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Ciguatoxins (CTXs) are neurotoxic marine biotoxins capable of contaminating marine organisms. Approximately 30 CTX analogues have been described; however, only a few have been documented in ciguatera poisoning (CP) outbreaks. Detecting CTXs from complex matrices at CP-relevant concentrations (<1 µg per kg seafood tissue) is analytically challenging. Analytical standards for CTXs are rare. Even after multi-step sample preparation (including liquid–liquid partition, defatting, and solid-phase extraction (SPE)), extracts can contain undesirable co-eluting matrix components. These limitations can exacerbate discrepancies between results obtained by LC-MS/MS and the N2a-bioassay, which are two common CTX detection methods. Herein, a rapid and simple fractionation method, based on normal phase SPE (silica), is demonstrated. Target CTXs were eluted using solvent mixtures of ascending polarity, passed through the column, and separated into eight fractions. To challenge the method, extracts with eleven naturally incurred CTX analogues among different structural CTX groups (e.g., CTX3C, CTX4A, and C-CTX group) were used. The most complex tissue matrix tested (viscera) was improved the most for extract purity and CTX detection, enhancing the correlation between LC-MS/MS and N2a-bioassay results. This workflow represents an advancement for characterizing CTXs in seafood products and CP outbreaks, irrespective of the responsible CTX analogue and where standards are lacking. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to assess if veterinary antibiotics, which are introduced to agricultural fields via fertilizer, are present in water systems (in both water and sediment) following overland transport via runoff and soil erosion. Materials and methods: Three water and sediment sampling schemes were conducted in Germany between June 2013 and June 2014 to examine the effects of (1) season, (2) heavy rainfall and (3) high veterinary antibiotic usage. The samples were analysed for 15 veterinary antibiotics using liquid chromatography tandem mass spectrometry. Results and discussion: Antibiotics were detected in all three schemes in trace quantities. This is the first time that veterinary antibiotics have been detected in German sediment. Conclusions: In particular, the presence of tetracycline in sediment taken from irrigation ditches in an agricultural area of high veterinary antibiotic usage offers proof that the overland transport of veterinary antibiotics is occurring. Due to the strong sorption of veterinary antibiotics (particularly tetracyclines) to soils, further research into their transport via soil erosion and assessment of their presence in sediment is advised. [ABSTRACT FROM AUTHOR]

Pharmaceuticals are widely distributed in low concentrations in the water compartment. Conventional sewage treatment plants (STP) are not capable to remove pharmaceuticals to the full extent, so alternative methods such as ozonization or photooxidation are tested as additional stage. However, it has been shown that in many cases no complete mineralization is achieved during these processes and transformation products occur. Up to now only little is known about these transformation products in terms of their structure and toxicity. The antibiotic sulfathiazole (STZ) is used in veterinary medicine and it can be detected in different water matrices such as STP effluent or surface water. We tested the ability of photooxidation for degradation of STZ using a medium pressure mercury lamp. LC-UV showed the formation of several minor compounds and one main transformation product which has been isolated subsequently. Using NMR and high resolution mass spectrometry techniques, we were able to reveal the structure of promizole, an antituberculotic, used in the 1950s. This compound showed a reduced microbial activity in Brilliant Black Reduction Maximum Residue Level test. Furthermore, there was no difference in the effects of promizole when compared to STZ on cell toxicity and cell proliferation in two murine cell lines. [ABSTRACT FROM AUTHOR]

Abstract: The sulfur metabolite dimethylsulfoniopropionate (DMSP) is the most important precursor of the climate relevant metabolite dimethylsulfide (DMS). It has thus gained interest in the context of climate change and several studies investigated the influence of elevated temperature and/or CO2 on DMSP in complex plankton communities. However, only little information about changes in response to these factors in single species is available. Therefore, we analyzed DMSP in different phytoplankton cultures (Thalassiosira pseudonana, Phaeodactylum tricornutum, Emiliania huxleyi) under the influence of increased temperature by 6 °C and elevated CO2 to 790 ppmv. In addition, we addressed glycine betaine (GBT) that fulfills a similar function as osmolyte like DMSP. In all cultures GBT concentrations increased at higher temperature and decreased at elevated CO2. In contrast, diatoms and prymnesiophytes revealed opposite trends for DMSP. In diatoms increased CO2 and temperature led to decreased DMSP concentrations, while rather elevated levels of this metabolite under the influence of these parameters were observed for E. huxleyi. [Copyright &y& Elsevier]

Abstract: A simple, derivatization free method for the direct determination of dimethylsulfoniopropionate (DMSP) using hydrophilic interaction liquid chromatography (HILIC)/mass spectrometry is introduced. DMSP is a zwitterionic osmolyte which is produced from marine plankton, macro algae and higher plants. Due to its central role in climate relevant geochemical processes as well as in plant physiology and chemical ecology there is a great interest in methods for its quantification. Since DMSP is labile and difficult to extract currently most protocols for quantification are based on indirect methods. Here we show that ultra performance liquid chromatography/mass spectrometry using a HILIC stationary phase is suitable for the direct quantification of DMSP from aqueous samples and microalgal extracts. The protocol requires minimal sample preparation and phytoplankton samples can be investigated after filtration of small volumes. The limit of detection is 20nM and the calibration curve is linear in the range of 60nM to 50μM. The use of [2H6]-DMSP as internal standard allows prolonged sample storage since it is transformed with the same kinetics as natural DMSP. This makes the method suitable for both laboratory and field studies. [ABSTRACT FROM AUTHOR]

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles. [ABSTRACT FROM AUTHOR]

Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell−1, and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices. [Display omitted] • Putative I/C-CTX-1 identified for the first time in a dinoflagellate extract. • Confirmation of 44-methylgambierone by LC-HRMS2 in G. balechii. • Interdisciplinary two-tiered approach for the dereplication of toxins. • Bio-guided fractionation of G. balechii extract by LC-HRMS and N2a-assay. [ABSTRACT FROM AUTHOR]