Brevisulcenal-F: a polycyclic ether toxin associated with massive fish-kills in New Zealand.

A novel marine toxin, brevisulcenal-F (KBT-F, from karenia brevisulcata toxin) was isolated from the dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata in Wellington Harbour, New Zealand, in 1998 was extremely toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders. An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of K. brevisulcata produced a range of novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column chromatographies. Chemical investigations showed that KBT-F has the molecular formula C(107)H(160)O(38) and a complex polycyclic ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD(50) (mouse i.p.) for KBT-F was 0.032 mg/kg.

Comment on "Effect of uncontrolled factors in a validated liquid chromatography-tandem mass spectrometry method question its use as a reference method for marine toxins: major causes for concern".

This recent paper by Otero and co-workers presents some data from analysis of okadaic acid group toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using different instruments, operating parameters, and solvent conditions. They question the suitability of this tool for quantitative analysis. This paper reveals a lack of understanding of critical factors for the successful use of LC-MS methodology in general as well as some specific proficiency issues with the work reported on the three toxins. We show that there are problems with the conduct and reporting of the experiments, including possible injector carry-over and lack of quality assurance/quality control (QA/QC) controls. Therefore the specific conclusions they draw from their data are considered invalid.

Determination of brevetoxins in shellfish by LC/MS/MS: single-laboratory validation.

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

Brevisulcenals-A1 and A2, Sulfate Esters of Brevisulcenals, Isolated from the Red Tide Dinoflagellate Karenia brevisulcata.

Two different types of polycyclic ether toxins, namely brevisulcenals (KBTs) and brevisulcatic acids (BSXs), produced by the red tide dinoflagellate Karenia brevisulcata, were the cause of a toxic incident that occurred in New Zealand in 1998. Four major components, KBT-F, -G, -H, and -I, shown to be cytotoxic and lethal in mice, were isolated from cultured K. brevisulcata cells, and their structures were elucidated by spectroscopic analyses. New analogues, brevisulcenal-A1 (KBT-A1) and brevisulcenal-A2 (KBT-A2), toxins of higher polarity than that of known KBTs, were isolated from neutral lipophilic extracts of bulk dinoflagellate culture extracts. The structures of KBT-A1 and KBT-A2 were elucidated as sulfated analogues of KBT-F and KBT-G, respectively, by NMR and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI TOF/TOF), and by comparison with the spectra of KBT-F and KBT-G. The cytotoxicities of the sulfate analogues were lower than those of KBT-F and KBT-G.

Widespread distribution and identification of eight novel microcystins in antarctic cyanobacterial mats.

The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [D-Asp(3)] MC-RR and [D-Asp(3)] MC-LR containing glycine [Gly(1)] rather than alanine and combinations of homoarginine [hAr(2)] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda(5)] substitutions. Nostoc sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing Nostoc sp. from multiple samples.

Comparative toxicity to mice of domoic acid and isodomoic acids A, B and C.

Seafood in many parts of the world may become contaminated with high levels of domoic acid and domoic acid isomers, and such seafood has been shown to cause toxic effects in humans and in marine animals. Domoic acid itself has been held responsible for the observed effects, although the possible contribution of the isomers to toxicity has not been investigated. In the present study, the acute intraperitoneal toxicity of isodomoic acid C in mice was found to be lower than that of domoic acid. Furthermore, the severities of the behavioural changes induced by isodomoic acids A, B and C were all much lower than that of domoic acid itself, suggesting that these substances pose relatively little risk to human or animal health.

First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand.

In November 2005, at least five dogs died rapidly after contact with water from the Hutt River (lower North Island, New Zealand). Necropsy performed 24h later on one of the dogs (a 20-month-old Labrador) revealed few findings of interest, except for copious amounts of froth in the respiratory tract down to the bifurcation of the trachea and large quantities of algal material in the dog's stomach. Low and relatively stable flows in the Hutt River during spring had resulted in the proliferation of benthic cyanobacteria that formed large black/brown mats along the river edge. Samples from the Labrador's stomach contents and cyanobacterial mats were analysed microscopically and screened using chemical and biochemical assays for cyanotoxins: anatoxin-a, homoanatoxin-a, cylindrospermopsins, saxitoxins and microcystins. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of the neurotoxic cyanotoxins anatoxin-a and homoanatoxin-a and their degradation products, dihydro-anatoxin-a and dihydro-homoanatoxin-a. This is the first report of homoanatoxin-a and associated degradation product in New Zealand. Based on morphology, the causative species was identified as Phormidium sp. Subsequent phylogenetic analysis of 16S rRNA gene sequences demonstrated that the causative organism was most similar to Phormidium autumnale. Further investigations led to the detection of homoanatoxin-a and anatoxin-a in cyanobacterial mats from four other rivers in the Wellington region (lower North Island, New Zealand). Access restrictions were placed on over 60% of river catchments in the western Wellington region, severely affecting recreational users.

Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand.

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141-150]. PTX-13 underwent slow deuteration at the 13beta-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study.

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).

Further characterization of glycine-containing microcystins from the McMurdo dry Valleys of Antarctica.

Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 á´…-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine.

Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.

Acute toxicity of gymnodimine to mice.

The acute toxicity of the phycotoxin gymnodimine to female Swiss mice by intraperitoneal injection and by oral administration has been determined. Gymnodimine was highly toxic by injection, the LD50 being only 96 microg/kg. Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects. Gymnodimine was also toxic after oral administration by gavage (LD50 755 microg/kg), but was much less toxic when administered with food. No signs of toxicity were seen in mice voluntarily ingesting food containing gymnodimine at a level sufficient to give a dose of approximately 7500 microg/kg. Pre-treatment with physostigmine or neostigmine protected against injected gymnodimine, suggesting that the latter exerts its toxic effects via blockade of nicotinic receptors at the neuromuscular junction. The low toxicity of gymnodimine when ingested with food suggests that this compound is of low risk to humans, a conclusion that is consonant with anecdotal evidence for the absence of harmful effects in individuals consuming shellfish contaminated with gymnodimine.

Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities.

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue (Mytilus edulis) or Greenshell (Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC-MS and NMR, and full (1)H and (13)C NMR assignments were obtained. Okadaic acid C(8)-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC-MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC-MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD(50)=219 microg/kg) but no effects were seen with PTX-2 SA at 5000 microg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 microg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.

Field dissipation of acetochlor in two New Zealand soils at two application rates.

The persistence of pesticides in soils has both economic and environmental significance and is often used as a key parameter in pesticide risk assessment. Persistence of acetochlor [2'-ethyl-6'-methyl-N-(ethoxymethyl)-2-chloroacetylanilide] in two New Zealand field soils was measured over two years and the data were used to identify models that adequately describe acetochlor persistence in the field. Acetochlor was sprayed onto six fallow plots (3 x 9 m each) at each site at the recommended rate (2.5 kg a.i. ha(-1)) and at twice that rate. Acetochlor concentrations were measured in soil cores. Simple first-order kinetics (Model 1) adequately described acetochlor persistence in Hamilton clay loam soil (Humic Hapludull, Illuvial Spadic) at the high application rate, but overestimated it at the low application rate. A quadratic model (Model 2), a first-order double-exponential model (Model 3), a first-order biphasic model (Model 4), or a two-compartment model (Model 5) better described acetochlor persistence at the low application rate. The time for 50% (DT50) and 90% (DT90) of initial acetochlor loss was approximately 9 and 56 d, and 18 and 63 d at low and high application rates, respectively. The more complex Models 2 through 5 also better described the biphasic dissipation of acetochlor in Horotiu sandy loam soil (Typic Orthic Allophanic) than Model 1, with Model 1 significantly underestimating acetochlor concentrations on the day of application at both application rates. The DT50 and DT90 values were 5 and 29 d and 7 and 31 d at low and high application rates, respectively. Overall, application rate significantly affected the DT50 and DT90 values in the Hamilton soil, but not in the Horotiu soil. Faster acetochlor loss in the Horotiu soil possibly resulted from the higher soil organic carbon content that retained more acetochlor near the soil surface where higher temperature and photolysis accelerated the loss.

Prediction of field atrazine persistence in an allophanic soil with Opus2.

A modified version of the model Opus was applied to measurements of soil water dynamics and atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) persistence in a Bruntwood silt loam soil (Haplic Andosol, FAO system) in Hamilton, New Zealand. The modified model, Opus2, is briefly described and parameter estimation for the simulations is discussed. Soil water dynamics were more accurately described by applying measured soil hydraulic properties than by estimating them using pedotransfer functions. A parameter sensitivity analysis revealed that degradation was the most relevant process in simulating pesticide behaviour by Opus2. The Arrhenius equation incorporated in Opus2 did not correctly describe the effect of temperature on degradation rates obtained at 10, 20 and 30 degrees C. However, as the Arrhenius coefficient is a very sensitive parameter and soil temperature variation was relatively narrow in the field, the Arrhenius coefficient was approximated from the laboratory study. The simulation results obtained were superior to modelling at constant temperature. Field measured persistence of atrazine in the topsoil was underpredicted using the half-life determined in the laboratory at 10 degrees C. Modelling with a lag phase followed by accelerated degradation by use of a sigmoidal degradation equation in Opus2 significantly improved the modelling results. Nevertheless, degradation processes in the laboratory under controlled conditions did not accurately represent field dissipation, however well the laboratory degradation data could be described by simple kinetic equations. The study indicates the importance of improving field techniques for measuring degradation, and developing laboratory protocols that yield degradation data that are more representative of pesticide dynamics in field soils.

Spatial variability of atrazine dissipation in an allophanic soil.

The small-scale variability (0.5 m) of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) concentrations and soil water contents in a volcanic silt loam soil (Haplic Andosol, FAO system) was studied in an area of 0.1 ha. Descriptive and spatial statistics were used to analyse the data. On average we recovered 102% of the applied atrazine 2 h after the herbicide application (CV = 35%). An increase in the CV of the concentrations with depth could be ascribed to a combination of extrinsic and intrinsic factors. Both variables, atrazine concentrations and soil water content, showed a high horizontal variability. The semivariograms of the atrazine concentrations exhibited the pure nugget effect, no pattern could be determined along the 15.5-m long transects on any of the seven sampling days over a 55-day period. Soil water content had a weak spatial autocorrelation with a range of 6-10 m. The dissipation of atrazine analysed using a high vertical sampling resolution of 0.02 m to 0.2 m showed that 70% of the applied atrazine persisted in the upper 0.02-m layer of the soil for 12 days. After 55 days and 410 mm of rainfall the centre of the pesticide mass was still at a soil depth of 0.021 m. The special characteristics of the soil (high organic carbon content, allophanic clay) had a strong influence on atrazine sorption and mobility. The mass recovery after 55 days was low. The laboratory degradation rate for atrazine, determined in a complementary incubation study and corrected for the actual field temperature using the Arrhenius equation, only accounted for about 35% of the losses that occurred in the field. Results suggest field degradation rates to be more changeable in time and much faster than under controlled conditions. Preferential flow is discussed as a component of the field transport process.

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method.

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.

A sensitive LC-MS/MS assay for brevisulcenal and brevisulcatic acid toxins produced by the dinoflagellate Karenia brevisulcata.

A toxic dinoflagellate, Karenia brevisulcata, devastated almost all marine life in Wellington Harbour, New Zealand during the late summer of 1998. Brevisulcatic acids (BSXs) and brevisulcenals (KBTs), both polycyclic ether toxins, have been identified as the causative agents. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the sensitive and specific determination of BSXs and KBTs in culture medium, seawater and shellfish. Acidified algal culture, or seawater, was extracted using reverse phase solid phase extraction cartridges. Shellfish tissue homogenate was blended with methanol-water (9:1) and partitioned with hexane to remove non-polar lipids. This extraction protocol is similar to that used for analysis of lipophilic shellfish toxins. LC-MS/MS (triple quadrupole) was used for quantitative analysis with gradient elution (acidic buffer), positive electrospray ionization and multiple-reaction monitoring. Purified toxins were available for 4 KBTs (KBT-F, -G, -H and -I) and 4 BSXs (-1, -2, -4, and -5), and were used to calibrate the instrument responses. Relative response factors were used for semi-quantitative analysis of BSX-3 and BSX-6, using BSX-1 and BSX-4 respectively. Calibration curves for all toxins monitored were linear over the concentration range tested (5-200 ng mL(-1)) with r(2) values >0.99. The method limit of quantitation was determined to be 2 ng mL(-1) for BSXs and KBTs, except KBT-I, which was 5 ng mL(-1). Validation data was generated for culture medium and shellfish. Toxin recoveries were typically >70% with relative standard deviations <20% across all of the matrices tested. In addition, toxins specific to K. brevisulcata were able to be detected in seawater at a cell concentration of 10,000 cells L(-1), which represents the suggested trigger level for this harmful algal species. This method shows suitable performance characteristics to be regarded a useful tool to monitor toxin levels in a variety of sample matrices during future bloom events.

Brevisulcatic acids, marine ladder-frame polyethers from the red tide dinoflagellate Karenia brevisulcata in New Zealand.

The isolation and structural determination of new marine ladder-frame polyethers, brevisulcatic acids-1 (1) and -4 (2) are reported. Brevisulcatic acids were isolated from the dinoflagellate Karenia brevisulcata, which was identified as the causative species of a major red tide event in New Zealand in 1998. The ether ring composition and a ß-hydroxy, γ-methylene valeric acid side chain of 1 and 2 are common, but 2 has a γ-lactone as the 5-membered A-ring while 1 is the seco acid analogue. Compound 2 has structural and bioactivity similarities to brevetoxin A.