Environmental Science and Pollution Research (2023) 30:27636–27649 https://doi.org/10.1007/s11356-022-24061-x RESEARCH ARTICLE Biochemical responses, feeding and survival in the solitary bee Osmia bicornis following exposure to an insecticide and a fungicide alone and in combination Cátia Ariana Henriques Martins1 · Ilaria Caliani2 · Antonella D’Agostino3 · Agata Di Noi4 · Silvia Casini2 · Martina Parrilli1 · Celeste Azpiazu5,6 · Jordi Bosch7 · Fabio Sgolastra1 Received: 11 August 2022 / Accepted: 2 November 2022 / Published online: 16 November 2022 © The Author(s) 2022 Abstract In agricultural ecosystems, bees are exposed to combinations of pesticides that may have been applied at different times. For example, bees visiting a flowering crop may be chronically exposed to low concentrations of systemic insecticides applied before bloom and then to a pulse of fungicide, considered safe for bees, applied during bloom. In this study, we simulate this scenario under laboratory conditions with females of the solitary bee, Osmia bicornis L. We studied the effects of chronic exposure to the neonicotinoid insecticide, Confidor® (imidacloprid) at a realistic concentration, and of a pulse (1 day) expo- sure of the fungicide Folicur® SE (tebuconazole) at field application rate. Syrup consumption, survival, and four biomarkers: acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), and alkaline phosphatase (ALP) were evaluated at two different time points. An integrated biological response (IBRv2) index was elaborated with the biomarker results. The fungicide pulse had no impact on survival but temporarily reduced syrup consumption and increased the IBRv2 index, indicating potential molecular alterations. The neonicotinoid significantly reduced syrup consumption, survival, and the neurological activity of the enzymes. The co-exposure neonicotinoid-fungicide did not increase toxicity at the tested concentrations. AChE proved to be an efficient biomarker for the detection of early effects for both the insecticide and the fungicide. Our results highlight the importance of assessing individual and sub-individual endpoints to better understand pesticide effects on bees. Keywords Biomarkers · IBRv2 index · Imidacloprid · Pesticides · Sub-lethal effects · Tebuconazole Introduction Pesticide use associated with agricultural intensification is considered one of the main drivers of pollinator declines (Goulson et al. 2015). Although most studies focus on sin- Responsible Editor: Giovanni Benelli gle products or active ingredients, pollinators are usually exposed to combinations of products (Woodcock et al. 2016; Cátia Ariana Henriques Martins and Ilaria Caliani contributed equally to this work. 4 * Agata Di Noi Department of Life Sciences, University of Siena, Via agata.dinoi@student.unisi.it Mattioli, 4, 53100 Siena, Italy 5 1 Institute of Evolutionary Biology (CSIC-Universitat Department of Agricultural and Food Sciences, Alma Pompeu Fabra), Passeig Marítim de La Barceloneta 37, Mater Studiorum Università Di Bologna, Viale Fanin 42, 08003 Barcelona, Spain 40127 Bologna, Italy 6 2 Universidad Politécnica de Madrid, 28040 Madrid, Spain Department of Physical, Earth and Environmental Sciences, 7 University of Siena, Via Mattioli 4, 53100 Siena, Italy CREAF, Universitat Autònoma de Barcelona, 3 08193 Barcelona, Bellaterra, Spain Department of Management and Quantitative Studies, University of Naples Parthenope, Naples, Italy 13 Environmental Science and Pollution Research (2023) 30:27636–27649 27637 Grab et al. 2019). Multi-pesticide exposure may occur due since they mechanistically interact with the nervous tissues to the application of tank mixtures, but also when differ- of organisms (Sanchez-Hernandez 2011). Other important ent products are applied sequentially. For example, residues biomarkers, such as glutathione S-transferase (GST) and of systemic insecticides from treatments performed before alkaline phosphatase (ALP), are involved in the biotrans- bloom (e.g., as seed coating) may appear in the flowers and formation and detoxification of pollutants, and were first get mixed with fungicides applied during bloom. Due to appointed as good candidates to monitor the defenses of their low toxicity for bees, many entomophilous crops are the honey bee by a neonicotinoid insecticide (Badiou- sprayed with fungicides at least once during bloom (Xavier Bénéteau et al. 2012). In the last years, the search for bio- et al. 2020; Almasri et al. 2021). Under this scenario, pol- markers indicative of sub-lethal effects to various organ- linators experience chronic exposure to residual concentra- isms has become a priority in ecotoxicological research tions of systemic insecticides and acute exposure to high (Tlili and Mouneyrac, 2021; López-Uribe et al. 2020); concentrations of fungicides. The levels of insecticides however, most studies on bees have only targeted the west- applied before bloom appearing in the pollen and nectar of ern honey bee, A. mellifera, and studies on solitary bees crop flowers are typically low (Zioga et al. 2020). Some are mostly lacking (Mokkapati et al. 2022). A research studies have reported that such concentrations pose no lethal effort on this topic is fundamental because solitary bees risk to bees (Maus et al. 2003; Faucon et al. 2005; Nguyen are more sensitive than honey bees to certain pesticides et al. 2009), but may cause sub-lethal effects. On the other (Arena and Sgolastra 2014; Sgolastra et al. 2017; Azpiazu hand, fungicides are not supposed to directly harm insects, et al. 2021) and have different routes and levels of expo- but sub-lethal effects, including genotoxicity (Caliani et al. sure (Sgolastra et al. 2019). In fact, the European Food 2021a) and alterations of the feeding behavior (Zhu et al. Safety Authority pointed out the necessity to include 2017a), have been found in Apis mellifera. In addition, sev- Osmia spp. as representative species of solitary bees in eral studies have demonstrated that some insecticide-fungi- pesticide risk assessment (EFSA 2013). Osmia bicornis is cide combinations induce synergistic toxicity effects in bees common European solitary bee that is managed for crop (Pilling et al. 1995; Thompson and Wilkins 2003; Johnson pollination in some areas (Sedivy and Dorn 2014), and et al. 2013; Thompson et al. 2014; Mengoni Goñalons and therefore is often exposed to pesticides. Farina 2018; Wang et al. 2020a, b a,b). In particular, sterol In this study, we conducted a laboratory experiment in biosynthesis inhibiting (SBI) fungicides have been shown which we combined a chronical exposure to a field-realistic to interact with neonicotinoids (Iwasa et al. 2004; Biddinger concentration of an insecticide (Confidor®, imidacloprid) et al. 2013; Sgolastra et al. 2017; Raimets et al. 2018; Iver- with a single exposure to a fungicide (Folicur® SE, tebu- son et al. 2019). This interaction occurs because SBI fungi- conazole) in females of the solitary bee O. bicornis. Despite cides modify the metabolic detoxification processes in bees the ban on the use of neonicotinoids (imidacloprid, thia- by inhibiting cytochrome P450-monooxygenase (Berenbaum methoxam, and clothianidin) in the European Union, their and Johnson 2015; Carnesecchi et al. 2019). presence in the environment is still reported due to the high Sub-lethal effects are not easy to detect over the course persistence of neonicotinoids (Botías et al. 2016; Winterm- of customary toxicological tests in the laboratory. Syrup antel et al. 2020), and therefore they may still pose a threat to consumption is an easy-to-measure fitness endpoint that pollinators. In addition, neonicotinoids are still widely used may provide insights on pesticide-induced changes at the in non-EU countries (Goulson 2020). We thus simulated a individual level. At the sub-individual level, the detoxifi- scenario in which bees foraging on flowers with residual cation energy costs related with enzymatic activity may concentrations of a systemic insecticide are exposed to a have repercussions on fitness-related traits (Castañeda high fungicide dose applied during bloom. Our study has et al. 2009). For this reason, biomarkers, which can pro- important implications for pesticide risk assessment: first, vide signals of early stage alterations at lower biological current risk assessment schemes are mostly based on sin- levels, represent an important tool to evaluate sub-lethal gle compounds (Rortais et al. 2017), even in the face of effects (Caliani et al. 2021a). Different biochemical and increasing evidence that pollinators are exposed to mixtures cellular biomarkers have been developed and applied in of pesticides (Sgolastra et al. 2020); second, current risk honey bees to assess ecotoxicological health status and the assessment schemes mostly overlook sub-lethal effects such sub-lethal effects of different pollutant compounds such as behavioral and physiological responses that may affect as pesticides, heavy metals, and PAHs (Badiou-Bénéteau bee health even when no effects on survival are detected et al. 2012; Carvalho et al. 2013; Zhu et al. 2017b; Han (Cresswell 2011; Azpiazu et al. 2019; Sandrock et al. 2014); et al. 2019; Caliani et al. 2021b). Acetylcholinesterase third, pesticide risk assessment has traditionally relied on a (AChE) and carboxylesterases (CaE) have been widely single species, the western honey bee, although pesticide used as biomarkers to assess the effects of different insec- effects may be species-dependent (Schmolke et al. 2021), ticide classes, such as organophosphates and carbamates, and extrapolation from honey bees to wild bees may not 13 27638 Environmental Science and Pollution Research (2023) 30:27636–27649 adequately reflect realistic scenarios due to colony resilience [Schmuck et al. 2001]; ornamental plants: < 1.2–5.7 ng ­g−1 in honey bees (Rundlöf et al. 2015). [Lentola et al. 2017]). For tebuconazole, we worked with the In this study, we measured syrup consumption and sur- potential concentration immediately after spray application, vival at the individual level, as well as a set of biomarkers calculated as the maximum field application rate of its com- covering various biological responses, including neurotoxic- mercial formulation (6.45 L ­ha−1) in orchards. ity (AChE and CaEs) and metabolic activity (GST and ALP). We also propose the development of an integrated biological Osmia bicornis and test conditions response (IBRv2) index (Sanchez et al. 2013) providing a measure of the overall response of the target organism to the Bees were supplied by Pollinature Srl. Cocoons were exposure of pesticides in O. bicornis. This index is based on shipped to the Department of Agricultural and Food Sci- the biomarker deviation from the reference site, allowing ences, University of Bologna, Italy, and kept at wintering the identification of how each selected biomarker contrib- temperatures of 3–4 °C and 55 ± 10% relative humidity. utes to the final toxicological status (Arrighetti et al. 2019). In May 2021, large cocoons, expected to be females, were To our knowledge, IBRv2 indexes have not been developed incubated at 22–23 °C until emergence. We worked with for insect pollinators except for honey bees (Caliani et al. newly emerged females (< 24 h old). Over a period of 5 days, 2021a, b a,b), but they are widely used to investigate the emerging females were distributed randomly and equally effects of different contaminants on other groups of organ- among the four exposure treatments (see below). Upon isms. Our goal was to assess whether the chronic exposure emergence, females were transferred to a Plexiglas flight to the insecticide, the fungicide pulse, and the insecticide- cage (50 × 50 × 50 cm) for meconium deposition and 24 h fungicide combination elicited some biomarker responses starvation. Two hundred and forty bees (60 bees per treat- that could be related to syrup consumption and survival. ment) were then transferred to individual cages (transparent plastic cups; volume: 150 cc), with perforated lids to allow air circulation. Each cup was provided with a syrup feeder Material and methods consisting of a 1-mL-calibrated syringe (BEROJECT® III, accuracy: 0.02 mL) inserted laterally and slightly inclined. Pesticides A petal of Euryops (Asteracea) was attached to the tip of the syringe to enhance prompt location of the feeder by the bee We used commercially available formulates, Confidor® (Sgolastra et al. 2018; Azpiazu et al. 2019). From emergence (imidacloprid 20% w/v) and Folicur® SE (tebuconazole until death, bees were maintained at 21–24 °C and 50–55% 4.35% w/v), rather than active ingredients. The two pesti- relative humidity under natural light, avoiding direct sun- cides were chosen because they are extensively used for pest light to reduce pesticide degradation. and disease control in bee-pollinated crops such as fruits, nuts, and vegetables. Many studies have documented co- Exposure conditions occurrence of the two active ingredients in nectar and pollen samples (Chauzat et al. 2006, 2009, 2011; Mullin et al. 2010; After 24 h of starvation, bees were divided into 4 groups: Pohorecka et al. 2012; David et al. 2015, 2016; Lentola et al. control (CTRL), insecticide Confidor® (INS), fungicide 2017; Ostiguy et al. 2019). Folicur® SE (FUNG), and the two pesticides (MIX). Bees of Stock solutions of each pesticide were prepared by dis- the CTRL treatment were fed regular syrup throughout the solving the products in distilled water at nominal concentra- experiment. Bees of the FUNG treatment were also fed regu- tions of 50 µg ­L−1 of Confidor® and 1850 mg ­L−1 of Foli- lar syrup throughout the experiment except on day 3 when cur® SE. The stock solutions were then diluted in a feeding they were offered syrup with fungicide. Bees of the INS solution (sugar and distilled water at 47.5% w/v; henceforth treatment were fed syrup with insecticide throughout the syrup) to achieve the desired concentrations of 5 µg L­ −1 and experiment. Bees of the MIX treatment were also fed syrup −1 185 mg ­L of imidacloprid and tebuconazole, respectively. with insecticide throughout the experiment, except on day The final concentration of the syrup given to bees was 38% 3 when they were fed syrup with insecticide and fungicide. w/v (Azpiazu et al. 2019). The concentration of imidaclo- In the treatment groups FUNG and MIX, the solution with prid was within the range of residues found in nectar col- fungicide was only offered for a period of 24 h to simulate a lected from flowers of different crops, either grown from pulse exposure. This exposure scenario represents a compro- imidacloprid-coated seed or treated via soil or spray applica- mise between a worst-case scenario, which does not account tions (citrus: 0.8–6.82 ng ­mL−1 [Byrne et al. 2014]; apples: for fungicide degradation during the 24 h, and a best-case 2–70 ppb [Heller et al. 2020]; cucurbits: 3.8–7.3 ng ­g−1 and scenario in which the fungicide is completely degraded in 6.7–16 ng ­g−1 [Dively and Kamel 2012], 5–14 ppb [Stoner one day. Tebuconazole is known to be a stable compound and Eitzer 2012]; sunflower: 0.0019 (± 0.001) mg ­kg−1 under hydrolytic and photolytic conditions (Lewis et al. 13 Environmental Science and Pollution Research (2023) 30:27636–27649 27639 2006; EFSA 2014). In all cases, bees were fed ad libitum CaE throughout the experiment. To avoid fungal proliferation, feeding solutions were freshly prepared every 3 days. The CaE activity was measured in the head extracts and quantified at 538 nm according to Caliani et al. (2021a). A mixture containing 100 mM sodium phosphate buffer Syrup consumption and survival (pH 7.4) and a 0.1-mL head extract was prepared and incu- bated at 25 °C for 5 min. The reaction was started by adding Syrup consumption and survival were monitored daily until 0.4 mM α-NA as a substrate. After 3 min, the reaction was all bees died. To account for potential evaporation, syrup stopped adding 1.5% SDS and 0.4 mg/L Fast Garnet GBC. levels were measured in eight cages without bees. After The products of the reaction were quantified spectrophoto- dead, the head width of each bee was measured under a metrically (Agilent CARY UV60) at 538 nm (25 °C) and the stereomicroscope with a micrometer as a proxy of body size enzyme activity was expressed as nmol α-NA ­min−1 ­mg−1 (Bosch and Vicens 2002). protein (ε = 23.59 × 103 ­mM−1 ­cm−1). Collection of tissue samples GST Ten bees per treatment were collected for biomarker assess- The GST activity was measured in the midgut samples fol- ment at two different time points: T1 (on the fourth day of lowing the method of Habig et al. (1974), modified. The exposure, that is 24 h after the fungicide pulse) and T2 (on reaction mixture consisted of 0.1 M sodium phosphate buffer the sixth day of exposure, that is 72 h after the fungicide (pH 7.4), 8 mM GSH (reduced glutathione), 8 mM CDNB, pulse). Bees were anesthetized in ice (4 °C) for 30 min and and 30 μL extract. The conjugation of GSH with 1-chloro- then the midgut and the head were removed and immediately 2,4-dinitrobenzene (CDNB) was recorded spectrophoto- frozen at − 80 °C. metrically (Agilent CARY UV60) at 340 nm (25 °C) and expressed as nmol CDNB conjugate formed m ­ in−1 ­mg−1 −1 −1 protein (ε = 9.6 × 103 ­mM ­m ). Biomarker analysis ALP For each specimen, the head and midgut were processed separately to obtain the extracts on which to perform the The ALP activity was assayed in the midgut samples fol- enzymatic tests. Nervous tissue extracts from the head were lowing the formation of p-nitrophenol, a product of the used to evaluate AChE and CaE, and midgut extracts were hydrolysis of the substrate (PNPP) due to the enzyme’s used to measure GST and ALP activities. Tissues were activity, according to Bounias et al. (1996), modified. The weighted, and extraction medium was added proportionally reaction mixture consisted of 100 mM Tris–HCl buffer (pH to the weight of the tissue at a ratio of 10% (w/v). The buffer 8.5), 100 mM MgCl2, 100 mM p-NPP as the substrate and contained 40 mM Na phosphate buffer (pH 7.4), a mixture a 25-μL gut extract. The reaction was monitored continu- of protease inhibitors enzymes and 1% Triton X-100. The ously for 5 min at 405 nm (25 °C) at the spectrophotometer samples were homogenized by a tissuelyser (Qiagen) at 20 F (Agilent CARY UV60), and the activity was expressed as for three periods of 30 s at 30-s intervals. The homogenates nmol p-nNPP ­min−1 ­mg−1 protein (ε = 18.81 × 103 ­mM−1 ­c were centrifuged at 4 °C for 20 min at 13,000 g and 15,000 g m−1 ­cm−1). for head and gut samples, respectively. The resulting super- natants were frozen at − 80 °C and used for the analyses. Protein concentrations AChE Protein concentrations were measured according to the method of Bradford (1976) by BioRad Protein Assay (Bio- The AChE activity was assayed in the head extracts accord- Rad), using bovine serum albumin (BSA) as standard. ing to Ellman et al. (1961) with modification from Caliani et al. (2021b). The reaction mixture was prepared in a 3-mL Statistical analysis cuvette and contained 0.1 M sodium phosphate buffer (pH 7.4), 10 mM DTNB, 41.5 mM acetylthiocholine, and 5 μL Statistical analysis was carried out with STATA (Stata- head extract. The activity was monitored continuously with Corp 2015) and data visualization with R software (Team a spectrophotometer (Agilent CARY UV60) for 5 min at R Core 2013). Only bees that consumed at least 10 µL on 410 nm (25 °C) and expressed in μmol−1 g ­tissue−1 min. 13 27640 Environmental Science and Pollution Research (2023) 30:27636–27649 the first 2 days of exposure were included in the analy- G1,1 to detect differences between treatments especially in ses. Individuals collected for biomarker analysis were not the intermediate section of the survival curves. included in the syrup consumption and survival analyses. We performed a Kernel regression (KR) to detect a possi- To avoid confounding effects of reduced syrup con- ble relationship between body size (measured as head width) sumption due to aging, we used daily syrup consumption and syrup consumption, and Cox regression model to detect data only up to the median survival date of each treat- the potential effect of body size on survival time. ment. Differences in daily consumption among treatments Biomarker data were first analyzed by comparing the were analyzed at three different times (the first 2 days of median of the two collection times (T1 and T2) for each bio- exposure or “pre-pulse,” the day of fungicide exposure marker and treatment. KW non-parametric test and Dunn’s or “pulse,” and between the day after fungicide exposure test were conducted. Spearman’s rank correlation coeffi- until the median survival date “post-pulse”). We used the cient was used to explore the relationship between pairs of Kruskall-Wallis (KW) non-parametric test to detect dif- biomarkers. Lastly, integrated biological response (IBRv2) ferences among treatments in daily syrup consumption at index (Sanchez et al. 2013) was used to quantify in a single each time. Dunn’s test (with Benjamini–Hochberg correc- value the overall degree of response to each treatment, in tion) was performed for pairwise multiple-comparison. A which higher IBRv2 values represent a higher stress level. Mann–Whitney U test for paired samples was used to test Results are reported with a significance level of 5%. for differences among pre-pulse and post-pulse periods and treatments. Bees that died before the post-pulse period were excluded from this analysis. Results Survival functions S(t) were estimated using a Kaplan- Maier estimator with no censoring. Accordingly, S(t) was Syrup consumption estimated as 1 − Fn (t), where Fn (t) is the empirical cumula- tive distribution function. Significant differences in syrup consumption were found The comparison of survival rates between CTRL and between treatments with and without the insecticide (Dunn’s the other treatments was performed using the Flem- test; p ≤ 0.0001; Fig. 1 and Table A.1); overall, bees from ing–Harrington test, belonging to the weighted log-rank INS and MIX consumed approximately 74% less syrup than test G𝜌,𝜆 class (Fleming and Harrington 2011). We used bees of the CTRL and FUNG treatments. The fungicide Fig. 1  Daily syrup consumption (µl ­day−1) up to the date of 50% day 3; POST, after the 3.rd day up to the date of 50% mortality within mortality within each treatment. CTRL, control (n = 25); FUNG, each treatment. Boxplots with asterisks are significantly different tebuconazole (n = 26); INS, imidacloprid (n = 26); MIX, tebucona- from the control (Dunn’s pairwise comparison, ****p < 0.0001) zole + imidacloprid (n = 24)l PRE, first 2 days of exposure; PULSE, 13 Environmental Science and Pollution Research (2023) 30:27636–27649 27641 pulse (FUNG) caused a decrease in feeding rate, which at both times compared to the control (Dunn’s test; T1, returned to control levels over the post-pulse period. Overall, p < 0.01; T2, p < 0.001); AChE was also significantly inhib- syrup consumption significantly (p < 0.0001) decreased from ited in FUNG treatment at T1 (Dunn’s test; p < 0.05). No sig- the pre-pulse to the post-pulse assessments in all treatments nificant differences were observed for CaE, GST, and ALP (see Table A.2 for Mann–Whitney U test results). Kernel activity, in none of the assessment times. Overall, we found a regression analysis indicates that body size had no effect on significant positive correlation between ALP and GST at T1 daily syrup consumption (Table A.3). (p < 0.05; ρ = 0.666) and at T2 (p < 0.001; ρ = 0.806) and a positive and significant correlation between syrup consump- Survival analysis tion and AChE activity at T1 (p < 0.05; ρ = 0.4072) and T2 (p < 0.01; ρ = 0.4710). Exposure to INS and MIX had an effect on survival of O. bicornis females. Survival significantly differed among treat- IBRv2 ments (p < 0.01) in the intermediate part of the distribution curves (Fig. 2 and see Table A.4 for results of Fleming–Har- The results of the integrated biological response (IBRv2) for rington tests). Median survival time dropped from approxi- each treatment are shown in Fig. 4. In the FUNG treatment, mately 21 days for CTRL and FUNG bees to 11 days for the IBRv2 value declined from T1 (6.26) to T2 (2.67). The INS and MIX bees. Body size had no effect on survival (see most discriminant factor for this treatment shifted from GST Table A.5 for Cox model results). at T1 to CaE at T2. Bees exposed to INS and MIX showed increasing IBRv2 values from T1 to T2, with CaE as the Biomarkers predominant factor in all the star plots. The MIX treatment showed the lowest IBRv2 at T1 (4.09). Biomarkers of neurotoxicity (AChE and CaE) and metabolic activity (ALP and GST) were assessed on days 4 (T1) and 6 (T2), that is 24 h and 72 h after the fungicide pulse, respec- Discussion tively. The results of the four biomarkers at T1 and T2 are shown in Fig. 3. The results of the descriptive statistics, the In this study, we tested the effects of oral co-exposure to a Kruskal–Wallis tests for the assessment of statistically sig- neonicotinoid, imidacloprid, and a SBI fungicide, tebucona- nificant differences among groups for each biomarker and zole, on adult O. bicornis females. Our first objective was syrup consumption at T1 and T2, and the Dunn’s pairwise to establish whether exposure to a fungicide pulse could comparison with the control group are summarized in the enhance the toxicity of low-level chronic exposure to the supplementary material (Tables A.6, A.7 and A.8). AChE neonicotinoid. Our second objective was to identify neuro- activity was significantly inhibited in the INS treatment toxicity and metabolic activity biomarkers that could act as early warning signals of sub-lethal effects. Our results clearly indicate a feeding reduction due to the continued feeding of imidacloprid, which agrees with the findings of other studies (Zhu et al. 2017a; Azpiazu et al. 2019). The fungicide pulse at the maximum field application concentration caused a temporary decrease in feeding rate but did not affect post-pulse syrup consumption. In addition, the fungicide pulse did not impact the feeding of bees of the MIX treatment. A study in which bumble bees were exposed to the combination imidacloprid-imazalil also failed to find synergistic effects on feeding rate (Raimets et al. 2018). The observed effect of imidacloprid on syrup consumption may be related to the reduction of bee mobility, leading bees to ingest less syrup (Medrzycki et al. 2003; Wu et al. 2017). In addition, the median survival time of bees exposed to imidacloprid at 5 µg ­L−1 (alone and in combination with the fungicide) was significantly shorter (e.g., 10 days) than Fig. 2  Survival curves of Osmia bicornis females orally exposed in bees from CRTL and FUNG treatments. In our study, to various pesticide treatments. CTRL, control (n = 38); FUNG, bees were chronically exposed to a constant concentra- tebuconazole (n = 31); INS, imidacloprid (n = 33); MIX, tebucona- zole + imidacloprid (n = 27). The dashed line indicates 50% survival tion of imidacloprid throughout their lifespan, an approach rate that does not account for pesticide degradation over time 13 27642 Environmental Science and Pollution Research (2023) 30:27636–27649 Fig. 3  Activity of biomarkers AChE (μmol−1 g ­tissue−1 min), CaE Measurements were taken at T1 (day 4 of exposure; A and T2 (day (nmol ­min−1 ­mg−1 protein), GST (nmol m ­ in−1 ­mg−1 protein) and 6 of exposure; B boxplots with asterisks are significantly different ALP (nmol ­min−1 mg.−1 protein) in Osmia bicornis females orally from the control (Dunn’s pairwise comparison, *p < 0.05; **p < 0.01, exposed to various pesticide treatments. CTRL, control; FUNG, ***p < 0.001) tebuconazole; INS, imidacloprid; MIX, tebuconazole + imidacloprid. or “dilution effect” due to visitation to uncontaminated Huang 2012) and may mitigate the negative impact of pes- flowers. However, levels of imidacloprid higher than the ticides (Castle et al. 2022). concentration tested in our study have been found in the In our study, the fungicide did not affect bee survival, flowers of crop and non-crop plants potentially extend- even when combined with the insecticide. Some oral acute ing the period of pesticide exposure beyond the bloom- exposure studies have found a synergistic effect of the fun- ing of the target crop (Botías et al. 2016; Wintermantel gicide propiconazole on the toxicity of the neonicotinoid et al. 2020). Interestingly, the toxicity of imidacloprid in clothianidin (Sgolastra et al. 2018, 2017). In contrast, the our study was higher than in a previous study that used tebuconazole pulse did not reduce the survival of O. bicornis a threefold higher concentration of the same commercial females chronically exposed to imidacloprid in our study. product, Confidor®, on O. bicornis females (Azpiazu et al. These results are in line with other studies in which honey 2019). Median mortality time in the control bees of the bees chronically exposed to imidacloprid-tetraconazole two studies was similar (19 and 20 days, respectively), but (Zhu et al. 2017b) and imidacloprid-difenoconazole (Pal the median mortality time of the group treated with imida- et al. 2022) mixtures did not yield synergistic effects; simi- cloprid was 10 days in our study compared to 16 days in larly, no interactions between imidacloprid (15 µg ­L−1) and Azpiazu’s study (Azpiazu et al. 2019). These differences myclobutanil were found following chronic oral exposure in could be explained by the different diet offered to the bees. O. bicornis (Azpiazu et al. 2019). Our bees were provided with syrup only whereas those of We assessed four selected biomarkers (AChE, CaE, GST, Azpiazu et al. (2019) also had access to pollen. Several and ALP) to determine the impact of the two pesticides at studies have shown that pollen feeding positively affects the neurological and metabolic levels. AChE, an important health and longevity in honey bees (Pasquale et al. 2013; enzyme responsible for the hydrolyses of acetylcholine at 13 Environmental Science and Pollution Research (2023) 30:27636–27649 27643 Fig. 4  Star plots of the integrated biological response (IBRv2) in dacloprid. Measurements were taken at T1 (day 4 of exposure) and Osmia bicornis females orally exposed to three pesticide treatments. T2 (day 6 of exposure). The dashed line indicates the control values FUNG, tebuconazole; INS, imidacloprid; MIX, tebuconazole + imi- the cholinergic synapses (Badiou-Bénéteau et al. 2012), by the imidacloprid (29% reduction, at T1 and 49% at T2), allows the control and modulation of neural transmission indicating a clear neurotoxic effect of the two pesticides and (Badiou et al. 2008). In our work, AChE was significantly confirming AChE as an excellent biomarker for the assess- inhibited by the tebuconazole (27% reduction, at T1) and ment of sub-lethal effects in O. bicornis. 13 27644 Environmental Science and Pollution Research (2023) 30:27636–27649 The levels of inhibition can be considered relevant in Low 1975; Srivastava and Saxena 1967). Although ALP is altering the proper functioning of the nervous system. AChE not involved in detoxification processes, its activity can be inhibition has been associated with exposure to some classes modulated in reaction to chemical stress. In our study, the of insecticides, such as carbamates and organophosphates ALP activity was not statistically inhibited by any treat- (Fulton and Key 2001; Rabea et al. 2010). To date, AChE ment or time, although we observed an overall decrease activity is also used for studying the neonicotinoids and their in its activity. Other studies showed a modulation of ALP metabolites neurotoxic effects (Boily et al. 2013; Shao et al. in honey bees exposed to insecticides, such as fipronil, 2013; Samson-Robert et al. 2015; Gyori et al. 2017). As spinosad, imidacloprid, or following infection by Nosema observed by Badawy et al. (2015), neonicotinoids such as (Dussaubat et al. 2012; Carvalho et al. 2013; Kairo et al. dinotefuran (nitro-substituted compound) and acetamiprid 2017; Paleolog et al. 2020). An inhibition was also found (cyano-substituted), enhance the inhibition of AChE activ- by Caliani et al.( 2021a), after honey bee exposure to ity in honey bees after exposure to different field relevant fungicides and heavy metals. A previous study (Almasri doses, even though AChE is not the target site of neonico- et al. 2020) on honey bees did not find ALP modulation tinoids. In our study, we also observed a neurotoxic effect after the administration of combinations of imidacloprid, positively correlated to alterations in the feeding behavior glyphosate and difenoconazole. We also found a positive of O. bicornis. Caliani et al. (2021b) found a neurotoxic correlation between GST and ALP at both times. The posi- effect of Amistar® Xtra (a.i., azoxystrobin), but no data tive correlation between these two enzymes could indicate were produced regarding syrup consumption. The inhibi- that both are affected by imidacloprid and tebuconazole. tory effect on the AChE activity by tebuconazole, related We used the IBRv2 index to integrate the responses with immobility, has been also reported in aquatic organ- of the selected biomarkers (AChE, CaE, GST, and ALP). isms (Altenhofen et al. 2017; Lebrun et al. 2021). The use This approach facilitates the visualization of the spatial of fungicides may also be associated with sub-lethal effects arrangement of different enzymatic responses and the disrupting the bee’s overall fitness and behavior (Artz and possible effects of different contaminant compounds. Pitts-Singer, 2015; Fisher et al. 2021). As for the MIX group, At T1, the FUNG treatment showed the highest IBRv2 in our study, non-statistically significant alterations in this value, followed by the INS treatment and finally the MIX enzyme activity were observed. We can hypothesize the treatment. This result indicates that the fungicide alone absence of a synergic effect of the two pesticides because induced a high oxidative stress, particularly expressed by we did not observe the highest inhibition in the MIX group. GST activity, although no difference was found between Yet, we cannot exclude an antagonistic or a predominant treatments. Previous studies also found increased GST effect of one compound over the other. response after fungicide exposure (Johansen et al. 2007; CaE are phase-I detoxifying enzymes that mainly hydro- Han et al. 2014). Since the fungicide was administered as lyse non-polar carboxyl esters (Badiou-Bénéteau et al. 2012; a pulse, we expected an improvement of the organisms’ Stone et al. 2002; Barata et al. 2005). Besides, they also play health status at T2 that was confirmed by the IBRv2 low- a role in the defense mechanism, protecting AChE from the est value. This result suggests that the bees are able to inactivation caused by organophosphates and carbamates. biotransform and detoxify when they are not chronically Several studies have also shown differential expression of exposed to the fungicide. On the other hand, the IBRv2 CaEs after exposure to pesticides (Badiou-Bénéteau et al. value increased from T1 to T2 in the INS treatment, as 2012; Zhu et al. 2017a,b). In our study, CaE was not modu- expected given the continuous exposure to the pesticide. lated by the fungicide or the insecticide. This result, together This treatment group is also the one that shows the high- with the AChE inhibition, leads us to hypothesize that the est IBRv2 value at T2. This could be due to the fact that AChE was the most affected enzyme. the bees of FUNG group at T2 were not exposed anymore The main role of the phase-II metabolizing GST isoen- to the fungicide, and they were recovering from the fun- zymes is to catalyze the reaction with reduced glutathione gicide pulse exposure, while bees of the INS treatment (GSH) and conjugate xenobiotic compounds, facilitating were in contact with the pesticide for a prolonged period their detoxification (Shi et al. 2012). The tendency for the of time. As with the INS treatment, the IBRv2 value of decreased of GST activity, in particular in INS treatment at the MIX treatment was higher at T2 than at T1. The MIX T1 and T2, could be indicative of an organism’s attempt to IBRv2 value confirms the results of FUNG at T2, indi- respond to an oxidative stress condition. This result could be cating recovery from the fungicide pulse. The obtained expected, since imidacloprid is known to induce metabolic MIX value is probably due to the insecticide action only. disruptions and oxidative stress in honey bees and other ani- These results confirm that biomarkers can be a useful tool mals (Nicodemo et al. 2014; Powner et al. 2016). in the framework of pesticide risk assessment as an early ALP is included in the final process of digestion and in warning signal of pesticide side effects on bees in post- the mechanism of active membrane transport (Cheung and registration monitoring programs. 13 Environmental Science and Pollution Research (2023) 30:27636–27649 27645 Conclusions Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- Our study demonstrates that exposure to the commercial bution 4.0 International License, which permits use, sharing, adapta- insecticide Confidor® and fungicide Folicur® may impact tion, distribution and reproduction in any medium or format, as long the solitary bee O. bicornis at different levels of biological as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes organization: from enzymatic responses to feeding rate and were made. The images or other third party material in this article are survival. Our results showed that (i) chronic exposure to included in the article's Creative Commons licence, unless indicated residual concentrations of imidacloprid affected feeding otherwise in a credit line to the material. If material is not included in and survival of this solitary bee; (ii) an acute exposure to the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will a fungicide, considered safe to use during bloom, had a need to obtain permission directly from the copyright holder. To view a temporary sub-lethal impact; (iii) contrary to our expecta- copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. tion, the pulse of fungicide did not exacerbate the effects of imidacloprid. As for the molecular tools, one of the four biomarkers tested, AChE, was inhibited by the fungicide References and the insecticide, showing promise as an indicator of sub-lethal effects in O. bicornis. The IBRv2 index proved Almasri H, Tavares DA, Pioz M, Sené D, Tehamitchian S, Cousin M, to be a powerful tool to describe the toxicological status Brunet JL, Belzunces LP (2020) Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic of O. bicornis, highlighting a good ability of the bees to physiological disturbances in winter Apis mellifera honey bees. recover from the fungicide pulse, while a chronic exposure Ecotoxicol Environ Saf 203:111013. https://​doi.​org/​10.​1016/j.​ to INS caused increased sub-lethal effects. No effects of ecoenv.​2020.​111013 the binary mixture were observed. Overall, this study pro- Almasri H, Tavares DA, Tchamitchian S, Pélissier M, Sené D, Cousin M, Brunet J-L, Belzunces LP (2021) Toxicological status changes vides evidence for improving the current risk assessment the susceptibility of the honey bee Apis mellifera to a single fun- procedures by including sub-lethal endpoints and other bee gicidal spray application. Environ Sci Pollut Res 28(31):42807– species in addition to A. mellifera. 42820. https://​doi.​org/​10.​1007/​s11356-​021-​13747-3 Altenhofen S, Nabinger DD, Wiprich MT, Pereira TCB, Bogo MR, Supplementary Information The online version contains supplemen- Bonan CD (2017) Tebuconazole alters morphological, behavio- tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11356-0​ 22-2​ 4061-x. ral and neurochemical parameters in larvae and adult zebrafish (Danio rerio). Chemosphere 180:483–490. https://​doi.​org/​10.​ Acknowledgements We appreciate the assistance of R. Spinelli and 1016/j.​chemo​sphere.​2017.​04.​029 K. Palpurtha during data collection. Arena M, Sgolastra F (2014) A meta-analysis comparing the sensitivity of bees to pesticides. Ecotoxicology 23(3):324–334. https://​doi.​ Author contribution Cátia Ariana Henriques Martins: writing—origi- org/​10.​1007/​s10646-​014-​1190-1 nal draft, writing—review and editing, methodology, data curation, Arrighetti F, Landro SM, Lambre ME, Penchaszadeh PE, Teso V visualization, and investigation. Ilaria Caliani: writing—original (2019) Multiple-biomarker approach in the assessment of the draft, writing—review and editing, conceptualization, methodology, health status of a novel sentinel mussel Brachidontes rodriguezii and investigation. Antonella D’Agostino: writing—review and editing, in a Harbor Area. Mar Pollut Bull 140:451–461. https://​doi.​org/​ visualization, data curation, and formal analysis. Agata Di Noi: writ- 10.​1016/j.​marpo​lbul.​2019.​02.​002 ing—original draft, writing—review and editing, investigation, and Artz DR, Pitts-Singer TL (2015) Effects of fungicide and adjuvant data curation. Silvia Casini: supervision, writing—review and edit- sprays on nesting behavior in two managed solitary bees, Osmia ing, conceptualization, and methodology. Martina Parrilli: data cura- lignaria and Megachile rotundata. PLoS ONE 10(8):e0135688. tion and investigation. Celeste Azpiazu: writing—review and editing, https://​doi.​org/​10.​1371/​journ​al.​pone.​01356​88 conceptualization, and methodology. Jordi Bosch: supervision, writ- Azpiazu C, Bosch J, Viñuela E, Medrzycki P, Teper D, Sgolastra F ing—review and editing, conceptualization, and methodology. Fabio (2019) Chronic oral exposure to field-realistic pesticide com- Sgolastra: supervision, writing—original draft, writing—review and binations via pollen and nectar: effects on feeding and thermal editing, conceptualization, and methodology. performance in a solitary bee. Sci Rep 9(1):1–11. https://d​ oi.o​ rg/​ 10.​1038/​s41598-​019-​50255-4 Funding Open access funding provided by Università degli Studi di Azpiazu C, Bosch J, Bortolotti L, Medrzycki P, Teper D, Molowny- Siena within the CRUI-CARE Agreement. This study was supported Horas R, Sgolastra F (2021) Toxicity of the insecticide sulfoxa- by a grant from the University of Bologna to Fabio Sgolastra (Grant flor alone and in combination with the fungicide fluxapyroxad in RFO2020_SGOLASTRA) and the doctoral scholarship to Cátia A.H. three bee species. Sci Rep 11(1):6821. https://​doi.​org/​10.​1038/​ Martins. s41598-​021-​86036-1 Badawy MEI, Nasr HM, Rabea EI (2015) Toxicity and biochemical Data availability The datasets generated by the current study will be changes in the honey bee Apis mellifera exposed to four insec- available upon request to the corresponding authors. ticides under laboratory conditions. 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