Characterization of microcystin-LR removal process in the presence of probiotic bacteria.

Toxic cyanobacteria have been reported in lakes and reservoirs in several countries. The presence of toxins in drinking water creates a potential risk of toxin transference for water consumers. Besides chemical and physical methods of cyanotoxin removal from water, biodegradation methods would be useful. The aim of the current study was to identify bacterial removal mechanisms of the hepatotoxin microcystin-LR. This was studied by testing the hypothesis of enzymatic degradation of microcystin-LR in the presence of probiotic lactic acid bacterial and bifidobacterial strains and the participation of the proteolytic system of the bacteria in this process. The results suggest that extracellularly located cell-envelope proteinases are involved in the decomposition of microcystin-LR. In particular, a correlation between proteolytic activity and microcystin removal was found and both these parameters were dependent on glucose as an energy source. In addition, EDTA, which was indicated as a main inhibitor of proteinases of the investigated strain, was shown to limit the rate of microcystin removal. The removal of microcystins was shown to be different from the known microcystin-degradation pathway of Sphingomonas. (14)C-labeled microcystin was not found inside the cells and bacterial cell extracts were not able to remove the toxin, which supports the involvement of extracellularly located proteinases. The results confirm the hypothesis of enzymatic degradation of microcystins in the presence of probiotic bacteria.

Legal and security requirements for the air transportation of cyanotoxins and toxigenic cyanobacterial cells for legitimate research and analytical purposes.

Cyanotoxins are now recognised by international and national health and environment agencies as significant health hazards. These toxins, and the cells which produce them, are also vulnerable to exploitation for illegitimate purposes. Cyanotoxins are increasingly being subjected to national and international guidelines and regulations governing their production, storage, packaging and transportation. In all of these respects, cyanotoxins are coming under the types of controls imposed on a wide range of chemicals and other biotoxins of microbial, plant and animal origin. These controls apply whether cyanotoxins are supplied on a commercial basis, or stored and transported in non-commercial research collaborations and programmes. Included are requirements concerning the transportation of these toxins as documented by the United Nations, the International Air Transport Association (IATA) and national government regulations. The transportation regulations for "dangerous goods", which by definition include cyanotoxins, cover air mail, air freight, and goods checked in and carried on flights. Substances include those of determined toxicity and others of suspected or undetermined toxicity, covering purified cyanotoxins, cyanotoxin-producing laboratory strains and environmental samples of cyanobacteria. Implications of the regulations for the packaging and air-transport of dangerous goods, as they apply to cyanotoxins and toxigenic cyanobacteria, are discussed.

Assimilation and depuration of microcystin-LR by the zebra mussel, Dreissena polymorpha.

Zebra mussels (Dreissena polymorpha) are an important component of the foodweb of shallow lakes in the Netherlands, amongst others in Lake IJsselmeer, an international important wetland. Large numbers of ducks feed on these mussels in autumn and winter. The mussels are filter feeders and are exposed to high densities of cyanobacteria in summer and autumn. Mussels and cyanobacteria both thrive in Lake IJsselmeer. Apparently the mussels are somehow protected against accumulation of harmful quantities of cyanobacterial toxins. In this study, we investigated the assimilation of the cyanobacterial toxin microcystin-LR (MC-LR) in zebra mussels when fed the toxic cyanobacterium Microcystis aeruginosa as sole food or in a mixture with the eustigmatophyte Nannochloropsis limnetica. After 3 weeks of assimilation we studied the depuration of MC-LR during 3 weeks when the food of the mussels was free of cyanobacteria. These assimilation/depuration experiments were combined with grazing experiments, using the same food treatments. Microcystins were analyzed using liquid chromatography-mass spectrometry (LC-MS); in addition, covalently bound MC were analyzed using the MMPB method. The mussels showed higher clearance rates on Microcystis than on Nannochloropsis. No selective rejection of either phytoplankton species was observed in the excretion products of the mussels. Zebra mussels fed Microcystis as single food, assimilated microcystin-LR relatively fast, and after 1 week the maximum value of free unbound microcystin assimilation (ca. 11 microg g DW(-1)) was attained. For mussels, fed with the mixed food, a maximum of only 3.9 microg g DW(-1) was recorded after 3 weeks. Covalently bound MC never reached high values, with a maximum of approximately 62% of free MC in the 2nd week of the experiment. In the depuration period microcystin decreased rapidly to low values and after 3 weeks only very low amounts of microcystin were detectable. The amount of toxin that accumulated in the mussels would appear to be high enough to cause (liver) damage in diving ducks. However, death by exposure to microcystin seems unlikely. Mussels seem efficient in minimizing the assimilation of microcystin. If it were not for this, mass mortalities of ducks in shallow lakes in the Netherlands would presumably occur on a much more widespread scale than is currently observed.

Uptake and accumulation of dissolved, radiolabeled nodularin in Baltic Sea zooplankton.

The mass occurrence of toxic cyanobacteria is a recurrent phenomenon in the Baltic Sea. Grazers may obtain toxins either through ingestion or by direct exposure to dissolved toxins. Despite this, there is little knowledge about the accumulation of cyanobacterial toxins in planktonic organisms present during these blooms. Toxin analyses of tissue samples are complicated to carry out and, because of the small size of microscopic planktonic organisms, often difficult to execute. Therefore, we wanted to use a precise and sensitive method to study toxin uptake and accumulation in zooplankton. We used chemically tritiated nodularin, (3)H-dihydronodularin, to study the uptake of dissolved nodularin, a cyanobacterial hepatotoxin produced by Nodularia spumigena. Cultures of the calanoid copepods Acartia tonsa and Eurytemora affinis, and an oligotrich ciliate Strombidium sulcatum were exposed to (3)H-dihydronodularin in filtered seawater, using naturally occurring concentrations of dissolved nodularin (5 microg L(-1)). All three species took up measurable amounts of radiolabeled nodularin. After 48 h we detected 0.37 +/- 0.22 microg toxin g C(-1) (mean +/- sd) in A. tonsa and 0.60 +/- 0.15 microg toxin g C(-1) in E. affinis, whereas 1.55 +/- 0.50 microg toxin g C(-1) was detected in S. sulcatum after 24 h. The minimum bioconcentration factor (BCF) of (3)H-dihydronodularin was 12 for A. tonsa and 18 for E. affinis. For S. sulcatum our results indicate a maximum BCF of 22. However, because the uptake studies for this species were done in the presence of bacteria, possible particulate transfer cannot be excluded. Nevertheless, our results indicate that dissolved nodularin can be taken up by planktonic organisms. Therefore, the vectorial transport of dissolved toxins to higher trophic levels seems possible, even if some planktonic grazers would avoid feeding on toxic cyanobacteria filaments.