Non-uniform sampling to enhance the performance of compact NMR for characterizing new psychoactive substances.

Efficient and robust analytical methods are needed to improve the identification and subsequent regulation of new psychoactive substances (NPS). NMR spectroscopy is a unique method able to determine the structure of small molecules such as NPS even in mixtures. However, high-field NMR analysis is associated with expensive purchase and maintenance costs. For more than a decade, compact NMR spectrometers have changed this paradigm. It was recently shown that a dedicated analytical workflow combining compact NMR and databases could identify the molecular structure of NPS, in spite of the lower spectral dispersion and sensitivity of compact spectrometers. This approach relies on 1H-13C HSQC to both recognize NPS and elucidate the structure of unknown substances. Still, its performance is limited by the need to compromise between resolution and experiment time. Here, we show that this strategy can be significantly improved by implementing non-uniform sampling (NUS) to improve spectral resolution in the 13C dimension of HSQC at no cost in terms of experiment time. Gains in the range of 3 to 4 in resolution are achieved for pure NPS and for a mixture. Finally, 2D HSQC with NUS was applied to improve the identification of NPS with the assistance of databases. The resulting method appears as a useful tool for the characterization of NPS in mixtures, which is essential for forensic laboratories.

Characterization of new psychoactive substances by integrating benchtop NMR to multi-technique databases.

New psychoactive substances (NPS) have become a serious threat for public health due to their ability to be sold in the street or on internet. NPS are either derived from commercial drugs which are misused (recreational rather than medical use) or whose structure is slightly modified. To regulate NPS, it is essential to accurately characterize them, either to recognize molecules that were previously identified or to quickly elucidate the structure of unknown ones. Most approaches rely on the determination of the exact mass obtained by high-resolution mass spectrometry requiring expensive equipment. This motivated us to develop a workflow in which the elucidation is assisted with databases and does not need the exact mass. This workflow combines 1D and 2D NMR measurements performed on a benchtop spectrometer with IR spectroscopy, for creating a multi-technique database to characterize pure and mixed NPS. The experimental database was created with 57 entries mostly coming from seizures, mainly cathinones, cannabinoids, amphetamines, arylcyclohexylamines, and fentanyl. A blind validation of the workflow was carried out on a set of six unknown seizures. In the first three cases, AF, AB-FUBINACA, and a mixture of 2C-I and 2C-E could be straightforwardly identified with the help of their reference spectra in the database. The two next samples were elucidated for the first time with the help of the database to reveal NEK and MPHP substances. Finally, a precise quantification of each characterized NPS was obtained in order to track NPS trafficking networks.

High-field and benchtop NMR spectroscopy for the characterization of new psychoactive substances.

New psychoactive substances (NPS) have become a serious threat to public health in Europe due to their ability to be sold in the street or on the darknet. Regulating NPS is an urgent priority but comes with a number of analytical challenges since they are structurally similar to legal products. A number of analytical techniques can be used for identifying NPS, among which NMR spectroscopy is a gold standard. High field NMR is typically used for structural elucidation in combination with others techniques like GC-MS, Infrared spectroscopy, together with databases. In addition to their strong ability to elucidate molecular structures, high field NMR techniques are the gold standard for quantification without any physical isolation procedure and with a single internal standard. However, high field NMR remains expensive and emerging "benchtop" NMR apparatus which are cheaper and transportable can be considered as valuable alternatives to high field NMR. Indeed, benchtop NMR, which emerged about ten years ago, makes it possible to carry out structural elucidation and quantification of NPS despite the gap in resolution and sensitivity as compared to high field NMR. This review describes recent advances in the field of NMR applied to the characterization of NPS. High-field NMR methods are first described in view of their complementarity with other analytical methods, focusing on both structural and quantitative aspects. The second part of the review highlights how emerging benchtop NMR approaches could act as a game changer in the field of forensics.

In Vivo Characterization of the Toxicological Properties of DPhP, One of the Main Degradation Products of Aryl Phosphate Esters.

BACKGROUND: Aryl phosphate esters (APEs) are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives, are poorly characterized. OBJECTIVE: Our aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs than direct assessment of their concentration and determine its potential deleterious biological effects in chronically exposed mice. METHODS: Conventional animals (FVB mice) were acutely or chronically exposed to relevant doses of DPhP or to triphenyl phosphate (TPhP), one of its main precursors. Both molecules were measured in blood and other tissues by liquid chromatography-mass spectrometry (LC-MS). Effects of chronic DPhP exposure were addressed through liver multi-omics analysis to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses. RESULTS: Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1mg/mL in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty acid catabolic processes centered on acylcarnitine and mitochondrial ß-oxidation in mice exposed to DPhP in comparison with those treated with vehicle. An interesting finding was that in mice exposed to DPhP, mRNA, expression of genes involved in lipid catabolic processes and regulated by peroxisome proliferator-activated receptor alpha (PPARα) was lower than that in vehicle-treated mice. Immunohistochemistry analysis showed a specific down-regulation of HMGCS2, a kernel target gene of PPARα. Overall, DPhP absorption disrupted body weight-gain processes. CONCLUSIONS: Our results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver is essential for controlling fast and feast periods, with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular pathologies and metabolic disease incidence deserves, in light of our results, further investigations. https://doi.org/10.1289/EHP6826.

A rapid and easy method based on hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS/MS) to quantify iodinated X-ray contrast in wastewaters.

This work proposes the first method based on hydrophilic interaction liquid chromatography coupled to multiple reaction monitoring with triple stage fragmentation (HILIC-MRM3) to quantify polar organic micropollutants in complex sewage waters. A fast HILIC-MRM3 analytical method, without sample preparation except a dilution step, was developed and validated to quantify seven iodinated contrast media (ICMs) in sewage waters, namely iohexol, iomeprol, ioversol, iopamidol, diatrizoic acid, iopromide and iopentol. Several chromatographic columns and mobile phase conditions were investigated and a good separation of the ICMs was obtained with a mixed-mode column (Acclaim Mixed-mode WAX) used in HILIC conditions. The validation was performed using a synthetic matrix: the limits of quantification (LOQ) were inferior to 1 µg/L and the linearity of each compound was comprised within the [0.5-50] µg/L range. The applicability of the HILIC-MRM3 method was assessed by the analysis of several raw waters. The results highlighted the presence of ICMs in most samples, at concentrations up to several mg/L in hospital sewage waters.

Non-targeted investigation of benthic invertebrates (Chironomus riparius) exposed to wastewater treatment plant effluents using nanoliquid chromatography coupled to high-resolution mass spectrometry.

Nanoliquid chromatography (nanoLC) was coupled to high-resolution mass spectrometry (HRMS) to perform a non-targeted investigation on benthic invertebrates, Chironomus riparius exposed to wastewater treatment plant (WWTP) effluents. Insect larvae represent a complex and low-weight matrix that required the use of a miniaturized Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method of extraction followed by nanoLC-HRMS to perform the analysis. The optimization of this coupling in terms of separation conditions including trapping step, detection conditions and data treatment provided reproducible fingerprints on insect larvae exposed to WWTP effluents with both in situ and ex-situ approaches. Statistical treatments such as principal component analysis highlighted the impact of WWTP effluents on the metabolome of insect larvae and showed the influence of exposure conditions. The identification of discriminating signals (m/z, tR) matched with several potential endogenous biomarkers. These are mainly fatty acids, indicating a change in lipid metabolism that can be correlated with exposure to WWTP effluents. Several xenobiotics have also been detected, including ibuprofen and propranolol, whose identities have been confirmed by analytical standards. This work demonstrates the effectiveness and sensitivity of nanoLC-HRMS based environmental non-targeted approaches in ecotoxicological studies and provides the first profiling data for a very small aquatic invertebrate.

High-Resolution Mass Spectrometry as a Tool To Evaluate the Sample Preparation of Sludge.

This study explored the sample preparation steps based on QuEChERS (quick, easy, cheap, effective, rugged, and safe) of a complex matrix, such as sewage sludge, regarding their impacts on the chemical signatures of the samples obtained via high-resolution mass spectrometry (HRMS). The effects on the HRMS signal of the intrinsic parameters of the QuEChERS method, such as the extraction solvent, the buffer salt, or the purification procedure, have been evaluated. Different strategies have been used to perform these evaluations, such as a comparison of the total number of HRMS signals, a comparison of the targeted screening based on more than 100 compounds, and a comparison of a nontargeted screening based both on the study of the most discriminating couples [mass-to-charge ratio (m/z); retention time (tR)] and on the most intense peaks. Moreover, two specific methods dedicated to the extraction of surfactants and drugs have been added to this comparison to visualize the impact of specific conditions on the chemical signature. This study provides the first information on the impact of sample preparation on the HRMS analysis of sludge. It shows that acetonitrile is the most effective solvent for obtaining maximum information from sewage sludge. The comparison of the different cleanup procedures emphasizes the significance of this step and demonstrates the connection between the intrinsic physicochemical properties of molecules and the operating conditions of the extraction, particularly the pH. Moreover, the study highlights that surfactants, i.e., quaternary ammoniums in the positive ionization mode and linear alkylbenzene sulfonates in the negative mode, are the most discriminating compounds.

An innovative and integrative assay for toxicity testing using individual fish embryos. Application to oxazepam.

This paper describes the development of an integrative embryo-toxicity assay in Japanese medaka allowing analysis of several toxicological endpoints together in a same individual. In this assay, embryos are topically exposed, and survival, hatching success, malformations, biometry, behaviour, and target gene expression are subsequently analysed in each individual. This assay was applied to oxazepam, an anxiolytic pharmaceutical compound currently found in wastewater treatment plant effluent. Even if oxazepam accumulation in embryos was very low, it caused spinal and cardiac malformations, delayed growth, erratic swimming and deregulation of genes involved in apoptosis, DNA repair and mitochondrial metabolism. Relationship between gene deregulation, abnormal behaviour, and developmental anomalies was demonstrated. This assay is sensitive enough to detect adverse effects at low chemical concentrations and at multiple endpoints in a unique fish embryo. This integrative embryo-toxicity assay is a powerful tool to characterize the spectrum of effects of new chemicals and also to link effects induced at different molecular, tissue and physiological levels.

Evaluation of the influence of surfactants in the bioaccumulation kinetics of sulfamethoxazole and oxazepam in benthic invertebrates.

The potential ecotoxicological effects of mixtures of contaminants in the aquatic environment are generating a global concern. Benthic invertebrates, such as the crustacean Gammarus fossarum, are key in the functioning of aquatic ecosystems, and are frequently used as sentinel species of water quality status. The aim of this work was to study the effects of a mixture of the most frequently detected surfactants in the bioconcentration kinetics of two pharmaceuticals in G. fossarum, evaluating their potential enhancing or suppressing effects. Laboratory exposure experiments for both pharmaceuticals and surfactants (concentration ratio 1:25) were set up for two individual compounds, the anxiolytic oxazepam and the antibiotic sulfamethoxazole. Gammarid samples were processed using microQuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction. Pharmaceuticals concentration in the organisms was followed-up by means of nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results indicated a similar mode of action of the surfactants in the bioconcentration kinetics of both drugs, decreasing the accumulation rate in the organism. Oxazepam showed a higher accumulation potential than sulfamethoxazole in all cases. Depuration experiments for oxazepam also demonstrated the high depurative capacity of gammarids, eliminating >50% of the concentration of oxazepam in <6h.

Occurrence of pharmaceuticals in WWTP effluents and their impact in a karstic rural catchment of Eastern France.

The occurrence of pharmaceuticals in freshwater ecosystems provokes increasing concern due to their potential risk to non-target organisms and to human health. Pharmaceuticals are used in both human and veterinary medicine and are essentially released into the environment via wastewater treatment plants (WWTPs) and from livestock. In this study, 31 pharmaceuticals were analyzed in effluent and surface water upstream and downstream of two WWTPs in the Loue-Doubs rural karstic catchment in Eastern France. Diclofenac (965 and 2476 ng L-1), sulfamethoxazole (655 and 1380 ng L-1) and carbamazepine (566 and 1007 ng L-1) displayed the highest levels in the effluents of both WWTPs. Diclofenac levels were also high in surface water samples 300 and 166 ng L-1 in the River Doubs and the River Loue, respectively, followed by paracetamol (273 and 158 ng L-1) and sulfamethoxazole (126 and 73 ng L-1). In both rivers, the most critical compounds were found to be the antibiotic sulfamethoxazole (risk quotient (RQ) from 23.7 to 51.1) and ofloxacine (RQ from 1.1 to 18.9), which reached levels inducing toxic effects in aquatic organisms. This study showed that WWTP effluents are the major sources of the pharmaceuticals, but raw discharges from human residences, pastures and livestock manure represent significant sources of contamination of surface water and groundwater. The aim of this study was to assist scientists and authorities in understanding occurrence and sources of pharmaceuticals in order to improve water quality management in chalk streams.

Quantification of emerging micropollutants in an amphipod crustacean by nanoliquid chromatography coupled to mass spectrometry using multiple reaction monitoring cubed mode.

An innovative analytical method has been developed to quantify the bioaccumulation in an amphipod crustacean (Gammarus fossarum) of three micropollutants regarded as anthropic-pollution markers: carbamazepine, oxazepam, and testosterone. A liquid-liquid extraction assisted by salts, known as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) was miniaturised and optimised, so it could be adapted to the low mass samples (approximatively 5mg dry weight). For this same reason and in order to obtain good sensitivity, ultra-trace analyses were carried out by means of nanoliquid chromatography. A preconcentration system by on-column trapping was optimised to increase the injection volume. In order to improve both sensitivity and selectivity, the multiple reaction monitoring cubed mode analyses (MRM(3)) were carried out, validated and compared to the classic MRM. To the best of our knowledge, this is the first time that MRM(3) is coupled to nanoliquid chromatography for the analysis and detection of organic micropollutants <300Da. The optimised extraction method exhibited recoveries superior to 80%. The limits of quantification of the target compounds were 0.3, 0.7 and 4.7ng/g (wet weight) for oxazepam, carbamazepine and testosterone, respectively and the limits of detection were 0.1, 0.3 and 2.2ng/g (wet weight), respectively. The intra- and inter-day precisions were inferior to 7.7% and 10.9%, respectively, for the three levels of concentration tested. The analytical strategy developed allowed to obtain limits of quantification lower than 1ng/g (wet weight) and to establish the kinetic bioconcentration of contaminants within G. fossarum.

Differential Isotope Labeling of 38 Dietary Polyphenols and Their Quantification in Urine by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry.

A large number of polyphenols are consumed with the diet and may contribute to the prevention of chronic diseases such as cardiovascular diseases, diabetes, cancers, and neurodegenerative diseases. More comprehensive methods are needed to measure exposure to this complex family of bioactive plant compounds in epidemiological studies. We report here a novel method enabling the simultaneous measurement in urine of 38 polyphenols representative of the main classes and subclasses found in the diet. This method is based on differential (12)C-/(13)C-isotope labeling of polyphenols through derivatization with isotopic dansyl chloride reagents and on the analysis of the labeled polyphenols by tandem mass spectrometry. This derivatization approach overcomes the need for costly labeled standards. Different conditions for enzyme hydrolysis of polyphenol glucuronides and sulfate esters, extraction, and dansylation of unconjugated aglycones were tested and optimized. Limits of quantification varied from 0.01 to 1.1 µM depending on polyphenols. Intrabatch coefficients of variation varied between 3.9% and 9.6%. Interbatch variations were lower than 15% for 31 compounds and lower than 29% for 6 additional polyphenols out of the 38 tested. Thirty seven polyphenols were validated and then analyzed in 475, 24 h urine samples from the European Prospective Investigation on Cancer and Nutrition (EPIC) study. Thirty four polyphenols could be detected and successfully estimated and showed large interindividual variations of concentrations (2-3 orders of magnitude depending on the compound), with median concentrations spanning from 0.01 to over 1000 µM for all 34 compounds.

MicroQuEChERS-nanoliquid chromatography-nanospray-tandem mass spectrometry for the detection and quantification of trace pharmaceuticals in benthic invertebrates.

Due to industrialization and the use of chemical products in everyday life, various types of drugs and pesticides are present in our environment, which threaten and cause negative impacts on aquatic ecosystems. The consequences of these pollutants are gradually becoming visible. Recent evidence confirms that long term exposure to environmental pharmaceutical concentrations can induce adverse effects in aquatic vertebrates and invertebrates such as reproductive impairments and collapse wild populations. Consequently, one of the challenges of environmental science is to evaluate the associated risks. In this context, a new methodology has been developed using nano-LC-nano-ESI MS/MS to quantify traces of two pharmaceuticals (a neuropharmaceutical drug, fluoxetine, and an anticonvulsant drug, carbamazepine) in two molluscs, Potamopyrgus antipodarum and Valvata piscinalis, which are both prosobranch gastropods. A simple and quick extraction method was developed based on a modified and miniaturized version of the QuEChERS method. The procedure involves the extraction of approximately 10 mg of wet mollusc tissue by 500 µL of a mixture of acetonitrile/water/hexane (50/20/30) and 100 mg of buffer salt. Thus, the extraction step was carried out on an individual scale. The sensitivity of this method allowed for the detection of levels as low as 18 ng/g and 128 ng/g for carbamazepine and fluoxetine, respectively, with recoveries of greater than 85% for the two targeted compounds. This method was then applied to both gastropod species exposed to fluoxetine under laboratory conditions. The results provide evidence of bioaccumulation in both P. antipodarum and V. piscinalis and reveal the inter-species differences.

A pragmatic approach to assess the exposure of the honey bee (Apis mellifera) when subjected to pesticide spray.

Plant protection spray treatments may expose non-target organisms to pesticides. In the pesticide registration procedure, the honey bee represents one of the non-target model species for which the risk posed by pesticides must be assessed on the basis of the hazard quotient (HQ). The HQ is defined as the ratio between environmental exposure and toxicity. For the honey bee, the HQ calculation is not consistent because it corresponds to the ratio between the pesticide field rate (in mass of pesticide/ha) and LD50 (in mass of pesticide/bee). Thus, in contrast to all other species, the HQ can only be interpreted empirically because it corresponds to a number of bees/ha. This type of HQ calculation is due to the difficulty in transforming pesticide field rates into doses to which bees are exposed. In this study, we used a pragmatic approach to determine the apparent exposure surface area of honey bees submitted to pesticide treatments by spraying with a Potter-type tower. The doses received by the bees were quantified by very efficient chemical analyses, which enabled us to determine an apparent surface area of 1.05 cm(2)/bee. The apparent surface area was used to calculate the exposure levels of bees submitted to pesticide sprays and then to revisit the HQ ratios with a calculation mode similar to that used for all other living species. X-tomography was used to assess the physical surface area of a bee, which was 3.27 cm(2)/bee, and showed that the apparent exposure surface was not overestimated. The control experiments showed that the toxicity induced by doses calculated with the exposure surface area was similar to that induced by treatments according to the European testing procedure. This new approach to measure risk is more accurate and could become a tool to aid the decision-making process in the risk assessment of pesticides.

Multi-residue analysis of emerging pollutants in benthic invertebrates by modified micro-quick-easy-cheap-efficient-rugged-safe extraction and nanoliquid chromatography-nanospray-tandem mass spectrometry analysis.

Aquatic ecosystems are continuously contaminated by agricultural and industrial sources. Although the consequences of this pollution are gradually becoming visible, their potential impacts on aquatic ecosystems are poorly known, particularly regarding the risk of bioaccumulation in different trophic levels. To establish a causality relationship between bioaccumulation and disease, experiments on biotic matrices must be performed. In this context, a multi-residue method for the analysis of 35 emerging pollutants in three benthic invertebrates (Potamopyrgus antipodarum, Gammarus fossarum, and Chironomus riparius) has been developed. Because the variation in response of each individual must be taken into account in ecotoxicological studies, the entire analytical chain was miniaturised, thereby reducing the required sample size to a minimum of one individual and scaling the method accordingly. A new extraction strategy based on a modified, optimised and miniaturised "QuEChERS" approach is reported. The procedure involves salting out liquid-liquid extraction of approximately 10-20mg of matrix followed by nano-liquid chromatography-nano electospray ionisation coupled with tandem mass spectrometry. The validated analytical procedure exhibited recoveries between 40 and 98% for all the target compounds and enabled the determination of pollutants on an individual scale in the ng g(-1) concentration. The method was subsequently applied to determine the levels of target analytes in several encaged organisms which were exposed upstream and downstream of an effluent discharge. The results highlighted a bioaccumulation of certain targeted emerging pollutants in three freshwater invertebrates, as well as inter-species differences. 18 out of 35 compounds were detected and eight were quantified. The highest concentrations were measured for ibuprofen in G. fossarum, reaching up to 105 ng g(-1).

Trace level determination of pyrethroid and neonicotinoid insecticides in beebread using acetonitrile-based extraction followed by analysis with ultra-high-performance liquid chromatography-tandem mass spectrometry.

Beebread is among the matrices suspected of contaminating honeybee. To better understand this contamination, the study aimed to develop an efficient, sensitive and reliable analytical method for the trace analysis of pesticides in beebread. This study focuses specifically on the insecticides pyrethroids and neonicotinoids and some of their metabolites. It describes the development and validation of an original analytical approach that consists of one simple extraction method based on modified QuEChERS followed by a selective and sensitive analysis by UHPLC-MS/MS to determine the target compounds in beebread. The method was validated using a quadratic fit. RSD values below 20% were obtained, except for 5-hydroxy-imidacloprid and imidacloprid at 0.5 ng/g, which exhibited RSDs of 25% and 21%, respectively. The intra-day precision was less than 10% for many of the investigated compounds. The inter-day precision varied between 2% and 36%, depending on the compound and the concentration. The recoveries varied from 53% to 119%, with averages of 83, 81 and 77% for the extraction of beebread samples spiked at 0.5, 5 and 10 ng/g, respectively. The LOD values for all the substances were below ng/g, with the exception of 6-chloronicotinic acid (LOD=1.7 ng/g). The method was then applied to the analysis of 32 beebread samples and revealed the presence of 7 of the target substances. The most frequently detected pesticides belonged to the neonicotinoid family and were generally present at low concentrations, but in some cases exceeded 170 ng/g (acetamiprid and thiacloprid). Some pyrethroids were also detected (lambda-cyhalothrine and bifenthrine), but at very low levels.

Widespread occurrence of chemical residues in beehive matrices from apiaries located in different landscapes of Western France.

BACKGROUND: The honey bee, Apis mellifera, is frequently used as a sentinel to monitor environmental pollution. In parallel, general weakening and unprecedented colony losses have been reported in Europe and the USA, and many factors are suspected to play a central role in these problems, including infection by pathogens, nutritional stress and pesticide poisoning. Honey bee, honey and pollen samples collected from eighteen apiaries of western France from four different landscape contexts during four different periods in 2008 and in 2009 were analyzed to evaluate the presence of pesticides and veterinary drug residues. METHODOLOGY/FINDINGS: A multi-residue analysis of 80 compounds was performed using a modified QuEChERS method, followed by GC-ToF and LC-MS/MS. The analysis revealed that 95.7%, 72.3% and 58.6% of the honey, honey bee and pollen samples, respectively, were contaminated by at least one compound. The frequency of detection was higher in the honey samples (n = 28) than in the pollen (n = 23) or honey bee (n = 20) samples, but the highest concentrations were found in pollen. Although most compounds were rarely found, some of the contaminants reached high concentrations that might lead to adverse effects on bee health. The three most frequent residues were the widely used fungicide carbendazim and two acaricides, amitraz and coumaphos, that are used by beekeepers to control Varroa destructor. Apiaries in rural-cultivated landscapes were more contaminated than those in other landscape contexts, but the differences were not significant. The contamination of the different matrices was shown to be higher in early spring than in all other periods. CONCLUSIONS/SIGNIFICANCE: Honey bees, honeys and pollens are appropriate sentinels for monitoring pesticide and veterinary drug environmental pollution. This study revealed the widespread occurrence of multiple residues in beehive matrices and suggests a potential issue with the effects of these residues alone or in combination on honey bee health.

Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.

This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at -20°C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD(50), 51.16 ng bee(-1)) and two sublethal doses, LD(50)/10 (5.12 ng bee(-1)) and LD(50)/20 (2.56 ng bee(-1)). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee(-1). We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides.

Multiresidue analysis of aromatic organochlorines in soil by gas chromatography-mass spectrometry and QuEChERS extraction based on water/dichloromethane partitioning. Comparison with accelerated solvent extraction.

A novel multiresidue method was developed for the simultaneous analysis of 34 organochlorines, including chlorobenzenes, chlorophenols, chlorinated hydrocarbons and chlorinated olefins, in soil by GC-MS, using a QuEChERS-based extraction. The conventional QuEChERS method was optimised and, for the first time, the use of a non miscible-water solvent was required. The method was compared to ASE extraction, versatile technique widely used for the soils' extraction and QuEChERS-based method was shown to be the most efficient in terms of recoveries, simplicity and rapidity. For ASE, recoveries between 42% and 85% were obtained for the majority of the compounds. However, due to the high pressure, all volatile compounds were lost. In opposite, QuEChERS extraction allowed detection and quantification of all the compounds with recoveries between 60% and 100%. Moreover, no additional clean up by dispersive SPE on PSA was necessary, which allowed reducing the cost of the analysis. Performance of the method was assessed. The method was linear over the range of concentration of 10-5000 µg kg(-1). Precision, expressed as intra-day precision and inter-day variation was verified at three concentrations. Limits of detection were from 2 to 50 µg kg(-1) and limits of quantification from 7 to 170 µg kg(-1) for the majority of the compounds (chlorobenzenes and chlorinated hydrocarbons and olefins), except for chlorophenols. The method was further applied to different soils coming from a contaminated industrial site, where a new environmental remediation process, using phytoremediation, was tested. The results showed that the method could be applied to any kind of soils (mineral or organic) and was appropriate to very volatile compounds which were not available with conventional technique.

Multi-residue analysis of 80 environmental contaminants in honeys, honeybees and pollens by one extraction procedure followed by liquid and gas chromatography coupled with mass spectrometric detection.

One of the factors that may explain nowadays honeybees' colonies losses is the increasing presence of chemicals in the environment. The aim of this study is to obtain a global view of the presence of environmental contaminants in beehives and, develop a fast, cheap and sensitive tool to analyze environmental contaminants in apiarian matrices. A multi residue analysis was developed to quantify 80 environmental contaminants, pesticides and veterinary drugs, belonging to different chemical classes, in honeys, honeybees and pollens. It consists in a single extraction, based on a modified "QuEChERS method", followed by gas chromatography coupled with Time of Flight mass spectrometry (GC-ToF) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The "QuEChERS method" combines salting-out liquid-liquid extraction with acetonitrile and a dispersive-SPE clean up. It was adjusted to honey and especially to honeybee and pollen, by adding a small fraction of hexane in acetonitrile to eliminate lipids that interfere with mass spectrometry analysis. This method, combined with accurate and sensitive detection, allowed quantification and confirmation at levels as low as 10 ng/g, with recoveries between 60 and 120%. Application to more than 100 samples of each matrix was achieved for a global view of pesticide presence in the honeybee environment. Relatively high percentages of honeys, honeybees and pollens were found to be contaminated by pesticides used to combat varroa but also by fungicides like carbendazim and ubiquitous contaminants.