ Entomol 48:351–362. https://doi.org/10.1093/ee/nvz007 Thompson H, Vaughan S, Mahlein AK, et al (2022) Is there a risk to honeybees from use of thiamethoxam as a sugar beet seed treatment? Integr Environ Assess Manag 18:709–721. https://doi.org/10.1002/ieam.4498 Thompson HM, Fryday SL, Harkin S, Milner S (2014) Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops. Apidologie 45:545–553. https://doi.org/10.1007/s13592-014-0273-6 Turgut C, Ornek H, Cutright TJ (2010) Pesticide residues in dried table grapes from the Aegean region of Turkey. Environ Monit Assess 167:. https://doi.org/10.1007/s10661-009-1037-z Unay A, Sabanci I, Cinar VM (2021) The effect of maize (Zea mays l.) / soybean (glycine max (l.) merr.) intercropping and biofertilizer (azotobacter) on yield, leaf area index and land equivalent ratio. Tarim Bilimleri Dergisi 27:. https://doi.org/10.15832/ankutbd.572495 Visscher PK, Crailsheim K, Sherman G (1996) How do honey bees (Apis mellifera) fuel their water foraging flights? J Insect Physiol 42:1089–1094. https://doi.org/10.1016/S0022-1910(96)00058-3 Whitehorn PR, O’connor S, Wackers FL, Goulson D (2012) Neonicotinoid Pesticide Reduces Bumble Bee Colony Growth and Queen Production Lab Field Week Cumulative weight gain (g) Downloaded from. www.sciencemag.org SCIENCE 336:. https://doi.org/10.5061/dryad.1805c973 Williams GR, Troxler A, Retschnig G, et al (2015) Neonicotinoid pesticides severely affect honey bee queens. Scientific Reports 2015 5:1 5:1–8. https://doi.org/10.1038/srep14621 Wirtz IP, Hauer-Jákli M, Schenke D, et al (2018) Investigations on neonicotinoids in guttation fluid of seed treated sugar beet: Frequency, residue levels and discussion of the potential risk to honey bees. Crop Protection 105:28–34. https://doi.org/10.1016/j.cropro.2017.11.004 Yalçin M, Kapiz I, Kösoglu M, et al (2021) Translocation of clothianidin to guttation fluid and its potential impact on honey bee, Apis mellifera anatoliaca Maa, 1953 (Hymenoptera: Apidae). Turk Entomol Derg 45:511–522. https://doi.org/10.16970/entoted.1017479 YALÇIN M, TURGUT C (2017) BAL ARILARINDA KOLONİ KAYBI. Adnan Menderes Üniversitesi Ziraat Fakültesi Dergisi 13:151–151. https://doi.org/10.25308/aduziraat.278388 (2015) The 2013 European Union report on pesticide residues in food. EFSA J Additional Declarations No competing interests reported. Supplementary Files ElectronicSupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8384368","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561692753,"identity":"5019a048-dbcc-4624-8f73-74d7418321c0","order_by":0,"name":"Zeliha Şimşek","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Zeliha","middleName":"","lastName":"Şimşek","suffix":""},{"id":561692754,"identity":"90ba8fea-f8c7-475d-9a37-3b43f0a7a9ca","order_by":1,"name":"Mustafa Kösoğlu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mustafa","middleName":"","lastName":"Kösoğlu","suffix":""},{"id":561692755,"identity":"b1568f30-a152-45b8-9143-c5905f649754","order_by":2,"name":"Melis Yalçın","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Melis","middleName":"","lastName":"Yalçın","suffix":""},{"id":561692756,"identity":"e2aedfd8-c50f-415a-9ab3-756377aaa0ae","order_by":3,"name":"Nalan Turgut","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Nalan","middleName":"","lastName":"Turgut","suffix":""},{"id":561692757,"identity":"07f061d6-36d3-4a5a-a93d-16e643dbd61a","order_by":4,"name":"Cafer Turgut","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBADOQaGBCDFRoxaiCIDY7gWHmK1JDYQrYV/fo/Zx69tf9L723MMGD6UHWawlz6AX4vEMR7j2bJtBrkzzrwxYJxx7jADD18Cfi0GbDzGzJJALQ03cgyYeduAWgi5DKYlXR6k5S+xWhg/thkkGIC0MBKjReJYWjEzwzljw41nnhUc7DmXzsNzhoAW/ubDmxl/lMnJyx1P3vjgR5m1HHsPAS0gwAxzygEGYmISBBh/EKVsFIyCUTAKRiwAAPmPOVrKSMvPAAAAAElFTkSuQmCC","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":true,"prefix":"","firstName":"Cafer","middleName":"","lastName":"Turgut","suffix":""}],"badges":[],"createdAt":"2025-12-17 10:08:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8384368/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8384368/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98624045,"identity":"170e7cce-7a99-4822-ac5f-0e7572e6dc0c","added_by":"auto","created_at":"2025-12-19 17:07:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":803914,"visible":true,"origin":"","legend":"","description":"","filename":"17122025MaizeHoneybee.docx","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/7c85d24459dda2e4235f811f.docx"},{"id":98487805,"identity":"f9a416ca-764e-4b6f-80e0-df7950bb6003","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6991,"visible":true,"origin":"","legend":"","description":"","filename":"b65134f29ad44aa9a12573a5bf19236f.json","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/0896ca3963bf9ed9cc27aae2.json"},{"id":98487809,"identity":"91544b13-faea-47e1-a7ff-e605874fdeca","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18204,"visible":true,"origin":"","legend":"","description":"","filename":"ElectronicSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/0f5ab831c546d2347a49b208.docx"},{"id":98487812,"identity":"3f3f14c6-e2a9-4757-a12b-1e6a7a0135df","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105949,"visible":true,"origin":"","legend":"","description":"","filename":"b65134f29ad44aa9a12573a5bf19236f1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/eb08bdee642c376a0b44f54c.xml"},{"id":98487821,"identity":"5f7d4f61-c34b-425f-81c2-f7420e492277","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"emf","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":506524,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.emf","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/3a0c51dbf43acd4e15d87d97.emf"},{"id":98487817,"identity":"849c8871-2b2b-4f4c-8f13-7d83c29d5952","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72156,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/19ce66eac602d8f51f421be5.png"},{"id":98487822,"identity":"53629444-7182-473f-a915-8e7f7cda9329","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77598,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/13dd285b0cd8678d5f4e0e3b.png"},{"id":98623937,"identity":"2def7450-a203-40ff-b4c1-756044bc1983","added_by":"auto","created_at":"2025-12-19 17:07:48","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21920,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/b56714901b5d8c0de40ab13d.png"},{"id":98624860,"identity":"092745af-9ce7-4b0a-a375-3e467e6d4f9a","added_by":"auto","created_at":"2025-12-19 17:08:46","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29313,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/bac6584111d92878b94091d5.png"},{"id":98487818,"identity":"1e26f027-e775-42ed-afb7-8cc7ea7fb9d0","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18257,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/119cae3efc4aac104e5370f3.png"},{"id":98624228,"identity":"6cb23235-bd0f-4c3c-a5a3-ac07759e50ff","added_by":"auto","created_at":"2025-12-19 17:08:10","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104359,"visible":true,"origin":"","legend":"","description":"","filename":"b65134f29ad44aa9a12573a5bf19236f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/f25616dd614aa57fa2c7399e.xml"},{"id":98487824,"identity":"8c6bc810-f66b-4538-b130-a44fbf365317","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112678,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/67c87d8dff3edc6a7f56e52f.html"},{"id":98487810,"identity":"0e7b7f5b-b847-48bf-b98a-1324d010a78f","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":351070,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of bees per hive collected on sample days in semi-field\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/1f8de0ede6cfd5c03f3c190b.jpeg"},{"id":98624229,"identity":"f3667044-8782-4de7-9b32-fde548fad077","added_by":"auto","created_at":"2025-12-19 17:08:10","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":357953,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of bees per hive collected on sample days in field\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/7b76785b8fde36ce9dd1d4ff.jpeg"},{"id":98624685,"identity":"7fd0934a-7afa-4e27-86d7-aaa901adde9c","added_by":"auto","created_at":"2025-12-19 17:08:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75823,"visible":true,"origin":"","legend":"\u003cp\u003ePesticide residues in guttation droplets (mg /L)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/71e162c39ea6eabcf677c6cd.png"},{"id":98487814,"identity":"95172beb-5a34-4faf-972d-20375d43b538","added_by":"auto","created_at":"2025-12-18 07:07:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102003,"visible":true,"origin":"","legend":"\u003cp\u003eToxicological effect of guttation fluid collected from maize plants with seeds coated in thiamethoxam\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/f761f3c2466c664f7b0a678d.png"},{"id":98624769,"identity":"089636b9-0b61-45f7-9ebf-7036a73846f4","added_by":"auto","created_at":"2025-12-19 17:08:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117204,"visible":true,"origin":"","legend":"\u003cp\u003eToxicological effect of guttation fluid collected from maize plants with seeds coated in Cyantraniliprole\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/71489a5b97d036f47a456d06.png"},{"id":98624650,"identity":"706f28dc-145b-423d-9b27-9e3b0245510f","added_by":"auto","created_at":"2025-12-19 17:08:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6348,"visible":true,"origin":"","legend":"\u003cp\u003eToxicological effect of guttation fluid collected from maize plants with seeds coated in Fludioxonil + metalaxyl M\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/21b7963926cda24a8916b806.png"},{"id":100360491,"identity":"eb3a6e97-5314-466f-8036-950d81fa091a","added_by":"auto","created_at":"2026-01-16 07:38:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1744306,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/efab2749-f5ad-468a-862f-52f0b59a640d.pdf"},{"id":98624901,"identity":"3bdb9828-4d5c-493c-9f8b-c5c7af73e4e6","added_by":"auto","created_at":"2025-12-19 17:08:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18204,"visible":true,"origin":"","legend":"","description":"","filename":"ElectronicSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8384368/v1/16079dbd5fdcafea8c1a55f7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Field-Level Evaluation of Honey Bee Exposure and Risk from Pesticides Used in Maize Production","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMaize (\u003cem\u003eZea mays\u003c/em\u003e L.) is a significant cereal crop, ranking third after wheat and rice, with an annual production of approximately 1\u0026nbsp;billion tons (FAO \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is used for food and livestock, and is also widely used to produce biofuel, starch, sweeteners, and additives. Maize is produced in many countries due to its adaptability to different climates (Unay et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePesticides are used to control pests, diseases, and weeds in maize production. Especially, seed coating is a key protection for seeds and emerging seedlings against soil-borne diseases and pests (Girolami et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yal\u0026ccedil;in et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thiamethoxam (THX), a neonicotinoid, and cyantraniliprole (CYN), a diamide, are among the most used systemic insecticides in maize seed treatments worldwide (Sparks et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSeed coating provides clear agronomic benefits by ensuring seed protection and early-stage pest control without the need for foliar spraying. These same systemic properties of seed-coated pesticides enable active compounds to persist within the plant and translocate to multiple tissues, including leaves, stems, and corn (\u0026Ouml;zşirvan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thiamethoxam and cyantraniliprole can be uptaken and translocated to the roots, stem, and leaves, and the bioconcentration factor for thiamethoxam reached up to 4500 during peak periods (\u0026Ouml;zşirvan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Such levels raise questions about unintended exposure pathways for non-target organisms.\u003c/p\u003e \u003cp\u003eThe translocated seed-coated pesticides occur in guttation, which is characterized by guttation droplets formed under conditions of high soil moisture and low transpiration. These droplets exude from hydathodes located at the leaf margins (Chen and Chen \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yal\u0026ccedil;in et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These droplets can contain substantial concentrations of systemic pesticides because of the upward flow of xylem sap (Girolami et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Reetz et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recent studies confirmed that maize guttation fluid can contain residues of thiamethoxam and cyantraniliprole at levels sufficient to pose both acute and chronic risks to honey bees and other beneficial insects (Analysis et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Reetz et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHoney bees, which are key pollinators of both wild flora and many agricultural crops, are known to forage for water sources near their hives to maintain colony thermoregulation and dilute honey stores (Visscher et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Joachimsmeier et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). When alternative water sources are limited, bees can collect guttation droplets directly from maize or other plants (Shawki et al. 2006). Single corn cultivation is practiced as monoculture, especially over large areas. Unlike nectar or pollen, this water is not stored but used immediately within the hive, meaning bees may be repeatedly exposed to any pesticides dissolved in guttation fluid (Whitehorn et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the environmental fate of neonicotinoids has been studied extensively in parts of Europe and North America, a notable lack of data remains for regions such as T\u0026uuml;rkiye, where local bee races and dense beekeeping activity may create unique exposure profiles (YAL\u0026Ccedil;IN and TURGUT \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The local agricultural conditions e.g. climate, soil type, and cropping practices, may influence the uptake and translocation of systemic pesticides in plant tissues (\u0026Ouml;zşirvan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Reetz et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to guttation, pesticide residues can accumulate in pollen and nectar, further elevating the risk to foraging bees. Multiple surveys have detected neonicotinoid residues in bee-collected pollen and wildflower samples adjacent to treated fields (Pohorecka et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; David et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The pesticide residues in pollen and nectar pose a risk to queen production and can cause colony collapse, even at sublethal concentrations (Whitehorn et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sgolastra et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, the simultaneous exposure to insecticides and fungicides can produce synergistic effects, amplifying toxicity (Thompson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite this growing body of evidence, studies examining the combined pathways of exposure, including guttation, pollen, nectar, and direct contact, remain limited in the Eastern Mediterranean and neighboring temperate regions. The lack of region-specific monitoring is concerning, given Turkiye\u0026rsquo;s status as one of the world\u0026rsquo;s largest honey producers (FAO \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the widespread reliance on local subspecies of \u003cem\u003eApis mellifera anatoliaca\u003c/em\u003e (Yal\u0026ccedil;in et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCyantraniliprole is a diamide insecticide from the anthranilic diamide class, known for its systemic action and strong efficacy against a wide range of chewing and sucking pests (\u0026Ouml;zşirvan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its mode of action targets ryanodine receptors, leading to uncontrolled release of calcium in insect muscle cells, which causes paralysis and death. While cyantraniliprole has been marketed as a selective compound with relatively low non-target toxicity, recent studies have highlighted that its systemic behavior allows it to move into guttation droplets, pollen and nectar, posing risks for pollinators that forage or collect water from treated crops (Girolami et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shepherd et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThiamethoxam, a neonicotinoid insecticide, exhibits systemic activity and is widely used for controlling a broad range of sucking and chewing insect pests. Its mode of action involves binding to nicotinic acetylcholine receptors in the insect central nervous system, leading to overstimulation, paralysis, and eventual death (Sandrock et al., 2013; Shareefdeen \u0026amp; Elkamel, 2024). Although thiamethoxam is valued for its high efficacy and relatively selective toxicity, its systemic properties facilitate translocation into guttation droplets, pollen, and nectar. This translocation raises concerns about potential adverse effects on pollinators that forage on treated plants or collect water from these sources (Stoner et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetalaxyl-M, a systemic phenylamide fungicide, is widely used to control oomycete pathogens that cause diseases such as downy mildew and late blight. Its mode of action involves inhibiting RNA synthesis in target fungi, thereby disrupting their growth and reproduction. Metalaxyl-M exhibits high efficacy and selectivity, with systemic properties that allow translocation within plant tissues to protect new growth. However, its systemic nature also raises concerns about the presence of residues in plant fluids, which may affect non-target organisms and environmental exposure. Although primarily targeting fungi, the potential for metalaxyl-M residues in pollen and nectar suggests a need to assess possible risks to pollinators foraging on treated plants.\u003c/p\u003e \u003cp\u003eFludioxonil, a non-systemic fungicide belonging to the phenylpyrrole class, is widely used to control a broad spectrum of fungal pathogens, particularly those causing post-harvest diseases and seed-borne infections. Its mode of action involves disrupting fungal signal transduction pathways, thereby inhibiting spore germination and mycelial growth. Unlike systemic fungicides, fludioxonil remains primarily on the plant surface, minimizing translocation within plant tissues. This characteristic reduces concerns about residues in internal plant fluids; however, its widespread use necessitates evaluation of potential environmental impacts, including effects on non-target organisms, such as pollinators.\u003c/p\u003e \u003cp\u003eField, semi-field, and laboratory studies generate valuable data to prevent colony losses and improve pest management strategies that protect pollinator populations and comparable agroecosystems. Therefore, the primary objective of this research is to identify the risks posed to honeybees by the translocation of seed treatment pesticides through guttation fluid, honey, and pollen, and to categorize these pesticides according to their associated risks.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Field and Semi-Field Experiments\u003c/h2\u003e \u003cp\u003eThe field studies were established in designated maize plots using a randomized complete block design with a single factor with three replicates per treatment. Maize seeds were planted using a 4-row precision pneumatic seeder. Prior to sowing, maize seeds were coated with the pesticides specified in ESM, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Untreated seeds served as the control group. The field and semi-field experiments were conducted at Aydın Adnan Menderes University, Faculty of Agriculture, Research and Field Trial Farm (37\u0026deg;45'32\" N 27\u0026deg;45'35\" E).\u003c/p\u003e \u003cp\u003eFollowing emergence, semi-field tunnels were installed over selected plots using galvanized steel frames at a height of 2.75 meters to ensure bee flight and control environmental exposure. Each tunnel was covered with fine-mesh netting (mesh size\u0026thinsp;\u0026le;\u0026thinsp;3 mm) that permitted air, light, and rainfall to pass through while preventing bees from escaping.\u003c/p\u003e \u003cp\u003eTwelve tunnels were covered for the semi-field maize trial: three for the untreated control and nine for the different seed treatment combinations. For the open-field plots, colonies were directly exposed without any enclosure to mimic realistic foraging conditions.\u003c/p\u003e \u003cp\u003eOne day prior to guttation sampling, one beehive was positioned at the midpoint of the upper left edge of each semi-field tunnel and at the center of each open-field plot. Colonies contained approximately 10,000 worker bees, headed by sister queens to maintain genetic consistency. Each hive comprised 5\u0026ndash;7 frames, including frames with stored honey and pollen for nutrition, as well as brood frames to ensure robust colony development. Brood and food resources were balanced across colonies at the start to maintain uniform strength.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Guttation Fluid Collection\u003c/h2\u003e \u003cp\u003eGuttation samples were collected daily from maize leaves during early morning hours, starting at sunrise (approximately 6:30 a.m.) and continuing until guttation droplets had fully evaporated under daylight. Fluid samples were drawn directly from leaf tips using sterile Pasteur pipettes and transferred into labeled Falcon tubes for laboratory analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Dead Bee Monitoring and Sampling\u003c/h2\u003e \u003cp\u003eDead bees were collected daily from designated collection boards placed at hive entrances and throughout the surrounding plot area. The dead bees were recorded in clean sample containers and stored in a deep freezer at \u0026minus;\u0026thinsp;20\u0026deg;C until extraction and analysis of pesticide residues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Toxicity Bioassays with Guttation Fluid\u003c/h2\u003e \u003cp\u003eTo assess the direct toxicity of maize guttation droplets, laboratory bioassays were performed in accordance with OECD Test Guideline 213 (OECD \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Anatolian honey bees (\u003cem\u003eApis mellifera anatoliaca\u003c/em\u003e) were used from healthy, disease-free colonies and carefully selected to ensure uniform worker age. Ten bees were housed in custom stainless-steel cages with glass panels for ventilation and observation.\u003c/p\u003e \u003cp\u003ePrior to exposure, the bees were acclimated for 24 hours at 25\u0026deg;C and 50\u0026ndash;70% relative humidity. After acclimation, 1\u0026ndash;2 mL of freshly collected guttation fluid was introduced into each cage through a sterile syringe, serving as the bees\u0026rsquo; sole water source. Mortality was monitored at 4-, 24-, 48-, and 72-hours post-exposure. All tests were conducted in triplicate, including untreated controls to account for natural mortality and environmental factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Pesticide Extraction and Residue Analysis\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Extraction from Guttation Fluid, bees, honey and pollen\u003c/h2\u003e \u003cp\u003eFor the determination of pesticide residues in guttation fluid, the modified QuEChERS method was used. A 5 mL aliquot of guttation fluid was mixed with 10 mL of acetonitrile containing 1% acetic acid, vigorously vortexed, and combined with 4 g MgSO₄ and 1 g sodium acetate. After shaking and centrifugation, the supernatant was cleaned with MgSO₄ and PSA, filtered through a Teflon membrane filter, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C prior to analysis.\u003c/p\u003e \u003cp\u003eDead bees (5 g), pollen (2 g), and honey (5 g) samples were weighed and processed with a modified QuEChERS method. After adding 10 mL of distilled water, the samples were vortexed and shaken. Then, 10 mL of acetonitrile was added, followed by MgSO₄, NaCl, and citrate buffers. After centrifugation and PSA cleanup, extracts were reduced, filtered, and analyzed by LC\u0026ndash;MS/MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Extraction of pesticides in soil\u003c/h2\u003e \u003cp\u003eSoil samples were taken three weeks prior to planting at five random locations per plot (at a 30 cm depth) to ensure that no pesticide residues were present before planting. Five grams of soil samples were placed in 50 mL Falcon tubes and mixed with a 1:1 acetone: dicloromethane solution. The tubes were then stored for one night with the lids closed. The next day, the mixture was extracted ultrasonically for 60 minutes and then centrifuged for 5 minutes at 4000 rpm. The supernatant was then transferred and filtered for pesticide analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Analytical Conditions\u003c/h2\u003e \u003cp\u003eAll residue analyses were conducted under Good Laboratory Practice (GLP) conditions using validated instruments (Shimadzu LC\u0026ndash;MS/MS). Pesticide identification was based on molecular ion masses, and quantification was performed by comparing sample peak areas to certified standards (Turgut et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Soydan et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Quality Assurance/Quality control\u003c/h2\u003e \u003cp\u003eThe analysis method used for thiamethoxam and cyantraniliprole, metalaxyl-M, fludioxonil has been validated and quality-controlled according to SANTE (SANTE \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The LOD and LOQ values were established to ensure the method's reliability (ESM, Tab. S2). Calculating LOD and LOQ typically involves statistical methods based on the response standard deviation and the slope of the calibration curve. This method assumes a linear relationship between analyte concentration and the analytical instrument's response. The signal-to-noise (S/N) ratio for the lowest analyte concentration was determined using a ratio of 1:3. Repeated analyses were performed to achieve consistent results (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Colony Development monitoring in bee colonies\u003c/h2\u003e \u003cp\u003eColony strength and brood area were assessed at three-week intervals during the trial. Bee-covered frames and brood areas were measured to monitor population dynamics and production efficiency, calculated according to the standard ellipse formula (K\u0026ouml;soğlu \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical evaluation of daily bee mortality was performed using the Wilcoxon signed-rank test to identify significant differences between treatments and controls. Other measured parameters were compared using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. All tests were conducted using IBM SPSS Statistics software at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Bee mortality in the semi-field and field experiment\u003c/h2\u003e \u003cp\u003eIn this semi-field experiment, daily and cumulative honey bee (\u003cem\u003eApis mellifera anatolica\u003c/em\u003e) mortalities were monitored to assess the effects of three pesticide treatments cyantraniliprole, fludioxonil\u0026thinsp;+\u0026thinsp;metalaxyl-M, and thiamethoxam compared with an untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the Wilcoxon test, all pesticide treatments caused significantly higher cumulative bee mortality than the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The total number of deaths clearly showed that the control group had the lowest bee losses (622), while the treated groups had notably higher totals: fludioxonil\u0026thinsp;+\u0026thinsp;metalaxyl-M (1231), cyantraniliprole (1523), and thiamethoxam, with the highest (1696).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBee mortality showed significant treatment- and time-dependent variation, with the guttation-active first 28 days representing the primary risk window in field studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During this early phase, all pesticide treatments produced markedly higher mortality than the control, with Thiamethoxam and Cyantraniliprole exhibiting the highest peaks (up to 125 and 173 bees per hive, respectively) and consistently elevated daily means relative to the control, which remained below 20 bees on most days. Fludioxonil\u0026thinsp;+\u0026thinsp;Metalaxyl-M also resulted in significantly greater mortality than the control, though at a lower magnitude than the other treatments. After Day 28, mortality declined across all groups; however, pesticide treatments continued to exceed control levels, indicating residual or chronic exposure effects. Cumulative mortality over the full observation period confirmed strong treatment effects, with totals of 1523 bees for Cyantraniliprole, 1231 for Fludioxonil\u0026thinsp;+\u0026thinsp;Metalaxyl-M, and 1696 for Thiamethoxam compared with 622 in the control, highlighting that guttation-related exposure substantially amplified early-phase mortality while longer-term impacts persisted at reduced intensity.\u003c/p\u003e \u003cp\u003eAll three pesticide treatments posed a significant risk compared to the control, but with different impact profiles. Thiamethoxam clearly caused the highest and most continuous daily mortality, while cyantraniliprole produced sharp local peaks that contributed to its high total mortality. The effect of fungicide coated seeds influence slowly but steady increase indicating that repeated low-level exposure can also accumulate to dangerous levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Pesticide residues in dead bees, honey, pollen, and guttation fluid\u003c/h2\u003e \u003cp\u003eThe residue of Metalaxyl-M was found only in dead bees collected from hives located in maize open-fields. Neither cyantraniliprole nor thiamethoxam was detected in any samples. The average residue level of Metalaxyl-M was significantly higher in hives in semi-field plots at 40\u0026thinsp;\u0026plusmn;\u0026thinsp;23.27 \u0026micro;g/kg, compared to 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;16 \u0026micro;g/kg in open-field plots. Statistical analysis confirmed this difference was significant (F\u0026thinsp;=\u0026thinsp;4.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for semi-field; F\u0026thinsp;=\u0026thinsp;18.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for open field) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePesticide residues in dead bees samples (\u0026micro;g/kg )\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetalaxyl-M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCyantraniliprole\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThiamethoxam\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFludioxonil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemi-field\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;23.27a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eField\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;16.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003end: not detected\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of honey samples from hives located in maize fields showed detectable residues of thiamethoxam, cyantraniliprole, and Metalaxyl-M. Fludioxonil was not detected in any of the samples. The mean concentration of thiamethoxam in semi-field plots was 13.213 \u0026micro;g/kg, while in open-field samples it was 10.458 \u0026micro;g/kg. The concentration of Cyantraniliprole was 8.461 \u0026micro;g/kg in semi-field plots and 7.423 \u0026micro;g/kg in the field. Metalaxyl-M concentration in honey samples was higher (14.023 \u0026micro;g/kg) in the samples from the field located hives compared to semi-field samples (0.210 \u0026micro;g/kg) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePesticide residues in honey bee samples (\u0026micro;g/kg)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePesticides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSemi-field\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThiamethoxam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.094\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyantraniliprole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.819\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetalaxyl-M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.02\u0026thinsp;\u0026plusmn;\u0026thinsp;4.858\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFludioxonil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of pollen samples showed that seed treatment pesticides can contaminate pollen collected by bees. Thiamethoxam was detected at 1.000 \u0026micro;g/kg in pollen from semi-field plots and 3.827 \u0026micro;g/kg in field pollen, indicating approximately four times higher exposure in open fields. Metalaxyl-M was also present in pollen, albeit at lower levels: 0.077 \u0026micro;g/kg in semi-field and 0.062 \u0026micro;g/kg in field (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePesticide residues in maize pollen (\u0026micro;g/kg)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePesticides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003esemi-field\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThiamethoxam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.827\u0026thinsp;\u0026plusmn;\u0026thinsp;0.391\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyantraniliprole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetalaxyl-M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.062\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFludioxonil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGuttation fluid in maize showed that thiamethoxam initially reached very high levels (2364 \u0026micro;g/L on day 1) and then gradually declined to 59\u0026ndash;73 \u0026micro;g/L by days 25\u0026ndash;27 after treatment. This decreasing trend reflects the systemic uptake and subsequent dilution or degradation of the compound as the plants developed. Cyantraniliprole was detected at 1\u0026ndash;30 \u0026micro;g/L during the first few days and was undetectable after day 10. The concentration of Metalaxyl-M was very low, starting at 5 \u0026micro;g/L and remaining at approximately 1 \u0026micro;g/L for most of the sampling period, whereas fludioxonil was not detected at any point, indicating negligible systemic transfer. Thiamethoxam residue was found in early guttation samples at levels as high as 2364 \u0026micro;g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Toxicity of guttation fluid to honey bees\u003c/h2\u003e \u003cp\u003eToxicity bioassays demonstrated that guttation liquid collected from maize plants grown from thiamethoxam-treated seeds poses a severe acute hazard to honey bees. During the first five days of the experiment, even 4 hours of exposure resulted in more than 80% worker bee mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMortality decreased slightly in samples collected after day 5, it remained consistently above 50% until day 10. Even in the later stages of plant growth, guttation liquid collected up to day 27 still caused high bee mortality. This shows that guttation remains a risk for an extended period. Bee mortality remained very high after 24 hours of exposure. When bees were exposed to guttation liquid collected during the first nine days, all of them died within 24 hours. In samples collected later, more than half of the bees still died within 24 hours. Longer exposures of 48 and 72 hours indicated that the risk continued to be severe. Guttation liquid from any day in the trial resulted in over 80% mortality after 72 hours in almost all cases, demonstrating that the toxic effect persisted throughout the study.\u003c/p\u003e \u003cp\u003eThe mortality levels showed clear temporal and day-to-day variability, but a consistent pattern of rapid and severe acute toxicity was observed throughout the experimental period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The mortality ranged between approximately 10% and 80%, with the highest peaks recorded on Days 1, 6\u0026ndash;10, and 24\u0026ndash;26 in 4 hours of exposure. On days 6\u0026ndash;9 and at 25 days, the mortality rate exceeds 70%, demonstrating strong acute toxicity even with early exposure durations. After 24 hours, mortality values generally increased by 10\u0026ndash;20%, maintaining high lethality above 60% on most days. At 48 and 72 hours, bee mortality continued to rise slightly or stabilized, suggesting that the compound exerts both acute and residual effects. In some periods (such as Days 17\u0026ndash;19 and 23\u0026ndash;27), cumulative mortality reached or surpassed 90%, confirming the persistence of toxic residues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMortality in honey bees exposed to the fungicide mixture of fludioxonil and metalaxyl-M varied considerably throughout the 27-day experimental period but consistently showed acute toxic responses during the initial days following exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The highest mortality levels occurred during the first four days, with 4-hour mortality reaching up to 90\u0026ndash;100%, indicating strong initial toxicity immediately after contact or ingestion. Although mortality decreased sharply after Day 5, periodic peaks were observed on Days 11, 15, 19, and 24\u0026ndash;25, suggesting that exposure intensity fluctuated with environmental or application conditions. The cumulative increase from 4 to 72 hours in most sampling days reflects both rapid and residual toxic effects of the fungicide mixture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHoney bee mortality was significantly increased in open-field studies by all three pesticide treatments compared with the untreated control, confirming the risks posed by systemic insecticides and fungicide seed coatings. Thiamethoxam-treated colonies exhibited the highest and most continuous daily mortality, with early peaks followed by sustained losses over multiple weeks, consistent with its neurotoxic effects documented under laboratory and field conditions (Laurino et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Krupke et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Cyantraniliprole also caused repeated mortality spikes, notably on day 26, indicating that newer generation insecticides can reach biologically relevant concentrations in foraging environments through guttation droplets or treated foliage (Cutler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; David et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Fludioxonil\u0026thinsp;+\u0026thinsp;metalaxyl-M produced lower but continuous daily deaths, highlighting that fungicide residues, often considered harmless, can accumulate and interact with other stressors to impair colony health (David et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These results demonstrate that both acute and chronic exposure under realistic agricultural conditions contribute to substantial colony-level mortality, emphasizing the limitations of laboratory-only toxicity assessments.\u003c/p\u003e \u003cp\u003eThe variation between the two experimental datasets further illustrates the influence of environmental factors on exposure outcomes. In semi-field studies, thiamethoxam mortality was similar to that of the control, whereas cyantraniliprole and the fungicide mixture continued to elevate losses, suggesting that local foraging behavior, flowering phenology, and weather conditions modulate exposure intensity. Sporadic mortality peaks in thiamethoxam and unusually high daily counts for fludioxonil\u0026thinsp;+\u0026thinsp;metalaxyl-M (e.g., day 56) imply that repeated or combined exposure exacerbates risks. Overall, these findings show that systemic insecticides and fungicide seed treatments can produce cumulative and potentially sublethal effects on colony structure, including reduced brood development and queen vitality. This underscores the need for integrated pest management, stricter residue monitoring, and practical mitigation strategies to protect honey bees and ensure sustainable crop production (Williams et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, continuous field-based risk assessments are essential to complement laboratory toxicity tests and inform pollinator conservation policies.\u003c/p\u003e \u003cp\u003eThe markedly higher Metalaxyl-M residues detected in dead bees from the semi-field (covered greenhouse) compared to the open-field plots suggest that restricted foraging conditions increased exposure and subsequent accumulation. Similar semi-field tunnel studies have shown that limited flight space can lead to higher pesticide and fungicide residues in bees due to prolonged contact within a confined environment (Shepherd et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)(Shepherd et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, the absence of cyantraniliprole, thiamethoxam, and fludioxonil in all samples is consistent with the possibility that these compounds were either not present at appreciable levels in the environment or were rapidly degraded or metabolized before they could accumulate in bee tissues (Cutler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The elevated levels of Metalaxyl-M in dead bees highlight a potential risk to honey bee health, underscoring the importance of considering pesticide application practices, environmental conditions, and bee foraging behavior when assessing exposure pathways and implementing measures to reduce risk to pollinators (M\u0026ouml;rtl et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe detection of thiamethoxam, cyantraniliprole, and Metalaxyl-M in honey from both semi-field and open-field maize plots indicates that foraging bees were exposed to these seed-applied pesticides either directly from treated maize plants or indirectly through contaminated environmental sources. The absence of fludioxonil across all samples may reflect low environmental presence or rapid degradation prior to incorporation into honey. Overall, the elevated pesticide residues observed here highlight the importance of considering crop management and exposure pathways when evaluating risks to honey bees and the quality of their products.\u003c/p\u003e \u003cp\u003eThe presence of thiamethoxam in maize pollen from both semi-field and open-field plots, with concentrations nearly four times higher in the open field, indicates that bees are more likely to encounter contaminated pollen when they can forage freely across larger areas. This aligns with previous findings showing that planter dust and contaminated pollen are primary exposure pathways for neonicotinoids originating from treated seeds (Cutler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The detection of Metalaxyl-M at low levels in both systems further demonstrates that fungicide residues also enter pollen, although to a lesser extent. The absence of cyantraniliprole and fludioxonil suggests limited systemic translocation into maize pollen or rapid degradation prior to bee collection. Recent studies have similarly shown that seed-applied insecticides can move into pollen and nectar at biologically relevant concentrations, potentially contributing to sublethal effects on bee behavior and colony performance (Thompson et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overall, the pollen residue patterns observed here reinforce that maize grown from treated seed can serve as a meaningful exposure source for honey bees, highlighting the need to consider pollen contamination when evaluating risks associated with seed-treatment pesticides.\u003c/p\u003e \u003cp\u003eThe high concentrations of thiamethoxam detected in maize guttation droplets during the first days after emergence demonstrate that guttation represents an acute and significant short-term exposure route for honey bees, particularly when residues reach levels far exceeding lethal doses (EFSA, 2015). The sharp initial peak followed by a steady decline is consistent with previous observations showing that neonicotinoid-treated maize can produce guttation droplets containing extremely high pesticide concentrations shortly after germination (Girolami et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tapparo et al., 2012). The rapid disappearance of cyantraniliprole after day 10 and consistently low levels of Metalaxyl-M, together with the absence of fludioxonil, indicate lower systemic mobility or faster dissipation of these compounds. Comparisons with earlier studies reveal that although the residue levels measured here fall within reported ranges, they remain sufficiently high to pose risks to bees collecting water from treated crops, reinforcing conclusions that guttation can be a critical exposure pathway in the early growth stages of seed-treated maize (Tapparo et al., 2012; Lin et al., 2021).\u003c/p\u003e \u003cp\u003eThe findings of this study show that guttation liquid from maize grown from thiamethoxam-treated seeds constitutes a sustained and severe hazard to honey bees, with mortality exceeding 80% during the first five days after emergence and remaining high throughout the 27-day sampling period. The rapid lethality observed within just a few hours matches earlier studies showing that neonicotinoid-contaminated guttation droplets can kill bees within minutes (Girolami et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tapparo et al., 2012), while the ongoing presence of toxicity over time supports field observations of extended neonicotinoid presence in maize guttation (Reetz et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Environmental factors such as temperature and humidity may have contributed to fluctuations in mortality levels, consistent with reports that guttation production and solute concentration vary with external conditions (Nikolakis; Wirtz et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Overall, the results reinforce the conclusion that guttation is an important but often overlooked exposure route for systemic insecticides, capable of delivering neonicotinoid concentrations that exceed lethal thresholds for honey bees under real agricultural conditions (Bonmatin et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Simon-Delso et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of this study show that guttation liquid from maize grown from cyantraniliprole-treated seeds poses a substantial and persistent hazard to honey bees, with mortality ranging from moderate to extremely high across all exposure intervals and throughout the 27-day sampling period. The rapid onset of toxicity observed within the first 4 hours\u0026mdash;often exceeding 60\u0026ndash;70% mortality on several days\u0026mdash;indicates that cyantraniliprole can exert strong acute effects shortly after ingestion, a pattern consistent with its systemic behavior that allows rapid translocation into guttation droplets, nectar, and pollen (\u0026Ouml;zşirvan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Girolami et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shepherd et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mortality remained high after 24, 48, and 72 hours, with cumulative values frequently approaching or surpassing 90%, demonstrating that bees continued to experience lethal exposure over time, even when residue levels in plant tissues may have declined. The observation that residues were sometimes undetectable in dead bees while mortality remained high aligns with recent findings that conventional residue analyses may underestimate real exposure because cyantraniliprole and its metabolites can act rapidly and degrade before detection. This supports the growing evidence that guttation represents a realistic and often overlooked exposure pathway for systemic insecticides, particularly during early-morning water foraging.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study clearly shows that seed-coated insecticides and fungicide seed treatments used in maize farming pose serious short- and long-term risks to honey bee colonies under real agricultural conditions. Treatments with thiamethoxam and cyantraniliprole resulted in high and ongoing bee mortality, with guttation droplets identified as a key exposure route that delivered lethal doses soon after the plants emerged. Fungicide residues, especially Metalaxyl-M\u0026thinsp;+\u0026thinsp;fludioxonil, also contributed to steady colony losses and built up more in environments with limited foraging space, highlighting their often-overlooked role in the decline of bee health.\u003c/p\u003e \u003cp\u003ePesticide residues in pollen and honey confirm that bees are exposed through multiple routes, meaning both direct contact and indirect contamination harm colony structure and vitality. This exposure involves a complex mix of environmental and behavioral factors that affect both the extent and duration of bee exposure. The presence of these pesticides in hive products raises broader concerns about pollination services and honey quality.\u003c/p\u003e \u003cp\u003eThese findings emphasize that laboratory toxicity tests alone are insufficient, as they fail to capture the complexity of real-world conditions. Differences in mortality and residue levels between open-field and semi-field studies demonstrate how factors such as foraging behavior, weather, and space constraints influence exposure.\u003c/p\u003e \u003cp\u003eGuttation droplets, especially from thiamethoxam-treated maize, pose an acute hazard, with residue concentrations far higher shortly after plant emergence, leading to rapid and severe mortality. Cyantraniliprole, often seen as low risk, also showed strong acute and chronic toxicity through guttation exposure, challenging assumptions about its safety.\u003c/p\u003e \u003cp\u003eGiven these diverse risks, the study highlights the need for integrated pest management strategies that reduce reliance on systemic insecticides and fungicide seed treatments. Improved residue monitoring that accounts for environmental variation and multiple exposure paths is crucial. Targeted mitigation measures such as adjusting application timing, developing bee-friendly formulations to protect pollinators and promote sustainable farming.\u003c/p\u003e \u003cp\u003eOverall, this thorough field-based research provides strong evidence that systemic pesticides in maize seed treatments threaten honey bee colonies through multiple exposure routes. It calls for ongoing, field-relevant risk assessments to complement lab studies and guide effective pollinator protection policies. Safeguarding honey bees from these chemical threats is critical not only for colony health but also for the resilience of agricultural ecosystems and food security.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eAcknowledgment\u003c/h3\u003e\n\u003cp\u003eThis research was funded by the Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK), project no 118O522. The authors sincerely thank T\u0026Uuml;BİTAK for its valuable support during the study and the development of this manuscript.\u003c/p\u003e\n\u003ch3\u003eAuthor contributions\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Cafer Turgut], [Zeliha Şimşek], [Mustafa K\u0026ouml;soğlu], [Melis Yal\u0026ccedil;ın] and [Nalan Turgut]. The first draft of the manuscript was written by [Cafer Turgut] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026rdquo;\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis study was funded by the Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK), project no 118O522.\u003c/p\u003e\n\u003ch3\u003eData Availability\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eData is provided in the supplementary information files\u003c/p\u003e\n\u003ch3\u003eConflict of interest\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eThe authors certify that they have no affiliations or involvement in any organization or entity with any financial or non financial interest in the subject matter or materials discusses in this manuscript.\u003c/p\u003e\n\u003cp\u003eEthical approval All applicable international, national and/or instutional guidelines for the care and use of animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnalysis P, Protection SP, Joachimsmeier I, et al (2012) Assessment of risks to honey bees posed by guttation. 11th International Symposium of the ICP-BR Bee Protection Group, Wageningen (The Netherlands) 437:87\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5073/jka.2012.437.020\u003c/span\u003e\u003cspan address=\"10.5073/jka.2012.437.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonmatin JM, Marchand PA, Charvet R, et al (2005) Quantification of imidacloprid uptake in maize crops. J Agric Food Chem 53:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf0479362\u003c/span\u003e\u003cspan address=\"10.1021/jf0479362\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen CC, Chen YR (2007) Study on laminar hydathodes of Ficus formosana (Moraceae) III. Salt injury of guttation on hydathodes. Bot Stud 48:215\u0026ndash;226\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCutler GC, Scott-Dupree CD, Sultan M, et al (2014) A large-scale field study examining effects of exposure to clothianidin seed-treated canola on honey bee colony health, development, and overwintering success. PeerJ 2:e652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.652\u003c/span\u003e\u003cspan address=\"10.7717/peerj.652\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid A, Bot\u0026iacute;as C, Abdul-Sada A, et al (2016) Widespread contamination of wildflower and bee-collected pollen with complex mixtures of neonicotinoids and fungicides commonly applied to crops. Environ Int 88:169\u0026ndash;178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envint.2015.12.011\u003c/span\u003e\u003cspan address=\"10.1016/j.envint.2015.12.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFAO (2023) FAO Statistical Yearbook 2023. In: Crop production statistics. Food and Agriculture Organization: Rome.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu Y, Wang Q, Zhang L, et al (2021) Ecotoxicology and Environmental Safety Dissipation, occurrence, and risk assessment of 12 pesticides in Dendrobium officinale Kimura et Migo. Ecotoxicol Environ Saf 222:112487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2021.112487\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2021.112487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGirolami V, Mazzon L, Squartini A, et al (2009) Translocation of Neonicotinoid Insecticides From Coated Seeds to Seedling Guttation Drops: A Novel Way of Intoxication for Bees. J Econ Entomol 102:1808\u0026ndash;1815. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1603/029.102.0511\u003c/span\u003e\u003cspan address=\"10.1603/029.102.0511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoachimsmeier I, Pistorius J, Heimbach U, et al (2012) Water collection by honey bees \u0026ndash; How far will foragers fly to use water sources like guttation drops? A first distance trial using cereals and oilseed rape. Julius-K\u0026uuml;hn-Archiv 437:82\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5073/jka.2012.437.019\u003c/span\u003e\u003cspan address=\"10.5073/jka.2012.437.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK\u0026ouml;soğlu M (2021) Honey Bee Breeding and Some Basic Approaches. MAS Journal of Applied Sciences 8:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.52520/masjaps.107\u003c/span\u003e\u003cspan address=\"10.52520/masjaps.107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrupke CH, Hunt GJ, Eitzer BD, et al (2012) Multiple routes of pesticide exposure for honey bees living near agricultural fields. PLoS One 7:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0029268\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0029268\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaurino D, Porporato M, Patetta A, Manino A (2011) Toxicity of neonicotinoid insecticides to honey bees: Laboratory tests. Bull Insectology 64:107\u0026ndash;113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026ouml;rtl M, Tak\u0026aacute;cs E, Kl\u0026aacute;tyik S, Sz\u0026eacute;k\u0026aacute;cs A (2020) Appearance of thiacloprid in the guttation liquid of coated maize seeds. Int J Environ Res Public Health 17:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijerph17093290\u003c/span\u003e\u003cspan address=\"10.3390/ijerph17093290\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikolakis A, JKMM\u0026amp; RS 2014. Neonicotinoid seed treatment products \u0026ndash; Occurrence and relevance of guttation for honeybee colonies, 160\u0026ndash;167ʺ.. 12th International Symposium of the ICP-PR Bee Protection Group (15\u0026ndash;17 September 2014, Ghent, Belgium), 450 pp.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOECD (1998) OECD GUIDELINES FOR THE TESTING OF CHEMICALS Honeybees, Acute Oral Toxicity Test\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;zşirvan G, Yal\u0026ccedil;ın M, Turgut N, et al (2024) Insights into the uptake, translocation, and accumulation dynamics of cyantraniliprole and thiamethoxam seed coating pesticides in maize plants. Environmental Science and Pollution Research 31:44900\u0026ndash;44907. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-024-34135-7\u003c/span\u003e\u003cspan address=\"10.1007/s11356-024-34135-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePohorecka K, Skubida P, Semkiw P, et al (2013) Effects of exposure of honey bee colonies to neonicotinoid seed-treated maize crops. J Apic Sci 57:199\u0026ndash;208. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2478/jas-2013-0029\u003c/span\u003e\u003cspan address=\"10.2478/jas-2013-0029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReetz JE, Schulz W, Seitz W, et al (2016) Uptake of neonicotinoid insecticides by water-foraging honey bees (Hymenoptera: Apidae) through guttation fluid of winter oilseed rape. J Econ Entomol 109:31\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jee/tov287\u003c/span\u003e\u003cspan address=\"10.1093/jee/tov287\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReetz JE, Z\u0026uuml;hlke S, Spiteller M, Wallner K (2011) Neonicotinoid insecticides translocated in guttated droplets of seed-treated maize and wheat: A threat to honeybees? Apidologie 42:596\u0026ndash;606. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13592-011-0049-1\u003c/span\u003e\u003cspan address=\"10.1007/s13592-011-0049-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSANTE (2019) Guidance document on analytical quality control and method validation for pesticide residues analysis in food and feed. Safety of the Food Chain Pesticides and Biocides European Commission 1\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSgolastra F, Medrzycki P, Bortolotti L, et al (2017) Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species. Pest Manag Sci 73:1236\u0026ndash;1243. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/PS.4449\u003c/span\u003e\u003cspan address=\"10.1002/PS.4449\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShepherd S, Park Y gyun, Krupke CH (2024) Effects of common co-occurring pesticides (a neonicotinoid and fungicide) on honey bee colony health in a semi-field study. Heliyon 10:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2024.e29886\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2024.e29886\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon-Delso N, San Martin G, Bruneau E, Hautier L (2018) Time-to-death approach to reveal chronic and cumulative toxicity of a fungicide for honeybees not revealed with the standard ten-day test. Sci Rep 8:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-24746-9\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-24746-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoydan DK, Turgut N, Yal\u0026ccedil;ın M, et al (2021) Evaluation of pesticide residues in fruits and vegetables from the Aegean region of Turkey and assessment of risk to consumers. Environmental Science and Pollution Research 28:27511\u0026ndash;27519\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparks TC, Wessels FJ, Lorsbach BA, Nugent BM (2019) The New Age of Insecticide Discovery - The Crop Protection Industry and the Impact of Natural Products. Pestic Biochem Physiol 161:12\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pestbp.2019.09.002\u003c/span\u003e\u003cspan address=\"10.1016/j.pestbp.2019.09.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoner KA, Cowles RS, Nurse A, Eitzer BD (2019) Tracking Pesticide Residues to a Plant Genus Using Palynology in Pollen Trapped from Honey Bees (Hymenoptera: Apidae) at Ornamental Plant Nurseries. Environ Entomol 48:351\u0026ndash;362. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ee/nvz007\u003c/span\u003e\u003cspan address=\"10.1093/ee/nvz007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson H, Vaughan S, Mahlein AK, et al (2022) Is there a risk to honeybees from use of thiamethoxam as a sugar beet seed treatment? Integr Environ Assess Manag 18:709\u0026ndash;721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ieam.4498\u003c/span\u003e\u003cspan address=\"10.1002/ieam.4498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson HM, Fryday SL, Harkin S, Milner S (2014) Potential impacts of synergism in honeybees (Apis mellifera) of exposure to neonicotinoids and sprayed fungicides in crops. Apidologie 45:545\u0026ndash;553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13592-014-0273-6\u003c/span\u003e\u003cspan address=\"10.1007/s13592-014-0273-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurgut C, Ornek H, Cutright TJ (2010) Pesticide residues in dried table grapes from the Aegean region of Turkey. Environ Monit Assess 167:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10661-009-1037-z\u003c/span\u003e\u003cspan address=\"10.1007/s10661-009-1037-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUnay A, Sabanci I, Cinar VM (2021) The effect of maize (Zea mays l.) / soybean (glycine max (l.) merr.) intercropping and biofertilizer (azotobacter) on yield, leaf area index and land equivalent ratio. Tarim Bilimleri Dergisi 27:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15832/ankutbd.572495\u003c/span\u003e\u003cspan address=\"10.15832/ankutbd.572495\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVisscher PK, Crailsheim K, Sherman G (1996) How do honey bees (Apis mellifera) fuel their water foraging flights? J Insect Physiol 42:1089\u0026ndash;1094. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0022-1910(96)00058-3\u003c/span\u003e\u003cspan address=\"10.1016/S0022-1910(96)00058-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhitehorn PR, O\u0026rsquo;connor S, Wackers FL, Goulson D (2012) Neonicotinoid Pesticide Reduces Bumble Bee Colony Growth and Queen Production Lab Field Week Cumulative weight gain (g) Downloaded from. www.sciencemag.org SCIENCE 336:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5061/dryad.1805c973\u003c/span\u003e\u003cspan address=\"10.5061/dryad.1805c973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams GR, Troxler A, Retschnig G, et al (2015) Neonicotinoid pesticides severely affect honey bee queens. Scientific Reports 2015 5:1 5:1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep14621\u003c/span\u003e\u003cspan address=\"10.1038/srep14621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWirtz IP, Hauer-J\u0026aacute;kli M, Schenke D, et al (2018) Investigations on neonicotinoids in guttation fluid of seed treated sugar beet: Frequency, residue levels and discussion of the potential risk to honey bees. Crop Protection 105:28\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cropro.2017.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.cropro.2017.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYal\u0026ccedil;in M, Kapiz I, K\u0026ouml;soglu M, et al (2021) Translocation of clothianidin to guttation fluid and its potential impact on honey bee, Apis mellifera anatoliaca Maa, 1953 (Hymenoptera: Apidae). Turk Entomol Derg 45:511\u0026ndash;522. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.16970/entoted.1017479\u003c/span\u003e\u003cspan address=\"10.16970/entoted.1017479\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYAL\u0026Ccedil;IN M, TURGUT C (2017) BAL ARILARINDA KOLONİ KAYBI. Adnan Menderes \u0026Uuml;niversitesi Ziraat Fak\u0026uuml;ltesi Dergisi 13:151\u0026ndash;151. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.25308/aduziraat.278388\u003c/span\u003e\u003cspan address=\"10.25308/aduziraat.278388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015) The 2013 European Union report on pesticide residues in food. EFSA J\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pesticides, Maize, Honey Bee, Residue, integrated pest management","lastPublishedDoi":"10.21203/rs.3.rs-8384368/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8384368/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaize seeds are coated with systemic fungicides and insecticides to control pests in maize production. The risk assessments are not well covered for their effects on non-target arthropods, e.g., honey bees, in real farm conditions. The effects of seed-applied thiamethoxam, cyantraniliprole, and a fludioxonil\u0026thinsp;+\u0026thinsp;metalaxyl-M mixture were investigated on honey bees using complementary field, semi-field, and laboratory experiments.\u003c/p\u003e \u003cp\u003ePesticide Residues were analysed in guttation fluid, honey, pollen, and dead bees, and acute toxicity bioassays were conducted with freshly collected guttation droplets from treated maize plants. The mortality rate was higher than in the control across all pesticide treatments in semi-field, and it was highest with thiamethoxam, followed by cyantraniliprole and the fungicide mixture. Field experiments showed lower overall effects, but cyantraniliprole and the fungicide mixture still caused significantly increased mortality. Thiamethoxam reached very high concentrations in guttation fluid (2364\u0026ndash;2565 \u0026micro;g L⁻\u0026sup1;) soon after plant emergence, resulting in rapid mortality of over 80% within 4 hours. Cyantraniliprole-contaminated guttation droplets also caused acute toxicity, with mortality often exceeding 60\u0026ndash;90% within 24 to 72 hours.\u003c/p\u003e \u003cp\u003eGuttation droplets are a critical, yet underappreciated, exposure route for seed-coated pesticides and can deliver lethal doses to honeybees in maize fields. Incorporating guttation exposure into pesticide risk assessment schemes and strengthening integrated pest management strategies are essential to reduce non-target impacts while maintaining effective pest control.\u003c/p\u003e","manuscriptTitle":"Field-Level Evaluation of Honey Bee Exposure and Risk from Pesticides Used in Maize Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 07:07:34","doi":"10.21203/rs.3.rs-8384368/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6c241480-dd4a-4d7d-ad3c-da9100be124a","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T13:25:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 07:07:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8384368","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8384368","identity":"rs-8384368","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}