High resolution mass spectrometry for quantitative analysis and untargeted screening of algal toxins in mussels and passive samplers

Measurement of marine algal toxins has traditionally focussed on shellfish monitoring while, over the last decade, passive sampling has been introduced as a complementary tool for exploratory studies. Since 2011, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been adopted as the EU reference method (No.15/2011) for detection and quantitation of lipophilic toxins. Traditional LC-MS approaches have been based on low-resolution mass spectrometry (LRMS), however, advances in instrument platforms have led to a heightened interest in the use of high-resolution mass spectrometry (HRMS) for toxin detection. This work describes the use of HRMS in combination with passive sampling as a progressive approach to marine algal toxin surveys. Experiments focused on comparison of LRMS and HRMS for determination of a broad range of toxins in shellfish and passive samplers. Matrix effects are an important issue to address in LC-MS; therefore, this phenomenon was evaluated for mussels (Mytilus galloprovincialis) and passive samplers using LRMS (triple quadrupole) and HRMS (quadrupole time-of-flight and Orbitrap) instruments. Matrix-matched calibration solutions containing okadaic acid and dinophysistoxins, pectenotoxin, azaspiracids, yessotoxins, domoic acid, pinnatoxins, gymnodimine A and 13-desmethyl spirolide C were prepared. Similar matrix effects were observed on all instruments types. Most notably, there was ion enhancement for pectenotoxins, okadaic acid/dinophysistoxins on one hand, and ion suppression for yessotoxins on the other. Interestingly, the ion selected for quantitation of PTX2 also influenced the magnitude of matrix effects, with the sodium adduct typically exhibiting less susceptibility to matrix effects than the ammonium adduct. As expected, mussel as a biological matrix, quantitatively produced significantly more matrix effects than passive sampler extracts, irrespective of toxin. Sample dilution was demonstrated as an effective measure to reduce matrix effects for all compounds, and was found to be particularly useful for the non-targeted approach. Limits of detection and method accuracy were comparable between the systems tested, demonstrating the applicability of HRMS as an effective tool for screening and quantitative analysis. HRMS offers the advantage of untargeted analysis, meaning that datasets can be retrospectively analysed. HRMS (full scan) chromatograms of passive samplers yielded significantly less complex data sets than mussels, and were thus more easily screened for unknowns. Consequently, we recommend the use of HRMS in combination with passive sampling for studies investigating emerging or hitherto uncharacterised toxins.

Obtenção de extratos de agaricus bisporus com ergosterol para incorporação em iogurtes e procedimentos de microencapsulação

Além de ser o cogumelo mais consumido no mundo, Agaricus bisporus é um dos cogumelos mais ricos em ergosterol, representando esta molécula quase 90% da sua fração de esteróis. Vários estudos têm atribuído ao ergosterol diferentes bioatividades, incluindo efeitos hipocolesterolémicos semelhantes aos exibidos pelos fitoesteróis. Isto torna o ergosterol uma molécula interessante para ser estudada como composto nutracêutico. Assim, este trabalho teve como objetivo avaliar o potencial de utilização dos extratos de A. bisporus ricos em ergosterol na produção de bebidas lácteas funcionais. Para o efeito, foram realizados testes de incorporação do extrato e do ergosterol puro em iogurtes que se compararam com bebidas lácteas funcionais comerciais (aditivadas com fitoesteróis). As amostras de A. bisporus foram submetidas a uma extração assistida por ultrassons e os extratos obtidos (IEXT), bem como a molécula de ergosterol em diferentes concentrações (IERG1 e IERG2), foram incorporados em iogurtes, e comparadas com amostras controlo (amostras de iogurte sem aditivos) (ICN) e iogurtes comerciais contendo fitoesteróis (ICP). Todas as amostras foram analisadas imediatamente após a incorporação (T0), e após sete dias de armazenagem a 4°C (T1), em relação aos parâmetros nutricionais, atividade antioxidante e propriedades citotóxicas em linhas celulares tumorais humanas e numa cultura primária de células de fígado de porco (não tumoral) para avaliação da toxicidade. O teor de ergosterol incorporado na forma pura, ou presente nos extratos, foi monitorizado por HPLC-UV. Adicionalmente, foi realizado um estudo de microencapsulação utilizando a técnica de coacervação, tendo o quitosano e o isolado proteico de soro como materiais encapsulantes. Num ensaio preliminar determinou-se o pH conducente a um maior rendimento de encapsulação e, seguidamente, verificou-se a influência da razão proteína:quitosano (P/Q) e da temperatura utilizada, no rendimento de encapsulação (Y1), na eficiência de encapsulação (Y2) e na carga (teor de ergosterol nas microesferas) (Y3). Posteriormente, o estudo foi realizado baseando-se nas melhores condições para encapsular ergosterol, sendo também avaliadas as respostas Y1, Y2 e Y3. Além de ser o cogumelo mais consumido no mundo, Agaricus bisporus é um dos cogumelos mais ricos em ergosterol, representando esta molécula quase 90% da sua fração de esteróis. Vários estudos têm atribuído ao ergosterol diferentes bioatividades, incluindo efeitos hipocolesterolémicos semelhantes aos exibidos pelos fitoesteróis. Isto torna o ergosterol uma molécula interessante para ser estudada como composto nutracêutico. Assim, este trabalho teve como objetivo avaliar o potencial de utilização dos extratos de A. bisporus ricos em ergosterol na produção de bebidas lácteas funcionais. Para o efeito, foram realizados testes de incorporação do extrato e do ergosterol puro em iogurtes que se compararam com bebidas lácteas funcionais comerciais (aditivadas com fitoesteróis). As amostras de A. bisporus foram submetidas a uma extração assistida por ultrassons e os extratos obtidos (IEXT), bem como a molécula de ergosterol em diferentes concentrações (IERG1 e IERG2), foram incorporados em iogurtes, e comparadas com amostras controlo (amostras de iogurte sem aditivos) (ICN) e iogurtes comerciais contendo fitoesteróis (ICP). Todas as amostras foram analisadas imediatamente após a incorporação (T0), e após sete dias de armazenagem a 4°C (T1), em relação aos parâmetros nutricionais, atividade antioxidante e propriedades citotóxicas em linhas celulares tumorais humanas e numa cultura primária de células de fígado de porco (não tumoral) para avaliação da toxicidade. O teor de ergosterol incorporado na forma pura, ou presente nos extratos, foi monitorizado por HPLC-UV. Adicionalmente, foi realizado um estudo de microencapsulação utilizando a técnica de coacervação, tendo o quitosano e o isolado proteico de soro como materiais encapsulantes. Num ensaio preliminar determinou-se o pH conducente a um maior rendimento de encapsulação e, seguidamente, verificou-se a influência da razão proteína:quitosano (P/Q) e da temperatura utilizada, no rendimento de encapsulação (Y1), na eficiência de encapsulação (Y2) e na carga (teor de ergosterol nas microesferas) (Y3). Posteriormente, o estudo foi realizado baseando-se nas melhores condições para encapsular ergosterol, sendo também avaliadas as respostas Y1, Y2 e Y3. As bebidas funcionalizadas com o extrato (IEXT) e com ergosterol na mesma concentração existente no extrato (IERG1) revelaram uma atividade antioxidante similar às bebidas comerciais com fitoesteróis. No entanto, as bebidas com ergosterol na mesma concentração do extrato de A. bisporus e de fitoesteróis (IERG2) revelaram uma atividade antioxidante superior. Além disso, apenas IEXT, IERG1 e IERG2 apresentaram um aumento na atividade antioxidante de T0 para T1, com destaque para a atividade exibida por IERG2, significando que o ergosterol e os extratos foram capazes de proteger a bebida láctea da oxidação, aumentando a vida de prateleira do produto. IERG2 foi a amostra que revelou a maior citotoxicidade para as linhas celulares tumorais, enquanto as bebidas com fitoesteróis mostraram a menor atividade, sem diferenças significativas entre T0 e T1. Os estudos de microencapsulação revelaram ainda que a técnica de coacervação permite obter cápsulas de distintos tamanhos e que as condições ótimas do processo ocorrem a pH 5,5, com temperatura de 55ºC e razão P/Q de 0,5, com um menor rendimento de encapsulação, mas com uma maior carga em ergosterol. Este trabalho contribuiu para o estudo do potencial da utilização de extratos de A. bisporus com ergosterol no desenvolvimento de novas bebidas funcionais. Constituiu um primeiro passo que necessita de estudos subsequentes relacionados com a avaliação da viabilidade da sua utilização ao nível industrial e demonstração clara da sua bioatividade in vivo.

Toxin and Growth Responses of the Neurotoxic Dinoflagellate Vulcanodinium rugosum to Varying Temperature and Salinity

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m−2 s−1. V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d−1) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts.

Areal rainfall estimation using moving cars - computer experiments including hydrological modeling

The need for high temporal and spatial resolution precipitation data for hydrological analyses has been discussed in several studies. Although rain gauges provide valuable information, a very dense rain gauge network is costly. As a result, several new ideas have been emerged to help estimating areal rainfall with higher temporal and spatial resolution. Rabiei et al. (2013) observed that moving cars, called RainCars (RCs), can potentially be a new source of data for measuring rainfall amounts. The optical sensors used in that study are designed for operating the windscreen wipers and showed promising results for rainfall measurement purposes. Their measurement accuracy has been quantified in laboratory experiments. Considering explicitly those errors, the main objective of this study is to investigate the benefit of using RCs for estimating areal rainfall. For that, computer experiments are carried out, where radar rainfall is considered as the reference and the other sources of data, i.e. RCs and rain gauges, are extracted from radar data. Comparing the quality of areal rainfall estimation by RCs with rain gauges and reference data helps to investigate the benefit of the RCs. The value of this additional source of data is not only assessed for areal rainfall estimation performance, but also for use in hydrological modeling. The results show that the RCs considering measurement errors derived from laboratory experiments provide useful additional information for areal rainfall estimation as well as for hydrological modeling. Even assuming higher uncertainties for RCs as obtained from the laboratory up to a certain level is observed practical.

MAGIC observations of the February 2014 flare of 1ES 1011+496 and ensuing constraint of the EBL density

Context. In February-March 2014, the MAGIC telescopes observed the high-frequency peaked BL Lac 1ES 1011+496 (z=0.212) in flaring state at very-high energy (VHE, E>100GeV). The flux reached a level more than 10 times higher than any previously recorded flaring state of the source. Aims. Description of the characteristics of the flare presenting the light curve and the spectral parameters of the night-wise spectra and the average spectrum of the whole period. From these data we aim at detecting the imprint of the Extragalactic Background Light (EBL) in the VHE spectrum of the source, in order to constrain its intensity in the optical band. Methods. We analyzed the gamma-ray data from the MAGIC telescopes using the standard MAGIC software for the production of the light curve and the spectra. For the constraining of the EBL we implement the method developed by the H.E.S.S. collaboration in which the intrinsic energy spectrum of the source is modeled with a simple function (< 4 parameters), and the EBL-induced optical depth is calculated using a template EBL model. The likelihood of the observed spectrum is then maximized, including a normalization factor for the EBL opacity among the free parameters. Results. The collected data allowed us to describe the flux changes night by night and also to produce di_erential energy spectra for all nights of the observed period. The estimated intrinsic spectra of all the nights could be fitted by power-law functions. Evaluating the changes in the fit parameters we conclude that the spectral shape for most of the nights were compatible, regardless of the flux level, which enabled us to produce an average spectrum from which the EBL imprint could be constrained. The likelihood ratio test shows that the model with an EBL density 1:07 (-0.20,+0.24)stat+sys, relative to the one in the tested EBL template (Domínguez et al. 2011), is preferred at the 4:6 σ level to the no-EBL hypothesis, with the assumption that the intrinsic source spectrum can be modeled as a log-parabola. This would translate into a constraint of the EBL density in the wavelength range [0.24 μm,4.25 μm], with a peak value at 1.4 μm of λF_ = 12:27^(+2:75)_ (-2:29) nW m^(-2) sr^(-1), including systematics.

Super-orbital variability of LS I+61 degrees 303 at TeV energies

Context. The gamma-ray binary LS I +61º303 is a well-established source from centimeter radio up to very high energy (VHE; E > 100 GeV). The broadband emission shows a periodicity of ∼26.5 days, coincident with the orbital period. A longer (super-orbital) period of 1667 ± 8 days was proposed from radio variability and confirmed using optical and high-energy (HE; E ¿ 100 MeV) gamma-ray observations. In this paper, we report on a four-year campaign performed by MAGIC together with archival data concentrating on a search for a long-timescale signature in the VHE emission from LS I +61º303. Aims. We focus on the search for super-orbital modulation of the VHE emission, similar to that observed at other energies, and on the search for correlations between TeV emission and an optical determination of the extension of the circumstellar disk. Methods. A four-year campaign has been carried out using the MAGIC telescopes. The source was observed during the orbital phases when the periodic VHE outbursts have occurred (φ = 0.55 – 0.75, one orbit = 26.496 days). Additionally, we included archival MAGIC observations and data published by the VERITAS collaboration in these studies. For the correlation studies, LS I +61◦303 has also been observed during the orbital phases where sporadic VHE emission had been detected in the past (φ = 0.75 – 1.0). These MAGIC observations were simultaneous with optical spectroscopy from the LIVERPOOL telescope. Results. The TeV flux of the periodical outburst in orbital phases φ = 0.5 – 0.75 was found to show yearly variability consistent with the long-term modulation of ∼4.5 years found in the radio band. This modulation of the TeV flux can be well described by a sine function with a best-fit period of 1610±58 days. The complete data, including archival observations, span two super-orbital periods. There is no evidence for a correlation between the TeV emission and the mass-loss rate of the Be star, but this may be affected by the strong, short-timescale (as short as intra-day) variation displayed by the Hα fluxes.

Multiwavelength observations of the blazar 1ES 1011+496 in Spring 2008

The BL Lac object 1ES 1011+496 was discovered at Very High Energy (VHE, E>100GeV) γ-rays by MAGIC in spring 2007. Before that the source was little studied in different wavelengths. Therefore a multi-wavelength (MWL) campaign was organized in spring 2008. Along MAGIC, the MWL campaign included the Mets¨ahovi radio observatory, Bell and KVA optical telescopes and the Swift and AGILE satellites. MAGIC observations span from March to May, 2008 for a total of 27.9 hours, of which 19.4 hours remained after quality cuts. The light curve showed no significant variability yielding an integral flux above 200 GeV of (1.3 ± 0.3) × 10^(−11) photons cm^(−2) s^( −1) . The differential VHE spectrum could be described with a power-law function with a spectral index of 3.3 ± 0.4. Both results were similar to those obtained during the discovery. Swift XRT observations revealed an X-ray flare, characterized by a harder-when-brighter trend, as is typical for high synchrotron peak BL Lac objects (HBL). Strong optical variability was found during the campaign, but no conclusion on the connection between the optical and VHE γ-ray bands could be drawn. The contemporaneous SED shows a synchrotron dominated source, unlike concluded in previous work based on non-simultaneous data, and is well described by a standard one–zone synchrotron self–Compton model. We also performed a study on the source classification. While the optical and X-ray data taken during our campaign show typical characteristics of an HBL, we suggest, based on archival data, that 1ES 1011+496 is actually a borderline case between intermediate and high synchrotron peak frequency BL Lac objects.

Insights into the emission of the blazar 1ES 1011+496 through unprecedented broadband observations during 2011 and 2012

Context. 1ES 1011+496 (z = 0.212) was discovered in very high-energy (VHE, E >100 GeV) γ rays with MAGIC in 2007. The absence of simultaneous data at lower energies led to an incomplete characterization of the broadband spectral energy distribution (SED). Aims. We study the source properties and the emission mechanisms, probing whether a simple one-zone synchrotron self-Compton (SSC) scenario is able to explain the observed broadband spectrum. Methods. We analyzed data in the range from VHE to radio data from 2011 and 2012 collected by MAGIC, Fermi-LAT, Swift, KVA, OVRO, and Metsähovi in addition to optical polarimetry data and radio maps from the Liverpool Telescope and MOJAVE. Results. The VHE spectrum was fit with a simple power law with a photon index of 3.69 ± 0.22 and a flux above 150 GeV of (1.46±0.16)×10^(−11) ph cm^(−2) s^(−1) . The source 1ES 1011+496 was found to be in a generally quiescent state at all observed wavelengths, showing only moderate variability from radio to X-rays. A low degree of polarization of less than 10% was measured in optical, while some bright features polarized up to 60% were observed in the radio jet. A similar trend in the rotation of the electric vector position angle was found in optical and radio. The radio maps indicated a superluminal motion of 1.8 ± 0.4 c, which is the highest speed statistically significant measured so far in a high-frequency-peaked BL Lac. Conclusions. For the first time, the high-energy bump in the broadband SED of 1ES 1011+496 could be fully characterized from 0.1 GeV to 1 TeV, which permitted a more reliable interpretation within the one-zone SSC scenario. The polarimetry data suggest that at least part of the optical emission has its origin in some of the bright radio features, while the low polarization in optical might be due to the contribution of parts of the radio jet with different orientations of the magnetic field with respect to the optical emission.

A space-time hybrid hourly rainfall model for derived flood frequency analysis

For derived flood frequency analysis based on hydrological modelling long continuous precipitation time series with high temporal resolution are needed. Often, the observation network with recording rainfall gauges is poor, especially regarding the limited length of the available rainfall time series. Stochastic precipitation synthesis is a good alternative either to extend or to regionalise rainfall series to provide adequate input for long-term rainfall-runoff modelling with subsequent estimation of design floods. Here, a new two step procedure for stochastic synthesis of continuous hourly space-time rainfall is proposed and tested for the extension of short observed precipitation time series. First, a single-site alternating renewal model is presented to simulate independent hourly precipitation time series for several locations. The alternating renewal model describes wet spell durations, dry spell durations and wet spell intensities using univariate frequency distributions separately for two seasons. The dependence between wet spell intensity and duration is accounted for by 2-copulas. For disaggregation of the wet spells into hourly intensities a predefined profile is used. In the second step a multi-site resampling procedure is applied on the synthetic point rainfall event series to reproduce the spatial dependence structure of rainfall. Resampling is carried out successively on all synthetic event series using simulated annealing with an objective function considering three bivariate spatial rainfall characteristics. In a case study synthetic precipitation is generated for some locations with short observation records in two mesoscale catchments of the Bode river basin located in northern Germany. The synthetic rainfall data are then applied for derived flood frequency analysis using the hydrological model HEC-HMS. The results show good performance in reproducing average and extreme rainfall characteristics as well as in reproducing observed flood frequencies. The presented model has the potential to be used for ungauged locations through regionalisation of the model parameters.

First metabolomic approach of the epiphytic bacteria-marine diatom Haslea ostrearia relationships

The marine diatom Haslea ostrearia [1] produces a water-soluble blue-pigment named marennine [2] of economic interest. But the lack of knowledge of the ecological conditions, under which this microalga develops in its natural ecosystem, more especially bacteria H. ostrearia interactions, prevents any optimization of its culture in well-controlled conditions. The structure of the bacterial community was analyzed by PCR-TTGE before and after the isolation of H. ostrearia cells recovered from 4 localities, to distinguish the relative part of the biotope and the biocenose and eventually to describe the temporal dynamic of the structure of the bacterial community at two time-scales. The differences in genetic fingerprints, more especially high between two H. ostrearia isolates (HO-R and HO-BM) showed also the highest differences in the bacterial structure [3] as the result of specific metabolomics profiles. The non-targeted metabolomic investigation showed that these profiles were more distinct in case of bacteria-alga associations than for the H. ostrearia monoculture Here we present a Q-TOF LC/MS metabolomic fingerprinting approach [3]: - to investigate differential metabolites of axenic versus non axenic H. ostrearia cultures. - to focus on the specific metabolites of a bacterial surrounding associated with the activation or inhibition of the microalga growing. The Agilent suite of data processing software makes feature finding, statistical analysis, and identification easier. This enables rapid transformation of complex raw data into biologically relevant metabolite information.