Identification of Pesticide Transformation Products in Surface Water Using Suspect Screening Combined with National Monitoring Data.

Pesticides are widespread anthropogenic chemicals and well-known environmental contaminants of concern. Much less is known about transformation products (TPs) of pesticides and their presence in the environment. We developed a novel suspect screening approach for not well-explored pesticides (n = 16) and pesticide TPs (n = 242) by integrating knowledge from national monitoring with high-resolution mass spectrometry data. Weekly time-integrated samples were collected in two Swedish agricultural streams using the novel Time-Integrating, MicroFlow, In-line Extraction (TIMFIE) sampler. The integration of national monitoring data in the screening approach increased the number of prioritized compounds approximately twofold (from 23 to 42). Ultimately, 11 pesticide TPs were confirmed by reference standards and 12 TPs were considered tentatively identified with varying levels of confidence. Semiquantification of the newly confirmed TPs indicated higher concentrations than their corresponding parent pesticides in some cases, which highlights concerns related to (unknown) pesticide TPs in the environment. Some TPs were present in the environment without co-occurrence of their corresponding parent compounds, indicating higher persistency or mobility of the identified TPs. This study showcased the benefits of integrating monitoring knowledge in this type of studies, with advantages for suspect screening performance and the possibility to increase relevance of future monitoring programs.

Seed coating with a neonicotinoid insecticide negatively affects wild bees.

Understanding the effects of neonicotinoid insecticides on bees is vital because of reported declines in bee diversity and distribution and the crucial role bees have as pollinators in ecosystems and agriculture. Neonicotinoids are suspected to pose an unacceptable risk to bees, partly because of their systemic uptake in plants, and the European Union has therefore introduced a moratorium on three neonicotinoids as seed coatings in flowering crops that attract bees. The moratorium has been criticized for being based on weak evidence, particularly because effects have mostly been measured on bees that have been artificially fed neonicotinoids. Thus, the key question is how neonicotinoids influence bees, and wild bees in particular, in real-world agricultural landscapes. Here we show that a commonly used insecticide seed coating in a flowering crop can have serious consequences for wild bees. In a study with replicated and matched landscapes, we found that seed coating with Elado, an insecticide containing a combination of the neonicotinoid clothianidin and the non-systemic pyrethroid ß-cyfluthrin, applied to oilseed rape seeds, reduced wild bee density, solitary bee nesting, and bumblebee colony growth and reproduction under field conditions. Hence, such insecticidal use can pose a substantial risk to wild bees in agricultural landscapes, and the contribution of pesticides to the global decline of wild bees may have been underestimated. The lack of a significant response in honeybee colonies suggests that reported pesticide effects on honeybees cannot always be extrapolated to wild bees.

TIMFIE Sampler-A New Time-Integrating, Active, Low-Tech Sampling Device for Quantitative Monitoring of Pesticides in Whole Water.

The need for inexpensive, time-averaged, quantitative determination of pesticides and other organic pollutants in whole water is not matched by the field sampling procedures available. Our new Time-Integrating, MicroFlow, In-line Extraction (TIMFIE) sampler comprises a low-tech syringe pump driven by a rubber band and connected to a flow restrictor enabling low microliter per minute water flow through a solid phase extraction (SPE) cartridge. This allows target compounds to be continuously extracted in the field over 1 week. The extracted water ends up in the syringe, where sample volume is accurately determined. TIMFIE followed by online SPE-LC-MS/MS determination was validated for 72 selected pesticides, and, except for three compounds, detection limit was 0.1-1 ng/L. In a field study, concentrations in TIMFIE samples and in grab samples were compared. Following TIMFIE sampling, on average 19 pesticides per sample were quantified, compared with nine pesticides per sample with grab sampling, as a result of the extra in-field concentration step. Duplicate TIMFIE sampling showed Pearson's correlation coefficient r = 0.998. Comparing concentrations from TIMFIE sampling to grab sampling resulted in ratios between 0.05 and 16.5 (mean 1.7; r = 0.532), demonstrating a discrepancy between the two sampling strategies and possible underestimation of chronic exposure by grab sampling.

Influence of blood/tissue differences in contrast agent relaxivity on tracer-based MR perfusion measurements.

PURPOSE: Perfusion assessment by monitoring the transport of a tracer bolus depends critically on conversion of signal intensity into tracer concentration. Two main assumptions are generally applied for this conversion; (1) contrast agent relaxivity is identical in blood and tissue, (2) change in signal intensity depends only on the primary relaxation effect. The purpose of the study was to assess the validity and influence of these assumptions. MATERIALS AND METHODS: Blood and cerebral tissue relaxivities r1, r2, and r2* for gadodiamide were measured in four pigs at 1.5 T. Gadolinium concentration was determined by inductively coupled plasma atomic emission spectroscopy. Influence of the relaxivities, secondary relaxation effects and choice of singular value decomposition (SVD) regularization threshold was studied by simulations. RESULTS: In vivo relaxivities relative to blood concentration [in s(-1) mM(-1) for blood, gray matter (GM), white matter (WM)] were for r1 (2.614 ± 1.061, 0.010 ± 0.001, 0.004 ± 0.002), r2 (5.088 ± 0.952, 0.091 ± 0.008, 0.059 ± 0.014), and r2* (13.292 ± 3.928, 1.696 ± 0.157, 0.910 ± 0.139). Although substantial, by a nonparametric test for paired samples, the differences were not statistically significant. The GM to WM blood volume ratio was estimated to 2.6 ± 0.9 by r1, 1.6 ± 0.3 by r2, and 1.9 ± 0.2 by r2*. Secondary relaxation was found to reduce the tissue blood flow, as did the SVD regularization threshold. CONCLUSION: Contrast agent relaxivity is not identical in blood and tissue leading to substantial errors. Further errors are introduced by secondary relaxation effects and the SVD regularization.

Ecological traits interact with landscape context to determine bees' pesticide risk.

Widespread contamination of ecosystems with pesticides threatens non-target organisms. However, the extent to which life-history traits affect pesticide exposure and resulting risk in different landscape contexts remains poorly understood. We address this for bees across an agricultural land-use gradient based on pesticide assays of pollen and nectar collected by Apis mellifera, Bombus terrestris and Osmia bicornis, representing extensive, intermediate and limited foraging traits. We found that extensive foragers (A. mellifera) experienced the highest pesticide risk-additive toxicity-weighted concentrations. However, only intermediate (B. terrestris) and limited foragers (O. bicornis) responded to landscape context-experiencing lower pesticide risk with less agricultural land. Pesticide risk correlated among bee species and between food sources and was greatest in A. mellifera-collected pollen-useful information for future postapproval pesticide monitoring. We provide foraging trait- and landscape-dependent information on the occurrence, concentration and identity of pesticides that bees encounter to estimate pesticide risk, which is necessary for more realistic risk assessment and essential information for tracking policy goals to reduce pesticide risk.

Greenhouse production contributes to pesticide occurrences in Swedish streams.

Greenhouse and other covered cultivation systems have increased globally over the past several decades, leading to considerably improved product quality and productivity per land area unit. However, there is a paucity in information regarding the environmental impacts of covered production systems, especially regarding pesticides entering the surrounding environment. Aiming to address this knowledge gap, we collected grab samples downstream of greenhouses from seven Swedish streams every 14 days during a 12 month period. In three of the streams, samples were also taken upstream of the greenhouses and in four of the streams time-integrated samples were collected by TIMFIE samplers in the period between grab sampling occasions. The samples were analyzed for 28 substances (27 that were permitted for use in greenhouse production systems in Sweden and one degradation product to a permitted substance). Pesticide use journals were collected from the greenhouse producers for the 12 month period. The results were examined for indications of greenhouse contributions to detection frequencies, maximum and average concentrations, and potential ecotoxicicity in several ways: (1) comparing locations downstream of greenhouses with registered use of a substance with those without registered use, (2) comparing results from this study with those from the Swedish environmental monitoring program of pesticides in surface water from catchments with no greenhouses from the same period and region, (3) comparing concentration trends with registered pesticide application times in the greenhouses, and (4) comparing up- and downstream concentrations. The results strongly suggest that greenhouse applications do contribute to pesticide occurrences, maximum and median concentrations for most of the pesticides included in this study, and to potential toxicity to aquatic organisms for several of them, most notably imidacloprid, acetamiprid, carbendazim, and pirimicarb.

Clothianidin seed-treatment has no detectable negative impact on honeybee colonies and their pathogens.

Interactions between multiple stressors have been implicated in elevated honeybee colony losses. Here, we extend our landscape-scale study on the effects of placement at clothianidin seed-treated oilseed rape fields on honeybees with an additional year and new data on honeybee colony development, swarming, mortality, pathogens and immune gene expression. Clothianidin residues in pollen, nectar and honeybees were consistently higher at clothianidin-treated fields, with large differences between fields and years. We found large variations in colony development and microbial composition and no observable negative impact of placement at clothianidin-treated fields. Clothianidin treatment was associated with an increase in brood, adult bees and Gilliamella apicola (beneficial gut symbiont) and a decrease in Aphid lethal paralysis virus and Black queen cell virus - particularly in the second year. The results suggest that at colony level, honeybees are relatively robust to the effects of clothianidin in real-world agricultural landscapes, with moderate, natural disease pressure.

Capillary microsampling in the regulatory environment: validation and use of bioanalytical capillary microsampling methods.

Capillary microsampling (CMS) has recently been introduced as a response to the demands for more ethical use of laboratory animals according to the 3R principles. In CMS, an exact volume of the blood, plasma or other biofluid is collected in a capillary from which it is washed out, resulting in a diluted sample that can be handled using the existing equipment in the bioanalytical laboratory. CMS differs from traditional large volume sampling as the microsample is diluted before further handling and analysis, and reanalysis is performed using the diluted sample. This has some implications for the validation and this report is an attempt to clarify how to validate and use CMS methods in a regulatory environment. CMS also shows some distinct new opportunities: labile analytes can be immediately stabilized at sample collection and the addition of the internal standard to the whole sample can improve analytical performance. The experiences from 5 years use of CMS of plasma and blood for determination of drug exposure in animal studies are reviewed.

Experimental treatment of superior venous congestion during cardiopulmonary bypass.

OBJECTIVES: Superior venous outflow obstruction affects cerebral perfusion negatively by reducing cerebral perfusion pressure (CPP). We present a randomized study designed to compare two alternative strategies to preserve the CPP during superior vena cava (SVC) congestion and cardiopulmonary bypass (CPB). METHODS: Fourteen pigs on bi-caval CPB were subjected to 75% occlusion of the SVC flow. CPP was restored either by vasopressor treatment (VP, n = 7) or by partial relief (PR) of the congestion (n = 7). The cerebral effects of the interventions were studied for 60 min with intracranial pressure (ICP) monitoring, cerebral blood flow measurement, the near-infrared light spectroscopy tissue oxygen saturation index (StO2), arterial and venous blood gas analyses and serial measurements of the glial cell damage marker protein S100ß. RESULTS: Both strategies restored the CPP to baseline levels and no signs of severe ischaemia were observed. In the PR group, the venous and ICPs were normalized in response to the intervention, while in the VP group those parameters remained elevated throughout the experiment. The haemoglobin oxygen saturation in the sagittal sinus (SsagO2) was increased by both VP and PR, while significant improvement in the StO2 was observed only in the PR group. The S100ß concentrations were similar in the two groups. CONCLUSIONS: Experimental SVC obstruction during CPB may reduce the CPP, resulting in impaired cerebral perfusion. Both vasopressor treatment and improved venous drainage can, in the short term, individually restore the CPP during these circumstances.

Capillary microsampling and analysis of 4-µl blood, plasma and serum samples to determine human α-synuclein elimination rate in mice.

BACKGROUND: The capillary microsampling technique was scaled down to enable repeated PK sampling of blood, plasma and serum from mice for the determination of the 14-kDa protein α-synuclein using the Gyrolab™ immunoassay platform. RESULTS: 4-µl plasma, serum or blood samples were taken from 36 mice, in total 648 samples were successfully collected and analyzed. Following intravenous administration of human α-synuclein to mice, the elimination of α-synuclein was rapid, with a half-life in plasma of 1.1 h. High endogenous levels in red blood cells in combination with some hemolysis led to a challenge in the evaluation of α-synuclein exposure in plasma and serum. CONCLUSION: The small sample volumes and flexibility in choice of liquid matrices using the capillary microsampling technique enable repeated sampling in mouse studies, as well as multi-matrix analysis if needed. Liquid microsampling is well suited for micro- and nano-liter scale immunoassays.

Validation of a bioanalytical method using capillary microsampling of 8 µl plasma samples: application to a toxicokinetic study in mice.

BACKGROUND: Capillary microsampling was recently introduced as a new technique for simplified collection and handling of small, exact volumes of liquid matrices. In this article, a bioanalytical method was developed and fully validated for 8 µl plasma samples and applied to a toxicology study in mice. RESULTS: The method was validated in the concentration range 0.06-30 µM. A procedure where 32 µl of blood was collected for preparation of 8 µl plasma was successfully implemented at the animal facility. All the results for the method and study validation met the requirements of a regulated assay. CONCLUSION: It is shown that 8 µl plasma microsamples can be sampled and analyzed with consistently excellent accuracy and precision. Capillary microsampling of plasma offers a possibility to combine ethical, scientific and economic benefits while still maintaining the advantage of having drug exposure data in plasma.

Capillary microsampling of 25 µl blood for the determination of toxicokinetic parameters in regulatory studies in animals.

BACKGROUND: Capillary microsampling (CMS) is a new technique for simplified collection, handling and analysis of small, exact volumes of liquid matrices. CMS was compared with conventional large volume sampling, in toxicology studies in rat and dog. RESULTS: Bioanalytical validation data were well within acceptance limits. Toxicokinetic (TK) parameters from microsampling were in agreement with data from conventional volume sampling. Clinical pathology parameters in rats measured 2 days after repeated microsampling were not affected when compared with rats not sampled. CONCLUSION: The fast collection and simple handling of small, exact volumes of liquid blood makes the CMS technique generic and flexible, as well as easily implemented and automated. Presented data support that TK measurements can be performed in main study rats, instead of dosing additional satellite animals only for TK sampling, giving both a higher scientific value and a substantial reduction of animal numbers in preclinical development.

Minimal safe arterial blood flow during selective antegrade cerebral perfusion at 20° centigrade.

BACKGROUND: Selective antegrade cerebral perfusion (SACP) enables surgery on the aortic arch, where cerebral ischemia may cause neurologic sequels. This study aims to identify the minimum arterial flow level to maintain adequate cerebral perfusion during SACP in deep hypothermia in the pig. METHODS: Two groups of pigs were subjected to SACP at 20(°)C α-stat. In group 1 (n = 6), flow was stepwise adjusted from 8-6-4-2-8 mL · kg(-1) · min(-1) and in group 2 (n = 5), flow was kept constant at 6 mL · kg(-1) · min(-1). Magnetic resonance imaging and spectroscopy were performed at each flow level together with hemodynamic monitoring and blood gas analysis. The biochemical marker of cerebral damage protein S100ß was measured in peripheral blood. RESULTS: Decreased mixed venous oxygen saturation and increased lactate in magnetic resonance spectroscopy was seen as a sign of anaerobic metabolism below 6 mL · kg(-1) · min(-1). No ischemic damage was seen on diffusion-weighted imaging, but the concentrations of S100ß were significantly elevated in group 1 compared with group 2 at the end of the experiment (p < 0.05). Perfusion-weighted imaging showed coherence between flow setting and cerebral perfusion, increase of blood volume across time, and regional differences in perfusion during SACP. CONCLUSIONS: The findings suggest an ischemic threshold close to 6 mL · kg(-1) · min(-1) in the present model. Regional differences in perfusion during SACP may be of pathogenic importance to focal cerebral ischemia.

Venous obstruction and cerebral perfusion during experimental cardiopulmonary bypass.

To investigate the effects on cerebral perfusion by experimental venous congestion of the superior vena cava (SVC) during bicaval cardiopulmonary bypass (CPB) at 34 °C, pigs were subjected to SVC obstruction at levels of 75%, 50%, 25% and 0% of baseline SVC flow at two arterial flow levels (low, LQ, high, HQ). The cerebral perfusion was examined with near-infrared spectroscopy (NIRS), cerebral microdialysis and blood gas analysis. SVC obstruction caused significant decreases in the NIRS tissue oxygenation index (TOI) and in SVC oxygen saturations (P<0.05, both groups), while the mixed venous saturation was decreased only in the LQ group. Sagittal sinus venous saturations were measured in the HQ group and found significantly reduced in response to venous congestion (P<0.05). No microdialysis changes were seen at the group level, however, individual ischemic patterns in terms of concomitant venous desaturation, decreased TOI and increased lactate/pyruvate occurred in both groups. The total venous drainage remained stabile throughout the experiment, indicating increased flow in the inferior vena cava cannula. The results indicate that SVC congestion may impair cerebral perfusion especially in the case of compromised arterial flow during CPB. Reduced SVC cannula flow may pass undetected during bicaval CPB, if SVC flow is not specifically monitored.

Selective antegrade cerebral perfusion at two different temperatures compared to hypothermic circulatory arrest--an experimental study in the pig with microdialysis.

Hypothermic arrest and selective antegrade cerebral perfusion (SACP) is widely used during aortic arch surgery. The microdialysis technique monitors biomarkers of cellular metabolism and cellular integrity over time. In this study, the cerebral changes during hypothermic circulatory arrest (HCA) at 20 degrees C and HCA with SACP at two different temperatures, 20 and 28 degrees C, were monitored. Twenty-three pigs were divided into three groups. A microdialysis probe was fixated into the forebrain. Circulatory arrest started at a brain and body temperature of 20 degrees C or 28 degrees C. Arrest with/without cerebral perfusion (flow 10 ml/kg, max carotid artery pressure 70 mmHg) lasted for 80 min followed by reperfusion and rewarming during 40 min and an observation period of 120 min. The microdialysis markers were registered at six time-points. The lactate/pyruvate ratio (L/P ratio) and the lactate/glucose ratio (L/G ratio) increased significantly (P<0.05), during arrest, in the HCA group. The largest increase of glycerol was found in the group with tepid cerebral perfusion (28 degrees C) and the HCA group (P<0.05). This study supports the use of SACP over arrest. It also suggests that cerebral metabolism and cellular membrane integrity may be better preserved with SACP at 20 degrees C compared to 28 degrees C.

Miniaturized dynamic liquid-liquid extraction of organophosphate triesters from blood plasma using the hollow fibre-based XT-tube extractor.

A miniaturized liquid-liquid extractor for bioanalytical sample preparation is described. The extractor consists of a polypropylene hollow fibre mounted inside polytetrafluoroethylene (PTFE) tubing by means of a cross (X) connector and a tee (T) connector. All parts are commercially available, inexpensive, and easily assembled. The aqueous sample, injected into a carrier flow, is pumped along the outside of the fibre and the organic phase, which also wets the pores of the hollow fibre wall, is pumped inside. Eight organophosphate triester (OPE) plasticisers/flame retardants were extracted from 50 microL spiked blood plasma that had been mixed with 50 microL formic acid to denature plasma proteins. The organic phase was a mixture of hexane and methyl tert-butyl ether (MTBE). A high concentration of formic acid in the sample and of MTBE in the organic phase had positive effects on the recovery of some OPE. When investigating the recovery as a function of extraction time it was found that the extraction reached a maximum after 10 min, at a flow-rate of 15 microL min(-1). Recoveries varied between 40 and 80% with RSD around 4% for most compounds. The whole 150- microL extract was injected into a GC-MS system equipped with a programmed-temperature vaporization (PTV) injector. With the MS in selected-ion monitoring (SIM) mode, the LOD for triphenyl phosphate and 2-ethylhexyl diphenyl phosphate were 0.3 and 0.2 ng mL(-1), respectively. More than 40 plasma extractions were performed with the same fibre without any detectable change in extraction efficiency.

The XT-tube extractor: a hollow fiber-based supported liquid membrane extractor for bioanalytical sample preparation.

A new supported liquid membrane extractor for bioanalytical sample preparation is presented. The extractor consists of a polypropylene hollow fiber mounted inside a PTFE tube by means of a cross-connector and a tee-connector. All parts are commercially available, inexpensive, and easily assembled. An organic solvent in the pores of the fiber forms a liquid membrane that separates the sample, which is pumped along the outside of the fiber, from the acceptor phase, which is pumped inside. The length of the hollow fiber may easily be varied to meet different demands on extractive surface and extract volumes. To test the system, the strongly acidic plasticizer/flame retardant metabolite diphenyl phosphate ester (DPhP), with a pKa value of 0.26, was extracted from urine. DPhP was protonated using 4 M hydrochloric acid and extracted into an acceptor phase at pH 9. Thirty extractions were made with the same liquid membrane without any decrease in extraction efficiency and with a relative standard deviation <7%. An analyte concentration enrichment of 5-10 times was achieved in the extraction step, giving a limit of detection (S/N = 3) of 0.014 microg/mL with LC/ESI-MS and 0.18 microg/mL with CE-UV. The effects on extraction efficiency using different sample pH, organic solvents, sample flow rates, and lengths of the fiber were evaluated.