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.

Adrenoceptor activity of muscarinic toxins identified from mamba venoms.

BACKGROUND AND PURPOSE: Muscarinic toxins (MTs) are snake venom peptides named for their ability to interfere with ligand binding to muscarinic acetylcholine receptors (mAChRs). Recent data infer that these toxins may have other G-protein-coupled receptor targets than the mAChRs. The purpose of this study was to systematically investigate the interactions of MTs with the adrenoceptor family members. EXPERIMENTAL APPROACH: We studied the interaction of four common MTs, MT1, MT3, MT7 and MTα, with cloned receptors expressed in insect cells by radioligand binding. Toxins showing modest to high-affinity interactions with adrenoceptors were additionally tested for effects on functional receptor responses by way of inhibition of agonist-induced Ca²âº increases. KEY RESULTS: All MTs behaved non-competitively in radioligand displacement binding. MT1 displayed higher binding affinity for the human α(2B)-adrenoceptor (IC50 = 2.3 nM) as compared with muscarinic receptors (IC50 ≥ 100 nM). MT3 appeared to have a broad spectrum of targets showing high-affinity binding (IC50 = 1-10 nM) to M4 mAChR, α(1A)-, α(1D)- and α(2A)-adrenoceptors and lower affinity binding (IC50 ≥ 25 nM) to α(1B)- and α(2C)-adrenoceptors and M1 mAChR. MT7 did not detectably bind to other receptors than M1, and MTα was specific for the α(2B)-adrenoceptor. None of the toxins showed effects on ß1- or ß2-adrenoceptors. CONCLUSIONS AND IMPLICATIONS: Some of the MTs previously found to interact predominantly with mAChRs were shown to bind with high affinity to selected adrenoceptor subtypes. This renders these peptide toxins useful for engineering selective ligands to target various adrenoceptors.

Nucleotide excision repair impairment by nodularin in CHO cell lines due to ERCC1/XPF inactivation.

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.

Effect of glucose and incubation temperature on metabolically active Lactobacillus plantarum from dadih in removing microcystin-LR.

Lactobacillus plantarum strains IS-10506 and IS-20506 isolated from Indonesian traditional fermented milk, dadih, were screened for their ability to remove the cyanobacterial toxin microcystin-LR (MC-LR) from aqueous solution (100 microg/L) at 22 and 37 degrees C. The objective was to study the main environmental factors influencing the metabolic activity of L. plantarum in MC-LR removal. Residual MC-LR was quantified using HPLC. Non-viable cells inactivated by boiling or acid showed only low MC-LR removal (<23 %). Viable L. plantarum strain IS-10506 at pH 7, at 22 and 37 degrees C was able to remove MC-LR, 64% and 43%, respectively, after 30 h. Strain IS-20506 at pH 7, at 22 and 37 degrees C removed 92% and 45 %, respectively, after 30 h. At 37 degrees C, the removal of MC-LR was lower than at 22 degrees C. Supplementation with glucose (1%, 2%, and 3%, w/v) resulted in faster and higher removal of MC-LR at 37 degrees C, while at 22 degrees C it did not improve MC-LR removal. In the presence of 1 % glucose, IS-10506 and IS-20506 demonstrated significantly the most efficient removal of 80% and 65% of applied MC-LR, after 25 and 20 h, respectively, at pH 7, 37 degrees C. Viable cells as well as active metabolism play important roles in removing MC-LR. This finding offers new and economical tools for decontaminating microcystin containing water.

Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution.

AIMS: The primary objective of this study was to compare the removal of cadmium, lead, aflatoxin B1 and microcystin-LR from aqueous solution by Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii shermanii JS and Bifidobacterium breve Bbi99/E8, separately and in combination. METHODS AND RESULTS: The removal of toxins and heavy metals was assessed in batch experiments. The removal of all compounds was observed to be strain specific. The removal of lead by a combination of all the strains used was observed to be lower than could be predicted from the removal by single strains (P < 0.05). A similar trend was also observed with the other compounds studied. CONCLUSIONS: The results show that the toxin-removal capacity of a combination of strains of lactic acid bacteria is not the sum of their individual capacities. Therefore, pure single strains should be used when the goal is to remove single compounds. The use of combinations of strains may be beneficial when several compounds are removed together. This needs to be studied in future experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria have been identified as potent tools for the decontamination of heavy metals, cyanotoxins and mycotoxins. The results of this study should be considered when selecting combinations of bacteria for the simultaneous removal of several toxic compounds.

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus.

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.

Development of new probiotics by strain combinations: is it possible to improve the adhesion to intestinal mucus?

We evaluated the ability of commercial probiotic strains (Lactobacillus rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve 99, and Propionibacterium freudenreichii ssp. shermanii JS) to adhere alone or in different combinations to immobilized mucus. Probiotic combinations were clearly able to enhance the adhesion of L. rhamnosus GG, L. rhamnosus LC705, and P. freudenreichii ssp. shermanii JS. For L. rhamnosus GG and P. freudenreichii JS, all the combinations significantly improved adhesion to intestinal mucus, from 29.7 to 34.9% and from 1.9 to 2.3%, respectively. The adhesion of L. rhamnosus LC705 was improved from 0 to 46.4%. The adhesion of B. breve 99 was improved only in combination with L. rhamnosus GG and P. freudenreichii JS. Our results suggest that probiotic combinations could increase the beneficial health effects as compared with individual strains. Combinations of probiotic strains may therefore have synergistic adhesion effects, and such combinations also should be assessed in clinical studies.

Transfer of nodularin to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus L.), and salmon (Salmo salar L.) in the northern Baltic Sea.

Nodularin (NODLN) is a hepatotoxin produced by the cyanobacterium Nodularia spumigena, which occurs regularly in the Baltic Sea. The primary aim of this study was to study the transfer of NODLN to three-spined stickleback (Gasterosteus aculeatus L.), herring (Clupea harengus membras L.), and salmon (Salmo salar L.), which were caught from the northern Baltic Sea between August 2002 and August 2003. Liquid chromatography mass spectrometry (LC-MS) was used for NODLN analysis. NODLN was found in both herring (0-90 microgkg(-1)dw) and three-spined sticklebacks samples (2.8-700 microgkg(-1)dw). The recovery for the spiked stickleback samples in vitro was 28%. Only 1 salmon of a total of 10 contained a small amount of NODLN (10 microgkg(-1)dw). However, the high concentrations in individual stickleback suggest that possible transfer to higher trophic levels deserves more research.

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.

Nodularin-induced genotoxicity following oxidative DNA damage and aneuploidy in HepG2 cells.

The problem of toxicity of Nodularia spumigena to animals and people is of increasing concern, as the incidence of such blooms grows. It was shown that nodularin is a liver carcinogen possessing both initiating and tumor-promoting activities. However, the mechanisms by which this toxin damages the DNA and induces liver cancer are not well understood. The aim of the present study was to investigate the DNA damaging properties of nodularin. The effect of different doses of nodularin (1-10 microg/ml) on DNA damage was determined in HepG2 cells after 6, 12, 24 and 48 h of the treatment. The modified comet assay in conjunction with Fpg (ROS-induced DNA damage) and FISH-micronucleus assay (clastogenic and/or aneugenic activities of nodularin) were applied. In addition the occurrence of apoptosis was estimated by the morphological analysis of chromatin condensation and the annexin method using flow cytometry. We found that nodularin induces oxidative DNA damage by oxidation of purines and increases the formation of centromere positive micronuclei due to aneugenic activity. In addition to genotoxic properties, nodularin exerts a cytotoxic activity by inducing apoptosis in HepG2 cells. These results suggest a causative role for nodularin in the process leading to the accumulation of genetic alterations which may be implicated in carcinogenesis.

Heterogeneity of nodularin bioaccumulation in northern Baltic Sea flounders in 2002.

The cyanobacterial hepatotoxin nodularin is abundantly produced by the cyanobacterium Nodularia spumigena in the Baltic Sea during July-August. Nodularin is a potent hepatotoxin and a tumour promoter, distributed in various Baltic Sea environmental compartments, especially food webs involving mussels. Flounders receive nodularin through consumption of blue mussels. In this study nodularin concentrations in individual flounders (liver) were examined between July and September 2002 (six sample sets, four to 10 samples/set), providing information about contribution of sampling on estimates of bioaccumulation intensity. Toxin was determined using liquid chromatography/mass spectrometry (LC/MS) and enzyme-linked immunosorbent assay (ELISA). Additionally, liver histopathology was examined. Observed toxin concentrations were ND-390 microg kg(-1) dw (LC/MS) and 20-2230 microg kg(-1) dw (ELISA), with maximum concentrations in September (ELISA). The ELISA protocol generally resulted in higher, up to approximately 10-fold, toxin concentrations than LC/MS, with increasing difference toward September. This difference may have originated from different extraction solvents in LC/MS and ELISA, ion suppression in LC/MS, and temporal increase in nodularin metabolites detectable with ELISA. The differences in toxin concentrations between individual liver samples were considerable with relative standard deviation values of 20-154% (LC/MS) and 28-106% (ELISA). Since the precision of the ELISA method employed was <25% and that of LC/MS <10%, it can be concluded that the largest source of error in bioaccumulation estimates may be an inadequate number of samples. Although there were tissue lesions in several liver samples, occurrence of lesions was not related to toxin concentrations.

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.

The toxicities of a polyunsaturated fatty acid and a microcystin to Daphnia magna.

Polyunsaturated fatty acids (PUFAs) are essential components of zooplankton diets. However, studies with PUFAs from cyanobacteria indicate toxic properties. We investigated the toxicity of the PUFA gamma-linolenic acid and the cyanobacterial peptide toxin microcystin-LR to Daphnia. The PUFA was acutely toxic at a concentration of 9 micrograms ml-1. The effect of microcystin-LR was not statistically significant at the concentration used (3 micrograms ml-1), but an additive effect with the PUFA was observed. Relative to LC50-values of well-known pollutants, the PUFA was intermediately toxic. The activity equaled that of microcystin-LR, which is commonly treated as one of the most potent cyanobacterial toxins. Our results suggest that the toxic properties of PUFAs deserve more attention.

Time-dependent accumulation of cyanobacterial hepatotoxins in flounders (Platichthys flesus) and mussels (Mytilus edulis) from the northern Baltic Sea.

There is only limited information about the accumulation of algal toxins in aquatic organisms in the Baltic Sea. In this study we measured total cyanobacterial hepatotoxin levels in blue mussel (Mytilus edulis) and flounderi (Platichthys flesus) tissues. Flounder were caught with gillnets from the western Gulf of Finland during July and August 1999. Blue mussels were collected from an enclosure at 3 m depth and from an artificial reef (wreck, 25-35 m depth) in the western Gulf of Finland between June and September 1999. Flounder liver and muscle samples and soft tissues of mussels were analyzed for the cyanobacterial hepatotoxins (nodularin, NODLN and/or microcystins, MCs) using an enzyme-linked immunosorbent assay (ELISA). Results showed a time-dependent accumulation of hepatotoxins in flounder and mussels. In flounder, the maximum concentration 399 +/- 5 (sd) ng NODLN or MC/g dry weight (dw) was found in the liver of specimens caught on 21 August 1999. No hepatotoxins were detected in muscle samples. The maximum concentration of 2150 ng +/- 60 (sd) ng hepatotoxin/g dw was found in the mussel soft tissues collected on 20 August 1999. Temporal NODLN or MC trends indicated depuration of cyanobacterial hepatotoxin from mussels at surface level and an increase in NODLN or MC concentrations in those from the sea bed. These studies showed that despite the low cyanobacteria cell numbers the cyanobacterial hepatotoxins can accumulate in flounder and mussels. This may allow the further transfer of cyanobacterial hepatotoxins in the food web.

Detection of nodularin in flounders and cod from the Baltic Sea.

The brackish water cyanobacterium Nodularia spumigena regularly forms waterblooms in the Baltic Sea. Many N. spumigena strains can produce nodularin, a hepatotoxic penta-peptide, which has caused several animal poisonings in the Baltic Sea area. To improve our understanding of nodularin bioaccumulation in aquatic organisms this study measured nodularin in flounder and cod caught from the Baltic Sea. Flounders were collected from the western Gulf of Finland in July 1996, September 1997, and September 1998, and from the Gulf of Bothnia in August 1997 and September 1998. Flounders were also collected from the coastal areas of Sweden in the Baltic Proper during September 1998. Cod were caught from the southern Baltic Sea in August 1998. Livers and muscles of the 1997 fish were isolated, extracted, and analysed for nodularin using high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 1 (PP1) inhibition assay. Approximately 30-70 ng of nodularin/g dry weight (maximum value 140 ng/g) were found in the liver tissue samples by ELISA and PP1 inhibition. These concentrations were below the detection limit of HPLC. PP1 assay showed inhibition also in muscle samples, but this may due to other compounds present in the muscle extracts rather than NODLN or due to matrix interference. The recovery of nodularin from liver tissue with ELISA and PP1 assays was about 30%. Nodularin concentrations in samples are not corrected for recovery. Although the concentrations of nodularin found in this study are low further studies of nodularin are needed to assess possible bioaccumulation in brackish water food webs.

High-performance liquid chromatographic separation of microcystins and nodularin, cyanobacterial peptide toxins, on C18 and amide C16 sorbents.

Four C18 columns and a novel amide C16 column were assessed in the HPLC separation of eight microcystins and nodularin-R. Gradient mobile phases of acetonitrile combined with trifluoroacetic acid, formic acid or ammonium acetate were compared. Special attention was paid to the resolution of four possible coeluting microcystin pairs. Generally speaking, the acidic mobile phases were superior to the ammonium acetate-based mobile phase in terms of resolution and selectivity. The amide C16 column had the best overall performance and unique selectivity properties.

A time-resolved fluoroimmunometric assay for the detection of microcystins, cyanobacterial peptide hepatotoxins.

An immunoassay based on the time-resolved fluorometry (TR-FIA) was developed for microcystins, cyanobacterial peptide hepatotoxins. The assay was performed in a competitive mode and it utilised the monoclonal antibodies raised against microcystin-LR, and a europium chelate of microcystin-LR as a competitive antigen. The sensitivity of the assay was 0.1microg/l. The detection method of TR-FIA was compared to a commercially available kit based on the enzyme-linked immunosorbent assay (ELISA). The same level of sensitivity could be obtained with TR-FIA (in a non-optimised system). The simplified method of TR-FIA leads to a shorter analysis time.

Production and specificity of mono and polyclonal antibodies against microcystins conjugated through N-methyldehydroalanine.

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae). Their toxicity is associated with specific inhibition of intracellular protein phosphatases type-1 and type-2A (PP1 and PP2A, respectively). We have developed a battery of antibodies to microcystins using chemical modification (aminoethylation) of one of its core amino acids, N-methyl-dehydroalanine. The developed antibodies displayed different reactivities to closely related MCs. Selected monoclonal antibodies were used for quantitative competitive ELISA assays. The analytical sensitivity of these assays was up to 1 ng/ml. Comparison of the developed ELISA tests with HPLC-based measurements of MCs in laboratory and field samples showed a good correspondence between the results yielded by these two methods. The antibodies developed by this technique provide the means for developing extremely sensitive and specific analytical assays for direct measurement of toxins in cyanobacterial or water samples.