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The control of food with regard to contamination with phycotoxins to comply strictly with national regulations is an important task. This chapter discusses the classes, incidence, chemical structure and toxicity of the two major groups of phycotoxins, the marine biotoxins and the cyanobacterial toxins. Analytical approaches for the determination of the different phycotoxins are also covered, with special focus on methods based on liquid chromatographic separation and tandem mass spectrometric detection (LC-MS/MS).

Contamination of water, foods and food supplements by various genera of cyanobacteria is a serious health problem worldwide for humans and animals, largely due to the toxic effects of microcystins (MCs) and nodularin (NOD), a group of hepatotoxic cyclic peptides. The toxins occur in variable structures resulting in more than 90 different MCs and 8 different NODs, many of them not having been investigated for their toxic potency. Potent MCs such as MC-LR have been shown to elicit their hepatotoxic potency via inhibition of hepatic protein phosphatases (PP) 1 and 2A leading to over-phosphorylation of vital cellular proteins. This mechanism of action is also thought to be responsible for the long term tumor promoting action of certain MCs and NOD in the liver. Here, we report on the isolation of certain MCs and NOD as well as a number of their desmethylated derivatives from algae bloom. Subsequently, we determined the cytotoxicity of these compounds in isolated primary human and rat hepatocytes in culture. In parallel experiments, we analyzed the inhibitory potency of these congeners on PP1 and 2A using commercially available enzymes. We found in primary rat hepatocytes that MC-LR, -YR and NOD were cytotoxic, namely in the 10 to >50 nM range, while MC-RR was not. The desmethylated congeners of MC-LR, -YR, and NOD were equally or more-toxic as/than their fully methylated counterparts. In primary human hepatocytes we could show that MC-LR, NOD and the desmethylated variants [³Asp]MC-LR, [⁷Dha]MC-LR and [¹Asp]NOD were cytotoxic in the 20 to >600 nM range. Inhibition data with human, bovine and rabbit protein phosphatases 1 and 2A were roughly in accordance with the cytotoxicity findings in human and rat hepatocytes, i.e. desmethylation had no pronounced effects on the inhibitory potencies. Thus, a variety of naturally occurring desmethylated MC and NOD congeners have to be considered as being at least as toxic as the corresponding fully methylated derivatives.

Microcystis aeruginosa isolated in 2005 from the shallow eutrophic Lake Chao (Anhui, China) was investigated in terms of growth parameters and microcystin production under varying nutrient concentrations (P, N) and pH values (abiotic factors) as well as under the influence of spent medium of the non-toxic cyanobacterium Synechocystis sp. (biotic factors). Stimulating effects on growth were observed at the alkaline pH value (10.5), whereas toxin production was significantly increased under phosphate-P limitation (0.6 mg L(-1) medium). Within a broad range of nitrate-N concentrations (41.2-247.2 mg L(-1) medium), no significant influence on cell growth and microcystin production was observed; however, N-starvation resulted in a typical decrease of growth and toxicity. In addition, cryopreservation of M. aeruginosa evidenced the decrease of toxin production by time-dependent exposure with the cryoprotectant dimethyl sulfoxide under thawing conditions without affecting the growth of the cyanobacterial cells.

Contamination of bivalves with yessotoxins (YTXs) has been reported since 1987 in several coastal environments. In our study we investigated the accumulation and the metabolism of YTXs in 2 bivalve species important to German fisheries and aquaculture. Mussels and oysters, Mytilus edulis and Crassostrea gigas, were exposed to the YTX-producing dinoflagellate Protoceratium reticulatum isolated from the North Sea. Toxin profiles from algae and bivalves were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). It was evident that 45-hydroxy YTX (45-OH YTX), carboxy YTX (COOH YTX), and the putative 45-hydroxy-carboxy YTX (45-OH-COOH YTX) were the dominant YTX analogues in M. edulis. The oyster C. gigas displayed a different toxin profile. YTX, 45-OH YTX, and 45-OH-COOH YTX were also present, but COOH YTX was not detectable. Furthermore, 2 unidentified analogues with the same MS/MS transition ((M-H) - > (M-H-SO3) - ) occurred in both bivalve species. Homo YTX and 45-hydroxy-homo YTX, which are regulated together with YTX and 45-OH YTX in 2002/225 EC were not detected. In general, we confirmed former field investigations on accumulation and metabolism of YTXs in M. edulis; however, this is the first study on accumulation and metabolism of YTXs in the oyster C. gigas.

International audience; Lake Chao, China, is highly eutrophicated and experiences recurrent dense cyanobacterial blooms. Its surface water is used as drinking water resource for Hefei city, hence the potential toxicity of those cyanobacteria was of interest. Sixteen isolated strains of Microcystis aeruginosa evidenced that non-toxic, toxic and highly toxic strains coexist in the lake. Microcystin variants within one strain ranged up to 11, the concentration up to 4.799 mg g DW1. Mass spectrometry analysis confirmed desmethylated microcystin variants.

Ciguatera fish poisoning characterizes the intoxication caused by consumption of fish from tropical and subtropical areas, which have accumulated ciguatoxins (CTXs). The observed pattern of ciguatoxins in fish highly depends on the marine region and the causative organisms. It is evident that differences exist between ciguatoxins produced by certain strains of the dinoflagellate Gambierdiscus toxicus and other Gambierdiscus spp. In this context cultured strains purchased from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) and strains from Vietnam were analyzed. Besides, lyophilized samples of several Gambierdiscus spp. from the National Oceanic and Atmospheric Administration (NOAA), USA and lyophilized samples of G. toxicus from Vietnam were analyzed. The latter has been cultured at different salinities. We observed differences between the toxin ratios of the analogues in the strain from Vietnam depending on the salinity. The CTX profiles of the Vietnamese samples were compared with cultures of Gambierdiscus spp. from CCMP and the National Oceanic and Atmospheric Administration (NOAA) resulting in an overview of toxins in cultures from different regions. Hence, it was obvious that the strain from Vietnam forms a characteristic CTX profile which is not directly comparable to CTX pattern observed in other tropical marine regions.

Sturgeon (Acipenseridae) have been subjected to intense fisheries pressure due to their highly-priced eggs. Despite the fact that all 27 species are highly endangered and have been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), regulating international trade, illegal catches still continue to threaten wild populations. In trade, the product is suspected to be camouflaged as caviar from aquaculture origin. Thus, tools to discriminate wild and aquaculture products have to be developed to enforce CITES regulation and thereby allow for the development of the aquaculture industry. Here, the feasibility to use food additives, namely fatty acids and vitamin E, is assessed for labelling caviar from aquaculture origin. Farmed female sterlet (Acipenser ruthenus) were fed on a diet supplemented with alpha-tocopherol, caprylic and capric acid at 15, 4 and 20 mg/g respectively. Marker concentrations in the caviar were determined from biopsies sampled via micro-incisions over a period of 90 days (0 d, 10 d, 60 d and 90 d) and compared to a control fed on a diet supplemented with 39 mg/g sunflower oil. To address the question whether the effectiveness in using the markers is dependent on strict timing with regard to gonad development and maturation, maturing (early vitellogenesis, 4 year old fish) and mature (late vitellogenesis, stage IV–V) females were studied. The latter represent fish that were ready to be used for caviar production. Neither alpha-tocopherol nor caprylic acid accumulated in the caviar and concentrations quantified by GC–MS were below the detection limit (

The accumulation of saxitoxins (STXs) in fish from freshwater aquaculture was investigated for the first time in the present study. Cyanotoxins have been monitored in liver and muscle samples of Oreochromis niloticus by chromatographic methods, both before and after the depuration process. The results show that tilapia can accumulate STXs. Our findings suggest that depuration with clean water is an alternative process to eliminate STXs from fish and, therefore, improve the safety of tilapia for consumers.

A comparative PCR analysis of the nodularin synthetase gene cluster (nda) of two closely related Nodularia strains from the Baltic Sea was performed. Unlike the nodularin-producing Nodularia spumigena Huebel 1988/306, all 11 tested nda sequences were absent in the non-toxic Nodularia harveyana Huebel 1983/300. Our results suggest that the benthic N. harveyana strain lacks the entire 48-kb nodularin synthetase gene cluster. We describe new primers and the requisite experimental conditions for screening different nda sequences.

Spirolides are biologically active macrocycles isolated first from scallops and phytoplankton from aquaculture sites in Nova Scotia, Canada. These compounds displayed "fast-acting" toxicity in the traditional bioassay. That phenomenon is related to the presence of a cyclic imine function in these compounds. Spirolides containing vicinal methyl groups in their seven-membered ring are suspected of being resistant to hydrolysis. We studied possible conversions of vicinal methyl groups wearing spirolides of Alexandrium ostenfeldii KO287 in enzymatic cell-free tissue extracts of Mytilus edulis, Pecten maximus and Crassostrea gigas. Our observations suggest that spirolides that contain an extra methyl group on the imine ring compared with spirolide A and B survive enzymatic hydrolysis conditions in shellfish and therefore may be toxic for human beings when shellfish is consumed.

Tetrodotoxin (TTX) and its analogs (TTXs), widely distributed among marine as well as terrestrial animals, induce dangerous intoxications. These highly potential toxins are also known as the causative agent of puffer fish poisoning. A newly developed highly sensitive method for determination of TTXs based on hydrophilic interaction chromatography and mass-spectrometric detection is presented. TTX, anhydrotetrodotoxin, 11-deoxytetrodotoxin and trideoxytetrodotoxin were determined in separated tissues of Bangladeshi marine puffers, Takifugu oblongus. TTX was predominant in skin, muscle and liver, whereas trideoxytetrodotoxin preponderated in the ovary. The toxicity of the various tissues was determined by a mouse bioassay.

The growth and toxin production of a Philippine Pyrodinium bahamense isolate in nutrient replete batch cultures were investigated under conditions affected by varying salinity, temperature and combined effects of salinity and temperature. Early exponential growth stage was reached after 7 days with a cell division rate of 0.26 div day(-1). The toxin content reached a peak of 298 fmol cell-1 at mid exponential phase and rapidly declined to 54 fmol cell-1 as it approached the death phase. Only three sets of toxins composed of STX, dcSTX and B1 were detected in which STX made up to 85-98 mol%toxincell-1. P. bahamense was able to grow in salinities and temperatures ranging from 26 per thousand to 36 per thousand and 23 to 36 degrees C, respectively. The optimum growth under varying salinity and temperature conditions was observed at 36 per thousand and 25 degrees C. Toxin content reached a peak of 376 fmol cell-1 at 25 degrees C and was lower (80-116 fmol cell-1) at higher temperatures (32-35 degrees C). Combined effects of salinity and temperature showed that P. bahamense was not able to grow at low salinity and temperature (i.e. below 26 per thousand-28 degrees C). Optimum growth was observed in higher salinities at all temperature conditions.

A novel method for paralytic shellfish poisoning (PSP) toxins which is based on the chromatographic separation of the toxins using a zwitterionic (ZIC) hydrophilic interaction chromatography (HILIC) column is presented. Efficient retention of the polar PSP toxins on the ZIC-HILIC column allowed their selective and sensitive determination by the application of mass spectrometric (MS/MS) detection or as derivatives after oxidation prior to fluorescence detection (FD). Low buffer concentrations and the omission of ion-pair reagents decreased the limits of detection (LODs) by MS/MS analysis and showed a good linearity for both methods of detection. This method can be applied for the qualitative and quantitative determination of PSP toxins in various types of phytoplankton, and for the routine analysis of seafood.

Many in vivo shellfish feeding experiments have been carried out in order to investigate the fate of PSP toxins in the marine food chain. A focal point of these studies concerns the species- and tissue-specific differences in toxin metabolism. However, tissue specific effects are often overlapped by selective toxin retention as well as transfer between individual compartiments. In in vitro experiments presented here digestive tissue and adductor homogenates of 10 shellfish species (bivalvia: Mytilus edulis, Crassostrea gigas, Cardium edule, Arctica islandica, Ensis ensis, Modiolus modiolus, Mactra stultorum, Pecten maximus as well as two snails: Littorina littorea and Buccinum undatum) were incubated with an extract of the toxic strain Alexandrium fundyense CCMP 1719. After incubation, changes in the toxin pattern could be observed in all samples with significant differences occuring between both the species and tissues. The greatest metabolic activity was found in digestive tissue samples. Among the organisms, the species with a non-filtering lifestyle, L. littorea and B. undatum, showed the highest conversion rates. Interestingly, the high metabolic transformation rate of the PSP toxins was accompanied with a fast reduction (up to 73%) of toxicity in the homogenates.

Thecadinium yashimaense was recorded for the first time in France, Great Britain, The Netherlands, and Germany. The invasion and establishment of the species in the German Bight was documented reliably and is presented here. The geographic expansion of the species from the North Pacific to the North Atlantic Ocean is discussed. This bloom-forming, marine, sand-dwelling dinoflagellate was shown to be non-toxic. Also Thecadinium kofoidii, the type species of the genus, was analyzed for potential toxin production and turned out to be non-toxic as well.

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins is diarrhetic shellfish poisoning (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs), and yessotoxins (YTXs). The most recently described shellfish poisoning syndrome, azaspiracid shellfish poisoning (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia.

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. Therefore, many efforts have been made to determine algal toxins using different chemical methods. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. , which have been traditionally separated by ion pair chromatography followed by derivatization and fluorescence detection. However, the ion pair reagents added to the mobile phases are not compatible with mass spectrometry, so a new chromatographic system had to be developed. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins are diarrhetic shellfish poising (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). The latter two groups are often associated with diarrhoea causing DTXs, but recently turned out not to have diarrhetic effects by themselves, which means in the strict sense they are no DSP toxins. For this reason the term “lipophilic” toxins rather than DSP is increasingly applied to for this group of toxins. The most recently described shellfish poisoning syndrome, azaspiracid shellfish poising (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, São Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in São Paulo.

In vitro experiments were performed with Gymnodinium catenatum Graham strains isolated from three locations in the Gulf of California to determine the variability in toxicity and toxin profiles. Strains were cultivated in GSe at 20° C ± 1° C, 150 μmol photons·m -2 ·s -1 (12:12 light:dark cycle), and harvested during different growth phases. Growth rates were higher than in previous studies, varying between 0.70 and 0.82 day -1 . The highest cell yields were reached at 16 and 19 days, with maximum densities between 1090 and 3393 cells· mL -1 . Bahia de La Paz (BAPAZ) and Bahia de Mazatlan (BAMAZ) were the most toxic (101 pg STXeq-cell 1 ), whereas strains from Bahia Concepcion (BACO) were the least toxic (13 pg STXeq·cell -1 ). A strain isolated from cyst germination was one of the least toxic strains. No significant changes in toxin content with culture age were observed (0.2 and 0.6pmol paralytic shellfish poisoning·cell -1 ). All strains contained neosaxitoxin (NEOSTX), decarbamoyl-saxitoxin (dcSTX), decarbamoyl-gonyautoxin-2,-3, (dcGTX2-3), N-sulfocarbamoylsaxitoxin (Bl), N-sulfo-carbamoylneosaxitoxin (B2), and N-sulfo-carbamoylgonyautoxin-2,-3 (C1-2). Bahia Concepcion strains had the highest content of Cl; BAPAZ and BAMAZ strains had a higher percentage of NEOSTX. Differences in toxin composition with culture age were observed only in BAMAZ and BAPAZ strains. Cultures with a higher percentage of long chains had more NEOSTX, while those with a higher proportion of short chains had a lower content of NEOSTX. Gulf of California strains are characterized by a high proportion of NEOSTX, and seem to have evolved particular physiological responses to their environment that are reflected in the toxin profile, suggesting different populations.

Over the past years the importance of food additives and the development of so-called novel food increased permanently. Especially, the application of dietary supplements was on the rise. Then, more and more new products based on plants hitherto not used for human consumption were launched. Algae products containing valuable amounts of essential nutrients such as amino acids and trace elements play a decisive role. On the other hand, some algae including the blue-green algae (cyanobacteria) are capable of synthesizing harmful substances depending on species and provenience. Therefore, methods must be available to evaluate possible risks caused by toxins in algae-based dietary supplements. There are different groups of toxins related to marine algae and cyanobacteria. However, both marine algae and cyanobacteria are able to produce Paralytic Shellfish Poisoning (PSP) toxins which are potential neurotoxins. Hence, analytical methods for PSP determination have to be developed. The method for PSP toxin determination described below is based on ion-pair chromatography of the underivatized PSP toxins followed by post-column oxidation and fluorescence detection (FD). The determination of very low amounts of PSP toxins in different matrices of novel food is possible. In addition, the method allows to compare PSP profiles of various algae-based dietary supplements.

Gymnodinium catenatum Graham is a paralytic shellfish poison (PSP) producer that was described for the first time from the Gulf of California in 1943. During the last decade, its distribution along the Mexican Pacific coastline has increased. In Bahia Concepcion, a coastal lagoon on the western side of the Gulf of California, G. catenatum has been linked to significant PSP concentrations found in mollusks. In this study, we describe the saxitoxin profile of 16 strains of G. catenatum , and catarina scallops ( Argopecten ventricosus ) from Bahia Concepcion. Toxins were analyzed by HPLC with post-column oxidation and fluorescence detection. The average toxicity of the G. catenatum strains was 26.0±6.0 pg and 28.0±18.0 pg STX eq/cell after 17 and 22 days of growth, respectively. Ten toxins were recorded, but only dcSTX, dcGTX2, dcGTX3, C1, and C2 were always present in all strains at both growth stages. Since toxin profiles in scallops were similar to the cultures, biotransformations are not significant in catarina scallop. NeoSTX, GTX2, GTX3, and B2 were present in some G. catenatum strains and their presence varied with the age of the culture. In scallop samples, dcSTX, dcGTX2, and dcGTX3 were the most abundant toxins, and from the C-toxin group, only C2 was found. This unique toxin profile can be used as a biomarker for this population, when compared with strains of G. catenatum from other geographic regions.

The frequency and intensity of harmful algal blooms (HABs) appear to be on the rise globally. There is also evidence of the geographic spreading of toxic strains of these algae. Consequently, methods had to be established and new ones are still needed for the evaluation of possible hazards caused by increased algal toxin production in the marine food chain. Different clinical effects of algae-related poisoning have attracted scientific attention; paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning are among the most common. Additionally, cyanobacteria (blue-green algae) in brackish waters often produce neurotoxic and hepatotoxic substances. Bioassays with mice or rats are common methods to determine algal and cyanobacterial toxins. However, biological tests are not really satisfactory because of their low sensitivity. In addition, there is growing public opposition to animal testing. Therefore, there has been increasing effort to determine algal toxins by chemical methods. Plankton samples from different European marine and brackish waters were taken during research cruises and analyzed on board directly. The ship routes covered marine areas in the northwest Atlantic, Orkney Islands, east coast of Scotland, and the North and Baltic seas. The first results on the occurrence and frequency of harmful algal species were obtained in 1997 and 1998. During the 2000 cruise an HPLC/MS coupling was established on board, and algal toxins were measured directly after extraction of the plankton samples. In contrast to earlier cruises, the sampling areas were changed in 2000 to focusing on coastal zones. The occurrence of toxic algae in these areas was compared to toxin formation during HABs in the open sea. It was found that the toxicity of the algal blooms depended on the prevailing local conditions. This observation was also confirmed by monitoring cyanobacterial blooms in the Baltic Sea. Optimal weather conditions, for example, during the summers of 1997 and 2003, favored blooms of cyanobacteria in all regions of the Baltic. The dominant species regarding the HABs in the Baltic was Nodularia spumigena. However, in addition to high concentrations of Nodularia spumigena in coastal zones, other blue-green algae are involved in bloom formation, with changes in plankton communities influencing both toxin profiles and toxicity.

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.

A 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.

A fast fluorimetric assay (FFA) for the detection of saxitoxins in plankton samples was compared with the analysis by high performance liquid chromatography (HPLC). The correlation between the results of the assay and those of the HPLC is significant for most of the carbamoyl saxi- toxins. During research cruises to the Orkney Islands and the Firth of Forth (Scotland) in 1997 and 1998, samples of toxic Alexandrium spp. blooms were analyzed successfully by FFA and HPLC. Because the toxin pattern of toxic Alexandrium spp. mostly consists of highly fluorescent saxitoxins (like STX and GTX3), the assay can be applied for monitoring toxic Alexandrium spp. blooms and is suggested as a tool for future prewarning systems of paralytic shellfish poisoning (PSP) events.

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.

The toxins of cyanobacteria include potent neurotoxins and hepatotoxins. Deaths after exposure by ingestion of cyanobacterial cells and toxins from freshwater sources have been reported. The hepatotoxins are cyclic peptides, which comprise microcystins (MC) and nodularin (NOD), and they cause poisonings with an established syndrome of human and animal illnesses. The identification and determination of these hepatotoxic peptides are challenges for analytical chemists. Methods based on solid phase extraction and size exclusion chromatography are available for the clean up of raw extracts. Both principles are also used for the enrichment of MC from low contaminated sample material. The separation of MC is achieved by high-pressure liquid chromatography methods with ultraviolet (UV) detection or mass spectrometric detection. The unambiguous identification of MC not available as standards is possible by mass spectrometry (MS) or, under optimal conditions, by UV spectrometry using diode array detection. To date, 60 different MC and four NOD have been described, but new compounds are frequently identified. Therefore, powerful tools for the elucidation of the structure of unknown MC are an important need. In addition to classical methods for structure elucidation (e.g. nuclear magnetic resonance spectrometry), microwave-assisted hydrolysis of cyclic peptides followed by enantio-selective determination of the amino acid profiles can be used. More recently, MS with collision-activated dissociation has become an important tool for obtaining structure information. The present paper discusses the advantages and disadvantages of the various analytical techniques used for monitoring of cyanobacteria in lakes, optimized or developed at the Institute of Nutrition, Jena, Germany. A pathway for handling MC-containing samples with regard to different analytical tasks (i.e. the identification and elucidation of structure followed by qualitative and quantitative determination) is proposed.

The elution orders of 20 hexa- to nonachlorobornanes and five hexa- to octachlorocamphenes were studied with normal-phase silica and amino phase HPLC, reversed-phase HPLC, as well as gel-permeation chromatography (GPC). Twenty-one compounds of technical toxaphene (CTTs) are commercially available and four were isolated from environmental samples. Structure-activity relationships and chromatographic properties were deduced from the data sets derived on these LC systems. The retention on silica (low-resolution LC and HPLC) increased with the polarity of the CTTs. The elution order of CTTs on amino normal-phase HPLC was, for the most part, the same as on silica normal-phase HPLC. The degree of chlorination determined the elution order of CTTs on C18 RP-HPLC. CTTs eluted from medium-pressure GPC with decreasing molecular size. Chlorobornanes with dichloro substituents on the six-membered ring eluted after the chloroboranes without geminal chlorine atoms on secondary carbons, indicating that these congeners are larger. Altogether, the results increase the knowledge of complex substance class and may serve as a tool in order to gain further standard components.

A new analytical technique was established to improve the performance of DSP toxin determination in different sample matrices. High performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algal cells and mussel tissue prior to the determination of DSP toxins by LC/MS. The proposed protocol can be performed totally automatically and enables fast and sensitive analysis of large sample numbers. The recovery of the entire method protocol (consisting of extraction, clean-up and LC/MS determination) was approximately 70% with good repeatability (standard deviation ranging from 1.9% to 5.0% in the concentration range analyzed).

Organochlorines (DDT, toxaphene, PCBs, chlordane, HCHs, dieldrin) were quantified in the blubber of eleven fur seals of the species Arctocephalus pusillus pusillus. The sample clean-up included microwave-assisted extraction (MAE), gel-permeation chromatography (GPC), adsorption chromatography on silica gel and quantitation with GC/ECD. Compounds of technical toxaphene (CTTs) were determined after separation from the PCBs. Highest levels arose for p , p ′-DDE (7–1000 μg/kg). A so far unknown compound labelled Q1 caused the second most abundant peak in the samples. Q1 was identified as a heptachloro compound by GC/MS in the electron capture negative ionisation and electron impact ionisation mode. Levels of Q1 were estimated relative to the ECD response factor of trans -nonachlor. These estimated Q1 levels ranged from 27 to 351 μg/kg being in the range or higher than the levels of PCBs (6–697 μg/kg), toxaphene (10–97 μg/kg), chlordane (3–159 μg/kg), and dieldrin (4–36 μg/kg). One sample which showed significantly higher DDT and PCB levels (but not toxaphene, dieldrin, Q1, and chlordane) than the others is most likely migrated from an area higher polluted with DDT and PCBs. Another one showed very low levels which were not explained. Except these samples, the levels determined in the blubber were among the lower levels reported for this kind of tissue. The data also confirmed that the organochlorine levels in Africa are varying from region to region, and that there is still a need for further research.

A 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...

The study of the total synthesis of toxaphene was the topic of the present investigation. Dichloromethyl groups should be introduced as carboxylic functions and later be converted. Strategy for the formation of the bornane backbone was the Diels-Alder addition of cyclopentadiene and anhydrous methylmaleic acid followed by Wagner-Meerwein rearrangement. However, the rearrangement failed, but chlorination of the double bond of the cycloadduct with MnCl2/acetic chloride, reduction of the carboxylic groups with LiAlH4 to the diol, subsequent esterification with p-tosyl chloride, and finally substitution of the tosylates by chloride using LiCl led to 5-exo,6-exo,8,10-tetrachlorodihydrocamphene and 5-exo,6-exo,8-trichlorocamphene. The mechanistic study revealed pathways for the formation of polychlorinated camphenes and dihydrocamphenes whose presence in toxaphene has been reported before but not mechanistically explained.

Microwave radiation has been successfully used for hydrolysis of the hepatotoxic cyclic peptides microcystins and nodularin. Set-up of the microwave device and the operating conditions for microwave hydrolysis were optimized. Results of the microwave hydrolysis were compared with results from conventional hydrolysis for 24 h at 110°C.

Enantioseparations of 26 chiral organochlorine compounds (α-hexachlorocyclohexane, toxaphene, chlordane, atropisomeric polychlorinated biphenyls) were studied with GC–electron-capture detection. The chiral stationary phases (CSPs) consisted of 35% heptakis (6 -O-tert. -butyldimethylsilyl-2,3-di -O- methyl)-β-cyclodextrin (β-TBDM) diluted in OV1701. One CSP was made of pure β-TBDM (purity>99%) while the other was randomly silylated β-TBDM. High-temperature gas chromatography showed that this CSP contained five products in almost equal amounts. Both β-TBDM columns were installed in parallel in the same GC system. On the randomly silylated β-TBDM, aromatic compounds had shorter and aliphatic compounds had longer retention times than on the purified β-TBDM. The randomly silylated β-TBDM resolved the enantiomers of 24 compounds (16 of them were baseline separated) and the purified β-TBDM only six (four of them were baseline separated). Thus, the purification of β-TBDM decreased the efficiency of the chiral resolution of organochlorine compounds on β-TBDM. The results clearly demonstrate that the randomly silylated β-TBDM contains one or more side products more suitable for the enantioseparation of organochlorine compounds than the purified β-TBDM.

Levels of organochlorines (PCBs, DDT, toxaphene, chlordane, hexachlorobenzene (HCB), hexachlo-rocyclohexanes (HCHs), dieldrin, Q1) were determined in eggs of both penguins (Adelie Pygoscelis adeliae, Chinstrap Pygoscelis antarctica, Gentoo Pygoscelis papua) and skuas (South Polar Skua Catharacta maccormicki, Brown Skua Catharacta antarctica lonnbergi, Mixed Pair Skua Catharacta maccormicki x lonnbergi) from the Antarctic. Focused open-vessel microwave-assisted extraction (FOV-MAE) was performed for the extraction of entire, partly lyophilised eggs (approx. 50 g). After gel-permeation chromatography (GPC) and adsorption chromatography on deactivated silica gel, the quantitation was performed by GC/ECD on two capillary columns of different polarity. Compounds of technical toxaphene (CTTs) were determined after separation of the PCBs. The sample clean-up method was validated with certified reference material SRM 1588. In general, skua eggs revealed higher organochlorine levels than penguin eggs. Mai...

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. (C) 1999 Elsevier Science Ltd, All rights reserved.

Enantioselective gas chromatography (GC) with 5 modified cyclodextrins was applied to chiral organochlorines. A prerequisite for determining GC elution orders of enantiomers is the availability of enantioenriched standard solutions. In addition to compounds reported before (e.g., α-HCH, PCB 174, oxychlordane), we determined the sign of optical rotation of enantioenriched solutions of e-aeee-pentachlorocyclohexene-1 (β-PCCH), perdeuterated α-HCH (α-PDHCH), perdeuterated β-PCCH, and the persistent compound of technical toxaphene—2-exo,3-endo,5-exo,9,9,10,10-heptachlorobornane (B7-1453)—by liquid chromatography (LC) with a chiral detector. An enantioenriched solution of (β-PCCH was obtained by enantioselective degradation of α-HCH with (-)-brucine. In addition to forming an enantiomeric excess of (-)-α- HCH, we formed enantioenriched (+)-(α-PCCH. In a similar study, α-PDHCH showed the same behavior with respect to enantioselectivity. Dextrorotation of an enantioenriched solution of B7-1453 was also confirmed by LC with a chiral detector. Enantioseparation of chiral organochlorines on 5 chiral stationary phases resulted in several reversed elution orders. These results indicate that a careful check of elution orders of organochlorine enantiomers is necessary prior to comparison of literature data for the study of enantioselective processes in the environment.

Improvements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins responsible for paralytic shellfish poisoning (PSP). The PSP toxin content of two strains of Alexandrium 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.

The separation of the enantiomers of the compounds of technical toxaphene (CTTs) on heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin (β-TBDM) has been studied by gas chromatography with electron-capture detection (GC-ECD). Enantiomers of eight of the nine CTTs under investigation were separated on this chiral stationary phase. Separations of the enantioners of CTTs have hitherto been achieved ontert-butyldimethylsilylated β-cyclodextrin (β-BSCD). The chiral resolution values and separation factors of the CTTs on β-TBDM have been compared with those obtained on β-BSCD. Although several components coeluted, enantioselective determination of three CTTs was possible in an extract of seal blubber. For each CTT the first-eluting enantiomer was enantioenriched. Enantioselective accumulation of2-endo, 3-exo,5-endo,6-exo,-8,8,9,10-octachlorobornane (B8-1412) in biota has been established for the first time.

Retention times of the 209 polychlorinated biphenyls (PCBs) were determined by gas chromatography in combination with electron ionization mass spectrometry on a newly developed stationary phase which was suitable to 380°C. The so-called Thermocap A phase consisted of 50% methyl–50% phenyl polysiloxane which was prepared by using an in situ process. On Thermocap A, the sum of the McReynolds constants was five times lower than on conventional equimolar methyl–phenyl phases distributed with the tradenames CP-Sil 24 or DB-17. The Thermocap A column showed some unique elution orders of PCB congeners. For example, PCB 133 eluted in front of PCB 136 which was not reported on any other stationary phase before. Most of the PCB congeners which are part of national regulations in Europe eluted without interference from the Thermocap A phase. The changed elution order and the high-temperature stability recommends the use of the Thermocap A phase for PCB analyses alone or as a confirmatory column.

Polychlorinated biphenyls (PCBs), oxychlordane, DDT and its metabolites, hexa-chlorobenzene (HCB), hexachlorocyclohexanes (HCHs), and compounds of technical toxaphene (CTTs) were quantified by gas chromatography and electron capture detection in livers of ten arctic foxes (Alopex lagopus) and in liver and adipose tissue of a polar bear (Ursus maritimus) sampled in Iceland. Enantiomeric ratios of oxychlordane and other chiral organochlorine were determined after gas chromatographic enantioseparation on two chiral stationary phases. The organochlorine pattern of the livers of arctic foxes was dominated by oxychlordane. The enantiomeric ratio of oxychlordane was >1 with the exception of the sample with the highest oxychlordane level. This result was confirmed by gas chromatography-mass spectrometry. PCB congeners and other organochlorines were only lowly abundant. PCB congeners were topped by PCB 180. The samples were from two independent populations, one feeding on the marine and the other feeding ...

A heptachloro compound of technical toxaphene (CTT), recently isolated from the technical mixture Melipax, was enantioseparated by application of gas chromatography/mass spectrometry (GC/MS) with a chiral stationary phase. Using tert-butyldimethylsilylated β-cyclodextrin and two different ionization techniques, the first eluted enantiomer of 2-exo,3-endo,5-exo,9,9,10,10-heptachlorobornane (B7-1453) was found in significantly more abundance. Furthermore, the enantiomeric ratio (ER) of 1.26 ± 0.03 was reproduced after GC-ECD analysis on another chiral stationary phase, heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin. This confirms that B7-1453 was not present in racemic composition in the technical product. At present it is not clear if other technical products also contain B7-1453 and other compounds in nonracemic composition. Several synthesized CTT standards, however, showed racemic composition. An ER of 1.0 for B7-1453 was determined in a cod liver extract. Assuming that B7-1453 was...

A precise quantification of toxaphene residues of environmental samples by gas chromatography/ electron capture detection (GC/ECD) requires the separation of the bulk of the polychlorinated biphenyls (PCBs) from the compounds of technical toxaphene (CTT) fraction. For this reason, a PCB/CTT group separation on silica was developed 88-1413 (Parlay #26) and B7-515 (Parlay #32) eluted as first and last out of ten important CTT standards, and can be used to determine the elution volume of the CTT fraction on silica. GC/ECD quantification of CTTs was possible after separation of PCBs on 8.0 g activated silica eluted with 48 mL n-hexane followed by quantitative elution of CTTs with n-hexane/toluene (65:35; v/v). This method is a compromise between separation efficiency and consumption of material. Finally, eight CTTs were quantified in cod liver samples from Iceland and the Baltic Sea.