[Show abstract][Hide abstract] ABSTRACT: 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 d-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.
[Show abstract][Hide abstract] ABSTRACT: 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 β-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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 r2 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract Portimine, a new polycyclic ether toxin containing a cyclic imine moiety, was isolated from the marine benthic dinoflagellate Vulcanodinium rugosum collected from Northland, New Zealand. The structure of portimine, including the relative configurations, was elucidated by spectroscopic analyses. The cyclic imine moiety consists of an unprecedented five-membered ring with a spiro-link to a cyclohexene ring. This is the only structural similarity to the pinnatoxin group of polycyclic ethers also produced by V. rugosum, which all contain a six-membered cyclic imine ring. The LD50 of portimine to mice by intraperitoneal injection was 1570 μg/kg, indicating a much lower toxicity than many other cyclic imine shellfish toxins. In contrast, portimine was highly toxic to mammalian cells in vitro with an LC50 to P388 cells of 2.7 nM, and activation of caspases indicating apoptotic activity.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of AOAC International 07/2012; 95(4):1097-105. DOI:10.5740/jaoacint.11-272 · 1.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three species of Kareniaceae, Karenia brevis, Karenia brevisulcata and Karenia mikimotoi were assessed for toxicity to juvenile Chinook salmon (Oncorhynchus tshawytscha), snapper (Pagrus auratus) and six species of marine invertebrate larvae (Greenshell mussel, pacific oyster, paua [New Zealand abalone], sea slug, sea urchin and brine shrimp). Karenia brevis was ichthyotoxic to juvenile salmon with a LT50 of 0.75 h at 0.18 × 10 cells L but juvenile snapper were less sensitive (LT50 of 4.5 h at 5.9 × 10 cells L). Karenia brevisulcata also showed differential toxicity to juvenile salmon and snapper with LT50 of 0.48 h at 11 × 10 cells L and 7.5 h at 16 × 10 cells L respectively. However, no acute symptoms were observed in the fish following 48 h exposure to K. mikimotoi at 3.4 × 10 cells L. Karenia brevis at the highest level tested (8.7 × 10 cells L) did not cause observable symptoms in any of the invertebrate larvae over 24 h. Karenia mikimotoi only affected paua larvae (LT50 10 h at 12 × 10 cells L). Karenia brevisulcata was highly toxic to mussel, sea urchin and paua larvae (LT50 1–6.1 h at 7.5–9.6 × 10 cells L), toxic to sea slug and oyster larvae (LT50 6 h at 80 × 10 cells L and 9.2 h at 34 × 10 cells L respectively) but did not affect brine shrimp. Analysis of test cultures by liquid chromatography-mass spectrometry confirmed that K. brevis produced brevetoxins (52 pg cell; mainly brevetoxin-2). Karenia brevisulcata produced brevisulcatic acids (BSXs) and the more toxic Kareniabrevisulcata toxins (KBTs). Karenia mikimotoi did not produce brevetoxin, BSXs or KBTs. The present study confirms the broad threats that blooms of K. brevisulcata pose to marine ecosystems and aquaculture, and the hazards of K. brevis to finfish and K. mikimotoi to abalone respectively.
New Zealand Journal of Marine and Freshwater Research 06/2012; 46(2-2):149-165. DOI:10.1080/00288330.2011.616210 · 0.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of the American Chemical Society 02/2012; 134(10):4963-8. DOI:10.1021/ja212116q · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Recent studies showing the presence of pinnatoxins in a range of shellfish from New Zealand and Australia (Selwood et al., 2010) prompted an investigation into historical mouse bioassay positives from Rangaunu Harbour, Northland, New Zealand. Eleven shellfish samples collected from the harbour between 1993 and 2008 were found to contain pinnatoxins as shown by liquid chromatography–mass spectrometric (LC–MS) analysis. Reanalysis of data from a quantitative mouse bioassay using an acetone extraction gave excellent correlation to pinnatoxin levels for seven of the nine samples tested. The cause of positive mouse bioassay results when using diethyl ether was not confirmed. A telephone survey of 22 Rangaunu Harbour residents confirmed that they had regularly consumed shellfish and other seafood collected from the harbour. Consumption was on average 2.6 times/week over 29 years. There were no reports of any associated illnesses.
[Show abstract][Hide abstract] ABSTRACT: Karenia brevisulcata (Chang), a new toxic dinoflagellate of the genus Karenia was isolated from a harmful algal bloom that occurred in Wellington Harbour, New Zealand in 1998. The bloom severely affected most marine biota resulting in long-term ecological damage and causing respiratory distress in harbour bystanders. Cultures of K. brevisulcata produced a range of novel toxins including ten lipid-soluble K. brevisulcata toxins (KBTs) and six water-soluble brevisulcatic acids (BSXs). Brevetoxins were not detected. KBT-F, KBT-G, BSX-1 and BSX-2 were isolated from 1450 L of bulk cultures and purified in mg quantities. Preliminary chemical and toxicological investigations show that KBT-F (M 2054 C107H160O38) and KBT-G (M 2084 C108H162O39) are complex polycyclic ethers with UVmax at 227 nm. NMR data gave characteristics of ladder frame polyether structures and a 2-methylbut-2-enal side chain, similar to gymnocins. The mouse i.p. LD(50)s for KBT-F and -G were 0.032 and 0.040 mg kg(-1), respectively. These KBTs were also highly cytotoxic and haemolytic. BSX-1 (M 916 C49H72O16) and BSX-2 (M 872 C47H68O15) are polycyclic ether dicarboxylates with UVmax 196 nm. BSX-4 and BSX-5, the lactone ring-closed analogues and the presumed primary toxins in the algal cells, were isolated in smaller quantities. Preliminary structural information from NMR and MS showed a carboxylated side chain and some similarities to brevetoxin-A. However, the structures have not yet been fully elucidated due to conformers confounding the NMR. The mouse i.p. LD50 for BSX-1 was 3.9 mg kg(-1) while no deaths were seen in mice injected with BSX-2 at 6.6 mg kg(-1). The LD(50)s for the lactones BSX-4 and -5 were 1.4 and 1.6 mg kg(-1) respectively. BSX-4 and -5 were agonists of voltage-gated sodium channels but only weakly haemolytic. Activities in the Neuro-2a cytotoxicity assay were ca 10% of dihydrobrevetoxin-2 and were fully antagonised by saxitoxin.
[Show abstract][Hide abstract] ABSTRACT: Vertebrate xenobiotic receptors are ligand-activated nuclear receptors (NRs) that bind exogenous biologically active chemicals before activating the transcription of genes involved in xenobiotic metabolism and excretion. Typically, xenobiotic receptors have ligand binding domains (LBDs) that can accommodate a structurally diverse array of molecules and in addition display high levels of inter-taxa sequence diversity suggestive of positive selection. Pursuing the idea that xenobiotic receptors may adaptively evolve to bind toxic chemicals commonly present in an organism's environment/diet, we examined ligand binding by a xenobiotic receptor orthologue of a marine filter-feeding organism. The solitary tunicate Ciona intestinalis (Phylum Chordata) genome encodes an orthologue of the vertebrate pregnane X receptor (PXR) and vitamin D receptor (VDR), here denoted CiVDR/PXRα. In a luciferase reporter assay the CiVDR/PXRα was activated, at nanomolar concentrations, by two of four natural marine microalgal biotoxins tested (okadaic acid, EC50 = 18.2 ± 0.9 nM and pectenotoxin-2, EC50 = 37.0 ± 3.5 nM) along with 1 of 11 synthetic toxicants (esfenvalerate: EC50 = 0.59 ± 0.7 μM). Two related C. intestinalis NRs, orthologous to vertebrate farnesoid X receptor and liver X receptors, respectively, along with the PXR of a freshwater fish (zebrafish, Danio rerio), were not activated by any of the 15 chemicals tested. In contrast, human PXR was activated by okadaic acid at similar concentrations to CiVDR/PXRα (EC50 = 7.2 ± 1.1 nM) but not by pectenotoxin-2. A common features pharmacophore developed for the CiVDR/PXRα ligand consisted of an off-center hydrogen bond acceptor flanked by two hydrophobic regions. The results of this study are consistent with the original hypothesis that natural toxins, present in the diet of filter-feeding marine invertebrates, may have acted as selective agents in the molecular evolution of tunicate xenobiotic receptors. Bioassays based on tunicate xenobiotic receptor activation may find application in marine environmental monitoring and bioprospecting.
[Show abstract][Hide abstract] ABSTRACT: A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4 L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ = 0.07 day−1 versus 0.05 day−1) and growth maximum (G
max, 169.106 versus 41.106 cells L−1). Maximal gymnodimine concentration (1200 μg L−1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell−1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L−1], though the yield [pg cell−1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793 μg day−1 over 58 days in continuous culture was achieved versus an average of 60 μg day−1 [carboy over 40 days] or 249 μg day−1 [batch mode] over 26 days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25 days, the yield of SPX-desMe-C (29 μg day−1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day−1 and 7.7 μg day−1 respectively). Similarly 5.4 L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12 μg day−1 versus 7 μg day−1). When the bioreactor system was upscaled to 52 L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.
[Show abstract][Hide abstract] ABSTRACT: Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.
Journal of Agricultural and Food Chemistry 05/2011; 59(10):5248-56. DOI:10.1021/jf200839b · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sampling and monitoring for cyanotoxins can be problematic as concentrations change with environmental and hydrological conditions. Current sampling practices (e.g. grab samples) provide data on cyanotoxins present only at one point in time and may miss areas or times of highest risk. Recent research has identified the widespread distribution of anatoxin-producing benthic cyanobacteria in rivers highlighting the need for development of effective sampling techniques. In this study we evaluated the potential of an in situ method known as solid phase adsorption toxin tracking (SPATT) for collecting and concentrating anatoxin-a (ATX) and homoanatoxin-a (HTX) in river water. Fifteen different adsorption substrates were screened for efficiency of ATX uptake, nine of which retained high proportions (>70%) of ATX. Four substrates were then selected for a 24-h trial in a SPATT bag format in the laboratory. The greatest decrease in ATX in the water was observed with powdered activated carbon (PAC) and Strata-X (a polymeric resin) SPATT bags. A 3-d field study in a river containing toxic benthic cyanobacterial mats was undertaken using PAC and Strata-X SPATT bags. ATX and HTX were detected in all SPATT bags. Surface grab samples were taken throughout the field study and ATX and HTX were only detected in one of the water samples, highlighting the limitations of this currently used method. Both Strata-X and PAC were found to be effective absorbent substrates. PAC has the advantage that it is cheap and readily available and appears to continue to sorb toxins over longer periods than Strata-X. SPATT has the potential to be integrated into current cyanobacterial monitoring programmes and would be a very useful and economical tool for early warning of ATX and HTX contamination in water.
[Show abstract][Hide abstract] ABSTRACT: A peridinoid dinoflagellate was newly identified as the producer of pinnatoxins E (0–3.7 pg cell−1) and F (0.3–20.1 pg cell−1), as determined by LC–MS analysis of extracts of eight strains of the organism. The cyst-forming, thecate dinoflagellate was isolated from surface sediments associated with eel grass beds and mangroves in Rangaunu Harbour, Northland, New Zealand. Extracts of mass cultures of the dinoflagellate were tested for toxicity in mice by intraperitoneal injection, gavage and voluntary consumption. The LD50 values were 1.33, 2.33 and 5.95 mg/kg respectively.
[Show abstract][Hide abstract] ABSTRACT: Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.