Toxin profiles of five geographical isolates of Dinophysis spp. from North and South America

This paper is not subject to U.S. copyright. The definitive version was published in Toxicon 57 (2011): 275-187, doi:10.1016/j.toxicon.2010.12.002.Marine dinoflagellates of the genus Dinophysis can produce toxins of the okadaic acid (OA) and pectenotoxin (PTX) groups. These lipophilic toxins accumulate in filter-feeding shellfish and cause an illness in consumers called diarrhetic shellfish poisoning (DSP). In 2008, a bloom of Dinophysis led to the closure of shellfish harvesting areas along the Texas coast, one of the first DSP-related closures in the U.S. This event resulted in a broad study of toxin production in isolates of Dinophysis spp. from U.S. waters. In the present study, we compared toxin profiles in geographical isolates of Dinophysis collected in the U.S. (Eel Pond, Woods Hole MA; Martha’s Vineyard, MA; and Port Aransas Bay, Texas), and in those from Canada (Blacks Harbour, Bay of Fundy) and Chile (Reloncavi Estuary), when cultured in the laboratory under the same conditions. For each isolate, the mitochondrial cox1 gene was sequenced to assist in species identification. Strains from the northeastern U.S. and Canada were all assigned to Dinophysis acuminata, while those from Chile and Texas were most likely within the D. acuminata complex whereas precise species designation could not be made with this marker. Toxins were detected in all Dinophysis isolates and each isolate had a different profile. Toxin profiles of isolates from Eel Pond, Martha’s Vineyard, and Bay of Fundy were most similar, in that they all contained OA, DTX1, and PTX2. The Eel Pond isolate also contained OA-D8 and DTX1-D7, and low levels (unconfirmed structurally) of DTX1-D8 and DTX1-D9. D. acuminata from Martha’s Vineyard produced DTX1-D7, along with OA, DTX1, and PTX2, as identified in both the cells and the culture medium. D. acuminata from the Bay of Fundy produced DTX1 and PTX2, as found in both cells and culture medium, while only trace amounts of OA were detected in the medium. The Dinophysis strain from Texas only produced OA, and the one from Chile only PTX2, as confirmed in both cells and culture medium.Funding was provided by NSF Grant OCE-0850421, the Ocean Life Institute and the Coastal Ocean Institute at the Woods Hole Oceanographic Institution, and the Woods Hole Center for Oceans and Human Health through NSF grant OCE-0430724 and NIEHS grant 1 P50 ES012742. MMT would like to thank the Ministry of Education, People’s Republic of China for financial support as a Grand Fostering Project (No. 707011) and the China Scholarship Council

Development and Evaluation of Passive Sampling and LC-MS Based Techniques for the Detection and Monitoring of Lipophilic Marine Toxins in Mesocosm and Field Studies

The consumption of shellfish that accumulate marine biotoxins produced by harmful Algae can result in severe gastrointestinal or neurotoxic illnesses depending on the nature of the toxin. Therefore, it is essential that suitable monitoring programs are implemented in order to protect public health. This PhD study focused on aspects of the characteristics and monitoring of azaspiracids and toxins from Dinophysis spp. Passive samplers were used in this study in parallel with both indigenous and transplanted mussels that did not contain toxins to investigate the accumulation of toxins on the West coast of Ireland during 2005. This approach allowed for a number of observations including the persistence and contamination characteristics of azaspiracids, high levels of okadaic acid toxins in the water which did not induce shellfish contamination and non-suitability of passive sampling for early warning of shellfish contamination when placed in the same location as the shellfish. Furthermore, several sampling materials were compared in mesocosm experiments (culture of Prorocentrum lima) where the adsorption and desorption behaviour of okadaic acid and dinophysistoxin-1 were examined. A rapid and efficient extraction method was developed for a wide range of toxins. The current EU reference method for the determination of lipophilic marine toxins is the mouse bioassay. Efforts toward the replacement of the non-ethical and non specific animal test has been suggested and LC-MS is perceived as an alternative method of choice. However, LC-MS methods suffer from highly variable matrix effects affecting quantitation of the toxins. This study has evaluated three approaches fro the evaluation of matrix effects in the analysis of okadaic acid, azaspiracid-1 and pectenotoxin-2. The influence of shellfish species, heat treatment and amount of dry residue in the shellfish extracts on the degree of matrix effects were evaluated. Additionally, a rapid Ultra-Performance LC-MS method for the analysis of 21 lipophilic marine toxins in 6.5 min was developed and matrix effects were evaluated. A phytoplankton survey was carried out in the Celtic Sea in July 2007. Dinophysis acuta was found at high concentrations. Seawater was sampled from specific depths. The toxin profile of the phytoplankton was established in samples collected over a 14 h cycles. Pectenotoxins, okadaic acid and dinophysistoxin-2 were detected in the phytoplankton and were not found to be produced at a particular time of the day. The toxins accumulated and their concentration ratios in passive samplers were identical to those observed in D. acuta. Passive samplers deployed at 110m depth and a phytoplankton sample from 80 m depth allowed for the establishment of the toxin profiles. In both cases, the toxin profiles and concentration ratios compared well with the samples obtained from other depths suggesting that D. acuta can occur and produce toxins in the absence of light. Recent developments in the field of chromatography have shown that the water octanol partition coefficient (log Pow) of chemicals can be evaluated from the retention time. The method uses a calibration curve from compounds with know logPow. The partition coefficient of toxins was investigated as an important physicochemical parameter governing the accumulation of toxins in passive samplers. Knowledge of the acidity constant (pKa) is essential as log Pow should be established in dissociated molecules

Strategies for the elimination of matrix effects in the liquid chromatography tandem mass spectrometry analysis of the lipophilic toxins okadaic acid and azaspiracid-1 in molluscan shellfish

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Chromatography A. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Chromatography A, [Volume 1217, Issue 45 (5 November 2010)] doi:10.1016/j.chroma.2010.09.020 http://www.sciencedirect.com/science/article/pii/S0021967310012409peer-reviewedConsiderable efforts are being made worldwide to replace in vivo assays with instrumental methods of analysis for the monitoring of marine biotoxins in shellfish. Analysis of these compounds by the preferred technique of LC-MS/MS is challenged by matrix effects associated with shellfish tissue components. In methods validation, assessment of matrix interferences is imperative to ensure the accuracy of analytical results. We evaluated matrix interferences in the analysis of okadaic acid (OA) and azaspiracid 1 (AZA1) in mollucscan shellfish by using a conventional acidic method on electrospray triple stage quadrapole (TSQ) and hybrid quadrupole time of flight (QToF) instruments, with matrix matched standards for several species. Using the acidic method, we found no matrix interferences for OA, and matrix suppression for AZA1, with the TSQ instrument; in contrast, we found matrix enhancement for OA, and no matrix interference for AZA1, with QToF. The suppression of AZA1 signal on the TSQ instrument was due to interfering compounds carried over from previous injections. The degree of suppression was dependent on the tissue type, ranging from 20 to 70%. Several strategies were evaluated to eliminate these interferences, including the partitioning of the extract with hexane, optimization of the chromatographi

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on oa and dtx1

NOTICE: this is the author’s version of a work that was accepted for publication in Harmful Algae. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Harmful Algae, [Volume 7, Issue 5 (August 2008)] doi:10.1016/j.hal.2007.12.008 http://www.sciencedirect.com/science/article/pii/S1568988307001928peer-reviewedIt has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads® SP850, Sepabeads® SP825L, Amberlite® XAD4, Dowex® Optipore® L-493 and Diaion® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media

The effects of growth phase and light intensity on toxin production by dinophysis acuminata from the northeastern united states

For many years, the study of toxic Dinophysis species was primarily restricted to field populations until it was recently demonstrated that some of these organisms can be mixotrophically cultured in the laboratory with the ciliate prey, Myrionecta rubra. which had previously been fed with cryptophytes of the genus Teleaulax and Geminigera. Here we investigated the influence of growth phase and light intensity on the production of diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTXs) in cultures of Dinophysis acuminata from the northeastern United States. The cell toxin content of okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and the okadaic acid diol ester (OA-D8) varied significantly with growth phase under all light treatments, at 6 degrees C. Each toxin quota remained low during middle and late exponential phases, but significantly increased by mid-plateau phase. DTX1 and OA-D8 were variable through plateau phase, while OA and PTX2 significantly decreased as the culture aged. Although maximum toxin content was not achieved until middle plateau phase, the rate of toxin production was generally greatest during exponential growth. The low and relatively constant cellular toxin levels observed during exponential and early-plateau phase indicate a balance between toxin production and growth, whereas in the middle-plateau phase, toxin production continues even though the cells are no longer capable of dividing, leading to higher toxin quotas. Light was required for Dinophysis growth and the production of all toxins, however, there was no significant difference in growth rates or toxin quotas between the higher light treatments ranging from 65 to 300 mu mol photons m(-2) s(-1). These results demonstrate that DSP production in D. acuminata is constitutive, and that specific toxins are differentially produced or accumulated during the cells' growth phase, possibly in response to changes to their environment. (C) 2010 Elsevier B.V. All rights reserved

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results - Fig 5

<p>A. Comparison of metabolite levels for centrifuged (no washing) in contrast to MxP® FastQuench (with washing) samples of NS0 cells. Impact of sampling on metabolite levels for selected metabolites with especially misleading results for centrifuged (no washing) in contrast to MxP^® FastQuench (with washing) samples. B Extracellular levels of glucose and lactate as well as cell viability and total cell number for comparisons</p