Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.

RATIONALE: Chlorothalonil (CHT), a broad-spectrum fungicide, has been employed widely to control foliar diseases, whereas with a major metabolite of polar 4-hydroxychlorothalonil (CHT-4-OH), only an acceptable nonpolar CHT residue is allowed by most countries. This study involves the method development for CHT residue in vegetables/fruits using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a novel modified discharge-adaptor (DA) interface. METHODS: CHT residue was analyzed using LC-MS/MS with DA interface (LC-DA-MS/MS), developed in our previous works. A DA was placed on the electrospray tip to switch the ionization modes. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to extract CHT residue of vegetables/fruits efficiently with less sample preparation time and analysis cost. RESULTS: CHT and CHT-4-OH spiked in four different vegetables/fruits were extracted using the modified QuEChERS method. After LC with isocratic elution, CHT and CHT-4-OH were separated within 3 min. Using LC-DA-MS/MS, the ion signals of CHT were improved two to three times, and the limit of quantification of 5 ng/g and linearity (r2 > 0.99) in the range of 5-200 ng/g were achieved using 10 g of vegetables/fruits. The precision and accuracy were within 15% each. The modified QuEChERS and LC-DA-MS/MS were applied to examine eight field-grown vegetables/fruits; 9.5 and 2588.9 ng/g of CHT were detected in two vegetables/fruits. CONCLUSION: LC-DA-MS/MS combined with modified QuEChERS was successfully applied to determine CHT residue <10 ng/g in vegetables/fruits and with satisfied validation results. The developed method could reduce both analysis cost and time, attributing to simplifications in modified QuEChERS, isocratic elution, and DA interface in LC-DA-MS/MS.

Fluorescent and Colorimetric Dual-Readout Immunochromatographic Assay for the Detection of Phenamacril Residues in Agricultural Products.

The fungicide phenamacril has been employed to manage Fusarium and mycotoxins in crops, leading to persistent residues in the environment and plants. Detecting phenamacril is pivotal for ensuring environmental and food safety. In this study, haptens and artificial antigens were synthesized to produce antiphenamacril monoclonal antibodies (mAbs). Additionally, gold nanoparticles coated with a polydopamine shell were synthesized and conjugated with mAbs, inducing fluorescence quenching in quantum dots. Moreover, a dual-readout immunochromatographic assay that combines the positive signal from fluorescence with the negative signal from colorimetry was developed to enable sensitive and precise detection of phenamacril within 10 min, achieving detection limits of 5 ng/mL. The method's reliability was affirmed by using spiked wheat flour samples, achieving a limit of quantitation of 0.05 mg/kg. This analytical platform demonstrates high sensitivity, outstanding accuracy, and robust tolerance to matrix effects, making it suitable for the rapid, onsite, quantitative screening of phenamacril residues.

Rapid and specific detection of thiabendazole: enzymatic digestion-enabled fluorescent aptasensor.

Thiabendazole, a widely used broad-spectrum fungicide in agriculture, poses risks to human health. To monitor its presence in water, we propose a fluorescent aptasensor utilizing Escherichia coli exonuclease I (Exo I). The findings demonstrate a linear correlation between thiabendazole concentrations and digestion percentage, with a detection limit (LOD) exceeding 1 µM and a determination coefficient (R2) of 0.959. This aptamer-based fluorescence spectroscopy detection system holds promise for a rapid, specific, and sensitive analysis of thiabendazole in environmental waters and food matrices.

Compound-Specific Isotope Analysis Provides Direct Evidence for Identifying the Source of Residual Pesticides Diazinon and Procymidone in the Soil-Plant System.

Compound-specific isotope analysis stands as a promising tool for unveiling the behavior of pesticides in agricultural environments. Using the commercial formulations of persistent fungicide procymidone (PRO) and less persistent insecticide diazinon (DIA), respectively, we analyzed the concentration and carbon isotope composition (δ13C) of the residual pesticides through soil incubation experiments in a greenhouse (for 150 days) and lab conditions (for 50-70 days). Our results showed that the magnitude of δ13C variation depends on pesticide specificity, in which PRO in the soil exhibited little variation in δ13C values over the entire incubation times, while DIA demonstrated an increased δ13C value, with the extent of δ13C variability affected by different spiking concentrations, plant presence, and light conditions. Moreover, the pesticides extracted from soils were isotopically overlapped with those from crop lettuce. Ultimately, the isotope composition of pesticides could infer the degradation and translocation processes and might contribute to identifying the source(s) of pesticide formulation in agricultural fields.

High-throughput semi-automated emulsive liquid-liquid microextraction for detecting SDHI fungicides in water, juice, and alcoholic beverage samples via UHPLC-MS/MS.

Herein, a High-Throughput Semi-automated Emulsive Liquid-Liquid Microextraction (HTSA-ELLME) method was developed to detect Succinate Dehydrogenase Inhibitor (SDHI) fungicides in food samples via UHPLC-MS/MS. The Oil-in-Water (O/W) emulsion comprising a hydrophobic extractant and water was dilutable with the aqueous sample solution. Upon injecting the primary emulsion into the sample solution, a secondary O/W emulsion was formed, allowing SDHI fungicides to be extracted. Subsequently, a NaCl-saturated solution was injected in the secondary O/W emulsion as a demulsifier to rapidly separate the extractant, eliminating the need for centrifugation. A 12-channel electronic micropipette was used to achieve a high-throughput semi-automation of the novel sample pretreatment. The linear range was 0.003-0.3 µg L-1 with R2 > 0.998. The limit of detection was 0.001 µg L-1. The HTSA-ELLME method successfully detected SDHI fungicides in water, juice, and alcoholic beverage samples, with recoveries and relative standard deviations of 82.6-106.9% and 0.8-5.8%, respectively. Unlike previously reported liquid-liquid microextraction approaches, the HTSA-ELLME method is the first to be both high-throughput and semi-automated and may aid in designing pesticide pretreatment processes in food samples.

Tracing the dissipation of difenoconazole, its metabolites and co-formulants in tomato: A comprehensive analysis by chromatography coupled to high resolution mass spectrometry in laboratory and greenhouse trials.

The study evaluated Ceremonia 25 EC®, a plant protection product (PPP) containing difenoconazole, in tomato crops, to identify potential risks associated with PPPs, and in addition to this compound, known metabolites from difenoconazole degradation and co-formulants present in the PPP were monitored. An ultra high performance liquid chromatography coupled to quadrupole-Orbitrap mass analyser (UHPLC-Q-Orbitrap-MS) method was validated with a working range of 2 µg/kg (limit of quantification, LOQ) to 200 µg/kg. Difenoconazole degradation followed a biphasic double first-order in parallel (DFOP) kinetic model in laboratory and greenhouse trials, with high accuracy (R2 > 0.9965). CGA-205374, difenoconazole-alcohol, and hydroxy-difenoconazole metabolites were tentatively identified and semi-quantified in laboratory trials by UHPLC-Q-Orbitrap-MS from day 2 to day 30. No metabolites were found in greenhouse trials. Additionally, 13 volatile co-formulants were tentatively identified by gas chromatography (GC) coupled to Q-Orbitrap-MS, detectable up to the 7th day after PPP application. This study provides a comprehensive understanding of difenoconazole dissipation in tomatoes, identification of metabolites, and detection of co-formulants associated with the applied PPP.

Novel litchi-like Au-Ag nanospheres driven dual-readout lateral flow immunoassay for sensitive detection of pyrimethanil.

Pyrimethanil (PYR) is a fungicide that is harmful to consumers when present in foods at concentrations greater than maximum permitted residue levels. High-performance immunoprobes and dual-readout strategy may be useful for constructing sensitive lateral flow immunoassay (LFIA). Herein, the prepared litchi-like Au-Ag bimetallic nanospheres (LBNPs) exhibited high mass extinction coefficients and fluorescence quenching constants. Benefiting from LBNPs and dual-readout mode, the limits of detection of LBNPs-CM-LFIA and LBNPs-FQ-LFIA for PYR were 0.957 and 0.713 ng mL-1, which were 2.54- and 3.41-fold lower than that of gold nanoparticles-based LFIA, respectively. The limits of quantitation of LBNPs-CM-LFIA and LBNPs-FQ-LFIA were 3.740 and 1.672 ng mL-1, respectively. LBNPs-LFIA was applied to detect PYR in cucumber and grape samples with satisfactory recovery (90%-111%). LBNPs-LFIA showed good agreement with LC-MS/MS for the detection of PYR in the samples. Accordingly, this sensitive and accurate dual-readout LFIA based on LBNPs can be effectively applied for food safety.

Designing a size exclusion-based hapten and the development of a quantitative and visual time-resolved fluorescence immunochromatography assay strip for detecting dimethomorph and flumorph in a group-specific manner.

Based on the size and surface properties of dimethomorph and flumorph, we used a computer simulation-assisted size exclusion hapten design strategy to develop group-specific monoclonal antibodies that can simultaneously recognize dimethomorph and flumorph. For this, we performed quantitative and visual semi-quantitative time-resolved fluorescence immunochromatography (TRFICA) to simultaneously detect dimethomorph and flumorph in potatoes and apples. In potato samples, the visual limit of detection (vLOD) for dimethomorph and flumorph was 4 ng/mL and 8 ng/mL, respectively, whereas the quantitative limit of detection (qLOD) for dimethomorph and flumorph was 0.26 and 0.33 ng/mL, respectively. The vLOD of dimethomorph and flumorph in apple samples was 8 ng/mL, whereas the qLOD of dimethomorph and flumorph was 0.17 and 0.38 ng/mL, respectively. The average recovery of potato and apple samples ranged from 77.5% to 121.7%, which indicated that the method can be used to rapidly detect dimethomorph and flumorph in food samples.

Current trends and mismatches on fungicide use and assessment of the ecological effects in freshwater ecosystems.

Despite increasing evidence of off-site ecological impacts of pesticides and policy efforts worldwide, pesticide use is still far from being ecologically sustainable. Fungicides are among the most sold classes of pesticides and are crucial to ensure global food supply and security. This study aimed to identify potential gaps of knowledge and mismatches between research and usage data of fungicides by: (i) systematizing the current trends in global sales of fungicides, focusing on the European context in particular (where they are proportionally important); (ii) reviewing the scientific literature on the impacts of synthetic fungicides on non-target freshwater organisms. Sales data revealed important global and regional asymmetries in the relative importance of fungicides and the preferred active ingredients. The literature review on the ecological effects of fungicides disclosed a mismatch between the most studied and the most sold substances, as well as a bias towards the use of single species assays with standard test organisms. To ensure a proper evaluation, risk scenarios should focus on a regional scale, and research agendas must highlight sensitive aquatic ecorreceptors and improve the crosstalk between analytical and sales data.

Occurrence and toxicological relevance of pesticides and trace metals in agricultural soils, sediments, and water of the Sogamoso River basin, Colombia.

Historical pesticide use in agriculture and trace metal accumulation have long term impact on soil, sediment, and water quality. This research quantifies legacy and current-use pesticides and trace metals, assessing their occurrence and toxicological implications on a watershed scale in the Sogamoso River basin, tributary of the Magdalena River in Colombia. Organochlorine pesticides (22), organophosphates (7), and azole fungicides (5), as well as trace metals cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), and zinc (Zn) were analyzed in croplands and along the river. Toxic units (TU) and hazard quotients (HQ) were calculated to assess the mixture toxicity. Organochlorines were detected in 84% of soils, 100% of sediments, and 80% of water samples. Organophosphates were found in 100% of soil and sediment samples, as well as in 70% of water samples. Azole fungicides were present in 79% of soils, 60% of sediments, and in 10% of water samples. Total pesticide concentrations ranged from 214.2 to 8497.7 µg/kg in soils, 569.6-12768.2 µg/kg in sediments, and 0.2-4.1 µg/L in water. In addition, the use of partition coefficient (Kd) and organic carbon fraction (foc) allowed the distribution analysis for most of the pesticides in sediments, suspended particulate matter (SPM), and water systems, but not for soils. Concentrations of trace metals Cu, Zn, Pb, and Zn exceeded international quality guidelines for agricultural soils in 16% of the samples. Furthermore, Cu and Zn concentrations exceeded sediment quality guidelines in 50 and 90% of the samples, respectively. These findings demonstrate the broad distribution of complex mixtures of trace metals, legacy organochlorines, and current-use pesticides across the basin, indicating that conventional agriculture is a significant source of diffuse pollution. Sustainable agricultural practices are needed to mitigate adverse impacts on ecosystems and human health.

Quick synthesis of CoFe-PBA@GO with electrochemical method as a novel, sensitive, and degradable nanocomposite applied in nanofibers for triazole extraction before HPLC-UV analysis.

Today, the wide use of triazole fungicides due to environmental damage and its side effects has raised global concern. Hence, in this research, poly-vinyl alcohol/polyacrylic-acid/CoFe-PBA@GO electrospun nanofiber was synthesized and applied as effective, degradable, and novel adsorbent at pipette-tip microextraction (PT-µSPE) method for the rapid and concurrent extraction of five of triazole fungicides in fruit and vegetable samples prior to quantitative analysis by high-performance liquid chromatography-ultraviolet. The incorporation of CoFe-PBA@GO with superporous structure and abundant functional groups in a polymer medium improves the extraction efficiency of nanofibers due to hydrogen bonding and π-π interactions formed between analytes and synthesized nano-adsorbent. Various important elements that affect the extraction yield of the target analytes were optimized utilizing a time-variable approach. Under the optimum conditions, dynamic range was attained in the range of 0.3-900.0 ng/mL with correlation coefficients ≥ 0.999. The identification limit of the PT-µSPE-HPLC-UV method ranged from 0.1 to 0.3 ng/mL.

Fungicide ingestion reduces net energy gain and microbiome diversity of the solitary mason bee.

Fungicides are frequently used during tree fruit bloom and can threaten insect pollinators. However, little is known about how non-honey bee pollinators such as the solitary bee, Osmia cornifrons, respond to contact and systemic fungicides commonly used in apple production during bloom. This knowledge gap limits regulatory decisions that determine safe concentrations and timing for fungicide spraying. We evaluated the effects of two contact fungicides (captan and mancozeb) and four translaminar/plant systemic fungicides (cyprodinil, myclobutanil, penthiopyrad, and trifloxystrobin) on larval weight gain, survival, sex ratio, and bacterial diversity. This assessment was carried out using chronic oral ingestion bioassays where pollen provisions were treated with three doses based on the currently recommended field use dose (1X), half dose (0.5X), and low dose (0.1X). Mancozeb and penthiopyrad significantly reduced larval weight and survival at all doses. We then sequenced the 16S gene to characterize the larvae bacteriome of mancozeb, the fungicide that caused the highest mortality. We found that larvae fed on mancozeb-treated pollen carried significantly lower bacterial diversity and abundance. Our laboratory results suggest that some of these fungicides can be particularly harmful to the health of O. cornifrons when sprayed during bloom. This information is relevant for future management decisions about the sustainable use of fruit tree crop protection products and informing regulatory processes that aim to protect pollinators.

Toxicological impact of strobilurin fungicides on human and environmental health: a literature review.

Fungicides are specifically used for controlling fungal infections. Strobilurins, a class of fungicides originating from the mushroom Strobilurus tenacellus, act on the fungal mitochondrial respiratory chain, interrupting the ATP cycle and causing oxidative stress. Although strobilurins are little soluble in water, they have been detected in water samples (such as rainwater and drinking water), indoor dust, and sediments, and they can bioaccumulate in aquatic organisms. Strobilurins are usually absorbed orally and are mainly eliminated via the bile/fecal route and urine, but information about their metabolites is lacking. Strobilurins have low mammalian toxicity; however, they exert severe toxic effects on aquatic organisms. Mitochondrial dysfunction and oxidative stress are the main mechanisms related to the genotoxic damage elicited by toxic compounds, such as strobilurins. These mechanisms alter genes and cause other dysfunctions, including hormonal, cardiac, neurological, and immunological impairment. Despite limitations, we have been able to compile literature information about strobilurins. Many studies have dealt with their toxic effects, but further investigations are needed to clarify their cellular and underlying mechanisms, which will help to find ways to minimize the harmful effects of these compounds.

Assessment of flusilazole residues on apples: Detection, quantification and health risk implications.

Apple, a major fruit of temperate Himalayas, is sprayed with chemical pesticides around 12 times during the cropping season. Various systemic and contact fungicides are applied for the management of major diseases. In order to manage disease, flusilazole 40 EC is frequently used. However, excessive chemical application has been found to be detrimental for consumer safety. Keeping in view consumer safety, risk assessment, the half-life and waiting period for flusilazole 40 EC were evaluated on the Red Velox variety of apple. The QuEChERS (quick, easy, cheap, effective, rugged and safe) method and high-performance liquid chromatography were adapted for sample processing and analysis, respectively. The recovery percentages of flusilazole at three fortification levels (0.04, 0.09 and 0.50 mg kg-1) were 98.85, 99.83 and 98.98%, respectively. Flusilazole at the recommended dose (80 g a.i. ha-1) left an initial deposit of 0.733 mg kg-1, which dissipated by 93.45% in 60 days and was non-detectable beyond this period. Meanwhile flusilazole at double the recommended dose (160 g a.i. ha-1) left an initial deposit of 0.913 mg kg-1, which dissipated by 93.43% in 70 days and was non-detectable beyond this period. Based on the maximum residue limit of 0.3 mg kg-1 as prescribed by the Codex Alimentarius Commission, a waiting period of 28.74 and 46.03 days was recorded for single and double doses, respectively. Moreover, in order to assess the consumer risk, theoretical maximum residue contributions (TMRCs) were derived using flusilazole residues (average and maximum) recorded at various time intervals and compared with the maximum permissible intake, which was found to be 0.42 mg per person per day. Based on the average per capita daily consumption of 6.76 g apple in India, the TMRC values were computed. Although the values of TMRC decreased below maximum permissible intake at the first day after application, indicating minimal consumer health risks, fruits sprayed with a double dose of flusilazole carried the risk even up to the tenth day after flusilazole application. The results of the present study will be valuable for safe and timely use of flusilazole on apple.

Mechanisms Underlying the Overlooked Chiral Fungicide-Driven Enantioselective Proliferation of Antibiotic Resistance in Earthworm Intestinal Microbiome.

From a "One Health" perspective, the global threat of antibiotic resistance genes (ARGs) is associated with modern agriculture practices including agrochemicals application. Chiral fungicides account for a considerable proportion of wildly used agrochemicals; however, whether and how their enantiomers lead to differential proliferation of antibiotic resistance in agricultural environments remain overlooked. Focused on the soil-earthworm ecosystem, we for the first time deciphered the mechanisms underlying the enantioselective proliferation of antibiotic resistance driven by the enantiomers of a typical chiral fungicide mandipropamid (i.e., R-MDP and S-MDP) utilizing a multiomic approach. Time-series metagenomic analysis revealed that R-MDP led to a significant enhancement of ARGs with potential mobility (particularly the plasmid-borne ARGs) in the earthworm intestinal microbiome. We further demonstrated that R-MDP induced a concentration-dependent facilitation of plasmid-mediated ARG transfer among microbes. In addition, transcriptomic analysis with verification identified the key aspects involved, where R-MDP enhanced cell membrane permeability, transfer ability, biofilm formation and quorum sensing, rebalanced energy production, and decreased cell mobility versus S-MDP. Overall, the findings provide novel insights into the enantioselective disruption of microbiome and resistome in earthworm gut by chiral fungicides and offer significant contributions to the comprehensive risk assessment of chiral agrochemicals in agroecosystems.

Synthesis and evaluation of a chitosan nanomaterial as efficient sorbent for determination of fungicide residues in waters and wine by liquid chromatography high resolution mass spectrometry.

In the present study a novel, cost-effective, environmentally friendly, and efficient analytical method was developed to analyze fungicide residues in water and wine. The method relies on the application of a newly developed sorbent nanomaterial named Nano-Cs-NAT, synthesized by modifying chitosan, a naturally occurring, low-cost polysaccharide, through grafting with two acrylic monomers and a cross-linker. Nano-Cs-NAT was introduced as analytical sorbent for Dispersive Micro Solid Phase Extraction (D-µ-SPE) before Liquid Chromatography-Orbitrap High-Resolution Mass Spectrometry (LC-Orbitrap HRMS) analysis of twelve fungicides commonly used in viticulture (among the others, triazoles, strobilurines and N-substituted imidazoles). Characterization of the sorbent was conducted, confirming the successful acrylation of chitosan. A multivariate approach was employed to optimize D-µ-SPE extraction parameters. The material was found to be highly effective in simultaneously purifying and concentrating the target analytes, enhancing overall analytical efficiency and sensitivity. The Nano-Cs-NAT-D-µ-SPE-LC-Orbitrap-HRMS method was thoroughly validated, exhibiting good recoveries (72-104%), reproducibility (average RSD ≤ 6%) and repeatability (average RSD ≤ 7%). It also achieved low limits of detection (LOD) in river water (average LOD of 0.04 µg L-1) and wine (average LOD of 0.72 µg kg-1), highlighting its potential for routine fungicide residue analysis. This developed method addresses environmental and food safety concerns by providing an efficient solution for detecting fungicide residues in waters and wine.

The molecular determinants of pesticide sensitivity in bee pollinators.

Bees carry out vital ecosystem services by pollinating both wild and economically important crop plants. However, while performing this function, bee pollinators may encounter potentially harmful xenobiotics in the environment such as pesticides (fungicides, herbicides and insecticides). Understanding the key factors that influence the toxicological outcomes of bee exposure to these chemicals, in isolation or combination, is essential to safeguard their health and the ecosystem services they provide. In this regard, recent work using toxicogenomic and phylogenetic approaches has begun to identify, at the molecular level, key determinants of pesticide sensitivity in bee pollinators. These include detoxification systems that convert pesticides to less toxic forms and key residues in insecticide target-sites that underlie species-specific insecticide selectivity. Here we review this emerging body of research and summarise the state of knowledge of the molecular determinants of pesticide sensitivity in bee pollinators. We identify gaps in our knowledge for future research and examine how an understanding of the genetic basis of bee sensitivity to pesticides can be leveraged to, a) predict and avoid negative bee-pesticide interactions and facilitate the future development of pest-selective bee-safe insecticides, and b) inform traditional effect assessment approaches in bee pesticide risk assessment and address issues of ecotoxicological concern.

Enantioselective fate and risk assessment of chiral fungicide pydiflumetofen in rice-fish and wheat farming systems.

Fungicides have been widely used for reducing the losses caused by plant diseases. Rice and wheat are the most basic food crops, and the potential risks after applying fungicides are worthy of attention. Especially rice-fish farming system is an ecological symbiosis system that is beneficial to both environmental and ecological protection. However, the application of pesticides will stress the ecosystem, and the pesticide residues in rice and fish would be transmitted along the food chain, which is harmful to human health. Here, the enantioselective behaviors of chiral pydiflumetofen in rice-fish and wheat farming systems were clarified. In the rice-fish farming system, pydiflumetofen enantiomers were preferentially attached to the plants, entering the paddy water and settling into the paddy soil, and then accumulating and dissipating in the fish. With the growth of rice, it was transported to rice fruits. The wheat farming system was similar. Enantioselective dissipation occurred in carp (Cyprinus carpio), brown rice and wheat soil, and S-(+)-pydiflumetofen was preferentially dissipated. In other words, R-(-)-pydiflumetofen showed higher concentrations, especially in carp, which meant R-(-)-pydiflumetofen was more easily accumulated in the environment, and posed a greater potential risk to the farming system. The pydiflumetofen residues in brown rice and wheat were lower than MRLs from the EFSA (0.02 mg/kg) and eCFR (0.3 mg/kg), respectively. What deserves attention is that the MRL of pydiflumetofen in fish is not clear. Meanwhile, pydiflumetofen in paddy soil and wheat soil had a persistent residual effect, and the risks could not be ignored. Combined with the previous research, developing S-(+)-pydiflumetofen products will help to reduce the dosage and reduce the risks to environment and people. This study evaluated the environmental fate and risk of chiral pydiflumetofen from the perspective of farming system, and would provide data support for its rational use and risk assessment.

Impact of landscape composition on honey bee pollen contamination by pesticides: A multi-residue analysis.

The honey bee is the most common and important managed pollinator of crops. In recent years, honey bee colonies faced high mortality for multiple causes, including land-use change and the use of plant protection products (hereafter pesticides). This work aimed to explore how contamination by pesticides of pollen collected by honey bees was modulated by landscape composition and seasonality. We placed two honey bee colonies in 13 locations in Northern Italy in contrasting landscapes, from which we collected pollen samples monthly during the whole flowering season in 2019 and 2020. We searched for almost 400 compounds, including fungicides, herbicides, insecticides, and acaricides. We then calculated for each pollen sample the Pollen Hazard Quotient (PHQ), an index that provides a measure of multi-residue toxicity of contaminated pollen. Almost all pollen samples were contaminated by at least one compound. We detected 97 compounds, mainly fungicides, but insecticides and acaricides showed the highest toxicity. Fifteen % of the pollen samples had medium-high or high levels of PHQ, which could pose serious threats to honey bees. Fungicides showed a nearly constant PHQ throughout the season, while herbicides and insecticides and acaricides showed higher PHQ values in spring and early summer. Also, PHQ increased with increasing cover of agricultural and urban areas from April to July, while it was low and independent of landscape composition at the end of the season. The cover of perennial crops, i.e., fruit trees and vineyards, but not of annual crops, increased PHQ of pollen samples. Our work highlighted that the potential toxicity of pollen collected by honey bees was modulated by complex interactions among pesticide category, seasonality, and landscape composition. Due to the large number of compounds detected, our study should be complemented with additional experimental research on the potential interactive effects of multiple compounds on honey bee health.

Residue dynamics of a contact and a systemic fungicide in pollen, nectar, and other plant matrices of courgette (Cucurbita pepo L.).

Pollen and nectar can be contaminated with a range of pesticides, including insecticides, fungicides, and herbicides. Since these matrices are important food sources for pollinators and other beneficial insects, their contamination can represent a key route of exposure. However, limited knowledge exists with respect to pesticide residue levels and their dynamics in these matrices for many crops and active ingredients (AIs). We used controlled glasshouse studies to investigate the residue dynamics of a systemic (cyprodinil) and a contact (fludioxonil) fungicide in the floral matrices and other plant parts of courgette/zucchini (Cucurbita pepo L.). We aimed to better understand the processes behind residue accumulation and decline in pollen and nectar. Each AI was applied to plants, either by spraying whole plants or by targeted spraying onto leaves only. Samples of pollen, nectar, anthers, flowers, and leaves were taken on the day of application and each subsequent morning for up to 13 days and analysed for residues using LC-MS/MS. Significant differences in residue levels and dynamics were found between AIs and floral matrices. The present study allowed for the identification of potential routes by which residues translocate between tissues and to link those to the physicochemical properties of each AI, which may facilitate the prediction of residue levels in pollen and nectar. Residues of the contact AI declined more quickly than those of the systemic AI in pollen and nectar. Our results further suggest that the risk of oral exposure for pollinators may be considerably reduced by using contact AIs during the green bud stage of plants, but application of systemic compounds could still result in a low, but continuous long-term exposure for pollinators with limited decline.