Patrick T. Holland

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC–MS/MS analysis

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.

FIRST REPORT OF THE CYANOTOXIN ANATOXIN‐A FROM APHANIZOMENON ISSATSCHENKOI (CYANOBACTERIA)1

The taxonomy and toxicity of a single‐filament isolate from a filamentous cyanobacterial bloom in Lake Hakanoa (New Zealand) were examined by microscopy and liquid chromatography–mass spectrometry. Based on a morphological examination of environmental and cultured material, strain CAWBG02 was identified as Raphidiopsis mediterranea Skuja; however, subsequent phylogenetic analysis of the 16S rRNA gene sequence demonstrated that CAWBG02 was most likely to be a single culture of Aphanizomenon issatschenkoi (Usacev) Proshkina‐Lavrenko. Toxin testing confirmed that the original bloom and A. issatschenkoi isolate produced anatoxin‐a but did not produce homoanatoxin‐a or any cylindrospermopsins, saxitoxins, or microcystins. Despite the absence of cylindrospermopsin production, genes implicated in the biosynthesis of cylindrospermopsin were successfully amplified from A. issatschenkoi strain CAWBG02. To our knowledge, this is the first confirmation of an anatoxin‐a‐producing species in the Southern Hemisphere and the first report of anatoxin‐a production by A. issatschenkoi.

Widespread Distribution and Identification of Eight Novel Microcystins in Antarctic Cyanobacterial Mats

ABSTRACT 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-Asp3] MC-RR and [d-Asp3] MC-LR containing glycine [Gly1] rather than alanine and combinations of homoarginine [hAr2] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda5] 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.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand

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.

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell(-1)). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.

Survey of cyanotoxins in New Zealand water bodies between 2001 and 2004

Abstract Contamination of drinking and recreational water bodies by toxic cyanobacteria is a significant water management issue in many countries. Until recently, knowledge of the occurrence of cyanotoxins and species responsible for cyanotoxin production in New Zealand was limited. In this study a total of 266 water and cyanobacterial mat samples collected from 227 different water bodies between 2001 to 2004 were analysed for cyanotoxins. Enzyme‐linked immunosorbent assays, liquid chromatography mass spectrometry, high performance liquid chromatography, and neuroblastoma assays resulted in the identification of microcystins/nodularins (102 samples from 54 different water bodies), anatoxin‐a (three samples from three different water bodies), and saxitoxins (48 samples from 41 different water bodies). The highest microcystin concentration was 36.5 mg litre‐1. The detection of anatoxin‐a is the first definitive report for New Zealand. Only low concentrations of saxitoxins were detected. Results indicate that cyanotoxins are more widespread in New Zealand water bodies than previously reported, in particular when cyanobacterial blooms or benthic cyanobacterial mats are present.

Identification of a sapogenin glucuronide in the bile of sheep affected by Panicum dichotomiflorum toxicosis.

Abstract Throughout the world, several hepatogenous photosensitisation diseases of ruminants are characterised by the presence of birefringent crystals in and about the bile ducts. These include photosensitisations caused by Tribulus terrestris (1) (geeldikkop of sheep in South Africa), Nurthecium ossifrugum (2).(alveld of lambs in Norway), Agave lecheguilla (3) (4), Brachiaria decumbens (5) and several Panicum species such as P miliaceum (6), P. coloratum (7), P. schinzii (8) (9), and P. dichotomiflorum (10).

A Chemical Approach to the Determination of Floral Sources of New Zealand Honeys

SummaryThe results of gas chromatagraphic analyses of the concentrated diethyl ether liquid-liquid extracts of 10 honey samples from a variety of floral sources are presented. The chemical composition was compared with the pollen composition and found to be related to the floral source. The gas chromatographic profile of honey extractives is proposed as a guide to the purity of unifloral honeys.

Development of solid phase adsorption toxin tracking (SPATT) for monitoring anatoxin-a and homoanatoxin-a in river water

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.

A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/ MS analysis of cleavage fragments from micro-scale oxidation

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.