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Nodularia spumigena Mertens ex Bornet & Flahault 1886 (Cyanophyceae) frequently forms harmful blooms in the Baltic Sea, and the toxin nodularin has been found in calanoid copepods during the blooms. Although nodularin has been found at higher trophic levels of the food web, no available information exists about the role of the microbial loop in the transfer of nodularin. We followed the transfer of nodularin to the copepod Eurytemora affinis during conditions that resembled initial 'pre-bloom' (Expt 1) and late stationary (Expt 2) phases of a N. spumigena bloom. The experiments were carried out using natural plankton communities spiked with cultured N. spumigena and grown in laboratory mesocosms, and E. affinis, which were isolated from the Baltic Sea and had no prior contact with nodularin. The plankton community was divided into 6 size fractions as follows: <150, < 45, < 20, <10, < 3 and < 0.2 µm, in which E. affinis was incubated for 24 h. Ingestion and clearance rates, food selection and faecal pellet production were based on microscopical analyses. Nodularin was measured with HPLC-MS with electrospray ionization in the copepods, as well as in dissolved and particulate fractions before and after incubation. We found that nodularin accumulated in copepods in all the plankton size fractions. The copepods contained nodularin concentrations of 14.3 ± 11.6 (mean ± SD) and 6.6 ± 0.7 pg ind.-1 after incubation in the <150 µm fraction in Expt 1 and Expt 2, respectively, while the range in the smaller size fractions was from 1.3 ± 2.8 to 5.7 ± 1.3 pg ind.-1 . Nodularin was transferred to the copepods through 3 pathways: (1) by grazing on filaments of small N. spumigena, (2) directly from the dissolved pool, and (3) through the microbial food web by copepods grazing on ciliates, dinoflagellates and heterotrophic nanoflagellates. The relative importance of direct grazing on small N. spumigena filaments varied from moderate to insignificant. The microbial loop was important in nodularin transfer to higher trophic levels. Our results suggest that the importance of the microbial loop in harmful algal bloom (HAB) toxin transfer may be underestimated both in marine and freshwater systems.

Key WordsColumn liquid chromatography-mass spectrometry Diarrhetic shellfish poisoning Amnesic shellfish poisoning Brevetoxins and pectenotoxins Spirohdes and gymnodimine SummaryA method is proposed for the simultaneous determination of amnesic shellfish poisoning (ASP) toxin, diarrhetic shellfish poisoning (DSP) toxins, spirohdes, azaspiracids (AZP), pectenotoxins (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 va riel",/of g rou ps was determined.

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