The analysis of lipophilic marine toxins : development of an alternative method

Lipophilic marine toxins are produced by certain algae species and can accumulate in filter feeding shellfish such as mussels, scallops and oysters. Consumption of contaminated shellfish can lead to severe intoxications such as diarrhea, abdominal cramps and vomiting. Methods described in European Union (EU) legislation to test for the presence of these toxins are based on a mouse or rat bioassay. These assays are unethical and have a poor sensitivity and selectivity. The aim of this thesis is to develop an alternative method based on liquid chromatography - tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of lipophilic marine toxins. LC-MS/MS methods described in literature for the determination of lipophilic marine toxins used an acidic chromatographic system. Under acidic conditions peak shape and separation of a number of toxins preferably analyzed in electrospray ionization negative (ESI–) and positive (ESI+) were poor. A LC-MS/MS method with alkaline chromatographic conditions in which we were able to analyze 28 different toxins in a single analysis in separated retention time windows operating in either ESI– or ESI+ was developed. Furthermore, a clean up procedure based on solid phase extraction (SPE) was developed to reduce the amount of matrix effects (ion suppression and enhancement). A combination of SPE clean up and alkaline chromatographic conditions resulted in reduced matrix effects for all matrices tested (mussel, scallop and oyster). The developed SPE & LC-MS/MS method was in-house validated at regulatory limits based on EU Commission Decision 2002/657/EC. With respect to accuracy, repeatability, reproducibility, decision limit, specificity and ruggedness the method performed well. The method also performed excellently in view of possible new limits that are four- to five-fold lower than current limits for some toxins. Finally a screening method based on LC orbitrap MS was developed for 85 marine toxins of which most are not stated in EU legislation. The screening used in-house developed software which made it possible to reduce the complex data files and screen for a large number of toxins within seconds. This thesis will contribute to the replacement of the animal assays that are still prescribed in EU legislation for the determination of lipophilic marine toxins in shellfish. </p

Nieuwe giftest spaart proefdieren

Oesters en mosselen kunnen van nature een gif bevatten dat door algen in zee ontstaat. Mensen kunnen diarree krijgen als ze zulke schelpdieren eten. Tot nog toe werden er ratten of muizen gebruikt om schelpdieren op de aanwezigheid van dat gif te testen. Arjen Gerssen, onderzoeker bij RIKILT, ontwikkelde een slimmere methode om de aanwezigheid van het gif aan te tonen

p3d: a general data-reduction tool for fiber-fed integral-field spectrographs

The reduction of integral-field spectrograph (IFS) data is demanding work. Many repetitive operations are required in order to convert raw data into, typically a large number of, spectra. This effort can be markedly simplified through the use of a tool or pipeline, which is designed to complete many of the repetitive operations without human interaction. Here we present our semi-automatic data-reduction tool p3d that is designed to be used with fiber-fed IFSs. Important components of p3d include a novel algorithm for automatic finding and tracing of spectra on the detector, and two methods of optimal spectrum extraction in addition to standard aperture extraction. p3d also provides tools to combine several images, perform wavelength calibration and flat field data. p3d is at the moment configured for four IFSs. In order to evaluate its performance we have tested the different components of the tool. For these tests we used both simulated and observational data. We demonstrate that for three of the IFSs a correction for so-called cross-talk due to overlapping spectra on the detector is required. Without such a correction spectra will be inaccurate, in particular if there is a significant intensity gradient across the object. Our tests showed that p3d is able to produce accurate results. p3d is a highly general and freely available tool. It is easily extended to include improved algorithms, new visualization tools and support for additional instruments. The program code can be downloaded from the p3d-project web site http://p3d.sourceforge.netComment: 18 pages, 15 figures, 3 tables, accepted for publication in A&

Observational Constraints on Disk Heating as a Function of Hubble Type

Current understanding of the secular evolution of galactic disks suggests that this process is dominated by two or more heating mechanisms, which increase the random motions of stars in the disk. In particular, the gravitational influence of giant molecular clouds and irregularities in the spiral potential have been proposed to explain the observed velocity dispersions in the solar neighborhood. Each of these mechanisms acts on different components of the stellar velocities, which affects the ratio of the vertical and radial components of the stellar velocity dispersion since the relative strengths of giant molecular clouds and spiral irregularities vary with Hubble type. A study of this ratio as function of Hubble type has the potential to provide strong constraints on disk heating mechanisms. We present major and minor axis stellar kinematics for four spiral galaxies of Hubble type from Sa to Sbc, and use the data to infer the ratios sigma_z/sigma_R in the galaxy disks. The results combined with two galaxies studied previously and with Milky Way data show that the ratio is generally in the range 0.5 - 0.8. There is a marginally significant trend of decreasing ratio with advancing Hubble type, consistent with the predictions of disk heating theories. However, the errors on individual measurements are large, and the absence of any trend is consistent with the data at the 1-sigma level. As a byproduct of our study, we find that three of the four galaxies in our sample have a central drop in their stellar line-of-sight velocity dispersion, a phenomenon that is increasingly observed in spiral galaxies. [ABRIDGED]Comment: 24 pages, LaTeX, 5 Postscript figures, to appear in AJ (Dec 2003

The stellar velocity dispersion in the inner 1.3 disk scale-lengths of the irregular galaxy NGC 4449

We present measurements of the stellar velocity dispersion in the inner 1 arcmin radius (1.3 disk scale-lengths) of the irregular galaxy NGC 4449 determined from long-slit absorption-line spectra. The average observed dispersion is 29 +/-2 km/s, the same as predicted from NGC 4449's luminosity. No significant rotation in the stars is detected. If we assume a maximum rotation speed of the stars from the model determined from the gas kinematics of Hunter et al. (2002), the ratio V_max/sigma_z measured globally is 3. This ratio is comparable to values measured in spiral galaxies, and implies that the stellar disk in NGC 4449 is kinematically relatively cold. The intrinsic minor-to-major axis ratio (b/a)_0 is predicted to be in the range 0.3-0.6, similar to values derived from the distribution of observed b/a of Im galaxies. However, V/sigma_z measured locally is 0.5-1.1, and so the circular velocity of NGC 4449 is comparable or less than the velocity of the stars within the central 1.3 disk scale-lengths of the galaxy.Comment: To be published in ApJ, Nov 200

GMOS IFU observations of the stellar and gaseous kinematics in the centre of NGC 1068

We present a data cube covering the central 10 arcsec of the archetypal active galaxy NGC 1068 over a wavelength range 4200–5400 Å obtained during the commissioning of the integral field unit (IFU) of the Gemini Multi-object Spectrograph (GMOS) installed on the Gemini-North telescope. The data cube shows a complex emission line morphology in the [O iii] doublet and Hβ line. To describe this structure phenomenologically we construct an atlas of velocity components derived from multiple Gaussian component fits to the emission lines. The atlas contains many features which cannot be readily associated with distinct physical structures. While some components are likely to be associated with the expected biconical outflow, others are suggestive of high velocity flows or disc-like structures. As a first step towards interpretation, we seek to identify the stellar disc using kinematical maps derived from the Mg b absorption line feature at 5170 Å and make associations between this and gaseous components in the atlas of emission line components

Hierarchical build-up of galactic bulges and the merging rate of supermassive binary black holes

The hierarchical build-up of galactic bulges should lead to the build-up of present-day supermassive black holes by a mixture of gas accretion and merging of supermassive black holes. The tight relation between black hole mass and stellar velocity dispersion is thereby a strong argument that the supermassive black holes in merging galactic bulges do indeed merge. Otherwise the ejection of supermassive black holes by gravitational slingshot would lead to excessive scatter in this relation. At high redshift the coalescence of massive black hole binaries is likely to be driven by the accretion of gas in the major mergers signposted by optically bright QSO activity. If massive black holes only form efficiently by direct collapse of gas in deep galactic potential wells with v_c > 100 km/s as postulated in the model of Kauffmann & Haehnelt (2000) LISA expects to see event rates from the merging of massive binary black holes of about 0.1-1 yr^{-1} spread over the redshift range 0 < z < 5. If, however, the hierarchical build-up of supermassive black holes extends to pre-galactic structures with significantly shallower potential wells event rates may be as high as 10-100 yr^{-1} and will be dominated by events from redshift z > 5.Comment: 8 pages, 4 postscript figures. Proceedings of the 4th International LISA Symposium, Penn State University, 19-24 July 2002, ed. L S Fin

Mechanisms of the Vertical Secular Heating of a Stellar Disk

We investigate the nonlinear growth stages of bending instability in stellar disks with exponential radial density profiles.We found that the unstable modes are global (the wavelengths are larger than the disk scale lengths) and that the instability saturation level is much higher than that following from a linear criterion. The instability saturation time scales are of the order of one billion years or more. For this reason, the bending instability can play an important role in the secular heating of a stellar disk in the $z$ direction. In an extensive series of numerical $N$-body simulations with a high spatial resolution, we were able to scan in detail the space of key parameters (the initial disk thickness $z_0$, the Toomre parameter $Q$, and the ratio of dark halo mass to disk mass $M_{\rm h} / M_{\rm d}$). We revealed three distinct mechanisms of disk heating in the $z$ direction: bending instability of the entire disk, bending instability of the bar, and heating on vertical inhomogeneities in the distribution of stellar matter.Comment: 22 pages including 8 figures. To be published in Astronomy Letters (v.29, 2003

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 µg kg-1 for OA, AZA1 and PTX2 and 1,000 µg kg-1 for YTX. For SPX1, 400 µg kg-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 µg kg-1 OA equivalents and 30 µg kg-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests

Finding Faint Intermediate-mass Black Holes in the Radio Band

We discuss the prospects for detecting faint intermediate-mass black holes, such as those predicted to exist in the cores of globular clusters and dwarf spheroidal galaxies. We briefly summarize the difficulties of stellar dynamical searches, then show that recently discovered relations between black hole mass, X-ray luminosity and radio luminosity imply that in most cases, these black holes should be more easily detected in the radio than in the X-rays. Finally, we show upper limits from some radio observations of globular clusters, and discuss the possibility that the radio source in the core of the Ursa Minor dwarf spheroidal galaxy might be a $\sim 10,000-100,000 M_\odot$ black hole.Comment: 10 pages, no figures, to appear in From X-ray Binaries to Quasars: Black Hole Accretion on All Mass Scales, ed. T. J. Maccarone, R. P. Fender, and L. C. Ho (Dordrecht: Kluwer