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Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E -nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients ( r ) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

This paper presents the results of a research aimed at establishing a novel method for the detection of primary and secondary micro- and nanoplastics (MNPs), by using the fluorescence properties of the dye Nile Red-n-heptane (NR-H). The method has been applied to the detection of laboratory degraded polymers (Polystyrene, PS and Polyethylene Terephthalate, PET) as well as traceable latex microspheres in aqueous environments, showing a remarkable detection capacity and avoiding the prior extraction or processing of MNPs in natural samples, with significant time savings compared to conventional methods. The study has been carried out on various types of water, including samples from wastewater treatment plants, boreholes, seawater and synthesized seawater. The effectiveness of the staining process was evaluated by scanning electron microscopy (SEM), dynamic light scattering (DLS) and optical microscopy. As a result, a novel standardizable protocol for the rapid detection of MNPs has been established, with the potential to improve environmental protection through fast in-situ detection and identification of plastic contaminants. The limitations of the protocol in the quantification of MNPs have also been identified and further studies are proposed to overcome these limitations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. IFTS-Project research reports financial support and administrative support were provided by Nouvelle-Aquitaine Regional Council. No conflict of interest to report. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02-0.06 h/cm 2 filter surface compared to 4.5-14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques., (© 2024. The Author(s).)

Agrochemical indoxacarb is an important tool for selective pest control in radish that be consumed globally. A rapid and sensitive analytical method UHPLC-MS/MS was developed for tracing indoxacarb in radish leaves and roots with LOQ of 0.001 mg/kg and RT within 2 min, which were confirmed the satisfied storage stability of indoxacarb in radish matrixes with degradation rates less than 30 %. The occurrence, pharmacokinetics dissipation and concentration variation of indoxacarb were reflected by the original deposition of 2.23-4.12 mg/kg, half-lives of 2.6-8.0 d and terminal magnitude of 0.17 × 10 -2 -25.46 mg/kg in radish, and the influencing factors were further illustrated in terms of climate factors, crop cultivars and soil properties. The highest residues of indoxacarb were 25.46 mg/kg in leaves and 0.12 mg/kg in roots, which were higher than international maximum residue limits. A probabilistic model, as well as deterministic model, were introduced to evaluated the health risks of indoxacarb offering a better description for uncertainty. The total chronic dietary risk values of indoxacarb were 146.961-482.065 % in 12 registered crops, of which ADI % in radish was accounted for 19.8 % with risk dilution effects. The unacceptable acute dietary risks of 121.358-220.331 % were observed at 99.9th percentile, whereas the high-potential non-carcinogenic effects were observed over 90th percentile (105.035-1121.943 %). The health risks should be continuously emphasized given the increasing applications and persistent characteristics of indoxacarb, which is vital to protect the human population from hazardous effects, particularly for vulnerable children., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The ban imposed by the European Union on the use of neonicotinoids as sugar beet seed treatments was based on the exposure of bees to residues of neonicotinoids in pollen and nectar of succeeding crops. To address this concern, residues of thiamethoxam (TMX) and clothianidin (CTD) were analyzed in soil collected from fields planted in at least the previous year with thiamethoxam-treated sugar beet seed. This soil monitoring program was conducted at 94 sites across Germany in two separate years. In addition, a succeeding crop study assessed residues in soil, guttation fluid, pollen, and nectar sampled from untreated succeeding crops planted in the season after thiamethoxam seed-treated sugar beet at eight field sites across five countries. The overall mean residues observed in soil monitoring were 8.0 ± 0.5 µg TMX + CTD/kg in the season after the use of treated sugar beet seed. Residue values decreased with increasing time interval between the latest thiamethoxam or clothianidin application before sugar beet drilling and with lower application frequency. Residues were detected in guttation fluid (2.0-37.7 µg TMX/L); however, the risk to pollinators from this route of exposure is likely to be low, based on the reported levels of consumption. Residues of thiamethoxam and clothianidin in pollen and nectar sampled from the succeeding crops were detected at or below the limit of quantification (0.5-1 µg a.i./kg) in 86.7% of pollen and 98.6% of nectar samples and, unlike guttation fluid residues, were not correlated with measured soil residues. Residues in pollen and nectar are lower than reported sublethal adverse effect concentrations in studies with honeybee and bumble bee individuals and colonies fed only thiamethoxam-treated sucrose, and are lower than those reported to result in no effects in honeybees, bumble bees, and solitary bees foraging on seed-treated crops. Integr Environ Assess Manag 2022;18:709-721. © 2021 SYNGENTA. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2021 SYNGENTA. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation., (© 2021 Wiley-VCH GmbH.)

Presently, most of anticancer drugs are high toxic for normal cells and, and as a result, they have severe side effects. Moreover, most of the formulations are lipophilic and have poor selectivity. To overcome these limitations, various drug delivery systems could be proposed. The aim of the current study was to fabricate novel polysaccharide nanocontainers (NC) by one-step ultrasonication technique and to evaluate their accumulation efficacy and cytotoxicity in 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. NC with mean sizes in a range of 340-420 nm with the core-shell structure are synthetized and characterized. The NC shell is composed from diethylaminoethyl dextran/xanthan gum polyelectrolyte complex, while the hydrophobic core was loaded with the lipophilic anticancer drug thymoquinone. To enhance NC accumulation in human breast adenocarcinoma MCF-7 cells, the NC surface was modified with poly-L-lysine (PLL) or polyethylene glycol. Cell uptake of the NC loaded with Nile Red into the tumor cells was investigated by laser scanning confocal microscopy, fluorescent flow cytometry and fluorimetry. Modification of the NC with PLL allowed to obtain the optimal drug delivery system with maximal cytotoxicity, which was tested by MTT-test. The developed NC are promising for lipophilic anticancer drug delivery., (© 2020 Wiley Periodicals LLC.)

This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

Rapid and efficient detection of indoxacarb (IXC), a common chemical contaminant, in environmental and biological samples is necessary. In this work, a modern optical sensor was developed for IXC, based on environmentally friendly molecularly imprinted polymer (MIP) coated on silica-carbon quantum dots (SiCQDs). A hydrothermal method was used to prepare highly fluorescence SiCQDs and, subsequently, MIP formed on surface (MIP@SiCQDs) using a sol-gel method. A linear relationship between the fluorescence quenching effect and increased IXC concentration was found for the range of 4-102 nM, under the optimal conditions, with a 1 nM detection limit. Precisions was of 4.5 and 2.3% for five replicate detections at 21 and 60 nM IXC, respectively. Applicability of the sensor for IXC quantification in environmental and biological samples was verified with recoveries in the range 95-106% with a relative standard deviation of <6.0%., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The fluorescence imaging technique has attracted increasing attention in the detection of various biological molecules in situ and in real-time owing to its inherent advantages including high selectivity and sensitivity, outstanding spatiotemporal resolution and fast feedback. In the past few decades, a number of fluorescent probes have been developed for bioassays and imaging by exploiting different fluorophores. Among various fluorophores, resorufin exhibits a high fluorescence quantum yield, long excitation/emission wavelength and pronounced ability in both fluorescence and colorimetric analysis. This fluorophore has been widely utilized in the design of responsive probes specific for various bioactive species. In this review, we summarize the advances in the development of resorufin-based fluorescent probes for detecting various analytes, such as cations, anions, reactive (redox-active) sulfur species, small molecules and biological macromolecules. The chemical structures of probes, response mechanisms, detection limits and practical applications are investigated, which is followed by the discussion of recent challenges and future research perspectives. This review article is expected to promote the further development of resorufin-based responsive fluorescent probes and their biological applications.

An efficient single quadrupole gas chromatography with mass spectrometry method was developed and validated for the determination of indoxacarb residues in tomato and soil. Residues were extracted from the samples using acetonitrile as extracting solvent and the extracts were purified through primary secondary amine and graphitized carbon black. Recoveries were obtained in the range of 92.12-110.51% with the relative standard deviation of 1.32-4.32%. Indoxacarb dissipated with half-life of 3.12-3.21 and 1.24-1.35d for tomato and soil, respectively following doses of indoxacarb 14.5% SC at 60, 90 and 120 g.a.i./ha. Safe waiting periods were found to be 1-3d. The residues were removed from tomato fruit was in the range of 16.73 to 54.32% using simple decontamination approaches. The present study suggest that the use of indoxacarb in tomato at recommended dose, does not seem to pose any dietary risk to the consumers. The soil RQ values indicated low level of risk to earthworms and arthropods., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1-5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C 18 UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%-99%) and IS (85%-103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A tetralactam macrocycle acts as a novel supramolecular adjuvant to capture a released resorufin dye and create a higher contrasting yellow/blue color change for enhanced naked eye interpretation of a colorimetric indicator assay.

Accurate analysis of pesticide residue in real samples is essential for food safety and environmental protection. However, a traditional electrochemical sensor based on single-signal output is easily affected by background noise, environmental conditions, electrode diversity, and a complex matrix of samples, leading to extremely low accuracy. Hence, in this paper, a ratiometric strategy based on dual-signal output was adopted to build inner correction for sensing of widely-used carbaryl (CBL) for the first time. By comparison, Nile blue A (NB) was selected as reference probe, due to its well-defined peak, few effects on the target peak of CBL, and excellent stability. The effects of a derivatization method, technique mode, and pH were also investigated. Then the performance of the proposed ratiometric sensor was assessed in terms of three aspects including the elimination of system noise, electrode deviation and matrix effect. Compared with traditional single-signal sensor, the ratiometric sensor showed a much better linear correlation coefficient (r > 0.99), reproducibility (RSD < 10%), and limit of detection (LOD = 1.0 μM). The results indicated the introduction of proper reference probe could ensure the interdependence of target and reference signal on the same sensing environment, thus inner correction was fulfilled, which provided a promising tool for accurate analysis.

The present paper describes the sono-assisted adsorption (sono-adsorption) of methylene blue (MB), methyl violet (MV), and Nile blue (NB) from aqueous solution by AC/CoFe 2 O 4 magnetic composite. FT-IR, TGA-DTG, VSM, XRD, TEM, SEM, EDX, Map, and Raman analysis were used to characterize the magnetic composite. The magnetization saturation value of AC/CoFe 2 O 4 magnetic composite was determined to be 53.06 emu/g. Dye sono-adsorption efficiency was increased by increasing adsorbent dose, pH value, and contact time, but not dye concentration. Pseudo-first-order, pseudo-second-order, and intra-particle diffusion models were used to study the kinetic behavior of the cationic dye sono-adsorption. The sono-adsorption kinetics was reasonably followed by pseudo-second-order model (R 2  > 0.998). The results showed that the Freundlich model (R 2  > 0.976) was more able to describe the sono-adsorption equilibrium behavior than Langmuir, D-R, and Scatchard models. The maximum sono-adsorption capacity of NB, MV, and MB was determined as 86.24, 83.90, and 87.48 mg/g, respectively. Based on the parameters derived from isotherm modeling (R L , n, and E), the sono-adsorption process of cationic dyes is desirable and physical. An increase in NaCl concentration reduced the sono-adsorption efficiency for all dyes. Also, the adsorption-desorption of AC/CoFe 2 O 4 magnetic was studied up to 10 stages, and it was confirmed that the sono-adsorption efficiency is acceptable up to the eight stage. AC/CoFe 2 O 4 magnetic composite is, therefore, an affordable and recyclable adsorbent to remove the molecule of NB, MV, and MB dyes from aqueous media.

We developed an analytical method using liquid-liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and quantify tebufenozide (TEB) and indoxacarb (IND) residues in animal and aquatic products (chicken muscle, milk, egg, eel, flatfish, and shrimp). The target compounds were extracted using 1% acetic acid (0.1% acetic acid for egg only) in acetonitrile and purified using n-hexane. The analytes were separated on a Gemini-NX C 18 column using (a) distilled water with 0.1% formic acid and 5 mm ammonium acetate and (b) methanol with 0.1% formic acid as the mobile phase. All six-point matrix-matched calibration curves showed good linearity with coefficients of determination (R 2 ) ≥0.9864 over a concentration range of 5-50 μg/kg. Intra- and inter-day accuracy was expressed as the recovery rate at three spiking levels and ranged between 73.22 and 114.93% in all matrices, with a relative standard deviation (RSD, corresponding to precision) ≤13.87%. The limits of quantification (LOQ) of all target analytes ranged from 2 to 20 μg/kg, which were substantially lower than the maximum residue limits (MRLs) specified by the regulatory agencies of different countries. All samples were collected from different markets in Seoul, Republic of Korea, and tested negative for tebufenozide and indoxacarb residues. These results show that the method developed is robust and may be a promising tool to detect trace levels of the target analytes in animal products., (© 2019 John Wiley & Sons, Ltd.)

Neonicotinoid insecticides such as imidacloprid, indoxacarb and thiamethoxam are widely used for control of a large number of insect pests of pomegranate crop. Their residue levels were evaluated on pomegranate fruits over 2 years during the same cropping season. The QuEChERS analytical method in conjunction with LC-MS/MS was validated to analyse the insecticides on pomegranate fruits with peel (whole fruit), without peel (aril) and in the field soil. The method performance was satisfactory with the limit of quantification (LOQ) of 0.005 mg/kg which was below the maximum residue limits (MRLs) in pomegranate for the 3 compounds. A first order reaction kinetics was observed for the three insecticides with the half -life of degradation of 8-11.1 days for imidacloprid; 7.4-8.4 days for indoxacarb and 9.8-14.2 days for thiamethoxam. Though the insecticides are systemic in nature, the residues in the edible pomegranate aril were always < LOQ. The maximum residue levels of imidacloprid on pomegranate was less than its MRL of 1 mg/kg, so the pre-harvest interval (PHI) required was 1 day only. For indoxacarb, 31-42 days PHI was needed for the residues to reduce to its MRL of 0.02 mg/kg. The PHI of thiamethoxam was 46-77 days, the time required for its residues to reduce to its MRL of 0.01 mg/kg. Higher rainfall possibly facilitated faster dissipation of imidacloprid residues from pomegranate whereas indoxacarb and thiamethoxam remained unaffected. The results of the study can be utilized to incorporate these three chemicals in the plant protection program of pomegranate and fixation of MRL in India., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this paper, the application of a multi-analytical approach for the characterisation of synthetic and natural dyes in a historical textile is presented. The work is focused on a historical dress of a Sicilian noblewoman, dating from about 1865-1870. Firstly, SERS on fibre was performed, in order to individuate the classes of dyes employed. The SERS spectra suggested the presence of two main dyes: mauveine and orcein. In order to confirm these preliminary results, two different extraction protocols were applied. The extracts obtained were analysed by ESI-MS, MALDI-ToF and UHPCL-MS analyses, confirming the SERS results. In particular, the application of the ammonia mild extraction technique allowed to selectively extract the phenoxazonic dyes, separating them already in the extraction step from the synthetic ones. Thanks to this multi-analytical approach, this dress could be considered as one of the first examples of employment of synthetic dyes in association with natural ones.

Estimations of consumer exposure to mycotoxins through surveillance of mycotoxins in the food trade are well described, but the exposure due to mouldy food in private homes is not known, and may result from removing visible mould on food and eating the rest. In this study, we followed the growth of Penicillium expansum on the surface of apple jam and Penicillium verrucosum on crème fraiche, as well as production and distribution of fungal metabolites throughout the sample (approx. 6 cm high divided into three equal layers), using a multianalyte method, over time (up to 28 days) and at 4, 8 and 15 °C. Growth rates and apparent lag times for P. expansum in apple jam at different temperatures were estimated by fitting to the Baranyi model. The growth rates were 1.7, 2.7 and 4.3 mm day -1 for storage at 4, 8 and 15 °C, respectively; apparent lag times decreased with increasing storage temperature and were 10.6, 7.9 and 2.6 days at corresponding temperatures. Patulin and roquefortine C were identified and quantified, among other fungal metabolites. Patulin was detected in all 2-cm layers of the apple jam at 15 °C. Concentrations in the upper two layers of the jar corresponded to exposures exceeding the health based guidance value (HBGV) for a normal serving size. Consequently, removal of the mouldy part is insufficient to avoid unhealthy exposure. In contrast to patulin, roquefortine C was also produced at 4 °C. The growth of P. verrucosum on crème fraiche was very restricted and could not be modelled. Despite the small colony (8 ± 0.5 mm in diameter), ochratoxin A and citrinin were detected after 21 days at 15 °C in the top 2 cm layer (including the fungal colony), and at concentrations in a normal serving corresponding to an exposure above the HBGV established by EFSA for both mycotoxins. Questiomycin A, an antibiotic, was also produced in crème fraiche but in contrast to the two mycotoxins, was detected throughout all layers of the crème fraiche and was produced also at 4 and 8 °C. As a complement to a previous study, we also present production and the distribution of major fungal metabolites in apple jam and crème fraiche for some additional fungal strains (P. crustosum, P. roqueforti and P. verrucosum on apple jam and P. expansum on crème fraiche). A pilot study investigating the effect of inoculation size on toxin production may have implications for the best inoculum to use in experimental studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Oleaginous microbes, which contain over 20% intracellular lipid, predominantly triacylglycerols (TG), by dry weight, have been discovered to have high oil content by many different protocols, ranging from simple staining to more complex chromatographic methods. In our laboratory, a methodical process was implemented to identify high oil yeasts, designed to minimize labor while optimizing success in identifying high oil strains among thousands of candidates. First, criteria were developed to select candidate yeast strains for analysis. These included observation of buoyancy of the yeast cell mass in 20% glycerol, and phylogenetic placement near known oleaginous species. A low-labor, semiquantitative Nile red staining protocol was implemented to screen numerous yeast cultures for high oil content in 96-well plates. Then, promising candidates were selected for more quantitative analysis. A more labor-intensive and quantitative gravimetric assay was implemented that gave consistent values for intracellular oil content for a broad range of yeast species. Finally, an LC-MS protocol was utilized to quantify and identify yeast triacylglycerols. This progressive approach was successful in identifying 30 new oleaginous yeast species, out of over 1000 species represented in the Phaff Yeast Culture Collection.

Over the last decade, finding bacterial strains with ability to accumulate high concentrations of lipids has gained increasing interest, since these lipids may be used in different industries. Here we describe two methods for evaluation of lipid accumulation in cyanobacteria, following by our personal reflection on issues surrounding the use of these methods. First, we present the Bligh and Dyer protocol as a traditional extraction method using organic solvents for quantitative determination of lipids and next Nile red, a selective fluorescent stain, that has been used as a rapid approach for both qualitative and quantitative measurement of lipids.

The aim of this study was to determine the insecticide residue processing factor (PF) from plums to prunes and the effect of the industrial processing of prunes residue concentrations. Our results show an increase of insecticide concentrations during plum dehydration that is explained by fruit water loss; however, the normalized insecticide residue concentration, based on plum dry weights to compensate dehydration, was reduced. The water washing and tenderizing of prunes produced insecticide residue reductions of 22.9 ± 4.5% and 21.9 ± 4.2%, respectively. PF were: 1.157, 1.872, 1.316, 0.192, 2.198, 0.775 and 0.156 for buprofezin, l-cyhalothrin, spirodiclofen, indoxacarb, acetamiprid, imidacloprid and emamectin benzoate, respectively, being directly related to water solubility, aqueous hydrolysis and degradation point and inversely related to molecular mass and melting point. In plums for the dehydrated agroindustry the final product is prunes, therefore, it is crucial to consider the PF to determine the specific preharvest interval for this important agroindustry., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, effects of low intensity electrical current (EC) and ultrasound (US) treatments on the reduction of some important pesticides (captan, thiamethoxam and metalaxyl) residues in tomato samples were investigated. Three different currents (200, 800 and 1400mA) of EC were applied at various time intervals (2, 4, 6, 8 and 10min). Two kinds of US treatments including ultrasonic bath (UB) at 40kHz and ultrasonic probe (UP) at 24kHz were tested for the determination of US effectiveness. In addition, synergistic effects of US on EC treatments were evaluated. The most effective conditions for reduction of captan, thiamethoxam and metalaxyl residues were 1400mA+40kHz, 800mA+24kHz and 1400mA+24kHz, respectively. The residues of captan, thiamethoxam and metalaxyl were reduced in the order of 94.24%, 69.80% and 95.06% by using these combinations. EC and US strategies can be considered as effective treatments in industrial scale in order to remove the pesticide residues from vegetables., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A chiral liquid chromatography-tandem high resolution mass spectroscopic method was developed for the analysis of indoxacarb enantiomers in rice plants, rice hulls and brown rice. Chiral separation of two enantiomers was carried out on a Superchiral S-OD column maintained at 20°C and eluted with 0.3 mL/min methanol. Samples were extracted by acetonitrile solution with ultrasound and cleansed by dispersive solid-phase extraction of 50 mg of primary secondary amine and 50 mg of C 18 . This method was successfully used to study the degradation and residues of two enantiomers with enriched S-indoxacarb (2.33S/1R) and pure S-indoxacarb in rice plants. The half-lives of R-indoxacarb and S-indoxacarb were 4.20-4.33 and 3.45-3.57 days in rice plants during the degradation of enriched S-indoxacarb in Guizhou and Hunan, respectively, whereas the half-lives of pure S-indoxacarb were 2.68 and 3.69 days in Guizhou and Hunan, respectively. The results indicated that preferential S-indoxacarb degradation occurred and that enantiomeric transformation was absent in the total experiment periods of pure S-indoxacarb in rice plants. The final residue concentrations of indoxacarb enantiomers in brown rice were significantly less than those in rice plants and rice hulls in the same rice field after applying indoxacarb SC and indoxacarb EC., (© 2018 John Wiley & Sons, Ltd.)

The U.S. EPA conducts dietary-risk assessments to ensure that levels of pesticides on food in the U.S. food supply are safe. Often these assessments utilize conservative residue estimates, maximum residue levels (MRLs), and a high-end estimate derived from registrant-generated field-trial data sets. A more realistic estimate of consumers' pesticide exposure from food may be obtained by utilizing residues from food-monitoring programs, such as the Pesticide Data Program (PDP) of the U.S. Department of Agriculture. A substantial portion of food-residue concentrations in PDP monitoring programs are below the limits of detection (left-censored), which makes the comparison of regulatory-field-trial and PDP residue levels difficult. In this paper, we present a novel adaption of established statistical techniques, the Kaplan-Meier estimator (K-M), the robust regression on ordered statistic (ROS), and the maximum-likelihood estimator (MLE), to quantify the pesticide-residue concentrations in the presence of heavily censored data sets. The examined statistical approaches include the most commonly used parametric and nonparametric methods for handling left-censored data that have been used in the fields of medical and environmental sciences. This work presents a case study in which data of thiamethoxam residue on bell pepper generated from registrant field trials were compared with PDP-monitoring residue values. The results from the statistical techniques were evaluated and compared with commonly used simple substitution methods for the determination of summary statistics. It was found that the maximum-likelihood estimator (MLE) is the most appropriate statistical method to analyze this residue data set. Using the MLE technique, the data analyses showed that the median and mean PDP bell pepper residue levels were approximately 19 and 7 times lower, respectively, than the corresponding statistics of the field-trial residues.

Because of the persistence and solubility of neonicotinoid insecticides (NNIs), there is concern that these compounds may contaminate sources of drinking water. The objective of this project was to evaluate the distribution of NNIs in raw and treated drinking water from selected municipalities that draw their water from the lower Great Lakes in areas of southern Ontario, Canada where there is high intensity agriculture. Sites were monitored using Polar Organic Chemical Integrative Samplers (POCIS) and by collecting grab samples at six drinking water treatment plants. Thiamethoxam, clothianidin and imidacloprid were detected in both POCIS and grab samples of raw water. The frequency of detection of NNIs was much lower in treated drinking water, but some compounds were still detected at estimated concentrations in the low ng L -1 range. Thiamethoxam was detected in one grab sample of raw drinking water at a mean concentration of 0.28 μg L -1 , which is above the guidelines for drinking water recommended in some jurisdictions, including the European Union directive on pesticide levels <0.1 μg L -1 in water intended for human consumption. Further work is required to determine whether contamination of sources of drinking water with this class of insecticides is a global problem in agricultural regions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A simple analytical method was developed to simultaneously determine thiamethoxam and its metabolite, clothianidin, in fresh tobacco leaf, soil and cured tobacco leaf using liquid chromatography with tandem mass spectrometry. Thiamethoxam and clothianidin in tobacco and soil samples were extracted with acetonitrile containing 0.1% formic acid and purified using an NH 2 -SPE column. The optimized method provided good linearity with coefficients of determination R 2  ≥ 0.9981. The limits of detection and quantification were between 0.006-0.12 and 0.02-0.4 mg/kg, respectively. Intra- and inter-day recovery assays were used to validate the established method. The average recoveries of thiamethoxam and clothianidin in fresh tobacco leaf, soil and cured tobacco leaf were 75.04-100.47%, 75.86-86.40% and 89.83-99.39%, respectively. The intra- and inter-day relative standard deviations were all <9%. The developed method was successfully applied for the analysis of thiamethoxam and clothianidin residues in actual tobacco and soil samples. The results indicated that the established method met the requirements for the analysis of trace amounts of thiamethoxam and clothianidin in fresh tobacco leaf, soil and cured tobacco leaf., (Copyright © 2018 John Wiley & Sons, Ltd.)

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 μM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Neonicotinoid insecticides (NIs) have recently been recognized as co-factors in the decline of honeybee colonies because most neonicotinoids are systemic and can transfer into the pollen and nectar of many pollinated crops. In this study, we collected pollen, nectar and leaves from a cotton crop treated with imidacloprid and thiamethoxam to measure the residue levels of these two NIs at different application doses during the flowering period. Then, the residual data were used to assess the risk posed by the systemic insecticides to honeybees following mandated methods published by the European Food Safety Authority (EFSA), and a highly toxic risk to honeybees was highlighted. Imidacloprid was found in both pollen and nectar samples, whereas thiamethoxam was found in 90% of pollen samples and over 60% of nectar samples. Analysis of the pollen and nectar revealed residual amounts of imidacloprid ranging from 1.61 to 64.58 ng g -1 in the pollen and from not detected (ND) to 1.769 ng g -1 in the nectar. By comparison, the thiamethoxam concentrations in pollen and nectar ranged from ND to 14.521 ng g -1 and from ND to 4.285 ng g -1 , respectively. The results of this study provide information on the transfer of two NIs from seed treatment to areas of the plant and provides an understanding of the potential exposure of the bee and other pollinators to systemic insecticides., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The accumulation of pesticides in vegetables may have serious effects on human health and ecosystems via food chains; therefore, it is of great importance to investigate the uptake and accumulation behaviours of pesticides in vegetable tissues. In the present study, the uptake, translocation and accumulation of five neonicotinoids, thiamethoxam (THIM), clothianidin (CLO), thiacloprid (THID), acetamiprid (ACE) and dinotefuran (DIN), in komatsuna (Brassica rapa var. perviridis, a vegetable) were investigated. The concentrations of neonicotinoids in vegetable tissues ranged from 0.068 ± 0.002 to 29.6 ± 2.5 mg/kg. During the cultivation (except for the first day), the concentration of each neonicotinoid in shoots was the highest, followed by roots and the soil. The concentrating of neonicotinoids from the soil to roots followed the order of THIM > CLO > THID > DIN > ACE, while the order of the ability of translocation neonicotinoids from roots to shoots was the just opposite. The difference in uptake and translocation behaviours of the test neonicotinoids seems to be not correlated with the octanol/water partition coefficient (logK ow ), water solubility or dissociation constant (pK a ), but significantly correlated with molecular weight. In addition, a greater concentration of the THIM-metabolite clothianidin (M-CLO) was detected in vegetable shoots than in roots and the soil., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An analytical method based on liquid chromatography coupled with mass spectroscopy/mass spectroscopy was developed and validated for the determination of thiamethoxam residues in banana fruit and stem tissue samples. In this study, Waters Alliance LC and Acquity TQD were used with an electrospray ionization interface in the positive ion mode. An isocratic flow of 0.5% HCOOH in water and 0.05% HCOOH in CH 3 CN was used for separation. Thiamethoxam residue was extracted from the samples using CH 3 CN and a dispersive solid-phase extraction method was used for subsequent cleanup. Linearity studies were conducted between 0.001 and 0.1 μg mL -1 of standard solution with three replicates for each concentration. Satisfactory recoveries (107.21 to 115.16% and 90.94 to 109.22%) and high precision (relative standard deviations of 3.71 to 12.83% and 3.24 to 10.78%) were obtained for the banana stem and banana fruit matrix, respectively. The lower limits of detection and quantification achieved were 0.002 and 0.008 μg g -1 for banana stem and 0.001and 0.005 μg g -1 for banana fruit, respectively. The developed method was used to analyze the banana stem and fruit samples collected from thiamethoxam-treated fields and stems from the local market.

To better characterize the transport of neonicotinoid insecticides to the world's largest freshwater ecosystem, monthly samples (October 2015-September 2016) were collected from 10 major tributaries to the Great Lakes, USA. For the monthly tributary samples, neonicotinoids were detected in every month sampled and five of the six target neonicotinoids were detected. At least one neonicotinoid was detected in 74% of the monthly samples with up to three neonicotinoids detected in an individual sample (10% of all samples). The most frequently detected neonicotinoid was imidacloprid (53%), followed by clothianidin (44%), thiamethoxam (22%), acetamiprid (2%), and dinotefuran (1%). Thiacloprid was not detected in any samples. The maximum concentration for an individual neonicotinoid was 230 ng L -1 and the maximum total neonicotinoids in an individual sample was 400 ng L -1 . The median detected individual neonicotinoid concentrations ranged from non-detect to 10 ng L -1 . The detections of clothianidin and thiamethoxam significantly increased as the percent of cultivated crops in the basins increased (ρ = 0.73, P = .01; ρ = 0.66, P = .04, respectively). In contrast, imidacloprid detections significantly increased as the percent of the urbanization in the basins increased (ρ = 0.66, P = .03). Neonicotinoid concentrations generally increased in spring through summer coinciding with the planting of neonicotinoid-treated seeds and broadcast applications of neonicotinoids. More spatially intensive samples were collected in an agriculturally dominated basin (8 sites along the Maumee River, Ohio) twice during the spring, 2016 planting season to provide further information on neonicotinoid inputs to the Great Lakes. Three neonicotinoids were ubiquitously detected (clothianidin, imidacloprid, thiamethoxam) in all water samples collected within this basin. Maximum individual neonicotinoid concentrations was 330 ng L -1 and maximum total neonicotinoid concentration was 670 ng L -1 ; median detected individual neonicotinoid concentrations were 7.0 to 39 ng L -1 ., (Published by Elsevier Ltd.)

Seed coatings are a treatment used on a variety of crops to improve production and offer protection against pests and fungal outbreaks. The leaching of the active ingredients associated with the seed coatings and the sorption to soil was evaluated under laboratory conditions using commercially available corn and soybean seeds to study the fate and transport of these pesticides under controlled conditions. The active ingredients (AI) included one neonicotinoid insecticide (thiamethoxam) and five fungicides (azoxystrobin, fludioxonil, metalaxyl, sedaxane thiabendazole). An aqueous leaching experiment was conducted with treated corn and soybean seeds. Leaching potential was a function of solubility and seed type. The leaching of fludioxonil, was dependent on seed type with a shorter time to equilibrium on the corn compared to the soybean seeds. Sorption experiments with the treated seeds and a solution of the AIs were conducted using three different soil types. Sorption behavior was a function of soil organic matter as well as seed type. For most AIs, a negative relationship was observed between the aqueous concentration and the log K oc . Sorption to all soils tested was limited for the hydrophilic pesticides thiamethoxam and metalaxyl. However, partitioning for the more hydrophobic fungicides was dependent on both seed type and soil properties. The mobility of fludioxonil in the sorption experiment varied by seed type indicating that the adjuvants associated with the seed coating could potentially play a role in the environmental fate of fludioxonil. This is the first study to assess, under laboratory conditions, the fate of pesticides associated with seed coatings using commercially available treated seeds. This information can be used to understand how alterations in agricultural practices (e.g., increasing use of seed treatments) can impact the exposure (concentration and duration) and potential effects of these chemicals to aquatic and terrestrial organisms.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity., (© 2017 Wiley Periodicals, Inc.)

We have developed a rapid, multi-compound analytical method for measuring residues of the pesticides thiamethoxam and its metabolite, clothianidin; fipronil and its three metabolites, fipronil sulfone, fipronil sulfide, and fipronil desulfinyl; and pyraclostrobin in unprocessed foods (rice, corn, cucumbers, tomatoes, apples, and bananas) by ultra-performance liquid chromatography coupled to tandem mass spectrometry. Acetonitrile was used as the extraction solvent, and an octadecylsilane-dispersive SPE was used to clean up the analytes, which were then separated through a UPLC HSS T3 column connected to a tandem mass spectrometer via an electrospray ionisation source. The linearity of this method for the target analytes was excellent (R 2  ≥0.990) in the concentration range of 5-1000 μg kg -1 . The average recoveries of the seven compounds at concentrations of 10, 100, and 1000 μg kg -1 from six spiked matrix samples ranged from 73.6 to 110.6%, all with RSD values of ≤19.7%. The limit of quantification was 10 μg kg -1 . The method validated the effectiveness of the method for routine monitoring the residue of these pesticides and their metabolites in foods.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

Hops used in the brewing process of beer for flavoring are known to contain estrogen active compounds (EAC) and to be the source of EAC in beer. The recently developed planar yeast estrogen screen (pYES) with the substrate resorufin-β-d-galactopyranoside (RGP) successfully was applied for the detection of EAC in ethanolic extracts of hops pellet samples. The only pYES positive compound was identified as the hop flavanone prenylnaringenin (PN) by thin-layer chromatography-mass spectrometry. The heat-induced formation of estrogen active PN from the inactive hop flavonoid desmethylxanthohumol was confirmed by simulation of wort boiling, extraction of both the hops' remainder and the supernatant water, and subsequent investigation of the extracts by pYES. By means of the dose-response curve of PN of a hops' remainder extract, the estradiol equivalent concentration (EEQ) and thus the estradiol equivalent amount (EEA) of PN in the hops' remainder after simulation of the wort boiling was determined to 39 μg/L and 52 μg/kg, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim was to assess the ability of microcosms (laboratory-scale shallow ponds) as a post polishing stage for the remediation of artificial textile wastewater comprising two commercial dyes (basic red 46 (BR46) and reactive blue 198 (RB198)) as a mixture. The objectives were to evaluate the impact of Lemna minor L. (common duckweed) on the water quality outflows; the elimination of dye mixtures, organic matter, and nutrients; and the impact of synthetic textile wastewater comprising dye mixtures on the L. minor plant growth. Three mixtures were prepared providing a total dye concentration of 10 mg/l. Findings showed that the planted simulated ponds possess a significant (p < 0.05) potential for improving the outflow characteristics and eliminate dyes, ammonium-nitrogen (NH 4 -N), and nitrate-nitrogen (NO 3 -N) in all mixtures compared with the corresponding unplanted ponds. The removal of mixed dyes in planted ponds was mainly due to phyto-transformation and adsorption of BR46 with complete aromatic amine mineralisation. For ponds containing 2 mg/l of RB198 and 8 mg/l of BR46, removals were around 53%, which was significantly higher than those for other mixtures: 5 mg/l of RB198 and 5 mg/l of BR46 and 8 mg/l of RB198 and 2 mg/l of BR46 achieved only 41 and 26% removals, respectively. Dye mixtures stopped the growth of L. minor, and the presence of artificial wastewater reduced their development.

Growing evidence for global pollinator decline is causing concern for biodiversity conservation and ecosystem services maintenance. Neonicotinoid pesticides have been identified or suspected as a key factor responsible for this decline. We assessed the global exposure of pollinators to neonicotinoids by analyzing 198 honey samples from across the world. We found at least one of five tested compounds (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) in 75% of all samples, 45% of samples contained two or more of these compounds, and 10% contained four or five. Our results confirm the exposure of bees to neonicotinoids in their food throughout the world. The coexistence of neonicotinoids and other pesticides may increase harm to pollinators. However, the concentrations detected are below the maximum residue level authorized for human consumption (average ± standard error for positive samples: 1.8 ± 0.56 nanograms per gram)., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In order to characterize the metabolic transformation of thiamethoxam (TMX) to clothianidin (CLO) in Helicoverpa armigera larvae and clarify its relationship with the insecticidal toxicity of TMX, method for determination of TMX and its metabolite clothianidin (CLO) residues in H. armigera larvae by solid phase extraction (SPE) combined UPLC-MS/MS was established. Following acetonitrile extraction and purification by SPE on florisil cartridge and C 18 cartridge sequently, and cleanup by PSA adsorption, TMX and CLO residues in H. armigera larvae were successfully determined by UPLC-MS/MS. By using the established method, the concentration-time curves of TMX and its metabolite CLO in H. armigera larvae in vivo and metabolism of TMX by microsome of H. armigera larvae midguts in vitro were studied. TMX was quickly eliminated from H. armigera larvae with the elimination half-life as 4.2h. Meanwhile, only a small amount of CLO was formed from TMX metabolism, with the maximum CLO level in H. armigera larvae only accounts for the metabolic transformation of 7.99% of TMX, at 10h after intravenous TMX administration. Our results suggested that the low insecticidal efficacy of TMX against H. armigera larvae was related with the rapidly elimination of TMX from H. armigera larvae, meanwhile, CLO as TMX metabolite at a very low level in vivo didn't contribute to TMX toxicity to H. armigera larvae. In H. armigera larvae, TMX didn't act as proinsecticide for CLO in insecticidal efficacy of TMX., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Garden centres frequently market nectar- and pollen-rich ornamental plants as "pollinator-friendly", however these plants are often treated with pesticides during their production. There is little information on the nature of pesticide residues present at the point of purchase and whether these plants may actually pose a threat to, rather than benefit, the health of pollinating insects. Using mass spectrometry analyses, this study screened leaves from 29 different 'bee-friendly' plants for 8 insecticides and 16 fungicides commonly used in ornamental production. Only two plants (a Narcissus and a Salvia variety) did not contain any pesticide and 23 plants contained more than one pesticide, with some species containing mixtures of 7 (Ageratum houstonianum) and 10 (Erica carnea) different agrochemicals. Neonicotinoid insecticides were detected in more than 70% of the analysed plants, and chlorpyrifos and pyrethroid insecticides were found in 10% and 7% of plants respectively. Boscalid, spiroxamine and DMI-fungicides were detected in 40% of plants. Pollen samples collected from 18 different plants contained a total of 13 different pesticides. Systemic compounds were detected in pollen samples at similar concentrations to those in leaves. However, some contact (chlorpyrifos) and localised penetrant pesticides (iprodione, pyroclastrobin and prochloraz) were also detected in pollen, likely arising from direct contamination during spraying. The neonicotinoids thiamethoxam, clothianidin and imidacloprid and the organophosphate chlorpyrifos were present in pollen at concentrations between 6.9 and 81 ng/g and at levels that overlap with those known to cause harm to bees. The net effect on pollinators of buying plants that are a rich source of forage for them but simultaneously risk exposing them to a cocktail of pesticides is not clear. Gardeners who wish to gain the benefits without the risks should seek uncontaminated plants by growing their own from seed, plant-swapping or by buying plants from an organic nursery., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

By recording the full fluorescence spectra and super-resolved positions of ∼10 6 individual polarity-sensing solvatochromic molecules, we reveal compositional heterogeneity in the membranes of live mammalian cells with single-molecule sensitivity and ∼30 nm spatial resolution. This allowed us to unveil distinct polarity characteristics of the plasma membrane and the membranes of nanoscale intracellular organelles, a result we found to be due to differences in cholesterol levels. Within the plasma membrane, we observed the formation of low-polarity, raft-like nanodomains upon cholesterol addition or cholera-toxin treatment, but found this nanoscale phase separation absent in native cells. The ultimate sensitivity achieved through examining the spectra of individual molecules thus opens the door to functional interrogations of intracellular physicochemical parameters at the nanoscale.

A rapid monitoring platform for sensitive voltammetric detection of thiamethoxam residues is reported in the present study. A β-cyclodextrin-reduced graphene oxide composite was used as a reinforcing material in electrochemical determination of thiamethoxam. Compared with bare glassy carbon electrodes, the reduction peak currents of thiamethoxam at reduced graphene oxide/glassy carbon electrode and β-cyclodextrin-reduced graphene oxide/glassy carbon electrode were increased by 70- and 124-fold, respectively. The experimental conditions influencing voltammetric determination of thiamethoxam, such as the amount of β-cyclodextrin-reduced graphene oxide, solution pH, temperature, and accumulation time, were optimized. The reduction mechanism and binding affinity of this material is also discussed. Under optimal conditions, the reduction peak currents increased linearly between 0.5 µM and 16 µM concentration of thiamethoxam. The limit of detection was 0.27 µM on the basis of a signal-to-noise ratio of 3. When the proposed method was applied to brown rice in a recovery test, the recoveries were between 92.20% and 113.75%. The results were in good concordance with the high-performance liquid chromatography method. The proposed method therefore provides a promising and effective platform for sensitive and rapid determination of thiamethoxam. Environ Toxicol Chem 2017;36:1991-1997. © 2017 SETAC., (© 2017 SETAC.)

We assessed whether resazurin was as efficient as XTT in the measurement of the metabolic activity of 209 clinical Staphylococcus aureus biofilm using an vitro model comparing the percentage of formazan and resorufin. The overall categorical agreement was 61.2% (r=0.024), which means that resazurin can not substitute XTT., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Streptococcus pneumoniae is a major human pathogen, causing around 1.6 million deaths worldwide each year. By optimizing a resazurin-based assay to detect S. pneumoniae growth in 384-well microplates, we developed a new high-throughput screening (HTS) system for the discovery of antipneumococcal molecules, which was unsuccessful using conventional absorbance measurements. Before applying our protocol to a large-scale screen, we validated the system through a pilot screen targeting about 7,800 bioactive molecules using three different S. pneumoniae serotypes. Primary screenings of a further 27,000 synthetic small molecules facilitated the identification of 3-acyl-2-phenylamino-1,4-dihydropquinolin-4-one (APDQ) derivatives that inhibited growth of S. pneumoniae with MIC 90 values <1 μM (0.03-0.81 μM). Five selected APDQ derivatives were also active against Staphylococcus aureus but neither Klebsiella pneumoniae nor Pseudomonas aeruginosa, suggesting that APDQ may act specifically against Gram-positive bacteria. Our results both validated and demonstrated the utility of the resazurin-based HTS system for the identification of new antipneumococcal molecules. Moreover, the identified new antipneumococcal molecules in this study may have potential to be further developed as new antibiotics.

Uptake and translocation of imidacloprid (IMI), thiamethoxam (THX) and difenoconazole (DFZ) in rice plants (Oryza sativa L.) were investigated with a soil-treated experiment at two application rates: field rate (FR) and 10*FR under laboratory conditions. The dissipation of the three compounds in soil followed the first-order kinetics and DFZ showed greater half-lives than IMI and THX. Detection of the three compounds in rice tissues indicated that rice plants could take up and accumulate these pesticides. The concentrations of IMI and THX detected in leaves (IMI, 10.0 and 410 mg/kg dw; THX, 23.0 and 265 mg/kg dw) were much greater than those in roots (IMI, 1.37 and 69.3 mg/kg dw; THX, 3.19 and 30.6 mg/kg dw), which differed from DFZ. The DFZ concentrations in roots (15.6 and 79.1 mg/kg dw) were much greater than those in leaves (0.23 and 3.4 mg/kg dw). The bioconcentration factor (BCF), representing the capability of rice to accumulate contaminants from soil into plant tissues, ranged from 1.9 to 224.3 for IMI, from 2.0 to 72.3 for THX, and from 0.4 to 3.2 for DFZ at different treated concentrations. Much higher BCFs were found for IMI and THX at 10*FR treatment than those at FR treatment, however, the BCFs of DFZ at both treatments were similar. The translocation factors (TFs), evaluating the capability of rice to translocate contaminants from the roots to the aboveground parts, ranged from 0.02 to 0.2 for stems and from 0.02 to 9.0 for leaves. The tested compounds were poorly translocated from roots to stems, with a TF below 1. However, IMI and THX were well translocated from roots to leaves. Clothianidin (CLO), the main metabolite of THX, was detected at the concentrations from 0.02 to 0.5 mg kg -1 in soil and from 0.07 to 7.0 mg kg -1 in plants. Concentrations of CLO in leaves were almost 14 times greater than those in roots at 10*FR treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue ® (resazurin) or by direct optical measurement of cell growth. Both methods can be miniaturized to allow screening of large numbers of samples, and can be performed using S. cerevisiae in 384 and 1536-well format. The alamarBlue ® approach achieves Z' values of >0.7 with signal to basal ratios of >6.5, and around 1.1 million low molecular weight compounds were screened, identifying approximately 25,000 primary hits. Dose response curves generated for a subset (1930) using both alamarBlue ® and optical density methods showed significant overlap. In genome-wide haploinsufficiency profiling (HIP), 572 of these hits demonstrated a diverse mechanism of action, affecting >25% of all yeast strains., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC 90 values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals., (Copyright © 2017 American Society for Microbiology.)

Numerous cellular and extracellular components should be analyzed in sections of atherosclerotic plaques to assess atherosclerosis progression and vulnerability. Here, we combined orcein (O) staining for elastic fibers and martius scarlet blue (MSB) polychrome to visualize various morphological contents of plaque in brachiocephalic arteries (BCA) of apoE/LDLR -/- mice. Elastic fibers (including broken elastic laminae and 'buried' fibrous caps) were stained purple and they could be easily distinguished from collagen fibers (blue). Orcein allowed clear identification of even the finest elastic fibers. Erythrocytes were stained yellow and they could easily be discerned from mature fibrin (red). Old fibrin tends to acquire blue color. The method of OMSB staining is simple, takes less than 1 h to perform and can be adapted to automatic stainers. Most importantly, the color separation is good enough to allow digital automatic segmentation of specific components in tissue section and quantitative analysis of the plaque constituents. OMSB was used to compare atherosclerotic plaques in proximal and distal regions of BCA in apoE/LDLR -/- mice. In conclusion, OMSB staining represents a novel staining that could be routinely used for qualitative and quantitative microscopic assessments of formaldehyde-fixed and paraffin-embedded sections of arteries with atherosclerotic lesions.

The strategy of formulating poorly soluble actives as nanosuspension has been explored by more than a thousand research papers, with some medicinal products for oral and intravenous use having reached the market or advanced clinical trials. Interestingly, there is a limited number of reports of nanosuspensions formulated for dermal and transdermal drug delivery. In the present work, a nanocrystals suspension of the fluorescent, water-insoluble dye Nile Red, is prepared through a media milling technique and exploited to characterize the fate of the nanosuspended drug when applied on the skin. More in detail, the accumulation of Nile Red nanocrystals inside the hair follicles is evidenced by scanning electron microscopy, and the diffusion of drug molecules in the different skin layers is evaluated by confocal microscopy and skin permeation studies. Overall, the combination of the analytical techniques provide a description of the mechanisms underlying dermal accumulation, and transdermal penetration of a drug formulated as a nanosuspension., (Copyright © 2017 Elsevier B.V. All rights reserved.)