Bangladesh is a tropical country of large alluvial plain with 1.3 million freshwater ponds and lakes and has a proper environment for luxuriant growth of cyanobacteria. Algal bloom of Microcystis aeruginosa occurred in an aquaculture pond in Gazipur, Dhaka. Bloom sample was collected and filtered through a glass fiber filter. Methanol-water extract of filtered cells was analyzed by high performance liquid chromatography (HPLC) with UV, MS and MS-MS detection, detected three types of microcystins viz., Microcystin-RR, Microcystin-YR and Microcystin-LR and those were confirmed by HPLC-MS. The amount of MC-LR was the highest (33.2 μg L -1 ) followed by MC-RR (9.03 μg L -1 ) and MC-YR (5.23 μg L -1 ). The concentration of microcystins was well above the WHO provisional guideline value of 1 μg L -1 MC-LR. Further investigations need to characterize other types of microcystins from bloom forming cyanobacteria and their effect on human health and cultured fish in Bangladesh.

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.

Occurrence and toxic profiles of paralytic shellfish toxins (PST) in the chocolata clam Megapitaria squalida were investigated. From December 2001 to December 2002, 25 clams were obtained monthly from Bahia de La Paz, Gulf of California. Additionally, net (20 microm) and bottle phytoplankton samples were also collected to identify toxic species. Toxins were analyzed by HPLC with post-column oxidation and fluorescence detection. Toxicity in the clam was low and varied from 0.14 to 5.46 microg/STXeq/100 g. Toxicity was detected in December, March, April, June, and August. Toxin profile was composed mainly by STX, GTX2, GTX3, dcGTX2, dcGTX3, C2, dcSTX and B1. Gymnodinium catenatum was the only PST-producing dinoflagellate identified in the phytoplankton samples throughout the study period. G. catenatum was observed mainly in net samples from December 2001 to December 2002; however, in bottle samples, G. catenatum was only observed in five months. Highest abundance (2600 cells l(-1)) was observed in March and the lowest (160 cells l(-1)) in June. G. catenatum mainly formed two-cell chains and rarely four or eight. The presence of PST in net phytoplankton samples support the fact that G. catenatum is the main source of PST in the clams. This study represents the first report of PST toxins in the chocolata clam from Bahia de La Paz.

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 saxitoxins. 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.

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.

Various microcystins (MCs) were isolated from extracts ofMicrocystis aeruginosa grown in lake Thang Cong, Hanoi, Vietnam.

Levels and distribution of eight compounds of technical toxaphene (CTTs) were determined in different marine species (seals, cetaceans, birds, and fish). The eight CTTs included six commercially available and two chlorobornanes prepared in our lab. These congeners were present in all investigated samples. In agreement with earlier studies, the octachlorobornane B8-1413 (P-26) and the nonachlorobornane B9-1679 (P-50) were the most abundant congeners in most of the samples. In seal blubber, B8-1413 (P-26) and B9-1679 (P-50) contributed with up to approximately 80% (Weddell seal) to the sum of the eight CTTs. In seals from the northern hemisphere the nonachlorobornane was more abundant while in those from the southern hemisphere (Antarctic and Namibia), the octachlorobornane B8-1413 (P-26) usually appeared at higher concentrations. Depending on the species the contribution of the other congeners varied significantly. B9-1025 (P-62) ranged from 2-20%, B8-1412 was found at 4-25% with highest contribution in birds, and B8-2229 (P-44) was found at 5-15%. The remaining three congeners B7-1453, B8-1414 (P-40), and B8-1945 (P-41) were lower abundant except B8-1414 (P-40) which was found at high contribution in liver and kidney of birds. The sum of the eight CTTs ranged from 4 microg/kg to 1.4 mg/kg, depending on the species and region. In most of the seal blubber samples, PCBs and DDT were more abundant (factor 2-20) but Antarctic Weddell seals showed higher CTT levels than PCBs and DDT.

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.

A Cl(7) component of technical toxaphene (CTT), previously detected in marine mammals and fish and referred to as "7-1", was isolated from contaminated estuarine sediment using preparative solid-liquid chromatography followed by reversed-phase HPLC. The structure of this compound, elucidated by GC/MS and (1)H NMR, was 2-endo,3-exo,5-endo,6-exo,8,8,10-heptachlorobornane (hereafter referred to as B7-1000). This newly identified CTT eluted in the nonpolar fraction from silica and shares the alternating endo-exo chlorine substitution pattern with other relatively nonpolar, persistent congeners (e.g., B8-1413 and B9-1679). Based on ECNI-MS response, levels of B7-1000 in tissue samples of various higher organisms including humans were as high as 16% of B8-1413. Enantioselective determination of B7-1000 using a modified cyclodextrin chiral stationary phase (beta-BSCD) resulted in enantiomer ratios that were depleted in adipose tissue of a marine bird (skua) and Weddell seal blubber (0.3 and 0.5, respectively), but not in elephant seal blubber (1.1). Elucidation of the structure of B7-1000 thus validates previous predictions of persistence based on structure-activity relationships, chromatographic properties, and molecular modeling.

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.

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.

A method based on microwave-assisted extraction (MAE) was developed to extract lipids from fish for the determination of the fatty acid composition. Microwave-assisted extraction was performed with an open-vessel extraction apparatus similar to the system of Soxhlet. The solvent was an equivolume mixture of ethyl acetate and cyclohexane. The solvent forms a ternary azeotrope with water, and water is separated after re-condensation in a water trap. With this technique, extraction can be performed without pre-drying of fish tissues. After lipid extraction, the esterification was performed with trimethylsulfonium hydroxide (TMSH) and the fatty acid methyl esters (FAMEs) were determined by GC/FID. The results were compared with those obtained after liquid-liquid extraction according to Bligh and Dyer. Fillets of mackerel (Scomber scombrus) and livers of cod (Gadus morhua) were used as the sample material. The water content (n=7) of the cod livers was 36.2±1.6% and that of mackerel fillets was 74.5±0.5%. The lipid contents (n=5) using the MAE method were 5.6±0.4% and 62.6±3.1% in mackerel and cod liver, respectively. Relative levels of 58 fatty acids (two co-eluted) were determined with GC/FID on two capillary columns with different polarity. These were 10 saturated, 24 unsaturated, and 24 unidentified peaks which are most likely unsaturated or branched-chain fatty acids. After MAE the composition of the 57 peaks was virtually the same as that obtained with the Bligh and Dyer method. The advantages are, however, that MAE is faster, requires less solvent, and avoids the use of chlorinated solvents and so is more environmentally friendly. Our results confirm that MAE is a well-suited alternative to the Bligh and Dyer method for the extraction of lipids followed by determination of the fatty acid profile.

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.

Enantiomer separation of chiral toxaphene components in biological samples was studied by application of different chiral stationary phases based on modified cyclodextrins. Several pairs of enantiomers were resolved on permethylated β-cyclodextrin (β-PMCD), among them 2-endo,3-exo,5-endo,6-exo,8,8,9,10-octachlorobornane (B8-1412), which was not enantiomerically resolved on tert-butyldimethylsilylated β-cyclodextrin (β-BSCD). The latter column was applied to determine the enantiomer ratios (ERs) of 2-endo,3-exo,5-endo,6-exo,8,8,10,10-octachlorobornane (B8-1413 or P-26) in brain tissue of three seal species. The ER of B8-1413 (P-26) in brain was virtually racemic as well as those of the two persistent and chiral components of technical chlordane, 1-exo,2,2,4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane (trans-nonachlor III or MC 6) and 1-exo,2-endo,3-exo,4,5,6,8,8-octachloro-3a,7,7a-tetrahydro-4,7-methanoindane (U82). In contrast, B8-1412 and 2-exo,5,5,8,9,9,10,10-octachlorobornane (B8-2229 or P-44) were significantly enantiomerically enriched in several samples of high trophic level biota. 2,2,5,5,8,9,9,10,10-Nonachlorobornane (B9-1025 or P-62), a chlorobornane metabolisable by seals and the presumable precursor of B8-2229 (P-44), was also enantiomerically enriched in seal blubber. These results confirm the assumption that some less persistent toxaphene components may be significantly degraded in biological samples. Enantioselective gas chromatography provides the information that such a degradation is happening by the characteristic change of the ratio of the two enantiomers in the respective tissues.

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).

Focused open-vessel microwave-assisted extraction (FOV-MAE), closed-vessel microwave-assisted extraction (CV-MAE), and accelerated solvent extraction (ASE) were used for extraction before determination of organochlorine compounds (polychlorinated biphenyls, DDT, toxaphene, chlordane, hexachlorobenzene, hexachlorocyclohexanes, and dieldrin) in cod liver and fish fillets. Wet samples were extracted without the time-consuming step of lyophilization or other sample-drying procedures. Extractions were performed with the solvent mixture ethyl acetate-cyclohexane (1 + 1, v/v), which allowed direct use of gel-permeation chromatography without solvent exchange. For FOV-MAE, the solvent mixture removed water from the sample matrix via azeotropic distillation. The status of water removal was controlled during extraction by measuring the temperature of the distillate. After water removal, the temperature of the distillate increased and the solvent mixture became less polar. Only the pure extraction solvent allowed quantitative extraction of the organochlorine compounds. For CV-MAE, water could not be separated during the extraction. For this reason, the extraction procedure for wet fish tissue required 2 extraction steps: the first for manual removal of coextracted water, and the second for quantitative extraction of the organochlorine compounds with the pure solvent. Therefore, CV-MAE is less convenient for samples with high water content. For ASE, water in the sample was bound with Na2SO4. The reproducibility for each technique was very good (relative standard deviation was typically <10%); the slightly varying levels were attributed to deviations during sample cleanup and the generally low levels.

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 chlorobornanes 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 strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.

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 μg/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 μg STX equivalent/100 g soft tissue), compared to the regulatory limit (80 μg 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.