C. Hummert

Discrimination of the toxigenic dinoflagellates Alexandrium tamarense and A. ostenfeldii in co-occurring natural populations from Scottish coastal waters

Blooms of the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech, a known producer of potent neurotoxins associated with paralytic shellfish poisoning (PSP), are common annual events along the Scottish east coast. The cooccurrence of a second Alexandrium species, A. ostenfeldii (Paulsen) Balech & Tangen is reported in this study from waters of the Scottish east coast. The latter species has been suspected to be an alternative source of PSP toxins in northern Europe. Recent identification of toxic macrocyclic imines known as spirolides in A. ostenfeldii indicates a potential new challenge for monitoring toxic Alexandrium species and their respective toxins in natural populations. In mixed Phytoplankton assemblages, Alexandrium species are difficult to discriminate accurately by conventional light microscopy. Species-specific rRNA probes based upon 18S and 28S ribosomal DNA sequences were developed for A. ostenfeldii and tested by dot-blot and fluorescence in situ hybridization (FISH) techniques. Hybridization patterns of A. ostenfeldii probes for cultured Alexandrium isolates, and cells from field populations from the Scottish east coast, were compared with those of rDNA probes for A. tamarense and a universal dinoflagellate probe. Alexandrium cell numbers in field samples determined by whole-cell in situ hybridization were much lower than those determined by optical microscopy with the Utermöhi method involving sedimentation chambers, but the results were highly correlated (e.g. r 2 = 0.94; n = 6 for A. tamarense). Determination of spirolides and PSP toxins by instrumental analysis on board ship demonstrated the presence of both toxin groups in plankton assemblages collected from surface waters near the Orkney Islands, and confirmed the association of A. ostenfeldii with spirolides in northern Europe. These results show that rRNA probes for A. tamarense and A. ostenfeldii are useful, albeit only semi-quantitative, tools to detect and discriminate these species in field studies.

A Modified HPLC Method for Analysis of PSP Toxins in Algae and Shellfish from China

SummaryImprovements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins reponsible for paralytic shellfish poisoning (PSP). The PSP toxin cotent of two strains ofAlexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

A Recent Shellfish Toxin Investigation in China

A shellfish toxin investigation along the Chinese coast has recently been conducted using both HPLC and mouse assay methods. The results showed that DSP was widely distributed in different shellfish species in China. 26 out of 89 samples had DTX1 (dinophysistoxin-1) or OA (okadaic acid) but the DSP content in most shellfish samples did not reach the regulatory limit for human consumption adopted in many countries (20 mu g/100 g soft tissue). PSP was also found in 5 out of 96 samples along the coast. One sample, Chlamys nobilis from Hong Kong contained high levels of PSP (320 mu g STX equivalent/100 g soft tissue), compared to the regulatory limit (80 mu g STX equivalent/100 g soft tissue). After the recent outbreak of red tide in Hong Kong waters, three further shellfish samples were collected within 40 days to investigate the impacts of this event, It was shown that high levels of PSP continued to exist in Hong Kong waters. This report provides the first report of DSP and PSP distribution along the Chinese coast

Analysis of the characteristic PSP profiles ofPyrodinium bahamense and several strains ofAlexandrium by HPLC based on ion-pair chromatographic separation, post-column oxidation, and fluorescence detection

SummaryA sensitive HPLC method for determination of paralytic shellfish poisoning (PSP) based on ion-pair chromatographic separation of PSP toxins, post-column oxidation with periodic acid, and fluorescence detection has been used to determine toxin profiles ofPyrodinium bahamense and several strains ofAlexandrium. The HPLC chromatograms revealed clear differences betweenPyrodinium bahamense andAlexandrium strains.

Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography

An efficient LC method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins based on ion-exchange chromatographic separation of the toxins followed by electrochemical post-column oxidation and fluorescence detection as well as mass spectrometric (MS) detection. The method can be applied to the determination of PSP toxins in phytoplankton and to control seafood for PSP content.

Simultaneous Analysis of Different Algal Toxins by LC-MS

SummaryA method is proposed for the simultaneous determination of amnesic shellfish poisoning (ASP) toxin, diarrhetic shellfish poisoning (DSP) toxins spirolides, azaspiracids (AZP), pectentotoxins (PTX), brevetoxins (PbTx), and gymnodimine. After extraction of all these toxins with one solvent only the crude extract is subjected directly to reversed-phase LC-MS with atmospheric-pressure ionization.The method was applied to bulk plankton samples obtained during a research cruise off the east coast of Scotland in May, 2000. Contamination of plankton samples from one spot with different toxins from a variety of groups was determined.

Determination of chloramphenicol in animal tissue using high-performance liquid chromatography with a column-switching system and ultraviolet detection

A rapid and universally applicable method for determination of chloramphenicol (CAP) residues in animal tissues using high-performance liquid chromatography with a column-switching system is presented. The clean-up procedure as well as the chromatographic conditions and detection are described. The linearity and repeatability of the data obtained by this method as well as the recovery rates of CAP in several farm animals are presented.

Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia, Germany).

The presence of microcystins in cyanobacterial samples collected from the Bleiloch reservoir, formerly an important drinking-water supply in Thuringia, Germany, was proven by application of a combination of recently developed analytical methods. The raw extracts were cleaned by size-exclusion chromatography (SEC) or solid-phase extraction (SPE). The determination of microcystins was achieved by different HPLC separation followed by the application of alternative detection methods (UV, diode array detection (DAD), and mass spectrometry (MS), respectively). Furthermore, the different results of clean-up by SPE and SEC are demonstrated. The identity of microcystins was verified by MS/MS measurements. In the cyanobacterial sample from 1998, microcystin-RR, -LR and -YR were found, whereas in 1999 only microcystin-LR and -YR were detectable. In addition to detection of cell-bound microcystins, in 1999 traces of dissolved microcystins in water from the Bleiloch reservoir were detected. It can be assumed that not only the Bleiloch reservoir is contaminated with hepatotoxins but also many similar lakes still used for drinking water supply.

Automatic HPLC-UV determination of domoic acid in mussels and algae

SummaryA rapid and sensitive automatic method is presented for the determination of domoic acid (DA) using HPLC with a column-switching system and UV-detection. Interfering peaks resulting from matrix protein components are excluded by use of an especially designed reversed-phase HPLC column for pre-separation. The method is suitable for extracts both from mussels and from algae. Sample material is extracted with pure water and the crude extract is injected directly. Application of a column-switching system eliminates the need for any further sample clean-up after extraction.

Rapid clean-up and effective sample preparation procedure for unambiguous determination of the cyclic peptides microcystin and nodularin

SummaryA new sample preparation strategy has been established to improve the identification and determination of nodularin and microcystins. The sample preparation consisted of enrichment of the analytes by solid phase extraction with C18 cartridges followed by clean-up of the enriched raw extracts by high performance size exclusion gel permeation chromatography. In contrast to established clean-up procedures based on polarity, related distribution of microcystins and nodularin in non-miscible phases (e. g. a C18 cartridge as stationary phase and a water-containing eluent as mobile phase) this strategy separates microcystins from interfering compounds by molecular size differences.The sample preparation procedure can be automated easily and was validated for both water samples as well as raw extracts of algal cells. The method was success-fully applied during an experiment with natural algae communities from the Baltic Sea to investigate the influence of different nutrient limitations on toxicity ofNodularia sp...