Low levels of per- and polyfluoroalkyl substances (PFAS) detected in drinking water in Norway, but elevated concentrations found near known sources.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous contaminants which are also found in drinking water. Concentration levels in drinking water vary widely and range from a very low contribution to total daily exposure for humans to being the major source of uptake of PFAS. PFAS concentrations in Norwegian drinking water has been rarely reported. We investigated concentrations of 31 PFAS in 164 water samples, representing both source water (i.e., before drinking water treatment) and finished drinking water. Samples were taken from 18 different water bodies across Norway. The 17 waterworks involved supply drinking water to 41 % of the Norwegian population. Only four of the waterworks utilised treatment involving activated carbon which was able to significantly reduce PFAS from the source water. Samples of source water from waterworks not employing activated carbon in treatment were therefore considered to represent drinking water with regards to PFAS (142 samples). All samples from one of the water bodies exceeded the environmental quality standard (EQS) for perfluorooctane sulfonic acid (PFOS) according to the water framework directive (0.65 ng/L). No concentrations exceeded the sum of (20) PFAS (100 ng/L) specified in the EU directive 2020/2184 for drinking water. Several EU countries have issued lower guidelines for the sum of the four PFAS that the European Food Safety Authority (EFSA) has established as the tolerable weekly intake (TWI) for PFOS, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS). Denmark and Sweden have guidelines specifying 2 and 4 ng/L for the sum of these PFAS. Only one of the 142 drinking water samples exceeded the Danish TWI and contained a sum of 6.6 ng/L PFAS. A population exposure model, for individuals drinking water from the investigated sources, showed that only 0.5 % of the population was receiving PFAS concentrations above the Danish limit of 2 ng/L.

Effect-Directed Analysis Based on Transthyretin Binding Activity of Per- and Polyfluoroalkyl Substances in a Contaminated Sediment Extract.

Only a fraction of the total number of per- and polyfluoroalkyl substances (PFAS) are monitored on a routine basis using targeted chemical analyses. We report on an approach toward identifying bioactive substances in environmental samples using effect-directed analysis by combining toxicity testing, targeted chemical analyses, and suspect screening. PFAS compete with the thyroid hormone thyroxin (T4 ) for binding to its distributor protein transthyretin (TTR). Therefore, a TTR-binding bioassay was used to prioritize unknown features for chemical identification in a PFAS-contaminated sediment sample collected downstream of a factory producing PFAS-coated paper. First, the TTR-binding potencies of 31 analytical PFAS standards were determined. Potencies varied between PFAS depending on carbon chain length, functional group, and, for precursors to perfluoroalkyl sulfonic acids (PFSA), the size or number of atoms in the group(s) attached to the nitrogen. The most potent PFAS were the seven- and eight-carbon PFSA, perfluoroheptane sulfonic acid (PFHpS) and perfluorooctane sulfonic acid (PFOS), and the eight-carbon perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), which showed approximately four- and five-times weaker potencies, respectively, compared with the native ligand T4 . For some of the other PFAS tested, TTR-binding potencies were weak or not observed at all. For the environmental sediment sample, not all of the bioactivity observed in the TTR-binding assay could be assigned to the PFAS quantified using targeted chemical analyses. Therefore, suspect screening was applied to the retention times corresponding to observed TTR binding, and five candidates were identified. Targeted analyses showed that the sediment was dominated by the di-substituted phosphate ester of N-ethyl perfluorooctane sulfonamido ethanol (SAmPAP diester), whereas it was not bioactive in the assay. SAmPAP diester has the potential for (bio)transformation into smaller PFAS, including PFOS. Therefore, when it comes to TTR binding, the hazard associated with this substance is likely through (bio)transformation into more potent transformation products. Environ Toxicol Chem 2024;43:245-258. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Identification of 24-O-ß-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-ß-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1-5, the 24-O-ß-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4-6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Fluorinated Precursor Compounds in Sediments as a Source of Perfluorinated Alkyl Acids (PFAA) to Biota.

The environmental behavior of perfluorinated alkyl acids (PFAA) and their precursors was investigated in lake Tyrifjorden, downstream a factory producing paper products coated with per- and polyfluorinated alkyl substances (PFAS). Low water concentrations (max 0.18 ng L-1 linear perfluorooctanesulfonic acid, L-PFOS) compared to biota (mean 149 µg kg-1 L-PFOS in perch livers) resulted in high bioaccumulation factors (L-PFOS BAFPerch liver: 8.05 × 105-5.14 × 106). Sediment concentrations were high, particularly for the PFOS precursor SAmPAP diester (max 1 872 µg kg-1). Biota-sediment accumulation factors (L-PFOS BSAFPerch liver: 22-559) were comparable to elsewhere, and concentrations of PFAA precursors and long chained PFAA in biota were positively correlated to the ratio of carbon isotopes (13C/12C), indicating positive correlations to dietary intake of benthic organisms. The sum fluorine from targeted analyses accounted for 54% of the extractable organic fluorine in sediment, and 9-108% in biota. This, and high trophic magnification factors (TMF, 3.7-9.3 for L-PFOS), suggests that hydrophobic precursors in sediments undergo transformation and are a main source of PFAA accumulation in top predator fish. Due to the combination of water exchange and dilution, transformation of larger hydrophobic precursors in sediments can be a source to PFAA, some of which are normally associated with uptake from water.

Paper product production identified as the main source of per- and polyfluoroalkyl substances (PFAS) in a Norwegian lake: Source and historic emission tracking.

The entirety of the sediment bed in lake Tyrifjorden, Norway, is contaminated by per- and polyfluoroalkyl substances (PFAS). A factory producing paper products and a fire station were investigated as possible sources. Fire station emissions were dominated by the eight carbon perfluoroalkyl sulfonic acid (PFSA), perfluorooctanesulfonic acid (PFOS), from aqueous film forming foams. Factory emissions contained PFOS, PFOS precursors (preFOS and SAmPAP), long chained fluorotelomer sulfonates (FTS), and perfluoroalkyl carboxylic acids (PFCA). Concentrations and profiles in sediments and biota indicated that emissions originating from the factory were the main source of pollution in the lake, while no clear indication of fire station emissions was found. Ratios of linear-to branched-PFOS increased with distance from the factory, indicating that isomer profiles can be used to trace a point source. A dated sediment core contained higher concentrations in older sediments and indicated that two different PFAS products have been used at the factory, referred to here as Scotchban and FTS mixture. Modelling, based on the sediment concentrations, indicated that 42-189 tons Scotchban, and 2.4-15.6 tons FTS mixture, were emitted. Production of paper products may be a major PFAS point source, that has generally been overlooked. It is hypothesized that paper fibres released from such facilities are important vectors for PFAS transport in the aquatic environment.

Selective Extraction and Purification of Azaspiracids from Blue Mussels ( Mytilus edulis) Using Boric Acid Gel.

Azaspiracids belong to a family of more than 50 polyether toxins originating from marine dinoflagellates such as Azadinium spinosum. All of the azaspiracids reported thus far contain a 21,22-dihydroxy group. Boric acid gel can bind selectively to compounds containing vic-diols or α-hydroxycarboxylic acids via formation of reversible boronate complexes. Here we report use of the gel to selectively capture and release azaspiracids from extracts of blue mussels. Analysis of the extracts and fractions by liquid chromatography-tandem mass spectrometry (LC-MS) showed that this procedure resulted in an excellent cleanup of the azaspiracids in the extract. Analysis by enzyme-linked immunoasorbent assay (ELISA) and LC-MS indicated that most azaspiracid analogues were recovered in good yield by this procedure. The capacity of boric acid gel for azaspiracids was at least 50 µg/g, making this procedure suitable for use in the early stages of preparative purification of azaspiracids. In addition to its potential for concentration of dilute samples, the extensive cleanup provided by boric acid gel fractionation of azaspiracids in mussel samples almost eliminated matrix effects during subsequent LC-MS and could be expected to reduce matrix effects during ELISA analysis. The method may therefore prove useful for quantitative analysis of azaspiracids as part of monitoring programs. Although LC-MS data showed that okadaic acid analogues also bound to the gel, this was much less efficient than for azaspiracids under the conditions used. The boric acid gel methodology is potentially applicable to other important groups of natural toxins containing diols including ciguatoxins, palytoxins, pectenotoxins, tetrodotoxin, trichothecenes, and toxin glycosides.

Pinnatoxin G is responsible for atypical toxicity in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from Ingril, a French Mediterranean lagoon.

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 µg kg(-1) in whole flesh, with a maximum in September 2010 of around 1200 µg kg(-1). These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 µg kg(-1) in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell(-1)). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC-MS with thiol derivatization.

Microcystins are cyclic heptapeptides from cyanobacteria which are responsible for poisonings of livestock and humans. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analysed by LC-MS. Thiol derivatization of the α,ß-unsaturated amide present in most microcystins was recently shown to simplify analysis of LC-MS chromatograms of a Microcystis culture, making it easier to identify peaks corresponding to microcystins in complex mixtures. This method was applied to analysis of extracts taken from a natural cyanobacteria bloom in Mwanza Gulf, Lake Victoria, Tanzania, in 2010, revealing the presence of numerous putative microcystin analogues in the sample. Results were verified using LC-MS², LC-MS/MS with precursor-ion scanning, and LC-HRMS, leading to identification of 8 major and 17 minor microcystins in the sample, including analogues of microcystin-RY, -RL and -RA. Microcystin-YR (2), -RR (3), and -RY (9) were isolated from bloom material from Lake Victoria, and the structure of 9 was confirmed by NMR spectroscopic analysis and NMR spectral comparison with 2 and 3. Confirmation of the structure of MC-RY (9) facilitated detailed analysis of its MS² spectrum, thereby supporting the structures of related analogues tentatively established on the basis of MS analyses.

Neurotoxicity of Penicillium crustosum secondary metabolites: tremorgenic activity of orally administered penitrem A and thomitrem A and E in mice.

Several cases of neurological disease in dogs after poisoning by food- and feed-borne Penicillium toxins in Norway during the last years have uncovered a lack of knowledge regarding the toxicity and mechanism of action of neuroactive mycotoxins. In the present study, the lowest tremor-inducing dose after single oral administration of penitrem A to mice was 0.50 mg/kg bw. The estimated half maximal effective dose (ED(50)) in respect to the visual tremor scale was 2.74 mg/kg bw. Mice receiving the maximum penitrem A dose (8 mg/kg bw) suffered severe spontaneous tremors and even convulsions. Thomitrem A and E are penitrem analogues lacking the C-16-C-18 ether linkage and possessing an olefin at C-18-C-19. Compared with penitrem A, the lowest tremor-inducing dose of thomitrem A was 16-times higher (8 mg/kg bw) and thomitrem E was found to be non-tremorgenic at the highest dose tested (16 mg/kg bw). During a recovery phase of two weeks post administration animals appeared restored and no changes in feeding and other biological processes were observed. An initial dose-related weight reduction was observed 2 days after penitrem A administration. Penitrem A was absorbed and distributed to gastrointestinal tract, liver, kidneys and brain in the mice. Elimination of penitrem A appeared to be mainly hepatic and the highest concentration levels were found 1 h post administration for all investigated organs. The relationship between liver and gastrointestinal tract concentration levels showed time-dependent linear correlation and a doubling within 1.5 h.

Thiol derivatization for LC-MS identification of microcystins in complex matrices.

Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,ß-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS(2) spectra was facilitated by addition of the mass associated with the thiol to the α,ß-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O'-methyl-hexa(ethylene glycol) (MEMHEG) (M(w)t. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system.

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA(A)-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC50 (half maximal inhibitory concentration) values of 11 and 9 µM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC50 values of 20 and 47 µM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA(A)-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.

Pinnatoxins and spirolides in Norwegian blue mussels and seawater.

Fast-acting cyclic imines belonging to the pinnatoxin and pteriatoxin group of toxins were originally identified in shellfish of the genera Pinna and Pteria in Japan, after food poisoning events in China linked to consumption of Pinna spp. Recently, a range of new and known pinnatoxin analogs has been identified in shellfish, sediment, and seawater samples from Australia and New Zealand. Although the structurally closely-related spirolide toxins are better known, and have a worldwide distribution including Norway and other parts of Europe, the presence of pinnatoxins has not been reported in European waters or shellfish. Here we report results from a survey of Norwegian blue mussels for the presence of pinnatoxins and spirolides, by LC-MS/MS analysis of extracts obtained as part of Norway's routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides and pinnatoxin G were widespread (pinnatoxin G (1), spirolide C (2), iso-spirolide C (3), 13-desmethylspirolide C (4), 13,19-didesmethylspirolide C (5), and 20-methylspirolide G (6) were detected in 69%, 13%, 60%, 22%, 33%, and 77%, respectively, of the shellfish samples) and, although levels were generally low, concentrations of up to 115 µg/kg of pinnatoxin G (1) and 226 µg/kg of 13-desmethylspirolide C (4) were detected. We also analyzed stored extracts from passive sampling disks deployed as part of a separate study in autumn 2007. All the stored extracts contained 20-methylspirolide G (which predominated at most locations), most contained pinnatoxin G (73%) and 13,19-didesmethylspirolide C (67%), but iso-spirolide C (36%) and 13-desmethylspirolide C (52%) were also detected in many of the samples. These results suggest that pinnatoxins may be much more widespread than previously suspected, and indicate that they or related compounds could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of these levels of pinnatoxins and spirolides is at present unclear. However, although pinnatoxins appear to be less toxic than spirolides by intraperitoneal injection in the mouse bioassay, recently published preliminary toxicological data indicate that pinnatoxins may be as much as an order of magnitude more toxic than spirolides by oral ingestion via food.

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation.

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography-mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.

Isolation and structure elucidation of secopenitrem D, an indole alkaloid from Penicillium crustosum Thom.

Chemical investigation of three isolates of Penicillium crustosum Thom cultures, one of which was derived from a recent dog poisoning investigation, has led to the isolation and structure elucidation of secopenitrem D (1). Penitrems A-F and roquefortine C were also present in the isolates analyzed. The structure of 1 was unambiguously assigned based on extensive 1D and 2D-NMR spectroscopic experiments, MS-aided structural studies and by comparison with structurally related congeners. Secopenitrem D lacks the C-16-C-18 ether linkage present in penitrems A-F.

Electron ionisation mass spectrometry of the pentafluoropropionate esters of trichothecene analogues and culmorin compounds from Fusarium species.

This paper reports the mass spectra, obtained after electron ionisation (EI) at 70 eV, of 34 trichothecenes and five culmorin compounds after acylation with pentafluoropropionic anhydride. The derivatised fungal metabolites were separated by gas chromatography, and the mass spectra were obtained by scanning of a single quadrupole mass filter in the scan range m/z 200-900. The fragmentation pathways of three trichothecenes (triacetyl-deoxynivalenol, 4,15-diacetoxy-scirpenol, T-2 toxin) have been studied in more detail by linked scan-high-resolution mass spectrometry. The most common trichothecenes are today more often routinely analysed using LC/MS-based methodologies. However, EI-MS may give complementary structural information, and the data that are summarised in this article may help to identify analogues of one of the largest class of mycotoxins, the tricothecenes, as well as culmorin compounds that are commonly co-produced by Fusarium culmorum and F. graminearum in cultures or naturally contaminated samples.

Detection of food-derived damaged nucleosides with possible adverse effects on human health using a global adductomics approach.

A range of damaged nucleosides, also found in digested dietary DNA, appear to be taken up by cells and incorporated into the cells' own DNA. Most incorporated damaged nucleosides will be repaired by cellular DNA repair systems. However, a small fraction of these will escape repair and thus ultimately create mutations. Over the long human lifespan this could be a mechanism that contributes to disease, cancer, and aging. This study analyzed damaged nucleosides derived from dietary DNA in a commercially successful fungus-based novel food, Quorn, and in two fungus-based food items with a history of safe use, button mushroom ( Agaricus bisporus ) and dried powdered brewers yeast ( Saccharomyces cerevisiae ). By using liquid chromatography combined with tandem mass spectrometry more than 90 putative DNA adducts were measured, showing that foods do contain a range of different DNA damages.

A structural basis for the reduced toxicity of dinophysistoxin-2.

Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic pi-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140 degrees so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.

A convenient and cost-effective method for monitoring marine algal toxins with passive samplers.

Passive sampling disks were developed based on the method of MacKenzie, L, Beuzenberg, V., Holland, P., McNabb, P., Selwood, A. [2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44, 901-918] and protocols were formulated for recovering toxins from the adsorbent resin via elution from small columns. The disks were used in field studies to monitor in situ toxin dynamics during mixed algal blooms at Flødevigen in Norway. Examples are given from time-integrated sampling using the disks followed by extraction and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (Mytilus edulis) were compared with the relative abundance of toxin-producing algal species. Results obtained showed that passive sampling disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to produce and convenient to use and, when combined with HPLC-MS or enzyme-linked immunosorbent assay (ELISA) analysis, provide detailed time-averaged information on the profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.

In vitro and in vivo hepatic metabolism of the fungal neurotoxin penitrem A.

Penitrem A is a potent neurotoxin produced by several species in the genus Penicillium, which primarily affects the central nervous system. The toxin has several effects on neurotransmitter release, both at the central and peripheral level, as well as on ion channels. We have evaluated the hepatic metabolism of penitrem A by rat hepatocytes and rat-liver microsomes in vitro. In addition, we have conducted an in vivo study in mice and determined metabolites in several organs. According to our results, penitrem A is extensively metabolized in the liver to at least five metabolites more hydrophilic than the parent compound.

Determination of cyclopiazonic acid in food and feeds by liquid chromatography-tandem mass spectrometry.

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol-water mixture, defatting with hexane and quantification using HPLC-MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 microg/kg while the limit of quantification was 20 microg/kg in the matrices investigated. The detector response was found to be linear over the range 25-250 microg/kg in feed and 25-500 microg/kg in wheat, peanuts and rice. The mean overall recoveries (n=18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n=3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.