Effective Extraction of Domoic Acid from Seafood Based on Postsynthetic-Modified Magnetic Zeolite Imidazolate Framework-8 Particles.

Domoic acid (DA) is a naturally occurring neurotoxin known to bioaccumulate in marine products. Despite its hypertoxicity, the enrichment and analysis of trace DA in complex marine organisms remains a challenge. We describe herein the fabrication of a postsynthetic-modified magnetic zeolite imidazolate framework-8 (Fe3O4 SPs@ZIF-8/Zn2+), based on Fe3O4 superparticles, for the adsorption of DA from complex biological matrices. The adsorption of DA is rapid (∼5 min) and occurs through strong electrostatic interactions and chelation with coordinatively unsaturated zinc sites on the surface of Fe3O4 SPs@ZIF-8/Zn2+. Employing our Fe3O4 SPs@ZIF-8/Zn2+ sorbent in a magnetic solid-phase extraction, followed by liquid chromatographic separation and tandem mass spectrometric detection, resulted in a facile, rapid, efficient, and sensitive method for the enrichment and detection of trace DA in marine products. After optimization, this method yielded satisfactory precision (relative standard deviation ≤3.4%; n = 5) with a high degree of linearity from 1.0 to 1000.0 pg mL-1 ( r2 = 0.9997) and a detection limit of 0.2 pg mL-1 (S/N = 3). Recoveries of 93.1-102.3% were obtained in spiked aquatic products. In addition, trace levels of DA (49.2 pg mL-1) were found in shellfish samples, confirming the applicability of our Fe3O4 SPs@ZIF-8/Zn2+ adsorbent for the detection of DA in seafood.

Pseudo-nitzschia, Nitzschia, and domoic acid: New research since 2011.

Some diatoms of the genera Pseudo-nitzschia and Nitzschia produce the neurotoxin domoic acid (DA), a compound that caused amnesic shellfish poisoning (ASP) in humans just over 30 years ago (December 1987) in eastern Canada. This review covers new information since two previous reviews in 2012. Nitzschia bizertensis was subsequently discovered to be toxigenic in Tunisian waters. The known distribution of N. navis-varingica has expanded from Vietnam to Malaysia, Indonesia, the Philippines and Australia. Furthermore, 15 new species (and one new variety) of Pseudo-nitzschia have been discovered, bringing the total to 52. Seven new species were found to produce DA, bringing the total of toxigenic species to 26. We list all Pseudo-nitzschia species, their ability to produce DA, and show their global distribution. A consequence of the extended distribution and increased number of toxigenic species worldwide is that DA is now found more pervasively in the food web, contaminating new marine organisms (especially marine mammals), affecting their physiology and disrupting ecosystems. Recent findings highlight how zooplankton grazers can induce DA production in Pseudo-nitzschia and how bacteria interact with Pseudo-nitzschia. Since 2012, new discoveries have been reported on physiological controls of Pseudo-nitzschia growth and DA production, its sexual reproduction, and infection by an oomycete parasitoid. Many advances are the result of applying molecular approaches to discovering new species, and to understanding the population genetic structure of Pseudo-nitzschia and mechanisms used to cope with iron limitation. The availability of genomes from three Pseudo-nitzschia species, coupled with a comparative transcriptomic approach, has allowed advances in our understanding of the sexual reproduction of Pseudo-nitzschia, its signaling pathways, its interactions with bacteria, and genes involved in iron and vitamin B12 and B7 metabolism. Although there have been no new confirmed cases of ASP since 1987 because of monitoring efforts, new blooms have occurred. A massive toxic Pseudo-nitzschia bloom affected the entire west coast of North America during 2015-2016, and was linked to a 'warm blob' of ocean water. Other smaller toxic blooms occurred in the Gulf of Mexico and east coast of North America. Knowledge gaps remain, including how and why DA and its isomers are produced, the world distribution of potentially toxigenic Nitzschia species, the prevalence of DA isomers, and molecular markers to discriminate between toxigenic and non-toxigenic species and to discover sexually reproducing populations in the field.

Evaluation of intracellular and extracellular domoic acid content in Pseudo-nitzschia multiseries cell cultures under different light regimes.

Pseudo-nitzschia multiseries is a diatom species associated with the production of domoic acid (DA), a water soluble neurotoxin that is easily transferred up in the food web, causing devastating effects on top marine organisms and humans. Despite studies on Pseudo-nitzschia are relevant to human health safety, partitioning of marine toxins between intracellular and extracellular fractions are poorly documented. This study aimed to determine the growth rates and DA content, both intracellular and extracellular, of Pseudo-nitzschia multiseries cultures at three different light settings (15, 120 and 560 µmol m-2 s-1). The optimal conditions for cell growth were observed at 120 and 560 µmol m-2 s-1, whereas DA production was observed in P. multiseries at 15 and 120 µmol m-2 s-1, ranging between 0.18-2.56 and 0.16-3.5 pg DA cell-1, respectively. Higher intracellular DA concentrations were found during the senescence phase at low light intensity and during the exponential phase at medium light intensity, while higher concentrations of dissolved DA were found at low and medium light intensities in the senescence phase reaching 3 and 10 ng DA mL-1 respectively. The amount of toxin released into the culture medium represents the most important fraction ranging between 63 and 98% during the exponential phase and nearly 99% during the senescence phase. In contrast, under low light intensity, dissolved DA was detected in the culture medium only during the senescence phase. This study confirms the importance of light intensity on DA production and clearly shows that dissolved domoic acid is an important fraction in Pseudo-nitzschia cultures, suggesting with the careful assumptions of results from static cultures extrapolated to bloom situations that waterborne exposure of marine organism should be considered during blooms of Pseudo-nitzschia multiseries.

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

Preparation and application of a molecularly imprinted monolith for specific recognition of domoic acid.

In this work, a molecularly imprinted monolithic column was synthesized by a facile procedure and was applied for specific recognition of domoic acid, an amnesic shellfish poison. The poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) molecularly imprinted monolith was synthesized in a stainless steel column by in situ polymerization. Pentane-1,3,5-tricarboxylic acid was used as a dummy imprinting template instead of the highly toxic and expensive target molecule. It is the first time that a molecularly imprinted monolith is introduced for separation and detection of domoic acid. After optimizing the preparation conditions, the prepared imprinted monolith was systematically characterized and exhibited excellent stability and permeability as a HPLC stationary phase. The results of chromatographic analysis demonstrated that the molecularly imprinted monolith exhibited specific retention and selective recognition toward domoic acid, with an imprinted factor up to 3.77. Furthermore, the molecularly imprinted monolith was successfully applied for selective enrichment of domoic acid from biological samples. Graphical abstract A molecularly imprinted monolith was prepared by using a dummy imprinted template and was successfully applied for specific recognition of domoic acid.

Rapid LC-MS Determination of Acromelic Acids A and B, Toxic Constituents of the Mushroom Paralepistopsis acromelalga.

A rapid LC-MS method was developed for determination of acromelic acids A and B, which are toxic constituents of Paralepistopsis acromelalga (=Clitocybe acromelalga), in mushroom samples. Acromelic acids were extracted twice with 50% methanol and the extract was passed through a syringe filter, and then analyzed by LC-MS. The LC separation was performed on a multi-mode ODS column. The recoveries of acromelic acids A and B spiked into blank mushroom samples at 2.5 µg/g were 93 and 74%, respectively. This method was applied to the remaining mushroom sample from a food poisoning case. Acromelic acids A and B were detected at 2.0 and 1.4 µg/g, respectively, in the remaining sample. Another toxic constituent, which appeared to be clitidine, was also detected in the sample.

Multipoint recognition of domoic acid from seawater by dummy template molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography.

Due to the high cost of domoic acid (DA), different carboxylic acid compounds including indole-3-acetic acid (IAA), pyrrole-2-formic acid (PFA), pyridine-2,3-dicarboxylic anhydride (PDA), trimesitinic acid (TA) and citric acid (CA) were investigated as dummy templates for the molecularly imprinted solid-phase extraction (MISPE) for selective isolation and pre-concentration of an amnesic shellfish poison (ASP), DA. The highest binding amount of the polymers towards DA was obtained when CA was used as dummy template owing to its high hydrophilicity. In addition, the "four-point" recognition site constructed by three COOH groups and a OH group in CA was also speculated to be the reason for the high binding amount of CA-MIPs and the rebinding of DA can be depend on the three COOH groups and a NH group with conformational change in the recognition process. Finally, the CA-MISPE column was chosen for DA isolation and pre-concentration and effective result was obtained with recoveries higher than 90% and relative standard deviation (RSD) less than 5% (n=3). This new polymer can be effectively applied to the monitoring and predicting the existence of trace DA.

Determination of domoic acid in shellfish extracted by molecularly imprinted polymers.

A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5-pentanetricarboxylic acid as the template molecule, 4-vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween-80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 µg/mL and apparent maximum adsorption capacity of 1249 µg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high-performance liquid chromatography. The detection limit of 0.17 µg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0-98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples.

Practical Total Syntheses of Acromelic Acids A and B.

Practical total syntheses of acromelic acids A (1) and B (2), which were scarce natural products isolated from toxic mushroom by Shirahama and Matsumoto, were accomplished in 13 (36% total yield) and 17 steps (6.9% total yield), respectively, from 2,6-dichloropyridine (8). Beginning with regioselective transformation of symmetric 8 by either ortho-lithiation or bromination, nitroalkenes 15 and 16 were provided. Stereoselective construction of the vicinal stereocenters at the C-3, 4 positions of 1 and 2 was performed by a Ni-catalyzed asymmetric conjugate addition of α-ketoesters to the nitroalkenes. Construction of the pyrrolidine ring was accomplished in a single operation via a sequence consisting of reduction of the nitro group, intramolecular condensation with the ketone, and reduction of the resulting ketimine.

Metal-organic framework UiO-66 modified magnetite@silica core-shell magnetic microspheres for magnetic solid-phase extraction of domoic acid from shellfish samples.

Fe3O4@SiO2@UiO-66 core-shell magnetic microspheres were synthesized and characterized by transmission electron microscopy, scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectrometry, vibrating sample magnetometry, nitrogen adsorption porosimetry and zeta potential analyzer. The synthesized Fe3O4@SiO2@UiO-66 microspheres were first used for magnetic solid-phase extraction (MSPE) of domoic acid (DA) in shellfish samples. Combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), a fast, simple and sensitive method for the determination of DA was established successfully. Under the optimized conditions, the developed method showed short analysis time, good linearity (r(2) = 0.9990), low limit of detection (1.45 pg mL(-1); S/N = 3:1), low limit of quantification (4.82 pg mL(-1); S/N = 10:1), and good extraction repeatability (RSD ≤ 5.0%; n = 5). Real shellfish samples were processed using the developed method, and trace level of DA was detected. The results demonstrate that Fe3O4@SiO2@UiO-66 core-shell magnetic microspheres are the promising sorbents for rapid and efficient extraction of polar analytes from complex biological samples.

Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system.

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1µg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC50's using this method were 1.9±0.1µg L(-1) MC-LR, 1.3±0.1µg L(-1) CYN, 61±4µg L(-1) ANA-a, 5.4±0.4µg L(-1) STX and 4.9±0.9µg L(-1) DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC-IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC-IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

Development and validation of the first high performance-lateral flow immunoassay (HP-LFIA) for the rapid screening of domoic acid from shellfish extracts.

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10 ppm (version 1) to 17.5 ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15 min after sample preparation. Stability of the aqueous extracts at room temperature (22 °C) at four time points (up to 245 min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10 ppm DA, and positive results at or above 17.5 ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20 ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12 min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5 ppm and positive results at or above 17.5 ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50 °C) was 100%. Validation for both versions included comparisons with results obtained using reference LC-UV methods.

Molecularly imprinted polymer for selective extraction of domoic acid from seafood coupled with high-performance liquid chromatographic determination.

In this work, a highly selective sample cleanup procedure that combining molecular imprinting technique (MIT) and solid phase extraction (SPE) was developed for the isolation of domoic acid (a fascinating marine toxin) from seafood samples. The molecular imprinting polymer (MIP) for domoic acid was prepared using 1,3,5-pentanetricarboxylic acid as the template molecule instead of domoic acid. 4-Vinyl pyridine was used as the functional monomer and ethylene glycol dimethacrylate was used as the cross-linking monomer. The obtained imprinted polymer showed high affinity to domoic acid and was used as selective sorbent for the SPE of domoic acid from seafood samples. An off-line molecularly imprinted solid phase extraction (MISPE) method followed by high-performance liquid chromatography (HPLC) with diode-array detection for the detection of domoic acid was also established. Good linearity was obtained from 0.5 mg L(-1) to 25 mg L(-1) (R(2)>0.99) with a quantitation limit of 0.1 mg L(-1), which was sufficient to determine domoic acid at the maximum level permitted by several authorities. The mean recoveries of domoic acid from mussel extracts were 93.4-96.7%. It was demonstrated that the proposed MISPE-HPLC method could be applied to direct determination of domoic acid from seafood samples.

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

Solid-phase extraction-fluorimetric high performance liquid chromatographic determination of domoic acid in natural seawater mediated by an amorphous titania sorbent.

The feasibility of using sol-gel amorphous titania (TiO2) as a solid-phase sorbent for the pre-concentration of domoic acid (DA), a potent amnesic shellfish poisoning (ASP) toxin, directly from seawater was explored. The sol-gel titania material is able to adsorb DA from seawater, via the formation of ester-linkage between the carboxylic moieties of DA and the Ti-OH groups on the sorbent surface, at low pH and desorb it at high pH. The chemisorption process is not significantly interfered by the seawater matrix. The optimum pH values for the adsorption and desorption of DA were found to be pH 4 and 11, respectively. The optimal sorbent loading for the batch-type solid-phase extraction of DA was 0.67 mg-TiO2 ng-DA(-1) and adsorption equilibrium was achieved in 2 h at room temperature. The desorbed DA in 500 microL of 0.1 M alkaline borate buffer can be directly derviatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in aqueous media for fluorimetric HPLC quantification. Analyte recovery, repeatability and detection limit of this titania SPE-fluorimetric HPLC determination are 89%, 6.2% and 120 pg-DA mL(-1) (n=7, P<0.05), respectively, for a sample volume of 30 mL. This titania SPE technique should also be applicable to the pre-concentration of other polar carboxylate- and phosphonate-containing biomolecules and pharmaceuticals in complex and interfering environmental sample matrices.

Production of isodomoic acids A and B as major toxin components of a pennate diatom Nitzschia navis-varingica.

In order to obtain more detailed information on the distribution of domoic acid-producing Nitzschia in Asian waters, Nitzschia-like diatoms were isolated primarily from the Philippines and established in culture for analysis by HPLC with fluorescence detection. Out of 58 isolates, 35 Nitzschia-like diatoms from the estuary areas of San Pedro Bay, Tacloban City and Manila Bay showed significant levels of domoic acid production (1.3-5.6pg/cell). These were identified as Nitzschia navis-varingica. Two isolates from the same locality did not produce domoic acid. Of the 21 isolates from Bulacan Estuary, Manila Bay, none produced domoic acid. They did, however, produce two substances that seemed to be domoic acid derivatives. One of the strains was mass cultured and the substances were extracted, purified and analyzed by LC-MS/MS, proton and (13)C NMR, and UV spectra. The produced substances were determined as isodomoic acids A and B. This is the first report of a diatom that produces isodomoic acids A and B as major toxin components.

Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid.

Domoic acid and its potent excitotoxic analogues glutamic acid and kainic acid, are synthesized by marine algae such as seaweed and phytoplankton. During an algal bloom, domoic acid may enter the food web through its consumption by a variety of marine organisms held in high regard as seafoods by both animals and humans. These seafoods include clams, mussels, oysters, anchovies, sardines, crabs, and scallops, among others. Animals, such as pelicans, cormorants, loons, grebes, sea otters, dolphins, and sea lions, which consume seafood contaminated with domoic acid, suffer disorientation and often death. Humans consuming contaminated seafood may suffer seizures, amnesia and also sometimes death. In addition to analytical measurement of domoic acid exposure levels in algae and/or seafood, it is useful to be able to identify the mode of toxicity through post-mortem evaluation of the intoxicated animal. In the present study, using the rat as an animal model of domoic acid intoxication, we compared histochemical staining of the limbic system and especially the hippocampus with degeneration-selective techniques (Fluoro-Jade and silver), a conventional Nissl stain for cytoplasm (Cresyl violet), a myelin-selective stain (Black-Gold), an astrocyte-specific stain (glial fibrillary acidic protein), early/immediate gene responses (c-Fos and c-Jun), as well as for heat shock protein (HSP-72) and blood-brain barrier integrity (rat IgG). The results demonstrate that the degeneration-selective stains are the biomarkers of domoic acid neurotoxicity that are the most useful and easy to discern when screening brain sections at low magnification. We also observed that an impairment of blood-brain barrier integrity within the piriform cortex accompanied the onset of domoic acid neurotoxicity.

Diatom cultivation and biotechnologically relevant products. Part II: current and putative products.

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.

Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid.

Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity. Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency. We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories.

Sea bird mortality at Cabo San Lucas, Mexico: evidence that toxic diatom blooms are spreading.

Domoic acid was found to be responsible for an isolated event involving the massive death of brown pelicans (Pelecanus occidentalis) in January 1996, at the tip of the Baja California peninsula. The death of these sea birds was the result of feeding on mackerel (Scomber japonicus) contaminated by domoic acid-producing diatoms (Pseudonitzschia sp.). The number of dead birds (150 animals) found during a period of 5 days caused alarm and called for a governmental task force that would help to implement emergency measurements to protect other species of bird. Also, local canneries were inspected to verify the safety of their recent production and prevent the toxin entering the human market. Fortunately, the timing, response and coordination of this task force enabled identification of the origin and nature of the toxin that provoked such a phenomenon. Future monitoring is recommended to avoid a larger impact of domoic acid spreading and the occurrence of similar toxic events.